in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (1), pp. 001-004, january, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. editorial why producing pigs in china is a big challenge egbabor oghale accepted 26 january, 2013 the chinese love pork – and in the future, more chinese will love even more pork. with many limiting factors, it is not hard to predict that pig production in china in the near future is going to become an increasingly difficult job. what challenges lie ahead? key words: pig, pig farming, pig industry, pork production, pork and processing the pig industry is considered of national importance to the chinese economy. pork production in china, however, is facing enormous challenges, and as a result, the chinese government is forced to implement changes to guide the industry to the future. the origin of these challenges lies in the balance between the limitations of the available water and land, suitable for agriculture in general (figure 1), the still enormous importance of backyard pork production and the steep rise in the level of urbanisation. pork production in china is enormous in numbers and in percentage of total meat consumed. in 2012 it was estimated that 50.7 million breeding sows and 470 million pigs were present in china. in 2012, the country's pork production amounted to 53 million tonnes – compare this to the total meat production, which was 84 million tonnes. a pig farm in china. the country had a dazzling 470 million pigs in 2012. within a relatively short period of time, the rapid urbanisation process will force the majority of pork to be raised by industrialised pork producers, replacing backyard production. commercial producers will feed the rapidly growing urban population who will purchase their ready-to-cook, pre-packed pork in local supermarkets. this rapidly growing urban population will also require vegetables and rice and water for domestic purposes, to go with their pork. exploring it may not come as a surprise that chinese businessmen have been exploring opportunities to acquire companies to produce basic food requirements outside of china. they could then export these food materials to china. shuanghui purchasing smithfield foods is a good example of this development. figure 2 (see next page) shows the potential of such a takeover. it involves around 24 million usa produced swine carcasses annually. changes the chinese government has announced changes that will affect their pork producing industry. by 2016 the chinese are planning to have at least a 100 breeding farms enrolled in their national nucleus breeding farm  programme, to improve the performance of their breeding farms. this is without neglecting their genetic potential present in their national breeds of which the meishan breed is a good example. another, and potentially major, new announcement of the chinese authorities relates to the proposed regulations to the environmental impact of pork production. with pollution in general being one of the bigger concerns in china, this new path to follow will for sure lead to an increase in production costs. as a consequence a certain number of small producers will be forced to step out of the pork business. this in turn will lead to a higher percentage of pigs that are kept on industrialised pork producing facilities, accelerating the consolidation in the swine industry in china. relatively small changes can be the origin of major shifts in the number of farms and the number of pigs per farm. all around the world we have seen the domino effect of this type of 'minor' changes. china's disease situation prrs virus is very commonly present in china and is causing a variety of different clinical problems. dr qigai he, college of veterinary medicine of the huazhong agricultural university in wuhan, spoke about this topic at the latest asian pig veterinary society (apvs) congress in ho chi minh city, vietnam. the main consequence of a prrs virus infection, he said, is the immune-deficiency that pigs are suffering from when infected with prrs virus leading to an increase in secondary (both bacterial and viral-) infections. in fact, in the beginning, when the high pathogenic prrs (hp-prrs) virus was devastating the pork industry in china (see figure 3), all kinds of other different pathogens were isolated apart from prrs virus. this high level of co-infections was also the reason for many discussions in that period and the reason for the prolonged time required to find the real cause of the problem. scientific work by baoqing guo and kelly lager, in 2012, using reverse genetics to restore the original hp-prrs field isolate, clearly showed the immune-suppressive characteristics of that original isolate. in these experiments, they also could show that the challenged pigs eventually died of infections with pathogens that were naturally present but harmless in immunologically normal or competent pigs. vaccines are only allowed to be used on farms where prrs virus is known to be present. live vaccines are preferred, as inactivated vaccines have failed to show efficacy under chinese conditions. more and more farms aim to rely on biosecurity. other diseases pcvd virus is of equal importance to the chinese pork industry as prrs. the main difference is of course that pcvd can be easily controlled by vaccination. on the chinese market a number of locally produced pcv2 vaccines have been available for some time now and recently a new pcv2d genotype has been described. on the chinese market five pcv2 vaccines can be found, based on different domestic isolates. classical swine fever (csf) or hog cholera control is making great progress in china, according to dr qigai he. all vaccines used are based on the c-strain and are either produced on cell culture or still making use of rabbit-derived material. also an st cells based vaccine is available. with african swine fever (asf) only a doorstep way, effective csf vaccines are important to exclude csf virus infections. this helps identifying a potential asf infection more rapidly. viral diarrhoea in piglets of three to ten days old, leading to high mortality figures, is regarded as a major problem in china. ped virus was frequently seen in these outbreaks but also tge virus and rota virus. aujeszky's disease virus or pseudorabies is controlled successfully in most breeding herds. a five step eradication plan, when completed successfully, leads to certification assuring potential buyers that they buy pigs free from ad virus. foot-and-mouth disease (fmd) virus will continue to be a major threat to the chinese pork industry. with so many fmd infected countries bordering china and with so much cross-border trade, new introductions and infections appear unavoidable. bacterial diseases are often secondary in importance to viral infections. main attention goes to haemophilus parasuis infections and of course to streptococcus suis because of its zoonotic aspects and the human casualties that were seen recently. for actinobacillus pleuropneumoniae, serotypes 1, 2, 3 and 7 are most frequently reported. it is stressed that housing conditions, poor ventilation, lack of all-in/ all-out management and overcrowding are considered to be the main reasons for app infections to occur. another major concern to pork producers, veterinarians and regulatory officials is the failing of antibiotic treatments due to increasing resistance problems. the enormous importance of pork production to china is reflected in the vast number of grants, research projects and other activities developed by the government to support the industry. at agricultural universities, numerous r&d projects are underway aimed at improving diagnostics, making better vaccines, etc. the lack of arable land and water, environmental problems, a growing demand of an ever-increasing urban population might force the  government to shift their focus a bit and allow for a part of their pork demand to be imported. this will solve some of china's land, water and environmental problems and will allow more regular supplies in the supermarkets should a new episode of hp-prrs virus infection occur. the impact on local pork prices in these exporting countries is subject to   speculation. one thing is sure, however – a small percentage for china is a giant amount for mankind. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (5), pp. 001-003, may, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. research paper an appeal against swine flu and its havoc a. n. pandey indian institute of spirituality (iis), india. e-mail: upanishad.an@gmail.com. tel: +91 9441877388, +91 9440944111, +91 4065698764. accepted 09 october, 2013 it is a saying that “prevention is better than cure”. the same principle has to be adopted in case of swine flu and its havoc as till there is absence of any medical and scientific development to prevent and cure the swine flu. after large death toll all over the world due to virus and its sign of spreading beyond human control, drew the attention of many to search any kind of remedial aspects to have some solace by preventing or controlling this. on 10 th evening, while i was meditating, i got a question “whether swine flu could be overcome or cured with the help of yoga or not?” after some time, an intuitive message came that “yes, there are methods available in yogic practice given by spiritual masters (rishis) and same can be used as a preventive approach against swine flu”. after meditation, i fortunately opened a book “a systematic course in the ancient tantric techniques of yoga and kriya” by swami satyananda saraswati of bihar school of yoga (the book reprinted in 2007; inscribed by nischalananda saraswati, uk; emendated by swami mangalmurti saraswati, australia). this book contains ancient spiritual wisdom along with modern scientific wisdom written, reviewed and amended by highly elevated spiritual masters. what is swine flu? in common man’s language, swine flu has following characteristics such as (i) running nose (ii) suffering from cough and cold (iii) having fever (iv) vomiting sensation (v) dizziness (giddiness). the symptoms number (i) and (ii) are mainly related to nostrils; symptom (iii) is mainly related to thyroid glands; symptom (iv) is mainly related to digestive system and symptom (v) is mainly related to respiratory system and central nervous system. the scientific methods of any kind, which can prevent these systems to get affected, will be of use to overcome or control swine flu. fortunately, scientific yogic practices explained in the above book can help as preventive measures for common people against the swine flu virus. at the same time they can be used as therapeutic practice for those who are affected at initial stage. in brief, yogic practices related to nostril, thyroid gland, digestive system, respiratory system and central nervous system are to be utilized to strengthen these systems of the body as a preventive measure to remain immune to the swine flu or related virus of this kind. ancient yogic practice against swine flu scientific yogic kriyas and practices are available to make the body system immune against this kind of virus. after intuitive signal, i thought to bring out those yogic practices available in scripture for the benefit of mankind. jala neti at first glance, i opened the topic of kriya like “jala neti” which provides the methodology to create resistance within human system to face the viruses like swine flu. jala neti is a process of inserting warm salt water through one nostril and let the same to come out through the other nostril under gravitational and natural flow of water. the advantage of neti is to prevent and eliminate colds. it is an effective cure for the common cold along with removal of breeding ground for any kind of viruses. how it works the indication of cold shows something significant when our body is in weak condition. the mucus due to cold around the inner portion of the nostrils makes the good ground for the dangerous and infectious virus to breed; like mosquitoes breed at stagnant water. jala neti cleans the mucus (breeding ground of infectious virus) and makes the capillaries of inner nostril strong to have pure flow of blood rich in oxygen. this will not allow the virus to further sustain for longer. while jala neti cleaning, the virus could be unable to penetrate the defenses of human system; our auto therapeutic could be strong enough to withstand such an attack. otherwise, the virus flourishes in nerve tissues, particularly the olfactory nerves in the nose. during virus attack, neti generally helps by removing the accumulated mucus in the nose which otherwise is a breeding ground for the virus. by neti, the nasal passages work at optimum efficiency and their by help to maintain a healthy body. it is better to remember that breathing through the mouth or insufficient treatment of the inhaled air prior to the entry into the lungs, due to nasal blockage and congestion, can encourage the onset of disease , by allowing germs to infect the lungs , or by generally weakening the state of the health of the body. the other advantages of the neti is to help in curing sinusitis , elements of eyes , nose and throat , tonsillitis , catarrh as well as inflammation of the adenoids and mucus membranes. it is effective in removing, headaches, insomnia and tiredness. jala neti has a subtle influence on the various nerves which end in nasal passages, such as the olfactory bulb and other adjacent nerves which innervate the eyes, ear etc. this has very soothing influence on the brain and can help to relieve such elements such as migraine, epilepsy, depression and tension. neti helps to prevent and cure lung diseases such as asthma , pneumonia , bronchitis , pulmonary tuberculosis etc .respiration becomes much easier which leads to an improved intake of oxygen , improved removal of carbon dioxide and consequently better health. the symptoms of fever and giddiness due to swine flu could be removed when lungs are kept in healthy condition. the above book describes how jala neti is helpful for removal of breeding ground of virus, making thyroid gland and lungs effective. if only this yogic kriya (jala neti) is regularly practiced by an individual (old, young and student), the virus like swine flu can be prevented. to have the firewall against swine flu virus, other techniques of yogic system could be used such as dhauti, simha mudra, relaxation and pranayama along with jala neti. simha kriya (lion’s yawn) another approach described in the yogic book is simha kriya (lion’s yawn).it is a simple process to sit erect, head little pushed back, inhalation in depth and while exhalation take out the tongue fully with sound of “aaah”. the tonsils can function effectively if blood supply around it is pure and rich in oxygen. the lion’s yawn removes the stagnant impure blood around the tonsils and allows having pure blood around it. the same kriya (action) provides the massages in throat region. it can squeeze the stagnant blood and send the same to kidney for purification. by this the blood vessels of the throat are relaxed. the rich oxygen supply in the blood in the tonsil region has wonderful rejuvenation effect; which in turn improves the health of the body and removes the cause of tonsillitis. how it works in the throat there are many vital organs including thyroid glands, the pharynx, the larynx and the salivary glands. all of them receive a wonderful massage with the help of lion’s yawn kriya. these organs provide the essential functions of the body, the improvement in their health through the simple practice of lion’s yawn (simha kriya) can have vast repercussions on the liver. neck is the vital crossroad of the nerves connecting body and brain. also, in the throat region there are various nerve plexuses which control the organs of the throat. this kriya stretches the nerves and remove stagnant blood. this inturn allow an influx of fresh blood. this increases the efficiency in the glands and organs of the body. it also improves the general throat elements. nadi shodhan pranayama (alternate nostril breathing) this practice helps to nourish the whole body with extra supply of oxygen and the carbon dioxide is removed effectively. this process purifies the whole blood system and increases the overall health of the body. it improves the power of the body to resist any kind of diseses. the deep and slow breathing encourages the removal of stagnant air from the lungs. the advantage of nadi shodhan pranayama is to achieve the calmness of the mind by regulating the flow of prana in the body. it helps to remove the blockage of nadis and there by allows the free flow of prana in them. kunjal kriya the process of this kriya is to drink warm salt water until stomach is completely full; then voluntarily expel the same through the mouth. the advantage of this kriya is to get ease feeling in the abdominal region. there is a tendency for the residue of undigested food to lie in the bottom of the stomach after all the easily digested food has passed through the intestine. this is particularly the case if one has a stomach with internal pressure at bottom of stomach. this acts as a reservoir in which fermentation takes place. when one eats the next meal this residue is mixed with incoming food and can pass into the intestines. this can contaminate and poison the system, for such it will be absorbed into the blood stream from the intestines. kunjal kriya throws this fermented waste out of stomach before it can cause the harm. this kriya is sure way of preventing auto poisoning of the body. this kriya expels the water (green colour) which indicates the presence of bile. bile is secreted from the gall bladder into the intestines below the stomach. this kriya provides the wonderful relief by eliminating the accumulation of nauseating and bitter tasting. in addition, it helps the asthmatic people to improve the lung function. this technique provides the best possible wash to the digestive system from the stomach to the mouth. it also helps to eradicate the accumulation of phlegm in the throat region which can become the breeding ground for the virus. relaxation there are various methods of relaxation described in yogic books. basically shavasana relaxes the physiological and psychological aspects. it relaxes the 3-h namely head, heart and hands of our human body mind configuration. a relaxed head allows us to see and relate the world and the people around us in a more realistic light. similarly we can carry our works (actions) more smoothly and attain the happiness in our day to day work. this happens at an emotional level. the emotional conflict is removed with the help of relaxation. otherwise the tensed and emotionally imbalanced mind implies a certain psychosomatic diseases. relaxation of mind/body complex helps to relieve and prevent diseases. the five steps described above are the full proof approach against infectious virus like swine flu. however, at least jala neti, lion’s yawn and relaxation techniques are recommended to have the preventive approach against swine flu. to build a firewall against the harmful virus, one has to adopt all the five steps. an appeal more than 3000 years ago, lord mahavir stated that environment consist of harmful and infectious viruses. the same can be encountered by wearing the mask on the nostril (normally jain monks wear) and remain in hygienic condition. this is not possible in the present era to wear the mask in busy day to day schedule. at least the practice of jala neti, sutra neti, dhauti, relaxation and pranayama can be adopted to encounter the dangerous viruses, specially the virus which spreads the infection. an appeal was made to the world citizens including indians to learn the easiest yogic method of jala neti or sutra neti ( another kind of cleaning the nose ) to have the prevention against any kind of virus , specially infectious. this is easy to learn. if required, any yogic institutions could be contacted for demonstration and learning purposes; as many yogic institutions are available throughout the world, specially the branches of swami vivekananda yoga anusandhana sansthan (deemed university) and bihar school of yoga (internationally known first university in yoga). in case of difficulty, interested persons can contact for any help (either related to clarification or demonstration) to the undersigned on the e-mail addresses thus: (i) upanishad.an@gmail.com, (ii) aiw.an@rediffmail.com and (iii) vedanta1232000@yahoo.co.in in the beginning jala neti practice could be done every alternate day for a fortnight and afterwards same can be done once in a week or fortnight to make the breathing system effective against the viral system. combined role of east and west the yogic practice can be implemented along with whatever precautions and practices given by world health organization (who) and indian medical association. till confidence is built, the importance of yogic practice should be understood and the result of the same could be experienced. the yogic practice works parallel to the vaccination against particular disease. when swine flu is spreading and there is no vaccine so far developed to prevent the infection caused by this; it is better to adopt the easiest yogic practice suggested by our spiritual masters (rishis) to encounter the same. i hope that the who, the common people, medical association, ngos and other government organizations can adopt this prevention without giving any colour of religion as yoga is meant for everyone. i appeal to adopt this practice of yogic kriya not with the sense of propagating yogic practice but with the feeling of utmost requirement of the same to prevent the virus generally not commonly known. reference a systematic course in the ancient tantric techniques of yoga and kriya” by swami satyananda saraswati of bihar school of yoga. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (4), pp. 001-003, april, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication antimicrobial susceptibility and genetic relatedness of salmonella enterica subsp. enterica serovar mbandaka strains, isolated from a swine finishing farm in greece g. filioussis1,2*, e. petridou3, a. johansson2, g. christodoulopoulos1 and s. k. kritas3 1 department of veterinary medicine, university of thessaly, karditsa, greece. 2 institute of veterinary bacteriology, university of bern, switzerland. 3 laboratory of microbiology and infectious diseases, school of veterinary medicine, university of thessaloniki, thessaloniki, greece. accepted 11 november, 2013 the current study investigated the antimicrobial susceptibility of salmonella enterica subsp. enterica serovar mbandaka (salmonella mbandaka) isolated from finishing swines in greece. pulsed-field gel electrophoresis (pfge) was used to examine the genetic relatedness of the isolates. the study was carried out for 1 year as part of a project focusing on antimicrobial resistance of salmonellae recovered from asymptomatic pigs. a total of 400 finishing pigs stabled in 20 swine farms in central greece were included in the study. fecal samples taken directly from the rectum, one sample from each pig, were cultured for salmonella spp. five of the 400 tested finishing pigs, originating from the same herd, were asymptomatic carriers of salmonella mbandaka. all five isolates were resistant to tetracycline, four were resistant to trimethoprim/sulfamethoxazole, and three to ampicillin and amoxicillin/clavulanic acid. in contrast, all five isolates were susceptible to cefuroxime and ceftriaxone, as well as to nalidixic acid, ciprofloxacin, and levofloxacin. all five isolates had indistinguishable pfge patterns. the present study confirms the existence of a nontyphoid salmonella serotype, salmonella mbandaka in asymptomatic carrier pigs in greece. further, the salmonella mbandaka isolates were found to be resistant to several antimicrobials. key words: salmonella enterica subsp. enterica serovar mbandaka, antimicrobial susceptibility, swine, pulsed-field gel electrophoresis (pfge), zoonotic. introduction salmonella spp. is recognized worldwide as important pathogens in the intestinal tracts of both animals and humans. infected animals are usually asymptomatic carriers, a fact that has a major effect on the spread of infection. the number of human cases of salmonellosis caused by the nontyphoid salmonella enterica subsp. enterica serovar mbandaka (salmonella mbandaka) increased during the 1990s. related outbreaks of human infection characterized by diarrhoea have been reported in the united kingdom (reid et al., 1993), italy (fantasia et al., 1989), australia (scheil et al., 1998), and the united states (gill et al., 2003). pigs infected with salmonella *corresponding author. e-mail: gfiliu@vet.uth.gr. tel: 00312106664688fax: 00302107496632. mbandaka, as well as pig products, have been implicated throughout pork production and processing in cases of human infection and clinical disease (davies, 1998). pou-try has also been considered an important source of salmonella mbandaka for humans (hoszowski and wasyl, 2001). a prominent reason for concern with regard to gastroenteritis-causing bacteria is the recognized emergence of antimicrobial resistance among key species. over the past decade, particularly in developing countries, the increase in resistance of animal origin nontyphoid salmo-nellae to broad-spectrum antibiotics such as cephalo-sporins, tetracycline, and quinolones has been extremely worrisome (streit et al., 2003). as a result, attempts are being made to trace salmonellosis outbreaks to contami-nated sources, and numerous typing methodologies have been used. information concerning the prevalence of s. enterica serotypes in greek finishing swine herds has previously been published (leontides et al., 2002; wong et al., 2003). in this study, we report the antimicrobial susceptibility and the genetic relatedness of five salmonella mbandaka isolates originating from healthy finishing swines in a greek herd. materials and methods study design asymptomatic carriers have a major effect on the spread of salmonella infections to humans (wong et al., 2003). therefore our study, carried out from march 2003 to october 2004, was focused on antimicrobial resistance and genetic variation of salmonellae recovered from asymptomatic pigs. samples were collected from 20 swine finishing farms, representing 10% of the swine finishing farms in central greece. twenty finishing pigs from each farm were sampled (almost 5% of the stabled animals), and 400 fecal samples were collected. faecal samples were taken directly from the rectum, one sample from each pig. samples were transported (4 o c) within 4 h to the laboratory. sampling and cultivation 25 g of each faecal sample was diluted in sterile plastic bags with 225 ml of buffered peptone water (bpw, merck kgaa, 64271 darmstadt, germany) and incubated in 37 o c for 24 h. after enrichment (41.5 o c for 24 h) in modified semi-solid rappaport vassiliadis ( msrv, merck kgaa, 64271 darmstadt, germany), salmonella spp. was isolated on xylose-lysine-deoxycholate (xld , merck kgaa, 64271 darmstadt, germany) and hektoen agar (emd, merck kgaa, 64271 darmstadt, germany, according to 6579:2002 iso. suspect colonies were sub cultured on nutrient agar (merck kgaa, 64271 darmstadt, germany ) and were characterized biochemically using triple sugar iron agar (tsi, merck kgaa, 64271 darmstadt, germany), lysine broth, urea broth, simmons citrate agar and tryptophan broth (merck kgaa, 64271 darmstadt, germany). serotyping was carried out by the national veterinary services laboratories (halkida, greece) according to the kaufmann-white scheme (popoff and le minor, 1997). antimicrobial susceptibility testing antimicrobial susceptibility of salmonella mbandaka isolates to nine antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefuroxime, ceftriaxone, nalidixic acid, ciprofloxacin, levofloxacin, tetracycline and trimethoprim/sulfamethoxazole) was determined by dilution antimicrobial susceptibility tests in accordance to the m100-s16 method and the interpretive criteria of the clinical and laboratory standards insitute (clsi, 2006). the mic was determined as the lowest concentration of antimicrobial agent that inhibited visible growth. escherichia coli attc 25922 and klebsiella pneumoniae atcc 700603 were used as quality control strains. pfge the genetic relatedness of the isolates was investigated by pulsedfield gel electrophoresis (pfge) as described in detail by gautom (1997). after cleavage with xbai, electrophoresis was performed at 6 v/cm with 1.0% agarosena agar (amersham biosciences, uppsala, sweden) by using the chef-dr ii system (bio-rad labo ratories, richmond, california, usa). running conditions for xbaidigested dna were 2.2 to 64 s for 20 h. lambda ladder with a size range of 0.13 to 194 kb (low range pfg marker, new england biolabs inc.), were used as molecular weight standard. the dna bands were visualized on a uv transilluminator and polaroid photographs of the gels were visually examined. results and discussion epidemiological data show that salmonellosis is a serious problem worldwide in pigs, cattles, horses, poultry, and even exotic animals. nontyphoid salmonellae are among the leading causes of food-borne disease in humans and are therefore of increased importance in public health. in the present study, five (1.25%) of the 400 tested finishing pigs were asymptomatic carriers of salmonellae, which were further serotyped as salmonella mbandaka. all five pigs originated from the same herd; the herd included 180 sows all of the same breed. the prevalence was lower than previously reported in other european countries (wong et al., 2003). no other salmonella serotypes were identified in these animals. therefore the present findings confirm the existence of the particular nontyphoid salmonella serotype in asymptomatic carrier swines in greece and therefore indicate a potential risk for trans-mission of the bacterium from swine to humans, directly or in meat products. concerning the susceptibility to antimicrobial agents all five isolates were found to be resistant to tetracycline (mic90 8 g/ml) four to trimethoprim/sulfamethoxazole (mic90 0.5 g/ml), and three to ampicillin and amoxicillin/clavulanic acid ( mic90 16 g/ml). in contrast, all five isolates were susceptible to cefuroxime and ceftria-xone, as well as to nalidixic acid, ciprofloxacin, and levofloxacin. given the zoonotic potential of salmonella mbandaka, research is needed to determine which antimicrobials might be efficacious against this pathogen. in the present study, susceptibility to some third-generation cephalosporins (cefuroxime, ceftriaxone) and quinolones (nalidixic acid, ciprofloxacin, levofloxacin) was detected in all five isolates. this susceptibility can be partially explained by the limited use of these antibiotics in greek farms (data not shown). however, an increasing resistance in salmonella mbandaka strains to some cephalosporin -lactams (cefotaxime, ceftazidime and aztreonam) has been observed by other researchers (szych et al., 2001). the resistance of salmonella mbandaka isolates to tetracycline, ampicillin, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole has previously been reported (streit et al., 2003) and reflects the use of these antimicrobials as growing factors in swine finishing farms. salmonella mbandaka sensitivity to antibiotics was examined in vitro in this study. in vivo confirmation of these results is necessary because persistent antibacterial effects at subinhibitory concentrations (postantibiotic effects), which facilitate removal of affected bacteria by host defense mechanisms, have been m 1 2 3 4 5 figure 1. pfge patterns of 5 salmonella mbandaka isolates. the patterns were obtained by the pfge protocol with xbai digestion. lane m, lambda ladder with a size range of 45.5 to 727.5 kb (high range pfg marker, new england biolabs inc.). lanes 1 – 5, the five salmonella mbandaka isolates analyzed. demonstrated for penicillins, cephalosporins, macrolides, tetracyclines, aminoglycosides, and several other antibac-terial agents (aiello s.e, 1998). in the case of increasing resistance to antibiotics, other methods such as identi-fication and elimination of carrier pigs might prevent the spread of this salmonella serotype. the xbai pfge patterns of the salmonella mbandaka isolates are presented in figure 1. all five isolates had indistinguishable pfge patterns. the results are not surprising as all the isolates were from the same farm. hoszowski and wasyl (2001) also demonstrated a high genetic relatedness among salmonella mbandaka strains from poultry and human origin. however, no studies concerning the genetic relatedness among swine and human isolates have been published so far. no studies have yet been published concerning isolation of salmonella mbandaka in any case of human disease in greece. therefore, we did not have the opportunity to include data relating to human isolates in our study. however, human cases in europe were mainly attributed to contact with asymptomatic carrier pigs. it is thus recommended that this particular salmonella serotype may be considered in investigations of future cases of animals and humans salmonellosis. acknowledgements we would like to thank h. simpson for technical support references performance standards for antimicrobial susceptibility testing of clinical and laboratory standards institute (2006). approved standard m 100s16. clinical and laboratory standards institute, wayne, pennsylvania, usa. davies pr, bovee fg, funk ja, morrow we, jones ft, deen j (1998). isolation of salmonella serotypes from faeces of pigs raised in a multiple-site production system. j. am. vet. med. assoc.15: 19251929. fantasia m, pontello m, fileciti e, aureli p(1989). salmonella mbandaka isolated in italy, 1979–1986. microbiologica. 12: 49-54. gautom rk (1997).rapid pulsed-field gel electrophoresis protocol for typing of escherichia coli o157:h7 and other gram-negative organisms in 1 day. j. clin. microbiol. 35: 2977-2980. gill cj, keene we, mohle-boetani jc, farrar ja, waller pl, hahn cg, cieslak pr (2003). alfalfa seed decontamination in a salmonella outbreak. emerg. infect. dis. 9: 474-479. hoszowski a, wasyl d (2001).typing of salmonella 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mbandaka outbreak investigation using a database to select controls. aust. n. z. j. public health, 22: 536-539. streit jm, jones rn, toleman ma, stratchounski ls, fritsche tr (2003). prevalence and antimicrobial susceptibility patterns among gastroenteritis -causing pathogens recovered in europe and latin america and salmonella isolates recovered from bloodstream infections in north america and latin america: report from the sentry antimicrobial surveillance program. int. j. antimicrobiol. agents 27: 367-375. szych j, cieslik a, paciorek j, kaluzewski s (2001).antibiotic resistance in salmonella enterica subsp. enterica strains isolated in poland from 1998 to 1999. int. j. antimicrobiol. agents 18: 37-42. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (12), pp. 001-004, december, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper passive and active immunity against parvovirus infection in piglets nenad stojanac*, mladen gagrčin, ognjen stevančević, ivan stančić and aleksandar potkonjak faculty of agriculture, department of veterinary medicine, university of novi sad, serbia. accepted 23 march, 2015 the aim of this study was to come to a closer understanding of the origination, dynamics of movement and cessation of colostral immunity to parvovirus infection in swine (ppv) on the basis of an analysis of antibody titres in the blood serum of piglets in their first 6 months. on the third day of life in the blood serum of newborn piglets, an average antibody titer of 13.37 was recorded. an antibody level of 13.30 was maintained until the 10 th day of life, when it started to decline to 12.02 on the seventeenth and to 11.80 on the twenty-fourth day of life. a seronegative result was ascertained on the 38 th and 45 th day of life (8.40 and 5.48). on the 55 th day of life, the titer increased to 10.86 and slowly continued to rise to 11.61 on the 180 th day of life. because negative results are the sign of a complete catabolism of colostral antibodies whose absorption was completed in the first 2 to 3 days of life, the antibodies recorded on the 55 th day are to be considered as a result of active immunological reaction formed between 31 st and 55 th day of life. the research was done on 60 piglets descending from vaccinated mothers and it was expected of the piglets to obtain enough immunity through colostrum which would protect them against ppv infection until they developed their own immunological response. on the basis of the given results, we conclude that colostral immunity to parvovirus infection in swine lasts for about one month and that antibodies found in the blood serum of piglets after the first month of life are a result of the activation of the immune system. key words: porcine parvovirus, colostral immunity, reproductive efficiency, antibody. introduction porcine parvovirus (ppv) infection is widely spread in swine around the world and has an enzootic character. the virus attacks swine at all ages and the most endangered categories are gilts before insemination due to the disappearance of passive immunity and of inadequately developed active immunity (mengeling, 2006). parvovirus infection is constantly present in serbia, especially in herds of clinically healthy swine in intensive breeding in the form of a persistent and in-apparent infection (došen et al., 2000). porcine parvovirus infection lowers reproductive efficiency which puts into question the continuity, amount, and feasibility of pig production in serbia. literature duly suggests the importance of the diagnosis of swine infection caused by *corresponding author. e-mail: stojanac.n@gmail.com. tel: +381638526510. parvovirus and the implementation of immunoprevention in order to inhibit its spread (antonis et al., 2006; oravainen et al., 2006). for these reasons, etiology, pathogenesis, and the route of the transmission of parvovirus infection in swine have been studied by many authors (clark, 1996; mengeling et al., 2000; rogan et al., 2002), with special emphasis on the investigation of a protective character of immunity achieved with seropositive and persistently infected swine without the clinical manifestation of the symptoms of the disease after vaccination with inactive vaccine against parvovirus, and the persistency of specific maternal antibodies in their piglets. newborn piglets are not protected against parvovirus infection because the specific maternal antibodies are only absorbed through colostrum in the first hours of life (dividich, 2007). colostrum is the only source of antibodies for piglets (damm et al., 2002; rooke et al., 2003), because many of the layered structures of the placenta do not allow transplacental transmission of antibodies against parvovirus from mother onto fetus, whereas pathogenic agents easily pass the placenta. the aim of this research was to follow immunity formation (active and passive) in piglets from the day they were born up until they were 6 months old. having in mind the data given by many authors that colostral immunity with parvoviral infections may last up to 5 months (mengeling et al., 1999; fenati et al., 2009), the main postulate about this type of immunity is a natural passive immunity whose effectors are synthesized in another organism of the same species. the carriers of this immunity are immunoglobulins of class g (igg). half life of these immunoglobulins is 15 days, which results in colostral immunity lasting for about 30 days, regardless of their concentration in colostrum itself. hence, we thought that the reasons for finding antibodies in blood serum of 5 months old piglets have to be searched for elsewhere. on the basis of this, it was decided to study the onset, dynamics of movement and ending of colostrum immunity in pigs. materials and methods experimental animals the experiment was performed on a pig farm, with a capacity of 2500 sows, with an intensive way of keeping the pigs infected with ppv enclosed. the experiment was performed on 60 piglets originating from mothers (5 gilts and 5 sows) of the breeds swedish landras hybrid (f1), large yorkshire, and swedish landrace. all experimental animals were clinically healthy and in good condition. from every mother, six piglets were randomly chosen for monitoring of the onset, development and length of passive and active immunity to parvovirus infection. during the experiment, a few piglets died, so the number of researched piglets dropped in time. all the piglets had tags on their ears and a tattooed number. blood sampling was performed on day 0, 3, 10, 17, 24, 31, 38, 45, 55, 65, 100, 130 and 180 of life. blood was taken by the puncture of the brachiocephalic plexus of the piglets. immunization of mothers a regular vaccination against ppv infection of all the mothers was performed on the farm. sows were vaccinated with inactivated monovalent vaccine against swine parvovirus according to the manufacturer’s instructions (intervet, holland) two weeks before insemination, while gilts were vaccinated twice, 8 and 2 weeks prior to insemination. the vaccine had inactivated swine parvovirus, subtype 014, which was diluted in water adjuvance. the vaccine was applied in 2 ml dosages, deeply intramuscularly, behind the ear. two milliliter dosage contained >2560 ha units. determination of the presence of antibodies antibodies against parvovirus were detected with a hi test (ašanin et al., 2006), with slight modifications: only guinea pig erythrocytes and v-bottom microplates were used and no bovine serum albumin was used for a clearer end-point. animals were considered to have low antibody levels when hi titres were ≤1:512. titres >1:512 were considered high. the scientific veterinary institute, novi sad diagnostic guidelines for ppv viruses, based on evaluations of vaccinated animals and field cases, are as follows: antibody titres ≤1:8 indicate that the animal has not seroconverted, 1:16 to 1:512 indicate intermediate seroconversion, and titres beyond this represent a high level of antibodies. statistical analyses during the processing of the results, antibody values characteristic of ppv were calculated on logarithm values log2. after log2 results, titres ≤1:512, were considered negative and were given 0, log2 titre 1:512 was 9, 1:1024 was 10, 1:2048 was 11, 1:4096 was 12, 1:8192 was 13 and 1:16384 was 14. after processing the results, and on the basis of referent values administered by accredited laboratories for testing, the obtained antibody titer results characteristic for ppv in blood serum of examined animals with the value less than 9, were considered as seronegative results. for the evaluation of the results, statistical methods were used: average and interval variation. data handling was done in excel 2007. the results were transformed to logarithmic values with the base logarithm 2 (log2). results the obtained results showed titre values of characteristic antibodies for ppv and represent the diluting of the serum where antibodies were detected, so, there is no unit in which they could be measured. the obtained results of the diluting were transformed into logarithmic values (log2). table 1 shows the levels of antibodies specific for ppv in blood serum of newborn piglets. before the uptake of colostrum, antibodies were not found in any piglet. in the blood serum of three days old piglets, antibodies specific for ppv were found. a total of 60 blood serum samples were checked, and the defined titre values of specific antibodies ranged between 11 to 14 (table 2). in three days old piglets, the antibody level was 13.37. with the same 10 days old piglets, an antibody level of 13.30 specific for ppv was diagnosed. in blood serum of 17 days old piglets, the average titer value of antibodies specific for ppv was 12.02, which is a lower level compared to an average level of antibodies specific to ppv in the blood serum of 10 days old piglets (13.30). from the results in table 2, it can be concluded that an average antibody titer specific for ppv in the blood serum of 24 days old piglets was 11.80. with 31 days old piglets, the determined antibody titer values specific for ppv was 8.70. the antibody titer value characteristic of ppv in blood serum of examined 38 days old piglets was 8.40. in blood serum of 45 days old fatlings, the lowest antibody titre value characteristic of ppv was found, and it measured 5.48. the average value of antibody titer characteristic of ppv with 55 days old fatlings was 10.86. the average antibody titer specific for ppv in blood serum of 65 days old fatlings was 10.63. with 100 days old fatlings, the antibody titre specific for ppv was found table 1. antibody titer specific for ppv in blood serum of piglets. titer number of piglets value *0 3 10 17 24 31 38 45 55 65 100 130 180 14 0 36 38 7 11 0 0 0 4 2 2 12 3 13 0 14 16 13 8 0 0 0 6 2 4 2 10 12 0 6 4 20 14 2 6 0 5 8 7 1 14 11 0 4 0 14 13 9 7 10 13 15 15 8 9 10 0 0 1 6 13 21 22 10 21 21 9 12 13 9 0 0 0 0 1 21 15 12 8 9 10 15 0 0 60 0 1 0 0 7 10 26 0 0 0 0 0 * age (days). table 2. the average antibody titer values specific for ppv in the blood serum of piglets. age (days) average interval 0 0 0 3 13.37 11-14 10 13.30 0-14 17 12.02 10-14 24 11.80 9-14 31 8.70 0-12 38 8.40 0-12 45 5.48 0-11 55 10.86 9-14 65 10.63 9-14 100 10.83 9-14 130 10.98 9-14 180 11.61 10-14 to be 10.83. the average antibody titer specific for ppv in the population of 130 days old fatlings was 10.98. within the examined population of 180 days old fatlings, antibody titer specific for ppv was found to be 11.61 (table 2). discussion before the uptake of colostrum, antibodies specific for ppv were not diagnosed in the blood serum of any piglet from the vaccinated mothers (table 1). this indicates no intrauterine infection (dividich, 2007). the average antibody titer specific for ppv in blood serum of 3 days old piglets was 13.37 which is a very high value and shows the efficiency of the transfer of colostral antibodies from sow to piglet (damm et al., 2002). nearly all the identical average antibody titer specific for ppv was diagnosed in the blood serum of 10 days old piglets. although it is a question of a relatively unexpected occurrence, it is possible that it is the case of a prolonged absorption of colostral immunoglobulins, which, in some cases, can last for 5 days (rooke et al., 2003). in this period, the first piglets without immunoglobulin appeared (table 1), which could be connected to immunoglobulins m (igm) which in a number of cases could be the colostral immune carriers. with 17 days old piglets, the decrease of the antibody titer specific for ppv was noted, compared to the antibody titre with the same piglets seven days before (from 13.30 to 12.02). the drop in antibody titer values of 10% is probably the consequence of the onset of the catabolism of colostral titre antibodies for about 10% (gagrčin et al., 1989) due to the decreased plasma concentration in a growing piglet (paul et al., 1981). antibody titer specific for ppv in blood serum of 45 days old piglets has a tendency to drop, and with 31 days old piglets, an increased number of seronegative animals occurred. with the same examined 45 days old fatlings, the lowest antibody titer specific for ppv was found to be 5.48. the antibody titer specific for ppv progressively increased from 55 until the 180 th day of fatling life. the antibody titer in 55 days old fatlings was 10.86, which is double the value compared to the one gained 10 days earlier. the reason for this abrupt raise of antibodies specific for ppv in the blood serum of fatlings should be sought in the fact that ppv infection of swine is widely spread with clinically healthy swine around the world. also, ppv is an enzootic infection, permanently present on the territory of serbia (došen et al., 2002), especially in clinically healthy swine herds in intensive breeding, in a form of inapparent persistent infection. all this is a consequence that the infection of fatlings whose level of protection from ppv infection was very low when they were 45 days old initiated immunological response which manifested itself with elevation of antibody titer specific for ppv. the results show that up till 45 days of life, piglet catabolism of colostral antibodies occurred, and the passive immunity seized to exist, which is evidenced by the fact that the bearers of passive immunity are immunoglobulin g (igg) (gagrčin et al., 1989; rooke et al., 2003). half-life of this class of immunoglobulin is 15 days (jerant-patic, 2000; tizard, 2000) which as a consequence has continuation of passive immunity for 3 to 40 days, regardless of their t it re o f a n ti b o d y figure 1. the dynamics of movement of antibody titer values specific for ppv in the blood serum of piglets. concentration in the colostrum itself. considering the data from many authors that colostrum immunity lasts up to 6 months (gradil et al., 1990; mengeling et al. 1999; fenati et al. 2009), the question that arises is “what is the class of immunoglobulin of which the half life would be 3 to 6 months?” because such an immunoglobulin class has not been determined, the reason for this statement should be sought in the activating of the self immunological response. in this study, a very high antibody titer specific for ppv in the blood serum of 130 and 180 days old fatlings was found (10.98 and 11.61). this high antibody titer specific for ppv has been confirmed in studies by mengeling et al. (1999) and fenati et al. (2009). they administered the findings of a high antibody titer with 3 to 6 months old piglets to passive immunity, that is, to colostral antibodies. the antibody titer specific for ppv in 40 days old piglets dropped to a low level since there was catabolism of colostral antibodies which gagrčin et al. (1989) explained in their research and which meant termination of passive immunity, upon which the self immune response happened (active immunity) (figure 1). references antonis fga, bruschke jmc, rueda p, maranga l, casal ji, vela c, hilgers atl, belt bgmp, weerdmeester k, carrondo jtm, langeveld pmj (2006). a novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure. vaccine, 24: 5481-5490. ašanin r, krnjajić d, milić n (2006). priručnik sa praktičnim vežbama iz mikrobiologije sa imunologijom. autorsko izdanje, beograd. pp. 63-66 clark lk (1996). epidemiology and management of selected swine reproductive diseases. anim. reprod. sci. 42:447-454. damm ib, friggens cn, nielsen j, ingvarsen lk, pedersen jl (2002). factors affecting the transfer of porcine parvovirus antibodies from sow to piglets. j.vet. med. series a, 49(9): p. 487. dividich lj (2007). the issue of colostrums in piglet survival: energy and immunity. nutri. biotech. feed food ind. pp. 89-102. došen r, gagrčin m, prodanov j, orlić d (2002). porcine parvovirus infection. vet. glasnik, 56(1-2): 13-19. fenati m, armaroli e, corrain r, guberti v (2009). indirect estimation of porcine parvovirus maternal immunity decay in free-living wild boar (sus scrofa) piglets by capture-recapture data. vet. j. 180(2): 262264. gagrčin m, popović m, ćirković d (1989). some aspects of colostral immunity in piglets against porcine parvovirus infection. vet. glasnik 44(7): 587-590. gradil cm, joo hs, molitor tw (1990). persistence of porcine parvovirus in swine infected in utero and followed through maturity. j. vet. med. b 37: 309-316. jerant-patić v (2000). viruses today and tomorrow. med. pregl. 53(1112): 547-558. mengeling wl (2006). porcine parvovirus. diseases of swine, iowa state university press, iowa, pp. 373-386. mengeling wl (1999). porcine parvovirus. diseases of swine, iowa state university press, iowa, pp. 187-200. mengeling lw, lager mk, vorwald ca (2000). the effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance. anim. reprod. sci. 60-61: 199210. oravainen j, hakala m, rautiainen e, veijalainen p, heinonen m, tast a, virolanen jv, peltoniemi oat (2006). parvovirus antibodies in vaccinated gilts in field conditions-results with hi and elisa tests. reprod. dom. anim. 41: 91-93. paul ps, mengeling wl, pirtle ec (1981). duration and biological halflife of passively acquired colostral antibodies to porcine parvovirus. am. j. vet. res. 43:8. rogan d, petrović t, lazić s (2002). novija saznanja o parvovirusnim infekcijama svinja, zbornik referata i kratkih sadržaja, 14. savetovanje veterinara srbije, zlatibor, pp. 49-58. rooke aj, carranca c, bland mi, sinclair ga, ewen m, bland cvi, edwards as (2003). relationship between passive absorption of immunoglobulin g by the piglet and plasma concentrations of immunoglobulin g at weaning. livestock prod. sci. 81: 223-234. tizard ir (2000). veterinary immunology. 6 th edition, london, wb sauders, 89: p. 223. http://www.ncbi.nlm.nih.gov/pubmed?term=%22fenati%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22fenati%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22fenati%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22fenati%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22corrain%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22corrain%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/18295517 in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (3), pp. 001-005, march, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effects of immune synergist of chinese medicinal herbs on the efficacy of vaccination against classic swine fever fuchuan wang1, yibo yan1*, yuhuan zhang1, yichao han1 and chao guan2 1 institute of animal husbandry and veterinary sciences, shanxi academy of agricultural sciences, taiyuan 030032, china. 2 modern agricultural research center, taiyuan 030000, china. accepted 16 november, 2014 two-month-old piglets were fed with 1, 1.5 and 2% immune synergist of chinese medicinal herbs together with vaccination against classic swine fever. serum igg and igm levels increased more than the control group on day 30 (p<0.05). b and t lymphocyte proliferation in piglets fed with 1.5 and 2% herbal immune synergist markedly increased on day 30 as compared to the control group (p<0.05). improvement was also observed in t lymphocyte cd3+, cd4+, cd8+ and cd3+/cd8+ levels. meanwhile, there were significant differences in sod activities and rate of neutrophil phagocytosis between synergist groups and the control group. these results suggest that herbal immune synergist of chinese traditional herbs prescription enhances the protective effect of classic swine fever vaccine. key words: immune synergist, chinese medicinal herbs, classic swine fever vaccine. introduction many chinese medicinal herbs are high efficacious immune synergist, including radix ginseng, astragalus, szechwan asiabell root, poria mushroom, chinese angelica and licorice root, which have been proven to possess immune-enhancement effects (hu, 1997; liu, 1998). these herbs contain plenty polysaccharides, saponins and flavones that are capable of modulating vertebrate cellular and humeral immunity, and disease resistance (liu et al., 2006; gao et al., 2000). because the prescription is made up of pure chinese medicinal herbs that are readily available, of low-cost and low side effects (kong et al., 2004, 2006; wang et al., 2005; ung et al., 2007), it has become a research focus recently. however, most studies are centered on single herb; little attention has been paid to compound herbs. we have previously reported the effect of chinese medicinal herbs synergist on chicken (wang, et al., 2006). here, we described its synergistic effects on vaccination against classic swine fever. *corresponding author. e-mail: aboluo2011888@163.com tel: +86-0351-7561528. fax: +86-0351-7561990. materials and methods animal selection and management piglets (n = 60) were provided by hongdong county great locust tree ecotech limited, china. the feed was manufactured by wuike feedstuff scientific limited, china, according to the formula provided by the research group. piglets were 50 to 60 days of age with body weight of 20 ± 2 kg. all animals were not previously exposed to any vaccine. piglets were managed with the same standard of nutrition and procedure. records and management were performed by designated personnel. compound herbs immune synergist ginseng (panax ginseng c.a.mey), milk vetch root (astragalus membranaceus bge var. mongholicus), szechwan asiabell root (codonopsis tangshen oliv), poria mushroom (poria cocos schw. wolf), chinese angelica (angelica sinensis oliv. diels), large head atractylodes rhozome (atractylodes macrocephala koidz), fructus amomi (amomum villosum lour), pericarpium citri reticulatae (citrus reticulata blanco), american ginseng (panax quinquefolium l.), and licorice root (glycyrrhiza uralensis fisch) were used. the herbs were supplied by taiyuan school of chinese traditional medicine. http://www.sxagri.ac.cn/lijy/english/animalvet.htm mailto:aboluo2011888@163.com table 1. effects of herbal immune synergist on serum igg content. group time (day) 10 20 30 40 a (1.0%) 606.25±43.04 b 612.17±40.55 b 618.46±34.95 b 616.65±44.81 b b (1.5%) 617.25±43.04 a 619.17±40.55 a 628.66±34.95 a 626.65±45.11 a c (2.0%) 618.02±42.34 a 619.19±41.35 a 629.12±33.85 a 625.35±44.21 a d (control) 561.25±43.04 c 598.17±40.55 c 609.46±34.95 c 607.65±44.81 c data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). table 2. effects of herbal immune synergist on serum igm content. group time (day) 10 20 30 40 a (1.0%) 320.31±21.45 b 329.65±27.01 b 332.87±26.68 b 330.60±25.36 b b (1.5%) 336.54±25.91 a 339.51±24.91 a 342.57±25.81 a 340.55±24.92 a c (2.0%) 336.88±26.19 a 339.89±26.21 a 343.21±24.92 a 41.33±23.71 a d (control) 309.51±24.42 c 318.60±25.61 c 322.58±26.11 c 319.96±24.61 c data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). vaccine and chemicals vaccine was bought from lanzhou biological pharmaceuticals of zhongmu industry limited china. sodium heparin, phytohemagglutinin (pha), trypan blue, lipopolysaccharide and 3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide (mtt) were produced by guangzhou medical industry institute, china. lymphocyte isolation solution was produced by chinese medical academy biotech institute. aluminum hydroxide gel was supplied by longkel biotech pharmaceuticals, china. calf serum, antibiotics, 0.8% nh4cl solution, wright’s dye and medium were prepared by the authors. experimental design randomized block design was used in this study. the 60 piglets were randomly assigned to 4 groups (n = 15 in each group). piglets in the synergist group were treated with classic swine fever vaccine and herbal immune synergist. starting from the first day of the experiment, piglets in the synergist group were fed, respectively with supplemented 1.0% (a group), 1.5% (b group) and 2.0% (c group) synergist prescription. on day 7, piglets were injected s.c. with vaccine, while piglets in the control group (n = 15) were treated with swine fever vaccine alone (d group). the whole experiment lasted for 60 days in which pretreatment lasted for 10 days and the treatment lasted for 50 days. blood samples were collected from each group at day 10, 20, 30 and 40 after immune synergist treatment. assay content of serum igg and igm was determined by immune transmission turbidity. peripheral t and b lymphocytes were measured with mtt colorimetric method. red blood cell (rbc) and hemoglobin (hb) were measured with general method. serum gpt, got and sod activity were measured with automatic biochemical analyzer (toshiba, tokyo, japan). analysis on subpopulation of t lymphocytes was used by flow cytometry (becton dickinson, rutherford, nj, usa). phagocytotic neutrophil assay was performed as follows: phagocytotic neutrophil phagocytosis rate = × 100% total neutrophil data analysis the data were subjected to one-way analyses of variance (anovas) using the statistical package minitab 15. where required, tukey multiple comparison tests were used following anovas to identify significant differences between individual treatments. results effects of herbal immune synergist on serum igg and igm contents serum igg and igm contents are shown in tables 1 and 2. there were significant differences in serum igg and igm contents on day 10 between the synergist groups and control (p < 0.05). on day 30, serum igg and igm reached a peak, where average igg levels in synergist groups were 2.54% higher than that in the control group (p < 0.05). group c had a higher igg content than group b, but no significant differences (p > 0.05). effects of herbal immune synergist on peripheral t and b lymphocyte proliferation groups b and c had a higher b and t lymphocyte proli table 3. effects of herbal immune synergist on peripheral b and t lymphocyte proliferation rate (od). group lymphocyte time (day) 10 20 30 40 a (1.0%) t 0.68±0.32 b 0.87±0.53 c 1.07±0.61 b 1.05±0.43 c b 0.69±0.41 c 0.88±0.56 b 1.09±0.60 c 1.08±0.46 c b (1.5%) t 0.81±0.23 a 0.98±0.49 b 1.18±0.57 a 1.13±0.41 b b 0.79±0.21 b 0.99±0.51 a 1.17±0.58 b 1.15±0.39 b c (2.0%) t 0.82±0.35 a 1.01±0.55 a 1.19±0.61 a 1.16±0.51 a b 0.83±0.32 a 1.00±0.60 a 1.20±0.63 a 1.18±0.50 a d (control) t 0.60±0.25 c 0.77±0.51 d 0.99±0.52 c 0.87±0.38 d b 0.62±0.31 d 0.78±0.48 c 0.98±0.54 d 0.89±0.37 d data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). table 4. effect of herbal immune synergist on t lymphocyte subpopulation on day 40. group item (x ± sd) cd3 + cd4 + cd8 + cd3 + / cd8 + a (1.0%) 45.38±0.32 b 27.55±0.23 c 21.96±0.35 c 1.34±0.25 a b (1.5%) 47.87±0.53 a 29.76±0.49 b 22.86±0.55 b 1.35±0.51 a c (2.0%) 47.88±0.61 a 29.78±0.57 a 22.89±0.61 a 1.35±0.52 a d (control) 44.75±0.43 c 26.79±0.41 d 21.92±0.52 d 1.31±0.38 b data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). feration rate (table 3). from day 10 to 30, proliferation rate showed an increasing tendency. on day 40, the lymphocyte proliferation decreased slightly. on day 30, b lymphocyte proliferation rates in groups b and c showed significant difference (p < 0.05), but t lymphocyte proliferation rates in group c were slightly higher than in group b, but there was no significant difference (p > 0.05). effect of herbal immune synergist on t lymphocyte subpopulation table 4 shows the effect of traditional chinese herbs prescription on subpopulation of t lymphocytes on day 40. the ratio of t lymphocyte subpopulation in the synergist group was markedly increased and improved. effects of herbal immune synergist on porcine red blood cell and hb content piglet red blood cell and hemoglobin contents are shown in table 5. there were no significant differences between synergist groups and the control group (p > 0.05). effects of herbal immune synergist on serum gpt, got and sod activity serum sod activities in synergist groups were significantly different from that of the control group on days 30 and 40 (p < 0.05) (table 6). got and got activities were similar between synergist groups and the control group (p > 0.05). effects of herbal immune synergist on rate of neutrophil phagocytosis rate of neutrophil phagocytosis showed a trend of increase in synergist groups, and reached a peak on day 30 (table 7). rate of neutrophil phagocytosis of synergist groups was higher than that of the control group (p < 0.05), but there was no significant difference between synergist groups b and c (p > 0.05). rate of neutrophil phagocytosis of synergist groups decreased slightly on table 5. effects of herbal immune synergist on porcine red blood cell and hb content. group item time (day) 10 20 30 40 a (1.0%) rbc 6.68±0.42 a 6.78±0.33 a 6.87±0.51 a 6.75±0.53 a hb 12.09±0.51 a 12.58±0.46 a 12.89±0.50 a 12.25±0.48 a b (1.5%) rbc 6.81±0.33 a 6.88±0.29 a 6.97±0.47 a 6.90±0.45 a hb 12.32±0.21 a 12.99±0.51 a 13.15±0.58 a 12.96±0.39 a c (2.0%) rbc 6.83±0.45 a 6.92±0.56 a 6.99±0.51 a 6.95±0.46 a hb 12.43±0.42 a 12.68±0.60 a 13.18±0.82 a 13.01±0.58 a d (control) rbc 6.59±0.35 a 6.63±0.46 a 6.82±0.58 a 6.80±0.58 a hb 11.62±0.61 a 11.88±0.48 a 12.38±0.46 a 12.71±0.67 a data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). table 6. effects of herbal immune synergist on serum gpt, got and sod activity. . group item time (day) 10 20 30 40 gpt 35.68±0.42 a 33.12±0.33 a 31.87±0.51 a 32.98±0.53 a a (1.0%) got 36.09±0.51 a 34.58±0.46 a 32.89±0.50 a 33. 05±0.48 a sod 58.56±0.51 a 98.55±0.46 a 102.89±0.50 b 98. 95±0.48 b gpt 35.51±0.33 a 33.08±0.29 a 30.27±0.47 a 30.80±0.45 a b (1.5%) got 36. 32±0.21 a 32.99±0.51 a 30.05±0.58 a 30.96±0.39 a sod 57. 82±0.21 a 98.99±0.51 a 123.15±0.58 a 118.96±0.39 a gpt 35.88±0.45 a 33.92±0.56 a 31.39±0.51 a 31.99±0.46 a c (2.0%) got 36.43±0.42 a 34.68±0.60 a 30.18±0.82 a 30.88±0.58 a sod 57.43±0.42 a 97.68±0.60 a 123.18±0.82 a 119.79±0.58 a gpt 35.59±0.35 a 34.13±0.46 a 33.82±0.58 a 35.80±0.58 a d (control) got 34.62±0.61 a 33.18±0.48 a 32.48±0.46 a 32.98±0.67 a sod 57.62±0.61 a 67.88±0.48 b 78.38±0.46 c 70.71±0.67 c data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different ( p < 0.05). gpt, glutamate-pyruvate transaminase; got, glutamate-oxaloacetate transaminase; sod, superoxide dismutase. day 40, which was still higher than that of the control group (p < 0.05). discussion animal immunity is closely related to disease resistance. when immune function decreases or certain killing mechanism is damaged, pathogens may invade animals, resulting to clinical symptoms. up to now, vaccination is still the most effective preventing measure. although, the strict immune program has been taken in the farms, some infectious diseases are still hard to be controlled chinese medicinal herbs has been proven to be possess immune-enhancing properties (xu et al., 2010, 2011). in addition, chinese medicinal herbs may have many advantages including extensive availability, lower cost, reliable efficacy, decreased risk of side-effects and toxicity. therefore, chinese medicinal herbs or their ingredients have become a hotspot in recent years. the veterinary immune synergist prepared in this study selected herbs containing polysaccharides, flavones and saponins that are capable of activating immune system and stimulating lymphocytes (jiang et al., 2010; meng et al., 2011; perera et al., 2010). in the study, our results demonstrate that supplementation of herbal immune table 7. effects of herbal immune synergist on rate of neutrophil phagocytosis. . group time (day) 10 20 30 40 a(1.0%) 0.068±0.21 b 0.152±0.43 b 0.216±0.51 b 0.212±0.43 b b(1.5%) 0.074±0.23 a 0.198±0.42 a 0.276±0.53 a 0.274±0.45 a c(2.0%) 0.075±0.22 a 0.199±0.45 a 0.276±0.47 a 0.275±0.51 a d(blank) 0.057±0.24 c 0.127±0.3 c 0.146±0.55 c 0.181±0.43 c data presented are means ± se of 15 replicates. for each column, means with the same letter are not significantly different (p < 0.05). synergist could increase serum igg and igm contents upon vaccination against classic swine fever, which is consistent with the report by kong et al. (2004) and hu et al. (2004). herbal immune synergist promotes t and b lymphocyte proliferation and improves t lymphocyte subpopulation composition, which is consistent with the results reported by chu et al. (2004). on the other hand, there were no significant differences for red blood cell and hb content between synergist groups and the control group, and there were no significant differences between synergist groups and the control group in gpt and got. it indicates that these chinese medicinal herbs are not toxic to the parenchymal of liver and heart. in addition, sod activities and rate of neutrophil phagocytosis were markedly increased by herbal immune synergist. although, 2.0% dose supplementation had a slightly better result than 1.5% dose, we recommend1.5% as the optimal dose. vaccine is an important method of animal infectious diseases prevention and control, the synergy and mechanism between the vaccine and immune synergist needs to be studied further. in addition, immune synergist is not effective against all diseases, therefore it is important to continue to develop new immune synergist, and to strengthen its mechanism research. chinese traditional medicinal herb is a potential candidate as a safe immune synergist for animal infectious diseases prevention and control. acknowledgement this work was supported by grants from key technologies research and development program of shanxi province (no. 983196). references chu yf, li xr, hu yl (2004). effects of chinese herbal medicinal ingredient on cells mediated immunity in mice. j. nanjing agric uni. 27(1): 97-100. gao xx, fei xf, wang bx, zhang j, gong yj, minami m, nagata t, ikejima t (2000). effects of polysaccharides (fi0-b) from mycelium of ganoderma tsugae on proinflammatory cytokine production by thp-1 cells and human pbmc (i). acta pharmacol sin. 21(12): 1179-1185. hu yl (1997). progress in the study of immunopharmacology of hu yl, chinese herbal medicine. chin j. immunol. 3: 96-98. kong xf, li xr, wang dy, liu jg, zhang bk, wang xt (2004). effects of ten chinese herbal medicinal ingredients on proliferation and resisting ndv infection of cef. acta veterinaria et zootechnica sinica. 35(3): 301-305． jiang mh, zhu l, jiang jg (2010). immunoregulatory actions of polysaccharides from chinese herbal medicine. expert opin ther target.14(12): 1367-1402. kong xf, hu yl, li xr, wang dy, zhang bk, liu jg, wang x (2004). effects of nine chinese herbal medicinal ingredients on serum hi antibody titer after vaccination of ndv ⅳ strain vaccine in chicks. acta veterinaria et zootechnica sinica. 35(4): 468-472． kong xf, hu yl, rui r, wang dy, li xr (2004). effects of chinese herbal medicinal ingredients on peripheral lymphocyte proliferation and serum antibody titer after vaccination in chicken. inter immunopharmacol. 4(7): 975-982. kong xf, hu yl, yin yl, wu gy, rui r, wang dy, yang cb (2006). chinese herbal ingredients are effective immune stimulators for chickens infected with the newcastle disease virus. poult sci. 85(12): 2169-2175. liu j, henkel t (2002). traditional chinese medicine (tcm): are polyphenols and saponins the key ingredients triggering biological activities?. curr med chem. 9(15): 1483-1485. liu jx, wang cy, bai ym, fang yp (1998). influence of astragalus and other nine natural medicines on newscastle disease antibody titer (nd-hi), coefficient of the immune organs and gains of chicks. chin j. vet. sci technol.12: 30-32. meng mb, wen ql, cui yl, she b, zhang rm (2011). meta-analysis: traditional chinese medicine for improving immune response in patients with unresectable hepatocellular carcinoma after transcatheter arterial chemoembolization. explore (ny). 7(1):37-43. perera pk, li y, peng c, fang w, han c (2010). immunomodulatory activity of a chinese herbal drug yi shen juan bi in adjuvant arthritis. indian j. pharmacol. 42(2): 65-69. ung cy, li h, kong cy, wang jf, chen yz (2007). usefulness of traditionally defined herbal properties for distinguishing prescriptions of traditional chinese medicine from non-prescription recipes. j. ethnopharmacol. 3: 21-28. wang d, hu y, sun j, kong x, zhang b, liu j (2005). comparative study on adjuvanticity of compound chinese herbal medicinal ingredients. vaccine. 23(28): 3704-3708.0 wang fc, fang cg, zhang yh, wang hb, han yc (2006). effects of compound chinese herbal immune synergist on immunity of egg laying chicken. acta veterinaria et zoot echnica sinica. 37(2): 187192. xu yg, ma r, yang xh, tang xd, sun sz (2010). effect of yiqi bushen granuleon the peripheral natural killer cell and γδ t-cell in the patients with minimal residual leukemia. chin j. integr. med. 16(5): 417-421. xu z, chen x, zhong z, chen l, wang y (2011). ganoderma lucidum polysaccharides: immunomodulation and potential anti-tumor activities. am. j. chin med. 39(1): 15-27. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (2), pp. 001-005, february, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper biodegradation of lagoma crude oil using pig dung yakubu, m. bello school of science, college of education, pmb 39, minna, nigeria. e-mail: bellyms@yahoo.com. tel: +23466-222205, +234-805046-7560. accepted 12 november, 2015 pig dung bacteria were isolated and screened for crude oil degrading capabilities. the pig dung was also investigated for enhancement of crude oil biodegradation. addition of chicken manure to oil polluted soil (at 10% (v/w) pollution level) stimulated the biodegradation of lagoma crude oil used in the present study. in the soil amended with pig dung, 68.2% of the crude oil was degraded, whereas only 50.7% of same oil was degraded in the unamended soil. the ph of the amended soil rose from 6.2 to 7.2. pig dung was found to contain 1.7 x 10 6 cfu g -1 crude oil degrading bacteria, and 1.8 x 10 8 cfu g -1 aerobic heterotrophs. the crude oil utilizing bacteria were identified as species of pseudomonas, proteus, klebsiella, bacillus and micrococcus. pig dung could therefore be an option for crude oil pollution mitigation project. key words: pig dung, biodegradation, crude oil, pollution, nutrients, amended soil. introduction oil released into the environment is a well-recognized problem in today's world. oil spills affect many species of plants and animals in the environment, as well as humans (plohl et al., 2002) . the involvement of microorganisms in the degradation of petroleum and its products in the environment has been established as an efficient, economic, versatile, and environmentally sound treatment (margesin and schinner, 2001). the microorganisms implicated in oil degradation are widely distributed in nature and have been isolated from soil and water ecosystems with their oil degrading potentials investigated. the search for effective and efficient methods of oil removal from contaminated sites has intensified in recent years, because microbial degradation that is responsible for clearing untreated oil spills is slow (grangemard, et al., 2001). one promising method that has been researched into is the application of chemical fertilizers to augment for the mineral elements, particularly nitrogen and phosphorous’ limitations in the soil during biodegradation (margesin and schinner, 1999). these fertilizers, especially in developing countries are not sufficient for agriculture, let alone for clearing oil spills. they also tend to result in soil hardening and fertility decline. it therefore, necessitates the search for cheaper and environmentally friendly options of enhancing crude oil biodegradation. the present study is therefore to investigate the potential of pig dung bacteria for crude oil degradation, as well as utilizing the pig dung for the probable stimulation of crude oil biodegradation in the soil. materials and method collection and processing of samples the crude oil used was lagoma light crude oil, which was collected in sterile sampling bottles from kaduna refining and petrochemical company, kaduna, nigeria. the soil sample used was collected from the main campus of wuhan university, wuhan, pr china, transported to the laboratory and processed for use in the bioremediation studies. the sample was air dried and sieved through 2 mm mesh size. the pig dung used was collected fresh from a pigsty at eastlake, wuhan, china. the dung was collected in thick polythene sheets and transported to the laboratory, and then sun dried for several days. after drying, the samples were ground and stored in the laboratory for further use. the biological and physicochemical properties of the pig dung used in this work were determined (ijah and antai, 2003) isolation and identification of bacteria isolation of bacteria was done following the modified methods of solano-solano et al. (2000). ten grams of air-dried pig dung were thoroughly suspended in 100 ml sterile tap water, and then the supernatant was serially diluted with sterile tap water. zero point one milliliter each of a serially diluted (10 -7 , 10 -5 , 10 -3 and 10 -1 ) pig table 1. biological and physicochemical properties of pig dung. descriptor value m ph 7.3 ± 0.2 moisture (%) 8.6 ± 0.9 nitrogen (%) 2.1 ± 0.6 phosphorous (%) 1.6 ± 0.3 calcium (%) 0.2 ± 0.1 magnesium (%) 0.5 ± 0.1 sodium (%) 1.9 ± 0.9 potassium (%) 1.8 ± 0.3 aerobic heterotrophic bacteria 1.8 x 10 8 cfu g -1 crude oil utilizing bacteria 1.7 x 10 6 cfug -1 m mean of three determinations. dung samples were spread inoculated onto nutrient agar (na) and mineral salts medium supplemented with the crude oil (oil agar, oa) for the isolation of bacteria and enumeration of crude oil degrading bacteria respectively. the mineral salts medium (msm) of bushnell and haas (1941) was employed, which has the following compo sition: (1.0 g kh po , 1.0 g k hpo , 1.0 g nh no , 0.2 g mgso .7h o, 0.05 g fecl , 0.02 g cacl .2h o in 1000 ml of distilled water at ph 7.0). the inoculated na plates were incubated at room temperature (28 ± 2 o c) for 48 h, while the oa plates were incubated for 3 to 5 days at the same temperature. the bacterial colonies that appeared on the plates were counted, and distinct colonies were picked and purified by repeated sub-culturing. a number of microscopic and biochemical tests were carried out for identification purposes. the tests included gram reaction, shape, spore, motility, catalase, indole production, oxidase, methyl redvoges proskauer (mr-vp), gelatin liquefaction, nitrate reduction, urease, citrate, starch hydrolysis, o/f of glucose, pigmentation and utilization of the following carbohydrates – glucose, sucrose, fructose, maltose, xylose, arabinose, mannitol and inositol. the isolates were identified by comparing their characteristics with those of known taxa, as recommended by cowan and lisbon (ijah and antai, 2003). utilization of crude oil by the bacterial isolates the ability of the bacterial isolates to utilize crude oil as the only source of carbon and energy was determined by the method of okpokwasili and okorie (1988). 0.1 ml of 24 h old nutrient broth culture was inoculated into each test tube containing 10 ml of sterile msm of bushnell and haas and 1% (v/v) crude oil. control test tubes were set up containing 10 ml of msm with 1% (v/v) crude oil, but had no added bacteria. the tubes were incubated at room temperature for sixteen days on an orbital shaker (hya, scientific instrument manufacturer, wuhan) at 120 rpm. at the end of the incubation period, the growth of the isolates was determined by visual observation of the oil medium for turbidity, as compared to the control tubes (olesnicky et al., 2002; okpokwasili and okorie, 1988). the extent of degradation of the incorporated crude oil by the bacterial isolates was determined by the gravimetric analysis method of odu (ijah and antai, 2003). the amount of crude oil left after the incubation time was determined by extracting the residual oil with 50 ml of toluene from the 10 ml culture. the mixture was separated using separatory funnel and then filtered off with what man filter paper. the optical density (absorbance) was read on a spectrophotometer (uv-120-02, shimadzu, japan) at 410 nm wavelength. using a previously prepared standard curve, the weight of the crude oil was determined. the amount of crude oil degraded was calculated by subtracting the weight of residual crude oil from weight of the added (initial) crude oil, divided by the weight of the initial crude oil and then multiplied by 100. measurement of crude oil biodegradation in soil amended with pig dung the rate of bacterial utilization of crude oil in the soil was assessed by using the the gravimetric method. 100 g of soil contained in screw-capped bottles in triplicates were each treated with 10% (v/w) crude oil. 10 g of pig dung were added to each bottle, and the soil moisture corrected by the addition of 10 ml of sterile double distilled water (roman et al., 1996). a control experiment, without added chicken manure was set up. all the bottles were incubated for 16 days at room temperature. at four days’ interval, the amount of weight loss of the crude oil was determined. 20 g of oil-polluted soil were weighed into sampling bottle and 100 ml of ccl4 added to each bottle. the oil-solvent mixes were separated using a separatory funnel. the extracts were dried by adding 0.1g anhydrous na2so4 (facundo, 2000; mulligan et al., 2001), and then filtered through whatman no. 1 filter paper. the ccl4 was allowed to evaporate off at room temperature in a fume hood. weight loss was determined as described above. gas chromatographic analysis was employed to confirm the results of the gravimetric method. the extractable crude oil was recovered and analyzed on gas chromatograph (hp, paw) equipped with a chromosorb, pa-aw 80 – 100 capillary column and flame ionization detector (fid). the operational parameters were: injection temperature of 400 o c, helium carrier flow of 28 ml per minute, injection 1 µl. the oven temperature was set initially at 50 o c min -1 . the major hydrocarbon compounds of the crude oil were identified on the basis of their retention time and by comparing them to those of analytical standards. to determine the ph, 10 g of the sample was mixed with 25 ml of sterile water in a beaker, stirred and allowed to stand for 30 min. the ph was then taken with the ph meter (hc21006, china). the mixture was stirred again, allowed to stand and ph retaken. a triplicate determination was performed. results the biological and physicochemical properties of the pig dung are presented in table 1. twenty-seven bacterial isolates were obtained from the pig dung as shown in table 2. the identification of the isolares revealed them to belong to the species of staphylococcus, pseudomonas, proteus, acinetobacter, campylobacter, streptococcus, klebsiella, enterobacter, bacillus, micrococcus and escherichia. bacillus species were the most frequent isolated bacteria, which constituted about 15% of the total isolates. the results in table 2 reveal the extent of growth, as well as the amount of crude oil degraded by the bacterial isolates from pig dung. twelve (44.4%) isolates, out of the total isolates were able to utilize crude oil as sole source of carbon and energy. a bacillus sp degraded the crude oil at a relatively high rate of 63.5% after the incubation time of 16 days. the other crude oil utilizers belong to the genera of pseudomonas, micrococcus, proteus, and klebsiella. it is however, table 2. extent of growth and degradation of crude oil by bacterial isolates from pig dung. growth in crude oil crude oil degraded bacterial isolates msm (%) m pgb07 bacillus sp +++ 63.5 ± 1.4 pgb12 pseudomonas ++ 43.4 ± 2.2 aeruginosa ++ 40.6 ± 1.3 pgb19 micrococcus sp ++ 39.7 ± 0.5 pgb27 bacillus sp ++ 37.6 ± 0.6 pgb03 bacillus sp + 22.8 ± 1.5 pgb05 pseudomonas sp + 15.5 ± 0.2 pgb09 micrococcus sp + 13.2 ± 0.8 pgb11 proteus sp + 13.2 ± 0.6 pgb15 klebsiella sp + 13.0 ± 0.4 pgb24 micrococcus sp + 11.5 ± 1.2 pgb23 proteus sp + 11.5 ± 1.1 pgb17 klebsiella sp 0 pgb01 escherichia coli 0 pgb02 bacillus sp 0 pgb04 campylobacter sp 0 pgb06 acinetobacter sp 0 pgb08 enterobacter sp 0 pgb10 proteus sp 0 pgb13 campylobacter sp 0 pgb14 enterobacter sp 0 pgb16 streptococcus sp 0 pgb18 staphylococcus sp 0 pgb20 staphylococcus sp 0 pgb21 campylobacter sp 0 pgb22 pseudomonas sp 0 pgb25 streptococcus sp 0 pgb26 staphylococcus sp +++ heavy growth; ++ moderate growth; + little growth; no growth. m mean of three determinations. table 3. the amount of crude oil degraded in soil amended with pig dung. incubation weight loss (%) m of crude oil in soil time (days) pig dung no pig dung 4 13.5 ± 0.5 10.4 ± 0.2 8 33.7 ± 1.8 24.3 ± 0.6 12 56.9 ± 2.2 38.6 ± 1.4 16 68.2 ± 1.9 50.7 ± 1.6 m mean of three determinations. shown that fifteen of the isolates were not able to degrade the crude oil. the results of the weight loss of oil from the soil treated with crude oil and amended with pig dung are presented in table 3. at the end of the incubation period, 68.2% of crude oil was degraded in soil amended with pig dung as against 50.7% of same oil in unamended soil after incubation period. these were confirmed by the results obtained from gas chromatograph in figure 1 (crude oil amended with pig dung) and figure 2 (control; crude oil not amended with pig dung), where it is shown that the bacterial population contained in pig dung utilized almost all the components of the crude oil. the fractions of isoprenoids (pristane and phytane) were however, not attacked by the bacterial community in the pig dung after 16 days of incubation. the pattern nonetheless shows that the microorganisms first attacked the lower and higher hydrocarbon chains and those of middle length were attacked later in the course of incubation. the ph values obtained in soils treated with crude oil and amended with pig dung reveal that the ph of control soil ranged between 5.6 and 5.8 (table 4) . while that of the unamended soil ranged between 5.7 and 6.0 after sixteen days of incubation. the ph of the amended soil increased from 6.2 at time zero of sampling to 7.2 over same period, which is from acidic to slightly alkaline ph (table 4). there were significant (p < 0.05) differences among the treatments and the period of incubation with respect to weight loss of the crude oil. a linear correlation was observed between the gas chromatographic rates of biodegradation and weight losses of crude oil in soils amended with chicken manure. discussion many types of microorganisms have been reported to be present in pig dung, which include bacteria and fungi (laukova, 2000; mansour et al., 1999). this is in addition to the mineral nutrients, which include nitrogen and phosphorous in the sample for microbial growth and metabolism. in this present study, eleven genera of bacteria were identified from the pig dung sample used, which were mainly enterics. though, bacillus species was the most predominant isolated bacterial species, it prevalence could be attributed to the fact that it forms spores, which help microorganisms to withstand harsh conditions, such as sun drying employed in this work. isolation of bacillus species from animal dung could also be attributed to its ubiquitous distribution in nature. mansour et al. (1999) reported the isolation of bacillus, acinetobacter, staphylococcus and enterobacter among other bacteria from pig dung. there are volumes of literature on bacterial degradation of crude oil in the ecosystem. however, what is interesting is the source of the oil degrading bacteria in the natural ecosystem. it is interesting to find that pig dung, in addition to being rich in mineral elements nitrogen and phosphorous (diez et al., 2001)] necessary for crude oil biodegradation, also contain bacteria with varying de table 4. the ph reactions of soil treated with crude oil and amended with pig dung. ph values m incubation non polluted, not crude oil polluted soil time (days) amended soil crude oil polluted soil amended with pig dung 0 5.6 ± 0.1 5.7 ± 0.1 6.2 ± 0.1 4 5.7 ± 0.0 5.8 ± 0.0 6.9 ± 0.0 8 5.8 ± 0.3 5.9 ± 0.1 6.9 ± 0.2 12 5.9 ± 0.1 6.0 ± 0.1 7.1 ± 0.4 16 5.8 ± 0.2 5.9 ± 0.2 7.2 ± 0.1 m mean of three determinations. figure 1. gas chromatograph of lagoma light crude oil amended with pig dung after, 4, 8, 12 and 16 days of incubation at 28 o c. figure 2. gas chromatograph of lagoma light crude oil (control) not amended with pig dung after, 4, 8, 12 and 16 days of incubation at 28 o c. grees of crude oil degrading capabilities. this capability is shown here to be further widespread among the bacteria present in the pig dung. the oil-utilizing bacteria isolated from the pig dung in the present work have previously been implicated in crude oil biodegradation, though from different sources (ijah and antai, 2003; ijah, 1998). stimulated biodegradation of crude oil is at present being encouraged because it ensures rapid remediation of oil-polluted ecosystems (ijah and antai, 2003). most often, chemical fertilizers are used to enhance microbial degradation of crude oil in the natural environments. mineral elements have been reported to be essential for crude oil biodegradation (mitchell, 1999; atlas, 1995). apart from the oil utilizing bacteria in animal dung, if manure is spread back on the land, not only will it add much needed nutrients, but also organic matter, which is not found in chemicals. therefore, the addition of pig dung to the soil enhanced the rates of crude oil biodegradation. also, the bacterial consortium in the pig dung attacked and degraded almost all the components of lagoma light crude oil, except the fractions of isoprenoids (pristane and phytane) that were resistant to bacterial attack. not a single bacterium can degrade all the component fractions of any crude oil, and significant oil biodegradation is normally achieved by microbial synergy. pig dung could, therefore, harbour both primary and secondary crude oil biodegraders. as such, the use of pig dung may be a promising potential in integrated oil spill abatement. furthermore, the pig dung raised the ph of the soil to a range between 6.2 and 7.2, which is ideal for most crude oil utilizing bacteria (vidali, 2001) . this indicates that the pig dung had a buffering effect on the soil, and since strong acidity is a limitation in biodegradation, it would have contributed to the enhanced crude oil degradation in the soil. the use of pig dung to stimulate crude oil biodegradation in the soil could be one of the severally sought environmentally friendly ways of eliminating petroleum hydrocarbon in the natural ecosystem. references atlas mr (1995). bioremediation of petroleum pollutants. inter. biodeterior. biodegrad. 35: 317-327. bushnell ld, haas hf (1941). the utilization of certain hydrocarbons by microorganisms. j. bacteriol. 41: 653-673. diez ja, de la torre ai, cartagena mc (2001). waste management evaluation of the application of pig slurry to an experimental crop using agronomic and ecotoxicological approaches. j. environ. qual. 30: 2165-2172. grangemard i, wallach j, marget-dana r, peypoux f (2001). licenysin: a more efficient cation chelator than surfactin. appl. biochem. biotechnol. 90:199-210. ijah ujj (1998). studies on relative capabilities of bacterial and yeasts isolates from tropical soil in degrading crude oil. waste manage. 18: 293-299. ijah ujj, antai sp (2003). the potential use of chicken-drop microorganisms for oil spill remediation. the environmentalist. 23: 89-95 laukova a (2000). in vitro treatment of different isolates from cattle dung and pig slurry by nisin. acta vet brno. 69: 147-151. mansour ma, bottefroy d linder a (1999). inhibition of bacillus lincheniformis spore growth in milk by nisin, monolaurin, and ph combinations. j. appl. microbiol, 86: 311-324. margesin r, schinner f (2001). bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area. appl. environ. microbiol. 67(7): 3127-3133. margesin r, schinner f (1999). a feasibility study for the in situ remediation of a former tank farm. world j. microbiol. biotechnol. 15: 615-622. mitchell jg (1999). in the wake of the spill: ten years after exxon valdez. natl. geogr. 195(3): 96-117. okpokwasili gc, okorie bb (1988). biodeterioration potentials of microorganisms isolated from engine lubricating oil. tribol. inter. 21: 215-217. olesnicky ec, miller l, fukunaga j, laskin ai (2002). biodegradation of brominated biphenyls by members of the genus sphingomonas (c). in: american society for microbiology. 102nd general meeting, salt lake city, utah. pp. 19-23. plohl k, leskovšek h, bricelj m (2002). biological degradation of motor oil in water. acta chim. slov. 49: 279-289. roman r, caballero r, bustos a, diez ja, cartagena mc, vallejo a. caballero a (1996). water and solutes movement under conventional corn in central spain: i. water balance. soil. sci. soc. am. j. 60: 1530-1536. solano-serena f, marchal r, casarégola s, vasnier c, lebeault jm, vandecasteele jp (2000). a mycobacterium strain with extended capacities for degradation of gasoline hydrocarbons. appl. environ. microbiol. 66(6): 2392-2399. vidali m (2001). bioremediation. an overview. pure appl. chem. 73(7): 1163-1172. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (7), pp. 001-005, july, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper the growth performance of growing pigs during feed restriction and re-alimentation in a humid tropical environment s. o. c. ugwu and a. e. onyimonyi* department of animal science, university of nigeria nsukka, nigeria. accepted 28 november, 2015 thirty two crossbred (large white x landrace) pigs were used in a 56 days restriction and 56 days re-alimentation study to evaluate the effect of restricting growing pigs at 90, 80 and 70% of the ad libitum feed intake of the control. the pigs averaged 35.23 ± 0.560 kg at the start of the study. there were four treatments (control, 90, 80 and 70%). each treatment had eight pigs allotted to 2 replications of four pigs each. the pigs received a 16% crude protein and 12.08 mjme/kg diet. results indicated that average daily gains (adg) of the control pigs were significantly superior to the others during the restriction period. however, at the end of the 56 days re-alimentation, pigs on the 80% and 70% feeding regime had superior adg than the pigs on the control and 90 percent feeding regime. data on the body length (bl) and height at shoulders (hs) followed the same trend as observed for adg. feed intakes of pigs were significantly affected during the restriction and re-alimentation period. there was a significant (p < 0.05) decrease in feed intake as level of restriction increased. also, during re-alimentation pigs on the 70% level had the highest feed intake. pigs on the 80% level of restriction had the best feed cost/kg gain values at restriction and realimentation. it is concluded that restricting growing pigs at 80% of the ad libitum intake of the control yields best performance and economic benefit. key words: growth, restriction, re-alimentation. introduction one of the major problems of food insecurity is the severe competition between man and farm animals for available food stuffs (steinbach, 1985). the type of animals mostly affected are the simple stomached species, pig and poultry, which depend on the same food needed by man to survive and produce. several reasons have been adduced for the short fall in food supply mostly experienced in developing countries and the most prominent appears to be the adoption of inadequate agronomic strategies including unsatisfactory storage programmes. this result in considerable loses in the field and after harvest such that supply of cereals and leguminous grains which are also the backbone of successful monogastric animal production is largely seasonal. to compound this situation is the uncontrolled *corresponding author. e-mail: onyimonyi@yahoo.com rise in human population in these countries resulting in continual reduction in food margins for animal feedings. the ultimate effect is the continual increase in feed costs to levels beyond 70% of total cost of animal production (onyimonyi and okeke, 2004). attempts to elevate feed supplies to animals in such countries mostly entailed the utilization of agroindustrial wastes which measure has largely proved not to be very effective due to availability and quality concerns. the concept of compensatory growth whereby an animal whose growth has been slowed by nutritional deprivation may exhibit enhanced growth after deprivation is stopped (wilson and osbourn, 1960; doyl and lesson, 2005) may be a reliable option in meeting challenges faced by pig farmers in food deficient countries. compensatory growth had been reported in pigs (prince et al., 1983; mersmann et al., 1987 and kyriazakis et al., 1991). prince et al. (1983) restricted pigs to 70 or 80% of ad libitum intake for either 2 or 4 weeks. table 1. daily feed intake feed/gain and feed cost/kg gain of growing pigs during feed restriction and post restriction. parameter control 90% 80% 70% sem (i) daily feed intake (kg/d) restriction period 2.36 a 2.18 b 1.84 b 1.57 c 0.17 post restriction period 2.34 2.42 2.57 2.65 0.58 whole period 2.54 2.44 2.36 2.43 0.41 (ii) feed/gain restriction period 3.74 3.65 3.57 3.68 0.25 post restriction period 3.92 3.87 3.76 3.83 0.40 whole period 3.89 3.68 3.67 3.74 0.37 (iii) feed cost/kg gain n restriction period 129.40 126.29 123.52 127.33 1.06 post restriction period 135.63 133.90 130.10 132.51 1.01 whole period 133.90 127.32 124.91 129.40 1.65 abc: row means with different superscripts are significantly different (p < 0.05). sem = standard error of a mean. the group restricted to 85% for 4 weeks performed the best. those restricted to 70% of ad libitum intake for 4 weeks were unable to freely compensate suggesting that the restriction was either too severe and/or too prolonged. mersemann et al. (1987) remarked that the magnitude of increase in growth rate following feed restriction may well be affected by the change in physiological status imposed by the weight loss during restriction. kyriazakis et al. (1991) observed that there were no significant difference in growth rate between either male or female pigs upon re-alimentation after feeding a low protein diet. the present study intends to investigate the extent of growth reduction in growing pigs subjected to feed restriction, the rate of recovery on re-alimentation and the benefits accruable to the farmer that may adopt this concept in the humid tropics where prevailing thermal condition may exert negative influence on growth performance of pigs. experimental procedures this study was conducted at the pig research and teaching unit of the department of animal science, university of nigeria, nsukka. the study lasted for 16 weeks. the location has a typical humid tropical climate with average day temperature ranging from 28°c in january (harmattan period), july and august to 34°c in february, march and april (hottest months of the year). humidity values ranged from 78% (november – february) to an average of 80% (march – october). thirty two crossbred (large white x landrace) pigs selected from litters of four landrace dams that farrowed within one week were allotted to four treatments made up of the control and three levels of feed restriction (90, 80 and 70% of ad libitum). the average live weight of the pigs was 35.23 kg at start of the restriction and they consisted of 16 castrates and 16 females. each treatment contained eight pigs (4 castrates and 4 females) separated into two replicates of four pigs each housed in a pen measuring 3.2 x 2.5 m equipped with concrete feeders separated into four feeding points with strong wooden planks to ensure that each pig had uninterrupted access to its ration. the pigs were fed a 16% cp and 12.08 mjme/kg diet made up of 20% cassava chips, 5.5% maize, 12% spent grain, 27.5% rice husk, 20.5% palm kernel cake, 10% groundnut cake, 1.5% palm oil, 2% bone meal, 0.5 common salt, 0.15 methionine 0.10 lysine and 0.25 vitamin premix. all pigs were weighed at the inception of the restriction and latter on weekly intervals. feed intake/unit of body weight was calculated for pigs on the control treatment (fed ad libitum) based on which the restricted levels of intake (90, 80 and 70% of ad libitum) were determined at the same average weight. before the commencement of the restriction phase (rst), pigs were allowed one week adjustment period in their new pens. thereafter, the restriction phase commenced and lasted for 56d. this was immediately followed by the re-alimentation phase (post – rst) which lasted for a maximum of 56d. all pigs were fed once per day at 7 h during both stages. pigs were weighed weekly in post – rst. other growth measurements taken in both stages at weekly interval were body length (bl); as t he length of pigs body from base of tail to base of skull (mersmann et al., 1987) and height at shoulder (hs) taken as the vertical distance from the floor of restraining cage to the highest point on the shoulder (lefaucheur et al., 1991). daily feed intake (table 1) was determined as the difference between the amount of feed fed and the amount left over in 24 h while feed/gain was calculated as the ratio of feed intake over weight gain. during post–rst all experimental measurements were discontinued within treatment when the average body weight of composite pigs reached 100 kg. all data on performance of animals at rst and post – rst were processed and analysed statistically according to the procedures of steel and torrie (1980) using a stats graphic computer package. statistically different means were separated by duncan’s new multiple range test (duncan, 1955) in the same package. results and discussion the results of the growth performance of pigs in the two phases of the experiment (rst and post – rst) are presented in table 2. there were significant (p < 0.05) table 2. performance of growing pigs during restriction (rst) and post restriction (post – rst) periods. parameter control 90% 80% 70% sem (i) body weight kg av. initial body wt. (rst) 42.27 41.90 42.32 41.21 av. final body wt (rst) 76.19 a 74.64 a 67.15 b 60.37 c 0.97 av. daily gain (rst) 0.62 a 0.57 a 0.41 b 60.32 c 0.03 av. daily gain (post-rst) 0.60 a 0.62 a 0.79 b 0.76 b 0.06 av. age at 100kg (d) 223 225 229 240 3.25 (ii) body length (cm) av. initial body length (rst) 56.13 55.92 56.21 56.87 av. final body length (rst) 77.25 a 75.20 a 73.31 a 70.19 b 2.30 av. daily gain in body length (rst) 0.38 a 0.34 a 0.30 a 0.23 b 0.02 av. body length at 100kg (post-rst) 89.65 88.24 88.27 87.46 2.31 av. daily gain in body length (post-rst) 0.30 0.32 0.35 0.33 0.04 (iii) height at shoulder (cm) av. initial height at shoulder (rst) 41.81 42.03 42.17 41.72 av. final height at shoulder (rst) 56.04 a 55.02 a 53.37 a 51.80 b 1.36 av. daily gain in height at shoulder (rst) 0.25 a 0.21 a 0.20 a 0.18 b 0.02 av. height at shoulder 100kg (post-rst) 64.26 63.63 63.65 63.76 1.78 av. daily gain in height at shoulder (post-rst) 0.19 0.20 0.25 0.23 0.04 abc: mean with different superscripts in a sow significantly (p < 0.05) different. sem = standard error of mean. decreases in body weight (bw), body length (bl) and height at shoulder (bs) at rst. the average gains in bw (adg), bl (adgl) and hs (adgh) also decreased significantly (p < 0.05). the control pigs were superior to the restricted groups in these parameters with pigs fed 70% of ad libitum growing at the lowest rates. growth reduction in pigs subjected to various levels of feed or nutrient deprivation has been widely reported in temperate climates (owen et al., 1971; wahlshom and libal, 1983; donker et al., 1986; mersmann et al., 1987; chiba, 1995). the extent of decrease in various components of body growth studied reflected the amount of feed made available to the restricted pigs which decreased significantly (p < 0.05) from control to 70% of ad libitum. the adg of 80 and 70% restricted pigs (0.41 and 0.32 kg/d respectively) were lower than 0.59 and 0.48 kg/d reported by prince et al. (1983) in growing pigs restricted to 85 and 70% of ad libitum consumption. the efficiency of feed conversion was generally low with values recorded for the restricted pigs being slightly lower than that of the control. steinbach (1985) observed that the exogenous and endogenous heat load affecting pigs in the humid tropics act through the neuro endocrine system to alter digestibility and absorption of nutrients, enzyme activity and metabolism of various nutrients, thereby reducing efficiency of feed conversion and growth rate. it is therefore likely that the lower gains obtained for 80 and 70% restricted pigs may not be as a result of the levels of restriction above but also involved the influence of prevailing environment. the pigs fed 90% of ad libitum were minimally affected by restriction with adg values (0.57 kg/d) differing only slightly from that of control (0.62 kg/d). cleveland et al. (1983) and mersmann et al. (1987) described this level as modest restriction since it caused reduction in fat deposition only with no change in gain of lean. when compared with the control, pigs fed 70 and 80% of ad libitum respectively lost 15.82 and 9.05 kg of bw respectively during the 56d restriction period while 90% restricted group lost 1.55 kg. a similar trend was observed in linear body measurements where for bl, pigs fed 70 and 80% of ad libitum lost 7.06 and 3.94 cm while the 90% group lost 2.05 cm. for hs, 4.01, 2.44 and 1.62 cm were the losses recorded by 70, 80 and 90% restricted groups respectively. minimal loss in bl was reported by mersmann et al. (1987) for growing pigs restricted for three weeks. significant reduction in bl was not recorded by these authors probably because of the short duration of restriction. it seems apparent that the pigs placed on 70% of ad libitum con-sumption received more severe treatment which resulted in significant retardation in growth of bone and lean tis-sue. doornenbal (1975) reported that after 12 weeks of age, pigs exhibit rapid lengthening of the backbone which accounts for the increase in body length. it is therefore felt that severe nutritional deprivation as was the case with pigs given 70% of ad libitum for 56d in the tropics where growth rate is generally low, could delay development of body length. similarly, the observed drop in hs measurement of the 70% restricted group may also have been the result of the prolonged nutritional assault on the pigs which are in the stage (30 – 159 kg) when proportionnal increases in muscle and length/diameter of the limb bones occur. the pigs exhibited different rates of recovery at post – rst with 80 and 70% restricted groups recording higher (p < 0.05) adg (0.79 and 0.76 kg/d) than the control and 90% restricted groups (0.60 and 0.62 kg/d). the post – rst adg of 80 and 70% groups were twice the rst gains and when compared with the control, the gains were higher by 0.19 and 0.16 kg/d respectively. however, the re-alimentation growth rates reported by mersmann et al. (1987) for control and restricted pigs (0.864 vs 1.005 kg/d) were much higher than the rates obtained in the present study. the gains reported by prince et al. (1983) for pigs fed 85 and 70% of ad lib for four weeks (0.83 vs 0.78 kg/d) were also higher than the post rst gains recorded in the present study. the 80 and 70% restricted groups also made up for lose in bl and hs at rst. the post – rst rates of increase in bl of 80 and 70% restricted groups (0.35 and 0.33 cm/d) were lower than 0.496 cm/d recorded by mersmann et al. (1987) for re-fed pigs in the temperate environment. the post – rst feed consumption of pigs were significantly (p < 0.05) affected during restriction. the highest intake was observed in the 70% restricted group (2.65 kg/d) while the lowest was in the control (2.34 kg/d). feed consumption in 80 and 70% groups were only a little higher than their intake at rst within the first week of re-alimentation but appreciated gradually from the second week reaching stable values from the third week till the end of that phase. this trend was not noticed in the 90% restricted group where the intake increased at the beginning of re-alimentation and following a period of feed restriction took several weeks to develop fully and persisted for some time. it is likely that the initial delay in attaining full consumption immediately on realimentation may be due to the inherent mechanism in pigs to adjust voluntary intake in response to previous restriction as earlier suggested by wilson and osbourn (1960) . it was also observed that feed consumption of control and restricted groups were low at post – rst and through the whole period. post – rst consumption ranged form 2.34 kg/d in the control to 2.65 kg/d in 70% restricted pigs while whole period consumption ranged from 2.6kg/d in 80% restricted to 2.54 kg/d in the control pigs. these rates were much lower than 3.26 kg/d reported by owen et al. (1971) for restricted re-alimentated pigs and 3.66 kg/d reported by mersmann et al. (1987) for restricted re-fed pigs in the temperate environment. it does appear that the generally low feed intake may be a defense mechanism developed by the pigs to cope with the high exogenous heat in the tropics and the resultant endogenous heat load which tend to increase to unmanageable levels following increased feed intake by pigs in this climate. feed intake of exotic pigs reared in the tropics was reported to de cline when ambient temperatures exceed 30°c (steinbach, 1976). the present study was conducted in the late dry season when average ambient temperatures averaged 34 o c condition is also felt to be partly responsible for the high post – rst feed/gain which ranged from 3.76 in 80% restricted to 3.92 in control groups. these values were higher than 2.44, 2.47 and 2.44 reported by donker et al. (1986) for pigs of similar body weight fed ad libitum or re-alimented after restriction at 85 and 70% of ad libitum respectively in the temperate environment. it therefore follows that both restricted and ad libitum fed pigs in this environment ate more feed per unit gain in weight. thus, the observed high feed/gain and the accompanying reduction in growth rate at both rst and post – rst culminated in the observed delay in age at 100 kg. contrary to 183.5d and 191.0d reported as extrapolated age at 100 kg for pigs fed ad libitum, 85 and 70% of ad libitum respectively (prince et al., 1983), the real age at 100 kg for control and restricted groups (90, 80 and 70% of ad libitum) were 233d, 235d 228d and 240d respectively. a striking finding of the present study is the feed cost/kg gain. pigs on the 80% level had significantly better values during restriction and realimentation. it follows that this level of restriction will guarantee better economic returns to a farmer. it is concluded that restricting pigs at 80% of the ad libitum intake of the control will ensure better performance and superior economic returns to a farmer. references chiba l (1995). effects of nutritional history on subsequent and over – all growth performance and carcass traits in pigs. livestock prod. sci. 41: 151-161. cleveland er, johnson rk, mandigo rw (1983). index selection and feed intake restriction is swine i. effect on rate and composition of growth. j. anim. sci. 560-567. donker ra, den hartogn la, brascamp ew, merks jwm, noordewier gj, buiting gaj (1986). restriction of feed intake to optimize the over-all performance and composition of pigs. livestock prod. sci. 15: 353-365. doornenbal h (1975). growth development and chemical composition of pigs iii. bone, ash moisture growth 39: 427-434. doyl f leeson s (2005). compensatory growth in farm animals: factors influencing response. technical paper, dept. anim. poultry sci. university of guelph, canada, pp. 1-10. duncan dp (1955). multiple range and multiple f-test. biometrics ii: 142. kyriazakis ic, stamataris gc emmans c, whittemore t (1991). the effects of food protein content on the performance of pigs previously given foods with low or moderate protein content. anim. prod. 52: 165-172. lefauheur lj, ledividich, j, mourot j, monin g, ecolan p, krauss d (1991). influence of environmental temperature on growth, muscle and adipose tissue metabolism and meat quality in swine. j. amin. sci. 69: 284-2854. mersmann hj, mac neil md seideman sc, pond wg (1987). compensatory growth in finishing pigs after fed restriction, j. anim. sci. 64: 765-773. onyimonyi ae, okeke gc (2004). performance of grower pigs fed cassava (manihot esculenta crantz) peel meal. trop. j. anim. sci. 7: 29-35. owen jb, ridgman jb, wyllie wjd (1971). the effect of food restriction on subsequent voluntary intake of pigs. anim. prod. 13: 537-546. prince tj, jungst sb, kuhlier dl (1983). compensatory response to short – term fed restriction during the growing period in swine. j. anim. sci. 56: 846-852. steel rgd, torrie jh (1980). principles and procedures of statistics, a biometric approach. 2 nd ed. mcgraw – hill publishers, new york, p 633. steinbach k (1985). impact of hot climate on feeding of farm animals: aspects of pig nutrition in tropical climates. 36 th annual meeting, eur. assoc. anim. prod. greece. pp. 1-25. steinbach k (1976). effecs of temperature on the growth of pig. in: progress in animal biometeorology, johnson hg (ed.) pp. 393-398. wahlstrom rc libal gw (1983). compensatory responses of swine following protein insufficiency in grower diets. j. anim. sci. 56: 118124. wilson pn, osbourn df (1960). compensatory growth after undernutrition in mammals and birds. boil. rev. 35: 324-363. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (9), pp. 001-006, september, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper dna damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol ming zhang1,2, hui yuan1*, zuping he3, liyun yuan1, jine yi1, sijun deng1, li zhu1, chengzhi guo1, yin lu1, jing wu1, lixinwen1, qiang wei1 and liqun xue1 1 college of veterinary medicine, hunan agricultural university, changsha, hunan 410128, people’s republic of china. 2 jiangxi biotech vocational college, nanchang, jiangxi 330200, people’s republic of china. 3 department of biochemistry and molecular & cellular biology, georgetown. university medical center, 3900 reservoir road nw, washington, dc 20057, usa. accepted 5 january, 2015 the study was designated to explore the toxic effects of gossypol on piglet sertoli cells. sertoli cells were isolated from piglet testes using a two-step enzyme digestion and followed by differential plating. piglet sertoli cells were cultured and classified into five groups, that is, group a, the control without gossypol, group b with 2.5 μg/ml gossypol, group c with 5 μg/ml gossypol, group d with 10 μg/ml gossypol and group e with 20 μg/ml gossypol. we found that sertoli cells’ growth was inhibited by gossypol at dose 2.5 μg/ml when compared with the control group. the oxidase activity of sertoli cell also decreased at 2.5 μg/m gossypol. moreover, dna damage of sertoli cells was observed at 5 μg/ml gossypol. putting this into consideration, our study suggests that exposure of gossypol to sertoli cells leads to an inhibition of sertoli cell growth and oxidase activity of sertoli cells at a low concentration, whereas gossypol results in dna damage of sertoli cells at a higher concentration. key words: gossypol, sertoli cells, oxidase, dna damage. introduction gossypol is a yellow polyphenolic compound isolated from the seed of cotton plants of the malvaceae family. it exists in two forms, free and bound. free gossypol is the major toxic ingredient of cottonseed meal, which is a source of protein for animal feed and its protein content is as high as 33.21 to 45.09%. however, the toxicity of gossypol has limited the use of cottonseed meal. gossypol is generally known as a male anti-fertility by suppressing spermatogenesis (dodou et al., 2005) and potential anticancer agent, which can induce the apoptosis of human breast cancer cells (ye et al. 2010). cytotoxic gossypol and its derivatives have been used for male contraception in humans (coutinho, 2002). ten micro *corresponding author. e-mail: yuanhui7269@yahoo.com.cn. abbreviations: sod, superoxide dismutase; gsh-px, glutathione peroxidase; mda, malondialdehyde; fcs, fetal calf serum; dntb, 5,5'-dithio-bis (2-nitrobenzoic acid) or ellman’s reagent. grams per day dose of gossypol, causes azoosper-mia and deleterious changes in monkey’s testicular morphology, especially in semi-iniferous tubules (sharma et al., 1999). in bulls, gossypol (8 mg/kg per day) induces testicular degeneration and significantly increases in primary and secondary sperm abnormalities and significant degeneration of germ cells between type b spermatogonia and pachytene primary spermatocytes (cerelli and johnson, 1999). in rats, gossypol with 15 mg/kg per day significantly decreases sperm concentration and increases the number of abnormal sperms (romualdo et al., 2002). this effect maybe caused by gossypol, which disrupts the important cell functions (ojha et al., 2006). recently, gossypol has been shown to impair the gap junctional intercellular communication in the cultured sertoli cells (zhou et al., 2008). in summary, animal experiments showed that gossypol damages animal spermatogenic epithelium, leads to abnormality and death of sperm, or even depletion of no sperm. thus, gossypol causes reduced reproductive capacity or male infertility. this study was designated to probe the toxic effects of gossypol on animal male repro duction using piglet sertoli cells in vitro as experimental model. materials and methods piglet testes testes were obtained from 3 to 4 weeks old piglets (changsha, hunan, china), placed in ice-cold phosphate-buffered saline (pbs) with 600 iu/ml penicillin-streptomycin and sent to the laboratory within 2 h. isolation, culture, and identification of testicular sertoli cells of piglets testicular capsule was removed under sterile conditions and semi-niferous tubules were isolated from piglet testis using mechanical dissociation and a one step enzymatic digestion with 1 g/l collagenase and 2.5 g/ trypsin, pursuant to the procedure as described previously (dym et al., 1995; he et al., 2007) with minor modifications. cell mixture containing male germ cells and sertoli cells were obtained using the second enzymatic digestion, collagenase iv, hyaluronidase and trypsin and sertoli cells were further separated from germ cells by differential plating according to the procedure described previously (he et al., 2007), dirami et al., 1999). for differential plating, germ cells and sertoli cells were placed into tissue culture dish in the dulbecco’s modified eagle’s medium (dmem)/f12 medium supplemented with 10% fetal calf serum (fcs) for 3 h at 34°c. sertoli cells were attached to the culture plates, whereas male germ cells remained in suspension and were removed. cell viability of sertoli cells was determined with 0.4% trypan blue exclusion assay. freshly isolated piglet sertoli cells were plated at a density of 2 × 10 6 cells/ml in dmem/f12 supplemented with 10% fcs in a humidified incubator with 5% co2 and 100% humidity for 24 h. sertoli cells were identified by oil red o staining and fas ligand (fasl) expression as assayed by immunocytochemistry when 80 to 90% of the dish was confluent with cells. experimental groups and mtt assay piglet sertoli cells were classified into five groups, which are: group a, the control without gossypol; group b with 2.5 μg/ml gossypol; group c with 5 μg/ml gossypol; group d with 10 μg/ml gossypol; group e with 20 μg/ml gossypol. after culture for 24 h, the prolife-ration of sertoli cells were determined using mtt assays with quadruplicate, according to the procedure as previously described (mosmann, 1983). determination of malondialdehyde (mda) level, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity of sertoli cells malondialdehyde (mda) level in the sonicated sertoli cells was measured by the thiobarbutiric acid method (heath and packer, 1968) and was presented as nmol per mg of protein. the activity of superoxide dismutase (sod) in the sonicated sertoli cells was measured by the xanthine oxidase method (mccord and fridovich, 1969) and presented as units per mg of protein. activity of glutathione peroxidase (gsh-px) in the sonicated sertoli cells was measured by the 5,5'-dithio-bis (2-nitrobenzoic acid) (dtnb) reaction test (flohe and gunzler, 1984) and presented as units per mg of protein. all the procedures were followed with the instruction on the kits from nanjing jiancheng company (china). determination of dna damage of sertoli cells by gossypol quantitation of dna damage in sertoli cells were performed using a single-cell microgel electrophoresis technique under alkaline conditions (singh et al., 1988) with certain modifications: the time of lysis, denature and electrophoresis were changed to 2 h, 1 h and 40 min, respectively. sertoli cells were observed for epifluorescence under fluophot microscope and 400 cells were counted in each group (4 parallel glass slides per group). being excited by ultraviolet light, the nuclear dna and the migration of dna in orange-red dna image (the comet tail) was clearly observed. statistical analysis all the data were presented as mean ± sem, statistically significant differences (p < 0.05) and extremely significant differences (p < 0.01) were determined among various groups by analysis of variance (anova) and tukey post-test using spss 12.0 statistical software. results isolation, identification and culture of piglet sertoli cells piglet sertoli cells were isolated by a 2-step enzymatic digestion with trypsin and collagenase and followed by differential plating. cell viability was up to 90% as assayed by trypan blue exclusion. after 24 h of culture, sertoli cells were attached to dish and formed a large columnar or irregular appearance with an elongated cellular body. sertoli cells were connected with each other and showed mosaic-like arrangement and intercellular irregular protrusions. on both sides of cells, there were several prominences and strong refraction. phagocytosed objects, varying sizes of vacuoles and the cell nucleus were observed in the cytoplasm of the cells. oil red o staining showed that, red lipid droplets occurred near the nucleus or at the two poles of cytoplasm (figure 1a). after 72 h of culture, high expression of fasl in sertoli cells was observed by immunocytochemistry (figure 1b). effects of gossypol on proliferation of piglets sertoli cells piglets’ sertoli cells were treated with different concentrations of gossypol for 24 h and the effect of gossypol on the proliferation of sertoli cells was assessed by mtt assay (table 1). table 1 showed that, with the increased concentration of gossypol, the sertoli cell proliferation decreased gradually. compared with the control group a, cell growth inhibition in group b was significant difference (p < 0.05), while groups c, d and e showed extremely significant difference (p < 0.01) when compared with figure 1. identification of the isolated piglet sertoli cells: (a) the oil red o staining showed that red lipid droplets (arrows) were presented near the nucleus or at the two poles of cytoplasm of the isolated piglet cells, which confirmed the identity of piglet sertoli cells. cell nuclei were counterstained with hematoxylin; (b) immunocytochemistry revealed that the isolated piglet cells were positive for fasl (arrows), further verifying the identity of piglet sertoli cells. magnification: (a) and (b) 400 ×. table 1. cell proliferation of piglet sertoli cells by mtt assay. group od value at 490 nm inhibition of proliferation (%) control (a) 0.66 ± 0.06 0 b 0.46 ± 0.03* 30.59 c 0.40 ± 0.05** 39.27 d 0.39 ± 0.01** 41.01 e 0.30 ± 0.02** 54.95 compared to the control group, “*” indicates significant difference (p < 0.05), “**” indicates extremely significant difference (p < 0.01). group a. the results suggest that, the effect of gossypol on growth inhibition of sertoli cells is in a dose-dependent manner. effects of gossypol on antioxidant enzymes activities superoxide dismutase (sod) and glutathione peroxidase (gsh-px) are two important enzymes in the antioxidant defense system. malondialdehyde (mda) is regarded as a major marker of lipid peroxidation in tissue. after expo-sure to gossypol, mda content, sod and gsh-px activities of sertoli cells were measured and the data was shown in table 2. compared with the control group a, the increase of malondialdehyde content in group b was significantly different (p < 0.05）.and the increase of mda content in groups c, d and e was extremely significant (p < 0.01). these results indicate that, the increase of mda content of sertoli cells by gossypol is also in a dose-dependent manner. the decrease of sod and gsh-px activity was obser ved in group b significant (p < 0.05) when compared with the control group a, but extremely significant decrease was observed in groups c, d and e (p < 0.01). notably, the decreases of sod and gsh-px activities were correlated with the concentration of gossypol. dna damage of gossypol on piglet sertoli cells comet tail length formed in comet assay is an important parameter in evaluating the dna damage. after uv excitation, sertoli cell dna from gossypol treated groups showed an orange-red color. dna fragments migration were observed in gossypol treated groups (figure 2a, b), suggesting that, gossypol induces dna damage in sertoli cells. table 3 showed that, 90.43% control cell were categorized at grade 0, no cells were categorized at grade 3 and 4. the increased gossypol concentration caused more severe dna damage. the rate of dna tail was used to show the degree of dna damage. an increased dna tailing rate was observed along with the table 2. effects of gossypol on malondialdehyde (mda) content, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activities of piglet sertoli cells. group mda (nmol/mgprot) sod (u/mgprot) gsh-px (u/mgprot) control (a) 2.67 ± 0.26 210.37 ± 24.63 18.87 ± 3.43 b 3.13 ± 0.47* 198.58 ± 21.32* 16.68 ± 3.26* c 5.19 ± 1.60** 178.86 ± 22.64** 13.96 ± 2.97** d 5.46 ± 0.88** 137.57 ± 21.57** 12.05 ± 2.18** e 11.24 ± 1.98** 105.13 ± 19.48** 9.63 ± 2.27** compared to the control group, “*” indicates significant difference (p < 0.05), “**” indicates extremely significant difference (p < 0.01). figure 2. the single-cell gel electrophoresis (comet assay) showed the dna damage of gossypol on piglet sertoli cells: (a) dna image of control group; (b) comet assay revealed that damaged dna of gossypol-treated group b contained strand breaks migrates farther in the gel than intact dna, creating an image resembling a celestial comet. magnification: 400×. table 3. effect of gossypol on dna damage of piglet sertoli cells. total cell number of different degree rate of dna group cell grade 0 grade 1 grade 2 grade 3 grade 4 tail (%) number control (a) 100 90.43 ± 4.34 5.17 ± 1.02 4.40 ± 1.23 0.00 ± 0.00 0.00 ± 0.00 9.57 ± 1.25 b 100 86.33 ± 4.51 6.29 ± 2.03 4.61 ± 1.62 1.65 ± 1.14 1.12 ± 0.53 13.67 ± 1.46 c 100 68.94 ± 5.73** 18.28 ± 3.79** 5.39 ± 0.97* 4.82 ± 1.13* 2.57 ± 0.46* 31.06 ± 3.13** d 100 60. 52 ± 3-48** 20.05 ± 2.68** 8.76 ± 1.55** 6.13 ± 1.35** 4.41 ± 1.3** 39.35 ± 2.57** e 100 40.12 ± 2.53** 24.06 ± 2.85** 12.24 ± 2.32** 13.56 ± 1.75** 10.02 ± 2.41** 59.88 ± 4.31** compared to the control group, “ increased gossypol concentration. these data indicates that, the overall level of dna damage gradually increased in an obvious dose-response to gossypol (table 3). discussion breeding domestic livestock and aquatic animals with cottonseed cake has significant economic benefits. however, gossypol has been found toxicity in male reproductive system as a feedstuff additive. this study showed that, gossypol inhibited the growth of piglet sertoli cells in a dose-dependent manner. thus, the current may shed insight into male reproductive toxicity of gossypol in clinic. we used 10% fetal calf serum (fcs) for the growth and survival of piglet sertoli cells. recently, we demonstrated that 10% fcs promotes proliferation of piglet sertoli cells (zhang et al. 2010). the control group and gossypoltreated groups were cultured with fcs and thus fetal calf serum has no effect for us to investigate the toxicity of gossypol on piglet sertoli cells. previous research of gossypol on male reproductive toxicity is mainly focused on the structure of testis, epididymis and spermatogenic cells. gossypol causes sparse array of testis seminiferous tubule, ambiguous or self-dissolving nucleus of epitheliums and reduces the amount of sperm, unclear sperm structure and swelling mitochondrial. gossypol inhibits testicular lactate dehydrogenase activity. thereby it undermines the animal spermatogenic epithelium, results in abnormality and death of sperm, or even no sperm. finally, it causes sire infertility, reduces mitochondrial function and results in infertility (monsees et al., 1998). many researches suggests that, apoptosis plays an important role in testis regression (furuta et al., 1994). apoptotic spermatogenic cells can be found at all levels, especially at spermatocytes and sperm cells. the apoptotic cells occasionally aggregate chain-like or piles. andrade et al. (2006), utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol (de andrade et al., 2006). sertoli cell junctional protein maybe the early target of toxins such as gossypol (fiorini et al., 2004). the effects of gossypol on sertoli cells are still unclear. oxidative stress can cause potential harm to animal organism. when oxidative stress is over generated or the enzymatic and non-enzymatic antioxidant defense systems are inefficient, it can stimulate some chain reactions causing cytotoxic effect. superoxide dismutase (sod) and glutathione peroxidase (gsh-px) are the two major part of oxidase defense system. superoxide dismutase is closely related to the cellular oxidative metabolism which widely exists in vivo. it is a natural scavenger of reactive oxygen species (ros) and superoxide anion radicals. in the antioxidant enzymes, sod is the first enzyme to combine with active oxygen free radicals. sod specifically combines with superoxide anions and acts synergistically with gsh-px in preventing cell membrane lipid peroxidation and damaging metabolites’ formation. they directly capture and eliminate free radicals (alul et al., 2003). through hydrogen atom donors’ effect of blocking or terminating radical consecutive reaction chain, superoxide dismutase prevents the action of free radicals, suppress cell membrane lipid peroxidation during pathological lesion and protect biological macromolecules and membranes from damage. glutathione peroxidase converts hydrogen peroxide (h2o2) into h2o. gsh-px has a strong ability of scavenging lipid peroxide and hydrogen peroxide which were induced by active oxygen species and hydroxyl radicals. thus, sod and gsh-px can protect biological macromolecules and membranes from damages. the values of sod, gsh-px and malondialdehyde (mda) content of oxidative metabolites malondialdehyde is often considered as the major hallmark of lipid peroxidation damage, which causes secondary damage to cell functions, genotoxicity and carcinogenesis (korchazhkina et al., 2003). dna damage is the major effect of gossypol in piglet sertoli cells. the tail ratio in gossypol treated groups from 5 μg/ml was remarkably different from the control, as detected by single cell gel electrophoresis. this result suggests that, gossypol can cause dna damage in piglet sertoli cells at high concentration. activities of superoxide dismutase, glutathione peroxidase and malondialdehyde content in gossypol treated groups b to e were significantly different from the control, whereas dna damage was observed in groups exposed to gossypol at concentrations above 5 μg/ml. the dna damage induced by gossypol could be as a result of its stimulation of hydroxyl radicals production (el-sharaky et al., 2009); gossypol also causes dnabreaking (li et al., 2000). thus, gossypol has a marked effect on the superoxide dismutase, glutathione peroxidase activities, malondialdehyde content and dna damage of 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cells and causes their dna damage, cell apoptosis and aberrant ultrastructure. reprod. biol. endocrinol. 8: p. 97. zhou dr, zhou yc, cui gh, guo x, qin j, gui yt, cai zm (2008). gossypol repressed the gap junctional intercellular communication between sertoli cells by decreasing the expression of connexin43. toxicol. in vitro. 22: 1719-1725. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (8), pp. 001-006, august, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper the effect of glutamine supplement on small intestinal morphology and xylose absorptive ability of weaned piglets c. b. hsu1, 2, h. j. huang1, c. h. wang1 h. t. yen3 and b. yu2* 1 kaohsiung animal propagation station, livestock research institute, coa, 372 tungang road, laupi village, neipu, pingtung 912, taiwan, r.o.c 2 department of animal science, national chung hsing university, 250 kuo kuang road, taichung 402, taiwan, r.o.c 3 animal technology institute taiwan, p.o. box 23, chunan, miaoli 350, taiwan, r.o.c accepted 05 february, 2015 the purpose of this study is to demonstrate the effects of glutamine (gln) supplement on small intestinal morphology, xylose absorptive and growth performance of weaned piglets. forty eight piglets weaned at 28 ± 2 days of age were randomly allotted to three treatment groups. a basal corn-soybean diet was formulated to contain 20.3% protein and 3450 kcal de/kg diet. glutamine was supplemented to the basal diet at 0% (control), 1% (gln 1%) and 2% (gln 2%). pigs were fed experimental diets for three weeks. the results showed that the villous height of the gln groups tended higher than the control group in duodenum and jejunum (p < 0.1). glutamine supplementation increased plasma net xylose absorptive concentration from 0.78 to 1.20 and 0.95 to 1.23 in gln 1% and gln 2% group, respectively, which were better than the control group (0.86 to 0.97) in day 7 to 14 after weaning. growth performance was not significantly affected by gln supplement; however, average daily gain was approximately improved from 21 to 28% by gln supplement compared to the control group during 21 days of experimental period. in summary, the results suggested that dietary supplementation of gln could be beneficial in small intestinal villous morphology and xylose absorptive capacity, and could have a slight contribution to the average daily gain of weaned piglets. key words: glutamine, growth performance, intestinal morphology, weaned piglets. introduction inadequate nutrient intake after weaning often causes damage to the intestinal villi resulting in poor growth of weanling pigs (van beers-schreurs et al., 1998). amino mucosa. one of them, glutamine (gln) is an essential *corresponding author. e-mail: byu@dragon.nchu.edu.tw. tel: +886-4-22860799. fax: +886-4-22860265. abbreviations: gln, glutamine; p5c, proline 5-carboxylate; de, digestible energy; cp, crude protein; si, small intestine; pbs, phosphate buffered saline;; edta, ethylenediaminetetraacetic acid;; tpn, total parenteral nutrition; iga, immunoglobulin a; vh, villous height; cd, crypt depth; bw, body weight; adg, average daily gain; adfi, average daily feed intake. acids provide the major energy source for the intestinal precursor for the synthesis of proteins as well as purine/pyrimidine nucleotides. moreover, it can be used as an energy source to support rapidly, the differentiation and proliferation of intestinal epithelial cells (newsholme et al., 2003; wu et al., 1996) and activated lymphocytes cells (wu et al., 1995). therefore, gln is the most abundant free amino acid found in the blood of animals and in the milk of sows (wu and knabe, 1994). at normal intakes, dietary gln is metabolized by the small intestine and essentially, all gln within the body is synthesized de novo through the action of glutamine synthetase. the major sites of net gln synthesis are lung, adipose tissue, and skeletal muscle and under some conditions, the liver. the intestine expresses proline 5carboxylate (p5c) synthase, which means that proline is table 1. the composition of basal diet. ingredient (%) basal diet maize, dent yellow 49.05 soybean meal, 44 of cp 23.70 dried skim milk 16.0 whey 5.0 soybean oil 1.0 dicalcium phosphate 1.60 limestone, pulverized 0.80 salt 0.50 vitamin premix 1 0.10 mineral premix 2 0.15 choline chloride, 50 0.10 maize starch 2.0 glutamine 0 calculated values crude protein 20.3 calcium 1.01 total phosphorus 0.77 lysine 1.17 1 supplied per kg of diet: vitamin a, 6,000 iu; vitamin d3, 800 iu; vitamin e, 20 mg; vitamin k3, 4 mg; vitamin b2, 4 mg; vitamin b6, 1 mg; vitamin b12, 0.02 mg; niacin, 30 mg; calcium pantothenate, 16 mg; folic acid, 0.6 mg; biotin, 0.01 mg; choline chloride, 50 mg. 2 supplied per kg of diet: fe (feso4.h2o), 140 mg; cu (cuso4.5h2o), 7 mg; mn (mnso4.h2o), 20 mg; zn (zno), 120 mg; i (kio3), 0.45 mg. an end product of intestinal gln catabolism (wu et al., 1995). previous studies have shown that gln is a conditionallyessential amino acid under weaning period as well as stressful conditions such as injury and infection (newsholme, 2001). yi et al. (2005) showed that gln is beneficial for maintaining muscular gln concentrations and normalizes lymphocyte function of escherichia colichallenged weaned pigs. furthermore, due to the effect of gln on regulation of systemic inflammation, it had been thought that it could be applied potentially to inflammatory diseases (singleton and wischmeyer, 2008). however, the endogenous gln is insufficient and consequently, animals need to increase their requirement (hall et al., 1996). therefore, the aim of this study is to investigate the effects of dietary glutamine supplementation on small intestinal villous morphology/structure, xylose absorptive ability and growth performance of weaned piglets. materials and methods animals and diets the animal feeding protocol of this research was approved by the animal care and use committee of kaohsiung propagation station, livestock research institute, council of agriculture. forty-eight crossbred pigs (landrace × yorkshire × duroc) weaned at 28 ± 2 days of age were obtained from 10 litters, and littermates were randomly allotted to 12 pens for three dietary treatments according to weight and sex. same sex was allotted to each pen. the treatments consisted of a control group (c), control diet supplemented with glutamine in replace of maize starch for 1% (gln 1%) and 2% (gln 2%). the control diet (table 1) was based on maize-soybean meal with digestible energy (de) 3450 kcal/kg, crude protein (cp) 20.3%, and lysine 1.17% according to the standards of nrc (1998). during the 21 days of experimental period, pigs were housed in a traditional nursery room with wire-floored pens. meal feed and nipple water were provided ad libitum. the individual pig weight, pen feed consumption, and pen feed efficiency (gain/feed) were recorded weekly. small intestinal morphology observation four pigs from each treatment (one pig per pen as duplicate) were sacrificed on day 14 post-weaning. pigs were anaesthetized by halothane inhalation. following intestinalectomy, the small intestine (si) was removed and the length was determined; the positions at 10, 50, and 90% of the length of the si were located at duodenum, jejunum and ileum, respectively. a 4 cm segments was taken from each portion for histological measurement. these samples were first rinsed with 0.1 m phosphate buffered saline (pbs) at ph 7.2, and then fixed with 10% neutral formaldehyde. after 24 h, the samples were removed from the fixative, cut into 1 cm 2 sections (two per location) and stored in fresh fixative. then, they were embedded in paraffin, sectioned at 6 µm thickness and stained with hematoxylin as well as eosin for a light microscopy examination. the villous height (vh) and crypt depth (cd) were measured based on 15 apparently intact villi from each section according to yu and chiou (1997). another 4 cm samples were taken from jejunum and ileum for morphological observation using a scanning electronic microscope according to the method of yu and chiou (1997). the gut samples were fixed in 10% buffered neutral formaldehyde, then rinsed in pbs 3 times and placed in 1% osmium tetraoxide overnight. they were rinsed again in pbs for 4 times. the samples were gradually dehydrated by increasing alcohol concentrations from 50 to 100%. they were then dehydrated, mounted on aluminum stubs, coated with gold for 30 min, and subsequently placed in the scanning electronic microscope (hitachi s-300) for scanning. xylose absorption ability on day 7 and 14 post weaning, eight pigs of each treatment group (2 pigs per pen) were carried to measure the ability of active absorption of small intestine. before the procedure, the pig fasted for 16 h, and then fed 10% d-xylose (sigma chemical inc, usa) solution at a dose of 1 ml/kg body weight by gavages. blood samples (6 ml) were withdrawn through anterior vena cava with tubes containing ethylenediaminetetraacetic acid (edta) pre and post 1 h gavages. plasma was obtained by centrifuging at 1500 g for 15 min and was stored at -20°c until analyzed for d-xylose concentration according to the procedures of trinder (1975). the ability of absorption was determined by measuring the difference of xylose concentration in plasma between pre and post feeding of xylose. another twelve pigs were randomly selected from the same herd at 28 day of age and their xylose absorption was measured as a reference data. statistical analysis data was analyzed by analysis of variance (anova) using the general linear model (glm) procedure of the statistical analysis table 2. effect of supplementing glutamine on the intestinal morphology of weaned pigs 1 . item control gln 1% gln 2% se contrast (c vs. gln) duodenum villus hight,µm 282 345 332 23 0.069 crypt depth,µm 266 282 260 32 0.914 vh/cd 1.06 1.21 1.27 0.20 0.941 jejunum villus hight,µm 306 377 397 33 0.068 crypt depth,µm 202 242 211 16 0.236 vh/cd 1.52 1.56 1.84 0.19 0.634 ileum villus hight,µm 260 349 302 33 0.121 crypt depth,µm 193 192 196 21 0.964 vh/cd 1.34 1.64 1.54 0.17 0.601 1 each value represents the mean of 4 pigs. vh, villous height; cd, crypt depth. figure 1. the scanning electron micrographs of the jejunal (a, b, c) and ileal (d, e, f) villi from control and glutamine supplement groups of pigs at 14 days postweaning. control group (a and d); gln 1% group (b and e); and gln 2% group (c and f). the boxed areas showed erosion of surface epithelium at the apex of the villi in control group. system (sas) programs (1999). duncan’s new multiple range test was applied for comparing the differences among treatments. the orthogonal contrasts were performed to compare the glutamine treatment effect. the difference was considered to be significant at p < 0.05, and p < 0.10 was considered as a trend. results intestinal morphology the morphology of the duodenum, jejunum and ileum at day 14 of the experiment is shown in table 2. the results showed that glutamine supplement groups numerically improved the villus height of duodenum and jejunum when compared to the control (p < 0.1). whereas, there were no difference between 1 and 2% gln supplement groups. figure 1 shows the scanning electron micrographs of the jejunal (figures 1a, b and c) and ileal (figures 1d, e and f) villi from the control and glutamine supplement groups, respectively. in general, blunted and folded villi were observed in most pigs (figures 1b, c, e and f). damaged villi with erosion of surface epithelium table 3. effect of supplementing glutamine on the plasma xylose concentration (mmol/l) of weaned pigs 1 . item control gln 1% gln 2% se day 7 preoral conc. 0.15 0.15 0.12 0.01 postoral conc. 1.01 0.93 1.06 0.22 net absorptive conc. 0.86 0.78 0.95 0.22 day 14 preoral conc. 0.16 0.09 0.14 0.02 postoral conc. 1.13 1.29 1.37 0.23 net absorptive conc. 0.97 1.20 1.23 0.22 1 each value represents the mean of 8 pigs. table 4. effect of supplementing glutamine on the growth performance of weaned pigs 1 item control gln 1% gln 2% se average bw (kg) initial 6.66 6.69 6.67 0.22 7 day (12) 1 7.26 7.28 7.37 0.24 14 day (12) 8.28 8.50 8.35 0.35 21 day (8) 9.78 10.35 10.36 0.58 adg (kg/head) 0 to 7 day 0.09 0.09 0.10 0.02 8 to 14 day 0.13 0.17 0.14 0.03 15 to 21 day 0.20 0.24 0.26 0.03 0 to 21 day 0.14 0.17 0.18 0.02 adfi (kg/head) 0 to 7 day 0.23 0.19 0.22 0.04 8 to 14 day 0.24 0.28 0.31 0.05 15 to 21 day 0.33 0.30 0.34 0.05 0 to 21 day 0.26 0.24 0.28 0.03 gain/feed 0 to 7 day 0.41 0.45 0.44 0.08 8 to 14 day 0.53 0.60 0.46 0.12 15 to 21 day 0.67 0.80 0.76 0.09 0 to 21 day 0.54 0.63 0.64 0.05 1 values are presented as means with the numbers of piglets given in parentheses. bw, body weight; adg, average daily gain; adfi, average daily feed intake. at the apex of the villi were detected in some pigs from the control group (figures 1a and d). the integrity of intestinal morphology was better in the glutamine supplementation groups than in the control group. absorption of xylose table 3 shows plasma xylose concentration of the three treatments. the average plasma xylose concentration at weaning was 0.17 mmol/l (data not shown). glutamine supplementation increased plasma net xylose absorptive concentration from 0.78 to 1.20 and 0.95 to 1.23 in 1 and 2% gln group, respectively, which were better than the control group (0.86 to 0.97) in day 7 to 14 after weaning. growth performance the growth performance is presented in table 4. no significant differences in average daily gain (adg), daily feed intake and gain/feed were observed regardless of the treatment group. however, adg was numerically improved (p > 0.05) 30 and 8% by gln 1% and gln 2% compared to the control from d 8 to 14 of the experimental period (p > 0.05) and 20 and 30%, respectively, from day 15 to 21. overall, adg was improved 21 to 28 % approximately by glutamine supplement compared to the control group during 21 days of experimental period. discussion weaning of piglets is known to be associated with gross changes in small intestinal morphology and structure such as villous atrophy, decreased villous height and increased crypt depth that will decrease the intestinal active absorption (van beers-schreurs et al., 1998). if the intestinal villous atrophy could be prevented, it would improve nutrient digestion and absorption and growth gap of weaned piglets (pluske et al., 1997). in the present study, glutamine supplementation showed a trend of improving villous heights of the duodenum and jejunum at day 14 of the weaning period (p < 0.1) and activation absorptive ability of xylose. the results agreed with the findings of wu et al. (1996) as well as the previous research that glutamine or glutamine-dipeptide supplementation to total parenteral nutrition (tpn) solution prevented gut atrophy in humans and rats (schroder et al., 1995). liu et al. (2002) suggested that the jejunal atrophy was prevented by 1.0% glutamine supplementation during the first week post-weaning piglets. yu et al. (2002) also suggested that a combination of 1.0% of glutamine and 1000 ppm of nucleotide in diet could improve feed intake and intestinal villus height. the reasons being that glutamine facilitated the survival and proliferation of intestinal mucosal cells and that glutathione synthesis from glutamine maintains the mucosal integrity and defenses. another explanation could be the glutamine-dependent protein expression of intestinal epithelial tight junction barrier and cellular localization in caco-2 cell monolayers (liu et al., 2002; wu et al., 1996). this mechanism may similarly relate to glutamine-mediated modulation of intestinal barrier function in stressed animals and humans (li et al., 2004; demarco et al., 2003). furthermore, enteral glutamine can stimulate the mucosal protein synthesis and preserve the paracellular permeability (coeffier et al., 2003; le bacquer et al., 2003) that will be helpful for maintaining the epithelial barrier function. our study indicated that the villi of the control pigs were damaged to some extent possibly due to inadequate nutrient intake and it may result in increase rate of cell turnover as well as decreasing villi height. the present study showed that the intestine xylose absorptive ability improved from day 7 to 14 after weanling in both gln supplement groups when compared to the control group. therefore, glutamine supplementation provided a beneficial environment for the proliferation of enterocytes, preventing intestinal atrophy and activation absorptive function. currently, glutamine supplementation had no significant improvement on the performance of piglets; however, it numerically improved 21 to 28% of adg compared to the control group during 21 days of experimental period. zou et al. (2006) found that pigs supplemented with 1% glutamine had a 12% lower feed/gain ratio during the first ten days after weaning and had a 27.8% higher adg during day 11 to 20 post-weaning. wu et al. (1996) had reported that 0.2 to 1.0% glutamine supplementation did not have any significant effects on the daily feed intake, adg, and gain/feed during the first week post weaning, but the gain/feed of pigs supplemented with 1% glutamine was 25% higher than the control pigs during the second week of post-weaning. lee et al. (2003) also found that 1.5% glutamine supplementation did not affect the feed intake, adg, and gain/feed of pigs weaned on 21 days of age, but the small intestinal development and bile immunoglobulin a (iga) production were improved. similar results were also reported by bartell and batal (2007) in chicken, their results showed 1% gln could improve growth performance, facilitate the health of gi tract, and increase the concentrations of sera igg and iga. in the study of yi et al. (2005), they indicated that 2.0% glutamine supplementation on pigs weaned at 17 days of age did not affect growth performance during 11 days of feeding, but glutamine supplementation had beneficial effects on alleviating growth depression of e. coli k88+-challenged weaned pigs, through maintaining intestinal morphology and function. focusing on glutamine supplementation for growth performance in weaned piglets, zou et al. (2006) explained the importance of glutamine on energy source for enterocytes, the necessary precursor for dna and protein synthesis, and the biological regulating function of glutamine metabolite. recently, research on molecular mechanisms had revealed the findings that dietary glutamine supplement would increase intestinal expression (120 to 140%) of genes that is necessary for cell growth and removal of oxidants (wang et al., 2008). however, the reason growth performance could not be consistently improved by glutamine might result from the fact that it may not be affected by glutamine alone, but with other uncertain environmental factors. therefore, from the results obtained, it is suggested that glutamine supplementation for weaned piglets seemed to lack the significant improving effects on growth performance, but posses positive and improving efficacy for the maintenance of the intestinal villous morphology and function. in conclusion, dietary supplementation of glutamine could be beneficial to small intestinal villous morphology, xylose absorptive capacity and slightly contribute to the average daily gain of weaned piglets. acknowledgements the authors would like to thank the council of agriculture of the republic of china for supporting the research and the ajinomoto co. inc. (yokyo, japan) for their generous gift of l-glutamine. the authors also appreciate dr. t. k. chung for his help to revise the manuscript. references bartell sm, batal ab (2007). the effect of supplemental glutamine on growth performance, development of the gastrointestinal tract, and humoral immune response of broilers. poult. sci. 86: 1940-1947. coeffier m, claeyssens s, hecketsweiler b, lavoinne a, ducrotte p, dechelotte p (2003). enteral glutamine stimulates protein synthesis and decreases ubiquitin mrna level in human gut mucosa. am. j. physiol. gastrointest liver physiol. 285: g266-273. demarco vg, li n, thomas j, west cm, neu j (2003). glutamine and barrier function in cultured caco-2 epithelial cell monolayers. j. nutr. 133: 2176-2179. hall jc, hell k, mccaulet r (1996). glutamine. br. j. surg. 83: 305-321. le bacquer ol, laboisse c, darmaun d (2003). glutamine preserves protein synthesis and paracellular permeability in caco-2 cells submitted to luminal fasting. am. j. physiol. gastrointest. liver physiol. 285: g128-136. lee dn, cheng yh, wu fy, sato h, shinzato i, cheng sp, yen ht (2003). effect of dietary glutamine supplement on performance and intestinal morphology of weaned pigs. asian-aust. j. anim. sci. 16: 1770-1776. li n, lewis p, samuelson d, liboni k, neu j (2004). glutamine regulates caco-2 cell tight junction proteins. am. j. physiol. gastrointest. liver physiol. 287: g726-g733. liu t, peng j, xiong y, zhou s, cheng x (2002). effects of dietary glutamine and glutamate supplementation on small intestinal structure, active absorption and dna, rna concentrations in skeletal muscle tissue of weaned piglets during d 28 to 42 of age. asian-aust. j. anim. sci. 15: 238-242. national research council (1998). nutrient requirements of swine. national academy press, washington, d.c. usa. newsholme p (2001). why is l-glutamine metabolism important to cells of the immune system in health, postinjury, surgery or infection? 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(1976) showed that these lc pigs not only matured earlier than improved european and american breeds but also had lower percentages of lean and bone and greater propor-tions of fat and skin as several reports have indicated that when protein intake is liberal, improved european and american breeds of pigs such as the lw and landrace (ld) have faster growth rate and better feed conversion efficiency than the unimproved lc pigs (vohradsky, 1968; ilori, 1974; fetuga et al., 1976, 1977). on the other hand, limited information from latin america shows that indigenous pigs of latin america tend to perform better than their european and american counterparts under conditions of poor protein nutrition (bressani, 1974). fetuga et al. (1977) reported on the comparative responses of large white x landrace (lw x ld) and lc *corresponding author. e-mail: abelokwori@yahoo.com. pigs to diets varying in protein level from 12 to 20% crude protein. increments in dietary protein improved both rate and efficiency of gain of the lw x ld pigs, the best performance being on the 18 and 20% crude protein diets. for the lc pigs, the best growth rates were obtained on the lowest two protein levels of 12 and 14% crude protein, while crude protein levels above 16% caused a decline in growth rate of the lc pigs. it was the objectives of the present study to compare the performance of lc, lw x lc and lw pigs fed different dietary protein levels. materials and methods tow feeding experiments were conducted using a 2 x 2 factorial design involving two genetic groups of pigs and two protein levels in each case. experiment 1 two isocaloric diets (3.1 mcal of me/kg) having 12 or 16% crude protein (table 1) were fed far seven weeks to a total of 24 barrows, 12 barrows each of the lc and the lw x lc genetic groups. there table 1. composition of experimental diets (experiment 1) % of diet ingredient 12% crude protein level 16% crude protein level maize 78.73 74.36 groundnut cake 2.00 14.40 fish meal 2.50 2.50 wheat middling 12.00 4.00 oyster shell 3.00 3.00 vitamin-mineral premix a 1.00 1.00 sodium chloride 0.50 0 dl methionine 0.27 0.24 total 100.00 100.00 analyzed composition dry matter (%) 96.55 97.40 crude protein (%) 12.03 16.40 ether extract (%) 9.00 10.35 crude fibre (%) 3.55 3.45 ash (%) 6.15 6.50 nitrogen-free extract (%) 65.82 60.70 a provided the following in units per kg of diet vitamin a, 5280 i.u.; vitamin d3, 704 i.u.; vitamin e, 70 i.u.; vitamin k, 3.52 mg; vitamin b12, 26 ug; riboflavin 5 mg; niacin, 28 mg; d-pantothanic acid, 21 mg; chlorine chloride 1,100 mg; biotin, 88 ug; thiamine, 2.2 mg; cu; 10 mg; fe, 160 mg; mn, 20 mg; zn, 100 mg; co, 2.5 mg; i, 2.5 mg. were six pigs in each of the four treatment combinations. the pigs were housed two per pen according to genetic group, on concrete floor pens. thus, there were three replicates per treatment. the lc and the lw x lc pigs had average initial weights of 7.3 and 10.2 kg, respectively. feed and water were offered ad libitum. animals were weighed at weekly intervals and feed consumption was measured by the weigh-back technique. feed samples were assayed for dry matter, crude protein, crude fibre, ether extract and ash by the a.o.a.c. (1975) method of analysis. data on feed intake, protein intake, growth rate, feed conversion ratio and protein efficiency ratio were obtained and subjected to a 2 x 2 factorial analysis of variance (snedecor and cochran, 1974). comparisons of treatment means were made by duncan’s multiple range tests (steel and torrie 1960). experiment 2 a total of 24 barrows, 12 each of the lw and the lw x lc genetic groups, were used in this experiment. they were housed two per pen, according to genetic group, an average floor pens and fed two isocaloric (3.1 mcal of me/kg) diets containing either 14 or 18% crude protein (table 2). thus, there were four treatment combinations (six pigs per treatment) and three replicates per treatment. the average initial weights of the lw pigs were 12.50 and 12.41 kg for those fed the 14 and 18% protein diets, respectively. average initial weights of the lw x lc pigs were 12.0 and 11.58 kg for those fed the 14 and 18% crude protein diets, respectively. feeding, weighing, analysis of feed and statistical analysis of data were as described for experiment 1. results experiment 1 the main affects of genetic group and dietary protein level on performance of pigs in experiment 1 are summarized in table 3. effects of genetic group the lw x lc pigs consumed 86.7 g more feed (p<0.05) and 12.8 g more protein (p<0.025) daily, and grew 33.4 g/day more rapidly (p<0.01) than the lc pigs. however, feed conversion ratios and protein efficiency ratios did not differ significantly (p>0.05) between the lc and the lw x lc genetic groups. effects of protein level feed intake was not significantly (p>0.05) affected by protein level but pigs fed the 16% crude protein diet consumed 39.5 g more protein and gained 30 g more weight daily (p<0.01) than those fed the 12% crude protein diet. feed conversion ratio was not significantly (p>0.05) affected by protein level but pigs fed the 12% crude protein diet gained significantly (p<0.01) more weight per gram of protein consumed than pigs fed the 16% crude protein diet. interactions between genetic group and protein levels the effects of the genetic group x protein level treatment combinations in experiment 1 are given in table 4. there table 2. composition of experimental diets (experiment 2). % of diet ingredient 14% crude protein level 18% crude protein level maize 76.25 72.22 groundnut cake 8.50 20.00 fish meal 2.50 2.50 wheat middlings 8.00 1.00 vitamin-mineral premix a 1.00 1.00 oyster shell 3.00 3.00 sodium chloride 0.50 0.50 dl methionine 0.25 0.28 total 100.00 100.00 analyzed composition dry matter (%) 95.60 96.00 crude protein (%) 13.78 18.16 ether extract (%) 10.20 11.00 crude fibre (%) 3.50 3.60 ash (%) 6.40 6.46 nitrogen-free extract (%) 61.72 60.78 metabolizable energy (kcal/kg, calculated) 3,070.0 3,108.0 a provided the following in units per kg of diet: vitamin a, 5280i.u.: vitamin d3, 704 i.u.; vitamin e 70 i.u.; vitamin k, 3.52 mg; vitamin b12, 26ug; riboflavin, 5 mg; niacin, 28 mg; d-panthothenic acid, 21 mg; choline chloride, 1,100 mg; biotin, 88 ug; thiamine, 2.2 mg; cu, 10 mg; fe, 160 mg: mn, 20 mg; zn, 100 mg; co, 2.5 mg; i, 2.5 mg. table 3. main effects of genetic group and protein levels on performance of pigs (experiment 1). genetic group crude protein level (%) parameter lc lw x lc s.e. 12 16 s.e. daily feed intake (g) 865.0 b 951.7 a 31.1 910.0 906.7 31.1 daily protein intake (g) 122.7 a 135.5 b 4.1 109.5 c 148.7 d 4.1 daily weight gain (g) 308.3 b 341.7 a 6.3** 310.0 d 340.0 c 6.3** feed (kg/gain kg) 2.81 2.84 0.12* 2.95 2.70 0.12* protein efficiency ratio 2.59 2.54 0.11** 2.84 c 2.28 d 0.11** a,b means of the main effects of genetic group having different superscripts are significantly different (p<0.05). c,d means of the main effects of protein level having different superscripts are significantly different (p<0.01). **significant genetic group x protein interaction (p<0.01). *significant genetic group x protein interaction (p<0.05). e protein efficiency ratio = weight gained (g) protein consumed (g). were no significant (p>0.05) genetic group x protein interactions and no significant treatment effects on feed intake. although protein intake different significantly (p<0.025) among treatments, these differences were not of interest because there were no significant (p> 0.05) genetic group x protein interactions on protein intake. average daily weight gains showed significant (p<0.01) genetic group x protein interactions. the lc pigs fed the 12% crude protein diet gained more weight per day (p<0.01) than the lc pigs fed the 16% protein diet whereas the lw x lc pigs fed the 16% protein diet gained more weight per day (p<0.01) than the lw x lc pigs fed the 12% protein diet. average (p<0.05) genetic group x protein interactions were also observed in feed conversion ratios and protein efficiency ratios (p<0.01). the lc pigs fed the 12% protein diet had better (p<0.05) feed conversion ratios than the lc pigs fed 16% protein diet but the lw x lc pigs which consumed the 16% protein diet had better (p<0.05) feed conversion ratios than the lw x lc pigs which consumed the 12% protein diet. weight gained by the lc pigs per gram of protein consumed was significantly (p<0.01) higher with the 12% protein diet than with the 16% protein diet. but weight gained by the lw x lc table 4. treatment effects of genetic group and protein levels on performance of pigs (experiment 1). lc lw x lc 12 crude 16 crude 12 crude 16 crude parameter protein level protein level protein level protein level s.e. p initial weight (kg) 7.30 7.30 10.20 10.20 final weight (kg) 23.14 21.67 24.74 29.15 daily feed intake (g) 876.7 853.3 943.3 960.0 44.0 (p>0.05) daily protein intake (g) 105.5 c 139.9 b 113.5 c 157.5 a 5.8 (p<0.025) daily weight gain (g) 323.3 b 293.3 c 296.7 c 386.7 a 8.9 (p<0.01) feed (kg/gain kg) 2.72 b 2.91 a 3.19 a 2.49 b 0.17 (p<0.025) protein efficiency ratio 3.07 a 2.10 c 2.61 b 2.47 bc 0.15 (p<0.05) table 5. main effects of genetic group and protein levels on performance of pigs (experiment 2). genetic group crude protein level (%) parameter lw lw x lc s.e. 14 18 s.e. daily feed intake (g) 1011.7 a 970.0 b 5.8** 1016.7 c 965.0 d 5.8** daily protein intake (g) 160.6 a 153.8 b 1.52** 140.1 c 175.2 d 1.52** daily weight gain (g) 383. 3 a 361.7 b 5.2** 346.7 c 398.3 d 5.2** feed (kg/gain kg) 2.73 2.69 0.05** 2.96 c 2.46 d 0.05** protein efficiency ratio 2.36 2.38 0.04** 2.47 c 2.26 d 0.04** a,b means of the main effects of genetic group having different superscripts differ significantly (p<0.01) c,d means of the main effects of protein level having different superscripts differ significantly (p<0.01). **significant genetic group x protein interaction (p<0.01) protein efficiency ratio = weight gained (g)/protein consumed (g). pigs per gram of protein consumed did not differ significantly (p>0.05) between the 12 and the 16% protein diets. experiment 2 the main effects of genetic group and dietary protein level on performance of pigs in experiment 2 are summarized in table 5. effects of genetic group the lw pigs consumed significantly (p<0.01) more feed and more protein and also gained (p<0.01) more weight than the lw x lc pigs. however, feed conversion ratios and protein efficiency ratios were not significantly (p>0.05) influenced by genetic group. effects of protein level the pigs consumed more (p<0.01) of the 14% protein diet than they did the 18% protein diet. but pigs fed the 18% protein diet consumed more (p<0.01) protein than those fed the 14% protein diet. the pigs fed the 18% protein diet also gained more weight and had better feed conversion ratios (p<0.01) than those fed the 14% protein diet. but weight gained per gram of protein con-sumed was more (p<0.01) with the 14% than with the 18% protein diet. interactions between genetic group and protein level the effects of the genetic group x protein level treatment combinations in experiment 2 are given in table 6. there were significant (p<0.01) genetic group x protein interactions in all the parameters measured in experiment 2. there were no significant differences (p>0.05) in feed intake between the lw pigs fed 14 or 18% protein diet but the lw x lc pigs fed 14% protein diet consumed 106.6 g more feed (p<0.01) per day than the lw x lc pigs fed the 18% protein diet. there were also significant (p<0.01) differences among treatments in protein intake. protein consumed per pay (184 g) by the lw pigs fed the 18% protein diet was more (p<0.01) than that (166.5 g) consumed by the lw x lc pigs also fed the 18% protein diet. protein consumption by the lw pigs fed the 14% protein (139.2 g/day) did not differ (p>0.05) from that of lw x lc pigs also fed the 14% protein diet (141 gm/day). the lw pigs fed the 18% protein diet had faster rates (p<0.01) of weight gain and better feed conversion ratios table 6. treatment effects of genetic group and protein levels on performance of pigs (experiment 2). lw lw x lc 14 crude 18 crude 14 crude 18 crude parameter protein level protein level protein level protein level s.e. p initial weight (kg) 12.50 12.41 12.0 11.58 final weight (kg) 27.85 34.62 30.62 28.40 daily feed intake (g) 1010.0 a 1013.3 a 1023.3 a 916.7 b 14.1 (p<0.01) daily protein intake (g) 139.2 c 184.0 a 141.0 c 166.5 b 2.2 (p<0.01) daily weight gain (g) 313.3 d 453.3 a 380.0 d 343.3 c 7.3 (p<0.01) feed (kg/gain kg) 3.23 a 2.24 c 2.70 b 2.67 b 0.07 (p<0.01) protein efficiency ratio 2.25 a 2.46 b 2.69 c 2.06 d 0.06 (p<0.05) a,b,c,d means having different superscripts differ significantly at the levels of probability indicated. protein efficiency ratio = weight gained (g)/protein consumed (g) (p<0.025) than those fed the 14% protein diet, whereas the lw x lc pigs fed the 14% protein diet gained weight faster (p<0.01) than those fed the 18% protein diet. there were no significant (p>0.05) differences in feed conversion ratios between the lw x lc pigs fed 14% protein and those fed 18% protein. the genetic group x protein interaction in protein efficiency ratio was such that the lw pigs gained more weight (p<0.01) per gram of protein consumed when they ere fed the 18% protein diet than when they were fed the 14% protein diet. the lw x lc pigs, on the other hand, gained more weight (p<0.01) per gram of protein consumed on the 14% protein diet than on the 18% protein diet. discussion there were significant (p<0.01) genetic group x protein interactions with respect to feed intake and protein intake in experiments 2 but not in experiment 1 in which feed intake was significantly (p<0.05) affected by genetic group but not by protein level and protein intake was significantly (p<0.05) affected by both factors. the significant genetic group x protein interactions with respect to average daily weight gains, feed conversion ratios and protein efficiency ratios in both experiments is worthy of note. in experiment 1, the lc pigs gained more weight and had better feed conversion ratios and protein efficiency ratios on the lower (12%) protein diet than on the 16% protein diet whereas the lw x lc pigs gained more weights and had better feed conversion ratios on the 16% protein diet than on the 12% protein diet. in experiment 2, the lw pigs gained more weight and had better feed conversion ratios and protein efficiency ratios on the higher (18%) protein diet than on the 14% protein diet whereas the lw x lc pigs gained more weight and had better protein efficiency ratios on the 14% protein diet, although they also had better feed conversion ratios on the 18% protein diet. fetuga et al. (1977) reported breed x diet interactions in daily live weight gains and efficiency of feed utilization for lc and lw x ld pigs fed protein levels varying from 12 to 20%. in that study, lc pigs had their fastest growth rate and best feed conversion ratios on the 12 and 14% protein levels whereas the lw x ld pigs had their best growth rates and efficiency of feed utilization on the 18 and 20% protein diets. the results of the present study are in consonance with these reports. significant breed x diet interactions have also been reported even among different breeds and strains of the improved european or american pigs in weight gain (bowland and berg, 1959; bayley and summers, 1968) and efficiency of feed utilization (hale and southwell, 1967). overall, the lc pigs performed better than the lw x lc pigs on the lower (12%) protein diet while the lw x lc pigs performed better than the lc pigs on the 16% protein diet. in experiment 2, the lw x lc pigs performed better than the lw pigs on the lower (14%) protein diet while the lw pigs performed better than the lw x lc pigs on the 18% protein diet. consequently, on all performance parameters, the lc pigs were inferior to the lw x lc pigs which were, in turn, inferior to the lw pigs. these observations are in line with the reports of cameron and ashton (1969), vohradsky (1968), ilori (1974) and fetuga et al. (1976, 1977). the difference in performance of the different genetic groups in response to differences in dietary protein may be related to the inherent differences in their ability to synthesize lean tissues. fetuga et al. (1976) had shown that muscle development proceeded at a slower rate in the lc pigs compared to the lw x ld pigs, while the proportion of fat to lean increased rapidly with increasing live weight in the lc pigs. it would appear that because of the lower potential for growth in the lc pigs, their dietary protein requirement would be lower than that of the lw x lc and lw pigs. the tendency of the growth rate of the lc pigs to decrease at high dietary protein levels may, therefore, be related to the fact that protein levels in excess of die tary requirements for optimum growth and feed efficiency depress growth. this has been shown to be the case in lw x ld pigs (cooke et al., 1972). the weight gained per gram of protein consumed by the lc pigs and the lw x lc pigs in experiment 1 and by the lw x lc pigs in experiment 2 decreased as the dietary protein increased, while for the lw pigs in experiment 2, the weight gained per gram of protein consumed increased as the level of dietary protein increased. these results would suggest that the 16% protein level probably exceeded the protein requirement of the lc and the lw x lc pigs while the 18% protein diet did not satisfy the protein requirement of the lw pigs. this view is supported by the evidence presented by cunha (1980) which shows that feeding the finishing pig 2 more percentage units of protein than required results in a decrease of 10 to 15% in the efficiency of converting dietary protein to tissue protein. work is continuing to establish the exact protein requirements of the lc, lw x lc and lw pigs in order to achieve optimum protein nutrition and optimum performance of these pigs in the tropics. acknowledgements the authors are grateful to messrs h. o. onu and e. e. ndukwe for technical assistance. references a.o.a.c. official methods of analysis (1975). (association of official analytical chemists, washington d.c. 12 th . edition. bayley hs, summers jd (1968). effect of protein level and lysine and methionine supplementation on the performance of growing pigs: response of different sexes and strains of pigs. can. j. anim. sci. 48: 181 -188. bowland jp, berg rt (1959). influence of strain and sex on the relationship of protein to energy in the rations of growing and finishing pigs. can. j. anim. sci. 39: 102-114. bressani r (1974). in annual report for 1973. (institute de nutricion da centro america, america y panama, incap, quatemala city. cameron cw, ashton gc (1969). the local black and large white breeds of pigs for meat production in ghana. legion j. agric. 2: 1932. cooke r, lodge oa, lewis, d (1972). influence of energy and protein concentration in the diet on the performance of growing pigs. response to protein intake on a high energy diet. anim. prod. 14: 3546. cunha tj (1980). action programme to advance swine production efficiency. j. anim. sci., pp. 1929-1433 fetuga bl, babatunde gm, oyenuga va (1976). comparative physical carcass characteristics in the indigenous nigerian and imported european pigs. niger. j. anim. prod. 3: 74-87. fetuga bl, babatunde gm, olabisi eo, oyenuga va (1977). comparative response of large white x landroce and the indigenous pigs to diets of varying protein concentration. niger. j. anim. prod. 4: 181-204. hale om, southwell bl (1967). differences in swine performance and carcass characteristics because of dietary protein level, sex and breed. j. anim. sci. 26: 341-344. igboeli g, orji bi (1980). female reproduction of tolerant breeds and the possibility of artificial insemination in west africa. paper presented at the fao/sida workshop on the breeding of trypanotolerant livestock. lome, togo. 26 nov. 50 5 dec. ilori jo (1974). assessing the production potentials of local breeds of pigs. 1. effect of protein levels on performance. proc. 1 st annual conf. nigerian soc. anim. prod., 1: 100 (abstr). th snedecor gw, cochran wg (1974). statistical methods. 5 edition. steel rgd, torriejh (1960). principles and procedures of statistics. (mcgraw hill book co., new york.1 vohradsky f (1968). the production of large white and local black sows in pure breeding and reciprocal crossing. legon j. agric. 1: 5965. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (5), pp. 001-006, may, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper rapid diagnosis of etec and hpi-harboring escherichia coli infection in newborn piglets with diarrhea cheng darong1*, zhu shan yuan2, chen xiao lang1, gao xiao pan1, ding wen wei1 and sun huai chang1 1 college of veterinary medicine, yangzhou university, yangzhou 225009, china. 2 jiangsu animal husbandry and veterinary college, taizhou 225300, china. accepted 03 november, 2014 diarrhea is a common and important disease in industrial pig farms and the pathogenic escherichia coli infection is the main cause of morbidity and mortality in newborn piglets. the preliminary diagnoses on this disease are mainly depending on clinical symptom and detailed body dissection. to further shorten the diagnosis time and increase the determination efficiency for newborn piglet diarrhea caused by e. coli, a rapid method was established based on the fast bacterial culturing followed by the pcr examining for the virulence factor genes, such as enterotoxin st1, st2, lt1 and high pathogenicity island (hpi). a total of 151 rectal swab samples were obtained from live diarrheic piglets from jiangsu province, china. following the first cultivation in lb broth at 37°c for 6 h, all the samples were detected by the pcr methods, and the data show that 95 cases (62.91%) were infected with hpi-harboring e. coli, 24 cases (15.89%) were infected with enterotoxigenic e. coli (etec) and 14 cases (9.27%) were infected with etec and hpi-harboring e. coli. in addition, 2660 bacteria isolates were picked from all the 133 bacterial cultures which contained hpi-harboring e. coli and/or etec and the data of pcr examination determined that only 57 isolates were hpi-harboring e. coli, 20 were etec and 3 were both etec and hpi-harboring e. coli. this research not only revealed that hpi-harboring e. coli and etec are the prevalent pathogen of newborn piglet diarrhea, but also suggested that the method used in this study is specific, easier and more rapid to perform in the diagnosis of the infection of diarrheagenic e. coli with high accurate rate than the bacterial isolation and identification. key words: piglet, diarrhea, escherichia coli, rapid detection, virulence factor, determination. introduction the newborn piglet diarrhea is frequently due to infection by one or the other pathogens, such as bacteria, viruses and parasites. after the first few days of life, increasing interaction with other animals and the environment, including introduction of artificial feeding, the risk of exposure to enteropathogens was increased (carpenter et al., 2005; oli et al., 1998; stevenson, 1999). as we all know, pathogenic escherichia coli, especially enterotoxi *corresponding author. e-mail: jsyzcdr@yahoo.com.cn. tel: +86-514-8797-2587. genic e. coli (etec), are a frequent, important and global cause of severe, watery diarrhea in the newborn (suckling) pigs. etec is defined as a pathogen containing e. coli isolates that elaborate at least one member of two defined groups of enterotoxins, namely: heat-labile (lt) and heat-stable enterotoxin (st) (nair et al., 1998; smith et al., 1970;). enterotoxins are extracellular proteins or peptides, which are able to alter the functions of enterocytes by increasing secretion and reducing absorption, and therefore they are the more efficient virulence attributes of etec (blanco et al., 1991; nagy et al., 2005, 1999). in addition, most etec isolated from diarrhoeic pigs can produce one or more of the following fimbriae: f4 (k88), f5 (k99), f6 (987p), f17, f18 and f41 (blanco et al., 1991; garabal et al., 1997; nagy et al., 1999; ojeniyi et al., 1994; wilson et al., 1986), which allow the bacteria adhering to the small intestinal epithelium but without inducing significant morphological changes (nataro et al., 1998). furthermore, the old topic of horizontal gene transfer (hgt) has become fashionable (syvanen et al., 1999; jain et al., 1999). from the overwhelming surge of genome sequence information, more and more candidates for horizontally transferred genes are being identified (coton et al., 2009; cruz et al., 2009). it is clear that genes have flowed through the biosphere, as in a global organism. there are a number of well documented examples in which bacterial adaptation has been influenced by hgt, such as the high-pathogenicity island (hpi) of pathogenic yersinia in e. coli (buchrieser et al., 1998; carniel et al., 1992, 1998; fetherston et al., 1994; hacker et al., 2000; perry et al., 1990; petermann et al., 2008; schubert et al., 1998), or the new “plasmidassociated” pathogenicity island pai2173 which encodes the tetb gene conferring tetracycline resistance (fekete et al., 2003). detection of hgt can provide an optimistic control strategy on the selected disease, as well as providing the foundation for considerations of a ubiquitous role of hgt in shaping modern eukaryotic species. the aims of the research were to establish a rapid method to further shorten the diagnosis time and increase the determination efficiency for newborn piglet diarrhea caused by hpi-harboring e. coli and/or etec, determine the relationship between the newborn piglet diarrhea and the pathogenic e. coli infection, as well as investigate the virulence factor genes of the diarrheagenic e. coli isolates. materials and methods collection of diseased samples from march to september, 2008, a total of 151 rectal swab samples were collected from live diarrheic piglets from 51 swine industrial farms in jiangsu province, china. all the diseased piglets with a mean age of 5.2 ± 3.6 days all suffered from watery diarrhea with a 1 3-day latency period and peaks around one week after birth, and the samples were collected within 36 h after diarrhea. bringing each test sample to a final volume of 500 l with lb broth (10 g tryptone, 10 g nacl, 5 g yeast extract, h2o to 1 l, ph 7.4), they were store at -70°c. primers the different sets of primers (table 1) used for pcr amplification were described previously (cheng et al., 2005, 2006; schubert et al., 1998) and synthesized by sangon biological engineering technology and service co. ltd. (shanghai, p. r. china). the primers that target the genes of sta, stb and lta were mixed together (named enterotoxin primers set, each 50 mmol/l) to detect etec, and the primers that target the genes of hmwp2 were mixed together (named hpi primers set, each 50 mmol/l) to detect hpi-harboring e. coli in the samples. the primers that target the genes of f4, f5, f6 and f41 were mixed together (named fimbria primers set i, each 50 mmol/l) to identify adhesion factor genes of the bacteria isolates, and the primers that target the f18 genes were mixed together (named fimbria primers set ii, each 50 mmol/l) to identify and distinguish f18ab + and f18ac + e. coli. rapid diagnosis of pathogenic e. coli infection in newborn piglets 100 l dilution of each rectal swab sample were transferred into separate tubes containing 2 ml of lb broth and grow the liquid cultures with vigorous agitation at 37°c for 6 h. 0.5 ml of the liquid cultures was transferred to labelled microcentrifuge tubes, and centrifuge at 10,000 rpm for 5 min. the supernatants were discarded and each pellet resuspended in 200 l of distilled water. after boiling for 10 min, the suspensions were chilled on ice for 5 min, centrifuged at 10,000 rpm, at 4°c for an additional 5 min and the supernatants were used directly or stored at 4°c before pcr detection. to detect etec and/or hpi-harboring e. coli in the liquid cultures of rectal swab samples, pcr assays were performed in microcentrifuge tubes for the applied biosystems 2720 thermal cycler america). all the reagents were purchased from takara biotechnology (dalian) co., ltd. the pcr mixture contained 5 l of 10× pcr buffer (mg 2+ plus), 5 iu of taq polymerase, 4 l of dntp mixture (each 2.5 mmol/l), 1 l of enterotoxin primers set or hpi primers set, 2 l of dna template, and deionized water to a final volume of 50 l. cycling parameters for each primer set are outlined in table 2. each sample underwent 32 cycles of pcr amplification. pcr products were separated by 1% agarose gel electrophoresis along with dl2000 dna markers and visualized after staining with ethidium bromide on a uv transilluminator. determination of the virulence factor genes of the bacteria isolates following rapid diagnosis of etec and/or hpi-harboring e. coli infection, all the positive samples were submitted to separative cultivation of bacteria by streaking on mackonkey agar plates. following incubation at 37°c overnight, twenty colonies were picked out from each sample and cultured on lb agar plates at 37°c for 24 h. all the 2660 bacteria isolates were submitted to pcr detection for the genes of enterotoxins (sta, stb and lta), hpi and fimbriae (f18, f4, f5, f6 and f41). the extraction of dna templates and pcr assays were performed as described above, while enterotoxin primers set was used for sta, stb and lta, hpi primers set for hpi, fimbria primers set i for f4, f5, f6 and f41, and fimbria primers set ii for f18ab and f18ac. in addition, all the etec and/or hpi-harboring e. coli were serotyped by using uni-factor serum of o antigen of e. coli. results rapid detection of pathogenic e. coli infection in newborn piglets the specificity of the pcr assays were described previously (cheng et al., 2006). among the 151 rectal swab samples from live diarrheic piglets, 95 (62.91%) samples (from 32 farms) only contained hpi-harboring table 1. primers used for pcr amplification in this study. rr position in open reading frame size of product primer sequence 5’-3’ (accession number in genbank) (bp) reference sta f gggttggcaatttttatttctgta 298-321 (m25607) r attacaacaaagttcacagcagta 457-480 (m25607) stb f atgtaaatacctacaacgggtgat 1-20 (m35729) r tatttgggcgccaaagcatgctcc 334-357 (m35729) lta f tagagaccggtattacagaaatctga 579-604 (ab011677) r tcatcccgaattctgttatatatgtc 835-860 (ab011677) hmwp2 f aaggattcgctgttaccggac 241-261 (l18881) r tcgtcgggcagcgtttcttct 507-527 (l18881) f4 f gatgaaaaagactctgattgca 35-56 (m29374, m29376), 407-428 (m25302) r gattgctacgttcagcggagcg 860-881 (m29374, m29376), 1226-1247 (m25302) f5 f ctgaaaaaaacactgctagctatt 70-93 (m35282) r catataagtgactaagaaggatgc 589-612 (m35282) f6 f gttactgccagtctatgccaagtg 707-730 (u50547) r tcggtgtacctgctgaacgaatag 1146-1169 (u50547) f41 f gatgaaaaagactctgattgca 254-275 (x14354) r tctgaggtcatcccaattgtgg 914-935 (x14354) f18 f1 atgaaaagactagtgtttatttctt 160-184 (m61713) f2 cgtgaacggtaaaacacaggg 504-524 (m61713) r ttacttgtaagtaaccgcgtaagcc 648-672 (m61713) 183 (cheng et al., 2006) 360 (cheng et al., 2006) 282 (cheng et al., 2006) 280 841 (cheng et al., 2006) 543 (cheng, et al. 2006) 463 (cheng et al., 2006) 682 (cheng et al., 2006) 513 or 516 (cheng et 169 al., 2005) e. coli, 24 (15.89%) samples (from 8 farms) just contained etec, 14 (9.27%) samples (from 5 farms) contained both etec and hpi-harboring e. coli, while 18 samples (from 6 farms) were not detected with etec or hpi-harboring e. coli (table 3). the data suggested that at least, 133 cases (from 45 farms) of the 151 diarrheic piglets were infected with pathogenic e. coli. in addition, among the 38 samples which contained etec, 34 were lta-positive, 9 were sta-positive and 25 were stbpositive. determination of the virulence factor genes of the bacteria isolates two thousand, six hundred and sixty bacteria isolates were picked from all the 133 bacterial cultures which contained hpi-harboring e. coli and/or etec, and the data of pcr examination (table 4) determined that only 57 isolates (from 55 samples) were hpi-harboring e. coli, 20 were etec (from 16 samples) and 3 were (from 3 samples) both etec and hpi-harboring e. coli. among table 2. pcr cycling conditions in this study. primer set time at denature at 94°c annealing time at extension at 72°c total no. of cycles enterotoxin primers set 30 s 60 –56°c for 30s 1 min 32 (decreasing 1°c every two cycles) hpi primers set 30 s 64–58 °c for 30s 1 min 32 (decreasing 1°c every two cycles) fimbria primers set i 30 s 66–62 °c for 30s 1 min 32 (decreasing 1°c every two cycles) fimbria primers set ii 30 s 66–62 °c for 30s 1 min 32 (decreasing 1°c every two cycles) table 3. the results of rapid detection of e. coli infection in newborn piglets with diarrhea. virulence factor type hpi lta sta stb numbers of samples + 95 + + + 10 + + 1 + + + 2 + + + + 1 + 11 + + 8 + + 1 + + 1 + + + 3 total 95 34 9 25 133 the 57 hpi-harboring e. coli isolates tested, one (26.24%) was f4+, 9 (3.75%) were f6+, while 10 were f4+ and f6+. among the 20 etec isolates, 8 (3.75%) were lta+, stb and f6+, 3 (3.75%) were lta+, stb, f4+ and f6+, 4 (3.75%) were lta+ and f6+, 2 (3.75%) were sta+, stband f6+, 2 (3.75%) were sta+, and one was lta+, sta, stb, f4+ and f6+. all the 3 both etec and hpiharboring e. coli isolates were lta+, stb and f4+. no single f5+, f41+and f18+ isolate was detected. in addition, all the 80 e. coli isolates were o serotyped with the most prevalent serotype been o138 accounting for 62.50% (50/80), followed by o65 (12.50%), o21 (8.75%), o139 (6.25%), o141 (5.00%), o9 (2.50%), o159 (1.25%) and o55 (1.25%). contrast between pcr detection and conventional diagnosis by bacteria isolation and identification the diagnosis time were first compared and the data show that the effective time of pcr detection method is not exceeding 10 h with 6 h bacterial cultivation, 0.5 h extraction of dna templates, 2 h pcr examination and 1.5 h electrophoresis of pcr products, while the working time of conventional diagnosis by bacteria isolation and identification usually was 3 to 4 days. then, the diagnosis efficiency were also analyzed and compared. the results of this research show that the etec and/or hpiharboring e. coli were found to exist in 133 (88.08%) of the 151 samples (45 of the 51 investigated farms) by pcr detection method, while only 57 isolates could be detected in 55 samples by bacteria isolation and identification, although in each sample was picked out 20 bacteria isolates. this suggested that the pcr detection methods used in this study is specific, easier and more rapid to perform in the diagnosis of the infection of diarrheagenic e. coli with high accurate rate than the conventional diagnosis by bacterial isolation and identification. discussion pathogenic e. coli is a common porcine enteric pathogen, causing diarrhea in newborn piglets or post-weaning porcine edema disease, and etec is considered to be table 4. summary of the o serotype and virulence factors of the 80 e. coli isolates from diarrheic piglets. virulence factor hpi lta sta stb f4 f6 numbers of isolates o serotype o138 + + + 8 o138 + + + + 3 o138 + + 4 o138 + + + 2 o138 + 2 o138 + + + + + 1 o138 + + + + 3 o138 + + + 5 o138 + + 4 o138 + 18 o65 + 10 o21 + 7 o139 + + 5 o141 + 4 o9 + + 1 o9 + 1 o159 + 1 o55 + + + 1 total 60 19 5 17 14 33 80 the main categories of diarrhoeagenic e. coli. but moreover, it is worth noting that the high-pathogenicity island has been identified in pathogenic e. coli strains causing diarrhea and dysentery in calves, rabbits, piglets and human (carniel et al., 1992; fetherston et al., 1994; paauw et al., 2009). on the other hand, most e. coli are the normal inhabitants of the intestinal tracts of animals (levine, 1987; martins et al., 2000), and it is very difficult to obtain the pathogenic e. coli in the rectal swab samples from live diarrheic piglets by bacteria isolation and identification. therefore, diagnosis of pathogenic e. coli infection has focused increasingly on the rapid detection method, although the conventional diagnosis method by bacteria isolation and identification has occupied a central place in the history. among many detection methods, pcr is a major advance in molecular diagnostics of e. coli infection for its good sensitivity and specificity (nataro et al., 1998). as a matter of fact, the clinical diagnosis of e. coli infection in piglets, especially in the rectal swab samples from live diarrheic animals, usually just need confirming whether the pathogenic e. coli was/were existed in the sample(s), and not always need bacterial isolation and identification anymore while it is so difficult to pick out the pathogenic isolate(s) . according to this point of view, a rapid detection method was established in this study based on the fast bacterial culturing followed by the pcr examining for the virulence factor genes, which could extremely shorten the diagnosis time and increase the determination efficiency for newborn piglet diarrhea caused by e. coli. this is perfectly supported by the data of this research fortunately. such as, in our research, a total of 151 rectal swab samples were obtained from live diarrheic piglets from 51 swine industrial farms, and the data of pcr method could confirmed that 88.08% diarrheic piglets (from 88.24% farms) were infected with etec and/or hpi-harboring e. coli. but among the 2660 bacteria isolates picked from the 133 bacterial cultures, only 80 isolates were hpiharboring e. coli and/or etec, while most isolates may were the important members of the normal microbiologic flora of piglets (moneoang et al., 2009). this research not only revealed that etec are still the prevalent pathogen of newborn piglet diarrhea, but also found that the hpi-harboring e. coli isolates were more frequently detected in the diarrhea samples. furthermore, most hpi + isolates (95.0%) were toxin negative which might contribute to the virulence of these pathogenic e. coli isolates that causes newborn piglet diarrhea, and this attractive hypothesis has to be verified by comparing the virulence of the parental strain and of the isogenic mutants in a suitable infection mode. by the way, the pathogenic e. coli associated with neonatal diarrhea belong to a limited number of serogroups with o138, o65, o21, o139, o141, o9, o159, and o55, while o138 being the most commonly found in etec and hpi isolates, which were different from the isolates from porcine post weaning diarrhea (pwd) and pig edema disease 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length research paper an efficient secretion of the protein fused to the agfa signal sequence in salmonella ho young kang1, ki hwan moon1, se won kim2, jeong dong bahk 2, sang wan gal3, kwangkeun cho4, chul-wook kim4, john hwa lee5 and sam woong kim6* 1 department of microbiology, college of natural sciences, pusan national university, busan 609-735, south korea. 2 division of applied life sciences (bk21-ebncrc), graduate school of gyeongsang national university, jinju 660-701, korea. 3 department of pharmaceutical engineering, gyeongnam national university of science and technology, 150 chilam dong, jinju 660-758, korea. 4 department of animal resources technology, gyeongnam national university of science and technology, 150 chilam dong, jinju, gyeongnam, 660-758, south korea. 5 college of veterinary medicine, chonbuk national university, chon-ju 561-756, south korea. 6 swine science and technology center, gyeongnam national university of science and technology, 150 chilam-dong, jinju, gyeongnam, 660-758, south korea. accepted 02 december, 2014 signal sequence (ss) of surface or secreting proteins plays an important function for protein secretion in bacterial system. the ss of various proteins may mediate different level of protein excretion yield of the proteins. in order to examine the effect of ss types in protein secretion, signal sequences of bla (– lactamase), agfa (thin aggregative fimbriae a), stfa (salmonella typhimurium fimbriae a) and ompw (outer membrane protein w) were selected for the secretion of pspa protein which was used as a test protein. the pcr-amplified dnas corresponding to each ss were cloned into the plasmid pya3342. a primer used in pcr was designed to insert a his6-tag at the cterminal of ss for the convenient detection of expressed ss. the 0.8 kb ecori-hin diii pspa gene was cloned into the recombinant plasmids, resulting pmmp66, pmmp67, pmmp68 and pmmp70. the s. typhimurium strains harboring the recombinant plasmids expressed the his6-tagged pspa, demonstrating in-frame fusion of pspa to each depending upon the type of ss, cell lysate as well as secreted pspa of each salmonella samples was found different. relatively, s. typhimurium containing pmmp67 (carrying agfa ss) secreted the highest level of pspa than others, and suggested that the agfa ss mediates efficient translocation of the pspa. conclusively, the agfa ss mediated secretion system in pmmp67 can be used in variety fields required for the high level of protein secretion, especially antigen delivery in recombinant attenuated salmonella vaccines. key words: –lactamase, thin aggregative fimbriae a, outer membrane protein w, salmonella typhimurium fimbriae a, signal sequence, secretion. introduction the conventional vaccines against pathogenic bacteria have been produced to majorly kill these bacteria or act *corresponding author. e-mail: swkim@gntech.ac.kr. tel: 82 55-751-3268. fax: 82-55-751-3280. as subunit vaccines (meeusen et al., 2007). however, the manufacturing of these vaccines are very complicated from isolation up to the final step. additionally, the prepared vaccines require stable storage to avoid any side effects or denaturation. these vaccines are most likely administrated to host through syringe, thereby making the child to be afraid. one way to resolve these table 1. bacterial strains and plasmids used for this study. strain or plasmid genotype or phenotype reference or source bacterial strains e. coli dh5 enda1 hsdr17 supe44 thi-1 reca1 gyra rela1 (laczya-argf)u169 ( 80lacz m15) new england biolabs 6212 f 80 (laczya-argf)enda1reca1hadr17deorthi-1glnv44gyra96 rela1 asda4 nakayama et al. (1988) salmonella 8554 hisg asda16 kang et al. (2002) plasmids pgem-t vector cloning vector promega pya3342 asd pbrori kang et al. (2002) pya3493 derivative -lactamase signal sequence-based periplasmmic secretion plasmid kang et al. (2002) pya3494 a pya3493 derivative containing pspa gene kang et al. (2002) pmmp58 a derivative of t vector containing bla signal sequence (ss) and his-tag this study pmmp59 a derivative of t vector containing agfa ss and his-tag this study pmmp60 a derivative of t vector containing ompw ss and his-tag this study pmmp61 a derivative of t vector containing stfa ss and his-tag this study pmmp62 a derivative of pya3342 containing ompw ss and his-tag this study pmmp63 a derivative of pya3342 containing stfa ss and his-tag this study pmmp64 a derivative of pya3342 containing bla ss and his-tag this study pmmp65 a derivative of pya3342 containing agfa ss and his-tag this study pmmp66 a derivative of pmmp63 containing pspa gene this study pmmp67 a derivative of pmmp65 containing pspa gene this study pmmp68 a derivative of pmmp64 containing pspa gene this study pmmp70 a derivative of pmmp62 containing pspa gene this study inconveniences is the development of attenuated bacterial vaccines. recombinant attenuated salmonella vaccines have been adapted to elicit mucosal and humoral immunities after oral administration. the elicited immune responses were augmented when the delivered antigen was translocated into cell envelop or out of the cells (kang and curtiss, 2003; kang et al., 2002; kim et al., 2007). multiple methods were applied to translocate the foreign antigen from cytosol into at least cell envelop or complete secretion (kim et al., 2007; wong et al., 1995). researchers inserted the target antigen or epitope into surface structure molecules such as flagella or fimbriae to present the antigen on cell surface (majander et al., 2005a, b; spreng et al., 1999). however, a limited size of the antigen can be inserted into surface molecule for stable conformation. another more convenient way is the secretion of the foreign protein into extracellular matrix through the use of a signal sequence (ss) (koshland and botstein, 1980; li et al., 2008). a previous study demonstrated that the pspa antigen was translocated into periplasmic space or outside of the cell through the fusion of -lactamase signal sequence (kang et al., 2002) . although this system has a merit to secret the protein to the extracellular matrix, a large portion (50%) of the expressed proteins exists still in the cytosol. due to the fact that more secreted antigen elicits the better immune responses, it is needed to develop a new efficient protein secretion system in live vaccine mediated antigen delivery. this study aimed to develop an efficient protein secretion system in salmonella. materials and methods bacterial strains and agents the bacterial strains, plasmids and oligonucleotides used for this study are listed in tables 1 and 2. salmonella enterica serotype typhimurium and escherichia coli were grown at 37°c using luriabertani (lb) or m9 minimal medium supplemented with 1.5% agar (bertani, 1952). the antibiotics were added in the following table 2. synthetic oligonucleotides used for this study. oligonucleotide name oligonucleotide sequence (5' 3') sagfa-f-bsphi tcatgaaacttttaaaagtggcag sagfa-r-his/ecori gaattcatggtgatggtgatgatggccgccgttatgattaccgc sbla-f-bsphi tcatgagtattcaacatttccgtg sbla-r-his/ecori gaattcatggtgatggtgatgatgttcagcatcttttactttca sompw-f-bsphi tcatgaaaaaatttacagtggcgg sompw-r-his/ecori gaattcatggtgatggtgatgatgtccggcttcgtgcgcgaacg sstfa-f-bsphi tcatgaatacagcagtaaaagctg sstfa-r-his/ecori gaattcatggtgatggtgatgatgaccggtaaaagtcaccgtac concentrations for each culture condition: ampicillin, 100 g/ml; streptomycin, 50 g/ml. dna manipulations general dna manipulations were conducted as described by sambrook et al. (1989). plasmids were introduced into e. coli competent cells by heat-shock with rbcl2 treatment (hanahan, 1983) and were introduced into salmonella competent cells by electroporation with 10% glycerol treatment (sambrook et al., 1989). nucleotide sequencing was conducted by using an abi 373 automatic sequencer (pe applied biosystems). cloning of genes for signal sequence the dna regions corresponding to signal peptide of bla, agfa, ompw and stfa was pcr-amplified from pya3493 or s. typhimurium 3339 chromosome as a template. the oligonucleotide primers used in pcr-amplification were designed to insert restriction sites for bsphi and ecori (table 2). the primers were also designed to include his 6-epitope at the c-terminal of each signal peptide, which allows the detection of expressed signal peptides by immunoblot with anti-his6-epitope antibody. pcr reaction conditions were as follows: denaturation at 95°c for 30 s, primer annealing at the temperature of primer’s melting temperature (tm) for 30 s, polymerization at 72°c for 30 s ~ 3 min depending on the length of dna fragment and a final extension at 72°c for 10 min. the amplified dna fragments were cloned into t-vector (promega) as a temporary step. the bsphi -ecori dna segment isolated from the recombinant t-vector was cloned into a plasmid pya3342 digested with ncoi and ecori. sds-page (polyacrylamide gel electrophoresis) and immunoblot bacterial culture broth was centrifuged for 10 min at 5,000 x g to separate the cells from the culture supernatant. the supernatants was collected and further used for the analysis of secreted proteins. the precipitated cell pellet was washed twice with 0.85% nacl and then resuspended in 10 mm tris hcl, ph 7.5. the suspended cells were mixed with sds gel loading buffer and lysed thermally. the 65 g proteins of total cell lysates were then separated through sdspage and the separated bands were visualized by coomassie staining (sambrook et al., 1989). for the analysis of secreted proteins, the cultural supernatant was concentrated for 1 h in icecold tca (trichloroacetic acid) solution (hoong et al., 1995; ma et al., 1996) and then 8 µg secreted proteins were separated by sds page. for immunoblotting, proteins separated by sds-page were electrophoretically transferred onto the nitrocellulose membranes. the immunoblotting was conducted in accordance with the protocols established by sambrook et al. (1989). the pspa protein and his6-tagged signal peptides were specifically recognized by the use of xi126 monoclonal antibody (mcdaniel et al., 1984) and his6tag monoclonal antibody (southern biotech), respectively. goat anti-mouse igg, horseradish peroxidase (hrp) conjugated antibody was used as secondary antibody and the 4-choloro-1naphthol was used for the substrate of hrp. results and discussion background of construction for new foreign antigencarrying systems and the results most of the bacteria operate general secretory pathway (gsp) to translocate proteins from cytoplasm to periplasmic space, outer membrane or extracellular matrix. the ss located at nterminus of the secreting protein leads the translocation of the protein to membrane. in a previous study (kang et al., 2002) a plasmid system (pya3493) was generated for the secretion of proteins through gsp. main idea of the pya3493 based expression system is the fusion of lactamase ss at the n-terminus of expressed proteins. in this study, we examined protein secretion capacity by the replacement of -lactamase ss with the ss of other proteins in pya3493. the plasmid pya3493 carries an aspartate semialdehyde dehydrogenase (asd) gene, which is required for the biosynthesis of mdiaminopimellic acid (m-dap), a component of bacterial cell wall. the “asd” gene in pya3493 complements “asd” deficiency of bacterial hosts, e. coli 6212 and s. typhimurium 8554 (table 1). multicloning sites (mcs) is positioned at the downstream sequence of ss sequence to fuse signal peptide with the protein of cloned gene. to examine the effect of the type of signal peptide for protein secretion, various dna fragments corresponding to the signal peptide of -lactamase (bla), agfa, ompw and stfa were localized at the upstream of mcs: “bla ss” in pmmp62, “agfa ss” in pmmp63, “ompw ss” in pmmp64 and “stfa ss” in pmmp65. the c-terminus of each signal peptides figure 1. schematic diagram of strategy for cloning of signal sequence (ss) dna. the signal sequences of bla (–lactamase), agfa (thin aggregative fimbriae a), stfa s. typhimurium fimbriae a) and ompw (outer membrane protein w) were used in this study. each pcramplified bsphi-eco ri ss dna was cloned into pya3342 digested with ncoi and ecori. gray box indicates a his6-tag contained at the c-terminus of each ss. exhibited his6 residues. it is possible to speculate that expression of each ss and his6 coding residue were regulated by ptrc promoter. a strategy for preparation of these vectors is shown in figure 1. in e. coli, the lactamase is secreted into periplasmic space through type ii sec-dependent secretion system, but in independent manner of secb (beha et al., 2003). agfa and stfa, major fimbrial proteins of s. typhimurium, agf and stf fimbriae are secreted into extracellular matrix by type iv sec-dependent secretion system. the mechanism of agf fimbrial protein secretion is unknown, whereas stf fimbrial proteins are secreted by similar manner to type ii sec-dependent pathway (humphries et al., 2003). the ompw (outer membrane protein w) is translocated through type ii secand secbdependent pathway (driessen et al., 2001; koch et al., 2003). identification of foreign antigen expression by pspa protein the pspa protein (streptococcus pneumoniae surface protein a) was selected as a testing protein, for protein secretion through the system developed in this study (briles et al., 1998). a 765 bp ecorihindiii pspa gene isolated from pya3494 was ligated with the plasmids pmmp62, pmmp63, pmmp64 or pmmp65 digested with ecori and hindiii (figure 2). the recombinant dnas were transformed into e. coli x6212 (kang et al., 2002). furthermore, the presence of recombinant plasmids were confirmed by analyses of restriction enzyme digestion figure 2. genetic maps of recombinant plasmids. the physical maps of each recombinant plasmid are indicated with their representative restriction enzyme sites: (a) basal vector, (b) pmmp62, (c) pmmp63, (d) pmmp64 and (e) pmmp65. the ptrc promoter region, ribosome binding site (rbs), each ss, his6-epitope and multi-cloning sites (mcs) are revealed in enlarged box. unique enzyme sites in mcs are designated by underlined bold letters. ptrc, trc promoter; 5st1t2, 5s rrna terminator; “asd”, aspartate semialdehyde dehydrogenase gene; ss, ompw ss (b), stfa ss (c), bla ss (d) and agfa ss (e). and nucleotide sequencing, and were finally designed as pmmp66, 67, 68 and 70 (table 1). the level of expressed pspa protein in the s. typhimuium 8554 host was examined by immunoblotting. a monoclonal antibody xi126 (mcdaniel et al., 1984) was used to detect pspa protein specifically. an anti-his6-epitope antibody (santacruz biotechnology, inc.) raised in mouse was used to detect expressed protein against his6-tagged signal peptide. an anti-mouse igg conjugated with hrp (horseradish peroxidase) (stressgen co.) obtained from goat was used as a secondary antibodies to recognize primary antibodies. the s. typhimuium 8554 carrying pya3494, pmmp66, pmmp67, pmmp68 or pmmp70 were cultured to od600 0.8 in lb broth at 37°c. total cell lysate of each sample containing 65 µg of protein was subjected for sds-page analysis. as seen in figure 3, all samples exhibited a major protein bands with approximately 37 kda size. a little size differences of the figure 3. expression of pspa in s. typhimurium containing recombinant plasmids. s. typhimurium strains harboring pya3494, pmmp66, 67, 68 and 70 were grown in lb broth until exponential phase (0.8 at od600). equal amounts of cell lysates were subjected to sds-page followed by immunoblot analyses with specific antibodies for each component. the goat anti-mouse conjugated with hrp was used as the 2nd antibodies. (a) sds page of total lysates. separated bands were visualized by coomassie staining. (b to c) immunoblot of total lysates. his6 tagged ss was detected by monoclonal antibody specific for his6 tag (b), and expressed pspa protein was detected by monoclonal antibody specific for pspa (c). (d) quantitative analysis of pspa protein. relative immuno-reactive intensities were analyzed by imagej program. arrow indicates the pspa protein. major bands between samples can be explained by the size difference of signal peptide which was fused to the pspa. the 37 kda proteins were reacted with anti-his6epitope antibody, which confirms that each 37 kda protein contains signal peptide designed in this study. the protein of s. typhimuium 8554 harboring pmmp67 exhibited a faint immune-reactive band, hypothesizing the access difficulty of h6-antibody due to protein conformation. as we expected, there was no his6-antibody reaction band in sample of s. typhimuium 8554 harboring pya3494, which does not carry his6-tagged signal peptide. immunoblot analyses with anti-pspa antibody demonstrated that the 37 kda protein seen in all samples figure 4. immunoblot of secreted pspa proteins. s. typhimurium 8554 harboring plasmids were cultured in lb broth until exponential phase (0.8 at od600). equal amount of cell-free culture supernatant were subjected to sds-page and immunoblot analyses. (a) sds page analysis. separated bands were visualized by coomassie staining. (b) immunoblot of secreted proteins. the pspa protein was detected by monoclonal antibody specific for pspa. (c) quantitative analysis of pspa protein. relative immuno-reactive intensities were analyzed by imagej program. arrow indicates the pspa protein. is the pspa protein. the highest amount of cell associated pspa protein was detected in salmonella containing pya3494, which was almost 2 fold higher levels than that in salmonella containing pmmp70 (carrying ompw ss) . the salmonella containing pmmp67 or pmmp66 expressed pspa 1.5 or 1.3 folds higher than salmonella containing pmmp68. an advantage of the system developed in this study is the detection of the expressed protein with commercially available his6-epitope antibody, without specific antibody for the expressed protein. identification of secretion ability according to signal sequences the protein expression system developed in this study is designed for the secretion of the pspa protein. each signal peptide fused to pspa may lead to the translocation of the cytoplasmic pspa to the cell envelop or to the outside of the cell. to examine the level of protein secretion, the s. typhimuium 8554 carrying pya3494, pmmp66, pmmp67, pmmp68 or pmmp70 were cultured to od600 0.8 in lb broth at 37°c. cell-free supernatant of each culture was examined for the secreted protein fractions. to eliminate the presence of salmonella cells entirely, culture supernatant after centrifugation was filtered through passing 0.2 µm filter. the proteins in 1 ml of supernatant were precipitated using 10% tca as described earlier. the precipitated pellets after washing twice were resuspended in 60 l of sds loading buffer. 8 g protein of each sample was subjected to sds-page. parts of samples were employed for immunoblot analysis after sds-page. all samples exhibited a major protein bands with approximately 37 kda size likely as seen in total lysates (figure 4). the 37 kda proteins were reacted with both anti-his6 epitope antibody and anti-pspa antibody, demonstrating pspa protein of the 37 kda protein. interestingly, a thick 37 kda protein band was observed in the secreted fraction of s. typhimuium 8554 carrying pmmp67. densitometer analysis of the secreted pspa in each sample shows that the secreted pspa in salmonella carrying pmm67 was 1.8 fold amount higher than that in salmonella carrying pmm70 which secrets the protein second level. the other three samples, salmonella carrying pya3494, pmm66 and pmm68 secrets the pspa with similar levels. although the pspa level in total cell lysate was similar for pya3494, pmmp67 and pmmp70 (figure 3), the highest amount of secreted pspa was detected in the supernatant of salmonella carrying pmm67. this discrepancy is not solely because of overexpression of the protein, but it might be due to the agf ss. thus, we speculate that the agfa signal peptide mediates pspa secretion more efficiently than any other signal peptides used in this study. taken all together, since signal sequences play roles as a leader sequence and a signal sequence, their existences raise individual expression and secretion of protein. the efficient protein secretion mediated by agfa signal sequence can extend its utility in various fields. especially, it can be applicable in the antigen delivery of recombinant attenuated salmonella vaccines (kim et al., 2009). a previous study suggested that salmonella vaccine secreting foreign antigen induces better immune responses than the vaccine localizing the antigen in cytoplasm (kang et al., 2002). therefore, more secretion of the foreign antigen may induce more immune responses in salmonella antigen delivery system. acknowledgements this work was supported by priority research centers program (2009-0093813) through the national research foundation of korea (nrf) funded by the ministry of education, science and technology of korea and by technology development program for (agriculture and forestry or food or fisheries), ministry for food, agriculture, forestry and fisheries, republic of korea. abbreviations: ss, signal sequencel; bla, 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farming vol. 1 (4), pp. 042-047, december, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper seasonal changes in body condition scores of pigs and chemical composition of pig feed resources in a semiarid smallholder farming area of zimbabwe orbert c. chikwanha 1 , tinyiko e. halimani 1 , michael chimonyo 2* , kennedy dzama 3 and evison bhebhe 4 1 department of animal science, faculty of agriculture, university of zimbabwe, p. o. box mp 167, mount pleasant, harare, zimbabwe. 2 department of livestock and pasture science, faculty of science and agriculture, university of fort hare, p. bag x1314, alice 5700, eastern cape, south africa. 3 department of animal sciences, stellenbosch university, p. bag x1 matieland, south africa. 4 department of paraclinical veterinary studies, university of zimbabwe, p. o. box mp 167, mount pleasant, harare, zimbabwe. accepted 23 august, 2013 there are few studies quantifying the productivity of rural pigs and evaluating the nutritive value of non-conventional feeds, such as weeds. the objectives of this study were to determine changes in body condition scores of boars and lactating sows and investigate changes in the chemical composition of commonly used pig feed resources in a smallholder farming area of zimbabwe. body condition scores (bcs) were measured monthly between october and april. commelina benghalensis (wandering jew) and richardia brasiliensis (mexican clover) were collected monthly, between october and march, for proximate and amino acid analysis. sows had lower bcs than boars (p<0.05). the neutral detergent fibre (ndf) and acid detergent fibre (adf) contents of c. benghalensis were lower (p<0.05) than those of r. brasiliensis. c. benghalensis had about twice the amount of crude protein (cp) compared to r. brasiliensis. lysine, methionine and cysteine, which are the most important amino acids in pig nutrition, were similar in groundnut hulls and r. brasiliensis. c. benghalensis had higher (p<0.05) levels of threonine, tryptophan, isoleucine, leucine, histidine, phenylalanine, tyrosine, valine, arginine, serine, aspartic acid, glutamic acid, glycine and alanine than r. brasiliensis. the proportion of essential amino acids (eaa) was significantly higher (p<0.05) in r. brasiliensis. a further study to determine the digestibility and growth performance of pigs fed on these non-conventional diets is needed. keywords: rural pig production, body condition scoring, commelina benghalensis, proximate analysis, richardia brasiliensis introduction rural pig farmers in southern africa rely on locally available feed resources to feed their animals. there is a general perception that feed supplies are seasonal and inadequate to meet pig maintenance and production feed *corresponding author. e-mail: mchimonyo@ufh.ac.za requirements throughout the year in rural areas (mashatise et al., 2005). however, there have been few attempts to quantify the productivity of local pigs, and the nutritive value of the common feeds that they consume, in order to improve smallholder pig production. it is assumed that animal performance during the wet season is high (mhlanga et al., 1999; holness et al., 2005). the influence of the production systems that the farmers use has often been ignored. there has been inadequate re chikwanha et al. 042 table 1. pig body condition scoring system used. body condition score description assessment of fat cover 0 emaciated exposed, no cover on bones 1 poor bones prominent, little cover 2 moderate bones easily felt without palm pressure 3 good bones only felt with firm palm pressure 4 fat bones cannot be felt with firm palm pressure 5 grossly fat further deposition of fat impossible source: holness (1991). search on locally available, non-conventional feed resources and their potential value as pig feeds (ly, 1993; lekule and kyvsgaard, 2003). efforts have been put into utilising non-conventional feedstuffs for pigs, including legume tree leaves (halimani et al., 2005), and maize cobs (chimonyo et al., 2006; chimonyo and dzama, 2007). attempts should be made to identify locally available feed resources for each area. the lack of information on changes in nutritive value during the growing season, when the bulk of the feeds are available, hampers the development of appropriate conservation strategies to maintain or produce pigs across the dry season, when both feed quantity and quality are low. use of non-conventional feed resources is particularly impor-tant since cereal grains are needed for human consump-tion and are not readily available for feeding pigs, espe-cially in rural areas of developing countries. there is a need to identify locally available non-conventional feed resources (for example weeds) for pigs and characterise their nutritive value including amino acid profiles. a ba-lanced and optimal proportion of amino acids are required for efficient protein deposition in the pig, along with a sufficient supply of energy (grala et al., 1999; kyriazakis and whittemore, 2006). nutritional status of rural pigs can be assessed by monitoring body weight changes and nutritionally-related blood parameters. be-cause scales are often not available in rural areas, adap-tation of the body condition scoring system, widely used in cattle, has been successful in assessing the perfor-mance of pigs (maes et al., 2004). the objectives of the current study were to determine changes in body condition scores of boars and lactating sows during the rainy season when feed is not likely to be a constraint, and investigate changes in the chemical composition of commonly used pig feed resources in a smallholder farming area of zimbabwe during the rainy and post-rainy seasons. the hypotheses tested were that the body condition and chemical composition of the commonly used feed resources do not change with during the rainy ad post-rainy seasons. materials and methods study site the study was conducted in chinyuni ward of chirumanzu district, in the midlands province, about 30 km to the north of masvingo, zimbabwe. it is located 19º34' s and 30º45' e, at an altitude of between 1300 to 1440 m above sea level. the district lies in natural region iii, a semi-arid agro-ecological zone where farming operations are extensive. the area receives mean annual rainfall of 650 to 800 mm. the mean maximum temperature ranges from 25 to 39ºc in cold (may to august) and hot seasons (september to november), respectively. the area experiences frequent seasonal droughts and severe dry spells during the growing season, which makes it generally unsuitable for cropping. the farming systems are largely based on livestock and drought-resistant fodder and food crop production. selection of pigs forty-five lactating sows and 11 mature boars were selected for body condition scoring from 38 households. the sows were nursing piglets ranging from one week to three weeks old. body condition scoring was done during the growing season (october 2005 to april 2006), the period when the majority of the sows are lactating. the selection of households was based on farmers with at least one mature pig. pig identification was reliant on farmers’ knowledge of their pigs, as is common practice in smallholder farming areas. these pigs were mostly free ranging and occasionally offered a supplementary diet of local feed resources when available. no commercial feeds, drugs or medicines were used but there was limited knowledge and use of ethno-veterinary medicine. the body condition scores (bcs) were measured using the scoring system from 0 to 5, as shown table 1. the assessment was based on the fat cover on the spine and transverse spinal process, and was assessed at the end of the month. one trained person assessed the condition of the pigs throughout the study. feed sample collection in a separate trial, the weeds commonly used by farmers in feeding their pigs were sampled for nutritive evaluation. the weeds, commelina benghalensis (wandering jew) and richardia brasiliensis (mexican clover), were collected on a monthly basis from five crop fields of one hectare each every month from october 2005 to 043 afr. j. pig farming figure 1. changes in body condition of pigs over time. march 2006. samples were cut at a height of 50 mm above the ground, air dried for 14 days and ground through a 2 mm sieve and stored at air temperature to await analyses. the plants were picked from four quadrants from each of the fields, two at the end and two at the middle of the field. about 100 g of maize dried brewers’ grain and groundnut hulls were also collected every month throughout the study period from farmers’ households. chemical analyses the dried brewers’ grain and groundnut hulls were analyzed for amino acid composition. weed samples were analysed for dry matter (dm), crude protein (cp), ash and ether extract (ee) as described by aoac (1990). neutral detergent fibre (ndf) and acid detergent fibre (adf) analyses were done according to the method described by goering and van soest (1970). amino acid profiles of r. brasiliensis, c. benghalensis and maize brewers’ dried grain and groundnut hulls were conducted at the agricultural research council, irene, south africa. amino acid analyses involved acid hydrolysis, pre-column derivatisation, separation by hplc and detection using fluorescence detector, as described by einarsson et al. (1983). tryptophan was analysed using enzymatic hydrolysis, separation by hplc and detection using fluorescence detector, as described by de vries et al. (1980). cystine was analysed as described by gehrke et al. (1985). the protein value was estimated by calculating the extent of deficit of each essential amino acid, in relation to that present in the ideal protein, using the egg as the standard. gross energy of the feeds was determined using the bomb calorimeter at the institute of mining research, university of zimbabwe, using methods described by aoac (1990). all analyses were performed in triplicate. statistical analyses the bcs of the pigs were tested for normality, and were skewed, even after transformation; they were then subjected to the friedman procedure (sas, 1996) and analysed using the following linear model: yij = µ + mi + eij; where: yij = change in body condition score; µ = overall mean common to all observations; mi = effect of the i th month (october… april); eij = random error distributed as n (0,i 2 e). the nutrient compositions of the feed resources were analysed using the proc mixed procedure of sas (1996). the linear model used was: yijk = µ + mi + wj + mi * wj + eijk; where: yijk = response variable (dm, cp, ash, adf, ndf and ee, amino acids); µ = overall mean common to all observations; m i = fixed effects of the i th month (october… april); w j = fixed effects of the j th weed (j = c. benghalensis and r. brasiliensis); mi * wj= interaction of month and weed; eijk = random error distributed as n (0,i 2 e). pair-wise comparisons between least square means were performed using the pdiff statement in sas (1996). results body condition scoring figure 1 illustrates the changes in the mean bcs for the lactating sows and boars. during october and november, the bcs of lactating sows were higher (p<0.05) than for boars. however, from december up to the end of the end of april, the bcs was significantly lower for lactating sows than boars. the sows maintained their condition during this period, while boars showed a marked increase (p<0.05) in condition. the main feeds that the pigs received during trial period were groundnut hulls, brewer’s dried grain and weeds (c. benghalensis and r. brasiliensis). the chemical composition of these feedstuffs was, therefore, evaluated. dry matter, ether extract and ash contents table 2 shows dm, ee, ash, ndf, adf and gross energy contents of c. benghalensis and r. brasiliensis. both month of sampling and species of weed influenced (p<0.05) chemical composition. across the six months, r. brasiliensis had a significantly higher dm than c. benghalensis. within each weed, dm content was constant (p>0.05) throughout the sampling period. ether extract contents were significantly affected by month, weed species and the interactions between the two. as with dm, in r. brasiliensis ee content remained constant throughout the monitoring period. the ee content for c. benghalensis fluctuated but was consistently higher (p<0.05) than for r. brasiliensis. r. brasiliensis had treble the amount of ash (p < 0.05) compared to c. chikwanha et al. 044 table 2. dry matter (dm, g/kg), crude protein (cp) and ether extract (ee) content, ash, neutral detergent fibre (ndf), acid detergent fibre (adf) (g/kg) and gross energy (ge) (mj/kg) of commelina benghalensis (cb) and richardia brasiliensis (rb) on dm basis. component month weed october november december january february march sem dm cb 823.7 a 823.0 a 820.5 a 825.8 a 823.3 a 821.3 a 52.06 rb 873.4 b 872.2 b 868.4 b 868.5 b 872.3 b 869.8 b cp cb 208.6 b 207.9 b 218.6 c 225.0 c 227.3 c 237.4 c 15.42 rb 100.8 a 106.3 a 109.9 a 104.8 a 111.9 a 134.7 a ee cb 18.4 c 18.5 c 18.2 c 18.7 cd 19.3 d 18.5 c 0.21 rb 16.0 a 16.2 a 15.9 a 16.5 b 16.4 b 16.2 a ash cb 22.6 a 25.4 b 23.9 a 22.6 a 23.8 a 25.3 b 1.90 rb 75.9 e 65.2 cd 63.7 c 65.5 d 62.8 c 63.6 c ndf cb 408.0 a 403.8 a 482.8 d 401.9 a 402.0 a 411.6 a 24.27 rb 492.0 c 498.3 c 471.6 b 482.8 b 470.0 b 467.6 b adf cb 196.1 a 195.2 a 187.2 a 184.8 a 185.4 a 188.0 a 10.25 rb 389.1 b 387.4 b 391.9 b 387.4 b 388.9 b 387.6 b ge (mj/kg) cb 13.1 c 11.8 b 14.4 c 7.6 a 14.9 c 13.9 c 1.16 rb 14.0 c 16.0 d 10.6 b 14.2 c 14.8 c 15.7 d abcd values with different superscripts for each component are different (p < 0.05). benghalensis . the mean ash contents were 66.1 and 23.9 g/kg for r. brasiliensis and c. benghalensis, respectively. the ash content, however, did not show consistent patterns within each weed. neutral and acid detergent fibre contents and gross energy levels the ndf and adf contents of c. benghalensis were lower (p<0.05) than those of r. brasiliensis. the differences in fibre contents were more marked for adf than ndf. r. brasiliensis had higher (p<0.05) energy levels than c. benghalensis . the highest gross energy levels of r. brasiliensis were recorded in november, while the highest gross energy levels for c. benghalensis were recorded in february. brewers’ dried grain had the highest gross energy (19.3 mj/kg), which did not vary across the sampling period. crude protein and amino acid profiles as shown in table 2, c. benghalensis had about twice the amount of cp than r. brasiliensis . the cp content remained constant at a mean of 108.1 g/kg for r. brasiliensis, but increased (p<0.05) from october to march for c. benghalensis. the highest cp content, observed in march, was 237 g/kg. the amino acid content of the feeds (weeds, brewers’ dried grain (bdg) and groundnut hulls (gnh) is shown in table 3. brewers’ dried grain had the highest cp content of 219.5 g/kg, followed by c. benghalensis, r. brasiliensis and groundnut hulls had the least cp content. brewers’ dried grain had the highest concentration of most of the individual amino acids. levels of lysine, methionine and cysteine, which are the most important amino acids in pig nutrition, were similar in groundnut hulls and r. brasiliensis. c. benghalensis had higher (p<0.05) levels of threonine, tryptophan, isoleucine, leucine, histidine, phenylalanine, tyrosine, valine, arginine, serine, aspartic acid, glutamic acid, glycine and alanine than r. brasiliensis. the proportion of essential amino acids (eaa) was significantly higher (p<0.05) in r. brasiliensis. no differences (p>0.05) existed in the ratio between eaa and non-essential amino acids (neaa) between c. benghalensis and groundnut hulls. levels of ho-proline were less than all other amino acids in all feedstuffs. table 4 shows the protein value of selected essential amino acids in the four feeds. the values for lysine, methionine, cysteine, threonine and isoleucine were highest for brewer’s dried grain. the c. benghalensis had higher (p<0.05) values for these amino acids than groundnut hulls and the highest tryptophan value of the four feeds. discussion although the use of body condition scoring in assessing nutritional status of pigs is not common, it has been shown to correlate well with the total amount of body fat (maes et al., 2004). measurement of back fat levels constitutes an important tool to evaluate the total amount of 045 afr. j. pig farming table 3. amino acid content (g/kg) for commelina benghalensis, richardia brasiliensis, brewers’ dried grain (bdg) and groundnut hulls (gnh). amino acid r. brasiliensis c. benghalensis bdg gnh sem lysine 6.7 a 9.0 b 11.2 c 6.8 a 0.76 methionine + cysteine 2.9 a 3.1 a 11.1 b 2.2 a 1.82 threonine 3.0 a 4.6 b 7.4 c 2.9 a 0.55 tryptophan 1.1 a 2.6 b 1.3 ab 0.8 a 0.08 isoleucine 3.9 a 5.6 b 10.3 c 3.7 a 0.67 leucine 6.1 a 9.0 b 30.5 c 6.0 a 0.85 histidine 4.2 a 5.1 a 12.3 b 4.1 a 0.45 phenylalanine + tyrosine 8.1 a 11.9 b 36.6 c 8.7 a 0.96 valine 4.4 a 6.1 b 11.6 c 4.4 a 0.53 arginine 4.2 a 7.6 b 10.1 c 5.6 a 1.12 serine 3.2 a 5.3 b 9.7 c 3.5 a 0.62 aspartic acid 8.9 a 21.4 c 12.8 b 9.6 a 1.46 glutamic acid 7.8 a 12.7 b 36.7 c 8.0 a 0.86 glycine 3.5 a 5.3 b 7.2 c 3.3 a 0.37 alanine 4.0 a 6.2 b 15.6 c 3.2 a 0.42 proline 3.1 a 5.0 b 16.5 c 5.1 b 0.53 ho-proline 0.4 a 0.7 a 0.7 a 2.8 b 0.26 eaa: neaa 1.04 c 0.92 b 0.84 a 0.93 b 0.05 abcd values with different superscripts within row differ (p < 0.05). table 4. protein value of r. brasiliensis (rb), c. benghalensis (cb), and brewers’ dried grain (bdg) and groundnut hulls (gnh). protein value amino acid rb cb bdg gnh sm 1 cm 1 lysine 9.6 a 12.9 b 16.0 b 9.7 a 17.1 25.7 methionine + cysteine 7.3 b 7.8 b 27.8 c 5.5 a 30.0 30.0 threonine 6.7 a 10.2 b 16.4 c 6.4 a 26.7 31.1 tryptophan 7.3 b 17.3 c 8.7 c 5.3 a 26.7 33.3 isoleucine 9.8 a 14.0 b 25.8 c 9.3 a 35.0 35.0 1 protein values for sunflower meal (sm) and cottonseed meal (cm) were derived from kyriazakis and whittemore (2006). abcd values with different superscripts within row differ (p < 0.05). of body fat, especially in rural areas where weighing scales, among other important equipment, are rarely available. maes et al., (2004) also showed that the number of stillborn piglets increased with decreasing back fat thickness at the end of gestation. the marked decline in bcs values for lactating sows from october onwards indicates that most of them receive inadequate nutrient during confinement. during confinement, the pigs were supplied with feed once a day (chikwanha, 2006). the steady decline in the condition of the lactating sows is as a result of suckling. in chirumanzu district, as in many other rural areas of southern africa, pigs scavenge for feeds during the dry season, and are confined during the rainy season to prevent them from destroying field crops (mashatise et al., 2005; chikwanha, 2006). the commonly held view that pigs get adequate nutrients during the rainy season does not apply in chirumanzu. the reasons why boars had low bcs in october is not clear. it could be possible that boars mate more females than is recommended, since the boar to sow ratio is extremely low in rural areas (chiduwa, 2006). there is a need to conduct more studies in characterizing rural production systems to develop sustainable and profitable pig enterprises that reduce po chikwanha et al. 046 verty among the poor. the steady increase in condition of boars from december indicates that nutritional status was improving, suggesting that their maintenance and production nutrient requirements were easily met during confinement. the nutritional quality of the commonly used feed resources was evaluated for their chemical composition. the feed resources that were predominantly fed to pigs throughout the year included c. benghalensis, r. brasiliensis, maize suckers, kitchen swill, maize cobs, vegetables, groundnut hulls, watermelons, hominy chops, brewers’ dried grain, pumpkins and some unidentified weeds. however, the chemical composition and amino acid profiles of the other feed resources are available in the literature, and for this reason only c. benghalensis, r. brasiliensis, brewers’ dried grain and groundnut hulls were collected and analysed. the observation that r. brasiliensis had a significantly higher dm than c. benghalensis was expected. c. benghalensis is a watery weed that has no distinct stem. the lack of month effects on the dm content within each weed demonstrates the high potential of these weeds as livestock feeds as most forages show an increase in dm as the season progresses (soest, 1987). ether extract contents also remained constant across the trial period. c. benghalensis had a consistently higher ee content than for r. brasiliensis , suggesting that the former contains a higher capacity to supply energy to pigs. on the other hand, r. brasiliensis had markedly higher ash content than c. benghalensis. these findings suggest that both weeds can complement each in supplying adequate nutrients to pigs (halimani et al., 2005). as expected, there was a considerable increase in fibre content for both weeds as they matured, which can have serious implications on the intake and digestibility of forages (van soest, 1987). however, local pigs have the capacity to utilize diets with higher fibre content than european breeds, such as the large white (kanengoni et al., 2002; 2004). the marked difference between the two weeds in fibre content should be noted. differences in gross energy levels were also observed. however, there is need to determine the digestibility and utilization of the nutrients. the relatively high cp content of the feeds under investigation imply that these weeds can be conserved for dry season of feeding pigs, and be used as protein supplements. this would require harvesting the weeds during the rainy season or to conserve them through silage or hay making. interestingly, c. benghalensis has a relatively higher cp than most leguminous leaf meals. the high cp levels of bdg agree with the study of yaakugh and tegbe (1990), who obtained 21% cp. the high cp levels of bdg agree with the study of yaakugh and tegbe (1990), who obtained 21% cp. the resultant amino acid products are either end products of bacteria or or other micro-organisms produced during the fermentation process, or from microbial matter attached to fibres in the brewers’ grains. our findings are consistent with those of varvikko (1986), who demonstrated that resi-dues of vegetable feed supplements in nylon bags could be markedly contaminated by microbes during rumen incubation. fermentation during the brewing process mimics rumen fermentation and, therefore, has a better amino acid profile. tryptophan quantities in r. brasiliensis and brewers’ dried grain fall in the lower range of requirements for weight gain and feed efficiency for pigs. the required levels range from 0.13 to 0.19% (nrc, 1998). however, c. benghalensis has a surplus of tryptophan. the amino acid profile for c. benghalensis and r. brasiliensis exhibited the usual profile of green plants (d’mello, 1995), whereby the ratio of glutamic acid and aspartic acid are higher in both weeds in comparison to the other amino acids. the high amino profile was expected since brewers’ dried grain is an end product of microbial fermentation and is most likely to have higher nutrient concentration as compared to the other feeds. the quantities of aspartic and glutamic acid were highest in c. benghalen-sis, r. brasiliensis and groundnut hulls. although, the feeds tested have reasonably high levels of cp, all the individual essential amino acids are below the ideal requirements (holness, 1991). brewers’ dried grain had an essential amino acid to nonessential amino acid (eaa: neaa) ratio almost similar to that observed by wang and fuller (1989) for optimum dietary amino acids for growing pigs. yaakugh and tegbe (1990) reported on the use of bdg as a potential feed for pigs. the high nutritive value of bdg makes it a more appropriate option for farmers and will also serve to reduce the competition for the same feed resources, especially cereals, between humans and pigs. the high protein content of c. benghalensis can complement the highenergy content in bdg. the supply of bdg is, however, intermittent since their availability depends on the number of people participating in brewing. usually each household brews beer about four times a year and produces an estimated 25 kg of brewers’ grain each time and, therefore, with these quantities it can only be used to alleviate food shortages. c. benghalensis and r. brasiliensis are troublesome weeds that need removing. feeding them to pigs repays the labour involved and provides useful feed. the integration of animal production using agricultural byproducts ensures that animals play a complementary role, rather than a competitive role with man in meeting their feed requirements. the use of diverse feed resources will ensure that pigs are able to meet their feed requirements (kyriazakis and whitte-more, 2006). of the four feedstuffs tested, brewers’ dried grain had slightly higher amino acid content than the other fermentation high potential as alternative feed resources for pigs. the low amino acid content does not meet the 047 afr. j. pig farming optimal pattern of essential amino acids required in the diet for efficient protein deposition in the pig (grala et al., 1999) . however, in terms of protein value (pv), the four feeds have relatively lower pvs in comparison to both sunflower meal and cottonseed meal (kyriazakis and whittemore, 2006) as shown for some of the individual essential amino acids. from these findings, it can be seen that bdg is close to those of sunflower meal and cottonseed meal. although weeds have a relatively low pv, under smallholder conditions they are usually fed fresh to the pigs. the only major limitation when fed fresh is the high water content, which can reduce intake (ly, 1993). there is now a need to conduct further research to assess the performance when pigs are given the weeds fresh, rather than dry. conclusions lactating sows lost body condition during the rainy season. the feeds tested are not able to meet the amino acids requirements for growth. therefore, there is a need to include these feeds in least cost feed diet formulation based on the use of locally available feed resources. there is also a need to conduct digestibility and growth performance trials with pigs fed on the non-conventional diets. acknowledgements we thank farmers in chirumanzu district for co-operating and keeping records for their pigs. funding was provided by the regional universities forum for capacity building in agriculture (ruforum) grant number 2005 ru cg 018. we also thank the department of livestock production and development, government of zimbabwe, for participating in the project. the amino acid profiles were analysed at the animal nutrition institute at the agricultural research council, irene, south africa. references associations of official analytical chemists (aoac) (1990). official methods of analysis of the association of official analytical chemists. 15 th ed. washington dc, usa. chiduwa g (2006). herd dynamics and contribution of indigenous pigs to the livelihoods of rural farmers in a semi -arid area of zimbabwe. msc thesis, department of animal science, university of zimbabwe, harare, zimbabwe. chikwanha oc (2006). seasonal changes in body condition scores of 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reversed-phase high-performance liquid chromatography. j. chromat. 282: 609-618. gehrke cw, wall ll, absheer js, kaiser fe, zumwalt rw (1985). sample preparation for chromatography of amino acids: acid hydrolysis of proteins. j. asso. anal. chem. 68 (5): 811-821. goering hk, van soest pj (1970). forage fibre analyses (apparatus, reagent, procedures and some applications). agriculture handbook ars-usda, washington dc p.379. grala w, verstegen mwa, jansman ajm, huisman j, van leeuwen p, tamminga s (1999). effects of ileal endogenous nitrogen losses and dietary amino acid supplementation on nitrogen retention in growing pigs. ani. feed sci. technol. 80: 207222. halimani te, ndlovu lr, dzama k, chimonyo m, miller bg (2005). metabolic response of pigs supplemented with incremental levels of leguminous acacia karroo, acacia nilotica and colophospermum mopane leaf meals. ani. sci. 81: 39-45. holness dh (1991). pigs. the tropical agriculturalist: pigs. coste, r. and a. j. smith ed. macmillan education limited, wageningen. holness dh, paterson r, ogle b (2005). livestock and wealth creation. improving the husbandry of animal kept by resource-poor people in developing countries. e. owen, a. kitalyi, n. jayasuriya and t. smith, ed. nottingham university press, nottingham, england. kanengoni at, dzama k, chimonyo m, kusina j, maswaure sm (2002). influence of level of maize cob inclusion on nutrient digestibility and nitrogen balance in the large white, mukota and f1 crossbred pigs. ani. sci. 74: 127-134. kanengoni at, dzama k, chimonyo m, kusina j, maswaure sm (2004). growth performance and carcass traits of large white, mukota and large white × mukota f1 crosses given graded levels of maize cob meal. ani. sci., 78: 61-66. kyriazakis i, whittemore ct (2006). whittemore’s science and practice of pig production. third edition, blackwell publishing ltd, oxford, united kingdom. lekule p, kyvsgaard cn (2003). improving pig husbandry in tropical resource-poor communities and its potential to reduce risk of porcine cysticer. acta. trop. 87 (1): 111-117. ly j (1993). the role of monogastric animal species in the sustainable use of tropical feed resources. in: proceedings of seventh world conference animal production. edmonton. pp. 95-117. maes dgd, janssens gpj, delputte p, lammertyn a, de kruif a (2004). back fat measurements in sows from three commercial pig herds: relationship with reproductive efficiency and correlation with visual body condition scores. liv. prod. scie. 91: 57-67. mashatise e, hamudikuwanda h, dzama k, chimonyo m, kanengoni a (2005). socio-economic roles, traditional management systems and reproductive patterns of mukota pigs in semi-arid north-eastern zimbabwe. bunda j. agri. environ. sci. technol. 3: 97-105. mhlanga fn, khombe ct, makuza sm (1999). indigenous livestock genotypes of zimbabwe. department of animal science, university of zimbabwe, harare. national research council (1998). nutrient requirements of swine: 10 th revised edition. the national academy of science, washington dc. sas (1996). statistical analysis systems user’s guide release 6.12. statistical analysis system inst. inc., cary, nc, usa. van soest pj (1987). nutritional ecology of the ruminant. cornell university press. pp. 117. varvikko t (1986). microbially corrected amino acid composition of rumen-undegraded feed protein and amino acid degradability in the rumen of feeds enclosed in nylon bags. brit. j. nutr. 56: 131-140. wang tc, fuller mf (1989). the optimum dietary amino acid pattern for growing pigs. brit. j. nutr. 62: 77-89. yaakugh idi, tegbe tsb (1990). performance carcass characteristics of grower and finisher pigs fed diets containing brewers’ dried grain. nigeria.agric.j.24:31-40. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (1), pp. 001-003, january, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper productive performance of piglets fed with different sources of lactose jimmy quisirumbay-gaibor*, luis delgado, césar oña, alexandra naranjo and eduardo aragón veterinary medicine college, central university of ecuador, quito, ecuador, jerónimo leiton s/n y gatto sobral. received 20 july, 2016; revised 10 august, 2016; accepted 14 january, 2017 and published 22 january, 2017 lactose is important to potentiate the health and growth of piglets before and after weaning. the aim of this study was to evaluate the effect of three sources of lactose on performance: 24 piglets from 7 days of age were divided into three groups (n = 8), 2 dietary phases were used: pre-weaning (7-35 days) and weaning (36-56 days). the source of lactose was the variable under study: crystallized lactose, lacto-whey and permeate. body weight (bw), average daily gain (adg) and feed conversion rate (fcr) were the parameters evaluated. the results were analyzed by anadeva and duncan. piglets receiving the permeate had a higher final weight 23.98 kg (p> 0.05) and higher adg 0.42 kg / d (p> 0.05). this experiment determined that any source of lactose can be included in feed processing for piglets however the inclusion of permeate cheaper production costs. keywords: piglet, feed, lactose, whey, permeate. introduction there is great interest in developing management and feeding strategies that stimulate the development and digestive health of piglets in order to enhance its growth and minimize the use of antibiotics (de lange et al., 2010). the use of dairy products for feeding piglets early in life has shown remarkable benefits thanks to the contribution of highly digestible nutrients such as the lactose (cromwell et al., 2007). the piglet at weaning (21-35 days old) suffers damage at the level of the villi of the intestinal mucosa which decreases the digestive capacity (lallès et al. 2007) and it is essential to avoid incorporating large amounts of plant nutrients which would aggravate the damage (pluske 2013). the inclusion of sources of lactose in the diet of pigs has shown an increase in daily weight gain in pigs in the weaning stage in approximately 17% (cera et al. 1988). the effect is associated with increased feed intake (gahan et al. 2009) and most nutrient digestibility ( tokach et al. 1989) thus decreasing the atrophy of the *corresponding author email: jrquisirumbay@uce.edu.ec tel: +593996720299 villi and pig productive performance is improved (dong and pluske, 2007). it has also been observed that diets that include lactose improve the digestibility of dry matter (dm) and crude protein (cp) thereby reducing the excretion of these nutrients (jin et al. 1998). whey, deproteinized whey (permeate) and lactose purified among others are used as source of lactose in feeding piglets. these vary greatly in their nutritional composition, lactose, protein, ash and its solubility properties and ph (nessmith et al. 1997). whey is the liquid fraction obtained after the coagulation of milk during cheese manufacture, it contains about 70% lactose but may vary depending the cheesemaking process (zadow, 2012). the crystallized lactose is obtained after evaporation and dehydration of serum (mainly composed of α-lactose) and pure lactose is obtained by centrifugation and purification of the crystallized whey: it contains more than 99% lactose. the permeate is the whey fraction deproteinized resulting from ultrafiltration process for the separation of whey protein concentrate and contains about 83% lactose (nessmith et al. 1997). the objective of this study was to evaluate the effect of dietary inclusion of three sources of lactose on the productive performance of piglets. materials and methods the experiment was conducted at the pig farm "san francisco" located in alangasí, pichincha-ecuador. 24 piglets from 7 days old landrace-york f1 were selected; 12 females and 12 males, weaning was performed at 21 days old. individuals were randomized into three groups of 8 animals each with an equal number of females and males (t1, t2 and t3). the study lasted 49 days and was evaluated in two diets: pre-weaning phase (7-35 d) and post-weaning phase (36-56 d) formulated based on the recommendations made by nrc 2012. the t1 group received pure lactose, t2 whey and t3 permeate. the nutritional composition of the diet study is detailed in table 1. table 1. calculated composition of the diets (as-fed basis). 7-35 d 36-56 d dm, % 91.3 89.6 lactose, % 15 7.5 cp, % 22.7 22.9 lys, % 1.60 1.56 thr, % 1.03 1.04 trp, % 0.27 0.27 met + cys, % 0.93 0.93 ca, % 0.78 0.80 p, % 0.71 0.69 bioavailable p, % 0.43 0.40 me, mcal/kg 3.42 3.43 fat, % 5.6 5.9 crude fiber, % 2.3 2.7 ash, % 7.6 7.3 provided per kilogram of diet: vitamin a, 11,600 iu; vitamin d3, 1,100 iu; vitamin e, 22 iu; vitamin k, 4 mg; riboflavin, 8.25 mg; d-pantothenic acid, 28 mg; niacin, 33 mg; and vitamin b12, 0.03 mg. provided per kilogram of diet: zn, 165 mg; fe, 165 mg; mn, 33 mg; cu, 16.5 mg; i, 0.3 mg; and se, 0.3 mg. production parameters: bw, adg and fcr were recorded from day 7 to day 56 of age. in addition a costbased economic analysis was carried out. statistic analysis anadeva and duncan test was used for variables, the analysis was performed using spss version 22.0 (spss inc., chicago, il). an α level of p <0.05 was considered statistically significant. results and discussion the values obtained for the evaluated parameters are presented in table 2. the final body weight at 56 days of age was similar in all three treatments and there was no significant difference (p> 0.05). pigs receiving permeate had a slightly higher average in adg (0.42 ± 0.030 kg / d) but no significant difference (p> 0.05). although there was no significant difference between treatments fcr was better in the t3 group (0.899 ± 0.06). in the economic analysis conducted for this study it was obtained as a result that the t3 group (permeate) the cost per kilogram of feed produced was lower which led to a higher net profit per kilo body weight produced (2.1 usd) when compared to t1 (1.99 usd) and t2 (2.04 usd). it is well established that the inclusion of lactose in the diet of piglets improves the yield of animals by stimulating the feed intake and palatability (mascarenhas et al. 1999), in the study is evidence that all animals showed uniformity in the weight at the end of the experiment and reached a greater weight than those estimated in the tables weight goals. in studies where higher percentages of lactose (25%) were included in the preference of animals by diet and overall higher consumption in the weaning it was evident, even at the stage of pre-weaning inclusion levels of lactose generated differences in consumption (de souza and landín., 2004). the adg was statistically similar in the 3 groups similar data were found in the work of jin et al. (1998) in which there were no differences in the adg in piglets receiving crystallized lactose (555.52 g/d) and those receiving whey (555.91 g/d) 0 to 21 days of age, as in the study nessmith et al. (1997) in which there were no differences in adg by comparing the same three sources of lactose in the period 0 to 35 days old. as for fcr was no difference between similar to what happened in the study by nessmith et al groups. (1997). however, in the study by jin et al. (1998) significant difference (p0.05) was found in both pure lactose ca between treatments (1.33) vs. dried whey (1.35). summary and conclusion replacing pure lactose whey or permeate in piglet diets phased pre-weaning and weaning not reflect significant differences on productive parameters evaluated in this study and represent suitable for supplying the nutrient sources, however whey can be very variable in composition and quality and is an important factor to consider for use in animal feeding. table 2. performance parameters. t1 t2 t3 pure lactose whey permeate piglets (n) 8 8 8 inicial body weight (kg) 3.51±0.32 a 3.43±0.14 a 3.26±0.30 a final body weight (kg) 23.13±1.81 a 23.24±2.59 a 23.98±1.77 a adg (kg/d) 0.40±0.036 a 0,41±0.049 a 0,42±0.030 a fcr 0.941±0.08 a 0.948±0.14 a 0.899±0.06 a average cost (usd/kg feed) 1.51 1.46 1.40 profit (usd/kg de pv) 1.99 2.04 2.1 same letters in the same row indicate no significant difference (p> 0.05). acknowledgement the authors thank the pig farm "san francisco" to allow the completion of this work; dr. nelson jaramillo for his contribution and leadership in the research, ing. roberto solorzano and the company "bioalimentar”. references cera, k. r., mahan, d. c., & reinhart, g. a. (1988). effects of dietary dried whey and corn oil on weanling pig performance, fat digestibility and nitrogen utilization. journal of animal science, 66(6), 1438-1445. cromwell, g. l., allee, g. l., & mahan, d. c. (2007). assessment of lactose level in the mid-to late-nursery phase on performance of weanling pigs1, 2. j anim sci, 2008(86), 127-133. dong, g. z., & pluske, j. r. (2007). the low feed intake in newly-weaned pigs: problems and possible solutions. asian-australasian journal of animal sciences, 20(3), 440-452. gahan, d. a., lynch, m. b., callan, j. j., o’sullivan, j. t., & o’doherty, j. v. (2009). performance of weanling piglets offered low-, medium-or high-lactose diets supplemented with a seaweed extract from laminaria spp. animal, 3(01), 24-31. jin, c. f., kim, j. h., moon, h. k., cho, w. t., han, y. k., & han, i. k. (1998). effects of various carbohydrate sources on the growth performance and nutrient utilization in pigs weaned at 21 days of age. asian australasian journal of animal sciences, 11, 285-292. lalles, j. p., bosi, p., smidt, h., & stokes, c. r. (2007). nutritional management of gut health in pigs around weaning. proceedings of the nutrition society, 66(02), 260-268. lallès, j. p. (2008). nutrition and gut health of the young pig around weaning: what news. archiva zootechnica, 11(1), 5-15. mascarenhas, a. g., ferreira, a. s., donzele, j. l., freitas, r., gomes, p. c., & oliveira, r. (1999). avaliação de dietas fornecidas dos 14 aos 42 dias de idade sobre o desempenho e a composição de carcaça de leitões. revista brasileira de zootecnia, 28(6), 13191326. de lange, c. f. m., pluske, j., gong, j., & nyachoti, c. m. (2010). strategic use of feed ingredients and feed additives to stimulate gut health and development in young pigs. livestock science, 134(1), 124-134. nessmith jr, w. b., tokach, m. d., goodband, r. d., & nelssen, j. l. (1997). defining quality of lactose sources used in swine diets. swine health and production, 5(4), 145. pluske, j. r. (2013). feed-and feed additives-related aspects of gut health and development in weanling pigs. journal of animal science and biotechnology, 4(1), 1. de souzaa, t. c. r., & landínb, g. m. (2004). preferencia alimenticia y comportamiento zootécnico en lechones alimentados con dietas formuladas con diferentes tipos de suero de leche deshidratado. téc pecu méx, 42(2), 193-206. tokach, m. d., nelssen, j. l., & allee, g. l. (1989). effect of protein and (or) carbohydrate fractions of dried whey on performance and nutrient digestibility of early weaned pigs. j. animal sci. 67(5), 1307-1312. zadow, j. g. (2012). whey and lactose processing. springer science & business media. african journal of pig farming vol. 1 (3), pp. 028-034, november, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper farmer perceptions of classical swine fever outbreak in communal pig production systems of south africa james madzimure 1 *, kerstin k. zander 2 , kennedy dzama 3 and michael chimonyo 1 1 discipline of animal and poultry science, university of kwazulu-natal, p. bag x01, scottsville 3209, pietermaritzburg, south africa. 2 research institute for environment and livelihoods, charles darwin university, darwin nt 0909, australia. 3 department of animal science, university of stellenbosch, p. bag x1, matieland 7602, south africa. accepted 25 september, 2013 after the outbreak of classical swine fever (csf) in the eastern cape province of south africa, policy makers are expected to make decisions on the restocking of pigs. the objective of this study was to investigate farmers’ perceptions of csf outbreak in coastal and inland communal production systems because of their differences in harbouring pathogens. data were collected from 288 farmers in two csf affected areas (one on the coast, one inland) and one unaffected coastal area. the majority of farmers in affected inland (73%) and coastal (89%) areas kept local pigs and non-descript crosses with imported pigs on backyard production system. significantly, more pigs were culled in the affected coastal area than inland area. in both areas, the culling of pigs affected pork availability and income generation. household heads that were residing on the farms that are educated, mature and located inland were less likely to experience disease challenges. to facilitate restocking and conservation of local pig genetic resources, farmers requested the government and stakeholders to assist with loans, breeding stock, proper housing structures and improved extension services. farmers in both coastal and inland areas perceived csf as destructive to pigs, thereby jeopardising their source of livelihoods. key words: pig genetic resources, conservation, disease outbreaks, pig culling, restocking. introduction utilisation of local pig genotypes in communal areas has the potential to increase food security, reduce poverty and improve livelihoods of the resource-poor farmers (halimani et al., 2010; madzimure et al., 2012). despite their importance, local pigs are threatened by sporadic disease outbreaks such as classical swine fever (csf) (world organisation for animal health, 2005). the outbreak of csf in south africa led to the culling of more than 335 000 pigs (more than two thirds of the pig population in the eastern cape province) and a loss of about 95% in production (south african press association (sapa), 2005). the loss of pig biodiversity affects the ability of future breeding programmes to respond to changing climate and consumer needs *corresponding author. e-mail: madzimurej@gmail.com. (halimani et al., 2010). culling also left few breeding males and females, thereby increasing the chances of related animals to mate each other. an obvious consequence is inbreeding. it is, therefore, important to understand the origins and impact of diseases in communal production systems, and tap on the indigenous knowledge on how to minimise their devastating effects. any intervention to reduce the impact of diseases assists in the conservation of pig genetic resources. the government of south africa should consider initiating a national restocking programme in the affected areas. there is risk of disease outbreak if farmers are left to restock using some of the pigs that were hid during the culling exercise. future outbreaks of contagious diseases such as csf (ruggli et al., 1996; liu et al., 2009; podgórska and stadejek, 2010) should be avoided since it has a huge financial implication on the national economy madzimure et al. 028 elundini ntabankulu ngqushwa figure 1. map showing study sites in the eastern cape province of south africa. economy as the government struggles to control the disease (edwards et al., 2000; leifer et al., 2005; rowlands et al., 2008). for example, the south african government paid more than r200 million to compensate the more than 83 000 affected farmers (national african farmers union (nafu), 2007). the disease slowed down the development of the pig industry, reduced trade at the national and international levels and resulted in job losses (sapa, 2005). the country lost potential revenue since it was banned from exporting pork up until it reached a csf-free status. the outbreak of csf first occurred in the western cape province before being detected in the eastern cape province in 2005 (penrith et al., 2011) where it was concentrated mainly around the informal free-roaming pig farming areas (nafu, 2007). the disease spread over large distances when human intervention such as illegal swill feeding facilitated its movement (edwards et al., 2000). most of the pigs that were culled were from areas located along the coast, perhaps because of the pattern of spread of the disease which emanated from a coastal town of centane (department of agriculture, 2006). the sampling of households for our study was, therefore, designed to represent the coastal and inland areas of the eastern cape. coastal areas are known to harbour many diseases because of the hot humid conditions when compared to inland areas (rowlands et al., 2008; jutla et al., 2010; ortiz-pelaez et al., 2010). restocking of commercial pigs is generally easy to implement, as imported pigs are widely available from renowned pig breeders. sourcing of local pigs after culling is, however, extremely difficult because there are no breeders. local pig genotypes are ideal for the resourcepoor communal farmers because they require low maintenance costs due to their foraging ability. designing restocking programmes for pigs without establishing the perceptions of the communal farmers is likely to cause passive resistance and prohibit co-operation by the communities. the objectives of this study were to investigate farmers’ perceptions of csf outbreak in coastal and inland communal areas and to reveal differences in the way farmers were affected by and dealt with the outbreak across areas and production systems. materials and methods study sites the study was conducted in communal production systems of elundini (inland), ntabankulu (coastal) and ngqushwa (coastal) municipalities in the eastern cape province of south africa (figure 1). the sites were chosen after the csf outbreak and policy makers needed data to restock pigs in the eastern cape province. the sites were selected with participation of state veterinary services, university of fort hare, councillors, farmer representtatives and government officials. in the whole of the eastern cape province, ntabankulu was the only municipality where pigs were not culled because the pigs tested negative against csf. in addition, farmers in ngqushwa municipality are generally less poor and market-oriented when compared to the subsistence-oriented farmers in elundini and ntabankulu municipalities. elundini municipality is situated 28° 25′ e; 30° 26′ s with an elevation of about 1600 m above sea level. the mean annual rainfall ranges from 800 to 1200 mm. the area has average minimum day temperature of 13°c and maximum temperature of 22°c. ngqushwa municipality is situated at 27° 7′ e and 33° 12′ s. the 029 afr. j. pig farming temperature ranges from -2 to 42°c with an average of about 18°c. the area receives an annual rainfall of about 450 to 900 mm with most of it occurring in summer. the area has deep loamy soils with vegetation greatly covered by the acacia karroo. ntabankulu municipality is situated at 29° 16′ e and 31 º 04′ s with an elevation of about 476 m above sea level. ntabankulu receives mean annual rainfall of 620 mm with most rainfall occurring during mid-summer. the average daily temperature ranges from 17.8°c in june to 25°c in january. in all municipalities, pigs formed integral components of mixed crop-livestock farming systems by providing manure or cash for the purchase of inputs for crop production. local pigs can also utilise resources that have few alternative uses, such as agricultural by-products. crops commonly grown by farmers in these areas include maize, beans, potatoes and pumpkins. data collection data were collected from three municipalities using individual structured questionnaires, in-depth interviews with key informants and direct observations of pigs and production practices. primary information about pig production was obtained from key informants. extension officers, veterinary specialists, local leadership (political and customary) and the elderly (over 70 years of age) provided the secondary data. secondary information was obtained from the department of agriculture. the study was conducted from august to december 2009. communities with many pig owning households were identified with the assistance of the national department of agriculture. the households with pigs were identified with the assistance of the local leadership and the snowballing technique was used to select participants who were willing to participate in the project. the key informants were interviewed to establish the pig production trends, factors affecting production levels and traits of economic importance, as a first step in designing a structured questionnaire. the questionnaires were administered in the vernacular xhosa language. farmers’ wealth status was categorized during interviews with key informants and was based on number of livestock species. any household owning more than five heads of cattle or more than 20 heads of small stock (sheep, goats and pigs) was considered as less poor while the other category of less privileged people was considered as poor. the number of households owning pigs that were interviewed in elundini, ngqushwa and ntabankulu was 122, 102 and 64, respectively. data were collected using structured questionnaires and included demographic data, pig rearing systems, number of culled per household, perceptions of farmers on the severity of csf and how the government should have controlled it. additional data included compensation price for different classes of pigs and whether farmers received it, farmers’ perceptions on whether the compensatory price was satisfactory and suggested compensation price. changes of pig prices with csf outbreaks, government’s effort in restocking and whether farmers were sending of dead pigs for post-mortem were also captured. direct observations were made to verify pig genotypes. the perceptions of the people on the need to conserve the local pig genetic resources were captured using a structured questionnaire. statistical analyses the generalised linear models procedure of sas (2006) was used to analyse the effects of farmers’ socio-economic profiles, area of location (coastal and inland) and pig rearing systems (backyard and scavenging) on the number of pigs culled. pair-wise comparisons of the least square means for culled pigs were performed using the pdiff option. information regarding demographic data, pig rearing system, pig breeds owned, farmer’s perceptions on the incidences, impact and control of csf was analysed using proc freq of sas (2006). an ordinal logistic regression (proc logistic) was used to estimate the probability of household experiencing csf (sas, 2006). the logit model fitted predictors such as area of location, pig rearing system, household size, pig housing, and head of household’s demographic factors such as age, education level, employment status, marital status and place of residence (stays mainly at home or works and stays away from home). the logit model used was: in [p/1−p] = β0 + β1x1 + β2x2 + β3x3… + βtxt + ε where: p = the probability of a household experiencing csf; [p/1−p] = odds ratio, which referred to the odds of household experiencing csf; β0 = intercept; β1x1...βtxt = regression coefficients of predictors; ε = random residual error when computed for each predictor (β1... βt), the odds ratio was interpreted as the proportion of households experiencing csf versus those that did not experience it. results farmers’ socio-economic profile the socio-economic profiles of respondents in the elundini, ngqushwa and ntabankulu municipalities are shown in table 1. the proportion of households headed by females in the three locations was equal. mean household size across all municipalities was 5.2 ± 4.63 (mean ± standard error) members. most of the interviewees in elundini, ngqushwa and ntabankulu municipalities were unemployed and survived on subsistence farming or social grants. there were more respondents with basic education (grade 1 to 7) than secondary or tertiary education in the three municipalities. the majority of the interviewees in elundini, ngqushwa and ntabankulu municipalities were christians, while the remainder were african tradition worshippers. most of the heads of households were resident on the farms in the three municipalities. in all three municipalities, most women over 60 years old were actively involved in pig rearing while men, boys and girls helped in the absence of women. across all the municipalities, pigs were mostly owned by women. the majority of farmers were using backyard production system where the pigs were free roaming in the yard and the remainder were using the free range or scavenging production system (table 1). about 86% of the farmers reported that the major field crop they grew was maize, largely for household consumption and as supplementary feed for livestock. the other minor crops grown for consumption, in order of importance, were beans, vegetables, potatoes and pumpkins. farmers’ perceptions on classical swine fever outbreak the impact of csf and the perceptions of farmers about madzimure et al. 030 table 1. socio-economic characteristics of respondents (percentage) in different locations. socio-economic characteristic elundini (inland) ngqushwa (coastal) ntabankulu (coastal) n = 122 n = 102 n = 64 male headed households 52 47 55 married respondents 67 63 73 women owning pigs 81 60 69 unemployed respondents 79 77 80 respondents with basic education (grade 1-7) 50 55 47 respondents that were christians 88 84 70 heads of households living on the farm 70 85 65 female pig keepers over 60 years of age 51 37 37 youths reported as interested in pig rearing 66 89 79 respondents using backyard production system 73 89 36 respondents keeping indigenous pigs 89 82 97 table 2. farmers’ perceptions (percentage) about classical swine fever disease outbreak. pig production and disease attributes elundini (inland) ngqushwa (coastal) ntabankulu (coastal) n = 122 n = 102 n = 64 respondents with culled pigs due to csf 97 93 0 respondents who hid some pigs from culling 17 22 0 respondents who never saw controllers of csf 2 3 16 respondents who send pigs for post-mortem 1 6 3 respondents who thought csf is dangerous for pigs 60 88 67 respondents who thought csf reduces pig production 10 13 12 respondents who thought csf decreases pig price 28 86 55 respondents who had no idea about csf impact 29 0 21 respondents who believed in vaccination against csf 71 50 66 respondents who believed housing controls csf 7 22 11 respondents advocating for educating people about csf 2 28 10 respondents who supported culling of pigs 14 0 10 respondents who supported compensation with pigs 50 0 38 respondents who received monetary compensation 25 71 0 respondents satisfied with compensation price 100 83 63 respondents supported a pig restocking programme 68 80 66 respondents who wanted loans for pig projects 0 36 20 respondents who demanded better extension services 56 64 56 the disease are shown in table 2. the majority of farmers in elundini and nqushwa municipalities had their pigs culled due to csf. there were differences (p < 0.05) in the number of culled pigs in elundini (4 ± 1.00), ngqushwa (8 ± 1.76) and ntabankulu (2 ± 1.00). generally, most farmers in the current study regarded csf as a dangerous disease for pigs, which reduce production and profit (table 2). more farmers in the csf affected areas hid their pigs whenever they saw government officials. most farmers in all the municipalities suggested the need for vaccination in order to control the disease (table 2). few farmers supported the culling of pigs as a control measure in all municipalities (table 2). there were more farmers in ngqushwa municipality than the other two municipalities who believed that housing pigs and educating people about csf would help in controlling the disease. all the respondents confirmed that they were aware of the government’s compensation price of r2000 per breeding sow. there were few respondents in elundini municipality who wanted the government to compensate them with uninfected pigs instead of money (table 2). farmers in ntabankulu municipality supported the notion that the government should restock any area that would have been affected by csf though they were not directly affected. ngqushwa municipality had the highest number 031 afr. j. pig farming table 3. odds ratio estimates, lower and upper confidence interval (ci) of a household experiencing classical swine fever. disease and parasites challenge odds ratio lower ci upper ci area of location (inland vs. coastal) 2.482 1.415 4.354 pig rearing system (free range vs. backyard) 0.766 0.408 1.437 age of head of household (young vs. old) 1.907 0.567 6.415 education (educated vs. uneducated) 2.202 1.186 4.089 employment status (employed vs. unemployed) 0.719 0.376 1.375 residence of household head (at the farm vs. away) 2.783 1.231 3.869 household size (large vs. small) 1.158 0.691 1.930 pig housing (pigs not housed vs. pigs housed) 0.485 0.230 0.824 marital status (married vs. not married) 0.989 0.775 1.280 the first category in parenthesis was used as the baseline (set to 1). of respondents who had been compensated for their culled pigs followed by elundini municipality (table 2). few farmers in ngqushwa and ntabankulu municipalities wanted loans for establishing pig projects. most people across the three municipalities requested better extension services from the government to prevent future disease outbreaks especially after restocking efforts (table 2). across the municipalities, most people were not sending their pigs for post-mortem. odds ratios for disease outbreak the odds ratios of a household experiencing csf were highest for head of household staying away from the homestead, followed by education level, age of head of household, municipality, size of household and production system (table 3). the odds ratio of 2.182 showed that heads of households who were staying away from the farm had a higher likelihood of experiencing csf. the uneducated farmers were more likely to experience csf than their educated counterparts. households headed by young people were affected by csf more than those led by old people. municipalities at the coastal areas were more challenged by csf than inland ones. prospects of restocking areas affected by csf on average, 22% of the farmers were resisting culling insisting that they were an important part of their livelihoods. most people (92%) mentioned that they were infuriated seeing government officials going around killing pigs. these farmers felt that the government was being inconsiderate by destroying their pigs without giving them immediate compensation to survive on. it was mainly piglets that were hid from government officials inside the houses while some tied mature pigs in the nearby bushes or mountain. all the farmers supported the idea of the government initiating a national restocking programme in the areas affected by csf. it was surprising that after the culling exercise in 2005, pig ownership was fast spreading in the communal households at the time of the study. the only thing that was limiting some farmers from venturing into pig production was shortage of breeding stock. discussion the culling of csf-infected pigs for the majority of respondents in ngqushwa and elundini municipalities meant that farmers’ welfare was affected since pigs are a source of livelihood (food and income generation) for these resource-poor farmers. the hiding of pigs in mountains or indoors and the self-initiated restocking revealed their importance to the respondents, the majority of whom were unemployed. women were more affected by the culling since they are the ones who rear more pigs than men for food and income generation (chiduwa et al., 2008). the continued upkeep of local pigs by the resource-poor communal farmers supports sustainable agricultural development (drucker and anderson, 2004). the majority of the youths were interested in helping with pig rearing implying pig production may continue into the future in communal areas. in the restocking programme, it can be suggested to fund women because they were the major owners of pigs as men were interested in large stock like cattle. the majority of the farmers regarded csf as dangerous since it reduces pig production and profits are supported by previous reports (widjojoatmodjo et al., 1999; sapa, 2005; fao, 2009). more farmers in ngqushwa municipality were affected in terms of price drop since they were keeping more pigs for the market when compared to the other municipalities surprisingly, most farmers did not support the government’s approach of culling pigs even though they accepted that the disease was dangerous. this could be due to the fear of losing pigs as their source of livelihood through generation of income and provision of pork. madzimure et al. 032 the government’s delays in compensating farmers may further explain the hiding of pigs by some farmers. according to the department of agriculture (2006), delays in compensation for rural farmers were orchestrated by lack of bank details for most of the rural farmers although efforts were made to pay them through the post office. unless market-related compensation for pigs slaughtered is paid promptly, farmers are tempted to evade the control measures and csf will continue to spread (penrith and thomson, 2004). the finding that some farmers in ngqushwa and ntabankulu municipalities were not satisfied with the compensatory price suggest that the government should pay them more for forced culling because it inconvenienced them. farmers in subsistence communal production systems might have appreciated compensation in the form of uninfected pigs instead of money. it could be that farmers feared failure to secure breeding stock when permission to restock is granted. this finding further supports the fact that csf causes loss of pig biodiversity. the south african government was justified in its culling action since it is recommended to stamp out infected and in-contact pig herds with destruction of the carcasses as one of the traditional control measure to achieve eradication of csf after an outbreak (elbers et al., 1999; garner et al., 2001). instead of culling, most farmers in elundini and ntabankulu municipalities suggested that vaccination could have been a better option. the farmers’ opinion was supported by some authors who reported that effective live-attenuated vaccines are available (wehrle et al., 2007; liu et al., 2009). vaccination is, however, not allowed in the export market such as the european union, since vaccinated and infected pigs are serologically indistinguishable (widjojoatmodjo et al., 1999; wehrle et al., 2007). the use of marker vaccines makes discrimination between vaccinated and infected animals possible (widjojoatmodjo et al., 1999; wehrle et al., 2007; kaden et al., 2008). the acceptability of marker vaccines rests with trade partners. the use of these marker vaccines might not be an option for south africa because they are costly to produce thus become expensive to the farmer. in addition, they are based on deoxyribonucleic acid (dna) and proteins, hence are not as efficacious as the live-attenuated vaccines (greiser-wilke and moennig, 2004). the south african government currently depends on serological surveillance to control csf; therefore, vaccinating pigs as suggested by farmers would interfere with the epidemiological tool (department of agriculture 2006; penrith et al., 2011). farmers need to be educated on the government’s reasons for the choice of the disease control programme so that they cooperate. the finding that farmers in ngqushwa municipality believed in educating people about the disease and housing pigs as control measures may be due to their market-orientation which makes them stricter. nafu (2007) reported that the spread of csf in eastern cape province of south africa was mainly due to free ranging pigs. farmers in ntabankulu and elundini municipalities, however, indicated that they could not afford the government’s recommended pig housing structure unless the government is to construct it for them. most farmers in these municipalities are resource-poor and, hence, largely depend on government. the continued free ranging of pigs increases the chance of them mixing with infected wild pigs thereby compromising the csf control effort (penrith et al., 2011). acutely infected pigs are a potent source of infection for other pigs (penrith et al., 2011). the disease is also transmitted from pregnant sow to foetuses (penrith et al., 2011) or from one farm to another through equipment, vehicles and people (van oirschot 2004). after the csf outbreak, farmers in affected areas were prohibited from slaughtering any pigs at the abattoirs to stop the spread of the disease through the food chain. generally, all farmers considered local pigs to be tolerant to disease challenges although it was not clear this includes csf. across all municipalities, the majority of farmers were not sending their pigs for post mortem which makes it difficult for the department of agriculture to quickly detect any disease outbreak especially in communal areas. delays in detecting outbreak of contagious diseases like csf (ruggli et al., 1996; liu et al., 2009; podgórska and stadejek, 2010) will result in the virus travelling long distances affecting many pigs hence increased costs of controlling the disease (edwards et al., 2000; leifer et al., 2005; rowlands et al., 2008). the odds ratios for a household experiencing csf were affected by demographic factors, area of location and production system as was also reported by mapiye et al. (2009). pigs for a household head who was resident at the farm were less likely to experience csf outbreaks because the farmer was always available to better take care of the pigs. a mature resident head of household’s pigs have less risk because the farmer may be more experienced and likely to comply with government’s recommendations to control diseases like csf such as confining the pigs. many people in each household may share responsibilities and better take care of pigs to avoid outbreak of diseases. the level of education of the farmer may also assists in better understanding and cooperation with the government in disease control. coastal municipalities like ngqushwa and ntabankulu are more likely to experience csf outbreaks because of their hot humid conditions which harbour diseases (rowlands et al., 2008; jutla et al., 2010; ortizpelaez et al., 2010). pigs that were free ranging were likely to experience csf than those that were either in the backyard or housed. when pigs are free ranging, they are likely to mix with infected herds and the disease will spread. the pigs that were owned by employed people were likely to be affected by csf probably because these farmers had little time available to take care of their pigs. 033 afr. j. pig farming municipalities wanted the government to come up with a restocking programme in affected areas in order to restore the local pig biodiversity. currently, the efforts from local municipalities are not capable of providing loans to all farmers who want to revive their piggery projects. farmers requested the government to promote the production of kolbroek, windsnyer and their crosses with imported breeds because they are hardy and resistant to diseases as was reported by halimani et al. (2010). farmers in ngqushwa municipality wanted the government to avail loans to individuals who could not be part of a cooperative but have pig structures and experience in pig rearing. in addition, these farmers wanted access to a reliable market so that they can boost pig production. coupled with restocking, the majority of farmers wanted the government to provide better veterinary and extension services in terms of disease control and pig husbandry. this will go a long way in preventing future disease outbreaks and supporting the conservation of threatened local pig genetic resources. conclusions the odds ratios showed that coastal areas are more likely to have csf disease outbreaks when compared to dry inland areas. more pigs were culled in coastal areas of ngqushwa municipality when compared to inland elundini municipality. the culling of pigs affected pork availability, income generation and caused ecosystem disturbance in the crop-livestock communal production systems. the same challenges were not witnessed in unaffected coastal area. to facilitate restocking or conservation of local pig genetic resources, farmers requested the government to assist with loans, local breeding stock, proper housing structure and improved extension services. we concluded that the risk of csf disease outbreak is high in coastal areas than inland areas but the disease can have equally devastating effects once there is an outbreak in an area. acknowledgements the authors are grateful to the national research foundation and govan mbeki research and development centre at the university of fort hare for funding this research. we acknowledge the assistance with field logistics from the eastern cape department of agriculture and agrarian reform. references chiduwa g, chimonyo m, halimani te, chisambara sr, dzama k (2008). herd dynamics and contribution of indigenous pigs to the livelihoods of rural farmers in a semi-arid area of zimbabwe. trop. anim. health prod. 37:333-344. department 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analysis of classical swine fever virus isolates from the epizootic in the netherlands in 1997-1998. vet. microbiol. 66:291-299. world organisation for animal health (2005). classical swine fever in south africa, follow up report number 4. disease information 18(50). http://www.oie.int/eng/. accessed 20 september 2010. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (11), pp. 001-009, november, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper characterisation of enterococci and escherichia coli isolated from commercial and communal pigs from mafikeng in the north-west province, south africa moneoang, modisane simon1* and bezuidenhout, cornelius carlos2 1 department of animal health, north-west university (mafikeng campus), private bag x2046, mmabatho, 2735, south africa. 2 school of environmental science and development, north-west university (potchefstroom campus), private bag x6001, potchefstroom, 2520, south africa. accepted 13 may, 2016 the aim of this study was to isolate enterococci and escherichia coli from faeces collected from commercial and communal pigs, and to characterise these isolates using antibiotic susceptibility profiles. enterococcus selective agar and eosin methylene blue lactose agar were used for enterococci and e. coli isolation, respectively. gram staining, api 20 strep and api 20e were used for identification of enterococci and e. coli, respectively. three-hundred-and-four enterococci and 208 e. coli were identified. the most prevalent enterococci species were enterococcus faecium (58%) and enterococcus gallinarum (23%). a large proportion of enterococci (62.5% to 100%) and e. coli (88.5 to 100%) were resistant to erythromycin, oxytetracycline and sulphamethoxazole. no vancomycin-resistant enterococci were found and pcr analysis for vana, vanb and vanc-1 were all negative. less than 7% of enterococci were resistant to ampicillin and amoxicillin, whereas 45% of e. coli isolates were resistant to the same antibiotics. antibiotic susceptibility tests and clustering patterns showed some similarities among these isolates. from the results, a common origin of the isolates or histories of antibiotic use among these farms was proposed. it could also be concluded that vancomycin-resistant enterococci were not present in pigs on these two farms. key words: vancomycin resistant enterococci, vancomycin susceptible enterococci, tetracycline, resistance genes, multiple antibiotic resistance. introduction enterococci form part of the normal flora of the intestinal tract of animals and humans (blondeau and vaughan, 2000; klein, 2003). among all the enterococci, enterococcus faecium and e. faecalis are the most prevalent species (takeuchi et al., 2005), and significant cause of nosocomial infections (blondeau and vaughan, 2000). escherichia coli also form part of the normal microflora of the gut of humans and animals (naylor et al., 2005). pathogenic e. coli strains have been recognised since the early 1900s, causing disease in both humans (naylor et and animals (blondeau and vaughan, 2000).multiple *corresponding author. e-mail: msmoneoang@nwpg.gov.za. tel.: +2718 299 6800.fax: +2718 293 3925. antibiotic resistance (mar) is common among various microorganisms (guan et al., 2002), and is nor-mally associated with the presence of antibiotic resistan-ce genes (maynard et al., 2003; bryan et al., 2004). antibiotic susceptibility tests are used to generate mar patterns (rota et al., 1996) and mar indices (kaspar et al., 1990; guan et al., 2002). these tests reflect the extent of selective pressures on the microbial floras of the gastrointestinal tract of humans and animals imposed by excessive antibiotic use (guan et al., 2002) . this technique could thus be used for classification and discrimination of isolates of the same or different location (kaspar et al., 1990; guan et al., 2002). enterococci have the ability to acquire and transfer resistance genetic markers to and from other bacterial species such as staphylococcus aureus (gambaratto et al., 2000). in the recent past, vancomycin has been used as the last resort to treat enterococcal infections (gambaratto et al., 2000). vancomycinresistant enterococci (vre) are now endemic worldwide. it was firstly reported in europe in 1986 and two years later in the united state of america (perl, 1999). furthermore, problems of vre have also become a reality in south africa (derby et al., 1998; von gottberg et al., 2000). according to blondeau and vaughan (2000), resistance to vancomycin may occur due to mutations, transposons and resistance genes. moreover, the extensive use of the growth promoter avoparcin in most of the european countries was also correlated to the high incidence of vre (wegener et al., 1999). in the united states, vres were thought to occur primarily by the unskilful application of vancomycin in hospitals (lemcke and bulte, 2000). in most cases, resistance to vancomycin is associated with the activity of the vana gene, which confers high levels of vancomycin and teicoplanin resistance (fluit et al., 2001). on the other hand, vanb confers moderate levels of vancomycin resistance and susceptibility to teicoplanin in vitro (fluit et al., 2001). another vancomycin resistance determinant is vanc. the genes encoding for this are intrinsic in enterococcus gallinarum (vanc1), e. casseliflavus (vanc2) and e. flavescens (vanc -3) (fluit et al., 2001). in the clinical environment, it may be imperative to determine the presence of these genes in order to establish the appropriate therapeutic and control measures (hanaki et al., 2004). studies have shown that pcr-based techniques are efficient and reliable for the surveillance of vres (bell et al., 1998; fluit et al., 2001). the goal of this study was to isolate and characterise enterococci and e. coli from a commercial and a communal pig farm in the north-west province (rsa). the purpose was to determine the antibiotic resistance patterns of these isolates, and to screen for the presence of vre or genes responsible for vancomycin resistance among the enterococci isolates. materials and methods sampling and methodology two hundred faecal samples were collected from mareetsane commercial pigs and the same number collected from tlapeng communal pigs. all the samples were collected using sterile gloves directly from the rectum. faeces (1 g) was inoculated into nutrient broth (5 ml) and incubated at 37°c for 24 h. these samples were sub-cultured on enterococcus selective agar and eosin methylene blue lactose agar (merck, rsa) for enterococci and e. coli isolation, respectively. the plates were incubated at 37°c for 16 24 h. colo nies showing typical characteristics of entero-cocci (black) and e. coli (metallic sheen) were selected for further analysis. gram staining was performed, then api 20 strep and api 20e (biomêrieux, france) to identify enterococi and e. coli, respect-tively. characterisation of isolates by mar phenotypes and mar indices the antibiotic susceptibility test was conducted by the disc diffusion method in mueller-hinton agar with and without 5% sheep blood for enterococci and e. coli , respectively (kirby-bauer, 1966) . the anti-biotics used are listed in table 2 and abbreviations of antibiotics were according to the manufacturer (mast diagnostics, u.k.). multi-ple antibiotic resistance phenotypes were determined for all the iso-lates that were resistant to three and more antibiotics (rota et al., 1996). the mar indices of isolates of a particular sampling station were also generated (kaspar et al., 1990). dna extraction and pcr genomic dna was extracted from selected mar enterococci, e. coli and vancomycin-susceptible enterococci using a modified ctab-pvp dna extraction method (doyle and doyle, 1990). briefly, this included incubation in ctab-pvp extraction buffer, followed by chloroform:isoamyl alcohol (24:1) extraction and precipitation of dna using an ethanol and sodium chloride step. the extracted dna was resuspended in te buffer (10 mm tris, 1 mm edta [ph 8.0]). the 16s rrna primer pair (table 1) was used to amplify 16s rrna gene fragment (positive control). the same dna was then used for screening of vancomycin resistance gene fragments using primer pairs as indicated in table 1. primers were synthesised by inqaba biotech (pretoria, rsa). all the amplifications were performed in a ptc-200 dna engine tm system (bio-rad, uk) . the pcr mix (25 µl) for amplification of 16s rrna consisted of 100 ng of dna containing 2x pcr master mix, 25 mm mgcl2, 16s primer (50 pmole), rnasednase free water (fermentas, us) with the add-ition of 1 u taq polymerase (promega, usa). the pcr conditions consisted of an initial denaturing period of 5 min at 95°c, followed by 35 cycles of the following: 95°c for 30 s (denaturing), 55°c for 30 s (annealing) and 72°c for 60 s (elongation), with a final elonga-tion step of 72°c for 5 min. for vancomycin resistance gene amplification, each pcr cycle consisted of an initial denaturing period of 2 min at 94°c, followed by 35 cycles of the following: 94°c for 60 s (denaturing), 62°c for 60 s (annealing) and 72°c for 60 s (elongation), with a final elongation step of 72°c for 10 min (bell et al., 1998). another set of pcr reaction conditions were also tested and consisted of 35 cycles at 94°c for 60 s (denaturing), 50°c for 60 s (annealing), 72°c for 60 s (elongation) and a final elongation step at 72°c for 10 min (donabedian et al., 2000). pcr products were resolved by gel electrophoresis on 1% (w/v) agarose that contained ethidium bromide (0.001 µg/µl). the electrophoresis buffer was 1x tae (40 mm tris, 20 mm acetic acid, 1 mm edta). electrophoresis was performed at 60 v for 60 min. a gene genius bioimaging system (syngene, synoptics, uk) was used to capture the image using genesnap (version 6.00.22) software. statistical analysis the complete antibiotic inhibition zone data set of both enterococci and e. coli were separately analysed using minitab release (version 13.31, france). these data sets were subjected to pearson’s correlation to determine the relationship of all the enterococci and e. coli isolated from the two farms. the correlation was considered significant at p<0.01. furthermore, a pooled data set of the total antibiotic inhibition zone diameters for e. faecium and e. coli were exported to statistica (version 7; statsoft software, us). the data were analysed by multivariate exploratory techniques using the ward’s clustering method and euclidean distances. the resultant dendrogram indicated clustering patterns and relationships (berge et al., 2003). table 1. oligonucleotide primers and their sequences used in this study. primer gene fragment sequence reference vanaf 5 gta ggc tgc gat att caa agc 3 vanar vana 5 cga ttc aat tgc gta gtc caa 3 bell et al., 1998 vanbf 5 gta ggc tgc gat att caa agc 3 vanbr vanb 5 gcc gac aat caa atc atc ctc 3 vanc1f 5 tgg tat tgg tat caa gga aac c 3 vanc1r vanc-1 5 aga ttg gag cgc tgt ttt gtc 3 gm5f 5 tac ggg agg cag cag 3 muyzer et al., 1995 907 r 16s 5 ccg tca att cct ttg agt tt 3 table 2. the numbers and percentages of various enterococci species and e. coli isolated from mareetsane commercial pigs and tlapeng communal pigs that were resistant to different antibiotics. mareetsane commercial pigs microorganis ap a e ot smx kf ne gm va c m 10µg 10µg 15µg 30µg 200u 30µg 30µg 30µg 30µg 30µg e. faecium nr 3 1 64 86 86 74 58 75 0 nt = 86 % 3.5 1.2 74.4 100 100 86.0 67.4 87.2 0 e. gallinarum nr 2 1 24 30 30 21 19 27 0 nt = 31 % 6.5 3.2 77.4 96.8 96.8 67.7 61.3 87.1 0 e. durans nr 0 0 2 8 8 0 2 5 0 nt = 8 % 0 0 25 100 100 0 25 62.5 0 e. avium nr 0 0 4 6 6 1 2 4 0 nt = 6 % 0 0 66.7 100 100 16.7 33.3 66.7 0 e. coli nr 29 31 95 99 92 23 33 8 18 nt = 104 % 27.9 29.8 91.3 95.2 88.5 22.1 31.7 7.7 17.3 tlapeng communal pigs e. faecium nr 1 0 45 57 64 43 46 44 0 nt = 64 % 1.6 0 70.3 89.1 100 67.2 71.9 68.8 0 e. coli nr 8 8 46 46 46 4 0 0 2 nt = 46 % 17.4 17.4 100 100 100 8.7 0 0 3 nt (number tested), nr (number resistant), a (amoxicillin), ap (ampicillin), gm (gentamycin), ot (oxytetracycline), ne (neomycin), smx (sulphamethoxazole), kf (cephalothin), e (erythromycin), va (vancomycin), c (chloramphenicol). results bacterial species identification three-hundred-andfour (76%) enterococci and 208 (52%) e. coli were positively identified from 400 faecal samples. during this study, e. faecium was the dominant species isolated and constituted 48.5% (tlapeng) and 58% (mareetsane) of the total enterococci population. the level of e. gallinarum was 9% among the tlapeng isolates and 23% among the mareetsane isolates. the other enterococci species contributed between 0.5 and 4% to the total enterococci population from both pig farms and the rest could not be identified to species level. antibiotic susceptibility tests the activities of nine different antibiotics were tested on both enterococci and e coli, and the results are represented in table 2. a large proportion of enterococci isolates (62.5 100%) showed resistance to oxytetracycline, sulphamethoxazole, gentamycin and erythromycin. among the enterococci, 70.3 to 100% of e. faecium isolated from both farms were resistant to erythromycin, oxytetracycline and sulphamethoxazole. a considerable percentage of these e. faecium isolates were resistant to aminoglycosides (67.4 87.2%) and cephalothin (67.2 86.0%). less than 7% of enterococci isolates were resistant to lactam antibiotics (ampicillin and amoxicillin). all the tested enterococci from mareetsane and tlapeng pigs were susceptible to vancomycin. to establish whether the genes responsible for vancomycin resistance (particularly low levels) might be present in the enterococci populations from mareetsane and tlapeng pig farms, pcr assays were employed. the genes targeted included vana, vanb and vanc-1 . all the identified e. faecium were screened for presence of vana and vanb genes, and 50% (8 from 16) of e. gallinarum from both pig farms were screened for presence of vanc1 gene. none of the enterococci species tested harboured either the vana, vanb and/or vanc -1 genes (results not shown). however, only an image of the positive control (16s rrna amplicons) was depicted in figure 3. susceptibility profiles for e. coli showed that the majority (88.5 100%) was resistant to erythromycin, oxytetracycline and sulphamethoxazole (table 2). similar results were observed in enterococci isolates (table 2). levels of -lactam (ampicillin, amoxycillin and cephalothin)-resis-tant e. coli isolated from faeces of pigs in mareetsane and tlapeng ranged from 17.4 to 29.8% (table 2). mar phenotypes and mar indices during this study, 150 e. faecium (86 and 64) and 122 e. coli (76 and 46) isolated from mareetsane commercial and tlapeng communal pig farms, respectively, were characterised for the mar phenotypes and mar indices. ten dominant mar phenotypes were observed from the 150 e. faecium tested. the predominant mar phenotype was e-ot-smx-kf-ne which occurred in 36% of the mareetsane isolates, and eot-smx-kf in 43.8% of the tlapeng isolates. thus, the majority of e. faecium isolates from both farms were resistant to e-ot-smx -kf. the mar indices were almost similar for mareetsane and tlapeng e. faecium isolates (0.5503 and 0.5121, respectively). six dominant mar phenotypes were found among 122 e. coli isolated from mareetsane and tlapeng pigs. the predominant mar phenotype was e-ot-smx, occurring in both mareetsane (36.8%) and tlapeng (65.2%) isolates. the mar indices for mareetsane and tlapeng e. coli isolates were 0.4369 and 0.3864, respectively. clustering analysis a total of 92 e. faecium isolates, as well as 92 e. coli were selected from both mareetsane and tlapeng pigs (46 per sampling station) in order to generate a dendrogram. among all 46 isolates selected, 23 were from the dominant mar phenotypes. the other 23 isolates were from the least dominant mar phenotypes. this selection criterion was decided upon to maximise the potential for determining similarities and differences between the two pig farms. the results of the clustering analysis for both e. faecium (92) and e. coli (92) are presented in figures 1 3, respectively. figure 1 indicates 5 major clusters from the 92 e. faecium isolated from mareetsane and tlapeng pigs. cluster i and iii were mostly represented by mareetsane isolates (78.3 and 57.1%, respectively). clusters iv and v were represented mostly by tlapeng isolates (78.9 and 76.9%). however, cluster ii was formed by equal proportions (50%) of e. faecium from both farms, suggesting a close relationship of these isolates. four major clusters were observed from e. coli isolated from both farms (figure 2). briefly, cluster i was mainly made up of mareetsane isolates (93.8%), cluster ii and cluster iv of tlapeng isolates (73.1 and 63.2%, respectively). in cluster iii a mixture of mareetsane and tlapeng isolates (54.8 and 45.2%), respectively, were found. discussion during this study enterococci and e. coli were isolated from faeces collected from both commercial and communal pigs. however, the numbers of these isolates were lower than the number of samples collected, since apis could not confirm the identities of some of the other putative isolates. results such as these are not uncommon and have also been reported previously (hayes et al., 2003). e. faecium was the most dominant enterococcal species identified in this study. the observation of this study is in agreement with klein (2003) in which e. faecium was the most frequent species isolated from pigs. high levels of e. faecium were also observed among the 981 retailed raw meat (chicken, turkey, pork and beef) samples randomly obtained from 263 grocery stores in iowa. among all these meat samples, 61% of the isolates were e. faecium, 29% e. faecalis and 0.7% e. gallinarum (hayes et al., 2003). a large proportion of enterococci were resistant to multiple antibiotics, with resistance mostly shown to erythromycin, oxytetracycline and sulphamethoxazole. these results suggest a possibility of high use or exposure of these antibiotics within these pig farms. less than 7% of all the enterococci isolates were resistant to -lactams (ampicillin and amoxicillin), indicating that these products could still be useful for enterococcal infection, particularly on the farms sampled. during this study, large proportions of enterococci were resistant to the macrolide antibiotic (erythromcin). however, all of these isolates were susceptible to glycopeptide (vancomycin). in most of the european countries, the prevalence of erythromycinand vancomycin-resistant bacterial species is mostly associated with the use of growth promoters such as the macrolide, tylosin (boerlin et al., 2001) and the glycopeptide, avoparcin (wegener et al., 1999). additionally, studies have shown that cross tree diagram for 92 cases ward`s method euclidean distances 9mcc 11mcca 51mcca 11tcn 22mcca 2mcca 35tcn 89mcc 5mcc 30mcc 3mcc 87mcc 24mcc 26tcn 24tcn 7mcc 64tcn 15tcn 36mcc 4tcn 33tcn 98mcca 48mcca 33mcca 33mcc 13tcn 8mcca 38mcc 1tcn 2tcn 40tcn 12tcn 61mcca 47tcn 32mcca 46tcn 10tcn 88mcc 44mcc 43mcc 2mcc 84mcc 10mcc 79mcc 6mcca 15mcc 42mcc 45mcc 8tcn 6mcc 7tcn 59mcca 63mcca 41mcc 83mcc 53tcn 19mcc 18mcc 16tcn 54tcn 84mcca 74mcca 69mcca 21tcn 80mcca 9tcn 25tcn 48tcn 38tcn 34tcn 19tcn 41tcn 6tcn 3tcn 20tcn 45tcn 39tcn 36tcn 28tcn 47mcca 32tcn 38mcca 56tcn 60tcn 27tcn 34mcca 55tcn 75tcn 57tcn 58tcn 61tcn 59tcn cluster i cluster ii cluster iii cluster iv cluster v 0 50 100 150 200 250 300 (dlink/dmax)*100 figure 1. a dedrogram showing clustering patterns for 92 e. faecium isolated from mareetsane and tlapeng pigs. the analysis is based on complete antibiotic inhibition zone diameter data. designation: mcca; mcc (mareetsane isolates), tcn (tlapeng isolates). selection and genetic linkage between erythromycin and vancomycin resistance occurs particularly when tylosin is used as a growth promoter (boerlin et al., 2001; jackson et al., 2004). e. faecium from these pigs were resistant to both erythromycin and vancomycin (boerlin et al., 2001). in our study, a large proportion of enterococci isolates from both farms were resistant to erythromycin. this is a cause for concern. even so, all the tested enterococci tree diagram for 92 cases ward`s method euclidean distances 9mcca 7mccb 76mccb 66mccb 11mcc b cluster i 18mccb 79mccb 36tcn 77mccb 57mcc b 88mcca 6mccb 15mcc b 56mccb 23mccb 59mccb 92mccb 61mccb 49mccb 99tcn 82mccb 42tcn 97tcn 93tcn 81tcn 89tcn 44tcn cluster ii 27mccb 2mccb 19mccb 1tcn2 91tcn gtcn1 ktcn2 ftcn 22tcn 56tcn atcn1 24tcn atcn2 utcn1 xtcn1 43mcca 1mcca 21mccb 100mcca 29mccb 90tcn 98tcn 32mccb 25mccb 22mccb 1tcn 24mcc b mtcn 47tcn cluster iii 28tcn ytcn1 44tcn1 ltcn2 vtcn1 93mccb 11mcc a 12mcca ctcn etcn2 27mcca 15mcca 17mccb 3mccb 10mcca 9tcn 79tcn 84mcca 34mccb 2mcca 28mccb 14mccb 95tcn cluster iv 94tcn 14mcca 21tcn rtcn 12mccb 100tcn 25tcn 96tcn 43tcn 82tcn 6tcn 79tcn2 ttcn1 0 20 40 60 80 100 120 140 160 (dlink/dmax)*100 figure 2. a dendrogram showing clustering patterns for 92 e. coli isolated from mareetsane and tlapeng pigs. the analysis is based on complete antibiotic inhibition zone diameter data. designation: mcca; mccb (mareetsane isolates), tcn, tcn1, tcn2 (tlapeng isolates). from mareetsane and tlapeng pigs were susceptible to vancomycin. the absence of vre is not uncommon. vancomycin susceptible enterococci (vse) were observed from faecal samples collected from pigs in sweden (van den bogaard et al., 2000). such a trend was also observed among enterococci isolated from retailed raw meat products in iowa (hayes et al., 2003). the farmers from both mareetsane commercial and tlapeng communal settings indicated that they do not use any growth promoters (tylosin and/or avoparcin) on figure 3. image of a composite agarose gel showing an amplified 16s rrna gene fragment from enterococci and e. coli. lane 1 (lambda dna-hind iii digest), lanes 2-3 (16s rrna; enterococci), lanes 4-5 (16s rrna; e. coli). their farms. they also indicated that they do not use vancomycin or any glycopeptides antibiotics for therapeutic purposes. thus, the results from this study support their claims. when antibiotic resistance data sets of e. faecium isolated from the two farms were compared, a strong positive correlation (r = 0.992; p < 0.001) was observed between these data sets. this result suggested that pigs from these two farms had a common history of antibiotic exposure. commercial and communal farmers in the north-west province, particularly those from areas around mafikeng, have indicated that they rely on tetracycline for therapeutic treatment of their animals. tetracycline is attractive to these farmers not only because of being a broad spectrum antibiotic, but also because it is a freely available over-the-counter product which is cost-effective. the results of the present study underscore the hypothesis that the continuous use of an antibiotic results in bacterial tolerance to that product. as such, high numbers of e. coli resistant to this antibiotic were isolated. this poses a potential threat to both animals and human health sectors. in a study conducted in canada, e. coli isolated from pigs were resistant to oxytetracycline (maynard et al., 2003; bryan et al., 2004). previous studies showed that a considerable large percentage of e. coli isolated from animals such as chickens, cattle, goats and sheep (bryan et al., 2004), as well as in human isolates (bartoloni et al., 2006) were resistant to tetracycline. a considerable percentage of e. coli isolated from pigs from both farms showed resistance to ß-lactam antibio tics (table 2). maynard et al. (2003) observed that between 15 30% of etec strains isolated from pigs in canada were also resistant to these ß-lactams. resistance to such antibiotics among e. coli isolated from pigs from mareetsane and tlapeng could be due to mutations in the penicillin-binding proteins and/or presence of blatem resistance genes (fluit et al., 2000). the higher percen-tages (29.8%) of these e. coli were resistant to the newer generation ß-lactam antibiotic, cephalothin, and this is a cause for concern. when comparing resistance results of e. coli isolated from mareetsane and tlapeng pig farms, a strong positive correlation (r = 0.992; p < 0.001) exis-ted, once again indicating common antibiotic usage regi-mes on these farms. during this study, a large percentage of enterococci and e. coli showed multiple antibiotic resistances. the most dominant mar phenotype pattern was e-ot-smxkf-ne for mareetsane e. faecium isolates with only the absence of one antibiotic (ne) for tlapeng isolates. the prevalent mar phenotype for e. coli isolated from both pig farms was e-otsmx. during this study, it appeared that e-ot-smx was the basis of all the phenotypes in both e. faecium and e. coli, suggesting a possibility of high use or exposure of these antibiotics in these pig farms. according to kaspar et al. (1990), isolates with similar mar phenotypes may have common origin or similar antibiotic exposure histories. furthermore, the mar phenotypes may sometimes suggest the extent of antimicrobials use in specific food animal production environments (hayes et al., 2003). when comparing the relationship of both e. faecium and e. coli using the complete antibiotic inhibition zone diameter data by the clustering patterns (figures 1 and 2), some close relationship between these isolates from mareetsane and tlapeng existed. a similar situation was shown by these isolates when considering the results of the resistance patterns (table 2), mar phenotypes and mar indices. these results support the deduction that the isolates from both farms may have a common origin. alternatively, these isolates may have had similar histories of antibiotic exposures. conclusion enterococci and e. coli were isolated and characterised in this study. it could be concluded that vres are currently not present in pigs on the farms that were tested. this observation is encouraging, particularly for the marketing of pork from these farms. however, further studies are essential to screen more farms in the northwest province to confirm the absence of vre among pigs in this region. furthermore, a large proportion of enterococci were susceptible to amoxycillin and ampicillin. these results indicate that -lactams could still be used as a synergy with other cell wallactive antibiotics such as vancomycin in the treatment of enterococcal infections in pigs, particularly in the areas sampled in the north-west province (rsa). the observations of this study provide some baseline data for management of antibiotic resistance among pigs in north-west province (rsa). a large proportion of enterococci and e. coli showed mar, and this should be considered a cause for concern for veterinary health authorities. this also suggests the need for changes in the use of antibiotics in both veterinary and human medicines, as some unusual events may affect propagation of antibiotic-resistant bacteria in the food environment and food supply. acknowledgements this work was made possible by a grant from the national research foundation (nrf) and funds from the department of animal health in the north-west university (mafikeng campus) . we also like to thank the farmers (mr. dryer and mr. molosi) for providing samples. our acknowledgements are also extended to the staff members, technicians, and colleagues within the department of animal health, north-west university (mafikeng campus). references bartoloni a, pallecchi l, benedetti m, fernandez c, vallejos y, guzman e, villagran al, mantella a, lucchetti c, bartalesi f, strohmeyer m, bechini a, gamboa h, rodríguez h, falkenberg t, kronvall g, gotuzzo e, paradisi f, rossolini gm (2006). multidrug-resistant commensal escherichia coli in children, peru and bolivia. emerging infectious disease, 12: 907-913. bauer aw, kirby wmm, sherris jc, turck m (1966). antibiotic 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am, bager f (1999). use of antimicrobial growth promoters in food animals and enterococcus faecium resistant to therapeutic antimicrobial drugs in europe. emerging infectious diseases 5: 329-335. african journal of pig farming vol. 1 (2), pp. 023-027, october, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper biodigestion of cassava peels blended with pig dung for methane generation oparaku, n. f., ofomatah, a. c* and okoroigwe, e. c. national center for energy research and development, university of nigeria, nsukka, enugu state, nigeria. accepted 16 september, 2013 biogas production from cassava (manihot esculentus) peels and pig dung under a mesophilic temperature condition was investigated. three blends of the wastes and a control labeled as b1, b2, b3 and c representing blend 1 (50:50 peel/dung), blend 2 (30:70 peel/dung), blend 3 (10:90 peel/dung) and control (pig dung alone) were used, respectively. biodigestion of the wastes blends and control was carried out simultaneously under the same environmental and operational conditions of 30 days retention period using four metallic biodigesters of 32 l capacity each. the biogas yield result shows that blend 2 yielded the highest cumulative biogas of 78.5 l, while the least yield of 61.7 l was obtained by blend 3. when compared with the control set up and biodigestion of cassava waste alone from literature, there was blending effect resulting in increase in yield of biogas over the sole digestion of cassava peel or pig dung. methane production leading to the combustibility of the biogas started at 6 th , 5 th , 5 th and 4 th days for b1, b2, b3 and c, respectively. this, in agreement with earlier studies show that better handling of cassava peels for energy production would be achieved by blending it with animal wastes in the right proportion. key words: cassava peel, biogas, co-digestion, anaerobic digestion, wastes blends, lag days. introduction one of the main environmental problems of today’s society is the continuous increase in production of organic wastes which are harmful to human existence. in many developing countries, sustainable waste management and reduction have become major issue due to lack of adequate technology and methodology to handle wastes generated from daily living activities. some of the waste types which are posing serious environmental threats to human and animal existence in these nations come from agriculture due to their degradable nature and lack of profitable technique to convert these ‘wastes’ to better manure quality or other useful means such as energy. with increase in farm (agricultural) operations, greater waste production is proposed. cassava is one of the major root crops produced in subsaharan africa. world cassava production in 2002 was estimated at 184 million tonnes (odoemenem and otanwa, 2011). as at 2002, africa exported only one ton of cassava annually (fao, 2001) but by 2007, out of “more than 228 million tonnes of cassava produced worldwide, africa accounted for 52% and nigeria produced 46 million tons making it the world's largest producer of cassava” (iita -1). it has been projected that total world cassava utilization would hit 275 million tons by 2020 (ifpri, 2008; arowolo and adaja, 2012) while some researchers estimate this number closer to 291 million tons. currently, there is increase in campaign for enlarging the cassava production scale in nigeria. the implication of this is increased waste production from cassava processing. according to fao (2001), about 250 to 300 kg of cassava peels is produced per tonne of fresh cassava *corresponding author. e-mail: ofomatony@yahoo.co.uk. tel: +2348034010807. oparaku et al. 023 root processed. this suggests huge sum of waste production in form of peels from cassava production and processing. hence, there is need to design and adopt a system capable of handling the huge waste accruing from this development and anticipated problems such as unpleasant odour production. one of the ways by which cassava peels can be managed in addition to using it as animal diet (okeudo and adegbola, 1993) is by anaerobic digestion for methane and bio-fertilizer production. from the literature, it is obvious that attempts have been made to convert cassava peels to energy. adeyanju (2008) demonstrated the effect of adding wood ash to the biodigestion of mixture of piggery wastes and cassava peels in a laboratory scale. it was found that the wood ash addition increased the biogas production of either the biodigestion of piggery wastes and cassava peels only or in combination of both wastes in different proportions. the period it takes to produce methane was not stated probably because it was only a laboratory set up. ofoefule and uzodimma (2009) compared the biogas production potential of cassava peels alone to the blends of the waste with animal wastes. it was found that cassava peels alone produced a total of 68.70 l of biogas which could burn after 58 days of digestion. when blended with cow dung, poultry droppings and pig dung, the volume of biogas produced increased to 146.5, 166.50, and 169.60 l, respectively, while the flammable gas was produced after 9 days in the first 2 and 11 days in the later. these imply that anaerobic digestion of cassava peels alone is by no means economical. however, in this work, the blending with animal waste was on equal ratio. the effect of different mix ratios in biogas production potential and flammability of the biogas is a gap to be filled up by further investigation. ezekoye and ezekoye (2009) combined cassava peels with rice husk in the ratio of 1:5 for biodegradation of the wastes. it is also gathered that this set up was inoculated with cow dung mixed with water. from the result, flammable biogas was produced after 30 days with a total of volume of 3.450 m 3 . it is not certain which of the substrates is responsible for the long retention period of 70 days and large volume of biogas recorded. however, it is obvious that combination of two plant biomass is not a favourable combination in anaerobic digestion since animal protein is important for microbial activity of the methanogens. adelekan and bamgboye (2009) have done a more detailed investigation from the existing works. the biogas productivity of cassava peels mixed in different ratios with fixed amount of animal wastes was investigated. in this work, 1, 2, 3 and 4 parts of cassava peels were mixed with one part each of poultry, piggery and cattle wastes. it was found that there was statistically significant effect of the mix ratios over biogas production. in this work also, it was found that the equal mass combination in all the animal waste types produced the highest biogas by volume when compared with other ratios. the effect of mix ratios on how long it takes to produce flammable biogas was not covered. combination of cassava waste with other biomass species of agricultural origin has continued to attract the interest of scholars. in addition to the above, ilaboya et al. (2010) blended cassava peels with pineapple and plantain peels in a laboratory set up. one of the aims of their work was to monitor the effect of alkaline addition to the substrate in biogas generation potential of the mixture. it was observed that addition of naoh (alkaline) solution resulted in increase in biogas production over no alkaline addition. also, there was positive effect in increase of biogas generation by different ratios of alkaline mixture. however, this work did not also address the effect of mixing the cassava peels in different ratios of other wastes. in order to fill some gaps existing in previous codigestion of cassava peel with other waste types, there is need to study the effect of varying the mix ratio of cassava peels vis-a-vis mix ratio of other wastes. in this work, quantity of cassava peel is varied with varying quantity of piggery dung in a batch anaerobic digestion. it is aimed at determining the mix combination that will yield the largest volume of biogas and the number of days it would take to produce flammable biogas while working with a particular waste. however, varying cassava peels in blends of multiple animal waste types can be investigated in another work. biodigestion of agricultural wastes is important as a way of maximizing the output of agricultural practice of developing nations. multiple products (benefits) can be derived. this includes energy and biofertilizer production. it has been found that biodigestion of agricultural wastes produces better manure than using the wastes as raw manures in farm practice (okoroigwe, 2007; okoroigwe et al., 2008). materials and methods the raw materials (cassava peels and the piggery waste) were obtained within university of nigeria, nsukka in enugu state of nigeria. prior to the biodigestion of the wastes, the cassava peels were partially fermented by soaking in water for 7 days in order to reduce the acid content of cassava peels which could be detrimental to the microbes. cyanide is an inhibitor to microbial activities (cuzin and labat, 1992; cuzin et al., 1992). standard methods were used to determine the proximate (moisture, ash) and chemical analyses of the samples prior to digestion. the cassava peels and the piggery dung were mixed in the ratio of 50% by weight of cassava peels to 50% by weight of piggery dung. this serves as blend 1 (b1). the second and third blends were achieved by 20% reduction and 20% increase in weight of cassava peel and piggery dung, respectively, in preceding blend. hence, b2 and b3 were 30:70 and 10:90 (cassava peels: piggery dung), respectively. a control set up contained only piggery dung mixed with water at equal weight. all the samples were added with equal weight of water to the combined weights of the waste types. this is required to achieve 5 to 10% total solids (ts) concentration. the set up was monitored for daily gas production by measuring the volume of gas using downward displacement method. in order to achieve 024 afr. j. pig farming table 1. proximate composition and physicochemical properties of the various wastes. parameter composition (%) b1 b2 b3 c crude protein 2.89 3.23 3.57 3.74 crude fat 1.17 0.87 0.57 0.42 crude fibre 1.17 0.87 15.05 0.42 ash 18.45 18.79 19.13 19.30 moisture 26.65 21.79 16.93 14.50 carbohydrate 37.69 41.22 44.75 46.52 total solids 63.43 67.86 72.29 74.50 volatile solids 35.00 35.07 35.15 35.19 carbon 22.72 21.22 19.71 18.96 nitrogen 0.46 0.52 0.87 0.60 c:n 49 41 23 32 homogeneity of the slurry and discourage scum formation in the system, daily stirring was carried out using the inbuilt stirring mechanism in the digesters. both slurry and ambient temperatures were measured at morning and afternoon hours. the average of both readings for any day becomes the reported slurry or ambient. results and discussion the physico-chemical parameters of the waste streams are presented in table 1. it shows that these wastes are good bioresources for methane generation. these values are close to values obtained by other researchers. the values however differ slightly because different waste combinations will give different composition of nutrients. it has been shown that biogas yield from ad of wastes depends on a number of factors such as ph, hrt and cn ratio (yadika et al., 2004). cn ratio is an important indicator for controlling biological treatment systems (wang et al., 2012). it has been pointed out that high c/n ratio indicates rapid nitrogen consumption by methanogens and leads to lower gas production while low c/n ratio results in ammonia accumulation and an increase in ph values, which is toxic to methanogenic bacteria (zhang et al., 2013). this is partly reflected in the biogas yield of the blends as b3 with the lowest c/n ratio yield the least gas among the blends and the b1 with largest c/n ratio yields moderate biogas as compared to b2 according to the explanation above but yadika et al. (2004), pointed out that during anaerobic digestion, microorganisms utilize carbon 25 to 30 times faster than nitrogen. thus, to meet this requirement, microbes need a 20 to 30:1 ratio of c to n. this may justify the values presented in table 1 as good for biogas generation. the ph of the blend substrates and the control are presented in figure 1. the result shows that the range of the ph values was 5 to 7.3 from the 3 rd day to the 30 th day. it shows that the b1 and c had initial acidic condition. this agrees with zhang et al. (2013) explanation on relationship between high c/n ratio and toxicity of the reacting medium. similarly, the ph values of other blends were responsible for the high biogas yield experienced by b2 and b3. figure 1 also shows that the reactions in all the blends and control have initial acid condition which neutralized as the reactions progressed to the 30 th day. table 2 presents the cumulative biogas yield of the different blends and the control, while figure 1 shows daily biogas production pattern of the waste blends and the control. from the result, blend 2 (b2) yielded the largest quantity of biogas (78.5l) followed by b1 (73.5l) while the least yield of 61.7l was obtained from the 3 rd blend (b3). when compared with the control set up of biodigestion of piggery dung alone, there was blending effect leading to the increase in biogas yield. this shows that anaerobic digestion of piggery waste can be enhanced by combination with other wastes such as plant biomass. the result is different if combustibility of the generated gas is of interest. biogas is not useful if it cannot be burnt in a combustor for energy and power production. even though, the lag day (period to produce flammable gas) is shortest in the control experiment, it is necessary to blend it with other wastes as there is not much difference in the lag day of b2 which produced the largest volume of biogas. the low volume of biogas in biodigestion of pig dung alone is due to high concentrations of nh4-n which inhibits the process of degradation of organic matter, causing a decrease in volume of biogas produced. when compared with literature value of anaerobic digestion (ad) of cassava peels only, the least blending yield of 61.7 l (b3) obtained in this work was close to 68.70 l obtained by ofoefule and uzodimma (2009). the higher yield however, could be because of larger volume of biodigester and pretreatment used by them. but it has been pointed out that cassava peels alone is a poor feedstock for ad due to its high acid content (cuzin and labat, 1992; cuzin et al., 1992). the results obtained here conform with other reports from other scholars (adeyanju, 2008; adelekan and bamgboye, 2009) showing blending effect. this result has shown that blending the cassava peel with animal wastes should be better done at co-variation of both waste types to obtain an optimum mix ratio that will yield larger volume of gas. the effect of microbial load (table 3) can be observed in the ph stabilization (figure 2) as the reactions progressed from 6 th to 30 th day. the multiplication of microbes in the system from 6 th to 21 st day is responsible for acid neutralization as there was progressive consumption of nutrients in the substrates. this was also responsible for the increase in the biogas yield in all the blends as microbial count increased. the peak production of biogas (figure 1) is observed around the 21st day in blend 2 due to peak microbial count about that oparaku et al. 025 8 7 6 ga s (l )o fg as (l ) 5 b1 4 b2 vo l o fv ol um e b3 3 c 2 1 0 0 5 10 15 20 25 30 35 40 time (days) figure 1. daily biogas production pattern in the wastes. table 2. cumulative biogas yield pattern of the wastes. parameter value b1 b2 b3 c lag days 6 5 5 4 cum biogas yield (l/gs) 73.50 78.5 61.7 55.1 mean biogas yield 2.45 2.62 2.05 1.83 table 3. microbial load of the different waste blends. day microbial count (x10 4 ) b1 b2 b3 c 1 2.0 2.2 1.9 1.9 6 2.2 2.3 2.1 2.0 11 2.3 2.5 2.3 2.2 16 3.8 3.9 3.2 3.0 21 2.0 22.0 1.3 12.0 26 3.2 3.3 2.2 2.0 30 2.5 1.7 1.9 1.9 time (table 3). the microbial load began to reduce from the 21 st day until the 30 th day due to extensive consumption of nutrients in all the blends as well as in the control. figure 3 shows the temperature distribution pattern in both waste slurries and the ambient. the temperature values were with 25 to 35°c in all cases within the 30 day test period. this is a mesophilic temperature range which enabled the micro-organisms to thrive favourably in the system for maximum performance. it therefore means that the volumetric result of biogas production is at its utmost yield condition. 8 7 6 5 sc a le b1 4 b2 p h b3 c 3 2 1 0 0 5 10 15 20 25 30 35 time (days) figure 2. ph variation in the wastes. 40 35 30 te m pt (o c )t em pe ra ur e° c ) 25 b1 b2 20 b3 c 15 ambient 10 5 0 0 5 10 15 20 25 30 35 40 time (days) figure 3. average slurry and ambient temperature variation. 026 afr. j. pig farming oparaku et al. 027 conclusion in this work, co-biodigestion of cassava peels with piggery waste was carried out in varying quantities of both wastes; there was blending effect over single biodigestion either of the plant and animal wastes. the b2 blend showing the combination of 30% by weight of plant waste (cassava peels) with 70% by weight of animal waste (piggery dung), yielded the largest volume of biogas on cumulative basis. flammable biogas (methane rich biogas) was produced on the 5 th day even though biodigestion of piggery waste alone produced flammable biogas on the 4 th day. the blending improved the methane production of cassava peels alone from about 59 days (literature value) to five days in combination with piggery dung. references adeyanju aa (2008). effect of seeding of wood-ash on biogas production using pig waste and cassava peels. j. eng. appl. sci. 3(3):242-245. arowolo aa, ji adaja (2012). investigation of manihot esculenta and manihot duleis production in nigeria: an econometric approach. j. appl. sci. environ. 3:18-24. cuzin n, farinet jl, segretain c, labat m (1992). methanogenic fermentation of cassava peel using a pilot plug flow digester. bioresour. technol. 41:259-264. cuzin n, labat m (1992). reduction of cyanide levels during anaerobic digestion of cassava. int. j. food sci. technol. 27:329336. ezekoye va, ezekoye ba (2009). characterization and storage of biogasproduced from the anaerobic digestion of cowdung, spent grains/cow dung, and cassavapeels/rice husk. pac. j. sci. technol. 10(2):898-904. fao (2001). proceedings of the validation forum on the global cassava development strategy. food and agriculture organization of the united nations international fund for agricultural development rome, 2001. p. 65. ifpri (2008). reaching sustainable food security for all by 2020. getting the priorities and responsibilities right. international food policy research institute, washington, d.c: ifpri. in aeowolo and adaja, 2012 iita -1 (http://www.iita.org/cassava, assessed 3/9/2013). ilaboya ir, asekhame ff, ezugwu mo, erameh aa, omofuma fe (2010). studies on biogas generation from agricultural waste; analysis of the effects of alkaline on gas generation. world appl. sci. j. 9(5):537-545. ofoefule au, uzodinma eo (2009). biogas production from blends of cassava (manihot utilissima) peels with some animal wastes. int. j. phys. sci. 4(7):398-402. okeudo nj, adegbola aa (1993). utilisation of dried caged-hen manure and cassava peels for intensive sheep production. trop. anim. health prod. 25(4):234-238 okoroigwe ec (2007). application of biomass technology in sustainable agriculture. trends appl. sci. res. 2(6):549-553. okoroigwe ec, eze ji, oparaku ou (2008). field comparison of biofertilizer and chemical fertilizer application on maize production. niger. j. solar energy. 19(1):55-57. wang x, yang g, feng y, ren g, han x (2012) optimizing feeding composition and carbon-nitrogen ratios for improved methane yield during anaerobic co-digestion of dairy, chicken manure and wheat straw. bioresour. technol. 120:78-83 yadvika, santosh, sreekrishnan tr, kohli s, rana v (2004). enhancement of biogas production from solid substrates using different techniques -a review. bioresour. technol. 95:(1)1-10. zhang t, liu l, song z, ren g, feng y, han x , yang g (2013). biogas production by co-digestion of goat manure with three crop residues. plos one 8(6): e66845. doi:10.1371/journal.pone.0066845. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (9), pp. 001-008, september, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper haematological and serum biochemical changes in pigs administered with ascorbic acid and transported by road for four hours during the harmattan season a.y. adenkola1*, j.o. ayo2, a.k.b. sackey3 and a. b. adelaiye4 1 department of physiology and pharmacology, college of veterinary medicine, university of agriculture, makurdi, nigeria. 2 department of physiology and pharmacology, faculty of veterinary medicine, ahmadu bello university, zaria, nigeria. 3 department of surgery and medicine, faculty of veterinary medicine, ahmadu bello university zaria, nigeria. 4 department of human physiology, faculty of medicine, ahmadu bello university, zaria, nigeria. accepted 31 december, 2015 this study was conducted with the aim of investigating the effect of ascorbic acid (aa) on haematology and serum biochemical responses of pigs transported for short journey (4 h) during the harmattan season. sixteen pigs administered with aa at a dose of 250 mg/kg per os and individually served as experimental animals, and 13 others each administered orally with sterile water served as control animals. the animals were then transported for 4-h at a speed of 4050 km/h covering a distance of 140 km. blood samples were taken early in the morning a day before transportation, immediately after and a week after transportation. the leucocyte count (15830.25 ± 1063.08 × 10 3 /µl) dropped (p < 0.05) in the experimental pigs after transportation and the value was significantly (p < 0.05) lower than the value of 22010.69 ± 1722.00 × 10 3 /µl obtained in the control pigs after the journey. the neutrophils: lymphocyte ratio obtained in the control animals increased from 0.61 ± 0.04 before transportation to 0.79 ± 0.17 immediately after transportation. there was a significant (p < 0.05) increase in total protein post– transportation in all experimental and control groups, but the increase was higher in the control than the experimental group. alkaline phosphatase and aspartate amino transferase values in the control pigs increased immediately after transportation. this study indicates for the first time the beneficial effect of aa administration on haematology of pigs transported by road during the harmattan season for short journey of 4 h. it is, therefore, recommended that pigs be administered with aa before transportation by road during the harmattan season in order to reduce the risk of adverse effects of transportation stress on health. key words: ascorbic acid, harmattan season, haematological and biochemical parameters, pigs, road transportation. introduction road transportation is a critical phase in animal production and utilization (odore et al., 2004; buckham et al., 2008a). the intensity and specialization of livestock production and the demand for livestock to be marketed and slaughtered outside places where they are being produced have necessitated animal transport all over the world (ayo and oladele, 1996; kannan et al., 2000; *corresponding author. e-mail: aadenkola@yahoo.com. tel: +2348054977696. perez et al., 2001) and it is often considered as one of the main causes of stress (giovagnoli et al., 2002; pineiro et al., 2007; adenkola et al., 2008; buckham et al., 2008b), adversely affecting production both in econo-mic and animal welfare terms (mormede et al., 1982). physical and psychic exertions occurring during transport of food animals disrupt their homeostasis and metabolism, and as a result of the exertion, road transport stress increases activity of enzymes and hormones (ayo and oladele, 1996; mstl and palme, 2002; buckham et al., 2008a). it has been shown that all stages of transporta tion to which pigs are subjected before slaughter or marketing constitute stress factors. they include loading, transport, unloading, regrouping of pigs and lairage. while pigs are undergoing these stages, they are simultaneously subjected to changes in their internal environmental conditions (ayo et al., 1996; santoro and faucitano, 1996). many pigs are transported in nigeria using vehicle not designed for swine transportation and often in the harmattan and hot-dry season. these conditions adversely affect the health or welfare of the animals and impair their homeostatic mechanisms (mstl and palme, 2002; buckham et al., 2008a) resulting in body dysfunctions which may be fatal. environmental stress causes oxidative stress and impairs antioxidants status in vivo (sahin et al., 2001). antioxidant supplementation, therefore, has been shown to be beneficial in attenuating the adverse effect of environmental stress (kafri and cherry, 1984) and stressinduced tissue damage (sen, 2001; minka et al., 2007a). adenkola and anugwa (2007) showed that ascorbic acid (aa) or vitamin c supplementation improved weight gain and better feed utilization in piglets and it is a naturally occurring antioxidant (sahin et al., 2001) and currently is the most widely used vitamin supplement throughout the world (naidu, 2003). haematological parameters are good indicators of the physiological status of animals (hawkey and dennett, 1989; adenkola and durotoye, 2004). it is also an excellent medium for the measurement of potential biomakers, because its collection is relatively noninvasive and it encompasses an enormous range of physiological process in the body at any given time (anderson and anderson, 2002; ginsburg and haga, 2006). currently in nigeria, there is paucity of information on the haematology and serum chemistry of pigs transported by road, especially during the harmattan season. the harmattan season occurs in the zone between late november and early march, and it is characterized by high ambient temperature (at) in the afternoon hours of the day and relatively at of about 10 o c in the evening and early morning hours of the day and the season is characterized by cold-dry and dust laden wind (igono and aliu, 1982).this zone is characterized by intensive livestock marketing and consequently, transportation. the aim of the present paper was to investigate the impact of road transportation during the harmattan season on haematology and serum chemistry of pigs administered ascorbic acid. materials and methods experimental site the experiment was performed at the livestock pen, faculty of veterinary medicine, ahmadu bello university, samaru-zaria (11 0 10 n, 07 0 38 e), located in the northern guinea savannah zone of nigeria during the harmattan season. experimental animals and management twenty nine local pigs, including males and non-pregnant, nonnursing females, weighing 20-48 kg, and ages ranging from 912 months were bought from different localities in zaria environs at least two weeks before the experimental day. they were kept at a stocking density of 0.8 m 2 per animals in a communal pen, made of concrete floor and iron walls with asbestos roofing. the pen measured 7.50 x 2.55 m with half the length of the wall to the roof without block work, which provided adequate ventilation. the pigs were not restrained inside the pen. they were kept under an intensive system of management and fed with maize offal, brewer’s waste and yam peel. they were given access to water ad libitum the pigs were pre-conditioned for two weeks before the commencement of the experiment. during the period, they were screened for haemoparasites and endoparasites by taking their blood and faecal samples for laboratory analyses. they were treated accordingly using oxytetracycline (kepro b. v ® , hollland) at the dose of 20 mg/kg by deep intramuscular route and thiabendazole (agvet ® , u.s.a.) at the dose of 25 mg/kg body weight per os. experimental design on the day of transportation, the experimental pigs (n = 16) were orally and individually administered with aa at 250 mg/kg (chervyakov et al., 1977) dissolved in 20 ml of water, while 13 pigs which served as control were given 20 ml of sterile water. the administrations were made immediately (15 min.) before loading the pigs into the vehicle. food and water were withdrawn 12 h before the journey and throughout the journey period, which lasted 4 h. the vehicle traveled along zaria-jos road at a speed of 40-50 km/h from faculty of veterinary medicine, ahmadu bello university, zaria (11 0 10 n, 07 0 38 e), on tarred smooth and rough road covering a total distance of 140 and back to the starting point. after completing the journey, the pigs were unloaded at the spot where they were original loaded. the animals were fed and watered as they had been prior to the journey. vehicle design, loading, and journey time a standard ford six wheeler bus, popularly used in northern nigeria in transportation of livestock was used to transport the pigs. the vehicle engine was in good condition, serviced and made ready for the journey. the floor of the vehicle was non-slippery and was covered with dry beddings before loading the animals. the inner compartment of the vehicle measured 3.22 x 1.67 x 1.2 m high. the sidewalls of the vehicle from the floor to the roof were made smooth with no protrusion of sharp edge and with a window, which provided adequate ventilation. each window measured 0.8 x 0.44 m on both sides of the vehicle, was at the height of about 1.0 m from the floor. a door which measured 1.4 x 1.2 m was provided at the rear end of the vehicle. other transportation procedures were carried out in accordance with the standard guidelines governing the welfare of pigs during road transportation (warris, 1998; lambooij, 2000) . the pigs were stocked at the density of 0.8 m 2 per animal. they were made to stand inside the vehicle in rows facing direction against the direction of the vehicle movement. the journey commenced at 8:00 am. blood sample collections blood samples were taken early in the morning a day before transportation, immediately after and a week post-transportation. ten millimeters of blood was taken aseptically from the anterior vena cava using a 10 ml syringe and 18 gauge x 1 1 /2 inch sterile needles from each animal. each blood sample for determination (4 ml) of haematological parameters was immediately poured inside a sample bottle, containing an anticoagulant, disodium salt of ethy-lene diaminetetraacetic acid (edta) at the rate of 2 mg/ml of blood (oyewale, 1992). after collection, the samples were transferred to clinical pathology laboratory, faculty of veterinary medicine, ahmadu bello university, zaria, where they were analysed for pack-ed cell volume (pcv) using microhaematocrit method, total leuco-cyte count using haemocytometer method as described by schalm et al. (1975). haemoglobin concentration and differential leucocyte count were also determined as described by schalm et al. (1975). the remaining (6 ml) meant for serum chemistry and serum elec-trolyte determination was centrifuged and the serum harvested. alkaline phosphatase was determined as described by chees-brough (1991), aspartate aminotransferase (ast) enzyme activity level was determined using the reitman-frankel ast method described by cheesbrough (1991), alanine amino transferase, total bilirubin, urea and total cholesterol was done as described by cheesbrough (1991), total protein by biuret method, serum albumin by bromocresol green method as described by cheesbrough (1991), serum globulin was determined by subtracting serum albu-min from total protein value. serum sodium and potassium ions was determined by flame emission spectrometry as described by cheesbrough (1991), while serum bicarbonate was done using titrimetric method as described by cheesbrough (1991) and chloride level was analylsed by the method of schales and schales (1941). statistical analysis the data obtained were analysed using graph pad prism package version 4.00 of 2003. data were subjected to student’s t-test and values of p < 0.05 were considered significant. results haematological parameters before, immediately after and 7 days posttransportation the values of haematological parameters obtained before the journey in experimental and control pigs were not significantly different (p > 0.05) . pcv values recorded on day 7 after the journey rose (p < 0.05) to 37.38 ± 1.39% in the experimental pigs, and the value was significantly (p < 0.05) higher than 33.54 ± 1.01% recorded in the control pigs post-transportation. the total leucocyte count dropped (p < 0.05) from 18920.00 ± 1200.00 x 10 3 /µl in the experimental pigs before the journey to 15830.00 ± 1063.00 x 10 3 /µl after the journey, and the value was significantly (p < 0.05) lower than 22010.00 ± 1722.00 x 10 3 /µl obtained in the control pigs after the journey. the values recorded on day 7 post-transportation in the experimental and control pigs were not significantly different. the absolute neutrophil count of 5246.93 ± 429.21 x 10 3 /µl obtained in the experimental pigs was significantly (p < 0.05) lower than the value of 8328.15 x 10 3 /µl recorded in control pigs immediately after the journey. although in the control pigs the absolute neutrophil dropped to 6667.07 ± 577.30 x 10 3 /µl on day 7 after the journey, the value was significantly (p < 0.05) higher than the corresponding value of 5058.87 ± 314.44 x 10 3 /µl recorded in the experimental pigs (table 1). the absolute lym phocyte count of 10450.00 ± 757.05 x 10 3 /µl obtained immediately after the journey in experimental pigs was significantly lower (p < 0.05) than 13510.85 ± 1032.04 x 10 3 /µl recorded in control pigs. immediately after the journey the absolute monocyte count rose when compared with pre-transportation values, but the increase in the value was not significantly different in experimental and control pigs (p > 0.05).the value increased to 134.76 ± 43.09 x 10 3 /µl in the control pigs on day 7 after the journey, and was significantly (p < 0.05) higher than the corresponding value of 35.62 ± 16.48 x 10 3 /µl obtained in the experimental pigs (table 1). the neutropils: lymphocyte ratio obtained in the control animals increased (p < 0.05) from pre-transportation value of 0.61± 0.04 to 0.79 ± 0.17 immediately after transportation. serum biochemical parameters before, immediately after and 7-days after four hour road transportation table 2 shows the response to serum biochemical changes in experimental and control pigs. there was no significant difference, in all the parameters between experimental and control pigs, before the journey (p > 0.05). immediately after the journey, the total protein in the experimental pigs did not increase significantly, from 67.94 ± 1.50 gm/dl to 69.69 ± 1.67 gm/dl, while in the con-trol it rose (p < 0.05) to 71.31 ± 1.93 gm/dl from 67.62 ± 1.29 gm/dl (table 2). albumin: globulin ratio of 1.06 ± 0.12 obtained immediately after transportation dropped to 0.87 ± 0.06 (p < 0.05), 7 days post-transportation from 1.11 ± 0.09 obtained before transportation in experimental pigs. the value thus increased from 1.30 ± 0.22 immediately after transportation to 1.34 ± 0.12 in the control pigs on day 7 post-transportation. however there was a significant (p < 0.05) difference in the recorded value of experimental and control pigs. the recorded hco3 value of 22.15 ± 0.75 mmol/l obtained immediately after transportation in the control pigs rose to a value of 24.15 ± 0.80 mmol/l on day 7 post-transportation (p < 0.05) while the corresponding value in the experimental pigs was 21.69 ± 0.84 mmol/l the cl value of 102.70 ± 1.17 mmol/l recorded in experimental pigs immediately after transportation decreased (p < 0.05) to 91.00 ± 1.79 mmol/l on day 7 post-transpor-tation, and this value was lower (p < 0.05) than the cor-responding value of 96.62 ± 1.22 mmol/l obtained in the control pigs. alkaline phosphatase value recorded in the experimenttal pigs dropped slightly from the pre-transportation value of 40.56 ± 2.85 i.u./l to 39.94 ± 2.85 i.u./l, while the value increased in the control pigs from the corresponding value of 40.92 ± 2.92 i.u./l before transportation to 48.23 ± 2.36 i.u./l immediately after transportation (p < 0.05) (table 2). however, the value dropped close to pre-transportation value on day 7 post-transportation in the control pigs. aspartate amino transferase increased significantly in the control pigs from 15.69 ± 1.08 i u./l table 1. haematological parameters of experimental (supplemented with ascorbic acid) and control (non-supplemented with ascorbic acid) pigs before short-term road transportation (mean ± sem). !" !" !" ! " #$$$ %& %# $ ! ' ($ &) * & + ! " #$$$ %& %# $ , % % $ % .'/ %& ) + # * .'/ 0 * #$ ) + # * .'/ ( $% .'/ 1 ) +% .'/ %& ) + # * 2 1 ) +% **values with asterisks are significantly different (p < 0.05). pre-transportation to 21.08 ± 1.09 i.u./l immediately after the journey (p < 0.05) . similar increase was observed in the values obtained in experimental pigs, which is not significantly differrent (p > 0.05). the values obtained 7 days posttransportation were not significantly (p > 0.05) different in the experimental and control pigs (table 3). the urea value recorded immediately after transportation rose (p < 0.05) in the control pigs from 4.67 ± 0.25 mg/100 ml pre-transportation to 6.79 ± 0.39 mg/100 ml. however, the value dropped to 4.14 ± 0.23 mg/100 ml in the control pigs 7 days after transportation (table 2), which was not significantly different from the corresponding value recorded in experimental pigs (p > 0.05). discussion the fact that there was no change in the pcv value immediately after transportation in the present study makes it agree with the result obtained by scope et al. (2002) who did not obtain significant change in the value of pcv of racing pigeons after 4-h road transportation. this is also similar to the observation of knowles et al. (1999b) who demonstrated that pcv value in transported cattle showed no consistency. the observed increase in the value of pcv on day 7 post-transportation in all the transported pigs was within the normal range for pigs while the higher value obtained in the experimental pigs may be attributed to the ability of aa to maintain the integrity of erythrocyte membrane in aa-treated group (candan et al., 2002). the higher value of leucocytes observed immediately post-transportation in control pigs is similar to the finding of buckham-sporer et al. (2008) who demonstrated leucocytosis in transported young beef bulls and that it is one of the bio-makers of transportation stress. the results of the study suggested that aa prevented the release of leucocytes from their pool in the body into peripheral circulation in experimental pigs, apparently due to its inhibitory role on circulating corticesteroids which is known to increase in animals under stress and cause leucocytosis. also a decrease in aa in the body tissues, especially in adrenal glands is known to be associated with corticosteroid release (whitehead and keller, 2003). the depletion of aa in the pigs was prevented in the present study by its exogenous supply prior to transportation of pigs. road transportation of pigs did not alter eosinophil count significantly in experimental and control pigs. this result disagreed with those obtained in goats by new et al. (1996) and minka and ayo (2007), who recorded an increase in eosinophil count following road transportation. the difference in the findings may be attributed to species difference and different conditions of transportation in the animals monocytosis was observed following road transportation in control pigs, but not in experimental pigs. again this finding disagreed with the results of scope et al. (2002), who did not observe any significant changes in monocyte count in transported racing pigeons for 4 h by road. the neutrophilia observed in the present study in the control pig was apparently due to the decrease effect of aa, which has been shown to increase the release of corticosterone into the peripheral circulation from the body pools. although the mechanism involved was not elucidated in this study, whitehead and keller (2003) has reported the release of corticosteroids in stressed layer chickens, which in turn is involved in the mobilization of neutrophils to peripherals circulation from the body pools. lymphopenia obser table 2. serum biochemical parameters of experimental (supplemented with ascorbic acid) and control (non-supplemented with ascorbic acid) pigs, before and immediately (30 min) after short-term road transportation (mean ± sem). before transportation after transportation parameters experimental (n= 17) control (n =12) experimental (n = 17) control (n =12) total protein (gm/dl) 67.94 ± 1.50 ns 67.62 ± 1.29 ns 69.69 ± 1.67 ** 71.31 ± 1.93 ** globulin (gm/dl) 33.81 ± 1.83 ns 30.85 ± 1.77 ns 35.56 ± 2.24 ns 33.77 ± 2.66 ns albumin (gm/dl) 34.75 ± 0.95 ns 36.77 ± 1.23 ns 34.13 ± 1.04 ns 37.54 ± 1.73 ns albumin:globulin 1.11 ± 0.09 ns 1.26 ± 0.11 ns 1.06 ± 0.12 ns 1.30 ± 0.22 ns na + (mmol/l) 140.70 ± 1.58 ns 141.90 ± 1.71 ns 144.80 ± 1.09 ns 144.50 ± 1.92 ns k + (mmol/l) 6.25 ± 0.19 ns 6.09 ± 0.21 ns 6.06 ± 0.19 ns 6.14 ± 0.16 ns hco3 (mmol/l) 24.83 ± 0.68 ns 24.62 ± 0.89 ns 21.25 ± 0.85 ns 22.15 ± 0.75 ns cl (mmol/l) 104.40 ± 1.46 ns 104.50 ± 1.15 ns 102.70 ± 1.17 ns 102.70 ± 1.72 ns alkaline phosphatase (i.u./l) 40.56 ± 2.85 ns 40.92 ± 2.92 ** 39.94 ± 2.85 ** 48.23 ± 2.36 ** aspartate amino transferase (i.u./l) 16.75 ± 1.04 ns 15.69 ± 1.08 ns 17.75 ± 1.04 ** 21.08 ± 1.09 ** alanine amino transferase (i.u./l) 32.13 ± 1.42 ns 32.69 ± 1.25 ** 26.06 ± 1.85 ** 26.54 ± 2.30 ** cholesterol (mg/100ml) 4.37 ± 0.26 ns 4.14 ± 0.24 ns 10.74 ± 3.31 ** 4.73 ± 0.22 ns bilirubin (mg/100ml) 9.06 ± 0.59 ns 9.23 ± 0.74 ns 8.81 ± 0.59 ns 8.92 ± 0.66 ns urea (mg/100ml) 5.29 ± 0.19 ns 4.67 ± 0.25 ** 5.29 ± 0.18 ** 6.79 ± 0.39 ** **values with asterisks are significantly different (p < 0.05). table 3. serum biochemical parameters of experimental (supplemented with ascorbic acid) and control (non-supplemented with ascorbic acid) pigs on day 7 after short -term road transportation (mean ± sem). parameters experimental (n = 16) control (n = 13) total protein (gm/dl) 73.50 ± 1.97 ns 67.77 ± 2.00 ns globulin (gm/dl) 40.19 ± 2.01 ** 30.00 ± 1.93 ** albumin (gm/dl) 33.31 ± 0.97 ** 37.77 ± 1.68 ** albumin:globulin 0.87 ± 0.06 ** 1.34 ± 0.12 ** na + (mmol/l) 139.40 ± 1.40 ns 142.80 ± 2.20 ns k + (mmol/l) 5.46 ± 0.13 ns 5.38 ± 0.20 ns hco3 (mmol/l) 21.69 ± 0.84 ** 24.15 ± 0.80 ** cl (mmol/l) 91.00 ± 1.79 ** 96.62 ± 1.22 ** alkaline phosphatase (i.u./l) 43.75 ± 2.63 ns 39.85 ± 1.42 ns aspartate amino transferase (i.u./l) 18.75 ± 1.24 ** 19.54 ± 1.44 ** alanine amino transferase(i.u./l) 32.88 ± 1.59 ** 26.77 ± 2.07 ** cholesterol (mg/100ml) 4.79 ± 0.25 ns 4.66 ± 0.20 ns bilirubin (mg/100ml) 9.38 ± 0.68 ns 9.46 ± 0.79 ns urea (mg/100ml) 4.50 ± 0.27 ns 4.14 ± 0.23 ns **values with asterisks are significantly different (p < 0.05). observed in the experimental pigs could be attributed to the depressive effects of road transportation on lymphoid tissues, which according to spain (1975) results in antibody depression, and impaired migration of phagocytic cells (spain, 1975). this is in agreement with the findings sudakov (1992), who showed that adrenocoticotropic hormone (acth) and glucocorticoids cause regression of lymphoid tissue due to stress. the increase in neutrophil:lymphocyte ratio after transportation agrees with the findings of rajion et al. (2001), who observed increase in the ratio following road trans portation in goats. the result also is in agreement with the established fact that the parameter is a good indicator of stress in goats (rajion et al., 2001; minka and ayo, 2007), calves (fraser and brown, 1990) and broiler chicken (zulkifli et al. , 2001). thus, an increase in neutrophil: lymphocyte ratio and a decrease in lymphocyte count obtained in the present study are consistent with the finding that neutrophilia, which occurs during stress, state stimulates the anterior pituitary gland to secrete acth. the circulating acth in turn induces the adrenal cortex to produce glucocorticoids, involved in the mobilisation of neutrophils from body pool into the pheripheral circulation. the increase in the neutrophil:lymphocyte ratio was more pronounced in control than experimental pigs because the control pigs that were not treated with aa appeared more stressed than the experimental pigs. neutrophilia has been shown to be necessary for increase in body resistance to stress situations (dohms and metz, 1991). the neutrophil: lymphocyte ratio is an indication of the activity of the hyphothalamus-adrenohypophyseal adreno-cortical-axis, and it increases with the degree of stress acting upon the body. the ratio has been shown to be one of the most sensitive and lasting indicators of physiological stress in poultry (whitehead and keller, 2003), goats and calves (fraser and broom, 1990). there was a significant (p < 0.05) increase in total protein (tp) post-transportation in all the experimental and control groups, but the value was higher in the control than the experimental group. this finding is in agreement with the observation of broom et al. (1999a) that pigs subjected to 24 -h road transportation suffered severe dehydration as the concentration of tp increased signifycantly. similar results were obtained in sheep by knowles et al. (1996) and in calves by (knowles et al., 1999a) and rajesh et al. (2003). the significant increase in the value of globulin and albumin recorded on day 7 post-transportation is in agreement with the findings of knowles et al. (1994), who reported a definite increase in total plasma protein, plasma albumin and plasma globulin due to transportation and that the concentrations of the proteins were restored relatively to normal values during the resting period in lairage. the high at and rh recorded during the transportation have been shown to cause heat stress, resulting in severe dehydration and, consequently, clinical haemo-concentration that may increase total protein; plasma albumin and globulin. excessive cortisol level released during stressful conditions may also cause diuresis to complicate the already aggravated situations. sodium, potassium and chloride are involved in various fundamental physiological processes, including the maintenance of normal osmotic equilibrium, maintenance of normal water balance and distribution, acid-base equilibrium and neuromuscular function. non-significance in the values of these parameters post-transportation in experimental and control pigs indicated that aa administration as well as 4h road transportation did not affect these important physiological parameters in pigs. the increase in chloride value in control pigs over experimental group on day 7 post-transportation may be attributed to an increase in capillary permeability with loss of colloidal protein into the tissues. the increase in alkaline phosphatase and aspartate aminotransferase activities observed in control pigs immediately after transportation may be due to increased hepatocellular destruction and muscular degeneration as a result of the road transportation stress. this finding is in agreement with the results obtained by hong et al. (2007) who observed changes in serum enzyme activities after transporting pigs due to tissue damage in transportstressed pigs. sahin et al. (2002) reported that the concentration of antioxidant aa in the serum and liver decreases with stress. the increased level of urea observed following transportation was, apparently, due to feed deprivation and elevated cortisol concentration as established by several authors (dalin et al., 1993; stull and rodiek, 2000; kannan et al., 2000; odore et al., 2004), and also as a result of increased catabolism of protein caused by hypoglycaemia. it has been shown that stress induces aa depletion in the adrenal glands, and this is associated with corticosterone release (sahota et al., 1995). maintenance of high adrenal gland concentrations of aa by dietary supplementation has been found to limit the rise in circulating corticosterone concentration under stress (pardue et al., 1985). aa is known to increase blood glucose level, which decreases in animals subjected to stressful conditions, especially during road transportation (sauberhich, 1994; hassanzadech, 1997). conclusion the results of the present study demonstrated for the first time the beneficial effect of aa administration on haematology and serum chemistry of pigs transported by road during the harmattan season for short journey of 4 h. it is, therefore, recommended that pigs be administered with aa before transportation by road during the harmattan season in order to reduce the risk of adverse effects of transportation stress on health. reference adenkola ay, durotoye la (2004). haematological study during prepartum and postpartum periods in brown savanna does in zaria, nigeria. proceedings 38 th annual conference agricultural society nigeria. pp 538-540. adenkola ay, anugwa foi (2007). effects of dietary supplement of ascorbic acid on performance of piglets in makurdi, benue state of nigeria. trop. vet. 25(1): 15-17. adenkola ay, ayo jo, sackey akb,minka ns (2008). ameliorative effect of ascorbic acid on rectal temperature of pigs transported by road for eight hours during the harmattan season. proc. 13 th ann. conf. anim. sci. assoc. nig. pp 177-181 anderson nl, anderson ng (2002). the human plasma proteome: history, character, and diagnostic prospects. mol. cell. proteomics.1:845-867 atkinson pj (1992). investigation of the effects of transport and lairage state on hydration state and resting behaviour of calves for export. vet. rec. 130: 413-416. ayo jo, oladele sb (1996). road transport stress in food animals in nigeria: a review. nig. vet. j. 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dm, diabetes mellitus; bha, butylated hydroxyanisole; bht, butylated hydroxytoluene; vc, vitamin c; ve, vitamin e; cl, luminous intensity. is the most active and also the most damaging to organisms (gutterridge 1994; tabatabaie et al. 2003; peng et al., 2005; zhang et al., 2005). common oxygen radical scavengers include antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase (fang et al., 2003), and endogenous and exogenous antioxidants such as vitamin c (vc), vitamin e (ve) and β-carotene. recent reports have indicated that the hydrolyzed product of soybean protein and casein had certain antioxidant activity (hou et al., 2008). swine blood is a valuable resource in china due to its high protein content. however, since the protein from dried blood powder is hard to digest and has poor palatability most of it is thrown out, which poses a serious threat to the environment (fang et al., 2004). to solve these problems, peptides from swine blood (psb) was obtained by the fermentation of swine blood (fang et al. 2005) followed by micro-filtration, ultra-filtration, and spray-drying. these peptides have been shown to have some biological activities. in this study, the components of psb were analyzed by a mass spectrometer, infrared spectrometer, and automatic amino acid analyzer. the ability of these peptides to scavenge ultra oxygen anion free radicals, hydroxy free radicals and hydrogen peroxide was examined by a chemical luminescence detection system. materials and methods materials bacillus subtilis strain a32 was obtained from a slaughterhouse (genbank, accession no. dq631809), psb was obtained by fermentation of swine blood with a32, micro-filtration, ultra-filtration, and spray-drying. luminol (chromatography grade) and catechin were obtained from sigma and hunan kinglong bio-resources products industry co., ltd, respectively. methods the molecular weight distribution of psb was measured by mass spectrograph (ms) 4307 (bruker, munich, german). mode of operation: reflection; extraction mode: delayed; polarity: positive acquisition; manual; accelerating voltage: 200000 v; grid voltage: 68%; acquisition mass range: 850-5000da; number of laser shots: 150/spectrum. the amino acid composition was measured by an automatic amino acid analyzer l 8800 (hitachi, tokyo, japan). column: na + exchange column (4.6 id × 40 mm); detector: uvvisible light detector; reagent: ninhydrin/sodium acetate buffer; buffer system: citric acid buffer b1 (ph3.2), b2 (ph3.0), b3(ph 4.0), b4(ph4.9), column temperature: 55°c, reaction temperature: 135°c, flow rate: 0.4 ml/min. the ability to scavenge o2 . was determined according to the method of xu et al. (2001). in this procedure, pyrogallol (10 μl mm) and luminol-carbonate buffer (940 μl and ph 10.2) were added to 50 μl samples and mixed completely, and the chemical luminescence power curve was measured for 20 s. the ability to scavenge . oh was analyzed according to the method of fan et al. (1998). thus, 50 μl mm cuso4, 50 μl mm 1,10-phenanthroline, and 700 μl boric acid-borax buffer (ph 9.0) were added to 50 μl samples with thorough mixing. next, 100 μl mm antiscorbutic acid and 50 μl 0.1 mm h2o2 were added, and the chemical luminescence power curve was recorded for 150 s. the ability to scavenge h2o2 was determined according to the method of xu et al. (2001). in this method, to 50 μl samples (distilled water as a control) were added 50 μl 0.15% h2o2 solution and 900 μl luminol-carbonate buffer (ph 10.2). after thorough mixing, the chemical luminescence power curve was measured for 60 s. the ability to scavenge oxygen free radicals was determined according to the method of wang et al. (2003). a certain level of luminous intensity (cl) is associated with the oxygen free radicalscavenging capacity. therefore, cl can be used to show the relative output of oxygen free radicals. furthermore, materials that can scavenge oxygen free radicals can reduce cl, so a material’s capacity to scavenge oxygen free radicals could be measured according to the decrease in cl: led inhibition rate = (cl control cl samples) / cl control a luminous inhibition curve was obtained with the sample concentration as the abscissa and the led inhibition rate as the ordinate. the ic 50 was the concentration at which the rate was 50%. a small ic 50 was associated with strong oxygen radical-scavenging capacity, and vice versa (wang et al. 2003). the assays were conducted in triplicate. the amino acid composition of psb was measured by an automatic amino acid analyzer l-8800 as described by li et al (2008) and kong et al (2008). results molecular composition of psb figures 1 and 2, respectively, show the molecular composition of swine blood powder and psb. psb was obtained from fermented swine blood by micro-filtration (0.05 μm ceramic membrane), ultra-filtration (1000 da), and spray-drying. the molecular mass of psb was less than 2000 da, and in most cases about 1000 da. amino acid content of psb figures 3 and 4 show that he contents of glutamine, glutaminic acid, valine, methionine, isoleucine, tyrosine, leucine, pheny-lalanine, lysine, histidine in psb were about 10 fold greater than those in swine blood powder. half-cystine was increased nearly four times, and threonine was about doubled. psb had a good balance of amino acids and abundant essential amino acids, and the imbalance of leucine and isoleucine was improved compared to that in swine blood powder. at the same time, the contents of taste-related amino acids such as glutamine (fresh) and glycine (sweet) were very high, and this improved the nutritional value and flavor of psb. ability of psb to eliminate free radicals figure 5a shows that all of the antioxidants tested decreased both the peak value and the area of luminous curves for h2o2. psb, catechin and vc could scavenge h2o2, with ic 50 values of 1.27, 0.45, and 1.31 mg·ml-1, respectively (table1). the ability of catechin to scavenge h2o 2 was obviously higher than those of psb and vc. the ability of psb to scavenge h2o2 was slightly stronger than that of vc, but this difference was not significant. figure 5b shows that all of the antioxidants tested could decrease both the peak value and the area of luminous curves for o2 . psb, catechin and vc could scavenge o2 ., with ic 50 values of 3.42, 1.19, and 0.45 mg·ml-1, respectively (table 1). the ability of vc to scavenge o2 . was obviously higher than those of catechin and psb. the scavenging capacity of psb was weaker than that of catechin. psb, catechin, and vc each reduced both the peak value and the area of luminous dynamics curves for ·oh. however, there were large differences in the ·ohscavenging capacities of these materials. the peak value and area of luminous dynamics curves of psb were much lower than those of caffeine and vc (figure 5c). their respective ic 50 values were 0.66, 14.23, and 16.74 mg·ml-1 (table 1). the ·oh-scavenging capacity of psb was nearly 22 times greater than that of catechin and 25 times greater than that of vc. discussion psb had a molecular mass of less than 2000 da, and most were about 1000 da. when the molecular mass of peptides is less than 850 da, the results obtained with the figure 1. the molecular weight distribution of swine blood which was spray-dried was measured by a mass spectrograph. figure 2. the molecular weight distribution of psb was measured by a mass spectrograph; psb was obtained by fermentation of swine blood with a32, micro-filtration, ultra-filtration, and spray-drying. i n t e n s i t y ( m v ) 400 m e t l y s 350 300 250 g l u p h e 200 150 100 a l a n h 3 50 v a l i l e l e u 0 t h r h i s 0 5 10 15 20 25 30 retention time (min) figure 3. the amino acid composition of psb was measured by an automatic amino acid analyzer l-8800. psb was obtained by fermentation of swine blood with a32, micro-filtration, ultrafiltration, and spray-drying. 80 g l u 60 40 m e t p h e l y s i n t e n s i t y ( m v ) a l a v a l 20 0 n h 3 t h r i l e u h i s a r g l e 0 5 10 15 20 25 30 retention time (min) figure 4. the amino acid composition of swine blood was measured by an automatic amino acid analyzer l-8800. swine blood was spray-dried. a 350000 300000 c d ) 250000 no u s( 200000 l u m i 150000 100000 50000 a 0 a 1 10 20 30 40 50 60 ti me( s) figure 5a scavenging of h2o2 by the various substrates used. cont r ol cat echi n psb vc b 60000 50000 c d ) 40000 （ u m i n o u s 30000 20000 l 10000 b 0 1 4 8 12 16 20 ti me（s） figure 5b. scavenging of o2 . by the various substrates used. cont r ol vc psb cat echi n applied bio-systems voyager system 4307 are not precise, since they are influenced by the instrument itself. thus, the optimum range for the molecular mass was chosen to be from 850 to 5000 da. psb with a different molecular weight distribution was also obtained in the ionization process, and the peak value of some peptides could not be scanned. therefore, the psb samples contained some pieces of peptides. psb could clearly scavenge o2 . , h2o2 and ·oh with ic 50 values of 3.42, 1.27, and 0.66 mg·ml-1. psb is rich in hydrogen, and can provide a hydrogen proton, which can reduce highly oxidized oxygen radical. thus, it can terminate the chain-reaction of oxygen radical and can scavenge or inhibit oxygen radical. the hydroxy free radical is the most active and dangerous among numerous oxygen radicals (rong 2001). psb had the strongest hydroxy free radical-scavenging activity, which suggests that psb may be useful as an oxidation inhibitor. in addition, the oxygen radical-scavenging activities of catechin and vc in this study were the same as those reported by hu (2004), but there was a difference in the ic 50 values. this discrepancy may have been due to the fact that the catechin and vc reagents were produced by different companies and the duration of recording the illumination curve also differed. at present, the use of chemical antioxidant additives like butylated hydroxyanisole (bah) and butylated hydroxytoluene (bht) has been limited due to considerations regarding food safety. the use of some natural antioxidants such as ve and herbal extracts has also been limited due to their high cost, their effects on food flavor and color. furthermore, oxidative stress can strongly c 70000 60000 （ c d ) 50000 40000 n o u s 30000 l u m i 20000 10000 0 1 c 15 30 c 60 75 90 105 120 135 150 ti me（s) cont r ol psb vc cat echi n figure 5c. scavenging of ·oh by the various substrates used. table 1. radical-scavenging activities of different antioxidants. antioxidants h2 o2 o2 . ·o h psb 1.27±0.13 a 3.42±0.23 a 0.66±0.14 a catechin 0.45±0.09 b 1.19±0.14 b 14.23±2.11 b vc 1.31±0.11 a 0.45±0.06 c 16.74±2.60 c abc values with different lowercase letters are significantly different (p < 0.05). values are given in ic 50(mg· ml -1 ). influence animal's growth. thus, the identification of minimally toxic and yet highly effective natural antioxidants is important for maintaining health, promoting animal growth and enhancing animal performance. psb may be produced by the fermentation of swine blood with strain a32. the production cost is very low, regardless of whether it is to be used as an antioxidant additive in food or feed. at present, there have been few reports on psb, and this promises to be a fruitful field of research. acknowledgements dr. jun fang is grateful for financial support from the china postdoctoral science foundation (20070410984), the department of science and technology of changsha city (k0904013-21 ） and hunan agricultural university (07yj07). references fan xb, li cj, sha dn (1998). detection of hydroxyl radical by phenanthroline chemiluminescence system. basic clin. med.18: 68-71. fang j, lu xy, ma mh, qiu yx (2004). research on microbial degradation of swine blood meal. food fermentation ind. 9: 93-95. fang j, lu xy, mo j, qiu yx, jiang hm, ma mh, li zj (2005). the primary filter of fermentable blood meal strains. j. hum. agric. univ. 3: 307-309. fang yz, yang s, wu gy (2003). free radicals, antioxidants, and nutrients in relation to health. spanish (spain-traditional sort), acta nutrimenta sinica, 25: 337-343. gutteridge jmc (1994). free radical in medicine. (10 september). bmj, 309: p. 678. hu wq (2004). the comparison of antioxidation capacity of soy protein and casein enzymatic hydrolysis. master's thesis, nanjing agricultural university. p. 11. hou zp, yu ly, rui lh, tie jl, rongqing h, yulan l, xin w, zhiqiang l, wence w, hua x, guo yw, liang-xi t (2008). rice protein concentrate partially replaces dried whey in the diet for early-weaned piglets and improves their growth performance. j. sci. food agric. 88: 1187-1193. kapalla m, yeghiazaryan k, hricová m, moenkemann h, pírek a, schild hh, golubnitschaja o (2005). combined analysis of biochemical parameters in serum and differential gene expression in circulating leukocytes may serve as an ex vivo monitoring system to estimate risk factors for complications in diabetes mellitus. amino acids, 28: 221-227. kong xf, yin yl, he qh, yin fg, liu hj, li tj, huang rl, geng mm, ruan z, deng zy, xie my, wu g (2008). dietary supplementation with chinese herbal powder enhances ileal digestibilities and serum concentrations of amino acids in young pigs. amino acids, 37:573-582 .-4.23 li tj, dai qz, yin yl, zhang j, huang rl, ruan z, deng z, xie m (2008) dietary starch sources affect net portal appearance of amino acids and glucose in growing pigs. animal, 2: 723–729. peng y, huang z, guo bj (2005). the scavenging effects of se enriched spiruline plantensis on oxygen free radicals. acta nutrimenta sinica, 27: 61-65. rong jh (2001). a study on peptides of soybean and its biological activity. master's thesis, huazhong agricultural university, p. 27. stadtman er, levine rl (2003). free radical-mediated oxidation of free amino acids and amino acid residues in proteins. amino acids, 25: 207218. tabatabaie t, vasquez-weldon a, moore r (2003). free radicals and the pathogenesis of type 1diabetes: â-cell cytokine-mediated free radical generation via cyclooxygenase-2. diabetes, 52: 1994-1999. http://springer.lib.tsinghua.edu.cn/content/104405/?p=d17664d6c71946bebfada3d808e64efb&pi=0 wang shao-ming, li ping, zhao ming-qiang (2003). determination of antioxidant activity of suanzaoren by biochemical luminescence. chin. trad. herbal drugs, 5: 417-419. wang sm, li p, zhao mq (2003). the biochemistry illumination law determines the antioxidation activeness of jujube seed. chinese traditional and herbal drugs, 34: 417-419. xu h, liu j, wang zt, ding jy (2001). the scavenging of freeradical oxygen speciesby crude drugs and cultured tissues of five species of dendrobium sw. j. plant res. environ. 10: 35-37. zhang j, ou sy, zhang n (2005). the scavenging capacity of free radicals by enzymes-hydrolyzed wheat bran in vitro. acta nutrimenta sinica, 27: 25-29. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (12), pp. 001-010, december, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review potential of bacterial fermentation as a biosafe method of improving feeds for pigs and poultry a. t. niba1*, j. d. beal2, a. c. kudi2 and p. h. brooks2 1 department of animal production, university of dschang, p. o. box 447, dschang, cameroon. 2 faculty of science, school of biological sciences, university of plymouth, drake circus, plymouth, devon pl 4 8aa, united kingdom. accepted 6 february, 2009 the use of fermented liquid feeds in monogastric animal nutrition is regarded as one of the biosafe methods of animal production. this paper examines bacterial fermentation of feed substrates for production of fermented liquid feeds for pigs and moist feeds for poultry. emphasis is placed on the interplay of factors affecting feed fermentation and their relationship to feed quality. the resistance of fermented feeds to enteropathogenic contamination prior to feeding and their potential contribution to african agriculture is highlighted. key words: fermented liquid feed technology, pigs, poultry. introduction there is considerable concern over the use of antibiotic growth promoters (agps) in animal production. their extensive usage has resulted in the selection for survival of resistant bacteria species or strains (doyle, 2001; montagne et al., 2003; khaksefidi and rahimi, 2005). this resistance can be transferred to other previously susceptible bacteria and can be hazardous to both animal and human health (montagne et al., 2003). the use of in-feed agps has been banned in the european union (wilkie et al., 2005; williams et al., 2005) and there are further attempts to reduce or remove in-feed agps worldwide (jin et al., 1998; yegani and korver, 2008) . this would have significant implications on gut microbial profiles (yegani and korver, 2008) as well as increase competition between gut microflora and the host for available nutrients (dibner and richards, 2005). however, there is an active search for alternatives to agps in animal feeding. this includes the use of probiotics, organic acids, prebiotics, minerals, enzymes, herbs, phenolic aromatic components and fermented feeds (ff) (knarreborg et al., 2002; reid and friendship, 2002; verstegen and williams, 2002; dahiya et al., 2006; steiner, 2006; missotten et al., 2007). ff is considered *corresponding author. email: at_niba@hotmail.com or atniba@yahoo.fr. as a biosafe method for replacing agp in pigs (knarreborg et al., 2002; kobashi et al., 2008) and poultry (e.g. heres et al., 2003a; heres et al., 2003b; heres et al., 2003d; niba, 2008). ffs are characterised by high numbers of lactic acid bacteria (lab) (approximately 10 9 cfu/ml of feed) and high concentrations of lactic acid (>150 mm) (heres et al., 2003a; niba, 2008). in chickens, the organic acid content of fermented feeds has been reported to improve foregut barrier function against pathogens by increasing acidity and lowering the ph (heres et al., 2003d; engberg et al., 2006). the proportion of chlortetracycline-resistant escherichia coli strains was significantly reduced in the gut of weaned piglets fed fermented liquid feed (flf) (22.2%) compared with dry feed (88.9%) (kobashi et al., 2008). this review, examines the potential of bacterial fermentation of feeds as a means of improving feeds for pigs and poultry. emphasis will be placed on the interplay of factors affecting feed fermentation and their relationship to feed quality. fermented liquid feed technology man has known the use of microbes for preparation of food products for thousands of years and all over the world a wide range of fermented foods and beverages contribute significantly to the diet of many people (achi, 2005). the use of liquid feeds in animals has created an opportunity for recycling of liquid co-products from the human food industry especially in the european pig industry (scholten et al., 1999; brooks et al., 2003a). this has considerably reduced the need for alternative methods of disposal of these products, like drying, disposal to land fill or burning (scholten et al., 1999). however, liquid feeds have the potential to serve as potent reservoirs of enteropathogens unless steps are taken to prevent their introduction and proliferation during storage and feeding (beal et al., 2002). brooks et al. (2001) also stated that liquidfeeding systems can easily become contaminated. they further observed that the development of computerised liquid-feeding systems capable of feeding pigs ad libitum has rekindled interest in the possibility of liquid feeding for weaner pigs. this, in addition to recent developments in the use of lactic acid bacteria in the accelerated fermentation of feed substrates for animal feeding as well as reducing the possibility of contamination by enteropathogens (beal et al., 2002; beal et al., 2005), has provided a good basis for improvement in pig nutrition. it is also having much promise in other farm animal species especially poultry (heres et al. , 2003b; skrede et al., 2003; niba, 2008) and aquaculture (refstie et al., 2005) . fermented liquid feed technology could make important contributions to african agriculture especially in semi-arid and hot areas (niba et al., 2008b). in such areas, ambient temperatures (appro-ximately 30°c) could support efficient lactic acid fermentation of feeds (beal et al., 2002; niba, 2008). furthermore, wet feeding in hot climates has been shown to improve feed intake and growth rates in poultry (forbes, 2003). according to beal et al. (2002), lactic acid bacterial fermentation of feeds provides a feed that has a ph of 3.8 4.0 and contains 150 250 mmol/l lactic acid. a similar range of fermented feed ph has been reported by geary et al. (1996) (3.8 4.2), christensen et al. (2007) (3.6 4.2), scholten et al. (1999) (3.5 4.5) and moran et al. (2006) (<3.8). the synergistic effect of a high lactic acid concentration and low ph is believed to act in concert to give fermented feeds their antimicrobial activity. this enables them to withstand contamination by pathogens like salmonella spp. (geary et al., 1996; van winsen et al., 2001a; beal et al., 2002; van winsen et al., 2002), campylobacter spp. (heres et al., 2004), and coliforms (russell et al., 1996). the mechanism of action of fermented feeds and fermented co-products in controlling enteropathogens both in vitro and in vivo has been reviewed extensively (brooks et al., 1996; scholten et al., 1999; hansen et al., 2000; van winsen et al., 2001a; beal et al., 2002; demeckova et al., 2002; hojberg et al., 2003; boesen et al., 2004; van immerseel et al., 2004; beal et al., 2005; moran et al., 2006). the antimicrobial effects of lactic acid are believed to be exerted by the ability of the undissociated acid to gain entry into the cell, disrupt ph homeostasis and consequently cause nucleic acid and protein damage (beal et al., 2002). according to moran (2001), the low ph, dissociation constant (pka value),and the molar concentration are factors that determine the inhibitory activity of lactic and acetic acid in fermented feed. while inside the cell, the acid dissociates and causes a drop in ph. this stops enzymatic processes and causes the proton motive force to collapse. the anion may also destroy the cell wall resulting in cell death (van winsen et al., 2001a; van winsen et al., 2001b). however, alakomi et al. (2000) earlier stated that disruption of the outer membrane by acids could involve both dissociated and undissociated forms. as indicated, the likely action on the outer membrane of salmonella could be protonation of anionic components such as carboxyl and phosphate groups. this consequently weakens the molecular interactions between outer membrane components thus increasing its permeability. in a recent review by brooks (2008) on fermented liquid feeds for pigs, increasing feed cost, withdrawal or reduction of antimicrobial growth promoters (agp) in feeds and quality assurance programmes related to salmonella in pig meat were given as reasons why producers should adopt liquid feeding. however, he indicated that the success of liquid feeding depended on; i.) microbial fermentations and selection of lab capable of generating lactic acid levels above 100 mmol/l that can significantly reduce numbers of enteropathogens and the incidence of salmonella. ii.) batch fermentation of the cereal portion of feeds with inoculants capable of generating high lactic acid concentrations to give more consistent results of fermentation. iii.) fermentations that could preserve the feed, improve the availability of nutrients, reduce the level of antinutrients and have lab with probiotic properties. meanwhile, the three principal components involved in the fermentation process are the fermenting microorganisms, the feed substrate and the enabling environment for fermentation (figure 1). influence of micro-organism and feed substrate on fermentation the selection of lab for feed fermentation to meet desired feed and production objectives has been highlighted in previous reviews (brooks et al., 2003b; brooks, 2008) . the choice of feed substrates to obtain high numbers of lab (10 9 cfu/g feed) and levels organic acids (>150 mmol/l) or a consistent fermentation product has also been researched (canibe et al., 2007a; niba et al., 2007; lyberg et al., 2008; niba, 2008; niba et al., 2008b; olstorpe et al., 2008) or reviewed (brooks, 2008). fermentation objectives that have influenced the use of lab have centred on; i.) selection for rapid production of organic acids (mainly lactic acid) to ensure biosafety (e.g. missotten et al., micro-organism -lab -yeasts and fungi environment -time (hours) substrate -temperature -carbohydrates -moisture content -proteins -air composition -fibres -feathers figure 1. interactions in the fermentation medium. fermentation conditions influence rate of fermentation, type of microbe and substrate quantity and quality affects the medium. 2007; missotten et al., 2008). ii.) selection for homolactic fermentation to improve feed palatability. iii.) breakdown of anti-nutrients and increased bioavailability of nutrients (e.g. bertsch et al., 2003; brooks et al., 2003a; oboh, 2006; skrede et al., 2007; lyberg et al., 2008; okpako et al., 2008). a summary of how some of these objectives relate is shown in table 1. the advantages of fermenting feeds can be summarised from the table as follows; i.) reduction in the level of anti-nutrients within the feed. ii.) improved bioavailability of minerals (e.g. p, ca, mg and cu). iii.) increase in protein contents (lysine, histidine and methionine). iv.) breakdown of indigestible carbohydrates. influence of fermentation length and conditions the length of steeping feed ingredients, the type of feed substrates and fermentation conditions influence the quality of the fermentation product. steeping time has been related to its effects on the activity of endogenous enzymes and the breakdown of anti-nutrients within the grain. according to choct et al. (2004a) the effects on growth and feed intake for weaner pigs resulting from steeping of feed for 15 h might be related to the release and activation of endogenous enzymes in the grain. the activation of these enzyme systems within the grain can act on cell wall structures in a similar way to exogenous feed enzymes (choct et al., 2004b). in reviewing the effect of steeping in liquid feeding systems, brooks et al. (1996) indicated that phytases that were naturally present in the pericarp of some grains (like cereals) could be activated by soaking. they also stated that soaking feed for 8 16 h before feeding increased the bioavailability of phosphorus, calcium, magnesium and copper. in another study (lyberg et al., 2008), the phytase activity for a cereal grain mix of wheat, barley and triticale was 1382 ftu /kg dm and inositol hexaphosphate boundphosphorus and total phosphorus were 2.2 and 3.7 g/kg dm. after fermentation, dietary inositol hexaphosphate was completely degraded to release phosphorus. fermentation of the carbohydrate-rich cereal components of the diet separately and combining them with the protein-rich components just before feeding has some practical and nutritional advantages (beal et al., 2002; brooks et al., 2003b; beal et al., 2005; moran et al., 2006; canibe et al., 2007a; brooks, 2008). fermenting the protein rich components produces undesirable endproducts, such as biogenic amines, which could affect the palatability of fermented liquid feed (canibe et al., 2007a). furthermore, some studies have reported the degradation of free amino acids added to diets during fermentation (handoyo and morita, 2006; canibe et al., 2007b). however, niven et al. (2006) demonstrated that the loss of lysine from fermented liquid pig feed was due to metabolism of lysine by e. coli present in the feed rather than its utilisation as an energy source by lab. it was observed that inoculation of feed with lab and 50 mmol/l lactic acid at the beginning of fermentation resulted in lysine levels remaining unaltered after 72 h fermentation. the addition of acid reduced or eliminated the e. coli and allowed the lysine to remain intact during table 1. effect of micro-organisms and feed substrates on feed fermentation. fermentation type substrate ph lactic acid acetic acid ethanol or source or effects or effects effects on on diet on diet diet l. plantarum, p. wheat and wheat by<3.8 <60 † <10 † <10 † (moran et al., pentosaceus, yeasts products 2006) 4.53 22.21* 22.42* 9.12* (beal et al., 2005) spontaneous barley 4.30 34.43* 27.34* 10.74* (beal et al., fermentation. 2005) (lab and yeasts) lactobacillus brevis soybean white flakes 4.8 elimination of indigestible carbohydrates (refstie et al., and lowered trypsin inhibitor activity 2005) lactobacillus sp. (strain barley and wheat and significant reduction in phytic acid, dietary (skrede et al., ad2), l. plantarum barley whole meal flours fibre and -glucans (33.5-18.4 g/kg in 2001; skrede et (am4). barley), alpha-amylase activity in barley al., 2002; skrede et al., 2003; skrede et al., 2007) lactobacillus acidophilus sesame seed meal phytic acid reduced to below detectable (mukhodhyay limits and tannin contents reduced from 20 and ray, 1999) to 10 g/kg lactobacillus plantarum complete diets of 3.6 reduction in feed dry matter and insoluble (christensen et cereals and soybean non-starch polysaccharides, increased al., 2007) meal viscosity of feed unspecified in vitro pig grower diet 4.9-5.3 reduced contents of total and free lysine (canibe et al., fermentation (g/kg crude protein, threonine and 2007c) (lab and yeasts) methionine lab fermentation phytic acid in cereals increase in apparent bioavailability of (brooks et al., phosphorus, calcium, magnesium and 2001; brooks, copper 2008) fermented fish silage increase in egg production and quality (zynudheen et supplementation in quail (haugh unit) al., 2008) diet kocuria rosea poultry feathers improved content and availability of amino (bertsch et al., (fermented feather acids, lysine 3.46%, histidine 0.94%, 2003; bertsch meal) methionine 0.69%. and coello, 2005) aspergillus nigeir and l. cassava peel meal increase in proteins (24.4%), ash (7.52%), (okpako et al., rhamnosus crude fibre (10.62%) and decrease in 2008) cyanide (7.35 mg/kg) saccharomyces cassava peel meal increase in protein content (21.5%) and (oboh, 2006) cerevisae and decrease in cyanide (6.2 mg/kg), and lactobacillus spp. phytate (789.7 mg/100 g). † g/kg dry matter, *mmol/l, wdg-wet wheat distiller’s grain. the fermentation process. the main goal of fermentation is a high lactic acid concentration (>150 mmol/l) and a low ph (<4.5). temperature affects fermentation rate and low temperatures may yield insufficient quantities of fermentation endproducts. according to carlson and poulsen (2003) an increase in fermentation temperature from 10 to 20°c improved the proportion of total barley phytate that was degraded during an 8 h fermentation from 48 to 55%. corresponding values for total wheat phytate degraded were 52 and 62%. at 38°c it required 2 h for 72% of the total phytate to be degraded. fermentation of a cereal grain mix at 10°c produced 8.6 gl -1 of lactic acid compared with 13.6 gl -1 at 20°c (lyberg et al., 2008). at low temperatures yeast predominates and produces ethanol (brooks, 2008). insufficient lactic acid concentra tion with 24 h fermentation cycles which are more practical on farms may be the case at low temperatures. furthermore, spontaneous fermentation of a cereal grain mix at 10°c required 7 days for the ph to drop to 4.0 compared with 5 days for 15 and 20°c. prolonged fermentation also results in considerable variation in species composition of fermented pig feed (olstorpe et al., 2008). fermentation at 30°c seems ideal as at 35 and 40°c there was no significant effect on lactic utilisation of feed nutrients like added synthetic lysine by and acetic acid concentrations (table 2) while butyric acid and ethanol concentrations were significantly increased (beal et al., 2005). lactic acid fermentation of sorghum and maize at 30°c produces high levels of lactic acid (>150 mmol/l) as fermented cereal-base for moist chicken feed (niba, 2008; niba et al., 2008a; niba et al., 2008b). liquid to feed ratios an important aspect of a successful liquid feeding regime is the liquid to feed ratio of the diet. this affects the dry matter content of diet and may also have implications for the intake and organic acid concentration of the feed. research to confirm the ideal dry matter content of liquid diets is limited (choct et al., 2004a). in pigs, a wide range of liquid to feed ratios (3:1, 4:1) (choct et al., 2004a), (2:1) (demeckova et al., 2002; choct et al., 2004a; xuan dung et al., 2005), (2.5:1)(russell et al., 1996; boesen et al., 2004), (3.5:1) (geary et al., 1996) have been used. in chickens, these ratios have been reduced to 1.3:1 (yasar and forbes, 1999) and 1.4:1 (heres et al., 2003a; heres et al., 2003b; heres et al., 2003c; heres et al., 2003d; heres et al., 2004). with lower liquid to feed ratios, fermented chicken feeds could be considered as fermented moist feeds rather than liquid feeds (niba, 2008). however, the dm concentration of feed has been shown to have little overall effect on the pattern of microbial activity (geary et al., 1996). meanwhile, increasing water to feed ratios improved both dm and energy digestibility of diets for pigs. however, since in commercial practice with pigs liquid to feed ratios can vary from 2:1 to 7:1 (choct et al., 2004a), performance is likely to be affected by dm intake (table 3). controlled fermentation using starter cultures successful fermentation results have also been found to be dependent on the type of fermentation adopted. a brief definition of the methods of fermentation is given in table 4. spontaneous (beal et al., 2005), backslopping (moran et al., 2006), inoculated or controlled fermentations (e. g. christensen et al., 2007; canibe et al., 2008) have been investigated as methods that could be used for production of fermented liquid feeds. spontaneous fermentation has been discouraged (brooks et al., 2003b; brooks, 2008) because in this system yeast, which can tolerate low ph and a low temperature, can predominate. yeast fermentation of starch will result in alcohol and carbon dioxide production. the production of co2 represents a loss of feed dry matter and energy value. such feeds could be unpalatable due to ‘off’ flavours resulting in reduced feed intake. secondly, spontaneous fermentation may not guarantee a rapid build-up of lactic acid in the feed, which is necessary for biosafety of the feed and to limit the pathogens (niven et al., 2006). lastly, since feed ingredients differ in their load of natural microflora, spontaneous fermentation of the same raw material at different times results in inconsistent endproducts. backslopping has been practiced on many farms (beal et al., 2002). the limitations of this method have recently been highlighted in the review by brooks (2008). in addition to these limitations, additions of fresh feed to a dynamic fermenting medium could have adverse implications on microbial balance and the ability of the feed to resist enteropathogens. temperature shifts during addition that are outside the optimal range of particular pathogens could provoke the secretion of cold-shock proteins (beal et al., 2002). such cold shock proteins could increase pathogen tolerance to lactic acid in feed fermented at 20°c compared with 30°c. controlled fermentation or inoculated fermented liquid feed would appear preferable for production of fermented liquid feeds for pigs or fermented moist feeds for chickens because more predictable results could be obtained. selection for lab that produce lactic acid rapidly, with high 24 h lactic acid (>150 mmol/l) contents (brooks, 2008), should be the primary objective. the selection of lab for other factors, such as probiotic properties is beyond the scope of this review. effects of fermented feed on performance of pigs in growing pigs, the daily weight gains (kg/day) of flf fed groups (0.572) and acidified feed (af) fed groups (0.567) were significantly higher (p < 0.05) than pigs on a dry diet (df) (0.515) and non-fermented liquid feed (nflf) (0.498) (xuan dung et al., 2005). plasma urea nitrogen was also significantly lower (p < 0.05) in flf fed pigs than the dry diet, nonflf and af. as observed by demeckova (2003), piglets from gilts fed flf were 300 and 450 g heavier than piglets from the gilts fed nflf (p < 0.01) and df fed gilts (p < 0.001). demeckova et al. (2002) reported that faeces from sows fed flf feed had significantly (p < 0.001) lower numbers of coliforms than sows fed nflf or df. they also reported that piglets from sows fed flf excreted faeces that were higher in lab (7.7 vs 7.3 log10 cfu/g, p < 0.01) and lower coliforms (7.5 vs 8.1 log10 cfu/g, p < 0.001) than faeces from piglets of df-fed dams. further table 2. effect of incubation time and fermentation temperature on feed fermentation. incubation temperature ph lactic acid or acetic acid or ethanol or effects source time (h) (°c) effects on diet effects on diet on diet 24 3.75 54.5 † yeast population yeast population (moran et al., increases 10fold increases 10fold 2006) 48 3.65 yeasts population yeasts population yeasts population stabilizes and stabilizes and stabilizes and coliforms coliforms eliminated coliforms eliminated eliminated mainly mainly by mainly by by backslopping backslopping backslopping 24 4.69 11.68* 17.22* 6.81* (beal et al., 48 4.34 31.92* 27.55* 10.62* 2005) 72 4.21 46.14* 30.75* 12.79* 30 4.47 30.16* 16.42* 11.69* 35 4.41 25.29* 26.57* 9.78* 40 4.36 28.64* 32.89* 8.42* 48 20 4.2 115* dvalue (min)-250 (beal et al., 72 20 3.9 164* dvalue (min)-164 2002) 96 20 3.8 167* dvalue (min)-137 48 30 3.8 161* dvalue (min)-45 72 30 3.8 196* dvalue (min)-38 96 30 3.8 203* dvalue (min)-34 0 a 10 nd b 5.5 c (olstorpe et 0 a 15 nd b 5.5 c al., 2008) 0 a 20 nd b 5.5 c 3 a 10 nd b nd c 3 a 15 nd b 5.3 c 3 a 20 2.1 b 5.6 c 5 a 10 nd b 3.8 c 5 a 15 2.1 b 4.6 c 5 a 20 3.1 b 4.9 c 7 a 10 2.0 b 4.5 c 7 a 15 2.1 b 6.2 c 7 a 20 2.1 b 5.7 c 0 20 <3.0±0.00 c 5.0±1.27 d 3.9±0.00 b (canibe et al., 6 20 <3.0±0.00 c 6.0±0.12 d <3.2±0.21 b 2007c) 24 20 8.1±0.75 c 7.1±0.56 d 3.6±0.16 b 48 20 9.5±0.34 c 6.7±0.87 d 3.7±0.95 b 0 20 nd 4.7±0.03 nd 6 20 nd 4.9±0.16 1.9±0.11 24 20 nd 5.9±0.42 6.8±0.14 48 20 91.2±27.66 20.9±6.21 15.5±1.31 17-19 a 10 1.8 10.4 1.2 (lyberg et al., 17-19 a 15 1.9 10.4 1.2 2008) 17-19 a 20 2.2 10.5 1.1 17-19 a 10 3.3 b 7.2 c 2.4 d 17-19 a 15 3.2 b 5.9 c 2.3 d 17-19 a 20 4.8 b 7.4 c 2.0 d † g/kg dry matter, *mmol/l, dvalue (min)-decimal reduction time (minutes) of salmonella in fermented feed, a days, b yeasts counts (cfu/g feed), c lab counts (cfu/g feed), d enterobacteriaceae counts (cfu/g feed), d moulds, nd-not detected. table 3. effect of liquid to feed ratios of diets on performance. liquid to feed ratio type of operation remarks source 2:1, 3:1,4:1 experimental trial no significant effect on growth and (choct et al., 2004b) performance parameters but fcr* of liquid diets higher (p<0.05) than dry weaner pig diets. 1.63: 1 to 3.25:1 experimental trial digestibility coefficient increase from 0.791 to (barber et al., 1991) 0.829 with increase in liquid to feed ratio in pigs. 1.5:1 to 2.25:1 experimental trial feed intake, weight gain and carcass weights (yalda and forbes, 1995) of chickens not significantly affected. 2.1:1 to 5:1 commercial farms good results with pigs. (choct et al., 2004b) 1.5:1 to 3:1 experimental trial no significant effect on pig performance (hurst, 2002) † dm 149 to dm 255 experimental trial little effect on microbial activity, dm intake, (geary et al., 1996) weight gain or dm fcr of pigs *fcrfeed conversion ratios; † diet dry matter concentrations (g/kg diet). table 4. definitions of types of fermentation. type of definition source fermentation spontaneous fermentation through the action of indigenous microflora present in the (brooks, 2008) feed backslopping a proportion of a previous fermentation is retained as an inoculum for (moran et al., 2006). fresh feed consecutive microbial re-inoculation with micro-organisms from the (häggman and salovaara, 2008) previous batch inoculated fermentation resulting from inoculation of feed with selected lactic acid (brooks, 2008) bacteria controlled fermentation resulting from inoculation of feed with selected lactic acid (e.g. beal et al., 2002) bacteria under controlled environmental conditions (e.g. temperature) table 5. economic analysis of fermented liquid feed for pigs (nguyen nhut xuan dung et al. 2005). parameter df nflf af flf feed cost, vnd/kg 3528 3528 5202 3528 total feed intake, kg 220 213 216 195 live weight gain, kg 50.9 49.6 55.4 55.0 feed cost/gain, vnd* 15.222 15.182 20.239 12.477 *vietnamese dong (currency), dfdry feed, nflf-non-fermented liquid feed, af-acidified feed, flffermented liquid feed. more, xuan dung et al. (2005) (table 5) has indicated that feeding flf to growing pigs is associated with the lowest feed cost/gain ratios compared with df, nflf and af for growing-finishing pigs in vietnam. effects of fermented feed on performance poultry research on the use of fermented moist feeds on the performance of chickens is limited. however, some stu dies have shown that wet feeding increases the feed intake and growth rate of chickens (yalda and forbes, 1995; yasar and forbes, 1999; mai, 2007). pre-soaking of broiler feeds for 12 and 24 h significantly increased dry matter digestibility and body weight gain in male broilers (25 40 days of age) compared with dry feed (yalda and forbes, 1996). bacterial fermentation of barley and wheat whole meal flours with -glucan-degrading lab has improved growth and early feed:gain ratio in broiler chickens (skrede et al., 2003). a 10% inclusion of fermented fish waste silage in poultry feed increased egg quality and production in japanese quails (zynudheen et al., 2008). early access to semi-moist diets for dayold chicks stimulates gastrointestinal (gi) development and prevents dehydration during transport from the hatchery (van den brink and van rhee, 2007) . rapid gi tract development after hatch is essential for optimisation of digestive function and underpins efficient growth and development as well as a full expression of the genetic potential for production traits (mitchell and moreto, 2006; 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prevalence of gastrointestinal nematodes in mukota pigs in a communal area of zimbabwe m. c. marufu1*, p. chanayiwa1, m. chimonyo2 and e. bhebhe1 1 faculty of veterinary science, university of zimbabwe, p. o. box mp167, mount pleasant, harare, zimbabwe. 2 department of livestock and pasture science, university of fort hare, p. bag x1314, alice 5700, republic of south africa. accepted 09 november, 2015 a one year monitoring study was conducted between november 2005 and october 2006 to determine the prevalence of gastrointestinal nematodes in indigenous mukota pigs in hama-mavhaire communal area of chirumhanzu district, zimbabwe. faecal samples from a total of 143 randomly selected pigs of both sexes and different ages (< 5 months, 5 -12 months and > 12 months) from 10 villages were collected from the rectum for identification and quantification of nematode eggs. of the 143 pigs, 58.7% were positive for gastrointestinal (gi) nematodes, 17.5% having mixed infections. four parasite species were identified; oesophagostomum species (54.6%) being the most prevalent followed by strongyloides ransomi (14%), ascaris species (7%) and trichuris suis (4.2%). month had an effect on the prevalence and mean egg counts of the four gi nematode species. however, pig class and the interaction between pig class and month did not have an effect on the prevalence and mean egg counts of the gi nematode species. the present work indicates that parasite prevalence in local indigenous pigs in the communal areas is moderate. further examinations are needed to determine the pathological importance and impact of parasitic infestations on indigenous pigs in the communal area. key words: ascaris, epidemiology, indigenous pigs, internal parasites, oesophagostomum. introduction the indigenous pig genotype of zimbabwe, generally known as the mukota, predominates in smallholder areas where it is kept under the free range system and thrives on low planes of nutrition (mashatise et al., 2005). these pigs are primarily scavengers (holness, 1991), utilising food scraps thrown away by people. the roaming of pigs favours the uptake of internal parasite eggs (roepstorff and nansen, 1994), making the pigs particularly sus-ceptible to infestation with internal parasites. moreover, the warm and humid conditions of the tropics and the in-frequent treatment of local pigs against parasitic dis-eases (mashatise et al., 2005) invariably cause them to carry heavy burdens of gastrointestinal (gi) nematodes (holness, 1991). gastrointestinal (gi) nematodes limit pig production; the direct losses caused by these parasites are attributed to acute illness culminating in death, premature slaughter *corresponding author: e-mail: chrismunya@yahoo.com and rejection of carcasses during meat inspection (pat-tison et al., 1980). indirect losses include decreased growth rate, weight loss in sows and reduction in litter size (pattison et al., 1980; taylor, 1999) . ajayi et al. (1988) reported that gi nematodes reduced average daily weight gain by up to 30% in indigenous pigs of all ages. the adult nematodes live in the intestines, grazing on the gut lining and ingesting particulate and liquid digesta, thus limiting nutrient uptake by the pigs. the damage caused by adult gi nematodes includes hemorrhagic gastroenteritis and anaemia. larval migration through tissues of the pigs results in spread of infectious orga-nisms from the gut as well as extensive tissue damage thus compromising organ function (kahn, 2006). the mukota pigs have been demonstrated to be less susceptible to internal parasites than exotic breeds (zan-ga et al., 2003). however, parasite prevalence and worm burden in these pigs under the free range system have not been evaluated. farmers do not keep pig health records, thus parasite prevalence and extent of infestation are not known in communal areas. the prevalence of gi nematodes in indigenous pigs in communal areas of zimbabwe is not known. moreover, the species of gi nematodes commonly affecting indigenous pigs in communal areas of zimbabwe have not been elucidated. knowledge about the prevalence of the nematodes is useful when formulating pig development and extension programmes for communal farmers. in addition, knowledge of the occurrence of particular parasite species enables the veterinary services to understand possible health threats and develop prophylactic measures to use to reduce parasite transmission among indigenous pig herds. therefore, the objective of the current study was to identify and determine the prevalence of gi nematodes in the indigenous pigs in the communal areas of zimbabwe. materials and methods description of study site the study was conducted in hama-mavhaire communal area of chirumhanzu district in masvingo province of zimbabwe located at 19º 83's and 30º 78' e. the area has a typical tropical climate with two distinct seasons. the warm rainy season starts in mid november and ends in early april. the cool dry season starts from april to mid november. the receives a mean annual rainfall of 394.5 mm. temperatures range from 16.0 to 31.5ºc with highest temperatures of average 31.1°c, being recorded during the hot months of october and november and lowest temperatures of on average 12°c, around late june to mid july. the altitude is about 400 m above sea level. sampling procedure a total of 143 free range mukota pigs were sampled (83 sows, 28 gilts, 15 boars and 17 piglets) over one year from all the10 villages in the hama-mavhaire communal area. since pigs were free ranging, faecal samples were collected from only those pigs that were found at the homesteads at the time of sampling. the same individual animals could not be monitored throughout the study period as farmers periodically sold or slaughtered some of their pigs hence; different individuals were sampled each month. only pigs that were not showing signs of gastrointestinal nematodiasis were sampled. sample collection and processing faecal samples were collected from indigenous pigs once a month from november 2005 to october 2006. the samples were collected by rectal palpation, using a glycerine lubricated latex glove for each pig. faecal samples were placed in an empty faecal pot, which was then stored in a cooler box at 4°c before being transported to the laboratory for analysis within 24 h. the modified mcmaster’s technique, as described by the ministry of agriculture, fisheries and food (1977) was used to prepare the faeces for identification and quantification of worm eggs in the faeces. whenever samples were positive for eggs characteristic for oesophagostomum species and hyostrongylus rubidus, a faecal culture was set up for identification at genus level (permin et al., 1999). faecal samples were mixed with some dry sterilized cow dung till a marshy consistency was reached this was done to improve aeration of the faecal sample. the faecal pots were then shut with a lid and placed in an incubator at 27c for seven days. the faeces dung mixture was turned once a day with a tongue depressor to aid in aeration of the samples before being returned into the incubator. after the seven days larvae were harvested using the technique described by the ministry of agriculture, fisheries and food (1977) upon which the nematode larvae were identified. nematode identification all nematode worm eggs were identified using a combination of keys given by soulsby (1982), uhlinger (1991) and foreyt (2001). nematode larvae were identified according to ministry of agriculture, fisheries and food (1977). statistical analyses the prevalence of each species of gi parasite was computed as: p  d n ; where: p is the prevalence, d is the number of individuals having the gi nematode at a particular point in time; and n the number of individuals in the population at risk at that point (thrusfield, 1995). the mean, median, standard deviation and range of the eggs per gram of faeces for each gi nematode species was also estimated. the log10 transformed data were analyzed using proc glm of spss 15.0 for windows ® (statistical package for social scientists, 2006). the effect of pig class and month, and their interaction on the prevalence and mean egg counts of gi nematode species was also determined. results a total of 143 pigs were sampled and 84 (58.7%) were positive for gi nematode eggs, 17.48% (25) had mixed infections, while 41.3% (59) of the pigs sampled were negative for gi nematode eggs. four parasite species were identified using the modified mcmaster flotation technique, namely ascaris spp, oesophagostomum spp, strongyloides ransomi and trichuris suis with the prevalences shown in table 1. the overall transformed (log10) range and mean egg counts for ascaris species, oesophagostomum species, strongyloides ransomi and trichuris suis are shown in table 1. month of sampling affected (p < 0.05) the prevalence and mean egg counts of the four gi nematode species. significantly higher (p < 0.05) overall monthly prevalences were recorded in april and may for s. ransomi and oesophagostomum spp respectively, compared to november for ascaris species and trichuris suis (table 2). there was a general decrease in overall monthly prevalence between may 2006 and october 2006 for all four nematodes species. mean monthly egg counts for all four parasite species had two major peaks, the first in november 2005 for all four nematode species; the second in february and march 2006 for ascaris species and trichuris suis respectively, and april and may 2006 for oesophagostomum species and s. ransomi, respectively (figure 1). pig class and the interaction between pig table 1. prevalence of common gastrointestinal parasites in mukota pigs of hama-mavhaire communal area. species* ascaris spp oes spp s. ransomi t. suis prevalence (%) 7.0 54.6 14.0 4.2 mean** 0.16 1.37 0.33 0.08 sd** 0.58 1.29 0.83 0.39 range** 0-2.95 0-3.61 0-3.11 0-2.00 *species names: ascaris species, oesophagostomum species, s. ransomi, trichuris suis. **log 10 transformed values. table 2. monthly prevalence of gi nematodes in the mukota pigs. month overall ascaris spp oesophagostomum spp strongyloides trichuris nov 05 55.6 38.9 b 38.9 b 5.6 a 22.2 b dec 05 23.0 0.0 a 23.0 a 7.7 a 7.7 a jan 06 60.0 0.0 a 60.0 b 40.0 b 0.0 a feb 06 43.8 12.5 a 37.5 b 18.8 a 0.0 a mar 06 64.7 5.9 a 58.8 b 32.3 a 0.0 a apr 06 100.0 0.0 a 90.91 c 45.5 b 0.0 a may 06 91.7 0.0 a 91.7 c 0.0 a 0.0 a jun 06 70.0 0.0 a 70.0 b 0.0 a 0.0 a jul 06 77.8 0.0 a 77.8 b 0.0 a 0.0 a aug 06 44.4 0.0 a 44.4 b 0.0 a 0.0 a sep 06 58.3 0.0 a 58.3 b 0.0 a 0.0 a oct 06 0.0 0.0 a 0.0 a 0.0 a 0.0 a abc indicates significant differences of the means. class and month did not have an effect on the prevalence and mean egg counts of the gi nematode species. discussion the current study is the first to be carried out for gi nematodes infesting free-range indigenous pigs under smallholder management in communal areas of zimbabwe. gastrointestinal nematodes were moderately prevalent in scavenging indigenous pigs in the hamamavhaire communal area of chirumhanzu district. apart from ascaris species, none of the parasites identified in the current study have been previously reported to affect mukota pigs in communal areas of zimbabwe. knowledge on the prevalence and significance of parasites in pigs in southern africa is rather limited, but our findings are, with regard to gi nematodes, largely in agreement with reports from other parts of africa (ajayi et al., 1988; salifu et al., 1990; esrony et al., 1997; permin et al., 1999; nsoso et al., 2000) . in nigeria, ajayi et al. (1988) examined faecal samples from 1140 pigs and observed that 97% excreted parasitic eggs. the range of species in their study is similar to our findings. interestingly, 90% of the pigs excreted a. suum compared to only 7% observed in the present study. salifu et al. (1990) made faecal examinations of about 1000 pigs in nigeria and observed the range of nematode species that is similar to the present study, but again with a higher prevalence for a. suum (60%). the high prevalence of a. suum in both nigerian studies might be due to the differrences in breed and production systems as reported by roepstorff et al. (1998). in tanzania, faecal samples from 424 local and crossbred pigs kept under different management systems, were examined (esrony et al., 1997). the coprological examination revealed that only 53% of the pigs excreted helminth eggs in their faeces. the range of species in this study is similar to the current study’s findings; however, the prevalence of s. ransomi is lower (9%) compared to our findings (14%). this might be attributed to the fact that the tanzanian study site included semi arid areas and as such these provide unfavourable environment for survival of strongyloides larvae as these larvae are susceptible to desiccation (melancon, 2003). faecal samples from 259 local cross-bred pigs in ghana were examined and 91% excreted parasitic eggs (permin et al., 1999). interestingly, s. ransomi, which was observed in the present study, was not identified in the ghanaian study. the 259 pigs sampled were all growers in the ghanaian study and s. ransomi usually affects younger piglets. in a study by nsoso et al. (2000), faeces 1000 ascaris spp 900 oes spp 800 strongyloides 700 trichuris c o u n t 600 500 e g g 400 300 200 100 0 nov dec jan feb mar apr may jun jul aug sept oct month figure 1. mean monthly egg counts of gi nematodes in mukota pigs. from 29 local and cross-bred pigs in botswana were examined. coprological examination revealed that 52% of the pigs excreted helminth eggs in their faeces. the eggs belonged to three different helminth species namely a. suum, trichostrongylus species and trichuris suis, with prevalences of 55, 24 and 8%, respectively. the prevalence of a. suum was markedly greater than that observed in the present study but a possible explanation to this finding could not be established. the prevalence of internal parasites is thought to be generally higher in young animals than in adults (visco et al., 1977; bugg et al., 1999); however, this was not observed in the present study. piglets are considered to be the most susceptible group and therefore it would not be surprising to find the highest egg counts in this age group. based on a study on production of indigenous pigs in the same area, chikwanha (2006) suggested the occurrence of high mortality in piglets in the hama-mavhaire communal area. nematode infections may be one of the contributing factors in piglet mortality. the pig-lets are undernourished and when exposed to heavily contaminated pastures, they acquire high levels of infec-tion with severe consequences. hence the few piglets sampled could be the only survivors that resisted infec-tion and so shed few gi nematode eggs thus giving a lower prevalence. among the adult animals only the boars had low mean egg counts that may be explained by age immunity (urqhart et al., 1996). clinical disease and death are not the common manifestations of internal parasitism and often develop only subsequent to malnutrition or stress (vassilev, 1999). though the impact of nematode infection was not determined, the moderate prevalence of gi nematodes and other helminth infections may be regarded as a real problem affecting productivity of the animals in the study site. our results suggest that antihelmintic treatment of animals should be carried out even if the levels of infection are only low to moderate. furthermore there should be extensive diagnostic and epidemiological studies on the internal parasites of pigs in this and surrounding communal farming areas, so that sound control programmes can be formulated. based on the present data, it is suggested that for the control of gastrointestinal nematodes, pigs in communal farming areas should be given an antihelmintic in april / may to destroy the worm burden acquired during the rainy season. this would also help the animals to withstand the nutritional stress of the dry season. a second treatment in the mid-rainy season may be advocated to reduce the build up of rangeland contamination during the rains. however any program should be applied at a locality level so that animals using the same rangeland are treated at the same time. it might be useful to treat the lactating sows to reduce the chances of contaminating piglets. it should be pointed out that any antihelmintic treatment regime would not be effective unless the nutrition of the animals is also improved (vassilev, 1999). knox and smith (2000) outlined the progress on vaccination against nematode parasite based on proteins isolated from the microvillar surface of the parasite enterocyte. this could be a new avenue in the control of gastrointestinal nematodes in the study site. in conclusion, our results show that parasite prevalence in local indigenous pigs in the hama-mavhaire communal area is moderate. the clinical examinations revealed that the majority of the animals were in good condition, sugg esting that the indigenous pigs have developed tole-rance to gi nematodes. further examinations are needed to determine the pathological importance and impact of parasitic infestations on indigenous pigs in the communal area. oesophagostomum species, s. ransomi, a. suum and trichuris suis with prevalences of 54.6, 14, 7 and 4.2% respectively, were the gastrointestinal nematode species observed to occur in indigenous mukota pigs of hama-mavhaire communal area of chirumhanzu district of zimbabwe. acknowledgements the authors sincerely appreciate the help of mr. r. kadewere, professor g. vassilev and the technical staff at the parasitology laboratory, faculty of veterinary science, university of zimbabwe in collecting and processing of faecal samples. the help of mr. cletos mapiye during data analysis is also recognized. references ajayi ja, arabs wl, adeleye ga (1988). helminths and protozoa of pigs on the jos plateau, nigeria: ocurrence, age, incidence and seasonal distribution. bulletin animal health and production in africa. 36:47. bugg rj, robertson id, elliot ad, thompson rc (1999). gastrointestinal parasites of urban dogs in perth, western australia. vet. j. 157(3): 295-301. chikwanha o (2006). msc thesis. university of zimbabwe, harare, zimbabwe. esrony k, kambarage dm, mutambo mma, muhairwa ap, kusiluka ljm (1997). helminthosis in local and cross-bred pigs in the morogoro region of tanzania. preventive veterinary medicine. 32: 41-46. foreyt wj (2001). veterinary parasitology reference manual, 5 th edition. blackwell publishers, iowa: pp.137 – 150. holness dh (1991). pigs. macmillan, london. p.16 kahn cm (2006). the merck veterinary manual, 9 th edition. merck and co. inc., whitehouse station, nj, usa. p.254 knox dp, smith wp (2001). vaccination against gastrointestinal nematode parasites of ruminants using gut expressed antigens. vet. parasitol. 100: 21-32. mashatise e, hamudikuwanda h, dzama k, chimonyo m, kanengoni a (2005). effects of corn cob-based diets on the levels of nutritionally related blood metabolites and onset of puberty in mukota and landracexmukota gilts. asian australasian j. anim. sci. 18(10): 1469-1474. melancon jj (2003). ivomec (ivermectin) brand products for swine: the parasite solution for today's pork producer. merial veterinary bulletin. mvb: pp. 3-12. ministry of agriculture, fisheries and food (1977). manual of veterinary parasitological laboratory techniques. crown printers: pp. 8-12. nsoso sj, mosala kp, ndebele rt, ramabu ss (2000). the prevalence of internal and external parasites in pigs of different ages and sexes in southeast district. botswana. onderstepoort journal of veterinary research. 67(3): 217-20. pattison hd, thomas rj, and smith wc (1980). veterinary record. 107: 415. permin a, yelifani l, bloch p, steehard n, hansen np, nansen p (1999). vet. parasitol. 87:63. roepstorff a, nansen p (1994). epidemiology and control of helminth infections in pigs under intensive and non-intensive production sys tems. vet. parasitol. 54: 69-85. roepstorff a, nilsson o, oksanen a, gjerde b, richter sh, örtenberg e, christensson d, martinsson kb, bartlett pc, nansen p, eriksen l, helle o, nikander s, larsen k (1998). intestinal parasites in swine in the nordic countries: prevalence and geographical distribution. vet parasitol. 76: 305-319. salifu da, mang tb, onyali io (1990). a survey of gastrointestinal parasites in pigs of the plateau and river states, nigeria. rev. elev. med. vet. pays. trop. 43(2): 193-196. soulsby ejl (1982). helminths, arthropods and protozoa of domesticated animals, 7th edition. lea & febiger, philadelphia. pp. 143-148, pp. 186-190. statistical package for social scientists (spss) 15.0 for windows, 2006. spss answernet. http://www.spss.com/tech/answer/ taylor dj (1999). pig diseases 7 th edition. glasgow. p. 49. thrusfield m (1995). veterinary epidemiology, 2 nd edition. blackwell science, london. pp. 39-41. uhlinger ca (1991). equine small strongyles: epidemiology, pathology and control. the compend. contin. educ. pract. vet. 13: 332338. urqhart gm, amour j, duncan jl, dunn am, jennings fw (1996). veterinary parasitology, 2 nd edition. blackwell science ltd, london. pp. 17-70. vassilev gd (1999). survey of trichinella spp. infection in pigs from commercial piggeries in zimbabwe. international – desepizooties. 18 (3): 753-757. visco rj, corwin rm, selby la (1977). effect of age and sex on the prevalence of intestinal parasitism in dogs. j am vet med assoc. 170 (8): 837-935. zanga j, chimonyo m, kanengoni a, dzama k, mukaratirwa s (2003). a comparison of the susceptibility of growing mukota and large white pig genotypes to infection with ascaris suum. vet res communic. 27: 653–660. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (7), pp. 001-004, july, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper prevalence of (high-pathogenicity island) hpi-harboring escherichia coli in diarrheic and healthy piglets darong cheng1*, xiaolang chen1, shanyuan zhu2, wenwei ding1, zhixia mu1 and jun zhou2 1 college of veterinary medicine, yangzhou university, yangzhou 225009, china. 2 jiangsu animal husbandry and veterinary college, taizhou 225300, china. accepted 22 october, 2014 one hundred and sixty four rectal swab samples were collected from 110 live diarrheic piglets and 54 healthy piglets during an investigation on the prevalence of hpi-harboring escherichia coli (hpi + e. coli) infection associated with porcine diarrhea. the data of polymerase chain reaction (pcr) revealed the presence of hpi + e. coli in 58.18% of swabs taken from diarrheic piglets, while 44.44% of samples taken from healthy pigs were so positive. further pcr examination of 600 bacterial isolates from diarrheic samples revealed that 25 isolates were hpi + e. coli while 4 (16%) were f4 + , 2 (8%) were f4 + and f6 + , 2 (8%) were f4 + and f6 + , and one (4%) was f6 + , lta + and stb + . among the 480 bacterial isolates from non-diarrheic samples, 20 isolates were hpi + e. coli, and only one (5%) was lta + and stb + , while absence of isolates belonging to the f4 + and f6 + groups was noted. o138 was the vast prevalent serotype among the hpi + e. coli isolates. it is suggested that hpi + e. coli maybe an opportunistic pathogen in swine. key words: escherichia coli, hpi, prevalence, piglet. introduction pathogenicity islands (pais) are a distinct class of genomic islands (geis), which are acquired by horizontal gene transfer. pais harbor virulence genes and some antibiotic resistance genes (oelschlaeger et al., 2004). the high-pathogenicity island (hpi) was previously described in yersinia pestis, yersinia pseudotuberculosis and yersinia enterocolitica (bearden et al., 1998; buchrieser et al., 1998a). in addition, hpi have been horizontally transferred to other bacterial species, such as shigella, citrobacter, klebsiella and escherichia (bach et al., 2000; karch et al., 1999; schubert et al., 1998). it was also reported that some hpi + e. coli isolates from patients or animals with diarrhea were closely correlated with clinical symptoms (xu et al., 2000; cheng et al., 2006). but the role of hpi + e. coli isolates play in the piglet diarrhea need to be deeply investigated and researched in detail. in this study, we detected hpi + e. coli infection in diarrheic and healthy piglets, and investigate the prevalence of the other virulence factors in the hpi + e. coli isolates. *corresponding author. e-mail: jsyzcdr@yahoo.com.cn. materials and methods collection of samples from march june, 2009, a total of 164 rectal swab samples were collected from piglets with a mean age of 6.5±3.4 days, including 110 live diarrheic piglets from 28 swine farms and 54 healthy piglets from 18 swine farms in the jiangsu province, china. this brings each test sample to a final volume of 500 μl with lb broth (10 g tryptone, 10 g nacl, 5 g yeast extract, h2o to 1 l, ph 7.4) and store at -70°c. bacterial pre-culturing and extraction of dna templates 100 μl dilution of each rectal swab sample was transferred into separate tubes containing 2 ml of lb broth and the liquid cultures were grown with vigorous agitation at 37°c for 6 h. 0.5 ml of the liquid cultures was transferred to labeled micro centrifuge tubes, and centrifuged at 10,000 rpm for 5 min. the supernatants were discarded and each pellet was resuspended in 200 μl of distilled water. after boiling for 10 min, the suspensions were chilled on ice for 5 min, centrifuged at 10,000 rpm at 4°c for an additional 5 min, and the supernatants used directly or stored at 4°c before pcr detection. pcr detection of hpi-harboring e. coli to detect hpi-harboring e. coli in the liquid cultures of rectal swab table 1. summary of the o serotype and virulence factors of the 25 hpi + e.coli isolates from diarrheic piglets. virulence factor numbers of lta sta stb f4 f6 isolates o serotype o138 12 o138 + 2 o138 + + 1 o138 + + + 1 o65 3 o139 + 2 o9 + 2 o55 + + 1 o141 1 total 1 0 1 4 7 25 samples, pcr assays were performed in micro centrifuge tubes for the applied biosystems (2720 thermal cycler america). the primers (f: 5’-aaggattcgctgttaccggac-3’, r: 5’tcgtcgggcagcgtttcttct-3’) were used for pcr amplifi-cation to detect hpi + e. coli (schubert et al., 1998) and synthesized by sangon biological engineering technology and service co. ltd. (shanghai, pr china). all the pcr reagents were purchased from takara biotechnology (dalian) co., ltd. the pcr mixture contained 5 μl of 10× pcr buffer (mg 2+ plus), 5 iu of taq poly-merase, 4 μl of dntp mixture (each 2.5 mmol/l), 1 μl of hpi primers set (each 50 mmol/l), 2 μl of dna template, and deionized water to a final volume of 50 μl. after denaturation at 94°c for 3 min, 30 cycles of the pcr was performed using the following program: denature-tion at 94°c for 30 s, annealing at 62°c for 30 s and elongation at 72°c for 60 s (10 min for the final cycle). the expected size of pcr products should be 287 bp following separation in 1% agarose gel electrophoresis along with dl2000 dna markers and visualization on a uv transilluminator after staining with ethidium bromide. virulence genes determination and o serotyping of hpiharboring e. coli following rapid pcr detection, 30 hpi-positive diarrheic samples and 24 hpi-positive healthy samples were submitted to separative cultivation of bacteria by streaking on mackonkey agar plates. after incubation at 37°c overnight, twenty colonies were picked from each sample and cultured on lb agar plates at 37°c for 24 h. all the bacterial isolates were submitted to pcr detection (cheng et al., 2005b, 2006) for the genes of hpi, enterotoxins (sta, stb and lta) and fimbriae (f18, f4, f5, f6 and f41). in addition, all the hpi-harboring e. coli were serotyped by using uni-factor serum of o antigen of e. coli. results among the 110 rectal swab samples from live diarrheic piglets, 64 (58.18%) samples (from 22 farms) were infected with hpi-harboring e. coli, while 24 (44.44%) samples (from 9 farms) were hpi-positive in the 54 healthy samples. six hundred bacterial isolates were picked out from the diarrheic samples, and the data for pcr examination determined that only 25 isolates (from 22 samples) were hpi + e. coli, thereinto, 4 (16%) were f4 + , 2 (8%) were f4 + and f6 + , and 1 (4%) was f6 + , lta + and stb + (table 1). in addition, the 25 hpi + e. coli isolates were o serotyped and o138 was the most prevalent serotype accounting for 64% (16/25), followed by o65 (12%), o139 (8%), o9 (8%), o55 (4%) and o141 (4%) (table 1). among the 480 bacteria isolates that were picked out from the non-diarrhoeic samples, just 20 isolates (from 18 samples) were hpi + e. coli, thereinto, only one (5%) was lta + and stb + , while no single f4 + and f6 + isolate was detected (table 2). furthermore, the 20 hpi + e. coli isolates were o serotyped and o138 also was the most prevalent serotype accounting for 60% (12/20), followed by o65 (25%), o21 (10%), o9 (5%), and o74 (5%) (table 2). discussions hpi was first discovered in pathogenic yersinia strains and has recently been found to be widespread in other enterobacteria (buchrieser et al., 1998b; hacker et al., 2000; carniel et al., 1992; fetherston et al., 1994; perry et al., 1990; schubert et al., 1998). hpi carries the gene fyua, which is specific for the pesticin receptor (fyua) and the irp (iron repressible protein, such as hmwp1 and hmwp2) loci encoding the siderophore yersiniabactin (carniel et al., 1992; guilvout et al., 1993; lucier et al., 1996). the hpi element is associated with asparaginespecific trna loci and carries an integrase gene int, often associated with a phage genome (buchrieser et al., 1998; rakin et al., 2001). but whether hpi could contribute to the virulence of e. coli isolates responsible for piglet diarrhea remains deeply a research question. in this research, the data revealed the presence of hpi + e. coli in 58.18% of swabs taken from diarrheic piglets, while 44.44% of samples taken from healthy pigs were table 2. summary of the o serotype and virulence factors of the 20 hpi + e.coli isolates from non-diarrheic piglets. virulence factor numbers of lta sta stb f4 f6 isolates o serotype o138 10 o138 + + 1 o65 5 o21 2 o9 1 o74 1 total 1 0 1 0 0 20 highly positive. therefore, we are suspicious of whether hpi could contribute to the virulence of e. coli isolates that causes piglet diarrhea. but after the analysis on the other hand, it was found that 78.57% diarrheic farms were infected with hpi + e. coli, while 50.00% healthy farms were confirmed to be hpi-positive. furthermore, some hpi + e. coli isolates from diarrheic samples more frequently contained other virulence-associated genes, such as toxin(s) and fimbria(e), while only one isolate from healthy samples was lta + and stb + , although the preva-lent o serotype all was o138 in diarrheic and healthy isolates. according to the previous investigation on the effects of different virulent factors of escherichia coli on pathogenesis (cheng et al., 2005a), it suggested that the hpi + e. coli most likely to be opportunistic pathogen. this attractive hypothesis has to be verified by comparing the virulence of the parental strain and that of the isogenic mutants in a suitable infection mode. it is well known that most e. coli are the normal inhabitants of intestinal tracts of animals (levine, 1987; martins et al., 2000), and it is very difficult to obtain the pathogenic e. coli in the rectal swab samples from live diarrheic piglets by bacteria isolation and identification. as a matter of fact, the clinical diagnosis of e. coli infection in piglets, especially in the rectal swab samples from live diarrheic animals, usually just need confirming whether the pathogenic e. coli was/were existed in the sample(s), and not always need bacterial isolation and identification anymore, while it is so difficult to pick out the pathogenic isolate(s). this is perfectly supported by the data of this research, such as 58.18% diarrheic rectal swab samples could be confirmed to be hpipositive by the rapid detection method, and only 22 hpi + e. coli were obtained in 600 bacteria isolates by bacteria isolation and identification; while most isolates were the important members of the normal microbiologic flora of piglets. the experience of this research could provide a constructive idea and use for the diagnosis of other pathogen infection. acknowledgement this work was supported by national natural science foundation of china (grant no. 30800821) and jiangsu province key laboratory of high technology researching program (grant no. bm2009701). we would like to give our thanks to all the staff of the veterinary microbiology laboratory of yangzhou university for their help with some experiments. references bach s, de almeida a, carniel e (2000). the yersinia highpathogenicity island is present in different members of the family enterobacteriaceae. fems bicrobiol. lett., 183: 289-294. bearden sw, fetherston jd, perry rd (1997). genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in yersinia pestis. infection immunity, 65: 1659-1668. buchrieser c, prentice m, carniel e (1998a). the 102-kilobase unstable region of yersinia pestis comprises a high-pathogenicity island linked to a pigmentation segment which undergoes internal rearrangement. j. bacteriol., 180: 2321-2329. buchrieser c, brosch r, bach s, guiyoule a, carniel e (1998b). the high pathogenicity island of yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn trna genes. molecular microbiol., 30: 965-978. carniel e, guiyoule a, guilvout i, mercereau-puijalon o (1992). molecular cloning, iron-regulation and mutagenesis of the irp2 gene encoding hmwp2, a protein specific for the highly pathogenic yersinia. molecular microbiol., 6: 379-388. cheng d, huang w, zhang y, zhang k, xu j (2005a). the detemintion of virulence of seven strains of different virulent of escherichia coli. progress in veterinary medicine, (in chinese), 12: 78-80 cheng d, sun h, xu j, gao s (2005b). prevalence of fimbial colonization factors f18ab and f18ac in escherichia coli isolates from weaned piglets with edema and/or diarrhea in china. vet. microbiol., 110: 35-39. cheng d, sun h, xu j, gao s (2006). pcr detection of virulence factor genes in escherichia coli isolates from weaned piglets with edema disease and/or diarrhea in china. veterinary vet. microbiol., 115: 320-328. fetherston jd, perry rd (1994). the pigmentation locus of yersinia pestis kim6 + is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and hmwp2. molecular microbiol., 13: 697-708. guilvout i, mercereau-puijalon o. bonnefoy s, pugsley ap, carniel e (1993). high-molecular-weight protein 2 of yersinia enterocolitica is homologous to angr of vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis. j. bacteriol., 175: 5488-5504. hacker j, kaper jb (2000). pathogenicity islands and the evolution of microbes. ann. rev. microbiol., 54: 641-679. karch h, schubert s, zhang d, zhang w, schmidt h, olschlager t, hacker j (1999). a genomic island, termed high-pathogenicity island, is present in certain non-o157 shiga toxin-producing escherichia coli clonal lineages. infection immunity, 67: 5994-6001. levine mm (1987). escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. j. infect. dis., 155: 377-389. lucier ts, fetherston jd, brubaker rr, perry rd (1996). iron uptake and iron-repressible polypeptides in yersinia pestis. infection immunity, 64: 3023-3031. martins mf, martinez-rossi nm, ferreira a, brocchi m, yano t, castro af, silveira wd (2000). pathogenic characteristics of escherichia coli strains isolated from newborn piglets with diarrhea in brazil. vet. microbiol., 76: 51-59. oelschlaeger ta, hacker j (2004). impact of pathogenicity islands in bacterial diagnostics. acta pathologica, microbiologica et immunologica scandinavica, 112: 930-936. perry rd, pendrak ml, schuetze p (1990). identification and cloning of a hemin storage locus involved in the pigmentation phenotype of yersinia pestis. j. bacteriol., 172: 5929-5937. rakin a, noelting c, schropp p, heesemann j (2001). integrative module of the high-pathogenicity island of yersinia. molecular microbiol., 39: 407-415. schubert s, rakin a, karch h, carniel e, heesemann j (1998). prevalence of the “high-pathogenicity island” of yersinia species among escherichia coli strains that is pathogenic to humans. infection immunity, 66: 480-485. xu jg, cheng b, wen x, cui s, ye c (2000). high-pathogenicity island of yersinia spp. in escherichia coli strains isolated from diarrhea patients in china. j. clin. microbiol., 38: 4672-4675. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (9), pp. 001-005, september, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper the cost of double standard risk communication during the swine-flu epidemic: reflections from norway john e. berg oslo university college, faculty of health sciences, p. o. box 4 st. olavs plass, 0130 oslo, norway and blakstad hosptial vestre viken hospital trust, 1309 rud, norway. e-mail: john@pong.no. tel: +47 22452000. accepted 22 march, 2014 the pandaemia of swine influenza 2009 released a multitude of reactions and actions of public authorities and private enterprises. after the first announcement of a probable new epidemic in may 2009 who issued a pandaemia warning in june 2009. norwegian health authorities reacted swiftly to the who recommendations and norway became the first country opting for buying vaccines for the whole population. the background of the strategy in norway is described using paper media communications and with reference to television broadcasts. the emanating maximizing and minimizing strategy followed by the norwegian health authorities was followed. tests of the swine flu virus were not performed on a scale sufficient to indicate the magnitude of the purported pandaemia. both strategies were followed simultaneously. who indicated in june 2010 that hitherto 18 337 people died of the h1n1 influenza virus compared to some 500 000 from the common flu each year (aftenposten 25 th july 2010, p. 2). more than $ 14 billion had been spent on vaccines and other medical tasks (> $ 750 000 per fatality). in norway the cost of 9.4 mill. doses of vaccines bought was equivalent to $ 3.7 mill. per fatality in 2009. an initial communication of immense risk and purchase of two vaccine doses for the total population led to rationing of vaccines when people subsequently wanted them. the cost of this decision was far above accepted limits for resource spending on health care. this was followed by communicating a small risk of infection for most of the population. experts exposed differing views on the developing epidemic. risk communication from high decision levels was inadequate. strategies for handling emerging high risk epidemics must be developed and applied in order to reduce fear in the population and huge costs to society. key words: swine influenza, pandaemia, risk communication, cost effectiveness of public health. introduction strategies for curbing communicable diseases are well developed and exercised, but new infections may not be handled in a cost efficient and supporting way for the public at risk. however, effective remedies are available and logistics may function according to needs in developed countries. during the early face of a pandaemia relevant information may be hard to get as shown in a study by norgaard and lazarus (2010). using pubmed as a source they found that only 27.8 records out of 44.3 potential records per week were relevant. search strategies are sensitive to inconsistent naming or changing nomenclature. if a pandaemia occurs, the risk for health care professionals at work, especially in hospitals, could increase. hospital effectiveness may thus, be hampered (damery et al., 2009). in some areas of the world access to and delivery of vaccines are restricted because of socio-economic, logistic or religious disparities. persons with influenza like virus diseases have been treated with prophylactic vaccines at a voluntary basis each year in norway, that is, the common flu or seasonal influenza. the death toll for a population of 4.7 million inhabitants has been fluctuating between less than hundred and 250 per year. vaccination of elderly and immune-depressed people has been recommended. the vaccines have been given free of charge for patients in institutions and rest homes. generally, doctors and nurses giving the vaccine did not always get themselves vaccinated. many employers, within healthcare organizations and in private companies have offered the vaccine to employees, in order to curb sickness absence when getting ill. the death toll of the common flu is not much referred to in media, whereas the new influenza with 29 persons certified deceased during 2009 in norway, was intensely focused in media (table 1). healthcare workers wanting to keep up to date consult information sources of differing quality and quantity, from scientific journals to public internet information sites. there are some evidence that prestigious journals which are associated with publications of papers partly funded by industry. there is no straightforward relation between study quality, concordance, funding and impact in studies of influenza vaccines (jefferson et al., 2009). the director general of world health oganisation (who) dr chan recently dismissed the allegations of influence over decisions at who from the pharmaceutical industry during the first face of the pandaemia (zaracostas, 2010). the british department of health renegotiated the initial deal over swine flu vaccine order when it turned out that much less was needed (mayor, 2010). a study of influenza pandemic preparedness has indicated low interest among many employers (watkins et al., 2007). another survey during the swine flu pandaemia showed an increased perceived risk (seale et al., 2010). the survey measured perceived risk, seriousness of disease, recent behavioural changes and acceptance of the vaccine in 627 respondents. when asked how much affected you would be 61.4% indicated somewhat affected. 57.7% thought the authorities were doing a good job and half the respondents thought that getting the flu would be out of their control. washing of hands was increased in half the respondents. there was a greater willingness of taking the vaccine among women compared to men. risk communication is thus difficult. the general public may have difficulties understanding standard epidemiological jargon. for instance, the difference between relative and absolute risks is not understood by many. in an online survey of 1290 us adults respondents from larger households reported stronger interest in taking medications and stick to hygienic measures (ibuka et al., 2010). perceived risk increased over time, but interest in pharmaceutical preventive measures and in precautionary hygienic activities decreased over time. a high risk in the state did not predict greater interest in pharmacological interventions. five hundred parents indicate a willingness to vaccinate against swine-flu if they had previously let their children be vaccinated (flood et al., 2010). generally, they would follow the advice of the gp. among parents who did not usually let their children get vaccines 44% even stated a perception of the vaccine giving influenza. cowling et al. (2010) sampled 12,965 hong kong residents in a telephone survey between april and november 2009 (cowling et al., 2010). respondents reported low anxiety levels throughout the epidemic. perceived severity was initially high, but declined during the epidemic. knowledge of modes of transmission did not increase during the epidemic. community mitigation activities had little effect. paradoxical risk perception was observed in the urban population of laos as a result of an educational campaign against the avian flu (barennes et al., 2010). after the campaign, the urban population considered risk to be decreased and unsafe behaviour increased. in the rural areas producing poultry, the reaction was opposite. health professionals should be able to evaluate epidemiological and biological research. in a web-based survey of 947 health–care workers in australia 59% were not convinced that australia was sufficiently prepared for the swine-flu (tebruegge et al., 2010). a third person would refuse to participate in screening suspected individuals. almost half of the group had a private supply of vaccines, and only half of the group had a realistic estimate of the mortality associated with an influenza epidemic. the sheer magnitude of people involved in an epidemic is not properly comprehended. knowledge about risk communication is limited or the practical application of it is clumsily handled (moore et al., 2008). a pandaemia has to be met by sufficient and effective countermeasures. however, a choice must be made between preemptive fast reactions on the basis of assumptions in the early face of an epidemic, and factbased reactions resting on more information. some would argue that a combined approach would be necessary, but such a strategy could be bewildering for the public (cornier et al., 2009). the observation of the strategy followed by norwegian authorities and its ensuing cost as depicted in newspapers and non-scientific journals was the aim of the present commentary. materials and methods the materials of this study were newspaper and broadcasted information of recommendations from the health authorities. the newspapers and journals read were both norwegian and european. the strategy of the world health organisation (who) emanated from some of the media references.the communicated recommendations and facts were analyzed using a framework of a dichotomized strategy of risk minimization and maximization as followed by the norwegian health authorities. a list of the articles from newspapers and periodicals used in the work may be obtained from the author results and observations data on the spread of common flu and swine flu in norway and usa during 2009 are depicted in table 1. the figures are not exact, as no country does test all who would have clinical signs of influenza, and post-mortem investigations are few and results not always reliable for viral diseases. swine-flu is a new version of influenza disease which has affected more young than old people. thus, it is suggested that the data for the case of swineflu would be more correct than those for the common flu. table 1. approximate death toll from common influenza and the swine-flu in 2009 in norway and usa. norway* norway usa# usa per 100,000 population per 100,000 population swine-flu 29 0.62 8330 -17160 2.71 – 5.60 common flu 100 2.13 36000 11.73 *) folkehelsa (state institute of public health), #) center of disease control and prevention. table 2. number of tests for swine flu virus (h1n1) and rhinovirus and percentage of positive tests according to month of testing in norway. h1n1 tests h1n1 % positive tests rhinovirus tests rhinovirus % positive tests august 2009 544 3.5 123 39.8 september 2009 440 0.7 67 56.7 october 2009 521 25.3 92 46.7 november 2009 1267 39.8 114 32.5 table 3. two strategies for risk communication to the population during an epidemic. strategy 1: minimize risk perception and calm the population strategy 2: maximize risk perception and keep the population alerted buy vaccines at same level as for the common flu face mask use discouraged keep schools open vaccination according to narrowly defined risk groups doubts about effectiveness of vaccination among virologists the economic impact of the swine flu is small front edge strategy: order and buy vaccines for the whole population (for vaccination twice) face mask use enhanced through free delivery close schools and kindergartens open vaccination of politicians and senior health officials first dissuade hugging in schools some 900 000 norwegians were allegedly infected. 2.2 million inhabitants got the vaccine. 1300 persons were remitted to hospital and 200 received intensive care. laboratory testing was initially recommended, but laboratory capacity was not sufficient and the authorities soon recommended not testing of persons without serious signs of disease. general practitioners sent the samples to the laboratory. the percentage of positive tests among those tested is shown in table 2. how did the norwegian health authorities meet this challenge? over time, the health authorities and health care politicians gave disparate and changing information, both according to scope and content. two different paradigms of information can be summarized as in table 3: i. pandaemia with less health effects than the seasonal influenza will develop, strategy 1 in table 3. this point, if true, would not generate a need for other actions than in a normal seasonal influenza year. no intervention would be recommended in schools and hospitals, and recommendations for vaccination would be the usual weak and immunosuppressed groups of patients. but early observations indicated a substantial mortality rate in younger age groups than for the well known seasonal influenza. one might suppose that, the health authorities would be afraid of being condemned as reckless if they maintained the advice of strategy 1, especially if media wrote extensively on the first child dying in the early face of the epidemic. this was the case in some of the newspaper articles listed below. the norwegian health authorities appeared on tv and radio informing the public on both strategies depicted in table 3, often within the same five minute broadcast. ii. pandaemia with serious health effects will develop strategy 2 in table 3. according to this strategy there is a need for harsh measures and immediate action. this was demonstrated in norway by a very rapid decision. the government bought 9.4 million doses of the vaccine under development, that is, enough to vaccinate the whole population twice. the price was 69 nok = $ 11 per dose, that is, $ 108 mill. early indications from who suspected a need for two vaccinations in every person under risk. as far as we know no other country did order so many vaccines. some members of the medical profession were interviewed in the media exposing recommendation with unclear epidemiological meaning. like stating that women should not get pregnant during the epidemic, although a risk in early pregnancy was not documented, and the uncertainty introduced by such a statement would make life intolerable for some. especially if inadvertently getting pregnant during the epidemic. information regarding putative actions in schools, rest homes and kindergartens had a double standard. one day no actions were advocated, the next day closing of schools was recommended. vaccination of health workers was initially done only in emergency medical units, not in emergency psychiatric units, not taking into account that persons with serious psychiatric illnesses would constitute a greater risk of getting infected or spreading the infection to others. by maximizing risk perception before enough vaccines were available, large queues and aggressive incidents occurred at general practitioner offices. conclusions the consequences of uncertain and dispersed advice from health authorities and its reception in media were: 1. huge costs of ordering swine flu vaccine at a time when it was not even put into production in order to secure delivery before other countries came up with a decision. who has indicated that hitherto 18 337 people died of the h1n1 influenza virus compared to some 500 0 who die of the common flu each year (aftenposten 25th july 2010, p. 2). more than $ 14 billion has been spent in the world on vaccines and other medical tasks (> $ 750 000 per fatality). the norwegian cost of 9.4 mill. vaccine doses in 2009 is equivalent to $ 3.7 mill per fatality that year. 2. untoward buying of two vaccines per person at risk. this turned out to be unnecessary. the cost could thus have been the half of the above mentioned. 3. creating havoc at vaccine offices, general practice offices and some hospitals as people rushed to get a rationed good. 4. vaccination of health workers in a sequence ignoring the risk of acutely ill persons with mental disorders. only health workers caring for acutely somatically ill persons were initially vaccinated. no compulsory vaccination of any group was instituted. top health authorities and members of the government got priority vaccinations. 5. trust in health authorities reduced. both the general public and medical and other experts had difficulties following and understanding advice given. 6. the death toll of common flu is according to data from 2009 about four times higher per 100,000 inhabitants than for the swine flu (table 1). 7. the media coverage contributed to two strategy initiatives focusing on the few severe illness episodes and the organization of distributing a rationed good. risk communication must be based on knowledge and given to the public in an understandable and coherent way. standard explanations of risk models should be available to all media as soon as an epidemic or other catastrophes occur, and they should be produced and ready in the relevant public relation offices of the health authorities. it would be difficult and probably unreliable what journalists themselves could produce at short notice. if media could run such pre-made broadcasts instead of repeating stories on extreme cases, both the public and health authorities would be able to absorb the different messages of table 3 in a meaningful manner. some newspaper articles (vårt land 17.11.09) indicated that the vaccine producing companies had a very strong negotiating power at the start of the epidemic, maybe contributing to the huge costs incurred. addendum after the main work with this paper was done reports have been published showing uncertainty regarding the decision on buying the vaccines (aftenposten 23.10.10). three levels of the health authorities disagree on who was responsible for the decision. references aftenposten (2010). hvem bestemte at vi skulle vaksineres? beslutning tatt for raskt, mener dsb” who decided the vaccination strategy? decision taken too soon, says the directorate of public security 2010., q.e.d. aftenposten (2010). “over og ut for svineinfluensaen” swine flu all ver. barennes h, harimanana an, lorvongseng s, ongkhammy s, chu c (2010). "paradoxical risk perception and behaviours related to avian flu outbreak and education campaign, laos." bmc infect. dis., 10(1): 294. cormier s, septimus ej, moody j, perlin jb (2009). a community health system response to the pandemic of novel h1n1. world. med. health. policy, 1(1): 35-46. cowling bd, ng dm, ip dk, liao q, lam ww, wu jt, lau jt, griffiths sm, fielding r (2010). community psychological and behavioral responses through the first wave of the 2009 influenza a(h1n1) pandemic in hong kong. j. infec. dis., 202(6): 867-876. damery ss, wilson s, draper h, gratus c, sheila greenfield, ives j, parry j, petts j, sorell t (2009). will the nhs continue to function in an influenza epidemic? a survey of healthcare workers in the west midlands, uk. bmc. public. health, p. 9. flood em, rousculp md, ryan kj, kathleen m. beusterien, victoria m. divino ba, toback sl, m sasané, sl block, hall mc, mahadevia pj (2010). parents' decision-making regarding vaccinating their children against influenza: a web-based survey. clin. ther., 32(8): 1448-1467. ibuka y, chapman gb, meyers la, m li, ap galvani (2010).the dynamics of risk perceptions and precautionary behavior in response to 2009 (h1n1) pandemic influenza. bmc. infect. dis., p. 10. jefferson t, pietrantoni cd, debalini mg, rivetti a, demicheli v (2009). relation of study quality, concordance, take home message, funding, and impact in studies of influenza vaccines: systematic review. br. med. j., pp. 338-354. mayor s (2010). department of health agrees deal with gsk to cancel part of swine flu vaccine order. br. med. j., p. 340. moore ra, derry s, hj mcquay, j paling (2008). what do we know about communicating risk? a brief review and suggestion for contextualising serious, but rare, risk, and the example of cox-2 selective and non-selective nsaids. arthritis research. therapy. norgaard o, lazarus jv (2010). searching pubmed during a pandemic. plos one, 5(4): 1-7. perceptions towards the pandemic (h1n1) 2009 vaccine." bmc infect. dis., 10: 99. seale hae, heywood ae, mclaws m, ward kf, lowbridge cp, van d, cr macintyre (2010). why do i need it? i am not at risk! public. tebruegge mpa, ritz n, connell t, bryant p, donath s, curtis n (2010). perception, attitudes and knowledge regarding the 2009 swine-origin influenza a (h1n1) virus pandemic among health-care workers in australia. j. paediatr. child. health, pp. (673-9). vårt land (2009). sterk lobby bak pandemibeslutning” a strong lobby behind decision to declare a pandaemia. about the disclosure of the special adviser of the who dr albert osterhaus having economic interests in vaccination production firms. vårt land (2009). influensaviruset kan komme tilbake” the influanza virus may return. interview with director of the health authority and the health minister. watkins re, cooke fc, donovan rj, cr macintyre, itzwerth r, plant aj (2007). "influenza pandemic preparedness: motivation for protection among small and medium businesses in australia. bmc. public. health, 7: 157. zaracostas j (2010). swine flu pandemic review panel seeks access to confidential documents between who and drug companies. br. med. j., p. 340. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 4 (6), pp. 001-009, june, 2016. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review energy and protein requirements of pigs and the utilization of fibrous feedstuffs in nigeria adesehinwa, a. o. k. swine research unit, livestock improvement programme, institute of agricultural research and training, obafemi awolowo university, p.m.b. 5029, moor plantation, ibadan. nigeria. e-mail: aokadesehinwa@yahoo.com. accepted 11 december, 2015 a sound nutrition programme, which allows the understanding of the interaction between nutrients and requirements for different levels of production, is essential for raising swine profitably. nutrient requirement is that amount of each essential nutrient that will result in maximum production with a minimum of feeding. hence, high quality feeds containing the essential nutrients in the amounts necessary to meet the animal’s requirements must be provided, in order to attain an optimal rate and efficiency of growth from birth to market. the formulation of balanced diets that provide the correct amounts and proportions of these nutrients is essential to support the requirements for maintenance, growth and reproduction of the animal. it is only then that the feeding programme can be said to have a major impact on the performance and the overall profitability of the swine herd. the scarcity of conventional feeds has hindered the growth and development of the livestock industry in nigeria. the general shortage of energy and protein feeds appear to be more severe for non-ruminants that depend to a great extent on compounded feeds, especially pigs, which are bulk feeders. there are a number of agro-industrial by-products, farm waste or crop residues that have been exploited as alternative feed sources for the high energy cereals. this has made a substantial contribution towards better and more economic feeding of non-ruminants. however, fibrousness, a feature of most locally available agro-industrial by-products and wastes has limited their use. key words: feeding programme, balanced diets, fibrous feedstuffs. introduction in recognition of the potential of pig as a prolific and fast growing animal, as well as a good converter of feed to meat, many nigerian farmers have embarked upon intensive production of pigs. this is an effort geared towards increasing animal protein supply, at reduced cost for human consumption (adesehinwa et al., 1998). pig production represents the fastest means of correcting animal protein shortage in africa. this is because, apart from their high rate of reproduction, poultry and pigs are characterized by the best efficiency of nutrient transformation into high quality protein (meat), although the cost of the transformation is very high (tewe and egbunike, 1988). therefore, nutrient supply has to be judiciously manipulated to ensure the production of meat at economic rates. there exists in country some agroindustrial by-products and crop residues that can be used as total or partial replacements for the conventional ingredients in finished livestock feeds. pigs are capable of converting these agro-industrial by-products or „wastes‟ of all kinds (which will normally be discarded by humans) into wholesome animal protein useful to the human being. the nrc (1988) recommendations of nutrient requirements, assumed to contain 90% dry matter and based on corn-soybean meal diet has been questioned in the tropics by the depressed feed intake, hence, depressed nutrient intake, resulting from the high ambient temperature and humidity prevalent in the tropical environment (tewe and adesehinwa, 1995a). rao et al. (1976) concluded that animals in the tropics performed better when fed on an energy level 10% lower than the nrc recommendations. fetuga (1984) in a similar study on energy, protein and amino acids requirements of pigs raised in the humid tropic concluded that a higher dietary protein is required. nutrient requirement was however reported (conrad, 1984) to be such that it gives an account of the amount of each essential nutrient that will result in maximum pro duction with a minimum of over-feeding. he observed that these requirements must include a factor of safety so that normal variation in the composition and nutritive value of feeds and in the functional capacities of animals will never result in under feeding. it should be noted that the safety cannot be introduced until minimum requirements have been established. the minimum requirements of the essential nutrients have been report-ed for all classes of pigs both in the temperate regions (cooke et al., 1972; wylie and owen, 1978) and the tropical areas of the world (devendra and parris, 1970; fetuga, 1972, 1984; babatunde et al., 1972; dividich and canope, 1978). energy requirements the energy requirements of pigs are very variable. they are higher with increasing body weight, as basal metabolism and maintenance requirements are proportional to live weight as well as to growth rate (serres, 1992). the more the animal grows, the more it requires energy to sustain this growth. from weaning until 20 kg, 1 kg of live weight represents about 3000 calories while at 90 kg, it is 5000 calories and it is beyond 6000 calories for fat pigs (serres, 1992). energy supplied to the animals may be measured as gross, digestible or metabolizable. the gross being that amount of energy liberated during complete combustion of the diet, which is of little practical value as a measurement. the digestible energy (de) is that proportion remaining after digestion and may be estimated from the difference between energy of food ingested and energy of faeces voided. while, the metabolizable energy (me) is the digestible less energy lost in the urine (serres, 1992). farrell (1978), agricultural research council (1981), and morgan and whittemore (1982) suggested that de is preferable in describing the energy content of swine feeds because de is more easily and precisely determined than me. the gaseous loss of energy in the digestive tract of swine is usually between 0.5 and 1.0% de (verstegen, 1971; fuller and boyne, 1972). the values are small and not measured hence they are ignored (nrc, 1988). the quality and quantity of protein in the diet was reported (may and bell, 1971; den hartog and verstegen, 1984) to affect the relationship between me and de. metabolizable energy decreases if protein is of poor quality and with excess protein because the amino acids not used for protein synthesis are catabolized and used as a source of energy and the nitrogen is excreted as urea (nrc, 1988). the energy level of the diet must be related to other components. longe (1988) reported that good diet formulation required that a balance be attained between the percentage crude protein and the number of energy units in the diets. this view was supported by fashina (1991) who reported the existence of a relationship between the dietary energy and protein requirements of pigs. energy : protein ratios that deviate from those that will make animals perform optimally will therefore result in marked difference in performance (longe, 1988). the net energy (ne) is the best measure of energy available to an animal for maintenance and production (nrc, 1988), and it is lower than the me because of losses associated with biochemical pathways within the animal (serres, 1992). it defines the efficiency of utilization of the productive purposes. the ne value is influenced by the composition of the feedstuff, level of feed intake, balance of nutrients in the diet, age, breed, sex, body condition of the pigs, environmental conditions under which the animal is maintained, and percentage of energy retained as protein (farrell, 1979; van der honing et al., 1985). the de requirement of the growing pig was described by nrc (1988) as the sum of its requirements for maintenance, protein retention, fat retention and cold thermogenesis. maintenance energy requirements include needs for all body functions and moderate activity and it is expressed on a metabolic weight basis. although the mean energy cost per kg of protein or fat deposited are approximately equal (wenk et al., 1980), 1 kg of lean muscle tissue is only 20 to 22% protein. therefore, the energy cost for muscle production is considerably less than that for fat production (nrc, 1988). the cold thermogensis influences energy requirements when the ambient temperature is below the critical temperature (which is the point below which an animal must increase heat production to stay warm). the energy content of the diet therefore generally controls the amount of feed consumed ad libitum daily (arc, 1981; cole, 1984) and pigs will compensate for decrease or increase in the nutrient density of the diet by increasing or decreasing their feed intake. this compensation normalizes energy intake within limits (nrc, 1988). however, voluntary feed intake varies considerably from day to day and among individual pigs (frank et al., 1983). the metabolic utilization of the metabolizable energy or heat increment was reported by noblet et al. (1994) to vary according to the diet‟s chemical characteristics and type of production (maintenance, growth, milk secretion, protein and fat deposition etc). the average efficiencies of utilization of the me for these different purposes have been shown to differ markedly in swine: approximately 80% for fat gain (kg) or maintenance; 60% for protein deposition; 75% for weight gain (kg) during growth and 70% for milk (arc, 1981; noblet et al., 1990, 1991 and 1993). protein requirements in swine nutrition, a good quality protein is one that provides the ten essential amino acids required for the normal body function in the amounts and proportions necessary for the particular need of the pig (adesehinwa and ogunmodede, 1995). traditionally, swine diets are formulated on the basis of crude protein. this refers to the nitrogen content of the feedstuff x 6.25 (nrc, 1988). amino acids are critical nutrients required by all classes of swine for the physiological processes of maintenance, growth, gestation and lactation (fashina, 1991). hence protein levels are established for the various weight classes of pigs so that the most limiting amino acid (lysine) will be present in adequate amounts (adesehinwa and ogunmodede, 1995). amino acids are reported to be the chemical components of protein and are generally supplied to the pig from the crude protein in the diet (fashina, 1991). failure to supplement a low protein diet or feedstuff with sufficient amounts of high quality protein source was reported by adesehinwa and ogunmodede (1995) to result in poor growth, inefficient feed utilization, increased carcass fatness, general unthriftiness and or reduced reproductive performance. the capacity of the diet or feedstuff to provide sufficient indispensable (essential) amino acids and nitrogen for the synthesis of dispensable (non essential) amino acids determines the adequacy of the dietary protein level (nrc, 1988). hence, the need for a nutritionally balanced ration in an economically viable pig production system (adesehinwa, 1992). lower levels of dietary protein are required to maximize growth and efficiency of gain for a protein source with well balanced amino acid profile (bender, 1975) in contrast to a protein source with poor amino acid pattern where more is required, to be efficiently utilized (adesehinwa, 1992). protein quality therefore becomes synonymous with amino acid balance. the amino acid requirements expressed as a percentage of the diet decrease as the pig becomes heavier, that is, the requirements are greatest during the rapidly growing stages of the young animal (conrad, 1984). the young animal was reported as not only growing at a more rapid rate as indicated by the percentage increase in the weight but that the proportion of the protein in the entire body weight is higher than during the finishing period. these changes in the rate of growth and body composition are the basis for recommending diffe-rent dietary protein levels to meet the amino acid requirements during the life of the pig (fanimo, 1991). fetuga (1984) reported the protein and amino acid requirements for the tropics to be higher than those recommended by nrc (1979) for the temperature zones. this view was also reported in an earlier work by babatunde et al. (1972). the variation in the recommended protein and amino acid allowances for different classes of pigs was attributed in part to variations that exist in the protein sources or protein qualities used. utilization of alternative feedstuffs by pigs livestock production in nigeria which had its boom in the late 70s is now taking a downward trend because of the high cost of production. commercial production takes two distinct forms or levels. there are large scale and small scale producers, each having its operational characteristics. however, for the successful take-off of either of the two scales of production, availability of feedstuffs in the right quality, quantity and price are very important. the astronomic rise in the price of livestock feeds has recently reached an alarming proportion. scarcity and outright non-availability of raw materials for feeds has hampered production and expansion of the livestock industry. stock population decrease is being embarked on by some farmers in order to meet the increasing feed bills and in most cases terminating in outright withdrawal from production. this upward trend in feed cost is due largely to steep prices of and diversified use of maize, increasing prices of guinea corn, groundnut cake, soybean products and fish meal. these shortages have either kept most feed mills in the country idle or operating, well below production capacity. these increases in prices over the years have also resulted in increased prices of some livestock products. apart from the high prices, other factors include, astronomical and uncontrollable shortage of basal feed ingredient, the cereal grains; growing shortage of oil seed cakes which nigeria used to produce and export in large qualities; adulteration of feed ingredients (particularly common with fish meal and groundnut cake, which suppliers now blend with brown sand and saw dust); lack of quality control body or the inefficiency of such body to monitor ingredient and finished feed composition and quality, and the seeming lack of government incentive to feed manufacturers or producers. while the shortages of raw materials for livestock feeds production, particularly poultry and pigs are numerous, however, the emphasis seems to be on the shortage of grains (basal energy feed) and protein supplements which together constitute about 70-80% of finished products (noblet et al., 1994). with the ban on their importation and the increasing demand for maize for industrial use as livestock feeds, beer production, baby foods, local consumption and staple food, the need to find substitutes to these items becomes urgent. such substitutes must be readily available or should be produced in commercial quantities at lower cost. this in essence means shifting to ingredients for which there is less competition by other secondary industrial users and producers. alternative energy sources maize is a major grain crop grown in nigeria and is used mainly in human foods as well as energy feed ingredient in animal production. its use in the brewing industry and production of composite flours for confectioneries has robbed the livestock industry of its fair share of the total maize available in the country (longe, 1988). this is re flected in the high prices of the finished feeds containing maize. since the feed crisis of 1983, efforts have been made to seek alternative energy sources to maize, and some other conventional feed ingredients. these alternative energy sources must be locally available, cheap and be able to replace a certain proportion, if not all of maize normally incorporated in diets of pigs without adverse effect on performance and product. most of these energy sources, if not all, are agro-industrial by-products that are low in energy, high in fibre and bulky in nature. they were hitherto used as energy diluents in swine ration (tegbe et al., 1995). generally, except for rice offal and cassava peel meal, the crude protein content of most of the agro-industrial by-products are higher that that of maize. their essential amino acid profiles also compare favourably with that of maize, thus showing that they could serve as sources of some essential amino acid in livestock feed (tegbe et al., 1995). feed ingredients which have been successfully incorporated into the diets of pigs as alternative energy sources include wheat offal (tegbe et al., 1984; tegbe et al., 1986), brewers dried grains (tegbe, 1983 and tegbe, 1985a), rice offal (tegbe et al., 1984 and tegbe, 1985b), rice mill by-product (attah et al., 1991), sorghum offal (adesehinwa, 1992; tegbe et al., 1995), cassava peel meal (tewe and oke, 1983; nghi, 1986; tewe et al., 1987; iyayi and tewe, 1988; tewe and egbunike, 1988; tegbe et al., 1992a; tewe and adesehinwa, 1995a), palm kernel cake (tegbe and jegede, 1988; tegbe et al., 1992b; jegede et al., 1994; tegbe et al., 1995) and maize offal (tewe, 1988a; longe and fagbenro-byron, 1990; ande, 1992). alternative animal and plant protein sources the alternative animal protein sources are animal byproducts obtained primarily from slaughter houses, surplus milk products and aquatic sources or wastes arising from animal production (sonaiya, 1988). virtually all these by-products can be used in the formulation of livestock feeds to supply protein or amino acids and minerals, in appreciable amount. they include meat and bone meal, blood meal, chicken offal meal, snail meal, prawn dust, shrimp meal, insect and fly larvae meal. other animal protein sources available for livestock feeds include meat scrap, hatchery by-product meal and, feather and hair meal. the sources of plant origin include soybean meal, groundnut cake sunflower meal and rapeseed meal. soybean meal is usually the most economical source of high quality plant protein available (adesehinwa and ogunmodede, 1995) because in terms of quality of amino acid content and ratio, it compares favourably with animal protein sources in most swine diets. most pig diets in the temperate countries have been al most exclusively based on maize and soybean meal, while in nigeria, the diets have been largely based on maize and groundnut meal or cake. it is only in recent times that the extensive use of soybean meal became prominent in pig feeding. the depletion of a large number of commercial pig and poultry stock has be reported to be due to the scarcity and high cost of groundnut cake, soyabean and fish meals (tewe, 1988b). utilization of fibrous feedstuff in pig nutrition various studies suggest that the pigs can utilize fibre for their growth, since fibre can be degraded by microbial fermentation. however, this process is reported to be confined to the lower part of the gastrointestinal tract and the products are volatile fatty acids, which can contribute significantly (5-30%) to the net energy requirements of pigs (kass et al., 1980; ehle et al., 1982; rerat et al., 1987). fibrousness of feedstuffs (mostly of by-products of plant origin) is important in relation to their feeding value to pigs. the fibrous components of plant materials are cellulose, hemicellulose and lignin. the influence of crude fibre on organic matter digestibility varies from feed to feed, depending on the special characteristics of the crude fibre in individual feeds (kidder and manners, 1978). the fibrous portion of feed, being fairly indigestible to pigs, influences the digestibility of the other constituents by excreting a protective action, encasing these constituents in a digestion-proof shield, as it were. however, for efficient pig feeding, some form of physical treatment of cereal grains is essential to the breaking down of the fibre encapsulating the more soluble constituents so that digestive secretions can penetrate more completely (kidder and manners, 1978). the addition of fibre to swine diets decreases the digestible energy (de) and metabolizable energy (me) concentration of the diet (kennelly et al., 1998; kennelly and aherne, 1980) and often results in bulk feeds. increased feed intake generally results as the pig attempts to maintain de intake (baird et al., 1975; agricultural research council, 1981; low, 1985). this could increase the length and weight of gastro-intestinal tract even when the fibre is eventually fermented (stanogias and pearce, 1985a and b). this change has been associated with physical properties of fibrous feeds such as its bulk (longe and fagbenro-byron, 1990). when dietary crude fibre exceeds 10-15% of the diet, feed intake may be depressed because of excessive bulk or reduced palatability (braude, 1967). low energy (as obtains for most high fibre diets) will support growth rates equal to those of pigs fed higher-energy diets at low temperature but usually depress the growth rate during periods of high temperatures (coffey et al., 1982; stahly, 1984). several researchers have tried to establish a relationship between de and the fibrous components of the diet (drennan and maguire, 1970; king and taverner, 1975; henry, 1977). longe and fagbenro-byron (1990) later reported the apparent digestible energy to be influenced by the level of dietary neutral detergent fibre (ndf), while henry (1977) showed it to have an inverse relationship to the fibre content, the former decreasing as the latter increases. however, the extent of the decrease in de varied with the feedstuffs, depending on the composition of the cell wall constituents, particularly the adf : ndf ratio (pond, 1987). utilization of crude fibre by non-ruminant has been shown to vary considerably depending on the fibre source (laplace and lebas, 1981), degree of lignification (forbes and hamilton, 1952), level of inclusion (farrell and johnson, 1970; just, 1979) and extent of processing (saunders et al., 1969; mcnab, 1975). the level of the fibrous feedstuff used in pig diets varies with the proportion of the cell wall constituents and it is generally thought that high fibre diet (>25% cell wall contents) will decrease growth efficiency in pigs (longe and fagbenrobyron, 1990). stahly and cromwell (1986) reported that the digestibility of ndf is optimized in pigs fed diets containing 15% or less ndf. fibre utilization is also influenced by the physical and chemical composition of the total diet (myer et al., 1975), level of feeding (cunningham et al., 1962) age and weight of animal (zivkovic and bowland, 1970), adaptation to the fibre source (pollman et al., 1979) and individual differences among pigs (keys et al., 1970 and farrell, 1973). when these factors are considered, it is not surprising that the digestibility of crude fibre has been shown to vary between 0 and 97% (rerat, 1978) and that the literature contains conflicting reports about effects of crude fibre on the digestibility of nutrients (nrc, 1988). there is disagreement concerning the influence of crude fibre on protein digestibility (nrc, 1988). several reports suggest that when the source of crude fibre does not contribute significant amounts of protein in the diet, then an increase in the level of fibre does not affect protein digestibility significantly (eggum, 1973; kennelly and aherne, 1980). other researchers have observed, however, that an increase in the dietary level of fibre decreases protein digestibility (kass et al., 1980; frank et al., 1983). just (1982) reported that an increase in dietary fibre by 11% depressed digestibility of gross energy by approximately 3.5%. longe and fagbenro-byron (1990) in their studies on fibrous waste and by-products for pig feeds in nigeria concluded that fibre sources in general, may be best suitable for adult pigs. this, they attributed to their requirement for lower dietary energy to obtain a desirable carcass lean: fat at slaughter. this agreed with the significant potential for fibre degradation in the pig large intenstine (varel, 1987). the appreciable contribution to the total digestible energy (rerat et al., 1987) encourages giving serious consideration to these fibrous materials and their potential as feed resources in pig production. though, longe and fagbenro-byron (1990) did not fail to add that the economics of production will need to be considered also in determining the suitability of fibrous materials as feed ingredients. however, high fibre diets were reported to be associated with some cardiovascular risks such as low density lipoprotein and total cholesterol (ande, 1992) and reduction in blood glucose and serum insulin concentrations (dodson et al., 1981). nutrient digestibility in pigs the pig is used to convert a variety of foods/feeds into meat for human consumption. the efficiency with which this conversion is carried out is of crucial importance to the pig industry. the efficiency of the conversion is dependent on the digestibility of the feeds and their constituents (kidder and manners, 1978). the proximate analysis of the feedstuffs provides vast amount of data on the crude composition of the feed (dry matter, ash, organic matter (dry matter minus ash), crude protein (n x 6.25), ether extract, crude fibre and nitrogen free extractives) (church, 1991). the measurement of the inputs and outputs of these components in feeds and faeces determine the extent of digestibility of the components. the concept of nutrients digestibility assumes that the feed residues found in feaces are merely the result of inefficient digestion and nothing else (kidder and manners, 1978). however, they reported that the position is more complex, because faeces contain undigested feed residues, unabsorbed digestive secretions, intestinal cells which are continually being sloughed off, as well as dead microorganisms and the products of microbial fermentation. the unabsorbed residues of secretions and cellular debris, predominantly proteineous in nature are of endogenous nitrogen source. generally, the digestibility measurements are reported in terms of apparent digestibility. this is so because, it is difficult to devise appropriate corrections for the amount of digestive secretions and other waste products which are irretrievably mixed with the undigested feed residues of which the faeces are largely composed (kidder and manners, 1978; church, 1991). the coefficient of apparent digestibility of dry matter provides a valuable index of the overall digestibility of the diet. however, a more complete picture of digestibility is obtained with the apparent digestion coefficients of the individual nutrient (crude protein, ether extract, crude fibre, ash and nitrogen free extractives) contained in the diet or feedstuffs. energy prediction in pig diets the cost of feed represents over 70% of the total cost of pig production (frape and tuck, 1977; adesehinwa and ogunbodede, 1995; tewe and adesehinwa., 1995), the energy component being the greatest proportion (frape and tuck, 1977; noblet and perez, 1993; noblet et al., 1994). therefore, it is important to know precisely both the energy requirement of the pigs and the energy value of feeds. the types of pig feeds used in the tropics vary greatly, and since many by-products are used in place of cereals, the variation in nutritive values between and within feeds may be considerable (lekule et al., 1990). the evaluation of the energy content of feeds is usually based on their digestible or metabolisable energy values (nrc, 1988; inra, 1989; noblet and perez, 1993). the energy value of feed ingredients or feeds enables the predictability of the amount of pig meat produced from a given feed and allows the achievement of the least-cost formulation of diets of given energy densities (frape and tuck, 1977). this assists the pig producer to produce a uniform carcass quality at a feed cost commensurate with maximum profit. the determinations of the metabolisable energy values are time-consuming and expensive, and the chemicals required are not readily available in many laboratories in the developing countries (lekule et al., 1990). hence, the apparently digested energy of feeds may be assessed from faecal output by crude chemical analysis of feed and faeces for crude protein, ether extract, crude fibre and nitrogen free extractives. the summation of these values, each multiplied by its characteristic coefficient yields a value representing the heat of combustion of the apparently digested product of 1 kg dietary dry matter (frape and truck, 1977). as consequence of the cost of biological determinations and because of the inherent variability between samples of the same ingredients, a number of equations have been derived to predict the digestible and metabolisable energies from easily determined chemical characteristics of the diets. it should however, be noted that these equations are based on some referenced sample feedstuffs. morgan et al. (1975b) predicted the following equations: de = 0.460cp + 0.625ee + 0.377nfe – 21.05 (r 2 = 0.95) me = 0.416cp + 0.605ee + 0.367nfe – 20.06 (r 2 = 0.94) where de is the digestible energy, me is the metabolisable energy, cp is the crude protein, ee is the ether extract and nfe is the nitrogen free extractives. however, it was reported that the starch content of the nitrogen free extract decreases as the fibre contents increase (nielsen, 1970). in the contribution of lekule et al. (1990), they showed the crude fibre to have the highest correlation to the me content and the relationship was shown as: me (mjkg -1 dm) = 16.81-0.031cf (gkg -1 dm) (r 2 = 0.68) where cf is the crude fibre and dm is the dry matter. the high correlation has also been shown by just et al. (1984), fernandez and jorgensen (1986) and morgan et al. (1987), among others. the best equation with two variables was reported by lekule et al. (1990) as: me (mjkg -1 dm) = 16.34 0.034cf (gkg -1 dm) + 0.003cp (gkg-1dm0 (r 2 =0.73) while they reported the best three-variable equation as: me (mjkg -1 dm) = 3.36 + 0.016cp + 0.029ee + 0.013sc (r 2 =0.83) where sc is soluble carbohydrate noblet and perez (1993), in their study later predicted thus: de = 4151 – 12.2ash + 2.3cp + 3.8ee – 6.4cf (r 2 = 0.89) me = 4168 – 12.3ash + 1.4cp + 4.1ee – 6.1cf (r 2 =0.88) the digestible energy of feed is by no means recoverable in useful animal products (frape and tuck, 1977). some workers, therefore, advocated the determination of metabolisable energy, which is that, available to the tissue for productive use. in pig, methane losses are small for normal diets, about 0.01 of the digestible energy (bowland et al., 1970) and therefore, in practice, the metabolisable energy is simply assumed to be that portion of the digestible energy not lost in the urine (frape and tuck, 1977; serres, 1992). urinary nitrogen losses were reported to increase as dietary protein is raised, so that high protein ingredients are likely to incur a greater handicap than cereals in the computation of their metabolizability. thus, metabolisable energy is affected by dietary protein level and by the way it is utilized and so, it is not a constant characteristic of an ingredient. metabolisable energy may, therefore, be calculated from the proximate composition of digestible dietary components as in the equation above, or as a proportion of the digestible energy: approximately 0.98de for cereals and 0.95de for protein concentrates (frape and tuck, 1977). morgan et al. (1975a) allowed for variation in protein intake, thus: me =de (0.997-1.88 x10 -4 cp1) (r 2 =0.88) where cp1 is crude protein in g/kg dietary dry matter. while, lekule et al. (1990) predicted: me (mjkg -1 dm) = 0.08 + 0.96de (mjkg -1 dm) (r 2 = 0.96). the relationship was found to be very close and of the same magnitude as that found by just (1982). the energy value of the diet could then be used to predict the amount of pig meat (pork) to be produced from a given amount of feed (which is an inverse of the amount of feed consumed per kg liveweight gained) or to achieve the least-cost formulation of diets of given energy densities. this is the major determinant of the nutritive value of a feedstuff and its assessment is therefore of great importance for diet formulation. this is of special practical importance in the developing countries where determination of proximate components in feed and feces are relatively more feasible compared to the use of sophisticated gadgets, required in other methods of energy value determinations, which are not available. references adesehinwa aok (1992). growth and protein utilization by grower pigs fed “dusa” and cashew nut testa. m.sc. theses, department of animal science university of ibadan, ibadan. adesehinwa aok, ogunmodede bk (1995). swine feeds and practical feed composition techniques, in: n.a.e.r.l.s. workshop training manual, moor plantation, ibadan, april 3-7, 1995) nat. pig prod. pp. 27-56. adesehinwa aok, dafwang ii, ogunmodede bk, tegbe tsb (1998). a review of utilization of some agro-industrial by-products in pig rations. niger. j. agric. ext. 11(1&2): 50-64. ande ko (1992). density and water holding capacity of diets containing graded levels of palm kernel meal and corn offal and dressing characteristics and gut dimensions of broilers fed these diets. m.sc. thesis, department of animal science, university of ibadan, ibadan. attah sto, tegbe tsb, olorunju sas, aduku ao (1991). the replacement alue of rice-mill by-products for maize on performance and carcass characteristics of pigs. niger. agric. j. 1(2): 93-102. babatunde gm, olomu mj, oenuga va (1972). determination of the optimum crude protein requirements of pigs in a tropical environment. anim. prod. 14: 57-67. baird dm, mccampbel hc, allison jr (1975). effect of level of crude fibre, protein and bulk in diets for finishing hogs. j. anim. sci. 41: 1039. bender ae (1975). nutritional aspects of amino acids. in: amino acid metabolism. john wiley and sons, london, 8: 192-202. braude r (1967). the effect of changes in feeding patterns on the performance of pigs. proc. nutr. soc. 26: 163. bowland jph, bickel hp, pfirter cp, wenk, schusrch a (1970). respiration calorimetry studies with growing pigs fed diets containing from three to twelve percent fibre. j. anim. sci. 31: 495-501. church dc (1991). livestock feeds and feeding, 3 rd ed. prentice hall, inc. englewood cliffs, new jersey. coffey mt, seerley rw, funderburke w, mccampbell hc (1982). effect of heat increment and level of dietary energy and environmental temperature on the performance of growing – finishing swine. j. anim. sci. 54: 95. cole dja (1984). the nutrient density of pig diets-allowances and appetite. in: fat in animal nutrition, j. wiseman, ed. london: butterworth, pp. 301-312. conrad jh (1984). feed formulation with soybean meal for efficient swine production. anim. nutr. res. highlight (special edition). quarterly publication of the american soybean association. cooke r, lodge ga, lewis d. 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(camb,) 90: 69-76. zivkovic s, bowland jp (1970). influence of substituting higher fibre ingredients for corn on the digesti9bility of diets and performance of sows and litters. can. j. anim. sci. 50: 177-184. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (8), pp. 001-005, august, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review swine flu: a threat to human health syeda samra iqbal jafri*, muhammad ilyas and muhammad idrees national centre of excellence in molecular biology, university of the punjab, lahore, pakistan. accepted 12 september, 2013 influenza virus has caused major global epidemics since 1918. although this commonly circulating viral strain is not virulent enough to cause mortality, but the re-assortment of viral genome to mutate at a very high rate can lead to the emergence of a highly virulent strain that may lead to next pandemics. swine flu is an example of re-assortment of two viruses in pig causing thousands of deaths in the whole world especially in the united states of america by zoonotic jumps. this review addresses the biological and epidemiological aspects of swine flu virus and efforts to have a control on the virus globally. key words: swine influenza, serotypes, pandemic, vaccine. introduction flu is an ancestrally old illness which started in birds initially and then slowly diversified to all warm blooded animals on earth in an era of thousands of years. global population affected by annual flu is estimated to be 5 15%. although most flu cases are mild but may cause severe illness in 3 5 million people and about 250,000 500,000 deaths worldwide. mostly chronically unhealthy people, the old and high risk populations of infants face severe illness and deaths in industrial countries (epi fact sheet, 2007). though the forms of animals are different organisms but flu viruses can travel from one organism to another organism of different species. humans are also included in this exchange of viruses. re-assortment is the process through which a virus organizes its genetic material and thus a hybrid variety of super-virus comes into existence. because this is a newly originated form so there is no innate or adaptive immunity present and flu spreads effectively and easily. swine flu is a respiratory disease caused by a reassortant virus and has elements *corresponding author. e-mail: milyaskh@hotmail.com. tel: +92-300-5740568. abbreviations: sars, severe acute respiratory syndrome; cdc, centre of disease control; who, world health organization; dhs, department of homeland security; fda, federal drug administration. of virus found in pigs. swine flu virus acts similar to the spanish flu, 1918, which caused the death of 20 million people; swine flu is showing the same age related mortality. swine flu caused a pandemic flu outbreak by spreading from human to human worldwide. because it is caused by a re-assortant virus so no one has immunity against this virus and is at risk of being caught by flu. so not only children, older people and diseased persons but also healthy people are at risk. swine flu virus is made up of tiny droplets and can spread through so many ways. epidemiology the key thing is going to be the epidemiology. swine flu virus is a “reassortant virus” containing genes from a number of sources. though the swine flu virus is specie specific but it can easily cross the specie barrier and zoonotic jumps were seen in swine flu virus from pigs to humans. once in humans, it can spread in whole population with ease. normally when virus jumps from one species to another, it gets illadapted due to its inability to multiply and spread in the foreign territory. but swine flu virus got well adapted to humans, enabling it to transmit efficiently from one person to the next. swine flu infection transmits to humans through pigs and then human-to-human transmission is possible but spread is table 1. pandemics of 20 th century due to influenza virus. pandemic due to flu prototype strain subtype infected people death rate case fatality rate virus and causative strain spanish flu a/fm1/47 h1n1 (yu-chia et 0.5-1 billion infected 20-100 million >2.5% al., 2006; li 2008) (who, 2005; (taubenberger et knobler et al., al., 2006) 2005; patterson et al., 1991) asian flu a/singapore/57 h2n2 (yu-chia et 2 million (who, <0.1% al., 2006) 2005) (taubenberger et al., 2006) hong kong flu a/hong kong/68 h3n2 (yu-chia et 1 million (who, <0.1% al., 2006) 2005) (taubenberger et al., 2006) seasonal flu mainly a/ h3n2, (5-15%) 340 million 250,000<0.05% a/h1n1 and b 1 billion (who, 500,000/year (taubenberger et 2009) (who, 2003) al., 2006) russian flu a/ussr/77 h1n1 limited to close contacts or closed groups of people. according to the working party report of uk government in 2000, “there’s something like 500,000 people airborne around planet earth at any instant in time”. this shows the potential of swine flu virus to spread far and wide very quickly and can lead to a pandemic. swine flu outbreak occurs in pigs in autumn and in winter in temperate zones. disease is considered as endemic. sporadic human infections and outbreaks are occasionally reported. global pandemics of 20 th century due to influenza virus three major global epidemics of the 20th century have been caused by influenza a virus, the spanish flu in 1918 1919, the asian flu in 1956 1958 and the hong kong flu in 1968 1969 by undergoing major genetic changes for which population did not poses significant immunity [who fact sheet; kilbourne, 2006]. over the past century several pandemic threats have been caused by h1n1 subtype, which include the 1947 pseudo-pandemic, swine flu outbreak 1976 and russian flu 1977 [kilbourne ed, 2006]. the sars epidemic by sars coronavirus in south east asia further increased the level of alerts. then the advent of h5n1 bird flu outbreaks further increased the level of preparedness though the strains have limited human-to-human transmission ability [yu-chia et al., 2006; beigel et al., 2005]. table 1 shows the effect of influenza virus on people in 20th century. swine flu swine flu, a global outbreak took its origin from a large pig farm in mexican town of la gloria in veracruz state with evidence that mexico was already in midst of an epidemic for months before the outbreak was recognized. it was first detected in mexico city on march 18, 2009 (kilbourne, 2006). this new strain was first diagnosed in two children, having no contact with pigs, by the cdc, first on april 14, 2009 in san diego country, california and a few days later in near by imperial county, california (new york times, 2009), which was not identified as a new strain in mexico until april 24, 2009 (morb mortal wkly rep, 2009). who has been notified of swine flu cases from the united states and spain. the government closed down most of the private and public offices and facilities of mexico city to stop the spread of disease. who declared it as pandemic in early june and noticed the moderate severity of disease. from mexico the virus spread with un-plecedented speed to southern hemisphere and less developed countries. outbreaks in north america, south america, europe (including the united kingdom, italy and sweden), africa (kenya), and in parts of the eastern asia including china and japan has also been reported. swine flu is a highly contagious acute respiratory disease of pigs, caused by swine influenza a virus, referred to as the novel h1n1. the most common subtype is h1n1 in case of swine flu but other serotypes e.g. h1n2, h3n1, h3n2 are also found circulating in pigs. it is thought that h3n2 strain was originally introduced into the table 2. various techniques used for the laboratory diagnosis of swine influenza. procedure influenza type detected acceptable specimen time for results (h) real time pcr a naso pharyngeal (np) swab, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum antigen detection a naso pharyngeal (np) swab, throat swab, nasal elisa wash, bronchial wash, nasal aspirate, sputum 2 2 3 pigs by humans [news report, 2009]. swine flu virus spreads by direct or indirect contact among pigs by aerosols and remains asymptomatic there. high morbidity and low fertility rate is observed in case of swine flu. symptoms swine flu shows clinical symptoms similar to seasonal flu and acute upper respiratory tract infection. the extent of this disease in humans is still unknown due to the escape of mild or asymptomatic case from recognition. an algorithm has been drawn up by the health protection agency in the uk as to how they intend to assess the people suffering with swine flu. geography is the first point on the algorithm according to which disease activity is seen in people coming from the same area. this obviously includes parts of the us but chiefly mexico. the us centres for disease control and prevention (cdc) noted that although the virus was very serious but the world wide cases were usually mild. cdc noted that the hospitalizations and deaths had been of persons that had underlying weekend immune system conditions, heart diseases, asthma, diabetes or obesity (pakspectator, 2009). according to symptom algorithm it is a temperature of 38 degrees or a history of a temperature of 38 degree not only respiratory symptoms e.g. runny nose or a headache, fever, cough, sore throat, body aches, chills, fatigue, but also diarrhea and vomiting have been observed in some cases. emergency warning signs in children and adults are thus highlighted. in children fever with rash, fast breathing or breathing trouble, bluish skin color, not drinking enough fluids, unbearable irritability in children, not waking up or interacting, flu-like symptoms improve but return with fever and cough. in adults shortness or difficulty in breathing, pressure or pain in abdomen or chest, sudden dizziness, confusion, persistant or severe breathing, flu-like symptoms improve but return with fever and cough (cdc, 2009). vulnerable group of people vulnerable group of people includes pregnant women, care takers of less than 6 months old infants, health care workers, adults with asthma and diabetes and people with immune-compromised systems. in a worst case scenario, 40% of the workforce are estimated by cdc might be unable to work at the peak of pandemic (los angeles times, 2009). spread of swine flu virus the actual transmission is from pig to pig but swine flu virus can also jump from pig to human and once in human, it can spread from human to human very easily through coughing, sneezing, touching nose, mouth or contaminated surfaces. currently h1n1 flu has been mild for most people; however it should not disregard the epidemic mild as is expected to come back and spread. everyone should be vaccinated against swine flu; however the vaccine will become available in batches and the entire population cannot be vaccinated at once. table 2 shows the various techniques used in the laboratory for the diagnosis of swine influenza. global spread of swine flu until 16 th april, 2010, worldwide more than 209 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza h1n1 2009, including at least 17,798 deaths. table 3 describes the deaths caused by swine flu in the world. swine flu in pakistan in pakistan, national centre of excellence in molecular biology has detected 3 cases out of 77 suspects while khan laboratory multan has detected 3 cases out of 7 suspects. total confirmed cases detected, using real-time pcr on who recommended kit, are 6 out of 84 suspects and out of these only 2 deaths have been reported until 16 th april, 2010 (idrees, 2010). table 3. deaths caused by swine flu in the world. region deaths* who regional office for africa (afro) 168 who regional office for the americas (amro) at least 8274 who regional office for the eastern mediterranean (emro) ** 1019 who regional office for europe (euro) at least 4776 who regional office for south-east asia (searo) 1757 who regional office for the western pacific (wpro) 1804 total* at least 17798 *the reported number of fatal cases is an under representation of the actual numbers as many deaths are never tested or recognized as influenza related. **no update since 7 march 2010 [pandemic (h1n1) 2009, 2010]. prevention and cure vaccination there are generally two kinds of vaccines, 1) inactivated virus vaccine and; 2) live attenuated vaccine (laiv). in august 2009, no vaccine was thought to be available before the end of 2009 by who. influenza virus changes quickly and it was thought that if virus mutated further then it could be more virulent and less susceptible to any new vaccine. when who declared the new h1n1 swine flu as the first pandemic of the 21 st century on june 12 th , a day after this declaration, novartis ag (swiss pharmaceutical company) announced the successful production of first batch of swine flu vaccine that was ready for sale in october, 2009. vaccination planning more than 30 countries have asked novartis to supply them with a swine flu vaccine. according to who several other drug makers have vaccine approved and ready for sale since september 2009 and cdc has already developed a vaccination plan. who should be vaccinated first? the public health agencies should work on priority list, deciding where the first batches should go. several articles have been published showing that, unlike seasonal flu, the new h1n1 flu strains attack younger people and new pandemic strain tend to be more deadly in younger patients as s-oiv infection can cause severe acute respiratory distress syndrome and death in young to middle aged (rogelio et al, 2009; chowell et al., 2009; mmwr, 2009). the descendants of the 1918 flu have genetically modified themselves to be better able to survive and spread (cdc, 2009; mmwr, 2009). therefore young people should be vaccinated first when a vaccine becomes available. it would protect not only them, but them, but also unvaccinated adults. second group on priority should be health workers as they need to be protected from infection by the virus. according to the morbidity and mortality weekly report a sample of 26 health-care workers found that half became infected while at work (cdc, 2010). next priority should be given to countries where the infection is more severe such as mexico. patients with chronic obstructive pulmonary disease, disease of the heart, and other parts of the body should be put on vaccination list before get vaccinate the whole population. antiviral medications cdc has issued recommendations for clinicians on the use of antiviral medications for the prevention and treatment of swine flu. there are two classes of medicines available which are effectively used in some countries for the prevention and treatment of the disease, 1) adamantanaes (amantadine and remantadine), and inhibitors of influenza neuraminidase (oseltamivir and zanamivir). swine flu virus developed resistance against amantadine and remantadine but are sensitive to oseltamivir and zanamivir. there are insufficient recommendations on the use of antivirals in the treatment and prevention of swine flu virus infection. based on virus’ susceptibility profile national and local authorities are recommending to use oseltamivir and zanamivir for the treatment and prevention of disease (cdc, 2010). fda has authorized emergency use of oseltamivir (tamiflu ®) and zanamivir (ralenza®) under certain circumstances not included in fda-approved uses or outside the fda-approved uses in response to the declaration of public health emergency involving 2009 h1n1 influenza a virus made by secretary of health and human services on april 26, 2009 (pan american health organization, 2009). in addition, fda has recently issued intravenous drug (paramivir) for the treatment of swine flu in certain hospitalized adults and pediatric patients. other preventive measures first prevention is to keep the infected children at home. children showing flu symptoms should be monitored; they may not have swine flu but can develop it. home treatment remedies including adequate liquid intake and rest, soup to ease congestion, aspirin intake for instance and antiviral drugs may help in the treatment of infection. an updated guidance for employers of all sizes was developed by the us department of health and human services (hhs) and the centres for disease control and prevention (cdc) with input from the us deapartment of homeland security (dhs) to use in response to 2009 h1n1 flu. to slow down the spread of infection, air line passengers in asia and other countries were quarantined with flu symptoms while some countries pre-screened passengers. disease can also be prevented by having no contact with pigs. initial screening and then activating various molecular tests, using tests that can detect the genetic material of the virus are seen to be useful for the early diagnosis of swine flu. swine flu virus can be killed by cooking the pig meat at 70°c and not transmitted through properly cooked pig meat (press release, 2009). so properly cooked pig meat is safe to eat (who, 2009). people getting flu are recommended to stay at home from work or school and to avoid crowds to avoid spread of virus further. facial masks are of little use and may benefit in case of close contact with infected people but can’t replace other precautions because they are not designed to filter viruses and other biological agents (mayo clinic, 2009). swine flu can be prevented by proper hand washing, balanced diet with fresh fruits and vegetables, whole grains and lean protein, sufficient sleep, regular exercise and avoiding crowds. is the swine flu a large medical scandal of 21 st century? it is said that swine flu is a large medical scandal of 21 st century. whether it is true or not, this is a contradictory matter. the purpose of this article is just to provide information to the readers. references beigel jh, farrar j, han am, hayden fg, hyer r, de jong md, lochindarat s, nguyen tk, nguyen th, tran th, nicoll a, touch s, yuen ky (2005). the writing committee of the world health organization (who) consultation on human influenza a/h5. n. engl. j. med., 353(13): 1374-85. cdc (2009). novel influenza a (h1n1) virus infections among healthcare personnelunited states. april – may 2009. morb mortal wkly rep (mmwr) 2009; 58(23): 641-645. centers for disease control and prevention (cdc) (2010). updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season. http://www.cdc.gov/h1n1flu/recommendations.htm chowell g, stefano mb, arantxa mc, hugo l, celia a, mauricio h, mark am (2009). severe respiratory disease concurrent with the circulation of h1n1 influenza. n. engl. j. med., 361: 674-679. epi fact sheet (2007). world health organization • searo • ivd• publish 27-jul-2007. global alert and response (gar) pandemic (h1n1) (2009) update 6 weekly update. idrees m (2010). unpublished data, national centre of excellence in molecular biology, pakistan. kilbourne ed (2006). influenza pandemics of the 20th century. emerging infectious diseases, 12 (1). knobler s, mack a, mahmoud a, lemon s (2005). the story of influenza. the threat of pandeic influenza: are we ready? workshop summary. washington, d.c. the national academies press, pp. 60-61. li fck (2008). finding the real case-fatality rate of h5n1 avian influenza. j. epidemiol. community health. 62(6): 555-559. los angeles times (2009). swine flu could kill hundreds of thousands in u.s. if vaccine fails. mayo clinic staff (2009). flu prevention. mayo clinic. accessed 200908-31. archived 2009-09-08 (unpublished). morbidity and mortality weekly report (2009). swine influenza a (h1n1) infection in two childrensouthern california. centers for disease control. new york times (2009). flu outbreak raises a set of questions. pan american health organization (2009). health surveillance and disease prevention and control, pandemic influenza a(h1n1), 2009. information for workers. pan american health organization. pandemic (h1n1) (2009). who programme office at national disease surveillance centre (ndsc) http://www.whoban.org/pdf/sitrep20on20pandemic20h1n12020092 0-201920april202010.pdf patterson kd, pyle gf (1991). the geography and mortality of the 1918 influenza pandemic. spring, bull. hist. med., 65(1): 4-21. press release (2009). "joint fao/who/oie statement on influenza a(h1n1) and the safety of pork", 7 may 2009. http://www.who.int/mediacentre/news/statements/2009/h1n1_200904 30/en/index.html q&a: what is swine flu? http://www.pakspectator.com/swine-flu-what-is it-and-how-to-cure-no-worries-pakistan/, swine flu: what is it and how to cure: no worries pakistan, 2009. rogelio p, daniela r, samuel p, mauricio h, francisco q, edgar b, alejandra r, jorge r, christopher eo, ariel c, anjarath h, edgar m, jose ac (2009). pneumonia and respiratory failure from swineorigin influenza a (h1n1) in mexico. n. engl. j. med., 361: 680-689. taubenberger jk, morens dm (2006). 1918 influenza: the mother of all pandemics". emerging infectious diseases (centers for disease control and prevention), january 2006, 12 (1). http://www.cdc.gov/ncidod/eid/vol12no01/05-0979.htm. who europe influenza (2009). world health organization. june 2009. http://www.euro.who.int/influenza. retrieved 2009-06-12. world health organization (2003). fact sheet n°211 revised march, 2003 http://www.who.int/mediacentre/factsheets/2003/fs211/en/ (accessed 7 sept. 2006). world health organization (2005). ten things you need to know about pandemic influenza. wkly epidemiol rec. 2005 dec 9; 80(49-50): 428-31. yu-chia h, tsung-zu w, ding-ping l, pei-lan s, luan-yin c, chun-yi l, chin-yun l, fu-yuan h, li-min h (2006).influenza pandemics: past, present and future. j. formosan med. assoc., 105(1): 1-6. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (12), pp. 001-007, december, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper a novel approach for very early pregnancy diagnosis in swine by anti-early pregnancy factor (epf) antiserum blocking enzyme-linked immunosorbent assay (elisa) kai quan1,2, xingxu zhao1*, changxing zhang2, qiuliang xu2, hongfang wei1, junjie hu1 and yong zhang1 1 college of veterinary medicine, gansu agricultural university, lanzhou, gansu 730070, china. 2 zhengzhou college of animal husbandry engineering, zhengzhou, henan 450011, china. accepted 09 april, 2014 early pregnancy factor (epf) is essential for the initiation and maintenance of pregnancy. early pregnancy factor activity monitoring has been reported to be the effective method for very early pregnancy diagnosis. in this study, three balb/c mice were immunized with the synthetic peptide segment corresponding to the amino acid sequence 36 to 55 of epf (ig20) for anti-epf antibodies. mouse anti-epf antiserum titers were evaluated by an indirect enzyme-linked immunosorbent assay (elisa), and the titers were 6.4 × 10 3 . serum samples were taken from 21 yorkshire × landrace crossbred sows (12 pregnant and 9 non-pregnant). the presence of epf in these serum samples was detected by a blocking elisa using the antigen-antibody (ag-abs) reaction between ig20-ovalbumin and mouse anti-epf antiserum for very early pregnancy diagnosis, blank was used as negative controls. the optical density (od) values were measured at 450 nm, and the od ratios of negative control/serum sample (n/s) >2.1 were considered positive, and n/s <2.1 negative. when the test serum samples were in 1/4 dilutions with pbs, twelve samples from pregnant swine were positive, nine non-pregnant serum samples were negative. very early pregnancy can be determined by using the mouse anti-epf antiserum blocking elisa in swine. key words: very early pregnancy diagnosis, early pregnancy factor (epf), rosette inhibition test (rit), blocking enzyme-linked immunosorbent assay (elisa). introduction early and accurate diagnosis of pregnant sows and gilts has potential economic advantages by reducing nonproductive days and increasing reproductive efficiency in pig production. currently, the porcine pregnancy diagnosis is mainly through the detection of return to estrus (almond et al., 1986, 1987), as well as the scan of embryo with amplitude-depth (a-mode) (pyorala, 1989), doppler and real-time (b-mode) ultrasonography (rtu) (zambelli et al., 2006; williams et al., 2008). however, these methods are only effective after 21 or 24 days of gestation (krüger et al., 2002), and could not be used for * corresponding author. e-mail: quankai1115@126.com abbreviation: epf, early pregnancy factor. very early pregnancy diagnosis (in pigs conceptuses 7 to 15 days) in sows (shaw et al., 1980). it is necessary to establish a fast and efficient method for very early pregnancy diagnosis in sows. early pregnancy factor (epf) is a pregnancyassociated protein, which was firstly detected by rosette inhibition test (rit) in mice serum as early as 4 h after mating (morton et al., 1974). early pregnancy factor has been shown to be essential for the initiation and maintenance of pregnancy, and it was assayed by the rit through the decreasing ability in the formation of rosettes (greco et al., 1992; straube et al., 1989). the activity of epf has been detected in maternal serum within 24 h of fertilization in all mammalian species tested and persisted for at least the first half of gestation (morton et al., 1987), and epf activity monitoring (rit) has been reported to be the only method suitable for very early pregnancy diagnosis (straube et al., 1988; mesrogli et al., 1988). however, rit was not a quantitative assay and has been proved cumbersome-and time-consuming (koch et al., 1985). the amino acid sequence of epf is fully identical to the extra-cellular form of chaperonin10 (cpn10) (alice et al., 1996; morton, 1998), human, bovine and swine share the same sequence of epf (englebretsen et al., 2002; fotini et al., 2003). in the present study, we have undertaken production of anti-epf antibodies to synthetic peptide segment corresponding to the amino acid 36 to 55 of epf (ig20). serum samples were collected from 12 pregnant and 9 non-pregnant sows, the presence of epf in these sera samples was detected by a blocking enzyme-linked immunosorbent assay (elisa) using the antigen-antibody (ag-abs) reaction between ig20 and mouse anti-epf antiserum, then, the optical density (od) values were measured at 450 nm, and pregnancy was determined by the od ratios of control/serum sample (n/s) (loffeld et al., 1989; shu et al., 2011; xie et al., 2007). materials and methods preparation of serum samples the blood samples (each 2 ml) were taken from the ear vein of 21 yorkshire × landrace crossbred sows (12 pregnancies and 9 nonpregnancies) after second parity. twelve blood samples of pregnant sows were collected on the sixth day after artificial insemination (ai), and pregnancy was confirmed by b-ultrasound after the 40 days of ai. nine blood samples of non-pregnant sows were collected on the sixth day after estrus (no insemination). these samples were allowed to clot for about 1 h at room temperature, and the sera were separated by centrifugation at 1 000 ×g for 30 min, and deactivated at 56°c for 30 min, then stored at -20°c until testing. rosette inhibition test the human lymphocytes were collected from one of the lymphocyte donors. lymphocytes were separated through a gradient centrifugation by the ficoll-hypaque (sigma chemical co., st. louis, mo, usa) method and processed according to the manufacturer’s instructions. the isolated lymphocytes were adjusted to 4 × 10 6 cells per ml with hanks balanced salt solution (hanks', ph7.4, ca 2+ and mg 2+ free). sheep red blood cells (srbc) were obtained from fresh defribrinated sheep blood according to the description of ghaffari et al. (2008), and srbc were adjusted to a final concentration (1 × 10 8 cells per ml) with hanks'. rosette inhibition test was performed as established by ghaffari et al. (2008) and athanasas-platsis et al. (2000) with minor modification. serum samples of each pig (100 µl) were mixed with human lymphocytes suspensions (500 µl) and incubated at 3°c for 60 min. the reaction solutions were divided into twelve equivalents (50 µl each sample), then 100 µl serially diluted (1 : 2 1 × 10 3 , 1 : 2 2 × 10 3 , to 1 : 2 12 × 10 3 ) rabbit antihuman-lymphocyte globulin (alg) were added, and incubated at 37°c for 60 min, washed 3 times with hanks', respectively. fifty micolitres srbc were added and centrifuged immediately at 800 ×g for 5 min. then one 100 µl 0.8% glutaraldehyde was added to fix the lymphocytes and erythrocytes. over 200 lymphocytes and rosettes were counted and recorded. each sample was repeated three times. the number of rosettes formed was determined and expressed as percentage of the number of rosettes formed without alg. results are recorded as rosette inhibition titers: the highest dilution of alg [log2 (reciprocal dilution × 10 -3 )] to give rosette formation of <75% (samples without alg = 100% rosette formation). preparation of immunogens a peptide segment (imlpeksqgkvlqatvvavg, ig20) corresponding to the amino acid sequence 36 to 55 of epf (accession no. np776771) was synthesized on 0.3 mmol/g fmoc wang resin (gl biochem ltd., shanghai, china) using a symphony ® multiplex peptide synthesizer (protein international technologies, inc., st. louis, mo, usa) (ahlborg, 1995). a cysteine residue was added at the n-terminal of the peptide segment to facilitate conjugation. the peptide was purified by reversed-phase high performance liquid chromatography (rp-hplc) coupled to pneumatically assisted electro spray ionization mass spectrometry (esi-ms) (quattro. micro tm api, waters corp., milford, ma, usa) with the solid-phase purification over 95% (sanz-nebot et al., 2001). the synthetic peptide was conjugated to a carrier protein of iggfree bovine serum albumin (bsa, sigma chemical co., st. louis, mo, usa) using a hetero-bifunctional cross-linker succinimidyl 4(nomaleinimido-methyl) cyciohexane-l-car-boxylate (sulfo-smcc, pierce biotechnology inc., rockford, il, usa) according to the method described by alexander et al. (1984). briefly, 4 mg of bsa was dissolved in 0.5 ml phosphate buffered solution (pbs, 0.1 m sodium phosphate, 0.15 m sodium chloride, 0.1 m edta, ph 7.2), then mixed with 1 mg sulfo-smcc dissolved in 50 µl dmso. the mixture was kept at room temperature for 30 min, and then the buffered solution dialysis was conducted at 4°c overnight. the dialysis fluid was changed three times to remove excess coupling agent. the smcc-activated bsa (smcc-bsa) solution was adjusted to a concentration of 5 mg/ml with pbs and stored at 20°c. two milligrams of ig20 were dissolved in 50 µl dimethylformamide (dmf) and added 150 µl conjugation buffer (5 mm edta buffered solution 0.01 m pb, ph 7.2). thereafter, 10 mg/ml of ig20 was mixed with smcc-bsa and kept at room temperature for 30 min, to have the peptide segment conjugated to carrier protein to form the immunogen ig20-bsa. the ig20-bsa was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). ovalbumin (ova, sigma chemical co., st. louis, mo, usa) was conjugated to ig20 (ig20-ova, coating antigen) as the similar procedure described earlier. preparation of mice antiserum to epf three balb/c mice (female, 8 weeks of age) were immunized with ig20-bsa conjugates. the first dose consisted of 50 µg of ig20bsa conjugates for injection subcutaneously as an emulsion of pbs and freund’s complete adjuvant (fca, sigma chemical co., st. louis, mo, usa). the boosters were given at two weeks intervals with the same dosage of ig20-bsa conjugates emulsified in freund’s incomplete adjuvant (fia, sigma chemical co., st. louis, mo, usa) (ebensen et al., 2007). blood samples (20 µl) were collected from tail veins 14 days after third immunization. anti-epf antibody titer was determined by the indirect elisa using the following procedure (kit et al., 1990; sun et al., 2007). 96 well elisa plates (costar) were coated with 1 µg/ml ig20-ova diluted in carbonate buffer, ph 9.6, in a 100 µl per well and incubated overnight at 4°c. thereafter, the plates were washed four times with pbst and repeat between each of the following steps, and blocked with 5% chicken serum at 37°c for 1 h, 50 µl 1/100 serial diluted anti-sera were added to the blocked plates and figure 1. the rosette inhibition titers (rit) range of pregnant and non-pregnant porcine serum samples. the rosette inhibition titers (mean ± sd). pregnant (◇): 8.0 ± 0.5**, non-pregnant (■): 6.0 ± 0.4. ** was p < 0.01 (t-test) between pregnant and non-pregnant serum samples. incubated at 3 °c for 15 min, then 50 µl 1000-fold diluted horseradish peroxidase (hrp)-conjugated rabbit anti-mouse igg (abnova corp., heidelberg, germany) was added and again incubated at 37°c for 30 min. the substrate-chromogen solution (tmb-h2o2) was added, and the od (450nm) value was measured using a micro-plate reader. 50 µl 1 µg/ml ova was used as negative controls and test was repeated three times. blocking elisa detection of epf presence in serum samples the procedures of blocking elisa was conducted as previously described (kit et al., 1990; sun et al., 2007). elisa plates were sealed with 5% chicken serum. hereafter, 50 µl of each porcine sera sample (9 non-pregnant and 12 pregnant sows, blank group served as negative control) were serially diluted (1/2 to 1/64) with pbs, then mixed with 50 µl 800-fold diluted mice anti-epf sera in each well, and incubated at room temperature for 30 min. the mixed solutions were transferred to another ninety-six well elisa plates which were coated with 100 µl 0.5 µg/ml ig20-ova and sealed with 5% chicken serum, incubated at 37°c for 15 min. horseradish peroxidase-conjugated rabbit anti-mouse igg was added, incubated at 37°c for 30 min, tmb-h2o2 was added and the od value at 450 nm was measured by micro-plate reader. the test was repeated three times. statistical analysis rosette-inhibition titers of 9 non-pregnant and 12 pregnant serum samples were analyzed using the spss statistics 17.0 anova and t-test. the od values of mouse anti-epf antiserum blocking elisa in serials diluted samples (nine non-pregnancies, twelve pregnancies and negative controls) were analyzed using the spss statistics 17.0 anova. results the rosette-inhibition test titers early pregnancy factor activities of 9 non-pregnant and 12 pregnant porcine serum samples were tested by rit using human lymphocytes. there were significant differences between pregnant and non-pregnant sera (p<0.01) (figure 1). evaluation of mice antiserum responses to epf the conjugations (ig20-bsa and ig20-ova) were identified by sds-page. the calculated molecular combined ratios of ig20/bsa and ig20/ova were 15:25 and 5:15 (figure 2), respectively. three balb/c mice were immunized with ig20-bsa, and the antibody responses to epf were measured by indirect elisa. the serum anti-epf antibody titers were 1:6.4 × 10 3 (figure 3). evaluation of blocking elisa when the test serum samples were diluted 1/4 with pbs, the od ratios of negative control/serum sample (n/s) were over 2.1 (positive) in 12 pregnant serum samples, and was less than 2.1 (negative) in nine non-pregnant serum samples (loffeld et al., 1989; shu et al., 2011; xie figure 2. the sds-page analysis of conjugated products. the molecular weight of ig20-bsa ranged from 97.4 to 110 kda, and ig20-ova was from 66 to 97 kda. figure 3. the titers of anti-epf serum antibody. when the od values of anti-epf serum antibody were 2.1 times negative, they were positive; and hence negative. et al., 2007). however, when the sera were 1/2 diluted, six of the nine non-pregnant serum samples were presented as a false-positive. the serum samples were 1/8 dilution or more, the non-pregnant and partly pregnant serum samples showed negative (figure 4 and table 1). discussion accurate and early detection of pregnant and nonpregnant sows has become a key to good breeding management because it is an essential factor for monitoring and controlling fertility in pigs. in commercial figure 4. the od ratios of n/s (negative control/porcine serum sample). n/s > 2.1 were considered positive (+), and n/s value < 2.1 negative (-). table 1. the od values of mouse anti-epf antiserum blocking elisa using the serum samples of serial dilution (the mean values of 3 times repeated for each sample). parameter number diluted time of sows’ serum sample negative 1/2 1/4 1/8 1/16 1/32 1/64 1 0.258* 0.380* 0.814 1.102 1.135 1.132 1.300 2 0.146* 0.397* 0.468 0.561 0.562 0.874 0.772 3 0.145* 0.183* 0.482 0.696 0.722 0.698 0.919 4 0.148* 0.298* 0.692 0.52 0.644 0.897 0.735 5 0.098* 0.440* 0.610* 0.704 0.835 1.021 1.321 pregnancy 6 0.114* 0.297* 0.544 0.663 1.061 0.574 0.565 7 0.125* 0.183* 0.388* 0.697 0.782 0.666 0.932 8 0.188* 0.268* 0.792 0.631 0.844 0.837 0.766 9 0.135* 0.480* 0.714 0.704 0.931 1.031 1.032 10 0.146* 0.357* 0.631 0.769 0.665 0.672 0.874 11 0.135* 0.283* 0.566 0.893 0.721 0.789 0.728 12 0.178* 0.252* 0.591 0.722 0.746 0.863 0.639 1 0.321* 0.438 0.603 0.794 0.761 0.862 0.835 2 0.342* 0.533 0.692 0.834 0.996 0.822 0.921 3 0.465 0.612 0.772 0.865 0.82 0.944 0.965 4 0.372* 0.552 0.685 0.903 0.804 0.835 0.932 non-pregnancy 5 0.467* 0.631 0.782 0.734 0.863 1.061 1.166 6 0.494 0.611 0.612 0.734 0.892 0.782 1.032 7 0.322* 0.461 0.722 0.776 0.7312 0.844 0.874 8 0.479 0.490 0.694 0.811 0.904 0.931 0.728 9 0.351* 0.589 0.731 0.921 0.869 0.665 0.939 * the od values < 2.1 times of negative, means positive. swine farms, the most common strategy for identification of non-pregnant females is detection of estrus via daily boar exposure from 17 to 23 days after breeding, followed by the use of ultrasound between 28 and 45 days of gestation (almond et al., 1986; zambelli et al., 2006). although, this regimen is often used with a high degree of accuracy, its usefulness is compromised by the inability to determine pregnancy status within 15 days after breeding (shaw et al., 1980). of course, the measurement of human chorionic gonadotrophin (hcg) was widely used for very early pregnancy diagnoses in human (thomas et al., 1986), and the plasma hormone (including prostaglandin-f2, hcg, progesterone and estrone sulfate) concentrations have been reported in very early pregnancy diagnoses in swine (almond et al.,1986; krüger et al.,2002), these plasma hormones concentrations had obvious changes from a non-pregnant to pregnant swine, but there was no statistical difference due to the high variation of the hormone levels between individuals (almond et al., 1987). hence, these hormones were not having the cut-off value as a neutral standard concentration range for identified non-pregnant or pregnant sows. the use of hormones as a diagnostic method may not be applicable in commercial units because of the accuracy in reducing the test. the earliest specific indication for fertilization and the continuing presence of a viable conceptus is a serum constituent, which was originally detected in mice (morton et al., 1974). this substance is known as the epf and has also been described in pigs (greco et al., 1992, koch et al., 1985). it has early appearance (within hours) after mating or insemination (shaw et al., 1980), and rapid disappearance following induced death or removal of the embryos (morton et al., 1987).these factors suggest that epf may be the most useful tool for very early pregnancy diagnosis. the objectives of this study were to evaluate the effectiveness of the epf test for detecting the pregnant sows, and to compare the reliability of rit and anti-epf antiserum blocking elisa for very early pregnancy diagnosis. the rosette inhibition titers of pregnant sows were significant higher than in non-pregnant gilts. this was consistent with the report of koch (1985). the rit test was based on the ability of alg to inhibit the formation of spontaneous rosettes between human lymphocytes and srbc. this means that the lymphocytes are spontaneously form rosettes, a flower-like arrangement in which a lymphocyte has several red blood cells attached to it. lymphocytes from pregnant animals form fewer rosettes than those from non-pregnant animals. therefore, with the aid of epf diagnostics, a pregnancy can be detected at a very early stage. however, rosette inhibition test is not a quantitative assay, and the assay is not suitable for high-throughput diagnostic and commercial applications, and rit proved cumbersome, time-consuming and was easy to disturb with a particular disadvantage which is the indirect character of the test and its possible interference by other substances (koch et al., 1985). to replace the rit with a more user-friendly assay, we developed an anti-epf antiserum blocking elisa for very early pregnancy diagnosis. the production of anti-epf antibody is indispensable for very early pregnancy diagnosis by anti-epf antiserum blocking elisa. to obtain anti-epf antiserum of high titer and specificity, the antigenic peptide (36 to 55, ig20) based on prediction of dnaman has been synthesized. three balb/c mice were immunized by ig20-bsa, and the mouse anti-ig20 antiserum titers were 6.4 × 10 3 . using anti-ig20 antiserum, a blocking elisa was constructed for very early pregnancy diagnosis in sows. when the porcine sera samples were 1/4 dilution, the pregnancy can be accurately determined (figure 4). the concen-tration of epf efficiently blocked the ag-abs reaction between ig20-ova and mouse anti-epf antiserum by a blocking elisa in pregnant serum samples. at the same time, the non-pregnancy ones were not obviously influenced by the od values of blocking elisa. the ones with pregnancy were positive and nonpregnant serum samples were negative, the pregnancy can be deter-mined by the anti-epf antiserum blocking elisa. however, some homologous substances of ig20 (hsig20) were in the porcine serum samples (betsou et al., 2003), when the serum samples were 1/2 dilution, with the concentration increases of hs-ig20, the cross-reaction was enhanced between mouse anti-epf antiserum and hs-ig20 (leder et al., 1994), and it was blocking the reaction between antibody and antigen (the coating material, ig20-ova). so, some non-pregnant serum sam-ples were presented as false-positive. on the contrary, when the porcine sera samples were 1/8 to 1/64 dilution, the concentration of epf was limited in serum samples, and it could not efficiently block the ab-abs reaction between ig20-ova and mouse anti-epf antiserum by blocking elisa, the presence of epf in these serum samples were not detected. in conclusion, the synthetic segment including the residue 36 to 55 of epf can be used as an antigen to produce the anti-epf polyclonal antibody. using this antibody, we developed an anti-epf antiserum blocking elisa for very early pregnancy diagnosis. when the test serum samples were in 1/4 dilutions, the very early pregnancy can be detected in sows. acknowledgements this study was supported by the basic and edge technology fund of henan, china (grant number: 082300433201). we would like to thank all the staff of henan key laboratory of animal immunology for their kind support. references ahlborg n (1995). synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using fmoc chemistry. j. immunol. methods. 179:269-275. alexander sm, peter o, gary k (1984). schoolnik. gal-gal pyelonephritis escherichia coli pili linear immunogenic and antigenic epitopes. j.exp.l med. 161:705-717 alice c, cavanagh a (1996). identification of early pregnancy factor as chaperonin 10: implications for understanding its role. reviews of reprod. 1: 28-32. almond gw, dial gd (1986). pregnancy diagnosis in swine: a comparison of the accuracies of mechanical and endocrine tests with return to estrus. j. am. vet. med. assoc. 189:1567-1571. almond gw, dial gd (1987). pregnancy diagnosis in swine: principles, applications, and accuracy of available techniques. j. am. vet. med assoc. 191:858-870. athanasas ps, corcoran c, kaye p, cavanagh a, morton h (2000). early pregnancy factor is required at two important stages of embryonic development in the mouse. ajri. 43:223-233. betsou f, borrego mj, guillaume n, catry ma, romao s, machadocaetano ja, sueur jm, mention j, faille n, orfila j (2003). crossreactivity between chlamydia trachomatis heat shock protein 10 and early pregnancy factor. clin. diagn. lab. immunol. 10:446-450. ebensen t, schulze k, riese p, morr m, guzman ca (2007). the bacterial second messenger cdigmp exhibits promising activity as a mucosal adjuvant. clin. vaccine immunol. 14:952-958. englebretsen dr, garnham b, alewood pf (2002). a cassette ligation strategy with thioether replacement of three gly-gly peptide bonds: total chemical synthesis of the 101 residue protein early pregnancy factor [psi (ch(ch (2)s)s)28) 28-29,56-57,76-77]. j. org. chem. 67:5883-5890. fotini b, maria j, nicolas g (2003). cross-reactivity between chlamydia trachomatis heat shock protein 10 and early pregnancy factor. clin. diagnostic laboratory mmunol. 10:446-450. 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(2007). a comparison of a blocking elisa and a haemagglutination inhibition assay for the detection of antibodies to avibacterium (haemophilus) paragallinarum in sera from artificially infected chickens. biologicals. 35:317-320. thomas cm, segers mf, leloux am, houx pc (1986). comparison of the analytical characteristics of ten urinary hcg tests for early pregnancy diagnosis. ann. clin. biochem. 23(pt 2):216-222. williams si, pineyro p, de la sota rl (2008). accuracy of pregnancy diagnosis in swine by ultrasonography. can vet. j. 49:269-273. xie l, wu xd, huang dz, chen hl, he lx, wang j, han dk(2007). clinical application and analysis of hepatitis c virus ns3 antigen detection by elisa in human serum. ch.in med. j. 120 (4):294-299. zambelli d, prati f (2006). ultrasonography for pregnancy diagnosis and evaluation in queens. theriogenology. 66:135-144. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (6), pp. 001-003, june, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. short communication a new alternative to treat swine influenza a virus infection: extracts from terminalia chebula retz hongbo ma1, yunpeng diao2,3, danyu zhao2, kun li4* and tingguo kang2* 1 department of nutrition, jilin medical college, jilin 132013, people’s republic of china, 2 college of pharmacy, liaoning university of traditional chinese medicine, dalian 116001, china 3 college of pharmacy, dalian medical university, dalian 116044, china 4 college of chemistry and chemical engineering, liaoning normal university, dalian 116029, china accepted 22 january, 2014 currently, a pandemic swine influenza a virus infection causes a huge negative impact on human beings all over the world. however, the methods of treatment are not satisfactory, therefore it is urgent for us to set up new theory and practice to fight against the intractable virus. terminalia chebula retz, as a kind of traditional chinese medicine, widely distributes and has multiple pharmacological effects. evidences in laboratory and clinic practice confirm us to the potential of terminalia chebula retz inhibiting influenza a virus infection. we thus hypothesize that acetone extracts (tannic acids, a) of terminalia chebula ret may as a new alternative treat influenza a infection based on holistic concept of traditional chinese medicine principle. key words: swine influenza a virus, terminalia chebula retz, hypothesis. introduction beginning in march 2009, an outbreak of influenza a (family orthomyxoviridae, genus influenzavirus a) has become the greatest pandemic disease threat to humankind [neumann et al., 2009]. the new strain of influenza virus designated influenza h1n1 2009, is a reassortant of swine, avian and human influenza viruses [brown, 2000; olsen, 2002; webby et al., 2000]. although the influenza a infection has less mortality, compared with other companions such as hiv-1, ebola, sars, the current transmit will not stop until pandemic eventually involve 80 percent of population all over the world according to who report. at present there are only two classes of antiviral drugs are approved to treat against influenza viruses including adamantanes and neuraminidase inhibitors such oseltamivir and zanamivir [schnitzler and schnitzler, 2009]. however, the effect of viral chemo-therapy that applies a single compound is limited with side effect such as diarrhea, dizziness or insomnia and this kind of therapy may cause drug resistant. terminaliae immaturus, the *corresponding author. e-mail: kangtg@126.com. tel:86-411 86110414 and lslikun@163.com. tel: 86-411-86110419. fruitlet of terminalia chebula fructus retz, which has been given the name of xiqingguo, mainly distribute in malaysia, thailand, india, pakistan and yunnan, tibet, guangdong, guangxi province of china [saleem et al., 2001; kusirisin et al., 2009; nariya et al., 2009; cai et al., 2008]. its active components which refer to acetone extracts (ta) contain digallic acid, chebulinic acid, chebulagic acid, terchebin and gallic acid with multiple pharmacologic actions such as anti-virus, anti-oxidation, cardiotonic action, antibacterial effect, anti-anaphylaxis, anti-tumor growth, [saleem et al., 2001; kusirisin et al., 2009; nariya et al., 2009; cai et al., 2008; feng et al., 2008; kusirisin et al., 2009; murali et al., 2007; lee et al., 2007; lee et al., 2005; juang et al., 2004; saleem et al., 2002; malekzadeh et al., 2001; sato et al., 1997; verma and raychaudhuri, 1970; cheng et al., 2003]. those biological activities are often in connec-tion with the high contents of tannic acids [kim et al., 2009]. the hypothesis to sum up the above statements, it is wise for us to get help from nature and from ethnopharmacological record or traditional medicine principle handed down from ancient times all over the world to battle with current pandemic influenza a virus. we will use acetone to extract from terminalia chebula retz to get a tannic acid mixture to inhibit pandemic influenza a infection. the theory of our hypothesis as we all know the influenza virus subtypes have a wide host range from avian to mammals including hens, pigs, horses and dogs. the genomes of influenza a virus are segmented and negative-sense rnas which can be translated into 11 functional proteins [schnitzler and schnitzler, 2009; fitzgerald, 2009; gatherer, 2009]. the main infective proteins contain the surface glycoproteins haemagglutinin (ha) binding virus to its purposive target cell, neuraminidase (na) facilitating virus release from infected cells and virulence factors ns1 antagonizing host interferon. there are 16 serotypes of haemagglutinin, and 9 serotypes of neuraminidase in total and according to these differences, the virus are classified. sialic acid is the receptor for haemagglutinin and sialyltransferases is expressed in human mucosal and respiretory tissues resulting in nglycans with -2,6 linked sialic acids. however in avian tissues, another structure of sialic acid is expressed and n-glycans are linked with 2,3-sialic acid. these different structures lead to virus specifying hosts, e.g. avian viruses mainly infect bird species. like other rna viruses, e.g. hepatitis c virus and hiv, influenza virus is characterized by genetic variability, resulting in frequent mutations and reassortment on account of influenza virus rna polymerase lack of proofreading abilities [reid and taubenberger, 2003]. thus the genetic material of current pandemic influenza a h1n1 virus is a combination of viruses that have infected pigs, birds and humans respecting swine tissues express both forms of sialic acid and can be coinfected with human and avian viruses [olsen, 2002]. it is reported that the rate of oseltamivir-resistant human seasonal h1n1 in the usa has increased to 98.5% [poland et al., 2009], up from 10% in the last year. although the instances of appearance of current pandemic influenza a h1n1 virus resistant to oseltamivir in denmark, japan and hong kong are only sporadic cases, the outbreak of large scale cases will happen inevitably if no effective actions are taken on. in addition, the supply with antiviral drugs is not sufficient for a pandemic and the cost of drugs is too expensive to afford especially for the developing countries. another alternative to defend virus infection is to appeal to our own immune system. on way the infected host immune system counters viral infection is with interferon, one of the principle functions of which is to interfere with viral multiplication without affecting the host cell itself. interferons, a group of small proteins, produced by virus-infected cells, react with plasma or nuclear membrane receptors of uninfected cells to induce synthesis of antiviral proteins. antiviral proteins are possessed of multiple functions to prevent further infection of virus including blocking initiation of virus protein synthesis, inhibiting virus polypeptide elongation and destroying viral mrna before translation. even through there do exists problems partially due to short term effectiveness of interferon, it typically plays a positive role against acute and short term virus infection especially influenza. therefore any measures that could improve our immune system response to secret sufficient interferon are suggested and approved. in laboratory test, the water decoction of fructus terminaliae immaturus exhibit obvious antibacterial effect inhibiting both gram positive bacterium including staphylococcus aureus, pneumococcus, streptococcus hemolyticus, bacillus diphtheriae and gram negative bacterium including escherichia coli, bacillus dysenteriae, pseudomonas aeruginosa, bacillus proteus, bacillus tphpi, bacillus typhi murium, helicobacter pylori. the alcoholic extracts of terminalia chebula retz demonstrated significant anti-virus effect in 2.2.15 cell line infected with hbv, and the extracts with a certain concentration of hydrochloric acid have a more powerful inhibitory effect on bacterium and fungus growth. chinese patent medicines containing terminalia chebula retz can inhibit acyclovir -resistant herpes simplex virus i in vitro and in vivo. in addition, in animal research, terminalia chebula retz was used to treat endotoxin sepsis shock because terminalia chebula retz can regulate immune response by making host cells release interferon and tnf as well as activating monocyte/macrophage system [cai et al., 2008; feng et al., 2008]. feasibility and prospects we will use acetone to extract from terminalia chebula retz to get a tannic acid mixture without purification further. base on traditional chinese medicine and chinese materia medica principle, we would like to emphasize the holistic concept, which means that every thing should be considered as a whole and there is synergistic effect of each component of a plant. the synergy is not the simple sum of several components, but rather mutually reinforcing role, with a single component can not be achieved. the aim of extraction is not intended to find a single anti-viral compound, but rather to remove impurities to enhance antiviral activity of terminalia chebula retz according to guiding role of holistic concept. the extracts combined with pandemic influenza a virus were inoculated into nonimmune chick embryo, and then the chick embryo and allantoic fluid were observed to evaluate the antiviral effect of the extracts. results natural medicine of the antiviral and enhancing immunity including extract or mixture, which may fight against influenza virus through different targets. conclusion the acetone extract (tannic acids, ta) of terminalia chebula ret may be considered as a effective method for human being fighting against pandemic swine influenza a virus on account of its low cost, easy preparation and significant therapeutic action. acknowledgement the authors thank the ph.d. zhang zhen for manuscript revision assistance. reference brown ih (2000). the epidemiology and evolution of influenza viruses in pigs. vet. microbiol. 74(1-2): 29-46. cai xh, xie b, du hj (2008). advances in research on chemical constituents and pharmacological action of terminaliachebula retz. prog. pharm. sci. 32(5): 212-215. cheng hy, lin tc, yu kh, yang cm, lin cc (2003). antioxidant and free radical scavenging activities of terminalia chebula. biol. pharm. bull. 6(9): 1331-1335. feng sx, ma xj, yan zg (2008). research progress on the chemical constituents and pharmacological action of termichebula retz. j. anhui. agric. sci. 36(25): 1093810939, 10941. fitzgerald da (2009). human swine influenza a [h1n1]: practical advice for clinicians early in the pandemic. paediatr. respir. rev. 10: 154–158. gatherer d (2009). the 2009 h1n1 influenza outbreak in its historical context. j. clin. virol. 45: 174–178. juang lj, sheu sj, lin tc (2004). determination of hydrolyzable tannins in the fruit of 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antiviral agents: a plea for rational use. clin. infect. dis. 48: 1254–1256. reid ah, taubenberger jk (2003). the origin of the 1918 pandemic influenza virus: a continuing enigma. j. gen. virol. 84: 2285-2292. saleem a, ahotupa m, pihlaja k (2001). total phenolics concentration and antioxidant potential of extracts of medicinal plants of pakistan. z. naturforsch. c. 56(11-12): 973-978. saleem a, husheem m, härkönen p, pihlaja k (2002). inhibition of cancer cell growth by crude extract and the phenolics of terminalia chebula retz. fruit. j. ethnopharmacol. 81(3): 327-336. sato y, oketani h, singyouchi k, ohtsubo t, kihara m, shibata h, higuti t (1997). extraction and purification of effective antimicrobial constituents of terminalia chebula rets. against methicillin-resistant staphylococcus aureus. biol. pharm. bull. 20(4): 401-404. schnitzler su, schnitzler p (2009). an update on swine-origin influenza virus a/h1n1: a review. virus. genes 39: 279–292. verma vs, raychaudhuri s (1970). effect of tannins from terminalia chebula retz. on the infectivity of potato virus x. acta. microbiol. pol. b. 2(2): 127-32. webby rj, swenson sl, krauss sl, gerrish pj, goyal sm, webster rg (2000). evolution of swine h3n2 influenza viruses in the united states. j. virol .74(18): 8243-8251. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (2), pp. 001-008, february, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper genetic diversity of escherichia coli isolated from commercial swine farms revealed by enterobacterial repetitive intergenic consensus li wan1, zhi-biao wang1, qi-gui yan1,2*, xu wang1, yan lei1, lan zuo1, yu cheng1, yu-peng ren1 and wan-zhu guo1 1 college of veterinary medicine, sichuan agricultural university, ya’an, 625014, p. r. china. 2 key laboratory of animal disease and human health of sichuan province, sichuan agricultural university, ya’an, 625014, p. r. china. accepted 09 november, 2014 the objective of this study was to use enterobacterial repetitive intergenic consensus pcr (eric-pcr) and repetitive extragenic palindrome pcr (rep-pcr) for the analysis of genetic diversity among escherichia coli strains isolated from commercial swine farms in sichuan province of china. thirty four strains of e. coli were selected by selective medium and conventional biochemical test from fresh stool samples of swines in five farms in sichuan province. the isolates were identified by 160 kinds of e. coli o serums. the results show that 30 strains were determined among 34 e. coli isolates, 12 kinds of o serogroups were obtained on the basis of the agglutination test. the predominant types are o23, o 113 and o120, representing 35.4%. furthermore, the genotypes and phylogenetic relationship of all isolates were analysed by enterobacterial repetitive intergenic consensus pcr (eric-pcr) and repetitive extragenic palindrome pcr (rep-pcr), 34 e. coli isolates were clustered to 19 eric-pcr genotypes and 13 rep-pcr genotypes. the isolates from the same farm or sharing the same serotyping showed different genotype. and the isolates which could not be serotyped were genotyped by eric-pcr and rep-pcr. the analysis of genetic type and original source revealed that isolates from different farms had different genetic types. the subtypes of e. coli are also different within a single farm. genetic variability with e. coli strains isolated from swine farms in china has been demonstrated. the presence of eric-pcr and rep-pcr sequences in the genome of e. coli was confirmed. eric-pcr and rep-pcr techniques are more rapid methods for molecular typing of e. coli strain. they are also useful methods for diversity survey of e. coli and the two methods analyzes genetic diversity of e. coli isolated in sichuan of china. key words: escherichia coli, serotype, enterobacterial repetitive intergenic consensus pcr (eric-pcr), repetitive extragenic palindrome pcr (rep-pcr). introduction escherichia coli is one of the strain widely distributed in nature and intestinal tract of animal. genetic diversity exists widely among strains, so that using traditional *corresponding author. e-mail: yanqigui@126.com. bacterial taxonomy proves problematic when differentiating between strains that share a close genetic relationship. the traditional method for subspecies typing among the e. coli has been serotyping. the serotype is based on the antigenic properties of the o-antigen (surface polysaccharide) and h antigen(s) (flagellar). typing the o-antigen itself denotes the serogroup, and table 1. the results of e. coli isolation. swine house serial number original king of sample number a a1 farrow stool 1 a a2 stag stool 1 a a3, a4, a5, a6 weaner stool 4 b b1, b3, b4 pregnancy sow stool 3 b b2, b5 farrow stool 2 b b6 stag stool 1 c c1, c2 pregnancy sow stool 2 c c3, c6, c7 weaner stool 3 c c4, c5 farrow stool 2 d d1, d6 pregnancy sow stool 2 d d2, d4, d7 weaner stool 3 d d3, d5 stag stool 2 e e1, e2, e6, e7, e8 pregnancy sow stool 5 e e3, e4, e5 weaner stool 3 total 34 the serotype is obtained by characterizing the flagella, which is often biphasic (popoff and le minor, 2001). although, all the methods are reliable, they present some disadvantages either in the time needed to perform the test or in the cost of sera and reagents. moreover, readings are not always easy to interpret. with the development of molecular biology, some classification methods based pcr technique had been recognized as having great ability to differentiate separate strains by producing dna fingerprints that are specific for individual strains. these methods were proved to be fast, sensitive and reliable for differentiation of microorganism. several of them had been used widely for studying genetic relationship of different bacteria such as the random amplified polymorphic dna pcr (rapd-pcr) (welsh and mcclelland, 1990), enterobacterial repetitive intergenic consensus sequence pcr (ericpcr) (wei et al., 2004; wenz et al., 2006; chen et al., 2010) and repetitive extragenic palindromic pcr (rep-pcr) (trombert et al., 2007; bonacorsi et al., 2009), other analysis methods such as pulsed-field gel electrophoresis (pfge) (mclellan et al., 2003; casarez et al., 2007), amplified fragment length polymorphism (aflp) (engstrom et al., 2003) and ribotyping also had been applied widely. rep consensus sequence consist of 38 bp inverted repeat sequence, eric sequences are 126 bp long and appear to be restricted to transcribed regions of the genome (versalovic et al., 1991). rep-pcr and eric-pcr were used for studying bacterial source tracking in the field and analysis of genetic diversity. identification of the spread of airborne escherichia coli using ericpcr and rep-pcr (dombek et al., 2000; duan et al., 2009; yuan et al., 2010) has been reported using eric-pcr fingerprinting to assess e. coli strains diversity isolated from natural water (casarez et al., 2007). sichuan is one of the biggest pig raising provinces in china. the infectious diseases caused by e. coli are very serious. analysis of e. coli strains genetic diversity is important for epidemiology. the objective of this study was to use reppcr and eric-pcr for the analysis of genetic diversity among e. coli strains isolated from commercial swine farms in sichuan province of china. materials and methods five commercial swine farms in sichuan were chosen, fresh stool samples were aseptically collected from sick pig that have being alo laxata from the five farms, respectively, a total of 40 stool samples were collected, they are listed in table 1. samples were diluted and spread onto the surface of macconkey agar plates which were incubated at 37°c for 18 to 20 h. colonies with typical pink on macconkey agar plates were picked and streaked onto eosin methylene blue (emb) agar plates. after overnight incubation at 37°c, one or two colonies with a metallic sheen on emb agar were selected and cultured in luria-bertani broth. all cultures were stored at -70°c in luria-berintani broth with 30% glycerol. then, the strains species identifications were made using conventional biochemical tests (not shown in the paper). a total of 34 e. coli strains were chosen due to their varying sources. serotyping procedures all the 160 standard e. coli o factor serums (o17, o23, o24 o198, etc) were kindly provided by china institute of veterinary drug control, and the serotyping procedures used including the production of heat-stable antigen extracts by heating at 121°c for 2 h have been described in detail elsewhere (biao et al., 2009). dna preparation genome dna of all e. coli was isolated by the ctab/naci miniprep protocol (barney et al., 2001) used for all pcrbased methods. all e. coli isolates strains were grown in 5 ml of luria table 2. the serological result of e. coli o antigen by slide agglutination. serotype strain numbe (%) o198 a1, a2, c1 3 (8.8%) o150 a3, b2, e4 3 (8.8%) o46 a5, d6, e7 3 (8.8%) o113 a6, c3, d3, e8 4 (11.8%) o26 b4 1 (2.9%) o120 b5, d2, e1, e2 4 (11.8%) o23 b6, c2, d4, e3 4 (11.8%) o17 c4, c5 2 (5.9%) o68 c6, c7 2 (5.9%) o24 d5 1 (2.9%) o78 d7 1 (2.9%) o129 e5, e6 2 (5.9%) bertani (lb) broth (oxoid) with moderate shaking for 18 h at 37°c. 1.5 ml lb broth was removed and centrifuged at 10,000 g for 2 min. the cell pellet was resuspended in 567 l te and mixed with 30 10% sds and 3 l 20 mg/ml protein k; and the mixture reacted for 1 h at 37°c; followed by 100 l abtc and 100 l 5mol/l nacl were added in the mixture, 65°c for 10 min, and dna templates were extracted by phenol/chloroform method, and dna preparation were stored at 20°c until used by pcr amplifications. three microlitres of dna preparation was used as the source of dna template in the pcr reaction. eric-pcr the primers 5’-atg taa gct cct ggg gat tca c-3’ (eric1); 5’-aag taa gtg act ggg gtg agc g-3’ (eric2) (versalovic el al., 1991) were synthesized by invitrogen. the pcr reaction (25 l) contained 10× buffer 2.5 l, dntps (2.5mmol/l) 2.5 l, taqdna polymerase (5 u/ l) 0.5 l, mgcl2 (25 mmol/l) 1.5 l, primers (10 mol/l) each 1 l, template dna 1 | and addition ddh2o to 25 l. amplification was performed in a thermal cycler (bio-bri) with different temperatures: denatured for 7 min at 94°c, followed by 40 cycles of 1 min at 94°c, 1 min at 48°c and 5 min at 72°c; and then extension at 72°c for 7 min. the reaction products were stored at 4°c until they were electrophoresed on a 2% (w/v) agarose gel containing 1×tae and 0.5 g ml -1 ethidium bromide together with the dl2000 dna markers (takara) for 2 to 3 h. rep-pcr the primers rep1r (5 -iii icg icg ica tci ggc-3 ) and rep2i (5 -icg ict tat cig gcc tac-3 ) (versalovic et al., 1991) were synthesized by invitrogen. the pcr reaction (25 l) contained 10× buffer 2.5 l, dntps (2.5mmol/l) 2.5 l, taqdna polymerase (5 u/ l) 0.5 l, mgcl2 (25 mmol/l) 1.5 l, primers (10 mol/l) each 1 l, template dna 1 l and addition of ddh2o (25 l) . the amplification was carried out by incubation of the mixture for 5 min at 95°c for predenaturation, followed by 35 cycles of denaturation at 94°c for 1 min, annealing at 45°c for 1 min and extension at 65°c for 8 min. a final extension was performed at 65°c for 16 min. the reaction products were stored at 4°c until they were electrophoresed on a 1.5% (w/v) agarose gel containing 1×tae and 0.5 gml -1 ethidium bromide together with the dna marker iii (takara) for 2 to 3 h. eric-pcr and rep-pcr fingerprints analyses eric-pcr and rep-pcr fingerprints of amplified dna fragments obtained from the agarose gel electrophoreses were recorded. the observed bands in the gels were evaluated based on the presence (coded 1) or absence (coded 0) of polymorphic fragments for the eric and rep-pcr primers. cluster analysis was performed with ntsyspc (version 2.10), a numerical taxonomy and multivariate analysis software package (rohlf, 2000), based on dice’s similarity coefficient (sd) with a 1% position tolerance and the unweighted pair group method using arithmetic averages (upgma). in addition, each isolate was considered as an operational taxonomic unit (otu). in order to reduce the number of otus in the dendrogram, to facilitate interpretation, isolates of 70% similarity were treated as a single isolate. results serotyping as noted earlier, 30 strains are determined among 34 e. coli isolates, 12 kinds of o serogroups were obtained on the basis of slide agglutination. the results are listed in table 2. the predominant types are o23, o113 and o120, representing 35.4%, but 3 strains are nontypeable by the test. the nontypeable isolates may either exist due to the fact that some isolates do not likely contain a sufficient level of serovar-specific antigens or that there exist novel serovars that have not been identified yet. the remaining serovars were detected at low frequencies, ranging from 2.9 to 8.8% (table 3). as indicated in table 3, 5 serovars were identified among 6 strains isolates from a farm, 4 serovars present in b farm, 7 serovars present in c farm, 6 serovars present in d farm, 8 serovars present in e farm, and otype 23 was reported to be the most prevalent serotype in b, c, d and e, followed by o113 (a, c, d and e). comparing the type strains, many field isolates with mixed serotypes showed that there are many kinds of serogroups in the same pig farm and they have predominant types in most pig farms. some predominant table 3. the distribution of o serogroups of e. coli in different pig farms. pig farm number of e. coli numberof identified serotype isolate isolate (number of isolate) a 6 5 o198(2) o150(1) o46(1) o113(1) b 6 4 o150(1) o26(1) o120(1) o23(1) c 7 7 o198 (1) o23(1) o113(1) o17(2) o68(2) d 7 6 o120(1) o113(1) o23(1) o24(1) o46(1) o78(1) e 8 8 o120(2) o23(1) o150(1) o129(2) o46(1) o113(1) figure 1. eric-pcr fingerprints obtained for e. coli isolate from a to e farms with primers eric1 and eric2. lane: m, dna marker dl2000; lane: a1 to a6, e. coli strains isolated from a pig house of sichuan; lane: b1 to b6, e. coli strains isolated from b pig house of sichuan; lane: c1 to c7, e. coli strains isolated from c pig house of sichuan; lane: d1 to d7, e. coli strains isolated from d pig house of sichuan; lane: e1 to e8, e. coli strains isolated from e pig house of sichuan. serogroups in different districts are identical, others are different. serogroups of different pig farms in the same district are generally different. eric-pcr the eric-pcr fingerprints analysis showed that there were 6 to 19 bands with molecular weight ranging from 100 bp to more than 3 kb generated by eric primers (figure 1), and a common intensive band of about 270 bp in most e. coli (figure 1). the data matrices based on the dna fragments and the dendrogram using the ntsyspc software were constructed, all e. coli isolates obtained in this study were grouped into different clusters or branches based on the eric-pcr pattern similarity. according to the upgma dendrogram (figure 2), all generated dna patterns are relatively complex. each subjects exhibited a unique banding pattern with similarity ranging from 0.17 to 0.93. these 34 isolates were grouped into 19 different groups with the similarity coefficients 0.78, designated as i-xix. the cluster x includes 5 strains isolated from d farm with similarity coefficients 0.83. this second level of clustering suggested that a remarkable polymorphism exists among all strains in these groups. the two samples collected at different farms showed much higher similarity, 0.89 between c6 and e5 and0.81 between a1 and b5. rep-pcr rep-pcr fingerprinting profiles and cluster figure 2. dendrogram established by the biostatistical analysis program ntsys-pc using the dice similarity coefficient and upgma on the basis of the eric-pcr profile of e. coli strains obtained with primers eric1 and eric2. analysis of 34 e. coli strains are listed in figure 3. with primers rep1r and rep2i, rep-pcr fingerprinting profiles were obtained. analysis of the strains with rep-pcr yielded one to fourteen bands depending on the strains. the size of these bands ranged from 200 bp to more than 4500 bp. rep-pcr fingerprinting profiles of e. coli strains were different from each other. according to upgma dendrogram, 34 e. coli strains were separated into thirteen types with 0.81 similarity named 1 to 13 (figure 4); however, e. coli strains of every pig house were not in the same type. figure 3. reppcr fingerprints for 34 e. coli strains a to e farms with primers rep1r and rep2i. lane: m, dna marker iii; a1 to a6, e. coli strains isolated from a pig house of sichuan; b1 to b6, e. coli strains isolated from b pig house of sichuan; c1 to c7, e. coli strains isolated from c pig house of sichuan; d1 to d7, e. coli strains isolated from d pig house of sichuan; e1 to e8, e. coli strains isolated from e pig house of sichuan. thirteen e. coli strains (a1, a4, a5, b1, b4, b6, c1, c3, c4, d7, e1, e4 and e5) sharing common band of about 700 bp were clustered together and belonged to type 1; among them a4, d7, e5 and b1, e4, e5 shared 1.0 similarity, however, they were not from simple pig house. type 2 only had one strain e7. four e. coli isolates d2, d4, d5 and d6 isolated from d pig house were clustered to type 3. other e. coli isolates sharing high similarity were separated into same type such as b2 and b3, e3 and e8, c5 and c6, a6 and c2.the remaining e. coli strains were unique. discussion rep-pcr and eric-pcr techniques that prove to be fast, sensitive and reliable for the differentiation of microorganism were applied for analysis of genetic diversity and epidemiology. rep-pcr and eric-pcr technique were established from highly conserved repetitive dna sequence distributed among the gene of microorganism. combined with pcr and gel electrophoresis, highly complex and specificity genome fingerprintings profile produced could discriminate spices and strains. rep-pcr and eric-pcr technique are widely used method for dna typing analysis and has been shown to successfully differentiate strains of bradyrhizobium japonicum (judd et al., 1993), lactobacillus johnsoni (ventura and zink, 2002) and clostridium perfringens (xiaoli et al., 2009). many genotyping studies were done on clinical isolates of e. coli, as well as environmental and veterinary isolates identified via biochemical and/or culture based approach (carvalho de moura et al., 2001; gordon and cowling, 2003; higgins et al., 2007). in this study, two molecular methods, rep-pcr and eric-pcr, were applied and their power to discriminate 34 e. coli strains was compared with serogrouping method. difference in pcr fingerprinting profile existed among identical swine farms. it was indicated that the diversity of e. coli strains of swine farms in sichuan province were high. genetic diversity of e. coli isolated from different five swine farms demonstrated in dendrogram based on eric-pcr fingerprinting profiles, namely, e. coli strains from different sources showed certain difference at the level of gene and were separated into different subgenetypes. e. coli isolated from the same swine farms were clustered into several groups, and had low cross with other farms. according to analysis of the dendrogram of eric-pcr, it can be seen that farm a was mainly concentrated in i, ii and xiv, farm b was mainly concentrated in iii, iv and v, farm c was mainly concentrated in ix, xii and xvi, farm d was mainly concentrated in x, xv and xiii, farm d was mainly concentrated in vi, vii, viii, x, xi, xvi, xvii and xix. when compared with eric-pcr, rep-pcr has lower discrimination power. although, genetic differences of e. coli of farms were demonstrated on dendrogram by reppcr, the sub-genetypes of identical farm has higher cross with others. especially, genetypes of e. coli from farm a were crossed with other farms, and it was difficult to analyze the major subgroup of e. coli of some farms. it was determined that the major groups of farm d were 3 and 6, farm e were 2, 5 and 8. each e. coli strain has only one o serovars, so it is usually chosen for serotype identification. the results show that e. coli o serovars diversity was presented in five swine house. the method of analysis of e. coli diversity by o serovars diversity is a practical method indeed. but the quantity of e. coli o serovars is much and it is difficult to collect all identified o serovars. accordingly, o serovars of 4 strains were not identified because only 160 kinds of o serovars were provided. figure 4. dendrogram established by the biostatistical analysis program ntsys-pc using the dice similarity coefficient and upgma on the basis of the rep-pcr profile of e. coli strains obtained with primers rep1r and rep2i. predicting salmonella enteric serotypes by when compared with both sides, o antigen study were quite complex, which made it difficult repetitive sequence-based pcr has been serotypes of salmonella strains studied by for eric-pcr and rep-pcr to identify the o reported recently successfully and it was albufera were relatively single (albufera et al., antigen serotypes of e. coli. for example, c6 and discovered that rep-pcr and padf analysis 2009; wise et al., 2009). e5 are closer that e6 and e5 on the dendrogram, have good correlation with o antigen serotyping. however, o antigen serotypes of e. coli in this but e5 and e6 belonged to the same serogroup and c6 to another. it was consistent with previous study. hence from this study, the results of pcr fingerprinting profiles with the primers rep and eric revealed that genetic variability of e. coli strains isolated from the five swine farms was obvious. it was indicated that the diversity of e. coli strains of swine farms in sichuan province were high. acknowledgements this study was supported by “program for changjiang scholars and innovative 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yu-chih wang1, yi-chih chang1,6, hsiao-li chuang2, chien-chao chiu2, kuang-sheng yeh4, chao-chin chang3, shih-ling hsuan5 and ter-hsin chen3,5* 1 department of veterinary medicine, national chung hsing university, taichung, 402, taiwan. 2 national laboratory animal center, national applied research laboratories, taipei, 115, taiwan. 3 the graduate institute of microbiology and public health, college of veterinary medicine, national chung hsing university, taichung, 402, taiwan. 4 department of microbiology and immunology, school of medicine, taipei medical university, taipei, 402, taiwan. 5 graduate institute of veterinary pathobiology, college of veterinary medicine, national chung hsing university, taichung, 402, taiwan. 6 department of medical laboratory science and biotechnology, china medical university, taichung, 402, taiwan. accepted 04 march, 2017 salmonella schwarzengrund is an infrequent serovar isolated from humans in taiwan. however, this serovar is which is highly invasive, is found in humans as report by two independent epidemiologic surveys. the present study delineated the widespread resistance to fluoroquinolone in s. schwarzengrund isolated from both poultry and swine carcesses in taiwan. we conducted the present study to investigate the prevalence and characteristics of s. schwarzengrund isolated from broiler chicken in central taiwan. a total of 187 s. schwarzengrund isolates was gotten from slaughtered chicken and pigs in central taiwan between june 2006 and march 2007. the percentages of resistance were as follows: ampicillin (90.74%), chloramphenicol (69.75%), florfenicol (24.07%), streptomycin (91.98%), trimethoprim and sulfamethoxazole (91.36%), nalidixic acid (96.30%), ciprofloxacin (8.02%), tetracycline (95.68%), amikacin (8.64%), ceftiofur (0.62%) and ceftriaxone (0%). four types of class 1 integrons were detected: 1.2 kb carried blapse-1, aada2 gene (n = 2), 1.2 kb carried blapse1, dfraa1 gene (n = 2), 1.0/1.2 kb carried blapse-1, aada2 ( n = 2) and 1.9 kb carried dfra12, aada2 gene (n = 152). xbai-digested pfge patterns generated related clusters implicated in the dissemination of integron. thirteen ciprofloxacin-resistant isolates were all detected in 5 identical mutations in the qrdr of gyra, parc and pare. our results suggest that there is a risk of transmitting multidrug resistant salmonella between pig and chicken farms, and also suggest that there is a need to prevent the transmission of this organism from a neighboring contaminated farm. key words: integron, multidrug resistance, salmonella schwarzengrund. introduction salmonella enterica is one of the most common causes of human foodborne infection. most serovars of salmonella infections result to self-limited gastroenteritis which is usually recovered from without treatment. however *corresponding author. e-mail: thc@dragon.nchu.edu.tw. tel: 886 4 22840894. ext: 407. some lead to severe invasive infections. invasive salmonella infections can be life threatening and usually require hospitalization and antibiotics treatment. antimicrobialresistant salmonella were associated with an increased rate of hospitalization compared with outbreaks caused by multidrug resistant (mdr) salmonella strains (boyd et al., 2002) . in taiwan, salmonella schwarzengrund was the 3rd or 4th frequent serotypes (chiu et al., 1999; lauderdale et al., 2006), and the high prevalence of resistance to extended-spectrum cephalosporins or fluoroquinolones found in salmonella schwarzengrund (chen et al., 2006; lauderdale et al., 2006; yan et al., 2005) has become an important concern. this serovar showed highly invasive rate (25 and 12.5%, respectively, for) report by two independent epidemiologic surveys (lauderdale et al., 2006; vugia et al., 2004). multidrug resistance of salmonella is linked to the presence of class 1 integrons (leverstein et al., 2003). the acquisition of integrons via horizontal gene transfer allows bacteria to rapidly evolve. class 1 integrons are the most common integrons found in clinical salmonella isolates (fluit 2005; kwon et al., 2002) . salmonella genomic island 1 (sgi1) is a 43 kb genomic island, which contains a complex integron. sgi1 is mobile and can be transferred between di erent salmonella serovars and other bacteria in the presence of a helper plasmid (doublet et al., 2005; khemtong and chuanchuen, 2008). studies suggest that broiler chickens are a major reservoir for salmonella schwarzengrund (aarestrup et al., 2007; bangtrakulnonth et al., 2004). however, epidemiology data of mdr salmonella schwarzengrund among chickens are limited. one potential health hazard associated with the veterinary use of antibiotics is the transmission of antibiotic-resistant pathogens from animals to humans, this study examined antibiotic resistance of salmonella schwarzengrund isolated from broiler and swine in taiwan. materials and methods sample isolates a total of 187 salmonella schwarzengrund isolates were obtained from slaughtered chickens from 5 chicken slaughterhouses (n = 159) in 2006 2007 and 2 pigs slaughterhouses (n = 28) in 2005 2006 in central taiwan. isolates were serotyped by using commercial salmonella o and h antisera purchased from s&a reagents laboratory (bangkok, thailand) and denka seiken (tokyo, japan). all isolates were stored in 20% glycerol at -80°c. bacteria were grown on tryptic soy agar/broth (difco, mi, u.s.a.) at 37°c. antibiotic susceptibility test antimicrobial susceptibility was tested by a standard disk diffusion method, and escherichia coli atcc 25922 was used for control. the antimicrobial agents used were ampicillin, chloramphenicol, florfenicol, streptomycin, trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, amikacin, ceftiofur, ceftriaxone and tetracycline. susceptible and resistant isolates were defined according to the criteria suggested by the clinical and laboratory standards institute. genotyping by pulsed-field gel electrophoresis (pfge) genotypes of all salmonella schwarzengrund isolates were determined by pulsed-field gel electrophoresis (pfge) analysis using the restriction endonuclease xbai to digest total genomic dna. the procedure for the pfge was performed according to the standard protocol developed by the centers for disease control and prevention (barrett et al., 2006). the digested dna was separated by the use of chef dr ii (bio-rad) in 0.5x tris-borateedta at 14°c for 20 h. pcr analysis, dna purification and dna sequencing dna templates used for pcr were prepared by boiling bacterial cultures or by using the qiagen genomic-tip system (qiagen). amplifications were performed in 25 l reaction mixtures containing 2.5 l of dna, 2.5 l 10x pcr buffer, 1.5 m mgcl2, 200 m each deoxynucleoside triphosphate, 2.5 u of taq dna polymerase (promega) and 1 m each primer. to amplify fragments larger than 3 kb, blend taq-plus polymerase (toyobo) was used instead of taq dna polymerase. the pcr products were visualized by ethidium bromide staining after agarose gel electrophoresis. amplification products were purified with the qiaquick pcr purification kit (qiagen) for sequencing. the resulting dna sequence data were compared to the genbank database using the blast algorithm available at the national center for biotechnology for biotechnology information website (www.ncbi.nlm.nih.gov). class 1 integrons were screened by pcr with primers 5’ -cs and 3’ -cs as described previously (lévesque et al., 1995). detection of salmonella genomic island 1 was done using primers corresponding to left and right junctions as described previously (cloeckaert et al., 2000). pcr mapping of the typical antibiotic resistance genes associated with sgi1 was performed using conditions and primers described previously (boyd et al., 2002; levings et al., 2005). for the detection of resistance-mediating mutations in the dna gyrase, topoisomerases iv and plasmidmediated quinolone resistance genes, the quinolone-resistance determining region (qrdr) of gyra, gyrb, parc, pare, qnra, qnrb and qnrs genes was amplified by pcr oligonucleotide primers which were amplified and sequenced. the sequences of the primers and pcr condition was described previously (casin et al., 2003; gay et al., 2006; ling et al., 2003). results antimicrobial susceptibility testing all isolates were sensitive to ceftriaxone and only one isolate was resistant to ceftiofur. eighty-six isolates (45.99%) displayed the pentaresistance phenotype (acssut). resistance to nalidixic acid, tetracycline, streptomycin, trimethoprimsulfamethoxazole and ampicillin was found in 96.30, 95.68, 91.98, 91.36 and 90.74% of the isolates, respectively. isolates from chickens resistant to ampicillin, trimethoprimsulfamethoxazole, nalidixic acid and tetracycline were more than the isolates from pigs. however, resistance to florfenicol, ciprofloxacin and amikacin of the isolates from pigs was higher than isolates from chickens (table 1). pcr detection of integrons and gene cassettes class 1 integrons were positive in 84.49% (158/187) table 1. percentages of salmonella schwarzengrund isolates resistant to antibiotics from broiler chickens and pigs. antibiotic % of resistant isolates from broiler(n = 159) pig (n = 28) overall (n = 187) ampicillin 93.06 (134) 72.22 (13) 90.74 chloramphenicol 69.44 (100) 72.22 (13) 69.75 florfenicol 18.75 (27) 66.67 (12) 24.07 streptomycin 91.67 (132) 94.44 (17) 91.98 trimethoprim-sulfamethoxazole 94.44 (136) 66.67 (12) 91.36 nalidixic acid 97.92 (141) 83.33 (15) 96.30 ciprofloxacin 4.17 (6) 38.89 (7) 8.02 tetracycline 96.53 (139) 88.89 (16) 95.68 amikacin 1.39 (2) 66.67 (12) 8.64 ceftiofur 0.69 (1) 0 0.62 ceftriaxone 0 0 0 table 2. characteristics of the serovar schwarzengrund strains tested in this study. size(kb) of integron i and variant genomic island name isolated from no. of strains identity of resistance gene broiler pig none 10 4 10 1.2blapse-1, aada2 sgi1 1 1 2 1.2blapse1, dfraa1 sgi1-f 1 1 2 1.2blapse-1, 1.0aada2 sgi1 2 0 2 1.9dfra12, aada2 130 22 152 isolates with 3 pcr amplification product patterns: 1,900 (152 isolates), 1,200 (4 isolates) and 1,000 + 1,200 bp (2 isolates) detected. sequence analysis of the integron pcr products showed the presence of classic gene cassettes in the integrons, including aada2 (which confer resistance to streptomycin), the dihydrofolate reductase gene cassettes dfrxii, dfra1 (which confer resistance to trimethoprim) and the beta-lactamase gene blapse1 (which confers resistance to ampicillin) (table 2). six isolates were positive to the sgi1. pcr mapping of the antibiotic resistance gene cluster demonstrated that 4 isolate contain the sgi1 and 2 isolate contain sgi1-f (table 2). chicken and pig sources. for all isolates of salmonella schwarzengrund, screening gave negative results for qnra, qnrb and qnrs genes. and 13 ciprofloxacin-resistant isolates were all detected as double mutations in the qrdr of gyra (s83f and d87g), double mutations in the qrdr of parc (t57s and s80r) and an additional mutation in the qrdr of pare (s458p). these 13 ciprofloxacin-resistant salmonella schwarzengrund isolates were isolated from 2 different chicken slaughterhouses and 2 different pig slaughterhouses. all these isolates had the same genotype as 2d. genotype of pfge eleven pfge profiles were observed from the 187 isolates in the study when total genomic dna was digested with xbai. genotype 2d was seen in 84 isolates (44.92%) and genotype 2b was represented by 44 isolates (22.53%) . the third common genotype was 1a which was found in 15 isolates (8.02%). a dendrogram generated from dice coefficients of similarity indicated that all isolates were highly related with dice values greater than 84% (figure 1). the predominant genotypes (1a, 2b and 2d) were observed in isolates from both discussion nontyphoidal salmonella which causes foodborne diseases has become an important public health problem worldwide. salmonella enteritidis and salmonella typhimurium were the most common causes of nontyphoidal salmonellosis. salmonella schwarzengrund is a less common cause of human salmonellosis worldwide. however, the incidence of salmonella schwarzengrund has increased in recent years (bangtrakulnonth et al., 2004; boyd et al., 2002). this serovar is commonly found from invasive salmonellosis patients in taiwan figure 1. pfge patterns of xbai-digested chromosomal dna of s. enterica serovar schwarzengrund. the dendrogram was constructed by use of the upgma algorithm and the dice similarity coefficient by using gelcompare ii software with 1% position tolerance. (boyd et al., 2002). this study showed a high frequency of antimicrobial drug resistance, including ampicillin, trimethoprimsulfamethoxazole, among salmonella schwarzengrund isolates from slaughtered chickens. in contrast, the frequency of resistance to florfenicol, ciprofloxacin and amikacin of isolates from pigs was higher. it may correlate the antibiotics used in the pig farms and broiler farms. 13 ciprofloxacin-resistant salmonella schwarzengrund isolates found in 2 different chicken slaughterhouses and 2 different pig slaughterhouses had the same mutations in the qrdrs of gyra, par and identical pfge pattern. molecular characterization suggests an epidemiological relationship between the swine and chicken salmonella schwarzengrund isolates. our results suggest that there is a risk of transmitting multidrug resistant salmonella between pig and chicken farms, and also suggest that there is a need to prevent the transmission of this organism from a neighboring contaminated farm. this study also found the widespread occurrence of class 1 integrons and sgi1 variants in multidrug resistant salmonella schwarzengrund isolates from broiler chickens and pigs. genes encoding for resistance to trimethoprim (dfra12) and aminoglycosides (aada2) were most commonly found. the presence of this 1.9 kb integrons has been found in many serovars worldwide (doublet et al., 2005; fluit 2005; gay et al., 2006; hsu et al., 2006). the transfer of conjugative plasmids is a common mechanism for genetic exchange between bacteria, transfer can occur both within bacterial species and between different species. therefore, the presence of the integron-carrying salmonella isolates detected in this study could have contributed not only to the spread of drug-resistant strains but also to the spread of mobile elements carrying drug resistance genes across bacterial species. references aarestrup fm, hendriksen rs, lockett j, gay k, teates k, mcdermott pf, white dg, hasman h, sørensen g, 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enterica typhimurium isolates in humans. emerg. infect. dis. 9: 1455 1457. chen th, wang yc, chen yt, yang ch, yeh ks (2006). serotype occurrence and antimicrobial susceptibility of salmonella isolates recovered from pork carcasses in taiwan (2000 through 2003). j. food prot. 69: 674 678. chiu ch, lin ty, ou jt (1999). predictors for extraintestinal infection of non-typhoidal salmonella in patients without aids. int. j. clin. pract. 53: 161 164. clinical and laboratory standards institute (2007). performance standards for antimicrobial susceptibility testing; seventeenth informational supplement, m100-s17. clsi, wayne, pennsylvania. cloeckaert a, sidi bk, flaujac g, imberechts h, d’hooghe i, chaslusdancla e (2000). occurrence of a salmonella enterica serovar typhi murium dt104-like antibiotic resistance gene cluster including the flor gene in s. enterica serovar agona. antimicrob. agents chemother. 44: 1359 1361. doublet b, boyd d, mulvey mr, cloeckaert a (2005). the salmonella genomic island 1 is an integrative mobilizable element. mol. microbiol. 55: 1911 1924. fluit ac (2005). towards more virulent and antibiotic-resistant salmonella? fems immunol. med. microbiol. 43: 1 11. gay k, robicsek a, strahilevitz j, park ch, jacoby g, barrett tj, medalla f, chiller tm, hooper dc (2006). plasmid-mediated quinolone resistance in non-typhi serotypes of salmonella enterica. clin. infect. dis. 43: 297 304. hsu sc, chiu th, pang jc, hsuan-yuan ch, chang gn, tsen hy (2006). characterisation of antimicrobial resistance patterns and class 1 integrons among escherichia coli and salmonella enterica serovar choleraesuis strains isolated from humans and swine in taiwan. int. j. antimicrob. agents 27: 383 391. khemtong s, chuanchuen r (2008). class 1 integrons and salmonella genomic island 1 among salmonella enterica isolated from poultry and swine. microb. drug resist. 14: 65 70. kwon hj, kim te, cho sh, seol jg, kim bj, hyun jw, park ky, kim sj, yoo hs (2002). distribution and characterization of class 1 integrons in salmonella enterica serotype gallinarum biotype gallinarum. vet. microbiol. 89: 303 309. lauderdale tl, aarestrup fm, chen pc, lai jf, wang hy, shiau yr, huang iw, hung cl, tsar hospitals (2006). multidrug resistance among different serotypes of clinical salmonella isolates in taiwan. diagn. microbiol. infect. dis. 55: 149 155. leverstein-van hall ma, blok m, donders ar, paauw a, fluit ac, verhoef j (2003). multidrug resistance among enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. j. infect. dis. 187: 251 259. lévesque c, piché l, larose c, roy ph (1995). pcr mapping of integrons reveals several novel combinations of resistance genes. antimicrob. agents chemother. 39: 185 191. ling jm, chan ew, lam aw, cheng af (2003). mutations in topoisomerase genes of fluoroquinolone-resistant salmonellae in hong kong. antimicrob. agents chemother. 47: 3567 3573. levings rs, lightfoot d, partridge sr, hall rm, djordjevic sp (2005). the genomic island sgi1, containing the multiple antibiotic resistance region of salmonella enterica serovar typhimurium dt104 or variants of it, is widely distributed in other s. enterica serovars. j. bacteriol. 187: 4401 4409. varma jk, greene kd, ovitt j, barrett tj, medalla f, angulo fj (2005). hospitalization and antimicrobial resistance in salmonella outbreaks, 1984-2002. emerg. infect. dis. 11: 943 946. vo at, van duijkeren e, fluit ac, wannet wj, verbruggen aj, maas hm, gaastra w (2006). antibiotic resistance, integrons and salmonella genomic island 1 among non-typhoidal salmonella serovars in the netherlands. int. j. antimicrob. agents 28: 172 179. vugia dj, samuel m, farley mm, marcus r, shiferaw b, shallow s, smith k, angulo fj (2004). emerging infections program foodnet working group. invasive salmonella infections in the united states, foodnet, 1996-1999: incidence, serotype distribution, and outcome. clin. infect. dis. 38: s149 156. yan jj, chiou cs, lauderdale tl, tsai sh, wu jj (2005). cephalosporin and ciprofloxacin resistance in salmonella, taiwan. emerg. infect. dis. 11: 947 950. african journal of pig farming vol. 1 (1), pp. 001-010, september, 2013. available online at www.internationalscholarsjournals.org © international scholars journals full length research paper nitrogen and phosphorus management on pig farms in northwest germany – nutrient balances and challenges for better sustainability d. otten* and h. f. a. van den weghe department of animal sciences, division: process engineering, georg-august university of goettingen, universitaetsstr. 7, d-49377 vechta, germany. accepted 26 september, 2013 the study analyses the nutrient flow of nitrogen (n) and phosphorus (p) on six intensive pig farms in northwest germany over a period of 5 years. the study investigated the farmers’ management of nutrient flows associated with intensive pig farming, focusing especially on the management of manure. it could be shown that in intensive pig production, high performance and performance-related feeding positively affects nutrient flow efficiency. however, on the crop production side of the farms, there existed a great disharmony between the farmers’ perception of manure used as (n, p) fertiliser on their crops and the real quantity of nutrients produced by their intensive pig farming. the farmers were unable to estimate the fertiliser effect of their manure correctly [underestimation n = 7.6% (10.6 kg/ha), p = 33.6% (11.6 kg/ha)]. the application of nutrients by mineral fertiliser was underestimated also [n by 4.1% (8.1 kg/ha), p by 12.7% (1.5 kg/ha)] and were inadequately attuned to the large amount of manure produced by their intensive pig production. as a result, the farms had large nutrient losses (104.5 kg n/ ha; 11.7 kg p/ha) and were unable to fulfil the minimum german legal requirements for fertiliser application. the study evaluated the critical aspects of nutrient management and discusses possible improvements for the future. key words: nutrient management, intensive pig production, northwest germany introduction sustainable food production places a great deal of responsibility on farm management to ensure a wellbalanced nutrient flow (koelsch and lesoing, 1999). nutrient management must always be considered with respect to the prevalent production conditions: the farm structure (e.g. husbandry, type of farmland, infrastructure), the biophysical conditions (e.g. climate, soil), the prevalent socio-economic conditions (e.g. availability and cost of intermediate goods or techniques) and other dependent variables. the ideal management strategy always depends on the individual situation (bosshard, 2000; koelsch and lesoing, 1999). bearing this in mind, the nutrient flow management in an intensive livestock area (ila) is associated with exceptional challenges. ilas are regions with a very high livestock concentration and often also with a higher productivity and degree of business organisation in livestock farming. pig farms in such regions often have a greater productivity, size and degree of specialisation than farms in other regions. this leads to a higher *corresponding author. e-mail: dennis.otten@agr.uni-goettingen.de. tel: 0049-4441-15524. otten et al. 002 material flow of individual nutrients per farm. in addition, ilas have a very high regional turnover of nutrients and the excessive production of manure is becoming an increasingly important regional challenge and environmental risk (ilea, 2009; warnecke et al., 2009; berkhoff, 2005; abdalla, 2002; tilman et al., 2002; wing and wolf, 2000). in the past, various authors have concerned themselves with nutrient flows in intensive pig production. abdalla (2002) has given a general historical overview about the industrialisation of livestock farming and its conflicts and discussed the consequences from a broader institutional economic perspective. cahoon et al. (1999) described the eutrophication threat to the river basins and other areas with expanding animal populations from the potential large nutrient loadings in north carolina. mc sweeny and shortle (1989) analysed the fertilisation problems from the perspective of a crop yield response to nitrogen application and the nitrogen content of manure in ilas. they analysed the effects of various policy prescriptions on fertilisation decisions by such farmers and showed the importance of understanding producer behaviour for the design of economically sound policies. the n and p consumption, utilisation and losses in pig production have also been analysed in the netherlands (van der peet-schwering et al., 1999; de boer et al., 1997; breeuwsma et al., 1995), france (basset-mens and van der werf, 2005; dourmad et al., 1999) and denmark (nielson and kristensen, 2005; fernandez et al., 1999). the various authors evaluated the overall status for reducing the environmental problems associated with pig production by using feeding and other management measures. they also discussed n and p management in relation to legislative measures. tamminga (2003) described the pollution caused by nutrient losses and its control in european animal production and suggested a combination of a better onfarm nutrient management and a maximum stocking density of 2.0 livestock units / hectare (lu/ha) (lu = livestock units; 1 lu = 500 kg). the german fertiliser ordinance prescribes this maximum stocking density also. additionally, a maximum overload (input – output) of 60 kg n/ha and 20 kg p2o5 (8.73 kg p)/ha is allowed. the maximum n fertilisation for manure is limited to 170 kg n/ha [german fertiliser ordinance (düngeverordnung – düv) 2007]. however, because of the dynamic regional concentration processes in ilas at present and the increased requirements for sustainable animal production, the current development in such regions has to be checked with respect to on-farm material flow management. with this in mind, the present study had the following specific research objectives: (i) to describe the flow of n and p through the individual farm materials and identify the important factors affecting efficiency nutrient management; (ii) to analyse the nutrient management of great quantities of manure; and (iii) to use the results to discuss any possible opportunities to improve nutrient flow management in ilas. materials and methods for the investigation, the total n and p flow (inputs, intra-farm and outputs) of all the materials on six pig farms [2x piglet production, 2x finishing pig production and 2x combination farms (piglet production and finishing pigs)] were analysed over a period of 5 years. due to this analysis of real farm data over a number of years, six farms were considered to be an adequate number of probands for a meaningful representation. in addition, it should be mentioned that a great deal of cooperation from the farmers is required to acquire all the data concerning a farm’s material flow and the farmer’s nutrient management. the six farms were located in northwest germany. table 1 gives an overview of the average structure of the six farms and information about the average stocking density of the whole region of northwest germany over the study period. the abbreviations in front of the farm number represent the type of pig farm: pp = piglet production, fp = finishing pig production and cf = combination farm. these abbreviations will be used throughout this paper. the average number of animals per farm [329 sows (pp + cf); 784 finishing places (fp + cf)] and the concentration of animals per unit area [2.73 lu/ ha (1 lu = 500 kg)] of the six farms reflected the regional conditions. their production was also typical of this region [weaned piglets/ sow and year = 21.9 (pp + cf); daily weight increase/ finishing pig = 707 g (fp + cf)]. the average area of the six farms was 68.4 ha. the crop production primarily involved cereals (36.3 ha), maize (25.6 ha), small amounts of oilseed rape (5.4 ha) and other crops (1.1 ha). the farms’ texture of soil was sandy or clayey and of medium quality [german soil quality index = 18-35; scale of index = 7-100 (bodschätzg, 2007)]. the analyses of the nutrient flow covered all the material inputs, the intra-farm transformations and the material outputs of all the farm’s goods. table 2 provides an overview of the audited materials and the calculations used in the investigation. the information about the inputs via the animals, feed and mineral fertiliser (the amount used and composition) was provided by the bills of sale from the producer or the supplier/ purchaser. the n and p flows of the animals were budgeted according to the german society of nutritional physiology (gfe, 2006). the values for the types and quantity of ammonia depositions in crop production (builtjes et al., 2011) and the emissions leaving the pig production (luft, 2002) were taken from other studies carried out in the region. in addition to the analyses of nutrient flow, the soil of each farm was analysed (vdlufa, 1991) over a period of 15 years (annual) to determinate its mineral makeup and any changes over time. a total of 15 to 30 samples were taken per ha and were mixed to give one sample. the depth of soil sample corresponded with the ploughing depth on arable land; grassland no ploughing (arable land = 0-30 cm; grassland 0-10 cm). the samples were analysed by the association of german agriculture analytic and research institutes (vdlufa 1991) [iso 11732 and 11885 (2005), din 19684 (2000), din 38414 (1985)]. the calculation period used for the pig production units was the standard german animal husbandry business year (01 july 30 june). for the crop production, each individual crop was analysed. the efficiency of the piglet production [output (piglets + slaughter sows) / input (lactation diet + gestation diet + piglet diet + gilts)] and the efficiency of the fattening pig production [output (fattening pigs) / input (fattening diet + piglets)] were calculated. the calculations for the combination farms included both these formulae. the efficiency of the crop production was also calculated [output (primary crops + secondary crops) / input (chemical fertiliser + manure + seeds + n deposition)]. 003 afr. j. pig farming table 1. structure of the six farms used in the study [pp: piglet production; fp: fattening pig production; cf: combination (piglets and fattening pigs)] parameter pp (i) 1) pp (ii) fp (i) fp (ii) cf (i) cf (ii) mean ± mean ± mean ± mean ± mean ± mean ± region lu/ha 2) 2.3 / 2.0 / 3.4 / 3.4 / 3.4 / 3.4 / farm lu/ha 4.5 / 2.0 / 3.7 / 2.0 / 1.6 / 2.6 / area of farm ha 83.3 7.2 67.6 0.3 50.1 3.1 33.7 0.8 106.8 2.5 68.9 3.5 crop production cereals ha 36.1 8.8 42.7 3.6 17.3 3.6 19.8 0.7 52.4 1.6 49.6 5.8 maize ha 47.2 8.6 7 4.6 29.8 2.8 12.7 1.3 46.7 3.3 10.6 5 oilseed rape ha / / 16.2 2.9 / / / / 7.7 3.9 8.2 2.5 other ha / / 1.8 2.6 3 3.2 1.1 / / / 0.6 0.9 pig husbandry pig breeding units 637.2 39 236.5 9 173.3 13.8 270.7 7.7 fattening units 1238 19 560 0 1143.3 222 199 18.5 production weaned piglets sow and year 23.7 1.1 22.1 1 20.8 0.4 21.1 0.6 no. of litters sow and year 2.4 0.0 2.2 0.1 2.6 0.4 2.3 0.03 body weight increase g/day 713 8.9 642 29 786.7 7.8 684 25.6 finished pigs/ unit p. a. 3) 2.6 0.1 2.4 0.1 3.1 0.0 2.8 0.1 1) (i) and (ii) are defined as farm one and two of the respective production system (pp, fp or cp). this definition is used throughout this study. 2) lu = livestock units; 1 lu = 500 kg. 3) p.a.: per annum. particular attention was paid to the farmers’ management of their manure. using a questionnaire about their crop production management, the farmers were asked about how many kg n and p they believed they were applying per hectare to each field. these values were then compared with the true audited amounts from the pig production and the differences determined (table 2). the differences between the perception of how much n (kg/ha) and p (kg/ha) were applied with mineral fertiliser and the true amount were determined also. subsequently, a stepwise linear regression was carried out to determine which factors significantly affected nutrient efficiency in the various forms of production (p < 0.05). in this procedure, the factors were implemented until there was no further increase in r 2 . the respective efficiency in the piglet production and the crop production were the independent variables and the different materials included in the nutrient flows the dependent ones. the factors were tested for their statistical significance (p< 0.05). the variables were standardised in order to analyse which ones had the greatest influence (standardised beta coefficient). the statistical analysis was carried out with the software program spss, version 19 (pasw statistics — spss 19. for windows). results the n and p flows and the important factors affecting their efficiency will be described, firstly with respect to the pig production and then to the crop production. lastly, the farmers’ management of the large amount of manure produced by their pig production will be elucidated. pig production table 3 illustrates the n and p flows in the pig production. the results for each type of pig production system [pp, fp and cf] over the whole study period have been summarised. the average total annual turnover [total (n, p) input; total (n, p) output] per sow in the piglet production varied between 54.7 kg (pp) and 54.9 kg (cf) for n, and between 10.5 kg (pp) and 10.6 kg (cf) for p. the average values per unit (place) in the finishing pig production ranged from 17.7 kg (cf) to 18.0 kg (fp) for n and 3.3 kg (fp) to 3.4 kg (cf) for p. the manure production was between 29.3 kg n/ sow (pp) and 29.6 kg n/ sow (cf) and 6.8 kg p/ sow (pp) and 7.0 (cf) kg p/ sow in the piglet production and 7.2 kg n/ unit (fp) to 7.6 kg n/ unit (cf) and 2.0 kg p/ unit (fp) to 2.2 kg p/ unit (cf) in the finishing pig production. the efficiency in the piglet production [n = 0.35 (pp) to 0.34 (cf); p = 0.34 (pp) to 0.33(cf)] was lower than in the finishing pig production [n = 0.39 (fp) to 0.36 (cf); p = 0.39 (fp) to 0.34 (cf)]. in the piglet production, the n and p concentrations of the different diets were rather similar on all six farms (table 3). this is typical of intensive pig farming in northwest germany as all the diets were industrially produced. since the mounting intensification of livestock otten et al. 004 table 2. the audited materials, the data sources and the calculations used in the inventory. material kind of calculation data diets animals primary crops,secondary crops, seeds mineral fertiliser ammonia emissions ammonia deposition losses and accumulation in soil manure bill of sale from producer or supplier about amount and concentrations information from farmer (bill of sale) for number and weight of pigs. then gfe 2006 to calculate nutrient flow information from producer or distributor about quantity (bill of sale). then german fertiliser ordinance [düngeverordnung (düv), 2007] to calculate nutrient flow (in g/kg) bill of sale from producer or distributor about amount and product contents technical instructions on air quality control (luft, 2002) builtjes et al. (2011) audited audited average amounts and concentrations (table 3) protein quantity (kg/pig) : < 30 kg body weight = 169 g/ kg body weight; >30 kg bodyweight = 0.168 x body weight 0.0000914 x body weight² p quantity (kg/pig): <80 kg bodyweight = 5.0 g/ kg bodyweight; > 80 kg body weight = 4.5 g/ kg body weight barley (12% cd 1 ) = 16.5 n; 3.5 p barley (13% cd) = 17.9 n; 3.5 p rye (11% cd) = 15.1 n; 3.5 p rye (12% cd) = 16.5 n; 3.5 p triticale (12% cd) = 16.5 n; 3.5 p wheat (12% cd) = 18.1 n; 3.5 p wheat (14% cd) = 21.1 n; 3.5 p crop straw = 5.0 n; 1.3 p rape (23% cd) = 33.5 n; 7.9 p grain maize (10% cd) = 13.8 n; 3.5 p grain maize (11% cd) = 15.1 n; 3.5 p silage maize (28% dm 2 ) = 3.8 n; 0.7 p silage maize (32% dm) = 4.3 n; 0.8 p grassland 1 cut/ year = 13 n; 2.6 p grassland 2 cuts/ year = 18 n; 3.1 p grassland 3 cuts/ year = 22 n; 4.1 p grassland 4 cuts/ year = 27 n; 4.4 p grassland 5 cuts/ year = 28 n; 4.4 p average amounts and concentrations (table 3) piglet production= 6.0 kg n/ sow (piglet production only) fattening pig production = 3.6 kg n/ unit 55 kg n/ha input (mineral fertiliser + manure + seeds + deposition) output (primary + secondary crops) input [different diets + animal inputs (piglets or gilts)] outputs: [animal outputs (sold animals and losses) + n emissions]. for input crop production: (10% n pit losses) (20% n losses during manure application) 1) cd = crude protein; 2) dm = dry matter. production in the mid-1990s, the farms have increasingly used crude-protein (cp) and phosphorus-reduced diets in order to reduce both n and p in the manure and the amount of ammonia emitted. the highest concentrations in the diets used on the six farms were lactation diet cp < 16.5%, p < 0.55%; gestation diet = cp < 14%, p < 0.45%; and piglet diet = cp <18%, p < 0.55%. because of the similar concentrations in the different 005 afr. j. pig farming table 3. the average (mean, sd) nitrogen (n) and phosphorus (p) flow in the animal production with their respective quantities (kg) and concentrations (g/ kg) over the whole study period. piglet production (pp) fattening pig production (fp) combination farm (cf) element unit amount concentration in amount concentration in amount concentration in n p product (g/kg) n p product (g/kg) n p product (g/kg) input "1) ± " ± n p " ± " ± n p " ± " ± n p lactation diet kg/sow 11.2 2.2 5.3 1.1 26.8 12.6 54.9 17.7 24.2 7.8 7.6 1.7 3.6 0.8 26.4 12.4 gestation diet kg/sow 20.4 5.2 9.5 2.4 22.7 10.4 23.9 2.1 10.8 0.7 22.8 10.3 piglet diet kg/sow 21.9 1.6 8.7 0.7 28.0 12.6 18.0 7.7 22.4 2.7 9.4 0.8 27.9 11.8 fattening diet kg/unit 15.9 0.9 6.8 0.3 24.9 10.7 15.6 1.7 6.9 0.7 26.1 11.6 gilts kg/sow 1.2 0.2 0.5 0.1 24.5 10.3 1.0 0.3 0.4 0.1 24.6 10.3 piglets kg/unit 54.7 24.0 2.1 0.1 0.8 0.1 26.9 11.5 2.1 0.4 0.9 0.2 26.9 11.5 output total (kg) 54.7 24.0 18.0 7.7 72.6 2) 32.1 2) fattening pigs kg/unit 7.0 0.3 3.0 0.1 24.2 10.3 6.3 1.0 2.7 0.4 24.2 10.3 piglets kg/sow 17.2 0.7 7.3 0.3 26.9 11.5 17.2 1.8 7.3 0.8 26.9 11.5 slaughter sows kg/sow 1.9 0.2 0.9 0.1 23.1 10.3 1.7 0.4 0.8 0.2 23.0 10.3 animal kg/sow 0.3 0.1 0.1 0.0 25.1 11.1 0.4 0.1 0.2 0.0 25.0 10.9 losses 0.1 0.03 0.05 0.01 26.3 11.5 0.1 0.0 0.0 0.0 26.2 11.5 kg/unit manure kg/sow 29.3 15.6 1.3 3.0 1.3 29.6 15.9 0.9 4.4 1.6 kg/unit 7.2 4.6 0.3 5.8 4.1 7.6 5.1 0.8 4.5 2.3 ammonia kg/sow 6.0 10.8 3.0 6.0 emissions kg/unit 25.4 8.3 3.6 3.6 ## 10.1 8.3 2.7 efficiency total (kg) 54.7 24.0 18.0 7.7 72.6 2) 32.1 2) efficiency (pp) 3) 0.35 0.34 0.34 efficiency (fp) 4) 0.39 0.39 0.36 0.34 total animal production efficiency 0.35 0.34 0.39 0.39 0.35 0.34 =1) = means; 2) = total inputs and outputs in cf: piglet production (pp): = 54.9 kg n, 24.2 kg p; fattening pig production 17.7 kg n, 7.9 kg p 3) = output (piglets + slaughter sows)/ input (lactation diet + gestation diet + piglet diet + gilts); 4) = output = (fattening pigs) / input = (fattening diet + piglets) diets used on the farms, the stepwise regression showed that it was particularly the farmers’ feeding management of the different diets which affected the nutrient efficiency [(model n efficiency: y = 1.434 * gestation diet 0.746 * lactation diet 0.347 * piglet diet, r 2 = 0.95; p efficiency model: y = 1.664 * gestation diet 1.067 * lactation diet 0.418 * piglet diet, r 2 = 0.85]. there was a lower use of gestation diet (from 40 g down to 25 g n/ kg produced piglet and from 8.5 g down to 5.0 g p/ kg produced piglet) and a higher use of lactation diet (from 13.0 g up to 20.0 g n/ kg produced otten et al. 006 table 4. the average (mean, sd) nitrogen (n) and phosphorus (p) flows in the crop production with their respective quantities (kg) and concentrations (g/ kg) over the whole study period. piglet production (pp) fattening pig production (fp) combination farm (cf) material unit amount concentration amount concentration amount concentration n p (g/ kg) n p (g/ kg) n p (g/ kg) input " 1) ± " ± n p " ± " ± n p " ± " ± n p chemical fertiliser kg/ha 100.6 36 6.6 4.4 257.8 20.4 52.8 11.8 6.9 3.2 231.8 30.4 107.2 30.5 5.1 1.7 182.8 10.8 manure kg/ha 85.0 11.7 33.8 5.1 3.0 0.6 85.3 27 36.6 6.7 5.8 1.8 100.6 7.77 36.2 1.5 4.4 0.7 seeds kg/ha 1.6 0.3 0.3 0.1 17.0 3.5 1.4 0.4 0.3 0.1 15.8 3.2 1.6 0.2 0.3 0.0 17.1 3.5 n depositions kg/ha 55.0 55.0 55.0 output total (kg) 242.2 40.7 194.5 43.7 264.4 41.6 primary crops kg/ha 106.1 13.3 23.9 3.1 16.6 3.7 108.1 16.2 25.1 3.9 9.7 2.0 130.5 12.6 28.0 2.3 0.0 25.6 secondary crops 2) kg/ha 12.6 6.6 3.8 1.4 0.6 0.2 12.1 3.3 3.2 0.8 0.6 0.2 18.1 2.2 4.7 0.5 12.2 4.0 losses and accumulations kg/ha 123.6 13.0 74.2 15.4 115.8 8.9 in soil efficiency total (kg) 242.2 ## 40.7 27. 194.5 12 43.7 28. 264.4 ### 41.6 3.7 total crop production efficiency 3) 0.49 0.68 0.62 0.65 0.56 0.79 1) = means 2) = input (different diets + animal inputs; piglets or gilts) outputs (animal outputs; marked, losses + n emissions) (10% n pit losses) (20% n losses during manure ___application)]; 3) = output (primary crops + secondary crops) / input (chemical fertiliser + manure + seeds + n-depositions). piglet and from 2.9 g up to 3.2 g p/ kg produced piglet) on the farms with a high nutrient efficiency. with the piglet diet, 35 g n and 6.9 g p was needed for each kg produced piglet. in the fattening pig production, the diets were different between the farms and their average n and p concentrations had a greater importance for nutrient efficiency than the feed conversion: model n efficiency: y = 0.894 * n amount in diet 0.401 * feed conversion, r 2 = 0.89; model p efficiency: y = 0.881 * p amount in diet 0.453 * feed conversion, r 2 = 0.89. with an average concentration of n= 25.5g/ kg and p = 4.8 g/kg, the more efficient farms had lower average dietary concentrations of both n and p than the less efficient farms (n = 27.9 g/kg, p = 5.1 g/kg). as in the piglet production, those factors which affected the n efficiency were rather similar to those affected the p efficiency. crop production the material flow of the crop production on the six pig farms and their n and p quantities (kg) and concentrations (g/kg) are shown in table 4. the average total annual turnover [total (n, p) input; total (n, p) output] per ha in the crop production over the whole study period lay between 233.7 kg n/ha and 42.3 kg p/ha, with an efficiency of 0.55 for n and 0.69 for p. the differences between the different production systems (pp, fp, cp) were not significant. the input via mineral fertilisers was 86.9 kg n and 6.1 kg p per ha. on average, 90.3 kg n and 35.5 kg p per ha was applied via manure. the average audited losses lay at 104.5 kg n and 11.7 kg p per ha and year. for the analysis of the factors affecting the nutrient efficiency in the crop production, the n and p flows will be described separately. n flows the stepwise regression showed that it was the inputs which particularly affected the n efficiency: model n efficiency: y = 0.508 * manure 0.449 * mineral fertiliser + 0.286 * primary crops (r 2 0.87). figure 1 shows how the fertilisation affected the n overload 007 afr. j. pig farming 250 200 h a ) (k g / 150 n -i n p u t 100 50 0 0 20 40 60 80 100 120 140 160 180 200 kg n overload/ ha (total input total output) total n fertiliser manure chemical fertiliser maximum legal total n overload (60 kg n/ ha) maximum legal total n fertilisation (170 kg n/ ha) figure 1. nitrogen (n) fertilisation on the farms (kg/ ha) and the legal thresholds for the maximum overload and maximum total fertilisation in germany [german fertiliser ordinance (düngeverordnung-düv, 2007)]. 80 70 s o i l 60 50 1 0 0 g 40 p / 30 g m 20 10 0 1995 1997 1999 2001 2003 2005 2007 2009 year figure 2. development of the soil p concentration (mg p/100 g soil) (median, 75% quartile, 25% quantile; maximum. minimum) on all 6 farms between 1995 and 20010. (losses). the figure also shows the thresholds for maximum n overload (loss) and n fertilisation (maximum) given by the german fertiliser ordinance (düngeverordnung – düv, 2007). there was a great deal of variation in the amount of n applied. the two legal thresholds overlap almost exactly by the total n input line. taking the standard method of fertiliser application on the farms into consideration, whereby there is a large input of chemical fertiliser, only a very small amount of manure can actually be applied to the fields before the legal thresholds are exceeded. looking at the intersection of the legal overload threshold and taking into consideration the farmers’ style of applying fertiliser, a maximum of 67 kg n/ha can be applied with chemical fertiliser in addition to 73 kg n/ha using manure (max. 140 kg/ha). p flows the p supply within the soil and its development over time is an important indicator for the evaluation of the p flow. figure 2 shows the p supply on the six farms over a period of 15 years (1995-2009). it became obvious that in the mid-1990s the intensification of pig production led to a high accumulation of p in the soil. since then, the soil p concentrations have been reduced over time so that they have almost reached optimum values at present (6 mg to 12 mg/100 g soil; fleischer 1998). in addition to the reduction in the mean p load of the farmland, there has also been a strong reduction in the variation (that is, standard deviation) in p load so that the farms have achieved a more uniform p condition in their soils. looking at the correlations of the p load in the soil with the distance of the field from the livestock buildings (table 5), it is apparent that in the 1990s the p supply within the soil was closely related to how near it was situated to the farm buildings: the manure was not distributed over the whole farm land evenly. with the intensification of animal production, the farmers tended to apply the manure to the closest fields, while the fields further away evinced little pollution. table 5 shows the correlation between the distance of the fields from the farm buildings and the p concentration between the years 1995 to 2009. the regressions make clear that the p concentration in soil has been improved; however, there is still a significant correlation between the distance of the field and the soil p concentration at present [(2007 – 2009) = r 2 0.19]. for the present-day p efficiency in the crop production, the stepwise regression showed that the nutrient efficiency was especially affected by the inputs. p efficiency model: y = 0.677 * manure + 0.474 * secondary crops + 0.187 * (r 2 0.91). it is obvi ous that, as with the n efficiency, manure management had the greatest influence. in contrast to the n efficiency, however, the mineral fertiliser was not included in the stepwise regression. figure 3 shows how the p fertilisation affected the p losses (overload). the minimum legal requirements in germany are also illustrated [german fertiliser ordinance (düngeverordnung-düv, 2007]. with a legal maximum threshold overload of 20 kg p2o5 (8.72 kg p), the farms can apply a total of 39.1 kg p/ha. at this threshold and the farmers’ present fertilisation practices, 33.2 kg p could be applied using manure and 5.9 kg p/ha using chemical fertiliser. primary crops otten et al. 008 60 50 h a ) 40 (k g / 30 p -o u tp u t 20 10 0 0 3 6 9 12 15 18 21 24 27 30 kg p overload/ ha (total input total output) total p fertiliser manure chemical fertiliser maximum legal total p overload (20 kg p2os / ha; 8.73 kg p/ ha) figure 3. phosphorus (p) fertilisation on the farms (kg/ ha) and the legal thresholds for the maximum overload in germany [german fertiliser ordinance (düngeverordnung-düv) 2007)]. % p kg/ ha % n kg/ ha -60 -40 -20 0 20 40 60 chemical fertiliser manure figure 4. the underestimation of the nutrient application by chemical fertiliser and manure. shown are the differences (% total, kg/ ha) in perception between the farmers’ declared fertiliser use and their true fertiliser use (mean, sd). the farmers’ perception of their application of nutrient via manure and/or mineral fertiliser from the crop production information, it is clear that the manure applications were very important for the nutrient efficiency. figure 4 shows the differences between the n and p quantities the farmers thought they had applied via manure and the true calculated quantity produced by the pig production. the figure shows also the differences between the farmer’s estimation of n and p application by mineral fertiliser and the applied amount. both the absolute differences (kg/ha) and the percentage differences (%) are illustrated. the figure summarises the information from all six farms over the whole study period. it is clear that the quantity of nutrient fertilisation – manure and mineral fertilisers – was underestimated. the farmers were unable to estimate the fertiliser effect of their manure correctly [underestimation n = 7.6% (10.6 kg/ha) p = 33.6% (11.6 kg/ha)]. the application of nutrients by mineral fertiliser was underestimated also [n = 4.1% (8.1 kg/ha) p = 12.7% (1.5 kg/ha)]. the differences between the farms were not significant. the high degree of standard deviation seen emphasises the high potential for improvement in the farmers’ estimation of their fertiliser use. discussion the intensive pig production on the six farms had positive effects on the nutrient efficiency in their livestock farming. as in other studies (aarnink and verstegen, 2007; fernandez et al., 1999; de boer et al., 1997), it was particularly the high performance and the requirementrelated diets which affected the nutrient efficiency positively. industrially produced diets with reduced crude protein and p contents or with supplements for better digestibility have been used for a long time in intensive pig production (aanrink and verstegen, 2007; de fernandez et al., 1999; de boer et al., 1997). the present study has been able to show that the farmers’ feeding management has the potential to improve nutrient efficiency even further. reducing p concentration in diets, strongly regulations (and controls of them) and strict fertilizing management were important factors for reducing the p load in soil in the past (fernandez et al., 1999; de boer et al., 1997). additionally, the increased cultivation of maize could be responsible for partly reduction. the cultivation of barley and rye were replaced by maize at this time very much. on average, maize requires a little more phosphorus of growth. because of a further reduction of p load is unnecessary in many places, the question arises as how much more p needs to be removed from diet. this reduction in p enables the farmers to have a higher stocking density and still be able to conform to the legislative requirements given by the german fertiliser ordinance (düngeverordnung-düv, 2007). from a sustainable point of view, the removal of p from the input side of animal production (that is, the diet) is questionable because it can only be done by using (energy-) costly methods and then any necessary p would have to be reapplied to the farm using by mineral fertiliser. as in other studies in ilas (cahoon et al., 1999; breeuwsma et al., 1995), there was a high accumulation of p in soil on the study farms with the increasing intensification of animal production in the middle of the 1990s. the high influence of the distance of the fields 009 afr. j. pig farming table 5. development of the relationship between the distance of the fields from the farm buildings and the soil phosphorus concentration (mg p/ 100 g soil) between the years 1995 – 2009. year regression function r2 f sig. y= mx + b 1995 1997 [-]2.1854x [+] 34.09 0.18 12.01 0.001 1998 2000 [-]0.4363x [+] 14.20 0.09 3.84 0.035 2001 2003 [-]0.6405x [+] 18.10 0.04 2.13 0.150 2004 2006 [-]0.4984x [+] 18.32 0.08 6.91 0.100 2007 2009 [-]0.4857x [+] 11.91 0.19 10.4 0.002 from the farm buildings on the soil p concentration in the 1990s illustrated that with any increase intensification of pig production, emphasis should not only be placed on the application technique (e.g. towing hose, direct incorporation into the soil, etc.) as had been done by the local government in northwest germany (ministry of food, agriculture, consumer protection and regional development, lower saxony, 2012). appropriate technologies are also necessary in overcoming longer transportation distances of manure. the present study had shown that there is a great potential for improving the nutrient efficiency in crop production, too. particularly, the contribution of the animal production (via the manure) to the nutrient supply in the crop production was not being taken enough into consideration with respect to determining the need for the mineral fertiliser input. this led to particularly high losses both of n (104.5 kg/ha) and p (11.7 kg/ha). with the present practise of fertiliser application on the study farms, a maximum of 68 kg n/ha could be applied by chemical fertiliser and 76 kg n/ha by manure to fulfil the legislative requirements of the german fertiliser ordinance (figure 1). using these values for the maximum fertiliser production (29.45 kg n/sow and 6.05 kg n/unit fattening pig), the maximum stocking density would have to be at 2.3 sows/ ha or 11.2 units fattening pigs/ha (0.97 lu/ha for sows or 1.79 lu/ ha for fattening pigs; sow including piglet rearing = 0.42 lu; fattening pig = 0.16 lu). with respect to the requirements of p fertilisation (overload = 20 kg p2o5; 8.72 kg p) a maximum of 33.2 kg p/ha could be applied by manure (figure 3). this corresponds to an equivalent of 4.8 sows/ ha (production = 6.9 kg n/sow) and 15.8 units/ ha (production = 2.1 kg p/unit) or 2.0 lu/ha for sows and 2.5 lu/ha for fattening pigs. these calculations of the maximum stocking density at the farms’ present practise of fertiliser application show that the main limiting factor is n. this means that the present practise of fertilisation and the large mineral fertiliser inputs are not compatible with the stocking density on any of the six farms. the underestimations in perception of the fertiliser application illustrated in this investigation emphasise the large managerial mistakes being made in the crop production. increasing ecological problems from pig production are also resulting at present from excessive n depositions from animal husbandry units. current values are already at 55 kg/ha (builtjes et al., 2011). beside the depositions caused by manure application, the exhaust air from livestock buildings is the most important form of n emission (dämmgen, 2009). mitigating such emissions is associated with a lot more costs and must be combined with regulatory measures. a major challenge of the future will be to prevent or reduce these emissions. the present investigation has shown that with respect to sustainable development particular attention needs to be paid to nutrient management practises on the crop side. even when economic advantages lie in the production of animals in ilas, the great amount of manure produced makes it necessary that a high degree of managerial attention should be paid to the crop production side of the business. amendments to the regulations governing fertiliser application would help to improve the situation. any monitoring of the situation must include an effective and exact controlling of the amount of fertiliser applied to any given area. the legal obligations for recording fertilisation set out in the present german regulations (düngeverordnung düv, 2007) do not fulfil this need. references aarnink aja, verstegen mwa (2007). nutrition, key factor to reduce environmental load from pig production. livest. sci. 109(1-3):194203. abdalla cw (2002). the industrialization of agriculture: implications for public concern and environmental consequences of intensive livestock operations. penn state environ. law rev. 10:175-191. berkhoff k (2005). nutrient modelling in an area of intensive livestock husbandry facing the demands of the wfd. in proceedings of the international conference "multifunctionality of landscapes", may 1819, 2005, justus-liebig university gießen, germany. p. 88. basset-mens c, van der werf hmg (2005). scenario-based environmental assessment of farming systems: the case of pig production in france. agric. ecosyst. environ. 105(1-2):127-144. bodschätz g (2007). gesetz zur schätzung des landwirtschaftlichen kulturbodens (bodenschätzungsgesetz – bodschätz g) [law for the estimation of agricultural crop land] (bgbl. i pp. 3150-3176. bosshard a (2000). a methodology and terminology of sustainability assessment and its perspectives for rural planning. agric. ecosyst. environ. 77:29-41. otten et al. 010 breeuwsma a, reijerink jga, schoumans of (1995) impact of manure on accumulation and leaching of phosphate in areas of intensive livestock farming. in: k. steele (ed.), animal waste and the land-water interface. boca raton (usa), lewis. pp. 239-249. builtjes p, hendriks e, koenen m, schaap m, banzhaf s, kerschbaumer a, gauger t, nagel h-d, scheuschner t, schlutow a (2011) erfassung, prognose und bewertung von stoffeinträgen und deren wirkung in deutschland. uba 38/2011 issn. pp. 1862-4804. cahoon lb, mikucki ja, mallin ma (1999). nitrogen and phosphorus inputs to the cape fear and neuse river basins to support intensive livestock production. environ. sci. technol. 33(3):410-415. dämmgen u (2009). calculations of emission from german agriculture national emission inventory report (nir) 2009 for 2007. vti agric.forest. res, isnb 978-3-86576-049-4, pp. 9-5. de boer ij, peters ht, grossman m, koops wj (1997). nutrient flows in agriculture in the netherlands with special emphasis on pig production. j. anim. sci. 75(8):2054-2063. din 19684 (2000). 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[first general administrative regulation pertaining to the federal immission control act] bundesministerium für umwelt, naturschutz und reaktorsicherheit [german federal ministry for environment, nature conservation and nuclear safety], carl heymanns verlag, berlin. tamminga s (2003). pollution due to nutrient losses and its control in european animal production. livest. prod. sci. 84:101-111. tilman d, cassman kg, matson pa, naylor r, polasky s (2002) agricultural sustainability and intensive production practices. nature 418:671-677. van der peet-schwering cmc, jongbloed aw, aarink aja (1999). nitrogen and phosphorus consumption, utilisation and losses in pig production: the netherlands. livest. prod. sci. 58 (3):213-224. vdlufa (1991). verband deutscher landwirtschaftlicher untersuchungsund forschungsanstalten (vdlufa) [association of german agricultural analytic and research institutes]. methodenbuch band i, die untersuchung von böden [method handbook 1: soil investigations] 4th edition, isbn 978-3-941273-139. warnecke sm, biberacher h-j, brauckmann broll g (2009). regionally optimised animal farm manure transports in an area with high intensity animal farming systems. in: van ittersum mk, wolf j, van laar hh (eds) proceedings of the “conference on integrated assessment of agriculture and sustainable development: setting the agenda for science and policy (agsap 2009)”. egmond aan zee, the netherlands, 10-12 march 2009. wageningen university and research centre, wageningen, pp. 312-313, isbn 978-90-8585401-2. wing s, wolf s (2000). intensive livestock operations, health, and quality of life among eastern north carolina residents. environ. health perspect. 108(3):233-238. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (10), pp. 001-009, october, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper real-time taqman polymerase chain reaction to quantify the effects of different sources of dietary starch on bifidobacterium in the intestinal tract of piglets zhentian xiang, hongwei qi, guoquan han, jingbo liu, zhiqing huang, bing yu and daiwen chen* institute of animal nutrition, key laboratory for animal disease-resistance nutrition of china ministry of education, sichuan agricultural university, ya’an, sichuan 625014, people’s republic of china. accepted 21 march, 2015 twenty-eight pic male piglets (similar birth and parity, weaned at 21±1.5 days) were used to study the effect of the different sources of dietary starch on the number of bifidobacteria in the digesta of the duodenum, jejunum, ileum, cecum and colon. pigs were randomly assigned to one of four diets formulated with corn starch, wheat starch, tapioca or pea starch. the determined ratio of amylose to amylopectin for these starches was 0.21, 0.24, 0.12 and 0.52, respectively based on the 16s rrna sequences of maximum species of bifidobacterium from genbank to design the primers and probe. taqman polymerase chain reaction was developed to quantify the number of bifidobacterium. we used this assay to detect genomic dna of bifidobacterium in the intestinal tract digesta of piglets, including duodenum, jejunum, ileum, cecum and colon. our results indicated that, developed new real-time quantitative pcr assays can be allowed for rapid, convenient, reproducible and steady quantification of the bifidobacterium in the intestinal content of piglets. additionally, the present study revealed that high amylose/amylopectin ratio of starches significantly enhanced the numbers of bifidobacterium in all segments of intestine. key words: starch, bifidobacterium, taq-man polymerase chain reaction (pcr), weaned pigs. introduction nowadays, weaned piglets undergo a transition from milkbased diet to adult-type plant-based diet. the transition combined with the stress of being transported to production farms is often followed by a period of low feed intake (brooks et al., 2003). the combination of these stresses can lead to intestinal malfunctions, intestinal microbiota in particular, which is subsequently followed by a reduced growth performance, diarrhea and mortality (mikkelsen et al., 2004a; spreeuwenberg et al., 2001a). the gastrointestinal tract (git) of pigs is harbored by dense and diverse bacteria (pryde et al., 1999). because there is a more stable microbial ecosystem when bacterial *corresponding author. e-mail: daiwenc@yahoo.com. tel: +86 835-2882088. fax: +86-835-2885106. diversity is increased, microbial diversity has been proposed as an indicator of the intestinal stability and health (zoetendal et al., 2004). the gut microbial communities are affected by various factors, including the diets (duncan et al., 2007a), environment (pluske et al., 2007) and so on. diet changes are likely to have a direct influence to place stress on the stability of bacterium and affect the entire git eco-physiology in lifestyle (spreeuwenberg et al., 2001b). certain dietary non-digestible carbohydrates can allow specific changes in the composition or activity of the intestinal microbiota (roberfroid, 2007) and selectively stimulate the growth of the health-promoting bacteria (bifidobacterium and lactobacillus) in the gastrointestinal tract (git) and potentially prevent or moderate intestinal infections (ebersbach et al., 2010; kaplan et al., 2000). indigenous bifidobacteria are believed to play an important role for animal and human health and immune function (orrhage et al., 2000) and it is considered to be the target organism due to their potential to inhibit the growth of pathogenic bacteria that may prevent intestinal disorders (mikkelsen et al., 2004b). yazawa et al. (1978) explained that, bifidobacteria are able to suppress pathogenic bacteria (escherichia coli) because they utilize oligoand polysaccharides that other intestinal bacteria cannot use. starches are the main source of carbohydrates in mixed diets and the major source of energy for monogastric animal and human (deng et al., 2009a). they are α-glucans and exist in two forms: amylopectin and amylose (cummings et al., 1995). starch are classified into rapidly digestible starch (rds), slowly digestible starch (sds) and resistant starch (rs) (englyst et al., 1999), where rds and sds are digested in proximal intestinal tract and the major digested products are absorbed in small intestine. however, the non-digestible starches (rs) together with a small quantity of digested products can reach the large bowel (aspvan et al., 1996; conlon et al., 2009). they have significant effects on the composition of the intestine microbiota, tending to increase bacteria associated with a healthy bowel and tending to decrease those potential diseases (conlon et al., 2009). pieper et al. (2009) reported that, the ratio of amylose to amylopectin and starch sources affected the abundance of bifidobacterium spp. in vitro, in particular high-amylose maize starch granules. consequently, the ratio of amylose to amylopectin of starch may enhance desirable intestinal bacteria and induce beneficial gut health. however, the underlying mechanism is still unclear. the aim of the present study was based on the 16s rrna sequences of different species of bifidobacterium from genbank to design the primers and probe. taqman polymerase chain reaction was developed to quantify the number of bifidobacterium. we used this assay to investigate the effects of different sources of dietary starch (cs; corn starch, ws; wheat starch, ts; tapioca starch and ps; pea starch) on the presence of bifidobacterium in the intestinal tract digesta of piglets, including duodenum, jejunum, ileum, cecum and colon. nowadays, weaned piglets undergo a transition from milk-based diet to adult-type plant-based diet. the transition combined with the stress of being transported to production farms is often followed by a period of low feed intake. materials and methods animals and housing twenty-eight pic male piglets (similar birth and parity), weaned at 21±1.5 days of age were used. they were fed in the animal center of animal nutrition institute in sichuan agricultural university (ya ’ an, china). they were kept in special metabolism pens over 24 days (3 days pre-experimental and 21 days experimental periods) in a thermo regulated environment (ambient temperature 22°c and relative humidity 55%). they had ad libitum access to feed or water. experimental design the experimental pigs were distributed into four blocks of seven piglets each according to body weight. treatments were arranged simple factor with four sources of starch (corn starch, wheat starch, pea starch and tapioca starch). experimental diets were formulated to meet nrc (1998) nutrient requirements for piglets weighing 5 to 10 kg. ingredients and chemical composition of experimental diets are presented in table 1. antibiotic growth promoters were not included in the diets. diets were balanced in essential amino acid concentration (lysine, methionine + cysteine and tryptophan) and had similar general energy (ge), crude protein (cp), starch (st) content, amylose content and amylopectin content. digesta sampling at the end of the experiment, the pigs was held under general anesthesia and killed by an intracardiac injection of sodium pentobarbital (30 mg/kg bw) after 2 h feeding. following euthanasia the abdominal cavity was opened from sternum to pubis to expose the gastrointestinal tract without damaging the wall of the digestive tract. the small intestine and large intestine were tied off respectively, and 5 to 10 cm sections of the duodenum, jejunum, ileum, cecum and colon were tied off. the digesta of the duodenum, jejunum, ileum, cecum and colon were removed immediately and stored at -80°c until further analyses. the small intestine was stripped free of its mesentery and further divided into 3 sections: (1) the ileum from the ileal-cecal junction to 80 cm anterior to this junction; (2) the duodenum, 80 cm posterior to the gastro-duodenal sphincter; (3) the jejunum constituted the regions between the ileum and duodenum (adeola et al., 2006). extraction of dna from digesta genomic dna from the bifidobacterium adolescentis (atcc15703) was extracted from the cultures with a takara minibest bacterial genomic dna extraction kit (takara, dalian, china), according to the manufacturer’s instructions. bacterial dna from the digesta samples was extracted using an e.z.n.a.tm stool dna isolation kit (omega bio-tek, doraville, ca) according to the manufacturer’s specifications. the final elution volume was 100 µl and the concentration was determined by spectrophotometer (beckman coulter du 800, fullerton, ca). designing and validation of primers primers and probe (table 2) were designed with primer express 3.0 (perkin elmer applied biosystem, san francisco, california) for quantitative detection of particular bifidobacterium. 16s rrna sequences of maximum species of each genus encountered in the swine intestinal tract were downloaded from the genbank database as well as embl and ddbj. the sequence of the bifidobacterium blocks of hyper variable regions was comprised with all other genera in order to avoid any non-specific amplification, the sequences of all the genera fetched from the database were submitted to dnastar (megalign) programme (dnastar, inc., madison, wi). these sequences were then submitted to second round of alignment where the maximum number of species belonging one genus was aligned and the regions showing conservations were selected as bifidobacterium genus-specific primers and probe. to further ensure that the oligonucleotide sequences were complementary pairing with the target genus only, they were checked with genbank program blast (ncbi blast, http://blast.ncbi.nlm.nih.gov/blast.cgi) and rdp program check table1. ingredients and nutrient levels in the experimental diets (%). ingredient cs diet ps diet ts diet ps diet corn starch (86%) 54.5 wheat starch (87%) 54.5 tapioca starch (86.5%) 54.5 pea starch (88%) 54.5 decupled soybean meal 2 2 2 2 soybean protein concentrate 17.83 17.83 17.83 17.83 soybean meal 10 10 10 10 whey 7.3 7.3 7.3 7.3 fish meal 6 6 6 6 calcium carbonate 0.55 0.55 0.55 0.55 monocalcium phosphate 0.7 0.7 0.7 0.7 sodium chloride 0.15 0.15 0.15 0.15 dl-met (98%) 0.17 0.17 0.17 0.17 l-thr (98.5%) 0.01 0.01 0.01 0.01 chromium oxide 0.4 0.4 0.4 0.4 premix* 0.39 0.39 0.39 0.39 total 100 100 100 100 nutrient levels (%) total starch 51.71 51.17 51.98 52.92 amylose 8.81 9.76 5.42 17.26 amylopectin 42.9 41.41 46.56 33.66 amylose/amylopectin ratio 0.21 0.24 0.12 0.52 cross energy(kj/g) 14.5 14.43 14.49 14.51 crude protein 19.9 19.87 19.87 19.99 lys 1.29 1.29 1.29 1.29 met+cys 0.74 0.74 0.74 0.74 thr 0.81 0.81 0.81 0.81 try 0.25 0.25 0.25 0.25 crude fiber 1.36 1.37 1.37 1.35 crude fat 2.6 2.53 2.58 2.62 *supplied per kg diet: 6.0 mg of cu as cuso4 5h2o, 100 mg of fe as feso4 7h2o, 4 mg mn of as mnso4 h2o, 100 mg of zn asznso4 h2o, 0.3 mg of se as na2seo3, 0.3 mg of i as ki, 1000 mg of choline chloride (50%), 200 mg of sweeteners, 0.04% of vitamin premix. table 2. sequences of oligonucleotide primers and probe. assay primer/probe name and sequence (5′-3′) product annealing reference size(bp) temperature (°c) all bacteria eub338f, actcctacgggaggcagcag 200 60 (fierer et al., eub518r, attaccgcggctgctgg 2005) bifidobacterium sb-p1, agggctcgtaggcggttcgtc 264 56.8 this study sb-p2, ccccacatccagcatcca sb-f, cgcgtccggtgtgaaag this study bifidobacterium sb-r, cttcccgatatctacacattcca 126 60 sb-p, (fma) attccaccgttacaccgggaa(bhq-1) table 3. reference strains used in this study and quantitative real-time pcr test results of reference strains. reference strain real-time pcr test result* sl-f,sl-r,sl-p eub338f, eub518r bifidobacterium adolescentis atcc15703(d) bifidobacterium animalis cicc6165(a) bifidobacterium suis sb08zy(e) bifidobacterium suis sb09nj01(e) bifidobacterium suis sb09nj02(e) lactobacillus acidophilus cicc6005(a) lactobacillus plantarum cicc6009(a) lactobacillus suis sl0501(e) lactobacillus suis sl0502(e) lactobacillus suis sl0503(e) bacillus subtilis atcc6633(d) bacillus cereus cmcc63302(d) bacillus subtilis bs01j(e) bacillus subtilis bs02j(e) bacillus pumilus bp0106(e) escherichia coli atcc8739(d) escherichia coli atcc25922(d) entero-hemorrhagic escherichia coli o157:h7(d) porcine pathogenic escherichia coli e.c0401(f) porcine pathogenic escherichia coli e.c0402(f) staphylococcus aureus atcc25923(c) staphylococcus aureus atcc6538(c) streptococcus hemolytic-β cmcc32210(d) + + + + + + + + + + + + + + + + + + + + + + + + + + + + a, purchased from china center of industrial culture collection; b, isolated from the attenuated live vaccine of salmonella choleraesuis; c, saved in laboratory of veterinary pharmacology of sichuan agricultural university; d, saved in laboratory of preventive veterinary medicine of sichuan agricultural university; e, isolated from healthy pig intestine by laboratory of preventive veterinary medicine of sichuan agricultural university; f, isolated from clinical samples by laboratory of preventive veterinary medicine of sichuan agricultural university. *amplification results with primers sl -f/sl-r and probe sl-p for validation specificity; *amplification results with primers eub338f/ eub518r for verification feasibility; ‘+’ represents positive test results; ‘-’ represents negative test results. probe (details about rdp data and analytical functions can be found at http://rdp.cme.msu.edu/). primers (table 2) for all bacteria were obtained from the published work (fierer et al., 2005). all the primers and probe were commercially synthesized from invitrogen (shanghai, china). reference strains, culture conditions and genome extraction the source of strain and reference strains used in this study were described in detail in table 3. briefly, five strains of bifidobacterium, five strains of lactobacillus, five strains of bacillus, five strains of e. coli, two strains of staphylococcus aureus, four strains of salmonella and four strains of streptococcus were used in this study. the strains were cultured anaerobically or aerobically in lb broth supplemented with 1% glucose at 37°c for 12 to 48 h. total genomic dna from the different reference strains was extracted and purified by using the method described in related kit manual (e.z.n.a. tm bacterial dna kit, omega bio-tek, usa). standard curve generation to quantify the number of bifidobacterium and all bacteria in test samples, the standard curves were produced by constructing two specific standard control plasmids. two amplicons were produced first. the 264 bp fragment was amplified, dna extracted from b. adolescentis (atcc15703), using the primers (table 2) sb-p1 and sbp2 and the following conditions: 95°c 5 min, followed by 35 cycles of 95°c 30 s, 56.8°c for 30 s, 72°c for 20 s, with a final extension of 72°c for 10 min. the 200 bp fragment was amplified, dna extracted from the test samples, using the primers (table 2) eub338f and eub518r (fierer et al., 2005) and the following conditions: 95°c 5 min, followed by 32 cycles of 95°c 30 s, 55°c for 30 s, 72°c for 30 s, with a final extension of 72°c for 10 min. the amplified products were eluted from the agarose gel using tianquick mini purification kit (tiangen, beijing, china) and cloned into the pmd19-t vector (takara, dalian, china). plasmids dna was purified using the e.z. n.a tm plasmid miniprep kit (omega bio-tek, usa). clones were screened for the inserts using the appropriate restriction enzymes and positive clones were sequenced. dna concentration of the plasmids preparation was determined by spectrophotometer (coulter du 800, beckman, usa) and the copy number calculated using the following formula: (dna concentration in µg/µl × 6.0233 × 1023 copies/mol)/ (dna size(bp) × 660 ×10 6 ). a 10-fold dilution series of the plasmid dna (1× 10 8 to 1× 10 1 copies/µl) was prepared and used to generate the standard curve. target copy numbers for each reaction were calculated from the standard curves. quantitative pcr conditions and validation of primers specificity all primers and probe used in this study are presented in table 2. real-time quantitative pcr was carried out with iq5 real-time pcr detection system (bio-rad, ca, usa) using optical grade 96-well plates in a final volume of 25 µl. reaction system was composed of 12.5 μl sybr premix ex taq (2×), 1 μl each of forward and reverse primers (100 nm), 9.5 μl ddh2o and 1 μl dna in each reaction for detecting all bacteria. the following cycling condition: 1 cycle of predenaturation at 95°c for 20 s; 40 cycles of denaturation at 95°c for 5 s; annealing at 60°c for 30 s and extension at 72°c for 50 s. melting curve conditions were 95°c for 0 s, 55°c for 1 min and 95°c for 1 min (temperature change velocity: 0.5°c/s). for bifidobacterium a primerscripttm pcr kit (perfect real time) (takara, dalian, china) was used with 100 nm of genus-specific primers and fluorescent probe. the reaction protocol was composed of 1 cycle of predenaturation at 95°c for 2min; 50 cycles of denaturation at 95°c for 15 s; annealing at 60°c for 30 s and extension at 72°c for 50 s. the genomic dna of each reference strain was detected using quantitative real-time pcr with primers sb-f and sb-r and probe sl-p for validation specificity and with primers eub338f and eub518r for verification feasibility. statistical analysis the dates were analyzed using spss12.0. a one-way anova procedure was carried out for all data. all results were expressed as means ± sd. the results were statistically analyzed using least significant difference test. p < 0.05 was considered significant. results specific verification of quantitative pcr products the primers of quantitative pcr were used for conventional pcr with b. acidophilus atcc15703 (d) dna templates for verifying the specific amplification. results showed that, the pcr produced an intense band with the expected 126 bp (data not shown), which indicated 100% specificity. quantitative pcr standard curve real-time quantitative pcr is the ability to quantitate bacterial abundance in various complex environmental samples. the correlation coefficient for the associated standard curve was 0.998 and pcr efficiency was 111.4%. amplification efficiencies, calculated using the equation: e = 10 [-1/slope] (amannludwig et al., 1995), indicating that, the crossing threshold values for the standards fell within an acceptable range. the numbers of dna copies for detecting samples was calculated by using the following equation: y= -3.075x + 47.94 (where, y is the threshold cycle (ct) and x is the log10 (copy number of 16s rdna)) (figure 1). reproducibility four different known concentrations of dna (1.2 ×10 9 -1.2 × 10 6 copies/μl) were amplified by performing the assay described earlier in triplicate for verifying the reproducibility between experiments. the results showed that, the assay was highly reproducible, because the coefficient of variation was statistically low, at < 1.5%. the threshold cycle for each concentration ranged from 1.2 × 10 9 copies/μl to 1.2 × 10 6 copies/μl and was different between 0.1 and 0.3 cycles (figure 2). specificity of the pcr all thirty bacterial strains were used to evaluate the specificity of the real-time quantitative pcr, which that indicated only bifidobacterium genomic strains can show positive results. however, there was no amplification with other bacteria group (figure 3). starch composition of experimental diets total starch content was basically similar among corn, wheat, tapioca and pea treatment. the ratio of amylose and amylopectin was 0.21, 0.24, 0.12 and 0.52, respectively (table 1). enumeration of bifidobacterium group and all bacteria real-time pcr analysis was performed to determine the numbers of bifidobacterium and all bacteria in the intestine content of all piglets. the copy numbers of all bacteria in the digesta of proximal intestine were not affected by the different dietary treatment (table 4). however, the copy numbers of all bacteria in the cecal and colon content of piglets fed ps was significantly lower than all other treatment (p < 0.05) (table 4). the copy numbers all bacteria and bifidobacterium was lowest for the duodenum and increased towards the colon (table 4). the numbers of bifidobacterium was the highest in all groups (p < 0.05) (table 4). however, there was a tendency for lower numbers of bifidobacterium in all segments of the intestinal tract of piglets fed ts. the copy numbers of all bacteria and bifidobacterium in all segments of the intestinal tract of piglets was not affected between the cs and ws group. figure 1. linear relationship between threshold cycles and the copy number of 1 6s rdna from bifidobacterium. the standard curve was developed by using the standard dna template with 10 fold serial dilutions. 1: 1 ×10 9 copies/μl; 2: 1 × 10 8 copies/μl; 3: 1 × 10 7 copies/μl; 4: 1 × 10 6 copies/μl; 5: 1 × 10 5 copies/μl; 6: 1 × 10 4 copies/μl. linear regression (r 2 =0.998) results in an equation of y= -3.075x + 47.94. figure 2. the values of two repeats of four different were showed to prove the reproducibility of developed real-time quantitative pcr. the percentage of bifidobacterium based on all bacteria the percentage of bifidobacterium (based on all bacteria) in all intestinal segments content were affected among dietary treatments (table 4). however, there was no effect between the cs group and ws group. the percentage of bifidobacterium (based on bacteria) in ps group was the highest among all treatments (p < 0.05) (table 4). meanwhile, the percentage of bifidobacterium (based on bacteria) in ps group was the lowest among all the treatments (p < 0.05) (table 4). discussion this experiment was conducted to evaluate the influence of dietary starch (different amylose/amylopectin ratio) on microbial populations of duodenum, jejunum, ileum, cecum and colon in piglets. in this study, the ge, cp, st, lysine, methionine + cysteine and tryptophan contents were all similar in the four experimental diets with the different starch sources, while there were differences in the amylose/amylopectin ratio of the starch. bifidobacterium is considered to be a beneficial component of the intestinal microbiota which can establish an figure 3. 1-5 are curves of bifidobacterium; 6-30 are curves of lactobacillus, bacillus, salmonella, e. coli, s. aureus and streptococcus (the strains were shown in table 3). table 4. the copy numbers of all bacteria, bifidobacterium and the percentage of bifidobacterium (based on all bacteria) in the all intestinal segments content of piglets among different dietary starch sources treatments. parameter corn starch wheat starch tapioca starch pea starch all bacteria duodenum （2.26±0.74）×10 9 （2.55±0.56）×10 9 （2.83±0.86）×10 9 （2.07±0.59）×10 9 jejunum （7.35±2.04）×10 9 （8.58±1.81）×10 9 （1.38±0.88）×10 10 （1.09±0.37）×10 10 ileum （7.74±2.93）×10 9 （8.28±2.29）×10 9 （7.06±1.19）×10 9 （6.60±3.20）×10 9 cecum （2.79±0.90a）×10 11 （2.33±0.63a）×10 11 （2.29±0.69a）×10 11 （1.67±0.81b）×10 11 colon （4.83±1.20a）×10 11 （5.28±2.12a）×10 11 （4.23±1.27a）×10 11 （1.84b±0.82）×10 11 bifidobacterium duodenum （5.49±0.69b）×10 6 （6.03±0.57b）×10 6 （3.52±0.76c）×10 6 （8.04±1.38a）×10 6 jejunum （1.53±0.42b）×10 7 （1.64±0.65b）×10 7 （4.14±0.59b）×10 6 （3.85±1.78a）×10 7 ileum （8.72±3.08b）×10 6 （1.04±0.22b）×10 7 （3.44±0.93c）×10 6 （2.32±0.31a）×10 7 cecum （5.91±2.26b）×10 8 （5.45±1.17b）×10 8 （2.86±1.05c）×10 8 （1.23±0.15a）×10 9 colon （3.76±1.21b）×10 8 （3.89±0.89b）×10 8 （7.56±2.80b）×10 7 （1.24±0.53a）×10 9 b/a (%)* duodenum 0.26±0.08b 0.25±0.05b 0.13±0.01c 0.41±0.07a jejunum 0.22±0.09b 0.19±0.08b 0.04±0.02c 0.35±0.09a ileum 0.12±0.02b 0.14±0.05b 0.05±0.02c 0.36±0.05a cecum 0.21±0.06b 0.24±0.05b 0.13±0.04c 0.74±0.05a colon 0.08±0.03b 0.09±0.04b 0.02±0.01c 0.69±0.17a the dates are expressed as mean values ± the standard deviation for all samples. a, b, c means with different superscripts in the row differed significantly (p < 0.05). * b/a (%): the percentage of bifidobacterium (based on all bacteria). efficient barrier to the invasion and colonization of the gut by putrefactive and pathogenic bacteria and produces a range of metabolic substrates such as short chain fatty acids, especially butyrate. it is used by the host and stimulates the immune system in a non-inflammatory manner (crittenden et al., 1999; lene lind et al., 2004; trevisi et al., 2008). in the present study, real-time taqman polymerase chain reaction assays is developed targeting the 16s rrna gene to quantify bifidobacterium groups in the intestinal tract content of weaned-piglets and thirty bacterial strains were used to assess the specificity of the pcr. positive results were only observed in bifidobacterium genomic strains, while there was no amplification with other spp. (figure 3). fluorescent quantitative has become a potential powerful method to quantify the population of gastrointestinal tract microbial due to its convenience, rapidity, reproducibility and accuracy (deng et al., 2008; deng et al., 2007). in recent studies, real-time quantitative pcr has been shown to be an available tool for quantifying bacterial abundance in many different kinds of complex environmental samples down to the genus and species level (skovhus et al., 2004; wellinghausenfrost et al., 2001; wilks et al., 2006). real-time quantitative pcr provides information on the relative and absolute abundance of a genus and species in complex environmental samples. such detailed information can often be difficult to obtain and investigate with conventional cell enumeration methods such as most probable number and direct cell counting methods (skovhus et al., 2004). the specific primer-probe combination is an available alternative for detecting the counts of intestinal bacterial species (dario de medici et al., 2003; deng et al., 2007). four different known concentrations of dna (1.2 ×10 9 -1.2 × 10 6 copies/μl) were amplified by performing the assay described earlier in triplicate. analysis of these values proved that the assay was reproducible. meanwhile, the detected results showed that the coefficient of variation was statistically low, at < 1.5%. the main advantage of fluorescent quantitative pcr is the ability to quantitate unknown samples. in summary, a new real-time pcr assays was developed that allowed for rapid, convenient, reproducible and steady quantification of the bifidobacterium group in the intestinal content of piglets. the gastrointestinal tract of pigs is colonized by a densely diverse bacterium and the intestinal microbiota has important influence on animal health and growth performance (leser et al., 2000; moore et al., 1987). meanwhile, the gut microbial communities are affected by various factors, such as the diets and environment (duncan et al., 2007b; pluske et al., 2007). generally, bifidobacterium can hydrolyze such polysaccharides, as starch and cellulose and so on. starches are the main source of carbohydrates and energy for monogastric animal and human (deng et al., 2009b). here, high amylose/amylopectin ratio starch (ps) can increase the number of bifidobacterium and the percentage of its (based on all bacteria) intestinal digesta of piglets. we also showed that, the low amylose/amylopectin ration starch (ts) can decrease the percentage of bifidobacterium (based on all bacteria). feeding high amylose starch can enhance the population of bifidobacterium and short chain fatty acids in the colon and fecal content of mammals (brown et al., 1997; chang et al., 2006; wang et al., 2002). chang et al. (2006) found that high amylose starch diet resulted in increased bifidobacterium growth compared with the low amylose starch diets in cecal contents of rats. brown et al. (1997) reported that, feeding high amylose starch to pigs increased fecal bifidobacterium population compared with feeding low amylose starch. pieper et al. (2009) reported that, the ratio of amylose to amylopectin and starch sources affected the abundance of bifidobacterium spp. in vitro model of porcine gastrointestinal tract. those results are in agreement with the present study in vivo of porcine intestinal tract. another results in this study suggested that, the high amylose/amylopectin ratio ps can increase the bifidobacterium numbers not only in the distal intestinal digesta but also in the proximate digesta. however, the high amylose/ amylopectin ratio ps decreased the number of all bacteria in the cecal and colon chymus of piglets, while the copy numbers of all bacteria in the digesta of proximal intestine were not affected by the different dietary treatment. these changes need a further study. in conclusion, we developed a new real-time taq-man pcr assays that allowed for rapid, convenient, reproducible and steady quantification of the bifidobacterium group in the intestinal content of piglets. additionally, the present study revealed that high amylose/amylopectin ratio of starches significantly enhanced the numbers of bifidobacterium in digesta of the all intestine segment. the new methodology used in this study provides an important tool for studying the influence of different starch diet on the endogenic bifidobacterium numbers in gastrointestinal tract of pigs. acknowledgements this work was supported by the program for changjiang scholars and innovative research team in university of china (irt0555-5) and the earmarked fund for modern agro-industry technology research system of china (cars-36). references adeola o,king de (2006). developmental changes in morphometry of the small intestine and jejunal sucrase activity during developmental changes in morphometry of the small intestine and jejunal sucrase activity during the first nine weeks of postnatal growth in pigs. j. ani. sci. 84(1): 112-118. amann r, ludwig w, schleifer k (1995). phylogenetic identification and in situ detection of individual microbial cells without cultivation. microbiol. rev. 59:143-169. asp ng, van amelsvoort jm, hautvast jg (1996). nutritional implications of resistant starch. nutr. res. rev. 9(1): 1-31. brooks ph (2003). factors affecting the voluntary feed intake of the weaned pig.in weaning the pig . wageningen: academic publishers. brown i, warhurst m, arcot j, playne m, illman rj, topping d l (1997). fecal numbers of bifidobacteria are higher in pigs fed bifidobacterium longum with a high amylose cornstarch than with a low amylose cornstarch. j. nutr. 127(9): 1822-1827. chang mj, soel sm, bang mh, park jh, kang ne, kim wk (2006). interactions of high amylose starch and deoxycholic acid on gut functions in rats. nutrition. 22(2): 152-159. conlon ma, bird ar (2009). interactive and individual effects of dietary non-digestible carbohydrates and oils on dna damage, scfa and bacteria in the large bowel of rats. br. j. nutr. 101(8): 1171-1177. crittenden rg (1999). prebiotics. in: tannock gw, ed. probiotics: a critical review . wymondham, uk: horizon scientific press. cummings jh, englyst hn (1995). gastrointestinal effects of food carbohydrate. am. j. clin. nutr. 61(4 suppl): 938s-945s. dario dm, luciana c, elisabetta d, simona dp,emma f, laura t (2003). evaluation of dna extraction methods for use in combination with sybr green i real-time pcr to detect salmonella enterica serotype enteritidis in poultry. appl. environ. microbiol. 69(6): 3456-3461. deng j, wu x, bin s, li tj, huang r, liu, z (2010). dietary amylose and amylopectin ratio and resistant starch content affects plasma glucose, lactic acid, hormone levels and protein synthesis in splanchnic tissues. j. anim. physiol. anim. nutr. 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specifically utilizable by bifidobacteria. chem pharm bull (tokyo). 26(11): 3306-3311. zoetendal eg, collier ct, koike s, mackie ri, gaskins hr (2004). molecular ecological analysis of the gastrointestinal microbiota: a review. j nutr. 134(2): 465-472. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (11), pp. 001-005, november, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper escherichia coli o157:h7 edl933 has a strong virulence to bama miniature pigs by injection and fails to colonize to their gastrointestinal tracts honglei ding1,2,3, rui zhang1, kaiyun liu1, linping huang1, maochun tian4, mingming jiang1, quanming zou1 and xuhu mao1* 1 department of clinical microbiology and immunology, faculty of medical laboratory science, third military medical university and national engineering technological research center of immunological biologicals, chongqing, china. 2 institute of sericulture and system biology, southwest university, chongqing, china. 3 chongqing productivity council, chongqing, china. 4 college of animal science and technology, southwest university, chongqing, china. accepted 21 july, 2017 detection of shiga toxin-producing escherichia coli o157:h7 from commercially grown pigs has been reported. furthermore, the e. coli o157:h7 colonized model of pig has been established and e. coli o157:h7 could be transmitted from infected donor pigs to naïve pigs directly and indirectly. in the present study, we want to know whether any e. coli o157:h7 strain can colonize to the alimentary tract of pig and the virulence of e. coli o157:h7 to pig by injection. bama miniature pig was infected with e. coli o157:h7 edl933 strain orally, but the organism could not be recovered from the feces and did not cause any tissue damage. nevertheless, this pathogen introduced serious clinical symptoms and pathological injuries by injection, especially the nervous system and the injected pig exhibited severe neurological symptoms, including synclonus tremens, ataxia, head-pressing and recumbency, etc. the pig did not excrete urine and feces and the abdomen became tympanous. these data suggested that only certain e. coli o157:h7 strains could colonize to the gis of pigs involved mechanisms that related to various factors. however, the organism has strong virulence to pig by injection mode and it is a risky pathogen to human health. key words: escherichia coli o157:h7, bama miniature pig, colonization, inoculation, pathological injury. introduction infection of shiga toxin -producing escherichia coli (stec) o157:h7 can lead to a spectrum of illnesses in human, including diarrhea, hemorrhagic colitis (hc) and hemolytic uremic syndrome (hus), which demonstrates as acute renal failure and may lead to death (besser et al., 1993; bruce et al., 2003; rivas et al., 2006). most cases are thought to occur as a result of the ingestion of ground beef (bell et al., 1994), unpasteurized milk (solomakos et al., 2009) and vegetables (besser et al., 1993), which are thought to have been contaminated *corresponding author. e-mail: maoxuhu@163.com, mxh95xy@mail.tmmu.com.cn. tel: 86-23-68752315. fax: 8623-68752315. with feces from infected cattle. outbreaks and sporadic cases have also been linked to water, animal-to-person and person-to-person transmission (swerdlow et al., 1992; belongia et al., 1993; shukla et al., 1995). cattle are considered to be the major reservoir of stec and the prevalence of e. coli o157:h7 in cattle is range (baker et al., 2007; wang et al., 2008; williams et al., 2008). e. coli o157:h7 has also been isolated from other ruminants, such as deer (garcía-sánchez et al., 2007; sánchez et al., 2009) and sheep (kudva et al., 1996). e. coli o157:h7 has occasionally been isolated from nonruminant animals, including poultry (baschkier et al., 2009; heuvelink et al., 1999), pigeons (cízek et al., 2000; kobayashi et al., 2002), wild birds (wallace et al., 1997; kobayashi et al., 2002) and raccoons (hancock et al., 1998), but the bulk of the data suggests that the, prevalence of stec is greater in ruminants than in other animals. recent studies have demonstrated that conventional pigs were permissive host for e. coli o157:h7 (booher et al., 2002; cornick and helgerson, 2004) and have established that e. coli o157:h7 could be shed by 3monthold pigs for 2 months. furthermore, these animals did not become clinically ill, and the duration of shedding in the feces was similar to that of ruminants experimentally infected with the same e. coli o157:h7 isolate (booher et al., 2002) and could transmit this pathogen to their offspring. meanwhile, some researches indicated that e. coli o157:h7 had been isolated from healthy pig in many countries (heuvelink et al., 1999; wang et al., 2008; oporto et al., 2008). nevertheless, the prevalence of the organism in these studies was generally low, except for the result from chile that the prevalence of e. coli o157:h7 was higher in pigs than in cattle, which suggested that pig might be an important source of this organism in some countries (borie et al., 1997). also, only one family outbreak has been specifically traced back to pork salami and the e. coli o157 isolated from the couple and the salami carried shiga toxin 1 (stx1), shiga toxin 2 (stx2) and e. coli attaching and effacing (eae) genes and shared the same pfge (pulsedfield gel electrophoresis) pattern (conedera et al., 2007) . to date, there is no explanation for the reason why the prevalence of e. coli o157:h7 in pig is generally lower than in ruminants. also, no research about the consequence of pig infected this organism in its organs or abdomen, though these animals did not become clinically ill infected this pathogen orally. we hypothesized that the gastrointestinal tract (gi) of pig did not facilitate the colonization of most e. coli o157:h7 strains and caused the low prevalence of e. coli o157:h7 in pig. in the study, e. coli o157:h7 edl 933 was selected, which obtained from a patient whose symptom, characterized by severe cramped abdominal pain, initially watery diarrhea and grossly bloody diarrhea (riley et al., 1983) and determined whether it was a suitable strain for the colonization to the gi of pig. also, the virulence of e. coli o157:h7 to pig was detected by injection. materials and methods e. coli o157:h7 strain e. coli o157:h7 strain edl933 (stx1, stx2, eaea, ehxa, tccp and espa positive) was originally isolated from patient with diarrhea (riley et al., 1983) and was kindly provided by prof. huaiqi jing (chinese center for disease control and prevention). e. coli o157:h7 cyb42 was isolated from diary cattle in chongqing (wang et al., 2008). these bacterial inocula were grown as previously described with minor modification (booher et al., 2002). briefly, a single colony from strain was picked and cultured overnight in luriabertani (lb) medium at 37°c with shaking (180 rpm) and the bacterial number was confirmed by direct plate counts. the inoculum was washed once with 0.1 m phosphate buffered saline (pbs) (ph 7.2) and adjusted to the appropriate concentration. animals and preparation bama miniature pigs were obtained from a commercial source at 1 month of age and housed in pens with cement floors at 2 pigs per pen under biohazard level 2 facilities. the pigs were acclimated to an antibioticfree feed (creep feed, chongqing zhengda company, chongqing) and water ad libitum for 2 weeks prior to inoculation. fecal samples were collected from each animal once prior to inoculation and injection and screened with sorbitol-macconkey agar supplemented with cefixime (2.5 mg . l -1 ) and potassium tellurite (0.05 mgl -1 ) (ctsmac) and polymerase chain reaction (pcr) to ensure that the pigs were not naturally colonized by e. coli o157:h7. inoculation of e. coli o157:h7 18 pigs were used in inoculation and were separated into 3 groups, of which 16 were inoculated with the edl933 and 2 were negative controls. after a 2 week acclimation period, groups 1 and 2 of each 8 pigs were inoculated with 1.0 × 10 8 and 1.0 × 10 12 clonal formation unit (cfu) of e. coli o157:h7 edl933 by adding the organism to a small amount of food placed in individual pans, respectively. on the 17th day after fed edl933 bacteria, groups 1 and 2 of each 4 pigs, selected randomly, were inoculated with 1.0 × 10 8 and 1.0 × 10 12 cfu of e. coli o157:h7 cyb42. group 3 of 2 pigs were inoculated with 10 ml 0.1 m pbs (ph 7.2). pigs were observed until the inoculum was consumed. injection of e. coli o157:h7 lysate 18 pigs were used in injection and were also separated into 3 groups. after a 2 week acclimation period, group 1 of 8 pigs were injected with 1.0 × 10 8 cfu of e. coli o157:h7 strain edl933 lysate intramuscularly; group 2 of 8 pigs were injected with 1.0 × 10 8 cfu e. coli o157:h7 edl933 lysate intraperitoneally; and group 3 of 2 pigs were injected with 10 ml 0.1 m pbs (ph 7.2) intramuscularly. fecal sampling individual fecal samples from inoculated pigs were collected on days 2, 3, 4 and at 2 weeks post inoculation (pi) (days 14, 15, 16) of edl933 and cyb42, respectively, and from injected pigs were collected on days 2, 3, 4 (booher et al., 2002). fecal samples were cultured as previously described (cornick and helgerson, 2004). briefly, 5 g samples were added to 20ml of pbs (ph 7.2) and mixed in a stomacher blender, and then serial 10 fold dilutions were made to use pbs (ph 7.2). samples were directly inoculated in triplicate into selective media ct-smac. enrichment cultures (10 g of feces in 100 ml lb plus 0.02% bile salts) were incubated overnight at 37°c, concentrated using immunomagnetic beads (dynabeads; dynal, oslo, norway), and plated into the selective medium described above. the sensitivity of the direct plating method was 50 cfu/g. colonies recovered on selective medium were confirmed as e. coli o157:h7 by using a commercial latex agglutination kit specific for the o157 lipopolysaccharide and pcr for rfbe (wang et al., 2008). necropsy the animals monitored closely to ensure their welfare after they were injected. both control and e. coli edl933 injected animals were euthanized and necropsied at the 4 th day when they behave severe clinical signs. meanwhile, after 2 weeks pi, control, e. coli edl933 (8 pigs) and e. coli cyb42 (8 pigs) inoculated animals orally were euthanized and necropsied. pigs were sedated with an injectable anesthetic (tiletamine hcl and zolazepaam hcl) and then euthanized with an intravenous overdose of sodium barbiturate (390 mg pentobarbital sodium and 50 mg phenytoin sodium/5 kg of body weight) (booher et al., 2002). the following types of tissue (approximately 5 cm length or 5 cm square) were collected from pigs: stomach, jejunum, ileum, distal colon, cecum, rectum, epencephalon, cerebrum, kidney, liver, and spleen. the sections of tissues were collected in neutral buffered formalin for histopathology, processed, and stained with hematoxylin and eosin (h and e). 5 g of rectal contents were also collected and cultured by using direct plating and enrichment broth as described above. western blotting serum samples were collected from both control and e. coli inoculated pigs prior to inoculation and at necropsy and detected stx2, intimin and espa neutralizing antibodies. the stx2, intimin and espa recombinant purified proteins were prepared in our lab (gu et al., 2009; ma et al., 2008). sds-page and western blotting were performed as described previously (cendron et al., 2009). for visualization of proteins after sds-page, gels were stained with coomassie brilliant blue r250. for the development of immunoblots, pvdf filters were blocked with blocking buffer (beyotime) and incubated with the respective antisera at a dilution of 1:1000. the membrane was washed 6 times with tbs containing 0.1% tween-20 (ttbs) (ph 7.5). horseradish peroxidaseconjugated anti-rabbit igg was used at a dilution of 1:10 000 to visualize bound antibody. results clinical response and bacterial culture of inoculated pigs none of the animals developed signs of intestinal or systemic disease following inoculation with the e. coli inoculum from the 2 groups that were inoculated the strain orally. e. coli o157:h7 was not recovered from any of the 16 pigs during the initial period or at 2 weeks after inoculation with 1.0 × 10 8 and 1.0 × 10 12 cfu. fecal samples from the rectalanal junction were also collected from these 16 pigs and no target organism was discovered. then, groups 1 and 2 of each 4 pigs were inoculated with 1.0 × 10 8 and 1.0 × 10 12 cfu of e. coli o157:h7 cyb42. however, e. coli o157:h7 cyb42 was still not recovered from the 8 inoculated pigs. no e. coli o157:h7 was investigated from any of the two control animals at any time during the experiment, and no symptom displayed. clinical observation and bacterial culture of injected pigs piglets challenged intramuscularly and intraperitoneally with edl933 typically developed neurological signs within 36 and 48 h, respectively, including anorexia, depression and paralysis of the hind limbs like goggy sitting. however, the feces are dry. 48 to 72 h following injection with e. coli o157:h7 strain, the piglets lay on one side, and went on to manifest severe neurological symptoms of synclonus tremens, ataxia, head-pressing and recumbency. simultaneously, the mouth was slightly open and jerked violently and spasmodically and the 2 fore limbs stroked like swimming. the pig did not excrete urine and feces and the abdomen became tympanous. when the anocelia was opened, a little of serous fluid leaked from the abdominal subcutaneous tissue, and the livers and spleen were adhered to the peritoneum. the intestinal wall was thinner than the normal one. there was too much urine in the bladder, which caused the tympanous abdomen. the blood became thick and reddish black with slow bloodstream. no stx2, intimin and espa neutralizing antibodies were detected in both e. coli o157:h7-infected and e. coli o157:h7-injected pigs. histological studies on e. coli o157:h7-infected and e. coli o157:h7-injected pigs we also studied the effect of e. coli o157:h7 injection on their main target organs, including stomach, intestine, epencephalon, cerebrum, kidney, liver and spleen by examining h and e stained-sections. the main pathological damage was observed at cerebrum (figure 1), epencephalon (figure 2) and large intestine (figure 3). the nerve cells of cerebrum became spindle-shaped and hydropic, accompanying coagulation necrosis and the nucleus was pycnotic. in addition, blood capillary was atresic, and perivascular space was broadened. examination at epencephalon revealed the obvious reduction of cells in tunicae granulosa and neuropile porous. the members of the research group could also detect karyopycnosis of purkinje cell. histological examination of ceca, recta of e. coli o157:h7 injected pigs showed multifocal areas of villous or surface epithelium degeneration, necrosis, or shedding, submucous membranous hydropsia, and extensive inflammatory cell infiltration, mainly the lymphocyte in proper coat. surprisingly, the small intestine and kidney (figure 4) only showed slightly pathological change with mild cell trauma. for instance, some glomeruli of kidney increased in volume, and the capsular space became narrow. proximal convoluted tubule cells exhibited denaturation and necrosis. meanwhile, the blood capillary was enlarged. conversely, e. coli o157:h7-infected group of pigs did show any pathology in their tissues. there were no abnormalities in the control animals as well. discussion during the experiment, it was found that edl933 could not infect bama miniature pigs orally, though the infectious dose is high. in order to further confirm this outcome, the researchers chose e. coli o157:h7 cyb42 that was isolated from diary cattle in chongqing toreinoculate the edl933-feeded pigs. but, the result was negative as previously. it demonstrated, to some extent, that some pig individuals were resistant to colonization and/or some e. coli o157:h7 strains could not colonize to the intestinal epithelium of pig. some researches indicated that many bacterial factors contributed to the colonization. some research data demonstrated that the flagellum of e. coli o157:h7, but not intimin, was important for persistence in poultry (best et al., 2005), whereas intimin (woodward et al., 2003) but not the flagellum was important in conventionally weaned lambs. this suggests that the function of both surface arrayed structures may be host dependent and may be linked to the availability of specific host-cell receptors. best et al suggested that the flagellum and intimin of a stx -negative e. coli o157:h7 isolate had little or no role to play in colonization of 14 week-old conventionally reared pigs (best et al., 2006). however, other reported e. coli o157:h7 virulence factors, such as long polar fimbriae, did contribute to the persistence in pig animal infection model (jordanet et al., 2004), and might have contributed to the persistent infection of pigs noted for the intimin and flagella deficient mutants reported. the strain, 86 24, which was isolated from an outbreak of human disease and caused attaching and effacing lesion, was used in most animal experiments and considered as a well established e. coli o157:h7 infected pathogen. the edl933 strain, but not 86 24, may be absent from the essential factors, which were necessary for the long-term colonization of the pigs' intestinal tracts. this could partially explain why edl933 could not adhere to bama miniature pigs' gi. the genetic difference of the 2 strains for colonization in animals needs further comprehensive researches. in this experiment, we also attempted to establish a bama miniature pig model infected steadily with e. coli o157:h7, for the small weight of the bama miniature pig of which the figure is thinner than susscrota domestica's. nevertheless, the edl933 strain could not colonize to the gis of bama miniature pigs. in the next plan, the research group will try to inoculate and acclimate edl933 strain into bama miniature pig through certain methods, such as serial passages in vivo, for procuring an adaptive colonization of e. coli o157:h7 strain. to identify whether e. coli o157:h7 was pathogenic to pigs, nonproliferative edl933 lysate were injected to bama miniature pigs intramuscularly and intraperitoneally. interestingly, the infected pigs exhibited identical clinical symptom, especial the nervous syndrome. however, the injected pigs did not manifest diarrhea, which was consistent to the histological findings. serious intestinal microvillus damage may be an indispensable process caused through intestinal infection of e. coli o157:h7. therefore, e. coli o157:h7 will be pathogenic when it 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semen characteristics of kolbroek and large white boars following computer aided sperm analysis ® (casa) matshidiso bailekae masenya 1,2 , m. l. mphaphathi 1,3 , m. h. mapeka 1,3 , p. h. munyai 1,4 , m. b. makhafola 1,2 , f. v. ramukhithi 1,3 , p. p. malusi 1 , d. o. umesiobi 2 and t. l. nedambale 1,3,4 * 1 agricultural research council, animal production institute, germplasm conservation & reproductive biotechnologies, private bag x2, irene, 0062, south africa. 2 department of agriculture, central university of technology, private bag x20539, bloemfontein, 9300, south africa. 3 tshwane university of technology, department of animal sciences, private bag x680, pretoria 0001, south africa. 4 university of the free state, department of animal, wildlife and grassland sciences, private bag x 339, bloemfontein, south africa. accepted 9 september, 2013 consistent estimates of boar fertility potential from objective semen evaluation could be a valuable tool for boar selection. the objective of this study was to evaluate semen characteristics of kolbroek and large white boars following computer aided sperm analysis ® (casa). eight ejaculates were collected separately from individual kolbroek (n = 4) and large white (n = 4) boars using the gloved-hand technique. following semen collection, semen was evaluated for macroscopic and microscopic characteristics. analysis of variance (anova) was used to test the differences between the breeds (p<0.05) . the bodyweight of kolbroek (154.7 ± 8.5) was significantly lower compared to large white (189.9 ± 7.7) boar. there was also a positive correlation between bodyweight and semen volume of both kolbroek (r = 0.2197) and large white (r = 0.2577) boar. however, no significant differences were observed in kolbroek and large white boar semen volume (140 and 170 ml), sperm concentration (0.727 and 0.761 × 10 9 sperm cell/ml), ph (7.0 and 7.0), total motility (95 and 91%) and morphology (84 and 82%). in conclusion, the bodyweight of kolbroek and large white boar was positively correlated with ejaculated semen volume. sperm characteristics of both kolbroek and large white boar were similar. sperm class analyser ® provided a precise and more objective information of sperm motility characteristics. key words: sperm, large white, kolbroek, motility rate, boar. introduction large white is the most popular exotic breed in south africa (arc, 1993) due to their superior fertility and growth rate (ncube et al., 2003). however, their high nutrient requirements and intensive management systems make them unsuitable for resource-poor rural farmers and harsh environmental conditions. kolbroek is a south african indigenous pig breed with unique genetic traits for diseases tolerance and adaptability in harsh *corresponding author. e-mail: lucky@arc.agric.za. tel: +2712 672 9210. fax: +2712 665 1604. environmental conditions (ramsay et al., 1994). they are considered appropriate breed for the resourcepoor rural farmers because of their tolerance to various diseases and capacity to utilize fibrous and poor quality feed resources compared to exotic breeds (halimani et al., 2010). a recent survey indicated a catastrophic collapse in the population of south african indigenous germplasm (fao, 2007). this collapse was attributed among others to unplanned breeding, crossbreeding and introduction of exotic germplasm (scholtz, 2005). mating and crossbreeding are largely unsupervised leaving these breeds vulnerable to inbreeding and uncontrolled genetic masenya et al. 011 admixture with other breeds (halimani et al., 2010). most researches have been focused on the imported genotypes which cannot be sustained under smallholder conditions (ncube et al., 2003). hence, there is a need to evaluate reproductive potential of imported boars in comparison with south african indigenous boars. the reproductive potential of the indigenous kolbroek boars has not been fully exploited in south africa compared to other pig genotypes. a proper semen analysis is empirical for boar selection in the herd and for preserving their genetic materials through ex -situ and in-situ. indigenous pigs were long regarded as unsuitable for intensive commercial breeding because of their slow growth and inadequate meat production (prolit, 2004). however, indigenous pigs exhibit well-established adaptations to severe environmental and management conditions (swart et al., 2010) . moreover, there is lack of accurate method of predicting the fertility rate of kolbroek boar sperm to determine their reproductive potential. sperm motility is known to be an important characteristic in predicting the fertility of male potential performances (holt et al., 1997; tardif et al., 1999; gadea, 2005). however, subjective microscope evaluation varied between 30 to 60% from the same ejaculates (amann, 1989). due to these biases, emphasis has been placed on the use of objective methods such as computer aided sperm analysis ® (casa) system (saikhun et al., 2011). therefore, the objective of this study was to compare south african indigenous kolbroek and exotic large white boar breeds on sperm characteristics following analysis by computer aided sperm analysis ® (casa) known as sperm class analyser ® (sca). materials and methods the study was conducted at the pig research unit of agricultural research council (germplasm conservation & reproductive biotechnologies unit), irene, south africa. the agricultural research councilirene campus is located at 25° 55' south; 28° 12' east. the institute is located in the highveld region of south africa and situated at an altitude of 1525 m above sea level. four indigenous kolbroek and four exotic large white boars were used for this study because of the scarcity challenge of finding kolbroek boars. the boars were between 2 to 3 years of age. the study was done in summer season (february to march, 2011). the boars were weighed using a km3 electronic weight indicator (rudoweigh ® ). the boars were in good health condition throughout the duration of the study. the diets were formulated to meet the nutritional requirements of the boars (crude protein (cp): 13% and digestible energy (de): 13 mj/kg) (national research council, 1998). water was given ad libitum throughout the duration of the study. semen collection and processing semen samples were collected from the experimental boars twice weekly from february to march. twelve ejaculates were collected separately from four kolbroek and four large white boars with the gloved-hand technique in a 300-ml glass beaker. the filtered semen fraction were sealed with a gauze filter inside a pre-warmed (39°c) insulated thermos flask. upon arrival at the laboratory, semen volume was measured by using the graduated falcon tube, ph was measured using the litmus paper, then sperm concentration was measured using the spectrophotometer (jenway 6310 spectrophotometer, bibby scientific, england) and was recorded in billions (× 10 9 /ml). experimental boars were cared for according to the guidelines for the agricultural research council, animal production institute ethics committee (ref: apiec10/01). sperm morphology semen was collected from kolbroek and large white boars and a 10× dilution was prepared by adding semen to 0.9% sodium chloride. one drop of 0.27% chicago sky blue and one drop of diluted semen were mixed on a slide. slides were air-dried in a near vertical position then put into a fixative in a jar for 2 min and then rinsed with tap and distilled water. slides were put into jars containing the giemsa staining solution and left for 20 h at room temperature. the slides were rinsed again in tap and distilled water for 2 min, air-dried in a near vertical position and cover slipped with methyl yellow. a drop of oil immersion (olympus, japan) was placed on the smeared microscope glass slide and 100 sperm were counted at 100 × magnification (figure 1a and b). a criterion was applied for the evaluation for abnormal sperm head (flat, sharp, double and if it is not oval); midpiece (proximal and distal cytoplasmic droplets); tail (coiled, double, broken) . a live sperm was white/pink in colour and a dead sperm was dark blue (kovács and foote, 1992). sperm motility rate the 10 µl of raw semen were placed into 500 µl of bo wash medium in 15 ml tube (falcon ® 352099, usa). the tube was then kept in co2 incubator (sanyo, japan) adjusted to 39°c. five micro litres of semen was placed on the warm glass slide (~76 × 26 × 1 mm, germany) and placed with a warmed cover slip (22 × 22 mm, germany) over the microscope-warm plate (omron) adjusted at 39°c. the sperm motility rates were evaluated by computer assisted sperm analysis system (sperm class analyzer ® [sca] 5.0, microptic, barcelona, spain) at the magnification of 10 × (nikon, china). the kinematic values recorded for each sperm included, in addition to the overall percentage of motile sperm, the velocity of movement, the width of the sperm head’s trajectory and the frequency of the change in direction of the sperm head (table 1). data analysis the analysis was done using genstat software. the experiment was designed as a completely randomised design with two treatments (kolbroek and large white boars). analysis of variance (anova) was used to test for differences between the treatments. the data were acceptably normal with homogeneous treatment variances. treatment means were separated using fisher’s protected t-test least significant difference (lsd) at a significant level of p<0.05 (snedecor and cochran, 1980). the correlation of the bodyweight with semen volume, concentration and sperm motility was performed using sas statistical software. the pearson two-sided was used to determine the correlation between bodyweight and the variables (snedecor and cochran, 1980). results the results of macroscopic evaluations are outlined in 012 afr. j. pig farming a b figure 1. (a): kolbroek live sperm; (b) kolbroek dead sperm. table 1. sperm class analyzer ® settings used to analyse sperm motility and velocity parameters. parameter setting contrast 169 brightness 470 image/second 50 optic ph chamber cover slide scale 10x particle size (µm 2 ) 10<70 slow (µm/s) <40 medium (µm/s) <80 rapid (µm/s) <120 progressivity (%) 40% of straightness circular (%) 50% of linearity connectivity 11 velocity on the average path points 7 number of images 50 table 2. macroscopic evaluation for kolbroek and large white boar semen. breed bodyweight (kg) semen volume (ml) semen ph semen concentration (× 10 9 sperm cell/ml) kolbroek 1 166.5 130.0 ± 26.5 7.0 ± 0.0 0.533.4 ± 90.8 kolbroek 2 150.5 126.7 ± 11.6 7.0 ± 0.0 1.0521 ± 283.3 kolbroek 3 147.0 100.0 ± 0.0 7.0 ± 0.0 0.9073 ± 333.1 kolbroek 4 155.0 205.0 ± 37.8 7.0 ± 0.0 0.4153 ± 174.2 averages 154.7 ± 8.5 a 140.4 ± 48.6 7.0 0.727 ± 340.8 large white 1 196.6 226.7 ± 100.2 7.0 ± 0.0 0.646.7 ± 82.2 large white 2 190.6 145.0 ± 42.7 7.0 ± 0.0 0.605.0 ± 328.0 large white 3 179.0 180.0 ± 45.8 7.0 ± 0.0 0.590.3 ± 135.9 large white 4 193.4 158.3 ± 18.9 7.0 ± 0.0 1.203.4 ± 487.9 averages 189.9 ± 7.7 b 177.5 ± 60.4 7.0 0.761. 0 ± 372.8 ab different letters indicate significant differences within columns (p<0.05). masenya et al. 013 table 3. sperm morphology and viability for kolbroek and large white boar semen (±sd). breed live (%) dead (%) abnormality (%) head midpiece tail kolbroek 1 88.7 ± 3.1 9.7 ± 4.0 0.7 ± 1.2 0.0 ± 0.0 1.0 ± 1.7 kolbroek 2 84.7 ± 7.5 10.0 ± 8.7 2.3 ± 0.6 1.7 ± 2.9 1.3 ± 1.2 kolbroek 3 82.0 ± 1.0 13.3 ± 6.0 2.7 ± 3.8 1.0 ± 1.7 1.0 ± 1.7 kolbroek 4 83.0 ± 10.0 9.7 ± 5.5 2.3 ± 2.3 1.0 ± 1.7 4.0 ± 3.5 averages 84.6 ± 6.1 10.7 ± 5.6 2.0 ± 2.1 0.9 ± 1.7 1.8 ± 2.3 large white 1 82.0 ± 9.6 7.0 ± 5.3 0.7 ± 0.6 0.3 ± 0.6 6.3 ± 6.0 large white 2 87.7 ± 6.7 5.3 ± 1.5 2.7 ± 1.5 1.0 ± 1.0 6.0 ± 6.0 large white 3 80.7 ± 5.5 9.3 ± 5.0 1.7 ± 2.1 0.0 ± 0.0 5.7 ± 7.4 large white 4 76.3 ± 3.5 17.3 ± 7.0 0.7 ± 0.6 1.3 ± 1.2 8.0 ± 4.4 averages 81.7 ± 7.1 9.8 ± 6.3 1.4 ± 1.4 0.7 ± 0.9 6.5 ± 5.2 table 2. the bodyweight of kolbroek (154.7 ± 8.5 kg) was significantly lower compared to large white (189.9 ± 7.7 kg) boar. there was a positive correlation between bodyweight and semen volume of kolbroek (r = 0.2197) and large white (r = 0.2577). conversely, there was a negative correlation between bodyweight and sperm motility rate (r = -0.9655) and concentration (r = -0.6600) of kolbroek. however, the bodyweight of large white was positively correlated with sperm concentration (r = 0.3721), but negatively correlated to total motility (r =0.1043). no significant differences were observed in kolbroek and large white boar volume (140 and 170 ml), semen ph (7.0 and 7.0) and sperm concentration (0.727 and 0.761 × 10 9 sperm cell/ml) . furthermore, no individual variation was observed for semen volume, ph and concentration. the results for kolbroek and large white sperm morphology are presented in table 3. the average percentage (±sd) of kolbroek and large white live sperm was 84.6 ± 6.1 and 81.7 ± 7.1%, respectively. there was no significant differences (p<0.05) in abnormal sperm morphology of kolbroek and large white. more also, the results of both kolbroek and large white sperm motility are presented in table 4. the average percentage (±sd) of kolbroek and large white sperm motility was 95.2 ± 4.2 and 91.4 ± 6.2%, respectively. however, a significant difference was observed for rapid sperm motility of kolbroek 4 (79.4 ± 2.6%) as compared to all the other boars including large white. no significant difference was observed for all other sperm motility and velocity parameters for kolbroek and large white boar. discussion this study demonstrates that the bodyweight of kolbroek (154.7 ± 8.5) was significantly lower compared to large white (189.9 ± 7.7) boar. there was also a bodyweight correlation to semen volume ejaculated by both kolbroek (r = 0.2197) and large white (r = 0.2577) . however, no significant differences were observed for kolbroek and large white boar semen volume (140 and 170 ml), sperm concentration (0.727 and 0.761 × 10 9 sperm cell/ml), ph (7.0 and 7.0), total motility rate (95 and 91%) and morphology (84 and 82%). similarly, it was previously reported that breed did not have a significant effect on boar sperm characteristics (kennedy and wilkins, 1984; rothschild, 1996; oh et al., 2003). kolbroek boars had a slightly lower semen volume as compared to the standard semen volume of 150 to 300 ml in exotic breeds (kondracki, 2003). egerszegi et al. (2008) reported similar results for hungarian indigenous mangalica boars (178 ml). in contrast, wolf and smithal (2009) found that czech large white and landrace had a slightly higher semen volume of 276 and 273 ml, respectively. furthermore, chimonyo et al. (2005) reported that indigenous pigs in southern africa are smaller in size compared to exotic pig breeds. this was evident in the present study as kolbroek boars had a lower bodyweight (154.8 kg) compared to large white boar (189.9 kg). larger breeds such as large white tend to produce higher semen volume (hughes and varely, 1980). similarly, same results were observed in the present study as semen volume of both boar breeds was influenced by bodyweight. although, kolbroek boar bodyweight was lower, the sperm concentration was higher. there was a negative correlation between bodyweight and sperm motility rate (r = -0.9655) and concentration (r = -0.6600); but positively correlated with volume (r = 0.2197) of kolbroek boar. however, the bodyweight of large white was positively correlated with volume (r = 0.2577) and sperm concentration (r = 0.3721), but negatively correlated to total motility (r = -0.1043). moreover, johnson et al. (2000) reported that the ph of raw boar semen varies between 7.0 and 7.5, irrespective of the boar breed. this is in agreement with the present table 4. sperm motility and velocity rates for kolbroek and large white boars (±sd). boar sperm motility sperm velocity tm (%) rap (%) pm (%) vcl (µm/s) vsl (µm/s) vap (µm/s) lin (%) str (%) wob (%) kolbroek 1 91.8 ± 6.6 a 35.5 ± 1.2 b 36.3 ± 25.8 a 135.2 ± 31.1 a 42.8 ± 21.2 a 82.7 ± 12.4 a 33.8 ± 20.8 a 51.4 ± 21.8 a 64.4 ± 21.2 a kolbroek 2 96.2 ± 3.4 a 54.0 ± 8.0 b 31.1 ± 4.6 a 133.6 ± 12.1 a 39.4 ± 5.8 a 97.7 ± 18.9 a 29.6 ± 4.7 a 40.8 ± 5.1 a 72.9 ± 9.3 a kolbroek 3 96.6 ± 2.6 a 52.1 ± 9.9 b 31.3 ± 9.9 a 132.7 ± 1.9 a 35.5 ± 3.0 a 84.0 ± 20.9 a 26.7 ± 2.0 a 43.3 ± 6.4 a 63.2 ± 14.8 a kolbroek 4 96.1 ± 3.5 a 79.4 ± 2.6 a 48.7 ± 13.3 a 171.7 ± 11.2 a 46.0 ± 5.4 a 98.0 ± 12.9 a 26.8 ± 2.2 a 47.7 ± 9.3 a 57.1 ± 7.0 a averages 95.2 ± 4.2 55.2 ± 17.3 36.8 ± 15.2 143.3 ± 22.8 40.9 ± 10.6 90.6 ± 16.1 29.2 ± 9.7 45.8 ± 11.5 64.4 ± 13.5 large white 1 94.8 ± 5.2 a 46.4 ± 21.2 b 27.9 ± 8.2 a 136.2 ± 37.2 a 36.0 ± 9.8 a 86.8 ± 27.3 a 26.8 ± 5.4 a 42.3 ± 9.0 a 63.3 ± 2.5 a large white 2 87.2 ± 10.6 a 39.2 ± 1.0 b 15.3 ± 3.6 a 121.2 ± 11.3 a 27.7 ± 1.0 a 77.7 ± 2.6 a 23.0 ± 3.1 a 35.7 ± 2.5 a 64.4 ± 4.1 a large white 3 93.5 ± 2.9 a 38.3 ± 11.8 b 24.4 ± 2.6 a 121.2 ± 16.0 a 33.8 ± 5.6 a 73.3 ± 10.5 a 28.6 ± 8.8 a 47.6 ± 15.6 a 60.4 ± 2.1 a large white 4 89.9 ± 3.7 a 44.0 ± 18.1 b 23.6 ± 6.6 a 137.7 ± 33.3 a 33.8 ± 8.9 a 86.7 ± 25.7 a 24.5 ± 1.2 a 39.4 ± 1.7 a 62.3 ± 5.1 a averages 91.4 ± 6.2 42.0 ± 13.4 22.8 ± 6.8 129.1 ± 24.8 32.8 ± 7.0 81.1 ± 17.7 25.7 ± 5.1 41.2 ± 9.0 62.6 ± 34 tm, total motility; rap, rapid; pm, progressive motility; vcl, velocity on the curve line; vsl, velocity on the straight line; vap, velocity on the average path; lin, linearity; str, straightness; wob, wobble. ab different letters indicate significant differences (p<0.05). observed ph results (7.0) in both breeds. however, a ph change (increase or decrease) is detrimental to both the sperm metabolism and motility. infection is usually associated with alkaline ejaculate (ph>8.0), which leads to diminished sperm motility and an increased proportion of altered acrosomes (althouse et al., 2000). in the present study, the boar semen ph did not negatively affect the sperm motility. in addition, no differences were observed for kolbroek and large white boar sperm concentration. variation in the number of sperm in an ejaculate has been described bet-ween different pig breeds (kommisrud et al., 2002), which is a first factor influencing semen dose production. not only differences in sperm concentration but also in sperm volume (kondracki, 2003), influence sperm concentration. the sperm concentration for indigenous kolbroek was higher (0.727 × 10 9 sperm cell/ml) as compared to the hungarian mangalica boar (0.490 × 10 9 sperm cell/ml) (egerszegi et al., 2008). the percentage of sperm with normal morphology was above 80% for kolbroek and large white boars. such percentages of normal morphology are correlated with fertility (sanchez et al., 1998; xu et al., 1998; alm et al., 2006). the results from this study also showed that there are no variations between individual boars, irrespective of the breed. similar findings were observed by borg et al. (1993) who reported that characteristics of sperm morphology did not differ among different boar breeds (duroc, meishan, fengjing and minzhu boars). kolbroek and large white semen showed a lower percentage of morphologically abnormal sperm (4.7 ± 2.0 and 2.9 ± 2.5%, respectively) as compared to other studies. wolf and smithal (2009) found a slightly higher percentage of abnormal sperm (11.4 and 11.2%) for czech large white and czech landrace boars, respectively. criteria for the maximum percentage of primary and secondary abnormalities in commercial pig ai-centres were determined as 10 and 20%, respectively (waberski et al., 1994; flowers, 1997). morphological abnormalities give an indication of aberrations in the spermatogenesis. morpho-logical abnormalities of sperm can also have a detrimental impact upon fertilization and embryonic development (walters et al., 2005; saacke, 2008). the average sperm total motility obtained for kolbroek and large white was 95 and 91%, respectively. these sperm motility results are an indication of an active metabolism and are con-sidered to be of great importance for fertilization to take place. lower motility percentages were reported (70.2 ± 8.8%) for czech hybrid ai boars (frydrychová et al., 2010). subjective method was used to evaluate sperm motility analysis. microscopic techniques have limitations including subjectivity, variability, the small number of sperm analysed and poor correlation with fertilizing potential (rijsselaere et al., 2005). subjective visual evaluation of motility is also prone to human error and biasness. hence, the computer -assisted 014 afr. j. pig farming masenya et al. 015 sperm analysis (casa) was initiated to reduce subjective bias on the motility assessment and to discriminate a series of motility patterns of boar semen (tretipskul et al., 2010). conclusion the bodyweight of kolbroek and large white boar was positively correlated with ejaculated semen volume. however, macroscopic and microscopic sperm characteristics of kolbroek were similar compared to large white boar. surprisingly, kolbroek boar sperm concentration and motility rate was negatively correlated to bodyweight compared to only large white sperm concentration. this is the first study that provided more information on sperm motility characteristics of both kolbroek and large white boar using sperm class analyser ® . it is recommended that further studies should be conducted with more number of boars to validate the sperm motility characteristics information following artificial insemination. acknowledgements the authors wish to acknowledge national research foundation, agricultural research council (arc) and department of agriculture, forestry and fisheries (daff) for funding the project. special thanks to prof. t. l. nedambale and arc-germplasm conservation and reproductive biotechnologies personnel for their assistance. references agricultural research council (1993). pig production in south africa. irene animal 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(2009). effects in genetic evaluation for semen traits in czech large white and czech landrace boars. czech j. anim. sci. 54: 349-358. xu x, pommier s, arbov t, hutchings b, sotto w, foxcroft g (1998). in vitro maturation and fertilization techniques for assessment of semen quality and boar fertility. j. anim. sci. 76: 3079-3089. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 2 (11), pp. 001-017, november, 2014. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evolutionary characteristics of swine-origin h1n1 influenza virus that infected humans from sporadic to pandemic lei han, wenying lu, yifang han, shuhua li, jianhua yin, jiaxin xie, tong su and guangwen cao* department of epidemiology, second military medical university, shanghai 200433, china. accepted 03 april, 2014 evolutionary process of swine-origin h1n1 influenza a viruses that infected humans from sporadic to pandemic is of high epidemiological significance but still remains obscure. to understand this process, we performed phylogenetic, bootscan, and adaptive evolution analyses using the sequences of the 8 gene segments from swine-origin h1n1 influenza a viruses that infected humans and the reference viruses. classic swine h1n1 viruses occasionally infected humans before 1998. sporadic human infection with the triple-reassortant swine-origin h1n1 viruses was firstly identified in 1998 and has become increasingly frequent since 2005. except genes encoding the neuraminidase and matrix protein of swine influenza viruses of eurasian lineage, other 6 genes of a/h1n1/2009 pandemic strain were most closely linked to those of a/iowa/ceid23/2005(h1n1), a representative swine-origin triple-reassortant virus that infected humans sporadically. potential positive selections acting on the haemagglutinin gene evolved from classic swine h1n1 viruses to the triple-reassortant h1n1 viruses and on the neuraminidase gene evolved from eurasian swine viruses to a/h1n1/2009 pandemic viruses might play a role in cross-species transmission and human infection. surveillance of genetic evolution of influenza a viruses in swine workers might provide useful clues of influenza pandemic. key words: swine, h1n1 influenza virus, evolution, sporadic, pandemic. introduction classic swine influenza a viruses cause sporadic human infection via animals to human transmission, especially in swine workers (myers et al., 2007; olsen et al., 2002). a swine influenza virus was firstly isolated from autopsy lung tissue of human in 1974 (smith et al., 1976). like the infections with avian influenza a viruses, sporadic cases of humans infected with classical swine influenza a viruses in recent decades have rarely resulted in humanto-human spread. pigs have receptors to swine, avian, and human influenza virus strains and act as a “mixing vessel” in which genetic material of the viruses can be exchanged (ito et al., 1998). the reassortment of swine, avian, and human influenza viruses in swine possibly *corresponding author. e-mail: gcao@smmu.edu.cn. tel: +86 21-81871060. fax: +86-21-81871060. results in interspecies transmission of influenza and generation of novel progeny viruses to which humans are immunologically naive and highly susceptible (scholtissek et al., 1985; webster et al., 1992). the emergence of pandemic h1n1/2009 influenza demon-strated that pandemic viruses could be generated in pigs. the novel h1n1/2009, triple-reassortant swine-origin influenza a virus, shows a strong ability to transmit from human to human and has caused influenza a pandemic worldwide since its first emergence in mexico in march 2009 (mmwr morb mortal weekly report, 2009). this novel virus contains 8 gene segments encoding haemagglutinin (ha), nucleoprotein (np), and nonstructural protein (ns) from classic swine influenza a virus of north american lineage, the polymerase basic 2 (pb2) and the polymerase acidic (pa) from avian influenza of north american lineage, the polymerase basic 1 (pb1) from human seasonal influenza a h3n2, and neuraminidase (na) and matrix protein (mp) from swine influenza a of eurasian lineage (babakir-mina et al., 2009; dawood et al., 2009; garten et al., 2009; lu et al., 2009). the reassortment of swine lineages may have occurred years before emergence in humans. however, the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic (smith et al., 2009). between the 1930s and the 1990s, classic swine h1n1 influenza a underwent little change. however, by the late 1990s, multiple strains and subtypes (h1n1, h3n2, and h1n2) of triple-reassortant swine influenza a (h1) viruses whose genomes included combinations of avian, swine, and human influenza virus gene segments had emerged in pigs (olsen, 2002). since 2005, the triple-reassortant swine-origin h1n1 influenza viruses have been frequently reported to be able to infect humans sporadically (gray et al., 2007; newman et al., 2008; shinde et al., 2009). in this study, we retrieved the representative dna sequences of swine-origin h1n1 influenza a viruses that infected humans and reference viruses from genbank and analyzed evolutionary relationships using phylogenetic analysis, bootscan analysis, and adaptive evolution analysis. the reassortment of swine-origin h1n1 with avian influenza virus of north america lineage and human seasonal influenza virus before 2005 in north america was found to be an important step during evolutionary process of a/h1n1/2009 pandemic virus. experimental searching for the sequences of influenza a viruses the genome sequences of the novel a/h1n1/2009 pandemic viruses were downloaded from the ncbi influenza virus resource (http://www.ncbi.nlm.nih.gov/genomes/flu/flu.html). we searched the pubmed database up to march 2009, the time point before the outbreak of novel influenza a viruses, using the searching terms “swine”, “influenza”, and “human”, to identify articles describing sporadic cases of human infection thought to be caused by swine influenza virus strains. reference lists from the articles selected by electronic searching were searched to identify further relevant articles. the 8 gene segments of the corresponding viruses were retrieved from genbank. the sequences of reference influenza a viruses sampled from human, avian and swine in north america, europe and asia during the period 1918 to 2009 were also retrieved from genbank, respectively. phylogenetic analysis sequences of the 8 gene segments of the reference viruses, those of all available swine-origin h1n1 influenza a viruses that infected humans sporadically in north america, and those of selected pandemic a/h1n1/2009 viruses were used for phylogenetic analysis, respectively. sequence alignments for the gene segments of pb2 (full-length 2,281 bp; partial 1,573 bp), pb1 (2,274 bp), pa (full-length 2,151 bp; partial 1,470 bp), ha (1,701 bp), np (1,497 bp), na (1,410 bp), mp (982 bp), and ns (842 bp) and phylogenetic analysis were performed using mega 4.0 software package (tamura et al., 2007). we deleted some of the phylogenetically closely related sequences from reference influenza viruses isolated in the same years and locations and then construct phylogenetic tree. a bootstrap resampling process (1000 replicates) using the neighbor-joining (nj) method was used to assess the robustness of individual nodes on the phylogeny. bootscan analysis full-length genomic sequences containing the pb2, pb1, pa, ha, np, na, mp, and ns gene segments in order of a given influenza isolate were generated using mega 4.0 software. bootscan analyses over full-length sequences were carried out by using simplot software (version 3.5) (200 bp window size, 20 bp step size, 100 bootstrap replicates, using gap-stripped alignments and neighbor-joining analysis) (cavinta et al., 2009). intact sequences of the test viruses, a pandemic a/h1n1/2009 strain (a/wisconsin/629-d01773/2009) and a representative triplereassortant swine-origin strain (a/iowa/ceid23/2005), were compared with those of the previously isolated reference viruses whose gene segments were the closest genetic neighbors of the test viruses. adaptive evolution analysis positive selection drives viral evolution during cross-species transmission and human infection. to investigate whether a/iowa/ceid23/2005 (h1n1) and a/h1n1/2009 viruses were driven by positive selections, the standard mcdonald–kreitman test (http://mkt.uab.es/mkt/mkt.asp) was applied to detect the natural selection (egea et al., 2008). for the analyses of the na and mp gene segments, the clades of the novel a/h1n1/2009 viruses were added into “species 1” and their nearest genetic clades (eurasian swine influenza strains) were added into “species 2”, respectively. for the analysis of the ha, two steps of the selections were performed. firstly, the clade of the novel a/h1n1/2009 viruses was added into “species 1” and the clade of the triple-reassortant swine h1n1 that sporadically infected humans in or after 2005 was added into “species 2”; secondly, the clade of the triple-reassortant swine h1n1 that sporadically infected humans in or after 2005 was added into “species 1” and the clade of the classic swine h1n1 that sporadically infected humans before 1998 was added into “species 2”. the neutrality index (ni) was used to indicate the extent to which the levels of amino acid polymorphism depart from the expected in the neutral model. a ni value < 1 indicated an excess of fixation of non-neutral replacements due to positive selection, 1 means under neutral selection, and > 1 reflected negative selection that prevented the fixation of harmful mutations (egea et al., 2008; ding et al., 2009). results genetic characteristics of swine-origin influenza viruses that sporadically infected humans before 2009 a total of 49 sporadic influenza patients caused by swineorigin influenza viruses were reported in north america. four infections occurred in 1980s and 5 in 1990s. however, 15 infections occurred between december 2005 and february 2009 and 10 of which occurred in or after 2007 (table 1). some nucleotide sequences of the 8 gene segments of swine-origin influenza viruses sporadically infected humans in north america were available in genbank. of the 49 cases, 46 were infected with h1n1 table 1. cases of human infections with swine-origin h1n1 influenza viruses in north america and their corresponding viral gene segments. patient reference year residence subtype access no. of gene segments in genbank pb2 pb1 pa ha np na mp ns 1 myers et al. (2007) 1974 minnesota h1n1 2 myers et al. (2007) 1975 wisconsin h1n1 3 myers et al. (2007) 1975 virginia h1n1 4 myers et al. (2007) 1975 virginia/ny h1n1 5 myers et al. (2007) 1975 tennessee h1n1 6 myers et al. (2007) 1976 missouri h1n1 7 myers et al. (2007) 1976 wisconsin h1n1 8 myers et al. (2007) 1976 wisconsin h1n1 9 myers et al. (2007) 1976 na h1n1 10-23 myers et al. (2007) 1976 new jersey h1n1 24 shinde et al. (2009) 1976 wisconsin h1n1 cy026146 cy026145 cy026144 cy026139 cy026142 cy026141 cy026140 cy026143 25 myers et al. (2007) 1979 texas h1n1 26 myers et al. (2007) 1980 texas h1n1 27 myers et al. (2007) 1982 nevada h1n1 28 shinde et al. (2009) 1988 ohio h1n1 cy024932 cy024931 cy024930 cy024925 cy024928 cy024927 cy024926 cy024929 29 myers et al. (2007) 1988 wisconsin h1n1 30 myers et al. (2007) 1991 maryland h1n1 cy039916 cy039915 cy039914 cy039909 cy039912 cy039911 cy039910 cy039913 31 myers et al. (2007) 1994 wisconsin h1n1 u53159* u53157* u53161* u53163 u53165* u53167* u53169 u53171 32 myers et al. (2007) 1994 wisconsin h1n1 u53158* u53156* u53160* u53162 u53164* u53166 u53168 u53170 33 myers et al. (2007) 1995 minnesota h1n1 34 myers et al. (2007) 1998 wisconsin h1n1 af342824* af342823 af342822* af342821* af342819 af342820 af342818 af342817 35 gray et al. (2007) 2005 iowa h1n1 dq889682 dq889683 dq889684 dq889689 dq889686 dq889687 dq889688 dq889685 36 newman et al.(2008) 2005 wisconsin h1n1 fj986619 37 olsen et al.(2006) 2005 canada h3n2 dq469955 dq469956 dq469957 dq469962 dq469959 dq469960 dq469961 dq469958 38 shinde et al. (2009) 2006 missouri h1n1 39 shinde et al. (2009) 2006 iowa h1n1 fj986618 40 shinde et al. (2009) 2007 ohio h1n1 fj986620 41 shinde et al. (2009) 2007 ohio h1n1 fj986621 42 shinde et al. (2009) 2007 michigan h1n2 fj986622 43 shinde et al. (2009) 2007 illinois h1n1 44 shinde et al. (2009) 2007 iowa h1n1 45 vincent et al. (2009) 2007 ohio hin1 eu604691 eu604692 eu604693 eu604689 eu604694 eu604690 eu604695 eu604696 46 bastien et al. (20098) 2007 canada h3n2 eu399758 eu399757 eu399756 eu399751 eu399754 eu399753 eu399752 eu399755 47 shinde et al. (2009) 2008 minnesota h1n1 48 shinde et al. (2009) 2008 texas h1n1 49 shinde et al. (2009) 2009 iowa h1n1 “-”, not available in genbank; “*”, partial sequences. influenza a. phylogenetic analysis with 30, 27, 27, 35, 28, 28, 30, and 30 full-length sequences of pb2, pb1, pa, ha, np, na, mp, and ns genes of the influenza viruses are performed, respectively (figure 1). all of the 8 gene segments of swine h1n1 viruses that sporadically infected humans in 1970s to 1990s clustered with those of classic swine influenza viruses of north american lineage, except a/wisconsin/10/98, an unreported isolate from a human case in wisconsin in 1998. partial pb2 (1,573 bp) and pa (1,470 bp) segments and other 6 fulllength gene segments of a/wisconsin/10/98 were retrieved from genbank. phylogenetic analyses with the partial sequences indicated that the pb2 and pa segments of a/wisconsin/10/98 were closely related to that of a/iowa/ceid23/2005(h1n1) (figure 2). the fulllength pb1 gene of a/wisconsin/10/98 was closely related to that of a/iowa/ceid23/2005(h1n1) (figure 1). bootscan analysis indicated that a/iowa/ceid23/2005 (h1n1), a representative swine-origin triple-reassortant influenza that sporadically infected humans in north america, shared 92.3 and 93.0% identities in the pb2 and pa genes with avian influenza virus of north american lineage, 94.6% identity in the pb1 gene with human h3n2 virus, and 92.8 to 96.5% identities in another 5 genes with classic swine influenza h1n1 virus of north american lineage, respectively, as shown in table 2. the 8 fragments of a strain isolated in 2007, a/swine/oh/511445/2007(h1n1), were phylogenetically linked to those of a/iowa/ceid23/2005(h1n1) (figure 1). the pb2 and pa segments, pb1 segment, and other 5 gene segments of swine-origin h1n1 viruses that infected humans sporadically between 2005 and 2007 (or 2009) were closely related to those of avian influenza viruses of north american lineage, human seasonal h3n2 influenza viruses, and classic swine influenza viruses of north american lineage, respectively. since human infections with the triple-reassortant swine-origin h1n1 viruses had been increasingly frequent several years before the outbreak of the novel h1n1 influenza in 2009, we hypothesize that introduction of the pb2 and pa genes of avian influenza viruses and the pb1 gene from human seasonal h3n2 viruses facilitate the sporadic infection of swine-origin h1n1 influenza a virus in humans since 2005. evolutionary relationship of swine-origin h1n1 influenza viruses that infected humans from sporadic to pandemic as shown in figure 1, the a/h1n1/2009 pandemic strains clustered in a unique clade for each of the 8 gene segments, respectively. within the clades of the nearest genetic neighbors, the pb2, pb1, pa, ha, np, and ns gene segments of the novel h1n1/2009 strains were most closely linked to those of a/iowa/ceid23/2005 (h1n1) and those of other triple-reassortant swine-origin viruses that sporadically infected humans since 2005 (figures 1 and 2). the pb1, np, and ns were also closely linked to those of a triple-reassortant swine-origin virus a/wisconsin/10/98 (h1n1) that sporadically infected humans. the ha was also closely linked to the gene of triple-reassortant swine viruses a/ohio/01/2007 (h1n1), a/ohio/02/2007 (h1n1), a/wisconsin/87/2005 (h1n1), and a/iowa/01/2006 (h1n1) that sporadically infected humans in north america. the na and mp were closely related to those of influenza a h1n1 viruses circulating in swine populations in eurasia. bootscan analysis showed that the a/h1n1/2009 pandemic strain shared great homology with a/iowa/ceid23/2005 (h1n1) in the pb2 (95.3%), pb1 (95.1%), pa (95.2%), ha (90.3%), np (95.2%), and ns (94.7%) genes. the na and mp genes of the a/h1n1/2009 pandemic strain were most closely related to those of a/swine/spain/50047/2003 (h1n1), a swine virus of eurasian lineage, sharing 90.2 and 94.4% identities, respectively, as shown in figure 3. the genetic components of the swine-origin h1n1 viruses that infected humans from sporadic to pandemic at different phases were shown in figure 4. positive selection drives the evolution of swine-origin h1n1 viruses that infected humans from sporadic to pandemic table 3 shows the results of the mcdonald– kreitman tests. a hi value of 0.817 was observed in the ha segment between the classic swine h1n1 viruses that occasionally infected humans before 1998 and the triplereassortant h1n1 viruses that sporadically infected humans in or after 2005, implying potential positive selection acting on this gene evolved from the classic swine h1n1 viruses to the triple-reassortant h1n1 viruses. however, positive selection was not observed on this gene between the triple-reassortant h1n1 viruses and a/h1n1/2009 pandemic viruses. moreover, a hi value of 0.773 was observed acting on the na gene between a/h1n1/2009 viruses and their nearest genetic neighbors (euroasian swine h1n1 viruses), implying a positive selection was acting on this gene before or during the reassortment of this gene segment into a/h1n1/2009 pandemic viruses. discussion the reassortments between gene segments are essential steps for the evolution of influenza viruses. this study indicated that swine-origin h1n1 influenza viruses that infected humans from sporadic to pandemic have experienced two critical steps of the reassortments. the first critical step is the introduction of the pb2 and pa genes of avian influenza viruses and the pb1 gene from human seasonal h3n2 viruses which might facilitate the pb2 100 a/karasuk/01/2010(h1n1) a/wisconsin/629-d01773/2009(h1n1) 100 57 a/beijing/718/2009(h1n1) a/ontario/rv1273/2005(h3n2) 98 91 100 a/ontario/1252/2007(h3n2) a/swine/oh/511445/2007(h1n1) 99 a/iowa/ceid23/2005(h1n1) 79 a/blue winged teal/la/b228/1986(h1n1) 100 a/duck/alberta/35/1976(h1n1) a/mallard/md/02-184/2002(h1n1) 93 98 a/mallard/minnesota/282/2000(h3n2) a/mallard/ohio/424/1988(h3n2) 90 a/alabama/ur06-0455/2007(h1n1) 100 100 a/albany/20/1978(h1n1) a/aa/huston/1945(h1n1) a/auckland/609/2002(h3n2) 80 a/swine/iowa/1/1976(h1n1) 96 88 a/wisconsin/301/1976(h1n1) a/swine/wisconsin/641/1980(h1n1) 100 a/maryland/12/1991(h1n1) a/swine/kansas/3024/1987(h1n1) 100 84 a/ohio/3559/1988(h1n1) 96 a/swine/bakum/idt1769/2003(h3n2) 100 a/swine/chonburi/niah9469/2004(h1n1) a/swine/cotes darmour/3633 100 a/mallard/netherlands/1/2007(h3n2) a/chicken/israel/1055/2008(h5n1) 100 100 a/cygnus olor/italy/808/2006(h5n1) 2009 a(h1n1) triple-assortment swine influenza north america avian human seasonal north america classic swine euroasian swine euroasian avian 0.02 a/mexico/48n/2009(h1n1) 100 pb1 a/new york/1669/2009(h1n1) 72 a/taiwan/206/2009(h1n1) 99 99 a/ontario/rv1273/2005(h3n2) 100 a/ontario/1252/2007(h3n2) 100 a/swine/oh/511445/2007(h1n1) 97 a/iowa/ceid23/2005(h1n1) 74 a/wisconsin/10/98 (h1n1) 68 a/denmark/35/00(h3n2) 100 a/alabama/ur06-0482/2007(h3n2) 91 a/ashburton/280/2004(h3n2) a/mallard/minnesota/282/2000(h3n2) a/blue-winged teal/ohio/31/1999(h3n2) 100 39 a/mallard/maryland/691/2005(h3n2) 54 a/swine/bakum/5/95(h1n1) 100 a/swine/jena/5/96(h3n2) a/swine/hungary/13509/2007(h3n2) 99 a/buzzard/denmark/6370/06(h5n1) a/chicken/hongkong/fy150/01-mb(h5n1) 100 100 a/goose/guangdong/1/96(h5n1) 100 a/alabama/ur06-0536/2007(h1n1) a/albany/20/1978(h1n1) 100 a/swine/arizona/148/1977(h1n1) 100 a/wisconsin/301/1976(h1n1) a/swine/alberta/56626/03(h1n1) 100 a/maryland/12/1991(h1n1) 92 99 a/ohio/3559/1988(h1n1) 0.01 2009 a(h1n1) triple-assortment swine influenza human seasonal h3n2 north america avian euroasian swine euroasian avian human seasonal h1n1 north america classic swine pa 100 a/taiwan/206/2009(h1n1) a/new york/1669/2009(h1n1) 100 95 a/mexico/48n/2009(h1n1) a/ontario/rv1273/2005(h3n2) 99 100 a/ontario/1252/2007(h3n2) 99 a/swine/oh/511445/2007(h1n1) 100 a/iowa/ceid23/2005(h1n1) 100 a/duck/ny/13152-13/1994(h1n1) a/black duck/carolina/675-075/2004(h3n2) 100 98 a/mallard/md/53/2003(h1n1) a/bar-headed goose/qinghai/12/05(h5n1) 100 a/chicken/hong kong/317.5/2001(h5n1) 100 a/beijing/01/2003(h5n1) 85 100 a/duck/italy/69238/2007(h1n1) a/swine/chonburi/niah9469/2004(h1n1) a/swine/italy/1513-1/98(h1n1) 100 93 a/swine/spain/42386/2002(h3n2) 100 a/wisconsin/301/1976(h1n1) a/swine/wisconsin/464/98(h1n1) a/maryland/12/1991(h1n1) 96 a/ohio/3559/1988(h1n1) 98 99 a/swine/iowa/1/1987(h1n1) 100 a/auckland/583/2000(h3n2) 74 a/boston/10/2008(h3n2) a/albany/1/1970(h3n2) 100 a/alabama/ur06-0455/2007(h1n1) 69 a/arizona/14/1978(h1n1) 0.01 2009 a(h1n1) triple-assortment swine influenza north america avian euroasian avian euroasian swine north america classic swine human seasonal ha 99 a/italy/05/2009(h1n1) a/new york/1669/2009(h1n1) 97 69 a/beijing/718/2009(h1n1) a/swine/oh/511445/2007(h1n1) 100 a/ohio/01/2007(h1n1) 84 a/ohio/02/2007(h1n1) 100 a/wisconsin/87/2005(h1n1) 92 a/iowa/ceid23/2005(h1n1) 100 a/iowa/01/2006(h1n1) a/swine/wisconsin/136/97(h1n1) a/maryland/12/1991(h1n1) 77 95 a/ohio/3559/1988(h1n1) 99 a/wi/4755/1994(h1n1) 95 a/wi/4754/1994(h1n1) 100 a/swine/indiana/1726/1988(h1n1) 60 a/swine/arizona/148/1977(h1n1) 96 a/wisconsin/301/1976(h1n1) a/aa/huston/1945(h1n1) a/ussr/46/1979(h1n1) 100 a/denmark/14/2001(h1n1) 97 a/norway/166/2008(h1n1) 64 100 a/michigan/09/2007(h1n2) 71 a/alabama/ur06-0536/2007(h1n1) 99 a/blue winged teal/tx/27/2002(h1n1) 99 a/mallard/md/390/2002(h1n1) a/black duck/ohio/95/1993(h1n1) 100 a/swine/beijing/21/2008(h1n1) 99 a/swine/france/wvl13/1995(h1n1) 93 a/duck/australia/749/80(h1n1) 63 a/duck/miyagi/66/1977(h1n1) a/swan/hokkaido/55/1996(h1n1) 99 a/anas crecca/spain/1384/2007(h1n1) 94 99 a/goose/italy/296426/2003(h1n1) 100 a/ontario/rv1273/2005(h3n2) a/ontario/1252/2007(h3n2) 2009 a(h1n1) triple-assortment swine influenza north america classic swine human seasonal north america avian euroasian swine euroasian avian 0.05 np a/italy/85/2009(h1n1) 100 a/new york/1682/2009(h1n1) 95 a/beijing/01/2009(h1n1) 93 100 a/ontario/rv1273/2005(h3n2) 99 a/ontario/1252/2007(h3n2) a/swine/oh/511445/2007(h1n1) 82 50 a/iowa/ceid23/2005(h1n1) 97 a/wisconsin/10/98 (h1n1) a/ohio/3559/1988(h1n1) 100 96 a/swine/california/t9001707/1991(h1n1) 39 a/swine/wisconsin/168/97(h1n1) 98 a/maryland/12/1991(h1n1) a/swine/ontaria/2/1981(h1n1) 100 a/swine/iowa/1/1976(h1n1) 100 a/wisconsin/301/1976(h1n1) 100 a/denmark/11/2001(h1n1) a/alabama/ur06-0536/2007(h1n1) 100 a/aa/huston/1945(h1n1) 100 a/iowa/1943(h1n1) 86 a/blue-winged teal/ohio/31/1999(h3n2) 100 a/mallard/maryland/712/2005(h3n2) a/duck/alberta/35/1976(h1n1) 100 100 a/bangladesh/207095/2008(h5n1) a/indonesia/286h/2006(h5n1) a/mallard/marquenterre/z237/1983(h1n1) 95 a/swine/beijing/21/2008(h1n1) 99 a/swine/england/wvl14/1996(h1n1) 99 98 a/swine/italy/839/1989(h1n1) 2009 a(h1n1) triple-assortment swine influenza north america classic swine human seasonal north america avian euroaisan avian euroasian swine 0.01 na 0.1 95 a/italy/05/2009(h1n1) 70 100 a/beijing/719/2009(h1n1) a/new york/1669/2009(h1n1) 97 a/swine/germany/siv05/2007(h1n1) 75 a/swine/denmark/wvl9/1993(h1n1) 96 95 a/swine/germany/vi5698/1995(h1n1) a/swine/cotes darmor/1515/1999(h1n1) a/swine/france/wvl8/1992(h1n1) 78 100 a/hongkong/156/97(h5n1) a/chicken/hong kong/220/97 (h5n1) a/egypt/10217-namru3/2007(h5n1) 62 81 a/anas crecca/spain/1402/2007(h1n1) 88 80 a/swan/hokkaido/55/1996(h1n1) a/chicken/scotland/59(h5n1) a/duck/ohio/194/86 (h1n1) 72 93 a/blue winged teal/tx/27/2002(h1n1) 99 a/alabama/20/2006(h1n1) a/kentucky/1/2005(h1n1) 70 a/iowa/ceid23/2005(h1n1) a/wisconsin/301/1976(h1n1) 96 a/wisconsin/10/98(h1n1) 96 a/maryland/12/1991(h1n1) 98 79 a/ohio/3559/1988(h1n1) 78 a/swine/oh/511445/2007(h1n1) 41 a/swine/north carolina/12869/2003(h1n1) 55 a/wi/4754/1994(h1n1) a/ontario/rv1273/2005(h3n2) 100 a/ontario/1252/2007(h3n2) 2009 a(h1n1) euroasian swine euroaisan avian north america avian human seasonal north america classic swine 81 a/ontario/rv1273/2005(h3n2) mp 95 a/swine/oh/511445/2007(h1n1) 100 a/ontario/1252/2007(h3n2) 97 a/iowa/ceid23/2005(h1n1) 85 a/wisconsin/10/98 (h1n1) 73 a/swine/minnesota/593/99 (h3n2) a/maryland/12/1991(h1n1) a/ohio/3559/1988(h1n1) 80 100 a/wi/4755/1994(h1n1) 67 a/wi/4754/1994(h1n1) 72 a/swine/indiana/1726/1988(h1n1) a/wisconsin/301/1976(h1n1) 99 a/swine/arizona/148/1977(h1n1) a/sw/shizuoka/120/97(h3n2) a/baylor/11735/82(h1n1) 100 a/switzerland/5389/95 (h1n1) 99 99 a/alabama/ur06-0536/2007(h1n1) 70 a/chicken/ny/11602-12/1998(h3n2) 93 a/mallard/ohio/424/1988(h3n2) a/mallard/md/168/2002(h1n1) 99 a/duck/italy/69238/2007(h1n1) 98 100 a/avian/italy/1485/1997(h5n1) a/chicken/hong kong/14/1976(h1n1) a/swine/italy/v.147/1981(h1n1) 99 a/swine/brno/1/1992(h1n1) 100 98 a/swine/schleswig-holstein/1/1993(h1n1) 68 a/swine/england/wvl10/1993(h1n1) 73 a/taiwan/t0724/2009(h1n1) a/new york/1669/2009(h1n1) 100 91 a/italy/05/2009(h1n1) triple-assortment swine influenza north america classic swine human seasonal north america avian euroaisan avian euroasian swine 2009 a(h1n1) 0.01 a/stockholm/34/2009(h1n1) ns 100 a/beijing/720/2009(h1n1) 80 a/new york/1669/2009(h1n1) 98 a/ontario/rv1273/2005(h3n2) 97 100 58 a/ontario/1252/2007(h3n2) a/swine/oh/511445/2007(h1n1) 78 a/iowa/ceid23/2005(h1n1) 95 a/wisconsin/10/98 (h1n1) 97 a/swine/california/t9001707/1991(h1n1) a/maryland/12/1991(h1n1) 65 a/wi/4754/1994(h1n1) 99 99 a/swine/indiana/1726/1988(h1n1) 69 a/wi/4755/1994(h1n1) 100 a/swine/iowa/1/1985(h1n1) 95 a/ohio/3559/1988(h1n1) a/swine/niigata/1/1977(h1n1) 100 a/wisconsin/301/1976(h1n1) 100 a/denmark/122/2008(h1n1) 51 a/california/ur06-0479/2007(h1n1) 100 a/united kingdom/157/1982(h1n1) a/hong kong/117/77(h1n1) 74 a/black duck/ohio/95/1993(h1n1) 100 a/mallard/ohio/424/1988(h3n2) a/mallard/maryland/802/2007(h5n1) 31 a/mallard/sweden/s90780/2005(h1n1) 99 75 a/turkey/england/1969(h3n2) a/chicken/scotland/59(h5n1) 91 a/swine/cotes d armor/1482/99(h1n1) a/swine/italy/1513-1/98(h1n1) 100 a/swine/england/wvl10/1993(h1n1) 59 0.02 2009 a(h1n1) triple-assortment swine influenza north america classic swine human seasonal north america avian euroaisan avian euroasian swine figure 1. phylogentic analyses of the 8 full-length gene segments (pb2, pb1, pa, ha, np, na, mp, and ns) of the selected pandemic a/h1n1/2009 strains, all available swine-origin influenza viruses that infected humans sporadically, and the selected reference influenza a viruses. ▲ indicates the strains infected humans sporadically. pb2 67 a/wisconsin/629-d01773/2009(h1n1) 100 a/beijing/718/2009(h1n1) 100 a/karasuk/01/2010(h1n1) 89 a/iowa/ceid23/2005(h1n1) 78 a/wisconsin/10/98 (h1n1) 94 a/blue winged teal/la/b228/1986(h1n1) 100 a/duck/alberta/35/1976(h1n1) a/mallard/md/02-184/2002(h1n1) 94 98 a/mallard/minnesota/282/2000(h3n2) a/mallard/ohio/424/1988(h3n2) 83 a/alabama/ur06-0455/2007(h1n1) 100 a/albany/20/1978(h1n1) 100 a/aa/huston/1945(h1n1) a/auckland/609/2002(h3n2) 77 a/swine/iowa/1/1976(h1n1) 89 84 a/wisconsin/301/1976(h1n1) a/swine/wisconsin/641/1980(h1n1) 100 a/maryland/12/1991(h1n1) a/swine/kansas/3024/1987(h1n1) 100 76 a/ohio/3559/1988(h1n1) 96 a/swine/bakum/idt1769/2003(h3n2) 100 a/swine/chonburi/niah9469/2004(h1n1) a/swine/cotes darmour/3633 100 a/mallard/netherlands/1/2007(h3n2) a/chicken/israel/1055/2008(h5n1) 100 100 a/cygnus olor/italy/808/2006(h5n1) 0.02 2009 a(h1n1) north america avian human seasonal north america classic swine euroasian swine euroasian avian pa 77 a/new york/1669/2009(h1n1) 100 a/mexico/48n/2009(h1n1) 100 a/taiwan/206/2009(h1n1) a/iowa/ceid23/2005(h1n1) 94 98 a/wisconsin/10/98 (h1n1) 78 a/black duck/carolina/675-075/2004(h3n2) a/mallard/md/53/2003(h1n1) 100 100 a/duck/ny/13152-13/1994(h1n1) 99 a/mallard/ohio/48/1986(h3n2) a/hongkong/156/97(h5n1) 100 a/hong kong/378.1/2001(h5n1) 64 a/bar-headed goose/qinghai/12/05(h5n1) 100 a/chicken/hong kong/317.5/2001(h5n1) 100 a/beijing/01/2003(h5n1) 79 100 a/duck/italy/69238/2007(h1n1) a/swine/belgium/wvl1/1979(h1n1) a/swine/italy/1513-1/98(h1n1) 100 a/swine/chonburi/niah9469/2004(h1n1) 93 50 a/swine/spain/42386/2002(h3n2) 97 a/ohio/3559/1988(h1n1) 96 a/swine/iowa/1/1987(h1n1) 92 a/maryland/12/1991(h1n1) 100 a/swine/wisconsin/464/98(h1n1) a/wisconsin/301/1976(h1n1) a/alaska/1935(h1n1) 83 a/alabama/ur06-0455/2007(h1n1) 100 a/arizona/14/1978(h1n1) 68 a/albany/1/1970(h3n2) 64 a/auckland/583/2000(h3n2) 100 a/boston/10/2008(h3n2) 0.01 2009 a(h1n1) north america avian euroasian avian euroasian swine north america classic swine human seasonal figure 2. phylogentic analyses of the partial pb2 (1,573 bp) and pa (1,470 bp) sequences of the selected pandemic a/h1n1/2009 strains, swine-origin h1n1 influenza viruses that infected humans sporadically, and the selected reference influenza a viruses. ▲ indicates the strains infected humans sporadically. table 2. sequence identities of the 8 gene segments of a representative swine-origin triple reassortant virus that infected humans in 2005 with those of selected reference strains using bootscan analysis. reference influenza virus strains identities (%) with a/iowa/ceid23/2005 (h1n1) full pb2 pb1 pa ha np na mp ns a/swine/spain/50047/2003 (h1n1) 80.6 81.0 83.4 84.3 64.5 80.9 77.2 85.5 78.9 a/swine/wisconsin/1915/1988 (h1n1) 86. 8 81.1 75.5 78.9 92.9 94.2 93.1 96.2 94.9 a/ohio/3559/1988 (h1n1)* 86.9 81.4 75.6 78.5 92.8 96.1 92.8 96.5 93.6 a/mallard/ohio/48/1986 (h3n2) 83.9 92.3 86.8 92.7 29.9 80.8 29.3 89.7 62.7 a/turkey/ontario/84/1983 (h5n1) 83.4 90.0 86.6 93.0 41.4 80.5 78.0 89.4 83.3 a/yucatan/me6057/2003 (h3n2)* 73.5 81.4 94.6 79.5 30.0 79.9 2.9 85.9 79.5 * infected humans sporadically. figure 3. bootscan analysis for full-length of a/h1n1/2009 pandemic strain with those of 3 representative reference strains. sporadic infection of swine-origin h1n1 influenza a virus in humans since 2005. the other step is the reassortment with the na and mp gene segments of swine h1n1 viruses of eurasian lineage which might enable the triplereassortant swine-origin h1n1 viruses to cause the pandemic in 2009. stochastic mutation in nucleotides and directional immune selection may play an important role in determining the direction of the evolution of influenza viruses. the reassortments between gene segments are essential steps for the evolution of influenza viruses. this study shows that swine-origin h1n1 influenza viruses that infected humans from sporadic to pandemic have experienced two critical steps of the reassortments. the gene segments of the swine h1n1 viruses that occasionally infected persons who ever exposed to pigs in north america before 1998 were phylogenetically linked to those of classic swine h1n1 influenza viruses. phylogenetic analyses with the partial pb2 and pa gene segments and the full-length pb1 gene segment of a/wisconsin/10/98 indicate that the triple-reassortant h1n1 virus started to infect humans in late 1990s. the swine-origin h1n1 viruses that sporadically infected humans between 2005 and 2009 were of the triplereassortant viruses (figure 4). the triple-reassortant virus may be considered as “backbone frame” of the a/h1n1/2009 pandemic strain or “intermediate” in evolutionary process of the a/h1n1/2009 pandemic swine h1n1 virus that sporadically triple -reassortant h1n1 virus infected humans in north america that sporadically infected before 1998 humans in no rth america in 1998 and between 2005 and 2009 pb2 pb2 pb1 pb1 pa pa ha ha np np na na m m pns pns avian, north american lineage human seasonal h3n2 classic swine, north american lineage eurasian swine lineage the novel h1n1 virus caused the pandemic in 2009 pb2 pb1 pa ha np na m pns figure 4. the genetic components of the swine-origin h1n1 viruses that infected humans from sporadic to pandemic at different phases. table 3. mcdonald-kreitman test for the natural selection of 3 gene segments of novel influenza a/h1n1/2009 viruses. gene neutral non-neutral neutrality proportion of adaptive p polymorphism divergence polymorphism divergence index (ni) substitutions ha* 206 13.30 89 7.02 0.817 0.182 0.677 ha** 126 32.79 71 17.15 1.077 -0.077 0.822 na 797 25.37 97 16.16 0.773 0.226 0.450 mp 50 14.60 15 4.01 1.091 -0.091 0.890 *the clade of the triple-reassortant swine h1n1 that sporadically infected humans in or after 2005 was added into “species 1” and the clade of the classic swine h1n1 that sporadically infected humans before 1998 was added into “species 2”; ** the clade of the novel a/h1n1/2009 pandemic strains was added into “species 1” and the clade of the triple-reassortant swine h1n1 that sporadically infected humans in or after 2005 was added into “species 2”. strain. as shown in figure 3, the h1n1/2009 pandemic strain shared great homology with a representative triplereassortant strain, a/iowa/ceid23/2005 (h1n1), in the pb2, pb1, pa, ha, np, and ns gene segments. the reassortment with the na and mp gene segments of swine h1n1 viruses of eurasian lineage might enable the triple-reassortant swine-origin h1n1 viruses to cause the pandemic. since the ha protein is critical for binding to cellular receptors and fusion of the viral and endosomal membranes (neumann et al., 2009), we investigated the natural selection acting on this gene of the swine-origin viruses that infected humans from sporadic to pandemic. a positive selection might exist during the evolution of the ha gene from the classic swine h1n1 viruses that sporadically infected humans before 1998 to the triplereassortant h1n1 viruses that sporadically infected humans from 2005 to 2009, possibly driving the adaptation of the classic swine h1n1 viruses to human at this stage. a positive selection might also act on the na gene segment from the swine h1n1 virus of eurasian lineage to the a/h1n1/2009 pandemic strain. the na protein facilitates virus release from infected cells by removing sialic acids from cellular and viral ha and na proteins (neumann et al., 2009). alterations of the na are needed for the emergence of pandemic influenza viruses (uhlendorff et al., 2009). the positive selection of the na gene might drive the adaptation of this gene segment to human. thus, in addition to the reassortment between gene segments of influenza a viruses of different lineages, nucleotide variations within a given gene segment and subsequent selection might play a role in the evolutionary process of swine-origin h1n1 viruses infected humans from sporadic to pandemic. however, there are population bottlenecks that might limit evidential power of the mcdonald-kreitman tests. this study has a number of limitations. firstly, the viral sequences of the swine-origin viruses used in this study were mostly from the united states because sporadic infections with classic swine h1n1 before 1998 and with triple-reassortant swine h1n1 influenza a viruses since 2005 in persons with exposure to pigs were welldocumented in this country, not in other countries. secondly, the patients with slight symptom and syndrome might not be reported, possibly resulting in underestimation of human infections with swine-origin h1n1 viruses before the outbreak. thirdly, not all sequences from the reported cases were available in genbank, which might have resulted in bias of the analyses based on the incomplete data. in conclusion, this study clearly characterized the evolutionary process of swine-origin h1n1 influenza viruses that infected humans from sporadic to pandemic. in addition to the present knowledge, this study indicates that a representative triple-reassortant strain with intact sequence data of the 8 gene segments, a/iowa/ ceid23/2005 (h1n1), may be a suitable “intermediate” in the evolutionary process of a/h1n1/2009 pandemic viruses. monitoring the evolutionary steps of animal-origin influenza a viruses that occasionally infect humans from swine workers is of epidemiological significance for the prediction of novel influenza pandemic. acknowledgements this work is supported by grant 06g65 (to guangwen cao) from the general logistics of the people’s liberation army, people’s republic of china. the 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humans. n. engl. j. med., 360: 2605-2615. ding n, wu n, xu q, chen k, zhang c (2009). molecular evolution of novel swine-origin a/h1n1 influenza viruses among and before human. virus genes., 39: 293-300. egea r, casillas s, barbadilla a (2008). standard and generalized mcdonald-kreitman test: a website to detect selection by comparing different classes of dna sites. nucleic. acids. res., 36: w157-162. garten rj, davis ct, russell ca, shu b, lindstrom s, balish a, gray gc, mccarthy t, capuano aw, setterquist sf, olsen cw, alavanja mc (2007). swine workers and swine influenza virus infections. emerg. infect. dis., 13: 1871-1878. ito t, couceiro jn, kelm s, baum lg, krauss s, castrucci mr, donatelli i, kida h, paulson jc, webster, rg, kawaoka y (1998). molecular basis for the generation in pigs of influenza a viruses with pandemic potential. j. virol., 72: 7367-7373. lu j, liu d, liu w, shi t, tong y, cao w (2009). genetic stability and linkage analysis of the 2009 influenza a(h1n1) virus 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(h1n1) virus infection mexico, march-april 2009. mmwr morb mortal wkly rep., 58: 467470. scholtissek c, burger h, kistner o, shortridge kf (1985). the nucleoprotein as a possible major factor in determining host specificity of influenza h3n2 viruses. virology, 147: 287-294. shinde v, bridges cb, uyeki tm, shu b, balish a, xu x, lindstrom s, gubareva lv, deyde v, garten rj, harris m, gerber s, vagasky s, smith f, pascoe n, martin k, dufficy d, ritger k, conover c, quinlisk p, klimov a, bresee js, finelli l (2009). triple-reassortant swine influenza a (h1) in humans in the united states, 2005-2009. n engl. j. med., 360: 2616-2625. smith gj, vijaykrishna d, bahl j, lycett sj, worobey, m, pybus og, ma sk, cheung cl, raghwani j, bhatt s, peiris js, guan y, rambaut a (2009). origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic. nature, 459: 1122-1125. smith tf, burgert eo, jr dowdle wr, noble gr, campbell rj, van scoy re (1976). isolation of swine influenza virus from autopsy lung tissue of man. n engl j. med., 294: 708-710. tamura k, dudley j, nei m, kumar s (2007) mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol., 24: 1596-1599. uhlendorff j, matrosovich t, klenk hd, matrosovich m (2009). functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses. arch. virol., 154: 945-957. vincent al, swenson sl, lager km, gauger pc, loiacono c, zhang y (2009). characterization of an influenza a virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the united states. vet. microbiol., 137: 51-59. webster rg, bean wj, gorman ot, chambers tm, kawaoka y (1992). evolution and ecology of influenza a viruses. microbiol. rev., 56: 152-179. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (2), pp. 001-006, february, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper assessment of probiotic properties of lactobacillus plantarum zlp001 isolated from gastrointestinal tract of weaning pigs jing wang, haifeng ji*, dongyan zhang, hui liu, sixin wang, dacong shan and yamin wang institute of animal husbandry and veterinary medicine, beijing academy of agriculture and forestry sciences, shu guang hua yuan middle road no. 9, haidian district, beijing 100097. accepted 09 october, 2017 the probiotic potential of lactobacillus plantarum zlp001, isolated from the gastrointestinal tract of a healthy weaning piglet, was assessed as a probiotic in vitro and in vivo. the survival rate of l. plantarum zlp001 when cultured in simulated gastric fluid with ph 2.0 and 3.0 and subsequent in intestinal fluid ph 8.0 was determined and the results show that this strain had resistance to ph 3.0 simulated gastric fluid and subsequent ph 8.0 intestinal fluid. bile salt resistance of this strain was examined in deman, rogosa and sharpe (mrs) broth containing oxgall concentration from 0.1% to 0.5%. the strain showed 85.3 and 61.4% bile tolerance under 0.1 and 0.3% bile salt, respectively, and was inhibited in 0.5% bile salt (9.4%). the sizes of the inhibitory zone of this strain against staphylococcus aureus, escherichia coli, and salmonella enterica were also determined. the result shows that this strain had high antimicrobial activity against selected pathogens. the probiotic strain was administered through the feed to 35-day old weaned piglets to estimate the effect of l. plantarum zlp001 on the growth performance. 80 piglets were selected and divided into five groups comprising of negative control without any supplementation, three treatments of different l. plantarum zlp001 levels (5.9×10 7 , 3.5×10 8 , and 1.8×10 9 cfu/g of diet), and positive control with antibiotic treatment (chlorotetracycline, 0.3% of diet). the results of feeding trial showed that l. plantarum zlp001 supplementation enhanced feed conversation rates in piglets compared with control. the present study implies that the strain l. plantarum zlp001 was considered to be a potential probiotic for weaned piglets. key words: lactobacillus plantarum, probiotic properties, in vitro assessment, in vivo trial. introduction piglets are faced with many new stressors during the post-weaning period which can lead to the risk of diarrhea, reduced growth rate, changes in gut morphology and microbial population numbers and an increased susceptibility to disease and death (hampson, 1994). antibiotics used as feed additives in pig production *corresponding author. e-mail: jhf309@sohu.com. tel: +86 10 51 503 865. fax: +86 10 88 433 070. abbreviations: mrs, deman, rogosa and sharpe; rrna, ribosomal ribonucleic acid; cfu, colony-forming units; od, optical density; bw, body weight. once became virtually universal owing to its obvious effects on growth promotion and diseases prevention. along with the concerns about antibiotic residues in animal products increase, the potential exists for the implementation of a complete ban of the use of antibiotics in animal feed all over the world. as a consequence, the development of alternatives to antibiotics is receiving considerable attention (turner et al., 2001). probiotics are described as ‘live microorganisms which, when administered in adequate numbers, confer a health benefit on the host’ (fao/who, 2001). direct-fed probiotic preparation plays an important role in the improving of gut microflora balance and consequently in the prevention of infections and better health condition. so, the addition of probiotics to the diet has gained increasing importance in pig nutrition in recent years and can become one potential alternative to antibiotic. the lactobacilli are major component of the gastrointestinal flora. besides other bacteria, lactobacilli establish early in the piglet intestine and remain there as a predominant part of the intestinal bacterial community throughout the pig’s lifetime (leser et al., 2002). the functional effects of lactobacilli such as protection against infections, stimulation of immune system, reduction of incidence of diarrhea have been demonstrated in many studies (ouwehand et al., 2002; koninkx and malago, 2008). however, not all strains of lactobacilli are equally resistant to the environment in the gastrointestinal tract and antimicrobial activity also varies between strains. more studies are still needed to evaluate the properties and effects on production performance of lactobacilli that comes from different sources and different strains. the main objective of this work was to examine the in vitro survival in simulated gastrointestinal condition, bile salt, and the ability to inhibit common porcine pathogens like escherichia coli, staphylococcus aureus and salmonella enterica. of lactobacillus plantarum zlp001 isolated from gastrointestinal mucosa of a healthy weaned piglet in our laboratory. in addition, the effects of the probiotic strain in vivo on the average daily weight gain, average daily feed intake, and feed conversion ratio in piglets were also determined. materials and methods in vitro assessment bacterial strain a probiotic lactic acid bacterial strain, l. plantarum zlp001 was originally isolated from the gastrointestinal tract of a healthy weaning piglet in our laboratory. strain was identified through standard morphological, biochemical, physiological tests and by 16s ribosomal ribonucleic acid (rrna) gene sequence analysis by the china center of industrial culture collection. stock cultures in deman, rogosa and sharpe (mrs) (oxoid, uk) broth were mixed with 20% sterilized glycerol (v/v) at a concentration of 4:1 and stored at -80°c. resistance to simulated gastric and intestinal fluids simulated gastric and intestinal fluids were prepared as described by fernández et al. (2003). simulated gastric fluid was prepared fresh daily by suspending 0.35 g of pepsin in 100 ml of 0.2% saline. the ph was adjusted to 2.0 or 3.0 with concentrated hydrochloric acid, and the fluid was sterilized by filtering through 0.22 um filter. simulated small intestinal fluid was prepared by suspending 0.1 g of trypsin and 1.8 g of bile salts in 100 ml sterile solution of 1.1 g of sodium bicarbonate and 0.2 g of sodium chloride. the ph was adjusted to 8.0 with 0.5 m sodium hydroxide. this solution was sterilized by filtering through 0.45 um filter. the bacterial strains were inoculated at 10% into the simulated gastric fluid at ph 2.0. the mixtures were mixed for 10 s and incubated at 37°c under anaerobic conditions and agitation to simulate peristalsis. aliquots of this suspension were taken at 3 h, and the total viable count of the bacteria was determined. then, the medium was removed by centrifugation, substituted with simulated intestinal fluid and incubated at 37°c anaerobically under agitation for additional 3 h. determination of colony-forming units (cfu) was performed on mrs agar by three day incubation (37°c) in anaerobiosis. bile salt resistance the effect of bile salts on the growth rate of l. plantarum zlp001 was determined by using the method described by lin et al. (2007). the mrs broth supplemented with 0.1, 0.3, and 0.5% (w/v) oxgall, respectively (oxgall bile b8381, sigma) and without oxgall were freshly prepared. the overnight suspensions of the l. plantarum zlp001 were inoculated (1%) into mrs broth. bacterial cell in the culture broth was measured by reading the optical density (od) at 620 nm after 4 h incubation at 37°c. at this time point, l. plantarum zlp001 strain growing in the mrs broth without oxgall was still in the logarithmic growth phase. the percentage of the bile tolerance was calculated by comparison of the od values of the bacteria cultures in mrs broth with oxgall to those in mrs broth without oxgall. antimicrobial activity assay the agar diffusion assay was used to test the antimicrobial activity of the suspending solutions of l. plantarum zlp001 according to the literature (ouoba et al., 2007) with some modifications. overnight cultures of the indicator microorganisms (0.1 ml, approximately 10 9 cfu/ml) were spread on 15 ml mrs agar in a square petri dish. a total of 100 µl suspending solutions were added into the oxford cup (a stainless cylinder, outer diameter 7.8 ± 0.1 mm, inner diameter 6.0 ± 0.1 mm and height 10.0 ± 0.1 mm) which was placed on the surface of the agar. the size of the clear zone around the cup (including that of the ‘oxford cup’ 7.8 mm) was measured and the results were reported in millimeter (mm). the antibacterial activities were classified as none (-), weak (+), middle (++), and strong (+++) inhibition, respectively, according to the diameters of inhibition zone of <5, >5, >10 and >15 mm (lin et al., 2006). the experiment was performed in triplicate. in vivo trials pigs and diets 80 piglets (large white × big white) with 8.35±0.79 kg initial body weight (bw) were selected from beijing jingdongyu farm (beijing city, china). the pigs were weaned at 35 days of age and randomly allotted to five groups by initial bw. there were four replicates per treatment and four pigs per pen. each pen was 1.65× 1.45 m 2 with mesh floor, a feeder and a water nipple. the pig barn was maintained at 25 to 28°c. all pigs had free access to feed and water throughout the four-week feeding trial. piglets were weighed and the feed intake was recorded every week in order to calculate average daily weight gain, average daily feed intake and feed conversion ratio. the basal diet (table 1) mainly contained maize and soybean meal, and the nutrient contents met or exceeded nutrient requirements recommended by nrc (1998). the dietary treatments consisted of the basal diet with no additives, the basal diet with antibiotic (chlorotetracycline, 0.3% of diet), and the basal diet with freeze dried l. plantarum zlp001 at 5.9×10 7 , 3.5×10 8 , and 1.8×10 9 cfu/g of diet. chemical analysis the diet samples were placed in a forced-air oven at 65°c for 48 to table 1. ingredient and composition of the basal diet. ingredient value maize (g/kg) 600 soybean meal (g/kg) 150 extruded soybean (g/kg) 60 fish meal (g/kg) 50 wheat bran (g/kg) 50 whey (g/kg) 50 premix 1 40 chemical composition digestible energy (mj/kg) 13.76 crude protein (g/kg) 182.9 lysine (g/kg) 11.8 methionine (g/kg) 4.2 calcium (g/kg) 9.8 total phosphorus (g/kg) 7.4 each kg of complete feed contains: vitamin a, 11,000 iu; vitamin d3, 3,300 iu; vitamin e, 16.5 mg; menadione, 3 mg; riboflavin, 7 mg; pantothenic acid, 10 mg; niacin, 50 mg; vitamin b12, 0.02 mg; mn, 100 mg; fe, 30 mg; zn, 80 mg; cu, 3 mg; i, 0.75 mg; se, 0.30 mg. 72 h. after drying, the samples were ground through a 0.42 mm screen in a mill and analyzed for crude protein, calcium and total phosphorus by the association of official analytical chemists method (aoac, 1992). the gross energy content was determined by total combustion of the sample with an adiabatic bomb calorimeter (model parr1281, parr instrument corp., us). the amino acid content of diet was determined by high performance liquid chromatography (hitachi l-8800 amino acid analyzer, tokyo, japan) according to the method of wang et al. (2006). statistical analysis in the in vitro assessment, the final results were expressed as the mean and standard deviation of three determinations. statistical analysis of the obtained data in the feeding trial was carried out using the proc general linear model procedure of sas (1997). the pen was considered the experimental unit. differences among means were tested using tukey’s test (zar, 1984) and probability of p<0.05 was used to denote significance. results and discussion resistance to simulated gastric and intestinal fluids probiotics delivered through the feed system have to firstly survive during transit through the upper gastrointestinal tract (huang and adams, 2004). the survival rate of l. plantarum zlp001 strains when cultured in simulated gastric fluid with ph of 2.0 and 3.0 are shown in table 2. the gastric fluid with ph 2.0 was inhibitory for this strain, as the number of cfu was reduced nearly 95% during 3 h incubation. at ph 3.0, the strain showed 81.28% survival rate after incubation after same time. the finding that the number of cfu was decreased faster at ph 2.0 gastric fluid than at ph 3.0 was similar with the findings of hacin et al. (2008) with the lactobacilli isolates from weaned piglets’ mucosa. incubation of the strain in gastric fluid with ph 2.0 and 3.0 for 3 h was an attempt to simulate the conditions that a probiotic would have to survive as it passes through the stomach of a pig. the fact that the strain kept more than 80% viable counts after 3 h of incubation suggest that a reasonable percentage of the strain should survive passage through the harsh environment of the pig stomach. another barrier that probiotic bacteria must survive is passage through the small intestine (huang and adams, 2004). the treatment with simulated intestinal fluid, which followed the incubation in gastric fluid, resulted in decreased viability when the cells were previously exposed to low ph 2 while lightly growth was observed under ph 3 conditions (table 2). the survival of strain l. plantarum zlp001 was considered satisfactory since it was comparable to the results obtained with strain l. gasseri k7 which was shown to survive well in vivo in piglets (rogelj and matijaš , 2006). bile salt resistance during passage through the gastrointestinal tract, ingested bacteria must face the challenge of toxic compounds such as bile. tolerance of bile salts seems table 2. resistance of l. plantarum zlp001 to simulated gastric and intestinal fluids. strain initial concentration ph of simulated gastric fluid concentration after 3 h concentration after 3 h (log cfu/ml) in gastric fluid (log cfu/ml) in intestinal fluid (log cfu/ml) l. plantarum zlp001 10.26±0.15 2 9.02±0.08 8.77±0.21 3 10.17±0.12 10.29±0.04 presented values are means of triplicate determinations ± standard deviation from the mean. to be an important character in strains envisaged as probiotics to grow and survive in the upper small intestine (de smet et al., 1995). growth of the strain was examined in mrs broth containing oxgall concentration from 0.1 to 0.5% in this experiment. a wide variation in survival was observed when the strains were subjected to different concentration of bile salt (table 3). after 4 h incubation, the strain showed 85.3 and 61.4% bile tolerance under 0.1 and 0.3% bile salt respectively, and was inhibited dramatically in 0.5% bile salt condition (9.4%) . the results of bile salt tolerance for l. plantarum were wide different owing to the different sources of strain (jacobsen et al., 1999) and different methods used to estimate the bile tolerance (cebeci and gürakan, 2003). antimicrobial activity one of the major probiotic properties for probiotic lactobacilli is its inhibitory effect on the growth of pathogenic bacteria. l. plantarum is known to produce antimicrobial substances, e.g. plantaricin, that are active against certain pathogens (cebeci and gürakan, 2003). the size of the inhibitory zone for the strain against the three indicator pathogens is shown in table 4. l. plantarum zlp001 strain was show high inhibitory activity against the gram-positive bacterium s. aureus and the gram-negative bacteria, e. coli and s. enterica. these bacteria are known as the main pathogens causing diarrhea in piglets (asai et al., 2002; fairbrother et al., 2005). therefore, the strain isolated in this experiment was thought to have potential to compete with pathogens and improve the balance of the microflora in the gastro-intestinal tract. the production of antimicrobials is considered one of the major mechanisms through which probiotics function and consequently is also one of the principle criteria for strain selection when screening potential probiotics (chang et al., 2001; hong et al., 2005). it will be good to undertake further studies on the antimicrobial activity of the l. plantarum zlp001 in order to isolate, characterize, and identify the antimicrobial compounds produced against the pathogens. performance trials the in vitro techniques for screening l. plantarum as potential probiotics are far from the requirements in practice for farmers. the strain for the in vivo trial was selected primarily for the tolerance to low ph, bile salts and antibiotics, as well as antimicrobial properties. the effects of dietary l. plantarum zlp001 supplementation on performance of weanling pigs are shown in table 5. over the four-week feeding trial, piglets of antibiotic group consumed significant higher feeds than the probiotics groups (p<0.05) and no additive group; this is similar to the results of chang et al. (2001) and it seemed to be positively affect the live-weight gain in them. differences between probiotic groups and no additive group in the mean daily feed intake were not significant. the piglets that received diets containing l. plantarum zlp001 supplements had the same live-weight gain daily compared with the antibiotic group ( p>0.05) and was significantly higher than the no additive group (p<0.05) except 1.8×10 9 cfu/g of diet supplementation diet. the result was different from earlier work with dose level dependence of lactobacillus in broiler chickens performance (choi et al., 2004). the difference may be due to not only the different strain and dose level of lactobacillus but also to the animal physical condition and environment. the feed conversion ratio in probiotic groups were significantly different (p<0.05) from those of the antibiotic and no additive groups. the rates in probiotics were better than those of the control groups. these results indicate that in terms of feed consumption, the probiotic groups consumed 10.6, 9.3, and 4.4% less than the antibiotic group to achieve the same weight, respectively. similar observations were made by chang et al. (2001) and francisco et al. (1995) that selected probiotic strains had increasing effect on feed conversion rate in piglets. during the feed trials, some sporadic case of diarrhea occurred in 28.5%, which corresponds to the study of the incidence in table 3. effect of bile salt on the growth of the selected l. plantarum zlp001. strain od 620 nm after 4 h incubation percentage of without bile salt with bile salt tolerance (%) 0.1% 0.806 85.3 l. plantarum zlp001 0.945 0.3% 0.580 61.4 0.5% 0.089 9.4 table 4. the inhibition zone of l. plantarum zlp001 when incubated with 3 indicator pathogens. strain inhibitory zone (mm) e. coli s. enterica s. aureus l. plantarum zlp001 21.58±0.4 19.88±0.5 21.00±0.8 values are the means and standard deviations of triplicate determinations; ±, standard deviation from the mean. table 5. effects on the growth performance in piglets by feeding of l. plantarum zlp001. parameter no additive antibiotic l. plantarum zlp001 (cfu/g of diet) sem p 5.9×10 7 3.5×10 8 1.8×10 9 average daily feed intake (g/d) 660 b 734 a 685 b 662 b 674 b 21.2 0.032 average daily gain (g/d) 357 c 390 a 402 a 385 ab 374 bc 10.7 0.018 feed conversion ratio 1.85 a 1.88 a 1.70 c 1.72 c 1.80 b 0.021 0.040 sem, standard error of the mean; mean within a row, lacking a common superscript letter differ (p<0.05). weaning piglets as 20 to 47% (backstrom, 1973). at the end of the trials, no death was recorded for the piglets. conclusion the study proved l. plantarum zlp001 for probiotic use in vitro by simulated gut environment tolerance tests and antimicrobial activity assay. the in vivo trial also encouraged that the use of l. plantarum zlp001 can improve feed conversion ratio and live-weight gain of piglets. the results show that l. plantarum zlp001 is a promising alternative to antibiotics for use as a feed additive in piglet diets. however, the exact mechanism through which l. plantarum may play an important role in the gastrointestinal tract remains uncertain, and further research is also needed to 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this article. full length research paper comparative utilization of two sources of expellerextruded soybean meal as a replacement for on-farm processed soybean in diets of growing-finishing pigs adesehinwa a. o. k. institute of agricultural research and training, obafemi awolowo university, p.m.b. 5029, moor plantation, ibadan. nigeria. e-mail: aokadesehinwa@gmail.com accepted 03 october, 2013 thirty pigs averaging 30 ± 1.1 kg were randomly assigned by origin and weight to one of three dietary treatments in a randomized complete block to determine the comparative utilization of two sources of expeller-extruded soybean meal as a replacement for on-farm processed soybean in diets of growing-finishing pigs. the result revealed that pigs fed diets containing the expelled soybean meal (esm) had decreased (p<0.05) average daily gain (adg) compared to pigs fed the on-farm processed soybean resulting in between 13-17% reduction in the adg. the expelled soybean meal samples resulted in comparable (p>0.05) gains even though slight numerical differences were observed. feed efficiency was 18 to 24% significantly (p<0.05) poorer for pigs fed the expelled soybean meal diets compared to the improvement observed in the feed conversion of pigs when fed the on-farm processed soybean. conclusively, the gains as well as the efficiency of feed utilization obtained with the on-farm processed soybean were significantly (p<0.05) superior compared to the expellerextruded soybean but the serum metabolites of the pigs were observed to be unaffected by the different processing methods. key word: expeller-extruded soybean, on-farm processed soybean, pigs, soybean processing, performance, serum metabolites introduction fish meal (fm) is presently the most expensive animal protein source in animal feeds (tacon, 1993). the increasing costs and unpredictable availability of fm necessitates the search for its replacement with cheaply and abundantly available plant protein feed stuffs (robinson and menghe, 2007). recent research trends indicate that there is an increased interest towards the search for alternative/additional protein source to meet the increasing requirements for protein source for the expanding livestock industry, especially in the developing countries (janardhanan et al., 2003). even though soybean and other common legume grains play a key role as protein source for human beings and animals alike, their production is not sufficient to meet the protein requirements of the increasing population and expanding livestock industries (vijayakumari et al., 2007). the heavy demand for these common legumes has given rise to a disproportionate increase in their prices, and consequently, the cost of the food and feedstuffs into which it is incorporated. use of raw soybean is limited due to the presence of endogenous and heat-labile anti-nutritional factors (anfs) that is, lectin, phyto-haemoglutinin, anti vitamins and protease (trypsin) inhibitors (liener, 1980). the most important of which are trypsin inhibitors, which seriously impair protein digestibility (balloun, 1980). there are both heat stable (saponins, tannins, estrogens, phytate) and heat-labile (protease inhibitors) in soy. protease inhibitors in soy are destroyed by heating but other factors also play a role, such as duration of heating, particle size and moisture conditions. thus, how soy products are processed will dictate how much of the protease inhibitor (also known as trypsin inhibitor) remains. the nutritive value of the protein in soybean actually increases with heating which destroy trypsin inhibitors (weingartner, 2008). roasting or cooking of raw soybean to a temperature of 240-260 degrees fahrenheit for 3 to 5 min destroys this inhibitor and ensures the utilization of soy table 1. diet composition. on-farm processed expelled soybean expelled soybean ingredient % soybean meal a(43%) meal b(41%) maize 30.00 29.30 28.54 maize offal 12.00 12.00 12.00 soybean meal (45%) 15.00 expelled soybean meal 15.70 16.46 palm kernel cake 20.00 20.00 20.00 wheat bran 20.00 20.00 20.00 bone meal 2.25 2.25 2.25 vit-min. premix 0.25 0.25 0.25 salt 0.50 0.50 0.50 total 100.00 100.00 100.00 calculated: crude protein 17.95 17.95 17.95 me kcal/kg 2773 2738 2735 bean as a protein supplement (vijayakumari et al., 2007). development of new processing technologies for the removal of anfs has facilitated its increased use as protein source in feeds (garg et al., 2002) . therefore, for the elimination of anfs, soybean seeds ( glycine max) could be hydrothermically processed before incorporating in diets. soybean meal (sbm) can be produced either by solvent extraction or expeller-extrusion. expeller-extruded sbm (ee-sbm) contains more oil but less crude protein than solvent extracted sbm (se-sbm) (powell et al., 2007). however, results of experiments indicate that eesbm had similar feeding value to se-sbm when the differences in nutrient values are considered in diet formulation. presently, solvent extraction is the most common method of extracting soybean oil today; leaving less than 1% fat, but, the expeller process is still the most commonly used industrial method in nigeria (fashina, 1991). this experiment was therefore conducted to determine the replacement value of expeller-extruded soybean meal for on-farm processed soybean in diets of growingfinishing pigs. materials and methods expeller-extruded soybean meal was purchased at two local industrial sales outlets in ibadan, nigeria. the two samples of expelled soybean meal and the on-farm processed soybean were analyzed for percentage protein (aoac, 1990) and diets formulated to be isonitrogenous and isocaloric (table 1). in the expeller process, the soybean are cracked, dried, and transported to a tempting device, which stirs them for uniform heat processing. the soybeans are then fed into an expeller barrel, which presses the oil from the beans. the soybeans leave the barrel and are ground. the expeller process leaves the beans with approximately 5% fat. however, boiling of soybean is the most conventionally and commonly used farm-adaptable processing method in nigeria (awosanmi, 1988). raw soybean packed in jute bags is lowered into half-drum of boiling water and allowed to boil for a time-period. the boiled seeds are then drained of water and sun-dried to less than 10% moisture content before being ground and stored (fanimo, 1996). the metabolisable energies were also determined with the equation predicted by morgan et al. (1975). me = 0.416cp + 0.605ee + 0.367nfe – 20.06 (r 2 = 0.94) where me is the metabolizable energy, cp is the crude protein, ee is the ether extract and nfe is the nitrogen free extract. lysine was assumed to be a fixed percentage of total protein in both soybean meals. the arrangement of treatments allowed for a direct comparison of the protein quality of the two expelled-soybean meal sources to the onfarm processed soybean, as well as assessing the effect of the potential variation in protein on pig performance. thirty pigs averaging 30 ± 1.1kg were assigned randomly by origin and weight to one of three dietary treatments in a randomized complete block. in each of the treatment groups, there were five replicates of two pigs per replicate. the pigs were allowed assess to feed and water ad libitum for a 42-day period. five of the ten experimental grower pigs in each of the three dietary treatments were randomly selected and bled at the end of the feeding trial. the bleeding was done in the morning before feeding and 10ml of the blood was obtained from the jugular vein into a sample bottle using a sterilized needle and syringe as described by adesehinwa (2007). the samples were allowed to clot before centrifuging to obtain the serum used in the determina-tion of some serum metabolites (total protein, albumin, globulin, urea nitrogen, creatinine, cholesterol and glucose) using methods described by toro and ackermann (1975) and kaneko (1989). all the data obtained were subjected to analysis of variance and where statistical significance were observed, the means were compared using the duncan’s multiple range (dmr) test. the sas computer software package (1988) was used for all statistical analyses. result and discussion pigs fed diets containing the expelled soybean meal (esm) had decreased (p<0.05) average daily gain (adg) compared to pigs fed the on-farm processed soybean (table 2) resulting in between 13 17% reduction in the adg. this could be attributed to a suspected higher fat content of the on-farm-processed soybean, which may have resulted in the higher caloric density of the diet, since, it was not defatted. the expelled soybean meal table 2. effect of expelled soybean meal on pig performance. on-farm processed expelled soybean expelled soybean ingredient, % soybean meal a(43%cp) meal b(41% cp) cv daily gain, kg 0.76 a 0.66 b 0.63 b 6.7 daily feed intake, kg 2.43 2.48 2.51 8.7 feed efficiency 3.27 b 3.59 a 3.73 a 9.0 a,b: means along the same row having different subscripts differ significantly at p<0.05 table 3. serum metabolites of growing pigs fed two sources of expeller-extruded soybean. metabolites on-farm processed expelled soybean expelled soybean meal b ±sem soybean meal a (43%cp) (41%) total protein (g/dl) 6.49 6.30 6.36 0.05 albumin (g/dl) 3.75 3.99 3.68 0.02 globulin (g/dl) 3.35 3.31 3.29 0.03 urea n (mg/dl) 44.13 41.25 43.38 1.84 creatinine (mg/dl) 1.49 1.57 1.44 0.05 cholesterol (mg/dl) 277.00 266.62 263.5 6.06 glucose (mg/dl) 85.13 84.46 90.13 2.75 samples resulted in comparable (p>0.05) gains which was significantly inferior to gains obtained with the onfarm soybean. the average daily fed intake was not significantly influenced by the different soybean samples, in accordance with the findings of awosanmi (1988) and bamgbose (1988), that processing of soybean may not affect the feed intake. hayward et al. (1953) had earlier reported inability of processed oilseed meals to promote good growth as not due to a lack of feed intake but difference in the nutritional value of their protein as a result of methionine deficiency. hence, it could be inferred that the nutritional value of the resultant protein contained in the onfarm processed soybean used in this study was superior to the expelled soybean taking into consideration the superior gains observed. balogun (1979) reported methionine deficiency in the rations of pigs to cause amino acid imbalance with consequent adverse effect on performance. feed efficiency was 18 to 24% significantly (p<0.05) poorer for pigs fed the expelled soybean meal diets compared to the improvement observed in the feed conversion of pigs when fed the on-farm processed soybean. however, the replacement of the on-farm processed soybean with the expellerextruded soybean resulted in neither beneficial nor adverse effect on the serum parameters. these results indicated that the expelled soybean meal used in this experiment was an inferior protein source compared to the on-farm-processed soybean due to the comparative adverse animal performance in terms of feed efficiency and weight gains. the effect of expeller-extruded soybean protein sources on the serum metabolites of the pigs is shown in table 3. the serum total protein, albumin and globulin of the pigs were observed to be unaffected by the different processing methods. these parameters are indications of protein reserves in animals (adesehinwa, 2007) and can be specifically influenced by protein shortage indicated by alterations in albumin content (gouache et al., 1991). the result of this study showed that the protein levels and the amino acid profiles of the isonitrogenous diets were able to support normal protein reserves in the experimental pigs. the influence of diets on haematological and serum biochemical variables have been established (ologhobo et al., 1993; otesile et al., 1991). the observed values were within the range reported by kaneko (1989) for this class of pigs. this could have been brought about by the efficient utilization of the soybean proteins contained in the diets thereby resulting in comparable (p>0.05) values. however, the gains as well as the efficiency of feed utilization obtained with the on-farm processed soybean were significantly (p<0.05) superior compared to the expeller-extruded soybean. it should be noted that the crude protein content of cakes have been reported to be dependent on the extent of dehulling and the efficiency of the oil extraction process (villamide and san juan, 1998). the efficiency of utilization of any protein source is affected by the level of protein in the diet, the digestibility of the protein, and the levels of essential amino acids, particularly the first limiting amino acid lysine (maina et al. 2002). the economic feasibility of feeding extruded soybeans to swine is affected by the differences in nutrient content compared to soybean meal, the need to increase the protein in diets containing whole soybeans, the value of extra fat and the cost of processing (hollis, 2008). references adesehinwa aok (2007). utilization of palm kernel cake as an energy source by growing pigs: effects on growth, serum metabolites, nutrient digestibility and cost of feed conversion. bulgarian j. agric. sci. 13: 593-600. aoac (1990). official methods of analysis (15 th ed.) association of official analytical chemists, arlington v.a. awosanmi vo (1988). detoxification and utilization of full-fat soybean (glycine max) for poultry feeding. ph.d. thesis, university of ibadan, nigeria. balloun sl (1980). soybean meal in poultry nutrition. american soybean association publication. st. louis missouri, u.s.a. balogun oo (1979). methionine and lysine requirements of imported european pigs in humid lowland tropics. ph.d. thesis. university of ibadan, nigeria. bamgbose am (1988). biochemical evaluation of some oil seed cakes and their utilization in poultry rations. ph.d. thesis, university of ibadan, nigeria. fanimo ao (1996). nutritional evaluation of full -fat soybean boiled for three time periods. pertanika j. trop. agric. sci. 19(2/3): 175-182. fashina ha (1991). utilization of soybean based rations by grower and finisher pigs in hot humid tropics ph d thesis, university of ibadan. nigeria. garg sk, kalla a, bhatnagar a (2002). evaluation of raw and hydrothermically processed leguminous seeds as supplementary feed for the growth of two indian major carp species. aquac. res. 33: 151-163. gouache p, lemoullac b, bleiberg-daniel f, aubert r, flament c (1991). changes in rat plasma apolipoproteins and lipoproteins during moderate protein deficiency: potential use in the assessment of nutritional status. j. nutr. 121(5): 653-662. hayward jw, steenbock h, bohstedt g (1953). the effect of heat as used in the extraction of soybean oil upon the nutritive value of the protein. j. nutr. 11: 219-233. hollis g (2008). swine management and nutrition faq http://faq.aces.uiuc.edu/ janardhanan k, vadivel v, pugalenthi m (2003) biodiversity in indian under-exploited/tribal pulses. in: improvement strategies for leguminosae biotechnology (editors: p k jaiwal and r p singh), pp. 353-405. kluwer academic publishers, britain. kaneko jj (1989). clinical biochemistry of domestic animals. 4 th edition. academic press, inc. n.york. liener ie (1980). toxic constituents of plant foodstuffs, academic press, new york, u.s.a., pp. 7-71. maina jg, beames rm, higgs d, mbugua pn, iwama g, kisia sm (2007). the feeding value and protein quality in high-fibre and fibrereduced sunflower cakes and kenya’s “omena” fishmeal for tilapia (oreochromis niloticus). livestock res. rural dev. 19(11). morgan dj, cole dja, lewis d (1975). energy values in pig nutrition 2. the prediction of energy values from dietary chemical analysis. j. agric. sci. (camb.) 84: 19-27. ologhobo ad, apata df, oyejide a, akinpelu ro (1993). a comparison of protein fractions prepared from lima beans (phaseolus lunatus) in starter diets for broiler chicks. j. appl. anim res. 4: 13-30. otesile eb, fagbemi bo, adeyemo o (1991). the effect of trypanosoma brucei infection on serum biochemical parameters in boars on different planes of dietary energy. vet. parasitol. 40: 207216. powell s, naranjo v, lauzon d, southern l, bidner t, parsons c (2007). evaluation of louisiana-produced extruded-expelled soybean meal for chickens (m18). book of abstacts, 2007 international poultry scienti c forum, georgia world congress center, atlanta, georgia january 22–23, p. 6. robinson eh, menghe hl (2007). catfish protein nutrition (revised). bulletin 1153. office of agricultural communications, mississippi state university, usa sas (1988). sas/stat user guide (release 6.03). sas inst. inc., cary., n.c. tacon agj (1993). feed ingredients for warm water fish. fishmeal and other processed feedstuffs. fao fishries circular no. 856, fao, rome, italy, p. 64. toro g, ackermann gp (1975). practical clinical chemistry little, brown and company, boston, massachusetts. vijayakumari k, pugalenthi m, vadivel v (2007). effect of soaking and hydrothermal processing methods on the levels of antinutrients and in vitro protein digestibility of bauhinia purpurea l. seeds. food chem. 103: 968-975. villamide mj, san juan ld (1998). effect of chemical composition of sunflower seed meal on its true metabolizable energy and amino acid digestibility. poult. sci. 77: 1884-1892 weingartner ke (2008). soybean utilization faq. http://faq.aces.uiuc.edu/. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (6), pp. 001-009, june, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper protease-activated receptor-2 (par-2) regulates enterotoxigenic escherichia coli-induced diarrhea during weaning in piglets xiao-ying pei1, ding-zong guo1*, dong-hai zhou1*, rui guo2, hong-mei gao1 and tian xia1 1 college of veterinary medicine, huazhong agricultural university, wuhan 430070, china. 2 key laboratory of animal embryo and molecular breeding, hubei academy of agricultural sciences, wuhan 430064, china. accepted 02 january, 2015 protease-activated receptor-2 (par-2) is a member of the g-protein-coupled receptor family. the proteases that activate par-2 are released during inflammation and injury, with par-2 regulating the response to these insults. in the gastrointestinal tract, par-2 is known to alter gastrointestinal secretion, motility, inflammation, and pain. in many cases, par-2 has been reported as proinflammatory and proliferative. paradoxically, par-2 is also suggested to be anti-inflammatory in some instances. weaning piglet diarrhea is severely detrimental to the porcine industry, being responsible for 11% of total piglet deaths, while those that survive from the disease experience developmental problems and fail to grow to the size of their healthy counterparts. thus, we sought to determine any correlation between par-2 and weaning diarrhea. we hypothesized that par-2 might represent a new target in the treatment of weaning diarrhea. the current study measured par-2 using immunohistochemistry on sections of piglet gastrointestinal tract mucosa and identified changes in receptor expression during the development and course of weaning. moreover, the effect of par-2 stimulation using lipopolysaccharide (lps) and heat-labile enterotoxin (lt) on il-6 and il-8 production in pig intestinal epithelial cells (iec) was determined. this study found that par-2 is expressed abundantly in the piglet gastrointestinal tract mucosa, and revealed that par-2 mrna and protein expression are both correlated with the severity of diarrhea. the generation of il-6 and il-8 by iecs was significantly increased following stimulation with par-2 agonists dose-dependently. thus, we suggest par-2 may be involved in the development of diarrhea during weaning in piglets. key words: diarrhea, weaning piglet, gastrointestinal tract, protease activated receptor-2, immunostaining, rtpcr, il-6, il-8. introduction protease-activated receptors (pars) are a family of gprotein-coupled, seven-transmembrane receptors, consisting of four known members, par-1 to par-4 (déry et al., 1998; macfarlane et al., 2001). pars are activated by proteolytic cleavage of their extracellular nh2-terminus. among the four pars identified to date, par-1, par-3, *corresponding author. +8627-87286251; e-mail: bigdefoot@ 163.com and par4 are known to be activated by thrombin, while trypsin and mast cell-derived tryptase are shown to activate par-2 (kanke et al., 2005). par-2 was first identified in 1944 and reported to be widely expressed in many cell types (dulon et al., 2005; saifeddine et al., 1996) such as enterocytes, endothelial cells of the lamina propria and the submucosa, fibroblasts, myenteric neurons, and immunological and inflammatory cells (including lymphocytes, neutrophils, and mast cells) (kawao et al., 2002; nystedt et al.,1996). the physiological and pathological roles of par-2 have been investigated in animal models in vivo using immunohistochemistry, pharmacology (employing synthetic par-2 activating peptide), and par-2 deficient (par2−/−) mice (kawagoe et al., 2002; vergnolle, 1999). par-2 was found to participate in leukocyte rolling, ad-hesion, and infiltration of neutrophils, as well as inducing nuclear factor kappa bdna binding (kanke et al., 2001). with regard to the role of par-2 in the intestine, hansen et al. (2005) reported par 2 to augment enteritis from many kinds of bacteria. par-2 can be activated by proteolytic enzymes secreted from bacteria, promoting substance p release, which binds and activates the neurokinin a receptor, which in turn induces tissue edema and effusion. acute activation of par-2 causes colitis characterized by gut wall edema, granulocyte recruitment, increased permeability and the release of proinflammatory cytokines such as interleukin-1 and tnf-α (cenac et al., 2002). other studies have shown activation of par-2 with chronic, systemic administration of a par-2 agonist to provide protection against a chronic model of inflammatory bowel disease (ibd). this protection was suggested to be the result of par-2-induced gastric mucus secretion and mucosal cytoprotection (fiorucci et al., 2001; kawabata et al., 2001). cytokines play a central role in the modulation of the intestinal immune system and can stimulate proinflammatory (il-1, tnf, il-12, il-6, il-8) or anti-inflammatory (il-4, il-10, il-1ra, il-11) responses. levels of both proinflammatory and anti-inflammatory cytokines are elevated in patients with ibd; however the ratio between proand anti-inflammatory cytokines becomes unbalanced, leading to inflammation. the major source of il-6 within the gut is from macrophages, although the primary source of il-6 in epithelial cells remains to be defined (rogler et al., 1997). il-6 is elevated in both the serum and mucosa of patients with ibd (murata et al., 1995). furthermore, il-8 (a powerful neutrophil chemoattractant and activator (daig et al., 1996) correlates with the level of inflammation in the colon of patients with ulcerative colitis (uc) (mazzucchelli et al., 1994) in which large numbers of neutrophils are found in crypt abscesses. our previous reports have demonstrated that direct activation of par-1 and par-2 in human endothelial cells leads to the production of il-6, and both lps and tnf-a markedly enhance par-induced activation (chi et al., 2001). par-2 activation stimulates the proliferation of endometriotic stromal cells (esc) and the secretion of il-6 and il-8 from the esc (hirota et al., 2005). at weaning, piglets are exposed to marked alterations in living conditions, such as shifting from milk to a cerealbased diet, becoming separated from the sow and mixed with other piglets. one of the consequences of weaning is the high risk of post weaning diarrhea (wellock et al., 2007). it has been reported that following three days of weaning, piglets become more susceptible to the detrimental effects of escherichia coli enterotoxin when compared to their unweaned littermates. further investigations report morbidity and mortality of enterotoxigenic escherichia coli (etec)-induced diarrhea to account for up to 56.2 and 24.7% of total diarrhea-induced morbidity and mortality, respectively (alexander, 1994; hampson, 1994; jin et al., 1998; stevens et al., 1972). characteristics of etec-induced disease include a rapid development and fast spread of infection resulting in either high mortality or chronically stunted growth. both outcomes seriously compromise productivity and animal yield for the porcine industry. thus, par-2 plays a potentially protective or pathogenic role in gastrointestinal tract mucosa when under pathological conditions, possibly activated in response to tissue injury or inflammation. many studies have found par-2 in humans and mice; however, few studies have found par-2 in pigs. the current study sought to determine the role of par-2 on diarrhea in weaned piglets. the results presented in this report demonstrate that par-2 is expressed abundantly in the piglet gastrointestinal tract mucosa, and reveals that par-2 mrna and protein expression are increased with the severity of diarrhea. direct activation of par-2 by both lps and lt in pig intestinal epithelial cells leads to enhanced production of il-6 and il-8. taken together we suggest that par-2 may be involved in the development of diarrhea during weaning in piglets. materials and methods regents and materials tryptone and yeast extract were obtained from oxoid (basingstoke, uk). goat anti-par-2 antibody and anti-goat horseradish secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca, usa). epithelial cells were cultured in high-glucose formulated dulbecco's modified eagle's medium (dmem, gibco). various concentrations of epidermal growth factor (egf; sigma), insulin (sigma), fetal bovine serum (gibco), glutamine (gibco), penicillin (sigma) and streptomycin (sigma) were also added to the medium to induce epithelial proliferation. the enzyme-linked immu-nosorbent assay (elisa) kits for il-6 were purchased from r and d systems (minneapolis, mn). par-2 agonist peptides -ser-leu-ilegly-arg-leu-nh2 (sligrl-nh2) and related hexa-peptides with an altered consensus sequence (lrgils-nh2) were synthesized by biotechnology corp. (xian, china). trypsin, the soy bean trypsin inhibitor, e. coli lps and e. coli lt were all obtained from sigmaaldrich. etec challenge strains the etec k88 o149 strain (provided by the china institute of veterinary drugs control, china) was grown in luria broth (lb) medium comprising: 1% tryptone, 0.5% yeast extract, and 1% nacl, final ph 7.0. after an overnight incubation at 37 c with shaking, the bacterial cells were then diluted to an optical density at 600 nm (od600) of 0.1 in fresh lb medium and allowed to grow to an od600 of ≈1.0. the cell cultures were then centrifuged at 4,000 g for 10 min at 4°c. the bacterial pellets were resuspended in 20% dextrose and 5% non-fat milk. the challenge dose consisted of an equal amount of each strain and was determined by serial dilution and plating to provide a total of 2×10 10 cfu/0.5 ml oral dose, twice daily with an 8-h interval for three days. animals animal care and procedures were in accordance with the national institutes of health recommendations for the humane use of animals. all experimental procedures were reviewed and approved by the appropriate animal use committee of huazhong agricultural university. 30 duroc piglets aged 25 28 days and weighing ~7 kg were provided by hubei academy of agricultural sciences china. 18 piglets were infected with ecet (enterotoxigenous e. coli), and 12 piglets suffering from diarrhea were selected as a diarrhea group. we killed 3 healthy piglets and 3 diarrheal pigs on the first, third, fifth and seventh day, respectively. all piglets were anaesthetized using sodium pentobarbital prior to opening the gut and dissecting the stomach, dodecadactylon jejunum ileum, cecum and colon. all sec-tions were washed 3 times in cold 0.9% nacl. half of the sections were fixed by immersion in 10% neutral-buffered formalin, dehydrated, embedded in paraffin wax, and stored at 4 c for immunohistochemistry. other sections were placed into liquid nitrogen for 3 h and maintained at -70 c for real-time rt-pcr. histopathology a subsection of samples were subject to histological analysis to verify the cellular changes within the gastrointestinal tract by h and e staining. immunostaining of par-2 in the piglet gastrointestinal tract mucosa immunostaining of par-2 was performed using a goat polyclonal antibody. briefly, sections of gut were deparaffinized, rehydrated, immersed in phosphate-buffered saline (pbs) for 15 min at 37 c and blocked in endogenous peroxidase (3% peroxide) for 5 min. after washing in pbs, blocking was performed with a 5% solution of normal rabbit serum for 30 min at 37 c. primary goat polyclonal antibody, diluted 1:100 in pbs, was applied to each section and incubated for 12 h at 4 c in a humidified chamber. after washing three times, the slides were flooded with biotinylated rabbit anti-goat linking antibody (1:200) for 20 min at 37 c. sections were then washed twice more in pbs, treated with peroxidase-conjugated avidin for 30 min, rewashed twice in pbs, and then incubated with 3-amino-9-ethylcarbazole for 25 min. gut sections were stained with mayer’s hematoxylin counterstain for 1 min, dewatered, mounted by neutral balsam, and then analyzed under a microscope (olympus, ix71, japan). sybr green real-time rt-pcr par-2 mrna expression was determined by sybr green i realtime quantitative polymerase chain reaction (rt-qpcr) analysis using an iq-5 real-time pcr detection system (bio-rad, america). total rna was extracted from the ileum, cecum and colon mucus using the trizol reagent (invitrogen). after verification of its integrity, rna was quantified spectrophotometrically with 1 ug processed for complementary dna (cdna) synthesis using superscript ii reverse transcriptase (toyobo, japan). specific primers for the par-2 gene were designed using primer5 software. the sequences of the primers used were: par2, sense: 5’-gca aca act ggg ttt acg gg -3’; antisense: 5’-ggt gtg atg tga agg gct gg -3’; gapdh, used as a housekeeping gene, sense: 5’-ggt gaa ggt cgg agt gaa cg-3’; antisense: 5’ ctc gct cct gga aga tggtg-3’. the efficacy of the rt-pcr primer pairs was determined by amplifying serial dilutions of cdna. the rt-pcr was performed using sybr green real-time pcr master mix (toyobo). each cycle consisted of three steps: denaturation for 30 s at 95°c, annealing for 30 s at 60°c, and 30 s of elongation at 72°c. the data acquired from each sample were normalized to those of gapdh. the specificity of the real-time reverse transcriptase was further confirmed by a regular rt-pcr followed by agarose gel electrophoretic analysis to verify the presence of a single band corresponding to the predicted size of the amplicon. relative ct (cycle time) values were obtained using the bio-rad iq5 optical system software data acquisition server. culture of pig intestinal epithelial cells newborn piglets were anaesthetized using sodium pentobarbital and killed. the small intestine was opened and sectioned into 2 3 mm lengths. tissue sections were transferred to a 25 ml flask and washed at least 8 times in 50 ml of fresh hanks’ balanced salt solution (hbss) with vigorous shaking. tissue was then placed on a petri dish and a sharp scalpel blade used to dice the tissue into <1 mm 3 pieces before being returned to a t25 ml flask with 20 ml of collagenase type 1 (sigma-aldrich). tissue was shaken vigorously for 2 h at 37 °c. 15 ml of dmem-s (dmem + 2.5% fcs +2% sorbitol) was then added to the flask and shaken again. the sedi-ment was allowed to dissociate from the supernatant under gravity for 60 s, and the supernatant carefully removed. this procedure was repeated twice. 10 ml of dmem-s was added to the supernatant before being vortexed and centrifuged at 200 300 rpm for 2 min. the supernatant was carefully removed and the pellet resuspended in 20 ml dmem-s. this procedure was repeated at least 5 6 times until the supernatant became completely clear and the pellet well defined. finally, the pellet was resuspended in the appropriate growth medium. assay of il-6 and il-8 production intestinal epithelial cells (1×10 5 ) were added to each well of a 12well microtiter plate and allowed to adhere for 24 h. following adherence, the medium containing serum was removed and serumfree medium added. selected concentrations of par-2 agonist peptides, trypsin, a soy bean trypsin inhibitor, e. coli lps, e. coli lt or medium were added to the monolayers. all incubations were carried out at 37°c in 5% humidified co2 for 24 h unless otherwise indicated. after incubation, the supernatant was collected and il-6 and il-8 levels were quantified by elisa according to the manufacturer’s protocol. statistical analysis data are displayed as the mean + sem. all statistical analyses were performed using spss statistical software. the means among different groups were compared by one way analysis of variance (anova).the par-2 positive area was calculated in pictures of immunohistochemistry using image-pro plus software (ipp6.0). the data from sybr green real-time rt-pcr was analyzed using the formula: -[mean ct of specific gene of diarrhea group mean ct of house-keeping gene of diarrhea] -2 δδct (2 [mean ct of specific gene of normal group mean ct house-keeping gene of normal group]). figure 1. representative h and e-stained histological sections of stomach, small intestine and large intestine from the diarrhea group (figure 1a, b and c) with slides d, e and f providing normal comparisons respectively. bar = 100 m. results microscopic examination of histopathology the most striking pathological changes of the diarrhea group was desquamation and denaturation of the epithelium mucosa, smooth muscle thinning of the bowels, infiltrated neutrophils and increased eosinophils exudated from the mucosal layer (figure 1). immunolocalization of par-2 in the piglet gastrointestinal tract mucosa par-2 was found expressed throughout the gastrointestinal tract, including in the mucosa of the stomach, duodenum, jejunum, ileum, cecum and colon (figure 2b1-b6). further observations revealed elevated staining of par-2 within the lamina propria and greater staining in intestinal mucosa of the diarrhea group compared with control (figure 2a1-a6). histochemical analysis using the serum from a non-immunized goat displayed no positive immunostaining for par-2 in the gastrointestinal tract mucosa (figure 2c1-c6). sybr green real-time rt-pcr using the 2 -δδct method to analyze the mean ct, our results display the ratio of par-2 mrna expression in pathogenic versus normal tissue over the development of diarrhea. during this time the ratio of par-2 expression was not found to significantly differ in the stomach mucosa (figure 3a), although it was significantly elevated in the ileum and colon. changes in the par-2 ratio were apparent from day 3 in the ileum and markedly so in the colon (figure 3b and c). effect of par-2 agonist peptides on il-6 and il-8 production by naive and lps+li-stimulated iec to examine the role of par-2 on intestinal epithelial cell figure 2. immunolocalization of par-2 in piglet gastrointestinal tract mucosa from healthy and diarrhea piglets using immunohistochemistry. strong staining was exhibited in the gastric gland of the stomach mucosa (a1), duodenum mucosa (a2), jejunum mucosa (a3), ileum mucosa (a4), cecum mucosa (a5) and colon mucosa (a6) compared to control cells (slides c1-c6 respectively) and healthy cells (b1-b6 respectively). arrows indicate positive staining for par-2, reference bar = 50 m. production of il-6 and il-8, the direct and amplifying effects of par-2 agonists on baseline and lps+ltactivated iec were studied. the data shown in figure 4 demonstrate il-6 and il-8 production by iecs during 24-h incubation with par-2 agonist peptides and the effect of agonists in the presence of lps+lt. non-activated iecs produced negligible amounts of il-6 and il-8. incubation of iecs with a par-2 agonist resulted in a low, but significant increase in il-6 and il-8 expression compared with controls. to examine the possible synergy between par-2 agonists and lps+lt on iec activation, iecs were incubated with trypsin and sligkv-nh2 in the presence concentrations of lps+lt incubation of iecs with the par-2 agonists in the presence of lps+lt resulted in marked potentiation of il-6 and il-8 production at all concentrations tested. the figure 3. expression of par-2 mrna in the mucosa of the stomach (b), ileum (c), and colon (d). data are displayed as the ratio of par-2 mrna in diarrheal tissue versus that of normal tissue using rt-pcr at four time points over a 1-week period. change in il-8 expression was more pronounced to that of il-6. discussion the present study demonstrates that par-2 is expressed abundantly in the piglet gastrointestinal tract mucosa, and reveals that both par-2 mrna and protein expression increases with the severity of diarrhea. in vitro analysis using piglet intestinal epithelial cells demonstrates that activation of par-2 leads to the production of il-6 and il8. the effects of par-2 agonists on the iec were greatly enhanced by concomitant stimulation by lps and lt. the mechanism of amplification of par-mediated il-6 and il-8 production when in the presence or absence of lps and lt is currently unknown. at least two possible mechanisms could explain the observed lps+ltenhanced iec activity through par-2. the first is that lps +lt induce expression of par-2 which, in turn, is activated by the agonists. second, the inflammatory activators prime par-2, causing amplification of figure 4. specificity of par-2 agonist on intestinal epithelial cell activation. iec monolayers (1×10 5 /well) were incubated with an agonist for 24 h in the presence or absence of lps+lt. after incubation, il-8 and il-6 expression in the culture medium was measured by elisa. values represent the mean ± sem of quadruplicate determinations. similar results were obtained in three replicate experiments (*p<0.05, **p<0.01). independent or common signal transduction pathways leading to increased cytokine gene expression. one report supports the initial hypothesis that lps independently induced the expression of par-2 on endothelial cells without affecting the expression of par1 (nystedt et al., 1996). the pathogenic mechanism of etec works by invading the intestine of a susceptible host. the pathogens pilus combines with the enterocyte microvillus and is accepted onto the cell surface where it adheres to the enteric mucosa to boycott enterokinesia and stimulate intestinal secretion. etec amplifies rapidly within the intestine and releases enterotoxin. the enterotoxin is highly toxic and effective in destroying the regulatory system of gastrointestinal mucous, pathologically remodeling the electrolyte/water balance, severely compromising digestive function, as observed by the current study (madec et al., 2000; wellock et al., 2007). taken together, our results suggest par-2 may be a regulatory factor in the pathogenesis of piglet diarrhea during weaning; one potential mechanism being the mediation of inflammatory factor production. par-2 is reported to be activated by multiple proteases such as trypsin, mast cell tryptase, and coagulation factors viia and xa. these factors may be activated and/or accessible to mucosal tissues including chief cells and sensory neurons during inflammation or tissue injury (camerer et al., 2000; kawabata et al., 1999). exogenous and endogenous par-2 activation has been shown to significantly reduce rat intestinal mucosal damage, but did not influence leukocyte infiltrates induced by intestinal ischemia/reperfusion injury (cattaruzza et al., 2006). with regard to the role of par-2 in piglet weaning diarrhea, we hypothesize that par-2 is activated by endogenous agonists and the production of inflammatory factors will increase further in the present of lps and lt, mediating the pathologenic processes of piglet diarrhea during weaning. the physiological dual role of par-2 in the gastrointestinal tract mucosa remains a topic of debate, with application of a par-2 agonist shown to facilitate pepsin secretion, as well as produce dose-dependent cytoprotection at low doses (kawao et al., 2002). the production of inflammatory factors is necessary for physiological function, and par-2 may have many physiological functions despite the pathophysiological mediation of diarrhea observed here; so we tentatively suggest par-2 functions as a double-edged sword in the gastrointestinal tract dependent upon the progressive state of diarrhea. for example, when activated par-2 initially promotes the release of mucus, providing protection for the mucosa; however, the mechanism regulating par-2 activity is not expounded in the present study. to the authors knowledge, this study is the first to exhibit that both par-2 mrna and protein expression are present throughout the gastrointestinal tract mucosa of weaning piglets. we found that par-2 mrna expression was significantly elevated in the intestine of piglets with weaning diarrhea, and par-2 mrna expression increased with the course of diarrheal development. direct activation par-2 in pig intestinal epithelial cells leads to the production of il-6 and il-8, and both lps and lt markedly enhanced parinduced activity. taken together it is suggested that par-2 may be involved in the development of diarrhea during weaning in piglets. future studies are required in which an exogenous or endo-genous agonist of par-2 will be administered into piglets with weaning diarrhea to further our knowledge of the disorder. the current study reveals a potentially novel therapeutic target in combating weaning piglet diarrhea. acknowledgments this work was supported by the national natural science fund of china under the contract number 30700588. we would like to thank mr. qianglin duan from tongji hospital of tongji university for critical reading of the manuscript. references alexander tjl (1994) neonatal 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rolling, adhesion, and extravasation in vivo. j. immunol., 163: 5064-5069. wellock ij, fortomaris pd, houdijk jgm, kyriazakis i(2007). effect of weaning age, protein nutrition and enterotoxigenic escherichia coli challenge on the health of newly weaned piglets. livestock sci., 108: 102-105. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 3 (4), pp. 001-005, april, 2015. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper prevalence and antimicrobial susceptibility of bacterial pathogens isolates from diseased swine in southwest, china xu-ting li*, bin wang, jin-liang li, fu-qiang zeng and xue gong sichuan animal science academy, chengdu, china, 610066, china. accepted 22 september, 2014 the purpose of this study was to investigate the prevalence of antimicrobial resistance in the clinical bacterial isolates from diseased swine in southwest, china during 2009-2010. a total of 504 bacterial isolates (19 species) were collected from the 364 clinical samples. the activity of 6-14 antibiotics to each bacterial species was examined. the sensitivity was tested by the disk diffusion method and performed according to clsi guidelines in mueller-hinton agar. the most common pathogens were escherichia coli (n=154; 30.56%), staphylococcus spp. (n=110; 21.83%), enterococcus faecalis (n=58; 11.51%), klebsiella pneumoniae (n=44; 8.73%), proteus mirabilis (n=43; 8.53%) and streptococcus suis (n=30; 5.93%). all isolates revealed high level of resistance to ampicillin (47.6-100%), amoxicillin (52.6-100%), cephalothin (29-100%), norfloxacin (52.6-83.3%), gentamicin (45.1-83.3%) and terramycin (61.9-100%). moreover, 93% of the isolates exhibited multiple drug resistance (mdr; resistance ≥ 3 antimicrobials). only ticarcillin/clavulanate exhibited very high activity against e. coli (98.1%), staphylococcus spp. (91.9%), k. pneumaniae (92.3%) and p. mirabilis (97.2%), respectively. these findings suggest that antimicrobial resistance of bacterial pathogens isolates is commonly present among diseased swine in southwest, china, and they also suggest the need for more prudent use of antibiotics by farmers and veterinarians. key words: identification, antimicrobial susceptibility, pathogen, swine. introduction as acute infections and outbreaks of infectious diseases in groups or herds become more common, use of an effective antimicrobial treatment as early as possible is critically important. the empirical treatment is generally based on knowledge of the resistance patterns of the different bacterial pathogens toward antimicrobial agents used in the particular animal species. uncontrolled usage of antimicrobial agents is recognized as the most important factor that favors the development and spread of resistant microorganisms (van den bogaard and stobberingh, 1999; white, 2002). the digestive tracts of *corresponding author. e-mail: lixutingzy@163.com. pigs can harbor antimicrobial-resistant bacteria among the commensal flora, which contain a reservoir of antibioticresistance genes potentially transmissible to humans through the food chain and environment (caprioli et al., 2000). in addition to the human health concerns, antimicrobial-resistant pathogens also pose a severe and costly health problem in that they may prolong illness and decrease productivity through higher morbidity and mortality (yang, 2004). however, data on the prevalence of antimicrobial-resistant veterinary pathogens are sparse, particularly in southwest, where animal husbandry was more developed than other area. therefore, the purpose of this study was to investigate the prevalence of bacterial infection and resistance to antimicrobial agents in the clinical isolates obtained from diseased swine in southwest, china. materials and methods clinical samples 364 samples, including lungs, lymph nodes, livers, hearts, spleens, kidneys, and blood (obtained between may 2009 and july 2010) were collected from 72 pig farms in southwestern china. all the samples were obtained from diseased pigs which had at least one of the following symptoms: septicemia, arthritis, enteritis, meningitis, pneumonia, pleuritis, diarrhea, edema, fever, pneumonia, endocarditis, and dysentery. before sampling, all samples surface were being asepsis by burning of ethanol. aseptically collected samples were appropriately processed and seeded in chocolate agar and blood agar. all of the samples were transported to the laboratory of sichuan animal science academy for bacterial counts and isolation within 6 h. bacteria isolation the strains were incubated at 37°c for 18-24 h. one loo p-full from each enrichment were streaked on tryptic soy agar, blood agar (5% fresh rabbit blood), macconkey agar (tianhe microorganism reagent co., ltd.) and salmonella-shigella agar respectively. all plates were incubated at 37°c in air for 24 h and pur ified by standard methods (murray et al., 2003). single colony was obtained from the isolates and stored in luria-bertani containing 20% glycerol, at –80°c until use. no replicate isolates f rom the same samples were used. all media agar were purchased from tianhe microorganism reagent co., ltd. bacteria identification identification was based on colony type and morphology, gram staining characteristics, and standard biochemical tests. all isolates were identified at species level by vitek (vitek system, biomerieux).they were also confirmed using primers 27f (5' aga gtt tga tcc tgg ctc ag 3’) and 1492r (5' tac ggc tac ctt gtt acg act t 3’) by the polymerase chain reaction (pcr) assay (wilson, 1990). the template dna was prepared by suspending an overnight cultured of bacteria in 400 µl milli-q water. the suspensions were heated at 95°c for 5 mi n and centrifuged at 13,000 rpm for 5 min. each 50 µl of pcr mixture consisted of 4 µl of template, 5 µl 10×pcr buffer, 1.5 mm mgcl 2, 200 µm dntp, 0.4 µm each of the seven primers and 2.5 u taq dna polymerase. pcr was performed in a dna thermal cycler (bio-rad, hercules, ca) using the following program: an initial denaturation step at 94°c for 5 min, 30 cycles of den aturation at 94°c for 1 min, annealing at 50°c for 1 min and exte nsion at 72°c for 1.5 min, followed by a final elongation at 72°c for 10 min. the amplified pcr products were analyzed on 0.8% (w/v) agarose gels. subsequently, the pcr product was sequenced by shanghai sangon bioengineering co., ltd. the nucleotide sequences were analyzed with software (blast) available from the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov). antimicrobial susceptibility susceptibility to 15 antimicrobial agents, ampicillin (amp; 10 µg), ampicillin/sulbactam (sam; 10/10 µg), ticarcillin (tic; 75 µg), amoxicillin (amo; 10 µg), amoxicillin/clavulanate (amc; 20/10µg), ticarcillin/clavulanate (tim; 75/10 µg), cephalothin (kf; 30 µg), ceftiofur (eft;30 µg), gentamicin (cn; 10 µg), amikacin (amk; 30 µg), terramycin (ot; 30 µg), erythromycin (e; 15 µg), clindamycin (da; 2 µg), norfloxacin (nor; 10 µg) and ciprofloxacin (cip; 5 µg), were tested by the disk diffusion method according to clinical and laboratory standards institute (clsi, 2009). the control strains used for all susceptibility tests were e. coli atcc 25922, pseudomonas aeruginosa atcc 27853 and staphylococcus aureus atcc 29213. detection of blatem resistance genes the template dna was prepared as described above. the forward primer atgagtattcaacatttccgtg and the reverse primer ttacca atgctta atcagtgag were used to amplify the blatem gene by following procedure: an initial denaturation step of 94°c for 5 min, 30 cycles of denaturation at 94°c for 1 min, annealing at 50°c for 1 min and extension at 72°c for 1 min, foll owed by a final elongation at 72°c for 10 min. amplified pcr products were analyzed on 0.8% (w/v) agarose gels. the pcr product was sequenced and analyzed as above. results bacterial isolates 504 bacterial isolates (which belong to19 difference species) were collected from the 364 clinical samples. the isolates were all obtained between may 2009 and july 2010 from diseased pigs, including suckling pigs, nursery pigs, grower pigs and grower-finisher pigs. the results revealed that e. coli (n=154; 30.56%) was the most widespread bacterial isolates, followed by staphylococcus spp. (n=110; 21.83%), e. faecalis (n=58; 11.51%), k. pneumoniae (n=44; 8.73%), p. mirabilis (n=43; 8.53%) and s. suis (n=30; 5.93%). only 7 h. parasuis (1.39%) and 6 p. multocida (1.19%) isolates were obtained from all clinical samples. antimicrobial susceptibility the majority of enterobacteria, including e. coli, k. pneumoniae and p. mirabilis, were resistant to ampicillin (88.4 to 100%), amoxicillin (92.3 to 100%), ticarcillin (50 to 76.9%), norfloxacin (61.5 to 83.3%), ciprofloxacin (67.3 to 83.3%) and terramycin (98 to 100%), and they were susceptibility to ticarcillin/clavulanate, ampicillin/sulbactam and amoxicillin/clavulanate. the gram-positive bacteria showed resistance to erythromycin (57.1 to 71.4%), clindamycin (79.6 to 85.7%), ampicillin (47.6 to 52.6%), amoxicillin (52.6 to 76.2%) and terramycin (61.9 to 84.2%) and were susceptibility to ceftiofur, ticarcillin/clavulanate, ampicillin/sulbactam and amoxicillin/clavulanate (table 1). in addition, most isolates from the swine were resistant to multiple classes of antimicrobial agents. four hundred and seventy (93.25%) isolates from diseased swine were resistant to at least 3 of the 15 antimicrobial agents. 50 (9.92%), 83 (16.47%), 133 (26.39%), 144 (30.56%) and 50 (9.92%) isolates were resistant to 3, 4, 5, 6 and 7 antimicrobial agents, respectively. blatem resistance genes detection based on the results of the susceptibility tests, 154 e. coli, 44 k. pneumoniae and 43 p. mirabilis were selected to amplify the blatem genes. the pcr analysis and sequencing showed that 140 (92.1%) e. coli, 44 (100%) k. pneumoniae and 42 (97.67%) p. mirabilis isolates harbored a blatem1 gene. discussion to investigate the prevalence of bacterial infections in swine in southwest, china, a total of 504 bacterial isolates (19 species) were collected and identified from the 364 clinical samples. the results revealed that e. coli and the staphylococcus spp. were the most widespread bacterial isolates. in swine, e. coli is an important pathogenic bacteria including enterotoxigenic (etec) and extracellular pathogenic e. coli (expec) strains which are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, pneumonia, neonatal meningitis, sepsis, neonatal and post weaning diarrhea and edema (yang, 2004; wada et al., 2004; nazareth et al., 2007; boerlin et al., 2005). e. faecalis (n=58; 11.51%), k. pneumoniae (n=44; 8.73%), p. mirabilis (n=43; 8.53%) and s. suis (n=30; 5.93%) were the another four frequent isolates. e. faecalis is intrinsical not as virulent as other gram-positive organisms, such as s. aureus, s. pneumoniae and s. suis type 2 (bittencourt, 2004; gaspar et al., 2009). it emerges as an opportunistic pathogen, nevertheless, it is known to cause serious infections such as bacteraemia, septicaemia, urinary tract infections, wound infections, meningitis and endocarditis (giacometti et al., 2000; hershberger et al., 2005; hällgren et al., 2003; hébert et al., 2007). k. pneumoniae is also an opportunistic pathogen that responsible for a wide range of infection in humans and animals, such as urinary tract infections, pneumonia, wound infections and septicemia (podschun and ullmann, 1998; brisse and duijkeren, 2005). p. mirabilis is also often found in human as opportunistic pathogens (zych et al., 2001). s. suis, especially the serotype 2, is an important swine pathogen causing meningitis, septicemia, endocarditis, and arthritis (marie et al., 2002; lun et al., 2007; domínguez-punaro et al., 2007; ma et al., 2009). in 2005, an streptococcal toxic shock syndrome (stss) human outbreak caused by s. suis serotype 2 was found in sichuan province with 38 human deaths and over 200 human infections, and more than 640 pigs were found to be severely infected (yu et al., 2006). although other zoonotic bacterial such as salmonella spp., h. parasuis, p. multocida and actinobacillus spp. were lesser, they showed higher mortality and morbidity. the traditional zoonotic e. coli, staphylococcus spp., s. suis and opportunistic e. faecalis, k. pneumoniae, p. mirabilis were the main pathogens in swine in southwest, china. the enterobacteria were resistant to β-lactams, tetracyclines and aminoglycosides as previously described in china (chang et al., 2002; yang et al., 2004; liu et al., 2007; tian et al., 2009). the e. coli isolates assessed in this study displayed similar levels of resistance to tetracyclines, ampicillin, gentamicin and fluoroquinolones as were previously reported for e. coli strains isolated from diseased swine in china by tian et al. (2009). high levels of resistance to tetracycline (~60-95%) have also been detected in e. coli isolates recovered from apparently healthy swine on-farm or at slaughter in other countries (kozak et al., 2003; kijima-tanaka et al., 2003; teshager et al., 2000; blake et al., 2003). most of e. coli isolates (67.3%) from swine were resistant to fluoroquinolones (e.g. norfloxacin and ciprofloxacin). somewhat similar findings have been reported in a recent study of clinical e. coli isolates from swine by wang et al., 2010. resistance to amoxicillin-clavulanic acid does not occur frequently in e. coli isolates from diseased swine in china before 2004 (yang et al., 2004), but 21.1% of our swine isolates were resistant to this antibiotic-inhibitor combination. in the present study, amikacin exhibited moderate activity against all strains tested. interestingly, fewer reports of antimicrobial resistance in k. pneumoniae isolated from swine have been published in china. in the present study, k. pneumoniae was the sixth most frequently encountered pathogen in swine and showed high resistance to antimicrobial. k. pneumoniae isolates may be naturally resistant to ampicillin, amoxicillin, carbenicillin, and ticarcillin, but not to extended-spectrum β-lactam antibiotics due to a constitutively expressed chromosomal class a β-lactamase (haeggman et al., 2004). in the present study, the degree of resistance to cephalothin and ceftiofur was 100 and 48.2%, respectively. the antibiotic-inhibitor combination revealed actively to k. pneumoniae. this could be due to the wide use of these classes of cephalosporins in husbandry activities and the considerable increase in prevalence of esbl-producing and multiple-antimicrobial-resistant isolates from pig farms (yang et al., 2004; tian et al., 2009). another important observation in this study is that the ceftiofur resistance has increased. ceftiofur is the only extended-spectrum cephalosporin approved for veterinary use in many countries (salmon et al., 1995). because ceftiofur-resistant organisms are cross resistant to ceftriaxone, the use of this antimicrobial agent in food animals has come under increasing scrutiny as a selective agent potentially responsible for the emergence and dissemination of ceftriaxone resistance in salmonella and other enteric pathogens (alcaine et al., 2005). the rate of resistance to ceftiofur were (9.6%) e. coli, (48.2%) k. pneumoniae, (30.1%) p. mirabilis and (23.8%) s. suis isolates in this study, respectively. these findings are table 1. antibiotic resistance in the clinical bacterial isolates. antimicrobial resistance(%) a agent e. coli k. pneumoniae p. mirabilis staphylococcus s. suis e. faecalis d others amp 88.4 100.0 100.0 51.0 47.6 52.6 67.7 aml 92.3 100.0 100.0 61.2 76.2 52.6 80.6 tic 73.0 76.9 50.0 61.2 sam 19.2 15.3 16.6 10.2 9.6 amc 21.1 15.3 16.6 10.2 12.9 tim 1.9 7.7 2.8 8.1 6.4 kf 40.3 100.0 35.2 40.8 52.3 29.0 eft b 9.6 48.2 30.1 12.2 23.8 16.1 e 65.3 57.1 71.4 da 79.6 85.7 nor 67.3 61.5 83.3 59.1 52.6 54.8 cip 67.3 69.2 83.3 51.0 36.8 45.1 cn 55.7 46.1 83.3 44.9 45.1 amk 44.2 46.1 16.6 30.6 51.6 ot c 98.0 100.0 100.0 83.6 61.9 84.2 87.1 a breakpoints are those recommended by the clinical and laboratory standards institute (clsi);. –, no clsi breakpoints were available, b according to cefotaxime, c according to tetracycline, d including shigella spp.,salmonella spp., h. parasuis, a. lwoffii, p. multocida (aarestrup, 2004). amp, ampicillin; aml, amoxicillin; tic, ticarcillin; tim, ticarcillin/clavulanate; sam, ampicillin/sulbactam; amc, amoxicillin/clavulanate; kf, cephalothin; eft, ceftiofur; e, erythromycin; da, clindamycin; nor, norfloxacin; cip, ciprofloxacin; cn, gentamicin; amk, amikacin; ot, terramycin. very significant difference to the previous reports which displayed high actively to ceftiofur (marie et al., 2002; yang et al., 2004; morioka et al., 2005; zhou et al., 2010: wang et al., 2010). the high incidence of ceftiofur resistance in the k.pneumoniae, p. mirabilis and s. suis isolates tested herein was somewhat unexpected, as this drug was introduce into veterinary clinics for use in china only 2 to 3 years ago. of all the 154 e. coli, 44 k. pneumoniae and 43 p. mirabilis, 140 (92.1%), 44 (100%) and 42 (97.67%) isolates harboured a blatem1 gene. the results of the study indicated that tem-1 was the most common β-lactamase gene among the k. pneumoniae and e. coli isolates, in agreement with previous studies that reported a high prevalence of the blatem-1 gene among animal e. coli isolates (liu et al., 2007; rayamajhi et al., 2008; li et al., 2007; chander et al., 2011). in conclusion, our results confirmed a different prevalence of bacterial infection in swine during 20092010, in southwest, china. the e. coli, staphylococcus spp., s. suis , e. faecalis, k. pneumoniae and p. mirabilis isolates are commonly present among diseased swine. here we have shown that bacterial pathogens from diseased swine exhibited high level 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268: 4346-4351. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (3), pp. 001-007, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper diversity of methanogens in the hindgut of grower and finisher pigs zhen cao1, xin di liao1*, juan boo liang2, yin bao wu1 and bin yu3 1 college of animal science, south china agricultural university, guangzhou, guangdong, 510642, china. 2 institute of tropical agriculture, universiti putra malaysia, serdang, 43400, malaysia. 3 agro-animal husbandry co., ltd. of shenzhen city, shenzhen, guangdong, 518023, china. accepted 07 january, 20119 this study examined the diversity of the methanogens in the hindgut of two different weight groups of pigs and correlated it with the amount of digested organic carbon (oc) and various components of dietary fiber. five grower (58.9 ± 1.15 kg) and five finisher (89.4 ± 0.85 kg) duroc × landrace × large yorkshire female pigs were allocated into two groups and individually housed in cages. during the experiment, feed intake and fecal output were recorded for determination of apparent digestibility of oc, crude fiber (cf), neutral detergent fiber (ndf) and acid detergent fiber (adf). at the end of the digestibility trial, pigs were sacrificed, and the contents of five segments of hindgut were sterilely collected to determine diversity of methanogens. total microbial dna of the hindgut contents was used as template for amplification of the methanogen16s rrna gene, and the pcr products were further subjected to denaturing gradient gel electrophoresis (dgge) analysis. results show that the number of dgge bands and shannon diversity index for the 90 kg pigs were higher (p<0.05) than those for the 60 kg pigs. methanogen communities did not alter along the different segments of the hindgut for the two weight groups. in addition, the amount of oc, cf, ndf and adf digested (g/d) for the 90 kg pigs (1018.77, 23.11, 268.86 and 99.16, respectively) was higher (p<0.05) than the respective values for the 60 kg pigs (669.27, 13.77, 222.31 and 69.07), indicating that the higher diversity of methanogens in the former group was related to the higher quantity of fiber materials fermented in the hindgut. the positive correlation (p<0.05) between number of dgge bands and shannon diversity index with quantity of digested oc and adf further reaffirmed the above suggestion. key words: methanogen, pig, shannon diversity index, polymerase chain reaction-denaturing gradient gel electrophoresis (pcr-dgge). introduction there has been an intense interest in rumen methanogenic archaea because hydrogen is used by methanogen to reduce carbon dioxide (co2) to methane (ch4) gas. it is well documented that diet influenced diversity and population of a wide range of bacterial *corresponding author. e-mail: xdliao2002@yahoo.com.cn. tel: +86-20-85280279. fax: +86-20-85280740. abbreviations: oc, organic carbon; cf, crude fiber; ndf, neutral detergent fiber; adf, acid detergent fiber; pcr, polymerase chain reaction; dgge, denaturing gradient gel electrophoresis. species (tajima et al., 1999, 2000; kocherginskaya, et al., 2001) and methanogen (zhou et al., 2007; zhou et al., 2010) in the rumen. due to its contribution as a greenhouse gas and loss of dietary energy for the host animals, enteric ch4 production from ruminant livestock has been extensively studied (johnson and johnson, 1995; lassey et al., 1997; moss et al., 2000; lassey, 2007; andy thorpe, 2009). on the other hand, studies on enteric ch4 emission from pigs are scarce (jørgensen, 2007; ji et al., 2011). however, due to the large population of pigs, particularly in china, enteric ch4 emission, which has been estimated to represent a 1.2% loss of the ingested energy (monteny et al., 2001) from hindgut fermentation in pigs, cannot be ignored. in addition, pigs mailto:xdliao2002@yahoo.com.cn are an appropriate animal model for gastrointestinal micro-ecological studies of monogastric animals including humans. butine and leedle, (1989) quantified methanogens in cecal and colonic contents of pigs using ruminal fluidbased broth medium and reported that the quantity of methanogens in colonic sample was 30 folds higher than those in the cecum without determining their diversity. although methanogenic archaea community in pig feces and in anaerobic bioreactors fed with pig feces were studied using different molecular techniques (ufnar et al., 2007; liu et al., 2009; zhu et al., 2011; mao et al., 2011), we do not know of any published data on diversity of methanogens in different segments of larger intestine (the major site of feed fermentation in pigs) of pig using polymerase chain reaction-denaturing gradient gel electrophoresis (pcr-dgge). methanogens are phylogenetically placed exclusively as members of the domain archaea (woese, 1987). due to the fact that methanogens are strictly anaerobes and difficult to isolate and culture, phenotypic characters are often insufficient for their identification (woese et al., 1990) and thus molecular ecology techniques, such as 16s rrna gene clone libraries, 16s rrna gene fingerprinting including pcr-dgge, quantitative real-time pcr, fluorescent in situ hybridization (fish) and dna microarray have been used in studies of gastrointestinal microbial communities (amann et al., 1992). pcr-dgge technology was used for the first time to analyze microbial diversity in soil by muyzer, et al. (1993), and then applied in micro-ecological studies of animal gastrointestinal tract (tannock et al., 2000; mccracken et al., 2001; donskey et al., 2003). the advantage of pcrdgge is its simplicity, that is, it can rapidly monitor the spatial-temporal variability of microbial populations by analyzing bands that migrates separately on dgge gel to study the structure and composition of intestinal microbes without using the conventional cultivation procedures. this study was designed to achieve three objectives: (i) to determine and compare the diversity of methanogens in hindgut between two different weight groups of pigs using pcr-dgge technique; (ii) to examine whether methanogen communities alter along the different segments of the hindgut, and (iii) to correlate the amount of digested dietary fiber with methanogenic diversity in hindgut of pigs. materials and methods animals and feeding five grower (mean body weight of 58.9 ± 1.15 kg) and five finisher (89.4 ± 0.85 kg) duroc × landrace × large yorkshire female pigs, purchased from a commercial farm near guangzhou, south china, were used for this study. the pigs were randomly assigned into individual cages (2.0 m long × 1.0 wide) with five animals as replicates per weight group. the experimental pigs were fed ad libitum with the same commercial diet as pigs in the respective weight groups in the farm, twice daily at 07.00 and 19.00 h. the composition and nutrient content of the experimental diets are shown in table 1. the study, carried out during winter with mean outdoor temperature of 20.3°c and the indoor temperature of 23.7°c, consisted of 11 days of adaptation and three days measurement of diet digestibility. pigs were weighed on day one and seven of the experimental period. fresh drinking water was available at all time. digestibility and sampling of intestinal content and feces during the digestibility trial, daily fecal output of each pig was collected, weighed and sampled (200 g) and stored at -20°c. fecal samples were separately dried at 60°c for 72 h and ground through 1 mm and followed by 0.45 mm sieve and the three days fecal samples were pooled for individual pig for determination of their nutrient contents, that is, organic carbon (oc), crude fiber (cf), acid detergent fiber (adf) and neutral detergent fiber (ndf). nutrient apparent digestibility was calculated as: apparent digestibility (%) = (nutrient intake – nutrient excretion) / nutrient intake ×100% and the amount of digested nutrient (g/d) = (nutrient intake – nutrient excretion). at the end of the digestibility trial, 10 pigs were sacrificed, approximately 3 h after feeding. the whole gastrointestinal tract was immediately excised; cecum, colon (proximal, medium and distal) and rectum were ligated and their contents sterilely collected separately into 50 ml sterile centrifuge tubes, and immediately stored at -20°c for later determination of diversity of methanogens using dgge procedure. chemical analysis cf, adf and ndf were determined according to van soest et al. (1991) using f57 filter bag in an ankom fiber analyzer (ankom220 fiber analyzer, ankom technology, usa). oc of diet and feces was analyzed following the method of bao (2000). dna extraction and pcr amplification the total microbial dna isolation from large intestinal contents were extracted using the e.z.n.a. stool dna kit (omega corp, usa) following the procedure provided by the manufacturer. total dna obtained was used as a template for pcr amplification of small subunit rrna gene sequences from the large intestinal contents for domain archaea community. the universal primer pair (wu et al., 2001) a934f (5’ aggaattggcgggggagca-3’) and 1390r-gc(5’ cgcccggggcgcgccccgggcggggcgggggcacgggcg gtgtgtgcaa-3’, with the underlined sequences are the gcclamp region) were used for pcr to amplify 16s rrna gene from members of the domain archaea with a pcr c1000 tm thermal cycler (bio-rad laboratories, inc., usa), using the following program: initial denaturation for 5 min at 94°c; 30 cycles of 94°c for 30 s, 56°c for 30 s, and 72°c for 2 min; and final extension for 10 min at 72°c. dgge analysis of methanogens the aforementioned pcr products were subjected to dgge using bio-rad d-code system. pcr products were separated using a table 1. ingredient (%) and chemical composition of the diets for two weight groups of pigs. the composition and nutrient content of diet diet 1 (60 kg grower pigs) diet 2 (90 kg finisher pigs) composition corn (%) 66 69 bean meal (%) 23 20 rapeseed meal (%) 4 4 ddgs a (%) 3 3 premix b (%) 4 4 nutrient content gross energy (mj/kg) 13.81 13.39 organic carbon (%) 47.04 46.40 crude protein (%) 17.00 16.10 lysine (%) 0.87 0.84 met+cys (%) 0.54 0.51 calcium (%) 0.60 0.53 phosphorus (%) 0.50 0.45 available phosphorus (%) 0.24 0.19 crude fiber (%) 3.90 3.77 neutral detergent fiber ((%) 17.97 16.23 acid detergent fiber (%) 7.99 7.04 a ddgs, soluble distiller’s dried grains. b commercial premix consists of trace elements (fe, cu, zn, mn, i and se), vitamin (a, d, k, e, b1, b2, b6, b12, c, folic acid and biotin), amino acids (lysine, methionine), ca, p and salts. 6.5% polyacrylamide gel in one tris-base, acetic acid and edta (tae) buffer (40 mm tris base, 20 mm glacial acetic acid, 1 mm edta) with a 40 to 65% linear denaturing gradient. the gels were initiated by pre-running for 10 min at 200 v and subsequently ran at 80 v for 21 h at 60°c. then the gels were stained for 10 to 15 min with sybr green and photographed using uv transillumination. gel images were analyzed using labwork 4.0 image analysis software. bands in dgge fingerprints were automatically identified. lanes were individually converted to filled plots by the program. after a background correction was made, the intensity of each band was measured by integrating the area under the peak and expressing the total area in the lane in percentage, and then the shannon diversity index of different lanes were calculated. the formula of shannon diversity index is as follow: s h ' pi ln pi i 1 where, s = numbers of band per lane, and pi = ratio of intensity of each band / total bands. statistical analysis data were analyzed using spss 15.0 (2005). t-test was used to compare treatment means. pearson correlation was adopted to analyze correlation of parameters, and 0.05 level of probability was used to identify differences. results 16s rrna gene fragments of methanogens were amplified by a934f/1390r primer using total microbial dna as template, and all pcr products were 500 bp. pcr products were then used for dgge analysis. pcrdgge profiles obtained from the large intestinal content samples of 60 and 90 kg pigs are presented in figures 1 and 2 and table 2. there were 5 dgge bands for the 60 kg pigs and the average shannon diversity index ranged from 1.33 to 1.38 between cecum to rectum. the higher shannon diversity index indicated higher diversity. the shannon diversity index of distal colon was lowest (1.33), followed by rectum (1.34), proximal colon (1.35), cecum (1.36) and medium colon (1.38) the highest. however, no differences (p>0.05) were observed in the number of band and shannon diversity index among the five segments, indicating no differences in diversity of methanogens throughout in the length of hindgut for the 60 kg pigs. for the 90 kg pigs, the averaged number of dgge bands in hindgut was 7.48 and shannon diversity index ranged from 1.45 to 1.69 for cecum to rectum. the mean number of dgge bands and shannon diversity index for rectum (6.47 and 1.45) were significantly lower (p<0.05) than the other four segments of hindgut, which were not statistically different (p>0.05). results of t-test (table 2) show that the number of dgge bands and shannon diversity index of 90 kg pigs were higher (p<0.05) than 60 kg pigs in four segments except rectum. only apparent digestibility of oc was higher (p<0.05) for the 90 kg pigs compared to the 60 kg pigs (92.05% vs. 89.01%), while no differences were detected in the figure 1. dgge profiles of 60 kg pig. dgge, denaturing gradient gel electrophoresis. figure 2. dgge profiles of 90 kg pig. dgge, denaturing gradient gel electrophoresis. apparent digestibility of the other nutrient between the two age groups. as expected (due to the heavier weight) the amount of digested oc, cf, ndf and adf (g/d) for 90 kg pigs was significantly higher (p<0.05) than those for 60 kg pigs (table 3). results of correlation studies show that the quantity of digested oc (p<0.01) and adf (p<0.05) significantly correlated with number of dgge bands and shannon diversity index (table 4). however, digested ndf only correlated (p<0.05) to number of dgge bands but not with shannon diversity index for the two weight groups (table 4). discussion the number of methanogenic pcr-dgge profiles from steer fed with different diets were reported to range from 22 to 28 (zhou et al., 2010) and 13 for swamp buffaloes fed ch4 mitigating agents, such as coconut oil and garlic powder (kongmun et al., 2011) while the present results recorded only between four to eight bands for pigs. mao et al. (2011) assessed the diversity of methanogens in feces of pig by constructing the 16s rrna gene clone libraries using primers met86f and met1340r and reported clones consisting of 10 phylotypes which belonged to three monophyletic groups. the lower value recorded for pigs could be because, being monogastric animals, most of the ingested feed are digested in the small intestine leaving only the undigested feed to undergo fermentation in the hindgut. this is in accordance with the much lower ch4 production in pigs (ji et al., 2011) compared to ruminants (yamaji et al., table 2. number of band and shannon diversity index between five segments of 60 and 90 kg pigs. segment / index cecum pro-colon med-colon dis-colon rectum number of dgge band for 60 kg 4.33±0.33 b 5.13±0.13 b 5.26±0.18 b 5.07±0.27 b 5.33±0.38 number of dgge band for 90 kg 8.00±0.28 aa 7.67±0.61 aa 7.88±0.48 aa 7.40±0.60 aa 6.47±0.26 b shannon diversity index for 60 kg 1.36±0.01 b 1.35±0.05 b 1.38±0.04 b 1.33±0.05 b 1.34±0.04 shannon diversity index for 90 kg 1.69±0.07 aa 1.67±0.01 aa 1.67±0.05 aa 1.59±0.04 aa 1.45±0.11 b a,b , different superscripts within the same row differed significantly (p<0.05) . a, b , different superscripts within the same column differed significantly (p<0.05). table 3. digestibility and daily quantity of oc, cf, ndf and adf digested for 60 and 90 kg pigs. parameter (kg) organic carbon crude fiber neutral detergent fiber acid detergent fiber digestibility (%) 60 89.01±0.85 a 22.09±2.45 77.36±3.32 54.10±3.31 90 92.05±0.60 b 25.71±2.99 69.46±2.66 59.06±2.88 digested nutrients (g/d) 60 669.27±5.94 a 13.77±1.54 a 222.31±9.97 a 69.07±4.08 a 90 1018.77±6.66 b 23.11±2.67 b 268.86±9.98 b 99.16±4.77 b a, b different superscripts within the same column for the same parameter differed significantly (p<0.05). table 4. the correlation of number of band and shannon diversity index with amount of digested oc, cf, ndf and adf (g/d). parameter digested oc digested digested digested number of shannon (g/d) cf (g/d) ndf (g/d) adf (g/d) band diversity index digested oc (g/d) 1.000 digested cf (g/d) 0.740* 1.000 digested ndf (g/d) 0.757* 0.796** 1.000 digested adf (g/d) 0.882** 0.648* 0.696* 1.000 number of band 0.910** 0.603 0.740* 0.814* 1.000 shannon diversity index 0.868** 0.652* 0.582 0.669* 0.878** 1.000 *p<0.05; **p<0.01. oc, organic carbon; cf, crude fiber; ndf, neutral detergent fiber; adf, acid detergent fiber. 2003; ipcc, 2007; zhou et al., 2007). liu et al. (2009) studied structure of the bacterial and archaeal community in a biogas digester using pig manure as substrate obtained from nine archaeal bands in dgge profile. the aforementioned finding further reaffirmed the lower diversity of methanogens in pigs compared to ruminants. we must emphasize that this study did not carry out gene sequence analysis of the dgge bands to identify the species of methanogens in the large intestinal content of pigs, thus the diversity of methanogens could have been over-or underestimated. however, we believe that the simultaneous use of dgge band and shannon diversity index procedures is sufficient for the primary objectives of this study; that is, to compare the diversity of methanogens in the hindgut between two different weight groups of pigs as well as whether methanogen communities alter along the different segments of their hindguts.fermentation of dietary fiber leads predominantly to the production of volatile fatty acids (vfa), gases (co2, h2, and ch4), ammonia and heat. methanogens obtained their energy by reducing co2 to ch4 using h2 (produced by catalyzing the terminal step in this anaerobic digestion) as the electron donor. results from our study suggest that the diversity of methanogens in hindgut of 90 kg pigs was higher (p<0.05) than that of 60 kg pigs. based on the higher quantities of cf, ndf and adf digested (g/d) in the heavier pigs (table 3), the present results seem to suggest that the higher methanogens diversity in the 90 kg pigs was related to the larger quantity of fermented fiber materials in their hindgut. this is further supported by the fact that the number of dgge bands and shannon diversity index were significantly correlated (p<0.05) with the amount of digested adf and oc (table 4) for the two weight groups. the aforementioed assumption is in agreement with previous study (noblet and goff, 2001) which reported that the ability of the pig to digest dietary fiber improved with the age and live weight and mainly due to changes in the composition of microbial population (without any reference to methanogens) of its hindgut. similarly, zhu et al. (1993) and jensen and jørgensen (1994) reported that the amount of ch4 production increased with increasing fiber content in the diet. recent study from our laboratory (ji et al., 2011) showed that daily enteric ch4 production from 90 kg (2.01 g/pig) was higher (p<0.05) than that from 60 kg (1.13 g/pig). available information from the literature seems to support our view that diversity of methanogenic archaea increased with body weight and/or quantity of fermented materials in the high gut of pigs. no regular pattern of change in the number of dgge bands and shannon diversity index among the different segments of the hindgut in the two weight groups was detected, thus indicating no differences in the diversity of methanogens along the hindgut of the two weight groups. it has been reported that the gastrointestinal tract bacterial community structure is susceptible to changes by the diet of the host animal (durmic et al., 1998; moore et al., 1987). for instance, bacterial community can adapt to the introduction of high levels of dietary fiber by increased growth of bacteria with cellulolytic and xylanolytic activities (varel et al., 1987). however, jensen and jørgensen (1994) found that the density of microorganisms was quite constant throughout the cecum and hindgut for pigs received high and low fiber diets, and they suggested that ch4 production from pig increased with increasing amounts of feed intake and dietary fiber in the diet because greater amount of undigested material would reached the hindgut and provided more substrates for the microorganisms to utilize. none of the aforementioned studies specifically referred to methanogens and thus could not be used for direct comparison with the present study. in conclusion, the number of dgge band and shannon diversity index for 90 kg pigs were higher (p<0.05) than those for 60 kg pigs, thus suggesting higher diversity of methanogen in the hindgut of the heavier finisher pigs compared to the lighter grower pigs. however, no differences in the diversity of methanogen among the different sections of the hindgut were detected in both weight groups of pigs. the amount of digested oc, cf, ndf and adf of the 90 kg pigs were higher (p<0.05) than those of the 60 kg group, indicating that the higher diversity of methanogen in the former group was due to the higher quantity of fiber materials fermented in the hindgut. the significant correlations (p<0.05) between number of dgge band and shannon diversity index with quantity of digested oc and adf further reaffirmed the above suggestion. we do not know of any published data on diversity of methanogenic archaea in the different segments of large intestine of pigs for direct comparison with the results of the present study. references amann ri, stromley j, devereux r, key r, stahl da (1992). molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. appl. environ. microbiol. 58: 614-623. bao sd (2002). chemical analysis 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different dietary and host feed efficiency conditions, as determined by pcr denaturing gradient gel electrophoresis analysis. appl. environ. microbiol. 12: 3776-3786. zhu cg, zhang jy, tang yp, xu zk, song rt (2011). diversity of methanogenic archaea in a biogas reactor fed with swine feces as the mono-substrate by mcra analysis. microbiol. res. 1: 27-35. zhu jq, fowler vr, fuller me (1993). assessment of fermentation in growing pigs given unmolassed sugar beet pulp: a stoichiometic approach. br. j. nutr. 69: 511-525. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (8), pp. 001-006, august, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of low protein diets and lysine supplementation on growth performance and carcass characteristics of growing pigs a. kumar1*, r. bhar1, a. b. mandal2 and s. k. mendiratta3 1 centre of advanced faculty training in animal nutrition, indian veterinary research institute, izatnagar – 243 122, india. 2 avian nutrition and feed technology, central avian research institute, izatnagar – 243 122, india. 3 division of livestock product technology, indian veterinary research institute, izatnagar – 243 122, india. accepted 08 may, 2019 the present study was to assess the effect of feeding low protein diet with or without supplemental lysine to meet nrc (1998) requirement on growth performance, carcass trait, meat composition, and meat quality of pigs. an experiment of 126 days was conducted on 21 crossbred landrace pigs (average weight 11.72 ± 0.57 kg, average age 7.59 ± 0.14 weeks). animals were randomly assigned to three dietary treatment groups t0, t1 and t2 each comprising 7 animals. a basal diet (t0) was formulated as per nrc (1998). basal diet (t1) reduced crude protein by 10% with supplemental lysine identical in t0; while without supplemental lysine in t2 (that is, reduced protein by 10% and lysine by 15%). all the diets were iso-caloric and offered in phases according to change in body weight (10 to 20, 20 to 50, 50 to 80 and >80 kg). growth performance, carcass parameters, meat composition and meat quality were evaluated at the end of the trial on four animals per group. the results show no significant difference on growth parameters and carcass and meat quality traits among the groups. thus, it was concluded that the crude protein and lysine concentration can be reduced safely by 10 and 15%, respectively to that of nrc (1998) in diet of crossbred landrace pigs without any compromise on performance during growing and fattening stage of production. key words: carcass trait, low protein, lysine, meat quality, pigs. introduction proteins and amino acids play a crucial role in the formulation of least cost ration, as they are essential for the normal growth of body tissues, synthesis of macromolecules involved in structural, metabolic and functional activities, reproduction and disease resistance of animals (kaur et al., 2006). the dietary provision of amino acids in correct amounts and proportions determines the adequacy of a dietary protein concentrate. feeding and growing pigs with diets containing higher crude protein level is a metabolic and *corresponding author. e-mail: ajitraj2k@gmail.com. tel: +919319424409. economically costly process. lysine is the first limiting amino acid while methionine is the second limiting amino acid in corn-soybean meal diet for pigs. lysine is the first limiting amino acid for pigs because of two main reasons. first, the concentration of lysine in muscles and other tissue is relatively high (about 7%) and second, many of the feedstuffs (maize grain, sorghum, wheat bran and barley) commonly fed to pigs are quite low in lysine (cromwell, 1996). the different advantages of adding lysine in the diets of pigs are protein sparing effect in pig diets (reduction of dietary cp level by up to 55 g/kg in association with adequate amino acids supplementation does not affect the level of performance and body composition of piglets; le bellego and noblet, 2002); environmental protection (decrease of nitrogen excretion by 15 to 20% with protein reduction of 2% on lysine supplementation; shriver et al., 2003); reducing energy expenditure as less energy is diverted for protein metabolism and elimination of excess nitrogen in the form of urea; reduction of feed cost, increased immuneresponsiveness, improved growth and feed efficiency and improved carcass trait and meat quality. therefore, quantifying its requirement followed by its supplementation in diet through commercial synthetic source (l-lysine hydrochloride 98.5%, pure l-lysine hydrochloride equivalent to 78.8% actual lysine) provides scope for reducing dietary protein supply. one of the major advantages of using low protein amino acid supplemented diet with lysine is the positive impact on environment. reducing cp by 4% unit with the addition of amino acid markedly decreased total n excretion by 40% without influencing growth performance and carcass traits (shriver et al., 2003). in addition, ammonia and other odorous nitrogenous emissions from manure are substantially reduced (carter et al., 1996). the use of lysine supplementation is also economically viable and cost effective, giving higher income over feed costs (main et al., 2008). many experimental trials done in the past, showed positive influence of lysine supplement on growth performance. average daily gain and gain: feed (g: f) ratio improves with increase in lysine to digestible energy (de) ratio (friesen et al., 1994; szabo et al., 2001; apple et al., 2004; main et al., 2008). there are also some reports of improvement in immunity with feeding high lysine diets. the carcass trait and lean percentage of meat also improves with increase of lysine content in diet (loughmiller et al., 1998; witte et al., 2000; szabo et al., 2001; main et al., 2008). therefore, the present research work was conducted to study the effect of feeding low protein diet with or without supplemental lysine to meet nrc (1998) requirement on carcass trait, meat composition, and meat quality of growing pigs. materials and methods experimental animal and diets an experiment of 126 days was conducted on 21 (9 males and 12 females) cross-bred landrace pigs (average weight 11.72 ± 0.57 kg, average age 7.59 ± 0.14 weeks) and were randomly assigned to three dietary groups (t0, t1 and t2). a basal diet (t0) was formulated (table 1) incorporating conventional feedstuffs (maize grain, soyabean meal, and fish meal and wheat bran), salt, mineral and vitamin supplements as per nrc (1998) specifications. the crude protein (cp) content of the t0 was reduced by 10% with supplemental lysine (as l-lysine hydrochloride) to meet the requirement in t1; while without supplemental lysine in t2 (that is, reduced protein by 10% and lysine by 15%). all the diets were isocaloric (3400 kcal/kg de) and offered in phases according to the change in body weight (10 to 20, 20 to 50, 50 to 80 and >80 kg) as per nrc (1998). carcass analysis at the end of the experimental trial, 4 pigs (2 males and 2 females) per group were sacrificed to study the carcass traits and meat quality following standard procedure. all the animals were kept in liarage after arrival at the slaughter house and deprived of feed overnight but with free access to water. they were weighed immediately prior to slaughter and recorded as pre-slaughter live weight of pigs. the pigs were slaughtered after proper stunning at 70 v, 250 ma by electricity. then, bleeding by heart puncturing with knife and wet scalding by hot water at 65°c for 5 to 6 min were performed followed by scrapping and removal of hairs. singeing was done by blow lamp followed by evisceration and removal of gastrointestinal tract (git), the weight was recorded as carcass weight. the weight of hot carcass was expressed as percent of preslaughter live weight to arrive at dressing percentage. carcass length was measured from the front of aitch bone to the middle of the front of first rib using a metal scale. back fat thickness was measured at first rib, last rib and last lumbar using plastic measuring scale and average back fat thickness was determined by taking the mean of all the three values. meat composition and quality analysis samples of longissimus muscle were taken from the carcass after dissection, sealed in polythene bags and stored at -20°c for further analysis. collected samples were analyzed for proximate principles after thawing. moisture, fat, protein and ash contents of dissected longissmus muscles were determined as per the procedures of aoac (1995). the ph of raw meat was determined as per the method described by trout et al. (1992). ten grams of sample were homogenized with 50 ml of distilled water for about a minute in ultra turrex t-18 tissue homogenizer (janke and kenkel, ika labor technik, usa). the ph was recorded by immersing the combined glass electrode of digital ph meter (elico, india, model: l1 114) directly into the meat suspension. the water holding capacity (whc) of raw meat was determined as per the method described by wardlaw et al. (1973) with little modifications. ten grams of finely minced meat sample was homogenized with 15 ml of 0.6 m nacl in a polycarbonate centrifuge bottle for about one minute in ultra turrex t-18 tissue homogenizer. after holding for 15 min at 4°c in order to allow the 0.6 m nacl to reach equilibrium, the meat slurry was again homogenized for 1 min and immediately centrifuged (remi centrifuge, t23, sl no. ggnc 338) at 5500 rpm for 10 min. the supernatant volume was measured and whc was expressed as ml of 0.6 m nacl retained by 100 g of meat. cooked pork chunks (from meat curry) were cut into 1.25 cm 3 cubes. the cut piece was then sheared in a warner-bratzler shear press (model: no. 81031307, g. r. elect. mfg. co., usa). the shear force was recorded (in kg/cm 2 ) as per the method of berry et al. (1980). ten observations were recorded for each sample to get the average value of shear force. statistical analysis results obtained from the study were subjected to one way analysis of variance (anova) as described by snedecor and cochran (1989) using statistical package for the social sciences, version 17(spss 17) software. the data were expressed as mean ± s.e., considering p<0.05 as significant. results and discussion the body weight gain during the entire experimental period and average daily gain (adg) are given in table 2. there was no statistical difference in total body weight and average daily gain among the dietary treatments, table 1. ingredients (% on as fed basis) and nutrient composition (as fed basis – 90.2%dm) of experimental diet as per nrc (1998) for growing pigs. ingredients% 10-20 kg 20-50 kg 50-80 kg 80-120 kg t0 t1 t2 t0 t1 t2 t0 t1 t2 t0 t1 t2 maize 51.19 58.51 57.70 59.10 65.50 64.60 66.79 72.10 71.40 72.90 77.40 76.90 soyabean meal (doc) 29.00 23.00 23.30 21.98 16.62 17.20 15.20 10.86 11.10 9.80 5.97 6.14 wheat bran 13.90 12.40 13.10 14.00 12.80 13.30 13.10 12.00 12.60 13.38 12.60 13.03 fish meal 4.00 4.00 4.00 3.00 3.00 3.00 3.00 3.00 3.00 2.00 2.00 2.00 limestone 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 salt 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 lysine 0.01 0.19 0.00 0.02 0.18 0.00 0.01 0.14 0.00 0.02 0.13 0.03 trace mm and vit premix a 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 total 100 100 100 100 100 100 100 100 100 100 100 100 calculated nutrient composition de (kcal/kg) 3399 3400 3399 3400 3399 3401 3400 3401 3400 3401 3400 3399 cp (%) 20.94 18.78 18.77 18.07 16.16 16.24 15.50 13.94 13.95 13.25 11.88 11.88 lysine (%) 1.15 1.14 1.00 0.95 0.94 0.81 0.77 0.76 0.66 0.61 0.60 0.53 true ileal dig. lys b (%) 1.01 1.01 0.87 0.83 0.83 0.70 0.66 0.66 0.56 0.52 0.52 0.44 true ileal dig. met b (%) 0.31 0.29 0.29 0.27 0.25 0.25 0.25 0.23 0.23 0.21 0.20 0.20 ca (%) 0.70 0.68 0.69 0.64 0.62 0.63 0.62 0.61 0.61 0.56 0.55 0.55 total p (%) 0.63 0.59 0.60 0.58 0.54 0.55 0.54 0.51 0.52 0.49 0.47 0.47 a trace min mix premix (0.1 kg) consisted of 10.8 g zno, 40 g feso4, 1.2 g mnso4, 2 g cuso4, 0.03g ki mixed as per quintal of feed and vit premix (0.1 kg) consisted of vitablend ad3 ( a = 50,000 iu/g, d3 = 5000 iu/g ) and b complex vitamins. b true ileal dig. lysine and methionine of different feed ingredient calculated by multiplying analyzed lysine and methionine to corresponding digestibility values of ingredient nrc (1998). though, treatment t2 showed the highest insignificant final body weight (89.38 kg) and body weight gain (77.65 kg). similar results were obtained by stahly et al. (1981) and asche et al. (1985), who found that the growth rate of pigs fed to appetite, were not significantly affected by dietary protein or non-essential nitrogen content when the levels of essential amino acids were adequately maintained to meet the requirements. moreno et al. (2008) also observed that the dietary lysine to ne ratio had no effect on the growth performance for late-finishing pigs. on the contrary, a positive linear effect of lysine: de on adg was observed by friesen et al. (1994). more recently, schneider et al. (2005) and lenehan et al. (2004) reported a linear increase in adg when diets were provided with 0.9 to 1.3% true ileal digestible lysine in exotic breed with higher growth rate. de la llata et al. (2007) and main et al. (2008) also found that the growth performance improved significantly with increase of dietary lysine to energy ratio. average daily gain in respective groups were not in accordance with the present study as pigs used in the study were crossbred landrace pigs with low growth potential and thus, had lower lysine requirement. the data obtained during the course of the carcass and meat study are presented in table 2. the preslaughter live weight (kg) of pigs did not differ significantly among treatment groups. the hot carcass weights (kg) and length (cm) taken were comparable with highest insignificant values (64.07 kg and 79.25 cm) respectively for t1, table 2. growth performance, carcass traits, meat composition and meat quality of growing pigs in different dietary treatments (t0standard protein and lysine as per nrc, 1998, t1reduced protein by 10% and standard lysine, t2reduced protein by 10% and lysine by 15%). parameter treatment t0 t1 t2 initial body weight (kg) 11.74 ± 1.21 11.73 ± 1.15 11.69 ± 0.71 final body weight (kg) 85.55 ± 3.79 89.38 ± 3.72 81.87 ± 2.11 total body weight gain (kg) 73.81 ± 3.43 77.65 ± 3.06 70.17 ± 2.05 average daily gain (kg) 0.59 ± 0.03 0.62 ± 0.02 0.56 ± 0.02 average daily feed intake (kg) 1.93 ± 0.11 1.99 ± 0.09 1.85 ± 0.05 daily dmi (kg) 1.72 ± 0.10 1.76 ±0.08 1.64 ± 0.03 feed : gain 3.30 ± 0.11 3.23 ± 0.09 3.33 ±0.11 dmi : gain 2.93 ± 0.09 2.85 ± 0.08 2.97±0.10 cpi : gain* 0.55 ± 0.02 a 0.48 ± 0.01 b 0.49 ± 0.02 b carcass traits pre slaughter live weight (kg) 93.63 ±1.34 92.88 ±2.11 89.00 ± 0.58 hot carcass weight (kg) 61.94 ±1.07 64.07±1.96 61.43 ±1.85 hot carcass length (cm) 74.00 ± 3.74 79.25 ± 1.70 74.5 ± 2.10 average back fat thickness (cm) 2.48 ± 0.42 2.83 ± 0.34 2.82 ± 0.07 leaf fat (kg) 1.25 ± 0.22 1.08 ± 0.17 1.49 ± 0.25 dressing percentage 66.18±1.29 68.97±0.95 68.99±1.64 meat composition % moisture 74.00 ±1.10 72.00 ± 0.83 71.90 ± 0.24 cp (n x 6.25) 22.21 ±1.23 23.72 ± 0.31 23.94 ± 0.49 ee 2.70 ±1.09 3.22 ± 0.99 3.08 ± 0.47 ash 1.08 ± 0.06 1.06 ± 0.22 1.08 ± 0.04 meat quality ph 6.03 ± 0.17 6.3 ± 0.04 6.15 ± 0.03 water holding capacity (ml/100 g) 32.5 ± 6.24 32.5 ± 4.79 27.5 ± 2.50 shear force (kg/cm 2 ) 5.53 ± 0.05 5.5 ± 0.07 5.5 ± 0.04 ab values in a row bearing different superscripts differed significantly (p<0.05).* p<0.05. irrespective of dietary treatments. the average back fat thickness (cm), leaf fat yield (kg) and dressing percentage also did not differ (p>0.05) due to the dietary treatments. these findings were in line with the observations of knowles et al. (1998) and shriver et al. (1999), who found similarity in carcass traits of pigs consuming low protein amino acid supplemented diet with those consuming the standard diet. the chemical composition (as percent on fresh basis) of longissimus muscle sampled from each carcass is given in table 2. the mean percent moisture, protein, ee and total ash content of longissimus muscle showed no significant difference (p>0.05) among the dietary treatments. castell et al. (1994) and cameron et al. (1999) also did not observe any changes in longissimus muscle moisture content with the increase of lysine: energy content in swine diets. however, goerl et al. (1995) reported that the moisture content in the longissimus muscle actually increased in response to increasing lysine or cp content in the diet. the cp content of longissimus muscle did not differ due to different treatments. these findings were in contrast to the findings of castell et al. (1994) and grandhi and cliplef (1997) who found that protein content in longissimus muscle increased with increasing dietary lysine level or cp (cromwell et al., 1978; goerl et al., 1995). dietary energy and protein content play an important role in the fat and lean tissue deposition (nrc, 1998). the numerical difference in ee is quite evident between the different groups giving trend of increase in fat deposition with decrease in protein in the diet. this finding is in accordance to those observed by castell et table 3. absolute and relative weight (% bw) of trimmed lean cut & organs of pigs under different treatments (t0standard protein and lysine as per nrc, 1998, t1reduced protein by 10% and standard lysine, t2reduced protein by 10% and lysine by 15%). parameter treatment t0 t1 t2 pre-slaughter live weight (kg) 93.63±1.34 92.88±2.11 89.00±0.58 carcass weight (%) 66.18±1.29 68.97±0.95 68.99±1.64 trimmed lean cuts head, kg 5.67±0.36 4.94±0.36 5.31±0.36 % bw b 6.06±0.38 5.32±0.13 5.97±0.15 leg, kg 2.20±0.28 2.12±0.14 2.18±0.23 % bw 2.35±0.28 2.28±0.11 2.45±0.25 jowl, kg 0.59±0.10 0.71±0.06 0.59±0.06 % bw 0.63±0.11 0.77±0.06 0.66±0.06 butt, kg 4.05±0.70 4.02±0.41 4.03±0.76 % bw 4.31±0.69 4.36±0.54 4.55±0.88 picnic, kg 4.37±0.34 4.39±0.19 4.05±0.44 % bw 4.68±0.40 4.73±0.13 4.55±0.47 belly, kg 4.14±0.13 3.96±0.28 3.83±0.26 % bw 4.43±0.14 4.25±0.23 4.30±0.27 ham, kg 7.21±0.34 6.95±0.09 7.00±0.37 % bw 7.69±0.31 7.50±0.25 7.87±0.41 loin, kg 10.39±0.88 11.70±0.93 9.93±0.97 % bw 11.06±0.78 12.60±0.99 11.16±1.09 organs liver, kg 1.54±0.08 1.36±0.08 1.57±0.12 % bw 1.65±0.07 1.46±0.08 1.77±0.12 heart, kg 0.32±0.03 0.30±0.02 0.33±0.01 % bw 0.34±0.03 0.33±0.02 0.37±0.01 kidney, kg 0.34±0.03 0.39±0.07 0.37±0.02 % bw 0.37±0.02 0.42±0.07 0.42±0.02 spleen, kg 0.23±0.03 0.20±0.01 0.21±0.02 % bw 0.25±0.03 0.22±0.02 0.24±0.02 lung, kg 0.80±0.13 1.01±0.16 0.82±0.14 % bw 0.86±0.15 1.09±0.18 0.92±0.16 git full (%) 7.69±0.59 7.41±0.94 7.51±0.39 % bw 8.25±0.71 7.93±0.86 8.43±0.40 b percent of pre-slaughter live body weight, (p>0.05). al. (1994) and blanchard et al. (1999). on the contrary, it has been reported that lean yield increased due to increase in lysine: energy ratio (grandhi and cliplef, 1997; szabo et al., 2001), lysine level (dourmad et al., 1996; witte et al., 2000) and cp content (cromwell et al., 1978) in pig. the findings related to ash contents were similar to the findings of goerl et al. (1995), who stated that increasing dietary lysine or cp had no effect on the proportion of ash in the longissimus muscle. the physico-chemical properties studied with longissimus muscle sampled from each carcass are given in table 2. the ph, whc (ml/100 g) and shear force (kg/cm 2 ) of the muscle samples ranged from 5.6 to 6.4, 20 to 50 and 5.3 to 5.6, respectively with no significant (p>0.05) difference recorded among dietary treatments. this finding is in accordance to earlier findings indicating no influence on ultimate muscle ph (goerl et al., 1995; witte et al., 2000; szabo et al., 2001), water-holding capacity (goerl et al., 1995) and firmness scores (friesen et al., 1994; grandhi and cliplef, 1997) in pigs fed with diets formulating based on cp, lysine, or lysine: energy ratio. absolute and relative weight (% bw) of trimmed lean cut and organs of pigs under different treatments are given in table 3. trimmed lean cut among different group did not differ significantly. the relative weights of visceral organs were also comparable. the body weight, carcass traits and yield of visceral organs were not affected by 10% reduced crude protein and 15% lysine comparable to nrc (1998). therefore, the results indicate scope for reduction of protein and lysine in diets of crossbred landrace pigs. conclusion from the ongoing findings, it is concluded that the crude protein and lysine concentration can be reduced safely by 10 and 15% respectively to that of nrc (1998) without any compromise on performance of the crossbred landrace pigs during growing and fattening stage of production. references aoac (1995). official methods of analysis (16th ed., vol. i). association of official analytical chemists. washington, d.c. apple jk, maxwell cv, brown dc, friesen kg, musser re, johnson zb, armstrong ta (2004). effects of dietary lysine and energy density on performance and carcass characteristics of finishing pigs fed ractopamine. j. anim. sci., 82: 3277-3287. asche gl, lewis aj, peo jr er, grenshaw jd (1985). the nutritional value of normal and high lysine corns for weanling and growing finishing swine when fed at four lysine levels. j. anim. sci., 60: 1412. berry bw, ray ee, stiffler dm (1980). effect of electrical stimulation and hot boning on sensory and physical characteristics of pre-rigor cooked beef roast. proceedings of the annual meeting of european meat research workers, colorado springs co. 26: 1. blanchard pj, ellis m, warkup cc, hardy b, chadwick jp, deans ga (1999). the influence of rate of lean and fat tissue development on pork eating quality. j. anim. sci., 68: 477-485. cameron nd, penman jc, fisken ac, nute gr, perry am, wood jd (1999). genotype with nutrition interactions for carcass composition and meat quality in pig genotypes selected for components of efficient lean growth rate. j. anim. sci., 69: 69-80. carter sd, cromwell gl, lindemann md, turner lw, bridges tc (1996). reducing n and p excretion by dietary manipulation in growing and finishing pigs. j. anim. sci. 74 (suppl. 1): 59 (abstr.). castell ag, cliplef rl, poste-flynn lm, butler g (1994). performance, carcass and pork characteristics of castrates and gilts self-fed diets differing in protein content and lysine: energy ratio. can. j. anim. sci., 74: 519-528. cromwell gc (1996). amino acid supplements for pigs. http://www.uky.edu/ag/animalsciences/pubs/aminoacidsupplementsf orpigs.pdf [accessed on april 26, 2012] cromwell gl, hays vw, trujillo-figueroa v, kemp jd (1978). effects of dietary protein and energy levels for growing finishing swine on performance, muscle composition and eating quality of pork. j. anim. sci., 47: 505-513. de la llata m, dritz ss, tokach md, goodband rd, nelssen jl (2007). effects of increasing lysine to calorie ratio and added fat for growingfinishing pigs reared in a commercial environment: i. growth performance and carcass characteristics. prof. anim. sci., 23: 417428. dourmad jy, guillou d, se´ve b, henry y (1996). response to dietary lysine supply during the finishing period in pigs. livest. prod. sci., 45: 179-186. friesen kg, nelssen jl, goodband rd, tokach md, unruh ja, kropf dh, kerr bj (1994). influence of dietary lysine on growth and carcass composition of high-lean-growth gilts fed from 34 to 72 kilograms. j. anim. sci., 72: 1761-1770. goerl kf, eilert sj, mandigo rw, chen hy, miller ps (1995). pork characteristics as affected by two populations of swine and six crude protein levels. j. anim. sci., 73: 3621-3626. grandhi rr, cliplef rl (1997). effects of selection for lower back fat, and increased levels of dietary amino acids to digestible energy on growth performance, carcass merit and meat quality in boars, gilts, and barrows. can. j. anim. sci., 77: 487-496. kaur s, mandal ab, singh kb, rajnarain. 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(2003). since it was only detected in bone, this group named this protein as “osteocrin”. they found that osteocrin was not only expressed in young bone cells, but also the expression was age-dependent; the expression level was higher in young animals than that in adults. moreover, vitamin d was found to be able to inhibit the expression of osteocrin in primary osteoblastic cells (thomas et al., 2003). one year later, musclin was identified from mouse skeletal muscle by nishisawa et al. (2004). this group showed that musclin expression was regulated by nutritional changes and insulin. musclin inhibited the insulin-induced glucose uptake and glycogen synthesis in myocyte (nishizawa et al., 2004). and further research indicated *corresponding author. e-mail: mdsys4439@hotmail.com. tel.:+86-29-87092164. fax: +86-29-87092164. abbreviations: rt-pcr, reverse transcription polymerase chain reaction; fas, fatty acid synthesis; ppar , peroxisome proliferator activated receptor ; lpl, lipoprotein lipase; tgh, triacyl glycerol hydrolase. that musclin was mainly related to muscle fiber fast glycolytic phenotypes (staiger et al., 2006; banzet et al., 2007) . all these evidences indicated that the musclin might play an important role in glycogen metabolism. the effect of musclin on lipid metabolism has not been systematically investigated, although nishisawa et al. (2004) claimed that in their preliminary experiments, an adenovirusmediated musclin expression significantly reduced fat mass in mice. here, we aimed to investigate the potential correlation between musclin and lipid metabolism. this preliminary data suggested that muslin might be involved in lipid metabolism. in this study, we examined the expression of musclin in pig fat tissue and primary adipocyte both in vitro and in vivo, and examined its effects on lipid metabolism and possible relationships with other lipogenetic genes. materials and methods experimental animals large white adult pigs, a local strain of chinese pig, were provided by shaanxi guangming pig farm. all animal studies were approved by the institutional animal care and use committee of the university of northwest agriculture and forestry. subcutaneous fat table 1. pcr parameter of primers and conditions. gene primers amplified dna (bp) tm°c mg 2+ (mmol/l) cycles musclin f: atggactggagactggcaag 374 56 3 34 r: cggtttctaccaatccgatc -actin f: actgccgcatcctcttcctc 399 53.8 2.5 28 r:ctcctgcttgctgatccacatc fas f: agtgtccaccaacaagcg 280 55.9 2.5 30 r: gatgccgtcaggtttcag ppar f: accactcgcattcctttgac 261 52.1 2.5 32 r: ccacagactcggcactcaat tgh f: cttggctccttgagatttg 455 53.3 2.5 30 r: agttggcaatgttgtcctg lpl f: gcaggaagtctgaccaataag 183 54.3 2.5 30 r:ggtttctggatgccaatac notes: f: forward; r: reverse and muscle tissue were quickly excised right after the animal was slaughtered, then frozen in liquid nitrogen, and stored at 80 for later use. methods total rna was extracted from adipose and muscle tissue with trizol reagent by following the manufacturer’s instructions (tiangen biothech co.). first strand cdna was prepared with revert aid tm first strand cdna synthesis kit (bio-tech). 20 l of reverse transcription polymerase chain reaction rt-pcr solution contained 6 l depc water, 5 g total rna, 4 l 5× reaction buffer, 1 l random hexamer primers (0.2 g/l), 1 l rnase inhibitor (20 /l), and 1 l mlv reverse transcriptase. the conditions for rt reaction were as follows: 25°c for 10 min, 42°c for 60 min, 70°c for 10 min (for enzyme inactivation), and 4°c for 5 min. the rt products were either used immediately for pcr or stored at 20°c. since there was no swine musclin gene sequence available. we used human and mice sequences as reference to design primers for amplification of pig musclin cdna. the pcr reaction conditions were summarized in table 1. pcr reaction was performed in a total volume of 25 l containing 1 l of tissue-specific cdna, 3 l mgcl2 (25 mmol / l), 0.25 l taq dna polymerase, 2 l dntps (2.5 mmol / l), 2.5 l 10 × buffer and 1 l of each primer (10 mol / l). the pcr product was examined by agarose gel electrophoresis (age). pcr fragment was purified from agarose gel and cloned into pmd 18t vector (takara bio inc.) for sequencing. bio-informatics analysis of swine musclin gene several online programs were applied to analyze signal peptide (http://www.cbs.dtu.dk/services/signalp/) and sub-cellular localization (http://www.cbs.dtu.dk/services/targetp/ and http://psort.nibb.ac.jp/form2.html) of musclin protein. pig preadipocyte and myocyte isolation and culture with pbs containing high concentration of mycillin and then digested with collagenases at 37 for 1 h. cells were then filtered through 40 micron nylon membrane to remove tissue debris and concentrated by centrifugation. isolated cell pellets were resuspended in dmem / f12. for each experiment, preadipocyte and myocyte were seeded in 12 well plates at a density of 10 5 cells per well. primary preadipocyte was grown for preparation of total rna at day 0, 2, 4, 6, 8 and 10 respectively. quantitative pcr musclin mrna from samples of adipose, muscle, primary preadipocyte (cultured for 0, 2, 4, 5, 6, 8 and 10 days) and myocyte (5 days) were determined by quantitative pcr. to determine the relationship between musclin and lipid metabolism, we examined the expression levels of fatty acid synthesis gene (fas), peroxisome proliferators-activated receptor gene (ppar ) and tricycle glycerol hydrolase gene (tgh) in adipose tissue, and lipoprotein lipase gene (lpl) and the gene ppar in primary adipocyte. to quantitatively determine the gene expression, we used -actins gene as the internal control. the primers and pcr amplification conditions were listed in table 1. limited by a lack of real -time pcr equipment, we used a simple and similar real-time pcr technique. briefly, for each sample, we prepared 6 tubes for pcr reaction, and the reaction was set as 26, 28, 30, 32, 34 and 36 cycles respectively. the pcr products were examined by agarose gel electrophoresis. data analysis software spss 13.0 was used for statistical analysis. musclin mrna expression under standard conditions was analyzed with one-way anova and lsd multiple comparison. pearson’s correlation coefficients were used to determine statistical linear associations between musclin and other genes involved in lipid metabolism. all data from samples was shown as means ± standard error (sem). primary pig stromal vascular cell (preadipocyte) and myocyte were results cloning of swine musclin gene obtained from new born pig subcutaneous fat tissue and skeletal muscle respectively, grown and differentiated as previously des we amplified the swine musclin gene using rt-pcr. cribed (hong-mei et al., 2007). in brief, tissues were initially washed figure 1. prediction results of musclin protein from online software. the signal peptide of musclin protein was predicted by neural network analysis from online server signal p 3.0 (a). the s-score is reported for every single amino acid position, with high scores indicating that the corresponding amino acid is part of a signal peptide. c-score should only be significantly high at the cleavage site. y-max is a derivative of the c-score combined with the s-score resulting in a better cleavage site prediction than the raw cscore alone, the high-peaking c-score is the true cleavage site. the s-mean and d-score is calculated separately for the length and the position of the predicted signal peptide. the subcellular localization of musclin protein was predicted by target p sever (b) (mtp: mitochondria, sp: the signal peptide secretion path, other: positioning expressed in cells of other locations agarose gel analysis of pcr product indicated a specific band and the sequencing result showed that the swine musclin cdna consists of 375 bp. the swine musclin cdna sequence was submitted to gen bank (accession no. eu122441). aligned with other species, swine musclin cdna shares the highest homology with human (86%), followed by cow (88%), sheep (87%), rat (78%), mouse (76%) and chicken (59%). bioinformatics analysis using neural network analysis from online server signal p 3.0, we found that swine musclin protein contains a signal peptide on the n-terminus, located from amino acid 1 to 26. a cleavage site was predicted between amino acid 26 27, which suggested that the musclin protein might be released by cell (figure 1a). as was shown in figure 1b, the target p 1.1 program analysis further indicated that musclin was a secreted protein, since it was very low in mitochondria and mostly distributed in the outside of cells, including the cell surface. all these data suggested that swine musclin might function as a cytokine in outside the cell or both extra cellular and intra cellular. different expression pattern of musclin in adipose tissue and muscle tissue, adipocyte and myocyte we compared the musclin gene expression in pig muscle and adipose tissue. the data suggested that musclin expression in adipose tissue was much higher than in muscle tissue (p < 0.01) (figure 2a). to further verify the musclin gene expression in muscle and adipose tissue, we next examined the expression of figure 2. the expression of musclin mrna in different tissues, primary cells (a, b), adipose tissues from pigs of different ages (c) and adipocytes cultured for different days (d), -actin was used as an internal control. the level of musclin mrna in adipose tissue is remarkably higher than muscle (a) (a, adipose tissue; m, muscle tissue). the difference of musclin mrna level between adipocyte and myocyte was not significant (b) (a, adipocyte; m, myocyte). the level of musclin mrna in adipose tissue of ten-month-old pig indicated much lower than a five-month-old (c). the level of musclin mrna in culture adipocytes of different days indicated an age-dependent manner (d). ** p < 0.01. the musclin gene in primary adipocyte and myocyte. the primary adipocyte and myocyte were prepared from young pigs and maintained with dmem / f12 medium for 5 days. the expressions of musclin gene in adipocyte and myocyte had no significant difference (figure 2b). this result confirmed that the musclin gene was indeed expressed in adipose tissue. the expression pattern of musclin gene both in adipose tissue and adipocyte we next examined the musclin gene expression in adipose tissue from different ages of animals. as shown in figure 2c, musclin gene expression in subcutaneous adipose of five-month-old. large white pigs was significantly higher than that in ten-month-old (p < 0.05), suggesting that musclin gene expression is higher in young pig than that in old pig. this expression pattern was further supported by primary adipocyte experiments. the primary adipocytes were prepared from young pig adipose tissues and maintained in dmem / f12 medium. as demonstrated in figure 2d, the expression level of the musclin gene in primary adipocyte decreased with culture time. this time-dependent expression manner of musclin suggested that musclin might be involved in the regulation of cell differentiation or adipose tissue development. correlation between musclin and some lipogenetic genes expressed in adipose tissue and primary cells to support the hypothesis that musclin is involved in adipocyte differentiation and adipose tissue development, we next investigated the relationship between musclin figure 3. the statistical correlation between musclin and lipogenetic genes was analyzed by spss software. the electrophoresis result of musclin and lipogenetic genes mrna level in adipose tissue, lanes 1 and 2: tgh (5-monthold and 10-monthold respectively), lanes 3 and 4 of second line: ppar (5-month-old and 10-month-old respectively), lanes 5 and 6 of second line: fas (5-month-old and 10-month-old respectively) (a). correlation analysis result of musclin and lipogenetic genes in adipose tissue (b). electrophoresis result of lipogenetic genes mrna level in adipocytes, lanes 1 and 2 of second line: lpl (5 days and 10 days respectively), lanes 3 and 4 of second line: ppar (5 days and 10 days respectively) (c). statistical correlation analysis results of musclin and lipogenetic genes in adipocytes (d). ** p < 0.01, *p < 0.05. expression and the expression levels of other lipogenetic genes in adipose tissue and primary adipocytes. fas, ppar and tgh are the main genes that play an important role in lipid metabolism in adipose tissue. applying pearson’s correlation analysis to the data obtained from the adipose tissue samples, we revealed that the expression level of musclin had a negative correlation with fas gene expression (r = -0.969, p = 0.01), had a positive correlation with ppar gene expression (r = 0.836, p < 0.05), and no correlation with tgh gene expression (r = 0.245, p > 0.05). in primary adipocytes, we examined the expression of lpl and ppar genes and compared their expression levels with musclin gene expression (figure 3c and 3d). musclin expression showed a negative correlation with ppar expression (r = 0.820, p < 0.05), and a positive correlation with lpl (r = 0.964, p < 0.01), suggesting a potential linkage between musclin and the lipolysis function of lpl. discussion the musclin gene was originally isolated from mouse bone and muscle tissue and therefore named “osteicrin” or “musclin”. it was initially believed that musclin was exclusively expressed in bone and muscle tissue in thomas et al. (2003) and nishisawa et al. (2004) research, separately. however, in this study, we found that musclin also expressed in adipose tissue and primary adipocytes. we cloned musclin gene from swine adipose tissue and examined its expression pattern in adipose tissues and primary adipocytes. it was found that musclin might influence the regulation of lypolysis (dong et al., 2008). with regard to musclin expression and its possible roles in lipid metabolism, we have addressed four questions. the first question concerned the tissue specificity of musclin gene expression. we found that the musclin expression level was higher in adipose tissue than in muscle. this discovery was completely different from the previous claim that muslin was exclusively expressed in muscle tissue (nishizawa et al., 2004). they reported musclin expression in adipose tissue was not significant. this is probably due to the different expression of musclin between swine and mouse species. the second question compared us in vivo versus in vitro experiments. we found that, between primary adipocyte and myocyte, the difference of musclin gene expression was not significant. this appeared inconsistent with our tissue test results. we hypothesized that the difference between the culture conditions in vitro and the physiological environments in vivo might contribute to the musclin expression pattern in primary cells. the in vitro data strongly suggested that musclin expression in adipose tissue is regulated by other factors. the third question is related to the musclin expression pattern. in the present study, we found that musclin expression in adipose tissue of young swine is higher than old swine. its expression decreased as age increased, suggesting that musclin might be involved in adipocyte proliferation and adipose tissue development. this result is similar to the previous research, which showed that osteocrin /musclin could be used as a marker of early osteoblast maturation due to its strong correlation with early stage of bone formation (thomas et al., 2003; bord et al., 2005). given the fact that fat mass gradually increases with animal growth, the musclin expression pattern implied that musclin might negatively regulate fat mass accumulation in adipose tissue. the fourth question was with regard to the potential function of musclin in lipid metabolism. previous research showed that musclin protein significantly inhibited fat mass accumulation in mice, suggesting musclin could be the underlying link between skeletal muscle and adipose tissue (nishizawa et al., 2004). moreover, the authors also found that insulin regulated the expression of musclin and musclin had feedback effect on insulin. recent studies showed that insulin resistance in skeletal muscle decreased muscle glycogen synthesis, resulting in an increase in plasma triglyceride concentration (petersen et al., 2007). although it was well known that obesity was associated with insulin resistance, much of the relationship between obesity and insulin resistance actually came from visceral and ectopic lipid accumulation (carr et al., 2004; xiao – rong et al., 2008). all these evidences implied that insulin-resistant induced fat mass and the abnormal process of fat accumulation in tissues other than fat tissue could increase the risk of obesity. how musclin is involved in insulin induced fat accumu-lation remains to be further investigated. intramuscular fat development had become a popular target for many scientists. a recent paper showed that 14 differentially expressed genes might participate in the development of intra muscular fat (lee et al., 2007). additionally, considering that musclin was originally identified in muscle, our results provided a new insight with the interaction between adipose tissue and muscle tissue, also musclin might be a candidate gene that participate intra muscular fat development. furthermore, a recent paper reported foxo1, a transcription factor involved in lipid metabolism, inhibited the expression level of the musclin gene (yasui et al., 2007). to further understand the relationship between musclin and obesity, we evaluated the correlation between musclin and key genes of lipid metabolism, and found that musclin had a negative correlation with fas, a key enzyme which regulates de novo lipogenesis and improves triglyceride accumulation in adipose tissue. many studies have shown that insulin, bile acids and feeding could increase fas mrna and protein levels (moustaid et al., 1993; matsukuma et al., 2006; ranganathan et al., 2006). musclin could suppress the function of insulin (nishizawa et al., 2004) . accordingly, we proposed that musclin might negatively regulate fas to restrain lipid synthesis and decrease fat mass. lipoprotein lipase (lpl) is an important enzyme which catalyses the hydrolysis of tricycle glycerol. thiazolidinediones affected adipocyte lpl production through activation of ppar , promoting the hydrolysis of triglyeride (schoonjans et al., 1996). furthermore, insulin might increase the activity and expression of lpl (albalat et al., 2007). therefore, based on the observation that musclin expression was positively correlated with lpl, it suggested that musclin might be involved in lipid degradation. since lipolysis function is a complicated procedure, further study on the interaction between musclin and other lipolysis genes will be conducted in our future experiment. ppar is a member of the nuclear receptor superfamily of transcription factors and previous studies showed that ppar responded to specific ligands by altering gene expression in a cell developmental and sexspecific manner (burns and heuvel, 2007). ppar regulates gene expression in many functional pathways. in this study, the correlation between musclin and ppar in adipose tissue was positive but in adipocyte was negative. this probably was due to some other factors involved in ppar expression on musclin, whereas in in vitro adipocyte culture, these factors were not present. how musclin regulates lipid metabolism via ppar remains to be further elucidated. in conclusion, in the present study, we found that musclin was expressed in both adipose tissue and primary adipocytes, and had a positive effect on lipolysis. our data suggested that musclin represented a novel potential factor which could be involved in lipid metabolism. further elucidation of the function and activity of musclin might provide new avenue for obesity therapeutic approaches. acknowledgements this work was supported by a grant from the national nature science foundation of china (30871785), the program for new century excellent talents in universities, chinese ministry of education (ncet-06-0865) and the project of young aged academic experts from northwest a and f university (yaab-05-22). references albalat a, saera-vila a, capilla e, gutierrez j, perez-sanchez j, navarro i (2007). insulin regulation of lipoprotein lipase (lpl) activity and expression in gilthead sea bream 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www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper genotyping of african swine fever virus (asfv) isolates associated with disease outbreaks in uganda in 2007 carmina gallardo1*, anna r. ademun2, raquel nieto1, noelina nantima 2, marisa arias1, elena martín1, virginia pelayo1 and richard p. bishop3 1 centro de investigación en sanidad animal, inia. valdeolmos, madrid 28130, spain. 2 ministry of agriculture, animal industry and fisheries. entebbe, uganda. 3 international livestock research institute (ilri), p. o. box 30709, nairobi, kenya. accepted 11 january, 2014 samples from infected domestic pigs associated with an outbreak of african swine fever (asf) in three districts of central uganda in 2007 were confirmed as being infected with african swine fever virus (asfv) using a p72 gene based polymerase chain reaction amplification (pcr) assay combined with restriction analysis. none of the sera collected from pigs with clinical symptoms were positive using the oie serological prescribed tests. however, seven haemadsorbing viruses were isolated in macrophage culture and genotyped by partial p72 and full length p54-gene sequencing. four of these viruses were isolated directly from serum samples. all the viruses were classified within the domestic-pig cycleassociated p72 and p54 genotype ix which also includes viruses responsible for asf outbreaks in kenya in 2006 and 2007 and uganda in 2003. to define virus relationships at higher resolution, typing was performed by analysis of tetrameric amino acid repeat regions within the central variable region (cvr) of the b602l gene. ugandan isolates sequences exhibited 100% identity to viruses isolated from outbreaks in kenya in 2007. the identity was greater than the viruses obtained from an earlier outbreak in kenya in 2006. this provides further evidence that genetically similar asfv virus within p72 genotype ix may be circulating between kenya and uganda. key words: african swine fever virus (asfv), restriction analysis, serological detection, genotyping, p72, p54, central variable region (cvr). introduction african swine fever (asf) is caused by a virus, ance-strally associated with argasid ticks and wild suids that can infect domestic pigs, frequently resulting in a rapidly lethal disease. however, depending on the combination of virus genotype and pig breed a range of outcomes varying from acute to chronic disease and apparently asymptomatic animals that are carriers of the virus can be observed. virulent genotypes induce acute haemor-rhagic disease, with symptoms including high fever, heamorrhages in the skin and internal organs and death within three to ten days. asfv is classified within the asfarviridae family, genus asfivirus (dixon et al., 2000, 2005). it is highly contagious and can spread very rapidly in pig populations by direct or indirect contact. the disease has been reported in *corresponding author. e-mail: gallardo@inia.es more than twenty african countries, where the virus is maintained either through a sylvatic cycle involving warthogs ( phacochoerus aethiopicus) and soft ticks in the genus ornithodoros or in a domestic cycle that involves pigs of local breeds, with or without tick involvement (haresnape and wilkinson, 1989; plowright and pierce, 1969; sanchezbotija, 1963; thomson, 1985). asf is endemic in most of sub-saharan africa, including the island of madagascar; the highest incidence of disease being recorded from the equator to the northern transvaal in southern africa. disease outbreaks have also occurred in europe, south america and the caribbean. in 2007, it was introduced into georgia, most probably through infected pig-meat that was unloaded from a ship, recycled and fed to local pigs and has since spread throughout the caucasus and into southern russia (rowlands et al., 2008). uganda, situated in the eastern-central part of africa, is divided into 80 districts and shares borders with kenya, the sudan, drc, rwanda and tanzania. currently, african swine fever is officially described as endemic in the whole country and outbreaks are reported sporadically, but without quantitative data on prevalence. although, no new asf outbreaks have been officially notified since 2004 (world animal health organizationworld organisation for animal health (who-oie), occurrence of hemorrhagic disease has been reported regularly to the ugandan veterinary authorities by pig farmers. a more recent asf outbreak occurred in uganda-kenyan border (busia district) in 2007, placing a significant proportion of the ugandan pig population at risk. in october 2007, an outbreak of virulent hemorrhagic disease was notified in three districts of central uganda, mukono, wakiso and nakasongola. following these outbreaks, blood, serum and tissue samples from pigs with clinical signs of hemorrhagic disease were collected for african swine fever diagnosis and molecular charac-terization. we report here the diagnosis and genetic characterization of asf viruses collected during outbreaks in uganda in 2007. the genotyping involved sequencing the 3’ end of the gene encoding the p72 protein (bastos et al., 2003; boshoff et al., 2007; lubisi et al., 2005) and the full length p54-gene (gallardo et al., 2009) to place isolates into major subgroups, followed by higher resolution sub-typing through analysis of tandem repeat sequences (trs) in the central variable region (cvr) of the asfv b602l gene (irusta et al., 1996; lubisi et al., 2007; nix et al., 2006; phologane et al., 2005). the very close genetic similarity of isolates associated with recent disease outbreaks in both uganda and kenya emphasizes the value of molecular epidemiology for tracing the source and dynamics of asf infections. materials and methods sampling during october 2007, the presence of virulent hemorrhagic disease suspected as being caused by asfv was described by uganda authorities in central uganda districts of mukono, wakiso and nakasongola. eighty four samples were collected by ministry of agriculture, animal industry and fisheries (maaif) staff from pigs with clinical symptoms and submitted to the animal health research centre (cisa-inia), valdeolmos, spain (eu asf reference laboratory). samples collected comprised sera from 39 pigs (wakiso n = 13; nakasangola n = 11; mukono n = 15), edta blood samples from 39 pigs (wakiso n = 11; nakasangola n = 9; mukono n = 19) and six tissue samples from one pig (wakiso n = 6). they were received at cisa-inia on 23 rd october 2007. asf diagnosis asf antibody detection 39 field sera were tested using oie (2008) prescribed tests (conventional elisa and immunoblotting assays) using as antigen a lysate of monkey stable (ms) cells infected with asf spanish isolate e70ms48 and protein-a/hrpo as the reporter (oie 2008). nucleic acid extraction and genomic dna amplification dna was extracted directly from 35 sera, 39 blood samples and 10% suspensions of 6 ground tissues (from one animal) using a nucleic acid extraction kit (nucleospin/ machery-nagel–cultek) following the manufacturer’s procedures. a polymerase chain reaction amplification (pcr) assay using the asf diagnostic primers ppa1/ppa2, which generates an amplicon of 257 bp within the p72 protein (aguero et al., 2003), was used to confirm the presence of asfv dna. the pcr products were analyzed by electrophoresis through 2% agarose gels visualized under uv light. restriction enzyme analysis the ndei restriction endonuclease (new england biolabs) was used to analyze the dna amplified by pcr. briefly, 10 µl of the pcr sample was digested for 1 h at 37°c with 8 to 20 u of restriction enzyme in a total reaction volume of 20 µl in the appropriate restriction enzyme buffer. aliquots of 10 µl of each reaction mixture were size fractionated on a 3% agarose gel and the restriction patterns were visualized under uv light. virus isolation primary leukocyte cultures were used for the isolation of samples recovered from naïve domestic pigs as previously described (malmquist and hay, 1960). briefly, cells were seeded into 96-well tissue culture grade microtitre plates (200 µl; 300,000 cells per well) in homologous swine serum and incubated in a humidified atmosphere containing 5% co2 at 37°c. three day cultures were infected at 1:10 dilution with serum or 10% suspensions of ground tissues supplemented with 5 µg/ml gentamycine sulphate (biowhittaker) and incubated for 24 h at 37°c. after inoculation, a preparation of 1% homologous red blood cells (rbc) in buffered saline was added to each well. the plates were examined for haemadsorption over a 6 day period. the samples were blind passaged three times. asf molecular characterization genomic amplification and nucleotide sequencing for molecular epidemiological analysis, dna was extracted from cell cultures and three separate polymerase chain reactions (pcrs) were set up; for p72 genotyping classification amplifying the cterminal region of p72 protein using the primers p72u/d as previously described (bastos et al., 2003) for p54 genotyping amplifying the complete gene encoding the p54 protein using the primers ppa722/ppa89 (gallardo et al., 2009); for cvr sub-typing using the primer pairs orf9l-f/9l-r to amplify the cvr located in the b602l gene (nix et al., 2006). amplicons of the expected size were excised, purified by quiaex gel extraction (qiagen) and cloned into a pgemt-easy vector (promega) according the manufacturer’s instructions. the nucleotide sequence of the purified products was determined using sp6/t7 primers specific for the pgemt vector using an automated 3730 genetic analyzer (applied biosystems). sequence analysis analysis of sequence data was performed with chromas (www.technelysium.com.au), bioedit (www.mbio.ncsu.edu/ bioedit/ bioedit.html) and clustalx version 1.83 (www.clustal.org) . for the trs, analyses including that of the cvr sequences and deduced amino acid sequences were manually aligned with gaps being table 1. asfv uganda isolates obtained from 2007 outbreak characterized in this study. isolate name id. sample kind of sample origin sequenced ug07.wak1 44a blood ug07.wak2 wakiso 46 sera ssebugwawo mixed farm, wakiso district kinoni ug07.wak3 wakiso 0656 sera ug07.wak4 1128 lymph node tissue ug07.mukono 20 blood katumba wamala farm, mukono district ug07.f7 63/fattener 7 f7 sera kinoni produce farm, nakasongola district ug07.f8 63/fattener 8 f8 sera inserted to optimize the alignment. two datasets were generated for phylogenetic analyses conducted using mega version 4.0 (kumar et al., 2001) (i) a p72-gene dataset comprising 73 taxa (404 characters) in which p72 nucleotide sequences generated in this study from the ugandan pig viruses were analyzed together with homologous sequences from at least two viruses representative of each of the 22 p72 genotypes identified in a previous study (boshoff et al., 2007), (ii) a p54-gene dataset comprising 155 taxa in which p54 sequences generated in this study were compared with homologous sequences available in genbank. neighbor joining (nj) and minimum evolution (me) p72 and p54 trees were constructed employing the p-distance nucleotide substitution model as implemented in the mega v4.0 program. to determine the degree of statistical support for each node in the resulting p72 and p54 trees, data were re-sampled 1000 times using the bootstrap method. results asf diagnosis among the 39 porcine serum samples received at cisa from the central ugandan districts, asfv antibody detection performed using the oie-prescribed elisa and immunoblotting (ib) assays revealed that, none of them were antibody-positive for asf. for asfv detection, pcr was performed on 35 serum samples, 39 blood samples and also 6 individual tissues from one of the sampled animals. following pcr amplification, a single major amplicon of approximately 260 bp was generated from 9 of the 35 sera, 17 of the 39 blood samples and all tissue samples examined. virus isolation was performed using homogenenised pooled tissues and also sera and blood from each pcr positive animal. seven asfv strains with a haemadsorption pattern typical of virulent asf viruses were isolated from the ugandan samples after three passages in leukocytes (table 1). in four cases these virus isolates were made directly from serum samples that were negative according to the oie elisa and ib (table 1). dna was extracted from the cell cultures and the presence of the asf virus was confirmed using diagnostic pcr. the sequencing and compared with sequences available in the genbank and with additional sequences obtained in a separate study from asf isolates recovered during asf outbreaks that occurred in 2003 and 2006 to 2007, in uganda and kenya, respectively (previously unpublished data held at cisa-inia). nucleotide sequence analysis of the genome region delimited by the ppa1/2 primers revealed an ndei restriction endonuclease site, which which could generate two fragments of 204 and 53 bp in length. ndei digestion of the amplicons from the ugandan asfv isolates resulted in generation of the two expected specific restriction fragments (figure 1). this site is conserved only in asfv isolates obtained from eastern african countries, while in sequences from european, caribbean, west and south african countries a mutation is present at bp 54 within the ndei restriction site (table 2). asf molecular characterization analysis of the p72-gene partial sequences from each of the seven uganda asfv isolates showed that, they were identical at the nucleotide level (results not shown). comparison of these sequences to the 22 (i-xxii) currently known major p72 genotypes (boshoff et al., 2007) identified the uganda 2007 sequences as falling within p72 genotype ix (figure 2). recent studies have demonstrated the value of p54-gene sequencing as an additional, intermediate-reso-lution, molecular epidemiological tool for typing of asfv viruses (gallardo et al., 2009) . amplification of the fragment containing the complete p54gene from all the ugandan isolates produced pcr products of 550 bp, which were identical in sequence. the sequences of the 7 uganda isolates were compared with 155 p54 asfv sequences available in genbank. as was the case with the p72genotyping, the uganda isolates were clustered together with ugandan and kenyan viruses obtained from domestic pigs (figure 3). in an attempt to discriminate genotypes at higher resoamplified products were characterized by nucleotide lution, the cvr sequences of viruses from uganda were figure 1. ndei restriction endonuclease analysis of ppa1/2 amplicons derived from different representative asfv isolates. lanes 1 and 1´, ug07.wak.1; 2 and 2´ ug07.wak4; 3 and 3´, ug07.mukono; 4 and 4´, ug07.f7; 5 and 5´, ken06.bus; 6 and 6´, ken07.eld1; 7 and 7´, ug03h1; 8 and 8´, e70; 9 and 9´, cam; 10 and 10´, za. lanes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 show amplification products. lanes 1´, 2´, 3´, 4´, 5´, 6´, 7´, 8´, 9´ and 10´ show amplification products after digestion with ndei. reaction conditions are described in the text. m, molecular weight marker vi. table 2. partial nucleotide sequences showing residues 1 to 60 within the p72gene pcr amplicon used for asfv diagnosis. the ndei restriction site conserved in kenya and uganda asfv isolates is highlighted in grey. isolate p72 country of 3´ 5´ nucleotide sequences from p72gene p72-based diagnosis reference genotype origin pcr diagnosis product genbank accession no. e70 i spain agttatgggaaacccgaccccgaacccactt ay578692 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt e75 i spain agttatgggaaacccgaccccgaacccactt ay578693 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt ba71v i spain agttatgggaaacccgaccccgaacccactt u18466 complete genome tgagtcaaatcgaagaaacacatttggtt haiti i haiti agttatgggaaacccgaccccgaacccactt ay578695 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt tengani62 v malawi aagtacgggaaacccgaccccgaacccactt ay578704 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg m1 iii south africa agttatgggaaacccgaccccgaacccactt ay578699 zsak et al. (2005) drc * tgagtcaaatcgaagaaacgcatttggtg za i agttatgggaaacccgaccccgaacccactt ay578708 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt mk i south africa agttatgggaaacccgatcccgaacccacttt ay578700 zsak et al. (2005) gagtcaaatcgaagaaacacatttggtg wart iv namibia agttatgggaaacccgaccccgaacccactt ay578706 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg cam i cameroon agttatgggaaacccgaccccgaacccactt ay578689 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt vic i zimbabwe agttatgggaaacccgatcccgaacccacttt ay578705 zsak et al. (2005) gagtcaaatcgaagaaacacatttggtg wb iii south africa agttatgggaaacccgaccccgaacccactt ay578707 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg cro1.2 iv south africa agttatgggaaacctgaccccgaacccacttt ay578690 zsak et al. (2005) gagtcaaatcgaagaaacacatttggtg cro 3.5 iv south africa agttatgggaaacctgaccccgaacccacttt ay578691 zsak et al. (2005) gagtcaaatcgaagaaacacatttggtg table 2. continued. f6 iii south africa agttatgggaaacccgaccccgaacccactt ay578694 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg k1 iii south africa agttatgggaaacccgaccccgaacccactt ay578696 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg o1 iii south africa agttatgggaaacccgaccccgaacccactt ay578701 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtt pr4 xx south africa agttatgggaaacctgaccccgaacccactt ay578702 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg pr5 xx south africa agttatgggaaacctgaccccgaacccactt ay578703 zsak et al. (2005) tgagtcaaatcgaagaaacacatttggtg ug03h.1 ix uganda agttatgggaaacccgaccccgaacccactt gq916933 this study tgagtcaaatcgaagaaacacatatggtg ug03p.4 ix uganda agttatgggaaacccgaccccgaacccactt gq916934 this study tgagtcaaatcgaagaaacacatatggtg ug07.wak1 ix uganda agttatgggaaacccgaccccgaacccactt gq916926 this study tgagtcaaatcgaagaaacacatatggtg ug07.wak2 ix uganda agttatgggaaacccgaccccgaacccactt gq916927 this study tgagtcaaatcgaagaaacacatatggtg ug07.wak3 ix uganda agttatgggaaacccgaccccgaacccactt gq916928 this study tgagtcaaatcgaagaaacacatatggtg ug07.wak4 ix uganda agttatgggaaacccgaccccgaacccactt gq916929 this study tgagtcaaatcgaagaaacacatatggtg ug07.mukono ix uganda agttatgggaaacccgaccccgaacccactt gq916930 this study tgagtcaaatcgaagaaacacatatggtg ug07.f7 ix uganda agttatgggaaacccgaccccgaacccactt gq916931 this study tgagtcaaatcgaagaaacacatatggtg ug07.f8 ix uganda agttatgggaaacccgaccccgaacccactt gq916932 this study tgagtcaaatcgaagaaacacatatggtg kenya1950 x kenya agttatgggaaacccgaccccgaacccactt ay261360 complete genome tgagtcaaatcgaagaaacacatatggtg ken06.b1 ix kenya agttatgggaaacccgaccccgaacccactt gq916935 this study tgagtcaaatcgaagaaacacatatggtg ken06.b2 ix kenya agttatgggaaacccgaccccgaacccactt gq916936 this study tgagtcaaatcgaagaaacacatatggtg ken06.b3 ix kenya agttatgggaaacccgaccccgaacccactt gq916937 this study tgagtcaaatcgaagaaacacatatggtg ken06.b4 ix kenya agttatgggaaacccgaccccgaacccactt gq916938 this study tgagtcaaatcgaagaaacacatatggtg ken06.b5 ix kenya agttatgggaaacccgaccccgaacccactt gq916939 this study tgagtcaaatcgaagaaacacatatggtg ken06.bus ix kenya agttatgggaaacccgaccccgaacccactt gq916940 this study tgagtcaaatcgaagaaacacatatggtg ken06.kis ix kenya agttatgggaaacccgaccccgaacccactt gq916941 this study tgagtcaaatcgaagaaacacatatggtg ken07.eld1 ix kenya agttatgggaaacccgaccccgaacccactt gq916942 this study tgagtcaaatcgaagaaacacatatggtg ken07.eld2 ix kenya agttatgggaaacccgaccccgaacccactt gq916943 this study tgagtcaaatcgaagaaacacatatggtg ken07.kia ix kenya agttatgggaaacccgaccccgaacccactt gq916944 this study tgagtcaaatcgaagaaacacatatggtg ken07.nak ix kenya agttatgggaaacccgaccccgaacccactt gq916945 this study tgagtcaaatcgaagaaacacatatggtg determined. amplification of the cvr fragment generated pcr products of approximately 350 b from all ugandan isolates. as in the case of the other genes, the uganda isolates characterized in this study clustered with the figure 2. minimum evolution (me) tree representing the 22 p72 genotypes that distinguish african swine fever viruses (labelled i-xxii) illustrating the phylogenetic position of the ugandan isolates from 2007. the optimal tree with the sum of branch lengths = 0.23816201 is shown. the percentage of replicate trees in which the associated taxa clustered together according to bootstrap analysis (1000 replicates) is marked adjacent to the nodes. the me tree was searched using the close-neighbor interchange (cni) algorithm at a search level of 1. a neighbor-joining algorithm was used to generate the initial tree. the seven ugandan viruses characterized in this study are highlighted in grey ( ) within genotype ix (marked in grey). figure 3. minimum evolution phylogenetic tree based on full length p54 gene sequences illustrating the position of uganda 2007 isolates relative to other asfv viruses. an unrooted minimum evolution tree was derived from alignments of complete p54-gene nucleotide sequences. the me tree was searched using the closeneighbor-interchange (cni) algorithm at a search level of 1. a neighbor-joining algorithm was used to generate the initial tree. the seven ugandan viruses characterized in this study are highlighted in black ( ). uganda isolates (uga 95/1; ug03h1-3, ug03p4-6) and kenya isolates (ken06b1-5, ken06.bus, ken06.kis, ken07eld1-2, ken07.nak, ken07.kia) within cvr subgroup xxiv. differences were identified mainly in the number of tetrameric amino acid repeats. when the tetrameric repeats within the b602l gene from the uganda isolates were compared with viruses included within the cvr sub-group xxiv (nix et al., 2006), the isolates from uganda obtained in october 2007, contained a minor change due to the absence of a single internally located tetrameric repeat (cadt) present in the viruses recovered from earlier uganda outbreaks that occurred in 2003 and in the kenyan asf visolates obtained in may 2006 (ken06.b1-5). however, they were identical to figure 4. amino acid sequence alignment of tetrameric tandem repeats within the central variable region (cvr) of gene b602l from ugandan and kenyan p72 group ix isolates associated with recent outbreaks. the isolates from the ugandan 2007 outbreaks are underlined. the ugandan 2007 isolate sequences determined in this study were aligned with additional kenyan and ugandan cvr sequences available in genbank (uga95/1, accession no. caj90783; ug03h1-3 and ug03p4-6 accession nos fj174339-44; ken06.b1-b5, ken06.bus, ken06.kis, ken07eld1-2, ken07.nak, ken07.kia, accession nos fj174329-39) belonging to b602l sub-group xxiv. those obtained from the second outbreak that occurred in western kenya in 2006 (ken06.bus) and to all isolates from subsequent outbreaks in the country (figure 4). discussion outbreaks of african swine fever have been reported in uganda sporadically, but regularly in specific areas since 1996 and the pig industry has been very significantly affected. the last major outbreak, according to the oie reports was in 2004 when an estimated 7,500 pigs were destroyed or slaughtered in twelve districts across the country. since then, outbreaks of asf have occurred on a regular basis in uganda, causing serious losses to both smallholder pig keepers and commercial pig farms. at present, asf is officially described as "present in the whole country but without quantitative data on the prevalence of the virus". in october 2007, a suspected african swine fever outbreak was reported by farmers in three central uganda districts. sampling in these areas in collaboration with the uganda maaif resulted in confirmation of gene based pcr and virus isolation at the cisa-inia eu reference laboratory. subsequent nucleotide sequence determi-nation of the pcr product allowed identification of a new ndei restriction site, specific for a particular group of viruses belonging to east african countries. the res-triction fragment pattern enables the specificity of the amplicons to be validated within hours. however, the method does not permit unambiguous classification of virus isolates because of the high variability observed in east african asvf isolates. although, nucleotide sequencing of the p72 amplicon would be more definitive, the technique is expensive, time consuming and less universally available in african laboratories. restriction digestion with nde1 therefore, represents a useful rapid method for confirmation of the presence of asfv following p72 pcr. interestingly, no positive antibody response was detected using oie-prescribed serological methods in any of the serum samples collected from these outbreaks. this data reconfirms previous studies that describe a low incidence of detectable serological response to asfv infection in pigs from east african countries using both recombinant antigen and oie-prescribed african swine fever serological methods (perez-filgueira et al., 2006). since the oie-prescribed methods are based on the use of total asf virus as antigen that includes many viral proteins, a possible explanation for the unexpectedly low seropositive response with east african sera may reside in the immunogenetics of the indigenous pig populations in the region and is unlikely to be due to polymorphisms in immunodominant viral antigens. the area of host-virus interaction, including possible differences in both innate and acquired immune responses therefore, represents an important area for future research on african swine fever (asf). to characterize the viruses responsible for the out-break of asf in uganda in 2007 and identify genetic relationships with recent asfv isolates from neighboring countries, the sequence of three regions of the asfv genome was determined. sequence analysis of the cterminal end of p72-gene and complete p54-gene placed the uganda isolates within genotype ix together with isolates from kenya and uganda. this is a domestic pig associated genotype that contains 8 isolates from western and eastern uganda (uga 1/95; ug03h1-3, ug03p4-6, uga2003/1), isolated from outbreaks in 1995 and 2003 (bastos et al., 2003; gallardo et al., 2009) and 11 isolates from western ands central kenya (ken06b1-5, ken06.bus, ken06.kis, ken07eld1-2, ken07.nak, ken07.kia) obtained from outbreaks in 2006 to 2007 (gallardo et al., 2009). one additional genetic type of asfv was also identified in this study as having occurred in uganda; this is classified within p72 genotype x that comprises viruses recovered from both domestic and wild pigs and the arthropod tick vector. the data generated using three genes indicated that, african swine fever outbreaks occurring in the three viruses whilst the 1995 outbreak was caused by two diffe rent viruses belonging to p72 genotypes ix and x (uga/1/95 and uga/3/95) (boshoff et al., 2007; lubisi et al., 2005). sequence analysis of the central variable region (cvr) from the ugandan isolates identified 22 different tetrameric amino acid units. the sequence of the cvr from the ugandan isolates was identical to the sequence of 6 isolates responsible for the second wave of infections that occurred in western and central kenya from october 2006 to january 2007, but was distinguishable from kenyan isolates recovered from an earlier outbreak in may 2006 and from ugandan viruses recovered from the outbreaks that occurred in western and eastern uganda in 2003. a deletion of one tetrameric repeat that was observed in viruses recovered from the 2007 ugandan outbreak was also present in viruses responsible for an outbreak that occurred in the uganda-kenya border district, subsequently spreading to central kenya, in 2006 to 2007. this is consistent with a single origin for the kenyan 2006 to 2007 and ugandan 2007 outbreaks, if there is rapid cross-border movement of the virus. the data from the cvr analysis are consistent with the hypothesis that, asfv virus exchange between the two countries has occurred on more than one occasion, although, an alternative explanation would be that identical mutations have occurred independently in the central variable region of two closely related virus lineages. such information should be proved useful to veterinary authorities for better disease control and highlight the requirement for improved regulation and monitoring of domestic pig movement and the pork product trade. acknowledgements the present work was partly supported under the grant ilri2003-001 funded by the spanish ministerio de innovacion y ciencia and by the european union through the network of excellence "epizone” and also the asfrisk project 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rowlands rj, michaud v, heath l, hutchings g, oura c, vosloo w, dwarka r, onashvili t, albina e, dixon lk (2008). african swine fever virus isolate, georgia, emerg. infect. dis. 2008 dec. 14(12): 1870-1874. sanchez botija c (1963). reservatoirios del virus de la peste porcina africana. investigacion del virus de la ppa en los atropodos mediante prova de la hemadsorcion. bull. off. int. epiz. 60: 895-899. thomson gr (1985) the epidemiology of asf: the role of free-living hosts in africa. onderstepoort j. vet. res. 52: 201-209. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (6), pp. 001-008, june, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper apolipoprotein d, apolipoprotein r and st6galnac4 genes respond to clenbuterol administration in pig adipose tissue jin zhang*, jiali lin, liangcai shen, fan zhang, yingbo zhu and libin zheng college of life science and technology, hebei normal university of science and technology, qinhuangdao, hebei province, 066004, china. accepted 05 october, 2017 pig fat accumulation can be reduced by feeding pigs with high dosages of clenbuterol, but the molecular mechanism has not yet been well characterized. cdna microarray and real-time pcr were used to identify the molecules potentially responding to clenbuterol in adipose cells. the mrnas of 17 genes were found to be differentially expressed more than 2 fold (ratios of the expression levels in the test pigs to those in the control pigs of more than 2 or less than 0.5) by microarray analysis, including three lipid metabolism related genes (apolipoprotein d, apolipoprotein r and st6galnac4). eight genes were verified by real-time pcr, with six of them reproducing the result from microarray analyses. the results showed that apolipoprotein d, apolipoprotein r and st6galnac4 genes respond to clenbuterol administration in pig adipose tissue and their functions may relate to fat accumulation reduction. key words: pig, clenbuterol, adipose, cdna microarray, real-time polymerase chain reaction (pcr). introduction adipose tissue plays critical roles in the regulation of energy homeostasis (gesta et al., 2007). excessive accumulation of adipose tissue has many negative effects (pospisilik et al., 2010). how to reduce adipose accu-mulation safely and effectively is of great importance both for human health and animal breeding. clenbuterol, a type of β2-agonist, can reduce body fat drastically (plant, 2003; sato, 2010). but clenbuterol is toxic both for animals and human and the usage has been banned for many years (kuiper et al., 1998; mitchell and dunnavan, 1998; shiu and chong, 2001). clenbuterol perturbs cell metabolism by binding to the β2adrenergic receptors and increasing the cyclic amp concentration in cells. in adipocytes, stimulation of βadrenergic receptors (by hormones) increases cyclic amp levels and activates protein kinase a (pka), which then stimulates lipolysis by phosphory-lating hormone-sensitive lipase and perilipin (londos et al., 1985; egan et al., 1990; greenberg et al., 1991; zhang et al., 2005). however, the molecular mechanism underlying the effect of clenbuterol on adipose *corresponding author. e-mail: zhangjin7688@163.com. tel: +86-335-8069613. fax: +86-335-8076166. accumulation is still not well understood. unveiling the mechanism will potentially identify new molecules that might play important roles in regulating adipose metabolism. dna microarray technology has been widely used as a powerful tool for functional genomics study in both basic science research and biomedicine research (petrik et al., 2006; sawada et al., 2010). however, the application of this technology to animal genetics and breeding related research is rare. in this study, the genes responding to clenbuterol were profiled by cdna microarray and real-time pcr techniques in fat tissue of chinese miniature pigs treated with/without clenbuterol. these genes are potential candidates to be used in developing high lean pig breeding and also are potential drug targets for human obesity treatment. materials and methods animal sampling, clenbuterol treatment and body composition analysis in total, 8 chinese miniature pigs were used in the experiments. four hogs and four sows, all at 4 weeks of age, were housed in the nutrition and metabolism laboratory at the china agriculture group a group b group c group d control hog1 sow1 hog3 sow3 treated hog2 hog2 sovw2 h hog44 s sow44 3-month-old 4-month-old figure 1. the groups of pig population. eight pigs were broken into four groups. there were two pigs in each group with the same gender. group a and b were slaughtered when they were 3month-old. group c and d were slaughtered at 4-month-old. university. they were raised under exactly the same conditions and were fed the same diets until 8 weeks old (average body weight 17.4±1.2 kg, table 3). they were broken into 4 groups named group a, b, c and d (figure 1), each group consisting of two pigs with the same gender. for the following 4 weeks, one pig in each group was fed 25 mg/kg clenbuterol twice daily in the diets, while the other was fed the same diet without clenbuterol as the control. pigs in group a and b were slaughtered for analysis (at 3 months old), while pigs in groups c and d were continued feeding with/without 50 mg/kg clenbuterol twice daily in their diets for another 4 weeks and then slaughtered for analysis. these two groups are referred to as the 4 month-old pigs. approximately, 1 g biopsies were taken from the back fat adipose tissues of each pig. the adipose tissue samples were rinsed in sterile water, snap frozen in liquid nitrogen and stored at -80°c. body composition analysis was done for each group just after the slaughter. the fat thickness were measured at the 10th rib by using standard procedures and the carcass lean content for each pig was also calculated (national pork board, 2000). hplc analysis for clenbuterol residue in different tissues the samples were sent to livestock and poultry quality inspection center of china ministry of agriculture and clenbuterol residue was analyzed according to ny/xq421-2003, which is the standard method for determination of clenbuterol residues in animal product with gc/ms. rna preparation, rna labeling and dna microarray hybridization total rna of the adipose tissue was extracted with trizol reagent (invitrogen, gaithersburg, md, usa). briefly, the sample was put into mortar with liquid nitrogen and grinded into power. rna was extracted from 100 mg of sample with 1000 ml trizol added and following the manufacturer’s instructions. the rna was purified using an rneasy mini kit (qiagen, valencia, ca, usa) according to the manufactures’ protocol. a cdna microarray representing 3358 pig genes was developed by our group (zhang, 2011). a cdna microarray hybridization analysis was performed in accordance with the standard protocol provided anywhere (zhang et al., 2011). dna microarray imaging and data analysis arrays were scanned with a scanarray express scanner (parckard bioscience, kanata, ot, u.s.) with the obtained images analyzed with genepix pro 4.0 (axon instruments, foster city, ca). the resulting microarray data was normalized using the space and intensity-dependent normalization in the lowess program (yang et al., 2002). each gene was represented in triplicate on each slide. the ratio presented for each gene was the mean of the “median of ratios” of 3 spots for this gene. for each slide, the positive genes were defined as those with gene ratios more than 2 (including 2) or less than 0.5 (including 0.5). to avoid dye bias, the experiments were performed in duplicate by dye swap with cy5-dctp first used with the test pig and cy3-dctp used with the control pig and then cy3-dctp used with the test pig and cy5-dctp used with the control pig. the positive genes in both dye swap slides were identified as the differentially expressed genes. differentially expressed genes in both the hogs and the sows of the same age were chosen for further analysis. quantitative real-time pcr to confirm the transcriptional differences observed by the microarray, fluorescent real-time pcr was done on an abi prism 9700 sequence detection system (applied biosystems, foster, ca, usa) using sybr green technology as described by li (2005). briefly, the pcr reaction mixture (20 µl) contained 10 µl of dynamo sybr green qpcr mix, 5 µl of primer (0.3 µm forward and 0.3 µm reverse) and 5 µl of cdna template (<10 ng/µl). the pcr protocol included uracil-n-glycosylase (ung) enzyme incubation at 50°c for 2 min and an initial denaturation at 95°c for 10 min. this was followed by 40 cycles of 10 s each at 94°c for dna denaturation, 20 s at different temperatures for annealing of primers, 20 s at 72°c for primer extension and 1 s at a different elevated temperature for data acquisition. the pcr primer sequences used for the real-time pcr are shown in table 1. the size of the pcr products was between 100 to 250 bp for the benefit of fluorescent signals accuracy. the annealing temperature of each gene was about 57±2°c so the pcr reaction for different genes could be done on the same plate. all primers were tested by none fluorescent pcr at first and the product size was confirmed by agarose gel. the quantification was normalized to an endogenous rna control glyceraidehyde-3-phosphate dehydrogenase (gapdh, a house sow 2 table 1. primers used for the real-time pcr analysis. genes and clone number primer sequence (5' to 3') pcr product size (bp) gapdh for. atggtgaaggtcggagtga 154 rev. atgggtagaatcatactgga apoliprotein d (rpfat_18926) for. agatcccagtgagctttgag 233 rev. cgtagttctcatagtcggtg prkar1a (rpfat_17661) for. ggcgacgaggtgctatcag 159 rev. atggcatcaaaaatatcac col1a1 (rpfat_8523) for. tcaagatgtgccactccgact 104 rev. gcctgtctccatgttgcagaa col1a2 (rpfat_16033) for. atatgcaccttggacatcggt 241 rev. cacgatgctctgatcaatcct col3a1 (rpfat_19990) for. cctgctggaaagaatggtgac 132 rev. acgttcaccggtttcacctt col1a2 (rpfat_17393) for. cctggctctagaggtgaacg 247 rev. agcaggaccaggattaccag col3a1 (rpfat_18309) for. tttcttttatggctccccctg 101 rev. gcgtgttcgatattcgaagac col3a1 (rpfat_19990) for. ctgctggaaagaatggtgac 132 rev. acgttcaccggtttcacctt scd (rpfat_16685) for. aaggaactagaaggctgctc 156 rev. tgtagagcagcagccatcac phpt1 (rpfat_15312) for. gaagacacagttgaggacac 110 rev. ggacattgttcggaggatag hsl (rpfat_11096) for. tccgaatggagtctgcactgt 128 rev. cttccactctgacctccaacg pmp22 (rpfat_18575) for. catgaacatttgcaccacttg 133 rev. gtcagcacctaatggtatgga for.: forward; rev.: reverse. table 2. clenbuterol residue in porcine blood and adipose. pig number (ng/ml) 3-month-old group 4-month-old group hog 1 hog 2 sow 1 sow 2 hog 3 hog 4 sow 3 sow 4 blood 0.00 10.00 0.00 26.44 0.00 60.57 0.00 176.08 adipose 0.00 0.00 0.00 1.62 0.00 5.32 -:not determined. keeping gene) which its expression level was unchanged to clenbuterol stimulation according to none fluorescent pcr analysis. results clenbuterol administration dramatically reduced adipose accumulation hplc analyses of the blood samples showed that the clenbuterol concentrations in the test pigs fed with clenbuterol were about 20 ng/ml in 3-month-old pigs and about 100 ng/ml in the 4-month-old pigs. clenbuterol could not be detected in the control pigs fed without clenbuterol (table 2). there was no difference in body weight between the test pigs and control pigs (table 3), but there was significant difference in body compositions (table 4). for 3month-old pigs, the lean meat percentage in the carcass was improved by about 2%, the back fat thickness was reduced about 0.2 cm and the eye muscle area was reduced by 4.7 cm 2 when treated with clenbuterol. for 4month-old pigs, the clenbuterol effects were more significant with the lean meat percentage in the carcass increased by 10.99%, the back fat reduce by table 3. body weight of pigs treated with/without clenbuterol. pig number 3-month-old group 4-month-old group hog1 hog2 sow1 sow2 hog 3 hog 4 sow3 sow 4 clenbuterol (mg/kg bw a ) 0 25 0 25 0 50 b 0 50 b 2-month bw (kg) 18.6 17.4 17.5 16.2 16.8 17.2 17.5 18.3 3-month bw (kg) 33 .2 32.6 30.6 29.8 30.5 32 31 32.2 4-month bw (kg) 45.6 46.6 46 46.4 increased bw (kg) 14.6 15.2 13.1 13.6 28.8 c 29.4 c 28.5 c 28.1 c a, bw : body weight; b, 25 mg/kg body weight for the 3 rd month and 50 mg/kg body weight for the 4 th month; c, increased body weight in the 3 rd month and increased body weight in the 4 th month. table 4. body composition of pigs treated with/without clenbuterol. clenbuterol administration* hog2 and sow2 hog1 andsow1 hog4 and sow4 hog3 andsow3 3-month-old 3-month-old 4-month-old 4-month-old (mg/kg body weight) 0 25 0 50 slaughter rate (%) 63.75 60.86 66.80 64.63 lean meat percentage of 52.40 55.24 41.45 52.44 carcass(%) thickness of back fat (m) 2.336 2.118 3.200 2.818 eye muscle area (cm 2 ) 19.488 24.199 23.083 25.270 *twice daily. the carcass lean meat percentage, the thickness of back fact and eye muscle area were all changed statistically between test pigs and control pigs in both 3 month old pigs and 4 month old pigs (p < 0.05). 1.02 cm and the eye-muscle area reduced by 2.18 cm 2 (table 4). seventeen genes were up-regulated in adipose tissue by clenbuterol treatment genes whose expression levels were changed by more than or equal to two fold in clenbuterol treatment group versus control group were selected as differentially expressed genes. there were 56 differentially expressed genes (35 up-regulated, 21 down-regulated) in the 3 month-old group and 58 differentially expressed genes (41 up-regulated, 17 down regulated) in the 4 month-old group (data not shown). 17 genes were differentially expressed in both groups (table 4). the purpose of this study was to found the key molecules by which clenbuterol reduce pig fat accumulation. therefore, the overlapping differentially expressed genes in 3 and 4month old group were considered as the stable responding genes for clenbuterol stimulation in pig adipose tissue and thus, selected for the first stage analysis. among the 17 genes, two genes related to signal transduction were found to be up-regulated these genes are camp dependent protein kinase type i regulatory gene (prkar1a) and rab30. up-regulation of prkar1a indicates increasing protein kinase a (pka) activity. three genes directly related to lipids metabolism were found to be up-regulated: apolipoprotein d (apod), apolipoprotein r (apor) and st6galnac4. furthermore, eight clones on the microarray were from three genes related to collagen protein synthesis which were up-regulated more than two folds: type i collagen alpha 1 (col1a1), type i collagen alpha 2 (col1a2) and type iii collagen alpha 1 (col3a1). eight differentially expressed genes were analyzed by real-time pcr to validate the microarray data (table 5). two of them were not detected by the real-time pcr, while the other six were also found to be differentially expressed more than 2 fold in the realtime pcr. discussion to identify the key molecules in regulating fat metabolism, pigs treated with or without clenbuterol were used as the animal model. the body composition of the pigs was the most affected by clenbuterol treatment. the difference between test and control pigs could come from individual difference since there are only two pigs in each group. however, the same trend was observed in all four comparison groups suggesting that clenbuterol does play a role in regulating adipose metabolism. the sample quality was sufficient for further analysis to identify genes with differential expression levels that impact adipose table 5. differentially expressed genes in adipose with the administration of clenbuterol. genbank access number gene name potential function induction fold change (clone no.) microarray real-time pcr nm_001647 (rpfat_18926) apolipoprotein d lipids metabolism 2.20±0 8.81±0.12 l06820 (rpfat_18262) apolipoprotein r lipids metabolism 2.47±0.23 ab035172 (rpfat_16328) st6galnac4 mrna for n--acetylgalactosaminide glycolipid metabolism 2.53±0.20 x05942 (rpfat_17661) camp dependent protein kinase type i regulatory signal transduction 2.0±0 7.0 u57092 (rpfat_19360) rab30 signal transduction 2.23±0.18 z74615 (rpfat_8523) mrna for prepro--alpha1(i) collagen (col1a1) cell structure and mobility 2.86±0 16.79 z74616 (rpfat_16033) prepro--alpha2(i) collagen cell structure and mobility 4.47±1.41 3.03±0.89 ab008683 (rpfat_12534) col1a2 mrna for alpha2(i) collagen cell structure and mobility 4.56±0.71 v00503 (rpfat_17393) mrna encoding pro--alpha--2 chain of type i procollagen cell structure and mobility 4.20±1.07 2.52±0.90 x06700 (rpfat_18309) mrna 3' region for pro--alpha1(iii) collagen cell structure and mobility 3.58±0.42 nd x14420 (rpfat_19990) pro--alpha--1 type 3 collagen cell structure and mobility 3.31±0.02 nd nm_000090 (rpfat_12646) collagen, type iii, alpha 1 cell structure and mobility 2.57±0.07 af017305 (rpfat_18326) deubiquitinating enzyme unpel (unp) mrna protein metabolism 2.35±0.03 aw656523 (rpfat_19130) 108820 marc 1pig cdna 5', mrna sequence. 2.90±0.68 ac087859.3 (rpfat_17641) h. sapiens chromosome 3 clone rp11-34l16 map 3p, complete sequence 3.74±0.37 rpig_3584 unknown est 2.35±0.13 rpfat_8229 unknown est 2.41±0.02 -: not analyzed with the real-time pcr; nd: not detected by the real-time pcr analysis. accumulation. to minimize the individual differences, only the genes that showed changes in all four comparison groups are used for further analysis. it has been suggested that clenbuterol works by binding to the β-androgenic receptor and transmitting signals into the cell alone the g-protein mediated camp signaling pathway. in this study, 17 genes were found to be consistently differentially expressed with clenbuterol treatment in the microarray analysis. among the 17 genes, two genes related to signal transduction were found to be up-regulated, the camp dependent protein kinase type i regulatory gene (prkar1a) and rab30. up-regulation of prkar1a correlates with increasing protein kinase a (pka) activity reported previously (domina et al., 2005). rab30 is a member of the ras oncogene family and involved in signaling pathway that controls the expression of a subset of yet-to-be-defined genes that are crucial for cell growth and differentiation. until now, there has been no reported physiologic function of rab30. only 3 genes (apolipoprotein d, apolipoprotein r and st6galnac4) directly involved in lipid metabolism were found to be differentially expressed. apod is a component of high density lipoproteins. apo-d is also closely associated with the enzyme lecithin: cholesterol acyltransferase, an enzyme involved in lipoprotein metabolism. apor is a 23 kda protein found on very lowdensity lipoproteins (vldl), on chylomicrons and in the d > 1.21 g/ml fraction of pig plasma (cooper and attie, 1992). the physiologic function of apo d and apo r in adipose tissue are unknown. the protein encoded by st6galnac4 is a type ii membrane protein that catalyzes the transfer of sialic acid from cmp-sialic acid to galactosecontaining substrates (kang et al., 2004). this protein is a member of glycosyltransferase family 29. st6galnac4 is involved in glycolipid metabolism. the functions of three genes in adipose metabolism were not well studied previously and might be worthy further characterization in regards to the reported findings here. the microarray analysis indentifies 17 genes to be up-regulated by the administration of clenbuterol in both the 3 and 4-month-old pigs, with no figure 2. the real-time pcr analysis of the mrna expression ratios of negative genes in the microarray results. microarray results: clear bars; real-time pcr results: solid bars. the present values are expressed as the ratio of the group mean levels in the test pigs to those in the control pigs. figure 3. the real-time pcr analysis of the mrna expression ratios of key genes in adipose metabolism. microarray results: clear bars; real-time pcr results: solid bars. the present values are expressed as the ratio of the group mean levels in the test pigs to those in the control pigs. genes found to be down-regulated in both groups. to further investigate whether some genes were downregulated by the administration of clenbuterol, phosphohistidine phosphatase 1 (phpt1) and peripheral myelin protein 22 (pmp22), which were found to be down-regulated in only one of the groups in the microarray results, were chosen for further analysis by real-time pcr (figure 2). the pcr results also showed that the mrna expressions of these two genes were not changed significantly (ratios between 0.5 and 2) which confirms that the unusual microarray result that no genes were suppressed. furthermore, some genes highly related to fat accumulation were not found to be differentially expressed by the administration of clenbuterol in the microarray data, such as stearoyl-coa desaturase (scd) and hormonesensitive lipase (hsl). real-time pcr analysis confirmed these results (figure 3). scd is a microsomal fatty acid modifying enzyme that catalyzes the introduction of the cis double bond between carbons 9 and 10 of saturated fatty acyl-coa substrates, resulting in the production of monounsaturated fatty acids. therefore, scd is the enzyme responsible for conversion of saturated fatty acids into monounsaturated fatty acids (mufa) in mammalian adipocytes (enoch et al., 1976). hsl hydrolyzes stored triglycerides to free fatty acids and is the rate-limiting enzyme of lipolysis. both of these enzymes were not found to be differentially expressed by the microarray or the real-time pcr. it was reported that the hsl expression level does not change but that its translocation rate increases when lipolysis is enhanced (lipolysis may be induced by this hormone) (egan et al., 1992; londos et al., 1999 a, b). such changes could not be detected by the techniques used in these tests. the microarray analysis and the real-time pcr analysis showed that the mrna of three collagen synthesis genes (col1a1, col1a2 and col3a1) was increased in adipose cells by the administration of clenbuterol. collagen protein expression increased in muscle tissue (including skeletal muscle and heart muscle) in mice fed with clenbuterol at high dosage, which indicates that collagen protein synthesis may be enhanced by clenbuterol (patiyal and katoch, 2005; bonnet et al., 2005). our data indicates that the collagen protein expression level also increased when the adipose cells were treated with clenbuterol. although, the biological functions of the differentially expressed genes are not completely known, higher expressions of these molecules in adipose tissue might contribute to the reduction of fat accumulation. it is possible to identify the key molecules which regulates adipose metabolism in these genes. conclusion in our study, cdna microarray and real-time pcr were used to identify 17 genes differentially expressed in pig adipose tissue when treated with clenbuterol. eight of the 17 genes and four of the negative genes were verified by real-time pcr and 10 genes got similar magnitudes as in the microarray results. apolipoprotein d, apolipoprotein r and st6galnac4 genes respond to clenbuterol administration in pig adipose tissue and their function may contribute to adipose tissue reduction. acknowledgements we thank state key laboratory of agrobiotechnology of china for offering the microarray printer and scanner. this work was supported by china natural science foundation (no. 30800778, 31072004 and 3094050), hebei natural science foundation (no.c2009000871), hebei educational foundation (s08337) and hebei excellent expert for oversea advanced training program (2009). references bonnet n, benhamou cl, brunet-imbault b, arlettaz a, 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j, shen l, hu x, li n (2011). development of a porcine cdna microarray: analysis of clenbuterol responding genes in pig different tissues. j. genet. genomics, (in press). in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (10), pp. 001-005, october, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper expression of protein-gene peptide (pgp) 9.5 in myocardial sleeves around the pulmonary artery and aorta in pigs jingping lu, dongsheng zhao, gang zhang, jie gen and qijun shan* department of cardiology, first affiliated hospital of nanjing medical university, 300 guangzhou road, nanjing, 210029, jiangsu province, china. accepted 13 july, 2019 a subgroup of outflow tract (ot) ventricular tachycardias (vt) originates from the aortic sinuses or the main stem of the pulmonary artery. myocardial sleeves onto the pulmonary artery (pa) and aorta (ao) have been recognized as a frequent site for the origin of arrhythmia. the aim of this study was to analyze pgp9.5 expression in pulmonary artery and aorta from 100 pigs hearts and relate it to the mechanism of arrhythmia. the pulmonary artery and aorta were obtained from 100 healthy pigs; longitudinal strips of tissue containing each cusp, aortic, pulmonary artery walls, left and right ventricular outflow tracts were excised and histologically analyzed. pgp9.5 was studied immunohistochemically in myocardial sleeves. myocardial sleeves were found in 88 of 100 aos (88%) and 20 of 100 pas (20%). pgp9.5 positivity was observed in 89 of 90 (99%) pigs, which had myocardial sleeves. pgp9.5 immunopositivity can be detected in most pig myocardial sleeves around the pas and aos; this finding may contribute to the substrate for ot vts. key words: myocardial sleeves, ventricular tachycardia, aortic sinus, pulmonary artery, protein-gene peptide (pgp) 9.5. introduction ventricular tachycardia arising from the right or left ventricular outflow tract is a recognized arrhythmia in individuals with structurally normal hearts. the left and right outflow tracts share a common embryonic origin, which explains the similarity of the tachycardias (lerman et al., 1996, 1997; stevenson, 2005; iwai et al., 2006; bunch and day, 2006). it has recently been shown that a subgroup of outflow tract ventricular tachycardias (ot vts) originated from the aortic sinuses or the main stem of the pulmonary artery (shimoike et al., 1999; ouyang et al., 2002; sadanaga et al., 1999; tada et al., 2004; kanagaratnam et al., 2001). a lot of studies have shown atrial myocardium extends into the pulmonary and caval veins plays a critical role in the initiation of atrial fibrillation (af) (haissaguerre et al., 1998; tsai et al., 2000; mansour et al., 2002). ventricular *corresponding author: e-mail: qjshan@njmu.edu.cn. tel: +8625-868136407. myocardial extensions onto the pulmonary artery (pa) and aorta (ao) beyond the ventriculo-arterial junction are relatively common. it seems that their mere presence does not predispose to ot vts. there are probably intrinsic arrhythmia properties in tissues specific to these regions in those patients who develop ot vts (hasdemir et al., 2007). the precise mechanism of these ventricular myocardial sleeves and their role in the causation of arrhythmia need to be explored further. pgp9.5 is a new cytoplasmic neuron-specific protein structurally and immunologically distinct from neuronspecific enolase. interestingly, pgp9.5 immunoreactivity has been described in morphologically dynamic myocardial regions during heart ontogenesis that may subsequently play a role in triggering arrhythmia. expression of pgp9.5 has also been hypothesized to correspond with abnormal ventricular automaticity. in addition, some authors used pgp9.5 as a marker of the conduction system (el et al., 2001; crick et al., 1999a, 1996). the aim of this study therefore was to analyze pgp9.5 expression in pulmonary artery and aorta from 100 pig hearts and relate it to the mechanism of arrhythmia. materials and methods anatomy a hundred adult chinese mongrel pigs of either sex weighing about 90 kg were used in this study. in all subjects, the heart was excised together with pa and ao. the ascending aos and pas were transected at the pericardial reflections. the aortic and pulmonary roots were opened by longitudinal incisions through the left aortic cusp and the anterior pulmonary cusp. the original strips of tissue samples were cut into three equal-sized pieces, containing each cusp, aortic and pa walls 20 mm above the ventriculo-arterial junction (vaj), and left and right ventricular myocardial 20 mm below the vaj, these were then fixed with 10% formalin. histology specimens were routinely dehydrated and embedded in paraffin using an automatic tissue processor. serial sections were cut at 4 to 5 μm thickness and mounted on poly l-lysine prepared slides. serial sections were stained with haematoxylin and eosin (h & e) stain. the presence of myocardial sleeves was evaluated in each section microscopically. immunohistochemistry immunohistochemistry was performed with antibodies against protein gene peptide 9.5 (pgp 9.5, rabbit polyclonal, dilution 1:200, abcam). deparaffnized sections were rehydrated and immunostained according to the following protocol: endogenous peroxidase activity was inhibited, 30 min incubation in 0.3% hydrogen peroxide and methanol followed by tap water wash, the slides were incubated in a retrieval solution for 15 min at 95 o c, which was cooled in the solution naturally and then washed in pbs (phosphate buffered saline) 3 times, 3 min each. subsequently, the sections were blocked with blocking buffer (from boster, sa1022) and incubated for 20 min at room temperature in a humidified chamber and finally with avidin biotin peroxidase complex (vectastain abc kit, ca, usa) for 1 h. peroxidase activity was detected by incubation of the slides with diaminobenzidine tetrahydrochloride (dab) and h2o2 for 5 min. after washing with distilled water, sections were counterstained with haemalum, dehydrated and mounted with dpx. for negative controls, the first antibody was replaced by pbs. staining intensity was evaluated as negative (-), faintly positive (+), moderately positive (++) and strongly positive (+++). results morphological characteristics myocardial sleeves were found in 90 of 100 (90%) pigs studied. ventricular myocardial extensions beyond the ventriculo-arterial junction were found in 108 of 200 (54%) of the great arteries examined.18 pigs had both aortic and pulmonary myocardial sleeves. myocardial sleeves were continuous in 70 (78%), discontinuous in 13 (14%), and both in 7 (8%). myocardial sleeves were oriented obliquely in 16 (18%) and longitudinally in 20 (22%), and both in 54 (22%). myocellular hypertrophy and fibrosis were present in 69 (77%) and fatty tissue between the myocardial sleeve was 63 (70%). myocardial sleeves were found in 88 of 100 aos examined (88%): 71 around the right coronary sinus, 15 around the left coronary sinus, and 59 around the noncoronary sinus (figure 1). myocardial sleeves were found in 20 of 100 pas examined (20%). myocardial sleeves in the pas were located in the anterior pulmonary sinus in 9, in the left pulmonary sinus in 12 and in the right pulmonary sinus in 1 (figure 2). expression of protein-gene peptide (pgp) 9.5 pgp9.5 positivity was observed in 89 of 90 (99%) pigs, which had myocardial sleeves (figure 3). pgp9.5 negative was found in 5 of 59 (8%) in noncoronary sinus, 4 of 71 (6%) in right coronary sinus, 1 of 9 (1%) in anterior pulmonary sinus. discussion ventricular tachycardia has diverse mechanisms and sites of origin. most vt originate from ventricular myocardium, however, a subgroup of ot vts originates from the aortic sinuses or the main stem of the pa (sadanaga et al., 1999; sekiguchi et al., 2005; hachiya et al., 2002). vt originating above the level of the semilunar valves has also been described in a small number of case studies. our study has shown that ventricular myocardial extensions extend into the aorta and pulmonary artery beyond the semilunar valves. ventricular myocardium extending into the great vessels above the semilunar valves may be a trigger for the arrhythmia, similar to that observed from the superior vena cava and pulmonary veins in patients with atrial fibrillation (haissaguerre et al., 1998; tsai et al., 2000; mansour et al., 2002). nevertheless, in some cases, these potentials may result from bystander tissue to getting passively activated from the ventricle. the precise mechanism of these potentials and their role in the causation of arrhythmia need to be explored further. pgp9.5 was expressed by cardiac nerves and conducting system components, and successfully used to quantify the innervation of the conducting system of the adult calf, pig, guinea pig and human heart. it serves as useful markers for the cardiac conducting system (crick et al., 1996, 1999a, 1999b). the sinus node, atrioventricular node, atrioventricular bundle and both left and right bundle branches all possessed a significantly higher density of pgp9.5-immunoreactive nerves relative to their surrounding myocardial tissues (el et al., 2001). pgp9.5 immunopositivity has been hypothesized to be associated with abnormal automaticity in human embryos, suggesting that this parameter could be used to figure 1. long axis view of the pulmonary root, stained with he stain, myocardial sleeves is shown by the arrow. identify foci with a potential for abnormal automaticity in ventricular myocardium. these findings indicate that a nonreentrant mechanism of vt, like automaticity or triggered activity, would be an explanation for the mechanism responsible for the vt in this study. however, ot vts that are based on a reentrant mechanism have been reported. the other hypothesis may reflect the fact that myocardial sleeves corresponding with pgp9.5 intensity may be a bystander tissue getting passively activated from the ventricle. rich pgp9.5 positive innervation of all the components of the conduction tissues displayed a homogeneous distribution. in this respect, the presence of regions that possessed a high density of pgp 9.5-immunoreactive nerve trunks and fibres in the myocardial sleeves extending into the aorta and pulmonary artery beyond the semilunar valves, may provide some evidence for the existence of 'preferential conduction pathways' through the atrioventricular nodal region. the true relationship of these 'nerve pathways' to the preferential conduction routes into the node is, however, highly controversial and merits further investigation. ventricular myocardial extensions extending into the aorta and pulmonary artery in pigs are relatively common. although pgp9.5 immunopositivity can be detected in some myocardial sleeves, this finding does not appear to reflect the prevalence of arrhythmogenic foci in ot vts. the precise mechanism of these myocardial sleeves and their role in the causation of arrhythmia need to be explored further. figure 2. long axis view of the aortic root, stained with he stain, myocardial sleeves is shown by the arrow. figure 3. myocardial sleeves with strong pgp9.5 immunopositivity in the compatible population of cells (black arrow). conclusions myocardial sleeves in pig arteries consist of myocytes different from normal ventricular myocytes, with abundant expression of pgp9.5, which serves as useful markers for the cardiac conducting system. the myocardial sleeves extending into the aorta and pulmonary artery may contribute to the substrate for ot vts. references bunch tj, day jd (2006). right meets left: a common mechanism underlying right and left ventricular outflow tract tachycardias. j cardiovasc. electrophysiol. 17:1059-1061. crick sj, anderson rh, ho sy, sheppard mn (1999a). localisation and quantitation of autonomic 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mansour m, ruskin j, keane d (2002). initiation of atrial fibrillation by ectopic beats originating from the ostium of the inferior vena cava. j cardiovasc. electrophysiol. 13:1292-1295. ouyang f, fotuhi p, ho sy (2002). repetitive monomorphic ventricular tachycardia originating from the aortic sinus cusp: electrocardiographic characterization for guiding catheter ablation. j. am. coll. cardiol. 39:500-508. sadanaga t, saeki k, yoshimoto t, funatsu y, miyazaki t (1999). repetitive monomorphic ventricular tachycardia of left coronary cusp origin. pacing clin. electrophysiol. 22:1553-1556. sekiguchi y, aonuma k, takahashi a (2005). electrocardiographic and electrophysiologic characteristics of ventricular tachycardia originating within the pulmonary artery. j. am. coll. cardiol. 45:887895. shimoike e, ohnishi y, ueda n, maruyama t, kaji y (1999). radiofrequency catheter ablation of left ventricular outflow tract tachycardia from the coronary cusp: a new approach to the tachycardia focus. j. cardiovasc. electrophysiol. 10:1005-1009. stevenson wg (2005). catheter ablation of monomorphic ventricular tachycardia. curr. opin. cardiol. 20:42-47. tada h, naito s, ito s (2004). significance of two potentials for predicting successful catheter ablation from the left sinus of valsalva for left ventricular epicardial tachycardia. pacing clin. electrophysiol. 27:1053-1059. tsai cf, tai ct, hsieh mh (2000). initiation of atrial fibrillation by ectopic beats originating from the superior vena cava: electrophysiological characteristics and results of radiofrequency ablation. circulation 102:67-74. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (3), pp. 001-007, march, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper factorial analysis of slaughter characteristics of fattening pigs fed different additives–enzyme and probiotic in mixtures blazenka popovic1*, branislav zivkovic2, radojka maletic1, zoran rajic1 and svjetlana jankovic-soja1 1 university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade zemun, serbia. 2 institute for animal husbandry, autoput 16, 11080 belgrade zemun, serbia. accepted 09 october, 2017 to successfully investigate slaughter characteristics of fattening pigs fed in different ways, this experiment was carried out on experimental farm of the institute for animal husbandry, belgrade-zemun. investigation of correlation between slaughter traits of pigs fed with different additives in their nutrition was done by factorial analysis. slaughter characteristics in three groups of fattening pigs fed in different ways were observed. the first group (variant 1) consisted of fatteners fed diets without any special additives. the second group (variant 2) consisted of pigs fed diets containing enzyme rovabio, and the third group (variant 3) probiotic lacture + microbond. this study was aimed at coming to conclusion based on the results of factorial analysis of the observed traits to the greatest extent which determined slaughter traits of pigs fed diets containing different additives. the results obtained in general, that is, the structure of separated factors showed that different slaughter characteristics are realized with different nutrition. key words: fattening pigs, slaughter characteristics, enzyme rovabio, probiotic lacture + microbond, factorial analysis. introduction probiotic preparations are used as food additives containing live micro organisms, used to replace or supplement micro flora of the digestive tract and protection of the animal health. the aim of their use is to reduce the utilization of antibiotics in animal nutrition, as much as possible, and that products are free of harmful residues. as a result, the following is realized: increase of milk performance and of body mass, animal resistance, better use of nutrients, products of higher quality are obtained and environment is protected. enzyme preparations are used in animal nutrition in order to improve the digestibility of food that is, increase the nutritional value of animal food. multi-applicative solutions contri-bute to improvement of animal performances, enable use of more raw materials in nutrition and reduce phosphorus *corresponding author. e-mail: blazenka@agrif.bg.ac.rs. tel: +381112615315. fax: +381113161730. emission on the farm (gardzińska et al., 2003; fililz et al., 2008; nortey et al., 2007; kosovac et al., 2006). the influence of different genetic characteristic, as well as of food additions, is a topic of numerous researches (collins et al., 2007 ostrowska et al., 2005; zivkovic et al., 2003c, 2006c) that are very important for improving slaughter characteristics of fattening pigs. considering our positive experiences with the use of probiotics in nutrition of sows and piglets (zivkovic et al., 2003a, b, 2004a, b, 2006b, d; radovic et al., 2007), as well as layer hens in production of table. the objective of this study was to investigate the effects of introduction of enzyme rovabio and probiotic lacture, based on yeast cultures saccharmoyces cerevisiae and bacteria of lactic acid, enzymes and manane oligosaccharides, as well as microbond, based on d-glucanes, in nutrition of fattening pigs (xin et al., 2007; angelov et al., 2010; winiarskamieczan and kwiecien, 2010). complete, clear and profound understanding of correlation between observed slaughter traits of fattening pigs can be achieved through the application of the method of factorial analysis, representing a set of mathematical-statistical procedures which enable that based on numerous mutually dependent and correlated variables less latent variables are determined which explain existing correlation. these fundamental variables which can elicit great number of mutually related variables are called factors. in this way, important task of factorial analysis is achieved as well as determination of homogeneity of observed characteristics (fulgosi, 1979). at the same time, this means reduction of the number of properties, which is also a significant contribution of the application of this method. if intercorrelation of traits is higher, factorization is more expressed, that is, number of factors is lower, and amount of information which they contain on initial traits is greater (ivanovic, 1977; maletic, 2000). materials and methods investigations were carried out on experimental pig farm of the institute for animal husbandry, belgrade-zemun. trial included total of 30 heads divided into three groups. according to present technology on the farm, and based on standard criteria of origin, sex and initial mass, animals were divided into three groups of 10, that is, three nutrition treatments. first, control group received diet of standard composition used on the pig farm. secondly, trial group of animals was fed the same diet supplemented with enzyme rovabio, and thirdly, trial group was fed standard diet supplemented with probiotic lacture and microbond. pigs received food ad libitum during entire trial period and received water from automatic waterers. all analyzes were done with software stat soft statistica 8.0 for windows. analyzed data relate to slaughter traits of pigs fed in three ways, that is, with different feed additives. 46 slaughter traits were observed for each of three nutrition methods, viz: x1body mass of pigs prior to slaughtering (kg); x2– mass of warm carcass sides (kg); x3– fat thickness, withers (mm); x4– fat thickness, back (mm); x5– fat thickness, rump (mm); x6– carcass length, atlas– pubis (cm); x7– carcass length, 1 st rib– pubis (cm); x8– dressing percentage (%); x9– meat yield (% ); x10leg (g); x11shoulder (g); x12back + fat tissue (g); x13 – rib fat (g); x14 – butt shoulder with neck (g); x15 – belly fat (g); x16– fore thigh (g); x17kidney (g); x18shank (g); x19 feet (g); x20– kidney fat (g); x21– head (g); x22– tender loin (g); x23– jowl (g); x24– front ribs in butt shoulder with neck (g); x25– meat for smoking in butt shoulder with neck (g); x26– cut meat in butt shoulder with neck (g); x27– fat tissue in butt shoulder with neck (g); x28skin in butt shoulder with neck ( g); x29– bones with 30% of meat in butt shoulder with neck, (g); x30mld loin part of the back (g); x31cut meat in loin part of the back (g); x32– fat tissue in loin part of the back (g); x33– skin in loin part of the back (g); x34– bones in loin part of the back (g); x35– meat in the leg (g); x36– fat tissue in the leg (g); x37– skin in the leg (g); x38– bones in the leg (g); x39– meat in the shoulder (g); x40– fat in the shoulder (g); x41– skin in the shoulder (g); x42– bones in the shoulder (g); x43– meat in rib fat tissue (g); x44– fat in rib fat tissue (g); x45– skin in rib fat tissue (g); x46– bones in rib fat tissue (g). statistical analysis for the obtained experimental data, descriptive statistics parameters have been defined first, and then the pearson's correlation coefficients, in order to detect the correlation in slaughter characteristics by using different feeding methods (popovic, 2001). the objective of the factorial analysis is to explain the mutual correlation between large number of variables with fewer fundamental variables (lakic, 1984; stankovic et al., 1985; popovic, 2005; ralevic et al., 2006). main task of the factorial analysis is to extract from initial set of indicators (x1,x2, ... xn) fewer (p<n) latent (hidden) factors (f1, f2 ... fp), so the model of factorial analysis can be written down in the following form p x j ∑ajkfkεj j = 1,2,  n k −1 where, ajk represent factorial load of k latent factor on j indicator, and εj so called specific factors, that is, residual random components. factorial analysis was carried out using the method of main factors where analysis of main components is applied on complete correlation matrix. based on established characteristic equations and their square roots by kaiser criteria, the number of factors which are maintained as relevant for further analysis is determined (only common factors with characteristic square root greater than 1 (one) are maintained). after that orthogonal rotation of primary factorial solution using kaiser varimax method was performed. by rotation, a factorial matrix was obtained whose elements are factorial ponders, and ponders of value above 0.7 are considered significant and based on their correlation with certain factor a factor interpretation was carried out. methodology reveals that in the first factor the highest proportional share of total variation of all 46 traits is located, and in subsequent factors this proportion decreases. results and discussion average level and variability of observed slaughter characteristics for all three nutrition methods are presented in table 1. variability of observed traits was expressed by variation coefficient and it is almost low and below 30%, which indicated that these are homogenous series of data. correlation analysis according to results of correlation analysis, correlation between slaughter properties realized in different nutrition treatments is similar. in case of first nutrition treatment, a very high negative correlation between the following indicators is observed: fat thickness at withers and rump fat thickness; belly fat and butt shoulder with neck; head and shank; jowl and body mass of pigs prior to slaughtering; kidney fat and front ribs in butt shoulder with neck; fat in rib fat tissue and fat thickness (withers) etc. very high positive correlation exists between the following characteristics: leg mass and body mass prior to slaughtering and mass of warm carcass sides; jowl and body mass of pigs prior to slaughtering; back fat and rump fat thickness; fat of the leg and carcass length (atlas-pubis); kidney fat and fat of the leg, etc. in case of t he second nutrition treatment, very high table 1. indicators of the average level and variability of slaughter characteristics. indicator variant 1 variant 2 variant 3 x s cv (%) x s cv (%) x s cv (%) x1 97.4 1.96 2.012 98.4 3.72 3.781 98.5 2.1679 2.201 x2 76.3 1.503 1.97 74.6 2.82 3.777 75.33 2.5232 3.349 x3 32.6 3.72 11.41 29.2 2.79 9.54 34.83 4.2622 12.24 x4 19.6 1.625 8.29 17.6 2.5 14.19 16.67 2.582 15.49 x5 20.6 3.382 16.42 20.8 3.43 16.49 16.67 3.6148 21.69 x6 97.0 1.789 1.844 95.6 3.61 3.777 96 1.7607 1.834 x7 80.7 2.088 2.587 79 5.4 6.84 80.5 0.8367 1.039 x8 78.342 0.876 1.118 75.826 1.52 2.009 76.49 2.1707 2.838 x9 41.706 0.888 2.13 42.668 1.2 2.821 43.57 1.0939 2.511 x10 8208 220.9 2.691 8055 346 4.291 8147 426.22 5.232 x11 4576 444.6 9.716 4704 323 6.859 4676 417.28 8.924 x12 5688 428.3 7.53 5729 333 5.812 5493 576.11 10.49 x13 4084 181.3 4.439 3984 341 8.553 3838 260.61 6.791 x14 5018 822 16.38 5159 146 2.835 5132 716.56 13.96 x15 2364 209.2 8.851 2227 142 6.381 2265 201.67 8.904 x16 729 26.91 3.691 772 62.4 8.085 765 38.859 5.08 x17 160 23.66 14.79 157 26.2 16.68 175.8 29.055 16.52 x18 1360 88.99 6.544 1445 155 10.73 1422 135.08 9.502 x19 684 37.2 5.439 707 47.9 6.777 702.5 36.021 5.128 x20 750 178.1 23.75 583 149 25.54 657.5 186.19 28.32 x21 1970 40.99 2.081 1840 201 10.91 2032 203.9 10.04 x22 337 41.9 12.43 349 35 10.02 345.8 30.069 8.695 x23 726 133.2 18.35 686 99.3 14.47 727.5 164.67 22.64 x24 537 70.11 13.06 589 65 11.03 511.7 42.151 8.238 x25 1412 168.9 11.96 1708 156 9.135 1632 200.44 12.28 x26 1068 77.05 7.214 1315 57.3 4.355 1240 262.81 21.19 x27 773 215.4 27.87 591 169 28.66 854.2 426.95 49.98 x28 193 51.34 26.6 141 25 17.72 238.3 127.97 53.69 x29 651 135.9 20.87 801 55.4 6.911 709.2 138.36 19.51 x30 2284 367.7 16.1 2262 269 11.89 2180 151.33 6.942 x31 939 68.29 7.273 1024 249 24.34 885 354.1 40.01 x32 1190 263 22.1 1024 249 24.34 1123 339.33 30.21 x33 234 42.12 18 251 44.3 17.66 256.7 68.093 26.53 x34 1019 114.8 11.27 1127 132 11.72 1028 174.64 17 x35 5951 367.9 6.182 5912 332 5.614 5955 417.65 7.013 x36 1315 281.1 21.38 1009 189 18.7 1108 161.54 14.58 x37 346 29.39 8.495 355 37.9 10.69 351.7 60.553 17.22 x38 708 39.19 5.536 752 52.7 7.006 702.5 30.943 4.405 x39 2909 325.7 11.2 2814 314 11.15 2937 262.42 8.936 x40 1133 339.6 29.97 1067 76.4 7.16 944.2 136.62 14.47 x41 289 36.8 12.73 285 44.3 15.53 301.7 134.19 44.48 x42 478 29.93 6.262 525 21.4 4.085 515 36.056 7.001 x43 2186 108.6 4.966 2275 231 10.17 2188 157.28 7.187 x44 1320 163.3 12.37 1073 214 19.94 1127 226.77 20.13 x45 304 28.71 9.443 335 40.2 12.01 285 70.64 24.79 x46 242 27.31 11.29 256 58.2 22.72 230.8 30.727 13.31 negative correlation was observed between the following characteristics: fat thickness (back) and meat yield; fat thickness (back) and leg; fat thickness (back) and meat in the leg; dressing percentage and fat in the shoulder; leg and bones in loin part of the back; dressing percentage and fat in shoulder, etc. high positive correlation was recorded between the following characteristics: body mass of pigs prior to slaughter and meat yield; body mass of pigs prior to slaughtering and leg meat; mass of warm carcass sides and leg; mass of warm carcass sides and shoulder; carcass length (atlas-pubis) and leg; meat yield and meat in the leg; shoulder and cut meat of butt shoulder with neck; shoulder and meat in the shoulder, etc. inter correlation of slaughter characteristics in case of third nutrition treatment is somewhat lower than in two previous cases, and very high negative correlation is recorded for: fat thickness (back) and meat yield; fat thickness (back) and leg; butt shoulder with neck and fat in shoulder. high positive correlation was recorded between the following characteristics: body mass of pigs prior to slaughtering and kidney fat; carcass length (ribpubis) and dressing percentage; dressing percentage and leg; meat yield and leg; shoulder and meat in shoulder, etc. factorial analysis factorization in the first and second variants of nutrition was finalized by the fourth factor, and in the third variant by fifth factor (since only common factors with characteristic square root over 1 are maintained) which include 100% of total variation of observed traits. distribution of total variability of slaughter properties per individual factors is presented in table 2. in regard to dispersion distribution per individual factors, it is obvious that most were included in the first factor, over 30%; the second factor included somewhat lower percentage of total variation varying from 25.78% in the third nutrition treatment, 27.54% in the first variant and 28.26% in the second nutrition variant. proportional share of remaining factors in total variation is increasingly lower, so that in the third factor it is approximately 20%, and in the fourth factor it varied from 10.34 to 14.38%. subsequent to rotation of initial factorial matrix, factors were obtained which were adequate for interpretation; and considering their correlation with initial indicators, they were interpreted. slaughter traits in the first nutrition treatment (variant 1) distributed themselves in four factors, where the first factor included 38.22% of total variability. in the structure of this factor, the following traits were dominant: fat thickness, withers; fat thickness, rump; belly fat; fat tissue in butt shoulder with neck; fat in loin part of the back; fat in rib fat tissue; so it can be concluded that the first factor was the one defining the fat. the second factor explained 27.54% of total variability, and in the structure of this factor the following traits are dominant: meat yield; tender loin; meat in the rib fat tissue; so the second factor can be defined as meat yield. the third factor explained 19.85% of total variability, and considering the correlation with the initial traits it can be defined as factor skin (skin in loin part of the back, skin in the shoulder, skin in the rib fat tissue). traits which were related with fourth factor mainly related to shoulder and butt shoulder traits, therefore this factor explained 14.38% of total variability. in the second nutrition treatment (variant 2), traits were distributed also within four factors, where the first factor explained 39.65% of total variability. this factor can be defined as meat yield since the following traits were related to this factor: meat yield; leg; shoulder; tender loin; leg meat; shoulder meat; rib fat tissue meat. the second factor could be defined as the fat factor since most of the traits related to this factor related also to fat (kidney fat; jowl; fat tissue in the leg). this factor explained 28.26% of total variability. the third factor was dominated by traits relating to skin and bones, and this factor explained 21.75% of total variability. the fourth factor explained 10.34% of total data dispersion and was mainly defined by traits relating to loin part of the back. the third nutrition treatment (variant 3), contrary to previous two, resulted in the separation of five factors. the first factor explained 31.53% of total variation, and most traits connected to this factor related to meat yield (meat yield; leg; butt shoulder with neck; meat in the leg; meat for drying in butt shoulder with neck). the second factor was dominated by traits related to fat tissue (fat thickness, withers; rib fat tissue; kidney fat; fat tissue in butt shoulder with neck; fat tissue in loin part of the back), and this factor explained 25.78% of total variability. traits connected to shoulder defined the third factor and this factor explained 18.91% of total variation. the fourth factor explained 12.97%, and the fifth 10.81% of total variability. traits related to these factors are very heterogeneous so it was not possible to define their names. part of total variance explained by separated factors for each observed slaughter characteristic and each nutrition treatment is presented in table 3. conclusion investigation of correlation between 46 slaughter characteristics of pigs fed diets containing different additives showed that observed traits do not present uniform set. by correlation analysis, it was determined that between observed traits there was expressed inter-correlation which resulted from extraction of fewer factors. in case of first and second nutrition treatment, four factors per each treatment were separated, and in the third treatment, due to somewhat lower correlation between indicators, five factors were separated. structure of formed factors table 2. distribution of variability of properties per individual factors variant 1 variant 2 variant 3 factor characteristic % of total cumulant characteristic % of total cumulant characteristic % of total cumulant square root variation (%) square root variation (%) square root variation (%) 1 17.58 38.22 38.22 18.24 39.65 39.65 14.50 31.53 31.53 2 12.67 27.54 65.76 13.00 28.26 67.91 11.86 25.78 57.31 3 9.13 19.85 85.62 10.00 21.75 89.66 8.70 18.91 76.22 4 6.61 14.38 100.00 4.75 10.34 100.00 5.97 12.97 89.19 5 / / / / / / 4.97 10.81 100.00 table 3. communalities (%) of slaughter characteristics for different nutrition treatments variant 1 variant 2 variant 3 trait for first for two for three for four for first for two for three for four for for two for three for four for first first factor factor factor factor factor factor factor factor factor factor factor factor factor x1 66.9 92.2 95.4 100 74.4 84.7 85.3 100 0.1 96.0 99.2 99.2 100 x2 33.7 86.1 94.0 100 91.1 91.4 96.2 100 60.4 86.2 91.1 91.1 100 x3 66.1 66.2 73.6 100 25.5 71.9 96.4 100 8.1 88.7 88.8 88.8 100 x4 0.0 72.8 94.1 100 53.6 57.2 62.8 100 92.2 93.0 98.1 98.1 100 x5 49.7 59.5 59.5 100 33.3 55.1 55.2 100 2.8 39.9 49.9 49.9 100 x6 0.0 0.4 0.7 100 86.7 87.9 95.3 100 34.8 50.0 51.5 51.5 100 x7 0.5 1.4 26.6 100 56.7 56.8 83.6 100 57.3 58.9 59.4 59.4 100 x8 19.4 33.2 99.7 100 3.6 54.7 87.3 100 79.2 81.8 83.5 83.5 100 x9 21.9 84.0 86.5 100 59.6 61.2 65.2 100 86.5 90.9 95.8 95.8 100 x10 49.2 80.2 80.4 100 81.9 88.1 88.1 100 87.0 92.4 95.5 95.5 100 x11 6.1 19.7 26.6 100 79.6 89.6 96.5 100 8.1 8.7 92.0 92.0 100 x12 0.9 77.4 91.9 100 14.6 16.6 99.8 100 1.1 47.7 53.9 53.9 100 x13 10.5 55.8 59.6 100 71.4 98.3 98.8 100 4.4 63.2 63.8 63.8 100 x14 45.4 62.4 88.2 100 6.8 63.9 88.5 100 60.6 72.1 74.1 74.1 100 x15 51.6 52.9 98.8 100 47.5 55.3 99.3 100 0.2 7.3 8.4 8.4 100 x16 95.5 97.6 99.5 100 19.8 91.0 93.8 100 15.2 17.9 18.9 18.9 100 x17 83.3 87.1 98.2 100 0.6 35.7 55.4 100 6.6 30.3 54.3 54.3 100 x18 0.1 90.5 91.1 100 35.3 44.6 90.5 100 4.4 8.5 77.3 77.3 100 x19 6.3 69.6 96.2 100 14.6 54.0 96.0 100 2.4 12.7 12.9 12.9 100 x20 2.9 3.6 4.4 100 3.1 92.1 99.1 100 2.0 98.1 99.0 99.0 100 x21 5.2 90.0 95.6 100 4.7 89.8 99.4 100 86.0 86.1 95.0 95.0 100 table 3.continues. x22 0.3 90.5 100.0 100 54.2 98.9 98.9 100 34.7 38.6 42.9 42.9 100 x23 86.2 99.2 99.9 100 8.7 59.1 79.2 100 4.2 5.6 15.5 15.5 100 x24 16.0 26.8 33.8 100 2.8 5.9 17.5 100 19.3 23.4 99.5 99.5 100 x25 16.8 17.3 99.9 100 21.8 24.5 93.0 100 90.1 91.2 97.0 97.0 100 x26 12.0 49.8 50.6 100 70.9 98.4 100.0 100 12.8 63.7 82.1 82.1 100 x27 56.2 65.3 71.1 100 0.4 1.4 94.4 100 20.3 70.9 80.2 80.2 100 x28 37.3 40.2 44.1 100 24.1 96.0 97.6 100 0.0 50.5 81.1 81.1 100 x29 5.0 6.6 100.0 100 0.4 25.7 97.5 100 17.2 25.1 68.6 68.6 100 x30 13.2 80.0 96.7 100 76.7 78.2 95.8 100 1.5 18.8 99.9 99.9 100 x31 87.8 97.1 97.3 100 11.2 11.9 12.1 100 28.7 31.4 54.3 54.3 100 x32 65.2 65.3 65.4 100 11.2 11.9 12.1 100 13.4 81.5 84.2 84.2 100 x33 6.0 15.8 94.5 100 3.9 37.4 87.2 100 4.8 70.3 78.0 78.0 100 x34 3.4 75.1 75.5 100 64.1 70.5 70.5 100 1.1 4.3 8.3 8.3 100 x35 88.55 89.4 92.7 100 58.7 59.1 59.5 100 70.1 83.3 88.2 88.2 100 x36 1.0 4.2 8.7 100 1.4 65.0 65.1 100 0.7 3.0 7.1 7.1 100 x37 80.1 80.2 90.9 100 43.0 44.0 60.5 100 0.3 0.9 37.2 37.2 100 x38 69.3 87.2 93.0 100 0.5 90.6 92.2 100 13.2 17.2 25.3 25.3 100 x39 59.5 91.9 97.6 100 82.1 82.1 99.5 100 1.3 1.7 87.5 87.5 100 x40 1.8 22.2 71.5 100 1.6 43.4 89.6 100 68.5 69.7 87.6 87.6 100 x41 8.9 13.6 99.9 100 12.4 80.4 88.9 100 0.5 4.9 94.5 94.5 100 x42 73.7 75.8 98.2 100 0.1 89.2 98.8 100 6.6 9.4 10.7 10.7 100 x43 0.7 85.7 99.8 100 51.3 54.8 87.8 100 48.4 63.9 72.4 72.4 100 x44 59.5 64.2 65.6 100 26.2 63.7 68.9 100 63.3 87.3 97.1 97.1 100 x45 0.3 24.9 99.8 100 3.7 9.6 62.7 100 3.1 42.0 42.1 42.1 100 x46 89.9 89.9 95.0 100 18.7 20.6 97.4 100 48.1 70.0 71.6 71.6 100 showed that in the first nutrition treatment, traits relating to fat tissue were more associated than those relating to meat yield, whereas in the two remaining treatments the situation is reverse. the third singled out factor in the first and second treatment were very similar and related to traits connected to skin and bones, whereas in the third treatment this factor related to traits connected with shoulder. fourth singled out factor was different for all three nutrition treatments which confirmed the fact that these additives in nutrition result in different slaughter characteristics of fattening pigs. acknowledgements this work is made possible with the help of previous researches funded by the ministry of science and technological development: “development and application of new and traditional technologies in the production of competitive food products with added value for domestic and world markets – create wealth from the wealth of serbia” and “rural labour market and rural economy of serbia income diversification as a toll to overcome rural poverty.” references angelov l, vrabcheva v, petrichev m, borisova l (2010). the effect of copper complex of methionine compared with copper sulfate in growing pigs turk. j. vet. anim. sci. 34: 1-5. collins cl, henman dj, dunshea fr (2007). reduced protein intake during the weaner period has variable effects on subsequent growth and carcass composition of pigs, aust. j. exp. agric. 47(11): 13331340. fililz a, özkan e, can k, hidir d (2008). effect of different diets on growth performance and feed efficiency in early weaned piglets. turk. j. 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online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper neonatal mortality of pigs in nsukka, southeast nigeria abonyi, f. o.1*, omeh c.v.o.1 and machebe, n. s.2 1 department of animal health and production, faculty of veterinary medicine,university of nigeria, nsukka, nigeria. 2 department of animal science, faculty of agriculture, university of nigeria, nsukka, nigeria. accepted 14 july, 2019 this study was conducted to investigate the causes of neonatal mortality among pig farms in nsukka local government area of enugu state, nigeria. forty (40) pig farms in the study area were randomly selected and used for the 20 weeks study duration. one week post partum was considered as the neonatal period. a total of 124 pregnant sows and 924 piglets farrowed during the study period were used. data were obtained from farm visitations/physical observations, farm records, oral interviews and structured questionnaires. feed samples were analyzed for their proximate contents. pregnant pigs were weighed prior, during and few days after farrowing, while piglets were weighed within seven days after farrowing. necropsy and bacteriological examinations of samples of feed, maternal and diseased or dying neonates were also carried out. results showed that mean litter size, piglet weight at birth and daily weight gain were 7.45 ± 0.02, 1.01 ± 0.03 and 0.21 ± 0.01 kg, respectively. 164 (17.75%) of total piglets farrowed died within the first week of life (neonatal period), whereas 144 (87.80%) of this number died within the first five days. in addition, 48 (29.27%) were over laid, 30 (18.29%) died of scouring caused by escherichia coli and salmonella organisms, 30 (18.29%) died of hypoglycaemia. on the other hand, 20 (12.20%) piglets were cannibalized by older pigs and or their dams, 16 (9.75%) were trapped within the drainage, 12 piglets (7.32%) died of chilling, while eight (4.88%) died of unknown causes. other contributory factors include inappropriate farm structures, absence or inadequate farm records, nutritional imbalance, improper management practices and inadequate veterinary services. it was concluded that neonatal mortality is a major impediment to optimum profit making from swine enterprise in nsukka, south eastern nigeria. key words: neonatal, piglets, mortality, nsukka urban, nigeria, sow performance. introduction pig production particularly in the tropics has high potentials for optimum profit making. unfortunately, this is often constrained by poor management practices including poor feasibility studies, inadequate feeds and feeding materials, technological application and neonatal mortality among others (schwartz and franzen, 1992; varley, 1995). frazer (1990) defined neonatal mortality in pig as death that occurs in piglets within few days of life. similarly, varley (1995) on observing the improvement in the reproductive performance of exotic pigs also noted high level of neonatal mortality up to 13%. according to pathiraja et al. (1987), neonatal mortality in small holder *corresponding author. e-mail: festus.abonyi@unn.edu.ng. tel: +2348035078356. farms at southern zaria, kaduna state, nigeria, was noted to be as high as 50%. in agreement, hughes (1993) noted that 50% of all pre-weaning death occurs within the first three days of life and that 90% of all were within one week of parturition. several studies had attributed neonatal mortality in pigs to be of multifactor causes including diseases such as colibacillosis, salmonellosis and piglet anaemia. other factors were low viability, chilling, maternal overlay and poor management practices (hrupka et al., 1998; john, 2004; damron, 2009). there is sparse information on the causes of neonatal mortality of pigs in nsukka area in particular and sub-saharan tropics in general. this situation no doubt has affected pig production in the study area. in order to examine the causes of neonatal mortality of pigs in nsukka and its environs, this study was aimed at; (i) identifying the possible causes of neonatal pig mortality in pig farms at nsukka urban and its environs, (ii) provide baseline data which will serve as a reference point for further studies and (iii) proffer solutions to identified problem(s) with a view to reducing pre-weaning mortality and hence increase swine productivity within the area. materials and methods the study area the study was carried out in pig farms located within nsukka local government area (lga). nsukka lga is located in the derived savannah zone ecological zone at latitude 6° 51 ′ 24°n and longitude 7°23 ′ 45°e at an elevation of 1,810 ft (550 m) above sea level. it covers a total land area of 17.52 sq miles (45.38 km 2 ) (https://en.wikipedia.org/wiki/nsukka#cite_note umuahia_population-0). the area experiences two distinct seasons; rainy and dry seasons. the 2006 population census report showed that nsukka had an estimated population of 309, 633. the indigenous people are involved in agriculture, basically crop and livestock farming activities (nwanta et al., 2011). the major animal production activities in the area are poultry and pig although other livestock like goat, sheep and cattle are produced (nwanta et al., 2011). a multi-stage sampling procedure was used to select towns and communities for the study. four communities namely; ehalumona, edemani, obukpa and obimo were randomly selected representing nsukka east (ne), nsukka west (nw), nsukka north (nn) and nsukka south (ns) of nsukka lga. a total of 10 pig farms were randomly selected from each community based on the willingness of the owners to engage in the study and also make available the farm records. in all, a total of 40 randomly selected pig farms were used and the farmers were all interviewed. these farms were selected based on the pig population ranging from 20 to 200 heads. generally, the research methodology used involved farm visitations/physical observations, farm records, oral interviews and the use of structured questionnaires. a total of 124 sows farrowed and 924 piglets produced were used in this study which lasted five months (april to september). on visiting a farm, information on date of mating and expected date of farrowing was recorded. physical observation of farm structure (presence or absence of basic facilities), the herd structure and compounded pig feed was also carried out on each visit. performance data namely live weight of gestating or lactating sows, piglet birth weight, litter size etc, were obtained from the selected pig farms by means of weekly or biweekly visits by a trained technician per farm. a total of 40 technical staff was used for the study. live weights of pregnant or lactating pigs were determined using weigh band (farmer’s boy®; dalton supplies limited oxon, england) on day 90 of gestation, at term, on day three and seven after farrowing. on the other hand, individual piglets were weighed using weighing balance (avery, england) on days one, three and seven of life. number of piglets that died within seven days at individual farms was recorded and possible causes of death determined at necropsy. samples collected for necropsy were rectal swabs, excised organs and feed samples from affected farms. feed samples were also randomly collected from 20 farms and analyzed for it proximate compositions. similarly necropsies were carried on dead piglets, bacteriological examinations were made on samples collected from some feed samples, unhealthy piglets and their dams using standard methods of counting bacteria. structured questionnaires, available farm records and oral interviews were used to obtain data covering incidence of diseases, treatment, breed, feeds and feeding and other management problems encountered in the farm. determination of composition of feed samples feed samples were randomly collected from 20 farms and analyzed for their proximate contents at the nutrition laboratory, department of animal science, faculty of agriculture and energy research and development centre both at the university of nigeria, nsukka. the analysis was carried out according to the methods of association of analytical chemists (aoac, 1990) with a view to determining their carbohydrate, crude protein, ash, ether extract, crude fibre, phosphorus and calcium contents, respectively; thus metabolizable energy was determined by gallenkamp ballistic bomb calorimeter; crude protein was determined by the micro kjeldahl technique, ether extract was determined by soxhlet fat extraction method, crude fibre was determined by the weende method, while calcium and phosphorus were determined using the atomic absorption spectrophotometer. statistical analysis mean statistics and standard deviation (sd), charts and percentages were used to analyze the data. statistical package for social science (spss) version 11 was the package used for the data analysis. results observations made on the farms showed that the herd structure comprised piglets, growers, gilts, sows and boars; usually they are not separated by sex, age or weight. generally, the pigs are genetically improved breeds (large white, landrace, duroc and their crosses). however, due to uncontrolled breeding programme practiced by the farmers, it was not possible to categorize them into a particular crossbred. there were no records of the presence of indigenous breed of pigs in the study area. the results on sow and litter performance are presented as means and standard deviation in table 1. the results show that mean gestation period was 113.18 ± 2.00 days, while dams’ mean weight at term, litter weight and litter size were 84.49 ± 1.94 kg, 1.01 ± 0.13 kg and 7.45 ± 0.02, respectively. our findings as presented in table 2 and figure 1 show that most piglets (29.27%) died as a result of crushing by their dams, this was followed by hypoglycaemia (18.29%) and diarrhoea or scouring (18.29%) caused by escherichia coli and salmonella organisms, while 4.88% piglets died of unknown etiologic agent. results of the daily neonatal pig mortalities among farms in the study area are presented in table 3. the study shows that most piglets lost due to maternal overlay died within four days of life. generally, high neonatal mortalities/death occurred on the second day of farrowing. the analytical composition of samples of feed made available to pigs in nsukka and its environs are presented in table 4. the results show that pig rations were grossly deficient in energy, protein and other essential table 1. performance of sows and litters within seven days of farrowing in pig farms surveyed in nsukka southeast nigeria (n = 40). parameter mean ± sd mean weight at day 90 of gestation (kg) 95.06 ± 2.48 gestation period (days) 113.18 ± 2.00 sow weigh at term (kg) 84.49 ± 1.94 litter size 7.45 ± 0.02 piglet weight at birth (kg) 1.01 ± 0.13 sow weight loss (kg/day) 0.54 ± 0.02 piglet weight gain(kg/day) 0.21 ± 0.04 number (n) of piglets lost (n/day) 23.43 (19.29%) total number of deaths 164 (17.75%) table 2. identified causes of neonatal deaths in pig farms surveyed in nsukka southeast nigeria (n = 40). cause number dead % mortality maternal over lay 48.00 29.27 scouring (escherichia coli and salmonella) 30.00 18.29 hypoglycaemia 30.00 18.29 cannibalism 20.00 12.20 trapped 16.00 9.76 chilling 12.00 7.32 unknown 8.00 4.88 total 164 100.00 nutrients. also, the feed had very high fibre content. this high content of fibre may be attributed to the high quantity of palm kernel cake (pkc) used in formulating the ration. it was observed that farmers depended on individually mixed feed formulated using locally available feed materials like pkc, brewer’s spent grain, cassava (peels and chips), bambara waste, kitchen waste, rice husk, common salt etc. adesehinwa et al. (2003) reported that pig farmers depended more on the use of nonconventional feedstuffs obtained from farm, household and agro-industrial wastes in formulating ration for pigs. the authors noted that these materials are indispensable for many of the farmers to keep operational cost, particularly average daily feeding cost within a range of one to twenty naira per pig. the results of the disease prevalence in pig farms in nsukka communities are presented in table 5 and figure 2. the results show that diarrhoea/scouring, trypanosomosis, helminthosis, swine erysipelas, ectoparasitism and foot rot are the prevalent diseases in pig farms in the study area. helminthosis was the most common disease (32.00 and 38.10%) followed by diarrhoea (16.00 and 19.05%) and ectoparasitism (12.00 and 14.29%), while foot rot (4.00 and 4.76%) was found to be the least prevalent diseases of pigs in nsukka environ. the farmer’s affirmative answers on common herd structure and management techniques identified in pig farms in nsukka southeast nigeria are presented in table 6. the results indicate that less than 15% of the farms identified had knowledge of fattening/isolation pens, herd health programme, identification of pigs, castration and/or stock pigs by sex or age. more than 65% of pig farms were identified to provide feed/water troughs for the pigs, keep other livestock and administers iron dextran to their piglets. the result also indicates that livestock replacement is common among farmers in nsukka area. discussion it is significant to note that performance of pigs in most farms within nsukka and environ is very low when compared to what is obtainable in pig farms in other parts of nigeria and other developed countries. consequently, mean litter size (7.45 ± 0.02), weight at birth (1.01 ± 0.13 kg) and dam gestation weight loss (0.54 ± 0.02 kg/day) are low when compared with litter size of 11.50 ± 0.20 reported by lay et al. (2001) and very low dam weight loss of 0.12 ± 0.03 kg/day noted by others (hrupka et al., 1998 and john, 2004). similarly, our finding on litter size of 7.45 ± 0.02 was less than 9.57 and 11.00 reported by omeke (1990) and iheukwumere et al. (2008) for pigs in north central and south eastern nigeria, respectively. figure 1. causes of neonatal mortality of pigs in nsukka communities. table 3. neonatal pig mortality in pig farms surveyed in nsukka southeast nigeria (n = 40). cause day 1 2 3 4 5 6 7 total scouring/diarrhoea 4 (2.44) 6 (3.66) 2 (1.22) 8 (4.88) 10 (6.10) 30 (18.29) crushing/maternal overlay 6 (3.66) 20 (12.20) 14 (8.54) 4 (2.44) 2 (1.22) 46 (28.05) trapped 6 (3.66) 8 (4.88) 2 (1.22) 16 (9.76) hypoglycaemia 30 (18.29) 30 (18.29) cannibalism 12 (7.32) 8 (4.88) 20 (12.20) chilling 4 (2.44) 6 (3.66) 4 (2.44) 14 (8.54) unknown 8 (4.88) 8 (4.88) total 6 (3.66) 70 (42.68) 48 (29.27) 10 (6.10) 10 (6.10) 18 (10.98) 2 (1.22) 164 (100.00) **figures in parenthesis represent percentages of the total. table 4. analytical composition of samples of pig feed surveyed in nsukka southeast nigeria (n = 20). nutrient composition (%) crude protein 12.18 ± 1.32 metabolizable energy (kcal/kg) 2048.10 ± 16.25 crude fibre 10.12 ± 0.15 ether extracts 3.43 ± 0.00 ash 2.51 ± 0.06 calcium 0.21 ± 0.05 phosphorus 0.18 ± 0.00 table 5. disease prevalence in 40 pig farms surveyed in nsukka southeast nigeria. disease number of occurrence occurrence (%) diarrhoea/scouring 16.00 19.05 helminthosis 32.00 38.10 trypanosomosis 8.00 9.52 swine erysipelas 8.00 9.52 ectoparasitism 12.00 14.29 foot rot 4.00 4.76 abortion/still born 4.00 4.76 diseases figure 2. disease prevalence in 40 pig farms in nsukka and its environs. low performance of pigs in this study is related to poor condition necessary to overcome the physiological stress infrastructure and management technique that are chathat accompanies lactation. racteristics of pig production practice in the area. for our observation of high piglet death rate (42.68%) for example, the observed farm structures showed poorly piglets within their first two days of birth is reminiscent to constructed houses which lacked basic facilities. the observations made by pathiraja et al. (1987). the similarly, proximate composition of feed available to the authors noted death rates of 50% among small holder pig pigs of all types was observed to be below the national farms in zaria, north central nigeria. from table 1, 164 research council (1998) nutritional recommended stanpiglets were lost within the study period. in financial dard. this may have been responsible for high number of terms, as much as n738, 000.00 ($4670.89) or n18, piglets (30.00 or 18.29%) loss resulting from hypo450.00 ($116.77) per farm was lost. this inference was glycaemia and significant daily weight loss by the dams based on the fact that a piglet was sold at an average during first seven days of lactation. johnston et al. (1997) price of n4500 ($28.48) and exchange rate of and damron (2009) recommended that lactating sows $1.00:n158.00 at the time of this research. this figure should be fed high energy dense diets in order to control represents a huge economic loss to pig farmers in nutritional deficiencies and maintain them in good body nsukka and its environs and a concomitant drop in the table 6. farmer’s affirmative answers on common herd management techniques identified in pig farms in nsukka southeast nigeria. technique number of farms percent of total (n=40) has a farm record 4.00 10.00 pig identification 2.00 5.00 stock pigs by age, sex or size 1.00 2.50 presence of fattening and or farrowing pen(s) 2.00 5.00 presence of isolation pen(s) 2.00 5.00 provision of feeding and drinking troughs 28.00 70.00 practice of creep feeding 0.00 0.00 mixture of other livestock 30.00 75.00 castration 4.00 10.00 any selection 0.00 0.00 administer iron dextran 28.00 70.00 consult veterinarian when animal is sick 14.00 35.00 knowledge of herd health 1.00 2.50 livestock off take/replacement 36.00 90.00 amount of pork supplied to the food chain of consumers in the country. on the other hand, susias (1994) observed low death rate of 14.50% over a period of five days. variations in piglets’ death rates reported by this author and that obtained in this study could be attributed to differences in breed, nutrition, climate and management. for instance, the authors may have made their observation among fairly managed farms in developed countries. varley (1995) had consequently highlighted high mortality rates for piglets within their first three days in life. this risky period demands special attention from farmers with a view to minimizing undue losses among piglets. hughes (1993) recommended that within this period when piglets had not fully developed peripheral body insulators such as fat, external thermoregulation method(s) need be applied. deaths following chilling could be avoided if farmers are enlightened on the provision of artificial heat source such as light, stove or beddings in form of wood shavings to keep the environment warm for the newly born pigs. similarly, death due to maternal overlay and cannibalism could be avoided by selecting breeder pigs with good mothering ability, provision of farrowing pens and adequate accommodation. trypanosomosis was observed to have occurrence rate of 9.52%. this figure was below 30.70% reported by onah (1991). differences in these observations might have been influenced by the fact the most farmers in nsukka area in an attempt to meet local demand now keep mainly crossbred pigs which was reported by omeke (1994) to be less susceptible to trypanosomosis infection than their pure breed counterparts. other management practices necessary for farmers to minimize neonatal mortality in piggery farms include provision of farrowing crates, assurance that piglets suckle their dam immediately after farrowing, provision of balanced diets to gestating and lactating dams and maintenance of high standard of hygiene within the farm premises. these are in agreement with recommendations of paul (1989), lay et al. (2001) and battaglia (2007). other prevalent diseases implicated as causes of neonatal mortality and low performance of pigs includes parasitism, scouring and trypanosomosis (table 5). these diseases not only predispose dam to abortion and mummification of foetuses, but also lead to neonatal death. these were noted through farm records, observations made and necroptic examinations conducted in the present study. several authors (pathiraja et al., 1987; radostits et al., 2003) have also observed significant neonatal losses due to piglet anaemia and scouring in line with the findings made in the present work. demand for pork in nsukka area is high and there are no cultural or religious barrier affecting its consumption. this may account for the high percentage (90%) of regular offtake and replacement of livestock observed in many farms investigated. thus, there is high potential for optimum profit making from pig farming in nsukka, south eastern nigeria. based on our findings, it is our recommendation that there is need to promote extension and herd health services by relevant professional and livestock personnel to potential and existing farmers in the area. the benefits of these will include good application of improved farming technique, proper utilization of agro-industrial wastes to formulate balanced and affordable diets for pigs, significant reduction in neonatal death and subsequent improvement in financial return to pig farmers. references adesehinwa aok, aribido so, oyediji go, obiniyi aa (2003). production strategies for coping with the demand and supply of pork in some peri-urban areas of southwestern nigeria. livestock research for rural development 15 (10). retrieved march 27, 112, from http://www.lrrd.org/lrrd15/10/ades1510.htm association of official analytical chemists (1990). official methods of analysis, 15 th edition. washington d.c. battaglia ra (2007). handbook of livestock management. 4 th edn. pearson educational inc. upper saddle river, new jersey. usa. pp. 561-562. damron ws (2009). introduction to animal science: global, biological, social and industrial perspective. 4 th edn. pearson education inc. upper saddle river, new jersey, usa. pp. 460-467. frazer d (1990). behavioural perspective on piglet survival. j. rep. fertil. 40(suppl.):355-370. hrupka bj,leibbramelt vd, creenshaw id, benevenga nj (1998). the effects of farrowing crate, heat lamp location on sow and pig patterns of lying on piglet survival. j. anim. sci. 76(12): 2996-3002. hughes pe (1993). piglet management. in: varley ma, williams pev and lawrence tij (eds). neonatal survival and growth. occasional publication no. 18. br. soc. anim. prod. leeds, uk. pp. 245-273. iheukwumere fc, ndubisi ec, etusiim pe (2008). the use of different sources of protein on growth and reproduction of pigs. pak. j. nutr. 7(4): 590-593. john g (2004). 1984-2004, the difference. pig progress, 20(9): p. 28. johnston lj, pettigrew je, shurson gc (1997). sow nutrition and reproduction. pig. news inform. (18): 261-264 lay dc, matteri ri, carol ja, fangman ji, safranski ij (2001). preweaning survival in swine. j. anim. sci. 80(suppl):74-86. national research council (1998). nutrient requirements of swine 10 th edition. washington national academy press washington d.c. nwanta ja, shonyinka svo, chah kf, onunkwo jo, onyenwe iw, eze ji, iheagwam cn, njoka eo, onyema i, ogbu ki, mgbegbu ec, nnadozie pn, ibe ec, oladimeji kt (2011). production characteristics, disease prevalence, and herd-health management of pigs in southeast nigeria. j. swine health prod. 19(6):331-339. omeke bco (1990). seasonal breeding performance in the field of landrace and large white pigs under humid environment. world rev. anim. prod. 25(4): 33-36. omeke bco (1994). pig trypanosomosis: prevalence and significance in the endemic middle belt zone of southern nigeria. revue elev. med. vet. pays trop. 47(4):381-386. onah dn (1991). porcine trypanosomiasis in nigeria: infection in local and exotic pigs in nsukka area of anambra state. trop. anim. health prod. 23:141-146 pathiraja n, oyedipe eo, alhasan ws (1987). pig production in nigeria. pig news inform. 8(2):165-170. paul s (1989). united kingdom welfare code recommendations. in: pig international, (19): p. 22. radostits om, gay cc, blood dc, hinchcliff kw (2003). veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goat and horses.9 th edn. saunders elsevier services limited. 90 tottenham court road, london. pp. 1505-1506. schwartz hj, franzen h (1992). potentials and limitations of biotechnology in livestock production in developing countries. proceeding of international symposium held at the institute of basic animal sciences, university of benin, edo state. nigeria. pp. 6-12. susias vc (1994). monitoring of piglet losses in intensive swine production. j. anim. sci. (48):83-91. varley ma (1995). factors affecting mortality rates. in: neonatal natal pig survival and development. cab international walling ford publishers uk. pp. 11-13. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (9), pp. 001-004, september, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper fresh cocoa pod husk as an ingredient in the diets of growing pigs e. o. k. oddoye1*, s. w. a. rhule2, k. agyente-badu1, v. anchirinah1 and f. owusu ansah1 1 cocoa research institute of ghana, p. o. box 8, new tafo-akim, eastern region, ghana. 2 animal research institute, p. o. box ah20, achimota. ghana. accepted 28 april, 2017 the growth rate, feed intake, feed to gain ratio and cost of gain of growing pigs fed diets containing 250 g kg -1 dry cocoa pod husk (control) (diet i) and 200 (diet ii) or 300 (diet iii) g kg -1 cocoa pod husk in the form of fresh (wet) cocoa pod husk were investigated in a feeding trial set up as a completely randomized design, at two (2) locations, with three (3) treatments replicated four (4) times and lasting 140 days. the variances at the two locations proved to be homogenous and as such data from the two (2) locations was pooled with the analysis being handled as a singlefactor design with treatment as the only factor. there was no significant difference (p > 0.05) between treatments for average daily weight gain, average daily feed intake and feed conversion ratio. cost of gain for 300 g kg -1 fresh (wet) cocoa pod husk treatment was, however, significantly higher (p < 0.05) than the other feeds. it was concluded that feeding fresh (wet) cocoa pod husk to growing pigs, up to 300 g kg -1 (on air-dry weight basis) of the diet had no deleterious effects on the pigs. key words: feeding trial, fresh cocoa pod husk, growing pigs, growth rate, feed to gain ratio. introduction the use of dried cocoa pod husk (cph) in poultry and livestock feeding is well documented (barnes et al., 1985; adomako et al., 1985; donkor et al., 1991; adomako and oseiamaning, 1996; adomako et al., 1999; agyentebadu and oddoye, 2005). cocoa pod husk is usually dried before storage and later use but the drying of the material has remained a challenge. the material is usually sun-dried. the greater part of the main harvesting season coincides with the rainy season, however, and therefore, sun-drying at this time has not been effective. initially the cocoa research institute of ghana (crig) employed a mechanical drier which operated on diesel fuel, for this purpose. the escalating cost of energy, however, made this method of drying uneconomical. these problems with drying have hampered efforts to popularize the use of cph as a feed ingredient for poultry and livestock. in the light of the above problems, the idea of feeding the material in its fresh (wet) state, without drying, was *corresponding author: e-mail: emmanuel_oddoye@yahoo. com. tel: 233-24-4574200. therefore considered as a viable option. this study aims at developing rations for growing pigs using fresh (wet) cocoa pod husk. materials and methods location the trial was carried out simultaneously at the acherensua (ashanti region) and worakese (central region) plantations of the cocoa research institute of ghana. experimental animals, treatments and design twelve large white growing pigs were used for the experiment at each plantation, and were fed for one hundred and forty (140) days, that is, from january 15, 2009 until june 03, 2009. all pigs were purchased from the kwame nkrumah university of science and technology, kumasi, ghana. there was an initial adjustment phase of ten days, from january 05, 2009 to january 14, 2009 to allow pigs to adjust to their new diets. pigs were individually housed at each plantation. pens were similar at each plantation and were constructed with cement blocks, had concrete floors with a rough finish and were roofed with aluminium roofing sheets. at each plantation, pigs were balanced for age, sex, litter and table 1. composition of experimental diets. experimental diet feed ingredient (g kg -1 ) diet i (control) diet ii 1 200 g kg -1 fresh cph diet iii 2 300 g kg -1 fresh cph air-dry cph 250 cassava peels 300 340 505 wheatbran 100 234 83.2 soya cake (local) 3 130 120 215 tuna meal 100 130 170 palm kernel cake 100 copra cake 150 oyster shell 13 17 17 salt 2.5 3 3.57 premix 1.25 1.75 1.8 michochem 4 1 1.25 1.43 lysine 2.25 3 3 total 1000 1000 1000 calculated analysis digestible energy (mj kg -1 ) 9.75 11.15 11.67 crude protein (g kg -1 ) 170.6 203.7 210.3 lysine (g kg -1 ) 12.3 14.2 16.6 methionine + cystine (g kg -1 ) 5.2 6.2 6.8 calcium (g kg -1 ) 12.1 13.0 14.5 available phosphorus (g kg -1 ) 4.3 7.1 5.6 cost (gh¢ metric tonne -1 ) 5 0.301 0.300 0.32 nb 1. the fresh/wet cph equivalent of 200 g kg -1 air-dry cph is fed with the concentrate (800 g kg -1 ) at feeding time. 2. the fresh/wet cph equivalent of 300 g kg -1 air-dry cph is fed with the concentrate (700 g kg -1 ) at feeding time. 3. soya cake produced locally by an expeller process. 4. michochem is a commercial mould fixing agent which is added to feeds at a rate of 1 kg per 1000 kg of feed. it binds mycotoxins in vivo preventing them from causing harm to the animals. 5. 1gh¢ = 0.697usd as at august 31, 2009. cost of feed includes the cost of the raw cocoa pod husk randomly allocated to one of three (3) treatments. diet i, which served as the control, was the normal diet fed to grower pigs and contained 250 g kg -1 dry cocoa pod husk (cph). diet ii contained the equivalent of 200 g kg -1 cph but fed fresh (wet) and diet iii contained the equivalent of 300 g kg -1 cph but fed fresh (wet). the experiment was laid out at each plantation as a completely randomized design with 3 treatments and 4 replicates making a total of 12 experimental units. feeds, feeding and management using diet i as a basis, two (2) concentrates were formulated to supply all nutrients in the control diet, less than what would be supplied by 200 g kg -1 airdry cph (diet ii) and 300 g kg -1 air -dry cph (diet iii), respectively (table 1). the cph portion of the diet was fed as the equivalent of fresh cph at feeding time. for example, at the start of the trials when pigs were being fed, an allowance of 1 kg day -1 , 0.8 kg and 0.7 kg of concentrate, with 0.2 and 0.3 kg equivalent of fresh cph was fed to pigs on diet ii and diet iii, respectively. the diets were, therefore, iso-energetic and iso-nitrogenous. a price of gh¢ 0.10 kg -1 was assigned to air-dry cocoa pod husk based on the prevailing price of wheat bran and the nutrient content of dried cocoa pod husk relative to wheat bran as at the time of the feeding trials. pigs were fed an allowance, equivalent to 5% of their body weight, once a day and water was provided ad libitum. any feed left over at the beginning of the next day was weighed and subtracted from that which had been fed the previous day to determine feed intake. feed allowance was adjusted at the end of each month after the pigs had been weighed. data collection and analysis feed intake was recorded daily for each pen and pooled for a month (28 days). this was then used in the computation of average daily feed intake. similarly, weights taken at the end of every month were used in the computation of average daily weight gain. the average daily feed intake, divided by the average daily weight gain was calculated as the feed to gain ratio, that is, the weight of feed needed to produce one kilogram of gain. similarly, the feed to gain ratio multiplied by the cost of a kilogram of feed was calculated as the cost of gain or the cost of feed needed to produce a kilogram of weight gain. samples of each feed and fresh cph were subjected to proximate chemical analysis (aoac, 2000). table 2. proximate analysis of experimental diets and fresh cph. diet i diet ii 200 g kg -1 fresh cph diet iii 300 g kg -1 fresh cph fresh (wet) cph dry matter (g kg -1 ) 897 914 873 130 g kg -1 dm organic matter 888 913 877 938 crude protein 205 229 263 78 ether extract 35 70 40 19 crude fibre 106 98 37 179 nitrogen-free extract 565 553 557 565 calcium 9.3 9.1 9.9 8.0 phosphorus 10 11 10.5 4.1 table 3. performance of pigs on experimental diets. diet i diet ii 200 g kg -1 fresh cph diet iii 300 g kg -1 fresh cph sed parameter average daily gain (kg day -1 ) 0.31 0.31 0.32 0.010 feed intake (kg day -1 ) 2.07 2.15 2.11 0.050 feed conversion ratio 6.69 6.63 6.69 0.066 cost (gh¢) 2.02a 2.01a 2.12b 0.020 n.b: means in a row with different postscripts are significantly different. sed – standard error of the difference between two means. statistical analysis the effects of the various treatments on average daily weight gain, average daily feed intake, feed conversion ratio and cost of gain were investigated using analysis of variance (genstat, 2007), with the initial weight of pigs serving as a covariate. variances at the two locations proved to be homogenous and as such data from the two locations was pooled and handled as a single factor model with treatment being the only factor and location being used as a block to allow the variation due to location to be accounted for (steel et al., 1997). results the composition of experimental diets and the results of proximate analysis are shown in tables 1 and 2, respectively. means for the various treatments for average daily feed intake, average daily weight gain, feed to gain ratio and cost of gain are shown in table 3. dietary treatment did not significantly (p > 0.05) affect average daily weight gain, average daily feed intake and feed conversion ratio. there was, however, a significant difference (p < 0.05) between treatments for cost of gain. diet iii was significantly different (p < 0.05) from the other two treatments. pigs consumed all the feed offered (both concentrate and fresh (wet) cocoa pod husk. discussion adjusted means (adjusted for initial weight) were quite similar and this points to the fact that the diets were comparable. the complete consumption of fresh (wet) cocoa pod husks by pigs indicates that, it was palatable to them. theobromine content of air-dry cph is about 2.0 g kg -1 as opposed to 10.0 g kg -1 in dry cocoa bean shells (seed testa) (obiakor and nwako, 1977; oddoye, 1984). there have been no previous reports of theobromine toxicity in experiments in which air-dry cph was fed to pigs and poultry. even though it was not possible to measure theobromine content of the fresh pod in this experiment, the performance of pigs and the fact that no health problems were observed during the 140 days of the trial suggest that the pigs were not adversely affected by the feeding of fresh cocoa pod husk. the results suggest that, it may be possible to feed fresh cocoa pod husk at a higher level. the problem with this will be the high cost of the concentrate to go with the higher level of fresh cocoa pod husk. at 300 g kg -1 fresh cph as fed in this trial, the high cost of the concentrate used reflected in significant (p < 0.05) differences in cost per gain with diet iii being significantly (p < 0.05) more expensive than the other two diets. this is because as the quantity of fresh cph increases, the concentrate feed will need to supply all other nutrients required in the growing pig’s diet apart from those contained in the fresh cph. as cph is not that nutritious (low metabolisable energy content due to high fiber content), this will require a concentrate with high level of nutrients (particularly metabolisable energy) to make up the deficit. there is a paucity of information on the feeding of fresh cocoa pod husk to pigs and it has therefore not been possible to compare this study to others. however, both barnes et al. (1985) and okai et al. (1985) fed air-dry cph at 150, 200 and 250 g kg -1 of the diet and reported comparable results (growth rate, feed efficiency and carcass characteristics) with the control feed which had no cph. they reported live weight gains ranging from 0.417 0.56 kg day -1 , which are much higher than that reported in this study (0.31 0.32 kg day -1 ). both of their diets contained maize as the major energy source whereas, this study used cassava peels. there may be the need to increase energy content of diets in future work, by including some maize or vegetable oils, to attain a higher growth rate. the results of this study will be of particular interest in cocoa growing areas where cocoa pod husks are usually left on the farm to rot. farmers could be encouraged to keep a few pigs for fattening, using cocoa pod husk as one of their feed resources, to improve their incomes and also to provide some animal protein in their diets. conclusion in conclusion, this study has confirmed the possibility of feeding fresh cph to form up to 300 g kg -1 (on air-dry weight basis) of their ration. for the most economical results, however, it should not form more than 200 g kg -1 (on air-dry weight basis). acknowledgements the assistance given by mr. dominic owusu, technical officer, in the feeding of pigs and collection of data is acknowledged. this paper is published with the permission of the executive director, cocoa research institute of ghana. references adomako d, okai db, osafo elk (1985). effect of different levels of cocoa pod husk on performance and carcass characteristics of finisher pigs. proceedings of 9 th international cocoa research conference, lome, togo pp. 455-459. adomako d, osei -amaning e (1996). livestock feeding trials with cocoa pod husk in ghana. cocoa research institute of ghana, tech. bull. 13: 23. adomako d, osei-amaning e, asante eg (1999). compound animal feed based on cocoa pod husk. trop. sci. 39: 120-123. agyente-badu k, oddoye eok (2005). uses of cocoa by-products. proceedings of 24 th biennial conference of ghana science association, university of ghana. legon pp. 115-127. aoac (2000). official methods of analysis. 17 th edition, association of official analytical chemists, gaithersburg, md. barnes ar, amega wk, manu m, rhule t (1985). utilization of cocoa husk meal by growing-finishing pigs. proceedings of 9 th international cocoa research conference, lome, togo pp. 449-454. donkor a, atuahene cc, wilson bn, adomako d (1991). chemical composition of cocoa pod husk and its effect on growth and food efficiency in broiler chicks. anim. feed sci. technol. 35: 161-169. genstat (2007). general statistics. release 7.2 de (pc/windows xp). lawes agricultural trust, rothamstead experimental station, uk. obiakor ek, nwanko sfn (1977). the effect of processing and drying techniques on the quality and theobromine content of cocoa beans. proceedings of 9 th international cocoa research conference, ibadan, nigeria pp. 568-573. oddoye eok (1984). the effect of dry cocoa husk meal and dry cassava meal on finishing pigs. b.sc. agriculture dissertation, university of ghana, legon. steel rg, torrie jh, dickey d (1997). principles and procedures of statistics. a biometrical approach. 3 rd edition, mcgraw-hill, new york. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (5), pp. 001-007, may, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper erythrocyte osmotic fragility of pigs administered ascorbic acid and transported by road for shortterm duration during the harmattan season a. y. adenkola1*, j. o. ayo2, a. k. b. sackey3 and a. b. adelaiye4 1 department of physiology and pharmacology, college of veterinary medicine, university of agriculture, makurdi, nigeria. 2 department of physiology and pharmacology, faculty of veterinary medicine, ahmadu bello university, zaria, nigeria. 3 department of surgery and medicine, faculty of veterinary medicine, ahmadu bello university zaria, nigeria. 4 department of human physiology, faculty of medicine, ahmadu bello university zaria, nigeria. accepted 12 october, 2016 the experiment was carried out with the aim of investigating the effect of an antioxidant ascorbic acid on erythrocyte osmotic fragility of pigs transported by road for 4 h during the harmattan season. 16 pigs administered with ascorbic acid at the dose of 250 mg/kg per os and individually served as experimental animals and 13 others administered orally with sterile water were used as control animals. the animals were then transported for 4 h at a speed of 40 50 km/h covering a distance of 140 km. blood samples for erythrocyte osmotic fragility determination which was done using standard procedure, were taken early in the morning a day before transportation, immediately after and a week after transportation. erythrocyte osmotic fragility decreased significantly (p < 0.05) at nacl concentration of 0.85, 0.80 and 0.70% in both experimental and control pigs following road transportation and the difference in the post-transportation values was higher (p < 0.05) in experimental compared to control pigs. the results indicated that ascorbic acid protected the integrity of the erythrocyte membrane in experimental pigs administered ascorbic acid following road transportation as demonstrated by lower percentage haemolysis immediately after road transportation and thus may alleviate the risk of increase in haemolysis due to road transportation stress in pigs during the harmattan season. key words: ascorbic acid, erythrocyte osmotic fragility, harmattan season, pigs, road transportation. introduction in many parts of the world, including nigeria, food animals are transported mainly by road. it has been established that road transportation is stressful to livestock (rajion et al., 2001; giovagnoli et al., 2002; adenkola and ayo, 2009). during stress, there is an increase in generation of reactive oxygen species (ros) in the body to a level that overwhelms tissue antioxidant defense systems (akinwande and adebule, 2003; powers and jackson, 2008). the mechanism of damage involves lipid peroxidation which destroys cell membranes with the *corresponding author. e-mail: aadenkola@yahoo. com. tel.: +234-805-4977696. release of intracellular compoents, such as lysosomal enzymes, leading to further tissue damage (demir et al., 2003). the ros play a vital role in cellular and tissue damage (tkaczyk and vizek, 2007) and they have been demonstrated to have adverse effects on erythrocytes (sumikawa et al., 1993; avellini et al., 1995). the magnitude of stress in the body depends on the ability of the tissues to detoxify ros (williams et al., 2008) that is, their antioxidant defence. the ros, including free radicals, initiate many reactions which are deleterious to body cells (sudha et al., 2007), if the ros “quencher” is not available to terminate the reactions (wulf, 2002; akinwande and adebule, 2003). antioxidant supplementation, therefore, has been shown to be beneficial in stress-induced tissue damage (senturk, 2001; adenkola and ayo, 2009). vitamin c or ascorbic acid is a naturally occurring antioxidant (sahin et al., 2001) and currently is the most widely used vitamin supplement throughout the world (naidu, 2003). ascorbic acid is an effective antioxidant because it has an important metabolic role as a result of its reducing properties and function as an electron carrier. it can give up 2 electrons, and it is converted to dehydro-l-ascorbic acid (rice, 2000; sahin et al., 2001). it has been established that ascorbic acid ameliorates heat stress and the adverse effects of stressful environmental conditions (tauler et al., 2003; adenkola and ayo, 2006; ayo et al., 2006; minka and ayo, 2007). the aim of the present study was to investigate the effect of ascorbic acid administration on the erythrocyte osmotic fragility of pigs transported by road for 4 h. materials and methods experimental site and meteorological conditions the experiment was performed at the faculty of veterinary medicine, ahmadu bello, university, samaru, zaria (11 0 10 / n, 07 0 38 / e), located in the northern guinea savannah zone of nigeria during the harmattan season. harmattan season in nigeria occurs between latenovember and early-march (igono et al., 1982; oladele et al., 2003). this zone is characterised by intensive livestock marketing and consequently, transportation of pigs, especially during the season. during the study period, wet and dry-bulb temperatures were determined before transportation at the experimental site using dry and wetbulb thermometers (brannan, england) and rela-tive humidity (rh) was calculated using the manufacturer’s standard manual attached. the dry-bulb temperatures and rh were also recorded at 06:00, 13:00 and 18:00 h for three consecutive days post-transportation. experimental animals and management 29 local pigs served as subjects. they comprised males and non-pregnant, non-nursing females of different age groups, ranging from 9 to 12 months and bought in zaria and its environs. the pigs were kept in a standard com-munal pen, made of concrete floor and iron walls with asbestos roofing. the pen measured 7.50 m x 2.55 m with half the length to the roof without block work, which provided adequate ventilation. the pigs were not res-trained inside the pen. they were kept under an intensive system of management. the pigs were pre-conditioned for 2 weeks before the commencement of the experi-ment. during the period, they were screened for haemo-parasites and endoparasites by taking their blood and faecal samples for analyses. pigs found to be infected were treated using oxytetracycline (kepro b. v ® , hollland) deep intramuscular at the dose of 20 mg/kg and thiaben dazole ((m.s.d agvet ® , u.s.a.) at the dose rate of 25 mg/kg, respectively. experimental design, transportation of animals and blood sample collection on the experimental day, 16 pigs were orally and individually administered ascorbic acid (juhel® nigeria ltd., enugu, nigeria) at 250 mg/kg (chervyakov et al., 1977) dissolved in 20 ml of water, while 13 pigs which served as control were given 20 ml of sterile water. these administrations were made immediately before loading the pigs into the vehicle. food and water were withdrawn 12 h before the journey and throughout the journey period. the vehicle travelled from faculty of veterinary medicine, ahmadu bello university, zaria on tarred road along zaria-jos road, covering a total distance of 140 km at a speed range of 40-50 km/h. the journey took 4 h (short-term duration), including stop-overs for police checkings. after completing the journey, the pigs were unloaded at the original loading point, fed and watered as they had been prior to the journey. blood samples were taken early in the morning a day before transportation, immediately and a week after transportation. 5 mm of blood was drawn aseptically via the anterior vena cava using a 10 ml syringe and 18 gauge x 1 1 /2 inch sterile needle from each animal. the blood was immediately poured inside a sample bottle, containing the anticoagulant, disodium salt of ethylene diaminetetra-acetic acid at the rate of 2 mg/ml of blood (oyewale, 1992). after collection, the samples were transferred to physiology research laboratory, department of physiology and pharmacology, faculty of veterinary medicine, ahmadu bello university, zaria, where erythrocyte osmotic fragility test was carried out as described by faulkner and king (1970). erythrocyte osmotic fragility determination sodium chloride (nacl) solution was prepared according to faulkner and king (1970) in volume of 500 ml for each of the samples in concentrations ranging from 0.05 to 0.85% at ph 7.4. a set of 10 test tubes, each containing 10 ml of nacl solution of concentrations, ranging from 0.05 to 0.85%, were arranged serially in a test tube rack. one set was used to analyse each sample. the test tubes were labeled with corresponding nacl concentration. 1 ml pipette was used to transfer exactly 0.02 ml of blood sample into each of the 10 test tubes. mixing was performed by gently inverting the test tubes for about 5 times. the test tubes were allowed to stand at room temperature (26-27 o c) for 30 min. the contents of the test tubes were maintained at ph 7.4. thereafter, the contents of the test tubes were re-mixed and centrifuged at 1,500 x g for 15 min. the supernatant of each test tube table 1. meteorological data from the study period pre-transportation. hour ambient temperature (°c) relative wind speed minimum maximum dry-bulb humidity (%) (km/ day) 06: 00 13 24 14 24 226.43 13: 00 23 24 23 20 226.43 18: 00 21 22 21 19 mean ± s. e. m 19.00 ± 3.1 23.33 ± 0.7 19.33 ± 2.7 21.00 ± 0.51 table 2. meteorological data from the study period post-transportation. hour ambient temperature (c) relative wind speed minimum maximum dry-bulb humidity (%) (km/ day) 06: 00 15 25 14 26 259.51 13: 00 24 26 24 20 259.51 18: 00 23 21 20 20 mean ± s. e. m 20.67 ± 2.85 24.00 ± 1.53 19.33 ± 2.91 21.00 ± 0.67 was transferred into a glass cuvette. the concentration of haemoglobin in the supernatant solution was measured using a spectrophotometer (spectronic-20, philip harris limited, shenstone, uk) at 540 nm by reading the absorbance. the same procedure was repeated for every blood sample of each pig used for the study. the percent haemolysis was calculated using the formula (faulkner and king, 1970). haemolysis (%) = (od of test/od of distilled water) x 100 erythrocyte osmotic fragility curve was obtained by plotting percent haemolysis against the saline concentrations. statistical analysis all data obtained were subjected to statistical analysis using student’s t-test. data were expressed as mean ± standard error of mean. values of p < 0.05 were considered significant. results meteorological data the meteorological data from the study period are shown in tables 1 and 2. the period was characterized by relatively low values of minimum ambient temperature of 19.0 ± 3.1 o c and low maximum ambient temperature of 23.3 ± 0.7 o c. the dry-bulb temperatures value obtained during the recordings was 19.3 ± 2.7 o c. the harmattan season was characterized by relatively low humidity of 21.00 ± 0.51%. the wind direction was north-east, and the speed was 226.43 km/ day. the meteorological data during the post-transportation period (table 2) were similar to those obtained during the pre-transportation period (p > 0.05) (tables 1 and 2). effect of ascorbic acid administration on erythrocyte osmotic fragility of pigs transported by road for 4 h the minimum and maximum haemolysis of erythrocytes occurred at 0.85 and 0.20% in the experimental pigs, while the corresponding values in the control pigs were at 0.85 and 0.50%, respectively, a day before the journey. all the values recorded at different concentrations before road transportation in experimental and control pigs (figure 1) were not significantly (p < 0.05) different. immediately after the journey, the minimum and maximum values of haemolysis were obtained at 0.85 and 0.50%, respectively in the control pigs; while in the experimental pigs, the corresponding values were obtained at 0.85 and 0.30%, respectively. at nacl concentrations of 0.70, 0.80 and 0.85%, significant (p < 0.05) differences existed between the values recorded in experimental and control pigs (figure 2). however, on day 7 after transportation, significance difference (p < 0.05) existed at the concentration of 0.40 and 0.50% between the experimental and control pigs (figure 3), with the minimum value occurring at 0.85% in both experimental and control pigs and the maximum haemolysis occurred at 0.1 and 0.50% in the experimental and control pigs, respectively. discussion the results obtained in the present study demonstrated that the transported pigs were subjected to a cold and 120 100 h a em o ly si s 80 60 % 40 20 0 0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 % nacl concentration figure 1. effect of ascorbic acid on erythrocytes osmotic fragility before 4 h of road transportation in pigs. 120 100 80 o ly s is h ae m 60 % 40 20 0 0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 % nacl concentration experimental control experimental control figure 2. effect of ascorbic acid on erythrocytes osmotic fragility immediately (within 30 min) 4 h of road transportation in pigs. % h a em o ly si s 120 100 80 60 40 20 0 experimental control 0.85 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 % nacl concentration figure 3. effect of ascorbic acid on erythrocytes osmotic fragility on day 7 after 4 h of road transportation in pigs. dust-laden wind with low ambient temperature, classical of the harmattan season in the northern guinea savannah zone of nigeria (ayo et al., 1998a,b). meteorological results obtained during the present study agree with the previous findings that the harmattan season is thermally stressful to pigs (adenkola and ayo, 2006; adenkola et al., 2007). the erythrocyte osmotic fragility in hypotonic solution has been studied in various mammals (olusanya and adepoju, 1979; oyewale, 1992) and it has been shown that it is related to geometric configuration of the erythrocytes, which in turn depends on the integrity of the cell membrane (schalm et al., 1975). before transportation, there was no significant difference between erythrocyte osmotic fragility of experimental and control pigs, but after the transportation the erythrocyte osmotic fragility was significantly different between the 2 groups. the re-sults showed, for the first time, that erythrocyte osmotic fragility test may serve as an indicator of road transport-tation stress in pigs and that it is of diagnostic value in stress due to road transportation of pigs during the har-mattan season. the maximum erythrocyte osmotic fragility obtained in this study disagreed with that of oladele et al. (2003), who reported maximum erythrocyte osmotic fragility at 0.15% nacl concentration in chickens and guinea fowls during the harmattan season. the difference in the values may be attributed to species difference. the result of this study showed that road transportation stress, apparently, mediates its adverse effects via oxidant mechanisms. thus immediately after transportation, haemolysis was higher in the control than experimental pigs. this finding was similar to those of sumikawa et al. (1993) and avellini et al. (1995) who observed that stress in-duced formation of free radicals which play a vital role in tissue damage and that they have deleterious effects on erythrocyte cytomembrane. mossad et al. (2000) showed that haematological complications and autoimmne haemolytic anaemia observed in malignant lymphomas were attributed to oxidative stress. thus, the results of the present study indicated that road transportation-induced stress enhances the generations of ros, shown to increase haemolysis (langsford and zydney, 1993). although free radicals were not measured directly in this study, it has been shown that they are generated in animals subjected to stress (halliwell, 1996; senturk et al., 2001; chihuailaf et al., 2002). the observed increase in erythrocyte osmotic fragility in transported pigs fur-ther supports this fact. the higher haemolysis recor-ded in the control than experimental pigs post-transportation may be due to established impaired homeostatic mecha-nisms induced in animals not supplemented with ascorbic acid. the increase in ros generation in the body, appa-rently, by road transportation stress on the body rendering cells more fragile and easily susceptible to hypotonic lysis. in contrast the lower percentage of haemolysis recorded in the experimental pigs was in agreement with observations of senturk et al. (2001) and candan et al. (2002) that ascorbic acid consolidates the integrity of erythrocyte membranes of and therefore reduces their oxidative damage. oxidative stress occurs when the antioxidant defence systems in the body are overwhelmed by free radicals (williams et al., 2008). ascorbic acid administration to experimental pigs apparently, reduced the intensity of oxidant stress by enhancing the antioxidant defense mechanisms and suppressing the transportation stress which greatly minimized the destruction of erythrocyte. therefore, an increase in osmotic fragility of erythrocytes, indicating a higher percentage of haemolysis obtained in control pigs may be due to exertional oxidant stress, prevented by ascorbic acid administration which increased the resistance of erythrocytes. it has been established that ascorbic acid ameliorates the adverse effects of environmental stress (tauler et al., 2003; adenkola and ayo, 2006; minka and ayo, 2007). ascorbic acid has also been shown to prevent injurious effects of oxidants by the reduction of reactive oxygen and nitrogen species to stable molecules (wilson, 2002). this fact may explain the finding in the present study that ascorbic acid administration in experimental pigs reduces erythrocyte osmotic fragility and the result is in agreement with that of candan et al. (2002) and chihuailaf et al. 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(2007). oxidative stress in the lung tissuesources of reactive oxygen species and antioxidant defence. prague medical report, 107(2): 105114. williams ca, gordon me, betros cc, mckeever kh 2008. apoptosis and oxidant status are influenced by age and exercise training i horses. j. anim. sci. 86: 576 – 883. wilson jx 2002. the physiological role of dehydroascorbic acid. fed. eur. biochem. soc. 57: 5 -9. wulf d 2002. free radicals in the physiological control of cell function. physiolo. rev. 82(1): 4795. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (7), pp. 001-008, july, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper identification of pig reproductive qtl genes based on gene set enrichment analysis of mouse microarray dataset kan he1,2, fan yang1,2, minghui wang1,2, qishan wang1,2, yuchun pan1,2 and yufang ma1,2* 1 school of agriculture and biology, shanghai jiao tong university, shanghai, china. 2 shanghai key laboratory for veterinary biotechnology, shanghai, 200240, china. accepted 23 january, 2018 nowadays, abundantly different transcripts related to reproductive traits using mouse model have been identified by microarray analysis with the robustness, which were needed of re-evaluation with quantitive real time pcr (qpcr). meanwhile, pig qtl databases were established but with large spans in the confidence interval for qtl location. therefore, a genome-wide comparative and functional analysis of the association between mouse microarrays analysis results and reported pig qtls is needed to better elucidate the genetic and physiological background of pig enhanced reproductive performance in genomics. here, we employed a microarray dataset of a high fertility mouse line and applied gene set enrichment analysis method to identify significant mouse genes in a pathway level. next we used a comparative genomic approach to find the homologous pig genes and locate them to the interval of the reported qtl for pigs’ traits based on animalqtldb. finally, we identified some pathways participating in pig high fertility, and some pig genes were further identified within the reported qtl location related to reproductive performance. combining microarray analysis of mouse high fertility dataset by gsea with pigqtldb will substantially help us to identify candidate genes in reported qtl regions related to pig reproduction that are eventually responsible for increased fertility performance in pig and are also helpful for understanding the genetic information of pig reproduction in genomics. furthermore, we can also provide some novel pig genes in identified pathways with relationship of reproduction. key words: reproductive, microarray, pig, pathway, endocrine. introduction it is well known that the number of ovulation is an important parameter for reproductive performance in polytocous species as mouse and pig. folliculogenesis, ovulation, and luteinization are regulated by some important endocrine factors including the hypothalamic gonadotropin releasing hormone, follicle stimulating hormone (fsh) and luteinizing hormone from the pituitary gland, and steroid and peptide hormones generated within the ovary as estrogen, progesterone, inhibin, activin, and follistatin (bastida et al., 2005; rice et al., 1993; emmen et al., 2005). nowadays, mice have been successfully used as models for swine selection systems. *corresponding author. e-mail: mayufang@sjtu.edu.cn tel: 86021-3420-6147. especially for studying the regulatory mechanism of reproduction in genomics, the expression profiles of animal model mouse reproductive organs have extensively been used to detect genetic factors involved in reproductive performance. however, abundantly different transcripts were identified by microarray analysis with the robustness, which were needed of re-evaluation with quantitive real-time pcr (qpcr). whereas dna microarray technology has extended to all fields of genomic research and has become practically the primary tool for gene expression analysis, the platforms of livestock are still not perfect, especially for pigs. therefore, comparative analysis of mouse model microarray datasets may help us to better understand the regulatory mechanism of pig reproductive performance. furthermore, there is a wealth of information that has been collated from many qtl studies over the last figure 1. six major steps in our analysis strategy for pig qtl genes identification. decade, summarized in resources such as animalqtldb (hu et al., 2005). however, even for relatively large effects with a saturated marker map, the confidence interval for qtl location by linkage analysis spans tens of map units, or hundreds of genes. this requires further linkage disequilibrium mapping to target their gene of interest (meuwissen et al., 2004). therefore, a genomewide comparative and functional analysis of the association between mouse microarrays analysis results and pig qtls is needed to better elucidate the genetic and physiological background of enhanced reproductive performance, including reproductive traits, reproductive organ, litter size and endocrine. currently the most well-known gene set enrichment analysis (gsea) method has been widely used to analysis gene expression profiles, especially to identify pre-defined gene sets which exhibited significant differences in expression between samples from control and treated (keller et al., 2007; subramanian et al., 2005). the algorithms calculate the statistical significance of the expression changes across groups or pathways rather than individual gene, thus allowing identification of groups or pathways most strongly affected by the observed expression changes. the analysis based on a group of relevant genes instead of on an individual gene increases the likelihood for investigators to identify the critical functional processes under the biological phenomena under study. gsea is likely to be more powerful than conventional single-gene methods in the study of complex diseases in which many genes make subtle contributions. the animal quantitative trait locus (qtl) database (animalqtldb, http://www.animalgenome.org/ qtldb) is designed to house all publicly available qtl data on livestock animal species for easily locating and making comparisons within and between species (hu et al., 2005). here, we employed a microarray dataset of a highfertility mouse line and applied gene set enrichment analysis method to identify significant pathways and genes in mouse. next we used a comparative genomic approach to find the homologous pig genes and locate them to the interval of the reported qtls for pigs’ traits based on animalqtldb. materials and methods there were six major steps in our analysis strategy showing in figure 1. data collection and preprocessing we searched the public database geo (www.ncbi.nlm.nih.gov/geo/) for the gene expression profiling studies related to reproductive performance. finally, we chose the dataset gse11113 for our re-analysis, which was contributed by vanselow et al. (2008) for mouse high fertility performance study. in this dataset, ovaries from 30 animals of line fl1 (selected for high fertility performance) and of the control line duksi were collected at the metestrous stage, combined to 6 pools each, and processed for microarray analysis. the samples of gsm280489 to gsm280500 were from line fl1 and gsm280501 to gsm280512 were from line duksi, which were hybridized onto affymetrix mouse expression 430a array. for the assessment of the influence of preprocessing on the comparison, data preprocessing was performed using software packages developed in version 2.6.0 of bioconductor and r version 2.10.1 (gentleman et al., 2004). each affymetrix dataset was background adjusted, normalized and log2 probe-set intensities calculated using the robust multichip averaging (rma) algorithm in affy package (gautier et al., 2004). during the previous study a high fertility mouse line fl1, which was generated by index trait selection over more than 130 generations was comparatively analyzed with the non-selected control line, duksi, and both lines have been derived from the same genetic pool (spitschak et al., 2007). the mean number of corpora lutea at the first day of fl1 pregnancy was 18.2 ± 6.8 (mean ± std, n = 10) compared to 12.2 ± 5.5 (n = 10) in the control line (p = 0.0437). the mean litter size and litter weight at birth increased during the selection period to 17.3 ± 7.7 (n = 60) and 27.8 ± 4.6 g, respectively in fl1 compared to 9.81 ± 2.03 (n = 75) and 15.1 ± 6.2 g in the control line (p < 0.0001 for litter size and weight). it suggested that the body weight of individual newborn pups however was not significantly reduced in fl1 despite of the strongly increased number of pups per litter. gene set enrichment analysis our gene set enrichment analysis of pathways and genes were performed using category package in version 2.6.0 of bioconductor (chiaretti et al., 2004). the goal of gsea is to determine whether the members of a gene set s randomly distributed throughout the entire reference gene list l or are primarily found at the top or bottom in a pathway level. one of the advantages of gsea is the relative robustness to noise and outliers in the data. the gene sets represented by less than 10 genes were excluded. the t-statistic mean of the genes was computed in each pathway. using a permutation test with 1000 times, the significantly changed pathways were identified with p-value less than 0.001. accordingly, the significant pathways and genes between line fl1 and line duksi were then identified. significance analysis of each pathway and retrieving the sequences of significant genes based on gsea, we have identified the significantly upor downregulated pathways and ranked genes list in each pathway. then we retrieved each significant gene’s entrez id and used biomart package (http://www.biomart.org/) to mine the sequence information of significantly identified genes in each significant pathway. comparative analysis with the pig genome and qtl mapping to identify pig genes homologous to the aforementioned mouse pathway targets and to predict their map locations in sus scrofa, the mouse sequences were blast searched against the pig genome using blast 2.2.23. the homologous gene was identified when the pig’s transcript or gene with the homology higher than 85% and alignment region longer than 300 bp. then we wrote a perl script to relatively locate genes to the interval of the qtl for pigs’ traits within pigqtldb. results and discussion significantly up or down regulated pathways by gsea of mouse dataset the previous gsea method was applied to the dataset of gse11113. based on the cutoff of the permutation pvalues, there were 5 up-regulated and 69 down-regulated pathways under the comparison of fl1 to duksi. the analysis results including significant genes in each identified pathway were summarized in appendices 1 and 2. mapping these ovarian genes to such pathway terms could elucidate the line-specific differences due to the genetic consequences of selection for reproductive performance. in total, the pathway categories of steroid/lipid metabolism, immune response, signaling pathways and other metabolic processes were significantly different in the selected high fertility mouse line fl1 compared to a non selected control line. on the other hand, numerous of the differently expressed genes in each significant pathway had also been identified by gsea which were known to be involved in female reproductive processes as folliculogenesis, ovulation, atresia, or luteinization and thus might play a role in increasing the ovulation number (in brown color in appendix 2). for example, we identified casp3 in the pathway of natural killer cell mediated cytotoxicity or huntington's disease or parkinson's disease, casp7 in alzheimer's disease, casp8 in rig-i-like receptor signaling pathway or toll-like receptor signaling pathway and casp9 in vegf signaling pathway. those members of casp family are well known to play a fundamental role during follicular atresia (valdez et al., 2005). members of gpx family including from gpx1 to gpx7 were mostly identified in the pathway of glutathione metabolism, which may be important factors in protecting cells of the cumulus-oocyte complex and luteal cells from oxidative stress inducing cell death (al-gubory et al., 2005; luciano et al., 2006). otherwise, h2-q7 was identified in the pathway of antigen processing and presentation whose functional role for reproductive performance is not yet clear. identification of pig reproductive qtl genes based on the previous identified mouse genes in each significant pathway, we then applied comparative genomic analysis to the pig genome with pigqtldb in order to locate homology genes to the interval of the qtl for pigs’ traits of reproduction. the results are shown in appendices 3 and 4, respectively for up or down regulated pathway genes to pig qtls. the information table 1. the number of pig down or up qtl genes related to traits of pig reproduction in pigqtldb. trait type trait name number of pig up qtl genes number of pig down qtl genes age at puberty 8 101 ejaculation duration 0 5 ejaculation times 0 3 reproduction traits gestation length 0 29 semen ph 0 1 sperm abnormality rate 0 2 sperm concentration 0 2 sperm per ejaculate 0 1 embryo survival 0 1 mummified pigs 0 4 litter size number of viable embryos 0 1 ovulation rate 6 50 total number born 1 8 total number born alive 0 7 epididymis weight 0 4 left teat number 0 10 nonfunctional nipples 10 115 number of corpora lutea 0 14 reproductive organ right teat number 0 5 seminiferous tubule diameter 0 2 teat number 3 192 testicular weight 2 20 uterine horn length 7 31 endocrine plasma fsh concentration 0 2 testosterone level 0 11 in pigqtldb, the traits of pig reproduction were classified into four types including reproduction traits, litter size, reproductive organ and endocrine. in each type, different traits were named. the numbers of pig up or down qtl genes were based on additional file 3 and 4. up or down qtl genes here represent that the genes located in reported pig reproduction qtl regions within identified up or down pathways. about the number of pig down or up qtl genes related to traits of pig reproduction is shown in table 1. in pigqtldb, the traits of pig reproduction were classified into four types including reproduction traits, litter size, reproductive organ and endocrine. in each type, different traits were named. obviously, more pig down qtl genes were found than up qtl genes as more down-regulated pathways were identified. for up qtl genes, most of them were related to age at puberty (one of the reproduction traits in animalqtldb), ovulation rate (litter size), nonfunctional nipples and uterine horn length (reproductive organ). by contrast, most of down qtl genes were related to teat number and nonfunctional nipples (reproductive organ), age at puberty (reproduction traits) and ovulation rate (litter size), each number was over 50. on the other hand, 13 down qtl genes were identified to be related to pig trait type of endocrine, including 11 genes for testosterone level and 2 genes for plasma fsh concentration (in green color in appendix 4), these related traits were not found in up qtl genes. for examples, uqcrc1 (ubiquinol-cytochrome c reductase core protein i) in pathways of alzheimer's disease and parkinson's disease and huntington's disease and oxidative phosphorylation, uba7 (ubiquitin-like modifier activating enzyme 7) in the pathway of parkinson's disease, gmppb (gdp-mannose pyrophosphorylase b) in the pathway of fructose and mannose metabolism, they were located in chromosome 13 and identified to be related to testosterone level (ren et al., 2009). oat (ornithine aminotransferase) in the pathway of arginine and proline metabolism and gk (glycerol kinase) in the pathway of glycerolipid metabolism were both located in chromosome x and identified to be related to plasma fsh concentration. totally, less pig genes were located in reported qtl regions than mouse identified pathway genes, which can ensure the accuracy of targets for pig reproduction. interestingly, there were more homology genes in latterly ranked pathway located to the interval of pig reproductive qtls especially for five up-regulated pathways (appendix 3). for example, there was only one pig qtl gene mpst (mercaptopyruvate sulfurtransferase) identified in the pathway of cysteine and methionine metabolism, which was located in chromosome 5 related to reproductive organ of teat number. mettl6 (methyltransferase like 6) might play a functional role in transferase activity, which was found in the pathway of selenoamino acid metabolism and located in chromosome 13 and with relationship of litter size of ovulation rate and reproductive trait of age at puberty and reproductive organ of nonfunctional nipples (rathje et al., 1997; jonas et al., 2008). another gene found in the same pathway was sephs1 (selenophosphate synthetase 1), located in chromosome 15 and related to both ovulation rate and nonfunctional nipples, which was well known in function of atp binding (rohrer et al., 1999). in the pathway of heparan sulfate biosynthesis, glce (glucuronic acid epimerase) in chromosome 1 and ext1 (exostoses 1) in chromosome 4 were identified to be related to age at puberty and ovulation rate respectively (jonas et al., 2008). in the last two up-regulated pathways including hedgehog signaling pathway and the pathway of arrhythmogenic right ventricular cardiomyopathy (arvc), there were almost the same pig qtl genes identified. for examples, csnk1e (casein kinase 1, epsilon) in chromosome 5 was found to be related to reproductive organs of both teat number and nonfunctional nipples. members of wingless-type mmtv integration site family including wnt1 and wnt10b and dhh in chromosome 5 were all found to be related to reproductive organ of uterine horn length (wilkie et al., 1999). bmp5 (bone morphogenetic protein 5) and a member of ras oncogene family rab23 in chromosome 7 were both identified to be related to age at puberty (cassady et al., 2001). btrc (beta-transducin repeat containing) and sufu (suppressor of fused homolog, drosophila) in chromosome 14 were found to be related to nonfunctional nipples (jonas et al., 2008). gli2 (gli family zinc finger 2) in chromosome 15 was identified to be related to both ovulation rate and nonfunctional nipples (jonas et al., 2008; rohrer et al., 1999). besides there were some novel genes in chromosome 1 and 5 related to age at puberty or uterine horn length. by contrast, there were more important pig qtl genes found in down-regulated pathways (the brown colors in appendix 4). for examples, in the pathway of natural killer cell mediated cytotoxicity or huntington's disease or parkinson's disease, casp3 (caspase 3, apoptosisrelated cysteine peptidase) was also identified as one of pig qtl genes, which was located in chromosome 15 and related to the traits of nonfunctional nipples, number of corpora lutea, and gestation length (jonas et al., 2008; wilkie et al., 1999). in some signaling pathways such as vegf signaling pathway, toll-like receptor signaling pathway, t cell receptor signaling pathway, b cell receptor signaling pathway and fc epsilon ri signaling pathway, the gene of map2k1 (mitogen-activated protein kinase kinase 1) in chromosome 1 was identified to be almost related to the traits of teat number, testicular weight and age at puberty (ren et al., 2009; rohrer, 2000; guo et al., 2008). besides there were also some novel genes identified to be related to pig traits of reproduction. for examples, in the pathway of fatty acid metabolism, a novel gene described as acyl coenzyme a synthetase long-chain 1 fragment (ec 6.2.1.3) in chromosome 15 was identified to be related to nonfunctional nipples, number of corpora lutea and gestation length (jonas et al., 2008; wilkie et al., 1999). nowadays, mice have been used as models for swine selection systems, the genetic parameters as the number of ovulation could be useful in determining alternative selection criteria for increasing the number born in mice, and potentially in swine (christenson et al., 1987; long et al., 1991). combining microarray analysis of mouse high fertility dataset by gsea with pigqtldb will substantially help us to identify candidate genes in reported qtl regions related to pig reproduction that are eventually responsible for increased fertility performance in pig and are also helpful for understanding the genetic information of pig reproduction in genomics. furthermore, we can also provide some novel pig genes in identified pathways with relationship of reproduction. acknowledgements our work is funded by national natural science foundation of china (grant no. 31072003 and 30871782) and the national high technology research and development program of china (863 project) (grant no. 2008aa101002 and 2006aa10z1e3). references bastida cm, cremades a, castells mt, lopez-contreras aj, lopezgarcia c, tejada f, penafiel r (2006). influence of ovarian ornithine decarboxylase in folliculogenesis and luteinization. endocrinology, 146: 666-674. rice vc, zusmanis k, malter h, mitchell-leef d (1993). pure fsh alone induces ovulation and subsequent pregnancy in the mouse resulting in fetal development. life sci., 53: 31-39. emmen jm, couse jf, elmore sa, yates mm, kissling ge, korach ks (2005). in vitro growth and ovulation of follicles from ovaries of estrogen receptor (er){alpha} and er{beta} null mice indicate a role for er{beta} in follicular maturation. endocrinology, 146: 2817-2826. hu zl, dracheva s, jang w, maglott d, bastiaansen j, rothschild mf, reecy jm (2005). a qtl resource and comparison tool for pigs: pigqtldb. mamm genome, 16: 792-800. meuwissen th, goddard me (2004). mapping multiple qtl using linkage disequilibrium and linkage analysis information and multitrait data. genet. sel. evol., 36: 261-279. keller a, backes c, lenhof hp (2007). computation of significance scores of unweighted gene set enrichment analyses. bmc bioinf., 8: 290. subramanian a, tamayo p, mootha vk, mukherjee s, ebert bl, gillette ma, paulovich a, pomeroy sl, golub tr, lander es (2005). gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. proc. nat. acad. sci. usa, 102: 15545-15550. vanselow j, nurnberg g, koczan d, langhammer m, thiesen hj, reinsch n (2008). expression profiling of a high-fertility mouse line by microarray analysis and qpcr. bmc genomics, 9: 307. gentleman rc, carey vj, bates dm, bolstad b, dettling m, dudoit s, ellis b, gautier l, ge y, gentry j (2004). bioconductor: open software development for computational biology and bioinformatics. genome biol., 5: r80. gautier l, cope l, bolstad bm, irizarry ra (2004). affy--analysis of affymetrix genechip data at the probe level. bioinformatics, 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738-744. long cr, lamberson wr, bates ro (1991). genetic correlations among reproductive traits and uterine dimensions in mice. j. anim. sci., 69: 99-103. appendices appendix 1 the list of significantly up or down regulated pathways by gsea of mouse dataset the data provided represent ranked list of significantly up or down regulated pathways by gsea of dataset gse11113. the numbers in the bracket after each pathway name represent the numbers of genes in each identified pathway. appendix 2 the list of significant genes in each identified pathway in mouse the data provided represent the list of significant genes in each identified pathway, including the information of probe set ids and gene symbols. up-regulated pathways are in red color and down-regulated pathways are in green color. appendix 3 pig qtl genes in up-regulated pathways the data provided represent the list of pig qtl genes in up-regulated pathways based on mouse data analysis and pigqtldb. appendix 4 pig qtl genes in down-regulated pathways the data provided represent the list of pig qtl genes in down-regulated pathways based on mouse data analysis and pigqtldb. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (2), pp. 001-006, february, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper cytogenetic characterization of nigerian indigenous pig oluwole, o. o.* and omitogun, o. g. department of animal science, obafemi awolowo university, ile-ife, nigeria. accepted 19 october, 2016 this work studied the chromosomal structure of the nigerian indigenous pig (nip). the karyotype was obtained and a detailed karyogram constructed. using sixty-two metaphases of the nip obtained from both mature male and female leucocytes culture, the chromosomal structure was studied. the karyotype obtained was based on the chromosome’s relative length, centromere position and arm (p/q) ratio. the cells had the usual diploid complement of 38 chromosomes (2n = 38) and a nombre fondamental of 64 as in the exotic. the karyotype of nip was characterized by submetacentric chromosome pairs 1 – 7; metacentric chromosome pairs 8 to 12 and telocentric chromosome pairs 13 to 18 with submetacentric xx for female and xy for male. a detailed karyogram was constructed from the obtained results. key words: karyotype, nombre fondamental, nigerian indigenous pig. introduction the black hairy nip is known with special traits such as greater resistance to african swine fever and heat tole-rance of high parasitic load, high percent of weaner weaned and low age at puberty (adebambo,1982). nip thrive better in extreme condition than exotic pig (ilori, 1974). due to extensive breeding with introduced exotic pig breed, there is a risk of loosing the gene pool of these traits (adebambo, 2003). from genome analysis point of view (galman et al., 1991), pig has the same genome compared to that of human (30 morgans or 3 x 10 9 bp in length). the karyotype of the domestic pig composed of 18 pairs of autosome and pairs of sex chromosomes (x or y) are already well characterized of which 13 are metacentic and 6 are acrocentric (cskss, 1988;cta, 1994; fairbank 1999). much cytogentic work has been done on the exotic pig bread but none has been done on the nip (omitogun, 2004). this paper therefore aims at filling this gap by investigating the characteristics of the nip at the chromosomal level and to compare its karyotype with the already published karyotype of the exotic pig. materials and methods animals were purchased from three remote areas of south western *corresponding author. e-mail: oluwafunmike@yahoo.co.uk. tel: +234(0)8053570807. nigeria; otan lle (llesa), lwo and lle-lfe and maintained at the teaching and research farm of obafemi awolowo university. blood was collected from the jugular veins using 18-gauge syringes and immediately transferred into heparinized bottles. the blood (0.7 ml) was cultured for 72 h in 10 ml of rpmi 1640 (sigma) supplemented with 0.1 ml of phytohaemaglutinin (sigma) and 10% fetal bovine serum (g1bco). 1 h before harvesting, colchicine 0.8 µg/ml was added to arrest the cells at metaphase stage. after centrifugation at 1000 rpm for 10 min, the blood lymphocyte layer was collected and transferred into clean dry test-tubes. 5-10 ml of 0.075% of hypotonic solution was added, recentrifuged and fixed three times with cold 3:1 methanol and glacial acetic acid (v/v) fixative. the fixed cells were stored at 4 0 c for 3 to 4 days. chromosome suspensions were dropped on a pre-cleaned microscope slides, then stained. stained and well-spread metaphase were analysed under the microscope, then photographed and karyotyped. out of the 62 metaphasic chromosome photographs, 10 photographs with well-spread chromosome from 5 male and 5 female pigs were selected for karyotyping. from these 10 karyotypes of the nip, means of each of 18 chromosomal arm lengths were obtained using a compass and a ruler to obtain the arm length ratios, centrometric indices and total chromosomal length which were required for classification of the chromosome (abraham and prasad, 1983). the karyogram showing size, variation and morphology of the chromosome were plotted using microsoft excel. results and discussion the karyotypic data obtained from the analysis of each chromosome of the nigerian indigenous pig (nip) and exotic pigs are shown in table 1 and 2. these showed the cytogenetic analysis of the 18 pairs of chromosome of the nip and exotic pigs. the short and long arm lengths table 1. the cytogenetic analysis of the 18 pairs of chromosome of the nigerian indigenous pig (nip). chromosome p (± sd) q (± sd) p + q (± sd) tcl arm ratio c.i c.t pair no. (µm) (µm) (µm) (%) p/q 100p/(p + q) 1 0.72 (± 0.15) 1.67 (± 0.15) 2.39 (± 0.53) 9.32 0.43 30.13 nsm (-) 2 0.50 (± 0.19) 0.95 (± 0.18) 1.45 (± 0.37) 5.65 0.53 34.48 nsm (-) 3 0.45 (± 0.09) 0.96 (± 0.21) 1.41 (± 0.30) 5.50 0.47 31.91 nsm (-) 4 0.45 (± 0.13) 0.92 (± 0.19) 1.37 (± 0.32) 5.34 0.49 32.85 nsm (-) 5 0.34 (± 0.05) 0.80 (± 0.14) 1.14 (± 0.19) 4.45 0.43 29.82 nsm (-) 6 0.39 (± 0.24) 1.29 (± 0.38) 1.68 (± 0.62) 6.55 0.30 23.21 nsm (-) 7 0.36 (± 0.07) 0.75 (± 0.12) 1.11 (± 0.19) 4.33 0.48 32.43 nsm (-) 8 0.59 (± 0.11) 0.72 (± 0.11) 1.31 (± 0.22) 5.11 0.82 45.04 nm 9 0.45 (± 0.08) 0.56 (± 0.11) 1.01 (± 0.19) 3.94 0.80 44.94 nm 10 0.43 (± 0.18) 0.59 (± 0.10) 1.02 (± 0.28) 3.98 0.73 42.15 nm 11 0.37 (± 0.10) 0.46 (± 0.12) 0.83 (± 0.22) 3.24 0.80 44.58 nm 12 0.36 (± 0.07) 0.38 (± 0.12) 0.74 (± 0.19) 2.89 0.95 48.65 nm 13 0 2.1 (± 0.19) 2.1 (± 0.19) 8.19 0 0 t 14 0 1.69 (± 0.28) 1.69 (± 0.28) 6.59 0 0 t 15 0 1.47 (± 0.22) 1.47 (± 0.22) 5.73 0 0 t 16 0 0.93 (± 0.25) 0.93 (± 0.25) 3.63 0 0 t 17 0 0.79 (± 0.14) 0.79 (± 0.14) 3.08 0 0 t 18 0 0.71 (± 0.14) 0.71 (± 0.14) 2.77 0 0 t x 0.35 (± 0.15) 1.38 (± 0.26) 1.73 (± 0.41) 6.75 0.25 20.23 nsm (+) y 0.38 (± 0.13) 0.38 (± 0.16) 0.76 (± 0.29) 2.96 1.0 50 m ct = chromosome type (abraham and prasad, 1983); tcl = total chromosome length percentage [(p + q)/σ(p + q)] x 100; ci = centromere index = (p/p + q) x 100; p = short arm length; q = long arm length; p + q = total chromosome length; nsm = nearly submetacentric; nm = nearly metacentric; m = metacentric; t = telocentric. are presented as well as the centromere index, the total chromosome lengths in percentage and the chromosome type. figure 1 and 2 showed the solid stained metaphasic chromosomes of the male nigerian indigenous pig and its karyotype. the figure showed the unbanded karyotype of nip having 38 chromosomes comprising of 19 pairs of autosomes, a large x and a very small y sex chromosomes. the karyogram of nip showing the morphology of the chromosomes is presented in figure 2 while the karyogram with size variation among chromosomes is presented in figure 3. chromosome number and nombre fondamental the result obtained (figures 1 and 2) showed that the nip has a chromosome number of 2n = 38 and nombre fundamental (nf) of 64, which are the same with those of the exotic sus scrofa domestica (cskss, 1988). nombre fondamental was coined by mathey (1945) as an index for quick estimation of the nature of the chromosome in a karyotype in relation to the presence or absence of arms. this nf value of 64 showed that 6 diploid chromosomes are telocentric, that is, chromosomes 13 – 18. the agreement of the nf value of the nip with that of the exotic pig indicates high degree of resemblance between the two. it is however believed that diversity in nf indicated species at different evolutionary levels with the more advanced ones showing an increase in metacentric chromosome at the expense of the telocentric ones (dallai and telluri, 1969) but this study does not show that. chromosome morphology the morphology of the nip chromosomes observed could be divided into three main groups according to their structures and sizes: group i: the chromosomes under this group are chromosomes 1-7 including the x sex chromosomes. they are submetacentric and distinct by identifiable in all metaphase spreads. group ii: this group consists of chromosomes 8-12 and y sex chromosome. they are metacentric chromosomes. group iii: these consist of chromosomes 13-18 and are telocentric. each chromosome description is as follows: group i chromosome 1: this is the longest submetacentric table 2. the cytogenetic analysis of the 18 pairs of chromosome of the exotic pigs. chromosome p q p + q tcl (%) arm c.i c.t pair no. (µm) (µm) (µm) ratio p/q 100p/(p + q) 1 0.72 1.40 2.10 10.40 0.50 33.30 nsm (-) 2 0.40 0.85 1.25 6.19 0.47 32.00 nsm (-) 3 0.40 0.60 1.00 4.95 0.67 40.00 nm (-) 4 0.30 0.60 0.90 4.46 0.50 33.30 nsm (-) 5 0.31 0.50 0.81 4.01 0.62 38.27 nm (-) 6 0.25 1.15 1.40 6.93 0.22 20.00 nsm (+) 7 0.25 0.70 0.95 4.71 0.36 26.30 nsm (-) 8 0.30 0.65 0.95 4.71 0.46 31.58 nsm(-) 9 0.50 0.60 1.10 5.45 0.803 45.45 nm 10 0.39 0.40 0.79 3.91 0.98 49.36 nm 11 0.30 0.35 0.65 3.22 0.86 46.45 nm 12 0.30 0.30 0.60 2.97 1.00 50.00 m 13 0 1.85 1.85 9.16 0 0 t 14 0 1.45 1.45 7.18 0 0 t 15 0 1.26 1.26 6.24 0 0 t 16 0 0.80 0.80 3.96 0 0 t 17 0 0.65 0.65 3.22 0 0 t 18 0 0.60 0.60 2.97 0.66 0 t x 0.43 0.65 1.08 5.35 1.00 39.81 nm y 0.25 0.25 0.50 2.48 1.00 50 m ct = chromosome type (abraham and prasad, 1983) tcl = total chromosome length percentage [(p + q)/σ(p + q)] x 100 ci = centromere index = (p/p + q) x 100 p = short arm length q = long arm length p + q = total chromosome length nsm =nearly submetacentric nm = nearly metacentric m = metacentric t = telocentric chromosome. it has p and q values of 0.72 and 1.67 µm respectively. chromosome 2: this chromosome is smaller than chromosome 1. it is nearly submetacentric. it has p and q values of 0.5 and 0.95 µm respectively. chromosome 3: this is also long but relatively shorter than chromosomes 1 and 2. it has p and q lengths of 0.45 and 0.96 µm respectively. chromosome 4: a nearly submetacentric chromosome that has p and q lengths of 0.45 and 0.92 µm respectively. chromosome 5: this also is a nearly submetacentric chromosome with p and q values of 0.34 and 0.80 µm respectively. chromosome 6: this is equally a nearly submetacentric chromosome with p and q values of 0.39 and 1.29 µm respectively. chromosome 7: this is a nearly submetacentric chromosome with p and q values of 0.36 and 0.75 µm respectively. chromosome x: this is nearly submetacentric. it has p and q values of 0.35 and 1.38 µm respectively. group ii chromosome 8: this is a long nearly metacentric chromosome with p and q values of 0.57 and 0.72 µm respectively. chromosome 9: this is a nearly metacentric chromosome. it has p and q values of 0.45 and 0.56 µm respectively. chromosome 10: this also is a nearly metacentric chromosome with p and q values of 0.43 and 0.59 µm respectively. chromosome 11: this is a short nearly median chromosome with p and q values of 0.37 and 0.46 µm respectively. chromosome 12: this is also a short nearly median chromosome with p and q values of 0.36 and 0.38 µm respectively. chromosome y: this is a very small metacentric chromo figure 1. the unbanded (solid stain) chromosomes of the male nip and its karyotype (38, xy). some with p and q values of 0.38 and 0.38 µm chromosome 16: this is a short telocentric chromosome respectively. with a q value of 0.93 µm. chromosome 17: this is also a short telocentric chromo some with a q value of 0.79 µm. group iii chromosome 18: this is the shortest telocentric chromo some with q value of 0.71 µm. chromosome 13: this is a long telocentric chromosome. it has a q value of 2.1 µm. chromosome 14: this also is a long telocentric chromosome, but shorter than chromosome 13. it has a q value of 1.69 µm. chromosome 15: this telocentric chromosome has a q value of 1.47 µm. the karyotypic data below is the karyotypic data obtained from the measurement of chromosomal arms, the p/q ratio, the centromere indices and the chromosome type of the nip are shown on table 1. the chromosome arm length and figure 2 the karyogram of nip showing the morphology of the chromosomes. figure 3. the karyogram of the nip showing the size variation among the chromosomes. p/q ratio of both were observed to be the same in chromosome 1, 2, 6, 9, 11, 12, 14 18 and y sex chromosome within the s.e measurement while differences were obtained in chromosome 3, 4, 5, 7, 8, 10, 13 and x sex chromosomes but these differences do not cause distinction in chromosome type, except in the x sex chromosome which showed distinction in chromosome type (oluwole, 2005). the karyotypic data obtained was also used to construct the karyogram showing the morphology of each chromosomes and size variation amongs the chromosomes (oluwole and omitogun, 2007). conclusion the nip has a chromosome number of 2n = 38 and a nombre fondarmental of 64 which tally with that of the sus scrofa domestica. similarities were found in p/q ratios nip and exotic pig in chromosomes 1, 2, 6, 9, 11, 12, 14 18 and y sex chromosome. differences were observed in the p/q ratios of nip and exotic pig in chromosomes 3, 4, 5, 7, 8, 13 and x sex chromosome. it is assumed that these information will be of tremendous benefit in pig breeding programme aiming at enhancing diseases resistance and better adaptation. acknowledgement our laboratory was funded by the obafemi awolowo university research council research grant (urc11812 avt). acknowledgements are due to ranjit vandjopathay, jackie hughes and o. mukangi of the international institute of tropical agriculture (iita), ibadan for the assistance in digital photomicrographic aspect of this research. references abraham z, prasad pn (1983). a system of chromosome classification and nomenclature cytology 48:95-101. adebambo oa (1982). evaluation of the genetic potential of nigerian indigenous pigs. proceeding 2 nd wld cong. genetic appl. livestock prod. madrid. pp. 543-553. adebambo oa (2003). animal breeds: a nation’s heritage. inaugural lecture, university of agriculture, abeokuta. committee for the standard karyotype of sus scrofa (1988). standard karyotype of the domestic pig heriditas 109: 151 – 157. cta (1994). animal breeding. mammalian publishers. pp. 18 – 24. dallai d, talluri (1969). a karyotypic study of three spiecies of scincidae (reptilia) chromosoma (berl) 27:86-94 fairbanks dj (1999). genetics: the continuity of life. pp. 304 – 309, 322 – 326. galman og, yerle m, echard g (1991). the high resolution gtg banding karyotype of the sus scrofa domestica. genet. set evol. 23: 113s – 116s. ilori ja (1974). assessing the productive potentials of local breeds of pigs. ann. conf. nig. soc. anim. prod. 1:100. mattey r (1945). l’evolution de la formulae chrosominale chez les vertebras experimentia (basel) 1: 78-86 omitogun og (2004). analysis of swine genome organization. evolution of g-t-g bands in porcine chromosomes for physical mapping. in: (o.j. oriyo c.o.n. ikeobi, m.o. ozoje, t. omoniyi, and o.b. kehinde, eds.)..proceedings of the 29 th annual conference of the genetics society of nigeria., nacgrab, moor plantation, ibadan pp. 54-57. oluwole oo (2005). ‘cytogenetic characterization of the nigerian indigenous pig’ unpublished msc thesis obafemi awolowo university, ile-ife. ‘ unpublished msc thesis obafemi awolowo university, ile-ife, pp. 1-61 oluwole oo, omitogun og (2007). ‘the karyomorphology of nigerian indigenouspig chromosomes prepared from leucocyte culture’ trop. anim. sci. 10(2):37-40. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (12), pp. 001-006, december, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper comparative proteomic analysis of the effects of dietary mugwort (artemisia iwayomogi kitamura) on the pig longissimus dorsi muscle byung uk kim1, mi ae jeong1, yeon sun ryu2, hwa chun park 3, jong hyun jung3, sam woong kim4, chul wook kim1, young min song1, ki hwa chung1, il suk kim1, sang keun jin1, sang suk lee5, in soon choi6 and kwang keun cho1* 1 department of animal resources technology, gyeongnam national university of science and technology, 150 chilam dong, jinju, gyeongnam, 660-758, republic of korea. 2 department of animal health management, woosuk university, 443 samnye-eup, wanju-gun, jeonbuk, 565-701, republic of korea. 3 dasan genetics, gasan-ri, unbong-ub, namwon-city jeonbuk, republic of korea. 4 swine science and technology center, gyeongnam national university of science and technology, 150 chilam-dong, jinju, gyeongnam, 660-758, republic of korea. 5 department of animal science and technology, sunchon national university, suncheon 540-742, republic of korea. 6 department of biological sciences, silla university, busan, 617-736, republic of korea. accepted 03 august, 2017 proteomic analysis by 2-dimensional electrophoresis (2-de) of the longissimus dorsi muscle in finishing pigs (ly × d) grown on a diet supplemented with mugwort powder found roughly 300 spots on a polyacrylamide gel image. among them, the expression levels of 12 spots were higher than those of the control group. from the results of peptide mass fingerprinting (pmf) analysis, nine proteins were characterized: myosin light chain 2v, elf-5a, myosin light chain 1, f1atpase chain d, peroxiredoxin-2, kelch-related protein 1, sla-7, glycine amidinotransferase, and tpi 1. the other three spots with increased expression were identified by chemically assisted fragmentation-matrix-assisted laser desorption/ionization (caf-maldi) sequencing as serum albumin precursor and two different types of myoglobin. taken together, these results indicate that mugwort powder has the potential to improve the quality of pork meat. key words: 2-de, longissimus dorsi muscle, meat quality, mugwort, pig, proteome. introduction because proteins play critical roles in transport, immune responses, storage, and structure, and serve as members of metabolic pathways, they are essential factors for the maintenance of life of all organisms. thus, proteomic tools were developed to investigate overall protein expression patterns (anderson and anderson, 1998; blackstock and weir, 1999; wilkins et al., 1996). from 2-de-based-proteomic analysis of proteins from white and red pork muscles, about 500 spots were obtained, and five spots were observed with different *corresponding author. e-mail: chotwo2@gntech.ac.kr. tel: 82 55-751-3286. fax: 82-55-751-3280. expression patterns including myoglobin, two slow-twitch isoforms of myosin light chain, and two small heat shock proteins (kim et al., 2004). oriental medicines have antibiotic, anti-cancer, antioxidant, and physiological activation properties, and there is a trend towards their increased utilization as feed additives because of the potential for improving intestinal microbiota as well as pig meat quality. in particular, mugwort grows naturally everywhere in south korea, so it may be an invaluable feed additive. the major components of mugwort are alkaloids, essential oils, vitamins, and various minerals (kang et al., 1995), and its pharmacological properties include anti-oxidative (lee et al., 1992; sim et al., 1992), anti-cancer (lim and lee, 1997; sun et al., 1992), and liver protective activities (gilanti et al., 2005). pigs fed mugwort powder improved their average weight gain, protein content, and the lightness of their meat and fat (kim et al., 2008). the improvement of pig meat quality through a mugwort powder diet should be accompanied by altered expression of the relevant genes. therefore, this study used comparative 2-de to investigate global changes in protein expression in longissimus dorsi muscle associated with dietary mugwort supplementation. materials and methods experimental animals and sample harvesting experimental diets included a control diet (c group), and a diet supplemented with 1.5% (w/w) mugwort powder as a diet additive (t group). among 20 selected growing landrace (lyxd) pigs of body weight 75  4 kg, half were grown for 50 days on the basal diet, whereas the other half was grown on the mugwort powder diet. longissimus dorsi muscle tissues of the carcass were harvested from four pigs that exhibited good breeding in each group, and these were then used for the experiment. protein extraction from longissimus dorsi muscle the harvested longissimus dorsi muscle tissues were sonicated for 10 s with a sonoplus (bandelin electronic, germany) and were then directly homogenized by a motor-driven homogenizer (powergen125, fisher scientific) in sample lysis solution composed of 7 m urea, 2 m thiourea, 4% (w/v) 3 [(3 -cholamidopropy) dimethyammonio]1 propanesulfonate (chaps), 1% (w/v) dithiothreitol (dtt), 2% (v/v) pharmalyte, and 1 mm benzamidine. after the homogenized product was centrifuged at 15,000 × g for 1 h at 15°c, the precipitated insoluble material was discarded, and a soluble supernatant fraction was collected and used for 2-de. protein quantity for sample loading was normalized by bradford assay (bradford, 1976). protein separation via 2-de and analysis of gel images immobilized ph gradient (ipg) dry strips were equilibrated for 12 to 16 h with equilibration buffer (7 m urea, 2 m thiourea, 2% chaps, 1% dtt, and 1% pharmalyte), and 1.1 mg of each sample was loaded onto a strip fixed in a strip module. isoelectric focusing (ief) was performed at 20°c using a multiphor ii electrophoresis unit and an eps 3500 xl power supply (amersham biosciences) according to the manufacturer’s instruction. the voltage for ief was linearly increased from 150 to 3,500 v over 3 h for sample entry into the gel followed by constant 3,500 v for complete focusing until 96 kv/h. prior to electrophoresis in the second dimension, the treated strips were incubated for 10 min each in equilibration buffer (50 mm triscl, ph 6.8, 6 m urea, 2% sds and 30% glycerol) plus 1% dtt and then equilibration buffer plus 2.5% iodoacetamide. the equilibrated strips were inserted into sds-page gels (20 × 24 cm, 10 16%). protein separation via sds-page was performed with a hoefer dalt 2d system (amersham biosciences) according to the manufacturer’s instructions. 2-de gels were run for 1,700 v/h at 20°c and then were stained with coomassie brilliant blue g250 solution as described by anderson et al. (1991). colloidal cbb staining of 2-de gels was used to delicately analyze the protein spots, and the solution was prepared with 177 ml 85% phosphoric acid, 150 g ammonium sulfate, 1. 8 g cbb-g 250, adjusted to 1,200 ml with water, and then finally adjusted to 1,500 ml with methanol. quantitative analysis of digitized gel images was carried out using the pdquest software (version 7.0, bio-rad) according to the protocols provided by the manufacturer. the quantity of each spot was normalized by total valid spot intensity. protein spots showing significant expression variation were selected as spots to exhibit critical changes between the control and treated groups. protein identification via pmf and caf-maldi sequencing protein identification via pmf was done as follows: protein spots were enzymatically digested with modified porcine trypsin in a manner similar to the method previously described by shevchenko et al. (1996). target spots were cut from separated gels by 2-de. the gel pieces were washed with 50% acetonitrile to remove sds, salt, and staining dye, dried to remove solvent, rehydrated with trypsin solution (8 to 10 ng/l), and then incubated for 8 to 10 h at 37°c. the proteolytic reaction was terminated by addition of 5 l 0.5% trifluoroacetic acid (tfa). tryptic peptides were recovered by combining the aqueous phases obtained from several extractions of gel pieces with 50% aqueous acetonitrile. after concentration, the peptide mixture was desalted using c18ziptips (millipore), and peptides were eluted in 1 to 5 lacetonitrile. an aliquot of this solution was mixed with an equal volume of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% aqueous acetonitrile, and 1 l of the mixture was spotted onto a target plate. protein analysis via pmf was performed by an ettan matrixassisted laser desorption/ionization-time of flight (maldi-tof; amersham biosciences). peptides were evaporated with an n2 laser at 337 nm, and a delayed extraction approach was used. they were accelerated with a 20-kv injection pulse for time of flight analysis. each spectrum is the cumulative average of 300 laser shots. the search program profound, which was developed by the rockefeller university (http://129.85.19.192/profound_bin/ webprofound.exe), was used for protein identification by pmf. spectra were calibrated with trypsin auto-digestion ion peaks m/z 842.510 and 2211.1046 as internal standards. caf-maldi sequencing was done by the following method described here briefly. protein digestion was performed by the same method as with pmf described above. 4-sulfophenylisothiocyanate (spitc, aldrich) reaction was done by the procedure of dongxia et al. (2004). a total of 10 mg/ml (final) spitc was dissolved in 20 mm nahco3, ph 9.5 and then treated with a half volume of spitc solution versus one volume of trypsin-digested solution. the reaction continued for 30 min at 55°c and was then terminated by adding 1 l 5% tfa. the subsequent procedures were performed via the same method as was used for pmf. statistical analysis statistical analysis was done by general linear model (glm) method of the sas program (statistics analytical system, usa, 1999). duncan's multiple range tests was used to compare the differences between the means of the investigated samples. the results of the statistical analysis are presented as mean  standard deviation (sd). results and discussion changes in protein expression patterns by mugwort powder mugwort powder contains various physiologically active materials, and the tissue of animals fed with the powder figure 1. full 2-de gel image of proteins from the longissimus dorsi muscle of pigs fed with mugwort powder. the 1st and 2nd gel runs were done by isoelectric focusing and sds-page, respectively. numerals at the bottom indicate pi values, and kda values at right side indicate the molecular weights of the separated proteins. is capable of undergoing changes in the expression of various proteins in response to the materials. therefore, in order to investigate the patterns of protein expression associated with a mugwort powder diet, 2-de analysis was performed using 1.1 mg protein extracted from pig l. dorsi muscle, which is one of the most important meat cuts for human beings. after 2-de separation of the proteins, quantitative colloidal cbb staining revealed roughly 300 spots (figure 1). the 2-des originating from the two different groups (c group, a basal diet and t group; a basal diet supplemented with 1.5% mugwort powder) were used to analyze the protein expression patterns associated with mugwort. a spot with different densities was selected through analysis of the gel images as a protein to show expression changes between groups. however, the spot selected as a protein showing an expression change was used only to show regular density changes of 2 to 3 spots among the four spots obtained per group. although most spots were of similar density between the c and t groups, 12 spots showed different expression levels, and all the spots were of greater density in the t group (figure 2). the spots with increased expression were described by mean intensity, as shown in table 1. the density of spot 7002 dramatically increased by 4.66-fold, while spots 1004, 5001 and 8002 increased more moderately by 2.51, 2.73 and 2.57 fold, respectively. the densities of spots 1106, 2001, 5002, 5602 and 7102 were marginally increased by 1.35, 1.41, 1.64, 1.69 and 1.60 fold, respectively. properties of the proteins identified by pmf the spots with higher expression levels identified by pmf are shown in table 2. myosin light chain 2v and eukaryotic translation initiator factor 5a (elf-5a) were obtained with 61 and 58% sequence coverage, respectively. myosin light polypeptide 3, f1-atpase, peroxiredoxin-2, kelch-related protein 1, mhc class 1 antigen 7 (sla-7), glycine amidinotransferase, and triosephosphate isomerase (tpi 1; http://www. ncbi.nlm.nih.gov/protein /38512111) were obtained with sequence coverage below 44%. when compared with known proteins, the proteins discovered by pmf were highly likely to be correctly allocated because of their isoelectric point (pi value), their 2-de position, and their molecular weight. myosin is the most abundant contraction molecule in mammalian skeletal muscle, and it relates directly to the quality of pork meat. although the heavy chain of myosin has been studied with respect to skeletal pork meat figure 2. comparative analysis of protein expression patterns with and without dietary mugwort powder. (a) and (b) indicate protein patterns of pig longissimus dorsi muscle grown on diets with and without mugwort. the enhanced spots are marked by numerals. table 1. proteins from pig l. dorsi muscle exhibiting changes in expression due to dietary mugwort powder. ssp 1) mw 2) pi 3) mean intensity mean intensity fold variation: of c 4) of t 5) t versus c 3 19.69 4.34 540 ± 235 6) 1055 ± 519 1.95 1004 15.58 4.68 309 ± 116 774 ± 366 2.51 1106 27.06 4.64 3292 ± 733 4442 ± 1773 1.35 1405 55.91 4.58 569 ± 161 1020 ± 765 1.79 2001 24.12 4.69 817 ± 128 1153 ± 521 1.41 2601 78.37 4.69 295 ± 153 574 ± 450 1.95 5001 19.98 6.23 357 ± 331 973 ± 825 2.73 5002 24.42 6.48 309 ± 417 508 ± 256 1.64 5602 76.14 5.98 551 ± 256 933 ± 530 1.69 7002 15.67 7.69 316 ± 65 1472 ± 1475 4.66 7102 28.79 7.95 2930 ± 73 4692 ± 889 1.60 8002 15.53 8.10 2550 ± 44 6559 ± 2333 2.57 1) ssp; standard spot protein, 2) mw; molecular weight in kda, 3) pi; isoelectric point, 4) pig fed basal diet without mugwort, 5) basal diet + mugwort powder 1.5%, 6) standard deviation. muscle by various methods (bee et al., 1999; gunawan et al., 2007; lin and hsu, 2005), studies involving other tissue types and myosin light chain have yet to be conducted. the protein elf-5a has been widely observed in all eukaryotic cells, and disruption of the gene encoding this protein causes the arrest of cell growth, cell death, and/or differentiation of cancer cells (jao and yu chen, 2002). f1-atpase mediates atp production in mitochondria (boyer, 1997). although f1-atpase chain d subunit d (zhang et al., 2000), is one of the components of this complex, we do not know the reason why only this subunit shows increased expression. if f1-atpase chain d acts to recruit the f1-atpase complex or enhance its activity, it is a reasonable hypothesis that chain d would have an effect on meat quality. muscle fiber is divided into types 1, 2a, and 2b. type 1 maintains a high number of mitochondria, whereas type 2b has fewer of this organelle (cieslak et al., 2000; kim et al., 2009). since f1-atpase is one of the major components of mitochondria, an increase of f1-atpase might lead to an increase of type 1 fiber containing abundant mitochondria. therefore, it was assumed that an increase in f1-atpase table 2. proteins identified by pmf. spot predicted protein accession number sequence pi/mw (source) coverage (%) (kda) 3 myosin light chain 2v np_998956 61 4.8/18.87 1004 eukaryotic translation initiation factor 5a np_001003658 58 5.1/17.04 similar to myosin light polypeptide 3 (myosin light chain 1, slow-twitch 1106 muscle b/ventricular isoform) (mlc1sb) (ventricular/slow twitch myosin xp_533849 43 5.0/22.52 alkali light chain) (cardiac myosin light chain-1) (cmlc1) isoform 1 chain d, the structure of bovine f1-atpase 1405 covalently inhibited with 1nbm_d 38 5.0/51.46 4-chloro-7-nitrobenzofurazan 2001 2601 5001 5002 peroxiredoxin-2 (thioredoxin peroxidase 1) (thioredoxin-dependent peroxide reductase 1) (thiol-specific antioxidant protein) (tsa) similar to kelch repeat and btb domain containing protein 10 (kelch-related protein 1) (kel-like protein 23) (sarcosin) isoform 2 mhc class i antigen 7 glycine amidinotransferase (l-arginine:glycine amidinotransferase) (transamidinase) (at) p52552 34 4.7/13.82 xp_535949 21 5.1/68.88 np_998933 21 9.0/46.05 p50441 14 6.2/44.63 7102 tpi 1 protein aah61781 44 7.1/ 27.21 pl, isoelectric point; mw, molecular weight. might induce a change in the quality of pig meat. since peroxiredoxin-2 exists in mitochondria and plays a role in electron transport (gromer et al., 2004), an increase in this protein could correlate with mitochondrial number, similarly to f1-atpase. kelch-related protein 1 has been reported to play a critical role in the maintenance of an ordered cytoskeleton (jiang et al., 2005). mhc class i immune responses react against foreign pathogens and transplanted tissue. mhc class i molecules are divided into classical and non-classical types, and sla-7 is included in the non-classical type (joyce et al., 2008). although this protein is induced as a result of immune responses due to physiological activating materials in mugwort, a definite function of the protein remains to be discovered. since glycine amidinotransferase relates to the biosynthesis of creatine in the liver and pancreas (engelke et al., 2009), we do not know the reason why this protein's expression is higher in this tissue. tpi protein is an enzyme that mediates the reaction from dihydroxyacetone phosphate (dhap) to glycerolaldehyde-3-phosphate, and it plays an essential role in the embden-meyerhof pathway (emp). since increases in this enzyme were assumed to correlate with mitochondrial numbers as mentioned previously, the increased amount of tpi would lead to increased sugar utilization by promoting the complete oxidation of sugar. therefore, the increased tpi protein might be involved in energy utilization owing to the increased growth rate and maintenance energy requirements. properties of proteins identified by caf-maldi sequencing the protein spots that could not be identified via pmf were analyzed by caf-maldi sequencing. as shown in table 3, serum albumin precursor and two types of myoglobins were identified by caf-maldi sequencing. generally, serum albumin, which has the signal peptide from serum albumin precursor consisting of 18 amino acids removed, plays an essential role in maintaining osmotic pressure for the approximate distribution of cellular fluidity in the extracellular matrix and between cells. serum albumin also plays other roles as a serum carrier via nonspecific interactions with hydrophobic steroid hormones as well as a transport protein for hemin and fatty acids (roche et al., 2008). therefore, the increase of this precursor might be involved in promoting physiological activation in pigs due to mugwort. myoglobin transfers oxygen to muscle cells. since the mitochondrial numbers in pigs fed with mugwort were assumed to indirectly increase as described in the cases of f1-atpase and peroxiredoxin-2, following the increase in the overall activation of oxidative electron transport, it was predicted that myoglobin in muscle helps to promote the supply of oxygen, which is the final electron acceptor. two myoglobin spots might have been obtained as derivatives originating from the chemical treatment process. in summary, the effects of mugwort treatment potentially include increases in growth, ordered arrangement of table 3. proteins identified by caf-maldi sequencing. spot identified protein accession number identified amino acid pi/mw (kda) (source) sequence 5602 serum albumin precursor p07724 dvflgtflyeysr 5.98/76.14 7002 myoglobin 1 1606205a hgbtvltalggilk 7.69/15.67 8002 myoglobin 2 1606205a hgbtvltalggilk 8.10/15.53 the cytoskeleton, immune responses, aerobic respiration, oxygen transport, and type 1 muscle fiber, which contains the highest amount of mitochondria. acknowledgements this work was carried 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chen sj, chen z (2000). cloning and functional analysis of cdnas with open reading frames for 300 previously undefined genes expressed in cd34+ hematopoietic stem/progenitor cells. genome res. 10(10): 1546-1560. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (4), pp. 001-006, april, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper polymorphism and association of microsatellite sj01 with birth weight and early growth traits in pigs y. j. zhang1,2, g. c. li1 and y. l. jiang1* 1 laboratory of animal molecular genetics, college of animal science and veterinary medicine, shandong agricultural university, taian 271018, china. 2 college of life science, linyi normal university, linyi 276005, china. accepted 22 november, 2016 myostatin is a negative regulator of animal skeletal muscle development and sj01 is a microsatellite locus flanking porcine myostatin gene. in the present study, the polymorphism of microsatellite sj01 in three pig populations, that is, duroc (n = 420), yorkshire (n = 414) and landrace (n = 119), were investigated, and its associations with birth weight and early growth traits were analyzed. the results indicated that genotype bb was predominant in yorkshire pigs and allele b was predominant in both yorkshire and landrace pigs, whereas allele a was more prevalent in duroc pigs. among the three pig breeds, duroc population had the highest heterozygosity and deviated from hardy-weinberg equilibrium (p < 0.05). the average daily gain from 28 d to 70 d in yorkshire pigs and the body weight at 70 d in landrace pigs were significantly different between sj01 genotypes (p < 0.05). these results suggest that microsatellite sj01 is a potential dna marker for early growth trait selection in yorkshire and landrace pigs as well as for refining qtl for early growth traits on ssc15. key words: pig, myostatin, microsatellite sj01, polymorphism, early growth traits. introduction myostatin is a negative regulator of skeletal muscle mass in mammals (mcpherron et al., 1997). natural mutations, such as deletions, point mutations in myostatin coding region, intronic region or 3′-untranslated region, bring about double-muscling traits in beef cattle (mcpherron and lee, 1997), sheep (clop et al., 2006; boman et al., 2009), human (schuelke et al., 2004) and dogs (mosher et al., 2007). in pigs differing in muscular traits, such as wild boar, pietrain and laiwu pigs, however, no mutation with similar effect in myostatin gene was reported (jiang et al., 2002a; stinckens et al., 2008). recent studies reported associations of polymorphisms in the promoter region of porcine myostatin gene with production traits (jiang et al., 2002b), meat quality traits (guimaraes et al., 2007) and mrna level (stinckens et al., 2008), suggesting that, in pigs, myostatin might also play important roles in the development of skeletal muscle and its related traits. microsatellite loci are frequently used as dna markers to locate quantitative trait loci (qtl), but usually to a *corresponding author. e-mail: yunliangjiang@gmail.com. relatively wide range of chromosomal fragment, in which many genes reside. to find the functional gene(s) or quantitative trait nucleotide (qtn) fine mapping with more markers is required. microsatellite sj01, which was identified by our previous study, is a locus located 42 kb downstream of porcine myostatin gene (jiang et al., 2004) whether it is associated with production traits and can be used as dna marker remain unknown. birth weight and early growth traits were two economically important traits in that, on one hand, they directly affect the mortality rate of weanling pigs and on another hand, are associated with growth rate and carcass traits on test. a qtl mapping study based on a f2 pedigree constructed with wild boar ×yorkshire suggested one qtl for average daily gain from birth to 70 d on ssc15 (50 cm, male; 78 cm, female) (knott et al., 1998), the mapping interval was partially overlapped with myostatin gene and sj01 locus. the production traits associated polymorphisms involve three sites in the promoter region of porcine myostatin gene (jiang et al., 2002a; guimaraes et al., 2007; yu et al., 2007; stinckens et al., 2008), the detection of which is not practical for use in molecular pig breeding. therefore, table 1. means and s.d. of birth weight and early growth traits. trait breed duroc yorkshire landrace bw0 (kg) 1.44 ± 0.31 1.49 ± 0.31 1.46 ± 0.25 bw28 (kg) 7.12 ± 1.52 7.53 ± 2.05 8.53 ± 2.15 bw70 (kg) 21.13 ± 5.27 21.78 ± 5.53 24.77 ± 5.65 adg1 (kg) 0.20 ± 0.05 0.20 ± 0.06 0.25 ± 0.08 adg2 (kg) 0.36 ± 0.10 0.50 ± 0.13 0.35 ± 0.07 bw0, bw28 and bw70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. adg1 and adg2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. it is necessary to find another closely linked marker. given the important role of myostatin in meat traits and the short physical distance between myostatin and sj01, we speculate that sj01 is likely associated with some production traits in pigs and therefore is a candidate marker. in this study, the polymorphism at sj01 locus and its associations with porcine body weight and early growth traits were analyzed in three pig breeds (duroc, yorkshire and landrace) which are currently used for worldwide pork production. materials and methods population and traits three western pig breeds, including 420 duroc, 414 yorkshire and 119 landrace individuals were randomly sampled from pig breeding center of shandong academy of agricultural sciences. animals were reared under the same environment and had free access to feed and water. data of the traits including birth weight (bw0), body weight on day 28 (bw28) and body weight on day 70 (bw70) of each individual were collected on farm. sampling was performed by taking a notch from the ear of pigs and put into a 1.5-ml eppendorf tube containing 70% ethanol and stored at −20ºc. the total number of production records was 2757 and the mean and standard deviation (s.d.) of the five traits are presented in table 1. isolation of genomic dna genomic dna from ear notch was isolated with phenol/chloroform method (sambrook and russell, 2001), dissolved in tris-edta buffer (ph 8.0) and stored at −20ºc. the concentration was checked by running 0.7% agrose electrophoresis and using uv spectrophotometer. genotyping primers for sj01 were from sts database of national centre for biological information (unists accession number: 81993): forward 5′-cagaacataaatgccaagag-3′ and reverse 5′-agtattta gtgaacacctcg-3′. pcr was performed by mixing 0.3 µl (50 100 ng) of genomic dna, 1.6 µl of dntps (2.5 mm each), 1.2 µl of mgcl2 (25 mm), 0.5 µl each of primers (10 µm), 0.1 µl of rtaq polymerase (5 u/µl, takara, dalian china) and 2.0 µl of 1×rtaq buffer in a 20 µl volume and running on a mastercycler gradient (eppendorf, germany) according to the following program: 95ºc for 3 min, 32 cycles of 95ºc for 30 s, 55ºc for 30 s and 72ºc for 30 s and final extension at 72ºc for 5 min. the pcr products were electrophoresed on 1.5% agarose gel to check amplification efficiency. genotyping of sj01 was carried out by running 12% polyacrylamide gels (arc：bis = 19:1) with 1×tbe as electrophoresis buffer at 180 v for 10 h. after electrophoresis, the gels were silver-stained and the genotype was determined according to the electrophoresis band patterns. sequencing the pcr products were purified with gel extraction system b (biodev, china), inserted into pmd-18t vector and was used to transform e. coli dh5 α competent cells. at least four clones were sequenced using the bigdye v3.1 kit (applied biosystem) by shanghai songon co. ltd (shanghai, china). statistics the genotype frequency, allele frequency, observed heterozygosity (ho), expected heterozygosity (he), average heterozygosity (ha), number of available alleles (na), number of effective alleles (ne) and polymorphic information content (pic) were obtained and the deviation from hardy-weinberg equlibrium was tested with popgen 32 software. associations of sj01 genotypes with bw0, bw28, bw70, average daily gain from birth to day 28 (adg1) and average daily gain from day 28 to day 70 (adg2) were performed separately in duroc,yorkshire and lanrace pigs with the proc glm procedure of sas 8.2 software (sas institute inc, 1998). in the statistics model, genotype, farm-year-season and sex were treated as fixed effects, litter size as co-variable to eliminate the influences of litter size on birth weight (model 1), birth weight as co-variable to eliminate the influences of birth weight on body weight (model 2) and start body weight as co-variable to eliminate the influences of start body weight on average daily gain (model 3). model 1: yijklm=µ+di+cj+sk+zl+eijklm; model 2: yijklm=µ+di+cj+sk+bl+eijklm; model 3: yijklm=µ+di+cj+sk+pl+eijkml; where yijklm is the birth weight (bw0), body weight (bw28, bw70) figure 1. genotyping and identification by sequencing of microsatellite sj01 locus. a. pcr amplification; b. genotyping. lanes 1-11 represent genotypes aa, bd, ab, ac, bc, be, dd, dd, cd, bb, ad and cc, respectively. c. sequencing result, indicating dinucleotide (tg) repeats of 16, 17 and 22, respectively. and average daily gain (adg1, adg2) of each individual for models 1-3 respectively; µ is the overall mean of population; di is the mean effect of genotype i; cj is the farm-year-season effect; sk is the effect of sex; zl is the effect of litter size (model 1); bl is the effect of birth weight (model 2); pl is the effect of start body weight (model 3); eijklm is the residual effect. the difference between genotypes was considered as significant when p < 0.05. ac, bc, be, dd, cd, bb, ad and cc is shown in figure 1b. among the five alleles, alleles a and b were reported by our previous study, which were composed of 13 and 19 repeats of dinucleotide (tg), respectively (jiang et al., 2004); alleles c, d and e were composed of 16, 17 and 22 repeats of dinucleotide (tg), respectively (figure 1c) and were reported for the first time by present study. results genotypes and alleles analysis of sj01 polymorphism revealed 13 genotypes (aa, ab, bb, bc, bd, be, ac, ad, ae, cc, cd, dd and ee) and five alleles (a, b, c, d and e) in duroc (n = 420), yorkshire (n = 414) and landrace (n = 119) pig populations. the electriphoresis result of genotypes aa, bd, ab, population genetic parameters at sj01 locus differences in genotype frequency and allele frequency were found between three pig populations of duroc, yorkshire and landrace (table 2). for genotypes with frequency exceeding 0.1, genotypes ab, aa and ad in duroc, ab, bb and be in yorkshire and bb and bd in landrace were identified. genotypes bc, ae, cc, cd and ee occurred at low frequencies in all of the three pig table 2. genotype frequency of microsatellite sj01 in duroc, yorkshire and landrace populations. breed (n) aa ab bb bc bd be ac ad ae cc cd dd ee duroc (420) 0.2167(91) 0.2905(122) 0.0762(32) 0.0048(2) 0.081(34) 0.0024(1) 0.0357(15) 0.2500(105) 0.0000(0) 0.0024(1) 0.0143(6) 0.0262(11) 0.0000(0) yorkshire(414) 0.0024 (1) 0.1232(51) 0.6594(273) 0.0000(0) 0.0773(32) 0.1232(51) 0.0000 (0) 0.0024 (1) 0.0048(2) 0.0000(0) 0.0000(0) 0.0024 (1) 0.0048(2) landrace(119) 0.0000 (0) 0.0000(0) 0.5210(620) 0.0000(0) 0.4034(48) 0.0084 (1) 0.0000 (0) 0.0000 (0) 0.0000(0) 0.0000(0) 0.0000(0) 0.0672 (8) 0.0000(0) table 3. allele frequency and population genetic parameters of microsatellite sj01 in duroc, yorkshire and landrace populations. breed a b c d e h-w test ho he ha na ne pic duroc 0.5048 0.2655 0.0298 0.1988 0.0012 0.0252 0.6786 0.6351 0.6343 5.0000 2.7347 1.1309 yorkshire 0.0676 0.8213 0.0423 0.0688 0.4226 0.3309 0.3148 0.3144 4.0000 1.4587 0.6618 landrace 0.7269 0.2689 0.0042 0.9308 0.4118 0.4010 0.3993 3.0000 1.6647 0.6080 table 4. least squares means for performances among genotypes of microsatellite sj01 in duroc populations. performance aa ab ac ad bd p-value bw0 1.41 ± 0.03 (n = 88) 1.47 ± 0.03 (n = 112) 1.41 ± 0.09 (n=13) 1.47 ± 0.03 (n = 101) 1.49 ± 0.05 (n = 34) 0.7686 bw28 7.18 ± 0.17 (n = 67) 7.28 ± 0.16 (n = 72) 7.12 ± 0.50 (n=8) 7.10 ± 0.18 (n = 57) 7.06 ± 0.26 (n = 27) 0.8664 bw70 21.31 ± 0.72 (n = 57) 21.51 ± 0.64 (n = 71) 19.39 ± 2.01 (n=7) 20.00 ± 0.73 (n = 56) 20.76 ± 1.13 (n = 22) 0.3297 adg1 0.20 ± 0.01 (n = 65) 0.21 ± 0.01 (n = 66) 0.20 ± 0.02 (n=7) 0.20 ± 0.01 (n = 55) 0.20 ± 0.01 (n = 27) 0.8664 adg2 0.36 ± 0.01 (n = 43) 0.36 ± 0.01 (n = 43) 0.36 ± 0.07 (n=2) 0.35 ± 0.02 (n = 29) 0.36 ± 0.02 (n = 17) 0.9808 bw0, bw28 and bw70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. adg1 and adg2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. populations. in yorkshire pigs, genotype bb was predominant, while in duroc and landrace pigs, no predominant genotype was found. as for allele frequencies, five, four and three alleles were detected in duroc, yorkshire and landrace pig populations, respectively and allele b was predominant in yorkshire and landrace, whereas allele a was more prevalent in duroc populations (table 3). at sj01 locus, yorkshire and landrace, but not duroc, were in a state of hardy-weinberg equilibrium (p > 0.05). information obtained from other genetic parameters of ho, he and pic (table 3) indicated that, at this locus, duroc is genetically more diverse compared to yorkshire and landrace pig populations. associations of sj01 genotype with birth weight and early growth traits the relationship of sj01 genotype with birth weight and some early growth traits, that is, body weight at 28 d and 70 d and average daily gain from birth to 28 d and from 28 to 70 d, were analy zed in present study. the results showed that, in duroc pig populations, differences in birth weight, body weight at 28 and 70 d and average daily gain traits were not significant between genotypes of sj01 locus (p > 0.1) (table 4); whereas in yorkshire pig populations, the least squares means for average daily gain from 28 to 70 d were significantly different among genotypes of sj01 locus (p < 0.05) (table 5) and in landrace pigs, the least squares means for body weight at 70 d were significantly different among genotypes of sj01 locus (p < 0.05) (table 6). the effect of sj01 on table 5. least squares means for performances among genotypes of microsatellite sj01 in yorkshire populations. performance ab bb bd be p-value bw0 1.44 ± 0.06 (n =51) 1.48 ± 0.02 (n = 272) 1.57±0.07 (n = 32) 1.56±0.05 (n = 51) 0.2361 bw28 7.54 ± 0.34 (n =34) 7.63 ± 0.17 (n = 161) 7.01±0.49 (n = 16) 6.80±0.37 (n = 29) 0.1581 bw70 20.45 ± 1.01 (n = 28) 22.05 ± 0.40 (n = 176) 22.48±1.21 (n = 19) 19.87±0.84 (n = 40) 0.0631 adg1 0.21 ± 0.01 (n = 30) 0.21 ± 0.00 (n = 149) 0.17±0.02 (n = 12) 0.19±0.01 (n = 28) 0.0910 adg2 0.42 ± 0.03 (n = 18) 0.49 ± 0.01 (n = 104) 0.53±0.04 (n = 9) 0.47±0.02 (n = 23) 0.0464 bw0, bw28 and bw70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. adg1 and adg2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. table 6. least squares means for performances among genotypes of microsatellite sj01 in landrace populations. performance bb bd dd p-value bw0 1.46 ± 0.03 (n = 61) 1.44 ± 0.04 (n = 48) 1.47 ± 0.10 (n = 8) 0.8384 bw28 8.35 ± 0.25 (n = 49) 8.51 ± 0.36 (n = 25) 7.14 ± 1.22 (n = 2) 0.5400 bw70 24.53 ± 0.83 (n = 27) 26.60 ±1.33 (n = 9) 33.02 ± 2.83 (n = 2) 0.0268 adg1 0.25 ± 0.01 (n = 48) 0.25 ± 0.01 (n = 25) 0.20 ± 0.04 (n = 2) 0.5400 adg2 0.38 ± 0.03 (n = 20) 0.35 ± 0.04 (n = 3) 0.6554 bw0, bw28 and bw70 represent birth weight, body weight at 28 d and body weight at 70 d, respectively. adg1 and adg2 represent average daily gain from birth to 28 d and from 28 to 70 d, respectively. other production traits of yorkshire and landrace pigs was not significantly different (p > 0.5). discussion myostatin is an important growth/differentiation factor regulating skeletal muscle development, therefore, is also associated with meat production and quality traits as well as with other related traits. porcine myostatin gene is mapped in a sw1065-(53.1)-sw1263(55.0)-myostatin(55.0)-swr1533 -(57.9) linkage group on ssc15q2.3 (sonstegard et al., 1998), the flanking microsatellite loci sw1263 and swr1553 were located at 55.0 and 57.9 cm, respectively. the microsatellite locus sj01 is located 42 kb downstream of myostatin gene, more close to myostatin gene than swr1553. compared with other microsatellite loci located in nonstructural gene region, the number of genotypes and alleles were rather few (jiang et al., 2004 and present study) and the heterozygosity and diversity were rather low for sj01, suggesting relatively higher selection force and more conservation in this region than microsatellite loci located in nonstructural gene regions. according to the results of sj01 polymorphism, of the three pig breeds used in this study, duroc pigs were more diverse and its genetic component was more complex. a qtl for average daily gain from birth to 70 d was mapped on ssc15 (50 cm, male; 78 cm, female) (knott et al., 1998), where myostatin gene and sj01 locus also reside. in this study, sj01 locus is found to be associated with average daily gain from 28 to 70 d in yorkshire pigs and with body weight at 70 d in landrace pigs. besides, a nearly significant association was also noted for sj01 with body weight at 70 d and average daily gain from birth to 28 d in yorkshire pigs. these data suggest that this region likely harbors a qtl affecting early growth and sj01 is a useful marker for refining this qtl. growth traits, such as body weight and average daily gain, are controlled by multiple genes. in addition to the aforementioned qtl for average daily gain from birth to 70 d on ssc15 (knott et al., 1998), other qtls for body weight of 10 weeks were found on ssc1, 4, 6, 7 and 9 and for average daily gain from 4 weeks to 13 weeks on ssc9 and ssc10, from 3 weeks to 10 weeks on ssc 1, 2, 4 and 7, respectively (http://www. animalgenome.org/ qtldb /pig.html). the effect of each gene or qtl on early growth traits is somewhat dependent on genetic background and environmental effect, giving rise to the differences between breeds concerning the relationship of sj01 genotype with these traits, as shown in this study. although three relatively large populations of duroc, yorkshire and landrace were used for association study, some genotypes were present at low frequency, that is, genotype dd in landrace population. the effect of sj01 genotype on birth weight at 70 d in landrace pigs requires further research with a large population containing more individuals with genotype dd, or in some pedigrees segregating at this locus. in conclusion, sj01 is a potential dna marker for average daily gain from 28 to 70 d in yorkshire and for body weight at 70 d in landrace pigs and is useful for refining qtl position for early growth traits. acknowledgements this work was supported by the 863 high-technology project of china (no. 2006aa10z1e1). we thank s. liang for helpful discussion. references boman ia, klemetsdal g, blichfeldt t, nfastad o, vage di (2009). a frameshift mutation in the coding region of the myostatin gene (mstn) affects carcass conformation and fatness in norwegian white sheep (ovis aries). anim. genet. 40(4): 418-422. clop a, marcq f, takeda h, pirottin d, tordoir x, bibé b, bouix j, caiment f, elsen jm, eychenne f, larzul c, laville e, meish f, milenkovic d, tobin j, charlier c, georges m (2006). a mutation creating a potential illegitimate microrna target site in the myostatin gene affects muscularity in sheep. nat. genet. 38(7): 813-818. guimaraes sef, stahl ch, lonergan sm, geiger b, rothchild mf 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sonstegard ts, rohrer ga, smith tp (1998). myostatin maps to porcine chromosome 15 by linkage and physical analyses. anim. genet. 29(1): 19-22. stinckens a, luyten t, bijttebier j, van den maagdenberg k, dieltiens d, janssens s, de smet s, georges m, buys n (2008). characterization of the complete porcine mstn gene and expression levels in pig breeds differing in muscularity. anim. genet. 39(6): 586-596. yu lz, tang h, wang jy, wu y, zou ll, jiang yl, wu cx and li n (2007). polymorphisms in the 5′ regulatory region of myostatin gene are associated with early growth traits in yorkshire pigs. science in china series c: life sci. 50(5): 642-647. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (10), pp. 001-006, october, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper mitochondrial genome of taiwan pig (sus scrofa) chia-hsuan chen1,2 , hsiu-lin huang1 , hsiu-ya yang 2, shan-hu lai1, neim-tsu yen2 , ming-che wu2 and mu-chiou huang1* 1 department of animal science, national chung hsing university. 250 kuo kuang road, taichung 402, taiwan. 2 livestock research institute, council of agriculture, executive yuan. 112, muchang, xinhua dist., tainan 712, taiwan. accepted 23 april, 2018 the purpose of this study is to investigate the complete nucleotide sequence of the mitochondrial genome of the taiwan lanyu pig (sus scrofa) and its phylogenetic relationships with other pig breeds. thirty-four forward and reverse primers were designed. sequencing was performed in both directions. the results showed that, the complete sequence of the mitochondrial genome of the taiwan lanyu (s. scrofa) is 16,747 bp, which was deposited in ncbi genbank (accession number dq518915). the complete sequence includes two rrna (12s and 16s), 22 trna and 13 mrna genes. the length of the d-loop region is 1,314 bp and there are 25 repeat sequences (5’-tacacgtgcg) in the region. it seems that there is a significant difference in the d-loop region between the lanyu and the european duroc (10 repeats), the landrace (13 repeats), the large white (6 repeats) or asian pig breeds such as the japanese wild boar (1 repeat) and the ryukyu wild boar (1 repeat). the phylogenetic relationships of the lanyu by comparing the sequence of the mitochondrial genome and the dloop region of asian and european pig breeds were investigated. it revealed that, the genetic distance of the lanyu is high when compared with both the european and asian pig breeds; the genetic exchange between the lanyu and other breeds is not frequent. there is also no evidence of genetic exchange or introgression caused by population migration. therefore, we conclude that the lanyu is an independent branch among the breeds. key words: complete genome, mitochondrial dna, phylogenetic relationships, genetic distance, pig. introduction mitochondrial dna has been widely used for evolution studies, because the evolution is more diversified than nuclear dna (brown et al., 1982, 1989; luikart et al., 2001). for evolution research, a number of studies of the d-loop region sequence mutation in mitochondrial dna have also been investigated (watanabe et al., 1985; lan and shi, 1993; huang et al., 1999; gongora et al., 2004). the lanyu pig is an indigenous breed form the lanyu islet. the island is located at the southeast of taiwan (jiang et al., 2008). since 1980, the livestock research institute of council of agriculture in taiwan introduced the lanyu small-ear strain and from then on, the pig group became isolated and did not cross with other breeds. lanyu pig skin colour and hair is black. the body weight at five months is less than 20 kg; mature pigs weight less *corresponding author. e-mail: mchunag@mail.nchu.edu.tw. tel: +886-4-22840366 ext 239. fax: +886-4-22860265. than 70 kg. the purpose of this study is to sequence the complete mitochondrial dna of the unique pig breed in taiwan. we also studied the phylogenetic relationships of the lanyu and other breeds from europe, japan, korea and china, in order to investigate the migration of pig populations. materials and methods blood collection and dna extraction 10 ml of blood was collected with anticoagulant from jugular vein of the lanyu boar reared in the livestock research institute, council of agriculture (taitung animal propagation station, lritaitung). dna extraction was performed according to the user manual of the kit (puregene gentra system, taipei, taiwan). primer design primers were designed according to the accession number table 1. primer pairs for pig complete mitochondrial dna sequencing. no. forward/reverse sequence no. forward/reverse sequence 1 caaccaaaacaagcattccattcg/ggtttggcaaggcgttataggg 18 aatccaactaacatccaaac/ccagtgaagaacagaacc 2 aatcgcccactctttccc/tgcctgctttcgtagcac 19 cacatagtaaacccaagccc/catctacgaagtgtcagtatcag 3 tcattcaaaccccccttaccc/atgtgaagcaccgccaagtc 20 ccacttcacatccaaccaccac/agcttcgcaggctgcaaac 4 ccacgaaagtgactctaataatcc/cccttacggtactatctctatagc 21 acgaatgaacccaaaaag/tagtataagggggaggag 5 tgggtactttgaaccaaagc/tgtttgccgagttccttttac 22 tctcacttaatatcttcactac/gtcgttctgtttggtttc 6 aagcctttctcctcgcacac/gaaaccgacctggattgctc 23 attataaccttcaccgccac/tgcaaacactcctaccactc 7 caactcaaccacaaagggataaaac/ggagattgaggatgtgcctg 24 tggcataatgcgaatcactac/cgattagattgatggatgggg 8 agaacagggcacattaggg/gctatgaagaatatggcgaaagg 25 cgtactgctaaaactcggagg/ggtagaatatgtagggctatgagg 9 ctctatcaaccctaatcacaacac/tccgattcagatgagtagtcag 26 acccccatccatcaatctaatc/ggactaggctgagagtgaag 10 agaggttcaaaccctcttatttc/ctctcaataggaggcctg 27 atcctacgccttcactctc/tctgttcgtccgtgtcatc 11 gagggctaaatcaaacccaac/ggatgggaacatagtcagtgag 28 ttcccgtagcactattcgtc/gctttatacagtccgcctatttttc 12 gcatcatcatgccaacactc/cagttaccaaaacccccaatc 29 aatagtgacaatcggcatcaac/taggcggtgtatgatggagg 13 atgtaattgttacagctc/caactaaatacttttactcc 30 acaacacaacctactacc/ttcttctaagccctctcc 14 tcaccgtaggaatagacgtag/tggaaagggtaagccatagag 31 caataccacaaccaactccac/gcggtaatgatgaatggcag 15 gtttcaagccaacgtcataacc/tgtttctacttcttgggcatcc 32 cacacgattcttcgccttc/gccctccttttctggtttac 16 ccctatatgcctctatggcttac/ttaaaccgagggatgggac 33 gcctccatcttatacttcctaatc/gtacttggcgttttggtttg 17 aacctggagaaatacgac/tgaatgagtgtggtagtag 34 acactaacatgaattggaggac/atgcacgacgtacataggg af034253 of ncbi genbank. a total of 34 primers were used (table 1). fragment amplification by polymerase chain reaction (pcr) and nucleotide sequencing a total volume of 25 ul of pcr mix consists of 2.5 µl pcr buffer (10 mm tris-cl, 50 mm kcl and 1.5 mm mgcl2), 1 ul of dntps, 0.5 µl of each forward and reverse primer (10 µm), 0.2 µl taq (takara taq dna polymerase, 5 u/µl), 100 to 500 ng of dna and 17.3 ul of 2 dh2o. the pcr program followed was: pre-denaturation at 94°c for 5 min, followed by 30 cycles of denaturation at 94°c for 30 s, annealing at 50 to 58°c based on the primer sequences for 45 s and an extension of 72°c for 45 s; then, a final extension at 72°c for 10 min. pcr products were purified according the manufacturer’s instructions using a spin column and gel and a clean extraction kit (biokit, hsinchu, taiwan). the pcr products were sequenced according to dideoxynucleotide chain termination method (sanger et al., 1977). the sequences were aligned using the program contigs express in vector nti suite 8 (informax, inc. wesconsin, usa). each sequence had overlap of at least 300 bp. sequences were analyzed in both directions for confirmation. phylogenetic analysis mitochondrial dna sequences of the lanyu pig (dq 518915) were compared with those of different pig breeds such as the large white (ay574048), hampshire (ay574046), berkshire (ay574045), landrace (af034253), duroc (ay337045), italian wild boar (af304201), yucatan (ab015093), meishan (d42181), korean wild boar (ay574047), jeju native pig (jnp8, dq334860; jnp10, dq334861), japanese wild boar (ab015085), ryukyu wild boar (ab015087) and the okinawa native pig (ab015092) breeds in ncbi genbank. multiple alignments between our sequence and the literature ones were performed using bioedit software and dambe software (data analysis in molecular biology and evolution version: 4.5.2) and transfer fas files were then, converted into a readable format of mega3.1 (molecular evolutionary genetics analysis, version 3.1) using data analysis in molecular biology and evolution. neighbor-joining methods and maximum parsimony methods were used to calculate the genetic distances and construct the phylogenetic tree (saitou and ne, 1987). results in present studies, the complete mitochondria genome of the lanyu pig (16,747 bp) was sequenced and deposited in ncbi genbank (accession no. dq 518915). the mitochondrial codons were also investigated. the complete sequence includes two rrna (12s and 16s), 22 trna and 13 mrna genes (table 2). using neighbor-joining methods, we investigated the phylogenetic relationship of the mitochondrial genome of the lanyu and other pig breeds (figure 1). the results showed that the berkshire, large white and the korean wild boar belongs to the asian type. in the hampshire, landrace and duroc breeds, the relationship is closer, which were called european type. the lanyu was classified as a new out-group. as the lanyu belongs to a breed that is unique in the taiwan islands, the question as to whether there is a phylogenetic relationship between the lanyu and other small-ear strains is worthy of study. by searching the sequences of pigs, the lanyu is found to be independently grouped compared with the korean wild boar and european breeds. this study has shown that, the lanyu breed is an independent out-group and is distant to any other breeds. also using neighbor-joining methods, we analysed the phylogenetic relationship of the mitochondrial d-loop region in the lanyu and other pig breeds (figure 2). the lanyu is an independent group from the two and is a separate out-group. its phylogenetic relationship with any other pig breeds is far from table 2. location of features in the mitochondrial genome of the lanyu pig (s. scrofa). name of gene location size (bp) start codon stop codon d-loop 1–1314 1314 repeat region 706–955 250 trna-phe 1138–1207 70 12s-rrna 1208–2163 956 trna-val 2164–2231 68 16s-rrna 2232–3794 1563 trna-leu(uur) 3801–3875 75 nadh1 3878–4837 960 atg tag trna-ile 4836–4900 65 trna-gln 4901–4973 73 l a trna-met 4975–5044 70 nadh2 5045–6088 1044 att tag trna-trp 6087–6154 68 trna-ala 6161–6227 67 l trna-asn 6230–6304 75 l or. l-stand repl. 6301–6347 47 trna-cys 6337–6402 66 l trna-tyr 6402–6467 66 l coi 6469–8013 1545 atg taa trna-ser (ucn) 8017–8087 71 l trna-asp 8093–8160 68 coii 8161–8848 688 atg t-b trna-lys 8849–8915 67 atpase8 8917–9120 204 atg taa atpase6 9078–9758 681 atg taa coiii 9758–10541 784 atg tab trna-gly 10542–10610 69 nadh3 10611–10956 346 ata ta trna-arg 10958–11026 69 nadh4l 11027–11323 297 gtg taa nadh4 11317–12694 1378 atg t- trna-his 12695–12763 69 trna-ser (agy) 12764–12822 59 trna-leu (cun) 12823–12892 70 nadh5 12893–14698 1716 ata taa nadh6 14698–15225 528 l atg taa trna-glu 15226–15294 69 l cyt b 15299–16438 1140 atg aga trna-thr 16439–16506 68 trna-pro 16507–16747 64 l a: (l), light-strand sense; nadh1–6 and nadh4l, subunits 1 to 6 and 4 l of nicotinamide dinucleotide dehydrogenase; atpase6 and 8, subunits 6 and 8 of adenosine triphosphatase; coi to coiii, cytochrome c oxidase subunits i to iii; cyt b, cytochrome b. b: ta-and t--: tnn indicates the incomplete stop codon, that is, amino acid translation is terminated when the gene forms a stop codon by posttranscriptional polyadenylation. european type, but its genetic distance is closer to the asian type. therefore, it can be counted as a cluster. pairwise sequence distances were used to analyse the genetic distance of the d-loop region among the pig breeds, which showed that the lanyu is an independent branch as the genetic distance is far from other pig breeds. in descending order, the lanyu has the biggest genetic distance with the landrace (0.102), hampshire (0.098), duroc (0.097), mexican yucatan, italian wild boar and jeju native pig (jnp10) (0.095), large white (0.092), figure 1. the phylogenetic relationship was analysed using neighbor-joining methods by comparison of the mitochondrial genome sequences of the lanyu and other breeds. kwbi, korean wild boar. figure 2. the phylogenetic relationship was analysed using neighbor-joining methods by comparison of the mitochondrial d-loop region of different strains of pig breeds. (1) jnp8, jnp10, jeju native pig; (2) kwbi, korean wild boar; (3) jw, japanese wild boar. korean wild boar (0.091), meishan (0.090), followed by the berkshire, jeju native pig (jnp8), japanese ryukyu wild boar (0.088). the shortest genetic distance is with the japanese okinawa native pig (0.087). of all the 15 pig breeds, the largest distance from the lanyu is the landrace (0.012), while the smallest is the berkshire and korean jeju native boar (jnp8) (0.002). discussion the complete sequence of lanyu pig contains two rrna (12s and 16s), 22 trna and 13 mrna (table 2). there are four overlapping regions in the pig mitochondria mrna and seven base pairs overlapping on nadh4l with nadh4. atpase6 has 43 and 1 bp overlapping with atpase8 and coiii, respectively. as these genes were from the same transcript in the mammals, there is a 1 bp frame shift during transcription. there are 1 bp overlapping in nadh5 and nadh6 and as they are transcribed from different strands of dna, there is no frameshift. there are 11 mrna genes starting the amino acid methionine in the lanyu; these were transcribed from 9 atg and 2 ata codons, respectively. the initiating codons of the other two genes were transcribed from att (isoleucine) and gtg (valine). there are four proteins (co ii, co iii, nadh3 and nadh4) in the stop codons; the stop codon is not taa and it is different from that in other breeds. the length of the mitochondrial d-loop region of the lanyu is 1,314 bp. twenty-five repeat sequences (5’-tacacgtgcg) in this region were found. there are obvious differences between the lanyu and other breeds, such as the duroc (10 repeats, ay337045.), landrace (13 repeats, af034253), large white (6 repeats, ay574048), japanese wild boar (1 repeat, ab015085) and the ryukyu wild boar (1 repeat, ab015087). we hypothesized that; these 25 repeat sequences in the d-loop region can easily form a hairpin structure in the com-plementary sequences. therefore, the copying process may easily introduce errors (mackay et al., 1993). similar cases occur in other mammals, such as the rabbit (dufresne et al., 1996), horse (xu and arnason, 1994), seal (arnason and johnson 1992), cat (lopez et al., 1996) and sheep (zardoya et al., 1995). many studies have also shown that, the d-loop region has the highest mutation rate in mitochondria. it is thought to be an im-portant region in the analysis of phylogenetic relation-ships and evolution of breeds. the lanyu pig is the small ear breed and lanyu lie in the off-shore island position of taiwan. the people living there rely mainly on the fact that the aboriginal reaches the yami of botel tobago. its development and the way taiwan and other aboriginals of this island build relevantly, have apparent difference. this study analysed the phylogenetic relationship of lanyu pigs and other pig breeds. figure 1 showed that, the berkshire and large white both belong to the asian type, a similar finding to that of kim et al. (2002). in kim’s study, the snps in the mitochondrial d-loop region were analysed and it was found that, chinese mainland pig breeds originated from southeast asia. in addition, they found that the berkshire and large white were both of the asian type. chinese mainland, korean and japanese local breeds have significant differences to the european type. we therefore deduced that, the european breeds may have originated from more than one breed. sequencing of both the complete mitochondrial genome and the d-loop region has given the same result that the berkshire and large white belong to the asian type. kijas and andersson (2001) used the phylogenetic relationship to analyse the mitochondria genome of four pig breeds in order to study the origin of domesticated pigs. using five pig breeds, including the chinese meishan, italian wild boar, sweden landrace and wild boar, they categorized the origin into three groups, a, e1 and e2. the genetic difference of these three groups is between 0.8 and 1.2%. branches a (chinese meishan) and e1 (european domesticated pigs) were probably separated 900,000 years ago. long term domestication started from about 9000 years ago. recent studies have shown that, branch a includes some of the major european breeds, such as the landrace and large white. it is speculated that asian breeds were introduced into europe by introgression in the 18 and 19th centuries. in present studies, both neighbor-joining methods and maximum parsimony methods showed that, the lanyu is of an independent branch in the phylogenetic analysis of both the mitochondrial complete genome and the d-loop region (figures 1 and 2). in 1980, the livestock research institute in taiwan introduced four boars and sixteen sows. they were then isolated and did not introgress with other breeds. in order to preserve the genetic resources of the local pig breeds, the taiwanese government listed lanyu pigs as a member of the taiwan conservation population. this study contributes to the knowledge of phylogenetic relationships of the lanyu with other pig breeds, which can further be applied to other pig breeds in the world. the lanyu is of an independent branch but probably still belongs to the asian type. this result implies that, the frequency of genetic exchange of the lanyu with other pig breeds is low and they therefore do not have a close relationship. this result also confirms that, after the introduction of the lanyu into east taiwan by the live-stock research institute, there was no cross with other pig breeds, the lanyu therefore becoming an isolated population with a unique genetic combination. whether the lanyu has a genetic relationship with the taiwan wild boar remains to be investigated. from ethnological and archaeological research, anthropologists have found that the tao people of lanyu island migrated from batan is-land, of the north philippines. we hypothesize that, lanyu pigs were introduced onto taiwan’s lanyu island by the early tao people from the batan island of the philippines. kim et al. (2002) used the d-loop region to analyse the jeju, chinese, japanese, european and mexican yucatan breeds. they indicated that, the berkshire and large white were of asian breeds. the chinese, japanese and korean local breeds were separated in recent years and based on some limiting factors; their classification may differ from the european type. by studying 48 local breeds, china mainland studies have shown that many breeds originated from southeast asia (kim et al., 2002). paszek et al. (1998) also calculated that, the genetic difference of the chinese meishan and european breeds probably originated around 2227 years ago. watanabe et al. (2003) collected 180 samples from 10 populations in shikoku, kyushu and honshu in japan. they used neighbor-joining methods to analyse 574 bp of the mitochondria region and found that, the ancestors of the japanese wild boar migrated from southeast to northeast asia in the pleistocene period. they deduced that, the japanese wild boar probably originated in mongolia. sus scrofa are widely distributed in asia, europe and north africa and their ancestors were processed into at least 16 subspecies. fossil records show that, wild boars existed in the pleistocene period in japan and that the japanese wild boar existed in shikoku, kyushu, honshu and ryukyu. kijas and andersson (2001) found that, the difference between the sweden wild boar and meishan is 1.21 ± 0.09%. this study found that, the phylogenetic relationship of the ryukyu wild boar and lanyu is similar to other breeds in the d-loop domain. as taiwan is close to ryukyu island, the appearance of the ryukyu wild boar is similar to that of the lanyu pigs. the ryukyu wild boar exists in the japanese amami islands, okinawa main island, ishigaki island and iriomote island. the ryukyu wild boar is small, with a dark brown to black hair colour and a thickset body and is nocturnal and omni-vorous. it belongs to the early asian wild boar branch. luetkemeier et al. (2010) indicated that, asian domestic populations were derived from multiple asian ancestral origins whereas the european domestic populations represent a single ancestral european lineage. the complete mitochondrial genome of taiwan lanyu pigs was sequenced; it is a reference for the sequence difference and phylogenetic relationship of other small-ear strains in the world. analysis showed that, the lanyu could be an independent branch among the other pig breeds. its far genetic distances with other asian and european breeds suggest that, the lanyu did not have a frequent genetic exchange with other breeds. therefore, there is no genetic exchange and cross between the lanyu and other breeds. references arnason u, johnsson e (1992). the complete mitochondrial dna sequence of the harbor seal, phoca vitulina. j. mol. evol. 34: 493505. brown wm, prager em, wang a, wilson ac (1982). mitochondrial dna sequences of primates: tempo and mode of evolution. j. mol. evol. 18: 235-239. brown dr, koehler cm, lindberg gl, freeman ae, mayfield je, myers am, schutz mm, beitz dc (1989). molecular analysis of cytoplasmic genetic variation in holstein cows. j. anim. sci. 67: 1926-1932. dufresne c, mignotte f, gueride m (1996). the present of tandem repeats and initiation of replication in rabbit mitochondrial dna. eur. j. biochem. 235: 593-600. gongora j, fleming p, spencer pbs, mason r, garkavenko o, meyer jn, droegemueller c, lee jh, moran c (2004). phylogenetic relationships of australian and new zealand feral pigs assessed by mitochondrial control region sequence and nuclear gpip genotype. mol. phylogenet. evol. 33: 339-348. huang yf, shi xw, zhang yp (1999). mitochondrial genetic variation in chinese pigs and wild boars. biochem. genet. 37: 335-343. jiang yn, wu cy, huang cy, chu hp, ke mw, kung ms, li ky, wang ch, li sh, wang y, ju yt (2008). interpopulation and intrapopulation maternal lineage genetics of the lanyu pig (sus scrofa) by analysis of mitochondrial cytochrome b and control region sequences. j. anim. sci. 86: 2461-2470. kijas jmh, anderson la (2001). phylogenetic study of the origin of the domestic pig estimated from the near-complete mtdna genome. j. mol. evol. 52: 302-308. kim ki, lee jh, li k, zhang yp, lee ss, gongora j, moran c (2002). phylogenetic relationships of asian and european pig breeds determined by mitochondrial dna d-loop sequence polymorphism. anim. genet. 33: 19-25. lan h, shi l (1993). the origin and genetic differentiation of nation breeds of pigs in southwest china: an approach from mitochondrial dna polymorphism. biochem. genet. 31: 51-60. lopez jv, cerario s, o’brien sj (1996). complete nucleotide sequences of the domestic cat (felis catus) mitochondrial genome and a transposed mtdna tandem repeat (numt) in the nuclear genome. genomics, 33: 229-246. luetkemeier es, sodhi m, schook lb, malhi rs (2010). multiple asian pig origins revealed through genomic analyses. mol. phylogenet. evol. 54: 680-686. luikart g, gielly l, excoffier, l, vigne jd, bouvet j, taberlet p (2001). multiple maternal origins and weak phylogeographic structure in domestic goats. proc. natl. acad. sci. usa, 98: 927-932. mackay sld, ghivizzani sc, madsen cs (1993). transcribed heteroplasmic repeated sequences in the porcine mitochondrial dna d-loop region. j. mol. evol. 37: 36-47. paszek aa, flickinger gh, fontanesi l, beattie cw, rohrer ga, alexander l, schook lb (1998). evaluating evolutionary divergence with microsatellites. j. mol. evol. 46: 121-126. saitou n, nei m (1987). the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol. biol. evol. 4: 406-425. sanger f, nicklen s, coulson ar (1977). dna sequencing with chain terminating inhibitors. proc. natl. acad. usa, 74: 5263-5467. watanabe t, hayashi y, ogasawara n, tomita t (1985). polymorphism of mitochondrial dna in pig based on restriction endonuclease cleavage patterns. biol. gene, 23: 105-113. watanabe t, ishiguro n, nakano m (2003). phylogeography and population structure of the japanese wild boar sus scrofa leucomystax: mitochondrial dna variation. zool. sci. 20: 1477-1489. xu x, arnason u (1994). the complete mitochondrial dna sequence of the horse, equus caballus: extensive heteroplasmy of the control region. gene, 148: 357-362. zardoya r, villalta m, lopez-perez mj montoya j, bautista jm (1995). nucleotide sequence of the sheep mitochondrial dna d-loop and its flanking trna gene. curr. genet. 28: 94-96. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (1), pp. 001-005, january, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review effect of stocking density on pig production jin ho cho and in ho kim* department of animal resource and science, dankook university, no. 29 anseodong, cheonan, choognam, 330-714, korea. accepted 11 september, 2017 numerous stressors such as environmental, nutritional, pathological or equipment related ones are operative in swine facilities. among many factors, socking density increases social stress and influences pig performance. many studies have reported that reducing space allowance could induce decreases in growth performance of pigs. in addition, high stocking density induces a behavioral problem and influences physiological stress during transport. thus, the optimum stocking density has to be defined for improving pig production. key words: performance, pigs, stocking density, stress. introduction under commercial conditions, because marginal profit increases with the size of pig operations (martin and kruja, 2000), the pork industry is shifting toward larger production units. housing pigs in large numbers and groups is a means of reducing housing costs and simplifying some aspects of management challenges. the provision of an adequate space allowance gives pigs sufficient space for drinking, lying and feeding. the main requirement for a pig during feeding is to be able to get to, and remain at the feed trough without feeling that their feeding space is threatened (baxter, 1985a). also, pigs will choose a resting area based on the security of that area (baxter, 1985b). from 25 kg to heavier body weights, pigs lie together most of the day. as they grow up, pigs begin to prefer recumbent lying postures (ekkel et al., 2003). space allowances should facilitate these behaviors (table 1). high stocking density may cause a behavioral problem for pigs. at higher stocking densities, the likelihood of heightened aggression, competition and disease outbreak rapidly rises, and when this happens, the negative relationship between space and growth becomes even worse (lebret et al., 2006). curtis (1996) reported that the reduction in feed intake found in large groups may be caused by an increase in social pressure in larger as compared to smaller groups. *corresponding author. e-mail: inhokim@dankook.ac.kr. tel: +82-41-550-3652. fax: +82-41-553-1618. stressors existing in swine production systems would include cold/hot environmental temperatures (stahly and cromwell, 1979), microbial infections (webel et al., 1997), insufficient space allowances (brumm and miller, 1996; wolter et al., 2000), social mixing (barnett et al., 1993; marchant et al., 1995) and nutritional deficiencies or imbalances (nrc, 1998). these stressors cause growth retardation, changes in hormone release, increases in disease susceptibility, and/or behavioral changes. also, physiological response to stressors (such as heat and spacial restriction) results in activation of the sympathetic nervous system and release of catecholamines and glucocorticoids reduce body weight (breinekova et al., 2006). pig performance being subjected to stressors is common in commercial swine production. stress may cause oxidative changes due to an increase in reactive oxygen species (ros) or a decrease in the antioxidant status (lykkesfeldt and svendsen, 2007). the primary goal of this review is to describe an optimum stocking density and its effects on growth performance and stress in pigs. space allowance space allowance is an important factor in the establishment of social rank (baxter, 1985a). when pigs are housed in space restricted environments, the dominance hierarchy becomes less stable (jensen, 1982). decreased stability most often results from the inability of a subordinate pig to retreat from a threat or act of table 1. categories of space requirements of pigs. group space requirement body-occupation space body-activity space social space system space a pig lying down occupies more space than one standing up, a supine pig occupies more space than one in the semi-sternum position. the space required for body posture changes, like getting up or down, lying supine, turning round/grooming itself. the space required for socialization with other pigs or access by stockpersons. the space required by different management systems, e.g. straw vs. slats, gestation stalls, group in yard, wet feeding vs. dry hoppers, etc. dead space the space required for partitions, passages, corners and pen furniture gadd (2003). aggression on behalf of the dominant pig. therefore, the dominant pig does not recognize submission, and a fight may continue unnecessarily or resume later (baxter, 1985a; turner et al., 2003). mcglone and newby (1994) attempted to determine the optimal amount of space required by a pig to achieve maximum performance. authors evaluated group sizes of 10, 20 and 40 pigs per pen during the grow-finish period (23 to 95 kg of bw) and found no difference in growth performance. however, in a group of growing pigs, those with a restricted space allowance (0.25 m 2 /pig), grew more slowly than pigs with a greater space allowance (0.56 m 2 /pig) (hyun et al., 1998) for each week of the four weeks study. pigs with the restricted space allowance showed reduction in feed intake at the 4th week. pigs with restricted floor space showed an increase uncharacteristically in the behaviours and amounts of aggression (hyun et al., 1998). as pigs become more aggressive, they use more energy and growth rates decline (hyun et al., 1998). increased aggression may also lead to increased injury levels and disease, and thus, increased stress. with an increase in stress, it is possible to reduce the gains potential, which in turn reduces appetite and average daily feed intake (chapple, 1993). furthermore, increased stress can increase the occurrence of stereotypic behaviors and vices, such as tail biting (baxter, 1985b). space allowance has traditionally been expressed empirically by categorizing pigs into a series of weight ranges and by designating space on a per animal basis (brumm and ncr-89 committee on management of swine, 1996). recommendations on space allowance for optimum feed intake have been set by the national research council (1988) with 0.6 m 2 /pig at 25 to 60 kg live weight and 1.0 m 2 / pig at 60 kg live weight. in korea, there is legislation on pig stocking density. also, in the eu, minimum stocking rates, listed in table 2, are the minimum space requirements mandated by eu law.however, in the us, there is no legislation on pig stocking density. the national pork board does, however, made recommendations in its swine care handbook (table 2). these recommendations are based on the minimum space required to achieve maximum performance. effect of stocking density on growth performance stocking density has a significant impact on growth performance. stocking rate can have a major effect on feed intake as shown by brumm and gonyou (2001) who found that a major response to space restrictions was a decrease in feed intake. stocking density allowances were seen excessively in pigs which have been shown to be necessary for maximum performance (edwards et al., 1988). when growing-finishing pigs are given less than optimal space per pig, feed intake always decreases (brumm et al., 2001), often resulting in a reduction in average daily gain (adg), with variable effects on the gain : feed ratio (g:f). social interaction with another pig reduces growth performance and feed intake regardless of stocking density. certain studies have demonstrated a reduction in growth performance with increasing stocking density (petherick et al., 1989; gonyou et al., 1992). swine producers try to maximize profits by minimizing both performance retardation and underutilized space. crowding stress deleteriously affect the growth performance of pigs. pigs, housed on deep-straw for six weeks in groups of 20 or 80, were provided with a low (50 kg/m 2 ) or high (32 kg/m 2 ) space allowance in turner et al. (2003) study. they reported that groups of 80 pigs had a lower adg than groups of 20 (0.684 vs. 0.732 kg). wolter et al. (2002) demonstrated that double stocking (0.64 m 2 /pig vs. 0.32 m 2 /pig) reduced growth rate to ten weeks after table 2. minimum recommended stocking densities for growing-finishing pigs in korea, eu and us. country live weight (kg) space allowance (m 2 ) 10-30 0.30 korea 1 30-85 0.60 85-110 0.90 <10 0.15 10-20 0.20 20-30 0.30 europe 2 30-50 0.40 50-85 0.55 85-110 0.65 >110 1.00 5.4-13.6 (12-30 lb) 0.15-0.23 (1.7-2.5 ft 2 ) 13.6-27.2 (30-60 lb) 0.27-0.37 (3-4 ft 2 ) u.s.a 3 27.2-45.6 (60-100 lb) 0.46 (5 ft 2) 45.6-68.0 (100-150 lb) 0.55 (5 ft 2) 68.0-market (150 lb -market) 0.74 (8 ft 2) 1 http://english.mifaff.go.kr; 2 httpp://www.dardni.gov.uk; 3 http://www.pork.org. weaning. wolter et al. (2003) investigated the subsequent effects of eight weeks space restriction in weanling pigs. for eight weeks, space restricted pigs showed growth retardation when compared with pigs provided with adequate space (27.4 vs. 29.3 kg of bw). smith et al. (2004) reported that nursery pigs with the greatest space allowance (0.35 m 2 /pig) were 5.6% heavier than pigs with the least amount of space (0.23 m 2 /pig). kerr et al. (2005) demonstrated that growing pigs maintained at low stocking density had a higher weight gain (8.23 kg) than their high stocking density counterparts (7.42 kg) for five weeks at the same room temperature. white et al. (2008) reported that reducing stocking density from 0.93 to 0.66 m 2 /pig resulted in 4.0% less body weight, 17.0% less adg, 10.7% less average daily feed intake (adfi) and a 7.8% less g : f ratio. recently, cho et al. (2010) reported that for the six-week nursery period, the crowding reduced adg of gilts (577: 0.50 m 2 /pig, 536: 0.25 m 2 /pig, and 558 g/d: 0.25 m 2 /pig) and barrows (578, 539 and 527 g/d). numerous previous studies evaluated the effects of space restrictions while using similar nutrient densities for all treatments (moser et al., 1985; ncr-89, 1993). in these studies, adfi reduced as a result of space restriction. however, kornegay et al. (1993) did not observe a space × lysine interaction in nursery pigs. further studies revealed no improvement in performance when finishing pigs were fed higher levels of energy and lysine with reduced space (nrc89, 1993) or amino acids (hahn et al., 1995). a study by brumm and miller (1996) indicated that added energy and lysine in growing-finishing pig diets did not overcompensate for the reduction in performance from a reduced space allowance. edmonds et al. (1998) suggested that pigs with lower feed intakes as a result of space restriction did not have higher cp requirements than those with more space. krohn et al. (2000) distributed pigs in nine groups with six pigs in each at three different stocking densities (0.27, 0.44 and 0.52 m 2 /pig). they reported that no significant differences within any of the different behavioral categories could be observed between the three housing densities. brumm et al. (2001) reported no residual effects of nursery crowding on growfinish performance. pigs that were crowded during the nursery period and uncrowded during the grow-finishing period had similar, although numerically rather lower, daily gains (849 vs. 867 g/d, during the grow-finishing period) than pigs that were uncrowded during both the nursery and growfinishing period. effect of different supplementation for reducing crowding stress marco-ramell et al. (2011) demonstrated that plasma proteins in animals stressed by increased stocking density (0.50 m 2 /pig vs. 0.25 m 2 /pig) were more oxidized and an increase in oxidative stress markers was detected in the high density animals.high stocking density is an agriculture-related situation which causes crowding stress in pigs and potentially affects their immune systems. protection against disease can be induced through immune system, enhancing defense mechanisms. stress has been generally shown to affect the immune function of animals (kelly, 1985), while vitamin c supplementation has been associated with enhanced immune system competence. vitamins e (150 vs. 120 mg/kg) and c (300 vs. 100 mg/kg) have been shown to depress the stress responses of pigs (25.1 ± 4.4 kg) undergoing vibration stimulation during transportation (peeters et al., 2005). also, essential oils have demonstrated ability to reduce stress, stimulate sluggish circulation, boost the immune system and induce uplifting or relaxing effects. wenk (2003) reported that the beneficial effects of essential oils on farm animals may arise from activation of feed intake and secretion of digestive juices, immune system stimulation and anti-bacterial, coccidiostatic, antiviral and antioxidant properties. effect of stocking density on reproductive performance rutledge (1980) reported that there was gilts allowance in small groups (six pigs) with an average of 11 piglets given birth to whereas gilts reared in a larger group (ten pigs) had an average of ten pigs given birth to. kuhlers et al. (1985) selected gilts at about 30 kg and reared them in pens of eight or 16 pigs. gilts reared in the smaller groups were more than the total pigs given birth to with 1.0. when comparing gilts raised in litters of either five or ten pigs, kirkpatrick and rutledge (1988) found that gilts in small litters had 1.1 more embryos at day 30 post-mating. stewart and diekman (1989) reported that gilts raised in litters of 6 had 0.3 more pigs in first parity than gilts raised in litters of 12. however, the impact of stress post-weaning has not been adequately studied to determine if group size or space allowance has a negative impact on reproduction. conclusion now, swine industry is changing on a large scale. high stocking density reduces the welfare of pigs. numerous results of researches show that the negative effects on performance was associated with large groups and the reduced floor-space allowance. according to this review, nursery, growing and finishing pigs require space allowances of >0.30, 0.60 and 0.90 m 2 /pig, respectively. future researches are needed to evaluate environmental configuration designs that may enhance growth and reproductive performance of pigs. references barnett jl, cronin gm, mccallum th, newman ea (1993). effects of ‘chemical intervention’ techniques on aggression and injuries when grouping unfamiliar adult pigs. appl. anim. behav. sci. 36: 135–148. baxter m (1985a). social space requirements of pigs. social space for domestic animals. kluwer academic publishers. hingham, ma, usa. pp. 116–127. baxter s (1985b). space and place. intensive pig production: environmental management and design. granada publishing ltd, london, uk. p. 210-254. breinekova k, svoboda m, smutna m, vorlova l (2006). markers of acute stress in pigs. physiol. res. 56: 323–329. brumm mc, miller ps (1996). response of pigs to space allocation and diets varying in nutrient density. j. anim. sci. 74: 2730–2737. brumm mc, ncr-89 committee on management of swine. 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83, 291–302. webel dm, finck bn, baker dh, johnson rw (1997). time course of increased plasma cytokines, cortisol, and urea nitrogen in pigs following intraperitoneal injection of lipopolysaccharide. j. anim. sci. 75: 1514–1520. wenk c (2003). herbs and botanicals as feed additives in monogastric animals. asian-aust. j. anim. sci. 16: 282–289. white hm, richert bt, schinckel ap, burgess jr, donkin ss, latour ma (2008). effects of temperature stress on growth performance and bacon quality in grow-finish pigs housed at two densities. j. anim. sci. 86: 1789-1798. wolter bf, ellis m, curtis se, parr en, webel dm (2000). group size and floor-space allowance can affect weanling-pig performance. j. anim. sci. 78: 2062–2067. wolter bf, ellis m, dedecker jm, curtis se, hollis gr, shanks rd, parr en, webel dm (2002). effects of double stocking and weighing frequency on pig performance in wean-to-finish production systems. j. anim. sci. 80: 1442–1450. wolter bf, ellis m, corrigan bp, dedecker jm, curtis se, parr en, webel dm (2003). impact of early postweaning growth rate as affected by diet complexity and space allocation on subsequent growth performance of pigs in a wean-to-finish production system. j. anim. sci. 81: 353–359. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (3), pp. 001-006, march, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper sequences polymorphism and variation of major histocompatibility complex drb exon 2 of black dahe pig lizhou tang1, long yu1, jiangang chen1, junjie wang1, mei ma1, weidong lu1* and tongzuo zhang2* 1 yunnan-guizhou plateau institute of biodiversity, college of biologic resource and environment science, qujing normal university, qujing, yunnan 655011, china. 2 key laboratory of the qinghai-tibetan plateau ecosystem and biological evolution and adaptation, northwest institute of plateau biology, chinese academy of sciences, xining, qinghai 810001, china. accepted 22 november, 2018 dna sequences of swine leukocyte antigens (sla) drb exon 2 from five populations were used to investigate genetic polymorphism of black dahe pig in yunnan province of china. our results showed that the sla-drb exon 2 of black dahe pig obtained high variable rate of 23.50%, which was higher than the values analyzed for cattles or other livestocks. the average g + c content at the third synonymous variable coding positions (gc3) was closed to 0.78. the amino acids composition rate and majority of relative synonymous codon usage were near the ranges of 0.00 to 11.27 and 1.00 to 2.00, respectively. these significant polymorphism, high gc3 content and differentiation of preferred codon usage could be resulted from over dominance selection, base compositional bias and the diversity of gene expression level. aforementioned results adequately testified the sla-drb exon 2 may be an important genetic marker for studies in genetic diversity, population genetics, breeding reproduction investigation and exploring new functional genes of black dahe pig. key words: black dahe pig, drb exon 2, genetic polymorphism, major histocompatibility complex, swine lymphocyte alloantigen, yunnan province of china. introduction major histocompatibility complex (mhc) gene is a member of the multigene families, which include three gene regions (classes i, ii and iii) and correlate with immunity function in vertebrate (benacerraf, 1981; gu and nei, 1999). recent studies showed that the compact chain, differ markedly in degree of expression and high polymorphism were the characters for some mhc genes, such as exon 2 of mhc-drb and mhc-dqb (kloch et al., 2010). previous researches of mhc gene mostly concentrated their attention on rats, and found that this gene was also discovered in different chromosomes of more vertebrates, such as sheeps and cattles (gorer, *corresponding author. e-mail: luweidonghxx@yahoo.com.cn, zhangtz@nwipb.ac.cn. tel: +86-874-8998627. fax:: +86-8748998627. 1937). some studies indicated mhc gene plays important role in the regulation of immune response, also could be related to the characteristics of disease resistance, reproductive capability, growth capability and fleshy quality in livestock and poultry (benacerraf, 1981). therefore, as an important candidate gene for investigation about disease resistance and susceptibility, the mhc gene has become a hotspot in the research area of genetic breeding, and has attracted the attention of some scientists, such as ecologists, geneticists and animal breeding scientists. the mhc gene of pigs, named swine lymphocyte alloantigen (sla), have been originally pitched on chromosome number 7 and have been finally located on the centromere of chromosome number 7 (rabin et al., 1985). other studies mostly investigated the relationship between sla genotypes and immunoreactions or disease table 1. the locations of sampled populations of black dahe pig. population site latitude (n) longitude (e) sample size 1 dahe town, fuyuan county, yunnan province 25°32'51.79" 104°18'10.16" 10 2 yingshang town, fuyuan county, yunnan province 25°28'37.52" 104°19'33.40" 10 3 zhongan town, fuyuan county, yunnan province 25°41'29.73" 104°14'52.72" 10 4 fucun town, fuyuan county, yunnan province 25°22'23.36" 104°26'29.68" 10 5 laochang town, fuyuan county, yunnan province 25°13'34.50" 104°31'15.63" 8 resistance (benacerraf, 1981; shia et al., 1995). more scientists subsequently studied differentiation of parasites resistance of sla genotypes in different pigs or tissues, and analyzed relationship between some economic characters and gene haplotypes based on molecular biologic techniques (lee et al., 2005). as a crossbreed hybridized by chinese dahe pig and england yorkshire pig, black dahe pig has been formally approved as an endemic new species by the national agriculture ministry of china, and distributed on districts of dahe-yingshang towns in qujing city of yunnan province. this species has become the material source for chinese famous ham brand (xuanwei ham) because of its excellent characters, such as better meat, high intramuscular fat, quick growth rate, strong reproductive ability, resistance of crude feed and disease resistance (huo et al., 2009). however, the piglets of black dahe pig were also killed by these familiar epidemic diseases, especially the yellow and white scour of piglets (huo et al., 2009). the clinic diseases could badly affect the pure reproduction, piglet supply and piglet production with high quality, and bring huge economic loses to the farmer. the medication therapy or vaccine inoculability could not settle the difficult problem of repetitive disease infection ultimately. but pig producing via the method of gene modification must satisfy modern people’s demands for food safety. therefore, we analyzed the sequence, amino acid substitution and polymorphism of exon 2 of sla-drb gene for endemic black dahe pig from yunnan province of china. the aforementioned acquired information can facilitate us to develop subsequent studies of disease resistance and breeding, and also to investigate the relationship between other economic characters and sla genotypes of this endemic breed. materials and methods population samples samples for genetic polymorphism analysis totaled 48 individuals from 5 populations of black dahe pig were collected and sequenced for this study (table 1). the sampled muscle tissues for each pig were immediately preserved in 95% ethanol and transferred to qujing normal university, for storage at -20°c. dna extraction, pcr amplification and sequencing total dna was isolated from ethanol-fixed muscle tissue after proteinase k digestion, followed by standard animal genome dna extraction (dingguo, beijing, china) and ethanol precipitation. the sequences of exon 2 of mhc-drb gene were amplified using primer pair drb1 (5’-taggatccctcacagcgcatttctt-3’) and drb2 (5’-gtgtctgcagtacgtgtcca-3’). pcr amplifications were performed in total reaction volumes of 15 µl, containing 1.5 µl of 10 × buffer (include mg 2+ ), 1 µl of mixed dntp, 1.2 µl of each primer (synthesized by sangon, shanghai, china), 1.5 µl (100 ng/µl) of template dna, and 0.12 µl taq dna polymerase (5 u/µl) (sangon, shanghai, china). the reaction mixtures were denatured at 94°c for 5 min and subjected to 30 cycles of 30 s at 94°c, 45 s at 60°c, 45 s at 72°c, and a final extension step of 8 min at 72°c. pcr products were purified using a geneclean purification kit, cloned and sequenced (xu et al., 2005; wu et al., 2007). data analysis all obtained sequences for black dahe pig were compared with the submitted sequences of sus scrofa in genbank (accession numbers gu263819, u52528). all sequences were aligned using clustal x (thompson et al., 1997) with the default settings and refined manually. the total number of mutations, sites with alignment gaps, polymorphic sites, singleton variable sites and parsimony informative sites were determined using dnasp (rozas et al., 2003). maximum likelihood estimates of transition/transversion bias were conducted in mega5 (tamura et al., 2011) based on using a user-specified topology and eliminating all missing positions. the nucleotide frequency, g + c content, relative synonymous codon usage, average nucleotide composition, amino acid composition and average nucleotide pair frequency were estimated in mega5 (tamura et al., 2011). results homologous comparison and clone sequencing as compared with exon 2 of sla-drb dna sequences of table 2. the maximum composite likelihood estimate of the pattern of nucleotide substitution. nucleotide a t c g a 5.04 5.71 12.94 t 5.48 15.86 7.86 c 5.48 14 7.86 g 9.01 5.04 5.71 rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics. the nucleotide frequencies are 22.73% (a), 20.93% (t/u), 32.64% (c), and 23.71% (g). the transition/transversion rate ratios are k1 = 1.645 (purines) and k2 = 2.778 (pyrimidines). the overall transition/transversion bias is r = 1.052, where r = [a*g*k1 + t*c*k2]/[(a+g)*(t+c)]. sus scrofa, the homologous percentage for all obtained black dahe pig sequences were close to 90 to 100%. a total of 29 haplotypes were identified in 48 pig individuals. there are 200 sites determined using clone sequencing. sequence polymorphism and variation of the 200 sites aligned in all individuals, 5 missing sites, 148 invariable sites and 47 polymorphic sites were observed respectively. these 47 polymorphic sites were constituted by two parts of 9 singleton variable sites (two variants) and 38 parsimony informative sites covered 29 two variants, 8 three variants and 1 four variants. the estimation of overall transition/transversion bias (r) was 1.052 when frequencies were observed for four nucleotides (a, 23.30%; t, 21.00%; c, 23.20%; g, 32.50%) (table 2). the molecular genetic indices analyses showed that 29 transitional sites and 30 transversional sites were estimated for all 48 pigs. among these substitutions, the rates of different transitions ranged from 9.01 to 15.86 with the highest t-c transition, while the transversional rates ranged from 5.04 to 7.86 with the highest of t-g and c-g transversion (table 2). codon usage in black dahe pig the overall codon usage analysis in coding sequences of 48 black dahe pig showed that the average g + c content at the third synonymous variable coding positions was 0.78. the amino acid composition, corresponding codon and their frequency and the relative synonymous codon usage were calculated for all pig individuals (table 3). the analysis of amino acids composition and their frequency indicated that amino acid compositional rate ranged from 0.00 to 11.27, and phe obtained the highest values followed by lesser adjacent rate of leu, glu, arg and gly respectively (table 3). there was clear heterogeneity of codon usage among different amino acid. firstly, estimated codon compositional frequencies obtained a range from 0.00 to 6.30, while the highest rate in codon uuc and the lowest on cuu, guu, gca, uaa, uag and gau. secondly, codon uuc was about six fold as frequent as uuu in the preferred phe amino acid, while the codon cca and ugu obtained a compositional rate of 1.00 in the secondly non-preferred amino acid of pro and cys. the trend of codon usage pattern indicated a significant biased codon usage because of majority of relative synonymous codon usage (rscu) values near the range of 1.00 to 2.00, but obvious exceptions were observed for four comparative large rscu in codons of gug, cca, cgg and agc (table 3). correlative analyses showed that there was a significant positive correlation between the codon frequency and rscu values (r 2 = 0.442) (figure 1). discussion we have presented evidence that the exon 2 of sla-drb of black dahe pig obtained the variable rate of 23.50%, which was higher than the values of 13.70% analyzed for cattles. the results also showed that the parsimony informative sites not only included two variants, three variants and four variants, but even accounted for nearly 80.85% of the 47 polymorphic sites. all the aforementioned results together indicated that there was higher polymorphism in the exon 2 of sla-drb gene of black dahe pig than in other class ii genes, which could be consistent with the supported viewpoint of previous studies (hughes and nei, 1989; kloch et al., 2010; spurgin and richardson, 2011). in this study, a better explanation for this high polymorphism could be the over dominance selection that emphasized enhancing the rate of amino acid substitution and increasing the heterozygosity and persistence time of polymorphic alleles as compared with those of neutral alleles (hughes and nei, 1989). the transitions of t-c and c-t pyrimidine achieved the highest values of 15.86 and 14.00 respectively among all transitional substitutions in this study (table 2). these may induce enhancing of amino acid substitution rate (table 3) instead of decreasing of disadvantageous selection, because the selection from advantageous mutations could reduce the level of nucleotide polymorphism (akashi, 1994; hughes and nei, 1989). this important genetic information of high table 3. the amino acid composition and relative synonymous codon usage. amino acid af codon f rscu amino acid af codon f rscu phe 11.27 uuu 1.00 0.27 ser 4.74 ucu 0 0 uuc 6.30 1.73 ucc 1.10 2.21 uca 0 0 leu 10.87 uua 1.40 1.21 ucg 0 0 uug 1.80 1.57 pro 1.59 ccu 0 0 leu cuu 0.10 0.11 ccc 0 0.13 cuc 1.20 1.05 cca 1.00 3.88 cua 0 0 ccg 0 0 cug 2.40 2.06 thr 4.63 acu 0 0 iie 0.00 auu 0 0 acc 1.40 1.89 auc 0 0 aca 1.00 1.38 aua 0 0 acg 0.50 0.73 met 0.05 aug 0 1.00 ala 3.89 gcu 0 0.05 val 7.03 guu 0.10 0.06 gcc 1.00 1.59 guc 0 0.03 gca 0.10 0.1 gua 0 0 gcg 1.40 2.26 gug 4.50 3.91 cys 1.59 ugu 1.00 2.00 tyr 5.08 uau 1.20 0.71 ugc 0 0 uac 2.10 1.29 ter uaa 0.10 1.50 ter uga 0 0 ter uag 0.10 1.50 trp 0.00 ugg 0 0 his 3.63 cau 1.70 1.42 arg 9.32 cgu 0.30 0.32 cac 0.70 0.58 cgc 1.00 1.03 cga 0 0 gb caa 0.30 0.41 cgg 2.90 2.86 cag 1.30 1.59 ser 4.74 agu 0 0 asn 4.74 aau 0.30 0.21 agc 1.90 3.79 aac 2.70 1.79 arg 9.32 aga 0.60 0.58 lys 4.59 aaa 0.80 0.54 agg 1.20 1.22 aag 2.20 1.46 gly 8.28 ggu 0 0 asp 6.03 gau 0.10 0.03 ggc 1.00 0.72 gac 3.80 1.97 gga 2.00 1.52 ggg 2.40 1.76 glu 10.22 gaa 0.90 0.27 gag 5.70 1.73 af, average amino acid frequency; f, codon frequency; rscu, relative synonymous codon usage. polymorphism in exon 2 of sla-drb of black dahe pig could provide us a good molecular marker for studying disease resistance breeding and correlative analyses of economic characters. the results also supported that there was a significant nucleotide base bias in the exon 2 of sla-drb gene of black dahe pig. the content of g + c in the 200 sites was nearly 55.70%, and increased to 78% at the third synonymous variable coding positions (table 2). this phenomenon of nucleotide bias could still influence codon usage and frequency of acid amino substitution, such as majority of rscu values near the range of 1.00 figure 1. the correlation between codon frequency and relative synonymous codon usage. the above and below lines indicate the 95% confidence interval. to 2.00, and the amino acid compositional rate ranged from 0.00 to 11.27 (table 3). this significant bias of rscu could be dominated by base compositional bias and also be associated with gene expression level (gupta and ghosh, 2001). such codon usage biases may be result from natural selection acting on silent changes in dna, or both mutational biases and natural selection (bulmer, 1991; akashi, 1994; duret and mouchiroud, 1999; grocock and sharp, 2002). the selective advantage may depend on increasing the translation efficiency, especially during periods of competitive exponential growth (duret and mouchiroud, 1999; grocock and sharp, 2002). in conclusion, the significant polymorphism, high gc3 content and preferred codon usage could indicate that the exon 2 of sla-drb gene of black dahe pig must be an important genetic marker of chromosome for studies in genetic diversity, population genetics, breeding reproduction investigation and exploring new functional genes for disease resistance. the acquired sequences information of sla-drb could be useful for the researches on the relationship between the sla haplotypes and the economic characters of black dahe pigs, despite fewer samples of pigs used in this study. acknowledgements this work was supported by the nsfc (30970366 to t-z zhang), the applied fundamental research project of yunnan provincial science and technology department (2010zc149 to l-z tang), and the key project of qujing normal university scientific research fund (2009zd004 to l-z tang). references akashi h (1994). synonymous codon usage in drosophila melanogaster: natural selection and translational accuracy. 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agric. biotech. 15: 606-611. xu rh, wang k, sun dx, ding xd, liu pq, zhang q (2005). sequences and their polymorphisms of swine mhc-dqb, drb proximal promoter region. acta genet. sin. 32: 282-288. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 8 (1), pp. 001-006, january, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper the influence of the different content of protein fractions in sows’ milk in piglet rearing ewa skrzypczak1*, karolina szulc1, monika demkowicz2, damian knecht3, anna jankowskamąkosa3 , janusz t. buczyński1 and marek babicz4 1 department of pig breeding and production, poznań university of life sciences, ul. wołyńska 33, 60-637 poznań, poland. 2 department of cattle breeding and milk production, the institute of animal breeding, wrocław university of environmental and life sciences, ul. j. chełmońskiego 38c, 51-630 wrocław, poland. 3 department of pig breeding, the institute of animal breeding, wrocław university of environmental and life sciences, ul. j. chełmońskiego 38c, 51-630 wrocław, poland. 4 department of breeding and technology of pig production, university of life sciences in lublin, ul. akademicka 13, 20 950 lublin, poland. accepted 15 june, 2019 the aim of this study was to investigate the influence of the percentage content of protein fractions in total protein of sow`s colostrums and milk and their influence on the traits related with piglet rearing. the animal specimens were 20 sows of the native złotnicka white breed. złotnicka pigs were subjected to the national genetic resources conservation programme. colostrum and milk were collected between the 20th and 24th h after parturition and on the 2nd, 3rd, 7th, 14th and 21st day of lactation. a total of 120 samples (60 colostrum samples and 60 milk samples) were collected from all active mammary glands. individual fractions of total protein were separated by means of electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate (sds). the piglets’ body weights average daily gains and mortality were checked consecutively after 24 hours after parturition and on the day 7 th , 14 th , and 21 st of lactation. 207 piglets were examined. a highly significant correlation between the number of piglets, daily growths and protein fractions was observed. the most favourable rearing results were obtained at the highest level (iii) of individual fractions. the study also proved most of the piglets are lost from the litters when the level of fractions is the lowest (i). key words: sows, milk, protein fractions, złotnicka white, piglet rearing. introduction sows’ colostrum and milk are the main sources of necessary nutrients during the first days of piglets’ lives (devillers et al., 2004; skrzypczak et al., 2012a). milk proteins are particularly significant due to the fact that they participate in all life processes (wheeler et al., 2007; stelwagen et al., 2009), they are *corresponding author. e-mail: ewa.skrzypczak@op.pl. abbreviations: sds, sodium dodecyl sulphate; lsm, least square means; se, standard errors. an important building block and play an enormous role in the development of the immune system (bernatowicz and reklewska, 2003; sangild, 2003; lipiński, 2007; płusa, 2009). the proteins whose role has only partly been recognised deserve attention. lactoferrin and βlactoglobulin exhibit the antineoplastic effect (mcintosch et al., 1998; szulc, 2010), β-lactoglobulin and immunoglobulins g also exhibit the supportive effect in viral and bacterial infections (pan et al., 2006, 2007; płusa, 2009), α-lactalbumin is a ca 2+ carrier and it is an anticarcinogenic, antibacterial, immunological and supportive factor. in stressful situations, it may lower the blood pressure (meisel, 1997; zimecki and arytm, 2005). figure 1. polyacrylamide gel with milk protein fractions. casein has above all anticoagulant properties and it inhibits thrombocyte aggregation and serotonin liberation (kuczyńska, 2008; płusa, 2009). therefore, investigation of the correlation between sows’ milk protein fractions and results of piglet rearing is justified. materials and methods animals the animal materials were 20 sows of the native złotnicka white breed, which were subject to the national genetic resources conservation programme ( szulc et. al., 2012). the research was carried out from october, 2009 to september, 2010. all experimental sows were housed in identical conditions meeting all welfare requirements. the sows were housed in single farrowing crates from about the 10 th day before parturition to the 4 th week of lactation. they were fed individually with standard total mixed rations according to polish nutrient requirements for pigs (1993). feed in the amount of 2 kg in one dose was supplied twice a day with ad libitum access to water. the sows in the experiment were naturally mated according to the mating plan approved on the farm and the offspring came from one boar. each farrowing it was supervised by personnel. the piglets’ body weight was checked consecutively after 24 h, on the 7th, 14th, and 21st day of lactation. altogether 207 piglets from 20 sows were examined. collection of materials for analysis and analytical methods colostrum and milk were collected after earlier intramuscular injection of 2 to 4 ml of oxytocin. the amount of oxytocin administered depended on the day of lactation. colostrum and milk were manually collected from all active mammary glands to test tubes with a preservative (milkostat). the samples were serum albumin α-casein β-casein κ-casein α-lactalbumin collected between the 20th and 24th h after parturition and on the 2nd, 3rd, 7th, 14th and 21st day of lactation. then the samples were cooled down to the temperature of -20°c. altogether 120 samples were collected (60 colostrum samples and 60 milk samples). the electrophoretic separation of individual fractions of total protein, that is, serum albumin, αcasein, βcasein, κcasein, immunoglobulins g and αlactalbumin and other protein structures were carried out according to laemmli’s method (1970) on polyacrylamide gel in the presence of sodium dodecyl sulphate (sds). the qualitative and quantitative analysis was carried out according to the electrophoretic separations developed by kim and jimenez-flores (1994). the percentage share of individual fractions of total protein was calculated by defining their sum as 100%. the electrophoretic separations were compared by means of a protein marker. the separations were archived by means of an optical scanner. bio rad 6 program was used for qualitative and quantitative analysis. figure 1 shows the vertical electrophoresis of colostrum and milk proteins on polyacrylamide gel. statistical analysis the obtained data was statistically processed with sas (2007) ver. 8.11. package with the use of the following methods: 1. normality test (univariate). 2. multivariate analysis of variance with proc glm lsm. 3. the results were presented as least square means (lsm) and standard errors (se). 4. for discrete random variables (the number of piglets aged 24 h, 7, 14 and 21 days) probit transformation described by żuk (1989) and lynch and welsh, (1998) was applied, which enables transformation from discrete random variables to continuous random variables. 5. the protein fractions were divided according to their levels in total protein (table 1). in the statistical models applied for the analysis apart from the table 1. protein fractions and their various medium levels. protein fraction protein levels (%) i (low) n = 40 ii (medium) n = 40 iii (high) n = 40 serum albumin ≤ 0.645 0.646 0.963 ≥0.964 αcasein ≤ 0.673 0.674 0.858 ≥0.859 βcasein ≤ 0.665 0.666 0.884 ≥0.885 κcasein ≤ 0.722 0.723 0.989 ≥ 0.990 αlactalbumin ≤ 0.651 0.652 0.881 ≥0.882 immunoglobulins g ≤ 0.879 0.880 1.212 ≥1.213 other fractions ≤ 0.860 0.861 1.407 ≥1.408 table 2. the influence of protein fractions on the average number of piglets protein fraction number of piglets (head) i ii iii serum albumin lsm 10.31 9.00 a 10.54 b se 0.68 0.48 0.42 α – casein lsm 10.21 9.00 a 10.64 b se 0.66 0.47 0.43 β – casein lsm 9.53 9.36 10.66 se 0.60 0.50 0.44 κ – casein lsm 10.25 9.06 a 10.56 b se 0.71 0.48 0.42 α lactalbumin lsm 10.36 9.07 a 10.54 b se 0.74 0.47 0.42 immunoglobulins g lsm 9.48 9.68 10.57 se 0.55 0.50 0.48 other protein structures lsm 9.92 10.59 10.47 se 0.59 0.52 0.49 means designated with small letters (a, b) statistically differ significantly at p ≤ 0.05. main effects under investigation, other effects were also taken into consideration: (i) season of sample collection (autumn, winter, spring, summer). (ii) sows’ lactation (according to the sow parity number) (iii) colostrum and milk collection (time specified). (iv) piglet’s sex (male, female). results tables 2 and 3 show characteristics of the leveldependent influence of individual protein fractions on the number and growth of piglets in the litter. the average number of piglets and their growth rate was proved to be the most favourable when the level of fractions in total protein was the highest. those differences were confirmed at the level p ≤ 0.01 and p ≤ 0.05. the lowest level of protein fractions (i) was also found to be decisive for the highest piglet mortality (table 4) and it was proved to affect the piglet’s mean body weight (table 5). the trait was proved to have statistically significant differences. discussion in contrast to many other species of animals and table 3. the influence of protein fractions on the growth of piglets protein fraction week's growth of piglets (kg) i ii iii serum albumin lsm 0.36 a 0.52 a 0.81 b se 0.14 0.10 0.09 α – casein lsm 0.44 a 0.51 a 0.80 b se 0.14 0.10 0.09 β – casein lsm 0.43 a 0.53 a 0.81 b se 0.13 0.10 0.09 κ – casein lsm 0.37 a 0.51 a 0.80 b se 0.15 0.09 0.08 α lactalbumin lsm 0.38 a 0.50 a 0.82 b se 0.16 0.10 0.09 immunoglobulins g lsm 0.47 a 0.52 a 0.84 b se 0.11 0.10 0.10 other protein structures lsm 0.51 a 0.53 a 1.03 b se 0.15 0.13 0.12 means designated with capital letters (a, b) differ statistically significantly at p ≤ 0.01. means designated with small letters (a, b) statistically differ significantly at p ≤ 0.05. table 4. the influence of protein fractions on the piglets mortality. protein fraction mortality of piglets (%) i ii iii serum albumin lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 α – casein lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 β – casein lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 κ – casein lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 α lactalbumin lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 immunoglobulins g lsm 21.00 a 9.00 b 11.75 se 3.69 2.61 1.85 other protein structures lsm 9.0 10.0 12.33 se 2.90 4.11 2.37 means designated with small letters (a, b) statistically differ significantly at p ≤ 0.05. table 5. the influence of protein fractions on the piglet`s body weight protein fraction average body weight of piglet (kg) i ii iii serum albumin lsm 5.13 aa 3.81 b 2.83 ba se 0.45 0.32 0.27 α – casein lsm 4.86 aa 3.83 a 2.83 bb se 0.44 0.32 0.29 β – casein lsm 4.86 aa 3.65 b 2.84 bb se 0.40 0.33 0.29 κ – casein lsm 5.19 aa 3.84 ab 2.83 bb se 0.47 0.31 0.26 α lactalbumin lsm 5.26 aa 3.85 ab 2.80 bb se 0.49 0.31 0.27 immunoglobulins g lsm 4.62 aa 3.62 b 2.76 bb se 0.36 0.33 0.32 other protein structures lsm 3.44 a 3.82 aa 2.17 bb se 0.41 0.36 0.34 means designated with capital letters (a, b) differ statistically significantly at p ≤ 0.01. means designated with small letters (a, b) statistically differ significantly at p ≤ 0.05. humans, pigs are in practice born without any immunological preparation for life in outer environment due to their placenta, which is impermeable to immune bodies. therefore, colostrum is the only possibility to obtain immunoglobulins from the mother (butler and kehrli, 2005; hurle and theil, 2011). later, when the level of immunoglobulins decreases and they can no longer be passively absorbed, the essential role of milk proteins is to provide the necessary amino acids for the needs of existence, growth and development of newly born animals (patureau-mirandi et al., 1990; xu, 1996; blӓttler et al., 2001; sauter et al., 2004; senda et al., 2011). therefore, obtaining appropriate growths has a considerable influence on piglets’ balanced weight when weaned and thus produces the best results of pig fattening. the authors’ own research proved a statistically significant correlation between individual protein fractions of colostrum and milk, and the number of piglets and their daily gains. the results are also confirmed by experiments on other species of animals. wroński and sosnowska (2008) proved that bulls fed with colostrum with higher specific weight and higher content of whey proteins and immunoglobulins gained higher body dimensions than those fed with colostrum with a lower content of those proteins. the experiment on deer calves made by landetecastillejos et al. (2001) confirms the fact that the body weight is strongly correlated with the content of protein in the milk (p ≤ 0.05). the authors also noted a high correlation between the protein and fat ratio and calf growth. buczyński et al. (2008) proved that the mean weight of the piglet (2.82 kg) and growths were higher in the group of sows whose milk had the highest content of protein. boruta et al. (2009) also arrived at similar conclusions in their studies on polish large white and polish landrace breeds and their crossbreeds. the authors proved that the piglets from the sows with the highest share of protein in their milk achieved better rearing results. they also noticed a correlation between piglet mortality and the share of whey proteins. a higher content of whey proteins increased piglets’ survival rate. the authors’ own studies confirm the fact that the highest mortality of piglets (up to 21%) was according to the lowest content of all protein fractions in their mothers milk (level i). the results were statistically confirmed. those results were not confirmed in an analogical experiment made by walkiewicz et al. (2004). they noted that crossbreeds piglets (puławska x duroc) from the sows with a higher level of protein grew worse than the piglets from the sows with a lower content of total protein. to sum up the importance of milk proteins in the processes of growth and development, it is necessary to add that they are not only decisive in piglets’ acquisition of immunity but they are also significant in piglet rearing. in view of those facts, it is particularly important to ensure the piglet’s access to its mother and feeding on colostrum as soon as possible after birth. this will guarantee minimisation of losses and achievement of high growths of piglets. references bernatowicz e, reklewska b (2003). the bioactive components of milk protein fraction. 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(in polish) pwril. państwowe wydawnictwo rolne i leśne warszawa. http://www.google.pl/url?sa=t&source=web&cd=1&ved=0cbcqfjaa&url=http%3a%2f%2fwww.phmd.pl%2f&rct=j&q=post%c4%99py%20hig%20med.%20do%c5%9bw.%20&ei=lhfbtojeccyq-gbot-dycq&usg=afqjcngwaq51uzbmzjlmp_dx-ge9tsfl0w&cad=rja http://www.google.pl/url?sa=t&source=web&cd=1&ved=0cbcqfjaa&url=http%3a%2f%2fwww.phmd.pl%2f&rct=j&q=post%c4%99py%20hig%20med.%20do%c5%9bw.%20&ei=lhfbtojeccyq-gbot-dycq&usg=afqjcngwaq51uzbmzjlmp_dx-ge9tsfl0w&cad=rja in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (4), pp. 001-008, april, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper improving the quality of pigskin leather texture images using enhanced image processing mohd hafrizal azmi, fakhrul zaman rokhani, raja syamsul azmir raja abdullah and m. iqbal saripan* department of computer and communication systems engineering, faculty of engineering, universiti putra malaysia, 43400, upm serdang, selangor, malaysia. accepted 22 january, 2019 in this paper, we propose a method to improve the quality of pigskin leather texture images using digital image processing approach. the proposed method intends to minimize the imbalance illumination problem that occurs in the images captured from consumer leather products. this method is compared with the other methods in the literature such as contrast adaptive binarization method, and the results showed that the proposed method yielded better results in terms of highlighting the special characteristics of pigskin in consumer leather products. key words: digital image processing, pig skin images, binarization, local global analysis, histogram. introduction pigskin leather has been used widely in consumer products such as leather jackets, shoes, handbags, etc. the use of pigskin in leather products has some advantages and disadvantages in terms of the quality, stiffness, toughness, smoothness and cost. for a rapid assessment of the type, digital image processing can be utilized to check the special characteristics of the pigskin, that is, a group of three hair pores. sometimes, these hair pores can be seen by human eyes, but at other times, they are hidden by the color. however, during the image capturing process, presence of variable illumination distorts the quality of the image. this variable illumination could be extracted as false patterns in the images, thus it could lead to a wrong interpretation. therefore, in this work, we use the digital image processing to improve the quality of the image by removing the effects of variable *corresponding author e-mail: iqbal@eng.upm.edu.my. tel: +603-89464344. fax: +603-86567127. illumination. when dealing with a raw image, the imbalance lighting effect is a common challenge that should be corrected before proceeding with further enhancement process (basri and jacobs, 2004) especially for the low-contrast image (chen et al., 2004). with the goal to enhance the texture image, imbalance illumination will affect the performance of any algorithm particularly for automatic image analysis such as segmentation, registration, quantification (basri and jacobs, 2004), recognition (du and ward, 2005; gonzalez, 2004) and classification thus could lead to false result. previously, we have proposed the localglobal block analysis to overcome the problem. however, the method leads to a new problem which is the occurrence of boundaries between blocks, also known as blockiness effect (liu et al., 2001). this new artifact will potentially worsen the quality of the output image after classification procedure. we have discovered the cause of the boundary existence and in this paper we propose a set of modifications to the local-global block analysis to minimize the blockiness effect. results presented later in this paper include the binarization of each output images of contrast adaptive, local-global block analysis and our proposed method to validate the performance of each method. related/previous work previously, there are numbers of image enhancement algorithms that have been developed to deal with illumination. generally, we can categorize the enhancement algorithms into two approaches. the former intends to increase the quality of the output image as a whole and may not perform well over some regions while the latter will perform the enhancement process independently on a group of neighbourhood pixels within the image (jack et al., 1990). the research to date has tended to focus on face recognition and optical character recognition rather than texture recognition and classification. the most popular and well established method used is homomorphic filtering (juhua et al., 2003). but we showed in (saripan et al., 2009) that localglobal analysis performs better in texture images. the other existing methods include wavelet transform (lee et al., 2001) logarithm and discrete cosine transform (mengling and yap-peng, 2004) nine points of light (ruiz-delsolar and quinteros, 2011) recursive least squares algorithm (chen et al., 2004) and many more elaborated in (du and ward, 2005). these methods involve complex calculation and computation-intensive which is not suitable for real-time automatic system. the most popular method used is homomorphic filtering. but we proved in (lee et al., 2001) that homomorphic filtering technique does not perform better than local-global block analysis. another interesting method is the local normalization using zero-mean and unit variance (tomaževič et al., 2002) which considers the quotient value of some parameters obtained within local area in the image. however, this method is mainly developed for face recognition field and its assumptions made are related to face recognition which is not suitable for texture. it assumes that the image of the face is a combination of small and flat facets, the light source is from one direction only and the face is in neutral expression. another method that attracts our interest is the contrast adaptive binarization (xudong and kin-man 2006). this method works well to improve contrast and remove the random noise of low quality document image but perform poorly in texture image. we will show the output images of this method as a comparison to the proposed method in the result section. in (saripan et al., 2009) local-global block analysis has been proposed to provide a fast and reliable method for illumination compensation. this technique is developed based on micro and macro levels analysis, a shown in figure 1. in digital image analysis, images are formed by a series of square pixels. the image could be divided into non-overlapped blocks in which each block contains group of pixels. local global block analysis method utilizes the information provided within the block. local is defined as the area within block while global is the area that covers the whole image. this method assumed that there is very little fluctuation of illumination component exists within local area. therefore, the size of the block must be small. for each local area, the local mean intensity will be calculated. for example, within 120 × 120 image, there will be 10 × 10 blocks in the image, assuming a block size of 12 × 12. with this configuration, the image is consists of 100 local mean values. if we assume that an image f(x,y) has a size of m × n, and divided into blocks of f(a,b), hence local mean could be expressed as : local a, b     m x a a   1 m a n y b b   1 nb f x, y  manb (1.1) where local a, b the local mean of the local blocks is  a , b is the index number of the blocks and m a  nb is the size of the local blocks. the global mean intensity can be calculated using this equation:  global   m x0 n y0 f ( x, y ) m  n (1.2) where global is the global mean of an image. then, the difference between local mean and global mean is given by this equation:   x, y   f x, y  local x a  , y  b (1.3) finally, to obtain the illumination compensated image, the local residual pixel is normalized to the position of the global mean. ˆ x, y    x, y global (1.4) f where ˆ x, y  is the normalized output image. this f technique however will produce one new artifact which is the blockiness effect. the blockiness effect is the existence of block pattern within the image, which is in figure 1. traditional local-global block analysis. figure 2. case a and case b of boundary pixels. this case, the boundary between certain adjacent local areas become visible (liu et al., 2001). the boundaries do not occur everywhere else in the image, except in the areas as shown in figure 2 this effect happens because of the intensity of certain pixels that lies at the boundary of the local block is closer to the adjacent local mean instead of their local mean. for this reason, we can eliminate the blockiness effect by applying a few ma nb local am , bn  local am , bn1  f  xi , y j  n α1 α2 m figure 3. local-global block analysis with horizontal neighbourhood improvement. modifications to the residual equation. proposed method as explained in previous chapter, blockiness effect occurs because of the intensity value of boundary pixels are closer to the adjacent local mean value instead of their own local mean. when this situation happen, the variations of intensity values across the boundary are obvious, therefore the block pattern is visible. in the traditional local-global block analysis technique, every pixel will be subtracted by their local mean and the residual value will be normalized to the global mean as shown in equation 1.3 and 1.4. but, in order to minimize the blockiness effect, pixels at the boundary will be subtracted by their local mean and adjacent local mean as shown by α1 and α2 in figure 3. in this paper, only horizontal neighbourhood block will be considered. so, instead of considering only their local mean as in equation 1.3, we should now also consider the adjacent local mean value by applying equation 1.5. we know that there are two cases of boundary pixels, either they are located along the column y  12n 1 or y 12n where n is integers from 1 to 9. we assume that the pixels located along y  12n 1 are known as case a while the other are case b. for case a, the residual pixel is calculated by subtracting the pixel intensity value to its right adjacent local mean value as shown in equation 1.5. for example, for pixel f xi , y j  that is located in block f am , bn  , the residual value can be calculated by subtracting its intensity value to the local mean, local a m , bn  . while for case b, as for example pixel f xi , y j 1  , its own local mean is given by local am , bn1  and the neighbour local mean that should be consider is local am , bn . ˆ c  b  1 for y  12 n 1 (1.5)   x, y   f x, y  local x a , y c c  b  1 for y 12n after applying equation 1.3 and 1.5, we got two residual values which is the difference between boundary pixels and its own local mean and the difference of the boundary pixels and it’s adjacent to local mean. now these values will be compared and the smaller value will be chosen to be normalized to the global mean using equation 1.4. however, by modifying only the border pixels of each local area, it is not sufficient to totally eliminate the blockiness effect. we need to observe the horizontal neighbourhood pixels of modified image until we found the pixel that has an intensity value closer to its local mean value. the equation to completely calculate all the residual values by considering the boundary pixels and their horizontal neighbor pixels is given as; case a: ˆ   f xi , y j   local x a  , y b 1 (1.6)   x, y j  j 1 case b: ˆ   f xi , y j  local x a  , y b 1 (1.7)   x, y j  j 1 where case a refers to boundary pixels located at position 12n-1 while case b refers to pixels located at position 12n. this situation is shown in figure 3, where pixel f  xi , y j  is assumed to be pixel that closer to adjacent mean value, local am , bn1  instead of its local mean value, localam,bnand the dots boxes show the potential pixel that needed to be modified. the modification will stop at pixel f xi , y j 3  with assumption that at the figure 4. pixel intensity for the local-global output image and proposed method output image. position xi , y j 4 , the pixel intensity value is closer to its local area mean. experimental results we have applied our proposed method to several pigskin images taken by digital camera under different illumination conditions with the block size of 12 × 12 pixels. the size of the block can vary based on the illumination condition and characteristic of texture that must be preserved after the process. in our case, 12 × 12 pixels of block size gave the most considerable result in terms of illumination compensation, characteristic kept and minimal blockiness effect. figure 4 shows the intensity value of pixels along the same selected row in the image after being applied with local-global block analysis with and without improvement. horizontal axis represents the position of pixels while vertical axis represents the intensity value of each pixel. the solid line shows the intensity value of unimproved image while the dots line shows the intensity value of improved image with proposed method. as we can see, there are improvements occur in several places on the graph. in the range within pixel 36 to 42, the rapid changes of intensity value of pixels that lead to blockiness effect is now reduced. the same trend occurs along the graph where the reductions of variations in pixel’s intensity are observed. this characteristic illustrates how the blockiness effect along the horizontal line of the image has been minimized. figure 5(a and b) highlight the obvious improvement occurrence after we applied the proposed method. it is noticeable that within region a in the graph, the variation of intensities is rapid, which shows the obvious difference level of intensity between pixel 36 and 37, hence leads to blockiness effect. but after we applied the improvement method, the difference value between these two pixels is now reduced. the same goes with region b shown in figure 5(b). the decrement of intensity variation means that the intensity across the boundary is now smoother, which reduced the visibility of block pattern. at the same time, the main characteristic of the graph is still maintained. it shows that the proposed method does not temper the original characteristic of the sample images. figure 6 shows the comparison of output images after being applied with several methods including our proposed method. excluding the contrast adaptive, the other output images are then binarized to show whether the methods applied can extract only the information needed of the texture image while sweeping away the unnecessary information. contrast adaptive comprises 5a 5b figure 5. a the variation of pixel intensities located from   to f   ; b .the variation of f 14, 32 14, 42   f   pixel intensities located from f 14, 47 to 14, 55 . the binarization process in its algorithm, thus we did not need to binarize its output images. the threshold value was manually chosen and set based on the sample image. each sample has its own unique threshold value and the value is applied to every methods. when a simple thresholding technique is applied directly to the original image, some regions in the image became black because they are affected by illumination and the texture on that region is now concealed. the contrast adaptive binarization method could not manage to preserve the texture of the image; hence it is not suitable to be used in extracting the information of texture images. local global block analysis succeeds in compensating the illumination, but it leads to blockiness effect. proposed method finally successfully removes the horizontal blockiness effect by reducing the large fluctuation of intensity amplitudes occur at the boundary of the blocks. furthermore, it gives the best result among other methods in terms of the occurrence of noise after binarization process. this is significantly helpful for future classification process. figure 6. output images. conclusion we have proposed a method to improve the visibility of the pigskin leather in consumer products using an improved method of local-global block analysis for illumination compensation by considering the horizontal neighbourhood local mean value. each pixel that lies at the boundary between blocks will be modified based on the information of adjacent block and it follows with the neighbourhood of the modified pixels. in comparison with contrast adaptive and traditional local-global analysis, the proposed method is proven to perform better for correcting the imbalance illumination for texture images. it preserves the characteristic of the texture and reduces noise occurrence after the binarization procedure. references basri r, jacobs d (2004). “illumination modeling for face recognition,” chapter 5, handbook of face recognition, stan z. li and anil k. jain (eds.), springer new york, pp. 89-111. chen w, er mj, wu s (2004). illumination compensation and normalization using logarithm and discrete cosine transform, proc. of ieee international conference on control, automation, robotics and vision 1, pp. 380-385. du s, ward r (2005). “wavelet-based illumination normalization for face recognition,” proc. of ieee int. conf. image proc., 2: 954-957. gonzalez rc, woods re (2004). “digital image processing,” prentice hall, new jersey. jack c, chung h, gary g (1990). leinenger, “compensation of nonuniform illumination and background variations using recursive least squares algorithm,” j. crystal growth, 103(1-4): 389-397. juhua z, bede l, stuart cs (2003). “general illumination correction and its application to face recognition,” proc. of ieee int. conf. acoustic, speech signal proc., 3: 133-136. lee k, ho j, kriegman d (2001). “nine points of light: acquiring subspaces for face recognition under variable lighting,” proc. ieee conf. comput. vision pattern recog., 1: 519-526, liu h, wen g, jun m, debin z, gang d, jintao l (2001). “illumination compensation and feedback of illumination feature in face detection,” ieee int. conf. on info-tech and info-net, beijing, 3: 444-449. meng-ling f, yap-peng t (2004). “contrast adaptive binarization of low quality document images,” ieice elect. express., 1(16): 501-506. ruiz-del-solar j, quinteros j (2011). “illumination compensation and normalization in eigenspace-based face recognition: a comparative study of different pre-processing approaches,” pattern recog. let., 29(14): 1966-1979. saripan mi, azmi mh, raja arsa, anuar lh (2009). “illumination compensation in pig skin texture using local-global block analysis,” j. modern appl. sci., 3(2): 89-93. tomaževič d, likar b, pernuš f (2002). “comparative evaluation of retrospective shading correction methods,” j. mic., 208(3): 212-223. xudong x, kin-man l (2006). “an efficient illumination normalization method for face recognition,” pattern recog. lett., 27(6): 609-617. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (1), pp. 001-004, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review greek experience of the use of phytogenic feed additives in organic pig farming v. g. papatsiros1*, e. d. tzika2, p. d. tassis2, d. kantas3, l. c. filippopoulos4 and d. s. papaioannou5 1 clinic of medicine, faculty of veterinary medicine, university of thessaly, 43100, karditsa, greece. 2 farm animal clinic, school of veterinary medicine, aristotle university of thessaloniki, greece. 3 department of animal production, technological educational institute of larissa, greece. 4 research associate technical manager, provet, vrago, aspropyrgos, attica greece. 5 department of obstetrics and reproduction, faculty of veterinary science, university of thessaly, greece. accepted 03 december, 2018 the purpose of this study is to present brief updating information about the use of phytogenic feed additives in greek organic pig farming. organic pig production systems in greece are growing in popularity over the last years due to the increasing interest of consumers for organic products. the national greek projects of organic pig farming started in 2002 and since then it has increased, representing the 15% of the total organic livestock production. phytogenic feed additives are plantderived products used in animal feeding to improve the performance of agricultural livestock. this class of feed additives has recently gained increasing interest, especially for use in pigs. during the last few years the number of greek pig organic farmers who use phytogenic feed additives as potential alternatives to antibiotics for prevention and control enteric diseases and as growth promoters, is increased. key words: organic farming, pig, phytogenic feed additives, greece. introduction in view of organic livestock farming, greece has many advantages in comparison to the other european countries, the temperate climate and the wide and rich ecosystems in countryside. for thousands of years, outdoor pig farming has been one of the most important livestock production sectors in greece. since the 60s, the introduction of high-yielding pig genotypes, under intensive production systems, has replaced almost to disappearance the traditional outdoor pig farms, which were based on the native pig breed. the practice of organic pig production systems in greece is growing in popularity over the last years due to the increasing interest of consumers for products considered as *corresponding author. e-mail: vpapatsiros@vet.uth.gr. traditional. the national greek projects of organic pig farming started in 2002 and since then it has increased, representing the 15% of the total organic livestock production. based on the data of greek ministry of rural development and foods, in 2002 the population of organic pigs was just 1,288, but in 2006 to 2007 it increased to a 175.000 (doa, 2009). the most common health problems that occur in the greek organic pig farming due to the different management strategies from conventional production in terms of, for example, feeding, access to outdoor areas, weaning age and no use of preventive medication with antibiotics (papatsiros, 2011). majority of them include mainly piglet mortality, parasitic infections, as well as respiratory, gastrointestinal and skin problems. causes of high piglet mortality are mainly various diarrhoea syndromes in suckling and weaning piglets and skin traumas or crushing of piglets by sows. escherichia coli, clostridium peifringens and parasites are involved in the risk factors of diarrhoea in weaned pigs. common are also respiratory problems (mainly due to mycoplasma hyopneumoniae, actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus), with usually sign in growing and finishing pigs (papatsiros, 2011). phytogenic feed additives (often also called phytobiotics or botanicals) are commonly defined as plant-derived compounds incorporated into diets to improve the productivity of livestock through amelioration of feed properties, promotion of the animals’ production performance and improving the quality of food derived from those animals (windisch et al., 2008). phytogenic feed additives include herbs, which are non-woody flowering plants known to have medicinal properties such as; herbs (flowering, non-woody, and non-persistent plants), spices (herbs with an intensive smell or taste commonly added to human food), essential oils (aromatic oily liquids derived from plant materials such as flowers, leaves, fruits and roots), or oleoresins (extracts derived by non-aqueous solvents from plant material) (windisch et al., 2008; jacela et al., 2010). two of the most common phytogenic substances evaluated in swine include the spices oregano and thyme (tsinas et al., 1998; jugl-chizzola et al., 2005; hagmuller et al., 2006; neill et al., 2006; windisch et al., 2008; papatsiros et al., 2009). the aim of this study is to present, for the first time, brief updating information about the use of phytogenic feed additives in greek organic pig farming. furthermore, it focuses on their beneficial effects on prophylaxis of swine diseases as well as on growth performance. phytogenic feed additives: beneficial effects on swine the mode of action of most phytogenic feed additives is still not fully understood, but antimicrobial, antioxidative, and growth-promoting effects in swine have already been reported (windisch et al., 2008; jacela et al., 2010). the most well known potential mechanisms of their action are as follows: antimicrobial effects oregano and thyme are among those which have received a great deal of interest (tsinas et al., 1998; hagmuller et al., 2006; neill et al., 2006; windisch et al., 2008; papatsiros et al., 2009). these plants contain the monoterpenes carvacrol and thymol, respectively, and have demonstrated high efficacy in vitro against several pathogens found in the intestinal tract (baratta et al., 1998; burt, 2004; namkung et al., 2004; jugl-chizzola et al., 2005). antioxidative effects phytogenic feed additives derived from plants high in terpenes (such as rosemary, oregano and thyme) have anti-oxidative properties, mainly due to their phenolic terpenes (aeschbach et al., 1994; jimenez-alvarez et al., 2008; windisch et al., 2008; frankic et al., 2009). in addition, phytogenic feed additives derived from the labiatae plant family containing herbal phenolic compounds to improve the oxidative stability of pork meat (janz et al., 2007). growth-promoting effects growth-promoting effects (increased feed intake, improved gut function and dietary palatability):their stimulatory effect on feed intake is probably due to the improvement in the dietary palatability of resulting from the enhanced flavor and odor, especially with the use of essential oils (kroismayr et al., 2007; frankic et al., 2009). recent studies indicated, also stabilizing effects of phytogenic feed additives (essential oils and oleoresins) on the ecosystem and the activity of gastrointestinal microbial flora of swine (manzanilla et al., 2004; namkung et al., 2004; castillo et al., 2006; kroismayr et al., 2007) associated with a decrease in microbial activity in the gastrointestinal gut. improvement in gut function is mainly attributed to the possible stimulatory effect of phytogenic substances on digestive secretions, such as digestive enzymes, bile, and mucus (platel and srinivasan, 2004). use of phytogenic feed additives in greek organic pig farming disease prevalence and veterinary treatments in organic animal production differ from conventional systems. since the use of antibiotics and antiparasitic drugs is undesirable in organic pig production, the main focus is on prevention of diseases and parasites and applying appropriate feeding and housing standards (european commission, 2004). therefore, among the alternatives to chemically synthesized allopathic veterinary medical products, plant extracts and essential oils also called phytogenics, have received greater attention in recent years. during the last few years the number of greek pig organic farmers using phytogenic feed additives as potential alternatives to antibiotics is increased. based on our experience, we support that the use of the above products can have significant antimicrobial activity against bacteria (especially gram, mainly escherichia coli), antioxidative action, as well as enhance dietary palatability, improve the gut functions and promote growth performance and carcass quality of pigs (papatsiros et al., 2009; tzika et al., 2010a,b,c; kantas et al., 2011). for example, the results of our field trial study, with the use flavouring material containing plant extracts of origanum vulgaris and allium sativum in the diet of weaning and growing pigs, indicated reduction of prevalence of lawsonia intracellularis in the intestine and the diarrhoea score (ds), as well as improvement of growth performance and carcass quality (increased body weight and average daily gain) (papatsiros et al., 2009). in a previous study, the use of a natural, plant-derived feed additive in the feed at inclusion rate of 50 ppm, had considerable beneficial effects on growth performance of weaned piglets, including a significant increase in body weight of piglets and the average daily gain as well as a reduction in feed conversion ratio (kantas et al., 2011). in addition, possible anti-inflammatory activity of larch sawdust, echinacea, pumpkin, salix, sinupret and grape seed, in order to reduce incidence of post-partum dysgalactia syndrome, were investigated (tzika et al., 2010a, b, c). the in-feed use of larch did not show a clear positive antimicrobial effect against l. intracellularis infection in growing pigs, although the 3rd group (adding larch in 2.5%) had less positive blood samples from pigs at 14th week of age relatively to the control group. however, for the overall trial period, productivity parameters and ds of larch-fed pigs were improved relatively to the negative controls (tzika et al., 2010a). moreover, the results of the trial showed that pumpkin at 1 and 2.5% inclusion rates did not show a clear positive antimicrobial effect against e. coli infection in weaners (tzika et al., 2010b). finally, the use of larch sawdust in the diet of sows results showed that sows fed with had an improvement tendency in performance and health parameters (for example, average litter body weight, post partum temperatures) compared to the control group. additionally, there was a significant decrease of serum il6 and plasma tumor necrosis factor-alpha (tnfα) concentrations at 24 and 72 h pp, probably due to larch sawdust anti-inflammatory activity (tzika et al., 2010c). conclusions and future directions the rapid growth of the popularity of organic farming can also be considered a major driving factor for the increased necessity of alternative control measures. in recent years, phytogenic feed additives have attracted increasing interest as an alternative feeding strategy to replace antibiotic growth promoters. this has occurred especially in the european union, where antibiotics have been banned completely from use as additives in livestock feed since 2006, because of a suspected risk of generating microbial floara with increased resistance to the antibiotics used for therapy in humans and animals. the organic pig farming is a new interesting market for phytogenic feed additives around the world and in greece, as these products could be used as alternatives to antibiotics for prevention and control of enteric diseases, as well as growth promoters. the phytogenic feed additives market will certainly continue growing in the next years but in our view, special attention needs to be paid to the following aspects: (a) focusing on well-designed clinical field trials to prove the safety and efficacy of phytogenic feed additives, in parallel to a cost-benefit evaluation. however, over the last decade, the global interest in organic livestock farming and phytogenic agents has attracted the attention of many pharmaceutical companies, and it is estimated that their efficacy and safety will be proved and improved in the near future. (b) promoting the use of phytogenic feed additives in organic pig farming, as it agrees with today consumer demands for more environmental friendly pig production, supporting at the same time farmers needs for increased and cost effective production. references aeschbach r, loliger j, scott bc, murcia a, butler j, halliwell b, aruoma oi (1994). antioxidant actions of thymol, carvacrol, 6gingerol, zingerone and hydroxytyrosol. food chem. toxicol., 32(1): 31-36. baratta mt, dorman hjd, deans sg, biondi dm,ruberto g (1998). chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils. j. essential oil res., 10(6): 618-627. burt s (2004). essential oils: their antibacterial properties and potential applications in foods – a review. int. j. food microbiol., 94(3): 223253. castillo m, martin-orue sm, roca m, manzanilla eg, badiola i, perez jf, gasa j (2006). the response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early weaned pigs. j. anim. sci., 84(10): 2725-2734. doa (directorate of organic agriculture), hellenic ministry of rural development and foods (2009). http://www.minagric.gr/greek/3.6.4.html. ec (european commission) (2004). 415 final. european action plan for organic food and farming. communication from the commission [com (2004) 415 final] of 10 june 2004. brussels, eu commission . frankic t, voljc m, salobir j, rezar v (2009). use of herbs and spices and their extracts in animal nutrition. acta. agric. slovenica, 94(2): 95-102. hagmuller w, jugl-chizzola m, zitterl-eglseer k, gabler c, spergser j, chizzola r, chlodwig f (2006). the use of thymi herba as feed additive (0.1%, 0.5%, 1.0%) in weanling piglets with assessment of the shedding of haemolysing e. coli and the detection of thymol in the blood plasma. berliner und munchener tierarztliche wochenschrift, 119: 50-54. jacela jy, derouchey jm, tokach md, goodband rd, nelssen jl, renter dg, dritz ss (2010). feed additives for swine: fact sheets prebiotics and probiotics, and phytogenics. j. swine health prod., 18(3): 132-136. janz jam, morel pch, wilkinson bhp, purchas rw (2007). preliminary investigation of the effects of low-level dietary inclusion of fragrant essential oils and oleoresins on pig performance and pork quality. meat sci., 75(2): 350-355. jimenez-alvarez d, giuffrida f, golay pa, cotting c, lardeau a, keely bj (2008). antioxidant activity of oregano, parsley, and olive mill wastewaters in bulk oils and oil-in-water emulsions enriched in fish oil. j. agric. food chem., 56(16): 7151-7159. jugl-chizzola m, spergser j, schilcher f, novak j, bucher a, gabler c, hagmuller w, zitterl-eglseer k (2005). effects of thymus vulgaris l. as feed additive in piglets and against haemolytic e. coli in vitro. berliner munchener tierarztliche wochenschrift, 118: 495-501. kantas d, tzika ed, papatsiros vg, tassis pd, kyriakis sc (2011). effect of a natural feed additive on performance and health of weaned piglets. ιnproc. 2nd greek vet. congress for farm animal medicine, food safety and hygiene, consumer protection, thessaloniki, greece, p. 152. kroismayr a, sehm j, pfaffl m, plitzner c, foissy h, ettle t, mayer h, schreiner m, windisch w (2007). effects of essential oils or avilamycin on gut microbiology and blood parameters of weaned piglets. j. land manage. food environ., 81: 1-4. manzanilla eg, perez jf, martin m, kamel c, baucells f, gasa j (2004). effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. j. anim. sci., 82(1): 3210-3218. namkung h, li m, gong j, yu h, cottrill m, de lange cfm (2004). impact of feeding blends of organic acids and herbal extracts on growth performance, gut microbiota and digestive function in newly weaned pigs. can. j. anim. sci., 84(4): 697-704. neill cr, nelssen jl, tokach md, goodband rd, derouchey jm, dritz ss, groesbeck cn, brown kr (2006). effects of oregano oil on growth performance of nursery pigs. j. swine health prod., 14(6): 312-316. papatsiros vg (2011). impact of animal health management on organic pig farming in greece. biotechnol. anim. husbandry, 27(1): 115-125. papatsiros vg, tzika ed, papaioannou ds, alexopoulos c, tassis pd, kyriakis sc, kyriakis cs (2009). εffect of origanum vulgaris and allium sativum extracts for the control of proliferative enteropathy in weaning pigs. polish j. vet. sci., 12(3): 407-414. platel k (2004). srinivasan k. digestive stimulant action of spices: a myth or reality? indian j. med. res., 119: 167-179. tsinas ac, giannakopoulos cg, papasteriades a, alexopoulos c, mavromatis j, kyriakis sc (1998). use of origanum essential oil as growth promoter in pigs. in: proc 15th ipvs congress, birmingham, uk, p. 221. tzika ed, papatsiros vg, tassis pd, koylialis d, kritas s, petridoy e, alexopoulos c, kyriakis sc (2010a). evaluation of the in feed use of larch sawdust in growing pigs for treatment and prophylaxis of proliferative enteropathies. ιn proc 21st ipvs congress, vancouver, canada, p. 711. tzika ed, tassis pd, papatsiros vg, koylialis d, petridoy e, alexopoulos c, kyriakis sc (2010b). evaluation of the in feed use of pumpkin for treatment and prophylaxis of escherichia coli postweaning diarrhoea. ιn proc 21st ipvs congress, vancouver, canada, p. 780. tzika ed, tassis pd, papatsiros vg, koylialis d, siochu a, toyplikioti p, alexopoulos c, kyriakis sc (2010c). evaluation of the in feed use of larch sawdust in sows for treatment and prophylaxis of postpartum dysgalactia syndrome. ιn: proc 21st ipvs congress, vancouver, canada, p. 1191. windisch w, schedle k, plitzner c, kroismayr a (2008). use of phytogenic products as feed additives for swine and poultry. j. anim. sci., 86(14): 140-148. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (9), pp. 001-005, september, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper utilization of sun-dried on-farm generated poultry litter as a feed resource for growing-finishing pigs a. o. k. adesehinwa*, o. o. obi, b. a. makanjuola, a. o. adebayo and e. s. durotoye institute of agricultural research and training, obafemi awolowo university, p.m.b. 5029, moor plantation, ibadan. nigeria. accepted 12 april, 2017 forty eight (48) growing-finishing pigs (36.11 ± 1.26 kg) were allotted to three dietary treatment groups of 0, 33.33 and 66.67% sun-dried on-farm generated poultry litter (sopl) as a replacement for 30% maize in diets of growing-finishing pigs on weight basis to assess its implication on the chemical composition of the feed, growth performance and blood chemistry of the pigs. each treatment was replicated four times with 4 pigs/replicate in a completely randomized design. the diets were formulated to contain 16 19% crude protein and the pigs housed in concrete floored pens containing feeding and watering troughs for the duration of the study. the results obtained in this study revealed some numerical differences in the crude protein and fibre contents of the sopl diets over the control diet. increases were also recorded in the acid detergent fibre (adf), neutral detergent fibre (ndf) and acid detergent lignin (adl) fractions of the diets as a result of the replacement levels of sopl. daily weight gains and the efficiency of feed utilization of the growing pigs were not significantly (p > 0.05) influenced by the graded levels of sopl. replacement of maize with 33 and 66% sopl resulted in 10.4 20.9% reduction in the cost of feed/kg. this in turn resulted in 15 25% reduction in the cost of feed required for an additional kg gain/pig when the graded levels of sopl was fed to the pigs. an increased level of up to 66% sopl resulted in a depression (p < 0.05) in the values of packed cell volume (pcv) and white blood cells (wbc) while the hemoglobin, serum glucose, urea, creatinine and glutamate pyruvate transaninase values of the pigs across the groups were unaffected (p > 0.05). however, variations recorded in the values of the red blood cells (rbc) and alkaline phosphatase did not follow any particular trend. the values of the serum total protein, albumin and cholesterol values increased significantly (p < 0.05). key words: acid detergent fibre, neutral detergent fibre, acid detergent lignin, sun-dried on-farm generated poultry litter. introduction research results of over 45 years in virginia polytechnic institute and state university, blacksburg, virginia, united states, on the nutritional value of feeding *corresponding author. e-mail: aokadesehinwa@yahoo.com. abbreviations: tdn, total digestible nutrient; sopl, sun-dried on-farm generated poultry litter; adf, acid detergent fibre, ndf, neutral detergent fibre; adl, acid detergent lignin; pcv, packed cell volume; wbc, white blood cells; alt, alanine transaminase; gpt; glutamate pyruvate transaminase; aibps, agro-industrial by-products; pkc, palm kernel cake; iaa, indispensable amino acids; hb, haemoglobin. poultry litter, performance of cattle fed litter, animal health status, quality of animal products and studies on residues in animal products have been satisfactory, with no toxic effect on the cattle. about 20 25% of the 5.6 million tons of poultry litter (dry matter) produced annually in the u.s. is fed to beef cows and stock cattle in virginia (fontenot and hancock, 2001). it was reported to be a rich source of essential minerals, with a crude protein content of between 25-50 and 55-60% total digestible nutrient (tdn) on dry matter basis, while the nutritional value was rated as similar to or higher than good quality legume hay. dehydration, with or without pelleting, has also been reported to be a satisfactory process, if the cost is not too high. poultry litter includes excreta, bedding (mainly wood shavings in nigeria), wasted feed and feathers (fontenot and hancock, 2001). hence it represents a vast reservoir of cheap nutrient. good management, including exclusion of dead birds/ other extraneous materials from the litter and appropriate processing prior to feeding, such as sun drying has been reported to prevent growth of microorganisms (fontenot and hancock, 2001). however, even in the face of the incidence of avian influenza, on-farm generated poultry litter in farms where integrated system is practiced, with no reported disease cases could be a potential cheap source of feedstuff for growing-finishing pigs. in recent years, the use of animal manure for livestock feeding has generated considerable interest due to the fact that the conventional feedstuffs can no longer adequately meet the need of the fast growing livestock industry (abeke et al., 2003). poultry waste has been successfully used in ruminant rations in egypt. the total bacterial count was considerably lower in sun dried poultry waste compared to the oven dried waste. aflatoxins were not detectable in the concentrate mixtures containing poultry litter (makkar, 2002). both feed intake and milk production in ewes was not affected by the inclusion of 14% poultry waste as a dietary supplement, suggesting that cottonseed meal and other high protein feed ingredients could be, at least partially replaced, by poultry waste without any loss in productivity. the weight and age at puberty of lambs fed a ration containing 17% poultry waste was similar to those given a ration without any poultry waste. similarly, poultry waste up to 20% in the diet had no detrimental effect on growth in cattle and buffaloes and on the reproductive performance in buffalo heifers evaluated. the inclusion of 15% poultry waste in mixed concentrate feed decreased the cost of feed by about 10% (makkar, 2002). it is noteworthy that a number of farmers are already involved in the use of poultry litter/manure in the feeding of pigs in some parts of nigeria. there is therefore the need for the right technology/package for the use of poultry litter as a feedstuff for growing pigs. the study was designed to investigate the effect of sun-dried onfarm generated poultry litter (sopl) (from a stock of growing pullets on the farm) as a replacement for maize in diets of growing-finishing pigs on the growth performance, some carcass traits, serum metabolites and heamatological indices in an attempt to reduce feeding cost using non-conventional feed resources. materials and methods on-farm generated poultry litter was processed by sun-drying in continuous sunlight (10-12 h/day) for three days during the dry period of the year (late november -late january) to a constant moisture content of 12% before being incorporated into the experimental diets. forty eight (48) growing-finishing pigs (36.11 ± 1.26 kg) were asigned to three dietary treatment groups of 0, 10 and 20% sopl table 1. gross composition of test diets fed to growing-finishing pigs. ingredients 0% sopl 33% sopl 66% sopl maize 30.0 20.0 10.0 sopl 0.0 10.0 20.0 palm kernel cake 30.0 30.0 30.0 wheat offal 18.0 18.0 18.0 groundnut cake 15.0 15.0 15.0 fish meal (65%) 3.0 3.0 3.0 bone meal 2.25 2.25 2.25 oyster shell 1.0 1.0 1.0 salt 0.50 0.50 0.50 premix* 0.25 0.25 0.25 *pfizer agricare grower premix supplied the following per kg diet: vit. a 10,000,000 iu; vit. d3 2,000,000 iu; vit. e 8,000 iu; vit. k 2,000 mg; vit. b1 2,000 mg; vit. b2 5,500 mg; vit. b6 1,200 mg; vit. b12 12 mg; biotin 30 mg; folic acid 600 mg; niacin 10,000 mg; pantothenic acid 7,000 mg; choline chloride 500,000 mg; vit. c 10,000 mg; iron 60,000 mg; mn 80,000 mg; cu 8,00 mg; zn 50,000 mg; iodine 2,000 mg; cobalt 450 mg; selenium 100 mg; mg 100,000 mg and anti oxidant 6,000 mg. (as a replacement for 0, 33.33 and 66.67% of the 30% maize content) in diets of growing-finishing pigs, on weight basis (table 1). each treatment was replicated four times with 4 pigs/ replicate in a completely randomized design. the diets were formulated to contain 16 19% crude protein (table 2) and the pigs housed in concrete floored pens containing feeding and watering troughs for the duration of the study. the pigs were allowed ad libitum access to the diets and water, served in concrete feeding and watering troughs, respectively. the growth performance and economy of production were monitored throughout the 42-day duration of the trial. proximate compositions of the test ingredients (sopl) and experimental diets, as well as separation of fibre fractions were done according to the methods of a.o.a.c. (1990). blood sample was randomly collected from 2 pigs/ replicate for the three treatment groups at the end of the feeding trial and analyzed for serum metabolites and hematological parameters. the bleeding was done in the morning before feeding and 10 ml of blood was obtained from the jugular vein into two sample bottles using a sterilized needle and syringe as described by adesehinwa (2007). the samples for the serum were allowed to clot before centrifuging to obtain the serum used in the determination of some serum metabolites (serum protein, albumin, glucose, cholesterol, creatinine, urea and hepatic serum enzyme activities of alanine transaminase (alt), glutamate pyruvate transaminase (gpt) and alkaline phosphatase) using commercial kits of span diagnostics, surat, india (tripathi et al., 2008). the haematological attributes were estimated in whole blood just after bleeding, using standard procedures (jain, 1986). data generated on growth performance, serum metabolites and hematological parameters of the pigs were subjected to statistical analysis using sas computer software package (sas, 1991). results and discussion chemical analysis proximate composition of the sun-dried on-farm poultry (growing pullets) litter (sopl) used in the study is table 2. proximate composition of sopl and experimental diets. parameters 0% sopl 33% sopl 66% sopl sopl dry matter (%) 88.59 88.65 88.72 88.61 crude protein (%) 16.58 19.67 19.85 23.76 crude fibre (%) 6.52 7.29 7.37 11.22 ether extract (%) 3.51 3.69 3.75 3.49 ash (%) 7.46 8.21 8.46 13.15 ndf (%) 34.97 49.77 51.28 42.68 adf (%) 15.87 31.24 33.54 22.87 adl (%) 5.12 13.58 13.76 8.77 table 3. performance, carcass and cost of feed conversion growing-finishing pigs. parameters 0% sopl 33% sopl 66% sopl sem (±) daily feed intake (kg) 1.39 1.44 1.37 0.07 daily weight gain (kg) 0.42 0.46 0.44 0.02 feed: gain 3.32 3.12 3.11 0.13 cost of feed consumed/day (n) 40.07 a 39.13 a 33.27 b 0.82 % reduction in cost of feed consumed 2.35 16.97 cost of feed/weight gain (n/kg) 105.35 a 90.09 b 78.94 b 4.23 % reduction in cost of feed/ gain 14.48 25.06 dressing percentage (%) 67.71 67.25 65.80 0.78 back fat thickness (cm) 2.35 2.28 2.38 0.11 $1 (us dollar) = n150.00 (nigerian naira). shown in table 2. slight increases were observed in the crude protein and fibre contents of the sopl diets over the control diet. aro and tewe (2007) reported a high fibre content for the dried poultry waste fed to barrows, while the higher protein content (23.76%) of the test ingredient (sopl) could be responsible for the higher values obtained for diets containing sopl compared to the maize-control diet. however, the crude protein of the litter used in this study was slightly lower than the range reported by fontenot and hancock (2001) for broiler birds. increases of about double were recorded in the acid detergent fibre (adf), neutral detergent fibre (ndf) and acid detergent lignin (adl) fractions of the diets as a result of the replacement levels of the sopl (table 2). this could be said to be in agreement with the findings of aro and tewe (2007), who reported high indigestible component even though the fibre fractions were not separated in their study with dried poultry waste for barrows. this is expected, taking into consideration the fact that wood shavings, which is generally used as beddings in nigeria is a highly fibrous material, being a by-product of the wood processing industry. heat treated sheep dropping inclusion in broiler finisher diet was reported by onu and otuma (2008) to result in increased crude fibre content with a resultant dilution in the energy values. growth performance daily weight gains and the efficiency of feed utilization (feed: gain) of the growing pigs were not significantly (p > 0.05) influenced by the graded levels of sopl (table 3). the performance results of the pigs fed with the waste (sopl) was similar to that of the maize-based control. however, the values obtained in this study were lower compared to the range of 550 610 g reported by aro and tewe (2007). processing of poultry litter (inclusive of sun-drying as obtained in this study) has been reported to destroy potential pathogens, improve handling and storage characteristics, as well as enhance palatability (fontenot and hancock, 2001), hence, its efficient utilization, as also shown in the findings of this study. there was no mortality recorded throughout the study period. the cost of feed consumed per day for the 66% sopl inclusion was significantly (p < 0.05) lowered by 17% when compared with the control diet (33% sopl). the resultant effect of the diets on the pigs in terms of cost per kilogramme gain in body weight was lowered by 15 and 25% at the two inclusion levels respectively. phillip (1984) reported that reducing feed cost was not only to obtain cheaper feed, but was also dependent on the production result obtained with this cheaper feed. the efficiency with which the feed was utilized was that which table 4. hematological indices of growing-finishing pigs fed graded levels of sopl. parameters 0% sopl 33% sopl 66% sopl sem (±) packed cell volume (pcv) % 37.63 a 36.13 ab 34.50 b 0.61 hemoglobin (hb) g/dl 11.93 11.30 10.60 0.29 red blood cell (rbc) x 10 6 /µl 805.00 a 648.75 b 736.25 ab 31.67 white blood cell (wbc) x 10 3 /µl 395.75 a 336.00 ab 251.25 b 24.93 was of major importance (phillip, 1984). the results obtained in this study is a pointer to the achievement of the objective of this study to reduce the cost of finishing pigs using agro-industrial by-products (aibps) or wastes. the dressing % and back-fat thickness were not significantly (p > 0.05) influenced by the increasing levels of sopl in comparison to the maize-based control diet (table 3). this is in agreement with the findings of fontenot and hancock (2001) who reported feeding of broiler litter not to adversely affect the carcass quality as observed in this study. the dressing % observed in this study was slightly lower compared to the values obtained by aro and tewe (2007). this may not be unconnected with the fact that a higher level of maize content (between 52 55%) was used in the diets utilized in this study. it should be noted that the sopl was employed in this study to replace maize as against its replacement of palm kernel cake (pkc) in their study, noting that pkc has higher fat content compaed to maize. blood analysis hematology and blood biochemistry are routinely used in veterinary medicine to evaluate the health status of animals and poultry (mafuvadze and erlwanger, 2007). an increased level of sopl up to 66% (that is, 20% sopl inclusion) resulted in a decrease (p < 0.05) in the values of packed cell volume (pcv) and white blood cells (wbc) while the hemoglobin (hb) values of the pigs across the groups were unaffected. the inclusion of sopl up to this level seemed to have reduced the values of the pcv and wbc. fluctuation in erythrocyte count, hematocrit (pcv) and hemoglobin concentration have been reported to be considerable during the growing period of the pig (mitruka and rawnsley, 1977). variations recorded in the values of the red blood cells (rbc) did not follow any particular trend (table 4). the value observed for the 66% sopl was comparable to those of pigs on the control diet. the influence of diets on haema-tological and serum biochemical variables have been established (ologhobo et al., 1993; otesile et al., 1991; olorede et al 1995). dietary protein quality is dependent upon the adequacy and balance of the ten indispensable amino acids (iaa). a deficiency of one or more of the iaa will not only result in poor growth and other productive functions but may also lower disease resistance (nrc, 1979). the urea values observed were comparable across the groups, as such, the animals could not have suffered muscular wastage but an efficient utilization of the diets, thereby resulting in high tissue deposition (adesehinwa, 2004) across the groups. this result showed that the protein levels in the diets were able to support normal protein reserves in the pigs resulting from efficient protein utilization (adesehinwa, 2007). reduction in concentrations of erythrocytic parameters (such as pcv, rbc counts and hb concentration) and elevation in mcv are indications of macrocytic (regenerative) anaemia emanating from increased destruction and subsequent enhanced erythropoiesis in the liver, spleen and kidneys (jain, 1986). however, the results obtained in this study were within the safe range reported in literature for this class of pigs (mitruka and rawnsley, 1977). the serum glucose, urea, creatinine and glutamate pyruvate transaninase values were unaffected by the treatment. however, the serum total protein, albumin and cholesterol values were increased while alkaline phosphatase did not follow any trend (table 5). protein deficiency has been reported to reduce most haematological and serum parameters (jain, 1986) through reduced or impaired synthesis of blood cells which are largely proteinaceous. serum urea and creatinine levels in animals are indicative of muscular wastage (fashina, 1991). higher urea and creatinine values may be brought about by the inadequacy or unavailability of the dietary protein, poor digestibility or inefficient utilization of the protein (adesehinwa and ogunmodede, 2004), but this was not reflected in the overall performance of the pigs used in this study. with these results, it is obvious that with good management, feeding sopl has no adverse effect on the health status of the pigs, taking into consideration the results of the serum metabolites and hematological indices observed in this study. rather it is beneficial for the poultry industry by helping to reduce the over-application of poultry litter to the soil which possibly results in high levels of nitrogen and phosphorus in soil water (dilger et al., 2004). conclusion from the above results, it could be inferred that growing table 5. serum metabolites of growing-finishing pigs fed graded levels of sopl. parameters 0% sopl 33% sopl 66% sopl sem (±) total protein (g/dl) 5.20 b 7.05 a 6.25 ab 0.38 albumin (g/dl) 3.98 b 4.20 b 5.63 a 0.27 glucose (mg/dl) 48.08 45.20 49.03 6.22 cholesterol (mg/dl) 87.48 ab 72.78 b 101.20 a 4.84 urea (mg/dl) 22.73 20.90 16.80 2.61 creatinine (mg/dl) 0.88 1.18 1.48 0.13 gpt (iu/l) 7.33 6.18 6.25 0.38 alkaline phosphatase (iu/l) 36.35 a 22.73 c 28.40 b 1.90 finishing pigs can tolerate the replacement of up to 66% of the 30 kg maize fraction of the diet (that is, 20 kg) with sun-dried on-farm poultry litter (sopl) without any adverse effect on the performance, cost of feed conversion and the overall health status of the pigs. references abeke fo, ogundipe so, sekoni aa, adeyinka ia, abubakar by, oni oo, nwagu bi (2003). response of laying hens to dietary levels of heat treated sheep manure (hsm). trop. j. anim. sci. 6: 111-116. adesehinwa aok (2007). utilization of palm kernel cake as a replacement for maize in diets of growing pigs: effects on performance, serum metabolites, nutrient digestibility and cost of feed conversion. bulg. j. agric. sci. 13: 593-600. adesehinwa aok (2004). implications of feeding high levels of maize offal as energy source on the growth and costs of feed conversion of growing pigs. j. food, agric. environ. 2(3and4): 95-98. adesehinwa aok, ogunmodede bk (2004). performance and serum metabolites of growing pigs fed diets containing “dusa” and cashew nut testa as protein sources. indian j. anim. sci. 74(1): 3-6. aoac (1990). official methods of analysis (15 th ed). association of official analytical chemists, arlington, v.a. aro so, tewe oo (2007). performance and carcass yield of barrows fed dried poultry waste as a replacement for palm kernel cake. afr. j. biotechnol. 6(4): 487-492. dilger rn, onyango em, sands js, adeola o (2004). evaluation of microbial phytase in broiler diets. poult. sci. 83: 962-970. fashina ha (1991). utilization of soybean based rations by grower and finisher pigs in hot humid tropics ph. d thesis, university of ibadan. nigeria. fontenot jp, hancock jw (2001). utillization of poultry litter as feed for beef cattle. paper presentation at fda public hearing, kansas city, mo, october 30, 2001, on animal feeding regulation animal protein prohibited in ruminant feedcode of federal regulations, titled 21 part 589, 2000. jain nc (1986). schalm’s veterinary haematology, 4 th edn. lea and febiger, philadelphia, pa, pp. 225-239. mafuvadze b, erlwanger kh (2007). the effect of edta, heparin and storage on the erythrocyte osmotic fragility, plasma osmolality and haematocrit of adult ostriches (struthio camelus). vet. arhiv. 77: 427434. makkar hps (2002). development and field evaluation of animal feed supplementation packages (afra project 11-17 raf/5/041) project summary. joint fao/iaea division of nuclear techniques in food and agriculture/international atomic energy agency p. 6. mitruka bm, rawnsley hm (1977). chemical biochemical and heamatological reference values in normal experimental animals. masson, n.y. national research council (nrc) (1979). nutrient requirements for domestic animals. 2. nutrient requirement for swine. 8 th rev. edn. national acad. press, washington dc. ologhobo ad, apata df, oyejide a, akinpelu ro (1993). a comparison of protein fractions prepared from lima beans (phaseolus lunatus) in starter diets for broiler chicks. j. appl. anim. res. 4: 13-30. olorede br, ajagbonna op, babatunde gm (1995). comparism of airdried poultry dropping in broiler rations: effect on performance, organ weight and haematological parameters. int. j. anim. sci. 10:289-293. onu pn, otuma mo (2008). utilization of heat-treated sheep dropping in the diets of broiler finisher chicks. int. j. poult. sci. 7(2): 169-173. otesile eb, fagbemi bo, adeyemo o (1991). the effect of trypanosoma brucei infection on serum biochemical parameters in boars on different planes of dietary energy. vet. parasitol. 40: 207216. phillips gd (1984). feed utilization: principles and new developments in physiology. can. j. anim. sci. 64: 543-549. sas institute (1991). sas® user’s guide: statistics. version 6.03 edition. sas institute, inc., cary., nc. tripathi mk, mondal d, karim sa (2008). growth, haematology, blood constituents and immunological status of lambs fed graded levels of animal feed grade damaged wheat as substitute of maize. j. anim. physiol. anim. nutr. 92: 75-85. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (11), pp. 001-007, november, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effect of cumulus-oocyte complexes (cocs) culture duration on in vitro maturation and parthenogenetic development of pig oocyte zai-dong hua1, xin-min zheng2*, qing-xin wei2, hou-qiang xu1, ya-gang wang2, ximei liu2, li li 2, hong-wei xiao2 and xian-feng qiao2 1 guizhou key laboratory of animal genetics, breeding and reproduction, college of animal sciences, guizhou university, guiyang 550025, china. 2 hubei key laboratory of animal embryo engineering and molecular breeding, hubei academy of agricultural sciences, wuhan 430064, china. accepted 27 july, 2018 we investigated and optimized the cumulus-oocyte complexes (cocs) culture duration for pig oocyte in vitro maturation and produced a number of high-quality metaphase-ii (m-ii) oocytes for generation of parthenotes. the present study graded the cocs into levels a, b and c according to layers of cumulus cells, which were cultured in vitro for 24, 32, 38, 44, 48 and 54 h, respectively. subsequently, the oocytes with extruded polar body at different time point were electrically activated. the rates of cleavage and blastocyst formation were assessed on day 2 and 7, respectively. the maturation rates of cocs of both level a and b arrived at the highest point at 44 to 48 h, which were statistically insignificant (p > 0.05), while level c cocs needed longer maturation time (54 h), for most of the oocytes to become mature. cocs of different levels were cultured in vitro for the same term, the maturation rates of levels a and b were not significantly different (p > 0.05), but both were significantly higher (p < 0.05) than that of level c. after parthenogenetic activation, there were no significant differences between the cleavage rates in the groups of 38 to 54 h maturation duration, whereas the developmental rate to blastocysts after 44 h (16.1%) and 48 h (16.5%) maturation duration were significantly higher than those from the other term groups (0, 2.2, 9.8 and 12.4% for 24, 32, 38 and 54 h, respectively, p < 0.05). cocs of levels a and b were more suitable for oocytes preparation in vitro, which led to a high maturation rate: 44 to 48 h duration was beneficial for maturation, cleavage and blastocyst formation. as for 38 h or less maturation duration, rates of blastocyst were extremely low although some oocytes could be seen with the polar body and cleaved to some extent (64.5% for 3 8h ivm), indicating that oocytes were not really matured. therefore, the developmental maturation of oocytes could not be judged only by excluding the first polar body. key words: cumulus-oocyte complexes (cocs), ivm, duration, polar body, activation. introduction since the cloned sheep “dolly” was produced by the transplantation of nucleus of sheep mammary gland cell *corresponding author. e-mail: anbit20@163.com. tel/fax: 0086-027-87380647. abbreviations: cocs, cumulus-oocyte complexes; m-ii, metaphase-ii; scnt, somatic cell nuclear transfer; nt, nuclear transfer; pff, pig follicular fluid; bsa, bovine serum albumin. into an enucleated oocyte (wilmut et al., 1997), the development of somatic cell nuclear transfer (scnt) has made great progress and success in generating cloned offspring in mouse, cattle and goat (cibelli et al., 1998; wakayama et al., 1998; baguisi et al., 1999; galli et al., 1999; wells et al., 1999). however, scnt in pigs has little progress and has been proven to be more difficult than in other livestock, so the somatic cell nuclear transfer offspring could not be produced successfully until 2000 (betthauser et al., 2000; onishi et al., 2000; figure 1. pig oocytes graded. polejaeva et al., 2000). animal cloning by nuclear transfer (nt) was dependent upon a range of factors, oocyte maturation has been seen as the most important step in scnt procedure, which directly impact the development of cloned embryos and the efficiency of scnt (wolf et al., 2001). therefore, the maturation conditions, especially the duration of culturing in vitro should be optimized. without enough maturation culturing may result in cytoplasm and nucleus premature and cleavage abnormalities. in turn, too long duration is likely to lead to age and apoptosis of the oocytes. the aim of this study was to investigate whether cumulus cells improved developmental ability of pig oocytes and screen the best lasting time for oocytes maturation in vitro for production of parthenotes. the oocytes with extruded polar body at different term were electrically activated using an electro cell manipulator 2001 (btx inc., san diego). the best term of oocytes maturation in vitro was assessed based on developmental ability to the cleavage and blastocyst stage. materials and methods chemicals dpbs was purchased from gibco company. pmsg and hcg were from hormone products factory in ningbo, china and other chemical reagents used for oocyte maturation, activation and embryo culture were purchased from sigma aldrich chemical co. (budapest, hungary) unless otherwise noted. preparation of pig follicular fluid the pig follicular fluid (pff) was collected from follicles (3 to 8 mm diameter) of pig ovaries using a 10-ml disposable syringe with a 16gauge needle. after centrifugation at 1600 rpm for 20 min at 4°c, the suspension was filtered through 0.22 µm syringe filters and stored at -20°c until use. cumulus-oocyte complexes (cocs) collection and classification pig ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory in a thermal container at 28 to 37°c in 0.9% nacl solution supplemented with penicillin (200 iu/ml) and streptomycin (150 iu/ml). the ovaries were washed 3 to 5 times with saline solution (28 to 37°c) until they were aspiration. pig follicular fluid was aspirated from follicles (3 to 8 mm diameter) using a 18-gauge needle fixed a 10 ml disposable syringe. cocs were washed three times in dpbs and graded into level a, b and c according to cumulus cells (a: cumulus cells of 5 or more layers, b: cumulus cells of 3 to 5 layers, c: cumulus cells of 1 to 2 layers) (figure 1). in vitro culture of pig oocytes after cocs were divided into level a, b and c, they were washed three times with a specified maturation medium-mtcm199 or ncsu-23 with pff and hormone, and transferred to a 500 µl drop of the same medium which had been previously covered with warm paraffin oil in a polystyrene culture dish and equilibrated at 39°c in atmosphere of 5% co2 in air overnight, and cultured for 24, 32, 38, 44, 48 and 54 h, respectively. during the first 22 h of maturation, the medium contained 10 iu/ml pmsg and 10 iu/ml hcg. culturing for the subsequent 22 h was performed in the same medium without hormone supplementation. the oocytes cultured in different terms were observed under stereo microscope at 4 × 10 times, and expanded cumulus cells were completely removed by treatment with 0.1% hyaluronidase and pipetted. the oocytes with uniform cytoplasm and emission of the first polar body (figure 2) were used for parthenogenetic activation. as in vitro culturing lasted longer, cumulus cells fell off on their own, the perivitelline space of oocyte was obviously widened, and some oocytes excluded the second polar body (figure 3). activation and embryos culture oocytes with the first polar body were washed three times with activation fluid (0.3 m mannitol, 1 mm cacl2, 0.5 mm mgso4 and 0.05 mg/ml bovine serum albumin, bsa), and transferred to a figure 2. cocs after 44 h of ivm. chamber containing the same fluid. activation were induced by application of an ac pulse of 10 v for 5 s followed by a single dc pulses of 1.6 kv/cm for 60 µs using an electro cell manipulator 2001 (btx inc., san diego). after each activation treatment, the embryos were washed five times with ncsu-23 containing 4 mg/ml bsa, and were then cultured in the same medium which had been previously covered with paraffin oil in a polystyrene culture dish and equilibrated at 38.5°c in an atmosphere of 5% co2. the rates of cleavage and blastocyst formation were assessed on day 2 and 7, respectively (figure 4). statistical analysis all data were obtained from five replicates. percentage data were analyzed by chi-square tests. a probability of p < 0.05 was considered statistically significant. results effect of in vitro culture duration on maturation of cocs of level a cumulus-oocyte complexes (cocs) of level a were cultured in vitro for 24, 32, 38, 44, 48 and 54 h, respectively, oocytes with extruded polar body were counted. the results showed that the maturation rates of oocytes cultured for 44 (79.4%), 48 (83.2%) and 54 h (74.2%) were significantly higher than those of those cultured for 38, 32 and 24 h (32.6, 15.0 and 4.0%, respectively, p < 0.05) (table 1). effect of in vitro culture duration on maturation of cocs of level b only oocytes with extruded polar body were selected. from table 2, the in vitro maturation duration of 44, 48 figure 3. cocs after 54 h of ivm. and 54 h led to more matured oocytes (the rates were 61.4, 65.7 and 63.9%, respectively) than those led to by duration of 24 ~ 38 h (3.1, 13.9 and 30.0, respectively for rates, p < 0.05) (table 2). effect of in vitro culture duration on maturation of cocs of level c after culturing cumulus-oocyte complexes of level c for 24 to 60 h, the number of oocytes with extruded first polar body was very small. clearly, under the same conditions, the maturation rates of level a and b were significantly higher than that of level c. meantime, the time that the maturation rates reached maximum (30.3%) extended to 54 h (table 3). the maturation rate of different levels of cocs in the same culture conditions, the maturation rates of levels a and b were not significantly different at the same time point, respectively (p > 0.05), but both were significantly higher when compared with level c (p < 0.05) (table 4). also, from figure 1, we can clearly see that cocs of levels a and b had better maturation ability than the cocs of level c. effect of maturation duration of cocs on parthenogenetic development oocytes with extruded polar body were activated after maturation culture of cocs for 24, 32, 38, 44, 48 and 54 h, respectively. the rates of cleavage and blastocyst formation were assessed after activation on day 2 and 7, figure 4. embryos of parthenogenetic activation. table 1. in vitro maturation of cocs of level a. ivm duration (h) number of cocs of level a number of matured oocytes (%) 24 600 24 (4.0) a 32 640 96 (15.0) b 38 614 200 (32.6) c 44 985 782 (79.4) d 48 856 712 (83.2) d 54 620 460 (74.2) d respectively (table 5). the results showed that the cleavage rates of oocytes after ivm for 38 to 54 h were significantly higher than for 24 and 32 h. although there were no significant differences in the cleavage rates within 38 and 54 h ivm groups, the developmental rate to blastocysts in 44 (16.1%), 48 (16.5%) and 54 h (12.4%) groups were significantly higher than at 38 h groups, as well as at 24 and 32 h groups (9.8, 0 and 2.2%, respectively, p < 0.05). from figure 2, we can clearly see the trend that the parthenogenetic development went and it was better with the ivm duration extending. table 2. in vitro maturation of cocs of level b. ivm duration (h) no. of cocs of level b no. of matured oocytes (%) 24 860 27 (3.1) a 32 920 128 (13.9) b 38 875 262 (30.0) c 44 914 586 (64.1) d 48 880 578 (65.7) d 54 906 579 (63.9) d no. = number. within the same column, values with same superscripts are not significantly different (p > 0.05). values with different superscripts are significantly different (p < 0.05). table 3. in vitro maturation of cocs of level c. ivm duration(h) no. of cocs of level c no. of matured oocytes (%) 24 420 7 (1.7) a 32 480 45 (9.4) b 38 498 92 (18.5) c 44 590 138 (23.4) c 48 504 142 (28.2) d 54 479 145 (30.3) d 60 405 106 (26.2) d no. = number. within the same column, values with same superscripts are not significantly different (p > 0.05). values with different superscripts are significantly different (p < 0.05). table 4. the maturation rate of different levels of cocs. cocs level percent of matured oocyte (%) 24 h 32 h 38 h 44 h 48 h 54 h a 4.0 a 15.0 a 32.6 a 79.4 a 83.2 a 74.2 a b 3.1 a 13.9 a 30.0 a 64.1 a 65.7 a 63.9 a c 1.7 b 9.4 b 18.5 b 23.4 b 28.2 b 30.3 b table 5. effect of maturation duration of cocs on parthenogenetic development. ivm duration (h) no. of embryos treated no. of embryos cleaved (%) no. of blastocyst (%) 24 58 15(25.9) a 0(0) a 32 272 104(38.2) b 6(2.2) a 38 572 370(64.5) c 56(9.8) b 44 1595 1247(78.2) c 256(16.1) c 48 1506 1230(81.7) c 248(16.5) c 54 1253 968(77.3) c 155(12.4) b no. = number. discussion it had been documented that in vitro pig oocyte maturation was accompanied by cumulus cell proliferation, which could regulate the transporation of a variety of chemical substances through oocyte membranes, and inhibit oocyte degradation by discharging adverse factors (chen et al., 2003; shirazi et al., 2007). in turn, the （% ） 100 80 c le av ag e 60 40 20 0 24 32 38 44 48 54 in vitro culture time（h） figure 5. the maturation rate of cocs level a, b and c. 100 % ） 80 60 r at e（ 40 20 0 24 32 38 44 48 54 in vitro culture time（h） figure 6. the rates of embryos cleavage and the blastocysts. a-level b-level c-level cleavage blastocyst growth factors released by pig oocytes can promote the development of cumulus cells (krisher et al., 2007; ge et al., 2008; zhang et al., 2010). however, details underlying this mutual in vitro process is yet to be discovered. here, we aimed to investigate the effect of granule cells upon in vitro maturation of cumulus-oocyte complexes (cocs) by grading cocs into a, b and c levels based on layers of cumulus cells. our results suggested that cumulus cells affected the maturation of pig oocyte in vitro. in the process of producing embryos by scnt, the first polar body (pbl) presentation was used as a sign of blind suction enucleation. under this kind of protocol, it was usually considered that oocytes developmental maturation can be judged only by excluding the first polar body. in our experiments, some oocytes could be seen with the polar body when ivm was cultured for 24 to 32 h, but the rates of cleavage and blastocyst were extremely low. even 38 h ivm duration was not enough which led to significant less blastocyst than 44 h or more, although the cleavage percent for 38 h group was substantial, these indicated that oocytes cultured for less than 38 h were not really matured. unmaturation of oocytes may signifi-cantly affect subsequent embryo development by reducing both the developmental rate and total cell number in blastocysts. yajuan et al. (2004) found that among the different ages of pig, eggs matured oocytes parthenogenetically developed with a great difference in rate, 48 h eggs obtained a high rate of development. in our study, oocytes with extruded first polar body were activated after ivm for 24, 32, 38, 44, 48 and 54 h, respectively. the results showed that although there were no significant differences in the cleavage rates for cocs culture in vitro for 38 to 54 h, the developmental rate to blastocysts of 44 and 48 h oocytes were significantly higher than of the other term groups (p < 0.05). so, oocytes cultured in vitro for 44 to 48 h were the best maturation duration and provided a number of highquality m-ii oocytes for production of parthenotes. if pbl emission was delayed, in other words, cocs were cultured longer in vitro, and pbl and metaphase chromosome location would be deviated which could lead to decrease of enucleation rate (kono et al., 1991, 1992) in scnt. on the one hand, this kind of aged oocytes may also compromise further development by inducing egg’s free ca 2+ increase, which leads to a lower level of maturation promoting factor mpf, and then starting oocytes’ uncompleted pre-activation (mtango et al., 2002; somfai et al., 2007). conclusion layers of cumulus cells have some influence on oocyte maturation; cocs of level a and b were more suitable for oocytes preparation in vitro, obtained a high maturation and cleavage rate. cumulus-oocyte complexes were cultured in vitro for less than 44 h and the rates of cleavage and blastocyst were extremely low, except for the cleavage rate for 38 h, although some oocytes could be seen with the polar body, indicating that oocytes were not really matured. at the same time, oocytes of the developmental maturation could not be judged only by excluding the first polar body. oocytes cultured in vitro for 44 to 48 h had the best maturation time and provided a number of high-quality metaphase-ii (m-ii) oocytes for production of parthenotes. acknowledgements this project was supported by subject of international cooperation foundation (no.2009bfa012) and national biological cultivation of new varieties in china (no.2008zx08006-003; no.2008zx08006-002; no.2008z x08010-003; no. 2008zx08011-004; no.2009zx08011030b). this study was supported from the project from hubei key laboratory (2010zd127). the authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. references baguisi a, behboodi e, melican dt, pollock js, destrempes mm, cammuso c, williams jl, nims sd, porter ca, midura p, palacios mj, ayres sl, denniston rs, hayes ml, ziomek ca, meade hm, godke ra, gavin wg, overström ew, echelard y (1999). production of goats by somatic cell nuclear transfer. nat. biotechnol. 17: 456461. betthauser j, forsberg e, augenstein m, childs l, eilertsen k, enos j, forsythe t, golueke p, jurgella g, koppang r, lesmeister t, mallon k, mell g, misica p, pace m, pfister-genskow m, strelchenko n, voelker g, wat s, thompson s, bishop m (2000). production of cloned pigs from in vitro systems. nat. biotechnol. 18: 1055-1059. chen xy, liu d, li qw (2003). collection method and in vitro maturation of pig acolytes of ovaries derived from slaughterhouse[j]. yunnan j. anim. sci. vet. med. 19: 75-78. cibelli jb, stice sl, golueke pj, kane jj, jerry j, blackwell c, ponce de león fa, robl jm(1998). cloned transgenic calves produced from nonquiescent fetal fibroblasts. science, 280: 1256-1258. galli c, duchi r, moor rm, lazzari g (1999). mammalian leukocytes contain all the genetic information necessary for the development of a new individual. cloning, 1: 161-170. ge l, sui hs, lan gc, liu n, wang jz, tan jh (2008). coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events. j. fertil. steril. 90: 2376-2388. krisher rl, brad am, herrick jr, sparman ml, swain je (2007). a comparative analysis of metabolism and viability in porcine oocytes during in vitro maturation. anim. reprod. sci. 98: 72-96. kono t, kwon oy, ogawa m, nakahara t (1991). development of mouse oocytes receiving embryonic nuclei and thymocytes, j. theriogenol. pp. 22-35. kono t, kwon oy, watanabe t, nakahara t (1992). development of mouse enucleated oocytes reciving embryonic a nucleus from different stages of the second cell cycle. j. reprod. fetil. 96: 275278. mtango nr, varisanga md, yajuan d, suzuki t (2002). development to blastocyst stage of pig oocytes matured fertilized and electroactivated in vitro j. arch. tierz. dummerstorf. 232: 178-183. onishi a, iwamoto m, akita t, mikawa s, takeda k, awata t, hanada h, perry ac (2000). pig cloning by microinjection of fetal fibroblast nuclei. scinece, 289: 1188-1190. polejaeva ia, chen sh, vaught td, page rl, mullins j, ball s, dai y, boone j, walker s, ayares dl, colman a, campbell kh (2000). cloned pigs produced by nuclear transfer form adult somatic cell. nature, 407: 85-90. shirazi a, sadeghi n (2007). the effect of ovine oocyte diameter on nuclear maturation. j. small rumin. res. 69: 103-107. somfai t, ozawa m, noguchi j, kaneko h, kuriani karja nw, farhudin m, dinnyés a, nagai t, kikuchi k (2007). developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage. j. cryobiol. 55: 115126. wakayama t, perry ac, zuccotti m, johnson kr, yanagimachi r (1998). full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. nature, 394: 369-374. wells dn, misica pm, tervit hr (1999). production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. biol. rep. 60: 996-1005. wilmut i, schnieke ae, mcwhir j, kind aj, campbell kh (1997). viable offspring derived from fetal and adult mammalian cells. nature, 385: 810-813. wolf dp, mitalipov s, norgren jr. rb (2001). nuclear transfer technology in mammalian cloning. arch. med. res. 32: 609-613. zhang x, miao yl, zhao jg, spate l, bennett mw, murphy cn, schatten h, prather rs (2010). porcine oocytes denuded prior to maturation can develop the to the blastocyst stage if provided a cumulous cell-derived co-culture system. j. anim. sci. 9: 25-27. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (7), pp. 001-003, july, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper antimicrobial residues screening in pigs and goats slaughtered in nsukka municipal abattoir, southeast nigeria ekene v. ezenduka* and chinyere ugwumba department of public health and preventive medicine, faculty of veterinary medicine, university of nigeria, nsukka, enugu state, nigeria. accepted 03 may, 2019 antimicrobial residue in animal food products is an important index of food safety. drug residues could result from chemotherapeutic or chemoprophylactic use of drugs in food animals. this occurrence of residue in animal food products has received enormous worldwide attention from some local, international and public health agencies. samples of tissues/organs from pigs and goats slaughtered at the nsukka municipal abattoir, southeast, nigeria were screened for the presence of antimicrobial residues. samples collected from the muscles, liver and kidney of 40 slaughtered pigs and 40 slaughtered goats were analysed for antimicrobial residues using the four plate agar diffusion method. 12 (30%) of the 40 sampled pigs and 10 (25%) of the 40 sampled goats were positive for antimicrobial residues. in pigs, antimicrobial residues occur more in the kidney, muscle and liver in that order, while in goats they occurred more and equally in kidney and liver than in the muscle. the result of the study clearly suggest that the rampant use of antimicrobial drugs in slaughter animals at the nsukka municipal abattoir coupled with non-adherence to withdrawal periods is grossly practiced in nigeria. this study further confirms the need to regulate the use of veterinary drugs in livestock production and to enforce laws guarding against drug residues in food animals. key words: residues, antimicrobial, pigs, goats. introduction meat is an essential part of mans’ diet and a significant percentage of meat and meat products consumed worldwide and in southeast nigeria in particular comes from pigs and goats. the food animals include goats for chevon and milk production, and pigs for pork production. challenges of global population growth have led to increased efforts at finding resources to enhance animal food production to its maximum (nonga et al., 2009). developments in animal husbandry in the past 50 years have resulted in the proliferation of intensive production units, especially for pigs and poultry where the animals are kept in limited spaces. such intensive units are prone to outbreak of diseases especially in underdeveloped countries where the practice of strict biosecurity *corresponding author. e-mail: ev.ezenduka@yahoo.com. tel: +2347061032840. measures is limited. routine prophylaxis with low level antibiotics was therefore found necessary to prevent disease outbreak. the use of antimicrobials in animals for therapeutic and prophylactic purposes closely follows their uses in humans (gustafason and bowen, 1997). these drugs are generally used today not only for treatment and prevention but to enhance growth and food efficiency (tollefson and miller, 2000). approximately 80% of all food producing animals currently receives medication in part or most of their lifetime (lee et al., 2001; pavlov et al., 2008). antimicrobial use in food animals may result to residues in their products (meat, milk and egg) especially when the stipulated withdrawal period of the agent is not observed. this use of antimicrobial drugs in food animals has recently become a very important public health issue (jafari et al., 2007). reports have shown that exposure to these antimicrobial residues in food animal products could result to transfer of resistant strains of microorganisms to humans, distortion of the intestinal microflora and bone marrow depression among other pathologies in humans (nisha, 2008; jafari et al., 2007). in southeast nigeria, pig, goat and chicken production is the major food producing livestock farming practiced. however, there is a dearth of data on the presence of antimicrobial residues in the meat of these food animals, hence the need for this study. materials and methods sample collection samples were collected from goats and pigs slaughtered at nsukka municipal abattoir, enugu state, southeast, nigeria. nsukka is one of the three agricultural zones in enugu state. 10 g each of meat (muscle), liver and kidney were collected from 40 goats and 40 pigs, totalling 120 samples from each species. the samples were packaged in polyethylene bags and labelled according to the animal from which they were collected. they were then taken to the laboratory and processed as described by oboegbulem and fidelis (1996), by cutting off approximately 2 g of 1 to 2 cm chunk of the 10 g collected from the abattoir. the samples were processed the same day as fresh samples. assay for antimicrobial residue the antimicrobial residue screening was done with the four plate agar diffusion test (fpt) using bacillus subtilis and staphylococcus aureus. the presence of antimicrobial agents in each of the samples was confirmed by a complete inhibition of microbial growth in an annular zone not less than 2 mm (0.2 cm) around the piece of meat. less than 2 mm of inhibitory zone indicate negative result. sensitivity test an initial sensitivity test to cephaloxin, norfloxacin, gentamycin, erythromycin, ciprofloxacin, amoxicillin, streptomycin oxytetracycline and clindamycin was done for the stock colonies of b. subtilis and s. aureus. preliminary test to assess the efficiency of the four plate test in detecting antimicrobial residue in tissue samples, a positive control as described by oboegbulem and fidelis (1996) was set up by injecting oxytetracycline intramuscularly into two chickens daily for three consecutive days. they were then sacrificed on the 4th day and samples from the breast/thigh muscles, liver and kidney were removed and tested by the fpt. results sensitivity test the stock b. substilis sample was found to be sensitive to cephaloxin, norfloxacin, gentamycin, erythromycin, ciprofloxacin, tetracycline, clindamycin, trimethoprin and streptomycin but less sensitive to amoxil and ampiclox. s. aureus stock sample was found to be sensitive to all the aforementioned antimicrobial agents except trimethoprin. preliminary test the preliminary test with oxytetracycline showed that all tissue samples were positive for tetracycline residue as each of the four plates showed a clear zone of inhibition for the growth of b. substilis and s. aureus. no such inhibition occurred in the negative control plate showing the efficiency and sensitivity of the fpt. antimicrobial residues in test samples from slaughtered pigs and goats the results of the antimicrobial residues in tissue samples from slaughtered pigs and goats showed that 12 (30%) of the 40 pig samples and 10 (25%) of 40 goat samples were positive for antimicrobial residue. antimicrobial residues in pig organs out of a total of 120 organs from slaughtered pigs comprising 40 samples from each of the three organs (muscle, liver and kidney), eight (20%) muscle samples, four (10%) liver samples and 10 (25%) kidney samples were positive for antimicrobial residues. antimicrobial residues in goat organs out of a total of 120 organs from slaughtered pigs comprising 40 samples from each of the three organs (muscle, liver and kidney), 10 (25%) muscle, 12 (30%) liver and 12 (30%) kidney samples were positive for antimicrobial residues. discussion pig and goat farming are the major food producing livestock farming in southeast nigeria. pigs in nsukka area are mostly reared intensively and most of the farmers do not employ the services of a veterinarian to take care of the animals. they (farmers) tend to treat the animals themselves due to uncontrolled availability of veterinary drugs with no restriction in purchase (purchase without prescription). goats slaughtered in nsukka abattoir on the other hand come from two sources, the northern part of nigeria and locally reared west african dwarf (wad) breed in nsukka metropolis. the occurrence of antimicrobial residue in goat tissues could be attributed to the practice of the indigenes selling off their mature sick goats after treating to mask clinical signs or selling off those that defied treatment. goat owners in the rural areas also tend to sell off their sick goats due to unavailable resources to employ the services of a veterinarian. most times these goats are treated in the lairage awaiting slaughter. the indiscriminate use of antimicrobial substances and non observance of withdrawal period which has been reported as the major cause of violative residues in animal food products (pikkemat et al., 2008; riviere and sundlof, 2001) have led to the availability of unsafe pork and chevon to consumers. few other works done in different parts of the country also detected antimicrobial residues in different animal species (ibrahim et al., 2010; ezenduka et al., 2011; dipeolu and alonge, 2002; kabir et al., 2002) and they also attributed the major cause of drug residue in food of animal origin to non-adherence to withdrawal periods and misuse and abuse of veterinary drugs. the lack of the enforcement of regulatory laws to veterinary drug use in nigeria has led to the abuse and misuse of drugs in food animals. the result of this work and other works that detected residue in food animals in nigeria will create awareness to the stake holders (government and consumers) gearing to necessitate the enforcement of adherence to withdrawal periods, effective monitoring and surveillance of drug residues in meat animals and control of drug use in food animals to prevent the occurrence of violative drug residue. the european fpt is essentially a multiresidue technique for screening animal products; it does not however identify the incriminating antimicrobial or the quantity of residue. despite its limitations, the use of bacteria in detecting residues has since been useful in the meat industry (okerman et al., 1998). references dipeolu ma, alonge do (2002). residues of streptomycin antibiotic in meat sold for human consumption in some states of south west nigeria. archivos de zootecnia 51:477-480. ezenduka ev, oboegbulem si, nwanta ja, onunkwo ji (2011). prevalence of antimicrobial residue in table eggs from farms and retail outlets in enugu state, nigeria. trop. anim. hlth and prodn. 43: 557-559. gustafason rh, bowen re (1997). antibiotic use in animal agriculture. j. appl. microbiol. 8:531-541. ibrahim a, junaidu a, garba m (2010). multiple antibiotic residues in meat from slaughtered cattle in nigeria. internet j. vet. med. 8(1). jafari m., khayamian t, shaer v, zarei n (2007). determination of veterinary drug residues in chicken meat using corona discharge ion mobility. spectro. anal. chim. acta 581:147-153. kabir j, umoh ju, umoh vj (2002). characterisation and screening for antimicrobial substances of slaughtered cattle in zaria, nigeria. meat sci. 64(4):435-439. lee hj, ryu pd, lee h, cho mh, lee mh (2001). screening for penicillin plasma residues in cattle by enzyme linked immunosorbent assay. acta vet. brno 70:353-358. nisha ar (2008). antibiotic residue, a global health hazard. vet. world 1(12):375-377. nonga he, mariki m, karimuribo ed, degala rh (2009). assessment of antimicrobial usage and antimicrobial residues in broiler chickens in morogolo munincipality, tanzania. pak. j. nutr. 8(3):203-207. oboegbulem si, fidelis ap (1996). detection of antimicrobial residues in poultry meat and slaughtered cattle in nigeria. meat sci. 43(1): 7174. okerman l, de wasch k, van hoof j (1998). detection of antibiotic in muscle tissue with microbial inhibition test: effects of the test matrix. the analyst. 123: 2361-2365. pikkemat mg, van-dijk so, schouten j, rapallini m, van egmond hj (2008). a new microbial screening method for the detection of antimicrobial residue in slaughter animals. the nouws antibiotic test (nat-screening). food control 19: 781-789. riviere je, sundlof sf (2001). chemical residue in tissues of food animals. in: veterinary pharmacol. therapeutics. ed. adams hr: 8th edition; blackwell publishing professional lowa pp.1166-1174. tollefson l, miller ma (2000). antibiotic use in food animals: controlling the human health impact. j. aoac. int. 83:245-256. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 5 (6), pp. 001-006, june, 2017. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effects of road transportation on excitability scores of pigs administered with ascorbic acid during the hot-dry season olayinka oluwafemi asala1*, joseph olusegun ayo2, adeshina yahaya adenkola3 and ndazo salka minka4 1 national veterinary research institute, p. m. b. 01, vom, plateau state, nigeria. 2 department of veterinary physiology and pharmacology, ahmadu bello university, zaria, nigeria. 3 department of veterinary physiology and pharmacology, university of agriculture, makurdi, nigeria. 4 college of agriculture and animal science, ahmadu bello university, mando, kaduna, nigeria. accepted 23 november, 2016 this study was carried out in order to determine the effect of eight-hour road transportation on the excitability scores of pigs administered ascorbic acid (aa) during the hot-dry season in northern nigeria. thirteen experimental pigs were administered with aa orally at 100 mg/kg, while ten control pigs were given only distilled water orally. excitability score of each pig was determined 30 min before and immediately after transportation by a single ‘blind’ observer during weighing. an excitability score of 4 indicated the highest excitability. percent excitability of experimental and control pigs with each score was also determined. post-transportation, an increase in the percentage of experimental pigs with excitability score of 4 was recorded (38.5 to 69.2%), while a decrease was obtained in the control pigs (40.0 to 10%). road transportation decreased the excitability scores and percent excitability in control pigs with high scores. in conclusion, administration of aa increased the nervous excitability of pigs transported by road during the hot-dry season in northern nigeria. key words: pigs, road transportation, stress, excitability scores, ascorbic acid, hot-dry season. introduction it has been established that road transportation has adverse effects on health and productivity of pigs as a result of vibration (perremans et al., 2001), regrouping of animals leading to fights (wellock et al., 2003) and other harmful effects (ishiwata et al., 2004). the negative effects of road transportation on pigs are further aggravated by concomitant subjection to heat stress, occurring when animals are transported during periods of high ambient temperature (at) and relative humidity (rh) (ayo et al., 1998; renaudeau et al., 2007), characteristic of the hot-dry season. the hot-dry season, also characterized by long duration of sunshine, has been shown to be thermally stressful to animals (ayo et al., 1998; sinkalu et al., 2008). the welfare of these animals can be *corresponding author. e-mail: ofcasala@yahoo.com. tel: +2348065690278 partially assessed by objective indicators of stress (william et al., 2004). livestock experience a variety of stressors provoked by abrupt social, nutritional and environmental changes, which induce homeostatic responses, modify behaviour and growth and leads to loss of performance (niekamp et al., 2007; obernier and baldwin, 2006; pineiro et al., 2007). transportation stress has been shown to activate the hypothalamo-pituitary-adrenal axis, releasing glucocorticoids from the adrenal cortex (yoshioka et al., 2004); thus resulting in decreased immunity and appetite (brown et al., 1999; renaudeau et al., 2007), hyperthermia (lambooij, 2000; minka and ayo, 2007) and decrease in nervous excitability (ayo et al., 2006). stress has been demonstrated to induce generation of free radicals in large quantities in the body such that the body‟s antioxidant defence mechanisms are overwhelmed or exhausted (iwolakun et al., 2004; meerson, 1986; tauler et al., 2003). the welfare of transported pigs and pork quality during slaughter of pigs have been shown to be improved by the administration of antioxidant vitamins before transportation (peeters et al., 2005; 2006). although, it has been demonstrated that pigs are capable of synthesizing ascorbic acid (aa), vitamin c, evidences have shown that during stress, the rate of the synthesis is grossly inadequate and plasma aa decreases considerably (de rodas et al., 1998). aa is a water-soluble antioxidant vitamin, non-toxic, sustainable and readily metabolized by the body of most domestic animals and humans (hickey et al., 2008; padayatty et al., 2003), when administered orally its absorption takes about 30 min (hickey et al., 2008; pardue et al., 1984; tsao, 1997). aa has been demonstrated to ameliorate heat and transport stresses in pullets (ayo et al., 2005; minka and ayo, 2008) and increase nervous excitability in transported goats (ayo et al., 2006). the aim of the present study was to determine the effect of eight-hour road transportation on the excitability of local pigs administered with aa during the hot-dry season in northern nigeria. materials and methods experimental site the experiment was conducted at the animal research pen of the faculty of veterinary medicine, ahmadu bello university, zaria (11 o 10′ n, 07 o 38′ e), located in the northern guinea savannah zone of nigeria. transportation of the pigs was carried out during the hotdry season in may, 2007 from zaria (11 o 10′ n, 07 o 38′ e), to pambegua (10 o 4' n, 08 o 16' e) and from pambegua back to zaria, covering a total distance of 400 km. the zone is characterized by three major seasons, namely: the hot-dry, rainy and harmattan seasons. of the three seasons, the harmattan has been described as the most thermally stressful to livestock (ayo et al., 1996; igono et al., 1982). this period marks the peak of the hot-dry season, just before the onset of the rainy season in the zone. animals and management twenty three (23), apparently, healthy adult local pigs comprising non-castrated males, non-pregnant and non-cycling females of about one-year-old and weighing between 40 50 kg served as subjects. the pigs were purchased from samaru, zaria four weeks before commencement of the experiment. they were housed in the animal pen of the faculty of veterinary medicine, ahmadu bello university, zaria. the pen was made of concrete floor, cement block wall and roof and consisted of two chambers. the pigs moved freely within the pen. they were managed intensively and fed a standard compounded feed, consisting of a mixture of maize bran and rice husk. water was given ad libitum. the pigs were preconditioned for two weeks before commencement of the experiment and they were dewormed using piperazine hydrochloride (everchem, china) at a dose of 110 mg/kg orally. they were also treated against ectoparasites using ivermectin r (merial, france) at a dose of 0.05 mg/kg subcutaneously. experimental design after the two-week period of pre-conditioning, each pig was identified by a numbered neck collar. a week to the day of commencement of transportation, pigs were assigned experimental (group i) and control (group ii) groups consisting of 13 and 10 pigs respectively. neck collars already numbered (123) were put in a bag and picked „blindly‟ one at a time without replacement. each collar picked was placed on the neck of individual pig caught at random. animals with collar numbers 1 to 13 were classified as experimental (group i) while those with collar numbers 14 to 23 were classified as control (group ii). on the day of transportation, 30 min before transportation, pigs in group i, which served as experimental animals were administered orally with 100 mg/kg body weight of aa (archy pharmaceuticals, nigeria; nafdac no: 04-5270, batch no:vcw 7024) dissolved in 20 ml of water. the 10 pigs in group ii which served as control were administered with 20 ml of distilled water, each orally. feed and water were withdrawn 12 h before and throughout the journey period, which lasted eight hours. all the pigs were transported on asphalt roads at an average speed of 50 km/h. after the completion of the journey, the pigs were unloaded at the same spot from where they were loaded previously. feed and water were then provided ad libitum. vehicle design and loading a standard bedford van (made in england) and popularly used in the northern guinea savannah zone of nigeria for transportation of pigs was used for the journey. the body of the vehicle was made of aluminium and the floor of steel. the inner compartment of the vehicle measured 5.5 m x 1.5 m. the side walls of the body of the vehicle to a height of 1.1 m was made of aluminium and above which had windows for ventilation. the vehicle had at its rear end a twin door with a rear wind screen. the upper side walls and roof are also covered with aluminium. the floor of the vehicle was covered with dry straw and covered with a thick rubber mat for secure footing. transportation procedures were carried out according to the guidelines governing animal transport welfare by road as des-cribed by knowles et al. (1998). briefly, pigs were loaded indivi-dually by two persons in a relatively calm condition. one person easily caught a pig at a time and carried it to the other person, who already was in the vehicle. the pigs were stocked at a rate of 0.3 m 2 /pig. measurement of meteorological parameters meteorological parameters of ambient temperature (at) and relative humidity (rh) were recorded using the wet and dry-bulb thermometer (ellab inc, u.s.a.) at the experimental site just before loading the pigs into the vehicle, during transportation and immediately after unloading. excitability score and live-weight of pigs excitability scores were recorded while weighing the pigs according to the adapted methods described by ayo et al. (2006), kannan et al. (2002) and voisinet et al. (1997). briefly, liveweights of pigs were measured using the conventional bathroom scale (philip harris, england). an individual whose weight was first recorded on the scale remained standing on the weigh scale, while another person restrained the pig and handed it to the person on the scale. the reading on the scale was taken again and the weight of the person on the scale was subtracted from the second reading to give the weight of the pig. a score of 1 to 4 was allocated to each pig by a single “blind” observer, with higher scores representing higher excitability. a score of 1 was allocated to a pig that was calm, with little movement during weighing. a score of 2 was allocated to a pig that occasionally shook itself in an attempt to escape. a score of 3 was assigned to a pig that table 1. ambient temperature and relative humidity at the experimental site for five days before transportation of pigs. hour ambient temperature ( o c) relative humidity (%) dry-bulb maximum minimum mean ± sem maximum minimum 06:00 23.5 ± 0.4 24.0 23.5 73.0 ± 8.0 95 57 13:00 37.0 ± 1.0 39.0 33.0 55.8 ± 6.0 78 43 18:00 35.6 ± 1.0 39.0 33.0 71.2 ± 6.0 82 50 overall mean ± sem 32.0 ± 4.2 34.0 ± 5.0 29.8 ± 3.1 66.7 ± 5.4 86.6 ± 6.7 50.0 ± 4.0 table 2. ambient temperature and relative humidity inside the vehicle during the journey period. journey dry-bulb relative duration (h) temperarure ( o c) humidity (%) 0.5 30.5 68.0 2 33.0 57.0 4 37.0 53.0 6 36.5 40.0 8 39.0 71 mean ± sem 34.0 ± 1.7 59.3 ± 4.7 table 3. meteorological data from the experimental site three days post-transportation of pigs. hour ambient temperature ( o c) relative humidity (%) dry-bulb maximum minimum mean±sem maximum minimum 06:00 22.0 ± 0.5 23.0 21.0 80.3 ± 4.9 89.0 72.0 13:00 29.6 ± 0.3 30.0 29.0 56.6 ± 1.6 60.0 55.0 18:00 28.0 ± 3.0 31.0 25.0 61.5 ± 11.5 73.0 50.0 overall mean ± sem 26.5 ± 2.3 28.0 ± 2.5 25.0 ± 2.3 66.1 ± 7.2 74.0 ± 8.3 59.0 ± 6.6 continuously attempted to free itself by shaking. a score of 4 was assigned to a pig that struggled violently throughout the weighing time. excitability scores and live-weight were measured just before loading and upon completion of tran-sportation, immediately after unloading of pigs. statistical analysis meteorological data obtained were expressed as mean ± standard error of the mean (mean ± sem) and were subjected to student‟s ttest. percentages of animals with each excitability score in experimental and control groups were calculated and the relationship between excitability score and percent excitability for each group was determined using pearson‟s correlation coefficient. p values < 0.05 were considered significant. results meteorological data the meteorological data from the study period are shown in tables 1 3. five days before transportation, the at recorded at the experimental site ranged between the maximum value of 39.0°c and the minimum value of 23.5°c, with a wide range of 16.5°c. the rh at the experimental site before transportation ranged between 43 95%. the dry-bulb temperature (dbt) was highest at 13:00 h with mean value of 37 ± 1.0°c (table 1). during the journey period and inside the vehicle, the dbt rose gradually from 30.5°c within the first 30 min of the journey and attained a peak value of 39.0°c at the 8 th h, when the journey was completed. during the journey, the mean dbt value inside the vehicle was 34.0 ± 1.7°c, while the rh ranged between 40 71%, with a mean value of 59.3 ± 4.7%. thus, the rh had a wide range of 29% during the journey period (table 2). the meteorological data during the post-transportation period are presented in table 3. the minimum and maximum at values were 21 and 31°c, respectively, while the mean dbt values at the experimental site ranged from 22.0 ± 0.5°c to 29.6 ± 0.3°c. the maximum and minimum rh values were 89 and 50%, respectively. excitability scores of pigs pre-transportation and post-transportation the excitability score of 1 was recorded in 15.0 and p e rc e n t e x c it a b ili ty ( % ) 80 70 60 50 40 30 20 10 0 1 2 3 4 excitability scores pre-transportation post-transportation figure 1. excitability score (%) of experimental (administered with ascorbic acid) pigs (n=13) pre and post-transportation. 30.0% of experimental and control pigs, respectively just before loading the pigs into the vehicle. however, immediately after eight-hour transportation, none of the experimental pigs showed excitability score of 1, but 70.0% of the control pigs showed an excitability score of 1. the excitability score of 2 was recorded in 7.7% of experimental pigs pre-transportation, but at post-transportation, the value increased to 23.0%. in the control pigs, the value of 20.0% was recorded for pigs with excitability of 2 for both preand post-transportation. the excitability score of 3 was recorded in 38.5% of the experimental pigs and 10.0% of the control pigs just before loading; but immediately after the 8-h journey, the values decreased to 7.7% and to 0.0% in the experimental and control pigs, respectively. before transportation, percent values of excitability score of 4 recorded in experimental and control pigs were 38.5 and 40.0%, respectively. post-transportation, the percent excitability in the experimental pigs rose to 69.2%, while in control pigs, the value decreased to 10.0%. the excitability score and percent excitability in pigs pre-transportation were strongly correlated (r = 0.8179; p < 0.001) in experimental pigs, but in control animals the relationships were weak (r = 0.2000, p > 0.05). imme-diately after transportation, the excitability scores and percentage excitability were significant (p < 0.001) and positively correlated (r = 0-795, p < 0.001) in experimental, but negative and significant in control pigs (r = -0.833, p < 0.001) (figures 1 and 2). discussion the results of the excitability scores in control pigs showed that road transportation for eight hours in the zone, with concomitant effect of high at and rh during the hotdry season, induced depression. this was evident in the high post-transportation percent excitability of 70.0% obtained in control pigs with an excitability score of 1 compared to the corresponding value of 30.0% recorded pre-transportation. and a percentage decrease 80 70 60 % e xc ita b ili ty 50 40 30 20 10 0 1 2 3 4 figure 2. excitability score (%) of control pigs (n =10) pre and post-transportation. pre-transportation post-transportation in the control pigs with an excitability score of 4 posttransportation. the results obtained from the study further confirm the previous findings in pigs (brown et al., 1999) and goats (ayo et al., 2006) that road transportation depresses the nervous system immediately after the journey in animals not administered with aa. the fact that the administration of aa before road transportation of pigs resulted in a decrease in the percentage of animals that were depressed (that is, those with the excitability score of 1) and an increase in the percentage of pigs that were excited (that is, those with excitability score of 4) after the journey demonstrated that aa activated the nervous system in experimental pigs by reverting the depression, observed in control pigs, back to excitation. the finding is in agreement with the previous result obtained by ayo et al. (2006) that aa activates the nervous system in goats transported by road. although, the mechanism of action of aa was not investigated in the present study, it has been shown that aa potentiate gamma amino butyric acid and inhibits the release of cortisol. aa was also shown to be involved in the synthesis of norepinephrine and 5-hydroxytryptamine which activate brain function and mood (balz, 2003). however, the fact that aa is found in high concentration in synaptic vesicles and the evidence that it could be released into the extracelluar space by neuronal activity suggested that aa might be a neurotransmitter, thus may cause excitation of the nervous system (karanth et al., 2000). the results obtained from the present study demonstrated that aa administration just before road transportation of pigs was beneficial in reducing stress induced by the transportation and heat stress caused by high at and rh experienced during the hot-dry season in northern nigeria. the results also confirmed the previous report that aa increases resistance to environmental stress (tauler et al., 2003). the results of the present study, for the first time, demonstrated that road transportation for eight hours during the hot-dry season in the zone is stressful and that aa administration abolished the fatigue and depression of the nervous system caused by the journey. conclusion in conclusion, transportation of pigs by road for eight hours in the northern guinea savannah zone of nigeria induces depression and aa administration increases nervous excitability in the transported pigs. references ayo jo, minka ns, fayomi a (2005). effects of ascorbic acid on rectal temperature of pullets transported by road during the hot-dry season in northern nigeria. trop. j. anim. sci. 8: 43-48. ayo jo, minka ns, mamman m (2006). excitability scores of goats administered ascorbic acid and transported during the hot-dry conditions. j. vet. sci. 7: 127-131. ayo jo, oladele sb, fayomi a (1996). effects of heat stress on livestock production. a review: nig. vet. j. 1: 49-57. ayo jo, oladele sb, fayomi a (1998). stress and its adverse effects on modern swine production. pig news info. 19, 51n-56n. balz f (2003). vitamin-c intake. nutr. dis.14:1-18. brown sn, knowles tg, edwards je, warris pd (1999). behavioural and physiological responses of pigs being transported for up to 24 hours followed by six hours recovery in lairage. vet. rec. 145: 421426. de rodas bz, maxwell cv, davis me, mandali s, broekman e, stoeker bj (1998). l-ascorbyl-2-polyphosphate as a vitamin c source for segregated and conventionally weaned pigs. j. anim. sci.76:16361643. igono mo, molokwu eci, aliu yo (1982). body temperature responses of savannah brown goat to harmattan and hot-dry season. int. j. biometeo. 26: 225-230. hickey s, roberts hj, miller nj (2008). pharmacokinetics of oral vitamin c. j. nutr. environ. med. 17: 169-177. ishiwata t, uetake k, tanaka t (2004). factors affecting agonistic interactions of weanling pigs after grouping in pens with a tire. anim. sci. j. 75: 71-78. iwolakun ba, adelakun om, magbagbeola oa, afolabi as, akinwande ai (2004). effects of aqueous extracts of allium sativum on some parameters of oxidative stress in mice brain. nig. j. nat. prod. med. 8: 13-18. kannan g, terril th, kouokou b, gelaye s, amoah ea (2002). stimulated pre-slaughter holding and isolation effects on stress responses and live weight shrinkage in meat goat. j. anim. sci. 80: 1771-1780. karanth s, yu wh, walczewska a, mastronardi c, mc cann sm (2000). ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone by scavenging nitric oxide. proc. natl. acad. sci. usa, 97: 1891-1896. knowles tg, brown sn, edward je, warris pd (1998). ambient temperature below which pigs should not be continuously showered in the lairage. vet. rec.143: 575-578. lambooij e (2000). transport of pigs. in: grandin, t. editor. livestock handling and transport (new york) cabi publishers. pp. 257-296. minka ns, ayo jo (2007). physiological responses of transported goats treated with ascorbic acid during the hot-dry season. anim. sci. j. 78:164-172. minka ns, ayo jo (2008). haematology and behaviour of pullets transported by road and administered with ascorbic acid during the hot-dry season. res. vet. sci. 85:389-393. meerson fz (1986). adaptation, stress and prophylactic. in: physiology of adaptation processes. 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(ed). marcel decker, inc. new york. voisinet bb, grandin t, o‟ connor sf, tatum jd, deesing mj (1997. bos-indicus-cross feedlot cattle with excitable temperaments have tougher meat and higher incidences of border-line dark cutter. meat. sci. 46: 367-377. wellock ij, emmans gc, kyriazakis i (2003). predicting the consequence of social stressors on food intake and performance. j. anim. sci. 81: 2995-3007. william la, kramfeld ds, hess tm, saker ke, waldron jn, granden km, hoffman rm, harris pa (2004). antioxidant supplementation and subsequent oxidative stress of horses during an 80-km endurance race. j. anim. sci. 82: 588-594. yoshioka g, imaeda n, torimoto y, ohtani t, hayashi k (2004). influence of transport stress on serum cortisol and thyroid hormones in pigs with halothane gene. anim. sci. j. 75: 451-456. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (9), pp. 001-010, september, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper analysis of pig serum proteins based on shotgun liquid chromatography-tandem mass spectrometry keshan zhang, yongjie liu, youjun shang, haixue zheng, jianhong guo, hong tian, ye jin, jijun he and xiangtao liu* state key laboratory of veterinary etiological biology, national foot and mouth disease reference laboratory, lanzhou veterinary research institute, chinese academy of agricultural science, xujiaping no.1, lanzhou, gansu, 730046, pr china. accepted 12 june, 2019 recent advances in proteomics technologies have opened up significant opportunities for future applications. we used shotgun liquid chromatography, coupled with tandem mass spectrometry (lc-ms/ms) to determine the proteome profile of healthy pig serum. samples of venous blood were collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and in-gel trypsin digestion. the peptides were then processed using shotgun lc-ms/ms. serum proteins were subjected to protein identification and bioinformatics analysis. a total of 392 proteins were identified, and 179 were annotated according to their molecular functions and biological processes, excluding 142 hypothetical proteins and 71 immune globulins. to the best of our knowledge, this represents the first porcine serum proteomics analysis based on shotgun lc-ms/ms. this method and the resulting proteomics information may prove valuable for ensuring good animal welfare practice and for monitoring swine health and disease status. key words: analysis, pig serum, shotgun coupled with tandem mass spectrometry (lc-ms/ms). introduction serum is a major body fluid. serum composition thus reflects the overall health status of the individual animal and is often used to monitor health and disease in farm animals (bendixen et al., 2011; eckersall et al., 1996). studies of protein distribution characteristics in serum may provide significant information to help unravel the mechanisms of disease and for the identification of biomarkers associated with new drug targets and early diagnosis (issaq et al., 2007; wan et al., 2006). human serum protein maps have already been established (millioni et al., 2012). detailed serum protein two *corresponding author. e-mail: hnxiangtao@hotmail.com. abbreviations: lc-ms/ms, liquid chromatography-tandem mass spectrometry; 2-d page, two-dimensional polyacrylamide gel electrophoresis; mws, molecular weights; pi, isoelectric point; prrsv, porcine reproductive and respiratory syndrome virus; pcv-2, porcine circovirus-2; prv, pseudorabies virus; hcv, hog cholera virus; fmdv, foot-andmouth disease virus. dimensional gel electrophoresis (2-de) identification maps have been described for healthy pigs, and 27 highto-medium-abundance plasma proteins, including some examples of infection/inflammation-regulated proteins in healthy landrace × large white pigs (miller et al., 2009). proteome analysis is most commonly accomplished using a combination of 2-de to separate and visualize proteins and mass spectrometry (ms) for protein identification (gygi et al., 2000a). however, the disadvantages of this technique include extensive sample handling, a limited dynamic range, and difficulties in resolving lowabundance proteins with extreme isoelectric points (pis) and molecular weights (mws), as well as hydrophobic proteins such as membrane proteins (corthals et al., 2000; gygi et al., 2000b; oh-ishi et al., 2000). liquid chromatography, coupled with tandem mass spectrometry (lc−ms/ms), represents a powerful technique for the proteomic analysis of complex samples, where peptide masses may still overlap, even with a highresolution mass spectrometer (adams and zubarev, 2005; wysocki et al., 2005). lc-ms/ms has been increasingly used for the accurate detection of changes in app:ds:early app:ds:diagnosis figure 1. separation of serum proteins by one dimensional sds-page. samples of 150 g of proteins were separated on 12% bis-tris gels and stained to allow protein identification. four sections were excised and subsequently used for digestion. protein profiles and to infer biological function (aebersold and mann, 2003; crockett et al., 2005). the shotgun lcms/ms proteomics method has been used to identify thousands of proteins in human body fluids, including blood, seminal plasma and tear fluid. in the present study, gel-lc-ms/ms and bioinformatics analysis methods were used to develop a pig serum protein profile. these results are expected to provide valuable information to assist in the practice of good animal welfare, and for monitoring swine health and disease status. materials and methods animals four landrace femal pigs, aged about six months, were bought from a local farm and bred in separated rooms. all the pigs were free of the following pathogenic agents: porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus-2 (pcv-2), pseudorabies virus (prv), hog cholera virus (hcv) and foot-andmouth disease virus (fmdv), which were detected by polymerase chain reaction or reverse transcription-polymerase chain reaction (datas not shown). the animal experiments were conducted in accordance with the international guiding principles for biomedical research involving animals, issued by the council for the international organizations of medical sciences. sample preparation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis separation samples of intravenous (iv) blood were collected, they were incubated at 37°c for 2 h, then 4°c for 6 h; at last serum were separated at 5000 rpm, for 5 min. the serum protein concentration was determined by quantitative kit (ge) according to the instructions and stored at -80°c until use (hsieh et al., 2006). four serum samples (150 g) were pooled and denatured at 100°c for 5 min in an equal volume of 2× protein loading buffer [0.1 m tris buffer, ph 6.8, 4% sodium dodecyl sulfate (sds), 0.2% mercaptoethanol, 40% glycerol, and 0.002% bromophenol blue], and subjected to sds-polyacrylamide gel electrophoresis. samples were separated using 12% homogeneous sds polyacrylamide slab gels and tris-glycine-sds buffer (10 mm tris, 50 mm glycine, 0.1% sds, ph 8.0) using a bio-rad mini-protean tera system (bio-rad). electrophoresis was carried out at a constant current of 15 ma/gel followed by 30 ma for about 1.5 h until the bromophenol blue reached the bottom of the gel. the gels were then stained with coomassie brilliant blue g250 (sigma, usa). images were acquired using a gs-800 densitometer (bio-rad, hercules, ca). in-gel digestion the in-gel trypsin digestion of proteins was conducted according to wilm et al. (1996). the protein lane of the stained gel was cut into four slices (a, b, c, and d), depending on protein molecular weight (mw) (figure 1). each slice was diced into 1 × 1 mm pieces and subjected to in-gel tryptic digestion. the gel pieces were rinsed three times using milli-q water and destained with 0.2 ml of 100 mm nh4hco3 in 50% acetonitrile for 45 min at 37°c, until complete depigmentation. the gel pieces were then dried in a vacuum centrifuge. 10 µl of 10 mm dithiothreitol in 100 mm nh4hco3, sufficient to cover the gel pieces, was added to the proteins at 56°c for 1 h. after cooling to room temperature, the dithiothreitol solution was replaced with the same volume of 55 mm iodoacetamide in 100 mm nh4hco3. after 45 min incubation at room temperature in the dark, the gel pieces were washed with 100 l of 100 mm nh4hco3 for 10 min, dehydrated in 100 l of acetonitrile, swollen by rehydration in 100 l of 100 mm nh4hco3, and shrunk again by adding the same volume of acetonitrile. the proteins were subsequently digested with 20 ng/l porcine trypsin (modified proteomics grade, sigma) overnight at 37°c. peptides were extracted by one change of 20 mm nh4hco3 and three changes of 5% formic acid in 50% acetonitrile (20 min for each change) at room temperature (li et al., 2009; zhang et al., 2007). shotgun lc-ms/ms analysis the extracted peptides from each gel piece were analyzed using an ettan mdlc system (ge healthcare, usa). in this system, samples were desalted on rp trap columns (zorbax 300 sb c18, agilent technologies, usa), and then separated on an rp column (150 m internal diameter, 100 mm long, column technology inc., fremont, ca). mobile phase a (0.1% formic acid in hplc-grade water) and mobile phase b (0.1% formic acid in acetonitrile) were selected. 20 g of tryptic peptide mixture was loaded onto the columns and separation was carried out at a flow rate of 2 l/min using a linear gradient of 4 to 50% b for 120 min. a finnigan ltq linear ion trap ms (thermo electron, usa), equipped with an electrospray table 1. numbers of peptides and proteins identified in porcine serum. parameter number of protein percentage (%) total peptides 5390 100 total proteins 848 15.7 protein groups 392 46.2 immune globulin 71 17.1 annotated proteins 179 45.7 hypothetical proteins 142 36.2 interface, was connected to the lc setup to detect the eluted peptides. data-dependent ms/ms spectra were obtained simultaneously. each scan cycle consisted of one full ms scan in profile mode, followed by five ms/ms scans in centroid mode with the following dynamic exclusion settings: repeat count 2, repeat duration 30 s and exclusion duration 90 s. each sample was analyzed in triplicate. protein identification and bioinformatics analysis peptides and proteins were identified using biowork 3.2 software (thermo finnigan, san jose, ca), which uses the ms and ms/ms spectra of peptide ions to search against the suina protein database. mascot protein scores (based on combined ms and ms/ms spectra) > 72 were considered statistically significant (p ≤ 0.05). we accepted individual ms/ms spectra with a statistically significant (confidence interval ≥ 95%) ion score (based on ms/ms spectra). the protein identification and annotation criteria were based on delta cn (≥ 0.1) and xcorr (one charge ≥ 1.9, two charges ≥ 2.2 and three charges ≥ 3.75). protein classification was performed using gene ontology annotation (goa; http://www.ebi.ac.uk/goa/), according to their molecular functions and biological processes. the subcellular locations of different proteins were predicated with psort (http://psort.hgc.jp/). results serum protein sds-page separation serum proteins were separated by one dimensional sdspage and the gel was cut into four pieces, according to mw, for shotgun lc-ms/ms analysis (figure 1). identification of proteins a total of 5390 peptides were detected by shotgun lcms/ms. a total of 848 proteins were identified, some of which belonged to the same peptides groups. thus, 392 proteins were finally identified (table 1). characterization of protein profile the molecular mass and pi value distributions of the 392 identified serum proteins were analyzed. their molecular masses ranged from 3.2 to 360 kda, with most being between 10 and 100 kda (figure 2a). the pis of the proteins ranged from 4 to 11.6 (figure 2b), with most being between 5 and 7. the predicted 2-de distribution (figure 3) showed that the pis of about 95% of the identified proteins were between 4 and 10, representing proteins that are usually difficult to separate by 2-de. about 40% of the identified proteins fell outside the typical limits of protein resolution obtained by 2-de. furthermore, about 14 proteins had higher pis (> 10), which are also usually difficult to separate by 2-de, but these proteins were also identified successfully by shotgun lc-ms/ms. bioinformatics analysis a total of 189 peptides were annotated according to the goa database and were classified on the basis of molecular function or biological process. they could be divided into about five functional molecular groups (figure 4a): the classical protein group (101, 32.6% of 189 annotated peptides) and the cellular protein group (88, 31.7%) were the most common. the classical serum protein group can be further sub-classified into five subgroups, based on their specific functions (figure 4b); most proteins were proteases or other enzymes (46, 47.9%), common circulating blood proteins (19, 19.0%), or coagulation and complement factors (18, 18.8%) which are important categories of classical serum proteins. the cellular protein group is also sub-classified into five subgroups according to their function or biological process (figure 4c): signaling channels, hormone regulation, the cytoskeleton, the nucleus, and cellular metabolic secretions. discussion serum contains many high-abundance proteins that perform various housekeeping functions, as well as numerous secreted or shed low-abundance proteins that are critical for signal transduction and regulatory events. during necrosis, apoptosis, and hemolysis, cell contents may be released into the serum. in a certain time period, the presence, absence or concentration of a specific protein from serum may be related with the http://www.ebi.ac.uk/goa/ http://psort.hgc.jp/ app:ds:on app:ds:on app:ds:on app:ds:on app:ds:basis app:ds:basis figure 2. distributions of molecular weights and pi values for proteins identified by lc-ms/ms, (a) distribution of molecular weights. (b) distribution of pi values. figure 3. theoretical 2-de distribution of proteins from porcine serum. the theoretical pis and mws of the proteins were calculated using compute pi/mw tools according to protein amino acid sequence or id. figure 4. categorization of 189 serum proteins by molecular functions or biological processes. (a) all identified proteins. (b) classical serum proteins. (c) cellular proteins. pathophysiological performance of body, and the presence of these components in blood reinforces the significance of a proteomic approach to identifying biomarkers for disease status. previous proteomic characterizations of pig serum have used two dimensional page (miller et al., 2009). result shows (figure 3) 40% proteins that are usually difficult to separate by 2-de. this study used the shotgun lcms/ms proteomics technique combined with informatics analysis to determine the proteome profile of pig serum. this technique represents an efficient strategy for swine serum proteomics research, and overcame the disadvantage of 2de that cannot separate polarity protein. to reduce the individual differences, serum from four pigs were merged together and used for gel electrophoresis separation. in order to identify proteins according to molecular weight, brand a and b mixed, and brand c and d mixed, respectively (figure 1). in this study, given profile of serum protein from porcine, we identified a total of 392 proteins (table 1), of which 189 (table 2) were annotated and classified based on their molecular function or biological process (figure 4). as we expected, besides immune globulin, albumin, apolipoprotein, hemoglobin and actin, which are both ubiquitous in the red blood cells were successfully identified. these represented the main serum proteins, and are involved in the combination and transportation of small molecules (alaupovic, 1996; bondarenko et al., 2002). few coagulation or complement factors associated with whole process of blood coagulation were identified successfully as well. sodium channel protein, transmembrane channel-like protein related to signaling pathways and receptors were identified, with important functions in signal transduction (naren et al., 1997). the identified proteins also included 46 kinds of proteases or enzymes related to many important biological processes, such as biosynthesis, metabolic regulation, nucleotide replication, damage repair, transcription and posttranslational modification. fibronectins involved in cell adhesion, cell motility, opsonization, wound healing, and table 2. list of 189 annotated serum proteins. protein name pepcount unique cover mw kd pi accession pepcount percent number common circulating blood protein 15 albumin 787 52 74.1 69.4 5.92 gi|833798 alpha-2-macroglobulin 91 37 32.2 167 5.58 gi|311256211 ceruloplasmin 71 26 33.7 121.8 5.72 gi|311269519 apolipoprotein b 31 22 12.3 300 6.19 gi|951375 apolipoprotein a-i 63 15 52.1 30.3 5.48 gi|461519 apolipoprotein a-ii 8 3 26 11.1 7.73 gi|297747304 apolipoprotein c-iii 4 2 28.1 107 4.76 gi|416627 hemoglobin subunit beta 33 9 72.1 16.2 7.1 gi|3041678 porcine hemoglobin 30 9 67.8 16 6.76 gi|5542425 haptoglobin 22 9 30.8 38.5 6.51 gi|41019122 hemopexin precursor 37 12 45.3 51.3 6.59 gi|47522736 apolipoprotein e 12 7 24.6 36.6 5.62 gi|461527 apolipoprotein d 3 2 13.7 21.5 4.76 gi|311269822 angiotensinogen-like 3 2 6.3 37.8 8.74 gi|311271188 spectrin alpha chain 2 2 1.7 284.9 5.2 gi|311246557 coagulation and complement factor 19 complement c3 272 55 46.8 186.8 6.09 gi|47522844 coagulation factor x protein 1 1 2.3 53.1 5.28 gi|113205818 coagulation factor ix 1 1 2.7 45.5 5.19 gi|60392241 complement component c3 70 12 60.5 33.4 5.69 gi|295656640 complement factor b 36 11 22.6 85.9 7.45 gi|162138242 complement component 4 65 20 19.5 192.5 6.8 gi|158537756 complement c2 1 1 3 83.3 7.95 gi|156120138 complement component c5 11 6 5.9 188.6 6.49 gi|37677940 complement component c6 5 3 5.1 105.3 6.92 gi|148226535 complement component c7 precursor 1 1 1.5 93.1 6.7 gi|47523630 complement component c8a 6 4 13.1 66 5.61 gi|147905213 complement component c8b 7 3 7.4 69.2 8.14 gi|148235410 component c8g 2 2 10.9 22.3 5.59 gi|148223227 complement component c9 5 1 3.3 62.3 5.92 gi|148233690 complement factor i 11 4 11.8 67.1 8.06 gi|311262683 complement c1 5 2 15.8 26.5 9.43 gi|51491906 blood coagulation factor xiv 9 4 13.9 51.8 6.23 gi|571399 coagulation factor xii 1 1 3.6 68 6.98 gi|35039077 galectin-8 1 1 6.3 36.3 7.86 gi|218664463 protease inhibitors 7 serpin a3-1 89 17 36.9 60.9 8.46 gi|311261515 inter-alpha-trypsin inhibitor 40 15 22.8 102.1 6.42 gi|48374067 inhibitor of carbonic anhydrase 34 13 26.9 77.6 5.88 gi|47523160 alpha-1protease inhibitor 16 6 26.8 47.2 5.54 gi|1703026 clusterin precursor 12 6 20.4 51.7 5.62 gi|47522770 plasma protease c1 inhibitor 7 2 6.5 54.6 6.77 gi|178056710 plasminogen activator inhibitor 2 1 4.7 44.8 8.6 gi|311259199 table 2. count’d. blood transport and binding proteins 9 serotransferrin 258 46 74.7 77 6.93 gi|136192 vitamin d-binding protein 12 6 24.3 24.5 5.02 gi|5186337 hemoglobin subunit alpha 10 6 57.4 150.3 8.76 gi|122465 transthyretin 7 5 48 16.1 6.29 gi|1717817 c4b-binding protein alpha chain-like 4 3 8.3 67.4 6.14 gi|311265150 transgelin-2-like 3 2 12.6 54.7 6.04 gi|311254018 polyadenylate-binding protein 4-like 2 1 3.8 70.5 9.33 gi|311258948 polypyrimidine tract-binding protein 1 1 1 5 59.9 9.24 gi|47523538 telethonin binding protein 1 1 9.6 18.9 5.38 gi|224809550 channel and receptor derived proteins 15 voltage-dependent anion-selective channel protein 1 3 1 6.7 30.7 8.62 gi|7505046 signal sequence receptor, alpha 2 1 5.2 32 4.36 gi|297632426 calreticulin 2 1 7 48.3 4.32 gi|290750002 signal recognition particle 68 kda protein 1 1 2.4 70.4 8.65 gi|311266756 lycine receptor subunit alpha-1 1 1 1.4 50.2 8.93 gi|311274089 sodium channel protein 1 1 0.8 206.8 4.92 gi|311266955 transmembrane channel-like protein 1 1 1.8 92.5 5.91 gi|311245910 phosphoinositide 3-kinase adapter protein 1 1 1 1.6 100.7 5.64 gi|194041783 insulin receptor substrate 4 1 1 2.3 53.1 8.83 gi|258590765 transient receptor potential cation channel 1 1 0.9 236.2 8.54 gi|311245919 sodium channel and clathrin linker 1 1 1 6.4 11 4.98 gi|311262562 mitochondrial import receptor subunit tom34 1 1 3.1 50.9 9.42 gi|311274903 calmodulin-like 1 1 14.8 16.8 4.09 gi|311252670 syntaxin-3-like 1 1 3.9 48.9 8.44 gi|311247613 protease or other enzymes 44 alpha-1-antichymotrypsin 2 32 9 40.2 46.7 6.28 gi|47523270 plasminogen 18 9 15.8 90.6 7 gi|146345485 fumarate hydratase 20 8 11.8 13.9 6.3 gi|47523636 antithrombin-iii 28 7 24.8 52.4 5.84 gi|194018664 prothrombin precursor 19 7 18.8 70.1 5.62 gi|172072659 membrane primary amine oxidase-like 12 6 15.7 78.3 6.61 gi|311267153 glyceraldehyde 3-phosphate dehydrogenase 18 4 29.4 35.8 8.57 gi|2407184 serum paraoxonase/arylesterase 1 8 4 18.8 39.9 5.29 gi|167621416 kininogen-1 isoform 2 5 4 11 43.8 6.64 gi|311269761 plasma kallikrein 8 3 7.9 72.3 7.78 gi|47522962 l-lactate dehydrogenase b chain 5 3 17.7 36.6 5.57 gi|1107387 beta-enolase 19 2 9.22 47.1 8.05 gi|113205948 pyruvate kinase isozymes 7 2 5.4 64.9 7.98 gi|311260850 atp synthase subunit alpha, mitochondrial 6 2 7.8 59.7 9.21 gi|297591975 atp-dependent rna helicase a 1 1 4.5 44.6 5.53 gi|311264941 alpha-1-antichymotrypsin 1 5 2 13.8 24.7 5.22 gi|9968809 transketolase 4 2 9.3 67.8 7.21 gi|162952052 carbonic anhydrase 1 3 2 12.7 29 6.67 gi|194037099 pig muscle 3-phosphoglycerate kinase 3 2 8.5 43.4 8.78 gi|13399644 adp/atp translocase 1-like isoform 1 3 1 6.7 24.7 10.89 gi|311254417 phosphoglycerate mutase 1-like isoform 2 3 1 10.5 28.9 6.51 gi|194041795 ribose-phosphate pyrophosphokinase 3 1 4.7 34.8 8 gi311276762| carboxypeptidase b2 2 1 4.3 48.6 6.83 gi|194040626 transmembrane protease serine 4-like 2 1 2.5 64.5 8.22 gi|311264000 table 2. count’d. bifunctional aminoacyl-trna synthetase 2 1 1 161.1 1.35 gi|311265228 glucosamine--fructose-6-phosphate 2 1 2.7 59.5 7.01 gi|311249541 aminotransferase 2 1 2.7 59.5 7.01 gi|311249541 polypeptide n-acetylgalactosaminyltransferase 2 1 4.8 64.2 8.63 gi|194042623 threonyl-trna synthetase 1 1 4 37.7 6.28 gi|311273548 nicotinamide n-methyltransferase 1 1 9 29.5 5.63 gi|118573081 phosphoinositide 3-kinase adapter protein 1 1 1.5 107.1 5.78 gi|28860138 mismatch repair endonuclease pms2 1 1 1.9 94 6.31 gi|311250873 alanyl-trna synthetase, cytoplasmic-like 1 1 23.5 8.6 6.12 gi|311257020 adenylosuccinate synthetase 1 1 2 50.1 8.72 gi|189031714 inorganic pyrophosphatase 1 1 6.6 27.5 5.44 gi|311271315 ubiquitin carboxyl-terminal hydrolase 22-a 1 1 2.2 94.4 7.34 gi|311276293 tyrosine-protein kinase 1 1 1 122.6 6.68 gi|311249266 ubiquitin-conjugating enzyme e2 l3 1 1 16.2 17.9 8.68 gi|297591969 serum paraoxonase/arylesterase 1 7 4 14.3 45.6 6.36 gi|118403912 cholinephosphotransferase 1 1 2.2 42.3 9.05 gi|311262709 serine/threonine-protein kinase 25-like 1 1 3.5 48.7 6.18 gi|311273415 serine/threonine-protein kinase nek5 1 1 0.1 79 8.67 gi|311266294 rho gtpase-activating protein 23-like 1 1 1 131.8 9.06 gi|311268532 carbonyl reductase [nadph] 3-like 1 1 6.1 30.7 5.57 gi|311270205 cytokines or homones 1 interleukin enhancer-binding factor 2 3 2 13.1 43.1 5.19 gi|311254260 other extracellular or secreted 55 spreading factor 6 4 10.7 52.6 5.6 gi|1351418 heparin cofactor 2 6 4 11.2 55.8 6.5 gi|194043402 matrin-3-like isoform 1 2 2 5.1 94.7 5.87 gi|311250254 elongation factor 1-alpha 2 6 1 6.3 50.2 9.33 gi|311263706 60s ribosomal protein l15 2 1 7.8 17.7 11.6 gi|6174950 40s ribosomal protein s5-like isoform 1 2 1 9.8 22.9 9.73 gi|311259613 vitamin k-dependent protein s 2 1 5.9 27.6 5 gi|311270126 leucine-rich alpha-2-glycoprotein 2 1 3.8 29.7 7.02 gi|311248408 macrosialin-like isoform 1 1 1 4.3 42.8 9.29 gi|311261974 fibronectin isoform 3 27 18 13.9 239.7 5.72 gi|311273025 histidine-rich glycoprotein 56 12 25.3 61.5 7.2 gi|311269757 gelsolin 30 11 25.7 84.8 5.93 gi|121118 alpha-1b-glycoprotein 42 10 30.8 54.4 5.99 gi|311259609 alpha-2-hs-glycoprotein 59 8 36.3 38.8 5.5 gi|311269753 60 kda heat shock protein 21 8 25.1 60.9 5.7 gi|194044029 actin 50 6 31.2 41.7 5.29 gi|311250866 heat shock cognate 71kda protein 26 5 25.9 50.4 5.41 gi|311264120 heat shock 70 kda protein 1b 18 5 17.5 70.1 5.6 gi|56748897 heat shock cognate protein hsp 90-beta 13 5 12.3 83.2 4.96 gi|31160516 heat shock protein hsp 90-alpha 8 3 7.4 84.7 4.93 gi|47522774 t-complex protein 1 subunit alpha-like 13 4 15.4 60.3 5.71 gi|194033404 vimentin-like 13 4 11.5 70.3 6.01 gi|257096532 complex of bdellastasin with porcine trypsin 27 3 22.4 23.4 8 gi|257472074 clathrin heavy chain 7 3 3.8 191.6 5.48 gi|224492556 fetuin-b-like 4 3 15.4 41.2 7.4 gi|31126975 zinc-alpha-2-glycoprotein-like 3 3 17.1 34.4 5.88 gi|311250971 eukaryotic translation initiation factor 3 1 1 16.8 12.6 4.84 gi|311253491 lumican-like 7 2 11.4 38.8 5.82 gi|194037683 table 2. count’d. ubiquitin-like modifier-activating enzyme 1 5 2 3.4 114.6 5.54 gi|311276235 elongation factor 1-gamma 5 2 11.4 50 6.15 gi|311247489 fascin 3 2 8.5 54.7 6.07 gi|226372953 40s ribosomal protein s15 3 2 28.3 17 10.39 gi|51338618 zinc finger protein 7 1 1 1.9 76.3 9.13 gi|311253237 zinc finger protein aebp2-like 1 1.4 1.4 54.2 5.13 gi|311250679 zinc finger protein 425-like 1 1 2.6 70.6 9.74 gi|311264767 polyubiquitin-c-like 1 1 1.2 80.8 9.36 gi|31127000 adiponectin 1 1 10.3 15.5 8.94 gi|33694199 troponin t 1 1 6.11 31.2 5.92 gi|66773803 transcription activator brg1 1 1 1.4 165 8.82 gi|311248656 mitogen-activated protein kinase 9 1 1 4 48.4 5.5 gi|311249537 cytotoxic t-lymphocyte protein 4 1 1 4.5 24.4 5.42 gi|12644505 centromere protein f-like 1 1 0.5 351.5 5.07 gi|311265008 transcription factor ap-2 gamma 1 1 4.9 49.1 7.69 gi|178056536 cell division control protein 42 homolog 1 1 6.8 21.3 5.76 gi122063302 94 kda glucose-regulated protein 1 1 2.1 92.5 4.75 gi|17865698 ubiquilin-4-like 1 1 3.3 63.9 5.14 gi|311254132 annexin a5-like 1 1 3.9 42.9 5.16 gi|311262609 apoptosis regulator protein 1-like 1 1 2.2 71.8 5.43 gi|311271288 c-reactive protein 1 1 11.3 24.9 5.75 gi|628999899 myosin-9 1 1 1 227 5.51 gi|311255169 adp-ribosylation factor 1-like 1 1 15 20.7 6.31 gi|311249487 golgi membrane protein 1-like 1 1 5.9 27.2 4.51 gi|311265509 40s ribosomal protein s28 1 1 17.5 9.1 11.03 gi|45268967 78 kda glucose-regulated protein 4 4 9.5 73.8 5.68 gi|194033595 leucine-rich ppr motif-containing protein 1 1 2.1 87 7.55 gi311252711 sytokeleton or nuclear related 24 heterogeneous nuclear ribonucleoprotein a1 10 4 20.9 34.2 9.27 gi|116175259 eukaryotic initiation factor 4a-i 11 3 14.5 46.1 5.32 gi|154147660 tubulin beta chain isoform 1 6 3 14 49.7 4.78 gi|194040122 histone h2a type 2-c-like 7 2 37.2 14 10.9 gi|311254405 plastin-2 isoform 1 3 2 6.4 70.2 5.25 gi|194040624 collagen alpha-3(vi) 1 1 0.8 32.1 7.33 gi|194043712 microtubule-associated protein 4-like 3 1 1.87 116.9 5.03 gi|311268808 heterogeneous nuclear ribonucleoprotein a/b 3 1 6.3 32 8.31 gi|162951821 histone h3.1-like 3 1 23.5 15.4 11.13 gi|311259879 rna-binding protein fus-like 3 1 6.2 52.5 9.4 gi|311251250 plastin-3 isoform 1 2 1 2.4 63.9 5.73 gi|311276826 heterogeneous nuclear ribonucleoprotein f 2 1 4.1 45.7 5.32 gi|311271228 vinculin 2 1 1.7 123.9 5.62 gi|50403675 heterogeneous nuclear ribonucleoprotein q 1 1 2.9 69.6 8.68 gi|194035295 actin related protein 1 1 8.3 19.7 8.53 gi|19556223 actin 50 6 31.2 41.7 5.29 gi|311250866 nucleolysin tiar isoform 2 1 1 4.6 43.4 8.1 gi|311271911 heterogeneous nuclear ribonucleoprotein d0 1 1 9.5 22.9 9.47 gi|311262905 small nuclear ribonucleoprotein e 1 1 27.2 10.8 9.46 gi|147903209 histone h3.2 1 1 23.5 15.4 11.27 gi|311254411 histone h1t-like 1 1 5.2 22.2 11.58 gi194039830 nucleophosmin-like isoform 1 1 1 4.8 32.6 4.61 gi|311273930 nuclear envelope pore membrane protein pom 1 1 7.3 31.2 11.43 gi|311265618 sister chromatid cohesion protein pds5 1 1 3.6 37.1 7.66 gi|311262027 maintenance of cell shape were identified (hakkinen et al., 2010). some organellar proteins were found (including 40s ribosomal protein s15 and 40s ribosomal protein s28), as well as a few eukaryotic translation initiation factors. heat-shock proteins (hsps) are specific proteins that can protect cells and play an important role in growth, development, differentiation and other physiological activities (arrigo and simon, 2010; burel et al., 1992). in a word, this overview map of pig serum protein provided a large number of reference information. according to function information of proteins, we can make a particular study of partially serum protein in some aspect of disease. additionally, as can be seen from the sub-cellular localization of identified proteins, these proteins distributed mainly in nucleus, cytoplasm, extracellular matrix, mitochondrion cytoskeleton, and perform their functions in these areas. therefore, subcellular localization of protein from serum has potential values in research on diseases. due to the limited number of pig proteins available in the public databases, protein annotation for some of the proteins was impossible and a number of peptide mass fingerprinting was unmatched effectively. the identification of total proteins in pig serum will be achievable as soon as the complete and accurately annotated genome and protein sequence databases for pig become available. 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nikola stanišić2, čedomir radović2 1 university of belgrade, faculty of agriculture, department of food technology, nemanjina 6, 11 080 belgrade, serbia. 2 institute for animal husbandry, autoput 16, p. box 23, 11080, belgrade-zemun, serbia. accepted 12 november, 2018 sremska sausage is a traditional dry-fermented sausage from serbia made from the meat of local, late maturing pigs of the mangalica breed, which had almost disappeared. sremska sausage is today produced from the meat of modern pig breeds. three variants were made from the meat and fatty tissue of 12-monthold white pigs (swedish landrace), moravka pigs and mangalica pigs. at the end of the production process, all sausages were characterized by a high level of fat and a low level of moisture. the content of non-protein nitrogen was higher in sausages made from the meat of landrace pigs, and so was its total increase during ripening and storage. the dynamics of total plate counts, lactic acid bacteria, gram positive, catalase positive cocci counts and electrophoretic profiles of proteins were very alike. at the end of ripening, most sensory parameters differed between breeds, however, sausages made from the meat of mangalica had a significantly (p <0.05) better odour and poorer cross-section appearance. the quality of sausages from the meat of mangalica was most consistent during storage, whereas in the sremska sausage made from the meat of moravka breed the majority of characteristics were significantly poorer. key words: sremska sausage, pig breed, biochemical parameters, sensory quality. introduction sremska sausage is a serbian dry-fermented sausage traditionally produced in the north-western part of serbia (srem region), where it was produced at village households. sremska sausage is of pronounced red colour, tender texture, slightly hot taste, with a fermented meat odour and a mild note of spices and smoke. it was made from pig meat and back fat cut into pieces by hand and mixed with salt and spices. the mixture was filled *corresponding author: e-mail: dule33@agrif.bg.ac.rs and zivdusan@yahoo.com. tel: +381112615315/272. abbreviations: rh, relative humidity; npn, non-protein nitrogen; sds-page, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; tpc, total plate count; lab, lactic acid bacteria; cfu, colony-forming units into pig small intestines, smoked and dried 14 to 21 days depending on ambient conditions. microflora of traditionally produced fermented sausages originated from the raw material or from the environment in which the sausages were made (borović et al., 2010). microorganisms responsible for the changes in the fermentation process are lactic acid bacteria (lab), coagulase negative cocci and some types of yeasts (hutkins, 2006). the dominant flora of sremska sausage are lactic acid bacteria and their number reaches the maximum from production days seven to 14 (borović et al., 2010; kozačinski et al., 2008). traditionally, sremska sausage was produced from the meat of local, late maturing, fatty pigs of the mangalica breed, extensively bred on pastures in oak forests. in time, the breed has almost but disappeared and sremska sausage is today made from the meat of modern breeds. however, in line with modern trends aimed at reviving and advancing traditional food production processes, autochthonous meat products, made from local breeds, are gaining on importance. moreover, numerous researches were conducted in order to determine the characteristics of traditional and natural fermented sausages throughout the world (comi et al., 2005; salgado et al., 2005; soyer et al., 2005; di cagno et al., 2008; el malti and amarouch, 2009; rašeta et. al., 2010; vuković et al., 2011).according to moretti et al. (2004) the chemical and sensory quality of products are related to the breed; they closely relate the quality of traditional salami sant angelo to nero siciliano breed. ortiz-somovilla et al. (2005) assert that iberian pork sausages owe their high quality to the specific quality of meat and fatty tissue of iberian pigs, slaughtered at a late age, with high weights and extensively fed. on the contrary, radman et al. (2005) claimed that kulen made from the meat of black slavonian pigs is of poorer sensory quality than kulen produced from the meat of modern pig breeds. toldra and flores (1998) link flavour development with the activity of muscle proteinases and lipases. the level and activity of certain endogenous enzymes in meat differs depending on the pig breed and pig weight (saraga et al., 1993; flores et al., 1994; toldra et al., 1996; armero et al., 1999). during the ripening of fermented sausages, acidification helps to create the colour and coagulate proteins, affects the increase of firmness and cohesiveness of the product and has an important role in the activation of muscle proteinases (molly et al., 1997; cenci-goga et al., 2008). proteolysis as a consequence of the activity of muscle and microbiological proteinases and peptidases increases the yield of polypeptides, peptides and free amino acids which contribute to taste and flavour (hierro et al., 1999; spaziani et al., 2009). lipolytic activity, as a consequence of microbiological and tissue lipases activity, causes the creation of flavour components such as free fatty acids, aldehydes, ketones and esters (casaburi et al., 2008). smoking and seasoning also contribute to the flavour of fermented sausages (johansson et al., 1994). the goal of this paper was to determine the chemical, sensory and microbiological characteristics and to detect potential differences in the sensory quality of sremska sausage produced from the meat of three pig breeds: mangalica, moravka and landrace. moravka breed was selected as an autochthonous serbia pig breed in terms of meat and fat, bred in the central part of the country in the same period as mangalica, while landrace was chosen as a typical commercial pig breed in serbia. materials and methods sausage manufacture and sampling for the production of sremska sausage, ham was used as well as shoulder meat and back fat in the ratio of 75:25 from 12-month-old: white pigs-swedish landrace (variant a), moravka pigs (variant b) and mangalica pigs (variant c). all animals were bred at the test farm of the institute for animal husbandry (belgrade). meat was processed 24 h after slaughter and cooling, and was frozen at the temperature of -20°c and stored for 10 days before production. the examined variants of sremska sausage were manufactured in a small processing plant of the institute for animal husbandry (belgrade). all variants were produced on the same day and in an identical manner. meat and fat were ground in a cutter (seydelman k60, germany) to 8 mm. the same amounts of ingredients were added to all sausage variants: 2.3 salt, 0.011 nano2, 0.3 dextrose, 0.20 garlic and 0.5% sweet red paprika. the mixture was filled in pig small intestines of around 32 mm diameter. after stuffing, the sausages were hung on sticks and the ripening was carried out in a drying chamber under controlled conditions (maurer, germany) and under the following regime: day 1, relative humidity (rh) 90% at 21°c; day 2, rh 88% at 20°c with smoking, day 3, rh 85% at 20°c; during the following days rh was reduced by 1% on a daily basis (until day 14) and the temperature was constant at 16°c. fermentation was spontaneous. after 14 days the sausages were vacuum packed and stored at 4 to 7°c up to 105 days. three sausages were taken from each variant for all analyses and each analysis was done in duplicate. sampling of all variants of sremska sausage was carried out on production days 0, one, three, seven and 14 and during storage on days 60 and 105 for microbiological analysis and to determine non-protein nitrogen (npn) and ph value. chemical analysis were conduct at the beginning (day 0) and end (day 14) of production. electrophoretic analysis was carried out at days 0, seven, 14 and 105. sausage colour was determined at the end of production. sensory evaluation of sausages was conducted at the end of production and during storage, on days 60 and 105. proximate composition and ph value analysis the chemical composition of meat was determined in the following manner: moisture content by drying samples at 105°c (iso 1442:1997); protein content by kjeldahl method and multiplying by factor 6.25 (iso 937:1978); total fat content by soxhlet method (iso 1443:1973), and ash content by sample mineralization at 550 ± 25°c (iso 936:1998). ph value was measured by ph-meter hanna, hi 83141 (hanna instruments usa). npn content was determined according to the method of hughes et al. (2002). microbiological analysis ten grams slices from each sausage variant were weighed aseptically, transferred to sterile saline diluent containing 1% peptone and homogenized for 2 min using stomacher 400 (seward, london, uk). appropriate decimal dilutions of the samples were prepared using the same diluent and plated in duplicate on different growth media. total viable counts were determined on plate count agar (pca) (merck, darmstadt, germany, incubated at 30°c for 72 h; gram positive, catalase positive cocci counts on mannitol salt phenol-red agar (msa, oxoid, cm 0085) at 37°c for two days; lactobacilli counts on de man rogosa sharpe (mrs, oxoid, cm 0361) agar, microaerophilic incubated (gas pack, bbl, germany) at 30°c for five days. microbiological data were transformed into logarithms of the number of colony-forming units (cfu g -1 ). sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) sarcoplasmic protein extracts were prepared according to the table 1. changes in the basic chemical composition of the sremska sausage during the ripening (%). ripening time variant proximate composition moisture protein lipid ash a 51.73 ± 1.09 a 16.60 ± 0.18 a 28.04 ± 1.43 a 3.40 ± 0.05 a day 0 b 49.26 ± 2.29 ab 15.85 ± 0.80 ab 31.29 ± 2.55 ab 3.60 ± 0.16 a c 48.91 ± 0.45 b 15.69 ± 0.52 b 32.29 ± 0.30 b 3.44 ± 0.07 a a 26.26 ± 0.29 ab 23.55 ± 0.98 a 44.25 ± 0.89 b 6.06 ± 0.20 a day 14 b 27.89 ± 1.00 a 21.46 ± 1.13 b 44.78 ± 0.09 b 6.24 ± 0.12 a c 25.11 ± 0.98 b 23.09 ± 1.04 a 44.98 ± 0.88 b 6.05 ± 0.18 a a,b mean values in the same column, for each day, with the different superscripts differ significantly (p<0.05). method of toldra et al. (1993). four grams of sausage was homogenised with 40 ml of 0.03 m potassium phosphate buffer (ph 7.4) for 5 min. the homogenate was centrifuged for 15 min at 10 000g at 4°c. the supernatant included the sarcoplasmic proteins. myofibrillar proteins were extracted from the resultant pellet by homogenising with a solution containing 8 m urea and 1% βmercaptoethanol for 2 min using a philips hr 2000 blender. the homogenate was recentrifuged under the same conditions and the supernatant contained the myofibrillar proteins. samples were diluted with sds-page sample buffer. samples were heated at 100°c for 5 min prior to electrophoresis. a 15% separating gel with 4% stacking gel was used for sarcoplasmic proteins, and a 12% separating gel with 4% stacking gel was used for myofibrillar proteins. both sarcoplasmic and myofibrillar fractions were analyzed by sds-page, according to the method of laemmli (1970), using 20.5 x 10 cm tv200yk twin-plate electrophoresis unit (consort, belgium) together with electrophoresis power supply ev202 (consort,belgium). after electrophoresis, the gels were stained with comassie brilliant blue r-250 (0.25%) in fixative (45% methanol, 10% acetic acid). the gels were destained using 45% methanol and 10% acetic acid. the molecular weights of the proteins were estimated by running standard proteins of known weight in each gel. the molecular weight standards used were phosphorylase b 97.4 kda, albumin bovine 67 kda, albumin egg 45 kda, carbonic anhydrase 29 kda, (serva electrophoresis gmbh, germany). 7 μl of both myofibrillar and sarcoplasmic protein solutions were applied onto the gels. working conditions were 80 ma and 300 v during 4 h for sarcoplasmic proteins and 3 h for myofibrillar proteins. during that time the smallest components of the protein standard mixture were lost in the running buffer. the molecular masses of the protein bands were calculated from the rf values by interpolation on the calibration curve constructed using the known markers. colour determination sausage colour was determined 14 days after the beginning of production by chromameter cr-400 (minolta co. ltd, tokyo, japan), in line with the cie l*a*b* system (l* – lightness, a* – redness (+/− red-green), b* – yellowness (+/− yellow-blue)). the colour of the surface in question was measured at the upper, middle and lower third of the sausage, and the colour of the cross section at three fresh cuts (three measurements each in both cases). the presented data are mean values of nine measurements. sensory analysis the evaluation of sensory characteristics of sausages was conducted by eight assessors with previous experience in the evaluation of dry-fermented sausages. prior to each evaluation, preparatory meetings were held to discuss in detail the defined characteristics of sausages which were to be evaluated. a numericdescriptive scale with nine-point system was used to evaluate sensory parameters such as appearance, cross section, colour, odour, texture and taste of sausages (1 – extremely unacceptable, 9 – extremely acceptable). the presented data are mean values of eight evaluations. statistical analysis the results were processed by single-factor analysis of variance (anova). the differences between individual averages were tested using tukey’s method. significant differences were considered for p < 0.05. calculations were done with software statistica 6.0 pl, for windows (statsoft inc.). results and discussion proximate composition the chemical composition of sremska sausage at the beginning and end of ripening is shown in table 1. sausages made from the meat of mangalica (variant c) at the beginning (day 0) had the lowest moisture content and protein content, but also the highest fat content, which is a statistically significant difference in comparison with variant of meat from landrace (p<0.05). the dis-crepancy was most likely a result of the different chemical composition of the meat of pigs of various breeds.at the end of ripening, sausages were characterised by a high fat content and low moisture content. moisture content decreased to the level of 25.11% to 27.89%. such a low moisture is typical for similar products in greece, hungary and croatia (kozacinski et al., 2008), and is a consequence of not only drying, but also of a higher fat content in the stuffing. at the end of ripening, all variants time (day) figure 1. changes in ph of sremska sausage during ripening and storage. figure 1. changes in ph of sremska sausage during ripening and storage. contained more than 40% of fat, which adds to the gentle texture of sremska sausage. ph during ripening, ph dropped moderately and reached the minimum on day seven of the process (figure 1). the dynamics of change was very similar in all variants, where a decrease of around 0.1 units a day was determined. the same dynamics and similar final ph values in traditionally produced sremska sausage, made from the meat of modern pigs, were reported by kozačinski et al. (2008). in variant c, the ph drop was less intensive and the minimal recorded value was 5.15. after day 7, ph began with a mild but constant increase. maximum values were recorded on day 105 and were very close 5.48 to 5.54. salgado et al. (2005) stated that the initial ph dropped in different raw cured sausages, varies in a broad interval of 0.16 to one ph unit or more. after the ph drop, there was an increase in ph values during ripening, usually in the range from 0.2 to 0.4 units, but larger variations were also possible. our experiment determined a ph increase of 0.36 units in c variant sausages, to 0.44 units in variant a. it is possible that the ph increase was caused by the formation of various alkaline compounds such as biogenic amines (roig-sagués et al., 1999). on the other hand, salgado et al. (2005) quoted authors who point out that ph increase in the latter stages of the ripening process appears to be more related to the decrease in lactic acid content than to the formation of low molecular weight nitrogen compounds. microbiological analysis total plate count (tpc) was approximately 5 log cfu g -1 for all variants (figure 2), which indicates that the raw mixture was produced in good sanitary conditions. tpc mostly stagnated on day one, as a result of the low temperature of raw materials, which during day one it slowly warmed up to 21 °c. after the first day of production, tpc slightly grew until day 14 of the process, when it reached 7.73 cfu g -1 (variant b) to 8.73 cfu g -1 (variant a). after the sausages were packed in vacuum bags, on day 14 of production a mild tpc drop was recorded until day 60, most likely caused by anaerobic conditions. during further storage, tpc stagnated and the recorded values were quite similar in all variants, from 7.29 cfu g -1 (variant b) to 7.56 cfu g -1 (variant a). lab count (figure 3) on day one either stagnated or slightly decreased, and during the next two days it marked intensive increase by around one logarithmic unit a day, reaching approximately 7 log cfu g -1 on day three. lab growth was slower until day seven, and especially between days 7 and 14. lab maximum was attained on production day 1 4 with values from 7.81 cfu g -1 (variant time (day) figure 2. total plate count growth dynamics during ripening and storage of sremska sausage. figure 2. total plate count growth dynamics during ripening and storage of sremska sausage time (day) figure 3. lactic acid bacteria growth dynamics during ripening and storage of sremska sausage. figure 3. lactic acid bacteria growth dynamics during ripening and storage of sremska sausage b) to 8.61 cfu g -1 (variant a). after day 14 the lactobacilli count reduced. moretti et al. (2004) and casaburi et al. (2007) made similar conclusions in italian slow-fermented sausages. micrococcus sp. reduced nitrate to nitrite and ensured colour development. they also contributed to the flavour of the fermented meat product (johansson et al., 1994) (johansson et al., 1994). gram positive, catalase positive cocci counts (figure 4) were lower than lab and at the beginning of the process they ranged from approximately 3.84 log cfu gˉ¹ for variant c and 4.41 log cfu gˉ¹ for variant a. their number during sremska sausage processing stayed at a similar level. the number of gram positive, catalase positive cocci in sremska sausage was lower than in similar dry-fermented sausages produced in italy (casaburi et al., 2007; spaziani et al., 2009), but it time (day) figure 4. gram positive and catalase positive cocci growth dynamics during ripening and storage of sremska sausage. figure 4. gram positive, catalase positive cocci growth dynamics during ripening time (day) figure 5. changes in npn* content throughout the ripening of sremska sausage *npn content as % of total nitrogen (tn). npn, non-protein nitrogen. remained within the framework cited by kozačinski et al. (2008). *npn content as % of total nitrogen (tn) non-protein nitrogen changes in npn content expressed as % of total nitrogen (tn) during the ripening are shown in figure 5. the content of npn increased during the first seven production days, which correlates with the ph drop in sausages. similar results were cited by flores et al. (1997). in the period from days seven to 14, the content of npn stagnated, and then during the storage period in day 0 day 7 day 14 day 105 figure 6. sds-page profile of sarcoplasmic proteins throughout the ripening and storage period of sremska sausage. sdspage, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. vacuum it increased but slightly due to low temperature completely disappeared after day seven of ripening. and anaerobic conditions which slowed down proteolysis. hughes et al. (2002) stated that endogenous muscle storage period of sremska sausage. enzymes and acid and salt induced denaturation were the intensity of proteolysis was very similar during the first seven days of ripening in all sausage variants. in the responsible for initial degradation of sarcoplasmic protein period between days seven and 14, proteolysis was fractions. depressed and a slight npn drop was recorded in all all variants of sremska sausage showed similar variants, however there were distinct differences between electrophoretic profiles of myofibrillar proteins during sausages made from the meat of various pig breeds. the ripening and storage (figure 7). mild acidification, intensity of proteolysis was the highest in variant a, minimum ph from 5.08 to 5.15, affected the low level of followed by b, while in variant c it was the lowest. total myofibrillar proteins degradation. verplaetse (1992) rise in the npn content in the period 0 to 105 days stood demonstrated a clear depressing effect of higher ph at 75.48, 36.84 and 23.61% of tn (a, b and c). rosell values on proteolysis. however, in the period from days 0 and toldrá (1998) also reported a more pronounced to 105, the degradation of a component which corresproteolytic activity in the meat of white pigs, when ponds to the myosin-heavy chain (mhc) was clearly compared to iberian breeds. observed. several authors have reported a decrease in myosin-heavy chain concentration during the ripening of dry sausages (spaziani et al., 2009) and even its sds–page complete degradation (hughes et al., 2002; casaburi et al., 2007). as a consequence of proteolysis of the the electrophoretic image of sarcoplasmic proteins myosin-heavy chain and comigration of other degra(figure 6) was very similar in all examined variants dation products, there was an increase in the intensity of throughout the experiment and it can be said that in this bands in the molecular mass zone of αactinin (97 kda) respect there were no significant differences between as well as of around 60 kda. sausage variants a, b and c. intensive degradation of degradation of actine (45 kda) and bands below it, sarcoplasmic components, especially in the zone of which most likely correspond to tropomyosin and myosinmolecular masses between 30 and 44 kda, was most light chain, was particularly intensive during the first intensive during the first seven days of ripening. seven days of ripening. actin degradation is a result of degradation of creatine kinase was pronounced, and it activities of endogenous proteinases (molly et al., 1997), corresponded to the molecular mass band of 44 kda, as but proteolysis is also intensified by proteinases of well as the molecular mass band of 14 to 16 kda, which microorganisms, which is in line with our results referring day 0 day 7 day 14 day 105 figure 7. sds-page profile of myofibrillar proteins throughout the ripening and storage of sremska sausage. sds-page, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. figure throughout the ripening and storage oftablesremska2.resultssausageofcolourassessment of sremska sausage. variant appearance cross-section lightness (l*) redness (a*) yellowness (b*) lightness (l*) redness (a*) yellowness (b*) a 30.4 ± 2.2 a 15.0 ± 1.2 a 16.7 ± 3.2 a 40.8 ± 1.8 a 17.0 ± 1.5 a 19.2 ± 2.4 ab b 29.4 ± 1.7 a 15.6 ± 1.8 a 16.1 ± 2.6 a 39.6 ± 2.8 a 17.8 ± 1.7 a 17.5 ± 1.9 b c 30.7 ± 3.1 a 15.3 ± 3.2 a 17.1 ± 4.7 a 41.7 ± 2.5 a 17.4 ± 2.4 a 19.5 ± 2.5 a a,b means with different superscripts in the same columns indicate significant difference (p<0.05). to the dynamics of lab count, ph reduction and npn increase during the first seven days of ripening. colour determination the surface of sausages (table 2) was of very similar lightness (l*), redness (a*) and yellowness (b*). sausage from the meat of moravka were somewhat lighter at the cross-section when compared to the variant made from the meat of landrace, and even more so to those made from mangalica. these results affirm the sensory colour evaluation, where colours a and b received similar grades, while variant c was graded a little lower. the measured values of the yellow colour (b*) were statistically less significant at variant b cross-section. sensory analysis results of sensory readings presented in table 3 indicate that breed affects the sensory characteristics of sremska sausage. the appearance of all examined sausage variants at the end of ripening was very good. the cross-section of sausages made from the meat of mangalica was assessed as the poorest. the coherency of meat and fatty tissue was poorer, which was most likely a consequence of higher ph value (5.15) at the end of fermentation, but also of the softer texture of mangalica fatty tissue. product colour correlated with the colour of the meat used in production. the meat of mangalica is darker than the meat of landrace and moravka. due to drying and oxidation processes, the colour of the product table 3. sensory evaluation results of sremska sausage. sensory time (day) variant characteristic a b c 14 7.62±0.52 aa 8.00±0.53 aa 8.25±0.71 aa appearance 60 7.00±0.53 ab 7.12±0.64 ab 8.12±0.99 ba 105 7.00±0.00 abb 6.00±0.53 ac 8.00±0.00 ba 14 7.87±0.64 aa 7.12±0.83 aba 6.62±0.74 ba cut appearance 60 7.37±0.92 aab 7.25±0.71 aa 6.87±0.99 aa 105 7.12±0.35 ab 6.87±0.35 aa 6.75±0.71 aa 14 7.50±0.75 aa 7.50±0.75 aa 7.12±0.35 aa colour 60 7.25±0.71 aa 7.25±1.03 aa 7.00±0.75 aa 105 7.62±0.52 aa 7.50±0.75 aa 6.87±0.35 aa 14 7.12±0.99 aa 7.50±0.75 aa 8.37±0.74 ba odor 60 4.75±0.71 ab 6.75±0.88 bb 7.62±0.52 cb 105 5.00±0.53 ab 6.37±0.52 bb 7.62±0.74 cb 14 7.87±0.35 aa 8.00±0.92 aa 7.87±0.99 aa taste 60 7.37±0.74 aa 5.37±0.74 bb 7.62±0.74 aa 105 7.75±0.46 aa 5.37±0.52 bb 7.62±0.74 aa 14 6.50±0.75 aa 5.75±0.71 aa 6.37±0.74 aa texture 60 6.37±0.74 aa 3.62±6.75 bb 6.75±0.71 aa 105 6.50±0.75 aa 3.62±0.52 bb 6.62±0.52 aa ab values in the same column for the same property, with different superscripts are significantly different (p<0.05). ab values in the same row with different superscripts are significantly different (p<0.05). grew darker during fermentation, therefore sausages made from the meat of mangalica were assessed as too dark, and received a somewhat lower grade. odour is a sensory indicator most affected by the choice of breed. the most typical and the best sausage was the one made from the meat of mangalica. the odour of this variant was rich and very pronounced, and received a much higher grade. the taste of all three variants was very similar and was graded from 7.87 to 8.00. texture is most poorly graded parameter in all variants, most notably in variant b 5.75. during storage, the overall sensory quality of the product reduced, but the dynamics of this change varied. sausages made from mangalica were of the most stable quality, and only a change of odour was statistically confirmed in this variant. on the other hand, in sausages made from the meat of moravka most of the qualities were significantly poorer. the most pronounced deterioration was observed in taste and texture. the texture of variant b was poor, insufficiently firm and mushy as soon as day 14, while during storage it deteriorated to an almost unacceptable level from the consumers’ point of view. it may be concluded that sausages made from the meat of mangalica are of very good sensory quality, which is in line with ortiz-somovilla et al. (2005) and moretti et al. (2004). sausages made from the meat of moravka had poorer sensory characteristics, as affirmed by a stance of radman et al. (2005) that the meat of certain breeds is not suitable for the production of fermented sausages. conclusion the results of this research indicate that pig breed affects the chemical and sensory characteristics of sremska sausage. proteolysis, in regard to the npn content as % of total nitrogen, was more intensive in sausages made from the meat of landrace when compared to the sausages made from the meat of old pig breeds. the breed affects most of the sensory characteristics, most notably the following: cross-section, odour and texture. based on the sensory analysis we may conclude that moravka is the least suitable pig breed for th production of this type of dry-fermented sausage because the sensory characteristics (texture, taste, odour and appearance) significantly deteriorate during storage. landrace and mangalica are very alike in terms of their sensory characteristics. sausages made from the meat of mangalica are superior in terms of odour and are of the most stable quality 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light and heavy pigs. j . sci. food agr. 71(1): 124-128. toldra f, rico e, flores j (1993). cathepsin b, d, h and l activities in the processing of dry-cured ham. j. sci. food agric. 62(2): 157-161. verplaetse a (1992). invloed van produktieparameters op het koolhydraat-en eiwitmetabolisme in droge gefermenteerde worst. in: ph.d thesis, universiteit gent, belgium. vuković i, saičić s, vasilev d (2011). contribution to knowledge of major quality parameters of traditional (domestic) kulen. tehnol. mesa 52 (1): 134-140. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (1), pp. 001-004, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. review review of somatic cell nuclear transfer in pig suchitra muenthaisong1,2, andras dinnyes1,2,3 and tshimangadzo lucky nedambale4,5,6* 1 molecular animal biotechnology laboratory, szent istvan university, h-2100 gödöllö, hungary. 2 biotalentum ltd, h-2100 gödöllö, hungary. 3 dept. of farm animal health, faculty of veterinary medicine, utrecht university, the netherlands. 4 agricultural research council (arc), animal production institute, germplasm conservation and reproductive biotechnologies, irene, 0062, south africa. 5 tshwane university of technology, department of animal sciences, private bag x680, pretoria 0001, south africa. 6 university of the free state, department of animal, wildlife and grassland sciences, p.o. box 339, bloemfontein, south africa. accepted 30 september, 2018 it is now more than 8 years, since the first cloned pig from nuclear transfer was reported. success of somatic cell nuclear transfer (scnt) in pig is still low compared to that in bovine. embryonic and neonatal abnormalities of cloned piglets are probably a result of incorrect or incomplete reprogramming of the transferred donor cell nuclei. recently, technical refinements have resulted in a considerable increase of scnt efficiency. the current level of efficiency is already sufficient for special purposes with high scientific and commercial impact including xenotransplantation, generation of human disease models and for biomedical studies. in this paper, we review current pig cloning methodologies for the technical and potential applications. key words: pig, somatic cell, nuclear transfer, oocyte and embryo. introduction to date, cloned offspring by somatic cell nuclear transfer (figure 1) have been successfully produced in a variety of mammals (wilmut et al., 1997; wakayama et al., 1998; kato et al., 1998; onishi et al., 2000). however, the success rate remains low and many important factors remain unclear especially in pig compared to other species (wu et al., 2001). usually, pig oocytes and embryos are very sensitive to stressors, physical change and chemical factors for nuclear transfer technology. the pig oocytes contain many lipids inside the cytoplasm which explain their sensitivity to environmental stressors. it seems that lipids in pig oocytes affect the development of embryos following somatic nuclear transfer technique (nagashima et al., 1995). several reports have indicated that the oocytes activation, embryo culture and embryo transfer and early embryo developmental stage seem to *corresponding author. e-mail: lucky@arc.agric.za. tel: +2712 672 9200. fax: +2712 665 1604. be the major problems of scnt in pigs. however, there is a considerable improvement on the efficiency of scnt in pigs and application such as cloned transgenic pigs to be used for xenotransplantation, are under intensive research (dai et al., 2002; lai et al., 2002b; kolbersimonds et al., 2004). the objective of this review was to provide an overview of the somatic cell nuclear transfer in pig and summarize recent improvement on the efficiency of pig scnt technology. in vitro maturation (ivm) of pig oocyte in scnt technique, the oocytes are needed to use for recipient cytoplasm. most of the recipient oocytes are matured in vitro or derived from in vivo. the first two reports using in vivo matured pig oocytes for scnt reported successfully application of scnt and they produced cloned piglet (onishi et al., 2000; polejaeva et egg donor nuclei donor propagate the single donor cell remove the egg nucleus inject the nuclei donor oocyte were electrical activated transferred reconstructed oocyte into surrogate mother surrogate mother cloned pigs figure 1. somatic cell nuclear transfer (scnt) in pig. al., 2000). as a result, cloned piglet was also born using in vitro matured oocytes from the ovaries derived from slaughter house (betthauser et al., 2000). the north carolina state university (ncsu) 23 and tissue culture medium (tcm) 199 have been compared for maturation of pig oocytes and no differences were found among them on supporting the development of cloned pig embryos (hyun et al., 2003a). several researchers are interested on improving oocyte and embryo developmental capacity by modify the culture media and culture conditions to enhance the successful development of cloned embryos in vitro. generally, the basic medium for pig oocytes is ncsu 23 or ncsu-37 (petters and wells, 1993). for in vitro maturation, pig follicular fluid (pff) was used to supplement in the ivm medium. the pff is usually added to maturation media of pig oocytes to protecting oocytes from oxidative stress through a higher level of radical scavenging activity, resulting in the enhancement of cytoplasmic maturation for development competence after fertilization. the maturation medium that supplemented with pff can enhanced development competence of pig maturation. the factors within the follicles may play an important role to support the oocytes during maturation (schoevers et al., 2003; algriany et al., 2004). however, pff can be contaminated from numerous undefined factors with viral pathogens (kim and dubovi, 2003). the addition of hormones, growth factors, vitamins, energy substrates, inorganic compound, cytokines and follicular fluid influences considerably maturation and subsequent development of pig oocytes (abeydeera, 2002). there is a report that epidermal growth factor (egf) plays an important role in nuclear and cytoplasmic maturation of pig oocytes (grupen et al., 1997; abeydeera et al., 2000). moreover, exposure of cumulusoocyte complexes (cocs) to dibutyryl camp (dbcamp) for maturation increased developmental competent of pig oocytes (funahashi et al., 1997). recently, defined systems for in vitro production of pig embryos using a single basic medium were reported for blastocyst production from chemically defined pig gamete medium (pgm) and pig zygote medium (pzm). the media based on the composition of pig oviductal fluid and appropriately supplemented for specific stage of embryo development thus can be used for in vitro maturation, fertilization and culture in pig (yoshioka et al., 2008). somatic cell nuclear transfer it is a process known as nuclear transfer (nt) in which the nucleus of a donor cell is transferred into an oocyte. the first pig cloned by scnt was reported by prather et al. (1989). they used pronuclei exchange zygote and blastomere as a donor cell. recipient cytoplasm derived from in vivo maturation was used and activated the reconstructed oocytes with electric before transfer to the oviduct for in vivo embryo culture. blastocyst embryos were transferred to the recipient for develop into term. they received seven live pigs born from pronuclei exchange zygote and one live pig born from blastomere at 4-cell stage. moreover, successful cloned pig by the use of somatic cell nuclear transfer technique has been reported by polejaeva et al. (2000). they used granulosa cells as a donor cell with two-stage nuclear transfer technique that donor cells were fused to enucleated oocytes in the first stage, and the pronucleus-like structures formed were then subsequently transplantedinto in vivo-produced, enucleated zygotes. however, the successful production of cloned pig from a single-step nuclear transfer and the method is still wildly used in several laboratories to produce the scnt pig (betthauser et al., 2000; onishi et al., 2000; park et al., 2001; de sousa et al., 2002; yin et al., 2002). several researchers also reported the successful production of pig cloned with different donor cell types (onishi et al., 2000; betthauser et al., 2000; bondioli et al., 2001; park et al., 2002) and also they attempt to modify donor cells to improve the efficiency of nuclear transfer. the donor cell in scnt can be prepared as synchronized (onishi et al., 2000; polejaeva et al., 2000) and non-synchronised donor cells (betthauser et al., 2000; bondioli et al., 2001). there success the nuclear transfer with both of in vivo-matured oocytes (onishi et al., 2000; polejaeva et al., 2000) and in vitro-matured oocytes (betthauser et al., 2000). many strategies to introduce the donor cell into cytoplasm of the recipient cytoplasm such as transferred by electrofusion (polejaeva et al., 2000), by piezo-microinjection of isolated donor nuclei (onishi et al., 2000), and also by whole cell injection (lee et al., 2003b). recently, the zona-free technique become of interest with zona removal before or after enucleation for fusion. (booth et al., 2001; kragh et al., 2004; du et al., 2005). artificial activation in an effort to establish the cloned embryos, activation is the part of important step to initial reconstructs oocytes and donor cells. since sperm-mediated activation is absent in scnt, an artificial activation is needed to initiate embryo development. mostly in mammals, oocytes are arrested at metaphase ii (mii) stage after ovulation and complete meiosis after fertilization. the mii arrest is characterized by mpf activity (nurse, 1990). since the sperm-induce the release from meiotic arrest by a signal transduction pathway, calcium, m-phase promoting factor (mpf) and its stabilizing molecules play a role during oocyte activation by inducing a transient increase in the intracellular free calcium concentration by artificial means. for in vitro culture of pig embryos, oocytes are matured for around 42-44 h after the start of ivm, thus on this stage, a calcium transient is able to stimulate development (machaty and prather, 1998). there are many activation protocols to stimulate the oocyte such as electrical, chemical or mechanical activation. simultaneous fusion/activation has been used for the generation of cloned pigs with targeted disruption of one or both alleles of the α-1,3-galactosyltransferase gene (dai et al., 2002; lai et al., 2002a; kolber-simonds et al., 2004). the transgenic cloned pig expressing the green fluorescent protein were produced by triggering fusion and activation at the same time with an electrical stimulus (park et al., 2001; lai et al., 2002; hyun et al., 2003b; lee et al., 2005). usually, high voltage direct current (dc) pulse is applied to generate an influx of extracellular calcium (machaty and prather, 1998). in the absence of extracellular calcium ions, the dc pulse induces membrane fusion only. in this case, high mpf levels in the oocyte cytoplasm cause to nuclear envelope breakdown and premature chromosome condensation. in the case of chemical activation, the oocytes were exposed to the substrates such as cytochalasin b (li et al., 2000; park et al., 2002; hoshino et al., 2005) to prevent the extrusion of the second polar body, cycloheximide (lee et al., 2003a) or 6dimethylaminopurine (dmap; de la fuente and king, 1998; hölker et al., 2005) to inhibit the protein kinase after an induced calcium transient to promote activation or ionomycin (betthauser et al., 2000; boquest et al., 2002) that it forms a complex with calcium ions and transports through the plasma membrane and it can also stimulate calcium and induce a calcium influx similar electrical activation (morgan and jacob, 1994). similarly, delayed activation was effective in supporting term development after demecolcine assisted enucleating (yin et al., 2002; kawakami et al., 2003) or mechanical activation when the donor nuclei were microinjected directly into the recipient cytoplasm (onishi et al., 2000; watanabe et al., 2005; takeda et al., 2006). it has been suggested that nuclear reprogramming is more successful if chromosomes of the nuclear donor cell are exposed to the recipient oocyte cytoplasm for an extended period of time (miyoshi et al. 2000; tani et al. 2001; shin et al. 2002). furthermore, development after nuclear transfer may be superior under delayed activation conditions compared to simultaneous fusion/activation (yin et al., 2003). all the activation protocols mentioned above stimulate embryonic development by triggering a single calcium transient. however, the availability of current methods to induce repetitive calcium transient in pig oocyte is limited. embryo culture and transfer in order to establish pregnancies, the pig embryos were transfer to the recipient in early embryo development stage between one-cell to eight-cell stage with a many number of embryo. most of embryo numbers were transferred more than 100 embryos to one recipient to avoid the negative effects of the in vitro system that seems to be effect to pig embryos more than other species. however, the pregnancy rate remains low and the fetus is almost lost during gestation (chae et al., 2006). numerous studies aimed to improve the culture medium for preimplantation of embryo that can produce the cloned pig after transfer to the recipient. the result is considered to better understanding of pig embryonic development. the culture medium for pig embryo culture is ncsu-23 (onishi et al., 2000; polejaeva et al., 2000), ncsu-37 (kikuchi et al., 2002) and pig zygote medium (pzm)-3 (yoshioka et al., 2002; im et al., 2004). recently, the result found that pzm-3 was superior in order to support the development of pig embryos, especially the embryos derived from scnt. it seems that the pzm-3 could better improve the efficiency of pig embryo to develop to preimplantation stage compared to ncsu-23 (im et al., 2004). moreover, there is a report to study effect of bovine serum albumin (bsa) and polyvinyl alcohol (pva) supplementation in ncsu-23. they found that both of them could enhance the production of pig embryo (roh and hwang, 2002). regarding to culture condition under temperature and gases control incubator, the pig embryos cultured in an atmosphere with 5% co2 in air showed the developmental rates and total cell numbers of blastocysts higher than embryos cultured under 5% co2, 5% o2 and 90% n2 (machaty et al., 1998). recently, there are reports showing that low oxygen concentration (fiveseven %) can enhance the pig embryo development from in vitro culture (im et al., 2004; sage et al., 2005). however, there were no differences developing in embryo development, total cell number and apoptosis incident under low and high oxygen concentration from in vitro fertilization and parthenogenetic pig embryos (ock et al., 2005). in addition, the modify culture conditions for cloned pig embryos by supplementation of various growth factors such as platelet activating factor (kidson et al., 2004), egf (lee et al., 2004) showed significant increased of blastocyst rate and total cell number in pig scnt embryos. moreover, drop and well of the well (wow) (vajta et al., 2000) including the drop culture technique are modified culture systems that seem to be beneficial to establish the pig scnt embryos (taka et al., 2005). regarding the attempts to modify the culture system for in vitro system, the ideal way used for cloned embryos in future experiment and in the physical environment may also have profound effect to in vitro development. pregnancy and farrowing although, the cloned pigs were established from in vitro production, the overall efficiency is still very low in pig compared to cattle, sheep and mouse. failures of scnt pregnancy are associated with placental abnormalities, such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium, and altered basement membrane (hill et al., 2000). pregnancy has already been achieved with non-surgical transfer of in vitro-produced pig embryos (suzuki et al., 2004). these may be due to intrinsic differences between pigs and these other species, or to differences in protocols for embryo production used. furthermore, the larger litter size in pigs may also limit fetal growth to a greater extent than in cattle and sheep (young et al., 1998). especially that the number of pig embryos transferred to the recipient is very high than in another species (koo et al., 2004). mostly pig embryos were transferred at the onecell; two-four-cell or four-eight-cell stages and 50-150 embryos were transferred to one recipient. in pigs, there is a need for a signal of three or more embryos to maintain pregnancy (king et al., 2002). in case, of scnt, this signal is weak, there are reports on the use of enhance and maintain the signal of pregnancy (polejaeva et al., 2000; lai et al., 2002b) or in vitro embryos by mating of recipient (onishi et al., 2000; lai et al., 2002b; pan et al., 2006) or by hormone injection after embryo transfer (walker et al., 2002; harrison et al., 2004; pan et al., 2006) that were successfully used in producing cloned pigs. application of pig somatic cell nuclear transfer (scnt) the mainly purpose of pig scnt is related on the biomedical applications to human and also for xenotransplantation to replace organs or tissues by using the pig as an animal models for human diseases to therapy study because pig has a relatively similar organ size, anatomy, and physiology to that of human. furthermore, their genome is much closer to human compared with mouse. the gene expression pattern of pigs is more closely similar to those of a human (forsberg, 2005). however, there is a need to improve and increase the efficiency of pig scnt and develop cloned pig with genetic modification that the organs will not cause an immunological response and destroy transplanted tissue when transferred to human. in addition, initial development and validation of the lab procedures, the availability of reliable and efficient 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onishi a (2008). defined system for in vitro production of porcine embryos using a single basic medium. j. reprod. dev. 54: 3: 208-213. yoshioka k, suzuki c, tanaka a, anas im, iwamura s (2002). birth of piglets derived from porcine zygotes cultured in a chemically defined medium. biol. reprod. 66: 112-119. young le, sinclair kd, wilmut i (1998). large offspring syndrome in cattle and sheep. rev. reprod. 3: 155-163. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 10 (2), pp. 001-009, february, 2022. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper appraisal of management and biosecurity practices on pig farms in makurdi, benue state, north central nigeria leo daniel ojabo* and moses ukwu enya department of animal health and production, college of veterinary medicine, federal university of agriculture, makurdi, benue state, nigeria. *corresponding author. e-mail: leojabo@yahoo.com. accepted 14 august, 2021 the role of biosecurity at farm level is to reduce the risk of introduction of diseases in pig farms and prevent the disease transmission between animals on farms. it is an important management practice that impacts the profitability of pig production ventures. a survey was conducted to assess the biosecurity practices in 50 farms in makurdi, benue state, nigeria, using a structured questionnaire and assessments through direct observations. descriptive statistics such as frequencies, percentages and tables were used to analyse and present data generated. the results revealed that majority of the farms were semi-intensive (54%), small scale producers keeping between 1 and 50 pigs formed 96% of surveyed farms and essentially raising indigenous and crossbreed (72%). only 12% of the farms were fenced and had gates closed to control unwanted visitors and stray animals. most of the farms (76%) had no footbaths indicating that the risk of disease introduction was high in such farms. the use of protective clothings and foot wears by employees was only observed on 24% of the farms. isolation of sick animal was practiced by 38% of respondents while 24% of respondents quarantined new animals to the farm. the use of disinfectants to clean the barns was practiced on 18% of the farms. overall, there was a low level of farm biosecurity in the study area. extension services to create awareness on the importance of biosecurity would help limit infections and boost pig production and profitability. key words: biosecurity, diseases, nigeria, pig farmers, production system. introduction pig production is one of the fastest growing livestock sectors in the world (fao, 2012a). according to oguniyi and omotosho (2011), pig production plays a vital role in small scale farming far beyond pork production and income generation. the animal is an asset of wealth or safety net in time of crisis when viewed from the economic perspective and serve as a source of protein nutritionally (fao, 2012b). pig production has been advocated as a short-term measure towards alleviating the animal protein and calorie deficit, especially where there are no religious edicts preventing their production and consumption (eusebio, 1984). apart from the shorter production time, pigs have been noted for their high conversion rate of feed to flesh 2 amongst red meat animals (fao, 2012b). pork represents high value animal proteins. swine production contributes significantly to the livelihood of many nigerians either directly or indirectly (ajala and osuhor, 2004; fao, 2006). benue state is one of the largest pig producing states in nigeria (more than 20% of the total country production) and the pig enterprise contributes significantly to the economy of the state with most of the pigs owned by small holders operating semi-intensive pig production system (umeh et al., 2015; asambe et al., 2019). nigeria is one of the african countries with significant pig population density (robinson et al., 2014). in the 1990s, the pig population was 3.5 million consisting of native black hairy pigs and exotic breeds (bourn et al., 1994). the latest population estimate was reported by the federal department of livestock (fdl) (2010) to have increased to 7.1 million, indicating that the population had doubled in about two decades. the pigs are reared in neighborhoods of villages and in semi-urban areas as small-scale enterprises having 12 50 pigs, but a few large-scale farms exist (ajala et al., 2006; saka et al., 2010; abiola et al., 2015). semi-intensive and extensive pig production systems occur in the northern, north central and niger delta regions of nigeria (bourn et al., 1994). intensive pig production exists mostly in southern nigeria (ajala et al., 2006; saka et al., 2010; nwanta et al., 2011) and consists of farms having each 50 – 200 pigs in concrete pens. commercial piggeries rear about 3% of the national pig population with usually more than five breeding sows. sustainable growth of the pig production industry in nigeria is adversely affected by factors such as unstructured pig marketing framework, fluctuation in the prices of pigs and pig products, cultural and religious prohibition of pork consumption, low demand for pork in parts of the country, high feed cost, inadequate extension services, slow integration of cost-effective equipment and genetically enhanced breeds and disease outbreaks (ajala and adeshinwa, 2008; ironkwe and amefule, 2008; fasina et al., 2010; anukwu and ebong, 2011; abiola et al., 2015; igbokwe and maduka, 2018). disease outbreaks represent the major constraints to profitable pig production and consumption in nigeria. important pig diseases reported in the country include african swine fever, foot-and-mouth disease, brucellosis, tryponosomosis, babesiosis, eperythrozoonosis, helminthosis, cocciodiosis and other parasitosis (igbokwe and maduka, 2018). these diseases impact on the production system by negatively affecting feed conversion efficacy, reproduction and growth rates as well as causing piglet and adult mortalities. the economic losses due to the disease burden and inadequate intervention strategies are current issues facing the pig production industry (igbokwe and maduka, 2018). thus, one of the important measures to increase pig productivity should focus on disease control. as such, implementation of biosecurity measures in pig production is of paramount importance to farm profitability (kouam et al., 2019). several pig diseases (intestinal and miscellaneous diseases) have been successfully controlled through the implementation of biosecurity practices in some eu countries (wallgren, 2009). the implementation of biosecurity measures is regarded as a powerful tool in the control of diseases on the farm; its main advantage is the potential to keep pathogens off the farm and to prevent pathogens from spreading to other farms. in the world, organisation of animal health (oie) terrestrial animal health code, biosecurity is defined as a set of management and physical measures designed to reduce the risk of introduction, establishment and spread of animal diseases, infections or infestations to, from and with an animal population (bellini, 2018). biosecurity measures are divided into three components: isolation, traffic control and sanitation (cardona and kuney, 2001; fao/oie/world bank, 2010). isolation can be regarded as measures related to physical barriers (fence, showers or footbaths) and distance between farms in order to limit contacts between infected animals and contaminated objects with disease-free farms (fao, 2008). traffic control can be considered as the restriction of feedstuff, human, equipment and animal movement onto the farm (fao, 2008). sanitation refers to the cleaning and disinfection of animal housing, people, material and equipment (cardona and kuney, 2001). successful implementation of biosecurity requires the adoption of a set of attitudes and behaviours by people to reduce interactions in all activities involving domestic, captive/exotic and wild animals (pigs) and their products (fao/oie/world bank, 2010). nyaga (2007) reported that biosecurity principles include simple procedures and practices which when applied prevent the entry of disease agents in a farm or exit of disease agent from infected premises. biosecurity entails a controlled/regulated movement of stock, persons, equipment and products into a clean farm and out of infected premises. it involves methods that enable the farm to remain in a state of sustained cleanliness referred to as sanitation. it has however been observed that many biosecurity measures are either partially observed or not observed at all, owing to several factors such as cost, inadequate veterinary extension and attitudinal dispositions (brennan and christley, 2012). the aim of this study was therefore to describe the pig production management system as well as associated biosecurity practices in pig farms at makurdi urban and peri-urban areas in benue state, north central nigeria. materials and methods study area the study was carried out on pig farms located in makurdi urban and peri-urban areas. makurdi is the capital city of benue state, nigeria. nigeria is a sub-saharan african country and is located in 3 figure 1. map of nigeria showing the location of benue state. the western part of the continent. it is the most populous country in africa and the most populated black nation on earth (akpotor and agbekabu, 2010). benue state is one of the 36 states in nigeria. it is located in north central nigeria (figure 1) and has a tropical climate with two distinct seasons: rainy and dry seasons. makurdi is a lowland area in the guinea savannah vegetational zone of nigeria and is located on longitude 08°31 ’ and latitude 07°14 ’ (abu, 2002). the rainy season starts in may and ends in october, while the dry season starts in november and ends in april. the annual rainfall ranges from 1270 to 1397 mm and the average annual temperature ranges from 22.43 to 33.41°c (abu, 2002). during the dry season between the months of february and march, temperatures may reach 35 to 40°c in makurdi town. the relative humidity ranges between 47 to 85% (tac, 2002). according to the 2006 census, benue state is estimated to have a population of about 4,253,641 inhabitants and a land size of about 33,955 km 2 (akaakohol and aye, 2014). the average population density is 99 persons per km 2 , which makes benue state the 9 th most populous state in nigeria. population distribution between male and females in benue state is almost equal (with 50.4% male and 49.6% female) (nigeria data portal, 2006). makurdi, the state capital has a population density of over 380 person per km 2 . agriculture is the backbone of benue state’s economy, and the state is the primary source of food in nigeria acclaiming the slogan ‘the food basket of the nation.’ benue state is endowed with fertile arable land and abundant raw materials and human resources with about 80% of the population directly involved in agriculture (akaakohol and aye, 2014). important cash crops include soybeans, rice, peanuts, mango varieties, citrus, etc. other cash crops include palm oil, melon, african pear, chili, cassava, sweet potato, beans, maize, millet, guinea corn, vegetables etc. there is very little irrigation agriculture and techniques. livestock species include pigs, small ruminants, cattle and poultry (wikipedia, 2020). study design and data collection fifty pig farms were randomly selected for the study based on the presence of pigs in their farms during the period of the study. fifty structured questionnaires and biosecurity assessment forms were used to obtain information from the farmers, while visits to the farm facilities for direct observation were also undertaken for data collection according to the methods described by augustine et al. (2010). each farm was visited twice to first distribute the data collection instruments and make observations, and secondly retrieve the completed instruments. data analysis data obtained from the study were analysed and summarised in 4 tables using simple descriptive statistics. this involved the use of statistical tools to generate frequency distribution and percentages (sas, 2003). results demographic characteristics data in table 1 shows that majority of the farmers were males (72%) and 28% females. the literacy level of the respondents was very high and most of the respondents were married. most (96%) of the respondents were aged between 21 years to 50 years. most of the farms were semi-intensive (54%), followed by 34% extensive farms (table 2). a minority of 12% farms were intensive. the most frequently used crossbreeds (40%) and 66% of the farmers used farmmixed feeds and kitchen wastes to feed their pigs. most of the farmers were relatively new to the industry with less than 5 years of farming experience. this study also revealed that the respondents are small-scale farmers and as such generally had small flock size. it was also observed that 54% of the farmers kept other livestock species in their farms. the farmers purchased 64% of their replacement stock for breeding locally from other farmers within their locality (within and around makurdi urban and sub-urban areas). the source of labour was divided into 2 categories (family and hired labour) but family was by far the greatest source of labour (62%). biosecurity practices on farms traffic control and isolation only 12% of farms were fenced and none had warning signs at their farm entrance forbidding access to piggeries for visitors (table 3). furthermore, functional footbaths were available in only 24% of farms, 16% of farms had feedstuff protected from rodents and 22% of the farms had a pest control programme in place. majority of the farms (66%) were properly ventilated. this study further revealed that 54% of the farmers also kept other domestic animals in their farms. only 24% of farmers kept newly arrived animals in quarantined. hygienic and sanitation use of protective clothings and boots was done by only 24% of the farmers (table 4). cleaning of facilities on a daily basis and the use of disinfectants were practiced by only 18% of farmers. when animals fell sick, 38.0% of the farms practiced isolation of sick animals. discussion the demographic characteristics of the pig farmers in this study showed that majority of the farmers were males suggesting that men who are relatively stronger are mostly involved in pig production in the study area. females in this study area were usually involved as helpers or suppliers of labour in light farm operations such as serving of feed, water or cleaning the piggery. umeh et al. (2015) and uddin and osasogie (2016) in previous studies also reported higher presence of male farmers. literacy level of the respondents was high as 92% of the farmers had attended high schools or higher learning institutions. according to moreki et al. (2014), educational level could influence farmers’ adoption of modern technologies needed for the improvement of productivity. the good educational background of these farmers could be beneficial in the areas of farm record keeping, reading of drug/vaccine prescription, adoption of innovations and other management functions to ensure productivity (uddin and osasogie, 2016). table 1 also revealed that most of the respondents were married and aged between 21 years to 50 years indicating that majority of the farmers were adults. okoro (2012) and egbule (2010) revealed in separate studies that majority of farmers in nigeria are married. this result shows that married people dominate pig production in the study area. married farmers could have more persons in the household to cater for and helping hands in taking care of pigs in their farms. animal husbandry was not the main occupation of pig farmers within the study area. other occupations also included civil/public servants, business/trader, crop farmers and retirees. this picture gives the impression that pig farming alone does not allow the farmers to make a living within the study area. the reason for this may be related to the recurrent epidemics of asf that has been decimating the herds in different localities of the country. the general pig husbandry characteristics in the study area as shown in table 2 revealed that most of the farms were semi-intensive (54.0%). a minority of 12.0% farms were intensive. family was by far the greatest source of labour. this finding is similar to that of kouam et al. (2019) in western cameroon. this study further reveal that the farmers are small scale farmers and as such generally have small flock size and hence family labour may be adequate. the low herd size may also be partly due to the common disease challenges faced by the pig industry in benue state, especially the regular outbreaks of contagious diseases, such as asf (asambe et al., 2017). in fact, the high mortality rate of these diseases (close to 100% for asf) in affected farms causes farm owners either to remain only with the few survivors, to restock the farms with few animals or to take less risk in investing for a larger farm (kouam and moussala, 2018). the results of this study show that the average farming experience of pig farmers was less than 5 years. this is in contrast to a previous study by umeh et al. (2015), who reported the average farming experience of pig farmers in benue state as 11.08 years. it should be noted however, that the study of umeh et al. (2015) covered all the three agricultural zones of benue state 5 table 1. demographic characteristics of the respondents in the study area. category frequency % gender male 36 72.00 female 14 28.00 educational status non formal education primary school completed 4 8.00 secondary school completed 9 18.00 tertiary education 37 74.00 occupation farmer 11 22.00 trader 7 14.00 business 10 20.00 retiree 8 16.00 civil/public service 14 28.00 age 11 20 years (under 20 years) 2 4.00 21-30 10 20.00 31-40 21 42.00 41-50 16 32.00 >50 1 2.00 marital status married 29 58.00 single 17 34.00 widower 3 6.50 widow 1 2.00 table 2. general characteristics of pig husbandry. category frequency % breed indigenous 16 32.00 exotic 14 28.00 crossbreeds 20 40.00 age (years) 0 1 16 32.00 1 2 13 26.00 2 3 14 28.00 3 4 7 14.00 > 5 purpose of production meat 2 4.00 income 19 38.00 both 29 58.00 6 table 2. cont’d number of pigs (flock size) 1-10 10 20.00 10-20 14 28.00 20-30 6 12.00 30-40 8 16.00 40-50 8 16.00 > 50 4 8.00 management system extensive 17 34.00 intensive 6 12.00 semi-intensive 27 54.00 source of pig pig market 18 36.00 other breeders 32 64.00 years of experience less than 3 30 60.00 3 5 years 19 38.00 > 5 1 2.00 feed source commercial 4 8.00 farm-mix 17 34.00 commercial/farm mix 13 26.00 kitchen (home remains) 16 32.00 other livestock species kept sheep 5 10.00 goat 7 14.00 cattle 1 2.00 rabbit 2 4.00 poultry 11 22.00 none 23 46.00 fish 1 2.00 source of labour hired 19 38.00 family 31 62.00 others while this present study covered only makurdi, the capital of the state. according to umeh et al. (2015), farming experience may increase technical efficiency as experience has positive influence on managerial ability as it tends to improve ability of the farmers to obtain and process information about technology which in turn increases efficiency. combining farm-mixing of feeds and kitchen wastes may be more economical than using commercial feeds alone. however, kitchen wastes are not properly heated before feeding it to animals, the risk of transmitting diseases such as african swine fever (asf) among other diseases is high (asambe et al., 2017). purchasing replacement stock for breeding within the same locality predisposes to inbreeding and subsequently low production, reproduction and poor health (kirima et al., 2017). concerning biosecurity practices, perimeter fence was present in only 12% of the farms and all the farm visited 7 table 3. biosecurity practices associated with traffic control and isolation. practices frequency % biosecurity information sign at entrance no information sign at entrance 100 100 perimeter fence and gate 6 12 no perimeter fence and gate 44 88 use of footbath 12 24 no footbath 38 76 maintain pest control programme 11 22 do not maintain pest control programme 39 78 farms with rodent proof feed store 8 16 farms without rodent proof feed store 42 84 farms with good ventilation 33 66 farms without good ventilation 17 34 other species are present on the farm yes 27 54 no 23 46 new animals are quarantined 12 24 new animals are not quarantined 38 76 table 4. biosecurity practices associated with sanitation practices frequency % protective clothing for employees 8 16 no protective clothing for employees 42 84 protective foot wears (boots) for employees and visitors 8 16 no protective foot wears (boots) for employees and visitors 42 84 disinfectants are used for cleaning 9 18 disinfectants are not used for cleaning 41 82 drinking water is treated with chemical (chlorine) drinking water is not treated with chemical (chlorine) 100 isolation of sick pigs 19 38 no isolation for sick pigs 31 65 did not have a sign board or any physical notice preventing access on farms for visitors. these results agree with the findings of kouam et al. (2019) in western cameroon. physical barriers are meant to protect the herd from stray animals or human intruder entering the farm. the risk of disease introduction in those farms without gates was therefore high. cobb avian 48 (2006) stated that each farm must have a perimeter fence to prevent unwanted entry of people, vehicles and animals. other animal species were found in 54.0% of farms, which is a serious biosecurity threat since cross transmission of pathogens between different species has been demonstrated (wall et al., 1995, kouam et al., 2019). footbaths were available in only 24% of the farms, which can be explained by the small number of farms under intensive system in this study. footbaths are essential for disinfection of foot wears and should be renewed on a daily basis. poor or absence of disease control strategies such as footbaths and inadequate management like replenishing footbaths after disinfection dries up are some of the factors that can increase disease outbreak. majority of the farms had poor pest control programme. rodents have been reported to be mechanical vectors for pathogenic microorganisms (annette and claes, 2012). there was a poor implementation of quarantine of new animals which might be explained by the fact that majority of the farmers are not experienced and have not been trained in pig farming and biosecurity practices. majority of the farms were properly ventilated (66.0%). the provision of good ventilation is an attempt to reduce the effect of heat stress as a result of hot and humid climate in the study area. this study also showed that most of the farmers do not 8 possess dedicated clothings and boots for use on the farm. the practice of owning dedicated clothing and boots was observed only in the intensive farms which are less represented in this study. farmers non-compliance with the practice of use of herd specific overall and boots is very risky as humans can act as mechanical vectors of diseases to pigs. the practice of cleaning and disinfection of farms was only practiced by 18.0% of farmers. this practice if properly implemented will help to break the cycle of diseases on the farm. the low implementation of use of disinfectant and daily cleaning indicate that most of the farmers do not understand the need to keep farms properly clean and disease free. the practice of isolating sick animals by farmers in this study was low (38.0%). disease can develop within a farm at any time, so farmers should practice separation of sick animals to protect the healthy animals. conclusion and recommendations this study has shown that the production system in the study area was dominated by the semi-intensive system of pig production. the most important biosecurity measures of concern and to improve are: (i) fencing of pig farms. (ii) training of pig farmers (iii) farmers compliance with the practice of use of herd specific overalls and boots. (iv) implementation of the use of disinfectants and daily cleaning. (v) keeping newly arrived animals in quarantine and the practice of isolation of sick animals. 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(m.sc. thesis). university of nigeria, nsukka, enugu state, nigeria. robinson tp, wint grw, conchedda g, van boeekel tp, erocoli v, palamara e, cinardi g (2014). mapping the global distribution of livestock. plos one 9(5):e96084 doi 10.1371/journal pone. 0096084. saka jo, adesehinwa aok, ajala mk (2010). incidence of african swine fever (asf) disease and its associated implications on pig production in lagos state, nigeria. bulgarian journal agricultural science 16(1):80-90 sas (2003). statistical analysis system. sas institute inc. cary north carolina. tac (2002). makurdi weather element. nigerian airforce tactical air command meteorological station, makurdi, nigeria. uddin io, osasogie d (2016). constraints of pig production in nigeria. a case study of edo central agricultural zone of edo state. asian research journal of agriculture 2(4):1-7, 2016; article no. arja. 30187. umeh jc, ogbaje c, adejo ma (2015). technical efficiency of pig production: a sustainable animal protein augmentation for nigerians. journal of advanced agricultural technologies volume 2, number 1, june 2015. wall pg, morgen d, lamden k, griffin m, threlfall ej, ward lr, rowe b (1995). transmission of multi-resistant strains of salmonella tryphimuriumfrom cattle to man. veterinary records 136:591-592. retrieved from https://doi.org/10.136/vr.136.23.591. wallgren p (2009). first out to ban feed additives in 1986. veterinary challenges within swedish pig production. part ii: intestinal and miscellaneous disease. pig journal 62:52-60. wikipedia (2020). benue state. https://en.wikipedia.org>wiki. accessed on april 20 th , 2020. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 8 (2), pp. 001-007, february, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper antibacterial effects of zinc oxide nanoparticles on escherichia coli k88 chao wang, lian-long liu, ai-ting zhang, peng xie, jian-jun lu* and xiao-ting zou* institute of feed science, college of animal science, zhejiang university, key laboratory of molecular animal nutrition, ministry of education, hangzhou zhejiang province, people’s republic of china 310058. accepted 14 october, 2019 this study was conducted to evaluate the antibacterial effects of zinc oxide nanoparticles in vitro. escherichia coli k88 was chosen as an indicator of pathogenic bacteria, because it could cause diarrhea in both children and in early-weaned piglets. in this study, the characterization of the nanoparticles was examined. antibacterial activities against e. coli k88 were evaluated by determining the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) and observing the effects on the values of the optical density (od) at 620 nm and the populations. results indicate that zinc oxide nanoparticles had strong antibacterial activity against e. coli k88. the activity increased as the concentration of the nanoparticles increased. the mic and mbc were 0.1 and 0.8 µg/ml, respectively. to study the antibacterial mechanisms, atomic force microscopy (afm) and scanning electron microscopy (sem) were used to observe morphological changes of e. coli k88 treated with 0.8 µg/ml zinc oxide nanoparticles. the results reveal that zinc oxide nanoparticles could damage cell membranes, lead to leakage of cytoplasm and kill the bacterial cells. our study indicates that zinc oxide nanoparticles could potentially be an antibacterial reagent to treat diseases caused by bacteria. key words: zinc oxide, nanoparticle, escherichia coli k88, antibacterial activity, atomic force microscopy (afm). introduction escherichia coli k88 is an important pathogen, which could cause diarrhea in both children and early-weaned piglets (guo et al., 2005; yu et al., 2011). antibiotics are the most common drugs to inhibit the growth and propagation of e. coli k88. however, the use of antibiotics has various side effects, such as the increase in bacterial resistance (baquero et al., 2011). the exploitations of novel substitutes to antibiotics, especially on inorganic nanoparticles, have recently attracted more attention. zinc oxide is an important inorganic material, which has multiple properties, such as semiconducting properties, antibacterial activity and growth promoter. it is widely applied in the field of optoelectronics (yu et al., 2003; gao *corresponding author. e-mail: jjlu@zju.edu.cn, xtzou @zju.edu.cn. tel: 86-571-88982729. fax: 86-571-88982650. abbreviations: mic, minimum inhibitory concentration; mbc, minimum bactericidal; od, optical density; afm, atomic force microscopy; sem, scanning electron microscopy. et al., 2005), pharmaceutics (baldwin et al., 2001), cosmetics (sheldon et al., 2000; mitchnick et al., 1999), food science (daniel et al., 2003) and agriculture (smith et al., 1997; carlson et al., 1999). the antibacterial activity of zinc oxide has been widely explored. it has been documented that concentration, size and healing temperature can affect the antibacterial activity. zinc oxide as an inorganic antibacterial reagent is more stable than the organic reagents (yamamoto, 2001; sawai, 2003; sawai et al., 1996a). several antimicrobial mechanisms of zinc oxide were supposed; i) hydrogen peroxide, which is generated from the surface of zinc oxide, can penetrate through the cell membrane, produce some type of injury, and inhibit the growth of the cells (yamamoto, 2001; sawai, 2003; sawai et al., 1996b, 1998); ii) the affinity between zinc oxide and bacterial cells is an important factor for antibacterial activity (stoimenov et al., 2002). combined with nanotechnology, zinc oxide nanoparticles can be prepared, which possess some unique characters, such as small particle size and large area surface. zinc oxide nanoparticles may exhibit stronger antibacterial activity than zinc oxide itself (yamamoto, 2001). therefore, the interactions of nanoparticles with microorganisms have recently attracted more attention and a wide range of antibacterial effects of zinc oxide nanoparticles have been reported (jones et al., 2008; reddy et al., 2007). moreover, zinc oxide nanoparticles have selective toxicity and are generally regarded as a safe reagent to humans and animals (reddy et al., 2007; liu et al., 2009; fu et al., 2005; berube, 2008), which could be an ideal potential antibacterial reagent to replace some antibiotics. however, few studies have been conducted to evaluate the antibacterial effects of zinc oxide nanoparticles on e. coli k88. moreover, the antibacterial mechanism of zinc oxide nanoparticles is still unclear. therefore, our study was undertaken to investigate the antibacterial activity against e. coli k88 in vitro. the atomic force microscope (afm) and scanning electron microscopy (sem) were used to study the mechanism. materials and methods characterization of zinc oxide nanoparticles zinc oxide nanoparticles were provided by institute of feed science, zhejiang university, zhejiang, china. the particle sizes of zinc oxide nanoparticles were determined by afm (afm, spm9500j3, shimadzu co., japan) and zetasizer nano-zs90 (malvern instruments) by the method of du et al. (2008). for afm, the zinc oxide nanoparticles suspended into water were placed onto cleaved mica and observed in contact mode with si3n4 probes. for zetasizer nano-zs90, the analysis was performed at a scanning angle of 90° at 25°c using samples diluted with water. the water used throughout this study was the reagent-grade water produced by milli-q sp ultra-pure-water purification system of nihon millipore ltd. (tokyo, japan). determination of minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) the minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were determined by a method recommended in nccls (2000) with some modifications. briefly, the sterile tubes were incubated aerobically at 37°c for 24 h, which contained 5 ml mullerhinton (mh) broth (difco, usa) with approximate 5×10 9 cfu bacterial cells and 0 (the control group), 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 µg/ml zinc oxide nanoparticles. the concentration of tube without visible growth of the bacterial cells was the mic. to evaluate the mbc, 100 µl of sample from each tube without visible growth was transferred into mh agar plate (difco, usa), and then incubated aerobically for another 24 h. the concentration of the tube without growth was the mbc (in this test, the population in agar plate less than 10 was regarded no growth). all the measures were triplicate. antibacterial effects on values of optical density (od) at 620 nm and the populations the sterile tubes contained 5 ml luria–bertani (lb) culture fluids, approximate 5×10 9 cfu bacterial cells and 0 (the control group), 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 µg/ml zinc oxide nanoparticles, and were incubated aerobically at 37°c for 24 h. then, samples were collected to measure the values of od at 620 nm and bacterial populations. values of od at 620 nm were determined using an ultra violet visible (uv-vis) spectrophotometer (hitachi ltd, tokyo, japan). a sample of 1 ml from each tube was serially diluted using phosphate buffer solution (pbs) for enumeration of bacterial populations. bacterial populations were enumerated on lb agar plate. all the measures were in triplicates. morphological changes of e. coli k88 afm (spm-9500j3, shimadzu co., japan) and sem (xl30-esem philips company, japan) were used to examine morphological changes of e. coli k88 treated with 0.8 µg/ml zinc oxide nanoparticles. for afm, the morphological changes were determined as described by du et al. (2008). briefly, the sterile tubes, which contained 5 ml, 0.8 µg/ml zinc oxide nanoparticles lb culture fluid and approximate 5×10 9 cfu bacterial cells, were cultured aerobically in a shaken thermostat (200 rmp/min) at 37°c. samples were collected from the tubes at 0 (the control), 0.5, 1.0, 2.0, 3.0, 4.0 h, dried on slices, and observed by afm in contact mode with si3n4 probes. for sem, samples were collected from the tubes at 0 (the control) and 0.5 h. the samples were immersed in the solution of 2.5% glutaraldehyde (merck em grade, auer bittman soulie ag, basel, switzerland) for 24 h and were washed by pbs. then, they were fixed by 1% oso4 solution for 2 h and dehydrated in a grade series of ethanol (50, 70, 80, 90, 95, 100, and 100%) for 15 min at each step. subsequently, the samples were treated with solution of ethanol and iso-amyl acetate(1:1) for 30 min, then treated with pure iso-amyl acetate for 2 h and dried by hitachi hcp-2 critical point dryer (hitachi ltd, tokyo, japan). the samples were then coated with gold using eiko ib-5 ion coater (eiko engineering co. ltd., tokyo, japan), after which, they were observed with sem. statistical analysis data were analyzed statistically by one-way analyses of variance (anova), using the spss statistical software package for windows (version 16.0, spss, chicago, usa). probability values below 0.05 were considered to be statistically significant. results and discussion particle size the primary sizes of zinc oxide nanoparticles are one of the important factors for the antibacterial activity (zhang et al., 2007a). in our study, zinc oxide nanoparticles were characterized by zetasizer nano-zs90 (malvern instruments) and afm. the sizes of zinc oxide nanoparticles were 75±20 nm as determined by zetasizer nano-zs90, which indicated that the zinc oxide nanoparticles in this study were small sized and could exhibit antibacterial activity of nanoparticles. images observed by afm are shown in figure 1. the images show that the zinc oxide nanoparticles were small sized, which were in line with the results of zetasizer nanozs90. moreover, figure 1b reveals that the morphologies of the zinc oxide nanoparticles were not quasi-spherical morphology, as reported by wu et al. (2010). the nonspherical morphology coming from the figure 1. afm images of mica surface (a) and zinc oxide nanopartcles (b). preparation method may influence the antibacterial activity, which could be studied in the future. antibacterial activity in this study, results of mic and mbc show that the mic and mbc were 0.1 and 0.8 µg/ml, respectively. the ratio of mbc/mic was 8. these results indicate that zinc oxide nanopaticles had antibacterial effects on e. coli k88, which is partly in accordance with the reports by soderberg et al. (1990), sawai (2003), hernandez-sierra et al. (2008), and liu et al. (2009). they reported that zinc oxide or its nanoparticles had antibacterial activity against staphylococcus aureus, streptococcus, e. coli 745 and e. coli o157:h7. however, the antibacterial concentrations were inconsistent. soderberg et al. (1990) revealed that 179 and 1790 µg/ml zinc oxide could exhibit a clear antibacterial effect on s. aureus. results of liu et al. (2009) indicated that 3 mmol/l zinc oxide nanoparticles could inhibit the growth of e. coli o157:h7 and 12 mmol/l or higher concentrations completely inhibited the growth. the main factors for the variable antibacterial concentrations may be that the different microbes and zinc oxide or its nanoparticles were used. interestingly, roselli et al. (2003) reported that 1 mmol/l zinc oxide (greatly exceeding the level of 0.8 µg/ml zinc oxide nanoparticles) did not affect the growth of e. coli k88, which partly explained the reports that zinc oxide nanoparticles exhibited stronger antibacterial activity than zinc oxide (yamamoto, 2001). it has been reported that the values of od at 620 nm can be an important indicator for bacterial growth (missotten et al., 2009; zhang et al., 2007b). therefore, effects of zinc oxide nanoparticles on values of od at 620 nm were measured and are shown in figure 2a, which indicated that 0.025 and 0.05 µg/ml zinc oxide nanoparticles did not affect the bacterial growth. compared with the control group, 0.1, 0.2, 0.4, and 0.8 µg/ml zinc oxide nanoparticles could significantly decrease the values of od at 620 nm, especially the 0.8 µg/ml. these results are consistent with the results of mic and mbc. figure 2a also reveals that the antibacterial activities increased as the concentration of zinc oxide nanoparticles increased, which are in line with results of brayner et al. (2006) and liu et al. (2009). effects of zinc oxide nanoparticles on the populations of e. coli k88 are shown in figure 2b, which indicates that compared with the control group, 0.05, 0.1, 0.2, 0.4 and 0.8 µg/ml zinc oxide nanoparticles significantly decreased the population of e. coli k88. these results are partly inconsistent with the results of the mic and effects on values of od at 620 nm, in which 0.05 µg/ml zinc oxide nanoparticles did not affect the bacterial growth. this difference may indicate that concentrations of zinc oxide nanoparticles in mh broth or lb media could affect the results of the mic and values of od at 620 nm. morphological changes of e. coli k88 the mechanisms of antibacterial activities of zinc oxide or its nanoparticles against some microbes have been explored. however, no study has been reported to investigate the mechanisms of antibacterial effects of zinc v al u e s o f o d a t 6 2 0 n m concentration of zinc oxide nanoparticles (μg/ml) co lo n ie s ex p re ss ed -f o rm in g u n it /m l) b a c t e r i a l 1 0 co lo n y n u m b e r o f (l o g concentration of zinc oxide nanoparticles (μg/ml) figure 2. effects of zinc oxide nanoparticles on values of od at 620 nm (a) and populations (b) of e. coli k88. data given represents means ± standard error of mean (n=3). oxide nanoparticles on e. coli k88. therefore, morphological changes were observed by afm and sem to investigate the preliminary mechanisms. the afm is a scanning probe technique. the probe allows imaging in any environment instead of only vacuum for conventional probes (binning et al., 1986). afm has been widely used in researches for yeast cells (touhami et al., 2003), biopolymers (morris et al., 2001), proteins (yan et al., 2003) and bacteria (kasas et al., 1994; doctycz et al., 2003; du et al., 2008; kailas et al., 2009). it is an ideal tool to determine the morphological changes of cells (camesano et al., 2000). in our present figure 3. afm images of e. coli k88 treated by zinc oxide nanoparticles for 0.0 (a), 0.5 (b), 1.0 (c), 2.0 (d), 3.0 (e), and 4.0 h (f). study, afm in contact mode was used to examine the morphological changes of e. coli k88. the images are shown in figure 3. figure 3a shows that e. coli k88 was a rod shape of about 1 µm wide and 2 µm long, and the membrane was intact. this result is similar to previous report (du et al., 2008). after 0.5 h of treatment with 0.8 µg/ml zinc oxide nanoparticles, the membrane of e. coli k88 was affected, and the boundary became blurry as shown in figure 3b. when treated for 1.0 h, the membrane of e. coli k88 was further affected, some cell contents leaked out of the cell and a lot of debris was found around the cell (figure 3c). results of the cells treated for 2.0 h are similar to the results for 1.0 h, as shown in figure 4. sem images of e. coli k88 without (a) and with (b) treatment of zinc oxide nanoparticles. figure 3d. after 3.0 h of treatment, the boundary of the cell was further damaged and almost disappeared, and only high concentrations of debris were left (figure 3e). when treated for 4.0 h, debris filled in the vision field instead of bacterial cells (figure 3f). in agreement with the afm image (figure 3a), sem image in figure 4a shows that most of the e. coli k88 in our present study were 1.0×2.0 µm rod shape and had no flagellum. however, after 0.5 h of treatment with 0.8 µg/ml zinc oxide nanoparticles, no intact cell was found as shown in figure 4b, which was different from the afm image (figure 3b). one of the reasons for no intact cell may be that the complicated treatments for sem images further damaged the structures of bacterial cells, and led them to debris. results of morphological changes show that zinc oxide nanoparticles could damage the membrane of e. coli k88, lead to the leakage of cytosolic components and kill the bacterial cells, which is partly consistent with the previous reports of sawai et al. (1996b, 1998), yamamoto (2001), stoimenov et al. (2002) and sawai (2003). they reported that zinc oxide could damage the membrane of bacterial cell by hydrogen peroxide or the affinity between zinc oxide and bacteria surface. from these studies, we preliminarily supposed that the mechanisms of antibacterial activities of zinc oxide nanoparticles against e. coli k88 were closely in line with those against other microbes. the unique characters of nanoparticles largely increased the surface of zinc oxide, or enhanced the affinity, so, zinc oxide nanoparticles could exhibit stronger antibacterial activity than zinc oxide (yamamoto et al., 1998; yamamoto, 2001). however, the concentration of nanoparticles should also exceed the mic to produce enough hydrogen peroxide or the affinity to damage the bacterial membrane. interestingly, results of sem images observed by liu et al. (2009) indicated that 12 mmol/l zinc oxide nanoparticles (the concentration which completely inhibited the bacterial growth) did not result in the morphological changes, which was inconsistent with our present results. reasons for the difference of the images are still unknown, which require further research. in addition, the mechanisms underlying the damage of bacterial membrane are yet to be studied. conclusion results in our present study indicate that zinc oxide nanoparticles had strong antibacterial activity against e. coli k88 and the activity increased as the concentration of zinc oxide nanopartices increased. the mechanisms may be that zinc oxide nanoparticles could damage the membrane, lead to the leakage of cytosolic components and kill the bacterial cells. in summary, our study reveals that zinc oxide nanoparticles could potentially be an antibacterial reagent to treat diseases caused by bacteria. in future, these nanoparticles might replace conventional antibiotics in 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administered with ascorbic acid during the hot-dry season o. o. asala1*, j. o. ayo2, p. i. rekwot3, n. s. minka4 and a. y. adenkola5 1 national veterinary research institute, p. m. b. 01, vom, plateau state, nigeria. 2 department of veterinary physiology and pharmacology, ahmadu bello university, zaria, kaduna state, nigeria. 3 national animal production research institute, shika, zaria, kaduna state, nigeria. 4 college of agriculture and animal science, ahmadu bello university, kaduna state, nigeria. 5 department of physiology and pharmacology, college of veterinary medicine, university of agriculture, makurdi, benue state, nigeria. accepted 09 november, 2016 experiments were performed in order to determine the rectal temperature (rt) responses of pigs to eight-hour road transportation and the effect of administration of ascorbic acid (aa) on the responses in transported pigs during the hot -dry season. twenty three experimental pigs were given aa orally and individually at 100 mg/kg prior to the transportation, while thirteen control pigs were administered with 20 ml of drinking water. the rts of the pigs were measured by inserting a clinical thermometer (hartman company, england) into the rectum via the anus. the ambient temperature (at) and relative humidity (rh) were measured concurrently with the rt. the at and rh values ranged between 23.5 39.0°c and 43 97% respectively. the values were outside the thermo-neutral zone for the pig, indicating that the season was thermally stressful. the at values, which fluctuated with the hour of the day and increased with the hour of the journey, were significantly (p < 0.001) and positively correlated with rt values in both experimental (r = 0.941) and control pigs (r = 0.942). the overall mean rt value of 39.8 ± 0.1°c recorded in the experimental pigs after 8 h road transportation did not differ (p > 0.05) with the corresponding value of 39.4 ± 0.3°c in control pigs, demonstrating that aa did not induce hypothermia in experimental pigs. in conclusion, transportation of pigs by road for 8 h in the zone induces hyperthermia and administration of aa in the experimental pigs did not induce hypothermia. optimum duration of road transportation of pigs is 6 h. this duration should be considered in the development of guidelines and welfare for pigs, transported by road during the hot-dry season. key words: transportation stress, rectal temperature, ambient temperature, hot-dry season, ascorbic acid, pigs. introduction it has been established that meteorological factors of high ambient temperature (at) and high relative humidity (rh), characteristic of the hot-dry season, cause heat stress which exerts adverse effects on livestock production (bianca, 1976; ayo et al., 1998a and b; renaudeau et al., 2007). during the hot-dry season *corresponding author. e-mail: ofcasala@yahoo.com. tel: +2348065690278. transportation of pigs is unavoidable, inspite of the unfavourable meteorological conditions prevailing during the season. of all the seasons prevailing in the northern guinea savannah zone of nigeria, the hot-dry season has been described as thermally stressful to livestock (igono et al., 1982; ayo et al., 1996, 1998a). efforts have been employed previously to ameliorate the adverse effects of road transportation stress in pigs these include the administration of hypnotics, sedatives (lopez-olevera et al., 2006) and tranquilizers (nyberg et al., 1988). administration of these drugs in transported pigs during the pre-slaughter period results in drug residues in the meat of such animals, especially in pigs that may be slaughtered compulsorily during the journey period as a result of stress (peeters et al., 2006). an increasing body of evidence has shown that free radicals are generated in poultry subjected to heat stress (sahota and gillani, 1995; altan et al., 2003; sahin et al., 2004), resulting in depletion of the antioxidant, vitamin c (ascorbic acid, aa) in tissues. tauler et al. (2003) and williams et al. (2008) showed that antioxidants in the body decrease or are overwhelmed due to exercise stress. road transportation stress in the body is ameliorated by administration of aa in goats (minka and ayo, 2007a and b) and pullets (ayo et al., 2006; minka and ayo, 2008). aa is a potent antioxidant vitamin that is readily available, relatively inexpensive and with virtually no toxic or residual effects (padayatty et al., 2003; tauler et al., 2003; sahin et al., 2004; peeters et al., 2006), and it is readily metabolized in the body (chervyakov et al., 1977). an important index in the evaluation of stress in domestic animals is rectal temperature (rt) (bianca, 1976; ayo et al., 1998a; sinkalu et al., 2008; adenkola et al., 2009). it is readily measured and shows the extent of thermal equilibrium in animals (bianca, 1976). the aim of the present study was to investigate fluctuation in rt responses in pigs transported by road during the hot-dry season, and the influence of aa administration on the fluctuation. materials and methods the experiment was conducted at the animal research pen of the faculty of veterinary medicine, ahmadu bello university, zaria (11° 10 n, 07° 38 e), located in the northern guinea savannah zone of nigeria. transportation of the pigs was carried out during the hotdry season in may, 2007 from zaria (11° 10 n, 07° 38 e), to pambegua (10° 4' n, 08° 16' e) and from pambegua back to zaria, covering a total distance of 400 km. the zone is characterized by three major seasons, namely: the hot-dry, rainy and harmattan seasons. of the three seasons, the harmattan has been described as the most thermally stressful to livestock (ayo and oladele, 1996; igono et al., 1982). this period marks the peak of the hot-dry season, just before the onset of the rainy season in the zone. animals and management twenty three (23), apparently, healthy adult local pigs comprising non-castrated males, non-pregnant and noncycling females of about one-year-old and weighing between 40 50 kg served as subjects. the pigs were purchased from samaru, zaria four weeks before commencement of the experiment. they were housed in the animal pen of the faculty of veterinary medicine, ahmadu bello university, zaria. the pen was made of concrete floor, cements block wall and roof, and consisted of two chambers. the pigs moved freely within the pen. they were managed intensively and fed a standard compounded feed, consisting of a mixture of maize bran and rice husk. water was given ad libitum. the pigs were preconditioned for two weeks before commencement of the experiment, and they were dewormed using piperazine hydrochloride (everchem, china) at a dose of 110 mg/kg orally. they were also treated against ectoparasites using ivermectin r (merial, france) at a dose of 0.05 mg/kg subcutaneously. experimental design after the two-week period of pre-conditioning, each pig was identified by a numbered neck collar. a week to the day of commencement of transportation, pigs were assigned experimental (group i) and control (group ii) groups consisting of 13 and 10 pigs respectively. neck collars already numbered (123) were put in a bag and picked ‘blindly’ one at a time without replacement. each collar picked was placed on the neck of individual pig caught at random. animals with collar numbers 1 13 were classified as experimental (group i) while those with collar numbers 14 23 were classified as control (group ii). on the day of transportation, 30 min before transportation, pigs in group i, which served as experimental animals were administered orally with 100 mg/kg body weight of aa (archy pharmaceuticals, nigeria; nafdac no: 04-5270, batch no:vcw 7024) dissolved in 20 ml of water. the 10 pigs in group ii which served as control were administered with 20 ml of distilled water each orally. feed and water were withdrawn 12 h before and throughout the journey period, which lasted eight hours. all the pigs were transported on asphalt roads at an average speed of 50 km/h. after the completion of the journey, the pigs were unloaded at the same spot from where they were loaded previously. feed and water were then provided ad libitum. vehicle design and loading a standard bedford van (made in england) and popularly used in the northern guinea savannah zone of nigeria for transportation of pigs was used for the journey. the body of the vehicle was made of aluminium, and the floor of steel. the inner compartment of the vehicle measured 5.5 x 1.5 m. the side walls of the body of the vehicle to a height of 1.1 m were made of aluminium and above which had windows for ventilation. the vehicle had at its rear end a twin door with a rear wind screen. the upper side walls and roof are also covered with aluminium. the floor of the vehicle was covered with dry straw and covered with a thick rubber mat for secure footing. transportation procedures were carried out according to the guidelines governing animal transport welfare by road as described by (knowles et al., 1998). briefly, pigs were loaded individually by two persons in a relatively calm condition. one person easily caught a pig at a time and carried it to the other person, who already was in the vehicle. the pigs were stocked at a rate of 0.3 m 2 /pig. measurement of meteorological parameters meteorological parameters of ambient temperature (at) and relative humidity (rh) were recorded using the wet and dry-bulb thermometer (ellab inc, u.s.a.). the dry bulb temperature (dbt) and rh were recorded at the experimental site at 06:00, 13:00 and 18:00 h every other day for 5 days before the commencement of the journey. the parameters were also measured just before transportation and during the journey at 30 minutes, 2, 4, 6 h and finally at 8 h, when the journey was completed. post-transportation, dbt and rh were recorded at 3 h after unloading and on days 1, 2 and 3. measurement of rectal temperature two weeks before commencement of the journey, the rt was determined every other day, during the measurement of the table 1. meteorological data from the study period before and after transportation. ambient temperature (°c) relative humidity (%) time, h before after before after 06:00 23.5 ± 0.4 22.0 ± 0.5 73.0 ± 8.0 80.3 ± 4.9 13:00 37.0 ± 1.0 29.6 ± 0.3 55.8± 6.0 56.6 ± 1.6 18:00 35.6 ± 1.0 28.0 ± 3.0 71.2 ± 6.0 61.5 ± 11.5 overall mean ± sem 32.0 ± 4.2 26.5± 2.3 66.7 ± 5.4 66.1 ± 7.2 table 2. ambient temperature and relative humidity inside the vehicle during the journey period. hour of journey, h dry-bulb temperature (°c) relative humidity (%) 0.5 30.5 68.0 2 33.0 57.0 4 37.0 53.0 6 36.5 40.0 8 39.0 71 mean ± sem 34.0±1.7 59.3±4.7 meteorological parameters, at 06:00, 13:00 and 18:00 h for a total of 5 days. on the experimental day, the rt was measured just before loading the pigs into the vehicle. thereafter as the vehicle kicked off, the rt was determined at 30 min, 2, 4, 6 and 8 h of the journey. on arrival, the rt was also measured after 3 h with the meteorological parameters after unloading and, subsequently, at 06:00, 13:00 and 18:00 h for 3 days consecutively posttransportation. all measurements were carried out in a relatively calm condition, and each pig was restrained lightly and properly for the measurements. the rt was recorded as an indicator of the body temperature using a digital clinical thermometer (the hartman’s company, plc, england). the thermometer was inserted 5 cm deep into the rectum of each pig via the anus (zaytsev et al., 1971) until an alarm sound was heard, indicating the end of the reading. statistical analysis all data are expressed as mean ± sem. data were analysed using student’s t-test and pearson’s correlation analysis. values of p < 0.05 were considered significant. results meteorological data the meteorological data are shown in tables 1 and 2. the at and rh recorded at the experimental site during the study period ranged between the maximum value of 39.0°c to the minimum value of 23.5°c, with a range of 16.5°c. the rh at the experimental site before transportation ranged between 43 97%. the dry bulb temperature (dbt) was highest at 13:00 h with mean value of 37 ± 1.0°c (table 1). during the journey period, the dbt inside the vehicle rose gradually from 30.5°c, 30 min into the journey and attained a peak value of 39.0°c at the 8 h of the journey with a mean of 34.0 ± 1.7 o c, while the rh ranged between 40 71% with a mean value of 59.3 ± 4.7%. thus, the at and rh had wide range values of 8.5°c and 29%, respectively during the journey period (table 2). the at ranged posttransportation between 21 31°c, while rh maximum and minimum values were 89 and 50% respectively (table 1). rectal temperature of pigs before the journey the rt of the experimental pigs measured for five days pre-transportation was lowest at 06:00 h and highest at 18:00 h, with the values of 36.8 ± 0.1 and 39.0 ± 0.1°c, respectively. the rt of the control pigs was also lowest at 06:00 h and highest at 18:00 h, with the values of 36.8 ± 0.1 and 38.9 ± 0.1°c, respectively. there was no significant difference (p > 0.05) between mean rt values of the experimental and control pigs pre-transportation, but the values were significantly different (p < 0.05) within the experimental and control groups between 06:00 h and 13:00 h, and between 06: 00 h and 18: 00 h (table 3). the hour of the day, maximum and minimum at values were positively correlated with rt values, both in experimental and control pigs before transportation. however, the rh was negatively correlated with rt in all the pigs before and after transportation. table 3. rectal temperature (°c) of experimental (13) and control (n = 10) pigs before and post transportation. pre-transportation post-transportation time, h experimental control experimental control 06:00 36.8 ± 0.1 36.8 ± 0.1 36.4 ± 8.0 36.4 ± 0.1 13:00 38.8 ± 0.1 38.7 ± 0.1 37.9 ± 0.2 37.6 ± 0.2 18:00 39.0 ± 0.1 38.9 ±0.1 38.3 ± 0.4 38.1 ± 0.4 overall mean ± sem 38.2 ± 0.7 38.1± 0.6 ns 37.5 ± 0.6 37.3 ± 0.5 ns ns = non significant (p > 0.05) difference. table 4. rectal temperature (°c) of experimental (n=13) and control (n=10) pigs during 8 h road transportation. mean±sem maximum minimum hour of the journey experimental control experimental control experimental control 0.5 39.1 ± 0.3 38.7 ± 0.1 41.0 39.6 38.4 38.2 2 39.1 ± 0.1 38.8 ± 0.1 39.8 39.5 38.6 38.3 4 39.5 ± 0.1 39.3 ± 0.1 39.8 40.0 39.0 38.9 6 40.3 ± 0.1 39.9 ± 0.1 40.7 40.6 40.0 39.3 8 40.9 ± 0.1 40.5 ± 0.1 41.2 41.4 40.5 40.1 overall mean±sem 39.8 ± 0.1 39.4 ± 0.3 ns 40.5 ± 0.2 40.2 ± 0.3 ns 39.3 ± 0.4 38.9 ± 0.3 ns ns = non significant (p > 0.05) difference. rectal temperature of the pigs during the 8 h road transportation during the journey period, the mean rt value fluctuated between 39.1 ± 0.3 and 40.9 ± 0.1°c in experimental pigs with an overall value of 39.8 ± 0.3°c, while in the control pigs the values fluctuated between 38.7 ± 0.1 and 40.5 ± 0.1°c. the overall mean value of 39.4 ± 0.3°c recorded in the control pigs were not significantly different (p > 0.05) from that of the experimental pigs. although the mean maximum and minimum rt values in the experimental pigs (40.5 ± 0.2 and 39.3 ± 0.4°c, respectively) were slightly higher than the corresponding values obtained in the control pigs (40.2 ± 0.3 and 38.9 ± 0.3°c, respectively), they were not significantly different (p > 0.05) (table 4). the rt values rose with duration of the journey both in experimental and control pigs. in the experimental pigs, the values rose (p < 0.001) from 39.1 ± 0.3°c after 30 min to 40.9 ± 0.1°c at the 8 th h of the journey. the rt value in the control pigs rose (p < 0.001) from 38.7 ± 0.1 39.4 ± 0.3°c at the 8 th hour of the journey. the dbt increased significantly (p < 0.001) and was positively correlated with the rt values. fluctuations in rectal temperature of pigs posttransportation the mean overall rt value three hours after the journey in the experimental pigs was 38.5 ± 0.2°c. the value was not higher (p > 0.05) than that of 38.1 ± 0.2°c recorded in control pigs. there was no significant difference between the mean values of rt in both experimental and control pigs recorded for three days post-transportation (table 3). discussion meteorological data pre-transportation the meteorological data obtained in the present study indicated that the dbt, which ranged between 23.5 39.0°c, was predominantly outside the thermoneutral zone of 12 25°c established for the pig in the temperate regions (bianca, 1976; bazhov and komlatsky, 1989). thus, the dbt recorded pre-transportation of the pigs was not optimum for their normal thermoregulation. similarly, the rh, which fluctuated between 43 97%, was outside the upper limit of the established normal rh values of 45 75% for the pig (agricultural research council, 2006), indicating that the rh which the pigs were subjected to was also not conducive. the dbt and rh values recorded from the study period were characterized by high values, established to induce heat stress in pigs (bazhov and komlatsky, 1989; ayo et al., 1998b; vecerek et al., 2006). thus, the conditions prevailing during the hotdry season in the northern guinea savannah zone of nigeria are not optimum for the rearing of pigs. the meteorological conditions showed that transportation of pigs during the hot-dry season was thermally stressful and that such transported pigs are inevitably subjected to concurrent actions of both heat and transport stresses. measures aimed at alleviating these stresses were, therefore, taken into consideration in the present study through the admini-stration of an antioxidant, aa demonstrated by ayo et al. (2005, 2006) in pullets and minka and ayo (2007a,b) in goats to alleviate transportation stress. meteorological data during and post-transportation the results of the dbt and rh obtained at the experimental site before and after the transportation of the pigs were not significantly different. this finding indicated that the dbt and rh values from the study period at the experimental site were similar, and that they exerted similar effects before and after the journey on the responses of the pigs to the transportation stress. there were considerable changes in the dbt values during the journey period, which rose from 30.5 o c at the first hour of the journey to a peak value of 39.0 o c at the 8 th hour of the journey when the transportation was completed. the dbt values were outside the established thermoneutral zone for the pig (14 25 o c) (bazhov and komlatsky, 1989). this finding indicated that the at prevailing during the transportation was also unfavourable to the pigs. similarly, there was a wide fluctuation in the rh values during the journey. the high fluctuation (40 71%) with a range of 31% was outside the normal range values of rh for the pigs (45 75%) (a.r.c., 2006). during the journey, the high overall at and rh values obtained have been shown to be thermally stressful to the pig (bianca, 1976; bazhov and komlatsky, 1989) . the at and rh values obtained in the present study were not significantly different from the corresponding values of 31.8 ± 2.6% and 65.2 ± 6.4 %, respectively obtained during the transportation of goats in the hot-dry season in the northern guinea savannah zone of nigeria (minka and ayo, 2007a). the mean maximum at value of 34.0 ± 1.7°c obtained in the present study was not significantly different from that of 34.1 ± 1.5°c recorded during the sixhour road transportation in pullets in the hot-dry season (minka and ayo, 2008). the rh value of 59.3 ± 4.7% obtained in the present study differed from that of 44.5 ± 1.4% obtained by minka and ayo (2008) during road transportation of pullets. the difference in the results on the rh value may be due to many factors including meteorological conditions, differences in species of animals used and the duration of the journey. this finding suggests that species variation may play some role in modulating the microclimatic conditions inside the vehicle during road transportation. the results further confirmed that the hot-dry season was thermally stressful to livestock during transportation by road in northern nigeria. thus, for pigs to be transported successfully during the season and perform optimally, efficient thermoregulatory (homeostatic) mechanisms must be switched on. rectal temperature of pigs five days pretransportation the rt values in both experimental and control pigs showed diurnal fluctuations, and this finding is in agreement with those of ayo et al. (1998a, 1998b), piccione and caola (2002) and sinkalu et al. (2008). according to piccione and caola (2002), such fluctuations are driven by a biologic clock in the mammalian brain. the overall mean and maximum rt values obtained in experimental and control pigs before the transportation fell within the established normal range of 38 40°c (zaytsev et al., 1971). however, the overall mean minimum values of 36.6 ± 0.8°c and 36.8 ± 0.7°c obtained in the control and experimental pigs, respectively were completely below the normal range of rt, established for pigs in temperate regions of the world. the rt in the pigs fluctuated between 36.6 and 39.8°c, with a range value of 3.2°c. according to zaystev et al. (1971), such a wide range in rt values (above 1.0 1.8°c) impairs body functions and may result in heat stroke. thus, the responses of the pigs demonstrated that the meteorological conditions prevailing during the hotdry season in the zone are not conducive both for rearing and road transportation of pigs. however, the animals, by switching on the homeostatic mechanisms, maintained their rt values within the normal range of rt values in the pig. furthermore, additional stress factors acting upon the pigs may completely impair their body functions and aggravate the incidence of road transportation-induced diseases. thus, the relatively low rt values of about 37.0°c, obtained in experimental and control pigs, may be a component of the adaptive and compensatory responses of the transported animals to the thermally stressful hot-dry season in the zone. rectal temperature of pigs during eight hours of road transportation the mean rt values obtained during the journey period rose (p < 0.001) with the hour of the journey in the experimental (r = 0.968) and control pigs (r = 0.987), and attained the peak at the 8 h when the journey was completed. the results clearly indicated that the journey time plays a crucial role in the rt values of transported pigs. the results showed that for normal thermoregulatory mechanisms to be maintained in the pig, the journey duration should be limited to six hours. this is because the mean rt values were maintained within the normal range in the pigs till the 6 th hour. thereafter, thermoregulatory mechanisms of the transported pigs were inadequate to maintain the rt values within the normal range. the results further showed that transportation of pigs within the zone is recommended to be conducted within 6 h. if the need arises for the animal to be transported above this duration, measures aimed at enhancing the relative maintenance of the rt values within the normal physiologic range must be ensured. it is worth noting that in the present study the rt values in both experimental and control pigs were not significantly (p > 0.05) different, indicating that aa did not exert a hypothermic effect on transported pigs during the hot-dry season. this finding contradicts the results of studies conducted by ayo et al. (2005) in pullets, ayo et al. (2006) and minka and ayo (2007a) in goats, and minka and ayo (2008) in pullets, who showed that aa exerted a hypothermic effect in livestock transported by road during the hot-dry season. the differences in the results may be due to species variation, and the fact the doses given to the animal species varied. this observation requires further investigation. furthermore, the study showed the need to increase the dose of aa in order to establish its anti-stress effect on transported pigs during the hot-dry season. this again requires further investigation. rectal temperature of pigs post-transportation the overall mean and the mean maximum rt obtained three days after the journey were within the normal range of rt values. the result agreed with the findings of minka and ayo (2007a, b) that rt values of transported animals were restored to the normal values shortly posttransportation. the results also showed that transportation of pigs by road for 8 h induced a transient stress, and that the rising of the rt above the normal value was short-lived. the overall mean and mean maximum rt values obtained three days posttransportation in both experimental and control pigs showed that the values were significantly reduced following road transportation. the transportation depressed the thermal responses in the pigs after the journey and even below the initial (pre-transportation) values. this finding agreed with that of ayo et al. (1998b) that road transportation in pigs induces an alarm response in the animals during the journey, which is later replaced by a stage of resistance. the stage of resistance observed in the present study was characterized by strong adaptation of the pigs, not only to the transportation stress, but also the heat stress characteristic of the hot-dry season in the northern guinea savannah zone of nigeria. conclusion in conclusion, transportation of pigs by road for eight hours in the zone induces hyperthermia and administration of ascorbic acid at 100 mg/kg in the experimental pigs did not exert any significant ameliorative effect. journey time and meteorological factors (at and rh) play significant role in rectal temperature responses of pigs transported by road. optimum duration of road transportation of pigs is six hours. this duration should be considered in the development of guidelines and welfare for pigs, transported by road during the hot-dry season. references adenkola ay, ayo 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animals. kolos publishing house, moscow p. 336 (in russian). 7 in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (5), pp. 001-011, may, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper effects of l-arginine on intestinal development and endogenous arginine-synthesizing enzymes in neonatal pigs lin huang1,2, zong-yong jiang2*, yin-cai lin2, chun-tian zheng2, shi-kui wang2, xue-feng yang1,2 and guo-yao wu3 1 college of animal science, south china agricultural university, guangzhou 510642, p. r. china. 2 key laboratory of animal nutrition and feed (south china), ministry of agriculture of p. r. china, institute of animal science, state key laboratory of livestock and poultry breeding, guangdong academy of agricultural sciences, guangzhou 510640, p. r. china. 3 department of animal science, texas a&m university, college station 77843, usa. accepted 07 november, 2017 this study aimed to investigate the effects of dietary l-arginine supplementation on the intestinal development of neonatal piglets and the underlying mechanisms. 36 neonatal piglets were randomly allocated into three diet groups: control group (supplemented with 0% l-arginine), 0.4 and 0.8% l-arginine groups. when compared with the control, dietary supplementation with l-arginine decreased (p<0.05) blood urea nitrogen (bun), and improved (p<0.05) serum t3 and insulin level of the piglets on day 11. arginine and its metabolites (citrulline and ornithine) were elevated, additionally, dietary supplementation with 0.8% l-arginine markedly enhanced jejunal villus height, villus area on day 11 and d-xylose absorption rate on day 19. dietary supplementation with 0.8% l-arginine increased (p<0.05) activities of maltose and lactose on day 18, respectively. this effect correlated with profound change in enzyme activities as inducible nitric oxide synthetase (inos), glutamine synthetase (gs) and ornithine decarboxylase (odc) were elevated on day 18. the concentrations of spermine was increased (p<0.05) by l-arginine supplementation on day 18. these results collectively suggest that dietary l-arginine supplementation improves protein synthesis and intestinal development of the neonatal pigs, the underlying mechanism includes dietary l-arginine supplementation which regulated the productions of intestinal polyamine in jejunum, and stimulated endogenous arginine-synthesizing enzymes in neonatal piglets. key words: neonatal pig, l-arginine, intestinal development, arginine-synthetases. introduction early weaning has been reported to correlate with villus atrophy, which depresses feed intake and growth performance in piglets (kelly et al., 1991; ou et al., 2007). adequate supply of nutrients from blood and intes-tine ensured the optimal development of villus (matheson et al., 2000). the small intestine was suggested to be the major organ to synthesize arginine in neonatal animals (wu et al., 1995). low arginine intake associated with * corresponding author. e-mail: jiangz38@gmail.com tel: +86 020 87596262. fax: +86 020 87503358. depresssed feed intake may be a major reason for increased intestinal epithelial damage in early-weaned pigs. previous studies indicated that arginine was strikingly deficient in milk-fed piglets, as the relatively low contents of arginine were found in sow’s milk (wu et al., 2000; o’quinn et al., 2002). arginine was therefore considered an essential amino acid for the optimal growth of neonatal pigs (wu and knabe, 1994, 1995; flynn et al., 2000; wu et al., 2004). dietary supplementation of l-arginine improved the growth of neonatal pigs (kim et al., 2004), mainly due to its beneficial effects in enhancing wound healing and angiogenesis, improving protein table 1. composition and nutrient contents of the basal diet (on an as-fed basis). ingredient (%) ratio (%) whole milk power (23% cp) 50.84 whey protein concentrate (34% cp) 22.00 plasma protein powder (78% cp) 5.00 whey power (4% cp) 14.32 coconut oil 5.00 l-alanine (98%) 1.67 dl-methionine (98%) 0.08 l-threonine (98%) 0.14 l-tryptophan (98%) 0.02 liquid chloride choline (70%) 0.12 vitamin-mineral premix 1 0.5 emulsifying agent 0.11 calculated composition crude protein (%) 26.34 digestible energy (mj/kg) 19.25 fat (%) 15.80 lactose (%) 32.20 calcium (%) 1.10 total phosphorus (%) 0.70 lysine (%) 2.20 methionine (%) 0.50 arginine (%) 0.72 1 providing the following (mg/kg powder diet): fe (as feso4.7h2o), 105; cu (as cuso4.5h2o), 10; mn (as mnso4.h2o), 5; zn( as znso4.7h2o), 110; retinyl palmitate, 13.2; cholecalciferol, 1.32; all-rac-α-tocopheryl acetate, 96; menadione sodium bisulfite, 1.50; biotin, 0.24; folic acid, 0.90; nicotinic acid, 60.0; calcium pantothenate, 36; riboflavin, 12; thiamin, 4.5; pyridoxine, 6; cyanocobalamin, 0.06. anabolism and immune functions as suggested by study in mice (cremades et al., 2004) and human (barbul et al., 1990). biochemically, arginine plays an important role as a substrate for protein synthesis, intermediates in the hepatic urea cycle, as well as precursors for the synthesisof various important metabolic molecules, including nitric oxide (no), polyamines and creatinine (wu and morris 1998; kim et al., 2007; flynn et al., 2002; cynober et al., 1995). wu and colleague suggested that l-arginine supplementation may boost the growth of young piglet possibly by enhancing the synthesis of no, proline and polyamines in animals (wu et al., 2004; kim et al., 2004). however, it remains unclear whether l-arginine supplementation has other effects besides these metabolic pathways and higher dosage of l-arginine may lead to beneficial effect or detrimental effect. in an attempt to unveil the underlying mechanisms of larginine supplementation and young piglet growth, we carried out this study with wider range of dosages and started during early neonatal period. we found that dietary arginine supplementation can prevent intestinal atrophy in early-weaned pigs, in addition to the impact on nitrogen metabolism as reported previously. the dosagedependent data suggested that higher arginine supplementation has more substantial effect on some but not all aspects of analysis, which may be of potential interest in applying such practice in livestock industry of china and the world. materials and methods experimental procedures in this study were approved by animal experimental committee of guangdong institute of animal science. 36, 4-day-old healthy male neonatal piglets (duroc, landrace x largininee white) were randomly assigned to three treatment groups, with four replicates of three piglets each. every replicate of piglets were housed in a pen in an air-conditioned room with ambient temperature of 32 ± 1°c and constant humidity. the basal milk-powder diets were formulated to meet required nrc 1998 minimal levels for 3 to 5 kg piglets (table 1). levels of exogenous l-arginine supplemented in diets of the three treatments were set at 0 (control group), 0.4 and 0.8% (on the basis of milk replacer powder). appropriate amounts of alanine were added to formulate isonitrogenous diets. the diets were mixed with water at a ratio of 1:4 freshly before feeding, and provided to the piglets every 3 h from 06:00 a.m and 12:00 p.m. piglets had easy access to water supplied by the semiautomatic device. experimental piglets were observed to become accustomed to the feeding method quickly and consumed each meal with no spillage. feed intake (as-fed basis) was calculated in accordance with the weight difference in feed trough before and after feeding. body weights (bw) of all piglets were individually measured on day 1, 11 and 18 of the experiment. average daily gain (adg), average daily feed intake (adfi) and feed : gain (f : g) ratio were calculated. sample collection blood samples of all piglets were obtained from anterior vena cava and both serum and plasma were separately kept for further determination on day 11 and 18 of the experiment. respectively, on day 11 and 18 of the experiment, one pig out of each experimental replicate was randomly selected and slaughtered after intraperitoneal injection of sodium pentobarbital (50 mg/kg bw). the small intestine from the pyloric sphincter to the ileocolonic junction was rapidly removed by cutting along the mesenteric border on an ice-cold metal plate and divided into three segments. the segment of small intestine proximal to the ligament of treitz was designated as the duodenum, with the stomach being removed. the remainder of the small intestine was divided into 2 equal portions; the proximal half was designated the jejunum and the distal half, the ileum (burrin et al., 2000). from the midline of each region, a 10 cm piece was slit along its length and mucosa was removed by gentle scraping with a glass slide. these samples were rapidly frozen in liquid nitrogen for analysis of the activities of disaccharidases (maltase, lactase and sucrase). on day 19 of the experiment, a d-xylose test was conducted on the remaining piglets. d-xylose was orally administrated to the piglets at dose of 1 ml/kg bw, blood samples were collected from anterior vena cava 2 h later for the determination of d-xylose. analysis procedures blood hormones (insulin, igf-1, t3 and t4) were measured through table 2. effect of dietary l-arginine supplementation on growth performance of piglets*. item control 0.4% arginine 0.8% arginine body weight (kg) day 1 2.07±0.01 1.99±0.07 2.08±0.08 day 11 3.28±0.12 3.36±0.04 3.37±0.03 day 18 5.54±0.32 5.79±0.09 5.76±0.19 daily weight gain (kg/d) days 1 to 11 0.12±0.01 0.14±0.01 0.13±0.01 days 12 to 18 0.32±0.02 0.34±0.01 0.34±0.11 days 1 to 18 0.22±0.01 0.24±0.01 0.24±0.04 daily feed intake (kg/d) days 1 to 11 0.12±0.01 0.12±0.01 0.12±0.01 days 12 to 18 0.26±0.01 0.26±0.02 0.27±0.04 days 1 to 18 0.19±0.01 0.19±0.01 0.19±0.01 feed : gain ratio days 1 to 11 0.99±0.12 0.86±0.03 0.91±0.02 days 12 to 18 0.82±0.05 0.75±0.05 0.79±0.02 days 1 to 18 0.86±0.04 0.78±0.04 0.82±0.01 *data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). radio-immuno method using reagent kits (tianjin jiuding bioengineering co. ltd). blood urea nitrogen (bun) was determined by automatic biochemistry analyzer (beckman instruments, fullerton, ca) at 340 nm using the assay kits from beckman coulter inc (fullerton, ca). plasma concentrations of arginine, glutamate, proline, glutamine, citrulline, ornithine, lysine, threonine, valine, histidine and alanine were analyzed by aminoacid autoanalyzer (l-8900, hitachi). nitric oxide synthetase (nos), no, disaccharidases and serum d-xylose were determined using colorimetric methods with a spectrophotometer (biomate 5, thermo electron corporation, rochester, ny). the assays were conducted using the assay kits purchased from nanjing jiancheng institute of bioengineering (nanjing, jiangsu, china) and the procedures were followed accordingly. activities of argininosuccinate synthetase (ass), pyrroline-5-carboxylate synthetase (p5cs) and spermine were determined using swine enzyme-linked immunosorbent assay kits (adlitteram diagnostic laboratories, usa). small intestinal morphology statistical analysis data were presented as means ± sd. statistical analyses were performed using the general linear model procedures of spss (version 11.5, spss institute). statistical comparisons were done by anova followed by the post-hoc newman–keuls multiple range test. difference was considered significant when p value <0.05. results growth performance the effect of dietary l-arginine supplementation on growth performance is summarized in table 2. when compared with the control group, arginine supplementation did not improve bw, adg and adfi, but slightly decreased f : g ratio of the piglets throughout the experiment (p>0.05). two centimeters segment of the jejunum was collected immediately after slaughter and processed as previously described (jensen et al., 2001). in brief, the samples were fixed with 10% neutral buffered formalin for 24 h, and trimmed to prepare paraffinembedded block for histological slides. three cross sections (5 µm thick) of each intestinal segment were stained with hematoxylin and eosin following standard protocol. the 10 straightest villus and their associated crypts from each segment were quantified. the villus height was measured from the tip to the base, and then the crypt depth was measured from the base of the villus to the base of the crypt. the villus area was calculated according to the published method (frankel et al., 1993). blood urea nitrogen (bun) bun levels of the piglets of 0.4 and 0.8% arginine groups were 39.4 (p<0.05) and 18.3% (p<0.05), both lower than that of the control group on day 11 (figure 1). no obvious difference was observed among bun contents of the three groups at day 18 (figure 1). blood hormones blood t3 levels of piglets obviously increased with b u n ( m m o l/ l ) 4.5 a control 4 ab 0.4% arginine 0.8% arginine 3.5 3 b 2.5 2 1.5 1 0.5 0 day 11 day 18 figure 1. effect of dietary arginine supplementation on serum urea nitrogen levels of piglets. bars represent the means ± sd. within an experimental phase, means without a common letter differ (p<0.05). table 3. effect of dietary l-arginine supplementation on serum hormone levels of piglets*. item control 0.4% arginine 0.8% arginine t3 (ng/ml) day 11 0.43±0.02 a 0.81±0.06 b 0.90±0.13 b day 18 0.64±0.08 a 0.97±0.05 b 0.87±0.02 b t4 (ng/ml) day 11 55.03±2.23 53.95±2.84 64.16±3.91 day 18 50.44±3.57 44.35±3.60 49.78±2.76 igf-1 (ng/ml) day 11 8723.2±1254.1 9511.6±2703.1 9494.8±957.3 day 18 12858.4±548.8 14166.6±752.0 13158.9±593.8 insulin day 11 8.46±1.46 a 21.24±3.28 ab 25.79±7.04 b day 18 6.93±0.43 7.51±0.76 6.73±1.53 *data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). arginine supplementation in contrast to those of control piglets on both days 11 and 18 (p<0.05; table 3). no significant difference in blood t4 or igf-1 content was seen among groups at either experimental phase, although, arginine supplementation did tend to increase blood igf-1 at both experimental periods. 0.8% arginine group possessed prominently higher level of blood insulin than the control on day 11 (p<0.05). small intestinal morphology jejunal villus height (p<0.05) and villus areal (p<0.05) of piglets were significantly higher in 0.8% arginine group than the control group on day 11. jejunal crypt depth was notably higher in 0.4% arginine group than the other two groups on day 18 (p<0.05, table 4). d-xylose absorption two hours after oral gavage of d-xylose, contents of plasma d-xylose of 0.4 and 0.8% arginine groups increased by 200 (p<0.05) and 140% (p<0.05), respectively than the control group (figure 2). table 4. effect of dietary l-arginine supplementation on jejunum histomorphology of piglets*. item control 0.4% arginine 0.8% arginine villus height (µm) day 11 364.9±19.3 a 414.1±21.6 ab 464.7±11.6 b day 18 452.7±20.3 501.0±33.3 479.2±41.5 crypt depth (µm) day 11 160.9±16.8 181.9±8.5 184.5±3.0 day 18 133.6±4.6 a 181.4±6.3 b 154.0±9.7 a villus areal (µm 2 ) day 11 138702±7244 a 180169±14027 ab 205545±15023 b day 18 184193±12304 187220±11190 167949±8587 *data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). p la s m a d -x y lo s e ( m m o l/ l ) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 b control 0.4% arginine b 0.8% arginine a day 19 figure 2. effect of dietary arginine supplementation on intestinal permeability of piglets. bars represent the means ± sd. within an experimental phase, means without a common letter differ (p<0.05). levels of jejunal disaccharidases in contrast to the control and 0.4% arginine groups, the activity of maltases in jejunal mucosa was notably elevated (p<0.05) in 0.8% arginine treatment on day 18 (table 5). both 0.4 (p<0.05) and 0.8% (p<0.05) arginine groups showed at least 2-fold higher contents of lactase in jejunal mucosa than the control group on day 11. strikingly, the enhancing effect of arginine decreased on day 18, although, there was still significant effect between two arginine supplementation groups and the control group. additionally, 0.8% arginine group also showed significant increase as compared to 0.4% arginine group. concentrations of jejunal mucosa sucrase did not differ among the three groups on day 11 as well as day 18. sucrase level significantly increased from 0.88 on day 11 to 3.55 on day 18 in the control group, while this change was not observed in arginine group with almost the same level of sucrase from day 11 to 18. concentration of plasma amino acids in comparison with the control group, 0.4 and 0.8% table 5. effect of dietary l-arginine supplementation on disaccharidases activities in jejunal mucosa of piglets (u/mg prot)*. item control 0.4% arginine 0.8% arginine maltases d 11 5.21±2.14 4.81±1.25 7.50±2.27 d 18 9.77±0.75 a 9.78±1.29 a 13.36±1.29 b lactase d 11 5.26±1.49 a 22.67±7.02 b 20.20±4.97 b d 18 3.41±1.12 a 4.66±0.84 a 6.94±1.2 b sucrase d 11 0.88±0.38 3.70±1.52 4.43±1.56 d 18 3.55±0.19 2.49±0.74 2.42±0.83 * data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). table 6. effect of dietary l-arginine supplementation on concentrations of plasma amino acid in piglets*. item control 0.4% arginine 0.8% arginine arginine day 11 57.44±5.06 a 89.08±6.96 b 92.17±10.05 b day 18 72.75±4.66 a 129.00±21.59 b 137.67±11.48 b aspartate day 11 21.75±1.11 25.75±5.12 29.75±5.54 day 18 18.50±2.66 a 24.75±2.7 ab 30.50±2.25 b citrulline day 11 76.25±8.64 a 115.25±8.14 b 77.75±8.19 a day 18 95.25±7.43 99.25±6.93 91.00±18.87 glutamate day 11 163.00±13.68 a 305.00±40.14 b 300.75±28.25 b day 18 247.00±49.79 306.75±46.96 350.50±32.72 ornithine day 11 54.25±7.73 52.50±4.73 58.00±8.75 day 18 56.75±6.34 a 95.75±14.94 b 111.00±11.1 b proline day 11 55.25±2.32 43.00±2.27 51.75±13.36 day 18 38.25±5.91 a 72.50±14.23 b 78.50±3.20 b *data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). arginine addition improved plasma concentrations of arginine respectively, by 29.53 (p<0.05) and 59.84% (p<0.05) on day 11, and 77.32 (p<0.05) and 89.24% (p<0.05) on day 18 (table 6). plasma citrulline level was higher in 0.4% arginine group than in the other two on day 11 (p<0.05), while it reduced to the similar level of the other two groups on day 18, suggesting some change of citrulline metabolism at early stage. contents of plasma ornithine (p<0.05) were notably raised by both 0.4 and 0.8% arginine supplementation than the control treatment only on day 18, but not on day 11. in comparison with the control group, both 0.4 and 0.8% arginine increased the level of plasma glutamate (p<0.05) on day 11, and those of plasma ornithine s p e rm in e ( µ g /u l ) 20 control 0.4% arginine b 18 a 0.8% arginine 16 14 12 10 a 8 6 4 2 0 day 11 day 18 figure 3. effect of dietary l-arginine supplementation on spermine concentrations in jejunal mucosa of piglets. bars represent the means ± sd. within an experimental phase, means without a common letter differ (p<0.05). table 7. effect of dietary l-arginine supplementation on concentration of no in small intestinal mucosa of piglets (u/mgprot)*. item control 0.4% arginine 0.8% arginine day 11 duodenum 0.36±0.02 b 0.60±0.11 a 0.34±0.05 b jejunum 0.37±0.04 0.31±0.04 0.30±0.03 ileum 0.40±0.06 0.45±0.07 0.29±0.04 day 18 duodenum 0.26±0.05 0.46±0.21 0.52±0.25 jejunum 0.19±0.03 0.22±0.02 0.26±0.07 ileum 0.25±0.02 0.22±0.03 0.21±0.03 *data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). (p<0.05) and proline (p<0.05) on day 18. contents of spermine in jejunal mucosa as revealed in figure 3, no noteworthy difference in jejunal mucosa spermine contents was observed among the three groups on day 11. as compared with the control and 0.8% arginine groups, concentration of spermine in jejunal mucosa of piglets was raised by 0.4% arginine supplementation at day 18 (p<0.05). concentration of no in small intestinal mucosa the level of no in the duodenal mucosa of piglets in 0.4% arginine group was higher than the other two on day 11 (p<0.05). contents of duodenal no in 0.4 and 0.8% arginine groups was respectively, 81.25 (p>0.05) and 101.95% (p>0.05) higher than the control group on day 18. no obvious difference in no concentration was observed in jejunum or ileum at either experimental phase (table 7). activities of nos, ass and p5cs in jejunal mucosa 0.8 and 0.4% arginine treatment prominently increased jejunal nos level (p<0.05) against the control group on day 18 (table 8), but no difference was observed on day 11 among three groups. in comparison with the control group, 0.4% arginine supplementation elevated jejunal table 8. effect of dietary l-arginine supplementation on enzyme activities in jejunal mucosa of piglets*. item control 0.4% arginine 0.8% arginine nos (u/mgprot) day 11 0.33±0.02 0.33±0.02 0.32±0.01 day 18 0.28±0.01 a 0.38±0.04 ab 0.44±0.05 b ass (mg/ml) day 11 8.19±1.53 a 20.43±0.85 b 17.24±5.33 ab day 18 5.05±0.83 6.16±0.48 8.32±2.18 p5cs (mg/ml) day 11 330.10±13.15 325.57±6.48 396.52±43.41 day 18 395.39±20.42 a 462.53±12.26 b 402.00±24.36 ab * data are means ± sd (n = 4). values in a row with different superscripts differ (p<0.05). nos, nitric oxide synthetase; ass, argininosuccinate synthetase; p5cs, pyrroline-5-carboxylate synthetase. ass activity on day 11 (p<0.05), while 0.8% arginine group showed no obvious difference from the other two groups. however, the effects of arginine supplementation seemed to be dampened on day 18 as similar level of ass was found in three groups. jejunal p5cs activity was improved by 0.4% arginine treatment on day 18 (p<0.05) against the control group, while 0.8% arginine treatment had no significant effect at either stage. discussion in this study, we systematically analyzed the effects of arginine supplementation on growth performance of young piglets, intestine morphology, enzymes related with arginine metabolism and some parameters related to amino acid metabolism. our result demonstrates that dietary arginine supplementation did not significantly improve bw, adg, adfi and feed conversion rate of neonatal piglets, which is contradictory to previous studies showing that dietary arginine supplementation, improved feed conversion rate, daily gain and bw of the weaned or early-weaned piglets (southern and baker, 1983). this discrepancy may be due to the different dose of arginine supplementation and the time course of piglet growth, in additional to different species of piglet studied. however, we did observe that blood urea nitrogen of both arginine groups were diminished through the experiment (figure 1) on day 11, indicating the improvement of protein utilization efficiency in vivo at early stage (sherry et al., 1978). arginine was reported to be effective in stimulating the secretion of insulin and growth hormone (flynn et al., 2000; wu and morris, 1998; yao et al., 2008). in this study, arginine treatment tended to increase blood igf-1 at both experimental periods (no significance, p>0.05), and 0.8% arginine administration significantly raised blood insulin on day 11. the results also reveal that blood t3 levels on both days 11 and 18 were increased by arginine (table 3). these findings indicate that hormone secretion might partly contribute to the enhancing tissue protein synthesis and the growthpromoting effect of arginine supplementation. in early infancy, the small intestine undergoes a period of rapid growth and development associated with multiple alterations in intestinal structure and function (butzner and gall, 1990). reduction of villus height was reported to retard the growth of weaned piglets (cera et al., 1988). our work reveal that jejunal villus height and villus area of piglets were improved by 0.8% arginine supplementation on day 11, and jejunal crypt depth was notably greater in 0.4% arginine group on day 18 than the control group, which were consistent with previous findings that 0.7% dietary l-arginine supplementation enhanced intestinal villus height on day 6 and 10 of experiment (zhan et al., 2008). absorption rate of d-xylose in both arginine groups were markedly higher than control group by the end of the experiment (figure 2), indicating that arginine supplementation improved the intestinal capability in both digestion and absorption in neonatal piglets. moreover, in contrast with the control group, both 0.4 and 0.8% arginine addition improved lactase level in jejunal mucosa on day 11, and 0.8% arginine supplementation elevated the activities of maltase and lactase in jejunal mucosa on day 18 (table 5). nevertheless, concentrations of small jejunum mucosa sucrase did not differ among the three groups on day 11 as well on day 18; it may change the pattern of sucrase level from day 11 to 18 period. these results show that the absorption efficiency of macromolecules and lactose of the intestinal mucosa was largely enhanced by arginine supplementation. as shulman et al. (2005) reported that changes in lactose absorption related primarily to lactase activity, previous work in miniature pigs also suggested that disaccharidase-specific activity together with changes in smallintestinal mucosal growth contributed to disaccharide digestibility (redel et al., 1997). our results indi-cate that arginine contributed to the maturational change in disaccharides digestion and absorption by im-proving the activities of lactase and maltase in the intestine. arginine family amino acids, including arginine, glutamine, glutamate, proline, aspartate, asparagine, ornithine and citrulline, were reported to be interconvertible via complex interorgan metabolism in most mammals, including pigs (wu et al., 2007). the small intestine is an essential organ for the inter-conversion of arginine family in pigs (wu, 1997). orni-thine and citrulline were two key amino acids involved in the endogenous synthesis of arginine in the enterocyte. citrulline and arginine, which were derived from glutamine, glutamate and proline in the small intestine of pigs, could be utilized for arginine synthesis by extrahepatic tissues and cells (wu et al., 1995, 2007). plasma arginine concentration was believed to be the most sensitive indicator of in vivo arginine status in neonates, including piglets (batshaw et al., 1984; johnson et al., 1972). in a research conducted in female mice, deficiency of dietary arginine reduced the concentration of plasma arginine, citrulline and ornithine (cremades et al., 2004). on the contrary, it has been reported that 0.48% dietary arginine supplementation significantly increased plasma arginine of the 28-day-old weaning piglets (southern et al., 1983). our study demonstrates that plasma content of arginine was dosedependently elevated by arginine treatment on both days 11 and 18 of the experiment (table 6), which was consistent with the findings of kim et al. (2004) and wilkinson et al. (2004). arginine, as well as citrulline, which were synthesized from glutamine, glutamate and proline in enterocytes (wu and knabe, 1994; wu et al., 1995), could be utilized for arginine synthesis by extrahepatic tissues and cells (wu et al., 1995, 2007). like the arginine, plasma content of citrulline was elevated by arginine treatment on day 11, indicating the possible improvement of de novo arginine synthesis (wu and knabe, 1995). ass was found to be a key enzyme which converted citrulline to arginine (morris, 2004). our work reveals that 0.4% arginine supplementation elevated jejunal ass activity on day 11 as well as p5cs level on day 18 against the control group, which was consistent with the changing trend of plasma citrulline of the piglets. as an important precursor of arginine synthesis in the enterocyte of neonatal piglets (wu and knabe, 1994; wilkinson et al., 2004; murphy et al., 1996), plasma glutamate was concordantly raised in the arginine groups on day 11, meanwhile, plasma ornithine, proline and aspartate levels were dose-dependently raised by arginine on day 18, which was consistent with the previous findings (southern et al., 1983). taken together, arginine supplementation improved the absorption of arginine, as well as the endogenous synthesis of arginine in the neonatal pigs. in addition, the longer period of arginine treatment (18 day) offered, the better availability of arginine was observed. apart from being an important precursor of arginine synthesis, ornithine also played a key role in synthesizing polyamines. as widely distributed organic cations, polyamines were involved in macromolecular synthesis as well as cell proliferation and differentiation in mammalian cell systems (hoshiai et al., 1981; morrison and seidel, 1995; wu et al., 2000; teixeira et al., 2002). polyamines were considered essential for endothelial cell proliferation (morrison and seidel, 1995). spermine is a polyamine involved in cellular metabolism found in all eukaryotic cells; it plays a significant role in cell proliferation and differentiation, especially in mucosal epithelial cells of the small intestine (tapiero and mathé, 2002). this study illustrates that jejunal spermine concentration was raised by 0.4% arginine treatment on day 18 in contrast to the other two groups (figure 3), which was believed to further improve the structural development and function of the intestine (hampson and kidder, 1986). no is a major endothelium-derived relaxing factor that plays an important role in regulating and maintaining vascular function (tangphaoa et al., 1999). as a lipophilic molecule, no diffuses easily into adjacent smooth muscle cells and activates soluble guanylate cyclase signaling pathway, which lead to vasodilation (furchgott and zawadzki, 1980; stuehr, 2004). it has been reported that physiological variations of plasma arginine could influence endothelial no production, thereby modifying vascular tone and platelet function (tangphaoa et al., 1999). arginine was catalyzed by nos to form endothelial no in the small intestine (moncada and higgs, 1993; berkowitz et al., 2003). in our study, 0.4% arginine supplementation improved the level of no in the duodenal mucosa of piglets on day 11, and both 0.4 and 0.8% arginine addition tended to improve duodenal no content on day 18. these findings were in line with those of clarkson et al. (1996), kharitonov et al. (1995) and urschel et al. (2007). likewise, the content of jejunal nos was obviously increased by 0.8% arginine supplementation on day 18 (table 8). in conclusion, our study demonstrates that dietary arginine supplementation was capable of improving protein synthesis and intestinal development, stimulating enzymes synthesis of intestinal mucosa, thereby promoting growth performance of the piglets. in all factors considered, the 0.8% arginine administration exhibited a slightly better effect in promoting the growth of piglets as compared to 0.4% arginine group, which has already showed many beneficial effects. acknowledgements this was supported by national science foundation of china (u0731002), international science and technology cooperation program of china (2009dfa31570) and national basic research program of china (2010cb35701). we thank the staff of institute of animal science, guangdong academy of agricultural science and guangdong public laboratory of animal breeding and nutrition for technical assistance. references barbul a, lazarou sa, efron dt, wasserkrug hl, efron g (1990). arginine enhances wound healing and lymphocyte immune responses in humans. surgery, 108: 331-336. batshaw ml, wachtel rc, thomas gh, starrett a, brusilow sw (1984). arginine-responsive asymptomatic hyperammonemia in the premature infant. j. pediatr. 115: 86-91. berkowitz de, white r, li dc, minhas km, cernetich a, kim s, burke s, shoukas aa, nyhan d, champion hc, hare jm (2003). arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels. circulation, 108: 2000-2006. burrin dg, stoll b, jiang r, chang x, hartmann b, holst jj, greeley gh, reeds pj (2000). minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough? 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vol. 6 (9), pp. 001-006, september, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper growing pigs fed cassava peel based diet supplemented with or without farmazyme ® 3000 proenx: effect on growth, carcass and blood parameters a. o. k. adesehinwa1*, o. o. obi1, b. a. makanjuola1, o. o. oluwole1 and m. a. adesina2 1 swine research unit, livestock improvement programme, institute of agricultural research and training, obafemi awolowo university, p.m.b. 5029, moor plantation, ibadan. nigeria. 2 national agricultural extension and research liaison services, south west zonal office, moor plantation, ibadan. nigeria. accepted 21 march, 2018 36 growing pigs (average initial weight of 22.74 ± 0.88 kg) were allotted to three dietary treatment groups of 30%maize-based control diet and 30%cassava-peel-based diet supplemented with or without farmazyme 3000 proenx. each treatment had three replicates of 4 pigs/replicate (12 pigs/treatment) in a complete randomized design. the pigs were allowed ad libitum access to the diets and water throughout the 42-day duration of the trial. the replacement of the 30% maize in the control diet with cassava peel resulted in increased bulkiness and crude fiber contents of the cassava peel-based diets, hence, lowered energy content. there was also a reduction in the dry matter intake of the pigs and the cost of feed per kg intake by 19.6 and 23.5% for the cassava peel based diet with and without farmazyme inclusion, respectively. the replacement of the maize content of the control diet with cassava peel resulted in 23 to 24% reduction in the cost of feed per kg live weight gain of the growing pigs. farmazyme resulted in enhanced utilization (p < 0.05) of the cassava peel-based diet in terms of the daily and overall weight gains as well as the serum total protein, albumin, urea and cholesterol. while the heamoglobin and red blood cell (rbc) of the pigs were significantly positively influenced by the inclusion of the enzyme, it had no effect on the packed cell volume (pcv). the blood minerals (na, ca, cl and p), relative organ weights and dressing percentage of the pigs were neither affected by the cassava peel replacement nor the enzyme inclusion but for the kidney, where lower values were obtained both for the control and farmazyme supplemented cassava peel-based diets. it could therefore be concluded that, inclusion of farmazyme 3000 proenx enhanced utilization of the cassava peel-based diet thereby, resulting in performance results comparable to pigs fed the maize-based control diet. key words: cassava peels, growing pigs, non-starch polysaccharides, exogenous enzyme, pig feeding. introduction cereal grains supply the bulk of livestock feed, especially for poultry and pigs. however, in developing countries like nigeria, cereal grains are in high demand for human *corresponding author. e-mail: aokadesehinwa@yahoo.com. abbreviations: rbc, red blood cells; pcv, packed cell volume; wbc, white blood cell. uses and the production has never been adequate to meet the need of the increasing population, consequently, there is little or no excess grain for livestock feed. when available, it is always very expensive. for instance, in the year 2005, the price of maize rose from about #30 to #70 per kilogramme (approx. $0.25 to $0.50). this continuous increase in the cost of conventional ingredients used in compounding livestock feed has necessitated intensive research into cheap alternative source of energy yielding unconventional ingredients. since the monogastric livestock industry constitutes the largest consumer of comercial livestock feeds in africa, it is imperative to find alternative feed resources to the expensive energy ingredients like the cereals. cassava (manihot esculenta) is an all-season crop of the humid tropics and ranks among the top 10 food crops in the world (oyebimpe et al., 2006). it is the highest supplier of carbohydrates among staple crops (fao, 1995). annual production estimate in nigeria was 34 million tonnes in 2002 (fao, 2002). cassava roots contain 30 to 40% dry matter, more than most roots and tubers. this depends on factors such as variety, soil type, moisture, climatic conditions and the age of the root at harvest. starch and sugar are the predominant components of the dry matter, approximately 90%, with starch being the most important (fao, 2002). although, the crude protein content of cassava root is 2 to 4% in dry matter, the true protein content is less than half this amount due to the fact that 50% of the nitrogen in the roots is in the form of non-protein-nitrogen. furthermore, the available true protein is deficient in the sulphurcontaining amino acids. the roots contain significant amounts of vitamins, particularly vitamin c, thiamine, riboflavin and niacin (fao, 2002). cassava products have been in use for a long time as an energy source in place of cereal grains for livestock (eruvbetine et al., 2003). cassava peels continue to constitute waste in the cassava processing industry. it accounts for 10 to 13% of the tuber by weight (oyebimpe et al., 2006). this is in spite of the potential of the by-product as an animal feedstuff (iyayi and losel, 2001). tewe and lyayi (1995) reported that, among the roots produced in nigeria, cassava from which cassava peel is obtained constitute 20% of total tubers produced annually. cassava peel meal could serve as a cheap source of energy for farm animals but should be fortified with additional protein source because of its low protein level (obioha and anikwe, 1982). considerable evidence has emerged for long of the possibility of using processed cassava peel as an energy source for pigs and poultry (longe et al., 1977; tewe, 1981; iyayi, 1986). higher inclusion of the by-product in monogastric feed or formulation of diets with cassava peels, as sole energy source is limited because of its fibrous nature. fakolade (1977) and arowora et al. (1999) have reported the occurrence of high amounts of nonstarch polysaccharides (nsps) in cassava peels. degradation of these carbohydrate compounds to simple sugars will further increase the energy value of cassava peels, hence, the need for the addition of exogenous enzymes when utilized in replacing cereals in diets for monogastric animals (adesehinwa, 2004). the aim of this study therefore, is to determine the effect of cassava peel supplemented with or without farmazyme 3000 proenx as a replacement for maize in the diets of growing pigs on the growth, some carcass traits, serological and heamatological indices of growing pigs. materials and methods the utilization of cassava peel based diet supplemented with or without farmazyme 3000 proenx (protease, fungal xylanase, fungal βgluconase, endo β-gluconase, α-amylase, β-gluconase (ph 7.530°c), β-gluconase (ph 5-30°c), hemicellulase, pentozanase, pectinase) for growing pigs was investigated using 36 growing pigs, with average initial weight of 22.74 ± 0.88. the pigs were allotted to three dietary treatment groups of (1) 30% maize-based control diet, (2) 30% cassava-peel-based diet without farmazyme inclusion and (3) 30% cassava-peel-based diet supplemented with farm-azyme 3000 proenx, as shown in table 1. cassava peels of mixed varieties were obtained within 24 h after peeling from a cassava processing industry in ibadan, nigeria. they were then sun dried for 5 days under intensive sunshine to constant weight and ground in a hammer mill before being incorporated in the experimental diets. the maize content of the maize-based control diet was totally replaced with cassava peel on weight for weight basis in the two treatment diets. each treatment had 12 pigs, comprising three replicates (with four pigs per replicate) in a complete randomized design. the pigs were allowed ad libitum access to the diets and water served in concrete feeding and watering troughs, respectively. the growth and economy of production were monitored throughout the 42-day duration of the trial. at the end of the trial, two pigs were randomly selected from each replicate for bleeding. 10 ml of blood was obtained from the jugular vein of each of the pigs into two sample bottles for serological, heamatological and blood mineral analyses (kaneko, 1989) using a sterilized needle and syringe. some carcass and organ weights were also taken from two pigs randomly selected per replicate for this purpose. the pigs were starved of feed for 16 h, weighed and slaughtered. the slaughtered pigs were properly bled, cleaned and eviscerated before dissecting into parts and weighed. all the data obtained were subjected to analysis of variance and where statistical significance was observed, the means were separated using the duncan’s multiple range (dmr) test. the sas computer software package (sas, 1999) was used for all statistical analysis. results and discussion effect on growth and feed conversion proximate composition of test ingredient (cassava peel) used and the formulated experimental diets is as shown in table 2. the replacement of the 30% maize in the control diet with cassava peel resulted in increased bulk and crude fiber contents of the cassava peel-based diets (table 2) hence, a lowered energy content is expected. the increased bulk could be said to be responsible for the reduction in the dry matter intake of the pigs fed the cassava peel based diet with and without farmazyme® supplementation in line with the earlier findings of adesehinwa et al. (2008a) inspite of the lowered energy content. there was a significantly (p < 0.05) lower quantity of feed consumed in the cassava peel-based diet treatments. even though the gains and feed conversion of pigs observed in the cassava peel-based diets were table 1. gross and proximate composition of diets fed to growing experimental pigs. ingredient maize-based cassava peel-based maize 30.00 0.00 cassava peel meal 0.00 30.00 palm kernel cake 30.00 30.00 wheat bran 18.00 18.00 groundnut cake 15.00 15.00 fishmeal 2.00 2.00 bone meal 3.00 3.00 oyster shell 1.25 1.25 salt 0.50 0.50 vit-min premix* 0.25 0.25 *pfizer agricare grower premix supplied the following per kg diet: vit. a 10,000,000 iu; vit. d3 2,000,000 iu; vit. e 8,000 iu; vit k 2,000 mg; vit b1 2,000 mg; vit. b2 5,500 mg; vit. b6 1,200 mg; vit. b12 12 mg; biotin 30 mg; folic acid 600 mg; niacin 10,000 mg; pantothenic acid 7,000 mg; choline chloride 500,000 mg; vit. c 10,000 mg; iron 60,000 mg; mn 80,000 mg; cu 8,00 mg; zn 50,000 mg; iodine 2,000 mg; cobalt 450 mg; selenium 100 mg; mg 100,000 mg; anti oxidant 6,000 mg. table 2. proximate composition of test ingredient and experimental diets (%). component maize-based diet cassava peel -based diet cassava peel dry matter 89.50 88.96 89.24 crude protein 18.20 17.33 3.15 crude fiber 5.50 9.89 33.96 ether extract 4.67 3.61 0.34 ash 6.57 7.13 1.44 table 3. performance characteristics and economy of gain of growing pigs fed diets with or without farmazyme 3000 proenx inclusion. parameter cassava peel-based sem maize-based without enzyme with enzyme (±) average initial weight (kg) 22.50 22.78 22.94 0.88 average final weight (kg) 39.28 37.17 39.44 1.13 average total weight gain (kg) 16.78 a 14.39 b 16.50 ab 0.36 average daily dry matter intake (kg) 1.63 a 1.42 b 1.49 b 0.02 average daily weight gain (kg) 0.40 a 0.35 b 0.39 ab 0.36 feed conversion (feed : gain) 4.51 4.65 4.20 0.13 av cost of feed consumed/day (n) 46.16 a 30.81 b 34.03 b 0.02 av cost of feed per gain (n) 115.40 a 88.03 b 87.26 b 0.05 a,b, means along the same row with different superscripts are significantly (p < 0.05) different from each other. to produce direct benefits of increase in body weight gain as a result of improved feed efficiency (adesehinwa, 2008b). the final weights of the pigs were com-parable across the groups over the 42-day duration of the study, as shown in table 3. there was also a reduction of the cost of feed per kg by 23.5 and 19.6%, respectively, as a result of the replacement of the 30% maize in the control diet with cassava peel supplemented without or with farmazyme inclusion (table 3). the replacement of the maize content of the basal diet with cassava peel resulted in comparable feed: gain, but a significant (p < 0.05) reduction of 23 to 24% in table 4. some carcass traits and relative organ weights of growing pigs fed diets with or without farmazyme 3000 proenx inclusion. parameter cassava peel-based sem (±) maize-based without enzyme with enzyme carcass slaughter weight (kg) 42.88 42.88 43.00 0.52 cold carcass weight (kg) 30.11 30.02 29.44 0.10 dressing percentage (%) 70.18 69.95 68.41 0.79 internal organs (%) kidney 0.30 b 0.39 a 0.35 ab 0.02 liver 2.28 2.45 2.23 0.05 heart 0.38 0.41 0.43 0.02 spleen 0.22 0.16 0.14 0.02 a,b, means along the same row with different superscripts are significantly (p < 0.05) different from each other. the cost of feed/kg live weight gain (n) for the growing pigs fed the cassava peel-based diets (table 3). farmazyme® resulted in enhanced utilization (p < 0.05) of the cassava peel based diet in terms of the daily and overall weight gains. dale (1997) reported addition of enzymes to diets to produce not only direct benefits of increased body weight gain and improved feed con-version but also reduction in feed costs. effect on carcass and organ weights the results of some carcass traits and relative organ weights of the growing pigs fed diets supplemented with or without farmazyme 3000 proenx is as shown in table 4. the dressing percentage of the pigs were com-parable across the groups showing that, the replacement of maize with cassava peel supplemented with or without the exogenous enzyme in the diets of growing pigs did not affect the resulting cold carcass weights of the pigs when slaughtered (table 4). addition of multi-enzyme, such as farmazyme®, has been reported not to affect edible organ weights and carcass characteristics such as dressing percentages except for abdominal fat content which decreased with enzyme addition (kilic et al., 2006). this was also in agreement with the findings of johri (2004) and torres et al. (2003) who also reported that, multi-enzyme addition did not affect carcass characteristics of broilers used in their study. in the present study, the relative organ weights of the pigs were neither affected by the cassava peel replacement nor the enzyme inclusion; but for the kidney, where lower values were obtained with the pigs on the control diet, the results were comparable (p > 0.05) to those on the cassava peel-based diet without enzyme supplementation (table 4). effect on serum metabolites, hematological parameters and blood minerals the inclusion of farmazyme resulted in enhanced utilization of the cassava peel-based diet in terms of increased daily and overall weight gains, comparable to those of pigs on the maize-based control diet (table 3). this also resulted in comparable values of serum total protein, albumin and urea obtained for the two groups (table 5). this indicated that the protein levels of the maize-based diet and the cassava peel based diet supplemented with farmazyme® was able to support the protein reserves of the pigs across the groups. the urea levels of pigs fed the cassava peel-based diet supplemented with farmazyme was also comparable (p > 0.05) to those of pigs on the maize-control but signifi-cantly lower (p < 0.05) compared with those of pigs fed with cassava peel-based diet without enzyme supplementation (table 5). high serum urea has been reported as an indicator of muscular wastage in animals (adesehinwa, 2008). in spite of the high fiber content of the cassava peel-based diets (cassava being highly fibrous), the diets seemed to have been efficiently utilized similar to the maize-based, thereby, resulting in high tissue deposition (adesehinwa, 2007) in pigs fed with both diets compared with the cassava peel-based diet without enzyme supplementation. the least cholesterol level was recorded for the enzyme supplemented diet, in line with the findings of kilic et al. (2006), who reported that enzyme significantly (p < 0.05) influence fat deposition in broilers. it could be observed that, the urea levels were inversely proportional to the live weight gains of the growing pigs (table 5). the hemoglobin and red blood cells (rbc) of the pigs were significantly (p < 0.05) increased by the inclusion of the enzyme, as the replacement of the maize content of table 5. hematological parameters, serum metabolites and blood minerals of growing pigs fed diets with or without farmazyme 3000 proenx inclusion. parameter cassava peel-based sem (±) maize-based without enzyme with enzyme heamatology packed cell volume (pcv) % 42.95 a 39.92 b 40.95 b 0.29 heamoglobin (hgb) g/dl 14.13 a 12.37 c 13.48 b 0.12 red blood cell (rbc) x10 6 /µl 7.17 a 5.97 b 6.95 a 0.07 white blood cell (wbc) x10 3 /µl 8.63 8.75 8.40 0.15 serum metabolites total protein (g/dl) 6.95 a 6.23 b 6.77 a 0.07 albumin (g/dl) 3.13 a 2.70 b 2.98 a 0.05 urea (mg/dl) 12.65 b 14.55 a 13.07 b 0.17 creatinine (mg/dl) 1.57 a 1.07 b 1.12 b 0.05 cholesterol (mg/dl) 124.15 a 113.03 b 102.65 c 1.06 glucose (mg/dl) 92.02 87.02 90.55 0.68 minerals sodium (na) meq/l 144.27 a 141.27 b 142.40 ab 0.38 potassium (k) meq/l 5.32 4.98 5.37 0.1 calcium (ca) mg/dl 10.18 a 9.53 b 10.37 a 0.10 chlorine (cl) meq/l 98.63 b 101.92 a 99.28 b 0.30 phosphorus (p) mg/dl 7.47 a 6.62 b 7.97 a 0.14 a,b,c = means along the same row with different superscripts are significantly (p < 0.05) different from each other. the basal diet with cassava peel significantly reduced both parameters. the enzyme had no effect on the packed cell volume (pcv) of the pigs, hence, the pcv of the pigs on the maize-control diet were significantly higher than those on the cassava peel-based diets (table 5). the white blood cell (wbc) values observed in this study were not significantly influenced by both treatments (cassava peel and enzyme inclusion). the enzyme had significant effect (p < 0.05) on all the blood minerals measured in this study but for k, resulting in values comparable to those of pigs on the maize-based control dietary group (table 5). conclusions it could be concluded that, even though the replacement of maize with cassava peel had no adverse effect on the dressing percentage and other parameters in some instances, inclusion of farmazyme 3000 proenx enhanced utilization of the cassava peel-based diet better; resulting in better performance comparable to pigs fed with the maize-based control diet. the result of this study therefore indicates that, the cassava peel meal, which is economically cheaper than maize and regarded as waste in some areas of nigeria can be used successfully to replace maize in conventional pig feed without any depression or adverse effect on the growth performance, health status and carcass characteristics. acknowledgements the authors thank the executive director of the institute of agricultural research and training, obafemi awolowo university, ibadan, for the funding of this work and general manager, farmavet international, istanbul, turkey, for providing the enzyme used in the study. we are also grateful to the staff of swine research unit of the livestock improvement programme for care of experimental animals. references adesehinwa aok (2004). growth performance and serum metabolites of young pigs fed maize offal as energy source. e. afr. j. life sci. 5(2): 87-96. adesehinwa aok (2007). utilization of palm kernel cake as an energy source by growing pigs: effects on growth, serum metabolites, nutrient digestibility and cost of feed conversion. bulg. j. agric. sci. 13: 593-600. adesehinwa aok (2008b). comparative utilization of two sources of expeller extruded soybean meal as a replacement for on-farm processed soybean in diets of growing-finishing pigs. afr. j. agric. res. 3(8): 574-577. adesehinwa aok, dairo fas, olagbegi bs (2008a). response of growing pigs to cassava peel based diets supplemented with avizyme® 1300: growth, serum and hematological indices. bulgarian j. agric. sci. 14(5): 491-499. arowora ka, tewe oo, fasein to, lamina dro (1999). carbohydrate constituents of cassava peel clones and their utilization in pig grower rations. trop. anim. prod. invest. 2: 29-34. eruvbetine d, tajudeen id, adeosun at, olojede aa (2003). cassava (m. esculenta) leaf and tuber concentrate in diets for broiler chickens. bioresour. technol. 86: 277-281. fao (1995). commodity review and outlook, 1994-95. economic and social development series. food and agriculture organization, rome italy. fao (2002). faostat statistics database. (http://aps.fao.org). june (accessed july). fakolade at (1997). effect of polysaccharide supplementation on the performance of layers maintained on 45% corn offal and 40% wheat offal diets. b.sc. dissertation, university of ibadan, ibadan, p. 84. iyayi ea (1986). varying dietary cyanide and protein levels on the performance of growing pigs. ph.d. thesis, university of ibadan, 1986. p. 306. iyayi ea, losel dm (2001). changes in carbohydrate fractions of cassava peel following fungal solid state fermentation. j. food technol. africa. 6(3): 101-103. johri ts (2004). dietary additives for enhancing nutritional value of feed. central avian research institute. http//www.fao.org/ docrep/article/agrippa/659-en-02htm#, pp. 97-9846. kilic u, saricicek bz, garipoglu av (2006). effect of enzyme supplementation to the rations in which soybean meal replaced by canola meal on performances of broilers. asian j. anim. vet. adv. 1(1): 76-81. longe og, famojuro ed, oyenuga va (1977). available carbohydrates and energy value of cassava, yam and plantain peels for chicks. e. afr. agric. for. j. 42: 408-413. obioha fc, anikwe pc (1982): utilization of sundried cassava peel by growing swine nut. rep. 26: 961-972. oyebimpe k, fanimo ao, oduguwa oo, biobaku wo (2006). response of broiler chickens to cassava peel and maize offal in cashewnut meal based-diets. arch. zootec. 55(211): 301-304. tewe oo (1981). grain replacement value of cassava peel in layers ration unpublished data. tewe oo, iyayi ea (1995). utilization of cassava peels in small holder pig and small ruminant production systems in nigeria. paper presented at the 6th triennial symposium of the istracar lilongwe. malawi, 22-28 october, 1995. torres dm, teixeria as, rodrigues pb, freitas rtf, santos ec (2003). the efficiency of amylase, protease and xylanase on broiler chicken performance. ciencia e agrotecnol. 27: 1401-1408. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (4), pp. 001-007, april, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. author(s) retain the copyright of this article. full length research paper comparison of growth and reproductive performance of exotic, indigenous and cross breeds of pigs in nepal shashi sharma1+*, chiranjibi pantha2+, prakash kumar yadav1, suman karki1 and nabaraj poudel1 1 nepal agricultural research council, nepal, fax 977-1-4262500. 2 department of livestock services, nepal, fax 977-1-5542915. accepted 10 march, 2019 the objective of this study is to evaluate growth and reproductive performance of the exotic, indigenous and cross breeds of pigs in nepal. a total of 42 female piglets were selected, 7 each from breed in hampshire, pakhribas black, nagpuri, hurrah, pakhribas black x nagpuri and hampshire x hurrah; and are kept under 6 treatments with 7 replications. mean body weight, age at first mating, litter size at weaning, litter weight at weaning and farrowing interval were significant (p<0.05%). weight at first mating and litter index sow/year were significant (p<0.01). age of first farrowing, litter size at birth, weight of piglets at birth and piglet mortality % were non significant. exotic breed hampshire has the highest adult body weight than synthetic breed pakhribas followed by cross breed pakhribas x nagpuri, hampshire x hurrah and local breeds nagpuri and hurrah. age at first mating, age at first farrowing and piglet mortality % were higher in local breeds. litter size at birth, litter size at weaning and weight of piglets at birth were higher in pakhribas. weight at first mating and litter weight at weaning were higher in exotic breed. farrowing interval was higher in local breed. litter index was higher in synthetic and exotic breeds. keywords: exotic, indigenous, cross, breed, farrowing, growth, reproductive, pig. introduction pig is a high value domesticated animal for meat as it gives quick return to the farmers by consuming waste food materials converting into valuable protein. the government through department of livestock services (dls) and different ngo/ingos have been distributing improved pig breeds in the country for many years. this approach has expanded the improved pig raising system in the rural farmers. regional agriculture research station (rars), tarahara, sunsari, nepal has been conserving different pig breeds viz. hampshire, pakhribas black, hurrah, nagpuri and their crosses for many years. the exotic and improved breeds maintained *corresponding author e-mail: timilsinashashi@gmail.com tel: +977-9841159515 + equal contributors as pure line parental (nucleus) seed stock. hurrah and nagpuri kept as conservation of native pig breeds and crossing each other for research purposes. materials and methods a total of forty two female piglets (hampshire n=7, pakhribas black n=7, nagpuri n=7, hurrah n=7, pakhribas black x nagpuri n=7 and hampshire x hurrah n=7) were kept under 6 treatments with 7 replications one pig considering as one replication. a study was conducted in regional agricultural research station (rars), tarahara, sunsari, nepal. the data for the present investigation were obtained from the pigs of exotic, indigenous and cross breeds of nepal over a period of 1 year from july 2017 to june 2018. the design used was crd. providing commercial feed upto twelve months file:///c:\users\user\downloads\www.internationalscholarsjournals.org of age, drenching and vaccination were routinely done in each pig. piglets from different groups were kept in indoor housing system with open running space under uniform management condition. data were analyzed using mstat. the observations were recorded from pedigree sheet of pig farm. mean body weight (kg) and other performance parameters like age at first mating (days), weight at first mating (kg), age at first farrowing (days), litter size at birth (nos.) , weight of piglets at birth (kg), litter size at weaning (nos.), litter weight at weaning (kg), farrowing interval (days), litter index /sow/year, piglet mortality % were compared. literature review nepalese farmers are nowadays raising various types of exotic breeds such as landrace, hampshire, duroc, yorkshire, etc. although chwanche, hurrah and nagpuri etc. are the indigenous breeds of pig which are the major component of livelihood for most of the deprived communities. pakhribas pig is a result of three-way crossing of exotic breeds (saddle back, fayuen and tamworth) developed at agricultural research station (ars), pakhribas (oli 1986, gatenby et al.,1990, aryal et al.,1992). the breed is very popular in the eastern hills of nepal because of its black color and a good litter size at birth and weaning (gatenby et al., 1990). for religious purposes, black color is preferred over other colors in the eastern hills. it is medium in size and can be maintained by the farmers in the hills. hurrah pigs are distributed in southern terai belt of nepal from east to west. the actual population of the hurrah breed is unknown at present but it was estimated that it constituted 23% of the total native pig population. they are rust brown in color, hardy and suitable for scavenging conditions. they reach puberty at an average age of 11 months, first farrowing at 14 months with average litter size 7 at birth and 6 at weaning. the average birth weight is 0.8kg and adult weight is 45kg (40-55kg). they have been characterized at phenotypic and genotypic level. their population is declining day by day and needs conservation measures. narc records (neupane and kadel 2008) show that hurrah pigs attained an average of 42kg body weight at one year of age with a body length 79 cm; 88 cm heart girth; 7 and 5.7 litter size at birth and at weaning respectively, with less than 6 months farrowing interval. according to dhakal (2012), pokhrel (2012), pandey (2011) and thapa (2009) least square mean and standard error of reproductive traits like age at first service (days), age at first farrowing (days), farrowing interval, litter size at birth (nos.) and litter size at weaning (nos.) were 241.73±2.13, 351.30±4.8, 180.30±0.3, 8.70±0.27 and 7.53±0.20 respectively. nagpuri or pundi breed is found in eastern hills and terai districts of nepal . they are black in color, short snout, wrinkled face and pendulous belly that resembles chinese mehisan breed. some believe that the breed is crosses between the chinese mehisan and local pig breeds which have been well established since long before but it is placed in local breed category in nepal. they are prolific with average weight of 40-50kg. under good management condition, the breed has potential to reach adult weight of about 70kg. the farmers of economically disadvantaged and some middle class farmers rear pundi (dhaubadhel and ghimire, 2002). this breed is said to have evolved from the crosses of meshain with local pig population. according to dhakal (2012), pokhrel (2012), pandey (2011) and thapa (2009) least square mean and standard error of reproductive traits like age at first service, age at first farrowing, farrowing interval, litter size at birth and litter size at weaning were 213.5±11.14 days, 335.54±9.91days, 170.96±7.28 days, 8.95±2.39 nos. and 8.14±2.10 nos respectively. hampshire breed having large body size, short legs and small erect ears originated from england. they are black in color with white color belt encircling the shoulder and forelegs. this breed is suitable for crossbreeding and has been very popular in eastern region (kayastha et al., 2014). hampshire is popular for pork production. it is well known for excellent growth rate and feed efficiency. it matures early, farrows 8-10 litters and is a good mother. mature boar weighs about 400kg and sow 350kg. according to tummaruk et al. (2001) age at first farrowing and farrowing interval of hampshire pig was found 386 days & 173.4 days respectively. similarly, adult mean body weight was 185kg (kumaresan et al., 2006). likewise thapa et al. (2018) who did research in production performance of crossbred pigs in government farm of bhutan farrowing interval, age at first service, weaning to service interval, litter size at birth and litter size at weaning of hampshire pig was 174 days, 301±42.42 days, 473 days, 11.77 days, 8.31 no. and 7.56 nos respectively. result growth performance of different pig breeds at rars, tarahara geometric means of body weight growth of different pig breeds at rars, tarahara has been presented in table 1. in the first and seconf month, the highest mean body weight was observed 8.9±1.12kg, 14.04±3.75kg in exotic breed hampshire followed by 8.5±1.50kg, 12.44±1.72kg in pakhribas (pac) breed and the lowest 3.9±0.29kg, 6.73±0.68kg in local breed hurrah. similarly, on third, fourth, fifth and sixth months, the highest mean body weight was found 18.65±3.73kg, 23.24±4.28kg, 30.97±4.83kg & 39.08±4.50kg in hampshire, hampshire, pakhribas × nagpuri & hampshire breeds respectively followed by 17.97 table 1. some production performances of indigenous breeds of pig (mean±sd). traits hurrah chwanche bampudke pakhribas birth weight (kg) weaning weight 0.8±0.08 6.1±0.09 0.7±0.06 5.7±0.08 0.6±0.08 1.05 6.19±1.77 age at first puberty (month) 10.8±0.99 7.3±0.85 6.2±0.12 age at first farrowing (month) 14.0±0.96 10.7±0.80 11.5±0.23 364.2±7.4 farrowing interval (month) 5.57±0.57 7.4±0.6 4.6±0.28 172.8±12.1 gestation length (days) 114.5±0.66 114.8±0.52 113.5±1.84 adult weight (kg) 45 35 20 350 litter size at birth (nos.) litter size at weaning (nos.) 7.04±1.26 5.73±1.25 7.33±1.28 6.0±1.0 4.7±0.27 3.4±0.30 9.24±2.87 8.43±2.7 source: annual report, abd (1997), pradhan (1999), neopane (2004), neopane (2006), neopane and kadel (2008). ±3.37kg, 22.62±3.88kg, 28.62±4.20kg, 37.62±6.34kg in pakhribas × nagpuri, pakhribas, pakhribas, pakhribas respectively and the lowest value was found 10.33±0.7kg, 14.33±0.57kg, 18.0±2.64kg, 21.33±3.21kg in local breed hurrah. table 2 shows on 7 th , 8 th and 9 th month, the highest mean body weight was observed 48.2±14.80kg, 66.0±15.78kg, 79.8±17.12kg in hampshire followed by 48.12±7.16kg, 60.87±8.38kg, 70.62±7.0kg in pakhribas and the lowest mean body weight was observed 26.16±2.24 kg, 31.33±2.51kg, 38.33±1.52kg in local breed hurrah. similarly, on 10 th , 11 th and 12 th month, the highest mean body weight was observed 98.0±13.54kg, 115.25±17.37kg and 127.6±21.49kg in exotic breed hampshire followed by 89.25±8.43kg, 108.87±9.03kg and 116.6±19.42kg in pakhribas breed and the lowest mean body weight was observed 41.66±3.78kg, 49.0±4.32kg and 55.9±4.0kg in local breed hurrah. the result of this research revealed that the highest body weight was observed in exotic breed hampshire followed by synthetic breed pakhribas, pakhribas × nagpuri, hampshire × hurrah, nagpuri and the lowest in hurrah. exotic breed like hampshire has higher weight than synthetic breed pakhribas, their crosses and local breeds. the f-test statistics showed significant during i st and 2 nd month at 1% (p<0.01) level of significance whereas this test statistics showed significant during 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th , 11 th &12 th months at 5% (p<0.05%) level of significance. reproductive performance of different pig breeds at rars, tarahara geometric means of reproductive performance of different pig breeds at rars, tarahara has been presented in table 3. the highest mean of age at i st mating & age at first farrowing were observed 335±26.8 days & 466±3.21days in hurrah & nagpuri breed respectively followed by 322±23.3 days & 462±5.59 days in nagpuri & hampshire breed respectively and the lowest 294±28.20 days & 408±6.9 days in pakhribas breed. similarly, the highest mean of weight at i st mating and litter weight at weaning were observed 95±10.10kg & 9.2±0.28kg in hampshire followed by 78±11.3kg & 8.1±0.75kg in pakhribas and the lowest 52±9.75kg & 5.54±0.97kg in and nagpuri & hurrah respectively. likewise, the highest mean value of litter size at birth & at weaning were observed 11.4±1.6 nos. & 9.4±1.01 nos. in pakhribas followed by 10.5±0.59 nos. & 8.28±0.96 nos. in hampshire and the lowest 7.56±1.32 nos.& 5.54±0.97 nos. in hurrah. table 3 shows the highest mean of weight of piglets at birth, farrowing interval, litter index & piglet mortality were observed 1.2±0.05kg, 207±12.32 days, 1.8±0.24 sow/year & 22±0.96% in hampshire, nagpuri, pakhribas & nagpuri respectively followed by 1.1±0.08kg, 197±7.65 days, 1.8±0.05 sow/year & 21±0.85% in pakhribas, hurrah, hampshire & hurrah respectively and the lowest 0.92±0.87kg, 184±32.9 days, 1.5±0.98 sow/year , 18±7.57% in hurrah, pakhribas, nagpuri & pakhribas respectively. among overall coefficient of variance (cv), the cv of litter index (sow/year) was 4.95 which was least cv and significant at 1% level of significance. the least value of cv among them indicates that it was more consistent and reliable than other cv. the f-test statistics showed significant in age at i st mating, litter size at weaning, litter weight at weaning and farrowing interval at 5% (p<0.05%) level of significance. similarly, this test statistics showed significant in weight at i st mating and litter index at 1% (p<0.01) level of significance whereas age of i st farrowing, litter size at birth, weight of piglets at birth and piglet mortality showed non significant. discussion the mean body weight of hampshire piglets in 1 st month was found higher 8.9±1.12kg compared to 8.5±1.50kg, 4.38±0.57kg, 3.9±0.29kg, 5.93±0.66kg and 6.23±0.97kg of pakhribas, nagpuri, hurrah, hampshire x hurrah and pakhribas x nagpuri piglets respectively. similarly, the mean body weight of hampshire piglets in 12 th month was found higher 127±0.10kg as compared to 116.6±18.42kg, table 2. mean body weight (kg) of different pig breeds upto age of 12 months. months pakhribas mean±sd hampshire mean±sd puri mean±sd hurrah mean±sd hamp× hurrah mean±sd pac×puri mean±sd ftest 1 st 8.5±1.50 8.9±1.12 4.38±0.57 3.9±0.29 5.98±0.66 6.29±0.97 * 2 nd 12.44±1.72 14.04±3.75 9.25±4.66 6.73±0.68 9.33±1.97 11.97±2.33 * 3 rd 17.29±±2.28 18.65±3.73 14.08±4.82 10.33±0.7 14.93±2.0 17.97±3.37 ** 4 th 22.62±3.88 23.24±4.28 17.6±4.82 14.33±0.57 17.78±2.68 22.39±4.32 ** 5 th 28.62±4.20 30.1±4.24 20.8±4.72 18.0±2.64 22.53±2.84 30.97±4.83 ** 6 th 37.62±6.34 39.08±4.50 25.2±6.14 21.33±3.21 30.33±1.52 37.33±3.33 ** 7 th 48.12±7.16 48.2±14.80 30.6±6.73 26.16±2.24 33.0±4.33 47.2±4.33 ** 8 th 60.87±8.38 66.0±15.78 35.64±7.36 31.33±2.51 44.0±5.29 52.31±3.32 ** 9 th 70.62±7.0 79.8±17.12 43.0±8.8 38.33±1.52 52.66±8.02 65.0±4.33 ** 10 th 89.25±8.43 98.0±13.54 48.4±8.58 41.66±3.78 69.65±5.33 73.33±5.97 ** 11 th 108.87±9.03 115.25±17.37 53.0±9.05 49.0±4.32 75.0±5.97 80.2±2.29 ** 12 th 116.6±19.42 127.6±21.49 59.8±8.4 55.9±4.0 81.0±7.9 85.33±4.29 ** notes: ns= non significant, *= significant at 1% level (p value < 0.01 ), **=significant at 5% level (p<0.05). 59.8±8.4kg, 55.9±4.0kg, 81.0±7.9kg and 85.33±4.29kg of pakhribas, nagpuri, hurrah, hampshire x hurrah and pakhribas x nagpuri respectively. the mean body weight of nagpuri was found higher than that of hurrah. similarly, the mean body weight of hampshire x hurrah and pakhribas x nagpuri was found higher than those of local breeds like hurrah and nagpuri. according to kumar et al. 2018, adult body weight of hampshire pig (9 th month) was observed 77.25±2.82kg which is almost similar with the finding of our research i.e. 79.8±17.12 kg. the highest mean of age at i st mating & age at first farrowing were observed 335±26.8 days & 466±3.21days in hurrah & nagpuri breed respectively and the lowest 294±28.20 days & 408±6.9 days in pakhribas breed. similarly, the highest mean of weight at i st mating and litter weight at weaning were observed 95±10.10kg & 9.2±0.28kg in hampshire and the lowest 52±9.75kg & 5.54±0.97kg in and nagpuri & hurrah respectively. likewise, the highest mean value of litter size at birth & at weaning were observed 11.4±1.6 nos. & 9.4±1.01 nos. in pakhribas and the lowest 7.56±1.32 nos.& 5.54±0.97 nos. in hurrah. the highest mean of weight of piglets at birth, farrowing interval, litter index & piglet mortality were observed 1.2±0.05kg, 207±12.32 days, 1.8±0.24 sow/year & 22±0.96% in hampshire, nagpuri, pakhribas & nagpuri respectively and the lowest 0.92±0.87kg, 184±32.9 days, 1.5±0.98 sow/year , 18±7.57% in hurrah, pakhribas, nagpuri & pakhribas respectively. according to neopane (2004), neopane (2006), neopane and kadel (2008), weight of piglet at birth and adult of pakhribas is 1.05kg and 350kg respectively that match the finding of our research in terms of birth weight 1.1±0.08kg but doesn't match the adult body weight 116.6±19.42kg. in this study, the lower value of adult body weight may be due to inbreeding in the herd. similarly, age of i st farrowing, litter size at birth, litter size at weaning and farrowing interval were observed 408±6.9 days, 11.4±1.6 nos., 9.4±1.01 nos. and 184±32.9 days which is higher than those of neopane (2006) 364.2±7.4 days, 9.24±2.87 nos., 8.43±2.7 nos. and 172.8±12.1 days respectively. the higher value may be due to inbreeding problems in pakhribas breed. in this research, piglet mortality was observed 18±7.57% which is higher than that table 3. reproductive performance of different pig breeds at rars, tarahara. parameters breeds pakhribas n=7 hampshire n=7 puri n=7 hurrah n=7 average n=28 cv% f test age at 1 st mating(days) 294±28.20 301±42.42 322±23.3 335±26.8 313.2±32.20 7.29 ** wt. at 1 st mating (kg) 78±11.3 95±10.10 52±9.75 58±8.75 70.8±9.63 11.25 * age of 1 st farrowing (days) 408±6.9 462±5.59 466±3.21 459±3.12 448.5±4.70 12.52 ns litter size at birth (nos) 11.4±1.6 10.5±0.59 10.7±0.98 7.56±1.32 10.04±1.11 11.57 ns wt. of piglets at birth (kg) 1.1±0.08 1.2±0.05 0.97±0.86 0.92±0.87 1.02±0.46 8.06 ns litter size at weaning (nos) 9.4±1.01 8.28±0.96 6.75±1.24 5.98±0.89 7.6±1.05 12.29 ** litter wt. at weaning (kg) 8.1±0.75 9.2±0.28 5.64±0.97 5.54±0.97 7.27±0.62 7.06 ** farrowing interval (days) 184±32.9 192±12.28 207±12.32 197±7.65 195.20±17.34 13.36 ** litter index sow/year 1.8±0.24 1.8±0.05 1.5±0.98 1.6±0.23 1.7±0.40 4.95 * piglet mortality % 18±7.57 19±7.75 22±0.96 21.±0.85 20.6±4.07 13.24 ns notes: ns = non significant * = significant at 1% level ( p value < 0.01 ), ** = significant at 5% level ( p<0.05). of 10.4% pre-weaning mortality (ghimire and aryal, 1999). panta (2015) reported higher mortality percentage 47%. season of birth and managemental stress significantly affected the health traits. the inbreeding affects fitness traits. the increase in piglet's mortality may be due to inbreeding. according to neopane and kadel (2008) weight of piglet at birth of hurrah local breed is 0.8kg and adult weight is 45kg which do not match the finding of our research in terms of birth weight 0.92±0.87kg and adult body weight 55.9±4.0kg. similarly, age of i st mating, age of i st farrowing and farrowing interval were 335±26.8 days, 448.5±4.70 days and 197±7.65 days respectively which is higher than finding of dhakal (2012), pokharel (2012), pandey (2011) and thapa (2009) i.e. 241.73±22.13 days, 351.30±4.8 days and 180.30±0.3 days respectively. the higher value may be due to inbreeding problem arise in pig of regional agriculture research station, tarahara, sunsari, nepal but litter size at weaning (nos.) and litter size at birth were almost similar to the finding of pradhan (1999), neopane (2004). according to pokharel (2012), pandey (2011), adult body weight of local nagpuri breed was 50-60kg which match the finding of our research i.e. 59.8±8.4kg. similarly, age at i st mating, age at i st farrowing, farrowing interval were 322±23.3 days, 466±3.21 days and 207±12.32 days which were observed higher than those of dhakal (2012) 213.5±11.14 days, 335.54±9.91days and 170.96±7.28 days respectively. according to tummaruk et al. (2001), age at i st farrowing and farrowing interval of hampshire pig were found 386 days & 173.4 days which were observed higher in this research finding i.e. 462±5.59 days & 192±12.28 days but litter size at birth & litter size at weaning were almost similar i.e. 10.5±0.59 nos. & 8.28±0.96 nos. respectively. litter size at birth & litter size at weaning were observed higher than those of kumar et al. (2018). similarly, according to thapa et al. (2018), farrowing interval , litter size at birth and litter size at weaning of hampshire pig were found 174 days, 8.31 nos. and 7.56 nos. which was lower than our research findings i.e. 192±12.28 days, 10.5±0.59 nos. and 8.28±0.96 nos. respectively. the adult mean body weight was 127.6±21.49 kg which was less than kumaresan et al. (2006). according to him, average daily weight gain of hampshire pig was 185 kg. this may be due to inbreeding problem and location of farm is in sunsari district which lie in terai region and climate over there is hot but hampshire breed performed better in cold places and could be suitable for temperate environment (thapa et. al., 2018). age at i st mating of hampshire was 301±42..42 days. age at i st service in any of the pig farms should be between 8 & 9 months and if it crosses more than 10 months , then it is advisable to cull these animals (rymer & grand, 2009). exotic breed hampshire has the highest body weight than synthetic breed pakhribas followed by pakhribas× nagpuri, hampshire× hurrah, nagpuri and hurrah. from above findings, the highest body weight was found in hampshire followed by pakhribas, pakhribas×nagpuri, hampshire×hurrah, nagpuri and hurrah. exotic breed like hampshire has higher body weight than synthetic breed pakhribas, their cross and local breeds. similarly, reproductive traits like age at i st mating, age of i st farrowing & piglet mortality were observed highest in local breeds nagpuri & hurrah followed by hampshire and the lowest in pakhribas. litter size at birth, litter size at weaning & weight of piglets at birth were observed highest in synthetic breed pakhribas followed by exotic breed hampshire & nagpuri and the lowest in hurrah. weight at i st mating & litter weight at weaning were higher in exotic breed hampshire followed by pakhribas and the lowest in local breed. farrowing interval was observed the highest in local breed followed by exotic breed and the lowest in synthetic breed pakhribas. litter index sow/year is higher in synthetic & exotic breeds and lower in indigenous breed. conclusion the result of this research revealed that the highest body weight was observed in exotic breed hampshire followed by synthetic breed pakhribas, pakhribas × nagpuri, hampshire × hurrah, nagpuri and the lowest in hurrah. exotic breed like hampshire has higher weight than synthetic breed pakhribas, their crosses and local breeds. similarly, reproductive traits like age at i st mating, age of i st farrowing & piglet mortality was observed the highest in local breed nagpuri & hurrah followed by hampshire and the lowest in pakhribas. litter size at birth, litter size at weaning & weight of piglets at birth were observed the highest in synthetic breed pakhribas followed by exotic breed hampshire & nagpuri and the lowest in hurrah. weight at i st mating & litter weight at weaning were observed the highest in exotic breed hampshire followed by pakhribas and the lowest in local breed. farrowing interval was observed the highest in local breed followed by exotic breed and the lowest in synthetic breed pakhribas. litter index sow/year is higher in synthetic & exotic breed and lower in indigenous breed. acknowledgment this study was supported through a grant from the nepal agricultural research council (narc). references abd 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existing farming system of indigenous hurrah breed of swine in nawalparasi district of nepal. b.v.sc.&a.h. internship final report (unpublished), tu/iaas, rampur, chitwan. 52. pradhan s m (1999). study of morphological characters of native pigs from salyan and pyuthan districts of mid western development region of nepal. proceeding of third national workshop on livestock and fisheries research in nepal. nepal agricultural research council khumaltar, lalitpur, nepal. 241-248. rymer ts and grant w (2009). latest international developments in genetics and research & development from the jsr technical team. jsr genetics limited. rymer ts, grant w (2009). latest international developments in genetics and research & development from the jsr technical team. jsr genetics limited. thapa l, timsina mp (2018). production performance of crossbred pigs in government farm. bhutan journal of animal science. 2,1,37-41. thapa db (2009). phenogenetic study on litter and reproductive traits of exotic swine breeds under farmers’ managed condition. m.sc. thesis (unpublished), tu/iaas, rampur, chitwan. 91. tummaruk p, lundeheim n, einarsson s, dalin am (2001). effect of birth litter size, birth parity number, growth rate, backfat thickness and age at first mating of gilts on their reproductive performance as sows. animal reproduction science. 66, 225-237. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (2), pp. 001-011, february, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs lifu zhao1, jianzhou li1, guoliang lv1, anye zhang 1, pengcheng zhou1, ying yang1, xiaoping pan1, xiaopeng yu1, yimin zhang1, shusen zheng2, yu chen1, yuemei chen1, chengbo yu1, weibo du1, tao song3, jiansheng xu3, yang yu3 and lanjuan li1* 1 state key laboratory for diagnosis and treatment of infectious diseases, first affiliated hospital, school of medicine, zhejiang university, hangzhou 310003, china. 2 department of surgery, state key laboratory of combined multi-organ transplantation, first affiliated hospital, school of medicine, zhejiang university, hangzhou 310003, china. 3 institute of electrical engineering, chinese academy of sciences, beijing 100190, china. accepted 25 october, 2018 an appropriate cell source is essential for the clinical application of bioartificial liver (bal) system. this study aimed to test a reversibly immortalized human hepatocyte line (hepli-4) in our newly validated choanoid fluidized bed bioreactor based bal in pigs with fulminant hepatic failure (fhf). 15 fhf pigs were allocated to three groups: a bal group receiving bal treatment with hepli-4 cells; a sham bal group receiving cell-free bal treatment; and a fhf group receiving intensive care only. expression of liver-specific genes in hepli-4 cells before and after bal was analyzed, adult human hepatocytes acted as a reference. in bal group, fischer index was higher and serum indirect bilirubin level was lower compared with two control groups. survival time in bal group was longer than that in two control groups, but the difference was not statistically significant. gene expression analysis showed that the transcript levels of liver-specific genes in hepli-4 were retained after bal, but significant variations were observed between hepli-4 and human hepatocytes. hepli-4 showed beneficial metabolic effects on fhf pigs in bal, but is still not an appropriate cell source for bal. more insights into interpreting the conditions for hepatocyte differentiation are needed. key words: reversible immortalization, hepatocytes, bioartificial liver, fulminant hepatic failure. introduction facing the world-wide donor organ shortage, bioartificial liver (bal) system has been anticipated to be a bridge to *corresponding author. e-mail: ljli@zju.edu.cn. tel/fax: 86-57187236759. abbreviations: bal, bioartificial liver; fhf, fulminant hepatic failure; ac, alginate-chitosan; perv, porcine endogenous retroviruses; sv40 lt, simian virus 40 large t antigen; bcaas, branched chain amino acids; aaas, aromatic amino acids; gs, glutamine synthetase; ugt1a1, uridine diphosphate glucuronyltransferase; alb, albumin; gst-p, glutathione stransferase p; β-actin, human beta-actin; he, hepatic encephalopathy; rt-pcr, reverse transcription-polymerase chain reaction. liver transplantation or to provide a chance for the native liver to recover in case of liver failure (strain and neuberger, 2002; carpentier et al., 2009). bioreactor and cell source are two main issues of bal (pless, 2010). fluidized bed bioreactor is promising for liver support (dore and legallais, 1999; coward et al., 2009; desille et al., 2001; david et al., 2004). we recently designed a bal system based on a choanoid fluidized bed bioreactor filled with alginate-chitosan (ac) encapsulated primary porcine hepatocytes and proved it could prolong the survival of pigs with fulminant hepatic failure (fhf) induced by d-galactosamine injection (lv et al., 2011). the potential risk of zoonotic transmission, however, might exist, which limits its clinical application (patience et al., 1997). researchers in our group also proved that porcine endogenous retroviruses (perv) can pass through the immune barrier in ac microbeads (yang et al., 2010). therefore, developing a safe cell source for bal is necessary. widespread clinical use of primary human hepatocytes in bal is almost impossible because healthy donor livers are so scarce that only organ or tissue discarded from transplantation are available. among non-primary cell sources, several human liver tumor derived cell lines, including gs-hepg2 (glutamine synthetase, gs), hepg2-gs-3a4 and flc-4, were used in bal in large animal models. prolongations of survival were achieved in these studies (kanai et al., 2007; enosawa et al., 2001, 2006; wang et al., 2005). however, none of them has been so far applied to clinical trials. poor differentiation and the potential risk of tumor transmigration might be the main hurdles (nyberg et al., 1994). only the c3a cell line, a subclone of hepg2, was used in a pilot-controlled clinical trial (ellis et al., 1996). unfortunately, no improvement in either survival or biochemical parameters is demonstrated. for several years, we have been dedicated to establishing immortalized hepatocyte line (pan et al., 2010; li et al., 2005). although these cell types have unlimited expansion capabilities in vitro, continuous expression of simian virus 40 large t antigen (sv40 lt) might be tumorigenic (kobayashi et al., 2001; hahn et al., 1999; woodworth et al., 1988). to solve this problem, researchers in japan established a reversibly immortalized human hepatocyte line (nknt-3) in the year 2000 (kobayashi et al., 2000). it shows that the immortalizing oncogene, e.g., sv40 lt, can be excised by a cre/loxp site-specific recombination. then, liver-specific genes expression can be increased later. however, obtaining a large number of reverted hepatocytes was hindered by the secondary gene transfer until the drug-mediated cre/loxp recombination was applied (totsugawa et al., 2007). with the drug-medicated cre/loxp recombination, we established another reversibly immortalized cell line (hepli-4) by transfection of primary human hepatocytes with the retrovirus containing sv40 lt flanked by a pair of loxp recombination targets. thus, enough number of reversibly immortalized hepatocytes can be achieved to equip our newly validated choanoid fluidized bed bioreactor based bal system. in order to demonstrate the clinical potential of our hepatocytes in bal, we carried out experiments in large animal models. materials and methods this study protocol was ratified by the animal ethics committee of zhejiang university and the ethics committee of the first affiliated hospital, zhejiang university school of medicine. large-scale cultivation and encapsulation of hepli-4 cells the hepli-4 cell line was developed by immortalization of primary human hepatocytes with the retrovirus containing sv40lt flanked by a pair of loxp recombination targets, and the hepli-4 cells were subsequently transfected with the recombinant vector containing cre-ert2 fusion protein gene derived from pcag-creert2 plasmid (kindly gifted by addgene, usa). reverted hepli-4 cells were capable of expressing liver-specific genes in vitro and prolonging the survival of common bile duct ligated mice after intrasplenic transplantation (pan et al., 2009). there was also no evidence for tumorigenesis of reverted hepli-4 cells in nude mice within three months after subcutaneous transplantation. the cultivation and reversion procedures were previously described in detail (totsugawa et al., 2007). briefly, hepli-4 cells at the 30th passage (figure 1a) were expanded in roller bottles (bellco, usa) with dulbecco modified eagle medium (dmem; gibco, usa) supplemented with 10% (v/v) fetal calf serum (gibco, usa) using a cell production roller bottle apparatus (bellco, usa) (figure 1b). after achieving the required number of cells, sv40 lt genes were removed by keeping the cells in culture media containing 500 nm 4-hydroxytamoxifen (4-oht; sigma-aldrich, usa) for five to seven days. to verify the efficiency of excision of the sv40 lt gene, the mrna expression of sv40 lt in immortalized and reverted hepli-4 cells was analyzed by reverse transcription-polymerase chain reaction (rt-pcr). the primer sequence and amplification condition were described in our previous study (pan et al., 2010). as shown in figure 1c, sv40 lt gene was almost not detected in reverted hepli-4 cells. thereafter, reverted hepli-4 cells were trypsinized. the cell viability assessed by trypan blue exclusion was above 95%. we still adopted the single-stage ac encapsulation procedure which had previously been reported by us (lv et al., 2011). briefly, about 3.0 × 10 9 hepatocytes were resuspended in 300 ml 1.7% sodium alginate (sigma-aldrich, usa) solution. the mixture was extruded through an electrostatic microencapsulator (300 µm nozzle; nisco, switzerland) into a 0.7% chitosan solution (jinan haidebei marine bioengineering co. ltd, china). the gelation process lasted for 30 min, of which microbeads of 600 to 1000 µm in diameter were obtained (figure 1d). experimental animals male chinese experimental miniature pigs weighting 10 to 15kg were purchased from china agriculture university. all pigs were kept in separate cages under standard conditions and fed standard laboratory chow. induction of fhf d-galactosamine (shanghai hanhong chemical co., ltd, china) induced pig model of fhf was used in this experiment. the experiment work flow is shown in figure 2. catheterization, which had been previously described (li et al., 2006), was carried out under general anesthesia achieved by continuous intravenous injection of diprivan (astrazeneca, italy) at a rate of 2.5 mg/kg/h. then, pigs awakened from anesthesia were sent back to their cages. twenty-four hours after the catheterization, without anesthesia, d-galactosamine was delivered to pigs via the venous catheter at a dose of 1.5 g/kg body weight. experimental groups as shown in figure 2, 15 fhf pigs were allocated to three groups: a bal group (n = 5), receiving bal treatment with hepli-4 cells; a sham bal group (device control, n = 5), receiving cell-free bal treatment; and a fhf group (baseline control, n = 5), only receiving intensive care under general anesthesia. these three types of interventions were initiated 18 h after d-gal injection and lasted for figure 1. large-scale cultivation and encapsulation of hepli-4 cells. (a) morphology of hepli-4 cells under optical microscopy. (b) large-scale cultivation of hepli-4 cells in roller bottles. (c) the mrna expression of sv40 lt in hepli-4 cells before and after reversion. sv40 lt mrna was detected in immortalized hepli-4 cells, whereas almost not detected in the reverted cells by rtpcr analysis. (d) optical micrograph of alginate–chitosan (ac) microbeads containing hepli-4 cells. cell density was approximately 1×10 7 /ml. scale bar = 500 µm. figure 2. experimental flow chart. 6 h according to previous studies (lv et al., 2011; li et al., 2006). bioreactor the bioreactor (figure 3a; chinese patent no: zl 200710070279.0) has a funnel-shaped structure with filters of 200 and 600 mesh/inch fixed to the bottom and top, respectively. to stop potential cell chips from entering the body, a 3 µm strainer was installed in the outlet. the volume inside the bioreactor was about 500 ml. other parameters were detailed in our previous report (lv et al., 2011). bal treatment the bal system (figures 3b and c) which had previously been figure 3. bioreactor and bal treatment. (a) schematic diagram of the choanoid fluidized bed bioreactor loaded with microbeads. 1, plasma inlet; 2, plasma outlet; 3, microbeads inlet; 4, microbeads outlet; 5, filters; 6, strainer (pore size: 3 µm). the solid arrows and the hollow arrows show the direction of plasma flow and the trajectory of microbeads in the bioreactor, respectively. (b) photograph of the bal treatment. (c) structural diagram of the bal system. bal, bioartificial liver. reported by us consisted of a plasma separation unit and a bioreactor unit (lv et al., 2011). both were driven by a series of roller pumps. under general anesthesia, arterial blood was drawn from pig’s body at 20 to 25 ml/min and pumped into the plasma separator (op-02w, asahi-kasei, japan) through which plasma was separated from the cellular components at 8 to 10 ml/min into the reservoir. in the bioreactor unit, plasma was perfused through the bioreactor containing 3.0 × 10 9 encapsulated hepli-4 cells at 25 to 35 ml/min. at the same time, the purified plasma was returned to the pig’s body at a rate equal to plasma separation. to prevent blood clotting, the first dose of heparin (100 u/kg) was administered intravenously and followed by sustained injection of heparin into the extracorporeal circulation at a rate of 40 u/kg per hour. in sham bal treatment, bioreactor contained cell-free microbeads with the same volume. observed respectively. trypan blue exclusion test was used to assess the viability of encapsulated hepatocytes before and after bal treatment. microbeads were lysed by a solution composed of 0.2 mol/l nahco3 and 0.06 mol/l na3c6h5o7•2h2o (ph = 7.8) (xue et al., 2004), then we used 0.4% trypan blue solution to dye dead cells according to the method described previously (david et al., 2004). also, comparison between the viability of encapsulated hepli-4 cells before and after bal was performed by a previously reported mtt method with some modifications (haque et al., 2005). in brief, 15 microbeads were incubated in a 96-well plate with 100 µl media and 25 µl mtt per well for 24 h. thereafter, the supernatants were substituted for 100 µl dimethyl sulfoxide (dmso). within 30 min, the absorbance of light at 595 nm was measured using a dtx 800 multi-mode detector (beckman coulter, usa). we repeated these experiments three times. test of microbead integrity and cell viability the percentage of intact beads before and after bal treatment was determined under light microscope. 100 beads in five samples were characterization of liver-specific genes microbeads containing hepli-4 cells before and after bal treatment were lysed by the solution mentioned above. total rna was table 1. microbead integrity and cell viability before and after bal treatment. paramater pre-bal post-bal p value integrity of microbeads (percentage of intact beads, %, n = 5) viability of encapsulated hepli-4 cells (trypan blue exclusion, percentage of live cells, %, n = 5) viability of encapsulated hepli-4 cells (mtt test, od value, n = 5) 99.2 ± 0.84 98.4 ± 1.14 0.24 92.2 ± 1.79 90.8 ± 1.92 0.27 0.73 ± 0.08 0.69 ± 0.08 0.45 values are expressed as mean ± standard deviation. difference between groups was analyzed using student's t-test. the experiments were repeated three times. extracted from hepli-4 cells using trizol reagent (invitrogen, usa). to provide a reference, adult human liver samples obtained during the course of graft reduction for liver transplantation were included in the analysis. rt-pcr containing 1 µg total rna was carried out using two-step rt-pcr kit (qiagen, usa) according to manufacturer's protocols. primer sequences for amplification of liver-specific genes and internal control gene, including gs (535 bp), uridine diphosphate glucuronyltransferase (ugt1a 1,495 bp), albumin (alb, 576 bp), glutathione s-transferase p (gst-p, 496 bp) and human beta-actin (β-actin, 610 bp), were detailed in previous report (totsugawa et al., 2007). the thermal cycle involved one cycle of initial denaturation at 95°c for 2 min, followed by 30 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s and elongation at 72°c for 40 s. amplification was concluded with a one cycle of extension program at 72°c for 5 min. the amplification products were run on 1% agarose gels stained with ethidium bromide. measurement of parameters the three blood sampling time points were the time just prior to the d-gal injection, before and after the 6-h intervention, respectively (0, 18 and 24 h after d-gal injection). biochemical parameters, including serum bilirubin, albumin, aminotransferases, total cholesterol, lactate, glucose, creatinine, urea nitrogen and prothrombin time, were examined in clinical laboratories. analysis of serum amino acids was performed by high-performance liquid chromatographic (hplc, hitachi, japan). the moment of d-gal injection was the starting point for survival time. death of animal was defined as the cessation of breathing, cardiac arrest and fixed pupil dilation. statistics values are expressed as mean ± standard deviation. group differences were analyzed using one-way analysis of variance (anova) and student's t-test. survival time was compared by kaplan-meier analysis (log-rank significance test). p value <0.05 was defined as significant. all data were analyzed using spss for windows version 15.0. results microbead integrity and cell viability as shown in table 1, having experienced the entire bal treatment, few beads lost its integrity (percentage of intact beads reduced from 99.2 ± 0.84 to 98.4 ± 1.14%, n = 5) (p>0.05). though the viability of encapsulated hepli-4 cells showed a slight downward trend by both trypan blue exclusion (%, from 92.2±1.79 to 90.8±1.92, n = 5) and mtt test (od, from 0.73 ± 0.08 to 0.69 ± 0.08, n = 5) after bal treatment, the differences were not statistically significant (p>0.05). expression of liver-specific genes as shown in figure 4, hepli-4 cells that had experienced the bal treatment still retained liver-specific gene expression. β-actin was an internal control, while the mrna levels of mature human hepatocytes acted as a reference. in hepli-4 cells, the mrna level of gs was not less than that in adult human liver, while the mrna level of ugt1a1 was somewhat lower. moreover, the alb mrna level was extremely lower and gst-p mrna level was extremely higher in hepli-4 cells compared with those in human liver. treatment process and survival throughout the 6-h bal or sham bal treatment process, no hemodynamic instability and bleeding episodes occurred in pig; no blood coagulation in extracorporeal circulation was observed. all pigs died of liver failure. as shown in figure 5, survival time in bal group (69.4 ± 16.5 h) was longer than that in fhf group (56.2 ± 13.3 h) and sham bal group (56.8 ± 18.2 h), although the difference was not statistically significant (p>0.05). biochemical parameters fischer index is calculated as a ratio of branched chain amino acids (bcaas; valine, isoleucine and leucine) to aromatic amino acids (aaas; /tyrosine and phenylalanine) in plasma. as shown in figure 6a, after d-galactosamine injection, fischer index declined progressively in all groups. bal treatment made the trend slow. at the end of the 6-h intervention (24 h after dgalactosamine injection), fischer index in bal group (2.32 ± 0.42) was significantly higher than those in sham bal group (1.46 ± 0.34) and fhf group (1.49 ± 0.28) (p<0.05). as shown in figure 6b, gradual increase of serum indirect bilirubin was observed in two control groups, while bal treatment figure 4. liver-specific genes expression in encapsulated hepli-4 cells before bal treatment (1), encapsulated hepli 4 cells after bal treatment (2) and adult human hepatocytes (3). bal, bioartificial liver. figure 5. survival curves for the fhf pigs in bal group, sham bal group and fhf group. the time of d-gal injection was the starting point for survival time. survival curves did not show significant prolongation of survival in bal group compared with two control groups by kaplan. figure 6. biochemical parameter changes caused by bal treatment. (a) fischer index in all groups (*p = 0.005 versus both control groups by one-way anova. fischer index = valine + isoleucine + leucine/tyrosine + phenylalanine. (b) serum indirect bilirubin levels in all groups (*p = 0.025 versus both control groups by oneway anova). bal, bioartificial liver; fhf, fulminant hepatic failure. prevented its further increase. the serum level of indirect bilirubin at 24 h after d-galactosamine injection in bal group (8.40 ± 2.97 µmol/l) was significantly lower compared with sham bal group (12.80 ± 1.92 µmol/l) and fhf group (12.60 ± 2.41 µmol/l) (p<0.05). biochemical parameters with non-statistically significant differences (p>0.05) between three groups are listed in table 2. among them, after d-galactosamine injection, table 2. biochemical parameters with non-statistically significant differences between three groups. parameter group baseline (0 h) pre-bal (18 h) post-bal (24 h) bal 3.26 ± 1.02 22.68 ± 6.48 34.24 ± 8.52 total bilirubin (µmol/l) sham bal 2.8 ± 0.45 24.6 ± 6.07 38 ± 13.82 fhf 2.38 ± 0.43 25.4 ± 9.11 37.88 ± 10.5 bal 11.56 ± 1.15 21.6 ± 2.75 65.98 ± 33.27 prothrombin time (s) sham bal 12 ± 2.32 23.16 ± 4.56 65.22 ± 32.75 fhf 11.62 ± 1.44 22.2 ± 2.32 53.38 ± 7.82 bal 2.44 ± 0.54 5.42 ± 0.8 6.46 ± 1.37 lactate (mmol/l) sham bal 2.18 ± 0.59 5.06 ± 0.89 6.28 ± 1.28 fhf 2.66 ± 0.41 5.88 ± 0.91 7.25 ± 1.25 bal 42.4 ± 9.96 100.6 ± 49.41 171.2 ± 47.52 alanine aminotransferase (iu/l) sham bal 43.6 ± 3.13 90 ± 11.94 164.4 ± 77.06 fhf 46.4 ± 9.56 92 ± 25.44 170.6 ± 40.16 bal 5.88 ± 1.15 4.54 ± 0.58 3.61 ± 1.59 glucose (mmol/l) sham bal 5.66 ± 0.51 4.7 ± 1.16 2.53 ± 0.4 fhf 5.45 ± 0.64 4.15 ± 0.44 2.71 ± 0.91 bal 2.18 ± 0.16 1.37 ± o.39 0.94 ± 0.3 total cholesterol (mmol/l) sham bal 1.89 ± 0.31 1.24 ± 0.4 0.81 ± 0.24 fhf 1.88 ± 0.16 1.27 ± 0.35 0.85 ± 0.18 bal 37.18 ± 5.6 38.12 ± 4.14 33.46 ± 3.5 albumin (g/l) sham bal 39.02 ± 4.78 37.72 ± 5.67 33.62 ± 5.28 fhf 38.76 ± 3.15 39.52 ± 4.36 37.58 ± 4.24 bal 37.2 ± 13.44 46 ± 5.96 44.8 ± 6.38 creatinine (µmol/l) sham bal 40.4 ± 14.29 41.8 ± 4.27 43 ± 7.84 fhf 39.4 ± 5.22 46.2 ± 11.65 44.6 ± 10.97 bal 3.35 ± 0.66 3.01 ± 0.44 2.98 ± 0.52 urea nitrogen (mmol/l) sham bal 3.91 ± 1.37 3.75 ± 0.52 3.58 ± 0.94 fhf 3.35 ± 0.6 3.2 ± 1.13 3.15 ± 0.97 values are expressed as mean ± standard deviation. difference between groups was analyzed using one-way anova. the levels of serum total bilirubin, prothrombin time, lactate and alanine aminotransferases increased gradually. on the other hand, the levels of glucose and total cholesterol decreased gradually, while the levels of albumin, creatinine and urea nitrogen did not change significantly. discussion to test the efficiency of a human derived hepatocyte line, we chose pigs as the animal model due to the high metabolic similarities between human and pig hepatocytes (donato et al., 1999). drug-induced fhf was applied to pigs because it met the criteria proposed by terblanche and hickman (1991). also, it closely resembled human fhf (kalpana et al., 1999). in this study, progressively prolonged prothrombin time increa-sed serum total bilirubin and lactate levels and decreased serum glucose level, and fischer index were observed in all pigs. histological examination showed massive hepatic necrosis in all of them (data not shown). it was therefore confirmed that all pigs died of fhf. studies related to the encapsulation of mammalian cells in ac microbeads have shown that ac membrane provides mechanical stability, mass transfer capacity and immunoisolation in vitro (baruch and machluf, 2006; yu et al., 2009; haque et al., 2005). in this study, cell viability and microbead integrity were maintained at acceptable levels after the 6-h perfusion of fhf pig’s plasma and this was consistent with our previous study (lv et al., 2011). also, the mrna levels of gs, ugt1a1, alb and gst-p in encapsulated hepli-4 cells were all retained after bal treatment. in this study, 3×10 9 encapsulated immortal hepatocytes were used to treat an fhf pig weighing 10 to 15 kg. the cell mass should be adequate assuming the cells were fully-functional (hoekstra and chamuleau, 2002). however, it was disappointing to note that the prolon-gation of survival by bal treatment was not statistically significant. this problem might lie mainly in cell functions. the results from rt-pcr analysis could explain partially. though we only adopted a semi-quantitative analysis, it could be intuitively seen in figure 4 that the expression of liverspecific genes of hepli-4 significantly varied with adult human hepatocytes, especially since the hepli-4 had an extremely lower transcript level of mature hepatocytes marker (alb) but a higher level of immature hepatocytes marker (gst-p) than human liver. thus, we concluded that hepli-4 cells were not fully differentiated after reversion, which was also found by other studies (deurholt et al., 2009; chamuleau et al., 2005). it was demonstrated by quantitative rt-pcr in these studies that the transcript levels of some mature hepatocytes markers (albumin, α-1-antitrypsin, and transferrin) in reverted nknt-3 cells were only equivalent to 0.1 to 1% of those in human liver, while the mrna level of gst-p was much higher. another reversibly immortalized human hepatocyte line, 16-t3, has never been compared with mature human hepatocytes at genetic level (totsugawa et al., 2007). in our previous studies, bals charged with primary porcine hepatocytes could reduce serum lactate and stabilize blood glucose in fhf pigs (lv et al., 2011; li et al., 2006). however, this phenomenon was not observed in the current experiment. this revealed that there are some other functional deficiencies in hepli-4 cells. although obvious defects existed, the relatively appropriate expression of detoxification-related genes (gs and ugt1a1) in hepli-4 cells prompted us to continue the study. gs, an enzyme responsible for ammonia elimination, was considered essential for liver function (kobayashi et al., 2000). some researchers had modified hepg2 cells to realize high expression of gs (tang et al., 2008; enosawa et al., 2000). although the expression of gs in hepli-4 cells was not weaker than that in human liver, we failed to reveal their ammonia removal efficiencies in fhf pigs due to the shortage of test equipments. ugt1a1 is the only physiological enzyme that can transform water-insoluble indirect bilirubin into water-soluble direct bilirubin by conjugating it with glucuronic acid (strassburg et al., 2008). thus, bilirubin can be excreted into bile duct and eliminated from the body. though the mrna level of ugt1a1 in hepli-4 cells was somewhat lower than human liver, we did observe a decrease of serum indirect bilirubin after bal treatment compared with two control groups. however, the levels of serum total bilirubin at 24 h after dgalactosamine injection showed no significant difference between three groups. this hurdle cannot be conquered because all of the current bioreactors lack a biliary system capable of collecting bile and moving it out of extracorporeal circulation. nevertheless, we proved that encapsulated hepli-4 cells could fulfill the function of bilirubin glucuronidation in our bal system. also, it can be inferred that ac membrane is permeable to pig serum albumin for bilirubin is an albumin-bound toxin (falkenhagen et al., 1999). in addition to bilirubin, ugt1a1 has several other substrates, such as hormones and drugs (strassburg et al., 2008). perhaps these functions benefited on correcting the metabolic disorders in fhf pigs, but they were not reflected in routine biochemical tests and survival. studies have shown that hepatic encephalopathy (he) is associated with an imbalance in plasma bcaas and aaas; fischer index and the grade of he are negatively correlated (koivusalo et al., 2008; fischer et al., 1976). in this study, bal treatment tended to normalize the fischer index in pigs with fhf. however, it is difficult to evaluate the mental status of an animal accurately, especially in the early stage of he. the method of reversible immortalization was once encouraging (kobayashi, 2009, 2000; totsugawa et al., 2007). however, our studies showed that hepatocytes generated by this method were poorly differentiated in vitro. the newly established immortalized human fetal hepatocyte line, cbal111, is facing the same dilemma (poyck et al., 2008; deurholt et al., 2009). even the stem cell derived hepatocytes, which are now considered promising sources for liver replacement therapy, still have functional deficiencies which are difficult to overcome (dalgetty et al., 2009; kung and forbes, 2009). no wonder some researchers hold the view that no cell source so far is suitable for bal support (pless, 2010; chamuleau et al., 2005). probably, clinical trials of bals can be carried out only on the condition that great progress is made in the promotion of hepatocyte differentiation in vitro. maybe future studies should focus on such issues as differentiation promoting factor, matrix and co-culture, so that an in vivo-like environment can be provided (chamuleau et al., 2005). in 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(in chinese with english abstract). yang q, liu f, pan xp, lv g, zhang a, yu cb, li l (2010). fluidizedbed bioartificial liver assist devices (blads) based on microencapsulated primary porcine hepatocytes have risk of porcine endogenous retroviruses transmission. hepatol. int. 4(4): 757-761. yu cb, lv gl, pan xp, chen ys, cao hc, zhang ym, du wb, yang sg, li lj (2009). in vitro large-scale cultivation and evaluation of microencapsulated immortalized human hepatocytes (hepll) in rollebottles. int. j. artif. org. 32(5): 272-281. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 7 (1), pp. 001-008, january, 2019. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper isolation, sequence identification and tissue expression profile of a novel ribokinase gene (rbks) from chinese banna mini-pig inbred line (bmi) jinlong huo1,2,3, pei wang2,3, yongwang miao3, hailong huo1,4, lixian liu4, yangzhi zeng2,3 and heng xiao1* 1 faculty of life science, yunnan university, kunming 650091, yunnan, china. 2 key laboratory of banna mini-pig inbred line of yunnan province, kunming 650201, yunnan, china. 3 faculty of animal science and technology, yunnan agricultural university, kunming 650201, yunnan, china. 4 department of husbandry and veterinary, yunnan vocational and technical college of agriculture, kunming 650031, china. accepted 23 november, 2018 the complete expressed sequence tag (cds) sequence of banna mini-pig inbred line (bmi) ribokinase gene (rbks) was amplified using the reverse transcription-polymerase chain reaction (rt-pcr) based on the conserved sequence information of the cattle or other mammals and known highly homologous swine ests. this novel gene was then deposited into ncbi database and assigned to accession number jf944892. sequence analysis revealed that the bmi rbks encodes a protein of 323 amino acids that has high homology with the ribokinase proteins of seven species: cattle (99%), horse (99%), orangutan (99%), human (89%), monkey (89%), rat (88%) and mouse (80%). the phylogenetic tree analysis revealed that the bmi rbks gene has a closer genetic relationship with the rbks genes of bovine and horse than with those of orangutan, human, monkey, rat and mouse. analysis by rt-pcr showed that bmi rbks gene was over-expressed in ovary and lung, moderately expressed in spleen, nerve fiber, large intestine and diencephalon, weakly expressed in heart, skin, muscle, small intestine, midbrain, kidney and fat, while almost silent in other five tissues. four microrna target sites were predicted in the cds of bmi rbks mrna for further study of this gene in the future. the 3d structure of the rbks by homology modeling was similar to that of human ribokinase (2fv7). our experiment will establish a foundation for further insight into this swine gene. key words: banna mini-pig inbred line (bmi), pig, ribokinase gene (rbks), tissue expression analysis, homology modeling. introduction ribokinase (rbks) is a key enzyme which catalyzes the phosphorylation of ribose to ribose 5-phosphate (r-p-5) using atp as a phosphate donor according to the * corresponding author. e-mail: xiaoheng@ynu.edu.cn. fax: +86-871-5033732. abbreviations: rbks, ribokinase gene; est, expressed sequence tag; cds, coding sequence; rt-pcr, reverse transcription-polymerase chain reaction; rmsd, root mean square deviation. reaction: exogenous ribose plays a number of roles in the biological metabolism. for example, ribose addition can help maintain higher levels of atp in rat heart and dog kidney during transplantation experiments (müller et al., 1998) and it leads to improvement of neurological symptoms in patients with adenylosuccinase deficiency (salerno et al., 1999). in order to trap them inside the cell and prepare them for further chemical reactions, ribose must first be converted into ribose-5-phosphate (bork et al., 1993). the phosphorylated ribose can then enter the pentose phosphate pathway (ppp) for energy production or be used as a carbon source for the synthesis of nucleotides, histidine and tryptophan (anderson and cooper 1969; lopilato et al., 1984). sequence analyses showed that the enzyme rbks belongs to the pfkb family of carbohydrate kinases (bork et al., 1993; wu et al., 1991). as another member, rbks contains two highly conserved sequence motifs, a glycine-rich area served as a substrate binding site near the n-terminus and a motif involved in atp binding and the catalytic center near the c-terminus (sigrell, et al., 1998, 1999). particularly, rbks is implicated in tissue-protective mechanisms against various ischemic insults. based on the above description of rbks gene, it is necessary to isolate this gene from pig for it is associated with energy metabolism, health and other important biological functions of animals. but until today, the porcine rbks has not been reported. surprisingly, the banna mini-pig inbred line (bmi) was exploited by yunnan agricultural university from 1980s based on the small-ear pigs at xishuangbanna, yunnan province, china. a pair of progenitors was a sow and her son. then, the propagation was conducted by means of highly full sibling or parent-offspring inbreeding and each generation underwent the strict selection. as heterozygotic genes were separated and recombined in the process of inbreeding, bmi has already owned six families and 18 substrains with different phenotypes and genotypes. due to their consistent genetic background and minor interindividual differences, bmi is considered as an ideal model organism for biological studies (crabbe et al., 2005; yu et al., 2004; zeng and zeng, 2005). the objective of this study was to isolate the full length coding sequence of bmi rbks gene according to the conserved sequence information of cattle or other mammals and highly homologous swine ests sequence information, conduct sequence analysis and some necessary function analysis of established nucleotide sequence, and finally examine the expression in a range of bmi tissues. these will provide a primary foundation for further research on this porcine gene. materials and methods samples collection, rna extraction and first-strand cdna synthesis three matured female bmi were slaughtered for sampling. fresh tissues (lymph node, midbrain, ovary, diencephalon, cerebrum, liver, kidney, spleen, heart, lung, nerve fiber, stomach, small intestine, large intestine, pancreas, skin, muscle and fat) were snap frozen in liquid nitrogen and stored at -80°c before use. the total rna was extracted using the rnaiso plus (takara, dalian) according to the manufacturer's instructions. to remove genomic dna contamination, total rna was digested with rnase-free dnase i (takara, dalian). three micrograms of rna were reverse transcribed with oligo (dt)18 primer and m-mlv reverse transcriptase (invitrogen, usa). the efficiency of reverse transcription was checked on 2% agarose gels stained with ethidium bromide. isolation of the bmi rbks gene the genbank rbks sequences for human (accession no. nm_022128), cattle (accession no. nm _001191271) and their highly homologous pig ests sequences: cx065544, cn154557, cn165374, ck461198, bx924148 and cn165735 were used to design a primer pair to amplify the complete coding sequence of rbks by using primer premier 5.0 software. the primers for bmi rbks gene were: 5'aat ggc cgc gtc tgg gga ac -3' and 5'-cag tca aaa cag gta aag ggg c -3'. reverse transcription-polymerase chain reaction (rtpcr) was performed to isolate the bmi rbks using the pooled cdnas from different tissues listed above. the 25 µl reaction system was: 2.0 µl cdna (25 ng/µl), 2.0 µl 2.5 mm mixed dntps (takara, dalian), 2.5 µl 10taq dna polymerase buffer (mg 2+ plus ), 0.5 µl 10 µm forward primer, 0.5 µl, 10 µm reverse primer, 0.25 µl taq dna polymerase (5 u/µl, takara, dalian), and 17.25 µl sterile water. the pcr program initially started with 94°c denaturation for 2 min, followed by 35 cycles of 94°c /30 s, 55°c /40 s, 72°c /1.5 min then 72°c extension for 10 min, and finally 4°c to terminate the reaction. after the pcr, the gene product was cloned into pmd18t vector (takara, dalian) and sequenced bidirectionally with the commercial fluorometric method. at least, five independent clones were sequenced. bioinformatics analysis sequence analysis of bmi rbks gene was performed using softwares in ncbi (http://www.ncbi.nlm.nih.gov) and expasy (http://www.expasy.org). the cdna sequence was predicted using the online genscan software (http://genes.mit.edu/genscan. html). putative protein theoretical molecular weight (mw) and isoelectric point (pi) prediction, signal peptide prediction, subcellular localization prediction and transmembrane topology prediction were performed using the compute pi/mw tool (http://us.expasy.org/ tools/pi_tool.html), signalp 4.0 server (http://www.cbs.dtu.dk/ services/signalp/), psort ii (http://psort.hgc.jp/) and tmhmm-2.0 server (http://www.cbs.dtu.dk/services/tmhmm-2.0/), respectively. web-based microrna (mirna) predicting program was used to locate conserved potential mirna targets (http://www.mirbase. org/). the blastp program and conserved domain architecture retrieval tool were used to search for similar proteins and conserved domain, respectively(http://www.ncbi.nlm.nih.gov/blast). the alignment of the nucleotide sequences and deduced amino acid sequences were computed using clusterx, and the phylogenetic tree was computed using the clustalx and mega 4.0 softwares with standard parameters. secondary structures of deduced amino acid sequences were predicted with sopma (http://npsa-pbil.ibcp.fr/). the 3d structures were predicted using discovery studio 3.1 software. semi-quantitative rt-pcr to characterize the rbks gene further, rt-pcr was conducted to determine its expression in 18 bmi tissues. to eliminate the effect of cdna concentration, we repeated the rt-pcr five times using 1, 2, 3, 4 and 5 µl cdnas as templates. we selected the housekeeping gene 18s rrna (nr_002170) as a positive control. the control primers used were: 5'ggacatctaagggcatcacag -3' and 5'aattccgataacgaacgagact -3'. the figure 1. rt-pcr result for bmi rbks gene. m, dl2000 dna marker; 1, pcr product. bmi rbks primers which were used to perform the semiquantitative rt-pcr for tissue expression profile analysis were the same as the primers for isolation rt-pcr above. the pcr reactions were optimized for a number of cycles to ensure product intensity within the linear phase of amplification. results cloning and identification of bmi rbks cdna through rt-pcr with pooled tissue cdnas for bmi rbks gene, the resulting pcr product was 976 bp (figure 1). this cdna nucleotide sequence analysis using the blast software at ncbi server revealed that bmi rbks gene was not homologous to any of the known porcine genes and it was then deposited into the genbank database under accession no. jf944892. the sequence prediction was carried out using the genscan software and results show that the 976 bp cdna sequence represents a single gene which encoded 323 amino acids. the complete cds and the encoded amino acids are presented in figure 2. physical and chemical characteristics of bmi rbks the theoretical isoelectric point (pi) and molecular weight (mw) were computed using the compute pi/mw tool. the pi and the molecular weight of bmi rbks are 5.16 and 34532.46, respectively. submitting the rbks protein sequence to signalp, the rbks protein had no nterminal signal peptide and is a non-secretory protein (petersen et al., 2011). using a hidden markov model algorithm, transmembrane topology prediction made by tmhmm program (moller et al., 2001), showed that bmi rbks was not a potential membrane protein. for subcellular localization analysis, the amino acid sequence was submitted to the psort ii program, and reinhardt’s method showed that bmi rbks was probably located in the cytoplasm with up to 94.1% probability (nakai and horton, 1999). prediction and analysis of structures and conserved domains of bmi rbks proteins often contain several domains, each of which had their own evolutionary origins and functions. examination using the conserved domain architecture retrieval tool of blast at the ncbi server (http:// www.ncbi.nlm.nih.gov/blast) indicated that bmi rbks contains one separated conserved domain-ribokinase (from 23 to 314 amino acid residues, figure 3). then, putative protein was analyzed using prosite (http:// expasy.org/prosite/) and smart (http://smart.emblheidelberg.de/) softwares. four kinds of sites were found, which were n-myristoylation sites (23-gscmtd-28, 53ggkgan-58, 56-ganqcv-61, 67-gaktsm-72, 238gaegcv-243, 267-gagdsf-272, 307-gtqssy-312), protein kinase c phosphorylation sites (33-tsr-35, 257tek-259), casein kinase ii phosphorylation sites (160tsle-163, 192-tlsd-195, 202-seae-205, 208-tgle-211, 246-srte-249, 287-slee-290) and n-glycosylation sites (200-nese-203, 285-nlsl-288). the prediction of secondary structure by sopma indicates that the deduced bmi rbks contains 105 alpha helices, 73 extended strands, 30 beta turns and 115 random coils (figure 4). homology modeling in order to better understand the detailed structures of bmi rbks, the homology modeling of rbks was performed to estimate its 3d structure using discovery studio 3.1 software. commonly, the root mean square deviation (rmsd) between corresponding atoms of the template protein and modeled protein is a used measure of similarity between two protein structures. the smaller the rmsd is between two structures, the more similar are these two structures. in protein structure prediction, one needs the rmsd between predicted and experimental structures for which a prediction can be considered to be successful. success is obvious only when the rmsd is as small as that for closely homologous proteins (<3 å) (reva et al., 1998). the rmsd value of bmi rbks and 2fv7b (b chain of human rbks) are 0.218 which indicates that the 3d structure of the bmi rbks was similar to that of the human ribokinase (2fv7 chain: b). furthermore, in the ribokinase domain of rbks, no differences were found among different species of animals in the shape and orientation in rbks on 3d structures (figure 5). the 3d structure analysis may provide a basis for further study of the relationship between structure and function of rbks. figure 2. the complete cdna sequence and amino acid sequence of the protein encoded by rbks (genbank accession number: jf944892). atg, start codon; tga, stop codon; capital letters, complete cds and amino acid sequence; gray highlighted nucleotide sequence, primers; pane, catalytic residues figure 3. the putative domains of the protein encoded by bmi rbks. location of potential mirna targets micrornas are noncoding single-stranded rna molecules of 17 to 24 nucleotides that can regulate gene expression by binding to the coding region of target mrnas (bartel, 2004; zeng et al., 2003). we used web-based microrna (mirna) predicting programs to locate conserved potential mirna targets: mirbase (http:// www.mirbase. org/). the results show that four sus scrofa micro rnas (ssc-mir-217, ssc-mir-196b-3p, sscmir1306-3p and ssc-mir-1306-3p) were found to have the predicted target sites (313ugcugcgacaggaacugcuucgau-336, 718ugaagggugugugaugcuguc738, 428accaucagcagagccaaagu figure 4. the secondary structure of the bmi rbks protein predicted by sopma. helices, extended strands,beta turns and random coils are indicated, respectively, with the longest, the second longest, the second shortest and the shortest vertical lines. figure 5. the tertiary structure of bmi rbks. (a) bmi rbks, (b) human 2fv7b, (c) the superimposition figure; bmi rbks (red); 2fv7b (green). 447 and 48-cggcggcaguggugguggug-67) in the bmi rbks sequence, respectively. analysis of sequence identity and evolutionary relationships of bmi rbks the deduced protein sequence of bmi rbks was submitted to generate blast reciprocal best hits, and similarity comparison revealed that bmi rbks protein has high homology with the rbks proteins of seven other species: cattle (91%), horse (91%), orangutan (91%), human (89%), monkey (89%), rat (88%) and mouse (86%) (figure 6). to evaluate the evolutionary relationships of bmi rbks with other species, we constructed a phylogenetic tree using dnastar, cluster, mega and dnaman softwares on the basis of the rbks amino acid sequences. the phylogenetic tree analysis revealed that the bmi rbks gene has a closer genetic relationship with the rbks genes of bovine and horse than with those of orangutan, human, monkey, rat and mouse (figure 7). mrna tissue-specific expression profile to check the relative expression levels of rbks mrna in various porcine tissues, semiquantitative rt-pcr was performed in 18 bmi tissues mentioned above. the continuously expressed gene, 18s, was used and served as figure 6. the alignment of the protein encoded by the bmi rbks and other seven kinds of rbks from cattle (np_001178200), horse (xp_001502121), orangutan (xp_002812214), human (np_071411), monkey (xp_001100564), rat (np_001102173) and mouse (edl37382). figure 7. the phylogenetic tree for several kinds of rbks protein from human, orangutan, monkey, bmi-pig, cattle, horse and mouse. figure 8. tissue expression profile of bmi rbks gene. the 18s expression level is used for the internal control. m, dl2000 dna marker; 1, lymph node; 2, midbrain; 3, ovary; 4, diencephalon; 5, cerebrum; 6, liver; 7, kidney; 8, spleen; 9, heart; 10, lung; 11, nerve fiber; 12, stomach; 13, small intestine; 14, large intestine; 15, pancreas; 16, skin; 17, muscle; 18, fat. an endogenous reference for determination of targeted mrna profiles. result reveals that bmi rbks gene was over-expressed in ovary and lung, moderately expressed in spleen, nerve fiber, large intestine and diencephalon, weakly expressed in heart, skin, muscle, small intestine, midbrain, kidney and fat and almost silent in lymph node, cerebrum, liver, stomach and pancreas (figure 8). discussion comparative genomics determines the relationship of genome structure and function of different species. researchers have learnt a great deal about the function of human genes by examining their counterparts in simpler model organisms such as the mouse and some results have revealed that virtually all (99%) of the protein-coding genes in humans align with homologues in mice, and over 80% are clear 1:1 orthologs (hardison, 2003; liu et al., 2008; yu et al., 2010; xi et al., 2011). this extensive conservation in protein-coding regions implied that the same protein-coding sequences may be expected in different mammals including pig. from the isolation of swine rbks gene, we can find that swine rbks is highly homologous with rbks of human, bovine and other mammals. this further validated that comparative genomics method is one useful tool to isolate the unknown genes especially the conserved coding region of genes for pig. from the alignment analyses for swine rbks protein, we also found that bmi rbks protein was not completely identical with human or other mammals. this implied that bmi rbks will have some differences in functions with those of human, bovine and other mammals. the phylogenetic tree analysis revealed that the bmi rbks gene has a closer genetic relationship with the bovine and horse rbks. therefore, we can use bovine and horse as model organisms to study the pig rbks gene or use pig as a model organism to study the bovine and horse rbks gene. most protein functions are regulated by phosphorylation/dephosphorylation, glycosylation/deglycosylation and bmi rbks protein has several kinds of functional sites (such as phosphorylation sites, glycosylation sites, myristoylation sites and ribokinase protein domain, which suggest that rbks protein plays important functional roles through these sites and domain. micrornas are small noncoding rna. they play a role in gene expression regulation by inhibiting translation of their target mrnas (bartel et al., 2004; zeng et al., 2003). their target predictions showed that four sus scrofa micrornas (ssc-mir-217, ssc-mir196b-3p, ssc-mir-1306-3p and ssc-mir-1306-3p) were found to have the corresponding target sites (313 ugcugcgacaggaacugcuucgau-336, 718-ugaagggugugugaugcuguc-738, 428-accaucagcagagccaaagu-447 and 48-cggcggcaguggugguggug-67) in the bmi rbks coding sequence. further investigation is needed to confirm whether corresponding mirna molecules can regulate the rbks gene expression in swine. rbks was discovered more than 50 years ago, and there has been much progress in understanding this important enzyme within the last decade, but the molecular mechanisms of its action and target remain a central unresolved problem for biochemists and pharmacologists. for this reason, the homology modeling of the bmi rbks protein was carried out using human ribokinase (2fv7 chain: b) as the template. our results indicate that the conserved domain (rbks, 17-323aa) of pig rbks protein mainly exists in the form of αβα sandwich domain and β-barrel. it suggests that these domains may play a pivotal role in rbks activity. this first pig structural model of rbks proved to be useful reference in designing studies aimed at understanding how rbks interacts with unknown protein partners for explaining its various functions. in this study, we not only cloned the cds sequences of the bmi rbks gene but also conducted the sequence analysis and tissue expression profiles analysis. from the tissue expression profile analysis, it can be seen that the gene was obviously differentially expressed in various tissues. as the researchers did not study functions at protein levels, there might be many possible reasons for differential expression of this porcine gene. the suitable explanation for this under the current conditions is that the biological activities associated with the functions of the gene were required in a different extent in different tissues at the same time. in summary, we firstly isolated bmi rbks gene and performed necessary functional analysis and tissue expression profile analysis. furthermore, several mirnas were found to have the corresponding target sites in the coding sequence of bmi rbks by theoretical prediction. the cdna clone, sequence information and function analyses of bmi rbks gene will be extremely important in elucidating the essential physiological function of rbks protein using bmi and other pigs as experimental animal models in the future. acknowledgment this study was supported by the national natural science foundation of china (grant no. 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scholars journals author(s) retain the copyright of this article. full length research paper in vitro antiviral activity of brazilian cerrado plant extracts against animal and human herpesviruses marina a. padilla1,2, isabela c. simoni1*, verônica moreira h. hoe1, maria judite b. fernandes1, clarice w. arns2, juliana r. brito3 and joão henrique g. lago4 1 centro de pesquisa e desenvolvimento de sanidade animal, instituto biológico, av. cons. rodrigues alves, 1252, cep 04014-900, são paulo, sp, brasil. 2 laboratório de virologia animal, universidade estadual de campinas, campinas, sp, brasil. 3 instituto de ciências ambientais, químicas e farmacêuticas, universidade federal de são paulo, são paulo, brazil. 4 centro de ciências naturais e humanas, universidade federal do abc, são paulo, brazil. accepted 16 january, 2023 the brazilian savanna known as “cerrado” is very rich in medicinal plants that are used by the local population for treatment of several illnesses. the herpesvirus is a serious problem worldwide, and affects both animal and human health. this work aimed to study the antiviral activity of eight extracts from plants natives of “cerrado” region against human (hsv-1), equine (eqhv-1) and swine herpesviruses (suhv-1). the results showed that all plant extracts: banisteriopsis variabilis, byrsonima intermedia, campomanesia xanthocarpa, erythroxilum deciduum, lacistema hasslerianum, ocotea pulchella, stryphodendron adstringens and xylopia aromatica presented antiviral activity against at least one herpesvirus. furthermore, it was observed a direct anti-herpes effect of extracts from b. variabilis and b. intermedia in non-toxic concentrations against all herpesviruses. b. intermedia crude aqueous extract showed the most promising results with selective index values of the 41.76 ± 0.04; 4.12 ± 0.1 and 193.97 ± 0.09 respectively against hsv-1, eqhv-1 and suhv-1. due to this, b. intermedia extract was also analyzed by hplc/ms allowing for the identification of gallic acid and quercetin as main compounds. key words: brazilian savanna, cerrado, byrsonima intermedia, antiviral, herpesvirus, gallic acid, quercetin. introduction the herpes viruses are a serious worldwide problem that can affect the health of both animals and human beings. the herpes simplex virus (hsv-1) is pathogenic to humans (wyler et al., 2017), whereas equine (eqhv-1) and swine herpesviruses (suhv-1), are responsible for causing serious diseases in horses and pigs respectively, resulting in large economic losses (wernike et al., 2013; gulati et al., 2016). these viruses belong to the subfamily alphaherpesvirinae, characterized by a rapid lytic viral cycle and the establishment of neuronal latency, which can be reactivated (riaz et al., 2017). the emerging of resistant virus strains to available drugs against human *corresponding author. e-mail: isabelasimoni@gmail.com, simoni@biologico.sp.gov.br. author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 herpesvirus is a growing problem, particularly in immunocompromised patients (vadlapudi et al., 2013). more recently, outbreaks of equine herpesvirus have had an impact on the equine industry, and stimulated interest in antiherpetic interventions (vissani et al., 2016). the suhv-1, also known as pseudorabies virus, is the causative agent of aujeszky‟s disease affecting other mammals besides pigs, is still present in wild boar and there are no antiviral drugs approved for veterinary use (zouharova et al., 2016). therefore, it is necessary to find new alternative drugs against these diseases that affect both humans and animals (martinez et al., 2015; nocchi et al., 2016). medicinal plants are natural resources, leading to valuable herbal products often used to treat a large number of various diseases in traditional medicine (katiyar et al., 2012; wachtel-galor and benzie, 2011). the south american flora represents one of the world‟s richest sources of material with pharmacological activity, and brazil has the biggest vegetal diversity (dutra et al., 2016). one of them is the “cerrado” (brazilian savannah) covering more than 2 million km 2 and land representing 25% of the area of the country (sawyer, 2016). this vegetation consists of more than 6,000 vegetal species, having many compounds such as saponins, tannis, steroids, and others (bessa et al., 2013), with biological activities including antifungal, antibacterial, antiprotozoal and antiviral ones (alves et al., 2000; mesquita et al., 2005, 2007; silva junior et al., 2009; brandão et al., 2010). many plant extracts have been described for antiviral activity against herpesviruses in vitro such as conocephalum conicum, polypodium glycyrrhiza and the water-soluble extract from the narcissus bulb flos verbasci, showed anti-hsv activity. the aqueous extracts of helichrysum aureonitens (asteraceae) shoots inhibited hsv-1 (chattopadhyay and khan, 2008). compounds in green tea extract could also inhibit hsv-1 and black tea extract enriched with theaflavins has the potential to prevent the spread of hsv-1 (cantatore et al., 2013). moreover, lithraea molleiodes, sebastiana brasiliensis and sebastiania klotzschiana used against infectious diseases showed antiherpetic activity with no cytotoxicity, as did the aqueous extract of beta vulgaris (betancourgalvis et al., 1999). the aqueous extracts from b. vulgaris and s. brasiliensis also exhibited antiviral activity against swine herpesvirus (koseki et al., 1990; simoni et al., 2014) and cecropia pachystachya, melochia villosa and p. acuminatum presented the most relevant results against bovine and swine herpesviruses (simoni et al., 2014). simoni et al. (2007) reported on the in vitro research for potential antiviral properties of the 16 species collected from the brazilian cerrado against bovine herpesvirus (bohv-1), infectious bursal disease virus (ibdv) and avian reovirus. they found 8 plant extracts active against bohv-1 including byrsonima intermedia, banisteriopsis variabilis, stryphodendron adstringens and xylopia aromatica. species of banisteriopsis are described as having antioxidant and antibacterial properties, while also inhibiting the activity of monoamine oxidases. banisteriopsis caapi is the most studied species of this genus because it is used by some religious groups in brazil as an ingredient of the drink locally known as ayahuasca. chemical compounds identified in this plant species include alkaloids, harmine and harmaline (wang et al., 2010; santos et al., 2017). b. intermedia belonging to the malpighiaceae family popularly known as “murici” has been used in folk medicine to treat fever, as diuretic, for skin infections and ulcers. b. intermedia is described mainly as being antimicrobial and as anti-inflammatory (moreira et al., 2011; sannomiya et. al., 2007). s. adstringens is one of the most widely used medicinal species by the brazilian population. the stem bark is usually used to treat leucorrhea, gonorrhea, vulvo-vaginal candidiasis, gastritis, sore throat, diarrhea and bleeding (costa et al., 2010). extracts from the leaves present trypanocidal activity associated with the chemical compound tannin (ishida et al., 2009). extracts from x. aromatica have been described for antibacterial, antifungal, insecticidal, antimicrobial, and antiparasitic activities. many compounds in this genus have been identified, but the triterpenoids are present in higher concentrations (stashenko et al., 2004; costa et al., 2013). the aim of this work was continue the search with these same eight positive plant extracts to evaluate the antiviral activity against equine, swine and human herpesviruses together with additional studies of antiviral and virucidal potential. additionally, the phytochemical profile of the extract from b. intermedia, which displayed higher antiviral activity, was performed by hplc/ms analysis. materials and methods plant materials the “cerrado” plant species b. variabilis b. gates, b. intermedia a. juss., campomanesia xanthocarpa o. berg, erythroxylum deciduum a. st.-hil., lacistema hasslerianum chodat, ocotea pulchella (nees) mez, s. adstringens (mart.) coville, and x. aromatica (lam.) mart were collected in mogi-guaçu (22º18‟s and 47º20‟w), são paulo state, brazil. the specimens were authenticated by a taxonomist in the herbarium of the instituto de botânica, são paulo, dr. eduardo luis martins catharino. preparation of crude extracts crude aqueous extracts were prepared by grinding dried leaves with de-ionized distilled water (10%, w/v) in a mixer and maintenance at 4ºc overnight. the aqueous extracts were filtered on whatman-1 filter paper and freeze-dried in flex-dry mp fts systems. lyophilized extracts were dissolved in equal parts of sterile de-ionized distilled water and eagle minimum essential medium (mem 2x) at a final concentration of 10,000, 4,000 or 2,000 3 µg/ml. the extracts were centrifuged at 2,500 g/10 min in a hettich zentrifugen model rotina r and sterilized by filtration (0.22 µm filter). hplc/ms analysis crude aqueous extract from b. intermedia was filtered on a seppak column using meoh as eluent. samples containing 1 ml of the crude extract was analyzed by hplc using a luna c-18 (phenomenex) column (5 mm, 250 x 4 mm), with a gradient from meoh:h2o:hccoh 1% 75:25 (0 min) to meoh 100% (30 min), flow rate 1.0 ml/min and detection at 254 nm. hplc/ms (negative form) were acquired on a bruker microtof-qii coupled to an apollo ion source set as follows: dry temperature at 180°c and voltage at 4.5 kv. the mass/charge ratios were detected in scan (m/z 100–1200 da) and product ion scan (m/z 50–1200 da) modes, using the same chromatographic method described above. cell cultures and viruses the equine herpesvirus 1 (ehv-1), strain a4/72 (moreira et al., 1998) and the herpes simplex virus 1 (hsv-1), strain kos (silva et al., 2010) were propagated in vero (african green monkey – atcc ccl 81). the swine herpesvirus 1 (suhv-1) strain np (nova prata) (fonseca et al., 2010) was propagated in mdbk (mardin and darby bovine kidney – atcc ccl 22). all cells were grown in mem with 10% fetal bovine serum (fbs) (simoni et al, 2007). cytotoxicity assay the assays were performed using 96well microtiter plates with 30,000 cell/well. after 24 h of incubation at 37ºc in a humidified 5% co2 atmosphere, each cell type was exposed to decreasing concentrations of plant extract in triplicate. any cell morphology alteration was observed at light microscopy during the next 3 days to determine the maximum non-cytotoxic concentration (mncc). monolayers of cells incubated only with mem were used as a control. antiviral assay for the assays, mdbk or vero cells were prepared in 96-well microplates at density of 3 × 10 4 cells per well and incubated for 24 h at 37ºc with 5% co2. after 24 h of incubation, the medium was poured off and 100 µl of extracts at dilutions corresponding at mncc were added. the cells were incubated for 1 h and after this period, 50 µl of logarithmic dilutions of viruses were inoculated for 96 h. mdbk cells were infected with suhv-1 and vero cells were infected with ehv-1 or hsv-1. controls consisted of untreated infected (virus titer), treated non-infected (extract control), untreated non-infected (cell control) cells. in addition, to verify if the antiviral activity of the extract remained below its mncc, a dose-response experiment was carried out using it at range of 250 until 1.9 µg/ml. direct anti-herpes assay the assay was described previously by simoni et al. (1996). briefly, 100 µl of each extract at mncc was mixed with 100 µl of logarithmic dilutions of each virus, incubated at 37°c in 5% co2 for 1 h. then, 50 µl of mixture was inoculated at the monolayers in each well of a 96-well plate. in addition, to verify if the direct antiherpes effect of the extract remained below its mncc, a doseresponse experiment was carried out using it at range of 250 until 1.9 µg/ml. mtt assay the mtt assay was performed to confirm qualitatively the mncc and the antiviral activity of the extracts that presented pi% more than 97%. cells in a 96-well microplate were incubated with 100 μl/well of increasing concentrations of crude extracts in quadruplicate with at least 5 different concentrations. thereafter, 100 tcid of virus was added. after incubation for 72 h, the medium was removed and added on cell monolayer 50 µl of mtt (1 mg/ml) to each well. the microplate was then incubated at 37ºc for 4 h. the supernatant was then removed from each well without disturbing the cell clusters containing formazan crystals. to solubilize the formazan crystal, 100 µl of sds was added and the microplate was incubated again overnight. the absorbance of the wells was read in a computer-controlled microplate reader (spectra max plus 384) at 540 nm wavelengths. the 50% cytotoxic concentration (cc50) of the test compound was defined as the concentration that reduced the absorbance of mock-infected cells by 50% of that of the control. the 50% antiviral effective concentration (ic50) was expressed as the concentration that achieved 50% protection of virusinfected cells from the hsv-1, ehv-1 and suhv-1 induced destruction (takeuchi et al., 1991). data and statistical analysis the antiviral and virucidal activities were based on reduction of viral titers using cpe criteria. values were expressed as titer (tcid 50 µl) (reed and muench, 1938) and viral inhibition index (vii) calculated as the difference of virus titer between treated and untreated infected control cultures. the vii was considered positive when ≥ 1.5 (barros et al., 2012). the 50% cytotoxic (cc50) and 50% inhibition (ic50) concentrations were calculated from concentration-effect curves obtained from nonlinear regression analysis of concentration-effect curves by the graphpad prism 5 demo program. the results were obtained from triplicate independent assays. the percentage of cytotoxicity was calculated as [(a – b)/a] × 100, where a and b are the od540 of untreated and of treated cells, respectively. the percentages of protection were calculated as [(a − b) × 100/(c − b)], where a, b and c indicate the absorbance of the extracts/fractions, virus and cell controls, respectively. each obtained ec50 value was defined as the effective concentration that reduced the absorbance of infected cells to 50% when compared with cell and virus controls. the selectivity index (si) was determined by the ratio of cc50 to ec50 and expressed as mean ± s.e.m. the statistically different effects of tested extracts on the inhibition of virus replication were compared with the control group using the student‟s t-test with p≤0.05 for significant result. results cytotoxicity the mnccs of the extracts were determined in two different cell lines, mdbk and vero and the concentrations used in bioassays are presented in table 1. in vero cells, the cc50 was lower than that one observed in mdbk cells. x. aromatica presented the lowest cytotoxic to vero and mdbk cells with mncc of 1250 and 625 µg/ml, respectively. the extract from o. pulchella was the most cytotoxic for both cell lines with 4 table 1. antiviral activity of extracts at mncc and cc50 against hsv-1, ehv-1 and suhv-1. plant extract mncc a vero cc50 b hsv-1 c ehv-1 mncc mdbk cc50 suhv-1 banisteriopsis variabilis 625 435.8 2.25 3.75 312 >2,000 3.24 byrsonima intermedia 31.2 141.1 3.25 4.87 250 782.1 5.45 campomanesia xanthocarpa 62.5 n.t 0 0.62 250 1,299 2.0 erythroxilum deciduum 125 n.t. 1.0 0.26 31.2 n.t. 1.16 lacistema hasslerianum 62.5 n.t. 0.5 0.26 62.5 821 2.0 ocotea pulchella 31.2 n.t. 0 0.26 31.2 290.9 1.76 stryphodendron adstringens 625 n.t. 2.75 1.0 62.5 >2,000 2.0 xylopia aromatica 1250 203.1 1.5 1.76 625 >2000 3.24 a: mncc: maximum non-cytotoxic concentration; hsv-1: human herpesvirus type 1 suhv-1: swine herpesvirus type 1; ehv-1: equid herpesvirus type 1; b: 50% cytotoxic concentration (μg/ml); c: vii: viral inhibition index; n.t.: cc50 not tested. table 2. direct anti-herpes effect of extracts at mncc against herpes simplex virus, equine herpesvirus and swine herpesvirus in cell cultures. plant extract vii a vii vii hsv-1 b ehv-1 c suhv-1 d banisteriopsis variabilis 3.0 3.67 3.34 byrsonima intermedia 4.5 4.0 5.22 stryphodendron adstringens n.t. n.t. 0.75 xylopia aromatica 1.5 0.84 1.67 a: vii: viral inhibition index; b: human herpesvirus type 1; c: equid herpesvirus type 1; d: swine herpesvirus type 1; n.t.: not tested. mncc: maximum non-cytotoxic concentration. mncc of 31.2 µg/ml. the other extracts showed mncc ranging between these values for both cells. for suhv-1and hsv-1, while the extracts from s. adstringens had action only on hsv-1. antiviral activity results are presented in table 1. the extracts from b. variabilis, b. intermedia, s. adstringens and x. aromatica presented vii greater than or equal to 1.5 for hsv-1. the extracts of b. variabilis, b. intermedia and x. aromatica were positive for ehv-1 while the extracts b. variabilis, b. intermedia, c. xanthocarpa, l. hasslerianum, o. pulchella, s. adstringens and x. aromatica were effective for suhv-1. direct anti-herpes effect assays to study the direct anti-herpes effect were done only with the extracts that were effective for at least two of the viruses tested in the antiviral assays. these results are presented in table 2. the extracts of b. variabilis and b. intermedia, presented an inhibition against the three viruses with vii greater or equal to 3.0. x. aromatica was only effective additional studies with byrsonima intermedia b. intermedia was selected for additional studies and to characterize its antiviral properties because it showed the highest values of vii against all herpesviruses. table 3 shows the results of the cytotoxic, antiviral activities and selectivity index (si) obtained from b. intermedia extracts. the mncc of b. intermedia extracts that did not cause alterations in the morphology of the cells visible under the optical microscope (table 2) were lower than that obtained with cc50 of 141.1 for vero cells and 782.1 for mdbk cells. b. intermedia presented si values of 41.76 ± 0.04; 4.12 ± 0.1 and 193.97 ± 0.09 respectively against hsv-1, eqhv-1 and suhv-1. figure 1 shows the antiviral activity of extract from b. intermedia at 8 set concentrations against the three viruses showing that treatment with b. intermedia extract resulted in reduced viral titers in dose-response curves. at 15.6 µg/ml, the initial reduction occurred firstly with suhv-1 and then with ehv-1 and hsv-1 at 62.5 µg/ml. at 250 µg/ml, the greater vii was also obtained with 5 table 3. selectivity index, cytotoxic and antiviral activities from b. intermedia aqueous extract against swine herpesvirus, equine herpesvirus and herpes simplex virus by the mtt assay. plant extract vero hsv-1 si c eqhv-1 si mdbk suhv-1 cc50 a ic50 b ic50 cc50 ic50 si byrsonima intermedia 141.1 3.37 41.76 ± 0.04 34.24 4.12 ± 0.1 782.1 4.03 193.97 ± 0.09 a: 50% cytotoxic concentration (μg/ml); b: 50% inhibitory concentration (μg/ml); c: si seletivity index ratio of cc50 to ic50. suhv-1: swine herpesvirus type 1; ehv-1: equid herpesvirus type 1; hsv-1: human herpesvirus type 1. 7 6 5 4 vii 3 2 1 0 1.9 3.9 7.8 15.6 31.2 62.5 125 250 suhv-1 eqhv-1 hsv-1 concentration (µg/ml) figure 1. antiviral activity of different concentration of extract from b. intermedia against swine herpesvirus type 1 (suhv-1); equine herpesvirus type 1 (ehv-1); human herpesvirus type 1 (hsv-1); vii: viral inhibition index. suhv-1 followed by ehv-1 and hsv. likewise, figure 2 shows the direct anti-herpes effect against suhv-1, ehv-1 and hsv-1 showing that treatment of virions with this various concentrations of extracts for one hour resulted in reduced viral titers. the extracts were active at the concentrations range 250 to 31.2 µg/ml. at 3.9 µg/ml, the reduction occurred firstly with suhv-1 and then with ehv-1 and hsv-1 at 7.8 µg/ml. at 250 µg/ml, the greater vii was obtained for suhv-1 and ehv-1 followed by hsv. phytochemical profile of byrsonima intermedia as follow-up, the b. intermedia extract was subjected in the current study to hplc/ms analysis (figure 3). the peak at rt = 10.2 min corresponds to a [m – h] quasimolecular ion peak at m/z 169, suggesting the occurrence of gallic acid (a). the second peak, detected at rt = 13.6 min corresponds to a [m – h] quasimolecular ion peak at m/z 301, suggesting the presence of flavonoid quercetin (b). discussion cytotoxicity the cytotoxic assay at the first stage of screening is very important to determine the maximum concentration of the aqueous extract that did not induce changes in cell morphology. this step is critical because the virus is an intracellular parasite and uses the cell machinery to replicate, so it must be ensured that the virus have the ideal conditions for their growth (cos et al., 2006). the study of cytotoxicity in different cell lines is important because one of the inherent drawbacks of in vitro antiviral testing is the environmental sensitivity of animal cells in culture, although the in vitro methodology is faster and less costly (mccutcheon et al., 1995). antiviral activity brazilian cerrado plants may lead to the study of a broad source of new natural compounds; however, most are 6 7 6 5 vii 4 3 2 1 0 1.9 3.9 7.8 15.6 31.2 62.5 125 250 hsv-1 suhv-1 eqhv-1 concentration (µg/ml) figure 2. direct anti-herpes effect of different concentrations of extract from b. intermedia against swine herpesvirus type 1 (suhv-1); equine herpesvirus type 1 (ehv-1); human herpesvirus type 1 (hsv-1); vii: viral inhibition index. being developed for use as antibiotics more than antiviral drugs. this is because the viruses present certain characteristics among others of replicating within the cell and persisting in the host. their combat must be more rational by the choice of species of plants that have already been used by the population to treat the diseases. many brazilian cerrado species have already been described as having antiviral activity. chattopadhyay et al. (2015) showed that byrsonima verbascifolia had strong anti-hsv activities, whereas maldini et al. (2011) found that b. crassifolia used as anti-inflammatory has been described as having compounds that also display antiviral activity against hsv-1 and hiv. in the present study, eight extracts reduced at least one of the viral titers, as compared to untreated control cells. b. variabilis, b. intermedia and x. aromatica species were active against all these herpesviruses. furthermore, a strong inhibition was obtained from b. variabilis and b. intermedia. s. adstringens extract also showed an effective inhibition against suhv-1 and hsv-1. extract from x. aromatica presented the highest values of vii against suhv-1. antiviral activity was also observed for extracts from c. xanthocarpa and o. pulchella only against suhv-1. frias et al. (2012) isolated from metanolic extract of b. variabilis, the flavonoids quercetin, rutin, and apigenin. quercetin is found in a wide variety of plants and studies showed that it could reduce infectivity of target cells and replication against herpesviruses. the results of our work indicated that the extracts of b. variablis showed inhibitory effect against the three herpesviruses and probably this inhibitory effect is due to presence of these flavonoids. felipe et al. (2006) described the inhibition bohv-1 replication of aqueous extracts from s. adstringens stem bark. simoni et al. (2007) showed that the leaves presented moderate activity against bohv-1. in the present work, leaves extracts from s. adstringens also presented moderate activity against ehv-1 but strong inhibition against hsv-1 and suhv-1. antiviral drugs have been reported for herpetic infections for both human and animal herpesviruses. although the regular use of vaccines helps in improvement of animal herds and avoids viral shedding, treatment of herpesvirus infections is not completely prevented by immunization (gulati et al., 2016; maxwell, 2017). thus, antiviral agents can be useful not only as preventives but also as therapeutic in herpesvirus infections which leads to a great interest in discovering new effective and safe drugs. the results of this present work suggest the importance of searching for new compounds for veterinary and human medicine in brazilian cerrado plants because they have shown potential for developing new antivirals. several plant extracts with promising results have been described for their antiviral potential against diverse herpes viruses. for example, cardamine angulata, conocephalum conicum and polypodium glycyrrhiza showed activity against bovine herpesvirus and hsv-1 (mccutcheon et al., 1995). aqueous extract from guettarda angelica seeds exhibited strong inhibition for three animal herpesviruses including bovine, swine and equine herpesviruses 1 (barros et al., 2012). the present results were promising especially for b. intermedia and indicating that materials derived from their 7 figure 3. hplc analysis of crude aqueous extract from b. intermedia leaves and identified compounds gallic acid (a) and quercetin (b). leaves exhibited antiviral activity and selectivity toward three herpesviruses hsv-1, eqhv-1 and suhv-1. in 2007, simoni et al. (2007) described the antiviral activity of the b. intermedia against bovine herpesvirus (bohv-1) and against avian reovirus indicating a broad spectrum of action of this plant species. direct anti-herpes effect to verify if the mechanism of action of extracts is also due to inhibition on the viruses in extracellular conditions, the selected extracts showing antiviral activity were studied. many plants may exhibit different mechanisms of viral inhibition acting on more than one target during viral replication. the opuntia streptacantha inhibited virus replication and inactivated extracellular virus, such as hsv, equine herpes virus, pseudorabies virus (jassim and naji, 2003). bidens pilosa extract was effective against hsv-1 and hsv-2 with potent virucidal activity (vadlapudi et al., 2013). virucidal activity has also been described in crude hydroethanolic extract from the stem bark of s. terebinthifolia and was effective against hvs-1 in the attachment and penetration stages (nocchi et al., 2016). moreover, hot water extract of b. pilosa showed potent inhibitory activity against hsv-1 and hsv-2 in vero cells. the authors suggest multiple targets of activity because this extract showed virucidal activity to block the binding of virus to host cells and viral cell penetration. the b. pilosa extract also inhibits virus adsorption to cells and affects some intracellular steps of viral replication (nakama et al., 2012). these findings are in agreement with those observed in this study. additional studies with byrsonima intermedia the extracts from b. intermedia were tested in their 8 maximum non-toxic concentrations in which no alteration of normal cell morphology was observed, and the cc50 for both mdbk and vero cells was obtained. therefore, the extract showed no toxicity even when used at high concentrations in the reduction of viral titers assay and direct anti-herpes assay. further, b. intermedia extracts showed anti-herpes activity in a concentration-dependent manner in both assays, suggesting that this effect may have occurred either acting directly on the viral particle or in other stages of the replicative cycle. additional studies should be done to study the mechanism of action of extracts of b. intermedia against those herpesviruses indicating at which stage the extract has activity against viruses. phytochemical profile using dereplication procedures by hplc/ms, the occurrence of gallic acid and quercetin in the active extracts from b. intermedia was identified. as previously reported, these compounds were detected in the meoh leaves extract from b. intermedia which displayed mutagenic activity (sannomiya et al., 2007). gallic acid is a phenolic compound present in many plants, fruits and vegetables. studies have demonstrated that gallic acid exhibits a strong inhibitory effect against several viruses such as the herpes simplex virus, hepatite c virus, hiv and enterovirus 71. therefore, the potential activity observed by ga suggests that this activity is due to the hydrophobic interaction between the functional group, (hydroxyl) and virion components of enveloped herpesvirus. in addition, gallic acid showed an inhibitory effect in a time-dependent manner, notably similar to the current study (govea‑ salas et al., 2016). quercetin, a flavonoid found in several plant species, including fruits and edible vegetables, displayed several biological potentials (sharma et al., 2018), including antiviral against hsv-1 (garrett et al., 2012). additionally, choi et al. (2009) described that quercetin-3-rhamnoside from houttynia cordata extract inhibit influenza a replication in the initial stage of virus infection by indirect interaction with virus particles. new studies are needed to bring the active substances present in b. intermedia extract and to identify in detail the ways in which they act. the other extracts that can be considered promising must also pass through the processes of fractionation and isolation of substances in order to verify the potential of this activity. all of them here have shown their potential to lead to the development of an alternative therapy for the treatment of such important infections worldwide. conclusion this study has demonstrated a broad antiviral activity of brazilian cerrado plants and indicated that they can be effective potential candidates for the development of new strategies to treat viral infections. b. intermedia was chosen for further research as well as gallic acid and quercetin all necessary to elucidate the mechanism of action of the extract or compounds both on herpesviruses replication and on direct antiviral effect. conflict of interests the authors have not declared any conflict of interests. acknowledgements the authors are thankful to the enrica g. d‟alessandro for reviewing english. references alves tma, silva af, brandão m, grandi tsm, smânia efa, smânia jr a, zani cl (2000). biological screening of brazilian medicinal plants. mem. inst. oswaldo cruz 95(3):367-373. betancour-galvis l, saez j, granados h, salazar a, ossa j (1999). antitumor and antiviral activity of colombian medicinal plants extracts. mem. inst. oswaldo cruz 94(4):531-535. brandão gc, kroon eg, dos santos jr, stehmann jr, lombardi ja, braga de oliveira a (2010). antiviral activity of bignoniaceae species occurring in the state of minas gerais (brazil): part 1. lett. appl. microbiol. 51(4):469-476. barros av, araújo lm, oliveira ff, conceicao ao, simoni ic, fernandes mjb, arns cw (2012). in vitro evaluation of the antiviral potential of guettarda angelica against animal herpesviruses. acta sci. vet. 4:1068. bessa ngfdei, borges jcm, beserra fp, carvalho rha, pereira mab, fagundes r, campos sl, ribeiro lu, quirino ms, chagas jr af, alves a (2013). prospecção fitoquímica preliminar de plantas nativas do cerrado de uso popular medicinal pela comunidade rural do assentamento vale verde-tocantins. braz. j. med. plants 15(4):692707. cantatore a, randall sd, traum d, adams sd (2013). effect of black tea extract on herpes simplex virus-1 infection of cultured cells. bmc complement. altern. med. 13:139. chattopadhyay d, khan mth (2008). ethnomedicines and ethnomedicinal phytophores against herpesviruses. biotechnol. annu. rev. 14:297-348. chattopadhyay d, ojha d, mondal s, goswami d. 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swine manure application on tifton 85 tangriani simioni assmann1*, alceu luiz assmann2, laércio ricardo sartor1, and talyta zortéa1 1 federal technological university of paraná, utfpr, dois vizinhos, pr, brasil. 2 agronomic institute of paraná, iapar, pato branco, pr, brasil. accepted 21 february, 2023 one major problem of swine production is the huge volume of manure generated; this involve difficulties in proper handling of the residue when applied to the soil, given that such elements can be toxic to the environment. this study examined the vertical movement of the p, k and mineral n in the soil profile cultivated with cynodon dactylon cv. tifton 85 which was submitted consecutively to rates of liquid swine manure (lsm) application (four years). the experiment was done using a randomized block design with four replications in a split-plot arrangement, where the whole plots were semiannual applications (november, 2002 to september, 2006) of increasing levels of lsm (0, 30, 60, 90, 120 and 180 m 3 ha -1 ); while the sub-plots were the soil samples at different depths (0-10, 10-20, 20-40, and 40-60 cm). the n-no3 leaching was observed when application of lsm exceeded 90 m 3 ha -1 twice annually or during the year, suggesting a limit level for fertilizer on tifton 85 pastures. phosphorous and potassium accumulation was observed at higher lsm rate, mainly at the 0-10 cm soil layer since the soil p levels increased up to the highest evaluated depth at the 180 m 3 ha -1 lsm level. lsm meets the tifton 85 nutritional requirement regarding n, p and k when applied semi-annually at the rate of 90 m³ ha -1 without causing pollution effects; although the grass production responds up to 180 m 3 ha -1 levels. key words: cynodon dactylon, environmental contamination, mineral nitrogen, organic fertilizers. introduction swine production is an important part of the brazilian agribusiness that consuming large quantities of grains and water, with daily water consumption of 9.05 to 22.05 l -1 pig -1 (nardi, 2009). consequently, this activity produces large quantities of residue, since each liter of ingested water generates 0.6 l -1 of liquid swine manure (oliveira, 1993); often times, distributed in small areas. majority of swine growers in the country are smallholders (mohedano et al., 2014). it is important to determine the liquid swine manure (lsm) effect as a soil pollutant under different crops in order to illustrate the rates its use would be acceptable long term (scherer et al., 2010; maccari et al., 2016). lsm is a nutrient and organic matter source for *corresponding author. e-mail: tangriani@utfpr.br. author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 cultivated soils, and lots of plant production systems can benefit from the agriculture cycling of swine manure (couto et al., 2013). its usage in pastureland, if conducted properly, can contribute to the system’s sustainability, given that it is capable of increasing production and quality of pastures (vielmo et al., 2011). in addition, lack of fertilization is one of the major factors contributing to the degradation of pasture land in brazil and the lms applications can remediate this kind of degradation. a significant amount of the macro and micronutrients ingested by the swine are excreted in dung and urine (berenguer et al., 2008). if the organic fertilization is cautious, it can replace chemical fertilization of crops in part or whole (ourives et al., 2010). however, when used irrationally, disregarding soil-support capability with no application rate limits, the lsm can become a great source of pollution for water springs (domene et al., 2007). approximately 70% of the n present in the lsm stocked in dunghills is in ammoniacal form (scherer et al., 1996). when applied to the soil, it is oxidized to nitrate by nitrifying bacteria (whitehead, 1995) in a relatively fast pace (aita et al., 2006). therefore, highly productive pastures like tifton 85 can exhibit higher utilization rates of the mineral n present in the soil arising from the lsm application. aita et al. (2006) observed higher mineral n in soils under fallow compared to soils cultivated with black oats under the 80 m 3 ha -1 of lsm rate. this behavior can be maximized by the use of perennial pastures like the cynodon ones. nevertheless, the swine manure application can be a possible source of soils and water contamination through nitrate (no3 ) (puig et al., 2017). this can result from excessive fertilizer application in the soil, independent of the fertilizer type, which can be dangerous to both human and animal health (robertson, 2005; bryan et al., 2012). various studies show n mobility to have higher depths when cultivated with annual crops (ceretta et al., 2010). such mobility can vary for soils under perennial forage species given their root system structure, greater cultivation period, and pasture utilization. besides n, elements that are deeply required by the cultures like phosphorus and potassium are added to the soil when the lsm is applied and are absorbed by the plants when available (ceretta et al., 2010) or can be lost by leaching, primarily p, which is the main element associated with eutrophication problems (sharpley et al., 2001; gatiboni et al., 2015). therefore, knowledge on n, p and k behavior in the soil profile under different rates and constant application of lsm is important to support the use of this organic fertilizer in pastures to ensure there is no environmental contamination. the research aim to evaluate the vertical movement of nitrate and ammonium as well as the p and k behavior in a soil cultivated with perennial summer grass (cynodon dactylon cv. tifton 85) under a semiannual application. this was done within four years of increasing rates of lsm and the forage production response of the pasture. materials and methods study area the research was conducted from november 2002 to september 2006, in an area located at 26° 07’ s and 52° 41’ w , at 700 m a.s.l, under a climatic condition that transits from cfb (temperate climate) to cfa (subtropical climate), according to the köppen climate classification (maak, 1968). the precipitation regime in the past 10 years was on an average of 2000 mm. the soil is classified as a dystrophic red oxisol (ferritic ferralsols, according to classification world reference base for soil resources, wrb, 2014). the soil is on a hilly topography with a clay texture and under a 10 year no-tillage planting system. sampling and experiment designs sprigs of cynodon sp cv. tifton 85 were planted in november 2002. the first lsm application was in march 2003, when the pasture was established and subsequent applications happened semi-annually; totaling six lsm applications with rates of 0, 30, 60, 90, 120 and 180 m 3 ha -1. the experimental design was in a randomized block with four repetitions in a split-plot. the treatments consisted of increasing lsm rates (0, 30, 60, 90, 120 and 180 m 3 ha -1 ) for the whole plots and the sampling depths as the sub-plots. in september 2006, soil samples were collected at different depths of (0-10, 10-20, 20-40, and 40-60 cm) to evaluate the n-no3 , n-nh4 + , p and k concentrations. in 2004, p and k concentrations were evaluated under the same depths. soil samples were collected at eight different sites per plot, which were later dried at 55°c for 72 h, and the n-no3 and n-nh4 + concentrations were determined through the method described by pavan and miyazawa (1996). the k and p contents were extracted by the double acid method melich-1, and later flame photometer, and the p determined the k by atomic absorption spectrophotometry. the dry matter production of tifton 85 evaluation started in november 2003 when the plant canopy in each plot was 30 cm high on average. samples were cut to a 10 cm stubble, from a 0.25 m 2 . four subsamples were taken in each plot, and mixed to represent one plot sample, which were dried at 55°c in a forced-air oven to constant weight. forage production was calculated (kg ha -1 de ms) annually (from january to december of each year). winter cuts were not performed due to minimal plant growth. statistical analysis an analysis of variance multifactorial was conducted. the variables considered homogeneous had their treatments evaluated with the f-test. when the results were significant at 5%, polynomial regressions were fitted for lsm rates, versus n-no3 , n-nh4 + , phosphorus and potassium concentration, for different depths, seeking the model with higher significance level. results and discussion the interaction between lsm rates and sample depth 3 figure 1. soil nitrate concentration at different depths as a function of liquid swine manure application rates. (figure 1) was observed for n-no3 soil concentration. after 46 months of the experiment establishment and six semiannual lsm applications according to stated rates, the highest n-no3 concentrations were observed in the topsoil layer (0-10 cm) using lsm application of 90 m³ ha 1 . for higher rates, n-no3 accumulation was observed for layers from 20 to 60 cm depth, which indicates n-no3 leaching potential for lsm applications from 90 to 180 m³ ha -1 (figure 1). therefore, these rates of lsm are not recommended for areas under c. dactylon cv. tifton 85 cultivation. the higher nitrate concentration in the 10 to 60 cm depth, as a consequence of the higher lsm rates, may result in contamination of the water table which depends on the leaching rate of n-no3 in the soil profile. the leaching rate of n-no3 is a consequence of the volume of infiltrated water and tifton 85 root system’s ability to absorb n-no3 in the deep layers. basso et al. (2005) observed higher n-no3 losses through percolated water when the lsm rate applied increased from 0 to 80 m 3 ha -1 , exposing that 40 m 3 ha -1 rates do not present n-no3 leaching concern. aita et al. (2006) have observed evidence of n-no3 leaching for layers beyond 60 cm depth using lsm at 80 m 3 ha -1 with annual species (corn, weeds and black oat). sacomori et al. (2016) verified that high doses of dls applied to the soil surface (200 m 3 ha -1 ) contributed to the leaching of nitric n at the depths of 40 and 80 cm. nikiéma et al. (2013), when assessing the application of dls in wheat cultivation up to the dose of 68 m 3 ha -1 in a sandy soil, verified that nitrate leaching is mainly related to annual rainfall. in years where rains were above average, dls n losses extended to 29.3%. in addition to the climatic conditions, the soil characteristics determine the intensity of the leaching process occurrence, since it is inversely proportional to the number of adsorption sites (mota et al., 2015). for the research presented here, this fact can also be stated for rates over 90 m 3 ha -1 applied semiannually, indicating that tifton 85 may present higher potential for n-no3 utilization when lsm is used. other studies evidenced the high potential of nitrogen utilization of organic fertilizers using grasses. franzluebbers and stuedemann (2005), after 5 years of application of different nitrogen sources (organic and inorganic), observed that there was little nitrate loss through leaching, despite the application of 200 kg n ha -1 year -1 ; this indicates that the uptake of n by bermudagrass (c. dactylon (l.) pers) was efficient to reduce the losses, mainly by the habit of vigorous growth of the roots in a way that potentiates the use of this element by the plants. the n-nh4 + concentration were fitted to a positive linear model to the lsm rates applied to the soil (figure 2), with an increase of 2.3 mg dm -3 , from the lower to the 4 (a) (b) figure 2. soil ammonium concentration as a function of lsm application rates (figure 2a) and for different soil depths (figure 2b). higher lsm rate without considering the high fertilizer amount. this is justified by the fast nitrification of the ammoniacal n applied via lsm (aita et al., 2006). higher n-nh4 + concentrations were observed at the topsoil layer (0 to 10 cm), significantly higher from the deeper layers of the soil profile; with lower n-nh4 + concentration at the 40 to 60 cm layer (figure 2). this nnh4 + increment with higher lsm rates and the higher concentration at the topsoil layer were expected, since the ammonium, being a cation, is stable in the soil, and is adsorbed by the soil negative charges; consequently, presenting low mobility (oliveira et al. 2011). therefore, ammonium does not contribute extensively to contamination problems of subsurface water. the effect of the interaction between lsm application rate and sample depth was not observed (figure 3) for potassium concentration in year 2004 and 2006. the highest k concentration of the experiment was observed under the 180 m 3 ha -1 rate, in the topsoil layer, with significant variations in the 0 to 10 cm layer and 10 to 20 cm due to lsm applied rates. during the two evaluation years, k concentration was higher in the topsoil layer and higher accumulations were observed with higher lsm rates. these results are not the same with those found by ceretta et al. (2003), who conducted an experiment in a chromic orthic alfisol with low clay percentage under natural grassland, and observed decrease of available k quantity in the topsoil layer with lsm application, when compared with the control treatment. results from this study suggest that the behavior of k present in the lsm in dystrophic red oxisol with high clay percentage differs from its behavior in sandy soils. queiroz et al. (2004), in a red-yellow sposol in rio grande do sul also observed exchangeable k accumulation in the topsoil layer when swine manure was applied. therefore, it is possible to state that there is a tendency of higher k accumulation in the topsoil layers in soils with high clay percentage, when increasing lsm rates are applied; leaving the nutrient available to the pasture or to leaching. when the lsm is applied at 90 m³ ha -1 , the k concentration at the 0-20 cm depth (figure 3), depth at which analysis and correction of the soil under pastures are recommended, is within that recommended for high productivity forage species, such as tifton 85. k accumulation in the soil was not observed from 2004 to 2006 in the respective lsm rates (figure 3). the only difference observed was between lsm rates and sample depths. scherer et al. (2007) did not observe lsm rate effects on the k concentration in the soil with lsm application from 0 to 115 m 3 ha -1 for three years, leading to decrease of k concentration with depth; which is similar to this research. lourenzi et al. (2016) observed increases in the available k content, mainly in the superficial layers of the soil after 6 years of applying organic pig waste (dls + shavings); reaching 159% k increase with application of 16 mg ha -1 of the compound in the 0-4 cm depth layer. p concentration at the beginning of the experiment was below that recommended for the forage species tifton 85, at approximately 5 mg dm -3 ; whereas the recommended is above 12 mg dm -3 (cqfsrs/sc, 2016), which is good for a soil located at the southwest of paraná and west of santa catarina. however, with the semiannual lsm application, an increase in p concentration was observed in the soil to levels that are considered high. the cqfsrs/sc (2016) considers that p concentration extracted by the mehlich -1 method, as 5 figure 3. soil potassium concentration at the depths of 0-10, 10-20, 20-40 and 40-60 cm, as a function of the 0, 30, 60, 90, 120, and 180 m 3 ha -1 of lsm application rates. year 2004 (figure 3a) and year 2006 (figure 3b). lsd: least significant difference. performed in this study, should not exceed 24 mg dm -3 since there is the possibility of fertilizer loss and waste, as well as surface water contamination by phosphates (berwanger et al., 2008). interaction between lsm rates and sample depth was observed for phosphorus concentration in the soil cultivated with tifton 85 in 2004 and 2006 (figure 4). the utilization of increasing lsm rates altered p concentration in the soil. higher concentrations of this element were found when 180 m 3 ha -1 lsm was applied. this increment in the concentration, as well as its tendency of higher concentration in the topsoil layer is consistent with results obtained by ceretta et al. (2003). there was p accumulation from 2004 to 2006 only in the 0-10 cm layer, for up to 120 m 3 ha -1 of lsm use (figure 4). under the application of 180 m 3 ha -1 , the p concentration also increased at deeper soil layers (0-60 cm). this high lsm rate suggests the possibility of p loss from runoff (ceretta et al., 2005) since the concentration remained the same between 2004 and 2006. although higher p concentration was observed at deeper layers, there is evidence of nutrient loss from runoff, which characterizes a rate with potential polluting effect (menezes et al., 2018). the accumulation at the topsoil layer was expected, since it presents low mobility in the soil. according to scherer et al. (1996), approximately two thirds of soil phosphorus is not soluble in water; potentially being part of organic structures and this contributes to the residual effect of manure. thus, it can be stated that this nutrient stays practically unavailable to plants right after its application, requiring microorganism participation to become available in larger quantities. phosphorus residual effect in the soil is therefore a consequence of that exposed. results published by berwanger et al. (2008) also point to p increments in deeper soil layers (0-15 cm), attesting nutrient mobility within the soil profile and a contamination risk to subsurface water (ceretta et al., 2003). some studies indicate that 90% of the p applied via lsm might be organic, that is insoluble (takalson and leytem, 2009), which contributes to the low leaching of the element in soil profile (figure 4), under applications that is up to 120 m 3 ha -1 and accumulation at the 0-10 cm soil layer. this is not the case for soluble mineral fertilizers which present higher leaching potential of soluble reactive p when compared with organic fertilizers (bertol et al., 2010). consequently, lsm stands as an option for fertilization of forage production fields. the increase in soil fertility, provided by successive lsm applications, led to higher levels of forage production of tifton 85 in 2003, 2004, 2005 and 2006 (figure 5), which was observed under higher lsm application rates. in 2003, measured forage production was lower when compared with subsequent years, possibly due to winter and first-year (establishment) carryover effect, even though fertilization responses were already noticeable. quantification of forage production started in november, 2003. for 2004, 2005 and 2006, the responses to the organic fertilizer were fitted to positive quadratic models (figure 5a). there was response to the forage production at 180 m³ ha -1 lsm rate, although n-no3 concentrations were observed at deeper layers of the soil. drumond et al. (2006) observed forage production 6 figure 4. soil phosphorus concentration at the depths of 0-10, 10-20, 20-40 and 40-60 cm, as a function of 0, 30, 60, 90, 120, and 180 m 3 ha -1 of lsm application rates. year 2004 (figure 4a) and year 2006 (figure 4b). lsd: least significant difference. figure 5. cynodon dactylon cv. tifton 85 dry matter production as a function of liquid swine manure application rates (0, 30, 60, 90, 120, 180 m 3 ha -1 ) for the years of 2003, 2004, 2005 and 2006 (figure 5a) and total accumulated during four years (figure 5b). iapar: research station of pato branco, 2006. (tifton 85) of 5828 dm kg ha -1 when 200 m³ of lsm were applied. their result is not in agreement with the findings of this study, since the lsm application of 180 m 3 ha -1 resulted in an annual production of 25000 to 30000 kg ha 1 of dry matter between 2004 and 2006 (figure 5). accumulated forage increase with lsm application rates in the four years of study (figure 5b), portraying the potential of lsm as a fertilizer for perennial forage species, enabling its use as nutrient source to these plants and an option for discard of the manure, which is a concern since it is an environmental contaminant. given the forage production levels obtained with the application lsm rates of 90 and 120 m 3 ha -1 , it is suggested that high quantities should not be used, given the n-no3 and p 7 potential for contamination, as shown in this study. conclusions n-no3 leaching occurs in tifton 85 pastures when lsm is applied semiannually, at the rate of 90 m 3 ha -1 . this rate is suggested as the limit to the use of this fertilizer in respective pasture. the lsm supplies the nutritional need of tifton 85 in respect to the availability of n, p and k at a 90 m³ ha -1 rate, applied semiannually without causing polluter effect. tifton 85 dry matter production responds up to 180 m³ ha -1 of lsm application rates. however, the fertilizer utilization efficiency decreases at higher lsm rates, when mineral n, p and k are accumulated in the soil with applications of 120 m³ ha -1 of the organic fertilizer presenting an environmental contamination risk. conflict of interests the authors have not declared any conflict of interests. references aita c, port o, giacomini sj (2006). dinâmica do nitrogênio no solo e produção de fitomassa por plantas de cobertura no outono/inverno com uso de dejeto líquido de suínos. the revista brasileira de ciência do solo 30:901-910. basso cj, ceretta ca, durigon r, polett n, girotto e (2005) dejeto líquido de suínos: ii perdas de nitrogênio e fósforo por percolação 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2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper salmonella diversity and antimicrobial resistance in burkina faso: an investigation of strains from various sources kagambèga a.1, 2, 3*, bouda s. c.2, bako e.2, cissé h.2, barro n.2 and haukka k.1, 4 1 bacteriology unit, department of infectious disease surveillance and control, national institute for health and welfare (thl), helsinki, finland. *corresponding author. e-mail: kagamas2007@yahoo.fr; kagambega.asseta@gmail.com. 2 laboratoire de biologie moléculaire, d’épidémiologie et de surveillance des bactéries et virus transmissibles par les aliments (labesta)/centre de recherche en sciences biologiques, alimentaires et nutritionnelles (crsban)/ecole doctorale sciences et technologies (edst)/université ouaga i professeur joseph ki-zerbo, 03 bp 7021 ouagadougou 03, burkina faso. 3 institut des sciences (ids), 01 bp 1757 ouagadougou 01, burkina faso. 4 department of food and environmental sciences, division of microbiology and biotechnology p. o. box 56, fi-00014 university of helsinki, finland. accepted 25 september, 2023 epidemiologic and traceback evidence of salmonella infection is not much in many developing countries including burkina faso. this study investigates the antimicrobial resistance and distribution of salmonella serotypes isolated from diverse sources in burkina faso. 615 salmonella serotypes isolated from beef meat, poultry carcasses, poultry, swine, cattle, hedgehog, fish, salad, channel and humans from 2009 to 2011 were analyzed to identify their diversity and distribution among the samples. the salmonella strains were subjected to antimicrobial sensitivity tests using disk diffusion methods, were analyzed and classified into 110 serotypes, with the most prevalent serotype being derby (91/615) found in beef meat, poultry carcasses, poultry and fish; muenster (48/615) found in cattle, swine, hedgehog, poultry, human and fish; chester (38/615) found in poultry carcasses, swine and poultry feces; hato (32/615) found in beef meat, poultry carcasses, cattle and poultry; drac (30/615) found in cattle, hedgehog and fish; and typhimurium (21/615) found in cattle, poultry, human and fish. among the 615 salmonella strains, 94% (581/615) were resistant to one or more antibiotics; resistance to streptomycin was the most common. the resistance pattern, str-sul-tet, str-tet and str-sul was dominant and found in 80% of the strains. about 3% of the strains were resistant to 5 or 6 antibiotics; their resistance pattern is amp-str-sul-tet-tmp or amp-chl-str-sul-tet-tmp. one salmonella strain, s. kentucky isolated from human stool was resistant to eight antibiotics; the resistance pattern is amp-strsul-tet-cip-gen-nal-mec. findings from this study can help define the guidelines for basic surveillance system of salmonella and other enteropathogenic bacteria circulating among humans, animals, food and environment. key words: salmonella, environment, human, risk. introduction salmonella spp. especially non-typhoid salmonella (nts) is a common source of foodborne diseases that cause morbidity and mortality worldwide (smith et al., 2016). it is estimated that salmonella spp. cause 93.8 million cases of gastroenteritis and 155,000 deaths each year worldwide. approximately 86% of these cases are 2 the result of foodborne infections (majowicz et al., 2010). in burkina faso, bacteriological results showed that the rate of salmonella contamination remains quite high (simporé et al., 2009). in fact, the infection progresses mostly periodically and rarely in an epidemic mode, and they are not reported due to lack of food borne pathogens surveillance system. salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is a great concern in public health. however, information on the diversity and occurrence of salmonella strains is very scarce (cui et al., 2008), making the ecology of these species remains unknown. food animals, including poultry, pigs, and cattle are the key reservoirs for human salmonellosis (hauser et al., 2011). in developing countries, wild and food animals are the sources of salmonella distribution in water, vegetables, salad and the products derived from these animals due to lack of hygiene (kagambèga et al., 2013). the emergence of antimicrobial resistant salmonella is mostly associated with the non-therapeutic use of various classes of antimicrobials in large quantities in food animals (marshall and levy, 2011; mir et al., 2015). researchers have reported a link between the use of antimicrobials in food animals and the emergence of antimicrobial resistance in pathogenic bacteria (ungemach et al., 2006; mir et al., 2015). the increasing number of multidrug-resistant nts strains is a global concern; this has made some countries and international organizations create surveillance systems which include collaboration between human health, veterinary, and food related sectors to monitor the spread of foodborne bacteria. unfortunately, these surveillance systems are missing in many developing countries like burkina faso. the absence of controlled reporting of salmonella serotypes through the who-gfn program in burkina faso inspired the compilation of data on the sporadic reporting of salmonella serotypes isolated from various sources. therefore, the purpose of the present study is to provide information on the diversity and antimicrobial resistance of salmonella strains isolated from environment, animals, food and humans. these data will allow one to follow the trends in salmonella enterica serotypes that provide information about sources of infection and the efficacy of prevention and control measures. materials and methods salmonella strains (n= 615) isolated from meat, poultry carcasses, poultry, cattle, pigs, hedgehog, water, salad and humans were collected in burkina faso from 2009 to 2011. sampling was done based on the microbiological conditions. the strains were isolated using standard bacteriological methods and serotyped according to kauffman white scheme (kagambega et al., 2013; bonkoungou et al., 2013; traoré et al., 2015). antimicrobial susceptibility of the isolates was tested by a standard disk diffusion method, and escherichia coli rhe 6715 (atcc 25922) was used for validating the antimicrobial test results (clsi, 2015). the antimicrobial agents used were ampicillin (10 µg), chloramphenicol (30 µg), streptomycin (10 µg), sulphonamides (3 µg), trimethoprim (5 µg), tetracycline (30 µg), gentamicin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), cefotaxime (30 µg), mecillinam (10 µg) and imipenem (10 µg). minimal inhibitory concentration (mic) for ciprofloxacin (0.002 to 32 μg/ml) was determined by e-test (ab biodisk, solna sweden) of the isolates resistant to nalidixic acid. mic breakpoint ≤ 1 μg/ml was interpreted as susceptible (clsi, 2009). ethics approval and consent to participate permission to conduct this study was obtained from the slaughterhouse authorities and the study protocol was approved by the ethical committee of burkina faso. results and discussion a total of 615 strains of salmonella isolated from meat, poultry carcasses, poultry, cattle, pigs, hedgehog, water, salad and humans were distributed into 110 different serotypes (table 1). the six most prevalent serotypes were derby, muenster, chester, drac, hato and typhimurium. salmonella derby (91/615) was found in beef meat (n=2), poultry carcasses (n= 34), poultry (n= 52) and fish (n = 3). our findings show that s. derby is the most often isolated serotypes in poultry. this is in contrast with results reported by some authors, showing that this serotype is most common in pig and also the 4 most frequently isolated serotype in humans in europe (kerouanton et al., 2013; hauser et al., 2011). salmonella muenster was the second most common serotype and was found in cattle, swine, hedgehog, poultry, human and fish. this finding shows that this serotype is not restricted to one host but can be found in animals, foods, water or humans. a documented food poisoning outbreak caused by s. muenster occurred in canada in 1982; it infected cheddar cheese made from unpasteurized milk (wood et al., 1984). salmonella chester was found in poultry carcasses, swine and poultry feces; this serotype has been reported in many countries: in canada, s. chester was responsible for an outbreak associated to frozen meals in 2010 (taylor et al., 2012); in 2014, six european countries (belgium, france, spain, germany, sweden and the uk) reported s. chester cases to the european centre for disease prevention and control (ecdc) associated with travel in morocco (whitworth, 2016). salmonella hato was found 3 table 1. salmonella serotypes and antimicrobials profile. salmonella beef poultry cattle swine hedgehog poultry human salad fish resrvoirs channel total resistance number of antibiotic number of resistant serotypes meat carcasses pattern resistered serotype s.abaetetuba 0 0 1 0 0 1 0 0 0 0 0 2 str 1 1 s.abony 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 s.adabraka 0 0 0 0 0 0 0 0 0 1 0 1 str 1 1 s.adelaide 0 1 0 0 0 1 0 0 0 0 0 2 0 0 0 s.agona 0 4 0 0 0 3 0 0 3 0 0 10 str 1 5 s.ahmadi 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.albany 0 0 2 0 0 2 0 0 0 0 0 4 str 1 2 s.anatum 0 1 0 0 0 1 0 0 0 0 0 2 str 1 2 s.angers 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.ank 0 0 0 0 4 1 0 0 0 0 0 5 str 1 5 s.antwepen 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.apeyeme 0 0 2 0 0 3 0 0 0 0 0 5 str 1 5 s.banana 0 3 1 0 1 2 1 0 0 0 0 8 str 1 5 s.bareilly 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.bargny 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.binningen 0 0 0 0 0 2 0 0 0 0 0 2 0 0 0 s.bochum 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.brancaster 0 1 1 0 0 3 0 0 0 0 0 5 str 1 4 s.bredeney 1 0 5 0 0 2 0 1 6 0 1 16 str 1 4 s.brive 0 0 1 0 0 0 0 0 1 0 0 2 str 1 1 s.carmel 0 0 1 0 0 0 0 0 0 1 0 2 0 0 0 s.carno 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.chandans 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 s.chester 0 5 1 0 0 31 0 0 0 0 1 38 str 1 37 s.chomedey 0 0 4 0 0 0 0 0 0 0 0 4 str 1 4 s.colindale 0 0 1 0 0 0 0 4 0 1 2 8 str 1 2 s.colobane 0 0 2 0 0 0 0 0 0 0 0 2 str 1 2 s.cubana 0 0 0 0 0 0 5 0 1 0 0 6 str 1 2 s.dahra 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 s.dakar 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 str,str-sul,str s.derby 2 34 0 0 0 52 0 0 3 0 0 91 tet,str-sul 5 57 tet,chl-str,str cip s.drac 0 0 26 0 1 0 0 0 3 0 0 30 str 1 20 s.dublin 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 4 table 1. contd. s.duisburg 0 0 0 0 0 1 1 0 0 0 0 2 str 1 2 s.ealing 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.eastbourne 0 1 2 0 0 2 0 0 1 0 3 9 str 1 5 s.eastglam 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.elisabethville 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.farakan 0 0 3 0 0 0 0 0 0 0 0 3 str 1 2 s.freetown 0 0 0 0 0 1 0 0 0 0 0 1 str 1 1 s.fresno 0 0 1 0 0 4 1 0 1 0 0 7 str 1 3 s.frintrop 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.fufu 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.galiema 0 1 0 0 0 2 0 0 0 0 1 4 str 1 4 s.gaminara 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.gerland 0 0 0 0 0 0 0 1 0 0 0 1 str 1 1 s.give 0 0 0 0 0 0 0 0 1 0 1 2 0 0 0 s.gokul 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 str, tet,str s.hato 1 4 5 0 0 22 0 0 0 0 0 32 tet,sul-tet,amp 5 15 str, amp-str sul-tet-tmp s.havana 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.hermannswerder 0 0 0 0 0 0 0 0 1 0 0 1 str-sul 2 1 s.hillingdon 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.hvittingfoss 0 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.ikeja 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.ilala 0 0 2 1 0 0 0 0 0 0 0 3 str 1 1 s.kaapstad 0 0 0 1 0 4 0 0 0 0 0 5 str 1 2 s.kalamu 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.kalina 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 amp-str-sul-tet s.kentucky 0 0 0 0 0 0 1 0 1 0 0 2 cip-gen-nal8 1 mec s.kiambu 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.kingston 0 0 2 0 0 3 0 0 0 0 0 5 str 1 2 s.kokomlemle 0 0 2 0 0 1 0 0 1 0 0 4 str 1 2 s.korlebu 0 0 2 0 0 0 0 4 1 0 0 7 str 1 5 s.lagos 0 0 4 0 0 2 0 0 0 0 0 6 str 1 4 s.llandoff 0 0 0 0 0 0 0 0 1 0 2 3 0 0 0 s.mbandaka 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 5 table 1. contd. s.minnesota 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.moero 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.monschaui 2 3 1 0 3 1 0 0 0 0 0 10 str 1 7 s.montevideo 0 0 0 0 0 0 1 0 1 0 0 2 0 0 0 str, str-sul, str s.muenster 0 0 17 3 11 6 4 0 6 0 0 47 tet, str, str-nal, 5 20 amp-str s.nima 0 1 3 0 0 0 0 0 0 0 0 4 0 0 0 s.nottingham 0 1 2 0 0 1 0 0 2 0 0 6 str-tet 2 1 s.offa 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.oranienburg 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.othmarschen 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.ouagadougou 0 0 0 0 0 0 0 0 0 0 1 1 str 1 1 s.ouakam 0 0 0 1 0 0 2 0 0 0 0 3 str 1 2 s.poona 0 0 2 0 0 1 3 0 2 1 1 10 str 1 5 s.rissen 0 0 1 0 0 0 0 0 0 0 1 2 0 0 0 s.rubislaw 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.ruiru 0 0 8 0 0 0 0 0 0 0 0 8 str,str-tet 2 5 str, str-tet, s.group b 0 1 1 0 0 0 2 0 1 0 0 5 amp-chl--str6 4 sul-tmp s.group c 0 1 1 0 0 9 0 0 0 0 0 11 str, str-sul 2 11 s.group¤ d 0 0 0 0 0 1 0 0 0 0 0 1 str 1 1 s.group¤ e 0 0 1 0 0 5 0 0 0 0 0 6 str, str-sul-tet 3 3 s.group¤ g 0 0 0 0 1 0 0 0 0 0 0 1 str 1 1 s.group¤ m 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.group¤ o:53 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 s.saarbruecken 0 1 0 0 0 0 0 0 0 0 0 1 str 1 1 s.saintpaul 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 s.salford 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.schwarzengrund 0 0 1 0 0 3 0 0 1 1 0 6 str 1 3 s.senftenberg 1 2 0 0 2 8 0 0 0 0 3 16 str, str-tet, str 3 6 sul-tet s.shangani 0 0 0 0 0 1 0 0 0 0 0 1 str-sul 2 1 s.shubra 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 s.soerenga 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.soumbedioune 0 0 4 0 0 0 0 0 0 0 0 4 str 1 3 s.stanley 0 0 0 0 0 0 1 0 0 0 0 1 str 1 2 6 table 1. contd. s.stanleyville 0 0 0 0 0 1 0 0 0 0 0 1 str-tet 2 1 s.tamberma 0 0 0 0 0 0 1 0 0 0 0 1 str-tet 2 1 s.tennessee 0 0 3 0 0 0 0 0 0 0 0 3 str 1 1 s.teshie 0 0 0 0 0 0 0 0 0 0 1 1 str 1 1 s.tilene 3 2 1 0 0 0 0 0 1 0 1 8 str 1 7 s.tounouma 0 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.trachau 0 0 1 0 0 1 0 0 0 0 0 2 str 1 2 s.typhi 0 1 0 0 0 0 1 0 0 0 0 2 str,amp-chl-str 6 2 sul-tet-tmp str,amp-chl-str s.typhimurium 0 0 3 0 0 4 13 0 1 0 0 21 sul-tet-tmp, 6 21 amp-chl-str-sul tmp s.umbilo 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.urbana 0 0 13 2 0 1 0 0 0 0 0 16 str, str-tet, chl 4 11 str-tmp s.vilvoorde 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.virchow 0 0 1 0 0 0 2 0 0 0 1 4 str 1 1 s.waedenswil 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.wagadugu 0 0 0 0 0 0 0 0 0 1 1 2 str 1 2 s.waycross 0 0 2 0 0 1 0 0 3 0 0 6 str 1 2 s.yoruba 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 salmonella sp. 0 0 1 0 0 0 0 0 0 0 2 3 str 1 2 total 10 68 161 8 23 193 48 10 56 6 25 608 359 amp, ampicillin; chl, chloramphenicol; str, streptomycin; sul, sulphonamides; tmp, trimethoprim; tet, tetracycline; nal, nalidixic acid; cip, ciprofloxacin; ftx, cefotaxime; mec, mecillinam. in beef meat, poultry carcasses, cattle and poultry; salmonella drac was found in cattle, hedgeghog and fish. salmonella typhimurium was found in cattle, poultry, human and fish. s. typhimurium has been implicated in many outbreaks worldwide (rayamajhi et al., 2008). this serotype has a wellcharacterized ability to infect various species (rabsch et al., 2002) and can survive for a long time in the environment (baudart et al., 2000); these 2 factors enhance the ability of this serotype to be one of the most common causes of salmonellosis in many geographically diverse regions and has caused many disease outbreaks (mather et al., 2013; cui et al., 2008; galanis et al., 2006). although serotype typhimurium was associated with several foods of animal origin, the most common was chicken (26% of outbreaks), indicating that chicken is a major route of exposure. this corroborates with our present results, where poultry is identified as the primary source of s. typhimurium. in contrast to the report of foley et al. (2008), typhimurium was the most common serotype among pork associated with outbreaks. this shows that serotype typhimurium has emerged as the predominant serotype in swine. after the six most prevalent serotypes, the presence of serotype like typhi (poultry carcasses and human), senftenberg, virchow, kentucky, stanley and kingston which have caused many outbreaks in diverse regions were noted. the presence of s. typhi in poultry carcasses shows that there is a cross-contamination during 7 carcasses handling since humans are the only reservoir of typhoid salmonella. this is also due to the fact that humans can be a chronic carrier of these salmonella (eng et al., 2015). carriers of s. typhi and s. paratyphi are responsible for the spreading of enteric fever in endemic regions, as the common transmission route is the ingestion of water or food, contaminated with the feces of chronic carriers (bhan et al., 2005). in this report, salmonella serotypes drac, banana, monschaui, muenster, seftenberg are found in wild hedgehogs. this animal has been the source of human salmonellosis in many europeans countries, used as a pet animal (nauerby et al., 2000). although little can be done to prevent the spread of salmonella in the environment amongst wild animals, the control should be done in food handling and drinking water. our results show that the same serotype of salmonella is found in diverse sources, meaning that this serotype can be transmitted to humans through many sources. in general, food animals such as swine, poultry and cattle are the prime sources of salmonella infections. the major dissemination routes of the pathogens involve trade in animals and uncooked animal food products. the slaughtering process of food animals at abattoirs is considered one of the important sources of organ and carcass contamination with salmonella (gillespie et al., 2005). in burkina faso, all serotypes of salmonella can circulate between animals, foods, fish, water and humans. this happens as animals shed feces in the environment which rain water carries to rivers, barrages and channel; aquatic animals like fish can be contaminated by diverse microorganisms coming from water. in addition, wastewater and/or untreated animal feces are used to grow salad and other vegetable from gardens. here, we can see how humans can be contaminated in this closed circle, since animals can contaminate meat, water, fish, vegetables and environment; the consumers are exposed to a high risk of contamination by salmonella and other pathogens. these findings are critical because burkina faso is facing serious problems in the area of water and sanitation due to demographic explosion and poor urban planning. this problem is increasing the risk of environmental and human contamination. in ouagadougou, the capital city of burkina faso, wastewater from channel, where we found many salmonella strains, is used to irrigate vegetables from gardens, and animals’ feces are also used in vegetable crops, which is a new source of growth for poor people. vegetables like salad are not nutritive media for microorganisms but can constitute mechanical vectors for transporting microorganisms to humans if hygienic condition in garden is not maintained well. based on our findings, as seen in figure 1, the salmonella transmission routes are based on their diversity and sources. the diversity of possible reservoirs of infection results in significant challenges for public health authorities to control the infections (dione et al., 2011). among the 615 strains, 94% (581/615) were resistant to one or more antibiotics, and resistance to streptomycin was the most common (table 1). the resistance pattern str-sul-tet, str-tet and str-sul was dominant and found in 80% of the strains. about 3% of the strains were resistant to 5 or 6 antibiotics; their resistance pattern was amp-str-sul-tet-tmp or amp-chl-strsul-tet-tmp. one salmonella strain, s. kentucky isolated from human stool was resistant to eight antibiotics with the pattern, amp-str-sul-tet-cip-gen-nal-mec. this serotype was found also in fish in this study. in contrast, centre for disease prevention and control (cdc) has reported that s. kentucky is typically found in cattle and poultry (cdc, 2011). in this study, streptomycin resistance was found in 94% of the salmonella serotypes. majority of aminoglycosides are bactericidal but salmonella use mechanisms to resist their antibiotic families such as expression of plasmidmediated aminoglycoside modifying enzymes against aminoglycoside (gebreyes and altier, 2002). the genes encoding theses enzymes have been found in varieties of salmonella subtypes like: agona, typhimurium, newport, typhimurium var. copenhagen, kentucky, blockely, bredeney, anatum, derby, give, enteritidis, heidelberg, saint paul, london, saintpaul, hadar, heidelberg, and 4,5,12:i: (levings et al., 2005). in this study, resistance to ampicillin was observed in salmonella serotypes: hato, kentucky, muenster, typhi and typhimurium. betalactams are generally considered bactericidal; but salmonella strains are now becoming resistant to ampicillin and methicillin due to their wide clinical use (angulo et al., 2000). in salmonella, the secretion of a beta-lactamase is the common mechanism of resistance to beta-lactamases. several authors reported betalactamases in a variety of salmonella serotypes including enteritidis, dublin, haadrt, anatum, muenchen, stanley, panama, virchow, and typhimurium (gebreyes and thakur, 2005; batchelor et al., 2005). in the present study, salmonella serotypes derby, hato, muenster, nottingham, ruiru, senftenberg, stanleyville, tamberna, typhi, urbana and typhimurium were resistant to tetracycline. resistance to tetracycline has been reported in several serotypes including typhimurium, saintpaul, enteritidis, hadar and choleraesuis, agona, anatum, blockley, bredeney, colorado, derby, give, haardt, heidelberg, infantis, orion, seftenberg, (frech and schwarz, 2000; pezzella et al., 2004). salmonella serotypes derby, hato, hermannswerder, kentucky, muenster, senftenberg, shangani, typhi and typhimurium were resistant to sulfonamide in the present study. many authors have been reported resistance to sulfonamide in a wide range of salmonella serotypes such as enteritidis, hadar, heidelberg, orion, rissen, agona, albany, derby, djugu, and typhimurium (antunes et al., 2005; doublet et al., 8 pets and wild animal water (rivers, barrage, channel, well, tap water) environment (wind, food animals insects, rodents, grass, birds) poultry vegetables, fish and other aquatic animals foods (meat and meat products, eggs, food staff and other food types) human figure 1.transmissions routes of salmonella. red color = more implicated group in pathogens transmission; orange color: the second more implicated group. 2004). in our report salmonella serotypes hato, urbana, typhi and typhimurium were resistant to trimethoprim. martinez et al. (2005) reported trimethoprim resistance in salmonella serotypes agona, djugu, hadar, neport, rissen albany, derby, and typhimirium. chloramphenicol resistance was found in salmonella serotypes, derby, typhi, typhimurium and urbana. this finding corroborates with the report of alcaine et al. (2005), where salmonella typhi, agona, derby, kiambo, albany, newport, haardy, enteritidis and typhimurium isolates have been found to harbor resistant genes for chloramphenicol. there are two mechanisms in which salmonella resistance to chloramphenicol is conferred: (i) by the plasmid-mediated enzymes called chloramphenicol acetyltransferases (cat) or nonenzymatic chloramphenicol resistance gene cm1a and (ii) efflux pump in which the antibiotic is pumped out of the cell. in this study, quinolone resistance was observed in salmonella serotypes derby, kentucky, muenster and typhimurium. the mechanisms of quinolone resistance for salmonella are all chromosomally mediated, so the numbers of quinoloneresistant salmonella can only increase in two ways: (i) the selection of a quinolone-resistant bacterium after exposure to a fluoroquinolone in humans or animals, or (ii) the spread of a quinolone-resistant bacterium to other animals or to humans (piddock, 2002). according to cdc, outbreaks caused by antimicrobial-resistant salmonella have been associated with an increased rate of hospitalization, and the rate of death was significantly greater in outbreaks caused by resistant strains (cdc, 2011). resistance can spread from non-human sources to human by various routes such as animal, water and contaminated foods (figure 1). resistance to combinations of many classes of antimicrobial agents in salmonella has led to the re-emergence of multidrug resistance salmonella (mdr) strains (o’brien, 2002). in this study, salmonella serotypes, derby, hato, hermannswerder, urbana, shangani, tamberma, kentucky, muenster, senftenberg, shangani, typhi and typhimurium were mdr. this is similar to the findings of many authors, who reported that mdr salmonella strains have been found to be of many serotypes such as agona, anatum, pullorum, schwarzengrund, choleraesuis, derby, dublin, heidelberg, kentucky, newport, senftenberg, typhimurium, and uganda (chen et al., 2004; zhao et al., 2008). all s. typhimurium reported in the present study were mdr and most of them were found to display a phenotype of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (acssut). these antibiotics are the 9 most common drug classes used in veterinary medicine (mulvey et al., 2006). conclusion the genetic make-up of the salmonella strains permits their adaptation in various environments, including human, animal and non-animal hosts. this increases the difficulty in eliminating the bacteria. moreover, the emergence of mdr salmonella strains poses a great challenge in terms of effective treatment of the infections caused by these strains. several preventive measures have been proposed to stop the spread of antimicrobial resistant salmonella infections, and the restriction of indiscriminate use of antibiotics in food animals is by far one of the most effective measures. this report can help international organization to understand salmonella data and trends and to develop more informed solutions for reducing salmonella contamination along the farm to table chain. the report of sporadic data about salmonella serotype distribution will highlight the importance of the potential source of salmonella infection to humans. the data obtained in this study can be used by the world health organizationglobal foodborne infections network (who-gfn) and public authority to define the guidelines for basic surveillance system of salmonella and other enteropathogenic bacteria circulating among humans, animals, food and environment. conflict of interests the authors have not declared any conflict of interests. acknowledgement this research was supported by the international foundation for science (ifs) grant e/5001-2f/4600 to ak. the efforts of all who contributed to the success of the research are highly appreciated, ranging from conducting of strain characterization and drafting of the manuscript, as well as supervision and participation in writing of 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© international scholars journals author(s) retain the copyright of this article. full length research paper tetracycline resistance genes in pasteurella multocida isolates from bovine, ovine, caprine and swine pneumonic lungs originated from different greek prefectures maria babetsa1,2, vassilios sandalakis4,5, christina vougidou3, antonios zdragas1, afroditi sivropoulou2, anna psaroulaki4,5 and loukia v. ekateriniadou1* 1 national agricultural research foundation, n.ag.re.f., veterinary research institute of thessaloniki, n.ag.re.f campus of thermi, 57001, thermi, thessalonki, greece. 2 department of biology, aristotle university of thessaloniki, campus of thessaloniki 54124, thessaloniki, greece. 3 institute of infectious and parasitic diseases of thessaloniki, ministry of rural development and food, 54627, thessaloniki, greece. 4 regional laboratory of public health of crete, 71110, heraklion, crete, greece. 5 laboratory of clinical bacteriology, parasitology, zoonoses and geographical medicine, whocc, university of crete, 71110, heraklion, crete, greece. accepted 13 october, 2019 the aim of the present work was to analyze the antibiotic resistance of pasteurella multocida isolates from sheep, goats, cattle and pigs originated from eight different greek prefectures, to determine the class(es) of the tet genes, to investigate any possible association with plasmids or/and chromosomes and to study the geographical distribution of the resistant isolates. eighteen out to one hundred isolates were resistant to tetracycline with a minimal inhibitory concentration (mic) higher than 128 μg/ml. the teth gene was found in thirteen (72.2%) and the tetb gene was found in four isolates (22.2%). after treatment with the restriction enzymes ecorv, ecori, psti and bsphi, the tetb gene showed a new polymorphism. partial sequencing of the teth and tetb genes revealed five polymorphisms in the tetb gene and two polymorphisms in the teth gene; phylogenetic and molecular evolutionary analyses were also conducted. both tetb and teth genes revealed patterns clearly differentiated from the rest of pasteurella species meaning that the genes could be transferred from a non pasteurella species. the results presented provide further knowledge and evidence of the role played by commensals as of resistance determinants. key words: pasteurella multocida, tet genes, multiple sequence analysis. introduction pasteurella multocida causes economically important diseases in food-producing animals and exhibits *corresponding author. e-mail: ekateriniadou@vri.gr. tel: +302310365392. fax: +302310365371. resistance to a large number of antimicrobial agents; as a result a limited number of antimicrobial agents is available for treatment including, sulphonamides, tetracycline and streptomycin. the use of tetracycline, for both the treatment and the prevention of diseases in which p. multocida is involved, results in high percentages of tetracycline-resistant strains (kehrenberg and schwarz, 2000). resistance to antimicrobial agents, including tetracycline depends on the host, the geographical origin and the antimicrobial pre-treatment. antimicrobial resistance has been reported in a wide variety of animal pathogens for example p. multocida from cattle; (mcewen and fedorka-cray, 2002). data collected from 13 european countries showed that isolates of p. multocida were resistant to different antimicrobial agents in eight countries. from 1987 to 2004, the percentage of tetracycline resistant isolates from cattle in different european countries ranged between 1% in england and 24% in italy (lizarazo et al., 2006; kaspar et al., 2007; wallmann et al., 2007; hendriksen et al., 2008). in greece, all p. multocida strains isolated from pigs and rabbits in 1984 to 1987 were susceptible to tetracycline (ekateriniadou, 1989). in the present study, the tetb, teta, tetm and teth genes were studied. the tetb gene which is part of the non-conjugative transposon tn10, has been found in genus pasteurella (kehrenberg et al., 2001) and it is the most frequently isolated tet gene in enterobacteriaceae. the teta gene, encoded also by the transposon tn10, is one of the most widespread tet genes in enterobacteriaceae (kehrenberg et al., 2001, 2005; millan et al., 2009) found in a variety of ecosystems including estuarine waters (henriques et al., 2008). the tetm gene is the most prevalent determinant in listeria species (chen et al., 2010) and common gene conferring tetracycline resistance in staphylococcus aureus http://jac.oxfordjournals.org/contnent/64/3/490.full and widely distributed among both gram-positive and gramnegative bacteria; this is probably due to the association of tetm gene with integrative and conjugative transposons as tn916 which can be expressed both in gram-positive and gram-negative bacteria (de vriest et al., 2009). the tetm gene has been detected in the chromosomal deoxyribonucleic acid (dna) of p. multocida isolates. the teth gene which seems to be indigenous to pasteurellae was detected on plasmids pvm111, pmht1, ppmt1 and ppat1 and on the transposon tn5706 (hansen et al., 1993; kehrenberg et al., 1998, 2001). the aim of our study was to determine the class(es) of the tet genes present in p. multocida isolates, to identify their association with plasmid or/and chromosome and to investigate the geographical distribution of the resistant isolates in greece. materials and methods bacterial isolates and antimicrobial susceptibility testing one hundred p. multocida strains were isolated from pneumonic tissue samples that originated from sheep, goats, cattle and pigs originated from eight different greek prefectures. the samples were inoculated on blood agar plates (5% v/v sheep blood) and were aerobically incubated at 37°c for 18 to 24 h. after isolation, microscopic examination and biochemical testing (oxidase, catalase and indole production) were used for the final identification of the strains (garity et al., 2004). the determination of tetracycline resistance was performed by the agar diffusion method using disks of tetracycline (30 μg/ml) and the phenotypic resistance of tetracycline was further analyzed by determining the mic according to the clsi’ broth micro dilution procedure, using two-fold dilution series in the range of 8 to 128 μg/ml. identification of the tet gene classes both genomic and plasmid dna extractions were carried out: genomic dna extraction was performed in the tetracycline resistance isolates using the purelink tm genomic dna kit (invitrogen) while plasmid dna extraction was performed using the purelink tm hipure plasmid dna purification kit (invitrogen). for the evaluation of the method applied in this study concerning the accuracy of the plasmid dna isolation and particularly whether fragments of chromosomal dna might be isolated with plasmids, closed circular plasmid dna from the tetracycline-resistant p. multocida strains was purified also by equilibrium centrifugation in csci-ethidium bromide gradients (sambrook et al., 2001). the purified plasmids were used to transform competent escherichia coli (xl1-blue) cells, which subsequently were plated in lb agar containing 50 μg/ml of tetracycline. the appearance of tetracyclineresistant strains in all cases, confirms the presence of tetracyclineresistant genes in the examined plasmids. further, in the above plasmids derived from the p. multocida strains were detected with polymerase chain reaction (pcr) the tetb, teth and teth genes, respectively, as in the method applied in the present study. additionally, for the evaluation of chromosomal dna isolation and particularly whether large plasmids might be segregated with chromosomal dna, plasmid curing was applied in two tetracyclineresistant p. multocida strains (706/14 and 806/8 p) by growing the bacteria on brain heart infusion agar in the presence of 0.002% sodium dodecyl sulphate (sds) and incubation for 24 h, at 42°c (sivropoulou et al., 2000). plasmid isolation from totally 30 colonies of p. multocida 706/14 p, revealed that two were free from plasmids, and from 45 colonies from p. multocida 806/8 p, five were free from plasmids. since the above colonies were remained resistant to tetracycline, the tetracycline resistant genes are located in the chromosome as detected with the method applied in the present study. for pcr analysis six class-specific primmer sets were used to amplify the teta, tetb, teth and tetm genes in all the resistant isolates (table 1). in order to optimize the methodology, three different pcr protocols were used: (a) a short protocol (sp) described by koike et al. (2007), (b) a two-temperature step-down protocol (tsp) characterized by a higher sensitivity described by aminov et al. (2002) and (c) a long time protocol (ltp) carried out to enhance a longer part of the tetb and teth genes (hansen et al., 1993; kehrenberg and schwarz, 2000). amplicons of the tetb and teth genes were of 1170 and 1076 bp, respectively. dna amplifications using the ltp protocol were carried out for dna analysis by the restriction fragment length polymorphism (rflp) method as well as by sequencing (fluit et al., 2001). dna amplification was performed in a ptc-200 peltier thermal cycler (mj research). non-amplified isolates by the sp protocol were further analyzed by the tsp protocol. analysis of the tetb and teth classes the isolates carrying the tetb and the teth genes were treated with the restriction enzymes ecorv, ecori, psti, bsphi to detect possible polymorphisms. reactions were performed in a total volume of 15 μl according to the manufacturer’s instructions. the resulting fragments were separated by 3% low melt agarose gel table 1. primers used for the identification of teta, tetb, teth, tetm genes of p. multocida isolates. primer set sequence (5'→3') annealing temperature (°c) protocol fragment size (bp) teta f΄ gcgcgatctggttcactcg 61 sp-stp 164 r΄ agtcgacagyrgcgccggc tetb f΄ tacgtgaatttattgcttcgg 61 sp-stp 206 r΄ atacagcatccaaagcgcac tetm f΄ acagaaagcttattatataac 58 sp-stp 170 r΄ tggcgtgtctatgatgttcac teth f΄ cagtgaaaattcactggcaac 61 sp-stp 185 r΄ atccaaagtgtggttgagaat tetb f΄ acgttactcgatgccat 55 ltp 1170 r΄ agcacttgtctcctgtt teth f΄ atactgctgatcacccgt 55 ltp 1076 r΄ tcccaataagcgacgct the tetb gene of this study is enclosed within the box. the gene from the greek isolate is clustered separately from other species of the pasteurella genus figure 2. phylogenetic tree of tetb gene. electrophoresis (ultrapure tm lmp agarose, invitrogen). sequencing analysis of the tetb and teth genes the tetb and teth genes were partially amplified using the ltp protocol and the primers tethltpf, tethltpr and tetbltpf, tetbltpr. sequencing analysis was performed on a sequence scanner version 1.0 (applied biosystems) and editseq, megalign modules of the lasergene ver.7.1 software (dnastar inc., madison, wi, usa). complementary alignments were also performed using clustalw2 multiple sequence alignment software (http://www.ebi.ac.uk/tools/msa/clustalw2). phylogenetic and molecular evolutionary analyses were conducted using mega version 4 (tamura et al., 2007). the tetb gene was aligned with the corresponding sequences from the bacteria haemophilus parainfluenzae, haemophilus influenzae, pasteurella aerogenes, pasteurella multocida and from plasmid r100. the sequence of the tetb gene in the tn10 transposon element (j01830.1, genbank) was used as reference sequence (figure 2). the teth gene was aligned with the corresponding sequences from two different strains of p. multocida, p. haemolitica, actinobacillus pleuropneumoniae, the teth gene of this study is enclosed within the box. the gene from the greek isolate is clearly separated from other species of the pasteurella genus figure 3. phylogenetic tree of teth gene. table 2. polymorphisms in tetb gene. accession number nucleotide position 511b 842b 902b 988b 1014b 1060b j01830.1 nt act gaa gat gag ttg act aa t e d e l t 706/p14tetb nt gct gga gtt cag ttg gct aa a g v q l a fq312002.1 nt gct gga gtt cag ttg gct aa a g v q l a aj627386.1 nt gct gga gtt cag ttg gct aa a g v q l a aj278685.1 nt gct gga gtt cag ttt gct aa a g v q f a ap000342.1 nt act gga gtt cag ttg gct aa t g v q l a eu252517.1 nt act gga gtt cag ttg gct aa t g v q l a *numbering is according to the highlighted is the reference sequence, nt-nucleotide sequence, aa-amino acid sequence. and p. aerogenes. plasmid pvm111 was used as reference sequence (figure 3). both amino acid sequences were aligned with the corresponding sequences (tables 2 and 3). the amino acid sequence of the tetb gene in the tn10 transposon element was used as reference sequence. nucleotide sequence accession numbers the nucleotide sequence of the 1069 bp segment of the tetb gene and the 987 bp segment of the teth gene were deposited in the embl database (accession no fr872822 and fr872823, respectively). results from the one hundred p. multocida isolates, eighteen showed resistance to tetracycline with mic higher than 128 μg/ml. the origin of the isolates is showed in figure 1. the size of the amplicons for each of the three pcr protocols used in the study is shown in table 1. the teth gene was found in thirteen (72,2%) and the tetb gene in four isolates (22,2%). in one isolate (5,5%) none of the four tet genes was detected. both genes were found in chromosomal as well as in plasmid dna. only one table 3. polymorphisms in teth gene. accession number nucleotide position 410b 755b 765b aj514834.1 nt gtt gcg ggt aa v a g 706/p8 teth nt gct gcg ggg aa a a g u00792.1 nt gtt gcg ggt aa v a g y16103.1 nt gct ggg ggt aa a g g y15510.1 nt gct gcg ggt aa a a g ay987962.1 nt gct gcg ggt aa a a g aj245947.1 nt gct gcg ggt aa a a g 1k 1t 1x 1c 10v isolates from pigs in the 1l prefectureofvoiotia (5 teth/4tetb/ not identified) 2i isolates teth from sheep in the prefecture of ilia 10v 1x isolate teth from pig in the 1a prefecture of xanthi 1c isolate teth from sheep in 2i the prefecture of chalkidiki ep 1a isolate teth from sheep in the prefecture of achaia 1k isolate teth from sheep in the prefecture of kilkis 1l isolate teth from sheep in the prefecture of larisa 1t isolate teth from sheep in the prefecture of thessaloniki figure 1. geographical distribution of the local isolates. isolate was found carrying the teth gene in plasmid but not in chromosomal dna. amplified tetb and teth genes were analyzed by the rflp method. digestion was performed with ecorv, ecori, psti and bsphi restriction enzymes. the amplicons of tetb gene resulted to the same rflps patterns as the reference sequence of transposon tn10 (j01830, genbank). only treatment with bsphi resulted in a different profile: in contrast to the local isolates, the reference strain showed a recognition site for the enzyme bsphi in position 951/955. no polymorphisms were identified in the amplicons of the teth gene. amplicons of the tetb and teth genes were partially (1060 bp and 987, respectively) sequenced. unique tetb and teth sequences were identified. analysis of the tetb gene and comparison with the corresponding sequences (table 2), revealed five single nucleotide polymorphisms (snps) (a538g, a892g, a929t, g1038c, a1087g). polymorphism a538g could be detected after digestion with the restriction enzyme tsp45i (gtsac). from the analysis of the teth gene two polymorphisms were found (c460t, g815t). polymorphism c460t was common (table 3) while polymorphism g815t was found for the first time. both amino acid sequences were aligned with the corresponding sequences. comparison between the tetb and transposon tn10, revealed five snps (t171a, e281g, d301v, e330q, t354a). polymorphism t171a was common among the tetb amino acid sequences of h. parainfluenzae, h. influenzae, and p. aerogenes. the other four polymorphisms (e281g, d301v, e330q and t354a) were common not only among h. parainfluenzae, h. influenzae and p. aerogenes but also among plasmid r100 and p. multocida. these four polymorphisms have not been found in tn10 transposon element. one polymorphism (v137a) was found in the teth aminoacid sequence. polymorphism v137a was common among strains of p. haemolytica, p. multocida, a. pleuropneumoniae and p. aerogenes. the phylogenetic trees for both genes were created using the neighborjoining method with bootstrap test (500 replicates). the evolutionary distances were computed using the kimura 2-parameter method (kimura, 1980). all positions containing gaps and missing data were eliminated from the dataset (complete deletion option). based on the tetb dendrogram (figure 2) we can observe the close clustering of the greek p. multocida isolates with species of shigella, salmonella, haemophilus and escerichia. the greek p. multocida isolates of the tetb gene were less homologous to those of p. aerogenes plasmid ppat2 and also clustered separately from the tetb of p. multocida plasmid pb1001. as shown in the dendrogram (figure 3), the teth gene of the greek p. multocida isolates has evolved and separated from the cluster of p. multocida ppmt1 plasmid (y15510.1), p. aerogenes plasmid ppat1, acinetobacter sp. and haemophilus somnus. furthermore, the teth gene of p. haemolytica plasmid ppht1 has followed an evolutionary process that differentiated it from the cluster forming a distinct branch. p. multocida u00792.1 and p. multocida partial plasmid pvm111, depicted in a common branch, also evolved separately from the rest of the entries. discussion antimicrobial resistance is a concern for animal health but little is known about the magnitude of this problem as the surveillance of resistance in exclusive animal pathogens (for example, moraxella bovis, actinobacillus pleuropneumoniae and pasteurella multocida) is poor compared with surveillance of enteric bacteria. knowledge and control of resistance in commensals is important as they can act as reservoirs of resistance determinants. the flora of the upper respiratory system of animals that have been treated with antimicrobial agents can also serve as a reservoir of resistance factors. tetracycline is the most frequently used antimicrobial agents in veterinary medicine. antimicrobial resistance of p. multocida strains has been reported in a wide variety of animal pathogens (mcewen and fedorka-cray, 2002; milan et al., 2009). data collected from 13 european countries showed that isolates of p. multocida from eight countries were resistant to different antimicrobial agents including tetracycline (kaspar et al., 2007; hendriksen et al., 2008). it is important that 18% of the strains isolated from clinically healthy animals in greece showed resistance to tetracycline higher than 128 μg/ml. the significant increase of the tetracycline resistant isolates contributes to the selection and spread of resistance in animals not only by the movement of carrier animals between herds but also by the assembly of susceptible animals in close confinement, and the movement of resistance determinants throughout the ecosystem (mcewen and fedorka-cray, 2002; adelowo and fagade, 2009). moreover, the spread of tet genes is often facilitated by their location on mobile genetic elements, such as plasmids and transposons. for the first time in greece, tet genes were investigated. the presence of the teth and tetb genes in both genomic and plasmid dna, suggests that they are carried on transposable elements as previous studies have shown for example, tetb gene with the transposon element tn10 (kehrenberg et al., 2001) and teth gene with the transposon element tn5706 (kehrenberg et al., 1998). the teth gene was found in the majority of the tetracycline resistant p. multocida isolates, which is not surprising as it is considered to be indigenous for the genus pasteurella (hansen et al., 1993) and it has been detected on plasmids pvm111, pmht1, ppmt1 and ppat1 and on the transposon tn5706 (hansen et al., 1993; kehrenberg et al., 1998, 2001). similarly, the tetb gene is the most frequently observed tet gene among enterobacteriaceae, as part of the nonconjugative transposon tn10 (chalmers et al., 2000). the wide distribution of the tetb across gram–negative genera, including escherichia, enterobacter, proteus, salmonella, actinobacillus, haemophilus, moraxella, and treponema indicates a great likelihood of horizontal gene transfer occurrence of tetracycline resistance (speer et al., 1992; roberts, 1996). moreover, the tetb gene was first detected in h. influenzae, a member of the pasteurelllaceae commonly isolated from the lungs. most of the polymorphisms can be detected in other strains or species (tables 1 and 2). glycine (255g) of the teth gene might have a substantial role in the functionality of the protein, thus, the observed mutation was synonymous. perhaps, this is why no mutations have been previously described at this site. based on the dendrogram, the tetb gene in the greek isolates of p. multocida is more homologous to species other than pasteurellae leading us in two explanations: the gene could probably has been transferred to the isolates from a non pasteurella species or it is still in an ancestral form which has not been altered as it has in other species of its genus, thus clustering separately from them. the local (greek) p. multocida teth gene revealed a different pattern of clustering. nevertheless, as in the case of tetb, the local isolates teth gene are clearly differentiated from the rest of pasteurella species. finally, the absence of teta and tetm genes suggests that a wide spectrum of tet genes should be used in future studies. although our results are similar to those of other researchers (claudio et al., 2003), further research on other genes involved in tetracycline resistance, such 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(1):1.691.71. sivropoulou a, haritidou l, vasara e, aptosoglou s, koliais s (2000). correlation of the insecticidal activity of the bacillus thurigensis a4 strain against bactrocera oleae (diptera) with the 140-kda crystal polypeptide. curr. microbiol., 41(4): 262-266. speer bs, shoemaker nb, salyers aa (1992). bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. clin. microbiol. rev., 5: 387–399. tamura k, dudley j, nei m, kumar s (2007). mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol., 24: 1596-1599. wallmann j, schröer u, kaspar h (2007). quantitative resistance level (mic) of bacterial pathogens (escherichia coli, pasteurella multocida, pseudomonas aeruginosa, salmonella sp., staphylococcus aureus) isolated from chickens and turkeys: national resistance monitoring by the bvl 2004/2005. berl. munch tierarztl., 120(9-10): 452-463. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 9 (1), pp. 001-006, january, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper seasonal emergence of swine erysipelas in hilly state nagaland, northeast india nagendra nath barman1*, debojyoti borkotoky2, biswajyoti borah1, anjan jyoti nath3, papiya das1 and durlav prasad borah1 1 department of microbiology, college of veterinary science, assam agricultural university, khanapara campus, guwahati-781022, assam, india. 2 subject matter specialist (animal science), kvk phek, icar-nrc on mithun, nagaland-797007, india. 3 department of microbiology, lakhimpur college of veterinary science, assam agricultural university, joyhing, north lakhimpur787 051, assam, india. *corresponding author. e-mail: nnbarman@gmail.com. accepted 20 september, 2019 seasonal outbreaks of swine erysipelas have been reported in back yard pig farms in the phek district of nagaland, india. the alpha haemolytic isolate of erysipelothrix rhusiopathiae was recovered on blood agar from the clinical samples. the organisms were confirmed microscopically, biochemical analysis as well as by polymerase chain reaction (pcr) amplification of 16s rrna gene and sequence analysis. these nagaland isolates (kt160358, kt160359) were closely related to the type spp. e. rhusiopathiae in phylogenetic analysis and forms the same clad with chineese isolates of swine and murine origin indicating an epidemiological link. the isolates were found to be most sensitive to oxytetracycline and responded to treatment. swine erysipelas occurred in phek district in a season due to sudden change of weather and temperature. pigs exposed to such predisposing factors probably favoured to propagation of already persisted organisms in pigs. this is the first confirmed case of e. rhusiopathiae infection from the ne states of nagaland, india. key words: swine erysipelas, erysipel othri x rhusiopathiae, pig, polymerase chain reaction (pcr) nagaland, india, oxytetracycline. introduction erysipelothrix rhusiopathiae, belonging to the family erysipelotrichac eae, is a non-motile, gram-positive, nonsporulating, non-acid-fast organism distributed worldwide affecting wide variety of vertebrate and invertebrate species including man (reboli and farrar, 1989). organisms in many occasions harbour by pigs in lymph nodes and shed along with feces, urine, saliva and nasal secretions (lee et al., 2011). affected pigs manifest the disease as (i) acute septic form, (ii) subacute urticarial form marked by reddis h-purple rhomboid spots or "diamonds" in the skin, (iii) joint or arthritic form, and (iv) chronic cardiac form (endocarditis) (reboli and farrar, 1989).various predisposing factors, change of environmental conditions and parasitic infestation lead to 2 reappearance of s wine erysipelothrix (se) infection in that population. seasonal outbreaks of swine erysipelas were investigated in phek district of nagaland, india during 2013-2015. materials and methods outbreaks of sw ine erysipelas – samples collection the disease of sw ine erysipelas w as reported from porba village (altitude 1985 msl), district phek of nagaland during the summer rainy season from 2013 to 2015. the village had a pig population of 552 cross bred and 200 local doom pigs as per the livestoc k census report 2012 (goi, 2012). far mers keep pigs as a bac k yard s mall unit mainly for meat purpose and fed them on household as w ell as hotel w aste. in every rainy summer season ( may – july) there w as disease outbreaks in pigs. affected animals (150) clinically w ere anorexic and recumbent w ith high fever (106°f) for 2-3 days. some animals developed erythematous patches. postmortem examination revealed haemorrhages in intestine, congestion of liver, spleen and kidney. during the course of investigation 3 more animals died w ithin 12 days. all affected pigs w ere also reported to be infested w ith pig louse haemat opinus suis. clinical sample like sw abs from the w ound and tissue biopsy from the affected areas were collected in sterile containers for isolation of the causative organis m. samples also included sloughed off tissues and biopsy samples preserved in 10% formalin for histopathological study . virological investigation tissue samples w ere processed for demonstration of classical sw ine fever using single step reverse transcription poly merase chain reaction ( rtpcr) (hoffmann et al., 2005) and sw ine pox as per the method of medaglia et al. (2011). identification and antibiotic activity of erysipelothrix rhusiopathiae the sw ab samples ( n=51) from the affected areas w ere inoculated into nutrient broth and incubated aerobically at 37°c for 48 h. sub-culturing w as done on blood agar plates, incubated at 37˚ c in presence of 5% co2 for 24 h. colony mor phologies w ere studied. gram’s staining and biochemical analysis w ere done to confirm the organis m. a panel of antibiotic discs containing amikacin (30 µg), amoxicillin (30 µg), ampicillin (10 µg), cefotaxime (30 µg), chloramphenicol (30 µg), c iprofloxacin (5 µg), cloxac illin (30 µg), co-trimoxazole (25 µg), enrofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), norfloxacin (10 µg), oxytetracycline (30 µg), streptomycin (25 µg) and tetracycline (30 µg) w as used to study the antibiotic sensitivity patter n of the isolates. the zone of inhibition was measured, recorded and interpreted according to the clinical and laboratory standards institute criteria (clis-mic). detection of nucleic acid of erysipelothrix rhusiopathiae and sequencing for detection of e. rhusiopathiae nucleic acid tissue samples w ere processed using dna sure® tissue mini kit ( nucleo-pore, cat.#np-61305) and poly merase chain reaction ( pcr) amplification for 16s r rna gene pr imers ( mo101 f agatgcca tagaaactggta and m0102 r ctgtatccgcca taa cta) of e. rhusiopathiae ( makino et al., 1994) w as used. the pcr conditions w ere optimized w ith a final volume of 25 µl at 94°c for 5 min follow ed by 30 cycle of 94°c for 30 s, 54˚c for 2 min and 72˚c for 45 s and final extension w as carried out at 72°c for 5 min. in pcr r eaction 28 ng/ µl total genomic dna w as taken along w ith positive and negative control. pr oducts of pcr w ere visualized in 2% agarose gel electrophoresis under geldoc ( kodak, usa). further pcr products w ere purified by qiaquick pcr purification kit protocol and sequenced. analysis of gene sequence and phylogenetic studies on erysipelothrix rhusiopathiae to deter mine the relationship of the nagaland isolates of e. rhusiopathiae w ith other isolates of this species, 16s r rna gene was amplified and sequenced (genbank accession number kt160358 and kt160359). sequence identity at nucleotide level was deter mined by clustal w method of meg-align program in dna sta r pac kage ( dnasta r inc., usa). the phylogenetic tree was constructed using other 16s r rna gene sequences available at ncbi (viz. strain zyl(kf811052.1), isolate eu188793.1, strain:kgbb1(ab055909.1), strain fujisaw a(nr_074878.1), isolate dq462571.1, strain: a tcc 19414(ab055905.1), strain jpb251209s( hm569359.1), chiba9393( ef494748.1), sp. t127_5( jq739693.1), sp. lv19(kj670316.1), strain er.gxlc1(kp063151.1), inopinata strain 143-02 ( inopinata strain 143-02) strain: jcm 8534(lc019778.1) and strain er.gxby -1(kp063149.1). sequences w ere aligned by clustalx version 2.1 (www .clustal.org), and the concatenated alignments w ere used for phylogeny inference ( mega5; www .megasoftw are.net) opting for the maximum parsimony and poisson correction. computed r eplicates for bootstrap support w as done and values w ere observed. histopathology for malin fixed tissues w ere processed as per standard protocol for histopathological studies. sections of 4-5 micron thickness w ere stained routinely w ith haematoxylin and eos in stain and observed under oil immersion objective of a low pow er light microscope. results and discussion tissue samples processed for detection of classical swine fever virus and swine pox virus were confirmed as negative for both viral agents. however, bacteriological investigation demonstrated association of e. rhusiopathiae infection in affected (5) as well as in dead (2) pigs. prevalence of erysipelothrix in many animals, mostly in pigs and birds has been reported throughout the world including from india (shank ar et al., 2009; arora et al., 2011). but there was a single report on swine erysipelothrix (se) from meghalaya (das et al., 2014). north eastern region has the highest pig population of the country. diverse geographical locations, varied climatic situations and frequent movement of pigs favour for spread of the disease through carrier pigs (leslie et al., 2015). present report is a thorough investigation on swine erysipelothrix occurred seasonally at nagaland, another ne state of india. clinical signs a total of 150 pigs during 2013-15 exhibited clinically high fever (105-107 0 f), anorexia, firm faeces, and animals 3 figure 1. character istic lesions of sloughed off skin from the cases of e. rhusiopathiae infected pigs. lying down. cutaneous lesions appeared after 5-7 days of illness initiating with erythematous patches followed by papules of 4-5 mm diameter, dark purple, raised, firm to touch giving square to rhom boid shape in entire body. no vesicular stage was noticed. although scabs developed in the entire body, prominent lesions were identified on head, face back, belly, limbs, tail and on ear (figure 1). in unattended cases scab lesions were sloughed off within 17-22 days leaving a large ulcerated area. thes e typical cutaneous lesions have been mostly seen in grower animals. in affected pigs it appears that cutaneous lesions were developed following acute stages of illness. considering the pathogenesis of e. rhusiopathiae, organisms gain access to the body, probably through the tonsils or other lymphoid tissue of the digestive tract and spread throughout the body. the bacteria produce neuraminidas e, an enzyme that cleaves mucopolysaccharides in cell walls which may mediate the wides pread vascular damage that accompanies se. vascular damage leads to thrombosis and interference with microcirculation in capillaries and venules at many sites. classic cutaneous rhomboid urticaria (diamond skin) occurs in a percentage of pigs shortly after the acute febrile stages. in younger pigs with acute erysipelas, signs are similar, with cyanosis of extremities, ears and snouts pronounced and urticaria less common (anonymous, 2016). seasonal occurrence of swine erysipelas in nagaland justifies the stress due to sudden change of temperature. as stated by amanda (2012)that stress factors such as overstocking, mixing pigs after weaning, and sudden changes in temperature can trigger clinical erysipelas. isolation and identification of organism on blood agar the organism produced small, circular and transparent αhemolytic colonies with a smooth glistening surface and edge. biochemically all the isolates were catalase, oxidase and urease negative, produces h2s and ferments glucose and lactose. all 7 isolates were identified as e. rhusiopathiae based on the cultural, morphological and biochemical characteristics. the nagaland isolates were sensitive in vitro to oxytetracycline, tetracycline, ampicillin, amoxicillin and cloxacillin; moderately sensitive to streptomycin, enrofloxacin, amikacin, co-trimoxazole, cefotaxime and ciprofloxacin; and resistant to gentamicin and norfloxacin, chloramphenicol and neomycin. based on the antibiogram, survived ailing pigs were treated with oxytertacycline at 10 mg/kg body weight, intramuscularly for one week. eight out of 13 treated animals responded promptly and recovered. treatment of swine erysipelas cases with penicillin (shankar et al., 2009) or other penicillin group of drugs such as the combination of amoxycillin and cloxacillin (das et al., 2014) have been frequently reported. molecular confirmation and characterization tissue samples were negative for swine fever virus and pox virus in pcr. a total of seven e. rhusiopathiae isolates recovered from tissue samples were subjected for pcr amplification using 16s rrna gene s pecific primer set. all isolates were found to be positive for e. rhusiopathiae with amplification products of 407bp. 4 500 bp 407 bp 100 bp figure 2. 2% agarose gel show ing pcr products. here, l1 100 bp marker, l2 ntc, l3& 4 pcr products. figure 3. phylogenetic tree of nagaland isolates of e. rhusiopathiae (kt160358, kt160359 as compared to the atcc 19414 type strain and other isolates. (figure 2). two nagaland isolates kt160358 and kt160359 were sequenced and showed high sequence identity with the sequences of other e. rhusiopathiae available in the genbank database. the nagaland isolates (accession numbers kt160358, kt160359) were in the same clad (figure 3) along with other strains originated from china. phylogeny based on the nucleotide and amino acid sequences of the viruses provides a better understanding of the molecular epidemiology of the isolates. the state 5 figure 4. histopathology of skin biopsy sample, h&e, 100x. congestion and presence of microthrombi in the capillar ies of the der mis along w ith infiltration of lymphoid cells and fibroblast w ere consistent finding. nagaland shares international boundaries with my anmar and having territorial link with other neighboring international countries like china, bhutan and bangladesh. the chinese strains used in the construction of phylogenetic tree viz. swine isolates kpo 63150.1, strain er.gxby-1(kp063149.1), strain er.gxlc-1(kp063151.1) and murine isolate dq4625711are forming same clad with nagaland isolates ( kt160358 and kt160359) indicated an epidemiological link. movements of animals and animal products might spread the infection to this locality. histopathology histopathologic alteration of affected skin showed extensive damage to the capillaries and venules of dermis with infiltration of lymphoid c ells and fibroblasts. congestion and pres ence of microthrombi in the capillaries were consistent finding in all skin biopsy samples investigated in the present study (figure 4). similar observations were also recorded by shankar et al. (2009). cultural characteristics, molecular confirmation and histopathological changes conclusively proved that swine erysipelas is prevailing in nagaland. pigs harbouring the infection manifest clinically at sudden change of climate. it is estimated that 30–50% of healthy swine harbour the organism in their tonsils and other lymphoid tissues (stephenson and berman, 1978). again, trans border movement of pigs and their products could facilitate spreading of se in this locality. further study on prevalence of swine erysipelas and identification of carrier pigs can provide actual guidelines to control the disease in this part of india. conclusion unorganized pig farms in the phek district of nagaland experienced high mortality of grower pigs during rainysummer season. a detail isolation and molecular investigation confirmed the association of swine erysipelas in this part of north eastern region of india. this is the only report on seasonal occurrence of swine erysipelas in the hilly low temperate climate of the ne states of nagaland, india. conflict of interests the authors have not declared any conflict of interests. references amanda l (2012). sw ine erysipelas. available online at http://www .dpi.nsw .gov.au/__data/assets/pdf_file/0003/436440/swine erysipelas.pdf, accessed on 12 th aug, 2016. anonymous (2016). erysipelas. veterinary diagnostic and production animal manual. college of vet. med. iow a state university. available from https://vetmed.iastate.edu/vdpam/fsvd/sw ine/indexdiseases/erysipelas. accessed on 12 th aug, 2016. arora n, rajora vs, prasad a, misra s (2011). urticarial form of swine erysipelas: a case report. vet. pract. 12(1):77. das s, ghatak s, bhattacharya u, puro k, amarjit k, kumar p, deepak verma kl, ahuja a, sen a (2014) . identification of erysipelothrix 6 rhusiopathiae infection from a pig farm in meghalaya, india. vet. pract. 15(1):52-54. hoffmann b, beer m, schelp c, schirrmeier h, depner k (2005). validation of a real-time rt-pcr assay for sensitive and specific detection of classical swine fever. j. virol. methods 130:36-44. lee jj, kim dh, lim jj, kim dg, chang hh, lee hj, kim sh, rhee mh, endalem, imada y, kim oj, kim s (2011). characterization and identification of erysipelothrix rhusiopathiae isolated from an unnatural host, a cat, with a clinical manifestation of depression. j. vet. med. sci. 73:149-154. leslie ee, christley rm, geong m, ward mp, toribio ja (2015). analysis of pig movement across eastern indonesia, 2009-2010. prev. vet. med. 118(4):293-305. makino si, okada y, maruyama t, ishikaw a k, takahashi t, nakamura m, ezaki t, morita h (1994). direct and rapid detection of erysipelothrix rhusiopathiae dna in animals by pcr. j. clin. microbiol. 32:1526-1531. medaglia mlg, de cassia pereira a, freitas trp, damaso cr (2011). sw ine pox virus outbreak, brazil. emerg. infect. dis. 17(10):1976-1978. reboli ac, farrar we (1989). erysipelothrix rhusiopathiae: an occupational pathogen. clin. microbiol. rev. 2:354-359. shankar bp, chandan s, madhusudan hs, ranjith d (2009). pathology of erysipelas infection in piglet. vet. world 2(6):234-235. stephenson eh, berman dt (1978). isolation of erysipelothrix rhusiopathiae from tonsils of apparently normal sw ine by two methods. am. j. vet. res. 39:187-188. 1 author(s) retain the copyright of this article. full length research paper rwandan smallholder households' challenges and the profitability of pig farming: a case study of the musanze district rutikanga alexis and sylvestre charles kigali independent university, kigali, 2280, rwanda accepted 7 april, 2025 insecurity is a risk associated with the growing human population, particularly with regard to animalbased foods. pigs contribute significantly to household income throughout all production levels. this study's primary goal was to examine rwandan smallholder households' pig farming profitability and restrictions. a multistage sampling strategy was used to ensure the research's success. five sections of the musnze district were used for the study. five sectors and the musanze district were chosen using a purposive selection technique. however, a sample size of 120 pig farmers who responded was obtained using a random sampling technique. the profitability of pig farming was assessed by calculating the costs and returns involved in production, and the factors influencing pig farming among small householders were identified using stata and a stochastic frontier production function. the results showed that the majority of respondents in the research area's pig farming production are men. according to the results of the regression analysis, pig farming in the study area was influenced by five factors: education level, market availability, off-farm generation, feed availability, and access to veterinary services. these factors were all positive and statistically significant at the p ≤ 0.01 level of probability. additionally, the results showed that net farm income (nfi), gross margin (gm), total revenue (tr), and total cost (tc) were all statistically significant at (p < 0.01). according to the research findings, pig farming is a lucrative enterprise in the region under investigation. regression analysis results also showed that, at the p < 0.01 level of probability, the three factors—income generation, employment prospects, and new job creation—had positive and statistically significant effects on pig farming in the research area. according to the study's findings, the most frequent obstacles to pig farming in smallholder households were inadequate initial capital, high feed costs, poor feed quality and sacristy, a lack of conservation facilities, pest and disease outbreaks, high piglet costs, a lack of farming knowledge, and substandard housing. notwithstanding the limitations mentioned by several study participants. in the research region, pig farming is a lucrative industry that can raise income, improve employment prospects, create new jobs, and obviously raise farmers' standard of living. the study's conclusions have led to recommendations for training and equipping pig farmers and veterinarians with new technology and innovations, as well as for promoting interventions that can help farmers or marketing organizations acquire initial start-up funding to help launch their farming operations. in order to support and enhance the underlying company progress or stunting, as well as to make wise decisions, encourage the examination of profitability, opportunities, and limits along the pig value chain. key words: pig farming, profitability, smallholder, households. introduction there will likely be a huge demand for food of animal origin due to factors like population expansion, urbanization, income development, and dietary changes (fao, 2006; thorton, 2010). if practical means of generating food are not taken advantage of, the growing human population poses a threat to food insecurity, particularly with regard to food originating from animals. as the primary source of essential animal protein, the livestock subsector of the agricultural sector plays a crucial role in the african journal of pig farming issn: 2375-0731 vol. 13 (1), pp. 001-009, april, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 national economy. the significance of the livestock subsector aligns with the fao's (2003) guideline that a man should consume 65–72 grams of protein per day on average, with 53% (about 35 grams) of that amount coming from animal sources. the livestock industry directly supports the livelihoods of more than 600 million impoverished smallholder farmers in the developing world, employs over 1.3 billion people worldwide, and accounts for 40% of global agricultural output (fao, 2011; thorton et al., 2006). worldwide, pig production is widely dispersed. with 56% of the world's total pork production, asia is the world's top producer, outpacing north america (17%) and europe (25%). 48% of the world's pork production comes from china alone (faostat, 2011). pigs' capacity to turn many types of feed, including household scraps, into meat is one of their main advantages (rahman et al., 2009). pigs are by far the most efficient farm animal when it comes to converting feed energy into body energy when considering overall feed conversion. another significant benefit of pigs is their high productivity rate, which ranges from 9.3 to 9.96 live piglets per sow. okoli (2006) and cta (1995). according to estimates, sub-saharan africa's yearly pork consumption is expected to rise by up to 155% between 2000 and 2030, while nations classified as low-income will see an even greater increase of 167% (fao 2011). however, unless the correct products are produced in the right location at the right price, profitable pig production will not be possible. in order to establish the most efficient method of producing pigs, it is crucial for the prospective pig farmer to comprehend the economic, physical, social, ethnic, and religious variables at play. pigs are still primarily kept for their meat production all throughout the world (fao, 2018). the producer and his family may use the pork, or it may be sold to generate revenue. additionally, processed meats like bacon sausage are being manufactured and are becoming more and more well-known. particularly in asian nations, by-products like pigskin and bristles are utilized in the production of light leather and brushes (fao, 2018). establishing intensive pig production in impoverished nations is quite simple if funding is available and sufficient feed supplies are guaranteed (ogunniyi and omoteso, 2011). pigs offer a consistent and immediate source of income to help rural households with daily necessities including school fees, medical bills, and farm inputs. in addition to being a great fertilizer, pig dung may be aerobically digested to provide cooking gas and promote the growth of plants and microorganisms that can be fed to freshwater ducks and fish (okoli, 2006). a vital and fundamental component of rwanda's agricultural economy, livestock production contributes to the creation of jobs, income for farmers, economic growth, vocation for farmers, and other multipurpose uses in addition to the direct production of food (fao, 2012; minagri, 2019). the primary species raised in rwanda include cattle (991,697 heads), goats (1,270,973 heads), sheep (371,766 heads), pigs (211,918 heads), poultry (2,482,124 heads), and rabbits (489,401 heads). the animal husbandry subsector accounted for around 8.8% of the gdp (minecofin, 2007). in terms of animal output, livestock production does not meet the nation's feeding needs (minagri, 2008). the primary reason limiting animal output today is the nutritional quality of feedstuffs, particularly forages, which is one of the many limitations facing animal husbandry. if the animal is not fed well, it will not show off its genetic potential or remain healthy (minagri, 2008). one significant subsector of the livestock business is pig farming. however, in rwanda today, pigs alone account for up to 21% of all meats produced and consumed. the production of pigs is special because it provides the fastest returns on investment and the highest turnover rate among animal operations (fao, 2011). livestock output in rwanda is currently increasing at an unparalleled rate. the production of livestock is increasing, and this trend is predicted to continue. this is especially because of the several policy initiatives that support food security. with 46% of the total meat production, cattle made a substantial contribution, compared to 21% for pigs (minagri, 2019). over time, there has been an increase in the production of animal products. this can be ascribed to many initiatives that assist the growth of the livestock industry, including the livestock intensification program and small stock development (minagri, 2019). priority is given to livestock infrastructure development, animal health, animal feeding, and genetic enhancement through these projects run by minagri, districts, and other stakeholders. in an effort to improve diet, the government started a few projects. a human diet must include animal protein, particularly for young children. the most effective way to generate protein on a limited amount of land—a precious resource in rwanda—is with small cattle (minagri, 2019). through investments, favorable policies for mediumto large-scale industrial operations, and subsidies to reduce poverty in low-income households and manage food security in vulnerable families, the government encourages increasing pig production and consumption (minagri, 2019). regarding livestock, 68% of rwandan homes own some kind of animal. musanze district, where 69% of all households own livestock, has seen a modest decline. this demonstrated that the number of families keeping cattle in musanze district is higher than the national average. there were 91,000 households in this district that raised livestock, mostly pigs. still, just a small percentage of musanze's population—roughly 3% of the nation as a whole—raise animals. because of the lack of livestock and the growth of its market, musanze is still dependent on agricultural output (eicv 3, nisr, 2011). despite certain challenges, rwandan pig production has expanded significantly over the past 10 years and is comparatively well-developed (fao, 2012). the high cost of feeds, restricted credit availability, diseases, poor marketing prospects, a lack of basic knowledge about pig management techniques, poor extension services, and a shortage of qualified veterinarians on pig diseases and preventive health are some of these issues (fao 2011; minagri, 2019). pigs contribute significantly to household income throughout all production levels. the selling of piglets and live adult pigs is how pig producers make money. in addition, pigs provide 3 food security, nutrition, manure, and waste disposal. many farmers believe that raising pigs is the livestock counterpart of cash crops and has the ability to enhance rural incomes because of the animal's short breeding cycle. the proceeds from the selling of pigs are used to pay for hospital expenses, school fees, clothing, food, and improved household systems (fao, 2012; dietze, 2011). pigs are permitted to forage on pasture and be supplemented with available farm waste by many pig farmers in underdeveloped nations and the tropics (rangoma, 2013). the majority of homes in the study region have some sort of kitchen waste that pigs can utilize, but it is insufficient and of poor quality to partially feed three mature pigs per family, whereas in sophisticated systems, pigs are completely contained and fed a balanced diet. the dietary needs of the various classes of pigs must be understood and satisfied in order to provide a balanced diet. therefore, buying pre-made meals from reputable commercial sources is wise for pig breeders (rangoma, 2013). significant obstacles prevent smallholder pig producers from taking advantage of improved marketing options and the growing demand for pork. lack of genetic and breeding strategies; poor slaughter technologies and limited value addition; low production and productivity levels; low farm-gate prices and distressed pig sales; lack of organized producer groups and organizational strategies to achieve economies of scale; low bargaining power; poor husbandry and farm management practices; poor quality feeds and pig nutrition practices; limited access to inputs, extension services, agricultural insurance, credit, and other financial services; and a lack of fair and effective market linkages for smallholder farmers are some of these challenges (tatwangire, 2013). the most crucial factor in pig production is feed cost. 75– 80% of the overall cost of raising pigs is spent on feed (smith, 2006; fao, 2012). pig meals consist of 55-70% grains (smith, 2006). however, a significant constraint in many developing nations is the lack of grain to feed both people and animals (petrus et al., 2011). high feed prices and shortages are issues for small-scale pig producers in developing nations (peters, 2004). in most research, the amount of feed and the frequency of feeding are not clear. age, the pigs' physiological condition, and feed availability all have a major impact on how much feed is given (hossain et al., 2011; kagira, 2010). pig farmers in rwanda often struggle with issues such adequate housing, pig feed supply, price, and accessibility. farmers must feed their pigs balanced commercial or homemade feeds in order to increase the pigs' weight and production and generate respectable income. this is rarely the case because the majority of pig farmers give their animals low-nutrient feedstuffs, such as agricultural waste. the majority of pig deaths happen when the piglets are young (fao, 2012). because pre-weaning mortality in piglets is frequently caused by exposure to adverse weather conditions, including cold, rain, and predators, housing is crucial. by offering suitable and safe housing, young pig mortality can be prevented (madzimure et al., 2013). a farmer may effectively raise 85% of all live-born piglets to market weight in the lowest amount of time with the aid of good housing, which also simplifies management (gikonyo, 2010). worm infestation is a major public health issue in rural areas due to inadequate husbandry measures and a shortage of meat inspectors (veary and monato, 2008). african swine fever (asf), porcine cysticercosis, mange, pediculosis, and infestations of worms, ticks, and jiggers are among the major diseases that affect pigs (fao, 2012). according to other research, pig productivity is significantly hampered by pests and diseases (muhanguzi et al., 2012). the absence of structured breeding programs to support genetic improvement is another obstacle to increased pig production in poor nations like rwanda and others (kahi et al., 2008). the absence of adequate, trustworthy records and sound pedigree structure has been a barrier to genetic improvement initiatives. the majority of pigs in the nation are extremely inbred, and this problem needs to be addressed immediately (kahi et al., 2008). although rwanda's existing system has been found to be ineffectual and unable to satisfy the demands of local farmers, agriculture extension is a potent instrument with a wealth of potential to empower and promote rural livelihoods (rola et al., 2002) (fao, 2012). in any industrial system, but especially in pig production, the development of a stable market is crucial (michael levy, 2014). in rural regions, pig farming is underdeveloped and fails to meet community requirements, such as improved earnings and food and nutritional security. these are caused by a number of limitations, including a lack of initial investment capital, the tiny amount of land set aside for livestock keeping, a lack of knowledge, and the fact that pig breeding and consumption are forbidden by the muslim faith (fao, 2018). currently, more and more people are interested in eating pig, despite the difficulties mentioned above. farmers now have more markets for their products since the cost of other meats is rising relative to pig and because there are more outlets, including small-scale processors and local butchers (fao, 2018). the majority of the clients in this area have strong demands for pig meat. even yet, the pig industry is important because of the nutritional content of pig meat, the contribution of pig production, and the range of agricultural operations. the profitability and limitations of pig farming in the study area have not been evaluated by any research. however, the purpose of this study was to examine the profitability and limitations of pig farming in rwandan smallholder households, using the musanze district as a case study. identifying the factors that influence pig farming in smallholder households in the study area, analyzing the profitability of pig farming in smallholder households in the study area, calculating the contribution of pig farming to smallholder households in the study area, and determining the constraints of pig farming in smallholder households in the study area are each of the specific objectives. 4 materials and methods description of study area the investigation was carried out in the musanze region of rwanda's five-pronged northern province. the musanze district is 1849 meters above sea level and is situated at latitude 1.50 and longitude 29.63. the majority of the volcanoes national park is located in this area, which is the most mountainous in rwanda. the majority of rwanda's mountain gorillas can be found in this district, which makes it the most visited tourist destination in the nation. the district, which has an average elevation of 1860 meters, is divided into two zones and, as a result, has two different types of soils: volcanic ash soils with lava-predominant stones and a volcanic area with moderate slopes (www.musanze.gov.rw.). steep hills with active erosion make up the last portion. the high-altitude tropical climate of musanze district features an average temperature of 20ºc and rainfall that ranges from 1400 to 1800 mm. the district's economy depends heavily on agriculture. agriculture employs at least 91% of the population. musanze is regarded as a national granary. the district has a total size of 5,304 km². musanze is separated into 432 settlements, 68 cells, and 15 sectors. there were 368,563 people living there in total in 2012, with a gross density of 695 people per km2. with 174,760 males and 193,803 females, it has an average annual growth rate of 1.8% (nisr, 2012). sampling technique and sample size the pig farmers in the musanze district were the study's population of interest. to find and examine the social and economic factors affecting smallholder households' pig farming, profitability, and social and economic impact on smallholder households, as well as the limitations in the study region, the study used a cross-sectional research design. a multistage sampling strategy was used to ensure the research's success. a purposive sample of the musanze district and its five sectors—rwaza, nkotsi, muko, kimonyi, and remera—was conducted in the first stage. in the second step, twenty-four (24) pig farmers were randomly selected from each of the selected sectors, for a total of 120 pig farmers. the district veterinary officer gave the list of pig farmers that were available in the district agriculture office. these sectors were selected because, in comparison to the other ten sectors in musanze, there are a greater number of pig farmers in them. structured questionnaires were used to gather primary data from the chosen pig farms. descriptive statistics, cost-benefit analysis, and the stochastic frontier production function were used to analyze the data collected from those farms. data analysis techniques the socioeconomic characteristics of the pig farmers, including gender, age, educational attainment, farming experience, occupation, marital status, and family size, were described in this study using descriptive statistics like frequencies and percentages. version 20 of the statistical package for social science (spss) was used to analyze this. however, the factors impacting pig farming among small homeowners were identified using stata's stochastic frontier production function, and the profitability of pig farming was then estimated using cost-benefit analysis and farm net revenue for pig production. if the bcr is greater than 1, pig production is lucrative. the profitability of the pig producing industry increases with the bcr. model specification to ascertain the viability of pig production, an analysis of the costs and returns related to pig farming was conducted. using budgetary methodologies, the cost and returns of pig production in the study area were calculated using net farm revenue and gross margin. the farm budgetary analysis assists in calculating the overall expenses and income incurred by the business within a given production period. where: gm = gross margin tr = total revenue tvc = total variable cost tfc = total fixed cost nr= net returns tc= total cost nfi= net farm income roi= returns on investment gr=gross ratio oer=operating expense ratio der=depreciation expense ratio nfir=net farm income ratio bcr=benefit cost ratio results and discussion socioeconomic features of pig farmers surveyed according to the results in table 1, the majority of respondents (59.2%) in the pig farming production industry are men, whereas the majority of pig farmers in the research region are women (41.8%). this suggests that men produced pigs at a higher rate than women. the study was backed by umeh et al. (2015), who found that men make up the majority of pig farmers in the study area and that sex may boost technical efficiency because male producers are typically the head of the household and are active in obtaining and managing production inputs. on the other hand, osondu et al. (2014) found that although males are more involved in pig production, females also helped with minor farm tasks including cleaning the pighouse and giving feed and water. according to the results in table 1, the majority of pig farmers in the research region are between the ages of 36 and 45 (37.5%), followed by respondents who are between the ages of 18 and 35 (34.2%), and those who are over 45 5 (25.7%). 2.5% of the respondents were under the age of 18, making them the lowest class. this indicates a decline in youth involvement in pig farming. this ought to be the result of the issue of insufficient startup funds for the pig industry. however, because they have beginning capital, the adults in the research region are more involved in pig farming production. according to table 1's findings, the majority of pig farmers in the study region are primary school graduates (46.7%), with secondary school graduates (24.2%) coming in second. the percentages for non-formal education and tertiary education are 10.8% and 18.3%, respectively. this suggests that pig farmers possess a degree of education that greatly aids in the adoption of new innovations and technology for improving the quantity and quality of pig products. according to the results in table 1, the majority of pig farmers in the research area—69.1%—are married, with 19.2% being single. however, 9.2% of the pig farmers in the study area are widows. the results of this study demonstrated that 2.5% of all respondents were divorced individuals who were involved in pig farming. the low participation of widows and divorced individuals may be the result of their inability to start this business with a pig or piglet because they always have different problems to handle on their own, whereas those families have both husband and wife to assist one another, as the saying goes. according to the results in table 2, the majority of pig farmers are between the ages of 1 and 5 (43.3%), followed by those between the ages of 6 and 10 (37.5%). the last class with the lowest percentage in the research region for pig farming productivity is families with more than 11 members (19.2%). this is because family members provided the labor force. this implies that the labor force involved in the various tasks necessary for pig farming production increases with the number of family members. according to their farming experience, the majority of pig farmers in the research region (51.7%) had between two and six years of experience, followed by those with seven to eleven years (28.3%). 6.7 percent of the population in the research region has 12–16 years of farming experience. nonetheless, the final group, which includes those over 17, accounts for 3.3%. this is because, in contrast to other livestock farming, particularly that of cattle, goats, and sheep, which are regarded as traditional activities, respondents said that pig farming was the new enterprise in the research area. the elements affecting smallholder household pig farming production a multivariate regression analysis was employed to identify the variables impacting smallholder families' pig farming output. age, gender, educational attainment, pig farming land size, farming experience, market accessibility, revenue generation, feed availability, feed cost, availability of credit facilities, availability of veterinary care, and information availability were the socioeconomic factors. five of the twelve variables— education level, market availability, off-farm generation, feed availability, and access to veterinary services—were statistically significant at the p ≤0.01 level of probability, according to the results of the regression analysis in (table). additionally, the results demonstrated that an r-squared of 0.76 indicates that 76% of independent factors account for the impact of these variables on the productivity of pig farms in the research area. the availability of feed, revenue generation, market accessibility, education level, and veterinarian services all exhibited positive coefficients. for example, this suggests that a 0.6% increase in pig farming production will result from a one-year increase in the respondent's educational attainment. additionally, it suggests that pig farming production will rise by 0.9% and 0.3%, respectively, for every unit increase in market and feed supply. this may be the case because education enables farmers to make wise and practical managerial and economic decisions by improving their understanding of the innovations brought to them in the production of pigs. the quality of farmers' skills, their capacity for allocation, and their level of knowledge about the advances and technology in their environment should all be influenced by their educational attainment. this is due to the fact that individuals with greater educational attainment typically adopt innovations in various fields more quickly. additionally, pig farming production was positively correlated with farming experience. this suggests that a one-year increase in the respondents' pig farming experience in the research area will lead to a 0.4% rise in high pig farming productivity. this ought to be the case since farmers with greater expertise are probably going to run the farm more effectively and make better decisions. the study by oluwatayo ib et al. (2008) validated the findings, which showed that farmers with more experience would be more productive, have a better understanding of the market and climate, and so be expected to run a more successful and efficient business. additionally, it confirms the findings of onyebinama ua (2004), who found that farmers with prior farm business management expertise are better able to detect production risks, allocate, combine, and use resources efficiently, and set realistic time and cost targets. indicating that the increasing off-farm revenue from other domains influenced pig farming productivity in the research area, the results showed that off-farm income was positive and significantly influenced pig farming production at the p≤ 0.01 level of probability. this suggests that for every 1% increase in respondents' off-farm income, pig farming output should rise by 1.1%. at the p < 0.05 level of probability, the results showed that gender had a positive and substantial impact on pig farming production, indicating that the research area's greater gender balance in pig farming had an impact on pig farming output. this suggests that a 0.3% increase in pig farming production will result from a unit increase in the number of women engaged in pig farming. osondu et al. (2014) provided support for the study by demonstrating that men 6 are more involved in pig production and that females also helped with light farm tasks like cleaning the piggery and serving feed and water. as the majority of pig farmers in the study area are male producers who are often the head of the household and actively involved in obtaining and managing production inputs, umea et al. (2015) also noted that men are primarily involved in pig production and proposed that sex may increase technical efficiency. according to the study's findings, the price of feed and the availability of credit facilities had a detrimental impact on the output of pig farms in the research region. for instance, this suggests that a 0.8% drop in the number of farmers and direct pig farming output will result from a unit rise in the price of pig feed. the reason for this is that as feed prices increased, more farmers left the domain since the prices were too high and there was little pig production in the area. pig farming's profitability in smallholder households using the cost-benefit method, the financial gains from pig farming in smallholder households were examined and contrasted. cost and profitability are two indicators of the economic health of pig producers. additionally, the results showed that net farm income, gross margin, total cost, and farm revenue were all statistically significant at p < 0.01. comparatively, the results in table 3 showed the economic returns and investment costs for a single female pig in a smallholder household unit region. according to calculations, the gross margin was 436167.5 and the net farm revenue per female pig was 172167.5rwf. this suggests that less educated and more seasoned farmers produced more pigs than newcomers to the field. this ought to be the result of a variety of school-based skills and knowledge as well as the expertise and instruction given by veterinary professionals and technicians who visited the current farmers in the study area. furthermore, the high cost of feeds brought on by covid-19 and the closure of international boards, especially those between rwanda and uganda, should be primarily blamed for the low economic return in the research area. additionally, the results showed that net farm income, gross margin, total farm revenue, and total cost were all statistically significant at (p < 0.01). according to the study's findings, the operating expense ratio—a gauge of the proportion of farm income devoted to variable operating expenses—was 60.3%. the net farm income ratio, however, was 15.7%. likewise, the benefit-cost ratio (bcr) was 1.2, indicating that 1.2 francs of revenue are generated for every franc invested in the research area. pig farming is a lucrative enterprise in the study region, as evidenced by the benefit-cost ratio (bcr) of 1.20, which is greater than one. in general, regular, appropriate feeding and immunization, which help to maximize the weight and health of pigs, could be credited with the high economic return connected with pig farming production. in the study region, proper feeding— especially that which is rich in various nutrients—also encourages the pig to be fully and well-developed, which leads to a higher production or number of piglets per mature female pig. furthermore, the investment yielded 0.2% profits, while the depreciation expense ratio was 24%. furthermore, the economic investigation and analysis showed that, in spite of certain negative effects, such as the high cost of depreciation, the issue of high feed costs in pig farming production as a business was prevalent in the study area because of the global covid-19 pandemic and the absence of international trade between nations. however, as farmers in the study area have indicated, in order to maximize net farm income and improve and increase efficiency in terms of technique and pig farming production, farmers should be encouraged to breed modern pigs and sell the piglet for approximately seven months, rather than selling piglets of two months. in order to maximize profit, the government should also establish long-lasting policies that make it easier for farmers to obtain agricultural loans. along with reducing pig and piglet mortality through improved knowledge and skills related to the factors of pig farming production, particularly the proper feeding, vaccination, and housing that in turn increase income from pig farming, it should also be better to expand extension and veterinary services. the profitability ratio technique used in the analysis shows that pig farming is a viable enterprise in the study area, despite the high prices of the feeds used. farming is a very alluring and lucrative business for pigs. a farmer must receive training and expand their knowledge of farming in order to properly raise pigs. this is due to the fact that in order to efficiently raise pigs, farmers must provide them with ideal housing. they ought to provide wholesome food and appropriate medical care. it is impossible to make a lot of money without raising pigs correctly. pig farming's impact on smallholder households in the research area pig farming has had a positive impact on smallholder households in burera and musanze districts on seven out of ten factors, according to the results of tobit regression analysis in table 4. these factors include income generation, animal manure, employment opportunities, protein supply, access to agricultural credit, information access, and infrastructures. at the p < 0.01 level of probability, the results in table 4 demonstrated that the three factors—income generation, employment prospects, and new job creation—had favorable and statistically significant effects on pig farming in the research area. pig farming in the research area was positively and significantly impacted by the four factors—animal manure, nutrition supply, access to agricultural loan, and information availability—at the p < 0.05 level of probability. infrastructure, for example, has a positive coefficient but is not significant when it comes to pig farming in the research area. on the other hand, pig farming in the research area was negatively impacted by two variables: poverty reduction 7 and food security control. numerous participants in the research region said that pig farming had a favorable impact on their standard of living. they claimed that the production of pigs, especially meat and oil, led to the creation of new jobs, particularly in the restaurant industry. according to their statement, they favored dining above other pursuits because of the daily rise in demand, particularly in urban regions where there are more tourists and visitors than in rural ones. for instance, a 1% increase in pig farming should result in a 0.5% increase in the study area's income output. this is due to the fact that the more demand there is for pig production (meat), the more supply there is at a competitive price, which in turn promotes pig farming in the research region. growing pig output also encourages the government and business sector to construct additional facilities, such as feeder roads, markets, electricity, and state-of-the-art slaughterhouses. many new jobs were made available to both educated and uneducated individuals in rural regions as a result of the construction of these new infrastructures, particularly for the workforce and labor day. additionally, it suggests that a 1% increase in pig farming should result in a 0.8% increase in employment opportunities, which will impact human livelihood. this is because there will be a need for graduate veterinarians to adhere to the standards of manufactured meat on a daily basis as the number of clients requesting meat rises, creating job opportunities. according to communities, the lack of interest in pig raising and consumption in the past, particularly among females, was mostly caused by the incompatibility with the culture of the largely uneducated and insecure population. these days, rural rwandans, especially those living close to musanze town, are content with the employment prospects in pig farming and selling pig meat in restaurants, despite the increased interest of men. some respondents, particularly young women from various parts of the nation, stated that they frequently take great pride in their pay. regression analysis results generally imply that pig farming is a lucrative enterprise in the studied area. generally speaking, the high economic return linked to pig farming output may be ascribed to consistent, appropriate feeding that is bolstered by high and quality production of various crops grown nearby that support feeds from the market. limitations of smallholder household pig farming pig farming is one of the most lucrative industries globally for a number of reasons, chief among them being the creation of revenue. the majority of rwandan farmers, both large and smallholder, do not now keep pigs for profit. a healthy flock of pigs or piglets depends on a number of elements, the two most crucial of which are management and environment. pigs that are in good health consume less food, rest for longer periods of time, and yield a desirable amount of meat in terms of both quality and quantity. when they have adequate shelter and a good feeder to feed and meet other needs, they are easier to care for and medical expenses are lower. furthermore, tatwangire (2013) pointed out that smallholder pig farmers have numerous obstacles that prevent them from taking advantage of improved selling prospects and the growing demand for pork. poor feeds and pig nutrition practices, a lack of genetic and breeding methods, inadequate slaughter technology, insufficient value addition, and restricted access to inputs, extension services, agricultural insurance, loans, and other financial services are some of these difficulties. the study's results, however, showed that the most frequent barriers to pig farming in smallholder households were the high cost of feed, insufficient initial capital (100%), feed sacristy and quality (9.5%), lack of conservation facilities (89.9%), outbreaks of pests and diseases (75.6%), high piglet costs (70.1%), lack of farming knowledge (65.7%), and substandard housing (50.4%). all respondents (100%) stated that the primary barriers to pig farming output in the research area were the high cost of feeds and insufficient beginning capital. in addition to lowering the quantity and quality of pig farmers, the high cost of feeds also lowers the number of farmers in the area. in the study area, it was also discovered that one of the things impeding pig farming production was the prevalence of pests and disease outbreaks (75.6%). this is because, when pigs and piglets are kept together in unsuitable pig homes, the disease can spread quickly among them. additionally, they share bowls for food and water, which can spread diseases and sickness from sick to healthy hens. ascaris, salmonellosis, colibacillosis, and pest pigs are the primary illnesses mentioned by research participants. according to the farmers, illness affects their livelihoods in a variety of ways, both locally and inside individual households. they claimed that because the piglets that would have been sold to survive or fed to guests or family are no longer available, priority needs shift within households. therefore, farmers have less capital accessible to them, particularly when they are dying and not producing. therefore, poverty affects rural households due to all of the aforementioned restraints, especially sickness. one of the main issues in the restrictions was the use of insufficient pig feed. it occasionally occurs when there is a shortage of human food. pigs currently have less access to food, which immediately lowers output. pig farming is generally less profitable due to the expensive expense of proper feeds and medications, particularly when there are insufficient veterinary and extension services. conclusion this study's primary goal was to examine rwandan smallholder households' pig farming profitability and restrictions. musanze district as a case study. the results showed that the majority of respondents in the research area's pig farming production are men. according to the results of the regression analysis, pig farming in the research area was influenced by five factors that were statistically significant at the p ≤0.01 level of probability: education level, market availability, off-farm generation, feed 8 availability, and access to veterinary services. additionally, the results showed that net farm income, gross margin, total cost, and farm revenue were all statistically significant at p < 0.01. according to the research findings, pig farming is a viable enterprise in the study area since the benefit-cost ratio (bcr) was (1.2) higher than a unit, indicating that one franc of investment in the study area produced 1.2 francs of revenue. it is impossible to make much money if pigs are not raised properly. a farmer must receive training and expand their knowledge of farming in order to properly raise pigs. this is due to the fact that in order to efficiently raise pigs, farmers must provide them with ideal housing. they ought to provide wholesome food and appropriate medical care. pig farming has had a favorable impact on seven out of ten criteria for smallholder households in the research region, according to the results of tobit regression analysis. however, at the p < 0.01 level of probability, the three factors—income generation, employment prospects, and new job creation—had a positive and statistically significant impact on pig farming in the research area. even though pig farming has many obstacles, the study's findings showed that the most prevalent ones for smallholder households were the high cost of feeds, insufficient initial capital, feed sacristy and quality, lack of conservation facilities, pest and disease outbreaks, high piglet costs, lack of farming knowledge, and substandard housing. notwithstanding the limitations mentioned by several study participants. several analysis techniques show that pig farming is a financially successful enterprise. in general, it contributes to the creation of new jobs, increases employment prospects, produces revenue, and unmistakably raises the level of living for farmers in the research region. the study's conclusions led to the following recommendations being made: encourage and educate farmer groups, particularly women, on the fundamentals of long-term accountability and participative group governance through fair elections. the unification of pig producers and marketing groups in the nation, as well as the development of powerful farmers' organizations, should result from this. coordination, promotion, and regulation of the pig industry's growth are aided by the union of pig producers and marketing organizations. encourage initiatives that can assist in giving farmers or marketing organizations the early startup funds they need to get their farming business off the ground. additionally, savings groups can be established to help generate funds for borrowing during peak markets and stressful times. encourage more direct involvement from the private sector and other suitable boundary partners who have a comparative advantage in the development of smallholder families' pig value chain operations in rwanda, particularly in the study area, in order to promote sustainable pig value chains. this might necessitate redefining goals and coming up with fresh ideas for increasing farmers' access to financing, training opportunities, credit services, and markets for pig products—especially meat and manure. pig farming output in the research area was profitable, as can be shown, however the most obstacles to this industry were discovered to be the growing shortage and high cost of feeds. rab and government organizations should, however, focus on ways to stabilize feed prices for smallholder households. since this region is the nation's granary, particularly for potatoes, pig farmers in the study area should effectively and economically learn how to formulate and expand their feeds by using local crop feedstuffs in order to minimize cost feeds. conflict of interest the author here declares that there is no conflict of 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recent trends, future prospects. philosophical transactions of the royal society b. umeh j. c., ogbanje c., adejo m. a. 2015. technical efficiency analysis of pig production: a sustainable animal protein augmentation for nigerians. journal of advanced agricultural technologies. veary, c.m. and manoto, s.n. 2008. neurocysticercosis: a possible cause of epileptiform seizures in people residing in villages served by the bethanie clinic in the north west province of south africa. journal of the south african veterinary association. www.musanze.gov.rw introduction 1 author(s) retain the copyright of this article. full length research paper the socioeconomic characteristics of pig farmers in the tropics as a determinant of pig production and profitability dozie de a. and tamunotonye v. university of abuja, abuja, nigeria accepted 22 january, 2025 in nigeria's imo state, the socioeconomic traits of pig farmers were examined as factors influencing their output and financial success. determining the socioeconomic characteristics of pig farmers, identifying the pig production systems in the study area, estimating the costs and returns associated with pig production, analyzing the constraints to pig production in the study area, and describing the socioeconomic characteristics of pig farmers will be the specific objectives. for a detailed investigation, 90 pig farmers will be selected using a multi-stage random sample process. structured questionnaires and secondary sources from conferences, seminars, journals, published and unpublished theses, and workshops provided the data for this study. the frequency distribution table and percentage response were used to record the first, second, and third objectives. gross margin analysis and the cobb douglas production function, respectively, were used to address objectives iii and iv. the socioeconomic characteristics of the sampled farmers were as follows: they were male, older, educated, had a large household, were more likely to use family labor, were experienced, and the majority of pig farmers used intensive rearing techniques, while the least number used extensive methods. additionally, the majority of farmers were involved in farrow to finish enterprises, with animal breeding being the least common. the cobb douglas production function revealed that piglet type, raising experience, organization membership, and pharmaceutical costs were positively correlated with the profitability of pig farming. because their return scale was 3.0141, which is higher than unitary, the pig farmers in the study area were working in stage 1 (irrational stage), which is not at their optimal production size. with net farm income of n334,542 and gross revenue of n740,000 per sampled farmer, pig production was profitable in the research area. high feed and housing costs, issues with product marketing, high labor costs, and limited financing availability were the main obstacles to pig production in the research area. among the suggestions made were the necessity of improving farmers' access to commercial bank credit, encouraging extension agents by covering their local transportation costs while doing their tasks, and guaranteeing that farmers have access to legitimate medications. key words: socioeconomic characteristics, pig, farmers, profitability, determinant, pig production. introduction a large percentage of the rural population in many subsaharan african countries suffers from hunger and malnutrition due to a lack of animal protein, a situation that has been extensively documented in various publications (ajala et al., 2007; food agriculture organization, fao, 2008, ume et al., 2019). for example, in many nigerian rural areas, the daily consumption of animal protein was 38 grams per caput, which is less than the food agriculture organization's (fao) minimum recommendation of 65 grams per caput per day. the aforementioned situation has a significant impact on their health, and the most frequently mentioned health issues in the research include poor mental capacity, low labor productivity, and slow national economic growth (fao, 2008, ewuziem, et al., 2010). one of the quickest ways to combat the lack of animal protein in the diets of the majority of people in rural parts of african journal of pig farming issn: 2375-0731 vol. 13 (1), pp. 001-008, january, 2025. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ developing nations is to increase the consumption of pigs. this may be due to the pig's inherent traits, such as its ability to adapt to situations in which other animals are unable to survive, its ability to efficiently use household waste and byproducts to produce fresh meat, its large litter size per sow, its superior carcass yielding ability over cattle, sheep, and goats, its high dressing percentage of roughly 70% as opposed to 52.5% for cattle and roughly 50% for sheep and goats, and its effective conversion of feed energy to body energy (okolo, 2011, osondu, et al., 2014). according to steinbach (1997) and bamiro et al. (2008), pig carcasses also have a larger proportion of edible meat and less bones, as well as high fecundity, high feed conversion efficiency, early maturity, short generation intervals, and a relatively small space demand.its meat (bacon), however, is the main source of income and foreign exchange earnings; pigskin and bristles are used to make light leather and brushes (john, 2007); pig manure is a valuable fertilizer; it can be aerobically digested to produce cooking gas; it may promote the growth of microorganisms and plants for aquatic animals to eat (osondu et al., 2013); and it can be used as a source of labor and employment (umeh et al., 2015). pig and poultry businesses are growing at the quickest rates in the livestock industry, while other emerging nations are seeing stagnant or declining growth (fao, 2008).pig production is declining, which could have a negative impact on the enterprise's profitability. this decline could be caused by a number of factors, including poor quality feeds from unbalanced rations, poor access to veterinary services, farmers' illiteracy, limited credit available for purchasing material inputs, poor housing due to the high cost of building materials, and occasionally pigs kept in makeshift shelters (pond and manr, 1998; ume, et al, 2018). moreover, the country's absence of a pig product processing business, high feed costs, inadequate infrastructure, a lack of genetically sound breeders, and the concern of a small market for pig products are all factors (ewusiem et al., 2008, getara, 2009, ume et al., 2017). the aforementioned issues have the potential to lower the production output of pig farmers, which would lower their profit margins. according to ironkwe and amefule (2008), successive governments at the state and federal levels have implemented various programs to boost animal production, including the farm settlement scheme, the agricultural development programme (adp), the better life program, and a microcredit scheme for livestock parent/foundation stock. however, these initiatives have not produced much. in actuality, many farmers have abandoned their operations, making the nation's population's protein intake deficiency much more complicated. however, research indicates that suitable disease control methods, such as appropriate medicine, adequate feed and feeding (drugs), good breeding selection, and acceptable housing practices, can achieve optimal pig development and profitability (agada, 1991, ajala, et al., 2007). therefore, the purpose of this work is to evaluate the socioeconomic traits of pig farmers as a factor that influences pig profitability in the research region. this could be useful because, with a thorough understanding of the socioeconomic characteristics of farmers and other swine production attendants, the management options they use or have access to, and the likely effects on production, it could help stakeholders and policy makers focus on the areas that require immediate intervention to improve the productivity and production of piggery enterprises and address the lack of protein in the diets of the majority of nigerians, especially those living in rural areas. the specific goals are to: (i) characterize the socioeconomic traits of pig farmers; (ii) identify the systems of pig production in the study area; (iii) ascertain the impact of the socioeconomic traits of pig farmers on their profit; (iv) calculate the costs and returns associated with pig production; and (v) identify and analyze the barriers to pig production in the study area. materials and methods the study area imo state, nigeria, which lies between latitudes 7056" and 6064'n of the equator and longitudes 6046' and 5049e of the greenwich meridian, was the subject of the study. anambra state borders the state to the north, abia state borders it to the east, and rivers and imo states border it to the south and west, respectively. its temperatures range from 26 to 440 degrees celsius, its rainfall ranges from 1500 to 2800 mm, and its relative humidity is moderate at 65%. imo is made up of numerous villages, numerous autonomous communities, and twenty-seven loc al government areas (lgas). with a population of 3,934 million, it occupies an area of 5100 km² (11). yam, cassava, maize, and cocoyam are among the crops grown by the imo people, who are mostly farmers. pigs, poultry, goats, lambs, rabbits, and snails were among the animals raised by the farmers. the farmers also worked as saloon workers, auto mechanics, traders, civil servants, and hostelers. sampling procedure and sample size local government areas (lga), communities, villages, and respondents were chosen using purposive and multi-stage random selection approaches. first, three lgas were specifically chosen for pig production due to their proximity to three-three (3-3) breweries. okigwe north, ideato north, and ideato south were the lgas that were chosen. second, from each of the chosen lgas, three communities were chosen at random out of five. this resulted in nine communities overall. third, out of the nine communities, ten villages were chosen at random. this increased the number of villages to 90. lastly, a total of 90 farmers were chosen for in-depth analysis, one from each of the 90 communities. method of data collection both primary and secondary sources provided the data for this investigation. structured questionnaires and informal or oral interviews with respondents were used to derive the primary data. information on labor costs, medication costs (drugs, disinfectants, and vaccines), years of farming experience, educational attainment, water use in pig production, household and flock sizes, and access to institutional and non-institutional credit were all gathered through the questionnaire. method of data analysis the frequency distribution table and percentage response were used to record the first, second, and third objectives. gross margin analysis and cobb douglas production function and factor analysis were used to meet objectives iii, iv, and v, respectively. model specification cobb douglas since the 1930s, the cobb-douglass theory of production has offered a crucial framework for calculating productivity and the use of production factors. under the presumption of constant returns to scale production, cobb and douglas modeled the increase of output in the american manufacturing sector between 1899 and 1922, where the output of commodities was determined by the combination of two factor inputs, namely labor and capital. the technological link between the quantities of two or more inputs (often labor and physical capital) and the output that can be produced in a production process is commonly denoted by the cobb-douglas production function. cobb-douglas is preferred in both developed and emerging nations since it can be used to analyze diverse economic policies and to function in numerous sectors of a nation's economy. it is used, for example, to rationally decide how much of each input factor to use in order to reduce production costs. it’s mathematically expressed as: y = alαkβ (1) where y = total output, l = units of labour, k = units of capital, and α and β are elasticity of labour and capital, and a is an efficiency parameter. the parameter a is the efficiency parameter. it serves as an indicator of the state of technology. the higher the value of a, the higher would be the level of output that can be produced by any particular combination of the inputs. the cobb douglas production function a, a and b are positive parameters where = a > o, b > o. the equation describes that productivity depends directly on l and c and that part of output which cannot be explained by l and c are explained by a which is the residual, often called technical change (hajkova andhurnik, 2007). the function was criticized from three fronts by economics scholars, namely on assumption of constant returns to scale which the model built its analysis, on the omission of technical change, thereby having the notion that technology i static within the duration of the study which is not possible in realities (fraser, 2002). furthermore, the neoclassical economists criticized the model on the basis that the productivity theory centered more of an pensiveness than a proven. in logarithms, the equation is: log y = log xo + log x1 + log x2……… + log xn (2) where; x1 = quantity of feed consumed in kilogram, x2 = labour ( mandays), x3 = cost of medication (drugs, disinfectants and vaccines) (n), x4 = years of rearing experience (years), . x5 = educational level (years), x6 = quantity of water(litres), x 7 = household size (no), x8 = flock size(no), x9 = credit (n) benefit cost ratio: this was used to estimate farm net revenue for pig production. theoretically, net revenue (nr) is the total revenue (tr) less the total cost(tc); nr = tr – tc ................................................ (3) total cost is the addition of the entire variable cost(vc) and fixed cost (fc) items; tc = tvc + tfc ........... (4) total revenue is the total amount of money that a farmer received from the sale of stock; tr = σpxqx ................................................ (5) gross margin (gm) = tr – tvc… (6) net farm income (nfi) = gm − tfc (7) the rate of return is a performance measure used to measure the amount of return on an investment relative to the investment cost. it is given by: rate of returns (ror) = nr/tc...................... (8) gross ratio (gr) = tc/tr ..............................(9) benefit cost ratio (bcr) = tr/tc (10) p = price per pig q = quantity of pig sold pig production is profitable if its bcr ≥ 1. the higher the bcr, the more profitable the pig production business is. depreciation was calculated using the straight line method factor analysis factor analysis was used to analysis the constraints to pig production in the study area using principal component factor analysis with varimax -rotation with factor loading of 0.3 was used. the constraints to pig production in the study area were categorized into three factors using varimax rotation and factor loading of 0.30. the principal component factor analysis model is stated thus r1 = y11 m1 + b12 m2 +----------yn1 mn ……………….. (11) r2 = y21 m2 + b22 m2 + ----------------y 2 nmn ………………..(12) r3 = y31 m3 + b32m2+ --------------------y3nmn ,………………(13) rn = yn1m1 +bn2m2 + -----------------------------ynnmn …………………….(14) where; r 1 = cn= observed variable /constraints in pig production pdts y1= yn = factor loading or correlating coefficients m1 = mn =unobserved underlying challenging factors facing pig production results and discussion socioeconomic characteristics of the farmers table 1 shows that 58.9 % of the respondents were more than 41 years and above, whilst 42.1% of them were within the age range of less than 41 years. table 1: distribution of respondents according to socioeconomic characteristics variable frequen cy percentag e mean age 2130 15 27.5 31 – 40 22 24.4 42 41 – 50 20 22.2 51 and above 33 36.7 educational level non formal education 15 16.7 primary education 25 27.8 secondary education 17 18.9 tertiary education 8 6.7 household size 1 – 5 18 20 6 – 10 35 39.9 7.2 11 -16 30 33.4 17 – 21 7 7.8 farming experience 1 – 10 20 22.2 1120 56 66.2 21 and above 14 15.6 11.4 drug/vaccine usage yes 90 100 no labour source family 41 45.6 hired 17 18.9 communal 6 6.7 hired & family 26 28.9 water usage yes 90 100 no rearing method intensive system 59 65.6 semi intensive 21 23.3 extensive 10 11.1 enterprises farrow 23 25.6 farrow finish 40 44.4 finishing operation 20 22.2 breeding 7 7.8 source; field survey; 2018 this suggested that older farmers dominated the production of pigs in the research area, and older farmers are typically knowledgeable and skilled managers who can run their businesses to generate large profits (duniya et al., 2013). this result contradicted the findings of rahman et al. (2008), who claimed that young people dominated their field of study. they noted that this farming group is typically creative and inspiring to advance the frontier of pig production and achieve high profitability. additionally, primary education was held by the majority of pig farmers (27.8%), followed by secondary education (18.7%), and university education (6.7%). farmers' educational attainment improves their managerial skills, openness to innovation, and comprehension and evaluation of new production technology, all of which contribute to increased farm productivity and business profitability (ironkwe and amaefule, 2008).according to table 1, 77.8% of the sampled farmers had more than 11 years of farming experience, whereas 22.2% had less than 11 years. this suggested that the farmers in the research region had a great deal of expertise raising pigs. according to ume et al. (2018), farmers with years of experience raising livestock are better able to set reasonable goals and use their resources wisely in order to increase their output, which might potentially result in significant profits. additionally, all of the pig farmers in the study area who were sampled employed drugs and vaccines to treat and prevent pig production diseases, respectively. the main issues with the usage of these medications and vaccinations are their scarcity at the farm level, their poor quality, and the fact that the majority of the vaccines are not maintained in the cold chain, which causes them to lose their viability. high mortality, low productivity, and low profitability are the results (ewuziem et al., 2009). according to table 1 above, the majority of respondents (38.9%) had households with 6–10 people, while the smallest (7.8%) had households with 16–20 people. pig production typically employs larger farming households with a larger number of labor-age members in order to lower production costs resulting from high labor costs and high profit margins (ezeibe, 2010). in addition, the chart shows that 45.6% of the farmers in the sample employed family labor to raise pigs, followed by family and hired labor (28.0%) and communal labor (6.7%). small-scale farmers in sub-saharan africa employ family labor in a unique way to reduce high production costs, which may be related to great profitability. additionally, water was employed in pig production by all of the sampled pig producers in the research area. according to studies, water makes up around 82 percent of the body weight of young pigs and 55 percent of the body weight of market hogs, making it the single greatest ingredient of the body (ewuziem et al., 2010). however, a high water content (86–98%) in pig dung raises storage and disposal costs, according to holness (1999). furthermore, the majority of respondents (65.6%) raised their pigs in an intense system, followed by those who raised them in a semi-intensive system (23.3%) and the respondents who raised them in an extended system of management (11.1%). according to studies, rearing practices are important for swine production since they facilitate management and enable farmers to successfully raise 85% or more of the animals in the lowest amount of time (pathraja and oyedipe, 1990).however, the following traits are present in pigs raised under intensive management: slow growth, insect and disease infestation, irregular sow breed, high piglet mortality, and low output due to inconsistent seasonal feeding (getara et al., 2009). according to table 11, the majority of respondents (52.2%) worked in the farrow and finish enterprise, while the smallest percentage (22.2%) worked in the finishing operation. compared to other prosperous businesses, the farrow and finish process required greater facilities (pond and manar, 1998). results of cobb douglas production function the results of cobb douglas production function is presented in table 2 as per the findings of ume et al. (2018), the profitability of the pig operation was negatively correlated with the age of the household head. contrary to the strength needed for pig production, the variable's sign identity may be linked to the waning of strength that comes with aging. the result is that these family heads use labor to carry out the pig producing activities, which hurts the farm's bottom line. furthermore, at a 1% significance level, the breed type of the piglets' coefficient correlates positively with the profitability of the pig farm. this suggests that piglets with traits like good litter sizes, leanness, muscle, current growth rates, and high feed conversion to pork efficiency are more likely to yield high profits than piglets with traits that contrast with the previously mentioned characteristics (bamiro et al., 2008).additionally, the labor cost coefficient was statistically significant at the 5% significance level and had a negative sign to the profit of pig producers. because pig production requires a lot of labor, only a small number of workers could volunteer, but at extremely high wages. the low worker acceptability may be due to their fear of pig bites and their allergy to the smell of the animal's produce. this outcome supported the findings of ogunfowona et al. (1990), who noted that raising pigs is a laborious process that requires effective management to lessen the odor connected with its production. additionally, there was a positive correlation between the farmers' level of profit and the rearing experience coefficients. this means that a one-year increase in the number of years of household farming experience might lead to a profit gain equal to the coefficient's size. mpofu and makuza's (2003) findings supported the aforementioned claim. they believed that farmers with a lot of experience are more likely to combine resources in the best way to increase productivity, which could result in a large profit. nevertheless, the household size coefficient was statistically significant at the 5% alpha level and positive, as predicted by a priori analysis. according to ume et al. (2017), "the more household members of labor age and availability the household head has, the more likely it is that they will use them to implement pig husbandry management technologies and save the money that could have been paid to hired laborers as part of the business profit." in order to alleviate the labor constraints that are typical in agricultural production, particularly during the peak farming season when labor is expensive and scarce, they proposed that families with large and mature household members have a greater chance of implementing labor-intensive technologies related to pig production. furthermore, at a 95% confidence interval, the feed cost coefficient demonstrated an indirect association with farm profit in pig production. this could imply that the higher the proportion of concentrates in pig diets compared to domestic and crop leftovers, the higher the outputs, but the lower the potential profit due to the high cost of the resource (concentrates). this result is consistent with a number of studies (bama et al., 2004; rahman et al., 2008; okolo, 2011) that show the detrimental effects of feeding pigs to grains, particularly in the majority of subsaharan african nations where grains are in short supply and fiercely compete with humans, resulting in low farm productivity and little profit. furthermore, as anticipated, the pig farmer's educational status coefficient was positive, meaning that a unit increase in the family head's years of education will result in a profit rise equal to the coefficient. the variable's positive sign may be explained by the fact that farmers with higher levels of education are better able to understand and respond to new and improved innovations, which can increase their output and profit on their farms compared to those with lower levels of education. however, research indicates that a sufficient extension delivery system could compensate for farmers' lack of education, particularly with regard to the adoption of new technology (rahman et al., 2008). additionally, at the 10% alpha level, the flock size coefficient was positive and statistically significant. the coefficient's sign matched the a priori hypothesis, indicating that farmers are more likely to make more money from pig sales if their flock size is larger. this result is consistent with ume et al. (2018)'s analysis of the economics of pig production in nigeria's ebonyi state's ezza north local government area. additionally, at the 5% alpha level, the drug cost coefficient was positive and significant. the outcome might make it necessary for pig farmers to have greater access to pharmaceuticals, disinfectants, and vaccines in order to increase their level of output and profit. on the other hand, the fao (2008) found that subpar and adulterated drugs and vaccines were flooding many markets in the majority of africa's developing nations. additionally, the high cost of these medications to farmers could result in high animal mortality and low profit margins. additionally, the organization's membership coefficient was significant at the 1.0% risk threshold and positively correlated with pig profitability in the research area. this suggests that farmers who belong to groups like cooperatives are more likely to turn a profit than those who do not. this could be explained by the cooperative's capacity to instill in its members a more positive attitude toward adopting innovations through information access, member interactions, and training (osundu et al., 2014). production elasticity and return to scale the return to scale of the production function is shown in table 2 the degree to which output responds to changes in inputs is measured by the elasticity of production. given the cobb douglas specification of the model, the direct elasticity of production for the different inputs serves as an estimate for the parameters of stochastic frontier production. pig production in the study area had a return to scale of 3.0141. since this number exceeds unity, the return to scale is increasing. this suggests that the farmers were not producing at their best scale and were instead functioning in stage 1, or the irrational stage. therefore, in order to increase their outputs, farmers must add additional resources or inputs to their production. table 2: production elasticity and return to scale variable elastici ty piglet type 0.6591 cost of feed 0.48632 cost of capital 0.5430 cost of medication 1.4390 cost of water 0.5741 flock size 0.8444 cost of labour -0.5591 return to scale 3.0141 source; field survey; 2018 costs and returns in pig production table 3 showed that the average total cost of production incurred by the respondents was n101,810. both variable and fixed costs make up the overall cost; according to the table, variable costs account for 86% of the production costs, while fixed costs make up 5.29%. furthermore, the cost of feed accounts for 50.5%, labor for 28.97%, and medications, disinfectants, and vaccines for 6.83%. each respondent's average gross revenue was n444, 000. for each respondent, the average gross margin was n350, 330. each respondent's average net farm income was n342,190. consequently, n19,010 was the average net farm income per pig. this suggested that raising pigs is a lucrative endeavor. according to the table, the total cost of production for ten (10) pigs in the study region, including total variable costs and total fixed costs, was n 383,458. eighty-six percent of the overall production costs were variable, and ninety-four percent were fixed. furthermore, the cost of feed accounted for 56.6% of the overall production costs, followed by the cost of piglets (17.2%) and the least expensive drug (1.5%). the competition for human consumption of the same ingredients may be the cause of the high cost of pig feed. as is frequently observed in african markets, the cheap cost of medication may be attributed to the fact that the majority of resource-poor farmers use indigenous known technologies (ikt), which are expensive and of poor quality (okolo, 2011). each farmer in the sample had an average gross revenue of n740,000, while their net farm income was n334,542. pig production had a 55% rate of return on investment, meaning that for every n1.00 invested, 54k was made. because the benefit-cost ratio (b c r) is greater than 1, it indicates that pig production is a lucrative enterprise. the gross margin ratio (g m r) was no different. the results of the expense structure ratio (e s r) also showed that the pig industry is financially sound. in conclusion, the business is profitable, according to the examination of the several profitability ratio methodologies. varimax-rotated factors against pig production table 4, three factors were take out based on the reaction of the respondents to the questionnaire issued to them, table 4 varimax-rotated factors against pig production in the study area. variable factor 1 factor 2 factor 3 feed -0.115 0.312 0.171 poor breed 0.216 0.320* 0.003 housing 0.236 0.093 0.339 marketing of products -0.308 0.414 0.367 lack of capital 0.346* -0.137 0.212 high labour cost 0232 0.329* 0.119 poor extension contact 0.318* -0.028 0.140 disease 0.122 -0.304 0.322* veterinary posts 0.007 0.128 0.349* inadequate equipment 0.307 0.190 0.326* lack of drugs 0.118 0.439* 0.127 source: computed from sas 2018. factor 1= economic/institutional factor, factor 2 = infrastructural factor and factor 3 = socio-financial factor (ikani and dafwang, 1995). only variable with factor loading of 0.30 and above at 10% overlapping variance were used in naming the factors.the factor loading of less than 0.30 and variables that loaded more than one factor were discarded. the variables that loaded more than one factor like inadequate equipment and marketing of product were revealed. in identification of the factors, ume, et al; (2018) opined that each factor is assigned a value considering their disposition. the limitations underneath the economic /institutional factor include capital problem (0.346) and poor access to extension services (0.318). the problem of poor access to credit has been a bane to agricultural development in nigeria and this could be correlated to lack of collaterals, high interest rates, shortterm repayment and ignorance of loan source by the farming population (ume, et al; 2018). in addition the problem of poor extension services to most pig farmers could be associated to high extension – farmers ratio, lack of technological information in pig farming and inadequate incentives to the change agents (ezeibe, 2010) the following variables are loaded under factor 2 (infrastructure factor): high labor costs (0.329), feed costs (0.312), disease costs (0.304), and medicine costs (0.439). since the majority of lowincome resource farmers use family labor in their pig operations, the high cost of labor has led to both a decrease in flock size and, as okolo (2011) asserts, a high cost of production. furthermore, the high cost of feed, particularly commercial components, forces farmers, especially those with limited resources, to feed their pigs with domestic or local food ingredients, which stunts their growth (duniya et al., 2013). furthermore, the majority of farmers abandon their farms due to low productivity to other economic activities because farm profits are no longer sufficient to support them due to the high cost of medications and vaccines, their inaccessibility at the farm level, and their lack of knowledge about how to use these medications (ume et al., 2019). additionally, the majority of pig-producing societies in developing nations continue to use local breeds of pigs due to their ability to withstand harsh weather conditions, low cost of piglets, resistance to pests and diseases, hardiness, and ability to be raised with minimal inputs through extensive rearing (bama et al., 2008). veterinary post (0.349), sickness (0.322), and housing (0.339) were the variables under socio-financial aspects. due to the problem of substandard housing, mice, insects, and odors have proliferated, polluting the near and neighboring environments. for example, pigs are kept in unfinished housing in most rural locations, close to residential buildings and natural materials like bamboos. this means that pigs are frequently unrestrained and harm home property and the environment (ajala et al., 2007). due to limited access to veterinary care in most rural areas, most farmers are forced to self-medicate, about which they lack sufficient knowledge, and seek the services of quacks, which has resulted in the complete or partial eradication of a significant portion of the farmers' flocks (agada, 1991). furthermore, a significant mortality rate in pig production, especially for piglets, may result from diseases such brucellosis, africa swine fever, diarrhea, and coccidiosis (adesechinwa et al., 2003). conclusion and recommendation conclusion these conclusions were reached in light of the data. the results of the socioeconomic characteristics revealed that the majority of the respondents were men, older, better educated, more likely to have a large home, more accustomed to family labor, and more experienced. the majority of pig farmers also worked in farrow-to-finish businesses and intensive rearing.additionally, piglet type, raising experience, organization membership, pharmaceutical costs, flock size, household size, and educational attainment all had an impact on the profitability of pig farms in the research area. the research area's pig production was further hindered by high feed and housing costs, issues with product marketing, high labor costs, limited financing availability, and weak extension contacts. recommendation the following suggestions were interpreted: workshops, seminars, and adult education should raise farmers' educational attainment. additionally, by offering superior breeds to increase their output, both new and experienced farmers could be persuaded to remain in the pig business. for easier access to pig material inputs like feed, veterinary medications, and vaccines, pig farmers should also be encouraged to establish or join cooperatives. in order to reduce manufacturing costs, large-family households are also urged to employ them as a source of labor. references 1. adesehinwa, a.o.k. makinde, g.e.o. and oladele, i.o. (2003). demographic characteristics of pig farmers as determinant of pig feeding pattern in oyo state, nigeria. proceedings 8th annual conference of animal science association (asan), september 16-18 in futo. 2. agada, e.s. (1991). economics of swine production: a study of two local government areaa in kaduna state. b.sc. project, unpublished. department of agricultural economics and farm management, university of ilorin, kwara state, nigeria. 3. ajala, m.k., adesehinwa, a.o.k. and mohammed, a.k. (2007). characteristics of small holder pig production in southern kaduna area of kaduna state, nigeria. american-eurasian journal of agriculture and environmental science, 2(2), 182-188, 2007. 4. bamiro o. m. (2008): technical efficiency in pig production in ogun state, nigeria. research journal of animal sciences 2(3): 78 – 82. 5. bawa g. s., balogun t. f., ega l., omage j. j. (2004): urban backyard swine production: a case study of kaduna, a nigerian metropolitan city. journal of animal production 31: 237 – 244. 6. duniya k. p., akpoko j. g., oyakhilomen o., nandi j. a. (2013): measurement of pig production profitability in zangonkataf and jema’a local government areas of kaduna state, nigeria. british journal of applied science and technology 3(4): 1455 – 1463. 7. ezeibe a. b. c. (2010): profitability analysis of pig production under intensive management system in nsukka local government area of enugu state, nigeria. international journal of economic development research and investment 1(2): 48 – 54. 8. ewuziem, j.e. onyenobi, v.o. and dronkwe, a.g. (2010). technical efficiency of pig farmers in imo state, nigeria. a trans log stochastic frontier production function approach. nigeria agric. journal 4(1):137143. 9. ewuziem, j.e., nwosu, a.c., amaechi, e.c.i. and anyaegbu, p.o. (2009). piggery waste management and profitability of pig farming in imo state, nigeria. nigeria agric journal 40(1):29-36. 10. fraser, i. (2002), the cobb-douglass production function: a antipodean defence? part 1. economic issues, 7, 39-58. 11. food and agriculture organization (2008): pig sector kenya: fao animal production and health livestock country reviews. no 3 fao, rome. accessed online on the 12 october, 2017 from http://www.fao.org/docrep/015/i2566e/i256 6e00.pdf 12. gekara, o., garner, j.o.o and dunbar, t.v (2009). evaluating production techniques to improve efficiency and profitability of hog farms in south east arkansas. university of arkansas press.pp: 321-324. 13. hajkova, d., hurnik, j. (2007), cobbdouglas production function: the case of a converging economy. czech journal of economics and finance, 57(9-10), 465-476. 14. hall, h.h. (1998), choosing an empirical production function: theory, nonnested hypotheses, costs of specifications, agricultural economics. research report no. 59. 15. holness, d.h. (1999). pigs: tropical agriculturists. gp maisonneurve et larose, is rue victor – cousin, 75005 paris, france. 16. ironkwe m. o., amefule, k. u. (2008): appraisal of http://www.fao.org/docrep/015/i2566e/i2566e00.pdf http://www.fao.org/docrep/015/i2566e/i2566e00.pdf indigenous pig procution and management practices in rivers state, nigeria. journal of agriculture and social research (jasr) 8(1):1 – 7. 17. miller, e. (2008), an assessment of ces and cobbdouglass production functions. available from: http://www.cbo.gov. 18. mishra, s.k (2007), a brief history of production function. munich personal repec archives (mpra, 5254). 19. mpofu, i. and makuza, s.m.m. (2003). pig production science and technology, 1st edition (ed.). mrshonhiwa up front publishing, uk. 20. national population commission (npc, 2006). national population census figure, abuja, nigeria. 21. ikani i.e. and dafwang, i.i. (1995): pig production technology for piggery farmers. extension bulletin,livestock series no 25. naerls, abu, zaria. 22. ogunfowora o, olayemi j k, fetuga b l and amogu u (1980)an evaluation of the state of development and economics of pig production in nigeria. report prepared for the federal livestock department, lagos. nigeria 23. okolo, c.i. (2011). tropical tips on intensive pig production: animal management and health issues. technical notes: tapas institute of scientific research and development. pp 240-246. 24. osondu c. k., ijioma j. c., anyiro c. o and obike k. (2014): economic analysis of pig production in abia state, nigeria. international journal of applied research and technology 3(3): 3 – 10. 25. pathraja, n. and oyedipe, e.o. (1990). indigenous pigs of nigeria. animal genetic resources information, 7:67-78. 26. pond, w.g and maner, j.h. (1998). swine production in temperate and tropical environment. freeman and company. second edition. rahman s., barthakur s., kalita g. (2008): pig production and management system in aizawl district of mizoram, india. healthcare 95, 5 p. 27. raval, d. (2011), beyond cobb-douglass: estimation of ces production function with factor augmenting technology. centre of economic studies, 2011, 5-11. 28. steinbach, j. (1997). effect of season and breed on sow performance in the seasonal equatorial climate of southern nigeria. journal of agricultural science (cambridge), 77:331-336. 29. ume, s i, ezeano, ci, gbughemobi, b o (2018)analysis of the environmental effect of pig production in okigwe local government area of imo state, nigeria international journal of environmental & agriculture research (ijoear) issn:[2454-1850] [vol-4, issue-6, june2018] 30. ume si, ezeano ci, onunka bn, (2018). technical efficiency of pig production in enugu north agricultural zone of enugu state, nigeria. international journal of research and review. 2018; 5(9):61-69. 31. ume smiles i., jiwuba, peter–damian, c., okoronkwo. mo and s. o. okechukw (2019)economics of pig production in ezza north local government area of ebonyi state, nigeria asian journal of agricultural extension, economics & sociology; 29(1): 1-11. http://www.cbo.gov/ introduction materials and methods the study area sampling procedure and sample size method of data collection method of data analysis model specification cobb douglas benefit cost ratio: where; results and discussion socioeconomic characteristics of the farmers results of cobb douglas production function varimax-rotated factors against pig production conclusion and recommendation conclusion 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 11 (1), pp. 001-009, january, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper application of gis in pig production system in nigeria taiwo omodele*, isaiah annayochukwu okere, mutiu olakunle oladele-bukola, adeboye joseph omole and ajoke oyegbami institute of agricultural research and training, obafemi awolowo university, p.m.b. 5029, moor plantation, ibadan. nigeria. accepted 20 november, 2022 this study aimed to identify the pig producers across various administrative regions and to determine the pig production system in nigeria through an intensive survey of pig farms with the use of global positioning system (gps). categorization of farms by proportion in nigeria showed that 81.1% of states had very low (<5) and low (5 to 50) number of pig farms, 8.1% of states had medium (50 to 100) number of pig farms and 10.8% of states had high (100 to 150) and very high (>150) number of pig farms. in nigeria, there are two separate pig production systems: small and medium scales. the small scale system is essentially characterized by very low to low scale of production. analysis of categories of production in nigeria showed that 45.9% of states recorded very low (<100 pigs) and low (100 to 1,000 pigs) scales of production, 46% of states were in the medium (1,000 to 10,000 pigs) scale of production and 8.1% of states had high (10,000 to 100,000 pigs) and very high (>100,000 pigs) scales of production. gis qualitative analysis showed that in the north, the production classes: very low, low, medium, high and very high scales of production by states were 40, 30, 25, 0 and 5%, respectively while in the south, the production classes were 0, 17.6, 70.6, 11.8 and 0%, respectively. a high number of northern states were identified in the low category of pig production scales while most southern states were in the medium category. the application of gis revealed that the pig sector in nigeria is dominated by small and medium scales of production while the harsh climate and its future change pose a major threat to pig farming in the northern nigeria. key words: data integration, extension services, farm survey, pig meat, qualitative analysis and rainfall distribution. introduction the importance of livestock sub-sector is in line with recommendation of the food and agricultural organization (fao) (2003) that on an average basis, a man’s daily protein intake should be between 65 to 72 g and 53% (about 35 g) of this should be animal based. the major sources of animal protein in nigeria are beef, pork, poultry, goats, fish and game animals (ajala, et al., 2007). pigs have some unique advantages over other domestic animals. they grow at a faster rate and are more prolific than cattle, sheep and goats (holness, *corresponding author. e-mail: modeltaiwo@yahoo.com author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 1991 and osaro, 1995). in addition, the pig sector provides employment and generates income. dwindling profit in a pig enterprise has been reported to be a function of poor quality feeds resulting from unbalanced ration (adesehinwa and ogunmodede, 1995). however studies on pig and poultry industries seem to reveal that the initial enthusiasm in these enterprises, especially pig production, is being constrained due largely to dwindling profit margins (ogunfowora, 1980). evidences abound in different parts of the world that pig sector is moving forward, unlike what is obtainable in nigeria. according to pig international (1997), a single integrated spanish company “vall company of spain” hit a production target of two million seven hundred thousand pigs, as early as 1986, with directors still making projection of making it four million by the year 2002. a similar report was given about farmers choice in kenya, a company that single handedly produces virtually all the national herd of sows in kenya, having 2500 sows in contract and another 2000 from internal production (pig international, 1999). it is however pathetic to compare all these figures with the meager pig population system in nigeria. the large muslim population of northern nigeria may also not favour profitable pig production in that part of the country (adetunji and adeyemo, 2012). in nigeria, the pig sector is dominated by small scale and medium scale holders. according to eusebio (1980), backyard pig farming and large scale pig production are more profitable than medium scale pig production. his claim was that large scale pig producers enjoy economies of scale which lowers the unit cost of production when compared to small scale of production. he further claimed that the cost of feeding is skipped. he discouraged medium scale production for the reason that purchasing of feed ingredients were made in small quantities. it is generally known that the more the quantity of production the more the profit. ojo (2000) said that the problem associated with small scale farming was that their scale of operation made it for them to obtain loan due to lack of collaterals. economic development is normally accompanied by improvements in a country’s food supply and the gradual elimination of dietary deficiencies, thus improving the overall nutritional status of the country’s population. furthermore, it also brings about qualitative changes in the production, processing, distribution and marketing of food (who, 2003). in spite of all policies that successive government made, the prevailing economic hardship in nigeria has also made the pig production system to remain underdeveloped as a result of the poor purchasing strength of the consumers. generally, livestock production in nigeria is not as efficient relative to the developed countries. apart from poultry, pig farming is a class of animal production that is not subjected to such heavy losses resulting from failure to follow good sanitation practices. despite the inherent productive capabilities of pigs, its production is low in nigeria and it is faced with a number of problems amongst which are inadequate supply of feed, water, worm infestation, good health management, religious, veterinary services, change in climate, housing and waste disposal, as well as government policy. social factors that could influence pig production in nigeria include a general preference for ruminant meat and lack of incentives for investing in large scale pig production due to economic, religious, political and climatic factors. the large moslem populations of northern nigeria may also not favour profitable pig production in that part of the country (adetunji and adeyemo, 2012). other social factors that have militated against pig production in nigeria include the belief by the general populace that pigs are dirty and constitute a health hazard. this is absolutely untrue for pigs that are produced under modern intensive production techniques since under suitable modern husbandry pigs can be very clean animals (ajala et al., 2007.) it is relatively easy to establish intensive pig production in a developing country like nigeria if capital are available and adequate feed supplies are assured (ogunniyi and omoteso, 2011). inadequate supply of feed poses the most critical problem. a number of pig farmers are faced with this problem leading to heavy losses due to malnutrition’s and increased death of embryo during early stage of pregnancy. hence, feed determines both productivity and growth performance of livestock. it has been identified as a major constraint in animal production. consequently, animal farmers opt for small scale production mainly to reduce cost of feeding. there are indications that pig production in the study area is mostly in the hands of small scale producers who may not have access to credit facilities because generality of the pig farmers are assumed to be peasants. the social factors that have militated against pig production in nigeria include the belief by the general populace that pigs are dirty and constitute a health hazard. this is absolutely untrue for pigs that are produced under modern intensive production techniques since under suitable modern husbandry pigs can be very clean animals (ajala et al., 2007.). therefore this study is expected to provide relevant information that would encourage pig farmers and new entrants to venture into pig farming, as nigerian population provides a readily available market. as the genetic potential of pigs is being improved, management intervention is also essential to help overcome the constraints on production set by variation in climate. the physical environment and the health hazards also pose threat to huge production of pigs in nigeria. heat stress is more common in the dry season and especially when the environmental temperature and relative humidity are high with prolonged exposure to direct sunlight (okoruwa, 2014). there is little or no robust data on the current facts about entrepreneurial characteristics and constraints to the development of pig 3 figure 1. spatial distribution of pig farms in the study area. enterprises in nigeria. previous research involving the effects of heat stress on reproduction has been conducted using dairy cows (west, 2002). amundson et al. (2006) also reported that of the environmental variables studied, minimum temperature had the greatest influence on the percent of cows getting pregnant. with all clarity, increases in temperature and/or humidity in the tropical regions of nigeria have the potential to affect and reduce conception rates of pigs not adapted to those conditions especially in the northern region of the country. if farmers are not making efficient use of existing technology, low production will be recorded both in food and livestock (ajibefun and daramola, 2003). this study will definitely provide succinct and invaluable information to policy makers, to help them design policies that will improve the pig sector as a control in filling the identified gaps in food security in nigeria. there is a continual need therefore to generate information on the facts about pig production and such information is needed for proper planning and regulation of the sector in nigeria. in this view, application of geographic information systems (gis) technology is needed to collect data, store, manage, analyse and produce useful information for timely monitoring of the pig sector in nigeria. gis capability in pig farming is achievable and useful in production level scaling, monitoring feed cost, investigating credit facility sources and disease spread analysis. materials and methods pig farm survey the survey was carried out in the year 2010 across all geopolitical zones in nigeria. the spatial dataset of identifiable pig farms were collected by surveyors with the use of gps for the identification of positions of the farms in the study area (figure 1). the data collected was accompanied by the administration of a set of questionnaire which was designed to obtain information on the production level of the sampled farms. the farm survey stage is the most important stage as its accuracy ascertains the production capacity estimate for the investigated boundaries. gis data integration for the purpose of data integration and querying, the logical structuring of the spatial and attribute data of both the farm and questionnaire were performed using arcgis ® capabilities. the geographical position (centroid) of each farm was defined as a feature data and linked with the attribute dataset of the respective farm to derive the farm production in a gis environment. the production scales of the farms resulted in the local to the national estimates of scales of production. the framework of the mapping procedure as adopted from omodele and okere (2014) is shown in figure 2. results and discussion analysis of production by states table 1 displays the production by states in nigeria. no records of pig farms and their production were made available for jigawa, kano, katsina, plateau, yobe and zamfara states. bauchi had some farms record but no production data. analysing table 1, the order of production by farms was profiled in figure 3. nassarawa state was discovered to have recorded the highest percentage production of pigs in nigeria followed by abia state, which had an appreciable level of production when compared with its percentage proportion of farms. enugu state was also discovered to have recorded a significant level of production. it was expected that kaduna state which had 4 figure 2. adopted mapping procedure from local to the national level. table. 1. pig production status per state in 2010 (pigs). s/n state no of farms production 1 abia 32 19235 2 adamawa 92 3026 3 akwa ibom 108 4589 4 anambra 40 5535 5 bauchi 12 0 6 bayelsa 9 1151 7 benue 29 6836 8 borno 15 225 9 cross river 16 472 10 delta 33 3390 11 ebonyi 30 4992 12 edo 45 3674 13 ekiti 27 1798 14 enugu 105 14659 15 federal capital territory 5 102 16 gombe 40 2466 17 imo 27 4955 32 jigawa 0 0 18 kaduna 157 4857 33 kano 0 0 34 katsina 0 0 19 kebbi 14 405 20 kogi 9 615 21 kwara 12 633 22 lagos 14 2961 23 nassarawa 122 104149 24 niger 13 789 25 ogun 72 5740 26 ondo 30 927 27 osun 66 4890 28 oyo 36 6700 35 plateau 0 0 29 rivers 7 552 30 sokoto 1 66 31 taraba 33 5192 36 yobe 0 0 37 zamfara 0 0 5 60 50 no of farms 40 production (% ) p e rc e n ta g e 30 20 ` 10 0 jigawa kano katsina plateau yobe fct rivers kogi bauchi kwara niger kebbi lagos borno river ekiti imo benue ebonyi ondo abia delta taraba oyo edo osun ogun enugu ibom zamfara sokoto bayelsa cross state anambra gombe adamawa akwa nassarawa kaduna figure 3. percentage production per state in nigeria. figure 4. pig farms proportion by states in nigeria. the highest number of farms in nigeria should have the highest proportion of pigs but this presumption was not achieved. the occurrence in kaduna state justifies the finding of omodele et al. (2014) that the high number or proportion of farms in an area does not guarantee a high production in such a locality. state-based analysis of farms proportion and production as displayed by figure 4 and expressed in figure 5, categorization of farm proportion in the states in nigeria showed 21.6% of states had very low (<5) proportion of 6 70 p e rc e n ta g e ( % ) 60 50 40 30 20 10 0 very low low medium high very high farms density figure 5. categories of percentage proportion of pig farms in nigeria. figure 6. pig production level by states in nigeria. farms, 59.5% of states had low (5 to 50) proportion of farms, 8.1% of states had medium (50 to 100) proportion of farms, 8.1% of states had high (100 to 150) proportion of farms and 2.7% of states had very high (>150) proportion of farms. the highest proportion or density of pig farms was discovered in the low density category. this connotes that pig farms are not many in nigeria, therefore pig production level is expected to be low because it is expected that the lower the proportion of farms the lower the quantity of production. by analyzing the categories of production in the states as shown in figure 6 and summarized by figure 7, the analysis showed 21.6% of states in the very low (<100 pigs) production class, 24.3% of states in the low (100 to 1,000 7 50 p e rc e n ta g e ( % ) 45 40 35 30 25 20 15 10 5 0 very low low medium high very high production level figure 7. categories of pig production in nigeria. figure 8. north and south assessment of pig production. pigs) production class, 46% of states in the medium (1,000 to 10,000 pigs) production class, 5.4% of states in the high (10,000 to 100,000 pigs) production class and 2.7% of states in the very high (>100,000 pigs) production class. the production analysis showed that the highest pig production level in nigeria is in the medium class. this indicated that the pig sector and its production systems in nigeria are absolutely in the small and medium scales. north and south qualitative production analysis as displayed in figure 8 and summarized in figure 9, further analysis of the qualitative performance of states in the northern and southern zones showed that in the 8 p e rc e n ta g e ( % ) 80 70 north south 60 50 40 30 20 10 0 very low low medium high very high production level figure 9. north and south pig production level. north the production classes: very low, low, medium, high, very high were 40, 30, 25, 0 and 5%, respectively while in the south, the production classes were 0, 17.6, 70.6, 11.8 and 0%, respectively. the most practiced production classes of pig meat by states in the north were in the low categories. in the south, the widely practiced class was the medium scale of production. there existed an appreciable acceptance and general production of pig meat in the southern part of nigeria as compared with the northern part. this approach has formed a platform on which further research could be made. climatic approach to pig production as climatic conditions vary across geographic boundaries in nigeria, the surrounding environmental conditions are expected to directly affect the rates of heat gain or loss by all animals. lack of prior knowledge of adequate conditioning to weather events most often results in these catastrophic losses. as noticed that the atmospheric temperature of the earth has been increased as a result of cumulative effects of greenhouse gases emitted into the atmosphere through different industrial and agricultural activities of human. the performance of both domestic and wild animals ranging from insects, amphibians, birds to mammals are expected to be affected by climate variation. therefore rainfall distribution pattern plays a crucial role in this regard. as in figure 10, the southern part of nigeria experiences a cooler atmospheric condition from the annual rainfall distribution. adopting the rainfall distribution patterns: <1000mm, (1000-1400)mm, (1401-1700)mm, (17012000)mm and >2000mm, table 2 and figure 11 showed that 5% of farms within <1000mm of rainfall had 0.8% of production, 46.2% of farms within (1000-1400)mm of rainfall had 69.9% of production, 14.3% of farms within (1401-1700)mm of rainfall had 0.6% of production, 15.3% of farms within (1701-2000)mm of rainfall had 11.5% of production and 19.2% of farms within >2000mm of rainfall had 17.2% of production. the extreme part of the moslem communities in the north with the lowest rainfall distribution pattern had the lowest farms proportion and production. apart from the religious belief/barrier, the harsh climate in the north poses another major threat to pig farming in that part of the country. conclusion and recommendation this study with the use of gis techniques has revealed that the pig sector in nigeria is dominated by small scale and medium scale holders. a high number or proportion of farms in an area does not guarantee a high production in such a locality or zone. there existed an appreciable acceptance general acceptance in general production of pig meat in the southern part of nigeria where a higher number of states had medium scale of pig production. a state and region-based agricultural development and monitoring of piggery is essential as it encourages grassroot awareness and sensitization, especially in the 9 forgotten pig meat production sector. introduction of modern hygienic practices could reduce the major constraints of religious and cultural restrictions in pig farming in nigeria. pig production is essential to food security in providing a fast and adequate protein level for the rapidly growing nigerian population. as the climate changes, substantial funding of the sector is essential especially for construction of modern housing system in the tropical regions of nigeria. therefore, application of gis techniques has assisted the decision makers and strengthened their horizon and perception of the pig farming system in nigeria. gis has also revealed the areas where possible developmental strategies, agricultural extension services and further research are most essential in the pig farming domain of nigeria. conflict of interests the authors have not declared any conflict of interest. acknowledgement the authors appreciate the availability of the pig farm data from the federal ministry of agriculture and rural development (fma&rd). references adesehinwa aok, ogunmodede bk (1995). swine feeds and practical feed composition techniques in national pig production training manual n.a.e.l.s/abu. pp. 27-56. adetunji mo, adeyemo ke (2012). economic efficiency of pig production in oyo state, nigeria: a stochastic production frontier approach. american journal of experimental agriculture 2(3):382394. ajala mk, adesehinwa aok, mohammed ak (2007). characteristics of small-holder pig production in southern kaduna area of kaduna state nigeria: american-eurasian journal of agricultural and environmental sciences 2(2):182-188. ajibefun ia, daramola ag 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farmers in: pig production workshop training manual naerls, abu, zaria, nigeria. pp. 100-130. pig international september (1997). europe’s biggest produce. 27(9)5761. pig international (1999). kenya’s dominants integrator. 29(12):26-30. west jw (2002). physiological effects of heat stress on production andreproduction.pages1–9inproc.tri-statedairynutr.conf., fort wayne, in. who (2003). diet, nutrition and the prevention of chronic diseases. who technical report series 916:13. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 11 (2), pp. 001-007, february, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper short-term amelioration of soil properties and maize yield enhancement using animal wastes in degraded hydromorphic soils of southeastern nigeria okenmuo f. c.1*, odii o. u.2 and okolo c. c.3 1 department of environmental management, pan african university of life and earth sciences, university of ibadan, nigeria. 2 national biotechnology development agency, nigeria. 3 department of land resources management and environmental protection, mekelle university, ethiopia. accepted 4 october, 2022 amending soil with animal wastes can be useful in food production as well as a means of waste disposal. it has been found to enhance physico-chemical properties and enhance yield. poultry manure (pm), swine waste (sw), cow dung (cd), and sewage sludge (ss) were added to a hydromorphic ultisol (sandy loam, typic haplusult at the rate of 10 tha -1 (12 kgplot -1 )). maize hybrid (oba super ii) was used as test crop. randomized complete block design (rcbd) with four replications was used in laying the experiment. data collected were analyzed using analysis of variance (anova) and means were separated using fishers’ least significant difference (f-lsd). physical properties of soil influenced by animal wastes include bulk density, total porosity, hydraulic conductivity, gravimetric moisture contents, aggregate stability, and rheological characteristics. amendments also enhanced soil organic matter, total nitrogen, available phosphorus, potassium, calcium, magnesium and sodium, ph, exchangeable acidity, cation exchange capacity, and base saturation were all higher relative to the unamended plots. productivity studies revealed that maize growth was significantly affected by amendments especially pm. this was observed in height, leaf area index and yield higher relative to the control. generally, the increase followed the order pm>sw>ss>cd. animal wastes especially from poultry sources are recommended for soil amelioration and for increased crop yield in the area. key words: cow dung, sewage sludge, swine waste, poultry manure, waste disposal. introduction sustainable agriculture is fast becoming the focus of the world today. sustainable agriculture is a method of farming that is not only humane, environmentally friendly and socially ethical, but can sustain itself. in the broadest sense, sustainable agriculture puts back into the earth what it takes out, making a cycle requiring no inputs from *corresponding author. e-mail: fokenmuo0255@stu.ui.edu.ng. author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 outside. for example, applying fertilizer derived from petroleum products is not sustainable, because the fertilizer was not produced within the agricultural cycle. composted manure from animals on the farm would be an example of a sustainable fertilizer. animal wastes may be suitable for amelioration of soils in the humid tropics which are characterized by poor native organic matter and low available nutrients productivity decline overtime when subjected to continous cultivation (zingore et al., 2003). in nigeria, the use of mineral ferilizers is common among farmers, probably for its ability to enhance yield, low cost of application and transportation. however, mineral fertilizers overtime have proved to be scarce, cost intensive, capable of causing pollution and toxicity and incapable of achieving soil conservation needs (kushwaha and ochi, 1999). for example, isherwood (2000) observed initial increase in yield with mineral fertilizer for only few years and followed by decrease in base saturation and acidification. futhermore, adeoye et al. (2008) associated decreases in yield, increases in soil acidity, and nutrient imbalance to continous use of mineral ferilizers in tropical soils. yield declines, pollution vulnerabilities and high cost of mineral fertilizers utilization has reawakened interests in organic wastes especially among the poor smallholder farmers. the use of organic inputs such as crop residues and manures have great potential for improving soil productivity and crop yield through improvement of the soil physical, chemical, microbiological and nutrient supply (abbasi et al., 2009). ofori and santana (1990) noted that cow dung improved the productivity of soil more than inorganic fertilizer owing to its slow release of nutrients. organic manures can also increase water infiltration, water holding capacity, water content and aeration (mccauley et al., 2017). many of the nutrients used by plants are held in organic manure until soil organisms decompose the material and release plant available nutrients. it is evident that a regular addition of organic manure is important as food for not only crops but also micro-organisms, insects, worms and other organisms. erosion will be reduced and root penetration and tillage operation will be enhanced when the soil is well aggregated (usda, 2003). addition of soil organic matter is also an important soil conservation measure that accomplishes soil carbon sequestration and mitigation of climate change (mccauley et al., 2017). even though there are many studies dealing with organic manure application in the abakaliki agro-ecology (mbah et al., 2004; mbah and mbagwu, 2006; nwite and alu, 2017), very little is known about their effects on hydromorphic soils and yet such soils are common in the area. the objective of this study was to evaluate the suitability of animal wastes (poultry manure, swine wastes, cow dung, and sewage sludge) on soil physicochemical properties and yield components of maize (zea mays l) in a degraded hydromorphic soil in abakaliki, southeastern nigeria. materials and methods description of study area the study was carried out at the teaching and research farm of the faculty of agriculture and natural resources management, ebonyi state university, abakaliki. the area lies within latitude 06° 4 1 n and longitude 08° 65 1 e in the derived savanna zone of southeastern nigeria. rainfall is bimodal; the rainy (april-october) and the dry season (november-march). there is usually a short break in august usually known as “august break”. the total mean annual rainfall ranges from 1700 mm for minimum to 2000 mm for the maximum, respectively. the annual temperature is between 27 and 31°c, while relative humidity is between 60 and 80% during rainy season (ofomata, 1975). the soil is hydromorphic and belongs to the order ultisol within the ezzamgbo soil association derived from shale and classified as typic haplusult (fdalr, 1986). field work the study was conducted in a land area of approximately 0.017 ha (13 × 13 m 2 ). the field was cleared manually of existing vegetation and debris removed. the area was demarcated into plots that measured 2 × 2 m 2 with 0.5 m space. the randomized complete block design (rcbd) was used in the experimental design. animal wastes consisting of poultry manure (pm), swine waste (sw) and cow dung (cd) obtained from the animal science section of ebonyi state university abakaliki and sewage sludge (ss) were obtained from sewage treatment plant of university of nigeria, nsukka. the animal wastes were dried, crushed, analyzed for their respective nutrient components and incorporated into the soil at 20 cm depth prior to planting. the treatments were 10 tha -1 (4 kg/plot) of each animal wastes (pm, sw, cd and ss) and a control. they were replicated four times to give a total of twenty experimental plots. maize (zea mays l. var oba super ii) was planted as test crop two weeks after incorporation of treatments. the seed rate was two seed per hole at a spacing of 25 × 75 cm 2 and depth of 5 cm. the seedlings were thinned down to one per hole at two weeks of germination and weeding was bi-weekly. agronomic data twelve plants constituting 25% of plant population per plot were tagged and used for agronomic measurements. plant height was measured with metric ruler from tallest leaf of a plant to base every two weeks till tasseling. the grain yield was determined by harvesting the cobs after drying of husks. the husks were removed, cobs shelled and maize grains were further dried and grain yield was adjusted to 14% moisture content. laboratory studies selected soil physical and chemical properties were determined after soil samples were collected with core samplers and augers, respectively auger at 0 to 20 cm. auger samples were composited, dried, ground, seived with 2 mm-mesh seive and labelled at pre-and post-planting. bulk density was determined using the method described by gee and or (2002). total porosity determination was done as described by obi (2000). the method of stolte (1997) was used to determine saturated hydraulic conductivity (ks). gravimetric 3 table 1. chemical composition of amendments used for the study. manure ph o.c tn (%) av.p exch. k exch. ca exch. na exch mg (kcl) (%) (mgkg -1 ) (cmol kg -1 ) (cmol kg -1 ) (cmol kg -1 ) (cmol kg -1 ) pm 7.5 23.0 3.67 0.4 0.55 3.27 0.33 1.52 sw 7.0 20.2 2.45 0.35 0.63 3.19 0.23 1.58 cd 7.0 26.2 3.21 0.34 0.47 2.81 0.11 1.22 ss 5.91 26.2 2.76 0.16 0.48 2.91 0.28 1.39 pm: poultry manure; sw: swine wastes; cd: cow dung; ss: sewage sludge; o.c: organic carbon; tn: total nitrogen; av. p: available phosphorus. moisture content determination was carried out as described by obi (2000). particle size distribution (fraction of sand, silt, and clay) was determined using hydrometer method (gee and or, 2002) with naoh as dispersant. mean weight diameter was determined using the method described by obi (2000). sower (1965) penetrometer and classical techniques were used to determine liquid limit and plastic limit, respectively. soil ph was determined in 1m kcl of 1:2.5 soil/water ratio. organic carbon was determined by the walkley and black dichromate oxidation method (nelson and sommers, 1982). organic matter was estimated as organic carbon × 1.729 (odu et al., 1986). and total nitrogen (tn) by kjeldahl method (bremner and mulvaney, 1982). available phosphorus by bray and kurtz (1945) (bray ii) method. after extraction with 1n ammonuim acetate solution, exchangeable na and k were determined by the flame photometry and ca and mg by an edta titration method. exchangeable acidity (al 3+ + h + ) was determined by the kcl displacement method and cation exchange capacity (cec) was obtained by the ammonuim acetate technique. percentage base saturation was calculated as total exchangeable bases divided by effective cec and multiplied by 100. all laboratory analysis was conducted at the soil science research laboratory, university of nigeria nsukka. exchangeable mg and k were dominant in swine waste (1.58 and 0.63 cmolkg -1 ), respectively as compared to other wastes. soil properties prior to amendment the properties of the soil at the initiation of the study are shown in table 2. sand fraction was dominant compared to clay and silt fractions leading to sandy clay loam textural class. the ph was slightly acidic (5.2) and organic carbon content (1.8%) was also low based on rating by landon (1991). total nitrogen (0.11%), available phosphorus (26 mgkg -1 ), cation exchange capacity (8.6 cmolkg -1 ) were rated low (landon, 1991). exchangeable calcium and magnesium dominated the exchange sites. data analysis all data collected were analyzed statistically using the analysis of variance technique (anova) and fishers least significant difference (steel and torrie, 1980) was used to separate means and significance was accepted at 5% probability level. results and discussion chemical composition of amendments used for the study table 1 shows the chemical composition of the animal wastes used for soil amendment. total n was higher (3.68%) in poultry manure following the order pm>cd>ss>sw. sewage sludge was slightly acidic (5.91) while other wastes were neutral to alkaline. although values of available p were low in the animal wastes based on landon (1991), it was highest in poultry manure (0.4 mgkg -1 ). the exchangeable ca values were 3.72, 3.19, 2.81 and 2.91 cmolkg -1 for poultry manure, swine waste, cow dung and sewage sludge, respectively and higher than mg, k and na values in the wastes. effect of treatments on soil physical properties table 3 shows the physical properties as influenced by animal wastes. in all treatments, the texture was sandy clay loam which may be attributed to nature of parent materials and high rainfall that could favor washing away and leaching of silt-sized and clay-sized fractions (igwe et al., 1999; akamigbo, 2010). the application of animal wastes significantly (p<0.05) decreased bulk density and increased total porosity. the implication of a lowered bulk density and increased total porosity are ease in root penetration, downward movement of water, more soil water retention, availability for greater water use efficiency by crops and lowered risks of compaction (ogbodo and chukwu, 2012; nwite and okolo, 2016). bulk density was lower in ss (1.45 gcm -3 ) amended plots relative to cd (1.51 gcm -3 ), pm (1.54 gcm -3 ) and sw (1.62 gcm -3 ) plots. this reflects the role that the different organic waste decomposition plays in soil loosening. however, saturated hydraulic conductivity obtained in pm amended plots were higher relative to other amendments, following the order pm>cd>ss>sw >c . pm relative to the control increased water transmission through the soil by 61.9%. similarly, gravimetric moisture contents of pm plots were higher relative to the amended and 4 table 2. pretreatment soil (0-20 cm) properties. particle size distribution value clay (%) silt (%) sand (%) textural class soil ph (kcl) exch. ca (cmol kg -1 ) exch. mg (cmol kg -1 ) exch. na (cmol kg -1 ) exch. k (cmol kg -1 ) cec (cmol kg -1 ) exch. acidity (cmol kg -1 ) tn (%) oc (%) av.p (mgkg -1 ) 21 20 59 scl 5.2 4.1 2.8 0.03 0.1 8.6 1.36 0.11 1.8 26 o.c: organic carbon; tn: total nitrogen; av. p: available phosphorus; exch ca: exchangeable calcium; mg: magnesium; na: sodium; k: potassium. table 3. physical and properties of the soil as influenced by wastes. treatment %sand %silt %clay tex bdg tp (%) hc gmc as sa mwd ll pl (cm -3 ) (cmh -1 ) (%) (%) (%) (%) control 59 20 21 scl 1.66 36.3 16 13.3 10.8 2.3 1.6 19.8 15.2 pm 59 23 18 scl 1.54 42.5 41 19.0 14.7 4.7 2.5 22.5 17.7 sw 58 23 19 scl 1.62 37.8 28 15.6 11.2 3.6 2.4 21.2 16.3 cd 58 22 20 scl 1.51 41.8 36 14.5 13.3 3.6 2.4 20.9 18.1 ss 60 21 19 scl 1.45 41.3 30 16.8 11.1 3.4 2.4 21.5 17.0 flsd (0.05) ns ns ns 0.07 3.3 ns ns ns ns ns 22.2 17.9 tex: soil texture; scl: sandy clay loam; bd: bulk density; tp: total porosity; hc: hydraulic conductivity; gmc: gravimetric moisture content; as: aggregate stability; sa: state of aggregate; mwd: mean weight diameter; ll: liquid limit; pl: plastic limit. unamended plots. there was no significant difference between the control and amended plots in terms of aggregate stability and state of aggregation. notwithstanding, what seems obvious are slight increase with amendment following the order pm>cd>sw>ss>c. this corroborates the findings of wang et al. (2016) who observed that organic matter is an indispensible component in soil aggregation. similarly, there were no significant differences among the treatments in terms of mean weight diameter. however, pm recorded the highest values (2.5%) as compared to other amendments and control (1.6%). this indicates that animal wastes enhanced mean weight diameter of soil. the liquid and plastic limits of amended soil were significantly (p<0.05) increased following amendments. higher plasticity values were recorded in cd (18.1), pm (17.7), ss (17.0) and sw (16.3) amended plots as compared to the control (15.2). the implication of this is that moisture contents of the soils were improved following the amendments. effect of treatments on soil chemical properties soil ph, organic matter, total nitrogen and available phosphorus soil ph was not significantly (p>0.05) affected following organic waste amendment as shown in (table 4). the highest value of 6.1 was obtained in plots amended with poultry manure while the lowest was 5.2 from the control. the increase in ph due to animal waste amendment have been widely reported in literature (darmordy et al., 1983; nwite et al., 2016). amendments increased p significantly (p<0.05). the highest value was obtained on poultry manure amended plots (40 mgkg -1 ) as compared to the lowest value (28 mgkg -1 ) recorded in the control (table 4). this agrees 5 table 4. chemical properties of soil at post harvest. treatment ph av. p tn om c:n ca mg k na ea cec bs (%) (kcl) (mgkg -1 ) (%) (%) ratio cmolkg -1 c 5.2 28 0.11 2.0 10 4.1 2.8 0.1 0.03 1.36 8.4 80 pm 6.1 40 0.13 2.3 11 6.1 3.2 0.20 0.07 1.22 10.9 86 sw 5.8 28 0.09 2.0 13 5.1 2.8 0.13 0.06 1.30 10.2 83 cd 5.9 30 0.12 2.2 11 4.9 2.8 0.11 0.06 1.28 9.4 83 ss 5.7 36 0.11 2.1 11 4.6 2.8 0.13 0.08 1.34 9.5 81 flsd (0.05) ns 7.5 ns ns ns ns ns ns 0.02 ns 1.65 ns c: control; pm: poultry manure; sw: swine wastes; cd: cow dung; ss: sewage sludge. table 5. effect of amendments on growth and yield of maize. treatment plant height (cm) leaf area index grain yield (tha -1 ) c 113.2 344 0.5 pm 161.7 519 1.6 sw 137.5 463 1.4 cd 129.4 397 0.9 ss 136.1 387 1.2 flsd (0.05) 20.3 154 0.2 c: control; pm: poultry manure; sw: swine wastes; cd: cow dung; ss: sewage sludge. with earlier findings by adeleye and ayeni (2009) that p content is increased by application of animal wastes. total n exhibited non-significant effect following wastes amendments. like p, the highest value of 0.13% was obtained in poultry manure amended plots, while the lowest value (0.11%) was recorded in the control. there was no significant difference (p>0.05) among the treatments. the increase in total n of amended plots is similar to the effects observed by khaliq and abbasi (2015) and attributed to build up of organic matter in the soil. there was no significant effect of amendments on soil organic matter. however, plots amended with poultry manure gave the highest values of 2.3%, while cd, ss and sw amended plots gave 2.2%, 2.1% and 2.0 as compared to the control plots 1.9%. this is consistent with reports of wang et al. (2014) soil organic matter was higher in organic waste amended soils and is dependent on its nature and rate of its decomposition by the microbial community. pm contains both solid and liquid excreta and tends to mineralize faster as compared to wastes from other sources where urine is lost-resulting in high solid excreta (amanullah et al., 2010). this explains the low c:n ratio of pm amended soils as an indicative faster release of nitrogen. exchangeable bases and exchangeable acidity amendments led to an increase in soil cec especially in pm plots (table 4). this implies better supply of nutrients, hence improved fertility in amended plots. the higher values in poultry manure amended plots for ca 2+ , k + and mg 2+ has been observed by other reseachers (hue and lucidine, 1999; adeniyi and ojeniyi, 2005). there is probably an increase in the amount of ca 2+ derived from caco3 due to formation of organic acids. increased ca 2+ will result in improved soil structure by forming cationic bridges between clay and soil organic matter (david and dimitrios, 2002). mg 2+ content tended to remain unaltered in all plots excluding the pm amended. the effect of amendments on na content was significantly increased. ss amended plots had the highest values for na + with value of 0.08 cmolkg -1 , while the control plots had the lowest values. this corroborates early findings (basta, 1996), that all wastes have high soluble salts in variable quantities and excessive salts in soils (natural or applied) can have detrimental effects on plants growth. exchangeable acidities were not significant (p>0.05). sewage sludge had the highest value of 1.86 cmolkg -1 . the changes may be due to al 3+ from soil exchange site acted upon by decomposing waste-humus having a weak acid nature (adediran et al., 2003). effect of treatments on growth and yield components plant height was significantly (p<0.05) increased (table 5) with the application of the amendments. the highest 6 result of 161.73 cm was 43% higher than the control plots. this was also observed in the leaf area index (51%) and yield (220%) higher relative to the control. it followed the order pm>sw>ss>cd for plant height, leaf area index and yield. this agrees with mbah (2008) and nwite et al. (2016) who observed that organic manure improves crops yield and is source-dependent. conclusion the study shows that animal wastes enhanced soil properties and maize yield of hydromorphic ultisols in abakaliki agro-ecology. the enhancement of soil properties and yield followed the order pm>sw>ss>cd. poultry manure having low c:n ratio had the ability to release faster, hence, performing better than other animal wastes in the short-term. cow dung, swine wastes and sewage sludge may have greater residual benefits. the application of sewage sludge may be beneficial in certain soil deficiency, but however has toxicity potentials. the application of these wastes at the rate used in this study will lead to restoration of degraded soils, thus ensuring that soil is healthy enough to perform its functions for agricultural sustainability. more studies are however needed using variable quantities and combination of wastes in hydromorphic soils. a long-term study of the wastes in order to bring out a cumulative effect of the amendments will also be a very useful practice. conflict of interests the authors have not declared any conflict of interests. references abbasi mk, mushtaq a, tahir mm (2009). cumulative effects of white clover residues on the changes in soil properties, nutrient uptake, growth and yield of maize crop in the subhumid hilly region of azad jamnu and kashmir pakistan. african journal of biotechnology 8(22):6191. adediran ja, taiwo lb, sobulo ra (2003). effect of organic wastes and method of compositing on compost, nutrient maturity, composition of compost and yield of two vegetable crops. journal of sustainable agricultural 22:95-109. adeleye eo, ayeni ls (2009). effects of soil preparation methods and organic wastes on soil nutrient status and yield of maize (zea mays) on an alfisol of southwest nigeria, american-eurasian journal of sustainable agriculture 3(3):460-467. adeniyi on, ojeniyi so (2005). effect of poultry manure, npk 15-15-15 and combination of their reduced levels on maize growth and soil chemical properties. nigerian journal of soil science 15:34-41. adeoye go, adeoluwa oo, ojelade m, sridhar e, makinde ea, olowoake aa (2008). comparative evaluation of organo-mineral fertilizers and mineral fertilizers (npk) on yield quality of maize (zea mays (l) moench). nigerian journal of soil science 18:132-137. akamigbo for (2010). soils: fundamental methods of soil resource survey, classification,interpretation, interpretation and application. published and printed by university press ltd. bookshop/bank buiding complex, unn. amanullah mm, sekar s, muthukrisnan p (2010). prospects and potential of poulty manure. asian journal of plant science 9:172-192. basta nt (1996). soil amendments: impacts in biotic systems. soil science 161(12):885-886. bray rh, kurt lt (1945). determination of total, organic and available forms of phosphorus in soils. soil science 59:39-45. bremner jm, mulvaney cs (1982). nitrogen total. in page, a.l.(ed) methods of soil analysis. no 9. part 2: chemical and microbiological properties, 2nd edn. am soc agron inc, madison, wi, usa. darmordy rg, foss je, mc itosh m, wolf dc (1983). municipal sewage sludge compost amended soils: some spatiotemporal treatment effects. journal of environmental quality 12:2. david r, dimitrios p (2002). diffusion and cation exchange during the reclamation of saline-structured soils. geoderma 107:271-279. fdalr (1986). reconnaissance soils survey of anambra state. nigeria soils report fdalr kaduna. gee gw, or d (2002). particle size analysis. in: dane, j. h. and topp, g. c. 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001-010, march, 2022. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper shea waste slurry as an organic soil amendment of tropical soils in the tamale metropolis, northern ghana abagale f. k.*, alazuga i. n. a. and osei a. r. school of engineering, university for development studies, tamale, ghana. *corresponding author e-mail: fabagale@uds.edu.gh. accepted 21 june, 2021 infertile and less productive soil due to continuous cropping, poor soil management and other forms of land exploitation are mostly amended using inorganic fertiliser. shea waste slurry (sws) as an organic soil amendment material was applied to soils in two locations and its effect on plant primary and secondary nutrients evaluated. one-way diagonal method was employed in sampling soil from fields measuring 25 m×40 m at depths of 0-30 and 30-60 cm for sws applied and non-applied soils. results showed increased ph resulting from sws application and ec increasing from 41.15±3.89 to 155.5±83.4 µs/cm in both locations. sws application also increased %n levels at depth of 0-30 cm from 0.03±0.0 to 0.56±0.2%, concentration of p increased from 3.47±0.62 to 262.0±176 mg/kg and k from 21.9±2.39 to 231.6±98 mg/kg. na levels increased from a low of 0.46±0.09 to a maximum of 2.81±1.0 meq/100 g in both study sites, mg increased from 0.80±0.3 to 8.51±4.86 meq/100 g whilst ca increased from 1.6±0.07 to 6.3±098 meq/100 g for the depth of 0-30 cm. soil %om and oc, respectively increased from 0.58±0.01 to 10.94±3.95 and 0.34±0.11 to 6.36 ±2.29% for the depth of 0 to 30 cm in both study locations. the study indicated a general increase in the levels of all the parameters analysed for the study at the sws applied soils as compared to the non-applied soils. the use of sws as a soil nutrient amendment in crop production as an organic material was observed to be very effective. key words: shea waste slurry (sws), organic soil amendment, tropical soil, plant nutrients. introduction the success of soil management to maintain soil quality depends on the understanding of how soils respond to agricultural use and practices over time (negassa and gebrekidan, 2004). ayoola (2006) reported improvement of environmental conditions and public health as well as the need to reduce costs of fertilising crops to also be important reasons for advocating increased use of organic materials. excessive and inappropriate use of chemical fertilisers has been reported by different authors as a major cause of nutrient imbalance and degrading soils (singh and agarwal, 2001; meena et al., 2003; mahajan et al., 2008; mukhtar et al., 2011). according to tran-thi et al. (2004), among available means to achieve sustainability in agricultural production, organic manure and bio-fertiliser play an important and key role because they possess many desirable soil properties and exert beneficial effect on soil physical, chemical and biological characteristics. crop yield increased from 30 to 50% resulting from application of commercial fertilisers as indicated by vlek (1990) and 2 stewart et al. (2005). shea butter is a product from the nuts of the shea tree (vitellaria paradoxa) mainly found in the savannah region of west africa. the butter is used for domestic consumption (mainly as edible oil) and in some products of the cosmetic and pharmaceutical industries (cof, 2016; akparanta et al., 2017). the shea butter processing industry is an increasing economic venture which serves as the principal source of income, particularly for many women in northern ghana (jibreel et al., 2013). according to teketay et al. (2003), processing of the shea butter from the shea fruit is labour-intensive activity mainly carried out using traditional techniques by women. the basic processes for the butter extraction begin with the collection of nuts, de-pulping, drying of nuts, dehusking, drying and smoking of kernels and pounding and grinding into past. next is mixing with water, treading, kneading and churning, floating and refining, solidifying and moulding (teketay et al., 2003). the tradition processing method generates significant quantities of both liquid and solid waste which is described by jibreel et al. (2013) as brown water and black sludge. the liquid fraction, according to ofosu (2009), constitutes suspended and dissolved om and oil, which might have a potential impact on the environment. shea waste-slurry (sws) as used in this study refers to the concentration of the remnant liquid (brown water) and solid (black sludge) as well as the oil at the end of the shea butter extraction processes (abagale et al., 2012). it is generally disposed of into the surroundings in the production communities which accumulate onto receiving soils. a survey conducted by jibreel et al. (2013) in the tamale metropolis realised that the solid waste-slurry is dried into cake for further use while almost 46% of the shea butter processors disposed of the liquid waste on bare land. production of shea waste-slurry is thus expected to increase as small-scale shea butter processing industries continues to gain much attention as a potential economic venture in the tamale metropolis. organic waste generally has a significant effect on soil properties (physical, chemical, and biological) which favours plant growth and development (hossain et al., 2017). according to jibreel et al. (2013), the intrinsic elements in waste-slurry might alter the structure and properties of receiving soils and affect crop development (jibreel et al., 2013). a study by abagale et al. (2012) highlighted the positive potentials of shea waste-slurry on improving soil physical properties such as the bulk density, infiltration rate, porosity, gravimetric and volumetric moisture content for crop development in northern ghana. however, the fertiliser potentials of sws for the recycling of essential plant nutrients, has over the years receive little attention (danikuu, 2016). the study, therefore, examined the effect of sws as an organic soil amendment material for enhancing soil nutrients for the small-scale subsistence farmer. materials and methods study area the study was conducted in two communities: kasaligu and jisonayilli in the tamale metropolis (731 km 2 ) and located on latitude 9°24′30.10″n and longitude 0°50′25.63″w of northern ghana. the northern region of ghana is characterised by a unimodal rainfall pattern with an average annual rainfall of 1,000 to 1,300 mm which begins from may to october with the peak occurring between august and september. a long dry period is experienced between the months of november and may. the region is one of the hottest in the country with an annual average temperature of 29 to 34°c. reference evapotranspiration (eto) is reported above 600 mm/year (kranjac-berisavljevic, 1999; armah et al., 2010; abdul-ganiyu, 2011). soils in the study areas are mostly sandstone, mudstone and shale and these have weathered into different soil grades. due to seasonal erosion, soils emanating from this phenomenon are sand, clay and laterite oxysols. the parent rock of the experimental field consists of sandstone and clay-shale and belongs to rock of the abosum bed of the lower voltaian formation. the soil belongs to kpalsawgu series and consists of yellow-brown clay and silt which are developed mainly from local colluvium (sari, 1993). soil sampling and analysis the study was carried out on soils that are used for the disposal of shea waste-slurry and cultivated for small-scale subsistence farming in the selected communities. these soils are manually tilled with simple farm tools (hoe and cutlass) at the onset of the raining season for the production of grains, cereals and vegetables. typical crops include capsicum annuum (pepper), zea mays (maise) and pennisetum glaucum (millet). the study considered soils with sws as the treatments while adjacent soils without the sws were used for the control. one-way diagonal method was employed in soil sampling towards the ends of the 25 m × 40 m diagonal fields for both sites of application of sws and non-application of sws as control. soil samples were taken uphill of about 200 m away from the application site of sws against gradient in each of the study areas to avoid interference from the application site. 200 g of soil samples were taken from 0-30 and 30-60 cm depths at both applied and non-applied sites of sws. sampling was carried out during the dry season before land cultivation. a total of 16 samples of soil were collected from the sampling sites for laboratory analysis. the gps coordinates of the various sampling points and the standard methods of laboratory analysis are presented in tables 1 and 2 respectively data analysis the analysis was carried out to determine and compared the concentration of the selected parameters for the different sampling sites (treatment and control) with respect to the depth (0-30 and 3060 cm). the variation in soil nutrients as influenced by sws was analysed using analysis of variance (anova) at probability (p) of <0.05. means at the various sites were compared for significant difference using the fisher pairwise comparisons (lsd) method at 95% confidence interval. data were analysed using minitab 17 and graphpad prism 8. 3 table 1. geo-references of soil sampling points. site latitude (º) longitude (º) altitude (m) kad1 n 09.40517 w 000.92334 169 kad2 n 09.40142 w 000.92448 166 kan1 n 09.40344 w 000.92555 163 kan2 n 09.40545 w 000.92452 167 jid1 n 09.45208 w 000.85558 163 jid2 n 09.45307 w 000.85557 165 jin1 n 09.45342 w 000.85529 168 jin2 n 09.45333 w 000.85629 167 kad = kasalgu applied site; kan = kasalgu non-applied site; jid = jisonayilli applied site, jin = jisonayilli non-applied site. table 2. laboratory methods of soil samples. soil parameter analysis method reference soil ph supernatant suspension of a 1:2.5 soil to water ratio using ph meter rhoades (1982) electrical conductivity (ec) 1:2.5 soil to water suspension was measured using conductivity meter motsara and roy (2008) organic carbon (oc) walkley-black method walkley and black (1934) organic matter (om) wet combustion with k2cr2o7 nelson and sommers (1982) total nitrogen (n) kjeldahl method bremner and mulvaney (1982) available phosphorus (p) olsen’s method olsen et al. (1954) potassium (k) flame photometer at 766.5 nm toth and prince (1949) calcium (ca) and magnesium (mg) ethylenediamine tetraacetic acid (edta) titration and use of aas cheng and bray (1951) sodium (na) flame photometry at 589 nm robbins and wiegand (1990) and helmke and sparks (1996) results and discussion effect of shea waste slurry on soil ph and ec the use of cover crops, the application of highquality compost, the return of crop residue and the application of other natural fertiliser have been reported to work together to neutralise soil ph (asfg, 2010). low soil ph stress is a major growth-limiting factor for crop production in many regions of the world and the optimum ph for crop growth can vary among crop cultivars and soils (fageria et al., 2011). according to liu and hanlon (2015), soil ph is one of the most important soil chemical properties which influences the solubility and bioavailability of essential plant nutrients. slightly acidic soils with ph levels ranging from 5.18±0.13 to 5.42±0.33 for both depths at kad and 5.49±0.08 to 5.97±0.3 at kan was recorded indicating how sws influences soil ph (table 3). for the jisonayili soils, average ph of 5.67±0.10 to 8.85±2.47 at jid were noted to be higher than that of jin with ph of 7.62±0.49 to 7.56±0.62 (table 3). though the effect of sws application to soils in the two study locations did not statistically influence the level of ph by the anova (table 3), the mean for the upper depth (0-30 cm) of kan was significantly higher than that of kad. 4 table 3. mean ph levels in the study area soils. site ph anova (α = 0.05) ec (µs/cm) anova (α = 0.05) 0-30 cm 30-60 cm f-stat f-pr 0-30 cm 30-60 cm f-stat fpr kad 5.18 ±0.13 b 5.42 ±0.33 ab 3.59 0.124 155.5±83.4 a 75.8 ±40.7 a 2.44 0.204 kan 5.97 ±0.3 a 5.49 ±0.08 ab 41.15 ±3.89 a 39.6 ±32.0 a jid 8.85± 2.47 a 5.67± 0.10 a 2.04 0.251 52.8± 53.0 a 47.4± 40.3 a 0.76 0.574 jin 7.62±0.49 a 7.56±0.62 a 110.2 ± 79.8 a 40.65± 7.28 a kad = kasalgu applied site; kan = kasalgu non-applied site; jid = jisonayilli applied site, jin = jisonayilli non-applied site. f-stat = f-statistics; f-pr = f-probability. means that do not share a letter ( a or b ) are significantly different. values after ± indicate standard deviation. liu and hanlon (2015) noted that ph range from 5.5 to 7.0 favours the solubility of essential nutrients thus indicating that the ph recorded for the two locations are suitable for the cultivation of crops. however, sws application to soils in the jisonayili resulted in increasing the ph from acidic to alkaline soils. a study by adeli et al. (2008) reported that long-term treatments with anaerobic swine lagoon liquid reduced the soil ph by 0.97, 0.11, and 0.88. sharpley et al. (2004) investigated soils with varying organic manure application histories and found that ph was significantly greater in manured soils than in untreated soils using poultry litter and swine slurry manure. similar results were also reported by oguike and mbagwu (2001) for soil ph using organic manure for soil fertility amendments. whalen et al. (2000) also observed a significantly higher ph for manure‐amended than unamended soil. another study by carmo et al. (2015) highlighted the potentials of organic wastes on increase soil ph and to elevated levels that may not be favourable for plant development. according to fageria et al. (2010), the most useful soil ph for acid soils, is the minimum ph above which liming will not increase crop yield. higher levels of ec of 155.5±83 and 41.15±3.89 µs/cm for the upper depth (0-30 cm) of kad and kan, respectively, reduced to 75.8±40.7 to 39.6±32.0 µs/cm at their respective lower depths (30-60 cm). similarly, ec at the upper depths (0-30 cm) of jid and jin, respectively decreased from 52.8±53.0 and 110.2±79.8 µs/cm to 47.4±40.3 and 40.65±7.28 µs/cm at their lower depths (30-60 cm). however, the observed variations for both sites were statistically insignificant. the application of sws as an organic soil amendment material thus increased the soil ec at kasalgu. a study by carmo et al. (2015), realised a slight increase in soil ec for medium-textured oxisol by the addition of organic wastes. the increase of ec levels by application of organic waste on soil have earlier been reported by different authors (tsadilas et al., 1995; topper and sabey, 1986; hinesly et al., 1982). plant primary nutrients in soils increasing the soil om as well as ph increases soil p availability. the optimum ph for p availability is 6.0 to 6.5. if ph is outside this range, supplementary p may be needed even if soil tests show adequate p. k regulates osmotic balance, opening and closing of stomata and cell turgor pressure, while stimulating rooting, photosynthesis, chlorophyll formation, starch formation and translocation of sugars. adequate k levels reduce plant susceptibility to insect and disease attack. as an essential component of chlorophyll, proteins, enzymes and hormones, n is essential for plant growth. n does not exist as a mineral element in the soil. it must be taken from the atmosphere, which is composed of approximately 78% n. however, plants cannot use atmospheric n until it is fixed into an available form such as ammonium or nitrate by free bacteria, algae in the soil and through symbiotic bacteria in nodules contained in the roots of legumes such as alfalfa and beans. artificial sources of n (chemical fertiliser) are fixed through the haber-bosch process of reacting hydrogen and atmospheric n under heat and pressure to form ammonium (asfg, 2010). table 4 presents changes in soil %n content due to the influence of sws. it can be observed from table 4 that n levels increased from a low of 0.03±0.0% at kan (0-30 cm) to 0.56±0.2% at kad while at the 30-60 cm, 0.015±0.02 and 0.01±0.0% were recorded for the kad and kan soils, respectively. the results of the anova at kasalgu site were statistically significant with f-pr of 0.012. the significant difference was observed for the mean concentration at kad (0-30 cm) over the other soil samples (table 4). n content increased from 0.04±0.01 at jin (0-30 cm) to 0.14±0.13% at jid while the 30-60 cm depth did not record any increment between the applied and the nonapplied soils. the variation among mean concentrations was statistically insignificant with f-probability values 0.274, respectively (table 4). at a depth of 0-30 cm, soil at kan and jin recorded lower than the 0.15% n-value recommended by brady and weil (1999) for cultivated soils resulting from the nonapplication of sws. comparison between both depths indicates that the level of concentration of %n was higher at the applied soils, thus presenting the 5 table 4. mean primary plant nutrients in experimental soils. site %n anova (α = 0.05) p (mg/kg) anova (α = 0.05) k (mg/kg) anova (α = 0.05) 0 30 cm 30 60 cm f-stat fpr 0 30 cm 30 60 cm f-stat fpr 0 30 cm 30 60 cm f-stat fpr kad 0.56 ± 0.2a 0.015 ± 0.02b 14.97 0.012 98.6 ± 21.9a 11.56 ± 12.29b 27.16 0.004 152.2 ± 14.3a 91.9 ±52.3ab 10.84 0.022 kan 0.03 ± 0.0b 0.01 ± 0.01b 3.47 ± 0.62b 3.83 ± 1.35b 21.9 ± 2.39b 18.60±7.17b jid 0.14 ± 0.13a 0.01 ± 0.0a 1.88 0.274 262.0 ± 176a 8.10 ± 2.09b 3.83 0.114 231.6 ± 98.0a 52.4 ±21.5b 7.43 0.041 jin 0.04 ± 0.01a 0.01 ± 0.0a 42.78 ± 1.52ab 11.00 ± 1.02b 43.96 ± 4.4b 21.98±7.17b kad = kasalgu applied site; kan = kasalgu non-applied site; jid = jisonayilli applied site, jin = jisonayilli non-applied site. f-stat = f-statistics; f-pr = f-probability. means that do not share a letter ( a or b ) are significantly different. values after ± indicate standard deviation. contribution of applied sws to increased concentration of %n. an experiment by kaniz and khan (2013) to reclaim saline soil using gypsum, rice hull, and rice straw resulted to an increase n content in plant straw which increase by the application of rice hull and sawdust as these treatments added organic matter, in turn, n to the soil. murmu et al. (2013) also found that organic manure increases crop productivity, n utilisation efficiency, and soil health compared to chemical fertiliser. a study by elbl et al. (2013) realised a significant decrease of n leaching by the simultaneous applications of soluble humic substances and compost to soils. according to li et al. (2014), the growth and development of plant may largely depend on the n supplying capacity of the soil. a higher p concentration of 98.6±21.9 mg/kg at 0-30 cm depth was recorded in soils of kad as compared to a low level of 3.47±0.62 mg/kg at kan. anova at 5% resulted in a statistically significant difference between the soils which received sws (specifically for kad at 0-30 cm) and soils which did not, with f-pr of 0.004 (table 4). according to hossain et al. (2018), the application of organic amendments at variable rates individually or in combinations showed a significant (p<0.05) positive influence on the p contents of rice plants grown under both field moist condition and standing water conditions. a higher p concentration of 262.0±176 mg/kg was recorded at jid (0-30 cm) while a relatively lower concentration of 42.78±1.52 mg/kg was noted for soils at jin (0-30 cm) resulting from the non-application of sws. however, p levels at jid (30 60 cm) was slightly lower (8.10±2.09 mg/kg) than that of jin (11.00±1.02 mg/kg). the variation was not statically significant by anova with f-pr of 0.114 (table 4). available p contents in the soil in tamale area ranges from deficient to marginal (5.0-7.0 mg/kg) in the top soil (agyare, 2004). the effect of sws in increasing the levels of p in tropical soils was evident from the results of the study and based on the recommended value of 20 ppm for crop cultivation (asfg, 2010), the addition of sws as an organic soil amendment material to increase p concentration in tropical soils has been realised. the higher level of p in sws applied soils could also be attributed to the findings of hinsinger (2001) which indicates that the distribution of p species (organic and inorganic) in soil is determined primarily by the ph of the soil solution. the study revealed that the ph of the soils at the sws applied site increased, leading to p availability. mokolobate and haynes (2002) and pypers et al. (2005) also reported that incorporation of organic residues can improve soil conditions making an increase in p availability possible. from table 4, soils from the sampling points of kasalgu recorded 152.2±14.3 and 21.9±2.39 mg/kg of k at 0-30 cm depth for kad and kan, respectively. concentration at the lower depth (30-60 cm) reduced to91.9±52.3 and 18.60±7.17 mg/kg for kad and kan, respectively. anova at 5% resulted in f-probability of 0.022, indicating a significant difference. k concentration at the upper depth (0-30 cm) of kad was significantly higher than the soils at kan (table 4). soils sampled from the jisonayilli community at 0-30 cm recorded an average of 231.6±98 and 43.96±4.4 mg/kg of k for jid and jin soils, respectively with a decrease to 52.4±21.5 and 21.98±7.17 mg/kg at their respective lower depths (30-60 cm). comparatively, results of the anova revealed that the mean concentration of k at the upper depth (0-30 cm) was significantly higher than the other samples at f-pr of 0.041 (table 4). according to hossner and juo (1999), most tropical soils are highly weathered, infertile, acidic and have deficient levels of k. however, the 6 table 5. mean levels of secondary plant nutrients in soil. site na (meq/100 g) anova (α = 0.05) mg (meq/100 g) anova (α = 0.05) ca (meq/100 g) anova (α = 0.05) 0 30 cm 30 60 cm f-stat fpr 0 30 cm 30 60 cm f-stat fpr 0 30 cm 30 60 cm f-stat fpr kad 1.76 ± 0.39 a 1.07 ± 0.46 ab 8.80 0.031 8.51 ± 4.86 a 1.47 ± 0.57 b 4.34 0.095 6.3 ± 0.98 a 1.87 ± 0.38 b 36.82 0.002 kan 0.46 ± 0.09 b 0.37 ± 0.03 b 1.19 ± 0.96 b 0.80 ± 0.38 b 1.6 ± 0.07 b 1.74 ± 0.19 b jid 2.81 ± 1.0 a 0.57 ± 0.18 b 9.19 0.029 3.21 ± 1.14 a 0.80 ± 0.38 b 5.74 0.062 6.14 ± 4.16 a 2.41 ± 0.38 a 1.58 0.327 jin 0.72 ± 0.09 b 0.53 ± 0.12 b 0.80 ± 0.3 b 0.81 ± 0.76 b 4.68 ± 0.19 a 2.27 ± 0.57 a kad = kasalgu applied site; kan = kasalgu non-applied site; jid = jisonayilli applied site, jin = jisonayilli non-applied site. f-stat = f-statistics; f-pr = f-probability. means that do not share a letter ( a or b ) are significantly different. values after ± indicate standard deviation. addition of sws to soils in the tropical lands have been noted to influence significantly, the amounts of k but this was noted for this study to have decreased with increasing soil depth. asfg (2010) reported that, at a depth of 0-30 cm, k is high in applied soils but low in non-applied soils while at 30-60 cm it was generally low in all the soils. secondary plant nutrients in soils a number of secondary nutrients such as iron, manganese, zinc, copper, boron, chlorine and molybdenum are known to influence plant growth and are usually required in small amounts for proper functioning of plant metabolism. the absolute or relative absence of any of these nutrients can hamper plant growth; alternatively, too high a concentration can be toxic to the plant or to humans (gruhn et al., 2000). as an organic soil amendment material, the study evaluated the effect of the application of sws on availability of mg, ca and na and results presented in table 5. na levels in soils of kad recorded higher levels of 1.07±0.46 to 1.76±0.39 meq/100 g while kan soils recorded levels ranges from 0.37±0.03 to 0.46±0.09 meq/100 g. a statistically significant difference of na concentration (table 5 and figure 1) was realised with fpr of 0.031. na concentration of the upper depth (0-30) of kad was significantly different from soils at kan (table 5). na concentration in soils of jid ranged from 0.57±0.18 to 2.81±1.0 meq/100 g while jin soils recorded 0.53±0.12 to 0.71±0.09 meq/100 g concentration levels. anova at 5% resulted in statistical difference with f-pr of 0.029. the maximum mean concentration of na at the upper depth (0-30 cm) of jid was significantly different over all the samples of the study site (table 5). mg levels of 8.51±4.86 and 1.19±0.96 meq/100 g reduced with depth to 1.47±0.57 and 0.80±0.38 meq/100 g for kad and kan, respectively (figure 2). anova at 5% significant level indicates that the difference in mg concentration is insignificant with f-pr of 0.095. however, the maximum mg concentration of kad at 0-30 cm was significantly higher than all the soil samples (table 5). soil mg concentration at jid reduced from 3.21±1.14 to 0.80±0.38 meq/100 g for 0-30 and 30-60 cm, respectively. at jin, mg concentration was virtually the same for both depths (figure 2). once more, the highest mg concentration recorded for sws at 0-30 cm was significantly different from all the soil samples though the results of anova was statistically significant with f-pr of 0.062 (table 5). the concentration of mg in soils applied with sws was noted to be influenced by high levels of soil ph. according to maathuis et al. (2011), concentration of mg is dependent on soil om content, ph and the presence of other cations. the availability of mg for plants is thus ultimately reduces by soil acidity (maathuis et al., 2011). the maximum soil ca of 6.30±0.98 meq/100 g recorded at the upper depth (0-30 cm) of kad showed a highly significant difference with all the other samples at the study site by anova with fpr of 0.002. despite the reduction in ca concentration at jisonayilli site, for the various depths, there was no significant difference among mean concentrations with f-pr of 0.327 by anova (table 5 and figure 3). an experiment by mubarak et al. (2009), found a significant increase (p 0.02) in ca and mg with the application of wool and hoof powder for amendment of sandy soils. however, residues of pigeon pea (dried or fresh) and baggase had no significant effects on soil ph 7 (m eq /1 00 g ) 5.0 4.0 2 .8 1 m ea nl ev el o fn a 3.0 2.0 1 .7 6 1 .0 7 0 .4 6 0 .3 7 5 7 0 . 1.0 0.0 kad kan jid study site figure 1. mean na level at the study areas. (m e q /1 0 0 g ) 15.0 8 .5 1 10.0 o f m g 1 .4 7 1 .1 9 8 0 0 . 2 1 3 . 8 0 0 . m ea n l ev el 5.0 0.0 kad kan jid study site 0-30 cm 30-60 cm 0 .7 2 0 .5 3 jin 0-30 cm 30-60 cm 8 0 0 . 8 1 0 . jin figure 2. mean mg level at the study areas. and the soluble cations (ca, mg and na). shea waste slurry effect on soil om and oc content use of cover crops, return of crop residue, addition of compost and/or composted manure and practices that maintain om on the surface of the soil all increase om levels (asfg, 2010). high levels of soil %om (0.35±0.09 to 10.9±3.95%) were recorded at kad while the kan fields recorded 0.29±0.02 and 0.58±0.01% (table 6). the variation among mean concentrations was statistically significant (f -pr 0.013) with %mo of kad at 0 30 cm being significantly higher than the other samples at the site. also, at jid, 0.29±0.03 to 2.45±2.15% of %om was recorded while 0.23±0.08 to 0.88±0.10% was measured for jin. the results of anova indicated no significant 8 table 6. mean percentage om content in the soil. site %om anova (α = 0.05) %oc anova (α = 0.05) 0 30 cm 30 60 cm f-stat fpr 0 30 cm 30 60 cm f-stat fpr kad 10.94±3.95 a 0.35±0.09 b 14.25 0.013 6.36±2.29 a 0.21±0.05 b 14.28 0.013 kan 0.58±0.01 b 0.29±0.02 b 0.34± 0.11 b 0.17±0.0 b jid 2.45 ±2.15 a 0.29±0.03 a 1.85 0.279 1.43 ± 1.25 a 0.17±0.03 a 1.84 0.280 jin 0.88 ± 0.10 a 0.23±0.08 a 0.51±0.10 a 0.14±0.05 a kad = kasalgu applied site; kan = kasalgu non-applied site; jid = jisonayilli applied site, jin = jisonayilli non-applied site. f-stat = f-statistics; fpr = f-probability. means that do not share a letter ( a or b ) are significantly different. values after ± indicate standard deviation. m e a n l e v e l o f c a ( m e q /1 0 0 g ) 15.0 10.0 6. 14 3 0 6 . 5.0 8 7 1 . 5 5 1 . 1 .7 4 2 .4 1 6 8 4 . 2 7 2 . 0.0 kad kan jid jin study site 0-30 cm 30-60 cm figure 3. mean ca level at the study areas. differences (f-pr 0.279) among the mean %om at the study site (table 6). comparison between both depths clearly indicates that the level of %om accumulation in the soils decreases with increasing depth in both soils. it is observed from the findings of the study that the om content is a clear improvement over the findings of fening et al. (2005) with the indication that soils in the interior savanna zone have low organic matter contents (1.0 ±0.4%) and low levels of the major plant nutrients. seçer et al. (2016), similarly recorded a significant increase in soil om with the application of organic waste products of oily oregano and cumin. soil organic amendments such as manures and plant residues are a significant source of oc to the soil. plants also promote microbial populations and subsequent turnover by exuding oc from their roots (merckx et al., 1985). the application of sws was observed to also influence the percentage levels of oc content of the study soils. high levels of %oc (table 6) were recorded in the two sites to which sws were applied. however, the difference in mean concentration of %oc was statistically significant at kpsalgu site only with a significantly higher concentration at the upper depth (0-30 cm) of kad (table 6). in a similar study, chen et al. (2019) observed an increase in soil oc when different organic waste was applied. landon (1991) reported an average of 0.5% of oc content as a requirement for tropical crops, whilst young (1976) also reported that tropical crops require an average range of 0.6 to 1.2% of carbon content for proper growth and development. the increased percentage oc content of soils was noted to have been influenced by the application of sws thus indicating it as a good source of organic manure for soil fertility amendments especially in degraded tropical soils. mekki et al. (2017), after observing a significant increase in soil om and oc from application of biowaste compost noted that soil oc content is critical for the maintenance of soil quality and balance of the terrestrial carbon cycling. conclusions the results of the study indicated that the application of 9 sws as an organic soil amendment material resulted in increased concentration levels of plant primary nutrients (n, p and k) and also secondary nutrients such as na, ca and mg. the application of sws also influenced soil ph, percentage om and carbon content percentage and soil ec by increasing their levels. the application of sws as an organic soil amendment material thus affected soil chemical properties positively and thus will translate to the availability of the necessary plant nutrients for plant growth and yield. comparison between both depths indicates that the level of %om accumulation in the soils decreases with increasing depth in both soils. the application of sws also influenced the percentage levels of oc content of the study soils. the study noted and concluded that sws has a nutrient influencing effect and therefore could be used as an organic soil amendment material for plant growth as well as soil physical properties improvement. conflict of interests the authors have not declared any conflict of interests. references abagale fk, abdulai n, ojediran jo (2012). effect of shea waste slurry on soil physical properties in peri-urban tamale, northern ghana. asian journal of science and technology issn: 0976-3376 4(12): 036-041. abdul-ganiyu s (2011). hydrological analysis of river basins: a case of nasia, a tributary of the white volta river basin of ghana. vdm publishing. adeli a, bolster ch, rowe de, mclaughlin mr, brink ge (2008). effect of long-term swine effluent application selected soil properties. soil science 173(3):223-235. aggrand soil fertility guide (asfg) (2010). amsoil, inc, winconsin-usa. agyare wa (2004). soil characterisation and modelling of spatial distribution of saturated hydraulic conductivity at two sites in the volta basin of ghana (vol. 17). cuvillier verlag. akparanta cd, abu ra, oloyede ao, obazce be (2017). international issues and agric. trade/marketing promoting shea nut and butter production in nigeria. conference proceedings of the 18 th annual national conference of the nigeria association of the nigerian agricultural economists held at federal university of agriculture, abeokuta, nigeria october, 2017. armah fa, yawson do, yengoh gt, odoi jo, afrifa ek (2010). impact of floods on livelihoods and vulnerability of natural resource dependent communities in northern ghana. water 2(2):120-139. ayoola o t (2006). effects of fertilizer treatment on soil chemical properties and crop yield journal of applied science research 2(12):1112-1116. brady nc, weil rr (1999). the nature and properties of soil. prentice hall of india pvt. ltd. new delhi, india, pp. 384-385. bremner jm, mulvaney cs (1982). nitrogen-total. in: methods of soil analysis. part 2. chemical and microbiological properties, page, a.l., miller, r.h. and keeney, d.r. eds., american society of agronomy, soil science society of america, madison, wisconsin, 595-624. carmo dld, lima lbd, silva ca (2015). soil fertility and electrical conductivity affected by organic waste rates and nutrient inputs. revista brasileira de ciência do solo, p. 40. chen x, wu j, opoku-kwanowaa y (2019). effects of organic wastes on soil organic carbon and surface charge properties in primary saline-alkali soil. sustainability 11(24):7088. cheng kl, bray rh (1951). 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difficulties, and possibilities. africa's pig population makes up 4.6% of all pigs worldwide. they are found all over africa, with the exception of northern africa, where pig farming is discouraged for religious and cultural reasons. smallholder farmers raise them mostly in rural areas of africa, which explains why indigenous breeds and their hybrids make up the majority of the pig population in most of the continent. pigs have cultural and social value and are essential to rural communities' ability to maintain their standard of living. africa's pig production system is primarily traditional, but it is expanding quickly and changing to become more modern. africa now produces more than 2 million tons of pork annually, up from less than a million tons in 2000. one of the primary factors limiting pig productivity in africa is the prevalence of illness outbreaks, particularly african swine fever. other factors include a lack of technical expertise and skills, high temperatures, restricted access to superior breeds, high feed and veterinary input costs, unwelcoming government regulations, prejudice based on religion and culture, inadequate processing facilities, and an underdeveloped value chain. the need for more food production is indicated by the estimated 2.5 billion people living in africa by 2050, rising urbanization, and a decline in the number of farmers. for increased productivity and easier exporting, the pig production systems in africa need developmental research, advances in housing, feed production and manufacture, animal health, processing, capacity building, and pig-friendly regulations. key words: farmers; policy; pork; smallholder; transformation; value-chain. introduction global cattle production has benefited from africa's enormous territory and growing population. with 1.4 billion people living there, africa is the second largest continent in the world and accounts for 17.9% of the global population [1]. the world's livestock sector is significantly shaped by africa's remarkable biological and geographical diversity as well as its sociocultural complexity. a third of the world's livestock production [2] and almost 40% of africa's agricultural gdp [3] come from the continent's livestock population. in sub-saharan africa (ssa), livestock will play a bigger role in the future as the region's population, affluence, and urbanization all increase demand for food derived from animals. by 2050, consumers with low and intermediate incomes are expected to require 5.5 million tonnes more milk and 107 million tonnes more meat than they did in 2005 and 2007 [4]. by 2050, ssa's per capita annual meat and milk consumption is projected to be 14 kg and 30 l, respectively [2]. one way that africa might satisfy its demand for animal protein and fulfill international demands through exports is by raising pigs. 1.67% of the world's pigmeat is produced in africa. in africa, pig production is a significant part of the livestock subsector of agriculture. it is impossible to overstate how important pig production is to the continent's ability to sustain livelihoods by providing a healthy source of protein, jobs, and revenue. pork is readily digested, has a high biological value, and contains vital amino acids. pork is referred to as "pink meat" because it shares the nutritional african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-008, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 characteristics of both red and white meats [5]. pig meat is a significant source of animal protein in human diets, according to the global trend in meat production and consumption from 2016 to 2020. according to estimates based on fao data [5,6], pig is the most consumed meat worldwide, making up 36% of global meat consumption, more than beef (24%), poultry (33%), and goats and sheep (5%). pig meat, however, is not the most popular meat in africa; instead, it comes in second after beef, mutton, fowl, and chevon. in the majority of africa, pigs are raised primarily under vast production systems, where they are neglected and left to fend for themselves [7]. however, the conventional extensive system is quickly giving way to semi-intensive and intensive manufacturing systems. pigs have the potential to significantly improve food and nutrition security in africa and around the world due to their inherent qualities, which include high fecundity, short generational intervals, superior feed conversion indices, and early maturity when compared to other livestock species. given its comparatively low production costs when compared to other significant livestock farming enterprises, investing in pig production has turned out to be one of the most lucrative livestock ventures [8]. pig farming is therefore a thriving industry that has the potential to close the gap in the supply of animal protein, offer reasonably priced meat, and support the economies of africa and the global community by creating jobs and revenue. despite africa's potential for pig production, the industry is hindered by a number of issues that keep it from reaching its full potential. with regard to the production system, population distribution, pork output, per capita consumption, problems, and future prospects, this research sought to analyze the current state of pig production in africa. present pig production status in africa pig production systems in africa the more than 70% of pigs in africa live in rural areas, where they are typically left to scrounge for food [9]. pigs are widespread throughout the continent. furthermore, 90% of nigeria's pig population is raised in vast systems, and the country contributes 65% of west africa's total pork production [10]. in many african countries, raising pigs is a source of income, particularly for young people and women engaged in small-scale farming. pig production systems in africa vary and are intricate among nations, each with unique potential and difficulties. the prevalence of disease outbreaks, particularly africa swine fever (asf), and rising input costs have had an impact on the pig business globally, affecting both production costs and output. africa has been impacted by these because of the widely used production systems. in africa, pig farming methods typically vary from smallholder, traditional scavenging systems to more intensive commercial setups [11,12]. the pigs are exposed to the risks of weather extremes, disease infection, and theft because the traditional arrangement permits them to move around and search for food. the majority of the animals raised in this method are native breeds. although they do not readily achieve market weight, this approach also uses crosses between native and exotic breeds. this is because scavenging operations take energy that should be used for growth and development, while traditional systems could not give enough nutrition for healthy growth [8]. animals that scavenge are typically linked to high rates of disease infection. the exotic breeds and their crosses are the primary foundation of the intense system. however, in order to meet the nutritional needs of the animals for optimal productivity in confinement, they must employ formulated diets, which can be costly because to the high cost of imported feed ingredients and the paucity of grains. more capital is needed for an intensive system of production since it also requires the supply of decent housing, health care, and facilities for disposing of waste for appropriate management. many african pig farmers, particularly smallholder farmers, use locally sourced materials like cane, bamboo, planks, or mud for walls or house demarcation, even if others are investing in the use of sophisticated pig farm equipment. although they are not exclusive, the common breeds of pigs raised under the traditional method include the indigenous breeds and their hybrids. despite the fact that large white, landrace, and their crosses are more popular, exotic breeds like large white, landrace, duroc, hampshire, and pietrain are common in the intense method of production [8]. disease propagation is facilitated by traditional systems' open scavenging and scant biosecurity precautions [11]. if properly maintained, intensive systems can use biosecurity techniques to reduce the risk of disease. however, small-scale pig producers in africa might find it difficult to afford the cost of putting biosecurity measures in place [11]. pig farm estates and clusters have recently been established for smallholder farmers in africa. they are sometimes arranged into cooperatives or farmer organizations. this is to make some facilities that large-scale farmers likewise enjoy easily accessible to smallholder farmers. farmers who belong to a cluster or cooperative can easily access resources including loans, government assistance, veterinary care, veterinary services, wholesale input procurement for member sharing, good infrastructure, and market accessibility. the oke aro pig farm cluster, the biggest pig farm estate in west africa, is located in lagos state, nigeria, and serves as a prime example [13]. this system's inability to consistently deploy and enforce sound biosecurity measures is one of its main problems. by exchanging labor and equipment among various units in the cluster, farmers and farm attendants violate biosecurity protocols, making it easier for diseases to spread and recur. technological developments, growing awareness of sustainable practices, shifting consumer preferences and perceptions, and initiatives to address issues like food security and disease management have all had an impact on pig production in africa 3 over the years [12]. pig breeds with enhanced disease resistance, growth rates, and reproductive performance that are well-suited to regional conditions have been developed. the utilization of better breeds and the implementation of intensive or modern production systems may be the reasons why south africa and nigeria appear to have the highest pork yield per animal in africa [1]. pig population distribution in africa as of 2020, there were 44 million pigs in africa, which accounts for about 4.6% of all pigs worldwide [1]. with 38% of africa's total pig population, eastern africa has the largest percentage of pigs among the continent's five regions (figure 2). because of the region's cultural-religious demography, which discourages the consumption of pork, northern africa has the smallest pig population in africa, accounting for less than 1% of the continent's total pig population. this has an impact on both the region's per capita consumption and production of pork. with corresponding contributions of roughly 33%, 17%, and 12% of the continental pig population, other regions such as western, central, and southern africa are significant producers of pigs [1]. the two african nations with the largest pig populations are nigeria and malawi (figure 1), with an actual population of over 7 million heads (table 1). together, the two nations account for about 35% of africa's entire pig population. malawi has the highest pig population density in africa, yet nigeria has the largest pig population. figure 3 shows that the number of pigs in africa has been growing over time, particularly since 1980. increased living standards and population expansion are to blame for this growth, as they are driving up the need for food. the pig population in northern and southern africa is an exception, despite the fact that the pig population in eastern, western, and middle (central) africa has grown more rapidly. in these two areas, the number of pigs has not grown significantly over time. the number of pigs in africa grew from 21 million to 43 million between 2001 and 2021, a 101% rise throughout the continent. pig meat yield in africa in 2021, more than 2 million tons of pork were produced in africa [1]. figure 4 shows the amount of pork produced (in tons) in various african regions from 1961 to 2021. figure 5 shows that, with the exception of northern africa, where pork production has decreased by 56%, pork production has increased over time in all of africa's regions. in western, southern, central, and eastern africa, the corresponding increases in pork production rates were 1,696%, 448%, 596%, and 1,396%. western africa produced 768,766.80 tons of pork in total in 2021. south africa is a large producer of pork in southern africa and had the highest per capita consumption of pork in africa (4.19 kg). its contribution to southern africa's pork output as of 2021 was over 320,450 tonnes, or 97% of the 329,710 tonnes of pork produced in the region (table 1). other nations in the region, primarily namibia, eswatini (swaziland), lesotho, and botswana, provided the remaining 3%. over the years, northern africa's pork output has followed an uneven pattern with little to no growth. reduced pig populations, which resulted from what can be called malicious culling of pigs, which claimed that pigs are reservoirs of avian influenza viruses after the disease outbreak in poultry in the region, are the cause of the dramatic decline in pork output in this region starting in 2008 [1]. from 3,893 tonnes in 2006 to 1,328 tonnes in 2011, and then to 1,234 tons in 2015, the production of pork fell [1]. nonetheless, the region's output figure increased once more to 1,610 tons in 2021, and it is anticipated to keep improving as some local farmers express interest in growing pork for the region's residents and visitors. the coptic christian minority, who make up only 10% of the population, are the main producers of pigs in egypt. nonetheless, nations like libya, sudan, and western sahara make up a very little portion of the region's pork production. the majority of pig farmers in morocco are poultry farmers who switched to raising pigs when avian flu outbreaks impacted their poultry farms. producing pork for visitors and certain foreign residents is often their goal. they also hope to close the gap on the importation of pork into the nation, which comes primarily from spain, belgium, and france. due to the significant expansion of the pig industry from 2008 to 2013, which was brought about by the arrival of new local farmers, morocco has also been known to export pigs to nations like the gambia and cyprus, where they are consumed by tourists and foreign residents. since then, however, morocco's pig population has not increased significantly; as of 2021, there were about 8,000 pigs and 630 tons of pig meat produced year [6]. per capita pork consumption in africa because of cultural, religious, economic, and geographic variables, pork intake differs greatly among african nations. in africa, pork is not the most popular meat. africa's per capita intake of pork (1.53 kg) is ranked below that of beef (5.22 kg), poultry (6.30 kg), and chevon and mutton (2.39 kg). africa consumes 1.55 kg of pork per person, which is less than the 14.35 kg consumed in asia and the 33.79 kg consumed in europe. due to shifting tastes and inclinations, meat consumption rises as wealth or income rises. like in certain other regions of the world, religious and cultural beliefs that forbid eating pork have a significant influence on how much pig is consumed in africa. the middle, western, eastern, and northern regions of africa consume 2.18, 1.97, 1.38, and 0.01 kg of pork per capita, respectively, whereas southern africa consumes the most (3.99 kg) [1]. pork is widely consumed in south africa, because the population is diversified and has a wide range of dietary preferences. pork consumption, however, is typically substantially lower in nations with sizable muslim or jewish populations. in many african countries, the patterns of pig eating are significantly influenced by these religious differences. because of islamic dietary prohibitions, countries with a majority of muslims, such egypt, tunisia, and algeria, usually have low per capita pork production and consumption [14]. by 2020, egypt had produced roughly 628 tons, 4 which is less than the more than 302,000 and 311,000 tonnes of pork produced in south africa and nigeria, respectively [1]. economically growing urban areas are known to see a shift in dietary diversity, with alternative protein sources being seen. the consumption of pork may be significantly impacted by this change. it has been recorded that multinational fast-food chains, especially those that serve pork-based products, have proliferated throughout most african urban centers [15]. marketing although live animal sales are the main marketing channel for pigs sold by producers, pig marketing methods in africa are found to be straightforward [13]. pig handling, transportation, and sale conditions are fraught with difficulties [16]. the majority of farmers are compelled to sell live animals to intermediaries, who then sell to butchers. to make as much money as possible, producers occasionally butcher their own animals and sell the fresh pork to the end user. this typically happens when middlemen attempt to drive down prices in order to exploit farmers who are under pressure to sell in order to increase profits. however, there are also out-growery systems, in which farmers produce for specific consumers or processors who then use the animals to make products. the market's ability to operate efficiently is hampered by a number of factors, including inadequate market knowledge, a shortage of capital, difficulty accessing official credit sources, poor roads, and excessive transportation costs. the real market and transaction costs are raised by these limitations. state support for pig products is not available. in most of africa, marketing is experienced similarly to how it is in rest of the world. thus, policy and infrastructure support from the government would improve pig marketing. challenges of pig production in africa common challenges pig farming in africa faces a variety of difficulties that differ from one nation to the next and even within regions. the following are some typical difficulties that pig farmers in africa face: i. policies and programs that are inconsistent both inside and across nations on the continent: south africa exports pork and is a significant participant in the continent's pork sector. shipment delays, product rejection, and harm to trade relationships may result from uneven or sudden changes to export restrictions, especially sanitary and phytosanitary standards. countries like kenya have seen changes in import laws pertaining to pork products during the past ten years. the stability of the local market is impacted by the implications of inconsistent import policies, which typically alternate between stringent prohibitions and lenient regulations. these shifts are difficult for importers and traders to handle, which causes supply interruptions and price volatility. another significant issue affecting pig production in various african nations is the inconsistent application of disease management measures. for instance, pig agriculture in africa and around the world has faced serious challenges due to asf outbreaks (figure 6). between 2018 and 2019, asf attacks caused the loss of about 25% of the world's pig population. both domestic and wild pigs are susceptible to this extremely widespread virus illness. a reduction in pig populations and an effect on farmers' livelihoods can result from the disease's high mortality rates, which can cause large financial losses and hinder pig output in impacted areas [13]. the recurrence of this disease has caused many farmers to quit their jobs because they receive little to no compensation. due to this, the majority of farmers who are afflicted with asf choose to keep to themselves and secretly sell the diseased animals, which further spreads the illness. pig farmers are often confused by inconsistent policies regarding disease management techniques, which makes illness prevention and control challenging. i. limited access to superior breeding stock: in the majority of african nations, there is limited access to superior genetics and genetically upgraded breeds. this has affected productivity and is the cause of the large differences in pig meat yield between nations such as south africa, which primarily produces better breeds or hybrids, and others that mostly raise non-distinct varieties. the profitability of the pig industry as a whole as well as the productivity and efficiency of pig farming systems can be impacted by access to quality breeds. ii. inadequate infrastructure: the growth of the pig business may be impeded by inadequate infrastructure, which includes waste management systems, transportation networks, cold storage facilities, and processing units. due to these infrastructure constraints, it is challenging to move pigs and pork products effectively, which raises costs and restricts farmers' access to markets. iii. exorbitant and fluctuating input costs, including those for medications and feed: one of africa's biggest problems is the cost and accessibility of high-quality pig feed and medications. commercial feed can be expensive, particularly if it contains imported ingredients. pig farmers in africa often have inadequate knowledge of the nutrients that pigs need and inadequate knowledge of how to formulate feed. given that pigs normally need a balanced combination of energy, protein, and other vital nutritional sources, this information gap affects the economics of pig farming. it is not necessary to offer a high-quality diet to native pigs that can survive on subpar feed. however, premium feed is necessary for imported breeds and hybrids. in the majority of african nations, high-quality commercial or branded meals for pigs are scarce. good breeds and the availability of high-quality feed or feed components will boost africa's pork production. furthermore, poor infrastructure and storage facilities for medications and pharmaceuticals, as well as feed ingredients, can cause feed spoiling, which reduces the materials' efficacy. iv. limited access to veterinary care: availability of high-quality veterinary in many regions of africa, access to services such as immunization programs, disease diagnosis, and treatment is frequently restricted [17]. this can make pig producers more susceptible to disease outbreaks and result in large financial losses by impeding attempts to prevent and manage disease. in pig farming, putting in place efficient biosecurity measures to stop the spread of illnesses is essential. however, it can be difficult to contain disease outbreaks and preserve healthy pig populations 5 due to a lack of knowledge, insufficient funding, and subpar biosecurity procedures. v. insufficient technical expertise: for small-scale pig farmers, who make up the majority in most countries, limited access to training and technical information about contemporary pig farming techniques and their adoption has remained a difficulty. lack of knowledge in areas like artificial insemination, breeding, nutrition, housing, and biosecurity measures has hindered productivity and made it more difficult to implement better agricultural methods, which has led to a decline in efficiency and productivity. vi. market and cultural factors: in several african nations, the demand for pork has been influenced by religious convictions and cultural preferences. pig farmers have had restricted market prospects due to cultural taboos surrounding the consumption of pork and preferences for other types of meat. additionally, weak value chains and restricted market access have made it difficult for the pig sector to flourish. this is due to the fact that the profitability and sustainability of pig production have been impacted by market accessibility issues, inadequate processing and storage facilities, and poor market relations. climate challenges high ambient temperatures, increased precipitation pattern variability, and other weather extremes are linked to an increase in greenhouse gas (ghg) production and concentration in the atmosphere [18,19]. the productivity of livestock species, including pigs, is impacted by these changes and related variations in climatic conditions throughout time, which are referred to as climate change. pig production and associated activities account for 10.1% of the entire animal contribution to ghg production, while livestock are consistently involved in producing 14.5% of all anthropogenic ghg emissions [18,19]. due to its excessive reliance on rain-fed agriculture [20], africa's agriculture, especially pig production, is susceptible to climate fluctuation, making it one of the world's most vulnerable regions to the negative consequences of climate change [21, 22]. climate change is expected to have a negative impact on water availability and use for crop (feed) and livestock production [23, 24]. while water availability is decreasing, the amount of water required per pig for intake and cooling—particularly to counteract the effects of rising temperatures—is increasing. the supply of plant-based feed resources, particularly grains, is also being adversely affected by the decrease in water availability, which is leading to shortages and higher commodity prices per unit. in certain regions of africa, drought and desert encroachment exacerbate the impact of climate change on pig productivity. for instance, namibia has been suffering from drought, which has had a detrimental impact on the nation's pig and other agricultural production activities [21, 25]. according to research, one of the main causes of rising ambient temperatures is climate change. this causes thermal stress, which has a greater detrimental effect on pig performance than cold stress, particularly in africa. pigs are particularly stressed by heat [26]. heat stress, which is brought on by high ambient temperatures and high humidity, reduces pig production productivity by influencing growth rate, reproduction efficiency, and the animals' overall health and mortality. one significant tactic that might be investigated to lessen the impact of climate change on pig production in africa is breeding for high adaptation and heat tolerance utilizing native breeds of pigs. adequate cooling equipment and management techniques are necessary to lessen the impacts of heat stress on pig production in africa, nevertheless, as animals might not be able to completely adapt to climate stressors alone [18,26]. farmers may make climatesmart farming decisions, prevent losses, and improve grain output and supply by forecasting seasonal climate variations and promptly communicating the information to them. in addition to being impacted by climate change, pigs contribute to it by producing greenhouse gases. therefore, effective management techniques to reduce greenhouse gas emissions from pig farming and associated activities and to lessen the impact of climate change on pigs are both crucial for increasing pig output in africa [18]. opportunities, prospects, and a catalyst for improved pig production in africa prospects and opportunities of pig production in africa in sub-saharan africa, pig farming offers a number of opportunities and potentials that can support local populations' better livelihoods, food security, and economic growth. building a strong and sustainable pig value chain is essential to achieving sub-saharan africa's full pig production potential. i. food security: unlike most livestock species, pigs are extremely prolific and transform feed into consumable meat. this makes them an ideal animal for producing high-quality and highquantity meat, particularly to address the problem of malnutrition caused by a lack of protein, which is common across most of africa. additionally, since pig meat is high in protein, raising pigs for meat can help diversify diets, providing customers with more meat options and improving food security. ii. employment opportunities: the breeding, management, processing, and marketing phases of the pig production value chain can generate jobs for both skilled and unskilled laborers from africa's rapidly expanding population. these include farmers, technicians, traders, farm managers, experts in various facets of animal production, processors, suppliers of inputs, craftspeople, facility managers, logistic operators, and local market sellers, to name a few. because pigs require less area and initial investment than other livestock species, women and young people can engage in pig production operations [27]. iii. foreign exchange earnings and income generation: pig production is a very profitable business with strong returns on investment. because it gives small-scale farmers a reliable source of income, pig farming has the potential to have a major impact on per capita income in sub-saharan africa. pigs' strong growth rate, quick generation interval, and quick reproduction rate enable regular sales of pork and piglets, which support a steady flow of revenue [28]. although most african nations raise pigs for their own consumption, exporting them to other nations or beyond the continent can also generate foreign exchange profits. the potential for foreign exchange profits from pig 6 production is indicated by the current discrepancy between the worldwide market's supply and demand for pork. potential for higher income generation through pig rearing is indicated by the anticipated rise in demand for pork products brought on by population growth and shifting dietary preferences [28]. iv. opportunities for investment: investment in pig farming has proven profitable due to the pigs' innate capacity for reproduction and development as well as advancements in production methods over time. despite its growth, africa's pig industry has not been able to maximize its production due to a lack of investment in infrastructure and technology. in many emerging nations, the demand for meat has also increased due to urbanization and shifting dietary habits [13]. this gives producers the chance to satisfy the demands of the local market while also maybe looking into international markets for pig products. africa's pig production offers investment potential in all stages of the value chain, including but not limited to breeding, housing, feed production, animal aggregation, processing or value addition, cold storage, transportation, and appropriate retailing locations. africa offers vast potential for pig value addition, but it hasn't been fully utilized. making sausages, cured goods, and other value-added products from pigs can increase profit margins, improving farmers' and processors' total revenue. this increases trade and economic activity by giving farmers the chance to reach both local and regional markets. there is potential for additional revenue development if the demand for pig products rises as a result of population growth and shifting dietary preferences [29]. the possibilities for pig production in north african countries are based on the growing demand for pork, which is fueled by tourists visiting the region and seeing pig production as a way to earn foreign cash by exporting it to countries where there is a growing need for pork. pointers to better future africa's population is expected to grow to around 2.5 billion people by 2050 [30], and they must be fed. this suggests that there is a greater need for food, including pig flesh. the necessity for modern or mechanized pig farming methods to boost productivity and production in response to the growing demand for pork has resulted from the rural-urban movement that has reduced the number of farmers. both government and commercial entities have recently boosted their investment. africa has the potential to become a global supplier of pork if it has adequate infrastructure, high-quality inputs, and sufficient agricultural land. after all, the united african company (uac) built the biggest pig farm in the world in 1943, and it was situated in kano, nigeria. in the 1950s and 1960s, the farm expanded to a very big size, and before being exported from the continent, the pig meat was rail-transported to lagos, which was then nigeria's political and commercial center. unfortunately, religious discrimination led to the pig farm's final closure in the late 1970s [13]. the demand for meat, especially pork, is rising due to variables such population expansion, urbanization, and incomes [31]. the need for greater investment in pig production is indicated by these rising demands. for instance, there is a sizable and expanding market for pork products due to the fast urbanization and population expansion that is occurring in east african cities and in nations like nigeria. opportunities to meet the growing urban demand for meat products, notably pork, are presented by this. for the advantage of both producers and consumers, coordinated efforts can ensure or guarantee the safe and legal commerce of pigs and pork products. changing the industry from the old practice of scavenging pigs to a more profitable manner is also necessary to increase pig production in africa to satisfy the demands of the growing population. this calls for the involvement of companies or actors who will offer farmers access to contemporary living facilities, better breeding stock, better feeding systems, and training on new pig production techniques like artificial insemination. the use of methods that are extensively utilized in africa is still low compared to europe and america because of a lack of infrastructure and expertise. opportunities for commercial feed production or input supply are also abundant in the feed business. commercial pig feed is rarely available to farmers, who are primarily ignorant of feed formulation. pigs are typically sold live by farmers to nearby slaughterhouses throughout the majority of africa, where the product receives little to no value addition. to realize the full potential of the business, significant investment is required in the processing and value addition of pigs in africa. private institutions and development partners' investments in pig production and processing have grown significantly in some african nations, including liberia and kenya. to spread best practices, disease control methods, and contemporary pig farming techniques to farmers, research and extension services must be funded [8,13]. abundance of agro-industrial by-product in africa pork and other livestock products are becoming more and more in demand as africa's population increases and becomes more urbanized. demand has increased as a result, placing strain on the supply and cost of traditional pig feed. african pig producers frequently purchase feed materials from local and international marketplaces. the availability and cost of imported feed ingredients, for instance, have been impacted by the disruption of global grain markets caused by the ukraine-russia conflict [32]. conflicts between farmers and herders, for example, have severely impacted the economy in nigeria and other west african nations by disrupting local agricultural production and lowering the supply of locally derived feed ingredients. pig farmers' finances have been stressed by the rise in feed costs brought on by interruptions in the global grain market. local disputes have also resulted in lower agricultural output, which has an impact on smallholder farmers' incomes and the availability of local feed ingredients. conflicts between farmers and herdsmen frequently result in the uprooting of farming 7 communities, when farmers are compelled to leave their lands or scale back agricultural operations because of insecurity [33]. due to interruptions in local and international markets, higher feed prices, and decreased agricultural production, the ukraine-russia conflict and farmer-herdsmen conflicts in nigeria have contributed to the scarcity of feed ingredients for pigs in africa. these crises demonstrate how regional and global forces interact to shape the difficulties african pig farmers experience in obtaining a sufficient and reasonably priced supply of feed components. to address these feed shortage issues, there is a chance to increase the usage of agro-industrial by-products as substitute pig feed sources. numerous by-products are produced by africa's agricultural industry from crops such as rice, oil palm, cassava, sorghum, corn, soybean, peanut, wheat, and sugarcane. these byproducts consist of materials that can be used as valuable pig feed ingredients, such as bagasse, rice bran, rice husk, soybean meal, corn bran, groundnut cake, palm kernel cake, brewer's grain, and wheat offal. since many of these agro-industrial by-products are high in fiber, protein, and energy, they can be fed to pigs [34]. on the other hand, their nutritional content can be increased with appropriate processing and supplementation. making use of these by-products helps to make agriculture more environmentally friendly and sustainable while also reducing waste. conclusion a common practice throughout africa, pig production has made significant contributions to the continent's agricultural economy, particularly as a source of income for many of its rural residents. pig production in africa still has the potential to become a major contributor to the continent's gdp and the achievement of the sustainable development goals on zero hunger and poverty eradication, despite the obstacles it faces. this is because pigs are a good source of animal protein that can help fill the protein gap in the rapidly expanding african human population. smallholder farmers must be given capacity building in the areas of appropriate feeding and feed management, health management, biosecurity, and value addition to boost productivity and the growth of the pig value chain throughout africa in order to unlock the continent's hidden pig production treasure. the expansion of pig production in africa depends on the implementation of suitable regulations that encourage the formation of private pig meat processing facilities or firms. it is crucial to do ongoing research on the creation of highly adaptable breeds, cost-effective feeding systems, and the utilization of alternative feed resources or agro-industrial byproducts. the expansion of the pig business in africa depends on the careful planning and implementation of disease surveillance, preventive, and control strategies. conflict of interest regarding the information covered in the manuscript, we attest that there is no conflict of interest with any financial institution. funding the authors received no financial support for this article. acknowledgments the authors would like to thank prof. jelili olaide saka for helping to read through a section of the article. references 1. faostat. crops and 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https://doi.org/10.20506/rst.25.2.1677 https://doi.org/10.3390/foods11142098 https://doi.org/10.3390/ani10010131 https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://doi.org/10.1111/tbed.13117 https://doi.org/10.1111/tbed.13117 introduction present pig production status in africa pig meat yield in africa per capita pork consumption in africa marketing challenges of pig production in africa common challenges climate challenges opportunities, prospects, and a catalyst for improved pig production in africa prospects and opportunities of pig production in africa pointers to better future abundance of agro-industrial by-product in africa conclusion conflict of interest funding references 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 11 (1), pp. 001-008, january, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper the response of pigs to diets containing varying levels of cocoa placenta meal (cpm) supplemented with an exogenous enzyme complex john dafaar damsere1*, michael boateng1, kwame owusu amoah2, yaw oppong frimpong3 and daniel boye okai1 1 department of animal science, faculty of agriculture, college of agriculture & natural resources, kwame nkrumah university of science and technology (knust), kumasi-ghana. 2 animal research institute, council for scientific and industrial research, accra, ghana. 3 department of animal production and health, school of agriculture and technology, university of energy and natural resources, sunyani, ghana. accepted 23 october, 2022 a nineteen-week experiment was conducted to establish the effects of an enzyme supplementation on growth performance, economics of production, carcass components and blood profile of pigs fed diets containing different levels of cocoa placenta meal (cpm). twenty-five large white grower pigs with mean initial live weight of 15.4 kg were randomly allocated to five treatments: t1 (0% cpm), t2 (5% cpm), t3 (10% cpm), t4 (15% cpm) and t5 (20% cpm) in a randomized complete block design (rcbd). diet t1 had no enzyme but diets t2, t3, t4 and t5 contained 35 g enzyme per 100 kg feed. each treatment had five pigs and each pig served as a replicate. feed and water were provided ad-libitum. pigs were slaughtered upon the attainment of a live weight of 70 ± 2.5 kg for carcass studies. blood samples were collected during slaughtering. feed cost (€ per kg) was inversely proportional to the inclusion level of the cpm + enzyme. pigs on the t1 and t2 diets utilized their feed more efficiently (p < 0.05) than those on the t3, t4 and t5. however, no differences (p > 0.05) were observed in the variations of the feed cost per kg gain values recorded. the cpm + enzyme inclusion resulted in decreased values (p < 0.05) for backfat thickness. there were no dietary (p ˃ 0.05) effects on the blood profile. dietary inclusion levels up to 20% cpm + enzyme can be fed to growing-finishing pigs without any detrimental effects on most of the growth performance and carcass criteria. key words: agro-industrial by-product, blood profile, carcass, growth performance. introduction much consideration has been drawn to the use of cheaper and less demanded alternatives such as agro-industrial byproducts and non-conventional feed resources (ncfrs) in the feeding of livestock (obirikorang et al., 2015) as a result of high cost of conventional feed ingredients. agro-industrial by-products such as dried brewers spent grains (dbsg), cocoa pod husk (cph), rice bran, and other ncfrs have been evaluated in *corresponding author. e-mail: damseredafaarja@yahoo.com. tel: +233 242817710. author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 ghana as potential feed ingredients for non-ruminant farm animals (atuahene et al., 2000; donkoh et al., 2013; nortey et al., 2015). moreover, some research done on these products have proved that their use in animals’ diets often reduce feed cost (okai, 1998). yet, there are other prospective by-products which have not been adequately studied. one of such is the cocoa placenta, a by-product of cocoa bean production. cocoa placenta is the slender, fibrous, rope-like tissue which holds the seeds (beans) in position inside the cocoa pod and also supplies nutrients to the cocoa seeds during the developmental stage of the cocoa fruit. the cocoa placenta accounts for about 3% of the cocoa fruit (atiemo, 2015), which on the average weighs 400 g and therefore a large quantity is produced during the fermentation process of the cocoa beans but are eventually removed and discarded haphazardly during sun drying of the fermented beans. atiemo (2015) reported that 30,966 metric tons of cocoa placenta is produced annually in ghana. this can be a nuisance as it invites a lot of houseflies, blocks drain and also pollutes water bodies via run off when scattered around the cocoa drying sites in the communities. one major challenge apart from the presence of theobromine in cocoa by-products is the high crude fibre content or non-starch polysaccharides (nsp). choct (2004) indicated that nsp are poorly digested by monogastric animals such as pigs because they do not produce enzymes that are capable of digesting these fibre components. according to bedford (2000), exogenous enzymes can be used to address the problems of some anti-nutritional factors and high fibre levels that limit feed value, thereby leading to a more economic and efficient utilization of aibp. this enzyme complex intended to increase the bioavailability of carbohydrates, proteins and fats in the diets of pigs and poultry. it has been suggested that it improves digestibility of feed ingredients and fcr. there is a dearth of information on its usefulness in monogastric diets in ghana. therefore, the objective of this study was to determine the growth performance, carcass traits and blood profile of grower-finisher pigs fed diets containing varying levels of cpm (0-20%) supplemented with an enzyme complex. materials and methods study location and duration of the experiment the study’s location was the livestock section of the department of animal science, kwame nkrumah university of science and technology (knust), kumasi, ghana. the feeding trial lasted for 19 weeks. source and processing of feed ingredients the wet cocoa placenta (wcp) were gathered from cocoa farmers inkukuom in the asunafo south district of the ahafo region and were sun dried on a raffia palm mat on a platform for 5-8 days, depending on the intensity of the sunshine and the humidity. the dried cocoa placenta (dcp) was ground in a hammer mill to produce cocoa placenta meal (cpm) whilst the other ingredients were obtained from rakeb company limited, kumasi. those that required grinding e.g. maize, were handled in the same way as the dcp proximate composition of cpm proximate analysis of the cpm was carried out using standard procedures outlined by the association of official analytical chemists (aoac, 2002). the nutrient compositions of other ingredients were obtained from the nrc (1998). experimental animals, diets and design twenty-five large white grower pigs (15 entire males and 10 gilts) with an overall mean initial live weight of 15.4 kg were selected and randomly allotted to five isonitrogenous (17.0% cp) dietary treatments (table 1) that is, 0% cpm, 5% cpm, 10% cpm, 15% cpm and 20% cpm replacing equal amounts of maize. adjustments of the wheat bran and soya bean meal levels were made to obtain the crude protein (cp) level desired and all diets contained the same level of fishmeal (5%). the allocations of the pigs were based on sex and live weight in a randomised complete block design. each treatment had three boars and two gilts and each pig represented a replicate. a kg of the exogenous enzyme complex containscellulase, 100,000,000 u; xylanase ds: 5,000,000 u; beta-glucanase: 70,000 u; amylase: 300,000 u; pectinase: 70,000 u; phytase: 1,450,000 iu; protease: 3,000,000 u; lipase: 10,000 u; arabinase, alpha galactosidase and hemicellulase. management of pigs prior to the commencement of the experiment, all the pigs were tagged and treated with tectin (ivermectin) inj. they were housed in a scrubbed and disinfected welded mesh, individual concretefloored cages (that is, 160×66×104 cm), constructed within an aluminium-roofed building. each cage was provided with a 43×12×10 cm concrete water trough. shallow feeding troughs measuring 46×23×13 cm were used during the first two weeks and they were replaced with deeper and heavier troughs measuring 54×24×27 cm (depth of 11cm at the feeding end) from the third week onwards. feed and water were provided without restriction throughout the study period. parameters measured growth performance and economics of production weekly feed intake and weight gain were measured and used to calculate the daily feed intake, daily weight gain and feed conversion ratio (fcr). total feed intake and weight gain were alsocalculated. cost per kg of each diet was computed by using the open market prices to estimate the cost of all ingredients used in the study. the cost of collecting, transporting and processing of cpm were estimated and added to the cost of the cpm diets. inaddition, the cost of the inclusion (¢/kg) of enzyme was added to the cpm diets. feed cost per kg gain for each diet was obtained by multiplying the cost per kg feed by the fcr. 3 table 1. composition (%) of the experimental diets. ingredients (%) 0% cpm 5% cpm + 10% cpm + 15% cpm + 20% cpm + cpm 0 5 10 15 20 maize 60 55 50 45 40 soya bean meal 15.5 13.8 12.1 10.5 8.7 wheat bran 18.5 20.2 21.9 23.5 25.3 fishmeal 5 5 5 5 5 dicalcium phosphate 0.25 0.25 0.25 0.25 0.25 vit-min. premix # 0.25 0.25 0.25 0.25 0.25 common salt 0.25 0.25 0.25 0.25 0.25 oyster shells 0.25 0.25 0.25 0.25 0.25 total 100 100 100 100 100 calculated composition (%) cp 17.0 17.0 17.1 17.0 17.0 cf 3.68 4.48 5.28 6.07 6.87 de (mj/kg) 15.9 15.5 15.1 14.7 14.3 calcium 0.51 0.52 0.54 0.57 0.56 phosphorus 0.74 0.73 0.87 0.92 0.98 + the enzyme was added at the rate of 35 g/100 kg to each of the cpm diets.#vit-min. premix per 100 kg diet: vitamin a (8×105u.i); vitamin d3 (1.5×104u.i); vitamin e (250 mg); vitamin k (100 mg); vitamin b2 (2×102 mg); vitamin b12 (0.5 mg); folic acid (50 mg); nicotinic acid (8x102 mg); calcium panthotenate (200 mg); choline (5×103 mg). trace elements: mg (5×103 mg); zn (4×103 mg); cu (4.5×102 mg); co (10 mg); i (100 mg); se (10 mg). antioxidants: butylatedhydroxytoluene (1×103 mg). carrier: calcium carbonate q.s.p (0.25 kg). carcass and internal organs measurement four animals (two males and two females) from each dietary treatment were slaughtered for carcass evaluation, upon attaining the targeted weight of 70 ± 2.5 kg after the weekly weighing. carcass parameters considered on the day of slaughter were; dressed weight, dressing percentage and weights of viscera, respiratory tract, full git, empty git, empty stomach, liver, spleen, heart, kidneys, trotters and head. after chilling the carcasses at 5°c overnight, the parameters measured were: chilled dressed weight, carcass length, meanback fat thickness, p2, loin eye area and weights of leaf fat, fillet, belly, loin, shoulder and thigh. an 8cmlength of the ileum, obtained between the caecum and the small intestine was taken for histological processing and microscopic observations for the villi count, height, width and villi area using the standard procedures outlined by baker and silverton (1976). haematological and serum biochemical studies two samples of blood were taken from each pig using heparinized vacutainer (venoject, lithium heparin, terumo europe, leuven, belgium) and sterilized micro tubes.the first sample from each pig was subsequentlyanalysed for haematological parameters whilst the serum obtained from the other sample was used for biochemical studies (tiezt,1995). in vitrodigestion an in vitro trial was conducted on the test ingredient (raw cpm) and the five diets (that is, 0% cpm, 5% cmp + , 10% cpm + , 15% cpm + and 20% cpm + ) to mimic the digestion process in the pig so as to ascertain the effect of the enzyme. parameters measured were sugar levels and the viscosities of the diets. statistical analysis all data collected were subjected to the analysis of variance procedure of the genstat statistical package version 11.1 (2009) and differences were deemed significant at p ˂ 0.05. ethical statement protocols used were in this study were approved by the animal ethics committee of kwame nkrumah university of science and technology, kumasi. results and discussion nutrient composition of the dried cocoa placenta (cpm) proximate composition of cpm (table 2) showed highervalues for most components than that reported byboatenget al. (2016) except the nfe and me values. boatenget al. (2016) reported nfe and me values of 63.17% and 3006.91 (kcal/kg) respectively on as-fed basis. torres-morenoet al. (2015) attributed variations of proximate values of cocoa beans and african locustbean pulp (albp) to varietal differences, geographicallocation, type of soils, maturity of fruit at harvest, method used in drying, processing and duration of storage period. the cocoa placenta used by boatenget al. (2016) was obtained from the plantations section of the department of crop and soil sciences, knust, kumasi, ghana. the cp value of the cpm obtained in this study is 4 table 2. proximate composition of the dried cocoa placenta meal (cpm). proximate composition (%) as-fed (%) dry matter (%) moisture 14.3 cp 16.0 18.6 cf 19.0 22.1 ee 3.02 3.52 ash 9.98 11.6 nfe 37.8 44.1 me (mj/kg) β 9.04 10.5 β metabolizable energy was calculated using pauzenga (1985) equation (that is, me = 37×%cp+81.8×%ee+35×%nfe) higher than the levels of cp in most of the conventional energy feed ingredients such as maize (8.3%) which is usually used in the diets of pigs in ghana. it is worth mentioning that, except cocoa bean meal (23.2% cp), the cpm (table 2) contained more cp than cocoa pod husk (8.4%) and cocoa bean shell (16.7%) in percentage dry matter terms (european food safety authority, 2008). the cf value of 19.0% for the cpm is higher than the cf values of most agro-industrial by-products (aibps) reported by rhule (2015) {that is, cassava peel (13.7 ± 0.23%), cocoa expeller cake (8.57 ± 0.22%), coconut chaff (13.8 ± 1.86%), copra cake (13.9 ± 0.65%), pineapple waste (14.7 ± 2.89%), pito mash (12.4 ± 2.84%) and brewer’s spent grains (16.1 ± 1.29%)}. therefore, the inclusion of the enzyme was to help degrade the high fibre in the cpm diets in order to release the nutrients that were bound in the fibre to the pigs. as a result of the fibrous nature of most aibp, several research studies using fibre-degrading exogenous enzymes have been undertaken in ghana (alemaworet al., 2009;nortey et al., 2015). growth performance of pigs there were no differences (p = 0.77) in the average daily feed intake among the different dietary treatments although the 20% cpm + diet recorded the least value. there was a trend of decreasing daily weight gain (p = 0.02) with increasing levels of the cpm. the 0% cpm and 20% cpm + diets recorded the highest and lowest average daily weight gain (adwg) value respectively. boateng et al. (2016) obtained a divergent result of the adwg (p ˃ 0.05) when rats were fed diets containing varying levels of cpm plus xzyme tm . the least adfi and adwg recorded by pigs on the 20% cpm + diet had an impact on the duration or number of days spent to reach the slaughter weight of 70±2.5kg because pigs on the 20% cpm + diet spent noticeably (p = 0.04) more days (116 days) compared to those on the 0% cpm (84 days) (table 3). contrarily, tengan et al. (2012) observed similar duration (p > 0.05) with varying levels of albp at the highest inclusion level (20%), where the pigs took 100 days compared with 105 days (no albp) to reach the target weight. the fcr values obtained implied that pigs on 0% cpm diet utilized their feed more efficiently (p = 0.001) than those on the 10, 15 and the 20% cpm + diets although the fcr was similar to the 5% cpm + diet. this study confirms the statement made by whittemore et al. (2003), that feed efficiency is directly affected by growth rate and feed intake and in all cases of feeding high cf diets, feed conversion efficiency decreased, with the decreases being more pronounced in young pigs. the feed cost decreased with increasing level of dietary cpm inclusion even with the addition of the enzyme. this implied that, the feed cost (gh₵ per kg) was inversely proportional to the inclusion level of the cpm + diets. however, there were no differences (p = 0.1) in the variations of the feed cost per kg gain values recorded among the dietary treatments and the variations did not follow any particular trend. carcass characteristics all the absolute and relative fat parameters studied (that is,backfat thickness, p2 measurement and leaf fat) recorded substantial differences (p < 0.05) between the control diet (0% cpm) and the cpm + diets except the mean back fat thickness which was similar (p > 0.05) withthe 5% cpm + diet. the cpm + inclusion in the diets resulted in decreased values of the fat parameters. for example, the p2 fat measurement was inversely proportional to the levels of cpm + in the diets. it may beinferred that pigs fed the cpm + diets converted their feed more into lean meat rather than fat deposits as a result oftheir high fibre levels but lower energy concentrations (table 1). amoah et al. (2017) observed a similar trend when they studied the performance of pigs at different phases of growth on sun-dried brewers spent grain (dbsg)-based diets. specifically, the diets with high and low metabolizable energy values (that is, 25% dbsg and30% dbsg) recorded lower values for fat parameters (that is, back fat thickness, leaf fat and p2 measurement). there were dietary influences (p ˂ 0.05) 5 table 3. growth performance of the experimental pigs. parameter (kg) 0% 5% 10% 15% 20% sem pvalue cpm cpm + cpm + cpm + cpm + initial weight 15.5 15.3 15.4 15.6 15.4 0.05 1.00 final weight 70.0 69.3 69.7 69.5 70.2 0.16 0.91 duration of trial, days 84.0 c 92.4 bc 107 ab 105 abc 116 a 5.66 0.04 daily feed intake 1.87 1.72 1.75 1.72 1.64 0.04 0.77 daily weight gain 0.65 a 0.60 ab 0.51 bc 0.52 bc 0.48 c 0.03 0.02 feed conversion ratio 2.87 bc 2.84 c 3.33 a 3.34 a 3.41 a 0.12 0.001 feed cost, € 0.26 0.24 0.23 0.22 0.20 0.01 feed cost/kg gain, € 0.74 0.70 0.77 0.75 0.68 0.02 0.10 a, b, cmeans on the same row bearing different superscripts are significantly different (p ˂ 0.05). table 4. absolute and relative carcass characteristics of the experimental pigs. parameter 0% 5% 10% 15% 20% sem p cpm cpm + cpm + cpm + cpm + value absolute (kg) warm carcass wt. 51.8 51.1 49.0 50.0 48.6 0.61 0.33 chilled carcass wt. 44.4 43.2 41.6 41.7 40.7 0.66 0.09 dressing percentage, % 73.9 73.8 70.9 71.8 69.6 0.83 0.15 loin eye area, cm 2 32.7 31.4 33.5 32.3 34.5 0.53 0.18 mean backfat thickness, cm 2.58 a 2.50 a 1.58 b 1.62 b 1.50 b 0.24 0.001 p2 measurement, cm 1.94 a 1.66 b 1.16 b 0.72 c 0.69 c 0.25 0.003 leaf fat 0.64 a 0.47 b 0.34 c 0.33 c 0.34 c 0.06 0.002 head 5.10 5.10 4.94 4.94 5.05 0.04 0.96 trotters 0.96 1.04 1.07 1.07 1.08 0.02 0.46 thigh 6.95 6.54 6.90 6.90 6.85 0.07 0.60 loin 5.80 5.66 5.69 5.69 5.84 0.04 1.00 fillet 0.41 0.41 0.41 0.41 0.40 0.002 1.00 viscera 12.1 b 12.2 b 12.3 b 12.3 b 14.6 a 0.48 0.02 full git 8.56 b 8.15 b 8.50 b 8.50 b 11.3 a 0.58 0.01 empty git 2.94 3.17 2.84 2.84 3.04 0.06 0.40 empty stomach 0.55 d 0.67 b 0.60 c 0.60 b 0.68 a 0.02 0.001 relative (%) leaf fat 0.91 a 0.67 b 0.49 c 0.47 c 0.49 c 0.08 0.002 head 7.27 7.37 7.16 7.09 7.23 0.05 0.96 trotters 1.36 1.50 1.55 1.53 1.55 0.04 0.39 thigh 9.91 9.45 10.0 9.93 9.80 0.10 0.59 loin 8.25 8.17 8.23 8.18 8.36 0.03 1.00 fillet 0.58 0.60 0.60 0.59 0.57 0.006 1.00 viscera 17.2 b 17.6 b 17.8 b 17.7 b 20.8 a 0.65 0.01 full git 12.2 b 11.8 b 12.3 b 12.2 b 16.1 a 0.80 0.01 empty git 4.19 4.57 4.12 4.09 4.36 0.09 0.38 empty stomach 0.78 c 0.96 a 0.87 b 0.87 b 0.97 a 0.03 0.001 a, b, c means on the same row bearing different superscripts are significantly different (p˂0.05). on the absolute and relative weights of viscera, full git and empty stomach of the experimental pigs. pigs on the 20% cpm + dietary treatment obtained the highest absolute and relative viscera, full git and empty stomach weights compared to the rest of the dietary treatments. according to jørgensenet al. (1996), pigs adapt to diets with increased fibre content by increasing gut volume and weight (table 4). 6 table 5. villi parameters of the experimental pigs. parameter 0% 5% cpm + 10% 15% 20% sem p-value cpm cpm + cpm + cpm + villi count, mm 2 1190 1476 1512 1602 1602 75.8 0.27 villi height, μm 1003 1237 1291 1362 1362 66.3 0.32 villi width, μm 187 239 221 239 239 10.1 0.76 villi area, μm 2 586287 948108 940540 1017699 1056279 83717 0.42 table 6. haematological and serum biochemical parameters of the experimental pigs. parameter 0% 5% 10% 15% 20% sem p-value cpm cpm + cpm + cpm + cpm + haematological assay* hct, % 42.9 46.3 43.8 49.4 45.7 1.13 0.67 hgb, g/dl 13.4 13.9 13.7 14.3 13.6 0.15 0.93 rbc, 10 6 /mi 6.98 8.01 7.30 7.73 7.68 0.18 0.72 lym, 10 3 /μi 12.8 12.6 12.9 10.9 11.9 0.37 0.95 mch, pg 19.2 17.6 18.7 18.6 17.8 0.30 0.25 mchc, g/dl 31.3 30.4 31.2 39.1 29.8 1.71 0.12 mcv, μm 3 61.4 57.9 60.0 63.9 59.7 1.00 0.25 mon, 10 3 /μi 2.90 0.51 0.43 0.33 0.60 0.49 0.58 neu, 10 3 /mi 5.72 9.41 11.6 15.5 8.85 1.62 0.22 plt, 10 3 /mi 234 278 203 247 301 17.1 0.73 wbc, 10 3 / m 19.7 24.3 25.8 27.6 22.4 1.37 0.43 bas, 10 3 /ml 0.24 0.23 0.33 0.19 0.13 0.03 0.24 serum biochemical assay total cholesterol, mmol/l 2.28 2.21 2.34 2.12 2.52 0.07 0.23 globulin, g/l 31.1 35.2 33.1 35.5 36.2 0.94 0.65 total bil, umol /l 2.90 2.90 2.90 2.58 2.86 0.06 0.69 total protein, g/dl 64.4 71.9 67.9 70.4 65.3 1.44 0.68 triglycerides, mmol/l 0.69 0.54 0.83 0.67 0.84 0.06 0.42 albumin, g/l 33.2 36.8 36.9 34.9 33.4 0.80 0.88 *hcthaematocrit, pcvpacked cell volume, hgbhaemoglobin, rbcred blood cell, lymlymphocytes, mchmean cell haemoglobin, mchcmean cell haemoglobin concentration, mcvmean cell volume, monmonocytes, neuneutrophils, pltplatelets, wbcwhite blood cell and basbasophils. the 20% cpm + diet had the highest crude fibre percentage (table 1) and that could have accounted for the highest weights of the viscera, full git and empty stomach. pigs that were fed a diet containing 30% distillers dried grains with solubles showed increased visceral organ mass relative to the control fed pigs (agyekumet al., 2012). some villi parameters montagne et al. (2003) had reported that, an increase in villi to crypt ratio leads to an increase in surface area for greater digestion and absorption of available nutrients to occur. however, the villi count, height, width and surface area measurements showed no dietary effects (p ˃ 0.05) among the dietary treatments studied (table 5). however, there were trends with the villi counts and heights; that is, as the inclusion rates of the cpm + increased the mean values increased. haematological and serum biochemical studies there were no dietary effects (p ˃ 0.05) on all the haematological and serum biochemical parameters (table 6) considered in the experiment. it is worth stating that, all the mean values obtained across the different dietary treatments fell within the physiological ranges for large white pigs raised in ghana as reported by okai et al. (1995). it can be deduced that the haematological and serum biochemical values obtained attest to the fact that 7 table 7. total sugars and viscosity of raw cpm and the dietary treatments. parameter raw cpm 0% 5% cpm + 10% 15% 20% sem p-value cpm cpm + cpm + cpm + sugar, mg/l 5450 f 13865 a 10546 b 9554 c 9248 d 9228 e 1103 0.001 viscosity, mm 2 /s 1.27 a 1.09 d 1.13 cd 1.13 cd 1.15 bc 1.17 b 0.03 0.001 a, b, c, d e fmeans on a row with different superscripts are significantly different (p ˂ 0.05). cpm plus enzyme-an exogenous enzyme complex, had no adverse effects on the physiology of the experimental pigs. in vitro digestibility dietary effects (p ˂ 0.05) were found in the mean values of the total sugar and viscosity measurements (table 7). the total sugar decreased clearly (p ˂ 0.05) as the inclusion level of cpm + increased. this trend could probably be attributed to the inability of the supplemented enzyme to satisfactorily degrade the cell structures of the cpm to release sugars (energy) bound in its cells (mcdonald et al., 2010). it has been reported that soluble fibre increases digest a viscosity and thereby slowdown the diffusion of the substrate and enzymes in the porcine small intestine, which hampers nutrient digestion and absorption (wenk, 2001). the highest viscosity values recorded by the 15 and 20% cpm + diets (table 7) could mean that the high crude fibre values recorded (table 1) may contain some soluble fibre components which led to increase in the viscosity values. it could also mean that there were important (p ˂ 0.05) enzymatic degradation on soluble fibres of the cpm + diets since the viscosity values seemed to have increased with an increasing in cpm inclusion. conclusions the growth rate and daily weight gain parameters of growing-finishing pigs were substantially lowered when cpm-based diets at 10-20% inclusion levels plus enzyme were fed. however, feed cost per kg body weight gain, haematological parameters and serum profiles were not affected by the inclusion of cpm + in the diets. all fat parameters considered were clearly lowered with the cpm-based diets containing 10-20%. in summary, the cpm + diets were not only cheaper but also resulted in leaner carcasses though such pigs took considerably longer to reach the market weight. conflict of interests the authors have not declared any conflict of interests. references agyekum ak, slominski ba, nyachoti cm (2012) organ weight, intestinal morphology, and fasting whole-body oxygen consumption in growing pigs fed diets containing distillers dried grains with solubles alone or in combination with a multi-enzyme supplement. journal of animal science 90(9):3032-3040. alemawor f, dzogbefia vp, oddoye eo and oldham jh (2009). enzyme cocktail for enhancing poultry utilisation of cocoa pod husk. scientific research and essay 4:555-559. amoah ko, asiedu p, wallace pa, bumbie gz, rhule swa (2017). the performance of pigs at different phases of growth on sun-dried brewers spent 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(1995). clinical guide to laboratory test, 3rd ed. (nw tietz; ed.). philadelphia: wb saunders 997p. torres-moreno m, torrescasana e, salas-salvadó j, blanch c (2015) nutritional composition and fatty acids profile in cocoa beans and chocolates with different geographical origin and processing conditions. food chemistry166:125-132. wenk c (2001). the role of dietary fibre in the digestive physiology of the pig. animal feed science and technology 90:21-33. whittemore ec, emanns gc, kyriazakis i (2003). the relationship between live weight and intake of bulky foods in pigs. animal science 76:89-100. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 6 (12), pp. 001-009, december, 2018. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper prevalence, haemolysis and antibiograms of campylobacters isolated from pigs from three farm settlements in venda region, limpopo province, south africa p. o. uaboi-egbenni1*, p. o. bessong2, a. samie3 and c. l. obi4 1 department of microbiology, university of venda, p.m.b. 5050, thohoyandou, limpopo province, south africa. 2 department of microbiology, university of venda, p.m.b. 5050, thohoyandou, limpopo province, south africa. 3 department of microbiology, university of venda, p.m.b. 5050, thohoyandou, limpopo province, south africa. 4 academic affairs and research, walter sisulu university, mthatha, eastern cape, south africa. accepted 14 may, 2018 we investigated the prevalence, haemolytic activities and antibiotic susceptibility profiles of campylobacter species isolated from pigs in three farms in the venda region, limpopo province, south africa. during the period of investigation, which spanned over one year, 450 faeces samples from pigs comprising 150 each from the three farms were collected and appropriately transported to the base laboratory at the department of microbiology, university of venda for analysis. in total, the prevalence rate of campylobacter isolates from all three farms was 30.2%, whereas, the rates of 25 26% were recorded for farms x, y and z, respectively. out of the isolated campylobacter species 10 (12.5%) were identified as campylobacter jejuni and 70(87.5%) as campylobacter coli. of the campylobacter isolates from the farms, 23, 22 and 35 were β-haemolytic from farms x, y and z, respectively. susceptibility of the isolates to 12 antibiotics was determined by disc diffusion technique. the overall rate of resistance determined in this study to most of the antibiotics was 84.2%. most campylobacter isolates from farm x (< 50%) were resistant to ciprofloxacin, cefexime, and erythromycin, while > 50% were resistant to tetracycline, (54.5%) to vancomycin, (100%) to ampicillin and methicillin. the rates of resistance to these antibiotics were not significantly different from farms x and y (p < 0.005). however, most isolates from farm y were more resistant to ciprofloxacin (57.1%) than isolates from farm x (36.4%) and z (40.7%). resistance rates to tetracycline by c. coli isolated from farm y (48%), farm z (45.5%) and farm x (44.4%) were noted. similar trend was observed for c. coli from farm y (40%), farm z (31.8%) and farm x (22.2%) for ciprofloxacin. all c. coli from farm z showed higher level of resistance to ampicillin (100%), y (64%) and x (55.6%). the c. jejuni isolates though few were markedly resistant to fluoroquinolone and the macrolide as well as aminoglycoside antibiotics. most of the isolates were resistant to at least seven (7) of the antibiotics tested. these observations indicate high levels of resistance to antibiotics commonly used in the swine industry (erythromycin and tetracycline) among the c. coli and c. jejuni isolates from pigs. erythromycin and ciprofloxacin are drugs for treatment of human campylobacteriosis. the high prevalence rate of resistance to these drugs among c. coli and c. jejuni isolates from pig faeces is of public health significance. key word: campylobacter, campylobacteriosis, haemolytic, susceptibility, resistance, profiles. introduction campylobacter, mainly campylobacter jejuni and *corresponding author. e-mail: uaboiegbenni@yahoo.com. tel: +27 15 962 8186. campylobacter coli, are recognized as major causes of acute bacterial food-borne gastroenteritis (world health organization, 2002). the incidence of human campylobacteriosis in the european union has increased over the past years and recently exceeded that of salmonella in many countries. although campylobacteriosis is usually a self-limiting diarrheal disease, severe complications such as septicemia, reactive arthritis, and guillain-barre syndrome sometimes occur (humphrey et al., 2007). c. jejuni colonize the intestinal tracts of a large number of mammals and birds. broilers are often carriers of c. jejuni. in thailand, campylobacter species were isolated from 12% of various food samples including pork, chicken and vegetables in bangkok (rasrinual et al., 1988; young, 2005), 40% poultry ceca in india (das et al., 1996), and 68-100% of poultry samples from retail markets in taiwan (shih, 2000). reports on campylobacter in pigs revealed higher proportions of c. coli than c. jejuni, whether they have enteritis or not (harvey et al., 1999; steinhauserova et al., 2001). in the u.s., campylobacter species were isolated from 76% gilts, 100% of pregnant sows, 57.8% of newborn piglets, and 100% of weaning pigs (young et al., 2000). in the netherlands, fifty-percent of piglets were infected with the same serotypes at seven days of age (weijtens et al., 1997). the average number of campylobacter colonizing the gut decreased toward the end of the rearing period (weijtens et al., 1999). a study in belgium reported the prevalence of campylobacter sp. on pig carcasses at slaughterhouses to be 2% (korsak et al., 1998), and campylobacter was found in 1.3% of samples from pork from a retail market in u.s. (duffy et al., 2001). although the poultry industry has often be considered primarily responsible for human campylobacteriosis, the pork industry has equally been identified as a potential source of human infection (allos, 2001; berndtson et al., 1996). uaboi-egbenni (2008) also documented the prevalence of campylobacters in dogs and guinea-fowl in nigeria. the gastrointestinal tracts of other food animal species have also been shown to be frequently colonized with campylobacters, particularly, c. jejuni and c. coli (minihan et al., 2004). reported rates of campylobacter carriage in food animals have varied widely between studies (busato et al., 1999). the high prevalence of campylobacters in pigs has been reported previously in numerous studies and dressed pig carcasses have been shown to be more frequently contaminated than either beef or sheep (nesbakken et al., 2003). this is most likely attributable to the fact that pig carcasses undergo a communal scalding process combined with the fact that the skin remains on the carcass following all of the dressing procedures (moore et al., 2005). thermotolerant campylobacters (c. jejuni/c. coli) constitute the most frequent cause of intestinal infections worldwide. the clinical spectrum of campylobacter enteritis ranges from a watery, nonbloody, noninflammatory diarrhea to a severe inflammatory diarrhoea with abdominal pain and fever. disease is less severe in developing countries than in developed countries. the disease is characterized by bloody stool, fever, and abdominal pain that is often more severe than that observed in shigella and salmonella infections. in developing countries the features reported are watery stool, fever, abdominal pain, vomiting, dehydration, asthenia, anorexia and presence of faecal leukocyte; patients are also often underweight and malnourished (rao et al., 2001; coker et al., 2002). stools remain positive for several weeks. treatment appears to be beneficial if it is administered early enough in the course of the disease (salazar-lindo et al., 1986). the recommended drugs are erythromycin, or amoxicillin or a fluoroquinolone (ciprofloxacin, norfloxacin) or tetracycline, provided the bacterium has not acquired a resistance. however, the emergence of antibiotic resistant strains has further opened a new dimension as to how to combat the disease together with the emerging resistant strains. in 1988, the emergence of fluoroquinolone resistance in campylobacter strains was first reported in spain, and since then the emergence of fluoroquinolone resistance has been identified in many countries, including finland, the netherlands, england, and canada (berndston et al., 1996), but not in south africa. in spain, 100% of campylobacter strains isolated from broilers and pigs were found to be resistant to fluoroquinolones (berndtson et al., 1996). there is a dearth of information and research on the prevalence, epidemiology and antibiograms of campylobacters in pigs in south africa in spite of the reports in europe and other parts of the world on pigs as potential sources of infections for humans. this study therefore ascertained the prevalence, antibiotic susceptibility profiles and haemolytic activities of campylobacters isolated from pigs in three farms in the venda region, limpopo province of south africa in order to provide updated information and data on the research subject. materials and methods collection of faeces a total of four hundred and fifty (450) freshly voided faeces comprising one hundred and fifty from each of the three farms were collected at random from pigs in sterile 50 ml plastic containers and were adequately kept in coolers packed with crushed ice, stored at 4°c and immediately transported to the laboratory for analysis. the faeces were immediately processed. about 2 gm of the samples was transferred to 6 ml of sterile phosphate-buffered saline and left to emulsify at room temperature for 10 20 min to release the bacteria. the suspension and bacteria were used directly for the identification of campylobacter. the three (3) sampling sites are separated from each other by about 200 km but are located in the same province. the three farms were code named x, y, and z for ethical reasons. isolation and identification of campylobacter sp. by conventional culture methods ten microlitres of the faecal suspension was spread on the surface of a charcoal cefoperazone deoxycholate agar plates (cm 739 [oxoid] with cefoperazone supplement sr 155e). the plates were incubated under microaerophilic conditions employing the campygen gas generating kit (oxoid cm025) at 42°c for 48 h. colonies suspected to be campylobacter were further purified on blood agar plates (blood agar base no.2 (oxoid) supplemented with 5% sterile laked horse blood). all the isolates were characterized by their catalase, oxidase reactions, hydrogen sulphide production, indoxyl acetate, hippurate and susceptibility to nalidixic acid by standard procedures (on and holmes, 1991, 1992). the resulting isolates were subsequently stored at -80°c in brain heart infusion broth with 15% glycerol until further investigation. confirmation of presumptive positive campylobacter strains presumptive identification of campylobacter isolates was done using the dryspot campylobacter test kit (oxoid basingstoke, hampshire england). the test is specific for pathogenic campylobacter strains belonging to c. jejuni, c. coli, campylobacter upsaliensis, and campylobacter lari. however, the other campylobacter species are not included in this identification scheme. the manufacturer’s instructions were strictly adhered to in this procedure. briefly, 48 h cultures of suspected campylobacter showing the usual cultural characteristics were selected. the reagents were removed from the refrigerator and allowed to attain room temperature according to the manufacturer’s instructions. the extraction tubes were placed in test tube rack. thereafter, one drop of extraction reagent 1 was added into the extraction tube. sufficient growth of the suspected campylobacter culture was removed to fill the inner diameter of a sterile loop. these cells were thoroughly suspended in a drop of reagent 1. the loop was allowed to stay in this reagent for 3 min. two drops of extraction reagent 2 was added to the previous mixture and mixed thoroughly using the retained loop. with the aid of a paddle pastette provided with the kit, 1 drop (50 µl) of the neutralized extract was placed onto the test circle and a drop onto the control circle. with the aid of the flat end of the paddle pastette, the extract was mixed into the dry control reagent spot until completely suspended to cover the reaction area. the same pastette was used to repeat this procedure for the test reagent. the test cards were rocked for about 3 min. this process was repeated for all suspected campylobacter isolates. agglutination under normal lighting condition indicated that the organism was campylobacter. the presumptive positive campylobacters were further subjected to mast diagnostic campylobacter kits consisting of urease, indoxyl acetate and hippurate test solution. briefly, 24 h cultures of the campylobacter were inoculated into the urease and hippurate test solutions according to the manufacturer’s instructions. these were then incubated for 4 h for colour development. for urease, development of pink colour was indicative of urease enzyme production (c. lari), while development of pink colour in hippurate solution indicated production of hippuricase enzyme (c. jejuni). in the case of indoxyl acetate solution, change of colour from colourless to blue/purple was indicative of the presence of c. jejuni or c. coli. the indoxyl acetate strips were impregnated with wet cultures and allowed to stay for 3 5 min. development of purple colour within this period was indicative of positive reaction (c. jejuni and c. coli) (popovic-uroic et al., 1990; on and holmes, 1992; elviss et al., 2008). blood haemolysis test to ascertain pathogenicity of isolates, the identified campylobacter sp. were subjected to haemolytic test according to the procedure of samie et al. (2007). briefly, a 24 h broth culture of campylobacter sp. were cultured by spread plate method on columbia agar supplemented with defibrinated sheep red blood cells. plates were incubated at 37 ± 2°c for 24 h. thereafter, plates were observed for complete, partial and no haemolysis. antimicrobial agents the antibiotics tested in this study were: trimethroprim (2.5 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), tetracycline (30 µg), ampicillin (10 µg), erythromycin (15 µg), streptomycin (10 µg), methicillin (µg), cefexime (30 µg), imipenem (µg), kanamycin (30 µg) and vancomycin (30 µg) (oxoid, unipath ltd, basingstoke, england). antimicrobial sensitivity testing the method of gaudreau and gilbert (1997) was used. briefly, the confirmed campylobacter isolates were inoculated into plates of mueller-hinton agar supplemented with 5% sheep red blood cells in plates carrying a maximum of five (5) antimicrobial discs. all plates were incubated at 35°c under a microaerophilic atmosphere obtained with a gas generator envelope (oxoid, unipath ltd, basingstoke, england) for 24 h. the resulting zone diameters were measured with a graduated metre rule. preparation of bacterial genome genomic dna was obtained by the whole-cell lysate method as described by marshall et al. (1999). briefly, cells from a 24 48 h culture grown on columbia blood agar were resuspended in sterile distilled water to an optical density of 2.5 at 540 nm. the suspensions were boiled 100°c for 20 min in eppendorf tube. the resulting templates were either used immediately for polymerase chain reaction (pcr) or were kept at 4°c for up to 1 month. pcr identification of campylobacter strains in order to ascertain if the dryspot positive campylobacter isolates were genuinely campylobacters, they were subjected to pcr identification using the general primers for the identification of campylobacter. these primers are also specific for other members of the campylobacteriaceae (helicobacter and arcobacter). however, arcobacter and helicobacter sp. show negative reaction to the campylobacter dryspot kit. hence, any amplification of the primer sequences at the 1,004-bp fragment within the coding region of 16s rrna confirmed such isolates as campylobacter sp. and not helicobacter or arcobacter sp. the pcr-restriction fragment length polymorphism (rflp) method used in this study was as previously described by marshall et al. (1999). briefly, amplification was done in 50-µl reaction volume containing 5 µl of whole-cell lysate, 1 µl each primer, 10x buffer (roche), 1.5 mm mgcl2, 200 µm each deoxynucleotide (roche) and 5u taq dna polymerase (roche). the pcr amplification was performed with a – thermocycler (esco swift mini thermal cycler version 1.0, esco technologies, philadelphia u.sa). the samples were subjected to an initial denaturation for 2 min at 95°c, followed by 30 amplification cycles, each consisting of 94°c for 30 s, 52°c for 30 s, and 72°c for 90 s. a final primer extension at 72°c for 10 min was included. oligonucleotides primers employed in this study were cah16s 1a (5’ – aat aca tca aag tcg aac ga – 3’) and cah16s 1b (tta acc caa cat ctg acg ac – 3’), respectively. the oligonucleotides used in this study were synthesized by roche applied science (manheim, germany). results cultural studies a total of four hundred and fifty (450) freshly voided c a m p y lo b a c te r is o la te s p e r fa rm n u m b e r o f 90 76.9 80 70 60 55.3 50 50 farm x farm y 40 30 farm z 30 21 19 20 12.8 10 2 3 5 0 0 0 5.3 7.9 0 0 0 0 c. jejuni c. coli c. lari % c. jejuni % c. coli % c. lari campylobacters isolated from pig faeces in farms x, y and z fig.1 : shows the number of c. jejuni , c. coli, c. lari and their percentages isolated from three pig farms in south africa 100 89.7 90 80 70 60.5 60 57.9 farm x 42.1 50 38 38 39 39 farm y 40 farm z 30 23 22 16 26.3 13.2 20 10.3 10 4 5 10 0 0 0 0 0 n o o f c am py lo ba ct er is o la te sp er fa rm n o of -β ha em ol yt ic n o of αh ae m ol yt ic % h a e m o ly ti c β % h a e m o ly t ic α n o no nha em ol yt ic % no nha em ol yt ic fig. 2: results of haemolysis of sheep red blood cells by campylobacter isolates faecal samples were processed for the detection of campylobacters over the course of the study. of the 138 presumptive isolates from pigs on mccda from the farms, 25.6% (115 out of 450) were confirmed as campylobacters. figures 1 and 2 show the percentage incidence of campylobacter strains from pig faeces in the three (3) farms. from, there was an equal percentage of occurrence of campylobacters in farms x and y with a value of 25.3%. however, there was a higher incidence of campylobacters among pigs in farm z (26%) than in farms x and y. it must be noted here that some strains that could not be resuscitated did not form part of the analysis and subsequent studies. with the aid of the campylobacter dryspot and mast diagnostic kits, 2(5.3%) of the isolates from farm x were identified as c. jejuni, while 21 (55.3%) were identified as c coli. in farm y, 3 (7.9%) were c. jejuni, while 19 (50%) were c. coli. in farm z, 5 (12.3%) were c. jejuni, while 30 (76.9%) were c. coli. overall, the prevalence of c. jejuni among pigs in the three farms was 10 (12.5%), while it was 70 (87.5%) for c. coli (figure 2). from this finding, there was a preponderance of c. coli compared with c. jejuni in all p e rc e n ta g e s u s c e p ti b il it y p ro fi le 100% 0 0 31.8 45. 5 50 50 90% 100 100 10 0 100 10 0 10 0 10 0 10 0 80% 68.2 54. 5 50 50 70% 0 4 0 0 0 0 0 0 farm z r farm z s 60% 40 48 48 64 64 52 40 60 72 farm y r 50% 96 10 0 10 0 40% 60 52 52 48 60 40 farm y s 36 36 28 farm x r 0 0 30% 22.2 44. 4 33.3 11.1 11.1 55.6 77.8 55. 6 farm x s 20% 88.9 10 0 10 0 88. 9 10 0 10 0 77.8 66.7 10% 55. 5 44.4 44. 4 0% 0 22.2 0 0 cip te cfm ipm va amp cn k met e w na antimidrobials to which c.coli isolates were exposed fig. 3: percentage susceptibility profile of campylobacter coli exposed to 12 antibiotics (key: s = susceptibility; r = resistance; cip= ciprofloxacin; te= tetracycline; cfm = cefexime; ipm = imipenem; va= vancomycin; amp = ampicillin; cn= gentamycin; k= kanamycin; met= methicillin; e=erythromycin; w= trimethoprim; na= nalidixic acid). farms. blood haemolysis of the 115 campylobacter strains from the pigs tested for sheep blood haemolysis, 23 were β-haemolytic, 10 were α-haemolytic and 5 non-haemolytic in farm x, 22 were βhaemolytic, 16 were α-haemolytic for farm y, 35 were βhaemolytic and 4 were α-haemolytic for farm z (figure 3). antimicrobial resistance sixty-six isolates were tested for their antibiotic susceptibility profiles. most campylobacter isolates from farm x (< 50%) were resistant to ciprofloxacin, cefexime, and erythromycin, while > 50% were resistant to tetra-cycline (54.5%), vancomycin (100%), ampicillin (54.5%), kanamycin (63.6%), and methicillin (100%). in farm y, ≥ 50% were resistant to ciprofloxacin (57.1%), tetracycline (50%), cefexime (53.6%), vancomycin (67.9%), gentamycin (53.6%), methicillin (75%), and erythromycin (53.6%), except imipenem and kanamycin which were 10.7 and 46.4% resistant, respectively. in farm z, > 50% but < 100% were resistant to tetracycline, gentamycin and erythromycin, while 40.7% were resistant to ciprofloxacin. approximately 100% of campylobacter species in this farm were resistant to cefexime, vancomycin, kanamycin, and methicillin. resistance to most of the antibiotics tested was more pronounced among isolates from farm z. majority of the isolates from farm y were more resistant to ciprofloxacin (57.1%) than isolates from farm x (36.4%) and farm z (40.7%) (figure 4 and 5). there were no significant differences in resistance profiles amongst campylobacter isolates in the farms (p < 0.005). study of individual species resistance showed that in farm x, all c. jejuni isolates were resistant to ciprofloxacin, tetracycline, cefexime, imipenem, vancomycin, gentamycin, kanamycin and methicillin. of the three c. jejuni isolates from farm y, ≤ 100% were resistant to all the antimicrobials except tetracycline (33.3%) and nalidixic acid in which all were susceptible. in farm z, ≤ 100% were resistant to 10 of the antimicrobials except ciprofloxacin to which 80% were resistant and nalidixic acid to which 100% were susceptible. for c. coli, in farm x < 50% were resistant to ciprofloxacin, tetracycline, cefexime, imipenem and erythromycin except vancomycin, ampicillin, gentamycin, kanamycin, methicillin and trimethoprim to which ≤ 100% were resistant. in farm y, < 50% of c. coli were resistant to ciprofloxacin, tetracycline, cefexime, imipenem and kanamycin, while ≤ 100% were resistant to vancomycin, ampicillin, gentamycin, methicillin, erythromycin and trimethoprim. in farm z, ≤ 50% were resistant to ciprofloxacin, tetracycline, gentamycin and erythromycin, while all c. coli isolates (100%) were resistant to cefexime, vancomycin, ampicillin, kanamycin, methicillin and trimethoprim. analysis of the zone diameters was done using clsi/nccls (2002) for enterobacteriaceae. the pcr micrographs of the dna from campylobacter strains from sheep are as indicated in. the purified dna from the campylobacter strains amplified at the 1004 bp, which is the specific region for the conserved 16s rrna for members of the genus campylobacter. the bands formed were confirmed as those for campylobacter. specific identification by the mast diagnostic kits 100% 0 20 90% 80 100 100 100 100 100 100 100 100 100 100 80% 80 farm z r 70% 20 0 0 0 0 0 0 0 0 0 farm z s 60% 66.7 33.3 66.7 66.7 66.7 farm y r 50% 100 100 100 100 100 100 100 66.7 farm y s 40% 33.3 33.3 33.3 33.3 0 0 0 0 0 0 0 farm x r 30% 50 50 20% 100 100 100 100 100 100 100 100 100 100 farm x s 10% 50 50 0% 0 0 0 0 0 0 0 0 0 0 cip te cfm ipm va amp cn k met e w na antimicrobials to which campylobacter jejuni were exposed fig.4: percentage susceptibility profile of campylobacter coli exposed to 12 antibiotics (key: s = susceptibility; r = resistance; cip= ciprofloxacin; te= tetracycline; cfm = cefexime; ipm = imipenem; va= vancomycin; amp = ampicillin; cn= gentamycin; k= kanamycin; met= methicillin; e=erythromycin; w= trimethoprim; na= nalidixic acid). 1004bp 1004bp fig.5: pcr products of amplified dna from pig campylobacter isolates aligning at the 1004bp of a 1.9kb ladder (a) pig and (b). (a) lane 1= 1.9kb ladder; lane 2,3,4,5,6,7, amplified bands of dna from pig campylobacter strains; (b) lane 1= 1.9kb dna ladder; lanes 2,3,4,5,6,7 are amplified bands of dna from pig (farm y) campylobacter strains. differentiated the isolates into c. jejuni and c. coli. discussion elucidating the shedding patterns and prevalence of campylobacter strains in the faeces of farm animals is a prerequisite for effective healthcare strategy against zoonotic infections. in the current study, the prevalence of campylobacters from pigs and their antimicrobial susceptibility pattern was determined. of the 450 fresh faecal samples processed for all farms, 84.6% were identified as campylobacters. there was an equal prevalence (25.3%) among pigs’ faeces in farm x and y, but a slightly higher predominance in faeces from farm z. in an epidemiological study carried out on 95 pigs originating from eight farms, weijtens et al. (1993) found 11% of the stomach samples of pigs to be positive for campylobacter, whereas, 85% of faeces were positive. a prevalence of campylobacters in pigs ranging from 63 100% has been reported in other studies (weijtens et al., 1993; saenz et al., 2000; young et al., 2000; pezzotti et al., 2003; payot et al., 2004). in our findings, none of the farms was free from campylobacter. there were no significant differences in the prevalence of campylobacter sp. in the three farms (x, y, z) with the average prevalence value at 25.3, 25.3 and 26% respectively (p < 0.005). weijtens et al. (1993, 1999) reported high counts of shedding of campylobacter genotypes in faeces of pigs. in terms of prevalence, c. coli were more predominant than c. jejuni in all farms. farm z had a higher incidence of c. jejuni and c. coli than the rest farms. this report is in line with previous reports which revealed that swine were predominantly contaminated by c. coli, whereas, c. jejuni were more frequently isolated from poultry (aarestrup et al., 1997; weijens et al., 1999; van looveren et al., 2001; pezzotti et al., 2003). the reason for this discrepancy in the distribution pattern of c. jejuni and c. coli in swine has not been elucidated. however, it is not implausible that host adaptation or differences in rearing practices may be significant factors. quite a large population of campylobacter isolates from pig faeces where β-haemolytic on 5% sheep redblood cells. this observation is in line with the report of samie et al. (2007). this is a confirmation that the campylobacter strains could be pathogens of medical significance. the predominance of c. jejuni in human infections has led to the disregard of the impact that less prevalence species like c. coli can play in human infections. nevertheless, a recent survey by tam et al. (2003) showed that the morbidity resulting from c. coli has actually been underestimated. the pcr micrographs of the dna from campylobacter strains from all animals were as shown in figure 1. the purified dna from the campylobacter strains amplified at the 1004 bp, which is the specific region for the conserved 16s rrna for members of the genus campylobacter. the bands formed are in line with those of marshall et al. (1999). after exposure to twelve (12) antibiotics, several strains showed multiple resistance to most of the antibiotics used. the resistance patterns displayed by campylobacter isolates from all farms to fluoroquinolone (ciprofloxacin) and macrolides (erythromycin) classified as second line and first line antimicrobials are of particular importance, since patients suffering from campylobacteriosis are usually treated with these antimicrobials agents. in the study, 22.2, 40 and 31.1% of c. coli were resistant to ciprofloxacin, while 11.1, 60 and 50% of c. coli were resistant to erythromycin in farm x, y and z, respectively. for ampicillin, 55.6, 40 and 100% of c. coli were resistant to this antimicrobial in farm x, y and z, respectively. norma et al. (2007) in their study in canada observed a high incidence of c. coli resistant to ciprofloxacin and erythromycin. the resistance of c. coli to ampicillin as exemplified in this study was higher than those reported from france and denmark (20 and 17%), respectively but lower than that reported from spain (65.9%) for farm x, y but not for farm z (100%). resistance to β-lactam antimicrobials in pathogenic bacteria develops through bacterial conjugation. this resistance-transfer mechanism is very crucial because it permits genetic exchange of information between species of bacteria (davis and conner, 1994; efsa, 2007; norma et al., 2007). in south africa, data on anti-microbial susceptibility of campylobacter isolated from pigs are scanty. due to high prevalence of resistance shown by campylobacter sp. to β-lactam antimicrobials (ampicillin) it is no longer recommended for use in farms (davis and conner, 1994). in this study, we observed resistance frequently to antimicrobials most commonly used in swine industry, tetracycline and erythromycin. different resistance patterns were observed for one aminoglycoside antibiotic for all isolates tested for c. coli. 77.8, 52 and 50% were resistant to gentamycin in farms x, y and z, respectively. however, the report of norma et al. (2007) in canada showed a low level of resistance (0.2%) of campylobacter coli to gentamycin. the recommendation of gentamycin as an alternative therapeutic antimicrobial against human campylobacteriosis by fernandez et al. (1994) in chile should be considered with caution judging from the high prevalence of resistance to gentamycin observed in this study. sato et al. (2004) and inglis et al. (2005) observed high levels of resistance to tetracycline (45%), which is in line with our observation of ≥ 50%. saenz et al. (2000) in their study reported no erythromycin resistance among c. jejuni isolates from broilers and high levels of resistance c. coli isolates from pigs (81.1%) compared to those from humans (34.5%). however, in our study, one c. jejuni isolate out of two was resistant to erythromycin in farm x; all three c. jejuni isolates in farm y were resistant to erythromycin, while one of the five c. jejuni isolate in farm z was resistant to erythromycin. in addition, more resistance was associated with c. coli than with c. jejuni. a study in quebec found that 61% of c. coli isolated from pigs but none of c. coli isolates from chickens were resistant to erythromycin (guevrement et al., 2006). resistance of campylobacter species to antibiotics may suggest the widespread use of antibiotics as growth promoters in farm animals (piddock et al., 2003; pezzotti et al., 2003; moore et al., 2001). resistance profiles to tetracycline and fluoroquinolones were very high in our study and this is consistent with other reports (saenz et al., 2000; norma et al., 2007). this may have stemmed from the fact that these antibiotics have been used in the swine industry for several years. all the campylobacter isolates were susceptible to nalidixic acid in this study, so that cross-resistance between nalidixic acid and ciprofloxacin was not found in all the quinolone susceptible campylobacter strains. this finding is however in contrast to the observation of saenz et al. (2000) who found cross-resistance between ciprofloxacin and nalidixic acid resistant strains in spain. moore et al. (2005) mentioned the use of mobile genetic elements in campylobacters as mechanism for the extrusion of antibiotics out of the bacterial cell. considering the feeding habits of pigs, it is plausible that they can ingest commensals carrying resistant genes from the feeds, facilitating transfer to pathogenic bacteria in the lumen of farm animals. such pathogenic acquisition of resistant genes from exogenous source can result in global prevalence of resistant pathogens. angulo et al. (2004) and blake et al. 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ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 8 (3), pp. 001-009, march, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper posterior paresis in pregnant gilts experimentally infected with trypanosoma brucei lushaikyaa allam1, david ogwu2, rowland ibrahim shehu agbede3 and anthony kojo bedu sackey2 1 veterinary teaching hospital, ahmadu bello university, zaria, kaduna, nigeria. 2 department of veterinary surgery and medicine, ahmadu bello university, zaria, kaduna, nigeria. 3 department of veterinary parasitology and entomology, ahmadu bello university, zaria, kaduna, nigeria. accepted 14 october, 2019 the effect of trypanosoma brucei infection on reproductive efficiency in gilts (n=12) was conducted. the gilts were bought as piglets aged eight weeks from piggeries in samaru village in zaria, nigeria. on attaining puberty, the gilts were divided into experimental and control groups, each group containing six animals. all the gilts were subsequently bred by fertile boars and they were confirmed pregnant. the pregnant gilts were then inoculated with about 1.8 × 10 6 trypanosomes via the anterior vena cava. the infected gilts developed clinical trypanosomosis after a pre-patent period of 2 to 3 days. the clinical signs observed were intermittent fever, short and moist cough, moist rales, mucopurulent ocular discharges, hyperaemia of the skin, reduced feed intake, loss of body condition, recumbency, uncoordinated movements, posterior paresis, loss of pregnancy and death. severe degeneration of the fibers of the hamstring muscles was observed along with elevated levels of serum potassium, aspartate amino transferase and creatine kinase. key words: trypanosomosis, infection, pregnant gilts. introduction the physiology of animals alters when they are infected with trypanosomosis (biryomumaisho et al., 2003). this is usually due to the wide range of biochemical changes that take place in the infected animals (katunguka– rwakishaya, 1996). hematological aberrations do occur in animals infected with trypanosomes (anosa and isoun, 1980; singla and juyal, 2000). the severity of the hematological and biochemical changes that take place in infected animals is determined by the strain of the infecting trypanosome and the host (anosa, 1983a, b). the evaluation of blood indices and parameters is required to enable the health status of animals to be determined (coles, 1986). this gives an indication of the *corresponding author. e-mail: doclu64@gmail.com. tel: +234803-701-2731. degree of degenerative changes that have occurred to host tissues as well as the severity of the infection (otesile et al., 1991). pigs like other domestic livestock are infected with several species of trypanosomes. the trypanosomes that cause infections in pigs are trypanosoma simiae, trypanosoma brucei trypanosoma congolense and trypanosoma suis (losos, 1986; sekoni, 1994; seifert 1996). infections in pigs by t. brucei have been reported to cause nervous signs (otesile, 1992; onah and ozuokwu 1991), uncoordinated movements and posterior paresis characterized by wobbling of the hind-legs (mumah, 1996; allam, 2004). the posterior paresis interfered with mating efficiency of gilts (allam et al., 2006). this paper intends to report the cause of the posterior paresis that was noticed in pregnant gilts infected with t. brucei along with other pathogenic effects of this parasitic infection. materials and methods experimental animals twelve cross breed piglets aged eight weeks were bought from piggeries in samaru village in sabon gari zaria local government area of kaduna state, nigeria. they were housed in clean fly proof pens in the faculty of veterinary medicine, ahmadu bello university zaria, nigeria. base line hematological data of the piglets were obtained on arrival and they were screened for endo and ecto parasites. the piglets were subsequently ear notched for identification and treated for nematodes and ectoparasites with ivermectin (ivomec®), at 200 µg/kg body weight sub-cutaneously. the piglets were fed compounded diet of 18% crude protein (composition: maize 36.8%, soya bean 5%, ground nuts 23.5%, rice bran 30%, beniseed 2%, bone meal 2%, premix 0.2%; table salt 0.5%) and water was provided ad libitum. strain of t. brucei used the t. brucei used in this present study was obtained from the nigerian institute of trypanosomiasis research vom, nigeria. it was originally isolated from a pure natural infection in cattle in federe, kaduna state nigeria. the parasite was inoculated into 2 mice and transported to the faculty of veterinary medicine, ahmadu bello university zaria, nigeria. inoculation of animals when the gilts were 7 months old, they were randomly divided into two groups of 6 experimental and 6 controls. about 2 ml of the infected rat blood containing approximately 1.8 × 10 6 t. brucei organisms were inoculated into each animal in the experimental group via the anterior vena cava while the ones in the control group were left intact. following inoculation, all the gilts were clinically examined, weighed and their blood samples collected daily using edta as anticoagulant. the blood was examined for levels of parasitaemia, packed cell volume (pcv) and serum biochemical parameters. the values obtained were recorded daily along with their weights and rectal temperatures. this continued until all the infected animals were positive for trypanosomes. subsequently, all the gilts were clinically examined weighed and their blood collected weekly to determine pcv levels, parasitaemia, and serum biochemical parameters until the experiment was terminated. gross pathological examination post mortem examination was carried out on the infected gilts that died during the study and also on the remaining ones that were sacrificed at the end of the experiment. histo-pathological examination the samples of the heart, brain and hamstring muscles were obtained from all the infected pigs. they were put into appropriately labeled bijou bottles and fixed in 10% formalin. the tissues were dehydrated and processed in tissue wax embedded in paraffin and were subsequently cut in to 5 µm thickness on slides and stained with hematoxyline and eosin. the slides were eventually examined using a light microscope. statistical tests used data obtained from the study were analyzed using student t test. values of p<0.05 were considered to be statistically significant (chatfield, 1983). results clinical observations all the pigs developed clinical trypanosomosis after a pre-patent period of 2 to 3 days. there was a steady increase in the levels of parasitaemia of the infected gilts which was followed by fluctuations. peak parasitemia was recorded between days 7 and 35 post infection (pi) (figure 1). the rectal temperatures of all the infected gilts increased during the course of the infection. this was also followed by fluctuations. the highest mean temperature attained by the infected gilts was 40.3ºc. during this period, the temperatures of the animals in the control group were within the normal range. the difference in the ranges of temperature between infected and the control animals was significant (p<0.05) (figure 2). there was a gradual decrease in the pcv of the infected gilts. this was first noticed on day 14 pi in one of the pigs. the lowest pcv value recorded during the experiment was 17%, the mean pcv values of the infected animals were significantly different from those of the control (p<0.05) (figure 3). the infected animals from day 28 pi were observed to be gaining less weight than the ones in the control group. the infected animals were gaining an average of 0.71 kg every two weeks whereas the controls were gaining an average of 1.5 kg during the same period. by day 42 pi, the infected animals started losing weight while the control gained weight steadily (figure 4). the difference in the weights between the infected and the control groups was found to be statistically significant (p<0.05). other clinical signs observed in the infected gilts were pale mucus membranes, short and moist cough, moist rales, mucopurulent ocular discharges, hyperemia of the skin, reduced feed intake, recumbency, uncoordinated movements, posterior paresis, loss of pregnancy and death. during the study, the mean aspartate amino transferase (ast) levels of the infected pigs increased significantly from day 35 pi and its level remained elevated until the end of the study. the highest mean level of 60.6 i.u/l was recorded on day 91 pi (figure 5). the mean serum levels of creatine kinase (ck) increased progressively from day 21 pi till the end of the experiment in the infected animals. the highest mean level recorded for this enzyme was 856.7 i.u/l on day 42 pi. the values of ck were significantly different from those of the control animals (p < 0.05) (figure 6). the mean serum values of potassium in the infected gilts increased progressively during the study. this m e a n p a ra s it e m ia ( + ) days pre and post infection figure 1. mean parasitemia of t. brucei infected gilts. t e m p e ra tu re v a lu e s ( ºc ) days pre and post infection figure 2. mean temperature changes of t. brucei infected and control gilts. days pre and post infection figure 3. mean packed cell volume of t. brucei infected and control gilts. b o d y w e ig h t ( k g s ) days pre and post infection figure 4. mean body weight changes of t. brucei infected and control gilts. a s p a rt a te a m in o t ra n s fe ra s e l e v e l (i . u .) days pre and post infection figure 5. mean aspartate amino transferase levels of control and t.brucei infected gilts. figure 6. mean creatine kinase levels of control and t. brucei infected gilts. p o ta s s iu m l e v e ls ( m m o l/ l ) days pre and post infection figure 7. mean potassium levels of control and t. brucei infected gilts. increase started on day 14 pi. the highest mean level recorded was 8.6 mmole/ l on day 63 pi. these levels were significantly different from those of the control animals (p < 0.05) (figure 7). histopathological lesions severe inflammatory lesions were observed in the heart of the infected gilts (figure 8). in some cases, necrosis of the myocardium with massive mononuclear cellular infiltration was observed. the blood vessels in the cerebrum were congested (figure 9). the fibers of the hamstrings muscle were degenerated. the perimysial tissue of the muscles had widened and had become edematous (figure 10). discussion the increase in ast levels noticed in this study agrees with the results obtained during an infection in sheep by t. brucei (taiwo et al., 2003), t. vivax infection of cattle and sheep (gray, 1963), t. congolense infection of goats (adah et al., 1992), and also in dogs infected with t. brucei (omotainse et al., 1994). however, it contradicts observations were made by taiwo et al. (2003) during an infected of sheep with t. congolense. the increase in the levels of ck agrees with results obtained in a t. cruzi infection in mice (cano et al., 2000), but disagrees with results obtained by (lunkins, 1992; chaudhary and iqbal 2000), who observed no change in ck values in animals with trypanosomosis. the causes of elevation of ast levels in the serum of animals are necrosis of the liver, skeletal muscles and kidneys, whereas ck is increased in skeletal muscle disease, myocardial injury or necrosis and cerebral cortical necrosis (lording and friend, 1991). conclusion in this study, the increase in ast and ck levels in the infected gilts could only have been due to the degeneration of skeletal muscle fibers that was observed histopathologically. even though histopathological lesions were also observed in the heart and brain of the infected animals, they were not that severe as to cause the increase in the levels of ast and ck as was noticed in this experiment. the hyperkalemia that was noticed is suggestive of massive leakages of this electrolyte from the skeletal days pre and post infection m m figure 8. photomicrograph of the heart of a t. brucei infected gilt. note the mononuclear cellular infiltration (m). h and e x 303. figure 9. photomicrograph of the brain of a gilt infected with t. brucei. note the congestion of the cerebral blood vessels (arrows). h and e x 303. f p figure 10. photomicrograph of the transverse section of the hamstrings muscle of a gilt infected with t. brucei. note the degeneration of muscle fibres in the fascicule (f) with widened and edematous perimysial tissue (p). h and e x147. muscles that were damaged during this infection. the low levels of this electrolyte in the skeletal muscles could have caused the decrease in neuromuscular excitability of the muscles. the pathology in the hampstring muscles could have caused the posterior paresis and the wobbling that was observed in the infected gilts. however, whether this abnormality could be reversed in treated cases or how the trypanosomes could have caused this problem was not within the scope of this study. further studies in these areas are therefore recommended. references adah mj, otesile eb and joshua ra (1992). changes in level of transaminases in goats experimentally infected with t. congolense. rev d’ elev. me’d vet. pays trop., 45 (3-4): 284-286. allam l (2004). the effect of trypanosoma brucei infection on reproduction in female pigs. msc thesis, ahmadu bello university zaria. allam l, ogwu d, agbede ris and sackey akb (2006). clinical and mating problems in gilts experimentally infected with trypanosoma brucei. j. trop. bios., 6: 28-31. anosa v0 (1983b). mammalian blood cells in health and in trypanosomiasis. trop. vet., 1: l77199. anosa v0 (1983a). diseases produced by trypanosoma vivax in ruminants, horses and rodents. zent fur vet b, 30 : 717-741. anosa vo, isoun tt (1980). haematological studies on trypanosoma vivax infection of goats 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36-39. onah dn, uzoukwu m (1991). porcine cerebral trypanosoma brucei brucei trypanosomiasis. trop. anim. health. prod., 23 (1): 39-44. otesile eb, akpavie so, fagbemi bo, ogunremi ao (1991). pathogenicity of trypanosoma brucei brucei in experimentally infected pigs. rev d’ elev. me’d vet. pays trop., 44 (3): 279-282. otesile eb, fagbemi bo, makinde mo, akinboade oa (1992). the response of pigs experimentally infected with t. brucei to isomethamedium® chloride therapy in relation to nutrition. vet-quart, 14 (3): 88-91. seifert shh (1996). vector born disease: trypanosomosis. in: tropical animal health. edited by kluver academic publishers, 152-169. sekoni vo (1994). reproductive disorders caused by animal trypanosomosis. a review therio. 42: 557-570. singla ld, juyal pd (2000). blood cellular responses in trypanosoma evansi infected and levamisole treated bovine calves. j. vet. parasitol., 14: 137-139, 2000 taiwo vo, olaniyi mo, ogunsanmi ao (2003). comparative plasma biochemical changes and susceptibility of erythrocytes to in vitro peroxidation during experimental trypanosoma congolense and trypanosoma brucei infections in sheep. isrl j. vet. med., 58 (4): 110. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-010, january, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper salmonella diversity and antimicrobial resistance in burkina faso: an investigation of strains from various sources kagambèga a.1, 2, 3*, bouda s. c.2, bako e.2, cissé h.2, barro n.2 and haukka k.1, 4 1 bacteriology unit, department of infectious disease surveillance and control, national institute for health and welfare (thl), helsinki, finland. *corresponding author. e-mail: kagamas2007@yahoo.fr; kagambega.asseta@gmail.com. 2 laboratoire de biologie moléculaire, d’épidémiologie et de surveillance des bactéries et virus transmissibles par les aliments (labesta)/centre de recherche en sciences biologiques, alimentaires et nutritionnelles (crsban)/ecole doctorale sciences et technologies (edst)/université ouaga i professeur joseph ki-zerbo, 03 bp 7021 ouagadougou 03, burkina faso. 3 institut des sciences (ids), 01 bp 1757 ouagadougou 01, burkina faso. 4 department of food and environmental sciences, division of microbiology and biotechnology p. o. box 56, fi-00014 university of helsinki, finland. accepted 25 september, 2023 epidemiologic and traceback evidence of salmonella infection is not much in many developing countries including burkina faso. this study investigates the antimicrobial resistance and distribution of salmonella serotypes isolated from diverse sources in burkina faso. 615 salmonella serotypes isolated from beef meat, poultry carcasses, poultry, swine, cattle, hedgehog, fish, salad, channel and humans from 2009 to 2011 were analyzed to identify their diversity and distribution among the samples. the salmonella strains were subjected to antimicrobial sensitivity tests using disk diffusion methods, were analyzed and classified into 110 serotypes, with the most prevalent serotype being derby (91/615) found in beef meat, poultry carcasses, poultry and fish; muenster (48/615) found in cattle, swine, hedgehog, poultry, human and fish; chester (38/615) found in poultry carcasses, swine and poultry feces; hato (32/615) found in beef meat, poultry carcasses, cattle and poultry; drac (30/615) found in cattle, hedgehog and fish; and typhimurium (21/615) found in cattle, poultry, human and fish. among the 615 salmonella strains, 94% (581/615) were resistant to one or more antibiotics; resistance to streptomycin was the most common. the resistance pattern, str-sul-tet, str-tet and str-sul was dominant and found in 80% of the strains. about 3% of the strains were resistant to 5 or 6 antibiotics; their resistance pattern is amp-str-sul-tet-tmp or amp-chl-str-sul-tet-tmp. one salmonella strain, s. kentucky isolated from human stool was resistant to eight antibiotics; the resistance pattern is amp-strsul-tet-cip-gen-nal-mec. findings from this study can help define the guidelines for basic surveillance system of salmonella and other enteropathogenic bacteria circulating among humans, animals, food and environment. key words: salmonella, environment, human, risk. introduction salmonella spp. especially non-typhoid salmonella (nts) is a common source of foodborne diseases that cause morbidity and mortality worldwide (smith et al., 2016). it is estimated that salmonella spp. cause 93.8 million cases of gastroenteritis and 155,000 deaths each year worldwide. approximately 86% of these cases are 2 the result of foodborne infections (majowicz et al., 2010). in burkina faso, bacteriological results showed that the rate of salmonella contamination remains quite high (simporé et al., 2009). in fact, the infection progresses mostly periodically and rarely in an epidemic mode, and they are not reported due to lack of food borne pathogens surveillance system. salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is a great concern in public health. however, information on the diversity and occurrence of salmonella strains is very scarce (cui et al., 2008), making the ecology of these species remains unknown. food animals, including poultry, pigs, and cattle are the key reservoirs for human salmonellosis (hauser et al., 2011). in developing countries, wild and food animals are the sources of salmonella distribution in water, vegetables, salad and the products derived from these animals due to lack of hygiene (kagambèga et al., 2013). the emergence of antimicrobial resistant salmonella is mostly associated with the non-therapeutic use of various classes of antimicrobials in large quantities in food animals (marshall and levy, 2011; mir et al., 2015). researchers have reported a link between the use of antimicrobials in food animals and the emergence of antimicrobial resistance in pathogenic bacteria (ungemach et al., 2006; mir et al., 2015). the increasing number of multidrug-resistant nts strains is a global concern; this has made some countries and international organizations create surveillance systems which include collaboration between human health, veterinary, and food related sectors to monitor the spread of foodborne bacteria. unfortunately, these surveillance systems are missing in many developing countries like burkina faso. the absence of controlled reporting of salmonella serotypes through the who-gfn program in burkina faso inspired the compilation of data on the sporadic reporting of salmonella serotypes isolated from various sources. therefore, the purpose of the present study is to provide information on the diversity and antimicrobial resistance of salmonella strains isolated from environment, animals, food and humans. these data will allow one to follow the trends in salmonella enterica serotypes that provide information about sources of infection and the efficacy of prevention and control measures. materials and methods salmonella strains (n= 615) isolated from meat, poultry carcasses, poultry, cattle, pigs, hedgehog, water, salad and humans were collected in burkina faso from 2009 to 2011. sampling was done based on the microbiological conditions. the strains were isolated using standard bacteriological methods and serotyped according to kauffman white scheme (kagambega et al., 2013; bonkoungou et al., 2013; traoré et al., 2015). antimicrobial susceptibility of the isolates was tested by a standard disk diffusion method, and escherichia coli rhe 6715 (atcc 25922) was used for validating the antimicrobial test results (clsi, 2015). the antimicrobial agents used were ampicillin (10 µg), chloramphenicol (30 µg), streptomycin (10 µg), sulphonamides (3 µg), trimethoprim (5 µg), tetracycline (30 µg), gentamicin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), cefotaxime (30 µg), mecillinam (10 µg) and imipenem (10 µg). minimal inhibitory concentration (mic) for ciprofloxacin (0.002 to 32 μg/ml) was determined by e-test (ab biodisk, solna sweden) of the isolates resistant to nalidixic acid. mic breakpoint ≤ 1 μg/ml was interpreted as susceptible (clsi, 2009). ethics approval and consent to participate permission to conduct this study was obtained from the slaughterhouse authorities and the study protocol was approved by the ethical committee of burkina faso. results and discussion a total of 615 strains of salmonella isolated from meat, poultry carcasses, poultry, cattle, pigs, hedgehog, water, salad and humans were distributed into 110 different serotypes (table 1). the six most prevalent serotypes were derby, muenster, chester, drac, hato and typhimurium. salmonella derby (91/615) was found in beef meat (n=2), poultry carcasses (n= 34), poultry (n= 52) and fish (n = 3). our findings show that s. derby is the most often isolated serotypes in poultry. this is in contrast with results reported by some authors, showing that this serotype is most common in pig and also the 4 most frequently isolated serotype in humans in europe (kerouanton et al., 2013; hauser et al., 2011). salmonella muenster was the second most common serotype and was found in cattle, swine, hedgehog, poultry, human and fish. this finding shows that this serotype is not restricted to one host but can be found in animals, foods, water or humans. a documented food poisoning outbreak caused by s. muenster occurred in canada in 1982; it infected cheddar cheese made from unpasteurized milk (wood et al., 1984). salmonella chester was found in poultry carcasses, swine and poultry feces; this serotype has been reported in many countries: in canada, s. chester was responsible for an outbreak associated to frozen meals in 2010 (taylor et al., 2012); in 2014, six european countries (belgium, france, spain, germany, sweden and the uk) reported s. chester cases to the european centre for disease prevention and control (ecdc) associated with travel in morocco (whitworth, 2016). salmonella hato was found 3 table 1. salmonella serotypes and antimicrobials profile. salmonella beef poultry cattle swine hedgehog poultry human salad fish resrvoirs channel total resistance number of antibiotic number of resistant serotypes meat carcasses pattern resistered serotype s.abaetetuba 0 0 1 0 0 1 0 0 0 0 0 2 str 1 1 s.abony 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 s.adabraka 0 0 0 0 0 0 0 0 0 1 0 1 str 1 1 s.adelaide 0 1 0 0 0 1 0 0 0 0 0 2 0 0 0 s.agona 0 4 0 0 0 3 0 0 3 0 0 10 str 1 5 s.ahmadi 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.albany 0 0 2 0 0 2 0 0 0 0 0 4 str 1 2 s.anatum 0 1 0 0 0 1 0 0 0 0 0 2 str 1 2 s.angers 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.ank 0 0 0 0 4 1 0 0 0 0 0 5 str 1 5 s.antwepen 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.apeyeme 0 0 2 0 0 3 0 0 0 0 0 5 str 1 5 s.banana 0 3 1 0 1 2 1 0 0 0 0 8 str 1 5 s.bareilly 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.bargny 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.binningen 0 0 0 0 0 2 0 0 0 0 0 2 0 0 0 s.bochum 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.brancaster 0 1 1 0 0 3 0 0 0 0 0 5 str 1 4 s.bredeney 1 0 5 0 0 2 0 1 6 0 1 16 str 1 4 s.brive 0 0 1 0 0 0 0 0 1 0 0 2 str 1 1 s.carmel 0 0 1 0 0 0 0 0 0 1 0 2 0 0 0 s.carno 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.chandans 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 s.chester 0 5 1 0 0 31 0 0 0 0 1 38 str 1 37 s.chomedey 0 0 4 0 0 0 0 0 0 0 0 4 str 1 4 s.colindale 0 0 1 0 0 0 0 4 0 1 2 8 str 1 2 s.colobane 0 0 2 0 0 0 0 0 0 0 0 2 str 1 2 s.cubana 0 0 0 0 0 0 5 0 1 0 0 6 str 1 2 s.dahra 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 s.dakar 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 str,str-sul,str s.derby 2 34 0 0 0 52 0 0 3 0 0 91 tet,str-sul 5 57 tet,chl-str,str cip s.drac 0 0 26 0 1 0 0 0 3 0 0 30 str 1 20 s.dublin 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 4 table 1. contd. s.duisburg 0 0 0 0 0 1 1 0 0 0 0 2 str 1 2 s.ealing 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.eastbourne 0 1 2 0 0 2 0 0 1 0 3 9 str 1 5 s.eastglam 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.elisabethville 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.farakan 0 0 3 0 0 0 0 0 0 0 0 3 str 1 2 s.freetown 0 0 0 0 0 1 0 0 0 0 0 1 str 1 1 s.fresno 0 0 1 0 0 4 1 0 1 0 0 7 str 1 3 s.frintrop 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.fufu 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.galiema 0 1 0 0 0 2 0 0 0 0 1 4 str 1 4 s.gaminara 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.gerland 0 0 0 0 0 0 0 1 0 0 0 1 str 1 1 s.give 0 0 0 0 0 0 0 0 1 0 1 2 0 0 0 s.gokul 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 str, tet,str s.hato 1 4 5 0 0 22 0 0 0 0 0 32 tet,sul-tet,amp 5 15 str, amp-str sul-tet-tmp s.havana 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 s.hermannswerder 0 0 0 0 0 0 0 0 1 0 0 1 str-sul 2 1 s.hillingdon 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.hvittingfoss 0 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.ikeja 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.ilala 0 0 2 1 0 0 0 0 0 0 0 3 str 1 1 s.kaapstad 0 0 0 1 0 4 0 0 0 0 0 5 str 1 2 s.kalamu 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.kalina 0 0 2 0 0 0 0 0 0 0 0 2 str 1 1 amp-str-sul-tet s.kentucky 0 0 0 0 0 0 1 0 1 0 0 2 cip-gen-nal8 1 mec s.kiambu 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.kingston 0 0 2 0 0 3 0 0 0 0 0 5 str 1 2 s.kokomlemle 0 0 2 0 0 1 0 0 1 0 0 4 str 1 2 s.korlebu 0 0 2 0 0 0 0 4 1 0 0 7 str 1 5 s.lagos 0 0 4 0 0 2 0 0 0 0 0 6 str 1 4 s.llandoff 0 0 0 0 0 0 0 0 1 0 2 3 0 0 0 s.mbandaka 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 5 table 1. contd. s.minnesota 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.moero 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.monschaui 2 3 1 0 3 1 0 0 0 0 0 10 str 1 7 s.montevideo 0 0 0 0 0 0 1 0 1 0 0 2 0 0 0 str, str-sul, str s.muenster 0 0 17 3 11 6 4 0 6 0 0 47 tet, str, str-nal, 5 20 amp-str s.nima 0 1 3 0 0 0 0 0 0 0 0 4 0 0 0 s.nottingham 0 1 2 0 0 1 0 0 2 0 0 6 str-tet 2 1 s.offa 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.oranienburg 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.othmarschen 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.ouagadougou 0 0 0 0 0 0 0 0 0 0 1 1 str 1 1 s.ouakam 0 0 0 1 0 0 2 0 0 0 0 3 str 1 2 s.poona 0 0 2 0 0 1 3 0 2 1 1 10 str 1 5 s.rissen 0 0 1 0 0 0 0 0 0 0 1 2 0 0 0 s.rubislaw 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.ruiru 0 0 8 0 0 0 0 0 0 0 0 8 str,str-tet 2 5 str, str-tet, s.group b 0 1 1 0 0 0 2 0 1 0 0 5 amp-chl--str6 4 sul-tmp s.group c 0 1 1 0 0 9 0 0 0 0 0 11 str, str-sul 2 11 s.group¤ d 0 0 0 0 0 1 0 0 0 0 0 1 str 1 1 s.group¤ e 0 0 1 0 0 5 0 0 0 0 0 6 str, str-sul-tet 3 3 s.group¤ g 0 0 0 0 1 0 0 0 0 0 0 1 str 1 1 s.group¤ m 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.group¤ o:53 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 s.saarbruecken 0 1 0 0 0 0 0 0 0 0 0 1 str 1 1 s.saintpaul 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 s.salford 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 s.schwarzengrund 0 0 1 0 0 3 0 0 1 1 0 6 str 1 3 s.senftenberg 1 2 0 0 2 8 0 0 0 0 3 16 str, str-tet, str 3 6 sul-tet s.shangani 0 0 0 0 0 1 0 0 0 0 0 1 str-sul 2 1 s.shubra 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 s.soerenga 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.soumbedioune 0 0 4 0 0 0 0 0 0 0 0 4 str 1 3 s.stanley 0 0 0 0 0 0 1 0 0 0 0 1 str 1 2 6 table 1. contd. s.stanleyville 0 0 0 0 0 1 0 0 0 0 0 1 str-tet 2 1 s.tamberma 0 0 0 0 0 0 1 0 0 0 0 1 str-tet 2 1 s.tennessee 0 0 3 0 0 0 0 0 0 0 0 3 str 1 1 s.teshie 0 0 0 0 0 0 0 0 0 0 1 1 str 1 1 s.tilene 3 2 1 0 0 0 0 0 1 0 1 8 str 1 7 s.tounouma 0 0 0 0 0 0 1 0 0 0 0 1 str 1 1 s.trachau 0 0 1 0 0 1 0 0 0 0 0 2 str 1 2 s.typhi 0 1 0 0 0 0 1 0 0 0 0 2 str,amp-chl-str 6 2 sul-tet-tmp str,amp-chl-str s.typhimurium 0 0 3 0 0 4 13 0 1 0 0 21 sul-tet-tmp, 6 21 amp-chl-str-sul tmp s.umbilo 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 s.urbana 0 0 13 2 0 1 0 0 0 0 0 16 str, str-tet, chl 4 11 str-tmp s.vilvoorde 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 s.virchow 0 0 1 0 0 0 2 0 0 0 1 4 str 1 1 s.waedenswil 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 s.wagadugu 0 0 0 0 0 0 0 0 0 1 1 2 str 1 2 s.waycross 0 0 2 0 0 1 0 0 3 0 0 6 str 1 2 s.yoruba 0 0 1 0 0 0 0 0 0 0 0 1 str 1 1 salmonella sp. 0 0 1 0 0 0 0 0 0 0 2 3 str 1 2 total 10 68 161 8 23 193 48 10 56 6 25 608 359 amp, ampicillin; chl, chloramphenicol; str, streptomycin; sul, sulphonamides; tmp, trimethoprim; tet, tetracycline; nal, nalidixic acid; cip, ciprofloxacin; ftx, cefotaxime; mec, mecillinam. in beef meat, poultry carcasses, cattle and poultry; salmonella drac was found in cattle, hedgeghog and fish. salmonella typhimurium was found in cattle, poultry, human and fish. s. typhimurium has been implicated in many outbreaks worldwide (rayamajhi et al., 2008). this serotype has a wellcharacterized ability to infect various species (rabsch et al., 2002) and can survive for a long time in the environment (baudart et al., 2000); these 2 factors enhance the ability of this serotype to be one of the most common causes of salmonellosis in many geographically diverse regions and has caused many disease outbreaks (mather et al., 2013; cui et al., 2008; galanis et al., 2006). although serotype typhimurium was associated with several foods of animal origin, the most common was chicken (26% of outbreaks), indicating that chicken is a major route of exposure. this corroborates with our present results, where poultry is identified as the primary source of s. typhimurium. in contrast to the report of foley et al. (2008), typhimurium was the most common serotype among pork associated with outbreaks. this shows that serotype typhimurium has emerged as the predominant serotype in swine. after the six most prevalent serotypes, the presence of serotype like typhi (poultry carcasses and human), senftenberg, virchow, kentucky, stanley and kingston which have caused many outbreaks in diverse regions were noted. the presence of s. typhi in poultry carcasses shows that there is a cross-contamination during 7 carcasses handling since humans are the only reservoir of typhoid salmonella. this is also due to the fact that humans can be a chronic carrier of these salmonella (eng et al., 2015). carriers of s. typhi and s. paratyphi are responsible for the spreading of enteric fever in endemic regions, as the common transmission route is the ingestion of water or food, contaminated with the feces of chronic carriers (bhan et al., 2005). in this report, salmonella serotypes drac, banana, monschaui, muenster, seftenberg are found in wild hedgehogs. this animal has been the source of human salmonellosis in many europeans countries, used as a pet animal (nauerby et al., 2000). although little can be done to prevent the spread of salmonella in the environment amongst wild animals, the control should be done in food handling and drinking water. our results show that the same serotype of salmonella is found in diverse sources, meaning that this serotype can be transmitted to humans through many sources. in general, food animals such as swine, poultry and cattle are the prime sources of salmonella infections. the major dissemination routes of the pathogens involve trade in animals and uncooked animal food products. the slaughtering process of food animals at abattoirs is considered one of the important sources of organ and carcass contamination with salmonella (gillespie et al., 2005). in burkina faso, all serotypes of salmonella can circulate between animals, foods, fish, water and humans. this happens as animals shed feces in the environment which rain water carries to rivers, barrages and channel; aquatic animals like fish can be contaminated by diverse microorganisms coming from water. in addition, wastewater and/or untreated animal feces are used to grow salad and other vegetable from gardens. here, we can see how humans can be contaminated in this closed circle, since animals can contaminate meat, water, fish, vegetables and environment; the consumers are exposed to a high risk of contamination by salmonella and other pathogens. these findings are critical because burkina faso is facing serious problems in the area of water and sanitation due to demographic explosion and poor urban planning. this problem is increasing the risk of environmental and human contamination. in ouagadougou, the capital city of burkina faso, wastewater from channel, where we found many salmonella strains, is used to irrigate vegetables from gardens, and animals’ feces are also used in vegetable crops, which is a new source of growth for poor people. vegetables like salad are not nutritive media for microorganisms but can constitute mechanical vectors for transporting microorganisms to humans if hygienic condition in garden is not maintained well. based on our findings, as seen in figure 1, the salmonella transmission routes are based on their diversity and sources. the diversity of possible reservoirs of infection results in significant challenges for public health authorities to control the infections (dione et al., 2011). among the 615 strains, 94% (581/615) were resistant to one or more antibiotics, and resistance to streptomycin was the most common (table 1). the resistance pattern str-sul-tet, str-tet and str-sul was dominant and found in 80% of the strains. about 3% of the strains were resistant to 5 or 6 antibiotics; their resistance pattern was amp-str-sul-tet-tmp or amp-chl-strsul-tet-tmp. one salmonella strain, s. kentucky isolated from human stool was resistant to eight antibiotics with the pattern, amp-str-sul-tet-cip-gen-nal-mec. this serotype was found also in fish in this study. in contrast, centre for disease prevention and control (cdc) has reported that s. kentucky is typically found in cattle and poultry (cdc, 2011). in this study, streptomycin resistance was found in 94% of the salmonella serotypes. majority of aminoglycosides are bactericidal but salmonella use mechanisms to resist their antibiotic families such as expression of plasmidmediated aminoglycoside modifying enzymes against aminoglycoside (gebreyes and altier, 2002). the genes encoding theses enzymes have been found in varieties of salmonella subtypes like: agona, typhimurium, newport, typhimurium var. copenhagen, kentucky, blockely, bredeney, anatum, derby, give, enteritidis, heidelberg, saint paul, london, saintpaul, hadar, heidelberg, and 4,5,12:i: (levings et al., 2005). in this study, resistance to ampicillin was observed in salmonella serotypes: hato, kentucky, muenster, typhi and typhimurium. betalactams are generally considered bactericidal; but salmonella strains are now becoming resistant to ampicillin and methicillin due to their wide clinical use (angulo et al., 2000). in salmonella, the secretion of a beta-lactamase is the common mechanism of resistance to beta-lactamases. several authors reported betalactamases in a variety of salmonella serotypes including enteritidis, dublin, haadrt, anatum, muenchen, stanley, panama, virchow, and typhimurium (gebreyes and thakur, 2005; batchelor et al., 2005). in the present study, salmonella serotypes derby, hato, muenster, nottingham, ruiru, senftenberg, stanleyville, tamberna, typhi, urbana and typhimurium were resistant to tetracycline. resistance to tetracycline has been reported in several serotypes including typhimurium, saintpaul, enteritidis, hadar and choleraesuis, agona, anatum, blockley, bredeney, colorado, derby, give, haardt, heidelberg, infantis, orion, seftenberg, (frech and schwarz, 2000; pezzella et al., 2004). salmonella serotypes derby, hato, hermannswerder, kentucky, muenster, senftenberg, shangani, typhi and typhimurium were resistant to sulfonamide in the present study. many authors have been reported resistance to sulfonamide in a wide range of salmonella serotypes such as enteritidis, hadar, heidelberg, orion, rissen, agona, albany, derby, djugu, and typhimurium (antunes et al., 2005; doublet et al., 8 pets and wild animal water (rivers, barrage, channel, well, tap water) environment (wind, food animals insects, rodents, grass, birds) poultry vegetables, fish and other aquatic animals foods (meat and meat products, eggs, food staff and other food types) human figure 1.transmissions routes of salmonella. red color = more implicated group in pathogens transmission; orange color: the second more implicated group. 2004). in our report salmonella serotypes hato, urbana, typhi and typhimurium were resistant to trimethoprim. martinez et al. (2005) reported trimethoprim resistance in salmonella serotypes agona, djugu, hadar, neport, rissen albany, derby, and typhimirium. chloramphenicol resistance was found in salmonella serotypes, derby, typhi, typhimurium and urbana. this finding corroborates with the report of alcaine et al. (2005), where salmonella typhi, agona, derby, kiambo, albany, newport, haardy, enteritidis and typhimurium isolates have been found to harbor resistant genes for chloramphenicol. there are two mechanisms in which salmonella resistance to chloramphenicol is conferred: (i) by the plasmid-mediated enzymes called chloramphenicol acetyltransferases (cat) or nonenzymatic chloramphenicol resistance gene cm1a and (ii) efflux pump in which the antibiotic is pumped out of the cell. in this study, quinolone resistance was observed in salmonella serotypes derby, kentucky, muenster and typhimurium. the mechanisms of quinolone resistance for salmonella are all chromosomally mediated, so the numbers of quinoloneresistant salmonella can only increase in two ways: (i) the selection of a quinolone-resistant bacterium after exposure to a fluoroquinolone in humans or animals, or (ii) the spread of a quinolone-resistant bacterium to other animals or to humans (piddock, 2002). according to cdc, outbreaks caused by antimicrobial-resistant salmonella have been associated with an increased rate of hospitalization, and the rate of death was significantly greater in outbreaks caused by resistant strains (cdc, 2011). resistance can spread from non-human sources to human by various routes such as animal, water and contaminated foods (figure 1). resistance to combinations of many classes of antimicrobial agents in salmonella has led to the re-emergence of multidrug resistance salmonella (mdr) strains (o’brien, 2002). in this study, salmonella serotypes, derby, hato, hermannswerder, urbana, shangani, tamberma, kentucky, muenster, senftenberg, shangani, typhi and typhimurium were mdr. this is similar to the findings of many authors, who reported that mdr salmonella strains have been found to be of many serotypes such as agona, anatum, pullorum, schwarzengrund, choleraesuis, derby, dublin, heidelberg, kentucky, newport, senftenberg, typhimurium, and uganda (chen et al., 2004; zhao et al., 2008). all s. typhimurium reported in the present study were mdr and most of them were found to display a phenotype of resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (acssut). these antibiotics are the 9 most common drug classes used in veterinary medicine (mulvey et al., 2006). conclusion the genetic make-up of the salmonella strains permits their adaptation in various environments, including human, animal and non-animal hosts. this increases the difficulty in eliminating the bacteria. moreover, the emergence of mdr salmonella strains poses a great challenge in terms of effective treatment of the infections caused by these strains. several preventive measures have been proposed to stop the spread of antimicrobial resistant salmonella infections, and the restriction of indiscriminate use of antibiotics in food animals is by far one of the most effective measures. this report can help international organization to understand salmonella data and trends and to develop more informed solutions for reducing salmonella contamination along the farm to table chain. the report of sporadic data about salmonella serotype distribution will highlight the importance of the potential source of salmonella infection to humans. the data obtained in this study can be used by the world health organizationglobal foodborne infections network (who-gfn) and public authority to define the guidelines for basic surveillance system of salmonella and other enteropathogenic bacteria circulating among humans, animals, food and environment. conflict of interests the authors have not declared any conflict of interests. acknowledgement this research was supported by the international foundation for science (ifs) grant e/5001-2f/4600 to ak. the efforts of all who contributed to the success of the research are highly appreciated, ranging from conducting of strain characterization and drafting of the manuscript, as well as supervision and participation in writing of 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www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper involvement of key stakeholders in controlling animal diseases in rural settings: experiences with african swine fever in uganda peter ogweng1*, charles masembe1, johnson francis mayega2, ibrahim keeya4, charles tumuhe2, rodney okwasiimire3 and vincent muwanika2 1 department of zoology, entomology and fisheries sciences, college of natural sciences (conas), makerere university, kampala, uganda. 2 department of environmental management, college of agriculture and environmental sciences (caes), makerere university, kampala, uganda. 3 central diagnostics laboratory, college of veterinary medicine, animal resources and biosecurity (covab), makerere university, kampala, uganda. 4 department of production, mukono district local government, mukono district, uganda. *corresponding author. e-mail: ogweng.peter@cns.mak.ac.ug. accepted 12 october, 2019 key stakeholders’ involvement in the design and enforcement of effective african swine fever (asf) biosecurity measures is very vital. unfortunately, many times key stakeholders are less involved in the policy designing process. this study analyzed information from stakeholders in mukono district, to assess the acceptability of enforcing asf biosecurity measures among key stakeholders. mukono district has a high density of pigs and a history of frequent asf outbreaks. key informants (n = 23) were identified and interviewed in four sub-counties to generate an asf control stakeholders’ list. eleven stakeholder groups were identified by the key informants. sixty participants representing different stakeholder groups identified through the key informant interviews participated in a workshop to assess stakeholders’ characteristics regarding strict enforcement of asf control measures. stakeholder grid analysis revealed 60% as drivers, and 40% as supporters. there were no blockers, abstainers, and bystanders. despite this, majority of the groups (90%) did not have adequate capacity to implement the intervention due to financial constraints and inadequate technical support. these results show that there is great support for enforcement of biosecurity measures if stakeholders are facilitated with financial and technical support thereby limiting outbreaks of asf in rural areas of uganda. key words: african swine fever, biosecurity, influence, power, roles, interests, stakeholder. introduction the agricultural sector is very important in the economy of uganda. it contributes up to 23.8% of the gross domestic 2 product (gdp), generating about 48% of export earnings (sebudde et al., 2018). the livestock sector alone contributes 15% of agricultural gdp (tatwangire, 2014). this sector registered a 3% increase in the number of livestock and poultry between 2009-2010 with 4.5 million households (70.8%) rearing at least one species of livestock (uganda beaural of stastics, 2016). the pig is one of the important livestock species in uganda in addition to sheep, goats, cattle and chicken (uia, 2009). uganda has the biggest pig population in east africa (faostat, 2018). the pig population in uganda has been steadily increasing over the years; from 0.19 million in 1980; to 1.7 million in 2002, to 3.2 million in 2008 and to 4 million in 2016 (ubos, 2016). pig production in uganda is widespread with about 1.1 million (17.8%) households owning at least one pig (tatwangire, 2014). unfortunately, this rapid growth in the pig sector is affected by many challenges. some of these include; inadequate quality extension services, high input costs, fluctuating feed sources and prices and pig diseases (muhanguzi et al., 2012). there are several pig diseases in uganda but african swine fever (asf) has so far proven to be the single biggest problem hindering the pig industry (muhanguzi et al., 2012). african swine fever is a highly contagious disease that affects both domestic and wild pigs (penrith et al., 2004). this pig disease causes enormous economic losses to domestic pig farmers (mulumba-mfumu et al., 2019) due to the high mortality rates that accompany outbreaks (arias and sanchezvizcaino, 2002; costard et al., 2009). asf has neither cure nor vaccine (costard et al., 2013, penrith et al., 2004). the only way to control the disease at the moment is through quick diagnosis and strict implementation of biosecurity measures (dione et al., 2017). in order to control animal diseases, there is need for both international and local animal disease control laws. uganda has good policies and laws that when well implemented can limit the spread of highly contagious diseases such as asf. for instance, the animal diseases act (government of uganda animal diseases act, 2014), provides for enforcement of quarantines in areas with animal disease outbreaks. the policy prevents the movement of animals and animal products from and into such areas and, animal owners (pigs inclusive) are required to keep them in proper buildings or paddocks constructed in accordance with the specifications. when well implemented, these requirements can potentially limit the spread of asf. however, despite the presence of these laws, asf outbreaks are still common (kalenzi atuhaire et al., 2013) because the enforcement of these regulations requires the cooperation of several stakeholders. stakeholder analysis has been effectively used in multiple disciplines to deduce solutions to various challenges. in the human health sector, stakeholder analysis led to the successful improvement of maternal and newborn health in uganda (namazzi et al., 2013). similarly, in the veterinary sector, effective control of highly contagious animal diseases such as asf can only be achieved if all key stakeholders are identified, involved in policy designing and implementation of the designed measures. in uganda, enhancing the capacity of all pig stakeholders through community sensitizations using drama and radio talk shows has been identified as being important in the control of asf (ouma et al., 2017). stakeholder capacity building ought to be preceded by a stakeholder analysis to develop a proper engagement framework. a proper stakeholder engagement framework determines who should participate, when and how (luyet et al., 2012). many times stakeholders are less involved in the policy designing process thus affecting policy implementation. in this study, we assessed the acceptability of enforcing asf biosecurity measures among key stakeholders in the control of african swine fever in mukono district, a rural area in central uganda. methodology study area this study was carried out in mukono district (0.2835° n, 32.7633° e), located in the central region of uganda. mukono district has a total area of 2,986.47 sq. km and is bordered by buikwe district in the east, kayunga district in the north, luwero district in the north west, kampala city and wakiso district in the south west and a shoreline on lake victoria to the south. mukono district has a high density of pigs and a history of frequent asf outbreaks. the district is composed of 13 sub-counties (scs), 72 parishes and 795 villages with a human population of 596,561 (289,804 males and 307,757 females) distributed in 144,160 households (hh) (uganda bureau of statistics (ubos), 2017). of these households, 63,079 (43.8%) are involved in livestock rearing. mukono district is the only district in uganda, which is piloting a community initiated and monitored asf control program where asf stakeholders implement biosecurity measures aimed at asf control. stakeholder analysis stakeholder analysis for this study was carried out in 3 phases: the first phase was carried out to generate a list of stakeholder groups who can influence the implementation of asf control measures in mukono district. this was followed by categorizing stakeholder groups based on their power and influence towards the successful implementation of asf control, and their (stakeholder groups) characteristics in terms of their roles and interests towards asf control. generation of asf pig stakeholder group list for mukono district this was carried out in four sub-counties (scs) of mukono district, central uganda. the sub counties were kasawo, namuganga, ntenjeru and mpunge (figure 1). in kasawo sc, key informant interviews were held with one veterinary officer in each of the 6 parishes (kitovu, kabimbiri, kakukuru, namaliri, kasana and kigogola). additionally, discussions were held with two veterinary officers in kasawo sc head offices making a total of eight key informant interviews in this sc. in namuganga sc, interviews were held with one veterinary 3 figure 1. a map of mukono district showing the study area. officer in each of the five parishes of kituula, kitale, namuganga, namanoga and kayini. in mpunge sc, one key informant interview was held with one lead pig farmer in each of the four parishes of ngombere, mpunge, lulagwe and mbazi parishes. in ntenjeru sc, five key informant interviews were held as follows: two veterinary officers and three parish asf control mobilizers. we also held one key informant interview with the mukono district veterinary officer (dvo). at this step, respondents were only asked about names of stakeholder groups within and outside mukono district and their descriptions in relation to asf control. consent of each respondent was sought before commencement of the interview to which they responded in approval and their responses were both hand written and audio recorded. the recorded audios were later transcribed and typed in microsoft word. based on these interviews, a stakeholder group list was developed indicating the name of the stakeholder group and a brief description of their relationship with asf control in mukono district. categorization of asf control stakeholder groups a stakeholder workshop was held at mukono zonal agricultural research and development institute (muzardi) on 20/3/2019. the workshop comprised of a total of 60 individual asf control stakeholders drawn from 13 different stakeholder groups. during the meeting, stakeholders from the same stakeholder group sat closest to each other to allow them to share ideas and report the consensus in case it was required. all necessary visual materials (elements that constitute the asf biosecurity program, analysis grids, and definitions of key words) were displayed in manila papers on the walls of the workshop room for easy reference by the participants. the stakeholder group list that was earlier developed during the initial steps of the study was displayed as well in manila papers for all participants to view and verify. the workshop facilitator introduced the purpose of the workshop and requested participants who consented to the publication of the workshop results to sign a consent form. fortunately, all participants consented and signed the consent forms. using a stakeholder analysis grid, participants classified stakeholders into five categories (of drivers, blockers, supporters, bystanders and abstainers) depending on their power and influence, and level of agreement with the enforcement biosecurity measures in asf control. the stakeholder categories were defined as follows: i) drivers: a stakeholder or group that has high power, influence and high agreement with the enforcement biosecurity measures in the control of asf and can champion it; ii) blockers: a stakeholder or group that has high levels of power and influence in the control of asf, but highly opposes the enforcement of control measures; iii) supporters: those that support the practices, but whose influence and power may be limited (on their own); iv) bystanders: those that disagree to the control practices but with low influence and support; v) abstainers: those who are neutral to the control practices, but may or may not have influence. all stakeholder groups were categorized into the five categories by selecting one and asking participants in this group to rate their degree of power and influence in asf control (using a scale of 1-3) by writing on pieces of paper. power of a stakeholder was defined as the extent to which a stakeholder was able to persuade other stakeholders to embrace and implement asf control measures. the influence of a stakeholder on the other hand was defined as the power a stakeholder can exert over the execution of asf control measures. each stakeholder group was later asked to assess their own level of agreement with the implementation of asf control measures using the same scale of 1-3. the level of agreement of a stakeholder was defined as the extent to which a stakeholder approves/ accepts the proposed asf control measures. the 4 research team analyzed these responses in microsoft excel (microsoft corp., washington, dc, usa) and developed the stakeholder categories. analysis of stakeholder group categories after categorization of all stakeholder groups, those categorized as abstainers, bystanders and blockers were separated from the other categories. these participants were asked to brainstorm in focus groups the reasons why they had abstained from or blocked the strict enforcement of asf biosecurity measures. the stakeholders who were categorized as supporters and drivers were further asked to reveal their roles and interests towards the control of asf. strategies to deal with each stakeholder group researchers brainstormed on the strategies to deal with each stakeholder group based on each stakeholder’s roles and category. the strategies were in terms of empowerment, continuous engagement, further involvement and further consultation. importance and relationships among stakeholder groups to determine the relationship among the different stakeholder groups, participants were requested to attach circles cut out of manila paper in three sizes as described by richards and panfil (2011) were the level of importance is directly proportional to the size of the cut manila paper. the three sizes were large, medium and small representing level of importance of the stakeholder group. importance of a stakeholder was defined as the roles of such a stakeholder in achieving anticipated results for the implementation of asf control measures. since all stakeholder groups were from mukono district all the circles were purple. for each stakeholder group, the participants described the importance of the stakeholder’s involvement in the control of asf and selected the corresponding size of the circle as summarized below. little importance = small circle (1 cm in diameter) some or moderate importance = medium sized circle (2 cm in diameter) very important = large circle (3 cm in diameter) the stakeholder group names were then inscribed on these circular cards. the circles were then arranged according to working relations among stakeholder groups. those with very close working relationships were put closest to each other and vice versa for those with little or no working relationship. since stakeholder groups located out of mukono district, failed to attend this workshop, the exercise was done for only stakeholder groups located within the district. the next stage was to attach triangular manila cards of three different sizes, large (3 cm × 3 cm × 3 cm), medium (2 cm × 2 cm × 2 cm) and small (1 cm × 1 cm × 1 cm) representing level of influence of stakeholder to the edge of the stakeholder circle. the outputs were reviewed by all participants, who later discussed the relative importance and influence of each stakeholder. the arrangement was then captured with a digital camera. the overlap of the circles represented the extent of the working relationship between stakeholder groups. results stakeholder groups in the control of asf in mukono the stakeholder groups that were identified by key informants were from both within and outside mukono district (table 1). the key stakeholder groups included those involved in animal healthcare such as veterinarians, pig production value chain actors (for example traders), law enforcement, regulatory and administrative agencies at both local and national levels. table 1 gives a brief description of each of these stakeholder groups. grid analysis of stakeholder group categories the stakeholder grid analysis revealed that 60% of the stakeholders were drivers and 40% were supporters. there were no blockers, abstainers, and bystanders (table 2). the categorization was in respect to the stakeholder’s level of agreement with the use of biosecurity measures in asf control, power, and influence in implementing asf control measures in mukono (table 2). the capacity of stakeholder groups to control the spread of asf all stakeholders discussed within their stakeholder groups, and each stakeholder group came up with a score between the scales of 1 to 3 to signify their own capacity in the control of asf, giving reasons for their score as indicated in table 3. it was observed that 90% of the stakeholders were incapacitated in the implementation of the asf intervention measures due to financial constraints and inadequate technical support. the roles of stakeholder groups in asf control in general, all stakeholders identified in the study were involved in asf control in one way or another as shown in table 4. farmers were identified to play significant roles in asf control since they are in direct contact with animals. these roles included reporting suspected asf cases to veterinarians, restricting animal movements, disinfecting pig sties and restricting visitors from their farms. interestingly, participants pointed out that security and judiciary support asf control strategies through enforcement of measures such as quarantines by restricting unlawful animal movements in the community in addition to apprehending and prosecuting quarantine defaulters. interest of stakeholder groups in asf control stakeholders had varying interests in asf control (table 5), but all were geared towards increasing pig production to improve both household nutrition and income. the interests of the stakeholders in the implementation of asf control measures reflected their roles in the community and occupation. 5 table 1. asf control stakeholder group list for mukono district, uganda. s/n name of stakeholder description 1 veterinarians 2 agricultural officers 3 veterinary/human drug shops 4 local council (lc) leaders 5 pig traders 6 pig farmers and pig farm workers 7 pig consumers 8 asf researchers 9 security and judicial agencies 10 media houses 11 district officials 12 pig feed suppliers 13 maaif 14 policy and law makers 15 dog owners 16 sub county and parish chiefs 17 church leaders 18 parish health coordinators 19 local ngos 20 pig processors 21 pig breeders 22 beach management units 23 community development officers all private and public (local government) veterinary doctors/ officers and animal husbandry officers within mukono. they treat pigs and provide technical advice to farmers. they oversee crop and animal production in the sc. sell animal drugs and give extension advice to farmers. human pharmacies also sell disinfectants (like jik) to pig farmers for asf control. include lc 1, 2 and 3 in the lower local government administrative units within mukono. they help in enforcing asf quarantines during outbreaks and information dissemination. butchers, buyers, ‘pork joint’ operators and transporters, locally termed as ‘babizzi’. they have a pig traders’ association with elected leadership. the traders come from within and outside mukono. both commercial and small-scale farmers involved in pig rearing with leaders in every village locally called ‘ssabalijja’ who coordinate asf control activities. kasawo and namuganga scs pork consumers are organized into a pork consumers’ association, that monitors the quality of pork being sold and the hygiene of the butcher. these include researchers from; covab and conas at makerere university, and ilri. these institutions are involved in asf research in mukono and uganda in general. police, crime preventers, the courts of law and prisons within and outside mukono, all help in enforcing asf control. include radios (sauti radio), television stations (bukedde), community radios with raised speakers (that cover 3-4 villages). these media stations, sensitize the community about asf disease control strategies. include the cao, dvo, owc /naads) officers. uwep and ylp officials involved in poverty alleviation by distributing animals including pigs to farmers. supply animal feeds, give advisory services to farmers regarding control of asf. they may also help in the spread of asf in case they sell contaminated pig feeds to farmers. government ministry in charge of agriculture, animal industry and fisheries resources in the country. provides technical guidance for formulation, review and implementation of animal production acts, policies and legislation. district council makes and approves community by-laws and ordinances while the parliament of uganda which enacts policy, regulations and acts e.g. the animal disease act. stray dogs are believed to aid in asf spread by moving asfv contaminated swill from one location to the other. government administrators at the sc and parish levels. they are involved programme/ activity such as asf control to be successful in their communities. church leaders play a pivotal role in the community, for example, the church of uganda (cou) in mukono is involved in pig projects. the church gives free piglets to christians. these work with veterinarians for information sharing regarding butcher health and hygiene. include feed the hungry (involved in buying and distribution of piglets to farmers in mukono), child fund (works in jinja and kampala) but buys piglets from mukono for distribution in jinja and kampala. include fresh cuts that is located in kampala, buy pigs from mukono farmers and process pig products. there is also wambizi, a pig abattoir, located in nalukolongo, kampala that receive pigs from all over uganda. these produce and sell high quality piglets and pig semen to farmers. kampala capital city authority (kcca) has a pig-breeding center in kyanja that has been in this business for five years. these are in areas bordering the lake such as; katosi and mpunge landing sites. they handle silver fish, locally called ‘mukene’ used in making pig feeds which when contaminated with asf can spread the disease. they sit at the sub county and heavily engage with all developmental activities, of which pig farming is one of them. cao: chief administrative officer, conas: college of natural sciences, covab: college of veterinary medicine, animal resources and biosecurity, ilri: international livestock research institute, maaif: ministry of agriculture, animal industry and fisheries, naads: national agricultural advisory services, owc: operation wealth creation, uwep: uganda women entrepreneurship programme, ylp: youth livelihood programme relationship, importance and influence of stakeholder groups according to the proximity of circle attached to the stakeholders, farmers, consumers and traders are all closely related as evidenced by the close grouping of their respective circles. in this cluster, farmers and consumers were seen as more important than traders since their circles were bigger than those of traders. interestingly, traders and farmers were seen as more influential than consumers because their triangles were bigger than that of consumers. district officials, security and judicial agencies were all closely related due to their circles being aggregated together but the district officials were seen as more important (bigger circle) than both the security and judicial agencies (smaller circles). 6 table 2. analyzed categories of stakeholder groups in the control of asf. stakeholder interest in the issue level of level of power and type of power category agreement influence pork consumers ensure that only clean and pork from healthy pigs is sold district officials (cao, lc5, supervise the veterinary officers owc, dcdo) pig trader generate income from pigs and pig products high high high low beneficiary high level supporter high decision maker high level driver high beneficiary high level driver religious leaders community spiritual overseers medium high opinion leader high level supporter community development officers ensure implementation of community high high development high level (cdo) development programs implementer driver agricultural officer provide agricultural extension service. medium low beneficiary and influence high level policy supporter security and judicial officials enforcement of community law and order medium high law and order enforcer high level supporter local council leaders veterinary officers pig farmer grassroot administrative structure of the community in charge and supervisor of animal welfare in the district earn a living from rearing pigs high medium high high opinion leader high level driver high beneficiary and influence high level policy supporter high beneficiary high level driver researchers design simple community-based asf control high high beneficiary and influence high level measures policy driver dcdo: district community development officer, lc5: local council five. on the other hand, district officials, security and judicial agencies were regarded as equally influential because their triangles were of equal sizes. veterinary officers, cdos, religious and local council leaders were all closely related as evidenced by their circles being grouped together. veterinary officers were the most important stakeholders in this group. comparatively, local council and religious leaders were both deemed moderately important while cdos were the least important. collectively, all stakeholders in this group were deemed highly influential owing to the equal sizes of their respective triangles. researchers were not related to any other stakeholder group as observed by the standalone circle. they were also not aggregated with any other stakeholder group but researchers were seen as moderately important since their circle had moderate size and are highly influential due to the large size of their triangle. agricultural officers were not directly mandated in animal production community outreach services although it was noted that in circumstances where no veterinarians existed, they provided minimal animal production services to farmers. in this respect, it was unanimously agreed by the participants that agricultural officers be excluded from this stage. however, through animal production trainings, agricultural officers would to some level provide animal production services to pig farmers in the absence of veterinarians (figure 2). strategies for dealing with the stakeholder groups following the categorization of stakeholder groups 7 table 3. stakeholder group’s capacity to control the spread of asf. stakeholder score reasons for their capacity potential solution veterinarians 2(moderate) inadequate facilitation and staffing for enforcement. increase funding and recruitment for field staff for community outreach. agricultural officer 1 (low) cdos 3 (high) farmers 2 (moderate) religious leaders 2 (moderate) security and judicial 1 (low) agencies inadequate technical knowledge among the staff on asf the lack of a close working relationship with other stakeholders minimizes their capacity in asf control. they have a close working relationship with the farmers stakeholders in the community (farmers, traders and butchers) in implementing asf control measures. inadequate funding and technical knowledge to implement recommended biosecurity measures. there is need for facilitation to enable religious leaders reach out to the community. inadequate funding to enforce quarantine. recruit more staff for community outreach and provide staff training on animal production. provide funding for adequate community outreach. sensitize farmers on asf control measures and subsidize prices of effective disinfectants. provide funding for community outreach. provide funding for quarantine enforcement. district officials 2 (moderate) local leaders 2 (moderate) pork consumers 2 (moderate) researchers 3 (high) limited human resources and funds humper the effective involvement of the district officials in asf control. limited facilitation to local leaders reduces their effectiveness in sensitizing the community on asf control measures. in ability to accurately identify asf sick pigs aids the spread of the disease. they have the funding for research and sensitization of all stakeholder groups in asf control. recruit more staff and increase supervision funds provide funds for community outreach. train consumers on simple animal disease diagnostics. train all stakeholder groups in disease control measures. (table 2), strategies were devised to deal with each of them based on their power, influence and importance. these strategies include empowerment, further consultation, continuous engagement and further involvement (table 6). discussion this study identified and evaluated stakeholders’ interest, capacity, roles and influence/power in relation to the implementation of biosecurity measures in the control of asf in rural settings. the results of this study revealed that all stakeholders were either drivers or supporters of the intervention. these are vital factors in determining the success of an intervention and could smoothen the implementation and acceptance of the measures among stakeholders. the high level of supporters and drivers may reflect the direct and indirect benefits of the interventions for stakeholders. the direct beneficiaries (farmers, traders, consumers and veterinarians) either earned an income or consumed quality pork while the indirect beneficiaries (district officials, religious and local leaders, judiciary and security) were happy to see an increased level of economic stability in the society. in that respect, community members get employment and reduction in the level of crime in the long run is achieved since most youth would be engaged in pig production. youth empowerment in agricultural sector would increase food production and household income, create stability in terms of security which leads to socioeconomic development of a country. the high level of drivers with high influence in particular at the district and community levels ensures the sustainability of the intervention programs. there is a need to involve stakeholders identified as drivers with high influence at the national level since they are very instrumental in policy formulation and influence (kanmiki and bempah, 2017). all the supporters, such as pork consumers, religious leaders, security and judiciary, and veterinarians are stakeholders who need to be empowered, involved further and continuously engaged (luyet et al., 2012) so as to become drivers of the intervention (table 2). the consumers for example should be sensitized on 8 table 4. the roles of stakeholder groups in asf control. stakeholder roles of stakeholders in asf control consumers pig farmers ensure that the pork butchers and the butcher environment have and maintain good hygiene. promote the consumption of only disease free pork. ensure proper farm hygiene by; disinfecting the farm and surroundings and restricting visitors entering the farm in addition to constructing improved pig sties. religious leaders agricultural officers pig traders cdo security /judiciary local leaders veterinarians district officials researchers sensitize people about asf, and instant reporting of outbreaks to veterinarians. sensitization farmers on asf and report outbreaks to the relevant authorities. instant reporting of outbreaks, slaughtering only on slaughter slabs, not buying of sick pigs and observing proper hygiene in farms and slaughter slabs by disinfecting. sensitize people about asf, mobilize the community and ngo, link farmers to asf technical people, coordinate all stakeholders involved in asf control and assist in enforcing quarantine. sensitization of the community and farmers on asf control measures and enforce quarantine. monitor, mobilize and sensitize farmers on asf control measures. sensitize, monitor, supervise, and create awareness among all stakeholders on asf control measures. regulate and enforce veterinary laws, coordinate and provide veterinary services and, initiate the institution of quarantines during asf outbreaks in their areas of operation. recommend for declaration of quarantine to maaif and enforcement of quarantine in the affected areas of the district. they also enact bylaws intended for asf control, sensitize farmers on asf control measures and oversee extension service provision to farmers. sensitization of all stakeholders in asf control in the pig production value chain. conduct asf surveillance and testing. solicit for funding to carry out research on asf. the dangers of zoonotic disease such as tuberculosis, anthrax, ebola and helminthiasis that one can contract from consuming uninspected pork although asf is non-zoonotic. consumers should also be empowered to form consumer protection associations with a strong voice to ensure the sale of clean and healthy pork. the religious leaders on the other hand are influential people in the community who can change the mindset of the masses and therefore need to be empowered and involved further to continuously remind the masses on the importance of implementing biosecurity. the security and judiciary play crucial roles in the implementation of quarantines during disease outbreaks. empowering them through formulation of policies with clear guidelines and providing financial support for activities during quarantines could greatly improve on the control of animal diseases in the community (larry, 2004). the veterinarians believe that recruitment and posting of staff at each parish would tremendously improve on surveillance and service delivery which eventually would lead to success in disease control. much as all stakeholders were either drivers or supporters, most of them needed technical support in the implementation of biosecurity measures in the control of asf. the capacity of almost all stakeholders was either low or moderate and hence needed improvement. as mentioned earlier, there is need for increased human resources to all technical stakeholders and funding for better service delivery (table 3). since most stakeholder groups felt that sensitization of farmers, traders and consumers (table 4) was their responsibility, there should be efforts to standardize and coordinate dissemination messages. the local government department of production is best suited to coordinate this activity since the mandate of improving production is it responsibility. by doing this mixed and confusing messages will be eliminated. this will promote early detection of asf and rapid implementation of biosecurity measures which to date are still the only effective means of asf control (gallardo et al., 2015; dione et al., 2017). it was generally agreed therefore that involving all stakeholders in developing the guidelines for biosecurity implementation would 9 table 5. interests of stakeholder groups in asf control in mukono district. stakeholder interests of stakeholders in the control of asf security and judiciary consumers district officials local leaders agricultural officers veterinarians pig farmers cdos pig traders religious leaders researchers are interested in a disease free pigs community, this creates employment, leading to decrease in crime rates in the community they are interested in good hygiene of the butcher and its immediate surrounding environment. they are also interested in the consumption of good and disease free pork. these want to fulfill their mandate to control asf epidemic and keep the reputation of their district. a good reputation will enable them lobby funds from donors for piggery projects in mukono district. they want to ensure that there is diversity of livestock enterprises for operation wealth creation (owc) program for people to have improved nutrition and increased household income. they want the pig sector to thrive by having increased number of improved pig breeds for people to get employment opportunities the local leaders are interested in increased household (hh) income among their electorates. they want to see their pigs multiply their animals and increase in number to; 1) provide market for pig feeds e.g. maize, 2) get organic manures and access enough pork in their area at affordable/reduced prices. they are interested in community members getting jobs in the piggery sector. they want farmers to have supplementary income from pig production. they want increased pig manure for crops from pig farmers. they want increased protein supply from pig farmers to consumers. they are interested in community members getting jobs in the pig sector. they want hh to have increased hh income. they want communities and hh to comfortably rear pigs for them be food secure. farmers aspire to increase their hh income by getting jobs from increased rearing of asf free pigs. they believe that higher incomes will reduce domestic violence in their homes. they to be more food secure and reduce malnutrition among their household members. they prefer having an asf-free community to motivate pig farmers and increase production and in turn supported government involved in livestock. they benefit from having an asf-free community since asf outbreaks are usually accompanied by several restrictions like quarantines which affect their business negatively. increased hh and church income from increased piggery production reduced domestic violence among the faithful in their congregation reduced crime rates among the faithful in their congregation they want control the spread of asf they want to increase the level of awareness about asf among all stakeholder groups involved in pig production. they want farmers to produce and sell asf disease free pigs. foster ownership of such a policy and subsequent sustainability. since financial constraint was mentioned as a challenge, costs of effective disinfectants and other farm inputs needed for disease control need to be subsidized by government to a rate that can be easily afforded by the farmers. the high costs of animal drugs and disinfectants hamper the implementation of biosecurity as a control measure in many developing countries (kouam and moussala, 2018). subsidizing farm inputs relevant to disease control as well as adherence to 10 farmers traders religious leaders local consumers council leaders veterinarians researchers cdo district officials security figure 2. relationship, power, influence and importance of stakeholders. circle = importance of stakeholder; triangle = power and influence. stakeholders whose circles are touching or closer indicate similarity in roles in the control of asf in mukono dis trict table 6. strategies to deal with each stakeholder groups. stakeholder justification/criteria strategy to deal with stakeholder power and influence importance agricultural officer low moderate provide animal production training and continuously engage traders high moderate consult further and continuously engage consumers low high empower veterinarians high high involve further and continuously engage cdos high low consult further and involve further security and judiciary high moderate consult further and continuously engage local council leaders high moderate consult further and continuously engage researchers high moderate consult further and continuously engage district leaders high high involve further and continuously engage farmers high high involve further and continuously engage religious leaders high moderate consult further and continuously engage practices reporting asf outbreaks, sale of only disease free pork and many other practices that do not require any drugs would play a great role in controlling the spread of asf. from this study, it was also revealed that funding of the other stakeholder groups was required to enable them play their roles effectively. provision of funding to religious leaders, district, and local council, and security and judiciary, veterinarians, agricultural officers and cdo would greatly improve on community outreach services leading to improvement on the implementation of control measures. in the same way, increasing the human resource for the veterinarians, cdos and agricultural officers would improve on the technical support provision to farmers while to the district, security and judiciary it would improve on supervision and monitoring of service providers to the farmers. the farmers in the stakeholder analysis meeting felt that 11 it would be proper to involve all stakeholder groups in designing the guidelines of implementing biosecurity measures. this concern arose since farmers in particular thought that their experience in asf outbreak management at the farm need to be amalgamated with that of the technocrats so as to develop a binding working document for asf control at farm and butcher levels. indeed, during the discussions, farmers pointed out some control measures such as each farmer having their own drugs, syringes and needles that the veterinarian can use while treating pigs on a particular farm to avoid transmission of infections from other farms. secondly, farmers also suggested that the manure from pig sties should be disinfected before use in the crop fields since that could be a potential source of disease spread. success in implementing biosecurity measures as a means of asf control in the rural setting, requires a number of changes in management and animal husbandry practices to be done at the farms, slaughter slabs and butchers. these include: the farmers should be encouraged to desist from free ranging system of management and embrace the use of raised housing or double fencing to reduce contact between their pigs and roaming pigs. secondly, traders and butchers should be told to stop buying and slaughtering asf sick pigs. thirdly, all the slaughter slabs should be fenced to prevent stray dogs, cats, free ranging pigs and birds from accessing swill which can be carried to pig farms and transmit disease. interestingly, during the meeting participants did come up with some vital and creative asf control measures. such measures included: 1) encouraging individual farmers to have their own injection syringes and needles for treating pigs in their own farms since those of the veterinarians may transmit diseases if not properly disinfected because they (veterinarians) treat pigs in many farms. 2) farmers were also encouraged to obtain both feeds and water from safe sources (asf free sources) because many times asf is spread to unsuspecting farms through african swine fever virus (asfv) contaminated feeds. 3) each pig farmer was encouraged to have their own breeding boar where possible or used artificial insemination instead of borrowing a boar from another farmer or using a community boar (where people take their sows to a boar that stationed in one of the farms for servicing). the use of a community boar and boar borrowing aid the spread of asf among farms. 4) the movement of pig farm wastes especially from pig sties was discouraged since it could spread asf among farms once one of the farms in infected with asfv. the results of this stakeholder analysis show that there is need to sensitize and mobilize the community before the implementation of biosecurity measures for any animal disease control or intervention to succeed. there is also need to consult the stakeholders in the pig value chain during the development of the biosecurity implementation guidelines to promote support and agreement to the intervention. lastly, it is crucial to promote a good working relationship and understanding among all the stakeholders for the success of the intervention (auvinen, 2003). methodological considerations we were not able to interview some categories of stakeholder groups who could have had an influence in the intervention, these include: pig feed manufactures, pork processers, law makers and maaif officials despite our endeavor to do so. the study was done in only one district and yet the situation in other districts may be varying. conclusion this stakeholder analysis has revealed that all key stakeholders in the pig value chain in a rural area in uganda were either drivers or supporters of the implementation of biosecurity measures in the asf control; although, there was need for financial and technical support to key stakeholders for the intervention to be implemented and enforced. these results show that there is great support for enforcement of biosecurity measures if stakeholders are facilitated with financial and technical support thereby limiting outbreaks of asf in rural areas of uganda. recommendation there is need for adequate funding for community outreach to facilitate sensitization activities especially to the veterinarians, cdos and district officials. ethics permission to carry out the study was granted by the ugandan national council for science and technology under the reference number a497. consent from the district veterinary officer was obtained prior to the start of interviews in mukono district. all participants were informed that the study was voluntary, confidential, and that they had the choice of ending their participation at any time of the interview. an informed consent was given by all participants prior to the implementation of the study. conflict of interests the authors have not declared any conflict of interests. orcid p ogweng https://orcid.org/0000-0001-5616-3819 12 acknowledgement this work was funded by african union grant agreement number aurg ii-1-196-2016. the authors thank the asf task force members of kasawo and namuganga sub counties, dr kiryabwire david, district veterinary officer of mukono, veterinary officers of kasawo, namuganga, ntenjero and mpunge, moses namitale and raymond ssekadde for driving the research team during the data collection period. finally we would like to sincerely appreciate all the farmers in the four sub counties for sparing their time to respond to the questions. references arias ml, sanchez-vizcaino j m (2002). african swine fever. in: trends in emerging viral infections of swine. iowa state university press pp. 119–139. available at: https://jcm.asm.org/content/jcm/41/9/4431.full.pdf auvinen am (2003). understanding the stakeholders as a success factor for effective occupational health care, occupational health 1:6-8. doi: 10.5772/66479. costard s, mur l, lubroth j, sanchez-vizcaino jm, pfeiffer du (2013). epidemiology of african swine 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https://hdl.handle.net/10568/34090 uganda beaural of stastics (ubos) (2016). national population and housing census 2014 – main report. uganda bureau of statistics. kampala, uganda. available at: https://doi.org/10.1017/cbo9781107415324.004 uganda bureau of statistics (ubos) (2017). national population and housing census: area specific profiles. kampala, uganda. 8–20. available at: https://www.ubos.org/wp content/upload/publications/2014censusprofiles/kiboga.pdf uia (2009). livestock sector profile. uganda investiment aouthority (vol. 2002). kampala, uganda. available at: https://www.ugandainvest.go.ug/uia/images/download_center/sect or_profile/livestock_sector_profile.pdf 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 12 (3), pp. 001-008, march, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper impact of pasture-raised pork farming on genes regulating lipid metabolism and meat quality n. okoli1, n. ceron-romero2, y. meng2 and m. o. ezekwe2* 1 department of chemistry, alcorn state university, 1000 alcorn ave., lorman, ms 39096, usa. 2 department of agriculture and applied sciences, alcorn state university, 1000 alcorn ave, lorman, ms 39096, usa. received 19 february, 2023; accepted 1 october, 2023 studies have shown that pasture-raised pork is fresher and of higher nutritional value than its conventional counterpart. this study was to determine the effect of grazing systems on meat quality, carcass traits, and on lipid metabolism gene expressions. control pigs were fed 100% commercial diet. fifty/fifty (50/50) group was placed on 50% of the diet consumed by control group plus free access to ryegrass-clover pasture. the twenty-five/seventy-five (25/75) group was fed 25% of the diet consumed by the control plus access to free pasture. the overall meat quality (flavor, overall acceptability and carcass traits (marbling, color)) were scored significantly higher (p<0.05) in the 25/75 group than in control or 50/50 group. back-fat thickness was lower in 25/75 group (p<0.05) than in control and 50/50 group. no differences were observed between the control and 50/50 in meat and carcass qualities. realtime polymerase chain reaction (pcr) revealed that peroxisome proliferator-activated receptor α (pparα), peroxisome proliferator-activated receptor γ (pparγ), lipoprotein lipase (lpl), and sterolregulatory-element binding protein 2 (srebp-2) responded differently in muscle and adipose tissues. the results indicated that pasture-based pork production could positively influence lipid metabolism genes important in meat and carcass quality traits, with pasture exposure and feed allowance. key words: pigs, lipid metabolism, meat quality, real-time polymerase chain reaction (pcr). introduction molecular biology approaches using gene expression have been reported that numerous genes are associated with leaner pigs carcasses, meat and carcass quality traits (albuquerque et al., 2017; ivanovic et al., 2015; tous et al., 2014; álvarez-rodríguez et al., 2016). many of these genes have also been found to be expressed during obesity-related diseases (georgiadi and kersten, 2012). currently, obesity is considered a public health disease. according to the world health organization (who), the obese population has doubled in number in less than 30 years (mitchell and shaw, 2015). consumers today are more concerned about their health and physical fitness when seeking ways to get a healthier source of protein. *corresponding author. e-mail: ezekwe@alcorn.edu. 2 therefore, the understanding of lipid metabolism in pigs could elucidate mechanisms underlining conditions such as hypertension, type 2 diabetes, cardiovascular diseases, cancer, and atherogenic dyslipidemia. this has led to increased interest in the study of grazing systems on meat quality and genes involved in lipid metabolism. animals raised in outdoor systems have been shown to contain higher intramuscular fat (imf) (högberg, et al., 2004), color (estévez et al., 2003; fraser et al., 2009) and leaner carcasses (álvarez-rodríguez et al., 2016; galvão et al., 2006) than animals in confinement. the content of polyunsaturated fatty acids (pufa) content on pastures, primarily essential linolenic acid (c18:3), and linoleic acid (c18:2) has been associated with pork of high nutritional value (ventanas et al., 2006; kim et al., 2009; nilzén et al., 2001; edwards, 2005). however, the impact of grazing systems on genes involved in lipid metabolism is still poorly studied in swine. dervishi (2012) reported changes in fatty acids profile of meat products and milk of grazing lambs as well as in genes responsible for stearoyl coa desaturase (sdc), and other lipid regulatory genes: peroxisome proliferatoractivated receptor α (pparα), eroxisome proliferatoractivated receptor γ (pparγ), and sterol-regulatoryelement binding protein 2 (srebp). ppars are a family of nuclear receptors acting as ligand-dependent transcription factors (pparα, pparδ, and pparγ) involved in fatty acid synthesis in pigs (wang, et al., 2016). pparα regulates cellular fatty acid uptakes, transportation, oxidation, ketogenesis and gluconeogenesis (könig et al., 2007). pparγ is considered an important regulator of adipogenesis (samulin et al. 2008). the increase in pparγ activates lipogenic genes such as lipoprotein lipase (lpl) and srebp-1 and srebp-2 (eberlé et al., 2004). the objective of this study was to determine the effects of a pasture-based pork production system on the expression of genes involved in lipid metabolism (lpl, pparα, pparγ, and srebp-2) and meat quality characteristics in pork. materials and methods the experiment was conducted at alcorn state university swine research and development farm, churchill, mississippi. the study was carried out during spring and summer (april-june). all the procedures of animal handling and treatment were approved by the animal use and care committee at alcorn state university. a total of 48 finishing pigs from yorkshire and hampshire crossbreed sows were randomly assigned to the three experimental groups. the commercial ration used for the experiment met the national research council (1998) requirements. the diet contains 15.5% cp of 12.04 kj of energy. the animals were assigned into six groups (8 animals each). the control groups and its replica received a finishing diet ad libitum. the rest of the groups were raised under pasture conditions and supplemented with a 50/50 and 25/75 of the control diet/pasture combination, respectively. the control groups were housed indoors on concrete slatted-floor pens (2.1 × 3.6 m). two pens were used to allocate 8 pigs each, approximately 1.2 m 2 per pig. the groups 50/50 and 25/75 were placed on a pasture fenced area of approximately 16200 m 2 divided in 8 paddocks (about 1300 to 1600 m 2 each). the pigs at the site were rotated among different paddocks and fed with a mixed pasture of ryegrass and red clover. a corrugated zinc hoop (4×3×2m) and an automatic refill water supply were established for each group. the animals were weighed weekly, body weights and feed intake recorded for later use in estimating growth performance. initial body weight (ibw), final body weight (fbw), average daily gain (adg), average feed intake (afi) and feed conversion ratio (fcr) were evaluated. pigs were slaughtered at a fbw of approximately 100 kg in southern university’s agricultural research and extension center (suarec) meat laboratory. the dressing percent was obtained using the final body weight and hot carcass weight. the carcass length was measured from the forward edge of the first rib to the forward edge of the aitch bone. backfat thickness was measured at the 10th rib at 7 cm off midline at last rib of the split carcass with a destron pg-100 fiber-optic probe. the estimated lean yield was calculated using the following formula: estimated lean yield (ely) = 55.07 (0.377 × fat depth) + (0.052 × muscle depth). measures of loin muscle meat quality include color, loin muscle score, loin muscle marbling score, and loin muscle firmness score. the major primal cuts from the carcass left side (shoulder, loin, ham, and belly) were trimmed to remove the fat and the weights were recorded to obtain net yield. the color was determined from chop cuts at the last rib level using a chromameter (c-300; minolta, dietikon, switzerland) and illuminant d65. thirty-three panelists were recruited to perform meat quality evaluation. roasted pork samples from the same muscle were given to the panelist from each treatment. additionally, red lights were used in the place to disguise color differences. the panelist evaluated the pork using the five-point hedonic scale (1 as extremely unpleasant and 5 extremely acceptable). sample collection and rna processing after slaughter, samples of muscle and adipose tissue (about 500 to 1,000 mg) were collected and immediately rinsed with depctreated water, then snap-frozen in liquid nitrogen, and then transferred to a -80°c freezer for future lipid metabolism analysis. total rna was extracted from the frozen tissue sample using trizol (sigma cat# t-9424). homogenization of the samples was done by quiagen-tissue laser. the rna precipitation was done following a standard protocol (thermo scientific). purity and concentration were analyzed by using a nanodrop nd-2000 spectrophotometer. the integrity of the samples was examined by gel electrophoresis. reverse transcription polymerase reaction and real-time polymerase chain reaction rna was treated with dnaase i. rt reaction was done using superscript® iii reverse transcriptase. then it was incubated at 50°c for 50 min. the reaction was inactivated by heating at 70°c for 15 min. the primers used were pparα, pparγ, srebp, and lpl (table 1). the reaction was developed using a modified protocol of kary mullis (1983). pcr reactions were run in an icycler (bio-rad, hercules, ca, usa) using perfecta® sybr® green fastmix® for iq (quanta biosciences, gaithersburg, md). the cdna cycling conditions were as follows: 3 min at 95°c, followed by 40 cycles of 15 s at 95°c, 1 min at 60°c; then followed by 80 cycles of 10 s at 55°c with increasing setpoint temperature after cycle 2 by 0.5°. changes in gene expression were conducted according to the protocol described by schmittgen and livak (2008). 3 table 1. primers used for quantitative real-time polymerase chain reaction. primer name forward primer: 5’ → 3’ reverse primer: 5’ → 3’ ppar-α caaggtctgaaaaagccaaa cttcttgatggcctcgtaaa ppar-γ aggtgcgatcttaactgtcg ccaacagcttctccttctca srebp gcaagagaaagtgcccatta cgcttctcaatgatgttgtg lpl tggacggtgacaggaatgta gtcccaccagcttggtgtat table 2. growth characteristics of pasture fed and control pigs at 103 days of the experiment. item diets control 50/50 25/75 initial body weight (kg) 29.61±6.03 29.77±3.07 25.04±2.33 final body weight (kg) 113.16±4.6 112.05±7.31 111.46±7.45 average daily gain (kg/day) 0.96±0.51 a 0.67±0.48 b 0.62±0.37 b average feed intake (kg/day) 3.07±0.82 a 1.83±0.24 b 1.22±0.23 c feed conversion ratio 3.19±0.75 a 2.73±0.48 b 1.97±0.30 c abc means ± sd for 8 pigs means with different superscripts within rows are significantly different (p<0.05). statistical analysis statistical analytic software (sas, 2013) was used to analyze the weights of the pigs and the relative expression levels of gene expression. mean squares were determined using the general linear model (glm) and mean separations were done using the duncan multiple range tests (sas ins., 1985). excel (microsoft office, 2010) was used to analyze the relative expression of the genes. the relative quantities of mrna expression were normalized to the relative quantities of a housekeeping gene and these normalized ratios were used for the quantitative determination of gene expression. results and discussion growth performance the rate of growth of the pigs across the treatment was evident (table 2). the different treatment groups were not varied in body weight since all the groups were slaughtered at similar market weight. however, average daily gain and feed conversion ratio differed (p <0.05) reaching the market weight 30 and 60 days earlier than 50 and 25% of commercial diet group, respectively. similar results were observed by heyer et al. (2006) who noted lower performance in crosses of duroc × large white and swedish landrace × large white when compared with indoor animals. also, hansen et al. (2006) reported a reduced average daily gain of the pigs under organic systems with restricted concentrate feeding. in contrast, lebret and guillard (2005) reported that outdoor sows showed a higher final body weight than the indoor sows. the reduced feed intake (p<0.05) in pigs from diets 50/50 and 25/75 groups was significantly lower than those of the controls due to the dietary fiber levels. kerr and shurson (2013) and noblet ans le goff (2001) reported that fibrous diets affected energy and nutrient digestibility and are associated with energy losses as methane, hydrogen, and fermentation heat. thus, the significantly lower fcr (p<0.05) observed on pasture fed animals were expected as long as the growth performance depends on the dietary energy-protein balance. similar results were observed by smit and beltrana (2017) in pigs fed a diet with camelina sativa cake inclusions, although a decrease in feed intake was observed, there was a significant increase of n-3fatty acids in adipose tissues. carcass and meat quality characteristics as shown in table 3, the diet had no effect (p>0.05) on primal cuts (length, shoulder, leg, rib). nevertheless, dressing percentage and loin were significantly (p<0.05) higher in the control group than in carcasses from pigs raised on pasture. similarly, rey et al. (2006), reported lower carcass weight and yield from iberian pigs raised on pasture in comparison to indoor animals. contrary to our findings, lebret and guillard (2005) and gentry et al. (2002) found that hot carcass weight, cold carcass weight, dressing percentage, and loin eye area were higher under organic feeding systems. in agreement with ahmed et al. (2016) who supplemented the diets of grower-finisher pigs using pomegranate, there was a significant reduction in backfat thickness while increasing the forage intake and 4 table 3. carcass and meat quality characteristics of pasture fed and control pigs. item a diets control 50/50 25/75 dressing % 86.20 ± 2.35ª 80.93 ± 0.71 b 75.60 ± 2.40 b firmness 2.00 ± 0.46 1.91 ± 0.25 2.00 ± 0.01 back fat (cm) 2.54 ± 0. 51 b 1.91 ± 0.67 b 1.02 ± 0.09 a color 1.31 ± 0.70 b 1.13 ± 0.29 b 2.5 ± 0.58ª marbling score 0.69 ± 0.50 b 0.81 ± 0.25ª b 1.31 ± 0.27ª loin (cm 2 ) 69.66 ± 3.56ª 58.47 ± 5.84 b 32.92 ± 3.33 c carcass length (cm) 79.76 ± 4.20ª b 72.33 ± 3.54 b 82.39 ± 1.93ª shoulder (kg) 10.70 ± 1.88 9.74 ± 1.06 9.65 ± 0.86 leg (kg) 9.56 ± 0.65 9.57 ± 0.96 8.97 ± 0.85 rib (kg) 9.52 ± 2.28 9.57 ± 1.64 8.96 ± 0.84 abc means ± sd for 8 pigs with different superscript within rows are significantly different (p<0.05). table 4. meat quality evaluation of roast pork from pasture fed and control pigs. item d diets control 50/50 25/75 tenderness 3.69 ± 1.10 3.48 ± 0.90 3.81 ± 1.30 flavor 3.33 ± 0.81 b 3.27 ± 0.79 b 3.97 ± 0.85ª aroma rating 3.93 ± 0.89 3.84 ± 0.83 4.04 ± 0.84 overall acceptability 3.90 ± 0.82ª b 3.73 ± 0.81 b 4.26 ± 0.85ª abc means ± sd for 35 participants with different superscripts within rows are significant (p<0.05). d based on the hedonic scale: 1 as extremely unpleasant and 5 extremely acceptable. restriction of feed. in the present study, the animals fed 25% of commercial diet developed leaner carcasses (p <0.05) with back fat results of 2.48, 2.22 and 1.01 cm for the control, 50/50 and 25/75 groups, respectively. strudsholm and hermansen (2005) also reported thicker back fat levels 1.7, 1.65 and 1.47 cm in control, outdoor with ad-libitum and outdoor with restricted intake, respectively. in addition, the significantly higher (p<0.05) intensity in color of pork from the 25/75 group consuming greater forage and potentially increasing the tocopherol levels was observed. consequently, myoglobin levels and a number of oxidative fibers might have improved too (högberg et al., 2004; rodríguez-estévez et al., 2009). these findings were also confirmed by gentry et al. (2002), tartrakoon et al. (2012) and álvarez-rodríguez et al. (2016). however, lebret and guillard (2005) reported decreased in redness in triceps brachii and bacon, whereas hansen et al. (2006) observed no influence of the production system on meat color. the marbling score, also known as intramuscular fat content (imf) presented a gradual tendency to increase in 25/75 (p>0.065). a possible explanation may be due to the monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) in meat, commonly as a result of high content of linolenic acid (c18: 3ω-3) and linoleic acid (c18: 2ω-6) present in forages (álvarez-rodríguez et al., 2016; nilzén et al., 2001). moreover, marbling in outdoor animals has been associated with an increased protein turnover rate and proteolytic activity in muscle (edwards, 2005). lipoprotein lipase (lpl) and indeed, intramuscular fat (imf) is the main determinant factor influencing sensory attributes of meat (araújo et al., 2011; lim et al., 2015). the pork aroma and tenderness was not significantly affected (p>0.05) among the treatments (table 4). however, a tendency for higher tenderness was observed on meat from 25/75 groups. in addition, flavor and overall acceptability were higher in 25/75 group (p<0.05), while differences were not observed between 50/50 group and the control. these results emphasized the positive effect of pufa content of the forages in the deposition in imf. a similar effect of pufa on pork quality was observed by tartrakoon et al. (2016) and nilzén et al. (2001) resulting in higher antioxidant levels and consequently increase oxidative stability. in a previous study, it was not observed that there was an association between pufa content with changes in the sensory characteristics of pork from korean native black pigs (kim et al., 2009b). gene expression levels of lpl, pparα, pparγ, and srebp-2 in muscle and adipose tissue forages like clover and ryegrass, are rich in pufa 5 figure 1. relative expression of targeted genes involved in lipid metabolism in adipose tissue from berkshire pigs finished on pasture with varying levels of commercial feed. peroxisome proliferator-activated receptor (ppar α, ppar γ), lipoprotein lipase (lpl), and sterol-regulatory-element binding protein 2 (srebp-2) were analyzed using real-time pcr. especially linolenic acid (c18: 3ω-3) and linoleic acid (c18: 2ω-6) (wyss et al., 2006). as previously mentioned, the effect of pufas on meat quality has been widely studied at the molecular level (ivanovic et al., 2015; joven et al., 2014; nilzén et al., 2001). moreover, new implications of pufa have been reported to influence genes involved in lipid metabolism and subsequently affect meat quality traits. in previous studies, trifolium pretense and plant extracts of mulberry, banana, and ginseng were shown to have significant implications in the activation of signaling pathways influencing meat quality traits, principally mediated throughout the ppars binding-activation (park et al., 2005; qiu et al., 2012). in this study, the pasture-based pork production system was compared with pigs fed on commercial feed to evaluate the meat quality and related lipid gene expression. real-time pcr was performed to detect gene expression variation of pparα, pparγ, lpl, and srebp-2. the relative expression levels of these genes are presented in figures 1 and 2. the expression levels differed significantly (p<0.05) among treatments and between tissue sample types. lpl, pparα, pparγ and 6 figure 2. relative expression of targeted genes involved in lipid metabolism in muscular tissue from berkshire pigs finished on pasture with varying levels of commercial feed. peroxisome proliferatoractivated receptor (ppar α, ppar γ), lipoprotein lipase (lpl), and sterol-regulatory-element binding protein 2 (srebp-2) were analyzed using real-time pcr. srebp-2 expression in adipose tissue were highly stimulated in 50/50 treatment than in the control and 25/75 group. however, lpl, pparα, and pparγ in muscle were significantly increased in 25/75 group (figures 1 and 2). peroxisome proliferator-activated receptor alpha (ppar-alpha), also known as nr1c1, is a nuclear receptor protein which in human is encoded by the pparα gene. pparα is activated under conditions of energy deprivation and is necessary for the process of ketogenesis. therefore, the increased relative expression level of pparα (p<0.05) in pasture animals could be related to a metabolic fasting process due to the reduced energy obtained from forages (yang et al., 2013). likewise, ppars signaling pathway is influential in meat quality traits (he et al., 2013; wang et al., 2016). it is known that activation of pparα promotes uptake, utilization and catabolism of fatty acids (vahmani et al., 2014). the leaner carcasses (p<0.05) on free-rearing systems might be explained by the transport and utilization of fatty acids to catabolic routes. luci et al. (2007), in their study using clofibrate, also confirmed an increase in pparα relative expression in adipose tissue, suggesting a direct relationship between the betaoxidation capacity of adipose tissue and pparα expression. on the other hand, a link between pparα and sterol regulatory element-binding protein-2 (srebp-2) has been found (könig et al., 2009). sterol regulatory element-binding proteins (srebps) are transcription factors that regulate lipid homeostasis. srebp-2 is related to cholesterol synthesis (eberlé et al., 2004). therefore, srebp-2 and its target genes relative expression could be attenuated by pparα resulting in lower cholesterol 7 concentration in plasma (könig et al., 2007, 2009). peroxisome proliferator-activated receptor gamma (ppar-γ or pparg), also known as the glitazone receptor, or nr1c3 is a type ii nuclear receptor, which in humans is encoded by the pparγ gene. the relation between imf content and pparγ activation is probably due to its important role in reducing glucose concentrations favoring adipogenesis. lipoprotein lipase (lpl) role is also considered essential in fat deposition (albuquerque et al., 2017). marbling score in 25/75 group was significantly higher than in the other two treatments. a possible explanation for increased imf (marbling score) deposition in animals under higher pasture consumption could be a possible activation of pparγ gene expression. conclusions the findings from the present study suggested that a reduction in swine diet by 25 to 50% plus ad-libitum grazing supplementation of rye-grass/red clover can maintain a sustainable pasture pork production of finishing pigs. the intense grazing and reduced rate of gain resulted in upload of lipid metabolic genes in muscle and adipose tissue, reducing backfat thickness while improving meat quality characteristics. this study showed that pasture-based pork production system could upregulate lipid metabolism genes expression that improved meat quality traits. 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www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper phylogenetic and pathogenetic analysis of streptococcus suis serotype 7 strain hw07 isolated from diseased pig in china shujie wang1,2, sen hu2, jiamin jin1,2, yonggang liu2, gang wang 2, yabin tu2, chenggang jiang2, xuehui cai2* and xiuying zhang1* 1 pharmacology laboratory, animal medical college, northeast agriculture university, harbin 150030, china. 2 state key laboratory of veterinary biotechnology, harbin veterinary research institute, chinese academy of agricultural sciences, harbin 150001, heilongjiang, people’s republic of china. accepted 08 december, 2019 a recent isolate of streptococcus suis serotype 7 (ss7), named hw07, was isolated from brains of pigs suspected with streptococcus suis (s. suis) infection. the sequence types (st) of this isolate was determined by multilocus sequence typing (mlst) method. in order to evaluate pathogenesis of hw07 isolate, zebrafish and pigs were inoculated with hw07 strain by peritoneal cavity and vein. the results demonstrate that the 50% lethal dose (ld50) of hw07 isolate was 1.25 × 10 5 colony forming units (cfu)/fish and could induce pig disease, the st number of hw07 isolate is a novel st-st335. molecular and phylogenetic analysis for the reca gene of hw07 isolate showed that it joins to the i group cluster of s. suis strains, the predominant genotype in china. key words: streptococcus suis serotype 7, multilocus sequence typing, the sequence type, phylogenetic analysis. introduction streptococcus suis is an important pathogen associated with a variety of pig diseases, including meningitis, septicaemia, arthritis, endocarditis and pneumonia and can cause severe zoonotic infection of humans. so far, 35 capsular serotypes of s. suis have been described (perch et al., 1983), serotype 2 strains are considered as the most virulent and often isolated from diseased pigs. however, epidemiological studies in our lab have indicated that the prevalence of ss7 strains from diseased pigs in china has significantly increased during the past 3 years (wang et al., 2012). therefore, in this study, we researched in ss7 strains hw07 isolated from brains of diseased pigs with classic clinical meningitis and arthritis symptoms of s. suis infection, which had resulted in 30% death rate of pigs in farm. pathogenicity can vary substantially both within and *corresponding authors. e-mail: aci139@sina.com, zxy0451@hotmail.com. tel: 86-18946066077, 86-451 55190674. fax: 86-451-55191200. among serotypes, and not all isolates of the same serotype cause the same disease (staats et al., 1997). previous studies have indicated that s. suis is a genetically diverse species (hampson et al., 1993). in order to determine the genotype or sequence of the hw07 isolates, mlst method was used in this study. mlst is a highly discriminatory and unambiguous method of characterizing bacterial isolates that has now been successfully employed in the characterization of several species (enright et al., 2001). mlst was used to investigate the genotype of s. suis as early as in 2002 (king et al., 2002). strains that have the same st number are identical at all of the sequenced loci and are considered to be members of the same clone, which means they have a recent common ancestor. up to august 2012, 665 s. suis strains have been recorded in the s. suis mlst database and classified into 334 sts which belong to 21 st complexes. the reca protein is a multifunctional enzyme that plays a role in homologous recombination, dna repair and induction of the sos response (selbitschka et al., 1991). reca sequencing is an adequate method to discriminate table 1. the primers for virulence-associated gene primer primer sequence(5’-3’) gene protein genbank accession number length of pcr plroduct (bp) gdh gagctcttctctacactt gdh glutamate dehydrogenase ay853916 1257 ttataccaaaccttgggc mrp atcagaatcaccacttttgg mrp muramidase-released protein x64450 [31] 885 tcatacccagtaaatacacg ef cgcagacaacgaaagattga epf extracellular factor x71881 [31] 744 aagaatgtctttggcgatgg sly gctttattgcgtgctgac sly suilysin z36907 [31] 1097 ctgttctccaccactccc among meat staphylococci, s. xylosus and s. equorum. the objective of the current study was to determine the genotype and pathogenesis of hw07 isolates and to investigate if it was the predominant s. suis strain in china by sequence and phylogenetic analysis of the reca gene. materials and methods strains the brain sample was collected from the infected pig and the bacteria was isolated using a method previously described (buddle et al., 1981). the s. suis suspected colonies. the strain 8074 is the international reference ss7 that was stored in our research institute. genomic dna extraction the isolate chromosomal dna was extracted using a method previously described (vaquero et al., 2004) . bacteria grow in thb at 37°c before chromosomal dna was extracted from overnight cultures using tianamp bacteria dna kit (tiangen, china) according to the manufacturer’s instructions. strain identification the s. suis suspected colonies were characterized by morphological, biochemical (api 20 strep, biomerieux, france) and based pcr assay. the presence of s. suis was confirmed by pcr with s. suis gdh gene primers (okwumabua et al., 2003): fp: 5' gcagcgtattctgtcaaacg-3 rp: 5' ccatggacagataaagatgg-3', the expected fragment was 688 bp. serotype 7 was confirmed by pcr with s. suis cps7h gene primers (okwumabua et al., 2003): fp: 5' aatgccctcgtggaatacag-3', rp: 5'tcctgacaccaggacacgta-3', the expected fragment was 378 bp. the identified ss7 clones were stored at 40°c. the virulence-associated genotype of hw07 isolate based on the presence of virulence-associate gene to characterize the ss7 strain, primers based on the following gene are listed in table 1: glutamate dehydrogenase (gdh), muramidase-released protein (mrp), extracellular factor (epf), and suilysin (sly) (silva et al., 2006). pcr was performed to characterize the virulence-associated genotype of hw07 isolate using the four virulenceassociated gene primers. mlst and phylogenetic analysis mlst was performed according to king et al. (2002). pcr amplification of the 7 housekeeping genes include aroa, cpn60, dpr, gki, reca, and thra was done with the primers shown in table 2. the amplified segments were sequenced at huada gene biotechnological co. ltd (beijing, china). mlst alleles and st number of hw07 isolate was analysed in s. suis mlst database (http://ssuis.mlst.net). eburst software (feil et al., 2004) was used to identify the phylogenetic position of strain and display the overall structure of the population. phylogenetic analysis for reca gene the partial reca gene nucleotide of hw07 isolate was sequenced and was sent to genbank, in which the accession numbers for reca was jx236275. sequence similarity searches in the genbank databases were carried out using basic local alignment search tool (blast), and aligned with the corresponding sequences of s. suis strains using the clustal w program in megalign of lasergene 7.0 software (dnastar inc. madison, wi, usa). then, the molecular and the phylogenetic analyses of reca gene was conducted by molecular evolutionary genetics analysis (mega) version 5.05 (tamura et al., 2011). the percentage of bootstrap confidence levels for internal branches, as defined by the mega program, was calculated from 1000 random resamplings. experimental animals 10-week-old inbred line zebrafish, which were purchased from national zebrafish resources of china (shanghai, china) and raised in solated fish bowls, were used to check the virulence of hw07 isolate. six 1-month-old specific pathogen free (spf) pigs, whose serology is table 2. primers used for amplification and sequencing of the seven loci in the s. suis mlst scheme. gene name primer sequence annealing tm (℃) length of pcr(bp) aroa-fp ttccatgtgcttgagtcgcta 55 482 aroa-rp acgtgacctacctccgttgac cpn-fp ttgaaaaacgtrackgcaggtgc 52 466 cpn-rp acgttgaaigtaccacgaatc dpr-fp cgtctttcagcccgcgtcca 50 434 dpr-rp gaccaagttctgcctgcagc gki-fp ggagcctataacctcaactgg 55 480 gki-rp aagaacgatgtaggcaggatt muts-fp cgcagagcagatggaagatcc 50 526 muts-rp cccatagctgttttggtttcatc reca-fp tatgatgagtcaggccatg 50 398 reca-rp cgcttagcattttcagaacc thra-fp gattcagaacgtcgctttgt 52 523 thra-rp aagttttcatagaggtcagc negative to s. suis, were purchased from dongsheng pig form (harbin, china). all animal work and experimental procedures were conducted with an approval of institutional animal care and use committee of heilongjiang, china. pathogenicity test of hw07 isolate to zebrafish the isolated bacteria were harvested from liquid cultures by centrifugation at 5,000 × g for 5 min prior to inoculation in the zebrafish, and were resuspended in phosphate-buffered saline (pbs, ph 7.4). strains of hw07 and 8074 were tested at 5 different doses from 5×10 7 to 5×10 3 cfu/fish, ten fish per dose. the zebrafish were injected through peritoneal cavity, and the control fish were injected with pbs. the infected zebrafish were monitored for 1 week. the test was repeated in triplicate and yielded reproducible results. the results were averaged and used to calculate the ld50 by the method of reed and muench (reed and muench, 1938). the results of biochemical tests (6.5% high salt gravy/ raffinose/ lactose/ sorbose/ mannose/ salicin/ serum dahlin/ hippurate/ esculin/ mushroom sugar: positive/ positive/ positive/ negtive/ positive/ positive/ negtive/ negtive/ positive/ positive) were consistent with the biochemical character of s. suis. the pcr result for isolate was gdh+/cps7h+. the virulence-associated genotype of hw07 isolate in order to determine the virulence-associated gene of isolate, four genes (gdh, mrp, epf and sly) were examined by pcr. as shown in figure 1, the virulenceassociated gene of isolate was gdh+/mrp+/epf--/sly-. pathogenicity test of hw07 isolate to piglet to evaluate the pathogenicity of the hw07 isolate, four-week-old piglets (3 piglets) were challenged with the hw07 isolate (dose of 2×10 8 cfu/piglet), and the control piglets (3 piglets) were challenged with pbs. clinical signs and rectal temperatures of the piglets were daily recorded until the piglets were euthanized with nembutal at the end of the experiment. all the tissue samples of infected piglets were obtained. bacteria were re-isolated from various tissues. results morphology and characteristics of recently ss7 isolate a wild-type ss7 isolate, named hw07, was isolated from the brain of the infected pig, which was gram positive chain shape coccobacteria by microscope observation. mlst and phylogenetic analysis partial sequences of 7 housekeeping genes for isolate revealed a low genetic variation, yielding one novel st number-st335 whose allelic profiles was 8, 30, 5, 34, 9, 3, 25. the closest matches are st129 (8, 30, 5, 34, 58, 3, 25 ), st83 (8, 30, 5, 34, 39, 3, 25 ) and st29 (8, 30, 5, 34, 30, 3, 25 ). from the above results we can conclude that five isolates come from a clone. the phylogenetic position of the isolate in the s. suis database and the overall structure of the population are displayed in figure 2. phylogenic and sequencing analysis of reca gene within hw07 isolate phylogenetic analysis of reca gene was conducted in hw07 isolate and 23 s. suis strains that were obtained 1 2 3 4 5 figure 1. pcr products of the virulence-associated gene of the hw07 isolate. lanes 2, 3, 4, 5 are pcr products using primers for sly, ef, mrp, gdh genes of strain hw07, respectively. lane 1 is dl 2000 marker. figure 2. analysis of the st25 clonal complex of s. suis. eburst groups were obtained from the entire s. suis public mlst database with the stringent (default) group definition; the eburst group that included st25 is displayed. the primary founder, st25 (bootstrap confidence value of 100%), and a major subgroup founder, st29, are labeled. from the genbank database. as shown in figure 3, hw07 isolate was grouped in one branch with chinese strain hb1001, s196 and bj0401, but in a different branch obviously with the chinese strain 40. the homologies for hw07 isolate and strains of hb1001, s196, bj0401 were higher with 95.2-95.5% identity. the reca gene of hw07 isolate showed a 94.1, 93.5, 93.2, 90.4% identity with japan strains dat301, dat289, figure 3. phylogenetic tree between reference s. suis strains from the genbank and hw07 isolate based on the nucleotide sequence of reca gene. table 3. ld50 of hw07 isolate and the reference strain 8074. strain infectious dose (cfu) total death rate ld50 (cfu) hw07 5×10 3 5×10 7 23/50 1.25×10 5 8074 5×10 3 5×10 7 10/50 3.70×10 7 control 0/10 ∞ dat291 and dat298, then 89.8 and 94.6% identity with two united kingdom strains, respectively. there was a poor homology between the nucleotide sequence of reca gene in hw07 isolate and canada strain atcc43765. the lowest homology of 81.6% was found between reca genes in hw07 isolate and chinese strain 40. pathogenicity test of hw07 isolate to zebrafish in order to test the virulence of hw07 isolate, zebrafish were challenged with hw07 isolate. as shown in table 3, control fish injected with pbs suffered no mortality. clinical symptoms showed that branchia and hypogastric region of the diseased fish bleed. the ss7 were reisolated from all organs of all dead zebrafish. results suggest that the pathogenicity of hw07 isolate was stronger than international standard ss7 strain 8074. pathogenicity test of hw07 isolate to piglet the piglets in isolate-infected showed body temperature elevated, ear purple, diarrhea and weight loss. no macroscopic lesions were observed in the control pigs. the piglets in infect showed sub-mandibular lymph node figure 4. pathological examination of tissues. (a) lung-diffuse interstitial pneumonia was observed with marked thickening of alveolar septa by infiltration of lymphocytes; (b) brain edema; (c) kidneya few of lymphocytes inflitrated in interstitial substance. (d) lung tissues from control group. (e) brain tissues from control group. (f) kidney tissues from control group. the tissues were stained with hematoxylin and eosin. bar, 100 um. lesions, including intumescentia and hemorrhage. the affected lung and kidney lesions appeared bleeding point on the surface. the control pigs showed no pathological lesions. the pathological brain lesions in infected pigs showed mild edema. the pathological lung lesions in infect pigs showed the following characteristics: intravascular thrombosis and widened alveolar septa with neutrophil suppurative lesions. the pathological kidney lesions showed lymphocytes infiltration (figure 4). bacteria that were isolated from all organs were identified as hw07 isolate. discussion in this research, a recent isolate come from s. suis infected pigs in heilongjiang province. the identification of the isolate had been confirmed by morphological biochemical and molecular analysis. mlst was used to determine the genotype of hw07 isolate. as a result, one novel st was found whose allelic profile was 8, 30, 5, 34, 9, 3, 25. allelic profiles of st129, st83 and st29 were the closest matches with allelic profile of hw07 isolate. from the eburst (figure 2), we can see that st25 is the primary founder of this group and st29 is the subgroup founder of this group. this group should be st25 clonal complex in which serotype 7 strain is the main representative strain. so, st335 belong to st25 complex, in which strains appeared less associated with human invasive disease (king et al., 2002). the result of pathogenicity experiment hinted that the hw07 isolate was virulent. in the research of pian et al.(2012). the s.suis-05zyh33-infected piglets die after infection 2 days. therefore, the possibility is suggested that the virulence of hw07 isolate was weaker than strain 05zyh33 from human invasive disease, stronger than international standard ss7 strain 8074. the reca protein is composed of about 350 amino-acid residues (karlin et al., 1995; roca and cox, 1997), which sequence is very well conserved among eubacterial species (cerutti et al., 1992) and the bacteria. however, in reca housekeeping genes of hw07 isolate, 2 points of deletion at position 340 and 334 leads to 7 amino acids substitution from 112 position to 118 position (substitution trp, tyr, ser, tyr, asn, gly, glu to gly, ile, ile, ter, ter, leu, gly). whether or not these 7 amino acids substitution affect virulence of hw07 isolate will be further researched in our next study. phylogenetic analysis for reca gene indicated that four clades of wild-type strains have existed in 23 reference s. suis strains. the reca gene phylogenetic relationships between hw07 isolate and other s. suis wild-type strains from genbank have been clarified in this research. in this report, hw07 isolate, identified from heilongjiang province in 2007, displayed the highest identity with chinese ss7 strain bj0401 and lowest identity with chinese strain 40 isolated from lung tissues with apparent hemorrhagic lesions in zhejiang province, thus, hw07 isolate can be classified into chinese predominant genotype. acknowledgments this work was supported by the national technology importance program for the 12th five-year plan (2012zx10004214-005-013), heilongjiang province brilliancy young science found (jc201020), and harbin tackle key problems in science and technology plan (2010aa6an083). references buddle jr, jones je, pass da, robertson j (1981). the isolation of streptococcus suis type ii from a pig with meningitis. aust. vet j. 57:437-438. cerutti h, osman m, grandoni p, jagendorf at (1992). a homolog of escherichia coli reca protein in plastids of higher plants. proc. natl. acad. sci. usa 89:8068-8072. enright mc, spratt bg, kalia a, cross jh, bessen de (2001). multilocus sequence typing of streptococcus pyogenes and the relationships between emm type and clone. infect. immun. 69:2416-2427. feil ej, li bc, aanensen dm, hanage wp, spratt bg (2004). eburst: inferring patterns of evolutionary descent among 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okwumabua o, chengappa m (1997). streptococcus suis: past and present. vet. res. commun. 21:381-407. tamura k, peterson d, peterson n, stecher g, nei m, kumar s (2011). mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol. 28:2731-2739. vaquero i, marcobal á, muñoz r (2004). tannase activity by lactic acid bacteria isolated from grape must and wine. int. j. food microbiol. 96:199-204. wang s, liu p, li c, tan y, cai x, zhou d, jiang y (2012). isolation and characterization of 89k pathogenicity island-positive st-7 strains of streptococcus suis serotype 2 from healthy pigs, northeast china. sci. world j. 2012:302386. 1 author(s) retain the copyright of this article. full length research paper northern ugandan pig farmers' utilization of labor jendyose islamic university in uganda, mbale, uganda accepted 8 december, 2024 pig (susscrofa domesticus) production is becoming a more significant source of food security and revenue for smallholder farmers in several sub-saharan nations. this is explained by the increased likelihood of market monitoring for pork, which is fueled by population expansion, urbanization, and the shift in diet toward a higher proportion of animal protein per person. consequently, one of the practical methods to lift smallholder farmers out of poverty and food insecurity is to increase pig production. few research have been conducted on the distribution of innovative behavior and the socioeconomic factors that affect labor use in the region, despite the fact that there are many studies on the aspects of pig production, such as feeding, breeding, and space requirements. finding the socioeconomic factors that affect the distribution of innovative behavior and the use of labor (family or hired) among pig farmers in northern uganda was the aim of this study. we characterized smallholder pig farmers in northern uganda based on the type of labor they employed to produce pigs using a cross-sectional survey and descriptive analysis. we also investigated how the four aspects of innovation behavior— exploration, experimentation, adaptation, and modification—were distributed among these farmers. the findings showed that young, educated farmers who had numerous pigs, were part of a farmer group, had a smaller household size, and had non-farm jobs were more likely to use hired labor than those who did not. the proportion of farmers who displayed the various aspects of innovation behavior varied significantly. therefore, the socioeconomic disparities among farmers that define the labor restrictions they experience should be taken into account when designing interventions to increase pig production through the use of hired labor. key words: innovation behaviour, labour utilisation, pig production. introduction the worldwide, pigs (susscrofa domesticus) play a significant role in the production of livestock. since 2000, the production of pork in sub-saharan africa (ssa) has more than tripled; uganda produces 12% of the region's pig meat from over three million pigs per year (harvestchoice, 2015). according to tatwangire (2014) and ndyomugyenyi and kyasimire (2015), the central area has the highest concentration of pig rearing households (56%), followed by the western (30.1%), eastern (28.8%), and northern regions (14.2%). pig production is increasing, which makes other agricultural sub-sectors more competitive for important production resources. pig farmers must therefore continually decide how much money, land, and labor to devote to each business in order to meet the household's overall goal of having enough money and food security. the most crucial production factor for pig farming households, aside from land, is labor, which places limitations on management and production when it is scarce (bedemo et al., 2013). in order to develop methods to boost the production of pork and other pig products, it is crucial to comprehend the labor allocation decisions made by farmers in the pig sub-sector. these treatments also call for innovation behavior, a crucial facilitator that enables farmers to maximize profits by optimizing combinations of agricultural inputs. the degree to which farmers, subject to social, economic, technological, and environmental restrictions, investigate, test, or modify a novel concept or technique, or enhance an established practice, is known as innovation behavior (schiederig et al., 2011; liao, 2017). its four dimensions are as follows: (i) exploration of new practices, which is the search for innovations and technology; (ii) experimentation of new practices, which is the tendency to test new technology and notice subtle changes; (iii) adaptation of new practices, which is the process of acclimating new tools, practices, and technologies to local conditions, resource endowments, and individual and community preferences; and (iv) modification of african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-006, december 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 existing practices, which is the enhancement of existing practices (popadiuk and vidal, 2009; tambo and wünscher, 2014; bragdon and smith, 2015). according to èandek-potokar et al. (2017), farmer innovation is a game changer in the efforts to commercialize smallholder pig farming. the development of labor-saving pig production technologies, such as indigenous microorganisms (imo) technology, which lowers the amount of labor needed to process and dispose of pig manure, is another way that innovation can lower labor demand. farmers must experiment with novel pig rearing techniques, adapt them, and/or change current ones in order to innovate. to what degree pig farmers experiment, research, adopt new pig rearing techniques, or alter current ones is unknown, though. although it is well known that a large number of smallholder farmers produce pigs primarily using family labor (ciaian et al., 2012), little research has been done on the socioeconomic determinants that make them more likely to employ hired or family labor. therefore, the goal of this study was to identify the socioeconomic factors that affect the distribution of the dimensions of innovation behavior among pig farmers in northern uganda, as well as the utilization of various forms of labor (hired or family). materials and methods study area between october and november 2018, this study was carried out in the northern ugandan districts of gulu and omoro. the districts are 600–1,200 meters above sea level and are located between latitudes 32.4467° and 32.4920° e and longitudes 2.7152° and 2.8186° n. the majority of homes in the area only retain six to twenty pigs, making smallholder pig farming the predominant practice. 60% of the labor is done by women, and 64% of the households raise native pig breeds, which are mostly handled through tethering (ikwap et al., 2014). the study was carried out in the paicho sub-county in aswa county in gulu, and in the koro sub-county in torchi county in omoro region. research design and sampling data from smallholder pig farmers were gathered for the study using a cross-sectional design. because it is a onetime study strategy, the design was selected because it is economical with regard to both time and money (levin, 2006). to choose study participants, a multi-stage sampling procedure was used. first, a purosive selection process was used to choose two districts, followed by one subcounty per district. due to reports of a profitable market and high turnover for pigs and pork in those administrative units, the sub-counties of paicho (gulu district) and koro (omoro district) were chosen (ikwap et al., 2014). through simple random sampling, three parishes were selected from each sub-county. in paicho sub-county, pagik, kal-umu and kalali parishes were selected; while in koro subcounty, pageya, labwoch and guna parishes were selected. a complete list of all pig rearing households in the selected parishes was obtained from the respective sub-county headquaters and used to select the study sample of 239 respndents; the number which was determined using yamane’s formula (yamane, 1967): = 239 ........……………………………..equation 1 where: n = population, n = sample size, e = degree of confidence level at 95%. the 239 respondents were distributed between paicho and koro in portions of 143 and 96 pig farmers, respectively, based on pig farmers total populations per district. data collection a semi-structured questionnaire that had been pre-tested was used to gather primary data. pre-testing was conducted on ten pig farmers in the unyama sub-county, which was not one of the sub-counties to be investigated despite being close to the study area and having a comparable number of pig farmers. following pretesting, questions that were consistently answered and understood by respondents were kept; questions that the researcher thought were crucial but had previously been left out were added. repetitive and/or unnecessary questions were eliminated. in several instances, questions were rearranged to guarantee logical sequence. the local dialect, acholi, was used to give the questionnaires, but english was used to record the answers. face-to-face interviews with pig farmers conducted at their houses were used to get the data. by building a relationship with the respondents, we were able to clarify any unclear responses and ensure that all questionnaire items were answered. the survey included both closed-ended and likert scale questions, asking respondents to score different items to make sure the questions were clear enough for them to answer. there were three sections to the questionnaire. routine sociodemographic data (table 1) and the type of labor employed in the pig enterprise were recorded in part 1. the variables included the number of casual and permanent laborers hired annually, the use of family labor in the pig enterprise (1 = yes, 0 = no), and the hiring of labor for the pig enterprise (1 = yes, 0 = no). data on pig production, marketing, and access to institutions and institutional services were collected in the second section of the survey. farming experience (years), current pig stock (number of pigs), distance to the closest market, access to extension services (1 = yes, 0 = no), and credit availability (1 = yes, 0 = no) were the variables in this case. data on farmer innovation behavior was gathered in part three and organized into four dimensions. a five-point likert scale, with 0 denoting not at all, 1 seldom, 2 occasionally, 3 frequently, and 4 always, was used to score each of the 12 items used to gather data on innovation behavior (sullivan and artino, 2013). since the used scale lacked a neutral point, the intensity of innovation was measured as it progressed from one end of a continuum to the other. (i) i am very interested in learning how to properly feed pigs; (ii) 3 i enjoy learning new ways to house pigs; and (iii) i like to take the chance of taking part in training on new methods of pig disease and parasite control were the items assessed on the likert scale for investigation. the following were the items for experimentation: (i) i enjoy trying out new pig feed management techniques; (ii) i am typically the first of my classmates to try out new pig rearing techniques; and (iii) i enjoy trying out new methods for building pig housing structures. the following things were included in the adaptation dimension: (i) i modify new pig feeding techniques to accommodate my circumstances; (ii) i modify new pig housing practices to accommodate my circumstances; and (iii) i modify new parasite and disease control procedures to accommodate my farming circumstances. the final items for changing current pig rearing practices were: (i) i know exactly how to change current pig housing practices; (ii) it's simple to change current pig parasite and disease control practices; and (iii) i apply new information to change current pig feeding practices on the farm. data analysis. gender, household head age (years), farming experience (years), household size, non-farm employment, distance to the closest market (kilometers), and education (years) were the independent variables examined for their ability to predict the usage of family or hired labor by smallholder pig farmers. while means, standard deviations, and t-tests were used to analyze continuous variables, frequencies, percentages, and chisquare tests were used to describe the categorical variables. for studying the distributions of the four dimensions of innovation behaviour, indices were computed from likert scale data from their respective items using the equation 2: to understand the level of exploration, experimentation, adaptation and modification in the study area, the farmers’ innovation behaviour with respect to these dimensions was classified into five groups; namely none, low, medium, high and very high based on each respondent’s exploration, experimentation, adaptation and modification index. no activity included farmers with a corresponding index of zero, 0.01-0.32 for very high innovation behaviour with regards to the dimension of interest. the described classification was adapted with modifications from tirfe (2014) and chopeva et al. (2015) results and discussion smallholder pig farmers' characteristics according to the kind of labor they employ in age, household size, education level, group membership, number of pigs and off-farm employment significantly (p< 0.05) influenced pigfarmers use of different types of labour (table 1). farmers who use both hired and family labour were significantly (p< 0.05) younger than their counterparts who use only family labour. this finding conforms to an earlier study in nigeria by echebiri and mbanasor (2003), which reported that in abia state, households who relied on only young people for provision of farm labour had less food production. this resulted from the fact that younger farmers were more likely than older farmers to engage in non-farm activities. young farmers in the current study are more likely to contract labor for pig production because they devote a portion of their time to jobs that generate revenue other than farming. this finding suggests that younger smallholder pig farmers are more likely than their older counterparts to generate more job possibilities for their peers. the size of the household had a substantial (p<0.05) impact on how much family labor was used by pig producers. large-family farmers typically relied entirely on their family members to provide the labor required, whereas small-family farmers employed outside help to raise pigs. this outcome is in line with research by nmadu and akinola (2015), who discovered that family size significantly influences the availability and use of farm labor among nigerian crop producers. according to this finding, smallholder farmers value having wives and/or kids in order to secure this labor source. therefore, households with larger family sizes have more labor available for pig production than households with smaller family sizes. however, the current study did not examine whether households made efficient use of the large number of workers provided by large families for the production of pigs. the utilization of family or hired labor was significantly impacted by education (p<0.01). compared to farmers who just employed family labor, those who used both hired and family labor had substantially higher levels of education. higher educated farmers are more likely to work outside the farm, according to reports (alassaf et al., 2011).as a result, these farmers are less involved in agricultural operations and are more likely to use hired labor to produce pigs. this finding suggests that education improves a farmer's capability to absorb, understand, and apply information that is pertinent to judgments on farm labor. group membership had a big impact on the kind of work that pig farmers did. while some employed both family and hired labor, the majority of farmers (41.00%) who were part of groups only used family labor. in acholi, the term "aleya i poto" (meaning "labor round") referred to the practice of farmers working together in groups. by increasing labor availability, this farmer peer support practice within groups lowers the demand for and perhaps lowers the cost of hired labor. therefore, among smallholder pig farmers in northern uganda, being a member of a farmer group may improve the supply of labor and lower labor costs. the usage of the two forms of labor by pig farmers was also considerably impacted by off-farm employment. only a small percentage of pig farmers with off-farm jobs (12.97%) employed only family labor; the majority (44.77%) used both hired and family labor. this is due to the fact that off-farm work provides pig farmers with extra cash that they can utilize to grow their pig production business, necessitating the hire of additional workers to augment family labor. the number of pigs, or the size of the pig herd, statistically affected how farmers used the various forms of labor. more labor 4 was needed by farmers who raised a large number of pigs to handle the different management tasks. as a result, they were more likely than individuals with little holdings to need hired labor. the ability of farmers to recruit and oversee additional labor to supplement family labor in specific situations should therefore be supported by interventions aimed at commercializing smallholder pig production. the type of labor employed by smallholder pig farmers did not change significantly according to gender, market distance, loan availability, pig rearing experience, or access to extension services. distribution of the dimensions of innovation behaviour. exploration, experimentation, and adaptation were all skewed to the left, but only changes to the current methods of pig rearing were distributed normally. all farmers demonstrated some degree of exploration, according to the results in figure 1, and the majority of them (86.61%) scored extremely highly on this innovation behavior characteristic. the proportion of farmers who displayed the various aspects of innovative behavior varied significantly (fig. 1). this indicates that neither mutual exclusion nor collective exhaustion existed in the distribution of innovation behavior aspects. this meant that a farmer could only explore and not try the other dimensions. however, a farmer does not have to investigate or test a method before changing an existing pig rearing practice. for agricultural researchers, extension agents, and policymakers, this finding has significant ramifications. smallholder farmers may not always adopt and utilize new technologies and innovations more effectively just because they are available (lemessa et al., 2018). as a result, implementing new agricultural technologies may need addressing other issues that restrict farmers' goals, like insufficient market connections and restricted financial availability. with a very high experimentation score, experimentation had a significantly lower number of farmers (47.28%) than exploration. since exploration is merely observing or listening to what other people are doing or saying without necessarily taking the initiative to try out or change the same, the majority of farmers (86.61%) were found to be quite exploratory. this finding suggests that smallholder pig farmers may find it simpler to attend trainings and/or extension meetings than to put what they have learned into practice (moschitz et al., 2015). as a result, in order to encourage farmers to try out new pig rearing techniques and to promote co-innovation and co-learning, innovators must actively involve farmers in technological development for the development of smallholder agribusinesses among pig farmers. in terms of adaptation and modification dimensions, the proportion of farmers who were inactive for these two aspects of innovative behavior increased (fig. 1). this is due to the fact that these two aspects of innovation behavior necessitate a significant amount of labor to modify what is seen or heard to one's preference or to accommodate one's living and working circumstances (wettasinha et al., 2014). this finding suggests that moving from one aspect of innovation behavior to the next takes work. the labor demand for the pig production business rises as a result of the effort required for a farmer to innovate by effectively progressing from exploration to testing and adaptation to modification. as a result, farmers with small family labor forces must also hire more workers. according to ndambiri et al. (2012), this suggests that families with larger family labor forces or those with hired labor will probably exhibit more innovative behavior. as a result, initiatives to commercialize smallholder pig farming through innovative farmer behavior ought to increase farmers' ability to recruit and oversee more workers. conclusion according to this study, young, educated farmers who have numerous pigs, a smaller household size, are part of a farmer organization, and work outside the farm are more likely to use hired labor than those who don't. the socioeconomic disparities among farmers, which have been shown to influence the labor limitations they encounter, should therefore be taken into account in programs aimed at increasing pig output through the use of hired labor. the proportion of farmers who displayed the various aspects of innovative behavior varied significantly. an expansion of this research will look at the variables that affect pig farmers' innovative behavior. acknowledgement the authors are grateful to the mastercard foundation and regional universities forum for capacity building in agriculture (ruforum) for funding research and publication cost through the transforming african agricultural universities to meaningfully contribute to africa’s growth and development (tagdev) program. references alassaf, a., majdalwai, m. and nawash, o. 2011. factors affecting farmer’s decision to continue farm activity in marginal areas of jordan. african journal of agricultural research 6(12): 2755–2760. doi: 10.5897/ ajar11.481. bedemo, a., getnet, k. and kassa, b. 2013. determinants of household demand for and supply of farm labour in rural ethiopia. australian journal of labour economics 16(3):351– 367. bragdon, s. h. and smith, c. 2015. smallscale farmer innovation. quaker united nations office. geneva: available at: https://quno.org/resource/2015/12/small-scale farmer-innovation. chopeva, m. a., nikolov, d. and yovchevska,p. 2015. farmers ’ adaptation: what factors affecting agricultural innovations? proceedings of the 147th european association of agricultural economists (eaae) seminar. institute of agricultural economics, sofia, bulgaria. 1:1–13. ciaian, p., falkowski, j. and kancs, d. 2012. access to credit, 5 factor allocation and farm productivity: evidence from the cee transition economies. agricultural finance review 72(1):22–47. doi: 10.1108/0002146 1211222114. èandek-potokar, m., nieto, r., pugliese, c., araujo, j. p., charneca, r., garcia casco, j. m., sanchez, g. e., hernandez-garcia, f. i., izquierdo, m., karolyi. d. and kušec. g. 2017. local pig breeds: nutritional requirements, innovative practices and local feeding resources as challenges in projecttreasure. agriculturae conspectus scientificus 82(2):127–131. echebiri, r. n. and mbanasor, j. a. 2003. rural age distribution and farm labour supply in crop production systems in abia state, nigeria. tropical and subtropical agroecosystems 2(1):129–136. harvestchoice. 2015. pig population (head, 2005). international food policy research institute, washington, dc and university of minnesota, st. paul, mn, usa. http:// harv estchoice.org/data/an05_pig. 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– a quantitative literature review. proceedings of he xxii ispim conference. available at: http:// ssrn.com/abstract=1846882. sullivan, g.m. and artino, a.r. 2013. analyzing and interpreting data from likerttype scales. journal of graduate medical education 5(4):541–542. doi: 10.4300/ jgme-54-18. tambo, j.a. and wünscher, t. 2014. more than adopters: the welfare impacts of farmer innovation in rural ghana. proceedings of the agricultural & applied economics association’s annual meeting 2014. minneapolis. tatwangire, a. 2014. uganda smallholder pigs value chain development: situation analysis and trends. nairobi, kenya: international livestock research institute (ilri). tirfe, a.g. 2014. smallholder farmers’ innovation and its determinants: the case of hirity mekan seed producers’ cooperative , tigray, ethiopia. developing country studies 4(21):104–115. wettasinha, c. waters-bayer, a., van veldhuizen, l., quiroga, g. and swaans, k. 2014. study on impacts of farmer-led research supported by civil society organizations. cgiar research program on aquatic agricultural systems. penang, malaysia. working paper: aas2014-40. yamane, t. 1967. statistics, an introductory analysis, 2nd ed. new york: harper and row. 886pp. 6 figure 1. levels of the dimensions of innovation behaviour among smallholder pig farmers in northern uganda. introduction acknowledgement 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-008, january, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper assessing nutrient leaching in red-yellow latosol: the role of swine water and irrigation water reuse talita dantas pedrosa1*, henrique takyiuki ozima2, roselene maria schneider2, adilson pacheco de souza2, ednaldo antonio de andrade1 and luciana vieira mattos1 1 institute of natural, human and social sciences, federal university of mato grosso sinop campus, alexandre ferronato avenue, 1200, 78557-267, sinop (mt), brazil. 2 institute of agrarian and environmental sciences, federal university of mato grosso sinop campus, alexandre ferronato avenue, 1200, 78557-267, sinop (mt), brazil. accepted 10 august, 2023 this study evaluates some microelements, phosphorus, copper and zinc leached in lysimeters with red-yellow latosol under different rates of reused water and irrigation water. this was done for a period of 40 days (august to october 2014) at the initial cycle of cauliflower cultivar ‘verona cms’, in sinop/mato grosso. reused swine water (0, 50 and 150 m 3 ha -1 ) was applied in one portion before transplanting. irrigation water of 100, 125 and 150% with crop evapotranspiration potential (etc) was used in a drip irrigation system daily. leachate samples were taken at 10, 20, 30 and 40 days after applying the reused water. with balanced leached phosphorus (p), a higher percentage of p retained in the soil was observed, indicating low mobility of this element. the concentrations of copper in the leachate were low at 150 m 3 ha -1 rate after the reuse of swine water, at day 20. 150 m 3 ha -1 used in the irrigated lysimeters with 100% etc is a good alternative for vegetable crops with a short cycle (less than 40 days), in sinop/mato grosso, as it does not exceed the limits of the conama resolution no. 396/2008. key words: cauliflower, evapotranspiration potential, lysimeters, irrigation. introduction the use of swine wastewater in agriculture is an alternative source of nutrients and organic matter (bertol et al., 2010); it grows combined with available elements such as phosphorus (p), copper (cu) and zinc (zn) (smanhotto et al., 2010). although these elements are essential for the growth and development of plants, when applied in excess, they can become sources of surface and groundwater pollution. phosphorus losses can occur via soil surface and subsurface, being potentially greater in sandy soils when subjected to high mineral or organic fertilizers. another problem associated with p is the eutrophication of surface water that results from superficial runoff and leaching. when leaching occurs in groundwater it may *corresponding author. e-mail: tdpedrosa2@yahoo.com.br. tel: (+55) 66 99622-0592. 2 affect surface waters (rivers and lakes) due to percolation. however, the soil has a high phosphate adsorption capacity, and its mobility is lower compared to other nutrients; in some cases, phosphorus loss by percolation in croplands is considered insignificant. bertoli et al. (2010) showed that phosphorus can be lost with greater sensitivity under reused swine water compared to fertilization with npk formulation, thus demonstrating that the transport of p is higher under organic sources. according to campos (2010), the movement of cu and zn in the soil profile depends on the physical and chemical properties of each type of soil and the physicochemical properties of the metal ion. cu and zn metals have low mobility in soil profiles and therefore tend to accumulate on the ground surface, reducing its leaching potential. however, excessive use of wastewater can cause losses in the subsurface (sistani et al., 2008). campos (2010) emphasizes that ph variation, biological processes, and chemical toxicity of the element and environment also play a fundamental role in the availability and mobility of these metals. according to rieuwerts et al. (2006), ph has an influence on cationic metal ions, as these ions are more mobile in acid ph conditions. however, in ph higher than six, these would possibly lead to the dissociation of h + of oh groups of the organic matter and fe and al oxides. this would thus increase the absorption of the metals with subsequent precipitation, resulting in the reduction of its bioavailabilities. in this sense, this study aims to evaluate phosphorus, copper and zinc transported in lysimeters with red-yellow latosol, after the application of swine reused water and irrigation water at different rates. materials and methods experiment location the research was conducted at the federal university of mato grosso, sinop university campus, located in 11°51’s and 55°29’w, from august to october 2014. precipitation occurred during the experiment. the soil of the experimental area is classified as red-yellow latosol. by the köppen climate classification, the prevailing climate of the north central region is aw (hot and humid tropical). it is characterized by the presence of two well defined seasons: rainy (from october to april) and dry (from may to september); with low annual temperature range (between 24 and 27°c) and average annual rainfall of 1974 mm (souza et al., 2013). lysimeters for the leaching study, twenty-seven lysimeters were built, and arranged in plate. the distance between each lysimeter was 0.50 m; they were inserted in trenches of approximately 1.20 m depth and 0.30 m diameter (figure 1). the structure of the lysimeters was hard pvc filled with soil, keeping the same sequence as in the original profile. the chemical and physical analysis of the soils was performed at two depths (0 to 20 cm and 20 to 40 cm) for subsequent filling of the lysimeters. to fill the lysimeters, an isolated trench was opened at approximately 1.0 m depth; undisturbed samples were taken from every trench at a distance of 10 cm to determine the soil density. the soil profile in the lysimeter was rebuilt to keep the densities of the respective layers. the lysimeter was provided with a collection system (funnel and bottle) positioned at the bottom of the trench, and wherein leachate was stored for later collection. chemical and physical soil analysis in the chemical analysis performed at soil layer of 0 to 20 cm, 2.46 to 32.00 mg dm -3 was determined for phosphorus and potassium, and 2.03 and 1.72 cmol dm -3 for calcium and magnesium, respectively. the micronutrients, zinc and copper concentrations were 3.90 and 0.59 mg dm -3 , respectively; the concentration of aluminum was zero, the ph of h2o was 5.4; cation exchange capacity (cec ph7.0) was 6.98 cmol dm -3 and organic matter content was 38.22 g dm -3 ; for the textural analysis, 462, 250 and 288 g dm -3 was identified for the clay, silt and sand, respectively. at 20 to 40 cm layer, for the same aforementioned variables, 4.61 and 55.00 mg dm -3 was obtained; 3.75 and 1.30 cmol dm -3 , 85 and 0.84 mg dm -3 was obtained for p, k, ca, mg, cu and zn, respectively; ph (h2o) was 5.9; cation exchange capacity (cec ph7.0) was 8.01 cmol dm -3 and organic matter content was 43.00 g dm -3 ; for the physical analysis of the clay, silt, and sand contents, 483, 167 and 350 g dm -3 respectively was obtained. chemical and physical characterization of reused water swine reused water was collected from a farm in the municipality of vera/mato grosso, after treating with biodigesters. the chemical and physical characteristics of the waste (reused water) were determined. the following was obtained: 6.85 ph, 4.970 ntu turbidity, electrical conductivity of 1.1 s m -1 , total dissolved solids concentration of 7.0 g l -1 , biochemical oxygen demand (bod) of 283.3, total kjeldahl nitrogen (tkn) of 308.7, nitrite (no2 ) of 154.7, nitrate (no3 ) of 811.36, total phosphorus (p) of 150.29, zinc (zn) of 35,90 and copper concentration (cu) of 10.88 mg l -1 . implementation of cultivation after the chemical and physical soil characterization, the supplement chemical fertilizer was calculated following the technical recommendations of zanuzo et al. (2013) for cauliflower cv. verona. in this context, the fertilizer used for cultivation corresponded with 10 g of urea, 15 g of potassium chloride, 20 g of simple superphosphate and 12.5 g of dolomite lime added to the surface of each of the lysimeter before transplanting the seedlings. transplanting of cauliflower seedlings was performed in (brassica oleracea l.) verona cms variety, manually in each lysimeter, in 08/03/2014. the spacing was set at 0.50 x 0.50 m (between plants and rows). after transplanting, drip irrigation system was installed. daily irrigation was done for 40 days by using dripped polyethylene hose with 25 cm space between emitters, outflow rate of 7.5 l h -1 m -1 and 10 mwc working pressure. reused water and water irrigation rates the reused water was applied once on the surface of the lysimeters, before transplanting the seedlings at three application rates of 0, 50 and 150 m 3 ha -1 yr -1 . the percentage rates of water 3 figure 1. construction scheme of the lysimeters and disposal in plat with a variation of water irrigation rates and reuse water rates. used for irrigation were 100, 125 and 150% of crop results and discussion evapotranspiration (etc), obtained from equation 2. the rates were determined according to the daily reference volume of water applied and collected evapotranspiration (et0), obtained by the method of class a tank. it considers the product between the evaporation of class a tank the highest percentage of etc generated higher volumes (eca) and the tank coefficient (kp), depending on the tank type, weather conditions and its location. with an average value, kp of water used for irrigation, regardless of the season; total (0.7795) was estimated for the municipality of sinop/mato gross. volume of 179.73, 224.67, 269.61 l of water led to the value of the crop coefficient kc (0.65) was used in the etc increments of 0.25 and 50% etc. however, there was a calculation. tendency to reduce the volume applied daily throughout et0  eca * kp the experimental time for all slides due to the reduction of (1) et0 (table 1). et  et * kc this research was conducted in an area with annual (2) rainfalls of 2,000 mm yr -1 , in seven months, from october c 0 to april (souza et al., 2013). thus, using irrigation water where, et0 is the daily reference evapotranspiration (l m -2 ); etc is rates (125 and 150%) higher than the 100% etc is the evapotranspiration of daily culture (l m -2 ); eca is the fundamental for the generation of leachate and evaporation of the daily class a tank (l m 2 ); kp is the coefficient of understanding the movement of p, cu and zn, in periods the tank; kc is the crop coefficient depending on the development of rainfalls higher than the demand for cauliflower. stage. as for the volume of water collected, the data showed a significant interaction between the irrigation rate and collection and samples analysis time, and also between water reuse rate and irrigation four leachate collections were done (10, 20, 30 and 40 days after rate. in table 2, it was observed that, the collection volume was greater with higher supply of water for application of the wastewater) with the experiment. the leached irrigation. in all the collection times, there was a reduction elements evaluated were p, cu, zn ions and h + concentration, and the volume of water applied and collected was monitored. the in the volume collected at 125 and 150% etc rates. this analyses were carried out in the waste treatment and integrated reduction is caused by irrigation since excess water laboratory for research in chemical sciences, following the promotes translocation of solid particles of soil (mainly methodology described in standard methods of water and clays), which in turn favors the process of soil storage wastewater (apha, 2012). within the lysimeters, reducing its permeability over time. however, even with the replacement of 100% etc, the evaluation and statistical analysis of data formation of leachate lysimeters was observed. the experimental design is a randomized block subdivided into a according to barros et al. (2009), the determination of the reference evapotranspiration (et0) by class a tank factorial plot of 3 x 3 x 4 (application rates x irrigation water rates x provides overestimation even when kp is regionally collection times), with three repetitions. the results obtained were calibrated. the total volume collected at 100, 125 and statistically evaluated and submitted to analysis of variance and f 150 etc was 8.86, 10.72 and 12.72% of the applied test; the means were compared by the scott knott test at 5% volume, respectively. significance. the statistical package used was sisvar 5.5 build 82. 4 table 1. volume of irrigation in liters subjected to different water irrigation rates and collection times. time (days) water irrigation rates (% etc)* 100 125 150 10 59.85 ca 74.81 ba 89.78 aa 20 55.18 cb 68.98 bb 82.77 ab 30 30.91 cd 38.64 bd 46.37 ad 40 33.79 cc 42.24 bc 50.69 ac total (l) 179.73 224.67 269.61 *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. table 2. the volume of collected water in liters, subjected to different water irrigation rates and collection times. time (days) water irrigation rates (% etc)* 100 125 150 10 5.14 ca 8.19 ba 11.96 aa 20 3.61 ca 5.87 bb 8.51 ab 30 3.50 ba 4.89 bb 6.89 ac 40 3.68 ba 5.14 bb 6.94 ac total (l) 15.93 24.09 34.30 *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. table 3. the volume of water collected in liters, submitted to different water irrigation rates and water reuse application rates. water irrigation rates (% etc) reuse water rates (m 3 ha -1 ) * 0 50 150 total (l) 100 3.80 ac 4.10 ac 4.10 ab 12.00 125 5.56 ab 6.06 ab 6.45 aa 18.07 150 10.34 aa 8.59 ba 6.79 ca 25.72 *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. table 3 shows the interactions of irrigation water rates and reused water rates, observing that the leached volume increased with an increase rate, regardless of the wastewater percentages applied. in the highest irrigated rate without wastewater, higher leached volumes occurred (10.34 l). the variation of the rates was significant only at 150%. in this case, there was a reduction in the volume collected with increased rates. the total collected volume was also higher for the greater rate (25.72 l). the difference between the volume of water applied and collected indicates that the rest of the remaining water content was required by the atmosphere for evaporation and/or was stored in the soil pores. ph the ph of the samples was assessed by the concentration of h + ions. the transport of ions showed a significant interaction for rate x time and residual water rate x rate. the transport of h + ions was significant only for 40 days after the application of wastewaters; it was the highest concentration observed at 150% etc rate (table 4). the ph is acidic throughout the experimental period, although it has been observed that the unfolding time results were only significant at 150% etc rate. in this case, the mineralization of organic matter and nitrogen, as well as the reduction of soil cec can cause 5 table 4. concentration of ion h + (l -1 mg) and ph values (in parentheses) submitted to different water irrigation rates and collection times. time (days) water irrigation rates (% etc)* 100 125 150 10 0.56x10 -06 aa (6.45) 0.84x10 -06 aa (6.21) 0.61x10 -06 ac (6.38) 20 0.37x10 -06 aa (6.56) 0.42x10 -06 aa (6.47) 0.22x10 -06 ac (6.75) 30 0.75x10 -06 aa (6.20) 1.58x10 -06 aa (5.93) 1.91x10 -06 ab (5.96) 40 1.08x10 -06 ba (6.02) 1.42x10 -06 ba (5.92) 3.81x10 -06 aa (5.68) *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. note: ph values were transformed into h + ions using [h + ] = 10^(-ph). table 5. concentration of h + ions (mg l -1 ) and ph values (in parentheses) submitted to different water irrigation rates and reuse water application rate. water irrigation rates (% etc) reuse water rates (m 3 ha -1 )* 0 50 150 100 0.81x10 -06 ab (6.25) 0.78x10 -06 aa (6.18) 4.64x10 -07 aa (6.50) 125 0.67x10 -06 ab (6.31) 1.70x10 -06 aa (5.90) 8.21x10 -07 aa (6.19) 150 2.66x10 -06 aa (6.06) 1.49x10 -06 ba (6.26) 7.57x10 -07 ba (6.40) *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. note: ph values were transformed into h + ions using [h + ] = 10^(-ph). solubilization of h + ions, causing its increase in leachate and reducing the ph over time. at 40 days, after the reuse of swine water it was observed that at 125 and 150% there was a higher concentration of h + ions. table 5 shows the data of the interaction rate x rate, corroborating the fact that the transport of h + ions was significant only at 150%, with higher and lower concentrations in the lysimeters that did not receive wastewater and with the highest rates, respectively (2.66x10 -6 ph value 6.06; 1.49x10 -6 ph value 6.26, and 7.57x10 -6 ph value 6.40). the evaluation of the h + ion transport in the rate x rate interaction indicates it was significant only in the lysimeters withot waste water, which increased with increased rates. soil acids interfere with the proper development of roots; being necessary to carry out liming for acidity correction. thus, acid in soils can be a concern considering that productivity can be affected. in this sense, the rate that provided higher transport of ions out of the zone of the root system of the plant was 150% etc, observed in 0 rate, with a concentration of h + ions of 2.66x10 -6 . therefore, 50 and 150 m 3 h -1 rates were similar to the effect observed in this interaction with 150% etc. phosphorus table 6 shows that phosphorus leached in irrigation water at days 10 and 30, after application of wastewater did not differ statistically. however, p leached significantly increased after 20 days with increased irrigation water percent. at day 40, this behavior differed from other samplings since there was an increase in the leaching of p between 100 and 125% etc, with a further reduction from 125 to 150% etc. p mobility in soil is very low, thus justifying the fact that the losses caused by leaching in arable soils are considered insignificant. the available phosphorus content usually tends to decrease with depth, following the content of soil organic matter. p applied at concentration exceeding the culture of demand can lead to leaching of this element in soil profile. according to maggi et al. (2011), evaluating the leachate impacts on drainage lysimeters at different times of collection, under different swine wastewater rates during the soy crop cycle observed quadratic regression models for phosphorus concentrations in the leachate over time. this approach confirms the data found in this work, which despite not having adjusted regression models showed that the p concentrations in the leachate increased and soon after decreased. at 100 and 125% etc, after the application of wastewaters at day 20, there were lower concentrations of p in the leachate over 150% etc. this result indicates that p concentrations subjected with diiferent rates were very heterogeneous with respect to the various collection times. table 7 shows the total average concentrations of the phosphorus obtained from the reused water and collected 6 table 6. concentration of p (mg l -1 ) submitted to different water irrigation rates and collection times. time (days) water irrigation rates (% etc)* 100 125 150 10 0.12 ab 0.10 ac 0.28 ab 20 1.30 ba 1.57 ba 2.32 aa 30 0.30 ab 0.31 ac 0.42 ab 40 0.08 bb 0.96 ab 0.01 bb *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. table 7. total p average concentration applied and leached (in 4 collections) and the average nutrient retained in the soil. water irrigation rates (% etc) reuse water rates (m 3 ha -1 ) p apllied (mg) p leached (mg) p in soil (mg) t0 0.00 1.82 l100 t50 13.53 1.68 11.85 t150 40.58 2.44 38.14 t0 0.00 3.89 l125 t50 13.53 3.58 9.95 t150 40.58 5.87 34.71 t0 0.00 8.37 l150 t50 13.53 6.96 6.57 t150 40.58 4.48 36.10 li: water irrigation rate; ti: reuse water rates; p: phosphorus. in lysimeters and average nutrient retained in the soil. from the results, a higher concentration of p was leached at 150% etc; at 0 m 3 h -1 rate, there was a concentration of 8.37 mg l -1 and at 100 and 125% etc, there was an increase of p rates in the leachate. at 150% etc, a decrease was observed in p leachate concentration due to increased concentration of p. the p concentration in the leachate was lower than the concentration applied, at a rate of 50 to 150 m 3 h -1 for the three evaluated rates. in the treatment without wastewater, p concentrations in the leachate were observed at the three irrigation water rates, resulting from leaching of existing sources in soil and additional chemical fertilization performed with superphosphate in the experiment. the balanced p applied and leached in lysimeters showed that most of the nutrient was retained in the soil and, therefore, available for the culture and various irrigation water rates l2 to l3. this led to a reduction of the p leached only in t150, ranging from 5.87 to 4.48 mg, respectively. the results found by chahal et al. (2011) corroborate with this research since phosphorus and potassium concentrations were observed in the leachate, and are lower than the wastewater concentrations used. copper table 8 shows cu concentration in the leachate under various wastewater rates and collection times. changes were observed in cu concentration in the leachate only at 20 and 40 days after the application of wastewater. the mobility of cu in this study was much reduced because the leachable concentrations did not exceed 0.044 mg l 1 , indicating that part of the metal applied by the effluent was retained in the soil particles inside the lysimeters and/or was absorbed by the used culture. the copper concentrations obtained were lower as observed by barros et al. (2003), in which swine effluents subjected to integrated treatment under soil deformed columns were applied. from their study, a leached maximum concentration of cu with value around 0.06 mg l -1 was obtained. messias et al. (2007) observed low movement of fe, zn and cu with sewage sludge contents, and cu concentrations observed in soil without wastewater proved to be uniform in depth. however, in the soils containing swine waste, the authors observed higher metal concentrations in surface layers (0 to 5.0 cm). according to oliveira and mattiazzo (2001), low movement may be related to mechanisms of 7 table 8. concentration of cu (mg l -1 ) submitted under different reuse water rates and collection times. time (days) reuse water rates (m 3 ha -1 )* 0 50 150 10 0.012 ab 0.013 ac 0.014 ab 20 0.015 bb 0.028 ab 0.021 bb 30 0.015 ab 0.017 ac 0.012 ab 40 0.031 ba 0.039 aa 0.044 aa *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. table 9. zn concentration (mg l -1 ) submitted to different water rates reuse and water irrigation rates. water irrigation rates (% etc) reuse water rates (m 3 ha -1 )* 0 50 150 100 0.139 bb 0.322 aa 0.228 bb 125 0.201 bb 0.306 aa 0.376 aa 150 0.486 aa 0.385 ba 0.349 ba *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. adsorption/desorption, precipitation/dissolution, complexation, and redox. the collection done at day 20 showed an increased concentration of the element at 50 m 3 ha -1 , while at day 40, the highest cu concentrations occurred in soils containing wastewater. there was a noted trend of increased cu concentration in all the three evaluated rates over time. in this case, the presence of cu metal regardless of the rate was increased by fertilization cultivation. according to messias et al. (2007), from the evaluation of iron, copper, zinc and cadmium movement in soil treated with sewage sludge, higher sludge dose increased the leachates elements concentration, except for copper. in short, the lower cu concentrations found in the leachate were observed at 0 rates in all collection times. at day 20, there were no differences between the rates of 0 and 150 m3 ha -1 , being characterized as the period and rates that provided lower losses of cu leaching. with observed low concentration of cu under the experimental conditions and considering that the use of waste can occur for decades, it is necessary to monitor groundwater to ensure metals do not concentrate on the water resource. zinc the zinc concentration showed an increased changes with increasing irrigation rate at 0 and 150 m 3 h -1 (table 9), ranging from 0.139 to 0.486 mg l -1 and 0.228 to 0.349 mg l -1 , respectively. the variation of reused water rates had a significant effect on metal leaching in any of the observed rates, and control plots. zn leaching was also observed, which might be due to fertilizer incorporated in the soil. since the plots did not receive an effluent and have a lower organic matter content compared to other lysimeters, the lowest content of organic matter may have provided greater leaching of zn. the presence of organic matter provides negative charges to the ground, and this in turn acts to maintain the positively charged elements adsorbed (as cu and zn), which consequently reduces mobility in the soil profile. the affinity of metals with soil varies according to the type, amount of organic matter, cation exchange capacity (cec), ph, clay quantity, mineralogy, and other characteristics; and it is also influenced by characteristics of metals. according to paganini et al. (2004), a large amount of zn can be fixed in the organic fraction of the soil, and may be temporarily fixed in microorganisms after the addition of organic matter in the soil. in this context, it is evident that treatment with a replacement of 100% etc, at 0 rates and 150 m 3 ha -1 led to lower losses of zn in the cauliflower. table 10 shows that the increase in reused water rates applied to the lysimeters was significant only for 40 days, with an increase in zn losses in the lysimeters under effluent application. this behavior can be explained based on ph (table 4), while campos (2010) states that 8 table 10. zn concentration (mg l -1 ) submitted to different water reuse rates and collection times. time (days) reuse water rates (m 3 ha -1 )* 0 50 150 10 0.201 ac 0.213 ac 0.183 ac 20 0.217 ac 0.268 ac 0.279 ab 30 0.402 aa 0.349 ab 0.397 aa 40 0.280 bb 0.521 aa 0.412 aa *means followed by the same lowercase letter in the columns and capitals in rows do not differ by the scott knott test at 5% probability. low ph values favor lixiviation and availability of metals in the soil. increase in ph increases the cec which allows the formation of chelates from the organic material, decreasing its mobility in soil. according to messias et al. (2007), evaluating the mobility of micronutrients there were no observed variations in the leaching of cu and zn over time (60 days), even for soils with a higher concentration of sewage sludge (75 mg h -1 ). in this sense, the rate of 150 m3 ha -1 reused water was a good choice for nutrient supply of cauliflower until 30 days. the data evaluated at 40 days showed higher zn concentrations compared to other evaluated periods. it is noteworthy that the continued application of reused water in the soil as a source of nutrients for crops can cause groundwater contamination, due mainly to changes in the concentrations of cu and zn based on the provisions of conama resolution no. 396/2008 (maximum values of 2.0 and 5.0 mg l -1 for copper and zinc, respectively). conclusion the volume of leaching increased with increasing amount of water supplied by irrigation; however, it decreased with increased swine wastewater rates. at 100 and 125% etc, there were higher concentrations of h + ions at day 40, after the application of swine wastewater. p leached concentrations were higher at day 20 after the wastewater application, regardless of the irrigation water rates. the smaller cu concentrations found in the leachate were observed at 150 m 3 ha -1 rates at 20 days after application of reused water. the application of 150 m 3 ha -1 and replacement of 100% etc can be indicated as a good alternative for short cycled vegetables (less than 40 days), as the concentrations of cu and zn did not exceed the limits of conama resolution no. 396/2008, at this interval, for redyellow latosol. conflict of interests the authors have not declared any conflict of interests. references american public health association (apha) (2012). standard methods for the examination of water and wastewater american public health association, american water works association, water environmental federation. 22. ed. washington, dc. 1496p. barros lss, amaral la, lucas júnior j (2003). pollution of swine wastewater after the use of an integrated treatment. braz. j. vet. res. anim. sci. 40:126-135. bertol oj, fey e, favaretto n, lavoranti oj, rizzi ne (2010). mobility of p, cu and zn in soil columns under direct seeding system submitted to organic mineral fertilizations. rev. bras. ciênc. solo 34:1841-1850. conama resolution (2008). resolution no. 396 of the national council for the environment – conama: provides for the classification and environmental guidelines for the groundwater framework and provides other measures. mma, brasília, brazil. campos mcc (2010). soil attributes and risk of leaching of heavy metals in tropical soils. ambiência 6:547-565. chahal mk, toor gs, nkedi-kizza p, santos bm (2011). effect of tomato packinghouse wastewater properties on phosphorus and cation leaching in a spodosol. j. environ. qual. 4:999-1009. maggi cf, freitas psl, sampaio sc, dieter j (2011). leaching of nutrients in cultivated soils under application of residual water of pig farming. rev. bras. eng. agríc. ambient. 15:170-177. messias as, silva ha, lima vn, souza jeg (2007). micronutrients mobility evaluation in soil treated with sewage sludge. rev. bras. gest. desenvolv. reg. 3:193-211. oliveira fc, mattiazzo me (2001). heavy metal mobility in a typic hapludox amended with sewage sludge and planted to sugarcane. sci. agric. 58:807-812. paganini ws, souza a, bocchiglieri mm (2004). assessment of the behavior of heavy metals in the sewage treatment by disposal in soil. eng. sanit. ambient. 9:225-239. rieuwerts js, ashnore mr, farago me, thornton i (2006). the influence of soil characteristics on the extractability of cd, pb and zn in upland and moorland soils. sci. total environ. 366:864-875. sistani kr, mclaughlin mr, brink ge (2008). soil nutrient evaluation from swine effluent application to five forage-system practices. nutr. cycl. agroecosyst. 82:265-271. smanhotto a, sousa ad, sampaio sc, nóbrega lh, prior m (2010). copper and zinc in the lixiviated material and in the soil application of swine wastewater on soil cultivated with soybean. engenharia agrícola. 30(2):347-357. souza ap, mota ll, zamadei t, martim cc, almeida ft, paulino j (2013). climate classification and climatic water balance in mato grosso state, brazil. nativa 1:34-43. zanuzo mr, ribeiro lm, lange a, machado raf, massaroto ja (2013). agronomic performance of cauliflower genotypes under climatic conditions of sinop. hortic. bras. 31:332-337. in ternationa l scholars journa ls african journal of pig farming issn 2375-0731 vol. 8 (2), pp. 001-004, february, 2020. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper molecular cloning and phylogenetic analysis of the e gene of transmissible gastroenteritis virus (tgev) isolated in china d. l. lan, j. p. gu, r. xing, c. l. yuan, l. cui, x. g. hua and z. b. yang* shanghai key laboratory of veterinary biotechnology, school of agriculture and biology, shanghai jiaotong university, shanghai 200240, china. accepted 15 september, 2019 porcine transmissible gastroenteritis caused by transmissible gastroenteritis virus (tgev) induces acute, high-contact intestinal infectious diseases in pigs. tgev encodes four structural proteins including spike (s), membrane (m), envelope (e) and nucleoprotein (n), of these, the genes of s, m and n respectively had been deeply elucidated. however, the real function of e protein was still unclear and sequence comparison of the e gene of different tgev isolates had not been reported so far. in this study, e gene of tgev strain isolated in china was molecularly cloned and sequenced. phylogenetic analysis confirmed this gene was relatively conserved. all the china tgev strains were brached in one clade together with the american strains, but showed relatively alien to the european strains. key words: transmissible gastroenteritis virus, e gene, phylogenetic analysis. introduction transmissible gastroenteritis virus (tgev) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas (zhou et al., 2007). as a member of the coronaviridae, tgev possesses a large 28.5 kb singlestranded sense rna genome in which genes are arranged in the order 5’-rep-s-3a-3b-e-m-n-7-3’(park et al., 2008). four of them, rep, 3a, 3b, and 7 encode non-structural proteins (penzes et al., 2001). the others encode four structural proteins including spike (s), membrane (m), envelope (e) and nucleoprotein (n). in these four structural genes, the genes of s, m and n respectively are being deeply elucidated, and some characteristics have been ascertained and explained (escors et al., 2001; gebauer et al., 1991; kapke and *corresponding author. e-mail: zbyang@sjtu.edu.cn. tel: +86 21-34205779. fax: +86-21-34205779. brian, 1986; krempl et al., 1997; laude et al., 1987). nevertheless, the functional action of e gene is poorlyunderstood currently. e gene is a small structural gene that encodes an 82 amino acid membrane-associated protein called e protein (formerly called sm)(baudoux et al., 1998). e protein is an integral membrane protein having a cexo-nendo orientation and binding with the envelope. the epitopes of this protein are localized within the last 21 c-terminal residues of the sequence, and the antibodies that are specific for e protein can be tested in pig sera infected tgev (godet et al., 1992). previous research suggested that e protein is an efficient inductor of alpha interferon (ifn-alpha) synthesis in vitro (baudoux et al., 1998; riffault et al., 1997). recently, it was reported that it is essential for viral reproduction and play a role in virion assembly or release (ortego et al., 2007). however, the real function of e protein is still unclear. moreover, to the best of our knowledge, sequence comparison of the e gene of different tgev isolates have been not reported so far. table 1. the characteristics of tgevs used in this study. strain name place of origin (abbreviation) genbank accession number h16 china fj755618 attenuated h china eu074218 sc-y china dq443743 ts china dq201447 tfi taiwan z35758 miller m60 the united states of america (usa) dq811786 miller m6 the united states of america (usa) dq811785 purdue the united states of america (usa) dq811789 purdue p115 the united states of america (usa) dq811788 pur46-mad the united states of america (usa) aj271965 96-1933 the united kingdom of great british (uk) af104420 fs772/70 the united kingdom of great british (uk) y00542 materials and methods virus and in vitro growth a chinese tgev strain named tges-1 was isolated from pigs in a small commercial pig farm that lies in shanghai suburb. the swine testis (st) cells were grown in dulbecco’s modified eagle medium (dmem, gibco, usa) supplemented with 10% foetal bovine serum (fbs, gibco, usa) and maintained in maintenance medium (dmem supplemented with 2% fbs) at 37°c in a 5% co 2 atmosphere. the tges-1 strain was passaged 6 times on st cells and the value of tcid50 was evaluated. virus infected st cells showing 90% cytopathic effects were frozen and thawed and cellular debris was removed by centrifugation at 10×l0 3 g at 4°c for 30 min. the clarified cell culture supernatant was collected and used for preparation of viral rna. rna isolation and reverse transcription-polymerase chain reaction (rtpcr) viral rna was extracted by use of trizol reagent (takara, china) according to the manufacturer’s instructions. the rt reaction was performed using primescript™ 1 st strand cdna synthesis kit (takara, china) following the manufacturer’s protocol. briefly, the mixture containing 5 µl of rna, 1 µl of random primers (50 µm), and 1 µl of dntps (10 mm) was heated at 65°c for 5 min. then, 4 µl of 5×primescript™ buffer, 0.5 µl of rnase inhibitor (40 u/µl), 1 µl of primescript™ rtase (200 u/ µl) and 7.5 µl of rnase free ddh2o were added to the mixture for a total volume of 20 µl. the rt reaction to synthesise cdna was performed at 30 ◦ c for 10 min, at 42°c for 60 min, followed by heating at 70°c for 15 min total volume of 20 µl. the rt reaction to synthesise cdna was performed at 30°c for 10 min, at 42°c for 60 min, fol lowed by heating at 70°c for 15 min. the cdna obtained was amplified with forward primer tgev-ef (5’-tttatgtattttcacaggagccc -3’) and reverse primer tgever (5’tagcacaatagcgttctccacat -3’) designed by ourselves. the pcr reaction mixture contained 25 µl of pcr premix (0.1u of taq polymerase/µl, 500 µm of dntpeach, 20 mm tris-hcl, 3 m mgcl2, 100 mm kcl) (tiangen, china), 1 µl of forward and reverse primers, and 5 µl of cdna. a 50 µl total reaction volume was obtained by adding distilled water. the pcr was performed by 35 cycles of 60 s at 94°c, 1 min at 50°c and 1.5 min at 72°c; followed by a final extension time of 5 min at 72°c. pcr products were electrophoresed in 2% agarose gels containing ethidium bromide (10 mg/ml) and confirmed using a uv-transilluminator (bio-rad, usa). cloning and dna sequencing the pcr products were gel purified using axyprep dna gel extraction kit (axygene, usa) according to the manufacturer’s instructions. the purified dna was ligated into the pmd-18 cloning vector (takara, china) and transformed into competent cells (top10, tiangen, china). the positive recombinants were selected with blue and white screening. in addition, the plasmids were extracted with extraction kits (axygene, usa) .then the identified positive recombinants were sequenced using dideoxynucleotide chaintermination procedures (shanghai, china), and the sequence of e gene of tges-1 strain was submitted to genbank of ncbi. sequence analysis the dna sequences were analyzed with the megalign software (dnastar inc., usa), and then compared with other 12 published tgev dna sequences (table 1) using clustal x v 1.82 programme (usa). a phylogenetic tree of the e gene was generated by the neighbour-joining method with 100 bootstrap replicates in a heuristic search with the mega 4.1 software programme (usa). the origin places and genbank accession numbers of tgevs used in this study are presented in table 1. results and discussion the tges-1 strain was passaged serially 6 times in st cells. the growth characteristics of tges-1 of each in vitro generation were almost the same. cytopathic effects (cpe) occurred 48 h post-infection. during this time the cells rounded, condensated, and gradually peeled off. the tcid50 of tges-1 strain was 10 -8.2 /0.2 ml. these results suggest that this isolated china strain is a virulent strain. then the complete e gene of tges-1 strain was amplified by rt-pcr, cloned, and sequenced. the figure 1. phylogenetic relationships based on nucleotide sequences of the gene e region among tgev strains used here. the phylogenetic tree was generated by the neighbor-joining method in a heuristic search with 1000 bootstrap replicates. complete e gene sequence of tges-1 strain was 249 bp (genbank accession number: gu250738). the nucleotide sequence comparison of tges-1 strain with other reference strains of tgev showed that there was no deletion or insertion in the e gene of the tges-1 strain. among these strains, tges-1 strain had high nucleotide identities of 98.4 -100% with other strains, specially had a 100% identity with china ts strain. comparison of these strains, only the taiwan strain tfi and the uk strain fs772/70 missing two bases in the e gene sequence, but these two strains are all virulent isolate, and whether this kind of point mutation will influence the viral reproduction, assembly, release or tropism of the virus need further study. likewise, the amino acid sequence comparison showed that tges-1 strain had a similarity between 95.1% (attenuated h) and 100% (ts) with other reference strains. in general, there is little different between each strain in e gene, and the e gene of tgev is relatively conserved. the precise function of the e gene product is not clearly understood, but it has been suggested that this gene might play a role in the pathogenicity of tgev (baudoux et al., 1998; ortego et al., 2007; riffault et al., 1997). however, interestingly, here we found that virulent strains have little difference with attenuated virulent strains in e gene. this result is contradicted to the previous studies and remained to be explored. we speculate that the virulence of tgev is related with e gene, but e gene is not the only determining factor. to investigate the evolutionary relationships between tges-1 strain and others, a phylogentic tree was constructed based on their e gene sequences (figure 1). the tree revealed the five china tgev isolates were clustered into three distinct groups. as expected, based on genetic homology, tges-1 and ts were clustered together in a group. sc-y strain (china) has closer relationship with purdue p115 and pur46-mad strains (usa), h16 strain and attenuated h strain could be clustered together in a group. notably, the phylogentic relationship of tgev strains from china was closed with the american strains, but alien to the european strains. in conclusion, the e gene sequences of tgev isolated from different regions are relatively conserved. all the china tgev strains possess extremely intimate relationship with the american strains and are relatively alien to the european strains. acknowledgement this work was supported by national key laboratory of veterinary biotechnology (sklvbf201001). references baudoux p, besnardeau l, carrat c, rottier p, charley b, laude h (1998). interferon alpha inducing property of coronavirus particles and pseudoparticles. adv exp med biol., 440:377-386. escors d, camafeita e, ortego j, laude h, enjuanes l (2001). organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core. j virol., 75:12228-12240. gebauer f, posthumus wp, correa i, sune c, smerdou c, sanchez cm, lenstra ja, meloen rh, enjuanes l (1991). residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein. virology, 183:225-238. godet m, l'haridon r, vautherot jf, laude h (1992). tgev corona virus orf4 encodes a membrane protein that is incorporated into virions. virology, 188:666-675. kapke pa, brian da (1986). sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. virology, 151:41-49. krempl c, schultze b, laude h, herrler g (1997). point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. j virol., 71:3285-3287. laude h, rasschaert d, huet jc (1987). sequence and n-terminal processing of the transmembrane protein e1 of the coronavirus transmissible gastroenteritis virus. j gen. virol., 68 (pt 6):1687-1693. ortego j, ceriani je, patino c, plana j, enjuanes l (2007). absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway. virology, 368:296-308. park jh, han jh, kwon hm (2008). sequence analysis of the orf 7 region of transmissible gastroenteritis viruses isolated in korea. virus genes., 36, 71-78. penzes z, gonzalez jm, calvo e, izeta a, smerdou c, mendez a, sanchez cm, sola i, almazan f, enjuanes l（2001). complete genome sequence of transmissible gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster. virus genes., 23:105-118. riffault s, grosclaude j, vayssier m, laude h, charley b (1997). reconstituted coronavirus tgev virosomes lose the virus ability to induce porcine interferon-alpha production. vet res., 28:77-86. zhou jf, hua xg, cui l, zhu jg, miao dn, zou y, he xz, su wg (2007). effective inhibition of porcine transmissible gastroenteritis virus replication in st cells by shrnas targeting rna-dependent rna polymerase gene. antiviral res., 74:36-42. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 9 (3), pp. 001-009, march, 2021. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper factors influencing success of artificial insemination of pigs using extended fresh semen in rural smallholder pig farms of rwanda munyaneza celestin1*, nyiramuhire valentine1, mubashankwaya isaac1,2, munyandamutsa fabrice1, ndisanze oscar3, bagaragaza françois1 and mujyambere jean marie vianey1 1 department of agriculture, faculty of agriculture, environmental management and renewable energy (faemre), university of technology and arts of byumba (utab), p. o. box 25, byumba, rwanda. 2 uganda martyrs university (umu), faculty of agriculture, p. o. box 5498 kampala, uganda. 3 animal resources office, rulindo district, p. o. box 6892, kigali, rwanda. *corresponding author. e-mail: munezacel@yahoo.fr. tel: +250784643802. accepted 8 february, 2020 the present study was conducted to analyse the factors influencing the success of artificial insemination (ai) in pigs using extended fresh semen in rural smallholder pig farms. one hundred female pigs from rulindo and gicumbi districts in northern region of rwanda were artificially inseminated using locally collected and extended fresh semen. the pregnancy was confirmed by using both no returns to estrus and abdomen palpation at 21 and two and half months post insemination, respectively. the factors influencing the success of ai were analysed using one way analysis of variance (anova) test. the results showed that the number of inseminations per conception (ic) and litter size were 1.89 ± 0.83 and 7.94 ± 2.24, respectively. the number of ai per conception was significantly influenced (p < 0.05) by intrinsic factors (age and parity of the female pig) and extrinsic factors (sex, age and education of household head, time of ai and distance between the farm and semen collection center). the litter size was significantly influenced (p < 0.05) by parity. from the results, we recommend establishing more semen production centers and providing more training on pig management and ai. key words: inseminations per conception, litter size, swine, fresh semen, smallholder farms, natural oestrus. introduction the world population continues to depend on pig meat as an important source of food and income. pig meat contributes around 36% (118.7 million tonnes) to the world meat output comprising bovine, pig, poultry and ovine meat (fao, 2018). indeed, pig meat is the second, after poultry meat, among the most consumed types of meat worldwide (on average, 12.3 kg retail weight equivalent/capita/year) (oecd-fao, 2018). the demand for meat including pig meat, is expected to increase due to the growing world population. the 2 projections have shown that the world population is expected to rise to 9.1 billion by 2050 (godfray et al., 2010). simultaneously, global pig meat consumption on a per capita basis remains stable: from the average of 12.3 to 12.2 kg retail weight equivalent/capita/year in 2015 to 2017 and 2027, respectively (oecd-fao, 2018). therefore, there is a need for increasing pig production in order to meet the growing demand in pig meat. the increase in pig production will be made possible through the use of improved technology in key areas such as genetics, nutrition, management, sanity and reproduction. among these areas, genetics play a primordial role in improving pig meat production and artificial insemination (ai) technology is among the best alternatives for its improvement. the use of ai in pig breeding presents great advantages, compared to natural mating, such as improving fertility, genetics, labour use efficiency and herd health which result in increased farm productivity and profitability (knox, 2016). artificial insemination enables to increase sire selection intensity by using the best sires available within individual breeds (ronald et al., 2013). in fact, the use of ai greatly increases the selection differential, where the genetic potential of the best sires can be extensively transferred to a large number of pigs using diluted semen (shimada et al., 2016). artificial insemination technique solves problems of infections due to the contact between boar and female pigs during mating (shimada et al., 2016). some infections caused mainly by viruses and bacteria can result in problems associated with sperm damage, pregnancy failure, abortion, embryo loss and endometritis (knox, 2016). artificial insemination reduces the use of the same male by a group of farmers, particularly within a village, which can lead to inbreeding (kumar et al., 2014). a standard farm boar to sow ratio is 1:20 using natural mating (kyriazakis and whittemore, 2006). hence, ai can reduce the ratio to 1:150 (roelofse, 2013). the reduced number of boars in a farm results in optimum use of the facilities and labour as well as reduction of feed supply and medicines; thus, monetary benefit (ronald et al., 2013; kadirvel et al., 2013). the use of ai in pigs has presented an enormous progress, particularly in europe and north america (gerrits et al., 2005; ronald et al., 2013). in some european countries, such as belgium, italy, the netherlands, norway and spain, more than 80% of the female pigs are bred using ai. in north america (usa, canada and mexico) and brazil, 75% of female pigs in large farm units are inseminated using ai (feitsma, 2009; roca et al., 2006). moreover, the conception rates in ai and natural mating in pigs are the same (kadirvel et al., 2013). indeed, the fertility rates of 80 to 90%, using ai, are common in many pig farms (roca et al., 2006) and artificial insemination can lead to better results in farrowing rate and litter size than natural mating (visalvethaya et al., 2011; am-in et al., 2010; kadirvel et al., 2013). although ai in pigs has more advantages compared to natural mating, it can have some limitations. for example, ai in pigs can result in low number of piglets per farrowing compared to natural mating; and the limited storage period of fresh semen are the main problems to its expansion in the field all over the world (shimada et al., 2016). therefore, maes et al. (2011) suggest that the success of ai in pigs is largely determined by the semen quality and the insemination procedure. some of critical issues for ai procedure involve oestrus detection in the gilt/sow, timing of insemination, applying strict hygiene measures, semen age and age of boar (maes et al., 2011; feitsma, 2009). practically, the success of ai can be gauged using specific parameters including fertility rate, number of inseminations per conception, farrowing rate, litter size, piglets born alive and stillborn piglets (vargas et al., 2009; niyiragira et al., 2018). despite its progress worldwide, the use of ai in pigs in rwanda is still limited. mbuza et al. (2016) reported that some pig farmers in rwanda are not aware of the existence of ai in pigs. moreover, niyiragira et al. (2018) suggested that reproduction performances such as litter size of weaned sows bred by natural service and ai are similar; and some factors like sow breeds and parity can significantly influence the outcomes of ai namely litter size. however, the study was conducted in an organized farm, where a large number of factors, particularly socioeconomic and management factors were controlled compared to smallholder pig farms in rural areas. currently, the literature shows that there is no study related to factors influencing the success of ai in smallholder pig farms in rural areas of rwanda. the present study aims to assess the factors influencing the success of ai using locally collected extended fresh semen in smallholder pig farms in rural areas of rwanda. materials and methods study area the study was conducted in rulindo and gicumbi districts in northern region of rwanda during the rainy and dry seasons from january to september 2018. the districts are mostly characterized by hills interspersed by valleys and swamps that border rivers. in the two districts, agricultural sector provides the main jobs and source of revenues; where 76 and 77% of working population in gicumbi and rulindo districts, respectively, are involved in this sector. around 14.7 and 14.9% of all households in gicumbi and rulindo districts, respectively, raise pigs. in this study, the semen were collected from centre de perfectionnement et de promotion agricole de kisaro (cppa) (1°37'41.99''s; 30°01'39.11''e), a private semen collection centre located in kisaro sector of rulindo district. figure 1 shows the location of gicumbi and rulindo districts and cppa de kisaro. semen collection, dilution and storage the semen was collected by the trained technician of the semen collection centre of centre de perfectionnement et de promotion 3 figure 1. location of gicumbi and rulindo districts and centre de perfectionnement et de promotion agricole de kisaro (cppa). agricole de kisaro (cppa)from the trained boars in a sterile beaker using a dummy sow. the motility and concentration of spermatozoa were assessed by using a microscope and spectrophotometer respectively. after estimating the concentration, the semen was immediately extended with the diluent to the final concentration of 3×10 9 spermatozoa per dose of 100 ml. then, the extended semen was stored in squeeze bottles at a temperature between 15 and 20°c. the storage period of the extended semen varied depending on appearance of heat in female pigs to be artificially inseminated, but it did not exceed five days before their utilization. artificial insemination and pregnancy detection a total of 100 female pigs from 100 farms were randomly selected on the basis of insemination service provider records. practically, an exhaustive list of the artificially inseminated female pigs during the study period was provided and the sample pigs were randomly selected. once a sow/gilt showed heat signs, the farmer informed the inseminator. then the inseminator transported the extended semen to the farmer’s place and inseminated the sow/gilt. a single ai per oestrus was performed for all sows/gilts using intra-cervical insemination (intra-cai) procedure, as described by kaysen (2013), 12 h after the pig had expressed oestrus signs. the pregnancy was confirmed by either no return to estrus after 21 days or abdomen palpation two and half months post ai. the inseminations were carried out by trained inseminators from caritas, a nongovernmental organization that was providing artificial insemination service in the study area during the study period. data collection and analysis the household and farm management data were collected using a structured questionnaire through interview with the farmers and personal observations. the farmers included 16 males and 84 females. the data related to ai were collected alongside the ai procedure using a predesigned form. the factors which were hypothesized to influence conception and litter size were categorized into those which are related to the female pig (intrinsic) and those which are not related to the female pig (extrinsic). the intrinsic factors were the age, parity and breed of female pig. the extrinsic factors were the age, the sex and experience in pig keeping of the household head, distance between ai centre and pig farm, time of ai, district and floor material of the pig house. a one way anova test was used to analyse the difference between means of number of inseminations per conception and litter size among groups. statistically significant difference between means was considered to exist if the p-value is less than 0.05. the data analysis was performed using spss-ibm 20 version software (ibm corp. released, 2011). results and discussion number of inseminations per conception the overall number of artificial inseminations per conception (ic) was 1.89 ± 0.83. moreover, the pigs that became pregnant after one, two, three and four ais were 37, 77, 97 and 100% of the total sample, respectively (figure 2). the average number of ais per conception was higher than the ones reported in the literature. for example, niyiragira et al. (2018) reported 1.17 ais per conception in rwanda while ronald et al. (2013) reported 1.00 ai per conception in india. this superiority could be explained by the fact that those studies were conducted 4 figure 2. number of artificial inseminations per conception using extended fresh semen in smallholder rural pig farms. in organized farms using two ais per oestrus contrary to one ai per oestrus in field conditions in this study. several authors including lamberson and safranski (2000), kaysen (2013), bortolozzo et al. (2005) and kumar et al. (2014) suggest that return to oestrus is higher in pigs artificially inseminated once than the pigs artificially inseminated twice per oestrus. the lower performance of ai in field condition compared to organized farm is due to the factors related to semen handling and farm management (kumar et al., 2014). extrinsic factors influencing the number of inseminations per conception the effects of extrinsic factors on the number of ic are shown in table 1. the number of ic was significantly lower in male household headed farms than in female household headed farms (1.38 ± 0.62 and 2.00 ± 0.83; p < 0.05, respectively). the difference could be explained by better farm management among male farmers compared to female farmers. in fact, mbuza et al. (2016) reported that, in rwanda, a large proportion of males is more involved in pig management activities such as feeding, cleaning, treating and record keeping compared to females; and good farm management is among the factors influencing fertility of pigs (merks et al., 2000). moreover, visalvethaya et al. (2011) argue that the male farmers have better management skills than female ones, especially in oestrus detection, where the males detect heat better than their female counterparts. similarly, visalvethaya et al. (2011) reported that male managed farms present better fertility than female headed ones in thailand. the number of ic significantly varied (p < 0.05) with household head age, particularly between middle age (from 35 to 49 years) and old farmers (50 years and above). the lowest number of ic (1.59 ± 0.67) was observed in the farms managed by a group of 35 to 49 years old farmers while the highest number of ic (2.00 ± 0.90 and 2.04 ± 0.87) was observed in the farms managed by young and older farmers (below 35 and above 50 years, respectively). this difference within age groups could be justified by the fact that age of the household head has a negative effect on the adoption of new technologies including ai (dhraief et al., 2018; bayei and nache, 2014; zanu et al., 2012) and innovations (howley et al., 2012). the old farmers tend to stick to what they used to and hardly venture into new ways of doing things including ai (bayei and nache, 2014). the poor performances in young farmers could be explained by the fact that the young have fewer financial resources and are more interested in other activities compared to the other groups of age. contrary, visalvethaya et al. (2011) in thailand reported that old farmers have better performances than young farmers. this difference between findings could be explained by the context specificity of the two areas. the highest number of ic (2.02 ± 0.78) was observed in farms managed by single household heads, followed by the farms managed by widows with 1.82 ± 0.89 ic. the lowest number of ic (1.82 ± 0.70) was observed in married household headed farms. although the difference was not significant (p > 0.05), the better fertility in married 5 table 1. effects of extrinsic factors on the number of inseminations per conception using extended fresh semen in rural smallholder pig farms. extrinsic factors n mean std. deviation sex of the household head p = 0.01 age of the household head p = 0.04 marital status of the household head p = 0.26 experience of the household head p = 0.12 education level of the household head p = 0.03 time of ai p < 0.01 distance from semen collection center to farm p < 0.001 floor material of pig house p = 0.08 district p < 0.001 male 16 1.38 b 0.62 female 84 1.99 a 0.83 young (less than 35) 11 2.00 ab 0.90 middle (35 to 49) 32 1.59 b 0.67 old (50 years and above) 57 2.04 a 0.87 single 45 2.02 a 0.78 married 10 1.60 a 0.70 widow 45 1.82 a 0.87 five years and below 82 1.95 a 0.83 more than five years 18 1.61 a 0.78 no formal education 3 1.33 ab 0.58 primary 27 2.26 a 0.86 secondary 41 1.85 ab 0.88 university 29 1.66 b 0.61 07:00 to 12:00 am 37 1.97 a 0.87 12:00 to 16:30 31 2.19 a 0.87 after 16:30 32 1.50 b 0.57 within 30 km 47 1.64 a 0.67 more than 30 km 53 2.11 b 0.89 concrete 76 1.97 a 0.88 timber 24 1.63 a 0.58 gicumbi 62 2.11 a 0.87 rulindo 38 1.53 b 0.60 total 100 1.89 0.83 a,b means in the same row with different superscripts significantly differ (p < 0.05). household compared to widow and single household headed farms could be explained by the fact that the married have more financial means and are more responsible, compared to the widows and single, which enable them to improve pig farm management and result in better reproduction performances. although, in the present study, the difference was not significant (p > 0.05), the number of ic decreased with the experience of the household head in pig management. the farmers with less than five years of experience presented higher number ic per conception (1.95 ± 0.83) than the farmers with five years of experience and more (1.61 ± 0.78). zanu et al. (2012) in ghana reported that adoptions of improved technologies are associated with farming experience. indeed, visalvethaya et al. (2011) suggest that experience is crucial for pig reproduction particularly in heat detection. although the lowest number of ic (1.33 ± 0.58) was observed in the farmers who do not have any formal education, it significantly increased (p < 0.05) with education level of the household head. the household head with primary, secondary and university levels of education presented on average 2.26 ± 0.86, 1.85 ± 0.89 and 1.66 ± 0.61 ais per conception. the present study results are consistent with the report by bayei and nache (2014) and dhraief et al. (2018) who suggest that the adoptions of new technologies are associated with education of the household head. indeed, ajala (2007) in 6 table 2. effect of intrinsic factors on the number of inseminations per conception using extended fresh semen in rural smallholder pig farms. intrinsic factors n mean standard deviation age of the female pig (years) p = 0.01 parity of the female pig p = 0.01 breed of the female pig p = 0.50 1 14 1.21 b 0.43 2 66 1.95 a 0.82 3 16 2.13 a 0.89 more than 3 4 2.25 0.96 1 12 2.42 a 0.79 2 64 1.92 ab 0.80 3 and more 24 b 1.54 b 0.78 landrace 21 2.00 a 0.90 large white 13 2.15 a 0.99 pietrain 5 1.80 a 0.84 cross 61 1.80 a 0.77 total 100 1.89 0.83 a,b means in the same row with different superscripts differ (p < 0.05). nigeria revealed that the level of education is positively correlated with good farm management, thus, better fertilization or conception rate. the number of ic significantly increased (p < 0.05) with the distance between the semen collection center and pig farm. the farmers located within 30 km from semen collection center presented the lower ic (1.64 ± 0.67) than the farms located at 30 km and more with 2.11 ± 0.90 ic. moreover, the average number of insemination per conception was significantly higher (p < 0.05) in gicumbi district than in rulindo district (2.11 ± 87 and 1.89 ± 60, respectively). the negative influence of distance to insemination center on conception rate was reported during ai in cows (nishimwe et al., 2015). on the contrary, am-in et al. (2010) revealed that distance to ai center does not have negative effect on farrowing rate and non-return to oestrus. this difference could be due to the fact that return to oestrus after ai depends on the timing of insemination relative to the time of ovulation (lamberson and safranski, 2000). hence, the long distance could compromise the precision in timing and risk to deterioration of semen due to shaking and increase in temperature, particularly when the inseminators use public transport. eventually, difference within the two districts could be explained by the fact that the semen collection center of kisaro is located in rulindo district. according to chanapiwat et al. (2014), the main limitation of the extended fresh semen is the short timing of its storage; therefore, the transport of the semen for long distances becomes difficult. although the difference was not significant (p > 0.05), the number ic varied with the type of floor material of pig house. the farms with floor in timber presented the lower average number of ais per conception (1.97 ± 0.88) than the farm with concrete floor (1.63 ± 0.58). in the present case, slatted timber floor is easier to clean than concrete floor which justify lower number of inseminations per conception. according to oravainen et al. (2006), better housing, particularly in terms of cleanness has a positive effect on fertility in female pigs. dee (1992) suggests that poor environmental hygiene is responsible of urogenital track diseases which result in poor farrowing rates. the number of ic significantly varied (p < 0.05) with the time of insemination. the pigs inseminated in the morning and evening (before 12:00 and after 16:30) showed the lowest number ic (1.97 ± 0.87 and 1.50 ± 0.57, respectively) while the pigs inseminated in the afternoon (from 12:00 to 16:00) presented the highest number of ic (2.19 ± 0.873). this difference could be explained by the cool temperatures observed during the morning and evening compared to the high temperature in the afternoon which can damage the fresh semen. intrinsic factors influencing the number of inseminations per conception the intrinsic factors influencing the number of ic are shown in table 2. the number of ic significantly increased (p < 0.05) with age of female pigs. the lowest number of inseminations per conception (1.21 ± 0.43) was observed in one-year old pigs while the largest number of ic (2.25 ± 0.83) was observed in female pigs aged of four years and more. this poor fertility in aged pigs could be associated with embryonic mortality. according to vanroose et al. (2000), older animals have 7 figure 3. litter size per female pig after artificial insemination (ai) using extended fresh semen in smallholder rural pig farms of rwanda. lower follicular activity and oocyte quality resulting in a decrease in embryos development. indeed, the older animals are, the more the quality of the endometrium gets deteriorated. the number of ic significantly (p < 0.05) decreased with parity of the female pig. the lowest number of ic (1.54 ± 0.78) was observed in the sows which had farrowed at least three times while the highest (2.42 ± 0.79) was observed in primiparous. the present findings are in line with the report by kaysen (2013) and vargas et al. (2009) who found the significant increase in fertility with parity. however, kaysen (2013) argues that the conception rate decreases when the parity goes beyond seven. large white breed presented the highest number of ic (2.15 ± 0.99), followed by landrace breed with 2.00 ± 0.89 ic. the lowest number of ic per conception (1.80 ± 0.837 and 1.80 ± 0.771) was observed in pietrain and cross breeds, respectively. however, there was no significant difference (p>0.05) between the number of ic among the breeds. similarly, kaysen (2013) and niyiragira et al. (2018) confirm that the breed of the female pig has no influence on the number of ic. litter size the overall litter size was 7.94 ± 2.24 piglets per sow and 33% of all pigs gave birth to 8 piglets par sow (figure 3). the litter size was comparable with 8.06 piglets per sow reported by niyiragira et al. (2018) in rwanda using imported fresh semen. the litter size was slightly higher than 7.2 piglets reported by mbuza et al. (2016) in rwanda using natural service. similarly, kumar et al. (2014) in india argue that litter size in higher when using ai than using natural service. effects of intrinsic factor on litter size table 3 shows the effect of intrinsic factors on litter size per female pig. the number of piglets per female pig significantly increased (p < 0.05) with parity of the female pig. female with 1, 2 and 3 and more parities presented the litter size of 6.75 ± 3.17, 7.81 ± 1.94 and 8.88 ± 2.19, respectively. lower ovulation rate and smaller uterine capacity than older sows are the possible reasons for smaller litter size in the first few parities (foxcroft et al., 2006). these results corroborate with the report by niyiragira et al. (2018) who observed that the litter size increased with parity. after the first parity, it increases gradually to a maximum in the third to fifth parity and slowly decreases through higher parities (tummaruk et al., 2000). however, the age of the female pig did not show any influence on litter size (p > 0.05). pietrain breed presented the highest average litter size (8.80 ± 1.92). however, the difference was not significant. on the other hand, niyiragira (2018) reported that pietrain breed has significantly the largest litter size than landrace and cross between landrace and pietrain breed. conclusions the present study shows that the conception rate using ai of pigs in rural smallholder farms of rwanda is lower 8 table 3. effect of intrinsic factors on litter size per female pig using extended fresh semen in rural smallholder pig farms. intrinsic factors n mean std. deviation landrace 21 7.86 a 2.06 female breed largewhite 13 7.92 a 2.36 p = 0.86 pietrain 5 8.80 a 1.92 cross 61 7.90 a 2.34 parity 1 12 6.75 b 3.17 2 64 7.81 ab 1.94 p = 0.02 3 24 8.88 a 2.19 age between 1 and 2 years 80 7.93 a 2.29 p = 0.89 above 2 years 20 8.00 a 2.08 total 100 7.94 2.24 a,b means in the same row with different superscripts significantly differ (p < 0.05). compared to the other studies done elsewhere and can be influenced by intrinsic and extrinsic factors. among the intrinsic factors influencing pregnancy rate, parity had a positive effect on pregnancy rate. for the extrinsic factors, the male managed farms presented better conception rates than female managed farms. farms managed by middle aged household heads presented better conception rates than the farms managed by young and old farmers. education level of the household head had positive effect on conception rate. inseminating in morning and evening results in better pregnancy rate than inseminating in the afternoon. reducing distance between insemination center and the farm had positive effect on conception rate. from the results, it is recommended to improve ai procedure in order to reduce the number of artificial inseminations per conception. this could be possible, for example, through increasing the number of ai centers in order to reduce the distance between the households and semen collection center, thus, optimize the success of ai in rwanda. indeed, more attention should be made regarding the pig farms managed by old, young, female farmers and farmers with only primary education to optimize the success of artificial insemination in pigs. conflict of interests the author has not declared any conflict of interests. references ajala mk (2007). analyses of factors affecting the management of pigs in kaduna state, nigeria. agricultural journal 2(2):343-347. am-in n, wichai t and mongkol t (2010). comparison of artificial insemination with natural mating on smallholder farms in thailand, and the effects of boar stimulation and distance of semen delivery on sow reproductive performance. tropical animal 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reproductive performance of purebred swedish landrace and swedish yorkshire sows: i. seasonal variation and parity influence. acta agriculturae scandinavica, section a animal science 50(3):205-216. vanroose g, de kruif a, van soom a (2000). embryonic mortality and embryo–pathogen interactions. animal reproduction science 6061:131-143. vargas aj, bernardi ml, bortolozzo fp, mellagi apg, wentz i (2009). factors associated with return to estrus in first service swine females. preventive veterinary medicine 89(1-2):75-80. visalvethaya w, wichai t, mongkol t (2011). the development of a model for artificial insemination by backyard pig farmers in thailand. tropical animal health and production 43(4):787-793. zanu hk, antwiwaa a, agyemang ct (2012). factors influencing technology adoption among pig farmers in ashanti region of ghana. journal of agricultural technology 8(1):81-92. 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 12 (2), pp. 001-008, february, 2024. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper optimizing pig artificial insemination with imported fresh semen: key success factors vincent niyiragira1, kugonza donald rugira1,2* and hirwa claire d’andre1 1 rwanda agriculture board (rab), p. o. box 5016, kigali, rwanda. 2 department of agricultural production (dap), school of agricultural sciences (sas), college of agricultural and environmental sciences (caes), makerere university, p. o. box 7062, kampala, uganda. *corresponding author. e-mail: aridian181@gmail.com. received 6 july, 2023; accepted 17 november, 2023 this study was conducted to assess the factors that affect pig litter size, proportion of live pigs at birth, number of inseminations per conception, and efficiency of artificial insemination. the main factors assessed were sow breed (n = 2), sire breed (n = 3), sow parity (n = 7) and insemination method (n = 2). the sow breeds used were landrace, ll (n = 27) and landrace × pietran crossbreds, lp (n = 37); boar breeds used were landrace (ll), pietran (pp) and landrace × pietran crossbreds (lp). sows were randomly inseminated either by boar or artificially, with semen freshly imported from belgium to rwanda. landrace (ll) sows had significantly smaller (p<0.05) litter sizes at birth (9.04±0.72) compared to lp sows (11.49±0.45). on the other hand, the litter size for ll (9.04±0.58) and lp (11.49±0.67) sires did not differ, while pp sires had the highest (p<0.05) litter size (13.37±1.43). interestingly, the method of insemination, whether use of a boar or artificially did not (p>0.05) affect the number of inseminations per conception, litter size, and proportion of piglets born live. sow parity was found to have a linear relationship with a mean litter size at birth of 6.9±0.43 piglets for primiparous sows and 15.2±1.12 piglets for a sow of parity 7. however, the number of piglets born dead increased with parity, peaking at 3.0±0.66 (parity 7), while it was only 0.2±0.2 for parity 1. therefore, the lp crossbred sows and pietran sires are recommended. artificial insemination should be promoted since it performed as well as natural insemination but provides other advantages such as african swine fever and inbreeding prevention, and avoids boar management costs. key words: breeding, genetic merit, performance, pietran, pigs. introduction the global pig population in 2017 was estimated at 784.83 million head (national hog farmer, 2017), with china leading in pig population at 435.04 million head, the top pork-producing country at 51.85 million tons (47.9% of world total), top pork-importers (40% of world total), and also the leading per capita pork consumer at 40.9 kg (national hog farmer, 2017; statista, 2018). in africa uganda leads the per capita pork consumption at a meagre 3.5 kg (birungi et al., 2015; kugonza et al., 2015), and hence a lot still has to be done on the 2 continent. pig production has been growing on the african continent, and particularly in rwanda; the growth has been consistent especially during the post-conflict years. the most recent census put the national pig herd at 706,000 animals (nisr, 2011), having risen by 35.4% during the period 2005 to 2010. the massive shifts are being driven by increased demand for pork and pork products such as sausages, bacon and special cuts. along with other livestock species, notably cattle, goats and sheep; pigs contribute 12% of the rwandan gdp, and a significant 32% to the agricultural gdp (nisr, 2017). pig farming in peri-urban areas is highly competitive when compared to goat and cattle farming (nabikyu and kugonza, 2016). this is attributed to pigs requiring less land per livestock unit for acceptable levels of production. farmers in peri-urban areas in much of east africa also have access to agro-industrial byproducts such as wheat pollards, brewers waste, molasses, and brans of maize, rice and wheat (mwesigwa et al., 2013; kugonza et al., 2015). these feedstuffs form the basal diet of pigs under commercial production. a major drawback to improving productivity is the breeding management, especially regarding unavailability and limitations in propagation of superior pig genetic material. the use of artificial insemination (ai) technique for pigs and non-bovine livestock is very limited in rwanda. the major driver of ai use is the need to disseminate superior genes within a given population at a reasonable cost. the greatest advantage of promoting ai use is that it will make possible, the widespread use of outstanding breeding sires and dissemination of valuable genetics to both big and small farms. this will lead to faster genetic improvement of the national pig herd. pig ai technology has been in use in northern rwanda at a private farm but is yet to be tried out at community level, where breeding boars are still communally used through natural service. small/medium scale farmers who do not own boars on their farms rely on a neighbour’s boar to breed their sows. this has also been reported in neighbouring uganda where over 52% of smallholder pig farmers do not own a breeding boar (bamundaga et al., 2018). however, this practice of communal boars promotes the spread of diseases especially african swine fever and various reproductive diseases because of the movement and contact between animals. also, the sharing of the boar by many farmers leads to its overuse which might explain the occurrence of small litter sizes at subsequent births. this ultimately leads to a low number of pigs per sow per year and economic loss. ai of the pig involves collection of semen from a boar and then introducing it into a sow or gilt later on by means of a catheter (ikani and dafwang, 1999; bamundaga et al., 2018). it differs from natural service which involves a boar mounting the sow and introducing his semen by copulation. sperm, the main ingredient in semen was first seen by leeuwenhoek and hamm in 1678 (foote, 2002). then, the first successful insemination was performed in dog by spallanzani in 1784 and over a century later, in 1897, ai in livestock specifically rabbits and horses was then reported (foote, 2002). pig ai was first performed by ivanoff in russia almost a century ago (ombelet and van robays, 2015) however, its wide commercial application in pig production is more recent. it is just over two decades ago when insemination protocols for pig ai were standardized (gadea, 2003), and related work continues. a contemporary study (bamundaga et al., 2018) has recently established that single and double ai protocols lead to non-varying conception rates (94.4 versus 89.6%), and litter sizes (8.16±0.34 versus 9.00±0.39 piglets). recent estimates put pig ai at nineteen million inseminations worldwide per year and of these, almost all (99%) are done using boar semen preserved at temperatures of 15 to 20°c (johnson et al., 2000). ai in pigs: (i) allows for the wider use and distribution of boars of high genetic merit; (ii) allows up to 25 sows to be served with semen from one boar ejaculate, each dose given to a sow containing 2 to 3 billion spermatozoa in 80 to 100 ml (maes et al., 2010); (iii) prevents the transmission of diseases from farm to farm by the movement of sows to and from the boar as well as the sale of diseased boars; (iv) helps to overcome the challenge of differences in size of males and females, especially the limited use of heavy boars which may be of high genetic caliber; (v) eliminates the need to purchase, house and feed boars especially on small scale agriculture; (vi) reduces the farmers risk of handling boars for use in natural service (ikani and dafwang, 1999). currently, the pig breeds reared in rwanda include large white (on 22.9% of pig farms), landrace (37.7%), pietrain (7.3%), duroc (1.6%), local (2.3%) and nondescript crossbred (28.1%) pigs (mbuza et al., 2016). the local pigs are black in colour or black in mixture with white. the productivity of the local pigs is still very low, characterised by low birth weight and slow growth rate. the average age at first farrowing of sows is between 18 and 24 months, while the number of piglets born from the black pigs range from 8 to 10 piglets, and a mature weight of 120 kg is attained in pigs aged 18 months (rarda, 2010). for the large white breed, the average weight of 70 kg is attained at 5 months, while first farrowing occurs at 12 months, with a litter size of 10 to 12 piglets. growth performance of the landrace breed is generally better than the large white although performance data is not readily available (rarda, 2010). pietrain pigs on the other hand generally have lower growth rates but produce predominantly lean carcasses when the effect of feed is accounted for. this study was therefore conceived to study the major factors that influence the success of an artificial insemination programme that uses imported fresh boar semen. 3 materials and methods ethical approval this research followed ethical standards and complied with regulations of the rwanda national council for science and technology. study location the study was conducted at centre de perfectionnement agricole et elevage de kisaro (1 o 37'41.99''s; 30 o 01'39.11''e), a private farm located at kisaro sector, rulindo district in the northern province of rwanda. the site is at around one and half hours’ drive from kigali city. experimental animals sixty-four sows of landrace breed (n = 27) and landrace × pietrain crossbreed (n = 37) were used in this study. the sows were of parities ranging from one to seven. semen or boars used were of landrace breed (n = 28), pietrain breed (n = 15) and landrace × pietrain crossbreeds (n = 21). forty of the total experimental sows were bred using artificial insemination, while twenty four were bred using natural mating with the boar. the experiment was conducted over eight month duration. source of semen for artificial insemination fresh semen was imported from hypor (a hendrix genetic company), a breeding company based in belgium (hypor belgium v, leie rechteroever19870, olsene belgique, t 014 63 53 47, f014 63 54 79, (www.hyvarselect.be). semen collection was performed the day prior to shipment to rwanda. semen was collected from several boars, pooled and then extended using standard protocols before being shipped. housing all pigs at the farm were kept indoors, adults in individual pens, while weaners and growers were reared in groups of up to ten. individual pens were made of a concrete floor, with walls of brick measuring up to 1.5 m in height. the average floor space area per adult pig was up to six square metres. farrowing pens measured 2.5 m by 2.5 m, boar pens measured 2.5 m by 1.25 m while grower pigs were kept in pens measuring 2.5 m by 1.25 m. pig house roofs were made of corrugated iron sheets. feeding the main diet of the pigs was a composite ration made of maize bran, wheat bran, bone meal, soybean meal, fish meal, vitamin mineral premix and salt. the wheat and maize used in feed formulation was largely produced on the farm. the pigs were fed twice a day, at 09:00 to 10:00 h, and at 15:00 to 16:00 h. on average, each mature pig was given 2.5 kg of the composite ration per day, with bigger sows getting proportionately higher amounts. water was provided ad libitum. piglets were farrowed in specialized farrowing pens (2.5 m x 2.5 m) and received an injection of iron on the 3 rd day of their lives. piglets stayed with their mother from birth until weaning at two months, though they had access to creep feed placed in a specialized creep area, accessible only to piglets. castration of male piglets was done within one to two and a half months of age. at weaning sows were removed from the pens and the weaned piglets were allowed to stay in the pens for extra one week. afterwards, the weaners were segregated by sex and relocated to grower pens (2.5 m x 1.25 m) in groups of up to ten. breeding and health management sows naturally came on heat 3 to 7 days after weaning their litters, and had never been induced using hormonal treatment, but were regular breeders. gilts were bred on their second heat, at the age of seven months. the sows were allocated individual pens next to adult boars to enable estrus detection. in addition, the back pressure test for standing heat reflex was performed and those that responded were considered to be in estrus. the sows were either taken to the boar for supervised service or were inseminated with imported extended fresh semen. twenty four sows were served by boars twice using the am-pm rule, so as to maximize on the conception rate. each boar was allowed to serve a maximum of four sows a week to avoid them being overworked. sows and gilts were served by boars in rotation to mimic ai where semen from several boars was pooled, in consideration of the desired breed. on the other hand, 40 sows were artificially inseminated twice, about 24 h between the inseminations using the am-pm rule. each dose inseminated by ai contained 2.5 billion spermatozoa in 80 ml. the intra-cervical method was used in all inseminations (darwin, 2007), with extra care taken to minimize backflow. conception was indicated by a non-return to heat after 18 to 22 days, the few gilts that did not conceive on first insemination were served again on the subsequent natural heat. the pigs were washed every week using water and soap. there was no practice of spraying against ecto-parasites, instead, doubleacting dewormer ivermectin ® was used for this purpose. the pigs were dewormed once every three months. disease outbreaks were described by the farm management as being very rare on the farm. record keeping records on each piglet farrowed were kept on the farm. records that were taken included farrowing date, sow breed, boar breed, parity of sow, litter size (number born alive and stillborn), method of insemination used, and live body weight (taken monthly). dates of treatment in cases of disease, inseminations and routine animal management practices were recorded as well. the farrowing rate recorded was 86% and above. study design, data collection and data analysis the study used a completely randomized design with sows that were recruited into the study randomly selected from the herd. also, sows that were subjected to artificial insemination were randomly selected from the experimental group so as to avoid bias. data were collected over an eight months period following specified/standard procedure, taking care to avoid stressing the animals, in consonance with the national provisions on animal welfare and ethics in handling experimental animals. data was checked for validity and was then entered into ms excel spreadsheets. it was then subjected to analysis of variance using the generalized linear model procedure of statistical analysis systems, version 9.2 (sas, 2004). the fixed effects were sow breed, boar breed, sow parity, and insemination method. the model used for data analysis was: yijklm = si + bj + pk + il + sbij + spik + bpjk + pikl + eijklm n (0, σ 2 e) (i) 4 table 1. least square mean litter size, piglets born alive, stillborn piglets, inseminators per conception. factor level litter size piglets born stillborn inseminations alive piglets per conception sow breed landrace × pietrain (n = 37) 11.49 a 10.81 a 0.67 1.02 landrace (n = 27) 9.04 b 7.79 b 1.25 1.11 landrace × pietrain (n = 21) 7.99 a 7.51 a 0.47 1.09 boar breed landrace (n = 28) 9.43 a 8.29 a 1.14 1.10 pietrain (n = 15) 13.37 b 12.09 b 1.27 1.01 1 st (n = 21) 6.91 a 6.76 a 0.15 a 1.06 2 nd (n = 15) 8.27 ab 7.77 a 0.50 a 1.12 3 rd (n = 9) 9.96 ab 8.75 ab 1.21 ab 0.98 parity 4 th (n = 6) 9.28 ab 8.28 a 1.00 ab 1.24 5 th (n = 5) 9.84 ab 8.66 ab 1.17 ab 1.01 6 th (n = 4) 12.29 ab 12.44 c -0.14 a 1.02 7 th (n = 4) 15.29 c 12.44 c 2.85 b 1.02 insemination method ai § (n = 40) 8.06 7.45 0.61 1.17 ns ∫ (n = 24) 12.47 11.15 1.31 0.97 sem 0.429 0.404 0.164 0.025 lsd 1.48 1.32 0.60 0.10 § ai = artificial insemination; ∫ ns = natural service. abc = means with similar superscripts within column and factor are not different (p<0.05) where, yijklm = observation of the variable for the a sow breed i, mated to boar breed j, for sow parity k and for insemination method l; µ = overall mean; si = effect of sow breed (i = 1,2); bj = effect of boar breed (j = 1,2,3); pk = effect of sow parity (k = 1,2,3,4,5,6,7); il = effect of sow insemination method (l = 1,2); sbij = effect of the interaction between sow and boar breed; spik = effect of the interaction between sow breed and parity; bpjk = effect of the interaction between boar breed and sow parity; pikl, = effect of the interaction between sow parity and insemination method; eijklm is the random effect on the trait, independently and identically distributed with mean = 0 and variance = e 2 . model (i) above was used in the preliminary analysis. due to the finding that the sow and boar breed interaction effect sbij, the sow breed and parity interaction effect spik, the boar breeds and sows parity interaction affect bpjk, and the sow parity and insemination method interaction effect pikl did not significantly affect the variables, they were eliminated from the model, subsequently, the model finally used is shown as follow: yijklm = si + bj + pk + il + eijklm n (0, σ 2 e) (ii) duncan’s multiple range test was used to separate means. results the mean number of piglets born alive was 39% higher for crossbred sows compared to pure landrace sows (table 1). however, the differences in number of piglets born live between boars of the two breed types were not significant; instead, it is the value for pietrain boars that stood out; 53% piglets more than the other categories. parity of the sow did not influence the number of piglets born alive, but it did significantly influence the total litter size. litter size clearly increased with parity, rising consistently from 6.91 piglets for sows of first parity to 15.29 piglets for sows of parity 7, a 121% increment. the litter size as well as the number of piglets born alive did not differ between sows bred using artificial insemination and those that were bred using natural service. the number of stillborn piglets was not affected by sow breed, boar breed, and insemination method; but was influenced significantly (p<0.05) by parity of the sow. in general, the number of stillborn piglets increased with parity (figure 1), rising from 0.15 for primiparous (first parity) sows to 2.85 for 7 th parity sows. considering the number of inseminations per conception, all the factors namely sow breed, boar breed, sow parity, and insemination method did not show significant differences (p>0.05). discussion this study focused on establishing the determinants of success in a pig artificial insemination programme using semen freshly imported from europe to central africa. sows inseminated artificially had a litter size of 8.06±0.42 piglets quite comparable to 9.06±0.4 piglets reported by bamundaga et al. (2018) and 10.8±2.2 (chanapiwat et al., 2014), and despite being in varying locations, the studies used landrace breed and its combinations with other breeds. improvements in the management of 5 18 16 14 y = 1.1807x + 5.54 o f p ig le ts 12 y = 0.9389x + 5.5443 10 8 n u m b e r 6 4 2 y = 0.2421x 0.0057 0 1 2 3 4 5 6 7 -2 parity figure 1. effect of parity on litter size, piglets born alive and those born dead. litter size piglets born live stillborn piglets inseminations per conception linear (litter size) linear (piglets born live) linear (stillborn piglets) artificial insemination imply that the technology can continue to impact the pig industry. despite chanapiwat et al. (2014) reporting that the main limitation of the extended fresh semen is the short timing of semen storage, and the likelihood that semen cannot be transported for a long distance, results of this study that used semen transported from northern europe shows that this is not an issue. instead, the chance of distributing a good genetic resource across countries and particularly, enabling locally raised sows to be bred with semen having the best estimated breeding values available in europe is very real. use of ai for breeding pigs has become instrumental for facilitating global improvements in fertility, genetics, labour, and herd health (bortolozzo et al., 2015; knox, 2016). the differences in number of piglets born live between boars of the two breed types in this study were not significant (table 1), instead, it is the value for pietrain boars that stood out over the other categories. the establishment of ai centers for management of boars and production of semen has allowed for selection of boars for fertility and sperm production using in vitro and in vivo measures (ringwelski et al., 2013; knox, 2016). parity of the sow did not influence the number of piglets born alive (table 1), but it did significantly influence the total litter size. second parity sows have been reported to produce an average litter size of 8.36±0.28 when artificially inseminated, and 10.6±0.64 when naturally served by the boar (ronald et al., 2013). while that study found the differences between the two methods were significant, this study found contrary results. though the superiority of natural mating in both studies is vivid and indeed, this study found a higher litter size (12.47) than that of the indian study, which was also done at a private farm. low parity females especially pregnant gilts and primiparous sows have also been reported elsewhere to have lower reproductive performance than sows in parities between second and fifth parity (koketsu et al., 2017). sow fertility is also closely associated with varying doses of semen volume and spermatozoa count for ai (apic et al., 2015). while stillbirths were found in both artificially and naturally inseminated sows, other studies (ronald et al., 2013) reported still births only in naturally inseminated sows. the variance between the two studies could be attributed to the parity factor, since the study had mixed parities, with older sows having high stillbirths and the values must have been responsible for swaying the overall mean. other contemporary studies also strongly support the position that number of piglets born and the number of live born piglets are highest in 2 to 3 parity sows and drop significantly in sows of more than five parities (wegner et al., 2014). while the number of still born piglets was lowest in second parity sows (0.5), wegner et al. (2014) found the lowest (0.8) in first parity sows, and the highest (1.44) in sows of more than five parities, slightly higher than 1.17 that was found in the current study. 6 conception failures in pigs have been known to result from effects of the season, mode of insemination, age of the sow, and the number of times the female is inseminated, and the females’ birth litter sex ratio (drickamer et al., 1997; wegner et al., 2014), this study differs having found that the mode of insemination and number of times the female is inseminated did not have significant effects. conclusion litter size of weaned sows bred by natural service or ai was the same. in addition ai has the significant advantage of enabling locally raised sows in rwanda to be bred with semen having the best estimated breeding values available in europe, from belgium. ai relying on imported semen should be promoted especially for introduction of superior genetics for which grade boars may not be manageable in tropical africa. acknowledgements the comments of the reviewers are strongly appreciated as they have contributed to improvements to the manuscript. conflict of interests the authors declare that there is no conflict of interests. references apic j, vakanjac s, stancic i, radovic i, jotanovic s, kanacki z, stankovic b (2015). sow fertility after insemination with varying doses of volume and spermatozoa count. turk. j. vet. anim. sci. 39:709713. bamundaga gk, natumanya r, kugonza dr, owiny do (2018). reproductive performance of single and double artificial insemination protocol in swine. bull. anim. health prod. afr. 66(1):143-157. birungi r, ouma e, brandes-van dorresteijn d, kawuma b, smith j (2015). pig marketing and institutional strengthening: uganda smallholder pig value chain capacity development training manual. ilri manual 11. nairobi, kenya: ilri. bortolozzo fp, menegat mb, mellagi apg, bernardi ml, wentz i (2015). new artificial insemination technologies for swine. reprod. domest. 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available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper antiviral activities of ellagitannins against bovine herpesvirus-1, suid alphaherpesvirus-1 and caprine herpesvirus-1 neli vilhelmova-ilieva1, ivo sirakov2, remi jacquet3, stephane quideau3 and angel s. galabov1* 1 department of virology, the stephan angeloff institute of microbiology, bulgarian academy of sciences, 26 g. bonchev str., bg-1113 sofia, bulgaria. 2 department of medical microbiology, medical faculty, medical university of sofia, 2 zdrave street, bg-1431 sofia, bulgaria. 3 universitéde bordeaux, institut des sciences moléculaires (cnrs-umr 5255), institut européen de chimie et biologie, 2 rue robert escarpit, 33607 pessac cedex, france. *corresponding author. e-mail: galabov@microbio.bas.bg. tel: +359 888 287 103. fax: +359 2 870 0109. accepted 23 july, 2021 in vitro antiviral activity of three ellagitannins castalagin, vescalagin and grandinin was investigated against replication of three of the most common herpes viruses infecting animals, namely bovine herpesvirus-1 (bohv-1), suid alphaherpesvirus-1 (suhv-1), and caprine herpesvirus-1 (caphv-1). the cytopathic effect inhibition test via the neutral red uptake assay in madin-darby bovine kidney (mdbk) cell culture was applied. the highest activity was demonstrated by castalagin followed by vescalagin against replication of suhv-1, strain a2, with selectivity index values of 336.8 and 309, respectively, which are close to that of acyclovir (si = 540). the activity of grandinin against the replication of suhv-1 a2 strain (si = 40.8) as well as the activity of all three substances against the replication of bohv-1 strain k22 was lower but still significant (castalagin si = 45; vescalagin si = 42.5; grandinin si = 32.3). against the e/ch strain of caphv-1, the three ellagitannins showed a moderate to weak activity: castalagin si = 19.3, vescalagin si = 18.8, grandinin si = 11.8. the results obtained characterize compounds tested as perspective antivirals. key words: bovine herpesvirus-1, suid alphaherpesvirus-1, caprine herpesvirus-1, ellagitannins, alpha acyclovir, antiviral activity. introduction among the viruses of the herpesviridae family, some are common pathogens for humans and animals. they are easily transmitted from an infectious to a healthy individual and also they have the ability to form a life-long latent infection. in the herpesvirus species that attack animals, it is important that the animals are not only a reservoir for viruses, but also that the quality of the meat from the infected animals of the point of view of food 2 production is deteriorated and this leads to great economic losses. among the most important herpesviruses infecting animals are the members of the subfamily suid alphaherpesvirinae bovine herpesvirus-1 (bohv-1), alphaherpesvirus-1 (suhv-1), and caprine herpesvirus-1 (caphv-1). the economic importance of these viruses lies in the high economic losses incurred due to infections and the clinical symptoms caused by them. bohv-1 infections lead to repeat breeding, rhinotracheitis in calves, vulvovaginitis-balanoposthitis in adult animals (haralambiev, 2002), infertility, abortions (yilmaz et al., 2016) and neonatal mortality (wyler et al.,1989). caphv1 causes a similar clinical picture, with neonatal losses of up to 80%, repeat breeding 15%, reproductive disorders in adult animals abortions 5% in affected flocks (sirakov 2012), and death in kids (saito et al., 1974). whereas bohv-1 and caphv-1 affect cattle and goats, suhv-1 causes infections in a range of various domestic and wild animals. in swine, it is associated with abortions (~35%) and mortality (3–5%) (yu et al., 2017), stillbirths and decrease in weight gain (gerdts et al., 1997). it affects ruminants, horses, dogs (quiroga et al., 1998), cats and panthers (glass et al., 1994). although some cases have been reported in humans (mravak et al., 1987; skinner et al., 2001) and that this virus has limited zoonotic potential (khan et al., 2013), suhv-1 to some extent has social importance, as well. to date, there is no in general effective therapy of herpesvirus infections, nor vaccines created to avoid the formation of latent infection. administration of anomalous nucleoside analogues is considered as the most potential by its efficacy chemotherapeutic agents used in the clinical practice, acyclovir been with largest application. a disadvantage of nucleoside analogues is the relatively faster development of drug-resistant mutants. limited scope of investigations was carried out on chemotherapeutic agents proved in humans against herpesvirus infections in domestic animals. treatment with cidofovir has been shown to give good relief of clinical symptoms and a decrease in the viral shedding titers in caphv-1 infection in goats (tempesta et al., 2008). chervenkov et al. (2014) demonstrated that aqueous extracts of melissa officinalis l. (lamiaceae) have a marked antiviral effect against suhv-1. antiviral activity against this virus, as well as against bohv-1, has also been shown by lambda-carrageenan prepared from red seaweed (diogo et al., 2015). moreover, lisov et al. (2015) demonstrated that 2,5-dihydrobenzoic acid– gelatin conjugate has an anti-bohv-1 effect and suggested that this agent would also be effective against other alphaherpesvirus subfamily members due to the similar mode of entry into the host cells. some studies on the spread and replication of herpesviruses have been conducted using natural substances found in many foods that a person consumes daily. recently a special interest as anti-herpetic agents are the tannins which are a group of polyphenols, divided into two groups of condensed and hydrolysable compounds. of the group of hydrolizable tannins are the ellagitannins. there is a lot of evidence in the literature that different types of ellagitannins show anti-herpesvirus activity (kurokawa et al., 2001; chattopadhyay et al., 2010; lin et al., 2011). in previous investigations we have also shown that three ellagitannins castalagin, vescalagin and grandinin, possess remarkable activity against human herpes simplex virus (hsv)-1 and hsv-2, sensitive and resistant to acyclovir strains (vilhelmova et al., 2011; vilhelmova– ilieva et al., 2014). in the present work we pay attention to the activity of these three substances on the replication of herpesvirus strains that are animal pathogens. materials and methods cells and viruses monolayer cultures of madin-darbey bovine kidney (mdbk) cells (cclv 1992, rie 261/ national bank for industrial microorganisms and cell cultures, sofia) were grown in dulbeco minimal essential medium (dmem) containing 10% bovine fetal serum (gibco brl, usa), supplemented with 10 mm hepes buffer (merck, germany) and antibiotics (penicillin 100 iu/ml, streptomycin 100 μg/ml), in co2 incubator (hera cell 150, heraeus, germany) at 37°c/5% co2. suid alphaherpesvirus 1 (suhv-1) strain a2, bovine herpesvirus 1 (bohv-1), strain k 22, and caprine herpesvirus-1 (caphv-1), strain e/ch (national diagnostic and research veterinary institute collection strains) were included in the study. the viruses were grown in mdbk cell cultivated in mem-eagle (sigma–aldrich, st. louis, mo, usa) and mem-hanks (sigma–aldrich, st. louis, mo, usa) supplemented with antibiotics (penicillin 100 iu/ml, streptomycin 100 iu/ml), essential amino acids and 2% fetal bovine serum (sigma–aldrich, st. louis, mo, usa). the virus titers were evaluated by the end-point dilution method, based on the cytopathic effect (cpe) inhibition and expressed as 50% cell culture infectious dose per ml (ccid50/ml). compounds tested nonahydroxyterphenoyl-containing c-glucosidic ellagitannins: castalagin, vescalagin and grandinin, extracted from powdered pedunculate oak (that is, quercus robur) heartwood and purified as previously described (quideau and feldman, 1996) were tested. the substances were dissolved in distilled water to a concentration of 0.01 m and then diluted in dmem to the required concentrations. acyclovir [9-(2-hydroxyethoxymethyl)-guanine] (acv) was also dissolved in dmem to the required concentration. antiviral activity assay cpe inhibition test was employed on confluent cell monolayer in 96well micro plates infected with 100 ccid50 in 0.1 ml (moi = 0.0025). after 1 h of virus adsorption, compounds were added in various concentrations and cells were incubated for 48 h at 37 °c. inhibition of cytopathic effect was determined using a neutral red 3 table 1. antiviral activity of ellagitannins castalagin, vescalagin and grandinin against replication of bovine herpesvirus-1 strain k 22, suid alphaherpesvirus 1 strain a2 and caprine herpesvirus-1 strain e/ch. compound suhv-1 a2 bohv-1 k 22 caphv-1 e/ch ic50 µm/ml si ic50 µm/ml si ic50 µm/ml si castalagin 0.16 ± 0.073 ***,^^^ 336.8 1.2 ± 0.12 ***,<<< 45.0 2.8 ± 1.61 ***,>>> 19.3 vescalagin 0.22 ± 0.068 ***,^^^ 309.0 1.6 ± 0.62 ***,<<< 42.5 3.6 ± 2.11 ***,>>> 18.8 grandinin 0.87 ± 0.24 ***,# 40.8 1.1 ± 1.07 ***,<<< 32.3 3.0 ± 3.2 ***,>>> 11.8 acv 2.4 ± 0.55 ^^^ 540.0 3.8 ± 0.84 <<< 341.0 18.4, >>> 70.4 *** p<0.0001, when comparing the values of each ellagitannin with that of acv for the same strain. ^^^ p<0.0001, when comparing the values against suhv-1 (a2) and bohv-1 (k22) strains of each substance. <<< p<0.0001, when comparing the values against bohv-1 (k22) and caphv-1 (e/ch) strains of each substance. >>> p<0.0001, when comparing the values against suhv-1 (a2) and caphv-1 (e/ch) strains of each substance. # p>0.05, when comparing the values against suhv-1 (a2) and bohv-1 (k22) strains of each substance. uptake assay (borenfreund and puerner, 1984). the ic50 values of ellagitannins were evaluated, that is, the concentrations that inhibited cpe development by 50%. the compounds cytotoxicity cc50 (50% cytotoxicity) values were used to determine the selective index (si) values of the compounds tested. cytotoxicity of ellagitannins and acv was determined on mdbk cells in our previous study (vilhelmova et al., 2011).the values of cc50 of the substances were as follows: castalagin 53.9 µm/ml, vescalagin 68.0 µm/ml, grandinin 35.5 µm/ml and acv 1296.0 µm/ml. statistical analysis data on compounds antiviral effects were analyzed statistically. the values of ic50 are presented as means ± sd. the significant differences between the effects of each ellagitannin on each virus strain are compared with the corresponding acyclovir value; also those between the effects of each compound on the different strains were done through the one-way anova where p-values of <0.05 were considered as significant. results the ic50 values of ellagitannins tested against the three herpesviruses (suhv-1, bohv-1 and caphv-1) are presented in table 1. all three ellagitannins show the highest activity against suhv, strain a2. among them the antiviral effect of castalagin attained a pronounced value, si = 336.8, followed by magnitude by the vescalagin’s effect, si = 309. the activity of both substances was close to that of acv si = 540. grandinin manifested a marked activity but was substantially weaker. the three ellagitannins demonstrasted a marked activity against bohv-1, strain k22, with close si values. the most active was castalagin with si = 45, followed by vescalagin with si = 42.5, and weakest activity was shown by grandinin si = 32.3. here, it should be noted that, unlike the results obtained against suhv-1, catalagin and vescalagin manifested significantly lower activity, while grandinin retained almost the same activity as that of acv against suhv-1. the weakest activity of the three ellagitannins was demonstrated against the replication of caphv-1, e/ch strain. the values of the selective indices of castalagin, vescalagin and grandinin were si = 19.3, si = 18.8, and si = 11.8, respectively. it could mark that the activity of acyclovir against this virus was lower than that established vs bohv-1 and suhv-1, although it was significantly superior (si = 70.4) compared to the effect of ellagitannins. discussion having in mind that the ellagitannins castalagin, vescalagin and grandinin, studied in the present work, were tested against viruses belonging to the subfamily alphaherpesvirinae of the herpesviridae family, it is not surprising that they manifested activities similar to that found against the subfamily members attacking humans (hsv-1 and hsv2) (vilhelmova et al., 2011). when comparing the activity of ellagitannins on the replication of the suhv-1 strain and their activity against human herpesvirus strains, it is noted that their activity is close to that observed with hsv-1 (victoria strain) (vilhelmova et al., 2011). on the other hand, the activity of the tested ellagitannins on replication of bohv-1 resembles their activity against hsv-2 (strain bja) (vilhelmova et al., 2011), although with lower values and that of castalagin and vescalagin was close to that of grandinin. evidently, anti-bohv-1 activity was considerably inferior when compared with the activity against suhv-1. similar dependence was observed in the ellagitannins activity against replication of caphv-1 strain, where the activity was significantly lower and the activity of grandinin can be said to be insignificant. different authors have conducted various experiments to determine the viral replication stage inhibited by ellagitannins. some ellagitannins have been shown to attack the extracellular virions (virucidal effect); others have an effect on the adsorption and entry of the virus into the host cell. there are ellagitannins that specifically inactivate viral dna polymerase and inhibit replication of viral dna or influence specific viral proteins necessary for viral replication (tan et al., 2013). the results of our 4 study on the mode of anti-herpesvirus action of castalagin on the model of hsv-1 (victoria strain) are in line with these data. the compound has been previously shown to have (i) a marked direct inactivating effect on extracellular virus; (ii) an inhibitory effect on virus adsorption; (iii) suppression of early steps in the viral replication cycle (vilhelmova-ilieva et al., 2013). there are conflicting reports about the effect of acyclovir as the sole antiviral therapy against caphv-1. elia et al. (2015) observed no antiviral effect, whereas camero et al. (2017) report good inhibitory activity. camero et al. (2017) explained these contradictory results to the different time of acyclovir application in the experimental setup. both studies, however, show good antivirus activity of acyclovir in combination with mizoribine. it was established that there is a synergistic combined effect of acyclovir and mizoribine on the replication of strain caphv-1 (elia et al., 2015, camero et al., 2017) and on the human hsv-1 (pancheva et al., 2002). in these previous experiments, acyclovir was used in sub-inactive concentrations, and with the addition of mizoribine a strong synergistic effect was obtained. this effect was mizoribine dose dependent. developing effective therapies to suppress herpes viral replication is important because the herpes infection of animals has not only economic significance for human infections but also for food production. in recent years, more and more attention has been paid to the ability of some herpes viruses to pass between species’ barrier. in these cases, a virus that specifically infects a particular animal can also infect other species, and in some cases it can also be transmitted to humans (reperant et al., 2016). data presented in this study enlarge the scope of ellagitannins’ effects on replication of herpesviruses of alphaherpesvirinae subfamily. it would be of interest to see the activity of these compounds against herpesviruses not belonging to this subfamily. among the pathogens with economic importance are gallid herpesvirus 1 (laryngotracheitis virus), ovine herpesvirus 2 (malignant catarrhal fever virus), equine herpesviruses 1, 4 and 9 and cyprinid herpesvirus 3. so, experimental chemotherapeutic investigations including these viruses could be considered as a target of primary importance. conflict of interests the authors have not declared any conflict of interests. references borenfreund e, puerner ja (1984). toxicity determination in vitro by morphological alterations and neutral red adsorption. toxicology letters 24:119-124. camero 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(ed.), herpesvirus diseases of cattle, horses and pigs. kluwer academic publishers, boston, dordrecht, london pp. 1-72. yilmaz v, coskun n, sahin m (2016). molecular detection of bovine herpes virus-1 (bohv-1), bovine herpes virus-4 (bohv-4) and bovine viral diarrhoea virus (bvdv) in aborted ruminant fetuses from kars province in northeast turkey. indian journal of animal research 50:551-556. yu x, zhou z, hu d, zhang q, han t, li x,tian k (2014). pathogenic pseudorabies virus, china, 2012. emerging infectious diseases 20:102-104. 1 author(s) retain the copyright of this article. full length research paper africa's pig industry: present situation, obstacles, potential, and prospects joseph k. ali laikipia university, nyahururu, kenya accepted 29 december, 2024 one of the profitable businesses in the livestock subsector of agriculture is the production of pigs. it makes a substantial contribution to the economy and the supply of animal protein, which improves food security in africa and around the world. the current state of pig production in africa was examined in this article, along with its opportunities, difficulties, and possibilities. africa's pig population makes up 4.6% of all pigs worldwide. they are found all over africa, with the exception of northern africa, where pig farming is discouraged for religious and cultural reasons. smallholder farmers raise them mostly in rural areas of africa, which explains why indigenous breeds and their hybrids make up the majority of the pig population in most of the continent. pigs have cultural and social value and are essential to rural communities' ability to maintain their standard of living. africa's pig production system is primarily traditional, but it is expanding quickly and changing to become more modern. africa now produces more than 2 million tons of pork annually, up from less than a million tons in 2000. one of the primary factors limiting pig productivity in africa is the prevalence of illness outbreaks, particularly african swine fever. other factors include a lack of technical expertise and skills, high temperatures, restricted access to superior breeds, high feed and veterinary input costs, unwelcoming government regulations, prejudice based on religion and culture, inadequate processing facilities, and an underdeveloped value chain. the need for more food production is indicated by the estimated 2.5 billion people living in africa by 2050, rising urbanization, and a decline in the number of farmers. for increased productivity and easier exporting, the pig production systems in africa need developmental research, advances in housing, feed production and manufacture, animal health, processing, capacity building, and pig-friendly regulations. key words: farmers; policy; pork; smallholder; transformation; value-chain. introduction global cattle production has benefited from africa's enormous territory and growing population. with 1.4 billion people living there, africa is the second largest continent in the world and accounts for 17.9% of the global population [1]. the world's livestock sector is significantly shaped by africa's remarkable biological and geographical diversity as well as its sociocultural complexity. a third of the world's livestock production [2] and almost 40% of africa's agricultural gdp [3] come from the continent's livestock population. in sub-saharan africa (ssa), livestock will play a bigger role in the future as the region's population, affluence, and urbanization all increase demand for food derived from animals. by 2050, consumers with low and intermediate incomes are expected to require 5.5 million tonnes more milk and 107 million tonnes more meat than they did in 2005 and 2007 [4]. by 2050, ssa's per capita annual meat and milk consumption is projected to be 14 kg and 30 l, respectively [2]. one way that africa might satisfy its demand for animal protein and fulfill international demands through exports is by raising pigs. 1.67% of the world's pigmeat is produced in africa. in africa, pig production is a significant part of the livestock subsector of agriculture. it is impossible to overstate how important pig production is to the continent's ability to sustain livelihoods by providing a healthy source of protein, jobs, and revenue. pork is readily digested, has a high biological value, and contains vital amino acids. pork is referred to as "pink meat" because it shares the nutritional african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-008, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 characteristics of both red and white meats [5]. pig meat is a significant source of animal protein in human diets, according to the global trend in meat production and consumption from 2016 to 2020. according to estimates based on fao data [5,6], pig is the most consumed meat worldwide, making up 36% of global meat consumption, more than beef (24%), poultry (33%), and goats and sheep (5%). pig meat, however, is not the most popular meat in africa; instead, it comes in second after beef, mutton, fowl, and chevon. in the majority of africa, pigs are raised primarily under vast production systems, where they are neglected and left to fend for themselves [7]. however, the conventional extensive system is quickly giving way to semi-intensive and intensive manufacturing systems. pigs have the potential to significantly improve food and nutrition security in africa and around the world due to their inherent qualities, which include high fecundity, short generational intervals, superior feed conversion indices, and early maturity when compared to other livestock species. given its comparatively low production costs when compared to other significant livestock farming enterprises, investing in pig production has turned out to be one of the most lucrative livestock ventures [8]. pig farming is therefore a thriving industry that has the potential to close the gap in the supply of animal protein, offer reasonably priced meat, and support the economies of africa and the global community by creating jobs and revenue. despite africa's potential for pig production, the industry is hindered by a number of issues that keep it from reaching its full potential. with regard to the production system, population distribution, pork output, per capita consumption, problems, and future prospects, this research sought to analyze the current state of pig production in africa. present pig production status in africa pig production systems in africa the more than 70% of pigs in africa live in rural areas, where they are typically left to scrounge for food [9]. pigs are widespread throughout the continent. furthermore, 90% of nigeria's pig population is raised in vast systems, and the country contributes 65% of west africa's total pork production [10]. in many african countries, raising pigs is a source of income, particularly for young people and women engaged in small-scale farming. pig production systems in africa vary and are intricate among nations, each with unique potential and difficulties. the prevalence of disease outbreaks, particularly africa swine fever (asf), and rising input costs have had an impact on the pig business globally, affecting both production costs and output. africa has been impacted by these because of the widely used production systems. in africa, pig farming methods typically vary from smallholder, traditional scavenging systems to more intensive commercial setups [11,12]. the pigs are exposed to the risks of weather extremes, disease infection, and theft because the traditional arrangement permits them to move around and search for food. the majority of the animals raised in this method are native breeds. although they do not readily achieve market weight, this approach also uses crosses between native and exotic breeds. this is because scavenging operations take energy that should be used for growth and development, while traditional systems could not give enough nutrition for healthy growth [8]. animals that scavenge are typically linked to high rates of disease infection. the exotic breeds and their crosses are the primary foundation of the intense system. however, in order to meet the nutritional needs of the animals for optimal productivity in confinement, they must employ formulated diets, which can be costly because to the high cost of imported feed ingredients and the paucity of grains. more capital is needed for an intensive system of production since it also requires the supply of decent housing, health care, and facilities for disposing of waste for appropriate management. many african pig farmers, particularly smallholder farmers, use locally sourced materials like cane, bamboo, planks, or mud for walls or house demarcation, even if others are investing in the use of sophisticated pig farm equipment. although they are not exclusive, the common breeds of pigs raised under the traditional method include the indigenous breeds and their hybrids. despite the fact that large white, landrace, and their crosses are more popular, exotic breeds like large white, landrace, duroc, hampshire, and pietrain are common in the intense method of production [8]. disease propagation is facilitated by traditional systems' open scavenging and scant biosecurity precautions [11]. if properly maintained, intensive systems can use biosecurity techniques to reduce the risk of disease. however, small-scale pig producers in africa might find it difficult to afford the cost of putting biosecurity measures in place [11]. pig farm estates and clusters have recently been established for smallholder farmers in africa. they are sometimes arranged into cooperatives or farmer organizations. this is to make some facilities that large-scale farmers likewise enjoy easily accessible to smallholder farmers. farmers who belong to a cluster or cooperative can easily access resources including loans, government assistance, veterinary care, veterinary services, wholesale input procurement for member sharing, good infrastructure, and market accessibility. the oke aro pig farm cluster, the biggest pig farm estate in west africa, is located in lagos state, nigeria, and serves as a prime example [13]. this system's inability to consistently deploy and enforce sound biosecurity measures is one of its main problems. by exchanging labor and equipment among various units in the cluster, farmers and farm attendants violate biosecurity protocols, making it easier for diseases to spread and recur. technological developments, growing awareness of sustainable practices, shifting consumer preferences and perceptions, and initiatives to address issues like food security and disease management have all had an impact on pig production in africa 3 over the years [12]. pig breeds with enhanced disease resistance, growth rates, and reproductive performance that are well-suited to regional conditions have been developed. the utilization of better breeds and the implementation of intensive or modern production systems may be the reasons why south africa and nigeria appear to have the highest pork yield per animal in africa [1]. pig population distribution in africa as of 2020, there were 44 million pigs in africa, which accounts for about 4.6% of all pigs worldwide [1]. with 38% of africa's total pig population, eastern africa has the largest percentage of pigs among the continent's five regions (figure 2). because of the region's cultural-religious demography, which discourages the consumption of pork, northern africa has the smallest pig population in africa, accounting for less than 1% of the continent's total pig population. this has an impact on both the region's per capita consumption and production of pork. with corresponding contributions of roughly 33%, 17%, and 12% of the continental pig population, other regions such as western, central, and southern africa are significant producers of pigs [1]. the two african nations with the largest pig populations are nigeria and malawi (figure 1), with an actual population of over 7 million heads (table 1). together, the two nations account for about 35% of africa's entire pig population. malawi has the highest pig population density in africa, yet nigeria has the largest pig population. figure 3 shows that the number of pigs in africa has been growing over time, particularly since 1980. increased living standards and population expansion are to blame for this growth, as they are driving up the need for food. the pig population in northern and southern africa is an exception, despite the fact that the pig population in eastern, western, and middle (central) africa has grown more rapidly. in these two areas, the number of pigs has not grown significantly over time. the number of pigs in africa grew from 21 million to 43 million between 2001 and 2021, a 101% rise throughout the continent. pig meat yield in africa in 2021, more than 2 million tons of pork were produced in africa [1]. figure 4 shows the amount of pork produced (in tons) in various african regions from 1961 to 2021. figure 5 shows that, with the exception of northern africa, where pork production has decreased by 56%, pork production has increased over time in all of africa's regions. in western, southern, central, and eastern africa, the corresponding increases in pork production rates were 1,696%, 448%, 596%, and 1,396%. western africa produced 768,766.80 tons of pork in total in 2021. south africa is a large producer of pork in southern africa and had the highest per capita consumption of pork in africa (4.19 kg). its contribution to southern africa's pork output as of 2021 was over 320,450 tonnes, or 97% of the 329,710 tonnes of pork produced in the region (table 1). other nations in the region, primarily namibia, eswatini (swaziland), lesotho, and botswana, provided the remaining 3%. over the years, northern africa's pork output has followed an uneven pattern with little to no growth. reduced pig populations, which resulted from what can be called malicious culling of pigs, which claimed that pigs are reservoirs of avian influenza viruses after the disease outbreak in poultry in the region, are the cause of the dramatic decline in pork output in this region starting in 2008 [1]. from 3,893 tonnes in 2006 to 1,328 tonnes in 2011, and then to 1,234 tons in 2015, the production of pork fell [1]. nonetheless, the region's output figure increased once more to 1,610 tons in 2021, and it is anticipated to keep improving as some local farmers express interest in growing pork for the region's residents and visitors. the coptic christian minority, who make up only 10% of the population, are the main producers of pigs in egypt. nonetheless, nations like libya, sudan, and western sahara make up a very little portion of the region's pork production. the majority of pig farmers in morocco are poultry farmers who switched to raising pigs when avian flu outbreaks impacted their poultry farms. producing pork for visitors and certain foreign residents is often their goal. they also hope to close the gap on the importation of pork into the nation, which comes primarily from spain, belgium, and france. due to the significant expansion of the pig industry from 2008 to 2013, which was brought about by the arrival of new local farmers, morocco has also been known to export pigs to nations like the gambia and cyprus, where they are consumed by tourists and foreign residents. since then, however, morocco's pig population has not increased significantly; as of 2021, there were about 8,000 pigs and 630 tons of pig meat produced year [6]. per capita pork consumption in africa because of cultural, religious, economic, and geographic variables, pork intake differs greatly among african nations. in africa, pork is not the most popular meat. africa's per capita intake of pork (1.53 kg) is ranked below that of beef (5.22 kg), poultry (6.30 kg), and chevon and mutton (2.39 kg). africa consumes 1.55 kg of pork per person, which is less than the 14.35 kg consumed in asia and the 33.79 kg consumed in europe. due to shifting tastes and inclinations, meat consumption rises as wealth or income rises. like in certain other regions of the world, religious and cultural beliefs that forbid eating pork have a significant influence on how much pig is consumed in africa. the middle, western, eastern, and northern regions of africa consume 2.18, 1.97, 1.38, and 0.01 kg of pork per capita, respectively, whereas southern africa consumes the most (3.99 kg) [1]. pork is widely consumed in south africa, because the population is diversified and has a wide range of dietary preferences. pork consumption, however, is typically substantially lower in nations with sizable muslim or jewish populations. in many african countries, the patterns of pig eating are significantly influenced by these religious differences. because of islamic dietary prohibitions, countries with a majority of muslims, such egypt, tunisia, and algeria, usually have low per capita pork production and consumption [14]. by 2020, egypt had produced roughly 628 tons, 4 which is less than the more than 302,000 and 311,000 tonnes of pork produced in south africa and nigeria, respectively [1]. economically growing urban areas are known to see a shift in dietary diversity, with alternative protein sources being seen. the consumption of pork may be significantly impacted by this change. it has been recorded that multinational fast-food chains, especially those that serve pork-based products, have proliferated throughout most african urban centers [15]. marketing although live animal sales are the main marketing channel for pigs sold by producers, pig marketing methods in africa are found to be straightforward [13]. pig handling, transportation, and sale conditions are fraught with difficulties [16]. the majority of farmers are compelled to sell live animals to intermediaries, who then sell to butchers. to make as much money as possible, producers occasionally butcher their own animals and sell the fresh pork to the end user. this typically happens when middlemen attempt to drive down prices in order to exploit farmers who are under pressure to sell in order to increase profits. however, there are also out-growery systems, in which farmers produce for specific consumers or processors who then use the animals to make products. the market's ability to operate efficiently is hampered by a number of factors, including inadequate market knowledge, a shortage of capital, difficulty accessing official credit sources, poor roads, and excessive transportation costs. the real market and transaction costs are raised by these limitations. state support for pig products is not available. in most of africa, marketing is experienced similarly to how it is in rest of the world. thus, policy and infrastructure support from the government would improve pig marketing. challenges of pig production in africa common challenges pig farming in africa faces a variety of difficulties that differ from one nation to the next and even within regions. the following are some typical difficulties that pig farmers in africa face: i. policies and programs that are inconsistent both inside and across nations on the continent: south africa exports pork and is a significant participant in the continent's pork sector. shipment delays, product rejection, and harm to trade relationships may result from uneven or sudden changes to export restrictions, especially sanitary and phytosanitary standards. countries like kenya have seen changes in import laws pertaining to pork products during the past ten years. the stability of the local market is impacted by the implications of inconsistent import policies, which typically alternate between stringent prohibitions and lenient regulations. these shifts are difficult for importers and traders to handle, which causes supply interruptions and price volatility. another significant issue affecting pig production in various african nations is the inconsistent application of disease management measures. for instance, pig agriculture in africa and around the world has faced serious challenges due to asf outbreaks (figure 6). between 2018 and 2019, asf attacks caused the loss of about 25% of the world's pig population. both domestic and wild pigs are susceptible to this extremely widespread virus illness. a reduction in pig populations and an effect on farmers' livelihoods can result from the disease's high mortality rates, which can cause large financial losses and hinder pig output in impacted areas [13]. the recurrence of this disease has caused many farmers to quit their jobs because they receive little to no compensation. due to this, the majority of farmers who are afflicted with asf choose to keep to themselves and secretly sell the diseased animals, which further spreads the illness. pig farmers are often confused by inconsistent policies regarding disease management techniques, which makes illness prevention and control challenging. i. limited access to superior breeding stock: in the majority of african nations, there is limited access to superior genetics and genetically upgraded breeds. this has affected productivity and is the cause of the large differences in pig meat yield between nations such as south africa, which primarily produces better breeds or hybrids, and others that mostly raise non-distinct varieties. the profitability of the pig industry as a whole as well as the productivity and efficiency of pig farming systems can be impacted by access to quality breeds. ii. inadequate infrastructure: the growth of the pig business may be impeded by inadequate infrastructure, which includes waste management systems, transportation networks, cold storage facilities, and processing units. due to these infrastructure constraints, it is challenging to move pigs and pork products effectively, which raises costs and restricts farmers' access to markets. iii. exorbitant and fluctuating input costs, including those for medications and feed: one of africa's biggest problems is the cost and accessibility of high-quality pig feed and medications. commercial feed can be expensive, particularly if it contains imported ingredients. pig farmers in africa often have inadequate knowledge of the nutrients that pigs need and inadequate knowledge of how to formulate feed. given that pigs normally need a balanced combination of energy, protein, and other vital nutritional sources, this information gap affects the economics of pig farming. it is not necessary to offer a high-quality diet to native pigs that can survive on subpar feed. however, premium feed is necessary for imported breeds and hybrids. in the majority of african nations, high-quality commercial or branded meals for pigs are scarce. good breeds and the availability of high-quality feed or feed components will boost africa's pork production. furthermore, poor infrastructure and storage facilities for medications and pharmaceuticals, as well as feed ingredients, can cause feed spoiling, which reduces the materials' efficacy. iv. limited access to veterinary care: availability of high-quality veterinary in many regions of africa, access to services such as immunization programs, disease diagnosis, and treatment is frequently restricted [17]. this can make pig producers more susceptible to disease outbreaks and result in large financial losses by impeding attempts to prevent and manage disease. in pig farming, putting in place efficient biosecurity measures to stop the spread of illnesses is essential. however, it can be difficult to contain disease outbreaks and preserve healthy pig populations 5 due to a lack of knowledge, insufficient funding, and subpar biosecurity procedures. v. insufficient technical expertise: for small-scale pig farmers, who make up the majority in most countries, limited access to training and technical information about contemporary pig farming techniques and their adoption has remained a difficulty. lack of knowledge in areas like artificial insemination, breeding, nutrition, housing, and biosecurity measures has hindered productivity and made it more difficult to implement better agricultural methods, which has led to a decline in efficiency and productivity. vi. market and cultural factors: in several african nations, the demand for pork has been influenced by religious convictions and cultural preferences. pig farmers have had restricted market prospects due to cultural taboos surrounding the consumption of pork and preferences for other types of meat. additionally, weak value chains and restricted market access have made it difficult for the pig sector to flourish. this is due to the fact that the profitability and sustainability of pig production have been impacted by market accessibility issues, inadequate processing and storage facilities, and poor market relations. climate challenges high ambient temperatures, increased precipitation pattern variability, and other weather extremes are linked to an increase in greenhouse gas (ghg) production and concentration in the atmosphere [18,19]. the productivity of livestock species, including pigs, is impacted by these changes and related variations in climatic conditions throughout time, which are referred to as climate change. pig production and associated activities account for 10.1% of the entire animal contribution to ghg production, while livestock are consistently involved in producing 14.5% of all anthropogenic ghg emissions [18,19]. due to its excessive reliance on rain-fed agriculture [20], africa's agriculture, especially pig production, is susceptible to climate fluctuation, making it one of the world's most vulnerable regions to the negative consequences of climate change [21, 22]. climate change is expected to have a negative impact on water availability and use for crop (feed) and livestock production [23, 24]. while water availability is decreasing, the amount of water required per pig for intake and cooling—particularly to counteract the effects of rising temperatures—is increasing. the supply of plant-based feed resources, particularly grains, is also being adversely affected by the decrease in water availability, which is leading to shortages and higher commodity prices per unit. in certain regions of africa, drought and desert encroachment exacerbate the impact of climate change on pig productivity. for instance, namibia has been suffering from drought, which has had a detrimental impact on the nation's pig and other agricultural production activities [21, 25]. according to research, one of the main causes of rising ambient temperatures is climate change. this causes thermal stress, which has a greater detrimental effect on pig performance than cold stress, particularly in africa. pigs are particularly stressed by heat [26]. heat stress, which is brought on by high ambient temperatures and high humidity, reduces pig production productivity by influencing growth rate, reproduction efficiency, and the animals' overall health and mortality. one significant tactic that might be investigated to lessen the impact of climate change on pig production in africa is breeding for high adaptation and heat tolerance utilizing native breeds of pigs. adequate cooling equipment and management techniques are necessary to lessen the impacts of heat stress on pig production in africa, nevertheless, as animals might not be able to completely adapt to climate stressors alone [18,26]. farmers may make climatesmart farming decisions, prevent losses, and improve grain output and supply by forecasting seasonal climate variations and promptly communicating the information to them. in addition to being impacted by climate change, pigs contribute to it by producing greenhouse gases. therefore, effective management techniques to reduce greenhouse gas emissions from pig farming and associated activities and to lessen the impact of climate change on pigs are both crucial for increasing pig output in africa [18]. opportunities, prospects, and a catalyst for improved pig production in africa prospects and opportunities of pig production in africa in sub-saharan africa, pig farming offers a number of opportunities and potentials that can support local populations' better livelihoods, food security, and economic growth. building a strong and sustainable pig value chain is essential to achieving sub-saharan africa's full pig production potential. i. food security: unlike most livestock species, pigs are extremely prolific and transform feed into consumable meat. this makes them an ideal animal for producing high-quality and highquantity meat, particularly to address the problem of malnutrition caused by a lack of protein, which is common across most of africa. additionally, since pig meat is high in protein, raising pigs for meat can help diversify diets, providing customers with more meat options and improving food security. ii. employment opportunities: the breeding, management, processing, and marketing phases of the pig production value chain can generate jobs for both skilled and unskilled laborers from africa's rapidly expanding population. these include farmers, technicians, traders, farm managers, experts in various facets of animal production, processors, suppliers of inputs, craftspeople, facility managers, logistic operators, and local market sellers, to name a few. because pigs require less area and initial investment than other livestock species, women and young people can engage in pig production operations [27]. iii. foreign exchange earnings and income generation: pig production is a very profitable business with strong returns on investment. because it gives small-scale farmers a reliable source of income, pig farming has the potential to have a major impact on per capita income in sub-saharan africa. pigs' strong growth rate, quick generation interval, and quick reproduction rate enable regular sales of pork and piglets, which support a steady flow of revenue [28]. although most african nations raise pigs for their own consumption, exporting them to other nations or beyond the continent can also generate foreign exchange profits. the potential for foreign exchange profits from pig 6 production is indicated by the current discrepancy between the worldwide market's supply and demand for pork. potential for higher income generation through pig rearing is indicated by the anticipated rise in demand for pork products brought on by population growth and shifting dietary preferences [28]. iv. opportunities for investment: investment in pig farming has proven profitable due to the pigs' innate capacity for reproduction and development as well as advancements in production methods over time. despite its growth, africa's pig industry has not been able to maximize its production due to a lack of investment in infrastructure and technology. in many emerging nations, the demand for meat has also increased due to urbanization and shifting dietary habits [13]. this gives producers the chance to satisfy the demands of the local market while also maybe looking into international markets for pig products. africa's pig production offers investment potential in all stages of the value chain, including but not limited to breeding, housing, feed production, animal aggregation, processing or value addition, cold storage, transportation, and appropriate retailing locations. africa offers vast potential for pig value addition, but it hasn't been fully utilized. making sausages, cured goods, and other value-added products from pigs can increase profit margins, improving farmers' and processors' total revenue. this increases trade and economic activity by giving farmers the chance to reach both local and regional markets. there is potential for additional revenue development if the demand for pig products rises as a result of population growth and shifting dietary preferences [29]. the possibilities for pig production in north african countries are based on the growing demand for pork, which is fueled by tourists visiting the region and seeing pig production as a way to earn foreign cash by exporting it to countries where there is a growing need for pork. pointers to better future africa's population is expected to grow to around 2.5 billion people by 2050 [30], and they must be fed. this suggests that there is a greater need for food, including pig flesh. the necessity for modern or mechanized pig farming methods to boost productivity and production in response to the growing demand for pork has resulted from the rural-urban movement that has reduced the number of farmers. both government and commercial entities have recently boosted their investment. africa has the potential to become a global supplier of pork if it has adequate infrastructure, high-quality inputs, and sufficient agricultural land. after all, the united african company (uac) built the biggest pig farm in the world in 1943, and it was situated in kano, nigeria. in the 1950s and 1960s, the farm expanded to a very big size, and before being exported from the continent, the pig meat was rail-transported to lagos, which was then nigeria's political and commercial center. unfortunately, religious discrimination led to the pig farm's final closure in the late 1970s [13]. the demand for meat, especially pork, is rising due to variables such population expansion, urbanization, and incomes [31]. the need for greater investment in pig production is indicated by these rising demands. for instance, there is a sizable and expanding market for pork products due to the fast urbanization and population expansion that is occurring in east african cities and in nations like nigeria. opportunities to meet the growing urban demand for meat products, notably pork, are presented by this. for the advantage of both producers and consumers, coordinated efforts can ensure or guarantee the safe and legal commerce of pigs and pork products. changing the industry from the old practice of scavenging pigs to a more profitable manner is also necessary to increase pig production in africa to satisfy the demands of the growing population. this calls for the involvement of companies or actors who will offer farmers access to contemporary living facilities, better breeding stock, better feeding systems, and training on new pig production techniques like artificial insemination. the use of methods that are extensively utilized in africa is still low compared to europe and america because of a lack of infrastructure and expertise. opportunities for commercial feed production or input supply are also abundant in the feed business. commercial pig feed is rarely available to farmers, who are primarily ignorant of feed formulation. pigs are typically sold live by farmers to nearby slaughterhouses throughout the majority of africa, where the product receives little to no value addition. to realize the full potential of the business, significant investment is required in the processing and value addition of pigs in africa. private institutions and development partners' investments in pig production and processing have grown significantly in some african nations, including liberia and kenya. to spread best practices, disease control methods, and contemporary pig farming techniques to farmers, research and extension services must be funded [8,13]. abundance of agro-industrial by-product in africa pork and other livestock products are becoming more and more in demand as africa's population increases and becomes more urbanized. demand has increased as a result, placing strain on the supply and cost of traditional pig feed. african pig producers frequently purchase feed materials from local and international marketplaces. the availability and cost of imported feed ingredients, for instance, have been impacted by the disruption of global grain markets caused by the ukraine-russia conflict [32]. conflicts between farmers and herders, for example, have severely impacted the economy in nigeria and other west african nations by disrupting local agricultural production and lowering the supply of locally derived feed ingredients. pig farmers' finances have been stressed by the rise in feed costs brought on by interruptions in the global grain market. local disputes have also resulted in lower agricultural output, which has an impact on smallholder farmers' incomes and the availability of local feed ingredients. conflicts between farmers and herdsmen frequently result in the uprooting of farming 7 communities, when farmers are compelled to leave their lands or scale back agricultural operations because of insecurity [33]. due to interruptions in local and international markets, higher feed prices, and decreased agricultural production, the ukraine-russia conflict and farmer-herdsmen conflicts in nigeria have contributed to the scarcity of feed ingredients for pigs in africa. these crises demonstrate how regional and global forces interact to shape the difficulties african pig farmers experience in obtaining a sufficient and reasonably priced supply of feed components. to address these feed shortage issues, there is a chance to increase the usage of agro-industrial by-products as substitute pig feed sources. numerous by-products are produced by africa's agricultural industry from crops such as rice, oil palm, cassava, sorghum, corn, soybean, peanut, wheat, and sugarcane. these byproducts consist of materials that can be used as valuable pig feed ingredients, such as bagasse, rice bran, rice husk, soybean meal, corn bran, groundnut cake, palm kernel cake, brewer's grain, and wheat offal. since many of these agro-industrial by-products are high in fiber, protein, and energy, they can be fed to pigs [34]. on the other hand, their nutritional content can be increased with appropriate processing and supplementation. making use of these by-products helps to make agriculture more environmentally friendly and sustainable while also reducing waste. conclusion a common practice throughout africa, pig production has made significant contributions to the continent's agricultural economy, particularly as a source of income for many of its rural residents. pig production in africa still has the potential to become a major contributor to the continent's gdp and the achievement of the sustainable development goals on zero hunger and poverty eradication, despite the obstacles it faces. this is because pigs are a good source of animal protein that can help fill the protein gap in the rapidly expanding african human population. smallholder farmers must be given capacity building in the areas of appropriate feeding and feed management, health management, biosecurity, and value addition to boost productivity and the growth of the pig value chain throughout africa in order to unlock the continent's hidden pig production treasure. the expansion of pig production in africa depends on the implementation of suitable regulations that encourage the formation of private pig meat processing facilities or firms. it is crucial to do ongoing research on the creation of highly adaptable breeds, cost-effective feeding systems, and the utilization of alternative feed resources or agro-industrial byproducts. the expansion of the pig business in africa depends on the careful planning and implementation of disease surveillance, preventive, and control strategies. conflict of interest regarding the information covered in the manuscript, we attest that there is no conflict of interest with any financial institution. funding the authors received no financial 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https://www.statista.com/statistics/1224205/forecast-of-the-total-population-of-africa/ https://www.statista.com/statistics/1224205/forecast-of-the-total-population-of-africa/ https://doi.org/10.20506/rst.25.2.1677 https://doi.org/10.3390/foods11142098 https://doi.org/10.3390/ani10010131 https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://rr-africa.woah.org/wp-content/uploads/2021/11/asf-ira-09-penrith.pdf https://doi.org/10.1111/tbed.13117 https://doi.org/10.1111/tbed.13117 introduction present pig production status in africa pig meat yield in africa per capita pork consumption in africa marketing challenges of pig production in africa common challenges climate challenges opportunities, prospects, and a catalyst for improved pig production in africa prospects and opportunities of pig production in africa pointers to better future abundance of agro-industrial by-product in africa conclusion conflict of interest funding references 1 author(s) retain the copyright of this article. full length research paper factors influencing agribusiness profitability: an analysis of smallholder pig farming in kenya's tharaka-nithi county kororia v. and chelule m. university of kabianga, kericho, kenya. accepted 30 december, 2024 aims: to increase the profitability of the pig subsector for farmers, particularly smallholder farmers, there have been concentrated efforts to commercialize it. smallholder farmers have not consistently made money in the field, despite advancements. profits for smallholder farmers have been inconsistent and disappointing. since the impact of institutional arrangements from a transaction cost perspective and managerial factors contributing to this inconsistency have not been thoroughly determined, the reasons for the disparate profitability have not been experimentally established. the study looked at how management practices and institutional structures affected the profitability of smallholder pig farming in kenya's tharaka-nithi county. research methods: eighty smallholder pig farmers were chosen using a two-stage sampling process. data was analyzed using the stochastic frontier production function and descriptive statistics after semi -structured interview guides were used. findings: according to the study, the majority of male respondents (75%) were of working age, had six years of experience raising pigs, and had only a basic education. findings from the stochastic frontier production, respondents' profit efficiency was negatively impacted by feed costs (p<0.01) and breed type (p<0.05), but positively by herd size (p<0.05) and veterinary and medication expenses (p<0.01). while information trust (p<0.05) and experience decreased inefficiency, gender (p<0.1) and the debt-to-asset ratio (p<0.01) increased it. conclusion: the research area's mean profit efficiency was 0.40, indicating low profit efficiency. by adopting current technologies, reducing transaction costs, and making better use of the resources already available, the efficiency level may be raised by 60%. adopting effective management techniques and marketing avenues would result in this acquisition. the gamma parameter (γ) was 0.63, indicating that profit inefficiencies account for 63% of the variation in net revenue. the study advances the subject of agribusiness and would enhance kenyan policies related to the growth of agribusiness. key words: smallholder farmers; institutional arrangements; management factors; stochastic frontier analysis; profit inefficiency. introduction the demand for animal protein has increased to an all-time high due to urbanization and population growth. the rate of food production in kenya is already out of step with the rate of demand. kenya generates an estimated 12,000 tons of pig meat for kes 1.2 billion, hence this has forced the country to import pork worth usd 700,000. the number of pigs killed in kenya has risen from 360,000 to 388,200 during the past five years, an increase of almost 8% [1]. one of the main causes of food disparity is the inability to supply the population's diets with the necessary quantity of animal protein [2]. the livestock industry in kenya accounts for 12% of the country's gdp and 50% of all jobs. dairy products, milk, meat, eggs, wool, hides, and skins make up the majority of this industry. over the past ten years, meat consumption has been rising quickly, and by 2025, it is predicted to reach 13.3 african journal of pig farming issn: 2375-0731 vol. 12 (1), pp. 001-007, december, 2024. available online at www.internationalscholarsjournals.org © international scholars journals http://www.internationalscholarsjournals.org/ 2 million tons [3]. the consumption of meat (beef, chicken, mutton, goat, pork, and camel) is predicted to continue rising from the present average of 19 kg per capita annually as most urban centers continue to expand [4]. through pig rearing, pork could be a significant factor in successfully lowering the inadequate amount of animal protein in diets. unfortunately, smallholder farmers in kenya lack proper agricultural methods and are poorly organized, which results in low yields and ultimately very low profits. additionally, shfs are not business-oriented, which means they do not run their agricultural operation as a business [5]. in kenya, the number of pigs killed has been continuously increasing over time. with a poverty rate of 65% in tharaka-nithi county, pig farming is crucial to smallholders' and households' risk diversification and livelihood security because they are a valuable asset that can be used to generate income for emergency cash needs, school fees, and the purchase of farm inputs [6]. when improved husbandry techniques and management abilities are used, the small-scale pig farming business has been shown to be highly profitable [2]. in order to promote genetic improvement and raise pig production in kenya, careful breeding stock selection and well-run breeding programs are required [6,7]. however, a variety of productivity and market-related barriers, such as illnesses, inadequate nutrition, and disorganized marketplaces, result in smallholder pig farmers in tharaka-nithi county earning inconsistent and meager returns from their business. pig output rises with effective institutional structures and managerial abilities, raising farmers' incomes and ultimately their profit margins. only if the pig subsector is operated like a business will this be possible [8]. since all of the major chain participants help one another to increase efficiency and competitiveness, the growth of the pig value chain is significant since it affects farmers' profitability [9]. the county's subsector is primarily unorganized, with a lack of technology, information, and services, as well as poorly managed markets. additionally, pig herds are at danger of disease during outbreaks due to a lack of feed quality control methods, which results in stunted growth and lower market value. the absence of farmer groups in the pig industry also hinders the exchange of useful information in the produce market. low-binding relationships between smallholder farmers and traders result in high transaction costs [10]. therefore, smallholder farmers must set up effective institutional arrangements in order to reduce these high transaction costs. a few studies have assessed how institutional structures and management characteristics affect the profitability of smallholder pig farmers. the majority looked at how institutional, social, cultural, technological, marketing, and farmer and farm conditions affected farm-level profit efficiency [11,12,13,14,15,16,17,18]. however, identifying the key elements that affect profit efficiency is a top research objective. this is because smallholder pig farmers' profit efficiency is influenced by more than just management characteristics and institutional structures. research is therefore required to determine which institutional arrangements and management aspects should be prioritized in order for smallholder pig farmers to be profitable. this study made an effort to close this knowledge gap as well. methodology study region, sampling method, and data gathering tools the study was conducted in kenya's tharaka-nithi county. a descriptive study design was employed. smallholder pig producers in the research area were chosen using a twostage sampling procedure. the maara constituency was purposefully chosen for the first stage due to the area's high concentration of pig farmers and its favorable agro-ecological conditions for pig rearing. the second step was a stratification random sample of 16 smallholder pig farmers from each of the five wards, for a total of 80 farmers. the farmers were traced within the stratified areas using the snowballing method. farmers were interviewed in-person using semi-structured interview schedules to gather primary data, which were then analyzed using the stochastic frontier production function and descriptive statistics. empirical model specification to analyze the data, the stochastic frontier production function (sfpf) was used. the stochastic frontier approach was utilized to determine which factors contributed to profit inefficiencies. it takes into consideration both the inefficiency component and random error [19]. by assuming a profit function that behaves in a way consistent with the stochastic frontier notion, this study applies the [21] model in accordance with [20]. in accordance with the work of [22], the functional form of the stochastic profit frontier was ascertained by fitting it with the less restrictive translog and evaluating the sufficiency of the highly restrictive cobbdouglas. equation 3, which is essentially the inputoutput transformation and transaction costs model [23], is the stochastic profit model that is employed. equation 4 represents the inefficiency model. the econometric model was typically defined to be: yi = xiβ+ ei ………. (1) yi = β0 + ∑ i= 1βi xi + vi ui (cobb-douglas function) (2) lnyi = β0 + β1lnx1+ β2lnx2 + β3lnx3 + β4lnx4 3 +β5lnx5+ β6lnx6+vi ui (translog function) (3) where: lny1 = normalized profit (net revenue per kilogram of output sold); x1= feed cost (kg); x2=wage rate (include wage rate for both hired and family labor) (man-days); x3 = breed type; x4= herd size; x5= search costs and x6= drug/veterinary costs (kshs); β0, β1,,,β5= parameters to be estimated; ui = degree of inefficiency which is halfnormal distributed (iid n|) (0, σu2). ui is closely related to the profit inefficiency which may arise from management factors and institutional arrangements. vi = statistical disturbance term that is caused by factors outside the scope of the farmers which is assumed to be identically and normally distributed with a mean of zero (iid) and constant variance of v~n ((o, σ2v) and independent of u. the coefficients of variables x1, x2, x3, x4, and x5 are estimated from the maximum probability of the profit function and are understood as the variables' elasticities. all of the coefficients have the proper sign. the link between the inputs used by the chosen smallholder pig farmers and their pig net revenue was ascertained using a stochastic frontier model. the inefficiency model was used to examine the study's goal, and profit inefficiency (u) was the dependent variable, while the independent variables were the inefficiency factors. the average county wage rate will be used in the study as a stand-in for the family workers' pay. the average salary is what the hired laborers in tharaka-niti county's pig farms make on average. this is calculated on the assumption that a worker would only be at the pig farm for two hours each day. results and discussion institutional structures and pig management techniques the institutional setup and management procedures of the smallholder pig sample was on a modest scale, which could be a result of the farmer's financial circumstances. according to the study, the majority of pig farmers used the semi-intensive management technique of penning (68%) and the intensive technique of stall-feeding (32%), which involved keeping the animals in a clean pigsty and providing them a balanced diet. in contrast to local consumers, who paid high prices with substantial search and contracting expenses, smallholder pig farmers in the study area sold their pigs to dealers directly 60% of the time, who provided exploitative prices. according to the study, 41% of smallholder pig farmers belonged to a farmer's group, whilst the majority (59%) did not. participants in farmer groups gained from trainings that forced them to embrace new technologies and adhere to management techniques suggested by trainers and extension agents. pig producers' profit efficiency in the research area table 2 displays the maximum likelihood estimates (mle) of the parameters in the stochastic frontier model. with the exception of labor and search costs, the majority of the inputs used were statistically significant at various levels, according to the production function results. with a coefficient of -0.255, the feed cost coefficient was correctly signed and statistically significant (p <0.01), indicating that a 1% increase in feed prices would result in a 25% drop in the enterprise's net revenue level, which was consistent with the findings of [18]. pigs' breed type coefficient was negative and statistically significant (p<0.05), with a coefficient of -0.100, meaning that a 10% decrease in net revenue would result from a 1% increase in the usage of subpar breeds. in line with the study of [27], the herd size was positive and statistically significant at the (p<0.005) level, with a coefficient of 0.080 despite being inelastic, meaning that a 1% increase in the number of pigs would result in an 8% increase in net revenue. finally, the cost of medications and veterinary care was positive and statistically significant at p <0.01. one of the main factors influencing the profit level of the pig firm in the research region seemed to be the coefficient, which was elastic in character. however, this suggested that a 1% increase in veterinary and medication costs would result in a 50% increase in the company's net revenue. this was consistent with the findings of [28], who discovered that a strengthened veterinary service system provided high-quality information about animal health and possible alternatives to antibiotic use, such as improved farm management, vaccines, and immunodulators. 4 although there was a clear correlation, the cost of labor coefficient of 0.444 was not statistically significant, suggesting that it was not a significant factor in determining the profit efficiency of the pig firm in the research area. the net revenue level rose by 0.444 for every unit rise in labor costs. this was consistent with the results of [29], which showed that labor had an inverse relationship with mustard yield but was not statistically significant. the inelastic nature of the search cost coefficient, which was 0.052, suggested that it was not a significant factor in determining the profit efficiency of the pig firm in the research area. as a result, the net revenue level decreased by 0.052 for every unit rise in search expenses. factors contributing to pig production's profit inefficiency gender was positive and significant at p < 0.10, according to table 2's inefficiency model results. according to the study of [6], this suggests that profit inefficiency rises with gender, indicating that households led by women are more profit-efficient than those headed by men since they were more involved in farm activities. lack of faith in market information was associated with a decrease in profit inefficiency, as evidenced by the negative and significant (p<0.05) trust in market information. farmers had to pay more to find better clients and pricing due to information asymmetry, which resulted in transaction costs. these costs included personal time, travel expenses, and communication costs. the results of [30], who discovered that knowledge asymmetry causes opportunism and mistrust among the participants in the milk value chain, supported this. the results also aligned with the research of [9], which found that the more trust between company partners in the marketing channel, the better the circumstances for successful commercial operations. [31] found that the sources of knowledge in flood farming were reliable. at p<0.01, the debt-to-asset ratio was statistically significant and positive. this suggested that when the debt-to-asset ratio increased, so did profit inefficiencies. the greater the ratio, the greater the liabilities of the farm business relative to the assets, which needed to be balanced. a high ratio rendered agricultural businesses insolvent and prevented them from obtaining additional credit [32]. [33] believed that dairy farms in the uk with a low debt-to-asset ratio were more productive. table 2 showed that the results of pig rearing experience were negative and significant at 5%. this implies that specialization evolved throughout time, resulting in better manufacturing techniques and increased profitability. this result was consistent with that of [34], who found that more agricultural production experience improves critical assessment of the applicability of superior production choices, such as the effective use of productive resources. with a coefficient of 0.119, the age variable showed a positive indication but was not statistically significant. these outcomes aligned with the conclusions of [18]. they found that older farmers are less likely to embrace modern inputs and innovative methods. additionally, since young farmers are more likely to have received some formal education, they may be better able to learn new techniques and gather information, which will increase technical and allocative efficiency and profit efficiency. years of formal schooling are typically used to measure there was no statistically significant correlation between schooling and either variable. this is in line with the research of [35], which found that farmers in the study area who had received formal schooling did not demonstrate greater levels of profit efficiency. distribution of profit efficiency the calculated stochastic frontier model's individual profit efficiencies for the sampled pig farmers are shown in table 3. the farmers' estimated profit efficiencies ranged from 0.094 to 1, which is a significant difference. the projections are left-skewed, with an estimated mean profit efficiency of 0.40. according to the study, the production level was almost 60% below the border, with pig farmers in the study region producing at about 40% of the possible production level. a study by [32] found that this was a sign of product waste brought on by farmers' inefficient use of resources. the results also indicated that by using more variable inputs to increase production and making better use of the resources already available, profit efficiency in pig farming in the research area could be raised by 60%. conclusion the purpose of the study was to evaluate the variables influencing the profitability of smallholder pig farmers in kenya's tharaka-nithi county. the aforementioned study suggests that pig profit inefficiency is negatively impacted by household head experience and faith in market information. also, the debt-to-asset ratio had a favorable impact on profit inefficiency on the farms in the research region. pig farmers were not entirely profit-efficient, according to the study, but there is a lot of room for further profitability. 5 recommendations 1. to achieve the required economies of scale, pig farmers should create groups like producer organizations or cooperative societies. this will lessen knowledge asymmetries and increase countervailing market power. 2. the study's conclusions lead to the following policy recommendations: sufficient pig production training (to introduce them to new developments) and fundamental financial management knowledge, such as the ideal debt-to-asset ratio and debt utilization. 3. in the research area, males predominate in pig production. pig farming, however, should be the starting point for programs aimed at empowering women because it offers substantial financial access prospects. to improve their livelihoods and raise their income, women must also be encouraged to work in the pig industry. to solve the fundamental issues smallholder pig farmers confront, the industry's major participants should develop a logical and comprehensive solution. to guarantee coordination and cooperation among various national institutions and agencies, both at the federal and municipal levels, as well as between private sector organizations, producer groups, and development partners, the government can collaborate with other stakeholders. suggestions for further research since the study concentrated on the variables that affect smallholder pig farms' profitability, it would be wise to conduct additional research on the use of smart farming in smallholder pig farming, since this could help to promote an integrative management strategy. capturing the effects of smart solutions in smallholder pig farming could be taken into consideration when redesigning the study. consent the author(s) have gathered and preserved the respondents' written consent in accordance with international or university standards. competing interests authors have declared that no competing interests exist. references 1. ilri. kenyan livestock sector to grow exponentially. livestock and development; 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2003. carroll j, newman c, thorne f. understanding the factors that influence dairy farm efficiency in the republic of ireland, rerc working paper series 07wpre-06; 2006. etim na, udoh ej. identifying sources of efficiency among resource poor indigenous vegetable farmers in uyo, nigeria. international journal of food and agricultural economics. 2014;2:33-39. weir s. working paper, csae, wps 99-7 centre for the study of african economics. university of oxford; 1999. introduction methodology results and discussion institutional structures and pig management techniques pig producers' profit efficiency in the research area conclusion recommendations suggestions for further research consent competing interests 1 in ternationa l scholars journa ls african journal of pig farming issn: 2375-0731 vol. 11 (2), pp. 001-011, february, 2023. available online at www.internationalscholarsjournals.org © international scholars journals author(s) retain the copyright of this article. full length research paper grass production tifton 85 and nutrient extraction with swine wastewater doses vinícius de oliveira rezende1*, luís césar dias drumond2, andré mundstock xavier de carvalho2, regina maria quintão lana3 and marcos vieira de faria3 1 department of agronomy, federal university of viçosa, brazil. 2 council for teaching, research and extension-rio paranaíba campus. federal university of viçosa, highway br 354 km 310 (at 1,300m) 38810-000 rio paranaiba, mg – brazil. 3 department of agronomy, federal university of uberlândia – ufu, brazil. accepted 13 november, 2022 swine wastewater (sw) is considered as a source of nutrients for agriculture. the quantities and frequencies applied may vary according to the soil class, the nature and composition of the waste, the climatic conditions and the cultivated plant species. therefore, this study aimed to evaluate the effects of application of five swine wastewater doses (0, 500, 1000, 2000 and 2500 m³ ha -1 ) in the production of components and grass nutrient extraction tifton 85 and changes in soil chemical properties after the end of the last application of swine wastewater. to achieve these goals, we evaluated the quantities of extracted nutrients, density and height of the pre-grazing grass, forage accumulation rate and carrying capacity. the experimental design was a randomized block. the production of dry matter was 18159.80 kg ha -1 , and the average height of 85 tifton was 34.83 cm. the higher carrying capacity, 10 au ha -1 was obtained with the dosage of 2500 m³ ha -1 . while, the dose of 2000 m³ ha -1 supplied the nutritional needs of tifton 85 in nitrogen, potassium, calcium, magnesium, copper, iron, zinc and boron. the quantities of extracted nutrients (kg ha -1 ) at grass tifton 85 grazed were: n = 405.14; p = 57.77; k + = 387.69; ca 2+ = 77.05; mg 2+ = 49.68; s-so4 -2 = 31.48; b = 0.33; cu 2+ = 0.20; fe = 2.88; mn 2+ = 4.61; zn 2+ = 2.86. the application of increasing doses of sw promoted a linear increase in grass production components tifton 85, as well, promoted changes in soil chemical properties and quantities of extracted nutrients. key words: pasture, liquid waste, nutrient extraction, plant growth. introduction brazilian swine production is increasing annually in order to meet both domestic and foreign market demand with regard to the quality of raw materials and environmental care (pinto et al., 2014). brazil is the fourth largest producer of pork in the world, this represents the equivalent of 3.7 million tonnes of pigs (emprapa, 2017). the main problem of this activity, therefore, is the generation of enormous quantities of manure that can *corresponding author. e-mail: msc.vinicius@gmail.com. author(s) agree that this article remain permanently open access under the terms of the creative commons attribution license 4.0 international license 2 corroborate to the pollution of terrestrial and aquatic ecosystems (segat et al., 2015). the fertilization of crops with swine wastewater is a common practice and is attractive for the reduction of natural resources and environmental pollution control. the feasibility of such use is due to the large volume of waste generated and the amount of nutrients that are easily mineralized when applied in soil (lucas et al., 2013). in this sense, an alternative that has been highlighted in the pursuit of sustainability in the recovery of degraded cultivated soils and pastures is the use of organic fertilizers or cover crops because the mineral fertilizers in this context is rather low due to its high cost and due to the low purchase power of most producers. for instance, mineral fertilizers in africa cost at the farm gate, two to six times as much as in europe, north america, or asia (sanchez, 2002). therefore, organic inputs are a viable alternative source of plant nutrients for resource-poor farmers. application of organic input usually leads to increased crop yields and pasture (ogundare et al., 2012; matthews, 2017). the acceptance by local farmers of the benefits of compost to the soil and crops would be a significant incentive to reduce the expensive mineral fertilizers and optimize their use (azim et al., 2017). thus, the use of sw is available as an alternative to replace the mineral fertilizer, demonstrating its efficiency in dry matter production and nutrient availability (zanine and ferreira, 2015; gomes et al., 2017; lucas et al., 2013). however, there is little information regarding the use of this waste in areas with degraded pastures (fogel et al., 2013). the supply of nutrients in adequate amounts via sw can increase the nutritional quality of the forage, enhancing performance and/or animal productivity (assmann et al., 2009). therefore, the use of swine wastewater (sw) as a source of nutrients in pasture areas is presented as an alternative to disposal of this waste (assmann et al., 2009; seidel et al., 2010). camargo et al. (2011) observed the effects of different doses of swine manure on forage tifton 85 and found that an increase in dry matter production and p content grew linearly with the doses, thus suggesting a dose of 100 m³ ha -1 to obtain about 3500 kg ha -1 dry weight in a period of 28 days. serafim and galbiatti (2012) discussed that an increase in the application of swine waste increased the supply of nitrogen and phosphorus to the soil, thus promoting plant growth and increasing the ratio of leaf/stem. thus, it is needed to find the proper management from a biophysical point of view to promote sustainable agriculture. however, it is also necessary that farmers accept new strategies that propose cultural and technical shifts (cerdà et al., 2018a). the search for information and knowledge to clarify how to use the sw in pastures is growing. to have a correct land management in intensive systems, it is important to know the nutrient extraction capacity for forage to mitigate the negative environmental impacts on the ground and to define the best application rates. in this way, there is a need to design proper policies to achieve sustainability, and for this, the scientific community should produce information in collaboration with land managers and other actors, which will guide policy makers to implement the most efficient managements and strategies (cerdà et al., 2018b). this study aimed to evaluate the effects of application of five sw doses (0, 500, 1000, 2000 and 2500 m³ ha -1 ) in the following production components: bearing capacity, dry matter yield, density, percentage of dry matter grass height, dry matter production per day and in grass nutrient extraction tifton 85 grazed and changes in soil chemical properties. materials and methods study area the study was conducted during the period from january to april, 2013, at the bonsucesso farm, which is located in, minas gerais state, brazil, at the geographical coordinates 19°05'17"s and 48°22'00"w, at an altitude of 820 m, at an dystrophic yellow oxisol, according to embrapa classification in 2006. according to the köppen and geiger (1928) system, the climate is characterized as aw (typical tropical, with average rainfall around 1600 mm per year, with moderate water deficit in winter and excessive rain in summer). before the experiment installation, it was determined that the chemical and physical soil characteristics of the area at different depths of 0.00-0.20 m and 0.20-0.40 m (table 1). for this, 10 soil samples to form a composite sample were collected at random in the experimental area with a dutch auger. the study was conducted on grazing tifton 85 and was installed five years ago. the experimental design was a randomized complete block design with five treatments and three replications. plots were 3 x 3 m, amounting 9 m², 1 m boundary among plot. the treatments consisted of the following wastewater doses of swine (sw): control (without application of sw), 500, 1000, 2000 and 2500 m³ ha -1 . the waste used in the experiment comes from a swine production system in the finishing phase, handled with biodigester pvc blanket and stabilization pond, being stored for about 20 days. after this period, the sw is applied in the grazing areas. there was the uniform grass height of tifton 85, 0.10 m tall, with hydraulic brush cutter before the start of the experiment. after the treatments (table 2) and data collection (21 day cycle), the remaining forage of each cycle was quantified and grazed up to a height of 0.10 m. the application doses of sw were performed manually with a 1inch diameter hose and distributed evenly over each plot. the doses were calculated due to the application of time on each plot. thus, the flow rate was set at 45 l min 1 and application times were 0 (no application), 2, 4, 8 and 10 min, respectively providing from the lower treatment dose to the higher dose. each dose of sw was split into five applications, always at the beginning of each 21day grazing cycle (table 2). this installment was not to exceed the field capacity in a single application. in each application date, they were collected a sample of 600 ml of sw and stored in refrigerator. the five sw samples were homogenized, then pulled out a sample that was sent to araxá environmental laboratory for chemical characterization. the average levels of nutrients are sw: n = 823.60 mg l -1 ; p = 20.46 mg l -1 ; = k + 509.40 mg l -1 ; ca 2+ = 51.54 mg l -1 ; mg = 33.53 mg l -1 ; organic matter = 331.80 mg l -1 , b = 0.55 3 table 1. chemical characteristics and grain size of the soil, in the depths studied the experimental area with the tifton 85 grass before application of treatments. depth ph p resin k+ s-so4 2 ca2+ mg2+ al3+ h+al (m) (water) ............ mg dm ³............... .......................cmolc dm ³ ......................... 0.00-0.20 5.50 39.90 94.0 1.77 1.00 0.30 0.15 2.60 0.20-0.40 5.50 18.20 56.0 2.02 0.70 0.10 0.20 2.10 b cu 2+ fe mn 2+ zn 2+ sb t t .......................................... mg dm ³................................... .............cmolc dm ³.......... 0.00-0.20 0.11 4.00 91.00 6.30 8.10 1.54 1.69 4.14 0.20-0.40 0.28 1.60 28.00 2.40 2.00 0.94 1.14 3.04 mo v m clay silt sand % % % ...................................... g kg ¹.................................. 0.00-0.20 2.3 37 09 153 25 822 0.20-0.40 1.6 30 17 165 17 818 potassium (k) = (hcl 0.05 mol l -1 + h2so4 0.0125 mol l -1 ); available p (resin); ca, mg, al, (kcl 1 mol l -1 ); h + al = (buffer solution smp ph 7.5); sb = basic sum; t = cec at ph 7.0; v = base saturation; m = saturation by aluminum; organic matter (m.o.) = colorimetric method. boron (b) = (0.0125% bacl2.2h2o); cu, fe, mn, zn = (dtpa 0.005 mol l -1 + tea 0.1 mol -1 cacl2 0.01 mol l -1 a ph 7.3). clay: pipette method. chemical analysis carried out according to methodologies described by embrapa (2009). table 2. total dose of sw, portions of value applied to each treatment and dates of split applications in grass tifton 85. dose total 05/01/13 26/01/13 16/02/13 09/03/13 30/03/13 ..................................................... m 3 ha -1 ................................................ 500 100 100 100 100 100 1000 200 200 200 200 200 2000 400 400 400 400 400 2500 500 500 500 500 500 mg l -1 ; cu 2+ = 4.33 mg l -1 ; fe = 6.34 mg l -1 ; mn 2+ = 0.91 mg l -1 and zn 2+ = 5.71 mg l -1 and ph cacl2 = 8.16. the methodologies used in determining the sw nutrients were based on standard methods for the examination of water and wastewater (apha, 2012). during the experiment, the rainfall and average temperature were measured daily and are displayed by means of each ten-day period (figure 1). for evaluation of forage growth, five successive cuts were performed (over 0.10 m high) in 21-day intervals (cycles). to collect the sample mass of dry (dm) forage, the sampling method square template, proposed by aguiar (2009), was used. the forage harvested in the area sampled per plot were determined a fresh pasture mass. subsequently, the identified subsamples were dried in an oven with forced air circulation at 65°c for 72 h to determine the mass of dry matter over 0.10 m high (gardner, 1986). the percentage of dry matter was then calculated and expressed in kg ha -1 dm. then, soil samples were crushed (willey mill) to determine the total content of nutrients. to determine the height of grass from the ground level to the highest part a top were used a scale and an x-ray paper were used to standardize the height of the plants, in 10 replications. the bulk density of forage was obtained by dividing the dry mass weight/height plants that was expressed in kg ha -1 cm -1 . the forage accumulation was calculated by subtracting the herbage mass in pre-grazing forage by the postgrazing mass. forage accumulation rate (over 0.10 m) was expressed in kg ha -1 day -1 dm. it was calculated by dividing the accumulation of forage for 21 days grazing cycle by 21, the number of days in the cycle. the pasture's carrying capacity was calculated considering an herbage allowance of 5 kg dm per 100 kg live weight. after the measurements of pasture, the cattle were put to graze, aimed at standardizing the grass height to 0.10 m. after 21 days of application of the last installment of sw, soil samples were taken at different soil depths such as 0.00-0.20 m and 0.20-0.40, originating six samples, randomly collected in the plot with a dutch auger. the ph in water, exchangeable acidity (al 3+ ), potential acidity (h + al) and soil organic matter (om), phosphorus (p), potassium (k + ), calcium (ca 2+ ), magnesium (mg 2+ ) copper (cu 2+ ), zinc (zn 2+ ), manganese (mn 2+ ) and iron (fe) was analyzed, according to the methodology described by embrapa (2009). the aerial parts of the plants were subjected to analysis of the n, p, k, ca, mg, s, fe, cu, mn, zn, b according to methodologies embrapa (2009) after each 21-day cycle. with the nutrient content of aerial parts of each cycle, the accumulation of these nutrients and recovery efficiency of each of the plant were evaluated. the accumulation of nutrients in the aerial part of each cycle was then used to define the nutrient uptake of the grazed pasture (kg ha -1 ) to 105 days of experiment. all results were analyzed using the barttlet and jarque-bera test (jarque and bera, 1980) to check the homogeneity of variances conditions and normality, respectively. analysis of variance and regression 4 figure 1. storm precipitation and average temperature during the period experiment. analysis for the sw doses were carried out, to determine levels of significance, using the saeg 9.1 program, 2007. results and discussion the results of analysis of variance for the production of components are shown in table 3. the application of different doses of sw provided a linear increase in all grass production components tifton 85 studied (figure 2). the average heights of tifton 85 ranged between 31 and 39 cm with a 21-day cycle (figure 2d). results similar to the aguiar (2009), an experiment in uberaba, with intensive management, which found an average height in the spring/summer of 36.4 cm and an annual average of 29.7 cm. the higher carrying capacity, 10 au ha -1 was obtained with the dosage of 2500 m³ ha -1 , whereas grazing efficiency of 50% (figure 2b). lupatinie hernandez (2006) showed, among various types of forage, the tifton 85 grass with high fertilization responded better to irrigation, with a carrying capacity of up to 10 au ha -1 . which, showed the great forage production potential of grass associating intensive management, adequate fertilization and irrigation. the accumulation rate of dry matter per day obtained averages 138-219 kg ha -1 day -1 (figure 2e). aguiar et al. (2005) showed an average annual accumulation rate of forage of 172 kg ha -1 day -1 . drummond et al. (2006) reached herbage accumulation rate of 148.2 kg ha -1 day -1 of dry matter in grazing tifton 85 fertilized with swine waste in the region of uberaba mg. research works on pastures have presented linear increases of forage dry matter using doses of wastewater (orrico junior et al., 2013; andrade et al., 2014; homem et al., 2016), with average accumulation doses of up to 170 kg ha -1 day -1 of dm in tifton 85 grass using swine wastewater (andrade et al., 2014). vielmo et al. (2011) used sw at doses of 0 to 320 m 3 ha 1 , cycle of 28 days on tifton 85, and verified a production of 151 kg ha 1 day -1 of dry matter at the highest dose. gomes et al. (2018) obtained higher productivity, equal to 189 kg ha 1 day -1 of dry matter using dose of 300 m 3 ha 1 . andrade et al. (2012) in an experimental area on the campus of rio paranaiba ufv evaluated the forage accumulation and managed in the intensive irrigated system. the ideal point of grazing and the chemical composition of forage produced in summer and autumn in pasture managed in intensive system with tifton 85 grass found herbage accumulation rates of 140.0 kg ha -1 day -1 of dm in the summer and 122.2 kg ha 1 day -1 of dm in the fall. the height of the ideal grass for grazing was 25.4 cm. the highest production of dry matter was obtained with an average dose of 2500 m³ ha -1 (figure 2a), which was 66. 5% higher than the average control, in the period of 105 days. the linear increase of production and grass 5 table 3. averages of the components related to production tifton 85 subjected to increasing doses of sw. components of production averages p-valor cv% carrying capacity (ua ha -1) production of dry matter (kg ha -1) -1 -1 density (kg ha cm of dm) dry matter (%) grass height (cm) accumulation rate of dry matter per day (kg ha -1) 7.69 0.000 5.90 18159.80 0.000 5.90 109.40 0.008 7.08 23.39 0.058 3.15 34.83 0.000 2.05 172.95 0.000 5.90 figure 2. components of production: production (kg ha 1 ) (a) carrying capacity (b), density (c) of the grass height (d) and accumulation rate of dry matter per day (e) the trial period with the tifton 85 grass, when subjected to increasing doses of sw. **: significant at 1% probability. 6 table 4. average macro content, micronutrients and chemical characteristics of the soil subjected to increasing doses of sw. depth variable p resin k+ s-so4 2 ca2+ mg2+ sb h+al t ............... mg dm -3 ............................... cmolc dm -3 ............ ............................ averages 31.27 101.00 3.67 0.64 0.44 1.34 2.29 3.63 0.00-0.20 m p-valor 0.385 0.468 0.011 0.230 0.034 0.092 0.594 0.344 c.v. % 58.97 22.62 39.83 24.71 22.92 17.82 14.07 10.67 averages 7.16 102.00 4.87 0.43 0.26 0.95 1.75 2.7 0.20-0.40 m p-valor 0.241 0.742 0.003 0.004 0.001 0.008 0.028 0.633 c.v. % 57.79 27.81 26.66 16.85 13.14 14.94 10.95 8.12 ph m.o. v b cu 2+ fe mn 2+ zn 2+ (water) % % .................................. mg dm -3 ........................ averages 5.49 1.69 36.82 0.10 3.10 54.40 7.22 5.27 0.00-0.20 m p-valor 0.089 0.328 0.168 0.096 0.464 0.679 0.029 0.652 c.v. % 3.44 15.64 14.91 15.18 26.26 42.83 23.21 42.75 averages 5.61 1.05 35.09 0.9 1.89 18.87 4.52 1.5 0.20-0.40 m p-valor 0.003 0.906 0.003 0.977 0.725 0.313 0.015 0.463 c.v. % 4.17 12.76 13.22 22.32 64.97 26.21 24.81 31.38 height influenced the dry matter density, which increased from 92.4 kg ha -1 cm -1 dm in witness to 125.2 kg ha -1 cm 1 dm at the highest dose (2500 m³ ha -1 ), an increase of 34. 5% in the grass density (figure 2c). with increasing doses of the sw, the ph did not show any variation in the two depths (tables 1 and 4), due to the alkaline characteristics of sw. according to the results this study, silva et al. (2014) observed that soil attributes related to the acidity did not suffer influence of successive applications of sw. these differed from queiroz et al. (2004) who observed a lowering of the ph with the application of sw. according to bouwer (2000), in soils receiving wastewater, there may be a decrease in ph due to the mineralization of organic compounds of the sw, which facilitates the production of co2 and organic acids. already, lucas et al. (2013) observed the ph increased from 5.47 to 6.77 in the soil (0 to 0.60 m) at 1015 days of sw application. increased ph values of soil were consistent with the high ph values of sw used, ranging from 7.08 to 7.70. the available potassium content (k + ) in the soil was similar at both depths with 101.0 and 102.0 mg dm -3 levels (table 4) and fall within the adequate availability class, according to cfsemg ribeiro et al. (1999). a significant increase in k + content at a depth of 0.20-0.40 m, indicated a percolation of k + for the deeper layers, because the soil characterization analysis before applying the sw doses (table 1) the levels were 94.0 and 56.0 mg dm -3 in these depths. by being a monovalent, k + has low retention on soil colloids, being susceptible to being leached from the surface layers for the subsoil layers. which with the application of sw doses, the amount of added k + was high than extracted by tifton 85 (figure 3c), favoring an increase in the levels of k + in the soil (table 4). the potassium content of the soil increased in this experiment because when compost is used, according to scherer (2001), the potassium k + from the mineral and organic fertilizer are similar. thus, there is no requirement to undergo any mineralization through the action of microorganisms. according to penha et al. (2015) long-term applications of pig slurry in a brazilian cerrado soil have shown to affect chemical characteristics of the soil. high pig slurry rates increased p contents only in the soil surface, while the contents of k increased throughout the soil profile. this fact shows the marked difference in terms of p and k behaviors in tropical soils, indicating that k is more prone to be leached in a cerrado soil following successive applications of pig slurry. the phosphorus (p) showed a higher concentration in the surface layer (table 4). this is because the positive organic radicals in sw adsorb p, favoring surface accumulation. also the clay in these soils are sesquioxidic which feature high phosphorus adsorption. the p in cerrado soils have low mobility focusing mainly in layers of 0.00 to 0.10 m. in a study on the use of swine wastewater in natural pasture, ceretta et al. (2003) showed no changes in phosphorus concentration, with high concentration of p in the surface layer, with increases of 580% to 8.3 months and 6.710% at 48 months of application liquid effluent from pig farms. these studies indicate the importance of monitoring when performing constant application of sw in the same area. 7 figure 3. extraction of the nutrients n (a), p (b), k (c), ca (d) and mg (e) by dry weight. ** and *: significant at 1 and 5% probability, respectively. the dotted line refers to the addition of nutrients to the soil by increasing doses of sw and the continuous line for extracting the forage. however, seganfredo et al. (2017), reports that the application of sw increases the ease of desorption of p and this makes it necessary to control its movement in the profile and transport through runoff. there was a significant effect of the sw application on the content of (mg 2+ ) available in soil (table 4). according to ribeiro et al. (1999), the levels observed in two depths remained low (0.44 and 0.26 cmolc dm 3 ). however, there was a significant increase in mg 2+ , in both evaluated depths, as in soil characterization analysis before applying the sw 8 table 5. medium of the leaf content of macro and micronutrients of tifton 85 subjected to increasing doses of sw. element n p k s ca mg b cu fe mn zn ................................. g kg -1 ............................... .........................mg kg -1 ........................ averages 22.62 3.12 21.28 1.69 4.18 2.66 18.11 11.19 151 242 144 p-valor 0.008 0.064 0.021 0.020 0.565 0.238 0.308 0.984 0.619 0.301 0.004 c.v. % 2.39 4.28 3.56 6.27 4.43 4.98 12.29 13.25 11.31 21.96 0.11 doses (table 1), the determined values were 0.30 and 0.10 cmolcdm -3 at a depth of 0.00 to 0.20 and 0.20-0.40 m, respectively. with the application doses of the sw the added amount of mg 2+ was higher than that extracted by tifton 85 (figure 3e), thus favoring the increase in mg 2+ content in soil (table 4). the content of calcium (ca 2+ ) at a depth of 0.00 to 0.20 m is 0.64 cmolcdm -3 , while the depth is 0.43 m 0.20-0.40 cmolcdm -3 (table 4), and these levels considered low by ribeiro et al. (1999). however, replacement of ca 2+ is less than the amount extracted at doses of 500 and 1.000 m³ ha -1 sw, however, higher in doses of 2000 and 2500 m³ ha -1 sw (figure 3d), not favoring the accumulation of calcium in two depths assessed (table 4). queiroz et al. (2004) observed no changes in ca 2+ levels in soil with wastewater. the base saturation (v) showed a significant increase in the depth of 0.20-0.40 m (35.09%) with the application of sw doses (table 4) in relation to the initial content (30%) (table 1). this result is related to the increase in potassium and magnesium content at this depth. with the application of sw, the levels of organic matter that were 2.3% at a depth of 0.00 to 0.20 and 1.6% in the depth 0.20-0.40 m (table 1), did not show any increase due to the application of the treatments (table 4), with no significant differences. mattias (2006) did not observe an increase of organic matter in the application of liquid effluent from swine farms. the lack of response to the increase of organic matter can be explained by assmann et al. (2006) who observed no increase in organic matter content by applying liquid swine waste. for, according to the authors, they must be considered inherent characteristics of manure used, where the quality of the organic compounds may determine a greater or lesser accumulation in the soil. the organic compounds present in the liquid manure of pigs have been easily digested by oxidizing in a few days or weeks and are favored for higher microbial activity. the microbial biomass is considered a vital part of the organic matter, composed of micro-organisms (bacteria, fungi and actinomycetes), comprising 2 to 5% carbon and up to 5% of total nitrogen (moreira and siqueira, 2003). there were no significant differences among treatments in nutrient contents fe, cu 2+ and zn 2+ (table 4). however, for lucas et al. (2013) and rosa et al. (2017a, b), the applications of swine wastewater favored the accumulation of copper and zinc. according to girotto (2007), successive applications of sw to the soil cause accumulation of zn 2+ in the surface layers that were found significant for the accumulation of zn 2+ layer to the depth of 0.10 m. the cu 2 + and zn 2+ elements are used in animal feed as a supplement, without being fully absorbed and therefore excreted in high amounts, remaining in sw (rosa et al., 2017b). the high capacity of oxisol adsorbs cu and zn and low mobility of these elements could be verified by (lopes et al., 2014). gomes filho et al. (2001) reported poor copper mobility in soil, stating that this element among the heavy metals is the most strongly adsorbed or complexed by the soil. furthermore, according to lopes et al. (2014) high adsorption of cu 2+ and zn 2+ may be the result of an increase in soluble organic compounds, which have the ability to complex these nutrients. the average leaf content of nutrients n, p, k, s and zn differed due to the application of increasing doses of sw (table 5). thus, the application of different doses of sw influenced the foliar contents of these nutrients. knowledge of the leaf content of an intensive system of production, mainly in a very demanding nutritionally species, such as tifton 85 grass, is very important for determining the amount of nutrient to be restored, reaching thus the mass production desired dry matter. silva (1999) described the range of suitable concentrations for tifton as follows, relative to macronutrients: c = 20.0-26.0; p = 1.5-3.0; k = 15.0-30.0; ca = 3.0-8.0; mg = 1.5-4.0; s = 1.5-3.0 g kg -1 . for optimal range micronutrient is: b = 5.0-30.0; cu = 4.020.0; fe = 50.0-200.0; mn = zn = 20.0-300.0 and 15.070.0 mg kg -1 . in this experiment the nutrient contents fall within the proper range suggested by silva (1999), except zn, wherein the average of treatments was 144 mg kg -1 . the extraction of nutrients by dry weight of the shoot (above 10 cm) was high, demonstrating that the tifton 85 grass has high nutrient extraction capacity (table 6). the extraction of macro and micronutrients of shoots of grass tifton 85 follows the following order: n> k> ca> p> mg> s> mn> fe> zn> b> cu (table 6). as the dm output followed a linear growth projection (figure 2a), all nutrients obtained the same trend (figures 3 and 4). the dose of 500 m³ sw ha -1 provided the soil 411.08 kg ha -1 of n and extraction was 358.01 kg ha -1 of n, which corresponds to 87.09% of the applied level. these results show that the synchronism between the availability of n from sw culture and demand generated 9 table 6. extraction of nutrients from the tifton 85 grass (dry matter), subjected to increasing doses of sw. element n p k s ca mg b cu fe mn zn ..................................................................................... kg ha -1 ........................................................................ averages 405.14 57.77 387.69 31.48 77.05 49.68 0.33 0.2 2.88 4.61 2.86 p-valor 0.001 0.001 0.000 0.005 0.017 0.008 0.057 0.025 0.007 0.078 0.050 c.v. % 7.73 6.25 5.34 11.23 9.65 8.98 10.50 13.30 8.49 27.09 15.36 figure 4. extraction of micronutrients cu (a), fe (b), mn (c) and zn (d) by dry weight. ** significant 10% probability. the dotted line refers to the addition of nutrients to the soil by increasing doses of sw and the continuous line for extracting the forage. a beneficial effect on the nutrient recycling process. this balance can reduce the mineral n concentration in the soil solution, promoting the sustainability of the production system. surely, this dose is the most appropriate in the environmental point of view with respect to n (figure 3a). sharpe and harper (2002) further justify about 35% of n sw is lost in the form of nh3 during application. the phosphorus provided by increasing doses of sw was lower than that extracted by the plant in all treatments (figure 3b), since the nutrient content in this sw is low (20.46 mg l -1 ). silva et al. (2012) conducted an experiment on the bonsucesso farm and found that the phosphorus content in sw was reduced by approximately 80% to pass the biodigester and the lagoon decantation, thus justifying the low content in sw. according to mattias (2006), the accumulation of p in soils treated with sw correlates with a lower n/p ratio of waste, since it meet the required amounts of n by plants it is necessary to simultaneously apply greater amounts of p resulting in accumulation and organic p moving in the environment. at a dose of 500 m³ ha -1 potassium applied via wastewater (254.69 kg ha -1 ) 10 was lower than that extracted by the plant (figure 3c). higher doses left nutrient residues in the soil (figure 3c and table 4). the calcium extractions (figure 3d) and magnesium (figure 3e) by tifton 85 grass were lower than the amount provided by the sw dose 2.000 m³ ha -1 . in this dose, the sw has provided the soil 103.8 kg ha -1 of calcium and magnesium 67.06 kg ha -1 and extracting the tifton 85 was 86.16 kg ha 1 of calcium and 55.92 kg ha -1 magnesium, this being the sw dose sufficient to provide the amount extracted at 85 tifton. there is a tendency to accumulate copper (cu) in soil with sw (figure 4a). cu average extraction obtained in the dry matter of grass tifton 85 was 0.2 kg ha -1 . the lowest dose of sw added tem times more than the cu extracted by forage, while the highest dose added 40 more times. figure 4 illustrates the low extraction by cu tifton 85 grass. lima and miyada (2003) conducted an experiment with cu in the form of cupric citrate, a more soluble source with greater utilization by the animal and less waste left in the excretions. thus, they concluded that cupric citrate may replace the cu sulfate, contributing to reducing this nutrient in the sw. sw in any of the doses could supply the manganese (mn) needed for the production of tifton 85 grass (figure 4c). it is necessary to monitor this nutrient in the soil and the plant, as part of nutrients extracted by grass after being consumed by cattle and returned to the soil through animal waste. zn supplied by the dose of 500 m³ ha -1 was 2.86 kg ha 1 approaches the extracted amount of and 2.72 kg ha -1 at tifton 85 (figure 4d). the view on organic waste recycling needs to be diversified, being recovery and recycling of nutrients from organic wastes are a possible solution. when organic waste recycling is complemented by nutrient extraction, some nutrient loops within society can be closes, enabling more sustainable agricultural production in future (kirchmann et al., 2017). conclusions the application of increasing doses of sw promoted a linear increase in carrying capacity, mass production of dry matter, density, height, and mass accumulation rate of dry matter per day of tifton 85 grass. the wastewater doses promoted changes in soil chemical properties. the dose of 2.000 m³ ha -1 supplied the nutritional needs of the grass tifton 85 in nitrogen, potassium, calcium, magnesium, 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