proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l corresponding author angelica stranieri angelica.stranieri@unimi.it abstract the aim of this study was to compare the diagnostic accuracy for feline infectious peritonitis (fip) of conventional clinic-pathological tests with that of molecular tests such as routine pcr and pcr followed by the sequencing of the spike (s) gene. blood, effusion and tissues specimens were collected from 21 fip suspected cats. in vivo examination consisted of cbc, serum protein electrophoresis, agp measurement, cytological and biochemical examination and the evaluation of the δtnc on effusions, and of molecular tests such the screening pcr (target: 3’utr region) and the pcr directed towards the s gene followed by the amplification products sequencing in order to detect the aminoacidic substitution recently considered diagnostic for fip1. these molecular techniques were applied to tissues collected during necropsy, which also allowed forming an fip group (13 cats) and a non-fip group (5 cats) based on histology and immunohistochemistry. the best test on tissues was immunohistochemistry (sens: 92.3%; spec: 100%), while the screening pcr suffered of low specificity (spec: 33.3%) and the s gene sequencing showed low sensitivity (sens: 69.2%).on effusions, the best tests resulted screening pcr and cytology (sens and spec: 100%) in comparison with the δtnc measurement (sens: 85.7 %; spec: 100%) and the s gene sequencing (sens: 42.8%; spec: 100%).on blood, the best test resulted agp measurement (sens: 81.8%; spec: 100%), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: 41.7%). screening pcr (sens: 55.6%; spec: 100%) and s gene sequencing (sens: 33.3%; spec: 100%) proved again low accuracy. references chang hw, egberink hf, halpin r, spiro dj, rottier pjm “spike protein fusion peptide and feline coronavirus virulence” emerg infect dis 18 (2012) 1089-1095 comparison between the diagnostic accuracy of clinico-pathological and molecular tests for feline infectious peritonitis (fip) a. stranieri, s. lauzi, c. giudice, v. cannito, a. giordano, s. paltrinieri department of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords animal model, sheep, imaging, glioblastoma corresponding author marco trovatelli marco.trovatelli@unimi.it journal home page riviste.unimi.it/index.php/haf sheep head frame validation for ct and mri studies marco trovatelli1*, fabio acocella1, stefano brizzola1, matteo ghiringhelli1, davide zani2 1university of milan, department of health, animal science and food safety, italy 2university of milan, department of veterinary medicine, italy abstract aim of eden 2020 (see references) project’s milestone 5 is the development of a steerable catheter for ced system in glioblastoma therapy. the vet group is involved in realization and validation of the proper animal model. in this part of the study two fresh sheep’s head from the local slaughter were used. the heads were located into an ad hoc frame system based on anatomical measures and ct images, producted by renishaw plc partner in this project. the frame was adapted and every components were checked for the ex vivo validation tests. ct imaging was taken in lodi at università degli studi di milano, facoltà di medicina veterinaria, with ct scanner and mri imaging was taken in la cittadina, cremona. system validation was approved by the ex vivo trial. the frame system doesn’t compromise the imaging acquisition in mri and ct systems. every system components are functional to their aims. the frame system is adapted to the sheep head. it is composed by elements able to lock the head during the imaging acquisition. frame system is characterized by a support base helping the animals to keep the head straight forward during imaging time, under general anesthesia. the design of these device supports the airways anatomy, avoiding damaging or obstruction to airflow during anesthesia period. the role of elements like mouth bar and ovine head pins is to lock the head in a stable position during imaging acquisition; fixing is guaranteed by v shape head pins that are arranged against the zygomatic arches. lateral compression forces to the cranium, and the v shape pins avoid the vertical shifting of the head and any kind of rotations. (fig. 1) http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: sheep head during ct imaging acquisition. the role of the frame is to preserve a physiological position of the head, along its median axis, without inclinations and rotations. references http://www.eden2020.eu/ http://cordis.europa.eu/project/rcn/200392_en.html http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author giuseppe federico labella, giuseppe.labella@unimi.it abstract thyreostats are orally active substances used in medicine and veterinary work to regulate the production of t3 and t4 hormones by the thyroid gland. they can be illicitly administered to livestock for fattening purposes in order to improve body-weight gain due to an important retention water in edible tissues and in the gastroenteric tract. their fraudulent utilisation is severely prohibited by the european union, which requires a precise monitoring. in 2007, european union of reference laboratories in the crl guidance paper proposed a recommended concentration of 10 ng ml-1 in urine, which has just been suggested to increase to 30 ng ml-1. in order to facilitate the detection of thyreostats in two bovine matrices, urine and thyroid glands, a method was developed without a previous derivatisation step, frequently used for hplc analysis of thiouracil. a salting-out assisted liquid–liquid extraction procedure for preparation was carried out to facilitate the movement of the thyreostats to the tert buthyl methyl ether phase. the hplc-ms/ms analytical procedure of the method was validated according to the guidelines of commission decision 2002/657/ec and has permitted to obtain satisfactory performance parameters and, decision limit and detection capability values for all the thyreostats lower than the recommended values hitherto mentioned. references j. vanden bussche, h. noppe, k. verheyden, k.wille, g. pinel, b. le bizec, h. f. de brabander . 2009. anal. chim. acta., 637, 2-12. commission decision 2002/657/ec of 12 august 2002 implementing council directive 96/23/ec concerning the performance of analytical methods and the interpretation of results. off. j. eur. commun. 2002, l221, 8-36. crl. crls view on state of the art analytical methods for national residue control plans. crl guidance paper, 2007. j. wauters, j. vanden bussche, b. le bizec, j. a. l. kiebooms, g. dervilly-pinel, s. prevost, b.wozniak, s. s. sterk, d.grønningen, d. g. kennedy, s. russell, p. delahaut, l. vanhaecke. j agr. food chem., 63, 1339-46. determination of thyreostats in bovine urine and thyroid glands by hplc-ms/ms g.f. labella 1 , l.m. chiesa 2 , e. pasquale 2 , s. panseri 2 , f. arioli 1 . 1 department of health, animal science and food safety, university of milan, milan, via celoria 10, 20133 milan, italy 2 department of veterinary science and public health, university of milan, milan, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords microwave sensor, dry matter, corn silage, dairy cows corresponding author vera perricone vera.perricone@unimi.it journal home page riviste.unimi.it/index.php/haf abstract dry matter (dm) intake in dairy cow is a central point to meet nutritional requirements and optimal performance, reducing the incidence of metabolic diseases (ingvartsen, 2006). dm content of some forages, such as silages, can undergo huge variations during storing, affecting the total daily dm consumed. reference laboratory method for dm assessment is time-consuming and cannot be applied to daily changes in diet composition. currently, new promising real-time technologies are available to monitor the dm content of feeds (nelson and trabelsi, 2004). the aim of the study was to test and calibrate a portable microwave sensor (ms) for dm content in corn silage samples. twentytwo samples were collected from the whole front of a corn silage trench, including the top and near the side walls, in order to collect as much as dm content variability as possible within the samples. ms readings were performed with 3 different methods for each samples: 1) directly on the silage front, 2) with the ms over the collected sample and 3) with ms placed under the sample. after the first ms reading, a correspondent silage sample was obtained by a silage corer for readings 2 and 3 and for the laboratory dm content assessment by drying in a 60°c forced air-oven to a constant weight. a linear regression analysis was performed (jmp, sas institute, cary, nc, 2015) on data obtained from a plot of ms readings against dm content. results evidence as the best ms reading method is represented by the probe burdening on the sample (r2=0.75) with respect to the other methods. the obtained results outlined as, with a correct reading method, ms could be valuable tool to determine dm content of corn silage directly at farm level. references ingvartsen, k. l., 2006. feedingand management-related diseases in the transition cow physiological adaptations around calving and strategies to reduce feeding-related diseases. animal feed science and technology. 126, 175–213. nelson, s.o. and trabelsi, s., 2004. principles for microwave moisture and density measurement in grain and seed. journal of microwave power & electromagnetic energy. 39(2), 107-117. real-time dry matter content of corn silage by a microwave sensor vera perricone1*, alessandro agazzi1, anna costa1, massimo lazzari1, giovanni savoini1, aldo calcante2, francesco m. tangorra1 1university of milan, department of health, animal science and food safety, italy 2university of milan, department of agricultural and environmental sciences production, landscape, agroenergy, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author marco tecilla, marco.tecilla@unimi.it abstract italian tortoises species are considered either endangered or near threatened according to international union for conservation of nature. when pet tortoises are abandoned or found injured or seized following illegal detention, they are sent to wildlife rehabilitation centers. from 2008, the testudo spp. population housed in the wwf vanzago’s oasis exhibited clinical signs compatible with testudinid herpesviurs 3 (tehv3) infection. by the end of 2012 all testudo had died. the presence of tehv3 was investigated by molecular biology and pathology. all the tortoises housed in vanzago resulted elisa positive for the presence of anti-tehv3 antibodies except one t. hermanni. of these, 12 animals died and were all necropsied. lesion frequency distribution was evaluate by histology. pcr was positive in 8/12 tortoises. to better complement the epidemiological evaluation of the virus in northern italy, 20 retrospective cases were selected from the archive of the university of milan. of these, 5 were tehv3 pcr positive. lesions closely resembled those of the vanzago’s population. these results are consistent with a high prevalence of tehv3 in northern italy. the finding of intranuclear inclusion bodies demonstrated to be specific but not sensitive. tehv3 diagnostic pathological lesions have been reported to vary according with host immune response and by the viral replicative status. molecular techniques were often necessary to confirm the infection. according to the literature and to our findings, t. hermanni spp. seems the species with higher mortality and lower antibody concentrations when infected with tehv3. references f. c. origgi. 2012. journal of herpetological medicine and surgery 22 ( 1-2): 52-54; e. r. jacobson., k. h. berry, f. c. origgi , j. f. jr wellehan, a. l. childress, j. braun, m. schrenzel, j. ye, b. rideout. 2012. journal of wildlife diseases 48(3): 747-57; f. c. origgi, c. h. romero, d. c. bloom, p. a. kelin, j. m. gaskin, s. j. tucker, e. r. jacobson. 2004. veterinary pathology 41(1): 50-61; j. f. soares,v. j. chalker, k. erles, s. holrby, m. waters, s. mcarthur. 2004. journal of zoo and wildlife medicine 35(1): 25-33; a. j. johnson, a. p. pessier, j. f. wellehan, r. brown, e. r. jacobson. 2005. veterinary microbiology 111(1-2): 107-16. tehv3 outbreak characterization in captive testudo spp. m. tecilla 1 , p. roccabianca 1 , g. grilli 1 , f. forlani 1 , f.c. origgi 2 1 department of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy 2 center for fish and wildlife health, vetsuisse-faculty of bern, laenggassstrasse 122 , 3012 bern, switzerland proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords transition goats, fatty acid, inflammation, immune response corresponding author greta farina greta.farina@unimi.it journal home page riviste.unimi.it/index.php/haf abstract as a new perspective in controlling cellular pathways, the aim of the trial was to investigate the expression of mirna implicated in adipogenesis and metabolic and endocrine functions in periparturient dairy goat fed saturated or unsaturated fatty acids. hepatic and adipose tissue samples were obtained from twenty-three second-parity twins-diagnosed alpine dairy goats were fed either calcium stearate (st, n:7), fish oil (fo, n:8) or a control diet without any fat supplement (c, n:8). dietary treatments lasted from one week before (30g/head/d of fatty acids) to three weeks after kidding (50g/head/d of fatty acids). st provided 26% c16:0 and 69.4% c18:0 while fo provided 10.4% epa and 7.8% dha. the expression of mir-26b and 155 (adipose infiltration of immune cells), mir-99a, 145 and 221 (inflammation and lipolysis) and mir-143 and 378 (pro-adipogenic function) was performed by rt-pcr on hepatic and adipose biopsies collected on day -7 and 7 and 21 from kidding. data were statistically analyzed by mixed and glm procedures of sas. no diet effect was found for all the mirna considered, but a significant effect of time for mir-155 (p= 0.028) and a tendency for mir-221 (p=0.083) were found with increased values from -7 to +21d. in particular, significant higher mir-155 expression (p<0.01) from -7 to 7d (-0.17 and 0.27, respectively) proved an inflammatory status in the first week after kidding in all the groups. obtained results for both mirna-155 and mirna-221 over the time copes with our previous findings on metabolic, productive parameters and mrna expression of genes related to lipid metabolism and inflammatory response (farina et al., 2016). these outcomes consolidate the hypothesis that lipogenesis takes place in dairy goat in the two weeks around calving, but it is reduced when compared to dairy cow (khan et al., 2013). references farina, g., agazzi, a., invernizzi, g., campagnoli, a., loor, j.j., and savoini, g., 2016. transcriptional regulation of lipid metabolism and inflammation in transition dairy goats by fish oil and stearate. international journal of health, animal science and food safety. 3 (1s) khan, m. j., hosseini, a., burrell, s., rocco, s.m., mcnamara, j.p.,and loor, j.j. 2013. change in subcutaneous adipose tissue metabolism and gene network expression during the transition period in dairy cows, including differences due to sire genetic merit. journal of dairy science. 96, 2171–2182 can lipid supplementation modulate inflammatory state and immune response in periparturient goats? a case study on hepatic and adipose mirna expression greta farina1*, alessandro agazzi1, guido invernizzi1, giovanni savoini1, j.j. loor2 1university of milan, department of health, animal science and food safety, italy 2university of illinois, department of animal sciences, urbana http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords equine asthma, pulmonary hypertension, vascular remodeling, vascular smooth muscle corresponding author serena ceriotti serena.ceriotti@unimi.it journal home page riviste.unimi.it/index.php/haf assessment of pulmonary vascular smooth muscle remodeling in severe equine asthma serena ceriotti1,2*, michela bullone2, francesco ferrucci1, jean-pierre lavoie2 1university of milan, department of health, animal science and food safety, italy 2 université de montréal, department of clinical sciences, sainthyacinthe, québec, canada abstract severe equine asthma (sea) is characterized by reversible airway obstruction and hypoxemia. pulmonary hypoxic vasoconstriction occurs during sea exacerbation, inducing pulmonary hypertension (ph). however, ph is only partially reversed by oxygen administration, and other etiological factors are uninvestigated (dixon, 1978). in rodent asthma models, airway inflammation is associated with pulmonary artery (pa) remodeling (rydell-tormanen et al., 2009), that could contribute to ph, as known in human chronic obstructive pulmonary disease (barbera et al., 2003). we investigated the presence of pa remodeling in sea, the involvement of vascular smooth muscle (vsm) alterations and their reversibility following long-term antigen avoidance or inhaled corticosteroids. using histomorphometry, the pa wall was measured on sections stained with hematoxylin-eosin saffron, collected post-mortem from different lung regions of 12 asthmatic horses and 6 controls. pulmonary vascular smooth muscle (vsm) mass was measured on sections stained for α-smooth muscle actin collected with in vivo thoracoscopy or post-mortem peripheral lung biopsy from 5 controls, 6 asthmatic horses in remission, and 11 asthmatic horses while exacerbation and after 1 year of antigen avoidance alone (5 horses) or treatment with fluticasone (6 horses). data were compared using unpaired t test with welch correction or paired t test (p<0.05). pa wall surface percentage was increased in apical (p=0.003) and caudo-dorsal (p=0.03) lung regions (respectively 50,66±13,16% and 50,56±15,02) of asthmatic horses, when compared to controls (respectively 35,38±7,06% and 38,13±10,18). similarly, vsm mass percentage was increased (p=0.03) in asthmatic horses (47,77±3,17%), compared to controls (41,07±6,22%). a tendency for normalization of the vsm mass was observed after treatment with antigen avoidance (p=0.05; 39,64±4,02%), but not with fluticasone (p=0.27; 45,35±14,98%). remodeling of the pa occurs in sea and the increase in vsm could lead to lumen narrowing and enhance hypoxic vasoconstriction, contributing to ph during exacerbation. vsm mass normalization is more effectively obtained by antigen avoidance than by corticosteroids. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references barbera, j. a., peinado, v. i. and santos, s., 2003. pulmonary hypertension in chronic obstructive pulmonary disease. eur. respir. j. 21(5), 892-905. dixon, p. m., 1978. pulmonary artery pressures in normal horses and in horses affected with chronic obstructive pulmonary disease. equine vet. j. 1(3), 195-198. rydell-tormanen, k., uller, l. and erjefalt, j. s., 2009. allergic airway inflammation initiates long-term vascular remodeling of the pulmonary circulation. int. arch. allergy immunol. 149(3), 251-258. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l aldosterone is a corticosteroid hormone that plays a pivotal role in homeostatic regulation of water and salt reabsorption, blood volume and pressure. aldosterone levels tent to rise in humans in hypertension, chronic and acute congestive heart failure (chf); detrimental effects are opposed by drugs like ace inhibitors and anti-mineralocorticoid. aldosterone has a pulsatile secretion, so measurement in serum is less indicative than in urine, where concentration can be indexed to creatinine ratio for estimation of the 24-h aldosterone excretion. few studies have evaluated aldosterone in canine urine patients, and none by elisa. aim of the study was to evaluate a commercial elisa kit for measuring aldosterone in dog’s urine. urine was collected by free catch from four dogs. two were healthy, one was affected by chf and prescribed anti-mineralocorticoid daily, one was affected by chronic kidney disease (ckd). urine was centrifuged (1250g/5 min) and supernatant frozen (-20°c). aldosterone was measured by a competitive elisa previously validated for dogs. twenty-four hours acid hydrolysis was performed on urinary samples before assay. the elisa standard curve in a semi-log plot was linear between 2.5 and 3.9 ng/ml. spike-andrecovery, linearity-of-dilution and parallelism experiments showed accuracy in measuring aldosterone in dog urine samples (syme et al., 2007). concentrations of urine aldosterone are reported in table 1. the intra-assay coefficient of variation showed good reproducibility of the assay. urinary samples are easy to collect, and the elisa used in this preliminary study seems promising in determining aldosterone in dog urine. its levels can be of great diagnostic and prognostic value for dogs affected by acute and chronic chf, in order to assess the best therapeutic strategy (gardner et al., 2007). this preliminary analysis will be followed by further studies in patients affected by acute and chronic chf. keywords competitive elisa, myxomatous mitral valve disease, aceinhibitors, angiotensin ii receptor blockers, heart failure. corresponding author alice savarese alice.savarese@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary evaluation of an elisa kit for the detection of aldosterone concentration in dog’s urine. a. savarese1*, c. locatelli1, v. borromeo1, a. berrini 1, a. galizzi1, m. crudo1, p.g. brambilla1 1 department of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 74 haf © 2013 vol. v, no. 1s issn: 2283-3927 patient aldosterone concentrations (ng/ml) healthy 1 2.593 ± 0.890 (cv 14.9%) healthy 2 2.737 ± 0.39 (cv 14.3%) chf 3.620 ± 0.44 (cv 12.2%) ckd 2.399 ± 0.42 (cv 17.5%) references syme, h.m., fletcher, m.g.r., bailey, s.r., elliott, j., 2007. measurement of aldosterone in feline, canine and human urine. journal of small animal practice. 48, 202–208. gardner s.y., atkins c.e., rausch w.p., defrancesco, t.c., chandler d.w., keene b.w. 2007. estimation of 24-h aldosterone secretion in the dog using the urine aldosterone: creatinine ratio. journal of veterinary cardiology 9, 1 7. table 1: concentrations of urine aldosterone measured in four dogs. values are mean ± sd of three determinations. in parenthesis the intra-assay coefficient of variation (cv%). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author valerio bronzo, valerio.bronzo@unimi.it abstract pentraxins are a superfamily of conserved molecules with immune functions such as complement activation and opsonization. ptx3 is the prototypic long pentraxin and is produced by different cell populations after proinflammatory stimuli. different studies have demonstrated the up-regulation of ptx3 during ruminant mastitis, but its role is still unknown. the aim of this study was to elucidate the role of ptx3 in the immune response to s. aureus intra-mammary infection (imi). given that no data are available on ptx3 expression in goat tissues, we first studied its pattern of expression in goat normal tissues, and then we investigated the role of ptx3 during mammary infection. six healthy goats were infused with pbs in the right udder and with s. aureus in the left udder. mammary biopsies from udders were collected 30h post infection, formalin fixed and routinely processed for microscopic evaluation or immediately stored in rnalater. tissue samples were collected at the slaughterhouse from healthy goats and were stored in rnalater. blood and milk were collected from healthy and infected goats; cells from blood and milk were isolated and processed for rna extraction or for cytospins. total rna from different organs, blood or milk cells, milk fat globules and mammary tissues was extracted and used as template in qpcr for ptx3. ptx3 expression was investigated by immunohistochemistry on formalin fixed paraffin embedded mammary tissue samples and on cytospins of isolated goat blood and milk cells. ptx3 mrna was expressed with very high levels in bone marrow, mammary gland, aorta, pancreas, skin and lung. given the high expression in the mammary gland, we investigated which cell population expressed ptx3. ptx3 was mainly expressed in the apical cytoplasmic portion of mammary gland epithelial cells, and in macrophages. during s. aureus infection ptx3 was up-regulated by epithelial cells. macrophages and mammary secretum didn’t show ptx3 modulation, but pmns recruited during infection were variably intensely positive. ptx3 mrna expression was low in healthy organs and tissues of goats as has been reported indeed the molecules commonly induced after pro-inflammatory stimulation. as expected, ptx3 was constitutively expressed in bone marrow, rich in pmns and monocytes, in aorta covered by endothelium and in the skin. ptx3 was up-regulated in epithelial mammary cells and in milk cells after s. aureus infection, demonstrating that it represents a first line of immune defense in goat udder. no modulation was observed in macrophages, in the secretum and in the ductal epithelial cells. further experiments are needed to elucidate the role of ptx3 in the pathogenesis of s. aureus infection. references daigo et al. 2014. immunology letters 161(1):38-43; brenaut et al. 2014. veterinary research 45:16; cremonesi et al. 2012. bmc genomics 13:540 ptx3 is up-regulated in epithelial mammary cells during s. aureus intra-mammary infection in goat j. filipe 1 , v. bronzo 2 , g. curone 1 , c. pollera 1 , l. turin 1 , p. dall’ara 1 , c. luzzago 1 , d. vigo 1 , a. casula 2 , p.roccabianca 1 , p.moroni 2 , f. riva 1 1 divet, dipartment of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy 2 vespa, department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords desaturase, cabannina, fatty acids, varzese, valdostana corresponding author giulio curone giulio.curone@unimi.it journal home page riviste.unimi.it/index.php/haf fatty acid profile, desaturase and atherogenic indices in milk of holstein friesian and italian autochthonous cattle breeds giulio curone1*, sara panseri2, carla colombani1, luca m. chiesa2, joel filipe1, massimo faustini1 1 university of milan, department of veterinary medicine, italy 2university of milan, department of health, animal science and food safety, italy abstract in the past decades, milk has been considered a mere supplier of nutrients, although its importance was considered paramount for the development and growth of newborns, a number of aspects regarding the biological functions of milk are still unknown. several positive functional properties of milk derive from fatty acids (fa), mainly unsaturated fatty acids (ufa), either monounsaturated (mufa) or polyunsaturated (pufa) fatty acids (mills, s. et al 2011; fedacko, j. et al 2007). in particular, ufas are considered functional components of food because of their positive effects on disease prevention (fao., 2010; connor, w.e.,2000; wijendran, v. and hayes, k.c., 2004). the objective of this study was to characterize the fatty acid profile, the desaturase index and the atherogenic index in milk of local italian bovine breeds (cabannina, varzese and valdostana) and in a cosmopolitan breed (holstein friesian) during the first period of lactation. a total number of 129 cows have been enrolled (friesian n=30, cabannina n=30, varzese n=30, valdostana n=39) from three dairy farms with similar management and feeding conditions. animals were chosen in order to have three classes of lactation stage: milk collections were carried out starting from 40±10 days (group a), 70±10 days (group b), and 130±10 days (group c). milk samples have been analyzed by gas chromatography to obtain the fatty acid profile, on the basis of these results, the desaturase and atherogenic indices were calculated. a number of differences between breeds have been found, in particular local breeds showed an higher percentages of ufa, mufa, pufa, and a higher ufa/sfa ratio, as well as lower desaturase indices (related to c14, c16 and c18) and atherogenic index, when compared to friesian cows. the results can add further information aiming to re-evaluate an almost lost local treasure in northern italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references connor, w.e., 2000. importance of n-3 fatty acids in health and disease. the american journal of clinical nutrition. 71(1 suppl), 171s-175s. fao, 2010. fats and fatty acids in human nutritionreport of an expert consultation. fao food and nutrition paper. 91, 1-166. fedacko, j., pella, d., mechirova, v., horvath, p., rybar, r., varjassyová, p., vargová, v., 2007. n-3 pufas-from dietary supplements to medicines. pathophysiology. 14(2), 127-132. mills, s., ross, r. p., hill, c., fitzgerald, g. f., stanton, c., 2011. milk intelligence: mining milk for bioactive substances associated with human health. international dairy journal. 21(6), 377-401. wijendran, v., hayes, k.c., 2004. dietary n-6 and n-3 fatty acid balance and cardiovascular health. annual review of nutrition. 24, 597-615. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords electrocardiography, dog, heart, rhythm, arrhythmia corresponding author alice savarese alice.savarese@unimi.it journal home page riviste.unimi.it/index.php/haf comparative analysis of a portable smartphone-based electrocardiograph (d-heart®) versus standard 6-leads electrocardiograph in the canine patient alice savarese1*, chiara locatelli1, niccolò maurizi2,3, nicolò briante3, paola g. brambilla1 1university of milan, department of veterinary medicine, italy 2university of florence, dept. of clinical and experimental medicine, italy 3 d-heart srl, genova, italy abstract d-heart® is a portable, smartphone-based device, which streams tracing via bluetooth, enabling multiple leads electrocardiograms (ecgs) acquisition, currently used in human cardiology (maurizi et al. 2017). the aim was to determine the accuracy of d-heart® compared with the gold standard non-portable 6-lead electrocardiograph in the evaluation of cardiac rhythm in dogs. standard 6-lead and d-heart® ecgs were acquired in conscious dogs. concordance between methods was assessed by weighted k cohen index, with its relative significance, taking as end point variable standard 6-lead ecg group. bland altman method (95% confidence level) was applied for p, pr, qrs, t and qt. since differences didn’t follow a normal distribution, a non­parametric approach was used to determine limits of agreement. p was significant when < 0.05 (maurizi et al. 2017). amplitude of waves was not considered because currently the software doesn’t allow voltage variation. 115 dogs of different weights and breeds admitted to the cardiology service of dimevet were enrolled. mean age was 7,5±4 years. most were intact males (45%, n=51). the most represented breed was mongrel (27%, n=32). weighted cohen's kappa test demonstrated excellent concordance in the evaluation of the heart rhythm (0.989, p<0.001), for st segment morphology (0.991, p<0,001) and for t wave morphology (0.838, p=0.040). there was a 100% concordance in p morphology determination. p, pr, qrs, t and qt intervals comparison with bland-altman showed an extremely good concordance for d-heart® measurements (95% limit of agreement ±0.9 ms for p, ±10 ms for pr, ±35 ms for qrs, ±5 ms for t wave). less concordance resulted for qt (±80 ms). in conclusion, d-heart® proved effective accurate recording of ecg comparable to standard 6-lead electrocardiographs, opening new perspectives to improve diagnostic tools in veterinary cardiology. future perspective will be the development of a telecardiology network and to improve arrhythmia’s diagnosis in small animal practice (bruining et al., 2014; haberman et al., 2015). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references bruining, n., et al., 2014. acquisition and analysis of cardiovascular signals on smartphones: potential, pitfalls and perspectives: by the task force of the e-cardiology working group of european society of cardiology. european journal of preventive cardiology. 21(2 suppl), 4-13 haberman, z.c., et al., 2015. wireless smartphone ecg enables large-scale screening in diverse populations. journal of cardiovascular electrophysiology. 26(5), 520-6 maurizi, n., et al., 2017. cardiovascular screening in low-income settings using a novel 4-lead smartphone-based electrocardiograph (d-heart®). international journal of cardiology. 236, 249-252 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords eels, perfluoroalkyl substances, lc-hrms corresponding author maria nobile maria.nobile1@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary study about the detection of perfluoroalkyl substances in eel samples of lake garda by liquid chromatography tandem mass high resolution mass spectrometry (lc-hrms). maria nobile1*, sara panseri1, francesco arioli1, luca m. chiesa1 1 university of milan, department of health, animal science and food safety, italy abstract perfluoroalkyl substances (pfass) are a large class of fluorinated aliphatic chemical of anthropogenic origin with high chemical stability even at high temperatures and in presence of alkalis, strong acids or oxidizing agents (lau et al. 2004). all these characteristics make them no biodegradable and very persistent in the environment, associated with adverse health risks (eriksen et al. 2010). food, especially fish and other seafood, is considered the main source of exposure to pfass (efsa, 2012). in this preliminary study we developed and validated a sensitive, selective and specific method by lchrms orbitrap to monitor the presence of 16 pfass in eel (anguilla anguilla) samples. the clean-up of the lyophilized samples consisted of a previous extraction step with acetonitrile to precipitate also proteins, followed by a purification step through oasis® wax spe (weak anionic exchange solid phase extraction) cartridges. the method applied to 45 farmed eel samples from lake garda showed the presence of several pfass, up to 10 in the same eel, in the order of ng/g (fig.1). the results provided a representative situation of the pfass contamination level of the lake, lower than those of others european countries (hoff et al. 2005, kwadijk et al. 2010). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: average concentrations (ng/g) of the pfass detected in the 45 eel samples of lake garda. pfba: perfluoro-n-butanoic acid; pfpea: perfluoro-n-pentanoic acid; pfhpa: perfluoro-n-heptanoic acid; pfoa: perfluoro-n-octanoic acid; pfna: perfluoro-n-nonanoic acid; pfos: sodium perfluoro-1-octanesulfonate; pfda: perfluoro-n-decanoic acid; pfuda: perfluoro-nundecanoic acid; pfdoa: perfluoro-n-dodecanoic acid; pftrda: perfluoro-n-tridecanoic acid; pfteda: perfluoro-ntetradecanoic acid. references efsa, 2012. perfluoroalkylated substances in food: occurrence and dietary exposure. efsa journal. 10 (6):2743, 1-55. eriksen, k.t., raaschou-nielsenb, o., sørensen, m., roursgaard, m., loft, s., møller, p., 2010. genotoxic potential of the perfluorinated chemicals pfoa, pfos, pfbs, pfna and pfhxa in human hepg2 cells. mutation research. 700, 39–43. hoff, p.t, van campenhout, k., van de vijver, k., covaci, a., bervoets, l., moens, l., huyskens, g., goemans, g., belpaire, c., blust, r., de coen, w., 2005. perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in flanders (belgium): relationships with biochemical and organismal effects. environmental pollution. 137, 324–333. kwadijk, c.j.a.f., korytar, p., koelmans, a.a., 2010. distribution of perfluorinated compounds in aquatic systems in the netherlands. environmental science & technology. 44, 3746–3751. lau, c., butenhoff, j.l., rogers, j.m., 2004. the developmental toxicity of perfluoroalkyl acids and their derivatives. toxicology and applied pharmacology 198(2), 231–241. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords mammalian fertility, oocyte, progesterone, pgrmc1, npr. corresponding author laura terzaghi laura.terzaghi@unimi.it journal home page riviste.unimi.it/index.php/haf effect of pharmacological inhibition of progesterone receptors pgrmc1 and npr on bovine oocyte meiosis laura terzaghi1*, alberto m. luciano1, trudee fair2, valentina lodde1 1university of milan, department of health, animal science and food safety, italy 2university college dublin, school of agriculture and food science, dublin, ireland abstract folliculogenesis is the process which leads to the acquisition of the oocyte developmental competence and to its maturation. both aspects are the result of oocyte and follicular cells interplay (luciano et al., 2004). recent studies in cattle describe progesterone (p4) as a key molecule acting during follicle development through different signaling pathways involving different receptors (aparicio et al., 2011, nilsson et al., 2009). the aim of this study is to evaluate the effect on oocyte meiotic maturation of inhibiting two p4 receptors: progesterone receptor membrane component 1 (pgrmc1) and the classic nuclear progesterone receptor (npr) respectively using the specific inhibitors ag205 and aglepristone. bovine cumulus cell-oocyte complexes (cocs) and denuded oocytes (dos) were in vitro matured with different concentrations of ag205. our results showed a decrease both in first polar body (pbi) extrusion and in the percentage of oocytes reaching mii stage in treated oocytes compared to controls (one way anova, p<0.05); these effects were more marked in dos, confirming pgrmc1 specific role in the oocyte. in ag205 treated oocytes aberrant meiotic figures were observed, including double metaphase plates or dna scattered in the ooplasm. in addition, aberrant meiotic plates showed irregular co-localization of pgrmc1 and aurkb; the proteins didn’t localize at the centromeric region of each chromosomes as previously described (luciano et al., 2013). this results suggests a p4 role in meiotic division mediated by pgrmc1 receptor. by contrast, aglepristone inhibition of npr didn’t affect dramatically the percentage of oocytes reaching mii stage of maturation. however, mii plates morphology analysis showed a significantly greater tubulin spindle length. this feature could account for the previously described reduced in vitro embryo development consequent to npr inhibition (aparicio et al., 2011). thus, p4 driven nuclear maturation could act on different oocyte development stages. further studies are in progress to elucidate p4 complex action in mammalian oocyte function. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references aparicio im, garcia-herreros m, o'shea lc, hensey c, lonergan p, fair t. 2011. expression, regulation, and function of progesterone receptors in bovine cumulus oocyte complexes during in vitro maturation. biology of reproduction. 84(5), 910-21. luciano am, modina s, vassena r, milanesi e, lauria a, gandolfi f. 2004. role of intracellular cyclic adenosine 3',5'monophosphate concentration and oocyte-cumulus cells communications on the acquisition of the developmental competence during in vitro maturation of bovine oocyte. biology of reproduction. 70(2), 465-72. luciano am, franciosi f, lodde v, tessaro i, corbani d, modina sc, john j. peluso. 2013. oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase b. biology of reproduction 88(3), 58. nilsson ee, skinner mk. 2009. progesterone regulation of primordial follicle assembly in bovine fetal ovaries. molecular and cellular endocrinology. 313(1-2), 9-16. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords sturgeon, stress, temperature, growth corresponding author lucia aidos marialucia.matelaaidos@unimi.it journal home page riviste.unimi.it/index.php/haf rearing temperature effect on the skeletal muscle fibers of acipenser baerii yolk-sac larvae lucia aidos1*, alessia di giancamillo1, marco lanfranchi3, daniela bertotto2, giuseppe radaelli2 and cinzia domeneghini1 1 university of milan, department of health, animal science and food safety, italy 2university of padua, department of comparative biomedicine and food science, italy 3società naviglio agricola ss, goito, italy abstract siberian sturgeon farming is important because it provides an alternative source of caviar and meat, but also for the conservation of the endangered natural stocks. farmed fish is continuously subjected to stress factors, of which, water temperature is considered a major one (schram et al., 2006). it has been demonstrated that physiological stress may have serious negative consequences on growth (wendelaar bonga, 1997) and that fish larvae appear less tolerant than adults to temperature variations (stefanovich et al., 2016). the present study aims at investigating the stress response and development in precocious stages of siberian sturgeon when subjected to different rearing temperatures, by analysing ontogeny, growth and stress response of yolk-sac larvae. this study was approved by the ethic committee of the university of milan (opba_20_2016). fertilized siberian sturgeon eggs were reared at 16°c, 19°c and 22°c until complete yolk-sac absorption. sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. water parameters and larvae development data were registered. histological, histochemical and immunohistochemical analyses were performed in order to assess ontogeny and stress biomarkers and whole body cortisol was measured by a specific microtitre radioimmunoassay (ria). statistical analysis was performed with sas software (v. 9.3, cary inc., nc). larvae subjected to the highest water temperature showed a faster yolk-sac absorption and larvae body weight significantly increased from hatching onwards. structural normal development considering the three different temperatures investigated from hatching until the end of the trial was observed. significant differences were found between temperatures regarding body weight and cortisol levels (p<0.01). a stronger expression of stress markers was noticed in larvae subjected to the lower temperature. even if this study indicates that lower rearing temperatures would appear more suitable for siberian sturgeon rearing, further studies would be necessary to evaluate the temperature effect on a mid-long term basis. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references schram e., van der heul j.w., kamstra a. and verdegen mcj. 2006. stocking density dependent growth of dover sole (solea solea). aquaculture, 252:339-47. stefanovich di, manzon la, mcdougall cs, boreham dr, somers cm, wilson jy, manzon rg. 2016. thermal stress and the heat shock response in embryonic and young adult of the year juvenile lake whitefish. comp. biochem. physiol. part a 193: 1-10. wendelaar bonga, s.1997. the stress response in fish. physiol.rev. 77:591–625. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords ixodes ricinus, midichloria mitochondrii, serological markers, tick bite corresponding author valentina serra valentina.serra@unimi.it journal home page riviste.unimi.it/index.php/haf serological evidence of midichloria mitochondrii circulation in humans parasitized by i. ricinus in germany and development of a marker for tick bite valentina serra1*, chiara bazzocchi1,2 1university of milan, department of veterinary medicine, italy 2university of milan, joint research centre for epidemiology and molecular surveillance of infections, italy abstract the tick ixodes ricinus transmits several microorganisms of medical and veterinary importance. midichloria mitochondrii (order rickettsiales; family midichloriaceae) is an intracellular symbiont present in the ovaries and salivary glands of 100% of adult i. ricinus females (sassera et al., 2008) and is transmitted to the vertebrate host during the tick bite (mariconti et al., 2012; bazzocchi et al., 2013) despite its infective role is not demonstrated. in this experimental study, a total of 324 human from different areas of germany were analysed in order to investigate the seropositivity against the flagellar flid protein of m. mitochondrii using an elisa approach. fifty sera were collected from patients living in non-endemic areas and used as negative controls while 274 sera were obtained from subjects exposed to tick bite and suffering from several symptoms referred to tick borne diseases and collected at the bca clinic in augsburg. since the positivity for the spirochete borrelia burgdorferi transmitted by i. ricinus is indicative for the tick bite, we considered also this additional information. the obtained results showed that 82 out of 274 sera were positive to m. mitochondrii and 42 out of 175 sera of subjects negative to b. burgdorferi were positive to m. mitochondrii to confirm the good property of flid protein as a tick bite marker. however, the high number (133 out of 274) of subjects parasitized by i. ricinus but negative to both bacteria prompted us to detect new more suitable i. ricinus/m. mitochondrii antigenic proteins to use as markers for tick bite. the next step to this purpose will be the setting up of a new elisa test for investigating the exposure of humans and animals to this tick species by using 12 synthetic peptides obtained from three i. ricinus proteins and one surface protein of m. mitochondrii. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references bazzocchi, c., mariconti, m., sassera, d., rinaldi, l., martin, e., cringoli, g., urbanelli, s., genchi, c., bandi, c., epis, s., 2013. molecular and serological evidence for the circulation of the tick symbiont midichloria (rickettsiales: midichloriaceae) in different mammalian species. parasites & vectors 6, 350-56. mariconti, m., epis, s., gaibani, p., valle, c. dalla, sassera, d., tomao, p., fabbi, m., castelli, f., marone, p., sambri, v., bazzocchi, c., bandi, c., 2012. humans parasitized by the hard tick ixodes ricinus are seropositive to midichloria mitochondrii: is midichloria a novel pathogen, or just a marker of tick bite? pathogens and global health 106, 391 396. sassera, d., lo, n., bouman, e.a., epis, s., mortarino, m., and bandi, c. 2008. "candidatus midichloria" endosymbionts bloom after the blood meal of the host, the hard tick ixodes ricinus. applied and environmental microbiology 74, 6138-6140. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l feed additives are commonly used to improve pig performance and health, but they need to be tested so new biomarkers for intestinal health, nonor minimally invasive, are under investigations. the quantification of myeloperoxidase (mpo) and pancreatitis associated protein (pap) in feces could prove useful to non-invasively monitor intestinal health (niewold, 2015). mpo is an enzyme that permits to quantify the number of inflammatory cells present in tissues and feces (prokopowicz et al., 2012) , while pap is a protein mainly produced in the small intestine with anti-inflammatory and bactericidal activity (cash et al., 2006; mukherjee et al., 2014). because of the lack of a commercial elisa kit for porcine pap detection, the main aim of this study was to develop and validate a new sandwich elisa test for the quantification of pap in pig fecal samples. our study consisted of two phases: test development (figure 1) and test validation. during the development phase we used polyclonal antibodies previously immunized from rabbit serum with a pure peptide containing the n-terminus of pig pap (soler et al., 2015). the validation of the test was then performed with fecal extraction samples derived from animals with known high or low growth performance. moreover, the temperature stability of pap in feces and the optimal extraction method was tested. even if only preliminary, our results seem to show a fair relationship between fecal consistency, used as health indicator, and pap fecal concentrations. furthermore, no relevant differences in pap concentration after 24h of incubation at 37 °c, 4°c or room temperature were detected. to date, the present results suggest that pap seems to be exceptionally stable in feces and is a very promising candidate as a non-invasive (fecal) biomarker for intestinal health and growth. keywords non-invasive biomarkers, pancreatitis associated protein, myeloperoxidase elisa, intestinal health. corresponding author elena mariani elena.mariani1@unimi.it journal home page riviste.unimi.it/index.php/haf non-invasive intestinal biomarkers: a new elisa test for pancreatitis associated protein detection in pig. e. mariani1,2,*, g. savoini1, t.a. niewold2 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 nutrition and health unit, department of biosystems, faculty of bioscience engineering, ku leuven. kasteelpark arenberg 30, b-3001 heverlee, belgium. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 52 haf © 2013 vol. v, no. 1s issn: 2283-3927 references cash, h.l., whitham, c.v., behrendt c.l., hooper l.v., 2006. symbiotic bacteria direct expression of an intestinal bactericidal lectin. science. 313, 1126-1130. mukherjee, s., zheng, h., derebe, m.g., callenberg, k.m., partch, c.l., rollins, d., propheter, d.c., rizo, j., grabe, m., jiang, q.x., hooper, l.v., 2014. antibacteria membrane attack by pore-forming intestinal c-type lectin. nature. 505, 103-107. niewold, t.a., 2015. intestinal health biomarkers in vivo. in: intestinal health. t.a. niewold (ed.). wageningen academic publishers. chapter 9. prokopowicz, z., marcinkiewicz, j., katz, d.r. and chain, b.m., 2012. neutrophil myeloperoxidase: soldier and statesman. archivum immunologiae et therapiae experimentalis. 60, 43–54. soler, l., miller, i., nöbauer, k., carpentier, s., niewold, t., 2015. identification of the major regenerative iii proteon (regiii) in the porcine intestinal mucosa as regiiiγ, not regiiiα. veterinary immunology and immunopathology. 167, 51-56. figure 1: standard curves with absorbance at 405 nm versus log 2 regiiiγ peptide concentration (µg/ml). capture antibody concentrations used were 10 µg/ml ( ); 5 µg/ml ml ( ); 3.125 µg/ml ( ) and 0 µg/ml ( ). detection antibody concentration used was 5 µg/ml. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords canine oral melanoma, canine cutaneous melanoma, mirna, ffpe sample corresponding author carlotta catozzi carlotta.catozzi@unimi.it journal home page riviste.unimi.it/index.php/haf expression analysis of micrornas in ffpe samples of canine cutaneous and oral melanoma by rt-qpcr carlotta catozzi1*, cristina lecchi1, chiara gini1, fabrizio ceciliani1, riccardo finotello2, lorenzo ressel3 1university of milan, department of veterinary medicine, italy 2university of liverpool, department of small animal clinical science, neston, united kingdom 3university of liverpool, department of veterinary pathology and public health, neston, united kingdom abstract microrna (mirna), a class of small, non-coding rna regulating post-transcriptionally protein expression are emerging as clinical biomarkers in many pathologies, including cancer (peng and croce, 2016). since mirna are supposed to represent fundamental key regulators, better understanding of melanoma tumour biology is essential to improve both disease grading and staging and, consequently, therapy options and prognosis. the aim of the study was to investigate whether mirna expression can vary in canine melanoma samples derived from formalin-fixed-paraffinembedded (ffpe) tissues. experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. two tissue slides were used for mirna extraction. the expression levels of seven mirna mir-145, mir-146a, mir-425-5p, mir-223, mir-365, mir-155 and mir-134 were detected and assessed by qpcr using taqman® probes (veramo et al, 2017; segura et al., 2012; wagner et al., 2013; sand et al.,2013). five mirna were significantly up-(n=3) or down-(n=2) regulated. in details, mir-146a and mir-155 abundance was increased as compared with control in both oral and skin melanoma (fig 1 b,e) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of mir-145 and mir-365 were lower (fig 1 a,d) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). mir-425-5p was upregulated (p = 0.039) only in skin melanoma (fig. 1 c). furthermore, functional analysis, carried out using mirnet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microrna (p = 6.95e-9); in particular, 21 target genes were associated with melanoma (p = 1.47e-5), including braf and cdk, e2f, fgf and pik3 families. in conclusion, mir145, mir-146a, mir-425-5p, mir-365 and mir-155 are differentially expressed in melanoma and healthy ffpe samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig 1: boxplots of the median (black line), the mean (+), 25%and 75%-quartile and range of the values measured, as well as statistical differences between groups are shown for mir-145, mir-146a, mir-425-5p, mir-365 and mir-155 in control, oral melanoma (oral m) and cutaneous melanoma (skin m) samples references peng and croce, 2016. the role of micrornas in human cancer. signal transduction and targeted therapy. 1, 15004 varamo et al., 2017. micrornaa role as potential biomarkers and key regulators in melanoma. genes, chromosomes and cancer. 56, 3-10 segura et al., 2012. microrna and cutaneous melanoma: from discovery to prognosis and therapy. carcinogenesis. 33, 1823–1832 wagner et al., 2013. comparison of non-coding rnas in human and canine cancer. frontiers in genetics. 4, 46-2 sand et al., 2013. comparative microarray analysis of microrna expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases, and benign melanocytic nevi. cell tissue res. 351(1), 85-98 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in equine sports medicine, the most considered performance parameter is the onset of blood lactate accumulation (obla), fixed at 4 mmol/l (courocè-malblanc et hodgson, 2014). nevertheless, in human medicine several methods to define individual lactate threshold (lt) were developed (faude et al., 2009). horses with poor performance were reported to have postexercise creatine-kinase (ck) activity higher than good performer (fraipont et al., 2011). aim of the study is to evaluate lts calculated by different methods in horses with increased postexercise ck activity. data from a cohort of standardbred racehorses that underwent clinical exercise testing, including incremental treadmill test with plasma lactate analysis at different speed steps, were retrospectively collected. a blood sample was collected 6hrs post-exercise and ck activity was evaluated with spectrophotometric method. a case group (group a) of 10 horses (3.1±1.0 y.o.) that did not present any other alteration potentially influencing performance than post-exercise ck activity greater than reference value (>735 u/l) was selected (valli, 2017). an age-matched and sex-matched control group (group b) of 10 horses with no alteration potentially influencing performance and normal post-exercise ck activity was selected. lactate concentrations obtained during treadmill test were analysed with a dedicated sotware (newell et al., 2009) and lt was determined by the following methods: a)inflection point, i.e. the point of intersection between two linear splines, b)lactate threshold by logaritmic transformation, c)obla and keywords racehorse, lactate, performance, obla, ck activity. corresponding author luca stucchi luca.stucchi@unimi.it journal home page riviste.unimi.it/index.php/haf effect of post-exercise increase in creatine-kinase activity on performance parameters in standardbred racehorses. l. stucchi1,*, c. valli2, f. ferrucci1,* 1 equine sports medicine laboratory “franco tradati”, department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2equine practitioner, lecco. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 82 haf © 2013 vol. v, no. 1s issn: 2283-3927 d)initial rise of 1 mmol/l post baseline. values of different lts from the two groups were compared by means of unpaired t-test. statistical analysis showed that obla was significantly lower (p=0.009) in group a when compared to group b. no differences between the two groups were observed for other lts values (figure 1). results confirm that horses with elevated post-exercise ck activity, probably due to muscle damage, have worse performance compared to controls. moreover, obla seems to be the more appropriate parameter for performance profiling in racehorses. obla g ro u p a g ro u p b 0 5 10 15 ** m /s references courocè-malblanc, a., hodgson, d.r. 2014 clinical exercise testing. in: hodgson d.r., mc keever k.h., mc gowan c.m. the athletic horse: principle and practice of equine sport medicine. 2nd edition, saunders, st. louis, 366-378. faude, o., kindermann, w., meyer, t. 2009 lactate threshold concepts: how valid are they? sports medicine, 39(6), 469–490. fraipont, a., van erck, e., ramery, e., richard, e., denoix, j.m., lekeux, p., art, t. 2011 subclinical diseases underlying poor performance in endurance horses: diagnostic methods and predictive tests. veterinary records 169(6) 154–154. newell, j., higging, d., madden, n., cruickshank, j, einbeck, j., mcmillan, k., mcdonald, k. 2007 software for calculating blood lactate endurance markers. journal of sports sciences, 25(12), 1403–1409. valli, c., 2017 determinazione degli intervalli di riferimento dell’enzima creatinchinasi (ck) prima e dopo esercizio massimale in una popolazione di cavalli trottatori in attività sportiva. degree dissertation. figure 1: difference in obla values between group a and group b (**=p<0,01) http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords flow cytometry, cat, lymphoma, sample quality, preanalitycal variability, needle size corresponding author serena bernardi serena.bernardi@unimi.it journal home page riviste.unimi.it/index.php/haf flow cytometry for feline lymphoma: a retrospective study about pre-analytical factors possibly affecting the quality of samples serena bernardi 1*, valeria martini1, stefano marelli 1, marzia cozzi1, stefano comazzi 1 1university of milan, department of veterinary medicine, italy abstract flow cytometry (fc) is an increasingly required technique on which veterinary oncologists rely to have an accurate, fast, minimally invasive lymphoma or leukemia diagnosis (burkhard and bienzle, 2013). fc has been studied and applied with great results in canine oncology (comazzi and gelain, 2011), whereas in feline oncology, the use of this technique is still to be experienced (guzera et al., 2016). this is mainly due to a supposed discomfort in sampling, because of the high prevalence of intraabdominal lymphomas (moore et al., 2012). the purpose of the present study is to investigate whether any pre-analytical factor might affect the quality of suspected feline lymphoma samples for fc analysis. ninety-seven consecutive samples of suspected feline lymphoma were retrospectively selected from the authors’ institution fc database. the referring veterinarians were recalled and interrogated about several different variables, including signalling, features of the lesion, features of the sampling procedure and the expertise of veterinarians performing the sampling. statistical analyses were performed to assess the possible influence of these variables on the cellularity of the samples and the likelihood of being finally processed for fc. none of the investigated variables significantly influenced the quality of the submitted samples, except for the needle size, the 21g needles providing the highest cellularity (table 1). notably, the samples quality did not vary between peripheral and intra-abdominal lesions. sample cellularity alone influenced the likelihood of being processed. about a half of the cats required pharmacological restraint. side effects were reported in one case only (transient swelling after peripheral lymph node sampling). fc can be safely applied to cases of suspected feline lymphomas, even for intra-abdominal lesions. 21g needle should be preferred for sampling. this study provides the bases for the spread of this minimally invasive, fast and cost-effective technique in feline medicine. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 table 1: cellular concentration of 52 samples of suspected feline lymphoma sent to the laboratory for flow cytometric immunophenotyping, according to the size of the needle used for sampling. samples collected through 21g needle show the highest cellular concentration. needle size (g) [number of samples] cellularity (x 103 cells / µl) mean ± sd median minimum maximum 18 [6] 12.67 ± 22.92 3.7 0.03 59.26 20 [1] 21.03 21 [4] 49.61 ± 36.72 51.90 4.75 89.88 22 [30] 9.49 ± 20.61 2.00 0.01 87.54 23 [8] 5.05 ± 8.32 1.83 0.63 21.99 25 [2] 20.19 ± 0.02 20.19 20.17 20.20 27 [1] 19.14 references burkhard, m.j., bienzle, d., 2013. making sense of lymphoma diagnostics in small animal patients. veterinary clinics of north america: small animal practice. 43(6), 1331-47, vii comazzi s., gelain m.e., 2011. use of flow cytometric immunophenotyping to refine the cytological diagnosis of canine lymphoma. veterinary journal. 188(2):149-55. doi: 10.1016/j.tvjl.2010.03.011 guzera, m., cian, f., leo, c., winnick, a. and archer, j., 2014. the use of flow cytometry for immunophenotyping lymphoproliferative disorders in cats: a retrospective study of 19 cases. veterinary and comparative oncology. 14 s1, 40-51 moore, p.f., rodriguez-bertos, a., kass. p.h., 2012. feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality. veterinary pathology. 49(4), 658-68 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords copy number variant, chicken, genetic variability corresponding author erica gorla erica.gorla@unimi.it journal home page riviste.unimi.it/index.php/haf genomic variability in mexican chicken population using copy number variation erica gorla1*, maria c. cozzi1, sergio i. roman-ponce2, felipe de jesus ruiz-lopez2, vicente e. vega-murillo2, silvia cerolini1, alessandro bagnato1, maria g. strillacci1 1 university of milan, department of veterinary medicine, italy 2instituto nacional de investigaciones forestales, agrícolas y pecuarias, mexico abstract copy number variants (cnvs) are polymorphisms which influence phenotypic variation and are an important source of genetic variability (mills et al., 2011). in mexico the backyard poultry population is a unique widespread creole chicken (gallus gallus domesticus) population, an undefined cross among different breeds brought to mexico from europe and under natural selection for almost 500 years (segura-correa et al.2004, rodriguez et al., 1996). the aim of this study was to investigate genomic variation in the mexican chicken population using cnvs. a total of 256 dna samples genotyped with axiom® genome-wide chicken genotyping array were used in the analyses. the individual cnv calling, based on log-r ratio and b-allele frequency values, was performed using the hidden markov model (hmm) of penncnv software on the autosomes (wang et al., 2007; peiffer et al., 2006). cnvs were summarized to cnv regions (cnvrs) at a population level (i.e. overlapping cnvs), using bedtools. the hmm detected a total of 1924 cnvs in the genome of 256 samples resulting, at population level, in 1216 cnv regions, of which 959 gains, 226 losses and 31 complex cnvrs (i.e. containing both losses and gains), covering a total of 47 mb of sequence length corresponding to 5,12 % of the chicken galgal4 assembly autosome. a comparison among this study and 7 previous reports about cnvs in chicken was performed, finding that the 1,216 cnvrs detected in this study overlap with 617 regions (51%) mapped by others studies. this study allowed a deep insight into the structural variation in the genome of unselected mexican chicken population, which up to now has not been never genetically characterized with snp markers. based on a cluster analysis (pvclust – r package) on cnv markers the population, even if presenting extreme morphological variation, does not resulted divided in differentiated genetic subpopulations. finally this study provides a cnv map based on the 600k snp chip array jointly with a genome-wide gene copy number estimates in mexican chicken population. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references mills r.e., walter k., stewart c., handsaker r.e., chen k., alkan c. et al., 2011. mapping copy number variation by population-scale genome sequencing. nature. 470, 59-65. segura-correa j.c., sarmiento-franco l., magaña-monforte j.g., santos-ricalde r., 2004. productive performance of creole chickens and their crosses raised under semi-intensive management conditions in yucatan, mexico, br poult sci. 45(3), 342-345. rodriguez j.c., allaway c.e., wassink, g.j., segura j.c., rivera t., 1996. estudio de la avicultura de traspatio en el municipio de dzununcàn, yucatàn. vet mex. 27(3), 215-219. wang k., li m., hadley d., liu r., glessner j., grant s., hakonarson h., bucan m., 2007. penncnv: an integrated hidden markov model designed for highresolution copy number variation detection in whole-genome snp genotyping data. genome res.17, (11):1665–1674. peiffer d.a., le j.m., steemers f.j., chang w., jenniges t., garcia f., et al., 2006. high-resolution genomic profiling of chromosomal aberrations using infinium whole-genome genotyping. genome res.16, 1136–1148. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in the search for the reduction of antibiotics in farm animals, it has been observed in piglets that medium-chain fatty acids (mcfa) with 6-12 atoms, exhibit an antimicrobic activity in particular against gram+ bacteria, at relatively high concentrations. however, mcfa, can be hardly used as such because of their repellent odour and taste and for their rapid absorption in upper gastrointestinal tract (git). these problems could be overcome by the generation of monoacylglycerol, but esterification is usually carried out on a silica base, which reduces the concentration of fa, therefore limiting the antibacterial effects. our hypothesis is that the saponification with calcium salts might positively affect mcfa concentration in the upper gastrointestinal tract. the aim of the present study was to examine the effects of laurate calcium soap (c12-ca soap) on growth performance and health status of post-weaning piglets. at weaning, 192 crossbreed topics piglets were assigned to 3 experimental groups consisting of 16 replicates (4 pigs/pen each): ctr (negative control), t1 (basal diet plus amoxycillin at 400 mg/kg), and t2 (basal diet plus c12-ca soap at 1 kg/ton). the basal diet, divided into pre-starter (administered from 0 to 14 days) and starter (administered from 15 to 42 days), was based on barley meal, maize meal and soybean meal. body weight, average daily gain and feed consumption did not differ among groups. feed efficiency was higher in t1 (0.61) and t2 (0.58) than in ctr (0.51) in the overall period (p <0.01) but also between 0-14 days and between 29-41 days (table 1). no mortality was observed in t1, while in t2 was 4,7% and in ctr was 10,9% (values calculated on 10 total dead animals). these preliminary results suggest that saponification of mcfa may be a valuable alternative to in-feed antibiotics, used for growth promotion, and for enhancing health in post-weaning piglets. keywords piglets, laurate, weaning, antibiotics, gut health. corresponding author silvia giorgi silvia.giorgi@unimi.it journal home page riviste.unimi.it/index.php/haf the saponification of lauric acid with calcium soaps as an alternative to infeed antibiotics in post-weaning piglets. s. giorgi1, m. comi1, m. ghiringhelli1, v. bontempo1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 64 haf © 2013 vol. v, no. 1s issn: 2283-3927 references decuypere, j. and dierick, n. (2003). the combined use of triacylglycerols containing medium-chain fatty acids and exogenous lipolytic enzymes as an alternative to in-feed antibiotics in piglets: concept, possibilities and limitations. an overview. nutrition research reviews, 16(02), p.193. desbois, a. and smith, v. (2009). antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. applied microbiology and biotechnology, 85(6), pp.1629-1642. hanczakowska, e. (2017). the use of medium-chain fatty acids in piglet feeding – a review. annals of animal science, 17(4). lallès, j., bosi, p., smidt, h. and stokes, c. (2007). nutritional management of gut health in pigs around weaning. proceedings of the nutrition society, 66(02), pp.260-268. zentek, j., buchheit-renko, s., männer, k., pieper, r. and vahjen, w. (2012). intestinal concentrations of free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial ecology and bacterial metabolic products in the digestive tract of piglets. archives of animal nutrition, 66(1), pp.14-26. table 1: feed efficiency of piglets fed basal diet without additive (ctr), with basal diet plus amoxycillin (t2) and basal diet plus laurate calcium soap (t2). feed efficiency was divided in different period: from 0 to 14 days, from 15 to 28 days, from 29 to 41 days and for the entire period of the trial. the results show that feed efficiency was higher in t2 and t1 groups than in ctr group. p-value ctr t1 t2 sem treat time treat*time feed efficiency 0-14 d 0,64 b 0,73a 0,65b 20,00 0,0134 <,0001 0,034 15-28 d 0,57 0,58 0,62 29-41 d 0,53 b 0,59a 0,53b overall 0,51 b 0,61a 0,58a 12,645 <.0001 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the intranasal (in) route shows promise for chemical restraint given the large area offered for drugs absorption. the nasal turbinates increase nasal mucosa surface, which have a greater blood flow than muscle, brain and liver tissue (dale et al. 2002). aim of the study is to compare the clinical effects and sedation scores following either in or intramuscular (im) administration of dexmedetomidine-methadone in dogs. twenty mixed-breed, client-owned, healthy dogs, undergoing soft tissue surgery or diagnostic procedures, were randomly allocated in two groups (n = 10) to receive dexmedetomidine (0.01 mg kg-1) together with methadone (0.4 mg kg-1) in (in-group) or im (im-group). temperament was evaluated before premedication (1 = calm and friendly, 4 = very excitable or nervous) (maddern et al. 2010). heart rate (hr), respiratory frequency (fr), body temperature, and side effects were recorded before (t0) and 10 (t10), 20 (t20) and 30 (t30) minutes after premedication. sedation was scored 3 times (every 10 minutes) after drugs administration using a descriptive sedation scale (0 = no sedation, 13 = extremely sedated) (gurney et al. 2009). induction was performed at t30 with titrate-to-effect propofol and the dosage was recorded. student t-test was performed. weight, age, temperament, body temperature and propofol dose were not different between groups (table 1). at each time point, excluding t0, im-group showed a statistically lower hr and fr compared to in-group. no undesirable effects were observed in both groups. sedation score in im-group was significantly higher compared to in-group at each time point. in conclusion, despite statistical differences, in administration produces a satisfactory clinical sedation with more gradual hemodynamic effects compared to im injection; this is probably due to a direct transport of drugs from cranial nerves (i-v) to brain with limited systemic absorption. however, the high variability recorded in sedation score between subjects in in-group (min 1/13; max 13/13 at t30) probably arises from a variable drugs conveyance from nasal mucosae to target cell in cns by in administration. keywords intranasal, sedation, dexmedetomidine, methadone, dog. corresponding author daniela gioeni daniela.gioeni@unimi.it journal home page riviste.unimi.it/index.php/haf clinical effects of dexmedetomidine combined with methadone after intranasal and intramuscular administration in dogs. d. gioeni1*, f. di cesare2, e.s. d’urso1, v. rabbogliatti1, g. ravasio1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 66 haf © 2013 vol. v, no. 1s issn: 2283-3927 references dale o, hjortkaer r, kharasch ed, 2002. nasal administration of opioids for pain management in adults. acta anaesthesiol scand 46, 759-770. gurney m, cripps p, mosing m, 2009. subcutaneous preanesthetic medication with acepromazine-buprenorphine is effective as and less painful than the intramuscular route. j small anim pract 50, 474-477. maddern k, adamns vj, hill na, leece ea, 2010. alfaxalone induction dose following administration of medetomidine and butorphanol in the dog. vet anaeth analg 37, 7-13. table 1: value of weight, age, temperament, body temperature and propofol dose presented as a mean ± standard deviation and p-value in in-group and im-group. in-group im-group p-value weight (kg) mean ± sd 23 ± 7 26 ± 10 p=0.1 age (months) mean ± sd 21 ± 13 31 ± 16 p=0.07 temperament (1-4) mean ± sd 2.6 ± 0.8 2.4 ± 0.9 p=0.3 body temperature (°c) mean ± sd 39,1 ± 0.1 39 ± 0.1 p=0.1 propofol dose (mg kg-1) mean ± sd 2.4 ± 1 1.7 ± 0.9 p=0.06 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l staphylococcus pseudintermedius (sp) has been associated with high antibiotic-resistance rates (e.g. methicillin) in european countries (flemming and wingender., 2010; osland et al., 2010; bardiau et al., 2013). this condition could be also related to the ability to produce biofilm (flemming and wingender., 2010). the aim of this study was to investigate the presence of methicillin-resistant sp strains and determine their ability to produce biofilm and some crucial virulence factors. forty-two sp strains, previously determined as multi drug resistant (mdr) by the disk diffusion method using a panel of 17 antimicrobial agents, were selected from our collection and tested phenotypically for the minimum inhibitory concentration (mic) of methicillin and genotypically for the presence of meca and blaz genes. the ability to produce biofilm was assessed phenotypically by two different assays: the congo red agar plates (cra) and the microtiter plate test (mtp) and genetically by the amplification of icaa and icad genes. three virulence factors genes coding for bicomponent leukocidin and enterotoxins (luk-i, sec, se-int) were searched. thirty-six strains revealed a value of mic major than 16 µg/ml (threshold for resistance); the remaining six isolates were sensible to this antibiotic. the 73% (31/42) were meca-positive and 86% (36/42) resulted blaz-positive; all the strains positive for meca were also positive for blaz. all sp strains resulted biofilm-producers by mtp assay (figure 1) and classified as weakly producers (4.7%), moderate producers (47.6 %) and strongly producers (47.6 %). in contrast, only 35.7% of all strains were considered biofilm-producers by cra method. the amplification of icaa and icad gene occurred respectively in 66.6% and 97.6%; only one strain was negative for both genes. almost all strains were positive for luk-i (95%), sec (74%) and se-int (84%). our data reveal the pathogenicity potential of sp strains from dogs, suggesting that they could be considered zoonotic potential agents and confirming other previous studies (osland et al., 2010; singh et al., 2013; stefanetti et al., 2017). in particular in this study we focused only on the dissemination of meca and blaz genes both coding for methicillin-resistance (meca is present in keywords biofilm, methicillin-resistant, staphylococcus pseudintermedius, icaa, icad. corresponding author gabriele meroni gabriele.meroni@unimi.it journal home page riviste.unimi.it/index.php/haf biofilm-forming ability and virulence factors of methicillin-resistant staphylococcus pseudintermedius from canine pyoderma. g. meroni1,*, p.a. martino1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 56 haf © 2013 vol. v, no. 1s issn: 2283-3927 chromosome while blaz in plasmid), but nowadays other authors reported the presence of methicillin-resistant sp strains mediated by mecc gene instead of meca. moreover could be observed a clear linkage between antibiotic-resistance and ability to produce biofilm. references bardiau, m., yamazaki, k., ote, i., misawa, n., mainil, j.g., 2013.characterization of methicillin-resistant isolated from dogs and cats. microbiology and immunology. 57, 496-501. flemming, h.c., wingender, j., 2010. the biofilm matrix. nature reviews microbiology. 8, 623-633. osland, a.m., vestby, l.k., fanuelsen, h.,schau slettemeas, j., sunde m., 2010. clonal diversity and biofilm-forming ability of methicillin-resistant staphylococcus pseudintermedius. journal of antimicrobial chemoterapy. 67, 841848. singh, a., waljer, m., rousseau, j., scott weese j., 2013. characterization of the biofilm forming ability of staphylococcus pseudintermedius from dogs. bmc veterinary research. 9, 93-98. stefanetti, v., bietta, a., pascucci, l., marenzoni, m.l., coletti, m., franciosini, m.p., passamonti, f., casagrande proietti, p., 2017. investigation of the antibiotic resistance and biofilm formation of staphylococcus pseudintermedius strains isolated from canine pyoderma. veterinaria italiana. 53, 289-296. figure 1: a) example of colourimetric appearance of colonies onto cra: very red (i), bordeaux (ii), almost black (iii), black (iv); (b) mtp assay for detection of biofilm production in some strains (column 11 and 12: negative controls). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l this paper analyses the long-term effects of neonatal calf diarrhoea (ncd) on the first milk production. a total number of 41 dairy heifers, belonging to two commercial dairy farms were admitted to our clinic for ncd between 2008 and 2015. survived animals, once returned to their farm, were followed until the end of the first lactation. as a treatment for ncd, we administered fluids and sodium bicarbonate intravenously according to the dehydration score described by boccardo et al. (2017) and blood-gas analysis results. the quantity of replacement fluid in liters was calculated as: replacement fluid (l)=dehydration (%) x bodyweight (kg). the required amount of sodium bicarbonate was calculated as: sodium bicarbonate (g) = body weight (kg) × base excess (mmol/l) × 0.6 (l/kg) × 0.084 (g/mmol). calves with a history of anorexia received 5 mg/kg glucose added to the saline solution. amoxicillin and clavulanic acid was administered sc at the dose of 10 + 2.5 mg/kg for 5 days to each calf. during lactation, we analyzed: milk production in a 305-day lactation, average fat percentage, average protein percentage, average somatic cell count and interval from birth to first calving. furthermore, days of hospitalization and severity of disease were considered (average calves age = 8,09 days, average body weight = 41,66 kg, average hospitalization = 10,61 days, average duration of treatment = 5,24 days). as a control, we considered non-hospitalized heifers (n.=238) with the same age, from the same herd, without clinical history of ncd. differences between the ncd group and control group were analyzed with general linear models. no statistic difference between the ncd group and control group was underlined (table 1). these findings differ from previous literature results. in fact, aghakeshmiri et al. (2017) found that the ncd increased the first calving age and heifer raising costs; svensson and hultgren (2008) showed that animals survived from ncd had a lower milk production. on the other hand, our results are similar to those reported by warnick et al. (1994;1995), even if in these works no data regarding the type of treatment and severity of clinical sings were considered. this study, while preliminary, suggests that the timely treatment of ncd could prevent irreversible damages and keywords neonatal calf diarrhoea, productive performance, reproduction performance, bovine. corresponding author giulia sala giulia.sala1@unimi.it journal home page riviste.unimi.it/index.php/haf long-term effect of neonatal calf diarrhoea on productive and reproductive performance: preliminary data. g. sala1,*, a. boccardo1, e. coppoletta2, a. belloli1, d. pravettoni1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. 2 veterinary teaching hospital, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 36 haf © 2013 vol. v, no. 1s issn: 2283-3927 ensure to reach a reproductive and productive standard during the first lactation. however the simple size needs to be increased. references aghakeshmiri, f., azizzadeh, m., farzaneh, n., & gorjidooz, m., 2017. effects of neonatal diarrhea and other conditions on subsequent productive and reproductive performance of heifer calves. veterinary research communications, 41(2), 107-112. boccardo, a., biffani, s., belloli, a., biscarini, f., sala, g., & pravettoni, d., 2017. risk factors associated with case fatality in 225 diarrhoeic calves: a retrospective study. the veterinary journal, 228, 38-40. svensson, c., and j. hultgren, 2008. associations between housing, management, and morbidity during rearing and subsequent first-lactation milk production of dairy cows in southwest sweden. journal of dairy science 91.4, 1510-1518. warnick, l.d., erb, h.n., and white, m.e., 1994. the association of calfhood morbidity with first-lactation calving age and dystocia in new york holstein herds. the kenya veterinarian 18, 177–179. warnick, l.d., erb, h.n., and white, m.e., 1995. lack of association between calfhood morbidity and subsequent first lactation milk production in 25 new york holstein herds. journal of dairy science 78,2819–2830. table 1: productive data in ncd and control groups. statistic differences were not recorded. herd 1 herd 2 ncd group (n.= 35) control (n.=182) p ncd group (n.=6) control (n.=56) p milk production (kg in 305 days) 9230 9122 0,768 8630 8682 0,934 fat percentage 3,7 3,8 0,350 4,0 4,7 0,033 protein percentage 3,4 3,5 0,175 3,4 3,8 0,026 scc (cells/ml) 155 204 0,568 234 388 0,733 first calving age (d) 960 990 0,135 794 798 0,920 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l predictable immobilization of wild zebras is challenging and there is massive variation in opiate response within different species. etorphine-azaperone combination is considered the protocol of choice, but no studies have investigated the physiological response to this procedure of immobilization in plains zebras. eleven free-ranging plains zebras (equus quagga) were immobilized for snares removal and translocations in kenya using a combination of etorphine 0.019 ± 0.003 mg/kg and azaperone 0.27 ± 0.05 mg/kg administered intramuscularly with a projectile dart. after recumbency, arterial blood was taken for gas analyses and physiological parameters were recorded for the duration of immobilizations (19 ± 6 minutes). ad hoc descriptive scores were given to the exertion resulting from high-speed chasing (table 1) and to quality of induction, immobilization and recovery. diprenorphine or naltrexone were used for opioid antagonism. the combination induced quick inductions within 3.5 ± 0.8 minutes and provided reliable recumbencies without attempts to stand for the duration of the immobilization. the average heart rates, respiratory rates and mean arterial blood pressure recorded were 102 ± 42 beats/minute, 18 ± 4 breaths/minute and 145 ± 28 mmhg respectively. arterial gas analyses demonstrated mild to severe and partially compensated metabolic acidosis and hypoxia, while electrolytes were within equids range. higher exertion levels during the chasing were significantly correlated to worse immobilization scores (p=0.008) and hyperthermia occurrence (p=0.0012) and non-significantly to severe acidosis. recoveries from anaesthesia were smooth, on average 121 ± 38 seconds after reversal. etorphine-azaperone combination keywords zebra, chemical immobilization, blood gas analysis, etorphine, physiology. corresponding author francesca vitali francesca.vitali@unimi.it journal home page riviste.unimi.it/index.php/haf physiological response to chemical immobilization: a case study of etorphine-azaperone in free-ranging plains zebra (equus quagga) in kenya. f. vitali1,*, e. kariuki2, m. njoroge2, t. kaitho2, g. curone1, d. mijele2, g. ravasio1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133, milan, italy. 2 department of veterinary and capture services, kenya wildlife service, nairobi, kenya. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 78 haf © 2013 vol. v, no. 1s issn: 2283-3927 produced physiological alterations in free-ranging plains zebra such as tachycardia, hypertension, metabolic acidosis and hypoxemia. however, these preliminary results indicate that high-speed chase might be responsible for physiological imbalance and that this drug combination does not suppress the compensatory response. regardless of the metabolic status, recover from immobilization was uneventful and all zebras went back to normal behavior thereafter. acknowledgements: the authors thank idexx laboratories, inc. (italy) for the donation of the vetstat electrolytes and blood gas analyzer and cartridges. references hoyer, m.j., de jong s., verstappen f. t. j. and wolters m. (2012): “standing sedation in captive zebra (equus grevyi and equus burchellii).” journal of zoo and wildlife. 43, 10-14. janssen, d.l., allen, j. l. (2015). equidae. in fowler's zoo and wild animal medicine, volume 8, 559-567. kock, m., burroughs, r. (2012). chemical and physical restraint of wild animals – a training and field manual for african species. international wildlife veterinary services. stemmet g. (2017). comparison of chemical capture efficacy of non-potent opioid drug combination to the preferred etorphine based combination in zebra (equus zebra). proceedings of sava wildlife group congress 2018, 80-81. west, g., heard, d., caulkett, n. (2014). nondomestic equids. in zoo animal and wildlife immobilization and anesthesia. (2nd edition). ames, iowa, usa: wiley blackwell publishing, 719-728. table 1: description of the scoring system used to categorise the amount of exertion resulting from high-speed chasing during vehicle-darting procedures in free-ranging plains zebra exertion score description 1 no reaction when approached by the darting vehicle or slow walking away (less than 30 seconds). no running. 2 suspicion over the darting vehicle. fast walking away (less than 60 seconds) or high-speed sprinting (less 10 seconds gallop). 3 running away within the herd when vehicle approach, no clearly individual chasing. medium/high-speed gallop up to 30 seconds. 4 jumpy reaction to approaching vehicle. individual high-speed chasing for 30 – 90 seconds. 5 extremely jumpy reaction to approaching vehicle. individual high-speed chasing for 90 – 180 seconds. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords staphylococcus epidermidis, implant-related infections, orthopedic infections, biofilm, genome sequencing corresponding author marta bottagisio marta.bottagisio@unimi.it journal home page riviste.unimi.it/index.php/haf phenotypic and genomic identification of staphylococcus epidermidis goi1153754-03-14 isolated from an infected orthopedic prosthesis marta bottagisio1,2*, alessio soggiu1, arianna b. lovati2, marco toscano3, cristian piras1, carlo l. romanò4, luigi bonizzi1, paola roncada5, lorenzo drago3,6 1university of milan, department of veterinary medicine, italy 2irccs galeazzi orthopedic institute cell and tissue engineering laboratory, milan, italy 3university of milan, department of biomedical science for health, italy 4irccs galeazzi orthopedic institute center for reconstructive surgery of osteoarticular infections milan, italy 5istituto sperimentale italiano lazzaro spallanzani, rivolta d'adda, cremona, italy 6irccs galeazzi orthopedic institute laboratory of clinical chemistry and microbiology, milan, italy abstract staphylococcus epidermidis goi1153754-03-14 is able to colonize orthopedic implants and to cause septic non-unions, as validated in a recent in vivo study (lovati et al., 2016). to the mechanisms leading to the biofilm formation on metallic implants, we carried out the phenotypic and genotypic characterization of the clinical isolate s. epidermidis goi1153754-03-14. the antimicrobial susceptibility and minimum inhibitory concentration (mic) of the strain were evaluated through the vitek2 system (biomerieux), as well as its ability to form biofilm in vitro through a spectrophotometric assay (stepanovich et al., 2000). the genomic dna was extracted by bacterial genomic dna isolation kit (norgen biotek corp.). libraries were prepared with the thruplex dna-seq (rubicon genomics) and then sequenced on the illumina miseq platform through the miseq reagent kit v3 (600-cycles) to produce 300 bp paired-end reads (illumina inc.). reads were quality-trimmed and gene annotated thanks to the rast software (aziz et al., 2008). the antimicrobial susceptibility along with the mic values are reported in table 1. the outputs resulted in 51 contigs (average = 50,720.6 mb) with 396x fold average coverage. the total genome is 2,586,753 bp long with a gc content of 31.84% and an n50 value of 7 bp. the whole genome is composed by 2,467 protein-encoding genes and 64 rnas (55 trnas and 9 rrnas). the entire genome sequence has been deposited in the european nucleotide archive (ena) under the accession no. fwcg01000000 (bottagisio et al., 2017). the genotypic and phenotypic characterization of the s. epidermidis goi1153754-03-14 will enable a better comprehension of the mechanisms involved in the biofilm formation on orthopedic implants, paving the way for innovative preventative and therapeutic strategies. moreover, the sequence of this clinical strain is mandatory to develop dedicated proteomics analysis in order to highlight functional mechanism of biofilm formation. acknowledgments this study was supported by the italian ministry of health (#rc 2016, l4083) http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 table 1: antimicrobial susceptibility and mic values antibiotic antimicrobial susceptibility mic benzylpenicillin resistant ≥ 0.5 oxacillin resistant ≥ 4 levofloxacin resistant 4 rifampicin resistant ≥ 4 gentamicin susceptible ≤ 0.5 erythromycin susceptible ≤ 0.25 clindamycin susceptible ≤ 0.12 linezolid susceptible 1 daptomycin susceptible 0.25 teicoplanin susceptible 4 vancomycin susceptible ≤ 0.5 tetracycline susceptible ≤ 1 tigecycline susceptible ≤ 0.12 fusidic acid susceptible ≤ 0.5 trimethoprim/ sulfamethoxazole susceptible ≤ 10 references aziz, r.k., bartels, d., best, a.a., dejongh, m., disz, t., edwards, r.a., formsma, k., gerdes, s., glass, e.m., kubal, m., meyer, f., olsen, g.j., olson, r., osterman, a.l., overbeek, r.a., mcneil, l.k., paarmann, d., paczian, t., parrello, b., pusch, g.d., 2008. the rast server: rapid annotations using subsystems technology. bmc genomics. 9, 75. bottagisio, m., soggiu, a., lovati, a.b., toscano, m., piras, c., romanò, c.l., bonizzi, l., roncada, p., drago, l., 2017. draft genome sequence of staphylococcus epidermidis clinical strain goi1153754-03-14, isolated from an infected knee prosthesis. genome announcements. 5, e00378-17. lovati, a.b., romanò, c.l., bottagisio, m., monti, l., de vecchi, e., previdi, s., accetta, r., drago, l., 2016. modeling staphylococcus epidermidis-induced non-unions: subclinical and clinical evidence in rats. plos one. e0147447. stepanovic, s., vukovic, d., dakic, i., savic, b., svabic-vlahovic, m.j., 2000. a modified microtiter-plate test for quantification of staphylococcal biofilm formation. journal of microbiological methods. 40, 175–179. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l bovine besnoitiosis, caused by besnoitia besnoiti, is a (re)emerging disease in europe (cortes et al.,2014), including italy (gazzonis et al.,2014;2017). however, its economic impact is scarcely considered and generally underestimated and there are still little studied aspects concerning both the parasite and the disease. following a natural outbreak of besnoitiosis in a dairy herd, a study was planned to characterize b.besnoiti infection in cattle through a multidisciplinary approach. suspicious abortions and clinical cases of besnoitiosis were reported in a dairy farm (september 2017, northern italy) housing 216 holstein cattle. blood samples were collected; haematological and serological analyses (elisa and confirmatory wb) were performed (fernandez-garcia et al.,2009). histology and molecular (endpoint its-1 pcr (cortes et al.,2007) and sequencing) analyses of tissues from a slaughtered cow with chronic besnoitiosis were carried out. out of 59 elisa-positive animals, 50 (23%) were confirmed by wb. b. besnoiti prevalence was higher in cows (41%) than in calves (12%); no heifer resulted positive. considering haematological parameters, a significant shift in the differential leucocyte formula from lymphocyte (l%) to granulocyte (g%) was recorded in infected cows (mean±s.d.:l=46.1±18.4,g=53.9±18.4) if compared to negative animals (student’s t-test,p=0.012). histology revealed a high load of b.besnoiti tissue cysts in skin, vulva, muzzle, sclera, eyelid, respiratory tract, emphasizing the possibility of parasite transmission through direct contact among animals. b.besnoiti was confirmed by pcr in other organs (heart, liver, aorta wall, tonsil) and especially in ovary, uterus and vulva, suggesting that the infection could affect cows’ fertility. parasite dna was also found in masseters posing an important question for food security, even if b.besnoiti is not considered zoonotic (figure 1). the study suggests that to investigate the dynamics of bovine besnoitiosis is mandatory to associate clinical and laboratory tests, including the genetic characterization of the parasite and its eventual correlation with the disease outcome. ethical statement: all procedures for collection of biological specimens from live animals were accomplished following good clinical practices in the respect of animal welfare according to keywords bovine besnoitiosis, serology, haematology, molecular biology, histology. corresponding author luca villa luca.villa@unimi.it journal home page riviste.unimi.it/index.php/haf investigating on besnoitia besnoiti (apicomplexa, sarcocystidae) in naturally infected dairy cattle by an integrated approach. l. villa 1,*, a.l. gazzonis1, s. mazzola1, s.a. zanzani1, c. perlotti1, g. sironi1, m.t. manfredi1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 70 haf © 2013 vol. v, no. 1s issn: 2283-3927 current legislation. the study was conducted with the approval of institutional animal care and use committee of università degli studi di milano (permission opba_34_2017). conflict of interest statement: the authors declare they have no conflict of interest. references cortes, h.c., reis, y., gottstein, b., hemphill, a., leitao, a., muller, n., 2007. application of conventional and real-time fluorescent its1 rdna pcr for detection of besnoitia besnoiti infections in bovine skin biopsies. veterinary parasitology. 146, 352-356. cortes, h.c., leitao, a., gottstein, b., hemphill, a., 2014. a review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by besnoitia besnoiti. parasitology. 141, 1406-1417. fernandez-garcia, a., alvarez-garcia, g., risco-castillo, v., aguado-martinez, a., marugan-hernandez, v., ortegamora, l.m., 2009. pattern of recognition of besnoitia besnoiti tachyzoite and bradyzoite antigens by naturally infected cattle. veterinary parasitology. 164, 104-110. gazzonis, a.l., alvarez-garcia, g., zanzani, s.a., garippa, g., rossi, l., maggiora, m., dini, v., invernizzi, a., luini, m., tranquillo, v.m., ortega-mora, l.m., manfredi, m.t., 2014. besnoitia besnoiti among cattle in insular and northwestern italy: endemic infection or isolated outbreaks? parasite and vectors. 7, 585. gazzonis, a.l., alvarez-garcia, g., maggioni, a., zanzani, s.a., olivieri, e., compiani, r., sironi, g., ortega-mora, l.m., manfredi, m.t., 2017. serological dynamics and risk factors of besnoitia besnoiti infection in breeding bulls from an endemically infected purebred beef herd. parasitology research. 116, 1383-1393. figure 1: (a) typical skin lesions in the chronic phase of besnoitiosis in a dairy cow. (b) histological section of skin: hyperkeratosis with lymphohistiocytic and eosinophilic flogosis. (c) pattern of recognition of besnoitia besnoiti tachyzoite antigens by sera from naturally infected cattle by western blot. (d) agarose gel-electrophoretic analysis of amplification products from conventional besnoitia besnoiti its1 rdna pcr on tissues from a slaughtered cow with chronic besnoitiosis http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords epigenetic, 5-aza-cr, 3d culture, micro-bioreactor, plasticity corresponding author elena f.m. manzoni elena.manzoni@unimi.it journal home page riviste.unimi.it/index.php/haf liquid marble micro-bioreactor promotes 3d cell rearrangement and induces, maintains and stabilizes high plasticity in epigenetically erased fibroblasts elena f.m. manzoni1*, tiziana a.l. brevini2, fulvio gandolfi1 1university of milan, department of agricultural and environmental sciences production, landscape, agroenergy, italy 2university of milan, department of health, animal science and food safety, italy abstract in the last years, many works demonstrated the possibility to directly interact with the epigenetic signature of an adult mature cell, through the use of epigenetic modifiers, (pennarossa et al., 2013; brevini et al., 2014, chandrakantan et al., 2016) and new mechanisms underlying this process have been recently described (manzoni et al., 2016). in particular, the small molecule 5-azacytidine (5-azacr) has been shown to induce a transient higher plasticity state in adult somatic cells, grown in standard 2d conditions. recent evidence have also shown the possibility to regulate and maintain cell pluripotency through the use of 3d culture systems. in the experiments here presented, we combine the two approaches and investigate whether the simultaneous use of a 3d micro-bioreactor and 5aza-cr is able to promote cell rearrangement, boost the induction of high plasticity and stably maintain it. to this purpose, fibroblasts were either plated on plastic dishes (2d) or encapsulated in a liquid marble (lm) micro-bioreactor (polytetrafluoroethylene (ptfe)), which has been previously shown to support the growth of living microorganisms, tumor spheroids, fibroblasts, red blood cells, and embryonic stem cells (ledda et al., 2016). cells were then erased with 5-aza-cr, for 18 hours and cultured in embryonic stem cell (esc) medium for up to 28 days. morphological analysis and pluripotency related gene expression levels were monitored for the entire length of the experiments. 2d cells, kept a monolayer pattern and acquired a pluripotent state that was, however, transient and lost by day 6. in contrast the use of a 3d system maintained and stabilized the high plasticity state in lm cells until the end of the experiments (fig. 1). the data obtained demonstrate that cell rearrangement and interactions may modulate 5-aza-cr induced plasticity and suggest a correlation between 3d mechano-transduction-related pathways and epigenetic regulation of cell phenotype. acknowledgments: supported by carraresi foundation. authors are members of the cost actions ca16119, bm1308 and cm1406. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: a) morphological changes in human erased skin fibroblasts plated on standard plastic dishes (plastic) or encapsulated in a lm micro-bioreactor (lm) at different time points. b) oct4, nanog and rex1 expression changes in mammalian skin fibroblasts plated on plastic dishes (blue) or encapsulated in a lm micro-bioreactor (red), exposed to 5-aza-cr for 18 hours (post 5-aza-cr) and subsequently cultured in esc medium up to 28 days (28d). gene expression levels are reported with the highest expression set to 1 and all other times relative to this. references brevini, t.a., pennarossa, g., rahman, m.m., paffoni , a., antonini, s., ragni, g., deeguileor, m., tettamanti, g., gandolfi, f., 2014. morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation. stem cell rev. 10(5), 633-42. chandrakantan v., yeola, a., kwan, j.c., oliver, r.a., qiao, q., kang, y.c., zarzour, p., beck, d., boelen, l., unnikrishnan, a., villanueva, j.e., nunez, a.c., knezevic, k., palu, c., nasrallah, r., carnell, m., macmillan, a., whan, r., yu, y., hardy, philip, grey, s.t., gladbach, a., delerue, f., ittner, l., mobbs, r., walkley, c.r., purton, l.e., ward, r.l., wong, j.w.h., hesson, l.b., walsh, w., pimanda, j.e., 2016. pdgf-abb and 5-azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. proc natl acad sci u s a. 113, 2306-2315. ledda, s., idda, a., kelly, j., ariu, f., bogliolo, l., bebbere, d., 2016. a novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor. j assist reprod genet. 33(4), 513-8. manzoni, e.f., pennarossa, g., deeguileor, m., tettamanti, g., gandolfi, f., brevini, t.a.l., 2016. 5-azacytidine affects tet2 and histone transcription and reshapes morphology of human skin fibroblasts. sci rep. 6, 37017. pennarossa, g., maffei, s., campagnol, m., tarantini, l., gandolfi, f., brevini, t.a.l., 2013. brief demethylation step allows the conversion of adult human skin fibroblasts into insulin secreting cells. proc natl acad sci u s a. 110, 89488953. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l animal production systems produce large quantities of manure, which is recognized as a significant source of heavy metals (hms) (hejna et al., 2018). some hms are essential nutrients and zinc oxide (zno) was often used at high doses to control the enteric disorders mainly in the swine post weaning phase (rossi et al., 2013, 2014). the general increase of hms content was registered in the livestock output with a negative impact on the environment. preliminary data showed that swine manure was an important source of zn, cu, mn and se to the environment reflecting the hms content in feeds (hejna et al., 2017a). the aim of this study was to evaluate the ability of typha latifolia (tl) and thelypteris palustris (tp) to bioaccumulate trace elements, from water as a cost-effective plant-based approach of wastewater remediation in pig livestock. the experimental design included four mesocosms (width: 4.0 m, length: 2.0 m, depth: 0.7 m; 695l of water, 210kg of soil): two controls, planted with tp (tpc) and tl (tlc) respectively and two treated, planted with tp (tpt) and tl (tlt) respectively. the treatment was represented by a mineral feed additive premix dissolved in tpt and tlt with the following final hms concentration: zn: 44.02mg/l; cu: 8.63mg/l; mn: 10.83mg/l; se: 0.09mg/l (figure 1). such high concentrations, corresponding to polluted wastewater, would be sufficient to reach the potential saturation limit of the substrates in the short experimental period. at day 0 (t0), day 15 (t1) and day 45 (t2) samples of roots, leaves, stems, soil and water were collected, dried and principal chemical component were estimated according to the official method of analysis of association of analytical communities (aoac). samples were also mineralized by an ultravawe single reaction chamber and analyzed using inductively coupled plasma mass spectrometry (icpms). obtained results showed that tl and tp tolerated high levels of zn, cu, mn and se with no visual toxicity signs or significant effects on growth during the entire experimental period. tp appeared more effective than tl at translocating elements from water to plant tissues. in particular, tpt showed a significant increase of zn and cu content in whole plants from t0 to t2 (p≤0.001). at t2 the mean zinc concentration was 409.26±342.33 mg/kg d.m. and 271.64±64.85 keywords heavy metals, swine livestock, phytoremediation, typha latifolia, thelypteris palustris. corresponding author monika hejna monika.hejna@unimi.it journal home page riviste.unimi.it/index.php/haf plant-based strategies to control the zinc output from swine production. m. hejna1,*, a. moscatelli2, n. stroppa2, s.r. pilu3, d. de nisi1, a. baldi1, l. rossi1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of biosciences, università degli studi di milano, via celoria 26, 20133 milan, italy. 3 department of agricultural and environmental sciences – production, landscape, agroenergy, università degli studi di milano, via celoria 2, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 60 haf © 2013 vol. v, no. 1s issn: 2283-3927 mg/kg d.m. in tpt and tlt plants respectively. in the plants of control mesocosms, differences between t0 and t2 were not observed. results suggested that both plant species were able to reduce the available amount of metals from the contaminated wastewater, thus tl and tp plants may be candidates for the phytoremediation approach to control hms output from the livestock wastewater. references hejna m., gottardo d., baldi a., dell’orto v., cheli f., zaninelli m., rossi l., 2018. review: nutritional ecology of heavy metals. animal. 1-15. hejna m., baldi a., onelli e., gottardo d., pilu s.r., dell’orto v., zaninelli m., rossi l., 2017a. evaluation of heavy metals in intensive animal production systems. italian journal of animal science vol.16:s1, 97. hejna m., stroppa n., moscatelli a., de nisi d., dell’orto v., pilu s.r., baldi a., rossi l., 2017b. phytoremediation as an innovative approach to control heavy metals output from livestock. italian journal of animal science vol.16:s1, 128. rossi l., dell'orto v., vagni s., sala v., reggi s., baldi a., 2014. protective effect of oral administration of transgenic tobacco seeds against verocytotoxic escherichia coli strain in piglets. veterinary research communications 38(1), 39–49. rossi l., di giancamillo a., reggi s., domeneghini c., baldi a., sala v., dell'orto v., coddens a., cox e., fogher c., 2013. expression of verocytotoxic escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model. journal of veterinary sciences 14(3), 263–270. figure 1: the schematic representation of the experimental mesocosms assembled in città studi botanical garden. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords spatial epidemiology, spatial statistics, one health, gis, genotype–environment association (gea), landscape genomics corresponding author stéphane joost journal home page riviste.unimi.it/index.php/haf spatial dependence of genetic data related to human health and livestock disease resistance: a role for geography to support the one health approach stéphane joost1,2,3* 1 laboratory of geographic information systems (lasig), school of architecture, civil and environmental engineering (enac), ecole polytechnique fédérale de lausanne (epfl), lausanne, switzerland 2 unit of population epidemiology, division of primary care medicine, department of community medicine, primary care and emergency medicine, geneva university hospitals, geneva, switzerland 3 group of geographic information research and analysis in public health (giraph; www.giraph.org) 1université de montréal ,department of clinical sciences, faculty of veterinary medicine, canada abstract the spatial dependence of located health and/or genetic data can be used to detect clusters likely to reveal disease prevalence or signatures of adaptation possibly associated with characteristics of the local environment (high temperatures, air or water pollution), be it in humans or animals (murtaugh et al. 2017). most often, geographic maps are produced to represent health data. medical information is transmitted through thematic choropleth maps. for instance administrative units are colored according to the variable of interest. but it is key to analyse health and/or genetic data by explicitly including geographic characteristics (distances, co-location) and also the potential and power of spatial statistics to detect specific patterns in the geographic distribution of disease occurrences (“make visible the invisible”). a classic example using clusters is the map produced by john snow (snow 1855) showing the number of deaths caused by a cholera outbreak in london. looking at a detail of snow's original map, it is possible to realize how he graphically represented the number of deaths, with short bold lines representing death occurrences (frequencies forming a kind of histogram) placed on the street at the addresses where it happened what we currently name georeferencing. a cluster of death people is an effect observed on the territory, and the existence of such a cluster depends on an infected water pump located at the same place (the cause). how can this spatial dependence be detected and measured? it is possible to identify spatial patterns in the geographic space by means of spatial statistics. we need to determine whether the variable of interest is randomly distributed or spatially dependent, and to check if the patterns observed are robust to random permutations. we also need to explore the data, to find out what is the range of influence of this spatial dependence. here we focus on the functioning of one among several measures of spatial autocorrelation named moran’s (moran 1950). moran’s i translates the global relationship between the behavior of points and of their neighborhood. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 measures of spatial dependence are key to detect and visualize spatial patterns in health and/or genetic data because spatial statistics can reveal signals that remain often hidden using thematic mapping. on the basis of the clusters highlighted by these exploratory methods, it is possible to formulate hypotheses about possible environmental or socio-economic causes and to test them with the help of confirmatory statistics. «ideas come from previous explorations» john tukey said in a paper published in 1980 in the american statistician, in a paper entitled «we need both exploratory and confirmatory» (tukey 1980). first explore and then confirm was already the reasoning applied by john snow to detect death "hot spots" in london, which then allowed him to hypothesize that a particular water pump was infected, and finally to take public health steps to check the cholera epidemic. two examples illustrate the use of these spatial statistics: first, a cohort named colaus and established in the city of lausanne was used to replicate the results obtained with 120’000 adults from the uk biobank study to test the hypothesis that high-risk obesogenic environments and behaviors accentuate genetic susceptibility to obesity (tyrell et al. 2017). our findings suggest that the obesogenic environment accentuates the risk of obesity in genetically susceptible adults. of the factors we tested, relative social deprivation (townsend deprivation index) best captures the aspects of the obesogenic environment responsible. we produced a map of lausanne showing the results of bivariate local indicators of spatial association (lisa) involving: 1) the value of the genetic risk score (grs) based on 69 genetic variants and associated with obesity as identified by the giant consortium (more than 330’000 individuals); 2) the townsend deprivation index (tdi), a composite measure of deprivation based on unemployment, non-car ownership, non-home ownership and household overcrowding. the analysis permits to identify clusters where a high grs depends on a high mean of the tdi calculated within a spatial lag of 800m. compared with a previous analysis applied to bmi in lausanne, we were able to delimit areas where genetic susceptibility and deprivation result in observed obesity. the second example is an application of landscape genomics (joost et al. 2007) to goat breeds in europe and to cattle in uganda to show how measures of spatial autocorrelation can be used to identify similarities or differences in genotype occurrences between neighboring individuals that cannot be explained by chance (stucki et al. 2016). in uganda, lisa indicators applied to genomic data in the ankole cattle breed reveal a pattern corresponding to the known geographic distribution of trypanosoma brucei gambiense. references joost s, bonin a, bruford mw et al., 2007. a spatial analysis method (sam) to detect candidate loci for selection: towards a landscape genomics approach to adaptation. molecular ecology, 16, 3955–3969. moran pap (1950) notes on continuous stochastic phenomena. biometrika, 37, 17–23. murtaugh mp, steer cj, sreevatsan s et al., 2017. the science behind one health: at the interface of humans, animals, and the environment. annals of the new york academy of sciences, 1395, 12–32. snow, j., 1855. on the mode of communication of cholera. john churchill. london. stucki s, orozco-terwengel p, forester br et al., 2016. high performance computation of landscape genomic models including local indicators of spatial association. molecular ecology resources, doi: 10.1111/1755-0998.12629. tukey, j.w., 1980. we need both exploratory and confirmatory. the american statistician, 34, 23–25. tyrrell j, wood ar, ames rm et al., 2017. gene–obesogenic environment interactions in the uk biobank study. international journal of epidemiology, dyw337. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en l keywords mycoplasma gallisepticum, ielisa, spa, serum, chicken. pages 59 – 66 references vol. 4 no. 1 (2017) article history submitted: august 10, 2017 accepted: december 01 2017 published: december 02 2017 corresponding author md zulfekar ali animal health research division, bangladesh livestock research institute (blri), savar, dhaka-1341, bangladesh mail: zulfekarvet@gmail.com, zulfekar@blri.gov.bd phone: +88 01711287146 journal home page riviste.unimi.it/index.php/haf article compared the effect of indirect elisa and serum plate agglutination (spa) test for the detection of mycoplasma gallisepticum in chicken md zulfekar ali 1, *, shirin sultana 2 , md. rezaul karim 1 , md. zakir hassan 1 , md. abu yousuf 1 , anowar hossen 3 , mohammed abdus samad 1 , md. giasuddin 1 and md. mostafizer rahman 4 1 animal health research division, bangladesh livestock research institute (blri), savar, dhaka-1341, bangladesh. 2 department of livestock services (dls), ministry of fisheries and livestock, dhaka-1215, bangladesh. 3 poultry production research division, bangladesh livestock research institute (blri), savar, dhaka-1341, bangladesh. 4 department of microbiology, hajee mohammad danesh science and technology university, dinajpur-5200, bangladesh. abstract mycoplasma gallisepticum (mg) is a highly economical and persistent threat of poultry industry in bangladesh. indirect elisa (ielisa) and serum plate agglutination test (spa) is available serological test for diagnosis of mg antibodies. the aim of this research was conducted on the basis of comparison on diagnosis results between ielisa and spa test for mg antibody in same sample in layer chicken. total 563 serum samples were collected and tested for mg antibody by both ielisa and spa test. out of 563 samples 363 (64.48%) samples were positive by ielisa and 316 (56.13%) samples were positive in spa test. the higher incidence of mg antibody was found in chicken at 50-56 weeks and flock size was 3000-4200 as 69.63% by ielisa and 61.21% by spa and in sonali breeds 69.08% by ielisa and 60.64% by spa. the results showed the comparatively higher number of positive results in ielisa test than spa test. so the findings of the study demonstrated that a significant (p<0.05) difference between ielisa and spa test present. the study may helpful for screening the flock for mg and small-holding farmers may use spa test rather than ielisa test due to rapid, easy and cost effective. md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 60 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction mycoplasma gallisepticum (mg) an organism under family mycoplasmataceae, affect chicken and turkey causes chronic respiratory disease (crd) resulting decrease food conversion ratio (fcr), dropping egg production with hatchability as well as increase production cost (kempf and gesbert, 1998; yoder, 1991). mg is the most common, constant and also an economical threat of poultry industry of bangladesh (arefin et al., 2011; ali et al., 2015). the degree of severity of mycoplasmosis depends on secondary bacterial infection mainly escherichia coli (oie, 2015) and also some viruses infection such as newcastle disease virus and infectious bronchitis virus. diagnosis of mg can be done by culture test, different biochemical test, serological test and molecular test. the most common and specific serological test for detection of subclinical also clinical mg in flock is indirect enzyme linked immunosorbent assay (ielisa) and serum plate agglutination (spa) test (avakian et al., 1988; jalilnia et al., 2011). the elisa test is more specific can detect up to very minute level of antibody by the help of elisa reader on the other hand the spa test is a rapid test where agglutination of antigen-antibody reaction can detect by necked eyes (kempf and gesbert, 1998). both serological tests were used for detection of specific antibodies against mg although there were differences in sensitivity and specificity of both methods (ley, 2008; arefin et al., 2011; feizi et al., 2013). the experiment was designed to know the comparison of results between ielisa and spa test and draw a conclusion between findings of two serological tests. 2 2. materials and methods 2.1 collection and preparation of samples: a total of 563 (2% of total population) blood samples were collected from 28,150 layer chickens rearing in 12 layer farms at northern region of bangladesh. the selected farms were on three layer breeds (sonali, isa brown and white leg horn) and the birds were not vaccinated against mycoplasma gallisepticum (mg). one ml blood samples were collected aseptically from wing vain by 3ml disposable plastic syringe with sterile needle without anticoagulant. individual syringe and needle was used for individual chicken. the syringe with blood samples were kept 1 hour at 450 angles in room temperature for clot the blood within syringe. the supernatant serum was decanted in a centrifuge tube for centrifuge at 2000rpm for 5minutes and straw color clear serum was collected in a sterile eppendorf tube. the serum samples were stored at -200c until the moment of serological test was performed. 2.2 indirect enzyme-linked immunosorbent assay (ielisa): the all collected serum samples were kept for ielisa test for detection of specific antibodies against mg by using commercially available indirect elisa test kit (biochek) manufactured by biochek uk ltd. co. uk (stipkovits et al., 1993; kempf et al., 1994). the elisa md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 61 haf © 2013 vol. iv, no. 1 issn: 2283-3927 test was performed according to manufacturer directions. briefly, serum samples were diluted with sample diluents as 1:500 ratio and dispended in the respective wells of antigen coated plate. then 100 μl of each negative control, positive control and reference control were added in a1-b1, c1-d1 and e1-d1 wells respectively. incubated at 220c temperature for 30 minutes and then washed by wash buffer. added 100 μl of conjugate in all wells and incubated at 220c temperature for 30 minutes then washed by same way. finally, added 100 μl substrate solutions in all wells and kept 15 minutes and then added 100 μl stop solutions in all wells to stop reaction. then the optical densities (od) were read by elisa reader (multiskan ® ex, usa) at 405nm light absorbance. the positive and negative results were interpreted by putting the od values in biochek elisa software. 2.3 serum plate agglutination (spa) test: the all serum samples were kept for spa test by using commercially available methylene blue stained antigen (lilli test mg rsa antigen, uk). the spa test of serum was performed according to standard protocol of kempf and gesbert, 1998. in brief, 50μl of serum was taken in white tiles and then added 50μl of methylene blue stained antigen on serum and then mixed homogenously by sterile tooth pick. then the results of agglutination for positive results observed under light source and negative result judged by no agglutination. 2.4 statistical analysis: at first the collected raw data were entered in microsoft excel and analyzed with sas software (sas, 1996). the confidence interval for comparative study between ielisa and spa were calculated according to the formula given in sas software and data were analyzed by χ 2 -test. p-value was considered significant if p<0.05. 3 results a total of 563 serum samples were collected from 3 layer breeds at 38-61 weeks of age. both serological tests were performed in all 563 samples for detection of specific antibodies against mg. the 363 samples were positive in ielisa test and 316 samples positive in spa test and the percentages were 64.48% and 56.13% respectively (table 1). by both serological tests the highest incidence shown in farm id 1 that was 71.43% and 63.10% for ielisa and spa test respectively. whereas the farm id 12 shown the lowest incidence as 50.00% by ielisa and 42.31% by spa test (table 1). the figure 1 shown comparisons between ielisa and spa test on age of layer chicken. in both tests higher positive was found in farms under 50-55 weeks of age that was 69.63% (149/214) and 61.21% (131/214) respectively. on the other hand lowest positive was found in farms under 56-61 weeks of age and results were 53.26% (49/92) and 44.57% (41/92). the highest and lowest positive of mg antibodies was found in sonali and white leg horn respectively in both tests among three layer breeds (figure 2). the mg antibodies were 69.08% md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 62 haf © 2013 vol. iv, no. 1 issn: 2283-3927 (172/249) by ielisa and 60.64% (151/172) by spa in sonali, 64.77% (114/176) by ielisa and 56.82% (100/172) by spa in isa brown, 55.80% (77/138) by ielisa and 47.10% (65/138) by spa in white leg horn (figure 2). the incidence of mg is related to flock size and highest incidence was found in larger flock size than smaller flock size by both ielisa and spa test. the lowest incidence was found 56.82% (50/88) and 48.86% (43/88) in flock size 1300-1600 by ielisa and spa test respectively and highest incidence was 69.63% (149/214) and 61.21% (131/214) by ielisa and spa test respectively in flock size 3000-4200 (figure 3). table 1: comparison between ielisa and spa test farm id age (weeks) no. of serum tested no. chicken/breed ielisa results spa results sonali isa brown white leg horn positive % positive % 1 55 84 221 182 160 60 71.43 53 63.10 2 59 30 16 53.33 14 46.67 3 61 36 20 55.56 16 44.44 4 46 54 35 64.81 31 57.41 5 50 70 48 68.57 42 60.00 6 49 46 28 60.87 24 52.17 7 38 40 27 67.50 24 60.00 8 53 60 41 68.33 36 60.00 9 43 35 23 65.71 20 57.14 10 40 32 21 65.63 18 56.25 11 44 50 31 62.00 27 54.00 12 56 26 13 50.00 11 42.31 total: 563 363 64.48 316 56.13 figure 1: comparison between ielisa and spa test on the basis of age of chicken. 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 38-43 44-49 50-55 56-61 p e rc e n ta g e age (weeks) ielisa results (%) spa results (%) md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 63 haf © 2013 vol. iv, no. 1 issn: 2283-3927 figure 2: comparison between ielisa and spa test on the basis of breeds of chicken. figure 3: comparison between ielisa and spa test on the basis of flock size. 4 discussion the objective of this study was comparison between two serological tests (ielisa and spa) for the diagnosis of mg antibody in commercial layer chicken in bangladesh. total 563 serum samples were collected during study period (2014-2016) from selected 12 commercial layer chicken farms in northern region of bangladesh. this study showed the comparative of results in between two serological tests. the overall sero-positive of mg was found 64.48% by ielisa and 56.13% by spa test. the comparatively number of positive sample was higher in 0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 sonali isa brown white leg horn p e rc e n ta g e breeds ielisa results (%) spa results (%) 0 10 20 30 40 50 60 70 80 1300-1600 1700-2000 2300-2700 3000-4200 p e rc e n ta g e flock size ielisa results (%) spa results (%) md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 64 haf © 2013 vol. iv, no. 1 issn: 2283-3927 ielisa test than spa test in same samples of the selected farms. similar findings were revealed by hossain et al. (2007) who reported overall seroprevalence of mg was 55.13% in chicken, higher prevalence was in young (72.72%) than adult (44.00%) and higher in larger flocks (62.86%) than smaller flocks (52.00%) by spa test in rajshahi district of bangladesh. these results also agreed with the findings of sikder et al. (2005) who reported overall seropositive of mg by spa test was 56.86% and 58.90% in layer chicken and breeder farm respectively in southern part of bangladesh. our results shown the number of seropositive samples were higher in ielisa (64.48%) than spa (56.13%) test and these results disagreed with the feizi et al. (2013), that reported seroprevalence of mg was higher in spa (42.22%) than elisa (33.33%) test in broiler breeder in iran. the breed variation, biosecurity, management practices and geographical location may the cause of disagreement with findings of feizi et al. (2013). the flock size is a factor that influences the spread of mg within flock. the results of this study showed the higher incidence of mg in larger flock than smaller flock which is strongly agreed with the findings of heleili et al. (2012) that revealed 76.97% incidence in large flock (18000 birds) and 20% in small flock (500-100 birds) by spa test of commercial poultry. the study showed significant (p<0.05) differences in results of both tests. feberwee et al. (2005) demonstrated a comparative study among serological tests for mg and found a high number of false positive results in both elisa and spa test. the authors also explained the factors that affect directly or indirectly to test results is cross reaction, lack of inactivation, age of chickens and applications of vaccines that are very similar with the present study. butcher (2002) indicated that the factors which play a partial role for high number of false positive results in spa test for mg is serum collected from vaccinated flock, contaminated serum, repeated thawing the serum before test, cross-reaction with mg antigen in ms affected flock. the use of elisa and spa test for mg antibody detection is only a screening tool for flocks but not for individual birds recommended by the world organization for animal health (oie). nascimento et al. (2000) carried out the detection of antibody by serological test are different, while spa test detect circulating igm antibody that develop 3-5 days of post infection (dpi) and persist 70-80 days but elisa and hi tests detect only igg antibodies develop 7-10 dpi and persists 180 days. luciano et al. (2011) reported the serological testspa, hi and elisa should be used only for screening tools of avian mycoplasmosis in poultry breeder flock. this recommendation based on different sensitivity and specificity of both tests (oie, 2015; pourbakhsh et al. 2010). researchers recommend that heating the serum at 56°c for 30 minutes could reduce false positive reactions (butcher, 2002). 5 conclusion although these tests were given different results so that it should be used as screening tests of mg routine monitoring for poultry flock health status. positive results obtained of spa test should be confirmed by additional tests such as hi, culture or molecular assays (pcr) because of the lack of specificity observed in spa. however, the ielisa and spa test may be md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 65 haf © 2013 vol. iv, no. 1 issn: 2283-3927 helpful for poultry farmers to know the mg status of the flock as well as pay an important role in use of anti-mycoplasma drugs, vaccination program and biosecurity of the farm. conflict of interest: the authors declare that there is no conflict of interest. acknowledgements: the authors proudly acknowledge to the lab workers of nourish poultry and hatchery ltd. dhaka, bangladesh for their kind help in this work. the financial support for this research was provided by the nst fellowship programme under ministry of science and technology, bangladesh. references ali, m.z., rahman, m.m., sultana, s. 2015. seroprevalence of mycoplasma gallisepticum antibody by elisa and serum plate agglutination test of laying chicken. veterinary world. 8(1), 9-14. arefin, m., begum, j.a., parvin, r., rahman, m.m., khan, m.a.h.n.a., chowdhury, e.h. 2011. development of slide agglutination test for the rapid diagnosis of mycoplasma infection in the chicken. bangladesh veterinarian. 28(2), 80-84. avakian a.p., kleven, s.h., glisson, j.r. 1988. evaluation of the specificity and sensitivity of two commercial elisa kits, the spat, and the hi test for antibodies formed in response to mycoplasma gallisepticum. avian diseases. 32, 262-272. butcher, g.d. 2002. factors to consider in serologic testing for mycoplasma gallisepticum (mg) and mycoplasma synoviae (ms). university of florida cooperative extension service, institute of food and agricultural sciences. 126, 1-2. available at: http://edis.ifas.ufl.edu/vm093. feberwee, a., mekkes, d.r., de wit, j.j., hartman, e.g., pijpers, a. 2005. comparison of culture, pcr and different serologic tests for detection of mycoplasma gallisepticum and mycoplasma synoviae infections. avian diseases. 49(2), 260-268. feizi, a., khakpour, m., nikpiran, h., kaboli, k., bijanzad, p. moggadam, a.r.j., hosseini, h. 2013. comparative evaluation of serological test in diagnosis of mycoplasma gallisepticum infection in iranian north-west rural poultry. advances in bioresearch. 4(3), 50-53. heleili, n., ayachi, a., mamache, b., chelihi, a.j. 2012. seroprevalence of mycoplasma synoviae and mycoplasma gallisepticum at batna commercial poultry farms in algeria. veterinary world. 5(12), 709-712. hossain, k.m.m., ali, m.y., haque, m.i. 2007. seroprevalence of mycoplasma gallisepticum infection in chicken in the greater rajshahi district of bangladesh. bangladesh journal of veterinary medicine. 5 (1 & 2), 09-14. md zulfekar ali et al. int. j. of health, animal science and food safety 4 (2017) 59 66 66 haf © 2013 vol. iv, no. 1 issn: 2283-3927 jalilnia, m., movassagh, m.h. 2011. a study on causes of poultry carcasses condemnation in east azerbaijan province (north west of iran) poultry slaughterhouse. annals of biological research. 2(4): 343-347. kempf, i and gesbert, f. 1998. comparison of serological tests for detection of mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens. veterinary microbiology. 60(2), 207-213. kempf, i., gesbert, f., guittet, m., bennejean, g., stipkovits, l. 1994. evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of mycoplasma gallisepticum antibodies. avian pathology. 23, 329-338. ley, d.h. 2008. mycoplasma gallisepticum infection, eds., saif, y.m. disease of poultry, wileyblackwell publishing, iowa, ia, pp: 807-834. luciano, r.l., cardoso, a.l.s.p., stoppa, g.f.z., kanashiro, a.m.i., de castro, a.g. m., tessari, e.n.c. 2011. comparative study of serological tests for mycoplasma synoviae diagnosis in commercial poultry breeders. veterinary medicine international. 2011, 1-5. nascimento, e.r. 2000. micoplasmoses in doenc¸a das aves, macari, m. and berchieri, a., jr., eds., campinas, sao paulo, brazil, pp. 217-224 oie. 2015. avian mycoplasmosis (mycoplasma gallisepticum, m. synoviae) in manual of diagnostic tests and vaccines for terrestrial animals. world organisation for animal health (oie), paris, france. pourbakhsh, s.a., shokri, g.r., banan, m., elhamnia, f., ashtari, a. 2010. detection of mycoplasma synoviae infection in broiler breeder farms of tehran province using pcr and culture methods. archives of razi institute. 65, 75-81. sas (1996). sas/stat user’s guide: statistics. version 6, 4th edn., sas institute inc., cary, nc. sikder, a.j., islam, m.a., rahman, m.m., rahman, m.b. 2005. seroprevalence of salmonella and mycoplasma gallisepticum infection in the six model breeder poultry farms at patuakhali district in bangladesh. international journal of poultry science. 4(11), 905-910. stipkovits, l., czifra, g., sundquist, b. 1993. indirect elisa for the detection of a specific antibody response against mycoplasma gallisepticum. avian pathology. 22(3), 481-494. yoder, h.w. 1991. mycoplasma gallisepticum infection. in: calnek, b.w., beard, c.w., barnes, h.j., reid, w.m., yoder, h.w., jr. eds., diseases of poultry. 9th 3dn. iowa state univ. press, ames, ia, pp. 198-212. proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l corresponding author federica maghin, federica.maghin@unimi.it abstract the effects of dietary supplementation with antioxidant mixture in medium-heavy swine on oxidative status, nutritional and sensory characteristics of longissimus dorsi (ld) muscle and smoked cured ham were evaluated. seventy-four pigs (pic x max grow), were assigned to two experimental groups: control (ct) and treated supplemented with antioxidant mixture (aox) for 45 days before slaughter. the total antiradicalic activity of blood (krl test) and carcass dressing percentage was positively affected (p<0.05) by aox supplementation. chemical composition of ld was not affected by dietary treatment. oxidative stability and colour indices were significantly affected (p<0.05) by dietary treatment and storage time (0, 6, 12, 15 days under modified atmosphere packs map). sensory analysis revealed that at 12 and 15 days of storage a loss of colour beside presence of off odors was higher (p<0.05) in ct than aox group. the seasoning losses of smoked cured ham tended to be lower (p=0.06) in aox group than ct. physical and chemical composition was not affected by dietary treatment. sensory analysis revealed a difference between ct and aox (p<0.05) in salty and sweet taste. furthermore, the consumer test revealed that smoked cured ham from aox were preferred (p<0.05) than ct. dietary supplementation with antioxidant mixture improves total antioxidant status, carcass dressing percentage and smoked cured ham seasoning losses. the oxidative, colour stability and sensory parameters of ld muscle was improved in aox groups during refrigerated storage in map. antioxidant mixture positively affect the consumer preference of smoked cured ham, without affecting other quality parameters. references shahidi, f., zhong, y. 2010. european journal of lipid science and technology 112, 930–940; franz, c., baserb, k. h. c., windischc, w. 2010. flavour fragrance journal 25: 327–340; falowo, a. b., fayemi, p. o., muchenje, v. 2014. food research international 64: 171–181; rossi, r., ratti, s., pastorelli, g., crotti, a., corino c. 2014. food research international 65: 88–94; antioxidant supplementation in pig nutrition: effects on shelf life of longissimus dorsi muscle and consumers’ preferences for smoked cured ham. f. maghin 1 , r. rossi 1 , s. ratti 1 , g. pastorelli 1 , c. corino 1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords perfluoroalkyl substances (pfass), residue, liquid chromatography tandem mass spectrometry, pork, wax cartridge corresponding author shihkuo lin shihkuo.lin@unimi.it journal home page riviste.unimi.it/index.php/haf abstract the perfluoroalkyl substances (pfass) residues, which come from environmental pollution, tend to accumulate in the food chain (efsa, 2008; guerranti et al., 2013). 17 chemicals of pfass family were selected for this study; only perfluorooctanoic acid (pfoa) and perfluorooctane sulfonate (pfos) are the most known compounds based on the literature works and european legislations. twenty frozen pork samples were collected from 7 european countries (austria, denmark, germany, holland, italy, poland and spain). one gram of pork sample was deproteined with acetonitrile, then extracted by waters® wax spe (solid phase extraction) cartridges. the wax spe cartridge was used because in the literature there are the best results for these analytes (taniyasu et at., 2005), but regards to pork meat there is a lack of information. this method was developed and optimized in the clean-up step for validation, according to commission decision 2002/657/ec (european community, 2002). all extracted samples were analyzed by liquid chromatography tandem mass spectrometry (lc-ms/ms), using methanol and 20 mm ammonium formate as mobile phases. for validation, we use 20 blank pork samples. the results of calibration curve of each pfas have high r2 values ranging from 0.9901 to 0.9993. the recoveries were in the range 80%-119%. the protocol of extraction was applied to the 20 european samples mentioned above and the average concentrations were from traces to 12.87 ng/g. these preliminary data don’t represent a risk or an indication of high pollution but we need more analyses to define a reliable risk assessment. references efsa, 2008. perfluorooctane sulfonate (pfos), perfluorooctanoic acid (pfoa) and their salts scientific opinion of the panel on contaminants in the food chain. efsa journal. 653, 1-13. european community, 2002. commission decision 2002/657/ec commission decision concerning the performance of analytical methods and the interpretation of results. official journal of the european union. 221 , 8-36. guerranti, c., perra, g., corsolini, s., focardi, s. e., 2013. pilot study on levels of perfluorooctane sulfonic acid (pfos) and perfluorooctanoic acid (pfoa) in selected foodstuffs and human milk from italy. food chemistry. 140, 197-203. taniyasu s., kannan k., so m. k., gulkowska a., sinclair e., okazawa t., yamashita n., 2005. analysis of fluorotelomer alcohols, fluorotelomer acids, and shortand long-chain perfluorinated acids in water and biota. journal of chromatography a. 1093, 89-97. preliminary results for the detection method of perfluoroalkyl substances (pfass) residues in pork shihkuo lin1*, maria nobile1, luca m. chiesa1 1 university of milan, department of health, animal science and food safety, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords nrf2, oxidative stress, reporter mice, omega-3 corresponding author elena mariani elena.mariani1@unimi.it journal home page riviste.unimi.it/index.php/haf abstract cells developed several mechanisms, including the transcription factor nuclear factor e2-related factor 2 (nrf2) to counteract oxidative stress (lee and johnson, 2004). the aim of the study was to evaluate the activation of nrf2 in transgenic mice fed saturated or polyunsaturated fatty acids and the anti-inflammatory effect of oestrogens on organism. forty-eight are cre omo reporter mice were divided into 3 groups, consisting of 16 animals, based on presence/absence of oestrogens (ovariectomized or sham female, ovx sh; male, ma). each group was further split in 4 subgroups of 4 animals each and fed different diets (7.5% lard, 7.5% tuna oil, 20.0 % lard and 20.0% tuna oil). two times a week animals were anaesthetized and injected intraperitoneally with 100µl luciferin 15 min before the imaging session. using the living image software, photon emission was mapped for selected body areas. on day 70, animals were sacrificed after a challenge with sodium arsenite. specific organs were dissected and immediately subjected to ex vivo imaging session. mixed and glm procedures of sas software were used for statistical analysis. dietary treatments did not affect body weight and feed intake as well as nrf2 expression in both preand post-challenge phases, with the exception of the abdominal region (p=0.031 pre-challenge); in this area, during the pre-challenge phase, ovx showed lower nrf2 activation (p<0.001). ex vivo results outlined a significant effect of the challenge on all the considered organs (p<0.001), while ovx subjects had higher nrf2 expression on urinary bladder and kidney (p<0.05) and high fat diet increased nrf2 in urinary bladder (p<0.05). the present trial shows how supplementation of saturated or polyunsaturated fatty acids in the diet do not exert significant effects on oxidative stress in mice, but confirms the protective role of oestrogens under physiological condition. references lee, j.m., and johnson, j.a., 2004. an important role of nrf2-are pathway in the cellular defence mechanism. journal of biochemistry and molecular biology. 37, 139-143. nuclear factor e2-related factor 2’s activation in transgenic mice fed with dosage of saturated or unsaturated fatty acids using in vivo bioluminescent imaging elena mariani1*, nicoletta rizzi2, guido invernizzi1, alessandro agazzi1, adriana maggi2, giovanni savoini1 1 university of milan, department of health, animal science and food safety, italy. 2university of milan, center of excellence on neurodegenerative diseases and department of pharmacological and biomolecular sciences, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author marcella de maglie, marcella.demaglie@unimi.it abstract silver nanoparticles (agnps) are widely used because of their antimicrobial properties in medical devices and in a variety of consumer products. the extensive use of agnps raises concerns about their potential toxicity, although it is still difficult to draw definite conclusions about their toxicity based on published data.our preliminary studies performed to compare the effect of the agnps size (10-40-100 nm) on toxicity, demonstrated that the smallest agnps determine the most severe toxicological effects. in order to best investigate the impact of physicochemical characteristics of 10 nm agnps on toxicity, we compare three different batches of 10 nm agnps slightly different in size distribution (batch a: 8.8±1.7 nm; batch b: 9.4±1.7 nm; batch c: 10.0±1.8 nm).mice were intravenously treated with two doses (5 and 10 mg/kg) of the 3 agnps. 24 hours after the treatment, mice were euthanized and underwent complete necropsy. tissues were collected for histopathological examination and total silver content was determined in tissues by inductively coupled plasma mass spectrometry (icp-ms). all batches induced severe hepatobiliary lesions, i.e. marked hepatocellular necrosis and massive hemorrhage of the gall bladder. the toxicity was dose-dependent and interestingly, the toxic effects were more severe in mice treated with batches a and b that contained smaller agnps.since the total silver mass concentration was similar, the observed batch-dependent toxicity suggest that even subtle differences in size may contribute to relevant changes in the toxicological outcomes, confirming the fundamental involvement of physicochemical features with respect to toxicity. references schäfer b, brocke jv, epp a, götz m, herzberg f, kneuer c, sommer y, tentschert j, noll m, günther i, banasiak u, böl gf, lampen a, luch a, hensel a.state of the art in human risk assessment of silver compounds in consumer products: a conference report on silver and nanosilver held at the bfr in 2012.arch toxicol. 2013 dec;87(12):2249-62. scenihr 2014, nanosilver: safety, health and environmental effects and role in antimicrobial resistance. european commission, 2013, 10.2772/76851 . dose and batch-dependent hepatobiliary toxicity of 10 nm silver nanoparticles after single intravenous administration in mice m. de maglie 1,2 , c. cella 3 , s.bianchessi 2 , s. argentiere 3 , e. scanziani 1,2 , c. recordati 2 1 department of veterinary science and public health, università degli studi di milano, via celoria 10,20133, milan, italy. 2 mouse & animal pathology laboratory, fondazione filarete, v.le ortles 22/4, 20139, milan, italy. 3 advanced biomaterials platform, fondazione filarete, v.le ortles 22/4, 20139, milan, italy. http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=sch%c3%a4fer%20b%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=brocke%20jv%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=epp%20a%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=g%c3%b6tz%20m%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=herzberg%20f%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=kneuer%20c%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=sommer%20y%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=tentschert%20j%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=noll%20m%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=g%c3%bcnther%20i%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=banasiak%20u%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=b%c3%b6l%20gf%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=b%c3%b6l%20gf%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=lampen%20a%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=luch%20a%5bauthor%5d&cauthor=true&cauthor_uid=23779146 http://www.ncbi.nlm.nih.gov.pros.lib.unimi.it/pubmed/?term=hensel%20a%5bauthor%5d&cauthor=true&cauthor_uid=23779146 proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords testudinid herpesvirus, tehv3, reptile immunology, testudo corresponding author marco tecilla marco.tecilla@unimi.it journal home page riviste.unimi.it/index.php/haf characterization of the immune response against testudinid herpesvirus 3: new insight marco tecilla1*, paola roccabianca1, paola pilo2, francesco origgi2,3 1university of milan, department of veterinary medicine, italy 2university of bern, institute of veterinary bacteriology, switzerland 3university of bern, centre for fish and wildlife health, switzerland abstract testudinid herpesvirus 3 (tehv3) is one of the most lethal viral agents in tortoises worldwide. although tehv3 have been extensively studied, only little information is available about hostpathogen interaction. tehv3 infections in different species of the genus testudo correlate with various lesions profiles, disease severity and clinical outcome, suggesting the existence of a complex host-pathogen interaction. this might reflect a possible viral-host coevolution (origgi, 2012). to study the host-pathogen interaction, we previously screened 5.000 clones from a bacteriophage library obtained from the tehv3 genomic dna using testudo graeca seropositive sera. of the six detected positive clones, only one was confirmed by f.a.c.s. selected clone was determined to be a concatamer of different tehv3 genomic fragments including the partial sequence of te17, ul15, major capsid protein (mcp), and glycoprotein b (gb) genes. after complete sequencing of the selected clone, the mcp and the gb were antisenses compared to the phagemid promoter. in order to assess which of the gene fragments among te17 and ul15 was encoding for the antigenic determinant that was recognized by the anti-tehv3 tortoise sera, distinct approaches were followed. te-17 and ul15 fragments were knock out from the original phagemid using the following approaches: a) directed-site mutagenesis, b) molecular cloning, and c) restriction enzymes cloning. all the modified constructs were cloned in two different e. coli cloning vectors (d5α and xl 1-blue). transformation of competent cells with the constructs described above did not yield any viable bacteria. among the different aspects might have influenced transformation success rate, construct size was probably the most relevant (about 9kb). furthermore, we could not entirely exclude that genomic dna editing might have induced mutations in the construct sequence causing toxic effects on the host bacterial cell. cloning of te-17 and ul15 gene fragments into different prokaryotic expression vectors is currently under way. references origgi, f.c., 2012. testudinid herpesviruses: a review. journal of herpetological medicine and surgery. 22, 42–54. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author marzia cozzi, marzia.cozzi@unimi.it abstract canine small clear cell lymphoma is a peculiar lymphoma entity with t-zone histopathological pattern andindolent clinical course. from an immunophenotypic point of view the main feature is the lack of cd45 staining by flowcytometry (fc), which accounts for >95% of cases. underlying mechanisms have never been investigated. aim of this work was to evaluate cd45 protein and mrna expression in small clear cell lymphoma. lymph nodes of 18 cases and 11 controls, with either reactive hyperplasia or cd45-positive high grade t-cell lymphoma, were investigated. fc was performed on lymph node fine needle aspiration and cd45 median fluorescence intensity (mfi) was then evaluated on small clear cells and normal residual t-lymphocytes. cd45 surface expression was also evaluated by immunohistochemical reaction on paraffin wax-embedded lymph node sections. quantitative real-time rt-pcr was performed on cases and controls. total rna was isolated from cell suspension in rna later. the generated cd45cdna was amplified and δδct method was used for the relative mrna quantification. cd45-mfi in neoplastic cells was <1% compared to normal residual t-lymphocytes in the same sample. cells were also negative for cd45 stain on histopathological preparations. rt-pcr showed a significantly lower amount of cd45 transcript in neoplastic samples compared to controls, likely due to the residual population. results showed the lack of cd45 surface antigen and the virtually absence of cd45-mrna in small clear cell lymphoma. we hypothesize a possible genomic/epigenomic aberration; further studies are in progress to investigate the pathogenesis of this aberrancy and the possible linkage to lymphomagenesis. references ponce f, marchal t, magnol jp et al. veterinarypathology 2010;47:414-433;martini v, poggi a, riondato f et al. veterinary and comparative oncology 2013 doi:10.1111/vco.12043 [epub ahead of print]; seelig dm, avery p, webb t et al. journal of veterinary internal medicine 2014;28:878-886; martini v, marconato l, poggi a et al. veterinary and comparative oncology 2015 doi:10.1111/vco.12155 [epub ahead of print]. evaluation of cd45 protein expression and transcript in canine small clear cell/t zone lymphoma. m. cozzi 1 , v. martini 1 , l. marconato 2 ,l. aresu 3 , s. comazzi 1 1 department of veterinary sciences and public health, university of milan, milan, italy 2 centro oncologico veterinario, sasso marconi, bologna, italy 3 department of comparative biomedicine and food science, university of padua, legnaro, padua, italy proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author francesca dell’orco, francesca.dellorco@unimi.it abstract honeybees (apis mellifera) play important roles in modern agriculture regarding zootechnical production and crop pollination. recently, honeybees have received more attention from the public, beekeepers and researchers due to emerging heath issues. thus, scientific interest for honeybee health and selection resistance to major pathogens is sharply increasing. honeybees evolved social immunity mechanisms consisting in the cooperation of individuals to control disease level in the hive, and in particular hygienic behavior (hb), as based on the uncapping and removal of dead, diseased or parasitized brood. hb is affected by heritable and environmental factors, and specific neurogenomic states can be inferred based on the coordinated brain expression of transcription factors and their predicted target genes, including mblk-1 (transcription factor that function in the mushroom body) and obp4 (sensitive olfactory detection in the antennae of adult bees). besides, micrornas are known to influence neurological status linked to age-related social behaviour in honeybees7. in order to investigate the relationship between microrna expression and hb, the present work performed the expression profile of selected honeybee brain microrna in individual’s honeybee from field colonies with high hb level compared to low hb level, in comparison with the expression profile of mblk-1 and obp4. the genetic information resulting from this project could help to understand the role of micrornas in hb and to drive honeybee selection schemes for production, health, and behavioral traits favoring pathogen control. references cremer et al. 2007. social immunity. current biology 17: r693–r702; cremer et al. 2009. philosophical transactions of the royal society of london b364: 129–142; robinson (2004). genomics. beyond nature and nurture. science 304: 397399; chandrasekaran et al. (2011). proceedings of the national academy of sciences 108: 18020-18025;takeuchi et al. (2001). insect molecular biology. 10: 487-494. forêt at al. (2006). genome research 16: 1404-1413. behura et al. (2010). insect molecular biology 19: 431-439. microrna expression correlated with hygienic behaviour in honeybees f. dell’orco 1 , m. loiacono 1 , f. albonico 1 , g. minozzi 1 , g. pagnacco 1 , m. mortarino 1 1 dept. divet università degli studi di milano proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords fip, feline coronavirus, microbiota analysis, immunity corresponding author sara meazzi sara.meazzi@unimi.it journal home page riviste.unimi.it/index.php/haf the gut microbiota and mucosal defenses in cats with coronaviruses: a pilot study sara meazzi1*, stefania lauzi1,2, angelica stranieri1, saverio paltrinieri1,2, alessia giordano1,2 1university of milan, department of veterinary medicine, italy 2university of milan, centro clinico-veterinario e zootecnico-sperimentale, lodi, italy abstract feline infectious peritonitis (fip) develops from a mutation of enteric feline coronaviruses (fcovs) and an imbalance of the host immune response. the wide polymorphism of fcovs is associated with the viral replication rate (licitra et al. 2013). changes in the composition of the gut microbiota may induce quali-quantitative modifications in fcovs and/or different immune profiles (weese et al., 2015). little information is available on feline gut microbiota and its relationship with systemic diseases (ramadan et al., 2014). the aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with fcov infected cats (with and without fip), in order to evaluate whether changes of gut microbiota may induce changes in fcov, in its genetic polymorphism and in the mucosal immunity. screening analyses have been performed on 22 cats: routine hematology and biochemistry on edta and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with fip) nested rt-pcr-3’utr on frozen faeces effusion evaluation fiv/felv serology due to strict inclusion criteria (cats younger than 2.5 years old, indoor and not treated with antibiotics in the previous two months) and based on the results obtained from the complete set of analyses, only 15 cats specifically 5 cats for each of the following 3 groups: fipaffected, healthy negative and positive for fcov have been recruited to perform the following analyses on frozen faeces: microbiota analysis through ngs of 16s rrna gene (v4 region) amplicons followed by bioinformatic analysis evaluation of secretory iga (elisa kit) phylogenetic analysis of fcovs s gene sequences http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 the differences among microbial communities will be compared and associated with the presence and genetic polymorphisms of fcov and mucosal defenses to establish possible significant correlations among these factors and susceptibility to fip. references licitra b.n., millet j.k., regan a.d., hamilton b.s., rinaldi v.d., duhamel g.e., whittaker g.r., 2013. mutation in spike protein cleavage site and pathogenesis of feline coronavirus. emerging infectious diseases. 19(7), 1066-1073 ramadan z., xu h., laflamme d., czarnecki-maulden g., li q.j., labuda j., bourqui b., 2014. fecal microbiota of cats with naturally occurring chronic diarrhea assessed using 16s rrna gene 454-pyrosequencing before and after dietary treatment. journal of veterinary internal medicine. 28, 59-65 weese j.s., nicholsb j., jalalia m., litsterb a., 2015. the rectal microbiota of cats infected with feline immunodeficiency virus infection and uninfected controls. veterinary microbiology. 180, 96-102 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l pyloric stenosis is rarely reported in horses (bart et al 1980; bezdecova et al. 2009). congenital and acquired conditions have been described (church et al 1986; heidmann et al 2004; laing et al. 1992; mcgill et al. 1984) clinical suspicion is based on clinical findings while definitive diagnosis is reached by exploratory laparotomy and gastroscopy. the use of other diagnostic techniques has never been described. two foals were admitted for recurrent abdominal pain. clinical and ultrasonographic (us) examinations were performed. foals underwent computed tomography (ct) of the abdomen, both native and helical hydro-ct. us revealed mild stomach distension, mild small bowel wall thickening; small intestine obstruction was suspected in both foals. case 1, two-month old thoroughbred filly 130 kg of weight: ct showed segmental concentric pylorus stenosis, at the pyloric duodenal junction level. mild liquid distension of the pyloric antrum and mixed gas and fluid distension of the cranial duodenum. during necroscopy the pyloric antrum showed stenosis due to an inelastic constricting ring reducing the lumen of the pyloric canal. the glandular part presented mild acute catarrhal gastritis. case 2, three-month old italian saddle colt 142 kg of weigh: ct showed small amount of intraluminal hyperattenuanting material within the gastric fundus and duodenum. hydro-ct highlighted the presence of mild pylorus narrowing, mild distension and moderate mucosal irregularities of the pyloric antrum. an acquired pyloric stenosis secondary to chronic gastritis of unknown origin was suspected. explorative laparotomy was performed; the antrum was mildly distended and the pylorus appeared narrowed and hard on palpation; gastrojejunostomy was performed. ante-mortem diagnosis of pyloric stenosis in horses is challenging because aspecific clinical signs. native ct keywords pyloric stenosis, horse, ct, contrast agent, hydro-computed tomography. corresponding author vanesssa rabbogliatti vanessa.rabbogliatti@unimi.it journal home page riviste.unimi.it/index.php/haf helical hydro-computed tomography in the diagnosis of pyloric stenosis in two foals. v. rabbogliatti1,*, m. di giancamillo1, d. de zani1, d. gioeni1, f. di cesare1, e.s. d’urso1, g. ravasio1, d. d.zani1 1 department of veterinary medicine, università degli studi di milano via dell’università 6 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 76 haf © 2013 vol. v, no. 1s issn: 2283-3927 allowed to investigate both the stomach and the small intestine and, in one case, outlined the presence of pylorus stenosis. in case 2, only helical hydro-ct allowed better evaluation of the pyloric thickness. ct and helical hydro-ct can be considered a useful diagnostic tool in foal with clinical suspicion of pyloric stenosis. references barth, a.d., barber, s.m., mckenzie, n.t. 1980. pyloris stenosis in a foal. can vet j. 21:234-236. bezdekova, b., hanak, j. 2009. pyloric stenosis in horses: a seven case reports. veterinarni medicina. 54:244-248. church, s., baker, j.r., may, s.a. 1986. gastric retention associated with acquired pyloric stenosis in a gelding. equine vet j. 18:332-334. heidmann, p., saulez, m.n., cebra, c.k. 2004. pyloric stenosis with reflux oesophagitis in a thoroughbred filly. equine vet educ. 16:172-177 laing, j.a, hutchins, d.r. 1992. acquired pyloric stenosis and gastric retention in a mare. aust vet j. 69:68-69 mcgill, c.a., bolton, j.r. 1984. gastric retention associated with a pyloric mass in two horses. aus. vet j. 61:190-195 figure 1: a) hyro-ct scan at level of the pyloric stenosis. pylorus was narrowed and pyloric margins appeared irregular (arrows). (b) in the second foal, the pylorus was regular in shape but mildly narrowed. antral walls were thickened and some heterogeneous hyperdense material was present in the gastric fundus. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords acute phase proteins, inflammation, oxidative stress corresponding author beatrice ruggerone beatrice.ruggerone@unimi.it journal home page riviste.unimi.it/index.php/haf validation of a paraoxon-based method for measurement of paraoxonase (pon-1) activity and establishment of ri in horses beatrice ruggerone1,2*, francesca bonelli3, alessia giordano1,2, irene nocera3, saverio paltrinieri1,2 ,micaela sgorbini3 1university of milan, department of veterinary medicine, italy 2university of milan , veterinary teaching hospital, lodi, italy 3university of pisa, department of veterinary sciences, italy abstract paraoxonase-1 (pon-1) is an anti-oxidant compound considered as negative acute phase protein in animals (rossi et al., 2013) and people (novak et al., 2010). the paraoxon-based method for measurement of pon-1 in equine serum has not yet been validated. the aim of this study is to validate a paraoxon-based method to measure pon-1 and to establish reference intervals (ris) in healthy horses and foals. 120 horses (40 geldings, 40 stallions, 40 mares; median age: 11 years; 57 warmbloods, 46 trotters) and 55 foals (27 females, 28 males; median age: 47 days; 22 warmbloods, 31 trotters) considered healthy after physical examination and biochemistry were examined. horses were grouped by breed: thoroughbreds, trotters, warmbloods, draft horses and ponies. serum pon-1 was measured with an automated spectrophotometer and an enzymatic method validated in other species (giordano et al., 2013). after the analytical validation (precision, accuracy, interference studies), ris were determined using the reference value advisor software, according to ascvp guidelines (friedrichs et al., 2012). the possible gender-, ageand breed-related differences were statistically investigated. the paraoxon-based method was precise (cvs <4.0%) and accurate (p<0.001 in linearity under dilution and spike-recovery testing) but is affected by interference from mild bilirubinemia, severe lipemia or hemoglobinemia. the ris recorded in the whole population was 38.1-80.8 u/ml. according to the harris and boyd test, separate ris are recommended only for adult females and for warmblood and trotter adults (figure 1). this study demonstrated that analytical performances of the paraoxon-based method for measurement of pon-1 in horses are acceptable. pon-1 is lower in horses than in other species. if future studies will demonstrate that oxidative stress induces a significant decrease of pon-1, this results will be useful to correctly classify healthy and sick horses; pon-1 could be used, as in human medicine, as a marker of oxidative stress. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig. 1: comparison of results obtained in adult horses vs foals, stallions vs mares vs geldings, trotters vs warmblood adult horses, male vs female foals, and trotter vs warmblood foals. the boxes indicate the i–ii interquartile range (iqr), the horizontal line indicates the median values, whiskers extend to further observation within quartile i minus 1.5 × iqr or to further observation within quartile iii plus 1.5 × iqr. ‘+’ indicates near outliers (i.e., values exceeding quartiles i or ii i minus or plus 1.5 × iqr). the asterisk indicates a significant difference (mares vs stallions and geldings; adult trotters vs warmbloods). references friedrichs, k.r., harr, k.e., freeman, k.p., szladovitz, b., walton, r.m., barnhart, k.f., blanco-chavez. j. 2012. asvcp reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. vet clin pathol. 41, 441-453. giordano, a., veronesi, m.c., rossi, g., pezzia. f., probo, m., giori, l., paltrinieri, s.,2013. serum paraoxonase-1 activity in neonatal calves: age related variations and comparison between healthy and sick animals. vet j.197, 499-501. novak, f., vavrova, l., kodydkova, j., novak, f.s., hynkova, m., zak, a., novakova, o. 2010. decreased paraoxonase activity in critically ill patients with sepsis. clin exp med. 10, 21-25. rossi g, giordano a, pezzia f, kjelgaard-hansen m, paltrinieri s. 2013. serum paraoxonase 1 activity in dogs: preanalytical and analytical factors and correlation with c-reactive protein and alpha-2-globulin. vet clin pathol. 42, 329-341. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords epigenetic conversion; pancreatic differentiation, matrices; 3-d culture systems; oxygen tension corresponding author alessandro zenobi alessandro.zenobi@unimi.it journal home page riviste.unimi.it/index.php/haf matrix stiffness and oxygen tension modulate epigenetic conversion of mouse dermal fibroblasts into insulin producing cells alessandro zenobi*1, fulvio gandolfi2 and tiziana a.l. brevini1 1university of milan, department of health, animal science and food safety, italy 2university of milan, department of agricultural and environmental sciences production, landscape, agroenergy, italy abstract in vivo, cells are surrounded by a three-dimensional (3-d) organization of supporting matrix, neighboring cells and a gradient of chemical and mechanical signals (antoni, et al., 2015). however, the present understanding of many biological processes is mainly based on two-dimensional (2-d) systems that typically provides a static environment. in the present study, we tested two different 3d culture systems and apply them to the epigenetic conversion of mouse dermal fibroblasts into insulin producing-cells (pennarossa, et al., 2013; brevini, et al., 2015), combining also the use of two oxygen tensions. in particular, cells were differentiated using the polytetrafluoroethylene microbioreactor (ptfe) and the polyacrylamide (paa) gels with different stiffness (1 kpa; 4 kpa), maintained either in the standard 20% or in the more physiological 5% oxygen tensions. standard differentiation performed on plastic substrates was assessed as a control. cell morphology (fig.1a), insulin expression and release were analyzed to evaluate the role of both stiffness and oxygen tension in the process. the results obtained showed that 1 kpa paa gel and ptfe system induced a significantly higher insulin expression and release than plastic and 4 kpa paa gel, especially in low oxygen condition (fig.1b). furthermore, comparing the efficiency of the two systems tested, 1 kpa paa gel ensured a higher insulin transcription than ptfe (fig.1c). recent studies show the direct influence of substrates on lineage commitment and cell differentiation (engler, et al., 2006; evans, et al., 2009). the evidence here presented confirm that the use of an appropriate stiffness (similar to the pancreatic tissue), combined with a physiological oxygen tension, promote β-cell differentiation, with beneficial effects on cell functional activity and insulin release. the present results highlight the importance of 3-d cell rearrangement and oxigen tension to promote in vitro epigenetic conversion of mouse fibroblasts into insulin-producing cells. acknowledgments: the research was funded by carraresi foundation. authors are members of the cost actions bm1308, cm1406 and ca16119. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: a) representative image of cells differentiated in ptfe (20% and 5% oxygen) and on paa gels (20% and 5% oxygen); b) insulin release in all samples after hyperglycemic stimulation with 20 mm of glucose. different superscripts indicate statistical differences among the samples (spss software, p≤0,05). insulin release is expressed as mean value ± sd; c) insulin expression in the more efficient experimental groups (ptfe 5% o2; 1 kpa 5% o2). different superscripts indicate statistical differences between the samples (spss software, p≤0,05). gene expression levels are reported with the highest expression set to 1 and the other relative to this. references antoni, d., burckel, h., elodie josset, e. and noel, g., 2015. three-dimensional cell culture: a breakthrough in vivo. international journal of molecular sciences, 16(3): 5517–5527; brevini, t.a.l., pennarossa, g., maffei, s., zenobi, a., gandolfi, f., 2015. epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts. jove, 109, e53880; engler, a.j., sen, s., sweeney, h.l. and discher d.e., 2006. matrix elasticity directs stem cell lineage specification. cell, 126(4): p. 677-89; evans, n.d., minelli, c., gentleman, e., lapointe, v., patankar, s.n., kallivretaki, m., chen, x., roberts, c.j. and stevens, m.m., 2009. substrate stiffness affects early differentiation events in embryonic stem cells. european cells & materials, 18: p. 1-13; discussion 13-4; pennarossa, g., maffei, s., campagnol, m., tarantini, l., gandolfi, f., brevini, t.a.l. 2013. brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. proceedings of the national academy of sciences, 110(22), 8948–8953. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords horse, local anaesthesia, nerve block, peribulbar anaesthesia, computed tomography corresponding author vanessa rabbogliatti vanessa.rabbogliatti@unimi.it journal home page riviste.unimi.it/index.php/haf abstract peribulbar block (ppb) has been used in humans as a safer alternative to retrobulbar block (rbb). pbb, depends on the diffusion of anaesthetic solution into the muscle across the connective tissue and it is performed introducing the needle within the extraconal space. the advantages are fewer complications and palpebral akinesia. in veterinary medicine few studies describe this technique in dogs (ahn j. et al., 2013) and cats (shilo-benjamini et al., 2013). based on literature the aim of the study is to determinate, in equine specimens, feasibility of inferior pbb with single needle injection, by using contrast medium (cm), and to evaluate thought computed tomography (ct) the distribution around the optic nerve (degrees). pbb was performed in 6 orbits. the mixture injected consisted of 20 ml of physiological solution and iodinated cm at 25%. each periorbital area underwent three ct scans. a basal acquisition to assess the needle position before the injection, a second and third scan were performed immediately after injection, and after application of pressure on the periorbital surface area to promote cm diffusion. the needle position was measured from the tip to the optic nerve with a mean distance of 2,27 mm ± 0,28. the mean volume distribution before pressure application was 23,56 cm3 ± 2,58 and after pressure application was 27,56 cm3 ± 4,8. the cm distribution, was defined (nouvellon et al., 2010) “successful” in 4 orbits (>270°) and “inadequate” in 2 orbits (<180°). the present study demonstrates feasibility of inferior pbb by single injection in horses for its simple and practical execution. inferior ppb is a potential alternative to systemic administration of neuromuscular blocking agents for ophthalmic surgery. however, this approach needs to be evaluated in clinical trials to assess its feasibility and effectiveness in clinical practice for standing procedures. references ahn, j., jeong, m., lee, e., et al. 2013. effects of peribulbar anesthesia (sub-tenon injection of a local anesthetic) on akinesia of extraocular muscle, mydriasis, and intraoperative and postoperative analgesia in dogs undergoing phacoemulsification. am j vet res 74: 1126-1132 shilo-benjamini, y., pascoe, p.j., maggs, d.j. et al. 2013. retrobulbar and peribulbar regional techniques in cats: a preliminary study in cadavers. vet anaesth analg 40, 623-631 nouvellon, e., cuvillon, p., ripart, j., viel, e.j. anaesthesia for cataract surgery; drugs and aging, 2010; vol. 10. issue 1 peribulbar block in equine isolated heads. development of a single needle technique and tomographic evaluation vanessa rabbogliatti1*; mauro di giancamillo1; donatella de zani1, daniela gioeni1, federica di cesare2, elisa silvia d’urso1, davide d. zani1, ravasio giuliano1 1 university of milan, department of veterinary medicine, italy 2university of milan, department of health, animal science and food safety, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords staphylococcus pseudintermedius, molecular typing, ribosomal spacers amplification, restriction fragment length polymorphism, random amplification of polymorfic dna corresponding author gabriele meroni gabriele.meroni@unimi.it journal home page riviste.unimi.it/index.php/haf molecular typing of staphylococcus pseudintermedius canine strains by three commonly used techniques gabriele meroni1*, stefano morandi2, milena brasca2, piera a. martino1 1 university of milan, department of veterinary medicine, italy 2 institute of sciences of food production (ispa – cnr ), italy abstract staphylococcus pseudintermedius is a newly described species of staphylococcus regarded as the main causative agent of canine pyoderma (devriese et al., 2005). s. pseudintermedius infection was recently described in humans. an important feature of this pathogen is the high genetic identity with two other species of staphylococci, namely s. intermedius and s. delphini, which are included all together in the staphylococcus intermedius group (sig) (fitzgerald, 2009). this scenario seriously hampers phenotypic differentiation of these three pathogens. despite this, only in 2008 was described the first molecular protocol for diagnostic identification of s. pseudintermedius (bannoehr et al., 2009). the aim of this work was to investigate the presence of different biotypes of s. pseudintermedius obtained from clinically relevant cases of pyoderma in dogs using three molecular methods commonly used to type bacteria: the ribosomal spacers amplification (rsa), the random amplification of polymorphic dna (rapd) and the restriction fragment length polymorphism (rflp). a total of 46 different strains were included in this work. the application of the rsa technique, which was applied here for the first time, identified the presence of s. pseudintermedius, although it did not allow any differentiation between biotypes. the rapd assay showed a single cluster that assembles all the interested strains that are grouped in three different sub-clusters (fig. 1). the rflp technique showed the most discriminative power, providing the opportunity to clearly identify this bacterium. in conclusion, the use of these three different techniques allows to clearly identify s. pseudintermedius and to observe the presence of different biotypes. in future it could be interesting to couple these results with the determination of the antibiotic resistance in order to verify if certain multi drug resistant strains have particular rsa and rapd profiles. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig. 1: dendrogram derived from the random amplification of polymorphic dna (rapd-pcr) profiles generated with primers m13 and opa a10. the rapd-pcr profile grouping was done with the gel compar 4.1 software package using the pearson productmoment correlation coefficient and unweighted pair group method with arithmetic mean (upgma) cluster analysis. references bannoehr j, franco a, iurescia m, battisti a, fitzgerald jr., 2009. molecular diagnostic identification of staphylococcus pseudintermedius. journal of clinical microbiology. 47(2), 469-471. devriese, l. a., vancanneyt, m., baele, m., vaneechoutte, m., de graef, e., snauwaert, c., haesebrouck, f., 2005. staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals. international journal of systematic and evolutionary microbiology. 55 (pt 4), 1569-1573. fitzgerald, j. r., 2009. the staphylococcus intermedius group of bacterial pathogens: species re-classification, pathogenesis and the emergence of meticillin resistance. veterinary dermatology. 20, 490-495. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords dexmedetomidine, methadone, trans-mucosal, pharmacokinetics, dog corresponding author federica di cesare federica.dicesare@unimi.it journal home page riviste.unimi.it/index.php/haf abstract oral-transmucosal (otm) drug delivery refers to noninvasive and painless administration of medical preparations through any oral cavity membrane to achieve systemic effects (sattar et al., 2014). regarding sedative drugs, otm administration is very attractive in veterinary medicine, especially for patients difficult to inject and restrain (messenger et al., 2016). this study aims to compare the pharmacokinetics of dexmedetomidine after otm and intramuscular (im) administration combined with methadone. after obtaining ethical committee approval and owner’s written consent, eight dogs, were administered with dexmedetomidine (10 g/kg) and methadone (0.4 mg/kg) by otm and other 4 dogs by im route. blood samples were collected at prefixed times up to four hours. dexmedetomidine was quantified by a validated hplc-ms method. on dexmedetomidine concentrations, a pharmacokinetic analysis was carried out with a noncompartmental approach (phoenix winnonlin® 7.0, pharsight, cary, nc). mean ± sd terminal half-lives of dexmedetomidine were 187.42 ± 109.66 and 94.78 ± 34.08 min after otm and im administration, respectively. maximum serum (cmax) concentrations were 0.83 ± 0.32 and 9.09 ± 2.46 ng/ml for otm and im administration, respectively. time to maximum concentration (tmax) were 44.38 ± 32.16 and 21.25±11.39 min by otm and im administration, respectively. area under the curve from 0 to the last measured concentration (auclast) were 103.75 ± 30.23 and 614.87 ± 77.15 min*ng/ml for otm and im administration, respectively. cmax, tmax and auclast values by otm route demonstrate a lower and delayed absorption of the drug compared to im. to complete the study, the pharmacokinetic analysis of methadone is foreseen, so as a clinical trial to compare the clinical effects of the combination of dexmedetomidine and methadone by otm and im administration and to establish an effective dosage of oral-transumucosal route in dogs for this association. references messenger, k., m., hopfensperger, m., knych, h., k., papich, m., g., 2016. pharmacokinetics of detomidine following intravenous or oraltransmucosal administration and sedative effects of the oral-transmucosal treatment in dogs. american journal of veterinary research. 77, 413420. sattar, m., sayed, o., m., lane, m., e., 2014. oral transmucosal drug delivery – current status and future prospects. international journal of pharmaceutics. 471, 498-506. pharmacokinetics of dexmedetomidine combined with methadone following oral-transmucosal and intramuscular administration in dogs federica di cesare1*, petra cagnardi1, daniela gioeni2, elisa s. d’urso2, vanessa rabbogliatti2, lorena lucatello3, roberta merlanti3, giuliano ravasio2, roberto villa1 1 university of milan, department of health, animal science and food safety, italy 2university of milan, department of veterinary medicine, italy 3 university of padua, department of comparative biomedicine and food science, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords honeybee, hygienic behavior, gene expression, brain corresponding author francesca dell’orco francesca.dellorco@unimi.it journal home page riviste.unimi.it/index.php/haf abstract honeybees (apis mellifera) evolved social immunity mechanisms based on coordinated behavioral mechanisms for reducing the spread of pathogens and parasites. in particular, hygienic behavior (hb) is an actively participation of family defense by uncapping and removal of dead, diseased or parasitized brood (cremer et al., 2007). currently, hb evaluation is performed only by in-field empirical assays, and suitable molecular markers that could allow the discrimination between families with high and non-high hb score are not yet available. the research activity was characterized by a transversal approach: from in-field phenotypic characterization, to expression profiling of selected coding genes in honeybee brains. the expression analysis was performed on pools of 15 days-old honeybees collected in spring 2017 from 10 different colonies (5 with high hb and 5 with low hb scores). the brains were dissected and total rna was purified. after quantitation and quality evaluation, the extracted rnas were reverse transcribed to cdnas and real time pcr was performed on different target genes (act5c, mblk-1, obp3, obp4, obp16 and obp18) previously showed by transcriptomic and proteomic studies to be involved in hb (cristino et al., 2014; takeuchi et al., 2001; chandrasekaran et al., 2011). the comparative analysis of rt-pcr threshold cycles (ct) showed that mblk-1 was the least expressed gene among the analyzed targets. most importantly, statistical analysis of expression data validated (p<0.05) the delta-ct between obp4 and act5c as a predictive index of high vs low hb score. these results confirmed the findings obtained through a preliminary experiment performed in spring 2016 (dell’orco et al., 2016). references chandrasekaran s., amentb, s. a., eddyc, j. a., rodriguez-zasd s. l., schatzb, b. r., pricea, n. d., robinsonb, g. e., 2011. behavior-specific changes in transcriptional modules lead to distinct and predictable neurogenomic states. proc. of the national academy of sciences 108, 18020-18025. cremer, s., armitage, s. a. o., schmid-hempel, p., 2007. social immunity. social immunity. current biology 17, r693–r702. cristino, a. s., barchuk, a. r., freitas, f. c. p., narayanan, r. k., biergans, s. d., zhao, z., simoes, z. l. p., reinhard, j., claudianos, c., 2014. neuroliginassociated microrna-932 targets actin and regulates memory in the honeybee. nature communications. 5, 5529. takeuchi, h., kage†, e., sawata, m., kamikouchi, a., ohashi, k., ohara, m., fujiyuki, t., kunieda, t., sekimizu, t., natori, .s., kubo, t., 2001. identification of a novel gene, mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type kenyon cells of the honeybee brain. insect molecular biology. 10, 487-494. dell’orco, f., facchini, e., cilia, g., rizzi, r., mortarino, m., 2016. candidate molecular markers of hygienic behaviour in honeybees (apis mellifera): an expression study. international journal of health, animal science & food safety, vol. 2s, proc. of the veterinary and animal science days, 8-10 june 2016 obp4 and act5c gene expression is related to hygienic behavior in honeybee families francesca dell’orco1*, elena facchini1, rita rizzi1, michele mortarino1 1 university of milan, department of veterinary medicine, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords tumor microenvironment, mouse models, histology, immunohistochemistry, digital image analysis corresponding author lucia minoli lucia.minoli@unimi.it journal home page riviste.unimi.it/index.php/haf tumor microenvironment in experimental models of human cancer: morphological investigational approaches lucia minoli1,2* , eugenio scanziani1,2 1university of milan, department of veterinary medicine, italy 2fondazione filarete, mouse and animal pathology laboratory (maplab), milan, itlay abstract tumor microenvironment (tme) is defined as the non-tumoral part of tumors. it is composed of different cell populations and structures (such as tumor-associated vasculature, immuneinflammatory cells, fibroblasts). tme could either promote or antagonize tumor growth and has a great potential as target for novel therapeutic strategies (hanahan and coussens, 2012). along with several methods (i.e. molecular assays), morphological techniques allow to evaluate the components of tme in the setting of their action. the aim of this work was to define valuable morphological approaches useful to investigate the tme. histological and immunohistochemical techniques, along with digital image analysis, were tested on experimental mouse models (both xenograft and genetically engineered mice) of four human tumors (ovarian cancer, pancreatic ductal adenocarcinoma (pdac), colon adenocarcinoma, thyroid carcinoma). concerning the vascular compartment, cd31 immunostaining and double-immunofluorescence with cd31 and α-sma (pericytes marker) allowed to respectively quantify vessels and evaluate their maturation degree. immunohistochemical detection of previously administrated pimonidazole, revealed variable extended areas of hypoxia in a consistent pattern between frozen and formalinfixed paraffin-embedded samples. concerning the stromal component, anti-human mhc i and species-specific markers for vimentin demonstrated the host-derivation of stroma in xenotumors, while sirius red histochemical staining allowed the quantification of desmoplasia in models of pdac. concerning immune-inflammatory cells, an immunohistochemical panel with cd3 (t-lymphocytes), b220 (b-lymphocytes), mpo (neutrophils) and iba-1 (macrophages), showed high reliability in characterizing the tumoral infiltrate. moreover, the application of markers specific for different macrophage subsets confirmed the higher prevalence of m2 (arginase i positive) on m1 (inos positive) macrophages. ym1 demonstrated low performance in detecting the m2 population (fig. 1). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 due to the microenvironmental heterogeneity which influence tumor development and behavior, a sole quantification is unreliable for characterizing the tme. considering that, morphological techniques proved to be a valuable approach, allowing the evaluation of spatial distribution and mutual interaction between the different elements. acknowledgments the authors wish to thanks dr. giavazzi (irccs-istituto di ricerche farmacologiche mario negri, milan, italy), dr. alessandra capuano (centro di riferimento oncologico, aviano (pn), italy), dr. greco (irccs-istituto nazionale dei tumori, milan, italy) and their teams. fig.1: mouse, subcutaneous xenograft of thyroid carcinoma, same area, 200x. hematoxylin&eosin (a). immunohistochemistry for iba-1 (b) revealed a macrophage infiltrate, which turn out to be also arginase i positive (c), demonstrating the prevalent m2 polarization of tumor-associated macrophages. conversely, immunohistochemistry for inos (d) show the absence of m1-polarized macrophages. references hanahan, d. and coussens, l. m., 2012. accessories to the crime: functions of cells recruited to the tumor microenvironment. cancer cell. 21, 309-322 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords pesticides residues, gc-ms/ms, food safety, environmental contaminants corresponding author giuseppe labella giuseppe.labella@unimi.it journal home page riviste.unimi.it/index.php/haf analyses of organochlorine pesticides residues in eels (anguilla anguilla) from lake garda using gas chromatography coupled with tandem mass spectrometry (gc-ms/ms) giuseppe f. labella1*, luca m. chiesa1, sara panseri1, francesco arioli1 1university of milan, department of health, animal science and food safety, italy abstract lake garda is located in one of the most populated and industrialized area of italy (camusso et al., 2001). therefore, the lake water, and also the fish species present, could be affected by environmental contamination. european eels (anguilla anguilla) are considered as suitable matrix for biomonitoring environmental contaminants in european water (belpaire et al., 2007), being widespread in many european waters and highly contaminated by lipophilic compounds, due to the high lipid content (up to 40%) (larsson et al., 1991). moreover, eel is an edible species (its farming currently supplies approximately 45,000 tons/year) (nielsen et al., 2008), so it also represents a public health issue. based on these considerations, the aim of this study was to evaluate the occurrence of fourteen organochlorine pesticides (ocs) in forty-five eels (anguilla anguilla) from lake garda, using accelerated solvent extraction (ase) procedure for the analytes extraction and gas chromatography coupled with tandem mass spectrometry (gc-ms/ms) for the analysis of ocs. gc-ms/ms analysis was developed and validated according to the sante/11945/2015 guidelines. uncontaminated eel sample (previously checked for the presence of ocs and considered blank with a concentration of compounds < limit of detection) were used for all procedure's optimization steps. for all the ocs, satisfactory results were achieved in terms of linearity (r2 higher than 0.985); recovery (ranging between 70–120 %) and repeatability (coefficient of variation % lower than 20 %). the results met the validation criteria required by eu guidelines. regarding eel samples, several pesticides were detected, but ddt and its metabolites were found with the highest prevalence (92 %). the concentration range was from not detected (n.d.) to 19000 ng g-1. although ddts levels in the environment are declining (albaiges et al., 2011), they continue to bioaccumulate in tissues of human and animal and biomagnify in food chains. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references camusso, m., balestrin, r., binelli, a. 2001. use of zebra mussel (dreissena polymorpha) to assess trace metal contamination in the largest italian subalpine lakes. chemosphere. 44, 263-270. belpaire, c., goemans, g. 2007. the european eel anguilla anguilla, a rapporteur of the chemical status for the water framework directive? vie milieu. 57, 235–252. larsson, p., hamrin, s., okla, l. 1991. factors determining the uptake of persistent pollutants in a eel population (anguilla anguilla l.). environ pollut. 69, 39-50. nielsen, t., prouzet, p. 2008. capture-based aquaculture of the wild european eel (anguilla anguilla). in a. lovatelli & p. f. holthus (eds.), capture-based aquaculture. global overview. fao fisheries technical paper. no. 508 (pp. 141– 168). rome: fao. albaiges, j., murciano, c., pon, j., 2011. hazardous substances in the mediterranean: a spatial and temporal assessment. in: unep/map, consultation meeting to review med pol monitoring activities athens, 22 e 23 november 2011, p. 106. annex ii. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords bone, histomorphometry, densitometry, microarchitecture, pig corresponding author maria elena andreis mariaelena.andreis@unimi.it journal home page riviste.unimi.it/index.php/haf swine cortical and cancellous bone: histomorphometric and densitometric characterisation maria e. andreis1*, marco cummaudo2, umberto polito1, alberto m. luciano1, cristina cattaneo2, mauro di giancamillo3, alessia di giancamillo1, silvia c. modina1 1university of milan, health, animal science and food safety, italy 2university of milan, department of biomedical science for health, italy 3university of milan, department of veterinary medicine, italy abstract swine bone morphology, composition and remodelling are similar to humans’, therefore they are considered good models in bone-related research (wancket et al., 2015; rubessa et al., 2017). they have been used for several studies involving bone growth, bone and cartilage fractures and femoral head osteonecrosis. nevertheless, the literature about pig normal bone features is incomplete (teo et al., 2005). this work aims to fill the literature gaps on the microarchitecture and bone mineral density (bmd) of swine femoral diaphysis and distal epiphysis and tibial plateau and diaphysis. five hind limbs were collected from slaughtered 80-100 kg pigs. microscopic analysis of cortical and cancellous bone from middle/distal femur and proximal/middle tibia was performed to determine basic histomorphometric parameters at different sites. dual-energy x-rays absorptiometry was also employed to evaluate bmd. anova and correlation between bmd, bone area (ba) and cortical thickness were performed. diaphyseal cortical bone was mostly plexiform both in the tibia and the femur; primary/secondary osteons without clear organization were also found. mean values for bone area, bone perimeter, trabecular width, number and separation and bmd at different anatomical sites were defined. no significant difference was found for these values at different anatomical sites. bmd proved to be positively correlated with cortical thickness (r=0,80; p<0,01). despite the small sample size, these results seem homogeneous. they could therefore represent reference values for normal bone parameters in pigs. applied anatomy and regenerative medicine, in fact, demand very precise information about bone micromorphology, composition and density to provide reliable indication in bone substitutes building. moreover, since the interpretation of bone abnormalities grounds on mastering normal bone characteristics, the definition of reference parameters is mandatory to avoid misinterpretation and allow comparative evaluation. the results of this study, although preliminary, may be considered a dependable starting point for the definition of normal bone features in pigs. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references rubessa m., polkoff k., bionaz m., monaco e., milner d.j., hollister s.j., goldwasser m.s., wheeler m.b., 2017. use of pig as model for mesenchymal stem cell therapies for bone regeneration. animal biotechnology mar (7),1-13. teo, j.c.m., shi-hoe, k.m., keh, j.e.l., teoh, s.h., 2005. relationship between ct intensity, micro-architecture and mechanical properties of porcine vertebral cancellous bone. clinical biomechanics 21, 235-244. wancket l.m., 2015. animal models for evaluation of bone implants and devices: comparative bone structure and common model uses. veterinary pathology 52(5), 842-850. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l quadriceps contracture is a debilitating and uncommon condition, mostly affecting young dogs. it can be congenital (stead et al., 1977) or acquired (leighton, 1981; bardet and hohn, 1983; 1984) and is reported to induce muscular hypotrophy/fibrosis, progressive degenerative joint disease, bone hypoplasia and limb hyperextension (bardet and hohn, 1983; 1984). the aim of this study was to elucidate anatomic, tomographic and biomechanical features of stifles affected by quadriceps contracture. seven 2-month-old dead dobermann pinschers with unilateral quadriceps contracture were included. before gross anatomic evaluation, all stifles underwent computed tomography before and after intra-articular administration of iodinated contrast medium (samii et al., 2004). images were acquired in double positioning (stifle extension and flexion) to identify articular cartilage, ossification centres’ (ocs) and menisci abnormalities, which were compared between affected and unaffected limbs (figure 1). in all affected limbs the stifle was back-turned, the distal femur was extra-rotated and the patella was luxated proximo-medially. severe lack of physiological stifle movements (rolling, gliding, spinning) was observed, so that affected joints could not be flexed. the articular cartilage of the femur was flattened and irregular in thickness, the femoral trochlea was hypoplasic and sloping, the menisci were misshaped. the oc of the distal femur and proximal tibia were misshaped; the tibial plateau was oriented caudodorsally-cranioventrally and significantly smaller (p<0,05). quadriceps contracture influenced stifle development. the action of quadriceps insertion on the tibia prevented normal development of the plateau, causing wedging and abnormal orientation. constant compression also induced external rotation of the distal femur (unable to develop distally) and patellar luxation, ending up in genu recurvatum. static compression was likely responsible for femoral trochlea hypoplasia, articular cartilage and meniscal deformation, due to the lack of physiological stifle movements. keywords quadriceps contracture, puppies, stifle, skeletal development, anatomy. corresponding author maria elena andreis mariaelena.andreis@unimi.it journal home page riviste.unimi.it/index.php/haf stifle anatomic, tomographic and biomechanical features of growing dogs affected by quadriceps contracture. m.e. andreis1,*, m. di giancamillo2, l.m. carnevale2, u. polito1, m.c. veronesi2, a. di giancamillo1, s.c. modina1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 54 haf © 2013 vol. v, no. 1s issn: 2283-3927 quadriceps contracture induces severe alterations of stifle development in affected puppies. histology, histochemistry and immunohistochemistry may better define the nature of such bone, cartilage and meniscal alteration. references bardet, j.f., hohn, r.b., 1983. quadriceps contracture in dogs. journal of the american veterinary medical association sep;183(6):680-685. bardet, j.f., hohn, r.b., 1984. subluxation of the hip joint and bone hypoplasia associated with quadriceps contracture in young dogs. journal of the american animal hospital association may/jun;20(3):421-428. leighton, r.l., 1981. quadriceps contracture (ischemic contracture of the quadriceps), in bojrab m (ed): pathophysiology in small animal surgery. philadelphia, lea and febiger, pp550-552. samii, v.f., dyce, j., 2004. computed tomographic arthrography of the normal canine stifle. veterinary radiology&ultrasound 45:402–406. stead., a.c., camburn, m.a., gunn, h.m., kirk, e.j., 1977 congenital hindlimb rigidity in a dog. journal of small animal practice 18:39-46. figure 1: gross anatomic evaluation of the stifle in a puppy affected by unilateral quadriceps contracture (distal epiphysis of the femur). a: affected stifle; b: unaffected stifle. in the affected stifle the femoral condyles are flattened, the trochlea is hypoplasic and sloping (black arrow). m: medial; l: lateral; edpl: extensor digitorum pedis longus. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l recently, many studies in livestock have focused on the identification of copy number variants (cnvs) using high-density single nucleotide polymorphism (snp) arrays, but few have focused on studying chicken ecotypes coming from many locations. cnvs are polymorphisms, which may influence phenotype and are an important source of genetic variation in populations (henrichsen et al., 2009). the aim of this study was to explore the genetic difference and structure, using a high density snp chip in 936 individuals from seven different countries (brazil, italy, egypt, mexico, rwanda, sri lanka and uganda). the dna was genotyped with the affymetrix axiom®600k chicken genotyping array and processed with stringent quality controls to obtain 559,201 snps in 915 individuals (stranger et al., 2007). the log r ratio (lrr) and the b allele frequency of snps were used to perform the cnv calling with penncnv software based on a hidden markov model analysis and the lrr was used to perform cnv detection with svs golden helix software. after filtering, a total of 19,027 cnvs were detected with the svs software, while 9,065 cnvs were identified with the penn cnv software. the cnvs were summarized in 7,001 copy number variant regions (cnvrs) and 4,414 cnvrs, using the software bedtool. the consensus analysis across the cnvrs allowed the identification of 2,820 consensus cnvr, of which 1,721 were gain, 637 loss and 462 complex, for a total length of 53 mb corresponding to the 5 % of the galgal5 chicken autosomes (table 1). only the consensus cnv regions obtained from both detections were considered for further analysis. the intersection analysis performed between the chicken gene database (gallus_gallus-5.0) and the 1,927 consensus cnvrs allowed the identification (within or partial overlap) of a total of keywords snp, cnv, genetic variability, chicken. corresponding author erica gorla erica.gorla@unimi.it journal home page riviste.unimi.it/index.php/haf genomic diversity using copy number variations in worldwide chicken populations. e. gorla1,2*, f. bertolini2, m.g. strillacci1, m.c. cozzi1, s.i. roman-ponce3, f.j. ruiz3, v.v. vega3, c.m.b. dematawewa4, d. kugonza5, a. elbeltagy6, c.j. schmidt7, s.j. lamont2, a. bagnato1, m.f. rothschild2 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milano, italy. 2 department of animal science, iowa state university, ames, iowa, usa. 3 instituto nacional de investigaciones forestales, agrícolas y pecuarias. 4 university of peradeniya, faculty of agriculture, department of animal science, 20400, sri lanka. 5 department of animal science, makerere university, kampala, uganda. 6 animal production research institute, department of animal biotechnology, dokki, cairo, egypt. 7 university of delaware, animal and food sciences, newark, de, usa. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 50 haf © 2013 vol. v, no. 1s issn: 2283-3927 2,354 unique genes with an official gene id (quinlan et al., 2010). the cnvrs identified here represent the first comprehensive mapping in several worldwide populations, using a highdensity snp chip. references stranger, b.e., forrest, m.s., dunning, m., ingle, c.e., beazley, c., et al., 2007. relative impact of nucleotide and copy number variation on gene expression phenotypes. science 315: 848–853. henrichsen, c.n., vinckenbosch, n., zollner, s., chaignat, e., pradervand, s., schutz, f., et al., 2009. segmental copy number variation shapes tissue transcriptomes. nat. genet. 41:424–429. doi: 10.1038/ng.345. quinlan, a.r., hall, i.m., 2010. bedtools: a flexible suite of utilities for comparing genomic features. bioinformatics 26, 841–842. doi: 10.1093/bioinformatics/btq033. http://penncnv.openbioinformatics.org/en/latest. http://goldenhelix.com. table 1: summary statistic of cnvr identified with of penncnv, svs software and consensus, divided according state into loss, gain or complex. cnvr count min length (bp) max length (bp) mean length (bp) total length (bp) coverage (%) svs all 7,001 493 571,113 15,541.61 108,806,878 11.68 gain 4,640 493 571,113 14,743.86 68,411,533 7.34 loss 1,573 699 146,115 11,288.70 17,757,126 1.9 complex 788 1,955 562,765 28,728.70 22,638,219 2.43 penncnv all 4,414 122 2,281,744 51,853.14 228,879,785 17.8 gain 3,281 122 750,953 47,193.85 154,843,028 12.04 loss 731 149 181,129 11,661.74 8,524,736 0.66 complex 402 472 2,281,744 162,965.22 65,512,021 5.09 consensus all 2,820 652 562,765 19,072.43 53,784,265 5.77 gain 1,721 652 544,758 17,181.11 29,568,693 3.17 loss 637 1,102 146,115 12,948.43 8,248,151 0.88 complex 462 2,696 562,765 34,561.51 15,967,421 1.71 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en l keywords cholesterol, cholesterol oxidation products, analysis, health diseases. pages 13 – 39 references vol. 5 no. 1 (2018) article history submitted: december 08, 2017 accepted: march 02 2018 published: march 03 2018 corresponding author bhavbhuti m. mehta dairy chemistry department, smc college of dairy science, anand agricultural university (aau), anand, gujarat, india mail: bhavbhuti5@yahoo.co.in . bhavbhutimehta@gmail.com phone: +91 9825807454 journal home page riviste.unimi.it/index.php/haf review cholesterol and its oxidation products: occurrence and analysis in milk and milk products krupaben m. shingla 1 and bhavbhuti m. mehta 1,* 1 dairy chemistry department, smc college of dairy science, anand agricultural university (aau), anand, gujarat, india abstract the cholesterol is one of the important components of biological membranes. it is associated with milk fat in milk and milk products. cholesterol present in animal origin foods undergoes autoxidation during processing as well as during storage yielding toxic products commonly known as cholesterol oxidation products (cops). the cops are significantly affected the human health such as atherosclerosis, inflammation, cancer, neurodegenerative diseases etc. various methods are reported in literature for determination of cholesterol and its oxidation products in milk and milk products. mailto:bhavbhuti5@yahoo.co.in mailto:bhavbhutimehta@gmail.com shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 13 haf © 2013 vol. v, no. 1 issn: 2283-3927 1 introduction the name cholesterol was derived from two greek words chole (means bile) and stereos (means solid). cholesterol is one of the components of biological membranes. cholesterol is found in all body tissues especially the brain and spinal cord (raguz et al., 2011). it is also found in many of the animal food products like eggs, dairy products, poultry, fish, lard and other fats. cholesterol, an unsaturated alcohol, undergoes oxidation resulting in the formation of toxic products. cholesterol undergoes auto-oxidation, photo-oxidation and enzymaticoxidation, producing relevant hydroperoxides (ubhayasekera et al., 2004). cholesterol oxidation products (cops) are similar to cholesterol, which contain an additional functional group, such as a hydroxyl, ketone or an epoxide group. the major factors that influence cops formation during food processing or storage are heat, ph, light, oxygen, water activity and the presence of unsaturated fatty acids. various analytical approaches have been described in the literature for example, colorimetric, gravimetric, chromatographic, infrared, and so forth for estimation of cholesterol in dairy and food products. for cholesterol estimation in dairy products, two main methods have been developed viz; direct and indirect. in the direct method, color is developed in anhydrous milk fat (i.e. ghee) dissolved in chloroform to a suitable dilution (bindal and jain 1973) whereas, in the indirect method unsaponifiable matter is used for color development (pantulu and murthy 1982). other methods like fourier transformed infrared (ftir) spectroscopy, high-performance liquid chromatography (hplc) are also reported. 2 structure and properties of cholesterol chemically, cholesterol is a fat-like compound, basically, an alcohol which, in its pure form appears as flakes. it is composed of 27-carbon atoms which form three fused cyclohexane (6carbon) rings, a cyclo-pentane (5 carbons) ring and a side chain of eight carbon atoms. its molecular weight is 386.66 and the molecular formula is c27h46 c. cholesterol contains the polar hydroxyl group at c-3, which gives cholesterol a slightly hydrophilic nature, can be esterified to a fatty acid, producing cholesterol ester. cholesterol is insoluble in water, sparingly soluble in cold alcohol or petroleum ether, and completely soluble in hot alcohol and in most other organic solvents. all the 27 carbon atoms of cholesterol are derived from acetyl coa through the 3-hydroxy3-methylglutaryl-coa (hmg-coa) pathway. first of all formation of mevalonic acid from three molecules of acetyl coa then biosynthesis of squalene from six molecules of mevalonic acid through a series of phosphorylated intermediates. subsequently the biosynthesis of lanosterol from squalene via cyclization of 2, 3-epoxysqualene, and modification of lanosterol to produce cholesterol (li and parish 1998). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 14 haf © 2013 vol. v, no. 1 issn: 2283-3927 3 significance of cholesterol cholesterol is more abundant in tissues having densely packed membranes, like the liver, spinal cord, and brain. there are mainly two types of cholesterol: high-density lipoprotein (hdl) cholesterol and low-density lipoprotein (ldl) cholesterol. if cholesterol is high, it builds upon the walls of the arteries leading to a heart attack or a stroke. cholesterol is a major determinant of membrane fluidity due to its hydrophobic and hydrophilic regions. cholesterol is a structural component of the cell membranes. approximately 80% of cholesterol is used by the liver to form the bile acids. cholic acid is the most abundant bile acid in human bile. eventually, bile acids get coupled to glycine and taurine to form salts of these substances which are water soluble and act as powerful emulsifiers, important in the digestion of fat. it is a structural component of the cell membranes particularly skin and the myelin sheath. it is a chemical ancestor of the hormones like progesterone, cortisone and estrogen containing 21, 19, and 18 carbon atoms, respectively. these hormones are found in the ovaries, testis and adrenal gland. it is a precursor for the synthesis of vitamin d (makwana 2007). 4 cholesterol in milk and milk products cholesterol is rich in animal food products like milk and milk products. cholesterol accounts for 0.25-0.40% of the total lipids in milk (jenness and patton 1959). in milk, it is present in the fat globule membrane (fgm), in the fat core and in association with milk protein particularly in skimmed milk (schlimme and kiel 1989). any process disrupting the membrane structure will result in the transfer of cholesterol along with ruptured membrane material to the aqueous phase. tylkin et al. (1975) reported 9 times higher cholesterol per gram fat in buttermilk than that in butter. vyshemirskii et al. (1977) reported that 80-90% cholesterol initially present in cream passed into butter and 10-20% to buttermilk. bindal and jain (1973) estimated free and esterified cholesterol in desi ghee and reported their contents as 0.288 and 0.038%, 0.214% and 0.056% in cow and buffalo ghee, respectively. the cholesterol in cheese was found to contain 69.3 mg/100 g of cheese (fuke and matsuoka 1974). the cholesterol content in unsalted butter and 10% cream were reported to contain 244 and 31.4 mg / 100g of product, respectively (aristova and bekhova 1976). arul et al. c) and reported that 80% of the total cholesterol content was present in the liquid fraction of the milk fat. bindal and jain (1973) estimated free and esterified cholesterol in desi ghee and reported their contents as 0.288 and 0.038%, 0.214 and 0.056% in cow and buffalo ghee, respectively. the cheese was found to contain 52.3 to 76.6 (average 69.3) mg of cholesterol per 100 g of cheese and 198 to 298 (average 273) mg of cholesterol per 100 g of cheese fat (fuke and matsuoka 1974). unsalted butter and 10% cream were reported to contain 244 and 31.4 mg of cholesterol per 100g of product, respectively (aristova and bekhova 1976). singh and gupta (1982) have estimated cholesterol content in different species and reported that goat ghee contains higher cholesterol (236 mg/100 g) than cow (230 mg/100 g) and buffalo (196 mg/100 g) ghee. pantulu et al. (1975) reported cholesterol content for buffalo milk fat in the range of 183 to 383 mg/100 shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 15 haf © 2013 vol. v, no. 1 issn: 2283-3927 g of fat. prasad and pandita (1987) reported the cholesterol content of ghee prepared from the milk of haryana, sahiwal and sahiwal x friesian cows and from murrah buffaloes as 303, 310, 328 and 240 mg/100 g fat, respectively. these workers also observed that seasons also affected the cholesterol content of ghee with the highest content in winter (301 mg/100g) and lowest in summer (291 mg/100g fat). 5 factors affecting the plasma cholesterol in dairy products milk contains a large number of bioactive compounds, but milk fat has the largest impact on plasma lipids. the lipid pattern in dairy fat is very complicated and more than 400 different fatty acids have been identified. about 70% of dairy fat contains saturated fatty acids (sfas) of which the majority (45%) are of 12–16 carbon chain length and 2.7% are transfatty acids (lindmarkmånsson et al., 2003), and these have the ability to raise plasma cholesterol. except for the concentration of different types of plasma lipoproteins that can be affected, their size and composition will change in response to different types of dietary fat. for example, it is suggested that larger sizes of lipoproteins are less atherogenic than smaller sizes (kawakami a and yoshida m 2005) and some of the fatty acids typically found in milk fat have been associated with less dense ldl particles (sjogren et al., 2004). other milk components like proteins, calcium, and lactose have been suggested to affect lipid metabolism directly or indirectly, but the strongest impact on plasma lipids emerges from the intake of milk fat. 5.1 saturated fatty acids palmitic acid is the major sfa in the diet and also in milk fat with a content of about 30%. palmitic acid raises the hdl cholesterol less than it raises ldl cholesterol (grundy 1994). myristic acid represents 11% of the dairy fatty acids and increase total cholesterol as much as palmitic acid, but does not affect total cholesterol: hdl ratio (fernandez and west 2005). lauric acid is the most potent fatty acid in raising plasma total cholesterol, but dairy content is only 3.3%. the increase in hdl cholesterol induced by lauric acid is higher than the increase in ldl and thus the total cholesterol: hdl ratio was decreased when lauric acid was used to replace carbohydrates (mensink 2003). stearic acid represents 12% of the dairy fatty acids and improves the plasma cholesterol profile by decreasing total/hdl cholesterol ratio compared to other sfas. but compared to polyunsaturated fatty acids (pufa), stearic acid increases ldl and decreases hdl and increase total/ hdl ratio. (hunter 2003). other sfas are short and medium chain length and are mainly considered to be cholesterol neutral(hayes and khosla 1992; lecerf 2009). in a recent meta analysis of prospective epidemiological studies, intake of sfa and risk of coronary vascular disease (cvd) was studied (siritarino et al. 2010). 5.2 unsaturated fatty acids unsaturated fatty acids are a minor component of milk fat. oleic acid is considered favorable since it has a cholesterol and triglyceride (tg) lowering effect compared with sfa, shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 16 haf © 2013 vol. v, no. 1 issn: 2283-3927 and meals containing olive oil increase the size of postprandial tg rich lipoproteins more than meals with butter (perezmartinezp et al. 2009), which is considered to be less atherogenic. 5.3 trans fatty acids of the seasonal variation of fat in bovine milk, trans fatty acids (tfa) have the largest variation and their concentrations are more than twice as high in summer milk as in winter milk (heck et al. 2009). milk fat is the major source of natural conjugated linoleic acid (cla) and the predominant isoform is cis 9, trans 11 cla, which accounts for 85–90%. the minor trans10, cis12 cla isomer has been shown to have a more detrimental effect on plasma cholesterol than cis 9, trans 11 cla since it increases total/hdl and ldl/hdl ratios. the effect of natural tfa, such as cla and trans 18:1, found in dairy. motardbelanger et al. (2008) suggested that ruminant tfa have little impact on cholesterol homeostasis. in this 4 week randomized, crossover controlled study on 38 healthy men, they found that a moderate intake of ruminant tfa had neutral effects on plasma lipids, whereas high amounts of both ruminant and industrial tfa increased both the total plasma cholesterol by 3 and 2%, respectively, and ldl cholesterol by 6 and 4.6%. in a quantitative review of 39 carefully selected original articles, only including randomized controlled trials in a parallel or crossover design, the authors conclude that all tfa increases the ldl/hdl ratio in a linear fashion (brouwer et al. 2010). the effect of ruminant tfa and cla on the ldl/hdl ratio was less than that of industrial tfa. in the transfact study, a randomized controlled trial, 46 healthy subjects consumed 11–12 g/day of either industrial or ruminant tfa (chardigny et al. 2008). the major finding was that the natural sources of tfa significantly increases hdl and ldl cholesterol concentrations in women but not in men, and the hdl lowering effect is mainly associated with industrial tfa. the differences in cholesterol concentrations observed in women were also associated with the size of lipoproteins and tfa from natural sources increases the proportion of larger particles. thus, reducing the intake of ruminant fat will decrease the plasma cholesterol concentration and an improvement of the ldl/hdl ratio is likely. the mechanisms underlying the gender effects and type of isoforms need further investigation. 5.4 medium chain fatty acids medium chain fatty acids (mcfa) are caproic acid (hexanoic acid, c6:0), caprylic acid (octanoic acid, c8:0), and capric acid (decanoic acid, c10:0). mcfa are present at about 6.8, 6.9, 6.6, and 7.3% (of total fatty acid) in butter, milk, yogurt, and cheese, respectively (nagao and yanagita 2010)). mcfa are rapidly hydrolyzed in the gastrointestinal tract (gi tract) and are directly transported to the liver and into the mitochondria of the hepatocytes for oxidation (aoyama 2007) but some, especially decanoic acid (c10:0) will be incorporated to chylomicron tg. several studies report effects such as increased lipid oxidation, decreased body weight, increased thermogenesis and energy expenditure from consumption of mcfa and a few studies report a lowering of total cholesterol, ldl, and an increase of ldl particle size (bourque et al. 2003 ; liu et al. 2009). whether the long term effects on plasma cholesterol levels and an improvement of ldl: hdl ratios are caused by weight reduction or are an actual effect of the mcfa itself is not fully clarified. in a randomized controlled intervention study on shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 17 haf © 2013 vol. v, no. 1 issn: 2283-3927 17 healthy individuals, a high intake of 70 g of mcfa per day for 3 weeks was, however, detrimental to plasma lipids with a 12% increase both in ldl cholesterol and ldl/hdl ratio (tholstrup et al. 2004). 5.5 proteins in the 1990s, it was suggested that whey proteins had more hypocholesterolemic effect than casein and soy proteins in rats (nagaoka et al. 1992). a decade later, four peptides formed from tryptic cleavage of bovine β-lactoglobulin: iiaek, gldiqk, alpmh, and vyveelkptpegdleillqk were found to inhibit cholesterol absorption by increasing fecal output of steroid. of the four peptides, iiaek had a higher effect than β-sitosterol. in a recent in vitro study on a human intestinal cell line (ncih716), the effects both of the specific amino acids leucine, isoleucine and valine, and of whey, skim milk and casein on the expression of lipid-regulating genes were examined (chen and reimer 2009). it was found that isoleucine, leucine, valine, and whey down-regulated niemannpick c1like 1 (npc1l1), a protein carrier with a critical role in intestinal cholesterol absorption. they conclude that dairy products such as whey, with a high content of branched-chain amino acids can have an effect on cholesterol absorption and possibly also on the plasma cholesterol concentration. the hypocholesterolemic effect of whey has now been confirmed also in humans (pal et al. 2010). 5.6 lactose in older human studies, ingesting 80 g of lactose per day as whey resulted in a decreased serum cholesterol (stahelin and ritzel 1979). furthermore, 50 g/day of added lactose to patients with cvd significantly reduced serum cholesterol over a 3 week period (agarwal et al. 1980). 5.7 fermented dairy products the effects of fermented dairy products have been studied since the 1970s. the first studies showed that unpasteurized yoghurt decreased serum cholesterol by 5–9%. later, results from some studies showed no effects of fermented products, but this was explained by the fact that the subjects in the study had a baseline cholesterol concentration of <5.0 mmol/l (ebringer et al. 2008). in a recent study on 34 women, an intake of 125 g of fermented milk three times a day for 4 weeks decreased plasma ldl cholesterol by 12.5–16%. there was also a significant decrease of hdl cholesterol of 10–12% (andrade and borges 2009). it is concluded that many strains of fermentation bacteria, but not all are viable enough to reach the lower part of the gut and exert effects on the microbiota, thereby increasing the amount of propionate that has a cholesterol lowering effect. the second effect by which bacteria may influence cholesterol level is by hydrolyzing glycine and taurin conjugated bile acids. by deconjugating bile acids, the excretion of bile acids in feces is increased, which promote the use of cholesterol for synthesis of new bile acids. the use of fermented milk instead of normal milk may therefore be a method to reduce or maintain the plasma cholesterol levels, but the effects on ldl/hdl ratio should be further investigated (nestel 2005; stonge et al. 2000). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 18 haf © 2013 vol. v, no. 1 issn: 2283-3927 6 methods for removal of cholesterol there are various methods and /or processes available in the literature for removal of cholesterol content in butter oil and foods in general and they generally categories into physical, chemical, biological and complexation processes. however, these processes are costly. 6.1 physical processes 6.1.1 vacuum steam distillation in this process, steam is bubbled through liquid milk fat under vacuum. the omega source corporation has acquired this proprietary technology and is currently producing 109.1 kg/day of decholesterolized anhydrous milk fat (boudreau and arul 1993). the process reduces free cholesterol by 93 per cent. firma hoche (speikem, germany) is also utilizing vacuum stem distillation technology on a commercial scale. the milk fat was reported to be almost neutral in smell and taste with 75 per cent less cholesterol content (boudreau and arul 1993). 6.1.2 short path molecular distillation short-path molecular distillation is based on the transfer of molecules from the hot surface of an evaporating liquid to the cooled surface of a condenser through a short path. lanzani et al. (1994) investigated a new short-path molecular distillation system for the reduction of cholesterol in butter fat and lard. this method however does not remove the high boiling cholesterol ester. 6.1.3 supercritical fluid extraction bradley (1990) have shown that it was technically feasible to produce a milk fat product (80 per cent plastic cream) for a reasonable cost, with at least 90 per cent of the cholesterol extracted using supercritical carbon dioxide. the finished milk fat retained its color and flavors. in the same study, he described a batch process that removed 90 per cent of the cholesterol from butter by this technology and subsequently yielded a soft spread product (bradley 1990). 6.2 chemical process gu et al. (1994) described chemical method based on the reaction between cyclic anhydride and the hydroxyl group of cholesterol. the reaction forms monoesters with acryl chains having a terminal acid group. this process could effectively and economically remove up to 40 per cent of the cholesterol from animal fats. wrezel et al. (1992) were issued a patent "method for removing cholesterol from edible oils" based on a procedure that was similar to that of gu et al. (1994). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 19 haf © 2013 vol. v, no. 1 issn: 2283-3927 6.3 biological process 6.3.1 cholesterol reductase cholesterol reductase is an enzyme that converts cholesterol to coprostanol which is poorly absorbed by humans. dehal et al. (1991) investigated several cholesterol reductases produced by bacteria or by extraction from leaves of several green plants. they found that cholesterol reductase could convert cholesterol to coprostanol in milk, cream, ground beef and pork. they concluded that these reductases from bacterial or plant sources could be used to decrease the cholesterol content of foods. 6.3.2 cholesterol oxidases several workers (christodoulou et al. 1994) have reported that cholesterol in foods could be nearly eliminated by cholesterol-degrading enzymes produced by some bacterial strains. these enzymes have been used by xiansheng et al. (1990). 6.4 complexation process 6.4.1 saponins saponins from plants can also be used to selectively bind and precipitate cholesterol. riccomini et al. (1990) reported an 80% cholesterol reduction in cream by simply mixing cream and saponins for 1 hour at 65 °c, filtering the mixture through celite and washing with water. the same treatment on anhydrous butterfat produced a 90%t reduction in cholesterol. no adverse flavors could be detected in the final products. micich et al. (1992) investigated polymer-supported saponins to improve the removal of cholesterol from butter oil. this new support was reusable and reduced the residual saponin in the butter oil. the 1 g polymersupported saponins can bind 3 mg pure cholesterol in hexane. it was also shown that cholesterol removal from butter oil (dissolved in hexane) was 10-40% less efficient than with pure cholesterol. 6.4.2 β-cyclodextrin s w βcyclodextrin (davidson 1990; ockenfull et al. 1991). yen and tsai (1995) studied cholesterol removal from a lard-w x w β-cyclodextrin, founded that about 90 per cent of cholesterol could be removed from lard. 7 formation of cops cholesterol present in animal origin foods undergoes autoxidation during processing as well as during storage yielding toxic products. cholesterol oxidation products (cop) are similar to cholesterol, which contain an additional functional group, such as a hydroxyl, ketone or an epoxide group in the sterol nucleus and/or on the side chain of the molecule. oxidation of shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 20 haf © 2013 vol. v, no. 1 issn: 2283-3927 lipids and sterol (cholesterol) follows the same oxidation patterns such as autoxidation, photooxidation and enzymic oxidation, producing relevant hydro peroxides. it is believed that the hydroperoxides derive from oxidation of unsaturated fatty acids play a significant role to x δ-5 double bond, which is more sensitive to oxidation (lercker et al. 2002). 7.1 autoxidation of cholesterol the main reaction involved in auto oxidation of cholesterol is a self-catalytic reaction with molecular oxygen cholesterol autoxidation usually starts at c-7 by abstraction of hydrogen following the addition of an oxygen molecule forming primary cop, isomers of 7hydroperoxycholesterol. the 7y xy 7α y xy 7β-hydroxycholesterol, which are commonly found in food (lercker et al. 2002). 7-ketocholesterol is formed by dehydration of isomeric 7-hydroxycholesterol in the presence of radicals. it is a major cop in the food matrix. formation of isomeric epoxy cholesterols occurs due to the interaction between cholesterol and hydroxyl radicals and these epoxy compounds can be hydrolyzed in acidic medium converting them into most toxic triols. the side chain oxidation occurs at c20 and c-25 positions resulting in the production of relevant hydroperoxide, 20-hydroperoxide and 25-hydroperoxide respectively, which can α -hydroxycholesterol and 25-hydroxy cholesterol (lercker et al. 2002). 7.2 photo oxidation of cholesterol in photooxidation of cholesterol, singlet oxygen is formed from triplet oxygen by light in the presence of an active sensitizer (natural pigment or synthetic colorant). cholesterol can react with singlet oxygen in the presence of photo-sensitizer, forming dominant hydroperoxide at c a y x α-hydroxycholesterol and the other part is further converted into stable 7-hydroxiperoxide and 6-hydroperoxide that are present in minor amounts. 7-hydroperoxides can be converted into isomeric 7hydroxycholesterol and into 7-ketocholesterol at the same time 5-hydroxycholessterol can be formed (lercker 2002). 7.3 enzymic-oxidation of cholesterol some enzymes in food oxidize cholesterol. available reports show that the conversion of α y x α-hp 7α-hp 7α-hp 7β-hpc, and formation of 7-hc epimers from the corresponding hydroperoxides occur by enzymatic reactions. but this has to be studied to a further extent due to the fact that these products can be formed by the usual non-enzymatic reactions (lercker et al. 2002). the main enzymes involved in the enzymatic oxidation of cholesterol are monooxygenase, dioxygenase, dehydrogenase, and x p 7α y xy y xy α hydroxycholesterol, (25r)-26-hydroxycholesterol, 22rhydroxycholesterol, are produced by enzymatic oxidation of cholesterol (lercker et al. 2002). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 21 haf © 2013 vol. v, no. 1 issn: 2283-3927 these oxides are produced endogenously in human tissues during conversion of cholesterol to bile acids and steroid hormones and they can oxidize cholesterol effectively in the presence of human gastric fluid and it acts as a better medium for cholesterol oxidation and produce cop in vivo (dobarganes et al. 2003). these enzymic and non-enzymic reactions can occur separately or/and simultaneously in food production, processing, distribution and storage (ubhayasekera et al. 2004; dobarganes and marquez-ruiz 2003). cholesterol oxidation products (cop) are similar to cholesterol but they contain an additional functional group like hydroxyl, ketone or an epoxide group in the sterol nucleus and/or on the side chain of the molecule. the cops can be formed by mainly 3 major oxidation pathway that is, autoxidation, photo-oxidation and enzymatic oxidation of cholesterol. the main reaction involved in autooxidation of cholesterol is a self-catalytic reaction with molecular oxygen. cholesterol autoxidation usually starts at c-7 by abstraction of hydrogen following the addition of an oxygen molecule. this will form primary cop, isomers of 7hydroperoxycholesterol. the 7y xy 7α y xy 7β-hydroxycholesterol. these 7αy xy 7β hydroxycholesterol are commonly found in food (lercker and rodriguez-estrada 2002). 7.4 cops in fresh milk and dairy products formation of cop in fresh milk or fresh dairy products is very low due to the low level of oxygen. bican (1984) studied cops content in raw milk using hplc but he did not find any detectable cops in fresh milk. kou and holmes in 1995 studied cops in fresh cream as well as fresh butter, but they did not find any detectable cops. 7.5 cops in heat-treated dairy products milk was heated under different time-temperature conditions, varying from pasteurization to uht-treatment but showed no (cleveland and harris 1987) cop formation. cops were detected in butter after different heat treatments. the higher the temperature used and the longer the storage time, the total cops were produced in more amount(pie et al. 1990).unsalted butter oil contained 300 mg/kg ketocholesterol and 200 mg/kg aepoxycholesterol which were two to three times higher than in salted butter oil (sander et al. 1989b). 7.6 cops in heat-treated and/or stored dairy products the spray dried skim milk powders were analyzed after storage of 13–37 months contained substantial amounts of total cops. they contained substantial amounts of total cops (between 20 mg/kg and 78 mg/kg in total lipids; (nourooz-zadeh and appelqvist 1988a). milk powder packaged in pe pouches had a total cops content of 15.6% of the original cholesterol content. samples packaged in glass vials with and without oxygen absorbers had cops contents of 0.7% and 5.9% of the original cholesterol, respectively. shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 22 haf © 2013 vol. v, no. 1 issn: 2283-3927 7.7 cops in dairy products after light exposure cholesterol in butter was oxidized during exposure to fluorescent light. the cops were more concentrated at the surface than throughout the ent y 7 w x c /30 days compared to day 0 (nielsen et al. 1995). 8 significance of cops on health 8.1 atherosclerosis and inflammation atherosclerosis is the most well-known condition that involves oxysterols. atherosclerosis means the accumulation of cholesterol deposits in macrophages in the walls of large and medium arteries. subsequent formation of atherosclerotic plaque gradually narrows vessel lumen, leads to thrombosis, and compromises oxygen supply to target organs, eventually causing heart attack and stroke-the major sources of mortality in the developed world. cholesterol present in atherosclerotic lesions comes mostly from low density lipoproteins. one of the popular hypotheses explaining atherogenesis (chisolm and steinberg, 2000) states that it is the oxidation of ldl that triggers its internalization by macrophages through the so-called scavenger receptor pathway (goldstein et al. 1979). significant amounts of 27-oh-chol, 7-ketochol, and 7a/b-oh-chol are often found in lesion and foam cells, where their levels are at least two orders of magnitude higher than in plasma (brown and jessup 1999). these oxysterols constitute 75–85% of all oxysterols in plaque and are present mostly (>80%) as monoand diesters. many other oxysterols, derived from diet, in vivo oxidation, or enzymatic reactions during cholesterol catabolism have been detected and are claimed to play an active role in plaque development. 8.2 neurodegenerative diseases n a ’ p ’ h ’ multiple sclerosis, constitute another set of conditions that are often related to oxysterols. the human brain, representing only 2% of total body weight, contains 25% of total body cholesterol, mainly in unesterified form, distributed between myelin (70%), glial cells (20%), and neurons (10%) (maxfield and tabas 2005). since the brain is isolated by the blood–brain barrier, its cholesterol has to be synthesized in situ, and its excess is eliminated after conversion to 24-ohchol by 24-hydroxylase (cyp46a1) (bjorkhem 2006). in contrast to cholesterol, this chainoxidized sterol can easily pass through the blood brain barrier and can be metabolized by the liver. also, 27-oh-chol, the most abundant oxysterol in human circulation, and 7a-hydroxy-3oxo-4-cholestenoic acid were shown to pass the blood brain barrier (bjorkhem et al. 2009). in this context it is not surprising that oxysterols play an active role in pathologic conditions related to the deregulation of brain cholesterol homeostasis (martins et al. 2009). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 23 haf © 2013 vol. v, no. 1 issn: 2283-3927 in the pathogenesis of ad, oxysterols were found to be involved in the alteration of cholesterol metabolism, modulation of neuroinflammation, aggregation and accumulation, g p ’ pd movement disorder. it is characterized by the aggregation of a-synuclein protein in lewy body inclusions and loss of dopaminergic neurons in the substantia nigra (rantham prabhakara et al. 2008). oxysterols (mainly 24s-oh-chol, 27-oh-chol, and secosterol) are particularly responsible for causing a-synuclein aggregation and destruction of dopamine-containing neurons (bosco et al. 2006). also, the levels of oxidative cholesterol metabolites are higher in the cerebral cortex of pd patients. the level of 24s-oh-chol in cerebrospinal fluid, but not in blood, correlates with the duration of the disease (bjorkhem et al. 2013. the antiviral properties of 25-oh-chol were first identified for enveloped viruses (that is, vesicular stomatitis virus, human immunodeficiency virus (liu et al. 2011b)) and later for nonenveloped ones (that is, human papillomavirus-16, human rotavirus, and human rhinovirus) (civra et al. 2014). recently, its antiviral action against hepatitis c virus has also been documented (anggakusuma et al., 2015; xiang et al., 2015). since both 25-oh-chol and statins have similar antiviral effects and mutual targets, it has been proposed that their synergy could be used in therapy (chen and peng 2015). 8.3 cancer oxysterols are associated with cancers of the colon, lung, skin, breast, prostate, and bile ducts. procarcinogenic oxysterols might exert their effect at three stages of carcinogenesis: by induction of dna damage, by induction of cyclooxygenase-2 expression, or by stimulation of tumor cell migration (zarrouk et al. 2014). in vitro, oxysterols were shown to interfere with proliferation and cause the death of many cancer cell types, while having little effect on senescent cells (de weille et al. 2013). certain oxysterols can exhibit anticancer effects, possibly via modulation of cholesterol efflux, protein kinase b, or lxrs. for example, treatment with 22r-oh-chol, 24s-oh-chol, 7a-oh-chol, 7b-oh-chol, 25-oh-chol, 5a, 6b-epoxychol, and 3b, 5a, 6b-3oh-chol suppresses the proliferation of human prostate, breast, colon, lung, and leukemia cancer cells (lin et al. 2013). 8.4 eye diseases in age-related macular degeneration (amd), cataracts, and opacified corneas, the involvement of oxysterols is also widely suspected (vejux and lizard 2009). the accumulation of 7-keto b ’ pigment epithelium (rpe) can have cytotoxic, pro-oxidant, and pro-inflammatory activities (rodriguez and larrayoz 2010). all those processes can contribute to amd, causing irreversible central vision loss and blindness. it seems that, to some extent, 7-keto-chol can be detoxified locally by enzymatic 25r, 26-hydroxylation in rpe (javitt 2008). 27-oh-chol was shown to cause ab accumulation and oxidative cell damage in rpe (dasari et al. 2010), linking the pathogeneses of amd and ad. during aging, oxysterols (that is, 7b-oh-chol, 7-keto-chol, 5a, 6a-epoxy-chol, 20a-oh-chol, and 25oh-chol) were shown to accumulate in human lenses, participating in clouding of the lens (cataracts), the dominant form of blindness (vejux and shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 24 haf © 2013 vol. v, no. 1 issn: 2283-3927 lizard 2009). cataracts are associated with smoking, diabetes, glucocorticoid therapy, and excessive exposure to sunlight, all of which are known to induce oxidative stress (zarrouk et al. 2014). an opacified cornea is also characterized by the accumulation of various oxysterols (that is, cholesta-3, 5-dien-7one, cholest-4-en-3-one) (vejux and lizard 2009). 8.5 other conditions the role of oxysterols has also been postulated in type 2 diabetes (sottero et al. 2015), sensorineural hearing loss (mal-grange et al. 2015), and chronic inflammatory processes of the gallbladder, anorexia, and chronic renal failure (sottero et al. 2009). intoxication with ethanol was found to increase the levels of 7-keto-chol, 7a-oh-chol, and 7b-oh-chol in the liver, skeletal muscle, jejunum, and heart of rats (sottero et al. 2009). estrogen signaling is critical for maintaining proper bone density. increased levels of 27-oh-chol were shown to decrease bone mineral density. these data provide evidence for interactions between estrogen signaling, cholesterol and metabolic disease, and osteoporosis (dusell et al. 2010). 9 methods for estimation of cholesterol and cops a number of methods are available in the literature and many of these are being tried in clinical and food laboratories. most of these methods for cholesterol estimation were initially developed for blood plasma and then applied to foods. various analytical approaches employed for cholesterol estimation are (1) gravimetric methods, (2) colorimetric methods (for example, liebermann-burchard reaction, o-phthaldehyde and ferric chloride method),(3) chromatographic methods (glc, hplc), (4) infrared based methods (ft-nir, mir), and (5) enzymatic method(makwana 2007). the quantification of cop in food is difficult because there are interruptions by large amounts of interfering cholesterol, triglycerides, phospholipids and other lipids. on the other hand technical difficulties associated with cholesterol oxide analysis play a major part due to similar chemical structures and presence of cholesterol oxides at trace levels. therefore, the extraction, purification, and detection methods that use to identify and quantify these products play a major role (ulberth and buchgraber 2002; guardiola et al. 2004). cops are fatsoluble compounds. many solvents or solvent combinations can be used to extract cop and the most important factor to be considered at this point is the complete recovery. the various combinations of the solvents are chloroform/methanol; n-hexane/2-propanol; dichloromethane/methanol and so forth. hplc is a better method to analyze cop due to the possibility of separation, detection and quantification, especially the thermo-labile molecules (for example, cholesterol hydro peroxides). the gc is the common method of cop determination using deivatized cop as trimethylsilyl ether (tmse) derivatives (ubhayasekera et al. 2004). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 25 haf © 2013 vol. v, no. 1 issn: 2283-3927 10 methods for estimation of cholesterol increasing awareness about health consciousness among consumers, formulation of mandatory guide lines which include the declaring of nutritional facts on the label of food packet have stressed much emphasis on estimation of cholesterol in food products. a number of methods are available in the literature and many of these are being tried in clinical and food laboratories. most of these methods for cholesterol estimation were initially developed for blood plasma and then applied to foods. various analytical approaches employed for cholesterol estimation are listed bellow and discussed in subsequent sections. 10.1 gravimetric methods windous (1910) presented a reaction between alcoholic solution of cholesterol and alcoholic solution of digitonin (c56h92o29) which results in precipitation of 1:1 molecular complex of cholesterol and digitonin as cholesterol digitonoids. this reaction became a platform for several gravimetric methods of estimating cholesterol in fats and oils. bloor (1916) observed that saponification was not necessary in the above method, as cholesterol in both forms gives colour. he suggested a method for extraction of cholesterol but the results obtained by his method gave much higher result than the windous method (1910). 10.2 colorimetric methods 10.2.1 liebermann-burchard (lb) reaction method ghee (0.2 to 0.25) g of ghee dissolved in chloroform (3 ml), 4 ml l.b. reagent (acetic anhydride: h2so4 :: 20:1, freshly prepared at 0 °c) was added and the mixture incubated at 25 °c for 12 min. the optical density was recorded at 650 nm in the colorimeter, taking care that the measurements were completed within 15 min after the addition of lb reagent. the amount of cholesterol was calculated by plotting a standard curve, prepared under same conditions simultaneously. they compared their results with that of gravimetric method of windaus (1910) and found that the method gave the comparable result with gravimetric method. cholesterol content in cow and buffalo ghee was found to be 310 and 270 mg per 100 g of ghee, respectively (bindal and jain, 1973) liebermann-burchard test is used for the colorimetric test to detect cholesterol, which gives a deep green color. the formation of a green or green-blue color after a few minutes mean is the positive result. this color begins as a purplish, pink color and progresses through to a light green then very dark green color. the color is due to the hydroxyl group (-oh) of cholesterol reacting with the reagents and increasing the conjugation of the unsaturation in the adjacent fused ring. in this reaction, the acetic acid in the liebermann reagent reacts with cholesterol in the sample, which gives a green color whose absorbance, can be determined by uv-visible spectrophotometer at 640 nm (burke et al. 1974). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 26 haf © 2013 vol. v, no. 1 issn: 2283-3927 10.2.2 o-phthaldehyde method bachman et al. (1976) used o-phthaldehyde method for determination of cholesterol in dairy products. accurately weighed dairy products were saponified and the unsaponifiable matter was extracted using 10 ml of hexane. an aliquot (4ml) of hexane extract was evaporated and 4 ml of o-phthaldehyde reagent (50 mg/100 ml in glacial acetic acid) was added and mixed properly. after 10 min, 2 ml of concentrated h2so4 was added and absorbance was taken after 10 min at 550 nm. these results were compared with glc method for cholesterol estimation and found good agreement between the two methods. using this method, they reported 13.2, 46.1, 104.1, 95.3, 97.4, 78.4 and 24.2 mg of cholesterol per 100 g of sample in milk, vanilla ice-cream, cheddar, swiss, processed cheese and creamed cottage cheese, respectively. recovery of the method was found to be 99.79%. 10.3 chromatographic methods 10.3.1 gas-liquid chromatographic methods cholesterol determination by gas-liquid chromatography (glc) is usually more accurate than colorimetric procedures (vanzetti 1964). numerous studies have been done on glc method of cholesterol determination, but in all cases, a trimethylsilyl (tms) ether derivatization of sterols is involved which is time-consuming. in the past, most studies focused on a quick, accurate and simple method to analyze cholesterol. for example, gas chromatography (gc) with capillary columns, in many cases, a method for routine analysis of sterols, especially for cholesterol (itoh et al. 1973). punwar (1975) developed a widely used approach for determining cholesterol in foods. the method involved lipid extraction, saponification and silyl derivatization followed by estimation through gas chromatography. however, the lipid extraction and saponification steps in this method were cumbersome, the derivatizing reagents were unstable and cholesterol was thermally decomposed in the gc column (kovacs et al. 1979). to overcome these problems, kovacs et al. (1979) proposed a method in which derivatization step of cholesterol was removed. they used a gas chromatograph equipped with flame ionization detector (fid) and glass column packed with gas chrom q and coated with 3% ov-17. the column w c using helium as carrier gas at a flow rate of 40 ml/min. injector and detector temperatures were 235 °c and 240 °c respectively. for α-cholestane was used as the internal standard and all sterols were identified by comparing their retention times with authentic standards. prior to glc analysis, sterols were saponified and extracted into hexane. tsui (1989) developed a method for rapid determination of total cholesterol in homogenized milk. the milk sample was saponified in ethanolic koh in the presence of an α-cholestane. cholesterol and cholestane were then isolated by solid phase extraction on a non-polar absorbent and eluted with the organic solvent. the evaporated extract was derivatized and analyzed by capillary gas chromatography. the average recovery of cholesterol added to milk prior to saponification was 95% and the average relative standard deviation for repeated analyses was 2%. the limit of detection for this method was 2 mg per 100 g. shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 27 haf © 2013 vol. v, no. 1 issn: 2283-3927 prior to gc analysis, cholesterol was saponified and extracted from the food sample (containing about 1 mg of cholesterol). unsaponifiable matter was extracted using n-hexane and tms ether derivatization of cholesterol was performed and dissolved in x μ hexane solution was injected into gc and glc was performed using the following analytical condition: injection port and detector block330 °c, column oven temperature program -250320 ˚ ˚ / 1.75 kg/cm2 at 250 ˚ 60:1. i α-cholestane) and tms ether derivative of cholesterol eluted at 16 and 20 min respectively, their peak area was determined and concentration of cholesterol was calculated. standard method of aoac (2005) for estimating cholesterol in foods involves saponification, extraction, derivatization, and gc analysis. in the method, 2-3 g of sample was saponified with 40 ml of 95% ethanol and 8 ml of 50% koh solution by heating on water bath for 70 ± 10 min. unsaponifiable matter was extracted into 100 ml toluene and it was washed with 110 ml of 1 m koh, 40 ml 0.5 m koh, and 40 ml water (washing with water was carried out for 3 times), respectively. then the toluene layer was passed through na2so4 and the solvent layer was allowed to stand for 15 min. the sterols present in the solvent layer were derivatized to trimethylsilyl esters (tms) using dimethylformamide(dmf), hexamethyldisilane hmds y m s α-cholestane was used i g y μ w j y hydrogen flame ionized detector was employed to detect the sterol peaks. the operating conditions maintained were; injector temperature 250 °c, detector temperature 300 °c, and column temperature 190 °c. 10.4 high performance liquid chromatographic (hplc) methods high-performance liquid chromatography (hplc) techniques involve the use of nonaqueous reversed phase systems with saponified or esterified derivatives to determine cholesterol in foods (hurst et al. 1983). the mobile phases in these reversed phase systems range from nonpolar that is, 2 propanol/hexane (newkirk and sheppard, 1981) to extremely polar that is, methanol (sugino et al. 1986). detection of cholesterol in extracted lipids by hplc is usually based on absorbance of shortwavelengths uv light (200-210 nm; duncan et al. 1979; carrol and rudel 1981). because of their simplicity and wide area of application, several liquid chromatographic methods have been developed for cholesterol determination coupling with uv-absorbing derivatives (newkirk and sheppard 1981). oh et al. (2001) developed a sensitive high-performance liquid chromatographic method to determine the cholesterol content in milk and milk products. they compared various extraction process and various mobile phases for separating cholesterol by hplc in order to develop a simple and accurate method. they observed that solid phase extraction (spe) had a high recovery with shorter extraction time and the process was highly reproducible in comparison to liquid-liquid extraction. further, acetonitrile: methanol: 2-propanol was found to be superior to the other mobile phase systems for separating cholesterol. hplc was performed using a multi y w μ j column. maintaining the flow rate of mobile phase at 1.6 ml/min, elution of cholesterol was shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 28 haf © 2013 vol. v, no. 1 issn: 2283-3927 monitored at 205 nm using uv detector method, again, accurately predicted the cholesterol content in a variety of dairy products. 10.4.1 methods based on infra-red spectroscopy paradkar and irudayaraj (2002a, b) developed two methods based on principle of ft-ir and ft-nir spectroscopy for determination of cholesterol in dairy products. in the ft-ir method, 1.5-2.5 g of sample was mixed with 9 ml of ethanol and 1 ml of potassium hydroxide solution (50 g per 100 ml) and vortex mixed for 20 sec. the capped tube was then placed in a water bath maintained at 60 °c for 1 hr in order to have complete saponification and stirred continuously at 200 rpm. after cooling to room temperature, 5 ml of deionized water and 10 ml of hexane were added and vortexed for approximately 2 min. the sample was then centrifuged for 3 min at 2000 rpm and the hexane layer was transferred into a clean tube. another 10 ml of hexane was added to the aqueous phase. the extraction and centrifugation steps were repeated. the combined hexane extract was then used for cholesterol determination. the hexane extract was evaporated to dryness and the sample was re-dissolved in 5 ml of chloroform and used for ftir analysis. they concluded that ftir (midinfrared) spectroscopy was successfully used for the rapid estimation of cholesterol in commercial dairy products such as milk, milk powder, yogurt, cheese, grated cheese, and p s p s w − − w r = w less expensive than the conventional method and could be accomplished in less than 5 min. 10.4.2 electrophoretic method xu et al. (2002) described a simple non-aqueous capillary electrophoresis (nace) method for the rapid quantification of cholesterol in egg yolk and milk. the samples were subjected to saponification and then quantified by nace, in which 100 mm sodium acetate-acetic acid in methanol was employed as the running buffer. the correlation coefficient between the cholesterol concentration and the corresponding peak area was 0.999. the detection limit of cholesterol was 5 g/ml (twice the signal-to-noise ratio). they recommended this method as a routine method for the rapid and sensitive determination of cholesterol in foods. 10.4.3 enzymatic method enzyme kits containing peroxidase, cholesterol esterase, and cholesterol oxidase have been employed satisfactorily to determine cholesterol in egg yolk (jiang et al. 1990; van elswyk et al. 1991). 11 analysis of cholesterol oxidation products the cop levels reported for similar food, sometimes not lie in the same range and sometimes a considerable difference could be observed. these differences are due to the differences in manufacturing technology and analytical procedure. this brings up a very important need of accurate quantification (mccluskey and devery 1993; guardiola et al. 2004). the quantification of cop in food is difficult because there are interruptions by large amounts shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 29 haf © 2013 vol. v, no. 1 issn: 2283-3927 of interfering cholesterol, triglycerides, phospholipids and other lipids (ulberth and buchgraber 2002). on the other hand technical difficulties associated with cholesterol oxide analysis play a major part due to similar chemical structures and presence of cholesterol oxides at trace levels (parts per million). therefore, the extraction, purification, and detection methods that use to identify and quantify these products play a major role (ulberth and buchgraber 2002). the analytical procedure should be designed in a way to guarantee efficient recovery of cop from food matrix or biological fluids and minimization of the generation of artifacts during sample clean-up and work-up steps. most common analytical protocol used for cop analysis, has more or less the same route: (i)extraction of cop from food matrix or biological system; (ii)purification of the sample extract; (iii) derivatization to suitable compounds and (iv)quantification with a suitable chromatographic method (ulberth and buchgraber, 2002; guardiola et al. 2004) 11.1 extraction, purification and enrichment cops are fat-soluble compounds. it is very difficult to isolate cops due to low levels from the other fat-soluble substances in food matrices, such as mono, di, tri acylglycerols, esterified and free cholesterol, free fatty acids and phospholipids. many solvents or solvent combinations can be used to extract cop and the most important factor to be considered at this point is the complete recovery. these preparation steps are very important due to their possible contribution to the formation of artifacts, cop recovery, peak resolution, detection efficiency, identification and quantification (rodriguez-estrada and caboni 2002). choose of a solvent or a solvent system play a significant role as the solvent system must be capable of disrupting the associative forces binding the cholesterol and lipids to non-lipid material in biological matrix. therefore no single solvent is capable of complete cholesterol extraction whereas a mixture of solvents is used (ulberth and buchgraber 2002). the most commonly reported methods for cholesterol extraction are: 1. methods involving a preliminary fat extraction followed by a saponification a. chloroform/methanol (2:1, v/v); (folch et al. 1957) b. n-hexane/2-propanol (3:2, v/v); (hara and radin 1978) c. dichloromethane/methanol (9:1, vol/vol); dry column method (higley et al. 1986; zubillaga and maerker 1991). 2. methods that directly treat the sample a. direct saponification or trans esterification (dionisi et al. 1998) apart from these methods, reports of usage of single solvents like chloroform and soxhlet extractions using solvents such as tert-butylmethyl ether and dichloromethane are also available (ulberth and buchgraber, 2002). very few reports are available on the comparison of different extraction methods. dionisi et al. (1998) have compared commonly used four lipid extraction methods, direct sapon m xw ’ ’ r ’ y x w y shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 30 haf © 2013 vol. v, no. 1 issn: 2283-3927 phase extraction (spe) column purification and gc-ms (gas chromatography coupled to mass spectrometric detector) quantification. d m xw ’ ’ b r ’ method had shown drastic low cop recovery, probably due to the apolar solvents usage in fat x i y r ’ w gives better recovery (ulberth and buchgraber 2002). accelerated solvent extraction (ase) is well known for high efficiency in lipid extraction but not many available reports on cop analysis. in a total lipid extract 11 triglycerides and/or phospholipids are major compounds whereas sterols are present in minor levels and cops are in trace levels. to analyze with less interference and for clear detection these cop need to be enriched. there are two enrichment methods such as saponification and trans esterification (ulberth and buchgraber 2002). saponification is employed to remove triglycerides, free fatty acids and water-soluble impurities during extraction of cop by converting them to water-soluble derivatives using methanoic or ethanoic naoh or koh. cop extraction to a suitable organic solvent is done after adding water to the saponified mixture. the unsaponifiable fraction contains cop. two saponification procedures, cold and hot saponification are often used. cold saponification at 25 °c for 18-22 hours has shown high recovery (tai et al. 1999) and low artifact formation. use of hot saponification at 60 °c for 45-60 minutes has reduced the saponification time. hot saponification leads to artifact formation by degrading 7-keto and isomeric epoxides. to overcome this problem purification of cop in silica gelor c18 cartridges have been used (ulberth and buchgraber 2002; guardiola et al. 2002). apart from the disadvantages of possible artifact formation, saponification also has some practical drawbacks. the saponified triglycerides form a soap solution giving bad separation of the evolved emulsions and micelle formation leads to loss of compounds of interest. (schmarr et al. 1996).the other method used to free the sterol and its oxides from the bulk of the accompanying lipids is trans esterification. the trans esterification is a method with mild conditions converting esterified oxysterols and triglycerides into fatty acid methyl esters. after esterification, the lipid primarily consists of acid methyl ester, free cholesterol and its oxidation products and some minor apolar and polar compounds. this mixture could be separated by a solid phase extraction column and stepwise elution with solvent of increasing polarity. no artifact formation is detected with this method (schmarr et al. 1996). solid phase extraction (spe) column uses the polarity differences in the matrix component to separate a mixture of compounds with different polarity. phospholipids, cholesterol and its oxidation products and cholesteryl esters are most, medium and least polar respectively. therefore, spe method helps to separate cop with minimum contamination with coeluted cholesterol and phytosterol, if present. the advantages of using a spe column are as follows: (i) no destruction of sensitive cop as no strong alkali is used (ii) artifact formation is minimized due to the removal of free cholesterol. the factors such as pressure of air, light, peroxides in solvents, heat and contact with reactive groups (for example, silicic acid) leads to artifact formation (ulberth and buchgraber 2002). there are several different matrices used in spe columns whereas the cop analysis has been reported with silica (si-), florisil, aminopropylmodified silica (nh2-) and octadecyl-modified silica (ods-) (ulberth and buchgrabe 2002). shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 31 haf © 2013 vol. v, no. 1 issn: 2283-3927 11.2 thin-layer chromatography (tlc) tlc is an efficient, rapid and simple technique used to separate, isolate and identify cop in crystalline cholesterol, food and biological samples. use of tlc is limited to the separation of cop but not for accurate quantification. tlc is capable of separating hydroxycholesterols but not cholesterol hydroperoxides (lebovics 2002).tlc is useful in confirming the identity of cop based on the color development after spraying sulfuric acid and observation under uv (tai et al. 1999). there are reports of separation of cop such as 7α-hc, 7β-hc, and 5,6-ce with tlc method and purification of cop (nourooz-zadeh and appelqvist 1988). the major drawbacks of tlc analysis are incapability of 5,6-ep isomer separation, tedious method, inaccuracy of quantification of cop based on the spot area, poor resolution, instability, time consuming procedure. there are many advantages of tlc such as parallel analysis of many samples, possibility of fast screening, ability to guess the amount of cop, purification to a certain level and low cost (lebovics 2002). 11.3 high performance liquid chromatography (hplc) cops are present in small quantities in food and quantification of them are challenging. the isomers of some cop show similar chemical, spectrometric and fragmentation characteristics. all these reasons stress the requirement of high sensitive methods and systems to identify and quantify cop. the choice of analytical tool is governed by, the type of matrix and the scope of the analysis (rodriguezestrada and caboni, 2002). hplc is a better method to analyze cop due to the possibility of separation, detection and quantification, especially the thermo-labile molecules (for example, cholesterol hydroperoxides) as it is a non-destructive technique. this is a versatile analytical system due to its ability to couple into many detector systems, and to run in normal or reverse phase modes using different column types and dimensions and mobile phase compositions, leading to different separation, identification and quantification. hplc coupled to ms detectors has become a very powerful tool over the past years due to the increment of the sensitivity (rodriguez-estrada and caboni 2002). detectors connected to hplc can give different results. in an hplc analysis with a spectrometric detection, it has been reported the inability in detection of some human harmful cop due to poor uv absorption careri et al. 1998). 11.4 gas chromatography (gc) the gas chromatography is the common method of cop determination using derivatized cop as trimethylsilyl ether (tmse) derivatives. this derivatization method avoids peak tailing and it improves the thermal stability of some hydroxy cholesterols. there are few factors affecting cop response in gc such as injection technique and conditions, reagents, and conditions use to get derivatives (guardiola, et al. 2002).gc gives an accurate quantification of cop with a flame ionization detector (fid), or with a mass selective detector (ms/msd). it has been observed a clear separation of cop with a good resolution when a silica capillary (dimethyl poly siloxane) column connected to gc (tai et al. 1999). the best combination of shingla and mehta int. j. of health, animal science and food safety 4 (2018) 13 39 32 haf © 2013 vol. v, no. 1 issn: 2283-3927 techniques to identify cop rapidly and sensitively is gc-ms with a selected ion monitoring mode (sim) and a capillary column. the outcome of this, the mass spectrum can be compared with the chemical library to identify cop with high accuracy (tai et al. 1999; guardiola et al. 2002). 12 conclusion cholesterol is one of the components of biological membranes and found in all body tissues. though cholesterol is vital to the body, the plasma cholesterol is associated with coronary heart disease. cholesterol undergoes autoxidation, photo oxidation, and enzymatic oxidation, producing relevant hydroperoxides. the cops are associated with various health diseases. accurate determination of cholesterol contents in foods is very essential for the regulatory aspects of food labeling, especially because of the fact that cholesterol is related to human 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(2002). quantification of cholesterol in foods using non-aqueous capillary electrophoresis. j. chromatogr b 768(2), 369-373. yen, g.c., tsai, l.j., (1995). cholesterol removal from a lard-w m x w βcyclodextrin. j. food sci 60, 561-565. zarrou a v j x a m j ’ y h m 'b n g 2014. involvement of oxysterols in age-related diseases and ageing processes. ageing res. rev 18, 148–162. zubillaga, m.p., maerker, g., (1991). quantification of 3 cholesterol oxidation-products in raw meat and chicken. j. food sci 56: 1194-1196. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l following the concepts of precision feeding, the right components balance (sova et al., 2014) and the correct particle size distribution (psd, khan et al., 2014) of total mixed ration (tmr) are essential for a complete homogeneity of the diet and are strongly influenced by adopted mixing time (mt, humer et al., 2018, schingoethe et al.,2017). the aim of the trial was to determine the influence of two mts (mt1≤7min and mt2>7min) on the chemical homogeneity and psd along the feeding alley (fa). diets were performed with a horizontal cutter-mixer wagon (gulliver 4016, sgariboldi), and tmr samples were collected from the beginning, middle and end of the feeding alley after discharge. triplicate samples of the diet were collected for chemical composition analyses (moisture, cp, ash, ee, ndf and adf) and for psd evaluation (heinrichs and kononoff, 2002) over two months (two sampling/week). statistical analysis was performed by a proc mixed for repeated measurements of sas (sas institute, cary, nc, 2015). mt1 evidenced a non-uniform distribution of moisture content along the feeding alley (p=0.05, figure 1): lower moisture was found at the end of the feeding alley compared to the beginning and the middle (47.55 vs 51.13 and 51.00%, respectively; p<0.01). no significant effects of mts were recorded for other chemical parameters. the psd showed trend to a higher retained amount of fibre in mt1 upper sieve (14.79 vs. 10.14%; p=0.06), while lower amount of feed was found in middle and bottom sieve than mt2 (38.9 and 12.81 vs 42.17 and 14.32%, respectively; p=0.08 and p=0.06). despite the trend for mt, mtxfa evidenced no significant differences for psd. day of sampling evidenced significant variation both in chemical and physical composition (p<0.05). obtained preliminary data evidenced the influence of mts on composition and on psd of the provided diet; results suggest to daily measure moisture of raw material in order to avoid negative changes in dry matter intake. acknowledgments: project co-funded in the framework of por fesr lombardia 2014-2020 (axis i, objective 1.b.1) keywords total mixed ration, dairy cows, mixing time, mixing wagon, particle size distribution. corresponding author vera perricone vera.perricone@unimi.it journal home page riviste.unimi.it/index.php/haf chemical homogeneity and particle size distribution of dairy cow tmr along the feeding alley with different mixing times. v. perricone1,*, a. agazzi1, a. costa1, m. lazzari1, g. savoini1, a. calcante2, f.m. tangorra1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of agricultural and environmental sciences production, landscape, agroenergy, università degli studi di milano, via celoria 2, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 68 haf © 2013 vol. v, no. 1s issn: 2283-3927 references heinrichs j. and kononoff p. 2002. evaluating particle size of forages and tmrs using the new penn state forage particle separator. pennsylvania state university, college of agricultural sciences, cooperative extension das, 02-42. humer e., petri r. m., aschenbach j. r., bradford b. j., penner g. b., tafaj m., südekum k.h and zebeli q. 2018. invited review: practical feeding management recommendations to mitigate the risk of subacute ruminal acidosis in dairy cattle. journal of dairy science. 101(2), 872-888. khan m. a., bach a., castells l., weary d. m. and von keyserlingk m. a. g. 2014. effects of particle size and moisture levels in mixed rations on the feeding behavior of dairy heifers. animal. 8(10), 1722-1727. schingoethe, d. j. 2017. a 100-year review: total mixed ration feeding of dairy cows. journal of dairy science. 100(12), 10143-10150. sova a. d., leblanc s. j., mcbride b. w. and devries t. j. 2014. accuracy and precision of total mixed rations fed on commercial dairy farms. journal of dairy science. 97, 562–571. figure 1: moisture content along the feeding alley by different mixing time. different letters refer to significant differences between different mixing time for p≤0.05. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author valentina lodde valentina.lodde@unimi.it the present studies were aimed to assess progesterone receptor membrane component-1 (pgrmc1) role in regulating bovine granulosa cells (bgc) mitosis. first, we performed immunofluorescence studies on in vitro cultured bgc collected from antral follicles, which showed that pgrmc1 localizes to the spindle apparatus in mitotic cells. then, to evaluate pgrmc1 effect on cell proliferation we silenced its expression with rna interference technique (rnai). quantitative rt-pcr and immunoblotting confirmed down-regulation of pgrmc1 expression, when compared to ctrl-rnai treated bgc (p<0.05). after 72h of culture, pgrmc1 silencing determined a lower growth rate (p<0.05) and a higher percentage of cells arrested at g2/m phase as assessed by flowcytometry (p<0.05). accordingly, live imaging studies revealed more aberrant mitosis and a delayed m-phase in pgrmc1-rnai treated cells compared to ctrl-rnai group (p<0.05). these data confirmed that pgrmc1 is directly involved in bgc mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. since pgrmc1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. to assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed pgrmc1 co-localization and direct interaction with clathrin. this is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. thus our studies set the stage for analysis aimed to further characterize pgrmc1’s mechanism of action in mitotic cells. funding: fp7-people-2011-cig-303640; fondo ricerca giovani ricercatori-15-6-3027000-54. references lodde v, peluso jj. a novel role for progesterone and progesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis. biology of reproduction 2011; 84:715-722. royle sj, bright na, lagnado l. clathrin is required for the function of the mitotic spindle. nature 2005; 434:11521157. progesterone receptor membrane component-1 in cell division: its role in bovine granulosa cells mitosis. l. terzaghi, a. m. luciano, v. lodde reproductive and developmental biology laboratory department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author raphaëlle teresa matilde maria prinsen, raphaelle.prinsen@unimi.it abstract genomic studies and their use in selection programs are having a strong impact in dairy cattle selection (e. liu et al., 2010). the first aim was to create a high resolution map of cnv regions (cnvrs) in brown swiss cattle and the characterization of identified cnvs as markers for quantitative and population genetic studies. cnvs were called in a set of 164 sires with penncnv and genocn. penncnv identified 2,377 cnvrs comprising 1,162 and 1,131 gain and loss events, respectively, and 84 regions of complex nature. genocn detected 41,519 cnvrs comprising 3,475 and 34,485 gain and loss events, respectively, and 3,559 regions of complex ones. consensus calls between algorithms were summarized to cnvrs at the population level. genocn was also used to identify total allelic content in consensus cnvrs. moreover, population haplotype frequencies were calculated. linkage disequilibrium (ld) was established between cnvs and snps in and around cnvrs. in this study the potential contribution of cnvs as genetic markers for genome wide association studies (gwas) has been assessed thanks to pic and ld values. the next aim is to investigate genomic structural variation in cattle using dense snp information in more than 1000 samples of the italian and swiss brown swiss breed genotyped on hd bovine beadchips. today there is still no cnv map available across brown swiss populations belonging to different countries. this study therefore expands the catalogue of cnvrs in the bovine genome, delivers an international based high-resolution map of cnvrs specific to brown swiss dairy cattle and will lastly provide information for gebv estimation with cnvs. liu ge, hou y, zhu b, cardone mf, jiang l, cellamare a, mitra a, alexander lj, coutinho ll, dell'aquila me, gasbarre lc, lacalandra g, li rw, matukumalli lk,,nonneman d, regitano lc, smith tp, song j, sonstegard ts, van tassell cp, ventura m, eichler ee, mcdaneld tg, keele jw: analysis of copy number variations among diverse cattle breeds. genome res 2010, 20:693-703. a high-resolution cnv map across brown swiss cattle populations. raphaëlle t.m.m. prinsen 1 , maria giuseppina strillacci 1 , karin schlangen 1 , fausta schiavini 1 , erika frigo 1 , maria cristina cozzi 1 , marlies alexandra dolezal 2 , birgit gredler 3 , alessandro bagnato 1 1 department of health, animal science and foodsafety, università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of biomedical sciences, university of veterinary medicine, veterinärplatz1, 1210, vienna, austria 3 qualitas ag, chamerstrasse 56, 6300 zug, switzerland proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords agrifood wastes, antioxidant capacity, phenolic compounds, functional feed corresponding author marta castrica marta.castrica@unimi.it journal home page riviste.unimi.it/index.php/haf abstract agri-food wastes (afw) and by-products can have the potential to be reprocessed as animal feed ingredients (bampidis, et al., 2006). the aim of this study was to determine the in vitro digestibility (ivd), the total phenolic content and the antioxidant capacity of 12 samples of afw and by-products. the ivd was assessed in all samples considered with the method developed by regmi et al. (2009). moreover, the total phenolic content and the antioxidant capacity were tested using two different extraction methods: chemical extraction and in vitro physiological extraction, simulating monogastric digestion. further, the polyphenolic content was assessed by folin–ciocalteu assay (attard, 2013) while antioxidant capacity was determined by 2, 2-azino-bis-3 ethylbenzothiazoline-6-sulfonic acid (abts) assay. soy and wheat samples were included as controls in all the experiments performed. the results showed that the ivd values ranged from 80 to 90% dm for afw and from 66 to 98% dm for the by-products. among afw and by-products considered, the grape marc exhibited the highest polyphenolic content with a value of 4.5% w/w, followed by camilina sativa cake (1.3%w/w), olive pomace (0.7% w/w), afw (1.3% w/w), orange (1.6% w/w) and strawberry dried (1.3% w/w). the grape marc showed a significantly (p<0.05) higher abts value (573.6 μmol trolox equivalent/g) compared to the other studied samples. the in vitro physiological extraction yielded high polyphenolic content and antioxidant capacity, suggesting that during the digestion the bioaccessibility of phenolic and antioxidant compounds was improved. the results obtained in this study indicate that afw and byproducts are relatively high digestible and they may represent a source of antioxidants and phenolic compounds when used as feed. acknowledgments: this research was supported by fondazione banco popolare di lodi. references attard, e, 2013. a rapid microtitre plate folinciocalteu method for the assessment of polyphenols. cent. eur. j. biol. 8(1), 48-53. bampidis, v., a., robinson, p., h, 2006. citrus by-products as ruminant feeds: a review. animal feed science and technology. 128, 175-217. regmi, p. r., ferguson, n. s., zijlstra r. t, 2009. in vitro digestibility techniques to predict apparent total tract energy digestibility of wheat in grower pigs. journal of animal science. 87, 3620–3629 valorisation of agri-food wastes and by-products as animal feed: digestibility, polyphenolic content and antioxidant capacity marta castrica1*, carlotta giromini1, raffaella rebucci1, davide gottardo1, valentino bontempo1, antonella baldi1 1 university of milan, department of health, animal science and food safety, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l stephanie mckay dr. stephanie mckay was born and raised in texas where she attended texas a&m university and received her undergraduate degrees in biochemistry and genetics. while working as a full time lab technician in a bovine genomics laboratory, dr. mckay worked on her m.s. in veterinary microbiology. after receiving her master's degree, stephanie ventured to the university of alberta in edmonton, alberta, canada where she received her ph.d. in animal science in 2007. dr. mckay has had varied research experience including generating transgenic mice, working in biohazardous disease related laboratories and predominantly working with the bovine genome. her work with the bovine genome started as an undergraduate student and has evolved with time as research with the bovine genome has grown. currently the mckay lab performs genetic, genomic, epigenetic and epigenomic work with beef cattle. our primary interest is identifying the proportion of epigenetic variation associated with complex traits. stephanie.mckay@uvm.edu to what extent does dna methylation affect phenotypic variation in cattle? stephanie mckay 1 1 university of vermont, department of animal veterinary sciences, 304 terrill hall570 main st. burlington, vt 05405usa. main lecture dna methylation is an environmentally influenced epigenetic modification that regulates gene transcription and has the potential to influence variation in economically important phenotypes in agricultural species. we have utilized a novel approach to evaluate the relationship between genetic and epigenetic variation and downstream phenotypes. to begin with, we have integrated rnaseq and methyl binding domain sequencing (mbd-seq) data in order to determine the extent to which dna methylation affects phenotypic variation in economically important traits of cattle. mbd-seq is a technique that involves the sample enrichment of methylated genomic regions followed by their next-generation sequencing. this study utilized illumina next generation sequencing technology to perform both rna-seq and mbd-seq. nextgene software (softgenetics, state college, pa) was employed for quality trimming and aligning the sequence reads to the umd3.1 bovine reference genome, generating counts of matched reads and methylated peak identification. subsequently, we identified and quantified genome-wide methylated regions and characterized the extent of differential methylation and differential expression between two groups of animals with extreme phenotypes. the program edger from the r software package (version 3.0.1) was employed for identifying differentially methylated regions and regions of differential expression. finally, partial correlation with information theory (pcit) was performed to identify transcripts and methylation events that exhibit differential hubbing. a differential hub is defined as a gene network hub that is more highly connected in one treatment group than the other. this analysis produced every possible pair-wise interaction that subsequently enabled us to look at network interactions of how methylation affects expression. (coexpression, co-methylation, methylation x expression). genomic regions of interest derived from this analysis were then aligned to previously identified qtl and gwas regions using animal qtl database. the approach described here has provided us with evidence that qtl and gwas regions overlay genomic regions where methylation may regulate transcription. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l tecalbo is an effective yet invasive surgical technique to treat end-stage internal and external otitis in rabbits (csomos et al 2016). the aim of this study is to describe a new locoregional analgesic approach, as until now only systemic analgesia was described for this surgery in rabbits. the first patient, a 7-year-old lop-eared neutered male weighing 2.1 kg, undergoing right tecalbo due to end-stage otitis with bulla empyema, received subcutaneous dexmedetomidine (60 µg/kg) and ketamine (10 mg/kg). propofol 0.5% (1 mg/kg) was administered for induction while titrate-to-effect isoflurane was administered for maintenance. after palpating the zygomatic arch, the mandibular nerve was blocked at the level of the temporomandibular joint. the auriculopalpebral nerve was blocked in the centre of a triangle identified between the base of the pinna and the occipital crest (ravasio et al 2008). ropivacaine 0.5% (1.5 mg/kg per block) was injected after neurolocation of the nerves. neither intraoperative (mean heart and respiratory rate 175 ± 22 and 25 ± 5 respectively) nor postoperative nociception were shown as no rescue analgesia was needed. full recovery and food intake occurred within 1 hour after awakening. the second patient, a 9-year-old dutch-belted intact female of 1.5 kg undergoing right tecalbo for end-stage otitis, received dexmedetomidine (40 µg/kg), ketamine (7 mg/kg) and midazolam (0.3 mg/kg) subcutaneously. propofol 0.5% (2 mg/kg) was administered for induction. titrate-to-effect isoflurane was administered for maintenance. mandibular and auriculopalpebral nerve blocks were performed as described. no intraoperative (mean heart and respiratory rate 195 ± 15 and 31 ± 7 respectively) nor postoperative nociception were recorded, despite delayed awakening and full recovery were shown (food intake occurred 2.5 hours after awakening) and were probably related to midazolam administration. in both cases, the combination of mandibular and auriculopalpebral blocks appeared to provide effective perioperative analgesia for tecalbo surgery in rabbits (figure 1). keywords tecalbo, rabbit, analgesia, locoregional anaesthesia, perineural block. corresponding author elisa silvia d’urso elisasilvia.durso@unimi.it journal home page riviste.unimi.it/index.php/haf locoregional anaesthesia for perioperative pain management in rabbits undergoing total ear canal ablation and lateral bulla osteotomy (tecalbo): description of two cases. e.s. d’urso1,*, d.d. zani1, m. dacerno1, d. gioeni1, v. rabbogliatti1, g. ravasio1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 72 haf © 2013 vol. v, no. 1s issn: 2283-3927 references csomos r, bosscher g, mans c, hardie r, 2016, surgical management of ear diseases in rabbits. vet clin exot anim 19, 189–204. ravasio g, carotenuto a, gobbi r, borghi l, boano s, greci v, mortellaro cm, fonda d, 2008, analgesic effects of trigeminal/facial nerve blockade by ropivacaine or bupivacaine in dogs undergoing total ear canal ablation/lateral bulla osteotomy: comparison with those obtained by morphine systemic use. proceedings of association of veterinary anaesthetists, ava meeting barcelona, spain, 14-16 october, 68. figure 1: neurostimulator connected to an insulated needle which is used to perform neurostimulated tecalbo blocks in rabbits. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l modern farming is increasingly relying on in vitro embryo production (ivp). however, in vitro culture systems are not yet capable to fully support oocyte and early embryonic development. recent studies in mice have revealed the importance of zinc in regulating oogenesis and early embryogenesis (bernhardt et al. 2012; tian & diaz, 2013). moreover, zinc transporters are differentially expressed in different cell types throughout several steps of oogenesis and embryogenesis in cattle (dieci et al. 2016; http://emb-bioinfo.fsaa.ulaval.ca/image/). nevertheless, zinc modulation during culture of oocytes and early embryos has not been yet implemented in cattle. thus, we assessed whether zinc is one of the essential players in the process of bovine oocyte competence acquisition and embryo quality by supplementing with 0.01-25 µg/ml of zinc the media used to 1) culture growing oocytes collected from small antral follicle, 2) mature fully-grown oocytes collected from middle-size antral follicles and 3) culture in vitro produced zygotes (figure 1). oocytes cultured under standard conditions served as control group. zinc supplementation during 24 hours of oocytes in vitro growth significantly improved the proportion of oocytes reaching the metaphase-ii stage of meiosis after subsequent standard in vitro maturation (ivm; 41.53±5.51 vs 57.79±4.32; p<0.05 onewayanova). on the contrary supplementation of ivm or embryo culture media had no effects on blastocysts rates and embryo quality, as assessed by cell number per embryo. in conclusion, zinc supplementation can greatly improve the exploitation of the ovarian reserve since it increases the meiotic competence of growing oocytes, which are usually discarded in standard ivp settings. these results are in accordance with in vivo studies in mouse, showing the detrimental effects of zinc deficiency before ovulation on subsequent maturation and embryonic development (tian & diaz, 2013). on the contrary, bovine fully-grown oocyte from seems able to compensate for zinc absence in the in vitro culture medium. keywords zinc sulfate, assisted reproductive technologies, oogenesis, embryogenesis, fertility. corresponding author rodrigo g barros rodrigo.garcia@unimi.it journal home page riviste.unimi.it/index.php/haf study on the effects of zinc supplementation during in vitro embryo production technologies in cattle. r.g. barros1,*, v. lodde1, c. dieci1, f. franciosi1, a.m. luciano1 1 reproductive and developmental biology laboratory, department of health, animal science and food safety, university of milan, via celoria 10 -20133, milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 38 haf © 2013 vol. v, no. 1s issn: 2283-3927 references bernhardt ml, kong by, kim am, o'halloran tv & woodruff tk 2012 a zinc-dependent mechanism regulates meiotic progression in mammalian oocytes. biol reprod 86 114. dieci c, lodde v, labreque r, dufort i, tessaro i, sirard ma & luciano am 2016 differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production. mol hum reprod 22 882-897. tian x & diaz fj 2013 acute dietary zinc deficiency before conception compromises oocyte epigenetic programming and disrupts embryonic development. dev biol 376 51-61. figure 1: schematic illustration of experimental design to assess the role of zinc during a) oocyte in vitro culture of growing oocytes (ivco) collected from small antral follicle, b) during in vitro maturation (ivm) of fully-grown oocytes collected from middle-size antral follicles and c) during in vitro embryo culture (ivc) after in vitro fertilization. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords normothermic extracorporeal perfusion, porcine, liver corresponding author matteo ghiringhelli matteo.ghiringhelli@unimi.it journal home page riviste.unimi.it/index.php/haf isolated slaughterhouse liver as model for normothermic perfusion after warm and cold ischemia: single case report matteo ghiringhelli1*, stefano brizzola1, filippo consolo3, alessia di giancamillo1, marco trovatelli1, piera a. martino2, camilla mocchi1, angelica stranieri2, tiziana vitiello2, eleonora fusi1, valentino bontempo1, fabio acocella1 1university of milan, department of health, animal science and food safety, italy 2university of milan, department of veterinary medicine, italy 3politecnico di milano, dipartimento di elettronica, informazione e bioingegneria, italy abstract liver transplantation is an ultimate procedure in patients suffering end-stage liver diseases. in these last years the donation after cardiac death (dcd) has increased the pool of potential liver donors. different studies and procedures are involved in the prevention of the main ischemic problems during the reconditioning and resuscitation of the marginal livers. normothermic extracorporeal liver perfusion (nelp) avoids prolonged cold storage damage that is the main cause of steatosis and biliary tract ischemia in transplanted patiens. different porcine models have been studied and developed to understand the ischemia mechanism and to select the better technique for nelp (bellomo et al., 2012). we conducted our study using a dcd pig liver model collected from slaughterhouse (grosse-siestrup et al., 2002). using extracorporeal membrane oxygenation, 2000 ml of total fluid containing autologous blood, lidocaine, heparin, antibiotics, glucose 10 % solution and flunixin, the nelp was achieved (guibert et al., 2011). the liver was perfused over 7 hours after 48 hours of cold storage (4c°), using eurocollins solution. during the liver withdrawal in the slaughterhouse 20 minutes were waited to simulate the warm ischemia (wi) time. histological samples, swab for bacterial grow, blood sample from venous drainage line, temperature and pulse oximetry saturation were collected to assess the liver viability and function. liver function, reflected by urea, albumin, total cholesterol, total bilirubin was seen during nelp. biochemical and microscopic assessment and an improved oxygen uptake revealed minimal injury sustained during perfusion. these analyses revealed stable metabolism throughout perfusion identifying a cycles 2 hours length, coinciding with recovery of oxygen uptake rates to fresh liver, as described in literature. in summary the preliminary established model of isolated hemoperfused slatherhouse liver reveals the important role of the relation between cold storage and normothermic perfusion. moreover this preliminary study justifies further investigation of the optimization of the treatment protocols and perfusion media. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: results obtained during normothermic preservation. (a) haematoxylin-eosin-stained liver histology, x 4 magnification. biopsies taken after 7 hours of perfusion. (b) graph displaying references bellomo, r., suzuki, s., marino, b., starkey, gk., chambers, b., fink, m.a., wang, b.z., houston s., eastwood, g., calzavacca, p., glassford, n., skene, a., jones, d.a., jones, r., 2012. normothermic extracorporeal perfusion of isolated porcine liver after warm ischaemia: a preliminary report. crit care resusc. 14(3), 173-6. guibert, e.e., petrenko, a.y., balaban, c.l., somov, a.y., rodriguez, j.v., fuller, b.j., 2011. organ preservation: current concepts and new strategies for the next decade. transfus med hemother. 38(2), 125-142. grosse-siestrup, c., pfeffer, j., unger, v., nagel, s., witt, c., fischer, a., groneberg, d.a., 2002. isolated hemoperfused slaughterhouse livers as a valid model to study hepatotoxicity. toxicol pathol. 30(6), 749-54. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in livestock, genomics has been used since a decade in combination with phenotypic information for the estimation of breeding values. in honey bees (apis mellifera), the advantage for including genomics in selective breeding programmes is represented by the possibility to reduce the generation interval and increase the accuracies of estimated breeding values resulting in higher genetic gain (brascamp et al., 2018). the limit for this application is dna extraction. extraction methods for small animals such as insects often rely upon destructive approaches. the challenge is to develop tissue sampling methods that permit the survival of the animal while providing adequate quality dna for genotyping. along with previous reports of dna extraction from several matrices, this study aims to contribute in developing suitable methodologies for genotyping honey bees queens using dna extracted from wing cuttings (chaline et al., 2004; gregory and rinderer, 2004; gould et al., 2011). the clipping of the queen wings in beekeeping is a common practice and it ensures the survival and normal activities of the animal (forster, 1971). a total of 57 queens with known pedigree were enrolled for this study. wings from each queen were cut and stored at -20°c until processed (figure 1). extractions were carried out using a modified protocol provided by qiagen (dneasy® blood & tissue). the modification consists in an initial incubation of the samples with proteinase k for 20 minutes, further steps are carried out following the manufacturer’s instructions. to test the suitability of the extracted dna for genotyping, pcr was performed on esterase fe4 like gene. although quantification with nanodrop™ resulted in <20 ng/μl of dna in solution, the extracted material was sufficient for pcr amplification of candidate genes for sequencing and genotyping. keywords honey bee, dna extraction, queen, wings clipping, sequencing corresponding author elena facchini elena.facchini@unimi.it journal home page riviste.unimi.it/index.php/haf dna extraction from wings as a suitable approach for queen bees genotyping. e. facchini1,*, r. rizzi1, s. chessa2 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 institute of agricultural biology and biotechnology, national research council, via einstein, 26900, lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 90 haf © 2013 vol. v, no. 1s issn: 2283-3927 our results show that it is possible to extract dna from wings’ cuttings permitting to implement genomic approaches in honey bee selective breeding. further work will analyse possible association between genetic variability and phenotypes of interest. references brascamp, e. w., t. h. v. wanders, y. c. j. wientjes, p bijma. 2018. prospects for genomic selection in honey-bee breeding. proceedings of the world congress on genetics applied to livestock productions. 11-16 february 2018. châline, n., f. l. w. ratnieks, n. e. raine, n. s. badcock, t. burke. 2004. non-lethal sampling of honey bee, apis mellifera, dna using wing tips. apidologie 35: 311-318. forster, i. w. 1971. effect of clipping queen honey bees' wings. new zealand journal of agricultural research. 14:2, 535-537. gould, e., m. taylor, s. j. holmes. 2011. a more consistent method for extracting and amplifying dna from bee wings. apidologie 42: 721-727. gregory, p. g., t. e. rinderer. 2004. non-destructive sources of dna used to genotype honey bee (apis mellifera) queens. entomologia experimentalis et applicata 111(3):173-177. figure 1: honey bee queen of apis mellifera. the picture shows how wings cutting were sampled. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l to date, knowledge about leukemias in feline patients is poor. classification of leukemias is mostly based on morphological, immunological and genetic aspects in dogs and humans (harvey, 2012). however, in cat poor literature about classification of leukemias via flow cytometric (fc) immunophenotyping is available and just a few publications of case reports are present (gelain et al., 2006; sharifi et al., 2007; shirani et al., 2011). we retrospectively selected 44 cases of leukemias from our fc service database from 2009 to 2017 with the aim to describe the major immunological features and define the prevalence of different subtypes. blood and/or bone marrow submitted for fc analysis with a prevalent hematological presentation were selected. cases with an evident lymph node enlargement or with clinical aspects suggesting a solid lesion (lymphoma) were excluded. cases were classified depending on antigen expression and hematologic features in five groups: chronic lymphocytic leukemias (cll), acute lymphoblastic leukemias (all), acute myelogenous leukemias (aml), acute undifferentiated leukemias (aul) and undetermined all vs lymphoma stage v. 26 cases (59%) were classified as acute leukemias whereas 11 cases (25%) were classified as chronic leukemias; 7 cases (16%) were classified as undetermined. among acute leukemias 10 (38.5%) were aml, 10 (38.5%) aul and 6 (23%) all. all all were of t cell origin (cd3+ or cd5+). among chronic leukemias, 10 (91%) were of t cell origin and among these, 80% expressed cd4 (t-helper lymphocytes), while 20% were double negative (cd4cd8-). these results confirmed that t cell leukemias are more frequent than b ones in the cat with a prevalent t-helper phenotypes as previously described (helfand and kisseberth, 2010). amls were highly represented, but the lack of an adequate panel of specific antibodies for myeloid lineage rendered a high number of aul in our caseload. similarly, the lack of an anti-feline cd34 antibody did not permit differential diagnosis of acute leukemia vs lymphoma with blood involvement in a remarkable percentage of cases without an evident nodal enlargement and without an extreme leukocytosis. keywords feline, flow cytometry, leukemia, classification. corresponding author serena bernardi serena.bernardi@unimi.it journal home page riviste.unimi.it/index.php/haf immunophenotypical and cytomorphological examination of feline peripheral blood in patients with suspect leukemia. s. bernardi1*, v. martini1, s. costanzo1, m. cozzi1, s. comazzi1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 58 haf © 2013 vol. v, no. 1s issn: 2283-3927 references gelain m.e., antoniazzi e., bertazzolo w., zaccolo m., comazzi s., 2006. chronic eosinophilic leukemia in a cat: cytochemical and immunophenotypical features. vet clin path. dec,35 (4): 454-9. harvey j.w., 2012. veterinary hematology: a diagnostic guide and color atlas. saunders, elsevier inc. helfand, c.s and kisseberth, w.c., 2010. general features of leukemia and lymphoma. schalm’s veterinary hematology. blackwell publishing ltd. p 455-465. sharifi h., nassiri s.m., esmaelli h., khoshnega j., 2007. eosinophilic leukemia in a cat. j feline med surg. dec,9(6): 514-7. shirani d., nassiri s.m., aldavood s.j., sheddig h.s., fathi e., 2011. acute erythroid leukemia with multilineage dysplasia in a cat. can vet j. apr,52(4): 389-93. figure 1: peripheral blood: evaluation of morphology of white blood cells in a blood sample with suspect acute leukemia. large mononuclear cells are present, with modest basophilic cytoplasm, rounded nuclei with a single large nucleolus and dispersed chromatin. fc analysis revealed a cd14 positivity and negativity to common lymphocyte markers: the final diagnosis was aml. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l aim of eden 2020 project is the development of a steerable catheter for ced system in glioblastoma therapy. the vet group is involved in realization and validation of the proper animal model. for surgical planning purpose a diffusion tensor imaging (dti) of white matter tracts in the sheep is necessary to identify the target points useful for the catheter introduction. the analysis of the sheep brain under a scanning electron microscope (sem) is required to understand any alterations due to the catheter introduction and to fluids injection during ced administration. animals were treated in accordance with the european communities council directive (86/609/eec), to the laws and regulations on animal welfare enclosed in d.l.g.s. 26/2014 a total of five 70 kg female, one year old, sheep were used for the study. all animals, under general anesthesia, underwent to magnetic resonance imaging (mri) acquisition. mri scanner used was philips ingenia 1.5 tesla system. once the dti imaging were acquired the animals were euthanased, sheep brain was collected and samples of white matter tracts obtained with disposable biopsy punches of 1.5-2 mm of diameter. the samples were fixed, stained in osmium tetroxide (oso4) and then embedded with two different protocols (cold curing vs thermal curing) in resin for the focused ion beam (fib) sem analyses. all the dti images were uploaded to trackvis software and major white matter fiber tracts analysed. corticospinal tract, visual radiation, fornix and fronto-occipital fasciculus were identified. corticospinal tract (figure 1) was identified as major white matter tract in sheep brain and useful as target area for the research aims. for the sem analysis the thermal protocol was recognised as better curing methods for the research purpose than cold curing one.the conclusions are missing while waiting for the data to be processed. the research is connected with researches of human medicine to improve the glioblastoma multiforme therapy. acknowledgment: the project has received funding from the european union’s eu research and innovation programme horizon 2020 (no 688279). keywords sheep, brain, animal model, dti, sem. corresponding author davide danilo zani davide.zani@unimi.it journal home page riviste.unimi.it/index.php/haf sheep brain atlas creation. diffusion tensor imaging and scanning electron microscope in sheep brain analysis m. trovatelli1, a. bernardini2, va pieri3, f. acocella1, s. brizzola4, d.d. zani*4 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of mechanical engineering, imperial college london, south kensington campus, london sw7 2az, united kingdom . 3 neuroradiology unit, vita-salute san raffaele university and irccs san raffaele scientific institute, via olgettina 58-60, 20132, milan, italy. 4 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 62 haf © 2013 vol. v, no. 1s issn: 2283-3927 references lee, w. lee, s, d. park, m, y. foley, l. purcell-estabrook, e. kim, h. yoo, s. 2015, functional and diffusion tenor magnetic resonance imaging of the sheep brain. bmc vet res,. 11: 262. assaf, y. pasternak, o. 2008, diffusion tensor imaging (dti)-based white matter mapping in brain research: a review. j mol neurosci,. 34(1): p. 51-61. saucier-sawyer, j. seo, y. gaudin, a. quijano, e. song, e. sawyer, a, deng, y. huttner, a. saltzman, w. m. 2016, distribution of polymer nanoparticles by convection-enhanced delivery to brain tumors. j control release. 232: 103-112. juratli, t.a., g. schackert, and d. krex, 2013. current status of local therapy in malignant gliomas—a clinical review of three selected approaches. pharmacol ther. 139(3): p. 341-58. figure 1: sheep corticospinal tract. in this picture is showed the corticospinal tract merged with a mri image acquired during the study. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the histopathological assessment of the first node receiving lymphatic drainage from a tumor – defined as sentinel lymph node (sln) – is essential to determine stage, therapy and outcome in oncological patients. both in human and veterinary medicine, lymphoscintigraphy is a recognized procedure for sln detection (mariani et al., 2004; tuohy et al., 2009; beer et al., 2017). in this study, we want to determine the most suitable pre-operative planar lymphoscintigraphy protocol for sln mapping in dogs with mast cell tumor (mct). we selected 5 dogs diagnosed with cutaneous mtc, with clinically negative lymph nodes and no distant metastasis, undergoing surgical tumor removal, and we obtained owner’s written consent. planar lymphoscintigraphy was performed in patients under general anesthesia, after subcutaneous peritumoral injection of different doses of technetium-99m (tc-99m) labelled colloid diluted reaching a 0.5 ml volume (worley, 2014). the megabecquerel value (mbq) of the syringe was measured before and after the injection. dynamic images (1 frame/second for 60 seconds) were taken at the moment of the injection, 3 and 8 minutes after the injection. ventrodorsal (vd) and lateral (l) static images (120 seconds/frame) were taken until the identification of sln had been made. if needed, the injection site was masked with a 2-mm lead foil. results are showed in table 1. in patient 1, the sln was not identify, probably due to a superimposition with the injection site. during the study, we increased the injected mbq dose, in order to better visualize lymphatic path and sln (balogh et al., 2002). in fact, the number of static images needed to identify sln have been reduced from 8,7 to 6. masking the injection site proved to be useful for a better visualization of sln. dynamic images showed to be unnecessary for the sln identification. for further studies, we suggest the injection of minimum 23,5 mbq tc-99m activity and the acquisition of vd and l static images with and without masking the injection site. keywords sentinel lymph node, mast cell tumor, lymphoscintigraphy, diagnostic imaging, oncology. corresponding author martina manfredi martina.manfredi@unimi.it journal home page riviste.unimi.it/index.php/haf planar lymphoscintigraphy for sentinel lymph node mapping in dogs with mast cell tumor: a pilot study. m. manfredi1,*, d. de zani1, d.d. zani1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 40 haf © 2013 vol. v, no. 1s issn: 2283-3927 references balogh, l., thuroczy, j., andocs, g., mathé, d., chaudhari, p., perge, e., biksi, i., polyák, a., király, r., jánoki, g., a., 2002. sentinel lymph node detection in canine oncological patients. nuclear medicine review. 5(2), 139-144. beer, p., pozzi, a., rohrer bley, c., bacon, n., pfammatter, n., s., venzin, c., 2018. the role of sentinel lymph node mapping in small animal veterinary medicine: a comparison with current approaches in human medicine. veterinary and comparative oncology. 16(2), 178-187. mariani, g., erba, p., villa, g., gipponi, m., manca, g., boni, g., buffoni, f., castagnola, f., paganelli, g., strauss, h., w., 2004. lymphoscintigraphic and intraoperative detection of the sentinel lymph node in breast cancer patients: the nuclear medicine perspective. journal of surgical oncology. 85(3), 112-122. tuohy, j., l., milgram, j., worley, d., r., dernell, w., s., 2009. a review of sentinel lymph node evaluation and the need for its incorporation into veterinary oncology. veterinary and comparative oncology. 7(2), 81-91. worley, d., r., 2014. incorporation of sentinel lymph node mapping in dogs with mast cell tumours: 20 consecutive procedures. veterinary and comparative oncology. 12(3), 215-226. table 1: distribution of mast cell tumor (mct) site, radioactive activity values of injected technetium-99m (mbq), number of dynamic (di) and static images (si) acquired and lymph node (ln) identified as the sentinel lymph node (sln). the patient 4 had 2 cutaneous mct injected for sln detection. patient mct site injected mbq di si identified sln pat.1 right shoulder 8,2 3 8 0 pat.2 left cranial abdomen 7,5 3 7 2 left accessory axillary ln pat.3 left flank 7,9 3 11 1 left accessory axillary ln pat.4a right knee 25,8 3 6 1 right inguinal ln pat.4b left thigh 26,7 3 6 1 left inguinal ln pat.5 right knee 23,5 3 6 1 right popliteal ln, 1 right inguinal ln http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the italian goat autochthonous breeds are appreciated for their milk and characteristics like the rusticity, frugality, fertility and longevity. therefore the local goat breeds play an important role in the livestock sector, and it is important to guarantee sanitary strategies control, prevention or treatment of diseases. the hematological parameters in goats undergo changes in relation with many factors like breed, age (piccione g. et al, 2014), physiological status, environment and stress (waziri m.a. et al, 2010). based on these differences it is necessary to establish appropriate physiological baseline values for every single breed in order to obtain a realistic evaluation of physiological or pathological status of the animal (arfuso f. et al, 2016). the aim of this work was to evaluate the differences between a local goat breed (verzasca) and a cosmopolite one (alpine) from the hematological point of view, and to establish hematological reference values. a total number of 71 healthy female goats, of alpine (n=37), and verzasca (n=34) were enrolled for this study, for a total of 716 blood samples. all animals were reared together and exposed to natural photoperiod. data were processed by a mixed model-repeated measures ancova in order to evaluate the effects of breed, parity, and season while baseline values for each breed have been calculated by evaluating the 2.5-97.5th percentile of variables distribution. the results showed that the breeds differ in a significant manner (table 1). verzasca goat shows significantly higher values in the erythroid parameters, whereas the alpine goat shows higher mean values of leucocyte count and absolute neutrophil count. a further interesting result is the neutrophil lymphocyte ratio which is 0,96 in the alpine and 0,57 in the verzasca. these results can add some knowledge to the definition of the health status of the two breeds, evidencing some environmental and physiological variation mechanisms. keywords goat, alpine, verzasca, biodiversity, hematology. corresponding author giulio curone giulio.curone@unimi.it journal home page riviste.unimi.it/index.php/haf blood baseline values in female alpine and nera di verzasca goats reared in italy. g. curone1,*, a. gazzonis1, s. zanzani1, d. ponzoni1, d. vigo1, m. t. manfredi1, f. riva1, p. moroni1,2, f.vitali1, m. faustini1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. 2 cornell university, animal health diagnostic center, quality milk production services, 14853 ithaca, ny, usa. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 44 haf © 2013 vol. v, no. 1s issn: 2283-3927 references arfuso f., fazio f., rizzo m., marafioti s., zanghì e., piccione g., 2016. factors affecting the haematological parameters in different goat breeds from italy. annals of animal science. 16, 743-757. piccione g., monteverde v., rizzo m., vazzana i., assenza a., zumbo a., niutta p.p., 2014. reference intervals of some electrophoretic and haematological parameters in italian goats: comparison between girgentana and aspromontana breeds. journal of applied animal research. 42, 434–439. waziri m.a., ribadu a.y., sivachelvan n., 2010. changes in the serum proteins, hematological and some serum biochemical profiles in the gestation period in the sahel goats. veterinarski arhiv 80, 215-224. table 1: hematological parameters in the two breeds of goats. sd-standard deviation; † p<0.001 difference between breeds. slopes and their statistical significances as a function of time in years are reported, as well as random factor significances. *p<0.05; **p<0.01; ***p<0.001. alpine verzasca age effect, slope subject effect variable mean sd mean sd alpine verzasca p<0.05 rbc (m/µl) 12.95† 1.85 14.37 1.35 -0.080 -0.320*** hgb (g/l) 86.60† 13.10 96.60 9.70 -0.400 -2.500*** pcv (%) 0.25† 0.03 0.27 0.03 0.001 -0.005*** mcv (fl) 19.72† 1.96 19.08 1.76 0.173** 0.129** mch (pg) 6.70 0.53 6.74 0.46 0.044** -0.026* mchc (g/dl) 34.20† 3.19 35.51 3.09 -0.062 -0.359*** rdw (%) 32.76† 4.80 34.91 4.27 -0.357** -0.757*** wbc (106/l) 8.60† 3.15 5.95 2.25 -0.365*** -0.491*** neu (106/l) 3.26† 2.07 1.80 1.11 -0.020 -0.061* lymph (106/l) 4.59 2.24 3.66 1.73 -0.327*** -0.399*** mono (106/l) 0.47 0.44 0.28 0.19 -0.031** -0.012** eos (106/l) 0.24 0.22 0.17 0.17 0.014* -0.021*** bas (106/l) 0.08 0.06 0.05 0.04 -0.001 -0.001 neu (fraction) 0.37 0.15 0.30 0.12 0.015*** 0.016*** lymph (fraction) 0.53 0.17 0.61 0.13 -0.019*** -0.017*** mono (fraction) 0.06 0.05 0.05 0.04 -0.003 0.002 eos (fraction) 0.03 0.03 0.03 0.03 0.003*** -0.002 bas (fraction) 0.01 0.01 0.01 0.01 0.001 0.005* n/l 0.96 1.63 0.57 0.38 0.053** 0.051*** http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in zoo practice, for physical examination or medical procedure in captive tigers, chemical immobilization is needed and ketamine (ket) in association with sedatives is an option frequently used (clark-price et al., 2015). aims of the study is the assessment of the pharmacokinetics of ket and its main metabolite, norketamine (norket), after its intramuscular administration in combination with dexmedetomidine in tigers. nineteen adult captive tigers, from different zoos, were scheduled for periodic physical examination or diagnostic procedures at the milan university facilities. all animals were administered with a combination of ket at 2 mg/kg and dexmedetomidine at 10 µg/kg, given intramuscularly through blowpipe darts. if necessary, tigers where re-administered with variable doses of ket and dexmedetomidine or other drugs. when animals were sufficiently sedated, blood samples were collected every 5-10 min for the time tigers were safely approachable. nine animals were assigned to standard protocol group (ket 2 mg/kg and dexmedetomidine 10 µg/kg) and ten animals to non-standard protocol group (tigers administered with different doses of ket, 2 – 2.5 mg/kg, and dexmedetomidine 10 – 30 µg/kg or with any other necessary drug, such as titrate-to-effect propofol and isoflurane, respectively for anaesthesia induction and maintenance). ketamine and norket were extracted from plasma according to a validated hplc-uv method (zonca et al., 2012). for pharmacokinetic assessment, ket and norket concentrations were analysed with a noncompartmental approach (phoenix® 7.0, pharsight). keywords ketamine, norketamine, dexmedetomidine, pharmacokinetics, tigers. corresponding author federica di cesare federica.dicesare@unimi.it journal home page riviste.unimi.it/index.php/haf pharmacokinetics of ketamine and norketamine following intramuscular administration combined with dexmedetomidine in tigers (panthera tigris). f. di cesare1,*, p. cagnardi1, g. ravasio2, m. capasso3, w. magnone4, r. villa1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. 3 zoo of naples, via john fitzgerald kennedy 76, 80125 naples, italy. 4 parco natura viva garda zoological park, loc. figara 40, 36012 bussolengo (verona), italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 86 haf © 2013 vol. v, no. 1s issn: 2283-3927 differences in the pharmacokinetic parameters between groups were statistically analysed (spss 25.0, spss inc.). results are reported in table 1. this is the first study that evaluates the pharmacokinetics of ket and norket in tigers. due to the harmful attitude of these animals, samples collection was limited to the period of sedation, a short time for a complete pharmacokinetic evaluation. nevertheless, we observed a favorable kinetic profile of ket and norket and, from a clinical point of view, all animals showed a good recovery, no adverse effects and a good level of sedation. standard protocol (mean ± s.d.) non-standard protocol (mean ± s.d.) ketamine hl_lambda_z min 77.62 ± 54.50 76.14 ± 67.32 tmax min 27.78 ± 7.90 49.70 ± 29.64 cmax ug/ml 0.63 ± 0.17 0.67 ± 0.19 auclast min*ug/ml 23.84 ±6.40* 35.97 ± 12.84* aumclast min*min*ug/ml 802.24 ± 331.03* 2054.97 ± 1018.88* mrtlast min 32.88 ± 5.71* 54.38 ± 19.71* norketamine tmax min 51.89 ± 8.95* 77.10 ± 24.41* cmax ug/ml 0.24 ± 0.07 0.23 ± 0.09 auclast min*ug/ml 7.30 ± 3.98 11.07 ± 5.46 aumclast min*min*ug/ml 291.94 ± 227.01* 701.87 ± 424.80* mrtlast min 36.95 ± 7.32* 58.65 ± 19.58* hl_lambda_z = elimination half-life; tmax = time to maximum concentration; cmax = maximum concentration; auclast = area under the curve to the last concentration; aumclast = area under the first moment curve to the last concentration ;mrtlast = mean residence time to the last concentration. references clark-price, s.c., lascola, k.m., schaeffer, d.j. 2015. physiological and biochemical variables in captive tigers (panthera tigris) immobilized with dexmedetomidine and ketamine or dexmedetomidine, midazolam and ketamine. veterinary record. 177, 570-574. zonca, a., ravasio, g., gallo, m., montesissa, c., carli, s., villa, r., cagnardi, p. 2012. pharmacokinetics of ketamine and propofol combination administered as ketofol via continuous infusion in cats. journal of veterinary pharmacology and therapeutics. 35, 580-587. table 1: pharmacokinetic parameters of ketamine and norketamine in nineteen adult captive tigers after intramuscular administration of 2 mg/kg of ketamine, with or without variation from the standard protocol, in combination with dexmedetomidine (with * are indicated results with p < 0.05). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l genetic selection of dairy cattle has classically been based on fertility and productivity parameters. in recent years a growing interest in characters related to health and efficiency of the animals has taken hold. the selection of animals with a high feed efficiency can bring benefits in terms of health, productivity and environmental impact. the aim of the trial was to create a dataset useful to build up genetic indexes based on feed efficiency. a first batch of 16 holstein heifers (mean age 12.63 ± 2.90 months) was selected and housed in a tie-stall of the centro zootecnico didattico sperimentale (czds) of lodi for the whole length of the trial (35 days). blood samples were collected and sent to the lab to perform genotyping of the animals. heifers were fed a composed ration of sorghum silage and total mixed ration, delivered daily on individual feeders to ensure each animal continuous access to the feed. animals had free access to the water. feed intake was obtained weighing the supplied ration and the residual the day after. individual body weight (bw), body condition score (bcs), heart girth (hg) and wither height (w) were measured weekly. feed-to-gain ratio (f:g) and residual feed intake (rfi) were calculated using dry matter intake (dmi) data. two sub-groups of 8 heifers each were ex post created based on rfi results (h-rfi, high rfi and l-rfi, low rfi; p < 0.01). data of the two groups were analysed by a mixed procedure of sas. preliminary results are reported in table 1. no significant differences were observed between h-rfi and l-rfi for age, weight, f:g and heart girth data, while lower rfi values were statistically related to lower dmi (p = 0.01) and higher wither height keywords feed efficiency, heifers, residual feed intake, genomics. corresponding author giovanni savoini giovanni.savoini@unimi.it journal home page riviste.unimi.it/index.php/haf feed efficiency of italian holstein dairy heifers in the genomic era – preliminary results. f. omodei zorini1, g. savoini1,*, r. finocchiaro2, m. cassandro3, g. invernizzi1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 associazione nazionale allevatori di razza frisona (anafi), via bergamo 292, 26100 cremona, italy. 3 dipartimento di agronomia animali alimenti risorse naturali e ambiente, università di padova, viale dell’università 16, 35020 legnaro, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 80 haf © 2013 vol. v, no. 1s issn: 2283-3927 gains (p < 0.05). future work will be focused on linking phenotypic results to genomic markers. to do so the trial will be performed on several batches to collect a larger statistical sample. moreover, the trial will be performed again on the same batch after the first parturition of the animals, so as to investigate the relationship between feed efficiency during the growth phases (use of feed energy for growth and maintenance) and the productive phases (use of feed energy for milk production). references waghorn, g.c., macdonald, k.a., williams, y., davis, s.r., spelman, r.j. 2012. measuring residual feed intake in dairy heifers fed an alfalfa (medicago sativa) cube diet. journal of dairy science 95(3):1462-71. williams, y.j., pryce, j.e., grainger, c., wales, w.j., linden, n., porker, m., hayes, b.j. 2011. variation in residual feed intake in holstein-friesian dairy heifers in southern australia. journal of dairy science 94: 4715-25. weller, j.i., ezra, e., ron, m. 2017. invited review: a perspective on the future of genomic selection in dairy cattle. journal of dairy science 100(11): 8633-44. table 1: average characteristics of the heifers with the highest (h-rfi; n = 8;) and the lowest (l-rfi; n = 8) residual feed intake (rfi) values. item h-rfi l-rfi sem p-value age (months) 12.56 12.67 1.06 0.94 initial weight (kg) 387.94 361.31 34.12 0.45 final weight (kg) 420 392.81 33.77 0.43 average daily gain (kg/d) 0.84 0.85 0.08 0.92 dry matter intake (kg) 182.36 146.84 8.72 0.01 residual feed intake (kg of dm/d) 0.35 -0.35 0.08 <0.01 feed-to-gain ratio 6.28 5.9 0.82 0.74 initial heart girth (cm) 171.94 166.56 5.59 0.35 final heart girth (cm) 180.88 174 5.52 0.23 heart girth gain (cm) 8.94 7.44 1.15 0.37 initial wither height (cm) 128.81 125.75 2.6 0.26 final wither height (cm) 131.75 130.88 2.7 0.75 wither height gain (cm) 2.94 5.13 0.71 <0.05 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords alpha1-microglobulin, urine, cat, validation, enzyme-linked immunosorbent assay, chronic kidney disease corresponding author marco giraldi marco.giraldi@unimi.it journal home page riviste.unimi.it/index.php/haf evaluation of a commercial elisa for measurement of feline urinary alpha1microglobulin. m. giraldia*, s. paltrinieria, p. scarpaa adepartment of veterinary medicine (dimevet) university of milan, via celoria 10, milano 20133, italy abstract urinary alpha1-microglobulin (a1m) in people is a biomarker of renal tubular damage (bazzi, 2001), but it has not yet been used in cats with chronic kidney disease (ckd). the aim of this study was to validate an elisa test marketed for the measurement of feline a1m. thirty-four urine supernatants collected from cats affected by or at risk for ckd were assayed by sdsage, to classify patients according to presence or absence of low molecular weight proteinuria suggestive of tubular damage. two samples with and one without tubular bands were used to evaluate intra-assay variability, linearity under dilution (lud) and spiking recovery test (srt) of the elisa. then, a1m concentration was measured in samples with (n°=10) or without (n°=25) tubular bands. the standard solution included in the kit was also assayed by sds-age. the intra-assay cvs was >20%. lud and srt showed that the test is not accurate. no significant difference was found between a1m concentration in samples with and without tubular bands (median values: 35.19 and 40.83 μg/ml respectively). sds-age on the standard solution failed to identify bands with molecular weight consistent with a1m but showed the presence of albumin. results of this investigation did not support the use of this test to measure a1m in cats likely due to the absence of a1m in the standard solution provided with the kit. references bazzi, c., petrini, c., rizza, v., arrigo, g., beltrame, a., pisano, l., & d'amico, g., 2001. urinary excretion of igg and alpha1microglobulin predicts clinical course better than extent of proteinuria in membranous nephropathy. american journal of kidney diseases, 38(2), 240–248. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l invited paper recently, a scientific interest in exercise in water in equine athletes, both from training and rehabilitation purposes has been developed. before an extended use of this practice in equine exercise physiology and medicine, the physiological adaptations to this type of exercise need to be elucidated. six horses were subjected to sessions of 40 minutes in water treadmill, with speeds of 5.5-6 km/h and with water at different depths: baseline (bl, exercise in water treadmill without water), and with the water at level of the metacarpophalangeal/metatarsophalangeal (mcpj; mtpj), tarsal (tj) and stifle joints (sj). hematology and blood lactate (la) were measured before and after each session, and heart rate (hr) was monitored with a pulsometer. horses also wore a portable gait analyzer, consisting of three orthogonal accelerometers, fixed at the sternum and at the sacrum, and the following parameters were measured: stride length (sl), stride frequency (sf), regularity (reg, measurement of the acceleration pattern similarity of successive strides in a period of time), symmetry (sym, measurement of the similarities between left and right acceleration patterns), dorsoventral displacement (dvd, displacement of the gravity center in a dorsoventral direction), dorsoventral dvp, propulsion pp, mediolateral mlp and total power tp, representing the amount of acceleration and deceleration along the dorsoventral, longitudinal and lateral axes and the sum of the three powers respectively. significant changes in hematological parameters and in blood la concentrations before and after each exercise session were not found. blood la concentrations after bl, mcpj, tj and sj sessions reached mean values of 1.03±0.41, 1.05±0.28, 1.05±0.52 and 1.11±0.22 mmol/l respectively. hr increased significantly in the four sessions, with significant differences after exercise between bl (66.13±8.92 bpm), tj (81.89±10.67 bpm) and sj (81.13±14.68 bpm) levels. further, significant differences were also found between exercise at the level of mcpj (71.09±8.90 bpm) and tj. keywords exercise, horses, performance, rehabilitation, training. corresponding author ana muñoz pv1mujua@uco.es journal home page riviste.unimi.it/index.php/haf exercise in water in the horse: physiological adaptations and potential benefits for training and rehabilitation. ana muñoz 1,* 1 department of animal medicine and surgery, equine sport medicine center cemede, school of veterinary medicine, university of córdoba, spain. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy vii haf © 2013 vol. v, no. 1s issn: 2283-3927 with the accelerometer fixed on the sternum, increased sl, dvd, dvp and tp together with a decreased sf was found with the water at the level of tj, compared to the other water depths. with the accelerometer fixed on the sacrum, significant changes were not observed in sf between the different water depths. the highest values for sl, dvd, dvp, pp, mlp and tp were found with the water at the level of the tj. our results reveal that exercise in wt, nevertheless of the water depth (at least until stifle joint), represents a mild aerobic exercise, with low hr and blood la under the aerobic threshold of 2 mmol/l. however, this type of exercise leads to profound changes in locomotion, mainly in stride length and frequency and in muscle power, with the highest values with water depths at the tarsal and stifle joints. consequently, the low cardiovascular and metabolic demands are expected to be of interest for the recovery of injured horses. further, the important effects on locomotion characteristics reflect a relevant future use for both training and rehabilitating horses. references becero, m., saitua, a., reguera, m., castejón-riber, c., herrera, r., muñoz, a. 2018. ejercicio en treadmill acuático en la rehabilitación de un caballo con tendinopatía del flexor digital superficial. ii congreso de veterinaria y ciencia y tecnología de los alimentos. herrera, r., saitua, a., becero, m., castejón-riber, c., muñoz, a. 2018. longitud de tranco y desplazamiento dorsoventral del centro de gravedad en caballos ejercitados sobre diferentes superficies, treadmill terrestre y acuático. congreso de la asociación de veterinarios especialistas en équidos. muñoz, a., castejón-riber, c., riber, c., esgueva, m., méndez-angulo, j., mateo, s., castejón, f. 2016. cambios locomotores determinados mediante acelerometría en caballos ejercitados en treadmill acuático a diferentes profundidades de agua. xvii congreso international de medicina y cirugía equina. riber, c., castejón-riber, c., esgueva, m., méndez-angulo, j.l., castejón, f., muñoz, a. 2016. cambios cinemáticos beneficiosos para rehabilitación en caballos ejercitados en treadmill acuático. congreso internacional de la sociedad española de cirugía veterinaria secive. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords honeybee, hygienic behaviour, genetic selection corresponding author elena facchini elena.facchini@unimi.it journal home page riviste.unimi.it/index.php/haf hygienic behaviour in honeybee: a comparison of two in-field assays for phenotypic characterization. e. facchinia*, m. mortarinoa, f. dell’orcoa, r. rizzia adept. dimevetuniversità degli studi di milano, via g. celoria 10, 20133, milano abstract the western honeybee, apis mellifera, represents a relevant productive livestock due to both hive products and to its indispensable role as commercial pollinator of many agricultural crops. in addition, honeybees contribute to the pollination of wild flowers, thereby helping the maintenance of natural ecosystems and biodiversity. recently, the number of managed honeybee colonies has declined in both north america and europe. beside environmental causes, e.g. the loss of forage as a consequence of agricultural intensification, another cause is the increasing relevance of pests and diseases affecting honeybee colonies. in the honeybee, hygienic behaviour (hb) is a heritable phenotype that confers to the colony resistance to foulbrood diseases, chalkbrood, and the parasitic mite varroa destructor. nurse bees manifesting hb are able to detect, uncap, and remove infested and/or parasitized pupae from the colony. the genetic and biochemical factors that drive the manifestation of this behaviour are under investigation. therefore, the selection of such trait still relies on field assays. heretofore, there are two main tests to measure hb: the pin killed brood (pkb) test and the freeze killed brood (fkb) test. concerning the fkb test, a comparison between the standard fkb test and a variant of this method (e. bonfanti) has been performed in order to optimize the methodology in terms of time, costs, feasibility and safety for the operator and to choose the best option for a subsequent large scale phenotypic characterization for genetic selection. references büchler, r., berg, s., le conte, y., 2010. breeding for resistance to varroa destructor in europe. apidologie. 41, 393–408. neumann, p., carreck. n. l., 2010. honey bee colony losses. journal of apicultural research. 49, 1-6. spivak, m., downey, d. l., 1998. field assays for hygienic behaviour in honey bees (hymenoptera: apidae). journal of economic entomology. 91, 64-70. spivak, m., reuter, g. s., 1998. honey bee hygienic behaviour. american bee journal. 138, 283. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:elena.facchini@unimi.it proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author cristina lecchi, cristina.lecchi@unimi.it abstract micrornas (mirnas) are small 21-25 nucleotide regulatory non-coding rnas that modulate gene expression in eukaryotic organisms. mirnas are complementary to the 3′-untranslated regions of mrna and act as posttranscriptional regulators of gene expression, exhibiting remarkable stability in extracellular fluids such as blood. turkey (meleagris gallopavo) farming is a species economically relevant but the lack of efficient protocols for the evaluation of commercial turkeys prevents to measure the impact of industry practices on birds productivity and welfare. in order to identify potential molecular biomarkers for monitoring stress in turkey’s handling, we investigated by taqman qpcr the abundance of five circulating mirna, namely mir-22, mir-155, mir-181a, mir-204 and mir-365, previously demonstrated to be involved in stress in chicken due to feed deprivation. road transportation related procedures were selected as stressful model for this study. the serum of twenty healthy animals was collected before and after 2h transportation. our results demonstrated that mir-22, mir-155 and mir365 are statistically more expressed after road transportation. receiver-operator characteristics (roc) analysis was used to estimate the diagnostic value of these mirnas to evaluate the stress in animals. the serum level of mir-22, mir-155 and mir-365 can discriminate stressed from non-stressed animals with an auc=0.763, 0.710 and 0.704, respectively, and the average expression of their combination has the same specificity (auc=0.745). mir-22, mir155 and mir-365 are stress-specific markers and can be considered as suitable biomarkers to identify turkeys stressed by road transportation. references ahanda m.l., zerjal t., dhorne-pollet s., rau a., cooksey a., giuffra e. 2014. plos one 9(12); wang s., ni a y., guo f., sun z., ahmed a., zhao r. 2014. domestic animal endocrinology 47:65–72; roberts t.c., coenen-stass a.m.l., wood m.j.a. 2014. plos one 9(2); marchewka j., watanabe t.t.n., ferrante v., estevez i. 2013. poultry science 92: 1467-1473. serum mirna disregulation during transport-related stress in turkey (meleagris gallopavo). a.t. marques 1 , f. ceciliani 1 , m. redegalli 1 , m. carisetti 1 , s. meani 2 , l.j. vinco 2 , v. bronzo 3 , c. lecchi 1 1 department of veterinary science and public health, università degli studi di milano, milano, italy 2 national reference centre of animal welfare, istituto zooprofilattico sperimentale della lombardia e dell’emilia romagna b. ubertini, brescia, italy 3 department of health, animal science and food safety, università degli studi di milano, milano, italy proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l feline infectious peritonitis (fip) is a fatal disease in which the definitive diagnosis is achieved by immunohistochemistry (ihc) on post-mortem biopsies (pedersen, 2007). the clinical suspicion is aroused by signalment, clinical signs and several laboratory tests, including alpha-1acid glycoprotein measurement for which the only validated kit is no longer available (paltrinieri et al., 2007). paraoxonase-1 (pon-1) is a serum enzyme with antioxidant activity, considered as a negative acute phase protein in several species (novak et al., 2010). since inflammation plays a major role in fip, and due to the high susceptibility of cats to oxidation, it could be of great interest the evaluation of this enzyme as a diagnostic marker for fip (tecles et al., 2015). the aim of this study was to measure paraoxonase-1 in healthy cats and cats with clinical signs consistent with fip (both wet or dry form), in order to evaluate the utility of this parameter in the diagnosis of fip. sixty-two cats were enrolled and divided into three groups: healthy (n=16), ihc-confirmed fip (n=22) and non fip with similar clinical signs (n=24). pon-1 was measured on one sample of serum, using an enzymatic method, already validated in cats (rossi et al., 2014). results showed significantly lower pon-1 activity in fip cats (mean ± sd: 29.1 ± 16.3 u/ml; median: 24.4; iqr: 16.638.3), compared with healthy cats (90.1 ± 24.1 u/ml; median: 86.0; iqr: 76.7-105.7; p<0.001) and with “non-fip” cats (55.9 ± 28.3 u/ml; median: 51.9; iqr: 35.7-68.8, p<0.001). a significant difference was also found between healthy and “non-fip” cats (p<0.001). the receiver operating characteristic (roc) curve demonstrated that pon-1 may discriminate cats with and without fip (auc 0.88; ci 95%, figure 1). at the cut-off that maximizes the diagnostic power of the test (40,70 u/ml), sensitivity and specificity for fip were 77% each, suggesting that pon-1 may be a reliable marker in association with other confirmatory tests and with signs consistent with the disease. keywords paraoxonase-1, feline infectious peritonitis, diagnostic marker, cut-off, cat. corresponding author sara meazzi sara.meazzi@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary data about paraoxonase-1 (pon-1) as a marker for feline infectious peritonitis (fip). s. meazzi1,*, r. ferriani3, s. paltrinieri1,2, a. giordano1,2 1 department of veterinary medicine, università degli studi di milano, via celoria, 10 – 20133 milan, italy. 2 veterinary teaching hospital, via dell’università, 6 – 26900 lodi, italy. 3 ospedale veterinario san francesco, via newton 2 – 20148, milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 48 haf © 2013 vol. v, no. 1s issn: 2283-3927 references novak f., vavrova l., kodydkova j., novak f.sr., hynkova m., zak a., novakova o., 2010. decreased paraoxonase activity in critically ill patients with sepsis. clinical and experimental medicine. 10(1), 21-25. paltrinieri s., giordano a., tranquillo v., guazzetti s., 2007. critical assessment of the diagnostic value of feline alpha1acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach. journal of veterinary diagnostic investigation. 19(3), 266-272. pedersen n.c. , 2014. an update on feline infectious peritonitis: diagnostics and therapeutics. the veterinary journal. 201, 133-141. rossi g., giordano a., costarelli e., moretti p., paltrinieri s., 2014. analytical validation of a paraoxon-based method to measure the activity of paraoxonase-1 in feline serum. 2014. acvp and asvcp annual meeting, atlanta, nov 8th-12th, 2014. published in: veterinary clinical pathology, 43:e18. tecles f., caldín m., tvarijonaviciute a., escribano d., martínez-subiela s., cerón j.j., 2015. serum biomarkers of oxidative stress in cats with feline infectious peritonitis. research in veterinary science. 100, 12-17. 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 t p f ( s e n si ti vi ty ) fpf (1 specificity) no discrimination pon-1 (0,883) figure 1: roc curve for paraoxonase-1. the furthest point from the no-discrimination curve is the one that maximizes the diagnostic power of the test, where sensitivity and specificity for fip were 77% each. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords lamp, feline coronavirus, pcr corresponding author angelica stranieri angelica.stranieri@unimi.it journal home page riviste.unimi.it/index.php/haf a reverse transcription loop-mediated isothermal amplification (lamp) assay for the detection of feline coronavirus. a. stranieria, s. lauzia, a. giordanoa, s. paltrinieria adepartment of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy abstract loop-mediated isothermal amplification (lamp) is a molecular method that amplifies dna under isothermal conditions. it relies on the use of 4 different primers recognizing 6 regions of the template sequence and on the use of a dna polymerase with strand displacement activity (notomi et al., 2000). the addition of two loop primers allows the reaction time to be of one hour only (nagamine et al., 2002). the aim of this study was to develop a reverse transcription lamp assay for an easy and inexpensive detection of feline coronavirus (fcov). six primers binding the conserved 3’utr region of the fcov were designed with the primer explorer software. thirty-two samples of rna (11 feces, 8 effusions, 9 blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (rt-pcr) for the 3’utr region was performed were used. the reaction was carried out in 25µl reaction volume and the mixture was incubated in a thermocycler at 63°c for 1 hour followed by 10 minutes at 80°c. lamp products were visualized under uv after electrophoresis migration on a 1.5% agarose gel stained with ethidium bromide, where they produce a ladder-like pattern if positive. results where compared with those obtained on standard pcr. sensitivity and specificity were respectively 60% and 100% on feces, 40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. the overall sensitivity and specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method, except for tissues. references notomi, t., okayama, h., masubuchi, h., yonekawa, t., watanabe, k., amino, n., hase, t., 2000. loop-mediated isothermal amplification of dna. nucleic acid research. 28(12), e63-e63. nagamine, k., hase, t., notomi, t., 2002. accelerated reaction by loop-mediated isothermal amplification using loop primers. molecular and cellular probes. 16(3), 223-229. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l invited paper in ruminants, changes in the amount and metabolism of foetal brown and post-natal white adipose tissues (at) contribute to adaptations or productive traits both at birth for neonate survival (especially in sheep), and in adult life for productive efficiency during the gestationlactation cycle of dairy females or for carcass yield and quality of meat animals (bonnet et al., 2010). the molecular pathways accompanying the growth (louveau et al., 2016) and metabolic adaptations (sauerwein et al., 2014) of at are complex and incompletely understood. indeed, they strongly depend on animal peculiarities (age, breed, sex), environmental factors (nutrition...) and they are dynamic (transition from foetal to post-natal age or from late pregnancy to lactation). the use of -omics methods, such as proteomics has begun to fill the gap of knowledge of the multifaceted regulations of at growth and metabolism by providing numerous information on pathways and functions. two examples illustrate the usefulness of proteomic approaches for ruminant issues by allowing knowledge acquisition and molecular phenotyping. in the first example, proteomics and measurements of chemical composition, cellularity, histology, enzyme activities, and gene expression were applied to fetal at at 110, 180, 210 and 260 days post conception (dpc) in blond d’aquitaine and charolais breeds. from 180 dpc in the two breeds, we identified (taga et al., 2012) proteins declared to be hallmarks of brown and white adipocytes in mice, that were underscored by the histological characterization of a mix of multilocular and unilocular cells, putatively brown and white adipocytes, respectively. these cellular and molecular features challenged the concepts on the largely brown nature of bovine foetal at (based on histological and metabolic features previously reported a few days before or after birth for perirenal at), and strongly suggested that fetal bovine at have much more in common with white than with brown at. keywords ruminants, adipose tissue, proteomics, adipogenesis, biomarkers of productive traits. corresponding author muriel bonnet muriel.bonnet@inra.fr journal home page riviste.unimi.it/index.php/haf proteomics of adipose tissue: from the molecular drivers of adipogenesis to the molecular phenotyping of ruminants. m. bonnet 1,* 1 université clermont auvergne, inra, vetagro sup, umr herbivores, f63122 saint-genès-champanelle, france proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy iii haf © 2013 vol. v, no. 1s issn: 2283-3927 the second example is an application of proteomics for biomarkers discovery of ruminant productive traits (fig. 1). for several years, proteomics has been used to identify adipose proteins differentially abundant between two groups of bovine differing mainly by their muscular or body adiposity or by their energy balance. we merged available proteomics data to provide a list of candidate biomarkers (first step in the process of biomarker discovery) of muscular adiposity that we declared robust candidates because they were identified in at least two publications differing by the breed, the age and the nutrition of bovine (ceciliani et al., 2018, bonnet et al., unpublished). very recently, targeted proteomics has become an approach of choice to validate and precisely/absolutely quantify protein biomarkers. we have evaluated and benchmarked three targeted methods (the selected reaction monitoring (srm), parallel reaction monitoring (prm) and sequential windowed acquisition of all theoretical spectral (swath-ms)) to precisely quantify adiposity biomarkers in muscle tissues of 64 cows (bons et al., 2018). the work in progress, is to use these absolute quantifications to bench test the quantification provided by much more rapide and cheaply methods for the the development of a final tool for bovine phenotyping. finally, public and available -omics data are very valuable for research purposes, since they can be aggregated and repurposed to provide insights in a context that may be entirely different from the original study. we have recently integrated and mined -omics data to computationally predict the large-scale “secretome” of adipose tissues and muscles in ruminants (bonnet et al., 2016). the knowledge gained from -omics studies in ruminant species will foster knowledge and minimize unnecessary redundancy in research efforts. references bonnet, m., cassar-malek, i., chilliard, y., picard, b., 2010. ontogenesis of muscle and adipose tissues and their interactions in ruminants and other species. animal 4 (7), 1093-1109. bonnet, m., tournayre, j., cassar-malek, i., 2016. integrated data mining of transcriptomic and proteomic datasets to predict the secretome of adipose tissue and muscle in ruminants. molecular biosystems 12 (9), 2722-2734. figure 1: use of mass spectrometrybased proteomics technologies in the biomarker discovery pipeline. 2-de : two-dimensional gel electrophoresis before mass spectrometry. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy iv haf © 2013 vol. v, no. 1s issn: 2283-3927 taga, h., chilliard, y., meunier, b., chambon, c., picard, b., zingaretti, m.c., cinti, s., bonnet, m., 2012. cellular and molecular large-scale features of fetal adipose tissue: is bovine perirenal adipose tissue brown? journal of cellular physiology 227 (4), 1688-1700 louveau, i., perruchot, m.h., bonnet, m., gondret, f., 2016. invited review: preand postnatal adipose tissue development in farm animals: from stem cells to adipocyte physiology. animal 10 (11), 18391847 sauerwein, h., bendixen, e., restelli, l., ceciliani, f., 2014. the adipose tissue in farm animals: a proteomic approach. current protein & peptide science 15 (2), 146-155. ceciliani, f., lecchi, c., bazile, j., bonnet, m., 2018. in proteomics in domestic animals: from farm to systems biology, pp. 233–254. springer international publishing, cham. bons, j., husson, g., delalande, f., cianférani, s., picard, b., bonnet, m., carapito, c., 2018. targeted proteomics method comparison:srm, prm and swath-ms to quantify proteins in bovine muscle tissues. in the procedding of xiith eupa congress, 16-20 june 2018, santiago de compostela, (sp), in press. proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords dietary supplementation, natural extracts, nutrition, rabbit does, reproduction corresponding author sara chiapparini sara.chiapparini@unimi.it journal home page riviste.unimi.it/index.php/haf effect of dietary natural extracts mixture on rabbit does reproductive performances: preliminary data sara chiapparini1*, francesco vizzarri2, raffaella rossi1, donato casamassima2, giuseppe maiorano2, carlo corino1 1 university of milan, department of health, animal science and food safety, italy 2 university of molise, department of agricultural, environmental and food sciences, italy abstract natural extracts have been widely reported to have antioxidant, anti-inflammatory and antimicrobial activities related to their phenolic content (pereira et al., 2009). in rabbit, the reproductive phase is critical, therefore, nutritional strategies are required (castellini et al., 2003; roche et al., 2000). the aim was to investigate the effect of dietary supplementation with natural extracts in rabbit does on reproductive parameters. the trial was performed at the research institute for animal production (nitra, slovak republic). sixty does, artificially inseminated, were divided into three experimental groups. the first fed a basal diet (c), the second (t1) and the third one (t2) received 0.3% and 0.6% of natural extracts mixture for gestation and lactation period. the mixture contains polyphenols from plants and seaweeds. does were allocated in individual flat-deck cages and at parturition, the number of kits and the litters weights were recorded. the data were analyzed by one way analysis of variance using spss (ibm. ssps statistics 24). dietary supplementation did not affect (p>0.05) number of kids born (8.0 ± 1.0 c vs 7.3 ± 0.97 t1 and 7.4 ± 1.0 t2) and birth weight (63 ± 2.0 g c vs 60.1 ± 2.3 g t1 and 61.0 ± 2.4 g t2). the administration of natural extracts in does did not improve (p>0.05) the kits average daily gain (20.54 ± 1.3 g/d c vs 21.92 ± 0.5 g/d t1 and 20.93 ± 0.9 g/d t2) and body weight at weaning (829 ± 16.6 g c vs 834 ± 26.6 g t1 and 826 ± 26.8 g t2). these preliminary data showed that at the present dosage, the natural extracts mixture is not able to affect does reproductive performance. however, further research is needed to confirm the present data and explore the mechanism of action of this natural mixture references castellini, c., dal bosco, a., mugnai, c., 2003. comparison of different reproduction protocols for rabbit does: effect of litter size and mating interval. livestock and production science. 83, 131-139. pereira, d.,m., valentão, p., pereira, j.,a., andrade, p.,b., 2003. phenolics: from chemistry to biology. molecules. 14, 2202-2211. roche, j.,f., mackey, d., diskin, m.,d., 2000. reproductive management of postpartum cows. animal reproductive science. 60-61, 703-712. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author adriano pilotto, adriano.pilotto@unimi.it abstract this work was aimed to investigate relationship between plasma vitamin e concentration and milk somatic cell count in healthy cows in commercial herds. 49 multiparous cows from two commercial dairy herds were monitored from the day of dry off until 90 dim. bcs was assessed and blood samples were collected at dry off, day 0, 30, 60 and 90 postpartum. plasma was analyzed for α-tocopherol content. quantification of nefa, bohb, zn and se was performed in serum samples. milk production and composition was obtained from routinely test-day of italian milk producers association. somatic cell score (scs) was calculated and included in the dataset. analysis of data was performed using mixed repeated and corr procedures of sas. we did not observe a correlation between plasmatic vitamin e and somatic cell score, and this can be explained by the low level of somatic cell score (averages 1.64 and 1.26). the lowest value of vitamin e was observed at parturition (1.64 µg/ml and 1.95 µg/ml). a significant (p<0.01) negative (-20%) correlation was observed between nefa serum content and α-tocopherol plasma concentration. serum selenium content was positively correlated (+42%, p<0.0001) to zinc concentration. grouping cows on the basis of their plasma α-tocopherol content higher or lower than 3 μg/ml at dry off, scs at 30 and 60 dim tended to be higher in lactating animals with lower content of α-tocopherol (1.12 vs. 1.72, p=0.18 at 30d; 0.92 vs. 1.72, p=0.07 at 60d). however, plasma α-tocopherol content at dry off could be usefully correlated with somatic cell count in fresh cows. references kommisrud e., osterds o., and vatn t. 2005. acta veterinaria scandinavica vol. 46, 225-240; ospina p. a., nydam d. v., stokol t., and overton t.r. 2010. journal of dairy science. vol.93, 1596–1603; politis i., bizelis i., tsiaras a., and baldi a. 2004. journal of dairy research. vol. 71, 273-278; politis i., theodorou g., lampidonis a. d., kominakis a., and baldi a. 2012. journal of dairy science vol. 95, 7331–7335; wilde d. 2006. animal reproduction science vol. 96, 240-249. plasma α-tocopherol content and its relationship with milk somatic cells count in italian commercial herds. a. pilotto 1 , g. invernizzi 1 , a. baldi 1 , g. savoini 1 1 department of health, animal science and foodsafety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords italian autochthonous cattle breeds, dairy cows, biodiversity, ketone bodies, reproduction, production. corresponding author giulio curone giulio.curone@unimi.it journal home page riviste.unimi.it/index.php/haf milk ketone bodies assessment in a local italian cow breed (modenese) vs. holstein and characterization of its physiological, reproductive and productive performances. g. curone*a, m. zanini a, s. panseri b, c. colombania, p. moronia, f. rivaa, m. faustinia adepartment of veterinary medicine (dimevet), university of milan, via celoria 10, 20133 milan, italy. bdepartment of health, animal science and food safety (vespa), university of milan, via celoria 10, 20133 milan, italy abstract several autochthonous cattle breeds characterized by a small territorial diffusion are farmed in northern italy. the technical data show that these animals have a good reproductive performance (communod et al 2010; communod et al 2011), disease resistance and resilience. the objective of this study was to characterize some productive, reproductive and metabolic parameters (ketone bodies) in the italian autochthonous cattle breed modenese, comparing them with those of holstein and their crossbred (f1=modenese x holstein; f2=modenese x f1) breed in the same farm in order to understand if there is a different metabolic picture that can influence the reproductive performances. milk samples have been collected at different times of lactation (20th, 40th, 90th day in milk) and analyzed by gas chromatography-mass spectrometry to obtain the ketone bodies concentration. the reproductive (open days period and number of services per pregnancy) and productive (percentage and kg of protein between the 40th and 90th dim) data have been recovered by the consultation of the farm registers and the apa (provincial breeder association) data. on days open, number of services per pregnancy, % of proteins in milk, and kg of proteins in milk; a spearman correlation analysis was applied. in all time points, the modenese breed showed a significant (p<0,05) lower ketone bodies concentration. the f1, f2 and modenese showed also better reproductive performances when compared to holstein, with 80-105 days of days open in average. in conclusion, the better resilience against the negative energy balance and his adverse effects of modenese cattle could be one of the phenomena underlying their better reproductive efficiency. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. ii, no. 2s issn: 2283-3927 references communod r., faustini m., munari e., colombani c., castagna g., comi m, torre m.l., chlapanidas t., lucconi g., lazzati m., vigo d. future perspectives of varzese breed in an innovative biodiversity enhancement process (2010). large animalreview, 16, 267-271 communod r., faustini m, chiesa l.m., torre m.l., lazzati m. and daniele vigo (2011). milk biodiversity: future perspectives of milk and dairy products from autochthonous dairy cows reared in northern italy. chapter proposal review book title: food production (isbn 979-953-307-284-4). doi: 10.5772/32759.edited by anna aladjadjiyan, isbn 978-953-307-887-8, hard cover, 270 pages, publisher: intech, published: january 20, 2012 under cc by 3.0 license, in subject agricultural and biological sciences http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords metals, icp-ms, mussels, maximum levels, tolerable intakes corresponding author federica ceriani federica.ceriani@unimi.it journal home page riviste.unimi.it/index.php/haf metals in mussels from italian mollusc culture plants federica ceriani1*, luca maria chiesa1, sara panseri1, francesco arioli1 1university of milan, department of health, animal science and food safety, italy abstract the beneficial effects on human health of seafood are well known. however, seafood is a major source of exposition for consumers of most of the contaminants due to human activities such as breeding, industries, mining and agriculture: the overall level in biota, therefore seafood and particularly molluscs, dramatically increased over this last two centuries (mozaffarian and rimm, 2016). this study evaluates the presence of cadmium, lead, mercury, arsenic, nickel and chromium in mussels from the italian mussel culture plants, and estimates the risk that italian consumer undergoes eating these molluscs. a total of 30 mussel samples was collected at the wholesale fish market of milan. the fishing area of origin were 37 fao marine area (corresponding to mediterranean sea), particularly from fao 37.2.1 ligury, 37.2.2. north adriatic, middle adriatic, puglia, 37.2.3 lazio and sardinia, and were collected from july 2016 to february 2017. (fig.1). the analyses were done by using an inductively coupled plasma mass spectrometer (icp-ms), according to the environmental protection agency (epa) 3050b method. the concentrations (mg kg-1 wet weight) were shown as 25th, 50th (median), 75th and 100th (maximum concentration) percentiles. the sample concentrations were below the maximum levels (mls) given by commission regulation (ec) no 1881/2006 (european union, 2006) for cadmium (0,15; 0,23; 0,08; 2,13 mg kg-1), lead (0,14; 0,20; 0,32; 0,79mg kg-1) and mercury (0,02; 0,03; 0,04; 0,16 mg kg-1), except one sample from south adriatic sea, that showed mercury concentration of 0.528 mg kg-1. arsenic, nickel and chromium mls are not stated by eu. arsenic concentration was 4,00; 4,73; 5,58; 13,36 mg kg-1 and nickel 0,27; 0,43; 0,69; 3,98 mg kg-1. chromium was found only in 5 of 30 samples analysed with a concentration 0,00; 0,00; 0,00; 0,64 mg kg-1. based on the tolerable intakes by efsa (efsa, 2014a, 2014b, 2015) and mls by eu, italian mussels do not pose a risk consumers. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig.1: origin fao areas of mussel samples fao 37.2.1 ligury, 37.2.2. north adriatic, middle adriatic, puglia, 37.2.3 lazio and sardinia; n=number of samples. references efsa, 2014a. dietary exposure to inorganic arsenic in the european population. efsa j., 12, 3597-3658. efsa, 2014b. scientific opinion on the risks to public health related to the presence of chromium in food and drinking water1. efsa j., 9, 11-25. efsa, 2015. scientific opinion on the risks to public health related to the presence of nickel in food and drinking water. efsa j., 13, 4002-4203. european union (2006) commission regulation (ec) no. 1881/2006 setting maximum levels for certain contaminants in foodstuffs. o.j.e.u., l 364, 5-24. mozaffarian, d., rimm, e.b., 2006. fish intake, contaminants, and human health: evaluating the risks and the benefits. jama, 296, 1885-1899. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l water buffaloes mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yield. the emergence of antibiotic resistance has led to investigate strategies in order to avoid or minimize the antibiotic use, especially during subclinical mastitis (sm) (moroni et al., 2006). lactobacillus rhamnosus is part of the normal gut microflora, having meanwhile an immunostimulatory activity (fong et al., 2016). the aim of this study was to investigate the change of milk microbiota after the therapeutic treatment of mammary gland quarters affected by subclinical mastitis with inactivated cultures of lactobacillus rhamnosus and antibiotics. a number of 43 quarters from 20 pluriparous animals and affected by subclinical mastitis (sm), with no signs of clinical mastitis and aerobic culture positive for udder pathogens were included in the study; milk samples were positive for coagulase-negative or positive staphylococci and/or streptococcus agalactiae. a total of 11 quarters were locally treated with antibiotic (amoxicillin trihydrate), 15 with lactobacillus rhamnosus and 17 with pbs as negative control, by means of intramammary injection. samples were collected at two time points, t0 (pre-treatment) and t5 (after 5 days post-treatment) and v4 region of 16s rrna gene was amplified by pcr and sequenced using ion torrent personal genome machine. the software quantitative insights into microbial ecology (qiime version 2) was used to analyse data. microbiota composition was evaluated in terms of taxonomy at phylum, family and genus level. microbiota structure was investigated through alpha and beta diversity analysis which take into account differences within and among samples, respectively. non-parametric test, namely wilcoxon signed rank and kruskal-wallis followed by dunn test, were used to perform statistical analysis for paired and unpaired groups, respectively. keywords water buffalo, milk microbiota, subclinical mastitis. corresponding author carlotta catozzi carlotta.catozzi@unimi.it journal home page riviste.unimi.it/index.php/haf effect of inactivated cultures of lactobacillus rhamnosus and antibiotics on subclinical mastitis quarter milk microbiota. c. catozzi*,1 , a. cuscó martí2, c. lecchi1, v. zamarian1, j. viñes pujol2, s. d'andreano2, a. martuccello3, g. cappelli3, c. grassi3, c. marianelli4, l. d’angelo3, e. de carlo3, d. vecchio3, a. sanchez bonastre5, o. francino5, f. ceciliani1. 1 department of veterinary medicine, università degli studi di milano, via celoria 10, milano, italy. 2 vetgenomics. ed eureka. pruab. campus uab, bellaterra, barcelona, spain. 3 istituto zooprofilattico sperimentale del mezzogiorno, national reference centre for hygiene and technologies of water buffalo farming and productions, via delle calabrie, salerno, italy. 4 unit of prophylaxis and control of bacterial zoonoses. department of veterinary public health and food safety. istituto superiore di sanità. viale regina elena 299, rome, italy. 5 molecular genetics veterinary service (svgm), veterinary school, universitat autònoma de barcelona, bellaterra, barcelona, spain http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 34 haf © 2013 vol. v, no. 1s issn: 2283-3927 regarding taxonomy, the microbiota composition of sm quarters showed no major changes after pbs treatment, while differed after antibiotic treatment where staphylococcus decreased its relative abundance from 41% at t0 to 3% at t5. lactobacillus rhamnosus induced a less dramatic change in milk microbiota, although the relative abundance of some genera was found to be modified, among which an increase of pseudomonas from 1.5% at t0 up to 4% at t5. the taxonomy at genus level is shown in figure 1. however, beta diversity showed no differences between the microbiota structure of quarters treated with lactobacillus rhamnosus and pbs (t0 vs t5). the effect of the treatment was different between antibioticvs pbstreated groups and antibioticvs lactobacillus rhamnosustreated groups. in conclusion, this study allowed to characterize the microbiota in milk from animals treated with lactobacillus rhamnosus and antibiotics; while changes in milk microbiota after antibiotic treatment were evident, changes after lactobacillus rhamnosus were more limited. following investigation will include the study of the microbiota changes of antibiotic-treated quarters during more than two time points, in order to investigate the colonization of mammary gland after antibiotic treatment during a time course. references moroni, p., sgoifo rossi, c., pisoni g., bronzo v., castiglioni, b., boettcher pj. 2006 relationships between somatic cell count and intramammary infection in buffaloes. j. dairy sci. 89, 998–1003. fong, fl., shah, np., kirjavainen, p., el-nezami, h,. 2016 mechanism of action of probiotic bacteria on intestinal and systemic immunities and antigen-presenting cells. int rev immunol. 35(3):179-88. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% sm_pbs_t0 sm_pbs_t5 sm_l_t0 sm_l_t5 sm_a_t0 sm_a_t5 low abunda nce taxa pseudomonadaceae pseudomonadaceae;g __pseudom onas mor axella ceae;g__psychrobacter mor axella ceae;g__a cinetobacter halomona da ceae;g__halomonas enteroba cteriaceae enteroba cteriaceae;g__escherichia idiomari na ceae rhodocyclaceae;g __hydrogenophilus comamonadacea e comamonadacea e;g__delftia sphing om onadaceae;g__sphing om onas xanthobacteracea e methylobacteria ceae;g__methylobacter ium methylobacteria ceae bradyrhizobiaceae bradyrhizobiaceae;g__bra dyrhizobium ruminococcaceae peptostreptococca ceae lachnospir aceae clostridiaceae [mogi ba cteriaceae] streptococca ceae;g__s treptococcus carnoba cteria ceae;g__granulicatella aerococcacea e aerococcacea e;g__facklam ia aerococcacea e;g__alkali ba cterium staphylococcaceae;g__staphylococcus staphylococcaceae;g__sali nicoccus staphylococcaceae;g__jeotga licoccus planococcacea e;g __soliba cillus planococcacea e;g __ly sinibacillus bacilla ceae;g__natronoba cillus xenococcacea e fla vobacteriacea e [weeksellaceae];g__chryseobacterium cytophag acea e;g __hym enobacter por phyromonadacea e bacteroidaceae;g__5-7n15 bacteroidaceae [pa raprevotellaceae];g__cf231 propionibacteria ceae;g__propionibacterium noca rdioidaceae noca rdiacea e;g __rhodococcus micrococcacea e micrococcacea e;g __nesterenkonia microbacteriaceae intraspor angiaceae dietziacea e;g__dietzia corynebacteri aceae;g __coryneba cterium actinomycetaceae deinococcacea e;g__deinococcus figure 1: microbiota composition at genus level (relative abundance > 1%). sm_pbs_t0: subclinical quarters treated with pbs (pre-treatment) sm_pbs_t5: subclinical quarters treated with pbs (post-treatment) sm_l_t0: subclinical quarters treated with lactobacillus rhamnosus (pre-treatment) sm_l_t0: subclinical quarters treated with lactobacillus rhamnosus (post-treatment) sm_a_t0: subclinical quarters treated with antibiotic (pre-treatment) sm_a_t5: subclinical quarters treated with antibiotic (post-treatment) http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords oral transmucosal, dexmedetomidine, methadone, butorphanol, dog corresponding author daniela gioeni daniela.gioeni@unimi.it journal home page riviste.unimi.it/index.php/haf evaluation of an oral transmucosal administration of dexmedetomidine-butorphanol and dexmedetomidine-methadone in dogs daniela gioeni1*, federica di cesare2, elisa s. d’urso1, vanessa rabbogliatti1, giuliano ravasio1 1 university of milan, department of veterinary medicine, italy 2 university of milan, department of health, animal science and food safety, italy abstract oral transmucosal (otm) delivery is a simple and painless method for sedative administration in veterinary medicine and allows a rapid absorption without a first-pass metabolism by the liver (porters et al., 2014.). otm is particularly useful in aggressive animals (santos et al., 2010). the aim of this study is to evaluate the efficacy of the otm route in dogs for sedative administration in comparison with intramuscular (im) injection. 24 mixed-breed dogs undergoing soft tissue surgery or diagnostic procedures were randomly divided in 4 groups (n = 6): two groups received otm administration of dexmedetomidine (10 µg/kg-1) together with butorphanol (0.2 mg/kg-1, btf-otm group) or methadone (0.2mg/kg-1, mtd-otm group); two groups received intramuscular (im) administration of dexmedetomidine (5 µg/kg-1) together with butorphanol (0.2 m/kg-1, btf-im group) or methadone (0.2 mg/kg-1, mtd-im). heart rate (hr), respiratory rate (rr), sedation score (gruney et al., 2009) and side effects were recorded 10 (t10), 20 (t20) and 30 (t30) minutes after premedication. induction was performed at t30 with titrate-to-effect propofol administration and the dosage required was recorded. at each time point btf-im group showed a statistically lower hr compared to btf-otm; rr was statistically lower at t10 in mtd-otm group (21.33 ± 8.64 pm) compared to btf-otm (46.16 ± 17.98); dogs in group mtd-im reached a higher sedation scores at each time point compared to mtdotm. the induction dose of propofol appears comparable among groups. marked vasoconstriction was observed after otm administration, as probably related to α2-agonists use. emesis and sialorrhea occurred in two subjects of mtd-otm group while only one dog presented sialorrhea in btf-otm group. in conclusion, otm administration appears effective and easy to perform; it takes a longer time to achieve a good sedation score, probably related to a gradual absorption of drugs that also leads to a more gradual hemodynamic effects. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references gurney m., cripps p., mosing m. 2009. subcutaneous pre-anaesthetic medication with acepromazine-buprenorphine is effective as and less painful than the intramuscular route. journal of small animal practice. 50, 474-477 porters n., bosmans t., debille m. et al. 2014. sedative a and antinociceptive effects of dexmedetomidine and buprenorphine after oral transmucosal or intramuscular administration in cats. veterinary anaesthesia and analgesia. 41, 90-96 santos l.c.p., ludders j.w., erb h.n. et al. 2010. sedative and cardiorespiratory effects of dexmedetomidine and buprenorphine administration to cats via oral transmucosal or intramuscular routs. veterinary anaesthesia and analgesia. 37, 417-424 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords holstein friesian, rendena, autochthonous cows, mammary immune response corresponding author joel f. soares f. joel.soares@unimi.it journal home page riviste.unimi.it/index.php/haf a comparison study of the inflammatory response in holstein friesian versus a local cattle breed (rendena) joel f. soares f.1*, giulio curone1, erminio trevisi2, massimo amadori3, lauretta turin1, paolo moroni1,4, daniele vigo1, maria f. addis1, federica riva1 1 university of milan, department of veterinary medicine, italy 2 università cattolica del sacro cuore, istituto di zootecnica, italy 3 istituto zooprofilattico sperimentale della lombardia e dell’emiliaromagna, laboratory of cellular immunology, italy 4 cornell university, animal health diagnostic center, quality milk production services, usa abstract the selective pressure for increased milk production brought about great difficulties in the adaptation of cows to their environment. however, not much is known about the biological mechanisms behind the relationship between genetic selection and higher risk of metabolic and infectious diseases (oltenacu and broom, 2010). it is well known that during the calving period, high-yielding dairy cattle are more susceptible to common environmental stressors, affecting disease occurrence and milk production levels (bach, 2011). in this study we compared innate immune response of 6 holstein friesian (hf) and 4 rendena (r) cows reared in the same farm and under the same management conditions. milk and blood samples were collected at dry-off (t1), 1 day after calving (t2), 7-10 days after calving (t3), and 30 days after calving (t4). milk samples were subjected to measurement of the inflammation marker cathelicidin and assessment of different innate immune-related mediators; blood samples were used for the analysis of plasma metabolites indicators of systemic inflammation. hf cows showed a more severe systemic inflammatory response at t2 and t3 in comparison with r cows (fig.1). concerning the milk protein abundance profile, higher levels in r cows were observed in the colostrum (t2). moreover, at all time points hf showed higher levels of the inflammation marker cathelicidin in milk. in addition, the expression of innate immune related genes were different in hf compared with r. our results suggest that hf cows develop a systemic and local mammary inflammatory response that confirms their higher susceptibility to disease compared with r cows. our findings reveal that fundamental effector activities of innate immunity in the mammary gland could be included in the breeding programs of hf cows and suggest the spread of autochthonous cow farming in order to maintain the biodiversity, reduce the antibiotic consumption and production of high quality dairy products. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig. 1: plasmatic concentration of metabolic markers. plasmatic concentration of total protein, globulin, total bilirubin, and haptoglobin in holstein friesian (hf=black line) and rendena (ren=grey line) cows at t1, t2, t3 and t4. significance of differences between groups at each time point has been indicated with: + for p<0.1, * for p < 0.05 and ** for p < 0.01. references oltenacu and broom, 2010. the impact of genetic selection for increased milk yield on the welfare of dairy cows. animal welfare. 19(s), 39-49 bach, a. 2011. associations between several aspects of heifer development and dairy cow survivability to second lactation. j. dairy sci. 94, 10521057 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author giovanni quintavalle pastorino, giovanni.quintavalle@unimi.it abstract interactions that animals experience can have a significant influence on their health and welfare. these interactions can occur between animals themselves, but also between animals and keepers, and animals and the public. human and non-human animals come into contact with each other in a variety of settings, and wherever there is contact there is the opportunity for interaction to take place. interaction with companion animals are well known, but human–animal interaction (har) (hosey, 2008) also occurs in the context of farms (hemsworth and gonyou, 1997; hemsworth, 2003), laboratories (chang and hart, 2002), zoos (kreger and mench, 1995) and even the wild (e.g. cassini, 2001). this project proposes a permanent monitoring scheme to record animal-human interactions and animal-animal interactions in zoos. this will be accompanied by a survey of animal personality for welfare, husbandry, breeding programs and reintroduction purposes. the pilot project is currently based on direct monitoring of animal behaviour, use of time lapse cameras and animal personality questionnaires completed by experienced keepers. the goal of this project is to create a network between zoos to explore the aforementioned interactions to produce husbandry protocols and explore personality and behavioural traits in multiple species. we present provisional data regarding polar bear (fasano zoosafari, italy), sumatran tigers, amur tigers and asiatic lion (zsl london and whipsnade zoo) interactions with humans and conspecifics. this data is collected across a broad range of environmental conditions and outlines the monitoring protocols developed to collect this data. the first year data show the great adaptability of these species to ex situ environments, low or absent negative impact of visitors’ presence and the relevance of individual personality in these interactions. references hosey, g., 2008. applied animal behaviour science 109:105–127; hemsworth, p.h., h.w. gonyou. 1997. in: appleby, m.c., hughes, b.o. (eds.), animal welfare. cab international, wallingford uk, pp. 205–217; hemsworth, p.h., j.l. barnett, g.j. coleman. 1993. animal welfare 2:33–51; chang, f.t., l.a. hart. 2002. ilar journal 43:10–18; kreger, m.d., j.a. mench, 1995. anthrozoos 8:143–158; cassini, m.h. 2001. applied animal behaviour science 71:341–346. factors influencing interactions in zoos: animal-keeper relationship, animal-public interactions and solitary animals groups g. quintavalle pastorino 1&2 ,r. preziosi 3 , m. albertini 1 1 department of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy, 2 zoological society of london, regent’s park, london nw1 4ry, united kingdom, 3 faculty of life sciences, the university of manchester, oxford road, manchester, united kingdom proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1 i ssn: 2283-3927 l invited abstract knowledge of host-pathogen interactions at molecular level is crucial for understanding of pathogenesis, disease prevention and cure. here borrelia is presented as a model neuroinvasive pathogen which employs vast immune evasion mechanisms like drastic change in antigenic proteins, complement regulatory protein binding, antigenic variation etc. it is proposed that borrelia may employ multiple strategies to evade host's complement system by binding complement regulatory proteins like factor h, vitronectin, c4bp and cd59. amplitude of these mechanisms, their pathogen species dependent disparity, and characterization of interacting proteins from both sides (host and pathogen) will be presented during vas presentations. borrelia is also capable of invading central nervous system (cns) in hide in the immune privileged site – the cns. to do so it must cross the blood brain barrier (bbb). the mechanisms of the bbb crossing of this organism, like many of the other cns invading pathogens, are still subject of ongoing research. our decade of research indicate the paracellular mechanism of the bbb penetration of borrelia. paracellular penetration of pathogen needs multiple protein:protein interactions between pathogen surface proteins and endothelial cells. the second part of my presentation in vas will be dedicated to mechanisms employed by borrelia to modulate cell signaling events in brain microvascular endothelial cells to cross the bbb. this presentation will also give sneak peak of several state-of-the-art technologies in genomics and proteomic used to understand the mechanisms of neuroinvasive and complement evasion by borrelia. i hope that doctoral fellows will take advantage of the experimental pipeline that will be presented in vas and apply successfully in their research. acknowledgment: work presented here was supported by apvv-14-0218 and vega 1/0258/15 keywords borrelia, host, interaction. corresponding author mangesh bhide bhidemangesh@gmail.com journal home page riviste.unimi.it/index.php/haf borrelia and host interaction: a tangled tale. m. bhide 1,* 1 laboratory of biomedical microbiology and immunology, university of veterinary medicine and pharmacy, komenskeho 73, kosice, slovakia. 2 institute of neuroimmunology, slovak academy of sciences. proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf1 l corresponding author claudia perrini, claudia.perrini@unimi.it microvesicles secreted from equine amniotic cells and their potential role in in vitro cell tendon repair. c. perrini 1 , a. lange-consiglio 1 , r. tasquier 1 , mc deregibus 2 , g camussi 2 , l. pascucci 3 , m.g. marini 4 , b. corradetti 4 , d. bizzaro 4 , f. cremonesi 1 1 large animal hospital, reproduction unit, università degli studi di milano; 2 research center for experimental medicine and center for molecular biotechnology, torino; 3 departiment of veterinary medicine, università degli studi di perugia; 4 department of life and environmental sciences, università politecnica delle marche abstract the regenerative mechanisms ascribed to mesenchymal stem cells (mscs) are classified into 3 categories: differentiating into damaged cell types, supplying nutrients, and improving survival/functions of the endogenous cells via paracrine actions. however, because of the inhospitable microenvironment of the injured tissues, a proportion of the implanted mscs may quickly die, suggesting that other mechanisms might be present. this notion is supported by the overlapping beneficial effect (in terms of time of healing) resulted after the injection of amcs or of amniotic mesenchymal cells conditioned medium (amc-cm) in equine spontaneous injured tendons and ligaments. microvesicles (mvs) released by cells are an integral component of the cell-to-cell communication network involved in tissue regeneration.in the present study, mvs secreted by amcs were investigated with nanosigth instrument and tem. then, the in vitro incorporation of mvs into equine tendon cells was studied by a dose-response curve. lastly, the ability of mvs to counteract an in vitro inflammatory process induced by lipolysaccaride on tendon cells was studied evaluating the expression of pro-inflammatory genes like metallopeptidase (mpp) 1 and 13, and prostaglandin-endoperoxide synthase 2 (cox2). results demonstrated that amcs secreted mvs ranging in size from 100 to 1000 nm with a prevalence of 100-200 nm large mvs. tendon cells were able to uptake them with an inverse relationship between concentration and time. the greatest incorporation was detectable at 40x106 mvs/ml after 72h. mvs induced down-regulation of mmp1 and mmp13, suggesting that they may have contributed, along with soluble factors, to in vivo tendon regeneration. references lange-consiglio, a., d. rossi, s. tassan, f. cremonesi, o. parolini. 2013. stem cell development 22:3015-3024; lange-consiglio, a., s. tassan, b. corradetti, a. meucci, r. perego, d. bizzaro, f. cremonesi. 2013. cytotherapy 10:1016-1023; camussi, g., m.c. deregibus, s. bruno, v. cantaluppi, l. biancone. 2010. kidney international 78: 838–848. proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords bovine, iatrogenic diseases, malpractice, medical error, zootechnical error corresponding author giulia sala giulia.sala1@unimi.it journal home page riviste.unimi.it/index.php/haf the role of iatrogenic disease of cattle in admission to veterinary hospital giulia sala1*, antonio boccardo1, eleonora coppoletta2, angelo belloli1, davide pravettoni1 1university of milan, department of veterinary medicine, italy 2university of milan, centro clinico-veterinario e zootecnico-sperimentale, clinica dei ruminanti e del suino, lodi, italy abstract iatrogenic diseases are due to negligence or malpractice (pezza et al., 2008). in human medicine, these conditions are widely described (weingart et al., 2000), mostly for insurance issues related to hospitalization, while in veterinary medicine only occasional cases are reported. 4155 clinical records related to cattle admitted to the clinic for ruminants and swine of the university of milan between 2005 and 2017 were analyzed. clinical cases that required admission because of an iatrogenic related disease were selected for this study. for case selection, 3 experienced veterinarians examined the clinical records, cross-compared the selection and pick 114 cases (2,7%). the iatrogenic diseases were primarily caused by farmers (93%) rather than veterinary practitioner (7%). iatrogenic diseases were caused mostly by erroneous administration of drugs (47,4%), excessive traction at birth (17,5%), improper milk or colostrum administration, frequently performed by oroesophageal tubing (16,7%) or by forced administration using a nipple bottle (12,3%). as verified by our study, farmers often perform medical, nursing and zootechnical procedures without adequate competences and sometimes choose medical treatment for sick animals without professional consultation of veterinarians. the veterinarian rule is fundamental in farmer education. clinicians, especially for some professional branches such as neonatology, should be more responsible of their assignments, avoiding delegation of specific procedures to unskilled staff. the importance of communication in improving management and health in dairy farms has been recently demonstrated (jansen and lam, 2012; jansen et al., 2010). effective communication has a key role in dairy herd health and communication strategies are required to support diseases control programs (lievaart et al., 2008). more attention to iatrogenic issue may have a positive impact on animal and public health. moreover, a decrease in unnecessary and injurious drug administration may result in a reduction of treatment costs and in prevention of antibiotic resistance. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references pezza, f., ruffo, g., & fossati, p. 2008. diritto e legislazione veterinaria. le point veterinaire italie. weingart, n. s., wilson, r. m., gibberd, r. w., & harrison, b. 2000. epidemiology of medical error. bmj. 320(7237), 774-777. jansen, j., lam, t.j., 2012. the role of communication in improving udder health. veterinary clinics of north america: food animal practice. 28, 363–379. jansen, j., steuten, c.d., renes, r.j., aarts, n., lam, t.j., 2010. debunking the myth of the hard-to-reach farmer: effective communication on udder health. journal of dairy science 93, 1296–1306. lievaart, j.j., noordhuizen, j.p.t.m., buckley, d., van winden, s.c.l., 2008. the marketing of herd health and production management services on dutch dairy farms: perceptions of dairy farmers and their veterinary surgeons. irish veterinary journal 61, 668–676. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords food safety, wild game meat, hunters, food supply chain, sustainable development, territorial marketing. corresponding author maria e. marescotti maria.marescotti@unimi.it journal home page riviste.unimi.it/index.php/haf development of a wild game meat supply chain: assessment of the food safety of large wild ungulates’ meat by interviews with hunters. m. e. marescottia*, a. gaviglioa, e. demartinia, a. pirania a department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy abstract despite the population of wild ungulates is rapidly growing (ramanzin et al., 2010) and the italian production has the predisposition to the high quality food, there is no food supply chain for hunted game in the italian territory. consequently, the customers’ demand is met by relevant amount of imported meat from other nearby markets. with the purpose of understanding the opportunities of an italian new supply chain, the present research aims at the collection of information about hunters and their behavior in order to assess the food safety of their product. the survey was carried out on a sample of 145 hunters of verbania (piedmont-italy), by using selfcompiled questionnaire. results show the interesting potentialities of the area in terms of quantity of salable product. on the other hand, the hunted meat still does not reach adequate hygienic and quality standards to be traded. in fact, the descriptive analysis show that 63% of the hunters do not provide a proper maturation of the meat and 21% of them do not bleed the game just after shooting. furthermore, 12% of the respondents use cellars or even the garages for the maturation. the wrong behaviors of the hunters are related to their cultural background, which probably represents the most important barrier for a change. our findings provide important inputs for the development of a real market for this type of meat. therefore, the research seems to be relevant at both scientific and practical level. it proposes a new conceptual and practical option for a more sustainable development of italian (and others) mountain areas. research funded by fondazione cariplo. project: “la filiera ecoalimentare progetto per la valorizzazione delle carni di selvaggina: la gestione di prodotto sostenibile come strumento di stimolo al miglioramento ambientale dei territori alpini”. references ramanzin, m., amici, a., casoli, c., esposito, l., lupi, p., marsico, g., mattiello, s., olivieri, o., ponzetta, m. p., russo , c., trabalza marinucci, m., 2010. meat from wild ungulates: ensuring quality and hygiene of an increasing resource. italian journal of animal science. 9:61, 318-331. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author valentino bontempo, valentino.bontempo@unimi.it abstract our study is focused on evaluation and use of the most effective and correct nutrients. in particular, our attention is directed to the role of certain amino acids in cachectic patients. during parenteral nutrition in humans, physician already associates in the pn-bags different formulations including amino acids, lipids and glucose solutions or essential amino acids solution alone or exclusively branched-chain amino acids (bcaa). studies investigated the effects of dietary bcaa ingestion on different diseases and conditions such as obesity and metabolic disorders, liver disease, muscle atrophy, cancer, impaired immunity or injuries (surgery, trauma, burns, and sepsis). bcaas have been shown to affect gene expression, protein metabolism, apoptosis and regeneration of hepatocytes, and insulin resistance. they have also been shown to inhibit the proliferation of liver cancer cells in vitro, and are essential for lymphocyte proliferation and dendritic cell maturation. oral or parenteral administration of these three amino acids will allow us to evaluate the real efficacy of these compounds during a therapy to treat malnutrition in subjects unable to feed themselves. references rajkumar rajendram, victor r. preedy, vinood b. patel, branched chain amino acids in clinical nutrition, new york, humana press, 2015. branched-chain amino acids m. ghiringhelli, f. acocella, v. bontempo department of health, animal science and foodsafety, università degli studi di milano, via celoria 10, 20133 milan, italy l keywords pigeon pea, fungi, mycological hazards, health, valorization, benin. pages 49 – 58 references vol. 4 no. 1 (2017) article history submitted: july 08, 2017 accepted: december 01 2017 published: december 02 2017 corresponding author euloge s. adjou laboratory of research and study in applied chemistry, polytechnic school of abomey-calavi, university of abomey-calavi, 01 p.o.b: 2009, cotonou, benin mail: eulogesenan@yahoo.fr phone: (+229) 95924907 journal home page riviste.unimi.it/index.php/haf article evaluation of the fungal microflora infesting pigeon pea (cajanus cajan l. millspaugh) in southern benin and associated mycological hazards euloge s. adjou 1, *, grace mètomè 1 , bertin a. gbaguidi 1 , edwige dahouenon-ahoussi 1 , dominique sohounhloue 1 1 laboratory of research and study in applied chemistry, polytechnic school of abomey-calavi, university of abomey-calavi, benin abstract pigeon pea is a perennial legume with a good nutritional value. unfortunately, it is also a substrate for fungi contamination. then, a qualitative semi-structured survey was carried out in the main production areas of pigeon pea in southern benin. this survey was coupled with samples collection. a total of 60 samples of pigeon pea were collected and analyzed for associated fungal microflora by using a taxonomic schemes primarily based on morphological characters of mycelium and conidia. obtained results indicated a low technological valorization of pigeon pea seeds in southern benin and their used only in direct consumption after cooking. microbiological analyses revealed the high contamination of pigeon pea seeds by fungi, with the most occurrence of aspergillus (71.42%), followed by fusarium (26.19%). fungal species such as aspergillus ochraceus, a. parasiticus, a. flavus and fusarium oxysporum were also detected in analyzed samples. taking into account the toxicity of the secondary metabolites produced by these fungi, mycological hazards are discussed and important methods for the control of mycotoxincontamination are further provided. more attention should be paid to the mycological quality of this legume, in order to protect the consumers’ health. euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 50 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction the nutritional value of legumes has considerable worldwide interest, due to the demand for healthy food (nestel et al., 2004). legumes are good sources of protein and dietary fiber with high levels of vitamins and minerals; and also contributed to the control of certain metabolic diseases (almeidacosta et al., 2006). pigeon pea (cajanus cajan l. millspaugh) is a legume belong to the family of fabaceae (wu et al., 2009). it is often cultivated in tropics areas, including the semi-arid one. its annual production, estimated at 3.1 million tons, accounts for about five percent of world seed legumes production. it has the fifth place among the legumes and contributes to thirty-three percent for the nitrogen requirements for human food (fossou et al., 2012). in developing countries, interest in the culture of pigeon pea is justified by many opportunities it offers to rural populations (fossou et al., 2012). indeed, it is one of the most attractive crops for african agriculture, due to its simple low-cost production adapted to subsaharan climates, its nutritional value, its exceptional capacity for soil regeneration and its diversified uses for men and cattle (pazhamala et al., 2015). it is grown in more than twenty five tropical and subtropical countries, either in monoculture or in rotation with cereals or other legumes. pigeon pea is mainly grown for its seeds, whose nutritional value is comparable to that of beans (phaseolus vulgaris) (fossou et al., 2012). indeed, known to be an excellent source of protein (21.7%), pigeon pea are also a good source of energy, vitamins and essential amino acids such as lysine, phenylalanine, valine, leucine and isoleucine (wu et al., 2009). the seeds are rich in fatty acids, such as linoleic and palmitic acids; and are also a good source of iron and calcium (fossou et al., 2012). unfortunately, like other legumes often grown in benin such as peanut and cowpea, pigeon pea is also a preferred substrate for parasites, such as fungi. this contamination could also be associated with mycotoxin production (sultan and magan, 2010). mycotoxins are secondary metabolites of low molecular weight, which are present in many food and feed products and can cause many diseases for humans and animals (wagacha and muthomi, 2008). these molecules are not destroyed during prolonged storage and are often resistant to thermal or chemical treatments (cahagnier et al., 1998). the main aim of this study was to evaluate the fungal microflora infecting pigeon pea seeds at post-harvest in benin, as well as associated mycological hazards. findings will serve the purpose of alerting consumers on the dangers of consuming poorly stored grains. 2 material and methods 2.1 survey a qualitative semi-structured survey was carried out in the main production areas of pigeon pea in southern benin, including six locations (kétou, pobè, agouna, klouekanmè, azovê and glazoué). a total, eighty (80) stakeholders of the sector, including farmers (production https://translate.googleusercontent.com/translate_c?depth=1&hl=fr&prev=search&rurl=translate.google.fr&sl=en&sp=nmt4&u=https://www.ncbi.nlm.nih.gov/pubmed/%3fterm%3dpazhamala%2520l%255bauthor%255d%26cauthor%3dtrue%26cauthor_uid%3d25741349&usg=alkjrhhmvgh3tlfz67zrfbpeycw1pvg5za euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 51 haf © 2013 vol. iv, no. 1 issn: 2283-3927 sites), traders and consumers (markets) were surveyed. the survey was carried out through individual interview by using a pre-established survey form. the searching information concerned: different types of varieties encountered in the areas investigated, parasitic factors of pigeon pea, conservation methods and different uses of the pigeon pea seeds. 2.2 collection of samples a total of 60 samples (each 500 g) of pigeon pea were purchased from the different investigated localities. the samples from ten points were collected. each sample was shelled in a sterile flow bench to obtain the pigeon pea grains which were kept at 4 °c until fungal enumeration. 2.3 fungal isolation and identification direct plating technique described by pitt et al. (1994) was used to examine samples. one hundred pigeon pea grains per sample were surface disinfected in 0.4 % active chlorine solution for 1 min at room temperature. then, they were placed directly on yeast extract sucrose agar medium (yes). plates were incubated at 25°c for 5 to 7 days. this method permits recovery of the fungi actually growing in the particles. the dilution plating method was also used in other to recovery of the fungi growing on the particles as described by nguyen (2007). fungi were purified by repeated subcultures. pure cultures of fungi were examined macroscopically and microscopically and their identification was carried out by using a taxonomic schemes primarily based on morphological characters using the methods given by singh et al. (1991), filtenborg et al. (1995), and tabuc (2007). 2.4 statistical analysis experiments were performed in triplicate, and data analyzed are means subjected to oneway anova. means are separated by the tukey’s multiple range test when anova was significant (p<0.05) (spss 10.0; chicago, il, usa). 3 results 3.1 results of survey the results of the qualitative semi-structured survey carried out in the production areas of pigeon pea in southern benin have revealed that there are mostly three types of pigeon pea varieties that are commonly encountered in investigated areas. these varieties are characterized by their color of seeds (whitish color, whitish color stained with black and brown color figure 1). however, the preference for each type of pigeon pea grain varies according to investigated zone. indeed, for grains of whitish color, the preference, according to the persons euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 52 haf © 2013 vol. iv, no. 1 issn: 2283-3927 surveyed, is 40% for ketou, 40% for pobè, 70 % for agouna, 70 % for azovè and 70% for glazoue (table 1). moreover, in the zone of klouekanmè, only the whitish color variety is mostly encountered. according to the questionary surveyed, this variety is much appreciated because its seeds have a faster cooking time with a more attractive taste. the surveys have also indicated that there is no other form of upgrading of pigeon pea in southern benin. the only form of use this legume is the direct consumption after cooking. besides pest’s attacks by weevils (callosobruchus maculatus), the survey also underlined the contamination of the seeds by fungi. this contamination is often characterized by the presence of black spot on the seeds, especially in post-harvest. the survey have also revealed that all farmers surveyed used chemical insecticides to control pest’s damage of pigeon pea in postharvest. figure 1. color varieties of pigeon pea commonly encountered in investigated areas awhitish color seeds bwhitish color stained with black seeds cbrown color seeds table 1: preference of surveyed people in each investigated area for different type of pigeon pea seeds (%) localities whitish color seeds whitish color stained with black seeds brown color seeds ketou 40 a 10 b 50 c pobe 40 a 00 b 60 c agouna 70 a 23 b 07 c kloukanmè 100 a 00 b 00 b azove 70 a 30 b 00 c glazoue 70 a 20 b 10 b values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. 3.2 results of identification of fungal microflora the result of microbial analysis and isolation of fungi in pure culture indicated that pigeon pea samples collected from investigated areas were highly contaminated by fungi (table 2). euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 53 haf © 2013 vol. iv, no. 1 issn: 2283-3927 the most prevalently fungi recorded, with statistically significant difference, are aspergillus ochraceus (23.80%), fusarium oxysporum (19.04%) and aspergillus flavus (16.66%) (table 3). the most prevalently genera was aspergillus (71.42%), followed by fusarium (26.19%). table 2: prevalence of isolated fungi taking into account the collection zones collection areas fungi isolated prevalence (%) agouna aspergillus ochraceus 33,33a aspergillus tereus 16,66b aspergillus niger 33,33a fusarium oxysporum 16,66c glazoué aspergillus ochraceus 25a aspergillus fumigatus 12,5b aspergillus ustus 12,5b fusarium oxysporum 37.5c fusarium spp. 11,11b azovê aspergillus flavus 22,22a aspergillus oryzae 11,11b aspergillus candidus 11,11b aspergillus ochraceus 22,22c fusarium oxysporum 22,22c fusarium verticilloides 11,11b klouekanmè aspergillus tereus 14,28a aspergillus parasiticus 14,28a aspergillus oryzae 14,28a aspergillus ustus 14,28a aspergillus ochraceus 28,57b fusarium oxysporum 14,28a kétou aspergillus niger 16,66a aspergillus flavus 33,33b aspergillus ochraceus 16,66a fusarium oxysporum 16,66b mucor spp. 16,66a pobè aspergillus flavus 66,66a aspergillus spp. 16,66b aspergillus ochraceus 16,66b fusarium verticilloides 16,66b values are means. the means followed by same letter in the same group (collection zone) are not significantly different according to anova and tukey’s multiple comparison tests. euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 54 haf © 2013 vol. iv, no. 1 issn: 2283-3927 table 3: prevalence of isolated fungi from collected pigeon pea fungi isolated nca prevalence (%) aspergillus ochraceus 10 23.80a aspergillus flavus 7 16.66b aspergillus niger 3 7.14c aspergillus oryzae 2 4.76d aspergillus terreux 2 4.76d aspergillus ustus 2 4.76d aspergillus parasiticus 1 2.38e aspergillus fumigatus 1 2.38e aspergillus candidus 1 2.38e aspergillus spp. 1 2.38e fusarium oxysporum 8 19.04b fusarium verticilloides 2 4.76d fusarium spp. 1 2.38e mucor spp. 1 2.38e nci : number of cases of isolation out of 60 samples. values are means. the means followed by same letter in the same column are not significantly different according to anova and tukey’s multiple comparison tests. 4 discussion increasing interest in finding new food sources to alternative malnutrition in developing countries has observed in recent time. according to naylor et al. (2004), some of africa’s native drought-tolerant crops are also some of the least researched worldwide and are thus referred to as “orphan crops’’. this study, which focus on the valorization of the neglected and under-utilized species of benin such as pigeon pea, has indicated that different varieties of this legume, often considered as a secondary crops, are not fully known by the beninese populations. it culture has not also been extensively focused by scientific researches as it is the case in some countries such as nigeria (ade-omowaye et al., 2015) and uganda (velay et al., 2001). however, cultivation of pigeon pea has been also reported in niger, mali (versteeg and koudokpon, 1993), ethiopia, zimbabwe (kamanga and shamudzarira, 2001), zambia (boehringer and caldwell, 1989), botswana (amarteifio et al., 2002), and south africa (swart, 2000). the results from the present study also indicated the low technological valorization of this food resource, which is used exclusively for direct consumption after cooking. therefore, these findings underlined the need to use scientific knowledge and biotechnological tools to value this important food resource. however, like other legumes, the results from this study also indicated the infestation of pigeon pea by insects as well as fungi. these findings are also in accordance of those reported by hubert et al. (2007) which underlined the implication of weevils in the contamination of euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 55 haf © 2013 vol. iv, no. 1 issn: 2283-3927 crops by fungi. the results from the isolation and identification of fungi in pure culture have indicated the contamination of pigeon pea collected in southern benin by fungi, with a high occurrence of aspergillus strains (71.42%). this contamination is characterized by the presence of aspergillus parasiticus, a. flavus and a. ochraceus (table 3). this high contamination by aspergilla strains could be in relation with the type of fungi presents in the soil of culture areas. the toxinogenic potential of these fungi have been reported by adjou et al. (2012) in peanut cakes and also in peanut seeds collected from benin (adjou et al., 2013). these fungi can produce a significant amount of mycotoxins, when food storage conditions are inadequate. review of scientific literature on mycotoxin related human diseases, clearly reveals a linkage between ingesting of mycotoxin-contaminated food and illness, especially hepatic, gastro-intestinal, carcinogenic and teratogenic diseases. among these mycotoxins, aflatoxins (afs) produced predominantly by aspergillus parasiticus and a. flavus are highly toxic as they are carcinogenic, teratogenic, and causing human liver and extra-hepatic cancers (castegnaro, 1999). other mycotoxins such as ochratoxin a (ota) and citrinin (cit) produced by aspergillus ochraceus, a. carbonarius and a. niger (pfohl-leszkowicz, 2002) are also detected in food. the co-contamination of fungi strains which can produce aflatoxins and ochratoxin a in pigeon pea seeds should be taken into consideration. this is particularly important in regard to possible synergism and additive effects of these mycotoxins. such co-contamination has been previously observed with other food samples, such as breakfast cereal (molinie et al., 2005), olives (el-adlouni et al., 2006) and peanut cakes (adjou et al., 2012). this makes it important to avoid the conditions that lead to mycotoxin formation at all levels of production, harvesting, transport and storage, which is not always possible and not always achieved in practice. according to fernández-cruz et al., (2010), environmental stress conditions such as insect infestation, drought, cultivar susceptibility, mechanical damage, nutritional deficiencies, and unseasonable temperature, rainfall or humidity can promote mycotoxin production in growing crops. in fact, changes in farming practices in the past few decades may result in increasing stress on plants and therefore enhance fungal invasion and mycotoxin contamination (fernández-cruz et al., 2010). 5 conclusion this survey underlined the low valorization of pigeon pea in southern benin and its contamination by fungi, which could be resulted in mycotoxin production. the control of mycotoxin-contamination in agricultural products must taking into account the good agricultural practices which include early harvesting, proper drying, basic sanitation measures (removal and destruction of debris from previous harvest) and proper storage (adequate drying, elimination of insect activity). the biological control through the development of atoxigenic bio-control fungi that can out-compete their closely related, toxigenic strains in field environments, could also be used. acknowledgments: the authors are grateful to the department of food engineering of polytechnic school of abomey-calavi university for the financial support. euloge s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 49 58 56 haf © 2013 vol. iv, no. 1 issn: 2283-3927 references ade-omowaye, b.i.o., tucker, g.a., 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l’ouganda. biotechnol. agron. soc. environ. 5 (2), 105–114. versteeg, m.n., koudokpon, v., 1993. participative farmer testing of four low external input technologies to address soil fertility decline in mono province (benin). agricultural systems, 42, 265 –276. wagacha, j.m., muthomi, j.w., 2008. mycotoxin problem in africa: current status, implications to food safety and health and possible management strategies. int. j. food microbiol. 124,1-12. wu, n., fu, k., fu, y.j., zu, y.g., chang, f.r., chen, y.h., liu, x.l., kong, y., liu, w., gu, cb., 2009. antioxidant activities of extracts and main components of pigeon pea (cajanus cajan l. millsp). leaves. molecule 14 (3), 1032-1043. l keywords broiler feed, fat source, feed additives, chicken burger, quality characteristics. pages 1 – 11 references vol. 5 no. 1 (2018) article history submitted: november 17, 2017 accepted: february 11 2018 published: february 15 2018 corresponding author engy, f. zaki animal breeding department, desert research center, , 1 matariya st., b.o.p.11753 matariya cairo, egypt mail: angyfayz@yahoo.com. phone: +202 26332846 fax: +202 26357858 journal home page riviste.unimi.it/index.php/haf article quality characteristics of chicken burger processed from broiler chicken fed on different types of vegetable oils and feed additives engy f. zaki 1, *, el faham a.i. 2 and nematallah g.m. 2 1 meat production and technology unit, animal breeding department, desert research center, cairo, egypt. 2 poultry production department, faculty of agriculture, ain shams university, cairo, egypt. abstract the objective of this study was to investigate the effect of feeding broiler chicken on different vegetable oils with commercial multienzyme feed additives on the quality characteristics of chicken burger. a total of 216 oneday-old chicks of (hubbard) strain were randomly assigned to six dietary treatments as (2×3) factorial designs where two sources of dietary oil contained soybean oil and palm oil with three levels of commercial multienzyme feed additives. treatments were: soybean oil only (t1), soybean oil+ zad (t2), soybean oil+ amphi-bact (t3), palm oil only (t4) , palm oil + zad (t5) and palm oil + amphibact (t6). results showed that chicken burger of t1 group had the higher ph value (6.22); slight difference was found in ph value of t3 group (6.18). no significant difference was found in burger of t5 and t6 group. burger processed from t1 group had the higher t.b.a value (0.115) followed by burger of t5 (0.076); while the lowest t.b.a value found in burger of t2 group (0.031). no significant differences were found in shrinkage measurements. burger processed from t6 group had the higher score of sensory attributes and overall acceptability, while the differences between the other burger groups were not significant. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 2 haf © 2013 vol. v, no. 1 issn: 2283-3927 1 introduction chicken has been considered an appropriate model in lipid nutrition studies, since it is highly sensitive to dietary fat modifications and many of the studies done with chickens deal with the degree of saturation or source type of the dietary replaced fat and how it influences the performance and carcass quality improvement of the animal (rymer and givens, 2005). vegetable oils are a widely used source of energy in broiler diets. however, most of the vegetable oils are mainly used for human consumption and also for biodiesel production. in this regard, interest is growing in using alternative fat sources in poultry nutrition rather than using crude oil sources, which would increase competition between bio fuel industry and food and feedstuff markets. palm oil or mixtures of palm oil, fatty acids distilled from the palm and calcic soap are sources of vegetal oils with a fatty acid profile that might replace animal fats without any kind of negative impact on carcass quality (rodriguez et al., 2002). the inclusion of soybean oil in broiler diets does not affect the moisture and ether extract in the breast and thigh muscles. furthermore, the deposition of fat on the breast muscle and viscera is not affected by the inclusion of the oil in the diet. dietary fat quality not only affects animal growth performance and health (lin et al., 1989; enberg et al., 1996) but also influences the quality of broiler meat and meat products (lin et al., 1989; asghar et al., 1989). lipid oxidation is a major cause of quality deterioration in meat and meat products and can give rise to rancidity and the formation of undesirable odours and flavours, which affect the functional, sensory, and nutritive values of meat products (gray et al., 1996). commercial enzyme preparations have been used widely to enhance nutritive value of wheat and rye-based diets because of high insoluble non-starch polysaccharides found in these feedstuffs which induce high digesta viscosity (lázaro et al., 2003). additionally, it was reported that enzyme cocktail feed additives improve bird's productivity (saleh et al., 2005) and digestibility of corn-soybean meal based diets, which in turn, induces less viscosity of ingested feed for broilers (olukosi et al., 2007). enzyme such as microbial phytase has been used as commercial feed additive in broiler feed production to improve nutritive values of plant based diets. addition of microbial phytase to broiler diet leads to hydrolysis of phytase, which bind phosphorus of the plant based diet (kies, et al., 2001). moreover, interest in the use of phytase as feed additive has now increased due to problems posed by phosphates in animal wastes. inclusion of exogenous enzyme in animal’s diet has been shown to improve broiler’s performance. but the effect on meat quality has to be determined as certain feed additives have been found to affect meat qualities (wang, et al., 2013; omojola, et al., 2014). therefore, this research aims to study the effect of using different vegetable oil sources and feed additives in finisher diets of broiler chicken, on the processing of chicken burger and its impact on the quality characteristics. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 3 haf © 2013 vol. v, no. 1 issn: 2283-3927 2 material and method 2.1 experimental design the experimental procedures were approved by the poultry production department, faculty of agriculture, ain shams university and as followed by the animal breeding department, animal and poultry production division, desert research center. the current study was conducted at poultry experimental unit, faculty of agriculture, ain shams university, located in agricultural research station, shalaqan, qalyobia governorate, egypt. the experiment was a 2 × 3 factorial design with two sources of vegetable oils (soybean oil and palm oil) with three levels of commercial multi-enzyme feed additives as shown in the table 1. a total of 216 one-day-old chicks of (hubbard) strain were used for this study, the chicks were randomly assigned to six treatment groups. each group consisted of six replicates and each replicate was made up of six chicks. the basal diet was formulated to meet the nutrient requirements of broiler chicken following the national research council (nrc, 1994) as shown in table 2. • starter: one-day-old till 11 days-of-age (basal diet – without additives all birds). • grower: 12 days till 22 days (basal diet without additives all birds). • finisher: 23 days till 35 days (experimental diets specified per treatment). chicks were housed in galvanized cages, where nine birds were allotted to a pen cage of 100 cm long, 40 cm width and 40 cm height. the farm building was aerated naturally. lighting program was controlled to provide 23 hours light and one hour dark daily by candescent bulb lighting system. room temperature was maintained around 32° c for the first week and was decreased by 3° c weekly afterwards. at the end of experiment, four chickens were randomly selected for slaughtering from each treatment to use in the processing of chicken burger. slaughtered birds were scalded in hot water bath, plucked and eviscerated manually. chicken meat from thigh and abdominal muscles were collected, packed and frozen at -18ºc until further analyses and processing of chicken burger were completed. table 1: experimental design type of oil feed additives without addition zad1 0.5kg/ton amphi-bact2 0.5kg/ton soybean oil treatment 1 (t1) treatment 2 (t2) treatment 3 (t3) palm oil treatment 4 (t4) treatment 5 (t5) treatment 6 (t6) 1 (zad) which contains bacteria (ruminococcus flavefaciens) with concentration of (28 x 104). also it contains a mixture of enzymes (cellulase xylanase α-amylase -protease). 2(amphi-bact), which contains bacteria (lactobacillus acidophilus) and (lactobacillus planterum) and (bifidobacterium bifidum) and extract ferment of both (bacillus subtilus) and (aspergillus niger) with concentration of 5 g / kg and also contains a mixture of enzymes that is estimated as 34.5 units / gram, that is equivalent to 2 g / kg (cellulase betaglucanase hemicellulase). engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 4 haf © 2013 vol. v, no. 1 issn: 2283-3927 table 2: feed ingredients and chemical analyses of experimental diets ingredients starter (0-11) grower (12-22) finisher (23-35) t1 t2 t3 t4 t5 t6 corn (grains) 52.05 55.91 56.80 56.80 56.80 56.80 56.80 56.80 soybean meal (44%) 31.50 30.00 28.25 28.25 28.25 28.25 28.25 28.25 corn gluten meal (62%) 7.20 4.86 4.40 4.40 4.40 4.40 4.40 4.40 soybean oil 3.00 3.65 5.00 5.00 5.00 palm oil 5.00 5.00 5.00 wheat bran 2.00 1.50 2.00 2.00 2.00 2.00 2.00 2.00 di-calcium phosphate 1.85 1.60 1.34 1.34 1.34 1.34 1.34 1.34 calcium carbonate 1.30 1.50 1.35 1.35 1.35 1.35 1.35 1.35 premix* 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 salt (nacl) 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 dl-methionine 0.29 0.28 0.21 0.21 0.21 0.21 0.21 0.21 l-lysine hcl 0.21 0.10 0.05 0.05 0.05 0.05 0.05 0.05 total 100 100 100 100 100 100 100 100 nutrient content (calculated) ** crude protein % 23.00 21.00 20.00 20.00 20.00 20.00 20.00 20.00 crude fat % 5.69 6.39 7.76 7.76 7.76 7.76 7.76 7.76 crude fiber % 3.88 3.75 3.70 3.70 3.70 3.70 3.70 3.70 me kcal/ kg diet 3029 3076 3171 3171 3171 3171 3171 3171 calcium % 1.00 1.01 0.90 0.90 0.90 0.90 0.90 0.90 available phosphorus % 0.50 0.45 0.40 0.40 0.40 0.40 0.40 0.40 lysine % 1.30 1.15 1.06 1.06 1.06 1.06 1.06 1.06 methionine & cystein % 0.97 0.93 0.84 0.84 0.84 0.84 0.84 0.84 * each 3 kg of premix contains: vitamins: a: 12000000 iu; vit. d3 2000000 iu; e: 10000 mg; k3: 2000 mg; b1:1000 mg; b2: 5000 mg; b6:1500 mg; b12: 10 mg; biotin: 50 mg; coline chloride: 250000 mg; pantothenic acid: 10000 mg; nicotinic acid: 30000 mg; folic acid: 1000 mg; minerals: mn: 60000 mg; zn: 50000 mg; fe: 30000 mg; cu: 10000 mg; i: 1000 mg; se: 100 mg and co: 100 mg. ** nutrient content calculated based on chemical analysis data of feedstuffs provided by nrc (1994). 2.2 preparation of chicken burger chicken meat from each experimental diet was ground through a 3mm plate grinder. chicken burger samples were prepared as follows ingredients; 7.5% onion, 0.5%black pepper, spices0.5%, salt 1.5% (mikhail et al., 2014). batches of 2kg of each dietary treatment were handily mixed and formed by using manual burger press machine (1cm thickness, 10cm diameter and 60±2g weight). burgers were placed in plastic foam trays packed in polyethylene bags and frozen at -18ºc±1until further analysis. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 5 haf © 2013 vol. v, no. 1 issn: 2283-3927 2.3 physical analysis 2.3.1 ph value ph of raw chicken burger was measured as described by hood(1980). ten grams of sample was homogenized with 100ml distilled water and measured using a digital ph-meter jenway 3310 conductivity and ph meter. ph values were done on three replicates per treatment. two burgers were used for each replication. 2.3.2 cooking measurements chicken burger samples of each treatment were cooked in preheated grill at110°c (to an internal temperature 72°c±2). all cooking measurements were done on four replicates per treatment. for each replication three burgers were examined for cooking loss, reduction in thickness, reduction in diameter and shrinkage. the cooking loss was determined as reported by naveena et al. (2006) as follows: 2.3.3 shrinkage measurements raw and cooked samples were measured for diameter and thickness of chicken burger as described by berry (1993) using the following equation: dimensional shrinkage was calculated using the following equation as reported by murphy et al. (1975): 2.4 t.b.a. value measurement of lipid oxidation: the extent of lipid oxidation in raw chicken burger was assessed by measuring 2thiobarbituric acid reactive substances (tbars), as described by aocs (1998).tba values were done on three replicates per treatment. three burgers were used in each replication. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 6 haf © 2013 vol. v, no. 1 issn: 2283-3927 2.5 sensory evaluation chicken burger was subjected to organoleptic evaluation as described by amsa (1995). ten trained panelists of staff members of food sciences department, faculty of agriculture, ainshams university were scored appearance, texture, juiciness, flavour, tenderness and overall acceptability using a 9-point hedonic scale. the mean scores of the obtained results of organoleptic evaluation were then statistically analyzed. 2.6 statistical analysis analysis of variance (anova) was used to test the obtained data using the general linear modelling procedure (sas, 2000). the used design was one way analysis. duncan’s multiple tests (1955) were applied for comparison of means. 3 results table 3 showed the physiochemical properties of chicken burger processed from broiler chicken fed on different types of vegetable oil and feed additives. chicken burger of t1 group had the higher ph value (6.22); slight difference was found in ph value of t3 (6.18) burger. no significant difference was found in burger of t5 and t6 group. however, burger of t2 and t4 group had the lower ph value (6.05). table 3: physicochemical properties of chicken burger treatments parameters ph cooking loss (%) t.b.a (mgmda/kg) t1 6.22 ± 0.06 a 36.21±2.95 a 0.115± 0.010 a t2 6.05±0.03 c 32.48±2.29 ab 0.031±0.017 d t3 6.18± 0.01 ab 31.75±4.37 b 0.061±0.011 c t4 6.05±0.04 c 31.68±2.20 b 0.063±0.010 c t5 6.14± 0.015 b 34.29±0.68 ab 0.076±0.010 b t6 6.14±0.02 b 35.83±1.53 a 0.065±0.010 c sem 0.020 1.30 0.003 a-d means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means data of cooking loss of chicken burger processed from broiler chicken fed on different types of vegetable oil and feed additives indicated that burger of t1 and t6 groups had the higher cooking loss, followed by burger of t2 and t5. no significant differences were found in burger of t3 and t4. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 7 haf © 2013 vol. v, no. 1 issn: 2283-3927 data of t.b.a value of burger processed from broiler chicken fed on different types of vegetable oil and feed additives were showed in table 3. burger processed from t1 group had the higher t.b.a value followed by burger of t5, while the lowest t.b.a value found in burger of t2 group. no significant differences were found in t.b.a value of other burger samples (t3, t4 and t6). data in table 4 showed the shrinkage measurements of chicken burger processed from broiler fed on different types of vegetable oil and feed additives. burger of t1 group had the higher reduction in diameter; no significant differences were found in burger of t5 group and burger of t6 group. also, no significant differences were found in burger samples of other dietary treatments. table 4: shrinkage measurements of chicken burger treatments parameters reduction in diameter (%) reduction in thickness (%) shrinkage (%) t1 23.30±2.65 a 28.51±5.70 a 26.64±2.80 a t2 16.61±1.31 c 27.85±2.58 a 16.46±2.58 d t3 14.74±1.14 c 21.02± 4.18 a 16.62±1.19 d t4 16.33±1.33 c 24.07±5.25 a 17.76±0.43 cd t5 19.33±1.30 b 23.24±6.38 a 20.23±1.71 cb t6 19.99±0.75 b 29.04±0.83 a 21.77±2.29 b sem 0.76 2.64 1.00 a-d means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means. from the same table 4, it can be found that no significant differences were found in the reduction in thickness% of chicken burger processed from broiler fed on different types of vegetable oil and feed additives. burger of t1 group had the higher shrinkage % followed by burger of t6. slight significant differences were found between the other burger samples. sensory evaluation of chicken burger processed from broiler fed on different types of vegetable oil and feed additives are showed in table 5. burger of t6 had the higher score for appearance and slight significant differences were found in burger of t2, t4 and t5 groups. no significant differences were found between burger of t1 and t3 which had the lower score. burger of t6 had the higher score of texture, juiciness and tenderness followed by burger of t2, t4 and t5 and no significant differences were found between the other burger samples. burger of t6 recorded the higher score for flavour while the lower score found in burger of t1 and no significant differences were found between other burger samples. however, burger processed from t6 had the higher score of overall acceptability, while the differences between the other burger samples were slightly significant. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 8 haf © 2013 vol. v, no. 1 issn: 2283-3927 table 5: sensory evaluation of chicken burger treatments appearance texture juiciness flavor tenderness overall acceptability t1 7.00± 1.41 b 6.71± 1.11 b 6.85±1.35 b 6.42±1.90 b 6.57±1.27 b 6.71±1.25 c t2 7.57±0.98 ab 7.14±1.35 ab 7.28±1.38 ab 6.71±1.80 ab 7.14±1.35 ab 7.42±0.98 abc t3 7.14±1.07 b 6.85±0.69 b 6.57±1.27 b 6.71±1.60 ab 6.57±0.98 b 7.28±0.49 bc t4 8.00±1.15 ab 7.28±1.60 ab 7.42±1.40 ab 7.85±0.69 ab 7.57±0.79 ab 7.71±0.76 ab t5 7.71±1.11 ab 7.28±1.70 ab 7.28±1.50 ab 6.85±1.21 ab 7.14±1.68 ab 7.14±1.07 bc t6 8.57±0.53 a 8.28±0.76 a 8.28±0.76 a 8.14±0.69 a 8.28±0.49 a 8.28±0.49 a sem 0.40 0.47 0.49 0.53 0.43 0.33 a-c means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means. 4 discussion addition of feed additives had no significant effects on ph value of t5 and t6 or between t2 and t4, while a slight different was found between t1 and t3. these results are close to that obtained by zakaria et al. (2010) they found that enzymes addition had no effect on ph value of broiler chicken meat. however, the effect of dietary enzyme on ph value of chicken meat was difficult to understand. these may be due to that enzymes are difficult to predict since enzyme action may be affected by many factors, including environment, amount of enzyme in the reaction, and interactions between enzyme and other substances, which are still not fully understood. type of dietary oil had a significant effect on cooking loss of chicken burger. these results are disagrees with that obtained by pekel et al. (2012) they indicated that dietary fat source did not affect cooking loss of chicken meat. although cooking loss decreased with the increasing levels of dietary fat, there were no significant differences between the dietary fat sources. the effects of feed additives on cooking loss of chicken burger were significantly different. addition of (zad with palm oil and soybean oil) had no significant effect on cooking loss, while addition of (amphibact with palm oil) caused a significant increase in cooking loss. omojola et al. (2014) found that chicken fed diets containing sesame and soybean diet supplemented with enzymes had higher cooking loss than those on sesame and soybean diet without enzymes. while zakaria et al. (2010) found that dietary enzyme had no effect on cooking loss of broiler chicken meat. data of t.b.a. values showed significant differences in chicken burgers processed from chicken feed different types of oils and feed additives. these results are close to that obtained by abdulla et al. (2015) they found that a significant difference in lipid oxidation was observed among the dietary oils. breast muscles from broilers fed a diet supplemented with palm oil (po) had a lower tbars value (p <0.05) compared with soybean oil (so) throughout the post-mortem storage. on the other hand, the present result disagrees with the findings of pekel et al. (2012) they found that no significant differences were found in t.b.a. value of thigh meat from broilers engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 9 haf © 2013 vol. v, no. 1 issn: 2283-3927 fed diets with different levels of fat from soybean oil (so) or neutralized sunflower soap stock (nss). data of shrinkage measurements showed that dietary fat sources had significant effect on reduction in diameter and shrinkage percentages of chicken burgers. however, addition of enzymes had no significant effect on shrinkage measurements. these results are consonance with that obtained by omojola et al. (2014) they reported that there was no significant effect on the meat characteristics of broiler chickens fed on diets (soybean and sesame) supplemented with or without microbial phytase. fat sources and addition of feed additives had no significant effects on reduction in thickness of chicken burgers. these results are close to that obtained by dalólio et al. (2015) they found that enzyme supplementation in diets based on corn and soybean meal did not influence the parameters of chicken meat quality. the same results were found by pekel et al. (2012). sensory evaluation of chicken burger processed from different vegetable oil and feed additives showed that the differences between sensory attributes were not significant, although the burger of t6 had the higher score in overall acceptability, but the differences between the other sensory attributes were not significant. these results are consonance with (stanaćev et al., 2014) they found that dietary addition of vegetable oils did not show any improvement of chicken breast meat sensory quality. also, kalakuntla et al. (2017) concluded that sensory attributes of chicken broiler meat were not influenced due to dietary incorporation of n-3 pufa oil sources. 5 conclusion the purpose of the current study was to evaluate the quality characteristics of chicken burger processed from broiler chicken fed on different type of vegetable oils and feed additives. the addition of soybean oil and palm oil as fat sources for use in chicken diets in combination with feed additives (enzymes) had no negative effects on the quality traits of chicken burger. further studies on the effects of feeding broiler chicken on dietary oils and commercial feed additives on the processing and quality characteristics of chicken meat products are suggested. references abdulla, n. r., loh, t.c., akit, h., sazili, a.q., foo, h.l., mohamad, r., abdul rahim, r., ebrahimi, m., sabow, a.b., 2015. fatty acid profile, cholesterol and oxidative status in broiler chicken breast muscle fed different dietary oil sources and calcium levels. south african journal of animal science. 45(2), 153-163. amsa, 1995. american meat science association. research guidelines for cookery, sensory evaluation and instrumental tenderness measurements of fresh beef. chicago, il, usa. aocs, 1998. american oil chemists' society. official method cd 19-90. 2-thiobarbituric acid value. direct method. in: firestone d, editor. official methods and recommended practices of the american oil chemists' society, 5th ed. champaign, p.3. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 10 haf © 2013 vol. v, no. 1 issn: 2283-3927 asghar, a., lin, c. f., gray, j. i., buckly, d. j., booten, a. m., flagal, c. j., 1989. influence of oxidized dietary oil and antioxidant supplementation on membrane bound lipid stability in broilers meat. british poultry science. 30, 815-823. berry, b.w., 1993. fat level and freezing temperature affect sensory, shear cooking and composition properties of ground beef patties. journal of food science. 58 (1), 34-42. dalólio, f. s., vaz, d. p., moreira, j., albino, l. f. t., valadares, l. r., 2015. carcass characteristics of broilers fed enzyme complex. biotechnology in animal husbandry. 31 (2), 153-162. duncan, d. b., 1955. multiple range and multiple ftests. biometrics. 11, 142. engberg, r. m., lauridsen, c., jensen, s. k., jakobson, k., 1996. inclusion of oxidized vegetable oil in broiler diets. its influence on nutrient balance and on the antioxidant status of broiler. poultry science. 75, 1003-1011. gray, j.i., gomaa, e.a., buckley, d.j., 1996. oxidative quality and shelf life of meats. meat science. 43, s111-s123. hood, d. e., 1980. factors affecting the rate of metmyoglobin accumulation in prepackaged beef. meat science. 4 (4), 47–50. kalakuntla, s., nagireddy, n. k., panda, a. k., jatoth, n.,thirunahari, r., vangoor, r. r., 2017. effect of dietary incorporation of n-3 polyunsaturated fatty acids rich oil sources on fatty acid profile, keeping quality and sensory attributes of broiler chicken meat. animal nutrition. 3, 386-391. kies, a.k., vanhemert, k.h.f., sauer, w.c., 2001. effect of phytase on protein and amino acid digestibility and energy utilization. world poultry science journal. 57, 109-126. lázaro, r., garcía, m., araníbar, m.j., mateos, g.g., 2003. effect of enzyme addition to wheat-, barleyand rye-based diets on nutrient digestibility and performance of laying hens. british poultry science. 44, 256-265. lin, c. f., asghar, a., gray, j. i., buckly, d. j., boome, a. m., crackel, r. l., flegal, c. j., 1989. effect of oxidized dietary oil and antioxidant supplementation on broiler growth and meat stability. british poultry science. 30, 855-864. mikhail, w.z.a., sobhy, h.m., khallaf, m.f., ali, h. m.z., el-askalany, s. a., ezz el-din, m. m., 2014. suggested treatments for processing high nutritive value chicken burger. annals of agricultural science. 59(1), 41–45. murphy, e. w., criner, p. e., grey, b. c., 1975. comparison of methods for calculating retentions of nutrients in cooked foods. journal of agricultural food chemistry. 23, 1153–1157. nrc, 1994. national research council. nutrient requirements of poultry 9th ed. composition of poultry feedstuffs. national academy press, washington, dc, usa. naveena, b. m., muthukumar, m., sen, a. r., babji, y., murthy, t. r. k., 2006. quality characteristics and storage stability of chicken patties formulated with finger millet flour (eleusine coracana). journal of muscle foods. 17 (1), 92–104. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 1 11 11 haf © 2013 vol. v, no. 1 issn: 2283-3927 olukosi, o.a., cowieson, a.j., adeola, o., 2007. age-related influence of a cocktail of xylanase, amylase and protease or phytase individually or in combination in broilers. poultry science. 86, 77-86. omojola, a. b., otunla, t. a., olusola, o. o., adebiyi, o. a., ologhobo, a. d., 2014. performance and carcass characteristics of broiler chicken fed soybean and sesame/soybean based diets supplemented with or without microbial phytase. american journal of experimental agriculture.4 (12), 1637-1648. pekel, a. y., demirel, g., midilli, m., yalcintan, h., ekiz, b., alp, m., 2012. comparison of broiler meat quality when fed diets supplemented with neutralized sunflower soapstock or soybean oil. poultry science. 91, 2361–2369. rodriguéz, m.a., crespo, n.p., cortés, m., creus, e., medel, p., 2002. efecto del tipo de grasa de la dieta en la alimentacion del broiler, con enfasis en los productos derivados del aceite de palma. selecciones avícolas. 44(10), 693-702. rymer, c., givens, d.i., 2005. omega-3 fatty acid enrichment of edible tissue of poultry: a review. lipids. 40, 121-130. saleh, f., tahir, m., ohtsuka, a., hayashi, k., 2005. a mixture of pure cellulase, hemicellulase and pectinase improves broiler performance. british poultry science. 46, 602-606. sas, 2000. user’s guide statistics. sas institute, inc., cary, n.c., usa. stanaćev, v. ž., milošević, n., pavlovski, z., milić, d., vukić vranješ, m., puvača, n., stanaćev, v. s., 2014. effects of dietary soybean, flaxseed and rapeseed oil addition on broilers meat quality. biotechnology in animal husbandry. 30 (4), 677-685. wang, w., wang, z., yang, h., cao, y., zhu, x., zhao, y., 2013. effects of phytase supplementation on growth performance, slaughter performance, growth of internal organs and small intestine and seum biochemical parameters of broilers. open journal of animal science. (3), 236-241. zakaria, h. a. h., mohammad, a. r. j., abu ishmais, m. a., 2010. the influence of supplemental multi-enzyme feed additive on the performance, carcass characteristics and meat quality traits of broiler chickens. international journal of poultry science. 9 (2), 126-133. l keywords broiler feed, fat source, commercial multienzyme, chicken meat, fatty acid, quality characteristics. pages 40 – 50 references vol. 5 no. 1 (2018) article history submitted: february 05, 2018 accepted: march 15 2018 published: april 20 2018 corresponding author engy, f. zaki animal breeding department, desert research center, , 1 matariya st., b.o.p.11753 matariya cairo, egypt mail: angyfayz@yahoo.com. phone: +202 26332846 fax: +202 26357858 journal home page riviste.unimi.it/index.php/haf article fatty acids profile and quality characteristics of broiler chicken meat fed different dietary oil sources with some additives engy f. zaki 1, *, el faham a.i. 2 and nematallah g.m. 2 1 meat production and technology unit, animal breeding department, desert research center, cairo, egypt. 2 poultry production department, faculty of agriculture, ain shams university, cairo, egypt. abstract the study was carried out to investigate the effect of feeding broiler chicken on different vegetable oils with feed additives on the quality characteristics of chicken meat. a total of 216 one-day-old chicks of hubbard strain were randomly assigned to six dietary treatments as (2×3) factorial designs where two sources of dietary oil with three levels of commercial multi-enzyme feed additives. treatments were: soybean oil only (t1), soybean oil+ zad (t2), soybean oil + amphi-bact (t3), palm oil only (t4), palm oil + zad (t5) and palm oil + amphibact (t6). results showed that feeding broiler chickens on different types of dietary oils had significant effect on the fatty acid profile of broiler chicken meat. ufa/sfa ration of broiler chicken fed palm oil were significantly lower compared with those fed soybean oil. broiler fed on soybean oil had significantly higher n-6: n-3 ration compared with broiler fed on palm oil. regardless of the source of dietary oil, significant differences were observed in the most of fatty acid profile in the chicken meat among levels of commercial multienzyme feed additives. meat of t5and t6 had the higher ph value, followed by meat of t1and t3 groups, while the lowest ph value found in meat of t2 and t4. the higher cooking loss was found in meat of t4 while, meat of t5had the lowest value. data of chilling loss indicated that the differences between dietary treatments were not significantly different except for meat of t6 which had the higher chilling loss. no significant differences were found in color measurements between dietary treatments. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 41 haf © 2013 vol. v, no. 1 issn: 2283-3927 1 introduction poultry feeding is one of the most important aspects of poultry production. therefore, for profitable poultry rearing, provision of economical and balanced feed is due. fats constitute the main energetic source for poultry and they have the highest caloric value among all the nutrients (anjum et al., 2004). poultry meat contains high and low total fat content and, more importantly a higher monounsaturated and polyunsaturated fatty acid (mufa and pufa) content than other meats (howe et al., 2006). currently, consumers are more concerned about their food, especially nutritional aspects. among the nutritional aspects of food, lipid content and fatty acid profile are the most important factors (bostami, et al., 2017). plant oils have commonly been used as energy sources in diets of broiler chicks (jalali et al., 2015). advantages of utilizing oils in poultry diet include decrease of feed dust, increase in absorption and hydrolysis of lipoproteins supplying the essential fatty acids, (nobakht et al., 2011). the fatty acid content of broiler meat depends on the type of diet intake by the birds (crespo & esteve-garcia, 2002). enzyme such as microbial phytase has been used as commercial feed additive in broiler feed production to improve nutritive values of plant based diets. inclusion of exogenous enzyme in animal’s diet has been shown to improve broiler’s performance (wang, et al., 2013 ) but the effect on meat quality has to be determined as certain feed additives have been found to affect performance and carcass characteristics (omojola, et al., 2014) . therefore, the objective of this study was to determine the effect of palm oil and soybean oil with or without addition of feed additives in finisher diets on the fatty acids profile of chicken meat and to examine the impact of these oils and feed additives on the quality characteristics of meat such as color measurements, ph value, chilling and cooking loss. 2 material and method 2.1 experimental design the experimental procedures were approved by the poultry production department, faculty of agriculture, ain shams university and as followed by the animal breeding department, animal and poultry production division, desert research center. the current study was conducted at poultry experimental unit, faculty of agriculture, ain shams university, located in agricultural research station, shalaqan, qalyobia governorate, egypt. the experiment was a 2 × 3 factorial design with two sources of vegetable oils (soybean oil and palm oil) with three levels of commercial multi-enzyme feed additives included (zad which contains bacteria (ruminococcus flavefaciens) with concentration of (28 x 104). also it contains a mixture of enzymes (cellulase xylanase α-amylase -protease). amphi-bact, which contains bacteria (lactobacillus acidophilus) and (lactobacillus planterum) and (bifidobacterium bifidum) and extract ferment of both (bacillus subtilus) and (aspergillus niger) with concentration of 5 g / kg and also contains a mixture of enzymes that is estimated as 34.5 units / gram, that is equivalent to 2 g / kg (cellulase beta-glucanase hemicellulase ). a total of 216 one-day-old chicks of hubbard strain were used for this study, the chicks were randomly assigned to six treatment groups. each group included six replicates and each replicate was made up of six chicks. the basal diet was formulated to meet the nutrient engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 42 haf © 2013 vol. v, no. 1 issn: 2283-3927 requirements of broiler chicken following the national research council (nrc, 1994) as shown in table (1). diets were offered in three feeding phases, starter from one-day-old to 11 days (basal diet – without additives all birds), grower from 12 to 22 days (basal diet without additives all birds) and finisher from 23 to 35 days (experimental diets specified per treatment). table 1: feed ingredients and chemical analyses of experimental diets starter (0-11) grower (12-22) finisher (23-35) ingredients t1 t2 t3 t4 t5 t6 corn (grains) 52.05 55.91 56.80 56.80 56.80 56.80 56.80 56.80 soybean meal (44%) 31.50 30.00 28.25 28.25 28.25 28.25 28.25 28.25 corn gluten meal (62%) 7.20 4.86 4.40 4.40 4.40 4.40 4.40 4.40 soybean oil 3.00 3.65 5.00 5.00 5.00 palm oil 5.00 5.00 5.00 wheat bran 2.00 1.50 2.00 2.00 2.00 2.00 2.00 2.00 di-calcium phosphate 1.85 1.60 1.34 1.34 1.34 1.34 1.34 1.34 calcium carbonate 1.30 1.50 1.35 1.35 1.35 1.35 1.35 1.35 premix* 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 salt (nacl) 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 dl-methionine 0.29 0.28 0.21 0.21 0.21 0.21 0.21 0.21 l-lysine hcl 0.21 0.10 0.05 0.05 0.05 0.05 0.05 0.05 total 100 100 100 100 100 100 100 100 nutrient content (calculated) ** crude protein % 23.00 21.00 20.00 20.00 20.00 20.00 20.00 20.00 crude fat % 5.69 6.39 7.76 7.76 7.76 7.76 7.76 7.76 crude fiber % 3.88 3.75 3.70 3.70 3.70 3.70 3.70 3.70 me kcal/ kg diet 3029 3076 3171 3171 3171 3171 3171 3171 calcium % 1.00 1.01 0.90 0.90 0.90 0.90 0.90 0.90 available phosphorus % 0.50 0.45 0.40 0.40 0.40 0.40 0.40 0.40 lysine % 1.30 1.15 1.06 1.06 1.06 1.06 1.06 1.06 methionine & cystein % 0.97 0.93 0.84 0.84 0.84 0.84 0.84 0.84 * each 3 kg of premix contains: vitamins: a: 12000000 iu; vit. d3 2000000 iu; e: 10000 mg; k3: 2000 mg; b1:1000 mg; b2: 5000 mg; b6:1500 mg; b12: 10 mg; biotin: 50 mg; coline chloride: 250000 mg; pantothenic acid: 10000 mg; nicotinic acid: 30000 mg; folic acid: 1000 mg; minerals: mn: 60000 mg; zn: 50000 mg; fe: 30000 mg; cu: 10000 mg; i: 1000 mg; se: 100 mg and co: 100 mg. ** nutrient content calculated based on chemical analysis data of feedstuffs provided by nrc (1994). the present trial was designed as testing dietary treatments was focused on only finisher stage and thus experimental diets will be offered for 12 days before marketing, from 23 days and up to 35 days. chicks were housed in galvanized cages, where nine birds were allotted to a pen cage of 100 cm long, 40 cm width and 40 cm height. the cages were kept in engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 43 haf © 2013 vol. v, no. 1 issn: 2283-3927 environmentally controlled rooms, where the temperature was maintained around 32° c for the first week and was decreased by 3° c weekly afterwards. lighting program was controlled to provide 23 hours light and one hour dark daily by candescent bulb lighting system. at the end of experiment, four chickens from each treatment were randomly selected based on similar body weight for slaughtering. slaughtered birds were scalded in hot water bath, plucked and eviscerated manually. chicken meat from thigh and abdominal muscles were collected, packed and frozen at -18ºc until further analyses. 2.2 determination of fatty acid profile the fatty acid profiles of broiler chicken meat were analyzed as describe by aoac (2012). the fatty acids were methylated with boron tri fluoride in methanol, extracted with heptanes and determined on a gas chromatograph with fid detector (pe auto system xl) with auto sampler and eȥchrom integration system. carrier gas (he); ca. 25psiair 450ml/minhydrogen 45mlsplit100 ml/min. oven temperature 200°c injector and detector 250°c. lipid extraction and direct methylation were performed in accordance with folsch et al. (1957). 2.3 physical analysis 2.3.1 ph value raw chicken meat ph values were determined as described by hood (1980). ten grams of sample was homogenized with 100ml distilled water and measured using a digital ph-meter jenway 3310 conductivity and ph meter. values of ph were determined in triplicate for each treatment. 2.3.2 cooking loss chicken meat samples of each treatment were cooked in a water bath at 85°c until the internal temperature reached 78°c (meek et al., 2000). cooked meat samples were cooled in running tap water for 1 h and then cooked samples were reweighed. all cooking measurements were done on three replicates per treatment. the cooking loss was determined as follows: 2.3.3 chilling loss chilling loss of broiler chicken meat was determined as describe by omojola et al. (2014). broiler chicken meat samples (200±10 g) were chilled at 4ºc for 24 hours, after which the meat was thawed and weighed .three replicates were done for each treatment. chilling loss was calculated as follows: engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 44 haf © 2013 vol. v, no. 1 issn: 2283-3927 2.4 color measurements color of raw chicken meat samples was measured by chroma meter (konica minolta, model cr 410, japan) calibrated with a white plate and light trap supplied by the manufacturer (cie, 1976). the color was expressed as l* (lightness), a* (the redness) and b* (the yellowness). the average of three spectral readings at different locations was obtained for each treatment. 2.5 statistical analysis analysis of variance (anova) was used to test the obtained data using the general linear modeling procedure (sas, 2000). the used design was one way analysis. duncan’s multiple tests (1955) were applied for comparison of means and the significance was defined as p<0.05. 3 results data of fatty acid profile of broiler chicken meat fed on different types of oil and commercial multienzyme additives are showed in table (2). it was observed that meat of chicken fed on diets containing palm oil (t3,t4and t6) had higher content of palmitic acid(c16:0) than those fed on soybean oil(t1,t2 andt3). meat of broiler chicken fed soybean oil had higher content of linoleic acid (c18:2ω6) than that fed on palm oil. also, it can be found that ufa/sfa ration of broiler chicken fed on palm oil groups (t4, t5adt6) were significantly lower compared with those fed on soybean oil (t1,t2 and t3). the polyunsaturated fatty acids content was significantly higher in broiler chicken meat fed on diets containing soybean oil than that fed on diets containing palm oil. broiler fed on soybean oil had significantly higher n6: n-3 ration compared with broiler fed on palm oil. regardless of the source of dietary oil, significant differences were observed in the most of fatty acid profile in the chicken meat among levels of commercial multienzyme feed additives. table (3) showed the physical characteristics of broiler chicken meat fed on different types of vegetable oil and feed additives. broiler chicken fed on palm oil (t5and t6) had the higher ph value, followed by broiler fed on soybean oil (t1and t3). also; it can be found that addition of commercial multi-enzyme feed additives had a significant effect on ph value of broiler chicken meat fed on soybean oil (t2 and t3), while no significant effect was found on ph value of those fed on palm oil with addition of commercial multi-enzyme feed additives (t5 and t6). data of cooking loss of broiler chicken meat fed on different types of oils showed that no significant differences were found between broiler fed on soybean oil t1 and t2; slight difference was found in cooking loss of t3 group, while significant differences were found in cooking loss of broiler fed on palm oil. addition of commercial multienzyme feed additives had significant effect on cooking loss. broiler chicken fed on palm oil supplemented with commercial multienzyme feed additives had lower cooking loss 21.59 and 26.40 %but slight difference was found in cooking loss of broiler chicken fed on soybean oil with commercial multienzyme feed additives. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 45 haf © 2013 vol. v, no. 1 issn: 2283-3927 table 2: fatty acid composition (% of total fatty acids) of broiler chicken meat fatty acids t1 t2 t3 t4 t5 t6 sem caproic acid c6:0 1.18 caprlyic acid c8:0 0.33 capric acid c10:0 0.73 lauric acid c12:0 0.13 0.16 0.16 1.80 myristic acid c14:0 0.67e 2.00b 0.79ed 1.31c 0.88d 3.62a 0.06 pentadecanoic acid c15:0 0.43 0.25 0.15 0.18 1.0 palmitic acid c16:0 24.70b 21.39e 23.34cd 33.60a 22.66d 23.99cb 0.37 heptadecanoic acid c17:0 0.20d 0.34c 0.48b 0.40c 0.40c 1.13a 0.01 stearic acid c18:0 5.50d 5.96cd 6.31bc 6.42bc 6.89b 10.19a 0.24 arachidic acid c20:0 0.25 0.14 0.71 0.20 0.33 behenic acid c22:0 0.50 ∑sfa 32.25b 30.54c 31.44bc 43.25a 31.38bc 43.14a 0.49 tetradecenoic acid c14:1ω5 0.15 c15:1ω6 0.38 palmiticoleic acid c16:1ω9 4.56 0.27 0.16 0.13 0.20 c16:1ω7 3.55 3.89 1.57 3.53 2.80 c16:1ω5 0.36 oleic acid c18:1ω9 37.30a 29.46d 36.94a 32.39c 33.49b 31.81c 0.35 vaccinic acid c18:1ω7 2.39c 3.91a 2.89b 2.39c 3.00b 0.48d 0.06 gadolic acid c20:1ω9 0.23 0.96 0.40 0.30 0.66 eicosaenoic acid c20:1ω11 0.27 c20:1ω7 0.40 eicosaenoic acid c20:1ω5 0.25 docosenoic acid c22:1ω11 0.20 0.10 0.26 c22:1ω9 0.24 ∑mufa 44.68a 38.60c 44.41a 37.15d 40.44b 36.96d 0.36 c16:2ω4 0.36 0.16 c18:2ω5 0.21 0.36 0.22 linoleic acid c18:2ω6 22.00b 24.98a 22.62b 14.39d 25.19a 16.62c 0.32 c18:2ω4 0.20 0.21 c20:2ω6 0.17 0.67 0.18 0.12 0.55 decatrienoic acid c16:3ω4 0.21 0.11 0.64 0.15 0.21 γ linolenic acid c18:3ω6 0.71 0.11 0.13 0.17 0.37 linolenic acid c18:3ω3 0.73d 1.57a 0.72d 1.20b 1.16b 0.89c 0.03 α octadectetraenoic c18:4ω3 0.27 0.30 eicosatrienoic acid c20:3ω6 0.51 0.13 0.36 arachidonic acid c20:4ω6 1.10 0.40 0.36 0.42 0.52 eicosapentaenoic c20:5ω3 0.16 1.06 0.17 ∑pufa 23.30c 30.45a 24.14c 18.29e 27.51b 20.27d 0.39 ∑ufa 67.84a 69.05a 68.55a 55.44b 67.96a 57.16b 0.63 ufa/sfa 2.10b 2.25a 2.17ab 1.27c 2.16b 1.32c 0.02 mufa/ sfa 1.38a 1.25b 1.40a 0.85c 1.28b 0.85c 0.02 pufa/ sfa 0.72d 0.99a 0.76c 0.42f 0.87b 0.46e 0.01 ∑ω6 22.17d 27.91a 23.31c 14.89f 26.03b 18.80e 0.34 ∑ω3 0.73d 1.74b 0.72d 2.23a 1.33bc 1.20c 0.13 n-6:n-3 30.55a 16.25bc 32.47a 6.88d 19.60b 15.68c 1.15 non identified 0 0.52 0.01 1.20 0.04 0 a-e means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means. . engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 46 haf © 2013 vol. v, no. 1 issn: 2283-3927 table 3: physical properties of broiler chicken meat treatments parameters ph cooking loss (%) chilling loss (%) t1 6.11±0.02 b 33.21±4.67 ab 3.64±0.42 b t2 5.96±0.06 c 31.77±1.14 ab 3.25±0.23 bc t3 6.08±0.01 b 30.14±0.94 b 3.13±0.05 c t4 5.99±0.01 c 33.89±1.90 a 3.37±0.11 bc t5 6.19±0.01 a 21.59±0.45 d 3.64±0.51 b t6 6.18±0.03 a 26.40±2.76 c 4.20±0.18 a sem 0.01 1.03 0.14 a-d means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means results of chilling loss of broiler chicken fed on different types of vegetable oil showed that the differences between broiler chicken fed on soybean oil were not significantly different. no significant differences were found between broiler chicken fed on palm oil except for t6 which had the higher chilling loss. addition of commercial multi-enzyme had no significant effect on chilling loss of broiler chicken meat except for broiler chicken fed on palm oil with multienzyme feed additives (t6)which had the higher chilling loss. color measurements of broiler chicken meat fed on different dietary oils and commercial multienzyme feed additives shown in table (4). no significant differences were found in l* value between dietary treatments. table 4: color measurements of broiler chicken meat parameters treatments l a b t1 51.82±4.37 11.80±2.02 20.05±3.24 t2 54.02±2.09 12.18±0.48 16.56±7.38 t3 53.74±2.70 12.89±2.36 19.53±1.26 t4 53.60±5.30 11.58±1.72 18.65±1.08 t5 52.41±1.25 13.55±0.33 21.06±2.96 t6 53.71±3.48 11.51±2.53 22.17±4.35 sem 2.00 1.03 2.30 means within the same column with different superscripts letters are different (p<0.05). t1, t2 and t3: treatments for soybean oil/ soybean oil with zad 0.5kg/ton and soybean oil with amphi-bact 0.5kg/ton. t4, t5andt6: treatments for palm oil/ palm oil with zad 0.5kg/ton and palm oil with amphi-bact 0.5kg/ton. means ± standard deviation. sem: standard error of means. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 47 haf © 2013 vol. v, no. 1 issn: 2283-3927 also, data showed no significant differences were found between a* and b* values of broiler chicken meat. addition of commercial multienzyme feed additives had no significant effect on color measurements. 4 discussion feeding broiler chicken on different types of dietary oils had significant effect on the fatty acid profile of broiler chicken meat. the higher content of palmitic acid (c16:0) in chicken groups fed on palm oil than that groups fed on soybean oil may be due to the high content of palmitic acid in palm oil. these results are in agreement with that obtained by abdulla et al. (2015) they found that the proportion of palmitic acid (c16:0) increased in meat from broiler fed palm oil (po) in comparison with those fed diets supplemented with soybean oil (so)and linseed oil(lo). the increase in the proportion of palmitic acid in chicken fed the palm oil diet could be owing the high palmitic acid content of palm oil (abdulla et al., 2015).this result is similar to the findings of hitn (2006) and smink et al. (2010) they reported that the levels of palmitic acid increased significantly in broiler breast muscle supplemented with palm oil compared with groups supplemented with soybean oil, coconut oil and sunflower oil. higher content of linoleic acid (c18:2ω6) was found in meat of broiler chicken fed soybean oil than that fed on palm oil. these results are agree with that found by abdulla et al. (2015) who reported that birds fed lo and so diets had significantly higher linoleic acid compared with those fed po. also, ayed et al. (2015) found that soybean oil caused large increase in the level of linoleic acid in broiler chicken meat compared with palm oil. the increasing level of linoleic acid is more pronounced because this acid is readily absorbed and deposited within the chicken’s fat depot (ayed et al., 2015). ufa/sfa ration of broiler chicken fed palm oil were significantly lower compared with broiler chicken fed soybean oil. these results are close to that obtained by abdulla et al. (2015) they reported that the proportion of total saturated of meat samples increased, while total ufa content decreased when palm oil (po) was incorporated in the diet, resulting in a significantly lower ufa: sfa ratio of the broiler breast muscle compared with those fed soybean oil (so) and linseed oil (lo) diets. the polyunsaturated fatty acids content was significantly higher in broiler chicken meat fed on diets containing soybean oil than that fed on diets containing palm oil. these results are agrees with ayed et al. (2015) they found that the polyunsaturated fatty acids content was significantly (p<0.05) higher in the group fed by ration containing soybean oil. the n-6: n-3 ratio significantly differs among sources of oil. these results are close to that obtained by bostami et al. (2017) they found that the higher n-6: n-3 ratio was found in broilers fed on soybean oil. significant differences were observed in the most of fatty acid profile in the chicken meat among levels of commercial multienzyme feed additives. responses to enzymes vary widely and are difficult to predict since enzyme action may be affected by many factors, including environment, amount of enzyme in the reaction, and interactions between enzyme and other substances, which are still not fully understood (zakaria et al., 2010). effect of feeding broiler chicken on different types of oils showed significant differences in ph values of chicken meat. these results are disagrees with that obtained by pekel et al. (2012) they found that the ph of breast meat did not differ between broilers fed on diets supplemented with soybean oil and the neutralized sunflower soapstock oil. addition of commercial multi-enzyme feed additives had no significant effect on ph value of broiler chicken meat fed on palm oil. these results are close to that obtained by zakaria et engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 48 haf © 2013 vol. v, no. 1 issn: 2283-3927 al. (2010) they found that enzymes addition had no effect on ph value of broiler chicken meat. however, the effect of dietary enzyme on ph value of chicken meat was difficult to understand. these may be due to that enzymes are difficult to predict since enzyme action may be affected by many factors, including environment, amount of enzyme in the reaction, and interactions between enzyme and other substances, which are still not fully understood. data of cooking loss indicated that addition of commercial multienzyme feed additives had significant effect on cooking loss of broiler chicken meat. broiler chicken fed on diets supplemented with commercial multienzyme feed additives had lower cooking loss than those fed on diets without feed additives. these results are disagrees with that obtained by omojola et al. (2014) they found that chicken fed diets containing sesame and soybean diet supplemented with enzymes had higher cooking loss than those on sesame and soybean diet without enzymes. while, zakaria et al. (2010) found that dietary enzyme had no effect on cooking loss of broiler chicken meat. however, the lower cooking loss in meat of t5and t6may be attributed to their ph values. low ph of meat is known to negatively affect the waterholding capacity of the meat and cooking loss is a function of the whc (warris and brown, 1987). the higher ph improved the whc and reduced the cooking loss. data of chilling loss indicated that the differences between dietary treatments were not significantly different except for broiler chicken fed on palm oil with commercial multienzyme (t6) which had the higher chilling loss. these results are close to that obtained by teye et al. (2015) they found that palm kernel oil residue inclusion up to 17.5% in broiler rations has no significant effects on chilling loss of broiler chicken meat. also, the results of the present study are consonance with that obtained by omojola et al. (2014) they reported that there was no significant effect on chilling loss of broiler chicken meat fed on diets (soybean and sesame) supplemented with or without microbial phytase. data of color measurements indicated that color characteristics of broiler chicken meat were not affected by the dietary oil types and feed additives. these results are close to that obtained by pekel et al. (2012) they found that breast meat color (l*, a*, b* values) were not affected by the dietary fat source on any of the measurement days (storage at 4°c for 0, 1, 2, and 5 days). also, dalólio et al. (2015) found that enzyme supplementation in diets based on corn and soybean meal did not influence the color parameters of chicken meat. l* was reduced when the dietary levels of fat increased. zakaria et al. (2010) they reported that dietary enzyme had no effect on the broiler chicken meat color. also, they reported that enzyme addition did not affect the different meat quality parameters which are related to each other, such as the ph value and color. woelfel et al. (2002) reported that there was a relationship between l* value and muscle ph in which l* value increased as the muscle ph decreased in broiler chicken meat. results of l* and ph values showed the same trend. 5 conclusion the results of the current study confirmed that using soybean oil and palm oil in broiler chicken diets would subsequently affect the composition of fatty acids in chicken meat. broiler fed on palm oil had higher total n-3 and lower n-6:n3 compared with broiler fed on soybean oil. however, palm oil can be used in broiler chicken feeding with positive effects on meat quality and human health. engy f. zaki et al. int. j. of health, animal science and food safety 5 (2018) 40 50 49 haf © 2013 vol. v, no. 1 issn: 2283-3927 references abdulla, n. r., loh, t.c., akit, h., sazili, a.q., foo, h.l., mohamad, r., abdul rahim, r., ebrahimi, m., sabow, a.b., 2015. fatty acid profile, cholesterol and oxidative status in broiler chicken breast muscle fed different dietary oil sources 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the influence of supplemental multi-enzyme feed additive on the performance, carcass characteristics and meat quality traits of broiler chickens international. journal of poultry science. 9 (2), 126-133. l keywords season, fertilizer, biomass production, chemical composition, weather. pages 12 – 23 references vol. 4 no. 1 (2017) article history submitted: january 17, 2017 revised: may 21, 2017 accepted: may 24 2017 published: may 30 2017 corresponding author nazmul huda, scientific officer, animal production research division, bangladesh livestock research institute savar, dhaka, bangladesh postal code: 1341 web: www.blri.gov.bd mail: hudanazmul1971@gmail.com phone: + +88019358388744 journal home page riviste.unimi.it/index.php/haf article seasonal weather impacts on biomass production of moringa oleifera at different fertilizer doses nazmul huda 1,* , khan shahidul haque 1 , biplob kumer roy 1 , nathu ram sarker 1 1 bangladesh livestock research institute, (blri), savar, dhaka, bangladesh abstract a year round agronomical trial was conducted on station (bangladesh livestock research institute) to investigate the impact of seasonal variations on biomass production of moringa oleifera at different fertilizer doses in bangladesh. a 6×3 factorial arrangement in a completely randomized design (crd) was applied on 20m×12m =240 m2 size plot, established in 2006 with a plant density of 13,500/hectare, was equally divided into 18 sub-plots and randomly grouped into three; 6 plots was treated as control, 6 plots was treated as medium with medium doses of fertilizer and rest 6 was treated as high fertilizer dose with the ratio of n.p.k was 90:30:15 and 160:60:40 kg/ha, respectively. the obtained result revealed that, the summer was the best season and autumn, monsoon and spring was also favorable for getting maximum yield of moringa oleifera with ambient temperature ranges 27-32 0 c and fertilization dose n:p:k= 90:30:15 is suitable for optimum moringa production and chemical composition of moringa varied with season and slightly with fertilization. n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 13 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction moringa oleifera, one of the fast-growing indian native trees (mekonnen, 2002) and the world’s most nutrient-rich plant (foidle et al., 2001) yet discovered, grows faster under the subtropical low lands in south asia and africa (yang et al., 2006) may be used as a cattle feed for increasing meat (roy et al., 2016) and milk (mathur, 2006) production and to improve nutritional status (kakengi et al., 2007) or as a goat feed for increasing their live weight replacing dietary conventional concentrates completely (sultana et al., 2015) in addition to its other uses like, growth enhancers of other plant (phiri & mbewe, 2010), water purifiers (sutherland, 1989), human herbal medicines (yongbai, 2011; sreelatha & padma, 2009), and a source of natural oil (farooq & rashid, 2007) or biodiesel (rashid et al., 2008). within 13 known species moringa oleifera lam. is the most common known and widely distributed utilized species (fahey, 2005). moringa, socially known to be a homestead vegetable plant in the country, may be produced as a tree fodder using appropriate agronomical practices (palada & cheng, 2003). getting biomass of foliage it could be grown through direct seeding, transplanting or using hard stem cutting (palada & cheng, 2003). once, twice or all year round flowering occurs depending with seasonal temperature and rainfall (parotta, 1993). for optimum production well draining soil is required but in most types of soil from acid to alkaline it can grow and prosper even in low nutrient soil that drains quickly having ideal ph 5.5-7.0 in range (duke, 1983). dry regions is particularly suitable for moringa production because it can tolerate full sun and very hot weather but doesn’t freeze or frost, rarely need watering as it favors rainwater for grown well without irrigation (saint & broin, 2010) except during very hot weather when it required irrigation just once a week (rajakrishnamoorthy et al., 1994). suitable climate for production is hot and humid ranges 25-35 °c temperature, exceedingly well growth is shown at temperature of 32.5-49.2 °c. below 21°c the plant growth become dormant and at more than 15°c fluctuating day-night temperature leaves may lose or become yellow and by freezing temperature it killed (lowell & sreeja, 2011; palada & cheng, 2003; parotta, 1993). without fertilizer moringa trees are able to grow successfully (ramachandran et al., 1980), once established, the deep and far-reaching root system of moringa can gather it’s nutrients from the soil efficiently (palada and chang, 2003). though, by applying 7.5 kg farmyard manure and 0.37 kg ammonium sulphate/tree pod yield is possible to increase threefold (lowell & sreeja, 2011). even, biodynamic compost helps to increase yield about 50% compared to ordinary compost (palada and chang, 2003). if some supplemental fertilizer and irrigation is applied, best yield can be got under dry and warm condition (radovich, 2009) with nine cuttings year (amaglo, 2006). however, bangladesh as a sub tropical moringa growing favored country. annual variations of weather and fertilizer doses may play a significant role on the availability of biomass and its quality and may mitigate the feed scarcity. season, weather, variety, fertilization and irrigation affects the yields vary widely. best yield can be get under dry and warm condition if some supplemental fertilizer and irrigation is applied (radovich, 2009). the highest (p<0.001) biomass (bm), dry matter (dm) and crude protein (cp) yield per hectare of moringa was obtained at 1 st cutting during autumn season than yielded that of 2 nd cutting. the higher ambient temperature (27 0 c) and humidity (81%) during 1 st cutting could be the reason of higher productivity of moringa than 2 nd cutting (average temperature 24 0 c; humidity 60%) the overall bm and dm yield per hectare were reported to be of 3.99 and 0.7 tons, respectively (huda et al., 2016). in the similar study, huda et al., (2016) n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 14 haf © 2013 vol. iv, no. 1 issn: 2283-3927 also reported that though application of fertilizer did not affects significantly on bm and dm yield but the medium doses of fertilizer (n:p:k at a ratio of 90:30:15) was suitable for optimum production of moringa in respect of bm, dm and cp yield during autumn season compared to control (without fertilizer) and high doses of fertilizer (n:p:k at a ratio of 160:60:40). here the result presented by the author was drawn only based on the autumn season. but in perspective of bangladesh considering agro-ecological variance, the overall seasonal impacts on productivity of m. oliefera at different fertilizer doses were unknown and needs to determine through year round in-depth study for building up the suited agronomic cultural practices of moringa oleifera. therefore, the present study was undertaken to determine the seasonal weather impact on biomass production of moringa oleifera at different fertilizer doses. 2 materials and methods the experiment was carried out in bangladesh livestock research institute (blri), savar, dhaka, bangladesh, located in middle of the young brahmaputra and jamuna flood plain & madhupur tract of agro-ecological zone having gray flood plain & madhupur tract soil. the soil was slightly acidic in reaction (ph 5.6-6.5) with silt loam texture. the land was flat, moderate drained and above flood levels. a moringa plot of 20×12 m2, established in 2006 with a plant density of 13,500/hectare, was equally divided into 18 sub-plots. keeping randomly 6 of them as a control, the rest 12 sub-plots were randomly grouped into two; one of them of 6 plots was fertilized with n:p:k at a ratio of 90:30:15, respectively; and the rest 6 plots with a ratio of 160:60:40, respectively. the experiment was conducted during autumn, 2013monsoon, 2014, and tripple super phosphate (tsp) and murate of potash (mp) were mixed with soil after spading (0.06 kg and 0.03 kg/plot, respectively) as basal doses. urea, splitting into two doses, was applied during spading of soil after each harvest, and again after 15 days of foliage growth. since the start of the experiment foliages were harvested at 33, 31, 48, 48, 36, 31, 31, 55 and 45 days of intervals on constant intercultural operations like, weeding, irrigation etc. except rainfall no irrigation were applied and stacked water of heavy rainfall was drained out and for enough precipitation desired moisture remains in the soil during almost the experimental time. the biomass (green foliage) was harvested in a single day each of the cutting from the foot of each stem where the shoots were pruned. fresh samples were collected and analyzed for determination of dry matter (dm), crude protein (cp), acid detergent fiber (adf), neutral detergent fiber (ndf), ether extract (ee) and ash according to aoac (1990). data on fresh biomass production and daily temperature, humidity and rainfall were recorded, and all the data of weather, biomass and its different nutrients as well were analyzed statically by an anova of a 6×3 factorial experiment in a completely randomized design (crd) using the glm procedures with spss, 17 computer software packages. n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 15 haf © 2013 vol. iv, no. 1 issn: 2283-3927 3 results all the year round readings of different weather parameters i.e.: temperature, relative humidity, precipitation, showed highly significant (p<0.001) difference among the season (table 1). highest and lowest temperature of the year was found in summer (32 0 c) and winter (19 0 c) season, respectively, differed significantly (p<0.001) with other four seasons; whereas the ambient temperature between autumn and monsoon season did not vary significantly (p>0.05). table 1: average daily weather readings of different season parameters season sed sig. autumn late a. winter spring summer monsoon tm (0c) 29a 24b 19c 27d 32e 29a 0.20 *** rh (%) 79a 64b 54c 48d 70e 77a 0.50 *** prec. (mm) 9.07a 2.38b 0.00c 0.92d 7.20e 11.64f 0.77 *** tm = temperature, rh = relative humidity, prec. = precipitation, *** = p<0.001, highly significant, abcd values with different superscripts in the same row differ significantly; se: standard error of mean the average relative humidity was significantly (p<0.001) higher in autumn and lower in spring season. however, the variation of relative humidity between autumn and monsoon was not significant (p>0.05). the average rainfall precipitation data vary significantly (p<0.01) among the seasons with highest at monsoon (11.64 mm) followed by autumn (9.07 mm), summer (7.20 mm), late autumn (2.38 mm), spring (0.92 mm) and winter (0.00 mm), respectively (table 1). variations on yield with season clearly visible in figure 1 showed that seasons as well as their ambient temperature has an impacts on fresh biomass, dry matter, and crude protein yield, and significant (p<0.05) polynomial relations (y= 0.138 x 2 0.8205 x + 2.4423, r²= 0.3217; y= 0.0185 x 2 0.0941 x + 0.3685, r²= 0.2357; y= 0.0051 x 2 -0.0247 x+ 0.0801, r²= 0.4229, respectively) were observed with the highest and lowest bm, dm and cp yield in summer and winter season, respectively; where autumn and monsoon showed comparatively higher (p<0.01) production than spring and late autumn season. this figure also shows that ambient temperature over 27 0 c favored to enhancing yield and at 32 0 c plants performed best. irrespective of fertilizer doses, the biomass yield of moringa oleifera in different season varied significantly (p<0.001) with the highest and lowest was in summer (2793.6 kg/ha) and winter (309.7 kg/ha). however, the bm yield of moringa oleifera did not differ significantly (p>0.05) among monsoon, spring and late autumn (1909.2, 1588.1 and 1387.8 kg/ha, respectively) and late autumn and spring (1387.8 and 1588.1 kg/ha) had similar non-significant (p>0.05) trend in between, with significant difference to other season (table 2). the dm yield of different season was almost similar as bm yield except little more production in spring than monsoon with nonsignificant difference, so that, highest dm yield was obtained in summer (588.6 kg/ha) followed by autumn (354.7 kg/ha), spring (340.7 kg/ha), monsoon (338.4 kg/ha), late autumn (239.7 kg/ha) and winter (59.2 kg/ha). in case of cp yield, highest and lowest production was also obtained in summer (127.3 kg/ha) and winter (12.5 kg/ha) season with significant (p<0.001) difference to other season; where autumn (70.9 kg/ha) and late autumn n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 16 haf © 2013 vol. iv, no. 1 issn: 2283-3927 (51.96 kg/ha) had no significant difference in between and autumn also had no significant difference with late autumn, spring (73.38 kg/ha) and monsoon (90.8 kg/ha) but differed with other season significantly (p<0.001) (table 2). figure 1: annual variations in moringa production figure 2: impact of weather on bm yield in different season with 3 doses of fertilizer. dm: y = 0.0185x2 0.0941x + 0.3685 r² = 0.2357 cp: y = 0.0051x2 0.0247x + 0.0801 r² = 0.4229 bm: y = 0.138x2 0.8205x + 2.4423 r² = 0.3217 0 0,5 1 1,5 2 2,5 3 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 1 2 3 4 5 6 7 b m y ie ld t o n /h e c d m , c p y ie ld t o n /h e c season with avg. daily temperature dm cp bm autumn late a. winter spring summer monsoon 29 0c 24 0c 19 0c 27 0c 32 0c 29 0c rh: y = 0.8929x2 5.3357x + 31.8 r² = 0.4214 tm: y = 4.25x2 29.69x + 104.8 r² = 0.8698 rain f.: y = 1.6x2 10.5x + 17.43 r² = 0.9532 0 10 20 30 40 50 60 70 80 0 0,5 1 1,5 2 2,5 3 3,5 autumn late autumn winter spring summer monsoon t e m p e ra tu re , h u m id it y & r a in fa ll b m y ie ld t o n /h e c; 3 t re a tm e n ts season control medium high tm rh rf n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 17 haf © 2013 vol. iv, no. 1 issn: 2283-3927 table 2: effect of seasons and fertilizer doses on yield of moringa oliefera season, fertilizer & their interactions parameters biomass yield (fresh; kg/ha) dm yield (kg/ha) cp yield (kg/ha) autumn fertilizer dose control 1951.23 339.95 64.63 medium 2247.43 404.17 87.18 high 1773.56 320.10 60.81 late autumn fertilizer dose control 1737.93 299.55 67.03 medium 1462.86 253.16 52.91 high 962.74 166.37 35.94 winter fertilizer dose control 411.35 72.62 15.47 medium 318.82 65.06 13.44 high 198.79 39.97 8.56 spring fertilizer dose control 1541.01 327.54 74.36 medium 1659.78 363.29 75.66 high 1563.51 331.35 70.14 summer fertilizer dose control 2910.72 643.11 132.41 medium 2996.37 622.27 135.26 high 2473.74 500.55 114.09 monsoon fertilizer dose control 1901.72 300.32 81.97 medium 2258.06 459.18 123.15 high 1567.89 255.81 67.39 season autumn 1990.75 a 354.74 a 70.87 ad late autumn 1387.84 d 239.69 d 51.96 a winter 309.66 b 59.22 b 12.49 b spring 1588.11 d 340.73 a 73.38 d summer 2793.61 c 588.64 c 127.25 c monsoon 1909.23a d 338.44 ad 90.84 d fertilizer dose control 1742.33a c 330.52 ac 72.65a c medium 1823.89b c 361.19 bc 81.27 bc high 1423.38 a 269.03 a 59.49 a sed 95.72 19.02 4.10 sig.lev. s *** *** *** f * * * s×f ns ns ns significant level= (non significant=p>0.05; *=p<0.05,significantly different; ***=p<0.001, highly significant), abcd values with different superscripts in the same row differ significantly; se: standard error of mean the application of fertilizer doses also had a significant (p<0.05) impacts on production in terms of bm, dm and cp yield of moringa oleifera. irrespective of seasons, significantly (p<0.05) higher bm (1823.9 kg/ha), dm (361.2 kg/ha) and cp (81.3 kg/ha) yield of moringa n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 18 haf © 2013 vol. iv, no. 1 issn: 2283-3927 oleifera was obtained by application of medium doses of fertilizer and the lower bm (1423.4 kg/ha), dm (269.0 kg/ha) and cp (59.5 kg/ha) yield was observed by applying the high doses of fertilizer. however, the bm, dm and cp yield of moringa oleifera did not differ significantly (p>0.05) between control and medium and control & high doses of fertilizer application. similarly, the interaction of season × fertilizer doses had no significant (p>0.05) effect on bm, dm or cp yield. with variant relative humidity, temperature and rainfall biomass yield of different season was varied significantly along with fertilizer doses. figure 2 shows that, except winter and late autumn, where the yield was little most, biomass yield was obtained most with medium doses of fertilizer in all other four season. and, it is also observed from fig 2 that, humidity, temperature and rainfall had a significant (p<0.05) polynomial relationship (y = 0.8929x2 5.3357 x + 31.8, r² = 0.4214; y = 4.25 x229.69 x + 104.8, r² = 0.8698; y= 1.61 x2 – 10.48 x + 17.43, r²= 0.9532; respectively) with yield of different treatment of fertilizer doses. the result of chemical composition of moringa plant fodder, irrespective of fertilizer doses, showed highly significant (p<0.001) differences among the season in all senses of dry matter, organic matter, crude protein, ash, acid detergent fiber, neutral detergent fiber and ether extract content (table 3). irrespective of fertilizer doses, the dm content in moringa plant fodder varied significantly (p<0.001) among the seasons. the higher and lower dm content in moringa plant fodder was observed in spring and late autumn, respectively. however, the dm content was not differ significantly (p>0.05) among the autumn, late autumn and monsoon seasons; and contained significantly (p<0.001) lower dm percentage as compared to winter, spring and summer seasons. the organic matter percentage of autumn and late autumn (92.61 and 92.78, respectively), winter and monsoon (91.06 and 90.79, respectively) & spring and summer (89.87 and 89.67, respectively) was nonsignificant inbetween but these three pairs of season differed significantly (p<0.001) with other pairs. logically, ash percentage maintained the same trend as of om percentage. the cp, adf, ndf and ee content in moringa plant fodder were also varied significantly (p<0.001) among the seasons. the highest and lowest cp content in moringa plant fodder was found in monsoon (26.78) and autumn (20.31), respectively which differed significantly (p<0.001) with each other. on the other hand, cp content in moringa plant fodder did not vary significantly (p>0.05) among the late autumn, winter, spring and summer seasons. the adf content vary (p<0.001) from 22.7 to 38.3 on percent dry matter among the different seasons. monsoon had the higher and late autumn had the lower adf content in moringa plant fodder. it was also observed an increasing trend of adf content from late autumn and continued up to monsoon season. in case of ndf and ee, no specific trend was observed in moringa plant fodder under this study. the higher value for ndf and ee was observed in autumn and monsoon and the lower value was in late autumn and summer, respectively. irrespective of season, the responses of fertilizer doses on nutrient composition of moringa plant fodder revealed that fertilizer doses had no impact (p>0.05) on om, cp, ash, adf and ee content except the dm and ndf content in moringa plant fodder. the dm and ndf content influenced significantly (p<0.001) by the application of different fertilizer doses. however, significantly higher dm content was obtained in moringa plant fodder when a medium dose of fertilizer was applied in the soil. but no differences (p>0.05) for dm content was observed between control and high dose of fertilizer applied in the soil. the highest ndf percentage was found with control fertilizer dose, which was differed significantly (p<0.001) with high dose of fertilizer but nonsignificant with medium dose of fertilizer. again, medium dose of fertilizer respond nonsignificantly with high dose of n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 19 haf © 2013 vol. iv, no. 1 issn: 2283-3927 fertilizer. except adf and ee, the interaction of season and fertilizer doses affected significantly on dm (p<0.05), om (p<0.05), cp (p<0.05), ash (p<0.05) and ndf (p<0.001) content in moringa plant fodder. table 3: effect of seasons and fertilizer doses on nutrient composition of moringa oliefera season, fertilizer & their interactions parameters % dm % om % cp % ash % adf % ndf % ee autumn fertilizer dose control 17.47 92.91 19.20 7.09 33.26 58.84 2.91 medium 17.94 92.54 21.77 7.46 33.37 57.69 2.76 high 17.97 92.37 19.95 7.63 29.24 57.49 2.76 late autumn fertilizer dose control 17.32 92.73 22.33 7.27 23.56 44.29 3.05 medium 17.34 93.11 20.94 6.89 22.19 37.81 3.02 high 17.25 92.50 21.40 7.50 22.33 32.05 2.91 winter fertilizer dose control 17.62 90.84 21.58 9.16 29.82 51.66 2.34 medium 20.14 91.45 20.93 8.54 32.36 49.42 2.23 high 19.97 90.88 21.58 9.12 28.53 49.43 2.63 spring fertilizer dose control 20.90 89.81 22.26 10.19 34.35 50.58 2.89 medium 21.85 89.94 20.80 10.06 34.97 52.19 2.85 high 21.67 89.86 20.86 10.14 36.25 51.83 2.39 summer fertilizer dose control 22.14 90.59 20.61 9.41 35.66 51.10 2.45 medium 20.90 89.66 21.80 10.34 33.73 54.12 2.22 high 20.43 88.76 22.95 11.24 35.56 53.49 2.48 monsoon fertilizer dose control 15.72 90.55 27.25 9.44 37.46 46.18 4.83` medium 20.88 90.32 26.78 9.68 38.99 45.46 4.45 high 16.36 91.50 26.31 8.50 38.55 42.63 4.84 season autumn 17.79a 92.61c 20.31b 7.39c 31.96ac 58.01a 2.81a late autumn 17.30a 92.78c 21.56c 7.22c 22.69b 38.05b 3.00a winter 19.24b 91.06a 21.37c 8.94a 30.24a 50.17c 2.40b spring 21.48c 89.87b 21.31c 10.13b 35.19d 51.53cd 2.71a summer 21.16c 89.67b 21.79c 10.33b 34.98cd 52.90d 2.38b monsoon 17.65a 90.79a 26.78a 9.21a 38.33d 44.75e 4.71c fertilizer dose control 18.53b 91.24 22.21 8.76 32.35 50.44a 3.08 medium 19.84a 91.17 22.17 8.83 32.60 49.45ab 2.92 high 18.94b 90.98 22.18 9.02 31.74 47.82b 3.00 sed 0.19 0.12 0.12 0.12 0.75 0.39 0.06 sig.lev. s *** *** *** *** *** *** *** f *** ns ns ns ns *** ns s×f *** * * * ns *** ns significant level= (non significant=p>0.05; *=p<0.05,significantly different; ***=p<0.001, highly significant), abcd values with different superscripts in the same row differ significantly; se: standard error of mean n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 20 haf © 2013 vol. iv, no. 1 issn: 2283-3927 4 discussion monsoon and autumn, these two seasons are the best for optimum production of moringa foliage (chandran, 2013) and suitable climate for production is hot and humid ranges 25-35 0 c temperature (lowell & sreeja, 2011), and even, fresh biomass have a partial correlation with temperature and rainfall averaged over time (nouman et al., 2013). according the observation of this experiment, summer is the best season for optimum moringa production, where autumn, monsoon and spring are favorable for around maximum moringa production and suitable climate for maximum production is hot and humid ranges 2732 0 c temperature, 70-79 percent relative humidity and 7.211.64 mm avg. daily rainfall. only spring favored good production with low humidity and rainfall (48% & 0.92 mm, respectively). the result also showed that, fertilizer doses also had a significant (p<0.05) impacts on bm, dm and cp yield and with medium doses of fertilizer (n: p: k= 90:30:15 kg/ha) highest yield could obtained. mendieta et al. 2013 reported that, n fertilization is a key factor for biomass production and other studies also have reported the positive effect of n fertilization on moringa yield (dash & gupta, 2009; radovich, 2009; jyothi & babu, 2007; pamo et al., 2005). most recently, larwanou et al. (2014) noted that actual yields of moringa vary widely, depending on season, variety, fertilization, and irrigation regime. in nigeria, nitrogen fertilization (urea) with a rate of 200 kg per hectare were found best for moringa biomass production (abdullahi et al., 2013) but here in bangladesh we found 90 kg per hectare is best for moringa production. the result of chemical composition of moringa plant fodder in this experiment, showed highly significant (p<0.001) difference among the season in all senses of dry matter, organic matter, crude protein, ash, acid detergent fiber, neutral detergent fiber and ether extract percentage but only dm and ndf percentage was fluctuated by different fertilizer doses. this finding is resembles to mendieta et al. (2013), who reported that, there were no obvious effects of n fertilization level on cp, adf and ash content. in this experiment it is also found that, season and fertilizer has an interaction effects on dm, om, cp, ash and ndf percentage. 5 conclusions from the above result, it may be concluded that, summer was the best season for optimum production of moringa plant fodder and autumn, monsoon and even spring were also favorable for maximum production with ambient temperature ranges 27-32 0 c and medium dose of fertilizer (n:p:k= 90:30:15) was suitable for optimum moringa production. the study also concluded that chemical composition of moringa plant fodder varied mostly with season and slightly with fertilization. references abdullahi, i.n., ochi, k., gwaram, a.b., 2013. plant population and fertilizer application effects on biomassproductivity of moringa oleifera in north-central nigeria. in peak journal of agricultural sciences, 1, 94-100. n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 21 haf © 2013 vol. iv, no. 1 issn: 2283-3927 amaglo, n., 2006. how to produce moringa leaves efficiently, retrieved 2013-11-19. association of official analytical chemists, washington d. c. chandran, r., 2013. growing moringa: issues and constraints. technologies and practices for small agricultural producers, fao. world wide web: http://teca.fao.org/discussion/growing moringaissues-and-constraints. dash, s., gupta, n., 2009. effect of inorganic, organic and bio fertilizer on growth of hybrid moringa oleifera (pkm 1). in science, 4, 630–635. duke, j.a., 1983. handbook of energy crops (moringa oleifera). center for new crops and plant products. purdue university, indiana, us. fahey, j., 2005. moringa oleifera: a review of the medical evidence for its nutritional therapeutic, and prophylactic properties. part 1. trees for life journal. 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[online] world wide web: http://www.tfljournal.org/article.php/ 20051201124931586. farooq, a., rashid, u., 2007. physicochemical characteristics of moringa oleifera seeds and seed oil from a wild provenance of pakistan. in pakistan j. bot., 39, 1443–1453. foidle, n., makkar, h.p.s., becker, k., 2001. the potential of moringa oleifera for agricultural and industrial uses. moringa oleifera world wide web: http://miracletrees.org/ moringadoc/the_potential_of_moringa_oleifera_for_agricultural_and_industrial_uses.pdf. huda, n., hashem, m. a., huque, k. s. and roy, b. k., 2016. study on effects of fertilizer on production performance of moringa oleifera (moringa oleifera lam.) in autumn season. int. j. agri. innovations and research., 5(1): 2319-1473. jyothi, g., babu, r., 2007. graded doses of nitrogen on drumstick (moringa pterygosperma goertn var pkm-1). in j. res. angrau, 35, 111–113. kakengi, a.m.v., kaijage, j.t., sarwatt, s.v., mutayoba, s.k., shem, m.n., fujihara, t., 2007. effect of moringa oleifera leaf meal as a substitute for sunflower seed meal on performance of laying hens in tanzania. in livestock research for rural development, 19, no. 8. larwanou, m., adamou, m.m., abasse, t., 2014. effects of fertilization and watering regimes on early growth and leaf biomass production for two food tree species in the sahel: moringa oleifera lam. and adansonia digitata l. in journal of agricultural science and applications. j. agric. sci. appl, 3, 82-88. lowell, j., fuglie, k.v., sreeja, 2011. cultivation of moringa. world wide web: http://miracletrees.org/growing_moringa_content.html. mathur, b., 2006. moringa for cattle fodder and plant growth. president, trees for life. world wide web: http://www.tfljournal.org/files/moringa%20for%20fodder%20%26%20spray%20 (screen). pdf. mekonnen, y., 2002. the multipurpose moringa tree: ethiopia (project 1996-2002). institute of pathobiology, addis ababa university. volume 10: examples of the development of pharmaceutical products from medicinal plants, 111-118. http://miracletrees.org/growing_moringa_content.html n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 22 haf © 2013 vol. iv, no. 1 issn: 2283-3927 mendieta-ararica, b., spo`rndli, e., reyes-sa`nchez, n., salmero`n-miranda, f., halling, m., 2012. biomass production and chemical composition of moringa oleifera under different planting densities and levels of nitrogen fertilization. in agroforest syst., received: 19 april 2011 / accepted: 21 may 2012, springer science+business media b.v. 2012. doi 10.1007/s10457-0129525-5. nouman, w., siddiqui, m.t., basra, s.m.a., farooq, h., zubair, m., gull, t., 2013. biomass production and nutritional quality of moringa oleifera as a field crop. in turkish journal of agriculture and forestry. turk j agric, 37, 410-419. palada, m.c., chang, l.c., 2003. suggested cultural practices for moringa. asian vegetable research & development center (avrdc). in international cooperators’guide, 3, 545. pamo, e., boukila, b., tonfack, l., momo, m., kana, j., tendonkeng, f., 2005. influence de la fumure organique, du npk et du me´lange des deux fertilisants sur la croissance de moringa oleifera lam. dans l’ouest cameroun. in livest. res. for rural dev, 17. parotta, j.a., 1993. moringa oleifera lam, reseda, horseradish tree, moringaceae, horseradish tree family. usda forest service, international institute of tropical forestry. retrieved 2013-11-20. phiri, c., mbewe, d.n., 2010. influence of moringa oleifera leaf extracts on germination and seedling survival of three common legumes. in int. j. agric. biol., 12, 315–317. radovich, t., 2009. farm and forestry production and marketing profile for moringa (moringa oleifera). permanent agriculture resources (par), po box 428, holualoa, hawai'i 96725, us, retrieved 2013-11-20. world wide web: http://eol.org/pages/486251/details. rajakrishanamoorthy, v., santhanabosu, s., duraisamy, v.k., rajagopal, a., 1994. drip irrigation in annual moringa. in madras agri. j., 81, 678-679. ramachandran, c., peter, k.v., gopalakrishnan, p.k., 1980. drumstick (moringa oleifera): a multipurpose indian vegetables. in economic bot., 34, 276-283. rashid, u., anwar, f., moser, b.r., knothe, g., 2008. moringa oleifera oil: a possible source of biodiesel. in bioresource technol., 99, 8175–8179. roy, b.k., bashar, m.k., hossain, s.m.j., huque, k.s., makkar, h.p.s., 2016. performance evaluation of moringa oleifera and available roughages (maize and australian sweet jumbo) on feeding values of growing blri cattle breed-1 (bcb-1) bull. in american j. experimental agric., 14, article no. ajea.29284. saint-saveur, a., broin, m., 2010. growing and processing moringa leaves. moringanews/moringa association of ghana. world wide web: http://miracletrees.org/ moringa.doc/moringa_book_growing_and_processing_moringa_leaves. pdf. sreelatha, padma., 2009. antioxidant activity and total phenolic content of moringa oleifera leaves in two stages of maturity. in plant foods human nutr., 64, 303–311. sultana, n., alimon, a.r., huque, k.s., sazili, a.q., yaakub, h., hossain, j., baba, m., 2015. the feeding value of moringa (moringa oleifera) foliage as replacement to conventional concentrate diet in bengal goats. in adv. anim. vet. sci., 3, 164-173. http://eol.org/pages/486251/details n. huda et al. int. j. of health, animal science and food safety 4 (2017) 12 23 23 haf © 2013 vol. iv, no. 1 issn: 2283-3927 sutherland, j.p., folkard, g.k., grant, w.d., 1989. seeds of moringa species as naturally occurring flocculants. in sci. technol. dev., 7, 191–197. yang, r.y., chang, l.c., hsu, j.c., weng, b.b.c., palada, m.c., chadha, m.l., levasseur, v., 2006. nutritional and functional properties of moringa leaves-from germplasm, to plant, to food, to health. in proceedings of the international workshop “moringa and other highly nutritious plant sources: strategies, standards and markets for a better impact on nutrition in africa”. accra, ghana.16-18, 1-9. yongbai, k.a., lewis, d.m., harris, p.l., 2011. application of phytodisinfectants in water purification in rural cameroon. in african journal of microbiology research. 5, 628-635. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords nrf2, oxidative stress, reporter mice, omega-3 corresponding author elena mariani elena.mariani1@unimi.it journal home page riviste.unimi.it/index.php/haf nuclear factor e2-related factor 2’s activation in transgenic mice fed with high dosage of fish oil. e. mariania *, n. rizzib, n. cesaric, a. maggib, g. savoinia a department of health, animal science and food safety (vespa), università degli studi di milano, via celoria 10, 20133 milan, italy b center of excellence on neurodegenerative diseases and department of pharmacological sciences, università degli studi di milano via balzaretti, 9, 20123 milan, italy c polo veterinario di lodi, università degli studi di milano, via dell’università 6, 20900 lodi, italy abstract some fatty acids, such as cla (conjugated linoleic acid) and n-3 fatty acids modulate immune and inflammatory response in ruminants and monogastrics; their supplementation alters fatty acids profile of meat and milk, enhancing their nutritional quality. however, it is still unclear if their addition causes oxidative damage to animals. nuclear factor e2-related factor 2 (nrf2) plays an important role in cellular defenses against oxidative stress, indeed it produces a rapid induction of its target genes involved in antioxidant response. the aim of the project is to investigate the activation of nrf2 in luciferase reporter mice fed different amount of n-3 pufa in the diet (7,5% lard, 7,5% tuna oil, 20 % lard and 20%tuna oil). forty-eight reporter mice are divided into three groups: male, intact female and ovariectomized female. each group is split in four subgroups fed different diets. oxidative status will be studied monitoring nrf2’s activation with in vivo bioluminescent imaging. the inflammatory and immune response will be assessed using calprotectin and lactoferrin levels in faecal samples that are non-invasive techniques. the trial is still in progress: on the 62nd day, animals will be sacrificed after a challenge in order to measure the different effects of diets and +/oestrogen on stress response. finally, the post mortem analysis will be carried on extract organs. data obtained will be analysed using statistical procedures and results will improve the knowledge about interaction between omega-3 fatty acids and animals’ oxidative status. references lee, j.m., and johnson, j.a., 2004. an important role of nrf2-are pathway in the cellular defence mechanism. j biochem mol biol 37, 139-43. maggi, a. and ciana p., 2005. reporter mice and drug discovery and development. nat rev drug discov. 4(3), 249-55. nguyen, t., nioi, p., and pickett, c.b., 2009. the nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. j biol chem 284, 13291-5. rossi, r., pastorelli, g., cannata, s., corino, c., 2016. recent advances in the use of fatty acids as supplements in pig diets: a review. animal feed science and technology, 162, 1-11 savoini, g., farina, g., dell’orto, v., and cattaneo, d., 2016. through ruminant nutrition to human health: role of fatty acids. advances in animal biosciences, in press. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords sturgeon, oxidative stress, muscle, temperature corresponding author lucia aidos marialucia.matelaaidos@unimi.it journal home page riviste.unimi.it/index.php/haf oxidative stress and lateral muscle development in siberian sturgeon (acipenser baeri): preliminary observations. l. aidos*a, a. di giancamilloa, m. lanfranchib, c. domeneghinia adepartment of health, animal science and food safetyuniversity of milan, italy bsocietà agricola naviglio, goito, italy abstract embryonic development in bony fish is strongly influenced by environmental factors, mainly by temperature and dissolved oxygen, but little is known about their influence on sturgeons. since 1998 the international trade of sturgeons has been regulated under cites due to depletion of wild stocks. sturgeon aquaculture as well as larvae production is, therefore, important because it may contribute to the repopulation of the wild stocks (bronzi et al., 2011; di giancamillo et al., 2012). the aim of this study is to monitor oxidative stress status and muscle development during embryonic and precocious larval phases in siberian sturgeon (acipenser baeri), when subjected to different incubating temperatures (13°, 16° and 19°c). siberian sturgeon eggs were subjected to different incubation temperatures. data regarding water quality parameters were recorded, as well as mortalities, embryonic period length, and precocious larvae behavior. sampling was performed in the same developmental stage for each temperature, at five time points: 48 hours post-fertilization, embryo movements, hatching, schooling and yolk-sac full absorption. the observed hatching rates were between 85.5% and 98.8% with significant differences concerning the different experimental temperatures (t19 vs t13 and t19 vs t16, p<0,05). histological, histochemical and immunohistochemical analyses will be performed to assess ontogeny of the lateral muscle and stress biomarkers (lushchak, 2011), and gene expression will be analyzed for muscle development (johnston, 2006). the obtained results will be compared with those concerning teleosts and will possibly contribute to better rearing conditions of sturgeons. this study has been approved by the ethic committee of the università degli studi di milano, with the following authorisation code: opba_20_2016. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. ii, no. 2s issn: 2283-3927 figure 1. precocious sturgeon larvae during the schooling phase. references bronzi, p., rosenthal, h. and gessner, j. (2011). global sturgeon aquaculture production: an overview. journal of applied ichthyology volume 27, issue 2, pages 169–175, april 2011. di giancamillo, a., martino, p.a., arrighi, s. and domeneghini, c. (2012). gut peculiarities of feed deprived white sturgeons (acipenser transmontanus, richardson 1836). open journal of veterinary medicine, 2012, 2, 52-59. johnston, i. a. (2006). environment and plasticity of myogenesis in teleost fish. in the journal of exp. biol. 209:2249-2264. lushchak, v.i. (2011). adaptive response to oxidative stress: bacteria, fungi, plants and animals. comparative biochemistry and physiology, toxicology and pharmacology. cbp vol.153, n.2, pp 175-190. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en http://onlinelibrary.wiley.com/doi/10.1111/jai.2011.27.issue-2/issuetoc proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author giada magro, giada.magro@unimi.it abstract mastitis represents the most important disease of dairy cattle. stabilized or primary epithelial cell lines have been extensively used to study the molecular mechanisms of the immune response in the udder. both models are considered reliable, but are constituted by a single cell population, i.e. epithelial cells, and changes in cell morphology and metabolism may occur, as a result of subculture, or even genetic instability. the aim of this study was to evaluate the immune response of an ex vivo model of the mammary gland, where the three-dimensional structure is maintained, assuming that the results were more comparable to the in vivo response. ex vivo 2 mm3 sections of a heifer udder were cultured under standardized conditions and treated for 3, 6, and 18 h with either e. coli lipopolysaccharide (lps) or with s. aureus lipoteichoic acid (lta). these molecules are constituent of the cell walls of gram-negative or -positive bacteria, respectively and are applied as an inflammatory stimulus in the research of mastitis pathogenesis. samples were analyzed for cell proliferation and apoptosis by immunofluorescence, in order to validate the model. thereafter, we analyzed the mrna expression of key molecules of the innate immune response (tnfα, il-1β, il-6, il-8 and lap) by quantitative real-time pcr. we used three reference genes validated by genorm application, as the better internal standard for mammary gland tissue. the model did not show proliferation and apoptosis increased during the culture period. the preliminary results showed diverse patterns for the different cytokines and lap: mrna expression was overall increased, but peaked at different time points. these data suggest that such ex vivo model could be a valid approach to study the mammary immune response to bacteria. references bonnet, m., l. bernard, s. bes and c. leroux. 2013. animal 7(8):1344–1353; günther, j., s.liu, k. esch, h. j. schuberth and h. m. seyfert. 2010.veterinary immunology and immunopathology 135: 152–157; griesbeck-zilch, b., h. h. d. meyer, ch. kuhn, m. schwerin, and o. wellnitz. 2008. journal of dairy science 91:2215–2224. an ex vivo model of the bovine mammary gland to study the pathogenesis of bacterial infections. magro g. 1 , brevini t.a.l. 2 , pasello l. 2 , piccinini r. 1 1 department of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of health, animal science and foodsafety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l mycobacteria in cats are responsible for cutaneous or visceral disease, often with systemic involvement (gunn-moore et al., 2011). infection can be sustained by tuberculous (e.g. m.bovis) and non-tuberculous mycobacteria (e.g. m. avium and m. lepraemurium) (lee et al., 2017). the aim of this study is to evaluate the distribution of the lesions in feline visceral mycobacteriosis. twenty-nine necropsy cases of feline visceral mycobacteriosis, conferred from 1965 to 2017, were studied. on histopathology hematoxylin-eosin and acid-fast stains were performed. mycobacteria strains were identified by microbiological and molecular methods. twenty-three out of 29 cases were submitted for necropsy in autumn and winter. when breed was known: nine cats were persian, 8 siamese and 10 crossbreed. in 17 out of 24 cases where age was known, it was lesser than 5 years. two cases had lesions confined to the digestive system, 3 to the respiratory system, while 24 cases were systemic forms. thirteen cases out of 29 were identified: 2 as mycobacterium spp, 7 as m. bovis and 4 as m. avium. siamese, persian breeds and young cats were overrepresented as already described (gunnmoore, 2014). the occurrence of mycobacterioses in our cases was higher during cold seasons, contrary to the man in which tuberculosis is a long term localized disease, more often diagnosed in summer even if acquired in winter; in cat the disease tends to generalize making shorter the course of the disease (fares, 2011). this is confirmed by lesions’ distribution that indicates a greater occurrence of systemic compared to localized forms. features of gastrointestinal lesions indicate the alimentary as the primary route of infection (figure 1). since the most recent cases were sustained exclusively by m. avium, while older cases were caused by m. bovis, a switching in the most diffused species or a different source of infection might occurred in the last years (pesciaroli et al., 2014). keywords cat, mycobacteriosis, visceral, tuberculosis, pathology. corresponding author claudio pigoli claudio.pigoli@unimi.it journal home page riviste.unimi.it/index.php/haf feline visceral mycobacteriosis. c. pigoli1,*, g. sironi1, m. caniatti1 1 department of veterinary medicine, università degli studi di milano, via dell’università 6, 26900 lodi, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 46 haf © 2013 vol. v, no. 1s issn: 2283-3927 references fares a., 2011. seasonality of tuberculosis. journal of global infectious diseases. 3(1), 46-55. gunn-moore d.a., mcfarland s.e., brewer j.i., crawshaw t.r., clifton-hadley r.s., kovalik m., shaw d.j., 2011. mycobacterial disease in cats in great britain: i. culture results, geographical distribution and clinical presentation of 339 cases. journal of feline medicine and surgery. 13(12), 934 944. gunn-moore d.a., 2014. feline mycobacterial infections. the veterinary journal. 201(2), 230-238. lee sh., go d.m., woo s.h., park h.t., kim e., yoo h.s., kim d.y., 2017. systemic mycobacterium kansasii infection in a domestic shorthair cat. journal of comparative pathology. 157(2-3), 215-219. pesciaroli m., alvarez j., boniotti m.b., cagiola m., di marco v., marianelli c., pacciarini m., pasquali p., 2014. tuberculosis in domestic animal species. research in veterinary science. 97, s78–s85. figure 1: cut section of severely enlarged mesenteric lymph nodes characterized by a white lardaceous parenchyma with no evidence of necrosis. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords craniometry, brachycephalic, mesaticephalic, dolichocephalic, puppies, dog corresponding author maria elena andreis mariaelena.andreis@unimi.it journal home page riviste.unimi.it/index.php/haf dog craniometry: a cadaveric study. m.e. andreisa*, m. di giancamillob, m. faustinib, m.c. veronesib, s.c. modinaa a* department of health, animal science and food safety università degli studi di milano, via celoria, 10 20133 milano, italy b department of veterinary medicine università degli studi di milano, via celoria, 10 20133 milano, italy abstract the morphology of canine head differs greatly among breeds, so that they have been categorized in 3 groups (brachycephalic, mesaticephalic and dolichocephalic) based on craniometric measurements. however, several dog breeds are still unclassified, and skull measurements, often analyzed in adult dogs, are rarely studied in dog puppies. the aim of this work is to clarify whether dog puppies can be classified as dolichocephalic, mesaticephalic and brachycephalic. the skulls of spontaneously dead dog puppies aged 0 to 57 days were studied by using the following anatomic and radiographic measurements and indices: skull length, cranial length, facial length, cranial width, skull width, cranial index (ci), skull index (si); radiographic condylobasal length, s-index and facial index were added. a new index, the modified-skull index, was created. pearson test, anova and neural nets were used in the statistical analysis. 173 dogs from 36 breeds were included in the study. anatomic and radiographic ci and si were significantly correlated (p<0,05). almost all the anatomic and radiographic measurement significantly differed between brachycephalic and mesaticephalic breeds (p<0,05), while dolichocephalic breeds showed intermediate features. the new modified skull index was significantly different among the three classes (p<0,05). the neural nets allowed to classify three previously unclassified breeds. with this work it was proved that many breeds can be classified as brachycephalic, mesaticephalic or dolichocephalic as early as up to 2 months after birth, and some previously unclassified breeds were also classified. a new useful craniometric index was introduced. finally, cadavers proved to be a very good model for dog craniometric studies. references alpak h., mutus r., onar v., 2004. correlation analysis of the skull and long bone measurement of the dog. annals of anatomy 186, 323-330. evans h. e., de lahunta a., 2013. miller’s anatomy of the dog. elsevier, 4° ed. koch d. a., wiestner t., balli a. montavon p. m., michel e., scharf g., arnold s., 2012. proposal for a new radiological index to determine skull conformation in the dog, schweizer archiv für tierheilkunde, 154(5):217-220. onar v., 1999. a morphometric study on the skull of the german shepherd dog (alsatian), anatomia, histologia, embryologia 28, 253-256. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords lymphoma, cd45, protein expression, transcription, dna, mrna corresponding author marzia cozzi marzia.cozzi@unimi.it journal home page riviste.unimi.it/index.php/haf loss of cd45 protein in canine small clear cell/t-zone lymphoma is due to absence of gene transcription. m. cozzia, v. martinia, l. aresub, m. mortarinoa, s. comazzia adepartment of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy bdepartment of comparative biomedicine and food science, university of padua, via dell'università 16, 35020 legnaro, padua, italy abstract canine small clear cell/t-zone lymphoma (tzl) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant cd45-negative phenotype, easily recognized by flow cytometry (fc). recent studies have described clinical presentation and behavior, but to date the mechanisms for cd45-negativity have never been investigated. aim of this study is to confirm the lack of surface protein using a different technique from fc and to investigate if cd45-absence in tzl is linked to the lack of the corresponding transcript and gene. 40 tzl cases and 17 controls (7 t-high grade lymphoma, 10 reactive lymphnodes) were included in the present study. immunohistochemistry was performed with a different antibody respect with fc to confirm cd45 surface protein absence. total rna and genomic dna were extracted from lymph-nodes aspirates. cd45 transcript amount was investigated by quantitative real-time rt-pcr and the corresponding gene fragment was analyzed by quantitative real-time pcr. δδct method was used for the relative quantification of transcript amount and dna load compared to housekeeping genes. all tzl cases were negative for cd45 at immunohistochemistry. cd45 transcript amount was significantly lower in tzl compared to controls (p=0.000). this difference was not significant (p=0.584) for cd45 dna load, that was similar between tzl and controls. these results highlight that cd45 protein is lacking on cell surface and gene transcription is absent in tzl, whereas the corresponding gene is not deleted. the data here reported support further studies for clarifying possible genomic or epigenomic factors involving cd45 gene transcription and for better clarifying the possible role of cd45 in lymphomagenesis. references martini v., marconato l., poggi a., riondato f., aresu l., cozzi m., comazzi s., 2015. canine small clear cell/t-zone lymphoma: clinical presentation and outcome in a retrospective case series. vet comp oncol. doi: 10.1111/vco.12155 [epub ahead of print]. martini v., poggi a., riondato f., gelain m.e., aresu l., comazzi s., 2013. flow cytometric detection of phenotypic aberrancies in canine small clear cell lymphoma. vet comp oncol. 13(3), 281-287. seelig d.m., avery p., webb t., yoshimoto j., bromberek j., ehrhart e.j., avery a.c., 2014. canine t-zone lymphoma: unique immunophenotypic features, outcome and population characteristics. j vet intern med. 28(3), 878-886. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:marzia.cozzi@unimi.it proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l although wild game meat constitutes a sustainable and healthy alternative to conventional meat and hunting contributes to the control of game populations (gaviglio et al., 2017; ramanzin et al., 2010; hoffman & wilkund, 2006), international studies on consumer attitudes towards this type of meat are still limited and no previous research has been focused on the italian population. for the development of successful marketing strategies and/or public policy intervention, the knowledge of consumers’ purchase behavior is a key factor. among all the determinants that can influence the behavior of consumers of hunted wild game meat (i.e. animal welfare, sustainability, ecological food choice, product safety, nutritional quality), the consumers’ awareness of hunting activity and their perceptions of wild game meat assume a crucial role (ljung et al., 2012; byrd et al., 2017). accordingly, in this paper an online survey on a sample of 741 italian meat consumers has been conducted to investigate the relationship between consumers’ purchase behavior and their awareness of hunted game meat and hunting practices (chi-square test, f-test). statistically significant differences were found among segments of consumers with different levels of wild game meat consumption frequency. the analysis shows that, as expected, the highest consumption level of wild game meat relates to the highest level of general awareness of wild game meat and hunting practices (figure 1). our findings are in line with previous literature, that links positive behaviors of consumers towards wild game meat and hunting to familiarity and experience with hunting and hunters keywords wild game meat, hunting, marketing strategies, consumer, survey. corresponding author maria elena marescotti maria.marescotti@unimi.it journal home page riviste.unimi.it/index.php/haf effect of hunting awareness on wild game meat purchase behavior. m.e. marescotti1,*, v. caputo2, e. demartini1, a. gaviglio1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of agricultural, food, and resources economics, michigan state university, 446 w. circle d., east lansing, michigan. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 88 haf © 2013 vol. v, no. 1s issn: 2283-3927 (ljung et al., 2012; byrd et al., 2017). nonetheless, the present study provides a deeper understanding of the italian consumers’ attitudes and perceptions of wild game meat and could suggests policy guidelines for the development of future targeted marketing strategies. acknowledgments: research funded by fondazione cariplo “bandi ambiente 2014” – progetto “la filiera eco-alimentare. progetto per la valorizzazione delle carni di selvaggina: la gestione di prodotto sostenibile come strumento di stimolo al miglioramento ambientale dei territori alpini. references byrd, e., lee, j.g., olynk widmar, n.j., 2017. perceptions of hunting and hunters by u.s. respondents. animals. 7(11): 83. gaviglio, a., demartini, e., marescotti, m.e., 2017. opportunities and limitation from an italian alpine case study. calitatea – access la succes. 18(2): 215-222. hoffman, l.c., wiklund, e., 2006. game and venison: meat for the modern consumer. meat science. 74, 197-208. ljung, p.e., riley, s.j., heberlein, t. a., 2012. eat prey and love: game-meat consumption and attitudes toward hunting. wildlife society bulletin. 36(4): 669-675. ramanzin, m., amici, a., casoli, c., esposito, l., lupi, p., marsico, g., mattiello, s., olivieri, o., ponzetta, m.p., russo, c., trabalza marinucci, m., 2010. meat from wild ungulates: ensuring quality and hygiene of an increasing resource. italian journal of animal science. 9(61): 318–331. figure 1: schematic representation of the framework of the analysis. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords leukotriene a4 hydrolase, immunohistochemistry, rt-pcr, dog, melanoma, oral cavity corresponding author laura nordio laura.nordio@unimi.it journal home page riviste.unimi.it/index.php/haf lta4h expression in canine oral melanomas: methodological set up and preliminary results. l. nordioa*, f. genovaa, v. serraa, c. giudicea a department of veterinary medicine, university of milan, via celoria 10, milan, italy abstract leukotriene a4 hydrolase (lta4h) is a hydrolytic enzyme which converts leukotriene a4 into leukotriene b4 inside the arachidonic acid cascade. besides playing a well-known role in inflammation, it has also been investigated for possible implications in different types of tumors, including canine uveal melanoma (chen et al., 2004; malho et al., 2013). in the present study, we set up rt-pcr and immunohistochemical protocols to investigate the expression of lta4h gene and protein, respectively, in formalin fixed paraffin embedded (ffpe) specimens of canine melanomas. 16 samples of canine oral melanomas were histopathologically evaluated and divided in two subgroups based on morphological criteria of malignancy. rt-pcr protocols for the target gene lta4h were set up on frozen and ffpe samples of the same tumor. immunohistochemical investigation of lta4h protein was performed with a mouse monoclonal antibody anti-lta4h (clone 1e9). preliminary tests were carried out to define the protocol: with and without bleaching, with different unmaskings, with different serial dilutions of the primary antibody. rt-pcr set up resulted in comparable good efficiency on both frozen and ffpe samples. final immunohistochemical protocol included: hydrogen peroxide bleaching, water bath unmasking and 1:100 dilution of the primary antibody. positive immunostaining for lta4h was present in neoplastic cells of all melanoma samples, with variable localization and intensity. these preliminary results encourage future applications of the studied techniques to quantify differential expression of lta4h in canine melanomas both at molecular and histological level. laboratory results will be compared with follow up data, in order to verify if lta4h can be proposed as a valuable prognostic marker. references chen, x., wang, s., wu, n., yang, c.s., 2004. leukotriene a4 hydrolase as a target for cancer prevention and therapy. current cancer drug targets 4, 267-283. malho, p., dunn, k., donaldson, d., dubielzig, r.r., birand, z., starkey, m., 2013. investigation of prognostic indicators for human uveal melanoma as biomarkers of canine uveal melanoma metastasis. j. small anim. pract. 54, 584-593. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in many countries of europe rabbit meat is consumed for its nutritional characteristics (dalle zotte, 2014; hernández and gondret, 2006). since the ban of the use of antibiotic as growth promoter, natural substances have been studied as alternative with antioxidant, antiinflammatory (korkina, 2007), antimicrobic and antiviral properties (hashemi and davoodi, 2011). the aim was to evaluate the effect of a dietary supplementation with natural extract mixture in growing rabbit on growth performances, carcass characteristics and longissimus lumborum (ll) muscle parameters. the trial was performed at the research institute for animal production (nitra, slovak republic) and lasted 42 days. at 35 days of age, 144 new zealand white rabbits were randomly selected and divided in 3 experimental groups (4 rabbits/cage). the first fed a basal diet (c), the second (t1) and the third one (t2) received 0.3% and 0.6% of natural extract mixture, containing polyphenols from plants and seaweeds. dietary integration with natural extract improve (p<0.05) growth performances: live weight (lw), average daily gain (adg), feed intake (fi) and feed conversion (fc) in t1 group (table 1). the fatty acid (fa) composition of ll muscle was positively affected (p=0.037) by natural extract supplementation with an increase of n-3 fa in t2 group than t1 and c. cholesterol content did not differ between dietary treatments (37.38 mg/100g c vs 26.72 mg/100g t1 vs 35.19 mg/100g t2). sensory analysis revealed that only the aroma was affected (p<0.05) by dietary treatments. overall, these results highlight that dietary supplementation with natural extract mixture, containing polyphenols from plants and seaweeds enhance growth performances, carcass weight, improving ll muscle nutritional parameter. keywords dietary integration, natural extracts, growing rabbit, growth performances, meat quality. corresponding author sara chiapparini sara.chiapparini@unimi.it journal home page riviste.unimi.it/index.php/haf dietary integration with natural extract in rabbit: effects on growth performances and meat quality. s. chiapparini1,*, f. vizzarri2, m. palazzo2, r. rossi1, d. casamassima2, c. corino1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 department of agricultural, environmental and food sciences, università degli studi del molise, via francesco de sanctis, 1, 86100 campobasso, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 42 haf © 2013 vol. v, no. 1s issn: 2283-3927 a, b, means with different letters are different (p<0.05), a, b means with different letters are different (p<0.01). references dalle zotte, a., 2014. rabbit farming for meat purposes. animal frontiers. 4, 62–67. hernández, p., and gondret, f., 2006. rabbit meat quality, in: recent advances in rabbit sciences, edited by: maertens, l. and coudert, p., ilvo, merelbeke, belgium. 269–290. hashemi, s.r., and davoodi h., 2011. herbal plants and their derivatives as growth and health promoters in animal nutrition. vet. res. commun. 35, 169–180. korkina, l.g., 2007. phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. cell. mol. biol. 53, 15–25. table 1: productive performances on growing rabbit fed basal diet (c) and diet supplemented with two levels of natural extract (t1, t2). c t1 t2 sem p-value lw, g initial 21d 42d 830.2 1860.9a 2655.9 846.0 1996.3b 2834.8 789.4 1825.3a 2725.2 21.02 44.15 52.40 0.161 0.024 0.066 adg, g/d 0-21d 21-42d 0-42d 49.1 37.9 43.5 54.8 39.9 47.4 49.3 42.9 46.1 1.848 2.192 1.156 0.062 0.284 0.067 fi, g/d 0-21d 21-42d 0-42d 154.9a 188.8 171.8 142.0ab 175.6 158.8 136.8b 192.6 164.7 5,07 8.99 6.54 0.046 0.382 0.379 fc, kg/kg 0-21d 21-42d 0-42d 3.20 5.03a 3.94aa 2.59 4.41b 3.35b 2.89 4.58ab 3.60b 0.17 0.18 0.11 0.057 0.049 0.003 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 1 haf © 2013 vol. ii, no. 2s issn: 2283-3927 figure 1 – chemical structures of the considered molecules. l introduction the council directive 96/23/ce allows the therapeutic use of the glucocorticoids prednisolone, methylprednisolone, betamethasone and dexamethasone, and the commission regulation no 37/2010/eu sets the maximum residue limits (mrls) in bovine liver, muscle, fat and milk. however, their possible use as growth promoters is well known, due to e.g. their ability to increase weight gain or their synergistic activity with β-agonists. no mrls are for urine so their presence at any concentration should not be allowed. in the last noughties, the frequency of detection of prednisolone in cow urine, especially in samples collected at the slaughterhouse, raised in italy. the possibility of an endogenous production of prednisolone after stress events as the slaughtering was demonstrated (pompa et al. 2011) on cows and its presence in bovine urine was shown to be related to a state of stress in the animals both at farm and slaughterhouse (dusi et al. 2012; bertocchi et al. 2013). the hypothesis that a contamination could lead to the presence of prednisolone in urine because of a likely microbiological dehydrogenase activity on cortisol was also shown in bovine (arioli et al. 2010) and human (bredehöft et al. 2012). in this study, saponins and sapogenins with steroidal nucleus were analogously considered as possible cause of the neo-formation of prednisolone in the faeces-contaminated urine and in feed. attention has been focused on α-solanine, a saponin contained in the solanaceae, and diosgenin, an important starting material to obtain steroidal drugs such as cortisol, testosterone, estradiol, contained in many herbaceous plants as dioscoraceae (figure 1). material and methods sample preparation 500 mg of faeces or feed from the market were suspended in 500 ml of 0.9% nacl, and gently mixed for a whole night. three aliquots of 40 ml were collected from each suspension. an aliquot was spiked (100 ng/ml) with diosgenin, one with α-solanine and one was the control. at the times: t = 0; 1; 2; 4; 8; 24; 48 and 72h, 1 ml was collected, heated at 80 °c (15 min), fortified with the internal standard (prednisolone d6, 10 ng/ml) and centrifuged at 3000xg (10 min). a 200 µl supernatant was collected and analysed. keywords saponins; sapogenins; prednisolone neoformation; faeces; feed corresponding author francesco arioli, francesco arioli@unimi.it journal home page riviste.unimi.it/index.php/haf are saponins and sapogenins precursors of prednisolone? preliminary results g.f. labella 1 , l.m. chiesa 2 , s. panseri 2 , f. arioli 1 1 department of health, animal science and food safety, università degli studi di milano, italy. 2 department of veterinary science and public health, università degli studi di milano, italy. article selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 2 haf © 2013 vol. ii, no. 2s issn: 2283-3927 sample analyses hplc-msn was made with a lcq deca xp ion trap mass spectrometer equipped with a pump and an autosampler lc surveyor (thermofinnigan, san jose, ca, usa). the column was a 100mm×2.1mm id, 3µm allure biphenyl (restek, bellefonte, pa, usa). the mobile phase consisted of an aqueous solution of formic acid 0.1% (a) and methanol (b). a gradient was used: 40% eluent a and 60% eluent b from 0 to 15 min and 100% eluent b from 20 to 40 min. the acquisition was in the electrospray ionization (esi) in the (+) mode for α-solanine and diosgenin, in the (-) mode for prednisolone. the data acquisition software was xcalibur® by thermofinnigan. the limit of detection (lod) were from 0.6 to 4 ng/ml, the limit of quantification (loq) were from 1 to 10 ng/ml. results diosgenin and α-solanine underwent a transformation in faeces. their concentrations decreased over time. from t = 48h diosgenin was not detected while α-solanine was detected at a concentration lower than 30% of the initial one. after 72h also α-solanine was not detectable. the faecal suspension spiked with diosgenin, at t = 8h showed the presence of prednisolone at the concentration of 7.1 ng/ml (figure 2). in the sample of faeces spiked with α-solanine, cortisol was detected at t = 24h with a concentration of 6.6 ng/ml (figure 3). in the feed solanine did not transform while diosgenin steadily decreased to the 15% of the initial concentration. no corticosteroids were detected. in the control samples, no transformations were observed. discussion and conclusions the presence of corticosteroids in two faecal suspensions could show that the neo formation of prednisolone and cortisol from diosgenin and α-solanine, even if sporadic, may be possible. it could be conceivable that the transformation event likely occurs in the presence of certain microorganisms, and is apparently random, as faeces are highly non homogeneous both in their rough composition and in the microflora present. further studies are ongoing to investigate these preliminary conclusions. references arioli f., fidani m., casati a., fracchiolla m.l., pompa g. (2010). investigation on possible transformations of cortisol, cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography-mass spectrometry, steroids, 75, 350–354. bertocchi l., dusi g., ghidelli v., hathaway t., nassuato c., casati a., fidani m., pompa g., arioli f. (2013), investigation on the origin of prednisolone in urine and adrenal glands of cows, food addi. contam., part a, 30, 1055-1062. bredehöft m., baginski r., parr m.k., thevis m., schänzer w. (2012) investigations of the microbial transformation of cortisol to prednisolone in urine samples. j. . steroid biochem. mol. biol., 129, 54–60. dusi g., arioli f., ghidelli v., casati a., hathaway t., pompa g., bertocchi l. (2012) investigations on the origin of prednisolone in cow urine “euro residue vii – conference on residues of veterinary drugs in food” 14-16 may 2012, egmond aan zee,the netherlands. pompa g., arioli f., casati a., fidani m., bertocchi l., dusi g. (2011) investigation of the origin of prednisolone in cow urine. steroids, 76, 104–110. figure 2 prednisolone neoformation in diosgenin-spiked faecal suspension. figure 3 cortisol neoformation in α-solanine-spiked faecal suspension. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l invited paper the thrifty phenotype hypothesis states that reduced foetal growth is strongly associated with the occurrence of a number of chronic non-communicable diseases such as coronary heart disease, stroke, diabetes, and hypertension in later life and during adulthood. this increased susceptibility results from adaptations made by the foetus in an environment limited in its supply of nutrients. since its conception, the thrifty phenotype hypothesis has always been tested exclusively in one of the genders, i.e. females. the evidence provided to prove the validity of this hypothesis has always been limited in experiments following mothers. furthermore, the majority of anecdotal evidence as well as epidemiological studies conducted have hardly been able to differentiate between the effect of factors such as nutrition, stress, climate changes, etc. on the environment in the womb during the periconception period and its effect on the embryo or the final maturational stages of female gamete either in ovary or in the womb. in this presentation, we discuss the tremendous potential provided by studying pregnancy in seahorses to test the effect of factors such as good and low-quality nutrition during the periconception period in pregnant males and its consequences on the health and quality of offspring produced. keywords foetal growth, pregnancy, nutrition, periconception, health. corresponding author alireza fazeli a.fazeli@sheffield.ac.uk journal home page riviste.unimi.it/index.php/haf when dads become pregnant, would they do a better job? a. fazeli 1,2, *, f. otero 3 , f. lättekivi 2 , s.koks 2 , m.izquierdo 3 , w. v. holt 1 1 academic unit of reproductive and developmental medicine, sheffield university, sheffield, united kingdom 2 department of pathophysiology, tartu university, tartu, estonia 3 grupo en biodiversidad y conservación, iu-ecoaqua, universidad de las palmas de gran canaria, crta. taliarte s/n, 35214 telde, spain proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author alessandro zenobi, alessandro.zenobi@unimi.it abstract epigenetic cell conversion overcomes the stability of a mature cell phenotype transforming a somatic cell in an unlimited source of autologous cells of a different type. it is based on the exposure to a demethylating agent followed by an induction protocol. in our work we exposed mouse dermal fibroblasts to the demethylating agent 5-azacytidine. cell differentiation was directed toward the endocrine pancreatic lineage with a sequential combination of activin a, retinoic acid, b27 supplement, its and bfgf. the overall duration of the process was 10 days. aim of this work was to evaluate the role of oxygen during differentiation of dermal fibroblasts derived from two different mouse strains, nod and c57 bl/6j. during differentiation, both cell lines were cultured either in the standard in vitro culture 20% oxygen concentration or in the lower and more physiological 5% of oxygen. our results show that c57 bl/6j cells are able to differentiate into insulin secreting cells in both oxygen tensions with a higher amount of insulin release in low oxygen conditions. on the other hand, cells of nod mice, which are physiologically predisposed to the onset of diabetes, differentiate in 20% of oxygen but not in low oxygen and they died after three days of culture. however, if these cells are moved to 5% of oxygen after their differentiation in high oxygen they remain viable for up to four days. furthermore, their capacity to release insulin remains unchanged for 24 hours. results suggest that genetic background has a profound effect on the role of oxygen during the in vitro differentiation process, possibly reflecting the different susceptibility to the disease of the strains used in the experiment. supported by efsd and carraresi foundation references pennarossa g., maffei s., campagnol m., tarantini l., gandolfi f., and brevini t.a.l. 2013. pnas 110(22):8948–8953; shi y., hou l., tang f. , jiang w., wang p., ding m, and deng h. 2005. stem cells 23(5):656–662. role of oxygen tension and genetic background during the epigenetic conversion of mouse fibroblasts into insulin secreting cells. zenobi a. 1 , gandolfi f. 1 , brevini t.a.l 1 . 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords chemonucleolysis, lumbosacral disk, fluoroscopy, cauda equine syndrome corresponding author maurizio longo maurizio.longo@unimi.it journal home page riviste.unimi.it/index.php/haf therapeutic chemonucleolysis of the lumbosacral disk in a cadaveric model: lateral vs sternal technique. m. longo*a, d. d. zania, d. de zania, p. riccabonia, m. di giancamilloa adipartimento di medicina veterinaria (dimevet), università degli studi di milano, az.polo veterinario di lodi, via dell’università n.6 lodi, italia abstract the widespread use of advanced imaging techniques has dramatically improved the diagnosis of cauda equina syndrome in geriatric small animal patients. two different techniques (sternal vs lateral recumbency) were tested in twelve cadavers for the l7-s1 disk injection under fluoroscopic guidance. twelve dogs were enrolled in the study: eight intervertebral lumbosacral disks were injected with a gelatinous radiopaque compound and the remnants four disks with an alcoholic radiopaque solution. the dogs were randomly positioned either in lateral recumbency with the lumbo-sacral joint in neutral position (six dogs), or in sternal recumbency with the hind limbs extended cranially along the body and the lumbosacral joint flexed (six dogs). the correct injection of the compounds within the lumbosacral disk was checked both by computed tomography (ct) and gross anatomic examination. the lateral technique required less time of execution and minor attempts for the correct positioning of the needle. all the injected disks were visible on ct, while necroscopy resulted satisfactory only in five patients. leakage of the compounds outside the disk was observed in two cases. the percutaneous injection in lateral recumbency under fluoroscopic guidance resulted rapid and feasible in a cadaveric model. future clinical trials are required to assess the safety of chemonucleolysis in diseased patients. references mackenzie sd, caswell jl, brisson ba, gaitero l, chalmers hj. comparison between computed tomography, fluoroscopy, and ultrasonography for guiding percutaneous injection of the canine intervertebral disc. vet radiol ultrasound. 2014 sep-oct; 55(5); 571-81. meij bp, bergknut n. degenerative lumbosacral stenosis in dogs. vet clin north am small anim pract. 2010 sep; 40(5): 9831009. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords dog, western blotting, carbonyl group proteins, oxidative stress, oxidants. corresponding author beatrice ruggerone beatrice.ruggerone@unimi.it journal home page riviste.unimi.it/index.php/haf analytical aspects of protein carbonyl group in oxidized canine serum. b. ruggeronea*, g. colombob, s. paltrinieria a department of veterinary medicine, university of milan, via celoria, 10 -20133 milan, italy b department of biosciences, university of milan, via celoria 26, 20133 milan, italy abstract oxidative stress (os) is an imbalance between oxidants and anti-oxidants and plays an important role in the aetiology and/or the progression of several diseases. protein carbonyl (pco) groups are so far used as biomarkers of os in humans (colombo et al, 2015). the aim of our study was to investigate whether pcos are present in canine serum and if they can be measured spectrophotometrically using a method not yet validated in dogs. the presence of pco was investigated by western blotting after separation by sds-page of serum at different dilutions (either before or after oxidation with 10% cigarette smoke extract). protein labelling with 2,4-dinitrophenylhydrazine (dnph, brady's reagent) was followed by a two-steps incubation with primary anti-dinitrophenyl-klh antibodies (rabbit igg fraction) and secondary goat anti-rabbit igg, hrp conjugate. signal was developed with enhanced chemiolumionescence (ecl). serum pco were quantified using a commercially available assay (protein carbonyl content assay kit abcam, uk), based on derivatization of proteins with 1,4-dinitrophenylhydrazine (dnph) and subsequent formation of protein-conjugated dinitrophenylhydrazones (dnps) with a peak absorbance at 366 nm. western blot showed an evident band of apparent mw of 69 kda, consistent with carbonylated dog serum albumin. the spectrophotometric assay failed to demonstrate any signal at 366 nm using the manufacturer’s instructions or modified protocols. this study demonstrated that pco are present in oxidized canine serum. however, the spectrophotometric assay employed in this study is not enough sensitive to detect pcos. further studies are needed to assess whether this depends on the poor re-solubilisation of dnps, or on the low concentration of pcos in dogs. references colombo, g., clerici, m., garavaglia, m.e., giustarini, d., rossi, r., milzani, a., dalle-donne, i., 2015. a step-by-step protocol for assaying protein carbonylation in biological samples. j chromatogr b. 103, 325-330. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords chromatin configuration, developmental competence, cocs morphology, pre-ivm treatment, transcriptomic analysis corresponding author cecilia dieci cecilia.dieci@unimi.it journal home page riviste.unimi.it/index.php/haf morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols cecilia dieci1, valentina lodde1, rémi labrecque2, irene tessaro1, marc-andré sirard2 and alberto m. luciano1 1university of milan, department of health, animal science and food safety, italy 2 universite laval, department des sciences animales, quebec, canada abstract oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation (luciano et al., 2014). moreover, several studies demonstrate that in vitro pre-maturation treatments (pre-ivm), aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle (luciano et al., 2011). this study aims at identifying correlations between cumulus-oocyte complex (coc) morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin (gv1) can benefit from pre-ivm treatment. cocs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (class-1, 2 and 3) as previously described (blondin & sirard, 1995). analysis of chromatin configuration revealed that only class-1 coc was enriched in gv1 oocyte, while class-2 and 3 presented a similar distribution of gv1, gv2 and gv3 oocytes, where gv2 and 3 oocytes are characterized by increased chromatin compaction (lodde et al., 2007). then cocs were divided into two groups, one containing class-1 cocs and the other containing class2 and 3 cocs and subjected to pre-ivm for 6 hours in presence of cilostamide and 10-4 ui/ml rhfsh. finally, cocs underwent standard in vitro maturation (ivm) for 22 hours, in vitro fertilization and embryo culture. blastocyst rate and embryos cell number were assessed at day 7. pre-ivm positively affected developmental competences of class-1, while in classes 2 and 3 pre-ivm had detrimental effects. in conclusion cocs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. these data could be useful in setting-up dedicated ivm protocols considering specific genes and pathways to improve ivp efficiency. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references blondin p., sirard m.a., 1995. oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. mol reprod dev. 41, 54-62. lodde v., modina s., galbusera c., franciosi f., luciano a.m., 2007. large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence. mol reprod dev. 74, 740-749. luciano a.m., franciosi f., modina s.c., lodde v., 2011. gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through camp-dependent mechanism(s). biology of reproduction. 85, 1252-1259. luciano a.m., franciosi f., dieci c., lodde v., 2014. changes in large-scale chromatin structure and function during oogenesis: a journey in company with follicular cells. anim reprod sci. 149, 3-10. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords natural extracts, livestock performance, lactating sow corresponding author federica maghin federica.maghin@unimi.it journal home page riviste.unimi.it/index.php/haf dietary supplementation with algae and polyphenols in lactating sows: effects on sows and piglets’ performance. f. maghina*, r. rossia, s. rattia, s. chiapparinia, c. corinoa adepartment of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. abstract the decrease of the use of antibiotic has promoted the scientific research towards the identification and study of natural substances able to improve animal performance and welfare (hashemi et al., 2011; daglia, 2012; maghin et al., 2014). the aim of this study was to investigate the effect of mixture of algae plus polyphenols (algatan mater®) on piglet and sow performance when used as dietary supplements to the sows. eighty-four sows (42 per batch) were divided in two groups fed a control and algatan mater® supplemented diet. feed intake, backfat thickness and blood samples at entry in farrowing room and at 21 days of lactation, and sows’ reproductive data until the next service were collected. haematochemical parameters and antioxidant status of sows were analysed. weight at birth, after cross-fostering and at 21 days of lactation were recorded to each litter. backfat loss tended to be lower (p=0.07) in sows treated than control. the administration of algae plus polyphenols in sows improved (p<0.05) the piglets average daily gain and body weight at 21 days of lactation (p=0.014, figure 1a.). haematochemical parameters and antioxidant activity of blood was not affected by dietary treatment. the total number of piglets born at the next farrowing from treated group was higher (p=0.04) than controls (figure 1b). the inclusion in lactating sows of algatan mater® improves lactating sows and piglets’ performance. further research is needed to explore the mechanism of action of this natural mixture. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. ii, no. 2s issn: 2283-3927 figure 1. (a) average daily gain of the piglets at 21 days of lactation and (b) total number of piglets born from control and algatan mater® group. references hashemi, s.r., davoodi, h., 2011. herbal plants and their derivatives as growth and health promoters in animal nutrition. vet res commun 35, 169-180. daglia, m., 2012. polyphenols as antimicrobial agents. curr opin biotechnol 23(2), 174-181. maghin, f., ratti, s., corino, c., 2014. biological functions and health promot-ing effects of brown seaweeds in swine nutrition. j dairy vet anim res 1(1), 00005. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords ultrasound, epidural catheter, dog, cadaver corresponding author elisa silvia d’urso elisasilvia.durso@unimi.it journal home page riviste.unimi.it/index.php/haf abstract several methods are described in veterinary medicine to perform and assess correct epidural needle placement to provide effective epidural analgesia (adami et al. 2017). the aim of this study is to evaluate the feasibility of an ultrasound longitudinal sagittal approach to epidural catheter placement using a biopsy needle guide. seven dog cadavers were used in the study. with the cadaver in sternal recumbency, a 5-8 mhz microconvex transducer provided with a 16-gauge biopsy guide was positioned to obtain a longitudinal sagittal scan of the spinal process of l7 and the sacral crest; the epidural space was identified between two parallel hyperechoic lines and, as the trajectory of the biopsy guide crossed them, a 17g tuohy needle was used to insert a 19g epidural catheter. correct catheter placement was visualised through a resection of the column between l2 and l3. firstly, an expert echographist (operator c1) visualised the ultrasonographic landmarks, while catheter placement was performed by an expert anaesthetist (operator a), a student (operator b) and another expert echographist (operator c2) (double-operator technique); secondly, operator a and c2 performed alone the whole procedure (single-operator technique); lastly all operators performed a blind procedure (jones, 2001). operator a failed 2/7 single-operator procedures; time to perform the blind technique was statistically lower than the double-operator technique (75 ± 132.4 vs 91.6 ± 79.3 seconds). operator c2 failed 3/7 blind procedures, scoring the higher total time of performance (329.3 ± 271.2 seconds), but was able to perform both the doubleand single-operator technique without significant difference with operator a, despite a faster time in positioning the probe. operator b showed a higher repositioning attempts of the needle with the double-operator procedure compared to the blind one. ultrasound guidance appears to be a promising technique to ease catheter placement also by operators inexperienced of locoregional techniques. references adami, c., gendron, k., 2017. what is the evidence? the issue of verifying correct needle position during epidural anaesthesia in dogs. veterinary anaesthesia and analgesia. http://dx.doi.org/10.1016/ j.vaa.2016.03.003 jones, r.s., 2001. epidural analgesia in dogs and cats. the veterinary journal. 161, 123–131 ultrasound-guided epidural catheter placement with a new technique: preliminary cadaveric study. elisa s. d’urso1*, stefano faverzani1, gabriele barella1, federica di cesare2, daniela gioeni1, vanessa rabbogliatti1, damiano stefanello1, giuliano ravasio1 1 university of milan, department of veterinary medicine, italy 2 university of milan, department of health, animal science and food safety, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l sturgeons, as well as paddlefishes, belong to the acipenseriformes group, which is one of the most primordial 57 orders of the osteichthyes that comprehends 25 species spread throughout europe, asia and north america (birnstein, 1993). the present study aims at investigating muscle growth and development as well as fatty acid profile in siberian sturgeon free-embryos when subjected to three different rearing densities. fatty acids, in particular polyunsaturated fatty acids of n-3 series, are generally known as key nutrients in fish larvae. this study was approved by the ethic committee of the university of milan (opba_22_2017). siberian sturgeon larvae were reared at 18°c, at three stocking densities until complete yolk-sac absorption: low (ld, 30 larvae/l), mid (md, 80 larvae/l) and high (hd, 150 larvae/l). sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage (ysa). sacrificed larvae were weighed and histological analyses were performed in order to assess muscle development as described elsewhere (di giancamillo et al. 2015; aidos, et al., 2017); fatty acid profile was determined by gc-fid analysis as described by vasconi et al. (2015). statistical analysis was performed with sas software (v. 9.3, cary inc., nc). at the end of the experiment, ld larvae presented a higher weight than larvae reared at the other two densities (p<0.05). within the schooling stage (fig. 1), total muscle area was lower for hd larvae (p<0.05); red and white muscle areas in schooling and ysa were higher than at hatching (p<0.05), regardless the density. concerning fatty acids, no statistical differences were recorded between different rearing densities, while during the development regardless the rearing density, there was a common pattern: linoleic and alfa linolenic acids, significantly decreased their relative keywords siberian stugeon, larvae, growth, muscle development, stress, density. corresponding author alessia di giancamillo alessia.digiancamillo@unimi.it journal home page riviste.unimi.it/index.php/haf effect of different stocking densities on growth, muscle development and fatty acid profile of acipenser baerii larvae. l. aidos1, m. vasconi1, m.lanfranchi2, a. di giancamillo1,* 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. 2 società naviglio agricola ss, goito, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 84 haf © 2013 vol. v, no. 1s issn: 2283-3927 content, while others, as arachidonic acid and dha, significantly increased. siberian sturgeon larvae reared at ld or md reveal an anatomically normal muscle development, while in the hd it is possible to observe a slowdown. what the aquaculture industry requires is a set of guidelines that allows the development of a sustainable industry, so that we tried to develop guidelines for stocking density in the very early stage of farming. as a conclusion, it would seem that mid density could be more suitable for this species in this stage of development. acknowledgments: the authors wish to thank the sturgeon farm “società naviglio agricola” for the supply of the fertilized eggs utilized for this study. references aidos l., valente l.m.p., sousa v., lanfranchi m., domeneghini c., giancamillo a.d., 2017. effects of different rearing temperatures on muscle development and stress response in the early larval stages of acipenser baerii.european journal of histochemistry 61,(4),2850. doi:10.4081/ejh.2017.2850. birnstein, v. j.,1993. sturgeon and paddlefishes: threatened fishes in need of conservation. conservation 298 biology 7, 773–787. di giancamillo a., rossi r., pastorelli g., deponti d., carollo v., casamassima d., et al. 2015. the effects of dietary verbascoside on blood and liver oxidative stress status induced by a high n-6 polyunsaturated fatty acids diet in piglets. journal of animal science 93, 2849-59. vasconi, m., caprino, f., bellagamba, f., busetto, m. l., bernardi, c., puzzi, c. and moretti, v. m. 2015. fatty acid composition of freshwater wild fish in subalpine lakes: a comparative study. lipids, 50: 283-302. doi:10.1007/s11745-014-3978 figure 1: siberian sturgeon larva in the schooling stage http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords fxr1, western blot, immunohistochemistry, oral melanoma, dog corresponding author laura nordio laura.nordio@unimi.it journal home page riviste.unimi.it/index.php/haf validation of anti-fxr1 antibodies in the canine species and application to an immunohistochemical study of canine oral melanomas laura nordio1*, andreia f. t. marques1 1university of milan, department of veterinary medicine, italy abstract fxr1 (fragile x mental retardation-related protein 1) is a cytoplasmic rna binding protein (siomi et al., 1995), which genetic expression has been related to metastatic potential in human melanoma. the aims of the present study were to validate two commercially available clones of polyclonal anti-human fxr1 antibody in dogs and to use them to investigate fxr1 expression in a group of canine oral melanomas. anti-fxr1 antibody was never validated before in the canine species. two different commercially available polyclonal anti-fxr1 antibodies (raised in goat and rabbit, respectively) were used. fxr1 protein in canine serum was identified by western blot after sds-page, using human serum as control. fxr1 immunohistochemical expression was tested in a series of normal tissues, that are expected to express fxr1, and in 31 cases of oral melanomas. the final immunohistochemical protocol used heat-induced unmasking and overnight incubation. fxr1 protein bands in canine serum were detected by validated antibodies. the rabbit antibody specifically identified a band around 65 kda (figure 1a), whereas the goat antibody reacted also with other not specific bands. fxr1 immunohistochemical staining was positive in all tested organs, with different levels of expression. fxr1 was also expressed in 31/31 tested melanomas, with variable intensity and percentage of positive cells (figure 1b). equal results were achieved with the two antibodies in 8 cases of melanoma, whereas there were variable differences in 22, and one case stained only with goat antibody. the rabbit antibody gave less background staining. this study validated anti-fxr1 antibodies for use in the canine species. this protein was identified in several normal tissues, as well as in the tested neoplasms. significance of different level of expression is undergoing evaluation with further studies. acknowledgments: francesca genova, valentina serra, maria lina longeri, chiara bazzocchi, cristina lecchi, fabrizio ceciliani, chiara giudice. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 figure 1. a) western blot, rabbit anti-fxr1 antibody, positive bands around 65 kda. b) immunohistochemistry, oral melanoma, cytoplasmic positivity of neoplastic cells to fxr1. rabbit anti-fxr1 antibody, aec chromogen, 40x. references siomi, m.c., siomi, h., sauer, w.h., srinivasan, s., nussbaum, r.l., dreyfuss, g., 1995. fxr1, an autosomal homolog of the fragile x mental retardation gene. embo j. 14: 2401-2408.ormation. journal of microbiological methods. 40, 175–179. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords gwas, copy number variation, brown swiss corresponding author raphaelle.t.m.m. prinsen raphaelle.prinsen@unimi.it journal home page riviste.unimi.it/index.php/haf genome-wide association studies using copy number variants in brown swiss dairy cattle. r.t.m.m. prinsena*, m.g. strillaccia, f. schiavinia, a. rossonib, b. gredlerc, m.a. dolezald, a. biebere, a. bagnatoa auniversità degli studi di milano, dimevet via celoria 10, milan, italy ba.n.a.r.b., località ferlina 204, bussolengo, italy cqualitas ag, chamerstrasse 56 zug, switzerland duniversity of veterinary medicine, veterinärplatz 1 wien austria eresearch institute of organic agriculture, ackerstrasse 113, frick, switzerland abstract detecting copy number variation (cnv) in cattle provides the opportunity to study their association with quantitative traits (winchester et al., 2009; zhang et al., 2009; hou et al., 2011; clop et al., 2012; de almeida et al., 2016;). the aim of this study was to map cnvs in 1,410 brown swiss males and females using illumina bovinehd genotyping beadchip data and to perform a genome-wide association analysis for production functional and health traits. after quality control, cnvs were called with the goldenhelix svs 8.3.1 and penncnv software and were summarized to cnv regions (cnvrs) at a population level, using bedtools. additionally, common cnvrs between the two software were set as consensus. cnvassociation studies were executed with the cnvruler software using a linear regression model. genes within significant associated cnvrs for each trait were annotated with a go analysis using the david bioinformatics resources 6.7. the quality control filtered out 294 samples. the goldenhelix svs 8.3.1 software identified 25,030 cnvs summarized to 398 cnvrs while penncnv identified 62,341 cnvs summarized to 5,578 cnvrs. a total of 127 cnvrs were identified to be significantly associated with one or more of the evaluated traits. the result of this study is a comprehensive genomic analysis of the brown swiss breed, which enriches the bovine cnv map in its genome. finally, the results of the association studies deliver new information for quantitative traits considered in selection programs of the brown swiss breed. references clop a., vidal o., amills m., 2012. copy number variation in the genomes of domestic animals. animal genetics 43(5):503 -17. de almeida santana m.h., junior g.a., cesar a.s., freua m.c., da costa gomes r., da luz e silva s., leme p.r., fukumasu h., carvalho m.e., ventura r.v., coutinho l.l., kadarmideen h.n., ferraz j.b., 2016. copy number variations and genome-wide associations reveal putative genes and metabolic pathways involved with the feed conversion ratio in beef cattle. j appl. genet. 10.1007/s13353-016-0344-7. hou y., liu g.e., bickhart d.m., cardone m.f., wang k., kim es., matukumalli l.k., ventura m., song j., vanraden p.m., sonste gard t.s., van tassell c.p., 2011. genomic characteristics of cattle copy number variations. bmc genomics 12: 127. winchester l., yau c., ragoussis j., 2009. comparing cnv detection methods for snp arrays. brief funct genomic proteomic. 8(5 ):353-66. zhang f., gu w., hurles m.e., lupski j.r., 2009. copy number variation in human health, disease, and evolution. annu rev genomics hum genet. 10:451 -81. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 8 haf © 2013 vol. ii, no. 2s issn: 2283-3927 l introduction intramammary infection (imi) can adversely affect, in dairy goats, milk quality and milk yield leading to high economical losses. although somatic cell count (scc) and microbiological tests could be valid approaches to detect imi, other methods of imi early detection may be useful to detect infected animals and to improve milk quality. in this respect, the evaluation of electrical conductivity (ec) of milk, by on-line sensors, represents an interesting approach. the concentrations of na+ and clin milk increase during an imi due to a change of the permeability of the udder tissue, influencing the ec of milk (tangorra et al. 2010). this effect has been largely studied in dairy cows and the use of ec has been adopted with successful results in terms of detection accuracy (de mol et al. 2001). on the contrary, in dairy goats, this has not been the case mainly because less research studies have been conducted. however, our research group has shown that a specificity of 65% and a sensitivity of 81% could be reached by a univariate model based on evaluations of time series of gland milk ec (zaninelli et al. 2015). these results are in accordance with other authors (diaz et al. 2011; romero et al. 2012) that suggest: to consider the intrinsic variation of animals (as in cows), to avoid the use of simple thresholds, and to use other significant factors in the model such as the ec values of milk in different milking fractions (romero et al. 2012). in agree with these latter suggestions, in another study our research group has tested a multivariate model, developed with the fuzzy logic technology, based on the ec of gland milk and the evaluation of the goats milk yield (zaninelli et al. 2014). the results obtained have shown an improvement in the detection accuracy of the model evaluated (specificity of 69% and a sensitivity of 81%). nevertheless, the reached values are not still enough high if compared with results obtained in dairy cows (de mol et al. 2001). further studies, on multivariate models that use more informative parameters derived from the ec of milk, are necessary. the aim of this study was to test a new multivariate model developed with the fuzzy logic technology and based on the milk ec acquired on-line for each gland by dedicated sensors and on new qualitative and quantitative indexes derived from the spectrum of the recorded signals. material and methods the experiment was carried out for two weeks. ten healthy saanen goats at second lactation were randomly selected from a herd of 400 dairy goats. the animals were milked twice a day (7:00 a.m. and 5:00 p.m.). milking parlor had: a low-line design, self-locking gates and 2 platforms with 16 milking units and milking posts per platform. milking parameters were: a pulsation rate of 90 cycles/min, a vacuum level of 40 kpa and a pulsation ratio of 60%. two individual milk samples were collected from each mammary gland, during the morning milking, and frozen. according to microbiological tests and scc performed on collected samples results were classified as: case 1 (c1) if scc<1,000,000 cells/ml and no pathogenic microorganisms were found; case 2 (c2) if milk samples had bacteriological tests positive for imi; and case 3 (c3) if scc>1,000,000 cells/ml in 2 or more consecutive sampling keywords electrical conductivity; fuzzy logic; dairy goat; corresponding author mauro zaninelli, mauro.zaninelli@unisanraffaele.gov.it journal home page riviste.unimi.it/index.php/haf use of electrical coductivity sensors to monitor health status and quality of milk in dairy goats m. zaninelli 1 *, l. rossi 2 , a. costa 2 , f.m. tangorra 2 , a. agazzi 2 , g. savoini 2 1 faculty of agricolture, università telematica san raffaele roma, via di val cannuta 247, 00166, roma, italy; 2 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy; article selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 9 haf © 2013 vol. ii, no. 2s issn: 2283-3927 days for non-physiological causes and without pathogenic microorganisms. subsequently, samples classified in c1 were associated with healthy glands, while those in c2 and c3 with non-healthy mammary glands. five experimental milking clusters were used to measure on-line the ec (ms/cm) of gland milk (zaninelli et al. 2015b). estimated ec values were then calculated through a moving-average model (using the ten previous values of ec observations) and the relative deviations of ec between measured and estimated values were calculated. from acquired electrical signals and through a customised matlab routine (the mathworks, usa) the fourier frequency spectrum of each signal was calculated and other qualitative and quantitative indexes were determined: the bandwidth length and the three highest frequency peaks of each spectrum (zaninelli et al. 2015b, 2015c). all the ec data were evaluated by a multivariate model developed using the fuzzy logic technology. input variables were: the ec deviations, the bandwidth length and the three highest frequency peaks of each frequency spectrum. after “defuzzification”, a couple of sensitivity and specificity of the model was calculated for different cut-off levels. finally, the cut-off level that allowed to reach a sensitivity of at least 80% was identified and the resulting pair of sensitivity and specificity was considered as the level of accuracy reached by the multivariate model. results and discussion the prevalence of bacteriological positive samples was 45.4% (n = 127). after the count of somatic cells in the milk samples, the resulting prevalence of glands with scc > 1,000,000 and without pathogenic microorganisms was 2.9% (n = 8). no cases of scc > 1,000,000 due to physiological causes were observed. the overall prevalence of samples from not healthy glands was 61.1% (n = 171) and no cases of clinical mastitis were observed. as expected, not healthy glands showed higher mean value of scc and of milk ec (diaz et al. 2011, romero et al. 2012, zaninelli et al. 2014, 2015a, 2015b, 2015c). the cut-off level for a sensitivity of at least 80% was equal to 0.7. with this cut-off level, the resulting specificity and sensitivity of the model were respectively: 73% and 81%. these results were better than those obtained by other multivariate and univariate models that use the ec of milk to detect healthy status of dairy goats. nevertheless, these results cannot be considered enough if compared with those obtained in dairy cows (de mol et al. 2001). however, “fuzzification” and “fuzzy inference” are the most important steps in the fuzzy logic technique since they affect the final accuracy of the model. in order to maximise the model accuracy, they need to use a big experimental data set. therefore, with the collection of further experimental data the accuracy of the multivariate model, evaluated in this preliminary investigation, should be improved. conclusions this preliminary evaluation demonstrated that the fuzzy logic model tested, based on the milk ec and on new qualitative and quantitative indexes derived from the spectrum of the recorded signals, could show better results than those already reached in dairy goat research. nevertheless, further experiment and more field data could be useful to reach the best possible accuracy that this multivariate approach could show. references de mol r. m. & ouweltjes w. (2001). detection model for mastitis in cows milked in an automatic milking system. preventive veterinary medicine, 49(1-2), 71–82. diaz j. r., romero g., muelas r., sendra e., pantoja j. c.f., paredes c. (2011). analysis of the influence of variation factors on electrical conductivity of milk in murciano-granadina goats. j. dairy sci., 94, 3885–3894. romero g., pantoja j.c.f., sendra e., peris c., díaz j.r. (2012). analysis of the electrical conductivity in milking fractions as a mean for detecting and characterizing mastitis in goats. small rumin. res., 107, 157–163. tangorra f.m., zaninelli m., costa a., agazzi a., savoini g. (2010). milk electrical conductivity and mastitis status in dairy goats: results from a pilot study. small rumin. res., 90, 109-113. zaninelli m., rossi l., tangorra f.m., costa a., agazzi a., savoini g. (2014). on-line monitoring of milk electrical conductivity by fuzzy logic technology to characterise health status in dairy goats. ital. j. anim. sci., 13, 340–347. zaninelli m., rossi l., costa a., tangorra f.m., agazzi a., savoini g. (2015a) monitoraggio dello stato di salute delle capre attraverso l’analisi on-line della conducibilità elettrica del latte. large anim. rev., 21, 81–86. zaninelli m., agazzi a., costa a., tangorra f.m., rossi l., savoini g. (2015b). evaluation of the fourier frequency spectrum peaks of milk electrical conductivity signals as indexes to monitor the dairy goats’ health status by on-line sensors. sensors, vol. 15, 20698-20716 zaninelli m., rossi l., costa a., tangorra f.m., agazzi a., savoini g. (2015c). signal spectral analysis to characterize gland milk electrical conductivity in dairy goats. italian journal of animal science, vol. 14, 362-367 proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords meniscus, pig, matrix corresponding author umberto polito umberto.polito@unimi.it journal home page riviste.unimi.it/index.php/haf meniscus matrix morphological composition: age-dependent evaluation in a swine model umberto polito1*, elena dell’era4, giuseppe m peretti2,3, irene tessaro2, cinzia domeneghini1, alessia di giancamillo1 1university of milan, department of health, animal science and food safety, italy 2irccs, istituto ortopedico galeazzi, milan, italy 3university of milan, department of biomedical sciences for health, italy 4dvm abstract menisci are fibro-cartilaginous structures interposed between femoral condyle and tibial plateau, which have multiple functions in the stifle joint: act as shock absorbers, bear loaders and allow joint stability, congruity and lubrication (sweigart et al., 2004; proffen et al., 2012). it is well known that meniscal injuries lead to osteoarthritis and for these reasons, menisci are considered important target of investigation. their important role in the knee wellness is only equalled by their deficiency in proper self-repairing. nowadays, the gold standard technique is not just to remove the damaged meniscus, but to rebuild it or to replace it. for these reasons, studies are necessary to increase the knowledge about these small but essential structures (streuli, 1999; deponti et al., 2013). composition and morphology are basic fundamental information for the development of engineered meniscal substitutes (di giancamillo et al., 2014). the analysis of the morphological, structural and biochemical changes, which occur during growth of the normal menisci, represent the goal of the present study. for this purpose, menisci from adult (7-month old), young (1-month old), and neonates (stillbirths) pigs were collected. cellularity and glycosamiglycans (gags) deposition were evaluated by elisa, while collagen-1 and collagen-2 were investigated by immunohistochemistry and western blot analyses. cellularity (p<0.01, all comparisons) and collagen-1 (p<0.05, neonatal-young vs adult) decreased from neonatal to adult stage while gags (p<0.01 neonatal vs young-adult) and collagen-2 (p<0.01 neonatal-young vs adult) showed the opposite trend. immunohistochemistry revealed similar changes occurring during animal growth thus revealing that cellular phenotype, cellularity and protein expression, as well as fibers aggregation in the matrix, are dissimilar in the three ages analysed categories. these changes reflect the progressive menisci maturation and hyper-specialisation. we observed the correlation between biochemical and phenotype properties of swine menisci follow age-dependent changes during growth: starting with an immature cellular and fiber pattern to the mature organised and differentiated adult menisci. acknowledgments this work was funded by the “finanziamento piano sviluppo ateneo linea 2a” http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references deponti, d., di giancamillo, a., scotti, c., peretti, g.m., martin, i., 2013. animal models for meniscus repair and regeneration. j tissue eng regen med. 9 (5), 512-527 di giancamillo, a., deponti, d., addis, a., domeneghini, c., peretti, g.m., 2014. meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth. j. cell. mol. med. 18 (10), 1964-1974. proffen, b.l., mcelfresh, m., fleming, b.c., murray, m., 2012. a comparative anatomical study of the human knee and six animal species. the knee. 19 (4), 493–499. streuli, c., 1999. extracellular matrix remodelling and cellular differentiation. current opinion in cell biology. 11 (5), 634–640. sweigart, m.a., zhu, c.f., burt, d.m., deholl, p.d., agrawal, c.m., clanton, t.o., athanasiou, k.a., 2004. intraspecies and interspecies comparison of the compressive properties of the medial meniscus. annals of biomedical engineering. 32 (11), 1569–1579. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en l keywords local turkey, plumage, indoor, outdoor. pages 01 – 15 references vol. 6 no. 1 (2019) article history submitted: november 07, 2018 accepted: december 03 2019 published: december 05 2019 corresponding author safiyu k.k. department of animal production and livestock management, michael okpara university of agriculture, umudike, abia state, nigeria, mail: safiyu.kamorudeenk@ pg.funaab.edu.ng phone: +2348060396895 journal home page riviste.unimi.it/index.php/haf article an exploratory study on the effects of rearing system and plumage colour on performance, carcass characteristics and meat quality of local turkeys safiyu k.k. 1,* , sogunle o.m. 2 , egbeyale l.t. 2 and shittu t.a. 3 1 department of animal production and livestock management, michael okpara university of agriculture, umudike, abia state, nigeria 2 department of animal production and health, federal university of agriculture, abeokuta, ogun state, nigeria 3 department of food science and technology, federal university of agriculture, abeokuta, ogun state, nigeria abstract in a bid to improve the productive potentials of local turkeys in developing countries, a total of 240 unsexed day-old poults arranged in a 2 × 2 factorial layout into 4 treatments with two rearing systems (indoor and outdoor) and two plumage colours (white and black) were used for this study. poults were brooded for 4 weeks followed by an acclimatization period of 2 weeks in the two different rearing systems before the commencement of the study which lasted 10 weeks. each treatment consisting of 60 birds was further sub-divided into six replicates of 10 birds per replicate. data obtained were subjected to analysis of variance in a completely randomized design. results on performance in the grower phase showed turkeys reared in indoor system recorded significantly (p<0.05) higher weight gain (29.39 vs. 105.19 g/bird/day) and daily feed intake (27.18 vs. 98.11 g/bird/day) compare to turkeys under outdoor system. in addition, weight gain was significantly (p<0.05) higher (29.16 g/bird/day) in turkeys with black plumage than (27.42 g/bird/day) recorded in turkeys with white plumage. however, in the finisher phase turkeys under outdoor system recorded significantly (p<0.05) higher weight gain than turkeys under indoor system. in the finisher phase, interaction effects showed best weight gain and feed conversion ratio (fcr) (39.22 g/bird/day and 4.60) in whiteplumaged turkeys reared in outdoor system. turkeys under outdoor system also had significantly (p<0.05) higher back and spleen percentages. however, proportions of thigh were significantly (p<0.05) higher in turkeys reared indoor. in addition, white-plumaged turkeys recorded significantly (p<0.05) higher (21.07%) cooking loss than 14.58% recorded in turkeys with black plumage. in conclusion, improved weight gain with best fcr at finisher phase as well as highest spleen portion and cooking loss in thigh meat was obtained in white-plumaged turkeys reared in outdoor system. mailto:safiyu.kamorudeenk@%20pg.funaab.edu.ng mailto:safiyu.kamorudeenk@%20pg.funaab.edu.ng safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 2 1 introduction in order to provide animal protein in adequate quantity and quality in developing countries, farmers as well as scientists are selecting poultry species with sufficient potentials for domestication and can supplement the availability of essential protein at cheaper cost (ironkwe et al., 2015). turkey production plays an important role in this aspect (amumueller, 2008). turkeys are excellent foragers that thrive better under arid conditions and tolerate heat when compared with broilers (yakubu et al., 2013). worldwide, indigenous turkey production is a highly profitable industry with an increased production quantity from 5.1 million ton in 2003 to 5.6 million ton in 2013 (faostat, 2013). also, the demand for turkey products is rising globally (yakubu et al., 2013); in fact turkey meat is one of the best options for alternative protein source in the tropics (asaduzzaman et al., 2017). according to karki (2005), the consumption of turkeys as white meat has increased worldwide and a similar trend also existed in developing countries. however, turkey production has not been fully exploited in developing countries despite its potential over other poultry species. turkey (meleagris gallopavo) is well known in the united states of america and europe, but in developing countries like nigeria, the rearing of local turkey in traditional production systems serves as an immediate source of meat and income for rural farmers (okoli et al., 2009; ekue et al., 2002). the traditional village poultry production systems are mainly based on scavenging indigenous poultry found in almost all households in the rural areas. they are characteristically an integral part of the farming systems requiring low-inputs with outputs accessible at household level (kitalyi, 1998). although the performances of local poultry are lower than exotic poultry breeds, its hardiness and disease resistance makes it more adaptable to the tropical environment (padhi, 2016). the importance of local poultry species in the national economy of developing countries and its role for improving the nutritional status and income of many smallholder farmers and landless communities has been very significant (creevey, 1991; fao, 1997). thus, the adoption of improved production systems is essential for the strategic increase in the productivity of local poultry flocks to improve household food security and alleviation of poverty in rural communities (awuni, 2002; case et al., 2010). in many parts of africa, local poultry have been characterized on different grounds; teketel (1986) characterized them on the basis of plumage colour, for example, kei (red) or tikur (black), tadelle (2003) and halima et al. (2007) both named on the basis of the geographic region of sampling with each local ecotype actually comprising chickens with a wide range of morphologic or genetic diversity. genetic diversity has been described in chickens using monogenic traits based on different pigmentation and comb types. these different pigmentations can be attributable to melanin which is responsible for the production of varieties of plumage colours in chickens (dana et al., 2010). the presence and level of melanin pigments such as trichochrome is related to feather colour and is considered to be indicative of genetic differences among certain plumage colours (smyth, 1990). though the bulk of research work on plumage colour is on chicken, dearth of information still exists on how differences in plumage colour influences performance and carcass components in local poultry. safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 3 in addition, plumage colour is second in importance to live weight in affecting market preference for chickens by consumers in developing countries (dana et al., 2010). in certain communities in africa, plumage colours have cultural and religious functions (gueye, 1998; leulseged, 1998). there are specific choices for plumage colours that affect preferences of different geographic markets around the world (jiang, 1999; smyth, 1990). producer, sellers and intermediary traders of chickens attach high market preference to plumage colour and feather distribution (aklilu, 2007). this clearly suggests that qualitative traits with specific characteristics should be carefully identified and considered for marketability of the local turkeys. based on this background, the objective of this research aimed to investigate the effects of rearing system and plumage colour on local turkey performances, carcass components and meat quality. 2 materials and methods 2.1 ethical statement this study was performed in accordance with the recommendations of the animal ethics committee guidelines of the federal university of agriculture, abeokuta. 2.2 experimental site the study was carried out at the poultry unit of the directorate of university farms (dufarms), federal university of agriculture, abeokuta (nigeria). 2.3 experimental design a total of 240 day-old unsexed turkey poults with two different plumage colours (white and black) from local turkey strain were used for this study. poults were purchased from a reputable hatchery and brooded for 4 weeks. afterwards, the poults were allotted on weight equalization basis into two different rearing systems (indoor and outdoor) for an acclimatization period of 2 weeks before the commencement of the study which lasted 10 weeks. the indoor pen was constructed with a stocking density of 0.4 m 2 per turkey with wood shavings used as bedding material. however, turkeys in outdoor system had access to runs for foraging with mini-shelters of stocking density of 4 m 2 per replicate. water and feed were provided ad libitum and the composition of experimental diets are presented in table 1. this study was arranged in a 2×2 factorial layout: the two factors, factor a (rearing systems) and factor b (plumage colours) with each taking two levels; factor a (indoor and outdoor) and factor b (white and black) resulted in four treatment combinations in total as shown in table 2. each treatment group consisting of 60 birds was further sub-divided into six replicates of 10 birds per replicate. safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 4 2.4 zootechnical evaluation the following growth performance parameters were collected at the grower phase (from 6 to 12 week) and finisher phase (from 12 to 16 week). birds in each replicate were weighed individually using a weighing scale (model: yongzhou yz-328+) and the total weight was divided by the number of weighed birds. the initial weights at the beginning of the experiment were recorded while subsequent body weights were recorded on weekly basis. wg (g/bird/day) = the daily feed intake in each replicate was estimated using the formula below: fi (g/bird/day) = – the feed conversion ratio (fcr) of birds in each treatment was determined by calculating the ratio of feed intake to weight gain and thus will be calculated as: fcr = table 1: composition (%) of experimental diets ingredient grower phase finisher phase maize 44.00 47.00 soyabean meal 31.00 20.00 wheat offal 10.00 25.00 fish meal (65% crude protein) 5.00 1.00 bone meal 5.60 3.50 oyster shell 3.00 2.50 salt (nacl) 0.45 0.25 dl-methionine 0.40 0.20 lysine 0.25 0.25 premix 0.40 0.30 total 100.00 100.00 calculated analysis metabolizable energy (mj/kg) 11.07 10.63 crude protein (%) 28.00 17.74 table 2: experimental design rearing system plumage colour white black indoor white turkeys under indoor system (n =60) black turkeys under indoor system (n =60) outdoor white turkeys under outdoor system (n =60) black turkeys under outdoor system (n =60) safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 5 2.5 meat quality after the 10 week study, two birds per replicate were selected for carcass analysis. selected birds were fasted for 12 hours before slaughtering by severing the carotid artery and the jugular vein. the birds were allowed to bleed completely followed by removal of feathers and viscera. the weights of the carcasses were recorded and the dressing percentages (dp) were estimated by dividing the dressed weight of carcasses by the live weight (lw) and multiplied by 100. the weight of cut-up parts (breast, back, thigh and drumstick) and organs (heart, spleen, liver, lungs, gizzard and proventriculus) were determined using an electronic scale and the values recorded in grams were expressed as a percentage of the live weight. cooking loss: this was determined by collecting a known amount (20 g) of meat from the breast and thigh regions of carcasses per replicate. samples were placed in an airtight polythene bag, labelled and immediately cooked in a water bath at 70 ° c for 15 minutes. thereafter, meat samples were allowed to cool at room temperature and weighed to determine the cooking loss. cooking loss (%) = × 100 chilling loss: twenty gram (20 g) of meat from the breast and thigh regions of carcasses from each replicate were placed in an airtight polythene bag; labelled and placed in a refrigerator at 7 ºc for 24 hours. chilling loss was determined as: chilling loss (%) = × 100 2.6 statistical analysis the data obtained were subjected to analysis of variance (anova) in a completely randomized design. tukey’s test as contained in minitab ® version 17.1.0 software (minitab inc., pa, usa) was applied for comparison of means at 5% probability level. the model of the study was; yijk = µ + αi + βj + (αβ)ij + ʃijk where: yijk = individual observation µ = population mean αi = main effect of rearing system βj = main effect of plumage colour (αβ)ij = interaction effects between rearing system and plumage colour ʃijk = residual error safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 6 3 results table 3 shows the effects of rearing system and plumage colour on growth performance of local turkeys. weight gain and feed intake measured at the grower phase were significantly (p<0.05) affected by rearing system. turkeys reared in indoor system recorded significantly (p<0.05) higher values (29.39 and 105.19 g/bird/day) for weight gain and feed intake than values (27.18 and 98.11 g/bird/day) recorded in turkeys under outdoor system. the effect of plumage colour significantly (p<0.05) influenced weight gain measured at the grower phase. weight gain was significantly (p<0.05) higher (29.16 g/bird/day) in turkeys with black plumage colour than (27.42 g/bird/day) recorded in turkeys with white plumage. weight gain and feed intake measured at the grower phase were significantly (p<0.05) influenced by rearing system and plumage colour interaction. weight gain was significantly (p<0.05) higher in statistically similar values (30.10, 28.68 and 29.64 g/bird/day) recorded in white-plumaged turkeys in indoor system, black-plumaged turkeys in indoor system and black-plumaged turkeys reared in outdoor system, respectively and lower (24.73 g/bird/day) in white-plumaged turkeys under outdoor system. feed intake was significantly (p<0.05) highest (107.71 g/bird/day) in white-plumaged turkeys reared indoor and lowest (95.54 g/bird/day) in whiteplumaged turkeys reared outdoor. in the finisher phase, turkeys under outdoor system recorded significantly (p<0.05) higher weight gain (36.05 g/bird/day) than (30.99 g/bird/day) recorded in turkeys in the indoor system. however, the effect of plumage colour on all performance parameters measured were not significantly (p>0.05) different. in addition, weight gain and feed conversion ratio were significantly (p<0.05) influenced at the finisher phase by the interaction between rearing system and plumage colour. weight gain was significantly (p<0.05) higher (39.22 g/bird/day) in white-plumaged turkeys under outdoor system than comparable means (30.05, 31.94 and32.88 g/bird/day) recorded in white-plumaged turkeys reared in indoor system and black-plumaged turkeys under indoor and outdoor systems, respectively. the most excellent fcr value (4.60) was recorded (p<0.05) in white-plumaged turkeys reared in outdoor system than 6.16 recorded in black-plumaged turkeys reared in outdoor system. the effects of rearing system and plumage colour on carcass yield of local turkeys are presented in table 4. rearing system significantly (p<0.05) influenced back, thigh and spleen percentages. turkeys under outdoor system had significantly (p<0.05) higher (13.24 and 0.21%) back and spleen percentages, respectively than values (11.80 and 0.10%) in turkeys reared in indoor system. however, proportions of thigh were significantly (p<0.05) higher (9.81 %) in turkeys reared in indoor and lower (8.53%) in turkeys reared in outdoor. the effect of plumage colour on all carcass traits measured was however not significantly (p<0.05) influenced. in addition, thigh and spleen were significantly (p>0.05) influenced by rearing system and plumage colour interaction. comparable means (9.57 and 10.05%) for thigh were recorded in white and blackplumaged turkeys, respectively reared in indoor system which were significantly (p<0.05) higher than similar means (8.52 and 8.55%) recorded in white and blackplumaged turkeys, respectively reared in outdoor system. also, spleen percentage was safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 7 significantly (p<0.05) highest (0.25%) in white-plumaged turkeys reared in outdoor system and lowest (0.09%) in white-plumaged turkeys reared in indoor system. table 5 shows the effects of rearing system and plumage colour on physical properties of breast and thigh meats from local turkey. rearing system had no significant (p>0.05) influence on all parameters measured. however, plumage colour effect significantly (p<0.05 affected the percentage cooking loss for thigh meat. whiteplumaged turkeys recorded significantly (p<0.05) higher (21.07%) cooking loss than 14.58% recorded in turkeys with black plumage. furthermore, rearing system and plumage colour interaction influenced cooking loss of breast meat which was significantly (p<0.05) highest (24.29%) in turkeys with white plumage reared in outdoor system and lowest (15.86%) in black turkeys reared in outdoor system. table 3: effects of rearing system and plumage colour on growth performance of local turkeys grower (from 6 to 12 week) finisher (from 12 to 16 week) rearing system iw wg fi fcr wg fi fcr indoor 774.40 29.39 a 105.19 a 3.63 30.99 b 170.25 5.68 outdoor 748.21 27.18 b 98.11 b 3.68 36.05 a 177.96 5.38 sem 7.69 0.45 1.93 0.11 1.06 6.42 0.24 p value 0.058 0.003 0.020 0.790 0.004 0.408 0.390 plumage colour iw wg fi fcr wg fi fcr white 750.60 27.42b 101.62 3.76 34.64 172.79 5.24 black 772.02 29.16a 101.68 3.55 32.41 175.42 5.82 sem 7.69 0.45 1.93 0.12 1.06 6.42 0.24 p value 0.066 0.014 0.985 0.217 0.156 0.775 0.106 rearing system plumage colour iw wg fi fcr wg fi fcr indoor white 757.10 30.10 a 107.71 a 3.63 30.05 b 168.44 5.88 ab black 791.70 28.68 a 102.67 ab 3.64 31.94 b 172.06 5.48 ab outdoor white 744.00 24.73 b 95.54 b 3.90 39.22 a 177.13 4.60 b black 752.40 29.64 a 100.68 ab 3.46 32.88 b 178.78 6.16a sem 10.90 0.64 2.73 0.17 1.50 9.08 0.34 p value 0.246 <0.001 0.041 0.195 0.014 0.915 0.010 ab means on the same column having different superscript are significantly different at 5% probability level iw—initial weight (g); wg—weight gain (g/bird/day); fi—daily feed intake (g/bird/day); fcr – feed conversion ratio safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -15 8 table 4: effects of rearing system and plumage colour on carcass characteristics of local turkeys cut-up parts 1 organs 1 rearing system lw (g) dp (%) breast back thigh drumstick heart spleen liver lungs gizzard proventriculus indoor 2835.00 74.89 18.72 11.80b 9.81a 9.98 0.45 0.10b 1.55 0.52 2.82 0.33 outdoor 2678.80 74.89 17.82 13.24a 8.53b 9.84 0.44 0.21a 1.69 0.49 2.54 0.30 sem 67.40 1.16 0.44 0.39 0.18 0.18 0.02 0.03 0.05 0.02 0.12 0.02 p value 0.121 0.999 0.165 0.019 <0.001 0.596 0.631 0.013 0.068 0.235 0.124 0.319 plumage colour lw (g) dp (%) breast back thigh drumstick heart spleen liver lungs gizzard proventriculus white 2746.10 73.69 18.28 12.26 9.05 9.82 0.43 0.17 1.58 0.50 2.54 0.31 black 2767.70 76.08 18.26 12.77 9.30 10.00 0.46 0.14 1.65 0.51 2.82 0.31 sem 67.40 1.16 0.44 0.39 0.18 0.18 0.02 0.03 0.05 0.02 0.13 0.02 p value 0.824 0.163 0.978 0.366 0.333 0.488 0.324 0.342 0.358 0.841 0.134 0.801 rearing system plumage colour lw (g) dp (%) breast back thigh drumstick heart spleen liver lungs gizzard proventriculus indoor white 2857.00 72.84 18.51 11.78 9.57a 9.86 0.44 0.09b 1.46 0.50 2.56 0.30 black 2813.00 76.93 18.92 11.81 10.05a 10.09 0.49 0.11ab 1.63 0.53 3.08 0.36 outdoor white 2635.20 74.54 18.04 12.74 8.52b 9.77 0.42 0.25a 1.71 0.50 2.52 0.33 black 2722.30 75.24 17.60 13.73 8.55b 9.91 0.46 0.16ab 1.67 0.48 2.55 0.27 sem 95.30 1.64 0.62 0.55 0.25 0.25 0.03 0.04 0.07 0.03 0.18 0.03 p value 0.501 0.317 0.496 0.393 0.397 0.865 0.906 0.171 0.151 0.287 0.168 0.051 abc means on the same column having different superscript are significantly different at 5% probability level 1: values are expressed as percentages of the live weight lw-live weight (g); dp-dressing percentage (%) safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -12 9 table 5: effects of rearing system and plumage colour on physical properties of breast and thigh meats from local turkeys breast thigh rearing system cooking loss (%) chilling loss (%) cooking loss (%) chilling loss (%) indoor 20.34 2.70 18.16 3.36 outdoor 20.08 5.10 17.49 3.18 sem 1.44 2.00 1.85 0.88 p value 0.898 0.410 0.803 0.889 plumage colour cooking loss (%) chilling loss (%) cooking loss (%) chilling loss (%) white 21.62 5.34 21.07 a 3.10 black 18.80 2.47 14.58 b 3.43 sem 1.44 2.00 1.85 0.88 p value 0.185 0.326 0.025 0.791 rearing system plumage colour cooking loss (%) chilling loss (%) cooking loss (%) chilling loss (%) indoor white 18.95 ab 3.29 21.13 3.09 black 21.73 ab 2.12 15.18 3.62 outdoor white 24.29 a 7.39 21.01 3.11 black 15.86 b 2.81 13.97 3.25 sem 2.04 2.84 2.62 1.25 p value 0.014 0.555 0.839 0.876 ab means on the same column having different superscript are significantly different at 5% probability level. 4 discussion this study revealed that growing turkeys under indoor system recorded significant higher weight gain and feed intake than turkeys reared outdoor. this is consistent with earlier reports by wang et al. (2009) and dou et al. (2009) who reported lower growth rate in growing birds in outdoor system than those in the intensive system. similarly, li et al. (2017) recorded significant changes in body weights and feed intakes of growing chickens reared on different housing system. however, rearing system had no influence on feed conversion ratio of growing turkeys in this present study. this contradicted safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -12 10 previous studies (hoop and rippinger, 1997; permin et al., 1999; zhao et al., 2014) indicating lower feed conversion for birds with outdoor access. this variation could be attributed to differences in poultry species reared, age of birds as well as welfare condition of rearing system between past and present studies. in addition, blackplumaged growing turkeys had higher weight gain than turkeys with white plumages. this agrees with reports of studies like yildiz and kesici (1997), petek et al. (2004), minvielle et al. (2005) and yilmaz and çağlayan (2008) indicating that white-feathered birds had less body weight than birds with wild breeds with different feather colours. contrary to the results obtained in the grower phase, weight gain obtained in the finisher phase was greater in turkeys reared in outdoor system than birds in indoor system. result is in line with the findings of batkowska et al. (2015) who observed higher weights in finisher broilers reared in extensive system than those reared in intensive system. santos et al. (2005) also reported higher weight gain in birds reared in semiconfined system than those in confined system. however, fortomaris et al. (2007) and sogunle et al. (2016) had contrary opinions; the authors respectively observed no significant differences in weights of poultry species monitored at the finisher phase in different housing systems. this variation could be attributed to differences in size and type of housing system in these studies. moreover, the effect of plumage colour had no impact on the growth performance of turkeys monitored at the finisher phase. this contradicted earlier reports by tarhyel et al. (2012), who observed significant differences in live weight of quails with different feather colours. inci et al. (2015) also revealed feather colour variations affected feed intake and feed conversion ratios of japanese quails at the end of fattening period. according to this study, rearing system had no impact on live weights and dressing percentages of local turkeys. wang et al. (2009), chen et al. (2013), batkowska et al. (2015) and sogunle et al. (2016) had reported similar findings. on the contrary, the reports of castellini et al. (2002) and feddes et al. (2002) revealed birds managed on outdoor houses had significantly higher dressing percentages compared to birds in indoor houses because of increased motor activity. however, back, thigh and spleen portions of turkeys reared in different rearing systems were significantly different in this study. this is in agreement with the findings of aline (2015) who observed significant differences in cut-up parts of broilers reared in indoor and free-range houses. similarly, li et al. (2017) observed differences in leg muscle yield in different production system. in addition, it was observed in this study that plumage colour had no effect on carcass characteristics of turkeys, which however contradicted the reports of inci et al. (2015) who observed significant variations in carcass weight, carcass yield, and carcass parts of safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -12 11 quails with different feather colour. this variation could be attributed to differences in genotype and species of birds in both studies. furthermore, rearing system had no influence on cooking and chilling losses of turkey meat in this study. similar findings on the influence of housing system were also reported by tong et al. (2015); the authors observed no differences in the physical qualities of meat from local chicken except meat colour. in addition, this study found significant differences in cooking loss of turkey thigh meat as influenced by plumage colour effect but literatures relating to this are limited. 5 conclusion from the findings of this study, improved weight gain with best feed conversion ratio at finisher phase as well as highest spleen and cooking loss in thigh meat was obtained in white-plumaged turkeys reared in outdoor system. references aklilu, h.a., 2007. village poultry in ethiopia: socio-technical analysis and learning with farmers. wageningen university, wageningen, the netherlands. 178 pp. (ph.d. thesis). aline, k., 2015. management systems and location effects on growth and carcass traits of kuroiler and local chickens. a thesis submitted to the directorate of research and graduate training in partial fulfilment of the requirements for the award of the degree of master of science in animal science of makerere university. 101 pages. amumueller, r., 2008. certified production of commercial turkeys. world poultry magazine production on turkeys. 10-18. asaduzzaman, m., salma, u., ali, h.s., hamid, m.a., miah, a.g., 2017. problems and prospects of turkey (meleagris gallopavo) production in bangladesh. research in agriculture, livestock and fisheries. 4(2), 77-90. awuni, j.a., 2002. strategies for the improvement of rural chicken production in ghana. in: characteristics and parameters in family poultry production in africa, (iaea: vienna). safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -12 12 batkowska, j., brodacki a., zieba, g., horbanczuk, j.o., łukaszewicz, m., 2015. growth performance, carcass traits and physical properties of chicken meat as affected by genotype and production system. archives animal breeding. 58, 325–333. case, l.a., miller, s.p., wood, b.j., 2010. factors affecting breast meat yield in turkeys. world's poultry science journal. 66(2), 189-202. castellini, c., mugnai, c., dal bosco, a., 2002. effect of organic production system on broiler carcass and meat quality. meat science. 60, 219-225. chen, x., jiang, w., tan, h.z., xu, g.f., zhang, x.b., wei, s., wang, x.q., 2013. effects of outdoor access on growth performance, carcass composition, and meat characteristics of broiler chickens. poultry science 92(2), 435-443. creevey, l.e., 1991. supporting small-scale enterprises for women farmers in the sahel. journal of international development. 3, 355-386. dana, n., dessie, t., van der waaij, l.h., van arendonk, a.m., 2010. morphological features of indigenous chicken populations of ethiopia. animal genetic resources. 46, 11–23. dou, t.c., shi, s.r., sun, h.j., wang, k.h., 2009. growth rate, carcass traits and meat quality of slow-growing chicken grown according to three raising systems. animal science papers and reports. 27, 361-369. ekue, f.n., pone, k.d., mafeni, m.j., nfi, a.n., njoya, j., 2002. survey of the traditional poultry production system in the bamenda area, cameroon. in characteristics and parameters of family poultry production in africa. fao/iaea co-ordinated research programme on assessment of the effectiveness of vaccination strategies against newcastle disease and gumboro disease using immunoassay-based technologies for increasing farmyard poultry production in africa. iaea vienna. faostat, 2013. livestock primary production data. retrieved from http://faostat.fao.org feddes, j.j.r., emmanuel, e.j., zuidhof, m.j., 2002. broiler performance, bodyweight variance, feed and water intake, and carcass quality at different stocking densities. poultry science. 81, 774–779. food and agricultural organization., 1997. guidelines for the inclusion of improved household poultry production. diversification component of the special programme for food security, fao, rome 76 pp. http://faostat.fao.org/ safiyu k.k. et al. int. j. of health, animal science and food 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performance characteristics and carcass yield of indigenous turkeys fed indomie waste-based diets. journal of agriculture and veterinary science (iosr-javs). 8(1), 88-91. jiang, x., 1999. broiler breeding: breeding goals, selection schemes and the usefulness of local breeds of china. wageningen, the netherlands, wageningen university. 185 pp. (ph.d. thesis). karki, m., 2005. growth, efficiency of utilization and economics of different rearing periods of turkeys. nepal agricultural research journal. 6, 89-88. kitalyi, a.j., 1998. village chicken production systems in rural africa:household and food security and gender issues, fao animal production and health paper 142, rome, italy. http://fao.org/docrep/003/w898e/w8989e00.htm. leulseged, y., 1998. study on production systems of indigenous and improved poultry in rural areas of north wollo. alemaya, ethiopia, alemaya university of agriculture. 102 pp. (m.sc. thesis). li, y., luo, c., wang, j., guo, f., 2017. effects of different raising 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white plumage colour mutation on hatchability and growth of quail hatched from breeders of different ages. british poultry science 45, 769-774. santos, a.l., sakomura, n.k., freitas, e.r., 2005. comparison of free-range broiler chicken strains raised in confined or semi-confined systems. brazilian journal of poultry science. 7, 85-92. smyth, j.r., 1990. genetics of plumage, skin and eye pigmentation in chickens. in r.d. crawford, ed. poultry breeding and genetics, pp. 109–168. amsterdam, elsevier science publishers. sogunle, o.m., ogundele, m.a., akinola, o.s., njoku, c.p., oso, a.o., 2016. effects of different housing systems on growth performance and carcass yield of two breeds of turkeys. bulletin of animal health and production in africa. 64(1), 83-93. tadelle, d., 2003. phenotypic and genetic characterization of local chicken ecotypes in ethiopia. berlin, humboldt university of berlin, 209 pp. (ph.d. thesis). tarhyel, r. tanimomo, b.k., hena, s.a., 2012. effect of sex, colour and weight group on carcass characteristics of japanese quail. scientific journal of animal science. 1, 2227. teketel, f., 1986. studies on the meat production potential of some local strains of chickens in ethiopia. geissen, germany, j.l. university of geissen. 186 pp. (ph.d. thesis). tong, h.b., cai, j., lu, j., wang, q., shao, d., zou, j.m., 2015. effects of outdoor access days on growth performance, carcass yield, meat quality, and lymphoid organ index of a local chicken breed. poultry science. 94, 1115–1121. safiyu k.k. et al. int. j. of health, animal science and food safety 6 (2019) 01 -12 15 wang, k.h., shi, s.r., dou, t.c., sun, h.j., 2009. effect of a free-range raising system on growth performance, carcass yield, and meat quality of slow-growing chicken. poultry science. 88(10), 2219-2223. yakubu, a., abimiku, k., musa azara, i.s., idahor, k.o., akinsola, o.m., 2013. assessment of flock structure, preference in selection and traits of economic importance of domestic turkey (meleagris gallopavo) genetic resources in nasarawa state, nigeria. livestock research for rural development. 25, 18. yıldız, m.a., kesici, t. 1997. examination of the genetic relationship between the genes that determine the yellow and stained white feather color in japanese quails (coturnix coturnix japonica). journal of lalahan livestock research institute. 37, 8490. yılmaz, a., çağlayan, t., 2008. egg weight, shape index and hatch weight in japanese quails (coturnix coturnix japonica) with different hair color. fırat university health sciences veterinary journal. 22, 5-8. zhao, z., li, j., li, x., bao, j., 2014. effects of housing systems on behaviour, performance and welfare of fast-growing broilers. asian-australasian journal of animal sciences, 27(1), 140–146. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l corresponding author yonglun luo alun@biomed.au.dk journal home page riviste.unimi.it/index.php/haf modeling and manipulating human diseases with induced pluripotent stem cells, pig models and precision gene editing. y. luoa a department of biomedicine, aarhus university (dk) abstract we have developed systems, e.g. c-check, that can be used to rapidly select and quantify crispr/cas9 nuclease activity and enrichment of genetically modified cells with desired mutations (zhou et al., 2016). to facilitate the simultaneous manipulation of multiple genes in cells, we have developed a system that allows concordant delivery of up to 30 sgrnas into one cell [johan vadnielsen et al., under review]. targeted insertion, fluorescent tagging or correction of endogenous genes is of great interest but greatly hampered by the technical difficulties and relatively low homology directed repair efficiency compared to the higher efficiency of nhej. thus, we have developed systems for rapid generation of gene targeting vectors (luo et al., 2014), lentivirusmediated gene targeting [yujia cai et al., elife, in revision], and recombinant cas9s to enhance hdr in mammalian cells. furthermore, to recapitulate the pathogenesis of human diseases, we have developed pig models of breast cancer and diabetes using gene editing and scnt, as well as human induced pluripotent stem cell models of mcadd. references zhou y, liu y, hussmann d, brøgger p, al-saaidi ra, tan s, lin l, petersen ts, zhou gq, bross p, aagaard l, klein t, rønn sg, pedersen hd, bolund l, nielsen al, sørensen cb, luo y. (2016). "enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system." cell mol life sci. [epub ahead of print] luo y, lin l, bolund l, sørensen cb. (2014). "efficient construction of raav-based gene targeting vectors by golden gate cloning." biotechniques 56(5): 263-268. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:alun@biomed.au.dk proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords beauvericin, caco-2 cells, deoxynivalenol, fumonisin b1, toxicity corresponding author marco albonico marco.albonico@unimi.it journal home page riviste.unimi.it/index.php/haf in vitro toxicity of beauvericin alone and combined with fumonisin b1 or deoxynivalenol on caco-2 cells. m. albonicoa*, c. cortinovisb, f. calonia a department of veterinary medicine (dimevet), università degli studi di milano, via celoria 10, 20133 milan, italy b department of health, animal science and food safety (vespa), università degli studi di milano, via celoria 10, 20133 milan, italy abstract beauvericin (bea) is a mycotoxin produced by fusarium species, frequently occurring in cereal grains in combination with fumonisin b1 (fb1) and deoxynivalenol (don). the aim of this study was to evaluate the in vitro toxic effects of bea alone and combined with fb1 or don on human intestinal caco-2 cells cultured on semi-permeable inserts (caloni et al., 2012). caco-2 cells were treated for 24 h with bea (1.5 µm) alone and combined with fb1 (1.5 µm) or don (3.5 µm) on both apical (ap) and basolateral (bl) sides. barrier impairment was assessed by measuring the trans-epithelial electrical resistance (teer) after 1 h, 2 h and 24 h of treatment. at the end of the experiment, the culture medium was collected for interleukin-8 (il-8) determination. the results indicate that teer was not significantly affected by ap or bl exposure to bea and fb1 alone, whereas a significant decrease (p<0.05) of teer was observed after exposure to bea in combination with fb1 for 1 h and 2 h. don was found to decrease (p<0.05) teer alone and combined with bea after bl application starting from the second hour of treatment. no significant release of the inflammatory mediator il-8 was observed after ap or bl exposure to bea and fb1 alone and in combination. on the contrary, don alone and combined with bea induced a significant (p<0.05) release of il-8 after both ap and bl exposure. further investigations are underway to better clarify the effects of bea on the intestinal epithelium and its interaction with other fusariotoxins. references caloni, f., cortinovis, c., pizzo, f., de angelis, i., 2012. transport of aflatoxin m1 in human intestinal caco-2/tc7 cells. front. pharmacol. 3, 111. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords pgrmc1, nucleolus, nucleolin, bovine granulosa cells, ovarian follicle corresponding author laura terzaghi laura.terzaghi@unimi.it journal home page riviste.unimi.it/index.php/haf progesterone receptor membrane component-1 (pgrmc1) nucleolar localization in bovine granulosa cells and its putative interaction with nucleolin. l. terzaghia*, a. m. lucianoa, s. c. modinaa, v. loddea a reproductive and developmental biology laboratory, department of health, animal science and food safety, university of milan, 20133 milan, italy abstract pgrmc1 is a multifunctional protein that is found in multiple subcellular compartments, suggesting a specific function at each site. among the several subcellular sites of expression, pgrmc1 was found in the nucleolus of human cells (ahmad et al. 2009) and bovine zygotes (luciano et al. 2010). however, the role at this site is not clear. therefore, the aim of this study is to assess whether pgrmc1 modulates nucleolar function. immunofluorescence experiments confirmed nucleolar localization in rat spontaneously immortalized granulosa cells, bovine granulosa cells (bgc) and bovine oocytes. moreover, in bgc pgrmc1 co-localizes with nucleolin, a well-known nucleolar marker exerting important functions within the nucleolus. additionally, sirna mediated gene knockdown experiments showed that when pgrmc1 expression is silenced, nucleolin localization shifts from the nucleolus to the nucleoplasm, suggesting a pgrmc1/nucleolin functional association. however, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate pgrmc1/nucleolin interaction. in conclusion, these studies suggest a function for pgrmc1 in nucleolar activity and set the stage for further investigations aimed at dissecting pgrmc1’s molecular mechanisms of action in the nuclear compartment. references ahmad, y., f. m. boisvert, p. gregor, a. cobley, and a. i. lamond. 2009. 'nopdb: nucleolar proteome database--2008 update', nucleic acids res, 37: d181-4. luciano, a. m., v. lodde, f. franciosi, f. ceciliani, and j. j. peluso. 2010. 'progesterone receptor membrane component 1 expression and putative function in bovine oocyte maturation, fertilization, and early embryonic development', reproduction, 140: 663-72. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l corresponding author arturo anadón anadon@vet.ucm.es journal home page riviste.unimi.it/index.php/haf pharmacokinetics, efficacy prediction indexes and residue depletion of antibacterial drugs. a. anadóna adepartment of toxicology and pharmacology faculty of veterinary medicine universidad complutense de madrid 28040-madrid abstract pharmacokinetics behaviour of the antibacterial in food producing animals, provides information on the rates of absorption and elimination, half-life in plasma and tissue, elimination pathways and metabolism. the dose and the dosing interval of the antimicrobial can be justified by considering the pharmacokinetic/pharmacodynamic (pk/pd) relationship, if established, as well as the severity of the disease, whereas the number of administrations should be in line with the nature of the disease. the target population for therapy should be well defined and possible to identify under field conditions. based on in vitro susceptibility data, and target animal pk data, an analysis for the pk/pd relationship may be used to support dose regimen selection and interpretation criteria for a clinical breakpoint. therefore, for all antibacterials with systemic activity, the mic data collected should be compared with the concentration of the compound at the relevant biophase following administration at the assumed therapeutic dose as recorded in the pharmacokinetic studies. currently, the most frequently used parameters to express the pk/pd relationship are cmax/mic (maximum serum concentration/mic), %t > mic (fraction of time in which concentration exceeds mic) and auc/mic (area under the inhibitory concentration– time curve/mic). furthermore, the pharmacokinetic parameters provide the first indication of the potential for persistent residues and the tissues in which they may occur. the information on residue depletion in food-producing animals, provides the data on which mrl recommendations will be based. a critical factor in the antibacterial medication of all food-producing animals is the mandatory withdrawal period, defined as the time during which drug must not be administered prior to the slaughter of the animal for consumption. the withdrawal period is an integral part of the regulatory authorities’ approval process and is designed to ensure that no significant drug residue is present in the animal at slaughter. drug residues in food-producing animals should comply with the mrl values for their target tissues in the animal species. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:anadon@vet.ucm.es proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author marco albonico, marco.albonico@unimi.it abstract fumonisin b1 (fb1) and beauvericin (bea) are fusariotoxins found to co-exist in food and feed commodities. the aim of this study is to evaluate the individual and combined effects of fb1 and bea on bovine granulosa cell proliferation and steroid production. granulosa cells (gc) from small bovine follicles (1-5 mm) were cultured for 48 hours in 10% fetal bovine serum followed by 48 hours in a serum-free medium containing 500 ng/ml of testosterone (as an estradiol precursor), 30 ng/ml of fsh and 30 ng/ml of igf-i with and without fb1 (3 µm) and bea (3 µm). at the end of the experiment, the numbers of gc were determined using a coulter counter (beckman coulter, usa) and concentrations of progesterone and estradiol in the culture medium were determined by radioimmunoassay. fb1 and bea, both individually and in combination, showed an inhibitory effect (p < 0.05) on gc proliferation. the number of gc decreased by 17.7%, 54.2% and 61.0% after exposure to fb1 (3 µm), bea (3 µm) and fb1 (3 µm) plus bea (3 µm) respectively. fb1 (3 µm) had no effect (p > 0.05) on estradiol and progesterone production, whereas bea (3 µm), both alone and in combination with fb1 (3 µm), was found to decrease (p < 0.001) the production of both steroids drastically. in conclusion, this in vitro study indicates that fb1 and bea, both individually and in combination, may affect gc proliferation to different extents and shows the drastic inhibitory effects of bea on steroid production. references cortinovis, c., pizzo, f., spicer, l.j., caloni, f. 2013. theriogenology 80:557-564; efsa (european food safety autority). 2014. efsa journal 12(8):3802; pizzo, f., caloni, f., schreiber, n.b., schutz, l.f., totty, m.l., albonico, m., spicer, l.j. 2015. ecotoxicology and environmental safety, 113:314-320; in vitro toxicological effects of fumonisin b1 and beauvericin on bovine granulosa cells m. albonico 1 , l.j. spicer 2 , c. cortinovis 1 , l. f. schutz 2 , f. caloni. 1 1 department of health, animal science and food safety (vespa), università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of animal science, oklahoma state university, stillwater, ok 74078, usa proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l corresponding author maria nobile, maria.nobile1@unimi.it abstract the european union regulations ban or regulate the use of substances with hormonal action. there are, however, some detectable steroids in biological matrices from cattle to which no administration were made. these are the so called “grey-zone” or “pseudoendogenous” substances (endogenously produced under certain circumstances). the administration of boldenone and androstadienedione to cattle is forbidden both for therapeutic or growth promotion purposes, while therapeutic use of prednisolone is permitted. the control of the use of these substances is hampered by their pseudoendogenous nature. we made a comparison between analyses performed on the classical matrix urine with bile (a novel matrix) from the same animals, with the aim to determine the better matrix for the above mentioned steroids. we tested the synthetic corticosteroid dexamethasone, too. the preliminary results do not show any difference between the two matrices, as concern the pseudoendogenous substances. the data about dexamethasone were instead very promising about the use of bile. the detection frequency and concentration levels were, in fact, higher in bile than in urine from the same animals. references european union. 2003. draft proposal; destrez, b., bichon, e., rambaud, l., courant, f., monteau, f., pinel, g., antignac, j.p. and le bizec, b. 2009. steroids, 74: 803–808; chiesa, l., nobile, m., panseri, s., sgoifo rossi, c. a., pavlovic, r., and arioli, f. 2014. analytica chimica acta, 852:137–145; ferretti, g., palleschi, l., marchiafava, c., delli quadri, f., fantozzi, l., ferranti, c., cammarata, p., macrì, a., montesissa, c. and draisci, r. 2007. analytica chimica acta, 589: 269–274; vanhaecke, l., antignac, j.-p., courtheyn, d., le bizec, b., and de brabander, h. f.2011. steroids, 76: 111–117. detection of boldenone, its sulfate and glucuronate forms, androstadienedione, cortisol, cortisone, prednisolone, prednisone and dexamethasone in bovine bile and urine by lc-ms/ms: preliminary results. m. nobile 1 ,l. chiesa 1 , r. pavlovic 1 , s. panseri 1 , f. arioli 2 1 department of veterinary science and public health, università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords dog, neonate, fetal fluids, biochemical composition corresponding author barbara bolis barbara.bolis@unimi.it journal home page riviste.unimi.it/index.php/haf abstract the full knowledge of the normal foetal fluid composition could be useful in the dog for the better management of newborns. the aim of the present study was to define the biochemical composition of amniotic fluid of puppies born by elective caesarean section (cs) at term of pregnancy. the study enrolled 24 purebred bitches, classified into small size (<10 kg) and large size breeds (>20kg). all the bitches were healthy and clinically monitored from mating until parturition. for all the bitches, an elective cs at term of pregnancy was performed (meloni et al., 2014). for each puppy, the amniotic fluid was collected, immediately centrifuged and frozen at – 20° c until analysis for albumin, amylase (amy), total bilirubin, cholesterol (chol), creatine kinase (ck), alkaline phosphatase, transaminases, lactate dehydrogenase (ldh), triglycerides, urea, glucose (glc), total proteins, creatinine , lipase, electrolytes, and globulins. data were analysed by analysis of covariance to verify the possible effects of parity, breed body size and newborn gender on amniotic biochemical composition. a total of 69 amniotic fluid samples were collected. the amniotic (mean ± standard deviation) and range (min-max) values for each parameter were defined. ldh value (p<0.01) and ck activity (p<0.05), as well as glc concentrations (p<0.0001) were negatively influenced by the parity. amy activity was significantly (p<0.05) higher in large sized (44.2±20.87 u/l) respect to small sized dogs (30.3±19.89 u/l), while a lower (p<0.05) chol amniotic concentrations were found in small sized (3.0±2.71 mg/dl) if compared to large sized (3.9±2.93 mg/dl) dogs. gender of the newborn did not influence the amniotic biochemical composition. the preliminary results of this study the suggested that, in dogs, some amniotic parameters could be influenced by breed body size and by parity. references meloni, t., comin, a., rota, a., peric, t., contri, a., veronesi, m.c., 2014. igf-i and nefa concentrations in fetal fluids of term pregnancy dogs. theriogenology. 8, 1307-1311. biochemical composition of amniotic fluid in normal puppies at term of pregnancy: preliminary data barbara bolis1*, magdalena schrank 2, jasmine fusi1, antonio mollo2, maria c. veronesi1 1 university of milan, department of veterinary medicine, italy 2university of padova, department of animal medicine, production and health, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords canine hepatocyte, bcaa, decellularizatio, liver, scaffold corresponding author matteo ghiringhelli matteo.ghiringhelli@unimi.it journal home page riviste.unimi.it/index.php/haf derivation of canine hepatocyte in vitro models to study branched-chain amino acid effects on liver functions. m. ghiringhellia*, g. pennarossaa, a. di giancamilloa, s. brizzolaa, t.a.l. brevinia, c. domeneghinia, f. acocellaa, v. bontempoa a department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy abstract branched chain amino acids (bcaa), have been shown to affect human gene expression, protein metabolism, apoptosis, and regeneration of hepatocytes. furthermore, they have been demonstrated to inhibit proliferation of liver cancer cells in vitro, and to be essential for lymphocyte proliferation. in veterinary medicine, the use of bcaas as integration of a normal dietary plan, is likely to be a valid choice for the same benefit found in human clinical nutrition, although this aspect is still debated. indeed, long-term oral supplementation with bcaas in the prevention of liver fibrosis and injury in the dog's liver is still unclear. aim of the present study will be to determine how bcaas preserve liver functions in vitro. to this purpose we have selected and set up three different in vitro models: hepatic dog cells and canine hepatocellular carcinoma cells plated in 2d monolayer and hepatic dog cells cultured onto 3d scaffolds, obtained from decellularized rabbit liver. all cells adhered and proliferated once plated. cells grown in monolayer quickly entered g0 end arrested growth, elisa test confirmed their ability to produce albumin. cells grown on scaffold vigorously replicated and showed their capability to recellularize ecm rabbit liver. these results, although preliminary, demonstrate that the culture conditions used well preserved the original phenotype and function and further support the possibility to use in vitro models to successfully study bcaa efficacy in dog. references boomkens, s.y., spee, b., ljzer, j., kisjes, r., egberink, h.f., van den ingh, t.s., rothuizen, j., penning, l.c., 2004. the establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns. comp hepatol. 3(1),9. duncan, a.w., dorrell, c., grompe, m., 2009. stem cells and liver regeneration. j gastroenterol 137(2), 466–81. kruitwagen, h.s., spee, b., schotanus, b.a., 2014. hepatic progenitor cells in canine and feline medicine: potential for regenerative strategies. bmc vet res. 10,137. rajkumar, r., victor, r.p., vinood., b.p., 2015. branched chain amino acids in clinical nutrition, new york, humana press. wakshlag, j.j., kallfelz, f.a., wakshlag, r.r., davenport, g.m., 2006. the effects of branched-chain amino acids on canine neoplastic cell proliferation and death. j nutr. 136(7), 2007s-2010s. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords innate immunity, forestomachs, inflammatory response, rumen corresponding author filipe joel joel.soares@unimi.it journal home page riviste.unimi.it/index.php/haf rumen fluid, a new diagnostic matrix in dairy cattle farms? j. filipe a*, e. trevisib, m. massaraa, a. minutib, p. banib, m. amadoric, f. rivaa a department of veterinary medicine, università degli studi di milano, milan, italy. b istituto di zootecnica, facoltà di agraria, università cattolica del sacro cuore, piacenza, italy. c laboratory of cellular immunology, istituto zooprofilattico sperimentale della lombardia e dell’emilia-romagna, brescia, italy. abstract production diseases of dairy cows are considered man-made problems caused by the inability of cows to achieve a sufficient feed energy intake (mulligan, 2008). a correct management of production diseases demands early diagnostic and prognostic parameters, in order to improve the management system and reduce the prevalence of clinical cases (ingvartsen, 2003). a previous study of our group indicated that forestomachs walls express immune receptors and cytokines, and the rumen liquor contains leukocytes able to produce ifn-γ (trevisi, 2014). our working hypothesis implied that ruminal fluids could be a source of diagnostic information for the identification of herds at risk for production diseases. we first demonstrated that the diet can influence the immune response in forestomachs. diverse leukocyte populations at low concentrations and ifn-γ were revealed in some samples of rumen fluids, with a clear inhibition of the response observed in the animals fed the maize-supplemented diet, compared to a normal and a soy-supplemented diet. we better characterized the leukocytes subpopulations in the rumen liquor, isolating b cells, monocytes and γδt cells. finally we performed a field survey in order to find correlation among the immune profile of the rumen liquor. clinically healthy animals showed a farm specific immunologic pattern of the rumen liquor: low cd45 mrna expression, low ifn-γ, few/absent b-cells. we can conclude that the epithelial cells of ruminant forestomachs can react to different stresses (metabolic, infectious, inflammatory) and the inflammatory response can be sustained by infiltrating leukocytes. our data points into the idea that dairy farms could be ranked according to a risk score using the inflammatory markers in rumen fluids, in addition to the traditional analysis. references ingvartsen, k.l., dewhurst, r.j., friggens, n.c., 2003. on the relationship between lactational performance and health: is it yield or metabolic imbalance that cause p roduction diseases in dairy cattle? a position paper. livest. prod. sci. 83, 277-308. mulligan fj, doherty ml. 2008 production diseases of the transition cow. the veterinary journal. 176:3–9. trevisi e, amadori m, riva f, bertoni g, bani p., 2014. evaluation of innate immune responses in bovine forestomachs. res vet sci. 96(1):69-78. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:joel.soares@unimi.it proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author stefano frattini, stefano.frattini@unimi.it abstract dna methylation and micrornas (mirna) are two important forms of epigenetic modifications that play an important role in gene regulation in animals. methylation at the carbon 5 position of cytosine residues is a fundamental layer of cellular differentiation through the control of transcriptional potential. mirna are small noncoding rna molecules that regulate gene expression. complete dna methylomes for several organisms are now available; at the present, methylome of the domestic goat is unexplored. there is also still limited knowledge about mirnas expression profiles in small ruminant species. therefore, to contribute information on epigenetic modification in capra hircus, we analysed the methylome and the mirna population of three tissues (hypothalamus, pituitary and ovary) from 3 adult saanen goats. we used methylated dna binding domain sequencing with enrichment of methylated dna fragments and next generation sequencing. we produced least 23 million reads per sample, which were aligned to the goat reference genome. further analyses were performed to identify peaks corresponding to hyper-methylated regions. we sequenced mirnas expressed in the three tissues with illumina high-throughput sequencing. reads were mapped on the capra hircus reference genome and both known and novel mirnas, and mirna target sites were identified using information collected in mirbase and using specific bioinformatic tools. this study produced a comprehensive mirna profile related to the biology of goat. furthermore, this is the first work dealing with methylome in capra hircus: our preliminary results could provide new information for a deeper comprehension of epigenetic mechanisms of this species. acknowledgement the research was funded by genhome project. references smith, z. d., and a. meissner, 2013, nat rev genet, 14: 204-20; schmiedel, j. m., s. l. klemm, y. zheng, a. sahay, n. blüthgen, d. s. marks, and a. van oudenaarden, 2015, science, 348: 128-32; shojaei saadi, h. a., a. m. o'doherty, d. gagné, é. fournier, j. r. grant, m. a. sirard, and c. robert, 2014, bmc genomics, 15: 451; mobuchon, l., s. marthey, m. boussaha, s. le guillou, c. leroux, and f. le provost, 2015, bmc genomics, 16: 285. a first glance on the epigenome of capra hircus s. frattini 1 , e. capra 2 , b. lazzari 2 , b. coizet 1 , d. groppetti 1 , p. riccaboni 1 , a. pecile 1 , s. arrighi 3 , s. chessa 2 , b. castiglioni 2 , a. giordano 1 , d. pravettoni 3 , a. talenti 1 , l. nicoloso 1 , j. l. williams 4 , p. crepaldi 1 , a. stella 2 , g. pagnacco 1 . 1 veterinary science and public health, university of milan, milan, italy. 2 institute of agricultural biology and biotechnology, national research council uos of lodi, lodi, italy. 3 department of health, animal science and foodsafety, university of milan, via celoria 10, 20133 milan, italy 4 school of animal and veterinary sciences, university of adelaide, roseworthy, australia proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords edible vaccines, nicotiana tabacum, vtec strain, avidity of antibody, streptococcus pneumoniae, monoclonal antibody corresponding author angela lombardi angela.lombardi@unimi.it journal home page riviste.unimi.it/index.php/haf avidities of human monoclonal antibodies derived from an adult immunized with pneumococcal polysaccharide vaccine. a.lombardia*, s. bandib, j. t. rahkolab, l. rossia, e. n. janoffb, a.baldia afaculty of veterinary medicine, department of health, animal science and food safety via celoria 10 20133 università degli studi di milano, milan. bschool of medicine division of infectious disease 12700 east 19th avenue, box b168 aurora, co 80045 university of colorado anschutz medical campus, aurora denver, usa. abstract plant based vaccines provide an instructive opportunity for immunologists. we have developed a plantbased oral vaccine against verocytotoxin–producing e. coli (vtec) in piglets (rossi et. al 2014). we engineered two independent lines of nicotiana tabacum plants for the seed-specific expression of vtec antigens, represented by the major subunit feda of the f18 adhesive fimbriae and the b-subunit of vt2e toxin respectively (rossi et. al. 2013). edible vaccines in particular are of interest as they are able to stimulate the mucosal immune system to produce secretory iga (s-iga) at mucosal surfaces and, potentially igg in the blood. the quality of the antibodies, such as avidity, should be considered in evaluating the efficacy of these vaccines. to develop this area, we determined avidity (strength of antibody-antigen binding) of igg to the capsule of another mucosal pathogen, streptococcus pneumoniae. using pneumococcal capsule-specific igg human monoclonal antibodies (hmab) cloned from single cells of a subject immunized with pneumococcal vaccine, we defined serotypes specificity and the avidity of these antibodies with ammonium thiocyanate (0, 4m, 2m, 1m 0.5m 0.025m) dissociation. igg with lower avidity to the capsule are dissociated at lower nh4scn levels, whereas igg with higher affinity require higher levels. we identified a range of avidities for 11 hmab’s (range x-y mnh4scn). we will evaluate the avidity of antibodies after immunization with edible vaccines against vtec strain in piglets about which little in known, but as demonstrated in granoff et al. the high-avidity antibodies are required in generating a more effective vaccine. references baxendale, h. e., goldblatt, d., 2006. correlation of molecular characteristics, isotype, and in vitro functional activity of human antipneumococcal monoclonal antibodies. infect. immun. 74:1025–1031. rossi, l., dell’orto, v., vagni, s., sala, v., reggi, s., baldi, a., 2014. protective effect of oral administration of transgenic tobacco seeds against verocytotoxic escherichia coli strain in piglets. vet res comm 38 (1), 39-49. rossi, l., di giancamillo, a., reggi, s., domeneghini, c., baldi, a., sala, v., dell’orto, v., coddens, a., cox, e., fogher, c., 2013. expression of porcine verocytotoxic escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model” jv 14(3), 263-270. schlesinger, y., granoff, d.m., 1992. vaccine study group. avidity and bactericidal activity of antibody elicited by dif ferent haemophilus influenzae type b conjugate vaccines. jama 1267, 1489–94. smith, k., muther, j., duke, a., mckee, e., zheng ny., wilson, pc., james, j.a., 2013. fully human monoclonal antibody from a ntibody secreting cells after vaccination with pneumovax®23 are serotypespecific and facilitate opsonophagocytosis. immunobiology; 218(5): 745–754. doi:10.1016/j.imbio.2012.08.278. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords midichloria spp., symbiosis, hard ticks, soft ticks. corresponding author alessandra cafiso alessandra.cafiso@unimi.it journal home page riviste.unimi.it/index.php/haf molecular screening for midichloria bacteria in hard and soft ticks (acari: ixodida). a. cafisoa, *, v. serraa, o.plantardb, c. bazzocchia,c a department of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy b bioepar, inra, oniris, route de gachet, 44307 nantes, france c joint research center for epidemiology and molecular surveillance of infections, university of milan, 20133 milan, italy abstract ticks can harbor complex and highly variable microbial communities. among these microorganisms, there are important pathogens of humans and animals that can be transmitted through the blood meal. less is known about the other members of the microbial community of ixodida, those that do not cause overt diseases. among these, midichloria mitochondrii, symbiont of the tick ixodes ricinus, is the first described member of the family midichloriaceae, order rickettsiales. this bacterium is present in 100% females and is vertically transmitted (sassera, 2008). the possibility of horizontal transmission is suggested by serological and molecular analyses showing positivity of mammalian blood and sera to m. mitochondrii (mariconti, 2012; bazzocchi, 2013). however, its role is still unknown. recent reports are expanding the view of this family, now including bacteria of great biological and medical interest, indicating a widespread distribution with an increasing range of hosts, with ticks being strongly represented (epis, 2008). here we present a molecular screening of 17 tick species (for a total of 92 individuals), detecting and quantifying bacteria closely related to m. mitochondrii in seven of them, including the first report of a midichloriacea in a soft tick species, ornithodoros maritimus. based on sequence identity and phylogenetic analysis we propose that these bacteria could constitute the genus midichloria. the performed screening highlights different prevalence levels in different tick species including one, ixodes aulacodi, where the bacteria is present in all examined individuals, like in i. ricinus. this result prompts us to hypothesize different roles of midichloria bacteria in different tick species. references bazzocchi, c., mariconti, m., sassera, d., rinaldi, l., martin, e., cringoli, g., urbanelli, s., genchi, c., bandi, c., epis, s., 2013. molecular and serological evidence for the circulation of the tick symbiont midichloria (rickettsiales: midichloriaceae) in different mammalian species. parasit vectors 6, 350–56. epis, s., sassera, d., beninati, t., lo, n., beati, l., piesman, j., rinaldi, l., mccoy, k.d., torina, a., sacchi, l., clementi, e., genchi, m., magnino, s., bandi, c., 2008. midichloria mitochondrii is widespread in hard ticks (ixodidae) and resides in the mitochondria of phylogenetically diverse species. parasitology 135, 485–94. mariconti, m., epis, s., gaibani, p., valle, c. dalla, sassera, d., tomao, p., fabbi, m., castelli, f., marone, p., sambri, v ., bazzocchi, c., bandi, c., 2012. humans parasitized by the hard tick ixodes ricinus are seropositive to midichloria mitochondrii: is midichloria a novel pathogen, or just a marker of tick bite ? pathogens and global health 106, 391–396. sassera, d., lo, n., bouman, e.a., epis, s., mortarino, m., bandi, c., 2008. “candidatus midichloria” endosymbionts bloom after the blood meal of the host, the hard tick ixodes ricinus. appl environ microbiol 74, 6138–40. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the current 30% of canine perinatal mortality (tonnessen et al., 2012) claims the need to deepen the knowledge about this phase, characterized by long-term hormonal and metabolic changes, that could benefit of long time-frame methods of investigation, reducing the quantity of samplings and thus respecting animal welfare (veronesi et al., 2015). indeed, apart from the wellknown invasiveness of blood samplings, the necessary repeated collection of feces, urine and saliva, providing punctual information, could be stressful for the newborns and their mothers with negative effects on health status and maternal behavior. given that sexual steroid hormones were reported to have an influence on health outcomes and development already from the perinatal period (frey et al., 2017), in this work 17-β-estradiol (e2) and testosterone (t) concentrations were assessed from the claws of dogs up to 60 days of age, to identify possible endogenous biomarkers. ten male and 10 female puppies, viable and healthy, born by elective cesarean section, were enrolled. samplings were performed by trimming claws at birth, and the regrowth at 30 and 60 days of age after the breeder or the owner signed an informed consent. then, e2 and t concentrations were analyzed by ria (veronesi et al., 2015) and a possible effect of gender evaluated by anova. all the hormonal claws concentrations showed a significant (p<0.001) drop from birth to 30 and 60 days of age, while no significant changes were observed between 30 and at 60 days of age (table 1). no influence of the newborns’ gender was found. because of the higher levels of e2 and t observed at birth and at 30 days of age, it could be supposed that a source of production could be the placental and maternal compartments. however, the direct involvement of the fetus itself could not be excluded, given the reported accumulation from the nail capillary bed of those hormones (de berker et al., 2007) and the production of sexual steroid hormones by feline fetal gonads (braun and jewgenow, 2017). keywords dog, puppy, claws, 17-ßestradiol, testosterone. corresponding author jasmine fusi jasmine.fusi@unimi.it journal home page riviste.unimi.it/index.php/haf 17-ß-estradiol and testosterone concentrations in claws of puppies up to 60 days of age. j. fusi1,*, t. peric2, c. bergamin3, m.c veronesi1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, milan, italy. 2 laboratory for environmental and life sciences, univerza v novi gorici, vipavska cesta, 5000, nova gorica, slovenia. 3 department of agrifood, environmental and animal sciences, university of udine, via sondrio, 2, udine, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 2 haf © 2013 vol. v, no. 1s issn: 2283-3927 references braun, b.c., jewgenow, k., 2017. expression of steroidogenic enzymes and steroid receptors in foetal gonads of domestic cat-sex similarities and differences. reproduction in domestic animals. 52, 130-136. de berker, d.a., andré, j., baran, r., 2007. nail biology and nail science. international journal of cosmetic science. 29, 241-75. frey, a.j., park, b.y., schriver, e.r., feldman, d.r., parry, s., croen, l.a., fallin, d.m., hertz-picciotto, i., newschaffer, c.j., snyder, n.w., 2017. differences in testosterone and its precursors by sex of the offspring in meconium. the journal of steroid biochemistry and molecular biology. 167, 78-85. tønnessen, r., borge, k.s., nødtvedt, a., indrebø, a., 2012. canine perinatal mortality: a cohort study of 224 breeds. theriogenology. 77, 1788-801. veronesi, m.c., comin, a., meloni, t., faustini, m., rota, a., prandi, a., 2015. coat and claws as new matrices for noninvasive long-term cortisol assessment in dogs from birth up to 30 days of age. theriogenology. 84, 791-6. table 1: claws concentrations of 17β-estradiol (e2), and testosterone (t) at birth, 30 and 60 days of age in the 20 puppies enrolled in the study. females (n=10) birth 30 days 60 days e2 (pg/mg) 9.2 ± 3.87a 3.3 ± 1.75b 1.9 ± 0.67b t (pg/mg) 11.2 ± 6.66a 4.3 ± 2.44b 2.3 ± 0.78b males (n=10) birth 30 days 60 days e2 (pg/mg) 8.3 ± 2.91a 2.4 ± 0.60b 1.7 ± 0.54b t (pg/mg) 8.7 ± 3.12a 3.7 ± 0.98b 1.9 ± 0.75b a, b within rows denotes a significant difference with p<0.001. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the indian subcontinent is incredibly rich in biodiversity and is home to many species of herpetological and conservation interest. the aim of this study was to provide a first assessment of hematological values in travancore tortoise (indotestudo travancorica) and red-crowned roofed turtle (batagur kachuga), two endangered species on which medical literature is still lacking. between may and june 2017, 19 healthy specimens of i. travancorica and 17 of b. kachuga were sampled. both populations were housed by the madras crocodile bank trust – centre for herpetology (tamil nadu, india). for each animal, physical examination and fecal analysis were performed. blood samples (0.5 ml) were obtained from the dorsal coccygeal vein, stored in a lithium-heparin test tube (bielli et al., 2015) at 10°c (50°f) and processed within three hours. regarding i. travancorica, from each sample was performed a complete red and white blood cell count (rbc and wbc) with the natt & herrik method using a neubauer chamber, and hematocrit values were assessed using microcapillaries (nardini et al., 2013). mean corpuscular volume (mcv) was calculated from pcv and rbc. regarding b. kachuga, complete rbc and wbc count with the same method was performed. due to the insufficient numerosity of both populations, only descriptive statistic was applied (friedrichs et al., 2012) (table 1). obtained values were compared with known references of species with similar ecological and biological characteristics with results consistent with those of geochelone elegans (klaphake et al., 2018) and mauremys sinensis (chung et al., 2009). this is the first study on hematological values of i. travancorica and b. kachuga. further studies will be necessary to assess actual reference values and to investigate other parameters such as wbc differential and erythrocytic indexes. keywords indotestudo travancorica, batagur kachuga, chelonian, hematology, reference values. corresponding author edoardo bardi edoardo.bardi@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary study on hematological values of two endangered turtle species: indotestudo travancorica and batagur kachuga. e. bardi1,*, e. lubian1,2, n. whitaker3, s. romussi1 1 department of veterinary medicine, university of milano, via celoria 10, 20133 milan, italy. 2 wildlife rescue centre crfs lipu “la fagiana”, via valle, 20013 magenta, italy. 3 madras crocodile bank trust, mahabalipuram, nadu 603104, india. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 6 haf © 2013 vol. v, no. 1s issn: 2283-3927 references bielli, m., nardini, g., di girolamo, n., savarino, p., 2015. hematological values for adult eastern hermann's tortoise (testudo hermanni boettgeri) in semi-natural conditions. journal of veterinary diagnostic investigation. 27, 68-73. chung, c., cheng, c., chin, s., lee, a., chi, c., 2009. morphologic and cytochemical characteristics of asian yellow pond turtle (ocadia sinensis) blood cells and their hematologic and plasma biochemical reference values. journal of zoo and wildlife medicine. 40(1), 76-85. friedrichs, k.r., hall, k.e., freeman, k.p., szladovits, b., walton, r.m., barnhart, k.f., blanco-chavez, j., 2012. asvcp reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. veterinary clinical pathology. 41(4), 441-453. klaphake, e., gibbons, p.m., sladky, k.k., carpenter, j.w., 2018. chapter 4 – reptiles, in exotic animal formulary, 5th edition (carpenter jw, editor), st. louis, mo, elsevier. nardini, g., leopoldi, s., bielli, m., 2013. clinical hematology in reptilian species. veterinary clinics of north america: exotic animal practice. 16, 1-30. table 1: median values (±sd=standard deviation) and range of hematological parameters in i. travancorica (19 specimens) and b. kachuga (17). rbc and wbc counts were obtained by the natt & herrik method using a neubauer chamber. pvc was assessed by centrifugation of microcapillaries. mcv was calculated from pcv and rbc. i. travancorica median (±sd) range rbc (106/µl) 0.597 (± 0.56) 0.16-1.97 wbc (103/µl) 5.38 (± 1.39) 3.91-7.82 pcv (%) 22 (± 8.73) 13.7-43.8 mcv (fl) 345.18 (± 176.59) 148.27-728.72 b. kachuga rbc (103/µl) 0.78 (± 0.34) 0.1-1.36 wbc (103/µl) 6.84 (± 5.03) 2.93-19.8 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author angela lombardi, angela.lombardi@unimi.it abstract two transgenic nicotiana tabacum plants, carrying respectively the f18 adhesive fimbriae and the b subunit of verocytotoxin genes from o138 verocytotoxic e.coli serotype were developed as a model of edible vaccine. tobacco plants were transformed by agroinfection according to rossi et al. (2013) stably. the f18 adhesive fimbriae and vt2e b-subunit were placed under control of the glob promoter for the seed-specific protein expression. agrobacterium tumefaciens binary vector system is an efficient tool to transform plant cells; however, the exogenous gene integrates at semi-random into the nuclear chromosome. pcr products, using specific oligonucleotides putatively encoding the b-subunit of vt2e-b and f18 fimbriae were identified on agarose gel (1.5% 0.9%) as bands with a length of 270 and 519 base pairs, respectively. we showed that the foreign vt2e-b and f18 fimbriae genes were stably integrated into the tobacco genome. northern blot and western blot analyses carried out respectively on total mrna and total soluble protein extract obtained from seeds. for each line, the obtained amount of antigens is sufficient for subsequent oral immunization trials. three lines of tobacco seeds (f18, vt2e-b, and wt) were seeded in homogeneous conditions and were harvested simultaneously. tobacco plants were analysed also by optical and electronic microscope in different phases of growth. germination of transgenic seeds were delayed of three/five days compared to wt in two replicated experiments, suggesting that genetic manipulation may influenced mechanisms leading to germination. in conclusion, the genes coding for vt2e-b and the f18 are stably maintained in the seeds and obtained tobacco seeds represent a valid strategy to ferry antigenic proteins to the gut and a promising noninvasive method of vaccination in pig industry. references rossi, l., di giancamillo, a., reggi, s., domeneghini, c., baldi, a., sala, v., dell’orto, v., coddens, a., cox, e., fogher, c. 2013 journal of veterinary science 14(3):263-270. rossi, l., fusi, e., baldi, g., fogher, c., cheli, f., baldi, a., dell’orto, v. 2013 journal of veterinary medicine, 3 73-78. rossi, l., pinotti, l., agazzi, a., dell’orto, v., baldi, a. 2014 italian journal of animal science 13 (1), pp. 23-29. rossi, l., dell’orto, v., vagni, s., sala, v., reggi, s., baldi, a. 2014 veterinary research communications. 38 (1), pp. 39-49. evaluation of nicotiana tabacum plants transformed for the expression of verocytotoxic escherichia coli antigens. a. lombardi 1 , l. rossi 1 , e. onelli 2 , a.moscatelli 2 , a. baldi 1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of biosciences, università degli studi di milano, via celoria 26, 20133 milan, italy proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords goat, snp, selection signatures corresponding author andrea talenti andrea.talenti@unimi.it journal home page riviste.unimi.it/index.php/haf detection of selection signatures for ear carriage in maltese goat breed andrea talenti1*, francesca bertolini2, stefano frattini1, giulio pagnacco1, max f. rothschild2, paola crepaldi1 and the italian goat consortium 1university of milan, department of veterinary medicine, italy 2iowa state university, department of animal science, usa abstract selection and breeding practices in goats have led to the fixation of several traits. this is probably due to the standardization of several peculiar morphological characteristics that have always been one of the major exclusion criteria of individuals from selection. among these, ear carriage is one of the most ancient and considered a signature of domestication in several species, such as the dog, pig, sheep and goat (boyko et al., 2010). the availability of improved genomic analyses tools for goats may provide useful information on genes involved in this trait. by studying, for example, the homozygosity decay of haplotypes (contiguous length of alleles) such information can be detected. in the current study, we focused on the maltese goat, a breed showing floppy ears, in comparison with other italian breeds using a goat medium density snp chip (nicoloso et al., 2015). a total 48,767 snp markers for 369 animals belonging to 16 breeds or populations were analyzed. genotypes were imputed within population excluding markers without known position on the current genome assembly (ars1, bickhart et al., 2017). population analysis using mds, admixture and faststructure confirmed the good differentiation among the populations. integrated haplotype score (ihs, sabeti et al., 2007) was performed for each population, comparing the regions detected on the maltese breed with the others considered to detect genes that may be involved into shaping ear morphology. these results may provide new insights into ear carriage phenotype by detecting genes that play a pivotal role in shaping the goat phenotypic diversity. acknowledgement the research was funded by innovagen project. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references bickhart, d.m.; rosen, b.d.; koren, s.; sayre, b.l.; hastie, a.r.; chan, s.; lee, j.; lam, e.t.; liachko, i.; sullivan, s.t.; burton, j.n.; huson, h.j.; nystrom, j.c.; kelley, c.m.; hutchison, j.l.; zhou, y.; sun, j.; crisà, a.; abel ponce de león, f.; schwartz, j.c.; hammond, j.a.; waldbieser, g.c.; schroeder, s.g.; liu, g.e.; dunham, m.j.; shendure, j.; sonstegard, t.s.; phillippy, a.m.; van tassell, c.p.; l smith, t.p., 2017. single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome. nature genetics, 49, 4–3. boyko, a.r.; quignon, p.; li, l.; schoenebeck, j.j.; degenhardt, j.d.; lohmueller, k.e.; zhao, k.; brisbin, a.; parker, h.g.; vonholdt, b.m.; cargill, m.; auton, a.; reynolds, a.; elkahloun, a.g.; castelhano, m.; mosher, d.s.; sutter, n.b.; johnson, g.s.; novembre, j.; hubisz, m.j.; siepel, a.; wayne, r.k.; bustamante, c.d.; ostrander, e.a., 2010. a simple genetic architecture underlies morphological variation in dogs. plos biology, 8, e1000451 nicoloso, l.; bomba, l.; colli, l.; negrini, r.; milanesi, m.; mazza, r.; sechi, t.; frattini, s.; talenti, a.; coizet, b.; chessa, s.; marletta, d.; d’andrea, m.; bordonaro, s.; ptak, g.; carta, a.; pagnacco, g.; valentini, a.; pilla, f.; ajmone-marsan, p.; crepaldi, p., 2015. genetic diversity of italian goat breeds assessed with a medium-density snp chip. genetics selection evolution, 47, 1–10. sabeti, p.c., varilly, p., fry, b., lohmueller, j., hostetter, e., cotsapas, c., xie, x., byrne, e.h., mccarroll, s.a., schaffner, s.f., lander, e.s., the international hapmap, 2007. genome-wide detection and characterization of positive selection in human populations. nature 449(7164), 913-918 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords food safety, food security, manual of good procedures for charitable organizations, food bank corresponding author marta castrica marta.castrica@unimi.it journal home page riviste.unimi.it/index.php/haf haccp (hazard analysis and critical control point) to guarantee safety in food recovery for social solidarity’s purposes. m. castricaa*, c. m. balzarettia, c. bernardia, a. baldia a department of health, animal science and food safety (vespa), università degli studi di milano, via celoria 10, 20133 milan, italy abstract fao estimates that each year 1.3 billion tons of food is wasted almost 1/3 of the total production intended for human consumption. the amount of food wasted each year in italy is around 5.1 million tons, of which only 500.000 tons recovered (9% of food surplus). during the last three years, in italy there was an important increase of the number of charitable organizations (ocs) active in recovering and distributing food for social solidarity’s purposes. the purpose of this study was to carry out hazards identification and potential risks analysis in the phases of recovery, collection, storage and distribution of food surplus with the aim to guarantee food safety among this supply chain. based on the results of the analysis, the main critical control points (ccps) were identified to guarantee the microbiological quality and safety of food recovery: (i) the best practices for managing the recovery of the different categories of food, (ii) the control of the correct storage temperatures and (iii) the volunteers required training on good hygiene procedures. the study of the ccps allowed defining the significant prerequisite programme (prp) that must be adopted by the ocs. all the prp have been collected in the manual of good procedures for charitable organizations validated by italian ministry of health in conformity to regulation (ec) n. 852/2004. thanks to the manual it has been also possible to write a simplified haccp system for the benefit of the centro di raccolta solidale per il diritto al cibo of lodi. references fao, 2013. global food losses and food waste. extent, causes and prevention. garrone p., melacini m., perego a., 2015. surplus food management against food waste. o’connor c., gheoldus m., jan o., 2014. comparative study on eu member states’ legislation and practices on food donation. parfitt, j., barthel, m., and macnaughton, s., 2016. food waste within food supply chains: quantification and potential for change to 2050. phil. trans. r. soc. b 365, 3065–3081. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:marta.castrica@unimi.it proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author francesco ferrucci francesco.ferrucci@unimi.it abstract ghrelin, the natural ligand for gh secretagogue receptors, influences the activity of both human and rat joint chondrocytes, modulating the synthesis of eicosanoids in the cartilage1. this project aims to assess the possible protective effects of ghrelin on equine cultured chondrocytes exposed to lipopolysaccharide (lps), as a model of osteoarthritic joint. in the preliminary step of our study, we have characterized equine chondrocyte primary cell cultures and standardized the detrimental effects lps on chondrocytes health. equine articular cartilage was collected with aseptic procedure and equine chondrocytes were isolated by overnight incubation. cells were cultured in monolayer culture to obtain third-passage (p3) chondrocytes and specific cartilaginous genes were identified by qualitative pcr. p3 chondrocytes were treated with increasing lps concentrations (1-100 ng/ml) and the effects of lps were assessed with the apoptosis-induction test and fluorescence microscopy after 1, 3, 6, 12, 24, 48 h of treatment. pcr evidenced type ii collagenases and aggrecan expression in isolated cells confirming the isolation of chondrocytes. healthy chondrocytes (hc) showed no fluorescence, necrotic chondrocytes (nc) red fluorescence, early apoptotic chondrocytes (eac) green fluorescence and advanced apoptotic chondrocytes (aac) mixed green-red fluorescence. in lps-stimulated chondrocytes, there was a trend throughout an increased percentage of aac that became equal to or overwhelmed the percentage of hc after 3 hours for all lps treatments and lasted for 6 hours after the highest lps concentration used. on the basis of these results, the concentration of lps 100 ng/ml for 6 h will be used for subsequent experiments. references caminos j.e., gualillo o., lago f. et al. 2005. endocrinology 146(3): 1285-92. effects of ghrelin on metabolic activity of equine cartilage affected with inflammatory degeneration: preliminary isolation, characterization of equine cultured chondrocytes and assessment of lpsinduced inflammatory stress. s. ceriotti 1 , a. lange consiglio 2 , l. casati 3 , f. cremonesi 2 , e. zucca 1 , v. sibilia 3 , f. ferrucci 1 1 department of health, animal science and food safety, school of veterinary medicine, università degli studi di milano ῀ milano 2 reproduction unit, large animal veterinary hospital (lodi), università degli studi di milano ῀ milano 3 department of medical biotechnology and translational medicine, università degli studi di milano ῀ milano proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords microbial competition, imaging mass spectrometry, specialized metabolites, food safety corresponding author isabella alloggio isabella.alloggio@unimi.it journal home page riviste.unimi.it/index.php/haf monitoring dynamics in bacterial competition by imaging mass spectrometry. i. alloggioa*, a. soggiua, c. pirasa, v. grecob, l. bonizzia, p. c. dorresteinc, p. roncadad adipartimento di medicina veterinaria, università degli studi di milano, italy; bfondazione santa lucia –irccs, rome, italy; cskaggs school of pharmacy and pharmaceutical sciences, university of california at san diego, la jolla, ca 92093, usa; distituto sperimentale italiano l. spallanzani, milano, italy abstract microbial competition is a mechanism that occurs when two or more microbial species compete for ecological niches to support their survival and growth (hibbing et al. 2010). different factors can contribute to the outcome of microbial competition, such as molecules exchanged between the competing organisms for the regulation of cell densities and the initial spatial configuration of the microbe–microbe interaction. specifically, production of compounds that kill or limit the growth of competing strains or species can promote niche monopolization (gonzalez et al. 2011). the released compounds include secondary metabolite antibiotics, bacterial peptides or low-molecular-mass organic compounds. in that sense, it is very important to develop tools that could capture metabolic interactions between two or more bacterial populations. imaging mass spectrometry (ims) enables the visualization of both spatial and temporal production of a huge number of metabolites from a single bacterial species and can observe the effects of multiple microbial signals in an interspecies interaction without using tags or labels (yang et al. 2009). this technique has the potential to be used for identification of novel metabolites and peptides that were previously undetected by other analytical methods. in this work, a combination of ims and lc-ms/ms was used to study the competition between listeria monocytogenes (lm) and lactococcus lactis (lac) to investigate the metabolic profile of each bacterium in the interacting microbial colonies. ims analysis revealed several interesting compounds during interaction of microbial colonies. at least six compounds are uniquely expressed during the interaction between lm and lac. these results could be useful to setup new molecular strategies in the control of bacterial species for a better food safety. references hibbing, m.e., et al., bacterial competition: surviving and thriving in the microbial jungle. nature reviews microbiology, 2010. 8(1): p. 15-25. gonzalez, d.j., et al., microbial competition between bacillus subtilis and staphylococcus aureus monitored by imaging mass spectrometry. microbiology, 2011. 157(9): p. 2485-2492. yang, y.-l., et al., translating metabolic exchange with imaging mass spectrometry. nature chemical biology, 2009. 5(12): p. 885-887. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy haf © 2013 vol. ii, no. 1s issn: 2283-3927 l corresponding author barbara bolis, barbara.bolis@unimi.it abstract during the last intrauterine fetal stage of development and the neonatal period, the hypothalamic-pituitary-adrenal axis (hpa) is a key system. apart of cortisol, the adrenals produce dehydroepiandrosterone (dhea), the major steroid produced by the fetus itself, so that it could be considered as a marker of offspring hpa activity. non invasive, long time-frame retrospective hormonal levels analysis were performed in the hair of humans and animals, but not in newborn puppies, and dhea never investigated in puppies. this study was aimed to assess dhea concentrations in the hair of newborn puppies, and to evaluate the influence of newborn age, gender, and breed size on dhea concentrations. the study enrolled 116 spontaneously dead puppies, grouped on the base of mother bodyweight to small or medium-large breeds, and on the base of age at death. hair samples were collected by shaving, and stored at room temperature until ria analysis. the overall hair dhea concentrations were 46.8±14.8 pg/mg. dhea levels were 48.6±15.66 pg/mg in females vs 45.1±13.73 pg/mg in males, without significant differences. dhea levels were 45.5±13.61 pg/mg in small size puppies and 47.8±15.61 pg/mg in medium-large size puppies, with no significant differences. dhea concentrations in premature puppies (52.5±15.12 pg/mg) were significant higher (p<0.05) than in puppies dead between 1 and 30 days after birth (44.5±17.78 pg/mg), but similar to fresh term borndead puppies (46.2±16.5 pg/mg). this study demonstrated that dhea is quantifiable in the hair of newborn dogs, and that dhea levels are significantly influenced by the puppy’s age. references beccaglia and luvoni, j small anim pract 2006;47:670-73; peric et al., proc 2nd dairycare conference 2015;cordoba,spain:68. 3) mongillo et al., res vet sci 2014;96:33-8. hair dehydroepiandrosterone (dhea) concentrations in newborn dogs b. bolis 1 *, t. peric 2 , m.c. veronesi 1 , m. faustini 3 , a. rota 1 *, m. montillo 2 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy; 2 department of food science, università degli studi di udine, via sondrio 2, 33100 udine, italy; 3 department of veterinary sciences and tecnologies for food safety, università degli studi di milano, via celoria 10, 20133 milan, italy; * ecar resident proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords nanoparticles, nanomedicine, mononuclear phagocyte system. corresponding author marcella de maglie marcella.demaglie@unimi.it journal home page riviste.unimi.it/index.php/haf role of the mononuclear phagocyte system in the uptake and accumulation of nanoparticles designed for biomedical applications. m. de magliea,b,*, silvia bianchessi b, c. recordatib, e. scanziania,b adepartment of veterinary medicine, università degli studi di milano, via celoria 10,20133, milan, italy. b mouse & animal pathology laboratory, fondazione filarete, v.le ortles 22/4, 20139, milan, italy. abstract the unique physical-chemical properties of nanoparticles (nps) make them suitable for many biomedical applications (drug delivery, imaging, cell tacking) (yildirimer et al, 2011). however, the design of nanoparticle-based platforms for nanomedicine is extremely challenging and must overcome a number of physiologically constraints, including the mononuclear phagocyte system (mps) (dawidczyk et al, 2014). the aim of this study was to assess the role of mps in the biodistribution and accumulation of metallic nps [iron-oxide nps and silver nps (agnps)] after single intravenous administration in mice. 24 hours after the treatment mice were sacrificed and underwent complete necropsy. liver, kidney, spleen and lung were collected, stained with hematoxylin and eosin (he), perls stain (for iron-oxide nps) or autometallography (for agnps), and evaluated under a light microscope. immunostaining for iba-1 (pan macrophage marker) was applied to localize the nps aggregates within mps cells. histologically, in all treated mice, intracytoplasmic brown granular material (consistent with iron or silver pigment, as confirmed by perls iron stain and autometallography) was found in stained sections of the liver (within kupffer cells), spleen (mps of marginal zone and red pulp) and lung (mps within alveolar septa). iron/silver deposits were mainly found in the cytoplasm of mps cells (iba1 immunostaining), indicating that most of injected particles were removed from blood circulation by phagocytic cells. these findings indicate that mps greatly influence nps biodistribution. for this reason, strategies able to increase bioavailability of nps by minimizing mps-mediated clearance are needed. moreover, the potential toxic effects due to the persistence of nps in mps should be further investigated. references yildirimer l, thanh nt, loizidou m, seifalian am. toxicology and clinical potential of nanoparticles. 2011. nano today. 6(6):585-607. dawidczyk cm, kim c, park jh, russell lm, lee kh, pomper mg, searson pc. state-of-the-art in design rules for drug delivery platforms: lessons learned from fda-approved nanomedicines. 2014. j control release. 187:133-44. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords goat, methylation, puberty corresponding author stefano frattini stefano.frattini@unimi.it journal home page riviste.unimi.it/index.php/haf the methylome of the hypothalamus of prepubertal and pubertal goats. s. frattinia, e. caprab, b. lazzarib, b. coizeta, d. groppettia, p. riccabonia, a. pecilea, s. arrighic, s. chessab, b. castiglionib, a. giordanoa, a. talentia, j. l. williamsd, s. d. mckaye, p. crepaldia, a. stellab, g. pagnaccoa adepartment of veterinary medicine, university of milan, milan, italy. binstitute of agricultural biology and biotechnology, national research council uos of lodi, lodi, italy. cdepartment of health, animal science and foodsafety, university of milan, milan, italy dschool of animal and veterinary sciences, university of adelaide, roseworthy, australia edepartment of animal and veterinary science, university of vermont, usa abstract puberty is the fulfillment of fertility, a process involving physiological and morphological development. it is well known that the increased hypothalamic secretion of the gonadotropin-releasing hormone (gnrh) is essential for the activation of this process, even if the elements coordinating the timing of puberty have not been fully identified1,2. recent studies provide proof that there is an epigenetic regulation of female puberty, and dna methylation, the most studied epigenetic modification, plays a major role in it3. we analyzed dna methylation patterns of 5 alpine goats at their prepubertal stage and 5 that reached puberty in order to highlight differences in their methylome. detection of methylated regions across the goat genome involved a methyl binding domain (mbd) enrichment followed by deep sequencing (hiseq2000 illumina). the software chipseeqer4 permitted the identification of peaks corresponding to hyper-methylated regions. we have observed a higher methylation level in prepubertal goats. the distribution of the methylation peaks across the genome and within cpg islands per chromosome per group of animals has been analyzed. furthermore, we have investigated differential methylation in genes associated with puberty. specifically, cbx7, coding for a core component of the polycomb group silencing complex, and gnrhr, the gene coding for gnrh receptor, showed a higher number of peaks into two intragenic fragments within prepubertal goats. these results, accompanied by transcriptome analysis, provide a foundation for elucidating the role of dna methylation in the complex mechanisms that drive puberty in goat species. the research was funded by genhome project. references lomniczi, a. et al.; 2013. epigenetic control of female puberty. nat. neurosci. 16, 281–289 mayer, c. et al.; 2010. timing and completion of puberty in female mice depend on estrogen receptor-signaling in kisspeptin neurons. proc. natl. acad. sci. 107, 22693–22698. lomniczi, a. et al.; 2015. epigenetic regulation of puberty via zinc finger protein-mediated transcriptional repression. nat. commun. 6, 10195. giannopoulou, e. g. & elemento, o.; 2011. an integrated chip-seq analysis platform with customizable workflows. bmc bioinformatics 12, 277. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords goat, roh, pakistan, italy, egypt corresponding author andrea talenti andrea.talenti@unimi.it journal home page riviste.unimi.it/index.php/haf initial genomic characterization of italian, egyptian and pakistani goat breeds. a. talentia*, f. bertolinib, s.frattinib, a. elbeltagyb,c, g. pagnaccoa, b. coizeta, a. m. aboul-nagac, j. m. reecyb, m. moaeen-ud-dind, m. f. rothschildb, p. crepaldia and the italian goat consortium. a department of veterinary medicine, university of milan, milan, italy. b department of animal science, iowa state university, ames, iowa 50011, usa. c animal production research institute, ministry of agriculture, cairo, egypt d laboratories of animal breeding & genetics, pmas-arid agriculture university, rawalpindi, pakistan abstract selection and breeding practices in goats have differed greatly among countries and populations. these processes, together with natural selection and regional drift, have shaped the phenotypic variability of goat breeds (kim et al., 2015). the availability of improved genomic analysis tools for this species may provide useful information on the history of selection, adaptation and differentiation of goats from different areas of the world, that can be evaluated by the study of gene frequencies and length of the runs of homozigosity (contiguous length of homozygous genotypes, roh; purfield et al., 2012). in current study, we examined using a goat medium density snp chip animals from three different countries: egypt (with lack of selection scheme), italy (with several standardized breeds; nicoloso et al., 2015) and pakistan (with several breeds showing peculiar phenotypes) to produce a genomic landscape of goats breeds in these countries. a total of 1,123 animals of 39 different populations, and 48,895 snp markers were analyzed. genotypes were imputed on a country-based approach, and markers without known position in the genome were excluded from the analysis. mds and admixture plots confirmed the good differentiation among populations from the three countries. runs of homozygosity (roh) were performed for each country and population allowed the detection of genomic regions with high homozygosity levels, common in at least two out of three sampling areas. these results provide new insights into goat genome structure within and among breeds and countries. the detection of conserved regions with different lengths may explain recent selection strategies or adaptation to different, extreme environmental conditions. the research was funded by innovagen project. support by iowa state university and the ensminger funds for ae and at as well as support by the fulbright foundation for ae are gratefully acknowledged. sampling from pakistan was funded by pak-usaid project. references kim, e-s, a r elbeltagy, a m aboul-naga, b rischkowsky, b sayre, j m mwacharo, and m f rothschild., 2015. multiple genomic signatures of selection in goats and sheep indigenous to a hot arid environment. heredity 116, 255–264. nicoloso, l., l. bomba, l. colli, r. negrini, m. milanesi, r. mazza, t. sechi, et al., 2015. genetic diversity of italian goat breeds assessed with a medium-density snp chip. genetics selection evolution 47:62. purfield, deirdre c, donagh p berry, sinead mcparland, and daniel g bradley., 2012. runs of homozygosity and population history in cattle. bmc genetics 13:70. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords acute phase proteins, animal welfare, chicken, gallus gallus corresponding author andreia t. marques andreia.tomas@unimi.it journal home page riviste.unimi.it/index.php/haf widespread extrahepatic expression of acutephase proteins in chicken (gallus gallus) tissues. a. t. marquesa,* , cristina lecchia, laura nordioa, chiara giudicea, guido grillia, helena ariño-bassolsa, fabrizio ceciliania auniversità degli studi di milano, department of veterinary medicine, via celoria 10, 20133 milano, italy. abstract acute phase proteins (app) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders, or stress. although app are produced mainly in liver, extrahepatic production has been described (marques et al., 2016; lecchi et al., 2012). the aim of this work was to study the extrahepatic gene expression of five app, namely α1acid glycoprotein (agp), serum amyloid a (saa), haptoglobin-like protein (pit54), c-rective protein (crp) and ovotransferrin (ovt) (o'reilly and eckersall, 2014) in different healthy chicken (gallus gallus) tissues by quantitative real time pcr (qpcr) and immunohistochemistry to detect the precise location of the proteins. app gene expression was higher in liver compared with other tissues. mrna coding for crp, ovt and saa was detected in all tissues involved in this study with a higher expression in gastrointestinal tract, respiratory system and lymphatic system. saa expression was particularly high in cecal tonsil, lung, spleen and meckel’s diverticulum, whereas ovt showed a high expression in lung, bursa of fabricius, pancreas, brain and adipose tissue. agp and pit54 was also detected in pericardial adipose tissue, spleen, kidney, lung, mucosa of proventriculus, mucosa of gizzard and pancreas but, oppositely to saa, their mrna was not detected in meckel’s diverticulum, cecal tonsil and bursa of fabricius. these results suggest that each tissue is able to express different amount of app even in healthy conditions and mount a local acute phase reaction. immunohistochemistry to detect the precise location for agp, ovt and saa using available antibodies is ongoing. references marques, a.t., lecchi c., grilli g., giudice c., nodari s.r., vinco l.j., ceciliani f., 2016. the effect of transport stress on turkey (meleagris gallopavo) liver acute phase proteins gene expression. res vet sci. feb 104:92-95. o'reilly, e.l., eckersall, p.d., 2014. acute phase proteins: a review of their function, behaviour and measurement in chickens. worlds poult. sci. j. 70, 27–44. lecchi, c., dilda, f., sartorelli, p., ceciliani, f., 2012. widespread expression of saa and hp rna in bovine tissues after evaluation of suitable reference genes. vet. immunol. immunopathol. 145 (1–2), 556–562. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 10 haf © 2013 vol. ii, no. 2s issn: 2283-3927 l introduction in nature, vitamin e is present under eight different forms, four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ). the α–tocopherol has the highest biological activity (dersjant & peisker, 2010). the natural form of α–tocopherol is composed of 100% rrr-α-tocopherol whereas the synthetic form (all-rac-αtocopherol) consists of a mixture of eight stereoisomers at equal amount. previous studies suggest that the bioefficacy of the different α-tocopherol forms should be reconsidered (vagni et al., 2011). at the cell level, α-tocopherol functions as an antioxidant (baldi, 2005). the main aim of this study was to investigate the in vitro relative bioefficacy of rrr-α-tocopherol (rrrα-t) versus all-rac-α-tocopherol (all-rac-α-t) to counteract the cytotoxic effect induced by hydrogen peroxide (h2o2). to this aim, bovine mammary epithelial cell line (bme-uv1) and madine darby canine kidney cell line (mdck) were used since they represent suitable and well characterized in vitro models in relation to their particular susceptibilities to oxidative damage. material and methods bme-uv1 and mdck cells were cultivated into 75 cm3 tissue culture flasks in complete medium. preliminary experiments were performed to determine h2o2 cytotoxicity and the lc50 were calculated in both cell lines. further a putative difference in the protective effect of the two forms of tocopherol against h2o2-induced stress was evaluated. cells were trypsinized and seeded into wells of 96 wells -cell culture plates (seeding density: bme-uv1 2 x 105 cells/ml; mdck 1 x105 cells/ml). cells were pre-incubated for 3 h with selected rrrα-t and all-racαt concentrations and then exposed to increasing h2o2 concentrations ranging from 250 to 750 µm in bme-uv1 cell line and from 125 to 175 µm in mdck cell line for the following 24h. the effects of rrrα-t and all-racα-t on both bme-uv1 and mdck cell lines in counteracting h2o2 toxicity were determined by the mtt test and the ldh test. at least three replicates at each incubation time were performed and all experiments were performed at least twice. results are expressed as mean and sd. the effect of various treatments were evaluated by one-way analysis of variance using the glm procedure of sas (sas institute inc, nc, usa). values significantly different from controls are indicated as p<0.05. results and discussion in bme-uv1, the proliferative effect induced by rrr-α-t form was higher at the lowest concentrations (1nm and 0.01um) than all-rac α-t (about 30%, which is consistent with the current bioactivity conversion factors). in mdck, no relevant effect on mitochondrial activity was observed (figure 1). lc50 values determined in bme-uv1 and mdck cells were 376µm and 140µm, respectively. keywords vitamin e, bioefficacy, in vitro models; corresponding author carlotta giromini, carlotta.giromini@unimi.it journal home page riviste.unimi.it/index.php/haf relative bioefficacy of rrr-α-tocopherol versus all-rac-α-tocopherol in in vitro models a. baldi 1 , c. giromini 1 , d. gottardo 1 , r. rebucci 1 , l. pinotti 1 , e. fusi 1 1 department of health, animal science and food safety, università degli studi di milano, italy article selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 11 haf © 2013 vol. ii, no. 2s issn: 2283-3927 pre-treatments with 100μm of rrr-α-t and 100μm all-rac-α-t were able to significantly (p<0.05) counteract the effect induced by 750μm of h2o2 in bme-uv1. in mdck the pre-treatment with 1nm of all-rac-α-t was able to significantly (p<0.05) reduce the effect of 125 and 150mm h2o2 (figure 2). in mdck cells, the pre-incubation with all-rac-α-t (1nm) determined a significant reduction of the membrane damage (ldh test), induced by 175 μm of h2o2. conclusions the dose-response curve experiments shown that rrr-α-t and all-rac-α-t tocopherols were able to maintain (mdck cells) and increase (bme-uv1 cells) the cell viability. it has been observed that adequate rrr-α-t and all-racα-t concentrations could reduce the oxidative damages induced by h2o2 in both bme-uv1 and mdck cells. differences detected in the two α-tocopherol forms, when present, were consistent with the conversion factors. however, in some cases all-rac was exhibited a higher antioxidant effect, compared with rrr-α-t. in conclusion, rrr-α-t and all-rac-α-t have shown the ability to counteract the oxidative effects of h2o2 in the cell lines considered. however, further investigation will help in describing their specific mechanism of action in vitro. references a. baldi, “vitamin e in dairy cows,” livestock production science, vol. 98, no. 1-2, 2005, pp. 117-122. doi:10.1016/j.livprodsci.2005.10.004 dersjant‐li, y., & peisker, m. (2010). a critical review of methodologies used in determination of relative bio‐availability ratio of rrr‐α‐tocopheryl acetate and all‐rac‐α‐tocopheryl acetate. journal of the science of food and agriculture, 90(10), 1571-1577 s. vagni et al., (2011). “vitamin e bioavailability: past and present insights”, food and nutrition science, 2, pp. 1088 -1096 acknowledgments this work was supported by dsm nutritional products ag. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords honeybee, hygienic behavior, microrna, gene expression, brain corresponding author francesca dell’orco francesca.dellorco@unimi.it journal home page riviste.unimi.it/index.php/haf candidate molecular markers of hygienic behavior in honeybees (apis mellifera): an expression study. f. dell’orcoa*, e. facchinia, g. ciliaa, r. rizzia, m. mortarinoa adipartimento di medicina veterinaria, università degli studi di milano, via celoria 10, milano abstract honeybees (apis mellifera) evolved social immunity mechanisms based on coordinated behavioral mechanisms for reducing the disease risk in the hive. among these, hygienic behavior (hb) leads to the uncapping and removal of dead, diseased or parasitized brood (cremer, 2007). the characterization of expression markers of hb could greatly assist in-field selection still incomplete. this work was characterized by a transversal approach: from in-field phenotypic characterization, to expression profiling of selected honeybee brain non-coding (micrornas) and coding (rnam) genes to assess differential expression of selected gene targets between selected honeybee families with high and low hb, respectively. after a preliminary work performed in 2015, during spring 2016 honeybee nurses were sampled from 28 hives simultaneously with the evaluation of hb score through freeze-killed brood assay performed with the cooperation of professional honeybee breeders (e. bonfanti, l. sesso). the nurses from 16 families (8 with the highest and 8 with the lowest hb scores, respectively) underwent expression analysis of the following target genes potentially linked to hb and/or learning processes: ame-mir-219, ame-mir-276, let-7a (behura, 2010) ame-mir-932 (cristino, 2014), and act5c, mblk-1 (takeuchi, 2001) and obp4 (chandrasekaran, 2011). the experimental workflow was as follows: 1) dissection and pooling of brains, 2) total rna extraction, quantification and retrotranscription, 3) rt-quantitative real time pcr. the differential expression analysis of the candidate markers, performed after selection of the normalizing gene through genorm and normfinder algorithms, highlighted a correlation of obp4 and act5c expression level with hb score. references cremer, s., armitage, s. a. o., schmid-hempel, p., 2007. social immunity. social immunity. current biology 17: r693–r702. behura, s. k., whitfield, c. w., 2010. correlated expression patterns of microrna genes with age-dependent behavioural changes in honeybee. insect molecular biology 19: 431-439. cristino, a. s., barchuk, a. r., freitas, f. c. p., narayanan, r. k., biergans, s. d., zhao, z., simoes, z. l. p., reinhard, j., claudianos, c., 2014. neuroligin-associated microrna-932 targets actin and regulates memory in the honeybee. nature comumunications 5:5529. takeuchi, h., kage†, e., sawata, m., kamikouchi, a., ohashi, k., ohara, m., fujiyuki, t., kunieda, t., sekimizu, t., natori, .s., kubo, t.,2001. blackwell science ltd identification of a novel gene , mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type kenyon cells of the honeybee brain. insect molecular biology. 10: 487-494. chandrasekaran s., amentb, s. a., eddyc, j. a., rodriguez-zasd s. l., schatzb, b. r., pricea, n. d., robinsonb, g. e., 2011. behavior-specific changes in transcriptional modules lead to distinct and predictable neurogenomic states.. proceedings of the national academy of sciences 108: 18020-18025. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l ad van vuuren ad van vuuren obtained his degree in veterinary medicine at utrecht university in 1974 and his phd in animal and environmental sciences at wageningen university in 1993. between 1975 and 2015, he was attached to wageningen ur livestock research as scientist in the digestive physiology of ruminants. his main topics of study were rumen health, nitrogen utilization & environmental protection, and nutrition management & welfare of dairy cattle around calving. ad van vuuren managed a large variety of contract research projects commissioned by governments, industries or the european union. he cosupervised various msc and phd students in dairy cow nutrition at wageningen and utrecht university and (co-)authored more than 70 peer-reviewed publications and numerous other international and national papers and documents. he also contributed to various international and national scientific commissions as well as to advisory and editorial boards. ad van vuuren received the adsa international diary production award in 2004 and is diplomate of the european college of veterinary and comparitive nutrition. ad van vuuren retired in june 2015. ad.vanvuuren@upcmail.nl confirmation bias: examples from dairy cow nutrition and their impact on evidence-based veterinary medicine ad van vuuren 1 1 archipel 1303, 8224 ga lelystad, the netherlands; main lecture in 1989 a boeing 737 crashed in the amazon jungle after running out of fuel 600 miles off course. the accident was due to human failures, being incorrect input of the required direction and misinterpreting external signals thereby (mis)confirming that the plane headed the right way and that the crew was infallible; a situation in psychology known as confirmation bias.confirmation bias is the tendency to search for, interpret, or recall information in a way that confirms one's beliefs or hypotheses. also in (veterinary) science external signals can be helpful to confirm one’s beliefs or hypothesis, but an open, critical attitude must be considered to prevent confirmation bias. therefore veterinary (and other) nutritionists should: -utilise facts and insights obtained in well-designed experiments to develop sound unbiased theories on the aetiology of nutrition-based animal diseases and to generate preferable interventions to prevent or treat such diseases. -reflect on the effect of dogmas in dairy cow nutrition, which may have consequences for preventing for example laminitis and metabolic disorders in early-lactation. this brings to evidence-based veterinary clinical nutrition whose development, advantages and disadvantages of evidence-based veterinary medicine (ebvm) have been reviewed recently (vandeweerd et al., 2012). similar to evidence-based medicine, ebvm distinguishes five sequential steps: (1) formulate answerable questions, (2) locate best evidence to answer the question, (3) assess internal validity of the obtained information, (4) integrate validated information with own clinical expertise and the unique situation of patient and owner and (5) assess effectiveness and efficiency of the therapy to improve future appraisals (schmidt, 2007, fajt et al., 2009, vandeweerd et al., 2012). in a contribution of roudebush et al. (2004) examples for applying ebvm in clinical nutrition have been presented, showing how this approach can improve patient outcomes. two examples will be analysed in detail: sub-acute ruminal acidosis and lipopolysaccharides as a cause of laminitis and postpartum low feed intake as a causes of metabolic disorders in early-lactation. we will see that to develop and update preventive and therapeutic interventions, a critical, unbiased approach is essential to deliver professional veterinary support to patients and owners coherent with the rapidly-evolving state of art. references fajt, v. r., d. brown, and m. m. scott. 2009. journal of veterinary medical education 36(2):186-195; roudebush, p., t. a. allen, c. e. dodd, and b. j. novotny. 2004. javmajournal of the american veterinary medical association 224(11):1766-1771; schmidt, p. l. 2007. veterinary clinics of north america-small animal practice 37(3):409-417; vandeweerd, j. m., n. kirschvink, p. clegg, s. vandenput, p. gustin, and c. saegerman. 2012. veterinary journal 191(1):28-34. mailto:ad.vanvuuren@upcmail.nl selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 3 haf © 2013 vol. ii, no. 2s issn: 2283-3927 l introduction plants have represent a promising alternative for biopharmaceutical proteins (ma et al., 2003; rossi et al., 2014). many plant based edible vaccines have been shown to be effective in inducing local immune responses (rossi et al., 2013). edible vaccines can activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. this dual effect would provide first-line defense against pathogens invading through the mucosa. the antigens are released in the intestines are taken up by m cells that are present over the payer’s patches (in the ileum) and the gut associated lymphoid tissue (galt). edible vaccines represent an important worldwide goal for the prevention of the enteric diseases, also in livestock. in particular, the enteric infections are a significant clinical problem in pigs. verocytotoxic escherichia (e.) coli strains are responsible for serious enterotoxaemia that causes important economic losses in the pig industry. the production of a vaccine for oral administration of transgenic seeds could be a practical and efficient system to prevent the infection and to reduce the antibiotic use. this study was focused on tobacco plants, previously transformed by agroinfection for the seed-specific expression of antigenic proteins (f18 adhesive fimbriae and the b subunit of the vt2e toxin) as model of edible vaccines against verocytotoxic e. coli strains. the dietary administration of transgenic tobacco seeds promotes a significant increase in the number of mucosal iga-producing cells of the tunica propria in both small and large intestine in mice (rossi et al., 2013). a protective effect of oral administration of transgenic tobacco seeds was also observed against verocytotoxic escherichia coli infection in piglets (rossi et al., 2014). the aim of this study was to assess the seedexpression stability, that is a important requirement in the vaccine production, of f 18 and vt2e-b heterologous genes into the progeny of transformed tobacco plants. material and methods production of transgenic seeds. seeds of control (not transformed nicotiana tabacum plants), f18 transformed plants (f18+) and vt2e-b transformed plants (vt2e-b+) were seeded in pots (3 seeds/pots) containing commercial soil and placed in fitotron® growth chambers with 200 mol photons m-2 s-2 illuminated, with a vapor growth lamp at 25 ± 2 c° and a photoperiod of 14h light and 10h night. the r3 generation was propagated in a greenhouse (80 plants for each line) protected by a polyethylene monofilament at the orto botanico "g.e. ghirardi" of the university of milan. during the growing period all the plants were regularly watered and fertilized in same conditions. evaluation of seeds seeds of r3 generation were collected from each plant and evaluated as described below. the plants were analyzed for the presence of foreign dna using pcr, (primers and conditions are described in table 1.) the total proteins were obtained from all mature transformed tobacco seeds by homogenization with liquid n 2 in a mortar with the solubilization buffer (50 mm tris, ph 8, 5 mm edta, 200 mm nacl, 0.1% tween 20). protein content was keywords edible vaccines; e.coli; tobacco seeds; pigs; enteric diseases corresponding author angela lombardi, angela.lombardi@unimi.it journal home page riviste.unimi.it/index.php/haf evaluation of antigens stability of tobacco seeds as edible vaccine against vtec strains l. rossi 1 , s. reggi 2 , m. zaninelli 3 , f. saccone 1 , d. gottardo 1 , a. crotti 1 , a. lombardi 1 , v. dell’orto 1 , r. rebucci 1 , a. baldi 1 1 department of health, animal science and food safety, università degli studi di milano, italy. 2 plantechno s.r.l. vicomoscano, cremona, italy. 3 faculty of agriculture, università telematica san raffaele roma, italy. article selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 4 haf © 2013 vol. ii, no. 2s issn: 2283-3927 1000 750 500 250 1 2 3 4 5 6 7 estimated by a bradford assay (biorad, hercules, usa) using bovine serum albumin as standard. the specific antibodies were obtained from rabbit polyclonal anti-vt2e-b serum and f-18 peptide polyclonal antibody (genscript, usa). the f18 and vt2eb proteins were purified and used as positive controls. the f18 fimbrae was purified as described by goddeeris et al. (2002) from o138 verocitotoxic e.coli strain. table 1. table of the oligonucleotides sequences for f18 and vt2e-b detected by pcr. gene oligonucleotide sequences pcr size (pb) pcr conditions f18 adhesive fimbriae 5’ ggatcc atgaaaagactagtgtttatttcttttg 3’ cgaatgcgccaatgaatgttcatt ctcgag 519 den. 95°c -1’; ann. 56°c -1’20”;ext. 72°c -1’30”; 35 cycles vt2e-b subunit 5’ ggatccatgaagaagatgtttatagcgg 3’ aacgggtccacttcaaatgattctcgag 270 den 95°c -1’; ann 50°c 1’ 20”; ext 72°c 1’30”; 35 cycles the vt2e-b protein were expressed by pet-system (novagen) in e.coli bl21 (de3) as described by rossi et al. (2014). a competitive indirect elisa was developed to quantify the vte2-b and f18 proteins in tobacco seeds. polyvinyl microtiter plates (thermo scientific) were coated overnight at 4°c with 100 µl of the respective purified protein (f18 and vte2b). the same protein purified, used for coating plates, was used to make a standard curve. plates were blocked with 100 µl/well of blocking buffer (2% bsa in pbs/tween 20) for 3 hours at 37°c. 100 µl of each standard solution and extracted samples were dispensed into the individual sterile tubes. 100 µl of diluted primary antibody (antif18 1:15.000; anti-vt2eb 1.10.000) were added to each tube and mix accurately (30 min of incubation at room temperature rt). the reaction was transferred (100 µl ) into the wells of coated plate. after a 30 min incubation at rt, the reaction was transferred into the plate. after four washing with washing buffer (pbst), 100 µl of peroxide-conjugated goat anti-rabbit (sigma) were added to the wells and incubated at 37° c for 30 minutes. the plates were then washed four times with pbst and wells 50 µl of tmb (sigma) were added in the wells. the reaction was stopped after 15 minutes by adding 150 µl of stopping buffer (0,4m hcl). optical density (od) was measured at 450 nm using a microplate reader (biorad). results and discussion the two lines of r3 generation of nicotiana tabacum transformed for e.coli antigenic proteins, the f18 adhesive fimbriae and the b subunit of verocytoxin, maintained the transgenes into plant genome. no cross-pollination was observed between the two transgenic lines as observed in figure 1. figure 1: electrophoresis of pcr products obtained using f18 oligonucleotides (samples 1, 2, 3) and vt2eb oligonucleotides (samples 5,6,7). lane 1: genomic dna from wild type nicotiana tabacum; lane 2: genomic dna from f18 positive nicotiana tabacum; lane 3: genomic dna from vt2e-b positive nicotiana tabacum plants. lane 4: marker (pb); lane 5: genomic dna from vt2e-b positive; lane 6: positive control; lane 7: genomic dna from wild the total soluble proteins extracted from 100 mg of seeds obtained from f18 positive plants were about 1,8 ug/ul, were estimated using bradford assay. f18 proteins was estimated about 66-74 ng/100 mg of tobacco seeds, by the developed elisa system. the total soluble proteins extracted from 100 mg of seeds obtained from vt2e-b positive plants were about 2.2 μg/ μl, evaluated with bradford assay. vte2-b proteins was estimated about 340370/100 mg of tobacco seeds, using elisa assay. the different amount of antigenic proteins in the two transgenic lines is due the agrobacterium-mediated transformation. in fact, agrobacterium tumefaciens binary vector system is an efficient tool to transform plant cells; however, the exogenous gene integrates at semi-random into the nuclear chromosomes. conclusions obtained data demonstrated that the genes coding for vt2e-b and the f18, represent important antigens and virulence factors of e. coli, therefore can be stably incorporated into the next generation of nicotiana tabacum genome. the foreign vt2e-b and f18 adhesive fimbriae genes , derived from verocytotoxic escherichia coli strain, selected papers from “l’innovazione scientifica per la nutrizione”, 15th october, 2015, lodi (mi), italy 5 haf © 2013 vol. ii, no. 2s issn: 2283-3927 stably incorporated into tobacco genome under control of seed-specific promoter, were well transcribed through the nuclear apparatus of the plant for specific expression in seed references ma k-c.; pascal m.w.; drake & paul christou. (2003) the production of recombinant pharmaceutical proteins in plants. nature reviews genetics 4, 794-805 2003 rossi l.; di giancamillo a.; reggi s.; domeneghini c.; baldi a.; sala v.; dell’orto v.; coddens a.; cox e.; fogher c. (2012). expression of porcine verocytotoxic escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model” jvs, 2013,14(3):263-270. rossi l.; fusi e.; baldi g.; fogher c.; cheli f.; baldi a.; dell’orto v. (2012). tobacco seeds by-product as protein source for piglets. ojvm, 2013, 3:7378. rossi l. pinotti l.; agazzi a.; dell’orto v.; baldi a. (2014). plant bioreactors for the antigenic hook-associated flgk protein expression. italian journal of animal science. ital j an sci, 13 (1):23-29. rossi l.; dell’orto v.; sala v.; reggi s.; baldi a. (2014). protective effect of oral administration of transgenic tobacco seeds against verocytotoxic escherichia coli strain in piglets. vet res comm, 38 (1): 39-49. l keywords birds, filaria heartworm; sarconema eurycerca; swans; europe; italy pages 20 – 27 references vol. 3 no. 1 (2016) article history submitted: february 12, 2016 revised: august 26, 2016 accepted: august 30 2016 published: september 05 2016 corresponding author claudia manno, istituto zooprofilattico sperimentale della sicilia, via g. marinuzzi 3, 90129 palermo, italy. mail: guidoruggero.loria@gmail.com phone: +39 091 6565111 fax: +39 091 6563568 journal home page riviste.unimi.it/index.php/haf article cardiac filariosis in migratory mute swans (cygnus olor) in sicily claudia manno 1,* , damer blake 2 , gabriele ghisleni 3 , marco tecilla 3 , giusi macaluso 1 , roberto puleio 1 , vincenzo monteverde 1 , guido r. loria 1 1 istituto zooprofilattico sperimentale della sicilia, via g. marinuzzi 3 90129 palermo, italy 2 royal veterinary college, hawkshead, north mymms, hertfordshire, al9 7ta, uk 3 sezione di anatomia patologica veterinaria e patologia aviare, dipartimento di patologia animale, igiene e sanità pubblica veterinaria università degli studi di milano, via celoria 10 20133 milano, italy abstract sarconema eurycerca is a common parasitic disease of north america swans and geese. the infection has been correlated with severe heart lesions, often resulting in cardiac failure and death of the animals. heartworms infections have been previously reported in european swans, and specifically in the united kingdom and nederland. both the countries are characterized by a cold temperate weather, similar to the one that can be found in swan wintering areas of u.s.a. and canada. the first record of cardiac filariasis associated with sarconema eurycerca infection in four swans in italy. twelve mute swans were examined during avian influenza surveillance activities on migratory birds. birds were collected in the year 2006, in wintering areas of eastern sicily (italy). four of the twelve swans showed necrotic-haemorrhagic myocarditis with intralesional nematodes. morphological characteristics identified the parasite as a filarial nematode. birds lungs samples were used for parasites dna extraction. the latter was used as template for polymerase chain reaction (pcr) amplification and sequencing of part of the 12s rdna gene. comparison of genomic dna extracted from a reference s. eurycerca isolate confirmed parasite identity and provided the first sequence resources for this species of value to future diagnostic and epidemiological studies c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 21 haf © 2013 vol. iii, no. 1 issn: 2283-3927 1 introduction heartworm disease in swans and geese is caused by sarconema eurycerca, a filarial nematode (spirurida, filarioidea, onchocercidae, lemdaninae) transmitted between birds by the biting louse trinoton anserinum (cohen et al., 1991). studies with s. eurycerca have shown it to be pathogenic to some geese and swan species including whistling swans (cygnus columbianus), trumpeter swans (cygnus buccinators), canada geese (branta canadensis) and white-fronted geese (anser albifrons) (holden and sladen, 1968; kluge, 1967; macneil, 1975). in these species massive infestation with s. eurycerca have been related to severe heart lesions resulting in cardiac failure and death (kluge, 1967; cole, 2013). mild infestation may not be pathogenic (holden and sladen, 1968) and the parasite may infect and develop within the bird with no clinical evidence (cole, 2013). disease caused by s. eurycerca is common in north america (u.s.a. and canada). to the authors knowledge the available literature pointed out the presence of s. eurycerca only in nederland and united kingdom, among all the europeans countries (cohen et al., 1991; de bruijn et al., 2009). s. eurycerca has an indirect life cycle that includes three larval stages. the larvae develop in an intermediate host (a louse) prior to infecting its definitive host (birds), where they become adult and reproduce. adult female heartworms release microfilariae into the blood stream of the definitive host bird. the microfilariae subsequently infect the biting louse t. anserinum during feeding upon the bird. a new host bird becomes infected when the louse bites it to feed on its blood and infective larvae move into the bird’s bloodstream. the larvae commonly migrate to the myocardium, develop to sexual maturity and release microfilariae into the blood stream (cole, 2013). the incidence of s. eurycerca in whistling swans in the western lakes area of the united states reported by quortup and holt (quortup & holt, 1940) was 18.5%, similar to the incidence of 16.2% reported in a field survey carried out by holden and sladen (holden and sladen, 1968). cole (cole, 2013) observed variable prevalence (4-20%) based upon microscopic examination of blood smears taken from apparently healthy birds. the occurrence of sarconema sp. in canadian swans is rarely reported (macneil, 1975). to our knowledge few data are available on pathology related to sarconema sp. infestation in european swans (de bruijn et al., 2009; oğuz et al., 2015) and currently no sequence data has been made publically available, hindering molecular identification and diagnosis. 2 material and methods case history. in wintering areas of migratory birds of eastern sicily (italy), in early february 2006 during surveillance activities against avian influenza (ai; as established by italian regulations, ministry of health n. 40129 dated november 11th 2005), twelve clinically suspected swans were trapped and humanely euthanized. the swans belonged a group of 19 birds where previously n. 8 were found to be positive for the pathogenic h5n1 strain. gross findings and histopathology. all swans were examined by a full necropsy. different tissues samples (lungs, heart, kidney, intestine, spleen, liver and brain) were collected from each bird for detection of ai virus and other differential laboratory investigations including histopathology. c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 22 haf © 2013 vol. iii, no. 1 issn: 2283-3927 four µm thick sections were obtained by formalin-fixed paraffin-embedded. the preparations obtained were dried overnight in an oven at 37 °c. it was proceeded with dewaxing by xylene for 20 min. after a descending alcohol series (100°, 95°, 75° and 50°), slides were washed in distilled water and then stained with haematoxylin and eosin. this was followed by the ascending scale of alcohols (50°, 75°, 95° and 100°) and clarification in xylene. after this phase, the slides were mounted in acrylic mounting medium (eukitt®, o. kindler gmbh). detection of ai virus. samples of the same organs collected for histology were used for quantitative real time polymerase chain reactions (qrt-pcr) for the gene m of the avian influenza virus as described elsewhere (spackman et al., 2002). 2.1 identification of parasites. histology. histological identification of parasites found in the lesions was performed according to published data (gardiner & poynton, 2006). polymerase chain reaction (pcr) and sequencing. dna samples were extracted from formalin fixed, paraffin embedded tissues from each infected animal. four 10 μm sections for each specimen were pre-treated according to the dneasy blood & tissue kit instructions as recommended by the manufacturer (qiagen gmbh; hilden germany) for formalin-fixed tissues. the dna extraction followed the manufacturer’s instructions for total dna purification from animal tissues. 12s rdna amplifications and sequences were obtained following casiraghi et al., (2004). in particular, dna sequences were amplified using the forward primer 12sf (5’-gttccagaataatcggcta-3’) and the degenerate reverse primer 12sr (5’-attgacggatg(ag)tttgtacc-3’). pcrs were performed in 20 µl volumes under the following conditions: 1x buffer, 1.5 mm mgcl2 (master taq kit, eppendorf™, hamburg, germany), 0.2 mm of each dntp, 1 µm of each primer, and 1 u of polymerase (master taq kit, eppendorf™, hamburg, germany). the thermal profile was the following: 94°c 45 sec, 50°c 45 sec, and 72°c 90 sec for 40 cycles. pcr products were gel purified (using the perfect prep gel clean-up, eppendorf™, hamburg, germany) and directly sequenced using abi technology. the four 12s rdna sequences were aligned with clustalx version 1.81 (thompson et al., 1997), manually curated and visualized with bioedit (hall, 1999). a blastn analysis was performed using the national center for biotechnology information (ncbi) web server (http://www.ncbi.nlm.nih.gov/blast) using default parameters and ‘genomes: other’, to identify similar annotated sequences. in parallel, genomic dna was extracted from a reference s. eurycerca sample collected previously from a swan during postmortem in january 1981 in berkshire, uk, and identified by the commonwealth institute of helminthology, st albans, uk, under the reference 3608 to provide a valid comparison. 2.2 phylogenetic comparison sequences found by blastn to be most similar to the 12s rdna sequences generated here were downloaded from genbank and used for phylogenetic comparison with dirofilaria immitis, a filarial heartworm, included as an out group. the optimal models for phyml and c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 23 haf © 2013 vol. iii, no. 1 issn: 2283-3927 mrbayes phylogenetic analyses were selected using topali v2.5 (milne et al., 2009), identifying tvm plus gamma (g) and gtr + g models respectively based on the akaike bayesian information criteria (aic and bic). a phyml phylogeny was constructed using 1,000 bootstrap replications. a bayesian phylogeny was constructed using 10,000,000 generations (10% discarded as burn-in) and sampled every 500. two replicate analyses were performed to ensure convergence and the results were then pooled. mega v5.1 (tamura et al., 2011) was used to construct a neighbor joining phylogeny with the kimura-2 parameter and 1,000 bootstrap replicates. 3 results gross findings. at necropsy, all swans found to be were in good body condition with thick subcutaneous and cavitary fat, and no external lesions. in four swans multiple, scattered, small (1-3 mm) yellowish-tan, myocardial (necrotic-haemorrhagic) foci were detected. quantitative real-time pcr for ai virus. all birds were negative for ai virus. histopathology. scattered heterophilic and macrophagic aggregates were observed in association with adult nematodes in the cardiac sections. adult nematodes observed in cardiac lesions were approximately 600-800µm in diameter, with a 2-3µm cuticle, pseudocoelom and coelomyarian muscular layer (figure 1). several microfilariae were detected in the distended uterus of adult female nematodes (figure 1). there were a few mineralization foci adjacent to the parasites (figure 1, inset). curvilinear nematode parasites (about 100µm in length) with discernable nuclear columns consistent with microfilarial forms were detected in the myocardium parenchyma and in the cardiac blood vessels (figure 2). interstitial fibrosis and degenerative changes were also observed associated with multiple haemorrhagic foci with deposits of hemosiderin (figure 2). scattered necrotizing vasculitis (fibrinoid necrosis) with infiltration of heterophils and macrophages were observed (figure 3). no remarkable changes were detected in the other organs. figure 1. sarconema eurycerca in the myocardium of a swan. note the latheral chords (c), the coelomyarian musculature (m) and the uteri (u) contain numerous microfilariae. haematoxylin and eosin, bar = 35µm. inset: the adult nematode is surrounded by mild inflammatory reaction, fibrosis (arrow) and mineral deposits (arrowheads). haematoxylin and eosin, bar = 35 µm. c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 24 haf © 2013 vol. iii, no. 1 issn: 2283-3927 figure 2. microfilariae in the myocardium (arrow), degenerative changes of myocardial cells and focal hemorrhages contained siderophages (arrowheads). haematoxylin and eosin, bar = 50 µm. figure 3. necrotizing vasculitis (fibrinoid necrosis). haematoxylin and eosin, bar = 50 µm. 3.1 parasite identification. histology. morphologic characteristics of the parasites observed in cardiac lesions included: 1) presence of microfilariae within the adult females; 2) coelomyarian musculature; 3) lateral chords; 4) small intestine; 5) evenly spaced, external longitudinal cuticular ridges. these morphological characteristics identified the parasite as a filarial nematode (gardiner and poynton, 2006), with features similar to those described for sarconema sp., possibly s. eurycerca (kluge, 1967). c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 25 haf © 2013 vol. iii, no. 1 issn: 2283-3927 pcr and sequencing. sequencing the 12s rdna amplified from genomic dna purified from paraffin embedded tissues of four specimens (cygnus olor) yielded four identical 483 bp sequences (100% identity, 100% similarity, blastn pairwise alignment). the sequence has been submitted to the embl data library according to the ebi procedure under the accession number am932375. comparison with the equivalent 483 bp amplicon derived from a reference s. eurycerca sample revealed 100% sequence coverage with 481/483 identical nucleotide matches (accession number ln812979). the next closest matches were the 12s small subunit ribosomal rna coding sequences from ascaridia galli (jx624728) and ascaridia columbae (jx624729), with 86%/84% similarity and 100%/85% query coverage respectively. the absence of single nucleotide polymorphisms (snps) and insertion/deletions (indels) between the four 12s rdna sequences indicate that no error was introduced by pcr or sequencing, and confirms that all the nematodes examined were the same species. blast comparison with the reference isolate sequence provided molecular confirmation of the histological identification as s. eurycerca. it is noteworthy that our submissions represent the first entries for the subfamily lemdaninae. following blastn comparison eight additional nematode 12s rdna sequences, including d. immitis as an outgroup, were downloaded and used for phylogenetic comparison with maximum likelihood (phyml), bayesian (mrbayes) and neighbor joining (nj) approaches (figure 4). the reference and candidate s. eurycerca were most closely related, supporting our sample identification. figure 4. phylogenetic comparison of 12s rdna coding sequences representing s. eurycerca with anisakis_physeteris, ascaridia columbae, ascaridia galli, contracaecum rudolphii, parascaris univalens, steinernema intermedium, toxascaris leonina and dirofilaria immitis as an out group (accession numbers as shown). branch values in excess of 70/0.7 are shown for maximum likelihood, bayesian and neighbor joining models. c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 26 haf © 2013 vol. iii, no. 1 issn: 2283-3927 4 discussion swans filariosis caused by s. eurycerca has been documented in north america, europe and asia. (holden and sladen, 1968; kluge, 1967; macneil, 1975; oğuz et al., 2015; woo et al., 2010) in europe, only few cases have been reported in nederland (de bruijn et al., 2009), turkey (oğuz et al., 2015) and united kingdom. (cohen et al., 1991) here, the presence of cardiac lesions associated with s. eurycerca confirmed the pathogenicity of filariosis in swans. the finding of necrotizing vasculitis suggests an immune complex-mediated (type iii) hypersensitivity pathogenic mechanism. furthermore, this is the first report of s. eurycerca in italy. the first reports of these parasites are recorded in boreal to warm-temperate areas such as north america and japan (cohen et al., 1991; macneil, 1975; woo et al., 2010). more recently the parasite has also been identified in warm temperate to sub-topical areas like turkey. (oğuz et al., 2015) the spreading of the parasite is suggesting that global climate changes may have played a role in the diffusion of the parasites and/or the intermediate host. nevertheless, the diffusion of these parasites can represent both a potential risk for domestic species and a risk for the european swan and geese population health. in domestic fowls preventions strategy should be practiced to avoiding the infestation. the use of proper restraining systems preventing direct contact between farmed and wildlife animals, and periodical treatment with insecticides should reduce the risk of diffusion of the intermediate host. to our knowledge, there are no data in the literature about the prevalence of sarconema sp. in italian swans. in this record cardiac filariosis was detected in 4 (33%) swans out 12. nevertheless, a larger population of swans should be examined, possibly from other italian or european wintering regions, to determine the prevalence of filariosis in european swans. in italy the warning and the emotional impact due to avian influenza risk and the outbreaks registered in wild species caused by virus h5n1-hpai (high pathogenicity strain avian influenza), has stressed the importance of knowledge on ecology and pathology of migratory species and particularly of mute swan (cygnus olor). this specie showed a primary role in avian influenza epidemic in europe (terregino et al., 2006). this experience underlined the importance of migrating species and their relevance in diffusion of high risk zoonosis and moreover, it has showed interesting data on less known parasitic diseases of birds which can potentially represent a potential risk also for domestic species. building on this we have established the first molecular sequence marker for s. eurycerca, providing a resource for the future identification of this parasite and discrimination from other closely related parasites. acknowledgement the authors would like to thank olivier sparagano for his intellectual guidance. c. manno et al. int. j. of health, animal science and food safety 3 (2016) 20 27 27 haf © 2013 vol. iii, no. 1 issn: 2283-3927 references de bruijn, n.d., velkers, f.c., and gröne, a. (2009). heartworm in a mute swan (cygnus olor). tijdschr. diergeneeskd. 134, 882–884. cohen, s., greenwood, m.t., and fowler, j.a. (1991). the louse trinoton anserinum (amblycera: phthiraptera), an intermediate host of sarconema eurycerca (filarioidea: nematoda), a heartworm of swans. med. vet. entomol. 5, 101–110. cole, r.a. (2013). chapter 31: heartworm of swans and geese. in field manual of wildlife disease — general field procedures and diseases of birds, (washington, d.c: u.s. dept. of the interior, u.s. geological survey), pp. 233–234. gardiner, c.h., and poynton, s.l. (2006). an atlas of metazoan parasites in tissue section (washington, dc: armed forces institute of pathology). hall, t. (1999). bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucleic acids symp. ser. 95–98. holden, b.l., and sladen, w.j.l. (1968). heart worm, sarconema eurycerca, infection in whistling swans, cygnus columbianus, in chesapeake bay. j. wildl. dis. 4, 126–128. kluge, j.p. (1967). avian parasitic (sarconema eurycerca) pancarditis. j. wildl. dis. 3, 114– 117. macneil, a.c. (1975). heartworm, sarconema sp. infection in a whistling swan, olor columbianus. can. vet. j. rev. vét. can. 16, 82–83. milne, i., lindner, d., bayer, m., husmeier, d., mcguire, g., marshall, d.f., and wright, f. (2009). topali v2: a rich graphical interface for evolutionary analyses of multiple alignments on hpc clusters and multi-core desktops. bioinforma. oxf. engl. 25, 126–127. oğuz, b., kilinç, ö.o., and değer, m.s. (2015). first reports of sarconema eurycerca and trinoton anserinum in the whooper swan (cygnus cygnus) in van, turkey. j. fac. vet. med. kafkas univ. 933–936. quortup, e., and holt, a. (1940). filariasis in wild swans. j. am. vet. med. assoc. 96, 543–544. tamura, k., peterson, d., peterson, n., stecher, g., nei, m., and kumar, s. (2011). mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol. 28, 2731–2739. terregino, c., milani, a., capua, i., marino, a.m.f., and cavaliere, n. (2006). highly pathogenic avian influenza h5n1 subtype in mute swans in italy. vet. rec. 158, 491. thompson, j.d., gibson, t.j., plewniak, f., jeanmougin, f., and higgins, d.g. (1997). the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids res. 25, 4876–4882. woo, g.-h., jean, y.-h., bak, e.-j., kang, s., roh, i.-s., lee, k.-h., hwang, e.-k., and lee, o.-s. (2010). myocarditis by nematodes infection, presumably sarconema eurycerca, in a wild whooper swan (cygnus cygnus) in korea. j. vet. med. sci. jpn. soc. vet. sci. 72, 1233–1235. 41 l keywords bacillus anthracis; anthrax; epidemic and papua new guinea. pages 41 – 48 references vol. 2 no. 1 (2015) article history submitted: april 12, 2015 revised: june 11, 2015 accepted: june 17, 2015 published: july 16, 2015 corresponding author johnson makaen emerging and environmental disease, papua new guinea institute of medical research, goroka, ehp, papua new guinea p o box 60 goroka, ehp 441 papua new guinea e-mail: johnson.makaen@pngimr.org.pg journal home page riviste.unimi.it/index.php/haf anthrax, fairly undetected in papua new guinea. johnson makaen 1* and lydia tasi 1 1 emerging and environmental disease, papua new guinea institute of medical research, goroka, ehp, papua new guinea. abstract. anthrax is caused by the organism bacillus anthracis. the organism is globally occurring and epidemics are reported the world over. it is an important infectious disease of domestic animals. b. anthracis can survive harsh conditions that would otherwise be drastic for other microorganisms. the inherently robust nature of the organism enables its extended survival and facilitates re-emergence. to date, no outbreak has been reported in the pacific island region except australia and new zealand where outbreaks are reported in both wildlife and livestock. papua new guinea has had sporadic (reported) instances of anthrax outbreak, but has not been scientifically established. it is still unclear if the anthrax causing organism is present in the environment or wildlife. it remains to be so until scientific evidence becomes available. this article aims to review any form of documented evidence of anthrax in the countr. makaen and tasi int. j. of health, animal science and food safety 2 (2015) 41 48 42 haf © 2013 vol. ii, no. 1 issn: 2283-3927 1 introduction anthrax is caused by the bacteria, b. anthracis. a gram-stained morphology of b. anthracis colony appears as gram positive rods, often linked at lengthwise to form a chain (murray et al. 1990; brooks et al. 1991; spencer 2003; schaechter et al. 2006; ma hornitzky and muller 2010; todar 2012). it is ubiquitous; survives in both soil and vegetation, and can resist adverse conditions (turnbull et al. 1998; murray et al. 1990; spencer 2003; schaechter et al. 2006; veterinary services 2012). anthrax is neither contagious nor invasive; however, it causes acute infection in susceptible animals (fasanella and garofolo 2008; beyer and turnbull 2009; stoltenow et al. 2010). the organism is vegetative in host tissues but sporulates when exposed to the environment (woodbury et al. 2002; spencer 2003; schaechter et al. 2006; stoltenow et al. 2010). sporulation shields the organism from unfavourable conditions and enables it to survive for years until it gets re-inoculated in another living host thereby continuing its vicious cycle of infection (massachusetts department of public health 2006; schaechter et al. 2006; fasanella and garofolo 2008; todar 2012) . in contrast to other zoonoses, anthrax affects a wide range of animals including human (murray et al. 1990; brooks et al. 1991; spencer 2003; beyer and turnbull 2009; stoltenow et al. 2010). it is believed that animals acquire the infection through ingestion of tainted feed or while foraging on contaminated field (tweet 2010; veterinary services 2012). nevertheless, human unintentionally contract it, usually through the skin while handling infected animals or their products (turnbull et al. 1998; ron parker et al. 2002; spencer 2003; edwards et al. 2005; beyer and turnbull 2009; todar 2012). inhalation of the contaminated aerosol or ingestion of partly cooked meat has also been observed (edwards et al. 2005; fasanella and garofolo 2008; stoltenow et al. 2010; todar 2012; hajramurni 2013). 2 epidemiology anthrax is caused by the bacteria, b. anthracis. a gram-stained morphology of b. anthracis colony appears as gram positive rods, often linked at lengthwise to form a chain (murray et al. 1990; brooks et al. 1991; spencer 2003; schaechter et al. 2006; hornitzky and muller 2010; todar 2012). it is ubiquitous; survives in both soil and vegetation, and can resist adverse conditions (turnbull et al. 1998; murray et al. 1990; spencer 2003; schaechter et al. 2006; veterinary services 2012). anthrax is neither contagious nor invasive; however, it causes acute infection in susceptible animals (fasanella and garofolo 2008; beyer and turnbull 2009; stoltenow et al. 2010). the organism is vegetative in host tissues but sporulates when exposed to the environment (parker et al. 2002; spencer 2003; schaechter et al. 2006; stoltenow et al. 2010). sporulation shields the organism from unfavourable conditions and enables it to survive for years until it gets re-inoculated in another living host thereby continuing its vicious cycle of infection (massachusetts department of public health 2006; schaechter et al. 2006; fasanella and garofolo 2008; todar 2012) . in contrast to other zoonoses, anthrax affects a wide range of animals including human (murray et al. 1990; brooks et al. 1991; spencer 2003; beyer and turnbull. 2009; stoltenow et al. 2010). it is believed that animals acquire the infection through ingestion of tainted feed or while foraging on contaminated field (tweet 2010; veterinary services 2012). nevertheless, human unintentionally contract it, usually through the skin while makaen and tasi int. j. of health, animal science and food safety 2 (2015) 41 48 43 haf © 2013 vol. ii, no. 1 issn: 2283-3927 handling infected animals or their products (turnbull et al. 1998 ; parker et al. 2002; spencer 2003; edwards et al. 2005; beyer and turnbull. 2009; todar 2012). inhalation of the contaminated aerosol or ingestion of partly cooked meat has also been observed (edwards et al. 2005; fasanella and garofolo 2008; stoltenow et al. 2010; todar 2012; hajramurni 2013). 3 acquisition anthrax is caused by the bacteria, b. anthracis. a gram-stained morphology of b. anthracis colony appears as gram positive rods, often linked at lengthwise to form a chain (murray et al. 1990; brooks et al. 1991; spencer 2003; schaechter et al. 2006; hornitzky and muller 2010; todar 2012). it is ubiquitous; survives in both soil and vegetation, and can resist adverse conditions (turnbull et al. 1998; murray, drew et al. 1990; spencer 2003; schaechter et al. 2006; veterinary services 2012). anthrax is neither contagious nor invasive; however, it causes acute infection in susceptible animals (fasanella and garofolo 2008; beyer and turnbull 2009; stoltenow et al. 2010). the organism is vegetative in host tissues but sporulates when exposed to the environment (parker et al. 2002; spencer 2003; schaechter et al. 2006; stoltenow et al. 2010). sporulation shields the organism from unfavourable conditions and enables it to survive for years until it gets re-inoculated in another living host thereby continuing its vicious cycle of infection (massachusetts department of public health 2006; schaechter et al. 2006; fasanella and garofolo 2008; todar 2012) . in contrast to other zoonoses, anthrax affects a wide range of animals including human (murray et al. 1990; brooks et al. 1991; spencer 2003; beyer and turnbull. 2009; stoltenow et al. 2010). it is believed that animals acquire the infection through ingestion of tainted feed or while foraging on contaminated field (tweet 2010; veterinary services 2012). nevertheless, human unintentionally contract it, usually through the skin while handling infected animals or their products (turnbull et al. 1998; parker et al. 2002; spencer 2003; edwards et al. 2005; beyer and turnbull 2009; todar 2012). inhalation of the contaminated aerosol or ingestion of partly cooked meat has also been observed (edwards et al. 2005; fasanella and garofolo 2008; stoltenow et al. 2010; todar 2012; hajramurni 2013). 4 pathogenesis the anthrax pathogenesis initiates when the spore enters the skin through lesions, lungs via inhalation or intestines from ingestion of contaminated meat (turnbull et al. 1998; spencer 2003; todar 2012). in the tissues, the spores are engulfed and ingested by resident or migratory macrophages. secured spores are then presented to regional lymph nodes (hanna 1993; bush et al. 2001; missiakas and md 2005; sherer et al. 2007). due to the capsulated cell wall, the spores evade phagocytosis and eventually vegetate. at this stage, germinated cells exhibit its virulence and toxin factors (spencer 2003; edwards et al. 2005; schaechter et al. 2006; todar 2012). productions of edema toxins induce blistering and necrotic lesions as in skins (murray et al. 1990; brooks et al. 1991; missiakas and md 2005). in the intestinal mucosa and alveoli, massive effusion and edema ensues leaving behind necrotising tissues the sum of makaen and tasi int. j. of health, animal science and food safety 2 (2015) 41 48 44 haf © 2013 vol. ii, no. 1 issn: 2283-3927 which triggers inflammatory responses. the combination of lethal toxins results in impaired immune response, host cell destruction and spread of bacterial infection (hanna 1993; spencer 2003; quinn et al. 2004; edwards et al. 2005; missiakas and md 2005; ebrahimi et al. 2011). in time, the bacterium reaches the blood stream causing septicaemia, respiratory distress, meningitis and eventually death. 5 control measures in the event of an anthrax outbreak as with other emerging infectious diseases, environmental and syndromic surveillance is crucial for prompt detection and control of anthrax outbreak in papua new guinea (paul horwood and greenhill 2012). the potential for re-emergence and sporadic occurrence of anthrax is high due its innate ability to survive in nature (turnbull et al. 1998; spencer 2003; durrheim et al. 2009). an outbreak in one kind of domestic animal can easily spread to other species since local animals in rural papua new guinea are free-ranging and often share the same surrounding (hide 2003; durrheim et al. 2009). environmental contamination of anthrax may arise from infected animal carcass being disposed off arbitrarily (fasanella and garofolo 2008; stoltenow et al. 2010; tweet 2010). this raises concern since the environment is a natural reservoir for b. anthracis or facilitates crossinfection of other animals that might feed on the carcass (stoltenow et al. 2010). in view of this, burying of infected animals should be discouraged as the organism will endure long after the carcass disintegrates and disappears. incineration of infected carcass in situ is important to minimise shedding of pathogenic agents in the environment that might serve as a reservoir for future infection (caledonia et al. 2002; fasanella and garofolo 2008; stoltenow et al. 2010; australia 2012; veterinary services 2012). isolation for vaccination or treatment and culling of infected animals, particularly the terminally ill or nearing dead are crucial to curb transmission and future outbreak of anthrax (tweet 2010). however, culling may not be practised in rural settings. even though the sudden death of one or more animal might stir inquisition, investigation of such occurrence by trained personnel is rarely expected. remoteness of sites and funding limitation tends to be the usual impediment to such effort. vaccination is an effective control measure as it offers valuable protection for susceptible animals. it has proven to be successful during outbreak in animal herds (gill 1993; turner et al. 1999; allan 2014; tweet 2010). human vaccination is purely recommended for individuals that deal with the organism as in laboratory facilities or livestock handlers where anthrax outbreak is endemic. most cases of human anthrax are attributed to direct contact with infected animals or its products (who 2008; stoltenow et al. 2010). therefore, vaccination of susceptible animals not only protects them, but prevents the basic route whereby humans contract the infection. nevertheless, animal vaccination is recommended just about the period when outbreak is detected (turnbull et al. 1998; gill 1993; turner et al. 1999; parker et al. 2002; hide 2003; spencer 2003; fasanella and garofolo 2008; stoltenow et al. 2010; hornitzky and muller 2010; tweet 2010; veterinary services 2012). proper personal protection equipment (ppe) is mandatory for any form of anthrax-related work or epidemiological response. they include: face masks or visor (if available but gas masks makaen and tasi int. j. of health, animal science and food safety 2 (2015) 41 48 45 haf © 2013 vol. ii, no. 1 issn: 2283-3927 are highly recommended), goggles, boots gloves and overalls with hoods. these are single-use equipment and should be incinerated afterwards or thoroughly sterilized if re-usable. 6 laboratory diagnosis it is a safety requirement that a laboratory facility dealing with anthrax should operate in a level 2 biosafety cabinet and the laboratory personnel thoroughly attired in ppe. level 3 bc is better still, if the amount of work is substantial or the potential for generating aerosols is high (turnbull et al. 1998). blood culture is routinely performed on virtually all suspected cases of b. anthracis infection (veterinary services 2012). initial presumptive identification of b. anthracis can be performed through rapid test kits such as immuno-chromatographic tests, using serum sample of infected animal or human (hornitzky and muller 2010). smears for staining and microscopic examination can be obtained by swab from lesions, tissue fluids or blood sample. heat-fixation is not applicable for b. anthracis as it not thoroughly bactericidal therefore; smears should be immersed in 40 percent potassium permanganate solution for at least 10 minutes (cheeesborough 2000). both gram stain and loeffler’s polychrome methylene blue staining are acceptable (turnbull et al. 1998; cheeesborough 2000; who 2008; australia 2012). however, loeffler’s polychrome methylene blue staining depicts the encapsulated bacillus which is a clear, but presumptive identification of b. anthracis (spencer 2003; who 2008). further work up and confirmation would require pure culture and isolation, elisa techniques, and if available pcr for definite diagnosis and genetic studies (hornitzky and muller 2010; australia 2012). 7 treatment options the acute nature of anthrax infection warrants prompt treatment. if delayed, it could lead to serious complications and even death. hospitalisation is necessary in virtually all cases of anthrax infection. the ‘cdc guidelines for the treatment of anthrax’ recommends the use of regular antibiotics including; ciprofloxacin, doxycycline, and penicillin for treatment (prevention 2014). a large dose, administered both intravenously and orally over couple months is suggested for very serious cases of b. anthracis infections as it takes about such length of time for spores to germinate (vyas 2013). the optimal antibiotic, dosage level, route of administration and the length of treatment may vary with patient and degree of infection. these basic antibiotic treatment options are available in papua new guinea. however, advance infections may necessitate the use of antitoxins (vyas 2013; administration 2012). the united states food and drug administration have recommended the use of monoclonal antibodies to counteract bacterial toxins which are responsible for the irreparable tissue damage caused during the course of infection (administration 2012). makaen and tasi int. j. of health, animal science and food safety 2 (2015) 41 48 46 haf © 2013 vol. ii, no. 1 issn: 2283-3927 8 conclusion no human case of anthrax has been reported in papua new guinea. if at all, there is no documentation or clinical evaluation of a patient that would otherwise suggest a probable infection. it is worth mentioning particularly, when papua new guinea is considered as anthrax endemic while other pacific island nations are deemed to be free (caledonia, community et al. 2002; fasanella and garofolo 2008; stoltenow et al. 2010). nevertheless, one case of an unknown disease outbreak in pigs in a remote area in papua new guinea has been reported. this report claimed that three people have died consuming pork from a carcass thought to have died from anthrax. however, it has never been established if b. anthracis was responsible. animal infections were reportedly observed in porcine and village pigs which are commonest form of domesticated animals in the highlands region of papua new guinea (hide 2003; fasanella and garofolo 2008; stoltenow et al. 2010; provet 2013). still, no human infection has been reported despite the fact that local highlanders live in close proximity to native animals. other than these, there is no documented evidence of infection in other animals including wildlife or livestock. concrete scientific data is needed to establish the presence of b. anthracis in native animals, wildlife or the environment in papua new guinea. references administration usfad: fda approves raxibacumab to treat inhalational anthrax. in: first monoclonal antibody approved using the animal efficacy rule. new hampshire. 2012. alla m.b.w., mohamed t.e, abdelgadir a.e., 2011. detection of antibiotics residues in beef in ghanawa slaughterhouse, khartoum state, sudan. african journal of food science. 5 (10): 574–580. animal health australia (2005). disease strategy: anthrax (version 3.2). australian veterinary emergency plan (ausvetplan), edition 3, primary industries ministerial council, canberra, act. brooks, butel, ornston: medical microbiology, 19th edn: appleton; 1991: 180-182 bush l. m., abrams b.h., beall a., johnson c.c. index case of fatal inhalational anthrax due to bioterrorism in the united states. the new england journal of medicine 2001. caledonia ipn, community sotp, organization wh: anthrax, 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international student edition edn: mosby; 1990: 180-183 prevention cfdca. guidelines for the treatment of anthrax. in: cdc 24/7: saving lives protecting people™. atlanta: centers for disease control and prevention 2014. price e.p., seymour m.l., sarovich d.s., latham j., wolken s.r., mason j., vincent g., drees k.p., stephen m., beckstrom-sternberg, adam m. phillippy et al. molecular epidemiologic investigation of an anthrax outbreak among heroin users, europe. emerg infectious disease 2012. provet. anthrax. in provet healthcare infromation. provet; 2013. quinn c.p., dull p.m., semenova v., li h. immune responses to bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax. the journal of infectious diseases. 2004: 190-199. schaechter m., ingraham j., neidhardt, microbe. f. bacillus anthracis. in microbe wiki. edited by ucar g., pogliano k., holzhauer d. washington dc: asm press; 2006. sherer k., li y., cui x., eichacker p.q. lethal and 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microbiology. 1999, 87(2): 294-297. tweet. preventing anthrax in livestock, hobby farms. 27 august 2010. veterinary services aha. australian veterinary emergency plan. 2012. vyas j.m.. anthrax. in medline plus. massachusetts: u.s. national library of medicine. 2013 williams a.a., parashar u. d., stoica a., ridzon. r: bioterrorism-related anthrax surveillance, connecticut, september–december, 2001. emerging infectious diseases. 2002, 8(10): 5. world health organization. anthrax in humans and animals. in 4 ed. geneva: who press, 2008. world health organization. b051 anthrax. in world health organization, guidelines for the surveillance and control of anthrax in humans and animals. l keywords cymbopogon citratus, essential oil, orange juice, conservation, benin. pages 01 – 12 references vol. 3 no. 1 (2017) article history submitted: december 15, 2016 revised: march 19, 2017 accepted: march 21 2017 published: march 27 2017 corresponding author euloge s. adjou, laboratory of study and research in applied chemistry, polytechnic school of abomey-calavi, university of abomey-calavi, 01, p. o. box 2009, cotonou, benin mail: eulogesenan@yahoo.fr phone: (+229) 95924907 journal home page riviste.unimi.it/index.php/haf article chemical composition and biological activity of essential oil from cymbopogon citratus leaves on the quality of fresh orange juice during storage euloge s. adjou 1,* , edwige dahouenon ahoussi 1 , rené g. dègnon 1 , chrystelle mongazi 1 , mohamed m. soumanou 1 , dominique sohounhloue 1 1 laboratory of study and research in applied chemistry, polytechnic school of abomey-calavi, university of abomey-calavi, benin abstract the present study aims to evaluate the effect of essential oil (eo) from cymbopogon citratus leaves against the spoilage flora of fresh orange juice. thus, the eo was extracted by hydrodistillation from fresh leaves of cymbopogon citratus collected in southern benin and its chemical composition was determined by gas chromatography, coupled to mass spectrometry (gc/ms). orange samples were collected from large production areas of south and central benin and juices were extracted by mechanical pressing. after identification of spoilage flora of fresh orange juice, antimicrobial tests were carried out with the eo of cymbopogon citratus to evaluate its antimicrobial activity on spoilage flora of fresh orange juice. results indicate that the spoilage flora of fresh orange juice is mainly composed of fungi belonging to the genera of cladosporium, penicillium and fusarium. bacteria such as enterobacter cloacae and enterobacter aerogenes were also identified in some samples. the major compounds identified in the eo by gc/ms are neral (33.0%) and geranial (41.3%) with a predominance of oxygenated monoterpenes (85.5%). antimicrobial tests have revealed a high antibacterial activity of the eo, with minimum bactericidal concentrations (mbc) between 0.1 and 0.15 μl.ml-1. antifungal tests revealed that fungi are also susceptible to this eo with minimum fungicidal concentration (mfc) between 0.15 and 0.25 μl.ml-1. results obtained during the evaluation of the physicochemical characteristics of the orange juice stored by adding eo, indicated a significant decrease in the ph and vitamin c content. however, with eo concentration of 0.250 μl.ml-1, the ph of stored juice was 6.4 ± 0.1 after 15 days of preservation, with a best vitamin c content of 28.06 ± 0.03 mg / 100ml. the eo of cymbopogon citratus, with high antimicrobial activity, could be used as an alternative in the preservation of fruit juices, replacing antimicrobials from chemical synthesis. e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 2 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction generally, fruit juices are a good source of minerals, vitamins, trace elements and antioxidants. fruits are also known to have nutritional and medicinal properties that can be attributed to their antioxidant effects and they can be used to fortify staple foods particularly for malnourished children (barminas et al., 1998). orange juice has great nutritional importance because of its richness in nutrients including vitamins, minerals, dietary fiber, organic acids and bioactive substances (tran and farid, 2004). however, because of this wealth, the orange juice is a preferred substrate for fermentation germs. to fight against these microorganisms, two preservation methods are often used by fruit juice producers in africa: pasteurization and use of chemical synthetic preservatives. pasteurization is a heat treatment to destroy pathogenic microorganisms, but also can change the nutritional quality of the juice, because of the sensitivity of certain nutrients like vitamins to heat. it is also known that the flavor of orange juice is influenced by heat treatment (bazemore et al., 1999). similarly, in the absence of heat treatment, antimicrobials from chemical synthesis also used in food preservation. however, application of high concentrations of these synthetic chemicals in a conservation of food, increases the risk of toxic residues in food. due to the increasing sensitivity of consumers in this residual pollution and toxic effects of many synthetic fungicides, the importance of using natural alternatives becomes necessary (hidalgo et al., 1998). similarly, the restriction imposed by the food industry and regulatory agencies on the use of certain synthetic food additives have led to renewed interest in the search for alternatives, such as natural antimicrobial compounds, especially those of vegetable origin (moosavy and basti 2008). essential oils and derivative compounds have important activities which the antimicrobial activity is the most studied (bankole et al., 2004). cymbopogon citratus, stapf (lemon grass) is a widely used herb in tropical countries. the essential oil of the plant is used in aromatherapy. early research reported that this essential oil, in agar plate, was active on bacillus subtilis, escherichia coli, staphylococcus aureus (melo et al., 2001). other studies reported that the oil is also active against food storage fungi (mishra and dubey, 1994). thus, the present study aims to evaluate the antimicrobial properties of the essential oil of cymbopogon citratus l. against the spoilage flora of fresh orange juice in benin. 2 material and methods 2.1 collection of plant leaves plant materials used for essential oil (eo) extraction were fresh leaves from cymbopogon citratus l. plants were collected at abomey-calavi (south benin) and identified at the benin national herbarium, where voucher specimens are deposited. e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 3 haf © 2013 vol. iv, no. 1 issn: 2283-3927 2.2 essential oil extraction the eo tested was extracted by the hydro-distillation method using clevenger-type apparatus. the oil recovered was dried over anhydrous sodium sulfate and stored at 4 °c until it was used (de billerbeck et al.,2001). 2.3 gas chromatography–mass spectrometry analysis the eo were analyzed by gas chromatograph (perkin elmer auto xl gc; waltham, ma, usa) equipped with a flame ionisation detector, and the gc conditions were equity-5 column (60 m x 0.32 mm x 0.25 µm); h2 as the carrier gas; column head pressure 10 psi; oven temperature program isotherm 2 min at 70 °c, 3 °c/min gradient 250 °c, isotherm 10 min; injection temperature, 250 °c; detector temperature 280 °c. gas chromatography–mass spectrometry (gc-ms) analysis was performed using a perkin elmer turbomass gc-ms. the gc column was equity-5 (60 m x 0.32 mm x 0.25 µm); fused silica capillary column. the gc conditions were injection temperature, 250 °c; column temperature, isothermal at 70 °c for 2 min, then programmed to 250 °c at 37 °c/min and held at this temperature for 10 min; ion source temperature, 250 °c. helium was the carrier gas. the effluent of the gc column was introduced directly into the source of ms and spectra obtained in the ei mode with 70 ev ionisation energy. the sector mass analyzer was set to scan from 40 to 500 amu for 22 s. the identification of individual compounds is based on their retention times, retention indices relative to c5 – c18 n-alkanes, and matching spectral peaks available in the published data (adams, 2007). 2.4 collection of oranges and juice extraction samples of oranges were collected in the main markets of localities of allada (south of benin), klouekanme (south of benin) and zakpota (center of benin) which are the major sales depot of oranges in benin. in each locality, four markets, were investigated and samples were purchased from five different points, and were mixed together to give a composite samples from each market which were used for the analysis. the juice was extracted by mechanical pressing after pulping and cleaning fruits. juices were collected and kept at 4 °c until microbiological analyses. 2.5 microbiological analysis for microbiological analysis, 25 g of each sample and 225 ml of peptone water was added and homogenized. from the initial concentration, appropriate decimal dilutions were prepared and aliquots were plated in duplicates on various media. plate count agar was used for the total bacterial count. plates were incubated at 30°c for 72 h. desoxycholate was used for the total coliforms count and plates were incubated at 30°c for 24 h. desoxycholate was also used for the faecal coliforms count. in this case, plates were incubated at 44°c and the identification was made using eosine methylene blue (emb) medium. tryptone sulfite neomycin agar was used for anaerobic sulfito-reducer (asr) count, and tubes were incubated at 37 °c for 24 h. e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 4 haf © 2013 vol. iv, no. 1 issn: 2283-3927 after incubation, the number of colonies was tracked, using a colony counter. the number of bacteria expressed as colony forming units per gram (cfu/g) was then determined by calculation, considering dilution factor. all media used for microbiological analysis were prepared as indicated by the manufacturer. after their isolation, bacteria were also controlled with api system (biomérieux france). 2.6 fungal isolation and identification the isolation of fungi from samples was performed using dilution plating method. ten gram of each juice sample were added separately to 90 ml of sterile water containing, 0.1% peptone water. this was thoroughly mixed to obtain the 10 -1 dilution. further, 10fold serial dilutions up to 10 -4 were made. 1 ml volume of each dilution was separately placed in petri dishes, over which, 10 to 15 ml of potato dextrose agar amended with 60 µg/ml chloramphenicol (pdac) was poured. the plates were incubated at 28 ± 2 °c for 7 days. fungal isolates from pdac were sub-cultured on malt extract agar (mea), and identification was carried out by using a taxonomic schemes primarily based on morphological characters, using the methods described by (singh et al., 1992). 2.7 biological assays 2.7.1 minimum inhibitory concentration (mic)-broth microdilution method to determine the mic, broth microdilution method proposed by bajpai et al. (2008) were used. the microdilutions on 96 well plates were used with mueller hinton broth (mhb) and 0.02 g/l phenol red. eo and mhb constitute the negative control. the positive one is bacteria strain plus mhb. the microplates were incubated at 37 ± 1 °c for 24 h, covered with a parafilm paper. 2.7.2 minimum bactericidal concentration (mbc) mbc were appreciated by method proposed by oussou et al., (2004). to determine the mbc, each microliter-plate well in which no color change occurred was used. the mixture of eo and the strain was isolated on sterile mha poured in petri dishes. these plates were incubated at 37 °c for 24 h. the mbc is the lowest concentration of eo which 99.9% of the microorganisms were killed. the tests were carried out in triplicate. 2.7.3 antifungal assay antifungal assay was performed by the agar medium assay (yehouenou et al., 2010). yeast extract sucrose (yes) medium with different concentrations of essential oil (0.015, 0.025, 0.050, 0.075, 0.100, 0.150, 0.200, 0.250 μl.ml -1 ) were prepared by adding appropriate quantity of eo and tween 20 to melted medium, followed by manual rotation of erlenmeyer to disperse the oil in the medium. about 20 ml of the medium were poured into glass petri dishes (9 cm). fungal isolates from orange juice on malt extract agar (mea) are transplanted (subcultured), using a disc of 6 mm in diameter which carries spores from the anamorph mold, on the surface of a petri dish containing the former medium yes and eo at different concentrations. positive e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 5 haf © 2013 vol. iv, no. 1 issn: 2283-3927 control plates (without eo and inoculated following the same procedure) and negative control plates were also used. plates were incubated at 25 °c for 5 days. 2.7.4 determination of the fongiostatic or fungicidal activity with the experimental concentrations where neither growth nor germination was observed, the fungiostatic or fungicidal activity was tested. this assay consisted by taking the mycelial disc not germinated at the end of the incubation of the petri dish and reintroducing it in a new culture medium (former one) without eo. if the mycelial growth is always inhibited, the plant extract is fungicidal at this concentration and allows the determination of the minimum fungicidal concentration (mfc). in the contrary case, it became fungiostatic activity which is related to the minimum inhibitory concentration (mic) (tomohiro, 1990). 2.8 conservation of orange juice with essential oil to evaluate the conservation potentiality of the eo of cymbopogon citratus l., juices extracted from oranges collected at allada (south of benin), klouekanme (south of benin) and zakpota (center of benin) were mixed together to give a composite sample which was used for the tests. five eo concentrations were tested. these are 0.025, 0.050, 0.062, 0.125 and 0.250 μl.ml -1 . these concentrations were chosen taking into account the high fragrant nature of the eo and different results about its antimicrobial properties, reported in the literature (esteve and frigola, 2008). a negative control (orange juice without eo) was also produced. samples were placed at 25 °c. after 15 days of conservation at this temperature, microbiological and physicochemical qualities of conserved juice were then evaluated. the ph of the samples were determined in 10ml of orange juice using a digital ph-meter. vitamin c (lascorbic acid) concentration was determined using method described by adjou et al. (2013). 2.9 statistical analysis experiments were performed in triplicate, and data analyzed are means ± se subjected to one-way anova. means are separated by the tukey’s multiple range test when anova was significant (p<0.05) (spss 10.0; chicago, il, usa). 3 results the result of microbial analysis and isolation of fungi in pure culture revealed that orange juices collected from the main markets of south and center of benin, were contaminated by microorganisms such as bacteria, yeast and fungi (table 1). bacteria isolates include e.coli, enterobacter aerogenes , enterobacter cloacae and staphylococcus aureus. high contamination rates of yeast and molds were obtained with the level ranging between 1x10 5 and 3x10 5 cfu / ml for yeasts and 1x10 2 and 1.5x10 2 cfu / ml for the mold, with the predominant presence of penicillium spp, and cladosporium spp. the results of the evaluation of the physicochemical characteristics of the fresh juice (table 2) indicated that the http://fr.wikipedia.org/w/index.php?title=enterobacter_aerogenes&action=edit http://fr.wikipedia.org/w/index.php?title=enterobacter_cloacae&action=edit e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 6 haf © 2013 vol. iv, no. 1 issn: 2283-3927 ph of the juice were between 6.2 ± 0.3 and 6.8 ± 0.1, with a vitamin c content ranged from 26.03 ± 0.08 mg / 100 ml to 31.03 ± 0.05 mg / 100ml. table 1: microbiological quality of investigated orange juice (cfu/ml). microbiological parameters localities investigated allada klouekanme zakpota total bacterial count 3.83 x 10 5 a 2.57 x 10 5 b 2.99 x 10 5 b total coliforms count 1 x 10 3 a 1 x 10 3 a 1 x 10 3 a e.coli 1 a 0 a 1 b enterobacter aerogenes 0 a 1 b 1 b enterobacter cloacae 1 a 1 a 1 a staphylococcus aureus 1.87 x 10 5 a 1.15 x 10 5 a 1.6 x 10 5 a yeast count 2 x 10 5 a 3 x 10 5 a 1 x 10 5 a fungi count 1 x 10 2 a 1 x 10 2 a 1.5 x 10 2 a values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. table 2: physicochemical quality of investigated orange juice physicochemical parameters localities investigated allada klouekanme zakpota ph 6.8 ± 0.1 a 6.2 ± 0.3 b 6.3 ± 0.1 b vitamin c (mg/100ml) 31.03 ± 0.05 a 26.03 ± 0.08 b 29.03 ± 0.04 b values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests. by hydrodistillation, fresh leaves of c. citratus yielded 1.31% of eo. chemical analysis by gc and gc-ms analysis of eo enabled the identification of 26 components, (table 3) representing 98.1 % of the eo. in the volatile extract, different groups of terpenes and terpenoids were detected. the eo has chemical composition characterized by myrcene (10.4 %), neral (33.0%) and geranial (41.3%) as major components. the eo exhibited pronounced antibacterial activity against the growth of eschericha coli, enterobacter aerogenes, enterobacter cloacae and staphylococcus aureus. mic value was 0.05 μl.ml -1 for all bacteria tested. however, mbc values were 0.1 μl x ml -1 for eschericha coli, enterobacter aerogenes, enterobacter cloacae and 0.15 μl x ml -1 for staphylococcus aureus. the eo exhibited pronounced antifungal activity against the growth of penicillium spp and http://fr.wikipedia.org/w/index.php?title=enterobacter_aerogenes&action=edit http://fr.wikipedia.org/w/index.php?title=enterobacter_cloacae&action=edit e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 7 haf © 2013 vol. iv, no. 1 issn: 2283-3927 cladosporium spp. the mic of the eo was found to be 0.15 μl x ml -1 for penicillium spp and 0.20 μl x ml -1 for cladosporium spp. the mfc was recorded to be μl x ml -1 for penicillium spp and cladosporium spp. table 3: chemical composition of investigated eo of cymbopogon citratus compound kovats index (ki) percentage (%) 6-méthyl-hep-5-en-2-one 985 1.2 myrcene 991 10.4 limonene 1031 (z)-β-ocimene 1036 0.2 (e)-β-ocimene 1047 0.2 6,7-èpoxymyrcene 1091 0.2 pirillene 1098 0.1 linallol 1100 0.5 2,2-octa-3,4-dienal 1106 0.1 cis-vervenol 1140 0.1 trans-verbenol 1144 menth-3-en-9-ol 1150 0.1 citronella 1153 0.4 cis-chrysanthenol 1162 0.5 epoxy rose furane 1170 0.2 nerol 1231 0.3 neral 1245 33.0 geraniol 1256 6.6 geranial 1276 41.3 formate of neryle 1285 0.1 acetate of geranyle 1378 2.4 β-caryophyllene 1419 oxyde of caryophyllene 1587 0.1 total 98,1 results obtained during storage tests of fresh juice with the eo of c. citratus at different concentrations (table 4) indicated a strong antimicrobial activity of the eo against the spoilage flora of fresh orange juice. indeed, with the essential oil concentration of 0.125 μl x ml -1 , there e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 8 haf © 2013 vol. iv, no. 1 issn: 2283-3927 was a good antibacterial activity, mainly against e. coli, enterobacter aerogenes, enterobacter cloacae, and staphylococcus aureus. table 5 presented the results of the evaluation of the physicochemical characteristics of the orange juice stored by adding eo after 15 days. these results indicated that, in orange juice stored with eo at the concentrations of 0.025 0.062 μl.ml -1 , there was a significant difference in ph and vitamin c content, after 15 days of storage. however, at the concentration of 0.250 μl x ml -1 , there was no significant difference in ph and vitamin c levels after 15 days of storage with a best vitamin c content of 28.06 ± 0.03 mg / 100ml. table 5: physicochemical quality of investigated orange juice after 15 days of conservation physicochemical parameters juice ‘s characteristics at the beginning of the conservation tests characteristics of the juices after 15 days of conservation concentrations of essential oil (μl/ml) 0 0.025 0.050 0.062 0.125 0.250 ph 6.2±0.3a 3.4±0.1 b 3.9±0.4b 4.4±0.6b 4.9±0.2c 5.3±0.6c 6.4±0.1d vitamin c (%) 29.02±0.01a 1.06±0.03 b 1.07±0.09b 8.01±0.04c 12.08±0.07c 21.06±0.08d 28.06±0.03e values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests table 4: microbiological quality of investigated orange juice after 15 days of conservation (ufc/ml). parameters concentrations of essential oil (μl/ml) 0 0.025 0.050 0.062 0.125 0.250 total bacterial count 2 x 10 7 a 3 x10 5 b 102 c 10 d 10 d 07 d total coliforms count 1 x 10 5 a 10 2 b 10 c 0 d 0 d 0 d e.coli 1 x 10 3 a 10 b 0 c 0 c 0 c 0 c enterobacter aerogenes 5 x 10 2 a 10 b 0 c 0 c 0 c 0 c enterobacter cloacae 2 x 10 2 a 10 b 0 c 0 c 0 c 0 c staphylococcus aureus 1.6 x 10 6 10 2 0 0 0 0 c yeast count 10 5 a 10 3 b 10 c 10 c 10 c 0 c fungi count 10 2 a 10 2 a 1.2 x 10 1 b 10 c 07 c 0 d values are means. the means followed by same letter in the same line are not significantly different according to anova and tukey’s multiple comparison tests http://fr.wikipedia.org/w/index.php?title=enterobacter_aerogenes&action=edit http://fr.wikipedia.org/w/index.php?title=enterobacter_cloacae&action=edit e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 9 haf © 2013 vol. iv, no. 1 issn: 2283-3927 4 discussion the beneficial activities of orange juice components for human health are primarily attributed to their antioxidant capacity (somogy, 1996). the results obtained on the evaluation of the physicochemical quality of fresh orange juice, indicated that they are a good source of vitamin c, natural and easily accessible by the public. indeed, vitamin c is an important vitamin and plays an important role in human health and preservation. it is valuable for its antioxidant effect, stimulation of the immune system and other health benefits that are being actively investigated and reported (adjou et al., 2012). the consumption of foods rich in antioxidant substance may contribute to the preservation of cell’s oxidation (fao, 1998). in the food industry, vitamin c is used as a food additive (leclerc et al., 2002). it is frequently added to fruit juices to preserve and protect them from any color changes. however, the ph of these fresh juices, being closely to neutrality, makes them subject to a potential risk of contamination by microorganisms. indeed, the results of microbiological analyzes on the quality of fresh orange juice indicated a contamination by bacteria from gastroenteritis tropism, such as, e. coli, enterobacter aerogenes, enterobacter cloacae and bacteria from mucocutaneous tropism, such as staphylococcus aureus. this high total bacterial and coliform count may have been as a result of the low level of hygiene maintained during the sale of oranges. indeed, the bad oranges handling conditions, induced the onset of injury to the pericarp, and the exposure of orange stock on ground for sale, promote contamination and penetration of microorganisms in oranges. the detection of escherichia coli, which is enteric bacteria, confirmed the poor hygienic practice during the sale of oranges. the isolation of coliforms from orange juices, pose a serious threat to food safety, due to the fact that fruit juice are ready to eat foods, which are consumed without further processing. similar results were found on the other street foods and the germs most identified in these foods were mainly staphylococci and enterobacteria (dahouenon-ahoussi et al., 2012). according to food and agriculture organization (fao)/ world health organization (esteve and frigola, 2008), epidemiological data in hospital showed a prevalence of 19% of diarrheal disease worldwide and bacterial diarrhea was estimated between 20 and 70% of the cases. the causes were related to poor hygiene found in the assessment of hazards and identification of critical points in the food processing chain (somogyi, 1996). however, fungal contamination of samples is higher, and can constitute a health risks to the consumer because of toxigenicity of molds, but also constitutes an important factor of impaired marketability of the product. these factors (yeasts and molds) are increasingly taken into account nowadays, when developing antimicrobial products to maintain quality of highly perishable foodstuffs eo are natural mixtures of hydrocarbons and oxygen (alcohols, aldehydes, ketones, carboxylic acids, esters, and lactones) containing organic substances of plants. their constituents and derivatives have a long history of application as antimicrobial agents in the areas of food preservation and medicinal antimicrobial production (voda et al., 2003). the present study also explores the bioefficacy of eo of c. citratus as the promising plant-based antimicrobial against oranges juice-infecting fungi and bacteria. this eo was found to be effective against bacteria, and fungi infecting orange juice. this bioefficacy may be due to the presence of some highly fungitoxic components in the oil such as terpenoids. indeed, terpenoids are a large group of antimicrobial compounds that are active against a broad e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 10 haf © 2013 vol. iv, no. 1 issn: 2283-3927 spectrum of microorganisms (dorman, 2000). their antimicrobial activities are linked to their functional groups and it has also been reported that the hydroxyl group of phenolic terpenoids and the presence of delocalized electrons are important for the antimicrobial activity (suhr et al., 2003). then, biological activities of eo depends on the qualitative and quantitative characteristics of their components (matasyoh, 2011). in our study, gc–ms data, depicted remarkable variation with the earlier reports on the oil (prakash et al., 2010). the chemical profile of essential oils, is also reported to be influenced by the harvest period, geographical, climatic conditions, and the amount of active constituents (soumanou and adjou, 2016). thus, the biologically active eo should be qualitatively standardized before their recommendation for practical exploitation as has been done in the present investigation. the results of conservation of orange juice with eo confirmed the bioefficacy potential of this eo and opened new perspectives in the use of natural plant extracts as an opportunity to avoid synthetic chemical preservatives, and offers novel approach to the management of storage fungi. it was a promising method for preserving stored products in rural areas, which do not have access to modern storage system. 5 conclusions this work underlined the bioactivity of essential oil of fresh leaves of c. citratus from benin as a promising plant-based antimicrobial against orange juice-infecting fungi and bacteria. different major components such as myrcene (10.4%), neral (33.0%) and geranial (41.3%) were present in the volatile extract. based on its antibacterial and antifungal activities, this natural plant product may successfully replace synthetic chemicals, and provide an alternative method in the stabilization of fresh orange juice, as well as other agricultural commodities of nutritional significance from microbiological spoilage alteration. acknowledgement the authors are grateful to the department of food engineering and technology of polytechnic high school of abomey-calavi university, for their financial support. references adams r.p., 2007. identification of essential oil components by gas chromatography / mass spectrometry. allured, carol stream. adjou e. s., amamion h., tchobo f. p., aissi v. m., soumanou m.m., 2013. extraction assistée par enzyme du jus de la pulpe fraîche du rônier (borassus aethiopum mart.) acclimaté au benin : caractérisation physico-chimique et microbiologique. int. j. biol. chem. sci. 7(3), 1135-1146. e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 11 haf © 2013 vol. iv, no. 1 issn: 2283-3927 adjou e.s., yehouenou b., sossou c.m., soumanou m.m., de souza c.a., 2012. occurrence of mycotoxins and associated mycoflora in peanut cakes products (kluiklui) marketed in benin. afr j biotechnol 11(78), 14354–14360. bajpai k.v., dung n.t., kwon j.o., kang s.c., 2008. analysis and the potential application of essential oil and leaf extracts of silene americana l. to control food spoilage and foodborne pathogens. j. food technol., 227: 1613-1620. bankole s., mabekoje o.o., 2004. mycoflora and occurrence of aflatoxin b1 in dried yam chips from markets in ogun and oyo states, nigeria. mycopathologia, 157, 111-115. barminas j.t., charles m., emmanuel d., 1998. mineral composition of nonconventional leafy vegetables. plant foods for human nutrition 53 (1), 29-36. bazemore r., goodner k., rouseff r., 1999. volatiles from unpasteurized and excessively heated orange juice analyzed with solid phase microextraction and gc-olfactometry. j. food. sci. 64 (5), 800-803. dahouenon-ahoussi e., degnon r. g., adjou e. s., sohounhloue d.c.k., 2012. stabilisation de la bière produite à partir de matières amylacées locales (sorghum bicolor et musa acuminata) par adjonction de l’huile essentielle de cymbopogon citratus. journal of applied biosciences 51, 3596 – 3607. de billerbeck v.g., roques c.g., bessière j.m., fonvieille j.l, dargent r., 2001. effect of cymbopogon nardus (l) w. watson. essential oil on the growth and morphogenesis of aspergillus niger. can j microbiol 47, 9–17. dorman h.j.d., deans sg. antimicrobial agents from plants: antibacterial activity of plant volatile oils, j. appl.microbiol., 88, 308–316. 2000. esteve j.m., frigola a., 2008.the effects of thermal and non-thermal processing on vitamin c, carotenoids, phenolic compounds and total antioxydant capacity in orange juice. tree and forestry science and biotechnology, 2(1), 128-134. fao/who, 1998. forty-ninth meeting of the joint expert committee on food additives. food agric. organ. united nation. rome, p. 140. hammer k.a., carson c.f., ridley c.v., 1999. antimicrobial activity of essential oils and other plants extract. journal of applied microbiology, 86, 985-990. hidalgo e., moor, d., patourel g., 1998.the effect of different formulations of beauveria bassiana on sitophilus zeamais. journal of stored products research, 34, 171–179. leclerc h., schwarrtzbrod l., dei-cas e., 2002. microbial agents associated with waterborne diseases. crit. rev. microbiol., 28,371-409. matasyoh j.c., wagara i.n., nakavuma j.l., kiburai a.m., 2011. chemical composition of cymbopogon citratus essential oil and its effect on mycotoxigenic aspergillus species. african journal of food science, 5(3), 138-142 melo s.f., soares s.f., costa d.r., silva d.c., oliveira d.m., bezerra r.j., 2001. effect of the cymbopogon citratus, maytenus ilicifolia and baccharis genistelloides extracts against the stannous chloride oxidative damage in escherichia coli. mutat res., 496, 33–41. e.s. adjou et al. int. j. of health, animal science and food safety 4 (2017) 01 12 12 haf © 2013 vol. iv, no. 1 issn: 2283-3927 misrha a.k., dubey n.k., 1994. evaluation of some essential oils for their toxicity against fungi causing deterioration of stored food commodities. appl environ microbiol., l60, 1101–1106. moosavy m.h., basti a.a., ali m., 2008. effect of zataria multiflora boiss. essential oil and nisin on salmonella typhimurium and staphylococcus aureus in a food model system and on the bacterial. international journal of food microbiology, 43, 69–76. oussou k.r., kanko c., guessend n., yolou, s., koukoua g., dosso m., n’guessan y.t., figueredo g., chalchat j-c., 2004. activités antibactériennes des huiles essentielles de trois plantes de côte d’ivoire. c.r. chim., 7, 1081-1086. prakash b., shukla r., singh p., mishra pk., dubey n.k., kharwar r.n., 2010. efficacy of chemically characterized ocimum gratissimum l. essential oil as an antioxidant and a safe plant based antimicrobial against fungal and aflatoxin b1 contamination of spices, food res. int., 10, 128 -132. singh k., frisvad j.c., thrane u., mathu s.b., 1991. an illustrated manual on identification of some seed borne aspergilli, fusaria, penicillia and their mycotoxins. heller up, denmark: danish government, instituteof seed pathology for developing countries. p 133. somogyi l.p., 1996. direct food additives in food processing. in l. p. somogyi, d. m. barrett, & y. h. hui (eds.), processing fruits: science and technology, biology, principles, and application. western hemisphere 7 technomic publishing, 1, 342-345. soumanou m.m. and adjou e.s., 2016. sweet fennel (ocimum gratissimum) oils. in:preedy, v.r. (ed.), essential oils in food preservation, flavor and safety. academic press, 765–773. suhr k.i., nielsen p.v., 2003. antifungal activity of essential oils evaluated by two different application techniques against rye bread spoilage fungi, j. appl. microbiol., 94(4), 665– 674. tomohiro s., 1990. laboratory manual for food analysis”, technical cooperation project of jomo kenyatta 76p. tran m. t. t., farid m., 2004. ultraviolet treatment of orange juice. innovative food science and emerging technologies 5, 495– 502. voda k., boh b., vrta-cnik m., pohleven f., 2003. effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot trametes versicolor and the brown-rot coniophora puteana, int. biodeterior. biodegrad., 51, 51–59. yèhouenou b., noudogbèssi j.p., sessou p., avlessi f., sohounhloué d., 2010. etude chimique et activités antimicrobiennes d’extraits volatils des feuilles et fruits de xylopia aethiopica (dunal) a. rich. contre les pathogènes des denrées alimentaires. j. soc ouest – afr. chim., 029, 19-27. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l corresponding author ioannis politis i.politis@aua.gr journal home page riviste.unimi.it/index.php/haf lipid mobilization, immune function and the paradigm of vitamin e in transition cows. i. politisa adepartment of animal science, agricultural university of athens, 11855 athens, greece abstract the number of metabolic disorders that dairy cows have to cope during the transition to lactation can be divided in three main categories. the first category includes disorders related to abnormal energy metabolism (ketosis, fatty liver, acidosis). the second and the third categories include disorders related to mineral metabolism (milk fever) and disorders related directly or indirectly to impaired immune function (mastitis, metritis, retained placenta), respectively. among the many physiological changes during the transition period, perhaps the most crucial, is an increase in the concentration of plasma non-esterified fatty acids (nefa). a portion of this increase in nefa is obligatory and it is under hormonal control while another portion is the result of a situation known as negative energy balance (difference between energy consumed and energy spent). in this presentation i will present data from a collaborative study between the university of milan and the agricultural university of athens which proves that negative correlations exist between blood concentrations of nefa and β-hydroxybutyrate with α-tocopherol. the adipose tissue contains two main categories of cells: adipocytes and immunocompetent cells mainly monocytes/macrophages. our research has tested the hypothesis that a cross-talk exists between adipocytes and monocytes/macrophages and this cross-talk is mediated by fatty acids released by adipocytes especially during the transition period. results indicate that all fatty acids tested (myristic, palmitic, palmitoleic, stearic and oleic) upregulate the expression of numerous pro-inflammatory genes by both monocytes but neutrophils, as well. the longer the carbon chain, the most potent is the effect. another hypothesis that we have tested is that vitamin e can interfere and block the cross talk between adipocytes and immunocompetent cells. against this notion, α-tocopherol does not interfere with the effect of fatty acids on expression of pro-inflammatory genes. thus, fatty acids compromise the function of two immunocompetent cells. vitamin e is unable to block this effect. even though vitamin e is unable to block this unwanted activation of immunocompetent cells, it certainly has a positive role because it helps to restore proper immune function during the transition period and evidence supporting this role will be presented. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en mailto:i.politis@aua.gr proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords term pregnancy, fetal fluids, cortisol, newborn outcome corresponding author barbara bolis barbara.bolis@unimi.it journal home page riviste.unimi.it/index.php/haf cortisol fetal fluids concentrations and newborn outcome in term pregnancy small-sized purebred dogs. b. bolis*b, t. pericc, a. rotab, faustini ma, veronesi mca adepartment of veterinary medicine, università degli studi di milano bresident department of veterinary medicine, università degli studi di milano cdepartment of food sciences, university of udine, center for biomedical science and engineering, university of nova gorica, slovenia abstract in order to provide further information about canine perinatology, and because of the scarce knowledge about fetal fluids composition in dogs, the present study was aimed to assess the cortisol concentrations in fetal fluids collected from small-sized purebred newborn puppies born by elective cesarean section, at term of pregnancy (meloni et al, 2014). furthermore we assessed possible correlations of amniotic and allantoic cortisol concentrations and newborn outcome at 24 hours of age and with the newborn gender. fetal fluids cortisol concentrations were also evaluated for correlation with maternal parity, litter-size, neonatal gender, birth weight and apgar score (veronesi et al, 2009). the study, performed on 50 born alive, normal weighed puppies, without gross physical malformation, showed that cortisol concentration was higher in allantoic than in amniotic fluid (p<0.01), even if a strong positive correlation between the two fluids cortisol concentration was found (p<0.0001; r=0.83). interestingly, higher amniotic (p<0.05) cortisol concentrations were associated to puppies not surviving at 24 hours after birth. therefore it could be suggested that this parameter may be useful for the recognition, at birth, of puppies needing special surveillance in the first day of age. in relation to the other evaluated parameters, no correlations with amniotic or allantoic cortisol concentrations were found. in conclusion, the present results showed that in small-sized purebred puppies, born at term by elective caesarean section, the evaluation of amniotic cortisol concentration seems useful for the detection of puppies that need special surveillance during the first 24 hours of age, and should be coupled to the newborn evaluation by apgar score. references meloni t, comin a, rota a, peric t, contri a, veronesi mc, 2014. igf-i and nefa concentrations in fetal fluids of term pregnancy dogs. theriogenology, 2014;81:1307-11. veronesi mc, panzani s, faustini m, rota a, 2009. an apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis. theriogenology; 2009; 72, 401–407. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords amnion, endometrium, microvescicles, mirna, bovine embryos corresponding author claudia perrini claudia.perrini@unimi.it journal home page riviste.unimi.it/index.php/haf secretome of bovine amniotic and endometrial cells: application for in vitro embryo production. c. perrinia*, a. lange consiglioa, p. espostia, d. compagnonia, f. cremonesia a reproduction unit, large animal hospital, università degli studi di milano, via dell’università 6, lodi, italy abstract some maternal mirnas are involved in early stage embryos [abd el naby, 2011]. microvesicles (mvs) have been suggested as carrier of mirnas for maternal-to-embryonic communication during the first days of early development [mondou, 2012]. mvs, together with soluble factors, are components of conditioned media (cm) produced by cells during their culture. aim of this study was to understand the role of cm, mvs and supernatant (sn, obtained from cm deprived of mvs) secreted by bovine endometrial and amnion cells on embryo development. in vitro produced embryos were cultured in sofaa alone (ctr) or supplemented on day 5 postfertilization with 10% of endometrial or amniotic cm or sn or 100x106 mvs/ml. the blastocyst rate obtained culturing embryos with amniotic cm and mvs was not significantly different from the ctr (34.17±3.29%, 32.82±3.26% and 35.45±2.53% respectively). the rate obtained by amniotic sn was 25.80±2.83% and statistically lower (p<0.05) than the other groups. the rate obtained by endometrial products were lower than ctr and the other conditions. the icm of embryos cultured in medium supplemented with amniotic components had a higher number of cells than the ctr group: 30.4±1.83 and 29.42±1.27 for cm and mvs respectively compared to 27.6±1.44 for ctr (p<0.05). our data showed that exposing the embryos to the amniotic secretome does not improve the blastocyst rate, but increases their quality. the hypothesis is that mirnas contained into mvs may contribute to the production of better quality embryos and that amniotic secretome supplies a more physiological environment for the embryo development. references abd el naby, w.s. ,hagos, t.h., hossain, m.m., salilew-wondim, d. , gad, a.y.,rings, f., cinar, m.u. , tholen, e., looft, c., schellander, k., hoelker, m., tesfaye., d., 2011. expression analysis of regulatory micrornas in bovine cumulus oocyte complex and preimplantation embryos. zygote. 21, 31–51. mondou, e. , dufort, i., gohin, m., fournier, e., sirard, m.a., 2012. analysis of micrornas and their precursors in bovine ea rly embryonic development. molecular human reproduction. 18(9), 425–434. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l corresponding author trudee fair trudee.fair@ucd.ie journal home page riviste.unimi.it/index.php/haf good egg or bad egg: developing markers of oocyte competence for assisted reproductive interventions. t. faira a school of agriculture and food science, university college dublin, ireland abstract the oocyte is the foundation of life. it develops from a single fertilized cell to a multicellular organism capable of an independent existence. competence to achieve this maximum potential is acquired following the protracted, but, highly co-ordinated process of growth and subsequently, maturation. the environment in which all or part of these processes occur, can ultimately have long-term consequences for female fertility and the health of resulting offspring. the pressure to identify and select oocytes or embryos with the highest developmental potential has intensified as the number of patients and the range of options available to them have increased. in particular, as in vitro maturation and single embryo transfer become more routine in assisted reproductive technology, selection is critical to a successful outcome. moreover, the identification of markers of oocyte health and quality is essential to monitor the impact of these technologies on gametes and embryos. technologies, such as transcriptomics, proteomics and metabolomics, offer more sophisticated methods for oocyte and embryo selection, with the emphasis on the predictive value of noninvasive protocols which profile follicular fluid, follicle/ granulosa cells and cumulus cells, for assessment of oocyte quality. using the bovine as a model, we have employed a range of approaches and identified many potential markers, such as oocyte and cumulus candidate proteins and transcripts and follicular fluid fatty acids and amino acids. the models, technologies, and future strategies will be discussed in detail. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords epigenetic conversion, pancreatic differentiation, glucose concentration, physiological environment corresponding author alessandro zenobi alessandro.zenobi@unimi.it journal home page riviste.unimi.it/index.php/haf high glucose concentrations are required for endocrine pancreatic differentiation of mammalian adult fibroblasts. a. zenobia *, f. gandolfia, t. a. l. brevini a a department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy abstract epigenetic conversion overcomes the stability of a terminally differentiated cell, allowing phenotype switch and providing an unlimited source of autologous cells of a different type. it is based on the exposure to an epigenetic modifier that increases cell plasticity, followed by a differentiation protocol. in our work we treat mammalian dermal fibroblasts with the demethylating agent 5-azacytidine. cell differentiation is directed toward the endocrine pancreatic lineage, with a sequential combination of key growth factors. the overall duration of the process is 36 days (pennarossa, 2013; brevini, 2015; brevini, 2015). however, this protocol, as well as all differentiation procedures described in the literature, uses high and non-physiological concentrations of glucose. here we report experiments aimed at investigating whether the use of lower glucose concentrations, that more closely mimic the in vivo physiological environment, can support fibroblast conversion into β-like cells. to do so, cells were cultured as described above, but using lower and more physiological glucose levels, namely 5.5 and 8.5 mm that correspond to normoglycaemia before and after meals (international diabetes federation, 2007). our results show that mammalian cells are not able to differentiate into insulin secreting cells in a low glucose environment. in particular, cells do not aggregate into pancreatic islet structures and display an altered gene expression pattern for several early pancreatic markers, when compared to the standard trend obtained with 17.5 mm of glucose. these results suggest that high glucose levels are essential for the achievement of the endocrine pancreatic differentiation process in mammalian cells and appear to be crucial for functional efficiency and morphological organization. references brevini, t.a.l., pennarossa, g., acocella, f., brizzola, s., zenobi, a., gandolfi, f., 2015. skin derived insulin-secreting cells as a potential therapy for diabetes in the dog. the veterinary journal 211, 52-56; brevini, t.a.l., pennarossa, g., maffei, s., zenobi, a., gandolfi, f., 2015. epigenetic conversion as a safe and simple method to obtain insulinsecreting cells from adult skin fibroblasts. jove 109, e53880; international diabetes federation, 2007. guidance for management of postmeal glucose; pennarossa, g., maffei s., campagnol, m., tarantini, l., gandolfi, f., brevini, t.a.l. 2013. brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. pnas 110(22), 8948–8953. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l corresponding author greta farina, greta.farina@unimi.it abstract the objective of the experiment was to understand the interaction between saturated or unsaturated fatty acids and genes involved in lipid metabolism in liver and subcutaneous adipose tissue. with this purpose, further gene expression assays were performed on obtained adipose and liver samples from a previous in vivo study where expression levels of adipoq, lpin1, lpl, pparg, srebf1 and thrsp were already determined. the study consisted on the administration of either a no fat-supplemented, or a stearic acid or fish oil supplemented diets to dairy goats from the last week of gestation until 21 days after kidding. fat-supplied goats received 30g/head/d extra fatty acids during the dry period and 50g/head/d during lactation. liver and subcutaneous adipose tissue samples were harvested at day -7, 7 and 21 relative to kidding and immediately snap frozen in liquid nitrogen. at the present moment, quantitative real-time rt-pcr of acat1, msmo1, cpt1, il6 on liver and acaca, lep, lpl, fasn, il6 and plin2 on adipose tissue are running. data obtained will be analysed using the mixed procedure of sas and results may increase the knowledge on the mechanism of action of saturated or unsaturated dietary fatty acid sources in the fatty acid metabolism changes during transition in dairy goats. references e. tsiplakou, e. flemetakis, c. kalloniati , g. zervas, small ruminant research (2011), 99, pp 110–115. l. bernard, c. leroux, j. rouel, m. bonnet and y. chilliard, british journal of nutrition (2012), 107, 1147–1159. p. g. toral, l. bernard, c. delavaud, d. gruffat, c. leroux, and y. chilliard, animal (2013), 7:6, pp 948–956. hepatic and subcutaneous adipose lipid metabolism genes modulation by dietary fish oil and stearate in transition goats f. greta 1 , a. agazzi 1 , j.m. caputo 1 , a. campagnoli 1 , m. ferroni 1 , j.j. loor 2 , g. savoini 1 , g. invernizzi 1 . 1 department of health, animal science and foodsafety, università degli studi di milano, via celoria 10, 20133 milan, italy 2 department of animal sciences, uiuc, illinois, usa. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords pesticide residues, analytical chemistry, mass spectrometry, honey. corresponding author giuseppe federico labella giuseppe.labella@unimi.it journal home page riviste.unimi.it/index.php/haf comparison between accelerated solvent extraction (ase) with clean up in-line and quick, easy, cheap, effective, rugged, and safe (quechers) extraction in honey. g.f. labella a*, k. bousova b, l. hollosib, f. ariolia a department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy b special solution center europe, thermo fisher scientific, im steingrund 4, 63303, dreieich, germany abstract honey is a popular bee product widely consumed and therefore it has to be assured that it is free of contaminants and safe for the consumers. many pollutants, including pesticides (chauzat et al, 2011) and heavy metals (tuzen et al., 2007) coming from the environment, could be the source of contamination for bee matrices. in this study the method performances of two solidliquid extraction techniques, accelerated solvent extraction (ase) with clean up in-line and quick, easy, cheap, effective, rugged, and safe (quechers) extraction, were evaluated and compared for the analysis of pesticide residues in honey. fiftythree pesticides, belonging to different chemical classes, were investigated in orange blossom honey from german local market using method based on gas chromatography coupled with tandem mass spectrometry (gc-ms/ms. the linearity (r2 > 0.985) in solvent and in matrix was achieved for most of the compounds with both extraction techniques, however showing a strong matrix effect. the criteria for a good repeatability (cv <20%) were satisfied for the 92 % of the compounds with quechers extraction and for the 74% of the compounds with ase extraction. the recovery (evaluated at three different concentration levels) was the same for the two techniques at the lowest concentration level, while was better for quechers than ase at the highest level. furthermore, quecheers method allowed a significant reduction of the time needed for sample preparation than ase extraction. references chauzat, m.p., martel, a.c., cougoule, n., porta, p., lachaize, j., zeggane, s., aubert, m., carpentier, p., faucon, j.p; 2011. an assessment of honeybee colony matrices, apis mellifera (hymenoptera: apidae) to monitor pesticides presence in continental france. environ. toxicol. chem. 30, 1-9 tuzen, m., silici, s., mendil, d., soylak, m.; 2007. trace elements levels in honeys from different regions of turkey. food chem. 103, 325-330. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf l abstract listeria monocytogenes (lm) is a foodborne pathogen responsible of listeriosis. in the spreading of this pathology, milk and dairy products are key reservoir for this pathogen1. food processing represents one of the major steps that could be linked to lm growth. inhibition of lm growth through competition of lactococcus lactis (lac) could represent a solution to this problem. exoproteome of lm and two different strains of lactic acid bacteria in co-culture have been studied in order to highlight mechanisms of bacterial competition useful to improve food safety. two different strains of lac and one strain of lm were cultivated in appropriate medium cultures (bhi), also in competition. filtrated cultures (secretome) were lyophilized and resuspended for proteomics analysis. shotgun analysis on each secretome was performed on nano uplc-ms system. obtained data reveal, during competition, the higher production by lm of moonlighting protein enolase and glucose 6 phosphate isomerase, of septation ring formation regulator ezra, involved into cell replication and the lower secretion of endopeptidase p60. in parallel, l. lactis produced higher amounts of secreted 45 kda protein and switched from lantibiotic nisin a production to nisin z production. in competition with lm, lac strain investigated produce higher amounts of secreted 45 kda protein with peptidoglycan lytic activity and the selective secretion of nisin z probably to improve lantibiotic solubility in less acidic environment. next step will be validation of obtained results in dairy products. these results are of interesting to design new strategies of fighting lm as contaminant in food from animal origin. work supported by ministry of health-ccm “milano expo 2015 project: garantire la sicurezza alimentare valorizzare le produzioni” references greenwood, m. h.; roberts, d.; burden, p., the occurrence of listeria species in milk and dairy products: a national survey in england and wales. international journal of food microbiology 1991, 12, (2), 197-206. corresponding author isabella alloggio, isabella.alloggio@unimi.it food safety: secretome of lactococcus lactis and listeria monocytogenes in competition. i. alloggio 2 , c. piras 2 , v. greco 3,4 , a. soggiu 2 , n. losio 5 , l. bonizzi 2 , a. urbani 3,4 , p.roncada 1,2 . 1 italian experimental institute l. spallanzani, milano, italy 2 department of veterinary sciences and public health (divet), university of milan, milan, italy 3 s. lucia foundation irccs, rome, italy 4 department of internal medicine, university of rome tor vergata, rome, italy 5 experimental zooprofilattico institute of lombardia and emilia romagna, brescia, italy proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy haf © 2013 vol. ii, no. 1s issn: 2283-3927 l corresponding author alessandra cafiso, alessandra.cafiso@unimi.it abstract red mark syndrome (rms) is a chronic skin disease of unknown aetiology affecting farmed rainbow trout oncorhynchus mykiss in europe that causes single or multiple bright red skin lesions. histological analysis showed acute inflammation in the area of the skin suggesting a bacterial infection. no aetiological agent has been unequivocally identified, although the involvement of a single transmissible agent has been suggested. the 16s rdna of a bacterium belonging to the family midichloriaceae (rickettsiales) was found in association with rms skin lesions. in this work, we present a novel specific method for absolute quantification of the midichloriacea associate d with rms in o. mykiss, based on a quantitative pcr approach. the qpcr method was tested on healthy skin, on lesions when present and on organ samples (heart, liver, spleen, intestine, and kidney) from ten fish. our work shows, for the first time, that the midichloriacea is present not only in skin lesions but also in organs of affected fish. further studies are needed to prove whether this bacterium is actually involved in the pathology. references oidtmann b., lapatra s.e., verner-jeffreys d., pond m., peeler e.j., noguera p.a., bruno d.w., st-hilaire s., schubiger c.b., snekvik k., crumlish m., green d.m., metselaar m., rodger h., schmidt-posthaus h., galeotti m., and feist s.w. 2013. journal of fish diseases 36: 931–937; schmidt-posthaus h., bergmann w., knusel r., heistinger h., and licek e. 2009. diseases of aquatic organisms 88:65–68. lloyd s.j., lapatra s.e., snekvik k.r., cain k.d., and call d.r. 2011. journal of fish diseases 34:701–709. metselaar m., thompson k.d., gratacap r.m.l., kik m.j.l., lapatra s.e., lloyd s.j., call d.r., smith p.d., and adams a. 2010. journal of fish diseases 33:849–858. montagna m., sassera d., epis s., bazzocchi c., vannini c., lo n., sacchi l., fukatsu t., petroni g., and bandi c. 2013. applied and environmental microbiology 79:3241–3248. molecular evidence for a bacterium of the family midichloriaceae (order rickettsiales) in skin and organs of the rainbow trout oncorhynchus mykiss affected by red mark syndrome a. cafiso 1 , c. bazzocchi 1 , u. mccarthy 2 1 department of veterinary science and pubblic health, università degli studi di milano, via celoria 10, 20133 milan, italy 2 marine scotland science, marine laboratory, aberdeen, scotland proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords cefazolin, dog, pharmacokinetic, pharmacodynamics corresponding author di cesare federica federica.dicesare@unimi.it journal home page riviste.unimi.it/index.php/haf cefazolin in dog: preliminary results for pharmacokinetic and pharmacodynamic parameters. f. di cesarea*, p. cagnardia, g. ravasiob, f. acocellaa, r. villaa adept. health, animal science and food safety, università degli studi di milano, italy; bdept veterinary medicine, università degli studi di milano, italy abstract cefazolin, a first generation cephalosporin, is commonly used in small animal surgery to avoid postoperative infections. the aims of the study were 1) assess cefazolin pharmacokinetics (pk) in dogs undergoing gonadectomy, 2) correlate pk and pharmacodynamics (pd) parameters and 3) attest the efficacy of dosage regimen through a pk/pd approach. thirty minutes before surgery, 25 mg/kg of cefazolin were administrated intravenously (iv) to 9 dogs (weight 22±7.5 kg; age 1.3±0.7 years). blood samples were taken at prefixed times from 0 to 8 h. quantification of cefazolin concentrations was performed through a validate hplc method with uv detection (kunicki, 2012). a two-compartmental model best described the pk profile of cefazolin. literature mic50 against canine pasteurella spp., staphylococcus spp. and streptococcus spp. ranged from 0.25 to 0.5 µg/ml (goldstein, 2012). distribution and elimination half-lives were 0.3 ± 0.2 h and 3 ± 1.6 h, respectively; area under the curve was 182.3±50.6 h*µg/ml; volume of distribution and clearance at steady state were 383.5±58.1 ml/kg and 150.2±49.8 ml/h/kg, respectively. the pk/pd index for cephalosporins efficacy is time above mic (t>mic) for 60% of dosing interval with values 4 times higher the mic50. our values of t>mic, calculated with mic 0.5 mg/ml, was for 100 % of the observation period from 2 to 6 times higher than mic. preliminary results showed a good efficacy of cefazolin against all the bacterial strains evaluated. therefore, a dosage regimen of 25 mg/kg iv every 8h might represent a valid tool in order to prevent surgical infections in small animal practice. references goldstein, e.j., citron, d.m., merriam, c.v., tyrrell, k.l., 2012. ceftaroline versus isolates from animal bite wounds: comparative in vitro activities against 243 isolates, including 156 pasteurella species isolates. antimicrobial agents and chemotherapy 56, 6319–6323. kunicki, p.k., wàs j., 2012. simple hplc method for cefazolin determination in serum validation and stability testing. journal of chromatography b 911, 133-139. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17t july, milan, italy riviste.unimi.it/index.php/haf l corresponding author andrea talenti, andrea.talenti@unimi.it abstract the recent availability of a medium density snps chip in goat offers the possibility to develop a useful and less expensive tool for parentage assessment. however, standard approaches of snp selection for parentage assignment are still ineffective due to a lack of information about markers position. in this study, we describe the identification of a 200 snps panel for parentage testing in goat. data on 350 goats of 14 different italian breeds genotyped with the illumina 50k snp array were provided by the italian goat consortium (igc). the 200 snps panel was identified by a three-step procedure, as follows: 1) parentage assessment by mendelian errors and genomic parentage to identify true parent-offspring pairs; 2) identification of informative snps by canonical discriminant analysis and 3) reduction by mendelian errors and stepwise regression. the 200 snps panel was tested on pairwise comparison of all animals at each locus. sensitivity, specificity and accuracy of the panel were assessed. the probability of exclusion (pe) and the probability of a random coincidental match inclusion (pi) for each breed were estimated. the panel showed good assessment power, with high sensitivity (0.9429), specificity (1.0) and accuracy (0.99997). pe values ranged from a minimum of 0.99999981 for maltese from sardinia to a maximum of 0.999999999996 for nicastrese. we further reduced panel size by stepwise regression to 174 snps showing the same performance of the 200 snp panel. the development of tools for parentage assessment could improv e breeding management also in species with low genetic information, as goat. acknowledgements dataset was provided by the italian goat consortium. the research was supported by the project "innovagen" (italian mipaaf ministry). references heaton, m. p., g. p. harhay, g. l. bennett, r. t. stone, w. m. grosse, e. casas, j. w. keele, t. p. l. smith, c. g. chitko mckown, and w. w. laegreid. 2002. selection and use of snp markers for animal identification and paternity analysis in u.s. beef cattle. mammalian genome 13(5):272-281; tosser-klopp, g., p. bardou, o. bouchez, c. cabau, r. crooijmans, y. dong, c. donnadieu-tonon, a. eggen, h. c. m. heuven, j. saadiah, j. abdullah johari, c. klopp, c. t. lawley, j. mcewan, p. martin, c. r. moreno, p. mulsant, i. nabihoudine, e. pailhoux, i. palhiere, r. rupp, j. sarry, b. l. sayre, a. development of a 200 single nucleotide polymorphism panel for parentage assessment for 14 italian goat breeds a.talenti 1 , e. l. nicolazzi 2 , l. nicoloso 1 , s. frattini 1 , b. coizet 1 , s. chessa 3 , g. pagnacco 1 , f. pilla 4 , p. ajmone-marsan 5 and p. crepaldi 1 1 department of veterinary science and public health, university of milan, milan, italy. 2 parco tecnologico padano foundation, lodi, italy . 3 cnr – ibba, milan, italy. 4 department of agriculture, environment and food, university of molise, campobasso, italy. 5 university cattolica del sacro cuore, piacenza, italy. proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords goat, lipidic metabolism, inflammation, gene expression, peripartum, adipose tissue, lipidic integration corresponding author alessandro agazzi alessandro.agazzi@unimi.it journal home page riviste.unimi.it/index.php/haf transcriptional regulation of lipid metabolism and inflammation in transition dairy goats by fish oil and stearate. g. farinaa, a. agazzi*a, g. invernizzia, a. campagnolia, j.j. loorb, g. savoini1 adipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare, università degli studi di milano, milano, italy bdepartment of animal sciences, uiuc, illinois, usa. abstract to better understand the interaction between saturated or unsaturated fatty acids and its effect on expression of genes involved in subcutaneous adipose tissue metabolism, 23 second parity alpine dairy goats were enrolled in the experiment and fed either a non fat-supplemented basal diet (c; n=8), the basal diet supplemented with stearic acid (st; n=7) or the basal diet supplemented with fish oil (fo; n=8). 30g/head/d supplemental fatty acids during the dry period and 50g/head/d during lactation were delivered starting one week before parturition up to 21 days in milk. subcutaneous adipose tissue samples were harvested at day -7, 7 and 21 relative to kidding and mrna levels of genes involved in inflammation were measured via qpcr. data were analyzed using the mixed procedure of sas. no significant effects for treatment were observed, however eight genes were significant for time. hp and saa3 expression peaked at day 7 postpartum, to then return at prepartum level around 21 d relative to kidding, while il8, il10, and il18 expression constantly increased along the transition period. vice versa, expression of il1β, il6r, and rxra decreased in response to kidding, with a subsequent increase at day 21. the obtained results led us to hypothesize that goats face a postponed lipomobilization after kidding, probably related to their reduced production. the next step will involve the analysis of mirna related to immune cell infiltration, adipocyte inflammation and lipolysis and positive regulation of adipogenesis to better understand the complex network of lipid metabolism in periparturient goats. references agazzi a., cattaneo, d., dell’orto, v., moroni, p., bonizzi, l., pasotto, d., and savoini, g., 2004. effect of administration of fish oil on aspects of cellmediated immune response in periparturient dairy goats. small ruminant research. 55, 77-83. calder, p.c., and grimble, r.f., 2002. polyunsaturated fatty acids, inflammation and immunity. europ. j. clincal nutr. 56, s14-s19. invernizzi, g., corbani, d., caputo, j.m., campagnoli, a., pisani, l.f., bronzo, v., agazzi, a., modina, s., and savoini, g., 2012. dietary fatty acids on subcutaneous adipose tissue modulation in transition dairy goats. page 312 in proc. xi international conference on goats, gran canaria, spain. mallard, b.a., dehhers, j.c., ireland, m.j., leslie, k.e., sharif, s., lacey vankampen, c., wagter, l., and wilkie, b.n., 1998. alteration in immune responsiveness during the peripartum period and its ramification on dairy cow and calf health. journal of dairy science. 81, 585–594. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords ixodes ricinus, midichloria mitochondrii, serological screening, mammalian hosts, marker. corresponding author valentina serra valentina.serra@unimi.it journal home page riviste.unimi.it/index.php/haf molecular and serological evidences of midichloria mitochondrii transmission to vertebrate hosts during the tick bite. v. serraa*, a. cafisoa, c. bazzocchia,b a department of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy b joint research center for epidemiology and molecular surveillance of infections, university of milan, 20133 milan, italy abstract the tick ixodes ricinus is vector of many pathogens important for human and animal health (parola and raoult, 2001; socolovschi, 2009). midichloria mitochondrii (order rickettsiales; family midichloriaceae) is an endosymbiont present in the salivary glands of 100% of ixodes ricinus females. two lines of evidence suggest a transmission of m. mitochondrii in mammalians during the tick bite: 1) detection of circulating dna in blood samples of different animal species; 2) seropositivity toward a m. mitochondrii protein (flid) in humans and dogs exposed to tick bite (mariconti 2012; bazzocchi 2013). here we present serological and molecular results demonstrating the circulation of m. mitochondrii also in capreolus capreolus (the host of choice for adult and nymph stages of i. ricinus), confirming that this host is a good subject to study the spread of tick-borne pathogens. based on these results, flid protein and other m. mitochondrii markers could thus be extremely useful to determine the risk of infection by i. ricinus pathogens in given areas, and for investigating the epidemiological association of a variety of pathological alterations with this tick. here we show results of the screening of 218 human sera (50 from non endemic areas used as negative controls and 168 from subjects exposed to tick bite) collected in different areas of germany. results showed that 48 out of 168 sera were positive to m. mitochondrii. these results have posed the basis for the development of a serological test for investigating the exposure of humans and animals to this tick species. references bazzocchi, c., mariconti, m., sassera, d., rinaldi, l., martin, e., cringoli, g., urbanelli, s., genchi, c., bandi, c., epis, s., 2013. molecular and serological evidence for the circulation of the tick symbiont midichloria (rickettsiales: midichloriaceae) in different mammalian species. parasites & vectors 6, 350–56. mariconti, m., epis, s., gaibani, p., valle, c. dalla, sassera, d., tomao, p., fabbi, m., castelli, f., marone, p., sambri, v ., bazzocchi, c., bandi, c., 2012. humans parasitized by the hard tick ixodes ricinus are seropositive to midichloria mitochondrii: is midichloria a novel pathogen, or just a marker of tick bite? pathogens and global health 106, 391–396. parola, p., raoult, d., 2001. ticks and tickborne bacterial diseases in humans: an emerging infectious threat. clinical infectious diseases 32, 897–928. socolovschi, c., mediannikov, o., raoult, d., parola, p., 2009. the relationship between spotted fever group rickettsiae and ixodid ticks. veterinary research 40, 34. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords epigenetic, 5-aza-cr, methylation, plasticity, tet genes, histone corresponding author elena f.m. manzoni elena.manzoni@unimi.it journal home page riviste.unimi.it/index.php/haf 5-azacytidine reactivates pluripotency gene expression, affects tet2 and histone transcription and modifies chromatin organization and morphology of mammal skin fibroblasts. e. f.m. manzonia* , t. a.l. brevinib, f. gandolfia adepartment of agricultural and environmental sciences production, landscape, agroenergy, università degli studi di milano, via celoria 2, 20122 milan, italy bdepartment of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy abstract phenotype expression is controlled by epigenetic regulations that guide cells through differentiation. the process is reversible and cells can be driven back to a higher plasticity state with the use of epigenetic modifiers. in this work we exposed skin fibroblasts to the demethylating agent 5-azacytidine (5-aza-cr), which is a well-known dna methyltransferase inhibitor, and has been recently shown to increase cell plasticity and facilitate phenotype changes in different cell types (pennarossa, 2013; brevini, 2014; pennarossa, 2014; brevini, 2016). although many aspects controlling its demethylating action have been widely investigated, the mechanisms through which 5-aza-cr acts on cell plasticity are still poorly understood. at the end of 5-aza-cr treatment, cells were divided in three experimental groups and cultured for 24 and 48 hours: a) cells were returned in standard fibroblast medium; b) cells were cultured in medium specific for pluripotency maintenance; c) cells were placed in a medium encouraging pancreatic differentiation. at the end of the culture period, as expected, we could observe a global demethylating effect. in parallel, however we detected a transient upregulation of the pluripotency genes oct4, nanog and rex1. increased transcription of tet2 and histones belonging to the 1,2,3 and 4 families, together with changes in the expression of enzymes controlling histone acetylation were also appreciated. interestingly, these results were accompanied by morphological and ultrastructural changes as well as by chromatin structure modifications. all together our findings indicate that 5-aza-cr induced somatic cell transition to a higher plasticity state involves novel cellular targets that activate multiple epigenetic regulatory pathways. supported by efsd and carraresi foundation. references brevini, t. a., pennarossa, g., rahman, m.m., paffoni, a., antonini, s., ragni, g., deeguileor, m., tettamanti, g., gandolfi, f., 2014. morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation. stem cell rev 10, 633-642. brevini, t. a., pennarossa, g., acocella, f., brizzola, s., zenobi, a., gandolfi, f., 2016. epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells. the veterinary journal 211, 52-53. pennarossa, g., maffei, s., campagnol, m., tarantini, l., gandolfi, f., brevini, t.a., 2013. brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. proc natl acad sci u s a 110, 8948-8953. pennarossa, g., maffei, s., campagnol, m., rahman, m.m., brevini, t.a., gandolfi, f., 2014. reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. stem cell rev 10, 31-43. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords testudinid herpesvirus, tehv3, reptile immunology, testudo. corresponding author marco tecilla marco.tecilla@unimi.it journal home page riviste.unimi.it/index.php/haf characterization of the immune response against testudinid herpesvirus 3. m. tecillaa*, p. roccabiancaa, p. pilob, f. origgic a department of veterinary science and public health (divet), school of veterinary medicine, university of milan, 20010, milan, italy. b institute of veterinary bacteriology, vetsuisse faculty, university of bern, laenggassstrasse 122, 3012, bern, switzerland. c centre for fish and wildlife health (fiwi), vetsuisse faculty, university of bern, laenggassstrasse 122, 3012, bern, switzerland. abstract numerous infectious diseases have been documented in reptiles, however minimal information is available concerning their immunological response. one of the most diffuse and lethal reptile pathogen is testudinid herpesvirus 3 (tehv3), a alphaherpesvirinae. all species of tortoises (testudinide) are considered susceptible to tehv3, however the virus is over represented in the genus testudo, which includes, among others, t. graeca, t. hermanni, t. marginata, and t. horsfieldii, that are popular pets in europe. incidence of tehv3-associated disease is highest right after hibernation (origgi, 2012). the aim of this work is to partially characterize the immunological response of t. graeca against tehv3. a bacteriophage library composed of about 5.000 clones containing genomic dna fragments of tehv3 was produced. bacteriophages were amplified in a specific strain of e. coli and were screened with tehv3-seropositive sera from t. graeca. phagemids were excised from the positive bacteriophages, sequenced, and compare with the tehv3 genome to identify the encoding genes. six different structural and non-structural proteins have identified as immune relevant. vero cells where transfected with phagemids of the positive clones, to confirm previous results. tehv3’s proteins expression was assessed by f.a.c.s using t. graeca seropositive sera. of all the six selected clones, only that expressing the partial sequence of the glycoprotein b (gb) showed a positive signal in the f.a.c.s. analysis. this result is consistent with the well-known immunogenicity of gb of other herpesviruses including those infecting humans and with the highly conserved role that gb plays in host-pathogen interaction across species and evolution (beals et al., 2016). references beals, c.r., railkar, r.a., schaeffer, a.k., levin, y., kochba, e., meyer, b.k., evans, r.k., sheldon, e.a., lasseter, k., lang, n., et al. (2016). immune response and reactogenicity of intradermal administration versus subcutaneous administration of varicella-zoster virus vaccine: an exploratory, randomised, partly blinded trial. lancet infect. dis. origgi, f.c. (2012). testudinid herpesviruses: a review. j. herpetol. med. surg. 22, 42–54 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords bovine, teeth, β2-agonists, residues, hplc-ms/ms. corresponding author maria nobile maria.nobile1@unimi.it journal home page riviste.unimi.it/index.php/haf detection of seven β2-agonists in teeth by lc-ms/ms: preliminary results. m. nobilea*, s. panseria, f. ariolia, l. m. chiesaa a department of health, animal science and food safety, university of milan, via celoria 10, 20133 milan, italy abstract β2-agonists are powerful tocolytic (the only use permitted in cattle) and bronchodilator agents, but may also be administered as growth promoters to improve the production of lean meat increasing also the lipolytic activity. although the european union (eu), china and other asian countries have banned the use of β2-agonists for growth promoting purposes (european union, 2003), the united states of america (usa) authorised ractopamine as a feed additive for swine, cattle and turkey. these veterinary drugs, generally show high clearance rates in the conventional biological matrices, as well as urine, liver and muscle, making difficult their detection (wu, 2014). for this reason, we suggested bovine teeth as a new unconventional matrix of accumulation in a more long-time window, for the detection of cimaterol, clenbuterol, isoxsuprine, mabuterol, ractopamine, salbutamol and terbutaline. in literature, the few studies on teeth are limited to human (andra, 2015) and are absent for veterinary medicine. the samples extracted by a simple liquid extraction step with ethyl acetate:tert-butyl methyl ether (4:1, v/v) after washing and pulverization of teeth, through a ball mill, were analysed using a liquid chromatography tandem mass spectrometry (lc-ms/ms) confirmatory method validated according to the commission decision 2002/657/ec criteria (european union, 2002). teeth from 8 veal calves, administered per os with 80 mg day-1 oral ractopamine for 32 days, and from seven random bovines from the food chain were collected at the slaughterhouse to test the suitability of this matrix. the results demonstrated ractopamine presence in teeth from the treated animals (average concentration 8.90 ng g-1). isoxsuprine was found in a control sample (13.67 ng g-1), demonstrating the effectiveness of this matrix as a powerful tool to ensure illegal treatment. references andra, s.s., austin, c., arora, m., 2015. tooth matrix analysis for biomonitoring of organic chemical exposure: current status, challenges, and opportunities. environ. res. 142, 387-406. european union, 2002. commission decision concerning the performance of analytical methods and the interpretation of results. off. j. eur. comm. l221, 8-36. european union, 2003. council directive 2003/74/ec amending council directive 96/22/ec concerning the prohibition on the use of stock farming of certain substances having a hormonal or thyrostatic action and of β-agonists. off. j. eur. union. l262, p.17. wu, j., liu, x., peng, y., 2014. determination of ractopamine in pig hair using liquid chromatography with tandem mass spectrometric detection. j. pharmacol. toxicol. meth. 69, 211-216. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords panthera leo, personality assessment, zsl, african lions, methodologies corresponding author silvia mazzola silvia.mazzola@unimi.it journal home page riviste.unimi.it/index.php/haf personality assessment and feline–keepers relationship in lions (panthera leo). g. quintavalle pastorinoac, n.r.p. soaresbc, s. mazzolaa*, r. preziosid, m. albertinia adepartment of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy bthe royal veterinary college, royal college street, london, nw1 0tu, united kingdom cinstitute of zoology, zoological society of london, regents park, london, nw1 4ry, united kingdom dfaculty of life sciences, the university of manchester, oxford road, manchester, m13 9pt, united kingdom abstract animal personality is a growing research area due to the increasing evidence of the impact that it has on welfare, health and management of animals in captivity (freeman and gosling, 2010). testing and improving existent methodologies, as well as develop new ones, to outline animal personality is an important step towards welfare, health and longevity of captive animals (phillips, 2007; whitham and wielebnowski, 2013). lions (panthera leo) were chosen for this study because the species is understudied compared to other felidae species in personality matters and because it displays a vast, diverse and well known behaviour repertoire (schaller, 1973). behavioural observations were conducted on the seven african lions housed in zsl whipsnade zoo. keeper-animal interactions were recorded and a personality questionnaire was given to the keepers in order to rate 28 personality traits (chadwick, 2014). sociogram, composite sociality index (csi) and spread of participation index (spi) were also used to assess personality traits (stanton 2015). data gathered in this pilot study was successfully used to produce id like personality traits profiles for all animals, to delineate personality traits, outline the unique characteristics of each subject. a sociogram consent to access and validate the dyad solitary/social and a spi to understand each animal enclosure usage and relate it to the trait triad affiliate/fearful/aggressive towards humans. we believe this approach is promising and could be applied to investigate keeper-animal interactions also in other species. the limitation of this study is the sample size of the animal considered; in future research we hope to test other animals in order to validate the methodologies. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. ii, no. 2s issn: 2283-3927 figure 1. whipsnade zoo african lion pride’s sociogram. the line that connects two animals is accompanied by a number that can range from 0 to 1, representing the closeness of relationship between the two: 1 means inseparable animals, while 0 represent animals that don’t cross their paths at all. references chadwick, c. 2014. social behaviour and personality assessment as a tool for improving the management of cheetahs (acinonyx jubatus) in captivity. [dissertation]. salford, salford university. freeman, hd., & gosling, sd. 2010. personality in nonhuman primates: a review and evaluation of past research. american journal of primatology, 72(8), 653-671. phillips, c., & peck, d. 2007. the effects of personality of keepers and tigers (panthera tigris tigris) on their behaviour in an interactive zoo exhibit. applied animal behaviour science, 106(4), 244-258. stanton, la., sullivan, ms., fazio, jm. 2015. a standardized ethogram for the felidae: a tool for behavioural researchers. applied animal sciences. whitham, jc., & wielebnowski, n. 2013. new directions for zoo animal welfare science. applied animal behaviour science, 147(3–4), 247-260. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l corresponding author maurizio longo, maurizio.longo@unimi.it abstract adrenal gland tumors are common in humans and in several animal species. studies concerning this neoplasia in human medicine indicate that clinical signs have a high variability. adrenal adenomas can be occasionally observed in asymptomatic patients during tomographic studies while estrogen-secreting tumors, known as "feminizing adrenal tumors" (fats), have been rarely reported. the aim of this study is to describe for the first time the imaging findings of a captivity lion affected by a neoplastic secreting adrenal tumour. an 8 year-old male lion with progressive lack of secondary sex characteristics, disorexia and weight loss was referred to our institution. the patient was chemically immobilized to undergo general clinical evaluation, hematologic, serum biochemical and hormonal profile, fiv and felv tests. three months later a total body computed tomography and abdominal ultrasonography were performed. liver and left adrenal lesions fnabs were performed. imaging findings showed the presence of an extended expansive neoplastic lesion on the left adrenal gland (40x39x37 mm) with right adrenal gland atrophy. generalized hepatopathy associated with a suspected intrahepatic cholestasis was confirmed by ultrasonography. cytological evaluation ruled out the presence of neuroendocrine cells without malignancy evidences compatible with the adenomatous nature of the lesion, associated with moderate degenerative hepatopathy. blood tests reported an estradiol concentration of 462 ng/dl. to our knowledge, this is the first description of adrenal mass in a lion associated with secondary feminization, inappetence and high values of hematic estradiol, referable to a feminizing adrenal tumor (fat). references feldman ec & nelson w r. canine and feline endocrinology and reproduction. 3rd ed, saunders, 2004, pp 251-357; 2) chidiac rm et al.endocrinol metab clin north am 1997,26:233–253; 3) moreno s, et al. ann endocrinol (paris) 2006;67:32–8; 4) gregori t, et al. vet radiol ultrasound. 2015 mar; 56(2):153-9 a case of adrenal tumour in a lion (panthera leo): tomographic and ultrasonographic findings. m. longo, g. ravasio, d. de zani, m. di giancamillo, v. rabbogliatti, d. zani centro clinico veterinario e zootecnico sperimentale, lodi (lo), department of health, animal science and food safety, università degli studi di milano http://www.ncbi.nlm.nih.gov/pubmed/25139015 proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords equine asthma, pulmonary vascular remodeling, pulmonary hypertension, histomorphometry corresponding author serena ceriotti serena.ceriotti@unimi.it journal home page riviste.unimi.it/index.php/haf pulmonary vascular remodeling occurs in severe equine asthma. s. ceriottia*, m. bulloneb, a. vargasb, m. leclèreb, f. ferruccia, j. lavoieb. adepartment of health, animal science and food safety, faculty of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milano, italy bdepartment of clinical sciences, faculty of veterinary medicine, université de montréal, 3200 rue sicotte, sainthyacinthe, québec, canada, j2s 6c7 abstract severe equine asthma (heaves) is a chronic genetic-environmental condition characterized by airway neutrophilic inflammation leading to airway obstruction, lung tissue remodeling and hypoxemia (bullone, 2015). hypoxemia triggers pulmonary vasoconstriction, resulting in higher vascular resistance and development of pulmonary hypertension (ph). ph-associated cardiovascular complications could be present in the end-stage disease, as it happens in human type 3 ph (tsangaris, 2012). pulmonary vascular remodeling is involved in the pathogenesis of type 3 ph (singh, 2016) and may represent a therapeutic target for preventing ph onset, both in human and in horses. in this study, we investigated the presence of pulmonary artery remodeling in severe equine asthma, as, to the best of our knowledge, this has not been yet studied. lung biopsy specimens were collected via thoracoscopy from 6 asthmatic and 5 age-matched control horses. histomorphometric assessment was performed on movat-russell stained histological sections, evaluating pulmonary artery wall area, intimal area, medial area and their correlations with the internal elastic lamina length (iel) (fernie, 1985). the total amount of smooth muscle within the artery wall and the density of proliferating smooth muscle cells were similarly evaluated using immunostaining for α-smooth muscle actin (α-sma) and proliferating cell-associated nuclear antigen (pcna). increased pulmonary wall area, increased amount of smooth muscle and loss of correlation between intimal area and iel were present in asthmatic horses, when compared to controls. in conclusion, pulmonary artery remodeling due to smooth muscle hypertrophy and intimal muscolarization may be induced by persistence of hypoxic vasoconstriction stimulus and release of cytokines from inflammatory cells infiltrating the annexed airways (barbera, 2003). references barbera j.a., peinado v.i., santos s., 2003. pulmonary hypertension in chronic obstructive pulmonary disease. eur respir j. 21(5), 892-905. bullone m., lavoie j.p., 2015. asthma "of horses and men"--how can equine heaves help us better understand human asthma immunopathology and its functional consequences? mol immunol. 66(1), 97-105. fernie j.m., lamb d., 1985. method for maximising measurements of muscular pulmonary arteries. j clin pathol. 38(12), 1380 -1387. singh i., ma k.c., berlin d.a., 2016. pathophysiology of pulmonary hypertension in chronic parenchymal lung disease. am j med. 129(4), 366-371 tsangaris i., tsaknis g., anthi a., orfanos s.e., 2012. pulmonary hypertension in parenchymal lung disease. pulm med. 2012, 684781. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l ticks are haematophagous ectoparasites of vertebrates habitually parasitizing avian species, which may contribute to tick dispersal across continents during migrations (hasle 2013; altizer et al., 2011). midichloria bacteria can be transmitted to the vertebrate host during the tick bite (bazzocchi et al., 2013; serra et al., 2018). although many avian species are common hosts of ticks harbouring midichloria (e.g. ixodes, hyalomma), the circulation of this bacterium in birds has never been investigated. the aims of this study are: 1) evaluate the presence of midichloria dna in h. marginatum ticks and blood collected from trans-saharan migratory birds; 2) quantify midichloria bacteria in ticks through a novel quantitative pcr (qpcr). a total of 256 h. marginatum ticks and 97 blood samples were collected from three different migratory species (phoenicurus phoenicurus, saxicola rubetra and sylvia communis) on ventotene island (central italy) and dnas were extracted. a nested-pcr targeting the 16s rrna gene of midichloria was used to detect bacterial presence. subsequently, primers targeting the gyrb gene of midichloria and the cal gene of h. marginatum were designed and used in a qpcr for midichloria quantification. results were expressed as gyrb/cal copy numbers ratio. 94% of hyalomma ticks harbored dna of midichloria belonging to the monophylum associated with ticks, while the bacterial dna was detected in 44.3% of blood samples. furthermore, engorged ticks showed significantly higher bacteria load than unengorged ticks (table 1; wilcoxon sum-rank test: z=3.14; p=0.0017), similarly to what has been observed for m. mitochondrii in i. ricinus ticks. this work provides evidence for the presence of circulating midichloria dna in long-distance migratory birds, suggesting an enhanced worldwide spread of these bacteria across haematophagous ectoparasite populations. future studies are necessary to increase the knowledge of midichloria role in the biology of this tick species. keywords hard ticks, midichloria, hyalomma marginatum, migratory birds. corresponding author valentina serra valentina.serra@unimi.it journal home page riviste.unimi.it/index.php/haf detection of a novel bacterium of the genus midichloria (family midichloriaceae) in avian-borne hyalomma marginatum ticks and their trans-saharan migratory hosts. v. serra1,*, c. bazzocchi1, i. di lecce2 1 department of veterinary medicine, university of milan, via celoria 10, 20133 milan, italy. 2 wild urban evolution and ecology lab, centre of new technologies, university of warsaw, banacha 2c, 02-097 warsaw, poland. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 18 haf © 2013 vol. v, no. 1s issn: 2283-3927 references altizer, s., bartel, r., han ba., 2011. animal migration and infectious disease risk. science. 331, 296-302. bazzocchi, c., mariconti, m., sassera, d., rinaldi, l., martin, e., cringoli, g., urbanelli, s., genchi, c., bandi, c., epis, s., 2013. molecular and serological evidence for the circulation of the tick symbiont midichloria (rickettsiales: midichloriaceae) in different mammalian species. parasites & vectors. 6, 350-56. hasle, g., 2013. transport of ixodid ticks and tick-borne pathogens by migratory birds. frontiers in cellular and infection microbiology. 3, 1-6. serra, v., cafiso, a., formenti, n., verheyden, h., plantard, o., bazzocchi, c., sassera, d., 2018. molecular and serological evidence of the presence of midichloria mitochondrii in roe deer (capreolus capreolus) in france. journal of wildlife diseases. doi: 10.7589/2017-09-241. table 1: range values of gyrb and cal copy numbers and of gyrb/cal ratios obtained in h. marginatum ticks through qpcr analysis. gyrb copy number range cal copy number range gyrb/cal range unengorged ticks 6.8 x 102 – 1.5 x 105 7.2 x 10 – 5.8 x 103 7.7 – 5.2 x 102 engorged ticks 2.2 x 102 – 1.8 x 105 6.6 x 10 – 6.2 x 102 1.5 x 10 – 8.2 x 103 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2015, 15th17th july, milan, italy riviste.unimi.it/index.php/haf 1 l nathalie beaujean dr. nathalie beaujean is group leader at the french national institute for agricultural research near paris. over the past 10 years, her team has developed studies on preimplantation development, genome reprogramming, and the control of pluripotency during early development, mainly in murine and nonmurine mammalian species. she is very experienced in the analysis of epigenetic reprogramming by powerful image analysis tools dedicated to the preimplantation embryos. one of her objectives is to better understand embryonic genome reprogramming after either fertilization or nuclear transfer (the cloning procedure). cloning indeed offers a unique opportunity to understand how the oocyte's environment dictates the reorganization of the introduced nuclei, and thereby the transcriptional programs that can have long‐term consequences. it should help to establish what constitutes a good nuclear organization and to understand how the environment shapes the epigenome. nathalie.beaujean@jouy.inra.frl histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture nathalie beaujean 1 1 inra, umr1198 biologie du développement et reproduction, f-78350 jouyen-josas, france main lecture in mammals, epigenetic markers are globally rearranged after fertilization: while gametes carry special epigenetic signatures and a unique nuclear organization, they attain embryo-specific patterns after fertilization. this “reprogramming” is promoted by the intimate contact between the parental inherited genomes and the oocyte cytoplasm over the first cell cycles of development. interestingly, histone post-translational modifications (ptms) are among the factors involved in this reprogramming. during the last few years, many studies focusing on epigenetic modifications have indeed shown that, immediately after fertilization, different histone ptm profiles create an asymmetry between the two parental genomes, although both parental genomes undergo global hyperacetylation and hypomethylation. thereafter, histone ptms reprogramming goes on (beaujean et al., mrd 2014). it is hypothesized that this ptms reprogramming is required for the embryonic genome activation (ega). recently, we for example put forward the importance of the prc1 complex that binds h3k27me3, for proper ega and development beyond the two-cell stage (posfai et al., 2012). by the stage of implantation (blastocyst stage) two cell subpopulations forms: an outer layer of epithelial trophectoderm cells (te) and the inner cell mass (icm) located eccentrically within the blastocoelic cavity. remarkably, some histone ptms have been found to differ between the icm vs. te and to correlate with specific gene expression in each of these cell types (dahl et al., 2010; vermilyea et al., 2009). on the other hand, it is well known that diverse parts of the genome have different types of chromatin configuration depending on their function (centromeric and telomeric heterochromatin for instance). interestingly, the mouse embryo presents a unique organization of the peri-centromeric heterochromatin that locates around the nucleoli. this configuration is rapidly acquired in the maternal pronucleus and more progressively in the paternal one (martin et al., 2006; aguirre-lavin et al., 2012), probably due to the specific epigenetic marks present only in the paternal chromatin. during the 2-cell stage, dissociation of pericentromeric heterochromatin from nucleoli begins, concomitantly with the major phase of embryonic genome activation, although the importance of this remodeling is not yet well understood. remarkably, it however seems that transcripts generated by pericentromeric satellite repeats are involved in this event and that interference with this phenomenon results in developmental arrest (probst et al., 2010; santenard et al., 2010; fulka & langerova 2014). altogether, it suggests that histone ptms may be closely correlated with the formation of a transcriptionally active or repressive state during early embryonic development and that they can modify chromatin organization and nuclear architecture during mouse embryonic development. it should also be mentioned that knock-outs of several histone modification enzymes have underlined the importance of ptms during preimplantation development. mailto:ad.vanvuuren@upcmail.nl proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords methicillin-resistant s. epidermidis, nonunion, mesenchymal stem cells, rat model, osteomyelitis corresponding author marta bottagisio marta.bottagisio@unimi.it journal home page riviste.unimi.it/index.php/haf evaluation of antibiotic and cell-based therapy in preventing s. epidermidis-induced nonunion in rats. m. bottagisio a, b *, l. drago c, d, c. romanò e, l. bonizzi a, a.b. lovati b a department of veterinary science and public health, university of milan, via celoria 10, 20133 milan, italy. b cell and tissue engineering laboratory, irccs galeazzi orthopaedic institute, via r. galeazzi 4, 20161 milan, italy. c laboratory of clinical chemistry and microbiology, irccs galeazzi orthopaedic institute, via r. galeazzi 4, 20161 milan, italy. d department of biomedical science for health, university of milan, via l. mangiagalli 31, 20133 milan, italy. e dipartimento di chirurgia ricostruttiva e delle infezioni osteo-articolari, irccs galeazzi orthopaedic institute, via r. galeazzi 4, 20161 milan, italy. abstract methicillin-resistant s. epidermidis (mrse) is responsible for biofilm-related infections (montanaro, 2011; romanò, 2013) and fracture nonunion, as recently demonstrated by our group (lovati, 2016). the present study aims to investigate the efficacy of antibiotic or cell-based therapies in preventing bacterial infections and nonunion establishment. under anesthesia, femoral fractures were performed in 30 rats, then the site of injury was injected with a clinical-derived mrse strain and, finally, synthesized with stainless steel plates. rats were differently treated as follows: mrse-infected controls (ic); systemically-injected vancomycin (s-vanc); local vancomycin-enriched hydrogel (l-hyd); systemically-injected bmscs (s-bmscs); and locallyinjected bmscs (l-bmscs). after 6 weeks, pro-inflammatory cytokines, quantitative micro-ct, histological and microbiological analyses were carried out to investigate the host response to the different treatments. half of the s-bmscs rats died closely to the systemic cell injection, thus excluded for further analyses. our results for the ic group were consistent with previously published data (lovati, 2016), showing signs of osteomyelitis and nonunion development. in s-vanc and l-hyd groups, micro-ct detected a good bony bridging and the microbiological counts were significantly lower with respect to the other groups. our study suggests that the association of s-vanc and l-hyd is an effective treatment to prevent biofilm-induced nonunions. differently, we cannot positively support cell therapies for this purpose due to the high risk related to the systemic cell injection, thus requiring further studies to be eventually proposed in clinics. references lovati, a.b., romanò, c.l., bottagisio, m., monti, l., de vecchi, e., previdi, s., accetta, r., drago, l., 2016. modeling staphylococcus epidermidis-induced non-unions: subclinical and clinical evidence in rats. plos one 11, e0147447. montanaro, l., speziale, p., campoccia, d., ravaioli, s., cangini, i., pietrocola, g., giannini, s., arciola, c.r., 2011. scenery of staphylococcus implant infections in orthopedics. future microbiol 6, 1329-1349. romanò, c.l., toscano, m., romanò, d., drago, l.,2013. antibiofilm agents and implant-related infections in orthopaedics: where are we? j chemother 25, 67-80. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l organochlorine pesticides (ocps) and polychlorinated biphenyls (pcbs) are synthetic chlorinated compounds classified as pops whereas only the penta e tetra-brominated polybromodiphenyl ethers (pbdes) are so defined by the stockolm convention (stockholm convention, 2005) in order to elimitate or restrict the use of pops. organophosphorus insecticides (ocps) represent important environmental and food contamination sources, widely used in agriculture. among polyciclic aromatic hydrocarbons (pahs), benzo[a]pirene is classified by iarc in group 1, as cancerogen and benzo[a] fluoranthene as a group 2b, as possible cancerogen (iarc, 2012; iarc, 2010). efsa (european food safety authority) has released a scientific opinion on the risks to public health related to the presence of brominated flame retardants in food (efsa, 2011) and in 2014 european commission has asked member states to monitor the presence of brominated flame retardants (bfrs) in food over the next two years (ec, 2014). due to their heir n-octanol/water partition coefficient (log kow), they accumulate in fat tissue, bioconcentrate and biomagnify in the animals at the higher trophic levels, possibly causing, through chronic exposure, endocrine disruption and cancer (wania et al., 1995; vallack et al., 1998). the aim of this study is to evaluate the presence of ocps, pcbs, pbdes and pahs in chamois and wild boar from eastern piedmont, italy. a total of 20 chamois and 20 wild boar muscle samples were collected during the hunting season 2017, from verbania cusio ossola (vco) (fig 1). the chemical analysis for the detection of ocps, pcbs, pbdes, and pahs was performed by gc-ms/ms on muscle samples purified and extracted using a quechers technique, validated according to sante 2017 (sante/11183/2017). these preliminary results show the ubiquitary presence of the studied contaminants. pcbs have been found more in chamois (45%) than in wild boar (35%). no pbdes were detected in wild boar but in chamois were found with a prevalence of 35% and concentration 0.25-1.52 ng g-1. about ocps, phorate and demeton were found in wild boar (55%-15%) and chamois (32%35%) with range concentrations 0.21-20.1 ng g-1. no pahs were detected, expect antharacene for one samples in wild boar (0.53 ng g-1). further studies are in progress in order to correlate environmental contamination and game animals. keywords pcbs, gc-ms/ms, game animals, ocps, pbdes. corresponding author federica ceriani federica.ceriani@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary studies on environmental pollutants in chamois and wild boar from eastern piedmont, italy. f. ceriani1,*, l. m. chiesa1, s. panseri1, f. arioli1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 4 haf © 2013 vol. v, no. 1s issn: 2283-3927 references commission recommendation (ec), 2014. on the monitoring of traces of brominated flame retardants in food. official journal of the european union. l 65/39. directorate-general for health and food safety (dg_sante), 2017. sante/11813/2017. guidance document on anylitical quality control and method validation procedures for pesticide residues and analysis in food and feed. efsa journal. 9(5), 2156. scientific opinion on polybrominated diphenyl ethers (pbdes) in food. efsa journal 9. international agency for research on cancer (iarc), 2010. iarc monographs on the evaluation on carcinogenic risks to humans.benzo[a]fluoranthene. 92, sup 7. international agency for research on cancer (iarc), 2012. iarc monographs on the evaluation on carcinogenic risks to humans. benzo[a]pyrene. sup 7, 92, 100f. stockholm convention on persistent organic pollutants. united nations environmental progamme. http://chm.pops.int/theconvention (accessed 24.04.18). vallack, h.w., bakker, d.j., brandt, i., broström-lundén, e., brouwer, a., bull, k. r., koch, r., 1998. controlling persistent organic pollutants–what next?. environmental toxicology and pharmacology. 6, 143-175. wania, f., mackay, d., 1995. a global distribution model for persistent organic chemicals. science of the total environment.12, 211-232. figure 1: collecting areas of chamois and wild boar. 1 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en http://chm.pops.int/theconvention proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l an early diagnosis of sepsis could allow a better prognosis and avoid the abuse of antibiotic administration; unfortunately, in veterinary medicine, specific and sensitive markers of sepsis are not available. because protein carbonyls (pcos), that result from protein oxidation, are widely used in human medicine as sepsis markers (abu-zidan et al., 2002; colombo et al., 2015), the aim of our study was to validate an elisa kit (enzolifesciences, 3v chimica, roma) on canine serum and to measure pcos, after a preliminary validation study, in three groups (homogeneous for age and size): healthy dogs without clinical or laboratory abnormalities (a, n=14), dogs with septic (b, n=14) and non-septic inflammation (c, n=12) at the first presentation and without previous treatments. moreover, paraoxonase-1, a negative acute phase protein with anti-oxidant properties (pon-1) was measured in each group with a method already validated in dogs (rossi et al., 2013). a kruskal-wallis test followed by a wilcoxon signed rank test was used to evaluate differences between groups. the elisa method for measuring pcos showed a very good precision (coefficient of variation <12%) and a good accuracy in spiking-recovery tests. compared with controls, the concentration of pcos was significantly higher either in dogs with sepsis (p<0.001) or in dogs with non-septic inflammation (p=0.005) but no significant differences were found between the two groups of sick dogs (figure 1a). conversely, pon-1 was significantly lower in sick dogs compared with controls (p<0.001 for both groups) and in septic dogs compared with dogs with non-septic inflammation (p=0.001) (figure 1b). a negative correlation between the two markers was found (p<0.001, r=-0.594) receiver operating characteristic (roc) curves demonstrated that both markers may discriminate dogs with sepsis with the other groups. however, pco was less sensitive than pon-1 in diagnosing sepsis. future studies should be focused on the association of pcos with other inflammatory markers, as well as the possible prognostic role of pcos based on the outcome of the enrolled patients. keywords elisa, oxidative stress, pco, pon-1. corresponding author beatrice ruggerone beatrice.ruggerone@unimi.it journal home page riviste.unimi.it/index.php/haf diagnosis of sepsis in dogs by measuring carbonylated proteins (pcos) and paraoxonase (pon-1). b. ruggerone1,2,*, r. troia3, e. murgia3, m. giunti3, f. dondi3, s. paltrinieri1,2 1 department of veterinary medicine, university of milan, via celoria, 10 20133, milano, italy. 2 veterinary teaching hospital, university of milan, via dell’università 6, 26900 lodi, italy. 3 university of bologna, department of veterinary medical sciences, ozzano emilia, bologna, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 22 haf © 2013 vol. v, no. 1s issn: 2283-3927 references abu-zidan f.m., plank l.d., windsor j.a., 2002. proteolysis in severe sepsis is related to oxidation of plasma protein. european journal of surgery. 168, 119-123. colombo g., clerici m., garavaglia m.e., giustarini d., rossi r., milzani a., dalle-donne i., 2015. a step-by-step protocol for assaying protein carbonylation in biological samples. journal of chromatography b. 1019, 178-190. rossi g., giordano a., pezzia f., kjelgaard-hansen m., paltrinieri s. 2013. serum paraoxonase 1 activity in dogs: preanalytical and analytical factors and correlation with c-reactive protein and alpha-2globulin. veterinary clinical pathology. 42, 329-341.. figure 1: distribution of pco (a) and pon-1 (b) values according to the group (a = healthy dogs, b = septic dogs, c = dogs with inflammation). the boxes indicate the i-iii interquartile range (iqr), the horizontal black line indicate the median values, whiskers extend to further observation within quartile i minus 1.5 x iqr or to further observation within quartile iii plus 1.5 x iqr. the symbol “+” indicates the near outliers. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l cytological evaluation of splenic lesions is a routine preoperative diagnostic technique. however, few studies have evaluated the utility of diagnostic cytology in canine splenic diseases (ballegeer et al., 2007; christensen et al., 2009; watson et al., 2011). our aim was to evaluate accuracy, sensitivity, specificity, positive and negative predictive value of cytology to diagnose canine splenic conditions using histopathology as the gold standard. splenic cytological samples obtained between january 1998 and 2018 were retrospectively evaluated. cases were included only when cytology and histology of the same lesion were available. all samples were blindly reviewed. ninety-two cases were included (65 neoplasms, 27 non-neoplastic lesions) and classified as: 36 true positive, 29 false negative, 26 true negative and 1 false positive. splenic cytology had a diagnostic accuracy of 67.39%, a sensitivity of 55.38%, a specificity of 96.3%, a positive and negative predictive value of 97.3% and 47.27% (table 1). to our knowledge, this is the first study reporting conjunctively accuracy, sensitivity, specificity, positive and negative predictive value of cytology in the diagnosis of canine splenic disorders. the major limit of splenic cytology was a reduced sensitivity related to a high number of false negative results that strongly correlate with lesion distribution, size and type and with the blood storage function of the spleen resulting in hematic samples (bertazzolo et al., 2005; o’brien et al., 2013). limitations were balanced by high specificity and positive predictive value making splenic cytology a valuable preliminary diagnostic tool to assist further diagnostic and therapeutic approaches in splenic disease. keywords dog, spleen, cytology, diagnostic accuracy, histology. corresponding author matteo gambini matteo.gambini@unimi.it journal home page riviste.unimi.it/index.php/haf diagnostic accuracy of cytology in canine splenic lesions. m. gambini1,*, a. forlani2, m. tecilla1, m. caniatti1, p. roccabianca1 1 department of veterinary medicine (dimevet), università degli studi di milano, via celoria 10, 20133 milan, italy. 2 idexx laboratories ltd. grange house, sandbeck way, wetherby, west yorkshire, ls22 7dn uk. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 14 haf © 2013 vol. v, no. 1s issn: 2283-3927 references ballegeer, e.a., forrest, l.j., dickinson, r.m., schutten, m.m., delaney, f.a., young, k.m., 2007. correlation of ultrasonographic appearance of lesions and cytologic and histologic diagnoses in splenic aspirates from dogs and cats: 32 cases (2002-2005). journal of the american veterinary medical association. 230(5), 690-696. bertazzolo, w., dell’orco, m., bonfanti, u., ghisleni, g., caniatti, m., masserdotti, c., antoniazzi, e., crippa, l., roccabianca, p., 2005. canine angiosarcoma: cytologic, histologic, and immunohistochemical correlations. veterinary clinical pathology. 34(1), 28-34. christensen, n.i., canfield, p.j., martin, p.a., krockenberger m.b., speilman, d.s., bosward, k.l., 2009. cytopathological and histopathological diagnosis of canine splenic disorders. australian veterinary journal. 87(5), 175-181. o’brien, d., moore, p.f., vernau, w., peauroi, j.r., rebhun, r.b., rodriguez jr., c.o., skorupski, k.a., 2013. clinical characteristics and outcome in dogs with splenic marginal zone lymphoma. journal of veterinary internal medicine. 27(4), 949-954. watson, a.t., penninck, d., knoll, j.s., keating, j.h., sutherland-smith j., 2011. safety and correlation of test results of combined ultrasound-guided fine-needle aspiration and needle core biopsy of the canine spleen. veterinary radiology and ultrasound. 52(3), 317-322. table 1: evaluation of diagnostic accuracy of cytology in the diagnosis of 92 canine splenic lesions. tp, true positive results; tn, true negative; fn, false negative; fp, false positive. parameter value (%) confidence interval (95%) accuracy (tp + tn / total cases) 67.39 (62/92 cases) 56.82 – 76.80 sensitivity (tp / tp + fn) 55.38 (36/65 cases) 42.53 – 67.73 specificity (tn / fp + tn) 96.30 (26/27 cases) 81.03 – 99.91 positive predictive value (tp / tp + fp) 97.30 (36/37 cases) 83.86 – 99.60 negative predictive value (tn / fn + tn) 47.27 (26/55 cases) 40.37 – 54.28 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l school catering services are characterized by a significant level of inefficiency regarding the food processed but not consumed during meals. this work analyses the meal supply in primary schools in italy in order to highlight new areas of inefficiency upstream of the food chain. a lack of conformity of food portions with nutritional guidelines can potentially lead to a double negative externality: overweight children and food waste. data were collected between april and june 2017 from the municipality website of each regional capital (rc) of the 20 italian regions. from the tendering process for primary school meal provision, data on the portions (in grams) of the most representative food categories were extracted and classified. to evaluate the degree of homogeneity amongst different regions, the average, minimum and maximum values, standard deviations and relative standard deviations of each individual food category were estimated. to verify the adherence to nutritional recommendations, anova was performed for multiple comparisons combined with duncan's multiple range test, with significance set at a p value < 0.05. the specific benchmarks for the evaluation of meal portion sizes were calculated based on the national recommended energy and nutrient intake levels. the results (table 1) show a great variability of food portions amongst the rcs analyzed. food categories with highest relative standard deviations values were cooked and raw vegetables (0.29 and 0.35 respectively) that indicate great levels of heterogeneity in food portions amongst italian regions. conversely, pasta and rice portions were more uniform (0.10 and 0.13), although on average above than the recommended portion. the only food categories characterized by a smaller mean portion than recommended are fish, raw vegetables and cooked vegetables. the educational role of eating at school can contribute to raising children's awareness about one of the most urgent environmental challenges food waste by introducing the best strategies for waste reduction, reuse and recycling. keywords portion size, food waste, sustainable public procurement, childhood obesity, school meals. corresponding author marta castrica marta.castrica@unimi.it journal home page riviste.unimi.it/index.php/haf meal portion sizes and their potential impacts on food waste: case study of school meals in italy. m. castrica1,*, c.m. balzaretti1, a.baldi1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 26 haf © 2013 vol. v, no. 1s issn: 2283-3927 references östergren, k., gustavsson, j., bos-brouwers, h., timmermans, t., hansen, o. j., møller, h., & easteal, s., 2014. fusions definitional framework for food waste. french parliament. loi n° 2016-138. lutte contre le gaspillage alimentaire. 2016; https://www.senat.fr/dossierlegislatif /ppl15-245.html. società italiana di nutrizione umana, (sinu). 2014. iv revisione dei livelli di assunzione di riferimento di nutrienti ed energia per la popolazione italiana (larn). available at: http://www.sinu.it/html/pag/tabelle_larn _2014_rev.asp. table 1: descriptive statistics of variables. 1 rsd (relative standard deviation) = std dev/mean. 2 av.delta portion= mean – larn portion. food category mean (g) std dev rsd1 min max larn portion (g) av. delta portion2 pasta 70.0 6.7 0.10 50 80 56 14.0 rice 69.5 8.9 0.13 50 80 56 13.5 bread 55.8 14.1 0.25 40 100 35 20.8 dried pulses 34.3 9.9 0.29 21 60 35 -0.7 meat 84.5 18.1 0.21 60 120 70 14.5 fish 98.5 22.2 0.23 60 120 115 -16.5 cooked / raw ham 52.3 10.1 0.19 35 75 35 17.3 cooked vegetables 127.9 37.5 0.29 75 235 140 -12.1 raw vegetables 101.0 35.5 0.35 35 150 140 -39.0 fresh fruit 150.5 18.5 0.12 130 200 105 45.5 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en https://www.senat.fr/dossierlegislatif http://www.sinu.it/html/pag/tabelle_larn proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords nerve block, electrolocation, mandibulectomy, analgesia corresponding author elisa silvia d’urso elisasilvia.durso@unimi.it journal home page riviste.unimi.it/index.php/haf proximal mandibular nerve block using electrolocation in 10 dogs undergoing mandibular surgery: a case series report. g. ravasioa, e.s. d’ursoa*, c. macchionia, d. stefanelloa adepartment of veterinary medicine, università degli studi di milano, via celoria 10, 20133, milan, italy abstract peripheral nerve block performed using electrical stimulation (i.e. electrolocation) is widely used for perioperative pain management during several surgical procedures in dogs (campoy 2008), but few data are reported concerning its application to invasive maxillofacial surgery (carotenuto et al 2011). the aim of this case series report is to evaluate the efficacy of proximal mandibular nerve block (pmnb) in perioperative pain management in dogs undergoing mandibulectomy. ten dogs of various breeds, (six spayed females and four neutered males of 10.353.09 years and mean weight of 19.5615.19 kg) presenting either neoplasia or mandibular fracture and scheduled for mandibulectomy were premedicated with intramuscular acepromazine maleate (0.02 mg/kg); after induction of general anaesthesia, bilateral pmnb was performed with ropivacaine 0.75% (2 mg/kg) inserting the stimulated needle in temporomandibular joint direction. whenever intraoperative nociception occurred, intravenous rescue analgesia was provided (fentanyl 3 g/kg). carprofen was administered subcutaneously as a sole postoperative treatment (3 mg/kg) and postoperative analgesia was assessed for at least 24 hours by a blind operator, accordingly to the glasgow composite pain scale (reid et al 2007); when it overcame a threshold of 5/24, intravenous rescue analgesia was administered (methadone, 0.2 mg/kg). in eight out of ten dogs no intraoperative nociception was shown, while in two dogs a single intravenous fentanyl administration was sufficient to provide additional analgesia. no acute and medium term complications were observed and postoperative analgesia lasted for 20.5±6.1 hours. pmnb seems to provide effective perioperative long-lasting analgesia leading to a reduction in intraand postoperative drug administration. references carotenuto am, ravasio g, fonda d, stefanello d 2011. proximal mandibular nerve block, using electrolocation, for rostral mandibulectomy in a geriatric dog. can vet j. may;52(5):515-8. campoy l 2008. fundamentals of regional anesthesia using nerve stimulation in the dog. in: recent advances in veterinary anes thesia and analgesia: companion animals, gleed r.d. and ludders j.w. (eds.). international veterinary information service, ithaca ny reid j, nolan am, hughes jml, lascelles d, pawson p, scott em 2007. development of the short -form glasgow composite measure pain scale (cmps-sf) and derivation of an analgesic intervention score. animal welfare, 16(s): 97-104 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en l keywords dairy sheep; greek breeds; ovardrb1 exon2 alleles; genetic polymorphism; parasites pages 1 – 19 references vol. 3 no. 1 (2016) article history submitted: november 20, 2015 revised: january 30, 2016 accepted: february 02 2016 published: february 08 2016 corresponding author panagiota koutsouli, department of animal breeding and husbandry, faculty of animal science and aquaculture, agricultural university of athens, greece. 75, iera odos, votanikos, athens 11855, greece. e-mail: panagiota@aua.gr phone: +30 210 5294440 fax: +30 210 5294442 journal home page riviste.unimi.it/index.php/haf article allelic polymorphism of ovardrb1 exon2 gene and parasite resistance in two dairy sheep breeds. stavros spetsarias1, panagiota koutsouli1*, georgios th eodorou1, georgios theodoropoulos2, iosif bizelis1. 1 department of animal breeding and husbandry, faculty of animal science and aquaculture, agricultural university of athens, 75 iera odos, votanikos, athens 11855, greece. 2 department of anatomy and physiology of farm animals, faculty of animal science and aquaculture, agricultural university of athens, 75 iera odos, votanikos, athens 11855, greece. abstract. the ovar-drb1 gene locus is one of the most polymorphic genes of the major histocompatibility complex (ovar-mhc) and holds a functional role to antigen presentation. the aim of this study was: a) to describe the ovar -drb1 locus variability in two dairy greek sheep breeds and b) to investigate associations between this variability with resistance to gastrointestinal parasitosis. blood and faecal samples were collected from 231 and 201 animals of arta and kalarrytiko breeds, respectively. the identification of alleles was performed using the sequence–base method. faecal egg counting (fec) of the gastrointestinal parasites and measures of blood plasma pepsinogen levels were performed in order to evaluate parasitological parameters. from this study in the overall examined animals, thirty-nine ovar-drb1 alleles were identified, among them, ten new alleles, reported for the first time in the literature. in arta breed a total of twenty-four alleles were found. among the detected alleles, ten were breed specific and five were new. regarding the kalarrytiko breed, twenty-nine alleles were found, fifteen of them were unique and nine were new. the studied breeds differed in their allelic profile, with only 12 common from the total of 134 different recorded genotypes. a higher number of animals with high parasitic load and high plasma pepsinogen values were found in kalarrytiko. associations between ovar-drb1 alleles with fec values were found with certain heterozygous genotypes to present significantly reduced fec values. the large number of detected alleles with low frequencies and the fact that the majority of animals were heterozygous, make hard to find strong associations. s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 2 haf © 2013 vol. iii, no. 1 issn: 2283-3927 1 introduction the major histocompatibility complex of sheep (ovarmhc) is a gen e complex associated mainly with immunological properties. οvar-drb1 en codes the b1 structural core of an ovarmhc class ii molecule, forming a large part of the binding region of the an tigenic peptide (pbr) of the molecule (marsh and bodmer, 1993). compared to all other gen es of the ovarmhc, the ovar-drb1 gene locus exhibits the largest variability (andersson and rask, 1988). this variability is localized at specific positions of exon 2 (270 bp). ovar-mhc class ii molecules present an tigenic peptides deriv ed from extracellular proteins and parasites on th e t-cell receptor (tcr) for cd4+t-lym phocytes (germain and margulies, 1993; fremont et al., 1996) which are expressed at higher concentrations on the surface of macrophages and b-lymphocytes (outteridge et al., 1996). since the polymorphic pattern of pbr amino acids determines its conformation and electric charge, strong hydrophobic bonds are created only with specific antigenic peptides (th orsby, 1999). thus, different ovar -drb 1 alleles favour the binding of differen t antigenic peptides in the pbr ovar-mhc class ii molecules (rudensky et al., 1991). consequen tly, the polym orphism of ovar-drb1 exon 2 locus is associated with the variability in resistance or susceptibility to diseases of a sheep population. many studies have shown that there is a correlation between ovar-drb1 alleles and parasite resistance, particularly to nematodes (outteridge et al., 1996; paterson et al., 1998; charon et al., 2002; sayers et al., 2005; hassan et al., 2011). this is also the case against other infectious agents, such as pathogenic bacteria and viruses (larruskain et al., 2010). gastroin tes tinal nematodes are th e most importan t source of infestation in sheep worldwide. anthelmin thic treatm ent with pharmaceu tical compounds is a relatively expensive m ethod of combating th e infestation, which leads in many cases to the developm ent of anthelmin thic resistance (roos, 1997). additionally, a growing concern related to residues of pharmaceu tical compounds which pass down into the food chain, have driven a large number of farmers to adopt breeding prog rams which rely less on drug deliv ery. for these reasons, the selection of sheep breeds and individuals resistan t to parasitic infestation is promoted in many countries. recen tly, ovarmhc loci hav e attracted the research interest as candidate d na markers since several gen es from all classes of ovar-mhc are involved in resistance or susceptibility to various sheep nematodes (dukkipati et al., 2006). generally, all research studies have mainly been carried out in breeds of meat and wool type. since g enetic differences exis t between th e indigenous greek and foreign sheep breeds (rogdakis, 2002), there was g reat interest in exploring the ovar-drb1 genetic variability and the possible iden tification of new alleles in th e greek breeds. for the pu rposes of this study, two dairy greek breeds with differen t management and productive characteristics were selected in order to describe th e variability of ovar-drb1 gene locus: i) arta, a breed with high milk yield and litter size and ii) kalarrytiko, a rare breed, with low milk yield bu t well adapted to harsh environmen tal conditions. considering that associations have been documented between certain ovar-drb1 alleles and parasitological param eters, there was further in terest to investigate, whether similar associations among ovar-drb1 alleles and parasites could be found in order to use this knowledge as a criterion for selection prog rams to create animals resistan t to gastrointestinal parasites, avoiding the adminis tration of an tiparasitic drugs. s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 3 haf © 2013 vol. iii, no. 1 issn: 2283-3927 2 materials and methods 2.1 animals and sample collection a total of 43 2 blood samples were taken randomly from two dairy greek sheep breeds. th e breeds reared at the region of western greece, in flocks w ith a mean size of approximately five hundred animals. the flock of arta breed (sample size=23 1) was kept at 414 m altitude (vassiliki, etoloakarnania prefectu re), grazing in private pastures throughou t th e year under a semi-extensive farming s ystem. the arta ew es were fed supplemen tary concen trates according to th e requiremen ts of their productive s tage, milked mechanically twice per day and produ ced on average 276.08 ± 9.73 l of milk within a whole lactation. the flock of κalarrytiko (sample size= 201) was kept at 838 m altitude (small gotista, ioannina prefecture) grazing in communal pastures under an extensive traditional farming system. during th e winter sufficien t herbage is not available and supplemen tary feed was given from october to march. kalarrytiko ewes were milk ed by hand and produced on average 80.45 ± 3.02 l of milk. blood samples were taken from each animal by jugular vein puncture, in order to use whole blood for genomic dna isolation and plasma for evaluation of pepsinogen levels. faecal samples were obtain ed from th e rectum of each animal to es timate parasitic burdens. 2.2 genotyping of ovar-drb1 exon2 region 2.2.1 identification of alleles by direct sequencing a touchdown pcr, using as substrate the g enomic dna from each animal, was used for th e amplification of a 340 bp long dna fragmen t con taining th e exon 2 of the ovar-drb1 locus. the total reaction volume was 25 μl and contained 110 -120 ng genomic dna, 10 pmol/μl of th e drb1-330 primers (forward prim er): attagcctcyccccaggagkc and drb1-329 (revers e primer): cacccccgcgctcacctcgccgc (ballingall and tassi, 2010) and 0.5 u phusion® high fidelity dna polymeras e (neb). initially, the reaction occurred in two hybridization cycles at the following temperatu res: 67°c, 66°c, 65°c, 64°c and con tinued with 30 cycles at 63°c. touchdown pcr products w ere directly sequenced at laboratories of cemia sa (larissa), with the genetic analyzer abi3730xl (applied bios ystems). the sequencing cycle kit bigdye terminator v3.1 (applied biosystems ) was used for the reaction, according to manufacturer’s guidelines. two suitable software packag es: “gen estudiotmprofessional sequence analysis software” and “nu cleotide blast basic local alignment search tool” w ere used for th e identification of alleles after sequencing. 2.2.2 identification of novel alleles by cloning and sequencing regarding the iden tification of candidate alleles (new alleles), cloning and sequencing was performed in th ree successive steps: at th e first step, for those animals presen ting new alleles, a simple pcr was performed with a differen t reverse prim er compared to th e respectiv e primer used in the touchdown pcr. a 364 bp long dna fragm ent containing the exon 2 locus of the ovar-drb1 gene was amplified. the total reaction volu me was 50 μl containing : a) 85-95 s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 4 haf © 2013 vol. iii, no. 1 issn: 2283-3927 ng genomic dna, b) 10 pmol/μl of the forward primer drb1-330, c) 10 pmol/μl of the revers e primer drb1-313: acacactgctccacactgg (ballingall and tassi, 2010) and d) 3,5 u taq dna polym erase (fermen tas). the reaction was pe rformed at +62°c, for 37 cycles. at the s econ d step, due to th e fact that the pcr product consisted of the allelic pair of genotypes, cloning was performed, using a culture of susceptible bacterial cells, in order to initially seg regate them, and subsequ ently to find the candidate allele considered as new. using th e ligation reaction, th e isolated 364 bp long dna fragm ent con taining th e exon2, was inserted to th e plasmid pbluescript sk (2961 bp) and used for the transformation of susceptible escherichia coli cells of the jm109 strain. the localization of cloned alleles of exon2 within a bacterial culture was performed with a colony pcr. the products of colon y pcr were incubated with 10u of th e restriction en zyme rsai (neb) in order to ascertain, from the dig es tion patterns in which of th e received transform ed bacterial colonies, clones of th e two alleles were contained. the third and final s tage of the process was th e selection of th e colonies containing the new alleles, from which the recom binant plasmids were isolated, in order to sequence th e inserts and to finally confirm the sequence of the alleged new allele as new indeed. the isolation of the plasmids was performed using standard reagent kitsnucleospin® plasmid (macherey nagel). 2.3 parasitological analyses 2.3.1 count of faecal parasite reproductive elements faecal egg counts (epg) w ere conducted for strongyle-type eggs cumulatively and for nematodirus sp., strongyloides sp., trichuris sp., and moniezia sp. eggs separately (thienpon t et al., 1986). faecal coun ts of coccidian oocysts were also enumerated. 2.3.2 estimation of pepsinogen levels in blood plasma plasma pepsinogen in each animal was determined by th e method of hirschowitz (1955) as modified by korot’ko & islyamova (1963). pepsinogen is the inactive p recursor of pepsin, on e of th e main proteolytic enzymes of digestiv e system. pepsin acts as peptidase, catalysing th e hydrolysis of peptide bonds from the aromatic amin o acids (tyrosine, phen ylalanine, tryptophan). in th e presence of parasites larvae th e intense inflammation of the gastric mucosa of the abomasum, affects the function of the gastric glands and hcl production is decreased. as a consequen ce pepsinogen could n ot be converted to pepsin and inserts in blood circulation, increasing the plasma peps inogen levels. in brief, blood plasma samples were collected from the supernatant after centrifugation (3000 rpm, 30 min) and six standard solutions of tyrosine were prepared. a s taining solution of f olin -ciocalteau was added in each sample. the optical density of blank, standard tyrosine solu tions and samples were m easured at 560 nm in a spectrophotometer. the concen tration of samples in pepsinog en was given as values of mi.u. tyrosine. s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 5 haf © 2013 vol. iii, no. 1 issn: 2283-3927 2.4 statistical analysis the genotypic frequencies (observed and expected estimations) and the allelic frequencies were estimated with genepop (rousset, 2008). the abov e software was also used to m easure the observed and expected heterozygosity and hom ozyg osity. the appropriate samplin g function z was used to test for significant differences betw een ratios of heterozygosity. th e null hypothesis was η0: pa–pκ=0 while the alternative one was η1: pa ≠ pκ. sample sizes were large en ough (>100) and th e null hypoth esis was rejected wh en z > z 0,05 or z < z 0,05. associations between genotypes and parasite categories were perform ed at first, with a χ 2 contingency pearson test but then th e simulated monte carlo method was preferred becaus e in many cases the number of observations was fewer than 5. th e epg values or f ec (faecal egg counts) and pepsinogen concen tration values (mi.u. tyrosine) declined from normality . their analysis was done using the bartlett’s tes t for checking homogen eity of the sample an d the non pa rametric tes t of kruskal-wallis. associations betw een ovar-drb1 genotypes and fec values of parasites were investigated using non parametric tests (mann-whitney or kruskal-wallis depending from the number of levels for th e specific alleles ) considering th e fixed effect of genotype for th e specific i allele with three lev els (firs t level: animals without the specific allele, second level: animals with on e copy of th e i allele and third lev el: animals with two copies of the i allele). the same analysis was perform ed in order to reveal associations between ovar-drb1 genotypes and pepsinogen levels. the above analyses were carried ou t a) on th e total examin ed animals for alleles which are common betw een the s tudied breeds and b) on a within breed basis for alleles which are unique for each breed. 33 results 3.1 genotyping analysis and polymorphism of ovar-drb1 exon2 gene 3.1.1 genotypic frequencies one hundred and thirty four gen otypes were recorded in the total of 432 examined animals. eighty-one (81) and sixty-five (65 ) differen t gen otypes w ere found among the 23 1 an d 201 individuals from arta and kalarrytiko breeds, respectively. twelv e common g enotypes were found between breeds which are presented in supplementary table a. among th e common genotypes only th ree were homozygous for alleles: drb1*0402, drb1*0901 and drb1*2101. homozygous genotypes among the examined animals were quite few, only eleven (11) and ten (10) in arta and kalarrytiko, respectively. a different set of genotypes with relative high frequency was observ ed in each breed. th e most common genotypes and in descending order for arta breed w ere: drb1*0101-drb1*0102, drb1*0102-drb1*1001, drb1*0102-drb1*2101, drb1*0101-drb1*0901, drb1*0102-drb1*0901, drb1*0102-drb1*0402. on the other hand in kalarrytiko breed the genotypes with higher frequ ency and in descending order w ere: drb1*0402-drb1*0702, drb1*0702-drb1*0702, drb1*0102-drb1*0402, drb1*0702-drb1*1602. am ong th e recorded genotypes th e s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 6 haf © 2013 vol. iii, no. 1 issn: 2283-3927 percentag e of new alleles was 23 % and 54 % for arta and kalarrytiko breed, respectively. a percentag e of 52 % and 49 % of arta and kalarrytiko genotypes, respectiv ely, w ere represen ted by only one individual. half of the g enotypes in both breeds had extrem ely low genotypic frequ encies below 0.01. taking into accoun t the above results it is evident that each breed is characteris ed by its specific g enotypic profile. 3.1.2 allelic frequencies a set of thirty-nine (39) alleles for both breeds w ere identified. the allelic frequencies for each breed and for th e total number of animals are presented in table 1. among the alleles found, a total of 29 are already known and registered in the database for ovar-mhc (ipd-mh c database embl-ebi), while the remaining ten (10) are reported for the first time. table 1 illustrates the number of identified alleles in arta (24 alleles) and in kalarrytiko (29 alleles ) flocks, the number of shared and the number of unique alleles for each breed. th e unique alleles for arta were ten out of 24 (drb1*0104, drb1*0302, drb1*0308, drb1*1001, drb1*1003, drb1*1501, drb1*1901, drb1*2001, drb1*2201 and drb1*12). also, in arta breed a subset of five alleles from the total of 24 (drb1*1606, drb1*1607, drb1*1608, drb1*2502 an d drb1*12) represented new alleles, reported first time in literature. the following a lleles: drb1*0102, drb1* 0101 and drb1*0402, were detected in descending order with frequencies of 0.232, 0.139 and 0.095, respectively. for the above alleles th eir cumulative inciden ce was equal to 0.465. in kalarrytiko a set of fifteen alleles, from the total of 29 detected, were breed specific (drb1*0403, drb1*0702, drb1*0803, drb1*1102, drb1*1301, drb1*1302, drb1*1502, drb1*160202, drb1*1604, drb1*1605, drb1*2003, drb1*2401, drb1*11, drb1*26 and drb1*30). a subset of 9 alleles, from the total of 29 detected, were new (drb1*160202, drb1*1606, drb1*1607, drb1*1608, drb1*2003, drb1*2502, drb1*11, drb1*26 and drb1*30). in kalarrytiko th e most frequent alleles were: drb1*0702, drb1*0402 and drb1*1606 with frequ encies 0.211, 0.192 and 0.107 respectively, and cumulati ve incidence equal to 0.510. 3.1.3 frequencies of homozygosity and heterozygosity deviations from hardy-weinberg equilibrium the percentag e of 84.49% in the overall examined animals (n=432) were classified as heterozygotes and the remaining 15.51% as homozygotes. estimates of observ ed homozygosity and heterozygosity (mean ± s.e.m.) within th e arta breed was pa=0.125±0.022 an d qa=0.875±0.022, respectively. for the kalarrytiko breed the relativ e es timat es were pk=0.189±0.028 and qk=0.811±0.028. the above estimates were not differen tiated betw een breeds (z = -1.82>-1.96, ρ>0.05). the unbiased estimation of expected heterozyg osity according to nei (1978) was hexpa=0.890 and hexpκ=0.886 in arta and kalarrytik o breed, respectively and the abov e estimations w ere n ot differen t between them (p>0.05). observed and expected estimations of heterozygosity differed significan tly in kalarrytiko breed reflecting deviation from hardyweinberg equilibrium. more specifically, the observed h eterozygote animals (qκ=0.811) were fewer than expected (hexpκ= 0.886) (p<0.05). s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 7 haf © 2013 vol. iii, no. 1 issn: 2283-3927 table 1. frequencies of ovar-drb1 alleles in arta breed (n =231), in kalarrytik o breed (n=201) and in the total examin ed animals (n=432). alleles allelic frequency arta breed kalarrytiko breed total drb1*0101 0.1385 0.0174 0.0822 drb1*0102 0.2316 0.0647 0.1528 drb1*0104 0.0043 0.0023 drb1*0302 0.0022 0.0012 drb1*0304 0.0498 0.0050 0.0289 drb1*0308 0.0043 0.0023 drb1*0311 0.0152 0.0522 0.0324 drb1*0402 0.0952 0.1915 0.1400 drb1*0403 0.0025 0.0012 drb1*0601 0.0498 0.0025 0.0278 drb1*0702 0.2114 0.0984 drb1*0801 0.0238 0.0025 0.0127 drb1*0803 0.0199 0.0104 drb1*0901 0.0844 0.0398 0.0637 drb1*1001 0.0887 0.0475 drb1*1003 0.0130 0.0069 drb1*1102 0.0025 0.0012 drb1*1301 0.0025 0.0012 drb1*1302 0.0025 0.0012 drb1*1501 0.0065 0.0035 drb1*1601 0.0130 0.0025 0.0081 drb1*1602 0.0697 0.0336 drb1*160202# 0.0373 0.0035 drb1*1604 0.0075 0.0046 drb1*1605 0.0100 0.0069 drb1*1606# 0.0022 0.1070 0.0081 drb1*1607# 0.0022 0.0697 0.0012 drb1*1608# 0.0152 0.0075 0.0463 drb1*1901 0.0130 0.0023 drb1*2001 0.0152 0.0023 drb1*2003# 0.0025 0.0035 drb1*2101 0.0801 0.0075 0.0174 drb1*2201 0.0043 0.0509 drb1*2401 0.0050 0.0336 drb1*2502# 0.0043 0.0025 0.0116 drb1*11# 0.0473 0.0220 drb1*12# 0.0433 0.0231 drb1*26# 0.0025 0.0012 drb1*30# 0.0050 0.0023 #: new alleles -: no allele found s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 8 haf © 2013 vol. iii, no. 1 issn: 2283-3927 3.2 parasitological analysis 3.2.1 faecal examination faecal samples were collected from 157 kalarrytiko and 224 arta sheep to obtain the faecal egg count of gastrointes tinal parasites. in th e total examined animals (n=381), a percen tage of 33% (126/381) was positively infected. specifically, eggs from various parasites were detected in 55.41% (87/157) and 17.41% (39/224) animals from kalarrytiko and arta breed, respectiv ely (table 2). a g reater variety of parasite reproductive elements was found in kalarrytik o: eggs of strongyles, nematodirus spp, strongyloides spp., trichuris spp., moniezia spp. and oocytes of coccidia while in arta breed w ere mainly detected, eggs of strong yles, strongyloides spp., capillaria spp. and coccidia. the above findings on fec and pepsinogen values between th e studied breeds could be considered as the possible effect of th e farming system com bined with the differen t pasture locations used by the flocks. table 2. prevalence (%) and faecal egg counts (fec) (epg) of gas troin testinal parasites and pepsinogen levels (m i.u. tyrosine) in sheep of the arta and kalarrytiko breeds. infection arta (n=224) kalarrytiko (n=15 7) n prevalence % fec median (qr) pepsinogen median (qr) n prevalence % fec median (qr) pepsinogen median (qr) helminths* 25 11.16 100 (200) 319.26 (356.46) 52 33.12 500 (1625) 228 (374) strongyles 13 5.80 100 (200) 405.20 (255.06) 46 29.30 500 (1175) 223 (345.5) strongyloides spp. 10 4.46 200 (200) 220.96 (517.99) 7 4.46 200 (1000) 138 (371) nematodirus spp. 0 1 0.64 n/a n/a trichuris spp. 1 0.46 n/a n/a 1 0.64 n/a n/a capillaria spp. 1 0.45 n/a n/a 0 moniezia spp. 0 7 4.46 300 (400) 212 (374) coccidia 17 7.59 200 (100) 317.13 (442.95) 57 36.31 200 (350) 381.5 (571.5) *: infection with all the investigated parasites except coccidian -: no parasite found n/a: not applicable 3.2.2 pepsinogen levels the percentage of animals with pathological values of tyrosine was higher in kalarrytik o breed (table 2). more specifically, 56.1% for kalarrytiko (88 animals from the total of 157 examined) and 43.6% for arta (89 animals from the total of 224 examin ed) exhibited >375 m s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 9 haf © 2013 vol. iii, no. 1 issn: 2283-3927 i.u. tyrosine. however, in kalarrytiko breed animals with values up to 500 m i.u. tyrosin e showed a very sharp increase in high levels up to 6.315 m i.u, while for arta breed the relativ e values showed a lower increase up to the value of 2.243 m i.u. tyrosine. the spearman correlation coefficient betw een f ec and pepsinog en values was nearly zero. 3.3 associations between genotypes, parasitic load, and pepsinogen levels investigation of possible associations between genotypes and f ec valu es was carried ou t for the genotypes of common alleles with relatively high frequ ency in the total examined animals or within each breed. we found few significan t associations betw een certain genotypes and fec values which are presented in table 3. animals heterozygous for alleles drb1*0101, drb1*0102 and drb*03 11 had significan tly lower fec valu es in comparison with th e remaining genotypes. in the case of drb1*0311 allele, within the group “heterozyg otes”, animals heterozygous for alleles drb1*0302, drb1*0304 and drb1* 0308, were also included, because th e above three alleles have a tigh t nucleotide resem blance with drb1* 03 11 (th ey belong to the same allelic family). on th e opposite direction the h eterozyg ous animals for drb1*0402 allele had high er f ec values in com parison with th e homozygotes and th e remaining genotypes. aditionally, animals carrying one or tw o copies of drb1*0702 allele (with th e highest frequency in kalarrytiko breed), had the higher m edian fec numbers in comparison with the remaining genotypes. table 3. associations between ovar-drb1 genotypes and faecal egg counts (f ec) (epg ). allele ovar-drb1 genotypes n fec median (interquartile range) p drb1*0102 no copy of the allele 265 0 (200) * heterozygote 106 0 (25) homozygote 7 0 (200) drb1*0101 no copy of the allele 316 0 (200) ** heterozygote 57 0 (0) homozygote 5 0 drb1*0402 no copy of the allele 281 0 (100) *** heterozygote 85 0 (350) homozygote 12 50 (175) drb1*0311 no copy of the allele 331 0 (200) ** heterozygote 47 0 (0) homozygote 0 n/a drb1*0702 no copy of the allele 319 0 (100) *** heterozygote 48 100 (300) homozygote 10 100 (42 5) *: significant association p<0.05 **: signifant association p<0.01 ***: significant association p<0.001 s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 10 haf © 2013 vol. iii, no. 1 issn: 2283-3927 pepsinogen levels did not differ significantly among the various parasites in the studied breeds (table 2). fu rth ermore, no significant association was found betw een genotypes an d pepsinogen levels with one exception for the h omozygote animals for drb1*0402 allele in th e total population which tend to have lower m edians values of pepsinogen levels in comparison with the remaining genotypes (p<0,07). 4 discussion the studied breeds differ considerably in their morphological and productive traits . arta is a breed of high milk production, kept in lowland of western greece in large flocks. kalarrytik o is a rare b reed of m ountain type of medium productivity with a small population size in comparison to arta breed (rogdakis, 2002) and this fact could explain the significant deficit of heterozygote individuals found in kalarrytiko. however, the fact that a greater number of alleles was found in kalarrytiko as well as, more n ew alleles were identified in the above breed, confirms the necessity to preserve the small and rare breeds because in their genetic pool, a considerable amount of gen etic variation, can be found. the large number of alleles and th e high levels of heterozygosity found in both breeds are expected findings, due to the fact that ovar-drb1 gene locus is the most polymorphic among th e οvar-mhc loci (andersson and rask, 1988). generally, relying on the number of detected alleles, th e results of the present study could be compared with relative studies using the sequencing method for the detection of ovar drb1 genetic variability. schwaiger et al. (1993, 1994) identified 47 alleles in flocks from perendale, coopw orth, landrace, merino and texel sheep. paterson (1998) iden tified 5 alleles in a population of soay feral sheep, while gruszczynska (1999) found 36 and 28 alleles in tw o flocks of merino sheep. from th e same scientific team (gruszczynska et al., 2005) 36 and 30 alleles were found respectively, in two flocks of polish heath sheep. additionally, charon et al. (2002) in another flock of polish heath breed found 20 ovar-drb1 alleles, while konnai et al. (2003) detected 28 alleles in suffolk, 14 in cheviot and 9 in corriedale sheep breeds. sayers et al. (2005) identified 8 alleles in texel and 7 in suffolk breeds. finally, in the study of balingall and tassi (2010) 38 new alleles were iden tified in a sample of 214 animals from 15 differen t breeds. considering all the above information, as well as the number of alleles found from th e presen t study, it is justified to argu e that the greek sheep breeds contain considerable am oun t of genetic variation in ovar-drb1 locus. there is substantial evidence that genetic factors affect the resistan ce of animals in gastrointestinal parasite numbers (bishop et al., 2004; sayers et al., 2005), although many other factors as the type, ag e and sex of the parasite, th e intensity of infection, th e age, die t and frequency of th e host defecation, act on the coun ted number of reprodu ctive elemen ts of gastrointestinal parasites. also, the fec values are affected by time of year and w eath er conditions (abbott et al., 2009). possibly, the large numbers of gastroin tes tinal parasit es in kalarrytiko animals occu rred due to a relatively large number of fertile and adult parasites, which act within the organism to enhance the immun e animal hypersensitivity reaction causing an increase of the perm eability of abomasum mucosa, resulting i n th e estimation of very hig h values mi.u. tyrosine (yakoob et al, 1983b). as already m entioned, the environmen tal factors s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 11 haf © 2013 vol. iii, no. 1 issn: 2283-3927 (climate and habitat) acting on th e studied breeds were differen t and do not allow a furth er comparison betw een fec valu es and the tyrosine values estimated for both breeds. pepsinogen levels in plasma were determin ed in order to assess the extent of damage in the gastric mucosa of animals by gastroin testinal parasites. the endoparasitic infection is associated with pathophysiological disorders such as the increase of plasma pepsinogen lev els, the albumin catabolism and plasma protein losses in the gastrointestinal tract (yakoob et al, 1983a). for this reason, the estimation of pepsinogen concentrations in the blood plasma has been proposed as an auxiliary diagnostic technique of endoparasitic infections (parasitic gastroen teritis) in ruminants (anderson et al., 1965). according to stear et al. (1995a, b) th e plasma pepsinogen concentration expresses the hos t response to infection by nem atodes, while fec values reflect th e behaviour of pes ts in th e hos t. in particular, th e above researchers found that in adult ewes infected naturally by gastroin tes tinal nematodes, the increase of plasma pepsinogen con centration was merely an expression of the immunological hos t response to infection and had no relation to the parasitic load. the identification of resistan t and susceptible sheep to nematodes is more effective when the estimation of fec values is don e in parallel with the estimation of plasma pepsinogen levels (stear et al., 1995a, b). in some regions where ostertagia spp is detected, serum plasm a analysis is a useful diagnostic tool. in g eneral, pepsinogen levels > 375 mi.u. tyrosin e correspond to clinical symptoms. however, interpretation problems may arise in immunized animals that are infected by gastroin testinal parasites. i n these animals, there are no clinical signs but, as mentioned, plasma pepsinogen lev els may be elevated because of immunological hypersensitivity reaction that occu rs in the abomasum mucosa. most studies on th e concen tration of pepsinogen in the blood plasma have been conducted in sheep experimen tally infected with nematodes (yakoob et al., 1983b; fox et al., 1988; mostofa et al., 1990; lawton et al., 1996) while there are few studies in sheep infected with nematodes in a natural way (yak oob et al., 1983a). besides th e determination of plasma pepsinogen levels when the genus haemonc hus spp dominates, hematocrit m easuring provides a rapid estimation of th e degree of anaemia which is the characteristic clinical sign of n ematodosis caused by the above parasite. also, in some countries the serological diagnosis (elisa) is used mainly for the species ostertagia spp and cooperia spp. however, so far there is insufficien t information on th e association betw een serological titres and parasitic load. generally, from this study neither significant differences of pepsinogen levels were foun d betw een heterozygote and homozygote ovar-drb1genotypes, nor an y relationship betw een the various genotypes with high or low pepsinogen levels. however, there was a trend for homozygous animals carrying drb1*0402 allele to have lower median values of mi.u. tyrosin e in comparison with the other genotypes. genetic factors play an importan t role in resistance to nematodes (bisset et al., 1992). as the es timated coefficient of heritability for f ec values rang es from 0.2 to 0.4 in various sheep breeds which have previously been infected with parasites (stear et al., 1997a, b), this suggests that a sheep s election prog ram of genetic resistance to nematodes can b e effectively implemen ted (stear et al., 2001). according to research studies of behnke et al. (2003, 2006) mhc polymorphism of class ii genes and mainly of tnf gen e regions determin es the resistance or th e host susceptibility to diseases caused by gastroi n tes tinal parasites. in the study of davies et al. (2006) in scottish blackface sheep, it was shown that specific qtls in chromosom es 2, 3, 14, and 20 are related with the resistance to infection of τeladorsagia s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 12 haf © 2013 vol. iii, no. 1 issn: 2283-3927 circumcincta. the analysis of ch romosom e 20 revealed that the ovar-mhc region is significantly correlated with resistan ce to gastroin tes tinal nematodes. furth ermore, qtls related to th e specific activity of th e iga immunoglobulins against nematodes are found on ch romosomes 3 and 20. associations of mhc drb1 alleles with resistance to n ematodes have been found not only in sheep (outteridge et al., 1996; sayers et al., 2005) bu t also in cattle (stear et al., 1988 & 1990 ; gasbarre et al., 1993) and mice (froeschke and somm er, 2005). early studies in sco ttis h blackface lam bs, based on th e hybridization of oligonucleotides in exon 2 of ovar-drb1 gene, have shown that, lambs infected natu rally with osterdagia circumcincta, had fec values associated with ovar-drb1 alleles (buitcamp et al., 1994; schwaiger et al., 1995; stear et al., 1996). in addition, the significan t effect of ovar-drb1 alleles on th e dras tic reduction of f ec values, was detected by substitution of the common allele i, with g2 allele, identical with th e allele ovar-drb1*0203, which confers resistance to nematode parasites. the association of g2 allele with low f ec values in scottish blackface sheep was con firm ed by th e study of stear et al. (2005). there was also, an association of g2 allele with reduced number of adult parasites of τ. circumcincta species, although no association was found with the length size of female parasites. a possible explanation is that the mhc effect on fec values functions th rough th e control of th e number of adult parasites and not th e control of th eir fertility. in the study of hassan et al., (2011) the g2 allele, renamed as ovar-drb1*1101, gave increased resistance against the nematode t. circumcincta in suffolk lambs. eviden ce was also, presen ted that th e resistance of animals, due to ovar-drb1*1101 allele, was more acquired and less innate an d furthermore it was depended from the excretion of adult parasites th rough the multiplication of mast cells of th e mucosa. in the study of sayers et al. (2005) the allele ovar-drb1*0203 was associated with reduced fec values in suffolk breed. the above strong association seems to be due, not only to an antibody production that protects the animals carrying g2 allele, but also to the genetic linkage of g2 allele with an allele of resistance mounted on another locus position. this probably occu rs because the loci close to on e another on the same chrom osome, tend to stay together during th e reduction and th erefore th ey are linked gen etically (mccririe et al., 1997). additionally, th e parasite trichostrongylus colubriformis in sheep appears capable to n egatively regulate many genes of immunologic importance, especially ovar-drb1 and dra, of th e migratory cells in lymph (knight et al., 2010). several genetic mechanisms have been proposed to explain the mhc allelic va riability. new alleles appear through point mutations, genetic recombination or genetic transmutation s (janeway and travers, 1996). the simultaneous antag onistic evolution (coevolution) betw een host and parasites is another proposed mechanism for maintenance of genetic variability both in host and parasitic species (paterson et al., 1998; cutrera et al., 2011). the h ypothesis that th e high levels of mhc gen etic polym orphism of hos t species, driven by th e parasites themselves, is the result of the action of balancing selection, interprets th e redu ction of homozygous and the increase of heterozygous frequencies in a particular population (h edrick and kim, 1998). the balan cing selection maintains g enetic polym orphism through th ree different genetic mechanisms: a) the heterozygote advantag e (overdominance) b) the advantag e of rare alleles and c) the variance in th e levels of selective pressure (charbonn el and pemberton, 2005). in the case of overdominance, selection will not fix or eliminate one or th e other allele, but will make a stable balance of heterozygotes, in which both alleles will be present in the population, s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 13 haf © 2013 vol. iii, no. 1 issn: 2283-3927 at frequ encies determin ed solely by the selection rates against the two homozygote genotypes. in this case, the deg ree of mhc heterozygosity increases the rang e of parasites which are recognized by the immun e system and thus increases th e relativ e darwinian fitn ess of mhc heterozygote genotypes compared to that of the homozygote (pa terson et al., 1998). in the case of the second m echanism (advantag e of th e rare allele) (hamilton, 1980; takahata and nei, 1990), the darwinian fitness of th e allele is reduced when th e frequency of allele in the population increases. genotypes with rare mhc alleles presen t a s trong selectiv e advantage as fewer pathogens hav e been expos ed and adapted to them (clark e and kirby, 1996). the mhc alleles are favou red at low frequencies, while an increase of their freq uencies could only cause a shift in the gen etic composition of parasitic population. thus , according to the case of rare allele, the interaction of host-parasite is considered as a dynamic procedure. the action of parasites decreases the darwinian fitness of the most common mhc alleles of the host with which in teracts (pa terson et al., 1998). the third proposed mechanism is related with variances on the rate of selective pressure. variations in type or size of the parasite population in th e area, lead to constant chang es in the intensity of selection and thus to maintenance of the polym orphism. this phenomenon is dependen t on the presen ce of th e same pathogen (hedrick, 2002; charbonnel and pemberton, 2005). the possibility that an in teraction between various genotypes, as this is reflected by th e different breeds and th e farming system could not be excluded although this was im possible to be investigated in th e present study. genetic selection schemes applied in australian conditions by selecting merino sheep with low faecal egg – counts gave good selection response and have not resulted in any n egative correlation response of th e economically importan t production traits. sheep resource flocks helmin th resistant (ryling ton merino) hav e been created providing significant contribution in understanding th e breeding for diseas e resistance (karlsson & greeff, 2012). from the oth er hand th e div ersity of environmen tal conditions in this continent, highlights the need of various strategies and makes difficult to give universal recommendations in order to con trol gastro-intestinal parasites (lars en, 2014). 5 conclusions our results suggest the considerable amount of g enetic variability in th e ovar-drb1 locus for the two greek dairy sheep breeds, which is underlined due to th e high number of detected alleles as well as the finding of new alleles from this study. in the kalarrytiko breed, a rare breed with small population size, a higher number of n ew alleles were iden tified than in th e arta breed. this finding suggests that rare greek sheep breeds poten tially are an importan t gene pool of new alleles. ou r results also indicated that the majority of detected alleles hav e low frequen cies and subsequently the majority of animals w ere in heterozyg ote state for both breeds. although certain genotypes of ovar-drb1 alleles were associated with lower median fec values, the abov e findings make more complicated th e selection of desirable g enotypes resistant to gastrointes tinal parasites. considering the central role that th e major histocompatibility complex (mhc) plays in the immune sys tem, our s tudy contributes to th e accumulated knowledge on mhc effects on parasite resistance, howev er furth er s tudies in th e future will be required with other genes involved in mhc region in order to cl arify th e genetic background of parasite resistan ce. s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 14 appendix: supplementary table table a. observed (obs) and ex pected (exp) g enotypes for ovar-drb1 all eles detected in the studied breeds genotypes obs (exp) genotypes obs (exp) genotypes obs (exp) arta drb1*0101-drb1*0101 6 (4.373) drb1*0901-drb1*0304 1 (1.946) drb1*1901-drb1*0101 1 (0.833) drb1* 0102-drb1*0101 14 (14.855) drb1* 0901-drb1*0311 # 2 (0.592) drb1*1901-drb1*0102 1 (1.393) drb1*0102-drb1*0102 8 (12.301) drb1*0901-drb1*0402 # 4 (3.722) drb1*1901-drb1*0402 2 (0.573) drb1*0304-drb1*010 3 (3.193) drb1*0901-drb1*0801 2 (0.931) drb1*1901-drb1*0601 1 (0.299) drb1* 0304-drb1*0102 # 7 (5.338) drb1*0901-drb1*0901# 2 (1.607) drb1*2001-drb1*0402 3 (0668) drb1*0304-drb1*0304 2 (0.549) drb1*1001-drb1*0101 7 (5.69 2) drb1*2001-drb1*0901 3 (0.592) drb1*0308 -drb1*0101 1 (0.278) drb1*1001-drb1*0102 12 (9.516) drb1*2101-drb1*0101 1 (5.137) drb1*0311-drb1*0102 1 (1.625) drb1*1001-drb1*0302 1 (0.089) drb1*2101-drb1*0102 12 (8.588) drb1*0402-drb1*0101 # 3 (6.109) drb1*1001-drb1*0304 1 (2.046) drb1*2101-drb1*0311 1 (0.562) drb1* 0402-drb1*0102 # 9 (10.213) drb1*1001-drb1*0311 1 (0.623) drb1*2101-drb1*0402 1 (0.532) drb1*0402-drb1* 0104 1(0.191) drb1*1001-drb1*0402 3 (3.913) drb1*2101-drb1*0801 1 (0.883) drb1*0402-drb1*0304 2 (2.195) drb1*1001-drb1*0601 2 (2.046) drb1*2101-drb1*0901 2 (3.130) drb1* 0402-drb1* 0308 1 (0.191) drb1*1001-drb1*0901 1 (3.469) drb1*2101-drb1*1001 8 (3.291) drb1*0402-drb1* 0311 # 1 (0.668) drb1*1001-drb1*1001 1 (1.779) drb1*2101-drb1*1901 1 (0.482) drb1* 0402-drb1* 0402 # 4 (2.052) drb1*1003-drb1*0101 1 (0.833) drb1*2101-drb1*2101# 2 (1.445) drb1*0601-drb1* 0101 3 (3.193) drb1*1003-drb1*0102 5 (1.393) drb1*2201-drb1*0102 2 (0.464) drb1* 0601-drb1*0102 8 (5.338) drb1*1501-drb1*0101 1 (0.417) drb1*2502-drb1*2502 1 (0.002) drb1*0601-drb1* 0304 1 (1.148) drb1*1501-drb1*0402 1 (0.286) drb1*12-drb1*0101 1 (2.777) drb1*0601-drb1*0402 3 (2.195) drb1*1501-drb1*0801 1 (0.072) drb1*12-drb1*0102 5 (4.642) drb1*0601-drb1* 0601 1 (0.549) drb1*1601-drb1*0101 4 (0.833) drb1*12-drb1*0104 1 (0.087) drb1*0801-drb1*0102 2 (2.553) drb1*1601-drb1*0402 # 1 (0.573) drb1*12-drb1*0304 1 (0.998) drb1*0801-drb1*0311 1 (0.167) drb1*1601-drb1*0601 1 (0.299) drb1*12-drb1*0601 1 (0.998) drb1*0801-drb1* 0402 # 1 (1.050) drb1*1607-drb1*0101 1 (0.139) drb1*12-drb1*0901 1 (1.692) drb1*0801-drb1*0601 1 (0.549) drb1*1608-drb1*0101 1 (0.972) drb1*12-drb1*1001 3 (1.779) drb1*0801-drb1*0801 1 (0.119) drb1*1608-drb1*0102 4 (1.625) drb1*12-drb1*2101 4 (1.605) drb1*0901-drb1*0101 10 (5.414) drb1*1608-drb1*0304 1 (0.349) drb1*12-drb1*1606 1 (0.043) drb1*0901-drb1*0102 # 9 (9.052) drb1*1608-drb1*2001 1 (0.106) drb1*12-drb1*12 1 (0.412) kalarrytik o drb1*0304-drb1*0102 # 1 (0.130) drb1*1601-drb1*0402 # 1 (0.192) drb1*1607-drb1*0102 1 (1.816) drb1*0402-drb1*0101 # 1 (1.344) drb1*1602-drb1*0102 2 (1.816) drb1*1607-drb1*0311 4 (1.466) drb1*0402-drb1*0102 # 11 (4.993) drb1*1602-drb1*0702 11 (5.935) drb1*1607-drb1*0402 3 (5.377) drb1*0402-drb1*0311 # 7 (4.032) drb1*160202-drb1*0402 2 (2.880) drb1*1607-drb1*0702 5 (5.9 35) drb1*0402-drb1* 0402 # 8 (7.297) drb1*160202-drb1*0702 1 (3.180) drb1*1607-drb1*1602 2 (1.955) drb1*0702-drb1*0101 4 (1.484) drb1*160202-drb1*1102 1 (0.037) drb1*1607-drb1*1606 4 (3.003) drb1*0702-drb1*0102 2 (5.511) drb1*160202-drb1*160202 5 (0.262) drb1*1607-drb1*1607 4 (0.943) drb1*0702-drb1*0402 13 (16.322) drb1*1604-drb1*0102 1 (0.195) drb1*1608-drb1*0402 1 (0.576) drb1*0702-drb1*0601 1 (0.212) drb1*1604-drb1*0702 2 (0.636) drb1*1608-drb1*0702 2 (0.636) drb1*0702-drb1*0702 11 (8.903) drb1*1605-drb1*1605 2 (0.015) drb1*2101-drb1*2101 # 1 (0.008) drb1*0801-drb1*0402 # 1 (0.192) drb1*1606-drb1*0101 2 (0.751) drb1*2401-drb1*0402 1 (0.384) drb1*0803 -drb1*0311 1 (0.419) drb1*1606-drb1*0102 5 (2.788) drb1*2502-drb1*0402 1 (0.192) drb1*0803 -drb1*0402 3 (1.536) drb1*1606-drb1*0304 1 (0.215) drb1*11-drb1*0402 3 (3.64 8) drb1*0803 -drb1*0702 1 (1.696) drb1*1606-drb1*0311 4 (2.252) drb1* 11-drb1*0901 1 (0.758) drb1*0803 -drb1*0803 1 (0.070) drb1*1606-drb1*0402 3 (8.257) drb1* 11-drb1*2003 1 (0.047) drb1*0901-drb1*0102 # 1 (1.037) drb1*1606-drb1*0403 1 (0.107) drb1*/11-drb1*2101 1 (0.142) drb1*0901-drb1*0311 # 1 (0.838) drb1*1606-drb1*0702 9 (9.115) drb1* 11-drb1*160202 1 (0.711) drb1*0901-drb1*0402 # 5 (3.072) drb1*1606-drb1*0803 1 (0.858) drb1* 11-drb1*1606 1 (2.037) drb1*0901-drb1*0702 1 (3.392) drb1*1606-drb1*0901 2 (1.716) drb1*11-drb1*/11 3 (0.426) drb1*0901-drb1*09 01# 1 (0.299) drb1*1606-drb1*1602 5 (3.003) drb1*26-drb1*1607 1 (0.070) drb1*1301-drb1*0702 1 (0.212) drb1*1606-drb1*2401 1 (0.215) drb1*30-drb1*0702 2 (0.424) drb1*1302-drb1*0702 1 (0.212) drb1*1606-drb1*1606 2 (2.252) symbol #: the common genotypes between the two breeds s. spetsa ria s et al. int. j. of health, animal science a nd food safety 3 (2016) 1 19 15 haf © 2013 vol. iii, no. 1 issn: 2283-3927 references abbott, k.a., taylor, m., stubbings, la., 2009. scops (sustainable control of parasites in sheep), sustainable worm control strategies for sheep, a technical 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commercial farm, growth performance, plasma parameters, weaned piglets. pages 50 – 57 references vol. 5 no. 1 (2018) article history submitted: may 08, 2018 accepted: october 23 2018 published: november 21 2018 corresponding author xianren jiang feed research institute, chinese academy of agricultural sciences, zhongguancun nandajie 12, beijing 100081, china mail: jiangxianren@caas.cn phone: +86 1062169968 fax: +86 1082106054 journal home page riviste.unimi.it/index.php/haf article effect of astragalus polysaccharide supplementation on growth performance and plasma parameters of weaned piglets under commercial condition bangmin liu a,1 , huiyuan lü b,1 , xianren jiang a,* , xilong li a a key laboratory of feed biotechnology of the ministry of agriculture, feed research institute, chinese academy of agricultural sciences, beijing, china b beijing traditional chinese veterinary medicine engineering research center, beijing centre biology co. ltd, beijing, china 1 these authors contributed equally to this study. abstract the aim of this study was to evaluate the effect of astragalus polysaccharide (aps) supplementation on the growth performance, plasma biochemical parameters, and plasma immune and antioxidant indexes of weaned piglets in a commercial swine farm. a total of 120 piglets weaned at 22 days and allocated to 2 groups, and fed a basal diet either without (ctr) or with 200 mg/kg of aps in a local commercial farm for a 42-d experiment. at end of the trial, one piglet from each pen was selected for blood sampling. the results showed that dietary aps decreased the feed conversion ratio (fcr) compared to the ctr group from day 14 to day 28 and day 0 to day 42 (p = 0.08 and 0.02, respectively). in addition, supplementation of aps had the tendency to increase the plasma superoxide dismutase activity and igg content of piglets compared to the ctr group on day 42 (p = 0.06 and 0.09, respectively). results in this study suggested that dietary aps might have a beneficial effect on growth performance and health status of weaned piglets under the commercial condition. bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 51 1 introduction there are significant changes in the histology and biochemistry of the gastrointestinal tract during the weaning phase that decrease digestive and absorptive capacity and contribute to post-weaning diarrhea (pluske et al. 1997). it is noteworthy that polysaccharide fractions of astragalus mongholicus and astragalus polysaccharide (aps) have been reported to reduce the incidence of diarrhea in animals (shao et al. 2004) and use as an immunomodulator (mao et al. 2005; zhuge et al. 2012). additionally, there is evidence that dietary supplementation with aps can improve growth performance (yuan et al. 2006; kang et al. 2010) and regulate the digestive and absorptive function of weaned pigs (yin et al. 2009). the antimicrobials usage as growth promoters or to prevent diseases provides great benefits for the growth and health of piglets, while it would also provides potential risks for human health through the transfer of resistant bacteria and associated genes via the food chain, direct contact or the environment (aarestrup et al. 2008). china has been limiting the usage of antibiotics for growth promoters in animal feed since that was banned by the european commission in 2006, while the productive performance in chinese commercial swine farms is still low. it has been reported that on average, chinese swine farms wean 22 piglets per sow per year (van dooren, 2018), which may be due to many issues such as equipment, management and feed quality. thus, the effective strategies aiming to improve health status of piglets and limit antibiotic use during the post-weaning period in swine production particular in the commercial swine farms are needed (stanton 2013). although aps has gained interests in medicine and health-related research in last decades, the information on efficacy of aps on growth performance and health status of weaned piglets in a chinese commercial swine farm is scarce. therefore, the objective of this study was to evaluate the efficacy of dietary aps supplementation on growth performance, plasma biochemical parameters, immune and antioxidant indexes of weaned piglets under the commercial condition. 2 material and method the animal protocol for this research was approved by animal care and use committee of the feed research institute of the chinese academy of agricultural sciences. bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 52 2.1 animals and treatments the study was conducted in a local commercial swine farm, langfang, heibei, china. a total of 120 crossed piglets (duroc × (landrace × yorkshire)) with an initial body weight of 7.38 ± 1.16 kg were used in a 42-day study. the pigs were weaned at 22 ± 2 days of age and allocated randomly on the basis of body weight to 2 treatments in a randomized complete block design. the dietary treatments were: the ctr group (basal diet) and the aps group (basal diet + 200 mg/kg aps). there were 60 piglets in each treatment, with 10 piglets per pen. the aps product was provided by beijing centre biology co., ltd. (beijing, china). each 2.00 × 2.00 m pen was equipped with a feeder and a water nipple to allow ad libitum consumption of feed and water. the temperature was kept at 28 ± 2°c, and relative humidity was maintained at 65-75%. feed intake was determined biweekly. piglets were weighed 1 h prior to feeding in the morning at the beginning of the experiment (day 0), day 14, 28 and the end of the experiment (day 42). the basal diet was formulated and manufactured before starting the trial, without the inclusion of any antibiotic growth promoters or antibiotic growth promoter alternatives. ingredients and chemical contents of the basal diets are summarized in table 1. all diets were formulated according to national research council (2012) recommended requirements of nutrients by swine. 2.2 experimental observations and measurements the body weight and feed intake were recorded at days 14, 28 and 42 for each replicate to determine the average daily gain (adg), average daily feed intake (adfi) and feed conversion ratio (fcr). on day 42, one piglet from each pen, close to the pen average weight, was selected to collect blood from jugular vein. blood samples were collected into heparinized test tubes to analyze the plasma biochemical parameters, antioxidant and immune status differences among dietary treatment, and were immediately centrifuged at 3000 × g for 10 min at 4°c to separate plasma. then the samples were stored at -20°c until analysis. the glutathione peroxidase (gsh-px) activity, total superoxide dismutase (t-sod) activity, and malondialdehyde (mda) content in plasma were analyzed using commercially available kits (nanjing jiancheng bioengineering institute, nanjing, jiangsu, china), and all the parameters were evaluated according to the manufacturer’s instructions. the contents of blood urea nitrogen (bun), total protein (tp) and glucose (glu) in plasma were determined by an automatic biochemistry analyzer (zhuoyue-310, shanghai kehua bio-engineering co., ltd., shanghai, china) in accordance with each kits (shanghai kehua bio-engineering co., ltd., china). quantification of immunoglobulin g bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 53 (igg) levels in plasma samples were assayed in duplicate using commercial porcine elisa kits according to the protocols provided by the manufacturer (nanjing jiancheng bioengineering institute, nanjing, china table 1. ingredient and chemical composition (g/kg) of the diets (as fed basis) item prestarter (day 0 to 14) starter (day 14 to 42) ingredients corn 563.1 617.5 soybean meal 337.6 282.2 corn protein powder (50% cp) 40.0 40.0 vegetable oil 17.3 23.1 dicalcium phosphate 19.0 13.9 limestone (caco3) 13.6 14.3 salt 3 3 dl-methionine 1.6 0.9 l-lysine hcl 1.4 1.7 vitamin and mineral premix1 2.4 2.4 choline chloride (50%) 1 1 nutrient composition2 me, mj/kg 12.13 12.55 crude protein 210 (209) 190 (187) calcium 10 9 available phosphorus 4.8 3.8 lysine 11 10 methionine 5 4 methionine + cysteine 8.2 7.2 1 premix supplied per kg of diet: retinyl acetate, 3.75 mg; cholecalciferol, 0.0625 mg; α-tocopherol acetate, 18.75 mg; menadione, 2.65 mg; thiamine, 2 mg; riboflavin, 6 mg; cobalamin, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; calcium pantothenate, 12 mg; niacin, 50 mg; manganese (mnso4·h2o), 100 mg; copper (cuso4 · 5h2o), 8 mg; zinc (zno), 75 mg; iron (feso4 · 7h2o), 80 mg; selenium (na2seo3), 0.15 mg; iodine (iodised nacl), 0.35 mg. 2 the nutrient levels listed in parentheses are determined values, others are calculated ones 2.3 statistical analysis all experimental data were analyzed by general linear model (glm) of spss 24.0 (spss inc., chicago, il). the model included the treatment effect (2 groups), and the pen represented the experimental unit for growth performance, while individual piglet was used as experimental unit for plasma parameters. treatment effects were considered significant at p ≤ 0.05, whereas a trend for a treatment effect was noted for p ≤ 0.10. bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 54 3 results and discussion the effect of dietary aps supplementation on growth performance of weaned piglets is shown in table 2. during the whole period of the trial, dietary aps decreased the feed conversion ratio (fcr) compared to the ctr group (p = 0.02). in addition, aps supplementation tended to reduce the fcr compared to the ctr group from day 14 to 28 (p = 0.08). in consistent with the observations in our study, previous studies indicated that supplementation of aps to the diet for weanling pigs enhanced feed efficiency (yuan et al. 2006; yin et al. 2009; kang et al. 2010). however, no difference in average daily gain (adg) between the ctr and aps groups was observed in all the periods of the trial (p > 0.05), which may be due to the filed condition and feeding phase. in our study, the improvement in adg by dietary aps was gradually enhanced with age of the piglets, while the adg of piglets in both groups was similar during the first 2 weeks post weaning. kang et al. (2010) added aps to the diet at a dose of 500 ppm of feed but failed to improve the growth performance of weaned piglets from day 0 to 14. yin et al. (2009) also observed that piglets fed with 1000 ppm aps did not increase adg during first 2 weeks post weaning. therefore, dietary aps might improve the growth of piglets not in the beginning period post weaning but in the late nursery phase. the effect of aps supplementation on the plasma biochemical parameters, antioxidant activities and igg content on 42 days of the trial are presented in table 3. results showed that supplementation of aps had the tendency to increase the plasma igg content compared to the ctr group (p = 0.09). yuan et al. (2006) suggested that dietary aps could be used as a potential immuno-modulating agent by affecting cellular immunity to improve growth and immune function of weaned pigs. adding aps diet tended to increase plasma t-sod activity (p = 0.06), and showed lower mean values in the contents of plasma mda (3.68 nmol/ml) though in this case differences compared with the ctr group (4.62 nmol/ml) were not significant (p = 0.21). zhao & shen (2005) suggested that the accumulation of ros might induce numerous disorders and cause a variety of impairments to tissues. similar with the observations in our study, zhong et al. (2012) reported that dietary aps exhibited antioxidant effects in newly weaned lambs by increasing t-sod activities in blood, and jia et al. (2012) concluded that aps could be used as a hepatoprotective and antioxidant agent in fish. in our study, we observed the decreased mean value in the content of bun of piglets fed the aps-containing diets compared to the ctr group (1.77 mmol/ml vs. 2.64 nmol/ml, p = 0.12), which in turn might have prevented or reduced the extent of the immune system activation in the group, thus allowing for a more efficient utilization of dietary protein and amino acid (aa) for growth or body protein deposition. yin et al. (2009) reported that aps might bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 55 ameliorate the digestive and absorptive function and regulate aa metabolism to beneficially increase the entry of dietary aa into the systemic circulation. table 2. effect of aps supplementation on growth performance of weaned piglets1 ctr aps s.e.m. p-value day 0 to 14 day 0 bw, kg 7.38 7.39 0.48 1.00 adg, g/d 132 133 13 0.96 adfi, g/d 242 232 16 0.64 fcr 1.895 1.771 0.097 0.40 day 14 to 28 adg, g/d 275 292 21 0.58 adfi, g/d 481 474 29 0.87 fcr 1.758 1.638 0.042 0.08 day 28 to 42 adg, g/d 444 470 27 0.52 adfi, g/d 722 712 45 0.88 fcr 1.641 1.511 0.058 0.18 day 0 to 42 adg, g/d 283 298 15 0.52 adfi, g/d 482 473 26 0.81 fcr 1.705 1.584 0.031 0.02 ctr = basal diet without additive; aps = astragalus polysaccharide; bw = body weight; adg average daily gain; adfi = average daily feed intake; fcr = feed conversion ratio. 1 n = 6 replicates/treatment table 3. effect of aps supplementation on the plasma biochemical parameters, igg content and antioxidant activities of weaned piglets 1 ctr aps s.e.m. p-value bun, mmol/l 2.64 1.77 0.36 0.12 tp, g/l 0.81 0.83 0.04 0.69 glucose, mmol/l 5.20 5.22 0.40 0.98 gsh-px, nmol/ml 596 677 70 0.45 mda, nmol/ml 4.62 3.68 0.49 0.21 t-sod, u/ml 62.48 73.01 3.34 0.06 igg, mg/ml 6.01 7.53 0.56 0.09 ctr = basal diet without additive; aps = astragalus polysaccharide; bun = blood urea nitrogen; tp = total protein; gshpx = glutathione peroxidase; mda = malondialdehyde; t-sod = total superoxide dismutase; igg = immunoglobulin g. 1 n = 6 replicates/treatment. bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 56 1 conclusions in conclusion, the supplementation of aps in weaned piglets’ diet has the potential to increase the growth performance of weaned piglets in the commercial swine farm but probably in the late nursery phase, and dietary aps may improve the plasma immune and antioxidant status of weaned piglets under the commercial condition. more studies on aps application may benefit the swine industry. acknowledgement: the authors gratefully appreciate the financially support provided by beijing natural science foundation (6174049) and fundamental research funds for central non-profit scientific institution (1610382017005, beijing, china). disclosure statement: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of this article. references aarestrup fm, oliver duran c, burch dg. 2008. antimicrobial resistance in swine production. animal health res rev. 9:135-148. jia r, cao l, xu p, jeney g, yin g. 2012. in vitro and in vivo hepatoprotective and antioxidant effects of astragalus polysaccharides against carbon tetrachlorideinduced hepatocyte damage in common carp (cyprinus carpio). fish physiol biochem. 38:871-881 kang p, xiao hl, hou yq, ding by, liu yl, zhu hl, hu qz, hu y, yin yl. 2010. effects of astragalus polysaccharides, achyranthes bidentata polysaccharides, and acantbepanax senticosus saponin on the performance and immunity in weaned pigs. asian-aust j anim sci. 23:750-756. mao xf, piao xs, lai ch, li df, xing jj, shi bl. 2005. effects of beta-glucan obtained from the chinese herb astragalus membranaceus and lipopolysaccharide challenge on performance, immunological, adrenal, and somatotropic responses of weanling pigs. j anim sci 83:2775–2782. pluske jr, hampson dj, williams ih. 1997. factors influencing the structure and function of the small intestine in the weaned pig: a review. livest prod sci. 51:215-236. bangmin liu et al. int. j. of health, animal science and food safety 5 (2018) 50 59 57 shao bm, xu w, dai h, tu pf, li zj, gao xm. 2004. a study on the immune receptors for polysaccharides from the roots of astragalus membranaceus, a chinese medicinal herb. biochem biophys res commun 320:1103–1111. stanton tb. 2013. a call for antibiotic alternatives research. trends microbiol. 21:111-113. van dooren k. 2018. yangxiang aims high with sows on many floors. pig progress. published on april 20, 2018. yuan sl, piao xs, li df, kim sw, lee hs, guo pf. 2006. effects of dietary astragalus polysaccharide on growth performance and immune function in weaned pigs. anim sci. 82:501-507. yin fg, liu yl, yin yl, kong xf, huang rl, li tj, wu gy, hou yq. 2009. dietary supplementation with astragalus polysaccharide enhances ileal digestibilities and serum concentrations of amino acids in early weaned piglets. amino acids. 37:263270. zhao rz, shen gx. 2005. functional modulation of antioxidant enzymes in vascular endothelial cells by glycated ldl. atherosclerosis. 179:277-284. zhuge zy, zhu yh, liu pq, yan xd, yue y, weng xg, zhang r, wang jf. 2012. effects of astragalus polysaccharide on immune responses of porcine pbmc stimulated with prrsv or csfv. plos one. 7:e29320. zhong rz, yu m, liu hw, sun hx, cao y, zhou dw. 2012. effects of dietary astragalus polysaccharide and astragalus membranaceus root supplementation on growth performance, rumen fermentation, immune responses, and antioxidant status of lambs. anim feed sci technol. 174:60-67. 32 l keywords fluoroquinolone residues; ciprofloxacin; norfloxacin; ofloxacin; consumer protection; meat safety. pages 32. – .40 references vol. 2 no. 1 (2015) article history submitted: march 15, 2015 revised: june 03, 2015 accepted: june 24, 2015 published july 11, 2015 corresponding author omotoso adekunbi b., animal products and processing unit, department of animal science, university of ibadan, ibadan, nigeria e-mail: kunbiadeshiyan@yahoo.com phone: +234 08033900828 journal home page riviste.unimi.it/index.php/haf fluoroquinolone residues in raw meat from open markets in ibadan, southwest, nigeria. omotoso adekunbi b.1* and omojola andrew b1 1 animal products and processing unit, department of animal science, university of ibadan, ibadan, nigeria. abstract. misuse of fluoroquinolones in livestock production may lead to the presence of their residues in tissues of meat animals after slaughter, constituting health hazards to consumers. the present study was designed to screen for residues of three fluoroquinolones (ciprofloxacin, norfloxacin and ofloxacin) in raw meat. microbiological assay, followed by high performance liquid chromatography (hplc) was used to screen three hundred and twenty samples of beef, chicken, pork and chevon purchased from open markets. initial screening by microbiological assay revealed that 50%, 55%, 40% and 40% of beef, chicken, pork and chevon, respectively were positive for residues of antibiotics. further analysis by hplc with uv detection revealed the presence of ciprofloxacin, norfloxacin and ofloxacin at varying concentrations in the meat samples. ofloxacin was the least in frequency and abundance in all meat types. results obtained in this study have implications for public health and will lead to steps that will further enhance the safety of animal foods in order to protect consumers and the animal production industry. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 33 haf © 2013 vol. ii, no. 1 issn: 2283-3927 1 introduction fluoroquinolones are syn thetic antimicrobials used in the treatment of severe and invasive infections in humans and animals. the presence of fluoroquinolone residues in meat has attracted extensive atten tion from national and international public health ag encies. this is because of th e risks associated with th e consumption of food con taminated with fluoroquinolone residues as they may be directly toxic or be a source of resistan t human pathogens, representing a possible risk to human health (juan -garcía et al. 2006). condition s such as phototoxic skin reactions in humans (klecak et al., 1997) and chondrotoxic effects in young animals (stahlmann et al., 2000) can be induced by fluoroquinolones. probably the mos t importan t threat to human health as a result of fluoroquinolon e use in animal production is th e developm ent of fluoroquinolone-resistant bacteria as it has been discovered that a major route of transmission of resistant micro-organisms from animals to humans is through th e food chain (h ernández-serrano, 2005). the abuse of veterinary drugs is one of the causes of drug residues in animal products (saleh zadeh et al, 2006, fagbamila et al., 2010). drugs belonging to the fluoroquinolone class such as enrofloxacin, n orfloxacin, pefloxacin, ciprofloxacin and ofloxacin are often used in livestock production to con trol diseas es, leading to the production of healthy animals, thereby reducing farmers’ losses. ofloxacin is a fluoroquinolon e of interest due to the resistance of a broad spectrum of microbes to it, especially as a result of its use as grow th promoter (naeem et al., 2006). low levels of ciprofloxacin, another fluoroquinolone, can strongly suppr ess facultative anaerobic human intestinal microflora (cerniglia and kotarski, 1999); it also leads to th e developmen t of resistant bacteria. similarly, exposure to norfloxacin has been shown to induce resistance to other fluoroquinolon es and even to the structurally unrelated aminoglycosides in coagulasenegative staph ylococci (deshmukh et al., 1997). detection and control of fluoroquinolon e residues in meat is important due to mark et globalization and food safety issues. methods developed for detecting f luoroquinolon e residues include microbiological assays, immunoassays, liquid chromatograph y, among others. microbiological inhibitory tests, which rely on inhibition of g rowth of the test organism by th e residual drug, are inexpensive and simple; however, most lack specificity and few are quantitative. microbiological assays are generally used for residue screening as part of an integrated s ystem with follow-up confirmatory analysis of suspicious samples (choi et al., 1999). high performance liquid chromatography (hplc) is used to separate, iden tify an d quantify different ch emical componen ts. it has been successfully applied to the detection of different drug residues in foods of animal origin (reig et al., 2006). in order to protect human health, regulatory bodies such as the european union and codex alim entarius commission, have established maximum residue limits (mrls) for veterinary drugs in differen t matrices of differen t animal species. th e eu mrl for the sum of enrofloxacin and ciprofloxacin is adopted for comparison in this study. the world health organization has been cam paigning for an tibiotic resistance to be tackled from a food safety perspective (wh o, 2011). there is th erefore a need to establish a comprehensive database for residues of relevan t antibiotics in the m eat production industry as a step towards con trolling th e ris e in antimicrobial resistance in africa. presen tly, several countries cannot assure consumers of the safety of th e meat products th ey consume with omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 34 haf © 2013 vol. ii, no. 1 issn: 2283-3927 respect to drug residue con cen tration. this study was set up to investigate the presence of ciprofloxacin, norfloxacin and ofloxacin residues in four commonly consumed m eat types in nigeria. 2 materials and methods a total of th ree hundred and twen ty raw meat samples (comprising 80 each of beef, chicken, chevon and pork ) were collected from four major markets in ibadan, nigeria. th ey were collected on ice and kept frozen at -30c until analyzed. the analyses w ere carried out a t the meat science laboratory of th e university of ibadan and national agency for food an d drug administration and control (nafdac) laboratory, agulu, anambra state, nigeria. th e samples were screened for three fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin . 2.1 qualitative analysis: microbiological screening initial screening was done using the one plate tes t (opt) as described by alla et al. (2011). the screening procedure has been previously reported in detail in a similar experimen t by omotoso and om ojola (2014). the tes t organism was escherichia coli (atcc 10536). zone of inhibition was measured with mm graduated ruler. negative samples were discarded while suspicious and positive samples were subjected to fu rther analysis for iden tification an d quantification of residual ciprofloxacin, norfloxacin and ofloxacin. 2.2 identification and quantitative analysis following residue extraction as described by ovando et al. (2004), the fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin were identified using hplc with uv detection. the hplc equipmen t was elite lachrom vwr, hitachi ch romatograph (hitachi, tokyo, japan) comprising l2200 autosampler, l2130 pump and l2350 column oven, equipped with l2400 uv vis detector and ezchrom elite software. th e separating column was elite c18 (250mmx4.6mmx5μm) (hitachi, tok yo, japan). reference standards of ciprofloxacin (99.6%), norfloxacin (99.5%) and ofloxacin (99.9) were provided by nafdac. details of th e extraction and subsequent analysis have been reported by omotoso and omojola (2014). 2.3 statistical analysis statistical analysis was accomplished by analysis of variance using spss (2005). treatm en t means were compared using duncan multiple range test and statis tical significance was set a t a probability of p ≤ 0.05. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 35 haf © 2013 vol. ii, no. 1 issn: 2283-3927 3 results frequency of antibiotic contamination in the different meat types is shown in figure 1. initial microbiological screening revealed that more than half of the chicken samples tes ted positive for antimicrobial residues. this was followed by beef with 50% positive samples. similarly, 40% of pork and chevon samples tes ted positive for antimicrobial residues. figure 1. percen tage of raw m eat samples positive for antibiotic residues . percentages of meat samples positive for ciprofloxacin, norfloxacin and ofloxacin residues are shown in figure 2. mos t chicken and beef samples had detectable levels of ciprofloxacin; however, less than half of pork and chevon contained residues of this popular fluoroquinolone. the m eat type with the high est frequency of n orfloxacin residue was chick en, follow ed by beef. ofloxacin occurred more frequen tly in beef (48.75%) than other meat types. it is worthy of note that pork, followed by chev on, had the least number of samples with detectable levels of an y of the th ree fluoroquinolon es. levels of ciprofloxacin, norfloxacin and ofloxacin residues in the tes ted raw meat samples are shown in table 1. in beef, m ean residual ciprofloxacin (231.08 ± 564.30) and norfloxacin (173.40 ± 154.57) were above th e adopted mrl of 100 µg/kg, while ofloxacin value (79.28 ± 183.70) was below the limit. in chicken, how ever, norfloxacin was the most abundan t fluoroquinolone, with a mean value that was sligh tly above 100µg/kg and significantly differen t from the levels of ciprofloxacin and ofloxacin. although ciprofloxacin in pork was very high, levels of norfloxacin and ofloxacin in this meattype were very low. high level of ciprofloxacin was also recorded in chevon (345.62 ± 796.35) but this had low er n orfloxacin and ofloxacin residues. percen tages of meat samples containing residues of ciprofloxacin, norfloxacin an d ofloxacin above adopted mrl are shown in table 2. 0 10 20 30 40 50 60 beef chicken pork chevon p e r c e n t (% ) p o s it iv e f o r a n ti b io ti c r e s id u e s meat type omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 36 haf © 2013 vol. ii, no. 1 issn: 2283-3927 figure 2. detection of ciprofloxacin, norfloxacin and ofloxacin in raw meat. table 1. mean concentration of fluoroquinolones in beef, chicken, pork and chevon. meat type mean concentrations (µg/kg) of fluoroquinolones p value ciprofloxacin norfloxacin ofloxacin beef 231.08 ± 564.30 173.40 ± 154.57 79.28 ± 183.70 0.536 chicken 67.85 ± 125.42b 113.59 ± 158.32a 13.55 ± 22.46c 0.022 pork 315.30 ± 834.35a 11.39 ± 28.31b 27.02 ± 64.48b 0.091 chevon 345.62 ± 796.35a 30.93 ± 129.18b 12.37 ± 30.64b 0.047 a, b, c : means in the same row with different superscripts are significantly different (p ≤ 0.05) 4 discussion several reports have shown that th e presence of fluoroquinolone residues in human food obtained from animal sources has reduced the effectiveness of these agen ts in human medicine. in addition, direct toxicity and other side effects of fluoroquinolones are w ell established in animals and humans (gru challa and pirm ohamed, 2006; khadra et al., 2012). thus it is importan t to monitor residues of fluoroquinolones in frequ ently consumed meat and mea t products. 0 10 20 30 40 50 60 ciprofloxacin norfloxacin ofloxacin p e r c e n t (% ) p o s it iv e s a m p le s fluoroquinolones beef chicken chevon pork omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 37 haf © 2013 vol. ii, no. 1 issn: 2283-3927 table 2. percentag e of raw meat samples with fluoroquinolone residues above maximum residue limit. fluoroquinolones meat type n/n % above mrl* ciprofloxacin beef 40/80 23.75 chicken 41/80 15.00 pork 28/80 20.00 chevon 23/80 38.75 norfloxacin beef 37/80 25.00 chicken 44/80 35.00 pork 24/80 6.25 chevon 20/80 5.00 ofloxacin beef 39/80 15.00 chicken 32/80 0.00 pork 20/80 10.00 chevon 11/80 0.00 n=number of positive samples; n = number of examined samples *100µg/kgeuropean union mrl (commission regulation (eu) 37/2010) for the sum of ciprofloxacin and enrofloxacin in poultry muscle. in the present s tudy, raw samples of fou r meat types comm only consumed in nigeria were tested for the pres ence of fluoroquinolones. approximately 50% of th e three hundred an d twen ty samples were positive for residues of at least on e antibiotic active against the tes t organism, escherichia coli. fifty-five percen t of chicken meat and fifty percen t of beef were positive for antibiotic residues. in a related study using samples obtained from open markets in sokoto, nigeria, 44% of slaughtered cattle were found to con tain residues of antibiotics (ibrahim et al., 2009). residues have been reported in 21% of meat samples in ghana (novais et al., 2010), and 70% in tanzania (kurwijila et al., 2006). tajick and shohreh (2006) also found that more than 50% of poultry m eat tissues tes ted in iran had residues of an timicrobials. in con trast, alla et al. (2011) analyzed beef samples in sudan and reported that only 3% of the muscles contained an tibiotic residue. findings in this study rev ealed that fluoroquinolon es are frequently administered in meat animal production in nigeria. results of microbiological screening were similar to findings in the screening of broiler m eat and beef sold in the markets of ankara, t u rkey by er et al., (2013 ) who discovered that 45.75% of chicken and 57.7% of beef were positive for quinolone residues; however, mean con centrations obtained in their study were below th e 100µg/kg mark (th e omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 38 haf © 2013 vol. ii, no. 1 issn: 2283-3927 mean lev els of quinolon e an tibiotic residue were found to be 30.81 ± 0.45 μg/kg in positiv e chicken samples and as low as 6.64±1.11μg/kg in positive beef samples ). in a similar experim en t to test for th e presence of fluoroquinolon es in chick en, omotoso and om ojola (2014) reported ciprofloxacin level of (354.83 ± 716.43 μg/kg) in imported chicken. the frequen cy of occurrence and high concentration of ciprofloxacin in th e present study may be due to the fact that ciprofloxacin is also a mark er residue for en rofloxacin. en rofloxacin is approved for use as a veterinary drug and when it is metabolized, its pharmacologically active metabolite, ciprofloxacin, is produced. hence detection of ciprofloxacin during marke t screening often reflects the use of both enrofloxacin and ciprofloxacin (navrátilová et al., 2011; botsoglou and fletouris 2001). mean norfloxacin residues in beef and chick en were higher than the mrl. ofloxacin concen tration was the lowes t in all tested m eat types except pork, which had higher ofloxacin than norfloxacin. generally, ciprofloxacin was the most abundant in all except for chicken meat which had the highest mean norfloxacin (173.40 ± 154.57). the level of abundance of th e individual agents in each meat type is as follows beef: ciprofloxacin > norfloxacin > ofloxacin chicken: norfloxacin > ciprofloxacin > ofloxacin pork: ciprofloxacin > ofloxacin > norfloxacin chevon: ciprofloxacin > norfloxacin > ofloxacin the study shows that ofloxacin is not administered as frequ ently as the other tw o antimicrobial agents in livestock production in the study area. no individual chicken or chevon sample had ofloxacin lev els above mrl (table 2). howev er, 15% and 10% of beef and pork respectively contain ed ofloxacin at levels above mrl. this shows that ofloxacin may no t necessarily be a drug of con cern in chicken m eat and ch evon. detection of fluoroquinolon e residues in meat samples from the city of ibadan, nigeria, corroborat es the findings of other researchers in other coun tries where residue monitoring is absent or inadequate. 5 conclusion in the absence of official m onitoring programm e on drug residues in developing countries, misuse of antimicrobials, including fluoroquinolon es, is inevitable. detection of residues of fluoroquinolones in half of tested meat samples shows the widespread use of these agen ts in cattle, poultry, hog and goat production in nigeria during the period of th e study. the pres en t situation can be con trolled as less frequent contamination and reduced concentration in contaminated samples may result when steps are tak en towards encou raging prudent use of fluoroquinolones. consumer protection can be ensured by screening food animals for residues of antibiotics before slaughter. data collected over a period of tim e will provide basis for legislations that will reduce th e risk to consumers. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2015) 32.-.40 39 haf © 2013 vol. ii, no. 1 issn: 2283-3927 6 acknowledgement the authors gratefully ackn owledg e the national agency for food and drug administration and control (nafdac), nigeria, for th e provision of materials, equipment an d technical support needed for th e 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meat using tlc. international journal of poultry science. 5 (7), 611–612. world health organization, 2011. tackling antibiotic resistance from a food safety perspective in europe. copenhagen: world health organization. l keywords camel milk; processing; rennet coagulation time; heat stability. pages 27 – 37 references vol. 3 no. 1 (2016) article history submitted: august 03, 2016 revised: december 08, 2016 accepted: december 12 2016 published: december 17 2016 corresponding author bhavbhuti m. mehta, dairy chemistry department, smc college of dairy science, anand agricultural university. anand-388 110, gujarat, india. mail: bhavbhuti5@yahoo.co.in phone: + 919825807454 journal home page riviste.unimi.it/index.php/haf article evaluation of camel milk for selected processing related parameters and comparisons with cow and buffalo milk shyam p. sagar 1 , bhavbhuti m. mehta 2,* , k.n. wadhwani 3 , v.b. darji 4 , k.d. aparnathi 2 1 executive qa, gcmmf, anand, gujarat, india 2 dairy chemistry department, smc college of dairy science, anand agricultural university (aau), anand, gujarat, india 3 department of livestock production and management, veterinary college, aau, anand 4 department of agricultural statistics, b.a. college of agriculture, aau, anand abstract cow and buffalo milk and camel milk were analyzed and compared for processing related parameters. the average heat stability of cow, buffalo and camel milk samples analyzed was 1807.4 seconds, 1574.6 seconds and 133.6 seconds respectively at 140 °c. thus, the heat stability of camel milk was significantly lower than the cow milk and buffalo milk. the average rennet coagulation time (rct) of cow, buffalo and camel milk was 310.6 seconds, 257.4 seconds and 604.2 seconds respectively. thus, rct of camel milk was significantly higher than the cow milk and buffalo milk. the camel, cow and buffalo milk samples showed negative alcohol stability. the rate of acidity was increased propositionally with time in camel milk with no curd formation and weaker body. s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 28 haf © 2013 vol. iii, no. 1 issn: 2283-3927 1 introduction camel (camelus dromedaries) milk and its products may be one of the economical ways to improve the social life of camel owners. camel milk is an important source of proteins for the people living in the arid lands of the world. various researchers have worked on camel milk which are related to production or composition aspects (farah 1993; wangoh 1997; konuspayeva et al. 2009; al haj and al kanhal 2010; yoganandi et al. 2015 a,b,c,d). camel milk is considered to have anti-cancer, hypo-allergic, and anti-diabetic properties (agrawal et al. 2003, magjeed 2005; shabo et al. 2005; konuspayeva et al. 2008). camel milk can be considered as a good food of high nutritive and therapeutic applications. in the western world, camel milk is experiencing a novel awareness in these days and even the fao has stepped in promoting camel milk (ramet 2001). various researchers have tried to prepared various milk products from camel milk like ice cream (abu-lehia et al. 1989), butter (farah et al. 1989), fermented products (farah et al. 1990; fguiri et al. 2015), probiotic frozen yoghurt (al-saleh et al. 2011) and cheese (inayat et al. 2007; el zubeir and jabreel 2008), khoa (chaudhary et al., 2016), ghee (parmar 2013). butter was reported to be only produced from camel cream at a high churning temperature of 20 ο c 25 ο c. these temperatures are higher than those values reported for bovine milk butter manufacture of 8 ο c 12 ο c (rüegg and farah 1991). chaudhary et al (2016) compared the various chemical compositions and characteristics of the khoa prepared from the camel milk with that prepared from the cow and the buffalo milk samples. the khoa prepared from the camel milk had the higher moisture, ash, acidity, soluble nitrogen, free fatty acids and peroxide value, but lower in fat, protein and lactose contents than that prepared from the cow and buffalo milk samples. chaudhary (2013) also prepared the burfi and gulabjamun from camel milk khoa. lad (2016) worked to enhance the quality of gulabjamun prepared from camel milk khoa. camel milk exhibits a two to three fold longer rennet coagulation time compared with bovine milk (farah and bachmann 1987). countries like india, uae having camel milk parlor in which various products prepared from camel milk are available which have high demand. however, publications dealing with the processing related parameters of camel milk are relatively scarce and much of the information is approximate and fragmental (farah and bachmann 1987; mohammed and larsson-raznikiewicz 1989; farah and atkins 1992; kouniba et al. 2005). up to the early 1970, research on camel milk was limited to studies on general composition and milk yields. much of the work so far has been carried out by the individuals with little institutional support. thus the research remained isolated with little impact on dairy camel production (farah 1993). development and research activities on domestic animals are mostly concentrated on species and breeds of animals available in asian countries. that leads to less concentration on several species of animals native to the countries. the camel is certainly one of the most neglected species of the domestic animals (knoess 1979). thus, fewer data on camel milk processing are available, compare with cow and buffalo milk. processing related parameters such as alcohol stability, heat stability, rennet coagulation time (rct) and rate of acid production are not well studied. camel (camelus dromedaries) population in gujarat state of india was reported to be 0.3 lacks which contributed 7.6% in india (4.0 lacks) and ranked 2nd position (livestock census 2014). however, the information on processing related parameters of camel milk produced in gujarat is not available. therefore, there is a need to undertake systematic study to generate s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 29 haf © 2013 vol. iii, no. 1 issn: 2283-3927 data. hence, the present study aimed to study the selected processing related parameters of camel (milk collected from anand and kheda district) and its comparison were carried out with cow and buffalo milk. moreover, this information will be beneficial to manufactures/industrial personnel as well as policy makers involved in processing of milk and milk products prepared from camel milk. 2 material and methods the three different milk samples of camel, cow and buffalo were studied. the pooled milk samples of camel milk (8 samples, from anand and kheda district of gujarat state, india) as well as cow milk (8 samples) and buffalo milk (8 samples) were collected from the local herd maintained in village nearby anand. samples were transported to the laboratory, where they were stored at 4 °c before its analysis. total 24 samples (8 each of camel, cow and buffalo milk) were analyzed for gross chemical composition of camel, cow and buffalo milk samples as described in bis handbook (sp 18: part xi, 1981). these samples were also analyzed for processing related parameters such as alcohol stability, heat stability, rennet coagulation time (rct) and rate of acid production. all the chemicals used for chemical analysis were of analytical reagent grade. 2.1 gross chemical composition of milk the milk fat content in all the milk samples were estimated by following the gerber method, solid not fat (snf) and total solids content were calculated by gravimetric method (sp 18: part xi, 1981). lactose content was determined using lane and eyon method, milk protein content was determined using micro-kjeldahl method of nitrogen estimation (percent total protein was obtained multiplying the percent nitrogen by a factor of 6.38), ash (a grey white residues obtained after incineration of milk at 500 to 550 c) content was determined using gravimetric method and acidity (% lactic acid) as described in bis handbook (sp 18: part xi 1981). 2.2 processing related properties processing related properties includes alcohol stability, heat stability, rennet coagulation time (rct) and rate of acid production. 2.3 alcohol stability in a test tube, 5 ml milk samples was taken and added an equal volume of ethyl alcohol (75% and 68% by volume for cow, buffalo and camel milk). formation of flakes or curd on the sides of the test tube indicated the positive test while no changed in milk considered as negative test (sp 18: part xi 1981). s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 30 haf © 2013 vol. iii, no. 1 issn: 2283-3927 2.4 heat stability heat coagulation time (hct) was determined in a thermostatically controlled oil bath at 140 °c according to the method of davies and white (1966). in a test tube, 3 ml of milk sample was taken and kept in controlled oil bath having 140 °c and noted down the time. gradually rotated the test tube in oil bath and observed for flakes formation (coagulation occurred) and noted down the time. the total time was considered as hct. 2.5 rennet coagulation time the time from rennet addition to the onset of gelation (rennet coagulation time, rct) is an important practical consideration in cheese making. the determination of rct involves measurement of the time elapsed between the addition of a known amount of rennet (diluted) to a known volume of milk at a given temperature and the onset of gelation (usually assessed visually). for determination of rennet coagulation time (rct), a macro film technique was used that is developed by sharma and bhalerao (1963). two test tubes of different diameter were taken. the five ml milk sample was taken in test tube with bigger diameter and brought its temperature to 40 °c, keeping it in water bath. 0.2 ml 1% rennet solution were added and started the stop watch soon after addition of rennet. mixed the content thoroughly and insert another test tube so that a film was formed in the annular space. incubated at 40 °c till the first appearance of clotting in the film was observe. noted down the time. 2.6 rate of acid production in the present investigation, rate of acid production in camel milk was measured by adding starter culture of streptococcus thermophilus in camel milk at the rate of 2% rate of milk and incubated at 37 °c for 24 hours and acidity (in percent lactic acid) was measured at every 2 hours interval (sp 18: part xi 1981). 2.7 statistical analysis the data obtained during investigation were subjected to statistical analysis using completely randomized design (rudolf et al., 2010). the data are mean (average) of 8 replicates for each type of milk. 3 results and discussion the gross compositions of camel, cow and buffalo milk were analyzed. the mean values of gross composition of different types of milk are shown in table 1. s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 31 haf © 2013 vol. iii, no. 1 issn: 2283-3927 table 1 types of milk gross composition (%)* ts fat snf protein lactose ash camel 11.89 4.39 7.46 2.93 4.15 0.72 cow 13.12 4.62 8.42 3.28 4.37 0.69 buffalo 15.56 6.41 8.94 3.82 4.63 0.70 *mean values of eight replications these data indicated that total solids content was lower in camel milk but ash content was relatively higher than other cow and buffalo milk. these data are well correlated with values reported by yoganandi et al. (2015a,d). 3.1 alcohol stability the alcohol test determines the susceptibility of milk to coagulate due to development of acidity, disturbed salt balance or high albumin-globulin content. the milk giving a positive alcohol test will, coagulates upon heat treatment. the alcohol stability of milk from cow, buffalo and camel milk were studied. ethyl alcohol of 68% by volume and 75% by volume were used to measure the alcohol stability. the collected cow, buffalo and camel milk samples showed negative alcohol stability i.e. there were no visible flakes/coagulation formation in all milk samples. no data is reported on alcohol stability of camel milk. thus, comparison is not possible. unnikrishnan et al (1988) found wide variation in the alcohol stability in both buffalo and cow milk. the buffalo milk coagulated in the ranged from 60 – 72% against 70 – 80% for cow milk. wang et al (2016) reported that goat milk exhibited a markedly lower alcohol stability than cow milk. the goat milk produced a much flocculated precipitate but the cow milk produced no flocculated precipitate. 3.2 heat stability heat stability of milk is defined as the time necessary to initiate coagulation in milk at definite temperature (generally at 140 °c). the coagulation is indicated by flocculation, gelation or changes in protein sedimentability (rose 1963). for bovine milk, the most widely used temperature for heat coagulation is 130 or 140 °c (farah and atkins, 1992). heat stability of cow, buffalo and camel milk samples was measured by heating milk at 140 °c in oil bath and data are shown in table 2. the range of hct of camel milk samples analyzed was 129 to 140 seconds at 140 °c. similarly in cow milk samples hct ranges from 1798 to1816 seconds and in buffalo milk it varied from 1563 to 1581 seconds. thus among all the milk samples analyzed, camel milk have poor heat stability (p<0.05) and such milk did not be withstand high heat treatments moreover, hct of buffalo milk was significantly lower than that of cow milk. s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 32 haf © 2013 vol. iii, no. 1 issn: 2283-3927 table 2. heat stability of cow, buffalo and camel milk type of milk heat coagulation time (hct) (seconds) range average cow 1798 – 1816 1807.4 buffalo 1563 – 1581 1574.6 camel 0129 – 0140 133.6 sem 2.8449 cd 8.7659 cv % 0.5428 5% level of significant (i.e., p<0.05). sem=standard error of mean, cd=critical difference, cv=coefficient of variance farah and atkins (1992) analyzed heat coagulation time for cow milk at 130 °c that is about 40 min at ph 6.7, whereas camel milk coagulates in 2 to 3 min at this temperature and ph. kouniba et al. (2005) carried out an experiment to check heat stability of camel milk. they found out that heat stability of camel milk was relatively lower at high heat treatments. heat coagulation time in the range of 100-130 ºc was too short (< 2 min.). compositional differences and heat-induced interaction between the caseins and whey proteins, particularly κ-casein and β-lactoglobulin, are reported to be responsible for these differences (haynes and fox 1975; fox and hoynes 1976). it is possible that camel casein contained so little k-casein that it escaped detection or was obscured by other casein fractions. the camel milk contains kcasein only 5 percent of total casein, compared to bovine milk (13.6%) (ramet 1991 ; farah 1993) and buffalo milk contains 12 percent of total casein. the evidence for presence of βlactoglobulin in camel milk is conflicting (farah 1986). the β lactoglobulin concentration in milk is reported 0.93 and 02-0.4 percent in buffalo and cow milk respectively (sahai 1996). the impact of other little-known factors in camel milk such as the level of soluble calcium and phosphate, as well as the concentration of colloidal calcium phosphate and the nature of its binding to casein, might also be considered (farah 1986). 3.3 rennet coagulation time (rct) in the investigation, rct of all three milk samples of camel, cow and buffalo was determined by adding 1% rennet solution to different milks and kept them at 40 °c water bath. the data obtained for rct content of camel, cow and buffalo milk are presented in table 3. the range of rct of camel milk samples analyzed was 595 to 618 seconds and having mean value of 604.2 seconds at 40 °c. similarly in cow milk samples rct ranged from 294 to 326 seconds and having mean value of 310.6 and in buffalo milk it was 243 to 270 seconds and having mean value of 257.4 seconds. thus rct of camel milk was significantly higher than the cow milk and buffalo milk. the observed data showed that range of rct of camel milk was approximately 2 times higher than that of cow and buffalo milk. similarly rct of buffalo milk was significantly lower than that of cow milk. s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 33 haf © 2013 vol. iii, no. 1 issn: 2283-3927 table 3 rennet coagulation time for cow, buffalo and camel milk type of milk rennet coagulation time (sec) range average cow 294-326 310.6 buffalo 243-270 257.4 camel 595-618 604.2 sem 4.881 cd 15.039 cv % 2.793 5% level of significant (i.e., p<0.05). sem=standard error of mean, cd=critical difference, cv=coefficient of variance mohammed and larsson-raznikiewicz (1989) studied the coagulation properties of somali camel milk using bovine chymosin. with the same chymosin concentration, the coagulation time for camel milk was 2 to 3 times longer than that for cow milk. farah and bachmann (1987) examined the rennet coagulation of 10 individual camel milk samples from northern kenya using commercial calf rennet powder. they observed that rennet coagulation time for camel milk at ph 6.65 is 840 seconds and rct of cow milk at ph 6.65 is 300 seconds. with the same amount of rennet the coagulation time of camel milk was two to three fold longer than that of cow milk. that may be because of low amount of kappa casein that is only about 5 percent of the total casein, compared with about 13.6 percent in bovine casein (farah 1993) and presence of β-lactoglobulin has not been clearly identified (farah 1986). renneting is probably low, because the mean size of casein micelles in camel milk is bigger than that of cow and buffalo milk. in comparison of these milk, the size of casein micelles is bigger camel milk (320 nm) followed by buffalo milk (110-160 nm) and cow milk (70-110 nm). bigger the size of casein micelles, less will be the κ-casein content. electron micrographs showed that the network formed at the coagulation point was less compact than in renneted cow milk, and the micelles were linked merely by contact adhesion, with little change in the original micellar structure, whereas the network formed in cow milk consisted of fused micelles (farah and bachmann 1987). 3.4 rate of acid production by starter culture fermented milk products are known for their taste, nutritive value and therapeutic properties. the preservation of food by fermentation is one of the oldest methods known to mankind. a typical example is lactic acid fermentation, which is widely used for the preparation of several fermented milk products, such as dahi (curd), yoghurt, acidophilus milk, shrikhand and various varieties of cheeses. for that starter culture was used. lactic acid bacteria like lactococcus, lactobacillus, streptococcus and leuconostocs are often called dairy starter cultures, which are used for the production of various fermented milk products. starter culture s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 34 haf © 2013 vol. iii, no. 1 issn: 2283-3927 should grow properly and should produce required acidity in limited time and should produce fine and firm curd. the figure 1 showed the graphical presentation of data on rate of acid production in camel milk. figure 1 rate of acid production in camel milk by streptococcus thermophilus the initial acidity observed was 0.144% and gradually the acidity was increased in camel milk. after 24 hours, acidity reached to 0.957% lactic acid but the curd formed was having very weak consistency and it was flow-able. magdi et al. (2010) carried out a research on biochemical changes occurring during fermentation of camel milk by selected bacterial starter cultures. the camel milk was inoculated with 5 different starter cultures that are streptococcus thermophilus 37, lactobacillus delbrueckii sub sp. bulgaricus ch2, lactococcus lactis, lactobacillus acidophilus and mixed yogurt culture (s. thermophilus and l. bulgaricus 1:1) and fermented at 43 °c for 6 h and changes occurred were observed. after 6 hours incubation lactic acid produced by these strains was 0.6, 0.73, 0.23, 0.47, and 0.85% lactic acid respectively but with weak curd formation. 4 conclusions the processing related parameters such as alcohol stability, hct, rct and rate of acid productions were studied. the camel, cow and buffalo milk samples showed negative alcohol stability. the heat stability of camel milk was significantly lower than the cow milk and buffalo milk but rennet coagulation time of camel milk was significantly higher than the cow milk and buffalo milk. the rate of acid production useful in fermented dairy products. the rate of acidity was increased propositionally with time in camel milk. after 24 hours, the camel became 0 0,2 0,4 0,6 0,8 1 1,2 0 4 8 12 16 20 24 % l a c ti c a c id hours rate of acid production % acidity s.p. sagar et al. int. j. of health, animal science and food safety 3 (2016) 27 37 35 haf © 2013 vol. iii, no. 1 issn: 2283-3927 thicker but the curd formed was having very weak consistency and it was flow-able. these parameters will be useful in manufactured of various dairy products from camel milk. 5 authors’ contribution shyam p. sagar has conducted the experiment and the laboratory analysis of the samples. bhavbhuti m. mehta helped in analysis the samples, analyzed the data as well as editing the paper. k.n. wadhwani helped in procuring the camel milk. v.b. darji has helped in statistical analysis of the data and k.d. aparnathi supervised the experiment. 6 references abu-lehia i.h.; al-mohizea i.s.; el-behry m. 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(2015c). comparative studies on selected enzymes activities of camel, cow and buffalo milk. journal of camel practice and research. 21(2):249-252. yoganandi j.; mehta b.m.; wadhwani k.n.; aparnathi k.d. (2015d). comparison of fat and snf contents of camel milk with cow and buffalo milk. indian j. dairy sci. 68(3): 298-302. unnikrishnan v.; bhavdasan m.k.; ramamurthy m.k. (1988). alcohol stability of buffalo milk. indian j. dairy sci. 41:421–426. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l pancreatic ductal adenocarcinoma (pdac) is one of the most lethal oncological malignancies in humans. not-specific symptoms and lack of early diagnostic strategies, frequently lead to late diagnosis which limited therapeutic possibilities (korc, 2007). the present study aimed at identifying novel potential serum biomarkers for early detection of pdac. in the first phase, two different mouse models of pdac were characterized: genetically engineered mice (gems) (hingorani et al., 2003) which developed panin (pancreatic intraepithelial neoplasia) lesions and three pdac patient-derived xenograft. in the second phase the two mouse models were used to evaluate the reliability of 3 circulating molecules as early diagnostic biomarkers of pdac. the plasma levels of matrix metalloproteinase-7 (mmp-7), tissue inhibitor of matrix metalloproteinases-1 (timp-1) and thrombospondin-2 (thbs-2) were tested on gems and pdac-pdxs bearing mice by elisa tests, during tumor development, and at sacrifice by immunohistochemistry performed on pancreatic tissue. the three established pdac-pdxs were found to better reproduce the tumor of origin after intra-pancreas transplantation compared to the subcutaneous ones, and to maintain molecular and morphological features over different passages. at sacrifice, histopathological analysis demonstrated different stages of panin lesions in gems and the presence of a well-developed pancreatic tumor in all the mice orthotopically inoculated with the pdac-pdxs. plasma levels of mmp-7, timp-1 and thbs-2 were progressively upregulated, over the time, in gems and in pdac-pdx bearing mice. in both animal models, immunohistochemistry revealed stromal immunoreactivity for timp-1 and thbs-2 (figure 1), while mmp-7 expression was mainly localized on epithelial cells. all the markers showed progressive increase of staining intensity along with panin progression. in conclusion, the investigated circulating molecules represent promising biomarkers for early diagnosis of pdac and to monitor the response to treatment in human patients. both tumoral keywords pancreatic cancer, circulating biomarkers, kc mice, patient derived xenograft, immunohistochemistry. corresponding author lucia minoli lucia.minoli@unimi.it journal home page riviste.unimi.it/index.php/haf identification and preliminary validation in mouse models of circulating biomarkers of pancreatic cancer. l. minoli1, 2,*, e. scanziani1,2 1 department of veterinary medicine, università degli studi di milano, milan, italy. 2 mouse and animal pathology lab, fondazione filarete, milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 30 haf © 2013 vol. v, no. 1s issn: 2283-3927 cells and associated stroma play a role in the production and release of such biomarkers which, in addition, represent also biologically relevant molecules in remodeling the tumor microenvironment, by a complex network of interacting molecules. acknowledgments: the authors wish to thanks dr. giavazzi and dr. belotti from mario negri institute (milan and bergamo, italy) and their research group for the collaboration. references hingorani, s. r., petricoin iii, e. f., maitra, a., rajapakse, v., king, c., jacobetz, m. a., ... & kawaguchi, y., 2003. preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. cancer cell, 4(6), 437450. korc, m., 2007. pancreatic cancer–associated stroma production. the american journal of surgery, 194(4), s84-s86. figure 1: mouse (gem model of pdac), pancreas. diffuse immunoreactivity for thbs-2 in the stroma surrounding a panin-1a lesion. immunohistochemistry for thrombospondin-2, 400x magnification. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l feline diffuse iris melanoma (fdim) is the most common primary intraocular neoplasm in cats (dubielzig, 2017). it is usually a malignant tumor, even if slowly progressive, thus representing an unique spontaneous model of the aggressive, although rare, human iris melanoma (demirci et al., 2002). in cats, the extent of the tumor within the eye, expressed as histological grade, is considered a good predictor of survival (kalishman et al., 1998). in the context of the neoplastic cells-tumor microenvironment interaction, matrix metalloproteinase-9 (mmp-9) is an endopeptidase able to digest the extracellular matrix with involvement in tumor invasion (nagase et al., 2006). mmp-9 expression has been positively correlated with metastasizing behavior in human posterior uveal melanoma (el-shabrawi et al., 2001). the present study investigates the expression of mmp-9 in a caseload of formalin-fixed paraffinembedded fdims in relation to the histological grade (kalishman et al., 1998) and mitotic index (mi) (threshold=7/10 hpf) (wiggans et al., 2016). sixty-one samples of fdim evaluated on light microscopy (figure 1) were selected (grade i n=22, grade ii n=20, grade iii n=19). immunohistochemical staining with standard abc method was performed using a mouse antimmp-9 antibody (porcellato et al., 2014). results were semi-quantitatively scored and compared by mann-whitney u test. mmp-9 was expressed in 59,1% grade i fdim, 90,0% grade ii and 80,0% grade iii. tumors with mmp-9 expression in more than 50% of neoplastic cells were 13,6% in grade i cases, 40,0% in grade ii and 36,8% in grade iii. mmp-9 was expressed in 71,4% of fdim with mi≤7 and 92,3% of fdim with mi>7. mmp-9 expression differed significantly between grade i and the other two grades, keywords feline diffuse iris melanoma, cat, immunohistochemistry, mmp9, tumor-matrix interactions. corresponding author laura nordio laura.nordio@unimi.it journal home page riviste.unimi.it/index.php/haf mmp-9 immunohistochemical expression is correlated with histologic grade in feline diffuse iris melanoma. l. nordio1,*, v. stornelli2, c. giudice1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, milano, italy. 2 private veterinary practitioner, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 8 haf © 2013 vol. v, no. 1s issn: 2283-3927 and between groups with low and high mi. in conclusion, intense expression of mmp-9 seems to correlate with the histological aggressiveness of fdim. references demirci, h., shields, c.l., shields, j.a., eagle, r.c. jr, honavar, s.g., 2002. diffuse iris melanoma: a report of 25 cases. ophthalmology. 109, 1553-1560. dubielzig r.r. tumors of the eye. in tumors in domestic animals. v ed. wiley blackwell, 2017. el-shabrawi, y., ardjomand, n., radner, h., ardjomand, n., 2001. mmp-9 is predominantly expressed in epithelioid and not spindle cell uveal melanoma, journal of pathology. 194, 201-206. kalishman, j.b., chappell, r., flood, l.a., dubielzig, r.r., 1998. a matched observational study of survival in cats with enucleation due to diffuse iris melanoma. veterinary ophthalmology. 1, 25-29. nagase, h., visse, r., murphy, g., 2006. structure and function of matrix metalloproteinases and timps. cardiovascular research. 69, 562-573. porcellato, i., giontella, a., mechelli, l., del rossi, e., brachelente, c., 2014. feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling. veterinary dermatology. 25, 86-94. wiggans, k.t., reilly, c.m., kass, p.h., maggs, d.j., 2016. histologic and immunohistochemical predictors of clinical behavior for feline diffuse iris melanoma, veterinary ophthalmology. suppl 1, 44-55. figure 1: feline diffuse iris melanoma, neoplastic melanocytes. hematoxylin and eosin, 40x . http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en 18 l keywords lead, toxicities, mechanisms, countermeasures. pages 18 – 31 references vol. 2 no. 1 (2015) article history submitted: april 25, 2015 revised: july 03, 2015 accepted: july 08, 2015 published: july 11, 2015 corresponding author aixin hou department of environemental sciences, 1285 energy, coast & environment building, louisiana state university, baton rouge, la, 70803, usa e-mail: ahou@lsu.edu phone: +01 2255784294 journal home page riviste.unimi.it/index.php/haf source of lead pollution, its influence on public health and the countermeasures. rui zhang 1 , vincent l. wilson 1 , aixin hou 1* , ge meng 2 1 department of environmental sciences, school of the coast & environment, louisiana state university, usa 2 school of pharmacy, xi’an jiaotong university, china abstract. lead is a well-known toxic heavy metal, which can have serious public health hazards at very low levels, especially for young children. this report summarized the background information on lead and its applications, pollution sources, poisoning pathways, biomarkers of exposure and effects, toxicities, poisoning mechanisms, preventive actions, decontamination strategies, and detoxification methods. zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 19 haf © 2013 vol. ii, no. 1 issn: 2283-3927 1 introduction 1.1 the discovery of lead lead is an abundant heavy metal in the earth, about 14 parts per million by weight or 1 part per million by moles. it rarely occurs in pure form in nature. lead is usually found in ores, mostly with copper, zinc and silver. the most common lead mineral is galena, which is lead sulfide (pbs). other minerals include lead carbonate (pbco3) and lead sulfate (pbso4) (holleman et al. 1985). lead is usually extracted from pbs by heating and processing. it was discovered and used in prehistoric times. the ancient romans used lead for plumbing and called it ‘plumbum’, which is what the symbol, pb, represents. 1.2 the extraction of lead the lead found in ore and other minerals, not in pure form. the separation of lead from other substances must go through a chemical refining process. the lead containing ore is first ground into small pieces, followed by dilution with water. then the mixture is dumped into the flotation chamber to produce slurry of ground ore and water. pine oil is added to the slurry to draw impurities. the slurry is continually stirred and shaken so that the ground ore can be separated from the impurities. next the slurry is processed through a filter to remove liquids. the remaining substance was processed by smelting, initially removing sulfur by roasting (heating) in an oxidizing environment to produce lead oxide (pbo), which is then reduced to elemental lead (avameg inc 2014). 1.3 the physical and chemistry characteristics of lead lead is gray color, soft and ductile metal. its density at 293 k (19.9 °c) is 11.34 g/cm 3 and it has a melting point of 621.5 °f (327.5 °c) and a boiling point of 3180 °f (1749 °c) (lide 2004). it is insoluble in water but dissolves slowly in a weak acid solution. it has four isotopes (rabinowitz et al. 1995; john et al. 1996). lead is a member of carbon group in the periodical table and is defined as an inert metal. with heat, pb will oxidize to produce pbo, and it will react with hno3 to produce pb(no3)2 (university of sheffield and web elements ltd 2014). 2 application of lead batteries: the major use of lead is making rechargeable storage batteries. the grey negative electrode is made of pb and the red colour on the positive electrode is pbo2 in the battery. airplanes, automobiles, electric vehicles, trucks, tanks, and broadcasting stations all use rechargeable storage batteries as the energy source for light. the manufacture of one battery requires dozens of kilograms of lead (crompton 2000). http://www.shef.ac.uk/chemistry/ zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 20 haf © 2013 vol. ii, no. 1 issn: 2283-3927 antiknock agent in gasoline: one of the most important organic compounds of pb, tetraethyl lead (tel, (ch3ch2)4pb) was invented as a very efficient and cheap antiknock additive to the gasoline by chemist thomas midgley, jr., in 1921. the components of gasoline are complex, but the main constituents are c4~c12 hydrocarbons. various grades of gasoline have different compositions, so their physical and chemical characteristics are different. commercial gasoline is rated by the octane numbers. the higher the octane number, the better the anti-knock capability of the gasoline. by adding a little tel, the octane numbers can be enhanced significantly (loeb 1995). from the 1920s to the 1970s, tel was universally added to the gasoline. following the discovery of the toxicities of pb in tel, its use in gasoline for automobile engines was phased out in the us. the time of cancellation of tel use in gasoline in different countries was disparate. while most countries have banned or at least restricted the selling of commercial leaded gasoline, it is still used in countries such as afghanistan and north korea. in the us a small amount of tel is still being used in marine and airplane engines. reaction tanks: lead is usually a grey colour since it can be easily oxidized into pbo by oxygen in the air. pbo forms a dense film, covering the outside layer and preventing deeper layers of metal from being oxidized. this feature together with its stable chemistry makes lead resistant for degradation, so it is often used to make pipes and reaction tanks in the chemical industries. the famous “lead chamber process” for making sulphuric acid (h2so4) was named due to the process of using a reaction tank made of lead (zumdahl 2009). soldering: soldering is used to join one or more metal pieces together such as in plumbing and other metal assemblies. alloys commonly used for soldering include tin and lead (sn/pb). since the discovery of toxicity of lead, lead-free alloys have gradually replaced lead containing alloys used for soldering. wire and cable insulation and jacketing: lead’s ductility and extrusion ability, along with its corrosion resistance enabled lead alloys to be widely used for sheathing the high voltage cables. radiation shields: lead’s characteristic of being able to obstruct radioactive rays makes it a good material for shielding radiation. when patients in the hospitals are being diagnosed with x ray, a piece of lead plate is set in front of their chest to protect them (bethesda 2004). personnel working in atomic reactor also wear outfits containing lead to shield against radiation. lead is also used in the glass of television and glass screens to shield the watcher from radiation. ammunitions: lead is inexpensive and it has a low melting point, which makes casting easy. its high density and weight can prevent bullets from being deflected by wind and air turbulence (trinogga et al. 2013). stiffener in candle wicks: lead was used as stiffener in some candles to ensure the candle burn longer and more even, although this kind of use has been banned in the us for many years (nriagu and kim 2000). zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 21 haf © 2013 vol. ii, no. 1 issn: 2283-3927 pigments: many lead compounds have beautiful colours, so they were widely utilized as pigments. the white pigment lead carbonate (pbco3) was the most extensively used, while other lead pigments include yellow (lead chromate (pbcro4)), gold (lead iodide (pbi)) and black (lead sulphide (pbs)). as health hazards produced by lead-based pigments were discovered, most countries have remarkably curtailed the lead content of paints and colorants. others: lead has also been used in other areas, including weights in sport instruments, water pipes, house and building roofs, various alloys, fuse wires, bearings, and lead crystal glassware (beattie et al. 1972; zietz et al. 2009). 3 sources of lead pollution lead has become widely distributed in the environment since it was discovered and used by humans for a long time (sturge and barrie 1989; sungmin et al. 1994; ingemar et al. 2000; branvall et al. 2001; pompeani et al 2013). natural lead pollution occurrs from volcanic explosions and forest fire. non-natural sources were from human activities, mainly referring to the lead emission from the industry and transportation. since the use of leaded gasoline, the auto vehicles were the main source of lead emissions to the air and several million tons of lead have been deposited in the soil in the united states. efforts in removing lead from gasoline substantially lessened the emissions of lead into the air and environment. the major sources of lead emissions to the environment today are from ore and the processing of metals, as well as leaded aviation gasoline (lin et al. 2011). the highest air lead levels occur close to lead smelters. other sources are from manufacturing batteries, coal burning, typecasting, and in older houses and buildings (american academy of paediatrics committee on environmental health 2005; woof et al. 2007; zhang et al. 2009). since lead is not degraded by microbial activity, it is persistent in the environment and accumulates in soils, water bodies and sediments through deposition, leaching and erosion (shotyk et al. 1998; maja-lena et al. 1999). apart from exposure to aerial lead released from legal industrial manufacturing, mining and smelting activities, other major exposure pathways also include intake of lead from consuming lead-contaminated water and food. as lead does not decompose, the longer fields have received effluent irrigation and fertilizer, the higher the level of lead accumulation in the soil. cultured crops therefore often contain increased levels of lead due to uptake during the growing process. importing food products from developing countries requires caution since lead contamination of food is more common in countries that do not have strict food standards. food can become contaminated with lead due to the soil and water in the growing environment, or materials used in processing or cooking or the containers used in storage. also, the lead concentration can be amplified through food chain. in addition, lead in dwellings is a well-known source of childhood lead exposure. it is common in lead-based paints, tap water, indoor and outdoor floor dust, and soil in and around old houses and buildings (lucas et al. 2012). other pathways include accidentally intake of lead from contaminated soil and dust, lead glazed ceramics, lead-based paint, lead acid batteries, electronic equipment with lead zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 22 haf © 2013 vol. ii, no. 1 issn: 2283-3927 containing components, water from lead soldered plumbing, leaded polyvinyl chloride (pvc) products, lead containing jewellery, toys, ammunition, lead contaminated herbal medicine, and cigarettes. hand-to-mouth behaviour is an important mechanism of lead intake among small children (day et al. 1975; bruce and klaus 1997). 4 biomarkers of lead exposure and effect blood lead reflects soft tissue lead, representing the lead burden of body and the doses absorbed. urine lead and plasma lead reflects recent lead exposure. lead can interact with some enzymes responsible for heme synthesis, inhibiting δ-aminolevulinic acid dehydratase (alad). variation in the concentrations of some metabolites such as δ-aminolevulinic acid in urine, blood or plasma, coproporphyrin in urine, and/or zinc protoporphyrin in blood can be used as the biomarkers for lead exposure. since the heme precursors from alad genotype alad1 have higher affinity to lead than alad2, alad1 homozygotes are more susceptible to heme biosynthesis disturbance by lead than alad2 carriers. decreased activities of pyrimidine nucleotidase and nicotinamide adenine dinucleotide synthetase in blood can also be used as the biomarkers for lead exposure effects (sakai 2000). 5 toxicities of lead since it cannot be degraded by microbial activity, lead is an environmentally persistent toxin, which accumulated upward the food chain. once taken into the body, lead can distribute in the blood throughout the body and accumulate in the bones and soft tissues leading to chronic toxicity. growing evidence supports the premise that no threshold exists for harmful effects from the lead exposure (jasna and gary 1988; lanphear et al. 2005). depending on the level of exposure, lead can cause neurological (thomson and parry 2006; woof et al. 2007), hepatic (singh et al. 1994), renal (goyer and rhyne 1973), hematological, circulatory, immunological, reproductive, developmental (davis and svendsgaard 1987), auditory (stevens et al. 2013), gastrointestinal, and cardiovascular pathologies (patrick 2006a, 2006b). generally speaking, lead harms children more than adults. infants and young children are especially sensitive to lower lead levels than adults, showing the lead poisoning effects such as neurological effects contributing to lowered intelligence quotient (iq) (hebert and constantine 1990; david et al. 1992; schwartz 1994; richard et al. 2003; lanphear et al. 2005, bellinger 2008), learning deficits, cognitive deficits (lanphear et al. 2000; mostafa et al. 2009), lower vocabulary and grammatical-reasoning ability (herbert et al. 1990), longer reaction time (herbert et al. 1990), poorer hand-eye coordination (herbert et al. 1990), deficits in psychological and classroom performance (herbert et al. 1979), hyperactivity and behavioural problems (oliver et al. 1972). the high lead poisoning prevalence in children may be caused by the facts that lead is efficiently absorbed and retained and the blood brain barrier is not fully developed in very young children (william et al. 1981; ekhard et al. 1978). high bone lead levels are associated with increased risk of somatic complaints, attention problems, social problems, as well as anxious/depressed, aggressive and delinquent behaviours among children zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 23 haf © 2013 vol. ii, no. 1 issn: 2283-3927 (needleman et al. 1996). chronic occupational exposure to lead was shown to increase the risk of parkinson’s disease (gorell et al. 1999; weisskopf et al. 2010). chronic exposure to lead can also cause tooth loss and the damage of hard dental tissues (cenic-milosevic et al. 2013). in addition, lead can cross the placental barrier, leading the impacts on fetal development, especially the developing baby’s nervous system. lead may also cause abortion and stillbirths. polluted ecosystems have adverse effects such as the losses in biodiversity, community components alteration, decreased pollen germination and seed viability, reduced growth and reproductive rates in plants and animals (briggs 1972; jens et al. 1979; bull et al. 1983; krishnayya and bedi 1986; myra et al. 2012; magwedere et al. 2013). lead toxicity also affects farm animals such as cows and horses, as well as animal pets. in addition, hunting ammunition causes toxicity in wild bird populations since lead pellets (lead shot) is ingested by waterfowls such as ducks and swan. predators consuming these lead contaminated birds are also at risk (ferreyra et al. 2009). lead shot has been banned for waterfowl hunting in several countries including us and canada (pokras and kneeland 2008; degernes 2008). 6 mechanisms of lead poisoning lead readily interacts with proteins causing various toxic symptoms and pathologies (goldstein 1990; goering 1993). it has been well recognized that the presence of lead can affect the homeostasis and physiological function of essential metals. lead may exert its toxicity via disturbing ca-mediated cellular processes by simulating and replacing calcium at the binding sites of functional proteins like calmodulin (pounds 1984; jasna and gary 1988; goldstein 1993), directly interrupting calcium transportation or storage involving ca2+ transport proteins or calcium gates, or by indirectly altering cell functions such as energy production and plasma membrane permeability (pounds 1984). interactions between pb and high-affinity metal-binding proteins have been shown to play a role in the inhibition of zncontaining enzymes and aminolevulinic acid dehydratase (goering 1993). in return, the shortage or surplus of some necessary metals in human can modify the absorption and elimination of lead. for example, increased dietary zinc and calcium can reduce the severity of lead toxicity (cerklewski and forbes 1976). other known proteins related to pb toxicity include nucleic acid binding proteins, protein kinase, carbonic anhydrase and calmodulin (goldstein 1990; goering 1993). lead also enhances oxidative reactions producing reactive oxygen species (ros), which contribute to lead related diseases by inhibiting sulfhydryl antioxidants production, heme impair and dna repair and inducing nucleic acid damage and peroxidation (patrick 2006a, 2006b). the toxicity of tetraethyl lead is 100 times higher than inorganic lead. tetraethyl lead can induce poisoning by contact and absorption through intact skin, inhalation, or by ingestion. following the combustion of leaded gasoline in the engine, 70% of tel is released to the atmosphere in the form of fine particles, while the remaining will be accumulated in the engines. forty percent of the lead particulates emitted to the air will fall to the ground quickly. http://europepmc.org/abstract/med/8247411/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/gterm?id=go:0046872 http://europepmc.org/abstract/med/8247411/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/gterm?id=go:0003676 http://europepmc.org/abstract/med/8247411/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=protein%20kinase%20c&sort=score http://europepmc.org/abstract/med/8247411/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=carbonic%20anhydrase&sort=score http://europepmc.org/abstract/med/8247411/?whatizit_url_gene_protein=http://www.uniprot.org/uniprot/?query=calmodulin&sort=score zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 24 haf © 2013 vol. ii, no. 1 issn: 2283-3927 while tel is suspended in the air, it can easily be inhaled into the lung through the human’s respiratory system. the accumulation rate of the inhaled lead is as high as 50%. these lead particulates are not easily excreted and have a half-life of 25 years in the human body. some of the absorbed lead will be transformed to triethyl lead, which can go through blood-brain barrier, damaging the central nervous system. 7 preventive actions and decontamination strategies advancement of analytic technologies: since lead toxicity has become well recognized, the main focus has shifted from high-dose effects of clinical symptoms to asymptomatic levels, specifically in young children. many research studies have substantiated that even very low lead levels can cause brain development problems in children and no threshold has been identified for this effect. children’s blood lead concentration should be measured and the development of more advanced analytical technologies will help detect very low blood levles (needleman 2004; american academy of pediatrics committee on environmental health 2005; maitreyi et al. 2011; mclaine et al. 2013). policies, regulations and supervisions: although high dose lead poisoning has become rare globally, long-term exposure to low levels of lead remains an issue for public health. more policies, regulations and supervisions on environmental lead level should be developed to improve the environmental safety and eliminate the existing problems (tong et al. 2000; david and andrew 2006). lead-free technologies: due to the harmful effects that lead can cause, replacement of the conventional lead-based materials by lead-free products was an inevitable trend. this trend includes the development of lead-free piezoelectric ceramics (tadashi et al. 1997; eric 2004; yasuyoshi et al. 2004; zang et al. 2006), lead-free solders (mulugeta and guna 2000; katsuaki 2001), lead-free glass, and lead-free electronic assembly. also, the development of new hydrometallurgical technologies significantly decreased the pd pollution during lead recovering processes (pan et al. 2013). industrial hygiene: good industrial hygiene and close monitoring should be adopted in a timely manner to detect improper or excessive lead emission before it leads to environmental hazards and human health impacts (thomson and parry 2006). reduction of indoor lead pollution: although the incidences of elevated blood lead levels in us children have decreased, a large number of children still dwell in old buildings with lead-based paint and are at risk of lead exposure with ensuing neurological damages (american academy of pediatrics committee on environmental health 2005; roberts et al. 2013). remediation for removing these lead sources from children’s home environments can help to reduce their lead exposure. workers involved in lead remediation or working with lead containing materials should take frequent showers and change clothes following work zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 25 haf © 2013 vol. ii, no. 1 issn: 2283-3927 activities before going home. indoor dust should be removed regularly and frequently for people living near lead industrial areas. decontamination strategies: phytoremediation is a more environmentally friendly improvement to the conventional chelating methodologies. phytoremediation technologies use plants to absorb lead from contaminated soils are emerging with increased frequency (huang et al. 1997; huang and cunningham 2006). materials like apatite and carbon nanotubes can also be used for phytoextraction, such as to clean lead from contaminated water or soil systems (ma et al. 1993; li et al. 2002). 8 lead detoxification taking chelating agents orally is a conventional therapy for acute lead intoxication (patrick 2006a, 2006b; george et al. 2010). people who have developed the lead poisoning symptoms can take in food rich in protein, fe, vitamin c, pectin, and/or garlicin to help detoxify and remove the lead. fe can replace pb to combine with the proteins in human, while protein can bind with lead to speed its metabolism and removal. vitamin c can interact with pb to produce water insoluble precipitates, which can be excreted through feces. foods rich in fe are greens and fruits such as spinach, celery, rape, radish, amaranth, shepherd's purse, tomatoes, oranges, peaches, plums, apricots, pineapples and red dates. edible sources of good quality proteins include eggs, milk and lean meat. vitamin c broadly exists in fruits, vegetables such as orange, lemon, dates, apple, strawberry, peppers, cabbage, garlic bolt, tomatoes and cauliflower. additional foods or ingredients for detoxification include yogurt, which can stimulate intestinal peristalsis and decrease pb absorption, pectin, which inhibits pb absorption (zhao et al. 2008), garlicin, which can bind with pb to produce a nontoxic compound and antioxidants like vitamins b6, c and e, taurine, n-acetylcysteine, and α-lipoic acid, which are able to lower ros generated cellular damages (patrick 2006a, 2006b). 9 conclusion as a summary, lead has been applied in a wide range of fields on our planet for thousands of years. as its toxicities are gradually uncovered, policies and regulations should be taken to reduce or stop its use. advanced strategies and technologies need to be developed to detect and cure the lead polluted environments. references amal a.h., manal m.z., 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zhang et al. int. j. of health, animal science and food safety 2 (2015) 18 31 31 haf © 2013 vol. ii, no. 1 issn: 2283-3927 zhang y., wanga x., chen h., yang x., chen j., allen j.o. 2009. source apportionment of leadcontaining aerosol particles in shanghai using single particle mass spectrometry. chemosphere. 74, 501-507. zhao z.y., liang l., fan x., yu z., hotchkiss a.t., wilk b.j., eliaz i. 2008. the role of modified citrus pectin as an effective chelator of lead in children hospitalized with toxic lead levels. altern ther health med. 14(6), 18. zietz b.p., lass j., dunkelberg h., suchenwirth r. 2009. lead pollution of drinking water in lower saxonomy from corrosion of pipe materials. gesundheitswesen. 71(5), 265-274. zumdahl s.s. 2009. chemical principles 6th ed. houghton mifflin company. p. a23. isbn 0-61894690-x. http://www.ncbi.nlm.nih.gov/pubmed?term=zhao%20zy%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=liang%20l%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=fan%20x%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=yu%20z%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=hotchkiss%20at%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=wilk%20bj%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed?term=eliaz%20i%5bauthor%5d&cauthor=true&cauthor_uid=18616067 http://www.ncbi.nlm.nih.gov/pubmed/19387929 http://en.wikipedia.org/wiki/international_standard_book_number http://en.wikipedia.org/wiki/special:booksources/0-618-94690-x http://en.wikipedia.org/wiki/special:booksources/0-618-94690-x proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l in the last years, many papers highlighted the possibility to use epigenetic modifiers to directly interact with the epigenetic signature of an adult mature cell (pennarossa et al., 2013; chandrakantan et al., 2016). in particular, the molecule 5-azacytidine (5-aza-cr), which is able to interfere with dna methylation, through both a direct and an indirect effect (manzoni et al., 2016), can be used to remove the epigenetic ‘blocks’ responsible for tissue specification and to facilitate cell transition to a different lineage. in parallel, recent evidence has also shown that epigenetic conversion is influenced by the 3d rearrangement and by the mechanical properties of the cellular microenvironment (pennarossa et al., 2017). in the experiments here presented, we investigated the effect of a selected 3d culture system on the conversion process. we used ins-egfp porcine fibroblasts, that express enhanced green fluorescent protein (egfp) under the control of insulin gene promoter, as experimental model, and wild-type pig fibroblasts, as control. both cell types, were plated either on plastic or on 1kpa polyacrylamide (paa) gel, that mimics the stiffness of pancreatic tissue in vivo. cells were erased with 5-aza-cr for 18h and exposed to specific differentiation stimuli for 36 days (pennarossa et al., 2014). the use of insegfp fibroblasts allowed real-time monitoring of cells progressing towards the pancreatic phenotype. morphological analysis and pancreatic marker expression were checked for the entire length of the experiment. paa gels encouraged the induction of islet-like structures, suggesting that the of tridimensional clusters may be a crucial aspect of pancreatic differentiation in vitro. moreover, the use of an adequate substrate accelerated cell differentiation process and anticipated insulin secretion ability. the results obtained demonstrated the direct implication of the yes-associated protein/transcriptional co-activator with pdz-binding motif (yap/taz) mechanotransduction-mediated pathway (figure 1), indicating that mechanical cues exert a key role in pancreatic phenotype definition. acknowledgments: supported by carraresi foundation. authors are members of the cost actions ca16119, bm1308 and cm1406. keywords epigenetic conversion, 5-aza-cr, 3d culture, paa gels, hippo signaling pathway. corresponding author elena fm manzoni elena.manzoni@unimi.it journal home page riviste.unimi.it/index.php/haf matrix stiffness boosts pancreatic differentiation via the yap/taz mechanotransduction mediated pathway. e.f.m. manzoni1,*, s. arcuri2, t.a.l. brevini2, f. gandolfi1 1 department of agricultural and environmental sciences production, landscape, agroenergy, università degli studi di milano, via celoria 2, 20122 milan, italy. 2 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 12 haf © 2013 vol. v, no. 1s issn: 2283-3927 references pennarossa, g., maffei, s., campagnol, m., tarantini, l., gandolfi, f., brevini, t.a.l., 2013. brief demethylation step allows the conversion of adult human skin fibroblasts into insulin secreting cells. proc natl acad sci u s a. 110, 8948-8953. chandrakantan v., yeola, a., kwan, j.c., oliver, r.a., qiao, q., kang, y.c., zarzour, p., beck, d., boelen, l., unnikrishnan, a., villanueva, j.e., nunez, a.c., knezevic, k., palu, c., nasrallah, r., carnell, m., macmillan, a., whan, r., yu, y., hardy, philip, grey, s.t., gladbach, a., delerue, f., ittner, l., mobbs, r., walkley, c.r., purton, l.e., ward, r.l., wong, j.w.h., hesson, l.b., walsh, w., pimanda, j.e., 2016. pdgf-abb and 5-azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells. proc natl acad sci u s a. 113: 23062315. pennarossa, g., maffei, s., campagnol, m., rahman, m.m., brevini, t.a.l., gandolfi, f., 2014. reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. stem cell rev, 10(1):31-43. manzoni, e.f.m., pennarossa, g., deeguileor, m., tettamanti, g., gandolfi, f., brevini, t.a.l., 2016. 5-azacytidine affects tet2 and histone transcription and reshapes morphology of human skin fibroblasts. sci rep. 6:37017. pennarossa, g., santoro, r., manzoni, efm, pesce, m., gandolfi, f., brevini, t.a.l., 2017. epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of lowstiffness substrate. stem cell rev. 10.1007/s12015-017-9799-0. figure 1: a morphological changes in ins-egfp porcine fibroblasts plated on standard plastic dishes (plastic) and paa gels (1kpa paa) at different time points of the endocrine pancreatic induction protocol. b insulin expression changes in porcine skin fibroblasts plated on plastic dishes (t0p, blue) and paa gels (t0g, red) and subjected to endocrine pancreatic induction (days 10, 14, 36). gene expression levels are reported with the highest expression set to 1 and all other times relative to this. different superscripts denote significant differences between groups (p < 0.05). c quantification of the nuclear/cytoplasmic ratio of yap in untreated fibroblasts (t0p, t0g), after 18-hour exposure to 5-aza-cr (post 5-aza-crp, post 5-aza-crg) and at the end of pancreatic induction (36p, 36g). bars represent mean ± sd of three independent replicates. different superscripts denote significant differences between groups (p < 0.05). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en 9 l keywords shelf-life; smoked herring; volatile compounds; microbiological analyses. pages 9 – 14 references vol. 1 no. 1 (2014) article history submitted: november 04, 2013 revised: january 29, 2014 accepted: february 03, 2014 published: february 10, 2014 corresponding author cristian bernardi, laboratory of food inspection, department of health, animal science and food safety università degli studi di milano via celoria, 1 – 20133 milan, italy e-mail: cristian.bernardi@unimi.it phone: +39 02 50318506 fax: +39 02 50318501 journal home page riviste.unimi.it/index.php/haf a case study: shelf-life of smoked herring fillets by volatile compounds analysis. cristian bernardi 1* , erica tirloni and patrizia cattaneo 1 laboratory of food inspection, department of health, animal science and food safety, università degli studi di milano. abstract. two different products of vacuum packed cold smoked herrings were analyzed at time intervals in order to evaluate the efficiency of the processing and product stability. microbiological total counts, lactic acid bacteria, total coliforms, ph, water activity, water content, salt content (wps) were determined. differences in hygienic conditions and salt content were found. principal components analysis (pca) of volatile compounds determined by gc-ms analysis allowed the differentiation of the processing. c. bernardi et al. int. j. of health, animal science and food safety 1 (2014) 9-14 10 haf © 2013 vol. i, no. 1 issn: 2283-3927 1 introduction the more widespread smoked herrings marketed in italy are beheaded with skin, vacuum packaged, silver and golden types; less frequent are skinned fillets, softer and with a milder taste, vacuum packaged, which meet more with the liking of the modern consumer. the cold smoked herring is at the limit of the group of the lightly preserved seafood (huss, 1994), comprising products with low salt level (<6% nacl-water phase salt wps), preservatives or smoke, ph value >5, mainly vacuum packaged, requiring refrigeration temperatures and typically consumed as ready-to-eat without heat treatment. the process must allow the survival of an adequate number of spoilage organisms, which have the important role to compete with the growth and toxin formation of c. botulinum type e and non-proteolytic types b and f (fda, 2001a). the control measures of potential hazards are based mainly on salt control (nitrite addition is not allowed by ec regulation in these products), on control of exposition to temperatures that favour c. botulinum throughout the processing steps, on maintenance at refrigeration temperatures of the final products. with regard to this, us haccp regulations suggest a critical limit for vacuum packaged cold smoked products of at least 3.5 % nacl –wps at  4.4°c, allowing for short time periods temperatures up to 10°c (fda, 2001b). the purpose of this investigation was to answer an importer’s request, that was to verify the established shelf-life of smoked vacuum packed herring fillets of a new producer (product a). with this aim traditional analytical techniques and spme headspace-gas chromatography-mass spectrometry were applied comparing product a to a traditional one (b) on the furnished packets. 2 materials and methods a total of 18 packets of smoked herring fillets, peeled and vacuum packed of two different producers were compared (10 of producer a, 8 of b). the packages were stored at 0-2°c for the whole period of the trial. analyses were performed as described in bernardi et al., 2009. water activity (aw), water content, nacl–wps were performed in quadruplicate. the enumeration of total psychrotrophic count (tpc), lactic acid bacteria (lab), total coliforms, ph and spme gcms analyses were performed at time intervals in duplicate. volatile compounds were tentatively identified by matching mass spectral data with the wiley and nist reference libraries of standard compounds. the identification was confirmed by comparison of the retention times and mass spectra (ms) with available authentic standards (as). semi-quantification of the compounds was based on arbitrary units of peak area counts divided by 105. 3 statistical analysis principal component analysis (pca) and statistical analysis were performed by the spss package version 9.0 (spss italia, bologna). c. bernardi et al. int. j. of health, animal science and food safety 1 (2014) 9-14 11 haf © 2013 vol. i, no. 1 issn: 2283-3927 4 results and discussion wps rate was amply above the 3.5 % nacl-wps; in product b, salt content was more variable; aw was similar in both, but more variable in product a, allowing to consider safe both products. product a had at the arrival (27 th days from production) a very high tpc (5.74 log cfu/g), afterwards tpc decreased and lab counts overcame 7 log cfu/g, but without causing detectable olfactory deterioration of flavour. product b showed lower tpc and lab than a (<3.70 and 5.78 log cfu/g, respectively at 9 th days). total coliforms were under the limit of detection (<2 log cfu/g) both in a and b (table n.1). in product b forty-five peaks were determined and identified by gc-ms. particularly, in product b benzaldehyde and furfural and small quantities of propionaldehyde and hexanal were present, while absent in a. phenol was present in both, without significant differences. acetic acid was found more in product b. dimethyl sulfoxide and dimethyl sulphide were more represented in a than in b. as a whole, product b showed an aromatic profile more complex and rich in qualitatively superior compounds, while product a showed a very little articulate profile. in figure n. 1 the analysed samples and the identified variables (volatile compounds) are represented in the space described by the two principal components, the first component explaining 78.3% variability among data, the second component explaining 9.2% variability. samples are clearly distinguished in two groups, coinciding with a and b productions. the second component allows to distinguish product a on 27 th day from the subsequent samples. on the “best before” date, product a had an initial softening with presence of fluid material in the pack, without other sensorial changes; product b maintained a better firmness, also for a lesser water content. the bacterial count at the first sampling of a was high, indicating bad hygienic condition, while the product b was hygienically better at about the same storage time. product a had a higher salt content; this was a negative aspect, not only from a nutritional point of view, but also for the consumer’s present preferences; its wps ratio allowed the same stability of the product b although a higher water content. wps more than 6% puts both products at the limit between lightly preserved and semi-preserved seafood (huss, 1994). the obtained results allowed to assume a different processing technology: brine salting for a, due to the high water level and the uniform salt content of the fillets, while for b a dry salting process with purging was probable, because of a considerably lower water content and a higher variability in the salt concentration (coefficient of variation 13.8%). in the verified conditions, the shelf life of product a is to be stated up to 4 weeks instead a “best before” date of 45 days; microbiological parameters of b were stable for the whole period of the trail. the exclusive presence in the product b of benzaldehyde and furfural, aldehydes known for giving a strong taste of smoke, confirmed the perception of a more typical aroma of the herring b. the clear finding of dimethyl sulfoxide and dimethyl sulphide only in the product a, well match with the microbial condition of a; these sulphur compounds may be the expression of bacterial activity besides of ripening (triqui, 1995). c. bernardi et al. int. j. of health, animal science and food safety 1 (2014) 9-14 12 haf © 2013 vol. i, no. 1 issn: 2283-3927 table 1. conservation parameters. sample a b m m mean sd cv m m mean sd cv aw 0.923 0.966 0.943 0.014 0.36 0.940 0.949 0.945 0.004 1.48 water content % 72.25 75.18 74.12 1.12 1.51 60.39 66.73 63.26 2.62 4.13 salt % 5.25 5.59 5.43 0.14 2.64 3.76 4.85 4.38 0.53 12.13 wps % 6.57 6.92 6.83 1.46 2.14 5.62 7.43 6.48 0.90 13.84 microbiological analyses a b day 27 45 49 55 60 9 26 30 41 tpc (log cfu/g) 5.74 <4.70 <3.70 <3.70 <3.70 <3.70 <3.70 <3.70 <3.70 lab (log cfu/g) 6.78 7.63 <4.70 7.38 7.30 5.78 6.48 6.54 6.90 coliforms (log cfu/g) <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 legend: m = minimum value m = maximum value cv = coefficient of variation tpc= total psychrotrophic count lab=lactic acid bacteria 5 conclusions the results related to the study of the volatile fraction confirmed what deduced by the more traditional analyses: in spite of the close outward likeness between the two products, their processing technologies were very different, resulting in distinct products as regards shelf life and quality. the reported case is an example of the result of the delocalization, due to economical reasons, of food processing in countries applying less advanced or less consolidated technologies or procedures with inferior process standard. c. bernardi et al. int. j. of health, animal science and food safety 1 (2014) 9-14 13 haf © 2013 vol. i, no. 1 issn: 2283-3927 figure 1. biplot of principal component scores and factor loadings from principal component analysis applied to volatile compounds of product a (scores l) and product b (scores d). volatile compounds identified in smoked herring (clupea harengus) by gc-ms: 1) 2-pentene; 2) heptane; 3) dimehyil sulphide; 4) methyl-cyclo-hexan; 5) propanal; 6) furan; 7) propanone; 8) methyl acetate; 9) 2-methyl-furan; 10) ethyl acetate; 11) 2-butanone; 12) 3-methyl-butanale; 13) ethanol; 14)benzene; 15) 2-ethyl-furan; 16) branched hydrocarbon; 17) 2-pentanone; 19) chloroform; 20) hexanal; 21) p-xylene; 22) cyclopentanone; 23) 2-methyl-cyclo-pentanone; 24) 3methyl-cyclo-pentanone; 25) pyrazine; 26) cyclohexanone; 27) acetoin; 28) 2-methyl-2cyclopentenone; 29) 3-furaldehyde; 30) acetic acid; 31) 2-furaldehyde; 32) acetyl-furan; 33) benzenaldehyde; 34) propanoic acid; 35) dimethylsulfoxide; 36) 5-metil 2-furaldehide; 38) gamma-butyrolactone; 39) butanoic acid; 40) 3,4-dimethyl-3-penten-2-one; 41) 5-metil-2(5h)furanone 44) 2pyranone; 45) 2-methoxy-phenol; 46) guaiacol; 47) phenol; 50) 2-furanone. c. bernardi et al. int. j. of health, animal science and food safety 1 (2014) 9-14 14 haf © 2013 vol. i, no. 1 issn: 2283-3927 references bernardi, c., ripamonti, b., campagnoli, a., stella, s., cattaneo, p., 2009. shelf-life of vacuum packed alaskan, scottish and norwegian cold-smoked salmon available on the italian market. international journal of food science and technology. 44, 2538-2546. fda, center for food safety & applied nutrition, 2001a. fish and fisheries products hazards and controls guidance. third edition, june. fda, center for food safety and applied nutrition, 2001b. processing parameters needed to control pathogens in cold-smoked fish. supplement to journal of food science. 66, (7), s1133. huss, h.h., 1994. assurance of seafood quality. fao fisheries technical paper. 334, fao, rome, italy. triqui, r., reineccius, g.a., 1995. changes in flavour profiles with ripening of anchovy (engraulis encrasicholus). journal of agriculture and food chemistry. 43, 1883–1889. 1 l keywords yoghurt serum powder, fattening lambs, growth, malondialdehyde. pages 1 – 8 references vol. 2 no. 1 (2015) article history submitted: november 12, 2014 revised: december 04, 2014 accepted: december 16, 2014 published: january 09, 2015 corresponding author panagiotis e.simitzis, faculty of animal science and aquaculture department of animal breeding and husbandry agricultural university of athens, greece 75 iera odos street, 118 55, athens, greece e-mail: pansimitzis@aua.gr phone: +30 121 05294449 fax: +30 210 5294442 journal home page riviste.unimi.it/index.php/haf effect of yoghurt serum powder dietary supplementation on growth performance and antioxidant status in fattening lambs. panagiotis e. simitzis1,*, stavros karas tamatis1, panagiota koutsouli1, iosif a. bizelis1, ioannis politis1 1 faculty of animal science and aquaculture, department of animal breeding and husbandry, agricultural university of athens, athens, greece. abstract. large quantities of serum are produced during the strained yoghurt manufacturing process, which is the predominant type of yoghurt in greece. however, the exploitation of this by-product as an alternative source of energy, protein and mineral elements in animal diets has not yet been examined. therefore, this experiment was conduc ted to determine the effects of dietary yoghurt serum supplementation on growth performance and antioxidant status in sheep. forty eight male 2 months old lambs of chios breed were randomly assigned to three experimental groups; control group was fed with a commercial basal diet, whereas the other two groups c onsumed the same diet, with the only difference that concentrated feed was uniformly supplemented with two levels of yoghurt serum powder (ys1: 25 g/kg feed or ys2: 50 g/kg feed). lambs were weighed in a weekly basis from the beginning until the end of the experiment and blood samples were c ollected to measure antioxidant status. no significant effect of y oghurt serum powder on growth performance of fattening lambs was demonstrated, even after 2 8 days of dietary supplementation (p > 0.05). at the same time, malondialdehyde (mda) levels in blood plasma were not significantly different among the experimental groups (p > 0.05) and no incidents of lambs with diarrhea were recorded. it can be concluded that yoghurt serum powder appears as a promising alternative of the cereals in the diets of fattening lambs, since no negative effects on growth performance and health status were observed. p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 2 haf © 2013 vol. 2, no. 1 issn: 2283-3927 1 introduction yoghurt production is continually enhanced worldwide; from 19263 tn in 1992 to 61246 tn in 2012, according to fao. with the g row th of the yoghurt manufacturing industry th e increasingly larger quantities of yoghurt serum have resulted in greater pollution hazards. yoghurt serum (may be called wh ey as well) is mechanically derived after the f erm entation of yoghurt and could be a strong environmen tal pollutan t when discharged in to streams, since its high organic matter content leads to a high biochemical oxygen demand (b od5) (alonso et al., 2010). although no data exists con cerning the use of yoghurt serum, the exploitation of th e diverse properties of wh ey, a by-product of cheese industry with similar composition, in a variety of food systems as source of protein, flavor enhan cer, egg white substitu te, and food binder for conventional food use is firmly established du ring the last decades (walzem et al., 2002). among others, whey could be an alternative source of energ y (high lactose conten t), nitrog en and mineral elem ents to cereals in animal diets (oba, 2011). wh ey derived from cheese industry is traditionally used in th e diets of pigs, but in areas unsuitable for pig breeding, this by-product could be used in dairy farms as liquid or after water evaporation (church, 1991). ferm ented, ammoniated, condensed whey could be a poten tial source of dietary non-protein nitrog en (alternative to urea) and could provide carbon skeletons for microbial protein syn thesis in sheep (boukila et al., 1995). howev er, special care must be taken that the animals do n ot consume excessive quantities (>20% of dry matte r) of dried whey, because it can cause digestive disorders and diarrhea incidents (anderson et al., 1974). since n o data exists regarding the utilization of yoghu rt s erum powder in the diets of fattening lambs, a preliminary study was conducted to examine the possible effects of yoghur t serum powder dietary supplem entation on their g row th performance and an tioxidan t status. 2 materials and methods 2.1 experimental design and diets forty eight male 2 months old lambs of chios breed were used in the present study . th e lambs were randomly assigned to three experimen tal g roups; con trol group was fed with a commercial basal diet (table 1), whereas th e treatment g roups consumed the same diet, with the only difference that con cen trated feed was uniformly supplem ented w ith tw o levels of yoghurt serum powder (ys1: 25 g/kg feed or ys2: 50 g/kg feed). yoghurt serum was mechanically derived after the ferm entation of au thentic greek yoghurt with lactobacillus bulgaricus and streptococcus thermophiles in a dry free flowing powder and contain ed 5.1% protein, 60% lactose, 12.5% galactose, 5.3% lactic acid and 18 g calcium, 6 g phosphorus, 6.6 g sodium, 24.7 g potassium, 1.7 g magnesium, 14.4 g chloride, 0.48 mg copper, 1.13 mg ferru m per kg (h ellenic protein a.e., athens, greece). lambs were fed in groups (control or ys1 or ys2) twice daily at 8:00 a.m. and 16:00 p.m. on average, each lamb consumed 550 g of concentrated feed mixture and 500 g of alfalfa hay per day, and the nutritional needs were estimated according to agricultura l and food research council (afrc, 1993 ). p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 3 haf © 2013 vol. 2, no. 1 issn: 2283-3927 2.2 growth performance and antioxidant status the duration of the experim ental period was 28 days and lambs were weighed in a weekly basis from the beginning until the end of the experim ent. in order to examin e the possible effects of yoghurt serum powder in take on th e health status of lambs, blood sa mples were obtained with minimal dis turbance by v enipunctu re of the jugular v ein on day 0, day 7 and day table 1: composition and analysis of control diet1 components (g/kg) control corn 465 wheat 120 soybean meal (44%) 210 sunflower meal 50 alfalfa meal 30 wheat bran 40 palm (rumen-protected) oil 25 molasses 15 sodium chloride (nacl) 9 calcium carbonate 18 monocalcium phosphate 14 vitamins & trace elem ents premix 42 analysis3 control alfalfa hay dry matter – dm (%) 88.0 89.0 net energy (mj/ kg) 7.3 4.0 crude protein – cp (%) 17.0 17.0 crude fiber (%) 5.4 30.0 ash (%) 8.0 10.0 fat (%) 5.0 calcium (%) 1.0 phosphorus (%) 0.7 sodium (%) 0.4 1. lambs were fed ad libitum; c ontrol group was fed with the commercial basal diet, whe reas the treatment groups consumed the same diet, with the only difference that feed was uniformly supplemented with two different levels of yoghurt serum powder (ys1: 25 g/kg feed or ys2: 50 g/kg feed) 2. premix contained per kg: 150 mg mg, 35 mg mn, 50 mg fe, 60 mg zn, 0.8 mg se, 0.75 mg co, 1.25 mg i, 60 mg se, 200 mg mo, 15 kiu vitamin a, 2 kiu vitamin d3, 2 5 mg vitamin e (kiu: 1000 international units). 3. according to aoac (1990) and van soest et al. (1991) p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 4 haf © 2013 vol. 2, no. 1 issn: 2283-3927 28 after th e beginning of the experimen t. the samples were transferred immediately to heparinized centrifuge tubes and plasma was separated by centrifu gation at 4°c within 30 min and was stored at -20°c. the lipid peroxidation values in plasma were determined according to the spectrophotometric method described by ohkawa et al. (1979). briefly, to each test tube, 0.5 ml of plasma, 0.5 ml of normal saline (0.9% sodium chloride solution ), 1 ml of 20% trichloroacetic acid (merck kgaa, german y) and 0.25 ml of thiobarbituric acid (tba) reagent 200 mg of tba (sigma chemical co, german y) in 30 ml distilled water and 30 ml of acetic acid (sds, france)were added. the test tubes were kept for boiling at 95 °c for 1 hou r. to each of the test tubes, 3 ml of n-butanol (chem-lab nv, belgium) was added and mixed well. th e tubes were cen trifuged at 3000 rpm for 10 min. the separated butanol layer was collected an d read in a spectrophotometer against reagen t blank at 535 nm. thiobarbituric reactiv e substances (tbars) content was expressed as nmol of malon dialdeh yde per ml of plasma. th e methods used in the presen t experiment w ere in accordance with the national legislati on an d the guidelines of the research ethics committee of the departmen t of animal science an d aquaculture of the ag ricultural university of ath ens. 2.3 statistical analysis data referring to body weights (kg ) and mda concentration values (nmol/ml) were analyzed using a mixed m odel procedu re appropriate for repeated m easuremen ts per subject, which included nutritional treatment as fixed effect (unstru ctured covarian ce structure). all model analyses were performed with sas/stat version 9.1.3 (2005). 3 results no significant effect of yoghurt serum powder on grow th performance (kg ) of fattening lambs was demonstrated, even after 28 days of dietary supplemen tation (24.16, 25.03 an d 25.63 for th e con trol, ys1 and ys2, respectively). on the oth er hand, weight of l ambs increased with age, irrespective of experim ental g roup (table 2). malondialdehyde (mda) lev els in plasma were not significantly differen t among th e experimen tal groups. howev er, a significant increase in mda values (nmol/ml) was observed in ys2 group on e week after the beginning of the dietary supplemen tation, an increase that was not so profound after 28 days of yoghurt serum powder dietary supplemen tation (0.714, 1.13 1 and 0.917 on day 0, 7 and 28, respectively) (table 3). 4 discussion although no data exist related to the use of yoghurt serum in animal production, th e feeding value of ch eese wh ey produ cts was extensively studied in the united states, especially during the late 1960s and early 1970s. dried whey derived from cheese industry can be us ed in small quantities in the diets of fattening lambs (< 6%) withou t negative effects on growth p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 5 haf © 2013 vol. 2, no. 1 issn: 2283-3927 performance, carcass characteristics and health status (larsen et al., 1963). boukila et al. (1995) reported a beneficial effect of ferm ented, ammoniated, cond ensed whey dietary supplementation at the level of 2-3% on feed intak e in sheep and suggested that may be a better alternative to urea as a source of non-protein nitrogen. whey permeate can also be successfully used as feed ingredien t (10%) in th e diet of highly productive lactating goats, since its inclusion in the diet results in a significant in crease of feed intake, possibly due to its hig h palatability (rapetti et al., 2002). the higher dry matter in take (+ 9. 2%) leads to a significantly higher fat, protein and raw milk production compared to goats fed no whey-supplemen ted diets (rapetti et al., 1995). schingoethe (1976) also indicated that ruminants can consume dried whey products up to 10% of dietary dry matter in high g rain diets without causing digestive disorders or n egatively affecting grow th and production. the incorporation of dried wh ey at the rate of 10-65% of th e table 2. effect of yoghurt serum powder dietary supplementation o n bo dy weights (kg) of lambs (mea ns ± s.e.m. ) experimental period (days) yoghurt powd er supplementation (g/kg) s.e.m. 0 25 50 0 20.03 20.41 20.38 0.783 7 21.22 21.56 21.56 0.784 14 22.06 22.56 23.41 0.797 21 23.50 24.37 24.72 0.833 28 24.16 25.03 25.63 0.815 no significant differences were found among the experimental groups (p> 0.05) table 3. effect of yoghu rt serum powder dietary supplem entation on malondialdeh yde (mda) values (nmol/ml) in blood plasma of fattening lambs (m eans ± s.e.m.) experimental period (days) yoghurt powder supplementation (g/kg) s.e.m. 0 25 50 0 0.855a 0.867a 0.714a 0.062 7 1.047a 1.093a 1.131b 0.106 28 0.947a 0.973a 0.917c 0.057 abc mean values with different superscripts within a column differ (p < 0.001). no significant differences were found among the experimental groups (p > 0.05) p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 6 haf © 2013 vol. 2, no. 1 issn: 2283-3927 concen trated feed or 15% of corn could also in crease feed intak e in cattle (galloway et al., 1992; morrill and dayton, 1974; schingoethe and skyberg, 1981). as it shown by the findings of th e presen t study and the existing literature, yoghurt serum, such as whey, could readily replace a part of the cereals in fattening rations for lambs, without negativ e implications on their growth. malondialdehyde (mda) lev els in plasma were not significantly differen t among th e experimen tal groups. howev er, a significant increase in mda values (nmol/ml) was observed in the lam b group fed with th e high lev el of yoghurt serum, especially on e w eek after th e beginning of the dietary supplemen tation. a possible explanation is that lactic acid in excess could penetrate the wall of th e rumen and may cause from slight alterations in some health indices, such as oxidation values in plasma, till serious metabolic disorders, such as acidosis and diarrhea (anderson, 1975). lactic acid is produced in the rumen by the breakdown of lactose by bacteria and protozoa and is further metabolized in to volatile fatty acids, mainly bu tyrate. feeding trials in cows show that wh ey dietary supplemen tation even at th e lev els of 10 -40% of dry matter intake does not have any detrimen tal effects on animal health, such as digestive disorders and diarrh ea inciden ts (schingoeth e et al., 1980; pinchasov et al., 1982; susmel et al, 1995). in th e presen t study, no in ciden ts of lambs with diarrh ea were also recorded, possibly as a result of the low dietary supplemen tation level of yoghu rt serum powder. as it is concluded, an adjustment period is therefore necessary to adapt the digestive system to this n ew form of feed and enable its consumption (anderson et al., 1974), especially when the concen tration of the yoghurt serum powder in the diet increases. in practice, according to the findings of th e presen t study, an adjustmen t period of about a week with th e low level of yoghurt seru m powder dietary supplem entation (25 g/kg) could alleviate the possible negative effects on animals’ health – displayed as in creas ed mda plasma concentration – caused by increasing th e level of supplemen tation (50 g/kg), at the age of 23 months in lambs. 5 conclusions yoghurt s erum powder, such as ch eese wh ey, is a source of en erg y that could readily replace a part of the cereals in fattening rations for ruminants and enhance th e use of n on protein nitrog en by the rumen microflora. it appears as a promising alternative for the diets of fattening lambs, although further experim entation is warran ted to elucidate its exact im pacts on th eir metabolism and to evaluate its economical convenien ce based on th e market values of feedstuffs. 6 acknowledgements the auth ors are grateful to hellenic proteins s.a. for providing the yoghurt serum powder. the statis tical expertis e of m. goliom ytis is also appreciated. p. e. simitzis et al. int. j. of health, animal science a nd food safety 1 (2015) 1 -.8 7 haf © 2013 vol. 2, no. 1 issn: 2283-3927 references afrc (ag ricultural and food research council) (1993 ). energy and protein requiremen ts of ruminants: an advisory manual. technical committee on responses to nutrients, alderman g, cottrill br, 100-106. cab in ternational, wallingford, uk. alonso s., herrero m., rendueles m., diaz m. 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(2002). whey compon ents: millennia of evolu tion create funcionalities for mammalian nutrition: what we know and what w e may be overlooking. crit. rev. food sci., 42: 353-375. 25 l keywords fluoroquinolones, residues, chicken, a ntibiotic resistance, nigeria. pages 25 – 34 references vol. 1 no. 2 (2014) article history submitted: september 26, 2014 revised: november 29, 2014 accepted: december 02, 2014 published: december 04, 2014 corresponding author omotoso adekunbi b., animal products and processing unit, department of animal science, university of ibadan, ibadan, nigeria e-mail: kunbiadeshiyan@yahoo.com phone: +234 08033900828 journal home page riviste.unimi.it/index.php/haf screening of fluoroquinolone residues in imported and locally produced broiler chicken meat in ibadan, nigeria. omotoso adekunbi b.1* and omojola andrew b.1 1 animal products and processing unit, department of animal science, university of ibadan, ibadan, nigeria abstract. the study was conducted to investigate residues of three fluoroquinolones (ciprofloxacin, norfloxacin and ofloxacin) in chicken meat sold in ibadan, nigeria. two hundred and ninety-seven (297) samples of imported frozen (99) loc ally produced frozen (99) and freshly slaughtered (99) broiler chicken meat products were screened for antibiotic residues by microbiological as say using escherichia coli as test organism. high performance liquid chromatography (hplc) with ultraviolet (uv) detection was used for the determination of ciprofloxacin, norfloxacin and ofloxacin in the positive samples. one hundred and sixty (160) samples, constituting more than half (53.87%) of total sample size tested positive for escherichia coli–sensitive antibiotic residues. positive samples were 54.55%, 56.57% and 52.53%, of freshly slaughtered, locally produced frozen chicken and imported frozen chic ken meat respectively. residues of investigated fluoroquinolones occurred more frequently in locally produced frozen chic ken than in imported frozen chicken. the concentrations were however consistently higher in imported chicken. among the three fluoroquinolones examined, the most abundant in imported frozen chicken was ciprofloxacin with mean 354.83±716.43µg/kg. norfloxacin was the most abundant in freshly slaughtered and locally produced frozen chicken meat, having mean values of 107.70±138.36µg/kg and 120.96±162.83µg/kg respectively while ofloxacin was the lowest in all categories. m ost frozen chic ken products imported into nigeria at the time of this study contain higher levels of residual fluoroquinolones than the locally produced chicken. in order to tackle fluoroquinolone resistance from a food safety perspective, proper usage and monitoring of fluoroquinolones in meat animals should be encouraged in developing countries. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 26 haf © 2013 vol. i, no. 2 issn: 2283-3927 1 introduction the world health organization (who) recognises that while an tibiotics can play a critical role in food production, th ere is need to balan ce their use to ensure they remain a valuable tool for both human and animal health (wh o, 2011b). antibiotics used in meat animal production do not pos e a health hazard provided they are used in accordance with th e recomm endations for th eir use, proper dosage, proper route of administration, proper species of animal and adequate withdrawal period before slaughter (dipeolu, 2010). quinolones are effective ch emotherapeutic agen ts with v ery good antibacterial activity that target dna synth esis. fluoroquinolones are derivatives of quinolon es which are typically fluorinated at c-6 or c-7 position of th e quinolon e ring. norfloxacin, ciprofloxacin, ofloxacin, lomefloxacin, enrofloxacin are second generation quinolones, exhibiting a broader activity against gram negative and gram positive bacteria, less protein binding, higher drug tolerance, lower toxicity and longer half life than the first generation (emami et al., 2010; somasundaram and manivannan, 2013). drug residues in foods have been said to cause allergic reactions, toxicity, techn ological problems in fermented products and the developmen t of an tibiotic resistance in human pathogens. it has been documen ted that a major rou te of transmission of resistant microorganisms from animals to humans is through the food chain (hernandez -serran o, 2005; canada-canada, 2012). quite unfortunately, poultry production has been implicated in th e emerg ence of quinolon e resistan t bacteria. resistance to fluoroquinolones in escherichia coli and salmo nella typhi is an increasing problem in nigeria and many oth er countries (daini et al., 2005). researchers hav e demonstrated the prevalence of bacteria which are resistan t to various fluoroquinolon es in different parts of nigeria, including lagos (aibinu et al., 2004), benin city (enabulele et al., 2006), oshogbo (olow e et al., 2008) and ibadan (makanjuola et al., 2012). antibiotic resistance has long been recognised as a food safety issue and all stakeholders in food production are responsible for the preven tion and con trol of antibiotic resistance through th e food chain. the rising prevalen ce of an tibiotic resistance is a particularly importan t problem in developing countries whe re there is limited con trol of th e quality, distribution an d use of antibiotics in human medicin e, veterinary medicine and food animal ag ricultu re (ok ek e et al., 1999). it is important for coun tries to monitor residues of antibiotics in their foods because the use of an tibiotics in on e sector, setting or coun try affects the spread of resistance in others. the wh o has noted that since animal products are traded w orldwide, th ey contribute to an tibiotic resistance in countries far from where the problem origin ates. hen ce the need to monitor both imported and locally produced animal products for residues of antimicrobials, especially highest priority critically importan t an timicrobials such as th e fluoroquinolones (wh o, 2011a ). ciprofloxacin and norfloxacin are am ong th e mos t commonly used fluoroquinolones in poultry production in ibadan m etropolis and ofloxacin is the common drug for treatin g tuberculosis in nigeria (daniel et al., 2011). this study seeks to examine samples of locally produced and imported chicken products for residues of three fluoroquinolone compounds (ciprofloxacin, norfloxacin and ofloxacin) because of th eir im portance in both animal an d human medicine. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 27 haf © 2013 vol. i, no. 2 issn: 2283-3927 2 materials and methods 2.1 study area the study was carried out in ibadan, oyo state, nigeria. samples were collected from small, medium and large scale farms as well as open frozen chick en market spread across th e eleven (11) local government areas that make up ibadan, the capital of oyo state, nigeria . 2.2 sample collection sample size was two hundred and ninety seven (297) comprising 99 samples of freshly slaughtered chicken (from 3 small scale, 3 medium scale and 3 large scale farms in each of eleven local governmen t areas), 99 samples of locally produced frozen chick en and 99 samples of imported frozen chick en thigh muscles. the samples were collected on ice and kept frozen at -30c at the meat science unit of the department of animal scien ce, university of ibadan, until th ey w ere further transported in padded coolers with ice packs to the agulu lab oratory of th e national agency for food and drug adminis tration and control (nafdac) where th e analysis was done. the samples were screen ed for three fluoroquinolones, ciprofloxacin, norfloxacin and ofloxacin with structural formulae as shown in figure 1. figure 1. structural formulae of ciprofloxacin, norfloxacin and ofloxacin. source: http://www.newdruginfo.com/pharmacopeia/usp28/v28230/usp28nf23s0.htm ciprofloxacin (b) norfloxacin (c) ofloxacin 2.3 microbiological screening one plate test (opt) as described by alla et al. (2011) was adapted for this study. microbiological screening using th e plate test depends on bacterial g rowth inhibition. the tes t organism was escherichia coli (american type tissue culture 11303 ). escherichia coli has been indicated as a reliable test organism for th e detection of fluoroquinolon es in animal products. to prepare the sub-culture, wire loop ch rom e passed through bunsen burner flam e was allowed to cool and used to s eed escherichia coli in the nu trient broth which was then incubated at 370c for 24 hours. th e culture m edium was prepared by suspending 34g of mueller hinton agar (merck kgaa, damstadt, germany) in 1 litre of demineralised water an d heating in a boiling water bath. it was th en autoclaved for 15 minu tes at 120 0c. the cultu re medium was sterilized in an autoclave at 121 0c for 20 minutes. javascript:modelesswin('imageviewer?doc='+parent.mytitle+'&img=uspnf/pub/images/v28230/g-1292.gif',500,500); omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 28 haf © 2013 vol. i, no. 2 issn: 2283-3927 sterilized stainless steel scissors, forceps and cork borers were used in handling meat samples. the work table was wiped down intermittently with 70% ethanol. 20ml of th e culture m edium was pou red in to each 90mm petri dish and left on th e work bench for abou t 15 minutes to solidify. with the aid of sterile swab sticks, escherichia coli was seeded in th e plates. holes were punched in to each aga r plate with a cork borer. sterile forceps were used to place meat discs inside the hole. th e plates w ere th en incubated a t 370c±2 for 24 hours. zon e of inhibition was measured with a mm -graduated ruler. samples with no clear zone or with clear zon es less than 1mm were taken as negative. clear zon es betw een 1mm and 2mm were considered doubtful while zones from 2mm upwards were positive. 2.4 confirmation and quantification of fluoroquinolones by hplc with uv detection negative samples w ere discarded while chick en samples that were either doubtful or positive for residues of an tibiotics were subjected to hplc uv for iden tification an d quantification. residue extraction was done as described by (ovando et al., 2004). 0.2g thigh muscle was homogenized with 2ml phosphate buffer (ph 7.2) prepared in the laboratory. 8ml dicholorom ethan e (sigma aldrich, st. louis, mo, usa) was added to the homogenate, which was mixed on a stuart scien tific sa8 vortex mixer (sigma aldrich, st. louis, mo, usa) at 1000rpm for 1 minute and centrifuged at 4000 rpm for 20 minutes. the upper aqueous layer was discarded, the organic phase was transferred to a clean tube and the tissue was again extracted with 6ml of dichlorom ethane. organic layers w ere combin ed and evaporated a t 300c under nitrog en stream. the extract was re-dissolved with 200μl of mobile phase an d 100μl was used for hplc analysis. the hplc equipmen t was elite lach rom vwr, hitachi hplc ch romatograph (hitachi, tokyo, japan) comprising l2200 autosampler, l2130 pump and l2350 column oven, equipped with l2400 uv vis detector and ezch rom elite software. th e separating column was elite c18 (250mmx4.6mmx5µm ) (hitachi, tokyo, japan). reference standards of ciprofloxacin (99.6%), norfloxacin (99.5%) and ofloxacin (99.9) were provided by nafdac. buffer phosphate solution 0.1m, ph 7.2 was prepared in th e laboratory. deionised water, passed through 0.45µm whatman filter was used throughout. the solvents, dichlorom ethan e, acetonitrile and triethylamin e were hplc grade. the mobile phase was water: acetonitrile: trieth ylamine (80:19:1), adjusted to ph 3.0 with phosphoric acid, filtered through 0.45µm nylon membrane. samples and separate s tandard solu tions were identified using the hplc ch romatograph with uv detector. ciprofloxacin, norfloxacin and ofloxacin were identified individually by comparing the retention time, area and spectra of peaks of unknown substance with respective s tandard substan ces. the quan tity of iden tified substan ces was calculated using th e formula adapted from naeem et al. (2006). omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 29 haf © 2013 vol. i, no. 2 issn: 2283-3927 2.5 statistical analysis statistical analysis was accomplished by analysis of variance using spss (2005). treatm en t means were separated by duncan multiple range test and statistical significance was set at a probability of p ≤ 0.05. 3 results results of microbiological screening show that out of a to tal sample size of 297 (tw o hundred and nin ety seven ), 162 (54.55%) w ere positive for at least one antibiotic to which escherichia coli was susceptible. samples were categorised as negative and positiv e based on the zon es of inhibition around m eat samples and confirmation with hplc (figure 2). figure 2. result of antibiotic residue screening in chicken meat pu rchased from differen t markets in ibadan. ciprofloxacin, norfloxacin and ofloxacin residues are present togeth er in most freshly slaughtered and locally produced frozen chicken samples. fewer samples contain ed eith er ciprofloxacin alone or ofloxacin alone. how ever, norfloxacin residues did not exist in isolation in any of the samples. distribution of identified fluoroquinolones in the three sample groups is shown in table 1. mean residual concen tration of ciprofloxacin in imported frozen chicken samples (354.83 ± 716.43 µg/kg) was significantly (p ≤ 0.05) higher than the con cen tration in locally produ ced frozen chick en (64.63 ± 120.77 µg/kg). ofloxacin follows th e same trend bu t no significan t difference was obs erved in concen tration of n orfloxacin residue in th e th ree categories. 54,55 43,43 52,53 45,45 56,57 47,47 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% freshly slaughtered locally produced frozen imported frozen p e rc e n ta g e o f sa m p le s class of chicken meat antibiotic-negative antibiotic-positive omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 30 haf © 2013 vol. i, no. 2 issn: 2283-3927 table 1: occurrence of three fluoroquinolones in chicken m eat sold in ibadan as confirm ed with hplc type of chicken meat freshly slaughtered locally produced frozen imported frozen samples with one of the fluoroqu inolones ciprofloxacin only 5 norfloxacin only ofloxacin only 3 3 samples with 2 of 3 fluoroquinolones ciprofloxacin & norfloxacin 5 18 10 ciprofloxacin & ofloxacin 5 norfloxacin & ofloxacin 5 3 9 samples with all three fluoroquinolones ciprofloxacin, norfloxacin & ofloxacin 44 32 20 total positive samples 54 56 52 total no of samples 99 99 80 % of total positive with at least one fluoroquinolone 54.55 56.57 52.53 4 discussion investigating residues of fluoroquinolon es in broiler chicken is of g reat public h ealth concern (al-mustafa and al-ghamdi, 2000; who, 2011b). it has been argued that small doses of fluoroquinolones inges ted from consumption of m eat products with residues of fluoroquinolones weak ens their effectiveness leading to drug resistance by bacteria. the national policy on food h ygiene and safety (npfh s) seeks to ensure that all foods consumed in nigeria, whether imported or locally produced, are wholesome, nutritious and free from contaminan ts (omotayo and d enloye, 2002). there is curren tly a ban on th e importation of poultry products in nig eria but im ported chick en parts are still freely sold in open markets, especially in the south western parts of th e country. since the products are illegally imported, not much atten tion is given to their inspection and quality con trol (dipeolu, 2010). omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 31 haf © 2013 vol. i, no. 2 issn: 2283-3927 table 2: fluoroquinolone residues in chicken m eat pu rchased from different markets in ibadan. fluoroquinolones mean concentration ± sd of fluoroquinolones (µg/ kg) p value freshly slaughtered locally produced frozen imported frozen ciprofloxacin 78.02 ± 202.38by 64.63 ± 120.77by 354.83 ± 716.43ax 0.000 norfloxacin 107.70 ± 138.36x 120.96 ± 162.83x 127.99 ± 279.63y 0.931 ofloxacin 15.18 ± 24.35bz 13.55 ± 22.46bz 42.33 ± 102.53ay 0.019 p value 0.007 0.000 0.049 a, b means in the same row with different superscripts are signific antly different (p ≤ 0.05) x,y means in the same column with different superscripts are significantly different (p ≤ 0.05) table 3: range of con centration of th ree flu oroquinolones in chicken meat purchased from different markets in ibadan, nigeria fluoroquinolones type of chicken meat range of concentration in positive sampl es (µg/kg) % above 100 (µg/kg)* ciprofloxacin freshly slaughtered 2.45 to 888.90 15.15 locally produced frozen 2.45 to 591.50 24.24 imported frozen 14.00 to 2767.00 32.32 norfloxacin freshly slaughtered 3.98 to 393.17 40.40 locally produced frozen 20.50 to 466.65 33.33 imported frozen 6.18 to 1081.27 20.20 ofloxacin freshly slaughtered 4.36 to 77.51 0.00 locally produced frozen 8.76 to 67.13 0.00 imported frozen 14.53 to 431.66 0.00 * european union mrl (commission regulation (eu) 37/2010) for the sum of enrofloxacin and c iprofloxacin in poultry muscle maximum residue limits (mrl) hav e been set for different antimicrobials in differen t food matrices by different organizations and countries. if th e residue of a chemical exceeds th e mrl in meat, it is considered unsafe (petrovic et al., 2006). mrl for antibiotics in foods of animal origin hav e not been s et in nigeria. thus the eu mrl for the sum of enrofloxacin an d ciprofloxacin is adopted for the pu rpose of this study. omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 32 haf © 2013 vol. i, no. 2 issn: 2283-3927 microbial inhibition assay was em ployed as a first qualitative screening step to sift ou t large number of compliant samples. overall, one hundred and thirty seven (137) representin g 46.13% of total sample size con tained no residues of escherichia coli sensitive antimicrobials. fluoroquinolone residues occu rred more frequ ently in locally produced frozen broiler chicken samples. positive samples were 54.55%, 56.57% and 52.53%, of freshly slaughtered, locally produced frozen chicken and imported frozen chicken respectively. norfloxacin residues are presen t in higher con centration in chicken produced within nigeria and are thus of more concern than ciprofloxacin and ofloxacin. h owever, ciprofloxacin is more abundant than norfloxacin and ofloxacin in imported products (table 2). in a similar experim ent, almustafa and al-ghamdi (2000) detected norfloxacin in raw chicken tissues from the eas tern province of saudi arabia at levels that were 2.7 to 34.3 folds higher than the mrl. naeem et al. (2006) observed residues of ciprofloxacin, norfloxacin an d ofloxacin in liver samples purchased from various mark ets in lahore, pa kistan, to be 2.45 to 245.00 µg/kg, 2 20 to 31.00 µg/kg and 2.05 to 22 µg/kg respectively in summer. in the pres en t study, there is wide variation in concentration (table 3) of flu oroquinolones in chicken samples. 5 conclusions if antibiotic resistance must be tackled from a fo od safety perspective, chick en meat, whether imported or locally produced, must be monitored for residues of antimicrobials, especially those that have been classified as critically important. th e present ban on importation of poultry m eat in nigeria should be properly implemen ted to redu ce th e exposure of consumers to fluoroquinolon e residues. as a matter of public health, it is importan t to con trol the use of these antimicrobials in the local production of chicken so as to maintain their potency for use in human medicine. 6 acknowledgements this study was technically supported by the national agency for food and drug administration and control (nafdac), nigeria. the authors are thankful for th e assistance of the staff of nafdac regional laboratory, agulu. references aibinu, i., adenipekun, e. and odugbemi, t., 2004. emergence of quinolone resistan ce amongst escherichia coli. nig erian journal of health and biomedical science. 3(2), 73 -78. http://www.ncbi.nlm.nih.gov/pubmed?term=al-mustafa%20zh%5bauthor%5d&cauthor=true&cauthor_uid=11260778 http://www.ncbi.nlm.nih.gov/pubmed?term=al-ghamdi%20ms%5bauthor%5d&cauthor=true&cauthor_uid=11260778 omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 33 haf © 2013 vol. i, no. 2 issn: 2283-3927 alla, m.b.w., mohamed, t.e and abdelgadir, a.e., 2011. detection of an tibiotics residues in beef in ghanawa slaughterhouse, khartoum state, sudan african journal of food science vol. 5(10), 574-58. al-mustafa z.h. and al-ghamdi m. s., 2000. use of norfloxacin in poultry produ ction in the eastern province of saudi arabia and i ts possible impact on public h ealth. international journal of environmental health research. 10, 291–299. barrow, g. i, feltham, r.k.a., 1993. cowan and steel’s manual for the iden tification of medical bacteria. london: cambridg e university press. cañada-cañada, f., espinosa-mansilla, a., jiménez, girón a., muñoz de la peña a., 2012. simultaneous determination of the residues of fou rteen quinolones and fluoroquinolon es in fish samples using liquid chromatograph y with photometric and fluorescence detection. czechoslovakia journal of food science. 30, 74–82. commission regulation (eu) no 37/2010 of 22 d ecember 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. daini, a., ogbolu, o. d. and ogunledun, a., 2005. quinolon e resistance and r plasmids of some gram negative en teric bacilli. african journal of clinical experimen tal microbiolog y. 6 (1), 14-20. daniel, o., osman, e., bakare, r., adebiyi, p., ige, o., ogiri, s., awe, e., kab ir, m., ogundahunsi, o., mourad, g. and declarq, e., 2011. ofloxacin resistance among mycobacteriu m tuberculosis isolates in tw o states of south-west nigeria. african journal of respiratory medicin e. 23, 18-20. dipeolu, m. a., (2010). healthy meat for wealth. unaab inaugural lecture series. 29, 12-49. emami, s., shafiee, a., foroumadi, a., 2010. quinolones: recen t structural and clinical developm ents. iran journal of pharmaceutical research. 4(3), 123 -136. enabulele, i.o., yah, s.c., yusuf, e.o., eghafona, n.o., 2006. emerging quinolon e resistant transfer gen es among gram-negative bacteria, isolated from faeces of hiv/aids patien ts attending some clinics and hospitals in the city of benin, edo state, nigeria. online journal of health and allied sciences. 3, 3. hernández serrano p., 2005: responsible use of an tibiotics in aquaculture. fao, rome. makanjuola, b.o, bakare, r.a and fayemiwo, s.a., 2012. quinolon e and multidrug resistant salmonella typhi in ibadan, nigeria. international jou rnal of tropical medicin e. 7, 103-107. naeem, m., khan, k. and rafiq, s., 2006. determination of residues of quinolones in poultry products by high pressure liquid chromatog raphy . journal of applied sciences . 6(2), 373379. okeke, i.n., lamikanra, a., edelman, r. 1999. socioeconomic and behavioural factors leading to acquired bacterial resistance to antibiotics in developing countries. emerging infectious diseases. 5, 18-27. olow e, o.a., ogungbamigbe, t.o., kolawole, s.o., olow e, r.a., and olayemi, a.b., 2008. hemolysis production and resistance to fluoroquinolones among clinical isolates of http://ascidatabase.com/author.php?author=khalida%20khanand%20samra%20rafiq&last= omotoso a nd omojola. int. j. of health, animal science a nd food safety 2 (2014) 25 34 34 haf © 2013 vol. i, no. 2 issn: 2283-3927 escherichia coli in osogbo metropolis, sou thwest nig eria. new york science jou rnal 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j.w.a., de cocq, a., nielen, m.w.f., van egmond, h.j., 2007. food additiv es and contaminants. 24, 8. somasundaram, s. and manivannan, k., 2013. an overview of fluoroquinolon es. annual review & research in biology 3(3 ), 296-313. stanier, r.y., ingraham, j.l, eelis, m.l., painter, p.p., 1986. the microbial world. 5th ed. new jersey: pris tice hall. world h ealth organization, 2009. report of th e first meeting of the wh o advisory group on integrated su rveillance of antimicrobial resistan ce (agisar), 15-19 june copenhagen geneva. world h ealth organization, 2011a. critically important an timicrobials for human medicine. wh o advisory group on integ rated surveillance of an timicrobial resistance (agisar), 3rd revision, 5-31. world h ealth organization, 2011b. tackling antibiotic resistance from a food safety perspective in europe who regional office for eu rope, copenhagen, denmark . proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords large wild game meat, short food supply chain, economic value, territorial marketing, sustainable development corresponding author maria e. marescotti maria.marescotti@unimi.it journal home page riviste.unimi.it/index.php/haf abstract currently in italy, in contrast to other eu countries, a supply chain (sc) for hunted game meat does not exist. nevertheless, there are the conditions for its development (gaviglio et al., 2017); in fact game meat dishes’ has always been part of alpine area’s culinary tradition and furthermore, management measures aimed at reducing the overpopulation of large wild ungulates leaded to an increase in the availability of their meat. in this context, the present research aims at analyse the dynamics of the value in the local non-existent sc of the large wild game meat by the application on the case study of valle ossola (piedmont). due to its representativeness among italian wild ungulates, the research focus on red deer meat. the data has been collected in 2016 through in-depth interviews (30) and a focus group with the stakeholders involved in the sc: hunters, transformers and restaurateurs. results show that for the hunter the red deer reach a hypothetical price of 6,00 €/kg. from a meat processing targeted at the maximum enhancement of the carcass, without any waste, the transformers can reach a hypothetical price of 9,80 €/kg. whereas for the restaurateur, the red deer meat can reach a final price range between 22,88 and 51,47 €/kg (hypothesizing maximum sales of high value-added course). through the maximization of the meat’s quality, hunter and transformers profits can increase significantly, with a redistribution of the added value throughout the sc. a limitation of this study is that the calculated values does not take into consideration the stakeholders’ production costs (that increasing along the sc). considering our findings, the development of sustainable sc of the local game meat could be economically interesting. thus, wild ungulates could represent an economic resource for the population rather than an environmental and social cost for the mountain areas. acknowledgments: research funded by fondazione cariplo. project: “processi di filiera eco-alimentare. la gestione di prodotto sostenibile per lo sviluppo dei territori alpini” references gaviglio, a., demartini, e., marescotti, m.e., 2017. opportunities and limitation from an italian alpine case study. calitatea – acces la succes. 18:s2, 215-222. the value of local italian supply chain of the large wild ungulates meat: the case of the red deer meat in alpine valleys maria elena marescotti1*, anna gaviglio1, eugenio demartini1 1 university of milan, department of health, animal science and food safety, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l corresponding author armand sánchez armand.sanchez@uab.cat journal home page riviste.unimi.it/index.php/haf why toll-like receptors genes are so polymorphic? a. sáncheza* adepartment of animal genetics, center for research in agricultural genomics (crag), molecular genetics veterinary service. veterinary school, universitat autònoma de barcelona, 08193 bellaterra, spain. abstract toll-like receptors (tlrs) are pattern recognition receptors (prrs) considered to be the primary sensors of pathogens in innate immunity. genetic variants could be associated to differences in breed innate immune response to pathogens and thus to susceptibility to infections or autoimmune diseases. tlrs are encoded by a multigene family and are conserved through evolution, from drosophila to mammals, because of its essential role in innate immunity. ten tlrs have been identified in most mammals. tlrs can be classified into groups, depending on the pamps detected and their cellular location. there are significant distinctions between intracellular and extracellular tlrs. intracellular tlrs theoretically can't accept much variability, because they have evolved under strong purifying selection. viruses can only be detected through their nucleic acids; therefore, intracellular tlrs have an essential non-redundant role in host survival. moreover, mutations in those tlrs could end up with an autoimmune disease against own nucleic acids or with high susceptibility to some viral infections. on the other hand, membrane or extracellular tlrs have evolved under less evolutionary pressure, due to they can recognize one pathogen through different pamps (immunological redundancy). so they show a higher rate of damaging nonsynonymous and stop mutations. although infective pressure that has reached these molecules is one of the main mechanisms of evolution, it is not the only one. in some domestic species nonadaptative evolution has also an important role, through genetic drift, bottlenecks and migratory routes. genetic variation in canine and porcine tlrs characterized in our laboratory using massive parallel sequencing will be presented and results discussed. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords dog, mitral valve disease (mvd), chronic heart failure (chf), serum iron deficiency (sid), tibc corresponding author alice savarese alice.savarese@unimi.it journal home page riviste.unimi.it/index.php/haf iron status in dogs with mitral valve disease. a. savaresea, m. proboa, c. locatellia, g. trainia, p.g. brambillaa, s. paltrinieria adepartment of veterinary medicine (dimevet), università degli studi di milano, via celoria 10, 20133 milano, italy. abstract in people, serum iron (si) deficiency (sid) is a frequent co-morbidity in chronic heart failure (chf), reducing quality of life and survival (belmar vega l, 2016). mitral valve disease (mvd), the most common canine acquired heart disease, lead to chf. (borgarelli, 2012). aims of the study were determine prevalence and characteristics of sid (si < 90 µg/dl) in mvd dogs, analyze differences in si among acvim classes, symptomatic and non-symptomatic patients, and study effects on survival. fifty-four privately owned mvd dogs (dimevet, january 2015 april 2016) with complete physical evaluation, chest x-ray, echocardiography and serum biochemical panel were included. iron status was evaluated measuring si, total iron-binding capacity (tibc) and percentage transferrin saturation (%sat). median age of dogs was 11 years (iqr 10 14), median body weight 11 kg (iqr 6 – 22). most were intact males (42%) mongrels (46%). non-symptomatic and symptomatic dogs were 64% (n=36) and 33% (n=18). the prevalence of sid was 18.5 % (10/54: 6 symptomatic and 4 non-symptomatic). only 3 patients (6%) presented anemia (hct ≤ 37%). tibc (nv: 270-496 μg/dl) was within or above the reference range in 6/10 dogs with sid, while %sat (nv: > 23%) was below the minimum level in 4/10 dogs. no differences in si were found between acvim classes, symptomatic and non-symptomatic patients. log-rank analysis showed significant shorter survival in mvd dogs with sid (p: 0.030), nevertheless multivariate cox analysis revealed that only the presence of chf symptoms affect survival (p: 0.001). tibc and %sat suggest that sid is most frequently true (primary) than functional (secondary). references belmar vega, l., de francisco, a., albines fiestas, z., serrano soto, m., kislikova, m., seras mozas, m., unzueta, m.g., aria s rodríguez, m., 2016. investigation of iron deficiency in patients with congestive heart failure: a medical practice that requires greater attention. nefrologia 16, in press, doi: 10.1016/j.nefro.2016.03.001 borgarelli, m., buchanan, j.w., 2012. historical review, epidemiology and natural history of degenerative mitral valve disease. j vet cardiol. 14, 93-101. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords horse, standardbred, treadmill, lactate, heart rate corresponding author luca stucchi luca.stucchi@unimi.it journal home page riviste.unimi.it/index.php/haf performance profiling of standardbred racehorses by means of treadmill exercise testing. luca stucchi1*, miranda dosi1, francesco ferrucci1 1university of milan, department of health, animal science and food safety, italy abstract treadmill exercise testing can be performed on a horse to evaluate the level of fitness with the aim of predicting performance (franklin and allen, 2014). the speed at 2 mmol/l of blood lactate (vla2), the speed at 4 mmol/l (vla4) and the speed at 200 bpm of heart rate (v200) are indices that have been related to performance (coroucé et al., 2002). aim of the present work is to analyze these parameters in a population of high performance standarbred racehorses. six healthy and at the same level of training standardbred racehorses (average age 3,3±2,0 y.o.) underwent an incremental exercise test (zucca et al., 2003) on a high speed treadmill (sӓto i, sato, sweden). during the test heart rate (hr) was monitored with a heart rate meter (polar horsetrainer, polar, finland). venous blood was collected with the aid of a 14g teflon venous catheter placed in the jugular vein. plasma lactate was measured with enzymatic colorimetric method lactate dry-fast kit for automatic system (cobas mira classic, roche, switzerland). data were analyzed with a dedicated software (lactate express, mesics, germany) and vla2, vla4 and v200 were calculated and statistically compared by t-student test for paired sample (prism, graphpad, usa). statistical significance was set at p<0,05. average vla2 was 8.3±0.5 m/s, average vla4 was 9.2±0.4 m/s, average v200 was 8.1±0.9. there was a significant difference between vla4 and v200 (fig. 1). no difference was observed between vla2 and v200 v200 is often reported to be close to vla4, and considered as correspondent to the onset of blood lactate accumulation (coroucé et al., 2002). according to our results, it may be argued that v200 is a measure that does not fit with the lactate threshold. these data could be used as control for further studies on racehorses with poor performance syndrome. acknowledgments: this study was supported by the italian ministry of health (#rc 2016, l4083). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 v l a 4 v 20 0 v l a 2 6 7 8 9 10 11 * * = p < 0,05 s p ee d m /s fig.1: difference between average vla2, vla4 and v200 references couroucé, a., chrétien, m., vallette, j.p. 2002 physiological variables measured under field conditions according to age and state of training in french trotters. equine veterinary journal. 34,91-97 franklin, s., allen, k. 2014 laboratory exercise testing. in: hinchcliff, k.w. , kaneps, a.j., raymond, j.g. equine sports medicine and surgery. saunders, st luis, usa. 11-20 zucca, e., ferrucci, f., di fabio, v., croci, c., ferro, e. 2003 the use of electrocardiographic recording with holter monitoring during treadmill exercise to evaluate cardiac arrhythmias in racehorses. veterinary research communication. 27(1), 811-814 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords asthma, horses, airway remodeling, therapy corresponding author jean-pierre lavoie jean-pierre.lavoie@umontreal.ca journal home page riviste.unimi.it/index.php/haf airway remodeling and its reversibility in equine asthma jean-pierre lavoie1 1université de montréal ,department of clinical sciences, faculty of veterinary medicine, canada abstract despite effective therapies for controlling its clinical manifestations, human asthma remains an incurable disease. it is now recognized that inflammation induced structural changes (remodeling) of the airways are responsible for the progressive loss of lung function in asthmatic patients. however, the peripheral airways, where most of the remodeling occurs in severe asthmatic patients, cannot be safely sampled in humans, and therefore, little is known of the effects of current therapies at reversing the established asthmatic remodeling, especially those occurring in the peripheral airways. animal models have been studied to unravel etiological, immunopathological, and genetic attributes leading to asthma. however, experiments in which the disease is artificially induced have been shown to have limited translational potential for humans. to the contrary, horses naturally suffer from an asthma-like condition which shares marked similarities with human asthma making this model unique to investigate the kinetics, reversibility, as well as the physiological consequences of tissue remodeling (bullone and lavoie 2015). we reported an increased deposition of smooth muscle, collagen and elastic fibers in the peripheral airways of affected horses, which was correlated with the lung function (herszberg et al., 2006; setlakwe et al., 2014). the airway subepithelial collagen depositions were almost completely reversed with 6 to 12 months of treatment with either antigen avoidance or inhaled corticosteroids (ics) administration, and there was a modest (30% on average) decrease in airway smooth muscle (leclere et al., 2011). a recent study also found that ics combined with long-acting ß2-agonists drugs (laba) and ics monotherapy similarly induced a 30% decrease of the airway smooth muscle mass at 3 months (buollone, 2017). however, only ics/laba and antigen avoidance decreased airway luminal neutrophilia. the findings indicate the enhance therapeutic effect of ics/laba over ics monotherapy at controlling asthma exacerbations in humans may be due to their anti-remodeling and anti-inflammatory effects. however, airway smooth muscle remodeling is only partially reversible with current anti-asthma medications. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references bullone, m., lavoie, jp., 2015. asthma "of horses and men"--how can equine heaves help us better understand human asthma immunopathology and its functional consequences? mol immunol. 66, 97-105 herszberg, b., ramos-barbon, d., tamaoka, m., martin, j.g., lavoie, jp., 2016. heaves, an asthma-like equine disease, involves airway smooth muscle remodeling. j allergy clin immunol. 118, 382-388 setlakwe, e.l., lemos, k.r., lavoie-lamoureux, a., duguay, j.d., lavoie, jp., 2014. airway collagen and elastic fiber content correlates with lung function in equine heaves. am j physiol lung cell mol physiol. 307, l252-260 leclere, m., lavoie-lamoureux, a., gelinas-lymburner, e., david, f., martin, j.g., lavoie jp., 2011. effect of antigenic exposure on airway smooth muscle remodeling in an equine model of chronic asthma. am j respir cell mol biol. 45, 181-187 bullone, m., 2017. reversibility of airway remodeling in equine asthma: contribution of anti-inflammatory and bronchodilator therapies. phd thesis, sciences vétérinaires: université de montréal http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en 1 l keywords soy; phagocytes; inflammation. pages 1 – 8 references vol. 1 no. 1 (2014) article history submitted: november 28, 2013 revised: january 14, 2014 accepted: january 14, 2014 published: january 28, 2014 corresponding author georgios theodorou, faculty of animal science and aquaculture department of animal husbandry agricultural university of athens 75 iera odos street, 118 55, athens, greece e-mail: gtheod@aua.gr phone: +30 210 5294450 fax: +30 210 5294442 journal home page riviste.unimi.it/index.php/haf association of dietary soy with expression of various pro-inflammatory genes in porcine phagocytes. georgios theodorou 1* , george papadomichelakis 2 , eleni tsiplakou 2 , roubini chronopoulou 1 , george zervas 2 and ioannis politis 1 1 faculty of animal science and aquaculture, department of animal husbandry, agricultural university of athens, athens, greece. 2 faculty of animal science and aquaculture, department of nutritional physiology and feeding, agricultural university of athens, athens, greece. abstract. soybean and whey are two common protein sources used in piglet feeding; however, their effects on pro-inflammatory responses remain unclear. the present study investigated the expression of various genes implicated in the activation/deactivation of porcine phagocytes post-weaning. eighteen piglets were divided into two groups based on the main protein source of their diet; soybean (sb) or whey proteins (wp). blood phagocytes were isolated at 72 days of age. expression of urokinase plasminogen activator (upa), u-pa receptor (u-par), plasminogen activator inhibitors 1 and 2, intercellular adhesion molecule 1 (icam-1), inducible no synthase (inos), cyclo-oxygenase-2 and interleukin-10 (il-10) in activated monocytes and neutrophils (except il-10) was determined by quantitative pcr. expression of u-par, icam-1 and inos were lower in both cell types obtained from sbfed piglets compared to wp-fed piglets. in conclusion, a sb-based diet, compared with a wp diet, is associated with reduced expression of crucial pro-inflammatory genes in porcine phagocytes. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 2 haf © 2013 vol. i, no. 1 issn: 2283-3927 1 introduction conflicting data concerning the effects of soy foods on expression of cellular adhesion molecules and production of pro-inflammatory cytokines have been published. a review of 14 independent studies concluded that soy-based diets have mixed effects on expression of cellular adhesion molecules, and do not affect production of crucial pro-inflammatory cytokines, such as interleukin 6 (il-6) and tumor necrosis factor-alpha (tnf-alpha) (beavers et al. 2009). however, there is evidence suggesting that soy protein peptides (spp) exert antiinflammatory activity (using a dextran sodium sulfate-induced pig model of intestinal inflammation) by down-regulating pro-inflammatory responses in vivo (young et al. 2012), and can reduce production of nitric oxide (no) and prostaglandin e2 (pge2) and the expression of inducible no synthase (inos) and cyclooxygenase-2 (cox-2) genes by macrophages activated by lipopolysaccharide in vitro (martinez-villaluenga et al. 2009). lunasin (a spp) inhibits inflammation through the suppression of the nuclear factor-kappab (nf-kb) pathway in the macrophage in vitro (de mejia and dia, 2009). our group has also shown that spp downregulated the expression of various pro-inflammatory genes in ovine phagocytes induced by fatty acids in vitro (politis et al. 2012). to the best of our knowledge, there is lack of studies investigating whether dietary soy proteins can modify the expression of various genes implicated in activation/deactivation of porcine phagocytes in vivo. taking into account the wide use of soybean meal and whey concentrates as protein sources in pig feeding (yang et al., 2007; yun et al., 2005), the investigation of their effects on pro-inflammatory responses would be of great importance; particularly, in the critical post-weaning period. therefore, the present study aimed to compare the expression patterns of urokinase-plasminogen activator (u-pa), u-pa receptor (upar), plasminogen activator inhibitor type 1 and 2 (pai-1, pai-2), intercellular adhesion molecule 1 (icam-1), interleukin 10 (il-10), inos and cox-2, in activated monocytes and neutrophils obtained from piglets fed diets with either soybean meal (sb-based diet) or whey proteins (wp-based diet) as the main crude protein source post-weaning. 2 materials and methods 2.1 animals and diets eighteen castrated male large white × duroc × landrace weaned piglets (29±2 days old) were used in compliance with the guidelines of the faculty of animal science and aquaculture of agricultural university of athens. animals were divided into 2 groups (with average body weight of 8.4 ± 0.68 kg per group). they were fed ad libitum one of two isocaloric (15.5 mj/kg dry matter) and isonitrogenous (230 g/kg dry matter) diets (nrc, 1998), which contained either soybean meal (sb) or a mixture of whey proteins [wp, 70% wheypro65 (650 g cp/kg) + 30% wheypro 80 (800 g cp/kg); hellenic proteins s.a., veria, greece] as the main crude protein (cp) source (table 1). g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 3 haf © 2013 vol. i, no. 1 issn: 2283-3927 2.2 chemical analyses feed samples were milled through 1-mm screen prior to analyses. feed dm was assessed in 5 g samples by oven drying at 105 o c overnight. routine procedures of aoac (1984) were used table 1. ingredients and chemical composition of the soybean meal-based (sb) and whey protein-based (wp) experimental diets. diets sb wp ingredients (g/kg) maize 621.0 754.0 soybean meal (440 g cp/kg) 342.0 whey proteinsa (660 g cp/kg) 210.0 l-lysine 80% 2.0 2.0 dl-methionine 99% 1.0 1.0 sodium chloride 5.0 4.0 calcium carbonate 13.0 14.0 monocalcium phosphate 13.0 12.0 mineral-vitamin premixb 3.0 3.0 analyzed chemical composition (g/kg dm) dry matter (g/kg) 888.0 903.0 crude protein 232.0 228.0 ether extract 33.1 40.4 digestible energyc (mj/kg) 15.5 15.4 a. mixture of whey proteins (hellenic proteins s.a., veria, greece); 70% wheypro 65 (650 g cp/kg) + 30% wheypro 80 (800 g cp/kg) designed to have a content of 660 g of crude protein/kg. b. mineral-vitamin premix (nuevo s.a., n. artaki, greece) provided per kg of diet: 15000 iu vitamin a (retinyl acetate), 2000 iu vitamin d3 (cholecalciferol), 100 mg vitamin e (dl-α-tocopheryl acetate), 3.5 mg menadione (vitamin k3), 2.5 mg vitamin b1, 6 mg vitamin b2, 3 mg vitamin b6, 25 μg cyanocobalamin, 25 mg nicotinic acid, 20 mg pantothenic acid, 2 mg folic acid, 250 μg biotin, 2 mg co, 4 mg i, 600 μg se, 300 mg fe, 100 mg mn, 100 mg mg, 320 mg cu and 240 mg zn. c. digestible energy values were calculated according to tabulated data (nrc, 1998). g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 4 haf © 2013 vol. i, no. 1 issn: 2283-3927 for ether extract (ee; 7.063). crude protein (cp) was determined as 6.25×kjeldahl nitrogen, using a kjeltec autoanalyzer unit (foss, sweden). all analyses were performed in duplicate. 2.3 blood sampling and cell isolation, activation blood samples were collected from all animals at the last day of the experiment (72 days of age). monocytes and neutrophils were isolated (politis et al., 2012), washed (×3) with hank’s balanced salt solution, and then activated by adding phorbol myristate acetate (pma; 81 μμ). after 30 min, cells were washed (×3) with hank's balanced salt solution (hbss) and kept at -80 ºc pending rna extraction and rt-pcr. 2.4 rna extraction and rt-pcr analysis total rna was extracted from 5 x 10 6 cells from both purified populations of blood cells. total rna extraction and reverse transcription were performed as described previously (politis et al., 2012). relative levels of mrna were quantified with real-time, quantitative rt-pcr using sybr green chemistry. a pair of primers for each of the genes used in this study was constructed using perlprimer software (marshall, 2004). all primer pairs are presented in table 2. the amount of sample rna was normalised by using b-actin as a housekeeping gene. real time pcr was performed in the myiq2 cycler (biorad) using the kapa sybr® fast qpcr kit (kapa biosystems) according to the manufacturer's protocol. each reaction contained 12.5 ng rna equivalents as well as 150-300 nm of forward and reverse primers for each gene. the reactions were incubated at 95 °c for 30 s followed by 40 cycles of 5 s at 95 °c and 15 s at 60 – 64 °c. this was followed by a melt curve analysis to determine the reaction specificity. each sample was measured in duplicates. the comparative ct method (livak & schmittgen, 2001) was used for relative quantification. 2.5 statistical analysis data were analyzed using the spss statistical package (version 17.0). comparisons between diets were conducted using a two-tail unpaired t-test and values are presented as means ± sem. 3 results the expression of u-par was lower (p<0.001) in both monocytes and neutrophils obtained from the sb fed piglets when compared to those obtained from the wp fed piglets, whereas the expression of u-pa, pai-1 and pai-2 did not differ between the two experimental groups (figure 1). the expression of icam-1 and inos was also lower (p < 0.05) in both monocytes and neutrophils obtained from sb fed piglets when compared with the corresponding values in phagocytes obtained from piglets fed the wp-based diet (figure 2). there were no differences g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 5 haf © 2013 vol. i, no. 1 issn: 2283-3927 in the expression of the cox-2 and il-10 between the two experimental groups (data not shown). table 2. sequences, corresponding accession numbers and relative positions of primers for urokinase plasminogen activator (upa), urokinase plasminogen activator receptor (upar), plasminogen activator inhibitors type 1 and 2 (pai-1 and pai-2), intercellular adhesion molecule 1 (icam-1), cyclooxygenase 2 (cox-2), inducible nitric oxide synthase (inos), interleukin 10 (il-10) and beta actin (actb) used in real time pcr. primer sequence 5’ to 3’ accession number position u p a sus_upa_f gtggctgtctgaatggagg nm_213945.1 202 318 sus_upa_r aggtttgcgatgtgtctatctc u p a r sus_upar_f atgggaaggaggtgagga xm_003127198.2 188 384 sus_upar_r aagcacattcaaggtaacgac p a i1 sus_pai1_f ttctgcccaagttctccc nm_213910.1 1027 1194 sus_pai1_r cattcacctcgatcttcacct p a i2 sus_pai2_f aacatcggatacttagcagacc xm_003121697.1 739 939 sus_pai2_r atacacctccacatcatcttcag ic a m -1 sus_icam1_f aacttatgtcctgccagcc nm_213816.1 671 841 sus_icam1_r ccattatgcgtgattgttagtgg c o x -2 sus_cox2_f ctgtactacacctgaatttctgac nm_214321.1 279 385 sus_cox2_r tgacaatgttccagactccc in o s sus_inos_f aagtttgaccataggacccag nm_001143690.1 344 488 sus_inos_r ctttgttaccgcttccacc il -1 0 sus_il10_f ctgtcatcaatttctgccctg hq236499.1 363 526 sus_il10_r agttcttcctcatcttcatcgt a c t b sus_actbf ctaccagttcgccatgga xm_003124280.2 78 252 sus_actbr cacgtaggagtccttctgg g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 6 haf © 2013 vol. i, no. 1 issn: 2283-3927 figure 1. relative mrna levels (means±sem) of urokinase plasminogen activator (u-pa), its receptor (u-par) and plasminogen activator inhibitors type 1 and 2 (pai-1 and pai-2) in blood monocytes and neutrophils from 72 day old pigs fed diets with soybean meal (sb) or whey proteins (wp). asterisks indicate significant differences between sb and wp (***: p < 0.001). 4 discussion the main finding emerging from the present study is that sb-based diets, when compared to wp-based diets, are associated with lower levels of expression of three crucial proinflammatory genes (u-par, icam-1 and inos) in phagocytes obtained from piglets. the u-par molecule plays a central role in the plasminogen activating cascade and a crucial role in cell migration. typically, u-par clusters are observed at the leading edges of migrating phagocytes. in an autocrine manner, the u-pa molecule produced by phagocytic cells themselves binds to its receptor (upar) and remains catalytically active; thus, it converts the inactive proenzyme plasminogen to active plasmin, which allows the migrating phagocytes to cross the endothelial barrier and reach the point of inflammation (de mejia & dia, 2009). the icam-1 encodes a cell surface adhesion glycoprotein, which is typically expressed by endothelial and phagocytic cells and it plays a crucial role in the process of diapedesis. on the other hand, expression of the inos gene results in the production of no (free radical), which is one of the main pro-inflammatory compounds secreted by activated phagocytes (de mejia & u-pa sb wp sb wp 0.0 0.5 1.0 1.5 monocytes neutrophils r e la ti v e e x p re ss io n u-par sb wp sb wp 0.0 0.5 1.0 1.5 2.0 monocytes neutrophils *** *** r e la ti v e e x p re ss io n pai-1 sb wp sb wp 0.0 0.5 1.0 1.5 monocytes neutrophils r e la ti v e e x p re ss io n pai-2 sb wp sb wp 0.0 0.5 1.0 1.5 monocytes neutrophils r e la ti v e e x p re ss io n g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 7 haf © 2013 vol. i, no. 1 issn: 2283-3927 dia, 2009). interestingly, the results of the present in vivo study have certain similarities with those of a previous in vitro study, which showed that soy protein hydrolysates (peptides) down-regulated almost the same set of genes (u-par, icam-1 and inos), but not that of cox-2 and il-10 in ovine phagocytes (politis et al., 2012). the reduced expression of the 3 key proinflammatory genes in the sb fed piglets can be reasonably attributed to the soybean meal proteins, without excluding a possible contribution of other nonprotein compounds, such as isoflavones (chacko et al., 2005). noteworthy, there were no differences between groups in the expression of pai-1, pai-2 and cox-2 in phagocytes, and il-10 in monocytes, which are genes with well-established roles in the inflammatory process (de mejia & dia, 2009; theodorou et al., 2010). the reasons behind the fact that certain pro-inflammatory genes were expressed in lower levels, whilst others were not, in phagocytes from piglets fed the sb-based diet are not known and require future research. figure 2. relative mrna levels (means±sem) of inter-cellular adhesion molecule 1 (icam-1) and inducible nitric oxide synthase (inos) in blood monocytes and neutrophils from 72 day old pigs fed diets with soybean meal (sb) or whey proteins (wp). asterisks indicate significant differences between sb and wp (*: p < 0.05, **: p < 0.01). 5 conclusions in conclusion, a sb-based diet, when compared with a wp diet, is associated with lower levels of three key pro-inflammatory genes (u-par, icam-1 and inos) in phagocytes obtained from piglets. future studies will focus on the potential mechanism of action through which soy may exercise its effects on immunocompetent cells. 6 acknowledgements the present study was partially supported by jsk, a company based in greece. icam-1 sb wp sb wp 0.0 0.5 1.0 1.5 2.0 * ** monocytes neutrophils r e la ti v e e x p re ss io n inos sb wp sb wp 0.0 0.5 1.0 1.5 2.0 * * monocytes neutrophils r e la ti v e e x p re ss io n g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 8 haf © 2013 vol. i, no. 1 issn: 2283-3927 references association of official analytical chemists, aoac, 1984. official methods of analysis, 10th edition. association of official analytical chemists, arlington, va, usa. beavers, k.m., jonnalagadda, s.s., messina, m.j., 2009. soy consumption, adhesion molecules, and pro-inflammatory cytokines: a brief review of the literature. nutrition reviews. 67, 213221. chacko, b.k., chandler, r.t., mundhekar, a., khoo, n., pruitt, h.m., kucik, d.f., parks, d.a., kevil, c.g., barnes, s., patel, r.p., 2005. revealing anti-inflammatory mechanisms of soy isoflavones by flow: modulation of leukocyte endothelial cell interactions. american journal of physiology. heart and circulatory physiology. 289, 908–915. de mejia, e.g., dia, v.p., 2009. lunasin and lunasin-like peptides inhibit inflammation through suppression of nf-kappab pathway in the macrophage. peptide. 30, 2388-2398. livak, k.j., schmittgen, t.d., 2001. analysis of relative gene expression data using real-time quantitative pcr and the 2 (delta delta c(t)) method. methods. 25, 402-408. marshall, o.j., 2004. perlprimer: cross-platform, graphical primer design for standard, bisulphite and real-time pcr. bioinformatics. 20, 2471-2472. martinez-villaluenga, c., dia v.p., berhow, m.a., bringe, n.a., gonzalez de mejia, e., 2009. protein hydrolysates from beta-conglycinin enriched soybean genetypes inhibit lipid accumulation and inflammation in vitro. molecular nutrition and food res. 53, 1007-1018. national research council (nrc), 1998. nutrient requirements of swine, 10 th revised edition. national academy press, washington, dc, usa. politis, i., theodorou, g., lampidonis, a.d., chronopoulou, r., baldi, a., 2012. soya protein hydrolysates modify the expression of various pro-inflammatory genes induced by fatty acids in ovine phagocytes. the british journal of nutrition. 108, 1246-1255. theodorou, g., daskalopoulou, m., chronopoulou, r., baldi, a., dell’orto, v., politis, i., 2010. acute mastitis induces upregulation of expression of plasminogen-activator genes by blood monocytes and neutrophils in dairy ewes. veterinary immunology and immunopathology. 138, 124-128. yang, y.x., kim, y.g., lohakare, j.d., yun, j.h., lee, j.k., kwon, m.s., park, j.i., choi, j.y., chae, b.j., 2007. comparative efficacy of different soy protein sources on growth performance, nutrient digestibility and intestinal morphology in weaned pigs. asian-australasian journal of animal sciences. 20, 775-783. young, d., ibuki, m., nakamori, t., fan, m., mine, y., 2012. soy-derived diand tri-peptides alleviate colon and ileum inflammation in pigs with dextran sodium sulfate-induced colitis. the journal of nutrition. 142, 363-368. yun, j.h., kwon, i.k., lohakare, j.d., choi, j.y., yong, j.s., zheng, j., cho, w.t., chae., b.j., 2005. comparative efficacy of plant and animal protein sources on the growth performance, nutrient digestibility, morphology and caecal microbiology of the earlyweaned pigs. asian-australasian journal of animal sciences. 18, 1285-1293. 15 l keywords feline; transfusion medicine; whole blood unit; packed red blood cell unit; ammonia; bacterial contamination. pages 15 – 23 references vol. 1 no. 2 (2014) article history submitted: september 23, 2013 revised: october 20, 2014 accepted: october 21, 2014 published: october 23, 2014 corresponding author eva spada, veterinary transfusion unit (rev) department of health, animal science and food safety (vespa) university of milan via celoria 10, 20133 milan, italy e-mail: eva.spada@unimi.it phone: +39 02 50318188 fax: +39 0250318171 journal home page riviste.unimi.it/index.php/haf ammonia concentration and bacterial evaluation of feline whole blood and packed red blood cell units stored for transfusion eva spada 1,* , daniela proverbio 1 , piera anna martino 2 , roberta perego, luciana baggiani 1 , nora roggero 1 1 veterinary transfusion unit (rev), department of health, animal science and food safety (vespa), university of milan, milan, italy 2 microbiology and immunology unit, department of veterinary science and public health (divet), university of milan, milan, italy abstract. ammonia concentrations increase in human, canine and equine whole blood (wb) and packed red blood cell (prbc) units during storage. the aim of this study was to determine the effect of storage on ammonia concentration in feline wb and prbc units stored in a veterinary blood bank and to evaluate possible correlations with bacterial contamination. ammonia concentration was evaluated in 15 wb units and 2 prbc units on day 1 and at the end of storage after 35 and 42 days, respectively. in an additional 5 wb units and 4 prbc units ammonia concentrations were determined daily until the day the normal reference range was exceeded and then weekly to the end of storage. all units were evaluated for bacterial contamination. ammonia increased markedly during storage as a linear function over time. on the 35th and 42th day of storage at 4±2°c (mean±sd) ammonia concentration reached 909±158 µg/dl and 1058±212 µg/dl in wb and prbc units, respectively. bacterial culture was negative in all units. high ammonia concentrations in stored wb and prbc units could result in toxicity, particularly in feline recipients with liver failure, portosystemic shunts or those receiving large transfusion volumes. clinical in vivo studies evaluating the effects on recipients should be performed. e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 16 haf © 2013 vol. i, no. 2 issn: 2283-3927 1 introduction in stored units containing rbcs as whole blood (wb) or packed red blood cell (prbc) units, deamination of adenine, and plasma and intra-erythrocytic protein leads to ammonia formation (conn, 1962). ammonia concentration has been shown to increase during storage of human (barta & babusikova, 1982), canine (waddell et al, 2001) and equine (mudge et al, 2004) wb, prbc and plasma units collected for transfusion purposes. the increased ammonia concentration may result in ammonia toxicity in the recipient of stored units, particularly in patients with liver failure, portosystemic shunts or in recipients receiving large transfusion volumes. ammonia concentration (ac) may increase because of contamination or deterioration of blood components during storage. ammonia is unstable in blood samples. in addition, after blood sample collection urea is exposed to air and begins to break down to ammonia (conn, 1966). certain aerobic bacteria such as escherichia coli, klebsiella, proteus, and pseudomonas spp. are known to be potent ammonia producers (washabau & day, 2013). in recent years feline transfusion medicine has become increasingly important with more cats receiving blood or blood components and more veterinary blood banks preparing and storing feline wb or prbc units worldwide for transfusion purposes. it is therefore useful to have information about the quality and characteristics of feline blood components. to the authors’ knowledge ac has not been evaluated in units of feline wb or prbc stored for transfusion purposes. the aim of this study was to investigate the effect of storage on ac in feline wb and prbc units prepared and stored for transfusion use, and to evaluate the influence of bacterial contamination on ac in wb and prbc units. our hypothesis was that ammonia concentration increases much more during storage in feline units containing rbcs than in the canine units due to the different blood collection method in cats (open system), that exposes blood to air during collection and different processing that increases the risk of bacterial contamination. 2 materials and methods 2.1 blood collection this prospective in vitro study was performed as internal quality control at the veterinary transfusion unit (rev) of university of milan. for this reason ethics approval by a specific committee is not required. informed owner consent was obtained to collect 10 ml/kg (with a maximum of 60 ml/cat) of blood from anesthetized (5 mg/kg tiletamine and zolazepam intramuscolarly, zoletil 100, virbac, milan, italy) healthy adult feline blood donors at the veterinary transfusion unit (rev) of university of milan. blood (10 ml/kg to a maximum volume of 60 ml) was collected, via a 19g butterfly needle, into 20-ml syringes containing citrate-phosphate-dextrose-adenine-1 (cpda-1) anticoagulant in a ratio cpda1 : blood of 1 : 7 (spada et al, 2014). e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 17 haf © 2013 vol. i, no. 2 issn: 2283-3927 cpda1 anticoagulant was taken from human sterile 450 ml blood bags (terumo cpda-1 single blood bag, terumo europe, leuven, belgium). 2.2 production and storage of wb and prbc units for wb units the anticoagulated blood was transferred to a 150 ml transfer bag (teruflex® transfer bag, terumo europe, leuven, belgium). prbc units were obtained after sedimentation of the rbcs in the three syringes, removal of plasma and transfer of rbcs into a 150 ml transfer bag filled with 10 ml of the nutritive additive solution contain saline, adenine, glucose and mannitol (sagm), taken from human sterile bags (terumo triple blood bag, terumo europe, leuven, belgium). blood units were stored in a controlled-temperature blood bank refrigerator (emoteca 250, fiocchetti & co, luzzara, italy), where the temperature was consistently maintained at 4±2°c. wb and prbc units were stored horizontally and mixed gently 2-3 times a week to maximize exposure of cells to the preservative solution. blood samples for all analyses were taken from segments attached to the blood bags. 2.3 ammonia concentration evaluation firstly, in order to confirm the hypothesis that ammonia increases in wb and prbc feline units at the end storage, ac was determined in 15 wb units (approx. 60 ml) and in 2 prbc units (approx. 40 ml) on the day of preparation (d1) and after 35 (d35) and 42 days of storage (d42), respectively. in a second study that aimed to verify the trend in ammonia increases, ac was measured in 5 new wb units and in 4 new prbc units on d1 (within 12 hours of donation) and daily until ac exceeded the feline (normal) reference range (100 µg/dl) (willard & twedt, 2012), and then weekly (at d7, d14, d21, d28) until d35 for wb units and d42 for prbc units. the products considered in the first and second part of the study were different and collected from different feline blood donors. the number of units tested depended on the units available during the study. ac was measured with a point-of-care portable analyzer (ammonia checker ii, menarini, florence, italy) based on a microdiffusion method. except for the first 15 wb units, all blood samples in which ac exceeded the upper limit of the assay (465 µg/dl) were diluted with bidistilled water and retested to obtain an ac end point value. internal validation of the point-ofcare portable analyzer for measurement of ammonia on feline whole blood was performed (data not shown). 2.4 bacterial evaluation to evaluate bacterial contamination microbiological analysis was performed on all wb and prbc units on d1, and on d35 and d42 of storage, respectively. from 200 to 500 microliters of blood samples were seeded aseptically in tryptic soy broth (oxoid, italy) with a ratio of 1:10. then tubes were incubated at 37 °c for 24 hours under aerobic atmosphere. after the e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 18 haf © 2013 vol. i, no. 2 issn: 2283-3927 incubation time, if the broth-culture was limpid (negative) it was incubated again at the same conditions for 24 hours (until 72 hours). otherwise, if the culture was positive (turbid), 100 microliters of the broth was plated onto blood-agar plates (oxoid, italy) and incubated at 37 °c for 24 hours under aerobic condition. then colonies, eventually grown on plates, were identified by macroscopic and microscopic evaluation (e.g. gram stain), biochemical tests and using selective media (e.g. macconkey agar for enterobacteriaceae, mannitol salt agar for staphylococcaceae). 2.5 statistical analysis results have been expressed as mean ± sd. data were tested for normality using the kolmogorov-smirnov test. in the first study on 15 wb units and 2 prbc units, mean acs was compared using a mann whitney rank sum test. in the second study (on 5 wb units and 4 prbc units), mean acs at each sampling point was compared using one way repeated measures anova for multiple comparisons. correlation and regression of ac and storage time were evaluated by pearson correlation and simple linear regression, respectively. mean ac values between wb and prbc units were compared using t-test. p<0.05 was considered statistically significant. data analysis was performed using statistical software (medcalc ® software, version 12.7.0, mariakerke, belgium). 3 results in the first study, in 15 evaluated wb units ac increased progressively from 99±78 µg/dl (range 28-251 µg/dl) on d1 to over 465 µg/dl on d35 in all units. acs, expressed as mean ± sd and range (min-max) in 5 wb units on different days of storage are reported in table 1. in these wb units ac exceeded the normal range on d1 in one unit, on d2 in three units and on d3 in one unit. there was a significant difference in the median ac measured every 7 days between d1 and d14 (p = 0.009), d21 (p = 0.004), d28 (p = 0.001), and d35 (p = 0.023), between d7 and d35 (p = 0.009), and between d14 and d28 (p=0.002), but not between other comparisons (p > 0.05). ac in wb units was highly correlated with storage time (r2 = 0.96, p < 0.0001, 95% ci 0.92 0.98) and increased over time in a linear fashion (y = 25x + 86, p < 0.0001). relative to the first 2 prbc units evaluated, ac increased from 87±2 µg/dl (range 86-89 µg/dl) on d1 to 898±210 µg/dl (range 750-1047 µg/dl) on d42 (p < 0.001). acs, mean ± sd and range in 4 prbc units on different days of storage are reported in table 2. in the prbc units considered in the second study, ac exceeded the normal range on d2 in three units and on d3 in one unit, and was not determined in two units. there was significant difference in the median ac measured every 7 days between d1 and d7 (p = 0.029), d14 (p = 0.002), d21 (p = 0.041), d28 (p = 0.007), d35 (p = 0.011), and d42 (p = 0.0002); between d7 and d14 (p = 0.015), d21 (p = 0.037), d28 (p = 0.002), d35 (p = 0.005), and d42 (p = 0.003); d14 and d28 (p = 0.018), and d42 (p = 0.001); d28 and d42 (p = 0.022), but not between other comparisons (p > 0.05). ac in prbc units was highly correlated with storage time (r2 = 0.93, p < 0.0001, 95% ci 0.86 0.97) and increased over time in a linear fashion (y = 25x + 98, p < 0.0001). e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 19 haf © 2013 vol. i, no. 2 issn: 2283-3927 table 1. mean ± standard deviation (sd), range (min-max) of ammonia concentration (µg/dl) in 5 feline wb units on different days of storage in a veterinary blood bank refrigerator days of storage (d) ammonia (µg/dl) mean ±sd range (min-max) d1 64 24 34-102 d2 121 30 77-158 d3 171 68 108-265 d7 296 135 177-288 d14 490 71 364-560 d21 532 67 471-613 d28 833 96 674-919 d35 909 158 738-1151 there was no significant difference (p > 0.05) between mean ac in wb and prbc at different points during storage (figure 1). no aerobic bacterial growth occurred in any wb and prbc unit analyzed on d1 and d35 and d42 of storage. table 2. mean ± standard deviation (sd), range (min-max) of ammonia concentration (ac, µg/dl) in 4 feline prbc units on different days of storage in a veterinary blood bank refrigerator. values at d1 and d42 were calculated considering also the 2 prbc units used in the first study. days of storage (d) ammonia (µg/dl) mean ±sd range (min-max) d1 66 23 35-89 d2 127 32 72-157 d7 243 41 202-293 d14 503 46 456-547 d21 665 234 421-872 d28 914 139 779-1068 d35 1018 65 959-1087 d42 1058 212 750-1412 e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 20 haf © 2013 vol. i, no. 2 issn: 2283-3927 4 discussion as in human (barta & babusikova, 1982), canine (waddell et al, 2001) and equine (mudge et al, 2004) blood and plasma units, the ac in stored feline wb and prbc units increased markedly during storage in this study. we hypothesized that ac in feline wb and prbc units collected and stored for transfusion purpose would increase much more than in canine blood units due to the open blood collection system used in cats. in an open system collection, blood is in contact to the air during collection and transfer. when urea is in contact with air after sample collection it begins to break down to ammonia. this breakdown is sufficient to elevate the ammonia concentration considerably resulting in spurious hyperammonemia (conn, 1966). open system collection may also predispose to bacterial contamination, and gram-negative enteric bacteria such as escherichia coli, klebsiella, proteus, and pseudomonas spp. are known to be potent ammonia producers (washabau & day, 2013). contrary to our hypothesis, the increase in ac in feline units was similar to that reported in canine prbc (barta & babusikova, 1982). no significant difference (p > 0.05 at t-test) was found between 4 canine prbc units stored in a similar controlled-temperature blood bank refrigerator in a previous study (barta & babusikova, 1982) and the mean values of feline prbc ac at d1, d7, d14, d21, d28 of our study. figure 1. mean ammonia concentrations (ac) in 5 feline wb units and 6 prbc units evaluated weekly during storage in a blood bank refrigerator. mean ac for prbc units was calculated using the two prbc units of the first study (d1 and d42) and the four prbc units of the second study (for the data on d1, d7, d14, d21, d28, d35 and d42). e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 21 haf © 2013 vol. i, no. 2 issn: 2283-3927 open system collection does not seem to predispose to bacterial contamination since no bacterial growth was achieved in any of the 26 units tested, as previously documented in feline units collected with an open system at the university of berlin (weingart et al, 2004), where blood products have been stored successfully without microbial growth if blood banking is done by experienced staff as in our study. high acs in our study do not appear to be the result of microbiological contamination with ammonia producing bacteria, since aerobic cultures were negative in all 20 feline wb and 6 prbc units analyzed on day of collection and on the expiration day of the units. ac may increase because of deamination of proteins such as glutamine, breakdown of adenyl pyrophosphate and/or adenylic acid, or hydrolysis of other ammoniogenic substances (conway & cooke, 1939). adenine, which is added to prbc in the red cell extender sagm, and in cpda anticoagulant as used in our study, may be the major substrate for in vitro ammonia formation in the feline wb and prbc units analyzed. the slightly greater increase in ac in our feline prbc units (that contain more adenine, which is present either in cpda-1 anticoagulant than in the nutritive solution sagm) compared to that in wb units (which contain only cpda-1) supports the theory that adenine breakdown could be a major factor in increased acs in stored blood bags. toxic ammonia levels are not known in cats (willard & twedt, 2012). in the feline wb units analyzed ac rapidly exceeded the normal feline range (within 3 days of storage) in all units evaluated. in this study the highest ac measured in a wb unit was 1151 µg/dl (d35) and 1412 µg/dl in a prbc unit (d42). assuming no metabolism or distribution from the intravascular space and a feline blood volume of 62 to 66 ml/kg (median 64 ml/kg) (wellman et al, 2012), the plasma ac in a 4 kg cat receiving one wb unit (approx. 60 ml) with an ac of 1151 µg/dl or one prbc unit (approx. 40 ml) with an ac of 1412 µg/dl would be expected to increase by 270 µg/dl and 220 µg/dl, respectively. samples for ac evaluation and for bacterial contamination in this study were taken from segments rather than the blood bags itself then that could represent a different microenvironment as demonstrated in a recent human study with different results for hematocrit, hemoglobin content, hemolysis, hematologic indices, and adenosine triphosphate concentration in samples taken from the bags and from the segments (kurach et al, 2014). the bags are gas permeable whereas the segment line is made of a different material. more importantly, if few bacteria made it into a blood unit then they are more likely to be in the bag rather than the line and so lack of contamination in the segment does not indicate lack of bag contamination. however, nondestructive testing offers several advantages, including the possibility of continuing storage of the units after testing without a shortened expiry time. this is a potential limitation of the current study and should be addressed in future studies. others limitations of this study were that anaerobic cultures were not performed although some anaerobes can synthesize ammonia. the accuracy of the ammonia analyzer used in this study has only been evaluated in dogs, although in this species results correlated well with the results from the standard enzymatic method in all ac ranges (sterczer et al, 1999). however, internal validation in our laboratory (data not shown) confirmed that the analyzer was also accurate in feline samples. finally, in our study blood units were stored in a blood-banking refrigerator with a temperature recorder and the refrigerator was not opened more than twice a day for the duration of storage. temperature has been demonstrated to be one of the most important factors affecting the increase in ac in stored blood bags (conn, 1962; waddell e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 22 haf © 2013 vol. i, no. 2 issn: 2283-3927 et al, 2001). this may be important since, in most veterinary private practices, temperature control may not be closely regulated during blood storage. it is possible that temperature fluctuations will be more significant in a small private veterinary practice refrigerator leading to higher ac in stored units. 5 conclusions in conclusion, this is the first study that shows that ammonia increases markedly, and linearly, with time of storage in feline wb and prbc units stored for transfusion purpose. there is a risk of ammonia toxicity in the recipient of stored units, particularly in feline recipients with liver failure, portosystemic shunts or those receiving large transfusion volumes. however, further clinical in vivo studies evaluating the effects on recipients need to be performed. 6 funding acknowledgment this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors 7 conflict of interest all the authors declare no conflicts of interest. references barta e., babusikova f., 1982. the concentration of ammonia in blood and plasma stored for transfusion. resuscitation. 10, 135-139. conn h.o., 1962. studies on the origin and significance of blood ammonia. i. effect of various anticoagulants on the blood ammonia determination. yale journal of biology and medicine. 35, 171-184. conn h.o., 1966. studies on the origin and significance of blood ammonia. ii. the distribution of ammonia in whole blood, plasma and erythrocytes of man. yale journal of biology and medicine. 39, 38-53. conway e.j., cooke r., 1939. blood ammonia. biochemical journal. 33, 457-478. e. spada et al. int. j. of health, animal science and food safety 1 (2014) 15-23 23 haf © 2013 vol. i, no. 2 issn: 2283-3927 kurach j.d.r., hansen a.l., turner t.l., jenkins c., acker j.p., 2014. segments from red cell units should not be used for quality testing. transfusion. 54, 451-455. mudge m.c., macdonald m.h., owens s.d., tablin f., 2004. comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation. veterinary surgery. 33, 475-486. spada e., proverbio d., bagnagatti de giorgi g., perego r., valena e., della pepa a., baggiani l., 2014. clinical and haematological responses of feline blood donors anaesthetised with a tiletamine and zolazepam combination. journal of feline medicine and surgery. published online before print july 11, 2014, doi: 10.1177/1098612x14542452. sterczer a., meyer h.p., boswuk h.c., rothuizen j., 1999. evaluation of ammonia measurements in dogs with two analysers for use in veterinary practice. the veterinary record. 144, 523-526. waddell l.s., holt d.e., hughes d., giger u., 2001. the effect of storage on ammonia concentration in canine packed red blood cells. journal of veterinary emergency and critical care. 11, 23-26. washabau r.j., day m.j., 2013. canine and feline gastroenterology. the liver. st louis, mo: elsevier saunders. 849-957. weingart c., giger u., kohn b., 2004. whole blood transfusion in 91 cats: a clinical evaluation. journal of feline medicine and surgery. 6, 139-148. wellman m.l., dibartola s.p., kohn c.w., 2012. applied physiology of body fluids in dogs and cats. in dibartola s.p. ed. fluid, electrolyte, and acid-base disorders in small animal practice. st. louis, mo: elsevier saunders. 2-25. willard m.d., twedt d.c., 2012. gastrointestinal, pancreatic and hepatic disorders. in willard m.d., tvedten h. eds. small animal clinical diagnosis by laboratory methods. st. louis, mo: elsevier saunders. 191-225. proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l the in vitro development of vitrified oocytes (vos) is still suboptimal (mandawala et al., 2016) and the traditional two-dimensional (2d) culture systems might not be adequate to fully exploit vos potential. the use of three-dimensional (3d) follicle-like structures, i.e. a combination of granulosa cells (gcs) and semipermeable 3d matrices, could mimic the physiological microenvironment and enhance vos maturation and embryo development. the aim of this study was to assess the steroidogenic ability (estradiol and progesterone secretion) of gcs encapsulated in 3d barium alginate microcapsules (follicle-like structure) compared to gcs cultured in a 2d monolayer and the maturation outcomes of vos cultured in these systems. after purification (simsek & arikan, 2015), cat gcs retrieved by mincing from isolated ovaries were in vitro cultured for 6 days in 3d microcapsules (vigo et al., 2005) or in 2d monolayers. on days 2 and 6, conditioned medium was collected and hormonal determination by enzyme-linked fluorescent assay was performed. on the same days, 3d (fig. 1) and 2d cultured gcs were used as artificial milieu for in vitro maturation of vos obtained by cryotop protocol. nuclear maturation was assessed by bis-benzimide staining. steroidogenesis was observed in 3d follicle-like structures as well as in 2d monolayers; hormonal concentration increased over time and on day 6 it was significantly higher in monolayers (p=0.02). vitrified oocytes resumed meiosis in presence of gcs cultured for 2 days (3d: 45.5%; 2d: 56.7%), while vos meiosis progression in gcs monolayers cultured for 6 days was significantly lower (21.7%, p=0.007); by contrast, 3d follicle-like structures cultured for 6 days better supported vos full maturation (26.7%, p=0.07). granulosa cells in 3d microcapsules maintained their physiological features and these folliclelike structures were able to restore vos developmental abilities. however, further advancements in vos culture methods would optimize the use of these valuable resources. keywords domestic cat, vitrification, oocytes, 3d culture, granulosa cells. corresponding author martina colombo martina.colombo@unimi.it journal home page riviste.unimi.it/index.php/haf follicle-like environment for domestic cat vitrified oocytes. m. colombo1,*, m.g. morselli1, g.c. luvoni1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 16 haf © 2013 vol. v, no. 1s issn: 2283-3927 references mandawala, a. a., harvey, s. c., roy, t. k., & fowler, k. e., 2016. cryopreservation of animal oocytes and embryos: current progress and future prospects. theriogenology. 86, 1637-1644. simsek, o., & arikan, s., 2015. effects of cholesterol, fsh and lh on steroidogenic activity of cat granulosa cells cultured in vitro. animal reproduction. 12, 931-938. vigo, d., villani, s., faustini, m., accorsi, p. a., galeati, g., spinaci, m., munari, e., russo, v., asti, a., conte, u., & torre, m. l., 2005. follicle-like model by granulosa cell encapsulation in a barium alginate–protamine membrane. tissue engineering. 11, 709-714. figure 1: domestic cat vitrified oocytes cultured in a three-dimensional barium alginate microcapsule containing conspecific granulosa cells. scale bar 100 µm. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2016, 8th10th june, milan, italy haf © 2013 vol. iii, no. 1s issn: 2283-3927 l keywords mammary gland, mastitis, mrna expression, cytokine, innate immune response, ex vivo corresponding author giada magro giada.magro@unimi.it journal home page riviste.unimi.it/index.php/haf an ex vivo model of heifer udder to study the innate immune response to bacterial infections. g. magroa*, j. filipea, f. rivaa, r. piccininia adepartment of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy abstract mastitis is the major concern for the dairy industry. intramammary infections cause a complex signaling network that activates the innate immune response, leading to inflammation symptoms. several types of cells, including endothelial cells, epithelial cells, resident macrophages and other leucocytes, are involved in the immune response. therefore we aimed to set up an ex vivo model of the mammary gland, where the three-dimensional structure is maintained, assuming that the results were more comparable to the in vivo response. two mm3 -sections were taken from the mammary gland of a heifer after slaughter and then incubated with either s. aureus lipoteichoic acid (lta) or with e. coli lipopolysaccharide (lps) for 1, 3, 6, and 18h. these molecules are constituent of the cell walls of grampositive or -negative bacteria, respectively, and are applied as an inflammatory stimulus in the research of mastitis pathogenesis. quantitative real-time pcr was applied to quantify the mrna expression of tumour necrosis factor (tnf-α), interleukin (il)-1β, il-6, il-8 (griesbeck-zilch 2008), pentraxin 3 (ptx3; lutzow, 2008), lingual antimicrobial peptide (lap) (günther, 2010;) and of interleukin-1 receptor 8 (il1r8; riva, 2012) and toll-like receptor 4 (tlr4; ibeagha-awemu, 2008). these molecules have been chosen as key factors of the innate immune response. preliminary results showed that in lps-treated cells, cytokine mrna expression increased between 1-3h, while tlr4, ptx3 and il1-r8 peaked at 3h and then decreased. lap displayed a different pattern, with the highest values at 3h, slightly increasing up to 18h observation. these data suggest that such ex vivo model could be a valid approach to study the mammary immune response to bacteria. references günther, j., liu, s., esch, k., schuberth, h. j., seyfert, h. m., 2010. stimulated expression of tnf-a and il-8, but not of lingual antimicrobial peptide reflects the concentration of pathogens contacting bovine mammary epithelial cells. vet. immunol. immunopathol. 135, 152–157. griesbeck-zilch, b., meyer, h.h.d., kuhn, c., schwerin, m., wellnitz, o., 2008. staphylococcus aureus and escherichia coli cause deviat ing expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells. j. dairy sci. 91, 2215–2224. ibeagha-awemu, e.m., lee, j.w., ibeagha, a.e., bannerman, d.d., paape, m.j., zhao, x., 2008. bacterial lipopolysaccharide induces increased expression of toll-like receptor (tlr) 4 and downstream tlr signaling molecules in bovine mammary epithelial cells. vet. res. 39, 2-11. lutzow, y.c.s., donaldson, l., gray, c.p., vuocolo, t., pearson, r.d., reverter, a., byrne, k.a., sheehy, p.a., windon, r., and tellam, r.l.,. 2008. identification of immune genes and proteins involved in the response of bovine mammary tissue to staphylococcus aureus infection. bmc vet. res. 4, 18. riva, f., bonavita, e., barbati, e., muzio, m., mantovani, a., garlanda, c., 2012. tir8/sigirr is an interleukin-1 receptor/toll like receptor family member with regulatory functions in inflammation and immunity. front. immunol. 3, 322. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l micrornas (mirnas) are a class of short non-coding rna, which interact with the 3’ utr region of complementary mrna to decrease or inhibit the translation of proteins (lai, 2002). mirnas regulate pathways in various pathophysiological status, and are regarded as biomarkers for early diagnosis of several diseases, including cancer (di leva et al., 2014). the study aims to evaluate the quality and purity of mirnas extracted from a) 11 archival formalin fixed and paraffin embedded (ffpe) samples of mast cell tumour (mct) at stage i, ii, iii and iv, and 8 intra-patient healthy controls; b) samples collected during surgery, including 6 samples of saliva, primary tumour biopsy and serum/plasma. the quality of mirna largely influence the downstream experiments, and must be carefully evaluated before performing for examples, the sequencing reaction. mirna extraction was carried out using commercial kits (qiagen) and quantify using small rna kit (agilent) on agilent 2100 bioanalyzer (figure 1). the results showed that the concentration of mirnas from ffpe, saliva, primary tumor biopsy and serum was acceptable with a median (me)= 56,91 ng/µl, me=10,30 ng/µl, me=3,44 ng/µl and me=0,71 ng/µl, and a mirna/small rna ratio of 48%, 61%, 17% and 76%, respectively. the concentration of mirnas from plasma was not detectable. studies reveal that plasma ranks as the first choice source for diagnostic purpose, much more than serum (aung et al., 2014), but the debate remains open and subsequent analyses are needed. the concentration of mirnas from ffpe and saliva samples is higher than that from other matrices. possible explanations include a) different quantity and quality of starting materials; b) nucleic acids fragmentation, due to the formalin fixation and paraffin embedded procedure; c) presence of nucleases in saliva, which produce small fragments recognized as mirnas or smallrnas. in conclusion, the quantity and the purity of mirnas, obtained using qiagen commercial kits, are reliable for further ngs analysis. keywords mirna extraction, veterinary oncology, mirna quantification, mast cell tumours, dogs. corresponding author valentina zamarian valentina.zamarian@unimi.it journal home page riviste.unimi.it/index.php/haf evaluation of quantity and purity of mirnas extracted from different matrices collected from dogs with mast cell tumours. v. zamarian1,*, c. lecchi1, f.ceciliani1, v. grieco1, d. stefanello1, r. ferrari1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 20 haf © 2013 vol. v, no. 1s issn: 2283-3927 references aung, k.l., donald, e., ellison, g., bujac, s., fletcher, l., cantarini, m., brady, g., orr, m., clack, g., ranson, m., dive, c., hughes, a., 2014. analytical validation of braf mutation testing from circulating free dna using the amplification refractory mutation testing system. the journal of molecular diagnostics. 16(3), 343-349. di leva, g., garofalo, m., croce, c.m., 2014. micrornas in cancer. annual review of pathology. 9, 287-314. lai, e.c., 2002. micro rnas are complementary to 3' utr sequence motifs that mediate negative post-transcriptional regulation. nature genetics. 30(4), 363-364. figure 1: example of electropherogram of a serum sample obtained from agilent 2100 bioanalyzer. it shows the number of nucleotides (x axis) and the fluorescence signal (y axis). the higher peak on the left corresponding to the marker, instead the others peaks represent the mirna (10-40 nucleotides region) and small rna (0-150 nucleotides region) present into the sample. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l harmonized data collection is essential to obtain a reliable picture of equine welfare conditions. effective education on how to assess and score welfare indicators plays a critical role in terms of inter-observer reliability. the horse grimace scale (hgs), a facial-expression-based pain coding system, is able to identify a range of acute pain conditions in horses (dalla costa et al., 2014, 2016; lecchi et al.,2018). this study aimed at evaluating the efficacy of a standardized training on hgs inter-observer reliability. students in veterinary medicine from the university of milan (n=46, second year course) and the university of teramo (n=31, fourth year course) were recruited. prior to any training, students were asked to score 10 pictures of horse faces using the six facial action units (faus) of the hgs: stiffly backwards ears, orbital tightening, tension above the eye area, prominent strained chewing muscles, mouth strained, strained nostrils. then, a 30-min training session was provided, including detailed descriptions and example pictures of each fau, as well as a discussion of five pictures previously scored by an experienced assessor. after training, students scored other 10 pictures. to determine the inter-observer reliability pre and post-training, intraclass correlation coefficient (icc) was used. students’ reliability was good even before training (icc=0,986 for the overall hgs score), with tension above the eye area, and strained nostrils appearing more challenging to be scored reliably. previous researches demonstrated that these faus are generally more difficult to score than the others (dalla costa et al., 2016). reliability improved after the 30 min training for the overall hgs score (icc=0,992) and for each fau (see table 1). according to cicchetti (1994), an icc score between 0.75 and 1.00 can be considered excellent. keywords horse, horse grimace scale, training, welfare, pain assessment. corresponding author francesca dai francesca.dai@unimi.it journal home page riviste.unimi.it/index.php/haf efficacy of a standardized training on horse welfare indicators: a preliminary study. f. dai1,*, e. d. costa1, m. minero1 1 department of veterinary medicine, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 10 haf © 2013 vol. v, no. 1s issn: 2283-3927 our results suggest that the hgs scoring system is easy to apply even without any training; however, the training method applied proved useful to improve the reliability of hgs scores. references cicchetti, d.v., 1994. guidelines, criteria, and rules of thumb for evaluating normed and standardized assessment instruments in psychology. psychological assessment. 6(4), 284–290. doi:10.1037/1040-3590.6.4.284. dalla costa, e., minero, m., lebelt, d., stucke, d., canali, e., leach, m.c., 2014. development of the horse grimace scale (hgs) as a pain assessment tool in horses undergoing routine castration. plos one. 9. dalla costa, e., stucke, d., dai, f., minero, m., leach, m.c., lebelt, d., 2016. using the horse grimace scale (hgs) to assess pain associated with acute laminitis in horses (equus caballus). animals. 3, 6(8). lecchi, c., dalla costa, e., lebelt, d., ferrante, v., canali, e., ceciliani, f., stucke, d., minero, m., 2018. circulating mir23b-3p, mir-145-5p and mir-200b-3p are potential biomarkers to monitor acute pain associated with laminitis in horses. animal. 12, 366–375. table 1: icc scores preand post-training for each facial action unit and total hgs score. facial action unit icc pre-training icc post-training stiffly backwards ears 0,993 0,999 orbital tightening 0,960 0,974 tension above the eye area 0,878 0,979 prominent strained chewing muscles 0,950 0,963 mouth strained 0,926 0,980 strained nostrils 0,851 0,918 tot hgs score 0,986 0,992 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l fipronil and amitraz are broadly used insecticides for the treatment or prevention for animal health, indoor pest control, and commercial crop protection. as the use of fipronil or amitraz on food-producing animals was not allowed by the eu legislation, the maximum residue limit (mrl) values of fipronil and amitraz were set at the detection limit of 5 ng ml-1 and 10 ng ml-1, respectively. according to the database of rapid alert system for food and feed (rasff), after the belgian authority reported fipronil residues in chicken eggs in 2017, there were 719 followup reports from 34 countries. fipronil and amitraz are included in the italian national residue program so it is necessary to develop a selective, sensitive, specific and rapid method. three extraction methods were evaluated on fresh egg blank samples to determine the presence of fipronil, as well as its metabolites and amitraz. in the solvent-salt method the sample was added by water, nacl and formic acetonitrile, followed by hexane to remove potential fat. in the quick, easy, cheap, effective, rugged, and safe (quechers) method the sample was extracted by superl® que citrate powder and acetonitrile, followed by superl® psa powder. in the waterassociated quechers method the sample was mixed with water and acetonitrile, followed by superl® que citrate powder, then the supernatant was collected and mixed with cacl2. the analyses of the extracts were performed with high performance liquid chromatography coupled to q-exactive orbitrap high-resolution mass spectrometer (lc-hrms). furthermore, thompson (2000) mentioned that the coefficient of variation(cv) is acceptable if it is lower than 22%. based on the obtained recovery values (72 to 113%) and cv (1.67 to 14.69&), the water-associated quechers method was selected because the recoveries rates obtained with the other methods were lower than 70%. calibration curves exhibited correlation values ranging from 0.9653 to 0.9999(figure 1); the limits of detection ranged from 0.08 to 1.21 ng ml-1, and the limits of quantification were from 0.28 to 4.04 ng ml-1. the preliminary results fulfilled the european criteria for the validation of the analytical methods. further analyses have been performed to evaluate the repeatability and reproducibility. keywords fipronil, amitraz, quechers, lchrms. corresponding author shih-kuo lin shihkuo.lin@unimi.it journal home page riviste.unimi.it/index.php/haf preliminary evaluation results of the extraction methods for fipronil and its metabolites and amitraz in chicken eggs. s.k. lin1,*, l.m. chiesa1, e. pasquale1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133 milan, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 28 haf © 2013 vol. v, no. 1s issn: 2283-3927 references european commission, 2002. 2002/657/ec: commission decision of 12 august 2002 implementing council directive 96/23/ec concerning the performance of analytical methods and the interpretation of results. official journal of the european communities. 221, 8-36. european commission, 2005. regulation (ec) no 396/2005 of the european parliament and of the council of 23 february 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending council directive 91/414/eec text with eea relevance. official journal of the european union. 70, 116. european commission, rapid alert system for food and feed (rasff), available: https://ec.europa.eu/food/safety /rasff_en [accessed 27 april 2018]. gupta, c. r. 2007. amitraz. in: gupta, c. r. veterinary toxicology. 3rd edition 2018. new york: elsevier; p514-517. poppenga, h. r. and oehme, w.f. 2010. pesticide use and associated morbidity and mortality in veterinary medicine. in: krieger, r. hayes' handbook of pesticide toxicology (third edition). london: elsevier; p285-301. thompson m. 2000. recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing. analyst. 125(3), 385-386. figure 1: the calibration curve of fipronil. the figure plots 5 concentrations (0.5, 1, 2, 5 and 15 ng g-1) in x-axis and instrument response (peak area value) in y-axis, respectively. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en https://ec.europa.eu/food/safety proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l copy number variations (cnvs) have become promising markers, representing a major source of genomic variation. cnv involvement in phenotypic expression and in different diseases onset have been widely demonstrated in humans as well as in many domestic animals. however, this genomic investigation is still missing in felis catus. this work is the first cnv mapping from a large data set of whole genome sequencing (wgs) data in the domestic cat. a total of 42 cats of 14 different breeds were sequenced on the illumina xten (washington university-st. louis) which generated approximately 30-fold genome coverage from 150 pairedend reads (99 lives initiative). maverix biomics mapped the reads on the v6.2 reference assembly. cnv detection was performed using cn.mops and cnvnator, two read depth method software. one cat was excluded as outlier while, on the 41 remaining individuals, 1640 cnvs were detected by both the software and used to obtain 2891 cnvrs with bedtools. cnvrs covered the 0.4% of the total cat genome, with 136 loss, 127 gain and 26 complex detected (figure 1). a total of 164 singletons were identified and 9 cnvrs mapped in at least the 50% of the individuals. the number of cnvs in each cat ranged from 12 to 83. the clustering analysis of the detected cnvs was performed with r package “pvclust” and shows that same breed individuals cluster together. this study has led to the genetic characterization of 14 main cat breeds. further analyses including other breeds and considering the genes located within these regions, could lead to better evaluate the relationship between the presence of a specific cnv and a specific breed trait. this study can be considered a starting point for genomic cnv identification in the domestic cat, which could be further developed using the new released felis catus vs9.0 reference aassembly. keywords cnv, domestic cat, genomic variability, whole genome sequencing, structural variants. corresponding author francesca genova francesca.genova@unimi.it journal home page riviste.unimi.it/index.php/haf genome-wide copy number variants mapping in felis catus using next generation sequencing data. f. genova1*, m. longeri1, c.m. cozzi1, a. bagnato1, m.g. strillacci1 and the 99lives consortium 1 department of veterinary medicine, università degli studi di milano, via celoria 10, milano, italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 24 haf © 2013 vol. v, no. 1s issn: 2283-3927 references henrichsen, c.n., chaignat, e., reymond, a., 2009. copy number variants, diseases and gene expression. hum mol genet. 18(r1): r1-8. clop, a., vidal, o., amills, m., 2012. copy number variation in the genomes of domestic animals. anim genet. 43(5): 503-17. klambauer, g., schwarzbauer, k., mayr, a., clevert, d.a., mitterecker, a., bodenhofer, u., hochreiter, s., 2012. cn.mops: mixture of poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate. nucleic acid res. 40(9): e69. abyzov, a., urban, a.e., snyder, m., gerstein, m., 2011. cnvnator: an approach to discover, genotype, and characterize typical and atypical cnvs from family and population genome sequencing. genome res. 21(6): 974-84. suzuki, r., shimodaira, h., 2006. pvclust: an r package for assessing the uncertainty in hierarchical clustering. bioinformatics. 22(12): 1540-2. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 0 15000000 30000000 45000000 60000000 75000000 90000000 105000000 120000000 135000000 150000000 165000000 180000000 195000000 210000000 225000000 240000000 f2 f1 e3 e2 e1 d4 d3 d2 d1 c2 c1 b4 b3 b2 b1 a3 a2 a1 figure 1: felis catus cnvrs map. the 6.2 assembly is represented as grey bars. gains, losses and complexes are shown as dots with different color http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy haf © 2013 vol. v, no. 1s issn: 2283-3927 l menisci are wedge-like structures interposed, in the knee joint, between the femoral and the tibial articular heads (kohn et al., 1995, greis et al., 2002). improving the articular surface, the cellular nutrition and the articular lubrication, they are essential structures for the prevention of gonarthrosis (proctor et al., 1989, makris et al., 2011). this study is focused upon the relationship between the contact forces at the femoral and tibial surfaces and the corresponding structure of these meniscal surfaces. for this purpose, 20 adult (~9 months old) female pigs (landrace x large white, average weight 75–90 kg; n=80 meniscal samples) were obtained from a local slaughterhouse and dissected to isolate the menisci. swine meniscal samples were evaluated from morphological (safranin-o, sirius red and collagen type i and ii) (di giancamillo et al. 2014), biochemical (dna and glycosaminoglycans, or gags, contents) and biomechanical (compression and traction tests) points of view at the level of femoral and tibial meniscal surfaces. results revealed a characterization of the meniscus which is biomechanicaldependent. the femoral surface, morphologically characterized by the interposition of radial and oblique fibers and biomechanically by the femoral condyles compression, sliding and rolling forces, shows a higher compressive modulus (p<0.05) and a greater amount of cells and gags deposition (p<0.01 for each analysis). on the other hand, results from traction test revealed a higher tensile modulus (p<0.05) in the tibial surface, characterized by a circumferential arrangement of the fibers and a poorer gags deposition and cellular distribution (p<0.01). results (summarized in the figure 1) from this work suggest that a biphasic “femoral-to-tibial” scaffold that mimic the different behavior and composition of the two meniscal surfaces could be useful in the light of meniscal replacement. in both animal models, immunohistochemistry revealed stromal immunoreactivity for timp-1 and thbs-2, while mmp-7 expression was mainly localized on epithelial cells. all the markers showed progressive increase of staining intensity along with panin progression. keywords menisci, meniscus, swine model, biomechanics, scaffolds. corresponding author umberto polito umberto.polito@unimi.it journal home page riviste.unimi.it/index.php/haf meniscal femoral and tibial surfaces characterization in the swine model. u. polito1,*, m. di giancamillo2, g.m. peretti3,4, m.e. andreis1, s.c modina1, f. boschetti3,5, a. di giancamillo1 1 department of health, animal science and food safety, università degli studi di milano, via celoria 10 -20133, milan, italy. 2 department of veterinary medicine, university of milan, via celoria 10 20133, milan, italy. 3 irccs, istituto ortopedico galeazzi, milan, via galeazzi, 4, milan, italy. 4 department of biomedical sciences for health, università degli studi di milano, ia carlo pascal, 36, 20133, milan, italy. 5 department of chemistry, material and chemical engineering department "giulio natta", politecnico di milano, piazza leonardo da vinci, 32, 20133, milan, italy.. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2018, 6th8th june, milan, italy 32 haf © 2013 vol. v, no. 1s issn: 2283-3927 in conclusion, the investigated circulating molecules represent promising biomarkers for early diagnosis of pdac and to monitor the response to treatment in human patients. both tumoral cells and associated stroma play a role in the production and release of such biomarkers which, in addition, represent also biologically relevant molecules in remodeling the tumor microenvironment, by a complex network of interacting molecules. references di giancamillo, a., deponti, d., addis, a., domeneghini, c. and peretti, g.m., 2014. meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth. journal of cellular and molecular medicine. journal of cellular and molecular medicine. 10: 1964-1974. greis, p.e., bardana, d.d., holmstrom, m.c., and burks, r.t. 2002. meniscal injury: i. basic science and evaluation. journal of american academy of orthopaedic surgery. 10(3):168-76. kohn, d., and moreno, b., 1995. meniscus insertion anatomy as a basis for meniscus replacement: a morphological cadaveric study. arthroscopy. 11(1):96-103. makris, e.a., hadidi, p., and athanasiou, k.a. 2011. the knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. biomaterial. 32(30): 7411–7431. proctor, c.s., schmidt, m.b., whipple, r.r., kelly, m.a., and mow, v.c. 1989. material properties of the normal medial bovine meniscus. journal of orthopaedic research. 7(6):771-82. figure 1: summarized results: femoral vs tibial meniscal surfaces. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords lymphoma, predisposition, breed, canine, european corresponding author marzia cozzi marzia.cozzi@unimi.it journal home page riviste.unimi.it/index.php/haf abstract canine breeds, being genetic clusters, are good models for studies on genetic predisposition. golden retriever (gr) has been described with a high incidence of both lymphoma overall (19%) and t zone lymphoma (tzl, 40%) with differences in different geographical areas in us. this breed predisposition is confirmed in japanese but not in european (eu) case series although specific studies are still lacking (modiano et al., 2005; seelig et al., 2014). aim of the present study is to investigate the prevalence of gr in a huge case series of canine lymphomas from different eu countries and to compare prevalence of different subtypes with studies in extra-eu countries, in order to support a possible different genetic predisposition. signalment data on 1734 consecutive cases of canine lymphoma collected from 9 different european countries are retrospectively analysed. when subtypes are available, cases are furtherly separated in three subtype groups: 1) b-cell lymphoma, 2) t-cell lymphoma-high grade, 3) tzl. odds ratio (or) for different lymphoma subtypes are calculated in comparison with mixed breed population, considered as control. overall prevalence of gr is 5.19% (range 1.59-7.32%) of lymphoma cases and differs from that reported in american and japanese caseloads. prevalence slightly varies among eu countries and no subtypes predilection is found if compared with mixed breed. concerning italian cohort, gr is not predisposed to develop a lymphoma when normalized for the breed prevalence (or=1.49, 95% confidence interval=0.87-2.55, p=0.14). prevalence of lymphoma in eu population of gr is much lower than that of us. no predisposition is identified in eu gr for tzl differently from us and japan. being genetic of european gr population quite different from american and japanese ones this suggest a possible different genetic predisposition. slight differences in gr lymphoma prevalence among european countries likely reflects different breed distribution rather than different genetic predisposition. references modiano et al, 2005. distinct b-cell and t-cell lymphoproliferative disease prevalence among dog breeds indicates heritable risk. cancer research. jul 1;65(13):5654-61 seelig et al, 2014. canine t-zone lymphoma: unique immunophenotypic features, outcome, and population characteristics. journal of veterinary internal medicine. may-jun;28(3):878-86 prevalence of golden retriever in european dogs with lymphoma: preliminary data marzia cozzi1*, stefano comazzi1, stefano marelli1, rita rizzi1 on behalf of the european canine lymphoma network 1university of milan, department of veterinary medicine, italy http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords hygienic behavior, odorant binding protein, antennae, gene expression, rt-pcr corresponding author elena facchini elena.facchini@unimi.it journal home page riviste.unimi.it/index.php/haf expression monitoring of relevant sensitivity genes in honey bee antennae and their relationship with hygienic behavior elena facchini1*, francesca dell’orco1, michele mortarino1, rita rizzi1 1university of milan, department of veterinary medicine, italy abstract honeybees are very important microlivestock, not only for the economic value of their productions but also for the crucial role they fulfill as pollinators. recently, colony losses have been recorded throughout europe and the reasons underpinning such phenomenon can be addressed to agrochemicals and pathogens. among the latter, the parasitic mite varroa destructor is considered the principal mortality cause. hygienic behavior (hb) in honeybee (apis mellifera) involves the detection and removal of brood affected by bacterial, fungal diseases, and parasitization. this behavior is part of a series of strategies evolved by social insects known as social immunity, which confers disease resistance thanks to the persistent elimination of pathogens and parasites from the hive, limiting their multiplication and the infection of other bees. it has previously reported that physiological changes in peripheral sense tissues of insects influence the behavioral state of individuals, and it has been suggested that changes in gene expression at antennal level can contribute to shifts in the behavioral states of honeybees (vergoz et al., 2009). moreover, it has previously reported that antennae hold a key role in the process of recognition of abnormalities in the brood by honeybees (mondet et al., 2015). the aim of the work was to investigate the expression level of selected genes (obp3, obp4, obp16, obp18, act5c, mblk-1) through realtime-pcr in honeybee antennae. these targets are reported as potential biomarkers of hb in previous transcriptomic and proteomic studies (mondet et al., 2015; guarna et al., 2015). hygienic behavior has been measured in the field through optimized freeze-killedbrood method on 10 colonies, from which workers of known age (15 days old) have been collected for molecular analysis (arathi et al., 2000). preliminary results show that obp3 is the least expressed among the tested genes, but its expression pattern is linked to the hb value; in particular, highly hygienic colonies express obp3 significantly at higher rate with respect to the lower hb group of colonies. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 references guarna m. m., a. p. melathoupolous, e. huxter, i. iovinella, r. parker, n. stoynov, a. tam, k. moon, q. w. t. chan, p. pelosi, r. white, s. f. pernal, l. j. foster. 2015. a search for protein biomarkers links olfactory signal transduction to social immunity. bmc genomics. 16, 63. mondet, f., c. alaux, d. severac, m. rohmer, a. r. mercer, y. le conte. 2015. antennae hold a key role to varroasensitive hygiene behaviour in honey bees. scientific resports. 5, 10454. arathi, h. s., i. burns, m. spivak. 2000. ethology of hygienic behavior in the honey bee apis mellifera l. (hymenoptera: apidae): behavioural repertoire of hygienic bees. ethology. 106, 365-379. vergoz, v., h. j. mcquillan, l. h. geddes, k. pullar, b. j. nicholson, m. g. paulin, a. r. mercer. 2009. peripheral modulation of worker bee responses to queen mandibular pheromone. pnas. 106(49), 20930-20935. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 l keywords cell culture, three-dimensional, tissue, model, assay corresponding author stefan przyborski stefan.przyborski@durham.ac.uk journal home page riviste.unimi.it/index.php/haf advanced cell culture technology for generation of in vivo-like tissue models stefan przyborski1,2 1durham university, department of biosciences, durham, united kingdom 2reprocell europe limited, netpark incubator, united kingdom abstract human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3d models of various tissue types that more closely resemble in vivo-like conditions (knight et al., 2011). the scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. this technology has been applied to generate numerous unique types of co-culture model. for example: 1) a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (fig.1) (hill et al., 2015); 2) a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (clarke et al., 2016); 3) formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ecm proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa). these organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. the addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in discovery, validation studies, and modeling disease. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en proceeding of veterinary and animal science days 2017, 6th8th june, milan, italy haf © 2013 vol. iv, no. 1s issn: 2283-3927 fig. 1 this figure showcases the potential of 3d cell culture technology and how it can be used to create tissue-like models. the histological image reveals the cellular structure of a human skin equivalent. the full thickness of the epidermis is shown resting on the underlying dermis. the model is built on the alvetex® platform that consists of a porous polystyrene scaffold in which human dermal fibroblasts are seeded. these cells produce exogenous collagens to form the dermal compartment. human keratinocytes are then seeded onto the surface of the dermal model that is subsequently raised to air-liquid interface where they differentiate, stratify and form a mature epidermis. the layers of cells in the 3d culture model replicate those in the real tissue, including the formation of the skin barrier and the surface stratum corneum (figure courtesy of s.bradbury, durham university) references clarke, k.e., tams, d.m., henderson, a.p., roger, m.f., whiting, a., przyborski, s., 2016. a robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition. neurochemistry international. 106, 74-84 hill, d.s., robinson, n.d., caley, m.p., chen, m., o'toole, e.a., armstrong, j.l., przyborski, s., lovat, p.e., 2015. a novel fully humanized 3d skin equivalent to model early melanoma invasion. molecular cancer therapeutics. 14, 2665-73. knight, e., murray, b., carnachan, r., przyborski, s., 2011. alvetex®: polystyrene scaffold technology for routine three dimensional cell culture. methods in molecular biology. 695, 323-40 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en 1 l 1 introduction keywords zinc; nutritional additive; in vitro model; gut; intestinal cells. pages 1 – 7 references vol. 1 no. 2 (2014) article history submitted: july 07, 2014 revised: july 14, 2014 accepted: july 14, 2014 published: july 18, 2014 corresponding author raffaella rebucci, department of health, animal science and food safety, university of milan via trentacoste 2, 20134 milano, italy e-mail: raffaella.rebucci@unimi.it phone: +30 02 50315735 fax: +30 02 50315746 journal home page riviste.unimi.it/index.php/haf effect of zinc oxide and zinc chloride on human and swine intestinal epithelial cell lines. luciana rossi 1 , eleonora fusi 1 , morena boglioni 1 , carlotta giromini 1 , raffaella rebucci 1* , antonella baldi 1 . 1 department of health, animal science and food safety, university of milan. abstract. zinc (zn) salts are often used as nutritional additives in order to promote gut health. the aim of the present study was to assess the effect of two widely used additives in feedstuff, on the intestinal epithelium. in particular, the effect of zinc oxide (zno) and zinc chloride (zncl2) was investigated in human (int-407) and porcine (ipi-2i) cell line models. the effect of zn sources on ipi-21 and int-407 cell lines was evaluated by a colorimetric viability test using an incubation period of 3 and 24 hours under serum-free conditions. int407 and ipi-2i showed to be a suitable model of the intestine and a simple tool to investigate the role of zn supplements. int407 showed to be the most sensible model to zn supplements considered, whereas ipi-2i were more resistant. the results of this study contribute to determine the role of zinc in human and swine intestinal epithelium. however, further in vivo experiments may be done to clarify the contribution of zn supplements in gut health and to improve zn supplementation in animal feed and in human formulations. l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 2 haf © 2013 vol. i, no. 2 issn: 2283-3927 1 introduction zinc (zn) is an abundant trace element in the body recognized as essential micronutrient. at intestinal level, zn is involved in the modulation of immune system, affecting both non-specific and acquired immunity (fraker et al., 2000; frieke, 2000), in mucosal resistance to infection, in the restoration of mucosal barrier integrity and the promotion of antibody production against intestinal pathogens (sargeant et al., 2011). the absorption of zn occurs primarily in the small intestine and is influenced by different dietary factors such as the presence of zn antagonists (e.g. calcium, phytate, fibers). low molecular weight binding ligands such as citrate, picolinate and aminoacids enhance its absorption (lönnerdal, 2000). zn is supplemented in human multivitamin formulations and in animal feedstuff in either inorganic or organic forms. in pig livestock, zn has been included as nutritional additive at concentration lower than 150ppm as indicated in the reg. ce 1831/2003. whereas, pharmacological concentrations of zn >1000ppm are commonly used as alternative to in-feed antibiotics (poulsen, 1995). generally, inorganic zn salts (e.g. zinc oxide) are the most commonly forms supplemented in animal diets. it has been previously demonstrated that in swine livestock the supplementation of 150ppm of zinc oxide (zno) improves gut health (rosselli et al 2005). also, zno at pharmacological concentrations reduces the incidence of diarrohea in the weaned piglets (owusu-asiedu et al., 2003). new approaches are necessary to optimize zinc supplementation in feedstuffs in order to improve animal health and to control zinc release in the environment. moreover, further investigations into the mechanism by which zinc supplementation improves intestinal swine condition may shed light on the role of zinc in human gut level. studies conducted in vitro reported that zno inhibits bacterial growth, plays a pivotal role in maintaining epithelial barrier integrity and function, may improve mucosal repair and paracellular permeability (roselli et al, 2003). nevertheless, despite the wide use of zn oxide in in vivo trials, few studies were focused on its effect at cellular level (sargeant et al., 2010; sargeant et al., 2011). the importance of intestinal cell models of human and pig origins in in vitro platforms for preclinical research is well recognised (langerholc et al., 2011). in this context, in vitro studies can provide evidences regarding the mechanism of action of specific dietary compounds at cellular level, as well as discover any cytotoxic effects of these molecules. cell culture models have been often used to evaluate the cellular mechanisms of several compounds (baldi et al, 2004; rebucci et al, 2007). in addition, in vitro animal and human cell models represent a suitable alternative to in vivo animal experiments (cencič & langerholc, 2010) for the determination of zn supplements effects. in light of that, the aim of the present study was to assess the effect of two widely used additives in feedstuff, on the intestinal epithelium. in particular, the effect of zno and zncl2 was investigated in human (int407) and porcine (ipi-2i) cell line models. 2 materials and methods 2.1 cell line and cell culture conditions the human embryonic intestinal cell line (int-407), a non-transformed epithelial cell line, originally derived from ileum of a 2-month-old human embryo, was obtained from the l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 3 haf © 2013 vol. i, no. 2 issn: 2283-3927 american type culture collection. int-407 were routinely cultivated into 75 cm 2 tissue culture flasks in rpmi-1640 medium supplemented with 200mm glutamine, 1% non-essential aminoacids (neaa) and 10% fetal bovine serum (fbs). cells used in this work were between passages 36-40. the ipi-2i cell line, derived from the ileum of an adult boar and immortalized by transfection with an sv40 plasmid (psv3) (kaeffer et al., 1993), was obtained from american type culture collection. ipi-2i were routinely cultivated into 75cm2 tissue culture in dmem-f12 supplemented with 4mm glutamine, 0.024ui/ml insulin and 10% fetal bovine serum (fbs). cells used in this work were between passages 5-12. cells were cultured in an atmosphere of 5% co2 at 37 °c until sub-confluence. cell monolayers were washed with phosphate buffered saline (pbs) and trypsinized with 0.25% trypsin-edta. after 48 hours, cells were detached and re-suspended in culture medium to a concentration of 2.5 x 105 cells/ml. portions (200 µl) of cell suspension were dispensed into sixty wells of a ninety-six-well tissue culture plates. 2.2 zinc oxide and zinc chloride solutions a stock solution of 400mm zno (zn content 80.34%) was prepared in 5% of acetic acid. starting from this stock, zno treatment solutions (50, 200, 1000 and 4000 µm) were dissolved in serum-free medium. a stock solution of zncl2 (zn content 47.97%) was prepared as described in merck index instructions (1989). in detail, 1 g of zncl2 was dissolved in 0.25 ml of 2% hcl. starting from this stock, zncl2 treatment solutions (50, 200, 1000 and 4000 µm) were dissolved in serum-free medium. ipi-2i and int-407 were exposed to treatment solutions in concentration mentioned above (200µl) of zno and zncl2 for the following 3 and 24h. in particular, for each cell lines we tested four concentrations of zn sources in triplicate (3 wells per treatment) at two incubation times (3 and 24h). moreover, at least two independent experiments were performed. the concentrations of zno and zncl2 were selected on the basis of preliminary assays (data not shown) whereas the times of exposure were selected on the basis of the intestinal transit time in according to roselli et al. (2003). 2.3 evaluation of the effect of different zinc sources on cell viability: mtt test the effect of zinc sources on ipi-21 and int-407 cell lines was evaluated by mtt test using an incubation period of 3 and 24 hours under serum-free conditions. in particular, after removing the treatment solutions, 150µl mtt stock solution (5mg/ml) in pbs was added to each well and the plates were incubated for 1.5 h in a humidified chamber. the reaction was accomplished by removing the incubation solution and adding 150µl dimethyl sulfoxide to dissolve the formazan. the optical density of dimethyl sulfoxide (540 nm) was determined on a biorad 680 microplate reader. cells incubated with culture medium alone representing 100% viability, were included as negative controls in all experiments. this assay measured the production of the chromophore formazan from (4,5 – dimethylthiazol – 2 yl) 2,5 l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 4 haf © 2013 vol. i, no. 2 issn: 2283-3927 diphenyltetrazoliumbromide (mtt) (sigma-aldrich). formazan was produced in viable cells by the mitochondrial enzyme succinate dehydrogenase. the percentage of cell viability induced by treatments was calculated as follows: 3 statistical analysis at least three replicates (3 wells per treatments) at each incubation time were performed and two independent experiment were conducted. the data are presented as means and standard error (se) and analyzed by one-way anova (sas, 2010). duncan’s post-hoc multiple range test was used, with p < 0.05 considered statistically significant. different concentrations of the two zinc sources were tested using a model including the systematic effects of the source of zinc (n = 2), concentrations of zinc (four levels). the effect of the assay (n = 2) was included as a blocking factor. 4 results int-407 cells have been exposed to a wide range of zno and zncl2 concentrations in order to establish the cell viability in response to zinc treatments (figure 1). in particular, int-407 cell line treated for 3h with 50µm of zno increased cell viability up to 120% of the control. the same tendency in term of enhancement of cell viability was observed after 3 and 24h of exposure to 50 µm of zncl2. higher concentrations of zno and zncl2 have affected cell viability in a dose dependent way. a significative (p < 0.05) reduction of cell viability was observed. as shown in figure 2, ipi-2i have shown a different susceptibility to zno and zncl2 treatment. in particular after 3h of incubation, zno did not significantly inhibited ipi-2i cell viability, which remained about 80% at 50, 200 and 1000µm. whereas, a significative (p < 0.05) reduction in ipi-21 cell viability was observed at the highest zinc oxide concentration tested (4000 µm). a significative reduction (p < 0.05) was observed at all concentrations of zno tested after 24h of incubation. a similar dose and time dependent effect was also observed when ipi-21 cells were treated with increasing concentrations of zncl2 for 3 and 24h. overall, a dose-response effect was observed after zno and zncl2 treatments in both the intestinal cell lines considered suggesting that in vitro cell models are suitable for studying the effect of zinc additives on cell viability. in particular, it was observed that after 3 hours of incubation, which correspond to the transit time for several nutrients, cell viability was maintained by the lowest zno and zncl2 presence in the medium. l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 5 haf © 2013 vol. i, no. 2 issn: 2283-3927 figure 1. effect of increasing concentrations of zno and zncl2 on cell viability of int-407 after 3h and 24h of treatment measured by mtt test. the graph shows the percentages of cell viability over control. values significantly different from proliferation obtained with 0 µm zn sources (control medium 100% viability) are indicated: a, p < 0.05 figure 2. effect of increasing concentrations of zno and zncl2 on cell viability of ipi-2i after 3h and 24h of treatment measured by mtt test. values significantly different from proliferation obtained with 0µm zn sources (control medium 100% viability) are indicated: a, p < 0.05 5 discussion and conclusion cell models are reaching growing interest among biological studies because of their wide use among the scientific community. they are becoming more realistic and representative of the in vivo environment providing an economic and ethical alternative to in vivo animal testing (langherolc, 2010). further, cell culture models meet the eu requirements for the reduction of the number of animal experimental models whenever possible, following the three r paradigm (reduce, refine, replace). intestinal in vitro model are of great importance in food and nutritional research, they represent an essential method to characterize the mechanism of action of several dietary compound in an easy and repeatable way. since intestinal cell model a a a a a a a a a a a a a a a a a a a a l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 6 haf © 2013 vol. i, no. 2 issn: 2283-3927 simplify biological systems, they are widely used in toxicological and bioavailability tests of a wide range of dietary compounds, which are inevitable for bringing products to the market (cencic & langherolc, 2011). mtt assay is a viability assay that measures the metabolic activity the cells. mtt assay used in this study represent a simple and useful method to preliminary qualify the effect of a large number of compounds (zn, tocopherol, probiotics) at cellular level (baldi et al., 2004; rebucci et al., 2007). faller et al. (2002) reported that this assay showed a better correlation with in vivo assay than other bioassays. cell-based model represents simplify biological system and may be used in the evaluation of nutritional additives effects. in this study, two intestinal epithelial cell lines from different species have been considered. this is essential in order to better characterize the response of dietary compounds on a specific epithelium. despite the wide use of zn as animal feed supplement, little studies have focused on its effect in vitro. the intestinal cell lines int-407 and ipi-2i showed to be suitable models of the intestine and represent a simple tool to investigate the role of zn supplements. int-407 showed to be the most sensible model to zn supplements considered in this study, whereas ipi-2i showed to be the most resistant. viability of ipi-2i cells is reduced at zno and zncl2 concentrations >1000um, whereas sargeant et al. (2010) found a reduction of ipec-j2 cell viability at concentrations >100µm of zno. however, the lowest concentrations of both zno and zncl2 considered in this study have maintained the viability of int-407 and ipi-2i underlining the beneficial role of zn on human and swine intestine. in particular, it was observed that after 3 hours of incubation, which correspond to the transit time for several nutrients, cell viability was enhanced by the lowest zno and zncl2 concentrations tested, indicating that these compounds may have a beneficial role for human and swine intestinal epithelium. overall, these results contribute to determine the role of zinc in human and swine intestinal epithelium. in general, this study confirm that the use of in vitro cell-based models for screening the biological activity of single compounds at specific concentrations and in a strictly controlled environment could offer novel insight in the field of human and animal nutrition, making in vitro models an essential tool in biological studies. however, cell-based models may not reflect the in vivo condition of the intestinal cells in their natural state in the intact organism, where bioavailability, metabolism, binding and transport proteins may influence the biological effect of zinc sources. therefore, further in vivo experiments are necessary in order to extend the results obtained in vitro, to clarify the contribution of zn supplements in gut health, and to improve zinc supplementation in animal feed and in human formulations. references baldi a., losio m.n., cheli f., rebucci r., sangalli l., fusi e., bertasi b., pavoni e., carli s., politis i., 2004. evaluation of the protective effects of alpha-tocopherol and retinol against ochratoxin a cytotoxicity. british journal of nutrition 91, 507-512. cencic a., langerholc t., 2010. functional cell models of the gut and their applications in food microbiology. international journal of food microbiology. 141, 4-14. l.rossi et al. int. j. of health, animal science and food safety 2 (2014) 17 7 haf © 2013 vol. i, no. 2 issn: 2283-3927 fraker p.j., king l.e., laakko t.,vollmer t.l., 2000. the dynamic link between the integrity of the immune system and zinc status. journal of nutrition. 130, 1399 – 1406. frieke c. 2000. function and mechanism of zinc. journal of nutrition 130, 1437s – 1446s. kaeffer b., bottreau e., velge p., pardon p., 1993. epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by sv40 plasmid. european journal of cell biology. 62, 152-162. langerholc t., maragkoudakis p.a., wollgast j., gradisnik l., cencic a., 2011. novel and established intestinal cell line models – an indispensable tool in food science and nutrition. trends in food science & technology. 22, 11-20. lönnerdal b., 2000. dietary factors influencing zinc absorption. journal of nutrition. 130, 13781383. owusu-asiedu a., nyachoti m.c., marquardt r. r., 2003. response of early-weaned pigs to an enterotoxigenic escherichia coli (k88) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc oxide, fumaric acid, or antibiotic. journal of animal science. 81, 1790-1798. poulsen h.d., 1995. zinc oxide for weanling piglets 45, 159-167. rebucci r, sangalli l, fava m, bersani c., cantoni c.a, baldi a., 2007. evaluation of functional aspects in lactobacillus strains isolated from dry fermented sausages journal of food quality 30, 187–201. regolamento (ce) n.1831/2003 del parlamento europeo e del consiglio del 22 settembre 2003 sugli additivi destinati all'alimentazione animale. gazzetta ufficiale dell'unione europea l 268/29-42. roselli, m., finamore, a., garaguso i., britti m.s., mengheri, e., 2003. zinc oxide protects cultured enterocytes from the damage induced by escherichia coli. journal of nutrition. 133, 4077–4082. roselli m., finamore a., britti m.s., bosi p., oswald i., mengheri e., 2005. alternatives to in-feed antibiotics: evaluation of probiotics, zinc or organic acids as protective agents for the intestinal mucosa. comparison of in vitro with in vivo results. animal research, 54, 1-16. sargeant h.r., shaw m.a, abuoun m., collins j.m., woodward m.j., la ragione r.m., miller h.m., 2010. the metabolic impact of zinc oxide on porcine intestinal cells and enterotoxigenic escherichia coli k88. livestock science. 133, 45–48. sargeant, h.r., miller, h.m., shaw, m. 2011. inflammatory response of porcine epithelial ipec j2 cells to enterotoxigenic e. coli infection is modulated by zinc supplementation. molecular immunology. 48, 2113–2121. l keywords cell culture, 3-d, epigenetics, environmental cues. pages 39 – 48 references vol. 4 no. 1 (2017) article history submitted: may 30, 2017 accepted: july 04 2017 published: november 26 2017 corresponding author tiziana a. l. brevini laboratory of biomedical embryology, centre for stem cell research, università degli studi di milano via celoria 10, milan, 20133, milan, italy mail: tiziana.brevini@unimi.it fax +39-02-50317980 journal home page riviste.unimi.it/index.php/haf review “bridging the gap between cell culture and live tissue” stefan przyborski 1 , fulvio gandolfi 2, 4 , tiziana a. l. brevini 2 , 3, * 1 department of biosciences, durham university, durham, dh1 3le, united kingdom. 2 unistem, centre for stem cell research, università degli studi di milano, milan, italy. 3 department of health, animal science and food safety, università degli studi di milano, milan, italy. 4 department of agricultural and environmental sciences-production, landscape, agroenergy, università degli studi di milano, milan, italy. abstract traditional in vitro two-dimensional (2-d) culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. in vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-d) organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (jong et al., 2011). in contrast, traditional in vitro culture, carried out on 2-d plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. it is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. in order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. this implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. in particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature. s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 40 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction traditional in vitro two-dimensional (2-d) culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. in vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-d) organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (jong et al., 2011). in contrast, traditional in vitro culture, carried out on 2-d plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. it is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. in order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. this implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. in particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature. 2 environmental cues of chemical nature 2.1 exosomes and non-coding rna exosomes are secreted vesicles that include membrane particles, microvesicles, ectosomes, exosome-like vesicles or apoptotic bodies (ostrowski et al., 2010), often found in physiological fluids such as normal urine (pisitkun et al., 2004), plasma (caby et al., 2005) and bronchial lavage fluid (admyre et al., 2003). they are secreted by several types of cell, such as dendritic cells (zitvogel et al., 1998), mast cells (raposo et al., 1997), t cells (peters et al., 1989), platelets (heijnen et al, 1999), schwann cells (fevrier and raposo, 2004), tumor cells (andre et al., 2001), endometrial cells (greening et al., 2016) and embryos (wydooghe et al., 2015). they contain evolutionarily conserved set of proteins but also have unique tissue/cell type-specific proteins that reflect their cellular source (mathivanan et al., 2010). although still controversial, they are believed to be important for intercellular communication. they are presently being investigated for their role as markers for disease and a possible use for diagnostic purpose has been postulated. furthermore, they represent a very promising biochemical cue in cell to cell communication and tissue engineering. in neuronal and embryo-maternal communication, inter-embryo communication and pathogenesis. s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 41 haf © 2013 vol. iv, no. 1 issn: 2283-3927 2.2 chemical gradient and macromolecular crowding gradients of distinct molecules play key roles in a variety of processes, affecting the commitment of cells as well as their role in vivo (keenan and folch, 2008) and a main need is to create in vitro methods that allow cells to be exposed to chemical gradients that may be tuned, quantified and controlled in such a way to mimic the gradients that are present in vivo (benny et al., 2016). it is obvious that a gradient across 3-d space more closely recreates the in vivo milieu and may be more advantageous to help elucidate biological processes are modulated by biomolecular gradients. an alternative strategy is based on the principles of macromolecular crowding (mmc), a biophysical phenomenon that directs the intraand extra-cellular milieu in multicellular organisms and increases thermodynamic activities and biological processes (zimmerman and harrison, 1987). mmc uses the principles of volume occupancy, where macromolecules take volumes larger than their ‘real’ volume owing to their high hydrodynamic radius, thereby reducing the space for other macromolecules belonging to the same system (ellis, 2001; lareu et al., 2007). several reports have recently shown the impact of these aspects. for instance, the addition of neutral or negatively charged molecules to the culture media was shown to increase collagen type i deposition in in vitro culture of human fibroblasts (lareu et al., 2007). this has been explained with the observation that in vivo cells are entrapped in highly crowded extracellular space, where the conversion of the de novo synthesised procollagen to collagen takes place rapidly, whereas in the diluted culture environment the conversion of procollagen to collagen is very slow (lareu et al., 2007). although the full applicability of this approach is under evaluation, a promising application of mmc for cell based therapies is addressed to the combination with cell sheet technology (l’heureux et al., 2007; peck et al., 2012) to accelerate the production of cell sheets rich in extracellular matrix (english et al., 2012). 2.3 epigenetic modifications cell differentiation processes are regulated by the expression of different sets of genes responsible for a distinct phenotype, under the control of regulatory mechanisms that include dna methylation and histone modifications. all these differences in gene expression drive development and differentiation, leading to the acquisition of epigenetic marks and the fixation of a specific fate that has been considered stable and potentially irreversible until recently. however, following the pioneering work carried out by jones (taylor and jones, 1979), in the last years, many groups reported that it is possible to directly interact with cell fate definition through the use of epigenetic modifiers (pennarossa et al., 2013; brevini et al., 2014; chandrakantan et al., 2016; manzoni et al., 2016) (figure 1). these studies are particularly intriguing, since they allow a better understanding of epigenetic restriction, and better clarify the mechanisms leading to the acquisition of a mature somatic phenotype. s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 42 haf © 2013 vol. iv, no. 1 issn: 2283-3927 figure 1: fibroblasts obtained from biopsies cultured on gelatin coated culture dishes (left) and exposed to epigenetic modification (right). striking changes in morphology are evident and are accompanied by a decrease in the global dna methylation. 3 environmental cues of mechanical nature in vivo, cells are surrounded by a complex 3-d organization of neighbouring cells and ecm, which interact to provide chemical as well as mechanical stimuli. integrin and surrounding matrix are not only in charge of ensuring physical attachment, but also convey chemical and mechanical signals from the outer environment (janmey and mcculloch, 2007), suggesting that the dimension where cells are grown is a key point for the determination of cell fate. until recently, cell culture has been performed in monolayers, that, although providing useful biological information, lack the ability to reproduce the morphology and 3-d architecture of the original tissue. the benefits of 3-d cell cultures are widely appreciated and more cell-based technologies are now becoming available that enable researchers to preserve the native 3-d structure of cells in vitro, offering many advantages over conventional monolayer culture. first of all, the mechanisms that underline the process of tissue formation, such as migration, proliferation, adhesion, differentiation and apoptosis can be better investigated. in addition, the 3-d environment enables cells to form cell-cell and cell-matrix interaction that may otherwise be precluded in monolayer culture (baker and chen, 2012). deconstructing the elements contributing to the 3-d microenvironment and the associated processes will aid us in better understanding the underlying mechanisms that guide cell growth in vivo. 3.1 technologies for 3-d cell culture the concept of 3-d cell culture is not new and has been around for over a century. what has changed more recently is the ease of access to new innovative technologies on the market that enable researchers to more readily practice 3-d cell culture. these can generally be categorized into three areas, namely aggregate/spheroid methods; hydrogels/extracellular matrix technologies; and solid scaffold products. there is no single solution, each approach s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 43 haf © 2013 vol. iv, no. 1 issn: 2283-3927 has strengths and weaknesses, and researchers must select the technology most appropriate for their needs to address the scientific questions that they are interested in. aggregate-based technologies promote bringing cells together to create 3-d tissue-like masses or spheroids, often called ‘micro-tissues’ by exploiting the biophysical properties acting on the media in which they are grown. cells grown as 3-d aggregates secrete their own extra-cellular matrix (ecm) and self organise creating multiple cell-cell interactions. different methods have been developed to generate this type of culture for routine use. the traditional hanging drop approach involves the culture of cells in a drop of medium suspended from the lid of a culture dish. these suspension cultures are adequate for cells that can proliferate in a non-adhesive environment where aggregation is favoured. an alternative strategy involves using attachment-resistant surfaces such as coatings with hydrophilic polymers (jo and park, 2000) or micro-patterned surfaces (yoshii et al., 2011). both methods inhibit cell adherence and forces cells to float in the medium, stimulating them to coalesce and form spheroids. regardless of how cell aggregates are formed, these methods make it possible to scale down experiments and work in smaller volumes and are therefore amenable for higher throughput applications. co-culture of different cells types is also possible, establishing signal rich environments to study the effect of paracrine signalling in real tissue (torisawa et al., 2009). hydrogels differ from solid scaffolds in terms of the strength of physical support. hydrogels are loose scaffolds consisting of cross-linked natural or synthetic materials within an aqueous environment for cell encapsulation. these highly absorbent matrices are better suited to modelling soft tissues basic of their tissue-like flexibility and viscoelasticity (tibbit and anseth, 2009). hydrogels can be derived from a variety of sources that in turn affect their compatibility and properties. for example, animal-derived hydrogels mainly use collagen, which is the most abundant protein in the ecm. matrigel® is an example popular commercially available hydrogel composed of tumour extract derived from mouse sarcoma cells. it is known to contain growth factors, a rich protein mix including collagen iv, laminin and entactin and other undefined constituents (vukicevic et al., 1992). matrigel® can promote cellular functions that would otherwise be unseen by providing a 3-d microenvironment and the necessary endogenous factors (benton et al., 2014). the use of defined synthetic hydrogels overcomes some of the issues of animal derived materials such as batch variation and unknown constituents. solid scaffolds were originally devised for applications in transplantation, however, there has been a growing interest to introduce scaffolds for routine use in 3-d cell culture. the materials used in the fabrication process are important in shaping the purpose of scaffoldbased technology. components of the native ecm including collagen, fibrin, and hyaluronic acid (ha) (gerecht et al., 2007) have been used effectively to create 3-d matrices to support cell growth. these constituents have the benefit of being biocompatible and possessing readily available adhesion sites that can increase the complexity of the tissue. despite being beneficial in the context of tissue engineering, working with biological materials in the laboratory can affect consistency. a partial solution has been to use biodegradable polymers such as poly(glycolic acid), poly(lactic acid) and their co-polymers poly(lactic-co-glycolic acid) (mikos et al. 1993). this is not ideal because their degradation results in the release of unwanted by-products that can alter cell behaviour. the added variability coupled with short shelf life and problematic storage make biodegradable materials unsuitable for standard use in 3-d cell culture. in light of these shortcomings, synthetic scaffolds with defined composition s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 44 haf © 2013 vol. iv, no. 1 issn: 2283-3927 have risen as a more consistent alternative. inert and non-degradable materials such as synthetic polymers can be carefully tweaked to capture the cellular niche, creating scaffolds suitable for cell culture (bokari et al. 2007; knight et al. 2011). the lack of biological activity and natural cell adhesion sites can be overcome by coating these substrates with ecm proteins such as laminin and fibronectin (knight and przyborski, 2014). despite providing physical support in the form of 3-d spaces where cells can proliferate, these voids have poor mass transfer since these cultures are static systems. for these reasons, scaffolds are usually engineered as thin membranes (e.g. 200μm) that permit sufficient exchange of nutrients and waste products. this in turn enriches the physiological accuracy of these models allowing researchers to study in vivo phenomena in a controlled in vitro setting. advances in technology have led to new opportunities for growing cells in culture and the creation of 3-d tissue-like constructs. this is primarily as a consequence of inter-disciplinary research between cell biology and the biophysical sciences, introducing new materials and methods of manufacture to create platforms tailored to support 3-d cell growth in vitro. the success of these technologies will depend on their adoption, validation and application. the creation of tissue-like constructs in a reliable and reproducible manner according to clearly defined protocols is essential. moreover, in-depth characterisation of the anatomy and physiology in vitro models alongside their native counterparts will be critical to convincing the scientific community and encourage users to adopt these new approaches (figure 2). figure 2: this figure showcases the potential of 3-d cell culture technology and how it can be used to create tissue-like models. histological images reveal the cellular structure of real human skin (left) and a human skin equivalent (right). the full thickness of the epidermis is shown resting on the underlying dermis. the model is built on the alvetex® platform that consists of a porous polystyrene scaffold in which human dermal fibroblasts are seeded. these cells produce exogenous collagens to form the dermal compartment. human keratinocytes are then seeded onto the surface of the dermal model which is subsequently raised to air-liquid interface where they differentiate, stratify and form a mature epidermis. the layers of cells in the 3-d culture model (right) replicate those in the real tissue (left), including the formation of the skin barrier and the surface stratum corneum. s. przyborski et al. int. j. of health, animal science and food safety 4 (2017) 39 48 45 haf © 2013 vol. iv, no. 1 issn: 2283-3927 there is no doubt that 3-d cell culture is a rapidly growing and important field of science and requires interdisciplinary research to be innovative and develop. these advances will continue to enable biomedical researchers to recreate the structure and function of human tissues in the lab for research, screening and safety assessment. this technology combined with human stem cell science will open new opportunities for tissue engineering in the lab where renewable sources of human cells can be generated to create robust and reproducible 3-d models of human tissues. furthermore, to reproduce the conditions in vivo requires many other factors such as oxygen control, perfusion, growth factors, cytokines, hormones, mechanical stiffness, etc. today we are applying technology to improve current practice, to make incremental advances over existing models, to enable greater insight into biological processes. as technology advances, we will further improve our cell culture models, edging closer to in vivo conditions, but researchers must always remember that it is a model they are studying in the lab and not real tissue. 3-d cell culture takes a big step towards achieving the goal of recreating the growth conditions cells experience in vivo. acknowledgments: the authors are members of the cost actions ca16119. talb participates to cost action cm1406. the authors thank dr. elena fm manzoni for her help in the preparation of this manuscript. references admyre, c., grunewald, j., thyberg, j., gripenbäck, s., tornling, g., eklund, a., scheynius, a., gabrielsson, s., 2003. exosomes with major histocompatibility complex class ii and costimulatory molecules are present in human bal fluid. eur respir j. 22(4):578-83. andre, f., andersen, m., wolfers, j., lozier, a., raposo, g., 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spheroid formation. biomaterials. 32, 6052–6058. zimmerman, s.b., harrison, b., 1987. macromolecular crowding increases binding of dna polymerase to dna: an adaptive effect. proc natl acad sci u s a. 84(7):1871-5. zitvogel, l., regnault, a., lozier, a., wolfers, j., flament, c., tenza, d., ricciardi-castagnoli, p., raposo, g., amigorena, s., 1998. eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. nat med. 4(5):594-600. 8 l keywords cervus elaphus, dermatophytes, fulgal flora, prevalence, red deer, stelvio national park pages 8 – 14 references vol. 1 no. 2 (2014) article history submitted: september 28, 2014 revised: september 30, 2014 accepted: october 03, 2014 published: october 21, 2014 corresponding author roberta perego, dipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare (vespa) università degli studi di milano via g. celoria 10, 20133, milano, italy e-mail: roberta.per ego@unimi.it phone: +39 02 50318188 fax: +39 02 50318171 journal home page riviste.unimi.it/index.php/haf prevalence of dermatophytes in red deer (cervus elaphus) in the stelvio national park, italy. roberta pereg o1, daniela prov erbio1, eva spada1, giada bagnagatti de giorgi1, emanuela valena1 and elisabetta f erro1 1 dipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare (vespa), università degli studi di milano, milano, italy. abstract. dermatophytosis has been described in wildlife, but the literature reporting dermatophyte prevalence in deer is incomplete. to determine the prevalence of dermatophytes and to evaluate the hypothetical role of asymptomatic carriers hair samples were collected from 30 legally hunted wild red deer (cervus elaphus) in the stelvio national park, italy. all deer were visually examined for dermatologic lesions and the coat was brushed using a modified mackenzie collection method. a small sample of hair was used for direct microscopical examination and subsequently fungal culture was performed on the hair samples. macroscopic and microscopic examinations were used to identify dermatophytes, saprophytic fungi and yeasts. none of the deer had visible cutaneous lesions. no dermatophyte spores or hyphae were found on direct microscopical examination and, when hair samples were cultured, dermatophytes were not demonstrated in any sample. only saprophytic fungi were grown, predominantly alternaria spp., mucor spp., cladosporium spp. these results did not reveal the presence of asymptomatic carriers of dermatophytes in the deer sample population of stelvio national park and suggest that it is unlikely that, at least in the investigated geographical area, the red deer act a reservoir for transmission of dermatophytes to other wild animals, livestock or people living locally. r. perego et al. int. j. of health, animal science a nd food safety 2 (2014) 8-14 9 haf © 2013 vol. i, no. 2 issn: 2283-3927 1 introduction dermatophytoses, also called ringworm, are common contagious infections of keratinized tissues caused by keratinophilic/keratinolytic fungi named dermatophytes which affect a wide range of mammals, including man (chermette et al. 2008). man y species of dermatophyte have been reported as animal pathogens, bu t these may also be carried as normal flora on th e haircoat of pets (cabanes et al. 1997; cafarchia et al. 2006; boyanowsky et al. 2000). dermatophyte fungi can also be found in farm animals and roden ts (cherm ette et al. 2008, cabanes et al. 1997; sargison et al. 2002; mahmoud 1995; moretti et al. 1998; papini et al. 2009). there have been previous reports of isolation of dermatoph ytes from th e haircoat of health y carriers (alteras et al. 1966; chabasse et al. 1987, marcianti et al. 1993) and cases of clinical dermatophytosis (alteras et al. 1968, knudtson et al. 1980, peano et al. 2008) also in wild animals. knudston and robertstad (1970) dem onstrated a positive specim ens prevalen ce of 26.8% for keratinophilic fungi in 224 animals tested from 30 wildlife species in south dakota. in a hair sample study in 2005 by gallo et al. (2005), th e alpine marmot (mar mota marmota) was identified as a reservoir for man y dermatoph ytes, including microporum canis; and dermatophytosis has previously been reported in water buffalo (b ubalus bubalis ) (pal et al. 1995). clinical dermatoph ytosis has been reported in mule deer (odocoileus hemionus ) (wobeser et al. 1983), barking deer (muntiacus muntjak) (pa l and thapa 1993) and rusa deer (cervus timorensis ) (le bec and beugnet 1994). conversely, in a recen t survey of fungal flora in white-tailed deer (odocoileus virginia nus ) in virginia (usa) no dermatoph ytes were identified (hall et al. 2011). rare studies of prevalence of dermatoph ytosis and ringworm episodes hav e also been signalled in wild animals in captivity (kuntze et al. 1967, janovitz and long 1984). however, th e literature reporting the prevalence of dermatophyte carriers in the majority of wild species is still incomplete, and th e role of th ese species as reservoir for these pathog ens is unclear. the red deer (cervus elaphus) is one of th e most represen ted animal throughou t the alps. measures have been tak en to preserve and manage thes e deer populations. curren tly, nearly 10,000 red deer are present in th e in the stelvio national park (pns) and adjacen t areas, of which around 60% liv e in a more or less stable community in the protected area that covers about 485 sq km. in 2000, th e consortium of pns started a project for the managem ent of deer populations in the pa rk allowing, in some protected areas, controlled culling (reduction in numbers of populations by con trolled hunting) to reduce th e density of th e deer population, since high densities have significant im pacts on ecosystems and cause economic damage to agricultu ral activities. the project, however, has also provided a mechanism for investigation of the health status of th e deer and their relationship with the ecos ystem and with man. in this regard wildlife surveillan ce (to assess the presen ce of poten tially zoonotic pathogens on th e skin of wild deer) is an important area for which data is lacking. to our know ledge no studies report dermatoph ytes prevalence from red deer populations. the aims of this study were to determin e the prevalence, and identify species, of dermatophyte fungi in a population of wild alpin e red deer in italy and to evaluate wh eth er asymptomatic deer may carry dermatoph yte fungi as part of the normal haircoat flora. given the importance of the d eer population in the stelvio national park this knowledge is importan t in identifying the risk of human and livestock exposure to this zoonotic path ogen. r. perego et al. int. j. of health, animal science a nd food safety 2 (2014) 8-14 10 haf © 2013 vol. i, no. 2 issn: 2283-3927 2 materials and methods under a scientific project pursuant to art. 11, paragraph 4 of law 6 december 1991 n. 394, and with permission of the italian ministry of environm ent and protection of land and sea (dpn 2010 – 13760, 17 june 201) and of the national institute for protection and environmen tal research (protocol n. 15387/t-a25, 6 may 2010), hair samples were collected from legally hunted red deer in stelvio national pa rk, italy. the culling programm e provided a redu ction in the density of th e red deer population by allowing hunting within a small wintering area of about 1400 h ectares, comprising the unit of managemen t referred to as "valfu rva-sondalo", belonging to th e forestry station of valfurva in the lombard sector of pns. this forestry station of about 24,600 hectares , the largest of the park, is of sufficient size to provide all th e needs of th e red deer (neighbourh oods for summering, wintering areas and breeding areas). hair samples were collected between november and december 2012. all deer tested had been killed no more than 12 hours before sampling. all deer sampled were visually examined for dermatologic lesions. samples obtain ed from 30 red deer consisted of 11 fawn s (5 females and 6 males) and 19 adult red deer (16 females, including 10 pregnan t females, and 3 males) ranging in age from yearling deer to mature deer (20 years). although several meth ods are available for dermatoph yte testing, including wood’s lam p examination, direct microscopic examination of hairs and fungal culture is considered the gold standard (moriello 2003 ) and was the m ethod used in this study. the deer coat was brushed using a modified mackenzie collection method (mack enzie 1963; moriello 2001). briefly, a new sterilized toothbrush was used to collect hair samples by brushing each deer for a minimum of 2 minutes (at least 30 strok es) ov er the face, neck , abdomen, thorax and limbs (hall et al. 2011) and immediately after brushing, all accumulated material was transferred to a sterile pou ch until it could be examined in the laboratory. in th e laboratory, a small sample of hair was collected from th e toothbrush with sterile, rubber covered, hemostats and examined under a microscope (direct hair examination). th e hairs were positioned in th e same orientation on a microscope slide, suspended in mineral oil, an d examined, with th e low-pow er objective of the microscope, for dermatoph yte hyphae an d arthrospores. 20% potassium hydroxide diaphanisation for 30 minutes of hair sampled was finally performed to microscopically detect ectotrix spores of trichop hyton verrucosum. the coat samples w ere th en subjected to fungal cultu re: sample toothbrushes w ere gen tly imprinted onto th e surface of 9 cm petri dishes con taining sabou raud dextrose agar (with chloramph enicol 0.5% and actidione 0.4% added) on one half and dermatophytes tes t mediu m (dtm) agar on the other half. an aliquot of samples was cultured on sabouraud dextrose agar supplemented with inositol and thiamine to obtain more easily the possible grow th of trichophyton verrucosum, a dermatophyte difficult to cultivate in normal dermatoph yte media (peano et al. 2008). the petri dishes were incubated upside down in two laboratory oven (micra 9t, i.s.co srl, pieve emanu ele, italy) in th e dark, both at a constant temperature of 25 °c and 37° c, and examined daily for 3 w eeks. after 3 weeks (4 weeks for dishes at 37 °c), th e dermatophyte colonies on th e medium were macroscopically and microscopically examined and identified to species level. macroscopic and microscopic examinations were also used to identify saprophytic fungi and yeas ts, but only to th e g enus level. f or each sample with r. perego et al. int. j. of health, animal science a nd food safety 2 (2014) 8-14 11 haf © 2013 vol. i, no. 2 issn: 2283-3927 saprophytic g rowth, the three predominant colony types w ere selected for saprophyte identification. 3 results no deer included in this study presen ted with cutaneous lesions oth er than those inflicted by gunshot. no dermatophyte spores or h yphae were identified in an y sample using direct hair examination and no abnormalities (at the level of the bulb, shaft or tip) w ere identified in an y hair samples. dtm colou r change did not occur with any sample of cultu red hair and no dermatophyte colonies were grown. only saproph ytic fungi were g rown predominantly alternaria spp., mucor spp., and cladosporium spp. (table i) samples from three subjects (10%) showed no fungal colon y g rowth, while two or more colonies were g rown in samples from 15 subjects (50%). table 1 saprophytic fungi isolated alone or togeth er on the sam e subject in th e study deer population. th e isolation could be pure or a deer could have more of a saproph yte . genus number of isolation % penicillum spp. 4 9.1 alternaria spp. 13 29.5 mucor spp. 10 22.7 cladosporium spp. 7 16.0 fusarium spp. 6 13.6 candida spp. 4 9.1 total 44 100 4 discussion this is the first study of the presence and prevalence of fungi on the haircoat of wild red deer and one of the first on the fungal flora carried ou t on wild deer. the results of our study did not reveal the presence of asymptomatic carriers of dermatophy tes in th e red deer sampled population of stelvio national pa rk; in the investigated geographical area seem s therefore unlikely that the red deer act as a source of dermatophyte transmission to other wild animals, livestock or people with whom th ey migh t com e into contact. due to th e absence of previous studies it is difficult to compare the results of this study with published literatu re. in a study published in 2013 by nemeth et al. on th e inciden ce, clinical manifes tations an d demograph y of bacterial and parasitic dermatological diseases in white-tailed deer in th e southeastern united states an incidence of 5.7% of fungi (n.5 subjects on 88 deer with positiv e r. perego et al. int. j. of health, animal science a nd food safety 2 (2014) 8-14 12 haf © 2013 vol. i, no. 2 issn: 2283-3927 isolation) was reported. how ever, the study did not specify wh eth er the fungal isolates were dermatophytes, or wh ether isolations came from asymptomatic animals or th ose with dermatologic lesions. th e on ly study of prevalence previously reported by hall et al. in 2011 studied 60 adult whitetailed deer in virginia (usa) but failed to isolate an y dermatophytes. . in our study fawns w ere well represen ted and th ere have been an ecdotal reports of outbreaks of dermatophytosis caused by fawns (hall et al. 2011). young animals are much more susceptible to infection, probably due to their naive immune system (stannard and white 2002). nevertheless, all th e fawns examined in our study, w ere n egative for dermatophytes by culture. the results of ou r study may be influ enced by th e sample group, g eographic limitations and season. the sample group consisted of only 30 animals. this low number of animals cannot be truly representative of the en tire deer population in th e park, (nearly 10,000 reed deer) notwithstanding the fact that th ere should have been a random selection of animals killed by hunters and the study group had a good representation of both sexes and the various age groups. all red deers were from a limited geographic region and therefore represen t a small subset of the italian red deer population. finally the deer w ere sampled in the late fall/early winter, during cooler weather, whereas dermatophyte flora is lik ely to be more prevalen t in hot, humid weather (stannard and white 2002). deer culling in the stelvio national park is always done in th e fall/ winter season because at this tim e th e deer population is closer to th e more accessible areas of the park wh ere the rangers place supplem entary sources of food. in calves of domes tic cattle, dermatoph ytosis is usually a self-limiting diseas e with a maximum duration of 4 m onths (stannard and white 2002). it is possible that in th e red deer population outbreaks occur in fawns during spring and summer and resolv e by the fall. the saproph ytic fungi isolated from th e haircoat of 90% of th e red deer in this study were similar to those reported in the usa on whitetailed deer (hall et al. 2011), showing that th e different g eographic area does not affect the commensal flora of the wild deer. 5 conclusion in conclusion, studies on a larger sample of red deer, or with hair samples taken in other seasons, would be needed to confirm that red deer population from the stelvio national park doesn’t act as a reservoir for dermatoph ytes in this area . it would be also in teresting to evaluate the prevalence of zoophilic dermatophytes and dermatophytosis in oth er wildlife, livestock and people living in the same g eographic area. 6 acknowledgements the auth ors thank ferruccio tomasi (president) and luca pedrotti (scientific coordinator) of the consortium of stelvio national pa rk and alessandro gugiatti and andrea zanoli of th e managemen t committee for th e lombardy region of th e consortium of stelvio national park for th eir technical assistance and collaboration. r. perego et al. int. j. of health, 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(b.p. smith, ed.). mosby in c., st. louis, missouri, 1213–1214. wobeser g., mitchum s.a., saunders j.r., hunt h.m., 1983. dermatom ycosis (ringworm ) in mule deer (odocoileus hemionus). can vet j. 24 (10), 316–317. 24 l keywords fresh cheeses, microbiological quality, unpasteurized milk, food safety. pages 24 – 31 references vol. 1 no. 2 (2014) article history submitted: september 24, 2014 revised: october 27, 2014 accepted: october 28, 2014 published: november 11, 2014 corresponding author erica tirloni, dipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare (vespa) università degli studi di milano via g. celoria 10, 20133, milano, italy e-mail: erica.tirloni@unimi.it phone: +39 02 50317855 fax: +39 02 50317870 journal home page riviste.unimi.it/index.php/haf concerns about the microbiological quality of traditional raw milk cheeses: a worldwide issue erica tirloni 1,* , simone stella, cristian bernardi 1 dipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare (vespa), università degli studi di milano, milano, italy. abstract. six types of unripened raw milk fresh (robiola, crescenza, primo sale and formaggella) and “pasta filata” cheeses (mozzarella and burrata) were evaluated for microbiological parameters. no listeria monocytogenes or salmonella spp. were detected, but high microbial counts were revealed. significantly higher total viable counts (tvc) (p=0.002) and enterobacteriaceae counts (p<0.001) were observed in “fresh cheese” than in “pasta filata” samples. values > 6 log cfu/g were found in 81.3% of fresh vs 50% in pasta filata for tvc and 65.6% vs 12.5% for enterobacteriaceae, respectively. an evident contamination by escherichia coli, coagulasepositive staphylococci and pseudomonas spp. was detected in all the cheeses: the causes could be the improper hygiene of the artisanal production practices and the permanence of the cheeses on the refrigerated shelves. a careful attention to the respect of the good manufacturing practices is suggested to avoid the presence of initial high bacterial loads. e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 25 haf © 2013 vol. i, no. 2 issn: 2283-3927 1 introduction recently, in south europe a number of small-scale dairies have been consolidating their market, especially those close to rearing facilities, and some of them use raw milk for cheese production. the production of raw milk dairy products has brought forward certain food safety concerns as unpasteurized milk is recognized to be responsible for foodborne diseases (de buyser et al. 2001; ryser 2001). with the dairy farms serving as possible reservoirs (jayarao et al. 2006), artisanal dairy products obtained from raw milk in small processing facilities could be extremely risky for the presence of potential pathogenic bacteria (schoder et al. 2003; latorre et al. 2009). environmental conditions such as temperature, ph, water activity, salt concentration and competing microflora are the main factors that influence the growth of pathogenic bacteria in raw milk and in dairy products. raw milk is known to be characterized by a complex microbial community: its high water content and neutral ph allow the growth of several microorganisms, including those of technological relevance like lactic acid bacteria (lab), but also several spoilage or potentially pathogenic species can affect the quality and the hygiene of dairy products with severe repercussions (oliver et al. 2005; mendonça moraes et al. 2009). in addition, many studies where milk was voluntarily inoculated with pathogenic bacteria have shown that these microorganisms are able to survive during the manufacturing process and/or the ripening period (d’amico et al. 2010). thus, the combination of high initial microbial loads (due to use of unpasteurized milk and to the application of improper traditional practices) and the lack of bacterial inactivation (due to the absence of ripening) can lead to an increase of microbial risk level of the products. the aim of the study was the microbiological evaluation of different types of unripened, raw milk cheese, produced in an artisanal small dairy plant in italy in order to measure the loads of spoilage microorganisms and detect the eventual presence of foodborne pathogens. 2 materials and methods 2.1 experimental design samples of unripened, raw milk cheeses were obtained from a small artisanal cheese dairy retail placed in italy and analysed in order to evaluate the microbial contamination. a total of 6 different cheeses were considered, grouped as “fresh cheeses” (crescenza, robiola, primo sale, formaggella) and “pasta filata cheeses” (mozzarella and burrata). a total of 8 samples for each type of cheese were collected, during four sampling sessions within the period mayjuly 2012. the samples were taken from the refrigerated shelf after a mean exposition time of 2 days (except for crescenza, that was sampled after 5 days of exposition), then transported to the laboratory at a standard refrigeration temperature (4°c) and immediately analysed. 2.2 microbiological analyses total viable count (tvc) was determined according to the iso 4833:2003 method. the number of enterobacteriaceae was determined by the iso 21528-2:2004 method. escherichia coli were enumerated according to the iso 16649-2:2001 method. coagulase-positive e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 26 haf © 2013 vol. i, no. 2 issn: 2283-3927 staphylococci were determined by the iso 6888-1:1999 method. pseudomonas spp. were enumerated on pseudomonas selective agar with cfc supplement (biogenetics, ponte san nicolò, i), incubated at 30°c for 48 hours. yeasts and moulds were enumerated according with iso 21527-1:2008 method. salmonella spp. detection was performed by the methods iso 6579:2002/cor 1:2004. for microbial counts, 10 g of each sample were homogenized in 90 ml of a diluent solution (0.85% nacl and 0.1% tryptone), and serial 10-fold dilutions were prepared. detection of l. monocytogenes was performed according to afnor brd 07⁄4–09⁄98 method. at the same sampling times, ph was measured by a ph meter (amel instruments, milano, i): three independent measurements were performed on each sample and means were calculated. 2.3 statistical analysis the values obtained from microbial counts were grouped in the two categories “fresh cheeses” and “pasta filata cheeses” and compared by student t test. the threshold for statistically significant differences was settled at p<0.05. 3 results and discussion the results obtained from the enumeration of total viable counts (tvc), enterobacteriaceae, escherichia coli, coagulase-positive staphylococci, pseudomonas spp., yeasts and moulds are reported in table 1: the tvc in fresh cheeses (crescenza, primo sale, formaggella and robiola) ranged from 4.8 and 8.2 log cfu/g while in pasta filata cheeses (mozzarella and burrata) ranged between 4.1 and 8.6 log cfu/g. considering enterobacteriaceae, values in fresh cheeses ranged from 4.1 and 7.6 log cfu/g while in pasta filata cheeses ranged between 2.8 and 8.0 log cfu/g. pseudomonas spp. in fresh cheeses ranged from 3.7 and 7.7 log cfu/g while in pasta filata cheeses ranged between 4.1 and 6.2 log cfu/g. considering coagulase positive staphylococci in fresh cheeses the loads were between the not detectable load (<2 log cfu/g) and 5.5 log cfu/g; log cfu/g, while in pasta filata cheeses these microorganisms ranged between <2 and 3.3 log cfu/g. yeasts in fresh cheeses ranged from 3.6 and 7.6 log cfu/g while in pasta filata cheeses ranged between <2 and 5.1 log cfu/g. no evident contamination of the products with moulds was detected, except for formaggella, that was the only product in which moulds were above the detection limit, with a mean value exceeding 5 log cfu/g. all the pathogens researched were not detected in any type of cheese analysed. based on the physical-chemical characteristics, the typologies of cheeses considered in our study represented an optimal substrate for bacterial growth. the richness in nutrients and water content were associated with ph values that were not sufficiently low to result in a microbial inhibition, both in “fresh cheeses” (mean values ranging between 5.25 – 5.55) and especially in “pasta filata cheeses” (mean values ranging between 6.44 – 6.46). considering the potential presence of pathogenic bacteria in cheese samples, we must consider that, according to reg. (ec) n. 1441/2007, in cheeses made from raw milk or milk that has undergone a lower heat treatment than pasteurisation, salmonella spp. should be absent in 25 g. moreover, in ready-to-eat foods able to support the growth of l. monocytogenes, like e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 27 haf © 2013 vol. i, no. 2 issn: 2283-3927 raw milk fresh cheeses, this pathogen must be absent in 25 g. in our study, none of these two microorganisms was detected in any sample. data from microbial counts are reported in table 1 (ranges) and in figure 1 (frequency distribution of raw data). generally, a poor hygienical quality of all the types of cheese was observed: 81.3% of fresh cheese samples (robiola, crescenza, primo sale and formaggella) and 50% of “pasta filata cheese” samples (mozzarella and burrata) were found to be characterized by tvc loads higher than 6 log cfu/g, and in particular 50% of fresh cheese samples resulted to be higher than 7 log cfu/g. crescenza samples were characterized by the highest loads: all the samples, showed values higher than 6 log cfu/g; similar situations were detected for the other “fresh cheese” typologies except for primo sale. in “pasta filata cheese” samples, the melting phase had probably determined a reduction of microbial loads, resulting in significantly lower (p=0.002) tvc values than those obtained from “fresh cheeses”. a similar trend was observed for enterobacteriaceae counts, with frequent presence of high values (fig. 1), especially in crescenza samples (87.5% of the values > 6 log cfu/g); also for this parameter, significantly lower counts (p<0.001) were detected in “pasta filata cheese” samples. high loads of pseudomonas spp. were detected in all the products analysed, but without significant differences among the cheeses; our data were comparable with those obtained by lanciotti et al. (2004) in crescenza cheeses obtained from homogenized milk after 6 days of storage where the counts reached 6.38 log cfu/g: these microorganisms, especially p. fluorescens, are considered the main responsible for the production of bitter peptides in traditional crescenza (ottaviani and disegna 1987). the contamination by yeasts was widespread in “fresh cheeses”, with 66.7% of the samples exceeding 5 log cfu/g; also for this parameter, the highest mean value was observed in crescenza samples, according to the studies of fleet (1990) and sarais et al. (1996), who detected in crescenza and other soft cheeses a large number of these microorganisms (>6-7 log cfu/g), responsible of cheese spoilage and unpleasant flavours. limited, significantly lower counts (p<0.001) were detected in “pasta filata” cheeses (table 1). our data were consistent with those obtained by brooks et al. (2012) who found presence of yeasts in 58.3% of analysed raw milk cheeses even if they found a lower rate (17.1%) of those that exceeded the load of 5 log cfu/g. table 1 ranges of microbiological loads (expressed as log cfu/g) and ph, obtained from analyses tvc enterobacteriaceae pseudomonas spp. e. coli cps yeasts moulds ph fresh cheeses crescenza 6.9-7.9 5.9-7.5 4.6-7.7 2.0-7.5 <2.0-5.4 4.6-7.6 <2.0 5.55 robiola 6.0-7.7 5.9-7.1 3.9-7.2 2.7-7.0 2.6-5.5 4.3-7.4 <2.0 5.25 primo sale 4.8-8.2 4.1-7.6 3.7-7.7 2.0-8.0 <2.0-3.0 3.6-6.3 <2.0 5.45 formaggella 5.8-7.5 5.6-6.9 6.4-7.0 <2.0-5.7 <2.0 4.0-6.8 4.8-5.7 5.50 pasta filata cheeses mozzarella 5.0-7.1 3.2-6.0 4.5-5.6 2.0-6.0 <2.0-3.0 <2.0-4.7 <2.0 6.46 burrata 4.1-8.6 2.8-8.0 4.1-6.2 <2.0-7.9 2.0-3.3 <2.0-5.1 <2.0 6.44 the mean values are calculated using the countable values. tvc= total viable count; cps= coagulase positive staphylococci e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 28 haf © 2013 vol. i, no. 2 issn: 2283-3927 figure 1: frequency distribution of microbial counts in the samples. c = crescenza, r = robiola, p = primo sale, f = formaggella, m = mozzarella, b = burrata considering coagulase-positive staphylococci, 75% of the samples of robiola and crescenza exceeded the “m” limit indicated in reg. (ec) n.1441/2007 for raw milk cheeses (4 log cfu/g), while in all the other cheeses all the samples were below this limit, with constant low values (<2 log cfu/g) in formaggella samples. these results should be carefully considered, as staphylococcus aureus counts between 3 and 5 log cfu/g are recognized to be able to produce amounts of enterotoxins that could be of concern for consumers. more limited counts were detected in “pasta filata cheese” samples, according to the data obtained by dambrosio et al. (2013) who found mean staphylococci counts of 3 log cfu/g in burrata samples produced in puglia (italy). raw milk cheeses are generally known as potentially contaminated by staphylococci and this characteristic is also well recognized by the eu regulation which settled a higher tolerance level if compared to fresh cheeses produced with e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 29 haf © 2013 vol. i, no. 2 issn: 2283-3927 milk that has undergone a lower heat treatment than pasteurization (m=2 log cfu/g) and pasteurized milk (m=1 log cfu/g). the counts of escherichia coli were very variable among the samples for all the cheese typologies, with the mean values ranging from 3.2 to 4.8 log cfu/g. in previous studies, coia et al. (2001) found that 98.6% of 735 raw-milk cheeses had e. coli counts below 4 log cfu/g and öksüz et al. (2004) detected that 82% of a total of 50 white pickled cheese manufactured from raw milk were characterized by loads below 3.8 log cfu/g, while in the current work, the rate of samples with e. coli counts <4 log cfu/g was evidently lower (42.3%). these parameters must be considered as potential hygienic criticisms, also if they cannot be linked to an actual risk for consumers. european legislation does not provide for limits for raw milk cheeses, even if a m limit of 2 log cfu/g is settled by reg. (ec) n. 1441/2007 for cheeses made from milk or whey that has undergone heat treatment. the high loads detected could be due to an improper hygiene of the artisanal production practices and to the permanence of the cheeses on the refrigerated shelves (2 to 5 days depending on the typology), that increases the possibility of microbial replication. improvements should be applied considering the usual transport and home storage procedures, that generally result in a further increase of microbial loads and in a limited residual shelf-life. a careful attention to the respect of the good manufacturing practices is suggested in order to avoid the presence of starting high bacterial loads. moreover, a punctual control of the refrigerator temperature, coupled with a reduction of the shelf permanence, especially in the warm season, when temperature fluctuations are more likely, should be recommended. 4 conclusion the picture emerged from the microbiological monitoring of 6 different types of raw milk cheeses obtained from a small dairy plant, representative of a popular dairy retail typology in italy, showed generally a very poor hygienical quality. an improvement of good manufacturing practices according with a more careful control of storage conditions is needed. 5 acknowledgements we would like to thank prof. patrizia cattaneo for her valuable review of this manuscript. references association francaise de normalisation – afnor, 1998. detection of listeria monocytogenes and listeria spp. afnor brd 07⁄04–09⁄98. http://www.sciencedirect.com/science/article/pii/s095671350300121x e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 30 haf © 2013 vol. i, no. 2 issn: 2283-3927 brooks j.c., martinez b., stratton j., bianchini a., krokstrom r., hutkins r., 2012. survey of raw milk cheeses for microbiological quality and prevalence of foodborne pathogens. food microbiology. 31, 154-158. cardello a.v., maller o., 1982. acceptability of water, selected beverages and foods as a function of serving temperature. journal of food science. 47, 1549-1552. coia j.e., johnston y., steers n.j., hanson m.f., 2001. a survey of the prevalence of escherichia coli o157 in raw meats, raw cow's milk and raw-milk cheeses in south-east scotland. international journal of food microbiology. 66, 63-69. d’amico d., druart m., donnelly c.w., 2010. behaviour of escherichia coli o157:h7 during the manufacture and aging of gouda and stirred-curd cheddar cheese manufactured from raw milk. journal of food protection. 73 (12), 2217-2224. dambrosio a., quaglia n.c., saracina m., malcangi m., montagna c., quinto m., lorusso v., normanno g., 2013. microbiological quality of burrata cheese produced in puglia region: southern italy. journal of food protection. 76, 1981-1984. de buyser m.l., dufour b., maire m., lafarge v., 2001. implication of milk and milk products in food-borne diseases in france and in different industrialised countries. international journal of food microbiology. 67, 1-17. european commission, 2007. reg (ec) n 1441/2007 of 5 december 2007 amending regulation (ec) no 2073/2005 on microbiological criteria for foodstuffs official journal of the european union. l 322, 7 december 2007. fleet g.h., 1990. yeasts in dairy products. journal of applied bacteriology. 68, 199-211. fox p.f., mcsweeney p.l.h., cogan t.m., guinee t.p. (eds), 2004. cheese: chemistry, physics and microbiology, third edition. elsevier academic press, london, 249. international organization for standardization – iso, 1999. microbiology general guidance for enumeration of staphylococcus aureus colony count technique iso 6888-1:1999. international organization for standardization – iso, 2001. microbiology general guidance for the detection of beta-glucuronidase-positive escherichia coli colony-count technique at 44 degrees c using 5-bromo-4-chloro-3-indolyl beta-d-glucuronide iso 16649-2:2001. international organization for standardization – iso, 2003. microbiology of food and animal feeding stuffs – horizontal method for the enumeration of microorganisms – colony count technique at 30 degrees iso 4833:2003. international organization for standardization – iso, 2004. microbiology of food and animal feeding stuffs horizontal methods for the detection and enumeration of enterobacteriaceae part 2: colony-count method iso 21528-2:2004. international organization for standardization – iso, 2004. microbiology of food and animal feeding stuffs horizontal method for the detection of salmonella spp iso 6579:2002/cor 1:2004. international organization for standardization – iso, 2008. microbiology general guidance for enumeration of yeasts and moulds colony count technique at 25 degrees c part 1: colony count technique in products with water activity greater than 0,95 iso 21527-1:2008. e. tirloni et al. int. j. of health, animal science and food safety 2 (2014) 24 31 31 haf © 2013 vol. i, no. 2 issn: 2283-3927 jayarao b.m., donaldson s.c., straley b.a., sawant a.a., hegde n.v., brown j.l., 2006. a survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in pennsylvania. journal of dairy science. 89, 2451-2458. lanciotti r., chaves-lopez c., patrignani f., paparella a., guerzoni m.e., serio a., suzzi g., 2004. effects of milk treatment with dynamic high pressure on microbial populations, and lypolitic and proteolytic profiles of crescenza cheese. international journal of dairy technology. 57, 19-25. latorre a.a., van kessel j.s., karns j.s., zurakowski m.j., pradhan a.k., zadoks r.n., boor k.j., schukken y.h., 2009. molecular ecology of listeria monocytogenes: evidence for a reservoir in milking equipment on a dairy farm. applied and environmental microbiology. 75, 1315– 1323. mendonça moraes p., nogueira viçosa g., keizo yamazi a., tassinari ortolani m.b., nero l.a., 2009. foodborne pathogens and microbiological characteristics of raw milk soft cheese produced and on retail sale in brazil. foodborne pathogens and disease. 6(2), 245-249. öksüz ö., arici m., kurultay s., gümüs t., 2004. incidence of escherichia coli o157 in raw milk and white pickled cheese manufactured from raw milk in turkey. food control. 15, 453-456. oliver s.p., jayarao b.m., almeida r.a., 2005. foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. foodborne pathogens and disease. 2, 115-129. ottaviani f., disegna l., 1987. muffe e lieviti nei prodotti e negli ambienti caseari. latte. 12, 779811. ryser e.t., 2001. public health concerns. in: marth, e.h., steele, j.l. (eds). applied dairy microbiology (2 nd ed). marcel dekker inc, new york, 397-546. sarais i., piussi d., aquili v., stecchini m.l., 1996. the behavior of yeast population in stracchino cheese packaged under various conditions. journal of food protection. 59, 541-544. schoder d., kareem a., baumgartner w., wagner m., 2003. a case of sporadic ovine mastitis caused by listeria monocytogenes and its effect on contamination of raw milk and raw-milk cheeses produced in the on-farm dairy. journal of dairy research. 70, 395–401. 9 l keywords apparent phosphorus digestibility; chicks; low-phosphorus; phosphorus output. pages 9 – 17 references vol. 2 no. 1 (2015) article history submitted: january 29, 2015 revised: march 29, 2015 accepted: march 31 2015 published: april 26 2015 corresponding author shu geng wu, feed research institute, chi nese academy of agricultural sciences, beijing, china zhongguancun nandajie 12, 100081, beijing, china e-mail: wushugeng@caas.cn phone: +86 1082106097 fax: +86 1082106054 journal home page riviste.unimi.it/index.php/haf effect of phytase supplementation on apparent phosphorus digestibility and phosphorus output in broiler chicks fed lowphosphorus diets. xian r. jiang1, 2, fa h. luo1, shu g. wu1*, hai j. zhang1, valentino bon tempo2, ming r. qu3, hong y. yue1, guang h. qi1 1 key laboratory of feed biotechnology of the ministry of agriculture, feed research institute, chinese academy of agricultural sciences, china. 2 dipartimento di scienze veterinarie per la salute, la produzione animale e la sicurezza alimentare, università di milano, italy. 3 animal science and technology college, jiangxi agricultural university, china. abstract. this study was conducted to evaluate the effect of supplemental phy tase in broiler chicks fed different low levels of total phosphorus (p) on the apparent phosphorus digestibility (apd) and phosphorus output (po) in the faeces and ileal digesta. after fed a standard broiler starter diet from day 0 to 14 post-hatch, a total of 144 male broiler chicks were allocated to 6 groups for a 7-d experiment with a 2 × 3 fac torial design comparing phy tase (supplemented without (ctr) or with 400 ftu/kg phytase (phy)) and total p levels (2.0, 2.5 and 3.0 g/kg). the faecal samples were collected from day 17 to 2 1 post-hatch. at 22 days of age, all the chicks were slaughtered and collected the ileal digesta. phytase supplementation signific antly (p < 0.01) increased apd and decreased po in the faeces and ileal digesta in comparison with the ct r group. in addition, po in the faeces expressed as g/kg dm diets and faeces (diet × p level, p = 0.047 and < 0.01, respectively) as well as po in the ile al digesta expressed as g/kg dm digesta (diet × p level, p = 0.04) were affected by diet and p level, which were due to the significant reduction (p < 0.01) by phy supplementation to the diets with 3.0 g/kg total p. the results evidenced that supplemental phytase improved the apd and po when chicks was fed 3.0 g/kg total p diet, while lower total p levels may limit exogenous phytase efficacy. x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 10 haf © 2013 vol. ii, no. 1 issn: 2283-3927 1 introduction monogastric animals, such as poultry and pig, have virtually no phytase activity of th eir own. thus, the availability of phosphorus (p) in feedstuffs of plant origin is generally very low , ranging from 30 to 40% (nelson et al., 1968). to increase p bioavailability, the mos t common ly used method is supplemen ting high dosage of inorganic p in feed, which leads the excretion of large amounts of p in animal manure. consequently, the cost of feed and the environmen tal adverse impact are increased. moreover, ph ytate limits th e availability of s everal oth er essential nutrien ts, such as minerals, protein and amino acids (bieh l and baker, 1996). man y studies showed that microbial phytase can be used to increase the availability of p and reduce its excretion (waldroup et al., 2000; paik, 2003). previous studies have mainly focused on th e utilization of 3-phytase (ec 3.1.3.8) derived from the aspergillus niger (panda et al., 2007) as feed additiv es for broilers. shieh and ware (1968) reported that 3 -phytase can catalyze th e conversion of myo-inositol hexakisphosphate and water to 1l-myo-inositol 1, 2, 4, 5, 6pentakisphosphate and orthoph osphate. the interes t in the supplem entation of ph ytas e in low p diets for monogastric animals has got great atten tion du e to environmen tal concerns and high cost of inorganic p. man y previous studies demonstrated that phytase supplemen tation to low -p diets improved performance and p utilization of broiler chicks (viveros et al., 2002; rutherfurd et al., 2004; 2012; jiang et al., 2013). however, it might be also interesting to investigate wheth er phytas e supplementation could consistently im prov e p use of broiler chicks fed th e p deficient diets even far from th e requiremen t for normal growth. therefore, we conducted this study to evaluate the effect of supplemen tal ph ytase on th e apparen t p diges tibility and p output in th e faeces and ileal digesta of broiler chicks fed differen t low -p lev els of diets. 2 materials and methods 2.1 bird husbandry and dietary treatments all the experim ental procedu res w ere approved by the animal care and use committee of the feed research institute of the chinese academ y of ag ricultural sciences. the feeding trial of this study was carried out in nan kou pilot base of the chinese academ y of agricultural sciences. two hundred arbor acres male broiler chicks were obtained from a local hatch ery, wing-banded, and reared in electrically heated battery brooders maintained at temperatures of 33°c from day 1 to day 7 pos t-hatch and 30° c from day 8 to day 14 post-hatch. during this time, chicks w ere provided free access to water and a standard maize soybean meal starter diet containing 230 g of cp/kg, 13.39 mj of men/kg, 10.0 g of ca/kg, and 6.8 g of total p/kg. at day 15 post-hatch, after overnigh t feed withdrawal, a total of 144 chicks were w eighed and divided in to 6 homog eneous g roups. six replicate cages (60×50×50 cm, length×width×h eight) were used per treatm ent with 4 chicks per cage. chicks had free access to feed and water from d 15 to 21 post-hatch, and battery temperatu re was maintained at 27°c du ring this period. lights w ere con tinuously on the first day post-hatch, after which a x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 11 haf © 2013 vol. ii, no. 1 issn: 2283-3927 23l:1d lighting schedule was maintained all through the duration of the feeding trial. feed and water were provided ad libitum throughout the trial. the experimen t was a 2×3 factorial arrangem ent of th e treatm ents with diet (unsupplemen ted control [ctr] or supplemented with 400 ftu/kg phytase [phy]) and 3 total phosphorus levels (2.0 g/kg [2.0p], 2.5 g/kg [2.5p] and 3.0 g/kg [3.0p] ). 1 ftu is the amoun t of enzym e which liberates 1 μmol of inorganic phosphate per minute from sodium phytate at ph 5.5 and 37°c. the phytase, whose type and source of extraction were 3 -phytase (ec 3.1.3.8) derived from aspergillus neiger, was purchased from basf vitamins co. , ltd., china with activity of 5,000 ftu/g. soybean meal was th e only ph osphorus source. diets (table 1) were semipurified, consisting primarily of cornstarch, dextrose, and soybean meal, and were formulated deceed the nrc (1994) recommendations to ensure maximum responses with phytase. chromium oxide (cr2o3) was incorporated into diets (3.5 g/kg, as-fed) to calculate nutrien t utilization via the index m ethod. diets in pellet form and water were provided a d libitum. mortalities were recorded daily. 2.2 sample collection and chemical analyses after two adaptation days at 15 and 16 days pos t-hatch, faecal samples w ere collected twice per day at 6 am and 6 pm from pans beneath each cage from day 17 to day 21 pos t -hatch for 5 days. at end of each collection period, cage samples were pooled. all the faecal samples were frozen and stored at -20°c before sending to laboratory for digestibility assays. at 22 day post-hatch, chicks were euthanized via carbon dioxide asph yxiation, and ileal dig esta samples were collected by dissecting a segm ent of the ileum defin ed as extending from meck el’s diverticulum to the ileocecal junction. contents of this segment were squeezed into a plastic container, pooled per cag e of 4 chicks, and subsequently lyophilized. freeze-dried digesta an d faecal samples and, along with air-dried diet samples, were g round through a 0.75-mm sieve using a grinding mill to facilitate chemical analyses. determination of dry matter (dm) was performed using the association of analytical communities (aoac, 20 05) official method aoac 930.15. the contents of phosphorus and chromium were analyzed as described by dilg er and adeola (2006). 2.3 calculations and statistical analyses apparent p digestibility (i.e. faecal and ileal digestibility) was calculated using the index method according to the following equation: apd (%) = 100 − [(cri/cro) × (po/pi) × 100] [1] where apd is apparent p diges tibility (calculated for ileal digesta or faecal samples ) expressed as a percen tage, cri is the chromium concentration of dietary in take, cro is th e chromium concentration of outpu t (as analyzed in ileal digesta or faeces), p o is the p x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 12 haf © 2013 vol. ii, no. 1 issn: 2283-3927 table 1. composition and nutrient content of th e experim ental diets (as fed basis) 1 ctr phy 2.0p 2.5p 3.0p 2.0p 2.5p 3.0p ingredient, g/kg corn starch 589.4 490.8 389.3 589.4 490.8 389.3 dextrose 90.0 90.0 90.0 90.0 90.0 90.0 soybean meal 294.2 379.8 465.5 294.2 379.8 465.5 soybean oil 12.0 25.0 40.0 12.0 25.0 40.0 limestone 4.8 6.0 7.0 4.8 6.0 7.0 salt 2.5 2.5 2.5 2.5 2.5 2.5 lysine 3.0 2.0 2.0 3.0 2.0 2.0 methionine 1.0 0.8 0.6 1.0 0.8 0.6 choline chloride 0.6 0.6 0.6 0.6 0.6 0.6 mineral premix 2 1.0 1.0 1.0 1.0 1.0 1.0 vitamin premix 3 1.5 1.5 1.5 1.5 1.5 1.5 phytase, ftu/kg --- 400 400 400 calculated and analyzed composition metabolizable energy, mj/kg 12.27 12.27 12.31 12.27 12.27 12.31 cp, g/kg 132.1 169.6 207.0 132.1 169.6 207.0 analyzed dm, g/kg 892.1 873.7 875.4 872.6 885.0 870.9 analyzed ca, g/kg 2.7 3.4 4.1 2.7 3.4 4.1 analyzed total p, g/kg 2.1 2.7 3.0 2.1 2.7 3.0 1. ctr = basal diet without phytase supplementation; phy = ctr + 400 ftu/kg phy tase; 2.0 p = 2.0 g/kg total p level; 2.5p = 2 .5 g/kg total p level; 3.0 p = 3.0 g/kg total p level. 2. provided the following per kg of diet: cu 8mg, zn 75 mg, fe 80 mg, mn 100 mg, se 0.15 mg, i 0.35 mg. 3. provided the following per kg of diet: re tinyl acetate, 4.3 mg; cholecalcipherol, 0. 0625 mg; dl-alpha-tocopherol, 18.75 mg; menadione, 2.65 mg; cyanocobalamin, 0.02 5 mg; biotin, 0.0325 mg; folic acid, 1.25mg; niacin, 50 mg; d -pantothenic acid, 12 mg; riboflavin, 6 mg; thiamin, 2 mg concen tration of output (as analyzed in ileal dig esta or faeces ), and p i is the p concentration of dietary in take. all analyzed values were expressed as grams per kilo g ram of d m. x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 13 haf © 2013 vol. ii, no. 1 issn: 2283-3927 calculation of p output, expressed on a dry matter in take (d mi) basis, utilized th e ratio of chromium intak e to chromium ou tput: po-dmi (g/kg) = po-dmo (g/kg) × (cri/cro) [2] where po-dmi and p o-dmo repres ent p ou tput concentrations (as analyzed in ileal digesta or faeces) on dmi and dm output bases, respectively, and cri and cro represen t chromiu m concen trations of intak e and output (of either ileal dig esta or faeces), res pectively. data were analyzed as a com pletely randomized block design by anova, as implemen ted in the mixed procedu re of sas v. 9.2 (sas institu te inc., cary, nc). a model for a 2×3 factorial design was applied. the model statemen t included th e effects of diet (ctr and phy), total p levels (2.0, 2.5 and 3.0 g/kg) and interactions among those factors. cage served as th e experimen tal unit for all statistical analys es. treatmen t differences were assessed by using th e least squares means with a tukey adjustmen t. treatment effects were considered significan t at p ≤ 0.05. 3 results graded lev els of dietary p in take and th e supplem entation of ph ytase, did not affect normal diges tive functions as was reflected by changes in apparen t dm diges tibility values (table 2). phytase supplem entation significantly ( p < 0.01) increased apd and decreased po in the faeces and ileal digesta in comparison with the ctr group, when these were expressed as g/kg dm diges ta, faeces or diets. compared with th e ctr chicks fed 3.0p, diet ary ph y increased apd in faeces (lin ear contrast, p = 0.02) and ileal digesta (lin ear con trast, p = 0.02) of 3.0p chicks. phy supplem entation decreased po in the ileal diges ta of birds fed 2.5 g/kg (lin ear contrast, p < 0.01) and 3.0 g/kg (linear contrast, p = 0.010) total p diets compared to the ctr chicks fed 2.5p and3.0p, respectively, when th ese were expressed as g/kg dm digesta. birds fed 2.0p had less po in the faeces than fed 3.0p (linear con tras t, p < 0.01), when these were expressed as g/kg dm faeces. however, compared to th e birds fed 2.0p, chicks fed 2.5p had an increased apd in th e ileal diges ta (lin ear contrast, p = 0.04), and chicks fed 3.0p had an increased apd in the faeces (linear contrast, p = 0.02). moreover, apd in th e faeces of 2.5p chicks and in the ileal digesta of 3.0p chicks had tendencies to be higher than in th e corresponding parts of 2.0p birds (lin ear contrast, p = 0.051 and 0.056, respectively). in addition, po in the faeces expressed as g/kg dm diets and faeces (diet × p level, p = 0.047 and < 0.01, respectively) as well as po in the ileal diges ta expressed as g/kg d m diets and digesta (diet × p lev el, p = 0.04) were affected by diet and p level, and po in th e ileal digesta expressed as g/kg dm diets had a tendency to be affected by diet and p lev el (diet × p level, p = 0.059). the interactions can be attributed to the significan t reduction (lin ear contrast, p < 0.01) by ph y supplementation to the diets with 3.0 g/kg total p, bu t no differences were found in others total p levels diets. x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 14 haf © 2013 vol. ii, no. 1 issn: 2283-3927 table 2. effect of ph ytase on total p ou tput, and apparent p digestibility in the faeces and ileal diges ta of chicks fed different levels of p ctr phy p-value 2.0p 2.5p 3.0p 2.0p 2.5p 3.0p sem phytase p level interaction no of pen 6 6 6 6 6 6 p intake, g/kg of dmi 2.3 3.1 3.4 2.4 3.1 3.5 faeces add, % 81.32 80.23 81.21 80.78 81.54 80.83 1.00 0.87 0.98 0.60 apd, % 57.33 63.52 60.22 60.89 69.17 74.77 2.94 <0.01 0.02 0.16 po g/kg of dmi 0.99b 1.15ab 1.34a 0.94b 0.96b 0.88b 0.08 <0.01 0.19 0.047 po g/kg of dmf 5.24bc 5.80b 7.12a 4.88c 5.16bc 4.60c 0.19 <0.01 <0.01 <0.01 ileal digesta add, % 80.07 78.84 77.95 78.89 82.55 78.66 1.20 0.28 0.15 0.14 apd, % 42.62 49.16 44.86 46.70 65.97 68.39 4.91 <0.01 0.03 0.16 po, g/kg of dmi 1.33 1.60 1.85 1.28 1.06 1.11 0.14 <0.01 0.41 0.059 po g/kg of dmd 6.79 ab 7.53ab 8.45a 6.05bc 6.03bc 5.15c 0.50 <0.01 0.69 0.04 a-c . data in the same row with different superscripts differ significantly (p<0.05). ctr = basal diet without phytase supplementation; phy = ctr + 400 ftu/kg phytase ; 2.0 p = 2.0 g/kg total p level; 2.5p = 2.5 g/kg total p level; 3. 0 p = 3.0 g/kg total p level; po = p output; add = apparent dry matter digestibility; apd = apparent p diges tibility; dmd = dry matter digesta; dmf = dry matter faeces; dmi = dry matter intake. 4 discussion the objectives of the s tudy w ere to determine wh ether dietary phytase added to th e low total phosphorus (p) diets of broiler chicks would consisten tly improve apparent p diges tibility (apd) and p ou tput (po) in faeces and ileal digesta, and whether th ere would be a significan t interaction between ph ytas e supplem entation and levels of low total p in th e diets. ou r previous work confirmed that dietary supplem entation of phytase can improve growth performance with a higher ash content in bon e of chicks fed with low-p diet (4.8 g/kg total p diets) during 8-21 days post-hatch period (jiang et al., 2013), which may be du e to th e improvem ent the availability and absorption of nutrients through increasing the dig estibility of the ingested diets (a budabos, 2012; attia et al., 2012). dilger and adeola (2006) also observed x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 15 haf © 2013 vol. ii, no. 1 issn: 2283-3927 that low-phytate sbm decreased p ou tput and increased p reten tion as compared to th e conventional sem. in the present study, positive results were obtained using 3 -phytas e supplemented to diets with low p (2.0, 2.5 or 3.0 g/kg total p) on apparen t p diges tibility and p output in both faeces and ileal digesta from day 14 to day 21 post-hatch. numerous studies have been conducted that confirm the finding of enhan ced p uptak e in and pou ltry after treatmen t with ph ytase (nelson et al., 1968; waldroup et al., 2000; paik, 2003; rutherfurd et al., 2004). the improvement in p utilization by supplemen ting phytas e may be related to th e increased dephosphorylation of ph ytate in th e small in testi n e. rutherfurd et al. (2004 ) reported that microbial phytase supplemen ted to the diets of chicks improved total p digestibility and increased ph ytate p disappearance in the ileum. in this study, higher po was observed in th e faeces of birds fed with 3.0 g /kg total p diets compared to 2.0 g/kg total p diets, which may be du e to the increased p intak e. however, th e interaction between diet and p level evidenced that the high po in the maximum total p level group was attributed by the unsupplemen ted g roup. mo reov er, the increased adp in th e faeces and ileal digesta of chicks fed with the diets containing 3.0 g/kg total p were due to th e 3-phytase supplem entation. in the present study, apd in the faeces were 57 -63% and in the ileal digesta were 42-49% in the unsupplemen ted chicks fed with low-p diets, which were similar as the finding by sebastian et al. (1996) and rutherfurd et al. (2004). whereas th e correspondin g value in the low-p diet supplem ented with 400 ftu/kg of 3-phytase were 61-75% and 47-68%, respectively. in the present study, phytase improved the adp and po in the faeces of broilers from day 14 to day 21 of ag e mainly reflected in th e birds fed with 3.0 g/kg total p diets, interestingly, the observations in the ileal digesta were in accordance with the findings in faeces. our observation is in agreemen t with the finding by dilger and adeola (2006) that low phytate sbm improved p ou tput (1.122 vs. 1.808 g/kg of dmi) and retention (65.5 vs. 54.1 %) compared to th e conven tional sem mainly in th e groups contained 528 g/kg sem (total p; 3.01 vs. 3.58 g/kg). the limited improvem ents of adp and po in the diets with 2.0 and 2.5 g/kg total p migh t be due to th e levels of th e dietary p concen trations. it is possible that chicks rapidly acclimated to these low p intak es in an attempt to main tain p homeostasis or even “buffer” body p homeostasis for long-term p deficiency. moreov er, th e low total p intak e may provide limited phytate p for dietary phytase to “recycle”, and consequen tly, rare improvemen ts in p digestibility and excrete w ere observed in the 2.0 and 2.5 g/kg total p diets even supplemen ted with 400 ftu/kg 3-phytase. however, it seems likely that the diets con taining 3.0 g/kg total p may possess enough phytate p for dietary phytase to hydrolyze and even tually result in th e significant improvem ents in th e p utilization. although dilger and adeola (2006) concluded that the p use and endogenous p loss were influenced by dietary ph ytate content when broiler chicks were fed p-deficient diets and the levels of total p used were in the ranges of 0.83-3.58 g/kg, it is well known that severe p reductions in starter chicks might have jeopardized th eir skeletal in teg rity, general health status and survival (scott et al., 1982). the levels of low total p in diets of th e studies which eviden ced the improvemen t in grow th performance and p utilization of broiler chicks by that phytase supplem entation were in the rang es of 4.8-6.4 g/kg (viveros et al., 2002; rutherfurd et al., 2004; 2012; jiang et al., 2013), which are much high er than the levels (2.0, 2.5 and 3.0 g/kg) used in this study. thus, phytase supplementation may not consisten tly improve p utilization during the early life stag e of a commercial chicken strain when they are fed severe p-deficien t diets. x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 16 haf © 2013 vol. ii, no. 1 issn: 2283-3927 5 conclusions the observations in this study demons trated that supplemen tation of phytase increased apparen t p diges tibility and deceased p output in both faeces and ileal diges ta when broiler chicks were fed the diets with 3.0 g/kg total p. however, the lower total p levels (2.0 and 2. 5 g/kg) may limit th e effect of exogenous phytas e on p utilization and could not be recomm ended to use. 6 conflicts of interest the authors declare that there are no con flicts of interest. 7 acknowledgements this work was granted by china agriculture research system poultry-related science and technology innovation team of peking, and the national key technology research an d developmen t program (2011bad26b04). references abudabos m.a. (2012). phytate phosphorus utilization and intestinal phytase activity in laying hens. ital. j. anim. sci., 11: 41-46. aoac (2005). official methods of analysis, 18th ed., association official analytical chemis ts, arlington, va, usa. attia y.a., el-tahawy w.s., abd el-hamid a.e., hassan s.s., nizza a., el-kelaway m.i. (2012). effect of phytase with or without multienzym e supplemen tation on performan ce an d nutrien t diges tibility of young broiler chicks fed mash or crumble diets. ital. j. anim. sci., 11: 303-308. biehl r.r., baker d.h. (1996). efficacy of supplem ental 1 alpha-hydroxycholecalciferol and microbial phytase for young pigs fed phosphorus or amino acid-deficien t corn-soybean meal diets. j. anim. sci. 74: 2960-2966. dilger r.n., adeola o. (2006). estimation of true phosphorus digestibility and endogenous phosphorus loss in growing chicks fed conventional and low -phytate soybean meals. poult. sci. 85: 661-668. jiang x.r., luo f.h., qu m.r., bontempo v., wu s.g., zhang h.j., yue h.y., qi g.h. (2013 ). effect of non-phytate phosphorus lev els and ph ytas e sources on the grow th performan ce, seru m biochemical and tibial parameters of broiler chick ens. ital. j. anim. sci. 12: 375-380. x.r. jia ng et al. int. j. of health, animal science a nd food sa fety 1 (2015) 9 17 17 haf © 2013 vol. ii, no. 1 issn: 2283-3927 national research council (1994). nutrien t requirem ents of poultry, 9th rev. ed. natl. acad. press, washington, dc, usa. nelson t.s., sheih t.r., wodzinski r.j., ware j.h. (1968). the availability of ph ytate phosphorus in soybean meal before and after treatmen t with a m old phytase. poult. sci. 47: 1842-1848. paik i.k. (2003). application of ph ytas e, microbial or plan t origin, to reduce phosphorus excretion in poultry produ ction. asian-aust. j. anim. sci. 16: 124-135. panda a.k., rama rao s.v., raju m.v.l.n., gajula s.s., bhanja s.k. (2007). performance of broiler chickens fed low non ph ytate phosphorus diets supplem ented with microbial phytase. j. poult. sci. 44: 258-264. rutherfurd s.m., chung t.k., morel p.c., moughan p.j. (2004 ). effect of microbial ph ytase on ileal digestibility of ph ytate phosphorus, total phosphorus, and ami no acids in a lowphosphorus diet for broilers. poult. sci. 83: 61-68. rutherfurd s.m., chung t.k., thomas d.v., zou m.l., moughan p.j. (2012). effect of a novel phytase on grow th performance, apparent metabolizable energy, and the availability of minerals and amino acids in a low-phosphorus corn-soybean meal diet for broilers. poult. sci., 91: 1118-1127. sas institute (2008). sas/stat user’s guide, version 9.2. sas institute, inc., cary, nc, usa. scott m.l., nesheim m.c., young r.j. (1982). nu trition of th e chicken. 3rd ed. scott m.l. & associates publ., ithaca, ny, usa. sebastian s., touchbu rn s.p., chavez e.r., lague p.c. (1996). the effects of supplemen tal microbial ph ytase on th e performan ce and utilization of dietary calcium, phosphorus, copper and zinc in broiler chickens fed corn-soybean diets. poult. sci. 75: 729–736. shieh t.r., ware j.h. (1968). survey of microorganisms for the production of extracellular phytase. j. appl. microbiol. 16: 1348-1351. viveros a., brenes a., arija i., centeno c. (2002). effects of microbial phytase supplem entation on mineral utilization and serum en zym e activities in broiler chicks fed differen t levels of phosphorus. poult. sci. 81: 1172–1183. waldroup p.w., kersey j.h., saleh e.a., fritts c.a., yan f., stilborn h.l., c rum jr r.c., raboy v. (2000 ). nonphytate phosphorus requirem ent and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphate corn with and withou t microbial phytase. poult. sci. 79: 1451-1459. 15 l keywords soybean meal; whey proteins; piglets; oxidation; meat quality. pages 15 – 26 references vol. 1 no. 1 (2014) article history submitted: november 28, 2013 revised: march 14, 2014 accepted: march 14, 2014 published: march 17, 2014 corresponding author panagiotis simitzis, faculty of animal science and aquaculture department of animal breeding and husbandry agricultural university of athens, greece 75 iera odos street, 118 55, athens, greece e-mail: pansimitzis@aua.gr phone: +30 121 05294449 fax: +30 210 5294442 journal home page riviste.unimi.it/index.php/haf effect of dietary protein source on piglet meat quality characteristics. panagiotis simitzis 1* , george papadomichelakis 2 , eleni tsiplakou 2 , georgios theodorou 1 , george zervas 2 , ioannis politis 1 1 faculty of animal science and aquaculture, department of animal breeding and husbandry, agricultural university of athens, athens, greece. 2 faculty of animal science and aquaculture, department of nutritional physiology and feeding, agricultural university of athens, athens, greece. abstract. an experiment was conducted to examine the effects of different dietary protein sources (soybean meal vs whey protein) on piglet meat quality characteristics. eighteen castrated male large white × duroc × landrace piglets were randomly assigned to 2 groups. piglets were kept in individual metabolic cages and fed ad libitum over a period of 38 days the following 2 diets: diet sb, which was formulated to meet the nutrient requirements of piglets using soybean meal as the main crude protein source and diet wp, where sb was totally replaced by a mixture of whey proteins on equal digestible energy and crude protein basis. at the end of the experiment, piglets were weighed and slaughtered. after overnight chilling, samples of longissimus dorsi muscle were taken and were used for meat quality measurements. no significant differences were observed in the values of ph, color, water holding capacity, shear force and intramuscular fat content of l. dorsi muscle between the dietary treatments. measurement of lipid oxidation values showed that dietary supplementation with different protein sources did not influence meat antioxidant properties during refrigerated storage. the sb piglets had lower c14:0 (p<0.01) and higher c18:3n-3 (p<0.001) levels in intramuscular fat in comparison with wp piglets. however, these changes were attributed to background differences in the dietary fa profile and not to a direct protein source effect. the results of this preliminary study indicate that the examined dietary protein sources do not have a significant effect on meat quality characteristics of piglets. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 16 haf © 2013 vol. i, no. 1 issn: 2283-3927 1 introduction pork is one of the most commonly consumed meats worldwide. according to fao databases, the last decade (2001-2011) the production of pig meat in the european union has increased from 21454984 to 23066700 tn (fao, 2013). during the last decades attempts have been made to improve pig meat products in line with the new dietary guidelines in order to make them more attractive to consumers. additionally, there has been an increased interest in finding ways to manipulate the fatty acid composition of meat, since it is the major source of fat and especially saturated fatty acids, which have been implicated in several diseases associated with the contemporary life in the developed countries. dietary interventions that influence lipid metabolism in farm animals have been therefore employed, since they represent a useful tool to modulate fat deposition and improve meat quality (wood et al., 2008). in pigs, an effort has been made to increase n-3 polyunsaturated fatty acids (pufas) and keep the favorable balance between n-6 and n-3 pufa (ratio of less than 4) (wood et al., 2003). soybean meal and milk products, such as dried whey concentrates are two protein sources widely incorporated in swine diets (mahan, 1993; yun et al., 2005; yang et al., 2007). inclusion of whey proteins in weanling piglets’ diets appears to have a beneficial effect on feed efficiency, nutrient digestibility and growth performance (tokach et al., 1989; grinstead et al., 2000). at the same time, soybean meal (40 %) could replace more expensive protein sources in nursery pig diets without affecting feed intake and growth performance (lenehan et al., 2007). in addition to the meat quality importance, the investigation of such dietary treatments in pigs could serve as a valuable model for studying the intermediary lipid metabolism in humans (carey, 1997). in this sense, numerous studies have focused in investigating the mechanisms underlying the influence of nutritional treatments on the lipogenic process. recent studies have indicated that the source of dietary protein has been shown to affect lipid metabolism. soy protein appears to reduce blood lipid levels and to have a positive effect in cholesterol levels and reduce the risk of diabetes, obesity, cardiovascular and coronary heart disease (sadler, 2004; torres et al., 2006). in detail soy protein, when compared to casein reduces the gene expression of lipogenic enzymes in liver (tovar et al., 2002; ascencio et al., 2004) and this effect is thought to be mediated by the decreased insulin to glucagon ratio, which has been attributed to the lower lysine to arginine ratio and the higher glycine content of soy proteins (tovar et al., 2002). at the same time, soy proteins may stimulate proliferator-activated receptor α (ppar-α) in liver and ppar-γ in adipose tissue increasing, thus, fatty acid oxidation (tovar et al., 2005) and fatty acid uptake from plasma (farmer, 2005), respectively. as it has been already stated, the diet has a direct effect on muscle composition and meat quality in pig, being a monogastric species. sensory properties in turn, such as flavor intensity, tenderness, juiciness and fat tissue firmness significantly influence the shelf life and the acceptability of pig meat products (wood et al., 2003; 2008). whey proteins and, to a lesser extent, soy proteins seem to be particularly attractive ingredients for the inhibition of lipid oxidation processes and the quality improvement in processed muscle foods (peña-ramos and xiong, 2003). however, little is known about the effect of these dietary protein sources on pig meat quality. the objective of this preliminary study was therefore to evaluate the effects of the above dietary protein sources; soybean meal or whey proteins on piglet meat quality characteristics. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 17 haf © 2013 vol. i, no. 1 issn: 2283-3927 2 materials and methods 2.1 animals and diets eighteen castrated male large white × duroc × landrace piglets weaned at 29±2 days of age were selected from a commercial farm located near the city of athens. upon arrival at the experimental facilities, they were allowed an adaptation period of 4 days and were then allotted into 2 groups (9 pigs/group) balanced for body weight (average bw of 8.4 ± 0.68 kg; mean ± sd). piglets were kept in individual metabolic cages (1.2 × 0.5 m) and fed ad libitum the following 2 diets, over a period of 38 days: diet sb, which was formulated to meet the nutrient requirements of piglets (nrc, 1998) using soybean meal (sb) as the main crude protein (cp) source and diet wp, where sb was totally replaced by a mixture of whey proteins [wp, 70% wheypro65 (650 g cp/kg) + 30% wheypro 80 (800 g cp/kg); hellenic proteins s.a., veria, greece], on equal digestible energy (de) and cp basis. the replacement of sb by wp aimed to alter the dietary lysine/arginine ratio (2.12 vs. 0.85; diet wp and sb, respectively) and glycine content (5.5 vs. 8.6 g/kg; diet wp and sb, respectively). the ingredient and chemical composition of the diets are summarized in table 1. at the end of the experiment (72 days of age) piglets were stunned and killed by exsanguination. after overnight chilling, a 10 cm loin section was removed from both sides of carcasses and was used for the determination of meat quality characteristics. handling and care of the experimental animals conformed to the guidelines of the faculty of animal science and aquaculture of agricultural university of athens. 2.2 meat quality measurements 2.2.1 ph24 and color ph was measured using a sentron 1001 ph system (roden, netherlands) model, with the electrode inserted into the centre of the longissimus dorsi muscle at the last rib 24 h after slaughter. the part of the muscle between 12th and 13th ribs was sliced across the fibers, left exposed to the air at room temperature for 30 min and meat color measured (3 measurements per sample) on the cut surface using a miniscan xe (hunterlab, reston, usa) chromameter set on the l*, a* and b* system (cie 1976, commission international de l’ eclairage). 2.2.2 shear force value samples (80 ± 2 g, 2 cm thickness) of l. dorsi muscle from each pig were placed in plastic bags and cooked in a water bath at 80°c for 50 min (internal temperature 72 ± 1°c), left under running water for 30 min and then placed in a refrigerator at about 4°c for 24 h. five sub samples with a cross section of 1 cm 2 were cut parallel to the muscle fibers and shear force value of the l. dorsi muscle was measured using a warner bratzler (wb) shear blade fitted to a zwick testing machine model z2.5/tn1s (zwick gmbh & co., germany). peak force values in newton were recorded. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 18 haf © 2013 vol. i, no. 1 issn: 2283-3927 table 1. ingredient and chemical composition of the experimental diets diet * sb wp in g re d ie n ts ( g /k g ) maize 621.0 754.0 soybean meal (440 g cp/kg) 342.0 whey proteins† (660 g cp/kg) 210.0 l-lysine 80% 2.0 2.0 dl-methionine 99% 1.0 1.0 sodium chloride 5.0 4.0 calcium carbonate 13.0 14.0 monocalcium phosphate 13.0 12.0 mineral-vitamin premix‡ 3.0 3.0 a n a ly z e d c h e m ic a l c o m p o si ti o n (g /k g d m ) dry matter (g/kg) 888.0 903.0 crude protein 232.0 228.0 ether extract 33.1 40.4 total weights of fa (mg/100 g dm) 31.4 38.7 in d iv id u a l f a ( % o f to ta l f a ) c14:0 0.08 2.91 c16:0 14.40 17.87 c16:1 cis9 0.16 0.40 c18:0 2.63 4.42 c18:1 cis9 21.71 24.27 c18:2n-6 54.28 41.42 c18:3n-3 3.35 1.32 c20:0 0.33 0.34 c20:1n-9 0.22 0.20 c22:2 0.60 0.77 c a lc u la te d c h e m ic a l c o m p o si ti o n (g /k g d m ) § digestible energy (mj/kg dm) 15.5 15.4 metabolisable energy (mj/kg) 14.7 14.6 sid §§ crude protein 166 177 sid §§ methionine+cystine 6.9 8.6 sid §§ lysine 11.2 14.2 sid §§ arginine 12.1 5.4 sid lysine/sid arginine ratio 0.93 2.63 sid §§ threonine 7.2 11.2 total glycine 9.7 6.1 calcium 9.2 9.1 total phosphorus 7.4 8.4 sodium 2.4 2.4 * sb, soybean meal as protein source; wp, whey proteins as protein source. † mixture of whey proteins (hellenic proteins s.a., veria, greece); 70% wheypro 65 (650 g cp/kg) + 30% wheypro 80 (800 g cp/kg) designed to have a content of 660 g cp/kg. ‡ mineral-vitamin premix (nuevo s.a., n. artaki, greece) provided per kg of diet: 15000 iu vitamin a (retinyl acetate), 2000 iu vitamin d3 (cholecalciferol), 100 mg vitamin e (dl-α-tocopheryl acetate), 3.5 mg menadione (vitamin k3), 2.5 mg vitamin b1, 6 mg vitamin b2, 3 mg vitamin b6, 25 μg cyanocobalamin, 25 mg nicotinic acid, 20 mg pantothenic acid, 2 mg folic acid, 250 μg biotin, 2 mg co, 4 mg i, 600 μg se, 300 mg fe, 100 mg mn, 100 mg mg, 320 mg cu and 240 mg zn. § digestible and net energy, macro-element, total amino acid and standardized ileal digestible (sid) protein and amino acid values for maize, soybean meal and whey proteins were adapted from tabulated data (fedna, 2003; nrc, 1988). §§ standardized ileal digestible nutrients. no data were available on glycine sid values. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 19 haf © 2013 vol. i, no. 1 issn: 2283-3927 2.2.3 drip loss the left part of l. dorsi muscle was sampled in two positions for each experimental animal and the drip loss was measured applying the ez-driploss method (christensen, 2003). in brief, the longissimus muscle of each experimental animal rested 4 h at approximately 7°c and was then cut into slices, each with a thickness of 2.5 cm. two cylindrical cuts were made with a fixed blade knife of diameter 25 mm. each sample was placed in a special ez-driploss container and remained for storage at 4°c for eight days. all containers were tared before use. the meat samples were then removed from the ez-driploss containers and each container with exudated meat juice was weighed on the scale used for the taring procedure. 2.2.4 measurement of total lipids and lipid oxidation – mda assay measurement of intramuscular total lipids (imf) was made according to the method first described by folch et al. (1957). tissue samples were homogenized with 2:1 chloroform– methanol mixture to a final dilution 20-fold the volume of the tissue sample. the crude extract was mixed with 0.2 its volume of water and it was separated into two phases. the lower phase contained the tissue lipids. lipid oxidation was assessed on the basis of the malondialdehyde (mda) formed during storage. in the present study, mda concentration in l. dorsi muscle samples was determined 1, 3, 6 and 9 days after storage at 4°c using a selective third-order derivative spectrophotometric method, previously developed by botsoglou et al. (1994). derivative versus conventional spectrophotometry was adopted because it offers improved sensitivity, specificity and reliability of the measurements, since it eliminates potential interferences from other reactive compounds. in brief, 2 g of each sample (2 samples per pig) were homogenized (edmund buehler 7400 tuebingen/h04, germany) in the presence of 8 ml aqueous trichloroacetic acid (tca) (50 g/l) and 5 ml butylated hydroxytoluene (bht) in hexane (8 g/l), and the mixture was centrifuged for 3 min at 3000g. the top hexane layer was discarded and a 2.5 ml aliquot from the bottom layer was mixed with 1.5 ml aqueous 2-thiobarbituric acid (tba) (8 g/l) to be further incubated at 70°c for 30 min. following incubation, the mixture was cooled under tap water and submitted to third-order derivative (3d) spectrophotometry (hitachi u3010 spectrophotometer) in the range of 500–550 nm. the concentration of mda (ng/g wet tissue) in analyzed samples was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3-tetraethoxypropane (tep), the malondialdehyde precursor. 2.2.5 fatty acid methylesters synthesis and determination the fa of diets and muscle tissue were hydrolyzed (with methanolic koh) and methylated (sulphuric acid catalysis) directly, according to o’fallon et al. (2007) in duplicate 1 g ground samples. the fa methylesters (fame) were extracted with clear nhexane and transferred into gas chromatograph (gc) vials. the fame were subsequently analyzed in a temperature programmed run using a perkin elmer autosystem xl gc equipped with a 30m×0.25mm i.d.×0.25μm film thickness ηρ-innowax capillary column (agilent technologies, j&w gc g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 20 haf © 2013 vol. i, no. 1 issn: 2283-3927 columns) and a flame ionisation detector (fid). the oven temperature was programmed for 1 min at 140 o c, raised by 2.5°c/min to 200 o c, then to 230 o c by 1°c/min and held for 1 min, and finally to 240 o c by 4°c/min and held for 10 min. helium was the carrier gas at a constant pressure of 18 psi and the temperature of both the injector and fid was set at 250 o c. fatty acids were identified by comparison with fame 37 component mix (supelco, sigma-aldrich co., usa) and quantification was achieved using the internal standard (13:0) added prior to hydrolysis. total weights of fa (mg/100g) in diets were calculated as the sum of areas for all fa peaks compared to area for 0.5 mg internal standard. individual fa were expressed as % by weight of total fa. 2.2.6 statistical analysis body weight (bw), intramuscular fat (%), fatty acids profile and meat quality characteristics, such as ph24, color parameters (l, a* and b*), cooking loss (%) and shear force value (n) measurements for the longissimus dorsi muscle were analyzed using the mixed model procedure which contained the fixed effect of the diet. malondialdehyde (mda) concentration and drip loss (%) were also analyzed using a mixed model appropriate for repeated measurements per subject, which included the diet as fixed effect. finally, power (1β) analysis for all tests was performed using α=0.05 (zar, 1996). all model analyses were performed by sas/stat (2005). 3 results no significant differences were observed in body weight at slaughter (kg), carcass weight (kg) and dressing out (%) between the two experimental groups (p>0.05). no effects of dietary protein source on feed intake and bw gain were also observed over the whole experimental period (30–72 days of age). average feed intake was 1.16±0.12 and 1.14±0.14 kg/day, and bw gain was 0.51±0.08 and 0.53±0.04 kg/day, resulting in an fcr of 2.41±0.20 and 2.10±0.17 for pigs fed the wp and sb diets, respectively. at the same time, meat quality characteristics did not significantly differ between sp and wp piglets (table 2). in detail, although instrumental ph and color values did not indicate meat of excessive acidity or darkness, respectively, and were within the normal range, these parameters were also not significantly influenced by the dietary protein source (p>0.05). moreover, dietary soybean meal or whey protein did not result in different values for shear force, intramuscular fat content, cooking loss and drip loss (table 2). refrigerated storage increased lipid oxidation in stored (4 °c) compared to fresh meat (p<0.001). however, dietary protein source appeared not to significantly influence lipid oxidation (mda formation) values in raw pork l. dorsi muscle after storage at 4°c for up to 9 days (table 3). with respect to the muscle fatty acid profile, lauric (c12:0) and myristic (c14:0) percentages were lower (p<0.05), whilst α-linolenic (c18:3n-3) and arachidonic (c22:4n-6) were higher (p<0.001 and p<0.05, respectively) for sb compared to wp fed piglets. however, total saturated fatty acids (sfas) and polyunsaturated fatty acids (pufas) were not affected by diet (table 4). g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 21 haf © 2013 vol. i, no. 1 issn: 2283-3927 table 2. effect of dietary protein source (whey proteins or soybean meal) on piglets’ meat quality characteristics (ls means ± s.e.m.) diet 1 s.e.m. wp sb body weight at slaughter (kg) 28.33 30.63 2.57 cold carcass weight (kg) 18.99 19.82 1.75 dressing out (%) 67.28 64.17 1.34 intramuscular fat content (%) 1.50 1.39 0.10 ph (24 h) 5.44 5.42 0.17 color parameters l 51.33 50.08 1.10 a* 4.08 4.90 0.39 b* 9.91 9.88 0.19 cooking loss (%) 36.40 36.21 0.48 shear force (n) 35.16 37.08 1.36 drip loss (%) (days) 2 1st 5.02 6.55 0.71 2nd 6.79 8.78 0.70 3rd 7.81 9.74 0.73 4th 8.75 10.45 0.73 5th 9.33 10.98 0.74 6th 9.87 11.34 0.76 7th 10.23 11.62 0.79 8th 10.74 12.04 0.79 1 sb, soybean meal as protein source; wp, whey proteins as protein source. 2 time effect was significant (p<0.001), but the effect of interaction of time with treatment (p=0.210) was not significant. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 22 haf © 2013 vol. i, no. 1 issn: 2283-3927 4 discussion no effect of dietary protein source on piglet meat quality was found. in detail, ph, color, shear force, intramuscular fat content, cooking loss and drip loss values were not significantly different between the wp and sb piglet group. data referring to the effects of the dietary protein source on pig meat quality are scarce. in general, soy proteins are more commonly used in processed meat products for their functional properties (waterand fat-binding ability, enhancement of emulsion stability) and relatively low cost compared with lean meat (chin et al., 2000). for example, soy protein isolates can be incorporated to chorizo raw sausage (porcella et al., 2001) or low-fat bologna (chin et al., 2000) without any detrimental effect on the organoleptic, physicochemical and microbiological properties of these meat products. moreover, dietary protein source (soybean meal, sunflower meal, pea and fish meal) had no effect on pig meat characteristics (intramuscular fat content, ph45 and ph24, drip loss and color) (szabo et al., 2001). dietary soy isoflavones also appear not to influence ph24, color parameters, drip loss, firmness-wetness and marbling of pig meat (payne et al., 2001). at the same time, the dietary protein sources used in the present study (soybean meal or whey protein) appeared not to affect differently lipid oxidation values in raw pork longissimus muscle after storage up to 9 days at 4 ºc (table 3). these findings are in contrast with the already demonstrated in vitro antioxidant activity of whey proteins (peña-ramos and xiong, 2003); hence, the lack of such a positive effect on muscle tissue (in vivo) is unclear. nevertheless, mda values for wp tended to be numerically lower compared to the sp group. finally, the limited differences in muscle fa profile; lower lauric and myristic and higher αlinolenic and arachidonic percentages for sb compared to wp fed piglets mainly depicted the incorporation of dietary fa into muscle lipids, i.e. lower c14:0 and higher c18:3n-3 in sb fed pigs and not a protein source effect. any other differences in fa observed in muscle between groups were more likely due to endogenous fa metabolism (wood and enser, 1997). table 3. effect of dietary protein source (whey proteins or soybean meal) and duration of refrigerated (at 4ºc) storage on lipid oxidation (mda , ng/g) of raw piglet longissimus dorsi muscle (ls means ± s.e.m.) storage period (days, at 4ºc) diet 1 s.e.m. wp sb 1 16.18 20.90 1.01 3 22.37 25.53 1.54 6 25.75 31.34 1.62 9 36.92 37.30 1.96 1 sb, soybean meal as protein source; wp, whey proteins as protein source. time effect was significant (p<0.001), but the effect of interaction of time with treatment (p=0.120) was not significant. higher levels of mda indicate higher rates of lipid oxidation. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 23 haf © 2013 vol. i, no. 1 issn: 2283-3927 table 4. effect of diet on total fatty acids (mg/100 g wet muscle) and fatty acid (fa) composition (% of total fa) of longissimus dorsi in piglets (ls means ± s.e.m.). fa diet 1 s.e.m. p-value 5 sb wp total fa 1027 1179 84.7 ns c12:0 0.08 0.11 0.007 * c14:0 1.12 1.34 0.049 * c16:0 22.31 22.73 0.261 ns c16:1 cis9 2.10 2.45 0.147 ns c18:0 11.54 11.41 0.208 ns c18:1 cis9 25.50 27.77 1.249 ns c18:1 cis11 3.32 3.27 0.074 ns c18:2n-6 16.66 14.48 0.863 ns c18:3n-3 0.40 0.20 0.027 *** c20:3n-6 0.53 0.46 0.028 ns c20:4n-6 3.78 3.19 0.237 ns c20:5n-3 0.33 0.32 0.044 ns c22:4n-6 0.51 0.40 0.025 * c22:5n-3 0.71 0.68 0.067 ns c22:6n-3 0.66 0.60 0.046 ns sfa 2 35.74 36.21 0.437 ns mufa 3 32.52 34.98 1.368 ns pufa 4 24.19 20.86 1.295 ns 1 sb, soybean meal as protein source; wp, mixture of whey proteins [wp, 70% wheypro65 (650 g cp/kg) + 30% wheypro 80 (800 g cp/kg); hellenic proteins s.a., veria, greece] as protein source. 2 sfa, total saturated fa, including individual fa which are not presented (c10:0 + c12:0 + c14:0 + c15:0 + c16:0 + c17:0 + c18:0 + c20:0). 3 mufa, total monounsaturated fa, including individual fa which are not presented (c14:1 + c16:1 cis9 + c16:1 cis7 + c17:1 + c18:1 cis9 + c18:1 cis11 + c20:1n-9). 4 pufa, total polyunsaturated fa, including individual fa which are not presented (c18:2n-6 + c18:3n-6 + c18:3n-3 + c20:2 + c20:3n-6 + c20:4n-6 + c20:5n-3 + c22:4n-6 + c22:5n-3 + c22:6n-3). 5 ns: not significant, *p<0.05, ***p<0.001 g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 24 haf © 2013 vol. i, no. 1 issn: 2283-3927 in conclusion, no significant differences between dietary soybean meal or whey protein diets on piglet meat quality characteristics (color parameters, ph24, cooking loss, intramuscular fat content and shear values) were observed. the extent of lipid oxidation in raw l. dorsi muscle stored at 4ºc for up to 9 days was also not influenced by the dietary protein source. the sb pigs had lower c14:0 (p<0.05) and higher c18:3n-3 (p<0.001) levels in intramuscular fat in comparison with wp pigs. however, these changes were attributed to background differences in the dietary fa profile and not to a direct protein source effect. the present results suggest that the two examined dietary protein sources (soybean meal or whey protein) do not have a different effect on meat quality characteristics of piglets. further investigation is warranted to elucidate the mechanisms underlying the potential effects of dietary protein source on meat quality in piglets and pigs. acknowledgements the authors are grateful to hellenic proteins s.a. for providing the whey proteins (wheypro 65 and wheypro 80). references ascencio, c., torres, n., isoard-acosta, f., gómez-pérez, f.j., hernández-pando, r., tovar, a.r., 2004. soy protein affects serum insulin and hepatic srebp-1 mrna and reduces fatty liver in rats. the journal of nutrition 134, 522–529. botsoglou, n.a, fletouris, d.j., papageorgiou, g.e., vassilopoulos, v.n., mantis, a.j., trakatellis, a.g., 1994. a rapid, sensitive and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissues, food and feedstuff samples. journal of agriculture and food chemistry 42, 1931-1937. carey, g.b., 1997. the swine as a model for studying exercise-induced changes in lipid metabolism. medicine and science in sports and exercise 29, 1437–1443. chin, k.b., keeton, j.t., miller, r.k., longnecker, m.t., lamkey, j.w., 2000. evaluation of konjac blends and soy protein isolate as fat replavements in low-fat bologna. journal of food science 65, 756-763. christensen, l.b., 2003. drip loss sampling in porcine m. longissimus dorsi. meat science 63, 469–477. farmer, s.r., 2005. regulation of ppargamma activity during adipogenesis. international journal of obesity 29, 13–16. folch, j., lees, m., stanley, s.g.h., 1957. a simple method for the isolation and purification of total lipids from animal tissues. the journal of biological chemistry 226, 497-509. g. theodorou et al. int. j. of health, animal science and food safety 1 (2014) 1-8 25 haf © 2013 vol. i, no. 1 issn: 2283-3927 fundación española para el desarrollo de la nutrición animal, 2003. in: de blas c., mateos g.g., rebollar p.g. 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(1996). biostatistical analysis, 4th edition, prentice-hall, englewood cliffs, nj, pp 665. haf © 2013 24 l keywords fatty acids, lambs, ewes, meat quality, pca. pages 24 – 38 references vol. 4 no. 1 (2017) article history submitted: may 24, 2017 revised: june 30, 2017 accepted: july 04 2017 published: july 21 2017 corresponding author roberto tocci dipartimento di scienze delle produzioni agroalimentari e dell’ambiente (dispaa) – sez. scienze animali, università degli studi di firenze. via delle cascine, 5. 50144 firenze, italy mail: roberto.tocci@unifi.it phone: +39 055 2755585 fax +39 055 321216 journal home page riviste.unimi.it/index.php/haf article quality characteristics of the musculus longissimus dorsi from pecora dell’amiata reared in tuscany roberto tocci 1,* , clara sargentini 1 , andrea martini 1 , matteo campostrini 1 , eleonora pippi 1 , valeria iaconisi 1 , antonio bonelli 1 , alessandro giorgetti 1 1 dipartimento di scienze delle produzioni agroalimentari e dell’ambiente (dispaa) – sez. scienze animali, università degli studi di firenze, via delle cascine, 5. 50144 firenze, italy. abstract the trial was performed with ewes and lambs deriving from the local breed pecora dell’amiata. in this work, the musculus longissimus dorsi (m. longissimus thoracis + lumborum) physical-chemical and nutritional characteristics of 23 ewes and 20 lambs were compared. the ewes of the trial were over 7 years old while the lambs were on average 80 days old. ewe meat has shown lower drip loss (4.14 vs 2.71%) and lightness (l* 38.6 vs 45.3) values, and higher ph (6.15±0.07), shear force (8.4 vs 2.31 kg), fat content (5.9 vs 2.0%). the lamb meat lipids had higher polyunsaturated fatty acid (pufa) content (14.58 vs 9.25%) and higher pufa/saturated fatty acids (sfa) ratio (0.31 vs 0.20). the principal component analysis (pca) identified two distinct groups regarding ewe and lamb meat respectively for the fatty acids composition and the health indices. ewe meat showed dietetic and nutritional characteristics similar to that of lamb meat. these characteristics may allow in the future, to the ewe meat valorisation, now not appreciated by tuscan and italian market. mailto:roberto.tocci@unifi.it r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 25 haf © 2013 vol. iv, no. 1 issn: 2283-3927 1 introduction 1.1 introduction pecora dell’amiata is an autochthonous tuscan sheep breed, registered in the regional population register of the autochthonous endangered sheep and goat breeds (d.m. 17444/2014), having meat and milk as main productions (giorgetti et al., 2012; assonapa, 2016). this breed may have an important role in ovine meat local market that valorises the regional supply chain. the ovine tuscan market follows the italian trend consumption; the sheep meat consumption occurs in particular in easter and in christmas time. the most consumed sheep meat is the light lamb meat (enough 30 days of age) from suckling lambs. this trend, constant during the years (istat, 2010; tocci et al., 2015), is in contrast with the european continental countries sheep meat consumption (kegalj et al., 2011). in the last years, the meat production and consumption decreased in italy, but the ewe meat consumption has been slowly increased, because of the spread of typical regional food (“arrosticini”, salami, ham sheep ecc…), and the new market needs: the west-northern african, and the near eastern immigrated populations appreciate this product (tocci et al., 2015). in this study, the qualitative characteristics of ewe and lamb musculus longissimus dorsi of pecora dell’amiata were considered. 2 materials and methods 2.1 animals this work dealt with the post mortem results of 23 over 7 years old (min. 8, max. 10. mean 9 years) ewes and 20 lambs slaughtered at 80 days (min. 70, max. 90, mean 80 days). the ewes were reared with a pasture-based semi-extensive system, with a supplementation of hay ad libitum and 400 gr of barley during the night housing. the lambs were raised with their dams on the pasture. before the slaughtering all animals were weighed. ewes and lambs of this trial were slaughtered following the council regulation of 24 september 2009 on the protection of animals at the time of killing animal welfare guidelines, which provide the head-only electrical as stunning method (european council, 2009). the ewe carcasses were weighed, and valuated for conformation and fat covering following the ue grid (commission regulation (ec) 2008). the lamb carcasses were weighed, measured, and valuated following the slaughter procedures of aspa (1991); carcasses were classified, according to the eu mediterranean grid, for carcass color and fatness score by experienced evaluators (eec, 1993). r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 26 haf © 2013 vol. iv, no. 1 issn: 2283-3927 2.2 laboratory analysis: in order to evaluate the physical-chemical and nutritional characteristics of the meat, four days after slaughtering, samples from the musculus longissimus dorsi were collected (rizzi et al., 2002). the carcasses were stored at 4 °c. on these samples, ph was measured in triplicate, through mettler toledo devengo sg2™ ph-meter (novate milanese, milano, italy) equipped with an inlab puncture electrode (mettlertoldedo, ltd). the mean value was considered. the water holding capacity (whc) was determined through the filter paper press grau and hamm method (1957), the drip loss, and the cooking loss. the filter paper press method is expressed as the ratio m/t, where m is the area (cm 2 ) of a cuboidal sample of 300 ± 5 mg kept for four minutes under a pressure of 50 kg/cm 2 , and t is the total wetted area of filter (cm 2 ) (destefanis et al., 1991; hofmann et al., 1982). the drip loss was performed on cubic samples of 30 grams kept at 4 °c for 48 h in a plastic container with double bottom. the cooking loss was carried out on parallelepiped samples of about 40 grams of weight kept in oven at 180 °c to an internal temperature of 75 ° c (poli et al., 1994). the meat colour was determined on three different homogeneous areas of each samples, which were kept 1 hour at room temperature; the meam value was considered. ph was measured through minolta chromameter cr 200, calibrated against a standard white tile in the cie l, a*, b* system, which measures the values of lightness (l*), redness (a*), yellowness (b*), chroma (colour saturation – (a2+ b2)1/2) and hue angle (arctan b/a) (renerre, 1982; aspa, 1996). the texture analyses (aussanasuwannakul et al., 2010) were carried out in raw and in oven cooked (1 cm x 1 cm) samples using a zwick roell® 109 texturometer (ulm, germany) with text expert ii software, equipped with a 1 kn load cell. the warner-bratzler shear test (wbshear force) consisted of a 3 mm thick steel blade which had a 73° v cut into it. the cut was perpendicular to the muscle fibre direction. the samples were placed on the table, under the v of the blade, and was cut through as the blade moved with a constant speed through the slit of the table (crosshead speed of 30 mm/min). the resistance of the samples to shearing was recorded every 0.01 seconds and plotted by a computer in a force deformation. maximum shear force, defined as maximum resistance of the sample to shearing (veland and torrisen, 1999) was determined. two raw and two cooked cores from each sample were submitted to wb-shear force; the mean value of both measures was considered. the chemical analyses were carried out on each sample of musculus longissimus dorsi determining dry matter, ether extract, crude protein and ash (aoac, 1990). the samples were analysed for total lipid concentration by gravimetric determination of total lipid extract according to folch et al. (1957). the tissue was homogenized with chloroform/methanol (2/1) to a final volume 20 times the volume of the tissue sample (1 g in 20 ml of solvent mixture). after dispersion, the whole mixture is agitated during 15-20 minutes in an orbital shaker at room temperature. the homogenate was either filtrated (funnel with a folded filter paper) to recover the liquid phase. the solvent was washed with 0.2 volume (4 ml for 20 ml) of water or better 0.9% nacl solution. after vortexing some seconds, the mixture was centrifuged at low speed (2000 rpm) to separate the two phases. after centrifugation and siphoning of the upper phase, the lower chloroform phase containing lipids was evaporated under vacuum in a rotary evaporator or under a nitrogen stream if the volume is under 2-3 ml (folch et al., 1957). the r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 27 haf © 2013 vol. iv, no. 1 issn: 2283-3927 samples were also analysed for quantitative fatty acid composition of total lipids by gas chromatographic separation of methyl esters, comprising c19:0 as internal standard, on capillary column oven temperature ranging from 164°c and 200°c with 3°c/min heat increment. the following health indices were also calculated: mufa (monounsaturated fatty acids)/sfa, pufa/sfa, ω6/ω3 pufa, epa (c 22:5 ω3)/dha (c22:6 ω3) ratio. 2.3 statistical analysis: data were submitted to one way anova through the means squares method, using jmp 10 statistical software (sas institute inch 2013), and considering as fixed factor the category (ewe meat and lamb meat). a pca was performed on single fatty acids and on fatty acid types; when many measures are used to assess meat quality and they are correlated, they can be replaced by fewer measures without a significant loss of information (karlsson, 1992). a bartlett test was performed: the bartlett’s test compares the observed correlation matrix to the identity matrix. this test checks if there is a certain redundancy between the variables that we can summarize with a few number of factors. if the variables are perfectly correlated, only one factor is sufficient. if they are orthogonal, we need as many factors as variables. the kaiser test was also performed: this test rotates only the factors with eigenvalues-greaterthan-one (kaiser, 1960). a varimax rotation was performed in order to maximize the factors variance (davis, 2002). the reduction in dimensionality is thus achieved by pca might be useful in visual interpretation of the data represented by two-dimensional graphics (kopuzlu et al., 2011). accordingly, a score plots and loading plots for sheep meat categories was performed. a correlation analysis between single fatty acids and between types of fatty acids was also performed. 3 results the live weight of ewes was 46.45±9.69 kg while that of lambs was 21.22 ± 1.13 kg. the carcass weight was 22.61 ± 0.74 kg and 10.96 ± 0.65 kg for ewes and lambs respectively, while the carcass conformation was r and c1. the fat score for the carcass ewes was 3. the chemical characteristics of grass and hay of the ewes’ diet are shown in table 1. the crude protein content was 15.4% and 8.5 % respectively for grass and hay, while the fibre content was 25.8% and 34.0%. table 1: chemical composition of the component ewes diet (%/dm) dry matter crude protein crude fiber grass % 20.0 15.4 25.8 hay % 88.5 8.5 34.0 barley % 89.9 11.2 6.1 r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 28 haf © 2013 vol. iv, no. 1 issn: 2283-3927 the physical characteristics of the ewe and lamb meat (table 2) have shown that the latest had lower ph, cooking loss and drip loss. the free water, physiologically bonded to the muscle fibres, was similar between meat types. lamb meat was tenderer, more brilliant, and more saturated in colour than the ewe meat. relatively to the chemical analysis (table 2), the lamb meat had higher crude protein content, and lower fat content. in table 3 the fatty acids were also shown: among the sfa, c12:0, c14:0, c15:0 and c16:0 contents were higher in the lamb meat, c17:0 and c20:0 contents were comparable between meat types, and c18:0 content (stearic acid) was higher in the ewe meat. among the mufa, c14:1 ω 5 and c16:1 ω7 contents were higher in the lamb meat, while c20:1 ω9 content (gadoleic acid) was higher in the ewe meat (table 3). the ω6 pufa, among which the linoleic acid, were higher in the lamb meat. c18:3 ω3 content (α-linolenic acid-ala) was comparable between the meats, while eicosapentaenoic acid (epa), and docosahexaenoic acid (dha) have shown higher content in ewe meat. the health indices indicated a better pufa/sfa ratio in the lamb meat, because of the higher pufa content. epa/dha was also favourable (lower values) on lamb meat. table 2: ewe and lamb musculus longissimus dorsi physical and chemical characteristics (mean±sem) ewe lamb sign. ph 6.15±0.07 5.61±0.05 *** water holding capacity cooking loss % 34.37±1.81 27.63±1.46 *** drip loss % 4.14±0.40 2.74±0.32 *** m/t 0.62±0.01 0.58±0.01 n.s. tenderness shear force on raw meat n 82.36±3.87 23.10±1.80 *** shear force on cooked meat in oven n 79.93±4.20 30.91±2.46 *** color lightness l* 38.63±0.69 45.34±0.72 *** redness a* 19.33±0.57 20.84±0.59 n.s. yellowness b* 6.20±0.63 7.84±0.66 n.s. chroma c* 20.30±0.64 22.50±0.67 ** hue angle h* (°) 0.30±0.02 0.35±0.02 n.s. chemical characteristics dry matter (d.m.) % 25.46±0.59 24.94±0.71 n.s. moisture % 74.53±0.59 75.05±0.71 n.s. ash % 1.20±0.06 1.29±0.07 n.s. crude protein % 19.04±0.40 21.25±0.48 *** fat % 5.12±0.43 2.09±0.51 *** 1 n.s.= not significant; ** p≤0.05; *** p≤0.00 r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 29 haf © 2013 vol. iv, no. 1 issn: 2283-3927 table 3: ewe and lamb musculus longissimus dorsi: % of fatty acids on total fatty acids (mean±sem) ewe lamb sign. c12:0 % 0.12±0.05 0.60±0.05 *** c14:0 % 2.26±0.32 5.91±0.30 *** c14:1 ω5 % 0.06±0.01 0.21±0.01 *** c15:0 ai % 0.20±0.01 0.26±0.01 ** c15:0 % 0.51±0.04 0.74±0.03 *** c16:0 iso % 0.19±0.01 0.23±0.09 *** c16:0 % 22.55±0.55 24.23±0.50 ** c16:1 ω7 % 1.41±0.08 1.97±0.07 *** c17:0 ai % 0.61±0.02 0.61±0.2 n.s. c17:0 % 1.15±0.04 1.12±0.03 n.s. c17:1 % 1.00±0.09 1.15±0.09 n.s. c18:0 % 17.57±0.60 13.40±0.54 *** c18:1 ω9 % 33.62±1.16 32.80±1.05 n.s. c18:2 ω6 cis % 5.48±0.37 7.81±0.34 *** c18:3 ω3 % 1.58±0.10 1.63±0.09 n.s. c20:0 % 0.11±0.01 0.10±0.01 n.s. c20:1 ω7 % 0.007±0.006 0.01±0.05 n.s. c20:1 ω9 % 0.21±0.01 0.10±0.01 *** c20:2 ω6 % 0.13±0.03 0.24±0.03 ** c20:3 ω6 % 0.10±0.01 0.18±0.01 *** c20:4 ω6 % 1.12±0.18 2.93±0.17 *** c20:5 ω3 % 0.40±0.07 0.95±0.06 *** c22:0 % 0.01±0.001 0.04±0.009 n.s. c22:5 ω3 % 0.42±0.05 0.83±0.04 *** c22:6 ω3 % 0.12±0.04 0.50±0.04 *** sfa % 45.49±0.78 47.47±0.71 n.s. mufa % 36.31±1.08 36.27±0.98 n.s. pufa % 9.25±0.71 14.58±0.64 *** ω3 % 2.40±0.20 3.41±0.18 *** ω6 % 6.84±0.55 11.17±0.50 *** mufa/sfa 0.80±0.33 0.77±0.03 n.s. pufa/sfa 0.20±0.01 0.31±0.01 *** ω6/ω3 3.12±0.17 3.32±0.15 n.s. epa/dha 3.90±0.26 2.07±0.31 *** n.s.= not significant; ** = p≤0.05; *** = p≤0.001 r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 30 haf © 2013 vol. iv, no. 1 issn: 2283-3927 on the fatty acids and on the health indices pca of fatty acids was carried out. the purpose of pca was to individuate the presence of fatty acids composition in the ewe meat and in lamb meat, through the axis deriving from the correlation matrix, and extracting the eigenvalues with different factorial load. the pca has shown 5 main factors with eigenvalue higher than 1 which explained almost 82% of the variability (table 4). pc1 only explained 35.67% and the pc2 20.69%. the score plot and the loading plot of the fatty acids composition for sheep meat types (figures 1 and 1a) have shown in the pc1 two distinct groups that identified the ewe and the lamb meat. table 4: fatty acids percentage in ewe and lamb meat: eigenvalue, cumulative percentage of variance and bartlett test number eigenvalue percentage cumulative percentage chi-sq. df sign. 1 9.63 35.67 35.67 1676.65 346.91 *** 2 5.58 20.69 56.35 1319.87 338.30 *** 3 3.46 12.82 69.18 1039.65 322.30 *** 4 1.90 7.03 76.21 816.68 302.65 *** 5 1.41 5.24 81.46 680.99 280.83 *** *** = p<.0001 figure 1. score plot of fatty acids composition for sheep meat categories figure 1a. loading plot of fatty acids composition for sheep meat categories r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 31 haf © 2013 vol. iv, no. 1 issn: 2283-3927 table 5: correlations between fatty acids c12:0 c14:0 c14: 1ω5 i-c15:0 aic 15:0 c15:0 ic16:0 c16:0 c16: 1 ω7 ai c17:0 c17:0 c17:1 c18:0 c18: 1ω9 c18: 2ω6 c18: 3 ω4 c18: 3ω3 c20:0 c20: 1ω9 c20: 1-n7 c20: 2ω6 c20: 3ω6 c20: 4ω6 c20: 5ω3 c22:0 c22: 5ω3 c22: 6 ω3 c12:0 1 c14:0 0.95 1 c14:1ω5 0.84 0.92 1 i-c15:0 0.57 0.51 0.41 1 aic15:0 0.70 0.58 0.42 0.85 1 c15:0 0.88 0.80 0.7 0.78 0.89 1 ic16:0 0.57 0.55 0.47 0.73 0.76 0.75 1 c16:0 0.71 0.74 0.63 0.58 0.47 0.62 0.37 1 c16:1ω7 0.73 0.82 0.88 0.50 0.42 0.66 0.55 0.69 1 ai c17:0 0.63 0.49 0.48 0.49 0.42 0.39 1 c17:0 0.63 0.57 0.59 0.54 0.38 077 1 c17:1 0.35 0.42 0.39 0.43 0.47 0.52 0.42 1 c18:0 -0.67 -0.76 -0.86 -0.49 -0.62 -0.85 1 c18:1-ω9 -0.46 -0.40 -0.63 -0.68 -0.62 -0.42 -0.36 -0.46 -0.56 1 c18:2-ω6 0.36 -0.39 0.36 0.57 -0.48 1 c18:3-n4 0.56 0.41 0.44 0.46 0.45 0.60 0.43 -0.44 1 c18:3-ω3 -0.52 0.42 1 c20:0 -0.38 0.45 0.36 1 c20:1-ω9 -0.59 -0.67 -0.66 -0.45 -0.46 -0.49 0.60 1 c20:1-n7 -0.44 1 c20:2-ω6 -0.37 1 c20:3-ω6 0.35 0.39 0.41 0.41 -0.50 0.83 0.43 -0.43 0.49 1 c20:4-ω6 0.38 0.45 0.51 -0.40 0.84 0.36 -0.55 0.54 0.90 1 c20:5-ω3 0.36 0.42 -0.36 0.77 0.52 -0.47 0.49 0.88 0.92 1 c22:0 -0.35 0.50 0.38 1 c22:5-ω3 0.37 0.35 -0.39 0.75 0.55 -0.50 0.60 0.84 0.89 0.92 1 c22:6ω3 0.62 0.59 0.46 0.40 0.50 0.57 0.53 0.66 0.61 0.46 0.57 1 r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 32 haf © 2013 vol. iv, no. 1 issn: 2283-3927 the varimax rotation has shown as 14 of 27 considered fatty acids represented the factor 1. the pc1 has shown as significant values the majority of sfa, while the pc2 has been identified in the pufa; mufa were equally distributed between pc1 and pc2. a great number of positive correlations between fatty acids were found (table 5). most of sfa characterized the lamb meat, while c18:0, c18:1 ω9, having negative correlations (table 5), identified the ewe meat. the pca analysis was also performed for the fatty acids types and for the health indices. in this case three eigenvalues higher than 1, explaining enough 90% of the cumulative variability, were observed. pc1 explained almost 47.75%, while pc2 27.41% (table 6). the score plot and the loading plot of the fatty acids types and health indices for ewe and lamb meat (figure 2 and 2a) have shown as both meat types were distinguished. the varimax rotation for the fatty acids types has shown as significant values in pc1 the pufa , both ω6 and ω3, and the pufa/sfa ratio; mufa, having negative correlation, showed lower value. pc2 identified for the sfa, for the mufa and for the mufa/sfa ratio. higher correlations were shown in mono and pufa, while the less correlated health index was ω6/ω3 that had negative correlation with pufa ω3 (table 7). mufa have shown the highest number of negative correlations. the pufa types and the pufa/sfa ratio identified in particular the lamb meat, while sfa, mufa, and mufa/sfa ratio, having almost always negative correlations, identified for the ewe meat. table 6. fatty acid types and healthy indices in ewe and lamb meat: eigenvalue, cumulative percentage of variance and bartlett test number eigenvalue percentage cumulative percentage chi-sq. df p 1 4.30 47.75 47.75 1404.66 35.82 > 0.01 2 2.47 27.41 75.17 1254.91 34.36 > 0.01 3 1.27 14.13 89.30 1106.86 29.51 > 0.01 figure 2. score plot of the fatty acids types and health indices for ewe and lamb meat figure 2a. loading plot of the fatty acids types and health indices for ewe and lamb meat r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 33 haf © 2013 vol. iv, no. 1 issn: 2283-3927 table 7. correlation between fatty acid types and health indices sfa mufa pufa-ω3 pufaω6 tot pufa mufa /sfa pufa /sfa ω6 /ω3 epa /dha sfa 1 mufa -0.62 1 pufaω3 -0.49 1 pufaω6 -0.40 0.79 1 pufa tot -0.43 0.88 0.99 1 mufa/sfa -0.81 0.96 -0.40 1 pufa/sfa 0.84 0.97 0.98 1 ω6/ω3 -0.40 1 epa/dha -0.37 -0.35 1 4 discussion live weight, carcass weight and the carcass evaluation identified ewes and lambs belonging to mediterranean ovine breeds. chemical composition of grass and hay of the sheep diet was typical of the central areas of tuscany. the pecora dell’amiata ewe meat has shown higher ph, lightness, redness, and chroma values, lower yellowness, and hue angle than the sarda ewe meat (mezzette et al., 2005). if compared with the merino ram meat (hopkins and toohey, 2006) the ewe meat of the trial has shown similar ph value, lesser tenderness and water holding capacity. if compared with the istrian lamb meat (piasentier et al., 2002), the pecora dell’amiata lamb meat had higher cooking loss, lightness and redness, while the shear force was lower; the ph values were similar between breeds. if compared with the bergamasca and suffolk lamb meat (sirtori et al., 2009) the lamb meat of the trial had lower cooking loss and higher chroma and hue angle. relatively to the chemical composition, the high crude protein and low fat content in lamb meat have met the normal physiology of the animal growth (russo et al., 2003; franci and gualtieri, 1988). the chemical composition of the ewe meat of this study was similar to that of the santa ines ewes, having the same carcass weight (costantino et al., 2014). if compared with the istrian lamb meat (piasentier et al., 2002) the pecora dell’amiata lamb meat had higher crude protein and ash, lower fat content, while the dry matter percentage was similar. the lamb meat of the trial has shown lower crude protein content than the bergamasca and suffolk lamb meat (sirtori et al., 2009). the pecora dell’amiata ewe meat and the sarda ewe meat (santercole et al., 2007), had similar sfa, pufa ω3 and ω6 content, while the mufa content was lower in the meat of the study. if compared with the istrian lamb meat (piasentier et al., 2002) the pecora dell’amiata lamb meat had lower pufa ω6 content, while if compared with the suffolk and bergamasca lamb meat (sirtori et al., 2009), had higher sfa and pufa ω3 percentage. the lamb meat of r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 34 haf © 2013 vol. iv, no. 1 issn: 2283-3927 the trial has shown higher pufa and mufa contents than the meat of santa ines and its crosses lamb meat (costa et al., 2015). if compared with the merino ram meat (greeff, 2007) the pecora dell’amiata ewe meat had lower c18:0 and c20:1 contents, while if compared with the sarda ewe meat (santercole et al., 2007) had higher c18:0 content, and similar c22:5 ω3 and c22:6 ω3 contents. if compared with the suffolk and bergamasca lamb meat (sirtori et al., 2009) the lamb meat of this study has shown a lower c18:0, c18:1 ω9 and c18:2 ω6 cis contents, and higher c20:5 ω3 (epa) content. the lamb meat of the trial has shown higher c22:5 ω3 and c22:6 ω3 contents than the meat deriving from romanov cross lambs (kuchtík et al., 2012). relatively to the healthy indices, some authors recommend a ratio of 1:1:1 in the diet for sfa:mufa:pufa (hayes 2002). western diets are poor in ω3 fatty acids and rich in ω6 fatty acids. a lower ratio of ω6/ ω3 fatty acids allows reducing the risk of many chronic diseases typical of western countries (simopoulus, 2008). simopoulos and cleland (2003) claim that the ideal ω6/ω3 is 4:1 for brain-mediated functions. some authors found that a similar 60/40 ratio of epa/dha eased depression somewhat in people with depression who didn’t have anxiety disorders (sarris, 2012). both meat types of this trial were characterised by a good ω6/ω3 ratio (3.12 and 3.32); in western countries diets this ratio is 15-16:1. very important for this index are the linoleic acid (la) (ω6) and the linolenic acid (ala) (ω3), which may compete with each other, because metabolised by the same enzyme, the δ6-desaturase. this issue is important for the human health, because an excess of la in the diet could reduce the δ6-desaturase available for the ala metabolism, with consequent risk of cardiac diseases increase (stanley et al., 2007). the pecora dell’amiata ewe meat has shown a favourable ω6/ω3 ratio if compared with simopoulos parameters (2008), and slightly higher of that of sarda ewe meat (santercole et al., 2007), while epa/dha ratio was similar between meats. if compared with the istrian lamb meat (piasentier et al., 2002) the pecora dell’amiata lamb meat had a slightly lower ω3/ω6 ratio, while if compared with the meat of santa ines lamb (costa et al., 2015), had a lower mufa/sfa ratio and a higher pufa/sfa ratio. epa/dha ratio was lower than that of romanov crosses lamb meat (kuchtík et al., 2012). the pca highlighted as the ewe meat was characterized by c18:0, while the total pufa and total sfa characterized the lamb meat. c18:0 characterized also the intramuscular fat deriving from the faroese ewes (jónsdóttir et al., 2001). as in the case of the faroese lamb meat, the higher sfa content (c12:0 and c14:0) derived from milk (jónsdóttir et al., 2001). also in bovines and in goats, younger animals showed higher c12:0 and c14:0 content (bednárová et al., 2013; beserra et al., 2004). 5 conclusion both ovine meat categories of this study have shown good dietetic characteristics with optimal health indices. the lamb meat has shown a higher pufa content, both ω3 and ω6, who determined a better pufa/sfa ratio, and both meat typologies had favourable ω6/ω3 ratio. the pca showed two different groups: lamb meat had higher saturated fatty acids that are derived from milk, and ewe meat was characterized by c18:0. lamb and ewe meat of r. tocci et al. int. j. of health, animal science and food safety 4 (2017) 24 30 35 haf © 2013 vol. iv, no. 1 issn: 2283-3927 pecora dell’amiata breed can be valorised in the local market. considering the dietetic and nutritional meat characteristics of the ewe meat, not so different from the lamb meat characteristics, it seems possible to valorise this product, now not required by the tuscan and italian market. acknowledgements: this work was supported by the regione toscana: “misura 124: cooperazione per lo sviluppo di nuovi prodotti, processi e tecnologie nei settori agricolo e alimentare e in quello forestale”. progetto “valorizzazione delle carni e dei sotto-prodotti della macellazione ovina tramite la realizzazione di prodotti innovativi e per nuovi mercati” (vacasopinum) (b15e13000170002) and vagal+ (valorizzazione dei genotipi animali autoctoni) programma di cooperazione 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riviste.unimi.it/index.php/haf abstract stress and wellbeing are psychological conditions that are mediated by the central nervous system. in the brain, stress is mediated mainly by the hypothalamus, which will activate the hypothalamicpituitary-adrenal (hpa) axis, leading to the secretion of cortisol, the paradigmatic stress hormone. other brain areas as the amygdala, the hippocampus or the prefrontal cortex (pfc) are involved in emotions such as happiness, anxiety and fear. communication between brain areas is achieved by chemical neurotransmitters (nts), which are secreted by presynaptic neurons to reach postsynaptic neurons, where they will cause a variation in membrane polarization and other cell signaling actions, leading to physiological responses. amongst these nts, catecholamines (noradrenaline and dopamine) and serotonin play an important role. on the other hand, the adverse effects of stress may be counteracted by housing the individuals under environmental enrichment conditions. this longterm situation should have an effect, not only on nts, but also on the brain proteome. under the hypothesis that different stress situations will lead to changes in nt composition that will be specific for crucial brain areas, we have tested the effects of transport stress, handling stress at the slaughterhouse, and the stress-susceptible genotype (ryr1) on the amine nt concentration in amygdala, hippocampus, pfc and hypothalamus of pigs. the effects of living under environmentally enriched or control conditions on the nt concentration in several brain regions and on the hippocampus proteome has been also analyzed. in conclusion, genetic factors as well as management conditions related to housing, transport and slaughterhouse alter in different degree the catecholaminergic and the serotoninergic neurotransmission in the brain, and give clues about how different individual types are able to react to external challenges. likewise, environmental enrichment leads to changes in the proteome especially related to protein translation in the hippocampus. brain neurotransmitters and hippocampal proteome in pigs under stress and environmental enrichment laura arroyo1 and anna bassols1* 1universitat autònoma de barcelona, department of biochemistry and molecular biology, faculty of veterinary, 08193 cerdanyola del vallès, spain http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en