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 2- 3 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. (2010). Residual yoghurt wh ey for lactic acid production. Biomass Bioenerg., 34: 931-938. Anderson M.J., Lamb R.C., Mickelsen C.H., Wiscombe R.L. (1974). Feeding liquid wh ey to dairy cattle. J. Dairy Sci., 57: 1206-1210. Anderson M.J. (1975). Metabolism of liquid whey fed to sheep. J. Dairy Sci., 58: 1856 -1859. AOAC (Association of Official Analytical Chemists ): Official m ethods of analysis (15th ed.). Washington, DC, USA: Association of Official Analytical Chemis ts. Boukila B., Seoane J.R., Goulet J., Bernier J.F., Petit H.V. (1995). Effect of feeding fermen ted, ammoniated, condensed whey perm eate on intak e, digestibility, rumen ferm entation, and acid-base balance in sheep. Can. J. Anim. Sci., 75: 135-143. Church D.C. (1991). Livestock Feeds and Feeding. Third edition. Prentice-Hall, Englewood Cliffs, NJ, 124-125. Galloway D.L., Goetsch A.L., Sun W., Forster L.A., Murphy G.E., Grant E.W., Johnson Z.B. (1992). Digestion, feed intak e, and live weight gain by cattle consuming Bermudagrass hay supplemented with wh ey. J. Anim. Sci., 70: 2533 -2541. Larsen R.E., Ewing S.A., Trenkle A., Vetter R.I., Burroughs W. (1963). Dried whey and lactose additions to finishing rations for lambs. J. Anim. Sci., 22: 1126. Morrill J.L., Dayton A.D. (1974). Effect of wh ey on calf starter palatability. J. Dairy Sci., 57: 430-433. Oba M. (2011). Review: Effects of feeding sugars on produ ctivity of lactating dairy cows. Can. J. Anim. Sci., 91: 37-46. Ohkawa H., Ohishi N., Yagi K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem., 95: 351-358. Pinchasov Y., Hasdai A., Gordin S., Katznelson D., Volcani R . (1982). Performan ce of high- yielding dairy cows fed liquid wh ey. J. Dairy Sci., 65: 28 -36. Rapetti L., Falaschi U., Lodi R., Vezzoli F., Tamburini A., Greppi G.F., Enne G. (1995). Th e effect of liquid whey fed to dairy goats on milk yield and quality. Small Ruminan t Res., 16: 215-220. Rapetti L., Crov etto M., Galassi G., Sandrucci A., Succi G., Tambu rini A., Battelli G. (2002). Effect of maize, rumen-protected fat and wh ey permeate on energy u tilization and milk fat composition in lactating goats. It. J. Anim. Sci., 1: 43-53. SAS/STAT (2005). Statistical analysis systems user’s guide, version 9.1.3. SAS Institute Inc., Cary, NC, USA. Schingoethe D.J. (1976). Whey u tilization in animal feeding: A summary and review. J. Dairy Sci., 59: 556-570. P. E. Simitzis et al. - Int. J. of Health, Animal science a nd Food safety 1 (2015) 1 -.8 8 HAF © 2013 Vol. 2, No. 1 ISSN: 2283-3927 Schingoethe D.J., Skyberg E.W. (1981). Lactation and growth of dairy cows and steers from large amoun ts of dried whey. J. Dairy Sci., 64: 1571-1578. Susmel P., Spanghero M., Mills C.R., Stefanon B. (1995). Rumen ferm entation characteristics and digestibility of cattle diets containing different wh ey:maize ratios. Anim. Feed Sci. Technol., 53: 81-89. Schingoethe D.J., Skyberg E.W., Bailey R.W. (1980). Digestibility, mineral balance, and rumen ferm entation by steers of rations con taining large amounts of lactose or dried whey. J. Dairy Sci., 63: 762-774. Van Soes t P.J., Robertson J.B., Lewis B.A. (1991). Methods for dietary fiber, neutral detergen t fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci., 74: 3583–3597. Walzem R.L., Dillard C.J., German J.B. (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.