B-3362 [1-6] Indian J. Anim. Res., *Corresponding author s

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1 B-3362 [1-6] Indian J. Anim. Res., Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Effect of replacing inorganic zinc with a lower level of organic zinc (zinc propionate) on performance, biochemical constituents, antioxidant, immune and mineral status in buffalo calves D. Nagalakshmi*, K. Sridhar, M. Satyanarayana, S. Parashu Ramulu, V.S. Narwade and L. Vikram Department of Animal Nutrition, College of Veterinary Science, P.V. Narasimha Rao, Telangana Veterinary Univesity, Hyderabad , Telangana, India Received: Accepted: DOI: /ijar.B-3362 ABSTRACT An experiment of 120 days was carried out to investigate the effects of replacing inorganic zinc (ZnSO 4 ; 80 ppm) with lower level (75% of inorganic levels; 60 ppm) of organic zinc (Zn propionate; Zn-Prop) on growth, serum biochemical constituents, immune response, mineral and antioxidant status in buffalo calves. Twelve buffalo calves (193.3±19.63kg; months) were randomly allotted to a control (80 ppm Zn) and experimental (60 ppm Zn) diets. All calves were weighed at fortnight interval. Animals were immunized against Brucella abortus (BA) and chicken RBC (CRBC) antigens on day 90 with a booster dose of 15 days interval and serum was collected on 7, 14, 21 and 28 d of post sensitization (PS) to study the antibody titres. Blood was collected on 114 d of experiment to prepare serum and haemolysate. Cell mediated immune response (CMIR) was determined against phytohemagglutinin-p (PHA-P) on day 118. The fortnightly body weights, average daily gain, dry matter intake, feed conversion ratio were comparable between the dietary groups. Similarly, serum albumin, globulin, creatinine and mineral (Zn, Cu, Mn, Fe) levels were comparable while higher (P<0.05) serum total protein and alkaline phosphatase activities were observed in organic Zn supplemented calves. After 7 d of PS, serum antibody titres (log 2 ) against BA were higher (P<0.05) with Zn-Prop supplementation, while the titres against CRBC were not affected by Zn source and dose. Lipid peroxidation and antioxidant enzyme activities (glutathione reductase, RBC catalase and superoxide dismutase) estimated in haemolysate were comparable between groups, while glutathione peroxidase (P<0.05) activity increased with Zn-prop supplementation compared to ZnSO 4. The CMIR was higher (P<0.05) with organic Zn. It can be concluded that the growth performance, antioxidant status, and immune response in calves fed lower levels of organic Zn (Zn-Propionate) (75% of inorganic mineral supplementation) was comparable to those fed 100% of inorganic zinc and the CMI response was higher in organic Zn supplemented calves. Key words: Antioxidant status, Buffalo calves, Immune response, Serum biochemical constituents, Zn propionate. INTRODUCTION Zinc (Zn) influences various biological functions by being a cofactor for more than 300 metalloenzymes (Chasapis et al., 2012). Besides this, Zn is essential for humoral and cell mediated immune responses (Gruber et al., 2013) and also plays an important role in antioxidant defense system (Oteiza, 2012). Similarly, Zn is required for female reproductive system and necessary for normal ovulation and fertilization (Tian and Diaz, 2011). Such an important mineral is critically deficient in various livestock feeds and fodders in most parts of the world (Nielsen, 2012), including India (Gowda et al., 2009 and Nagalakshmi et al., 2009b). Furthermore, in spite, Asia having million buffaloes contributing 96.4% of world buffalo population (FAO, 2010) and accounting for about 96.8% and 91.9% of the world s buffalo milk and meat production, respectively (FAO, 2010), no separate mineral requirement standards (including Zn) are available for this species. Series of experiments (in vitro *Corresponding author s dnlakshmi@rediffmail.com and in vivo) were conducted (at College of Veterinary Science, Hyderabad, India) which indicated 80ppm zinc supplementation to be optimum for growth and digestibility while higher immune response and antioxidant status was observed with 140 ppm Zn supplementation (Parashuramulu et al., 2013 a & b and 2015). Several researchers have also noticed beneficial effect of higher levels of Zn supplementation than recommended levels on antioxidant status, immunity (Shinde et al., 2007, Nagalakshmi et al., 2009a and Nagalakshmi et al., 2015b) and reproduction (Laar and Jongbloed, 2010 and Nagalakshmi et al., 2016). But it s over supplementation could lower the absorption and increases the mineral excretion of other minerals such as Cu (Spears, 1996), hence it causes mineral imbalance which could adversely affect the animal performance. To avoid this problem the concept of organic minerals was developed in which mineral is in a chemically inert form, more stable and less prone to interactions, so absorbed and

2 2 INDIAN JOURNAL OF ANIMAL RESEARCH circulated to target tissues very efficiently (Spears, 1996) thereby decreasing the mineral excreted and hence scope for lowering the levels of supplementation. Based on this, the present preliminary study was undertaken to investigate the effect of reducing the Zn requirements by 25% when supplemented as Zn-propionate (Zn-Prop) (60ppm) on growth performance, serum biochemical parameters, minerals retention in serum, antioxidant status and immune response in buffalo calves by comparing with 80ppm Zn supplemented from inorganic source (ZnSO 4 ). MATERIALS AND METHODS Animal and feeding management: Twelve buffalo calves (average body weight of 193.3±19.63 kg; months) were randomly divided in to two groups of six animals each in completely randomized design. A basal diet (BD) was prepared using locally available feed ingredients (Table 1) to meet the nutrient requirements (NRC, 2001) except Zn. The dietary treatments varied in the source and level of Zn supplementation in the BD. Control diet was supplemented with 80 ppm Zn from ZnSO 4, and experimental diet was supplemented with 60 ppm Zn from Zn propionate (Zn- Prop). All the experimental animals were housed in a wellventilated animal shed with the provision for individual feeding and watering. Strict hygienic and management practices were followed throughout the experimental period of 120 days. Deworming was done for both endo and ecto parasites before start of the experiment. Blood collection: Blood was collected aseptically from jugular vein with the help of sterilized needles in clean sterilized glass tubes and heparin coated vacutainers from all animals on day 114 of feeding trial for serum and haemolysate preparation, respectively. For serum, glass tubes were kept in slanted position at room temperature for 45min and then the tubes were centrifuged at 3000 rpm for 5 minutes. The clearly separated serum was transferred to 5 ml Eppendorf tubes and stored at -20 o C for estimation of various biochemical constituents and Zn content. Serum biochemistry: Serum alkaline phosphatase (ALP) activity, total protein, albumin and glucose concentration were determined by the method of Kind and King (1954), Reinhold (1953), Gustafsson (1976) and Cooper and Mc Daniel (1970), respectively. The globulin was determined by subtracting albumin from total protein content. The minerals concentration in serum was analysed by Atomic Absorption Spectrometer (Varian AA 240) according to the procedure described by Arenza et al. (1977). Haemolysate (RBC lysate) preparation and estimation of antioxidant enzyme activities: The blood collected in clean heparinized vacutainers was centrifuged at 2000 rpm for 15 minutes at 4 o C to separate buffy coat and erythrocyte pellet. The erythrocytes were washed thrice with phosphate buffer saline (PBS) (PH 7.4). Equal volume of PBS added to the obtained packed RBC and then diluted it as per requirement with distilled water. This prepared haemolysate was stored at C for further analysis of lipid peroxidation (Placer et al., 1966) and antioxidant enzyme activities viz., RBC catalase (Bergmeyer, 1983), glutathione peroxidase (Paglia and Valentine, 1967), superoxide dismutase (SOD) (Madesh and Balasubramanian, 1998) and glutathione reductase (Carlberg and Mannervik, 1985) enzyme activities. The Hb and protein concentrations in haemolysate were estimated colorometrically as per procedures of Cannan (1958) and Lowry et al. (1951), respectively. IMMUNITY Humoral immune response: Humoral immune response was assessed by using heat killed Brucella abortus S 19 and chicken RBC as immunogens. Heat killed phenolised suspension of Brucella abortus S 19 was centrifuged at 10,000 rpm for 15 min. Sediment was resuspended in normal saline and was repeated three times to get the turbidity of the cell suspension that was adjusted to McFarland tube number four (i.e. 1.2 x 10 9 cells/ml). For preparation of chicken RBC, freshly collected chicken whole blood in Elsevier s solution was centrifuged at 2500 rpm for 5 minutes. Cell pack was resuspended with sterile physiological saline to make 20% suspension. Prior to sensitization, all the heifers were screened for Brucellosis with the help of Rose Bengal Plate Test (RBPT). On day 90 of experimental feeding, all heifers were sensitized with heat killed Brucella abortus S 19 and 20% chicken RBC cell suspension, administered intramuscularly as per the body weight and a booster dose of antigen was given on 14 th d post sensitization (PS). The serum was collected from sensitized calves on 7, 14, 21 and 28 th day of PS to assess antibody titres against the antigens. The antibody response against Brucella abortus antigen was measured by standard tube agglutination test (STAT) (Alton et al., 1975). The immune response against chicken RBC was measured by direct haemagglutination assay (DHA) (Wegmann and Smithies, 1966). Cell mediated immune response: The cell mediated immune response (CMIR) was assessed at the end of Table 1: Ingredient composition (%) of the basal diet Ingredient Composition Sorghum straw 40 Maize 40 Soya bean meal 6.5 Molasses 8.5 Red gram chunni 1.41 Urea 1.0 Limestone powder 0.90 Mono calcium phosphate (MCP) 1.35 Salt 0.21 Trace mineral and vitamin premix* 0.21 *Trace mineral premix provided (mg/kg diet): Iron, 41; manganese, 21; copper, 10; cobalt, 0.1; iodine, 0.27; selenium, 0.3. Vitamin A, D and E were provided to supply 2927 IU; 1097 IU and 39 IU per kg diet, respectively.

3 experiment (on day 118) in all animals by in vivo delayed type hypersensitivity (DTH) reaction against phytohaemagglutinin phosphate (PHA-P). The skin on both sides of the neck was cleaned and shaved with help of sterile razor 24 h prior to injection so that any inflammation set during shaving or due to abrasion may subside. Next day, 150µg of PHA-P in 100 µl of Phosphate buffer saline (PBS) (ph 7.4) was injected intra-dermally into shaved area on one side of neck and 100µl of PBS was injected on other side of neck as a control. The injected sites were measured in millimeters using vernier calipers prior to injection and at 24, 48 and 72 h post injection. Inflammatory response was measured as a change in skin thickness as per the procedure described by Quist et al. (1997). Statistical analysis: The obtained data were analyzed statistically by one way ANOVA as completely randomized design using statistical package for social sciences (SPSS) 16 th version and means were compared with t test (Snedecor and Cochran, 1994). RESULTS AND DISCUSSION Performance: Optimum dietary Zn supplementation is essential for growth of the animals and lower levels of Zn in the diet is associated with negative affect on growth performance and feed intake (Jadhav et al., 2008). However, in current study, reducing the dietary Zn (60ppm) supplementation by 25% as Zn-prop had no effect on negative effect on fortnightly body weight changes, DMI and FCR throughout the feeding trial of 120 days compared Vol. Issue, () to control group animals (Table 2). This might be due to higher bioavailability of organic Zn compared to inorganic Zn (Spears, 1996; Nagalakshmi et al., 2015a&b). Similarly, reduced Zn supplementation up to 25% of recommendation, resulted in no adverse effect on growth performance in broilers (Sridhar et al., 2014). Serum biochemical parameters: The serum creatinine, albumin and globulin levels were comparable between treatments, except for total protein that was higher (P<0.01) in Zn-Prop supplemented calves compared to ZnSO 4 (Table 3). The ALP activity, used as an indicator of animal Zn status (Miller and Cragle, 1965) was higher (P<0.05) with organic Zn (60 ppm) supplementation (Table 3). Similarly, Nagalakshmi et al. (2009 a) and Devi et al. (2014) observed higher serum ALP activity in lambs and kids, respectively, with organic Zn supplementation compared to inorganic Zn supplementation. Mineral profile: Reducing the Zn supplementation in diet by 25% and supplemented from Zn-Prop did not affect the Zn concentration in serum, as well as other minerals also (Cu, Fe and Mn) and was comparable to ZnSO 4 supplemented at 80ppm (Table 4). Similarly, Cope et al. (2009) observed comparable serum Zn levels in dairy cows supplemented with either recommended level (60 ppm) of Zn from ZnO or lower levels (36 ppm) from chelated Zn. Present study results are in agreement with the findings of Sridhar et al. (2015) who observed that, reducing Zn supplementation up to 25% of NRC recommendation using organic Zn (Zn glycinate) Table 2: Body weight changes (kg), dry matter intake (kg) and feed conversion ratio (intake/gain) in buffalo calves fed Zn propionate supplemented diets Fortnight ZnSO 4 Start st nd rd th th th th th Weight gain Average daily gain (g) Average DMI DMI/100kg BW FCR DMI: Dry matter intake; FCR: Feed conversion ratio (kg intake/kg gain); SEM: Standard error mean Table 3: Serum biochemical constituents in buffalo calves fed zinc propionate supplemented diets Attribute ZnSO 4 ALP (U/L) a b Total protein (g/dl) 5.81 a 7.97 b Globulin (g/dl) Albumin (g/dl) Creatinine (g/dl) ab Means with different superscripts in a row differed significantly: P<0.05; SEM: Standard error mean

4 4 INDIAN JOURNAL OF ANIMAL RESEARCH Table 4: Serum mineral concentration in buffalo calves fed zinc propionate supplemented diets Mineral ZnSO 4 Zinc (ppm) Copper (ppm) Manganese (ppm) Iron (ppm) SEM: Standard error mean had no significant influence on the serum Zn and other minerals (Cu, Mn, Fe) concentration in broiler chicken. The Zn retention in rats supplemented with either recommended levels (ZnCO 3 ; 12ppm) or lower levels (Zn-nicotinate; 9 or 6 ppm) using organic Zn (Nagalakshmi et al., 2015a) corroborating with the present findings. Antioxidant status: Zn is a cofactor for superoxide dismutase (SOD) enzyme (Marklund et al., 1982) and also considered as marker for Zn bioavailability (Paik et al., 1999). This enzyme activity was comparable between dietary treatments. No significant effect with variation in source and dose of Zn supplementation was observed on glutathione reductase and RBC catalase activities (Table 5). However, the glutathione peroxidase activity which play a vital role in antioxidant defense system (Prasad, 2014) was higher (P<0.05) in Zn-prop supplemented calves than ZnSO 4 group (Table 5). In agreement, Nagalakshmi et al. (2015a) also observed higher GPx activity in rats supplemented with 9 ppm (75% of recommended levels) organic Zn (Zn nicotinate) compared to control group rats supplemented with 12 ppm Zn as ZnSO 4. The lipid peroxidation (LPx) which can be treated as best marker of oxidative stress (Sies, 1997) was comparable between dietary group animals though reducing the Zn supplementation by 25% as Zn-prop compared to control (Table 5). Immune response: Supplementing Zn at lower level in the form of Zn-prop did not affect the antibody titres in buffalo calves against chicken RBC at 7, 14, 21 and 28 days of PS. Similarly, the STAT titres against B.abortus were comparable between the treatments on day 14, 21 and 28. However on day 7 after immunization, higher (P<0.05) STAT titres were observed in Zn-prop supplemented calves compared to ZnSO 4 supplemented calves (Table 6). The CMIR against PHA-P expressed as DTH reaction was higher (P<0.05) in Zn-Prop supplemented calves compared to ZnSO 4 at both 24 and 48h of post sensitization (Table 7). Current findings are in consistent with the results of Sridhar et al. (2016) who observed better (P<0.05) cell mediated immunity in Table 5: Lipid peroxidation and antioxidant enzyme activity in buffalo calves fed zinc propionate supplemented diets Attribute ZnSO 4 Lipid peroxidation ( µmol MDA/mg Hb) Glutathione peroxidase (µmole NADPH oxidized/g Hb/min) 7.64 a b Glutathione reductase ( µmol/mg Hb) RBC Catalase (mmol/mg Hb) SOD (IU/mg protein) abc Means with different superscripts in a row differed significantly: P<0.05; SEM: Standard error mean Table 6: Humoral immune response against B. abortus titers and chicken RBC-HA titers (log 2 ) in buffalo calves fed zinc propionate supplemented diets Days of post sensitization ZnSO 4 B. abortus a 9.40 b Chicken RBC abc Means with different superscripts in a row differed significantly: P<0.05; SEM: Standard error mean Table 7: Cell mediated immune response against PHA-P (increase in skin fold thickness, mm) in buffalo calves fed zinc propionate supplemented diets Hours post sensitization ZnSO 4 24h 2.24 a 3.65 b h 1.37 a 2.12 b ab Means with different superscripts in arrow differed significantly: P<0.05; SEM: Standard error mean. PHA-P: Phytohemagglutinin- Phosphate

5 broiler chicks with 30 ppm Zn (75 % of recommended levels) supplementation in organic form compared to birds supplemented with 40 ppm Zn (recommended level) from ZnSO 4. Similarly, Nagalakshmi et al. (2009a) and Wang et al. (2013) observed better immune response with organic Zn supplementation compared to inorganic Zn supplementation in lambs and dairy cows, respectively. Vol. Issue, () Based on the results it can be concluded that, zinc supplementation in the diets of buffalo calves could be reduced to 60 ppm with supplementation from organic Zn (Zn propionate) with performance and immune response similar to those supplemented with 80ppm Zn from ZnSO 4. REFERENCES Alton, G.G., Jones, L.M. and Pietz, D.E. (1975). Laboratory techniques in brucellosis: 2nd edition: WHO Monograph Series, 55: WHO: Geneva. Arenza, J.S., Hathi, S.D., Singh, B. and Verma, P.N. (1977). Status of some micro minerals in neonatal buffalo calves and their mothers. Indian J. Dairy Sci., 30: Bergmeyer, H.U. (1983). Catalase. In: Methods of Enzymatic Analysis, Vol 2, pp (Weinheim, VerlagChemie). Cannan, R.K. (1958). Proposal for a certified standard for use in hemoglobinometry. Clin. Chem., 4: 246. Carlberg, I. and Mannervik, B. (1985). Glutathione reductase. Methods in Enzymol., 113: Chasapis, C.T., Loutsidou, A.C., Spiliopoulou, C.A. and Stefanidou, M.E. (2012). Zinc and human health: an update. Arch.Toxicol., 86(4): Cooper, G.R. and Mc Daniel, V Assay methods. In: Mc Donald R.P. (Ed.) Standard methods for clinical chemistry. John Wiley and Sons, New York Cope, C.M., Mackenzie, A.M., Wilde, D. and Sinclair, L.A. (2009). Effects of level and form of dietary zinc on dairy cow performance and health. J. Dairy Sci., 92(5): Devi, J., Goswami, J., Sarmah, B.C. and Sarma, K. (2014). Effect of zinc supplementation on serum enzym.tic activities of Assam local kids. Indian J. Small Rumin. 20(1): FAO (2008) Production Yearbook. Gowda, N.K.S, Pal, D.T., Gupta, R., Prasad, C.S. and Sampath, K.T. (2009). Feed resources and feeding practices in different agroeco zones of India. NIANP, Bangalore. Gruber, K., Rink, L., Calder, P.C. and Yaqoob, P. (2013). The role of zinc in immunity and inflammation. Diet Immun. Inflam., 1: Gustafsson, E.J. (1976). Improved specificity of serum albumin determination and estimation of acute phase of reactants by use of the bromocresol green. Quinn Chem. 22: Jadhav, S.E., Garg, A.K. and Dass, R.S. (2008). Effect of graded levels of zinc supplementation on growth and nutrient utilization in male buffalo (Bubalus bubalis) calves. Anim. Nutr. Feed Technol., 8(1): Kind, P.R. and King, E.J. (1954). Estimation of plasma phosphatase by determination of hydrolyzed phenol with amino-antipyrine. J. Clinic. Path. 7: Laar, H.V. and Jongbloed, A.W. (2010). Effect of zinc and copper on health and fertility in cattle, internal report ,Wageningen UR Livestock Research Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193(1): Madesh, M. and Balasubramanian, K.A Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J. Biochem. Biophysics, 35: Marklund, S.L., Westman, N.G., Lundgren, E. and Roos, G. (1982). Copper and zinc-containing superoxide dismutase, manganesecontaining superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res., 42: l. Miller, J.K. and Cragle, R.G. (1965). Gastrointestinal sites of absorption and endogenous secretion of zinc in dairy cattle. J. Dairy Sci., 48:370. Nagalakshmi, D., Dhanalakshmi, K. and Himabindu, D. (2009 a). Effect of dose and source of supplemental zinc on immune response and oxidative enzymes in lambs. Vet. Res. Commun., 33(7): Nagalakshmi, D., Reddy, M.R. and Prasad, M.R. (2009b). Mineral status of dairy animals in high altitude and tribal zone of Andhra Pradesh. In: Proceedings of ANA world conference, 14th -17 th February, 2009, New Delhi. Nagalakshmi, D., Sridhar, K. and Parashuramulu, S. (2015a). Replacement of inorganic zinc with lower levels of organic zinc (zinc nicotinate) on performance, hematological and serum biochemical constituents, antioxidants status, and immune responses in rats. Vet. World, 8(9): Nagalakshmi, D., Rao, K. S., Aruna, G. and Sridhar, K. (2015b). Effect of replacing inorganic zinc with lower levels of organic zinc on zinc retention and follicular population in rats. J. Anim. Res., 5(4): Nagalakshmi, D., Rao, K.S., Kumari, G.A., Sridhar, K. and Satyanarayana, M. (2016). Comparative evaluation of organic zinc supplementation as proteinate with inorganic zinc in buffalo heifers on health and immunity. Indian J. Anim. Sci., 86(3): Nielsen, F.H. (2012). History of zinc in agriculture. Advances in Nutrition: An Int. Review J., 3(6):

6 6 INDIAN JOURNAL OF ANIMAL RESEARCH NRC. (2001). Nutrient Requirements of Dairy Cattle. 7 th Revised Edition, Subcommittee on Dairy Cattle Nutrition, Committee on Animal Nutrition, Board on Agriculture and Natural Resources, National Research Council, National Academy Press, Washington, D.C. Oteiza, P.I. (2012). Zinc and the modulation of redox homeostasis. Free Radic. Biol. Med., 53(9): Paglia, D.E. and Valentine, W.N. (1967). Studies on the quantitative and qualitative of erythrocyte glutathion peroxidase. J. Lab. Clin. Med., 70: Paik, H.Y., Joung, H., Lee, J.Y., Lee, H.K., King, J.C. and Keen, C.L. (1999). Serum extracellular superoxide dismutase activity as an indicator of zinc status in humans. Biol. Trace Elem. Res., 69(1): Parashuramulu, S., Nagalakshmi, D., Srinivasa Rao, D and Kishan Kumar, M. (2013a ). Effect of zinc supplementation on growth performance and nutrient digestibility in Murrah buffalo calves. Indian J. Dairy. Sci., 66(6): Parashuramulu, S., Nagalakshmi, D. and Rao, D.S. (2013b). Dose response effect of dietary zinc on in vitro digestibility and gas production in sorghum based diet for buffaloes. Indian J. Anim. Nutr., 30(4): Parashuramulu, S., Nagalakshmi, D., Rao, D.S., Kumar, M.K. and Swain, P.S. (2015). Effect of zinc supplementation on antioxidant status and immune response in buffalo calves. Anim. Nutr. Feed Technol., 15(2): Placer, Z.A., Cushman, L.L. and Johnson, B.C. (1966). Estimation of product of lipid peroxidation (Malonyl Dialdehyde) in biochemicalsystems. Anal Biochem., 16: Prasad, A.S. (2014). Zinc: An antioxidant and anti-inflammatory agent: Role of zinc in degenerative disorders of aging. J. Trace Elem. Med. Biol., 28(4): Quist, C.F., Howerth, E.W., Bounous, D.I. and Stallknecht, D.E. (1997). Cell mediated immune response and IL-2 production in white triated deer experimentally infected with haemorrhage disease viruses. Vet. Immunol. Immunopathol., 56: Reinhold, J.G. (1953). Standard Methods of Clinical Chemistry C, [(Ed.) Rynner, M], Academic Press. New York. p. 88. Shinde, P.L., Garg, A.K., Das, R.S., Chaturvedi, V.K. and Kaushik, P. (2007). Effect of dietary supplementation of zinc on humoral immunity Effect of dietary supplementation of zinc on humoral immunity in guinea pigs. Indian J. Anim. Nutr., 24(1): Sies, H. (1997). Oxidative stress: oxidants and antioxidants. Experiment Physiol., 82(2): Snedecor, G.W. and Cochran, W.G. (1994). Statistical methods, 8th ed., (Iowa State University Press, Ames, Iowa, USA). Spears, J.W. (1996). Organic trace minerals in ruminant nutrition. Anim. Feed Sci. Technol., 58(1): Sridhar, K., Nagalakshmi, D., Rao, D. S. and Rao, S.V. (2014). Effect of dietary addition of organic zinc on performance and carcass traits in commercial broiler chicken. Indian J. Poult. Sci., 49(2): Sridhar, K., Nagalakshmi, D., Rao, D.S. and Rao, S.V. (2015). Effect of supplementation of graded levels of organic zinc on nutrient utilization and retention of minerals in Broiler Chicken. Indian J. Anim. Nutr., 32(1): Sridhar, K., Nagalakshmi, D., Srinivasa Rao, D. and Rama Rao, S.V. (2016). Effect of graded concentration of organic Zinc (Zinc glycinate) on antioxidants status and immune response in commercial broilers. Indian J. Anim. Res., 50(4): Tian, X. and Diaz, F.J. (2011). Zinc depletion causes multiple defects in ovarian function during the periovulatory period in mice. Endocrinol., 153(2): Wang, R.L., Liang, J.G., Lu, L., Zhang, L.Y., Li, S.F. and Luo, X.G. (2013). Effect of zinc source on performance, zinc status, immune response, and rumen fermentation of lactating cows. Biol Trace Elem. Res., 152(1): Wegmann, N.T.G. and Smithies, O. (1966). A simple haemagglutination system requiring small amounts of red cells and antibodies transfusion. Philadelphia, 6: 67.