'~, 'k ..!Ai;

1 ST Mediterranean Summitof WPSA

Advances and Challengesin PoultryScience

Book of Proceedings

Editors:A. Tserveni-Goussi

A. YannakopoulosP. FortomarisG. ArsenosE. Sossidou

07 - 10 MAY2008Porto Carras, Chalkidiki

I

Greek Branch

j,'-",,>

.r I, . .~I' Jt, >

';, ,&

Il~.~ :,.~_':"

-.

-

.-I

.,11.1'- ,.1, :>"\~I~"""W ~ "{#~ 'tI~..,

.i' .,

'..1< '~-'-.

AVIANHEALTHANDBIOSECURITYIN THEPOULTRYINDUSTRY

T EFFECTOFVITAMIN CSUPPLEMENTATIONON PERFORMANCEANDIMMUNE RESPONSEOFBROILERCHICKS

A. Hesabi Nameghi1, H. NassiriMoghaddam2

1 Agricultural and Natural ResourcesResearchCenterof Khorasan,Mashhad,Iran2 AgricultureFerdowsyuniversityof Mashhad,Iran

Correspondingauthor.Email:hessabi@kanrrc.ac.ir

Keywords: Vitamin C, immune response, performance, broiler chicks

ABSTRACT

In order to evaluatethe effect of supplementalvitamin C (VC) levelson perfor-mance and immune response of broiler chicks, 300 day-old Ross male broiler chickswere allocated in complete randomized design with 5 treatments(500 and 1000ppmin diet, 500 and 1000 ppm in drinking water and control). Immunity was assessedas antibody production to Infectious Bronchitis virus (IBV) and Newcastle Diseasevirus(NDV), mitogenic response to phytohemagglutininA (PHA) and concanavalinA(conA), cutaneous basophil hypersensitivity (CBH) to PHA. VC ( 500 and 1000ppm in drinking water and 1000ppm in diets) increased (P

pt Mediterranean SummitofWPSA.Advances and Challenges in PoultryScience

arrangement of two levels ofVC ( 500 and 1000 ppm) in drinking water and di-etary with control (five treatments) . VC was provided by Basfco LTD.

Humoral immune response. Chicks were vaccinated against, respectively in-fectious bronchitis virus (IBV) and Newcastle disease virus (NDV) by intraocularon d 6 (attenuated live virus) and i.m inoculation on 18 d of age. Blood was col-lected from the wing vein at 6 and 12 d after vaccination. Serum was isolated andstored at -20C until analyzed. Pooled samples representative of the pen, were ob-tained by mixing equal volumes of the serum from vaccinated birds from the samepens (n=3). Serum antibody titers were determined by ELISA antibody test kitl ac-cording to the manufacturer's directions.

Antibodies specific for NDV were detected in the serum of chicks by means ofa haemagglutination inhibition (HI) test (Allan et ai., 1978).

In Vitro Proliferation of isolated Blood Lymphocytes in response to phyto-hemagglutininA (PHA) and Concanavalin A (Con A). At 26 days of age, three birdsper pens were randomly assigned for lymphoblastogenesis assay in vitro. Bloodsamples (3mLlchick) were withdrawn with a EDT A syringe and diluted with equalvolume of PBS. Blood from all birds in each pens was pooled and The peripheralblood lymphocytes (PBLs) were separated from the whole blood by Ficoll (AtlantaBiologicals, Norcross, GA) density gradient by centrifuging at 2000 rpm for 30 min.The cells were washed twice and cell count was adjusted to 2x105/mL in RPMI-1640 growth medium (Fisher, Norcross, GA) supplemented with 5% heat inacti-vated fetal bovine serum and 1% antibiotic. One hundred ~L PBLs, containing2xl05 cells from each sample were added to each of the four wells of a 96-well plate.In a preliminary experiment, the dose response of the mitogen-induced lympho-cyte proliferation was determined by incubating cells with different concentrationofConA2 or pHA3. The PHA and ConA concentrations that gave maximal prolif-eration responses were choosen for testing experimental samples. All experimen-tal samples were incubated in triplicate in 96-well flat-bottom plates with eitherPHA-P (25 flg/mL), Con A (15 flg/mL), or cell culture media (nonstimulated). Theplates were incubated at 37° C in a humidified incubator in the presence of 5% CO. After 72 h of incubation, At the end of the incubation period, 20 flL of MTT4 wasadded to each well. The plate was incubated again for 4 h at 37°C with 5% C02,after which 150 flL of supernatant was pipetted from each well.,,-

One hundred fifty microliters of 10% saponin in PBS was added into each wellto lyse the cells. The plate was shaken by a plate shaker for 10 min and centrifugedat 1,500 rpm for 10 min. One hundred seventy-five microliters of supernatant waspipetted from each well,175flL of acid isopropanol solution was added, and the platewas shaken again for 10 min. The solutions in each well were then mixed by a mul-tichannel pipette until sedimentation disappeared, and the plate was then centrifugedat 1,500 rpm for 10min. One hundred microliters of supernatant from each well wastransferred to a new 96-well flat-bottomed plate. The optical density (OD) was readon a plate reader at 540 nm wavelength. Proliferative responses oflymphocytes wereexpressed as absorbance as described by Maslak and Reynolds (1995).

550

.J

P 0 S T E R PRESENTATIONS

In Vitro phytohemagglutininA (PHA) and Concanavalin Exposure in wholeblood: At 26 days of age, three birds per pens were randomly assigned for lym-phobiastogenesis assay in vitro. Blood samples (3mL/chick) were withdrawn witha EDTA syringe and diluted blood from allbirds in each pens was pooled .Pooledwholeblood (4~) and PHA(25~g/ml)or Con A(10~g/ml)were added to microtiterplates containing a 200 ~l of RPMI 16405and antibiotic. The cell were plated, in-cubated, harvested, and analyzed as described for isolated lymphocytes.

Data is reported as differencebetween skin thickness of the PHA and saline in-jected sites.Data were analyzed using the SASsoftware computer package (SAS,1989).A complete random design was used.

RESULTSAND DISCUSSION

VC addition did not influence food consumption (0-21d). Chicks fed additionalVC (lOOOppm) exhibited a significant beneficial effect for body weight gain (42d),daily weight gain and feed consumption (21-42d). VC is involved in growth bypromoting collagen synthesis, calcium and vitamin D3 metabolism, carnitine syn-thesis for oxidation of fatty acids, oxidation of amino acids, electron transport inthe cells, and scavenging of free radicals (Combs, 1992). Kidneys, which are theprincipal organs for chickens to synthesize AA, cannot synthesize adequate amountsof AA until after 15 d of age (PuIs, 1994). Therefore, kidneys of chickens at 21 d ofage are functionally and morphologically competent to synthesize sufficient amountsofVC to supply the tissues.

Table 1. Effectof vitamin C supplementation on performance in broiler chicks

Chicks drank 500and 1000ppm of added VC had significantly(P < 0.05) high-er IBV and NDV antibody titers than chicksdrank normal water. This finding wascompatible with those of previous reports that chickens supplemented with AA at

43_(4,5-dimethylthiazol-2-yl)-2,5 diphenol tetrazolium bromide135 Sigma Chemical Co., St. Louis.

551

Treatment weight gain Feed consumption Feed conversion Chick body(g/chicks/day) (g/chicks/day) ratio weight(g)0-21 21-42 0-21 21-42 0-21 21-42 21 d 42d

Vitamin C (ppm)control 20.7b 65.9ab 47.1 141.9a 2.01 2.15a 457.8b 1861.8b

500 (soluble) 21.4ab 65.4b 43.1 135.4b 2.01 2.06c 491.4ab 1865.4b

1000(soluble) 22.2a 66.3ab 42.7 138.3ab 1.91 2.08bc 507.8a 1901.6ab

500( in diets) 22.1a 65.6b 41.8 14().lab 1.89 2.13ab 504.4a 1883.4b

500( in diets) 22.1a 68.8a 43 140.9ab 1.94 2.04c 505.2a 1950.6aSEM 0.311 0.967 0.96 1.91 0.036 0.019 6.553 20.1

a-bMeansin a column with no common superscript differ significantly (P < 0.05).

pt Mediterranean SummitofWPSA .Advances and Challenges inPoultryScience

500 ppm had increased antibody titers to infectious bronchitis virus (Tuekam etaI., 1994). The increase of antibody titer VC-supplemented chickens may have beendue to the antioxidant property ofVC in that VC was able to protect immaturelymphocytes from damage by free radicals due to oxidation, thus enhancing theimmune response.

Table 2. Effect of vitamin C supplementation on Anti-NDV and IBV 6 and 12 d post vac-cination

TreatmentAnti-IBV antibody (Log10)

6 d 12 dAnti- NDV antibody (Log2)

6 d 12 d

Vitamin C (ppm)control

500 (soluble)1000 (soluble)

500 (in diets)500 (in diets)SEM

3.513.51

3.523.53.530.0106

3.67b

3.66b3.71a3.71a3.74a0.0107

4c4c

4.58b5.06a4.94a0.076

3.76c

3.86c5.14a4.86b5.36a0.085

a-bMeansin a column with no common superscript differ significantly (P < 0.05).

chicks drank 1000 ppm ofVC had significantly greater (P < 0.05) in vitro lym-phocyte proliferative responses to ConA and PHA than those of chicks drank nor-mal water. proliferation with mitogen and control (without mitogen) of wholeblood lymphocytesin the mitogens PHA and Con A was affectedwith VC (Table3).

Table 3. Effectof vitamin C supplementation on the proliferation lymphocyte in purifiedlymphocyte and whole blood (OD in 540nm)

Lymphocyte proliferation in response to mitogens is correlated with the abili,.ty of the host to mount a cellular immune response. It has been suggested that d-

552

Purified lymphocyte Whole blood

Without Mitogen Without Mitogen

Mitogen PHA Con.A Mitogen PHA Con.ATreatment (Control) (Control)

.control 0.343c -' 0.402d 0.374c 0.222b 0.294c 0.277b

500(soluble) 0.496b 0.587b 0.641b 0.237b 0.351b 0.31b

1OOO(soluble) 0.414b 0.514c 0.45c 0.244b 0.284c 0.277b

500( in diets) 0.455b 0.646b 0.467c 0.162c 0.25c 0.266b

500( in diets) 0.636a 0.896a 0.739a 0.355a 0.584a 0.424aSEM 0.023 0.024 0.03 0.0128 0.0169 0.015

a-bMeansin a column with no common superscript differ significantly (P < 0.05).

P 0 S T E R PRESENTATIONS

ifferential reactivity to mitogens reflects either maturational or functional differ-ences in the responsive lymphocytes. VC enhances lymphocyte proliferation byimproving the responsivenessofT lymphocytesto mitogens (Johnston and Huang,1991)and its antioxidant activity (Jacob, 1995;Retskyand Frei, 1995).Bendich etal. (1984) reported that the responses ofT and B lymphocytes of guinea pigs wereenhanced by diets containing higher-than-standard levels of vitamin C supple-mentation.

In summary, VC supplementation (500and 1000ppm in drinking water) mayimprove body weight gain. Our study demonstrated that VC enhances the anti-body response to some antigens, improve the proliferative response of lympho-cytes to polyclonal mitogens.

REFERENCE

Allan, W.H., J. E. Lancaster, and B.Toth. 1978.Newcastlediseasevaccines, their produc-tion and use. FAG Animal Production and Health Series,No. 10.

Bendich, A., P. D. Apolito, E. Gabriel, and L. J.Machlin. 1984.Interaction of dietary vita-min C and vitamin E on guinea pig immune responses to mitogens. J. Nutr.114:1588-1593.

Combs, G. F., 1992.Vitamin C. Pages 223-249 in Vitamins. Academic Press, Inc., NewYork, NY.

Jacob,R A., 1995.The integrated antioxidant system.Nutr. Res. 15:755-766.Maslak, D. M., and D. L.Reynolds. 1995.Mitogenicresponses of the head-associated lym-

phoid tissues of the chicken. AvianDis. 39:1-8.National Research Council. 1994.Nutrient Requirements of Poultry. 9th ed. National A-

cademy Press,Washington, DC.PuIs,R, 1994.Vitamin levelsin animalhealth. Page 78 in:DiagonisticData and Bibliogra-

phies. Sherpa International, Clearbrook,BC,Canada.Retsky, K. L., and B. Frei. 1995.Vitamin C prevents metal iondependent initiation and

propagation oflipid peroxidation in human low-density lipoprotein. Biochem. Bio-phys. Acta. 1257:279-287.

SASInstitute, Inc. 1989.SAS/STATUser's Guide.Version6, Vol. I. SASInstitute Inc.,Cary,NC.

Tuekam, T. D., R D.Miles,and G. D. Butler. 1994.Performance and humoral immune re-sponse in heat-stressed broilers fed;m ascorbicacid supplementation. J.Appl. Anim.Res.6:121-130.

553