Effect of Dietary Protein on Egg Production and Immunity Responses of Laying Hens During Peak Production Period

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1 Interntionl Journl of Poultry Science 4 (9): , 2005 ISSN sin Network for Scientific Informtion, 2005 Effect of Dietry Protein on Egg Production nd Immunity Responses of Lying Hens During Pek Production Period hiypoom unchsk, Knokn Poosuwn, Rttn Nukrew, 1 2 Knchn Mrkvichitr nd pssr hoothes Deprtment of niml Science, Fculty of griculture, Deprtment of Physiology, Fculty of Veterinry, Ksetsrt University, ngkok, Thilnd, strct: The study ws completely rndomized design. Six hundred nd seventy two commercil lying hens (cock -308) from 21 to 33 weeks of ge were used. The hens were divided into 3 groups; ech group consisted with 6 replictions of sixteen lyers ech, nd then two hens were kept in multiple-cge locted in evportive cooling house system. Feed nd wter were offered d liitum. ccording to the experimentl groups, 3 levels of dietry protein (14, 16 nd 18% P) with similr energy content (2,750 ME kcl/kg) were given to the hens in order to investigte effects of dietry protein on their production performnces, liver triglyceride, serum non-esterified ftty cid (NEF) nd immunity responses during pek production period. The results showed tht hens received 14% P diet hd significntly poorer in production performnces thn the 16 nd 18% P groups. Liver weight of hens fed 14% P diet ws smller thn those of 16 nd 18% P diets (P<0.05). However, the protein conversion rtion ws significntly improved s decrese of protein consumption (P<0.01), while feed intke ws not significntly ffected y dietry protein levels. There were tendencies of increse of liver triglyceride nd NEF due to high protein consumption. For the immunologicl spect, Newcstle disese (ND) titre of hens fed 18% P diet ws significntly higher thn those of hens fed 16 nd 18% P diets (P<0.05). Except lph-gloulin nd rtio of lumin : gloulin tht tended to decline, ll serum protein frctions nd serum totl protein were tended to increse s protein levels incresed. Spleen size ws not ffected y dietry protein levels. Key words: Dietry protein, production performnce, non-esterified ftty cids, immunity responses Introduction Thilnd is locted in the tropicl zone. During summer o seson, the temperture is round This high temperture cn normlly induce het stress nd loss of resistnce to infectious diseses. lthough the rising lying hens in Thilnd hs een commercilized nd evportive cooling system re commonly implemented in order to minimize those prolems. However, in generl, hens produce high egg production need more nutrients consumption nd susceptile to lterntion of the environments. Thus, nutrient nd environment fluctutions during pek production period my hve serious implictions on defensive system nd nutrients metolism of the hens. Inpproprite mount of nutrients consumption cn modulte quntittive nd qulittive spects of the immune response to pthogens. In poultry, it hs een shown tht deficiency or excess of dietry protein (Glick et l., 1981, 1983; Pyne et l., 1990) or mino cids (hrgv et l., 1970; Tsige et l., 1987,) chnges immune responses. Protein deficiency inhiited ntiody production nd the development of ntiodyproducing cells in response to T-dependent ntigens (rlomgno et l., 1980). Glick et l. (1983) showed tht diet deficient in protein (33% of requirement) could reduce numers of lymphocytes in the thymus of chickens. However, the responses re vried y strin (Mnzoor et l., 2003), environment, stress, production stte nd helth sttus. Thus, protective immune responses require supply of nutrients t the pproprite times nd mounts (Humphrey et l., 2002). Regrding to the mesurement of protein induced immune nd lipid metolism ltertion, pproprite hemtologicl, immunologicl tests, liver triglyceride nd NEF in serum hve een recommended (Mrgret, 2001 nd Poosuwn nd unchsk, 2004). The present study, therefore, ws designed to ssess the effect of vrious dietry protein levels on production performnce, liver triglyceride, serum NEF nd serum protein frctions of commercil hens during pek production period. ntiody response to ND vccine ws mesured to ssess humorl-medited immunity, while the serum protein frctions were used s sensitive indictors of protein deficiency responses relevnt to immune function. Mterils nd Methods Hens nd diets: The study ws designed with completely rndomized design. Six hundred nd seventy two commercil lying hens (cock -308) from 21 to 33 weeks of ge were used. The hens were divided into 3 groups; ech group consisted with 6 replictions of 701

2 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Tle 1: omposition of experimentl diets Ingredients (%) 14% P 16% P 18% P orn Soyen mel (44% P) Fish mel (58% P) Extrcted rice rnd L-lysine Monoclciumphosphte (P 21%) Oyster shell Slt / Premix Totl (kg) rude Protein (%) Metolizle energy (kcl/kg) / Premix: Lutvit Mix NK004 consist of Vitmin 4.80 MIU; D MIU; E 3.20 g; K g; g; g; g; g; Pntothenic cid 3.80 g; Nicin 6 g; Folic cid 0.20 g; iotin g; Se 0.04 g; Fe 24 g; Mn 24 g Zn 16 g; u 2.40 g; I 0.14 g; Feed preservtive sustnce 2.50 g; Flvor 10 g nd crrier dded to 1.00 kg premix. sixteen lyers ech, nd then hens were kept in protein conversion rtio ws clculted s grm feed multiple-cge locted in evportive cooling house nd protein per dy per hen divided y grm egg mss system. Environmentl temperture nd reltive per dy per hen, respectively. o humidity were set round nd 75-80%, t the end of experiment, fter overnight feed deprivtion, respectively. ge dimensions were 41 cm 46 cm one hen from ech repliction tht hd ody weight 2 equling 1,886 cm totl floor spce. With 2 hens per closed to the replicte men were chosen, 30 ml of 2 cge, ech ird hd pproximtely 943 cm of floor lood were tken from wing vein. Susequently, the spce. The lighting period ws provided for 16 hours hens were killed to evlute the size of spleen nd liver. from 05:00 to 21:00 dily. Wter nd feed in msh form o The livers nd serum were stored t -20 until the were supplied d liitum throughout the experiment. chemicl nlysis. To test the effects of dietry protein, three dietry protein levels (14, 16 nd 18% P) were given to the hens Smple preprtion nd chemicl nlysis: Serum from 21 to 33 weeks of ge. ccording to the suggestion ws seprted from the 30 ml of lood smples y of Poeikhmph nd unchsk (2004), the centrifugtion t 3,000 rpm for 15 minutes. Then, serum metolizle energy of ech experimentl diet ws o ws kept t -20 efore chemicl nlysis. Serum totl 2,750 kcl/kg diets. To eliminte the influence of protein ws ssyed using iuret method (Test kit; Er methionine nd mino cid pttern (reltive to lysine), Dignostics Mnnheim GmH). mount nd frctions of rtio of crude protein to methionine of ech experimentl serum protein such s lumin, lph-gloulin, etgroup ws kept t nd 0.11% of synthetic L-lysine gloulin nd gmm-gloulin were investigted y ws supplemented to the 14% P diet. Electrophoresis (cellulose cette technique). Other nutrients in diets were djusted ccording to the Hemgglutintion-Inhiition test (HI-test) ws used to recommendtion of NR (1994) (Tle 1 nd 2). Feed evlute the level of ND-titre. Non-esterified ftty cid smples were collected nd susequently ground using (NEF) in serum ws determined ccording to the 1-mm screen in grinder. ll diets were nlyzed for description of Smith (1975). protein, ft, fier, sh, clcium nd phosphorus Livers were retined for histopthologicl study. ccording to the O (1990) methods. Gross energy Determintion of triglyceride content in the liver ws contents in diets were determined y om-clorimeter. prepred ccording to the method of Sutton et l. (1984). mino cids composition of feedstuff nd experimentl One grm of liver ws extrcted with 5-fold of cetonediets were determined using Ner-Infrred ethnol (1:1 v/v) y homogeniztion t 20,000 rpm for 1 Spectroscopy (NIR) (Jinguo et l., 2002). min. fter the homogeniztion, the smples were centrifuged t 3,000 x g t room temperture for 10 min, Mesurements: Hen-dy egg production nd feed nd then superntnt ws tken for triglyceride intke were recorded dily wheres egg weight were determintions using Enzymtic colorimetric method determined 4 weeks intervl (3 periods), regulrly in the (Er Dignostics Mnneim GmH s test kit). sme dy of period. The hens were weighed t the eginning nd finl dte of the experiment. Egg mss Sttisticl nlysis: ll dt were sttisticlly nlyzed ws clculted y multiplying egg weight y hen-dy egg using nlysis of vrince (NOV). The differences production percentge. Feed conversion rtio (FR) nd etween the mens of groups were seprted y 702

3 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Tle 2: nlyticl nutrients composition in experimentl diets Nutrients 14%P 16%P 18%P Moisture (%) Dry mtter (%) Ft (%) Fier (%) sh (%) Gross energy (Kcl/Kg) Protein (%) lcium (%) Totl phosphorus (%) Lysine (%) Methionine (%) Tryptophn (%) Threonine (%) rginine (%) Isoleucine (%) Vline (%) Leucine (%) Totl rnch chin mino cids (%) Phenyllnine (%) Histidine (%) Duncn s Multiple Rnge Test (Duncn, 1955). ccordingly, Humphrey nd Klsing (2004), Keshvrz Sttements of sttisticl significnce re sed on nd Jckson (1992) nd Penz nd Jensen (1991) P<0.05. ll sttisticl nlyses were done in ccordnce reported tht poultry fed diets deficient in protein, lysine, with the method of Steel nd Torrie (1980). methionine, threonine, or rginine did not show ny chrcteristic depression in feed intke. In contrst, our recent study showed tht hens fed with low P diet (14% P) hd significntly lower feed intke thn the 16% P group (unchsk nd Silpsorn, 2005). In ddition, lowering feed intke of roiler chicks due to indequte methionine consumption lso hs een reported y unchsk et l. (1996; 1997). Since the present study ws conducted in n evportive cooling system, while the previous study ws conventionl house system. Difference results of feed consumption my cused y the environmentl differences. Results nd Discussion Production performnce nd mino cids intke: Effect of dietry protein on production performnces nd mino cids consumption re presented in Tle 4 nd 5, respectively. Dietry protein levels did not significntly ffect to egg production, ut higher protein contents (16 nd 18% P) tend to hve etter percentge of egg production thn the 14% P. Egg mss of hens fed the 16 nd 18% P diets were significntly higher thn the 14% P group due to hevier egg weight (P<0.05). There ws no effect of dietry protein levels on feed, energy consumptions nd mortlity rte of the hens. Essentil mino cids nd protein intke were linerly incresed s increse of protein content in diets (P<0.01). However, feed intke ws not ffected y dietry protein levels. The results were in greement with generl phenomen tht egg production of hens received dietry high in oth qulity nd quntity re commonly incresed. ompring recommendtion of NR (1994), hens fed 14% P diet hd deficient in protein, methionine, isoleucine, vline nd tryptophn consumption, while ll essentil mino cids consumption in the 16 nd 18% P groups were meet or higher thn hens requirement. For tht reson, production performnce of 16% P group ws not different from the 18% P group. The study showed tht decrese of dietry protein levels from 18 to 14% did not ffect to feed intke of the hens. 703 Liver triglyceride, serum NEF, spleen nd ND titre: Liver weight, liver triglyceride, serum NEF, spleen weight, ND titre nd totl serum protein re shown in Tle 6. Hens fed 14% P diet hd smller liver weight (expressed s % of ody weight) thn the 16 nd 18% P diets (P<0.05). onsequently, liver triglyceride (mg/liver) of hens fed 18% P diet ws significntly higher thn those of 14 nd 16% P diets (P<0.05). It seems tht liver triglyceride, NEF, spleen weight nd totl serum protein were elevted s the increse of dietry protein levels, lthough significnt differences were not found. Hens received 18% P diet hd significntly higher ND titer thn the hens fed 14 nd 16% P diets (P< 0.05). In vin species, ft synthesis nd ccumultion in the liver re minly ffected y hormone estrogen which synthesized from n ovry (ki et l., 1982). In the 18%

4 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Tle 3: nlysis of chemicl nutrients composition of feedstuff orn Soyen mel Fish mel Extrcted rice rn Moisture (%) Protein (%) lcium (%) Totl phosphorus (%) sh (%) Lysine (%) Methionine (%) Tryptophn (%) Threonine (%) Isoleucine (%) rginine (%) Leucine (%) Vline (%) Tle 4: Effects of dietry protein on production performnces of lying hens during weeks of ge P (%) P-vlue Finl ody weight (kg) 1700± ± ± ody weight chnge (g) 390± ± ± Egg production (%) 91.9± ± ± Egg weight (g) ± ± ± Egg mss 51.8 ± ± ± Feed intke (g/hen/d) 110.1± ± ± Energy intke (kcl/hen/d) ± ± ± Feed conversion (feed intke : egg mss) 2.12 ± ± ± Protein conversion rtio 0.29 ± ± ± (protein intke : egg mss) Mortlity (%) 0.33± ± ± ,, c Mens within row with no common superscripts differ significntly (p<0.05). Vlues reported represent n verge± SE. P group, incresing liver triglyceride content my e (triglyceride). These phenomen re confirmed y cused y high estrogen synthesis in ovry in order to reported of unchsk nd Slipsorn (2005) nd support high egg production. However, the unchsk et l. (1996, 1997). onversely, Pyne et l. lipolysis (mount of NEF) ws tended to increse; this (1990) reported tht deprivtion of protein in chickens cn e hypothesized tht during pek production period, reduced numers of lymphocytes in the circultion nd lipid turnover of high productive hens (18% P group) the spleen. Kenney et l. (1968) studied effect of chronic my e higher thn those gve lower egg productions. protein deprivtion on ntiody forming cpcity nd In ddition, ody weight ws inversely relted with the reported tht spleen size ws decresed with fewer vlues of NEF. This cn e postulted tht since feed cells, less RN nd more DN per cell, nd mounts of nd energy intke of ll experimentl groups were not circulting ntiodies ws lso depressed. Moreover, significntly different, high lipolysis rte my e Konshi et l. (2000) reported tht mino cid stimulted in order to supply energy for high egg deficiencies my preferentilly ffect cell-medited production nd triglyceride synthesis. immune responses reltive to development of the Spleen is identified s the secondry lymphoid tissue lymphoid orgns nd ntiody production in chickens. (Glick, 2000), while ll plsm proteins, except However, the results my e vried ccording to the immunogloulins, re mnufctured in the liver. Spleen durtion of protein deprivtion nd the severity of the size ws not significntly ffected y dietry tretments; deficiency. this indicted tht deprivtion of protein level in diets Moderte protein deficiency hs een intensively from 18% to 14% did not give ny hrmful effect on the exmined for its effect on immune response nd secondry lymphoid tissue, wheres higher protein infectious disese resistnce. Pyne et l. (1990) intke my induce igger liver size due to high protein reported tht susceptiility to Newcstle disese virus synthesis (serum protein frctions) nd ft synthesis ws incresed drmticlly in protein-deprived chicks, 704

5 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Tle 5: Protein nd mino cids intke of the hens P (%) P- vlue (g/hen/d) Protein ± ± ±0.127 <0.001 Methionine 0.30 ± ± ±0.002 <0.001 Lysine 0.89 ± ± ±0.007 <0.001 Isoleucine 0.59 ± ± ±0.004 <0.001 Leucine 1.33 ± ± ±0.011 <0.001 Vline 0.73 ± ± ±0.006 <0.001 rnch-chin mino cids 2.65 ± ± ±0.022 <0.001 Threonine 0.58 ± ± ±0.005 <0.001 rginine 0.93 ± ± ±0.008 <0.001 Histidine 0.46 ± ± ±0.004 <0.001 Phenyllnine 0.74 ± ± ±0.006 <0.001 Tryptophn 0.16 ± ± ±0.001 <0.001,, Mens within row with no common superscripts differ significntly (p<0.01). Vlues reported represent n verge± SE. Tle 6: Effect of dietry protein levels on liver triglyceride, NEF, ND titer nd totl protein in serum t 33 weeks of ge P (%) P-vlue Serum non-esterified ftty cid (micro mole/liter) 1210± ± ± Liver weight (% of ody weight) 1.67 ± ± ± Liver triglyceride (mg/liver) 934 ± ± ± Liver triglyceride (mg/g liver) 32.7± ± ± Ftty degenertion score* Spleen weight (% of ody weight) 0.10± ± 0.14± ND Titre (HI-test) 2.20 ± ± ± Totl serum protein (mg/ml) 3.38 ± ± ± ,,c Vlues within row with different superscripts re significntly different (p<0.05). Ftty degenertion score; 0 = norml, 1 = mild, 2 = undnt. * ochrn-mentel-henzel Sttistic the sme s serum ntivirl ntiody responses were Therefore, we suggest tht deficient in methionine my lso reduced significntly. Similrly, the present study minly ffected to negtive immunity responses of the lso showed tht hens received high protein (18% P) hens. Likewise, it seems tht mino cids requirement hd significntly higher ntiody titre of ND thn the 14 for egg production is lower thn the requirement for nd 16% P diets. Reduction of this humorl immunity humorl immune responses. my e cused y the deficiencies of methionine, isoleucine, vline nd tryptophn in the 14% P group. Serum protein frctions: Effect of dietry protein on Severl investigtors hve reported the negtive effects serum lumin nd gloulin re presented in Tle 7. of indequte essentil mino cids in diets on immune Dietry protein levels did not significntly ffect to responses. For exmple, hrgv et l. (1970) nd mount of serum lumin. For gloulin frctions, et- Mzij et l. (1982) showed tht the shortge of vline (it gloulin ws highest in hens fed 18% P diet (P<0.05). mkes 7% of poultry gmm-glouline), lysine, Gmm-gloulin nd totl serum gloulin were methionine or methionine + cysteine hd significnt incresed when protein levels in diets were incresed negtive effects on the immune response to ND vccine (not significnt). In contrst, hens fed 14% P diet tend (HI titre). mong mino cids deficiency, Konshi et l. to hve higher lph-gloulin thn the hens fed 16 nd (2000) reported tht rnch-chin mino cids () 18% P diets, whilst protein intke seems to reduce the hd the gretest potentil to modulte immune rtio of lumin : gloulin. responses in chickens. In current study, lthough vline Grndhi et l. (1975) reported tht serum protein profiles nd isoleucine were insufficient for the requirement of were vried with reed, strin nd ge; for exmple, the hens fed 14% P diet, ut totl ws higher thn percentge of gmm-gloulin frction ws greter in hens requirement due to high level of leucine. dwrf 1 wk. old nd lying hens of the White Leghorn 705

6 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Tle 7: Effect of dietry protein levels on lumin nd serum protein frctions of lying hens during weeks of ge P (%) P-vlue lumin (g/dl) 1.14± ± ± lph-gloulin (g/dl) 0.55± ± ± et-gloulin (g/dl) 1.74 ± ± ± Gmm-gloulin (g/dl) 0.05± ± ± Gloulin (g/dl) 2.20± ± ± lumin : Gloulin 0.60± ± ± ,, c Mens within row with no common superscripts differ significntly (p<0.05). Vlues reported represent n verge± SE. reed when compred with the norml hens. However, ntigenic stimultion, usully from infectious gents the serum protein profiles in present study were in (Mrgret, 2001). ho nd Lee (2001) hve exmined greement with the report of Odunsi (2005) who showed the ssocition etween the level of serum gmmtht lumin, gloulin nd lumin : gloulin of gloulin nd reproductive performnce or immunity of roiler chicks were round , nd Tiwn ountry chicken. They found tht the high serum , respectively. gmm-gloulin level is geneticlly ssocited with low In vin species, serum totl proteins re consisted with fertility. ccording to Person nd Heron (1982) who lumin nd gloulins nd these prmeters re reported tht high protein intke induced low fertility nd commonly used in nutritionl studies. Since the htchility of roiler reeder hens. In greement with quntities of essentil mino cids nd protein our study, the dt lso showed tht hens received 18% consumption of 18% P group were higher thn the P hd highest level of the gmm-gloulin. requirements of rown lying hens (110 g/d of feed intke) tht recommended y NR (1994). Thus, the cknowledgements results of high serum proteins of this group re not The uthors grtefully cknowledge tht its primry surprised. lthough gede nd letor, (2003) hve funding comes through the Ntionl enter for Genetic reported tht totl serum protein, lumin nd gloulin Engineering nd iotechnology (IOTE), Thilnd. We syntheses were not ffected y sources of dietry protein lso pprecite the support of severl stff from (qulity of protein). urrent study grees with Eggum, Deprtment of niml Science t Khmphengsen (1989) nd Tewe, (1985) who stted tht totl serum mpus, Ksetsrt University. protein, gloulin nd lumin were directly responsive to oth protein quntity nd qulity. Reference lumin is serves s the mjor reservoir of protein nd gede, J.O. nd V.. letor, Evlution of fish involved in colloidl osmotic pressure, cid-se mel replced with lef protein concentrte from lnce, nd it cts s trnsport crrier for smll gyricidi in diets for roiler chicks: Effect on molecules such s vitmins, minerls, hormones nd performnce, muscle growth, hemtology nd ftty cids (Mrgret, 2001). The results of production serum metolites. Int. J. Poult. Sci., 4: performnce tht ssocited with serum lumin ki, Y., L.S. Jenson,.R. r nd R.R. Kreling, confirmed the generl knowledge. The gloulins re Plsm estrdiol, thyroid hormones nd liver composed of three frctions, designted lph, et nd lipid content in lying hens fed different isocloric gmm. In this study, serum et nd gmm-gloulin diets. J. Nutr., 112: were incresed s increse of protein levels, while the O., Officil Method of nlysis. 15th ed. result of lph-gloulin seems to e contrsted. ssocition of Officil griculturl hemists, Inc., lph-gloulins re group of proteins mnufctured Virgini, 1422 p. lmost entirely y the liver. Normlly, these proteins hrgv, K.K., R.P. Hnson nd M.L. Sunde, increse with cute nephritis, severe ctive heptitis, ctive, usully systemic inflmmtion, mlnutrition nd in nephrotic syndromes (Mrgret, 2001). Thus, the tendency of increse of lph-gloulin in the 14% P group indicted n incidence of mlnutrition (deficient in protein nd mino cids). The gmm-gloulin frction contins most of the immunoproteins, including IgM, Ig, IgE nd IgG. These usully elevte with ongoing Effects of methionine nd vline on ntiody production in chickens infected with Newcstle disese virus. J. Nutr., 100: unchsk,. nd T. Silpsorn, Effects of dding methionine in low-protein diet on production performnce reproductive orgns nd chemicl liver composition of lying hens under tropicl conditions. Int. J. Poult. Sci., 5:

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8 unchsk et l.: Effect of Dietry Protein on Egg Production nd Immunity Responses Poosuwn, K. nd. unchsk, Effect of dietry protein on production performnce, triglyceride nd non-esterified ftty cid level in lying hen. P. 7. In nnul Meeting of the Thi Society for iotechnology 16st, Nresun University. (st.) Smith, S.W., New slting-out technique for clorimetric free ftty cid ssys. nly. iochem., 67: Steel, R.G.D. nd J.H. Torrie, Principle nd procedures of sttistics. Mc Grw Hill Pulishers. nd Newyork. 2 edn. Sutton,.D., W.M. Muir nd G.E. Mitchell, holesterol metolism in the lying hen s influenced y dietry cholesterol, cloric intke, nd genotype. Poult. Sci., 63: Tewe, O.O., Protein metolism in growing pigs fed corn or cssv peel sed diets contining grded protein levels. Res. Vet. Sci., 29: Tsige, V.K., M.E. ook,.e. Hrper nd M.L. Sunde, Efficcy of cysteine in replcing methionine in the immune responses of roiler chickens. Poult. Sci., 66: Tsige, V.K., M.E. ook,.e. Hrper nd M.L. Sunde, Enhnced immune responses in roiler chickens fed methionine-supplemented diets. Poult. Sci., 66: