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1 WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI Helsinki, Finland info@world-food.net Journal of Food, Agriculture & Environment Vol.11 (2): Effects of maternal dietary 25-hydroxycholecalciferol and progeny dietary vitamin premix on progeny performance, immune responses and bone quality of broiler chicks Huan-wei Peng 1, Xue-mei Ding 1, Shi-ping Bai 1, Yu-heng Luo 1, Qing Zhu 2 and Ke-ying Zhang 1 * Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, 2 College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 25014, China. * zkeying@yahoo.com Received 22 January 2013, accepted 2 April Abstract The experiment was conducted to investigate the effects of maternal dietary 25-hydroxycholecalciferol (25-OH- ) supplementation on egg production, hatchability, progeny performance, immune function and bone quality, when the progeny was fed diets with or without vitamin premix. A 2 2 factorial arrangement was used. A total of 1152 breeder hens aged 43 wk were selected and allotted to the 2 treatments with or without 25-OH- addition ( mg/kg). The eggs at 18 wk of trial were hatched and 0 newly hatched progeny from each breeder treatment were randomly divided into 2 groups with replicates of 10 birds and fed the diets with or without progeny vitamin premix (NRC recommendation for starter) for 28 d. As results, the maternal dietary 25-OH- did not affect the reproductive performance of breeder hen, but increase the Ca level in progeny tibia at 1 d of age. Vitamin premix in progeny diet improve the growth performance, immune characters, and bone quality of progeny broilers, and decrease the variation of body weight among the individuals. Key words: Broiler breeder, broiler, progeny, reproductive performance, growth performance, bone quality, vitamin, 25-hydroxycholecalciferol, immune response, immune organ index, antibody. Introduction As a primary dietary source of vitamin D, vitamin is required for growth, health and bone development in the chick. Comparing with vitamin, dietary 25-hydroxycholecalciferol (25-OH- ) may be more efficient for conversion to 1,25(OH) 2 -, enhancing the functions that vitamin D metabolites serve within the body such as improving production efficiency 1,2 and bone quality 3. It is of great necessity to supply with 25-OH- in broiler diet when the liver was impaired because of infection stresses 4 or immaturity of enzyme development required for vitamin D absorption in the young chick 5. Currently, 25-OH- is commercially available for using in poultry diets with the the trade name of Hy.D. In previous studies, positive effects of dietary 25-OH- on body weight, breast muscle yield in broilers, as well as chick embryo development 7, was found to be several times than that of vitamin. The vitamin status of newly young chick at starter phase depends, to great degree, on the level of vitamin in the breeder hen diet 8. With development of broiler growth, young chick was more dependent on its own formulated diet, as the nutrient in chick embryo has been gradually consumed. It was supposed that the broiler performance may not only depends on the level of vitamin fed to the breeder hen, but also depends on the level fed to the young bird. Therefore, this research was to investigate the effects of maternal dietary 25-OH- on production performance of breeder hens, and the effects of maternal dietary 25-OH- or/and the progeny vitamin premix on progeny growth performance, immune function and bone quality. Materials and Methods Animals: The experimental protocol was approved by the Animal Policy and Welfare Committee of Sichuan Agricultural University under the Chinese Council on Animal Care guidelines. A total of 1152 breeder hens aged 43 wk were selected and allotted to two treatments based on the hatchability of 7 days ahead in a completely randomized design and fed with or without 25-OH- addition ( mg/kg). Two breeders were raised in a stainlesssteel cages in the same house with 1 h light for 18 wk. Breeders were free access to water by nipple drinkers, but with restricted feed intake of 0 g per day for 1 to wk, and 3 g per day for 7 to 18 wk. The breeders were artificial insemination per every 3 days. The eggs at 18 wk of trial were collected and hatched. A total of 0 newly hatched progeny from each breeder treatment were randomly divided into 2 groups with replicates of 10 birds according to initial average body weight and fed the diets with or without vitamin premix for 28 d. All the progeny were free access to feed and water. Diets and feeding: The commercial vitamin premix with or without 25-OH- for breeder and vitamin premix for progeny were provided as gifts by DSM Nutritional Products Ltd. (Shanghai, P. R. China). All diets were corn-soybean-based, formulated to meet or exceed current NRC recommendation (National Research Council, 1994) for breeder hens, or meet the recommendation of domestic nutrition requirement of China for young broilers (NY/T ). The composition of basal diet for breeder hens and young broilers was shown in Tables 1 and 2, respectively. The composition and Journal of Food, Agriculture & Environment, Vol.11 (2), April

2 levels of vitamin premix for breeder hens was shown in Table 3. The 25-OH- levels of mg/kg in breeder hen diets were according to the recommendation. The progeny vitamin premix was designed according to NRC recommendation for starter. Hatching eggs: Eggs were incubated for 21±0.5 d (temperature 37.5 C and relative humidity 85%) and transferred to a hatcher at 18 d with separate baskets. The hatchability, chick BW, and malonaldehyde (MDA) in serum of broilers were assessed for each maternal dietary treatment group. Table 1. Composition and nutrient levels of basal diet for breeder hen. Ingredients Contents (g/kg) Nutrients component Calculated levels Corn 57.7 CP (g/kg) Soybean meal ME MJ/kg 11.5 Rapeseed meal 20.0 Lys(g/kg) 8.1 Soybean oil.0 Met(g/kg) 3.9 Calcium carbonate 75.0 Ca(g/kg) 32.1 Dicalcium phosphate 15.0 TP(g/kg).2 DL-Met 1.5 AP(g/kg) 4. Lys-HCl 0.8 Salt 4.0 Choline 1.5 Vitamin premix 1.0 Mineral premix * 1.5 Total 1000 *Mineral premix (per kg diet): 80 mg Fe; 1 mg Cu; 80 mg Mn; 0 mg Zn; 0.3 mg Se; 0.3 mg I in respective form of FeSO 4 O, CuSO 4.5H 2 O, MnSO 4 O, ZnSO 4 O, Na 2 SeO 3 and KI. Table 2. Composition and nutrient levels of basal diet for broiler. Ingredients 0-4 wk Starter (g/kg) Nutrient component 0-4 wk Starter Corn ME (MCal/Kg).2 Soybean meal CP (g/kg) Rapeseed meal 30.0 Lys (g/kg) 10. Soybean oil 30.0 Met (g/kg) 4.5 Calcium carbonate 13.0 Ca (g/kg) 9.5 Dicalcium phosphate 1.0 AP (g/kg) 4.2 Salt 3.0 DL-Met 1.5 Choline 1.0 Vitamin premix 1.0 Mineral premix* 1.5 Total 1000 *Mineral premix (per kg diet): 80 mg Fe; 8 mg Cu; 80 mg Mn; 0 mg Zn; 0.15 mg Se; 0.35 mg I in respective form of FeSO 4 O, CuSO 4.5H 2 O, MnSO 4 O, ZnSO 4 O, Na 2 SeO 3 and KI. Table 3. Composition and levels of vitamin premix in diet of breeder hens. Vitamin Vitamin level (per kg of diet) Control Vitamin A, IU/kg Vitamin, IU/kg OH-, mg/kg 0 Vitamin E, IU/kg Vitamin K 3, mg/kg 5 5 Vitamin B 1, mg/kg Vitamin B 2, mg/kg 8 8 Vitamin B, mg/kg 3 3 Vitamin B, mg/kg Niacin, mg/kg Pantothenic acid, mg/kg Folic acid, mg/kg Biotin, mg/kg Progeny performance: One hundred and twenty progeny chicks from each maternal treatment were randomly allocated to 2 treatments with pens of 10 birds. The BW of broilers was measured at 1, 7, 14 and 28 d. Feed consumption and feed conversion ratio were assessed on the pen basis at each phase, Individual BW were obtained at 28 d to determine the coefficient of variation within the treatment. Ca and P contents in tibia of progeny at 1d and 28 d of age were determined, respectively. Immune response of progeny broilers: At 7 d of age, birds were immunized with Newcastle disease (ND) virus. At the end of experiment, two birds per pen selected based on average BW were killed. Blood was collected into tubes for centrifugation and then the serum was collected for ND antibody titers measurement using an ELISA kit (Breedervac, Intervet Inc., Delaware, USA). Bursa of Fabricius, thymus and spleen were quickly excised and weighed to calculate the organ index. Statistical analysis: The difference between the breeder hen dietary treatments were assessed using t-test of paired samples. The other data were analyzed as a 2 2 factorial with 2 maternal dietary treatments and 2 progeny dietary treatments with GLM procedure of SPSS 1.0 with the main factors including the maternal diet, progeny diet, and their interaction at a probability of P<. Values are expressed as mean values with their standard errors. Results Reproductivity performance of breeder hen: The maternal 25- OH- did not affect significantly egg production(data not shown), the fertility of total eggs set (Table 4), the hatchability of total fertile eggs (Table 5), and the hatchability of total eggs set (Table ). The body weight of hatched progeny were not affected significantly by maternal 25-OH- except decreased significantly at 54 wk of breeder (Table 7). There was no significant difference of MDA level in progeny at 1 d of age between treatments (P = 0.138) (Table 8). The maternal 25-OH- increased significantly the level of Ca in tibia of progeny at 1 d of age with the trend to increase the level of P in tibia (P = 0.081) (Table 9). Progeny growth performance: The maternal 25-OH- increased the F/G of broiler from 0 to 7 d(p<), and did not significantly affect the BWG, AFI of broiler at every week from 0 to28 d. The addition of vitamin premix in progeny diet improved the BWG, AFI(except at 0-7d), and FCR (except at 0-7d)of progeny(p<) with no significant interaction with maternal 25-OH- on BWG, AFI, and FCR (Table 10-). The addition of progeny vitamin premix was decreased the variation of body weight of broiler at 28d (Table 13). Progeny immune response: The addition of progeny vitamin premix increased significantly the index of bursa of Fabricius and thymus gland, while the maternal dietary 25-OH- increased the spleen index. The interaction effects on spleen index of broiler between maternal and progeny vitamin premix were significant. Without addition of progeny vitamin premix, the maternal 25-OH- decreased the spleen index (P<) (Table 14). Serum antibody responses to ND in broiler at 28 d of age were affected significantly by addition of vitamin premix in progeny diets (Table 15). 702 Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013

3 Table 4. Effects of maternal dietary 25-OH- on fertility of total eggs set (%). Treatment 4 wk 50 wk 54 wk 0 wk 4-0 wk - 25-OH OH Table 5. Effects of maternal dietary 25-OH- on hatchability of total fertile eggs (%). Treatment 4 wk 50 wk 54 wk 0 wk 4-0 wk - 25-OH OH Table. Effects of maternal dietary 25-OH- on hatchability of total eggs set (%). Treatment 4 wk 50 wk 54 wk 0 wk 4-0 wk - 25-OH OH Table 7. Effects of maternal dietary 25-OH- on average body weight of hatched progeny (g). Treatment 4 wk 50 wk 54 wk 0 wk 4-0 wk - 25-OH OH Table 8. Effects of maternal dietary 25-OH- on levels of MDA in serum of breeder hen at 0 wk of age and progeny at 1d of age. Serum MDA (nmol/ml) Treatment Hen Progeny MEAN SEM MEAN SEM - 25-OH OH Table 9. Effects of maternal dietary25-oh- on Ca and P contents in tibia of progeny at 1 d of age. Percentage of Ca and P in total tibia Treatment Ca (%) P (%) MEAN SEM MEAN SEM - 25-OH OH Table 10. Effects of maternal dietary 25-OH- and progeny vitamin premix on BWG of progeny (g). 0-7d 8-14d 15-21d 22-28d 0-28d n - Premix - Premix 37.1ab 35.0a 38.9b 38.b a 0.3a 82.0b 80.7b a 58.4a 130.4b 134.1b a 1.5a 188.9b 197.5b a 215.2a 440.1b 450.9b Journal of Food, Agriculture & Environment, Vol.11 (2), April

4 Table 11. Effects of maternal dietary 25-OH- and progeny vitamin premix on AFI of progeny (g). Progeny 0-7d 8-14d 15-21d 22-28d 0-28d n - Premix - Premix - Premix Maternal a 139.4a 174.0b 170.7b a 170.9a 328.4b 390.2b a 194.4a 37.5b 401.1b Table. Effects of maternal dietary 25-OH- and progeny vitamin premix on FCR (F/G) of progeny. Maternal Progeny -Premix -Premix - Premix 27.4a 588.2a 93.5b b d 8-14d 15-21d 22-28d 0-28d n 2.28ab 2.44b 2.18a 2.33ab a 3.37a 1.99b 2.03b a 2.78a 2.19b 2.32b Table 13. Effects of maternal dietary 25-OH- and progeny vitamin premix on BW, and BW coefficient of variation (CV) of progeny at 28d of age. n MEAN (g) SDM CV (%) Maternal vit a 259.2a 483.3b 493.7b Ca, P level in broiler tibia: The levels of Ca and P in tibia of broiler at 28d were increased by addition of vitamin premix in progeny diet, but were not significantly affected by maternal 25- OH- and their interaction (Table 1). Discussion Providing the broiler breeder hen with a more available source of vitamin D, such as 25-OH-, may increase the absorption efficiency of Ca from the gut, allowing more Ca to be available for eggshell formation. In addition, maternal supplementation of 25- OH- allows more precursors to be available during egg formation and may result in increased egg hatchability and improved chick quality which could in turn result in better growth and feed conversion. In general, 25-OH- was effective in improving performance at lower levels of dietary vitamin D compare with higher cholecalciferol levels. In this study, there was no significant improvement on production performance when broiler breeders were supplemented with 25-OH- at 4 wk of age (P>). The reason may be that the V level (3000 IU/kg) in maternal diet is enough for maternal production need. This result is in agreement with Coto et al. 9 who observed the 25-OH- effected on egg shell and hen-day production were not obtained at higher levels of vitamin D. Atencio et al. 10 reported a significant improvement in hatchability as hen breeders received increasing levels of vitamin D up to 4000 IU/kg, and also a numerical effect of vitamin D source on hatchability/fertility (P=0.07). Birds receiving low levels (35 ng/kg) of vitamin supplemented with 25-OH- (35 or 500 ng/kg) had a higher hatchability than those birds fed without Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013

5 Table 14. Effects of maternal dietary 25-OH- and progeny vitamin premix on immune organ index* of progeny at 28d of age. Bursa of Fabricius Thymus gland index Spleen index n index g/kg BW g/kg BW g/kg BW MEAN SEM MEAN SEM MEAN SEM -25-OH-D3 Maternal vit. 2.84ab 2.19a 3.4b 4.03b a 4.74a 8.92b 8.22b a 1.23b 1.5ba 1.0ba *immune organ index = immune organ weight (g)/ body weight (kg). Table 15. Effects of maternal dietary 25-OH- and progeny vitamin premix on serum antibody to ND of progeny at 28d of age. n Titers of antibody to ND in serum (log 2 ) MEAN SEM OH- supplemented 11. In this experiment, the maternal 25-OH- did not affect the progeny BW except that at 54 wk significant lower BW was found (Table 7). Parameters of mineralization, bone quality and monitoring of vitamin D plasma levels are better indicators of vitamin D status. We found that maternal 25-OH- increased significantly the level of Ca in tibia of progeny at 1 d of age with the trend to increase the level of P in tibia (Table 9), and kept the same trend at 28d broiler but no significant difference. Previous work investigating the effect of increasing maternal dietary levels of vitamin (from 0 to 2000 IU or from 5 IU to 4000 IU of vitamin 14, and from 300 to 2700 IU vitamin 15 ) on bone ash of the broilers found that the higher level of maternal dietary vitamin, the greater bone ash and reduction in tibial dyschondroplasia (TD) score of the progeny. A significant effects of progeny vitamin premix on progeny growth performance was obtained with higher BWG (Table 11), AFI (Table ), BW (Table 13) at 28d (P<). But these performance parameters were not significantly affected by maternal dietary 25-OH- (Table 11-13) (P>). On the contrary, Frits and Waldroup 13 reported that performance parameters of broilers were improved when 25-OH- was supplemented at lower level of vitamin D in broiler diet. Conclusions The addition of 25-OH- ( mg/kg) at maternal diet with V 3000 IU/kg could not improve the maternal production, but increase the Ca level in progeny tibia at 1 d of age. The addition of vitamin premix at NRC recommendation in progeny diet for 1-28d improve the growth performance, immune characters, and bone quality of progeny broilers, especially for the progeny with no maternal 25-OH- addition. Table 1. Effects of maternal dietary 25-OH- and progeny vitamin premix on percentage of Ca and P in total tibia of progeny at 28d. n Ca (%) P (%) MEAN SEM MEAN SEM 11.01a a a a b b b b Journal of Food, Agriculture & Environment, Vol.11 (2), April

6 Acknowledgements The authors would like to acknowledge the financial support by China Agriculture Research System (CARS-41-K0), the Key Scientific and Technological Project of Sichuan Science and Technology Department (2011NZ007320NZ0037, National Development Project and Erlangshan Chick Expert Academy cooperated by Sichuan Agricultural University and Ya an Government. We also sincerely appreciate the kind supply of vitamin premix by DSM Nutritional Products Ltd. (Shanghai, P. R. China). References 1 Mitchell, R. D., Edwards, H. M. Jr. and McDaniel, G. R The effects of ultraviolet light and cholecalciferol and its metabolites on the development of leg abnormalities in chickens genetically selected for a high and low incidence of tibial dyschondroplasia. Poult Sci. 7: Aburto, A., Edwards, H. M. Jr. and Britton, W. M The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25-hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens. Poult. Sci. 77: Atencio, A., Edwards, H. M. Jr. and Pesti, G. M Effect of the level of cholecalciferol supplementation of broiler breeder hen diets on the performance and bone abnormalities of the progeny fed diets containing various levels of calcium or 25-hydroxycholecalciferol. Poult. Sci. 84: Waldenstedt, L Nutritional factors of importance for optimal leg health in broilers: A review. Anim. Feed Sci. Technol. : Ward, N. E Consideration of vitamin absorption may be needed. Feedstuffs 43:3-37. Yarger, J. G., Saunders, C. A., McNaughton, J. L., Quarles, C. L., Hollis, B. W. and Gray, R. W Comparison of dietary 25- hydroxycholecalciferol and cholecalciferol in broiler chickens. Poult. Sci. 74: Bar, A., Sharvit, M., Noff, D., Edelstein, S. and Hurwitz, S Absorption and excretion of cholecalciferol and of 25- hydroxycholecalciferol and metabolites in birds. J Nutr. 110: Mattila, P., Lehikoinen, K., Kuskinen, T. and Purnen, V Cholecalciferol and 25-Hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed. J. Agric. Food Chem. 47: Coto, C., Cerate, S., Wang, Z., Yan, F., Min, Y., Cotsta, F. P. and Waldroup, P. W Effect of source and level of vitamin D on performance of breeder hens and the carryover to progeny. Int. J. Poultry Sci. 9: Atencio, A., Edwards, H. M. Jr., Pesti, G. M. and Ware, G. O The vitamin requirement of broiler breeders. Poult. Sci. 85: Atencio, A., Pesti, G. M. and Edwards, H. M. Jr Twenty-five hydroxycholecalciferol as a cholecalciferol substitute in broiler breeder hen diets and its effect on the performance and general health of the progeny. Poult. Sci. 84: Driver, J. P., Atencio, A., Pesti, G. M., Edwards, H. M. Jr. and Bakalli, 70 Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013