Study On The Influence Of Vitamin E On The Reproductive Performance And It’s Mechanism In Breeding Hens

This study was carried out to investigate the effects of different levels of vitamin E(Toadd vitamin E acetate, hereinafter VE) on production performance， reproductiveperformance, oocyte vitellogenesis receptor gene (OVR) expression and proliferation andapoptosis of granulosa cell cultured in vitro in Hy-line brown parent females to explain theeffect of VE on breeding hens reproductive performance and the regulation mechanisms ofVE from the organism, cellular and molecular levels. This study was carried out to find asuitable amount of VE of breeding hens to get the best reproductive performance, trying toclarify the mechanism and provide rationale and guidance for future production.Experiment1was conducted to investigate the productive and reproductiveperformance effects of VE in breeding hens diet. A total of28848-week-old Hyline brownbreeding hens were selected and divided into six groups randomly, with four replicationsper group. Diets with the form of dl-α-tocopheryl acetate were added on0,20,50,80,160or320mg of VE.kg-1. Preliminary trial period was2weeks and experimental time was8weeks. Results were as follows(:1)The laying rate can be improved significantly with anincrease in VE concentration of diet, of which160mg/kg group was the highest (P<0.01),while the320mg/kg group showed a downward trend comparing with the160mg/kggroup.(2)There was no effect in egg weight, egg shape index, Haugh unit, thickness ofeggshell, eggshell relative weight, yolk weight and egg yolk color (P＞0.05).(3)Withthe effects of the increase of VE content, follicular development and fertilization rate of thebreeding eggs, hatchability of fertile eggs, hatchability of setting eggs, birth weight,healthy chick rate increased. The group of160mg/kgwas the best concentration tofollicular development(P<0.05)and the group of320mg/kgwas the best concentration ofincubation results(P<0.05).(4)Different VE dosage in diet had no significant effect onserum reproductive hormones (P<0.05).Experiment2was carried out to investigate the effect of VE on α-tocopherol(α-T)deposition of egg yolk and tissues of hens. A rapid method for the determination of α-Tcontent in egg yolk and tissues were established, and to be used to analyze effects ofdifferent doses of VE in diet on α-T deposition of egg yolk and tissues.The results showedthat:(1)The method of ultrasonic treatment20min after methanol extraction was tested tobe simple and with less α-T losted loss. The extraction efficiency is significantly higherthan saponification method and the method of placement at room temperature for12h aftermethanol extraction. The method had a good linear relationship in the range of oncentration, with R0.9999,10h at20℃extraction recovery of100.8%~109%;(2)α-Tcontent in egg yolk, liver and heart showed a linear increase with the increase of VEcontent and the maximum added concentration was320mg/kg, but had no significantincrease of renal deposition.Experiment3was conducted to investigate the effect of VE on OVR gene expressionin breeding hens. Furthermore the regression analysis were implemented about expressionof OVR gene with VE deposition of yolk, production performance and reproductiveperformance. Result was as follows: The hens OVR gene fragments were amplifiedsuccessfully, to be about110bp; The OVR mRNA expression for40,80mg/kg add group(Ⅲ, Ⅳ) is significantly higher than control group(P <0.05), while the160and320mg/kg(Ⅴ, Ⅵ group) add group expression decreased;There was reciprocal relationship betweenthe expression of OVR gene and VE deposition，fertilization rate and birth weight(P＜0.05).Experiment4was conducted to investigate the effect of VE on granulosa cellsproliferation level of breeding hens. The growth trend of cells and proliferation wereobserved through primary culture of granulosa cells of breeding hens，and the regulation ofVE on the proliferation of granulosa cells was studied. Result was as follows:(1)Adaptive phase of granule cells cultured in vitro was24h, and reached the peak at72h,after that the cells came into stasis.(2)Along with the prolongation of culture time within48h, proliferation rate of cells in each group was increased with the VE concentration, andreached the peak at48h, while the growth rate started to decline after48h. Training to72h,10mg/L group maintained a higher number of cells,while250mg/L group turned negativein cell proliferation, to began to inhibit cell growth, and the other groups decreasedremarkably with the dosage increased.Experiment5was conducted to investigate the effect of different concentrations ofVE on hen granulosa cells apoptosis caused by serum-free culture. Based on the test offour, concentration of10mg/L of VE was the most obvious proliferation in hen granulosacell，and the protective effects of10mg/L concentration of VE on hen granulosa celloxidation damage and apoptosis was studied，with flow cytometry to detect granule cells’apoptosis and cell cycle changes. Result was as follows: vitamin E helps promotegranulosa cells from G0/G1phase to S phase, so that more granular cells passed throughG1/S period limit, thereby hen granulosa cell apoptosis induced by serum-free culture wasinhibited.The experimental results show that the diet of VE in breeding hens can improve thereproductive performance, such as egg fertilization rate, hatching rate through promotingthe concent of egg yolk; VE can promote the proliferation and reduce the apoptosis offollicular granulosa cell, thus reduce the rate of follicular atresia and improve the layingrate and reproductive performance; Dietary VE content on the reproductive performance ofbreeding hens does not depend on the high expression of OVR gene, and there are othermechanism.