【作者】 张宏馨；

【导师】 黄仁录；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 在海兰褐父母代种母鸡饲粮中添加不同剂量的维生素E（以维生素E醋酸酯形式添加，以下简称VE），通过研究VE对种母鸡生产性能、繁殖性能、卵母细胞卵黄生成受体（OVR）基因表达和体外培养的卵泡颗粒细胞增殖和凋亡的影响，从机体、分子和细胞三个层面来解释VE对种母鸡繁殖性能的影响及其调控机制，旨在探索种母鸡获得最佳繁殖性能的VE适宜添加剂量，明确其作用机制，为生产提供理论依据和指导。试验一研究饲粮VE对种母鸡生产及繁殖性能的影响。试验选用48周龄海兰褐种母鸡288只，随机分成6组，每组4个重复。试验组以VE醋酸酯的形式添加VE，其添加水平分别是0、20、40、80、160和320mg/kg，预试期2周，试验期8周。结果表明（1）增加饲粮VE浓度，可以显著提高产蛋率，其中160mg/kg组最高（P<0.01），而320mg/kg组相对160mg/kg组有下降趋势；（2）改变饲粮中VE的浓度对种蛋蛋重、蛋形指数、哈氏单位、蛋壳相对重、蛋黄相对重和蛋黄颜色等蛋品质未见影响（P＞0.05），但是160和320mg/kg的添加剂量使蛋壳厚度降低。（3）随饲粮VE添加量的增加，卵泡发育情况和种蛋受精率、受精蛋孵化率、入孵蛋孵化率、初生重、健雏率随之提高，160mg/kg对卵泡发育最好（P<0.05），而320mg/kg的孵化成绩最佳（P<0.05）；（4）饲粮VE的添加剂量对血清生殖激素无显著影响（P＞0.05）。试验二研究饲粮VE对种蛋蛋黄和鸡体组织中α-生育酚沉积（α-T）的影响。建立了测定鸡蛋黄及组织中α-T含量的快速测定方法，进而以此方法分析了饲粮中不同剂量VE对种鸡蛋黄及组织中α-T沉积量的影响。结果发现（1）对样品以甲醇提取后超声处理20min的预处理方法，操作简单，α-T损失少，提取效率明显高于皂化法和甲醇提取后室温静置12h的方法，在所考察的浓度范围内具有良好的线性关系，r为0.9999，20℃浸提10h加标回收率在100.8%～109.00%；（2）随VE添加量的增加，蛋黄、肝脏、心脏中-生育酚含量呈线性增加，添加320mg/kg浓度达到最大，而肾脏沉积增加不明显。试验三研究VE对种母鸡OVR基因表达的影响，并对OVR基因表达量与卵黄的VE沉积、生产性能及繁殖性能进行了回归分析。结果：成功扩增出种母鸡卵泡OVR基因片段，大小约110bp；对于OVR mRNA的表达量，40、80mg/kg添加组（Ⅲ、Ⅳ组）较对照组显著升高（P＜0.05），而160和320mg/kg（Ⅴ、Ⅵ组）添加组表达量反而降低；OVR基因的表达量与蛋黄VE沉积量、受精率和初生重之间呈倒数关系（P＜0.05）。试验四研究了VE对种母鸡等级前卵泡颗粒细胞增殖的影响。通过对种母鸡颗粒细胞进行原代培养，观察细胞的生长趋势和增殖情况，并在此基础上研究VE对颗粒细胞增殖的调控作用。结果发现：（1）颗粒细胞体外培养的适应期为24h，72h达到高峰，之后细胞进入停滞期。（2）48h内，随着培养时间的延长，各组细胞增殖率随VE浓度的增加而升高，48h时达到高峰，之后增殖率开始下降，培养至72h，10mg/L组维持较高的细胞数量，250mg/L组的细胞增殖率变为负值，即开始抑制细胞生长，其他各组随着添加剂量的升高有显著的降低。试验五研究了不同浓度VE对种母鸡卵泡颗粒细胞无血清培养所致细胞凋亡的影响。在试验四的基础上，观察对种母鸡颗粒细胞的增殖作用最明显的10mg/L浓度VE对种母鸡卵泡颗粒细胞氧化损伤和凋亡的保护作用，以流式细胞仪检测颗粒细胞的凋亡情况和细胞周期变化。结果发现：VE有助于促进颗粒细胞由G0/G1期进入S期，使更多颗粒细胞通过G1/S期限制点，从而抑制无血清培养所诱导的种母鸡颗粒细胞的凋亡。试验结果表明，种母鸡饲粮VE可以通过促进种蛋蛋黄中的VE沉积，提高种蛋受精率、孵化率等繁殖性能；通过促进卵泡颗粒细胞的增殖，减少其凋亡，从而降低卵泡闭锁率，提高母鸡产蛋性能和繁殖性能；种母鸡饲粮中VE对其繁殖性能的促进作用并不依赖于OVR基因的高表达量，还有其它机制存在。更多还原

【Abstract】 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.更多还原