【作者】 应诗家；

【导师】 王锋；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2012， 博士

【摘要】 本文研究了在同一生殖和营养生理状态下湖羊黄体期不同发育阶段卵泡内代谢产物和激素含量的变化关系,探讨了黄体期不同饲喂量对湖羊卵泡发育、血液和卵泡内代谢产物和激素浓度、卵泡和黄体细胞类固醇调节基因表达的影响,旨在揭示黄体期不同饲喂量影响卵泡发育的机制,为规模化羊场空怀母羊的科学饲养以及进一步研究营养影响绵羊繁殖性能的精细信号通路提供一定的参考。试验共分5个部分：试验一、湖羊不同发育阶段卵泡内代谢产物和激素含量的比较研究选择体重40kg左右的湖羊11头,同期发情结束后第12天屠宰,按不同大小卵泡分离卵泡液。试验结果显示,与≤2.5mm卵泡相比,>2.5mm卵泡内葡萄糖浓度显著提高(P<0.05),胰高血糖素浓度显著降低(P<0.05),乳酸脱氢酶活性和睾酮浓度极显著降低(P<0.01),雌二醇浓度极显著提高(P<0.01),血氨、游离脂肪酸、尿素、胰岛素和孕酮浓度差异不显著。雌二醇浓度与LDH活性极显著负相关(P<0.01),与葡萄糖浓度显著正相关(P<0.05),与胰高血糖素浓度显著负相关(P<0.05),与睾酮浓度极显著负相关(P<0.01),与孕酮浓度接近正相关(P=0.051)。试验结果表明,卵泡内代谢产物和激素共同参与调节卵泡发育。试验二、黄体期不同饲喂量对湖羊卵泡发育的影响选择体重40kg左右,3-4岁的经产健康母羊28头,随机分三组：R组(n=11),C组(n=6)和S组(n=11),同期发情结束后第6天分别按0.5倍体重维持需要量(0.5×M),1×M和1.5×M饲喂,第12天每组屠宰6只,分离不小于1.0mm的卵泡,分别计算每头羊卵泡直径为1.0-2.0mm,2.0-2.5mm,2.5-3.5mm和≥3.5mm卵泡数和卵泡体积,剩下的湖羊用于观察发情。分别在放栓、拆栓、不同饲喂量处理和屠宰时称重和评定体况。试验结果显示,整个试验过程中体重和体况无显著性差异。随着饲喂水平提高,≥3.5mm卵泡数显著增加(P<0.05),2.5-3.5mm卵泡数显著降低(P<0.05),平均发情周期缩短(P<0.05),>2.5mm卵泡平均体积显著增大(P<0.05)。试验结果表明,黄体溶解前限饲6天抑制卵泡发育。试验三、黄体期不同饲喂量对湖羊血液理化指标的影响发情周期第12天屠宰后,测量器官重量,在不同饲喂量处理期每天采血。试验结果显示,随着饲喂水平降低,血液尿素,胆固醇,低密度脂蛋白、游离脂肪酸、FSH、雌二醇和孕酮含量及孕酮与雌二醇比值和胰岛素和胰高血糖素比值显著升高(P<0.05),葡萄糖、甘油三酯、三碘甲状原氨酸、胰岛素和胰高血糖素含量显著降低(P<0.05)；血液尿酸,血氨,高密度脂蛋白和谷草转氨酶和谷丙转氨酶活性差异不显著(P>0.05),但尿酸、血氨、低密度脂蛋白含量和谷草转氨酶活性具有时间效应(P<0.05)。R组亮氨酸、缬氨酸、异亮氨酸、酪氨酸、丝氨酸、乙醇胺、必须氨基酸、必须氨基酸衍生物、氨基酸代谢物和总氨基酸在饲喂期间低于S组,而牛磺酸、瓜氨酸和氨能代谢产物与总氨基酸比值高于S组。与S组相比,R组脾脏重,脾脏与体重比值显著升高(P<0.05),肝脏重、小肠重、肝脏与体重比和胃重与体重比显著降低(P<0.05)。试验结果表明,绵羊黄体期不同生理阶段具有不同的营养需求和代谢特点；黄体期限饲抑制卵泡发育与能量负平衡引起蛋白质和脂类合成降低,分解增强以及氨基酸代谢和内分泌激素分泌模式改变相关。试验四、黄体期不同饲喂量对湖羊卵泡液微环境的影响发情周期第12天屠宰后,取每头羊卵巢,分离1.0-2.0mm组,2.0-2.5mm组和单个>2.5mm卵泡卵泡液。试验结果显示,与S组相比,R组增加了卵泡内雌二醇、磷酸乙醇胺、尿素(P<0.05)和δ-羟赖氨酸浓度(P=0.085),减少了鸟氨酸浓度、必须氨基酸与必须氨基酸衍生物比值(P<0.05)和氨能代谢产物与总氨基酸比值(P=0.075)。限饲和补饲组卵泡内胰岛素水平相似,但高于对照组(P<0.05)。不同大小卵泡显著影响卵泡液葡萄糖、雌二醇、睾酮、胰岛素、胰高血糖素和乳酸脱氢酶水平(P<0.05),但仅在R组茎2.5mm卵泡液和>2.5mm卵泡液乳酸脱氢酶活性和睾酮浓度差异不显著。试验结果表明,黄体期限饲影响了发育后期卵泡内氨基酸代谢,增加雌二醇和睾酮浓度以及乳酸脱氢酶的活性,抑制了卵泡发育。试验五、黄体期不同饲喂量对湖羊卵泡细胞类固醇调节基因表达的影响发情周期第12天屠宰后,取每头羊卵巢,分离1.0-2.0mm组,2.0-2.5mm组和单个>2.5mm卵泡细胞以及黄体细胞。试验结果显示,不同饲喂量仅影响ESR1、CYP17A1和CYP19A1mRNA表达水平(P<0.05)。对>2.5mm卵泡细胞,R和C组CYP17A1mRNA表达水平显著低于S组(P<0.05),R组CYP19A1mRNA表达水平显著高于S组(P<0.05),ESR1mRNA表达水平显著低于C和S组(P<0.05)。不同大小卵泡显著影响VLDLR、ESR2、FSHR (P<0.05)、CYP17A1和CYP19A1(P <0.01) mRNA表达水平,在S组>2.5mm卵泡细胞ESR2mRNA表达水平显著低于≤2.5mm卵泡细胞。试验结果表明,黄体期限饲抑制卵泡发育的机制可能是限饲影响发育后期卵泡内CYP17A1、CYP19A1和ESR1mRNA表达水平。更多还原

【Abstract】 The present paper comparatively studied the follicular fluid metabolites and hormones concentrations form different-sized follicles at the same reproductive and nutritional physiology, investigated the effects of short-term different nutritional intake on folliculogenesis, plasma and follicular fluid metabolites and hormones concentrations and sterol-regulatory genes expression of follicular and luteal cells during luteal phase in Hu sheep. The objectives of this study were to reveal the mechanism in which short-term different nutritional intake influenced folliculogenesis, and to provide certain references for scientifically rearing barren ewe in large-scale sheep farms and further studying precise signal pathway by which nutrition affected ovine reproductive performance. This study contained the following5parts:Experiment1. A comparative study of follicular fluid metabolites and hormones concentrations from different-sized follicles in Hu sheep11multiparous Hu sheep of proven fertility were used. After estrus synchronization, all ewes were slaughtered and follicular fluid was collected according to different-sized follicles on day12of estrous cycle. The results showed that there were no significant effects of follicle size on plasma serum ammonia, NEFA, urea, insulin and progesterone concentrations, however, compared with follicles<2.5mm in diameter, the follicular fluid glucose and estradiol concentrations in follicles>2.5mm in diameter were increased (P< 0.05) and the testosterone and glucagon concentrations and LDH activity were decreased (P<0.05). The follicular fluid concentration of estradiol was positively correlated with glucose (P<0.05) and progesterone (P=0.051) and negatively correlated with LDH (P<0.01), glucagon (P<0.05) and testosterone (P<0.01). In conclusion, follicle development was co-regulated by intrafollicular metabolites and hormones.Experiment2. Effect of different levels of short-term feed intake on folliculogenesis in Hu sheep during the luteal phase28multiparous Hu sheep (3-4years) weighed approximately40kg were selected and randomly assigned to3groups:the control group (n=6) received a maintenance diet (M) while the supplemented group (n=11) and restricted group (n=11) received1.5xM and0.5×M, respectively, on days6-12of their estrous cycle. After estrus synchronization,6ewes per group were slaughtered on day12of the estrous cycle. The ovarian follicles>1.0mm were dissected free and the number and volume of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe were calculated, respectively. The remaining ewes were detected for estrous behavior. During the experimental period, body condition and body weight were measured four times:at pessary insertion, at pessary removal, at the start of different feeds and on the day they were killed. The results showed that there were no significant differences in the body weight and body condition score among times or treatments, however, compared with restriction, supplementation shortened the estrus length (P<0.05), decreased the number of follicles2.5-3.5mm (P<0.05), increased the number of follicles>3.5mm (P<0.05) and augmented the volume of follicles>2.5mm (P<0.05). In conclusion, short-term dietary restriction for6days before Iuteolysis inhibits folliculogenesis.Experiment3. Effect of different levels of short-term feed intake on plasma physiochemical indexes in Hu sheep during the luteal phaseAfter estrus synchronization, all ewes were slaughtered on day12of the estrous cycle and live and organ weights were determined. Blood samples were taken during experiment period. The results showed that as dietary intake decreased, plasma urea, total cholesterol, LDL-C, NEFA, FSH, estradiol, progesterone concentrations and progesterone:estradiol ratio and insulin:glucagon ratio increased (P<0.05), and plasma glucose, triglyceride, insulin and glucagon concentrations decreased (P<0.05). There was no significant effect of treatment on plasma uric acid, plasma ammonia and high density lipoprotein and spartate transaminase, alanine transaminase activities (P>0.05), however, there were significant effect of day on plasma uric acid, ammonia, low density lipoprotein concentrations and aspartate transaminase activity (P<0.05). Plasma leucine, valine, isoleucine, tyrosine, serine, ethanol amine, essential amino acid (EAA), essential amino acid derivatives (EAAD), amino acid metabolites (AAM) and total amino acids (TAA) concentrations in R group ewes were lower, while plasma taurine and citrulline concentrations and ammonia/energy metabolites (AEM):TAA ratio than those in S group ewes during experiment period. Restricted group ewes exhibited greater spleen weight and spleen weight per live weight, and less liver and small intestine weights, liver and stomach weights per live weight than supplemented group ewes (P<0.05). In conclusion, different nutritional requirement and metabolic feature were present in different physiological periods during ovine luteal phase and the mechanism by which dietary restriction inhibited folliculogenesis may involve responses to increased the capacity of lipolysis and protein degradation, decreased the capacity of lipid and protein synthesis, changed amino acids metabolism and varied levels of metabolic and reproductive hormones.Experiment4. Effect of different levels of short-term feed intake on follicular fluid microenvironment in Hu sheep during the luteal phase6ewes per group were slaughtered on day12of the estrous cycle and the follicular fluid of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe was collected, respectively. Compared with supplementation, restriction increased follicular fluid estradiol, phosphorylethanolamine, urea (P<0.05) and8-hydroxylysine (P=0.085) concentrations, decreased ornithine concentration, EAA:EAAD ratio (P<0.05) and AEM:TAA ratio (P=0.075). Restricted ewes had higher intrafollicular insulin concentration than C group ewes (P<0.05), but it was similar to that of supplemented ewes. There were significant follicle size effect on intrafollicular glucose, estradiol, testosterone, insulin, glucagon and lactate dehydrogenase activity (P<0.05), however, only in restricted ewes were intrafollicular lactate dehydrogenase and testosterone concentrations in follicles>2.5mm not different from those in follicles<2.5mm. In conclusion, the mechanism by which short-term dietary restriction inhibits folliculogenesis may involve responses to intrafollicular influenced amino acids metabolism, increased estradiol, testosterone and lactate dehydrogenase levels in late-stage follicles.Experiment5. Effect of different levels of short-term feed intake on sterol-regulatory gene expression of follicular and luteal cells in Hu sheep during the luteal phase6ewes per group were slaughtered on day12of the estrous cycle and the follicular and luteal cells of follicles1.0-2.0mm,2.0-2.5mm,2.5-3.5mm and>3.5mm per ewe was collected, respectively. The results showed that short-term different nutritional intake affected the genes expression of ESR1, CYP17A1and CYP19A1in follicular cell (P<0.05). Compared to S group ewes, R and C group ewes decreased the CYP17A1mRNA expression (P<0.05) and R group ewes increased CYP19A1mRNA expression (P<0.05) and decreased ESR1mRNA expression (P<0.05) in follicles>2.5mm. There is follicle size effect on VLDLR, ESR2, FSHR (P<0.05), CYP17A1and CYP19A1(P<0.01) mRNA expression, however, only in S group ewes, were follicles>2.5mm less ESR2mRNA expression than follicles≤2.5mm (P<0.05). In conclusion, the mechanism in which short-term restriction inhibited folliculogenesis during luteal phase is that restriction influenced CYP17A1, CYP19A1and ESR1mRNA expression in late-stage follicles.更多还原