【作者】 李卓昭；

【导师】 吴德；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 本试验旨在研究早熟和晚熟母猪品种(梅山和长大母猪)繁殖轴(下丘脑-垂体-卵巢组织)kiss1/GPR54基因表达差异及其激素的变化,并进一步考察日粮能量水平对晚熟品种母猪初情启动的影响。研究内容如下：试验一、分别选取70日龄、初情启动的梅山和长大母猪(n=4),采用ELISA法测定母猪血液中Leptin和E2水平,采用RT-PCR法克隆两品种Kiss1基因序列,荧光定量PCR法检测母猪繁殖轴上Kiss1/GPR54基因,下丘脑LeptinR和ERα,以及卵巢上LHR基因表达的情况。结果发现：(1)基因分析发现,梅山和长大母猪品种Kiss1基因序列一致。(2)梅山和长大母猪平均初情日龄分别为100天和199天,梅山母猪繁殖轴Kiss1表达量显著高于长大母猪(P<0.05),但受体GPR54 mRNA表达量差异不显著(P>0.5)。两个品种母猪繁殖轴Kissl/GPR54 mRNA表达量均随其日龄增加而升高(P<0.05)。(3)梅山母猪血液中Leptin浓度在70日龄时显著高于长大母猪(P<0.05),但初情启动时差异不显著(P>0.05)；梅山猪血液中E2浓度初情启动时显著高于长大母猪,70日龄未发现两品种存在差异(P>0.05)；随日龄的增加,母猪血液中leptin和E2浓度都显著升高(P<0.05)。(4)基因检测发现,梅山母猪下丘脑ERa mRNA表达显著高于长大母猪(P<0.05)；而下丘脑LeptinR mRNA表达量仅显著高于70日龄长大母猪(P<0.05),初情时LeptinR mRNA表达量无显著差异(P>0.05)；梅山母猪卵巢LHR mRNA表达量显著高于70日龄与初情长大母猪(P<0.05)；两品种母猪下丘脑LeptinR、ERαmRNA及卵巢LHR mRNA表达量均随其日龄增加而升高(P<0.05)。(5) Person相关性分析发现,除卵巢上GPR54基因外(P>0.05),母猪血液中leptin水平与HPG轴上Kissl和GPR54基因表达之间存在显著正相关关系(P<0.05)。除下丘脑Kissl基因外(P<0.05),母猪血液中E2水平与HPG轴上Kissl和GPR54基因表达之间不存在显著正相关关系(P>0.05)。试验二、选取体重50±4.5kg,日龄和体况相近的LY母猪20头,按照配对试验设计原则分配到两个处理组,能量限饲组和NRC水平组(10个重复/处理,每个重复/1头猪)。NRC水平组日粮能量水平参照NRC(1998)后备母猪推荐水平,能量限饲组能量供给为NRC水平组70%,其它养分摄入量均一致。母猪50 kg,80 kg,100kg和初情启动时收集血液,采用ELISA法测定血液中E2和leptin水平。母猪超过80 kg后,每天两次进行发情观察,母猪首次出现阴户红肿并伴有稀薄粘液定义为初情启动。分别考察两个处理组母猪的初情日龄以及初情期的体重和P2点背膘厚度,结果如下：(6)与能量限饲组相比,NRC水平组母猪能及早启动初情期(177.7±3.4 d VS 190.1±10.2d, P<0.05),并具有较高的初情体重、日增重和P2背膘厚度(122.6±7.3kg VS 108.4±4.1 kg、0.85±0.02 kg/d VS 0.65±0.15 kg/d和13.50±1.38 mm VS 10.57±1.27mm; P<0.05)(7)母猪血液中Leptin和E2水平随日龄的增加而提高,母猪在100kg阶段和初情期时显著高于50kg和80kg体重阶段(P<0.05)；能量水平对80kg后母猪血液Leptin和E2水平有影响,能量限饲组显著低于NRC水平组(P<0.05)。同时NRC水平组母猪下丘脑Kiss1基因表达量显著高于能量限饲组(P<0.05),但两能量水平组GPR54基因表达无显著差异(P>0.05)。根据以上结果,可以得到以下结论：(1)梅山与LY母猪Kiss1基因序列一致,但母猪不同品种和日龄在繁殖轴上kiss1基因的表达存在差异,繁殖轴kiss1的高度表达有利于促发母猪初情启动；(2)母猪血液中高Leptin和E2水平可上调繁殖轴Kiss1基因表达,梅山母猪血液中高Leptin和E2水平有利于其初情及早启动,日粮高能量水平可提高母猪血液Leptin和E2水平而促发初情的启动。更多还原

【Abstract】 The main objective of the present study was to investigate the difference of Kissl/GPR54 gene expression level on Gonadal (hypothalamus-pituitary-ovary) axis and related hormones in different breed gilts (Meishan and LY), Additionally, whether dietary energy level could induce variation for the age of puberty onset in the breed of late-maturing gilts. Two experiments was conducted:In experiment 1, meishan gilts (at estrus, n=4) and LY gilts (non-estrus, n=4) at age of 70 days were used to collect samples for determination the concentration of serum Leptin and E2 by ELISA, Kiss1 gene sequence clone by RT-PCR, and Kiss1/GPR54 expression in reproductive axis, LeptinR and ERαin hypothalamus, and LHR in ovary by RT-PCR.(1) DNA sequence of Kiss1 gene between meishan and LY gilts were not different.(2) The average puberty onset age of Meishan and LY was 100d and 199d separately, Gene expression encoded by Kiss1 in reproductive axis (including hypothalamus, pituitary and ovary) were significantly higher in LY gilts (P<0.05), but GPR54 mRNA expression level was similar between two breeds (P>0.05). Gene expression level encoded by Kissl/GPR54 in reproductive axis were gradually higher as the advent of first estrus in both meishan and LY gilts (P<0.05)(3) Leptin concentration in meishan gilts at age of 70 days was higher than in LY gilts (P<0.05), but circulating concentration of Leptin at estrus was similar between two breeds (P>0.05). E2 concentration in meishan gilts at estrus was significantly higher than that in LY gilts (P<0.05), but was similar at 70 days of age (P>0.05). Both Leptin and E2 concentration was gradually higher as the increase of age (P<0.05)(4) ERa mRNA expression level was higher in meishan gilts than LY gilts in hypothalamus (P<0.05). LeptinR mRNA expression in hypothalamus was higher in meishan gilts than LY gilts at 70 days of age (P<0.05), but LeptinR mRNA expression was not different between two breeds at estrus (P>0.05). LHR mRNA expression in ovary at estrus was higher than that in LY gilts at 70 days of age and at estrus (P<0.05). LeptinR and ERa mRNA in hypothalamus, LHR mRNA expression in ovary was gradually higher as the increase of age (P<0.05)(5) Person correlation analysis revealed that circulating Leptin concentration was positively correlated with Kiss1 and GPR54 expressions (with exception for GPR54 in ovary) in HPG axis (P<0.05). Circulating E2 concentration was positively correlated with Kissl expressions in hypothalamus (P<0.05), but not for Kissl and GPR54 expression in HPG axis (P>0.05). In experiment 2, twenty LY gilts with initial bodyweight of 50±4.5 kg were randomly allocated to 2 treatment groups based on paired experimental design, ten gilts were fed energy restriction of 70% energy requirement (L) of NRC(1998), remaining ten LY gilts were fed 100% energy requirement (H) of NRC(1998), notably, these two groups of gilts were fed similar nutrients except for the difference of energy intake. Circulating blood samples were collected at the age of 50 kg,80 kg,100 kg and at estrus, for the determination of serum E2 and Leptin concentrations. Estrus check twice per day were conducted when bodyweight of gilts over 80 kg. Occurrence of pink vaginal orifice and mucosal fluid leak was defined as estrus. The age, bodyweight and backfat P2 thickness at estrus initiation were determined.(6) Compared with gilts in L group, gilts in H group was 13 days earlier to reach estrus (177.7±3.4d VS 190.1±10.2 d, P<0.05). Gilts in H group had higher bodyweight, average daily gain, P2 fat thickness(122.6±7.3 kg VS 108.4±4.1 kg; 0.85±0.02 kg/d VS 0.65±0.15 kg/d; 13.50±1.38 mm VS 10.57±1.27 mm, P<0.05).(7) Circulating Leptin and E2 concentrations was higher as the increase of age, which was evidenced by higher Leptin and E2 concentration at bodyweight of 100kg than that at 50 kg and 80 kg (P<0.05). Leptin and E2 concentrations were affected by dietary treatment. Leptin and E2 concentration in L group was significantly lower than H gilts (P<0.05). Kissl expression in hypothalamus was significantly higher than L gilts (P<0.05), but GPR54 expression was not affected by diets (P>0.05)Conclusion:(1) There was no difference of Kissl DNA sequence in meishan gilts and LY gilts, but Kissl/GPR54 expression in reproductive axis was different between breeds, the higher Kissl expression could be beneficial for the initiation of estrus.(2) Higher circulating Leptin and E2 could up-regulate the Kissl expression, and higher Leptin and E2 concentration could promote the initiation of estrus. Dietary high energy level could promote the puberty onset through higher Leptin and E2 level.更多还原