梅山母猪论文：梅山母猪LY母猪初情期Kiss1基因瘦素雌激素

梅山母猪论文：梅山母猪 LY母猪初情期 Kiss1基因瘦素雌激素
【中文摘要】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(P0.5)。

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(P0.05)；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(P0.05)；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(P0.05),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
(P0.05)。

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, P0.05)。

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。

【英文摘要】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 (P0.05). 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 (P0.05). E2 concentration in meishan gilts at estrus was significantly higher than that in LY gilts (P0.05). Both Leptin and E2 concentration was gradually higher as the increase of age (P0.05). LHR mRNA expression in ovary at estrus was higher than that in LY gilts at 70 days of age and at estrus (P0.05). 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, P0.05)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.
【关键词】梅山母猪 LY母猪初情期 Kiss1基因瘦素雌激素
【采买全文】1.3.9.9.38.8.4.8 1.3.8.1.13.7.2.1 同时提供论文写作一对一辅导和论文发表服务.保过包发.
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【英文关键词】meishan gilts LY gilts puberty Kiss1 leptin E2
【目录】不同品种猪繁殖轴Kiss1/GPR54表达差异及能量水平对
母猪初情日龄的影响摘要5-7ABSTRACT7-8本文专业名词缩
写符号9-12 1 前言12-13 2. 文献综述13-21 2.1 动物初
情的机理13-14 2.2 Kiss1/GPR54与发情启动14-16 2.3
Kiss1/GPR54在繁殖轴(下丘脑-垂体-卵巢)的表达16-18 2.3.1
Kiss1/GPR54基因在下丘脑的表达16-17 2.3.2 Kiss1/GPR54基因
在垂体的表达17 2.3.3 Kiss1/GPR54基因在卵巢的表达
17-18 2.4 Kiss1/GPR54基因表达及调节18-21 2.4.1 性激素
对Kiss1基因的表达调节19-20 2.4.2 能量对Kiss1/GPR54基因
表达调节20-21 3 存在问题及研究目的和意义21-22 3.1 有待
研究的问题21 3.2 本研究的目的和意义21-22试验一早熟与
晚熟品种猪繁殖轴Kiss1/GPR54表达及相关血液繁殖激素的差异研
究22-47 4.1 试验动物22 4.2 样品采集22 4.3 测定指标
和方法22-30 4.3.1 主要器材22-23 4.3.2 主要试剂
23-24 4.3.3 Kiss1基因的克隆测序24-29 4.3.4 基因检测
29-30 4.3.5 血液激素测定30 4.4 数据处理30-31 4.5 试
验结果31-39 4.5.1 梅山猪与LY母猪Kiss1基因PCR扩增结果
31 4.5.2 梅山与LY Kiss1基因克隆测序结果31-32 4.5.3 梅
山与LY母猪相同日龄与生理阶段血清激素水平变化32-33 4.5.4
Real Time PCR33-39 4.5.4.1 管家基因β-actin的检测结果
33-34 4.5.4.2 梅山和LY母猪繁殖轴Kiss1基因的检测
34-35 4.5.4.3 梅山和长大母猪繁殖轴GPR54基因的检测结果
35-36 4.5.4.4 梅山和长大母猪下丘脑LeptinR基因检测结果
36-37 4.5.4.5 梅山和长大母猪下丘脑ERα基因的检测结果
37-38 4.5.4.6 梅山和长大母猪卵巢LHR基因的检测结果
38 4.5.4.7 繁殖轴Kiss1和GPR54基因与血液相关性分析
38-39 4.6 讨论39-47 4.6.1 Kiss1/GPR54基因序列与初情日龄差异39-40 4.6.2 梅山猪与LY母猪下丘脑Kiss1/GPR54表达变化40-41 4.6.3 梅山与LY母猪垂体Kiss1/GPR54表达变化
41-42 4.6.4 梅山猪与LY母猪卵巢Kiss1/GPR54表达变化
42-44 4.6.5 繁殖轴Kiss/GPR54基因的表达调控44-47试验二能量水平对后备母猪初情日龄及下丘脑Kiss1/GPR54基因表达的影响47-54 5.1 动物选择及日粮处理47 5.2 饲养管理
47 5.3 样品采集47-48 5.4 测定指标与方法48-49 5.5 数据处理49 5.6 试验结果49-51 5.6.1 能量水平对体重、背膘厚度、日增重和初情日龄的影响49-50 5.6.2 不同能量水平对后备母猪血液激素水平的影响50 5.6.3 不同能量水平对下丘脑
Kiss1/GPR54基因的影响50-51 5.7 讨论51-54 5.7.1 能量水平对长大母猪初情日龄的影响51-52 5.7.2 能量水平对血液leptin、E_2水平及下丘脑Kiss1/GPR54基因表达的影响52-54 6 结论54-55参考文献55-60致谢60。