【作者】 陈辉；

【导师】 黄仁录； 杨宁；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 褪黑素具有广泛的生理和药理作用，能影响多个系统（包括免疫系统、内分泌系统、神经系统），同时也对生物的昼夜节律、性成熟及生殖、免疫反应、代谢、肿瘤、衰老等均有调节作用。性成熟是一个复杂的过程，参与触发青春发育启动的物质或机制目前尚不十分清楚，褪黑素对下丘脑-垂体-性腺轴的作用机制目前未有确切的研究成果。瘦素具有广泛的生物学效应，在能量代谢、脂肪代谢、繁殖等方面具有重要的调控作用，褪黑素可抑制瘦素的产生，瘦素受体在代谢与生殖内分泌之间起重要作用。本文为了探索光照周期和能量调节蛋鸡生殖轴的效应机理，设计了光照周期、外源褪黑素、饲喂水平和外源瘦素四个影响因子，采用分子生物学手段，研究对光敏感的神经内分泌高位调节器松果腺与下丘脑-垂体-性腺轴的交互作用，揭示生殖节律的环境依赖依据及光照和能量调整生殖节律的机理。光照周期处理组选用10周龄海兰灰商品蛋鸡240只，随机分为3组，试验Ⅰ组光照周期为8L:16D，试验Ⅱ组光照周期为12L:12D，试验Ⅲ组光照周期为16L:8D，试验Ⅲ组光照周期为16L:8D，每组设4个重复，每个重复20只鸡。褪黑素处理组选用10周龄海兰灰商品蛋鸡320只，随机分为4组，每组设4个重复，每个重复20只鸡，试验Ⅰ组为8小时光照对照组，试验Ⅱ组为16小时光照对照组，试验Ⅲ组基础日粮中添加低剂量褪黑素（20mg/kg），试验Ⅳ组基础日粮中添加高剂量褪黑素（200mg/kg）。取得的主要结果如下：（1）体重与跖长、龙骨长、耻骨间距、鸡冠长厚高呈显著正相关；（2）光照试验中16小时光照处理组与8小时光照处理组相比，育成鸡开产日龄提前了21.75天，性成熟体重降低了87.1g。试验2中，外源褪黑素促使开产日龄推迟，性成熟体重增加，腹脂绝对重和相对重增加；（3）血浆甘油三酯浓度在性成熟时显著增加，光照周期和外源褪黑素对性成熟过程中蛋鸡血浆胰岛素和葡萄糖浓度影响差异不显著；（4）短光照抑制GnRH受体mRNA表达，长光照促进GnRH受体mRNA表达；下丘脑GnRH受体mRNA表达量与年龄呈负相关；（5）在性成熟前FSHR基因mRNA表达呈现出波动性增长趋势；给予外源褪黑素可降低性成熟前垂体FSHR基因mRNA表达，对性成熟后FSHR基因mRNA表达无显著影响；（6）不同光照周期或给予外源褪黑素对垂体LHR mRNA表达无显著影响，结果提示在卵巢功能的调节中，除了下丘脑－垂体－卵巢轴外，还存在着卵巢局部多因子调节系统。（7）性成熟前卵巢ERmRNA表达量呈现随年龄增长而增加的趋势；（8）垂体MELR1A mRNA基因表达量随年龄增长呈下降趋势；不同光照周期对下丘脑、垂体、卵巢MELR1A mRNA基因表达无显著影响；长光照促进了性成熟时腹脂MELR1A mRNA表达；外源褪黑素促进性成熟垂体MELR1A mRNA表达；外源褪黑素并未达到显著抑制性成熟腹脂MELR1A mRNA表达的效果。MELR1B mRNA在下丘脑表达量最高，其次为垂体、卵巢和腹脂；性成熟后下丘脑MELR1B mRNA表达呈现下降趋势，垂体、卵巢和腹脂MELR1B mRNA表达量无显著变化；（9）下丘脑和垂体LEPR基因表达量随年龄增加呈递减趋势，卵巢LEPR和ER基因表达量随年龄增大呈增加趋势；（10）研究推测性成熟过程中褪黑素受体与下丘脑—垂体—卵巢轴的成熟具有相关性，主要是通过MELR1A抑制中枢神经系统启动，或与LEPR在垂体进行整合，或与其他HPG调节的信号相互作用影响繁殖性状。饲喂水平和光照周期对蛋鸡卵巢和腹脂LEPR、MELR1A和MELR1B基因表达影响：选用10周龄海兰灰育成鸡480只，采用2（自由采食，自由采食量80%）*3（8L:16D，12L:12D，16L:8D）双因子设计，每组4个重复，每个重复20只鸡。结果如下：饲喂水平和光照周期对14周龄和18周龄蛋鸡腹脂LEPR、MELR1A、MELR1B基因表达不存在互作效应，对14周龄、18周龄和性成熟蛋鸡卵巢ER、LEPR、MELR1A、MELR1B基因表达不存在互作效应，仅对性成熟时蛋鸡腹脂LEPR基因表达存在互作效应；限饲显著抑制了14周龄、18周龄和性成熟时卵巢ER、LEPR和MELR1B基因表达。14周龄时，长光照处理组腹脂LEPR基因表达量显著低于短光照处理组，性成熟时短光照周期和限饲抑制了蛋鸡腹脂LEPR基因表达；光照周期对卵巢LEPR基因表达影响差异不显著，饲喂水平对卵巢LEPR基因表达影响非常显著，表明在蛋鸡性成熟启动过程中，LEPR基因影响繁殖调控主要受饲喂水平和体脂水平的影响。外源瘦素对禁食后蛋鸡生殖功能恢复调控研究：25只体重相近（1560g±114g）的蛋鸡随机分为5组，每组5只鸡，I组自由摄食组；II组禁食对照组；Ⅲ组禁食+低浓度leptin；Ⅳ组禁食+中浓度1eptin；V组禁食+高浓度leptin。4个禁食组于第1天和第2天禁食，之后恢复自由摄食。第3天到第5天每天早上08:00-08:30，3个注射组的蛋鸡通过跖部静脉注射用0.75%PBS-BSA（1mg/mL）稀释后的商品化重组小鼠leptin。2个对照组在同样的时间注射0.75%PBS-BSA（lmg/mL）。3个leptin处理组的leptin注射量分别为50（F+L），200（F+M）和400（F+H）μg/只。主要结果如下：2天禁食导致了产蛋期蛋鸡卵巢功能的受损，适当浓度的外源瘦素可以促进蛋鸡受损卵巢的恢复；外源注射中浓度小鼠重组瘦素促进了垂体MELR1A表达，抑制了垂体LEPR基因表达；禁食可以抑制卵巢LEPR基因表达，而瘦素可以促进卵巢LEPR基因的表达，抑制MELR1A和MELRA1B基因表达，研究推测外源瘦素可以通过中枢神经系统调控卵巢机能的恢复或直接与其作用来参与调控蛋鸡卵巢功能的恢复。更多还原

【Abstract】 Melatonin has a wide range of physiological and pharmacological effects, can affect multiplesystems (including the immune system, endocrine system, nervous system), regulate biologicalcircadian rhythm, sexual maturation and reproduction, immune response, metabolism, cancer andsenium. Sexual maturity is a complex process, substances or mechanisms involved in triggering pubertyis not very clear, the mechanism of melatonin on the hypothalamic--pituitary--gonadal axis was not veryclear. Leptin has a wide range of biological effects, plays an important role in the regulation of energymetabolism, fat metabolism, reproduction, and so on. Melatonin could inhibit leptin secretion, leptinreceptor played an important role between metabolism and reproductive endocrinology. In this paper, inorder to study the effects of photoperiod and energy on regulating the reproductive axis mechanism, fourinfluencing factors were designed in this research: photoperiod: exogenous melatonin, feeding level andexogenous leptin. Using the molecular biology methods, we studied the interactions between thelight-sensitive nerve endocrine regulator--pineal gland and hypothalamus--pituitary--gonadal axis, inorder to reveal the mechanism of photoperiod and energy regulating the reproductive rhythms.Photoperiod treatment group selected10-week-old240Hyline gray hens, randomly divided intothree groups, photoperiod was8L:16D in group Ⅰ, photoperiod was12L:12D in group Ⅱ, photoperiodwas16L:8D in group Ⅲ, each group had four replicates of20chickens in each replicate. Melatonintreatment group selected10-week-old320Hyline gray hens, randomly divided into four groupswith fourreplicates of20chickens, the hens were maintained on8-h photoperiods(group Ⅰ),16-hphotoperiods(groupⅡ), and16-h photoperiods supplemented with20mg/kg melatonin (groupⅢ),200mg/kg melatonin(groupⅣ).Effect of photoperiod and melatonin on hypothalamic--pituitary--gonadalaxis, the main results obtained showed as follows:(1) There was a significant positive correlationbetween body weight and plantar long, long keel, pubic spacing, comb length, comb height and combthickness;(2) In experiment1, age of first egg in16-hour photoperiod group was21.75d earlier than8-hour photoperiod group. Body weight in16-hour photoperiod group was87.1g lighter than8-hourphotoperiod group. In experiment2, exogenous melatonin could delay age of first egg, increase sexualmaturity weight, absolute and relative abdominal fat weight;(3)There was a significant increase inplasma triglyceride concentrations at sexual maturity. photoperiod and exogenous melatonin showed nosignificant differences on plasma insulin and glucose concentrations at sexual maturity;(4)The shortphotoperiod inhibited GnRH receptor mRNA expression, long photoperiod promote GnRH receptormRNA expression; hypothalamic GnRH receptor mRNA expression was negatively correlated with senium;(5) FSHR mRNA gene expression showed a volatility increasing trend; given exogenousmelatonin reduced the mRNA expression of pituitary FSHR before sexual maturity, FSHR mRNA geneexpression was not significant different at sexual maturity;(6) Photoperiod and exogenous melatoninhad no significant effect on the pituitary LHR mRNA expression, which suggestted that, in addition tothe hypothalamic--pituitary--ovary axis, there were also some other multi-factors regulating systemsexisted in the ovary;(7) Ovary ER mRNA expression level showed an increasing trend with theincreasing age;(8) Pituitary MELR1A mRNA gene expression decreased according with the increasingage; Photoperiod did not show significant difference on MELR1A gene expression of hypothalamus,pituitary and ovary; long photoperiod promoted abdominal fat MELR1A mRNA expression at sexualmaturity; melatonin promoted pituitary MELR1A mRNA expression, but did not inhibit abdominal fatMELR1A mRNA expression at sexual maturity. MELR1B mRNA expression was highest in thehypothalamus, followed by the pituitary, ovary and abdominal fat; MELR1B mRNA expression in thehypothalamus reduced at sexual maturity, but did not change significantly in the pituitary, ovary andabdominal fat;(9) LEPR gene expression in the hypothalamus and pituitary reduced with the senium,while LEPR and ER mRNA expression in ovary increased with the senium;(10) In conclusion,melatonin receptor affected the HPG ’s maturity, MELR1A could inhibit hypothalamus and pituitarymelatonin receptor expression, or integrated with LEPR gene in the pituitary, or played a role from othersignal ways.Effects of feeding level and photoperiod on LEPR, MELR1A and MELR1B gene expression ofovary and abdominal fat.10weeks480Hy-Line Gray commercial henswere randomly divided into6groups, using2(ad libitum, voluntary feed intake80%)*3(8L:16D,12L:12D,16L:8D) two-factor design,each group of4replicates of20chickens. The results showed as follows: there is no interaction effectbetween photoperiod and feeding level on abdominal fatLEPR, MELR1A, MELR1B gene expression at14w and18w, there is no interaction effect between photoperiod and feeding level on ovaryER, MELR1A,MELR1B gene expression at14w and18w, but ovary LEPR gene expression showed interaction effect atsexual maturity. Restricted feeding significantly inhibited ovary ER, LEPR and MELR1B geneexpression at14weeks,18weeks and sexual maturity. At14-week-old, abdominal fat LEPR geneexpression was significantly lower in long photoperiod group compared with the short photoperiodgroup; At sexual maturity, short photoperiod and feed restriction group reduced abdominal fat LEPRgene expression; photoperiod did not show significant differences on ovary LEPR gene expression,feeding level showed a sinificant difference on ovary LEPR gene expression, the resutls indicated thatLEPR gene regulating sexual maturity mainly affected by feeding levels and body fat level.25Hy-Line Gray commercial hens (1560g±114g) were randomly divided into five groups:(I) adlibitum control group (n=5);(II) fasted control group (FC; n=5);(III) fasted+low concentration ofleptin (F+L; n=5);(IV) fasted+middle concentration of leptin (F+M; n=5);(V) fasted+highconcentration of leptin (F+H; n=5). All four fasted groups were fasted for2days and then ad libitumand the hens were treated with leptin at dose of0(control and FC),50(F+L),200(F+M) and400(F+H)ug/kg/d. on day3-5.The results of exogenous leptin on reproductive function of fasting hens were showed as follows: ovary function was affected by two-day fasting, appropriate concentrations ofexogenous leptin could recover the damaged ovary; exogenous injection of recombinant mouse leptincould promote pituitary MELR1A expression, suppressed pituitary LEPR gene expression; fasting couldinhibit ovary LEPR gene expression, and exogenous leptin could promote ovary LEPR gene expression,inhibit ovary MELR1A and MELRA1B gene expression. The results suggested exogenous leptin couldregulate ovary function’s recovery by central nervous system or directly through ovary regulatingsystem.更多还原