【作者】 杨海明；

【导师】 丁家桐； 王志跃；

【作者基本信息】 扬州大学 ， 特种经济动物饲养， 2014， 博士

【摘要】 作为季节性繁殖家禽,鹅的繁殖性能受季节影响较大。扬州鹅是我国培育的第一个鹅品种,其繁殖具有典型的部分长日照型特点,因此对扬州鹅的繁殖进行光照调控具有重大生产意义。本文通过光照调节来研究光照对扬州鹅繁殖性能和繁殖生理等方面的影响,并采用抑制性消减杂交技术,筛选影响种鹅季节性繁殖的相关基因,为种鹅稳产、高产的环境控制和高产品种鹅的选育提供参考依据。一、自然光照条件下种鹅产蛋规律及血清生化指标、生殖激素水平的变化规律试验鹅全程采用自然光照,公母鹅混群饲养,每月统计鹅群的产蛋率,并于每月11-15日上午9：00-10：30随机取15只母鹅、10只公鹅,进行翅静脉采血,离心收集血清,用于相关血液指标的测定。同时随机取6只种母鹅和3只种公鹅屠宰,取下丘脑和垂体,用于测定相关基因的表达量；分离心脏、肝脏等内脏器官、称重,分离生殖系统的组织、器官,进行重量或长度测定；取输卵管放入福尔马林溶液,用于组织切片制作。结果表明：1、种鹅2月份的产蛋率最高,随后逐渐下降,7-10月份停产,11月份又重新产蛋,且产蛋率逐渐上升。2、光照延长或缩短所引起的公母鹅血清总蛋白和白蛋白含量的变化规律是一致的。光照延长时母鹅在各时间点的血清总蛋白含量均高于光照缩短时。类似地,光照延长时种鹅前3个月的白蛋白含量也明显高于光照缩短时。此外,公母鹅血清尿素氮浓度变化随光照时间的延长而增加,反之亦然。3、在光照延长或缩短3个月后,公鹅的血清促卵泡素(FSH)浓度均处于较高水平,而母鹅则均处于相对较低水平,且血清的FSH水平变化不大。对于血清促黄体素(LH)水平,种鹅在光照长度变化后的第2个月出现较大变化,其中公鹅明显升高,母鹅明显下降。光照延长或缩短在公母鹅均引起血清催乳素水平的变化,但变化趋势相反；然而,光照延长或缩短引起的母鹅血清雌二醇(E2)浓度变化规律却相似。4、公母鹅的心脏指数均随光照时间的延长而发生变化,但变化趋势相反。母鹅的卵巢指数随光照时间的延长而不断下降,但整个延长期均大于光照缩短时的卵巢指数。光照延长或缩短引起的公鹅睾丸指数变化趋势相反；在光照延长的前4个月,公鹅睾丸指数大于光照缩短时的公鹅睾丸指数,而4个月后则小于光照缩短时的公鹅睾丸指数。光照长度变化引起的母鹅输卵管指数和输卵管长度变化趋势一致,且光照延长时输卵管指数和输卵管长度均明显大于光照缩短时；光照延长时母鹅的卵泡数逐渐减少,而光照缩短时母鹅的卵泡数逐渐增多,两者变化趋势相反。5、在光照延长或缩短的前4个月,种鹅垂体中FSH的相对表达量均呈现升高的趋势,只是光照延长时种鹅FSH表达量升高的幅度更大。然而,在第5、6个月,两者变化趋势相反。6、在光照延长的前3个月,种鹅输卵管膨大部腺体具有明显的管腔,在第4个月腺体管腔则明显缩小,第5、6个月时细胞质中均出现空泡,腺体管腔变模糊。当光照缩短时,输卵管膨大部组织结构变化趋势与光照延长时相反。此外,光照延长和缩短引起的种鹅输卵管峡部、子宫部组织结构变化规律与膨大部相似。二、不同光照和温度处理对母鹅血清生化指标、生殖激素水平、繁殖机能的影响采用二因子方差分析,将120只成年母鹅随机分成6组,每组20只,分别是组1(0-5℃、8hL)、组2(0-5℃、12hL)、组3(0-5℃、16hL)、组4(25-30℃、8hL)、组5(25-30℃、12hL)、组6(25-30℃、16hL)。所有试验鹅饲养于密闭鹅舍。试验期每隔10天,于上午9：00-10：30随机取5只鹅进行翅静脉采血,静置、离心后收集血清,用于相关血液指标的测定。同时每个处理随机取3只鹅,屠宰,取下丘脑和垂体,用于相关基因表达量的测定；分离心脏、肝脏等内脏器官并称重,分离生殖组织或器官,进行重量或长度测定,同时取输卵管放入福尔马林溶液,用于组织切片制作。结果表明：1、不同光照、温度处理的种鹅前20天的血清总蛋白、白蛋白、碱性磷酸酶、尿素氮含量变化稍小,但30天后变化幅度明显加大。2、不同光照、温度处理第20天时,各组间生殖激素呈现出较大差异,其中组1和组2鹅血清LH浓度明显升高；第30天后,各组鹅血清FSH浓度呈现出较大差异,其中组3鹅血清FSH浓度相对较低,组4鹅血清FSH浓度相对较高；不同光照、温度处理前20天鹅血清PRL浓度均较高,且各组间差异较大,而后逐渐减低并趋于一致；不同光照、温度处理下各组间鹅血清E2的浓度差异随着时间的延长逐渐增大,第50天时达到最大,其中组2和组6鹅血清E2的浓度变化相对小一些。3、不同光照、温度处理影响鹅心脏指数和肝脏指数,其中组3变化相对大一些,组5变化相对小一些；组4、组5鹅肝脏指数随时间的变化较小,但组5鹅肝脏指数基本都大于组4。不同光照、温度处理对鹅繁殖系统产生了较大影响,其中组4和组5鹅卵巢指数、卵泡指数均较大,组1和组3较小；组4、组5鹅的卵泡数较多,组3鹅的卵泡数较少；组4、组5鹅的输卵管指数、输卵管长度较大,组1和组3的较小。4、不同光照和温度处理对处理30天的鹅血清生化指标影响较大,组5的鹅血清总蛋白含量显著高于组1、组2、组3(P<0.05),组4鹅血清碱性磷酸酶含量显著高于组1、组3(P<0.05),组5鹅血清尿素氮浓度显著高于组2(P<0.05),极显著地高于组1、组3、组6(P<0.01)。12h光照处理组鹅血清尿素氮浓度显著高于16h组(P<0.05)。5、不同光照和温度处理对处理30天的鹅血清激素指标产生了一定影响,其中组6鹅血清LH含量显著高于组2、组4(P<0.05),组6鹅血清PRL含量显著高于组1、组2、组3、组5(P<0.05)。高温处理组鹅血清LH、PRL含量均极显著高于低温组(P<0.01)。6、不同光照和温度处理对处理30天的鹅内脏器官影响较小,只是组3鹅心脏指数显著大于组2、组4(P<0.05),其他各组间肝脏指数、脾脏指数差异均不显著(P>0.05)。7、不同光照和温度处理对处理30天的鹅繁殖系统产生较大影响,其中组2、组4、组5鹅卵巢指数较大,均显著大于组6(P<0.05),组4鹅卵泡指数显著大于组3、组6(P<0.05),组4鹅卵泡数显著大于组3、组6,组2、组4鹅输卵管指数显著大于组3(P<0.05),组2、组5鹅输卵管长显著大于组1(P<0.05)；12h光照处理组鹅卵巢指数、卵泡指数均极显著大于16h组(P<0.01),8h光照处理组鹅卵泡数显著影响大于16h组(P<0.05),12h光照处理组鹅输卵管的长度显著大于8h组和16h组(P<0.05)；高温处理组鹅输卵管指数极显著大于低温组(P<0.01)。8、光照和温度处理30天,8h光照处理组鹅垂体FSH基因表达量显著高于12h组和16h组(P<0.05),16h光照处理组鹅垂体PRL基因表达量显著高于8h组和12h组(P<0.01)。三、恒定光照对种鹅血清生化指标、生殖激素水平、繁殖机能的影响于2月份,取自然繁殖期的扬州鹅种鹅7只,其中6只母鹅、1只公鹅,饲养于密闭鹅舍,施以11h/d的恒定光照处理,定期采集血样,用于测定血清生化指标和激素指标,饲养至6个月,随机取5只母鹅屠宰,同时随机取5只自然光照下的种母鹅、屠宰,取下丘脑和垂体,用于相关基因表达量测定及免疫组化检测；分离心脏、肝脏等内脏器官并称重,分离生殖系统,进行重量或长度测定；取输卵管放入福尔马林溶液,用于组织切片制作,结果表明：1、恒定光照下母鹅血清总蛋白、白蛋白、胆固醇含量变化趋势趋于一致,均是先升高再下降,又逐渐升高；雌二醇浓度在第2个月大幅升高且达到最高点,然后大幅下降并维持在相对较低水平。2、恒定光照处理6个月鹅的血清白蛋白、谷草转氨酶含量均显著高于自然光照组(P<0.05)；两处理组鹅生殖激素指标虽有差异,但未达到显著水平(P>0.05)。3、恒定光照处理6个月鹅的肝脏重显著大于自然光照组(P<0.05),卵巢重、卵泡重、卵泡数、输卵管重、输卵管长均显著大于自然光照组(P<0.05)。4、经免疫组化染色的下丘脑切片,恒定光照处理组、自然光照组均检测到棕黄色或黄褐色形态不一的促性腺激素释放激素(GnRH)、褪黑素(Mel)免疫反应阳性细胞,只是恒定光照处理组GnRH平均灰度值高于自然光照组。5、恒定光照处理6个月的鹅输卵管膨大部的腺体内具有明显的管腔,血管网密集,峡部初级褶明显,子宫部具有明显的子宫褶,而自然光照组鹅输卵管膨大部的细胞胞质中有空泡出现,腺体管腔模糊,峡部结缔化严重,子宫部血管较少,腺体少,黑色素较多。四、自然休产期产蛋鹅与休产鹅下丘脑基因差异表达分析于6月下旬选自然光照下相同家系的半同胞母鹅3对(产蛋鹅3只、休产鹅3只),取下丘脑放于液氮中冷冻保存,用于基因差异表达分析。结果表明：1、从休产鹅文库和产蛋鹅文库各随机取205个阳性克隆、测序。：利用NCBI数据进行BLASTx比对,结果有101条ESTs找到与之匹配的同源序列,以筛选到的E值小于1E-5的片段视为功能已知基因,从休产鹅文库中获得21条功能基因,产蛋鹅文库中获得24条功能基因。2、对筛选出的差异表达基因进行在线功能聚类分析,通过细胞定位、分子功能、生物学功能发现,休产鹅特异性文库中的基因SMARCA4-201参与雄激素受体结合的过程,产蛋鹅特异性文库中的淋巴增强结合因子参与雌激素受体活性、雌激素受体结合的过程。更多还原

【Abstract】 Goose is a seasonally reproductive poultry and its reproductive performance is influenced by season. Yangzhou goose is the first cultivated breed of goose in China with typically partial long-day breeding characteristics, thus light regulation on the reproduction of Yangzhou goose may have an important implication in goose industry. The objective of this study was to investigate the effect of light regulation on the reproductive performance and physiology of Yangzhou goose. Also, we employed the SSH technology to screen and identify the major genes associated with goose reproductive performance. This study may provide useful information for stable high-yield production of goose and genetic selection on goose reproductive performance.1. The Influence of Natural Light on Laying Pattern, Serum Biochemical Parameters and Reproductive Hormone Levels in Breeding Yangzhou GeeseThe geese were sexually mixed and raised in natural light. Eggs laid by the female geese were collected and counted every month, and laying rate was then calculated. For blood sampling,15female geese and10male geese were randomly selected. Blood was collected from these geese at9:00-10:30am during the11th to15th of a month. Serum biochemical parameters were subsequently determined using the blood samples. Also, other six female geese and ten male geese were slaughtered for harvesting tissues and organs. The hypothalamus and pituitary gland were used for gene expression analysis. The weights of internal organs and reproductive tissues and the lengths of reproductive tissues and organs were measured. The results are showed as follows:①The laying cycle of Yangzhou geese started in November, reached the peak of laying rate in February, and stopped around July to October.②The levels of serum total protein and albumin changed with the length of lighting time. The level of serum total protein in the male geese during the lengthening daytime was higher than that in the geese during the shortening daytime. Similarly, the content of albumin in the breeding geese during the lengthening daytime was significantly greater than that in the geese during the shortening daytime at the first3months of treatment. In addition, the serum uric nitrogen level in the geese treated with the lengthening daytime also changed in the same trend; but in the geese treated with the shortening daytime the trend became opposite.③The serum FSH of male geese was at higher level after3months of light treatment, while the serum FSH of the female geese was at a low level and remained little changed with the length of lighting time. In the second month, the serum LH level of breeding geese greatly changed with the length of lighting time. The LH level in female geese was significantly increased, while that in in male geese was significantly decreased. The serum E2level was in the same trend, while the serum PRL level was in an opposite trend.④The change of heart index with the length of lighting time was opposite between female and male geese. The ovarian index of female geese were in decline during the lengthening daytime, but the ovarian index of geese treated with the lengthening daytime were significantly higher than those geese treated with the shortening daytime. For testis index of male geese, the change patterns were absolutely opposite between the lengthening daytime and the shortening daytime, and the testis index of geese treated with the lengthening daytime were greater than the geese treated with the shortening daytime before4months of treatment. However, the trend of change in testis index was opposite after4months. The index and length of oviduct were increased with the length of lighting time. In addition, the follicle number in the ovary oppositely changed with the length of daytime.⑤The expression of pituitary FSH was on the rise at the first4months of light treatment, and the geese treated with the lengthening daytime had a larger range in the expression of pituitary FSH than those treated with the shortening daytime. However, the expression pattern was changed in an opposite way during5or6months of light treatment.⑥At the first3months of light treatment, the glands in dilated fallopian tube during the lengthening daytime had obvious lumen, but during the5or6months of light treatment, the lumen was obviously decreased, the vacuoles could be founded in magnum cells, and the gland lumen became fuzzy. In contrast, the structure of dilated fallopian tube changed in the opposite trend during the shortening daytime. The effect of lighting time on isthmus and uterus was similar to the fallopian tube.2. Effects of Light and Temperature on Serum Biochemical Variables, Reproductive Hormone Concentrations and Breeding Function in the Breeding Yangzhou Geese In a two factorial arrangement,120adult female geese were randomly divided into six groups group1(0-5℃,8h L), group2(0-5℃,12h L), group3(0-5℃,16h L), group4(25-30℃,8h L), group5(25-30℃,12h L) and group6(25-30℃,16h L), each group included twenty birds. All the geese were kept in a closed house. During the experiment,5geese were randomly selected for collecting blood at9:00-10:00am every10days, and the blood was sit for a while before centrifugation to separate the serum from the blood. The serum was used for biochemical analysis. For each treatment,3geese also were selected and slaughtered for collecting their hypothalamus and pituitaries. These organs were quick-frozen in liquid nitrogen and stored in-70℃for later use. The expression levels of some related genes in these organs were determined subsequently. In addition, some organs including heart and liver were separated and weighed, the reproductive tissues and organs were also separated for length or weight measurement. The results are showed as follows:①Serum total protein, albumin, alkaline phosphatase and urea nitrogen slightly changed with different lighting times and temperatures before20days of treatment, but they were significantly increased after30days of treatment.②There was big difference in the levels of reproductive hormones among the groups at20days of both light and temperature treatments. For example, the serum LH level was increased significantly in group1and2but not in groups. Also, there was a big change in the serum FSH level across the groups at30days of treatment. The serum level of FSH was lower in group3but higher in group4. Moreover, the serum concentrations of PRL were higher at20days of treatment and the difference across groups was large, however, the level of PRL was gradually reduced and remained the same. At last, the change in the serum E2level was gradually increased with the time of light and temperature treatments, and serum E2level was the highest at50days of treatment. However, serum E2level in group2and6only slightly changed.③Different light and temperature had an effect on the heart index of the geese. Heart index in group3was greater, while that in group5was smaller. Different light and temperature also affected the liver index. The change of liver index in group4and5was smaller across the time of treatment, while the liver index in group5was larger than that in group4. In addition, those treatments had an obvious impact on reproductive system of the geese. For example, group4and5had a bigger ovarian index and follicle index, though group1and3had smaller index. The number of follicles in group4and5were also larger than that in group3, and he index and length of oviduct in group4and5were bigger than those in group1and3.④There was a significant influence on serum biochemical parameters of breeding geese at 30days of light and temperature treatments. Serum total protein content was higher in group5than that of group1,2and3(P<0.05), serum ALP content was higher in group4than that in group1and3(P<0.05), the serum urea nitrogen in group5was significantly higher than that in group2(P<0.05), and significantly higher than group1,3and6(P<0.01), Serum urea nitrogen of geese under12h light was significantly higher than that under16h light.⑤There was a certain influence on serum hormone indicators at30days of light and temperature treatment. The group6had a significant higher serum LH levels than group2and4(P<0.05), and the serum PRL levels were higher than that in group1,2and5(P<0.05). Serum LH and PRL of geese in high-temperature group were significantly higher than that in low-temperature group (P<0.01).⑥The change in the internal organ index was smaller at30days of light and temperature treatment. Heart index was significantly larger than that in group2and4(P<0.05). Additionally, there were no significant difference in liver or spleen index (P>0.05).⑦There was a big influence on reproductive system at30days of light and temperature treatment. The ovarian index was bigger in group2and4, both of them were significantly greater than that in group6(P<0.05), the number and index of follicles in group4was significantly greater than that in group3and6(P<0.05), the tubal index in group2and4was significantly greater than that in group3(P<0.05), the tubal length in group2and5was significantly greater than that in group1(P<0.05), the number and index of follicles of geese under12h of light were significantly higher than that in group under16h of light (P<0.05), the number of follicles under8h of light was significantly more than under16h of light (P<0.05), the tubal length of group under12h of light was significantly greater than under8and16h of light (P<0.05). Tubal index of geese under higher temperature was greater than that under lower temperature (P<0.01).⑧At30days of light and temperature treatments, the geese under8h of light had a significantly higher expression of pituitary FSH than that under12and16h of light (P<0.05). In contrast, the geese under16h of light had a significantly higher expression of pituitary PRL than that under8and12h of light (P<0.01).3. The Effect of Constant Light on Serum Biochemical parameters, Reproductive Hormone Concentrations, and Breeding Function in Breeding Yangzhou GeeseIn February, seven geese were selected (6female and1male) and raised in hermetic farm with constant light of12h/d. Blood were collected regularly to measure the serum biochemical variables and hormone concentration. Geese were fed for6months, then five female geese under natural light were slaughtered for hypothalamus and pituitary gland to determine the expression of some related genes. The weights of internal organs and reproductive tissues, and the lengths of reproductive tissues and organs were also measured. The results are showed as follows:①Under constant light, the changes in total serum protein, albumin and cholesterol of breeding geese appeared to be in accordance:rose at first, declined later, and gradually rose again. The estradiol concentration was increased markedly in the second month, reached its peak, followed by a big drop, and remained at a relatively low level.②The concentrations of serum albumin, glutamic oxalacetic transaminase of the breeding geese treated with6month’s constant light were significantly higher than the natural light group (p<0.05). There was a difference in the reproduction hormone index between the two groups of breeding geese, though it didn’t reach the remarkable level.③Liver weight, ovary weight, follicles weight, follicular number, tubal weight and tubal height of the breeding geese treated with6month’s constant light were significantly higher than the natural light group (P<0.05).④Tan or brown positive immunoreactive cells of GnRH and Mel with different forms could be determined in both constant light and natural light group by observing the biopsies of hypothalamus with immunohistochemistry, but the mean gray value of GnRH in constant light group were higher than that in another group.⑤The dilated fallopian tube glands of breeding geese treated with6months constant light had an obvious lumen and intensive vascular network, and the isthmus primary fold was obvious and the uterine folds of uterus also was quite clear, but there were some vacuoles in oviduct magnum cells of the geese under natural light, some appearance such as the fuzzy gland lumen, serious connective in the isthmus, and less vascular and glandular but more melanin in the uterus were also presented,.4. Differentially Expressed Genes in Hypothalamus between laying and Resting GeeseThree pairs of half-sibling geese (3laying geese,3resting geese) were selected and raised under natural light. The geese were in the same family. In the last ten days of June, their hypothalamus tissue samples were harvested and kept in fluid nitrogen for later gene expression analysis. The results are showed as follows:⑥Two hundred and five positive clones were chosen randomly from laying geese library resting geese library. The clones were sequenced. Use the NCBI data to do the BLASTx contrast,101ESTs were found its homologous sequence. Among the sifted piece that had an E value smaller than1E-5as the function-known gene,21functional genes were got from the resting geese library and24from the laying geese library.②The online function clustering analysis was conducted using the sifted and differentially expressed genes. The cellular localization, molecular function and biology function were also predicted. It was shown that the SMARCA4-201gene from the specific library of resting geese could participate in the process of androgen receptor binding, while the LEF1gene from the specific library of laying geese could participate in the process involving estrogen receptor activation and binding.更多还原