【作者】 庞训胜；

【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， 2009， 博士

【摘要】 本文调查了黄淮山羊高繁殖力母羊不同胎次与产羔数之间的关系,比较了高低繁殖力母羊在发情周期和卵巢摘除初期外周血P4和E2变化的特点,探讨了不同繁殖性状母羊在黄体期早期的卵泡、黄体及其卵泡闭锁的差异。通过抑制消减杂交技术建立起了高繁殖力母羊卵泡发育颗粒细胞差异表达基因消减cDNA文库,旨在揭示高繁殖力母羊卵泡发育调控及生殖内分泌特点,为进一步研究高繁殖力母羊的遗传育种提供一定的参考依据。1黄淮山羊高繁殖力母羊产羔调查与分析为了探究高繁殖力母羊多胎繁殖性状的特点,对江淮地区427只具有高繁殖力黄淮山羊母羊(窝产羔数至少一次产羔≧5)的产羔进行了调查和分析。结果表明,随着胎次的增加,高繁殖力母羊的产羔数增加,直至第5胎次,之后产羔数下降。在所有胎次中,高繁殖力母羊在第5胎次的产羔数达到最大,平均窝产羔数3.78±0.06羔,与其他胎次的产羔数相比较,呈极显著差异；相比之下,母羊在第1胎次的产羔数极显著低于其他胎次。根据不同窝产羔数的母羊所占比例分析,在第1胎次,随窝产羔数的增加,其母羊的比例依次降低,但在之后母羊所占的比例中,窝产1羔和2羔的比例急剧下降；而窝产4～7羔的母羊比例呈递增趋势延至第4～5胎次,其中随着窝产羔数的增加,其母羊所占比例的最大值在不同胎次间的分布依次后移。以上结果提示,高繁殖力母羊的多胎性状在不同胎次间具有明显的差异,其母羊性成熟后在初次繁殖时卵巢卵泡成熟与排卵及其妊娠生理等已经具备了极端高繁殖力的生理特点。2在发情周期中高低繁殖力母羊外周血孕酮和雌二醇浓度变化的比较选择生殖机能正常、空怀、经产的黄淮山羊高繁殖力(窝产羔数至少一次产羔≧5)和低繁殖力(每次窝产羔数≦2)母羊各4只。以间隔12 d肌注氯前列烯醇0.1 mg/只·次诱导同期发情后,在自然发情周期中,连续两个情期每天以间隔12 h和在黄体中期和发情前期间隔12 min密集性采集颈静脉瘘管血样,测定外周血E2和P4含量。结果显示,在黄体期的大多数时间内,高繁殖力母羊外周血P4量高于低繁殖力母羊,在黄体期早期的P4量上升速度,高繁殖力羊早于低繁殖力羊；两组母羊在P4的平台期,P4量呈显著性差异。在整个发情周期中,高繁殖力母羊平均E2浓度均高于低繁殖力母羊；在黄体期早期,高繁殖力母羊出现2个E2峰值,而低繁殖力母羊仅有1个。高低繁殖力母羊外周血P4和E2的量均是以非典型的脉冲式变化。以上结果表明,在黄体期早期,高繁殖力母羊外周血P4量的上升速度和卵泡波卵泡发育的周转均高于低繁殖力母羊,而在E2含量上,高繁殖力母羊维持在较高水平。3高低繁殖力母羊在黄体期早期卵巢卵泡和黄体的比较高低繁殖力母羊各4只,在氯前列烯醇同期发情后的第3个发情周期7～9 d,通过颈静脉瘘管采集羊的外周血液,测定E2和P4含量,并于第7和第9 d应用羊用腹腔内窥镜和腹部手术方法,观察和测定母羊卵泡和黄体大小。结果显示,在发情周期7～9 d的卵泡发育中,高低繁殖力母羊卵巢小卵泡和大卵泡数均处于减少趋势中,但差异均不显著,而中卵泡数以及总卵泡数均增加,其中高繁殖力母羊的中卵泡数平均增加了7.50个/只,低繁殖力母羊平均增加了4.80个/只。高繁殖力母羊外周血P4和E2浓度分别高于低繁殖力母羊,P4浓度在高低繁殖力母羊之间呈显著性差异；在发情周期第9d,高繁殖力母羊卵巢小卵泡数和Ⅱ型黄体数均分别显著高于低繁殖力羊16.43%和27.73%。以上结果表明,在发情周期7～9 d的卵泡发育中,黄淮山羊高低繁殖力母羊卵巢小卵泡、中卵泡和大卵泡数的发育趋势基本处于相同的生理变化。高繁殖力母羊外周血P4浓度显著高于低繁殖力母羊,促进了高繁殖力母羊卵泡的周转；高繁殖力母羊排卵卵泡以体积相对较小的成熟卵泡增多,卵巢卵泡的共优势化明显,Ⅱ型黄体数增加。4卵巢摘除初期高低繁殖力母羊外周血孕酮和雌二醇浓度变化的比较高低繁殖力母羊各4只,在发情周期的第9 d,通过腹部外科手术摘除双侧卵巢,并应用颈静脉瘘管连续5 d和卵巢摘除后第1和第5 d密集性采血。结果显示,母羊卵巢在摘除后的E2和P4分泌,均以非典型性的脉冲式释放。在卵巢摘除后的第1d,高繁殖力母羊外周血液P4和E2浓度均分别大于低繁殖力母羊。高低繁殖力母羊在卵巢去除后的1～3 d的P4水平均呈下降趋势；高繁殖力母羊的E2除在第1 d极显著性高于低繁殖力羊外,在其后的2～5 d,E2的平均值总体呈低于低繁殖力母羊的趋势,并在第5 d呈现差著性差异。以上结果表明,母羊非性腺相关组织在分泌E2和P4时呈现非典型的脉冲式。在外周血E2量上,高低繁殖力母羊之间存在呈现显著性差异,非性腺组织所产生的E2可能参与了母羊的生殖过程。5山羊黄体期闭锁卵泡的组织定位观察高低繁殖力母羊各4只,在发情周期第9 d通过腹部外科手术摘除双侧卵巢,迅即投入液氮中保存,冷冻切片,TUNEL检测法结合苏木精染色法,观察卵巢闭锁卵泡的组织学特点。结果显示,在高低繁殖力母羊卵巢闭锁的无腔卵泡中,均极少见到凋亡的卵母细胞。有腔卵泡的闭锁,多表现在卵泡腔的壁颗粒细胞层中有大量颗粒细胞凋亡,表现出明显的荧光反应,并多处于近腔颗粒细胞；而部分卵泡在颗粒层中出现凋亡细胞偶有零星存在的现象,但是在高低繁殖力母羊之间均无明显差异。在高繁殖力母羊的卵巢中,仅一膜之隔的相邻卵泡之间闭锁和非闭锁同时显现。以上结果表明,TUNEL检测结合原片苏木精染色的鉴定为卵巢组织凋亡细胞的定位提供一个较好的方法。在闭锁的有腔卵泡中,卵泡多表现出近腔细胞的凋亡特征,高低繁殖力母羊在闭锁卵泡的组织定位上无显著性差异。6高繁殖力母羊大小卵泡颗粒细胞差异表达基因消减cDNA文库的构建和筛选以氯前列烯醇肌肉注射2只高繁殖力母羊40 h后剪取卵巢,在20 min内计数卵泡、测量卵泡直径,以组织钝性分离和收集颗粒细胞,并剪取取样后的卵泡部分组织置入液氮内保存。从颗粒细胞中提取总RNA的大卵泡(5～6 mm)和小卵泡(3～4 mm)均未发生凋亡,总RNA经过双链cDNA的合成和扩增,以抑制消减杂交技术(SSH)构建了山羊卵泡颗粒细胞正向和反向cDNA文库。在正反向文库中,分别随机挑选了190个克隆进行斑点杂交。根据斑点杂交信息,在正反向文库中共选择了110个克隆单向测序,获得了109个EST,聚类后经过Blastn比对后发现,共有10个与已知功能基因相似,16个与已知但功能未知基因相似,65个为新的EST。这些差异基因的发现有助于揭示山羊卵泡发育和分化的调控机制,为进一步研究高繁殖力母羊卵泡共优势化的生殖机理提供一定的参考依据。更多还原

【Abstract】 The present paper investigated relationship between different parities and litter size of Huanghuai goats at high prolificacy. This research also compared peripheral blood concentrations of progesterone and estradiol of Huanghuai goats with poor prolificacy during the estrous cycle and in the early days after ovariectomy. The different prolific goats were also employed to analyse differences in number and size of their ovaries antral follicles and corepus luteum and in follicular atresia. In this study, a subtractive cDNA library of different expression genes in follicular granulosa cells in Huanghuai goats of high prolificacy was constructed by suppression subtractive hybridization. The objective of this study was to reveal the mechanism of follicles development regulation and their feature of reproduction endocrine in goat of high prolificacy, and to provide certain references for identifying the candidate genes and for its heredity and breeding of their high prolificacy.1. Investigation of Little Size of Huanghuai Goats with High ProlificacyThe little sizes of 427 highly prolific Huanghuai goats (HP,≥5 kids born per litter on at least one occasion) distributed in the Huanghuai plain were investigated to approach characteristic of their little size as following different parities. The results showed that litter size increased up to parity 5 and then decreased. At all parity levels, parity 5 HP does have the highest mean litter size (3.78±0.06) and parity 1 goats have the smallest. Based on analyzing the percentage of females at different little size, the females decreased at parity 1 as little size increased, and the proportion had 1 or 2 kids born per litter decreased rapidly after parity 1.However, the percentage of goats with 4 to 7 little size increased up to parity 4 to 5, furthermore as little size increased, the maximum number of the does delayed in order at different parity. It was concluded that there were obvious differences in little size at different parity in prolific does, and the limit facts of prolificacy probably was their uterus capacity. 2. Comparison of Concentrations of Progesterone and Estradiol in Peripheral Plasma during the Estrous Cycle between the Goats of High and Poor ProlificacyEight Huanghuai goats with 3-5 years old, non-lactating, parity 4-5 were selected to study two sequential natural estrous cycles of does with high prolificacy ((?)5 kids born per litter on at least one occasion, n=4) or poor prolificacy (<3 kids born per litter on any occasion, n=4) by initial synchronization achieved using two i. m. of 0.1 mg of a synthetic analog of prostaglandin F2 alpha given 12 days apart. Jugular blood samples were collected twice a day during the natural estrus by jugular syrinx and at 12-minute intervals for 3.2 hours over 2-3 days of proestrus and mid-luteal phase to assay for progesterone and estradiol. The results showed that progesterone concentrations began to rise earlier and were higher in the HP than in the PP goats on most days of the luteal phase but not during the follicular phase of the cycle. There was a significant difference between the two groups in the magnitude of the progesterone plateau. Mean estradiol concentrations in the HP group remained significantly higher than in the PP group during the estrus cycle. There were two estradiol peaks in the HP goats during the early luteal phase, but only one in the PP group. Progesterone and estradiol were secreted in a pulsatile atypia manner in HP and PP goats. It was concluded that the increase of progesterone in peripheral plasma and the turnover of follicles growth of waves in HP goats were higher than in PP group during the estrus cycle, and were the same for the overall mean estradiol concentrations.3. Comparison of Number and Size of Follicles and Corpora Lutea in Early Days of Luteal Phase in Huanghuai Goats of High or Poor ProlificacyFour HP and four PP goats were employed to collect Jugular blood samples individually at seven to nine days in third estrus cycle after synchronization achieved using a synthetic analog of prostaglandin F2 alpha and to assay for progesterone and estradiol. Meanwhile, follicles and corpora lutea were observed and measured with laparoscopy and operation on abdominal region at day 7 and day 9 in estrus cycle respectively. The results showed that number of small and large follicles had a decreasing tendency during 7 to 9 days of estrus cycle, but no difference was observed respectively. Both middle and all follicles increased, namely the mean middle follicles did to 7.50 per HP goat and to 4.48 per PP goat. Concentration of P4 and E2 in peripheral blood in HP does were higher than in PP goats, respectively, however there was no difference in P4 between two groups. Small follicles and typeⅡof corepus lutea in HP goats had obviously higher by 16.43%and 27.73%respectively compared with the PP does at day 9 of estrous cycle. It was concluded that there was a nearly same tendency in physiology of small-, middle- and large-follicles development during 7 to 9 days of estrus cycle in the HP and PP goats. The higher concentration of P4 in HP does promoted the turnover in follicles compared wich PP goats. There were more ovulation follicles characterizing smaller follicle in volume when matured in HP goats, and obvious co-dominance of follicles, and moreⅡtype of corepus lutea after ovulation, compared with those of PP does.4. Comparison of Concentrations of Progesterone and Estradiol in Peripheral Plasma in Early Days after Ovariectomy between the Goats of High and Poor ProlificacyFour HP and four PP goats were used to make ovariectomy of both ovaries respectively at day 9 of estrus cycle and to collect Jugular blood samples twice daily from 1 to 5 days after ovariectomy and at 12-minute intervals for 3.2 hours at day 1 and 5, in order to assay for progesterone and estradiol. The results showed that progesterone and estradiol were secreted in a pulsatile atypia manner in HP and PP goats. At day 1 after ovariectomy, the E2 and P4 concentrations were significantly higher in the HP than in the PP goats. Then there were a decreasing tendency in P4 lever in two groups during 1 to 3 days. The overall mean E2 concentrations in HP goats were lower than in the PP group from 2 to 5 days, significant differences were found at day 1 and 5 but their different concentrations were opposite. It was concluded that progesterone and estradiol from nonovaries were secreted in a atypia form of pulse. There existed a significant difference in E2 concentration in peripheral blood between HP and PP goats, furthermore the E2 from nonovaries probably involved in reproduction process in female.5. Observation of Histological Localization of Atretic Follicle in Luteal Stage in the GoatFour HP and four PP goats were employed to make ovariectomy of both ovaries respectively at day 9 of estrus cycle. The ovaries were preserved into liquefacient nitrogen quickly and made cryostat section to observe the histological characteristics of atretic follicle by TUNEL detecting and haematoxylin staining method. The results showed that very few oocytes with atresia were found in preantral follicles in the HP and PP goats. Apoptosis displayed more in a number of wall granulose cell near follicle antrum characterized appearance of fluorescence response. Few and individual apoptosis cell was found in granulose cell in some follicles, but no difference between HP and PP does. Follicles that were closed together with only a membrane separated, could have different physiological state at same time, one atresia another nonatresia in HP goats. It was concluded that the method of TUNEL assay and haematoxylin staining could provide a correct way for precisely detecting and localizing apoptotic cells in ovarian section. Apoptosis displayed more in a quantity of wall granulose cell near follicle antrum in atretic antrum follicles. There was a strictly physiological barrier between two adjacent follicles in HP goats.6. Construction and Selection of Subtracted cDNA Library of Granulosa Cell in Small and Large Follicles in Early Days of Heat in Huanghuai Goats of High ProlificacyTwo HP goats were used to collect both ovaries individually 40 hours after synchronization achieved using a synthetic analog of prostaglandin F2 alpha. Counting and measuring follicles and collecting granulose cells by blunt dissection and preserving a fraction of follicle used into liquid nitrogen were within 20 min. Total RNA was extracted from granulose cells of large (5-6 mm) and small (3-4mm) follicles that were not found atresia, and were synthesized and amplified, then a forward- and reverse-subtractive cDNA library were constructed by suppression subtractive hybridization. A total of 190 clones randomly selected from the library were proved to be differentially expressed in the granulose cells, and 110 clones were selected out to be sequenced in one way based on the dot blot analysis. Among of these,109 EST obtained, and 10 exhibited high homology to be known genes,16 exhibited high homology to be found but its function not known genes,65 identified as new EST by Blastn checking after clustering. These different genes could conduce to reveal the regulation mechanism of follicles development in goats, meanwhile provide some reference for identifying the candidate gene of HP goats.更多还原