【摘要】 猪的卵母细胞对外界环境非常敏感,造成猪的胚胎学相关研究是家畜繁殖学研究领域中最困难的方面之一。猪卵脆弱的主要原因是其胞质中含有大量的脂肪滴,但目前其具体生物学功能还不清楚。众所周知,猪的繁殖表现和环境密切相关,例如,温度、湿度、光照等。本研究目的是描述季节的变化对体外成熟卵母细胞发育能力的影响。连续两年从当地屠宰场获取初情期前猪卵巢,抽取猪卵丘卵母细胞复合体,并在不含BSA的NCSU-23(添加10 ng/mL EGF,10 ng/mL leptin,0.57 mm o l/L cysteine,10 IU/mL PM SG和10 IU/mL hCG)中培养44 h,然后用透明质酸酶脱去卵丘。部分卵母细胞经过电击进行孤雌激活,部分卵母细胞用来进行体细胞核移植。对于猪体细胞核移植,使用长白猪胎儿成纤维细胞做为核供体,上述脱去卵丘的卵母细胞作为胞质供体。克隆胚和孤雌胚在添加4 m g/mL的BSA液滴中培养,第2天和第6天分别计算卵裂和囊胚率(D 0:融合或激活的当天)。其中每个实验至少重复3次,所有实验都经过SPSS(13.0)统计分析。比较了不同季节每对卵巢获得A级卵(外包3层以上致密的卵丘细胞并且卵胞质均匀的卵母细胞为A级卵)和B级卵(外包2~3层卵丘细胞并且胞质均匀的卵母细胞为B级卵)的数量以及卵母细胞核成熟的比例(实验1),统计分析了不同季节孤雌(实验2)和核移植(实验3)胚胎发育能力的差异。实验1中发现春季(3~5月)COC s/ovary pairs的比例最高[7.6±3.5(春)vs 6.4±3.3(夏),6.7±2.9(秋),6.7±3.4(冬),P<0.05]。然而,没有发现卵母细胞的MⅡ百分比有显著性差异[75.3%±3.3%(春),73.5%±2.3%(夏),73.0%±4.2%(秋),76.5%±6.7%(冬),P<0.05]。实验2中观察到夏季孤雌胚胎的囊胚率显著性降低[10.2%±2.7%(春)vs 27.2%±3.5%(夏),24.2%±3.2%(秋),22.3%±3.4%(冬),P<0.05]。实验3,猪体细胞克隆胚胎在春季囊胚率获得提高[12.2%±1.3%(春)vs 10.3±1.1%(秋),8.1±1.4%(冬),P<0.05]。以上结果表明,季节变化影响体外成熟卵母细胞的发育能力。更多还原

【Abstract】 Porcine embryology is one of the most demanding fields in reproductive research of domestic animals due to its environment-sensitive oocytes.Plenty of lipid droplets in cytoplasm of porcine oocytes are the main cause of fragility,but their specific biological functions are still unknown.It is well recognized that environmental influence,such as high/low temperature and humidity,are associated with the reproduction performance in pigs.The goal of this study is to describe a seasonal variation in the developmental ability of porcine oocytes matured in vitro.Prepubertal gilt ovaries were collected more than two years(from spring to winter every year) from a local shambles to collect cumulus-oocytes-complexes(COCs) to mature for 44h in BSA-free NCSU23 supplemented with 10 ng/ mL EGF,10 ng/mL leptin,0.57 mmol/L cysteine,10 IU/mL PMSG and 10 IU/mL hCG.After cumulus cells removal,and the others were subjected to parthenogenetic activation,and the others were used to conduct somatic cell nuclear transfer(SCNT).In porcine cloning,cell lines established from 35~40 d Landrace fetus were used as nuclear donors,and the oocytes matured in vitro as cytoplast recipients.Embryos were in vitro cultured in NCSU23 supplemented with 4 mg/mL BSA.Cleavage and blastocyst rates were assessed on D2 and D6(D0:the day fusion and activation conducted).All the experiments were replicated at least three times.All data were analyzed by using SPSS(13.0) with one-way ANOVA.In this study,we compared the number of COCs(Grade A&B,Oocytes surrounded by at least 3 layers of compacted cumulus cells and with uniform cytoplasm were defined Grade A. Oocytes surrounded by 2~3 layers of cumulus cells and with uniform cytoplasm were defined Grade B.) every ovary pair collected in different seasons(COCs/ovary pair) and meiotic maturation ability of in vitro-matured oocytes(experiment 1) and developmental competence of the parthenogenetic activated oocytes(experiment 2) as well as somatic cell nuclear transfer embryos(experiment 3) using oocytes collected from four different seasons.In experiment 1,we found that COCs/ovary pairs was significantly increased in spring(March to May) in comparison with the other seasons [7.6±3.5 vs 6.4±3.3,6.7±2.9,6.7±3.4 in summer(June to August),autumn(September to November),winter(December to Feberary),respectively,P<0.05].However,no significant differences were found among the rates of oocytes progressing to MⅡ in four seasons (75.3%±3.3%,73.5%±2.3%,73.0%±4.2%,76.5%±6.7% in spring,summer,autumn,winter, respectively).In experiment 2,we saw that the rate of blastocyst formation of parthenogenetic embryos in summer prominently decreased compared to the other seasons(10.2%±2.7% vs 27.2%±3.5%,24.2%±3.2%,22.3%±3.4% in spring,autumn,winter, respectively,P<0.05).In experiment 3,increased blastocyst rate of cloned porcine embryos were found in spring when compared to autumn and winter(12.2%±1.3% vs 10.3%±1.1%,8.1%±1.4%,P<0.05).The results suggest there is a seasonal influence on the developmental competence of porcine oocytes matured in vitro.更多还原