分离方法及卵巢状况对猪次级卵泡采集数量的影响

Effects of isolation methods and status of ovary on the collection of porcine secondary follicles

【目的】探讨影响猪腔前卵泡分离的因素,建立有效分离猪腔前卵泡的方法。【方法】以猪次级卵泡为对象,比较4种分离方法和11种卵巢状况对猪次级卵泡采集数量的影响。【结果】针刮法分离猪次级卵泡的数量（（14.37±2.29）枚）极显著高于剪碎法（（5.62±2.67）枚）、显微分离法（（8.28±2.53）枚）和酶消化-机械分离法（（8.86±1.91）枚）,而且针刮法的处理时间（（15.29±1.82）min）较其他3种方法短（（20.51±1.99）min（、59.24±4.59）min（、40.59±2.67）min）,分离得到的次级卵泡体外培养3 d后,存活率在针刮法（53.29%）、剪碎法（51.08%）、显微分离法（49.87%）之间无明显差异,但均极显著高于酶消化-机械分离法（28.31%）。从不同质量卵巢中采集的次级卵泡数量有较大差异,质量为〉3.0～≤8.0 g（（15.36±2.32）枚）的卵巢得到的次级卵泡数量较≤3.0g（（6.31±2.19）枚）和〉8.0 g（（7.83±2.71）枚）多。卵巢有无黄体与次级卵泡的采集数量无明显关系;卵巢上不同大小的可见卵泡数量和分布与腔前卵泡的采集数量相关,卵巢上可见卵泡均衡分布,中、小卵泡较多而无大卵泡,无论黄体存在与否,均可获得较多次级卵泡。【结论】针刮法是分离猪次级卵泡的一种有效方法;在分离卵泡时,要选择质量在3.0～8.0 g,且有可见中、小卵泡均衡分布而无大卵泡的卵巢。

【Objective】 The objective of this study was to define the factors influencing isolation and to establish effective isolation methods of porcine preantral follicles.【Method】 By using secondary follicle as an object,the effects of four isolation methods and eleven status of ovary on the collection of porcine secondary follicles were compared.【Result】 The number of porcine secondary follicle isolated by needle scraping method（14.37±2.29） harvested significantly more than cutting（5.62±2.67）,microdissection（8.28±2.53） and mechanical-enzymatic combination methods（8.86±1.91）.The average handling time of needle scraping method for each ovary was less than that of other three methods（（15.29±1.82） min VS（20.51±1.99） min,（59.24±4.59） min,（40.59±2.67） min）.The suruival rate of secondary follicles by needle scraping（53.29%）,cutting（51.08%） and microdissection（49.87%） isolation methods after three days in vitro culture was not significantly different,but the livability of secondary follicles by three isolation methods was highly significant more than that of mechanical-enzymatic combination method（28.31%）.The number of secondary follicles collected from the different weight ovaries was remarkablly different,but ovaries of 3.0～≤8.0 g（15.36±2.32）weight ranges were more than ≤3.0 g（6.31±2.19） and 8.0 g（7.83±2.71）.The presence or absence of corpus luteum did not affect the number of isolated secondary follicles.The distribution of antral follicles on the surface of ovary was related to the number of secondary follicles isolated.The ovaries with balanced distribution of superficial small and middle antral follicles,but with no big follicles,regardless of the presence or absence of corpus luteum,could give rise to a large number of the secondary follicles.【Conclusion】 The needle scraping method can be used effectively to isolate intact secondary follicles from porcine ovaries.Ovaries with the weight 3.0-8.0 g and balanced distribution of superficial small and middle antral follicles without big follicles should be collected.