动物腔前卵泡分离培养的研究
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摘要
1．探讨了水牛和小鼠的各级腔前卵泡在卵巢中的分布规律、形态特征及超微结构特点。(1)水牛和小鼠卵巢中各级卵泡的分布具有明显的区域性，原始卵泡和初级卵泡分别位于血管稀少的皮质最外层和中层，而次级卵泡位于富含血管的皮质最内层。(2)水牛成年牛的原始卵泡的直径明显大于胎牛和青年牛(p＜0.05)，而三者的卵母细胞直径和颗粒细胞数没有差别；成年牛的初级卵泡的直径和卵母细胞直径显著大于胎牛和青年牛，颗粒细胞数也显著较多(p＜0.05)。胎牛的次级卵泡的直径和卵母细胞直径显著小于成年牛和青年牛，颗粒细胞数也显著较少(p＜0.05)，次级卵泡的颗粒细胞在2—4层时分布不均，一侧较多，而另一侧较少。(3)7d、14d和28d小鼠的原始卵泡的直径、卵母细胞直径和颗粒细胞数均没有差异，赤道面颗粒细胞数约为10个。7d小鼠初级卵泡和次级卵泡直径和卵母细胞直径显著小于14d和28d小鼠，颗粒细胞也少(p＜0.05)。(4)水牛和小鼠腔前卵泡的基膜由两层薄而光滑的纤维状结构组成，线粒体中有帽状结构，卵母细胞的细胞器集中于细胞核附近。
2．探讨了不同分离方法对水牛腔前卵泡分离效果的影响。梳刮法平均每个水牛卵巢回收所得的腔前卵泡数(152.35±44.81)明显高于剪碎法(32.62±14.81)和显微分离法(8.95±3.44，p＜0.05)，且其平均每个卵巢的处理时间(39.05±4.27min)明显少于剪碎法(46.43±4.15min)和显微分离法(44.55±7.82min，p＜0.05)。梳刮法分离所得的腔前卵泡，其中原始卵泡最多(占41.25％)，其次为初级卵泡(占38.79％)，而次级卵泡最少(仅占19.95％)。结果表明，梳刮法能有效地分离回收水牛卵巢的腔前卵泡。
3．探讨了水牛胎牛、青年牛和成年牛卵巢腔前卵泡的分离效果。每个胎牛、青年牛和成年牛的卵巢分别获得943.43±143.13、249.50±40.11和128.50±19.99个腔前卵泡，胎牛卵巢获得的腔前卵泡数量明显多于青年牛和成年牛(p＜0.05)，表明水牛胎牛卵巢可分离得到较多的腔前卵泡。
4．探讨了剪碎结合酶消化法的水牛卵巢腔前卵泡分离效果。水牛卵巢先用眼科手术剪刀将其剪碎，然后分别用浓度为0(对照)、0.02％、0.04％、0.08％的胶原酶消化20min回收腔前卵泡，每个卵巢分别获得33.56±6.15、40.38±6.12、47.75±6.84和52.69±6.15个腔前卵泡，酶消化组的腔前卵泡数量明显多于单纯剪碎组(p＜0.05)，表
中文摘要
明酶消化有助于提高剪碎法的腔前卵泡分离效率。
5.探讨了不同分离方法对小鼠卵巢腔前卵泡的分离效果的影响。梳刮法平均每个
小鼠卵巢回收所得的腔前卵泡数明显高于剪碎法和显微分离法(61.00士9.12vs
26.30士506和14.1肚4.61，p<0.05)，且其每个卵巢的平均处理时间明显少于剪碎法和
显微分离法(30.80士3.glmin vs 39.10士4.olmin和38.40士3.50min，p<0.05)，梳刮法分
离所得的小鼠腔前卵泡，次级卵泡最多(占83.61%)，其次为初级卵泡(占12.13%)，
而原始卵泡最少(仅占4.26%)，表明梳刮法也能有效地分离回收小鼠卵巢的腔前卵泡。
6.探讨了分离方法和水牛年龄对水牛腔前卵泡的体外发育能力的影响。用梳刮
法、剪碎法和显微分离法获得的水牛卵巢腔前卵泡体外培养48h和72h，其存活率差
异不显著(p>0.05)。体外培养72h，胎牛腔前卵泡存活率明显低于青年牛和成年牛
(34.69%vs 52.67%、52.03%，P<0.05)。剪碎结合酶消化法分离得到的水牛卵巢腔前
卵泡在体外培养72h后的存活率随着胶原酶浓度的增加而逐渐下降，浓度达到0.08%
时下降达到显著性水平。结果表明，分离操作方法对水牛腔前卵泡的培养存活率没有
影响，但酶消化处理则降低腔前卵泡分离后的培养存活率;水牛胎牛分离得到的腔前
卵泡比青年牛和成年牛分离得到的腔前卵泡更难培养。
7.探讨了卵泡培养密度对水牛腔前卵泡的体外发育能力的影响。培养到IOd，单
独培养(每孔放1个卵泡)的卵泡存活率(30.36%)与直径增长(7 .71士2.29林m)显
著低于群体培养A(每孔放2一3个卵泡，51.61%、10.19士3.24林m)和群体培养B(每
孔放4一5个卵泡，52.24%、10.94士3.80林m，p<0.05)，表明水牛腔前卵泡的群体培养
效果优于单独培养效果。
8.探讨了卵泡大小对水牛卵巢腔前卵泡的体外发育能力的影响。体外培养10d，
起始直径为101一120和扒21林m的卵泡的存活率显著高于三60、61一80林m和
81一10如m的卵泡的存活率，起始直径扒21林m的卵泡的直径增长显著高于且20林m的
卵泡的直径增长(p<0.05)，表明直径较大水牛腔前卵泡的体外培养效果优于直径较小
的腔前卵泡。
9.确定了水牛腔前卵泡体外培养胎牛血清(F Cs)的最佳浓度。体外培养10d，
10%FCS组的卵泡存活率显著高于对照组和1%FCS组(p< 0.05)，卵泡的直径增长了
14.36士4.90林m，亦显著高于对照组和1%FCS组、5%FCS组和20%FcS组的直径增长
(P<0.05)，表明水牛腔前卵泡体外培养的适宜FsH浓度是10%一巧%。
10.比较了不同培养方法的水牛腔前卵泡体外培养效果。培养10d，三维培养法
的卵泡存活率显著高于微孔板培养法和二维培养法(p<0.05)，且卵泡直径平均增长了
13.03士5.37林m，亦显著高于微孔板培养法和二维培?
1. The distribution, histological and ultrastructural characterization of buffalo and mouse preantral follicles in ovaries was studied. Primordial follicles, which had one layer of squamous granulosa cells, were always located in the outer part of ovary cortex. Primary follicles, which had a single layer of cubical granulosa cells, were found in the middle cortex. Secondary follicles with two or more layers of cubical granulosa cells in symmetric distribution were located only within the inner region of the cortex with many large blood vessels. The diameter of primordial, primary and secondary follicles and their oocyte diameter in adult buffalo ovaries was larger than those in heifer and fetal ovaries. Adult buffalo primordial, primary and secondary follicles also had more ganulosa cells in comparison with heifer and fetal follicles. The diameter of primordial follicles and oocytes and granulosa cell number from 7d, 14d and 28d of mice did not differ significantly, but the diameter of primary and seconda
ry follicles and their oocytes from 7d mice was shorter than that of 14d and 28d mice, granulosa cell number of primary and secondary follicles from 7d mice was also less than that of 14d and 28d mice (p<0.05). The basement membrane of buffalo and mice follicles was made of two layers of thin and smooth fiber-like material. Mitochondria had cap ultrastructure. Organelle of oocyte distributed near the nucleus.
2. Methods for isolation of buffalo and mouse preantral follicles were investigated. Comb-scrape method harvested significantly more preantral follicles (152.35+44.81/ovary) than cutting (32.62+ 14.81/ovary) and micromanipulation method (8.95+3.44/ovary, p<0.05). The average manipulative time for each ovary (39.05+4.27min) was also less than cutting (46.43+4.15min) and micromanipulation method (44.55+7.82min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were primordial follicles (41.25%) and primary follicles (38.79%), and very few of secondary follicles (19.95%). These results indicated that comb-scrape method was suitable for isolating buffalo preantral follicles.
3. Fetal ovaries harvested significantly more preantral follicles (943.43+143.13/ovary) than heifer (249.50+40.11 /ovary) and adult ovaries (128.50+19.99/ovary) (p<0.01) , indicating that more preantral follicles can be isolated from buffalo fetal ovaries.
4. Digestion of small ovary tissue pieces with 0.02% 0.04% and 0.08% collagenase harvested significantly more preantral follicles in comparison with without digestion (40.38+6.12 47.75+6.84 and 52.69+6.15 vs 33.56+6.15 per ovary, p<0.05), indicating that combination of cutting and enzymatic method can harvest more buffalo preantral follicles than cutting method alone.
5. Comb-scrape method harvested significantly more mouse preantral follicles
(61.00+9.12/ovary) than cutting (26.30+5.06/ovary) and micromanipulation method (14.10+4.16/ovary, p<0.05). The average manipulative time for each ovary (30.80+3.91min) was also less than cutting (39.10+4.01min) and micromanipulation method (38.40+3.50min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were secondary follicles (83.61%) and primary follicles (12.13%), and very few of primordial follicles (4.26%). These results indicate that comb-scrape method is also suitable for isolating mouse preantral follicles.
6. Effects of isolation methods and buffalo ages on the growth of preantral follicles in vitro culture were studied. The survival rate of preantral follicles isolated using the comb-scrape method at 48 and 72h after in vitro culture did not differ significantly from that of cutting and micromanipulation methods (p>0.05). The survival rate of buffalo fetal preantral follicles was significantly lower than that of heifer and adult preantral follicles (34.69% vs 52.67% and 52.03%, p<0.05) after in vitro culture of 72h. The survival rate of buffalo preantral follicles isolated by 0.08% collagenase treatment was significantly lower than the follicles insolated by 0, 0.02% and
2．探讨了不同分离方法对水牛腔前卵泡分离效果的影响。梳刮法平均每个水牛卵巢回收所得的腔前卵泡数(152.35±44.81)明显高于剪碎法(32.62±14.81)和显微分离法(8.95±3.44，p＜0.05)，且其平均每个卵巢的处理时间(39.05±4.27min)明显少于剪碎法(46.43±4.15min)和显微分离法(44.55±7.82min，p＜0.05)。梳刮法分离所得的腔前卵泡，其中原始卵泡最多(占41.25％)，其次为初级卵泡(占38.79％)，而次级卵泡最少(仅占19.95％)。结果表明，梳刮法能有效地分离回收水牛卵巢的腔前卵泡。
3．探讨了水牛胎牛、青年牛和成年牛卵巢腔前卵泡的分离效果。每个胎牛、青年牛和成年牛的卵巢分别获得943.43±143.13、249.50±40.11和128.50±19.99个腔前卵泡，胎牛卵巢获得的腔前卵泡数量明显多于青年牛和成年牛(p＜0.05)，表明水牛胎牛卵巢可分离得到较多的腔前卵泡。
4．探讨了剪碎结合酶消化法的水牛卵巢腔前卵泡分离效果。水牛卵巢先用眼科手术剪刀将其剪碎，然后分别用浓度为0(对照)、0.02％、0.04％、0.08％的胶原酶消化20min回收腔前卵泡，每个卵巢分别获得33.56±6.15、40.38±6.12、47.75±6.84和52.69±6.15个腔前卵泡，酶消化组的腔前卵泡数量明显多于单纯剪碎组(p＜0.05)，表
中文摘要
明酶消化有助于提高剪碎法的腔前卵泡分离效率。
5.探讨了不同分离方法对小鼠卵巢腔前卵泡的分离效果的影响。梳刮法平均每个
小鼠卵巢回收所得的腔前卵泡数明显高于剪碎法和显微分离法(61.00士9.12vs
26.30士506和14.1肚4.61，p<0.05)，且其每个卵巢的平均处理时间明显少于剪碎法和
显微分离法(30.80士3.glmin vs 39.10士4.olmin和38.40士3.50min，p<0.05)，梳刮法分
离所得的小鼠腔前卵泡，次级卵泡最多(占83.61%)，其次为初级卵泡(占12.13%)，
而原始卵泡最少(仅占4.26%)，表明梳刮法也能有效地分离回收小鼠卵巢的腔前卵泡。
6.探讨了分离方法和水牛年龄对水牛腔前卵泡的体外发育能力的影响。用梳刮
法、剪碎法和显微分离法获得的水牛卵巢腔前卵泡体外培养48h和72h，其存活率差
异不显著(p>0.05)。体外培养72h，胎牛腔前卵泡存活率明显低于青年牛和成年牛
(34.69%vs 52.67%、52.03%，P<0.05)。剪碎结合酶消化法分离得到的水牛卵巢腔前
卵泡在体外培养72h后的存活率随着胶原酶浓度的增加而逐渐下降，浓度达到0.08%
时下降达到显著性水平。结果表明，分离操作方法对水牛腔前卵泡的培养存活率没有
影响，但酶消化处理则降低腔前卵泡分离后的培养存活率;水牛胎牛分离得到的腔前
卵泡比青年牛和成年牛分离得到的腔前卵泡更难培养。
7.探讨了卵泡培养密度对水牛腔前卵泡的体外发育能力的影响。培养到IOd，单
独培养(每孔放1个卵泡)的卵泡存活率(30.36%)与直径增长(7 .71士2.29林m)显
著低于群体培养A(每孔放2一3个卵泡，51.61%、10.19士3.24林m)和群体培养B(每
孔放4一5个卵泡，52.24%、10.94士3.80林m，p<0.05)，表明水牛腔前卵泡的群体培养
效果优于单独培养效果。
8.探讨了卵泡大小对水牛卵巢腔前卵泡的体外发育能力的影响。体外培养10d，
起始直径为101一120和扒21林m的卵泡的存活率显著高于三60、61一80林m和
81一10如m的卵泡的存活率，起始直径扒21林m的卵泡的直径增长显著高于且20林m的
卵泡的直径增长(p<0.05)，表明直径较大水牛腔前卵泡的体外培养效果优于直径较小
的腔前卵泡。
9.确定了水牛腔前卵泡体外培养胎牛血清(F Cs)的最佳浓度。体外培养10d，
10%FCS组的卵泡存活率显著高于对照组和1%FCS组(p< 0.05)，卵泡的直径增长了
14.36士4.90林m，亦显著高于对照组和1%FCS组、5%FCS组和20%FcS组的直径增长
(P<0.05)，表明水牛腔前卵泡体外培养的适宜FsH浓度是10%一巧%。
10.比较了不同培养方法的水牛腔前卵泡体外培养效果。培养10d，三维培养法
的卵泡存活率显著高于微孔板培养法和二维培养法(p<0.05)，且卵泡直径平均增长了
13.03士5.37林m，亦显著高于微孔板培养法和二维培?
1. The distribution, histological and ultrastructural characterization of buffalo and mouse preantral follicles in ovaries was studied. Primordial follicles, which had one layer of squamous granulosa cells, were always located in the outer part of ovary cortex. Primary follicles, which had a single layer of cubical granulosa cells, were found in the middle cortex. Secondary follicles with two or more layers of cubical granulosa cells in symmetric distribution were located only within the inner region of the cortex with many large blood vessels. The diameter of primordial, primary and secondary follicles and their oocyte diameter in adult buffalo ovaries was larger than those in heifer and fetal ovaries. Adult buffalo primordial, primary and secondary follicles also had more ganulosa cells in comparison with heifer and fetal follicles. The diameter of primordial follicles and oocytes and granulosa cell number from 7d, 14d and 28d of mice did not differ significantly, but the diameter of primary and seconda
ry follicles and their oocytes from 7d mice was shorter than that of 14d and 28d mice, granulosa cell number of primary and secondary follicles from 7d mice was also less than that of 14d and 28d mice (p<0.05). The basement membrane of buffalo and mice follicles was made of two layers of thin and smooth fiber-like material. Mitochondria had cap ultrastructure. Organelle of oocyte distributed near the nucleus.
2. Methods for isolation of buffalo and mouse preantral follicles were investigated. Comb-scrape method harvested significantly more preantral follicles (152.35+44.81/ovary) than cutting (32.62+ 14.81/ovary) and micromanipulation method (8.95+3.44/ovary, p<0.05). The average manipulative time for each ovary (39.05+4.27min) was also less than cutting (46.43+4.15min) and micromanipulation method (44.55+7.82min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were primordial follicles (41.25%) and primary follicles (38.79%), and very few of secondary follicles (19.95%). These results indicated that comb-scrape method was suitable for isolating buffalo preantral follicles.
3. Fetal ovaries harvested significantly more preantral follicles (943.43+143.13/ovary) than heifer (249.50+40.11 /ovary) and adult ovaries (128.50+19.99/ovary) (p<0.01) , indicating that more preantral follicles can be isolated from buffalo fetal ovaries.
4. Digestion of small ovary tissue pieces with 0.02% 0.04% and 0.08% collagenase harvested significantly more preantral follicles in comparison with without digestion (40.38+6.12 47.75+6.84 and 52.69+6.15 vs 33.56+6.15 per ovary, p<0.05), indicating that combination of cutting and enzymatic method can harvest more buffalo preantral follicles than cutting method alone.
5. Comb-scrape method harvested significantly more mouse preantral follicles
(61.00+9.12/ovary) than cutting (26.30+5.06/ovary) and micromanipulation method (14.10+4.16/ovary, p<0.05). The average manipulative time for each ovary (30.80+3.91min) was also less than cutting (39.10+4.01min) and micromanipulation method (38.40+3.50min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were secondary follicles (83.61%) and primary follicles (12.13%), and very few of primordial follicles (4.26%). These results indicate that comb-scrape method is also suitable for isolating mouse preantral follicles.
6. Effects of isolation methods and buffalo ages on the growth of preantral follicles in vitro culture were studied. The survival rate of preantral follicles isolated using the comb-scrape method at 48 and 72h after in vitro culture did not differ significantly from that of cutting and micromanipulation methods (p>0.05). The survival rate of buffalo fetal preantral follicles was significantly lower than that of heifer and adult preantral follicles (34.69% vs 52.67% and 52.03%, p<0.05) after in vitro culture of 72h. The survival rate of buffalo preantral follicles isolated by 0.08% collagenase treatment was significantly lower than the follicles insolated by 0, 0.02% and
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