绵羊卵母细胞体外成熟与体外受精的初步研究
摘要
本研究对绵羊卵母细胞体外成熟及其体外受精进行了初步探讨。切剖法平均每个卵巢可采集得到可用卵母细胞数3.95枚(1953枚／494卵巢)，显著高于抽吸法的1.96枚(219枚112卵巢)。卵母细胞体外成熟后，在含5％、10％和20％发情羊血清的受精液中进行体外受精，其分裂率分别是73.79％、73.25％和77.61％，显著高于不含发情羊血清的受精液(0％，P＜0.01)。用含5％发情羊血清的受精液为基础液，添加5IU／ml和10IU／ml的肝素，受精后的分裂率分别为86.63％和75.53％，5IU／ml肝素组的分裂率与不加肝素组(86.64％)差异不显著，而10IU／ml高浓度肝素组的分裂率则显著低于其他两组(P＜0.01)。用含5％发情羊血清的受精液为基础液，以添加3mg／ml BSA为试验组，不加BSA为对照组，受精后两组分裂率差异不显著(73.79％vs70.83％，P＞0.05)。
以上结果表明：(1)切剖法采集屠宰绵羊卵巢卵母细胞回收效率明显高于抽吸法；(2)非繁殖季节诱导发情的发情绵羊血清对绵羊卵母细胞的体外受精具有促进作用；(3)当受精液含有发情绵羊血清时，肝素和BSA对绵羊卵母细胞的体外受精效果无明显影响，且过高浓度的肝素(10IU／ml)会降低其体外受精效果。
The present study was conducted to investigate factors affecting the in vitro fertilization of sheep oocytes matured in vitro so as to establish a preliminary IVF procedure for sheep. Follicle cutting harvested significantly more usable cumulus-oocytes complexes (COCs) (3.95/ovary) in comparison with follicle aspiration (1.96/ovary, P<0.05) from surface-visible follicles of slaughtered ovine ovaries. After maturation in vitro for 23-25 h, oocytes were fertilized with frozen-thawed ovine sperm in the SOF supplemented with or without estrous sheep serum (ESS). Addition of ESS (5%, 10% and 20%) to the SOF resulted in a significant increase in the cleavage rate of oocytes (73.79%, 73.25% and 77.61%) compared with the oocytes fertilized in SOF without ESS (0%, P<0.01), although there was no significant difference in the cleavage rate among the three ESS groups (P>0.05). Supplementing the fertilization medium containing 5% ESS with 5IU/ml heparin did not affect the cleavage rate of oocytes (86.63% vs. 86.64%), but the cleavage rate decreased significantly when the concentration of heparin was increased to 10IU/ml (75.53%, P<0.05). Moreover, addition of 3mg/ml bovine serum albumen (BSA) to the fertilization medium containing 5% ESS also did not affect the cleavage rate of oocytes at 48h after IVF (70.83% vs. 73.79%, P>0.05).
In conclusions, (1) Follicle cutting is more effective than follicle aspiration in recovering COCs from ovine ovaries; (2) ESS from estrus ewes induced in anoestrus season can improve IVF efficiency of sheep oocytes matured in vitro; (3) Heparin and BSA are unnecessary for the IVF of ovine oocytes when fertilization medium contains ESS, and high concentration of heparin (10IU/ml) may play a negative role in the process of sheep IVF.
以上结果表明：(1)切剖法采集屠宰绵羊卵巢卵母细胞回收效率明显高于抽吸法；(2)非繁殖季节诱导发情的发情绵羊血清对绵羊卵母细胞的体外受精具有促进作用；(3)当受精液含有发情绵羊血清时，肝素和BSA对绵羊卵母细胞的体外受精效果无明显影响，且过高浓度的肝素(10IU／ml)会降低其体外受精效果。
The present study was conducted to investigate factors affecting the in vitro fertilization of sheep oocytes matured in vitro so as to establish a preliminary IVF procedure for sheep. Follicle cutting harvested significantly more usable cumulus-oocytes complexes (COCs) (3.95/ovary) in comparison with follicle aspiration (1.96/ovary, P<0.05) from surface-visible follicles of slaughtered ovine ovaries. After maturation in vitro for 23-25 h, oocytes were fertilized with frozen-thawed ovine sperm in the SOF supplemented with or without estrous sheep serum (ESS). Addition of ESS (5%, 10% and 20%) to the SOF resulted in a significant increase in the cleavage rate of oocytes (73.79%, 73.25% and 77.61%) compared with the oocytes fertilized in SOF without ESS (0%, P<0.01), although there was no significant difference in the cleavage rate among the three ESS groups (P>0.05). Supplementing the fertilization medium containing 5% ESS with 5IU/ml heparin did not affect the cleavage rate of oocytes (86.63% vs. 86.64%), but the cleavage rate decreased significantly when the concentration of heparin was increased to 10IU/ml (75.53%, P<0.05). Moreover, addition of 3mg/ml bovine serum albumen (BSA) to the fertilization medium containing 5% ESS also did not affect the cleavage rate of oocytes at 48h after IVF (70.83% vs. 73.79%, P>0.05).
In conclusions, (1) Follicle cutting is more effective than follicle aspiration in recovering COCs from ovine ovaries; (2) ESS from estrus ewes induced in anoestrus season can improve IVF efficiency of sheep oocytes matured in vitro; (3) Heparin and BSA are unnecessary for the IVF of ovine oocytes when fertilization medium contains ESS, and high concentration of heparin (10IU/ml) may play a negative role in the process of sheep IVF.
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