聚乙烯醇和卵泡液添加方式对猪卵母细胞体外成熟及孤雌胚胎发育的影响

Effect of Different Ways of PFF and PVA Add into Maturation Medium on Porcine Oocyte Maturation and Parthenogenetic Embryonic Development in vitro

本试验研究目的在于探讨在猪卵母细胞体外成熟(IVM)过程中,聚乙烯醇(polyvinyl alcohol,PVA)和猪卵泡液(porcine follicular fluid,PFF)添加方式对猪卵母细胞体外成熟及孤雌激活早期胚胎发育的影响。卵母细胞、卵丘细胞复合体(COCs)在含有HCG和PMSG的改良TCM-199+10%FBS+10%猪卵泡液(PMH-PFF)或改良TCM-199+10%FBS+0.1%PVA(PMH-PVA)成熟液中培养22~23 h;再移至无HCG和PMSG的PM-PFF或PM-PVA的成熟液中成熟培养至44 h。试验:(1)处理组1:使用PMH-PFF培养22~23 h后,更换PM-PFF继续培养至44 h;(2)处理组2:使用PMH-PFF培养22~23 h后,更换PM-PVA继续培养至44 h;(3)处理组3:使用PMH-PVA培养22~23 h后,更换PM-PVA继续培养至44 h;(4)处理组4:使用PMH-PVA培养22~23 h后,更换PM-PFF继续培养至44 h。将在不同成熟体系中IVM 44 h后的卵母细胞进行固定染色,鉴定卵母细胞核和细胞质成熟情况;对在不同处理组的成熟液中成熟培养44 h的卵母细胞进行孤雌激活后放入PZM-3中培养,分别于第2天、第7天统计分裂率和囊胚发育率。结果表明:经44 h成熟培养后,处理组1卵母细胞核成熟率(42.00%)显著低于处理组2(64.67%)、处理组3(69.00%)、处理组4(64.33%)核成熟率(p<0.05)。处理1、处理2、处理4的卵母细胞皮质颗粒分布类型Ⅲ比例(62.00%/54.67%/46.67%)均高于处理组3(28.67%),且处理组1、处理组2卵母细胞皮质颗粒分布类型Ⅲ比例显著高于处理组3卵母细胞皮质颗粒分布类型Ⅲ比例(p<0.05)。处理组1、处理组2、处理组4孤雌分裂率(81.92%/76.00%/73.33%)均高于处理组3(69.00%)。处理组3孤雌激活囊胚率(9.67%)显著低于处理组1(23.33%)、处理组2(25.00%)、处理组4(23.00%)(p<0.05)。结果表明,在本研究条件下,处理组2的各项指标均优于其它组,IVM液中添加猪卵泡液培养22~23 h后更换添加有PVA的IVM液继续培养至44 h,更有利于促进IVM猪卵母细胞核成熟、胞质成熟以及早期孤雌胚胎发育。

The objective of this study was to investigate the effect of supplementation method of PFF(porcine follicular fluid) and PVA(polyvinyl alcohol) in maturation medium on porcine oocytes maturation and further embryonic development after parthenogenesis. Porcine cumulus-oocyte complexes(COCs) were cultured in the medium of TCM-199+10% FBS+10% PFF(PMH-PFF) or TCM-199+10% FBS+0.1% PVA(PMH-PVA) with HCG and PMSG for 22~23 h; and then cultured in the medium of PM-PFF or PM-PVA without HCG and PMSG for 22 h.Experiments:(1) Group 1, the COCs were cultured in PMH-PFF for 22~23 h, and then cultured in PM-PFF for 22 h;(2) Group 2, the COCs were cultured in PMH-PFF for 22~23 h, and then cultured in PM-PVA for 22 h;(3) Group 3,the COCs were cultured in PMH-PVA for 22~23 h, and then cultured in PM-PVA for 22 h;(4) Group 4, the COCs were cultured in PMH-PVA for 22~23 h, and then cultured in PM-PFF for 22 h. After cultured 44 h in maturation medium, the oocytes were fixed and stained to evaluate the stages of meiotic and cytoplasmic maturation. The other matured oocytes were parthenogenetic activation, and then cultured in PZM-3 medium 7 days for development.The cleavage rate was examined on 2nd day and the blastocyst rate was examined on 7thday. The results showed that: after 44 h maturation, the nuclear maturation rates of group 1(42.00%) was significantly lower than that of group 2(64.67%), group 3(69.00%) and group 4(64.33%)(p<0.05). The ratio of oocytes with type Ⅲ CGs distribution of group 1, group 2 and group 4(62.00%, 54.67%, 46.67%) were higher than that of Group 3(28.67%), and the ratio of oocytes with type Ⅲ CGs distribution of group 3 had significantly lower than that of group 1 and group 2(p<0.05).The cleavage rate of group 1, group 2 and group 4(81.92%, 76.00%, 73.33%) were higher than that of experimental group 3(69.00%); and the blastocyst rate of group 3 had significantly lower than that of group 1, group 2 and group4(9.67% vs 23.33%, 25.00%, 23.00%, p<0.05). In summary, the evaluated parameters of group 2 was better than those of other groups. The COCs were cultured in PMH-PFF for 22~23 h, and then cultured in PM-PVA for 22 h, it can improve the porcine oocytes maturation and further embryonic development after parthenogenesis.