水牛分离精子胞质内显微受精的初步研究
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摘要
本研究主要探讨了经流式细胞分离仪分离的水牛精子对水牛卵母细胞ICSI后胚胎发育的影响。体外成熟22—24h的水牛卵母细胞注入水牛的分离精子,经激活处理后培养在颗粒细胞单层共培养系统中,48h时检查分裂率,6—9d记录囊胚发育情况。结果发现,水牛分离精子ICSI卵子用Ionomycin(5min)+CDM(3h)+6-DMAP(3h)联合激活较Ionomycin(5min)+6-DMAP(3h)效果好,前者的囊胚形成率显著高于后者(X-ICSI:35.4%vs.28.42%;Y-ICSI:36.27%vs.26.53,p<0.01);分离后的死精子经超声断尾处理后进行ICSI,获得了与不经超声断尾的活精子ICSI后相同的受精与胚胎发育效果;分离精子与未分离精子ICSI后的卵裂率(75.31%vs.80.13%)及囊胚发育率(34.57%vs.35.26%)二者之间差异不显著;分离精子ICSI后的卵裂率和囊胚发育率均高于IVF组(76.92%vs.48.64%;33.85%vs.18.91%,P<0.05)。用含10μg/ml肝素的受精液处理ICSI激活卵4-6h,比用不含肝素受精液处理的ICSI激活卵的分裂率及囊胚率高(73.42%vs.67.16%;30.38%vs.26.87%,P<0.05);将肝素浓度从10μg/ml提高到100μg/ml对其ICSI后胚胎的18h雄原核的形成率(32.91%vs.33.8%,P>0.05),分裂率(73.42%vs.74.65%,P>0.05),囊胚率(30.38%vs.32.4%,P>0.05)均无显著影响。随机挑选的3头公水牛的分离精子的受精率及胚胎发育率,在不同公牛之间都有所不同。结果表明:1)水牛分离精子ICSI后用Ionomycin(5min)+CDM(3h)+6-DMAP(3h)方法激活处理能提高其胚胎发育率;2)分离后的死精子经超声断尾也能用于水牛卵母细胞的ICSI;3)分离精子的ICSI效果不低于未分离精子,但分离精子的IVF效果却低于未分离精子;4)肝素能促进分离精子ICSI后的胚胎发育;5)分离精子ICSI后的胚胎发育率在不同公牛之间有差异,选择能产生高质量精子的公牛进行精子分离,可以提高ICSI的效率。
Factors affecting the fertilization of buffalo oocytes with flow cytometrically sorted buffalo sperm and embryo development of the oocytes after ICSI were investigated in this study. Buffalo oocytes matured in viro for 22-24 h were fertilized by injecting the sorted buffalo sperm, and then were activated by Ionomycin (5min) +CDM (3 h)+ 6-DMAP(3 h) compared with Ionomycin (5min) + 6-DMAP(3 h). The blastocyst rate of oocytes activated by Ionomycin (5min) +CDM (3h)+ 6-DMAP(3 h) was higher than those activated by Ionomycin (5min) + 6-DMAP(3 h) ( X-ICSI: 35.4%vs.28.42%; Y-ICSI: 36.27%vs.26.53, p<0.01). The same results of fertilization and development of embryos produced by ICSI with dead or live ultrasoniced sorted sperms were obtained; There was no significant difference either in cleavage (71.43%vs.71.79%) or blastocyst development rates between ICSI with sorted sperm and unsorted sperm(75.31 %vs.80.13%; 34.57%vs.35.26%, p>0.05); The rates of cleavage (76.92%vs.48.64%) and blastocyst (33.85%vs.18.91%) of embryos produced by ICSI with sorted sperm were higher(P<0.05 )than that of IVF embryos; The cleavage rate and blastocyst rate of embryos with 4-6 h heparin treatment were higher than that which without it, but there were no significant difference in the rates of male pronuclus formation (32.91%vs.33.8%), cleavage (73.42%vs.74.65%) and the blastocyst (30.38%vs.32.4%) of ICSI oocytes using different treatments of heparin concentration from 10 μg/ml to 100 μ g/ml. The fertilization rate and the embryo development rate with sorted sperms were variant among 3 random selected buffalos. In conclusions: 1) The treatment that buffalo occytes are cultured in Ionomycin (5 min) +CDM (3 h)+ 6-DMAP(3 h) after ICSI with sorted sperm can improve their subsequent embryonic development; 2) The dead ultrasoniced sorted sperm can be used for ICSI; 3) The result of ICSI with sorted sperm is similar to that with unsexed sperm, but the result of IVF with sorted sperm is lower than that with unsorted sperm; 4) The treatment of heparin after ICSI with sorted sperm can improve the blastocyst development rate; 5) The development rate of blastocyst with sorted sperm was variable among buffalos indicating that sorting high quality sperm from specific buffalos can increase
the efficiency of ICSI.
Factors affecting the fertilization of buffalo oocytes with flow cytometrically sorted buffalo sperm and embryo development of the oocytes after ICSI were investigated in this study. Buffalo oocytes matured in viro for 22-24 h were fertilized by injecting the sorted buffalo sperm, and then were activated by Ionomycin (5min) +CDM (3 h)+ 6-DMAP(3 h) compared with Ionomycin (5min) + 6-DMAP(3 h). The blastocyst rate of oocytes activated by Ionomycin (5min) +CDM (3h)+ 6-DMAP(3 h) was higher than those activated by Ionomycin (5min) + 6-DMAP(3 h) ( X-ICSI: 35.4%vs.28.42%; Y-ICSI: 36.27%vs.26.53, p<0.01). The same results of fertilization and development of embryos produced by ICSI with dead or live ultrasoniced sorted sperms were obtained; There was no significant difference either in cleavage (71.43%vs.71.79%) or blastocyst development rates between ICSI with sorted sperm and unsorted sperm(75.31 %vs.80.13%; 34.57%vs.35.26%, p>0.05); The rates of cleavage (76.92%vs.48.64%) and blastocyst (33.85%vs.18.91%) of embryos produced by ICSI with sorted sperm were higher(P<0.05 )than that of IVF embryos; The cleavage rate and blastocyst rate of embryos with 4-6 h heparin treatment were higher than that which without it, but there were no significant difference in the rates of male pronuclus formation (32.91%vs.33.8%), cleavage (73.42%vs.74.65%) and the blastocyst (30.38%vs.32.4%) of ICSI oocytes using different treatments of heparin concentration from 10 μg/ml to 100 μ g/ml. The fertilization rate and the embryo development rate with sorted sperms were variant among 3 random selected buffalos. In conclusions: 1) The treatment that buffalo occytes are cultured in Ionomycin (5 min) +CDM (3 h)+ 6-DMAP(3 h) after ICSI with sorted sperm can improve their subsequent embryonic development; 2) The dead ultrasoniced sorted sperm can be used for ICSI; 3) The result of ICSI with sorted sperm is similar to that with unsexed sperm, but the result of IVF with sorted sperm is lower than that with unsorted sperm; 4) The treatment of heparin after ICSI with sorted sperm can improve the blastocyst development rate; 5) The development rate of blastocyst with sorted sperm was variable among buffalos indicating that sorting high quality sperm from specific buffalos can increase
the efficiency of ICSI.
引文
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