水牛和黄牛同种内及种间显微受精的初步研究
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摘要
本研究探讨了水牛和黄牛显微受精的影响因素及其种间受精的可行性。体外成熟22-24小时的水牛和黄牛卵母细胞分别注入冷冻尼里-拉菲水牛和利木赞黄牛精子，然后培养16-18h固定染色检查原核形成情况，培养48h检查分裂率，继续培养7-9d记录囊胚发育情况。结果发现，水牛同种(水牛+水牛)和水牛异种(水牛+黄牛)显微受精的双原核率(47.73％ vs 36.67％)，分裂率(68％ vs72.63％)和囊胚发育率(16.44％ vs 22.39％)均无显著差异(p＞0.05)；黄牛同种(黄牛+黄牛)和黄牛异种(黄牛+水牛)显微受精的双原核率(60％ vs 45.28％)，分裂率(73.33％ vs 71.31％)和囊胚发育率(28.57％ vs 33.70％)亦无显著差异(p＞0.05)。当用添加有100μg／mL溶血卵磷脂的受精液预处理水牛精子时，水牛同种ICSI后的分裂率(80.41％ vs 87.25％)和囊胚发育率(6.7％ vs 10.78％)无明显影响(p＞0.05)。黄牛卵子和水牛精子异种受精的囊胚经常规冷冻保存，解冻后24h的存活率为63.89％(23／36)，培养48h后的囊胚孵化率为30.56％(11／36)，与未冷冻的囊胚孵化率(34.78％，16／46)无显著差异(p＞0.05)。
以上结果表明：①水牛精子注射到黄牛卵子和黄牛精子注射到水牛卵子的种间受精胚胎可以体外发育到囊胚阶段，且其获得的囊胚具有较好的抗冻性。②黄牛卵子无论同种和异种显微受精的效果均优于水牛卵子。③溶血卵磷脂预处理水牛精子对其ICSI效果没有明显影响。
Factors affecting the fertilization of buffalo and bovine oocytes by sperm injection were examined, and the possibility of intraspecific fertilization between buffalo and cattle was also explored in this study. Buffalo and bovine oocytes matured in vitro for 22-24h were fertilized by injection of buffalo or bovine frozen-thawed spermatozoa. At 16-18h after injection, a portion of oocytes were fixed in acetic ethanol to examine the pronuclear formation, others were cultured in vitro to evaluate their cleavage rate and embryonic developmental ability. There was not difference in the two pronuclear formation rate (47.73% vs 36.67%), cleavage rate (68% vs 72.63%) and blastocyst development (16.44% vs 22.39%) of buffalo oocytes fertilized with buffalo (intraspecies) and bovine sperm (interspecies). (p>0.05). The two pronuclear formation rate (60.00%), cleavage rate (73.33%) and blastocyst rate (28.57%) of bovine oocytes fertilized with bovine sperm (intraspecies) were also not different from those (45.28%, 71.31%
and 33.70% respectively, p>0.05) of bovine oocytes fertilized with buffalo sperm (interspecies). Treatment of buffalo sperm with 100ug/mL lysophosphatide did not affect the percentage of buffalo oocytes cleaved (80.41% vs 87.25%, p>0.05) and developed to blastocysts (6.7% vs 10.78%, p>0.05) after ICS1. Sixty-four percent of blastocysts derived from bovine oocytes fertilized with buffalo sperm survived and 30.6% of them hatched after freezing thawing, which was similar to the hatching rate (34.78%, p>0.05) of blastocysts unfrozen.
In conclusions, 1) buffalo oocytes fertilized with bovine sperm and bovine oocytes fertilized with buffalo sperm can develop to blastocysts, which are proved to be freezable; 2) the developmental capacity of bovine oocytes is higher than baffalo oocytes as evidenced by their proportion cleaved and developed to blastocysts after intraspecific and interspecific fertilization by ICSI; 3) treatment of buffao sperm with lysophosphatide does not affect its ICSI efficiency.
以上结果表明：①水牛精子注射到黄牛卵子和黄牛精子注射到水牛卵子的种间受精胚胎可以体外发育到囊胚阶段，且其获得的囊胚具有较好的抗冻性。②黄牛卵子无论同种和异种显微受精的效果均优于水牛卵子。③溶血卵磷脂预处理水牛精子对其ICSI效果没有明显影响。
Factors affecting the fertilization of buffalo and bovine oocytes by sperm injection were examined, and the possibility of intraspecific fertilization between buffalo and cattle was also explored in this study. Buffalo and bovine oocytes matured in vitro for 22-24h were fertilized by injection of buffalo or bovine frozen-thawed spermatozoa. At 16-18h after injection, a portion of oocytes were fixed in acetic ethanol to examine the pronuclear formation, others were cultured in vitro to evaluate their cleavage rate and embryonic developmental ability. There was not difference in the two pronuclear formation rate (47.73% vs 36.67%), cleavage rate (68% vs 72.63%) and blastocyst development (16.44% vs 22.39%) of buffalo oocytes fertilized with buffalo (intraspecies) and bovine sperm (interspecies). (p>0.05). The two pronuclear formation rate (60.00%), cleavage rate (73.33%) and blastocyst rate (28.57%) of bovine oocytes fertilized with bovine sperm (intraspecies) were also not different from those (45.28%, 71.31%
and 33.70% respectively, p>0.05) of bovine oocytes fertilized with buffalo sperm (interspecies). Treatment of buffalo sperm with 100ug/mL lysophosphatide did not affect the percentage of buffalo oocytes cleaved (80.41% vs 87.25%, p>0.05) and developed to blastocysts (6.7% vs 10.78%, p>0.05) after ICS1. Sixty-four percent of blastocysts derived from bovine oocytes fertilized with buffalo sperm survived and 30.6% of them hatched after freezing thawing, which was similar to the hatching rate (34.78%, p>0.05) of blastocysts unfrozen.
In conclusions, 1) buffalo oocytes fertilized with bovine sperm and bovine oocytes fertilized with buffalo sperm can develop to blastocysts, which are proved to be freezable; 2) the developmental capacity of bovine oocytes is higher than baffalo oocytes as evidenced by their proportion cleaved and developed to blastocysts after intraspecific and interspecific fertilization by ICSI; 3) treatment of buffao sperm with lysophosphatide does not affect its ICSI efficiency.
引文
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