Preliminary Studies On Intracytoplasmic Sperm Injection (ICSI) With Flowcytometrically Sorted Buffalo Sperm

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 increasethe efficiency of ICSI.