| Intracytoplasmic sperm injection (ICSI) is a very important assistant reproductive technology for treatment of man infertility and fundamental research. Success in ICSI in human and other animals has recently been achieved, however, there is no any information available on ICSI in buffalo. The present pilot studies were conducted to investigate factors influencing ICSI in buffalo so as to establish a preliminary procedure for ICSI in buffalo.Buffalo oocytes were collected from slaughterhouse ovaries and matured in vitro for 22-24h. Buffalo frozen-thawed sperm was injected into an oocyte by assistance of a micromanipulation system. Development of injected oocyte to pronuclear, cleavage and blastocyst stages was examined at 18h, 48h and 7d following ICSI, respectively, depending on different treatment designs. Five experiments were conducted in these studies to investigate factors affecting ICSI in buffalo. In experiment 1, two concentrations (10% and 5%) of polyvinylpyrrolidone (PVP) in the sperm injection vehicle solution were compared. There was no effect of the two PVP concentrations on either survival rate of oocytes after sperm injection (100% vs. 97.8% for 10% and 5%, respectively, P>0.05) or blastocyst rate (23.5% vs. 24.2%, for 10% and 5%, respectively, P>0.05); however, cleavage rate was significantly higher with 5% than that with 10% (88.6% vs. 79.4%, P<0.05). In experiment 2, two positions (6 and 12 o'clock) of the first polar body (PB) located in oocyte at ICSI were examined. There was no significant difference in ihe cleavage rate (89.5% vs. 86.2% for 6 and 12 o'clock, respectively, P>0.05) between the two positions of the PB, but significantly higher blastocyst rate was found in oocytes injected when the PB was located at 6 than at 12 o'clock (43.4% vs. 27.5%, P<0.05). In experiment 3, in order to polarize the ooplasm lipids, treatment of oocytes with or without centrifugation before ICSI was studied. There was no significant difference in either cleavage or blastocyst rates (94.3% vs. 91.3% and 23.4% vs. 27.5%; P>0.05 for with and without centrifugation, respectively). In experiment 4, oocytes after ICSI were treated with different chemicals for activation as followings. 1) Oocytes injected without sperm and without activation; 2) Oocytes injected with sperm but without activation; 3) Oocytes injected with sperm and activated with 7% ethanol for 5 min; 4) Oocytes injected with sperm and activated with 5[iM ionomysin for 5 min: 5) Oocytes injected without sperm and activated with 5^iM ionomysin for 5 min followed by 1.9mM 6-DMAP for 3 hours; 6) Oocytes injected with sperm and activated with the same treatment as group 5. Significantly higher cleavage and blastocyst rates were obtained from group 6 than that from groups 4, 3 and 2 (85.8% vs. 49.0% vs. 46.9% vs. 6.4% and 30.3% vs. 3.1% vs. 3.1% vs. 0%, P<0.05-0.001, for groups 6, 4, 3 and 2, respectively). Interestingly, a blastocyst rate of 16.7% was also observed in group 5, it might be due to parthenogenesis. In Experiment 5, pretreatment of sperm with or without DTT before sperm injection was performed. Significantly higher rate of blastocyst were found in the DTT- treated group than without (42.8% vs.27.5%, P<0.05). However, no significant difference was found in two-pronuclei formation and cleavage rates (20.3% vs.11.2%, P>0.05 and 87.0% vs. 84.4%, P>0.05, respectively) between the two treatments.These results indicate that (1) properly reducing the PVP concentration to 5% in the sperm injection vehicle solution can improve the development capability of oocytes after ICSI. (2) Oocytes should be injected with the polar body at 6'oclock. (3) The opacity of oocytes does not affect the results of ICSI. (4)Activation is necessary for ICSI in buffalo, the optimal combination of chemicals for activation is lonomysin + 1.9mM 6-DMAP, however, activation may also cause parthenogenesis. (5) Efficiency of ICSI in buffalo can be improved by sperm pretreatment with DTT.
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