Abstract：This study was conducted to investigate factors influencing intracytoplasmic sperm injection(ICSI) of x-sorted frozen-thawed sperm in dairy cow so as to establish a preliminary procedure for ICSI in dairy cow. In experiment 1, two different sperm states (motile sperm and immotile sperm) were used in ICSI, the higher cleavage and blastocyst rate of ICSI zygotes were obtained from the motile sperm (86.4% and 28.1%) than that those from the immotile sperm (82.3% and 25.7%), but there was no significant different (P>0.05). In experiment 2, three different concentrations (5%, 8% and 10%) of pdyvinyl pyrrolidone(PVP) in the sperm injection vehicle solution were compared. There was no effect of the three PVP concentrations on cleavage rate after sperm injection, but regular cleavage rate and blastocyst rate were descended with increasing concentration of PVP, regular cleavage rate (78.4% and 69.1%) and blastocyst rate (29.7% and 20.4%) were significantly higher with 5% than 10% PVP(P< 0.05). In experiment 3, two positions (6 and 12 o'clock) of the first polar body (PB) located in oocytes at ICSI were examined. There was no significant difference in the cleavage rate (P>0.05) between the two positions of the PB, but regular cleavage rate (76.4% and 66.7%) and blastocyst rate(28.6% and 19.0%) were significantly improved at 6 o'clock of the polar body(P<0.05). The result indicated that the motile sperm be used in ICSI and the sperm was treated in the manipulation medium with 5% PVP, and the oocytes should be injected with the polar body at 6 o'clock, can improve the in vitro development of the ICSI zygotes in dairy cow.
