Study On Intracytoplasmic Sperm Injection (ICSI) In Bovine

Intracytoplasmic sperm injection( ICSI )is a very important assistant reproductive technology for the treatment of man infertility and fundamental research. Success in ICSI of human and other animals has recently been archieved, hower, the efficiency of bovine ICSI is very low. To investigate the factors influencing the fertilization of bovine ICSI, the present studies were conducted.Bovine oocytes were collected from ovaries and matured in vitro for 22-26h. Bovine frozen-thawed sperm was injectied into an oocyte by the assistance of a micromanipulation system. Development of injected oocytes was examined and recorded respectively depending on different treatment designs. Six experiments were performed in these studies to investigate factors affecting ICSI in bovine. In experiment 1, two kinds of injection pipettes were compared, and it was found that the survival rate of oocytes after sperm injection, the cleavage rate and blastocyst rate were higher when the sperm was injected with 3-5μm injection pipette (84.80% vs 76.56%, 57.55% vs45.92%, 22.64% vs 14.29%) (p<0.05). In experiment 2, when heparin was added into fertilization medium or embryo culture medium respectively, both the cleavage rate and blastocyst rate were significantly improved (56.88% and 54.31% vs 35.79%, 22.94% and 18.97% vs 8.42%) (p<0.05). But when the 10μg/mL heparin was added in both fertilization medium and embryo culture medium, the cleavage rate and blastotocyst rate was not higher than the treatments when the heparin was added in any of them (57.14% vs 56.88% and 54.31%, 21.43% vs 22.94% and 18.97%) (p>0.05). In experiment 3, oocytes were matured for different time in vitro and then used in ICSI, and it was found that the cleavage rate and blastocyst rate were higher when oocytes were matured for 24h or 26h other than 22h (54.55% and 56.19% vs 42.06 %, 22.73% and 23.81% vs 12.15%) (p<0.05). So the oocytes used for ICSI need a longer in vitro maturation time than 1VF. In experiment 4, the live or dead sperm and sperm head were used for microinjection and the results showed that the cleavage rate and blastocyst rate was higher when oocytes were fertilized with the live sperm other than the sperm or sperm head (57.84% vs 42.71% and 41.28 %, 21.57% vs 12.5% and 9.52%) (p<0.05), but the oocytes survival rate after ICSI was higher when the sperm head was used for microinjection (57.84% vs42.71% and 41.28%,21.57% vs 12.5% and 9.52%) (p<0.05). In experiment 5, oocytes were centrifuged before microinjection and it was found centrifuge of oocytes before microinjection did not affect the survival rate, cleavage rate and blastocyst rate of the injected oocytes when compared with the control (85.16% vs 87.12%, 52.29% vs 54.78%, 21.10% vs 18.02%) (p>0.05). In experiment 6, sperm was pretreated with DTT before microinjection, and it was showed that DTT pretreatment of the sperm can improve both the cleavage rate and blastocyst rate of the injected oocytes (62.81% vs 54.05%, 28.10% vs 18.02%) (p<0.05) .