Improving Bovine Intracytoplasmic Sperm Injection

Despite large efforts, there has been minimal improvements in bovine intracytoplasmic sperm injection (ICSI) in the last few decades. In this study, we investigate methods to improve embryo development rates with ICSI in cattle through addressing sperm and oocyte factors and modifying injection methods. Inducing the acrosome reaction in bull sperm prior to injection with progesterone increased cleavage (46.2% vs 16.2%) and blastocyst (21% vs 3.8%) rates compared to controls, respectively. Supplementing glutathione during in vitro maturation of the oocyte did reduce free reactive oxygen species (ROS) in the oocyte, but did not improve embryo development rates with ICSI or in vitro fertilization (IVF). However, modifications of the injection protocol did result in increased embryo development from fresh and vitrified oocytes compared to conventional injection without the need for artificial activation. Modified vigorous injection in fresh oocytes resulted in 82.6% cleavage and 31.9% blastocyst development compared to conventional injection with 20.9% cleavage and 15.2% blastocyst rates. Similar results were seen in vitrified oocytes with vigorous injection producing 78.9% and 33.9% cleavage and blastocyst rates compared to conventional injection with 22.2% and 3% cleavage and blastocyst rates, respectively. Embryo transfer was performed and ICSI embryos from both fresh (1/7) and vitrified (2/3) oocytes maintained pregnancy to full term, resulting in the birth of one live bull calf from ICSI in a vitrified oocyte. ROS demand of in vitro produced embryos was examined with fresh and vitrified oocytes with IVF and ICSI. Vitrification significantly increased ROS within oocytes through to the blastocyst stage compared to embryos generated from ICSI in fresh oocytes. However, blastocysts from ICSI in vitrified oocytes had similar levels of ROS to IVF blastocysts, with ICSI in fresh oocytes producing lowest levels of ROS at the blastocyst stage. Despite advances with ICSI protocol, IVF produced higher cleavage and blastocyst (86.5% and 52%) rates than either fresh (76.1% and 34.7%) or vitrified (72.5 and 28.1%) ICSI group. However, these results show ICSI can be improved through modifications to the injection protocol to produce viable embryos from fresh and vitrified oocytes which are capable of establishing and maintaining pregnancy.