Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI)

The objective of this study was to develop a reliable method for intracytoplasmic sperm injection (ICSI) in bovine oocytes. Oocytes recovered from abattoir-derived ovaries were centrifuged for 5 min at 6000xg to facilitate sperm injection. Sperm were pre-treated in vitro with 5mM dithiothreitol (DTT), and diluted (approximately 1:5) with 5% polyvinylpyrrolidone (PVP) in 0.9% saline. After sperm injection, various activation procedures were compared. Initially, 3 h after activation with 5muM Ionomycin (A23187), oocytes with second polar bodies were selected and treated with 1.9mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with Ionomycin and DMAP was higher than with Ionomycin alone (62.1 vs. 27.3%, p < 0.05). In sham-injected control oocytes treated with Ionomycin and DMAP, the cleavage rate was approximately six times higher than that of oocytes treated with Ionomycin alone (44.3 vs. 7.4%, p < 0.001). Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70.2%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (54.6%), while one and three pronuclei were seen in 8 of 33 (24.2%) and 7 of 33 (21.2%), respectively. In sham-injected DMAP-treated control oocytes, 6 of 15 (40.0%) had one pronucleus while 9 of 15 (60.0%) had two pronuclei. Since a single Ca2+ stimulation provided insufficient activation and DMAP treatment could result in triploidy, activation by multiple Ca2+ stimulations following ICSI was tested. Three Ca2+ stimulations were given at 30-min intervals. Pronuclear formation was observed in 16 of 41 (39.0%) oocytes at 16 h after sperm injection, with one and two pronuclei found in 4 of 16 (25.0%) and 12 of 16 (75.0%), respectively. Although one pronucleus was formed in 3 of 33 (9.1%) sham-injected control oocytes treated with three Ca2+ stimulations, two pronuclei were not seen in any of these oocytes. Due to the low rate of pronuclear formation after 5muM Ionomycin, 50muM Ionomycin was compared in the same multiple treatment protocol. The higher dose (50muM) resulted in a higher incidence of pronuclear formation (40.9 vs. 26.3%) after ICSI. The rate of metaphase III (MIII) arrest was lower and the rate of pronuclear formation was higher in sperm injected oocytes suggesting that the sperm did contribute to the activation process. Further improvements are required in order to increase the rate of normal activation following ICSI in the bovine oocyte.