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

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A series of in vitro studies were conducted to evaluate the effect of poly-l-lysine concentration, molecular weight, and encapsulation temperature on the post encapsulation survivability of spermatozoa. Viability of spermatozoa encapsulated at 2012 C using four poly-l-lysine concentrations (.05%, .15%, .25%, and .35%) did not differ over the 8 h incubation period. However, the viability of each of the four treatments was lower than that of the unencapsulated control (p<.05 and p<.01; percentage motility and percentage intact acrosomes, respectively), indicating spermatozoal damage occurred during the encapsulation process. Capsule wall thickness and integrity for the .15%, .25%, and .35% concentrations were greater (p<.Ol) than that of the .05% capsules. / Master of Science