Development of an efficient method to produce uniformly haploid parthenogenones

Bovine ovaries (paired by cow) were obtained from a local abattoir and cumu lus oocyte complexes were aspirated within six hours of slaughter. Two meth ods for activation [(1) calcium ionophore (ionomycin) alone (n = 191); and (2) ionomycin followed by the protein synthesis inhibitor cycloheximide (n = 207)] were evaluated for production of bovine parthenogenones. Activation with ionomycin alone resulted in a development rate of 33%, while activati on with ionomycin and cycloheximide sequentially resulted in a development rate to two-cell stage of 49%. A procedure was developed to expedite accura te evaluation of activated oocytes for uniformly haploid development. Unifo rmly haploid parthenogenones that cleaved at least once in four days of in vitro culture were individually prepared for genetic analysis. Three techni ques: (1) phosphate buffered saline; (2) TL-HEPES with 0.2% ovine serum alb umin; and (3) TL-HEPES with 0.2% polyvinyl pyrrolidone were compared to har vest parthenogenones for genetic analysis. The only effective method that d id not create spurious results during later genetic analysis was TL-HEPES w ith 0.2% polyvinyl pyrrolidone. Based on the results of this study, we esti mate that an average of 5-7 uniformly haploid bovine parthenogenones can be realized from each donor (using pairs of ovaries). These parthenogenones, when maintained as family units, will be valuable for accomplishment of fem ale-specific genetic linkage analysis. J. E3Gp. Zool. 284:112-118, 1999. (C ) 1999 Wiley-Liss, Inc.