Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry

The present study examined the ability,to establish pregnancies after trans fer of pig embryos derived from in vitro fertilization (IVF) of in vitro ma tured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flo w cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU- 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 mu g/mL) and FSH (0.5 mu g/mL) for 22 h, then the oocytes were cultured without hormonal supplements for a n additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0 .4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium con taining 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transf erred to the uterus of recipient gilts. Insemination of IVM pig oocytes wit h X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 us 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furtherm ore, no difference was observed between cleavage rates at 48 h after IVF (X , 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 ma le piglet. Similarly, transfer of embryos derived from Y-bearing sperm cell s to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered . The results show that X- and Y-bearing spermatozoa sorted using USDA sper m sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex. (C) 1998 by Elsevier Science Inc.