title: 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.
abstract: The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow 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 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an 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 containing 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 transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, 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 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.
authors.affiliation: Department of Animal Sciences, University of Missouri, Columbia 65211, USA.
authors.forename: L R
authors.initials: LR
authors.lastname: Abeydeera
authors.forename: L A
authors.initials: LA
authors.lastname: Johnson
authors.forename: G R
authors.initials: GR
authors.lastname: Welch
authors.forename: W H
authors.initials: WH
authors.lastname: Wang
authors.forename: A C
authors.initials: AC
authors.lastname: Boquest
authors.forename: T C
authors.initials: TC
authors.lastname: Cantley
authors.forename: A
authors.initials: A
authors.lastname: Rieke
authors.forename: B N
authors.initials: BN
authors.lastname: Day
mesh_terms: D000818:Animals; D002469:Cell Separation; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005434:Flow Cytometry / Q000379:methods*; D008297:Male; D011247:Pregnancy; D012743:Sex Preselection / Q000662:veterinary*; D013094:Spermatozoa / Q000648:ultrastructure*; D013552:Swine; D014960:X Chromosome; D014998:Y Chromosome