The objective of this study was to reestablish an extinct strain of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Twenty female carriers of the trait, produced in a previous study (Bormann et al. 2006 Reprod. Fertil. Dev. 18, 201202), were backcrossed using 3 straws of semen from their affected sire using either IVF or multiple ovulation embryo transfer (MOET). Eleven oocyte donors were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Nine MOET donors were synchronized using CIDRs (14 days), superovulated with a declining dose of FSH (184 mg) BID for 3 days with pregnant mare serum gonadotropin (PMSG; 200 IU) given with the final dose of FSH, and given 1000 IU of hCG 12 h post-CIDR removal. Recipient ewes were synchronized using sponges (Ovakron, HeriotAgvet, Rowville, Victoria, Australia) containing 30 mg of flugestone acetate (14 days) and given PMSG (400 IU) at sponge removal, followed by 1000 IU of hCG 12 h post-sponge removal. Oocytes were collected via follicular aspiration during midventral laparotomy and matured as previously reported. Semen for IVF was prepared by centrifugation on a Percoll gradient. Oocytes and sperm were incubated in mTALP with 20% estrus sheep serum (modified from Bavister et al. 1977 Bio. Reprod. 16, 228237) for 20 h, then vortexed to remove cumulus cells, and cultured in G1.3 medium (Vitrolife, Englewood, CO) with BSA until transfer. Embryos were surgically transferred into oviducts of recipients 24 to 48 h following IVF. The 9 MOET donors were surgically inseminated at the uterotubal junction with approximately 12.0 106 spermatazoa. Oviducts of eight of these ewes were flushed 48 h post-insemination with warm M199 containing Hanks salts, 25 mm HEPES, 10% FBS, and 0.5 g mL1 gentamicin. MOET embryos were surgically transferred to synchronized recipients within 5 h. One MOET donor was not flushed due to poor response and did not produce an offspring. Utilizing 140 ova, IVF produced 54 embryos for an embryo/oocyte rate of 38.6%. All IVF embryos were transferred into 15 recipients resulting in 3 lambs for a lamb/embryo rate of 5.5%. The MOET donors produced 38 embryos and 13 apparently unfertilized ova, generating an embryo/oocyte rate of 74.5%. MOET embryos were transferred into 21 synchronized recipients. MOET produced 16 lambs for a lamb/embryo rate of 42.1%. Co-transfer of 1 IVF and 1 MOET embryo into a single recipient produced one offspring. Utilizing multiple reproductive technologies over a two-year period, 8 hemophilic offspring (7 females and 1 male), 6 carrier females, and 6 unaffected males were produced. This strain of sheep will be used to produce affected offspring for stem cell-based therapies.