187 RE-ESTABLISHMENT OF AN EXTINCT STRAIN OF SHEEP FROM A LIMITED SUPPLY OF FROZEN SEMEN

Abstract

The objective of this study was to restore a line of sheep that exhibits spontaneous X-linked factor VIII deficiency closely mimicking human hemophilia A. Six straws of frozen semen from an affected Alpine White male were obtained from Switzerland. In the first experiment the straw of semen thawed was of poor quality. Two ewes were synchronized for use as embryo donors (MOET) by means of CIDRs for 14 days and superovulated with declining doses of FSH (184 mg) twice daily for 3 days. PMSG (200 IU) was given with the final dose of FSH and 1000 IU of hCG 12 h post-CIDR removal. The ewes were surgically inseminated 24 h later. Oviducts were flushed 48 h post-insemination producing 13 unfertilized ova (UFO). Spermatozoa were used for intracytoplasmic sperm injection (ICSI) utilizing oocytes collected from superstimulated ewes by laporatomy. These ewes were synchronized with CIDRs (15 days) and superovulated with a declining dose of FSH (204 mg) twice daily for 3.5 days. Utilizing 236 oocytes, ICSI produced 189 embryos, an 80% embryo/oocyte rate. Embryos were transferred surgically to the oviducts of 17 synchronized recipients. Recipients were synchronized using sponges (Ovakron; Heriot Agvet, 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. Eleven recipients produced 17 lambs for a lamb/embryo rate of 8.9%. The straw of semen utilized for the second experiment was of higher quality. Three ewes were superstimulated for use as MOET donors, as above, with increased doses of FSH (228 mg) and PMSG (500 IU). Donors were surgically inseminated and oviductal flushes were performed 40 h post-insemination, yielding 19 UFO and 12 embryos for an embryo/oocyte rate of 38.7%. Embryos were transferred to four recipients, synchronized as above with an increased dose of PMSG (600 IU). These MOET recipients produced nine lambs for a lamb/embryo rate of 75%. Semen was used to produce embryos via in vitro fertilization (IVF) using oocytes collected from superstimulated ewes (as above with an increase of FSH to 252 mg). IVF produced 91 embryos from 247 oocytes for an embryo/oocyte rate of 36.8%. Embryos were transferred to 20 recipients 24 to 48 h post-fertilization. Seven recipients maintained pregnancy and produced 10 lambs with a lamb/embryo rate of 11%. ICSI was also utilized, producing 54 embryos from 98 oocytes, an embryo/oocyte rate of 55.1%. Embryos were transferred to eight recipients; none maintained pregnancy. Through the use of multiple reproductive technologies, 36 lambs (22 carriers) were produced from two straws of semen. Carriers will be bred back to their sire in a similar program to produce affected lambs.

The authors would like to acknowledge J. Liu and M. Ridha for their contributions. This work was supported by NIH Grant HL073737-12.