194 BIRTH OF KITS AFTER STORAGE IN CULTURE AND TRANSFER OF IN VIVO EMBRYOS IN THE FARMED EUROPEAN POLECAT MUSTELA PUTORIUS)

Abstract

The effect of in vitro culture on viability of pre-implantation stage embryos in the farmed European polecat was studied, aimed at developing assisted reproductive technology for conservation of endangered mustelids, particularly the European mink (Mustela lutreola). Embryo storage in culture would enable embryo recovery and transfer in different locations. Ferret (Mustela putorius furo) kits have been produced from embryos that were cultured for 3 days in serum-containing medium (Li et al. 2001 Reproduction 122, 611–618). In our earlier studies, polecat embryos were maintained for 24 h in culture conditions (Lindeberg et al. 2003 Theriogenology 60, 965–970). Fourteen estrous donors were kept in the same cage with a fertile male overnight and sacrificed 3 days after the start of mating for recovery of embryos from the oviducts. Embryos were flushed with Emcare^™ Complete ultra flushing medium (ICPBio, Auckland, New Zealand), washed twice in it, washed once in Emcare^™ embryo holding solution and transported in the holding solution at room temperature for 1 h to the laboratory. Embryos of seven donors were pooled and cultured in 30-μL drops of TCM199 + glutamax I (GIBCO^™) supplemented with fatty acid-free albumin (FAFBSA, Sigma-Aldrech, Helsinki, Finland) under a cover of paraffin oil (Medicult) for 3 days in a humidified atmosphere (39°C) and in 5% of O₂. At the end of the culture, the embryos were evaluated and the ones that had developed at least to morula stage were chosen for transfers. The selected embryos were transported at room temperature in Emcare^™ embryo holding solution for 1 h to the farm where they were surgically transferred under general anesthesia into seven recipients. The recipients had been mated the same way as the donors but with vasectomized males either on the same day as the donors (the first set: 7 donors, 3 recipients) or one day later than the donors (the second set: 7 donors, 4 recipients). Five embryos were cultured a total of 6 days to the blastocyst stage and stained for a count of cell numbers. A total number of 169 one- to 16-cell-stage embryos were recovered. At the end of the 3-day culture period, a total of 139 (139/169, 82%) had developed to morula (56.6%), compact morula (9.8%), early blastocyst (30.3%), or blastocyst stage (3.3%). Of these 139 embryos, a total of 102 were surgically transferred. Five of the 7 recipients delivered one to 5 kits each 43 to 45 days after the mating. Altogether 21 kits were born and the success rate was 21% (21 kits/102 transferred embryos). Cell numbers of the five Day 6 blastocysts varied from 130 to 430. In conclusion, this preliminary trial confirms that polecat embryos can be stored in culture for 3 days. In this study polecat embryos were cultured in 5% oxygen and without addition of serum which resulted in considerably better cell numbers for Day 6 blastocysts than in our earlier studies (90 to 165 cells; Lindeberg et al. 2003 Theriogenology 60, 965–970).