85 DOMESTIC CAT KITTENS BORN AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERM

Abstract

For successful application of sperm sorting technology to a particular species, the various assisted reproductive techniques required for in vivo or in vitro embryo production must be established. Previously, we have demonstrated the in vivo developmental competence of cat embryos derived by IVF 1) with presumptive X-chromosome bearing sperm and 2) after controlled-rate cryopreservation. Here, our intent was to determine in vivo viability by transfer to recipients of cryopreserved cat embryos produced by IVF with flow-sorted sperm. Semen collected from a single male using an artificial vagina was extended in electrolyte-free solution and shipped overnight (4°C) to the sorting facility. After sorting immediately upon arrival, sperm were concentrated by centrifugation, suspended in TEST yolk buffer, and return-shipped to the IVF laboratory at 4°C. At 48 h after semen collection, in vivo matured oocytes (n = 64) recovered from 2 gonadotropin-treated donors were inseminated in vitro with presumptive X-chromosome-bearing sperm. At 5 h post-insemination, oocytes were rinsed and placed in modified Tyrodes+BSA and NEAA (IVC-1) at 38°C in a humidified atmosphere of 5% O₂, 5% CO₂, and 90% N₂. At 27 h post-insemination (Day 1), 43 two-cell embryos (67% cleavage rate) were equilibrated (3 steps) in 1.4 M propylene glycol, 0.125 M sucrose, and 10% dextran 70, cooled at a slow controlled rate (0.3°C min^–1) from –6°C to –30°C, and plunged into liquid nitrogen. After 26 to 43 months in cryostorage, 4 straws, each containing 8 or 9 embryos, were thawed in air (22°C) and cryoprotectant was removed from the embryos by a 5-step rinse. Then, embryos were cultured for 17 to 20 h in IVC-1, as described earlier. On Day 2, 35 embryos were transferred (8 or 9 per recipient) by laparoscopy to the oviduct of four Day 1 gonadotropin-treated recipients at 24 h after laparoscopic oocyte retrieval (16 to 25 oocytes/donor). Most embryos (83%) were at the four- to eight-cell stages at transfer. Three recipients established pregnancies and delivered, without assistance, litters of 1, 2, and 3 live kittens (27% embryo survival) between Day 64 and Day 67 of gestation. Four kittens were female, and two were male. Birth weights ranged from 73 to 133g (mean = 117 g). Overall, of 18 kittens [12 from fresh (2009 Theriogenology, 71, 864) +6 from frozen embryos] born after transfer of embryos produced by IVF with sex-sorted sperm, 16 (89%) have been female. The variation in sex ratios between kittens born from fresh v. cryopreserved embryos probably is a reflection of the sort purity of the different sperm samples used for IVF. The ability to produce kittens after transfer of cryopreserved embryos derived by in vitro insemination with sex-sorted sperm represents an expansion in the assortment of assisted reproductive techniques that may be useful for selective propagation of biomedically relevant and endangered felids.