267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA

Abstract

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4°C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 × 10⁶ sperm mL^–1), adjusted to 50 × 10⁶ sperm mL^–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo^® flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P > 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.