66 DEVELOPMENT OF IN VITRO-PRODUCED CAT EMBRYOS AFTER VITRIFICATION AND NONSURGICAL EMBRYO TRANSFER: PRELIMINARY RESULTS

Abstract

There are no refereed reports on vitrification of domestic cat embryos derived from in vitro-matured oocytes and transferred using a nonsurgical embryo transfer technique. The aim of this study was to verify the effects of vitrification on the in vitro and in vivo developmental ability of in vitro-produced (IVP) cat blastocysts. Oocytes recovered from minced ovaries were matured, fertilized, and cultured in vitro as previously reported (Merlo B et al. 2005 Theriogenology 63, 2032–2039). On Day 7 of in vitro culture (IVC), blastocysts were selected and vitrified in straws (Cristal ET 0.25 mL, 133 mm, IMV-Technologies, Paillette Crista, France). For vitrification (modified from Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200–1214), the embryos were transferred in 1 mL of V1 [ethylene glycol 3.5 m in HEPES synthetic oviductal fluid (HSOF)] for 3 min, and then in 10 μL of V2 (ethylene glycol 7 m, galactose 0.5 m, Ficoll 70 18% in HSOF) for 20 s. Finally, the embryos were loaded in straws preloaded with 190 μL of dilution solution (galactose 0.5 m in HSOF). Straws were heat sealed and immediately plunged into liquid nitrogen. Vitrified embryos were warmed in air for 10 s, and then in a waterbath at 37°C for 30 s. For developmental ability and in vitro evaluation, 27 embryos were warmed and immediately examined: 25 re-expanded, 2 did not re-expand, and 1 had damaged zona pellucida. Re-expanded embryos were cultured in SOF plus amino acids, 16 mg mL^–1 BSA, and 5% fetal bovine serum at 38.5°C in 5% O₂, 5% CO₂, 90% N₂. After 24 h of IVC, only 4 blastocysts were expanded, and after 48 h, embryos were clearly degenerated or shrunk. in vivo developmental ability was tested by nonsurgical embryo transfer of 8 vitrified-warmed embryos and 6 IVP fresh embryos into 2 natural estrus queens, injected with 200 IU of hCG i.m. (Day 0) for induction of ovulation. Ovulation was confirmed by plasmatic progesterone assay on Day 5. Nonsurgical embryo transfer was made on Day 8 using the catheter proposed by Zambelli et al. 2001 for transcervical insemination in the cat. The catheter was connected to a 1-mL syringe and loaded with the embryos. Then, it was inserted in the vagina and transrectally guided into the uterus, where the embryos were deposited. To assess pregnancy status, abdominal ultrasonography was done on recipients on Day 13, 25, and 40. On Day 13, an embryonic vesicle was observed in both queens, although a smaller diameter than expected was detected in the recipient of the vitrified embryos. On Day 25, a viable embryo was detected only in the recipient of fresh IVP embryos. On Day 40, the gestational chamber was still present but no sign of a viable embryo was detected. Further studies are in progress to improve the nominal incidence of pregnancy and frequency of embryo survival after vitrification. Nevertheless, the preliminary results obtained using an AI catheter for nonsurgical embryo transfer are encouraging, and the improvement of the technique could make it reliable in the cat.

Supported by Animal Stem Cells Laboratory, Regione Emilia Romagna, PRRIITT Project Number M-404AIWTSV.