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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. more...

The potential for rescuing immature oocytes from the ovaries of females of rare species of felids that die or undergo ovariohysterectomy has been reported in only two studies (Johnston et al. 1991 Biol. Reprod. 45, 898-906; Jewgenow et al. 1997 J. Reprod. Fertil. Suppl. 51, 33-39), in which oocytes were maturated in complex media (Eagle's MEM, M199, respectively). The domestic cat is used as a research model for endangered species but there may be some differences that perturb the adaptation of in vitro maturation (IVM), fertilization, or culture methods of lion oocytes compared to cat oocytes. Therefore, in the present study we evaluated the in vitro response of lion oocytes to our system for in vitro maturation of domestic cat oocytes in a simple medium (Merlo et al. 2005 Theriogenology 63, 2032-2039). A 14-year-old lioness, referred to our clinic because of pyometra, underwent ovariohysterectomy. After premedication with acepromazine 0.1 mg/kg i.m. (Prequillan; ATI, Bologne, Italy) and ketamine 5 mg/kg i.m. (Ketavet 100; Intervet, Boxmeer, The Netherlands), anesthesia was induced with ketamine 0.05 mg/kg i.v. and diazepam 0.02 mg/kg i.v. (Diazepam; Intervet) and maintained with isoflurane (Forane; Abbott, Rome, Italy) after intubation. Ovaries were removed and stored in saline solution at room temperature until collection of oocytes (within 1 h). A total of 53 small follicles (2-4 mm), five corpora lutea and a 15-mm follicle were present on the ovaries. Oocytes were collected by aspirating visible follicles of each ovary with a 21-ga needle connected to a vacuum pump (K-MAR-5100; Cook Australia, Brisbane, Australia) at -75 mmHg; then CL were removed and the ovaries were minced with a scalpel blade in a 60-mm petri dish containing HEPES-SOF (H-SOF) for recovery of additional oocytes. A total of 45 oocytes were recovered, of which 19 were degenerate (42.2%); of the remaining 26, 12 were fully surrounded by cumulus cells, 9 had only corona radiata, and 5 were denuded. Degenerate oocytes were discarded and all other oocytes were washed twice in H-SOF and matured in a 35 mm petri dish containing SOFaaBSA 5 mg/mL plus 0.1 IU of porcine FSH-LH (Pluset; Laboratorios Calier, Barcelona, Spain), 25 �L/mL insulin-transferrin-selenium (ITS) (Sigma, Madrid, Spain), 1.2 mm l-cysteine (Sigma), and 25 ng/mL epidermal growth factor (EGF) (Sigma) for 24 h in a humidified atmosphere of 5% CO2 in air at 38.5�C. After maturation, cumulus cells were removed by pipetting oocytes into a 0.25% trypsin solution for 2 min. Then, denuded oocytes were washed once in H-SOF plus 10% FCS to inactive trypsin and twice in H-SOF before being stained with Hoechst 33342 (10 �g in 10 mL PBS) for 30 min at room temperature. After washing in PBS, oocytes were observed using fluorescence microscopy to determine maturation rate. Oocytes in telophase I or metaphase II were considered mature. Of 26 oocytes, 19 (73.1%) were mature and 7 (26.9%) were at the GV stage. These results demonstrated that lion oocytes can undergo successful IVM at a frequency that is similar to that of cat oocytes cultured in the same system (76.9%, P > 0.05). Furthermore, the maturation rate obtained in a simple medium was higher or similar to those previously reported (mean: 22.9% and 69.7% respectively, by Johnston et al. 1991 and Jewgenow et al. 1997). more...