38 BIRTH OF AFRICAN WILD CAT CLONED KITTENS

The African wild cat (AWC, Felis silvestris lybica; 2n = 38) is one of the smallest wildcats, and it’s future is threatened by hybridization with domestic cats (Felis silvestris catus; 2n = 38). Nuclear transfer (NT) is a potentially valuable tool for retaining genetic variability, and could assist in the continuation of species with few remaining individuals. Inter-species nuclear transfer into domestic cat (DSH) supports development of somatic cell nuclei from AWC (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Therefore, the purpose of the present study was to evaluate the in vivo developmental competence of nuclear transfer embryos derived by fusion of African wildcat fibroblasts with domestic cat cytoplasts, after transfer into domestic cat recipients. In vivo- and in vitro-matured domestic cat oocytes were mechanically enucleated in modified Tyrodes salt solution supplemented with 20 µg mL-1 of cytochalasin B (CCB) and 2 mg mL-1 of sucrose, and reconstructed with AWC fibroblast cells derived from an adult male; cultured and passaged 1 to 3 times before serum-starved with DMEM + 0.5% FBS and cultured for 5 additional days before use. Fusion took place in fusion medium (0.3 M mannitol and 0.1 mM Mg + 2), and membrane fusion was induced by applying a 3 s AC pre-pulse of 20 V, 1 MHz; followed by two 30 µs DC pulses of 240V/mm at intervals of 0.5 s. Fused couplets were activated 2–3 h after fusion by placing the couplets between two electrodes in a fusion chamber containing 3 mL of fusion medium and exposing them to two 60 µs DC pulses of 120 V/mm. Then, couplets were incubated in 30 µL drops of Tyrodes solution containing 1% MEM nonessential amino acids, 3 mg mL-1 BSA (IVC-1 medium), and supplemented with 10 µg mL-1 cycloheximide and 5 µg mL-1 CCB at 38°C in 5% CO2 for 4 h. After activation, cloned embryos were cultured in 500 µL of IVC-1 medium until the day of the transfer. Derived AWC NT embryos were transferred into the oviducts (Day 1) or uteri (Days 5, 6, 7) of 36 gonadotrophin-treated DSH recipients on Day 1 after ovulation or on Days 5, 6, or 7 after oocyte aspiration, respectively. Pregnancy was assessed by ultrasonography on Days 21 to 23. One domestic cat was still pregnant and ongoing on Day 60. Kittens were delivered by Cesarean section in each of the seven pregnant recipients on days 61 to 67 of gestation. The kittens weighed an average of 86.2 g (50.0 to 103 g) and died within 36 h after delivery. The post-mortem pathology reports revealed that most of them had an immature respiratory system. The clonal status of the kittens was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. Results from these assays confirmed that the AWC kittens had originated from the AWC donor somatic cell line and were not related to the DSH recipient cats. In summary, these results indicate that AWC cloned kittens can be produced by ET of embryos derived from AWC cells into DSH cytoplasts. Research was funded partially by the John & Shirley Davies Foundation. Table 1

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