IN-VITRO FERTILIZATION IN DOMESTIC AND NONDOMESTIC CATS INCLUDING SEQUENCES OF EARLY NUCLEAR EVENTS, DEVELOPMENT IN-VITRO, CRYOPRESERVATIONAND SUCCESSFUL INTRASPECIES AND INTERSPECIES EMBRYO-TRANSFER

The domestic cat may be used as a model for developing assisted reprod uction techniques including in vitro fertilization (IVF), embryo cultu re, cryopreservation and embryo transfer (ET) for application to threa tened and endangered species of non-domestic cats. Interoestrous domes tic cats were injected with a total of 1.0-6.0 mg follicle-stimulating hormone (FSH) daily for 4 days and with 100 iu human chorionic gonado trophin (hCG) on day 5. Follicular oocytes recovered at 26 +/- 1 h aft er hCG were co-incubated for 4-6 h at 38-degrees-C in 5% CO2 with sper matozoa (1-2 x 10(5) motile spermatozoa ml-1) collected by artificial vagina. To determine the timing of sperm penetration and early fertili zation events in vitro, oocytes were fixed and examined at intervals f rom 0.5 to 10 h after sperm exposure. The penetration rate of metaphas e II (MII) oocytes at 0.5-3 h was equivalent to that at 3-6 h (95 vers us 96%). Second polar body extrusion, pronuclear formation and apposit ion were observed at 2, 6-8 and 10 h, respectively. Simple (Tyrode's) and complex (F-10, M-199 and CMRL-1066) culture media with 10% fetal c alf serum were compared for their ability to support development to th e morula or blastocyst stage during culture periods of 96-168 h after IVF. The average number of cells per embryo was similar (P > 0.05) in the various media at each interval except that CMRL-1066 reduced (P < 0.05) development at 96 h if it was used before the two-cell stage. In F-10, neither the presence of intact cumulus cells nor changing to fr esh F-10 medium at 48 h affected development at 96 h. Blastocyst devel opment at 168 h was similar in both F-10 (18%) and Tyrode's (26%). To determine developmental ability in vivo, IVF-derived embryos (n = 586) were transferred at 96 or 120 h to recipients (n = 49) that had under gone synchronous oocyte recovery as donors. The percentage of recipien ts producing kittens after transfer of embryos cultured for 96 or 120 h in F-10 was 31 and 25, respectively, compared with 55% of 120 h reci pients receiving embryos cultured in M-199 or Tyrode's. Overall, more pregnancies occurred following transfer of greater-than-or-equal-to 12 embryos (11/26) than if < 12 embryos were transferred (6/23). Two- to four-cell IVF embryos frozen in propanediol (1.4 mol l-1) and sucrose at either 0.125 or 0.25 mol l-1 developed to the morula/blastocyst st age at a rate (65%) equal to that of unfrozen control embryos (65%). T he IVF/ET procedures developed in domestic cats have been applied to s everal species of small non-domestic cats, including the Indian desert cat (IDC, Felis silvestris ornata), jungle cat (JC, Felis chaus), fis hing cat (FC, Felis viverrinus) and black-footed cat (BFC, Felis nigri pes). The same methods were used except that the gonadotrophin dose wa s often higher, ranging from 2.0 to 10.0 mg FSH (FSH-P) and from 100 t o 300 iu hCG. All non-domestic oocytes that developed in vitro after I VF were transferred at 96 or 120 h. In IDC (n = 3), an average of 8.8 mature oocytes were recovered and the fertilization rate was 67% (53/7 9). Two IDC kittens were born 68 days after IVF to a domestic cat reci pient following the interspecies transfer of four IDC embryos and intr aspecies transfer of ten domestic embryos. A total of 51 additional ID C embryos were transferred to seven domestic cats (n = 46) and one IDC (n = 5) but no IDC offspring were born. Of 85 JC mature oocytes (aver age 11.8 per female), 49 (58%) developed in vitro and were transferred with one IVM/IVF embryo: 39 were to five domestic cat recipients and 11 were auto-transferred, but no pregnancies were produced. Two FC IVF procedures resulted in a total of 94 mature oocytes; however, only 21 (22%) developed after IVF and no pregnancies followed transfer to two domestic cat recipients. A BFC female produced 20 mature oocytes, but no fertilization was achieved. The reduction in (FC) or failure of (B FC) IVF was possibly caused by the rapid loss of motility and poor sur vival of the spermatozoa in vitro.