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ContentsOur objectives were (i) compare in vitro development of early cleavage stage domestic cat embryos after cryopreservation by minimal volume vitrification vs a standard slow, controlled-rate method, (ii) determine viability of vitrified domestic cat embryos by oviductal transfer into synchronous recipients and (iii) evaluate in vivo survival of black-footed cat (BFC, Felis nigripes) embryos after intra-and inter-species transfer. In vitro-derived (IVM/IVF) cat embryos were used to evaluate in vitro development after controlled-rate cryopreservation vs vitrification vs controls. Blastocyst development was similar in both groups of cryopreserved embryos (22-26%), but it was lower (p < 0.05) than that of fresh embryos (50%). After embryo transfer, four of eight recipients of vitrified embryos established pregnancies -three of six (50%) and one of two (50%) that received embryos from in vivo-and in vitromatured oocytes, respectively. Three male and two female kittens weighing from 51 to 124 g (mean = 88 g) were delivered on days 61-65 of gestation. In BFC, four intra-species embryo transfer procedures were carried out -two recipients received fresh day 2 embryos (n = 5, 8) and two recipients received embryos that had been cryopreserved on day 1 (n = 6) or 2 (n = 8). A 2-year-old recipient of cryopreserved embryos established pregnancy and delivered two live male kittens. Subsequently, five cryopreserved BFC embryos were transferred to a domestic cat recipient. On day 29, the recipient was determined to be pregnant and delivered naturally a live, healthy female BFC kitten on day 66. In summary, in vivo survival of vitrified domestic cat embryos was shown by the births of kittens after transfer into recipients. Also, we demonstrated that sperm and embryo cryopreservation could be combined with intra-and inter-species embryo transfer and integrated into the array of assisted reproductive techniques used successfully for propagation of a rare and vulnerable felid species, the black-footed cat.

Contents Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black‐footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but ‘inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent‐related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black‐footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black‐footed cat donor cells and reconstructed embryos with epigenetic‐modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Efficient production of cloned embryos and live offspring is dependent on the ability of the nuclear-donor cell to be reprogrammed to direct normal conceptus development. Results of comparative studies indicate that embryonic and fetal cells are more successful candidates for nuclear transfer (NT) than terminally differentiated cells. Comparison of donor-cell efficiency is difficult to interpret across laboratories and from donor animals of varying genetic backgrounds and tissues of origin. This study was undertaken to determine the effect of the stage of differentiation of adult somatic donor cells derived from one-cell lineage of an individual donor animal. The follicular cell lineage including preantral follicle (PAFC), cumulus (CC), granulosa (GC), and luteal cells (LC) was chosen as several cell types in the lineage have previously proven successful for NT. Donor cell cultures were established from a 3-year-old Holstein cow. Embryos were reconstructed using confluent, early passage cultures. For each replicate, embryos were produced from two donor cell types in randomized block design (14 trials). Viable embryos were transferred to recipient females after 7 days of in vitro culture. Pregnancy and fetal viability were monitored weekly by ultrasonography from Days 30-100 of gestation and by rectal palpation thereafter. Embryos reconstructed from PAFC were less likely to develop to the blastocyst stage on Day 7 than embryos derived from CC or LC (Table 1, P < 0.05). Pregnancy rates at Day 30 were similar across donor cell types. A greater proportion of PAFC embryos were viable at Day 60 of gestation than embryos derived from CC and GC (P < 0.05). To date, normal cloned calves have been delivered at term from CC and GC, and two pregnancies (n = 3 fetuses) are ongoing from PAFC. The fetus cloned from LC, the terminally differentiated cell type in the lineage, was spontaneously aborted at day 211 with congenital abnormalities. Results from comparative studies of development of mouse embryos cloned from embryonic stem (ES) cells v. somatic donor cells indicate that ES-derived clones are less efficient in blastocyst formation, but survival to term is greater (Humpherys D et al., 2002 PNAS 99, 12 889-12 894). Likewise, our results in cattle suggest that PAFC, the least differentiated cells in the lineage, result in fewer cloned blastocysts, but blastocysts that do develop are more likely to progress through implantation and into later stages of pregnancy. Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683-700). On Day 8, degenerated embryos were ...