The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst dev...

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.

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 ...

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 20μgmL−1 of cytochalasin B (CCB) and 2mgmL−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 (0.3M mannitol and 0.1mMMg+2), and membrane fusion was induced by applying a 3s AC pre-pulse of 20V, 1MHz; followed by two 30μs DC pulses of 240V/mm at intervals of 0.5s. Fused couplets were activated 2–3h after fusion by placing the couplets between two electrodes in a fusion chamber containing 3mL of fusion medium and exposing them to two 60μs DC pulses of 120V/mm. Then, couplets were incubated in 30μL drops of Tyrodes solution containing 1% MEM nonessential amino acids, 3mgmL−1 BSA (IVC-1 medium), and supplemented with 10μgmL−1 cycloheximide and 5μgmL−1 CCB at 38°C in 5% CO2 for 4h. After activation, cloned embryos were cultured in 500μL 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 86.2g (50.0 to 103g) and died within 36h 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 &amp; Shirley Davies Foundation. Table 1

The oviduct epithelium undergoes marked morphological and functional changes during the estrous cycle. It has been shown that a dramatic change in the frequencies of ciliated and non-ciliated cells occurs during the estrous cycle. At estrus the epithelium consists of secretory and ciliated cells and at diestrus mainly of ciliated cells. The oviduct provides the microenvironment for sperm capacitation, fertilization, and early cleavage-stage embryonic development. At the molecular level, only a few genes or proteins are known that change during the estrous cycle and which may be important for fertility, so as the bovine oviduct-specific glycoprotein, the major secretory protein in the oviduct. Therefore, we studied systematically the changes in gene expression in bovine ipsilateral oviduct epithelial cells at estrus and diestrus. To identify differentially expressed genes, a combination of subtracted cDNA libraries and cDNA array hybridization was used. Two subtracted libraries were produced to enrich cDNAs of upregulated genes at estrus and at diestrus. A total of 1536 cDNA clones of each library were analyzed with radioactively (33-P) labeled probes generated from the oviduct epithelial cells of six Simmental heifers, three of them slaughtered at Day 0 (estrus) and three at Day 12 after standing heat (diestrus). After normalization of the raw data and statistical analysis, all cDNAs showing significant differences in their expression levels at estrus compared to diestrus were sequenced. Sequencing revealed 84 different cDNAs; 42 of them matched bovine genes or their human/mouse homologs with known functions, and 42 matched genes without a known function. Half of the genes (n = 42) were expressed at a higher level at estrus; for the other (n = 42) expression levels were higher at diestrus. The regulated genes or their products represented a variety of functional classes, such as genes of the secretory pathway, genes involved in transcription regulation, cell-surface proteins, cell-cell interaction proteins, secreted proteins, members of signal transduction pathways, immune-related proteins, and some enzymes. The identification of genes differentially regulated in ipsilateral oviduct epithelial cells at estrus v. diestrus is the first step of a systematic analysis of differential gene expression during the estrous cycle. Further studies will follow, focusing on different compartments of the bovine oviduct and additional times of the estrous cycle. EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY Z. Beyhan and N.L. FirstDepartment of Animal Science, University of Wisconsin-Madison, WI, USA. email: zbeyhan@wisc.edu Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to ...