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The aim of the study was to investigate the relationship between ooplasm morphology, lipid content, glucose-6-phosphate dehydrogenase activity (G6PDH) and maturation potential of domestic cat oocytes. Cumulus-oocyte complexes were classified according to ooplasm morphology: evenly dark (dCOC), heterogeneous/mosaic (hCOC), or light/transparent (lCOC), however only dCOCs are thought to be the best-quality, the remaining ones are usually rejected, therefore little is known about their intracellular properties. Lipid droplets (LDs) were visualized and quantified using Oil Red O. G6PDH activity was assessed before in vitro maturation (IVM), using the brilliant cresyl blue (BCB) test. IVM-control oocytes underwent IVM without BCB staining. The dCOCs and hCOCs had different patterns of LD spatial distribution, but similar amounts of lipid, although this tended towards being lower in hCOCs. Low G6PDH activity (BCB+) was observed in 74 %, 60 % and 24 % (P < 0.01) of dCOCs, hCOCs, and lCOCs, respectively. Significantly more BCB+ /oocytes than BCB–/oocytes reached the metaphase II stage in all groups. The maturation rate of BCB+ /hCOCs was higher than that of IVM/hCOC-controls (40 % v.s. 20 %, P < 0.001), and was comparable to that of BCB+ /dCOCs (54 %; P > 0.05). lCOCs were the smallest (P < 0.01), contained fewer (P < 0.01) lipids than dCOCs or hCOCs, and displayed reduced maturational potential. Overall, LD content and distribution, as well as G6PDH activity, in cat oocytes were strongly associated with ooplasm morphology and oocyte maturational competence. Deeper understanding of the intrinsic properties of oocytes with different ooplasm morphology using the domestic cat model, may be particularly important in the context of the conservation of endangered felids.

Cell cycle synchronization of donor cells is an important step in mammalian somatic cell nuclear transfer (SCNT). This study was designed to compare the efficiency of serum starvation (Ss) and contact inhibition (cI) on cell cycle synchronization of jaguarundi, manul, and domestic cat skin fibroblasts, in the production of G0/G1 cells suitable for SCNT in felids. Ss was performed after the growing (G) cells reached 40–50% (G50+Ss), 60–70% (G70+Ss) and full confluency (Fc), i.e. in association with cI (cI+Ss). Frozen-thawed cells were cultured to the given state of confluency (d0; controls), and subjected to Ss or cI for 1, 3, and 5 days (d). In manul, the effect of Ss on arresting fibroblasts in the G0/G1 phase was noted after just 1d of culture at G70 confluence, while G50+Ss and cI+Ss were effective after 5d of treatment. In jaguarundi, 1–5d of G50+Ss and 5d of G70+Ss increased the percentage of G0/G1 cells versus d0 (P<0.01), with 5d of G70+Ss producing more (P<0.05) quiescent cells than after the same period of G50+Ss, cI+Ss and cI. In the domestic cat, Ss was efficient only after 3 and 5d of G50+Ss. In all species, cI alone failed to increase the proportion of G0/G1 cells compared to d0, however in the domestic cat, 5d of cI was more efficient than the same period of G50+Ss. In jaguarundi, >93% of cells were already in G0/G1 phase at d0 of Fc, suggesting that culture to Fc could be also a valuable method for fibroblast cell cycle synchronization in this species. In contrast to cI, prolonged Ss generated cell loss and could induce apoptosis and/or necrosis. In conclusion, Ss was the more efficient method for skin fibroblast cell cycle synchronization at the G0/G1 phase in manul, jaguarundi and the domestic cat. The response of cells to the treatments was species-specific, depending on cell confluence and duration of culture. This research may find application in preparing donor karyoplasts for SCNT in felids.

Dermal fibroblasts are commonly used as donors of genetic material for somatic cell nuclear transfer in mammals. Basic fibroblast growth factor (bFGF) is a cytokine that regulates proliferation and differentiation of different cell types. The study was aimed at optimizing the cell culture protocol for cat dermal fibroblasts by assessing the influence of culture media and different doses of bFGF on proliferation of fibroblasts and their viability in terms of cell banking and somatic cloning of felids. In Experiment I, skin biopsies of domestic cats were cultured in DMEM (D) and/or DMEM/F12 (F), both supplemented with 5 ng bFGF/ml (D-5, F-5, respectively). After the primary culture reached ~80% of confluency, the cells were passaged (3–4 times) and cultured in media with (D-5, F-5) or without (D-0, F-0) bFGF. To determine the optimal doses of bFGF, in Experiment II, secondary fibroblasts were cultured in DMEM with 0 (D-0), 2.5 (D-2.5), 5 (D-5) or 10 (D-10) ng bFGF/ml. The results showed that in D-5 the cells proliferated faster than in D-0, F-5 and F-0. Due to their poor proliferation, passages IV were not performed for cells cultured in F-0. In experiment II, a dose-dependent effect of bFGF on proliferation of cat dermal fibroblasts was found. In D-5 and D-10, the cells exhibited higher (P<0.05) proliferation compared with D-0. In D-2.5 the cells showed a tendency to proliferate slower than in D-5 and D-10 and at the same faster than in D-0. In conclusion. DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12. Supplementation of culture medium with bFGF has a beneficial effect on cat dermal fibroblast proliferation and could be recommended for addition to culture media.