No abstract

The objective of this study was to evaluate the eCG stimulation on domestic cat oocyte competence during the non-breeding season. Four experimental groups were made: (a) untreated cycling cats (Breeding season group; BS), (b) untreated anestrous cats (Non-breeding group; NB), (c) anestrous cats treated with 200 IU of eCG (eCG group) and (d) anestrous cats treated with 200 IU of eCG and 100 IU of hCG four days later (hCG group). In the BS, NB and eCG groups, grade I and II immature cumulus-oocyte complexes (COCs) were subjected to in vitro maturation or used for the gene expression analysis of FSHR, LHCGR, EGFR, EGR1, ESR2, PTGS2, GDF9, BMP15 and GATM. The in vitro matured oocytes from the BS, NB and eCG groups and the in vivo matured oocytes from the hCG group were subjected to parthenogenetic activation. The grade I and II COCs from the eCG group had an increased expression of FSHR, LHCGR, EGFR, EGR1 and ESR2 and a higher maturation rate than the BS and NB groups (p < 0.05). After parthenogenetic activation, the blastocyst rate from the hCG, eCG and BS groups was higher than the NB group (p < 0.05). However, no significant improvement was observed in the blastocyst rate in the hCG group compared to the eCG group (p > 0.05). In conclusion, the eCG treatment increases the expression of specific genes improving the oocyte competence during the cat non-breeding season, which is reflected in an enhanced in vitro maturation and in vitro embryo development after parthenogenetic activation.

The objective of this research was to evaluate the effect of eCG on morphological quality and gene expression profile of cumulus-oocyte complexes (COC) recovered from anestrous cats. For this purpose, 3 experimental groups were made. Group 1 consisted of 11 oestrous cats (oestrous); Group 2 had 13 anestrous cats (anestrous); and Group 3 was made up of 11 anestrous cats treated with a single subcutaneous dose of 200 IU of eCG. In oestrous and anestrous groups the ovaries were obtained directly by ovariohysterectomy, whereas in the eCG group this was achieved 4 days after the dose injection. In all groups, each cat corresponded to an individual biological replicate, whereby the COC recovered from each cat were classified and processed separately for the experiments of gene expression analysis and in vitro maturation (IVM). The COC were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. For gene expression analysis, pools of 8 to 10 grade I and II immature COC were made, resulting in 7 pools for each group. Quantitative RT-PCR was performed for gonadotrophin receptor genes (FSHR and LHCGR), FSH-induced genes (EGFR, EGR1, ESR2, and PTGS2), and genes related to oocyte competence (GDF9, BMP15, and GATM). The gene SDHA was used as the internal control. The total remaining proportion of grade I and II COC were used for IVM, and maturation rate was measured by visualisation of the first polar body. Statistical analysis was performed using the Kruskal–Wallis test. No differences were found in the total number of COC (mean ± standard deviation) recovered per cat among the oestrous (56.8 ± 20.5), anestrous (80.2 ± 35.2), and eCG groups (96.5 ± 62.0; P > 0.05). With respect to morphological quality of COC, the eCG group had a higher proportion of grade I COC (33.6 ± 11.0%) than the oestrous and anestrous groups (16.5 ± 8.7 and 8.9 ± 6.0%, respectively; P < 0.05). However, the anestrous group had a higher proportion of grade II COC (26.8 ± 6.4%) than the eCG group (21.1 ± 6.6%; P < 0.05). On the other hand, the eCG group had a lower proportion of grade III and IV COC (45.3 ± 12.8%) than the anestrous group (64.3 ± 9.1%; P < 0.05), without differences from the oestrous group (57.1 ± 12.0%; P > 0.05). Concerning to gene expression analysis, COC from the eCG group had a higher relative expression of FSHR, LHCGR, and EGFR than COC from the oestrous and anestrous group (P < 0.05). Furthermore, the COC from the eCG group had a higher relative expression of EGR1 than COC from the anestrous group and a higher expression of ESR2 than COC from the oestrous group (P < 0.05). However, COC from the eCG group had a lower relative expression of GATM and PTGS2 than COC from the oestrous group and a lower expression of GDF9 and BMP15 than COC from the anestrous group (P < 0.05). Although a higher number of oocytes with a first polar body could be seen in the eCG group after IVM, no significant differences in the maturation rate were found among the eCG (55.3 ± 13.2), oestrous (43.7 ± 11.1), and anestrous groups (47.9 ± 13.6; P > 0.05). In conclusion, the treatment with eCG improved the morphological quality of COC recovered from anestrous cats, which agrees with an increased relative expression of FSHR, LHCGR, EGFR, EGR1, and ESR2 and might be related to an enhanced competence of COC.

The domestic cat is a valuable model for developing assisted reproductive technologies in endangered felids. However, the in vitro production of cat embryos is negatively affected during the anestrous season. It seems that a low FSH level or the few FSH receptors in the granulosa cells might affect oocyte quality. The objective of this research was to evaluate the effect of ovarian stimulation of anestrous cats with FSH on oocyte quality and developmental capability after parthenogenetic activation. For this, two experimental groups were made. The first (FSH group) consisted of 9 anestrous cats treated with a subcutaneous dose of 5 mg of porcine FSH, every 24 h, for 4 days. The second (control group) consisted of 10 untreated anestrous cats. The ovaries were recovered by ovariohysterectomy. Cumulus-oocyte complexes (COCs) were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. Grade I and II COCs were matured in vitro during 30 h and matured oocytes were used for parthenogenetic activation. Matured oocytes were activated using 7% ethanol for 5 min, followed by incubation in 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 5 h. Presumed zygotes were in vitro cultured in SOF for 8 days, and blastocyst and hatching blastocyst rates were assessed. The relative expression of pluripotency (OCT4, SOX2, and NANOG) and differentiation markers (CDX2 and GATA6) were evaluated by RT-qPCR in the blastocysts. The results were normalized using the geometric mean of GAPDH and SDHA. Statistical analysis was performed using the Wilcoxon non-parametric test. A total (mean ± s.d.) of 741 COCs (82.33 ± 36.61 per cat) were recovered from the FSH group and 660 COCs (66 ± 20.67 per cat) from the control group (P > 0.05). The FSH group had a higher rate of grade I COCs and a lower rate of grade III and IV COCs (22.28% and 50.62%, respectively) than the control group (13.2% and 60.08%, respectively; P < 0.05). However, no significant differences were found in grade II COCs between FSH (26.21%) and control groups (27.01%; P > 0.05). Thereafter, 190 matured oocytes were activated in the FSH group and 198 in the control group. The FSH group had higher blastocyst and hatching blastocyst rates (30.52% and 13.15%, respectively) than the control group (13.13% and 1.01% respectively; P < 0.05). The blastocysts from the FSH group had a higher cell number (± s.d.) than their counterparts from the control group (199.9 ± 53.6 and 121 ± 34.91, respectively; P < 0.05). Furthermore, the blastocysts from FSH group had an increased relative expression of OCT4 and GATA6 (P < 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improves oocyte quality and embryo developmental capability, which agrees with an increase in relative expression of OCT4 and GATA6.