103 Ovarian Stimulation of Anestrous Cats With Follicle-Stimulating Hormone Improves Oocyte Quality and Developmental Capability After Parthenogenetic Activation

Abstract

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.