Abstract

The domestic cat is a valuable model for the development of assisted reproductive techniques that might be used in the conservation of endangered felids. However, the efficiency of the in vitro embryo production in the domestic cat remains low compared to other species. In the bovine and equine, the zona pellucida removal enhance the developmental competence of the embryos generated in vitro , allowing the birth of live offspring (Gambini et al., 2012; Rodriguez et al., 2008). The objective of this research was to evaluate the effects of zona pellucida removal in domestic cat embryos generated by in vitro fertilization (IVF). To achieve this purpose, two experimental groups were made, 1) domestic cat embryos generated by IVF and in vitro cultured (Zona-included), 2) domestic cat embryos generated by IVF and cultured without zona pellucida (Zona-Free). To evaluate the effect of the zona removal, the developmental capacity and morphological quality of the embryos generated in the Zona-free group were compared against the Zona-included group. For this, the ovaries of domestic cats were collected by ovariohysterectomy and the cumulus-oocyte complexes (COCs) were recovered by slicing. The COCswere in vitro matured in supplemented TCM-199 Earle’s salts medium for 26-28 hours, in a 5% CO2, 5%O2 and 90% N2 atmosphere, at 38.5ºC. The IVF was realized using epididymal refrigerated sperm. 1.5 –2.5 x 106 spermatozoa /mL were incubated with 20-30 COCs in supplemented TALP medium for 18hours, in a 5% CO2 atmosphere, at 38.5ºC. After cumulus cell removal, the zona pellucida of the presumed zygotes was removed by 2-4 minutes incubation in 2 mg/mL of pronase (Sigma-Aldrich,P8811, USA). The presumed zygotes were cultured using the well of the well system (Vajta et al., 2000)in supplemented SOF medium, in a 5% CO2, 5% O2 and 90% N2 atmosphere, at 38.5ºC, for 8 days. The cleavage, morula and blastocyst rates were estimated at day 5 and 8. The Zona-included and Zona-free groups were performed at different times, with nine and six replicates respectively. The diameter and total cell number of the blastocysts were evaluated. The Wilcoxon non-parametric test was used to analyze the developmental competence and the t-test was used to analyze the diameter and total cell number. Regarding to the results, no statistical differences were observed between the Zona-included and Zona-free groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morula rate: 115/155(74.2%) and 68/116 (58.6%), and blastocyst rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P >0.05). No differences were observed in the total cell number (mean ± SD) of the blastocysts generated in the Zona-included (279.9 ± 148.1) and Zona-free group (313.1 ± 164.9) (P > 0.05). Finally, the diameter(mean ± SD) of the blastocysts from the Zona-free group (253.4 ± 83.3 μm) was significantly higher than the diameter of the blastocysts from the Zona-included group (210.5 ± 78.5 μm). In conclusion, the zona pellucida removal did not affect negatively the morphological quality and developmental competence of domestic cat embryos in our cultured conditions. However, more studies are needed to evaluate the in vivo competence of these embryos.