Use of synthetic polymers improves the quality of vitrified caprine preantral follicles in the ovarian tissue.

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.

Natural antioxidants in the vitrification solution improve the ovine ovarian tissue preservation.

The present study evaluated the effect of the addition of antioxidants anethole (AN) and robinin (RO) in the vitrification solution, and the in vitro incubation (IVI) medium of ovine ovarian tissue. Ovarian fragments were vitrified without antioxidant (VWA) or with different concentrations of AN (30, 300 and 2000 µg/mL) or RO (0.125, 0.25 and 0.50 mg/mL), followed by IVI (24 h). Histological analyses showed that the percentage of morphologically normal preantral follicles (MNPF) in AN 2000 did not differ from RO 0.125 or fresh ovarian tissue (CTR). Subsequently, ovarian fragments were vitrified in the presence of AN 2000 and RO 0.125 followed by IVI without or with (AN 2000+ and RO 0.125+) the same antioxidants. The follicular activation in all treatments was significantly increased as compared to the CTR. The stroma cell density (SCD) in all the vitrified fragments was significantly lower than the CTR. However, in the AN 2000 and RO 0.125 this parameter was significantly higher when compared to the VWA. The reactive oxygen species (ROS) in the ovarian cortex of the AN 2000 or AN 2000+ were significantly reduced in comparison with the CTR while the intracellular ROS levels of AN 2000 and CTR were similar. The total antioxidant capacity (TAC) in RO 0.125 was significantly higher than that of VWA, AN 2000 and AN 2000+. According to the results, the use of antioxidants (AN or RO) only in the vitrification solution of ovine ovarian tissue is recommended, due to their better preservation of the SCD. Moreover, AN 2000 best maintains the follicular morphology, while RO 0.125 has a high TAC.