Vitrification of ovarian tissue of Brazilian North-eastern donkeys (Equus asinus) using different cryoprotectants.

Abstract

The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3M or 6M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3M+EG 3M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p<0.05). When comparing treatments, the use of DMSO 3M (81.7±37.5%), EG 3M (83.7±27.4%) and the combination of both DMSO 3M+EG 3M (81.8±46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6M (69.8±16.5%) and EG 6M (72.3±18.0%; p<0.05). When vitrified using the DMSO+EG combination, a higher percentage (62.5±29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p<0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3M+EG 3M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3M+EG 3M (p>0.05). We concluded that the combination DMSO 3M+EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.