Comparison of different intracellular cryoprotectants on the solid surface vitrification of red-rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue.

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.

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

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 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). 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 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (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 3 M + EG 3 M 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 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + 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.