RESUMEN
The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs.
Asunto(s)
Artiodáctilos/fisiología , Crioprotectores/farmacología , Ovario/fisiología , Conservación de Tejido/veterinaria , Vitrificación/efectos de los fármacos , Animales , Supervivencia Celular , FemeninoRESUMEN
The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.