In vitro culture of canine preantral follicles after slow freezing

The objective of this study was to analyse the effect of slow freezing on the morphology and viability of canine isolated preantral follicles. Ovaries were collected from ovariohysterectomy bitches being in different ages. Preantral follicles were isolated from ovarian cortex using collagenase-based digestive solution and randomly divided into two groups: fresh control and slow frozen. Fresh and frozen/thawed preantral follicles were cultured individually in 100 µl drops of culture medium for 10 days at 38.5 °C with 6.5 % CO₂, and half of the medium was changed and samples from culture media were collected on Day 2, Day 5 and Day 10 for hormonal analysis. Post-thaw live cell rate, normal morphology rate, area change, and estradiol and progesterone production were examined. Frozen/thawed follicles have lower number of live cells than that of fresh ones (94.69 % ( ± 6.97) vs 98.58 % (± 1.71), respectively [p < 0.05]). Normal morphology rate was different only on Day2 (91.2 % in fresh and 50 % in frozen/thawed, p < 0.05) and showed decreasing tendency in both groups. Differences in follicular growth were found on Day5 and 10, when fresh follicles showed lower area than that of frozen/thawed ones (5.84 vs12.62 mm² [p < 0.005] and 6.47 vs 10.75 mm² [p < 0.05], respectively). Regarding the hormonal production, estradiol and progesterone concentrations were lower in frozen/thawed samples than that of fresh follicles throughout the culture period. In conclusion, our data suggest that slow freezing can provide canine follicular survival, however, the quality and viability of the follicles are reduced.