Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane.

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1-2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems.

BACKGROUND: A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. METHODS: Ovarian cortical tissues (1-2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. RESULTS: In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). CONCLUSIONS: The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.