In ovo culturing of turkey (Meleagris gallopavo) ovarian tissue to assess graft viability and maturation of prefollicular germ cells and follicles

Biobanking of turkey ovarian tissue appears to be the most cost-effective method for the long-term preservation of female genetics. However, to ensure the successful transplantation of biobanked ovarian tissue for breed or line revival, the transplantation and development of fresh ovarian tissue must be evaluated. To assess transplantability, ovaries from poults 1 to 15 days posthatch (dph) were cultured in ovo in chicken eggs for 6 d and compared with the equivalent fresh tissue. The viability of cultured ovarian tissue was evaluated visually, whereas the level of late-stage apoptosis was measured via the TUNEL assay. In addition, the diameter and density of prefollicular germ cells and follicles (primordial and primary) were measured to assess maturation. Results showed that all cultured grafts (74/74), on surviving chicken chorioallantoic membrane, were viable with low levels (0.8 ± 0.1%) of late-stage apoptosis. The diameter of prefollicular germ cells in cultured ovaries from poults at 5 and 7 dph were larger (P < 0.002) than that of their preculture counterparts but were not able to reach their in vivo size. No significant follicular growth was observed in ovaries cultured in ovo; however, prefollicular germ cell density was over 4-fold greater in ovaries cultured from 7 dph poults (81,030 ± 17,611/mm(3)) than in their in vivo counterpart (16,463 ± 6,805/mm(3)). Interestingly, cultured ovaries from all other ages displayed equal or lower (P ≤ 0.05) prefollicular germ cell densities than their in vivo counterparts. Cultured ovaries from poults at 5 and 7 dph also exhibited an increase (P ≤ 0.05) in follicle density compared with their preculture counterparts; whereas, cultured ovaries from 15 dph poults had decreased densities (P < 0.001) compared with their preculture counterparts. This study demonstrated that, although age of ovarian tissue cultured in ovo did not affect the overall viability, 7 dph ovaries appeared to have a better cellular morphology after culturing in ovo than other ages. In addition, we also demonstrated for the first time that avian follicles can form during tissue culturing in ovo.