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

Authors: HALL, GEORGE - University Of Guelph; Long, Julie; WOOD, BENJAMIN - Hybrid Turkeys; BEDECARRATS, GREGOY - University Of Guelph

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2020
Publication Date: 12/1/2020
Citation: Hall, G.B., Long, J.A., Wood, B.A., Bedecarrats, G.Y. 2020. In-ovo culturing of turkey (Meleagris gallopavo) ovarian tissue to assess graft viability and maturation of prefollicular germ cells and follicles. Poultry Science. https://doi.org/10.1016/j.psj.2020.09.014.
DOI: https://doi.org/10.1016/j.psj.2020.09.014

Interpretive Summary: Storing frozen gametes (sperm, eggs) or embryos in germplasm repositories is a proven method to save today’s genetic resources for the future, as well as provide safeguards for important populations in the event of unforeseen circumstances such as disease outbreaks. While these methods work well for many mammalian livestock species, there are challenges associated with preserving germplasm from poultry. One challenge is that the genetic makeup of birds is opposite from that of mammals. In mammals, sperm contain either XX- (female) or XY- (male) bearing chromosomes; whereas, in birds, it is the female’s chromosomes within the egg that determine if offspring will be male or female. Therefore, in order to preserve the complete genetic complement of a bird breed or line, the female germplasm must be cryopreserved. One method to effectively save female germplasm is to cryopreserve immature ovaries, with the goal of transplanting the ovaries into recipient birds who would serve as surrogates. This methodology has been used in chicken and quail, but not turkeys. In this paper, the optimal age of the donor tissue was evaluated for the ability to mature in a culture system by comparing the cultured ovarian tissue to tissue collected directly from age-matched counterparts (1 to 15 days old). Results indicated that donor tissue collected from immature birds 7 days old adapted the best to the culture system and, further, continued to mature during culture. This data demonstrates differences among poultry species in that the optimal donor age in chickens is 1 day of age and provides the foundation for developing ovary cryopreservation and transplantation in the turkey.

Technical Abstract: 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 days and compared to 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) compared to their preculture counterparts but weren’t 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 four-fold greater in ovaries cultured from 7 dph poults (81,030 +/- 17,611/mm3) compared to their in-vivo counterpart (16,463 +/- 6,805/mm3). 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 to their preculture counterparts; whereas, cultured ovaries from 15 dph poults had decreased densities (P < 0.001) compared to their preculture counterparts. This study demonstrated that, while 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. Additionally, we also demonstrated for the first time that avian follicles can form during tissue culturing in-ovo.