No abstract

This study determined, for the first time, the different subpopulations of germ cells and stereological changes within the cortex of the functional left ovary during germ cell nest breakdown, and formation of the primordial follicle pool in the domestic turkey. This was accomplished by measuring the size, density, and count of prefollicular germ cells and primordial follicles in turkey poults between 1 and 35 days posthatch ( dph ). The percent volume ( PV ) of germ cells and follicles within the cortex was also calculated as a means of validating the counting technique. The total percent volume of germ cells and primordial follicles within the cortex ranged between 42 and 84%, suggesting that the counting technique was valid. Our findings show that before germ cell nest breakdown (5 dph), there were roughly 1,000,000 prefollicular germ cells within the cortex of the left ovary and that germ cell nest breakdown initiated between 5 and 7 dph, characterized by a decrease ( P ≤ 0.001) in prefollicular germ cell density and the subsequent appearance of primordial follicles. Nest breakdown is followed on day 9 by the first increase ( P ≤ 0.05) in size of prefollicular germ cells. These cells continue to grow throughout nest breakdown. The majority (>90%) of germ cell nest breakdowns concluded by 15 dph; although, the primordial follicle pool was not fully established until 35 dph, as determined by a total lack of prefollicular germ cells. At this point, the pool was comprised of an estimated 60,000 primordial follicles and shows that during nest breakdown and follicle pool formation, ∼94% of germ cells were lost. This 94% decrease in the number of germ cells during nest breakdown in the turkey is comparable to the domestic chicken but is greater than the average two-thirds which are lost in mammalian species.

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 .

No abstract

Biobanked ovaries collected from recently hatched poults can only be revived through transplantation, using a recipient bird. The main hurdle in transplantation is preventing graft rejection, which appears as lymphocytic infiltration upon histologic evaluation of the graft. In this study, the condition of the transplants [immunological compatibility (auto- vs. allotransplants), donor age, time in holding media, and temperature of holding media] and treatment of recipient poults with varying immunosuppressants [mycophenolate mofetil (MFM), cyclophosphamide (CY), and cyclosporin A (CsA)] were studied to determine which factors could reduce lymphocytic infiltration, during the first 35 days post-transplantation. Lymphocytic infiltration was determined via cytoplasmic CD3 (T cell) and nuclear PAX5 (B cell) expression. There was no significant difference in the percent of cytoplasmic CD3 or nuclear PAX5 immunostained area between the unoperated group and the autotransplants, by 6 days post-transplantation. However, the allotransplants had more (P &lt; 0.05) positive cytoplasmic and nuclear immunostained areas compared to autotransplants, irrespective of donor age, time in holding media or temperature of the media. By 14 days post-transplantation, the CsA 25 and 50 mg/kg/day treatment groups had less (P &lt; 0.05) CD3 and PAX5 positive areas in their allotransplants, compared to the unsuppressed group. At 35 days post-transplantation, the CsA 25 mg/kg/day allotransplant group also had less (P &lt; 0.05) CD3 and PAX5 positive areas compared to the unsuppressed group. The CsA 25 mg/kg/day transplants also had a similar ovarian follicular size compared to the unoperated group, although they contained fewer (P &lt; 0.05) follicles based on follicular density. Donor age, duration in holding media, temperature of media, and treatment of recipients with MFM or CY had no effect on reducing lymphocytic infiltration. However, immunological compatibility was associated with decreased lymphocytic infiltration, as autotransplants had little lymphocytic infiltration. Treatment of recipients with CsA at 25 mg/kg/day was also associated with reduced lymphocytic infiltration and allowed transplants to develop normally during the first 35 days post transplantation.