47 THE AVIAN CHORIO-ALLANTOIC MEMBRANE: A SUITABLE SHORT-TERM CULTURE SYSTEM FOR BOVINE OVARIAN TISSUE

Successful cryopreservation of bovine ovarian tissue holds enormous potential for long-term maintenance of female gametes to preserve genetic diversity by tissue banking. Traditionally, in vitro culture followed by histopathological examination has been used to assess the post-thaw viability of cryopreserved tissues. Recently, in ovo transplantation of mammalian tissues on the chorio-allantoic membrane (CAM) of a growing chicken embryo has emerged as an alternative method for short-term culture. The purpose of this experiment was to compare CAM culture of bovine ovarian tissue over a 5-day period with the in vitro culture system. Fertilized White Leghorn eggs were incubated at 37°C and 62% relative humidity. A window (1 × 2 cm) was cut into the eggshell on Day 3 of incubation. Ovaries were retrieved from a local abattoir and brought to the laboratory within 6 h. Ovarian cortex pieces (1–2 mm3) were randomly assigned to control, CAM-culture, or in vitro-culture groups. Control-group tissues were fixed immediately in 4% paraformaldehyde. The CAM was traumatized on Day 10 of incubation to expose the underlying blood vessels, and tissue pieces were grafted at the site (one graft per egg). For in vitro culture, the ovarian cortex pieces were placed on tissue culture inserts within 6-well plates containing TCM199 with 1% insulin-transferrin-selenium, 100 mIU mL–1 of FSH, 100 IU mL–1 of penicillin, and 50 μg mL–1 of streptomycin and incubated at 38°C in 5% CO2. Ovarian tissues from the CAM and in vitro culture group were removed on Day 1, 3, and 5 of grafting/culture, fixed, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin-eosin, and analysed under a light microscope. The numbers of normal and degenerated follicles (indicating follicle survival) and number of blood vessels containing bovine and avian red blood cells (indicating angiogenesis) were counted using standard stereological procedures. All ovarian cortex grafts from surviving chick embryos showed adhesion with the CAM and a marked neo-vascularization in the graft areas. Gross and histological examination revealed the circulation of avian blood cells in ovarian stromal vessels with a concomitant decrease in the number of bovine blood vessels over the incubation period. Total follicle densities (mean ± s.e.m.) on Day 1, 3, and 5 were 13.3 ± 5.9, 27.9 ± 6.7, and 36.9 ± 7.3 in the in vitro-cultured group and 36.7 ± 13.0, 73.6 ± 24.0, and 44.02 ± 12.67 per millimeter cubed in the CAM-cultured group, respectively. Overall, total follicle density was higher in the CAM-cultured group (P < 0.05). Likewise, the normal follicle densities on Day 1, 3, and 5 were 10.4 ± 4.9, 15.5 ± 3.6, and 20.7 ± 6.3 in the in vitro-cultured group and 30.5 ± 8.5, 45.7 ± 18.4, and 22.7 ± 7.3 per millimeter cubed in the CAM-cultured group (P > 0.05). In conclusion, in ovo CAM grafting system was as successful as the in vitro-culture system and may be considered an acceptable alternative to the traditional in vitro-culture system for bovine ovarian tissue.