234 POTENTIAL SEASONAL INFLUENCE ON THE RECOVERY OF IN VIVO-MATURED CANINE OOCYTES BY FLUSHING FALLOPIAN TUBES

Abstract

Reproductive physiology, especially the gamete physiology of bitches, hinders the success of in vitro maturation procedure. Assisted reproductive techniques (ART) in canine greatly depend on in vivo matured oocytes. The study was designed to evaluate the potential of flushing fallopian tubes to collect in vivo-matured oocytes for ART by laparotomy and to study the seasonal effects and the parity of donor bitches on the success of oocyte retrieval. A total of 124 bitches, 39, 44, 25, and 16 in spring, summer, autumn and winter, respectively, were used. The average temperature (°F) and relative humidity (%) were 55.29 and 57.17, 75.67, and 72.83, 59.65 and 64.67, and 33.55 and 58.67 for spring, summer, autumn, and winter, respectively. Eighteen nulliparous and 18 multiparous bitches were used to determine the effect of parity. Vaginal cytology and serum progesterone concentration were checked daily to determine ovulation time. Laparotomy was performed under general anesthesia after 72 h when progesterone initially reached 4.0 nm/mL or more. About 7 mL of TCM-199 supplemented with HEPES (Gibco, Grand Island, NY, USA) was used to flush each individual fallopian tube. After morphological assessment under a stereomicroscope, the oocytes were denuded in 0.5% (W/V) hyaluronidase solution (Sigma, St. Louis, MO, USA) and stained with 5 µg/mL bisbenzimide (Sigma). Nuclear status was then observed under ultraviolet light. Based on the nuclear stage, cumulus cell layers, color, and homogeneity of ooplasm, the oocytes were categorized as good, fair, poor, immature, or aged. The good and fair oocytes were considered usable and poor, aged, and immature oocytes were considered unusable for ART. In vivo-matured oocytes can be collected all year around by flushing fallopian tubes. A significantly higher average number of oocytes per bitch were observed during the spring (11.21 ± 0.56), compared to the other seasons (7.88 ± 0.63 to 9.27 ± 0.35) (P < 0.05). A comparatively higher percentage of usable oocytes were collected during autumn. The percentage of oocyte recovery was 74.67, 67.51, 71.57, and 62.70 for autumn, spring, summer, and winter, respectively (P > 0.05). Both the number and the quality of oocytes were influenced by the parity of the donor bitch. Significantly more oocytes were collected from the multiparous bitches compared to nulliparous bitches (10.4 ± 0.54 and 7.7 ± 0.86, respectively). The percentage of usable oocytes was larger in multiparous bitches compared to nulliparous (70.75 and 64.74, respectively) (P > 0.05). Collection of in vivo-produced oocytes could be a potential source of matured oocytes for ART in canine.