269 EFFECTS OF REPRODUCTIVE STATUS AND OOCYTE QUALITY ON THE DEVELOPMENTAL COMPETENCE OF OOCYTES FOLLOWING IN VITRO PRODUCTION IN DOMESTIC CAT

Abstract

A recent study showed that the developmental competence of cat oocytes after IVM and IVF was affected by the estrous cycle stage of the donor (Freistedt et al. 2001 Biol. Reprod. 65, 9-13). This study was conducted to examine the effect of the cat reproductive cycle stage and the oocyte quality on the developmental competence of the oocyte following IVF production. Cat ovaries were collected at veterinary clinics and stored at 4°C for 24 h. Based on the presence or absence of follicles and corpora lutea, the ovaries were classified into the luteal, follicular, or inactive stage. Cumulus oocyte complexes (COCs) from the different stage ovaries were separated at recovery into three ranks (A, B, and C) according to pigmentation, uniformity and smoothness of ooplasm, and amount of surrounding cumulus cell mass and were cultured separately in 100 ¼L drops of maturation medium (TCM-199), supplemented with 0.4% bovine serum albumin, 0.1 IU/mL (human menopausal gonadotropin (HMG), 10 IU/mL HCG, 1 ¼g/mL 17²-estradiol, and 100 ¼g/mL gentamicin) for 24-h at 38°C, 5% CO₂ in air. After 24-h in vitro culture, the oocytes were transferred into 100 ¼L sperm microdrops of Brackett-Oliphant medium (2 × 10⁶ sperm/mL) for fertilization and were co-incubated for 12 h. Subsequently, presumptive zygotes were transferred into a modified Earle's balanced salt solution (MK-1) supplemented with 4 mg/mL BSA and 50 ¼g/mL gentamicin. Three days after insemination, all embryos were transferred into culture medium of MK-1 supplemented with 5% (v/v) fetal bovine serum and 50 ¼g/mL gentamicin. The cleaved embryos were further cultured for 5 days to evaluate their ability to develop to the blastocyst stage. Data were analyzed by ANOVA. The percentages of COCs of A (35.5%, 31.8%, 27.7%), B (41.5%, 39.9%, 42.9%), and C (23.0%, 28.3%, 29.4%) ranks did not show significant differences (P > 0.05) among the reproductive stages of ovaries (luteal, follicular, and inactive, respectively). There were significant differences in the percentages of cleavage (P < 0.05) among the A, B, and C ranks of oocytes from ovaries classified as luteal and follicular stages (50%, 35%, 1.8%; 52%, 35%, 3.9%, respectively, P < 0.05). However, there were no significant differences between the A and B ranks of oocytes obtained from inactive stages of ovaries (47% vs. 44%, respectively; P > 0.05) but there was a significant difference for C rank (1.9%). The percentages of blastocyst formation were significantly different (P < 0.05) among the ranks of oocytes obtained from luteal, follicular, and inactive stages (27%, 14.6%, and 0% for A; 24%, 11.7%, and 0.6% for B; 35%, 21.6%, and 2.9% for C). However, there were no significant differences (P > 0.05) among the reproductive cycle stages of ovaries in the three oocyte ranks with respect to the percentages of cleavage and blastocyst formation.These results indicate that the reproductive stage of donor cat ovaries stored at 4°C for 24 h has no apparent effect on the developmental competence of the oocyte following IVF, but development is affected by oocyte quality.