328 EPIDERMAL GROWTH FACTOR ENHANCES MEIOTIC MATURATION OF CANINE OOCYTES IN THE PRESENCE OF BSA

Abstract

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of (1) ovarian estrous stage on oocyte quality, and (2) epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. Reproductive tracts from normal, client-owned bitches aged approximately 7 months were collected after routine ovariohysterectomy at private veterinary clinics. The ovaries were removed, washed free of blood with PBS, and repeatedly cut with a scalpel blade at room temperature in HEPES-buffered Tyrodes medium containing 0.1% (w/v) polyvinyl alcohol. Each group of 10 cumulus-oocyte complexes (COCs) was matured in North Carolina State University (NCSU)-17 medium supplemented with 1 �g/mL FSH, 10 IU/mL hCG, and 1 �g/mL �-estradiol under paraffin oil for 48 or 72 h at 37�C. Follicular ovaries contained the largest number of oocytes (more than 300), and the number of oocytes e100 �m in diameter was also significantly greater in follicular ovaries than in ovaries at other stages (P < 0.001). Luteal ovaries had the smallest number of both total and large oocytes. After 72 h of culture, significantly more oocytes recovered from follicular ovaries were in GVBD than oocytes from anestrous or luteal ovaries. After 72 h of culture, oocytes recovered from follicular ovaries had a significantly lower percentage reaching the GV stage (59.7 � 5.2%) and more in GVBD (40.3 � 3.1%) compared with both anestrous (GV, 75.1 � 5.2%; GVBD, 24.9 � 3.1%) and luteal (GV, 79.8 � 5.2%; GVBD, 22.0 � 3.1%) ovaries (P < 0.05). However, maturation to MI/MII was not statistically different between the stages of the estrous cycle. BSA or FBS supplementation did not improve meiotic maturation when compared to PVA supplementation. In the presence of 100 nM epidermal growth factor (EGF), either FBS or BSA enhanced the proportion of oocytes in GVBD/MI (59.7 � 2.2% [FBS] and 51.7 � 2.2% [BSA] vs. 36.1 � 2.2% [PVA], P < 0.05). In addition, 0.4% BSA significantly increased the percentage of oocytes in MII (18.5 � 0.8%) compared to 10% FBS (7.1 � 0.8%; P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic maturation of oocytes cultured in vitro, and EGF increases the meiotic maturation of canine oocytes in the presence of BSA in vitro. This work was funded by a grant from the Ministry of Science and Technology of Korea and the National Research Laboratory Program.