347 EFFECTS OF NEWBORN CANINE SERUM ON IN VITRO MATURATION OF CANINE OOCYTES

Abstract

Research on in vitro maturation (IVM) of canine oocytes has been scant, due to the fact that canine oocytes are at an immature stage (geminal vesicle stage) during ovulation. The most commonly used protein source for IVM of canine oocytes is fetal bovine serum (FBS), estrus bitch serum (EBS), or bovine serum albumin (BSA). However, the IVM rate of canine oocytes is still low. One of the reasons may be that the culture medium is not appropriate enough for canine oocytes in IVM. Therefore, we evaluated the effects of four different protein sources, BSA, FBS, EBS, and newborn canine serum (NBCS), on IVM of canine oocytes. The NBCS was collected from 3 puppies that had induced euthanasia within 30 min after delivery. The entire volume of blood was collected by heart puncture, through which an 18 G needle was attached to a 10-mL sterile plastic syringe (without anticoagulant), and then transferred into a 10-mL plastic conical tube. The samples were stored overnight at 4°C to allow clotting to occur. To separate the serum, the sample was centrifuged for 10 min at 3000 rpm; the NBCS was heat inactivated at 56°C for 30 min and stored at -70°C for later use. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and the oocytes collected were cultured in NCSU37 supplemented with 1 mg mL^-1 cysteine, 0.2 mM pyruvic acid, 20 µg mL^-1 E₂, and various protein sources (0.3% BSA, 10% FBS, 10% EBS, and 10% NBCS) at 38.5°C, 5% CO₂ in air for 72 h. Cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were then fixed in 4% paraformaldehyde supplemented with 10 µg mL^-1 Hoechst 33342 at room temperature for 40 min. Nuclear status was observed using fluorescence under a UV light. The proportion of oocytes at the MII stage was significantly higher in the NBCS group than in the EBS, FBS, and BSA groups (14.6 vs. 10.4, 8.8, and 3.3%; P < 0.05). These results indicate that the supplementation of NBCS in IVM medium can enhanced the development of canine oocytes to the MII stage.

This work was supported by KOSEF (Grant M10525010003-05N2501-00310) and ARPC (Grant 203121-03-2-SB010).