331 DEVELOPMENT OF CANINE SYNTHETIC OVIDUCT FLUID (cSOF) MEDIUM AND EFFECT OF THE cSOF IN IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

Abstract

A bitch ovulates a primary oocyte that undergoes both maturation and fertilization within the oviduct fluid for 3 days. In an attempt to define the physiologically appropriate conditions for oocyte maturation in the bitch, in vitro conditions based upon the oviductal environment need to be established. The present study was conducted to develop canine synthetic oviduct fluid (cSOF) by investigating the composition of canine oviduct fluid, follicular fluid, and bursa fluid. The bursa and oviduct fluid were collected at Days 1 and 3 of ovulation, respectively. Before ovulation, follicles were punched and the fluid was collected by aspiration. Biochemical parameters (Ca, P, Mg, albumin, total protein, and glucose) were measured using a chromatographic enzymic method. Quantitative determination of electrolytes (Na, Cl, K) concentration in the follicular, bursa, and oviductal fluids was performed using an Electrolyte 5 Analyzer (Nora Biomedical, Waltham, MA, USA). The concentrations of sodium, potassium, and chloride were similar among oviduct (153.5, 5.2, and 121.5 mmol/L, respectively), bursa (149.5, 4.3, and 123 mmol/L, respectively), and follicular (147, 4.2, and 120.5 mmol/L, respectively) fluids. Glucose concentration was different in oviduct, bursa, and follicular fluids (1.09, 3.75, and 3.94 mmol/L, respectively). Total protein and magnesium concentrations were not different among the three fluids, but phosphorus concentration differed in oviduct, bursa, and follicular fluids (0.001, 0.044, and 0.024 g/L, respectively). The oviduct fluid showed lower concentrations of albumin and calcium (0.001 g/L and 1.372 mmol/L, respectively) compared to bursa (0.023 g/L and 2.532 mmol/L, respectively) or follicular fluid (0.025 g/L and 2.632 mmol/L, respectively). The cSOF1 and cSOF2 were developed on the basis of the oviduct and follicular fluids, respectively. Canine oocytes were recovered by slicing ovaries collected after ovariohysterectomy in bitches at follicular stages, and in vitro nuclear maturation of canine oocytes cultured in cSOF1 or cSOF2 were compared to that of intra-oviduct (in vivo) culture. For in vivo culture, the canine oocytes were transferred and cultured in intra-oviduct for 72 h and were recovered by intra-oviduct flushing using surgical methods. For in vitro culture, canine oocytes were cultured in cSOF1, cSOF2, or TCM-199 (control) for 72 h at 38.5°C in a humidified atmosphere of 5% CO₂ in air. The experiment was replicated three times and statistical analysis was carried out by ANOVA with GLMs in the statistical analysis system program (SAS Institute, Inc., Cary, NC, USA). Nuclear maturation of canine oocytes to MII was not different in cSOF1, cSOF2, or intra-oviduct (2.5%, 2.5%, and 2.2%, respectively) compared to the control (1.6%). However, the degeneration rates were significantly higher in oocytes cultured in intra-oviduct (48.2%) compared to control, cSOF1, and cSOF2 (1.6%, 6.8%, and 7.5%, respectively). In conclusion, the present study analyzed the components of the oviduct, bursa, and follicular fluids and developed two canine synthetic oviduct fluids (cSOF1 and 2). In addition, the present study demonstrated that cSOFs can be used for in vitro maturation of canine oocytes.