42 DIFFUSION OF THE CRYOPROTECTANTS ETHYLENE GLYCOL AND GLYCEROL INTO DAY 9 TO 11 EQUINE EMBRYOS: PRELIMINARY RESULTS

Abstract

The impermeability of the capsule to cryoprotectants (CPA) has been blamed for poor cryosurvival of equine embryos. We hypothesised that ethylene glycol, with its lower molecular weight and higher permeability, would be preferred over glycerol for equine embryos. The purpose of this study was to determine concentrations of glycerol and ethylene glycol within equine blastocoele fluid of Day 9 to 11 equine embryos following 30 min of exposure to a 1.5 M solution. Fourteen grade 1 embryos were recovered from May through September, using standard transcervical lavage techniques. Day of ovulation (Day 0) was determined as first appearance of the corpus luteum by daily transrectal ultrasonography, following insemination and administration of an ovulatory induction agent (Chorulon, 2500 IU; Intervet, Canada). Embryos were graded, measured, and washed in holding media (emP3^TM Partnar Animal Health, Ontario, Canada), blocked by age, and randomly assigned to either 1.5 M ethylene glycol in holding solution (EG; n = 7) or 1.5 M glycerol in holding solution (Gly; n = 7) each containing 1% dimethyl sulfone as the internal standard, for 30 min at room temperature. Following CPA exposure, embryos were moved to a sterile, dry dish, all fluid was removed with a micropipette, and the dish dried with fine tissue. The embryonic capsule was punctured, and aspirated blastocoele fluid was analysed for CPA concentration by gas chromatography using a CP-3800 gas chromatograph (Varian Canada Inc., Mississauga, Ontario, Canada). The CPA concentration was determined by comparing the internal standard count to the accumulated CPA in millimoles per liter by gas chromatograph. A two-sample Kolmogorov-Smirnov test for equality of distribution was used because data were not normally distributed. Post-hoc 2-way t-test with unequal variances was used to compare mean effective CPA concentration across treatments, with significance set at P = 0.05. Collected embryos ranged from 1470 μm to 9240 μm in diameter, but there was no difference in embryo size between groups (P = 0.59). Mean concentrations of EG from blastocoele fluid were significantly higher than Gly (71.58 ± 18.9 mmol L^–1 v. 50.71 ± 11.2 mmol L^–1, respectively; P = 0.016). This represents 4.8 and 3.4% of available CPA, respectively. Mean EG concentrations by embryo age were not different (Day 9: 68.3 ± 23.1 mmol L^–1; Day 10: 83.0 ± 15.4 mmol L^–1; Day 11: 71.0 ± 21.9 mmol L^–1; P = 0.75). Mean Gly concentrations by embryo age were not different (Day 9: 47.0 ± 12.7 mmol L^–1; Day 10: 46.0 ± 7.1 mmol L^–1; Day 11: 56.3 ± 13.6 mmol L^–1; P = 0.35). The low concentrations of EG and Gly found in this preliminary study are in agreement with indirect evidence from other studies. Ethylene glycol reached higher concentrations inside the embryo than glycerol because of lower molecular weight and higher permeability. However, permeation appeared to be minimal for both agents despite exposure to solutions of high CPA molarity.