68 OSMOTIC RESPONSES OF EQUINE EMBRYOS WITH AND WITHOUT CAPSULES TO CRYOPROTECTANTS

Equine embryos become more difficult to cryopreserve as they get larger, which may be due in part to the presence of the capsule. We hypothesized that presence of the capsule will alter osmotic responses of equine embryos. Embryos were collected from mares of light horse breeds. Ovulation was confirmed by daily transrectal ultrasonography, and uterine flushes were performed 7 or 8 days post-ovulation. At collection, embryos were evaluated for size and morphology and graded on a scale of 1 to 5, 1 being excellent and 5 being degenerate/dead. Only embryos of grades 1 or 2 were used. Boyle Van’t Hoff plots were created for embryos <300 (n = 2), 300–600 (n = 5), or >600 μm (n = 2) in diameter from data collected after incubating embryos in a series of increasingly concentrated sucrose solutions of 250, 500, 1000, and 2000 mOsm in Syngro® (275 mOsm) at room temperature (27–30°C). Embryo diameters were measured every minute for at least 60 min. Volumes were calculated, and relative volumes calculated against that were measured immediately after the embryo was flushed from the uterus (100%) while the embryo was in Syngro®. Regression analyses were used to estimate the y-intercept from which the percentage volume that is osmotically active water was calculated [91.9 for embryos < 300 μm (P = 0.287), 85.7 for 300–600 μm (P = 0.0005), and 80.0% for >600 μm (P = 0.003)]. To evaluate osmotic responses of equine embryos to 3 cryoprotectants, Day 7 embryos between 300 and 600 μm with or without a capsule were transferred to 5 mL of 1.5 M methanol (n = 3 with capsule, n = 2 without), 1.5 M glycerol (n = 5 with, n = 2 without), or 0.75 M ethylene glycol + 0.75 M methanol (n = 6 with, n = 3 without) in Syngro®. Diameters were measured every min for the first 30 min and every 5 min thereafter. Upon removal of the capsule and before exposure to cryoprotectants, embryos lost and did not recover on average 22% of their initial volume (range 9 to 30%) after 15 min of incubation in Syngro® at room temperature (27 to 30°C). When incubated in 1.5 M methanol, embryo volume increased ∼10%, and then decreased to ∼95% original volume after 1 h incubation for embryos that had intact capsules; embryos without capsules remained ∼5% larger than initial volume throughout incubation. When incubated in 1.5 M glycerol, embryos with capsules decreased to ∼40% within 2 min and did not recover volume during incubation; embryos without capsules decreased to ∼75% of original volume gradually over 60 min. The volume decrease of embryos with a capsule is similar to previous reports for embryos of similar size in 1.5 M glycerol. Embryos incubated in 0.75 M ethylene glycol + 0.75 M methanol without a capsule initially lost ∼15% of their volume and remained at ∼87% of initial volume for the duration of the experiment; embryos with capsules lost nearly 30% of their original volume but then recovered volume to ∼92% of original volume by the end of 1 h. These experiments suggest that the equine capsule may impede movement of glycerol and, to a lesser extent, ethylene glycol into the embryo.