Cryopreservation procedures for Day 7-8 equine embryos.

Larger grade 1 or 2 (1 = excellent,.... 4 = degenerate) equine embryos that ranged in diameter from 300 to 680 microm and were recovered from mares on Day 7 or 8 after ovulation, were randomly assigned to 3 widely divergent cryopreservation treatments. Treatment 1 consisted of cooling from -6 degrees C to -35 degrees C at 0.5 degrees C per min followed by plunging into liquid nitrogen, with a one-step addition and a 4-step removal of 1.0 M glycerol. Treatment 2 (step-down equilibration) consisted of a 2-step addition of glycerol to 4.0 M followed by a decrease to 2.0 M prior to freezing, with galactose present in the final step and in all glycerol removal steps and with a cooling protocol identical to Treatment 1. Treatment 3 was a standard vitrification protocol with step-wise addition of ethylene glycol up to 11.9 M, and step-wise removal of cryoprotectant with decreasing concentrations of galactose in the dilution medium. The cryoprotectants were removed at 20-21 degrees C. After the final dilution, the embryos were cultured in 5% CO2-in-air for 36 h at 38.5 degrees C on equine oviductal epithelial cell monolayers. Their morphology was then evaluated, their capsules were removed mechanically, and they were fixed prior to staining with 1% w:v orcein in 45% acetic acid to assess the morphology of their nuclei and cytoplasm. All 7 embryos in Treatment 1 degenerated during thawing or culture. Of the 6 embryos included in Treatment 2, 4 were graded 1, one was graded 2 and one graded 3 after culture in vitro. Of the 7 embryos in Treatment 3, one was graded 2, one was graded 3 and the remaining 5 were degenerate (P<0.01 among treatments). The average changes in initial diameter exhibited by the frozen/thawed embryos during culture after thawing were: Treatment 1, -91 microm; Treatment 2, +179 microm; Treatment 3, +20 microm (P<0.05). Two 26-day pregnancies were established following transfer of 6 Treatment 2 embryos (step-down equilibration method) to recipient mares.