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Cryopreservation procedures for Day 7–8 equine embryos
Author(s) -
YOUNG C. A.,
SQUIRES E. L.,
SEIDEL G. E.,
KATO H.,
McCUE P. M.
Publication year - 1997
Publication title -
equine veterinary journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.82
H-Index - 87
eISSN - 2042-3306
pISSN - 0425-1644
DOI - 10.1111/j.2042-3306.1997.tb05111.x
Subject(s) - cryoprotectant , cryopreservation , glycerol , ethylene glycol , embryo , vitrification , chemistry , dilution , liquid nitrogen , andrology , chromatography , acetic acid , staining , biology , biochemistry , medicine , physics , genetics , organic chemistry , microbiology and biotechnology , thermodynamics
Summary Larger grade 1 or 2 (1 = excellent, …. 4 = degenerate) equine embryos that ranged in diameter from 300 to 680 μm 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°C to –35°C at 0.5°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°C. After the final dilution, the embryos were cultured in 5% CO 2 ‐in‐air for 36 h at 38.5°C on equine oviductal epithelial cell monolayers. Their morphology was then evalued, 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 μm; Treatment 2, +179 μm; Treatment 3, +20 μm (P < 0.05). Two 26‐day pregnancies were established following transfer of 6 Treatment 2 embryos (step‐down equilibration method) to recipient mares.