42 Glycerol Dilution in the Straw by Mixing Methanol and Sucrose for Bovine Embryo Cryopreservation

Abstract

This report focuses on post-thawing dilution of glycerol within the straws using a solution of methanol and sucrose. Dilution media contained 1.5 or 2.0 M methanol and 0 or 1.0 M sucrose (Suc). Straws of 0.25-mL were loaded as follows: (1) 15 mm of 10% glycerol (Gly) solution; (2) 7 mm of air layer; (3) 7 mm of Gly solution; (4) 7 mm of air layer; (5) 10 mm of Gly solution with an embryo; (6) 12 mm of air layer; and (7) 50 mm of dilution medium. Straws were then heat sealed. The ratio of Gly layer to dilution media layer was 1:6 after the solutions were properly loaded into the straw (post-thawing, 10% Gly then was diluted to 1.6%). For freezing, straws were held with cotton plug down and immersed in an alcohol bath to the column containing the embryo. The dilution media layer was not immersed in the bath immediately. When temperature reached –10°C or lower, the straw was taken out the alcohol bath and shaken towards a cotton plug, and the whole straw was immersed into the bath. By this operation, the air layer above the already frozen column containing the embryo was removed, and the dilution medium was directly mixed with Gly. For thawing, straws were kept in air for 5 to 8 s, and then immersed in a water bath at 25°C with cotton plug down for 3 or 10 min. All 18 fresh embryos exposed to 1.5 M methanol + 1.0 M Suc for 40 min at room temperature continued to develop for 48 h in TCM-199. Thawed embryos from diluting glycerol in the straw with methanol + Suc survived well regardless of sucrose concentration. All 28 embryos diluted in the straw with 1.5 M methanol + 1.0 M Suc, and all 14 from 2.0 M methanol without sucrose survived in culture. From 2009 to 2016, the direct transfer of in-straw glycerol diluted embryos using the 1.5 M methanol + 1.0 M Suc treatment led to 2,358/3,702 (63.7%) pregnancies. The mixtures of methanol and sucrose seemed to have no adverse effect on either fresh or frozen embryos. The hydrophilic methanol may absorb water from embryos at the same time that it antagonizes movement of glycerol into the embryo. This may allow gentle intracellular and extracellular glycerol equilibration.