Effect of addition of ascorbate, dithiothreitol or a caspase‐3 inhibitor to cryopreservation medium on post‐thaw survival of bovine embryos produced in vitro

Abstract

Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 μM) and z-DEVD-fmk (0, 50, 100 or 200 μM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated.Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

Creating Ex Situ Protected Bioreservoirs as a Powerful Strategy for the Reproductive Biotechnology-Mediated Rescue of Threatened Polish Livestock Breeds

“…Two basic strategies are frequently utilized for the cryopreservation of oocytes and embryos in liquid nitrogen. The first strategy involves the method of slow freezing, in which germplasm-carrying bioresources are sequentially subjected to treatment with relatively low concentrations of cryoprotectants displaying a high capability to permeabilize the plasma membranes [91][92][93]. Some of the most efficient, most potent and most permeable cryoprotectants are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG) and propylene glycol, while their non-permeable counterparts are monosaccharides, e.g., glucose and fructose, and disaccharides, e.g., sucrose [88,90,94].…”

“…Such a finding can be explained by the fact that the viability of murine oocytes that had been subjected to vitrification under the conditions of a very slow cooling rate and high warming rate increased to a large extent, as compared to the viability of oocytes undergoing vitrification characterized by a combination of highly rapid kinetics of cooling and subsequent slow kinetics of warming [86,87,100,101]. Nevertheless, such a negative correlation has not been confirmed in the investigations aimed at the vitrification of oocytes and embryos in selected livestock species (rabbits, pigs and cattle), which exhibit a particular oversensitivity to cooling rates related to cryopreservation procedures [92,99,101,102].…”