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

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.

Effect of addition of l-carnitine to media for oocyte maturation and embryo culture on development and cryotolerance of bovine embryos produced in vitro.

The objective of these experiments was to determine the effect of l-carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1-3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l-carnitine. There was no effect of l-carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l-carnitine treatment on embryo development, but embryos treated with l-carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l-carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l-carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l-carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l-carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l-carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos.