Abstract
The protocol of in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size ( ? 180 ?m) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro -cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi 2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development between both IVP protocols, the use of different protocols under the same laboratory conditions did not afect the in vitro and in vivo embryo viability after vitrification into hand-pulled glass micropipettes.
Highlights
Bovine embryos generated by distinct in vitro production (IVP) systems can respond differently to cryopreservation approaches [1,5,36]
The embryo cryotolerance largely depends on the IVP conditions, which is a determinant of embryo quality, but cryosurvival can be improved by adjusting or improving the cryopreservation procedures
Cumulusoocyte complexes (COCs) isolated by ovary slicing were evaluated under a stereomicroscope and only COCs with evenly granulated cytoplasm and surrounded by a compact and dense cumulus cell layer were used in two distinct IVP protocols
Summary
Bovine embryos generated by distinct in vitro production (IVP) systems can respond differently to cryopreservation approaches [1,5,36]. It is difficult to compare data from different laboratories due to the diverse experimental and enviromental conditions. This variability may be determined by a multitude of factors such as water quality [21,22], type of oil overlay [36], oocyte quality [15,28], and culture systems [18,19,27], among others. The rapid cooling and warming rates in the OPS system may be accompanied by variations in the vitrification container [3,20] In this view, the use of glass micropipettes may offer physical advantages over plastic straws. Methodologies that allow the direct transfer of the embryos after the in-straw cryoprotectant dilution [16,34] have not been tested with glass micropipettes
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