Abstract
Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae.
Highlights
Cryopreservation of bovine embryos is widely used for trade of genetically superior animals and conservation of genetic diversity
Using FlexArray software, total 64 genes differentially expressed in blastocysts developed from vitrified compared to control morulae; and 1 and 165 genes differentially expressed in blastocysts developed from slow-frozen compared to control morulae at fold change ! ± 2 and ! ± 1.5, respectively (P < 0.05; Table 2)
The current study revealed the downregulation of genes involved in steroid biosynthesis, pregnenolone biosynthesis and eicosanoid signaling (cytochrome P450 subunit 11 type A 1 (CYP11A1), 3-beta hydroxy steroid dehydrogenase delta-isomerase type 1 (HSD3B1), ATP-binding cassette subfamily C-2 (ABCC2) and prostaglandin synthase 2 (PTGS2) / cyclooxygenase 2 (COX2) in blastocysts developed from vitrified morulae
Summary
Cryopreservation of bovine embryos is widely used for trade of genetically superior animals and conservation of genetic diversity. Bovine embryos are commonly frozen with either slow freezing or vitrification method [1]. Both techniques differ in concentration of cryoprotectants and cooling rates [2]. It damages mammalian embryos due to intracellular ice formation and toxic effect of the permeating cryoprotectants (~1–2 M). The attempts have been made to minimize damage to morulae/blastocysts caused by cryoprotectant-associated toxicity and intracellular ice formation during cryopreservation [1,6]. Vitrification includes the use of highly viscous solution of cryoprotectants (~7–8 M) to achieve a glass-like state with ultra-rapid cooling rates (>5000 ̊C/min) avoiding the intracellular ice formation [7,8]. Vitrification and slow freezing cause intracellular/extracellular fractures in freezing planes, acute shrinkage in cell volume and organelle damage in mammalian embryos [1,14,15]
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