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Sugars are commonly supplemented into vitrification solution to dehydrate cells in order to reduce the formation of fatal intracellular ice crystals. Natural honey is a mixture of 25 sugars (mainly fructose and glucose) that have different biological and pharmacological benefits. The present study was designed to determine if honey can be used as a nonpermeating cryoprotectant in vitrification of bovine oocytes. In the first experiment, denuded-MII oocytes were exposed to 0.25, 0.5, 1.0, 1.5 or 2.0 M of honey or sucrose. Natural honey and sucrose caused similar ooplasm dehydration. A significant relationship existed between time and ooplasm volume change (P < 0.05), during dehydration and rehydration phases, in both honey and sucrose solutions. In the second experiment, the immature cumulus-oocyte complexes (COCs) were vitrified in an EG/DMSO-based vitrification solution containing honey (0.5, 1 or 1.5 M) or sucrose (0.5 M) as a gold standard. The vitrified-warmed COCs were matured in vitro and evaluated for nuclear maturation. The maturation (MII) rate was greater in nonvitrified control (81%) than vitrified groups (54%, P < 0.05). In the third experiment, COCs were either remained nonvitrified (control) or vitrified in 1.0 M honey or 0.5 M sucrose, followed by IVM, IVF and IVC (for 9 days). Cleavage rate was greater in control (74%) than in vitrified groups (47%, P < 0.05), without significant difference between sugars. Blastocyst rate was 34, 13 and 3% in control, honey and sucrose groups respectively (P < 0.05). In conclusion, natural honey acted as a nonpermeating cryoprotectant in vitrification solution and improved the embryonic development in vitrified bovine COCs. [ABSTRACT FROM AUTHOR]

Egg yolk is a common ingredient of mammalian semen extender to protect sperm against initial cold shock. However, egg yolk has biosecurity risks. Our main objectives were to cryopreserve bull semen without egg yolk using exogenous cholesterol and to study the protective role of glycerol in egg yolk-free semen extender. Other objectives were to compare protein profiles and in vitro fertilization potential of bull sperm frozen with and without egg yolk. In first experiment, semen was either diluted in conventional tris-egg yolk glycerol (TEYG control) extender or first treated with cholesterol-cyclodextrin complex (CC, 2 mg/ml semen) followed by dilution in egg yolk-free tris-glycerol (TG) extender (collectively called as "CC+TG") at 22°C or 4°C, and frozen. Post-thaw sperm motion characteristics were similar between CC+TG and TEYG control extenders, and temperature of glycerol addition. In second experiment, semen was frozen in CC+TG extender varying in glycerol concentration (7 to 0%; v/v). Post-thaw sperm quality decreased with the decline in glycerol concentration in TG extender, even higher concentration of CC complex (3 or 4 mg/ml semen) could not protect sperm in the absence of glycerol in TG extender. In third experiment, SDS electrophoresis of proteins from fresh sperm and sperm frozen in CC+TG, and TEYG control extenders was conducted. Protein profiles in fresh sperm and CC+TG frozen sperm were almost similar. Egg yolk proteins bound tightly with sperm plasma membrane. In fourth experiment, in vitro fertilization potentials of sperm frozen in TEYG control and CC+TG extenders were tested. Cleavage and blastocyst rates of semen frozen in CC+TG and TEYG control extenders were similar. In conclusion, cholesterol-cyclodextrin replaced egg yolk from the semen extender; glycerol remained essential for egg yolk-free sperm cryopreservation; and CC+TG extender did not modify sperm plasma membrane CC+TG whereas egg yolk extender changed the plasma membrane composition of bull sperm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage. [ABSTRACT FROM AUTHOR]

Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (−171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification. [ABSTRACT FROM AUTHOR]