Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes

Abstract

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.

Natural honey acts as a nonpermeating cryoprotectant for promoting bovine oocyte vitrification

“…Several attempts were made to improve the blastocyst rate following vitrification of germinal vesicle (GV)-stage bovine oocytes. Cholesterol-loaded cyclodextrin improved the survival of bovine oocytes [24] but could not improve the blastocyst development [25]. Similarly, the pretreatment of GV oocytes with cytoskeletal relaxant cytochalasin B did not improve the blastocyst rate of vitrified GV oocytes [26].…”

Cryopreservation of farm animal gametes and embryos: recent updates and progress

“…Other chemicals, such as conjugated linoleic acid [75] , L-carnitine [76] , glutathione [77] and a cAMP agonist [78] have also improved outcomes. Cholesterol, coenzyme Q10, BAPTA-AM (Ca 2+ chelator) and ruthenium red have also improved the freezability of in vitro matured bovine oocytes [79][80][81] . Liquid helium vitrification of immature bovine oocytes had better outcomes for reducing injury to the cytoskeleton structure and improving the viability compared with liquid nitrogen vitrification [82] .…”

Features of the preparation of biological material for genome editing in cattle

“…Follicles of from 2 to 8 mm are aspirated with a 18G needle attached to a 5-10 ml syringe [18,24]. Transportation is done in special incubators with constant temperature 30-35 °С [11,24]. According to the data obtained by L. V. Golubets and co-authors, the optimum delivery time of oocytes tissue or aspirated oocyte-cumulus complexes at temperature of 37 °С is not more than 3 hours, whereas formation of morulas/ blastocytes can be up to 27 %.…”

“…For thawing, the device for vitrification is placed in 2-5 ml of medium containing 0.5-1 mol/L saccharose or 0.3 mol/L trehalosa at temperature of 37 °С for 1 minute. After that oocytes/zygotes are transferred to the medium with reduced concentration of saccharose or trehalosa for 3-5 minutes in each medium [11,14]. Gradual changes in concentration of penetrating cryoprotectors is necessary to prevent excess increase of cell volume and cell lysis during influence of large osmotic gradients on the membrane.…”