Abstract
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.
Highlights
Sugars are commonly used as energy substrate and/or nonpermeating cryoprotectants (CPs) in cryoprotective solution
The specific objectives of this study were to determine osmolalities of different concentrations of honey and sucrose, to compare the volumetric change in bovine oocytes in different concentrations of natural honey and sucrose solutions, and to investigate in vitro maturation (IVM), fertilization (IVF) and embryo development (IVC) of germinal vesicle (GV)-stage oocytes vitrified in honey and sucrose solutions
The time required for maximum dehydration in bovine oocytes was 60 s in all concentrations of honey and sucrose (Fig 2)
Summary
Sugars are commonly used as energy substrate and/or nonpermeating cryoprotectants (CPs) in cryoprotective solution. Sugars, being nonpermeating CPs, cause an osmotic gradient across the cell membrane that enhances cell dehydration before freezing to reduce the quantity of intracellular water. This reduction in water quantity decreases intracellular ice formation and minimizes lethal freezing injuries [1, 2]. Honey and cryopreservation of bovine oocytes compartment, decrease the concentration of permeating CPs and lessen the associated intracellular toxicity [3, 4]. Vitrification solution containing sugars improved the survival of bovine blastocysts and human immature oocytes following vitrification [5, 6]
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