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

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.

Fig 1. Relationship between molal concentrations and osmolalities of 0.25, 0.5 and 1 M honey and sucrose solutions.

Fig 2. Effect of honey (H) and sucrose (S) concentrations (0.25, 0.5, 1, 1.5 and 2 M), time (0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 and 180 s) and their interactions on ooplasm volume (μ³ × 1000) during dehydration and rehydration phases.

Each point represents a mean ± SEM (n = 3 oocytes per treatment group per replicate × 3 replicates).

Fig 3. Relationship between molal concentrations of honey and sucrose, and ooplasm shrinkage at 60 s during dehydration phase (n = 3 oocytes per treatment group per replicate × 3 replicates).

Fig 4. In vitro maturation of bovine oocytes (GV-stage) vitrified in 0.5 M sucrose and different concentrations of honey solutions.

Each bar represents mean ± SEM from 5 replicates. Different letters on bars represent differences among treatment groups (P < 0.05). Letter n represents the number of COCs used in control and vitrified groups; MII, metaphase-II.

Fig 5. In vitro cleavage and blastocyst rates in oocytes (GV-stage) vitrified in 0.5 M sucrose or 1 M honey solution.

Each bar represents mean ± SEM from 5 replicates. Within an embryonic stage, different letters on bars represent difference among treatment groups (P < 0.05). Letter n represents the number of COCs used in control and vitrified groups.