Natural honey as a cryoprotectant to improve viability of vitrified bovine oocytes

Abstract

The main objective of this study was to investigate if natural honey can be used as a cryoprotecting agent (CP) in vitrification medium to improve the viability of vitrified-warmed bovine oocytes. The first study was conducted to investigate the dehydration capability of natural honey compared with sucrose, and to determine the proper concentration of honey-based medium and the optimum time for sufficiently safe dehydration of bovine oocytes. Matured cumulus-oocyte complexs (COCs) were denuded and introduced individually into different concentrations (0.25, 0.5, 1.0, 1.5 or 2.0 M) of honey and sucrose-based medium followed by rehydration in control media (TCM). Video images were recorded during dehydration and rehydration, and oocyte images were captured at 12 time intervals to calculate oocyte-volume changes during dehydration and rehydration. Results demonstrated that, in honey-based media, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25M, 0.5M and 1.0M concentrations; while at higher concentrations 1.5M and 2.0M, the maximum dehydration occurred at 30 and 20 seconds respectively. In sucrose-based medium, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25 or 0.5M concentrations. However, at higher concentrations (1M, 1.5M or 2M), the maximum dehydration occurred at 30, 20 and 10 sec. For rehydration, oocytes dehydrated in honey or sucrose-based medium were able to regain their original volume within 60-120 sec. However, oocytes dehydrated in higher concentrations (2M honey, and 1.5M and 2M sucrose) were rehydrated back to their original volume within 20 sec. This study concluded that natural honey and sucrose caused similar cell dehydration. Only oocytes dehydrated in 1M honey-based media reached maximal dehydration after 60 sec and equally regained original volume. Therefore, 1M of honey-based medium is suggested for sufficient and safe oocyte dehydration during vitrification. The second study was conducted to determine in vitro maturation (IVM), in vitro fertilization (IVF) and embryonic development of bovine oocytes vitrified in honey-based vitrifcation media. In Experiment 1, bovine COCs were randomly distributed in control group (non-vitrified; G1), 0.5M sucrose group (second control; G2), and 0.5M, 1M and 1.5M honey groups (G3, G4 and G5 respectively). The COCs were exposed to equilibration solution 1 (VS1) at ~ 22 oC for 5 min and to vitrification solution 2 (VS2) for 1 min, mounted on Cryotops and plunged into LN2. COCs were warmed in TCM and honey/sucrose medium at 38.5oC for 1 min, washed, matured in vitro (IVM), denuded, and immunostained to evaluate maturation. Maturation rate was significantly higher (80.7%) in control group (G1) than in vitrified groups (56, 52, 55 and 51% in G2, G3, G4 and G5, respectively) (P<.0001), whereas there was no significant difference among the vitrified groups (P>0.05). In Experiment 2, bovine COCs distributed in control (not vitrified, G1) and vitrified groups using 1M honey and 0.5M sucrose (G2 and G3 respectively), underwent for IVM, IVF and in vitro culture (IVC) for 9 days. Cleavage rate was significantly higher (P<.0001) in the control group (74%, G1, n=183) than rates of vitrified groups (51% in G2, n=137; and 42% in G3, n=131), whereas no differences among vitrified groups (P=0.0723). Rate of blastocyst formation was significantly higher (34%) in G1 than in the vitrified groups (P<.0001); however, blastocyst formation rates in the honey group were significantly higher (P=0.0026) than in the sucrose group (13% and 3% respectively). Addition of natural honey (1.0M; or 21.7%w/v) in vitrification medium can safely and sufficiently dehydrate bovine oocytes during vitrification procedure. The vitrification of bovine oocytes in 1M honey improved their post-warming maturation abtility and embryonic development.