213 THE EFFECTS OF SERICIN SUPPLEMENTATION IN IN VITRO CULTURE MEDIUM ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

Abstract

The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus–oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22 h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002 AU mL–1 of FSH, and 1 μg mL–1 of oestradiol-17β at 38.5°C under an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n = 400) or sericin at 3 different concentrations (wt/vol; 0.05%: n = 493; 0.1%: n = 419; or 0.15%: n = 520; sericin groups) at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 for 5 days. They were then transferred into each IVC medium supplemented with 0.1 mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n = 51, 0.05% sericin group: n = 56, 0.1% sericin group: n = 44, 0.15% sericin group: n = 44). They were frozen in m-PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose, 20% calf serum, and 4 mg mL–1 of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol under the same atmosphere used for IVC for 72 h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72 h after thawing. Rates of cleavage and blastocyst formation were expressed as mean ± SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher’s exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6 ± 15.1 and 22.1 ± 3.5%; 0.05% sericin: 71.6 ± 12.0 and 19.4 ± 6.7%; 0.1% sericin: 70.3 ± 4.7 and 18.4 ± 5.6%; 0.15% sericin: 66.9 ± 10.3 and 17.9 ± 6.9%, respectively). There were no significant differences in post-thaw survival rates after 24 h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72 h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P < 0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.