Effect of culture-medium supplementation with α-mannosidase and/or β-N-acetyloglucosaminidase on in vitro bovine embryonic development

Glycosidases are enzymes with a potential role in embryonic development. The objectives of this study were to assess: (a) whether in vitro bovine embryonic development is affected by the addition of β-N-acetyloglucosaminidase (β-NAGASE) and/or α-mannosidase to the culture medium and (b) whether these enzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were produced using standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50 μl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 °C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) β-NAGASE, (2) α-mannosidase, (3) β-NAGASE plus α-mannosidase, and (4) control (no supplement). Embryos were evaluated and samples of culture medium collected and frozen prior to assay for glycosidases at day 7 of culture. The experimental design was a randomised block arrangement of 4 treatments × 7 replicates with 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean ± S.E.M. The osmolarity of the control culture medium was 272 mOsm. This was increased to 279 mOsm by the addition of α-mannosidase, 424 mOsm by β-NAGASE and 337 mOsm with a combination of the two enzymes. The β-NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages (P < 0.002) relative to control medium (35.7 ± 8.4%). Embryo development was also reduced to 21.9 ± 3.2 (Ρ < 0.002), relative to control, by α-mannosidase supplementation. The reduced embryo development in the β-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize α-mannosidase because its concentration decreased from 600.95 ± 174.03 IU/l in drops without zygotes/embryos to 211.01 ± 71.59 IU/l in drops with zygotes/embryos. Other culture media supplementation showed no significant differences between droplets, with or without zygotes/embryos. It was concluded that β-NAGASE increased medium osmolarity, embryos utilized α-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.