title: Effect of culture-medium supplementation with alpha-mannosidase and/or beta-N-acetyloglucosaminidase on in vitro bovine embryonic development.
abstract: 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 beta-N-acetyloglucosaminidase (beta-NAGASE) and/or alpha-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 microl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 degrees C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) beta-NAGASE, (2) alpha-mannosidase, (3) beta-NAGASE plus alpha-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 x 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 alpha-mannosidase, 424 mOsm by beta-NAGASE and 337 mOsm with a combination of the two enzymes. The beta-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 (P< 0.002), relative to control, by alpha-mannosidase supplementation. The reduced embryo development in the beta-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize alpha-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 beta-NAGASE increased medium osmolarity, embryos utilized alpha-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.
authors.affiliation: NAGREF, Veterinary Research Institute of Thessaloniki, Ionia, Thessaloniki 57008, Greece. tsiligianni@nagref.gr
authors.forename: Theodora
authors.initials: T
authors.lastname: Tsiligianni
authors.forename: Leen
authors.initials: L
authors.lastname: Vandaele
authors.forename: Aart
authors.initials: A
authors.lastname: de Kruif
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002417:Cattle / Q000196:embryology*; D003470:Culture Media; D046149:Embryo Culture Techniques / Q000662:veterinary; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000201:enzymology; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D009994:Osmolar Concentration; D043323:alpha-Mannosidase / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D006026:Glycoside Hydrolases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase