title: A simple and high-throughput method to assess maturation status of bovine oocytes: comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique.
abstract: A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4',6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.
authors.affiliation: Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Saskatchewan, Canada.
authors.forename: J R
authors.initials: JR
authors.lastname: Prentice-Biensch
authors.forename: J
authors.initials: J
authors.lastname: Singh
authors.forename: B
authors.initials: B
authors.lastname: Alfoteisy
authors.forename: M
authors.initials: M
authors.lastname: Anzar
mesh_terms: D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes; D007211:Indoles; D034904:Lamin Type A / Q000276:immunology*; D008677:Metaphase; D009865:Oocytes / Q000502:physiology / Q000648:ultrastructure*; D010078:Oxazines; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D034904:Lamin Type A; D010078:Oxazines; C018437:aceto-orcein; C007293:DAPI