title: Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes.
abstract: Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro.
authors.affiliation: Department of Farm Animal and Equine Medicine and Surgery, Royal Veterinary College, University of London, Hatfield, UK.
authors.forename: D A
authors.initials: DA
authors.lastname: Hewitt
authors.forename: P F
authors.initials: PF
authors.lastname: Watson
authors.forename: G C
authors.initials: GC
authors.lastname: England
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods / Q000662:veterinary; D002467:Cell Nucleus / Q000502:physiology; D004396:Coloring Agents / Q000737:chemistry; D004285:Dogs / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006651:Histocytochemistry; D008858:Microscopy, Phase-Contrast / Q000662:veterinary; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D010053:Ovary / Q000166:cytology; D010078:Oxazines / Q000737:chemistry; D011374:Progesterone / Q000097:blood; D012710:Serum Albumin, Bovine / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods / Q000662:veterinary*; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary*
chemical_list: D004396:Coloring Agents; D010078:Oxazines; C018437:aceto-orcein; D012710:Serum Albumin, Bovine; D011374:Progesterone