NUCLEAR STAINING AND CULTURE REQUIREMENTS FOR IN-VITRO MATURATION OF DOMESTIC BITCH OOCYTES

Oocyte nuclear staining and culture requirements for in vitro maturati on (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear mate rial (573 oocytes). The most favorable method involved fixation plus a ceto-orcein staining and light microscopy. The influence of serum supp lementation 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 ves icle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 o r 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 metaphas e I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectivel y. 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 concentra tions of FCS appear to be optimal for bitch oocyte maturation in vitro . (C) 1998 by Elsevier Science Inc.