MOLECULAR REPRODUCTION AND DEVELOPMENT 74:870–877 (2007) Requirement for, and Patterns of, Pyruvate and Glutamine Metabolism in the Domestic Dog Oocyte In Vitro N. SONGSASEN, 1 * R.E. SPINDLER, 2 AND D.E. WILDT 1 1 Department of Reproductive Sciences, Smithsonian’s National Zoological Park, Conservation & Research Center, Front Royal, Virginia 2 Reproductive Program, Toronto Zoo, Scarborough, Ontario, Canada ABSTRACT Supplementation of energy sub- strates to culture medium is essential for resumption and completion of meiosis in vitro for many mamma- lian species. Objectives were to study the dog oocyte, specifically the influences of pyruvate and glutamine on maturation and the utilization of these two substrates at various developmental stages and incubation times. Ovarian oocytes (n¼681) were obtained from spayed bitches and cultured for 48 hr in TCM 199 medium containing various concentrations of pyruvate (0–2.5 mM) and glutamine (0–4 mM) before being assessed for nuclear status. For analyzing metabolic activity, 259 dog oocytes were cultured for 0, 12, 24, 36, or 48 hr, assessed for pyruvate and glutamine metabolism using the hanging drop method and then evaluated for nuclear status. Neither pyruvate nor glutamine had influence (P > 0.05) on oocyte maturation in vitro (IVM). However, both culture interval and meiotic status influenced pyruvate uptake (P < 0.05). Specifically, pyruvate uptake declined as the oocyte progressed from the germinal vesicle (GV) to metaphase II (MII) stage. Glutamine oxidation de- creased as culture duration progressed (P < 0.05). In summary, pyruvate or glutamine is not required to promote successful IVM of dog oocytes. But, both substrates are being metabolized, and in patterns different to the domestic cat, another carnivore species. Pyruvate played an important role earlier in the maturational process, and less glutamine was oxidized as the oocyte neared nuclear maturation. These variations emphasize the importance of defining species specificities in carnivores before expecting consistently successful IVM/IVF. Mol. Reprod. Dev. 74: 870–877, 2007. ß 2006 Wiley-Liss, Inc. Key Words: canine oocyte; nuclear maturation; energy substrate; metabolism INTRODUCTION Assisted reproductive techniques have significant potential as tools for propagating and managing genetically valuable and rare populations and/or species (Pukazhenthi and Wildt, 2004), including canids. To date, embryo transfer has never been consistently successful in the dog because of inadequate knowledge to produce embryos in vitro. It is generally accepted that embryo technologies will become viable given that intraovarian oocytes can be recovered and successfully matured and fertilized in vitro (Rodrigues and Rodri- gues, 2006; van Wagtendonk-de Leeuw, 2006). Interest- ingly, none of these essential techniques (oocyte IVM, in vitro fertilization [IVF], and embryo culture) has been established in the domestic dog, despite the popularity of this species as a companion animal or as a model for studying human genetic disorders (Patterson, 2000) or conserving endangered wild canids (Farstad, 2000; Goodrowe et al., 2000). For example, there are more than 21 human-related genetic disorders that have benefited from basic studies of the domestic dog (Patterson, 2000). Meanwhile, of the 19 species that comprise the Canidae Family, 9 are formally listed as ‘‘vulnerable,’’ ‘‘threatened,’’ or ‘‘critically endangered’’ by the IUCN-World Conservation Union (IUCN, 2006). In short, fundamental information on ovarian, follicu- lar, and oocyte biology in the domestic dog has relevance to propagating and conserving rare canid genotypes, but much more research is required. Despite there being an impressive history of IVM investigations of canine oocytes (Mahi and Yanagima- chi, 1976; Hewitt and England, 1997, 1998, 1999a, 1999b; Hewitt et al., 1998; Otoi et al., 1999, 2000, 2001, 2002; Saint-Dizier et al., 2001; Bolamba et al., 2002; Songsasen et al., 2002, 2003; Rodrigues and Rodrigues, 2003; Luvoni et al., 2005), overall efficiency remains comparatively low (Kim et al., 2004, 2005; De los Reyes et al., 2005; Luvoni et al., 2005; Rodrigues and Rodrigues, 2006). Success is substantially less than in ß 2006 WILEY-LISS, INC. Grant sponsor: National Institutes of Health; Grant number: 1KO1 RR020564-01. *Correspondence to: N. Songsasen, Conservation & Research Center, Smithsonian’s National Zoological Park, 1500 Remount Road, Front Royal, VA 22630-5972. E-mail: songsasenn@si.edu Received 7 September 2006; Accepted 20 September 2006 Published online 21 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20667