CSIRO PUBLISHING Reproduction, Fertility and Development, 2009, 21, 538–548 www.publish.csiro.au/journals/rfd The effects of hCG and growth factors on in vitro nuclear maturation of dog oocytes obtained during anoestrus A. K. Alhaider A,B and P. F.Watson A,C A Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK. B Present address: Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, PO Box 1757, Al Ahsa 31982, Kingdom of Saudi Arabia. C Corresponding author. Email: pwatson@rvc.ac.uk Abstract. The effects of human chorionic gonadotrophin (hCG) and a combination of growth factors on the develop- mental competence of canine oocytes during in vitro maturation was examined. Oocytes recovered from domestic dog ovaries at routine ovariectomy were cultured in a basic tissue culture medium with 0.3% BSA, 7 μg mL −1 progesterone and antibiotics. After the appropriate culture periods (up to 96 h), they were fixed and labelled by double-antibody immunoflu- orescence for tubulin and with propidium iodide for chromatin. Human chorionic gonadotrophin increased the proportion of oocytes resuming meiosis and reduced the degeneration rate. Supplementing with hCG in declining concentrations was of no superior benefit but the presence of a combination of growth factors (growth hormone, insulin-like growth factor-1, transforming growth factor-α and fibroblast growth factor) improved both the resumption of meiosis and the degeneration rate. No particular synergisms between pairs of growth factors could be demonstrated. Human chorionic gonadotrophin and growth factors together gave poorer results, implying that hCG inhibited the beneficial effects of the growth factors. A growth factor combination is the present most successful treatment, with 49% of total oocytes (inclusive of degenerated) recovered from anoestrous bitches at MI or MII by 96 h of culture. This is the highest result so far demonstrated for cultured dog oocytes. Introduction Assisted reproduction technology (ART) has been successfully used in many species. Healthy offspring have resulted from IVF and intracytoplasmic sperm injection (ICSI) techniques utilis- ing in vitro-matured oocytes in many species and are regularly used in clinical settings. In vitro oocyte maturation in the dog, however, has not been very efficient. Farstad (2000) quoted mat- uration rates varying from 0% to 58% for oocytes matured to MI, anaphase I and MII. A low maturation rate could be due either to low meiotic competence of the oocytes or to suboptimal cul- ture conditions. This low success of oocyte IVM has hampered other ART in canids, such as in vitro embryo production and embryo transfer. The capacity of oocytes to complete nuclear and cytoplasmic maturation in vitro and to support embryonic development after IVF is greatly affected by the components of the maturation medium. Gonadotrophins are regarded as of paramount importance in oocyte maturation. In addition, pep- tide growth factors have been reported to have an autocrine and paracrine regulatory role in ovarian function (Tonetta and diZerega 1989). Peptide growth factors include, among others, growth hormone (GH), insulin-like growth factor 1 (IGF-1), transforming growth factor-α (TGF-α), fibroblast growth factor (FGF) and epidermal growth factor (EGF). Unlike other mammals, canids ovulate eggs in the first mei- otic division, and therefore their eggs may require strategies for in vitro maturation that differ from those used in other species (Bolamba et al. 1998; Durrant et al. 1998). In most mammals, resumption of meiosis is associated with LH release from the anterior pituitary and ovulation occurs after oocytes have arrested at MII (Florman and Ducibella 2006). In the bitch, ovulation of immature oocytes at the GV stage (Andersen and Simpson 1973; Tsutsui 1975; Hyttel et al. 1990) occurs 48 to 72 h after the LH surge (Holst and Phemister 1971; Tsutsui 1975), which itself takes place over 48 h (Badinand et al. 1993). Around ovulation, the oocytes are exposed to high concentrations of progesterone due to the early luteinisation of the pre-ovulatory follicle (Concannon et al. 1977). The oocyte takes 2 to 5 days to complete maturation to MII in the oviduct (Holst and Phemister 1971; Renton et al. 1991). Several reports have investigated hormonal supplementation to improve canine oocyte IVM. However, no positive effect was reported when gonadotrophins were added to the culture medium during the entire culture period (Srsen et al. 1998; Hewitt and England 1999; Songsasen et al. 2002; Rodrigues and Rodrigues 2003). Communication between cumulus cells and the oocyte is a key factor in the acquisition of maturation competence (Luvoni 2000). In rat follicles, LH has been shown to regulate the expression and phosphorylation of connexin-43, the ovarian gap-junctional protein (Granot and Dekel 1994). Gonadotrophin supplementation has been shown to increase © CSIRO 2009 10.1071/RD08167 1031-3613/09/040538