Influence of gonadotrophin supplementation on the in vitro maturation of bitch oocytes

IN VITRO maturation and fertilisation techniques are becoming increasingly popular for the manipulation of reproductive function in domestic animals. In carnivores, there is a need for basic investigations into oocyte maturation and the interaction of male and female gametes so that these techniques can be applied to the conservation of endangered species. Currently, two wolf and three fox species are considered to be at risk according to CITES (1997): the grey wolf (Canis lupis), the red wolf (Canis rufus), the Simien fox (Canis [Simenia] simensis), the San Joaquin kit (Vulpes macrotis mutica) and the northern swift fox (Vulpes velox hebes). These in vitro techniques may provide useful information for gamete salvage programmes, and may also allow the establishment of assays that can be used to assess the functional capacity of sperm. Despite the fact that mammalian oocytes released from the follicular environment can mature spontaneously in vitro and subsequently fertilise, their developmental ability is often lower than in vivo (Leibfried and Bavister 1983, Saeki and others 1991). This indicates that the culture systems used are inadequate models of the physiological environment and may require modification by supplementation with various factors. The intrafollicular oocyte is exposed to changing concentrations ofluteinising hormone (LH) and follicle-stimulating hormone (FSH), especially during the pre-ovulatory period. Such exposure may be essential for pre-ovulatory development of the oocyte. When in vitro maturation studies are performed, oocytes are removed from the follicular environment, and therefore from any priming effect of these gonadotrophins. The in vitro maturation studies that have been performed in the domestic bitch have generally used standard culture techniques without hormonal supplementation (Mahi and Yanagamachi 1976, 1978, Shimazu and others 1992, Yamada and others 1992, 1993). In one study, however, oestradiol was added routinely, but no effect on oocyte maturation was described (Nickson and others 1993). Previous work by the present authors also showed that there was no benefit in adding oestradiol and progesterone, alone or in combination, to a culture medium for canine oocyte maturation (Hewitt and England 1997). This short communication describes an investigation into the effect of supplementing oocyte culture medium with LH and FSH, to try to establish the effect of these factors on the follicular canine oocyte in an attempt to optimise the conditions for in vitro maturation and fertilisation. Cumulus oocyte complexes (cocs) were harvested from ovarian tissue from healthy bitches aged one to seven years. Ovarian and uterine tissue and blood samples were processed for histological analysis and plasma progesterone determination, respectively, allowing the categorisation of bitches according to the stage of the oestrous cycle at the time of ovariohysterectomy (Andersen and Simpson 1973, Eckersall and Harvey 1987). The cocs were harvested in modified HEPES-199 culture medium supplemented with 0-3 per cent bovine serum albumin (BSA) and antibiotics. The cocs were graded (Hewitt and others 1998) and Grade 1 cocs were selected for further use. The cocs were cultured in groups of up to 10 with others from the same bitch in 400 St droplets of modified HEPES199 supplemented with 0-3 per cent BSA. To the medium, either: 1 [ig/ml LH; 1 jg/ml FSH; 1 [ig/ml LH + 1 [ig/inl FSH; or no hormonal supplement was added. The LH and FSH were donated by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK, USA). From each bitch, one half of the oocytes were cultured for 48 hours and the remaining half were cultured for 96 hours. Following culture, fixing and staining were performed as previously described (Hewitt and others 1998). Oocytes were examined using light microscopy and classified according to their degree of nuclear maturation (Hewitt and others 1998). These were germinal veside breakdown (GVBD) and metaphase I/anaphase I/ metaphase II (MI/ AI/MII). A total of 238 grade 1 cocs were obtained from 10 bitches. The stage of the oestrous cycle when each sample was obtained was determined, showing that oocytes were obtained from bitches in pro-oestrus (two), oestrus (three) and metoestrus (five). A total of 205 oocytes had identifiable nuclear material following culture. The oocyte maturation data for the 48and 96-hour culture periods were assessed using three orthogonal contrasts. These were: the overall effect of LH; the overall effect of FSH; and the interaction between these two effects. The effect of each of the treatments upon resumption (oocytes undergoing GVBD) and completion (MI/Al/MII) of meiosis were analysed separately. The data were initially assessed for the 48and 96hour culture periods combined to investigate any effect of time, treatment or their interaction (Table 1). Since there was no evidence of any of these effects for oocytes undergoing GVBD, these data were considered for the two culture periods combined. In contrast, there was evidence of an effect oftime upon oocytes maturing to MI/Al/Mll, so this analysis was performed for the 48and 96hour data separately (Table 2). It was shown that GVBD occurred in each of the culture treatments. This meant that chi-squared analysis was appropriate. In contrast, no oocytes matured to MI/AI/MII in five of the eight treatment groups: the control medium after both 48 and 96 hours; the FSH treatment after 48 hours; and both LH