Sessional Committee on Environment and Sustainable Development Written Submissions Received Volume 2 Issues associated with the progressive entry into the Northern Territory of Cane Toads October 2003
Tabled Paper 1123
Tabled Papers for 9th Assembly 2001 - 2005; Tabled papers for 9th Assembly 2001 - 2005; Tabled papers; ParliamentNT
Tabled by Delia Lawrie
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Dr Mahony Written Submissions Cane Toad Inquiry Report Volume 2 172 Hormonal induction of ovulation and in vitro fertilisation with testicular spermatozoa are established procedures that have been used for many years with Anurans (Rugh, 1962; Hollinger & Corton, 1980; Fontdevila et al., 1991). Induction of ovulation is a critical event for IVF in Anurans as only oviductal oocytes are capable of fertilisation. Ovulation in Xenopus laevis and the Bufonidae has been achieved by either injection of gravid females with homologous pituitaries (usually 1 to 6 pituitaries, depending on the species, sex and season) or with mammalian gonadotrophin such as HCG (Rugh, 1962; Carbada et al., 1989; Verhoeff-de Femery & Griffin; Omata, 1993). For the Bufonidae (e.g. Bufo japonicus and B. arenarum) recent IVF work has tended to rely on homologous pituitaries for induction of ovulation (Carboda et al., 1989; Omata, 1993). At least seven species of the Bufonidae have been successfully ovulated using pituitary extracts (Rugh, 1962; Omata, 1993). Step two In vitro fertilisation of eggs with sperm suspensions. We have developed the necessary protocols in our laboratory with the cane toad. Collection of motile, viable spermatozoa for IVF is generally achieved by the maceration of testes into amphibian Ringer's solution of low osmotic pressures (Hollinger & Corton, 1980) in the region of 50-100m Osm kg-1. Very high fertilisation rates (in the order of 90%) can be obtained in Bufonidae and Xenopus with IVF using hormonal induced oocytes and testicular sperm (Hollinger & Corton, 1980). We have established basic IVF as a routine technique with Bufo marinus using pituitary extracts for ovulation and testicular sperm. IVF procedures work well with B. marinus after initial experimental work to optimise conditions. Work we have conducted shows that viable and motile testicular sperm from B. marinus may be collected at any time of the year (and activated in media of low osmotic pressure). However, mature oocytes are only available from females during the breeding season (August - March). We will attempt to further refine our basic IVF procedures for B. marinus by attempting to induce ovarian growth and oocyte maturation in non-seasonal females using gonadotrophin and oestradiol treatments (Wallace & Bergink, 1974; Wallace, 1985; Kwon et al., 1991). We will also investigate other hormonal procedures for the induction of ovulation and testicular sperm release using HCG (Hollinger & Corton, 1980; Verhoeff-de Fremery & Griffin, 1989), progesterone (Wright, 1961; Schuetz, 1971), and dopamine and adrenalin (Minucci et al 1993), as well as investigating the use of arginine vasotocin (AVT)(=oxytocin) to induce oviposition (La Pointe, 1977). Step three We have developed the necessary protocols in our laboratory with the cane toad. Shock treatment of eggs immediately following fertilisation to prevent the extrusion of the second polar body from the egg.
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