Birth of Koalas Phascolarctos cinereus at Lone Pine Koala Sanctuary following artificial insemination

This paper documents the successful development of an artificial insemination (AI) programme for the Koala Phascolurctos cinereus. The protocols for trials involving two methods to induce ovulation and two insemination techniques are described. In Trial 1, interrupted coitus using a 'teaser'♂ successfully induced ovulation in nine Koalas. Five ♀♀ were inseminated while conscious using a modified 'foley catheter' (Cook insemination catheter) resulting in the births of two offspring. The other four ♀♀ were anaesthetized and inseminated using a technique which allowed visualization of the most cranial portion of the urogenital sinus, where semen was deposited using a 3.5 Fr. 'Tom-cat catheter' (urogen-itoscopic insemination). Three of the four ♀♀ inseminated by this technique produced pouch young. Microsatellite analysis of DNA from the pouch young excluded the teaser ♀♀ as possible sires, confirming that all offspring were sired by donor sperm. In Trial 2, eight ♀♀ were induced to ovulate by injecting them with 250 International Units of human chorionic gonadotrophin (hCG). A luteal phase was confirmed in all eight ♀♀ but only one gave birth following urogenitoscopic insemination. The Koala pouch young in this study are the first of any marsupial to be conceived and born following A1 procedures. Details of the A1 procedures used are presented and the significance of A1 to the conservation biology of P. cinereus discussed.

Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system

Two methods were compared for determining the concentration of penetrative biomass during growth of Rhizopus oligosporus on an artificial solid substrate consisting of an inert gel and starch as the sole source of carbon and energy. The first method was based on the use of a hand microtome to make sections of approximately 0.2- to 0.4-mm thickness parallel to the substrate surface and the determination of the glucosamine content in each slice. Use of glucosamine measurements to estimate biomass concentrations was shown to be problematic due to the large variations in glucosamine content with mycelial age. The second method was a novel method based on the use of confocal scanning laser microscopy to estimate the fractional volume occupied by the biomass. Although it is not simple to translate fractional volumes into dry weights of hyphae due to the lack of experimentally determined conversion factors, measurement of the fractional volumes in themselves is useful for characterizing fungal penetration into the substrate. Growth of penetrative biomass in the artificial model substrate showed two forms of growth with an indistinct mass in the region close to the substrate surface and a few hyphae penetrating perpendicularly to the surface in regions further away from the substrate surface. The biomass profiles against depth obtained from the confocal microscopy showed two linear regions on log-linear plots, which are possibly related to different oxygen availability at different depths within the substrate. Confocal microscopy has the potential to be a powerful tool in the investigation of fungal growth mechanisms in solid-state fermentation. (C) 2003 Wiley Periodicals, Inc.