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Reproductive techniques

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

Johnston, SD, MacCalum, C, Blyde, D, McClean, R, Lisle, A & Holt, WV 2006, Cryobiology, vol. 53, no. 1, pp. 218-228.

Cryopreservation of spermatozoa is far less successful for koala sperm compared to the sperm of the closely-related common wombat. After thawing, the survival of spermatozoa was greatest when samples were frozen slowly in a 14% glycerol solution. Koala sperm also has low osmotic tolerance compared to that of the wombat. The authors aimed to determine the factors responsible for these differences in post-thaw survival in the two marsupials and predicted that the presence of the microfilamentous protein F-actin in koala spermatozoa might cause inflexibility in the sperm plasma membrane. As F-actin was not detected, however, it is suggested that some other factor may be responsible for the low success rates of koala sperm cryopreservation.

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Antibiotics for the preservation of the koala (Phascolarctos cinereus) semen

Johnston, SD, O’Boyle, D, Frost, AJ, McGowan, MR, Tribe, A & Higgins, D 1998, Australian Veterinary Journal, vol. 76, no. 5, pp. 334-338.

From samples of koala prepuce and ejaculate collected using electroejaculation and artificial vagina methods, a range of bacteria was identified in koala semen, predominantly of the Corynebacterium genus but of unknown species. The antibiotics penicillin G at a dose rate of 1000-2000 IU/mL, and gentamicin at a dose rate of 100 to 200 µg/mL, had no detrimental effects on sperm motility within a 24-hour incubation period. Furthermore, the antibiotics inhibited the growth of bacterial contaminants in the semen.

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Artificial insemination in the koala: the how and why

Johnston, SD & O’Callaghan, P 2001.

The first birth of a koala, and indeed a marsupial, as a result of artificial insemination (AI) occurred at Lone Pine Koala Sanctuary in 1998, and an additional five koalas were later confirmed to be born following AI. In this presentation, Johnston and O’Callaghan provide an overview of their many developments in establishing protocols and techniques for the successful production of young in captive koalas via AI.

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Artificial insemination the koala: its role in conservation biology and impact on current wildlife legislation

O’Callaghan, P & Johnston, SD 1998, Lone Pine Koala Sanctuary.

Assisted breeding technologies, and specifically artificial insemination, present exciting prospects for the management of both captive and wild koala populations. Artificial insemination techniques have been successfully applied in previous case studies with several eutherian species in captivity, but have only recently shown success in marsupial species.

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Assisted breeding technology for the conservation and propagation of Phascolarctos cinereus or how to make a koala pouch young

Johnston, SD, McGowan, MR & O’Callaghan, P 1999, Proceedings of the Postgraduate Committee in Veterinary Science, Sydney, pp. 199-217.

This report presents an overview of the scientific developments that have contributed to the early success of artificial insemination (AI) in the koala. In the AI process, there are three major hurdles to be overcome: (1) collecting, manipulating and preserving semen; (2) selecting a suitable time for insemination; and (3) depositing inseminate at a suitable site to increase the likelihood of conception. Johnston, McGowan and O’Callaghan share their insights regarding these challenges and present a rationale for the ex situ conservation of koalas via artificial breeding programs.

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

Johnston, SD, McGowan, MR, O’Callaghan, P, Cox, R, Houlden, B, Haig, S & Taddeo, G 2003, International Zoo Yearbook, vol. 38, no. 1, pp. 160-172.

In May 1998, Lone Pine Koala Sanctuary announced the birth of the first koala produced by artificially inducing ovulation and artificial insemination. The success of this procedure was a first not only for the koala, but any marsupial species. An additional five koalas were also confirmed to be produced by artificial insemination.

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Characteristics of koala (Phascolarctos cinereus adustus) semen collected by artificial vagina

Johnston, SD, O’Callaghan, P, McGowan, MR & Phillips, NJ 1997, Journal of Reproduction and Fertility, vol. 109, no. 1, pp. 319-323.

An artificial vagina is recommended as a simple, reliable and non-invasive method of semen collection from koalas in captivity. Out of 90 attempts, 40% resulted in collection of a complete ejaculate comprising a rubbery copulatory plug fraction and a sperm fraction with high motile spermatozoa content. No semen was collected in 38% of trials, while only partial ejaculates were collected in 14.5% of trials. Urine was detected in 4.5% of samples, and 3% of semen samples were collected after the koala’s ejaculation behaviour had ended.

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Individual variability in post-thaw sperm survival in a captive koala population

Zee, YP, Holt, WV, Nicolson, V, Pyne, M, Johnston, SD 2009, Cryobiology, vol. 59, no. 1, pp. 69-74.

The tolerance of koala sperm obtained using electroejaculation to cryopreservation exhibits considerable variation at an individual level, supporting the hypothesis that variation in post-thaw sperm viability exists in a population. Single koala spermatozoa samples obtained from 22 captive koalas revealed that sperm viability and quality differed less between individuals prior to the cryopreservation process than after. This suggests that cryo-tolerance and post-thaw viability are dependent upon individual biophysical sperm characteristics.

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Koala (Phascolarctos cinereus) captive husbandry guidelines

Jackson, S, Perry, L, O’Callaghan, P, Spittal, D, Romer, L & Reid, K 1999

These guidelines for the husbandry of koalas in captive populations represent the combined insights of keepers from Lone Pine Koala Sanctuary, Healesville Sanctuary, Taronga Zoo and Currumbin Wildlife Sanctuary. The authors report on best practice principles in multiple aspects of the care and management of the species.

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Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus)

Johnston, SD, McGowan, MR, O’Callaghan, P, Cox, R & Nicolson, V 2000, Journal of Reproduction and Fertility, vol. 120, no. 1, pp. 59-64.

A luteal or ovulation phase in the oestrous cycle of the female koala can be effectively induced by both the physical act of mating with a male or pharmacologically following a dose of the reproductive hormone human chorionic gonadotrophin (hCG).

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Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Johnston, SD, McGowan, MR, Phillips, NJ & O’Callaghan, P 2000, Journal of Reproduction and Fertility, vol. 118, no. 1, pp. 273-281.

Under the same physical and chemical conditions in short-term liquid storage, koala spermatozoa respond similarly to eutherian mammal spermatozoa, except that koala spermatozoa are more resilient to exposure to rapid changes in temperature.

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Seasonal reproduction in wild and captive male koala (Phascolarctos cinereus) populations in south-east Queensland

Allen, CD, de Villiers, DL, Manning, BD, Dique, DS, Burridge, M, Chafer, ML, Nicolson, VN, Jago, SC, McKinnon, AJ, Booth, RJ, McKee, JJ, Pyne, MJ, Peng Zee, Y, Lundie-Jenkins, G, Theilemann, P, Wilson, RJ, Carrick, FN & Johnston, SD 2010, Reproduction, Fertility and Development, vol. 22, no. 1, pp. 695-709.

Male koalas exhibit seasonal changes in anatomical and physiological characteristics relating to fertility and reproduction. These seasonal variations have implications for artificial insemination (AI) programmes, as semen samples collected from wild koalas during winter appear to retain the highest quality after thawing from cryopreservation.

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Semen-induced luteal phase and identification of a LH surge in the koala (Phascolarctos cinereus)

Johnston, SD, O’Callaghan, P, Nilsson, K, Tzipori, G & Curlewis, JD 2004, Society for Reproduction and Fertility, DOI: 10.1530/rep.1.00300

The successful induction of a luteal phase in female koalas following insemination and no other stimulus suggests that koala semen possesses characteristics able to induce ovulation.

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Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Miller, RR Jr, Sheffer, CJ, Cornett, CL, McClean, R, MacCallum, C & Johnston, SD 2004, Cryobiology, vol. 49, no. 1, pp. 137-148.

Koala cauda epididymidal spermatozoa have low membrane cholesterol levels and a high ratio of unsaturated/saturated membrane fatty acids compared to other studied species, including other marsupials. In particular, docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid and the most predominant membrane fatty acid in the studied marsupial species, was found in high levels in koala spermatozoa and is thought to be related to its cryogenic tolerance.

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The effects of gestagen implants on the behaviour of free-ranging female koalas

Hynes, EF, Handasyde, KA, Shaw, G & Renfree, MB 2011, Applied Animal Behaviour Science, vol. 134, pp. 209-216.

Population control in koalas can be achieved using hormonal contraception; however, hormonal changes can have unexpected effects on koala behaviour. Three groups of free-ranging female koalas were treated with either levonorgestrel implants, etonogestrel implants or control to evaluate fertility and effect on behavior. The most prominent behaviour observed was koalas travelling large distances of up to 11 kilometres. Koalas observed to travel these distances were those receiving levonorgestrel implants and with no dependent young.

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The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein

Kitchener, AL, Kay, DJ, Walters, B, Menkhorst, P, McCartney, CA, Buist, JA, Mate, KE & Rodger, JC 2009, Journal of Reproductive Immunology, vol. 82, pp. 40-47.

Zona pellucida, a membrane that surrounds mammalian ova, was used to immunise koalas against pregnancy. Three treatments were applied: zona pellucida from pig (PZP), zona pellucida protein from brushtail possum (recBP-ZP3) and a control. In 29 koalas for which fertility after immunisation was evaluated, both PZP and recBP-ZP3 were found to raise antibodies in the koala, but only PZP was effective as contraception.

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