Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, and makes available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for artificial insemination is complicated because koalas are induced ovulators, and it is thought that ovulating factors are present in the semen, so that semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of this study were designed to determine whether artificial insemination using undiluted, extended, and extended-chilled semen collected by electroejaculation was capable of inducing a luteal phase and/or the production of pouch young. In Experiment 1, 1 ml undiluted electroejaculated semen, 2 ml diluted (1:1) semen, and 1 ml diluted (1:1) semen resulted in seven of nine, six of nine, and six of nine koalas showing a luteal phase, respectively; four pouch young were produced in each treatment. A second artificial insemination experiment was conducted in which 2 ml diluted (1:1) semen was administered in three groups of nine koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, and the final group received semen that had been chilled for 72 h. In the first group, five females had a luteal phase, but none became pregnant. In group 2, two of the five females that had a luteal phase gave birth, whereas in group 3, four of the six females that had a luteal phase produced pouch young. In addition, experiment 3 was conducted to determine whether it was possible to produce pouch young by naturally mating koalas that were in the latter stages of their behavioral estrus; this information is important to the logistics of transporting koala semen for artificial insemination by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on Day 8 of estrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on Day 10 of estrus produced pouch young. The majority of females (21 of 22) in experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended, or extended-chilled semen to produce koala offspring up to Day 8 of estrus at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The pouch young produced in this study represent the first marsupials born following artificial insemination of extended-chilled semen and bring the total number of koalas produced by artificial insemination to 31.

The effects of breeding season (late spring to early autumn) on south-east Queensland male koala fertility were examined to improve the efficacy of the AI procedure and to determine the practicality of using free-range animals as semen donors for a genome resource bank. Seasonal changes in male koala reproductive function were assessed in a wild free-range population (n = 14; obtained every 6 weeks from January to November 2005), a necropsied healthy wild population (n = 84; obtained monthly from September 2004 to August 2005) and a captive population (n = 7; obtained monthly from October 2005 to October 2006). Reproductive parameters investigated included bodyweight, coat score, sternal gland area and activity, testosterone secretion, reproductive anatomy volume and semen quality (before and after cryopreservation). Collectively, these findings show that reproduction in male koalas from south-east Queensland changes seasonally and that winter appears to be the optimal season in which to collect semen samples by electroejaculation. While it was possible to repeatedly collect semen from free-range koalas for future genetic management via potential storage in a genome resource bank, the survival of these spermatozoa after cryopreservation was poor and will require further improvement.

The aim of the present study was to determine whether analogues of gonadotrophin-releasing hormone (GnRH) could be used to both induce an acute testosterone response and suppress anterior pituitary function in male koalas, and induce a luteal phase in female koalas. Experiment 1 characterised the steroidogenic response of male koalas to administration of 30 microg (4.3 microg kg(-1)) natural-sequence GnRH. Intra-muscular injection of natural-sequence GnRH induced the release of LH and testosterone with peak concentrations at 30 min (3.7 +/- 1.9 ng mL(-1)) and 2 h (5.4 +/- 0.5 ng mL(-1)), respectively. In Experiment 2, a single injection of the GnRH antagonist acyline (100 microg (14.3 microg kg(-1)) or 500 microg (71.4 microg kg(-1))) did not influence the testosterone response to subsequent injections of natural-sequence GnRH. In Experiment 3, 4 microg (~0.67 microg kg(-1)) of the GnRH agonist buserelin induced a luteal phase in five female koalas based on a LH surge, secretion of progestogen, and a normal-length oestrous cycle. The findings have shown that (1) natural-sequence GnRH can be used to test gonadotroph cell function and determine the testosterone-secreting capacity of male koalas, (2) the GnRH antagonist, acyline, at the dose rates used, does not suppress the pituitary-testis axis in male koalas, and (3) the GnRH agonist, buserelin, induces a normal luteal phase in female koalas.

Seasonal changes in male reproductive function were assessed in a wild free-range population (n = 10; obtained every six weeks from January to November 2005), a deceased wild free-range population (n = 84; obtained monthly from September to August 2005) and a captive population (n = 7; obtained monthly from October 2005 to October 2006) of koalas in south-east Queensland. This study also determined the practicality of using free-range wild male koalas as potential semen donors for genome resource banks. Examination of a range of reproductive variables initially revealed no significant seasonal change in the 3 koala populations; however, when the data were adjusted to account for individual koalas, their size and/or their health status, the majority of reproductive parameters showed evidence of seasonal variation that was supported by statistical modeling. Relationships between variables were based on simple polynomials, up to a cubic for some variables. Total testicular volume changed throughout the year in the wild and captive populations with an increase over spring and summer and a decrease in autumn and winter; no such change was detected in the deceased population. Maximum area of the sternal gland stain occurred in spring in both the deceased and captive populations but in winter for the wild free-range population. Total bulbo-urethral gland volume in the deceased population showed an increase over spring, a decrease over summer and autumn and then an increase towards the end of winter. The steroidogenic capacity of the koala testis (testosterone secretion) in both the wild free-range (live) and captive populations showed a peak during spring and a nadir in autumn. The quality of semen samples collected by electroejaculation from the wild (live) and captive koala populations showed evidence of being influenced by season. Initial percentage motility of the wild population decreased marginally throughout the study and initial rate of sperm movement was highest in winter. Motility of spermatozoa after thawing from the wild koala population was also highest in winter as was the percentage of cryopreserved spermatozoa with intact plasma membranes collected from the captive population. This study has shown that male koala reproduction in south-east Queensland is seasonal and that it is possible to repeatedly collect semen from free-range koalas as potential genetic donors. Nevertheless, semen quality from captive and wild caught animals appears to be susceptible to seasonal change and winter appears to be the optimal season in which to collect such samples. We are grateful to the veterinary staff, zookeepers, and volunteers at Dreamworld for their assistance with general animal husbandry. We also sincerely thank the many veterinarians, volunteers, and Queensland Parks and Wildlife Service staff that assisted with this project whose help was invaluable for the accomplishment of this project. This project was funded by an Australian Research Council (ARC) Grant, the School of Animal Studies (SAS) The University of Queensland, and the Queensland Government’s Koala Enhanced Genetic Exchange Program (KEGEP).