Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

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.

Koala sperm mitochondria were examined by cryomicroscopy using the fluorescent probe JC-1, which distinguishes high (red) and low (green) mitochondrial membrane potential (MMP). At normal body temperature, approximately 70% of live and untreated spermatozoa exhibited high MMP whereas <3% of live untreated spermatozoa exhibited low potential. A third class, in which single midpieces contained mixed mitochondrial populations, was also detected. Heterogeneity was noted in the level of MMP between individual koalas, individual spermatozoa and even between mitochondrial gyres within single midpieces. MMP of the live sperm population was not significantly affected by glycerol but was suppressed by freezing and thawing treatments. After thawing, MMP declined significantly during rewarming, especially as the temperature increased from 5 to 35 degrees C. The distribution of the ganglioside GM1 was examined using fluorescent-labelled cholera toxin B. In fresh, untreated koala spermatozoa GM1 was detected on the head and midpiece, but not on the principal piece. No significant redistribution of GM1 was observed after chilling and cryotreatment. Phosphatidylserine translocation across the plasma membrane was examined using fluorescent-labelled annexin V. Few fresh spermatozoa exhibited phosphatidylserine translocation (approximately 1%); this was not increased by chilling or cryopreservation, thus implying that cryotreatment had little effect on plasma membrane lipid asymmetry.

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.