Refinement of Artificial Insemination in the Koala (Phascolarctos cinereus) with an Emphasis on Male Factor Fertility

Abstract

The overarching theme of this thesis was to gain a better understanding of male koala reproductive biology that would lead to improved outcomes for the application of artificial insemination (AI) in this species. The primary aims of the thesis were centred around (1) the male's contribution to the success of AI with respect to any influence of breeding season (Chapters 2 and 3), (2) the ovulation-induction capacity of the semen in regard to the effect of its dilution and preservation (Chapter 4) and (3) the preliminary development of methods for controlling anterior pituitary (AP) function, leading to the control of oestrus and ultimately the timing of insemination (Chapters 4 and 5). Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating plasma testosterone after injection of a gonadotropin releasing hormone (GnRH) agonist or human chorionic gonadotropin (hCG). Hence, studies conducted in Chapter 2 examined fluctuations in testosterone secretion of the koala (n = 6) over a 24 h period and then characterised testosterone secretion after injection of the GnRH agonist buserelin (4 μg mL-1) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 h but there was no apparent diurnal pattern of testosterone secretion (P > 0.05). Injection of buserelin or hCG resulted in an increase (P < 0.05) in blood plasma testosterone concentration. Near maximal concentrations of plasma testosterone occurred at around 60 min after injection of exogenous hormone. There was a tendency for plasma testosterone to decline after 90 min with buserelin but concentrations following administration of hCG remained near maximum for 240 min. There were strong positive correlations between the average testosterone concentration for each individual koala over 24 h and the maximum observed testosterone concentration after stimulation with GnRH or hCG (GnRH, r = 0.772; P = 0.07 and hCG, r = 1.0; P < 0.01). These findings showed that individual male koalas can show large fluctuations in plasma testosterone concentrations over time and that a GnRH agonist and hCG can be used in the koala to obtain an index of the prevailing steroidogenic capacity of the testes. This technique was then used in Chapter 3 as part of larger study to investigate seasonal changes in male koala reproduction in south-east Queensland (SEQ). The effects of breeding season on male koala fertility have not been investigated in detail so that a better understanding of this phenomenon should help to improve the efficacy of the artificial insemination procedure in this species. Seasonal changes in male reproductive function were assessed in a wild free-range population (n = 14; obtained every six weeks from January to November 2005), a deceased wild 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 SEQ. In addition to improving AI procedures, this study was also used to determine 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 modelling. Relationships between variables were based on simple polynomials, up to a cubic for some variables (Chapter 3, Figures 3 – 5 and the corresponding discussion). 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 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 and captive populations showed a peak during spring and a nadir in autumn. The quality of semen samples collected by electroejaculation (EE) from the wild 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 SEQ appears to be seasonal and that it is possible to repeatedly collect semen from free-range koalas as potential genetic donors. Nevertheless, the semen quality of captive and wild caught animals may be susceptible to seasonal change and winter seems to be the optimal season in which to collect such samples. 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, as well as making available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for AI is complicated by this species being an induced ovulator and it is thought that ovulating factors are present in the semen; hence semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of Chapter 4 were designed to determine whether AI using undiluted, extended and extended-chilled semen collected by EE was capable of inducing a luteal phase and/or the production of pouch young (PY). In Experiment 1, 1 mL of undiluted EE semen, 2 mL of diluted (1:1) semen and 1 mL of diluted (1:1) semen resulted in 7 out of 9, 6 out of 9 and 6 out of 9 koalas showing a luteal phase respectively; 4 PY were produced in each treatment. A second AI experiment was conducted in which 2 mL of diluted (1:1) semen was administered in 3 groups of 9 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, while the final group received semen that had been chilled for 72 h. In the first group, 5 females had a luteal phase but none became pregnant. In group 2, 2 of the 5 females that had a luteal phase gave birth, while in group 3, 4 of the 6 females that had a luteal phase produced PY. In addition, Experiment 3 was conducted to determine whether it was possible to produce PY by naturally mating koalas that were in the latter stages of their behavioural oestrus; this information is important to the logistics of transporting koala semen for AI 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 oestrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on day 10 of oestrus produced PY. The majority of females (21 out 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 oestrus, 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 PY produced in this study represent the first marsupials born following AI of extended-chilled semen and bring the total number of koalas produced by AI to 31. The final series of experiments conducted in Chapter 5 explored the efficacy of GnRH analogues to control the koala AP as a preliminary investigation for the development of methods of oestrus control. The aim of Chapter 5 was to determine whether analogues of 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 μg (4.3 μg kg-1) natural sequence GnRH. Injection IM of natural sequence GnRH induced the release of LH and testosterone with respective peak concentrations at 30 min (3.7 ± 1.9 ng mL-1) and 2 h (5.4 ± 0.5 ng mL-1). In Experiment 2, a single injection of the GnRH antagonist acyline [100 μg (14.3 μg kg-1) or 500 μg (71.4 μg kg-1)] did not influence the testosterone response to subsequent injections of natural sequence GnRH. In Experiment 3, 4 μg (≈0.67 μg 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 (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. Overall, this thesis has resulted in an improved understanding of koala reproductive biology as it relates to the further refinement of AI and the contribution of the male. A total of 31 koala PY, 18 conceived during this study, have now been produced following AI, making the koala AI program one of the most successful assisted breeding programs for wildlife species in the world. The findings of this thesis are essential for the next step in the development of this technology, the production of koala pouch following insemination of frozen-thawed spermatozoa and the establishment of a functional genome resource bank for the species