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

2008

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…

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