Studies towards the development of an artificial insemination protocol in the koala, Phascolarctos cinereus

Abstract

The objective of this thesis was to develop an artificial insemination (AI) program in thenKoala (Phascolarctos cinereus). The success of the program was dependent onnsystematic investigation of 3 key areas, (1) semen collection and preservation, (2)nstudies to determine the most appropriate timing of insemination and (3) examinationnof female reproductive anatomy to determine the most appropriate site for thendeposition of the inseminate. The Koala's husbandry and propagation in captivity arenwell established and make it ideal for reproductive studies. Whilst the species is in nonimmediate threat of extinction, its maintenance in captivity in commercial wildlife parksnwill benefit from an effective AI procedure, and it will provide a model for assistednreproduction applications in some threatened marsupial taxa.SEMEN COLLECTION AND PRESERVATIONCollecting Koala semen with an artificial vagina (AV)Semen was successfully collected from 19 of 25 males, the majority of which requirednno training to ejaculate into the AV. The AV procedure was repeatable, non-invasivenand required no anaesthesia. The seminal characteristics of ejaculates collected by AVnin this study probably provide the most reliable parameters of marsupial semen qualitynso far described.Antibiotics for the preservation of Koala semenThe normal microbial flora of the Koala ejaculate and prepuce was determined, whichnenabled the selection of antibiotics for addition to semen diluents for the preservationnof Koala spermatozoa. The predominant organisms isolated from the prepuce andnsemen were Corynebacterium spp; none of which could be assigned to any recognisednspecies. Based on sensitivity testing results, the addition of penicillin G and gentamicinnto PBS diluent at dose rates of 1000 - 2000 I.U. / ml and 100 - 200 mg / ml respectively,nresulted in no adverse effect on sperm motility and prevented growth of bacterial contaminants over a 24 h storage period.Manipulation of koala semenIncreasing serial dilution of Koala spermatozoa in PBS medium resulted in a decline ofnsperm motility. While semen diluted 1:1 had a tendency to coagulate, a dilution rate ofnno more than 1:8 should be used for electroejaculated semen samples. It is alsonrecommended that diluents for Koala sperm manipulation, be buffered in a pH range ofn7 to 8 and have an osmolality of 280 to 320 mmol / kg. While Koala spermatozoanexposed to a rapid decrease in temperature from 35oC to 15oC showed no reduction innmotility, rapid cooling of spermatozoa from 35oC to 5oC, resulted in a significantndecline in motility. This appears to be the first evidence of qcold shockq reported fornmarsupial spermatozoa. The effect of cryoprotectant type and concentration on thenmotility of Koala sperm stored at 35oC was investigated as a means of identifyingnalternative cryoprotectants that might be less cytotoxic than glycerol. At highnconcentrations (2.14M / 3.21 M) glycerol and ethylene glycol had a less adverse effectnon Koala sperm motility than did DMSO or propanediol.Semen preservationTris Citrate Glucose diluent (TCG) was selected and compared with PBS for its abilitynto maintain sperm motility of Koala sperm over a range of incubation temperaturesn(35oC, 25oC, 15oC, 5oC). Reduction in sperm motility was directly related tontemperature, with motility being sustained for the longest duration when stored at 5oC.nThe TCG diluent was clearly superior to PBS as a preservation diluent at allntemperatures, with Koala sperm remaining motile even after 42 d storage at 5oC innTCG. Straws of Koala semen (0.25 ml) diluted in TCG medium containing 12%nglycerol were frozen in either a programmable freezer at -6oC / min (slow freeze) ornsuspended in liquid nitrogen vapour 4 to 5 cm above the liquid nitrogen surface (fastnfreeze). Spermatozoa frozen at the slower rate of -6oC / min had significantly highernpost-thaw motility than semen frozen 4 to 5 cm above the liquid nitrogen surface.TIMING OF INSEMINATIONCharacterisation of the oestrous cycleNon-mated and mated Koala oestrous cycles were characterised by means ofnbehavioural oestrus, changes in urogenital cytology, morphology of external genitalianand changes in the peripheral plasma concentrations of oestradiol-17b andnprogestogen. The mean (p s.e.m.) length of the non-mated oestrous cycle andnduration of oestrus for all Koalas examined was 31.8 p 0.9 d (n = 31) and 10.2 p 0.8 dn(n = 24) respectively. As progestogen concentrations remained basal throughout theninteroestrous period, non-mated cycles were considered to have no luteal phase andnpresumed anovulatory. By contrast, peak oestradiol-17b concentrations coincided withnbehavioural oestrus and partial comification of the urogenital epithelium. However,nthere were no reliable changes in the morphology or appearance of the Koala clitoris,npericloacal region, pouch or mammary teats that could be used to characterise thennon-mated cycle or predict the onset of oestrus. n