The reproductive biology of the common wombat, vombatus ursinus : studies towards the development of an artifical insemination protocol

The primary objective of this thesis was to gain a better understanding of the basic reproductive biology and management of the male and female common wombat in captivity, and to develop protocols that would lead to the use of artificial insemination in this species.         Gross reproductive anatomy of the male common wombat reported in this study was similar to that described for the male southern hairy-nosed wombat. It possessed a non pendulous scrotum, a relatively large carrot-shaped prostate and three distinctive pairs of bulbourethral glands. The caudae epididymides of this species were tightly enclosed within a separate compartment of the tunica vaginalis; the functional significance of which is unknown.          Quantitative histology of the testis of the common wombat was described for the first time. The proportion of Leydig cells (14.6 ± 1.12%) was significantly lower than that reported for the koala. Seminiferous tubule diameter was 234.0 ± 4.6 µm (n=10). Eight stages of the seminiferous epithelial cycle were clearly identified and their histology and relative frequencies determined. Stage 1B of the seminiferous cycle possessed unique rod shaped condensing spermatids not previously reported in marsupial spermiogenesis. Unlike the koala, common wombat Sertoli cells did not possess enlarged nuclei or crystalloid inclusions.            The electro-ejaculation procedure reported in this thesis was a safe, reliable and repeatable method for collection of semen from the common wombat. High quality spermic ejaculates were successfully recovered on 58 of 64 attempts. Seminal characteristics such as sperm motility, rate of sperm movement and percentage of live spermatozoa were obtained from both captive and wild populations of common wombats by electro-ejaculation and epididymal recovery respectively. The seminal characteristics of cauda epididymidal spermatozoa recovered in this study were found to be similar to that of electro-ejaculated semen (including sperm concentration) and may, therefore, be equally useful for the purposes of artificial insemination.             Seven spermatozoal morphotypes were identified in the common wombat. Mid-piece and principal piece structural abnormalities of spermatozoa were observed in both electro-ejaculates and in epididymal recovered samples. A unique possible sperm abnormality was described for the first time, in which a "droplet or swelling" was observed at the proximal region of the principle-piece immediately distal to the annulus; the functional significance of this abnormality on fertility remains to be determined.           Male common wombats in captivity at Western Plains Zoo did not appear to be seasonal breeders in terms of body weight, testosterone secretion or electro-ejaculate quality (% motile, rate, % live). There was, however, a significant increase in testicular volume (p < 0.05). Lack of distinct seasonal variation in male reproduction was also evident in wild common wombats in the Southern Highlands of NSW. There was no significant difference between wild wombats collected in June or November with respect to their body weight, peripheral plasma testosterone concentration, testicular volume, epididymidal sperm quality, prostate size or bulbourethral gland volume. The relative proportion of Leydig cells present in the testes of the wild common wombat population was higher in November (breeding season) than in June (non-breeding season) (p <0.05). There was no corresponding change in the size of seminiferous tubule diameters of these same animals.               The preservation of common wombat spermatozoa was subdivided into 2 areas of investigation; the physio-chemical tolerance of wombat spermatozoa and the cryopreservation of wombat sperm. This thesis revealed that; (1) The plasma membrane of common wombat spermatozoa is tolerant of hyper-osmotic conditions up to 1410 mmolkg-1, whereas hypo-osmotic environments cause significant damage to the sperm membrane; (2) Cytochalasin D at 5 µM was largely ineffective at improving osmotic tolerance of wombat sperm across the range of osmolalities tested. Cytochalasin D did, however, have a significant effect on the percentage of coiled sperm tails at 104 mmolkg-1 (p < 0.01), indicating that there may have been some degree of interaction of the this compound with the cytoskeleton of the sperm; (3) Moderate and rapid changes in temperature had no significant effect on the motility, rate of sperm movement or percentage of live spermatozoa with intact plasma membranes, confirming resistance to cold shock injury in marsupial spermatozoa; (4) Tris-citrate-fructose (TCF) diluent was the optimal diluent for maintaining common wombat sperm motility and the percentage of live spermatozoa across three storage temperatures (4°C, 25°C and 35°C).     The most successful protocol for the cryopreservation of common wombat spermatozoa in 0.25 ml straws reported in this study, involved semen that was initially equilibrated to 4°C, loaded into a programmable freezer and cooled at 6°C/min in a TCF diluent containing a final concentration of 7.5% egg yolk and 14% glycerol. This method was significantly better than other protocols that used a more rapid freeze rate and lower concentrations of glycerol (8%). This thesis also reports for the first time, a successful pellet method of freezing wombat spermatozoa; pellet volumes of 0.25 ml and 0.5 ml resulted in higher post-thaw survival when compared to pellet volumes of 0.1 ml.                In an attempt to develop a gamete recovery protocol for the northern hairy-nosed wombat, spermatozoa were recovered from the caudae epididymides of common wombats and frozen following a variety of pre-freeze storage conditions. Spermatozoa stored for 3 d at 4°C within 'intact' epididymides, tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability compared with spermatozoa that were recovered from the cauda epididymis and frozen on the day of post mortem. The effect of post-thaw dilution with TCF on the survival of epididymidal common wombat sperm was also investigated. The motility (p < 0.05), rate of sperm movement (p < 0.01) and percentage of live spermatozoa (p < 0.05) were all significantly greater when epididymal spermatozoa were thawed and immediately diluted in TCF, than when thawed without dilution.             Urogenital cytology smears, changes in pouch condition, external genitalia, peripheral plasma and faecal metabolite concentrations were used to detect patterns of cyclicity and seasonal breeding activity of a captive population of common wombats. No reliable changes in the appearance of the clitoris, pericloacal region or pouch morphology could be used to characterise cyclic activity or predict the onset of oestrus. Changes in the proportion of epithelial cells (Karyopyknotic Index) in the urogenital smears could be not be accurately used to define the length of the oestrous cycle but were broadly indicative of ovarian activity. Oestrous cyclic activity was observed in all months of the year in two individual wombats. Oestrous cycle length of these two animals as determined by plasma progesterone and faecal progestagen concentration was approximately 50.7 ± 2.0 d (range: 47 to 54 d) and approximately 52.1 ± 1.9 d (range: 44 to 69 d) respectively. From this study it can be concluded that female common wombats are polyoestrous, ovulate spontaneously and do not have a specific period of breeding activity in captivity.        Gross observations and measurements of reproductive tracts from 10 anoestrous females were made. The ovary is enclosed within a fold of translucent peritoneum (ovarian bursa) making it difficult to clearly visualise primary structures on the ovarian surface. An intact muscular septum divides the vaginal culs-de-sac throughout its entire length. No patent birth canal was observed. Lateral vaginae ostia arise from lateral folds of the urogenital margin, and the caudal extremity of the lateral vaginae ostia narrow significantly as they enter the terminal portion of the urogenital sinus.                The use of the "Cook Koala Insemination Catheter" for Al in the common wombat represents a non-invasive, non traumatic rapid method of insemination for facilitating the entry of semen into both sides of the reproductive tract at the same time. The length and width of the urogenital sinus of the common wombat is twice that of the koala for which it was originally designed. Consequently, the catheter requires lengthening to accommodate the dimensions of the wombat urogenital sinus with a corresponding increase in cuff diameter to prevent retrograde loss of semen. The reproductive anatomy of the lower reproductive tract described in this study suggests that direct catheterisation of the lateral vaginae ostia may be difficult.