C. MacCallum

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat ( Lasiorhinus krefftii ), spermatozoa were removed from the cauda epididymides of four common wombats ( Vombatus ursinus ) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P < 0.01; rate of movement P < 0.01; % live P < 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P < 0.05), rate of sperm movement (P < 0.01) and the percentage of live spermatozoa (P < 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5 mL resulted in higher post-thaw survival compared with 0.1-mL pellets.

9. Lack of seasonal variation in male reproductive parameters measured in a captive and wild population of common wombats in New South Wales

Despite their abundance in the wild, common wombats (Vombatus ursinus) do not breed regularly in captivity, such that there is little published about their captive reproductive management. As part of the development of an artificial insemination program in this species and in an attempt to establish important baseline reproductive information, 4 captive male common wombats at Western Plains Zoo (Dubbo, NSW) were systematically examined over a 12-month period in order to assess whether male reproductive function was seasonally dependent. The reproductive parameters investigated included, peripheral plasma testosterone secretion, testis volume and quality of semen (% motility, % live and % normal) collected by electro-ejaculation. An attempt was also made to determine the extent of seasonal change in male reproductive function of common wombats in wild populations. Plasma samples and reproductive tracts were collected from 12 wild male common wombats from the Kangaroo Valley district (NSW) in June (n = 7) and November (n = 5); plasma testosterone secretion, epididymidal sperm characteristics (% motile, % live and % normal) and measurements of testis, prostate and bulbourethral glands were compared. Our results indicated that male common wombats in captivity at Western Plains Zoo did not appear to be seasonal breeders in terms of testosterone secretion or electroejaculate quality (% motile, % live and % normal); there was, however, a significant increase in testis size between the months of June to October (F = 4.04; P = 0.05). Lack of distinct seasonal variation in male reproduction was also evident in wild common wombats in Kangaroo Valley. There was no significant difference (P > 0.05) between wild wombats collected in June or November with respect to plasma testosterone concentration, epididymidal sperm quality or testis, prostate and bulbourethral gland dimensions.