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Keeley T.,University of New South Wales | McGreevy P.D.,University of New South Wales | O'Brien J.K.,University of New South Wales | O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center
Theriogenology | Year: 2012

Effective sperm cryopreservation protocols are limited to a small number of marsupial species. In this study, postmortem gamete rescue (PMGR) epididymal sperm samples from Tasmanian devils (N = 34) euthanized due to the fatal Devil Facial Tumor Disease were used to develop long-term sperm storage techniques for the species. Cryoprotectant toxicity associated with equilibration of sperm samples in a TEST yolk diluent (TEST; 189 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 85 mM Trizma base [Tris], 11 mM glucose, 20% vol/vol egg yolk; pH 7.1, and 315.0 ± 5.0 mOsm/kg) with a final concentration of 0.06 M trehalose, or 4%, 10%, and 18% vol/vol of either glycerol or dimethyl sulfoxide (DMSO), was examined over 12 h at 15 °C. Trehalose supplementation resulted in an immediate decline (P < 0.05) of total motility. After 12 h, total motility was reduced (P < 0.05) in treatments containing 18% glycerol, and 10% and 18% dimethyl sulfoxide. The effects of final glycerol concentration (4% and 10%), glycerol equilibration duration (10 min 1 h, or 3 h) prefreeze, freezing rate and the addition of 0.10 M lactose or a combination of 0.10 M lactose and 0.11 M raffinose were assessed during three experiments on the cryopreservation of postmortem gamete rescue samples in TEST. In all experiments, motility and viability were reduced (P < 0.01 postthaw). Samples cryopreserved in TEST supplemented with lactose or lactose with raffinose using a fast freezing rate (-8 °C/min from 4 to -40 °C, then -65 °C/min until -165 °C) produced the highest (P < 0.05) postthaw motility (18.6 ± 5.5% and 16.9 ± 8.5%, respectively), which represented 35% to 48% retention of prefreeze motility. These results apparently were the best postthaw results of dasyurid sperm reported to date and will help lay the foundations for developing assisted reproductive technologies for marsupial species. © 2012 Elsevier Inc.

Osborn S.,SeaWorld San Antonio | Dalton L.,SeaWorld San Antonio | Dold C.,SeaWorld Parks and Entertainment | Robeck T.,SeaWorld Parks and Entertainment | Robeck T.,SeaWorld and Busch Gardens Reproductive Research Center
Journal of Zoo and Wildlife Medicine | Year: 2012

A 22-yr-old multiparous beluga, Delphinapterus leucas, with consistently elevated serum progesterone concentrations postartificial insemination was diagnosed with viable twins at 149 days postconception. Twins were of similar size at least until day 264, the last point when ultrasound measurements of both twins were made. However, ultrasound was used to determine the viability of both fetuses on days 365, 393, and 400. After 90% of normal gestation, or 434 days, steroids were administered to encourage fetal lung maturation. Seven days later a 40.9-kg live female calf was delivered headfirst. A second 22.7-kg stillborn calf was delivered in fluke-first presentation 8 hr later. Immediately after birth, the live calf was administered surfactant intratracheally. The next day, it was given beluga immunoglobulin intramuscularly, and started on oral antibiotics and provided nutritional support via an oral gastric tube. The calf started nursing voluntarily on day 3. Antibiotic and nutritional support was discontinued on day 10. Bimonthly weight and length measurements demonstrated that after an initial increased growth rate, the calf has grown within normal parameters after birth. This calf represents the first known successful surviving twin of any cetacean species and sets an important precedent for treatment modalities that may be available to assist the premature cetacean neonate. Copyright © 2012 by American Association of Zoo Veterinarians.

Keeley T.,University of Sydney | McGreevy P.D.,University of Sydney | O'Brien J.K.,University of Sydney | O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center
Reproduction, Fertility and Development | Year: 2012

Devil facial tumour disease (DFTD) is the cause of the rapid decline of wild Tasmanian devils. Female devils are seasonal breeders with births peaking during autumn (i.e. March) but the degree of reproductive seasonality in male devils is unknown. The objective of this study was to examine the potential effects of season and DFTD on reproductive function in male devils (n≤55). Testicular (1.900.23g) and epididymal (0.900.06g) weights were maximal during autumn and spring (P0.05), whereas prostate (3.710.74g) and Cowper's gland (0.680.22; 0.520.21g) weights peaked during autumn (P0.001). The motility of spermatozoa from the cauda epididymides extracted post-mortem was similar (P0.05) across season and disease state (31.513.1% total motility). Testicular and epididymal weights were no different between animals displaying late or early-stage DTFD signs or disease-free animals (P0.1). The accessory sex glands were larger in late-stage DFTD animals than in animals with early-stage disease signs or which were disease-free (P0.01) but effects of season on this result can't be excluded. Serum testosterone concentrations peaked during summer (0.250.18ngmL-1) but values were not different from the preceding and subsequent seasons (P0.05), nor influenced by disease stage (P0.1). Seasonal and DFTD-related changes in serum cortisol concentrations were not evident (P0.1). Male devil reproduction does not appear to be restricted by season nor inhibited by DFTD. © CSIRO 2012.

Keeley T.,University of Sydney | McGreevy P.D.,University of Sydney | O'Brien J.K.,University of Sydney | O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center
Theriogenology | Year: 2011

The Tasmanian devil is suffering from a severe population decline due to the fatal Devil Facial Tumour Disease (DFTD). The development of assisted reproductive technologies such as AI and long-term sperm storage could facilitate genetic management of captive insurance populations. The aim of this study was to characterise semen samples collected post-mortem, and to develop a suitable diluent for short-term preservation of devil sperm. Low numbers of sperm (1.33 0.85 10 6 sperm per male) were extracted from the epididymides of 17 males. Devil sperm sample characteristics such as concentration and morphology were similar to other dasyurids. The most commonly observed morphological abnormalities were midpiece separation, tail curling, and tail twisting (on the axial plane). Changes in motility occurred throughout the regions of the epididymis with (mean SD) 29.4 16.8, 46.8 13.6 and 29.4 18.1% of sperm exhibiting motility, and 88.9 11.4, 32.0 24.3 and 0.1 0.2% of motile sperm exhibiting forward progressive motility in the cauda, corpus and caput, respectively. Sperm from the cauda and corpus epididymis maintained 31.7 26.6 and 80.6 85.9%, respectively, of initial motility after 12 h at 15 ;C in a TEST yolk buffer diluent. These findings provided new information regarding devil sperm biology and short-term sperm storage; such information is necessary for future development of long-term sperm preservation methods in the Tasmanian devil. © 2011 Elsevier Inc.

Keeley T.,Taronga Conservation Society Australia | Keeley T.,University of Sydney | O'Brien J.K.,University of Sydney | O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center | And 3 more authors.
General and Comparative Endocrinology | Year: 2012

Numbers of wild Tasmanian devils are declining as a result of the fatal, transmissible Devil Facial Tumor Disease. A captive insurance population program has been initiated but current captive breeding rates are sub-optimal and therefore the goal of this project was to increase our understanding of the estrous cycle of the devil and elucidate potential causes of failed male-female pairings. Temporal patterns of fecal progestagen and corticosterone metabolite concentrations were examined for females (n= 41) in three categories of reproductive status (successful: viable young, n= 20 estrous cycles; unsuccessful: paired with a male but no young confirmed, n= 44 estrous cycles; non-mated: no access to a male during estrus, n= 8 estrous cycles) but substantial differences were not found. Females were more likely to produce pouch young if pairing with the male extended into late proestrus (P< 0.05), thereby decreasing the time between pairing and presumed ovulation. The interval between the end of proestrous elevation in progestagen metabolite concentrations and the beginning of the luteal phase was 7.6 ± 2.3. days in successful females. The length of the luteal phase in successful females was 12.5 ± 1.4. days which was not different from unsuccessful or non-mated females (P> 0.05). Unsuccessful females had 1-3 estrous cycles within a single year. Successful females were predominantly wild-caught (17/19, 90%) and most produced young following the first estrous cycle of the season (18/20, 90%). Unsuccessful females were predominantly captive born (20/27, 74%) in this study. It is possible that a proportion of females that do not produce pouch young achieve conception but the timing of reproductive failure continues to be elusive in this species. © 2012.

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