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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.

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.

Montano G.A.,SeaWorld and Busch Gardens Reproductive Research Center | Montano G.A.,Texas A&M University | Kraemer D.C.,Texas A&M University | Love C.C.,Texas A&M University | And 3 more authors.
Reproduction | Year: 2012

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNAwas low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI. © 2012 Society for Reproduction and Fertility.

PubMed | SeaWorld and Busch Gardens Reproductive Research Center and Center for Conservation and Research of Endangered Wildlife
Type: | Journal: Animal reproduction science | Year: 2016

The goal of this study was to identify factors that influenced the ability to successfully rescue sperm post-mortem from rhinoceroses maintained in North American zoos. Factors considered included procedural technicalities, individual rhinoceros characteristics and timing. Gross testicular pathology was noted in 17.4% of males (4/23) but did not impact sperm recovery except in one case of azoospermia (4.3%). Of the males in which sperm recovery was attempted (n=21), 62% yielded quality samples considered adequate for cryopreservation ( 30% motility with 2.0 forward progressive status). A high percentage of males (70.6%; 12/17) from which reproductive tissue was removed an d cooled 4 h after death yielded quality sperm samples, whereas only 25% (1/4) of males from which tissue was removed>4h after death yielded quality samples. Quality samples were recovered 1-51 h post-mortem from rhinoceroses 8 to 36 years old. Neither type of illness (prolonged or acute), or method of death (euthanasia or natural) affected the ability to harvest quality samples (P > 0.05). The Indian rhinoceros yielded significantly more sperm on average (40 10(9)) than the African black rhinoceros (3.6 10(9); P < 0.01) and the African white rhinoceros (3.2 10(9); P < 0.05). Across all species and samples assessed (n = 11), mean post-thaw sperm motility (41%), was only 15% less than pre-freeze motility (56%) and only decreased to 22% during the 6h post-thaw assessment period. Rhinoceros sperm rescue post-mortem is relatively successful across a wide range of variables, especially when tissues are removed and cooled promptly after death, and should be considered standard practice among zoos.

Steinman K.J.,SeaWorld and Busch Gardens Reproductive Research Center | Steinman K.J.,Smithsonian Conservation Biology Institute | O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center | Monfort S.L.,Smithsonian Conservation Biology Institute | Robeck T.R.,SeaWorld and Busch Gardens Reproductive Research Center
General and Comparative Endocrinology | Year: 2012

Recent, successful application of assisted reproductive technologies in captive beluga has resulted from the extensive study of male beluga reproductive biology. Optimization of assisted reproduction requires additional detailed knowledge of the female estrous cycle. Our specific objectives were to: (1) validate urinary immunoassays for use in this species; (2) elucidate annual ovarian cycle dynamics through the combined use of hormone excretion patterns and transabdominal ultrasound; and (3) establish whether ovulation in this species is spontaneous or induced by male factors. Ovulation was observed in four of 15 estrous cycles monitored in four adult female beluga maintained in a single-sex group. After introduction of a breeding male, ovulation was observed in six of seven estrous cycles. All estrous cycles occurred from March through June. For spontaneous ovulations (n= 4), the inter-estrous interval was 34. d (range 33-35. d), with a follicular phase length (FPL) of 25 ± 8. d (mean ± SD). For all ovulatory estrous cycles (with and without a breeding male), urinary estrogen conjugates (EC, 15.3 ± 7.9. ng/mg Cr) and ovulatory luteinizing hormone (ovLH, 17.1 ± 6.6. ng/mg Cr) concentrations both peaked on Day 0, and EC concentrations returned to baseline 8 ± 7. d later. For non-conceptive cycles, urinary progestagen (Pg) concentrations increased on Day 0 (3.5 ± 1.7. ng/mg Cr), peaked on Day. +. 19 (19.7 ± 17.1. ng/mg Cr), and were elevated above baseline for 27 ± 4. d. Preovulatory follicular diameter and circumference on Day -2 ± 2 (range: Day -4 to -1) from peak EC were 2.5 ± 0.7 and 7.8 ± 1.3. cm, respectively. The FPL in non-ovulatory estrous cycles (n= 11) lasted 24 ± 10. d and EC concentrations gradually declined to baseline over a 21 ± 10. d interval following the EC peak (27.8 ± 28.8. ng/mg Cr). Non-ovulatory estrous cycles were characterized by the absence of an ovLH surge and no concomitant increase in Pg concentrations above baseline excretion; the mean follicular diameter at or near peak EC was 3.1 ± 0.8. cm on Day 2 ±3. d from peak EC (range: -1 to +5. days from peak EC). Overall, these data confirm that captive beluga exhibit reproductive seasonality and demonstrate that the species is a facultative-induced ovulator. © 2011 Elsevier Inc.

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.

O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center | O'Brien J.K.,University of Sydney | Robeck T.R.,SeaWorld and Busch Gardens Reproductive Research Center
Theriogenology | Year: 2012

Bottlenose dolphins (Tursiops truncatus) undergoing natural breeding and artificial insemination (AI) were examined to characterize serum progesterone concentrations and determine relationships among age, parity, and reproductive outcome. Progesterone profiles of five cycle types (n = 119 total cycles from 54 animals) were characterized as follows: (i) conception and production of a live term calf (conceptive-term, n = 73); (ii) conception and abortion after Day 60 (conceptive-abortion, n = 12); (iii) unknown conception status with prolonged, elevated progesterone and absence of a fetus (conceptive-unknown, n = 14); (iv) conception failure with normal luteal phase progesterone concentrations (non-conceptive, n = 14, AI cycles only); and (v) conception failure with progesterone insufficiency occuring after spontaneous ovulation or owing to premature ovulation induction using GnRH (non-conceptive-PI, n = 6, AI cycles only). By Day 21 post-insemination (PI), progesterone concentrations were similar (P > 0.05) among conceptive-term, conceptive-abortion and conceptive-unknown, and higher (P < 0.05) for conceptive-term than non-conceptive and non-conceptive-PI cycles. Progesterone concentrations of known conceptive cycles peaked by Week 7 PI (P < 0.05) and remained elevated for the remainder of pregnancy (Weeks 8 up to 54, ≥5 days pre-partum). During midpregnancy (Days 121-240), conceptive-term cycles had higher (P > 0.05) progesterone concentrations than conceptive-abortion and unknown conception status cycles. Parity was not associated with reproductive outcome based on cycle type (P > 0.05). Age of females in conceptive-unknown (26.5 ± 10.1 yrs) and conceptive-abortion (22.1 ± 9.4 yrs) groups was higher (P < 0.05) than in conceptive-term (15.7 ± 7.2 yrs). The conceptive-unknown cycle type possibly represents undetected early embryonic loss occurring before Day 60 PI. Length of gestation using known conception dates was 376.1 ± 11.0 days and the range of this parameter (355-395 days) has implications for peri-parturient management procedures for the species. © 2012 Elsevier Inc.

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.

PubMed | SeaWorld and Busch Gardens Reproductive Research Center and Loyola University
Type: Journal Article | Journal: Zoo biology | Year: 2016

The in vitro and in vivo functionality of cryopreserved spermatozoa was examined over two breeding seasons in a zoological colony of Magellanic penguins (Spheniscus magellanicus). Frozen-thawed semen was inseminated into five anesthetized females, over a total of eight egg production cycles, with a different male used for each artificial insemination (AI) within each season. Females were maintained within the colony in cordoned nest sites to prevent copulation with their paired male, and were inseminated every 3-10 days until the first oviposition. Semen frozen from seven males using a straw method retained 39.8%, 25.7%, 74.0%, and 52.1% of its initial total motility, progressive motility, average path velocity, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P<0.05) during freeze-thawing from 76.7% immediately prior to freezing to 65.3% post-thawing. Conceptive females received 1.60.2 inseminations before the first oviposition, with 19.21.610(6) motile, morphologically normal spermatozoa per insemination. Overall fertility was 53.3% (8/15 eggs), hatchability was 50.0% (4/8), and genetic analyses confirmed that all embryos and hatchlings were sired by the AI male. Fertile eggs were laid at 4.0-12.1 days after AI, indicating that frozen-thawed spermatozoa resided in the female reproductive tract for up to 7.2days prior to fertilization. Results demonstrate that frozen-thawed Magellanic penguin spermatozoa are fully functional in vivo and support the use of genome banking and AI as tools for managing the sustainability of zoological penguin populations. Zoo Biol. 35:326-338, 2016. 2016 Wiley Periodicals, Inc.

O'Brien J.K.,SeaWorld and Busch Gardens Reproductive Research Center | Robeck T.R.,SeaWorld and Busch Gardens Reproductive Research Center
Zoo Biology | Year: 2014

Research was conducted to examine seasonal seminal traits and to establish short-term and long-term sperm preservation methods in the king penguin (Aptenodytes patagonicus) for use in genome banking and artificial insemination (AI). Spermic ejaculates (n=87) obtained using a cooperative method were collected across multiple (n=6, Male 1) and a single (Male 2) breeding season(s). Non-contaminated ejaculates (n=69) were 0.36±0.32ml at 56.3±62.7×107sperm/ml with 85.3±10.6% total motility (TMot), 52.5±12.9% progressive motility (PMot), 86.6±24.3μm/sec average path velocity (VAP) and 92.3±3.7% plasma membrane intact. In vitro quality of chilled semen was best maintained over 48hr at 5°C than 21°C, with decreased (P<0.05) motility and morphology parameters observed by 24 and 6hr, respectively. A comparison of two freezing methods (straw [STR] vs. directional [DF]) demonstrated similar effects on post-thaw quality at 0 and 3hr, with the exception of plasma membrane integrity which was higher (P<0.05) at 0hr for DF (48.7±6.5%) than STR (41.2±7.0%). At 0hr post-thaw, DF samples retained 46%, 69%, and 52% of their initial PMot, VAP, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P<0.05) during freeze-thawing from 84% post-collection to 37% and 34% at 0 and 3hr post-thaw, respectively. Results indicate that chilled and cryopreserved semen from the king penguin has potential for use in AI. © 2014 Wiley Periodicals, Inc.

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