No description available.

The pallid sturgeon (Scaphirhynchus albus) is a federally endangered species, endemic to the Mississippi River drainage, stretching from the upper Missouri River in Montana to the Mississippi River, and continuing to the Gulf of Mexico. They are largely sympatric throughout this range with a close congener, the shovelnose sturgeon (S. platorhynchus), although speciation may have occurred when they were isolated in different refugia. In this study, we examined gene expression differences among pallid and shovelnose sturgeon families in response to heat stress. Gene expression can be considered a phenotype, and therefore, variability in expression can have an adaptive role in species. Additionally, we compared our results to a previous expression study that utilized RNA-Seq. We developed viable primer pairs for five genes in order to conduct RT-qPCR assays. There were significant differences in heat stress response between pallid and shovelnose sturgeon, potentially indicative of different evolved stress response pathways. Our species results contrasted with results from the previous study, indicating that further research is needed to improve the robustness of the results. Additionally, we found that offspring of hatchery and wild pallid sturgeon demonstrate different responses to heat stress, and potentially general stress that can occur in a hatchery environment. Overall, this study lays the groundwork for future research that can incorporate a larger suite of families to improve the robustness necessary to make actionable management recommendations.

This PhD thesis aims to improve the ex situ conservation of threatened and endangered species by investigating the effects of captivity and improving the methodology of current conservation techniques. The use of reintroduction as a tool for the purpose of conserving species is becoming increasingly popular. Since many wild populations are declining, captive-bred stock are frequently used to restore or supplement wild populations. Evidence suggests that captive-bred animals are less successful than their wild counterparts, but this has not been recently reviewed and there is limited research into investigating what aspects of captivity may be affecting success. Here, I conduct a review of carnivore reintroductions for projects carried out post 1990, which shows that captive-born animals are less likely to survive a release into the wild than their translocated wild-caught counterparts. A case study species, the endangered red panda (Ailurus fulgens), is used to investigate how a species involved in captive breeding for conservation responds to life in captivity. Results from analyses of lifetime reproductive success (and related variables) showed that both adaptation to captivity and inbreeding depression are occurring in the global captive red panda population. An investigation into behavioural adaptation to captivity was less revealing, although only generations three to seven from the wild were observed. The effects of captive environment and husbandry regime were also investigated and revealed that the size of the useable area and amount of human contact were among the factors influencing the behaviours of red pandas. How these findings contribute to a greater understanding of effects of captivity is discussed. The use of selection criteria based on temperament was also investigated in order to improve the likelihood of survivorship upon release into the wild. This method needs to be tested in practice, but based on the selection criteria used, there was evidence that unsuitability for release was positively predicted by generation time in captivity. Implications for the future use of captive red pandas in efforts to conserve the species in the wild are discussed, as well as how these findings can be utilised for other species involved in conservation efforts.

Genetic analyses of increasing power are now regularly incorporated into wildlife management assessments of threatened and endangered species. Genetic data provide valuable information regarding taxonomy, kinship, and population size and structure. Recently transformed by the advent of powerful technologies that expand our view from single genes to the entire genome, the field of conservation may be on the verge of another revolution with the emergence of epigenetics as a promising means of surveying environmental response in natural populations. In this dissertation, I present my doctoral research upon population genetics and epigenetics of pronghorn (Antilocapra americana). Considerable effort has been undertaken to conserve pronghorn, particularly in the periphery of its range in the southwestern United States and northwestern Mexico. Translocation is regularly used to supplement and re-establish populations of the wide-ranging A. a. americana subspecies while captive breeding has been established for two endangered pronghorn subspecies, A. a. sonoriensis found in Arizona and Sonora, Mexico and A. a. peninsularis of the Baja Peninsula. The primary goal of my doctoral work was to provide pronghorn managers with current estimates of genetic diversity, relatedness, and structure within and between pronghorn subspecies in the desert southwest. My work shows that conservation measures for A. a. sonoriensis have successfully maintained genetic diversity within this endangered subspecies. My estimates of population structure within A. a. americana in northern Arizona reveal the influence of translocation and habitat fragmentation and demonstrate the successful reestablishment of gene flow following the removal of highway fences. With the purpose of guiding future release of captive pronghorn, I explored the subspecies status of pronghorn extirpated from a portion of their range in southern California and northern Baja California. My analyses of museum specimens indicate that the historical range of A. a. peninsularis may have extended as far north as the international border while specimens collected just north of the border share more genetic identity with A. a. sonoriensis. To follow my interests in epigenetics, I also conducted the first ever conservation epigenetics study with Arizona pronghorn. I found that pronghorn are more epigenetically than genetically diverse and this is an indicator that further epigenetic study will reveal the signature of response to environmental factors, as it has with other species demonstrating this pattern.

The recent decline and disappearance of many amphibians around the world is thought to be the sign of an impending sixth mass extinction that is driven by disease, habitat loss and pollution. Reproductive technologies are now required to establish captive colonies followed by reintroduction into suitable habitats. The AMPHIPLEX method is a hormone mixture that has successfully stimulated spawning in several amphibians. However, its extensive application requires further experimentation and knowledge regarding the basic neuroendocrine control of reproduction in amphibians. The role of the catecholamine neurotransmitter dopamine in the regulation of spawning and gonadotropin synthesis was investigated using multiple time- and dose-related approaches in the field and laboratory. These end points were explored in two distantly-related frog species: the Northern leopard frog (Lithobates pipiens) and the Western clawed frog (Silurana tropicalis). Northern leopard frogs were injected during the natural breeding season with three doses of a gonadotropin-releasing hormone agonist (GnRH-A) (0.1 μg/g , 0.2 μg/g and 0.4 μg/g) alone and in combination with two doses of the selective dopamine receptor D2 antagonist metoclopramide (MET) (5 μg/g and 10 μg/g). Injected animals were allowed to breed in mesocosms in an outdoor field. Time to amplexus and oviposition were assessed, and egg mass release, incidences of amplexus, egg mass weight, total egg numbers and fertilization rates were measured. The results revealed no statistically significant interaction between GnRH-A and MET on amplexus and oviposition. A series of GnRH-A dose-response spawning studies were conducted in the Western clawed frog. The current findings indicate that partial ovulation, male sexual behavior and fertilization can be induced by 4 μg/g of GnRH-A alone and in combination with 10 μg/g of MET. This represents a first step towards understanding basic neuroendocrine reproductive mechanisms in this species. These spawning results were paired with a second end point which explored the molecular mechanisms of gonadotropin synthesis in response to GnRH-A and MET alone and in combination. Pituitary gene expression results in the Northern leopard frog indicate a potentiating action of MET when combined with GnRH-A on the mRNA levels of gonadotropin subunits 36 hours following injection. The postulated mechanisms of action are through the upregulation of gonadotropin-releasing hormone receptor 1 and the downregulation of dopamine receptor D2. Such gene expression pathways were similarly explored in the Western clawed frog, however no significant changes in pituitary gonadotropin and receptor gene expression were present at 12 hours post-injection. The hypothesized inhibitory action of dopamine was supported by pituitary gene expression analysis, but not by spawning outcome. The results from this study provide a fundamental framework for future time- and dose-response investigations to improve current spawning methods in amphibians.

The genetic status of the critically endangered Mississippi sandhill crane (Grus canadensis pulla) was analyzed using 2009 studbook data from the U.S. Fish and Wildlife Service managed captive breeding and release program. Microsatellite DNA data provided information on shared founder genotypes, allowing for refined analysis of genetic variation in the population, and informed breeding recommendations. The genetic variation observed in the Mississippi sandhill crane was contrasted with variation observed in the Florida sandhill crane (Grus canadensis pratensis). Results show far less variation in the Mississippi population. Results also suggest that while gene flow no longer occurs between the two populations, the introduction of cranes from the Florida population would help to increase the observed genetic diversity of the Mississippi sandhill crane population.

Developmental studies on elasmobranchs are challenging due to the difficulties in obtaining sufficient numbers of animals of different age cohorts. The brown banded bamboo shark, Chiloscyllium punctatum is a good model in this regard as it is abundant and readily available in the wild, is quick to mature, is frequently housed and bred in captivity and is a relatively small species of shark. Whilst there are important factors that must be considered when comparing the retinal development of animals raised in captivity to those caught from the wild, the use of C. punctatum represents an outstanding opportunity to study the development of the elasmobranch visual system from pre-hatching embryonic to adult life stages. In this study, the developing eye and retina of C. punctatum were studied using light and electron microscopy, electroretinography (ERG) and microspectrophotometry (MSP). To provide a source of early-stage animals, and to investigate the effects of environmental factors (such as temperature) on physical development, a captive breeding program was established at the University of Queensland. Sharks sourced from this facility were supplemented with animals bred at UnderWater World, Sea World and caught from the wild. Monitoring the fecundity, embryonic development, growth and viability of captive C. punctatum showed that females lay an average of 115.3 eggs, 38 of which were viable and 21.4% of which hatched. Embryos have an average gestation of 153 days post deposition (dpd; temp: 21 - 25º C) and embryonic growth is most rapid from 99 dpd until hatching. The eye of C. punctatum develops early in embryogenesis, with visible optic vesicles bulging at 27 dpd. Recent advances in fixation and processing techniques for transmission electron microscopy (TEM) have yielded improved levels of ultrastructural detail in a variety of tissue types. Consequently, in addition to conventional chemical fixation (CF) methods, the retina of C. punctatum was also processed using microwave chemical fixation (MCF) and high pressure freezing (HPF), and the resulting ultrastructure compared. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF, evidenced by higher resolution of ultrastructural detail and fewer artefacts. MCF provided the best, consistent ultrastrucutral results. By examining the time-course of retinal cell differentiation, it was found that ganglion and Müller cells are the first to differentiate, at approximately 81 dpd. The interneurons differentiate next, beginning with the amacrine cells (81 dpd), followed by the bipolar cells (101 dpd) and horizontal cells (124 dpd). The adult retina is duplex and rod and cone photoreceptors are differentiated and synaptic connections are formed by 124 dpd. Topographic analysis of retinal neuron sub-types reveals that C. punctatum undergoes rapid changes in ganglion cell distribution during embryogenesis. High levels of apoptosis, especially around the retinal periphery, result in relatively higher cell densities in the central retina, which progressively extend nasally and temporally to form a meridional band. C. punctatum develops a horizontal streak and shows only minor changes in topography during growth. Only basal levels of apoptosis are seen post-hatching. In the adult shark, the total ganglion cell number reaches 547,881. The mean and highest retinal ganglion cell densities reach a peak around hatching (3,228 cells mm-2 and 4,983 cells mm-2, respectively). Using measurements of lens focal length and ganglion cell density, the calculated maximum spatial resolving power (assuming a hexagonal mosaic) increases from 1.47 cycles degree-1 during embryogenesis to 4.29 cycles degree-1 in adults. The addition of a high ganglion cell density area within the visual streak and an increasing spatial resolving power over post-hatching development suggest an increased prey targeting and capture ability for this species. Using ERG, it is shown that C. punctatum becomes responsive to light at 127 dpd and light sensitivity peaks around the time of hatching, with a slight decrease post-hatching. C. punctatum maintains a flicker fusion frequency (FFF; an indicator for temporal resolution) at 7 - 22 Hz through juvenile stages), which is relatively low compared to other marine predators. ERG results suggest that this species is adapted to low light vision with low temporal resolution. The early differentiation, development and functionality of the visual system in C. punctatum allows for a period of synaptic maturation and potentially the ability of embryonic predator avoidance. The retina of C. punctatum contains a rod visual pigment with a wavelength of maximum absorbance (λmax) at 500 nm and cone visual pigment with a λmax at 532 nm; the max values of these pigments do not change during development. Rod and cone outer segments differentiate at 113 days post deposition (dpd), lengthen during embryogenesis and accumulate pigment throughout life. Although the photoreceptors develop and differentiate well in advance of hatching, there is considerable variation in outer segment length and pigment density during embryogenesis, which suggests that these cells are developing up until hatching. C. punctatum does not appear to have the potential for colour vision based on the lack of two cone photoreceptor types each containing a visual pigment maximally sensitive to different parts of the visual spectrum, but appears specialised for dim-light contrast vision.

The Mississippi gopher frog (MGF, Lithobates sevosa) is a critically endangered amphibian which requires the use of assisted reproductive technologies (ART) to successfully reproduce in captivity. Several challenges to successfully breeding MGFs were investigated to improve reproduction in this species. Accurate identification of biological sex is a necessary first step to reproducing species, thus several non-invasive techniques for sex identification were compared in the weakly dimorphic MGF. Reliably obtaining eggs from female MGFs has also proved difficult, therefore, four exogenous hormone therapies were compared for their success to induce ovulation. Ultrasound imaging was used alongside hormone treatments to improve understanding of ovarian response to hormone treatment. Lastly, short-term cold storage of spermic urine was studied to determine the feasibility of this technique for MGF gamete storage. Findings from these research projects are important for the successful conservation of the MGF by aiding in the production offspring for future reintroduction programs.

These chapters were written as three separate papers for publication. After the island fox (Urocyon littoralis) underwent a period of severe population decline, the National Park Service began a captive breeding program to increase their numbers. Here, I identify the factors influencing the breeding success of pairs in captivity to formulate strategies that could increase productivity. I compiled a database of variables including litter size, reproductive success, distance to nearest occupied pen, subspecies, exposure, female age, male age, age difference, female and male origin (wild vs. captive born), years paired, previous reproductive success by the pair, previous reproductive success by the female, mate aggression related injuries reported, and previous involvement in an aggressive pair. I performed linear multiple regression to identify factors related to litter size, and logistic regression to predict the probability of reproductive success. A larger interpen distance, higher male age, less exposure, and a smaller intra-pair age difference positively affected litter size. The probabilities of reproductive success increased with interpen distance, lower female age, fewer years paired, and less exposure. Comparatively, pairs with wild born females (vs. captive born females), previously successful pairs (vs. previously unsuccessful and new pairs), and pairs with a previously unsuccessful female (vs. untried and previously successful females) were most likely to be successful. These results suggest that the ideal situation and pairing would be to pair young wild-caught females with older males in sheltered pens that are as far from other pens as possible, while maintaining successful pairs and repairing unsuccessful pairs.

No description available.