Sex Ratio Assessment Of Endangered Kemp’s Ridley Sea Turtle Foraging Populations: Validation Of A Testosterone ELISA For Juvenile Sex Determination

Currently all species of sea turtles are listed as threatened or endangered with extinction under the U.S. Endangered Species Act. In order to effectively construct management approaches we need as much information on various sea turtle populations as possible including demography, genetic origin, and critical habitat. One demographic piece of data that is lacking is the sex ratio of turtle populations in foraging habitats, as this information is integral in determining overall population abundance. Because secondary sex characteristics (i.e. males have longer tails) are not evident until turtles start to reach sexual maturity, the sex of juvenile turtles cannot be easily determined externally. The least invasive way to determine the sex of juvenile turtles is through hormone analysis (testosterone) of the blood plasma. There are several methods for determining hormone concentration in turtle plasma; we used enzyme-linked immunosorbent assays (ELISA), which are the most cost effective and user friendly technique available. The testosterone ELISA has recently been validated for use with green sea turtle Chelonia mydas plasma but has yet to be validated for the other sea turtle species. My project focused on the endangered Kemp’s ridley sea turtle Lepidochelys kempii that is only found in the Gulf of Mexico and U.S. Atlantic seaboard. We validated the ELISA testosterone technique through demonstrating ‘parallelism’ to prove that the assay is measuring the same antigen (i.e. testosterone) in the plasma extracts and the standard controls (provided in the testosterone assay kit). We then determined the sex of approximately 140 juvenile turtles

Sea turtle hatchling sex ratios determined via hormone assay: implications of climate change?

Currently all species of sea turtles are listed as threatened or endangered with extinction under the U.S. Endangered Species Act. Due to their status, sea turtle conservation is a high priority for the U.S. National Marine Fisheries Service and U.S. Fish and Wildlife Service. One major challenge conservationists face is the lack of a noninvasive, cost efficient method for determining the sex of hatchling sea turtles. Because secondary sex characteristics (i.e. males have longer tails) are not evident until turtles start to reach sexual maturity, the sex of hatchlings is not easily determined. The least invasive way to determine the sex is through hormone analysis of blood plasma. The testosterone enzyme-linked immunosorbent assay (ELISA) has been validated for use with all six sea turtle species plasma and has been shown to be an effective method of sex determination in juvenile sea turtles. We have validated two new high sensitivity ELISA’s (testosterone and estradiol) for use with loggerhead sea turtles (Caretta caretta) and will subsequently use these to explore whether sex can be assigned to live hatchlings. We will apply both ELISA’s to small plasma volumes from known-sex loggerhead hatchlings and examine the ratio of testosterone to estradiol to determine sex. If applied over multiple nesting seasons, this may facilitate subsequent studies to identify the degree to which climate change may impact sex ratios of annual hatchling cohorts at key beaches in the US and beyond

Malam puisi Riong buka tirai di UMP

MALAM Puisi Riong membuka tirai pertamanya tahun ini di Universiti Malaysia Pahang (UMP) dan menjadikan masalah pembuangan dan pembunuhan, fenomena membimbangkan mutakhir ini sebagai tema

Species and Population Specific Gene Expression in Blood Transcriptomes of Marine Turtles

Background: Transcriptomic data has demonstrated utility to advance the study of physiological diversity and organisms\u27 responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations.ResultsWe generated high quality blood transcriptome assemblies for hawksbill (Eretmochelys imbricata), loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance.ConclusionsObtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles

Species and population specific gene expression in blood transcriptomes of marine turtles

Background: Transcriptomic data has demonstrated utility to advance the study of physiological diversity and organisms’ responses to environmental stressors. However, a lack of genomic resources and challenges associated with collecting high-quality RNA can limit its application for many wild populations. Minimally invasive blood sampling combined with de novo transcriptomic approaches has great potential to alleviate these barriers. Here, we advance these goals for marine turtles by generating high quality de novo blood transcriptome assemblies to characterize functional diversity and compare global transcriptional profiles between tissues, species, and foraging aggregations. Results: We generated high quality blood transcriptome assemblies for hawksbill (Eretmochelys imbricata), loggerhead (Caretta caretta), green (Chelonia mydas), and leatherback (Dermochelys coriacea) turtles. The functional diversity in assembled blood transcriptomes was comparable to those from more traditionally sampled tissues. A total of 31.3% of orthogroups identified were present in all four species, representing a core set of conserved genes expressed in blood and shared across marine turtle species. We observed strong species-specific expression of these genes, as well as distinct transcriptomic profiles between green turtle foraging aggregations that inhabit areas of greater or lesser anthropogenic disturbance. Conclusions: Obtaining global gene expression data through non-lethal, minimally invasive sampling can greatly expand the applications of RNA-sequencing in protected long-lived species such as marine turtles. The distinct differences in gene expression signatures between species and foraging aggregations provide insight into the functional genomics underlying the diversity in this ancient vertebrate lineage. The transcriptomic resources generated here can be used in further studies examining the evolutionary ecology and anthropogenic impacts on marine turtles

Refinement of Artificial Insemination in the Koala (Phascolarctos cinereus) with an Emphasis on Male Factor Fertility

The overarching theme of this thesis was to gain a better understanding of male koala reproductive biology that would lead to improved outcomes for the application of artificial insemination (AI) in this species. The primary aims of the thesis were centred around (1) the male's contribution to the success of AI with respect to any influence of breeding season (Chapters 2 and 3), (2) the ovulation-induction capacity of the semen in regard to the effect of its dilution and preservation (Chapter 4) and (3) the preliminary development of methods for controlling anterior pituitary (AP) function, leading to the control of oestrus and ultimately the timing of insemination (Chapters 4 and 5). Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating plasma testosterone after injection of a gonadotropin releasing hormone (GnRH) agonist or human chorionic gonadotropin (hCG). Hence, studies conducted in Chapter 2 examined fluctuations in testosterone secretion of the koala (n = 6) over a 24 h period and then characterised testosterone secretion after injection of the GnRH agonist buserelin (4 μg mL-1) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 h but there was no apparent diurnal pattern of testosterone secretion (P > 0.05). Injection of buserelin or hCG resulted in an increase (P < 0.05) in blood plasma testosterone concentration. Near maximal concentrations of plasma testosterone occurred at around 60 min after injection of exogenous hormone. There was a tendency for plasma testosterone to decline after 90 min with buserelin but concentrations following administration of hCG remained near maximum for 240 min. There were strong positive correlations between the average testosterone concentration for each individual koala over 24 h and the maximum observed testosterone concentration after stimulation with GnRH or hCG (GnRH, r = 0.772; P = 0.07 and hCG, r = 1.0; P < 0.01). These findings showed that individual male koalas can show large fluctuations in plasma testosterone concentrations over time and that a GnRH agonist and hCG can be used in the koala to obtain an index of the prevailing steroidogenic capacity of the testes. This technique was then used in Chapter 3 as part of larger study to investigate seasonal changes in male koala reproduction in south-east Queensland (SEQ). The effects of breeding season on male koala fertility have not been investigated in detail so that a better understanding of this phenomenon should help to improve the efficacy of the artificial insemination procedure in this species. Seasonal changes in male reproductive function were assessed in a wild free-range population (n = 14; obtained every six weeks from January to November 2005), a deceased wild population (n = 84; obtained monthly from September to August 2005) and a captive population (n = 7; obtained monthly from October 2005 to October 2006) of koalas in SEQ. In addition to improving AI procedures, this study was also used to determine the practicality of using free-range wild male koalas as potential semen donors for genome resource banks. Examination of a range of reproductive variables initially revealed no significant seasonal change in the 3 koala populations; however, when the data were adjusted to account for individual koalas, their size and/or their health status, the majority of reproductive parameters showed evidence of seasonal variation that was supported by statistical modelling. Relationships between variables were based on simple polynomials, up to a cubic for some variables (Chapter 3, Figures 3 – 5 and the corresponding discussion). Total testicular volume changed throughout the year in the wild and captive populations with an increase over spring and summer and a decrease in autumn and winter; no such change was detected in the deceased population. Maximum area of the sternal gland stain occurred in spring in both the deceased and captive populations but in winter for the wild free-range population. Total bulbo-urethral gland volume showed an increase over spring, a decrease over summer and autumn and then an increase towards the end of winter. The steroidogenic capacity of the koala testis (testosterone secretion) in both the wild free-range and captive populations showed a peak during spring and a nadir in autumn. The quality of semen samples collected by electroejaculation (EE) from the wild and captive koala populations showed evidence of being influenced by season. Initial percentage motility of the wild population decreased marginally throughout the study and initial rate of sperm movement was highest in winter. Motility of spermatozoa after thawing from the wild koala population was also highest in winter as was the percentage of cryopreserved spermatozoa with intact plasma membranes collected from the captive population. This study has shown that male koala reproduction in SEQ appears to be seasonal and that it is possible to repeatedly collect semen from free-range koalas as potential genetic donors. Nevertheless, the semen quality of captive and wild caught animals may be susceptible to seasonal change and winter seems to be the optimal season in which to collect such samples. Artificial insemination in the koala using chilled, electroejaculated semen provides for a marked improvement in the reproductive and genetic management of captive koala colonies in Australia and internationally, as well as making available the option of using semen collected from wild populations to expand restricted gene pools. Dilution of koala semen for AI is complicated by this species being an induced ovulator and it is thought that ovulating factors are present in the semen; hence semen extension for preservation purposes might be anticipated to result in a failure to induce ovulation. The first two experiments of Chapter 4 were designed to determine whether AI using undiluted, extended and extended-chilled semen collected by EE was capable of inducing a luteal phase and/or the production of pouch young (PY). In Experiment 1, 1 mL of undiluted EE semen, 2 mL of diluted (1:1) semen and 1 mL of diluted (1:1) semen resulted in 7 out of 9, 6 out of 9 and 6 out of 9 koalas showing a luteal phase respectively; 4 PY were produced in each treatment. A second AI experiment was conducted in which 2 mL of diluted (1:1) semen was administered in 3 groups of 9 koalas. The first group received semen that had been collected and diluted immediately without chilling, the second group was deposited with semen stored chilled for 24 h, while the final group received semen that had been chilled for 72 h. In the first group, 5 females had a luteal phase but none became pregnant. In group 2, 2 of the 5 females that had a luteal phase gave birth, while in group 3, 4 of the 6 females that had a luteal phase produced PY. In addition, Experiment 3 was conducted to determine whether it was possible to produce PY by naturally mating koalas that were in the latter stages of their behavioural oestrus; this information is important to the logistics of transporting koala semen for AI by establishing the maximum time frame in which females might be expected to shed a fertile oocyte. Of the 12 females mated on day 8 of oestrus, 6 gave birth, whereas only 3 of the 10 females naturally mated on day 10 of oestrus produced PY. The majority of females (21 out of 22) in Experiment 3 showed evidence of a luteal phase. Together, these experiments have shown that it is possible to use undiluted, extended or extended-chilled semen to produce koala offspring, up to day 8 of oestrus, at conception rates similar to those achieved following natural mating. These findings represent a significant advancement in the use of reproductive technology in marsupials and provide the basis for the shipment of koala semen over long distances. The PY produced in this study represent the first marsupials born following AI of extended-chilled semen and bring the total number of koalas produced by AI to 31. The final series of experiments conducted in Chapter 5 explored the efficacy of GnRH analogues to control the koala AP as a preliminary investigation for the development of methods of oestrus control. The aim of Chapter 5 was to determine whether analogues of GnRH could be used to both induce an acute testosterone response and suppress anterior pituitary function in male koalas, and induce a luteal phase in female koalas. Experiment 1 characterised the steroidogenic response of male koalas to administration of 30 μg (4.3 μg kg-1) natural sequence GnRH. Injection IM of natural sequence GnRH induced the release of LH and testosterone with respective peak concentrations at 30 min (3.7 ± 1.9 ng mL-1) and 2 h (5.4 ± 0.5 ng mL-1). In Experiment 2, a single injection of the GnRH antagonist acyline [100 μg (14.3 μg kg-1) or 500 μg (71.4 μg kg-1)] did not influence the testosterone response to subsequent injections of natural sequence GnRH. In Experiment 3, 4 μg (≈0.67 μg kg-1) of the GnRH agonist buserelin induced a luteal phase in five female koalas based on a LH surge, secretion of progestogen, and a normal length oestrous cycle. The findings have shown (1) natural sequence GnRH can be used to test gonadotroph cell function and determine the testosterone secreting capacity of male koalas, (2) the GnRH antagonist, acyline, at the dose rates used does not suppress the pituitary-testis axis in male koalas, and (3) the GnRH agonist, buserelin, induces a normal luteal phase in female koalas. Overall, this thesis has resulted in an improved understanding of koala reproductive biology as it relates to the further refinement of AI and the contribution of the male. A total of 31 koala PY, 18 conceived during this study, have now been produced following AI, making the koala AI program one of the most successful assisted breeding programs for wildlife species in the world. The findings of this thesis are essential for the next step in the development of this technology, the production of koala pouch following insemination of frozen-thawed spermatozoa and the establishment of a functional genome resource bank for the species

Use of a GnRH agonist and hCG to obtain an index of testosterone secretory capacity in the koala (Phascolarctos cinereus)

Testosterone secretion in mammals typically occurs in random pulses such that a single blood sample provides limited information on reproductive endocrine status. However, it has been shown in several species that an index of the prevailing testosterone biosynthetic capacity of the testes can be obtained by measuring the increase in circulating testosterone after injection of a GnRH agonist or human chorionic gonadotrophin (hCG). Hence, the aims of the present study were to examine fluctuations in testosterone secretion in the koala (n = 6) over a 24-hour period and then characterise testosterone secretion after injection of the GnRH agonist buserelin (4 mu g) or hCG (1000 IU). The latter was used to establish an index of the prevailing testosterone biosynthetic capacity of the koala testis. Individual koalas showed major changes in blood testosterone concentrations over 24 hours, but there was no apparent diurnal pattern of testosterone secretion (P >.05). Injection of buserelin and hCG resulted in an increase (

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells

The relationship between sperm morphology and chromatin integrity in the koala (Phascolarctos cinereus) as assessed by the sperm chromatin dispersion test (SCDt)

Koala (Phascolarctos cinereus) sperm nuclei show a tendency to swell after cryopreservation, but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, we validated a modified version of the sperm chromatin dispersion test (SCDt) for use with koala spermatozoa, which is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion, and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa with the use of a lysing solution that did not contain dithiothreitol. DNA fragmentation of lysed and nonlysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of 3 different koalas. Spermatozoa were classified into 3 distinct koala sperm morphotypes (KSMs) after the SCDt: 1) KSM-1, rod-shaped cells with no halo of DNA; 2) KSM-2, rounded nuclei with various degrees of halo formation about a dense chromatin core; and 3) KSM-3, rod-shaped or rounded nuclei consisting,of an inner chromatin core but with large dispersed halos of stellar chromatin. Although ISNT after the SCDt did not label KSM-1, both KSM-2 and KSM-3 stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3. Although application of the SCDt to the spermatozoa of another 3 koalas showed no difference in the percentage of the 3 sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35 C for 2 hours showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 represent nuclei that show either no, or only limited, sperm DNA fragmentation, respectively. It is likely that the halos formed around KSM-2 are from DNA that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulfide bonds in marsupial sperm DNA. "True" sperm DNA damage is most likely associated with KSM-3, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei after the SCDt. Further studies are required to determine whether DNA damage found in KSM-2 is indicative of single-stranded DNA breakage associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether KSM-3 is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species