Increasing incubation temperature impacts species with temperaturedependent sex determination suc... more Increasing incubation temperature impacts species with temperaturedependent sex determination such as green sea turtles. Jensen et al. combined genetic and endocrine techniques to show that an important green turtle population has produced primarily females for two decades, suggesting that complete feminization is possible in the near future.
The development of cryopreservation techniques has led to important changes in animal reproductiv... more The development of cryopreservation techniques has led to important changes in animal reproductive biotechnology. However, these techniques are associated with cellular and molecular damage, affecting the mitochondrial function and quality of spermatozoa; moreover studies in fish are limited. In this work, the effects of cryopreservation on ultrastructure, mitochondrial function and antioxidant defences in Atlantic salmon (Salmo salar) spermatozoa were assessed, along with intracellular calcium (Ca 2+) i , mitochondrial DNA sequence and sperm function (motility and fertilization rate). Significant ultrastructure alterations of the middle piece and mitochondria were observed in cryopreserved spermatozoa as compared to controls. Oxygen consumption and ATP were also significantly different in cryopreserved spermatozoa, and in
Following the Deepwater Horizon (DWH) oil spill, reproductive success rates in 2 northern Gulf of... more Following the Deepwater Horizon (DWH) oil spill, reproductive success rates in 2 northern Gulf of Mexico (GoM) bottlenose dolphin stocks exposed to oil were evaluated for 4 yr during and after the spill (2010 to 2015) in efforts to assess population-level reproductive health. Pregnancy was determined from either (1) ultrasound examinations of the reproductive tract during capture-release health assessments, or (2) endocrine evaluations of blubber tissue collected from dart biopsies of free-ranging dolphins. Follow-up photo-identification was then used to track the status of pregnant females and any associated neonatal calves for a minimum of 1 yr after the initial pregnancy detection (IPD). For all pregnant females observed following IPD, individuals seen with a calf (reproductive success) and without one (reproductive failure) were recorded. The resulting estimated reproductive success rates for both GoM stocks (19.4%; 7/36) were less than a third of those previously reported in other areas not impacted by the spill (i.e. Sarasota Bay, FL; Indian River Lagoon, FL; and Charleston Harbor, SC) using similar techniques (64.7%; 22/34). We also evaluated the relationships between reproductive success and 13 potential covariates, including stock, ordinal date, progesterone, cortisol, thyroid hormone concentrations, leukocyte count, lung health score, and total body length. Among these, the results only provide strong evidence (Bayes factor > 20) of a relationship between reproductive failure and the total leukocyte count covariate. The high reproductive failure rates measured in both GoM stocks following the DWH oil spill are consistent with mammalian literature that shows a link between petroleum exposure and reproductive abnormalities and failures.
The hypothalamic-pituitary-adrenal (HPA) axis undergoes critical developments during adolescence.... more The hypothalamic-pituitary-adrenal (HPA) axis undergoes critical developments during adolescence. Therefore, stressors experienced during this period potentially have long-term effects on adult HPA axis function. We hypothesized that adolescent intermittent ethanol (AIE) exposure would affect adult HPA axis function, resulting in altered responses to an alcohol challenge in young adults or adults. To test these hypotheses, male rats were exposed to alcohol vapor for 6 h per day from post-natal day (PND) 28-42, then acutely challenged with alcohol intragastrically (3.2-4.5 g/kg) in young adults (PND 70) or adults (PND 90). Overall, we observed blunted HPA axis responses to an alcohol challenge due to AIE exposure. Specifically, AIE tended to inhibit the alcohol-challenge-induced increase in plasma corticosterone (CORT) concentrations in young adult and adult rats. As well, AIE significantly blunted the alcohol challenge-induced arginine vasopressin (Avp) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus of adult rats. Results of the present study are similar to what we have previously shown, that these changes in PVN responsiveness may result from AIE-induced alterations in adrenergic neurons in brain stem regions C1-C3 known to project to the PVN. AIE elevated the number of colocalized cfos/phenylethanolamine N-methyltransferase (PNMT)-positive cell bodies in the C1 region of adult rats. Together, this data suggests that AIE exposure produces alterations in male HPA axis responsiveness to administration of an acute alcohol challenge that may be long-lasting.
Juvenile green turtle (size of an adult Kemp's ridley) Good correspondence between the two assays... more Juvenile green turtle (size of an adult Kemp's ridley) Good correspondence between the two assays Testosterone assay measured the same antigen in the standard controls and plasma extracts Female Male
The overarching theme of this thesis was to gain a better understanding of male koala reproductiv... more 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…
We are particularly grateful to Matthew Godfrey who reviewed the entire document. xi 3.4. Assessm... more We are particularly grateful to Matthew Godfrey who reviewed the entire document. xi 3.4. Assessment of Significant Portion of its Range (SPR) .
Additional file 2: Table S2. Annotation and orthogroup information for green turtle blood transcr... more Additional file 2: Table S2. Annotation and orthogroup information for green turtle blood transcriptome assembly transcripts. Transcripts that did not match to a Gene Ontology (GO) ID are not included.
Additional file 3: Table S3. KEGG pathway analyses results. KEGG pathways for the green turtle tr... more Additional file 3: Table S3. KEGG pathway analyses results. KEGG pathways for the green turtle transcriptome and the leatherback multi-tissue assembly.
Additional file 10: Figure S1. Green turtle GO slim plots. Bar plots representing the number of g... more Additional file 10: Figure S1. Green turtle GO slim plots. Bar plots representing the number of genes in each Gene Ontology (GO) slim functional category from the green turtle blood transcriptome.
Additional file 4: Table S4. Annotation and orthogroup information for leatherback multi-tissue t... more Additional file 4: Table S4. Annotation and orthogroup information for leatherback multi-tissue transcriptome assembly transcripts. Transcripts that did not match to a Gene Ontology (GO) ID are not included.
Additional file 5: Table S5. Species-specific orthogroups. Orthogroups and the transcripts that b... more Additional file 5: Table S5. Species-specific orthogroups. Orthogroups and the transcripts that belong to each orthogroup from each species-specific blood transcriptome.
The Editorial Staff of Herpetological Conservation & Biology would like to acknowledge the follow... more The Editorial Staff of Herpetological Conservation & Biology would like to acknowledge the following 360 peer reviewers for their expert opinions in 2014-2015. Your efforts, dedication, and professionalism have ensured that Herpetological Conservation & Biology continues to be a success.
When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of r... more When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measure-ments exhibited appropriate quality characteristics when analyzed via a bootstraped step-wise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 – 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75–150mg; r2 = 0.012, p = 0.78, slope = 0.022ngcortisol deviation/ultissue extract added). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality ...
Background Transcriptomic data has demonstrated utility to advance the study of physiological div... more 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 traditionall...
Increasing incubation temperature impacts species with temperaturedependent sex determination suc... more Increasing incubation temperature impacts species with temperaturedependent sex determination such as green sea turtles. Jensen et al. combined genetic and endocrine techniques to show that an important green turtle population has produced primarily females for two decades, suggesting that complete feminization is possible in the near future.
The development of cryopreservation techniques has led to important changes in animal reproductiv... more The development of cryopreservation techniques has led to important changes in animal reproductive biotechnology. However, these techniques are associated with cellular and molecular damage, affecting the mitochondrial function and quality of spermatozoa; moreover studies in fish are limited. In this work, the effects of cryopreservation on ultrastructure, mitochondrial function and antioxidant defences in Atlantic salmon (Salmo salar) spermatozoa were assessed, along with intracellular calcium (Ca 2+) i , mitochondrial DNA sequence and sperm function (motility and fertilization rate). Significant ultrastructure alterations of the middle piece and mitochondria were observed in cryopreserved spermatozoa as compared to controls. Oxygen consumption and ATP were also significantly different in cryopreserved spermatozoa, and in
Following the Deepwater Horizon (DWH) oil spill, reproductive success rates in 2 northern Gulf of... more Following the Deepwater Horizon (DWH) oil spill, reproductive success rates in 2 northern Gulf of Mexico (GoM) bottlenose dolphin stocks exposed to oil were evaluated for 4 yr during and after the spill (2010 to 2015) in efforts to assess population-level reproductive health. Pregnancy was determined from either (1) ultrasound examinations of the reproductive tract during capture-release health assessments, or (2) endocrine evaluations of blubber tissue collected from dart biopsies of free-ranging dolphins. Follow-up photo-identification was then used to track the status of pregnant females and any associated neonatal calves for a minimum of 1 yr after the initial pregnancy detection (IPD). For all pregnant females observed following IPD, individuals seen with a calf (reproductive success) and without one (reproductive failure) were recorded. The resulting estimated reproductive success rates for both GoM stocks (19.4%; 7/36) were less than a third of those previously reported in other areas not impacted by the spill (i.e. Sarasota Bay, FL; Indian River Lagoon, FL; and Charleston Harbor, SC) using similar techniques (64.7%; 22/34). We also evaluated the relationships between reproductive success and 13 potential covariates, including stock, ordinal date, progesterone, cortisol, thyroid hormone concentrations, leukocyte count, lung health score, and total body length. Among these, the results only provide strong evidence (Bayes factor > 20) of a relationship between reproductive failure and the total leukocyte count covariate. The high reproductive failure rates measured in both GoM stocks following the DWH oil spill are consistent with mammalian literature that shows a link between petroleum exposure and reproductive abnormalities and failures.
The hypothalamic-pituitary-adrenal (HPA) axis undergoes critical developments during adolescence.... more The hypothalamic-pituitary-adrenal (HPA) axis undergoes critical developments during adolescence. Therefore, stressors experienced during this period potentially have long-term effects on adult HPA axis function. We hypothesized that adolescent intermittent ethanol (AIE) exposure would affect adult HPA axis function, resulting in altered responses to an alcohol challenge in young adults or adults. To test these hypotheses, male rats were exposed to alcohol vapor for 6 h per day from post-natal day (PND) 28-42, then acutely challenged with alcohol intragastrically (3.2-4.5 g/kg) in young adults (PND 70) or adults (PND 90). Overall, we observed blunted HPA axis responses to an alcohol challenge due to AIE exposure. Specifically, AIE tended to inhibit the alcohol-challenge-induced increase in plasma corticosterone (CORT) concentrations in young adult and adult rats. As well, AIE significantly blunted the alcohol challenge-induced arginine vasopressin (Avp) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus of adult rats. Results of the present study are similar to what we have previously shown, that these changes in PVN responsiveness may result from AIE-induced alterations in adrenergic neurons in brain stem regions C1-C3 known to project to the PVN. AIE elevated the number of colocalized cfos/phenylethanolamine N-methyltransferase (PNMT)-positive cell bodies in the C1 region of adult rats. Together, this data suggests that AIE exposure produces alterations in male HPA axis responsiveness to administration of an acute alcohol challenge that may be long-lasting.
Juvenile green turtle (size of an adult Kemp's ridley) Good correspondence between the two assays... more Juvenile green turtle (size of an adult Kemp's ridley) Good correspondence between the two assays Testosterone assay measured the same antigen in the standard controls and plasma extracts Female Male
The overarching theme of this thesis was to gain a better understanding of male koala reproductiv... more 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…
We are particularly grateful to Matthew Godfrey who reviewed the entire document. xi 3.4. Assessm... more We are particularly grateful to Matthew Godfrey who reviewed the entire document. xi 3.4. Assessment of Significant Portion of its Range (SPR) .
Additional file 2: Table S2. Annotation and orthogroup information for green turtle blood transcr... more Additional file 2: Table S2. Annotation and orthogroup information for green turtle blood transcriptome assembly transcripts. Transcripts that did not match to a Gene Ontology (GO) ID are not included.
Additional file 3: Table S3. KEGG pathway analyses results. KEGG pathways for the green turtle tr... more Additional file 3: Table S3. KEGG pathway analyses results. KEGG pathways for the green turtle transcriptome and the leatherback multi-tissue assembly.
Additional file 10: Figure S1. Green turtle GO slim plots. Bar plots representing the number of g... more Additional file 10: Figure S1. Green turtle GO slim plots. Bar plots representing the number of genes in each Gene Ontology (GO) slim functional category from the green turtle blood transcriptome.
Additional file 4: Table S4. Annotation and orthogroup information for leatherback multi-tissue t... more Additional file 4: Table S4. Annotation and orthogroup information for leatherback multi-tissue transcriptome assembly transcripts. Transcripts that did not match to a Gene Ontology (GO) ID are not included.
Additional file 5: Table S5. Species-specific orthogroups. Orthogroups and the transcripts that b... more Additional file 5: Table S5. Species-specific orthogroups. Orthogroups and the transcripts that belong to each orthogroup from each species-specific blood transcriptome.
The Editorial Staff of Herpetological Conservation & Biology would like to acknowledge the follow... more The Editorial Staff of Herpetological Conservation & Biology would like to acknowledge the following 360 peer reviewers for their expert opinions in 2014-2015. Your efforts, dedication, and professionalism have ensured that Herpetological Conservation & Biology continues to be a success.
When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of r... more When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measure-ments exhibited appropriate quality characteristics when analyzed via a bootstraped step-wise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 – 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75–150mg; r2 = 0.012, p = 0.78, slope = 0.022ngcortisol deviation/ultissue extract added). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality ...
Background Transcriptomic data has demonstrated utility to advance the study of physiological div... more 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 traditionall...
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