Assessing stress physiology within a conservation breeding program for an endangered species.

Conservation breeding programs typically involve the management of individuals both in and ex situ, so it is vital to understand how the physiology of managed species changes in these environments to maximize program outcomes. The Vancouver Island marmot (VIM; Marmota vancouverensis) is one species that has been managed in a conservation breeding program to recover the critically low wild population. Previous research has shown there are differences in hair glucocorticoid concentrations for VIMs in different managed groups in the program. Therefore, we used >1000 blood samples collected since the program's inception to assess the neutrophil to lymphocyte (N:L) ratio among captive, pre-release, post-release and wild populations as another metric of stress. In situ VIM populations were found to have a significantly higher N:L ratio than ex situ populations, suggesting that the wild is a more physiologically challenging environment than managed care. Moreover, the effect of age, sex and the month of sampling on the N:L ratio were found to be different for each population. Age had the greatest magnitude of effect in the wild population, and sex was only significant in ex situ populations. This study provided previously unknown insights into the physiology of VIMs and increased post-release monitoring will be useful in the future to fully understand how physiology may be contributing to differences in survival of VIMs in the program.

Variation of hair cortisol in two herds of migratory caribou (Rangifer tarandus): implications for health monitoring.

Migratory caribou (Rangifer tarandus sspp.) is an ecotype of conservation concern that is experiencing increased cumulative stressors associated with rapid climate change and development in Arctic Canada. Increasingly, hair cortisol concentrations (HCCs) are being used to monitor seasonal hypothalamic-pituitary-adrenal axis activity of ungulate populations; yet, the effect of key covariates for caribou (sex, season, sampling source, body location) are largely unknown. The objectives of this research were 4-fold: first, we assessed the impact of body location (neck, rump) sampling sites on HCC; second, we assessed key covariates (sex, sampling method, season) impacting HCCs of caribou; third, we investigated inter-population (Dolphin and Union (DU), Bluenose-East (BNE)) and inter-annual differences in HCC and fourth, we examined the association between HCCs and indices of biting insect activity on the summer range (oestrid index, mosquito index). We examined hair from 407 DU and BNE caribou sampled by harvesters or during capture-collaring operations from 2012 to 2020. Linear mixed-effect models were used to assess the effect of body location on HCC and generalized least squares regression (GLS) models were used to examine the impacts of key covariates, year and herd and indices of biting insect harassment. HCC varied significantly by body location, year, herd and source of samples (harvester vs capture). HCC was higher in samples taken from the neck and in the DU herd compared with the BNE, decreased linearly over time and was higher in captured versus hunted animals (P < 0.05). There was no difference in HCC between sexes, and indices of biting insect harassment in the previous year were not significantly associated with HCC. This study identifies essential covariates impacting the HCC of caribou that must be accounted for in sampling, monitoring and data interpretation.

Effects of EGF and melatonin on gene expression of cumulus cells and further in vitro embryo development in bovines.

Despite previous research demonstrating the benefits of including growth factors and antioxidants to maturation medium to support embryo production, to date the effect of epidermal growth factor (EGF) and melatonin (Mel) on oocyte competency has not been studied. This study supplemented in vitro maturation (IVM) medium with EGF (10 ng/ml) and Mel (50 ng/ml) alone, or in combination, and evaluated cumulus cell (CC) gene expression and the development and quality of parthenogenetic blastocysts. No differences in CC gene expression levels indicative of developmental potential were found among the treatment groups. Antioxidant gene CuZnSOD was significantly (P < 0.05) decreased in CCs from the Mel group. Moreover, blastocyst rates on day 7 were significantly increased in EGF or Mel (P < 0.05), but not EGF+Mel. Significant decrease (P < 0.05) in GPX1, CuZnSOD, SLC2A1 and HSPA1A (P = 0.07) mRNA levels was observed in blastocysts from the Mel group. OCT4 gene expression was significantly increased (P < 0.05) in EGF+Mel and confirmed using immunofluorescence. Our results indicate that, despite the lack of changes of competence-related genes in CCs, IVM medium supplemented with Mel improved the culture environment sufficiently, resulting in improved blastocysts. Moreover, EGF and Mel combined during maturation increased OCT4 gene and protein expression in blastocysts, indicating its potential for stem cells.

Hormonal correlates of male dominance rank, age, and genital colouration in vervet monkey (Chlorocebus pygerythrus).

Primates are the most colourful members of the Mammalian clade. In vervet monkeys (Chlorocebus pygerythrus), males are characterized by their red penis and blue scrotum. Such colour signals are often used in conspecific communication, and thus could be used to convey signaller condition. We quantified scrotal and penile colour characteristics using digital photographs between May-June 2016 from males in two neighboring groups along the shores of Lake Nabugabo, Uganda. We examined the relationship between fecal hormones, male dominance rank, age (adult vs. immature), and colour. Adult males were higher ranking than immatures, but there were no rank or age differences in fecal hormone levels. Glucocorticoids and androgens were positively correlated in immature, but not adult males. All scrotal characteristics were predicted by age, with adult males having more teal (i.e., less blue, more green) and more luminant scrota. Within adult males, those with higher androgens levels had more saturated blue scrotal colouration and higher-ranking males were more luminant. Penile colouration was also associated with age and rank. High-ranking males had a more saturated red penis, and adult male penile colour was more luminant and bluer than in immature males. Our findings are consistent with previous reports that scrotal colouration advertises sexual or reproductive maturity (i.e., age), but we also find that within adult males, colour also advertises dominance rank and may be mediated by androgen levels. Penile colouration also appears to signal information about male age and dominance rank but does not appear to be mediated by hormones.

Validation of reference genes for use in untreated bovine fibroblasts.

Proper normalization of RT-qPCR data is pivotal to the interpretation of results and accuracy of scientific conclusions. Though different approaches may be taken, normalization against multiple reference genes is now standard practice. Genes traditionally used and deemed constitutively expressed have demonstrated variability in expression under different experimental conditions, necessitating the proper validation of reference genes prior to utilization. Considering the wide use of fibroblasts in research and scientific applications, it is imperative that suitable reference genes for fibroblasts of different animal origins and conditions be elucidated. Previous studies on bovine fibroblasts have tested limited genes and/or samples. Herein, we present an extensive study investigating the expression stability of 16 candidate reference genes across 7 untreated bovine fibroblast cell lines subjected to controlled conditions. Data were analysed using various statistical tools and algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder. A combined use of GUSB and RPL13A was determined to be the best approach for data normalization in untreated bovine fibroblasts.

Qiviut cortisol reflects hypothalamic-pituitary-adrenal axis activity in muskoxen (Ovibos moschatus).

Muskoxen (Ovibos moschatus) are increasingly exposed to a broad diversity of stressors in their rapidly changing Arctic environment. There is an urgent need to develop validated tools to monitor the impact of these stressors on the hypothalamic-pituitary-adrenal (HPA) axis activity of muskoxen to help inform conservation actions. Here, we evaluated whether muskox qiviut (dense wooly undercoat) cortisol accurately reflects changes in HPA axis activity. Two repeated pharmacological challenges, involving weekly administrations of saline (control group) or adrenocorticotropic hormone (ACTH) during five consecutive weeks, were done on captive muskoxen, in winter (no hair growth) and summer (maximum hair growth). Pre-challenge qiviut cortisol levels were significantly higher in the shoulder than in the neck, but neither differed from rump concentrations. Qiviut cortisol levels significantly increased (p < 0.001) in response to the administration of ACTH during the hair growth phase, but not in the absence of growth (p = 0.84). Cortisol levels in the qiviut segment grown during the summer challenge increased significantly over a six-month period in the ACTH-injected muskoxen with a similar trend occurring in the control animals. Finally, cortisol levels in shed qiviut were significantly higher and not correlated to those of fully grown qiviut shaved three months earlier. Our results show that cortisol is deposited in qiviut during its growth and that qiviut cortisol can thus be used as an integrated measure of HPA axis activity over the period of the hair's growth. Differences in qiviut cortisol across body regions, significant differences in qiviut segments over time, and differences between shed qiviut versus unshed qiviut, highlight the importance of consistent design and methodology for sample collection and analyses in order to account for sources of variation when using qiviut cortisol as a biomarker of HPA axis activity in muskoxen.

Evidence of degradation of hair corticosterone in museum specimens.

Researchers increasingly rely on non-invasive physiological indices, such as glucocorticoid (GC) levels, to interpret how vertebrates respond to changes in their environment. Recently, hair GCs have been of particular interest, because they are presumed stable over long periods of storage, which may facilitate the study of large-scale spatial and temporal patterns of stress in mammals. In the current study, we evaluated the stability of hair corticosterone levels in museum specimens, and the potential effects of different museum curation treatments. Using deer mouse (Peromyscus maniculatus) specimens collected from Vancouver Island (11 sites, 82 individuals) over 76 years, we found that specimens collected earlier in the 20th century had lower hair corticosterone than more recently collected specimens. These results suggest that hair hormone levels may not be stable over decades of storage time. We then subjected hair samples collected from white-footed mouse (Peromyscus leucopus, n = 36) to 3 different museum curation treatments, and found that borax lowered hair corticosterone levels relative to control samples, but air drying samples, or treating them with turpentine had no effect. Our results present a source of concern for the use of museum specimens for hair hormone analysis, and for studying long term trends in glucocorticoid levels.

Choosing a culture medium for SCNT and iSCNT reconstructed embryos: from domestic to wildlife species.

Over the past decades, in vitro culture media have been developed to successfully support IVF embryo growth in a variety of species. Advanced reproductive technologies, such as somatic cell nuclear transfer (SCNT), challenge us with a new type of embryo, with special nutritional requirements and altered physiology under in vitro conditions. Numerous studies have successfully reconstructed cloned embryos of domestic animals for biomedical research and livestock production. However, studies evaluating suitable culture conditions for SCNT embryos in wildlife species are scarce (for both intra- and interspecies SCNT). Most of the existing studies derive from previous IVF work done in conventional domestic species. Extrapolation to non-domestic species presents significant challenges since we lack information on reproductive processes and embryo development in most wildlife species. Given the challenges in adapting culture media and conditions from IVF to SCNT embryos, developmental competence of SCNT embryos remains low. This review summarizes research efforts to tailor culture media to SCNT embryos and explore the different outcomes in diverse species. It will also consider how these culture media protocols have been extrapolated to wildlife species, most particularly using SCNT as a cutting-edge technical resource to assist in the preservation of endangered species.

Parasites, stress and reindeer: infection with abomasal nematodes is not associated with elevated glucocorticoid levels in hair or faeces.

Stress hormones (glucocorticoids), incorporated into hair/fur and faeces, have been proposed as biomarkers of overall health in wildlife. Although such biomarkers may be helpful for wildlife conservation and management, their use has rarely been validated. There is a paucity of studies examining the variation of stress hormones in mammals and how they relate to other health measures, such as parasitism. Parasites are ubiquitous in wildlife and can influence the fitness of individual animals and populations. Through a longitudinal experiment using captive reindeer (Rangifer tarandus tarandus), we tested whether animals infected with Ostertagia gruehneri, a gastrointestinal nematode with negative impacts on fitness of the host, had higher stress levels compared with those that had been treated to remove infection. Faecal samples were collected weekly for 12 weeks (June-September) and hair was collected at the start and end of the study; glucocorticoids were quantified using enzyme immunoassays. Contrary to what was expected, infected reindeer had similar levels of cortisol in hair and slightly lower glucocorticoid metabolites in faeces compared with uninfected reindeer. Faecal corticosterone levels were higher than faecal cortisol levels, and only corticosterone increased significantly after a handling event. These results suggest that reindeer may use a tolerance strategy to cope with gastrointestinal nematodes and raise the question as to whether moderate infection intensities with nematodes are beneficial to the host. By removing nematodes we may have altered the gut microbiota, leading to the observed elevated faecal glucocorticoid metabolite levels in the treated reindeer. These findings demonstrate the importance of considering both cortisol and corticosterone in physiological studies, as there is mounting evidence that they may have different functionalities.

Enzyme immunoassays as a method for quantifying hair reproductive hormones in two felid species.

Non-invasive monitoring of wild felid reproductive states is important, given that many species reproduce poorly in captivity. Despite extensive work in faecal hormone analysis in felids, continued development of techniques is necessary, particularly with wild populations. In this study, our aims were as follows: (i) biochemical validation of enzyme immunoassays for estrogen, testosterone and progesterone in Canada lynx and domestic cat hair extracts; (ii) assessment of the use of hair reproductive hormones to differentiate between reproductive states (intact, estrus, pregnant and spayed/neutered), using domestic cats as a model; and (iii) assessment of the use of hair reproductive hormones to differentiate between age and sex, accounting for potential regional variability in wild lynx populations. Analysis of hair hormone levels showed prospective value in detecting pregnancy states, with pregnant domestic cats having higher levels of progesterone than spayed females. However, intact and pregnant cats did not differ in progesterone levels. Yet, two female domestic cats had higher levels of hair progesterone following a 38-day oral progestin treatment, perhaps providing a preliminary pharmacological validation of the method. Estrogen and testosterone did not differ statistically according to reproductive states of domestic cats, although intact males had higher levels of hair testosterone than neutered males. When we applied these techniques to lynx fur, we determined that hormone levels were not sufficiently precise to differentiate age classes. Hair reproductive hormone ratios differed between sexes, with the estrogen-to-progesterone ratio demonstrating the highest accuracy in differentiating males from females. Hair hormone levels differed regionally for wild lynx, indicating that spatial variability should be a consideration in wildlife hormone studies spanning large spatial scales. We conclude that use of hair hormone analysis by enzyme immunoassay may hold promise for differentiating sex in felids, but the technique will require further refinement and validation before it can be applied broadly and reliably.

Influence of adrenocorticotrophin hormone challenge and external factors (age, sex, and body region) on hair cortisol concentration in Canada lynx (Lynx canadensis).

Land use changes are a significant factor influencing the decline of felid populations. However, additional research is needed to better understand how these factors influence populations in the wild. Hormone analysis can provide valuable information on the basic physiology and overall health of an animal, and enzyme immunoassays (EIA) are generally used for hair hormone analysis but must first be validated for the substrate of choice and species of interest. To date, hormone assays from hair have not been validated for Felidae, despite that the method holds considerable promise for non-invasive sampling of free-ranging animals. We sought to: (1) evaluate whether increased adrenocorticotrophin hormone (ACTH) during the period of hair growth results in elevated hair cortisol; (2) validate the enzyme immunoassay used; and (3) identify any variations in hair cortisol between age, sex and body regions, using Canada lynx. We quantified hair cortisol concentrations in captive animals through an ACTH challenge and collected samples from legally harvested lynx to compare variability between body regions. An EIA was validated for the analysis of hair cortisol. Lynx (n=3) had a qualitative increase in hair cortisol concentration following an ACTH challenge in captive animals (20 IU/kg of body weight weekly for 5 weeks), thereby supporting the use of an EIA to quantify cortisol values in hair. Based on our analysis of sampled lynx pelts, we found that hair cortisol did not vary between age and sex, but varied within the foot/leg region to a greater extent than between individuals. We recommend that future studies identify a standardized location for hair cortisol sampling.

In vitro development of bison embryos using interspecies somatic cell nuclear transfer.

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear-cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34-33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45-12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80-87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

Disorders of sexual development in wild and captive exotic animals.

Disorders of sexual development (DSDs) are an increasing concern in both captive and free-ranging wildlife species. Partial or complete reduction in fertility that results from intersex conditions or gonadal dysgenesis is detrimental to the reproductive potential of wildlife populations, and consequently, to their long-term survival. Compared to the wealth of information available on humans and domestic species, a better understanding of the factors influencing sexual development in wildlife is essential for developing and improving population management or conservation plans. This review attempts to bring together the different facets of DSDs as studied in the fields of reproductive physiology, endocrinology, ecotoxicology, wildlife biology, and environmental health.

Investigation into developmental potential and nuclear/mitochondrial function in early wood and plains bison hybrid embryos.

Studies to date have shown that bison embryo development in vitro is compromised with few embryos developing to the blastocyst stage. The aim of this study was to use bison-cattle hybrid embryos, an interspecific cross that is known to result in live offspring in vivo, as a model for assessing species-specific differences in embryo development in vitro. Cattle oocytes fertilized with cattle, plains bison and wood bison sperm were assessed for various developmental parameters associated with embryo quality, including cell number, apoptosis and ATP content. Decreased development to the blastocyst stage was observed in hybrid wood bison embryos compared with the other treatment groups. Although both wood bison and plains bison hybrid blastocysts had significantly lower cell numbers than cattle blastocysts, only wood bison hybrid blastocysts had a greater incidence of apoptosis than cattle blastocysts. Among the treatment groups, ATP levels and expression profiles of NRF1, TFAM, MT-CYB, BAX and BCL2 were not significantly different in both 8- to 16-cell stage and blastocyst stage embryos. These data provide evidence of decreased developmental competence in the wood bison hybrid embryos, owing to inadequate culture conditions that have increased apoptotic events.

Cytogenetics of wild and captive bred non-domestic animals.

Cytogenetics of wild and captive bred non-domestic animals provides us with valuable information that can be implemented in wildlife management and species conservation strategies. In this review, we summarized the data published to date describing a range of chromosome abnormalities observed in non-domestic animals and their effect on phenotype. Two important factors that can potentially have drastic effects on captive breeding programs are discussed: presence of classic chromosome abnormalities, spontaneously-occurring and inherited, and intraspecific variations in chromosome number. Short-term consequences, primarily reduced reproductive efficiency, and long-term consequences, such as changes in population dynamics, are examined.

Early pregnancy diagnosis by serum progesterone and ultrasound in sheep carrying somatic cell nuclear transfer-derived pregnancies.

Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)-derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus-synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post-estrus (day 7 sample) and 19 days post-estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.

The impact of chromosomal alteration on embryo development.

Chromosome alterations, such as those affecting telomere erosion, predictably occur with each cell division, others, which involve changes to the expression and replication of the X-chromosome occur at particular stages of development, while those that involve loss or gain of chromosomes occur in a random and so far unpredictable manner. The production of embryos in vitro and by somatic cell nuclear transfer (SCNT) has been associated with altered expression of marker genes on the X-chromosome and an increased incidence of chromosomally abnormal cells during early development. In the case of SCNT embryos chromosome abnormalities may be associated with the nuclear donor cell. Telomere rebuilding subsequent to SCNT appears to vary according to species and type of donor cell used. It is speculated that the rate of telomere erosion and incidence of chromosome abnormalities affects developmental potential of early embryos and may be potential predictors of developmental outcome.

Different culture media requirements of IVF and nuclear transfer bovine embryos.

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.