Tracking reproductive events: Hoof growth and steroid hormone concentrations in hair and hoof tissues in moose (Alces alces).

Measurements of reproductive and stress-related hormones in keratinous tissues (e.g. hair, claws, hooves, baleen) can provide a record of stress and reproductive response in wildlife. We evaluated a method to collect keratin tissue from hooves of immobilized moose (Alces alces) and validated enzyme immunoassays for measuring cortisol and progesterone in hooves and hair. We also measured the annual growth and wear rates of moose hooves. Progesterone (range: 1.0-43.7 pg/mg) and cortisol (range: 0.05-2.9 pg/mg) were measurable and showed variation among hoof samples and moose. Pregnant females had twice as high progesterone concentrations (18.00 ± 3.73 pg/mg) from hoof sample locations post breeding compared to non-pregnant moose (9.40 ± 0.25 pg/mg). Annual hoof growth differed between the front (5.58 ± 0.12 cm) and rear (4.73 ± 0.13 cm) hooves and varied by season with higher growth rates during summer which decreased into autumn and winter. Adult female hooves represented between 1.6 and 2.1 years of growth and included up to two reproductive cycles. We established a method to estimate hoof growth rate and applied this to postmortem samples and were able to detect previous pregnancies. Shoulder guard hairs grew between August and March including during late gestation; however, hair progesterone concentrations (range: 2-107.1 pg/mg) were not related to reproductive state. Hair cortisol concentrations in our study (range: 0.2-15.9 pg/mg) were within the range of values previously reported for cervids. Our study supports the use of hooves for longitudinal sampling and measuring reproductive and stress-related hormones, providing a new tool for tracking reproductive events and understanding what variables may contribute to population level changes in reproduction.

Whiskers as a novel tissue for tracking reproductive and stress-related hormones in North Pacific otariid pinnipeds.

Keratinized tissues, including whiskers, are ideal for acquiring a record of physiological parameters. Most tissues provide a snapshot of physiological status; however, whiskers may support longitudinal sampling for reproductive and stress-related hormones, if hormones are incorporated as whiskers grow and concentrations change with physiological state. Whiskers from female Steller sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus) were serially sectioned and pulverized and steroid hormones were extracted. Standard methods were used to validate enzyme immunoassay kits for cortisol, progesterone, 17ß-estradiol and testosterone. All hormones were measurable in whisker segments from both species with progesterone concentrations showing cyclical patterns, which appear to signify previous pregnancies or luteal phases. Yearly progesterone concentrations were greater in years a pup was produced compared with years when no pup was observed. Free-ranging female Steller sea lions had reproductive rates between 0 and 1.0 (0.53 ± 0.33, n = 12) using a yearly progesterone concentration of 30 pg/mg or greater to classify a reproductive year as producing a pup and below 30 pg/mg as non-reproductive. Cortisol concentrations were greater near the root and rapidly declined, lacking any obvious patterns, throughout the rest of the whisker. Progesterone and testosterone concentrations were able to help determine sex of unknown individuals. Immunohistochemistry revealed that steroid hormones most likely do not leach out of whiskers based on the deposition patterns of progesterone and cortisol being present throughout the whisker length. Overall, measuring steroid hormones in whiskers can reveal individual reproductive histories over multiple years in sea lions and fur seals. Cyclical patterns of δ15N were useful for identifying periods of up to ~10 years of growth within whiskers, and measuring both stable isotopes and hormones may be useful for differentiating periods of active gestation from diapause and potentially track multi-year reproductive histories of female otariids.

Regional, seasonal and age class blubber fatty acid signature analysis of harbour seals in Alaska from 1997 to 2010.

Alaskan harbour seal populations are currently listed as a species of special concern. Although there is evidence of recent stabilization or even partial recovery of harbour seal numbers in areas of historic decline, most populations have not made substantial recoveries. To date, few data exist regarding spatial and seasonal changes in blubber fatty acids (FAs) for Alaskan harbour seal populations. The purpose of this study was to qualitatively investigate harbour seal blubber FA profiles for regional, seasonal and age class differences. Blubber FA concentrations were analysed using MANOVA and linear discriminant analysis (LDA) from 760 individual harbour seals across Bristol Bay, Kodiak, Prince William Sound and Southeast Alaska from 1997 to 2010. Our results suggest spatial and seasonal differences are largely driven by monounsaturated FAs, most notably 14:1n-5, 16:1n-7 and 18:1n-7. In addition, our data revealed a progression in blubber FAs from pups to adults, with a shift from saturated FAs and short-chained monounsaturated FAs in the pup blubber to more long-chain monounsaturated FAs and polyunsaturated FAs in adults. Lastly, harbour seals pups had elevated saturated FA 16:0 concentrations when compared to other age classes, regardless of location or period. With this vast spatial and seasonal FA information, we believe future sampling of blubber FAs from Alaskan harbour seal populations could be a useful tool in assessing the response of this species and its ecosystem to changes associated with natural and anthropogenic pressures.

Stress-related and reproductive hormones in hair from three north Pacific otariid species: Steller sea lions, California sea lions and northern fur seals.

Assessing the physiological impact of stressors in pinnipeds is logistically challenging, and many hormones are altered by capture and handling, limiting the utility of metabolically active tissues. Hair is increasingly being used to investigate stress-related and reproductive hormones in wildlife populations due to less-invasive collection methods, being metabolically inert once grown and containing multiple biomarkers of ecological interest. We validated enzyme immunoassays for measuring aldosterone, cortisol, corticosterone, and testosterone in lanugo (natal hair grown in utero) samples collected from Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), and northern fur seals (Callorhinus ursinus). We applied laboratory validation methods including recovery of added mass, parallelism and dilution linearity. We found no effects due to differences in alcohol- versus detergent-based cleaning methods. Further, there were no significant differences in hormone concentrations in hair samples collected immediately after the molt and the subsequent samples collected over 1 year, indicating steroid hormones are stable once deposited into pinniped hair. We found no sex differences in any hormone concentrations, likely due to the lanugo being grown in utero and influenced by maternal hormone concentrations. For Steller sea lion and California sea lion pups, we found hormone concentrations significantly differed between rookeries, which warrants future research. Hair provides a novel tissue to explore the intrinsic or extrinsic drivers behind hormone measurements in otariids, which can be paired with multiple health-related metrics to further investigate possible drivers of physiological stress.

Organochlorine contaminant concentrations in blubber of young Steller sea lion (Eumetopias jubatus) are influenced by region, age, sex, and lipid stores.

Contaminant exposure is particularly important for species and populations of conservation concern, such as the Steller sea lion (Eumetopias jubatus). We used blubber samples (n = 120) to determine organochlorine concentrations, including polychlorinated biphenyls (PCBs) and dichloro-diphenyl-trichloroethane (DDTs), and blood samples (n = 107) to estimate total body lipids based on the hydrogen isotope dilution method. We assessed the influence of age, sex, condition, and geographic area on contaminant concentrations in blubber and contaminant body load. The concentration of ΣPCBs was highest in pups (<6 months) from the Aleutian Islands, and the concentrations in males were higher than females in all regions. The ΣPCBs and ΣDDTs concentrations and loads decreased with increasing mass in pups, however, there were no regional or sex differences in contaminant load. Within each of the five age classes, the concentrations of ΣPCBs and ΣDDTs decreased with increasing mass, but overall these OCs increased with age. Further, accounting for the lipid content, a potential proxy for energy balance, in the animal load reduced or removed the regional and sex effects present in age models for contaminants. We propose, that adjusting OCs concentration by the lipid content of the blubber sample alone may not fully account for the variability in OC concentrations associated with differences in condition or energy states between young Steller sea lions.

Organochlorine contaminant concentrations in multiple tissues of free-ranging Steller sea lions (Eumetopias jubatus) in Alaska.

The relationships of selected organochlorine (OC) contaminants between blubber, blood, feces, and milk of young, free-ranging Steller sea lions (Eumetopias jubatus) were examined. Both between and within each tissue there was considerable individual variation. In spite of the variation, similar patterns were observed across the tissues for most of the selected PCB congeners. In all four tissues, the major PCB congeners were PCB101, PCB118, PCB138, and PCB153. The most prominent congener, both as a weight (ng/g lipid) and as a percentage of summed PCBs (∑PCBs), was PCB 153. Comparisons between paired tissues showed that ∑DDTs in blubber samples were related to concentrations in blood, feces, and milk. The ∑PCBs in blubber were related to concentrations in milk and fecal samples, though the relationship with feces was weak. Our findings show milk samples, in particular, are useful for assessing OCs in young sea lions. Blubber concentrations of PCB101, PCB118, and PCB138 were an order of magnitude higher than those in milk, supporting the biomagnification of these PCB congeners in SSL tissues. The findings indicate alternative tissues may be used as indicators of relative contaminant exposure in lieu of surgical blubber biopsy.

Endocrine and immunological responses to adrenocorticotrophic hormone (ACTH) administration in juvenile harbor seals (Phoca vitulina) during winter and summer.

There is increasing interest in measuring endocrine and immune parameters in free-ranging seals and sea lions, but there is a lack of understanding in how an acute stress response, often associated with capture and handling, influences these parameters of interest. The main objective of this study was to assess the impact of a simulated stressor on both endocrine and immune parameters. During two seasons, exogenous adrenocorticotrophic hormone (ACTH) was administered to seven female juvenile harbor seals and the response of several hormones (cortisol, aldosterone, total and free thyroxine and total triiodothyronine) and immunological parameters (total and differential leukocyte counts and peripheral blood mononuclear cells (PBMC) proliferation) were assessed. Cortisol peaked at 165 min (winter 203.1±84.7 ng/ml; summer 205.3±65.7 ng/ml) and remained significantly elevated 240 min after ACTH infusion in both seasons. Aldosterone peaked at 90 min (winter 359.3±249.3 pg/ml; summer 294.1±83.7 pg/ml) and remained elevated 240 min after administration of ACTH in both seasons. An increase in circulating total white blood cells was driven primarily by the increase in neutrophils which occurred simultaneously with a decrease in lymphocytes leading to an overall increase in neutrophil to lymphocyte ratio. These findings demonstrate that a simulated stress response in juvenile harbor seals results in a predictable increase in both cortisol and aldosterone concentrations, and were associated with altered immunological parameters.

Body condition and endocrine profiles of Steller sea lion (Eumetopias jubatus) pups during the early postnatal period.

Body condition indices have been useful in assessing the health of domestic and free ranging populations of terrestrial mammals. Given the high energy demand and rapid growth during the postnatal period of Steller sea lion (Eumetopias jubatus) (SSL) pups, body condition was expected to be related to concentrations of a suite of hormones (cortisol, aldosterone, thyroxine, triiodothyronine, leptin) previously associated with changes in body mass and composition in developing pinnipeds. Blood samples were collected from free ranging SSL pups of known ages and sex. A body condition index (BCI) previously developed for SSL pups based on a mass and length relationship was applied to 61 SSL pups ranging in age from 5 to 38days old. BCI was not related to pup age. Overall, male pups were larger than females and older pups were larger than younger pups. Aldosterone was negatively correlated with BCI only in female pups, whereas no relationship was observed between aldosterone and BCI in males. Further, male pups had higher aldosterone concentrations than females. Concentrations of cortisol, total and free thyroxine (T4), and total triiodothyronine (T3) decreased when regressed against the elapsed time between researchers' arrival on the rookery and removal of pup from the holding corral for blood collection. While the overall variation attributed to the rookery disturbance was low (r(2)<0.293), it may be of significance for future studies on free ranging pinnipeds. This study adds to the current knowledge of the postnatal changes in free ranging SSL pups by providing ranges of the BCI and several hormone concentrations from an apparently stable population.

Steller sea lion (Eumetopias jubatus) pups undergo a decrease in circulating white blood cells and the ability of T cells to proliferate during early postnatal development.

Postnatal changes in circulating immune components and peripheral blood mononuclear cell (PBMC) proliferation were assessed in Steller sea lion pups (SSL; Eumetopias jubatus). Blood samples were collected for complete blood cell counts including total and differential white blood cell (WBC) counts from 46 pups ranging in age from 5 to 38 days old. Total WBC and neutrophil counts decreased in association with increased age of the pups. The ability of PBMC to proliferate was assessed by in vitro exposure to concanavalin A (ConA) or lipopolysaccharide (LPS) in 21 pups ranging in age from 7 to 32 days old. All SSL pups responded to in vitro stimulation with ConA and LPS 055:B5 indicating peripheral T and B cells are capable of responding to an antigenic challenge. ConA-induced T cell proliferation decreased with age while there was no change in spontaneous proliferation of PBMC or B cells exposed to LPS. The decreases in total WBC, neutrophil counts and T cell proliferation indicates that SSL undergo a period of postnatal development in cell-mediated immune function which is comparatively longer than phocid pups and consistent with other otariids.

Neuronal fiber composition of the corpus callosum within some odontocetes.

Odontocetes (toothed whales) evolved from terrestrial mammals approximately 55 million years ago and have since remained on a unique evolutionary trajectory. This study used formalin-fixed tissue and light microscopy to quantify the size and number of fibers along the corpus callosum of the bottlenose dolphin (n = 8). Two other species, the Amazon River dolphin (n = 1) and the killer whale (n = 1), were included for comparison. A large amount of variation in the shape and area of the corpus callosum was observed. The odontocete corpus callosum is a heterogeneous structure with variation in fiber size and density along the length of the corpus callosum in all specimens examined. Using the species with the largest sample size, the bottlenose dolphin, comparisons by sex and age (sexually mature verses immature) were made for the area of the corpus callosum, five subregions, and fiber densities. Although no sex differences were detected, age appeared to affect the size, shape, and fiber composition of the bottlenose dolphin corpus callosum.