Influence of season, age, sex, and time of day on the endocrine profile of the common bottlenose dolphin (Tursiops truncatus).

Understanding baseline hormone levels, the magnitude of intra-individual variability, and their variation as a function of life history is difficult in toothed whales (e.g. dolphins and porpoises) because of the effects of capture stress. To determine the endocrine profile of the common bottlenose dolphin (Tursiops truncatus) as a function of season, time of day (TOD), age, sex, and reproductive status, blood corticosteroids, thyroid hormones, and catecholamines were repeatedly measured in a managed-care population exposed to ambient light and water temperatures of San Diego Bay. Additionally, fecal hormone metabolites were assessed for cortisol, aldosterone, and triiodothyronine. Samples were collected at two to four-week intervals over a period of two years, and multiple times within a day at monthly intervals over a year. Samples were collected through the voluntary participation of the dolphins in the blood draws and fecal collections in order to avoid the effects of handling stress. All serum hormones except aldosterone significantly varied with season and all serum hormones except total thyroxine significantly varied as a function of TOD. Fecal glucocorticoid metabolites significantly correlated with circulating cortisol levels, and there was a significant seasonal effect on triiodothyronine fecal metabolites. Strong seasonal effects demonstrated complex interactions with age and sex suggesting that contextual information is critical to interpreting differences in endocrine profiles. Strong circadian patterns further suggest that sampling design is important to the interpretation of blood or fecal collections, particularly since diurnal changes in some serum hormone levels are similar to the magnitude of seasonal differences. Despite potential impacts of feeding schedules on diurnal patterns, managed care populations can provide important insights into seasonal and age-related endocrine changes in toothed whales.

Isolation of DNA from small amounts of elephant ivory: Sampling the cementum with total demineralization extraction.

Genotyping ivory samples can determine the geographic origin of poached ivory as well as the legality of ivory being sold in ivory markets. We conducted a series of experiments to determine where the DNA is most concentrated in ivory samples and how best to increase DNA yield from groups of samples likely to vary in DNA concentration. We examined variation in DNA amplification success from: the layer(s) of the tusk (cementum and/or dentine) being extracted, demineralization temperature and time, and the concentration of eluates. Since demineralization of the pulverized sample produces a pellet and supernatant, we also assessed DNA amplification success from the pellet, the supernatant, their combination, as well as variation in the respective amounts used for extraction. Our results show that the outer cementum layer of the tusk contains the highest concentration of DNA and should be separated and used exclusively as the source material of ivory processed for extraction, when available. Utilizing the combined demineralized lysate improves extraction efficiency, as does increasing demineralization time to 3 or more days, conducted at 4°C. The most significant improvements occurred for low template DNA ivory samples followed by medium quality samples. Amplification success of high quality samples was not affected by these changes. Application of this optimized method to 3068 ivory samples resulted in 81.2% of samples being confirmed for both alleles at a minimum of 10 out of 16 microsatellite loci, which is our threshold for inclusion in DNA assignment analyses.

CONSERVATION. Genetic assignment of large seizures of elephant ivory reveals Africa's major poaching hotspots.

Poaching of elephants is now occurring at rates that threaten African populations with extinction. Identifying the number and location of Africa's major poaching hotspots may assist efforts to end poaching and facilitate recovery of elephant populations. We genetically assign origin to 28 large ivory seizures (≥0.5 metric tons) made between 1996 and 2014, also testing assignment accuracy. Results suggest that the major poaching hotspots in Africa may be currently concentrated in as few as two areas. Increasing law enforcement in these two hotspots could help curtail future elephant losses across Africa and disrupt this organized transnational crime.

Validation and application of noninvasive glucocorticoid and thyroid hormone measures in free-ranging Hawaiian monk seals.

We validate fecal glucocorticoid (GC) and thyroid (T3) hormone metabolite measures in the Critically Endangered Hawaiian monk seal for the first time, and examine variation in the concentrations of these hormones in individuals across the species' range. We test hypotheses that monk seals from declining subpopulations have relatively high GCs and low T3 on average suggesting impacts of food limitation, and that this hormone pattern is more apparent in immature animals compared to adults, as food limitation is specifically indicated as a principal cause of poor body condition and survival of juvenile monk seals. We opportunistically sampled scat from 84 individually identifiable monk seals during the 2010 breeding season from two geographic regions, the main Hawaiian Islands (MHI) and the Northwestern Hawaiian Islands (NWHI). The MHI subpopulation of monk seals is growing, whereas subpopulations at many sites in the NWHI are in decline. Best fit general linear models predicting variation in GCs and T3 (examined separately) were similar (after accounting for significantly elevated hormone concentrations associated with molt and possibly lactation); both included sample date, region, and monk seal age as predictors. GC concentrations were significantly lower in MHI versus NWHI monk seals and decreased as the breeding season progressed. T3 concentrations were significantly lower in immature monk seals compared to adults. GC and T3 concentrations were positively correlated at 4 NWHI sites; prey may be adequate for physiological growth or maintenance at these sites but relatively stressful to acquire. GCs were highest at French Frigate Shoals, (a NWHI site) while T3 was relatively low here, indicating a possible signal of food limitation. GCs were lowest in the MHI. Disturbance associated with living near a high human population in the MHI appears to impact monk seal physiology less than other stressors encountered in the remote and highly protected NWHI where human presence is extremely low.

Glucocorticosteroid concentrations in feces and hair of captive caribou and reindeer following adrenocorticotropic hormone challenge.

Climate change and industrial development are contributing to synchronous declines in Rangifer populations across the Arctic. Chronic stress has been implicated as a proximate factor associated with decline in free-ranging populations, but its role in Rangifer is unspecified. Analysis of glucocorticosteroid (GC) concentration in feces, and more recently in hair, is a non-invasive method for monitoring stress in wildlife. Adrenocorticotropic hormone (ACTH) released from the pituitary gland stimulates GC release from the adrenals and can be administered to reflect adrenal activation. In this study, we assessed concentrations of GC metabolites in feces and cortisol in hair of Alaskan caribou (Rangifer tarandus granti) and reindeer (R. t. tarandus) following ACTH treatment. We predicted that ACTH challenge would increase concentrations of fecal GCs, but not hair cortisol because steroid deposited into the hair shaft occurs over an extended period of time (months) and is likely insensitive to acute adrenal stimulation. Adult caribou (n=10; mean age, 6.5 years old) exhibited a peak increase in fecal GCs 8h following a 2 IU/kg dose of ACTH compared to pre-injection concentrations. In contrast, sub-adult reindeer (n=10, 0.8 years old) elicited a diminished response to the same dose. Quadrupling the dose (8 IU/kg) prolonged the fecal GC response in female reindeer, but male reindeer were unresponsive. Hair cortisol was unaffected by a single ACTH challenge. Further investigation is required to ascertain whether subspecific differences in adrenal sensitivity are attributed to age or sex differences, or historical selective pressures from semi-domestication and/or sedentary life cycle in reindeer.

Fecal triiodothyronine and thyroxine concentrations change in response to thyroid stimulation in Steller sea lions (Eumetopias jubatus).

Variation in concentrations of thyroid hormones shed in feces may help to identify physiological states of animals, but the efficacy of the technique needs to be validated for each species. We determined whether a known physiological alteration to thyroid hormone production was reflected in hormone concentrations in the feces of Steller sea lions (Eumetopias jubatus). We quantified variation of triiodothyronine (T3) and thyroxine (T4) concentrations in feces following two intramuscular injections of thyrotropin (thyroid-stimulating hormone, TSH) at 24h intervals in four captive female sea lions. We found fecal T3 concentrations increased 18-57% over concentrations measured in the baseline sample collected closest to the time of the first TSH injection (p=0.03) and 1-75% over the mean baseline concentration (p=0.12) for each animal of all samples collected prior to injections. Peak T3 concentrations were greater than the upper bound of the baseline 95% confidence interval for three animals. The peak T3 response occurred 48h post-injection in three animals and 71h in the fourth. Post-injection T4 concentrations did not differ between the baseline sample collected closest to the time of the first TSH injection (p=0.29) or the mean baseline concentration (p=0.23) for each animal. These results indicate that induced physiological alterations to circulating thyroid hormone concentrations can be adequately detected through analyses of fecal T3 concentrations and that the technique may provide a means of non-invasively detecting metabolic changes in Steller sea lions.

Long-term impacts of poaching on relatedness, stress physiology, and reproductive output of adult female african elephants.

Widespread poaching prior to the 1989 ivory ban greatly altered the demographic structure of matrilineal African elephant (Loxodonta africana) family groups in many populations by decreasing the number of old, adult females. We assessed the long-term impacts of poaching by investigating genetic, physiological, and reproductive correlates of a disturbed social structure resulting from heavy poaching of an African elephant population in Mikumi National Park, Tanzania, prior to 1989. We examined fecal glucocorticoid levels and reproductive output among 218 adult female elephants from 109 groups differing in size, age structure, and average genetic relatedness over 25 months from 2003 to 2005. The distribution in group size has changed little since 1989, but the number of families with tusked old matriarchs has increased by 14.2%. Females from groups that lacked an old matriarch, first-order adult relatives, and strong social bonds had significantly higher fecal glucocorticoid values than those from groups with these features (all females R(2)= 0.31; females in multiadult groups R(2)= 0.46). Females that frequented isolated areas with historically high poaching risk had higher fecal glucocorticoid values than those in low poaching risk areas. Females with weak bonds and low group relatedness had significantly lower reproductive output (R(2)[U]=0.21). Females from disrupted groups, defined as having observed average group relatedness 1 SD below the expected mean for a simulated unpoached family, had significantly lower reproductive output than females from intact groups, despite many being in their reproductive prime. These results suggest that long-term negative impacts from poaching of old, related matriarchs have persisted among adult female elephants 1.5 decades after the 1989 ivory ban was implemented.

A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species.

Noninvasive fecal glucocorticoid analysis has tremendous potential as a means of assessing stress associated with environmental disturbance in wildlife. However, interspecific variation in excreted glucocorticoid metabolites requires careful selection of the antibody used in their quantification. We compared four antibodies for detecting the major fecal cortisol metabolites in yellow baboons following (3)H cortisol administration, ACTH challenge, and HPLC separation of fecal glucocorticoid metabolites. The most effective antibody (ICN corticosterone RIA; Cat. No. 07-120102) demonstrated relatively high cross-reactivities to the major cortisol metabolites present in feces during peak excretion, following both radiolabel infusion and ACTH challenge. This same antibody also detected increased fecal glucocorticoid metabolites after ACTH administration in the African elephant, black rhinoceros, Roosevelt elk, gerenuk, scimitar-horned oryx, Alaskan sea otter, Malayan sun bear, cheetah, clouded leopard, longtailed macaque, and northern spotted owl. Results suggest that (1) fecal glucocorticoid assays reliably detect endogenous changes in adrenal activity of a diverse array of species and (2) where comparisons were made, the ICN corticosterone antibody generally was superior to other antibodies for measuring glucocorticoid metabolites in feces.

Lifetime reproductive success, longevity, and reproductive life history of female yellow baboons (Papio cynocephalus) of Mikumi National Park, Tanzania.

The relationship between longevity and lifetime reproductive success (LRS) was studied in free-ranging female baboons of Mikumi National Park, Tanzania. A severe population decline occurred between the 12th and 20th years of the study. The total sample consisted of 72 females born and reaching adulthood before the start of the population decline. There were 27 females who were adult at the start of the study and 45 who became adult within the 12 years prior to the decline. The subjects were studied until all 72 were dead and all of their offspring were either dead or at least six years old; this took 24 years. The relationship of longevity to LRS was statistically significant for the total sample and for both sub-samples, with 70% of the total variance in LRS accounted for by longevity. Longevity was linked to LRS via a chain of statistically significant relationships: The longer the life span, the longer the reproductive life; the longer the reproductive life, the more offspring produced; the more offspring produced, the higher the LRS. Mean LRS, life span, and reproductive longevity all differed between the two sub-samples. Since the sub-samples were time-linked to a population decline affecting longevity, either sub-sample separately would fail to reflect the broader picture. This illustrates the importance of appreciable sample sizes from long-term studies in helping understand the dynamics between life history estimates and ecological conditions in variable environments.

Serum prolactin concentrations in the captive female African elephant (Loxodonta africana): potential effects of season and steroid hormone interactions.

Research was conducted to determine whether seasonal changes in prolactin secretion occur in nonpregnant female African elephants and to examine potential functional interrelationships between secretion of prolactin, cortisol, and progesterone. Weekly blood samples were taken for 18 months from four female African elephants and the sera were analyzed by RIA for progesterone, cortisol, and prolactin concentrations. There was no significant effect of season on serum concentrations of prolactin. Estrous cycles averaged 14 weeks in length and were composed of a 9-week luteal phase and a 5-week follicular phase (based on progesterone concentrations consistently >200 and <200 pg/ml, respectively). Estrous cycle synchronicity was evident between pairs of elephants. Serum concentrations of prolactin (3.91 +/- 0.69 ng/ml; range: 0.84-15.8 ng/ml) were significantly lower during the luteal, compared with the follicular, phase (P < 0.0001; t test) and were positively correlated with serum concentrations of cortisol (r = 0.14; P < 0.05). Mean (+/-SE) serum concentration of cortisol was 5.7 +/- 1.3 ng/ml (range: 1.4-19.3 ng/ml), and concentrations of this adrenal steroid were negatively correlated with progesterone concentrations (r = -0.15; P < 0.01). Increased serum concentrations of prolactin detected during the follicular phase suggest that this hormone may be regulated by ovarian estrogens and may play a role in modulating ovarian function in the elephant.

Stress and reproductive failure: an evolutionary approach with applications to premature labor.

This article takes an evolutionary approach to the study of reproductive failure, drawing attention to the importance of reproductive failure among mammals as a means of timing reproduction in response to a variable environment. These concepts also apply to the diagnosis and treatment of prematurity, because before the advent of modern medicine most premature births probably constituted failed reproduction. Problems in the diagnosis and treatment of reproductive failure are described, with specific parallels drawn between the diagnoses and treatments of infertility and prematurity.

Ageing and social rank effects on the reproductive system of free-ranging yellow baboons (Papio cynocephalus) at Mikumi National Park, Tanzania.

Ageing and social status effects on reproductive condition are examined in 106 wild female baboons from 1974 to 1997. The mean duration of each reproductive state (follicular and luteal phase components, pregnancy and lactation) was examined per female, using a repeated measures analysis of variance across each of four age classes (3-6, 7-10, 11-14, and 15-20+ year olds) and dominance rank quartiles. The sex-skin swelling portion of the follicular phase increased whereas the fully swollen portion decreased in duration with advancing age and with low dominance rank. The detumescing portion of the luteal phase increased with advancing age, being most pronounced in lowest ranking females. The number of cycles to conception decreased with age, with no dominance rank effect. However, owing to variation in lactation duration, the interbirth interval was unaffected by age but was shorter in high ranking females. The miscarriage rate was unaffected by age but increased as female rank declined. Age and rank effects became significantly more pronounced in response to a crash in the study population between 1987 and 1993. In conclusion, age- and rank-related reproductive changes in baboons, like those in humans, appear to represent attempts to compensate endocrinologically for accelerated ovarian atresia and increased environmental hardship.

Longitudinal fecal steroid excretion in maned wolves (Chrysocyon brachyurus).

This study used a fecal steroid monitoring technique to evaluate reproductive cycles in male (4) and female (15) maned wolves, endangered South American canids. A radiolabeled testosterone infusion on a male revealed a fast and predominantly fecal route of excretion for this steroid. Testosterone was also excreted as eight unidentified metabolites, which was not the primary form of this steroid quantified in our assays. Fecal steroid concentrations (estradiol, E2; progestins, P; testosterone, T) in males and acyclic, nonpregnant (pseudo-pregnant), and pregnant females were monitored over four breeding seasons (October-January). Significant differences were detected between longitudinal P profiles of cyclic and acyclic females during estrus, luteal phase, and after birth/end of pseudo-pregnancy. Concentrations of P were also significantly higher in pregnant, compared to nonpregnant females, from proestrus to the end of the pregnant luteal phase. Although levels of T were higher in males than in females throughout the breeding season, no cyclicity in male fecal T concentrations was detected. Values of fecal P, T, and the ratio P/T were useful for differentiating gender and detecting pregnancy in females. Similarities to available data on other canids and the management and conservation implications of these findings were discussed.

Validity of an estimator of reproductive success.

Lifetime reproductive success is a major component of individual fitness and a central dependent variable for the study of natural selection. For long-lived animals, such as apes or baboons, assessment of lifetime reproductive success requires observations of identified individuals in continuous, long-term studies from which it is difficult and often impossible to obtain an adequate sample of necessary reproductive and survival data. This situation can be alleviated by the availability of a valid measure that uses incomplete reproductive histories to estimate the lifetime reproductive success of individuals. The validity of one such estimator was tested by determining if, after 10.5 years of studying free-ranging female baboons, it predicted lifetime reproductive success obtained from full reproductive histories after 21.5 years. Validity was evaluated for seven criteria of success, ranging from the number of a female's live births to the number of her offspring that reached the age of 72 months. Moderate to good prediction of lifetime reproductive success by the estimator was found for criteria of offspring living to 36 months or more. After 10.5 years, complete reproductive life spans were available for only eight females. Using the estimator, analytic potential, via sample representativeness and size, was improved at 10.5 years by an increase from a sample of eight to between 34 and 62, depending upon the criterion used, and at 21.5 years from 39 to 70. With a valid estimator, the opportunity to study lifetime reproductive success of a long-lived species is substantially improved without having to depend upon rarely available, uninterrupted data collection for 20-60 years.

Techniques for application of faecal DNA methods to field studies of Ursids.

We describe methods for the preservation, extraction and amplification of DNA from faeces that facilitate field applications of faecal DNA technology. Mitochondrial, protein encoding and microsatellite nuclear DNA extracted and amplified from faeces of Malayan sun bears and North American black bears is shown to be identical to that extracted and amplified from the same individual's tissue or blood. A simple drying agent, silica beads, is shown to be a particularly effective preservative, allowing easy and safe transport of samples from the field. Methods are also developed to eliminate the risk of faecal DNA contamination from hair present in faeces.

Utility of follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol and FSH:LH ratio in predicting reproductive age in normal women.

The relative efficacy of follicle stimulating hormone (FSH), luteinizing hormone (LH), FSH:LH ratio and oestradiol is evaluated as a predictor of ovarian reserve (reproductive age) in normal women. Serum levels of FSH, LH, oestradiol and FSH:LH ratios were measured during menstrual cycle days 1-4 in younger (20-25 years; n = 23) and older (40-45 years; n = 32) reproductive age women with regular menstruation and normal reproductive function. On days 1-4, mean levels of FSH, oestradiol and FSH:LH ratios were significantly higher in older compared with younger women. FSH increased in concentration across cycle days in both age groups. A significantly lower LH value in younger versus older women was found only on day 1. Oestradiol showed no change across days in the younger group, but increased significantly from day 1 to day 4 in the older group. FSH values on days 1 or 2 were the best single predictor of age differences. However, the best prediction of age differences was obtained by using the combination of FSH and LH (as opposed to the FSH:LH ratio) on day 1 of the menstrual cycle.

Reproductive control in wild baboons measured by fecal steroids.

Measurements of reproductive hormones (progestins and estrogens) excreted in feces were used to discriminate between 25 conceptive and 76 nonconceptive (including undetected early abortion) cycles of free-ranging yellow baboons at Mikumi National Park, Tanzania. Conceptive cycles had significantly higher luteal-phase progestin and estrogen concentrations than nonconceptive cycles as early as Day 4 postovulation. However, mean early luteal-phase progestin concentrations in conceptive cycles were lower when conception occurred during ecologically optimal vs. suboptimal times, and among females of high compared to low dominance rank. Mean estrogen concentrations in conceptive cycles showed the opposite dominance rank pattern: mean luteal-phase estrogen concentrations were higher in conceptive cycles of high-compared to low-ranking females. None of these relations existed for nonconceptive cycles. These data suggest that successful implantation is facilitated by relatively high early luteal-phase progestin and estrogen concentrations. However, long-term environmental cues predicting the probability of offspring survival appear to influence the amount of progesterone required for successful implantation; progesterone concentrations necessary to facilitate successful implantation are higher during suboptimal seasons or among females of low dominance rank-cues that also suggest that offspring survival conditions are relatively poor. This may act as a reproductive filter, restricting conception to females whose immediate condition (e.g., low social stress and good physical health) enables them to compensate physiologically and behaviorally for effects associated with these relatively harsh offspring survival conditions.

Excretory fate of estradiol and progesterone in the African elephant (Loxodonta africana) and patterns of fecal steroid concentrations throughout the estrous cycle.

We developed and validated a noninvasive method to quantify fecal estrogens and progestins as a tool for monitoring long-term ovarian activity in free-ranging African elephants. The lag times between iv injection of [(3)H]estradiol and [(14)C]progesterone and peak excretion of radioactivity in urine and feces were approximately 4 hr and 48 hr, respectively. The majority of progesterone metabolites recovered was excreted in feces (55%) versus urine (45%), whereas comparatively little of the recovered estradiol metabolites were excreted in feces (5%) compared to urine (95%). Intrasample variation in fecal hormone concentrations was extremely high but could be substantially reduced by extracting well-mixed fecal powder from freeze-dried samples, taken from the central or premixed portion of the wet sample. This method resulted in a close correspondence between matched serum and fecal progestins (mean correlation = 0.81, range 0.61-0.94) collected from five nonpregnant adult females over a 7-month period. Fecal estrogen profiles were more ambiguous, tending to overlap with those of fecal progestins. We conclude that analyses of fecal progestins can provide an effective, noninvasive means of characterizing ovarian activity in free-ranging African elephants.