GnRH Vaccine Could Suppress Serum Testosterone in Stallion Mules.

Stallion mules have been used as working equids in several countries. Aggressiveness under the influence of testosterone results in the necessity for surgical castration before work training. The gonadotropin-releasing hormone (GnRH) vaccine may be an alternative method for immunocastration in mules. The objective of this study was to evaluate the effect of the GnRH vaccine on anti-GnRH antibody concentration, serum testosterone concentration, clinical adverse effects, and behavioral changes in response to receiving selected physical manipulations from humans. Twenty-five mules were separated into three groups: Control-intact, Control-castrated, and Treatment. The Treatment group was further divided according to condition (intact or unilateral cryptorchid) and age. The Treatment group received 195 µg of the GnRH vaccine intramuscularly at weeks 0, 4, and 8. The anti-GnRH antibody concentrations increased at weeks 6 and 10, and then they gradually decreased to baseline at week 24. The Treatment-intact-young group had the highest concentration of anti-GnRH antibody. The serum testosterone concentrations in the Treatment group were lower than before vaccination from weeks 6 to 14. Subcutaneous edema adjacent to the injection site was detected in the Treatment-intact group after booster vaccination. In conclusion, the mules responded to the GnRH vaccine, which could temporarily suppress testosterone for up to 14 weeks.

Characterization of Longitudinal Testosterone, Cortisol, and Musth in Male Asian Elephants (Elephas maximus), Effects of Aging, and Adrenal Responses to Social Changes and Health Events.

The conservation of endangered species and sustainability of managed populations requires considerations to ensure the health and welfare of individuals. Male elephants experience a biological phenomenon called "musth", which is characterized by increased testosterone production, temporal gland secretion and urine dribbling, heightened aggression and sexual behavior, and therefore can pose unique challenges for human safety and animal welfare. This study characterized longitudinal (9 to 22 years) patterns of circulating testosterone and cortisol in relation to musth in four adult Asian elephant bulls spanning ages from 12 to 54 years. Age-related effects on musth activity and adrenal responses to social changes and clinical health events were also examined. All bulls exhibited regular annual musth cycles. Circulating cortisol covaried positively with testosterone and musth, highlighting intrinsic patterns that should be considered when evaluating the impact of social, health, and environmental changes on adrenal glucocorticoid activity. Except for an end-of-life cortisol increase in one bull, there was no clear evidence of chronically elevated cortisol secretion outside of musth in any individual. Testosterone decreased with age in sexually mature bulls, whereas age-related changes in cortisol varied across individuals, with the three older bulls showing the greatest rate of change during musth versus inter-musth periods. In contrast to physiological factors, there was no evidence of social factors, such as addition of a new male and death of male herdmates, impacting adrenal glucocorticoid activity in these bulls in the short term. Changes in cortisol were associated with treatment for Mycobacterium tuberculosis (M. tb) in two bulls, increasing after start of treatment and decreasing with cessation of treatment, but were not clearly associated with activation of disease. This study highlights the importance of longitudinal hormone monitoring to track changes in physiological function and responses to social, health, and environmental change in elephant bulls, which is important for making more informed decisions on how to manage male elephants under varying degrees of human care to ensure welfare and safety.

A Longitudinal Study of Hematology and Stress Biomarker Profiles in Young Asian Elephants (Elephas Maximus) in Relation to Elephant Endotheliotropic Herpesvirus (EEHV) in Thailand.

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is a virulent disease that causes severe hemorrhage and sudden death in Asian elephant calves. A change in hematology profiles is one indicator of infection before clinical signs appear; however, to be effective, individual baselines and age-matched reference values are needed. Stress has been speculated to be a factor in clinical EEHV cases, but relationships have not been demonstrated empirically. This study evaluated blood hematology and several stress response markers-salivary cortisol, fecal glucocorticoid metabolites (FGM), salivary Immunoglobulin A (SIgA), and fecal IgA (FIgA) in samples collected for 1 year from three healthy calves with no EEHV history (non-EEHV), and six that had previously been infected, developed clinical signs and survived (prior-EEHV). Hematology values between non-EEHV and prior-EEHV elephants were not different and within published reference ranges. Concentrations of salivary cortisol, FGM, SIgA, and FIgA also were variable and showed seasonal differences, but no relationships to prior EEHV status. One of the prior EEHV calves became re-infected, developed hemorrhagic disease (HD), and died during the study period. That calf exhibited lymphocytopenia, monocytopenia, and thrombocytopenia. Additionally, all stress biomarker concentrations were lower in the 12 days before viremia was observed. Thus, as in other studies, changes in hematology occur with EEHV infection, while preliminary data in one calf suggests that stress-response measures might also be informative and should be studied further.

Reproductive performance of captive Asian elephants (Elephas maximus) in large tourist camps in Thailand.

In Thailand, many elephants are used in tourism, with populations sustained by breeding of animals that are in captive habitats. Even though there are programs to promote breeding, there is not success in all camps. In this study, there was summarization of reproductive performance data of 407 elephants (150 males, 257 females) at seven tourist camps based on 4-21 years of breeding records. Age pyramid structures for elephants varied among camps. Reproductive rates averaged 21.6 ± 6.17% and varied among camps (2.8-45.0%). Based on parity, 77.4% of elephants were nulliparous, 8.2% produced one calf, and 14.3% were multiparous, with there being camp differences. There were 1.10 ± 0.46 (range, 0.03-3.55) births per year, with a total of 19.6 ± 9.3 (1-71) calves per camp. Age at first calving was 19.2 ± 1.1 years (range, 8-40 years), mean inter-birth interval was 4.4 ± 0.2 years (range, 1.8-7.9 years), and average gestation length was 653.9 ± 6.9 days (range, 578-743 days). Rates of abortions/stillbirths averaged 12.4% and ranged from 3.5%-66.7%. There were no obvious differences in management (e.g., number of males, estrous detection methods, work activities) that when evaluated explained the range in breeding success, although lack of male interest in females was a common problem. While informative and useful for designing future studies, results of this study indicate there is a lack of precise breeding records that makes it difficult to evaluate effects of management practices on reproductive performance of captive elephants in Thailand.

Effect of Tourist Activities on Fecal and Salivary Glucocorticoids and Immunoglobulin A in Female Captive Asian Elephants in Thailand.

Asian elephants have been an important part of wildlife ecotourism in Thailand for over two decades. Elephants in tourist camps are exposed to a variety of management styles and daily activities that can potentially affect health and welfare. This study investigated relationships between a novel welfare biomarker, immunoglobulin A (IgA), and daily camp activities, and compared results to glucocorticoid (GC) measures. Often no-riding camps are portrayed as providing better welfare than camps that offer riding. Therefore, we predicted that elephants at no-riding camps would have lower GC and higher IgA concentrations, and a low GC/IgA ratio. Forty-four female elephants from six elephant camps were divided into three groups based on riding activities: saddle-riding, bareback-riding, and no-riding. Fecal and salivary samples were collected monthly for 1 year along with evaluations of body condition, foot health, and wounding. Camp environment and management varied among camps, although the major difference was in riding activities. Concentrations of GCs and IgA varied among the working groups, but not always consistently between sample matrices. Overall fecal glucocorticoid metabolite concentrations were lowest in the saddle-riding group. Only in one bareback-riding camp did the elephants exhibit a potentially positive welfare response with a low GC/IgA ratio over time. Other results varied between the two biomarkers, with considerable variability across camps, suggesting there is more to good welfare than whether elephants participate in riding or not. Several other human-induced stressors, like chaining, ankus use, and limited social opportunities are likely to be impacting well-being and should be considered to ensure management practices meet physical and psychological welfare needs.

Circadian Rhythm of Salivary Immunoglobulin A and Associations with Cortisol as A Stress Biomarker in Captive Asian Elephants (Elephas maximus).

Salivary immunoglobulin A (sIgA) has been proposed as a potential indicator of welfare for various species, including Asian elephants, and may be related to adrenal cortisol responses. This study aimed to distinguish circadian rhythm effects on sIgA in male and female Asian elephants and compare patterns to those of salivary cortisol, information that could potentially have welfare implications. Subjects were captive elephants at an elephant camp in Chiang Mai province, Thailand (n = 5 males, 5 females). Salivette® kits were used to collect saliva from each elephant every 4 h from 06:00 to 22:00 h for 3 consecutive days (n = 15 samples/elephant). Enzyme immunoassays were used to quantify concentrations of IgA and cortisol in unextracted saliva. Circadian rhythm patterns were determined using a generalized least-squares method. Both sIgA and cortisol followed a circadian rhythm, although the patterns differed. sIgA displayed a daily quartic trend, whereas cortisol concentrations demonstrated a decreasing linear trend in concentrations throughout the day. There was no clear relationship between patterns of sIgA and salivary cortisol, implying that mechanisms of control and secretion differ. Results demonstrate for the first time that circadian rhythms affect sIgA, and concentrations follow a daily quartic pattern in Asian elephants, so standardizing time of collection is necessary.

Proinflammatory cytokines and lipopolysaccharides up regulate MMP-3 and MMP-13 production in Asian elephant (Elephas maximus) chondrocytes: attenuation by anti-arthritic agents.

BACKGROUND: Osteoarthritis (OA), the most common form of arthritic disease, results from destruction of joint cartilage and underlying bone. It affects animals, including Asian elephants (Elephas maximus) in captivity, leading to joint pain and lameness. However, publications regarding OA pathogenesis in this animal are still limited. Therefore, this study aimed to investigate the effect of proinflammatory cytokines, including interleukin-1 beta (IL-1ß), IL-17A, tumor necrosis factor-alpha (TNF-α), and oncostatin M (OSM), known mediators of OA pathogenesis, and lipopolysaccharides on the expression of cartilaginous degrading enzymes, matrix metalloproteinase (MMP)-3 and MMP-13, in elephant articular chondrocytes (ELACs) cultures. Anti-arthritic drugs and the active compounds of herbal plants were tested for their potential attenuation against overproduction of these enzymes. RESULTS: Among the used cytokines, OSM showed the highest activation of MMP3 and MMP13 expression, especially when combined with IL-1ß. The combination of IL-1ß and OSM was found to activate phosphorylation of the mitogen-activated protein kinase (MAPK) pathway in ELACs. Lipopolysaccharides or cytokine-induced expressions were suppressed by pharmacologic agents used to treat OA, including dexamethasone, indomethacin, etoricoxib, and diacerein, and by three natural compounds, sesamin, andrographolide, and vanillylacetone. CONCLUSIONS: Our results revealed the cellular mechanisms underlying OA in elephant chondrocytes, which is triggered by proinflammatory cytokines or lipopolysaccharides and suppressed by common pharmacological or natural medications used to treat human OA. These results provide a more basic understanding of the pathogenesis of elephant OA, which could be useful for adequate medical treatment of OA in this animal.

Management factors affecting adrenal glucocorticoid activity of tourist camp elephants in Thailand and implications for elephant welfare.

Elephant camps are among the most popular destinations in Thailand for tourists from many countries. A wide range of management strategies are used by these camps, which can have varied impacts on health and welfare of elephants. The objectives of this study were to examine relationships between FGM (fecal glucocorticoid metabolite) concentrations and camp management factors (work routine, walking, restraint, rest area, foraging), and to other welfare indicators (stereotypic behaviors, body condition, foot health, and skin wounds). Data were obtained on 84 elephants (18 males and 66 females) from 15 elephant camps over a 1-year period. Elephants were examined every 3 months and assigned a body condition score, foot score, and wound score. Fecal samples were collected twice monthly for FGM analysis. Contrary to some beliefs, elephants in the observation only program where mahouts did not carry an ankus for protection had higher FGM concentrations compared to those at camps that offered riding with a saddle and shows. Elephants that were tethered in the forest at night had lower FGM concentrations compared to elephants that were kept in open areas inside the camps. There was an inverse relationship between FGM concentrations and occurrence of stereotypy, which was not anticipated. Thus, assessing adrenal activity via monitoring of FGM concentrations can provide important information on factors affecting the well-being of elephants. Results suggest that more naturalistic housing conditions and providing opportunities to exercise may be good for elephants under human care in Thailand, and that a no riding, no hook policy does not necessarily guarantee good welfare.

Survival analysis of confirmed elephant endotheliotropic herpes virus cases in Thailand from 2006 - 2018.

The elephant endotheliotropic herpesvirus (EEHV) has been a known cause of death of young elephants in Thailand for over a decade. In this study, we report on the demography, disease characteristics and mortality of 58 elephants with confirmed EEHV hemorrhagic disease between January 2006 and August 2018 using retrospective data subjected to survival analysis. Median age of EEHV presentation was 29 months, and the mortality rate was 68.97% with a median survival time of 36 h. Most EEHV cases occurred in the north of Thailand, the region where most of the country's captive elephants reside. The hazard ratio analysis identified application of medical procedures and antiviral medications as being significant factors correlated to the risk of death. Our results indicate a need to focus EEHV monitoring efforts on young elephants and to follow current protocols that advise starting treatments before clinical signs appear.

Effect of time and temperature on stability of progestagens, testosterone and cortisol in Asian elephant blood stored with and without anticoagulant.

The value of biological samples collected in the field is compromised if storage conditions result in analyte degradation, especially in warmer climates like Thailand. We evaluated the effects of time and temperature on immunoactive steroid hormone stability in Asian elephant (Elephas maximus) blood stored with and without an anti-coagulant before centrifugation. For each elephant (5 male, 5 female), whole blood was aliquoted (n = 2 ml each) into 13 red top (without anticoagulant) or purple top (with anticoagulant) tubes. One tube from each treatment was centrifuged immediately and the serum or plasma frozen at -20°C (Time 0, T0). The remaining 12 aliquots were divided into stored temperature groups: 4°C, room temperature (RT, ~22°C), and 37°C, and centrifuged after 6, 24, 48 and 62 h of storage. Serum and plasma concentrations of progestagens in females, testosterone in males and cortisol in both sexes were quantified by validated enzyme immunoassays. Steroid concentration differences from T0 were determined by a randomized complete block ANOVA and Dunnett's tests. The only evidence of hormone degradation was cortisol and testosterone concentrations in serum stored at 37°C. Testosterone concentrations declined by 34% at 48 h and 52% at 62 h, cortisol was decreased by 19% after 48 h and 27% after 62 h at 37°C, respectively. None of the other aliquots displayed significant changes over time at any temperature. In conclusion, steroids appear to be stable in blood for nearly 3 days at room or refrigeration temperatures before centrifugation; steroids in samples with ethylenediaminetetraacetic acid were particularly stable. However, warmer temperatures may negatively affect steroids stored without anti-coagulant, perhaps due to red blood cell metabolism. Thus, under field conditions with no access to cold or freezer temperatures, collection of plasma is a better choice for elephants up to at least 62 h before centrifugation.

Associations among tourist camp management, high and low tourist seasons, and welfare factors in female Asian elephants in Thailand.

This study investigated how camp management and tourist activities affect body condition, adrenocortical function, lipid profiles and metabolic status in female tourist elephants. We compared twice monthly serum insulin, glucose, fructosamine, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), and fecal glucocorticoid metabolite (FGM) concentrations to body condition scores (BCS) at five camps with different management styles (e.g., tourist activities, work type, diet) between the High (November-February) and Low (March-October) tourist seasons. There were significant camp effects on health parameters, with BCS, TC, HDL, insulin and glucose being among the highest, and G:I being the lowest (less heathy) in elephants at an observation camp compared to those at camps where elephants received exercise by providing rides to tourists. Differences between High and Low tourist season months also were found for all measures, except TG and FGM concentrations. Both work time and walking distance were negatively correlated to glucose, fructosamine and insulin, while walking distance was negatively related to FGM concentrations. By contrast, positive associations were found between tourist number and BCS, TG, and insulin, perhaps related to tourists feeding elephants. Quantity of supplementary diet items (e.g., bananas, sugar cane, pumpkin) were positively correlated with FGM concentrations, glucose, fructosamine, and insulin. This study provides evidence that body condition, adrenal activity, metabolic markers, and lipid profiles in captive elephants may be affected by visitor numbers, work activities, and the amount of supplementary foods offered by tourists. Some activities appear to have negative (e.g., feeding), while others (e.g., exercise) may have positive effects on health and welfare. We conclude that camps adopting a more hands-off approach to tourism need to ensure elephants remain healthy by providing environments that encourage activity and rely on more natural diets or foraging.

Influence of season, tourist activities and camp management on body condition, testicular and adrenal steroids, lipid profiles, and metabolic status in captive Asian elephant bulls in Thailand.

We previously found relationships between body condition and physiological function affecting health and welfare of female tourist camp elephants in Thailand, and used that approach to conduct a similar study of bull elephants in the same camps (n = 13). A body condition score (BCS) was done every other month, and fecal glucocorticoid metabolite (FGM) concentrations were measured twice monthly for 1 year. Effects of season, camp management and tourist activity on lipid profiles [total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG)] and metabolic factors [insulin, glucose, fructosamine, glucose to insulin ratio (G:I)] were determined and correlated to measures of body condition, testosterone and FGM. Positive correlations were found between BCS and TG, between FGM and TG, HDL and glucose, and between testosterone and HDL, whereas BCS and testosterone were negatively associated with the G:I. There was a significant positive relationship between FGM and testosterone. Elevated FGM concentrations were associated with altered lipid and metabolic profiles and were higher in winter compared to summer and rainy seasons. Insulin and glucose levels were higher, while the G:I was lowest in the winter season. Strong positive associations were found between TC and HDL, LDL and HDL and glucose, and glucose and insulin. By contrast, negative relationships were found between the G:I and HDL and glucose, and between insulin and G:I. Differences also were found between High and Low tourist season months for FGM, insulin, and G:I. Last, there was notable variation among the camps in measured parameters, which together with tourist season effects suggests camp management may affect physiological function and welfare; some negatively like feeding high calorie treats, others positively, like exercise. Last, compared to females, bull elephants appear to be in better physical health based on normal BCSs, lower insulin levels and higher G:I ratios.

Changing trends in elephant camp management in northern Thailand and implications for welfare.

BACKGROUND: Elephant camps are among the most attractive destinations in Thailand for tourists from many countries. A wide range of management strategies are used by these camps, which can have varied impacts on health and welfare of elephants. METHODS: This study surveyed 33 camps with 627 elephants in northern Thailand to quantify the types of management practices and work activities experienced by captive elephants. The survey consisted of an interview with camp owners, and direct observations of camp operations. RESULTS: Data revealed considerable variation in elephant demographics, work activities, elephant care (i.e., housing, restraint, nutrition, health care, and breeding), and mahout management among the camps. In general, older camps (those in existence for >16 years) were involved in more intensive activities, like riding with saddles and shows. By contrast, newer camps provided more one-on-one activities for tourists and elephants, and emphasized more intimate, relaxing experiences (e.g., feeding, bathing, walking) than entertainment. A demographic shift also was observed, with elephants 20 years of age and younger having a sex ratio closer to 1:1 compared to elephants in older age categories (1:4.1-1:9.8). DISCUSSION: Shifts in elephant management to less intensive activities were observed, which could have positive implications for elephant welfare. The shifting sex ratio suggests successful captive breeding is resulting in the birth of more males, which could present new welfare challenges in the future, because bulls can be more difficult to manage and socialize, and are more likely to be kept isolated during musth. Ultimately, the goal is to understand how camp activities affect welfare, and to develop science-based guidelines and standards to aid in the management of both male and female elephants used in tourism.

Effect of Artificial Piglet Suckling Sounds on Behavior and Performance of Piglets and Adrenal Responses of Sows.

Operation of the farrowing house is essential to the productivity of a swine farm, requiring not only good management but also knowledge of the behavior of sows and piglets. Stress can negatively affect production in farm animals and could be a factor in production indexes. The objective of this study was to investigate the effect of artificial sucking sounds on the behavior of piglets and fecal glucocorticoid (FGM) concentrations of sows. A total of 30 sows were divided into two groups: a treatment group (15 sows) was exposed to artificial sucking sounds and a control group (15 sows) was not. Both groups received the same management; the two open-house system locations were separated by a distance of about 270 meters. The study had three key objectives: to compare farrowing indexes and to observe the sucking behavior of piglets using CCTV cameras. Fecal samples were collected daily for 21 days from the period after parturition to weaning to assess adrenal activity. The treatment group had a significantly higher average number of times piglets came to a sow's udder, and sows had a shorter onset time for the first piglet to come to the sow's udder than the control group (both P<0.05). The patterns and levels of FGM between the two groups were not different (both P<0.05), but the treatment group had better farrowing indexes than the control group (P>0.05), particularly for litter weight gain and percent preweaning mortality. In addition, the weaning to first service interval of the treatment group was shorter than the control group (P<0.05). This indicates that the artificial suckling sound probably has no adverse effect on adrenal responses of pig; however, it improves production indexes of postparturition sows.

Body condition and adrenal glucocorticoid activity affects metabolic marker and lipid profiles in captive female elephants in Thailand.

Studies in western zoo elephants have found relationships between body condition and physiological function, and identified mitigating management strategies to optimize health and welfare. A similar methodological approach was used in this study, which evaluated a body condition score (BCS; 1 = thinnest, 5 = fattest) every other month and fecal glucocorticoid metabolite (FGM) concentrations twice monthly in 33 tourist camp elephants in Thailand for a 1-year period to assess seasonal variations, and determine how lipid profiles [total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG)] and metabolic parameters [insulin, glucose, fructosamine, glucose to insulin ratio (G:I)] related to measures of body condition and adrenal function. The most prevalent BCS was 3-3.5 (60.6%), with 27.3% at BCS = 4 (overweight) and 12.1% at BCS = 4.5-5 (very overweight); no elephants had a BCS <2. BCSs were higher in rainy and winter seasons compared to summer, with FGM, TG, HDL, LDL, and insulin also higher in the rainy and/or winter seasons (p<0.05). By contrast, TC and glucose were lowest in the rainy season. FGM measures were negatively associated with two environmental factors: temperature and rainfall, but not humidity. Positive correlations were found between BCS and TC, LDL, and HDL, and between FGM and TC, HDL, glucose, and insulin (p<0.05), whereas BCS and FGM were both negatively associated with the G:I (p<0.05). However, there was no relationship between BCS and FGM among the camp elephants. Using BCS and FGM measures as outcome variables in separate regression models, this study found high BCS and elevated FGM concentrations were associated with altered lipid profiles and metabolic status in elephants. Furthermore, more work hours/day was associated with better body condition and health measures. Thus, being overweight and exposed to factors that increase adrenal activity could adversely affect health status, requiring alterations in management for some individuals, whereas exercise appears to have a protective effect.

ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS ASSOCIATED WITH CLOSTRIDIUM PERFRINGENS INFECTION IN TWO ASIAN ELEPHANT ( ELEPHAS MAXIMUS) CALVES.

Elephant endotheliotropic herpesvirus (EEHV) is an infection associated with fatal hemorrhagic disease in young Asian elephants ( Elephas maximus). This brief communication describes the postmortem evaluation of two Asian elephant calves diagnosed with EEHV4 and EEHV1A in conjunction with Clostridium perfringens infection. Case 1 was a 7-mo-old, male captive-born Asian elephant that developed diarrhea and died 2 days after clinical presentation. Examination of the heart, lungs, liver, and spleen revealed predominantly basophilic intranuclear inclusion bodies in the endothelial cells of the blood vessels. Case 2 was a 3-mo-old, female wild-born Asian elephant that showed signs of lethargy, anorexia, and convulsions and died 6 hr after clinical presentation. No intranuclear inclusion bodies were observed. The heart, lung, liver, and spleen of both calves tested positive for EEHV by polymerase chain reaction. Phylogenetic analysis identified EEHV4 and EEHV1A in Case 1 and 2, respectively. Additionally, liver, spleen, and hemorrhagic intestinal tissue samples tested positive for C. perfringens α, ß, and ε toxins. This is the first reported case to describe coinfection of EEHV and C. perfringens in Asian elephant calves.

Assessment of faecal glucocorticoid metabolite excretion in captive female fishing cats (Prionailurus viverinus) in Thailand.

There is little information on the endocrinology of fishing cats (Prionailurus viverinus), an endangered species in Southeast Asia, especially that pertaining to adrenal function. This study characterized faecal glucocorticoid metabolites in female fishing cats housed at Chiang Mai Night Safari to investigate seasonal and age relationships in hormone patterns. Faecal samples were collected 3 days/week for 1 year from seven females ranging in age from 4.5 to 9.6 years. A corticosterone enzyme immunoassay was validated for fishing cats by showing increases (∼60%) in faecal glucocorticoid immunoactivity above pre-treatment baseline levels within 1-2 days after an adrenocorticotrophic hormone injection. Faecal glucocorticoid metabolite concentrations were not related to age (P > 0.05), but there was a seasonal effect, with concentrations being higher (P < 0.05) during the winter (1.54 ±â€…0.04 µg/g) and rainy season (1.43 ±â€…0.04 µg/g) compared with the summer (1.22 ±â€…0.05 µg/g). Significant relationships were found between faecal glucocorticoids and rainfall (positive) and day length (negative), but not a temperature-humidity index. This is the first study to assess adrenal steroidogenic activity in female fishing cats, and we found that glucocorticoid metabolite production was influenced by seasonal factors, but not by age. We conclude that weather patterns should be taken into consideration in future studies of glucocorticoid activity in this endangered species, especially those studies aimed at improving captive management to create self-sustaining and healthy populations.

Use of handheld X-ray fluorescence as a non-invasive method to distinguish between Asian and African elephant tusks.

We describe the use of handheld X-ray fluorescence, for elephant tusk species identification. Asian (n = 72) and African (n = 85) elephant tusks were scanned and we utilized the species differences in elemental composition to develop a functional model differentiating between species with high precision. Spatially, the majority of measured elements (n = 26) exhibited a homogeneous distribution in cross-section, but a more heterologous pattern in the longitudinal direction. Twenty-one of twenty four elements differed between Asian and African samples. Data were subjected to hierarchical cluster analysis followed by a stepwise discriminant analysis, which identified elements for the functional equation. The best equation consisted of ratios of Si, S, Cl, Ti, Mn, Ag, Sb and W, with Zr as the denominator. Next, Bayesian binary regression model analysis was conducted to predict the probability that a tusk would be of African origin. A cut-off value was established to improve discrimination. This Bayesian hybrid classification model was then validated by scanning an additional 30 Asian and 41 African tusks, which showed high accuracy (94%) and precision (95%) rates. We conclude that handheld XRF is an accurate, non-invasive method to discriminate origin of elephant tusks provides rapid results applicable to use in the field.

Differential testosterone response to GnRH-induced LH release before and after musth in adult Asian elephant (Elephas maximus) bulls.

Bull elephants exhibit marked increases in testosterone secretion during musth, and studies have shown a heightened sensitivity of the testis to GnRH-stimulated testosterone production in musth compared to nonmusth males. However, activity of the hypothalamo-pituitary-gonadal axis before or soon after musth has not been studied in detail. The aim of this study was to evaluate LH and testosterone responses to GnRH challenge in nine adult Asian elephant (Elephas maximus) bulls during three periods relative to musth: premusth, postmusth, and nonmusth. Bulls were administered 80 µg of a GnRH agonist, and blood was collected before and after injection to monitor serum hormone concentrations. The same bulls were injected with saline 2 weeks before each GnRH challenge and monitored using the same blood collection protocol. All bulls responded to GnRH, but not saline, with an increase in LH and testosterone during all three periods. The mean peak LH (1.76 ± 0.19 ng/mL; P < 0.001) and testosterone (6.71 ± 1.62 ng/mL; P = 0.019) concentrations after GnRH were higher than the respective baselines (0.57 ± 0.07 ng/mL, 3.05 ± 0.60 ng/mL). Although basal- and GnRH-induced LH secretion were similar across the stages, evaluation of the area under the curve in GnRH-treated bulls indicated that the testosterone response was greatest during premusth (2.84 ± 0.76 area units; P = 0.019) compared to postmusth (2.02 ± 0.63 area units), and nonmusth (2.01 ± 0.46 area units). This confirms earlier reports that GnRH stimulates LH release and subsequent testosterone production in bull elephants. Furthermore, although the hypothalamo-pituitary-gonadal axis is active throughout the year, the testis appears to be more responsive to LH in terms of testosterone production in the period leading up to musth, compared to the nonmusth and postmusth periods. This heightened sensitivity, perhaps as a result of LH receptor up-regulation, may prime the testis for maximal testosterone production, leading to the physiological and behavioral changes associated with musth.

Elemental Analysis of Asian Elephant (Elephas maximus) Teeth Using X-ray Fluorescence and a Comparison to Other Species.

Elemental composition in bone of the different species has variation depending on genetic and environmental factors especially their food habitat. The aims of this study were to conduct an elemental analysis of Asian elephant teeth, both deciduous (first molar, second molar, and tusk) and permanent (molar and tusk), and compare the elemental composition of permanent teeth among 15 species, mostly mammalian. These teeth were analyzed using X-ray fluorescence at two voltages: 15 and 50 kV. In Asian elephants, deciduous tusk showed a lower Ca/Zn ratio compared to permanent tusk, because of the lack of Zn in permanent molars. Ca/Fe ratio was higher in deciduous than permanent molars. For permanent teeth, elephant molars presented a high Ca/Pb ratio but no Ca/Zn, Ca/Sr, and Zn/Fe ratios because of the lack of Zn and Sr in the samples tested. The key elemental ratios for differentiating elephant deciduous and permanent tusk were Ca/P and Ca/Zn. The considerable variation in elemental ratio data across 15 species was observed. All tooth samples contained Ca and P, which was not surprising; however, Pb also was present in all samples and Cd in a large majority, suggesting exposure to environmental contaminants. From discriminant analysis, the combination of Ca/P+Ca/Zn+Ca/Pb+Ca/Fe+Ca/Sr+Zn/Fe can generate two equations that successfully classified six (dog, pig, goat, tapir, monkey, and elephant) out of 15 species at 100 % specificity. In conclusion, determining the elemental profile of teeth may serve as a tool to identify the tooth "type" of elephants and to potentially classify other species.