Androgen Metabolite Concentrations Research Articles

The dissection of a despotic society: exploration, dominance and hormonal traits.

Naked mole-rats (Heterocephalus glaber) live in large colonies with one breeding female (queen), one to three breeding males (BMs) and the remainder are non-reproductive subordinates. The animals have a linear dominance rank with the breeders at the top of the hierarchy. We investigated how dominance rank in naked mole-rats differs with exploration (the propensity to explore a novel environment) and related endocrine markers. Exploration behaviour, faecal progestagen metabolite (fPM), faecal glucocorticoid metabolite (fGCM), faecal androgen metabolite (fAM) and plasma prolactin concentrations were quantified in breeding, high-, middle- and low-ranked females and males from five naked mole-rat colonies. There were no significant differences between the dominance rank and exploration behaviour. Interestingly, the queens and high-ranking females had higher fGCM and fAM concentrations compared with middle- and low-ranked females. The queens had significantly higher fPM concentrations than all other ranked females, since they are responsible for procreation. In the males, the BMs had higher fGCM concentrations compared with high- and low-ranked males. In addition, BMs and middle-ranking males had overall higher prolactin levels than all other ranked males, which could be linked to cooperative care. Overall, the results suggest that physiological reproductive suppression is linked to high dominance rank.

FRI382 High Androgen And Progesterone Metabolite Concentrations In Naturally Matured Follicles Are Concurrent With High Serum Anti-Müllerian Hormone Concentrations

Abstract Disclosure: T. Du Toit: None. M. Von Wolff: None. A.C. Swart: None. M. Groessl: None. C.E. Flueck: None. Infertility in women is routinely evaluated with the assessment of circulating steroid levels. Profiling steroid levels in follicular fluid (FF) would provide a focused evaluation of the ovarian endocrine milieu and is therefore better suited to identify key hormones potentially involved in dysfunctional follicular growth. The anti-Müllerian hormone (AMH) level in serum is a well-established clinical marker to estimate the ovarian reserve in assisted reproductive technology, while the AMH level in FF serves as a new prognostic marker for oocyte developmental potential. However, AMH-linked changes in the follicular steroid milieu have not been fully explored. We conducted an AMH-dependent analysis of the FF endocrine milieu. Hormones were quantified using LC-MS (n=42) and ELISA (n=4) in FF of 83 women undergoing natural cycle in vitro fertilisation. Patients were age-matched and classified into 4 statistically different (p<0.0001) groups according to serum AMH levels: group A (<1.1 pmol/L, n=20); group B (1 - 7.14 pmol/L, n=21); group C (7.14 - 35.5 pmol/L, n=21); and group D (>35.5 pmol/L, n=21). Group D was designated as our polycystic ovary syndrome (PCOS)-like group, as high serum AMH is a marker for PCOS. We characterised androgen and progesterone metabolic pathways in FF. Androstenedione (A4) and testosterone (T) levels differed significantly between the groups (p<0.01) and were highest in group D at 60 nmol/L and 4.7 nmol/L, respectively. The levels of 11-ketoandrostenedione (11KA4) and 11β-hydroxyandrosterone (adrenal-origin) also differed between the groups (p<0.05), with higher 11KA4 levels (±2.7-fold) quantified in group D. In parallel with serum AMH, FF AMH differed between the groups (p<0.0001), together with luteinizing hormone/FSH, A4/dehydroepiandrosterone, estradiol(E2)/T and E2/A4 ratios. In comparison to groups A, B and C combined, the PCOS-like group D showed significantly increased downstream progesterone metabolite levels –11α-hydroxyprogesterone [OHP4], 17α,20α-diOHP4, 16α-OHP4 and 5α-pregnanolone, with lower FSH levels. Our results show that the follicular milieu is significantly different when high AMH levels are measured compared to when AMH levels are low, marked by: (i) higher progesterone metabolite levels in follicles and (ii) higher A4 and T levels, concomitant with unchanged E2 levels suggesting that androgens were not fully converted to estrogens in these follicles. Our study furthermore corroborates other studies reporting an intrafollicular hyperandrogenic state in women with increased circulatory AMH levels. In summary, our study not only provides data based on a broad steroid profile that the follicular androgen endocrine milieu is affected in women with high serum AMH levels, but it also provides a model for the premature progesterone increase in PCOS women which leads to a reduction in the embryo implantation rate. Presentation: Friday, June 16, 2023

Non-Invasive Quantification of Faecal and Urine Reproductive Hormone Metabolites in the Naked Mole-Rat (Heterocephalus glaber).

The naked mole-rat (Heterocephalus glaber) occurs in colonies with a distinct dominance hierarchy, including one dominant, breeding female (the queen), 1-3 breeding males, and non-reproductive subordinates of both sexes that are reproductively suppressed while in the colony. To non-invasively evaluate reproductive capacity in the species, we first had to examine the suitability of enzyme immunoassays (EIAs) for determining progestogen and androgen metabolite concentrations in the naked mole-rat, using urine and faeces. A saline control and gonadotrophin-releasing hormone (GnRH) were administered to twelve (six males and six females) naked mole-rats which were previously identified as dispersers and housed singly. The results revealed that urine is possibly not an ideal matrix for progestogen and androgen metabolite quantification in naked mole-rats as no signal was detected in the matrix post GnRH administration. A 5α-Progesterone EIA and an Epiandrosterone EIA were identified as suitable for quantifying faecal progesterone metabolites (fPMs) and faecal androgen metabolites (fAMs) in males and females, respectively. The results suggest that there are individual variations in baseline fPM and fAM concentrations, and only two out of six females and no males exhibited an increase in fPM concentrations greater than 100% (-20% SD) post GnRH administration. Conversely, only four out of six females and three out of six males had an increase in fAM concentrations greater than 100% (-20% SD) following GnRH administration. These results imply that some naked mole-rat individuals have a reduced reproductive capacity even when they are separated from the queen.

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species’ reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

Measures of Adrenal and Gonadal Hormones in Relation to Biological and Management Factors among Captive Red Pandas in Indian Zoos.

Animals in human care are affected by stressors that can ultimately reduce fitness. When reproduction is affected, endangered species conservation programs can be severely compromised. Thus, understanding factors related to stress and reproduction, and related hormones, is important to ensure captive breeding success. Red pandas (Ailurus fulgens) are endangered, and populations in the wild are threatened with extinction. A global captive breeding program has been launched to conserve the species with the goal of reintroduction. However, there is little information on how stressors impact physiological aspects of the species. This study measured fecal glucocorticoid (fGCM), progestagen (fPM), and androgen (fAM) metabolite concentrations in 12 female and 8 male red pandas at 3 zoos in India to determine predictors of adrenal and gonadal steroid activity, and the influence of fGCM on reproduction. Based on the generalized linear mixed model (GLMM), fGCM concentrations were positively correlated with the number of visitors, number of nests and enclosure areas, and negatively related to frequency of feedings, log density, and social time, while fPM concentrations were negatively associated with enclosure areas. A confounder for enclosure areas and number of nests was the fact that these spaces were relatively barren, with limited hiding spaces, compared to the smaller enclosures. By contrast, no significant relationships were found for fAM, perhaps due to the smaller sample size. A negative relationship between fGCM and fPM was observed, indicating increasing adrenal hormones may decrease reproductive function among female red pandas. Results suggest that zoo management should consider increasing feeding frequency, providing larger enclosures with more enrichment and more nests in larger spaces, and regulating visitor numbers to support good welfare and potentially improve reproductive fitness of red pandas in captivity.

Egg production and endocrine profiles of female whooping cranes (Grus americana) maintained ex situ are improved in naturalized enclosures

Whooping cranes (Grus americana) are naturally seasonal breeders and rely on wetland habitats throughout the annual cycle. However, captive cranes are commonly housed in dry outdoor pens, which may lack key environmental stimuli that in turn compromise reproduction. Our study sought to first, assess seasonal patterns of reproductive hormones in successful versus non-successful bird pairs and second, determine endocrine responses to alterations in enclosure environments. Fecal samples were collected from eight crane pairs year-round for 3 consecutive years, once a week during non-breeding season and three times a week during the breeding season. During Year 1, all pairs were housed in traditional dry pens, whereas during January of Year 2 all pairs were moved to either new dry pens (control, n = 4) or ponded pens (wetland, n = 4) and remained in the same pen until the end of the study. Fecal samples were assessed for gonadal (year-round) and adrenal (breeding season only) hormones. Estrogen and progestagen metabolite concentrations were low during non-breeding season in all females. However, as birds transition into a breeding season, gonadal hormone concentrations significantly increased (p < 0.001) in laying females but not in non-laying individuals. Androgen metabolite concentrations during breeding seasons were higher (p < 0.001) in males paired with non-laying females, with no variations observed during non-breeding months and within birds of the same reproductive output. We observed significant effect of enclosure environment on ovarian function of female whooping cranes. Specifically, mean estrogen metabolite concentrations increased after birds were moved from dry pen to wetland enclosures (Year 1: 349.1 ± 83.4 ng/g feces; Year 2: 382.7 ± 82.9 ng/g feces; Year 3: 556.5 ± 85.4 ng/g feces, p = 0.008 and 0.019 respectively,), whereas those of the control females remained constant. Further, estrogen concentration assessed during breeding season of Year 3 in females housed in wetland pens was higher than birds housed in a dry pen (556.5 ± 85.4 vs 311.7 ± 85.12 ng/g feces; p = 0.019). The number of eggs laid increased in three of the four pairs housed in the wetland pens, while there was no change in egg production in control birds (9 vs. 2 combined number of eggs produced by all pairs in each respective group in Year 3). Finally, moving birds to an enclosure that mimic natural environment did not impact androgen or glucocorticoid excretion. The findings demonstrate that differences in gonadal hormone production between laying and non-laying whooping crane females exist primarily during the breeding season, and that a more natural environment can have a positive influence on ovarian function in female whooping cranes.

11-Oxygenated Androgen Metabolite Concentrations Are Affected by Pubertal Progression and Obesity

Introduction: 11-oxygenated C19 steroids (11-oxyandrogens) have been shown to rise during adrenarche and remain higher throughout adulthood than in early childhood. The patterns of circulating 11-oxyandrogens throughout normal puberty have not yet been described. Methods: We conducted a secondary analysis of healthy youth participants, both males and females, enrolled in six prior endocrine studies (N = 249). Participants were classified according to Tanner stage and body mass index (BMI). Concentrations of three adrenal-specific 11-oxygenated androgens, 11β-hydroxyandrostenedione (11OHA4), 11β-hydroxytestosterone (11OHT), and 11-ketotestosterone (11KT), were measured in fasting serum samples. Results: 11OHA4 and 11OHT increased modestly between early and late puberty in youth with normal weight (p < 0.05), whereas increases in 11KT did not reach statistical significance (p < 0.06). 11KT levels differed between sexes throughout puberty (p < 0.01), and changes in 11-oxyandrogens were small compared to the marked increases for estradiol in girls or testosterone in boys. The trajectories of 11KT and 11OHA4 changes throughout puberty differed by BMI category (p < 0.05). Conclusion: Beyond adrenarche, 11-oxyandrogens continue to rise during pubertal development. The differences in 11KT trajectories in males and females are small compared to changes in testosterone for males and estradiol for females during puberty. Obesity appears to influence the trajectories of 11-oxyandrogens during puberty.

SENSITIVITY, SPECIFICITY, AND PREDICTIVE VALUE OF URINARY ANDROGEN METABOLITES FOR THE DIAGNOSIS OF POLYCYSTIC OVARY SYNDROME

The objective of this study was to determine if urinary androgen metabolite concentrations measured using an at-home dried urine sampling method and an accompanying gas chromatography-tandem mass spectrometry (GC-MS/MS) assay could be used to confirm or rule out polycystic ovary syndrome (PCOS).

Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal – CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

Characterizing zoo-housed Bactrian camel (Camelus bactrianus) reproduction using gonadal steroid metabolite analysis in feces

Millions of people globally depend on camelids, which demands an increased knowledge of their reproduction. We used zoo-housed Bactrian camels (Camelus bactrianus) to better understand camelid reproductive physiology. Our specific objectives were to: 1) validate the use of fecal hormone metabolite analysis to characterize camel reproductive physiology during sexual maturity and pregnancy; and 2) determine the influence of season on male and female reproduction. We collected fecal samples from 1 male and 3 females housed at Lincoln Park Zoo (Chicago, IL, USA) 1 to 2 times per week for 3.5 years. Extracted hormones were analyzed using enzyme immunoassays for progestogen (FPM), estrogen (FEM), and androgen (FAM) metabolite concentrations. One female sexually matured during our study as evidenced by increased FEM baseline. Results demonstrated seasonal effects on male androgen production with FAMs higher (P < 0.05) January to June (mean ± SEM: 664.6 ± 22.6 ng/g wet feces), compared to July to December (401.6 ± 17.5 ng/g wet feces). One female experienced a persistent corpus luteum, a reproductive abnormality, which was identified by prolonged elevated FPM. FPMs increased during pregnancy for two females (452.9 ± 24.9 and 294.4 ± 19.8 ng/g wet feces) with a gestation of 404 d and 442 d, respectively. The third female never conceived. The FEMs varied (P < 0.05) during the year with no clear seasonal patterns (monthly mean range: 213.1–371.0 ng/g wet feces). Fecal hormone metabolite analysis is a validated method for assessing male seasonality and female pregnancy in the Bactrian camel and can for their management and conservation in zoos and the wild.

Group structure, but not dominance rank, predicts fecal androgen metabolite concentrations of wild male mountain gorillas (Gorilla beringei beringei).

Androgens are important mediators of male-male competition in many primate species. Male gorillas' morphology is consistent with a reproductive strategy that relies heavily on androgen-dependent traits (e.g., extreme size and muscle mass). Despite possessing characteristics typical of species with an exclusively single-male group structure, multimale groups with strong dominance hierarchies are common in mountain gorillas. Theory predicts that androgens should mediate their dominance hierarchies, and potentially vary with the type of group males live in. We validated the use of a testosterone enzyme immunoassay (T-EIA R156/7, CJ Munro, UC-Davis) for use with mountain gorilla fecal material by (1) examining individual-level androgen responses to competitive events, and (2) isolating assay-specific hormone metabolites via high-performance liquid chromatography. Males had large (2.6- and 6.5-fold), temporary increases in fecal androgen metabolite (FAM) after competitive events, and most captured metabolites were testosterone or 5α-dihydrotestosterone-like androgens. We then examined the relationship between males' dominance ranks, group type, and FAM concentrations. Males in single-male groups had higher FAM concentrations than males in multimale groups, and a small pool of samples from solitary males suggested they may have lower FAM than group-living peers. However, data from two different time periods (n = 1610 samples) indicated there was no clear relationship between rank and FAM concentrations, confirming results from the larger of two prior studies that measured urinary androgens. These findings highlight the need for additional research to clarify the surprising lack of a dominance hierarchy/androgen relationship in mountain gorillas.

Scales of our lives: Sex identification of Temminck’s pangolin (Smutsia temminckii) using scales retrieved out of the illegal wildlife trade

Pangolins are the most trafficked wild mammals, with their scales in high demand. Scales are often the only part of the animal confiscated from the trade, but they represent accessible material for forensic investigations, including for sexing. This study aimed to develop a sexing tool for Temminck’s pangolin, using scales for hormone quantification. Scales from males and females were liquidised using keratinase and the resulting suspension analysed for progestagen and androgen metabolite (scPM and scAM) concentrations. Scale PM and scAM concentrations were compared between sexes, while overall median values for scPM and scAM, as well as a ratio of scPM to scAM (P/A) were used as boundary values for sex identification. Neither scPM nor scAM concentrations were significantly different between the sexes and concentrations of a juvenile and sub-adult male overlapped with females, possibly indicating later sexual maturity in males. Boundary values for scAM concentrations and the P/A ratio predicted sex with 100% accuracy for females and 78% for males, while the accuracies for the scPM boundary value were lower. When only adult individuals are considered, scAM and P/A ratio boundaries are 100% accurate for both sexes. Therefore, scale hormone ratios show promise as a sex identification tool for Temminck’s pangolin, particularly applicable in forensic investigations on the pangolin trade.

Habituation of common eland (Taurotragus oryx) to intensive routine handling, and the effect of immunocastration thereon

The temperament of captive common eland (Taurotragus oryx) during handling is crucial for their management, as they have been identified as an ideal antelope for game meat farming, and are well-represented in captive antelope populations world-wide. Generally speaking, common eland are considered undomesticated, and thus maintain their natural prey instincts, making it necessary to habituate them to routine handling. Immunocastration can be implemented as part of the herd management strategy in mixed or single-sex eland groups, and might reduce aggressive behaviour of males by blocking the production of testosterone, thereby improving docility for ease of handling. Within this study, data was collected to determine the influence of routine handling and immunocastration on the temperament of common eland during handling. Twenty-nine common eland were divided into two groups: sub-adults (n = 15; ca. 2 years old; 182.9 ± 59.37 kg) and juveniles (n = 14; ca. 6 months old; 94.18 ± 24.76 kg), and they were routinely handled every two weeks over a four-month period (10 handling events). Within each age group, females, males, and immunocastrated males were present. Immunocastration treatment was administered during the second and fourth handling events (2 mL Improvac®/ animal). During handling, animals were individually driven through the raceway and finally restrained in a squeeze chute, where routine biological samples and measurements were collected. Faecal androgen metabolite (fAM) concentrations were measured for each event, for males only. Each animal was focally sampled for specific behaviours during the handling. A temperament score was obtained by observing the general state of the animal from the point of entering the raceway, until its exit from the squeeze chute. Generalized linear mixed models tested the influence of immunocastration, week, group, fAM concentration, and body weight on the temperament score. Immunocastration did not affect the temperament score, the general state in the squeeze chute, the order of entering the handling system, and the chute exit. However, the temperament score and the state in the squeeze chute improved overtime (with animals being less nervous). Moreover, animals with low temperament scores (calmer) entered the system earlier, and exited the system calmly over the repeated handling period. The results show that routine handling through a raceway and squeeze chute system leads to habituation, and improves the ease of handling in common eland. However, immunocastration did not affect the temperament of the male common eland during handling.

Effects of Lupron and surgical castration on fecal androgen metabolite concentrations and intermale aggression in capybaras (Hydrochoerus hydrochaeris).

To curb agonistic interactions in a bachelor group of three male capybaras (Hydrochoerus hydrochaeris), a single dose of leuprolide acetate (Lupron®) was used in an attempt to chemically sterilize the males. Concurrently, fecal androgen metabolite (FAM) concentrations were quantified via enzyme immunoassay to monitor changes in testosterone production after injection of the gonadotropin-releasing hormone agonist. When Lupron proved ineffective in suppressing intraspecific aggression, surgical castration was performed on two males, with continued noninvasive endocrine monitoring. In all three capybaras, FAM concentrations increased initially as a result of the luteinizing hormone surge, but then decreased significantly following chemical sterilization. Surgical castration resulted in further, persistent declines in FAM concentrations in two males, while the third, intact male demonstrated a rise in FAM to pre-Lupron concentrations at 8.5 and 9.5-month postadministration. Despite successful suppression of sperm and testosterone production, intermale aggression continued, ultimately necessitating separation of the animals and transfer to other holding institutions. Under this set of conditions, a single Lupron dose was inadequate for suppressing intraspecific aggression in a group of three males with a pre-established history of aggression.

Reproductive events and respective faecal androgen metabolite concentrations in captive male roan antelope (Hippotragus equinus).

Understanding the reproductive biology of the roan antelope (Hippotragus equinus) (É. Geoffroy Saint-Hilaire, 1803) is crucial to optimise breeding success in captive breeding programmes of this threatened species. In this study, the pattern of faecal androgen metabolite (fAM) production related to reproductive events (calving or birthing, mating, gestation, and lactation), sexual behaviours as well as environmental cues were studied in captive adult male roan antelope. Faecal sample collection and behavioural observations were carried out from August 2017 to July 2018 for three reproductive males participating in a conservation breeding programme at the Lapalala Wilderness Nature Reserve in South Africa. As a prerequisite, the enzyme immunoassay used in this study was biologically validated for the species by demonstrating a significant difference between fAM concentrations in non-breeding adults, breeding adults and juvenile males. Results revealed that in adults males, the overall mean fAM levels were 73% higher during the breeding period compared to the non-breeding periods, and 85% higher when exclusively compared to the lactation/gestation periods, but only 5.3% higher when compared to the birthing period. Simultaneously, fAM concentrations were lower during the wet season compared to the dry season, increasing with a reduction in photoperiod. With the exception of courtship, frequencies of sexual behaviours monitored changed in accordance with individual mean fAM concentrations in male roan antelope, the findings suggest that androgen production varies with the occurrence of mating activity and may be influenced by photoperiod but not with rainfall.

Congenital Adrenal Hyperplasia in Children: A Pilot Study of Steroid Hormones Expressed as Sex- and Age-Related Standard Deviation Scores

Introduction: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease predominantly caused by 21-hydroxylase deficiency. Clinical management in children includes glucocorticoid and often mineralocorticoid treatment alongside monitoring outcomes such as an­thro­po­metry, pubertal status, blood pressure, and biochemistry. Objective: The objective of this pilot study was to present the use of 17-hydroxyprogesterone (17-OHP) and androgen metabolites expressed as standard deviation (SD) scores rather than actual concentrations as a tool in the management of children with CAH as well as in research settings. Methods: The study was a retrospective, longitudinal study that took place in a single, tertiary center and included 38 children and adolescents aged 3–18 years with CAH due to 21-hydroxylase deficiency. Biochemical measurements of 17-OHP, androstenedione, dehydroepiandrosterone-sulphate (DHEAS), and testosterone using liquid chromatography-tandem mass spectrometry were expressed as SD scores, and outcomes such as genotype, height, bone maturation, blood pressure, and treatment doses were extracted from patient files. Results: The majority (86%) of CAH patients had 17-OHP measurements above +2 SD during standard hydrocortisone therapy, receiving an average daily hydrocortisone dose of 12.6 mg/m². Androstenedione concentrations were mostly within ±2 SD, whereas DHEAS values were below –2 SD in 47% of patients. Conclusions: Applying sex- and age-related SD scores to 17-OHP and androgen metabolite concentrations allows for monitoring of hydrocortisone treatment independent of age, sex, assay, and center. We propose that 17-OHP and androgen metabolites expressed as SD scores be implemented as a unifying tool that simplifies research and, in the future, also optimal management of treatment.

Endocrine correlates of gender and throat coloration in the southern ground-hornbill (Bucorvus leadbeateri).

The southern ground-hornbill (SGH) is a cooperatively breeding bird endemic to eastern and southern Africa, but is endangered in its southern distributional range. The national conservation restoration program harvests redundant chicks for captive breeding and reintroduction; with sexing and social grouping of the species evaluated by throat-skin coloration, with adult males displaying a completely red color compared to dark blue within the red observed in adult females. However, recent findings indicate that dominant and subordinate adult males exhibit patches of blue throat-skin. To optimize SGH management practices, it is vital to determine the role of red and blue coloration, as well as the possible drivers thereof. As a prerequisite, an enzyme immunoassay for monitoring fecal androgen metabolite (fAM) concentrations in SGH was established. Following this, fresh fecal samples were collected from 78 SGH, of various demographics and origin, across 12 captive institutions, to determine whether fAM concentrations differ between blue (B), partially blue (sB), and fully red (R) throat-skin colored SGH. Furthermore, fAM concentrations were compared between males housed in different social groups of different age and sex classes. Individual median fAM concentrations of B, sB, and R adult males did not differ significantly but were considerably higher in B and sB males compared to R males. Social dynamics within captivity, for example, dominance, played no role as a driver of male gonadal activity or throat skin coloration. The results of the study indicate that androgens and apparent social dynamics are not primary determinants of throat coloration in male SGH.

Maternal phthalate exposure associated with decreased testosterone/LH ratio in male offspring during mini-puberty. Odense Child Cohort

BackgroundPhthalates are plastic softeners with anti-androgenic properties. Prenatal exposure has led to lower testosterone (T) levels and smaller testicles in adult rats. To our knowledge, no studies have examined associations between prenatal phthalate exposure and sex hormone concentrations in infants. ObjectiveTo study associations between phthalate exposure in Danish pregnant women and T, luteinizing hormone (LH), follicle stimulating hormone (FSH), Δ4-androstenedione (adion), 17α-hydroxyprogesterone (17-OHP) dehydroepiandrosterone sulfate (DHEAS) concentrations in their infants (N = 479) during mini-puberty. MethodsConcentrations of 12 phthalate metabolites from six phthalate diesters were measured in urine samples collected from 2010 to 2012 from 479 pregnant women participating in the Odense Child Cohort at gestational week 28 (range 20.4–30.4). Serum T, LH, FSH, adion, 17-OHP, DHEAS, weight and height were measured approximately three months after expected date of birth. Associations between prenatal phthalate exposure and gonadotropin and androgen metabolite concentrations were estimated in boys and girls separately in adjusted linear regression models. ResultsT concentration was lower in boys prenatally exposed to phthalates. Maternal urinary concentrations of summed mono-iso-butyl and mono-n-butyl phthalate (∑MBPi+n) and summed metabolites of di-iso-nonyl phthalate (∑DiNPm) were associated with lower T/LH ratio in male offspring and a dose-response association was found. FSH was 14% (95% CI: 1; 25) lower among male offspring from mothers exposed to ∑DiNPm in the highest compared to the lowest tertile. No association was found for girls. ConclusionEven in these low exposed children, we found a significant decrease in T/LH ratio during mini-puberty in boys prenatally exposed to phthalates, which may suggest impairment of Leydig cells. The children will be followed as they approach adrenarche and pubarche in order to assess if long-term adverse effects persist.

Anogenital distance as a measure of male competitive ability in Rwenzori Angolan colobus.

Anogenital distance (AGD) is positively correlated to fetal androgen exposure and developmental masculinization in mammals. Independent of overall body size, AGD shows a strong positive correlation with male fertility and in rodents, AGD is a good indicator of male competitive ability and is associated with female choice. We hypothesized that AGD will also predict male competitive ability in non-human primates. To test this, we measured AGD noninvasively with a parallel laser in a wild population of Angolan colobus monkeys (Colobus angolensis ruwenzorii) in Uganda and correlated to it to their social structure. C. angolensis ruwenzorii form a multilevel society with both one-male/multifemale units (OMUs) and multimale/multifemale units (MMUs). We compared AGD in males from five OMUs and six MMUs and related it to their fecal androgen metabolite concentrations, dominance rank and body size, and to the number of females in their unit. Males in OMUs had greater access to females, so were predicted to have longer AGDs, but this was not found. AGD also did not correlate overall with mean fecal androgen metabolites in MMUs. However, AGD was correlated with dominance rank in MMUs, demonstrating that higher-ranking males in these multimale units had longer AGDs. Body size did not show the same relationship with dominance rank, suggesting that male rank was not just a reflection of absolute male size. Our findings indicate that AGD predicts male competitive ability in this species and that it may be a useful correlate throughout the non-human primates. These results also support the idea that prenatal androgen exposure increases the likelihood of the expression of behaviors that maintain high dominance rank.

Linkage between fecal androgen and glucocorticoid metabolites, spermaturia, body weight and onset of puberty in male African lions (Panthera leo).

There is limited physiological information on onset of puberty in male lions. The aim of this study was to use longitudinal non-invasive monitoring to: 1) assess changes in steroid metabolite excretory patterns as a function of age and body weight; 2) determine correlations between fecal androgen (FAM) and glucocorticoid (FGM) metabolite concentrations; and 3) confirm spermiogenesis non-invasively through urinalysis. Specifically, FAM and FGM metabolites were analyzed in samples collected twice weekly from 21 male lions at 17 institutions (0.9–16 years of age) for 3.8 months– 2.5 years to assess longitudinal hormone patterns. In addition, body weights were obtained approximately monthly from 10 individuals at five zoos (0.0–3.0 years), and urine was collected from six males at two facilities (1.2–6.3 years) and evaluated for the presence of spermatozoa. An increase in overall mean FAM occurred at 2.0 years of age, at which point concentrations remained similar throughout adulthood. The onset of puberty occurred earlier in captive-born males (<1.2 years of age) compared to wild-born counterparts (<2.5 years of age). Additionally, males in captivity gained an average of 7.3 kg/month compared to 3.9 kg/month for wild males over the first 2–2.5 years of age. Sperm (spermaturia) was observed in males as young as 1.2 years in captivity compared to 2.5 years in the wild (ejaculates). There was no difference in FAM or FGM concentrations with regards to age or season. Overall, this study demonstrates that: 1) captive male lions attain puberty at an earlier age than wild counterparts; 2) onset of puberty is influenced by body weight (growth rate); and 3) spermiogenesis can be confirmed via urinalysis. Knowledge about the linkage between body weight and onset of puberty could facilitate improved reproductive management of ex situ populations via mitigating the risk of unintended breedings in young animals.