Skip to main content
NCBI home page
Conservation Physiology logoLink to Conservation Physiology
. 2024 Nov 14;12(1):coae079. doi: 10.1093/conphys/coae079

Validating enzyme immunoassays for non-invasive reproductive hormone monitoring in Temminck’s pangolin

Juan Scheun 1,2,✉, Andre Ganswindt 3, Raymond Jansen 4,5, Kim Labuschagne 6
Editor: Christine Madliger
PMCID: PMC11562634  PMID: 39544460

Abstract

Gonadal hormones play a central role in reproductive function and success. As such, quantifying reproductive hormones non-invasively in threatened, vulnerable and endangered wildlife species offers an ideal tool for assessing general and individual reproductive patterns in situ. Whilst the use of faeces as a hormone matrix is often preferred in these cases, the required enzyme immunoassays (EIAs) for measuring faecal androgen (fAM), oestrogen (fEM) and progestagen metabolite (fPM) concentrations must first be validated if a species gets investigated for the first time to ensure biologically relevant patterns can be observed. In this study we aimed to biologically validate the EIAs for monitoring fAM, fEM and fPM concentrations in Temminck’s pangolin, Smutsia temminckii. Hormone metabolite concentrations derived from each EIA tested were compared between different age and sex classes. An epiandrosterone EIA effectively measured androgen levels in males, distinguishing between adult and juvenile individuals, as well as both female age classes. Similarly, the tested oestrogen EIA successfully distinguished between adult and juvenile female fEM concentrations, and both tested progestagen EIAs demonstrated adequate differences between fPM concentrations of adult and juvenile females. The now-validated EIAs offer robust tools for a non-invasive monitoring of reproductive activity in Temminck’s pangolin. The development of such techniques will allow researchers to assess reproductive hormone patterns of the species in situ, whilst also paving the way for further studies in this field. Despite the small sample size due to the species’ conservation status, the study provides a foundation for future research using a robust, validated, non-invasive monitoring tool. The latter can now be implemented in long-term monitoring with larger sample sizes to yield more comprehensive data, aiding in the conservation of Temminck’s pangolin.

Keywords: Conservation, endocrine monitoring, non-invasive, pangolin, reproduction

Introduction

Hormone monitoring has emerged as an important technique in the field of wildlife conservation, offering invaluable insights into stress and reproductive physiology in wildlife species (Möstl and Palme, 2002; Hodges et al., 2010; Sheriff et al., 2011). This has enabled researchers to gain a better understanding of animal behaviour, reproductive status, population dynamics, survival and health (Lipschitz, 1997; Schwarzenberger and Brown, 2013; Cain and Cidlowski, 2017). In terms of reproduction, it is the hypothalamic–pituitary–gonadal (HPG) axis that is responsible for initiating, regulating and coordinating most of the reproductive functions in vertebrate species (Saltzman et al., 2011). In many free-roaming species a change in specific seasonal parameters, such as temperature or rainfall, act as indicators of the approaching breeding season and triggers breeding activity, both on a behavioural and physiological level (Bronson and Heideman, 1994; Bronson, 2009). Monitoring the reproductive physiology of a species enables researchers to decipher the complex mechanisms that underlie courtship, mating and other reproductive behaviours (Christensen et al., 2012; Watts, 2020). Here, researchers often monitor the so-called ‘male (androgen) and female (oestrogen and progestagen)’ reproductive hormones, which are important for the development of secondary sexual characteristics, the activation of reproductive behaviours, sperm production in males as well as cyclicity and pregnancy maintenance in females (Beehner et al., 2006a; Beehner et al., 2006b; Muller, 2017).

Although blood collection and analyses are often regarded as the gold standard of reproductive endocrine monitoring, providing real-time gonadal hormone concentrations (Kumar and Umapathy, 2019), the method requires researchers to trap and restrain individuals for extended periods of time. Not only can this result in animal and/or researcher injuries, but prolonged stressful events to the individual can also impact on reproductive processes and foetal development, should such events occur regularly (Wu et al., 2020; Fogel et al., 2023). Due to the invasive nature of endocrine monitoring using blood, this technique has fallen out of favour with many members of the research community, especially those working on threatened species (Kersey and Dehnhard, 2014). This is especially relevant to pangolin species, who roll into a tight ball as part of its defensive behaviour; as a result of this, individuals must be anaesthetised (Hooijberg et al., 2021), which is not only invasive but leads to extended handling and recovery times. Considering these difficulties, many researchers have opted for examining endocrine patterns using non-invasive monitoring techniques. Reproductive steroid hormones are predominantly secreted by the HPG axis into the bloodstream and are metabolized by the liver and excreted as conjugates in bile and ultimately faeces (Grow, 2002; Rhyu and Yu, 2021). As such, monitoring steroid hormone metabolite concentrations in faeces can provide a robust proxy of gonadal function in an organism (Schwarzenberger, 2007; Hodges et al., 2010).

Despite the numerous advantages of using non-invasive endocrine metabolite monitoring, hormone secretion, metabolism and excretion are species- and sex-specific (Goymann, 2005; Goymann, 2012). As a result of this, any enzyme immunoassay (EIA) hormone matrix combination, employed to monitor the reproductive physiology in a species for the first time, must be validated to ensure a reliable quantification of respective male and female reproductive biomarkers (Touma and Palme, 2005; Palme, 2019). Such EIA validations can be conducted via physiological or biological means. Physiological validations include the injection of a substance to hyperactivate the HPG axis; faecal samples are collected prior to and following the injection and analysed with specifically chosen EIAs for each hormone class in order to determine which assays can reliably detect biologically relevant changes in faecal hormone metabolite concentrations (Pribbenow et al., 2016; Scheun et al., 2018). However, due to its invasive nature, it is not always possible to implement a physiological validation, especially in threatened species (Touma and Palme, 2005; Eckardt et al., 2016). In these instances, a biological validation can be used to successfully validate an appropriate EIA system for measuring the reproductive physiology in a species. Here, sex- and age-specific differences in hormone metabolite concentrations can be used to validate EIAs for monitoring reproductive endocrine patterns in a species (Wielebnowski and Watters, 2007). Generally, adult and pregnant females have higher faecal oestrogen (fEM) and progestagen metabolite (fPM) concentrations than juvenile and adult non-pregnant females or males of any age class; similarly, adult males have higher faecal androgen metabolite (fAM) concentrations than juvenile males or any female age class (Pineda-Galindo et al., 2017; Scheun et al., 2017).

Once respective monitoring systems are in place, they can be used to enhance our understanding and drive further research into the reproductive biology of the species in situ, specifically by determining the reproductive status and sex determination in the field (Tubbs et al., 2014; Scheun et al., 2016; Brown, 2018). Importantly, endocrine metabolite monitoring can be used as a key indicator of the ability of a species to reproduce and/or adapt to changing environmental conditions. Non-invasive endocrine monitoring offers an easy-to-use technique to assist in the management and conservation of endangered and vulnerable wildlif species.

Temminck’s pangolin (Smutsia temminckii), is listed as globally vulnerable by the International Union for Conservation of Nature (Pietersen et al., 2019). The species is facing several anthropogenically driven threats, including the illegal wildlife and medicine trade, habitat loss, electrical fences and overutilization (Pietersen et al., 2014a). In addition to these threats, individuals retrieved from the wildlife trade go through prolonged rehabilitation and release processes (Wright and Jimerson, 2020). Despite the often-prolonged rehabilitation processes, no information has been gathered on reproductive physiology in the species. Rehabilitation efforts, coupled with direct monitoring of populations, are critical to understanding and enhancing their survival prospects (Challender et al., 2019). These initiatives help bridge gaps in our understanding of their ecological needs and reproductive cycles, crucial for formulating effective conservation strategies such as conducting a Population Viability Analysis. To assist in this regard, it is important that new methods for determining the reproductive status sex determination be developed. As such, the aim of this study was to validate EIAs for a reliable monitoring of fAM, fEM and fPM concentrations in Temminck’s pangolin.

Materials and Methods

Study animals

Due to the conservation status and pattern of population decline throughout the species’ distribution range, it is not possible to capture and/or hold Temminck’s pangolin for extended periods of time in order to conduct research. Furthermore, as pangolins are highly susceptible to stress (Wicker et al., 2020), long-term housing for research purposes may lead to a decrease in health and reproductive function in captive individuals (Dobson and Smith, 2000).

Owing to their low population densities, low detection rates and the inability to maintain them in full captivity for any length of time, there is a dearth in physiological data. As such, we depended on opportunistic samples collected at various facilities involved in the rehabilitation and release of confiscated individuals. Though this provided us with an opportunity to collect faecal samples from several male and female adult and juvenile individuals, it should be noted that the rehabilitation process puts an emphasis on limiting any human–pangolin interaction. As such, long-term sample collection from individuals was not possible. A trained veterinarian ensured individual health and welfare of all individuals used in this study. In addition to this, pregnancy status was also determined by the veterinarian through abdominal palpation and abdominal sonar.

Participating facilities and animal care

The Johannesburg Wildlife Veterinary Hospital, Loskop Dam Nature Reserve and The Munywana Conservancy all contributed samples to this study. Temminck’s pangolins are not held at these facilities but at an offsite location for the safety of the individuals and staff members. Pangolin are kept in large individual pens (>8 m2) and all care, including daily walks ranging from 2 to 6 h per individual, as well as feeding are conducted by facility staff as per established pangolin rehabilitation and care protocols.

Ethical clearance

The study was performed with the approval of the University of South Africa’s Animal Research Ethics Committee (2020/CAES_AREC/109).

Biological validation process

Due to restrictions on retaining individuals for research purposes, and the impracticality of conducting physiological validations, we chose to employ biological validations for determining the most suitable EIAs to monitor reproductive physiology in Temminck’s pangolin.

To biologically validate the most appropriate EIAs for monitoring fEM, fPM and fAM concentrations in Temminck’s pangolin, we compared the respective median metabolite concentrations between adult (>6 kg) and juvenile (<6 kg) male and female individuals (Pietersen et al., 2014b). A total of three samples were collected from each of the 10 study animals, resulting in a total of 30 samples (five females (two adults, three juveniles) and five males (three adults and two juveniles)). Sample collection occurred over a year period. As Temminck’s pangolin are not seasonal breeders (Pietersen et al., 2014b), this span should not influence observed endocrine patterns over this period. A single faecal sample was collected from a pregnant female (as determined by the veterinarian). All fresh faecal samples were placed into individual sample collection bags, sealed and stored at −20°C until transported to the SANBI Biobank.

Faecal sample extraction and analysis

All frozen faecal samples were extracted at the SANBI Wildlife Biobank following the methods used by Long et al. (2021). Faecal samples were lyophilized, pulverized and sieved through a thin mesh to remove any non-faecal matter that might be present. Subsequently, 1.5 ml of 80% ethanol was added to 0.050–0.055 g of faecal powder and vortexed for 15 min, before centrifuging the samples at 1500 × g for 10 min. The supernatant was then transferred to clean, clearly marked 2.5-ml microcentrifuge tubes, sealed and stored at −20°C until EIA analyses at the Endocrine Research Laboratory (ERL), University of Pretoria, South Africa. EIA analyses occurred within 3 months post-extraction.

All faecal extracts were measured for immunoreactive fEM, fPM and fAM concentrations, using an (i) oestrogen EIA, two progestagen EIAs ((ii) progesterone and (iii) 5α-progesterone) and two androgen EIAs ((iv) epiandrosterone, (v) testosterone). Details of the assays, including cross-reactivities of the antibodies, are described by Palme and Möstl (1993) for oestrogen, testosterone and epiandrosterone, Schwarzenberger et al. (1996b) for progesterone and Dehnhard et al. (2010) for 5α-progesterone. Antibody, conjugate and standard for the 5α-Progesterone EIA were purchased from Leibniz-Institute for Zoo Biology and Wildlife Research, PF 601103, D-10252 Berlin, Germany. Antibodies, conjugates and standards for the remaining EIAs (Oestrogen, Testosterone, Epiandrosterone and Progesterone) were purchased from the Department of Biomedical Sciences/Physiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria (contact: Associate Prof. Dr R. Palme, Rupert.Palme@vetmeduni.ac.at). Information on the conjugates is available in the cited publications. Plates were coated (Corning Product Number 9018) with AR-IgG (Arbor Assays, USA) using a standard procedure. Serial dilutions of extracted samples gave displacement curves that were parallel to the respective standard curves in all assays (Table 1). The intra- and inter-assay coefficient of variance (CV), determined by repeated measurements of high- and low-value quality controls, as well as assay sensitivities are shown in Table 1. Concentrations measurements in the faecal extracts were carried out in different dilutions, all within the parallel ranges of the respective EIAs. Diluted Standards were used as quality controls, at 20 and 80% binding for the respective EIAs. The standards can be provided by the suppliers of the antibodies and conjugates. As the faecal extract dilutions could be increased, no maximum detection value was reported.

Table 1.

The intra- and inter-assay CV, assay sensitivity, as well as parallelism test results and dilution ranges for the faecal oestrogen, progestogen and androgen metabolite enzyme immunoassays

Enzyme immunoassay Intra-assay CV Inter-assay CV Assay sensitivity ng/g faecal dry mass Parallelism test Dilution ranges used in the parallelism test
Testosterone 6.58 and 7.59% 8.02 and 14.93% 0.4 <4% 1/20–1/8000
Epiandrosterone 4.96 and 5.09% 6.89 and 9.67% 2.4 <4% 1/50–1/8000
Oestrogen 5.85 and 6.27% 6.65 and 13.20% 0.1 <5% 1/50–1/2000
Progesterone 5.55 and 5.73% 9.12 and 10.76% 3.2 <5% 1/50–1/2000
5α-progesterone 5.32 and 6.36% 5.03 and 6.99% 2 <5% 1/50–1/8000
Open in a new tab
a

Sample pools and individual samples were used for the parallelism tests depending on concentrations and volumes of the individual samples

Data analysis

No analytical statistics were conducted due to the limited number of samples collected. The median value for each individual was calculated and compared with the different age and sex classes across the fPM, fEM and fAM results. Similarly, sex+age group median values were calculated and compared when possible. For the fAM assay validation adult males should have higher fAM values than juvenile males, whilst both male age classes should have higher fAM values than females. For the fEM and fPM assay validations, adult females should have higher fEM and fPM values than juvenile females, whilst both female age classes should have higher values than either male age class. Similarly, fPM and fEM of the pregnant female should be higher than all other female and male age classes. All values are presented as median and interquartile range (IQR). The IQR provides a robust measure of variability that is less sensitive to extreme values than the range or standard deviation.

Results

Faecal androgen metabolite measurements

The testosterone EIA only detected low fAM levels in both male and female individuals across all age groups. Additionally, testosterone metabolite concentrations in males (range: 0.05–0.98 ng/g dry weight (DW)) and females (range: 0.02–0.79 ng/g DW) exhibited significant overlap, with just one male sample exceeding the highest female sample (1.98 vs 0.79 ng/g DW). Therefore, the assay was deemed inadequate for detecting biologically relevant differences in fAM levels between sexes. In contrast, the epiandrosterone EIA revealed a substantial difference in fAM ranges between males (7.17–43.34 ng/g DW) and females (0.80–13.14 ng/g DW) in this study. Median epiandrosterone concentrations were notably higher in adult males compared to juvenile males, and both male age groups had higher median fAM levels than adult and juvenile females (Table 2).

Table 2.

The median and range values for faecal testosterone and epiandrosterone metabolite concentrations between all age and sex classes

ID Assay Median (ug/g DW) Range (ug/g DW)
Adult female 1 Testosterone Epiandrosterone 0.33 4.80 0.15–0.44 2.30–9.30
Adult female 2 Testosterone Epiandrosterone 0.06 8.72 0.05–0.15 6.44–13.14
Juvenile female 1 Testosterone Epiandrosterone 0.03 1.02 0.02–0.03 0.80–1.24
Juvenile female 2 Testosterone Epiandrosterone 0.72 1.52 0.56–0.79 0.80–1.95
Juvenile female 3 Testosterone Epiandrosterone 0.51 3.07 0.31–0.66 1.57–3.77
Adult male 1 Testosterone Epiandrosterone 0.58 21.47 0.43–1.98 19.86–43.34
Adult male 2 Testosterone Epiandrosterone 0.11 24.72 0.08–0.13 12.92–24.92
Adult male 3 Testosterone Epiandrosterone 0.38 19.85 0.10–0.71 12.07–24.94
Juvenile male 1 Testosterone Epiandrosterone 0.13 9.94 0.05–0.24 7.17–22.07
Juvenile male 2 Testosterone Epiandrosterone 0.11 14.45 0.08–0.29 7.37–21.24
Open in a new tab

Faecal oestrogen metabolite measurements

Male fEM levels were at or below the detection limit of the oestrogen EIA, ranging from 0.01 to 0.02 ng/g DW. In contrast, female fEM levels were distinctly higher, ranging between 0.02 and 0.62 ng/g DW. Median fEM levels for individual adult females were notably higher than those of juvenile females (Table 3). Similarly, the fEM levels were considerably higher for the pregnant female sample than all other age and sex classes (Table 3).

Table 3.

The median and range values for faecal oestrogen metabolite concentrations between adult and juvenile females as well as the pregnant female. All adult and juvenile male values were <0.02 and thus excluded from this table

ID Median (ug/g DW) Range (ug/g DW)
Adult female 1 0.42 0.42–0.62
Adult female 2 0.33 0.19–0.36
Juvenile female 1 0.12 0.03–0.48
Juvenile female 2 0.09 0.02–0.24
Juvenile female 3 0.07 0.04–0.14
Pregnant female 0.83 N/A
Open in a new tab

Faecal progestagen metabolite measurements

Both progestagen EIAs effectively measured adequate fPM levels in Temminck’s pangolin. In both assays, fPM ranges were significantly higher in females (Progesterone EIA range: 1.51–12.64 ng/g DW; 5α-progesterone EIA range: 1.12–19.03 ng/g DW) compared to males (Progesterone EIA range: 0.95–2.42 ng/g DW; 5α-progesterone EIA range: 0.74–3.09 ng/g DW). Across both assays, median fPM levels for individual adult females were higher than those of juvenile females, and both adult and juvenile females had median fPM levels distinctly higher than all male study animals (Table 4). Finally, group-specific median fPM levels were higher in adult females compared to juvenile females, whilst both female groups had higher median values than adult and juvenile males. The pregnant female had an fPM level higher than all other age and sex classes (Table 4).

Table 4.

The median and range values for faecal progesterone and 5α progesterone metabolite concentrations between all age and sex classes including the pregnant female

ID Assay Median (ug/g DW) Range (ug/g DW)
Adult female 1 Progesterone 9.39 2.40–10.73
5α Progesterone 9.60 3.08–13.92
Adult female 2 Progesterone 7.49 1.51–12.64
5α Progesterone 9.19 2.05–19.03
Juvenile female 1 Progesterone 3.11 2.32–6.08
5α Progesterone 4.69 3.34–9.49
Juvenile female 2 Progesterone 3.82 3.18–5.18
5α Progesterone 4.07 3.11–4.28
Juvenile female 3 Progesterone 3.32 1.98–3.84
5α Progesterone 1.45 1.12–2.56
Pregnant female Progesterone 165.36 N/A
5α Progesterone 233.44 N/A
Adult male 1 Progesterone 2.04 1.54–2.42
5α Progesterone 1.98 0.94–1.98
Adult male 2 Progesterone 1.29 1.11–2.30
5α Progesterone 1.56 1.55–3.09
Adult male 3 Progesterone 1.80 1.32–2.16
5α Progesterone 1.58 1.26–1.59
Juvenile male 1 Progesterone 0.95 0.92–1.10
5α Progesterone 0.94 0.74–0.95
Juvenile male 2 Progesterone 0.93 0.84–1.25
5α Progesterone 1.14 0.94–1.50
Open in a new tab

Discussion

This is the first study to validate EIAs for a reliable determination of gonadal endocrine patterns using faeces as a matrix in Temminck’s pangolin. The use of a biological validation process was sufficient to validate the respective EIAs. Reproductive activity can now be monitored in male and female Temminck’s pangolin via non-invasive endocrine monitoring.

The epiandrosterone EIA was successfully validated for measuring fAM concentrations in the Temminck’s pangolin. This finding confirms the applicability of this EIA for monitoring reproductive function in pangolins, as it has also been deemed suitable for quantifying fAMs in the Taiwanese pangolin (Manis pentadactyla pentadactyla; Arora et al., 2020). Adult male Temminck’s pangolin had considerably higher fAM levels than juvenile males, highlighting the ability of the epiandrosterone EIA to discriminate between different maturation stages in males of this species. Similarly, the epiandrosterone EIA exhibited the capability to distinguish between male and female individuals across all age classes, revealing only a minimal overlap observed between juvenile males and adult females. The detection of such fAM differences has been observed and utilized as a biological validation tool in various mammalian species (Pineda-Galindo et al., 2017; Scheun et al., 2017), further affirming the reliability of employing this assay in Temminck’s pangolin.

The tested total oestrogen EIA was also successfully validated for quantifying fEMs in Temminck’s pangolin. As the assay can measure various oestrogens, such as estrone (E1), estradiol (E2) and estriol (E3) (Denver et al., 2019), to name a few. As such, the antibody of the applied assay shows a recognizable cross-reactivity with a number of common oestrogens. However, Arora et al. (2020) suggested that estradiol-17β might be one of the primary oestrogens present in the faeces of pangolins. Whether this is the case for Temminck’s pangolin remains to be determined. Much like the discerning ability of fAM levels in males, the oestrogen EIA demonstrated its effectiveness in distinguishing between different age classes in females, affirming its ability to differentiate between various maturation stages in Temminck’s pangolin. Consistent with the outcomes of this study, previous research has also noted variations in female oestrogen levels associated with age (Kanematsu et al., 2006; Musey et al., 1987). Furthermore, fEM levels in male Temminck’s pangolin, when measured with the oestrogen EIA, were below the measurement threshold of the tested oestrogen EIA, indicating the ability of the EIA to differentiate between the sexes. Several studies have shown that males have lower oestrogen levels than their female counterparts (Barbosa-Moyano et al., 2024; Gesquiere et al., 2014). Therefore, the applicability of the oestrogen EIA in monitoring fEM concentrations in female Temminck’s pangolin was confirmed, demonstrated by its capability to distinguish not only between female age classes but also between male and females.

Both progestagen EIAs successfully discriminated between different age and sex classes; in addition to this, both EIAs also allowed for the determination of pregnancy. As a result, both progestogen EIAs are equally suitable to quantify fPMs in Temminck’s pangolin. Arora et al. (2020) reported comparable findings in the context of the Taiwanese pangolin, highlighting the prevalence of the 5α-configuration within the observed pregnane series. In this study, however, both assays demonstrated an ability to distinguish between adult females and their younger counterparts (i.e. able to differentiate between maturation stages). This ability is crucial when validating an EIA through biological means. Such differences in fPM levels between different age classes have been observed in previous research (Greenberg et al., 2022) and supports the findings of this study. Finally, there were clear differences in fPM concentrations between females and males, with minimal overlap between juvenile females and adult males. This pattern has been observed in previous studies (Pineda-Galindo et al., 2017) and support the validity of using either EIA to monitor fPM concentrations in Temminck’s pangolin.

Finally, although only one sample was available, both the fEM and fPM concentrations of the confirmed pregnant female Temminck’s pangolin were considerably higher than these metabolite concentrations in all other age and sex classes. An elevation in fPM and fEM concentrations during pregnancy has been observed in numerous species (Schwarzenberger et al., 1996a; Nagl et al., 2015; Scheun et al., 2016) and used to validate the most appropriate EIAs for monitoring fEM and fPM levels in several species (Dehnhard et al., 2008; Knott et al., 2013; Mithileshwari et al., 2016; Bleke et al., 2021). This lends further support to our biological validations performed for both the fEM and fPM assays in Temminck’s pangolin.

Conclusion

The EIAs validated in this study offer a powerful tool set for understanding the species’ reproductive patterns whilst also allowing for sex determination. In addition to this, information gathered can provide necessary parameters required to perform a robust Population Viability Analysis, which will be useful at present. Future research should focus on using these EIAs to determine female cyclicity and explore the connection between gonadal hormones and breeding behaviour. One limitation of the current study is the small sample size, a constraint due to the species’ conservation status as well as general ecology. Increasing the number of animals in future studies, along with long-term monitoring, would contribute significantly to our understanding of the reproductive patterns of this species.

Acknowledgements

We would like to thank the Munywana Conservancy (previously known as andBeyond Phinda Private Game Reserve), the Johannesburg Veterinary Hospital and Loskop dam Nature Reserve for their assistance.

Contributor Information

Juan Scheun, Department Nature Conservation, Tshwane University of Technology, Staatsartillerie Rd, Pretoria West, Pretoria 0183, South Africa; Faculty of Natural and Agricultural Sciences, Mammal Research Institute, University of Pretoria, Lynnwood Rd, Hatfield, Pretoria 0002, South Africa.

Andre Ganswindt, Faculty of Natural and Agricultural Sciences, Mammal Research Institute, University of Pretoria, Lynnwood Rd, Hatfield, Pretoria 0002, South Africa.

Raymond Jansen, Department of Environmental, Water and Earth Science, Tshwane University of Technology, Staatsartillerie Rd, Pretoria West, Pretoria 0183, South Africa; IUCN SSC Pangolin Specialist Group, c/o Zoological Society of London, London NW1 4RY, UK.

Kim Labuschagne, SANBI Wildlife Biobank, South African National Biodiversity Institute, 232 Boom St, Daspoort 319-Jr, Pretoria 0001, South Africa.

Author contributions

J.S.: Conceptualization, resources, funding acquisition, data curation, methodology, formal analysis, visualization, writing (original draft preparation, review and editing).

A.G.: Conceptualization, resources, methodology, writing (original draft preparation, review and editing).

K.L.: Resources, supervision, methodology, writing (original draft preparation, review and editing).

R.J.: Resources, methodology, data curation, writing (original draft preparation, review and editing).

Conflicts of interest

The authors have no conflicts of interest to declare.

Funding

This work was supported by the National Research Foundation, South Africa [grant number: 138099].

Data availability

The data underlying this article are available in the article and in its online supplementary material.

References

  1. Arora B, Jai-Chyi Pei K, Feng Weng C, Ching-Min Sun N (2020) Measuring fecal metabolites of endogenous steroids using esi-ms/ms spectra in Taiwanese pangolin, (order pholidota, family manidae, genus: Manis): a non-invasive method for endangered species. Gen Comp Endocrinol 299: 113607. 10.1016/j.ygcen.2020.113607. [DOI] [PubMed] [Google Scholar]
  2. Barbosa-Moyano H, Zacariotti RL, de Oliveira CA (2024) Validation of enzyme immunoassays for quantifying sex steroid hormones in tropical screech owls (Megascops choliba). Gen Comp Endocrinol 345: 114386. [DOI] [PubMed] [Google Scholar]
  3. Beehner JC, Bergman TJ, Cheney DL, Seyfarth RM, Whitten PL (2006a) Testosterone predicts future dominance rank and mating activity among male chacma baboons. Behav Ecol Sociobiol 59: 469–479. 10.1007/s00265-005-0071-2. [DOI] [Google Scholar]
  4. Beehner JC, Nguyen N, Wango EO, Alberts SC, Altmann J (2006b) The endocrinology of pregnancy and fetal loss in wild baboons. Horm Behav 49: 688–699. 10.1016/j.yhbeh.2005.12.016. [DOI] [PubMed] [Google Scholar]
  5. Bleke CA, Gese EM, French SS (2021) Variations, validations, degradations, and noninvasive determination of pregnancy using fecal steroid metabolites in free-ranging pronghorn. Gen Comp Endocrinol 312: 113841. 10.1016/j.ygcen.2021.113841. [DOI] [PubMed] [Google Scholar]
  6. Bronson FH (2009) Climate change and seasonal reproduction in mammals. Philos Trans R Soc B 364: 3331–3340. 10.1098/rstb.2009.0140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bronson FH, Heideman PD (1994) Seasonal regulation of reproduction in mammals. In Knobil E, Neil JD, eds, The Physiology of Reproduction. Raven Press, New York, pp. 541–581. [Google Scholar]
  8. Brown JL (2018) Comparative ovarian function and reproductive monitoring of endangered mammals. Theriogenology 109: 2–13. 10.1016/j.theriogenology.2017.12.004. [DOI] [PubMed] [Google Scholar]
  9. Cain DW, Cidlowski JA (2017) Immune regulation by glucocorticoids. Nat Rev Immunol 17: 233–247. 10.1038/nri.2017.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Challender DW, Ades GW, Chin JS, Sun NC-M, lian Chong J, Connelly E, Hywood L, Luz S, Mohapatra RK, de Ornellas P (2019) Evaluating the feasibility of pangolin farming and its potential conservation impact. Global Ecology and Conservation 20: e00714. [Google Scholar]
  11. Christensen A, Bentley G, Cabrera R, Ortega H, Perfito N, Wu T, Micevych P (2012) Hormonal regulation of female reproduction. Horm Metab Res 44: 587–591. 10.1055/s-0032-1306301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dehnhard M, Fanson K, Frank A, Naidenko SV, Vargas A, Jewgenow K (2010) Comparative metabolism of gestagens and oestrogens in the four lynx species, the Eurasian (Lynx lynx), the Iberian (l. Pardinus), the Canada lynx (l. Canadensis) and the bobcat (l. Rufus). Gen Comp Endocrinol 167: 287–296. 10.1016/j.ygcen.2010.03.023. [DOI] [PubMed] [Google Scholar]
  13. Dehnhard M, Naidenko S, Frank A, Braun B, Göritz F, Jewgenow K (2008) Non-invasive monitoring of hormones: a tool to improve reproduction in captive breeding of the Eurasian lynx. Reprod Domest Anim 43: 74–82. 10.1111/j.1439-0531.2008.01145.x. [DOI] [PubMed] [Google Scholar]
  14. Denver N, Khan S, Homer NZM, MacLean MR, Andrew R (2019) Current strategies for quantification of oestrogens in clinical research. J Steroid Biochem Mol Biol 192: 105373. 10.1016/j.jsbmb.2019.04.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dobson H, Smith RF (2000) What is stress, and how does it affect reproduction? Anim Reprod Sci 60-61: 743–752. 10.1016/S0378-4320(00)00080-4. [DOI] [PubMed] [Google Scholar]
  16. Eckardt W, Stoinski TS, Rosenbaum S, Umuhoza MR, Santymire R (2016) Validating faecal glucocorticoid metabolite analysis in the Virunga mountain gorilla using a natural biological stressor. Conserv Physiol 4: cow029. 10.1093/conphys/cow029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fogel AS, Oduor PO, Nyongesa AW, Kimwele CN, Alberts SC, Archie EA, Tung J (2023) Ecology and age, but not genetic ancestry, predict fetal loss in a wild baboon hybrid zone. Am J Biol Anthropol 180: 618–632. 10.1002/ajpa.24686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gesquiere LR, Ziegler TE, Chen PA, Epstein KA, Alberts SC, Altmann J (2014) Measuring fecal testosterone in females and fecal estrogens in males: Comparison of ria and lc/ms/ms methods for wild baboons (papio cynocephalus). Gen Comp Endocrinol 204: 141–149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goymann W (2005) Noninvasive monitoring of hormones in bird droppings physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. Ann N Y Acad Sci 1046: 35–53. 10.1196/annals.1343.005. [DOI] [PubMed] [Google Scholar]
  20. Goymann W (2012) On the use of non-invasive hormone research in uncontrolled, natural environments: the problem with sex, diet, metabolic rate and the individual. Methods Ecol Evol 3: 757–765. 10.1111/j.2041-210X.2012.00203.x. [DOI] [Google Scholar]
  21. Greenberg JR, Montgomery TM, Holekamp KE, Beehner JC (2022) Validation and effects of drying and prey hair on fecal hormone concentrations in spotted hyenas. J Mammal 103: 394–406. [Google Scholar]
  22. Grow DR (2002) Metabolism of endogenous and exogenous reproductive hormones. Obstet Gynecol Clin North Am 29: 425–436. 10.1016/S0889-8545(02)00009-8. [DOI] [PubMed] [Google Scholar]
  23. Hodges K, Brown J, Heistermann M (2010) Endocrine monitoring of reproduction and stress. In Kleiman DG, Thompson KV, Baer CK, eds, Wild Mammals in Captivity: Principles and Techniques for Zoo Management. University of Chicago, Chicago, pp. 447–468 [Google Scholar]
  24. Hooijberg EH, Lourens K, Meyer LCR (2021) Reference intervals for selected hematology and clinical chemistry measurands in Temminck's pangolin (Smutsia temminckii). Frontiers in Veterinary Science 8: 654529. 10.3389/fvets.2021.654529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kanematsu N, Jin W, Watanabe G, Taya K (2006) Age-related changes of reproductive hormones in young meishan boars. J. Reprod. Dev. 52: 651–656. [DOI] [PubMed] [Google Scholar]
  26. Kersey DC, Dehnhard M (2014) The use of noninvasive and minimally invasive methods in endocrinology for threatened mammalian species conservation. Gen Comp Endocrinol 203: 296–306. 10.1016/j.ygcen.2014.04.022. [DOI] [PubMed] [Google Scholar]
  27. Knott KK, Roberts BM, Maly MA, Vance CK, DeBeachaump J, Majors J, Riger P, DeCaluwe H, Kouba AJ (2013) Fecal oestrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation. Reprod Biol Endocrinol 11: 83. 10.1186/1477-7827-11-83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kumar V, Umapathy G (2019) Non-invasive monitoring of steroid hormones in wildlife for conservation and management of endangered species - a review. Indian J Exp Biol 57: 307–314. [Google Scholar]
  29. Lipschitz DL (1997) Effects of estradiol-17b and progesterone on mating behaviour in female lesser bushbabies (Galago moholi). Horm Behav 32: 73–84. 10.1006/hbeh.1997.1405. [DOI] [PubMed] [Google Scholar]
  30. Long C, Tordiffe A, Sauther M, Cuozzo F, Millette J, Ganswindt A, Scheun J (2021) Seasonal drivers of faecal glucocorticoid metabolite concentrations in an African strepsirrhine primate, the thick-tailed greater galago (Otolemur crassicaudatus). Conserv Physiol 9: caob081. 10.1093/conphys/coab081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mithileshwari C, Srivastava T, Kumar V, Kumar A, Umapathy G (2016) Non-invasive assessment of fecal progestagens and pregnancy detection in Himalayan musk deer (Moschus chrysogaster). Theriogenology 85: 216–223. 10.1016/j.theriogenology.2015.09.009. [DOI] [PubMed] [Google Scholar]
  32. Möstl E, Palme R (2002) Hormones as indicators of stress. Domest Anim Endocrinol 23: 67–74. 10.1016/S0739-7240(02)00146-7. [DOI] [PubMed] [Google Scholar]
  33. Muller MN (2017) Testosterone and reproductive effort in male primates. Horm Behav 91: 36–51. 10.1016/j.yhbeh.2016.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Musey VC, Collins DC, Musey PI, Martino-Saltzman D, Preedy JRK (1987) Age-related changes in the female hormonal environment during reproductive life. Am J Obstet Gynecol 157: 312–317. [DOI] [PubMed] [Google Scholar]
  35. Nagl A, Kneidinger N, Kiik K, Lindeberg H, Maran T, Schwarzenberger F (2015) Noninvasive monitoring of female reproductive hormone metabolites in the endangered European mink (Mustela lutreola). Theriogenology 84: 1472–1481. 10.1016/j.theriogenology.2015.07.023. [DOI] [PubMed] [Google Scholar]
  36. Palme R (2019) Non-invasive measurement of glucocorticoids: advances and problems. Phys Behav 199: 229–243. 10.1016/j.physbeh.2018.11.021. [DOI] [PubMed] [Google Scholar]
  37. Palme R, Möstl E (1993) Biotin-streptavidin enzyme immunoassay for the determination of ooestrogens and androgens in boar faeces. Adv Steroid Anal 93: 111–117. [Google Scholar]
  38. Pietersen D, W, Jansen R, Connelly E (2019). Smutsia temminckii, The IUCN Red List of Threatened Species pp e.T12765A123585768. [Google Scholar]
  39. Pietersen DW, McKechnie AE, Jansen R (2014a) A review of the anthropogenic threats faced by Temminck's ground pangolin, Smutsia temminckii, in southern Africa. S Afr J Wildl Res 44: 167–178. 10.3957/056.044.0209. [DOI] [Google Scholar]
  40. Pietersen DW, McKechnie AE, Jansen R (2014b) Home range, habitat selection and activity patterns of an arid-zone population of Temminck’s ground pangolins, Smutsia temminckii. Afr Zool 49: 265–276. 10.1080/15627020.2014.11407642. [DOI] [Google Scholar]
  41. Pineda-Galindo E, Cerda-Molina AL, Mayagoitia-Novales L, Matamoros-Trejo G, de la O C (2017) Biological validations of fecal glucocorticoid, testosterone, and progesterone metabolite measurements in captive stumptail macaques (Macaca arctoides). Int J Primatol 38: 985–1001. 10.1007/s10764-017-9992-7. [DOI] [Google Scholar]
  42. Pribbenow S, Wachter B, Ludwig C, Weigold A, Dehnhard M (2016) Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus). Gen Comp Endocrinol 228: 40–47. 10.1016/j.ygcen.2016.01.015. [DOI] [PubMed] [Google Scholar]
  43. Rhyu J, Yu R (2021) Newly discovered endocrine functions of the liver. World J Hepatol 13: 1611–1628. 10.4254/wjh.v13.i11.1611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Saltzman W, Tardif SD, Rutherford JN (2011) Hormones and reproductive cycles in primates. In Norris DO, Lopez KH, eds, Hormones and Reproduction of Vertebrates. Academic Press, London, pp. 291–327 [Google Scholar]
  45. Scheun J, Bennett NC, Nowack J, Ganswindt A (2017) Reproductive behaviour, testis size and faecal androgen metabolite concentrations in the African lesser bushbaby. J Zool 301: 263–270. 10.1111/jzo.12420. [DOI] [Google Scholar]
  46. Scheun J, Greeff D, Ganswindt A (2018) Non-invasive monitoring of glucocorticoid metabolite concentrations in urine and faeces of the sungazer (Smaug giganteus). PeerJ 6: e6132. 10.7717/peerj.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scheun J, Nowack J, Bennett N, Ganswindt A (2016) Female reproductive activity and its endocrine correlates in the African lesser bushbaby, Galago moholi. J Comp Physiol B 186: 255–264. 10.1007/s00360-015-0947-z. [DOI] [PubMed] [Google Scholar]
  48. Schwarzenberger F (2007) The many uses of non-invasive faecal steroid monitoring in zoo and wildlife species. Int ZooYearb 41: 52–74. 10.1111/j.1748-1090.2007.00017.x. [DOI] [Google Scholar]
  49. Schwarzenberger F, Brown J (2013) Hormone monitoring: an important tool for the breeding management of wildlife species. Wien Tierarztl Monatsschr 100: 209–225. [Google Scholar]
  50. Schwarzenberger F, Möstl E, Palme R, Bamberg E (1996a) Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Anim Reprod Sci 42: 515–526. 10.1016/0378-4320(96)01561-8. [DOI] [Google Scholar]
  51. Schwarzenberger F, Tomasova K, Holeckova D, Matern B, Möstl E (1996b) Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes. Zoo Biol 15: 159–171. . [DOI] [Google Scholar]
  52. Sheriff M, Dantzer B, Delehanty B, Palme R, Boonstra R (2011) Measuring stress in wildlife: techniques for quantifying glucocorticoids. Oecologia 166: 869–887. 10.1007/s00442-011-1943-y. [DOI] [PubMed] [Google Scholar]
  53. Touma C, Palme R (2005) Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation. Ann N Y Acad Sci 1046: 54–74. 10.1196/annals.1343.006. [DOI] [PubMed] [Google Scholar]
  54. Tubbs C, McDonough CE, Felton R, Milnes MR (2014) Advances in conservation endocrinology: the application of molecular approaches to the conservation of endangered species. Gen Comp Endocrinol 203: 29–34. 10.1016/j.ygcen.2014.02.013. [DOI] [PubMed] [Google Scholar]
  55. Watts HE (2020) Seasonal regulation of behaviour: what role do hormone receptors play? Proc R Soc B 287: 20200722. 10.1098/rspb.2020.0722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wicker LV, Cabana F, Chin JS-C, Jimerson J, Lo FH-Y, Lourens K, Mohapatra RK, Roberts A, Wu S (2020) Chapter 28 - captive husbandry of pangolins: lessons and challenges. In Nash HC, Waterman C, eds, Challender DWS. Academic Press, Pangolins, pp. 443–459 [Google Scholar]
  57. Wielebnowski N, Watters J (2007) Applying fecal endocrine monitoring to conservation and behavior studies of wild mammals: important considerations and preliminary tests. Isr J Ecol Evol 53: 439–460. 10.1560/IJEE.53.3.439. [DOI] [Google Scholar]
  58. Wright N, Jimerson J (2020) The rescue, rehabilitation and release of pangolins. In Challender DWS, Nash HC, Waterman C, eds, Pangolins. Elsevier, Cambridge, UK, pp. 495–504 [Google Scholar]
  59. Wu Y, Lu Y-C, Jacobs M, Pradhan S, Kapse K, Zhao L, Niforatos-Andescavage N, Vezina G, Plessis AJ, Limperopoulos C (2020) Association of prenatal maternal psychological distress with fetal brain growth, metabolism, and cortical maturation. JAMA Netw Open 3: e1919940–e1919940. 10.1001/jamanetworkopen.2019.19940. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data underlying this article are available in the article and in its online supplementary material.

Articles from Conservation Physiology are provided here courtesy of Oxford University Press

RESOURCES