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Volume 12 • 2024 10.1093/conphys/coae048
Toolbox
Nailing it: Investigation of elephant toenails for
retrospective analysis of adrenal and
reproductive hormones
Garrett Rich1,Rebecca Stennett2,Marie Galloway3,Mike McClure2,Rebecca Riley3,Elizabeth W. Freeman4
and Kathleen E. Hunt1,5,*
1Department of Biology, George Mason University, 4400 University Drive, Fairfax, VA 22030, USA
2The Maryland Zoo in Baltimore, 1 Safari Place Baltimore, MD 21217, USA
3Smithsonian’s National Zoo and Conservation Biology Institute, 3001 Connecticut Ave NW, Washington, DC 20008, USA
4School of Integrative Studies, George Mason University, 4400 University Drive, Fairfax, VA 22030, USA
5Smithsonian-Mason School of Conservation, 1500 Remount Road, Front Royal, VA 22630, USA
*Corresponding author: Colgan Hall, George Mason University, Sci-Tech Campus, 10900 University Blvd, Manassas, VA. Email: kehunt@gmu.edu
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Hormone monitoring of at-risk species can be valuable for evaluation of individual physiological status. Traditional non-
invasive endocrine monitoring from urine and faeces typically captures only a short window in time, poorly reecting long-
term hormone uctuations. We examined toenail trimmings collected from African (Loxodonta africana) and Asian (Elephas
maximus) elephants during routine foot care, to determine if long-term hormone patternsare preserved in these slow-growing
keratinized tissues. We rst measured the growth rate of elephant toenails biweekly for one year, to establish the temporal
delay between deposition of hormones into nail tissue (at the proximal nail bed) and collection of toenail trimmings months
later (at the distal tip of the nail). In African elephants, toenails grew ∼0.18 ±0.015 mm/day (mean±SEM) and in Asian
elephants, toenails grew ∼0.24 ±0.034 mm/day. This slow growth rate, combined with the large toenail size of elephants,
may mean that toenails could contain a ‘hormone timeline’ of over a year between the nail bed and nail tip. Progesterone,
testosterone and cortisol were readily detectable using commercial enzyme immunoassays, and all assays passed validations,
indicating that these hormones can be accurately quantied in elephant toenail extract. In most cases, variations in hormone
concentrations reected expected physiological patterns for adult females and males (e.g. ovarian cycling and musth) and
matched individual health records from participating zoos. Progesterone patterns aligned with our calculations of temporal
delay, aligning with female ovarian cycling from over six months prior. Unexpectedly, male testosterone patterns aligned with
current musth status at the time of sample collection (i.e. rather than prior musth status). Though this sample type will require
further study, these results indicate that preserved hormone patterns in elephant toenails could give conservationists a new
tool to aid management of elephant populations.
Key words:Elephants, hormones, keratin, noninvasive, reproduction, stress
Editor: Christine Madliger
Received 30 October 2023; Revised 25 June 2024; Editorial Decision 3 July 2024; Accepted 15 July 2024
Cite as: Rich G, Stennett R, Galloway M, McClure M, Riley R, Freeman EW, Hunt KE (2024) Nailing it: Investigation of elephant toenailsfor retrospective
analysis of adrenal and reproductive hormones.Conserv Physiol 12(1): coae048; doi:10.1093/conphys/coae048.
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Too lbo x Conservation Physiology • Volume 12 2024
Introduction
Endocrinology techniques are a valuable tool for the conser-
vation and management of wildlife. Evaluation of hormonal
states can provide researchers with critical information about
an individual’s reproductive physiology and responses to
stress. In mammals, the steroid hormones progesterone and
testosterone often reflect reproductive states including preg-
nancy, sexual maturity and reproductive cycles (Lasley and
Kirkpatrick, 1991;McCormick and Romero, 2017;Melica
et al., 2021), while the glucocorticoids (cortisol, corticos-
terone) provide insight into exposure to stressors and the
resulting impacts on health (Sheriff et al., 2011;Matas et al.,
2016). Evaluating hormonal states of in situ and ex situ
wildlife populations can be accomplished through a wide
variety of sample matrices. Traditional endocrine sampling
relies on collection of serum or plasma, but alternative sample
types such as faeces and urine are increasingly used due to
the potential for minimally invasive or non-invasive collection
from living individuals in captivity and the wild (Lasley and
Kirkpatrick, 1991;Sheriff et al., 2011;Heimbürge et al.,
2019;Palme, 2019).
Elephant physiology is marked by notably long-term hor-
monal changes corresponding with reproductive and poten-
tially stressful events. In female elephants, the 3-month-long
ovarian cycle is characterized by a prolonged elevation of
progestagens during a 10-week luteal phase (Hess et al., 1983;
Plotka et al., 1988). Pregnancy in elephants is characterized
by elevated progestagens continuing after the luteal phase
and throughout the 22-month gestation period (Hess et al.,
1983;Hodges, 1998). In male elephants, the unique male
reproductive state of musth can be detected via a significant
elevation in circulating androgens (testosterone, etc.) that can
last weeks to months. Musth has an unpredictable onset in
different individuals and involves changes in behavior that
can pose challenges for management of male elephants ex
situ (Poole et al., 1984;Lincoln and Ratnasooriya, 1996;
Brown, 2000;LaDue et al., 2021). Glucocorticoids, which
can become detrimental to health if elevated over extended
periods of time (Moberg, 2000;Sheriff et al., 2011), are often
monitored as a tool for management of elephant populations
(Ahlering et al., 2011;Fanson et al., 2013;Brown et al., 2019).
Although monitoring hormones can, in theory, facilitate man-
agement decisions, collection of samples from elephants is
often challenging. Blood collection ex situ requires training
for minimally invasive collection and from in situ individuals
is logistically difficult if not dangerous. Urine and faecal
samples can be challenging to match to the individual, and
repeated samples across months are not always available,
particularly in situ, which challenges ability to monitor the
prolonged (multi-month) reproductive cycles of elephants.
A potential alternative endocrine matrix for evaluation is
the keratin tissues (e.g. hair, feathers, scales, nails), which
may enable retrospective evaluation of endocrine events
that occurred in prior months or years (Matas et al., 2016;
Kalliokoski et al., 2019). All vertebrate keratin tissues studied
to date have been shown to accumulate steroid hormones
as they grow. Some of these tissues grow continuously in a
linear fashion, extending distally from a well-vascularized
epidermal growth zone (e.g. hair follicle, nail bed), and these
tissues can represent an endocrine ‘time series’ with points
along the keratin structure containing hormones that were
deposited at different times. Thus, an entire sample can be
used to reconstruct a detailed individual endocrine history
that spans the time period of tissue growth (e.g. whale baleen,
Hunt et al., 2014,2017b; human fingernail, Izawa et al.,
2021; seal vibrissae, Keogh et al., 2021). Further, keratin
samples can often be collected non-invasively (e.g. shed hair)
or with minimal invasiveness (e.g. clipping of distal parts
of hair or nail samples). The dry matrix of keratin has
also been shown to preserve steroid hormones for decades
even at room temperature (e.g. Hunt et al., 2017a;Beattie
and Romero, 2023). Long-term preservation of hormones
could thus enable historic keratin samples from natural
history museums to be used to reconstruct endocrine patterns
of past populations, enabling comparison to present-day
populations and enhancing ability to understand the effects
of anthropogenic impacts (Koren et al., 2002).
In elephants, keratin tissues such as toenails and tail hairs
could allow evaluation of prolonged (multi-month) patterns
of hormones, while providing a much broader timeframe than
serum, faecal or urine sampling. While elephant tail hair has
been investigated as a potential endocrine sample type for
elephants, specifically for cortisol (Pokharel et al., 2021), to
our knowledge, no studies have evaluated elephant toenails
as a potential sample matrix for any hormones. In ex situ
populations, toenail trimmings are frequently removed from
the distal part of the nail during routine foot care. Trimmings
are usually discarded but could be used to retrospectively
evaluate the endocrine status of the elephant. Natural history
museums, as well, contain historic specimens of toenails
that could be studied to evaluate endocrine patterns of past
populations. Nails of humans (Palmeri et al., 2000;Warnock
et al., 2010;Izawa et al., 2015;Fischer et al., 2020) and claws
of other mammalian and non-mammalian species (turtles,
Baxter-Gilbert et al., 2014; seals, Karpovich et al., 2020,Crain
et al., 2021; wolves, Roff ler et al., 2022) have proven to be
informative endocrine sample types. However, each species’
unique nail or claw growth rate must be evaluated alongside
endocrine evaluations in order to estimate the date of growth
for the distal trimmed piece.
The overarching goal of our study was to assess the utility
of African and Asian elephant toenails as a non-invasive
hormone matrix. We hypothesized that the steroid hormones
progesterone, testosterone and cortisol would be detectable
in elephant toenail extract and that patterns in hormones
across successively collected toenail trimmings would reflect
the elephant’s endocrine status from the prior months or
years, with a temporal lag determined by nail growth rate
and length of the nail. Specific goals were to (i) measure the
growth rate of elephant toenail in African and Asian elephants
of both sexes, across a full year; (ii) determine whether
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Tab le 1 : Details of individual elephants sampled in this study. Toenail trimmings were sampled from African (n=3) and Asian (n=4) elephants
once a month. Maryland Zoo = The Maryland Zoo in Baltimore; NZCBI= the Smithsonian’s National Zoo and Conservation Biology Institute.
Individual Species Zoo SSP # Sex Age Number of samplesa
AnnabLoxodonta africana Maryland Zoo 138 Female 47 9
FelixcL. africana Maryland Zoo 339 Female 39 10
Samson L. africana Maryland Zoo 561 Male 14 10
KamalacElephas maximus NZCBI 145 Female 47 12
MaharanicE. maximus NZCBI 307 Female 32 14
Spike E. maximus NZCBI 141 Male 41 15
SwarnabE. maximus NZCBI 146 Female 47 13
The number of toenails from that individual that were assayed for hormone content, i.e. excluding any samples that were collected but not assayed, suchassame-day
replicates (i.e. multiple toenail trimmings collected from the same day), samples with very low mass <10 mg (known to result in inaccuratehormone data for dry sample
types; ‘small sample eect’ (Hayward et al., 2010,Berk et al., 2016,Fernández Ajó et al., 2022), or samples with intractable shape for pulverization.
Irregular/acyclic female.
Cyclic female.
progesterone, testosterone and cortisol are present and
detectable in elephant toenail extract; (iii) validate (par-
allelism, accuracy) commercial enzyme immunoassay kits
to quantify hormones in elephant toenail matrix; and (iv)
perform preliminary biological validations by comparing
patterns in toenail hormones to documented physiological
status from keeper records.
Materials and methods
Sample collection
Toenail trimmings were collected monthly from two female
and one male African elephants (Loxodonta africana) at the
Maryland Zoo in Baltimore (September 2020 to August 2021;
Table 1) and from three female and one male Asian elephants
(Elephas maximus) at the Smithsonian’s National Zoo and
Conservation Biology Institute (November 2020 to December
2021; Table 1). All elephants were well habituated to routine
foot care by animal care staff, voluntarily allowed their feet to
be handled, received positive reinforcement via food rewards
and were not anesthetized or restrained for toenail trimming.
Toenails were collected from front or hind feet based on
the management practices of both zoos; specifically, African
elephant toenail samples were collected from the hind feet and
Asian elephant samples from the front feet. Upon collection,
toenail trimmings were stored at −20◦C for up to 6 months
until transfer to George Mason University for analysis. Both
participating zoos are accredited by the Association of Zoos
and Aquariums and follow all recommended best practices
for housing and husbandry of elephants in human care. This
study followed all applicable local, state and federal regula-
tions and was approved by both participating zoos and by
the Institutional Animal Care and Use Committee of George
Mason University.
Animal care staff routinely keep a log of reproductive
events (e.g. indications of ovarian cycling or musth) and
note any potentially stressful events. As not all female ele-
phants experience regular ovarian cycling (Brown, 2000;
Freeman et al., 2009;Brown, 2019), females in this study
were classed as either ‘cycling’ (i.e. experienced regular cycles
of predictable occurrence and duration) or ‘irregular/acyclic’
(i.e. irregular cycles of unpredictable occurrence, or acyclic),
based on keeper records and veterinary records of serum
progesterone (Table 1). Finally, both males experienced one
musth episode during the sample collection period, based on
indicators of urine dribbling and temporal gland secretions,
behavior, and/or endocrine status from faeces (LaDue et al.,
2022). These individual records were used for biological
validations, e.g. comparison of known or inferred endocrine
status (as described above) to patterns of hormone concentra-
tions observed in toenail samples.
Determination of toenail growth rate
The toenail growth rate was determined by marking a shallow
(∼1 mm deep) horizontal groove on the surface near the
cuticle of one toenail of each elephant. This groove did not
penetrate through the dead outer layer of the nail and was
similar to (but visually distinct from) surface markings that
normally occur on elephant toenails during normal activity.
Approximately every 2 weeks, the distance of the groove from
the cuticle was photographed and measured to the nearest
millimetre with a tape measure. Growth rate was evaluated
in two ways: (i) biweekly growth rate, calculated for each
2-week period separately, as the distance between each new
measurement and the prior measurement, divided by elapsed
number of days (Fig. 1) and (ii) total growth rate across
the entire study, calculated as the distance of groove from
original measurement at the end of the study, divided by
total number of days of the study period. All growth rates
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Too lbo x Conservation Physiology • Volume 12 2024
Fig. 1: General methodology for determining growth rate of
elephant toenails. (A) An initial marking was made near the cuticle of
the elephant toenail at the start of the study date (indicated in red).
(B) Every 2 weeks, the distance the mark travelled from the cuticle
was measured (indicated by the red arrow). Growth rate was
determined by dividing the distance travelled by the time interval
between the initial mark and the new position. Image credit: Chase
LaDue.
were converted to daily growth rates, millimetres per day,
to standardize results.
Toenail preparation and hormone
extraction
Frozen toenail trimmings were transferred to George Mason
University’s Sci-Tech Campus (Manassas, VA), stored at
−80◦C until drying and then air-dried in a fume hood.
Samples were weighed daily starting on the seventh day of
drying and considered dry when mass of the sample remained
consistent (≤0.5 mg variation) across successive daily mea-
surements. Dried toenail samples were then cleaned of any
surface contamination with 70% isopropyl alcohol. Toenails
were pulverized to a fine powder via a Dremel model 3000 (a
powered rotary grinder) with flexible shaft attachment using
a tungsten-carbide cylindrical tip. The Dremel, other tools
and workspace were thoroughly cleaned with a 70% alcohol
solution between samples to prevent cross-contamination. A
Sartorius Entris II digital scale was used to weigh 10 ±1mg
of toenail powder (recorded to the nearest 0.1 mg); this target
mass was selected following published findings for minimum
sample mass of other mammalian keratin samples (Hayward
et al., 2010;Berk et al., 2016;Fernández Ajó et al., 2022).
During weighing of dried toenail powder, an Ohaus Ion 100A
ionizer was placed next to the scale to reduce the effects of
static charge on apparent sample mass. The weighed toenail
powder was then transferred to a 16 ×100 mm borosilicate
glass extraction tube, 4.00 ml of 100% methanol was added
and tubes were vortexed for1honarack shaker (Glas-
Col Large Capacity Mixer, at a speed setting of 40) and then
centrifuged at 3000 rpm for 15 min (Sorvall ST4R centrifuge).
Following centrifugation, 3.50 mL of the methanol extract
(containing hormones) was pipetted into to a 13 ×100 mm
borosilicate glass tube (final hormone data were corrected
for percentage of supernatant that was not recovered), and
the methanol was evaporated via a Thermo Scientific Savant
SpeedVac rotary vacuum concentrator (model #SPD1030) at
45◦C under vacuum until all samples were dry. Once dry,
500 μl of assay buffer (#X065; Arbor Assays, Ann Arbor,
MI) was added to each tube, and the dried hormones were
resuspended via vortexing for 1 min at medium speed on the
rack shaker, sonication for 5 min (Branson Ultrasonic Bath
M3800) and a final manual vortex for 20 sec at high speed
on a Vortex Genie 2. The resulting extract was considered
the ‘1:1’ or full-strength extract. Extracts were pipetted to O-
ring-capped vaporproof cryovials for storage at −80◦C until
assayed within 6 months.
Hormone assays
Hormone concentrations from toenail samples were deter-
mined via commercial enzyme immunoassay (EIA) kits for
progesterone, testosterone and cortisol (catalog # K025,
K032 and K003, respectively; Arbor Assays, Ann Arbor, MI;
arborassays.com). Parallelism was assessed via comparison of
the slopes of the binding curve of serially diluted extract pools
(eight dilutions spanning 1:1–1:128) for each sex and each
species to the slope of the hormone standards. An appropriate
dilution for each hormone for each species was then selected
based on the 50% binding point of the parallelism results, as
follows: progesterone was assayed at a 1:4 dilution for both
species; testosterone was assayed at 1:51 for African elephants
and at 1:4 for Asian elephants; and cortisol was assayed at 1:4
for African elephants and 1:1 for Asian elephants. Dilutions
were the same for males and females of the same species. Due
to generally low cortisol content of elephant toenail extract,
cortisol parallelism was assessed with a unique sample
pool consisting specifically of high-apparent-cortisol samples
from both sexes. That is, some samples were first assayed
individually in order to identify those that had relatively
high cortisol concentration, and then a ‘high-hormone pool’
was created using just those samples, thereby ensuring
that the pool would have enough cortisol for parallelism
assessment. Further, cortisol parallelism in Asian elephant
also necessitated the use of a 1:5 dilution of assay antibody
and conjugate to improve precision of the assay at very low
concentrations; this 1:5 dilution has been comprehensively
tested for low-cortisol samples in our lab and produces
improved precision at high percent bounds while retaining
acceptably low inter- and intra-assay variation (see below).
After successful parallelism validations, accuracy (matrix
effect) validations were then performed to assess the ability
of each assay to accurately distinguish high from low con-
centrations in the presence of sample matrix (toenail extract).
Accuracy validations were performed at the aforementioned
dilutions, by comparing hormone concentrations in a set of
standards spiked with pooled diluted extract (equal volumes
of standard and pool) to a standard curve spiked only with
assay buffer (equal volumes of standard and buffer). Due to
limited sample volume, assay accuracy was assessed for each
hormone using pools from both males and females of the same
species and not for each sex separately.
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All assays followed the manufacturer’s protocols (available
at www.arborassays.com), with two changes. First, one
additional low-dose standard was added to each assay by
extending the standard curve by one additional dilution, using
the same process used to create the previous standards (for
progesterone and cortisol assays, 250 μl buffer +250 μl
of previous standard; for the testosterone assay, 300 μl
buffer +200 μl of previous standard). Second, the cortisol
assay was run in X065 buffer (rather than its usual X053
buffer) after consultation with the manufacturer, in order to
streamline extraction methodology. Standards, pure hormone
controls and samples were assayed in duplicate, with non-
specific binding (‘NSB’) wells and zero-dose wells (‘maximum
binding’) wells in quadruplicate. Each elephant’s samples
were assayed in the same 96-well microplate with a full
standard curve and control. Percentage binding of each
sample (percentage of label bound) of each well was then
used to interpolate the concentrations of the sample extracts
with four-parameter logistic curve fits using Prism v.9 for
OSX (www.graphpad.com). Cross-reactivities are reported in
the manufacturer’s protocols (www.arborassays.com) and are
further described in Hunt et al. (2017b). All assays were
inspected for good fit of standard curve, normal NSBs, normal
NSB/zero ratio, and coefficient of variation (%CV) of optical
densities <10% for all standards and samples (i.e. to identify
any cases of pipetting error; any sample with %CV >10%
was re-assayed). Assay precision was assessed via calculation
of %CV of interpolated hormone concentrations of African
and Asian elephant toenail extract pools (i.e. one pool for each
species) assayed in six different assays (inter-assay variation)
and also assayed six times within one assay (intra-assay
variation); all intra-assay %CVs were below 4% and all inter-
assay %CVs were below 7% (see Table S1 in Supplementary
Information for details). Any single anomalous standard was
excluded from the standard curve; any assay with two or
more anomalous standards was re-assayed. Final data were
expressed in nanograms per gram of hormone per toenail
powder.
Statistical analyses
Toenail growth rate data were inspected for anomalous
results. Four measurements that resulted in apparent negative
growth rates were assumed to be due to measurement or
data recording error and were subsequently removed from
the dataset. An average of the biweekly growth rate for
each individual was calculated for comparison to their
total growth rate. Each individual was assigned an overall
average growth rate by averaging that elephant’s biweekly
average growth rate with that elephant’s total growth rate.
Species growth rate was then estimated for African elephants
and Asian elephants separately by averaging results for
all individuals of that species (i.e. averaging the individual
averages). Finally, African and Asian elephant growth rates
were averaged to produce a single estimated toenail growth
rate for Elephantidae. Differences in average growth rate
between species were assessed via Welch’s t-tests.
Assay parallelism was evaluated via F-test to compare
slope of the serially diluted toenail extract to the slope of
the binding curve (percent bound vs. log[concentration]) of
the hormone standards. Assay accuracy was evaluated via
graphing apparent concentration vs. standard concentration
and inspecting goodness of fit of the linear regression line
(r2>0.95) and assessing whether the slope was between 0.7
and 1.3 (ideal slope = 1.0).
Baseline hormone concentrations for each elephant were
estimated via an iterative process by removing hormone data
points that were >2 SD) above the mean, until none of
the remaining samples exceeded this limit. The average of
remaining samples is considered to represent ‘baseline’ for
that hormone in that elephant. Any samples more than two
times this baseline was termed ‘elevated’, and any samples
also exceeding 2 SD above this baseline was termed a prob-
able ‘peak’, i.e. with ‘peaks’ conceptualized as unusually
high elevations. Hormone data are continuous in nature, and
physiologically relevant peaks are not always easily identifi-
able; our thresholds for ‘elevated’ and ‘peak’ were intended
for initial exploratory assessment of toenail hormone pro-
files but are not intended as a definitive determination of
physiological relevance. Average concentration of hormones
between species were compared using Welch’s t-test using the
hormone concentrations of all samples from each individual.
Pearson correlations were measured between the hormone
concentrations within samples from the same individuals.
Hormone concentrations were log-transformed before these
statistical tests, due to non-normal distribution.
Lastly, hormone and growth rate data were combined to
assign an estimated date to each hormone data point. The
estimated date was determined by dividing the length of
the toenail (in mm) by the individual’s average growth rate
to yield an approximate number of days since growth (i.e.
since emergence from the nail bed). The number of days
was then subtracted by the date of toenail collection to yield
an estimated growth date. Hormone ‘peaks’ and estimated
growth date were compared to known physiological data for
biological validations.
Averages and Welch’s t-tests were calculated using
Microsoft Excel version 2309. F-test parallelism and
Pearson correlation tests were conducted using Graphpad
Prism version 9.3.1 for Windows (www.graphpad.com).
Individual growth rates and hormone results are expressed
as mean ±SD (mean ±SEM for species growth rates and
Elephantidae growth rate). The significance threshold was set
at alpha = 0.05.
Results
Growth rate
The average toenail growth rate for African and Asian ele-
phants combined was 0.21 ±0.030 mm/day (mean ±SEM).
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Tab le 2 : Toenail growth rate results from African elephantsa(n= 3) and Asian elephantsb(n= 4). Average growth rate was calculated as the grand
average of the biweekly growth rate and the total growth rate
Elephant/Species Biweekly growth rate (mm/day) Total growth rate (mm/day) Average growth rate (mm/day)
Annaa0.15 ±0.09 0.15 0.15
Felixa0.17 ±0.10 0.18 0.18
Samsona0.20 ±0.09 0.20 0.20
African elephant average 0.18
Kamalab0.22 ±0.13 0.19 0.20
Maharanib0.18 ±0.10 0.17 0.18
Spikeb0.36 ±0.24 0.31 0.33
Swarnab0.24 ±0.18 0.22 0.23
Asian elephant average 0.24
For African elephants, the average growth rate was
0.18 ±0.015 mm/day with variation among individuals
ranging from 0.15 to 0.20 mm/day (Table 2).For Asian
elephants, the average growth rate was 0.24 ±0.034 mm/day
with variation ranging from 0.18 to 0.33 mm/day (Table 2).
Asian elephant toenail growth rates tended to be faster than
African elephants, but this difference was not significantly
different (P= 0.1825, n= 3 African elephants, n= 4 Asian
elephants; t=−1.6109, df = 4, Welch’s t-test). Five of seven
elephants exhibited higher growth rate in warmer months
(March–September), though low sample size and the 1-year
study duration precluded formal statistical evaluation of
potential seasonal changes in growth rate.
Assay validations
Progesterone and cortisol assays demonstrated good par-
allelism for toenail extracts of both species (Table S2 in
Supplementary Information). The testosterone assay, how-
ever, demonstrated parallelism for African elephant males but
non-parallelism in African elephant females and both sexes
of Asian elephants (Table S2 in Supplementary Information).
The slopes of the central portions of the curves, however,were
generally similar (Fig. 2, middle panel), interpreted here to
indicate presence of a probable androgen that binds relatively
well to the testosterone assay antibody. Finally, all assays
passed accuracy testing, with observed vs expected dose
curves being linear and possessing a slope within a desired
range of 0.7–1.3 (Table S3 in Supplementary Information).
Hormone concentrations
Our final sample size was n= 83 toenail trimmings, all of
which had quantifiable concentrations of immunoreactive
progesterone, testosterone and cortisol. One sample was
found to be considerably lower in hormone than the rest
of the samples but was not omitted from the data set. Across
all elephants, toenail progesterone content ranged from 0.5
to 588 ng/g, testosterone ranged from 8 to 2930 ng/g, and
cortisol ranged from 2 to 116 ng/g. Average concentrations
of all three hormones were higher for African elephant toenail
samples than in Asian elephant toenail samples (progesterone:
Africans, 139 ±139 ng/g; Asians, 54 ±18 ng/g; testosterone:
Africans, 404 ±745 ng/g; Asians, 144 ±306 ng/g; cortisol:
Africans, 32 ±25 ng/g; Asians, 12 ±5 ng/g). These species
differences were significant for progesterone, testosterone
and cortisol concentrations (all P<0.0001, n= 29 samples
for the African elephants, n= 54 for the Asian elephants;
progesterone: t= 4.5404; df = 55. Testosterone: t= 4.5077;
df = 62. Cortisol: t= 5.8709, df = 44, Welch’s t-test). Female
African elephants had significantly higher average toenail
progesterone (120 ng/g) than Asian elephant females (51 ng/g;
P= 0.0004, n=19 samples from the African females, n=39
samples from the Asian females; t= 3.8571, df = 43, Welch’s t-
test). Average toenail testosterone was higher for the African
male (890 ng/g) than the Asian male (328 ng/g), but this
difference was not significantly different (P= 0.1319, n=10
samples from the African male, n= 15 samples from the Asian
male; t= 1.5784, df = 18, Welch’s t-test).
Longitudinal hormone profiles for each individual
elephant, with estimated dates of growth at the nail bed, are
presented in Figs 3 and 4. With a few exceptions (Table 3),
samples with high reproductive hormone concentrations also
tended to be high in cortisol (P<0.05). For the two acyclic
females, testosterone and cortisol were not correlated (Figs 2
and 3;Table 3). One of the three cycling females, African
female ‘Felix’, displayed high variation in hormones, with
higher concentrations than other females, and estimated
growth dates of peak samples corresponding with her known
ovarian cycles (Fig. 3). The remaining females all had similar
toenail hormone concentrations (range: 0.5–90 ng/g) with
fluctuations that did not resemble apparent ovarian cycling
(Figs 3 and 4). Some toenail samples from the two males
had high testosterone concentrations (Fig. 5), but estimated
growth dates did not correspond with prior musth cycles.
Rather, for both these males, the high-testosterone toenail
samples were collected during a current musth episode (i.e.
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Fig. 2: Parallelism results for enzyme immunoassays tested with serially diluted pools of elephant toenail extract for progesterone (top),
testosterone (middle) and cortisol (bottom).
the individual was in musth when that toenail sample was
collected). Additionally, when an elephant was in musth,
toenail trimmings collected from female elephants at the same
zoo also showed minor elevations in testosterone (see Fig. S1
in Supplementary Information).
Discussion
Toenails of African and Asian elephants contained detectable
and quantifiable concentrations of progesterone, testosterone
and cortisol when analyzed with commercial EIA kits. All
assays passed validation tests of parallelism and accuracy
for toenail extract of both elephant species (albeit with one
case of minor non-parallelism), indicating presence of likely
hormones in toenail extract (i.e. extract contains substances
that bind well to the assay antibodies) as well as good mathe-
matical accuracy of the assay across a range of concentrations.
Prior studies have demonstrated that claws of some other ver-
tebrates, and fingernails of humans, also contain detectable
steroid hormones. To our knowledge, our study is the first
to test elephant toenails for steroid hormone content. In
combination with prior studies, our results suggest that claws
and nails of many other vertebrates, as well as potentially hoof
of artiodactyls and perissodactyls, may also contain steroid
hormones.
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Fig. 3: Concentration of progesterone and cortisol graphed against
estimated toenail growth date for female African elephants (L.
africana) Anna (top) and Felix (bottom). Sampled toenails of African
elephants reected expected acyclicity (Anna) and historical ovarian
cycling (Felix). Coloured bars indicate independently conrmed luteal
phases. aThis luteal phase was classed as ‘abnormal’by zoo sta.
The temporal period represented by any keratin sample
type requires careful consideration, as there can be a
substantial lag in time between deposition of hormone in
the growth zone and emergence of (or collection of) the
sample distally at a later time point. Though there have
been at least three prior efforts to estimate the toenail
growth rate in elephants (Seilkopf, 1959;Benz, 2005;Fowler
and Mikota, 2006), none of those reports have been peer-
reviewed. Our study included a year-long effort at evaluating
typical toenail growth rate for multiple individuals and
both sexes of two elephant species. Based on our estimated
average growth rate for Elephantidae of 0.21 mm/day, a
large toenail could contain over a year’s worth of hormone
concentrations deposited across the nail from proximal
cuticle down to the distal tip. Growth rate ranges of
0.17–0.33 mm/day reported by Fowler and Mikota (2006)
Fig. 4: Concentration of progesterone and cortisol graphed against
estimated toenail growth date for female Asian elephants (E.
maximus) Kamala, Maharani and Swarna. Asian elephant toenails did
not show evidence of present or historical ovarian cycling, with
minimal uctuation between samples.
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Tab le 3 : Pearson correlation statistics of log-transformed hormone concentrations
within each elephant. Signicance is denoted via superscripts.
Elephant Progesterone–
testosterone
Progesterone–
cortisol
Testosterone–
cortisol
Anna 0.3331 0.8056∗−0.1754
Felix 0.7771∗0.7228∗∗ 0.8548∗
Samson 0.9479∗∗∗ 0.8719∗∗∗ 0.8817∗∗∗
Kamala 0.4254 0.7411∗0.7062∗∗
Maharani 0.4707 0.8193∗∗∗ 0.7304∗
Spike 0.7768∗∗∗ 0.6735∗0.8103∗∗∗
Swarna 0.1408 0.7985∗0.3022
∗P<0.01;
∗∗P<0.05;
∗∗∗P<0.001.
were similar to our observed ranges: 0.15–0.33 mm/day.
Our estimated growth rates are also similar to those of Benz
(2005; unpublished thesis) for African (5.4 mm/28 days) and
Asian (7.0 mm/28 days) elephants. Benz (2005) additionally
found that Asian elephants had higher toenail growth rate
compared to Africans. We also observed growth rate variation
between species and individuals and some indication of
seasonal differences (data not shown). However, additional
samples, individuals and years would be necessary to
determine if these differences truly reflect variation by sex,
season or time of year.
It is possible that substrate and locomotory patterns may
affect toenail wear and growth rate. Substrate and locomotion
may vary seasonally, as elephants in North American zoos
often spend more time within barns in winter than in summer.
The anatomy of the elephant foot is complex and is believed to
house a pressure mechanism that increases circulation to the
foot when actively walking (Fowler and Mikota, 2006); it is
possible, therefore, that locomotion may affect circulation to
the nail bed and hence may directly affect toenail growth (i.e.
through mechanisms other than increased nail wear). These
questions await further study. It may be fruitful to combine
studies of locomotion and growth rate with studies of clinical
care, as locomotion is an important element in foot health in
elephants (Fowler and Mikota, 2006).
Biweekly growth rates were evaluated in order to inspect
potential variation in nail growth rate during the observation
period, but were predicted to have greater measurement error,
while the total growth rate was considered to have less
measurement error (due to the greater length of toenail being
measured), but measurement error does occur even with total
growth rates. Due to the difficulties inherent in measuring
distances with ∼1 mm precision on a living, unrestrained,
elephant’s foot, some measurement error is inevitable with
both methods. We therefore considered the most accurate
estimate of growth rate to be the combined average of both
of these methods.
Concentrations of hormones in toenails showed varia-
tion between samples, individuals, sexes and species. Corti-
sol concentration tended to correlate with testosterone and
progesterone, with only a few cortisol elevations that did
not match reproductive hormone elevations. This notable
correlation among all steroid hormones is a common finding
in all vertebrate sample types and is thought to be due in
part to the fact that progesterone is the precursor to all the
steroids, and in part because reproduction is itself a type
of stressor. For example, cortisol and androgens typically
show positive correlations across musth cycles in males (e.g.
LaDue et al., 2023), presumably to help animals cope with
the energetic burden of reproductive behavior and reproduc-
tive physiology. In female elephants, diverse positive, neg-
ative and no correlations have been noted between gluco-
corticoids and the reproductive steroids, varying with the
nature of the reproductive event (e.g. Bechert et al. 1999;
Brown et al., 2004;Oliveira et al., 2008;Fanson et al.,
2014;Kajaysri and Nokkaew, 2014;Glaeser et al., 2020;
Towiboon et al., 2022). Generally, it is believed that positive
correlations between glucocorticoids and the reproductive
hormones can occur during normal reproductive cycling in
vertebrates, while negative correlations are more likely to
indicate occurrence of unpredictable, non-reproductive or
unusually severe stressors (reviewed in Romero & Wing-
field 2016). Overall, in this study, cortisol patterns did not
suggest occurrence of any unusual stressful events during
the study period or the prior year. We hypothesize, though,
that acute stress (short-term stress) may not be reflected in
toenail endocrine data, due to the short period in which
glucocorticoids would be deposited into the toenail. It is also
possible that the strong correlations observed here across
hormones may represent variations in deposition rate of
steroids into toenail matrix that may not necessarily reflect
circulating concentrations. Parallel studies of plasma and
toenails collected from the same elephants across two or
more years could shed further light on this issue. Such studies
should be possible, since many elephants in human care are
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Fig. 5: Concentration of testosterone and cortisol graphed against
the toenail collection date for two male elephants, African elephant
(L. africana) Samson (top) and Asian elephant (E. maximus)Spike
(bottom). Toenail samples containing high testosterone were
collected while individuals were actively in musth (coloured bars).
habituated to routine blood collection as well as routine
foot care.
The reproductive hormones progesterone and testosterone
showed variable patterns. Testosterone was elevated in toenail
samples trimmed while the male was actively in musth, rather
than reflecting prior occurrence of a historical musth cycle.
This finding suggests that even after an increment of toenail
is grown and emerges from the nailbed, it can still acquire
hormone through some as-yet-undetermined route. Male ele-
phants in musth routinely dribble urine onto their legs (urine
dribbling is one of the diagnostic indicators of musth), and
urine is one of the excretion routes for testosterone, and
thus urine may have deposited testosterone onto the external
nail surface. While it would be expected that urine dribbling
would deposit testosterone primarily onto hind feet, increased
testosterone was observed in toenails from both front and
hind feet. Alternatively, hormones could be entering nails
simply due to the elephant walking through areas of substrate
containing urine or faeces (i.e. affecting both front and hind
feet), or via some other route, such as skin oil or from the
underlying nail bed (Palmeri et al., 2000). Intriguingly, when a
male was in musth at a given zoo, toenail trimmings collected
from nearby females from the same zoo also had minor
elevations in testosterone. It is not clear whether this simply
represents widespread contamination of all the elephants’
feet and toenails, or whether other elephants might react
physiologically to the nearby presence of a musth male.
In contrast to testosterone data, progesterone data of at
least one female corresponded to prior ovarian cycles and
not current ovarian cycles. Toenails believed to have grown
during two of African elephant Felix’s luteal phases showed
elevated progesterone. Thus, toenail progesterone content
may indicate whether a female experienced a luteal phase in
the prior year. However, one Felix toenail believed to have
grown during a luteal phase did not show elevated proges-
terone, though this particular luteal phase had been noted by
keepers as “abnormal” (Fig. 3, superscript a). Felix was the
only one of three cycling females that showed clear evidence
of such a pattern (i.e. high toenail progesterone corresponding
to a documented prior ovarian cycle). The other two cycling
females, and both of the acyclic/irregular females, had much
lower progesterone with erratic but not notable elevations.
Therefore, further study will be necessary to verify whether
toenail progesterone is a reliable indicator of female ovarian
cycling in the prior year. The slow growth rate of toenail
may mean that toenail endocrine data are more suitable for
determination of the very long progesterone elevations of
pregnancy, rather than the briefer elevations of luteal phases.
Follow-up studies examining toenail progesterone patterns
across full pregnancies would be fruitful.
Additional research is needed to further evaluate the utility
of monitoring hormone concentration in toenails for elephant
conservation. For future studies, we recommend sampling
the largest toenail on a foot; comparing front to back feet
(especially for males, across musth cycles); regularly measur-
ing the whole length of the toenail; comparing freeze-drying
to air-drying nails; and evaluating additional hormones (e.g.
estradiol, thyroid hormones). Possible effects of season or
locomotion on toenail growth also should be considered.
Studies of more individuals, ideally including multiple cycling
females as well as musth males, will enable better assessment
of whether reproductive status of the prior year is consistently
reflected in toenail endocrine data. Such studies could be
paired with ongoing plasma collections and accelerometer
locomotion studies.
New tools and techniques for wildlife endocrinology could
allow for the enhancement of methodologies and research
of in situ and ex situ elephants. Examination of hormone
patterns in toenails could allow conservationists to look into
the past at physiological profiles of elephants both living
and deceased. Toenails are regularly trimmed as a part of
regular care for ex situ individuals, and the trimmings can
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be readily collected and can be stored indefinitely at room
temperature once dried. Thus, toenail samples could function
as a long-term hormone bank for conservation programs
to retrospectively evaluate individual physiology. Toenails
collected from deceased individuals in captivity, the wild or
in archived museum samples can be trimmed into successive
pieces to reconstruct a hormone profile and physiological
history leading up to the individual’s death. It is possible,
too, that wild elephants tranquilized for other procedures
could be sampled by means of collecting small samples from
the toenail surface from top to bottom to assemble hormone
histories. Other analyses commonly employed on keratin
samples (e.g. drug analysis, stable isotopes, mineral status;
Palmeri et al., 2000;Trueman et al., 2019;Sach et al., 2020)
may allow for insight into relationships among physiolog-
ical state, diet and other relevant biological information.
Continued work is needed, but elephant toenails could pro-
vide conservationists with a new tool to monitor individual
physiology.
Acknowledgements
We thank Chase Ladue, Trent Grasso, Allison Case and
Rebecca Evey for their advice and assistance in the laboratory.
We would also like to thank keepers and animal care staff for
collection of growth rate data and samples. This study was
approved by animal care and research committees at both
participating zoos.
Author contributions
G.R., E.W.F. and K.E.H. secured funding, conceived the ideas
and designed methodology; R.S., M.G., M.M. and R.R.
revised methodology and study plan and collected samples,
measurement data and photographs; G.R. performed
laboratory analyses, analyzed data and produced figures
and tables; GR, E.W.F. and K.E.H. interpreted data; G.R.
led the writing of the manuscript with edits on early drafts
from E.W.F. and K.E.H. All authors contributed critically to
subsequent drafts and gave final approval for publication.
Conicts of interest
The authors declare no conflicts of interest in relation to this
study.
Funding
This work was supported by the George Mason University
Faculty Research Development Award to K.H.and E.F. and by
two George Mason University Office of Student Scholarship,
Creative Activities, and Research (OSCAR) Undergraduate
Research Scholars Program (URSP) awards to G.R., K.H.
and E.F.
Data availability
Data utilized for this article can be accessed by requesting
access from the corresponding author.
Supplementary material
Supplementary material is available at Conservation Physiol-
ogy online.
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