Journal of Mammalogy, 97(3):852–860, 2016
DOI:10.1093/jmammal/gyw013
Published online March 10, 2016
Interaction of hibernation and male reproductive function in wild
Tasmanian echidnas Tachyglossus aculeatus setosus
Gemma E. Morrow,* Susan M. Jones, and Stewart C. Nicol
School of Biological Sciences, University of Tasmania, Private Bag 5, Hobart, Tasmania 7001, Australia
* Correspondent: [email protected]
The short-beaked echidna Tachyglossus aculeatus is a seasonally breeding mammal with a near ubiquitous
distribution throughout Australia. In Tasmania, breeding follows a period of deep hibernation, and males begin
mating approximately 30 days after the termination of hibernation. The echidna has exceptionally large testes,
which may reach a maximum of 1% of body mass during the mating period. As involution of gonads is considered
a prerequisite for entering hibernation and hibernation typically suppresses all reproductive function, this raises
questions about the timing of testes recrudescence in the Tasmanian echidna. We measured plasma testosterone
concentrations and used ultrasonography to measure testicular and crural gland volume through the annual
cycle in wild Tasmanian echidnas. Testes were at their minimum size (0.06% of body mass) in December (early
summer); testes recrudescence occurred prior to entry into hibernation when plasma testosterone concentrations
were low; and testes were maintained at 75% of their maximum volume throughout the hibernation period.
The crural glands, which are secondary reproductive structures in the echidna, also exhibited an annual pattern
of recrudescence and involution, with recrudescence occurring after males emerged from hibernation, when
plasma testosterone was rising. We suggest that the unusual strategy of testes recrudescence occurring prior to
hibernation in the Tasmanian echidna is a consequence of extremely high competition between males.
Key words: echidna, hibernation, monotreme, Tachyglossus, testes, testosterone
© 2016 American Society of Mammalogists, www.mammalogy.org
Hibernation places constraints on the endocrine system and
function of reproductive organs in mammals (Hoffman 1964;
Hudson and Wang 1979; Barnes et al. 1986). As many crucial reproductive functions are reliant on hormone-mediated
processes, which are normally depressed at lower body temperatures, reproduction and hibernation were long considered
to be mutually exclusive processes (Wimsatt 1969). However,
a number of birds and mammals, including members of all 3
mammalian subclasses, enter torpor or hibernation during their
reproductive period (reviewed by McAllan and Geiser 2014).
The discussion of the overlap of hibernation and reproduction often insufficiently distinguishes between the different
consequences for male and female reproduction although the
profile of energy expenditure in male reproduction is very different from that of females. For female mammals, mating itself
does not incur large energetic costs (Gittleman and Thomson
1988) with the major daily energetic costs being incurred
during late pregnancy and lactation (Bronson 1985). In some
mammals, pregnancy and lactation may extend over a long
period, so that the daily energetic costs can be quite low and
compatible with hibernation (McAllan and Geiser 2014). By
contrast, in males that do not show parental care, the energetic
costs of reproduction are largely associated with mating behavior—seeking out females and physically competing with other
males (Bronson 1985). The major physiological cost of reproduction for males appears to be spermatogenesis, hence the
majority of seasonally breeding species undergo 40–90% atrophy in testis mass after the breeding period (Wedell et al. 2002).
Spermatogenesis seems to be incompatible with deep hibernation, and male hibernators require several weeks at euthermic
temperatures for testicular recrudescence and spermatogenesis before they can produce viable sperm (Barnes 1996). In
bat species in which the male is able to produce viable sperm
immediately on arousal from hibernation, the sperm are produced before entry into hibernation and stored in the epididymis (Crichton 2000).
The short-beaked echidna (Tachyglossus aculeatus) is a myrmecophagous monotreme with a near ubiquitous distribution
throughout Australia. It breeds in winter–early spring following
a period of inactivity that ranges from short periods of torpor in
mild climates (e.g., Kangaroo Island—Rismiller and McKelvey
1996) to prolonged deep hibernation in cooler areas (Beard
et al. 1992; Beard and Grigg 2000; Nicol and Andersen 2002).
Tasmania represents the southernmost extent of the echidna’s
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MORROW ET AL.—HIBERNATION AND REPRODUCTION IN ECHIDNAS853
range and therefore the region where seasonal influences on
the timing of reproduction are likely to be the greatest. In
Tasmania, the breeding season extends from mid-June to midSeptember following a period of deep hibernation during which
body temperature (Tb) typically falls below 10°C, depending on
substrate temperature, with the lowest Tb recorded being below
5°C (Nicol and Andersen 2007b). Sexually mature echidnas do
not mate every year: females mate only every 2nd year, while
25% of males hibernate throughout the mating season (Nicol
and Morrow 2012). Males enter hibernation in late January to
early February (late summer) and individuals in their breeding years terminate hibernation between early May and late
June (late autumn–early winter). As in all deep hibernators,
in echidnas, the period of reduced Tb is broken by periods of
spontaneous arousal during which body temperatures return to
normothermic levels (Nicol and Andersen 2002). Arousals are
generally less than 24 h in duration (Nicol and Andersen 2000)
and hence echidnas spend the majority of their hibernation season with Tb below 10°C.
Involution of gonads is generally considered to be a prerequisite for mammals to enter hibernation (Hoffman 1964;
Darrow et al. 1988) and gonadal recrudescence is inhibited at
the low body temperatures experienced during torpor (Wimsatt
1969). However, in our study population of echidnas, males
have been observed mating approximately 30 days after they
terminate hibernation (Morrow et al. 2009). As echidna testes
are reported to reach 1% of an echidna’s body mass during
the breeding season (Griffiths 1984) compared with 0.18% in
woodchucks, a comparable sized eutherian hibernator whose
testes recrudesce after emergence from hibernation (Kenagy
and Trombulak 1986), this raises questions about the timing
of testes recrudescence in the Tasmanian echidna: in order to
reach this large size are testicular growth and testosterone production initiated during hibernation?
There is very little information about the physiological state
of hibernating echidnas in the period preceding mating when
testes recrudescence would be expected to occur. Male echidnas exhibit seasonal changes in plasma testosterone concentrations (Dean 2000; Nicol et al. 2005) and testes size (Johnston
et al. 2007), and testes mass and seminiferous tubule diameter
reach their maximum during the breeding season (Griffiths
1978). Reproductively mature male echidnas have a keratinous
spur on each ankle joint with a central canal connected by a
duct to the crural gland located in the popliteal fossa (Krause
2010). Although the crural glands have been described as
vestigial (Augee et al. 2006), Griffiths (1968) noted that they
increase in size during the breeding season, reaching a diameter
of 2 cm, while histological examination demonstrated that they
are functional in the winter breeding months but involuted for
the greater part of the year (Carmichael and Krause 1971). We
have observed that during the breeding season a milky fluid can
be expressed from the spur by compressing the ampulla at its
base (G. E. Morrow, pers. obs.).
While these studies provided some useful information,
echidnas are testicond, making accurate measurement of testes size difficult: Griffiths (1978) collected data on testes mass,
seminiferous tubule, and crural glands by lethal sampling
(Carmichael and Krause 1971). More recently, Johnston et al.
(2007) used ultrasonography to investigate variation in testes
size of Queensland echidnas, but these were captive animals
that did not hibernate, so any extrapolations to wild populations
must be made with caution. Since 1996 we have been studying
a wild population of echidnas in the Tasmanian Midlands and
many aspects of their seasonal physiology and reproduction are
now well understood (Nicol et al. 2005; Nicol and Andersen
2007a; Morrow et al. 2009; Nicol and Morrow 2012; Sprent
et al. 2012).
The overall aim of the current study was to investigate the
temporal relationships between reproduction and hibernation
in male echidnas in this population. Using ultrasonography to
measure testes and crural gland volume, we investigated the
relationships between these parameters, plasma testosterone
concentrations, and hibernation state throughout the year.
Materials and Methods
Field site.—This study was conducted on a 12 km2 site
(42°28′S, 142°14′E) in the Tasmanian Midlands approximately
50 km north of Hobart, Tasmania. The study site is part of a
grazing property, consisting of native and improved pasture as
well as remnant Eucalyptus amygdalina woodland. The site has
variable topography with altitudes ranging from 200 to 400 m
above sea level; mean daily minimum and maximum temperatures are −1°C to 10°C in winter and 7°C to 23°C in summer
(Australian Bureau of Meteorology).
Animals and radiotelemetry.—Over the duration of this study
(2008–2010), 14 reproductively mature males were monitored,
9 of these for more than 1 year, resulting in the accumulation of 28 individual years of data (breeding and nonbreeding
years). As not all males in the population breed in a given year,
data collected during nonbreeding years have been excluded
from this study. All echidnas were implanted with a passive
implantable transponder (PIT tags; LifeChip, Destron-Fearing,
St. Paul, Minnesota) on their right ventral side under light isoflurane anesthesia and for convenience are referred to by the
last 4 digits of their tag number. Transmitters (Bio Telemetry
Tracking, St. Agnes, Australia) were attached between spines
on the animal’s lower back using 2-component epoxy glue.
A small external temperature logger (iButton, DS1922L,
Maxim Integrated Products Inc., Sunnyvale, California, resolution 0.5°C) was attached to the transmitter. These loggers
record temperature at 1-h intervals and could be downloaded
in the field. When the echidna is hibernating, the logger falls
to substrate temperature: the record is clearly distinguishable
from that of an active animal, allowing determination of the
date and time of entry into and arousal from hibernation (Nicol
and Andersen 2008; Morrow and Nicol 2009).
This work was carried out under a permit from the Tasmanian
Department of Primary Industries, Water and Environment, and
the University of Tasmania Animal Ethics Committee; complies with the Tasmanian and the Australian Code of Practice
for the Care and Use of Animals for Scientific Purposes (2004);
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and meets the guidelines approved by the American Society of
Mammalogists (Sikes et al. 2011).
Blood samples.—Blood samples (approximately 1 ml) were
taken from the rostral sinus using a 21G needle and a plastic
syringe, while the animal was under light isoflurane anesthesia. All samples were collected within an hour of animal capture and were stored on ice until centrifuged in the laboratory
at 13,000 rpm for 10 min to separate plasma. Plasma was then
stored at −20°C until analysis. Plasma testosterone was measured via radioimmunoassay (RIA) as described in Nicol et al.
(2005). The sensitivity of the assay was 6 pg authentic testosterone (~0.15 ng ml−1 plasma). Assay accuracy and precision
were monitored by including 3 levels of commercially available
human control serum (CON 6, Diagnostic Products Corporation,
Los Angeles, California) in each assay. All samples collected
from the same individual were analyzed in a single assay.
Ultrasonography.—Ultrasonography was performed in
the field using an ESAOTE (Caris Plus; Esaote Biomedica,
München, Germany) ultrasound unit with a LA523 linear probe
(13–4 MHz), while the echidna was under light isoflurane anesthesia. The animal was placed in dorsal recumbency and clippers were used to remove hair from the cloaca to the lower
chest and the inside of the hind legs from the spur to the point
where the leg joins the body. Conductance gel was then applied
directly onto the animal.
The dimensions of the testes and the crural glands were measured by capturing images when the organs appeared at their
maximum size on the screen and using the calliper system integrated into the ultrasound machine. Each testis and crural gland
was measured twice, with 2 different images used to measure
dimensions. The volume of each testis or crural gland was calculated according to the volume of a simple rotation ellipsoid
accommodating the symmetrical shapes of the organs (as seen
in Goeritz et al. 2003; Hermes et al. 2005). Total testes and
crural gland volumes were calculated by adding the average left
and right dimensions together.
During the postbreeding phase, when males were feeding
most heavily, it was not always possible to visualize the right
testis using ultrasonography due to interference from ingestafilled intestinal loops. As there was no significant difference in
volume between the right and left testis (t108 = 1.37, P = 0.17),
total testes volume was calculated by doubling the left testis
value in cases when the right testis was not visible.
Testis density.—In order to calculate testes mass from the
measured volume, we measured testes density using autopsied specimens collected opportunistically through the study:
5 were roadkill echidnas, and 2 were echidnas that died from
natural causes at our study site. Only males with adult male
spurs were included in the analysis. Density was calculated (as
mass/volume) by removing the testes and measuring their mass
and volume.
Frequency of sample collection.—Samples were collected
throughout the year from males that were being radiotracked.
Blood samples were collected between 2008 and 2010; once the
machine became available, ultrasound images were taken from
June 2009 until December 2010. Body mass was measured using
portable weighing scales (OHAUS Navigator XT NVT10001,
Ohaus Corporation, Port Melbourne, Australia, resolution < 1 g)
each time an animal was handled. We aimed to collect at least
1 set of samples (blood sample and ultrasound data) per month
from all males fitted with radiotransmitters, but it was not always
possible to collect and sample all individuals in each month.
Opportunistic samples were collected from males not fitted with
radiotransmitters that were found in mating groups. Only males
with 2 or more samples collected within a year were included
in the analyses. If a male either lost his transmitter or his transmitter failed and that male was not subsequently observed in a
mating group, samples collected from that individual were discarded because we could not determine whether or not that animal mated in that year. If more than 1 blood sample or ultrasound
was collected from an individual in any 1 month, mean monthly
values for each variable were used for analysis.
Cameras.—Infrared motion-triggered cameras (Scoutguard
SG550, HuntingCamOnline, Gadsden, South Carolina) were
set up at different angles around mating groups to record mating behavior. Colored tubing pushed over spines allows accurate identification of animals on camera footage, at night (on
infrared camera footage), the location of the tags and radiotransmitters allowed individuals to be identified.
Statistical analysis.—Differences in testes and crural gland
volume, plasma testosterone concentrations, and body mass
between months were analyzed using linear mixed effects
models (Pinheiro and Bates 2000) using the “nlme” package
(Pinheiro et al. 2013) in R (Team R. C. 2012). Response variables were log transformed to improve normality when required
(plasma testosterone and crural gland volume only), and the
modeling procedure followed Zuur et al. (2009) using likelihood
ratio tests and inspection of residuals and diagnostics. Initially, a
full model with random effects for animal ID, month and year,
and fixed effects for month and year was fitted, but only random
effects for animal ID were retained (all P for “year” and “month”
random effects > 0.10). Models were refitted using Maximum
Likelihood to simplify the main effects (Zuur et al. 2009), and
“Year” was eliminated from all models (all P > 0.10). The final
models included month as a fixed effect and animal ID as a random effect. We used restricted (or residual, or reduced) maximum likelihood with our linear mixed effects models to compare
values between months. The significance level was P < 0.05,
N = number of samples, and n = number of animals.
Results
Over the duration of this study, we collected a total of 196
blood samples from males during their mating years, which
yielded 104 monthly testosterone values from 13 individuals.
Some individuals were sampled during more than 1 year, giving a total of 22 breeding years of data from those 13 males.
A total of 75 monthly testes volumes and 70 monthly crural
gland volumes were collected via ultrasonography from 10
individual males. Ultrasounds were performed on several individuals for multiple years, resulting in 15 breeding years of
ultrasound data.
Frequency of breeding.—All monitored males mated during
at least 1 year of the study (2008–2010) except for 1 individual
MORROW ET AL.—HIBERNATION AND REPRODUCTION IN ECHIDNAS855
with no right testes: with the majority of males (75%) mating
during at least 2 breeding seasons. A total of 9 males were monitored for consecutive breeding seasons: 1 male mated every
year of the 3-year period. Due to transmitter loss or failure,
other males monitored over the duration of this study have
some missing data and we do not always know if an individual
male participated in mating groups.
Testosterone.—Plasma testosterone concentrations from the
13 males sampled during their breeding years varied significantly throughout the year. Plasma testosterone concentrations
were significantly higher in June than in May (P < 0.001), with
testosterone concentrations increasing after males terminated
hibernation in May. Plasma testosterone concentrations peaked
in June (0.99 ± 0.15 ng ml−1, SEM, n = 8) just prior to the breeding season and decreased progressively throughout the breeding season (Fig. 1). Plasma testosterone decreased significantly
from June to July (P < 0.05), from July to August (P < 0.001),
and from August to September (P < 0.001). Mean plasma testosterone concentrations were the lowest in March (0.13 ± 0.014 ng
ml−1, SEM, n = 4). The highest plasma testosterone concentration measured was 3.30 ng ml−1 from an individual sampled 4 h
after he located a female within her hibernaculum: a camera
trap setup over that female demonstrated that mating had not
yet occurred.
Plasma testosterone was low at the end of hibernation but
increased after arousal, reaching peak levels after approximately 30 days of euthermia (see Fig. 2). The mean interval
from a male terminating hibernation to being observed in a
mating group was 34 ± 6.5 days (SD, range 32–44 days, n = 5).
Testes.—Mean total testes volume varied significantly with
month. Testes volume during the period February to August
was significantly larger than testes volume in January (P
<0.05). Testes recrudescence was initiated in December after
the summer solstice, with testes volume increasing throughout
January and February. Testes volume was significantly larger
in February than January (P < 0.01). Mean hibernation entry
date was 17 February ± 3.6 days (n = 14—Nicol and Morrow
2012), and testes were at 75% of peak volume in March shortly
after males entered hibernation. Total testes volume did not
change significantly during hibernation and testes volume during hibernation was not significantly different from peak testes volume. Total testes volume peaked in June (19.0 ± 2.2 cm3,
SEM, n = 6) just prior to the breeding season and decreased
throughout the breeding season until the testes reached minimum size in December (2.05 ± 0.41 cm3, SEM, n = 7). Testes
volume decreased significantly between July and August (P
< 0.01), August and September (P < 0.0001), and September
and October (P < 0.01). Figure 3 shows ultrasound images of
the left testis from 1 individual during the breeding season (in
which he mated) and in December, prior to the initiation of
recrudescence.
Testis density varied significantly throughout the year.
Testis density was the greatest during the breeding season
(1.73 ± 0.32 g cm3) and the lowest (1.15 ± 0.02 g cm3) during the
postbreeding period, when the testes were also at their smallest volume. Testis density increased after recrudescence was
Fig. 1.—Seasonal changes in combined testes volume (a), crural gland
volume (b), body mass (c), and plasma testosterone (d) in breeding
male echidnas. The gray background shows the hibernation period,
while the shaded area shows the breeding period. Error bars are SEs
calculated using a linear mixed effects model for month and animal
ID. Body mass is represented as % of adult lifetime average mass.
initiated in late December so that testes density during hibernation (1.42 ± 0.62 g cm3) was greater than during the postbreeding period.
Total testes mass peaked at 0.84 ± 0.05% of body mass during
June and July and was the lowest in November and December
(0.06 ± 0.04%). Five males sampled between June and August
had testes mass greater than 1% of their body mass; in 1 individual sampled in July, the testes reached 1.16% of body mass.
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Testes mass was the lowest in October and December (0.02%
of body mass).
Crural glands.—Crural gland volume varied significantly
with month. Recrudescence did not occur until after the termination of hibernation. There was a significant increase in crural
gland volume between May and June (P < 0.01), with crural
gland volume increasing after males terminated hibernation
in May. Crural gland volume peaked in July (3.42 ± 0.33 cm3,
SEM, n = 13) during the breeding season and decreased as the
Fig. 2.—Plasma testosterone concentration (ng ml−1) plotted against
the number of days since the final termination of hibernation (represented by a gray vertical line) for 5 breeding males (N = 90). When
males terminated hibernation, testosterone concentrations were low,
but increased with increasing days of euthermic Tb.
breeding season continued (see Fig. 1). There was a significant
decrease in crural gland volume between August and September
(P < 0.001). Crural glands were enlarged only during the breeding season (June–August) and were at their smallest during the
period October to May. There was a significant positive relationship between crural gland volume (y) and plasma testosterone concentration (y = 1.46 + 1.05 x, r2 = 0.179, P < 0.0005,
where x is plasma testosterone concentration). Figure 3 shows
ultrasound images of crural glands from 1 individual during the
breeding and nonbreeding periods.
Body mass.—To assess seasonal changes in body mass, lifetime average adult mass for each individual male was calculated
from data collected between 1996 and 2012, and body mass
then expressed as a percentage of his long-term mean (Nicol
and Morrow 2012; Sprent et al. 2012). The smallest male used
in the study had a long-term mean mass of 3.52 ± 0.42 kg (range
2.97–4.4, n = 16) over 16 years, and the largest 4.28 ± 0.38 kg
(range 3.14–4.84, n = 51) over the same time. During the study
period, there was a significant annual variation in body mass.
Body mass in May, July, August, and September was significantly less than in January (P < 0.05). Males reached maximum body mass in December (108.7 ± 1.99% of adult average
mean mass, SEM, n = 4). Body mass decreased significantly
between February and March (P < 0.05) and continued to
decrease throughout the hibernation period. At the end of
hibernation (May), males were below their average adult body
mass (95.0 ± 0.40% of adult mean mass, SEM, n = 4). Body
mass increased to their adult average in June (see Fig. 1), but
males lost weight during the breeding season, with body mass
Fig. 3.—Ultrasound images of left testis in December prior to testes recrudescence being initiated (a), left testis in June at the beginning of the
breeding season (b), right crural gland in December (c), and right crural gland in July (d) from individual 7A59. All images were taken at the same
scale, and white crosses indicate linear measurements used to calculate volume.
MORROW ET AL.—HIBERNATION AND REPRODUCTION IN ECHIDNAS857
decreasing significantly between June and July (P < 0.02).
Males were at minimum body mass (92.7 ± 2.3% of adult mean
mass, SEM, n = 12) in August at the end of the breeding season.
At the termination of the breeding season, males began to forage extensively so body mass increased significantly between
September and October (P < 0.01) and October and November
(P = 0.05).
Discussion
Although involution of the gonads is often considered a prerequisite for mammals to enter hibernation (Hoffman 1964;
Darrow et al. 1988), in Tasmanian echidnas, testes recrudescence begins in late December, after the summer solstice, so that
males enter hibernation in February with testes that are already
75% of peak testes volume. This unusual pattern of testes
growth beginning prior to the hibernation period can be related
to the echidna diet and resulting physiological constraints on
life history, as well as the mating system. The diet of Tasmanian
echidnas is primarily ants (Sprent 2012), a food with relatively
low nutritional content (Redford and Dorea 1984) and the
energy density is reduced further by the ingestion of soil. The
low energy diet means that echidnas must maximize energy
savings and in Tasmania hibernation seems to be an essential
part of this strategy (Nicol and Andersen 2002; Schmid et al.
2003; Grigg et al. 2004; Nicol and Andersen 2007a). Because
mating occurs in mid-winter, hibernation must begin very early,
and males enter hibernation in late summer by seeking out cool
hibernacula (Nicol and Andersen 2007b).
Throughout their range, echidnas mate in winter, with breeding beginning later in more northerly populations (Morrow
et al. 2009). Although the gestation and incubation periods are
very short, the lactation period is very long. Echidnas have a
very low metabolic rate, limiting the maximal lactation rate,
and mid-winter mating ensures that peak lactation and thus
maximum growth of the young occurs at the time of the greatest ecosystem productivity—late spring to early summer, and
that young are weaned before the next winter (Morrow and
Nicol 2012; Nicol and Morrow 2012).
In our study population, there is a highly skewed operational
sex ratio (OSR): the ratio of the number of sexually receptive
females to sexually active males, resulting from females breeding less frequently than males (Nicol and Morrow 2012). In
any given breeding season, the OSR is 1.55 males: 1 female,
and this skewed OSR results in intense intermale competition
for access to receptive females. Females mate with a number
of males in a breeding season (Morrow 2013) and consistent
with a mating system where both sexes are promiscuous and
with intense sperm competition, male echidnas have large testes relative to body size. Relative testes size provides a useful measure of the strength of sperm competition in a species
(Simmons and Fitzpatrick 2012). In all amniotes, there is negative allometric relationship between body mass and relative
investment in testes mass (MacLeod and MacLeod 2009), so
comparisons are best made between animals of similar size.
In male echidnas with a body mass of about 4 kg, testes mass
during the breeding season averaged 0.84% of body mass, considerably larger than the value of 0.34% predicted for eutherian
mammals of the same mass using the allometric relationship
found by Kenagy and Trombulak (1986), and much larger than
the value of 0.18% they measured in 4 kg woodchucks, which
are comparable sized eutherian hibernators whose testes recrudesce after the emergence from hibernation in parallel with testosterone production (Baldwin et al. 1985a). As competition by
echidna males for females is intense, it is important that males
emerge from hibernation in a reproductive state that allows
them to mate as soon as possible.
If testes recrudescence occurred following hibernation, male
echidnas would have to enter hibernation even earlier, which
would not be possible, as echidnas require a substrate temperature < 16°C to enter hibernation (Nicol and Andersen 2007b),
or mating would have to occur 2 to 3 months later—the period
needed for 75% testes growth before entry into hibernation.
Later mating would delay the weaning of young by a similar
amount unless the lactation period was reduced, which may not
be possible, as the lactation period in Tasmania is only 125–145
days, compared with 150–165 days in southeast Queensland,
and 200–212 days on Kangaroo Island (Morrow et al. 2009;
Morrow and Nicol 2012). By initiating testes recrudescence
prior to entering hibernation, males maximize hibernation time
and are able to mate shortly after the emergence.
The high costs of spermatogenesis mean that maintaining
mature testes is energetically expensive (Wedell et al. 2002)
hence the majority of seasonally breeding species undergo
40–90% atrophy in testis mass after the breeding period. Testes
growth at the start of the breeding period and involution afterward appears to be less energetically expensive than the maintenance of mature size testes (Kenagy and Trombulak 1986).
For species with large testes relative to body mass, and a high
degree of sperm competition (Wedell et al. 2002), the costs
must be very high. In our echidna population, mean testes volume was 0.84% of body mass during the breeding season, with
some males having testes greater than 1% of their body mass
during the breeding season, and testicular atrophy postbreeding
in echidnas must result in significant energy savings.
Unlike the vast majority of seasonally breeding mammals,
in the Tasmanian echidna, testes recrudescence occurs before
entry into hibernation and at basal levels of testosterone.
Testes recrudescence prior to entering hibernation has been
documented in reptiles such as the horned lizard Phrynosoma
cornutum, P. douglassi, P. modestum (Howard 1974) and the
sagebrush lizard Sceloporus graciosus (Goldberg 1975), but is
highly unusual for a mammal. Although it had been reported in
the golden hamster Mesocricetus auratus (Smit-Vis and Smit
1970) and the Arctic ground squirrel Urocitellus parryii (Hock
1960), later studies on both species found that testes recrudescence only occurred after the hibernation was completed (SmitVis 1972; Boonstra et al. 2001). Small amounts of testicular
growth and testes recrudescence during periodic arousals in the
latter stages of hibernation have been reported in golden-mantled ground squirrels (Callospermophilus lateralis—Barnes
et al. 1986), woodchucks (Marmota monax—Baldwin et al.
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1985b), European hedgehogs (Erinaceus europaeus—Saboureau and Dutourné 1981; Dutourné and Saboureau 1983),
and black bears (Ursus americanus—Erickson et al. 1964 cited
in McMillin et al. 1976). Why is the echidna the only mammalian hibernator that initiates testes recrudescence prior to entering hibernation? Echidnas have testes that are approximately 5
times the size of those of a comparable sized eutherian hibernator, the woodchuck (Kenagy and Trombulak 1986), but have
a much lower metabolic rate, and hence the time required for
testicular recrudescence is substantial, 2–3 months. The minimal costs of maintaining large testes during hibernation, when
whole body metabolic rate is reduced to about 16% of resting metabolic rate (Nicol and Andersen 1993), is clearly outweighed by the advantage of being able to breed in mid-winter.
In the Tasmanian echidna, recrudescence is initiated after
the summer solstice: presumably the long photoperiod stimulates the release of gonadotropin-releasing hormone (GnRH)
from the hypothalamus and stimulates the secretion of folliclestimulating hormone (FSH). While testosterone is required to
maintain germ cells within the testis and for spermatid maturation (Young and Nelson 2001), it is not required for the initiation of recrudescence in the echidna as testes volume and
density increased prior to plasma testosterone concentrations
increasing. Hence seasonal increases in testes volume and
plasma testosterone concentrations are not synchronized in the
Tasmanian echidna. A similar pattern of testes recrudescence
in the absence of an increase in plasma testosterone concentration is seen in the Djungarian hamster (Phodopus sungorus—
Schlatt et al. 1995). Spermatogenesis and spermatid maturation
is reliant on testosterone (McLachlan et al. 1996), and therefore it is likely that spermatogenesis is not initiated until male
echidnas resume euthermic Tb as plasma testosterone concentrations only increased after males terminated hibernation.
However, Griffiths (1978) found echidnas in the southeast of
mainland Australia with large testes exhibiting spermiogenesis
in April and May. Therefore, echidnas may not be reliant on
increased concentrations of plasma testosterone for the early
stages of spermatogenesis. It is possible that the echidnas sampled by Griffiths (1978) were from a region where echidnas
do not enter deep hibernation, as testes size did not increase
until April. Whether spermatogenesis is initiated prior to males
entering hibernation in our study population cannot be determined without the use of invasive techniques, although the
increase in testes density prior to entering hibernation observed
in our study suggests an increase in seminiferous tubule diameter and the maturation of Sertoli cells (Muñoz et al. 2001).
The pattern of testes recrudescence occurring in late
December after the summer solstice in Tasmanian echidnas is
in sharp contrast to the study conducted on captive echidnas
from Queensland in which testes volume only began to increase
in late autumn (Johnston et al. 2007) but these animals did not
hibernate in captivity (S. Johnston, University of Queensland,
School of Agriculture and Food Sciences, pers. comm.), and in
the wild not all echidnas from warm coastal regions hibernate or
enter torpor prior to the reproductive period (Grigg et al. 2004).
In subspecies that do not show hibernation or torpor prior to the
breeding season, it is likely that testes recrudescence is initiated
in April–May (late autumn) so that males are in breeding condition in June as reported by Griffiths (1978). Initiation of testes
recrudescence in late autumn has also been reported in echidnas
from coastal Victoria (M. Augee, pers. comm.). In contrast, in
our population of echidnas in Tasmania, testes are enlarged for
6 months of the year but for 3 of these months males are in
deep hibernation when metabolic rates of all tissues are reduced.
Variations in the timing of testes recrudescence in echidnas from
different geographical regions demonstrate the adaptive plasticity of the annual life cycle in this widely distributed species.
Although echidna crural glands have been described as vestigial (Augee et al. 2006), we have demonstrated that the echidna
crural glands exhibit a distinct circannual pattern of recrudescence and involution consistent with the observations of Krause
on Victorian echidnas (Krause 2010). Recrudescence of crural
glands in Tasmanian echidnas occurs after males emerge from
hibernation, and they are at maximal size during the breeding
season, when plasma testosterone is the highest. A similar correlation between testosterone and crural gland size has been
demonstrated in the platypus (Ornithorhynchus anatinus—
Temple-Smith 1973; Grant and Temple-Smith 1998). The function of crural glands in the echidna remains unknown, but we
suggest that they may have a role in chemical communication,
which is known to be very important in echidnas (Harris et al.
2014). Camera trap footage of echidnas in mating groups shows
that males spend a considerable amount of time grooming themselves (using their grooming claws on their hind legs), and it is
therefore possible that males are spreading crural gland secretions on themselves, and possibly females, as a form of communication. The spurs of both male platypus and echidnas have
a small diameter central canal that opens on the side of the tip
of the spur. This canal connects to a duct that leads to the crural
gland (Krause 2010). Because crural glands only become active
during the breeding season, the presence or absence of fluid
within the male spur is a clear indicator of reproductive activity.
The Tasmanian echidna is an atypical hibernator, utilizing
the unusual strategy of initiating testes recrudescence prior to
entering hibernation to optimize reproduction around an obligatory hibernation period. This strategy can be linked to the low
metabolic rate, the low energy diet, and a requirement to hibernate to maximize energy savings and to the large relative size
of echidna testes associated with intense levels of intermale
competition. By initiating testes recrudescence prior to entering
hibernation, males maximize hibernation time while ensuring
that they emerge from hibernation in a state that allows them to
mate within 30 days.
Acknowledgments
We thank R. Harris for all her assistance in the field, and S.
Muir for teaching GEM to ultrasound, coming out in the field
and for assistance in converting ultrasound files. Thanks to L.
Barmuta for his statistical advice and to S. Troy for running the
linear mixed effects models. We also thank the McShane family
for allowing us to access their property. This work was carried
MORROW ET AL.—HIBERNATION AND REPRODUCTION IN ECHIDNAS859
out under permit FA08053, FA09048, and FA10045 issued in
accordance with Section 29 of the Nature Conservation Act
2002. This work was supported by grants from: The University
of Tasmania Institutional Research Grants Scheme (grant
N0016573; http://www.research.utas.edu.au/); The National
Geographic Committee for Research and Exploration (grant
49286; http://www.nationalgeographic.com/field/grants-programs/cre.html); Holsworth Wildlife Research Endowment
(grant M0017093; http://www.anz.com/documents/au/aboutanz/HolsworthWildlifeGuidelines2007.pdf);
Australian
Geographic (grant N0017888; http://www.australiangeographic.
com.au/society/sponsorship/2013/11/apply-for-sponsorship).
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Submitted 4 May 2015. Accepted 17 January 2016.
Associate Editor was Chris R. Pavey.