Non-invasive collection and analysis of
semen in wild macaques
Ruth Thomsen
Primates
ISSN 0032-8332
Primates
DOI 10.1007/s10329-013-0393-z
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Primates
DOI 10.1007/s10329-013-0393-z
ORIGINAL ARTICLE
Non-invasive collection and analysis of semen in wild macaques
Ruth Thomsen
Received: 21 February 2013 / Accepted: 16 October 2013
Ó Japan Monkey Centre and Springer Japan 2013
Abstract Assessments of primate male fertility via
semen analyses are so far restricted to captivity. This study
describes a non-invasive method to collect and analyse
semen in wild primates, based on fieldwork with Yakushima macaques (Macaca fuscata yakui). Over nine mating
seasons between 1993 and 2010, 128 masturbatory ejaculations were recorded in 21 males of 5 study troops, and in
11 non-troop males. In 55 %, ejaculate volume was
directly estimated, and in 37 %, pH-value, sperm vitality,
numbers, morphology and swimming velocity could also
be determined. This approach of assessing semen production rates and individual male fertility can be applied to
other primate taxa, in particular to largely terrestrial populations where males masturbate frequently, such as
macaques and baboons. Furthermore, since explanations of
male reproductive skew in non-human primate populations
have until now ignored the potential role of semen quality,
the method presented here will also help to answer this
question.
Keywords Non-invasive semen collection Fertility analysis Masturbation Macaques Macaca
fuscata yakui Yakushima
R. Thomsen (&)
Institute of Biology, University of Leipzig, Talstrasse 33,
04103 Leipzig, Germany
e-mail: rthomsen@uni-leipzig.de; ruth.thomsen@ucl.ac.uk
R. Thomsen
Department of Primatology, Max Planck Institute for
Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig,
Germany
Introduction
Semen parameters of wild primate males are still poorly
researched, although comparative studies of semen quality
in primates started almost two decades ago (Møller 1988).
In wild non-primate mammals, semen quality can differ
between individuals and populations (e.g., red deer, Cervus
elaphus hispanicus: Malo et al. 2005; Namibian cheetah,
Acinonyx jubatus: Crosier et al. 2007), thus rendering
sperm an important source to assess male fertility.
The lack of semen analysis in primate field research may
be due to two reasons. First, traditional studies on the
reproductive physiology of primate males concentrated on
comparison of testicle sizes at the inter- and intra-specific
level, assuming that larger testes produce larger volumes of
semen, and that large volumes of semen are the crucial
factor during sperm competition (Harcourt et al. 1981;
Short 1981; Bercovitch and Rodriguez 1993). These results
are still relevant and have generated insights into the
evolution of male reproductive strategies in different social
systems. The semen itself, however, was not studied,
although it is apparently the parameter containing the most
valuable information concerning male fertility. During the
next two decades, knowledge regarding the sophisticated
biology of sperm cells has accumulated rapidly (reviewed
in Birkhead and Montgomerie 2009). For instance, a large
midpiece is suggested to be beneficial for sperm motility
(Anderson and Dixson 2002) and in primate species where
females mate with multiple males in short succession, such
as chimpanzees (Pan troglodytes) and rhesus macaques
(M. mulatta), sperm cells have evolved to swim much
faster than in less promiscuous species such as gorillas
(Gorilla gorilla) and humans (Nascimento et al. 2008).
Secondly, field primatologists often need to rely on noninvasive sampling methods. Captive primates are
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anaesthesized and semen is sampled by electro-ejaculation
or direct penile electro-stimulation (e.g., Platz et al. 1980;
van de Voort 2004). Both methods require capture and
immobilisation of the target animal and these methods are
often unsuitable under field conditions since the trapping of
a single monkey also stresses group members. Subsequent
fertility analysis was also presumed to be restricted to the
use of laboratory facilities that, for instance, contain an
incubator to release sperm from the plug part of the semen
(van Pelt and Keyser 1970; Matsubayashi 1982; Schaffer
et al. 1992; Seier et al. 1996; Gago et al. 1999; Kuederling
et al. 2000; Dixson 2012). Given the stressful and invasive
character of electro-ejaculation and the putative dependence on laboratory facilities, semen analysis has never
been considered viable in wild primates.
However, during the last decade, it has become
increasingly known that primate males of a considerable
variety of species masturbate as a common component of
their sexual behaviour. Particularly in macaques, mangabeys, baboons and guenons, male masturbation is, beside
heterosexual copulations, probably the most frequently
observed sexual behaviour (Thomsen et al. 2003; Frearson
2005; Dixson 2012). This offers the possibility of estimating semen volumes by direct observation (Thomsen
and Soltis 2004; Inoue 2012) and to determine male fertility under field conditions. Non-invasively collected
semen has also proven to be a good source for energetic
studies (Thomsen et al. 2006), the scanning of viruses
including HIV/SIV types (Dejucq-Rainsford and Jégou
2004), and for DNA paternity analyses (Soltis et al. 2001;
Hayakawa 2008). However, fresh semen originating from
masturbation is likely to be most appropriate to assess male
fertility and the fertility of populations.
Here, a method developed for non-invasive semen collection and semen analysis in wild macaques is presented
and its potential for future studies in field primatology is
discussed.
2007), and frequent male masturbation behaviour (Thomsen and Soltis 2004). In Japanese macaques semen production starts at the age of 4 years and ceases at
approximately 20 years of age (own unpublished data).
Spermatogenesis is restricted to the breeding season
between August/September and January, with a peak in
October/November and ceases completely during the nonbreeding season (Thomsen and Soltis 2004). In the genus
Macaca, residual sperm from the previous breeding season
that have been retained for months in the cauda epididymis
and vas deferens are non-motile and structurally abnormal
(M. mulatta: Zamboni et al. 1974; M. fuscata: personal
observations). Therefore, only semen samples from the
peak of the breeding season were used in this study.
Study troops
Data were collected between 1993 and 2010 from males
residing in four well-habituated troops (B, KZ, NINA-A, S)
and one partially-habituated troop (H), as well as from
several non-troop males. The four former troops ranged in
coastal subtropical rainforest at the Hanyama study site on
the western coast of the island (0–350 m a.s.l., Maruhashi
1980; Soltis et al. 2001), whereas H-troop ranged in the
Ooko area in a deciduous forest at 900–1100 m a.s.l.
(Thomsen 1996).
Troop males (TM) and non-troop males (NTM)
Methods
The Yakushima population is characterized by a large
number of NTMs that can comprise up to 50 % of all males
in the population during the breeding season (Sprague 1991;
Hayakawa 2007). In contrast to TMs, NTMs are less- or unhabituated to human observers. They appear on the edges of
bisexual troops, trying to entice oestrous females to follow
them or taking part in sneaky copulations. NTMs achieve
40 % of all copulations observed during daytime (Sprague
et al. 1998) and successfully reproduce with this strategy
(Soltis et al. 2001; Hayakawa 2008). Since both male types
were observed to masturbate, data from both are included.
Study site and species
Male age
The study took place on Yakushima Island (30°N, 130°E),
the site of several long-term projects on macaque socioecology (Maruhashi 1980; Yamagiwa 2010). The Yakushima macaque is a seasonally breeding subspecies of the
Japanese macaque (M. f. fuscata) endemic to the island,
which lies at the southern limit of the species’ distribution
(Yamagiwa 2008; Nakagawa et al. 2010). The population
is characterized by a polygynandrous mating system, high
degrees of female and male promiscuity, severe sperm
competition (Sprague 1991; Soltis et al. 2001; Hayakawa
The age of the 32 males studied ranged between 5 and 16
years (mean ± SD = 9.1 ± 3.6). Age was estimated using
morphological criteria (e.g., body size, face colour, coat
thickness, scars) validated during over 50 years of studies
on Japanese macaques (Soltis et al. 2001; Yamagiwa 2010).
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Ambient temperature
Whenever masturbation with ejaculation was observed, air
and ground temperatures were measured in °C (infrared
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Fig. 1 a Masturbation by a troop male during daytime. b Masturbation at night by two non-troop males. c Fresh ejaculate on the forest floor. The
discernible white parts contain the plug, while the transparent fluid has seeped into the leaf litter
thermometer; Voltcraft IR-280) to enable proper treatment
and storage of the semen sample (see below).
Estimating semen volume
Macaque males masturbate with their hands (Fig. 1a and b;
Thomsen et al. 2003; Thomsen and Soltis 2004; Inoue
2012). Whenever males were seen to masturbate sitting on
the ground (earth, rocks, tarmac, etc.) volumes of the
ejaculated semen were estimated (Fig. 1c). Since volumes
are likely to be reduced by consecutive ejaculations
(chimpanzees: Marson et al. 1989), only data from observation days when no oestrous female with swollen perineum (see for details: Soltis et al. 2001) had been seen in or
near the troop were used. Semen volume was visually
estimated as well as collected from the ground (see below).
Estimated volumes were noted directly after ejaculation
and included both the liquid and the solid plug portions
(judged in intervals of 0.1 ml, from 0.1 to 5.0 ml). Volumes
\0.1 ml were not utilised, due to the imprecision inherent
in visual estimates.
Semen collection
Masturbating males that had ejaculated were closely
approached and displaced by the researcher to prevent them
from feeding on the specimen. The fluid portion of macaque
semen consists of two parts: the dropped-down fluid and the
fluid that is stored inside the plug. The dropped-down fluid
of the ejaculate was collected using a volumetric pipette
(Eppendorf Research plus 200 lm, Sarstedt pipette tips)
and stored in Eppendorf tubes until analysis. The plug
(Fig. 1c) was collected with tweezers and stored in a plastic
Ziplock bag (10 9 15 cm). To squeeze the remaining fluid
out of the plug without laboratory facilities such as an
incubator (e.g., Kuederling et al. 2000), the plug was
carefully pressed inside the bag until its fluid was released.
To quantify the volume of the squeezed plug it was placed
in a tube filled with M199 (Sigma Corp. M 7528), a medium
which allows sperm cells to survive for a further 5–8 h.
Then the volume of M199 displaced was measured to define
the plug’s volume. If the fluid that was released from the
plug was\1.0 ml it was carefully diluted drop by drop in a
ratio of 1:2 with M199 of appropriate temperature to prolong the survival of sperm cells.
Temperature and sample storage
During peak breeding season, daytime air temperatures in
Hanyama ranged between 20 and 25 °C, and 4 and 18 °C
in Ooko. Night temperatures ranged between 10 and 18 °C
in Hanyama and 5 and 15 °C in Ooko (Japan Meteorological Agency 1971-2012; own measurements). Temperature of the ground where samples dropped varied with
surface. The tarmac road, for instance, rose to 45 °C or
more at noon, a temperature at which macaques’ sperm
cells instantaneously stop progressive movements (own
data). During night hours, the road and rocks were typically
between 30 and 35 °C. As semen is approximately 32 and
35 °C, sample temperatures were either reduced by dilution
with cooled M199 or kept in a pocket close to the observer’s body to keep the specimen warm.
Analysis of semen samples
The pH-value, sperm motility, sperm swimming velocity,
sperm morphology and number of sperm per semen sample
were assessed. Common protocols for semen analysis
collected by electro-ejaculation established for laboratory
macaques were adapted to field conditions and supplemented with protocols from small-sized mammals such as
common marmosets (Callithrix jacchus) and domestic cats
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(Felis catus), species producing semen volumes of only
0.01–1.0 ml.
Table 1 Possible morphological defects in primate sperm cells
(World Health Organisation 1999; combined with own protocols)
pH-value
Primary
defects
Secondary
defects
Handling
defects
Defects of
unknown origin
Macrocephalic
Cytoplasmic
droplet
Detached tail
Detached
acrosome
Microcephalic
Bent midpiece
Detached
midpiece
Malformed head
Double-head
Bent tail
Broken tail
Detached head
The pH-value of the seminal fluid was measured using
indicator paper (range 6.4–8.0) for human semen (Merck
KgaA, Germany).
Sperm swimming velocity
Coiled tail
Double tail
To analyse sperm swimming velocity in the field, a
microscope (1009, 4009, 12509 incl. oil inversion) was
carried in the backpack while following the macaques. The
swimming speed of 100 sperm cells was assigned to one of
three categories: 2 = rapidly progressive, 1 = progressive
and 0 = non-progressive (adapted from WHO 1999).
Vitality
If a sample contained more than 60 % non-progressive
sperm, an Eosin (1 %) staining test was performed. One
hundred sperm cells were classified as either alive or dead
by distinguishing between non-progressive and dead
(stained red) sperm cells.
Sperm morphology
To examine sperm morphology, two smears of each sample
were stained. A dilution of 0.1 ml fluid and H20dest. (ratio
1:1) was smeared on pre-stained slides used for human
haematology (Testsimplets, Boehringer Ingelheim Corp.,
Germany). One hundred sperm cells per sample were
examined under light microscopy (91250, oil inversion)
and classified into normally or abnormally shaped forms.
Abnormal forms were further divided into four categories
of potential defects (Table 1).
Concentration of sperm cells
Sperm concentration (mio/ml) was determined by diluting
0.01 ml of the sample with 5 % saline solution (ratio 1:20),
which stops sperm movements. Sperm numbers per 1.0 ml
were calculated with a Makler cell count chamber (SefiMedical Instrument, Haifa, Israel).
Results
Visual estimates of semen volumes
In total, 21 TMs and 11 NTMs were observed to masturbate and produced 128 ejaculations, with each male
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Table 2 Semen analysis in wild Yakushima macaques, based on
ejaculates of 32 males of 5 troops (H, S, B, NINA-A, KZ) collected
during 9 breeding seasons between 1993 and 2010
Parameter
Mean ± SD
Range
N
Estimated volume (ml) after
masturbation
2.2 ± 1.2
0.1–5.0
71
Volume of collected samples
(ml)
1.5 ± 0.7
0.5–3.2
48
pH
7.1 ± 0.3
6.4–7.9
48
529.4 ± 265.4
12.5–990
48
48
6
Sperm concentration (910 /ml)
% Viable spermatozoa
59.6 ± 30.5
0–100
% Normal spermatozoa
94.4 ± 5.2
61–100
Viable spermatozoa per
ejaculate (9106)
1102.1 ± 1079.8
0–4950
48
48
contributing at least two semen samples. Estimates were
undertaken for 71 (55 %) of 128 cases (Table 2). In the
remaining cases volume could not be estimated due to poor
visibility (dawn, dusk, rainy or cloudy periods, obscuration
by vegetation), or because the male ingested the semen
from its hand.
Semen collection
Forty of the 48 samples (83 %) used for detailed semen
analysis were collected from TMs during the early morning
hours or before noon (Fig. 1a). The remaining eight samples (17 %) were collected during night hours from NTMs
while they were resting in close proximity to sleeping
troops (Fig. 1b). Bare and dry rocks, branches and roots of
large fig trees, leaf litter (including fern) and road tarmac
were the best substrates, while sand, mud, salty rocks at the
shoreline or urine-contaminated areas were unsuitable. The
most important factor affecting collection of live sperm
cells was the temperature of the ground where the sample
dropped. If it exceeded approximately 38 °C, vitality
almost always reached 0 % and thus motility also could no
longer be determined; however, these samples were still
suitable for determining all other sperm parameters
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(Table 2). The best sampling conditions were the early
morning hours when ambient air temperatures were still
cool at 15–20 °C, or during the night when males remained
constantly on the boulders. It was often not easy to displace
a male before he started to feed on the fresh semen, and
success was best with well-habituated and younger males.
Slight signs of anxiety or disturbance, such as a threat face
directed toward the observer, sometimes occurred with
poorly-habituated TMs or non-habituated NTMs.
Semen analysis
Semen analysis was performed no later than 30 min after
ejaculation. The majority (94 %) of sperm cells were
morphologically normal. Defects included abnormalities of
sperm head, midpiece or tail, cytoplasmic droplets located
at the midpiece or tail, microheads (but no macroheads),
and/or (slightly) detached acrosomes. In the samples of 4
males, up to 39 % of all sperm cells were defective (bent
midpieces, bent or broken tails and microheads). In 6 of 48
samples (12.5 %), no progressive sperm movements were
recorded (velocity = 0). In the remaining 42 samples,
sperm movements were classified as progressive [velocity = 1; 14 samples (29.2 %)] or rapidly progressive
[velocity = 2; 28 samples (58.3 %)]. Within the samples,
no differences were detected in the degree of sperm
progressiveness.
Discussion
This study provides pioneering information on the collection and analysis of semen in wild primates. Weather
conditions and the substrate upon which semen fell crucially influenced collection success. The quality and fertilizing potential of semen that came into contact with sand,
mud, or sun-baked rocks might have been underestimated,
because viable sperm cells were probably quickly
destroyed. However, this method is still likely to produce
better results than semen analysis following electro-ejaculation in macaque spp. (e.g., Harrison 1980; Matsubayashi
1982; Sarason et al. 1991; Gago et al. 1999). Compared to
naturally ejaculated semen, electro-ejaculation tends to
flush out unripe sperm with large amounts of cytoplasmatic
droplets (Matsubayashi 1982), personal observation; Morrell et al. 1996. For this study, semen was analysed in situ
in the forests of Yakushima Island and the results provide a
basis for future studies in wild primates. The wild Yakushima population, while of low genetic diversity (Hayaishi
and Kawamoto 2006), seems to possess very good fertility
compared with captive Japanese macaques (Matsubayashi
1982). Sperm concentration and the percentage of normally
shaped sperm cells is remarkably high (Table 2). The lack
of laboratory conditions entails some restrictions (e.g.,
acrosome reaction tests cannot be performed; vitality might
be at times underestimated). Nevertheless, volume estimates via direct observation constitute the perhaps most
promising part of this new method.
Estimating semen volumes in wild primates
In 55 % of observed masturbations, semen volumes could
be estimated. Thus, even when semen cannot be collected,
its volume can be estimated through direct observation.
Human semen volumes likely vary with rates of sexual
intercourse or masturbation, health condition, and time of
day (Cagnacci et al. 1999). Factors that may influence
semen volumes in non-human primates that live in the wild
and breed seasonally are not yet known. Still, the current
method produces better information on male reproductive
performance than measurements of testicular volumes
(Harcourt et al. 1981; Møller 1988). Since it is non-invasive, volumes can be repeatedly assessed and without harm
to the target subjects or group members. Depending on
body size of the species concerned, in smaller species
classifying the volume of semen is recommended. If estimation to 0.1 ml precision is not possible, discrete categories could be used (for example: small \0.5 ml,
middle = 0.5–2.5 ml, large [2.5–5.0 ml, very large
[5.0 ml). In smaller species, such as Diana monkeys
(Cercopithecus diana) and squirrel monkeys (Saimiri
sciureus), two categories (small versus large) would be
sufficient to assess semen volumes over longer time periods
and for comparative studies.
Male fertility
Human males differ strongly in semen quality (e.g.,
Lipshultz et al. 2009). Nevertheless, field primatologists
seem to assume that all males exhibit similar fertilizing
potentials as inter-individual differences in semen quality
are not taken into account when potential causes of male
reproductive skew are discussed (e.g., Widdig et al. 2004;
Dubuc et al. 2011). This shortcoming seems to reflect the
traditional opinion that male reproductive success is not so
much influenced by semen quality but by factors such as
cryptic female choice (Eberhard 1996), female choice of
specific males (Klinkova et al. 2005) or multiple males
(Clarke et al. 2009), and male ability to time mate-guarding
in correspondence with temporary female fertility
(Deschner et al. 2004; Dubuc et al. 2012). However,
individual differences in semen quality have been found in
captive primates (Papio anubis: Schaffer et al. 1992;
M. nigra: Thomson et al. 1992; M. fascicularis: Gago et al.
1999; Callithrix jacchus: Kuederling et al. 2000), a finding
that is supported by the current study of wild Yakushima
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macaques. Sperm concentration, for instance, ranged
between 12.5 and 990.0 mio/ml among the 32 males surveyed, and estimated semen volumes were not uniform in
size, but differed between 0.1 and 5.0 ml (Table 2). It is,
therefore, very likely that variance in semen influences the
reproductive success of males more than commonly suggested. Thus, semen quality may exert an important influence on reproductive skew among males, especially when
individuals mate with multiple partners and when dominance rank is not closely associated with reproductive
success (reviewed in Cowlishaw and Dunbar 1991; Radespiel et al. 2002; Marvan et al. 2006; Dubuc et al. 2011). In
plain language: not the highest ranking males, but those
with superior semen might sire the most offspring.
Promising future research arenas
The potential to collect semen non-invasively from wild
primates other than macaques depends on whether or not
males masturbate, habituation level, environmental conditions, and degree of terrestriality. Masturbation by males
has been reported in approximately 60 primate species to
date. It occurs in 91 % of cercopithecoid genera (Thomsen
et al. 2003; Dixson 2012). Beside macaques, baboons
(Papio spp.), mandrills (Mandrillus sphinx) and drills
(M. leucophaeus) are most promising for semen collection
as these species are largely terrestrial and several habituated
populations are available (e.g., Chacma baboons, P. ursinus: Moscovice et al. 2010; gelada baboons, Theropithecus
gelada: Le Roux et al. 2011; mandrills: Setchell et al. 2011).
Standardising methods
Fertility analysis of human semen has been conducted by
the World Health Organisation (1999, 2012) since 1972.
However, data quality is often poor (Pacey 2009) and the
standard methodology has often been modified (e.g., Chia
et al. 1998; Cooper et al. 2010). To avoid such pitfalls, field
primatologists should develop a standard protocol before
expanding the scope of semen analysis in wild primate
populations.
Acknowledgments The study was funded by a grant (HSP III-D/97/
16290) from the German Academic Exchange Service (DAAD), a
stipend from the City of Berlin (Berliner Programm zur Förderung der
Chancengleichheit für Frauen in Forschung und Lehre, N-16/04), the
Japanese Society for the Promotion of Science (JSPS; pilot-1994/96),
Shimazu Kyoto (2009–2011), Suzuki Corp. (1999–2001), and a private donor (1997 and 2010). I thank Volker Sommer, Joseph Soltis,
Mike Huffmann and Ute Radespiel for their encouragement during
different stages of this project. Particular thanks go to Kiyoaki
Matsubayashi, Juichi Yamagiwa, Hideki Sugiura, Sachiko Hayakawa,
Miki Matsubara, Goro Hanya, and two field assistants as well as to all
members of the Yakushima Research Group for support of my work
in Japan and on Yakushima over the last two decades. I am grateful to
123
Bettina Wachter and two anonymous reviewers for helpful comments
on an earlier draft and to Anahita Kazem for proofreading the
manuscript.
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