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Functional assessment of sexual maturity in male macaques (Macaca fascicularis)

C.M. Luetjens, G.F. Weinbauer ⇑Covance Laboratories GmbH, 48163 Münster, Germany

a r t i c l e i n f o

Article history:Received 16 January 2012Available online 10 May 2012

Keywords:SemenEjaculateSpermatogenesisTestis histologyCynomolgus monkeyLong-tailed macaquePubertyMaturity

a b s t r a c t

Selection of suitable criteria for assessing sexual maturity in the male long-tailed macaque (Macaca fas-cicularis) has yielded conflicting results. The present retrospective work investigates whether the solepresence of sperm in the baseline semen sample unequivocally (i.e. for every animal) hallmarks completetesticular maturation. For 956 animals providing the baseline semen sample, neither age, body weightnor testes volume unequivocally predicted the presence of sperm in that sample, and for 322 animalsthese parameters failed to predict testicular histology. In contrast, the presence of sperm in the baselinesemen sample correlated with mature testis histology at study termination in every single animal(n = 197/322). Surprisingly, for the 125/322 animals without sperm in the baseline semen sample, sper-matogenesis was also mature in 95 animals. Thus, the mere provision of a semen sample without sperm –implying peripheral reproductive tract maturation – was associated with mature spermatogenesis inapprox. 75% of animals. Interestingly, testicular maturation occurred approx. 2 years earlier in Mauritiancompared to Asian mainland animals. In conclusion, a single semen sample that contains sperm providesunequivocal evidence for mature spermatogenesis and, thus, is suggested as a functional parameter forsexual maturity assessment in this species.

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1. Introduction

Nonhuman primate (NHP) models play a critical role in preclin-ical safety assessment, and this holds particularly true for thedevelopment of biopharmaceuticals, e.g. monoclonal antibodies(Baldrick, 2011; Chapman et al., 2009; Chapman et al., 2007).Among the various NHP species available, the long-tailed macaque(Macaca fascicularis, synonymous with crab-eating macaque orcynomolgus monkey) currently is most frequently used for safetyassessment (Baldrick, 2011; Chapman et al., 2010; Pentsuk andvan der Laan, 2009). In few cases, rhesus monkeys or marmosetmonkeys were also observed to be the most relevant animal model(Martin et al., 2009; Martin and Weinbauer, 2010). Given theincreasing number of biopharmaceuticals in development, it is ex-pected that NHP models with focus on long-tailed macaques willbe increasingly required for preclinical safety testing and it is man-datory to optimize the use of NHP during drug development (Chap-man et al., 2010). The revised ICH S6(R1) (ICH, 2011) biotechnologyguideline addresses the use of NHP models at various levels. A par-ticularly relevant item that triggered the current investigation isthat ‘‘. . ., when the NHP is the only relevant species, the potentialfor effects on male and female fertility can be assessed by evalua-tion of the reproductive tract (organ weights and histopathological

evaluation) in repeat dose toxicity studies of at least 3 monthsduration using sexually mature NHPs’’ (ICH S6(R1) (ICH, 2011). Un-like in rodents, however, primates undergo a prolonged postnatalperiod of reproductive axis quiescence followed by a resumptionof brain-gonadal axis activity (Plant and Ramaswamy, 2009) ulti-mately resulting in sexual maturity and fertility. Hence, for reasonsof availability and cost, preclinical safety assessment in NHPs istypically done using immature/juvenile animals except for specialreproductive toxicity investigations. The ICH S6(R1) (ICH, 2011)provides the option to avoid these special reproductive toxicitystudies, in which case, however, it is essential to use sexually ma-ture NHPs.

Overall, the postnatal development of the male reproductivetract including peripubertal events and attainment of sexual matu-rity appear strikingly similar between men and male macaques (forreview: Plant et al., 2005; Plant and Witchel, 2006; Irfan et al.,2011). Moreover, a number of papers has specifically addressedthe physiology and endocrinology of testicular development andmaturation in the long-tailed macaque (M. fascicularis, Fouquetet al., 1984; Steiner and Bremner, 1981; Meusy-Dessolle and Dang,1985; Dang and Meusy-Dessolle, 1984; Kluin et al., 1983). Human/macaque development is characterized by a relatively long periodof postnatal reproductive quiescence, commonly known as prepu-bertal hiatus, with very low to minimal reproductive hormone lev-els and very little proliferative activity of testicular cells. The onsetof pubertal events is essentially triggered by hypothalamic factors(e.g. kisspeptin, gonadotropin-releasing hormone) which in turn

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⇑ Corresponding author. Address: Covance Laboratories GmbH, Kesselfeld 29,48163 Münster, Germany. Fax: +49 (251) 78 46 97.

E-mail address: gerhard.weinbauer@covance.com (G.F. Weinbauer).

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activate pituitary gonadotropin secretion followed by testicularsteroid production and activation of the spermatogenic process.At the testicular level, organ growth is initially driven by expansionof peritubular cells and Sertoli cells with the latter cells exhibitinga fast and remarkable proliferative activity associated with sub-stantial testicular growth and onset of spermatogenesis. Testicularandrogens are needed for maturation of the peripheral reproduc-tive tract including semen formation whilst both androgens andfollicle-stimulating hormone are needed to achieve complete sper-matogenesis throughout the testis.

Unfortunately, in primates, the sexual maturation processshows enormous individual variability both in terms of the initia-tion age as well as the duration of the process that can extend overmonths and years: In boys, genital enlargement starts between 9.5and 13.5 years and maturity is not reached until 13–17 years of age(Rapaport, 2004), and in macaque models the peripubertal phasehas been reported to occur over an age range of 2.5–5.5 years (Irfanet al., 2011). These particular features of primate sexual matura-tion require firm and reliable criteria to unequivocally assess sex-ual maturity for every single animal to be used in safetyassessment and toxicological studies. Another hurdle is the lackof validated criteria for assessing sexual maturity under in vivoconditions, particularly for the male. It is apparent, that the avail-ability of a valid indicator of sexual maturation with 100% predic-tivity for every single NHP would be highly desirable from theviewpoint of animal supply, optimized study conduct, and animalwelfare. Recognition of sexual maturity is comparatively easierachieved in female long-tailed macaques by collecting daily vagi-nal swabs for identification of menstrual bleedings and ample lit-erature is available on this approach (Weinbauer et al., 2008). Inclear contrast, assessment of sexual maturity in male animals isless obvious and pertinent information is scarce and evenconflicting.

For example, to ensure that an animal has a 90% probability ofsexual maturity, it has been recommended that body weight shouldbe at least 5.3 kg and age at least 5 years 5 months (Smedley et al.,2002). According to Lawrence and Saladino (Lawrence and Saladino,2009), a 90% probability for sexual maturity was associated withbody weights of 4.52 kg or heavier. On the other hand, testicular vol-umes over 20 mL were considered to be compatible with sexualmaturity (Ku et al., 2010) whereas in other studies testis size deter-mination alone was insufficient for predicting sexual maturity(Smedley et al., 2002; Vogel, 2000). In boys it has actually beennoted previously that genital growth and testis size are highly var-iable, and a lack of a direct correlation between testicular size andspermarche has also been observed (Nielsen et al., 1986). Testicularsize was not necessarily an indicator of sexual maturity whilst in an-other report testicular volume was considered to be the most dis-criminative parameter for spermarche (Schaefer et al., 1990).These differences are potentially related to the methodology for tes-tis size determination. Using hormone analysis, e.g. testosteroneconcentrations, as a criterion to define sexual maturity, requiresspecial and sophisticated sampling schedules owing to assay vari-ability plus the pulsatile and circadian hormone synthesis/releasepatterns and the very spurious hormone peaks at the onset of thepubertal process (Bercu et al., 1983; Meusy-Dessolle and Dang,1985; Steiner and Bremner, 1981). A correlation between testoster-one levels and spermatogenic status in developing long-tailedmacaques revealed significant individual variation (Dreef et al.,2007). A potential disadvantage of using increasing cut-off levelparameters for sexual maturity determination, is that many animalsthat are already sexually mature at lower ages, body weights, testissize, etc., will go unnoticed.

Whilst menarche is an accepted milestone in female develop-ment and constitutes a functional endpoint for unequivocal sexualmaturity determination, a similar milestone, i.e. spermarche, has

not been described originally for male development (Laron, 2010)for details). It has been suggested earlier for NHP models that proofof sperm in the ejaculate might be a better indicator of sexualmaturity compared to other parameters (Korte et al., 1995). In-deed, presence of sperm would provide a functional criterion forassessing testicular maturation and sexual maturity and has mean-while been recommended as a milestone of pubertal developmentin boys (Laron, 2010; Nielsen et al., 1986). The ability to produce asemen sample also implies the maturity of the various componentsof the male reproductive tract since sperm transport and semenproduction are dependent on a variety of factors including repro-ductive hormones. For the conduct of safety assessment/toxicitystudy, enrolment of ‘‘a sexually mature male’’ NHP should ideallyguarantee complete testicular maturation and reproductive tractmaturation. From the viewpoint of the analysis and interpretationof such studies, it is pivotal that spermatogenesis is complete byhistological analysis since the purpose of using such animals is tobe able to detect adverse effects on the reproductive system. It isevident from the foregoing that neither age, body weight, testissize, nor peripheral testosterone level provide unequivocal identi-fication of sexual maturity, e.g. complete spermatogenesis in NHPmodels.

For the purpose of the present investigation, sexual maturity isdefined as completion of testicular gametogenesis and the abilityto provide a semen sample containing sperm since this confersthe potential for fertility. In this laboratory - throughout a numberof years – long-tailed macaques were selected on the basis of thepresence of sperm in semen samples for studies with sexually ma-ture animals. The present work is a retrospective investigation andaddresses the question whether the sole presence of sperm in thebaseline semen sample – irrespective of sperm number – indeedcorrelates with a mature testis, i.e. complete testicular spermato-genesis based upon histopathological analysis. Such unequivocalcriterion for sexual maturity is highly desirable for optimizingthe use of NHP both in terms of study quality, animal supply,and animal welfare. Our data demonstrate that – without excep-tion – the presence of sperm in the baseline semen sample wasassociated with complete gametogenesis as evidenced by histopa-thological analysis of the testis. Hence, a single semen sample thatcontains sperm provides unequivocal evidence for sexual maturity– at least in the long-tailed macaque - in the context of preclinicalNHP studies.

2. Materials and methods

2.1. Animals

This investigation comprises data from 956 male long-tailedmacaques (cynomolgus monkey, Macaca fascicularis). These ani-mals underwent baseline investigations prior to nonclinical safetyassessment studies and were selected for this evaluation sincethese animals provided semen samples. Baseline data for age andbody weight ranged from 2.8–11.9 years and 3.0–11.0 kg, respec-tively, and are presented in Table 1. Animals were either of main-land Asian origin (China or Vietnam) or Mauritian origin. Datawere collected between 2004 and 2010. Animals were single- orgroup-housed at Covance facilities (Münster, Germany) under a12:12 h light:dark cycle and in a controlled environment with > 10air changes/hour and temperature and relative humidity ranges of19–25 �C and 40–70%, respectively. Twice daily the animals wereoffered a commercial pellet diet for primates (Ssniff P10, SsniffSpezialdiäten GmbH, Soest, Germany) supplemented with freshfruit and bread added as food supplement. Tap water was providedad libitum. Housing and handling of animals was in accordancewith the German Animal Welfare Act.

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Tables 1 and 2 provide an overview of the available baseline andterminal data for age, body weight, duration between baseline andtermination, seminal sperm presence/absence and animal origin.Among the 956 animals, sperm were absent in semen samples of230 animals and sperm were present in semen samples of 726 ani-mals. These data were used to analyze baseline differences for ageand body weight relative to the presence of sperm in a semen sam-ple. For 322 animals, terminal data were available for testicularhistopathology analysis, the key parameter to be related to the ab-sence/presence of sperm in baseline semen samples. This testiculardiagnosis data came from 125 animals without sperm in the base-line semen sample and from 197 animals with sperm in the base-line semen sample. Terminal data from other animals in whomtesticular histology analysis was not available and/or a test item-related effects could not be ruled out with certainty were excludedfrom analysis.

2.2. Semen collection and analysis

Semen samples were obtained during baseline periods usingrectal probe electro-stimulation based upon previous reports(Weinbauer et al., 1994)). Semen samples were only inspectedmicroscopically to verify the presence of sperm. No quantitativesemen analyses such as determination of ejaculate weight, spermmotility, or sperm number were undertaken. Up to three attemptswere made to obtain semen samples. If a semen sample was suc-cessfully obtained, no further attempts were made to collect addi-tional semen samples.

2.3. Organ weights and testicular histology

At termination, body weight was recorded and – depending onstudy protocols – various organs were excised, weighed and pre-pared for histological analysis. For the purpose of this investiga-tion, body weight, testis weight (left and right combined) andseminal vesicle (left plus right combined; blotted-dry) were used.Testes were fixed in modified Davidson solution, embedded in par-affin, and sectioned at 3 lm for light microscopical analysis. Sec-tions were stained with hematoxylin–eosin and in someinstances also with periodic acid Schiff0s base/hematoxylin and across-section throughout the testis were examined as part of thestandard histopathology examination. Spermatogenesis was histo-logically classified as mature (complete spermatogenesis in allseminiferous tubules), adolescent-immature (a mixture of seminif-erous tubules with complete and incomplete spermatogenesis in

the same testis cross-section) and immature (incomplete sper-matogenesis in all seminiferous tubules). Details on organ weightavailability and testicular diagnosis are provided in Tables 3 and 4.

2.4. Testes volume

This parameter was available for 683 animals at baseline andfor 14 animals in whom baseline and terminal measurements oftesticular volume could be used. Testis volume was determinedas described previously by sonography using measurements ofthe length and width of the testis (Weinbauer and Kamischke,2003). An ultrasound instrument (ALOKA SSD-1000/1400)equipped with a 7.5 MHz probe was used. Data are presented ascombined volume of left and right testis.

2.5. Data analysis

Data are expressed as mean ± standard deviation (SD), range,and/or median. Statistical comparisons were made using paramet-ric tests (student’s t-test, ANOVA) and non-parametric tests(Mann–Whitney Rank Sum Test, Kruskal–Wallis ANOVA) depend-ing on the data distribution. A two-sided p-value below 0.05 wasconsidered statistically significant. All statistical calculations wereperformed using Sigmaplot version 11.00 (Systat Software, Inc.).

3. Results

3.1. Baseline data

Baseline data for age and body weight for the 956 animals areprovided in Table 1 and Fig. 1. Animals with sperm in a baseline se-men sample (n = 726) were significantly older (p < 0.001) and weresignificantly heavier (p < 0.001) compared to animals withoutsperm in the baseline semen sample (n = 230). On average, theage difference was 1.1 years and the body weight difference was1.2 kg. Ages were also significantly different (p < 0.001) dependingon animal origin (Asia vs Mauritius) irrespective of whether spermwere present or absent in the baseline semen sample (Table 1). Forexample, for animals with sperm present, Mauritian animals were,on average, 1.9 years younger compared to Asian animals. In con-trast, body weight difference was less pronounced between Mauri-tian and Asian animals. Mauritian animals with sperm, on average,weighed 0.4 kg less (p < 0.001) than Asian animals with sperm. Foranimals without sperm, body weight differences averaged at 0.2 kgand did not attain statistical significance (p > 0.05) between animal

Table 1Baseline age and body weight of 956 long-tailed macaques with or without the presence of sperm in the baseline semen sample.

Parameter All animals Mauritian Asian

Sperm absent Sperm present Sperm absent Sperm present Sperm absent Sperm present

Number of animals 230 726 174 505 56 221

Age [years]Number of animals 230 726 174 505 56 221Mean ± SD 4.1 ± 0.9* 5.2 ± 1.2 3.8 ± 0.6*,** 4.6 ± 0.7** 5.1 ± 1.2* 6.5 ± 1.3Range 3.0–7.7 2.8–11.9 3.0–5.8 2.8–7.1 3.2–7.7 3.9–11.9Median 3.8 5.0 3.7 4.6 4.9 6.3

Body weight [kg]Number of animals 222 726 166 505 56 221Mean ± SD 4.6 ± 1.0* 5.8 ± 1.1 4.6 ± 1.1* 5.7 ± 0.9** 4.4 ± 0.9* 6.1 ± 1.3Range 3.0–7.5 3.2–11.0 3.0–7.5 3.7–8.7 3.1–6.7 3.2–11.0Median 4.4 5.7 4.5 5.7 4.2 6.0Age/body weight ratio (median based) 0.86 0.88 0.82 0.81 1.17 1.05

Animals of Asian origin were from Chinese or Vietnamese breeding facilities.* p < 0.001 sperm present vs sperm absent.** p < 0.001 Mauritian vs Asian.

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origins. Regression analysis indicated that Mauritian animals growby 1.7 kg/year whilst Asian animals grow by 0.9 kg/year (basedupon Fig. 1).

The baseline age and body weight data for those animals withtesticular histology diagnosis available are summarized in Table 2and are depicted in Fig. 2. Overall, the data are comparable to thosepresented in Table 1 in that animals with sperm in the baseline se-men sample were older (by 1.2 years, on average), and heavier (by1.3 kg, on average), compared to those animals without sperm inthe baseline semen sample (p < 0.05). Baseline age and bodyweight were also lower in animals without sperm in a semen sam-ples irrespective of their terminal testicular histology diagnosis(Fig. 2). These data indicate that the subset of animals with avail-able testicular histology is not different from the entire set of ani-mals under investigation. Also, whilst Mauritian animals weresignificantly younger (p < 0.05) compared to Asian animals, thebody weight differences between origins did not attain statisticalsignificance (p > 0.05).

Mauritian animals aged 3–4 years had sperm present in thebaseline semen sample whilst for Asian animals this was seen onlyin animals older than 4 years (except for one animal aged 3.9 years,Table 1, Fig. 1). The data also suggest that animals weighing lessthan approx. 4 kg combined with an age below approx. 4 yearsmay provide semen samples that do not contain sperm (Figs. 1and 2).

3.2. Terminal data - age, body weight, and duration

Study termination data are summarized in Table 2. Data analy-sis was confined to those 322 animals with available testicular his-tology diagnosis. The time interval from baseline semen collectionuntil termination is denoted as duration. For animals withoutsperm in the baseline semen sample, duration was, on average,8.3 months (range: 1.0–18.9 months) vs 6.9 months (range: 1.0–43.4 months) in animals with sperm in the baseline sample(p < 0.05). For body weight, animals without sperm in the baselinesample, tended to gain more weight over baseline when comparedto animals with sperm present in the baseline semen sample.Based on all animals, body weight gain was, on average, 0.7 kg(p < 0.05) for animals with sperm absent vs 0.2 kg (p > 0.05) foranimals with sperm present; and the body weight ratio of base-line/terminal was 0.84 vs 0.97 for sperm absent vs sperm present,respectively.

3.3. Testicular histology and reproductive organ weight

Testicular histology diagnosis was available for 322 animals(Table 3). Spermatogenesis and testicular morphology were ma-ture in all 197 animals with sperm present in the baseline semensample. Thus, the presence of sperm in the baseline semen samplecorrelated entirely with mature testis histology at study termina-tion in every single animal. Interestingly, among the remaining125 animals with sperm absent in the baseline semen sample,spermatogenesis and testicular morphology were also rated as ma-ture in 95 animals, adolescent–immature in 12 animals and imma-ture in the remaining 18 animals. Thus, the provision of a semensample – even without sperm present – correlated with maturespermatogenesis in approx. 75% of animals. Conversely, basedupon all 322 animals, spermatogenesis and testicular morphologywere mature in approx. 90% (n = 292) of animals that provided asemen sample irrespectively of the sperm content (Table 3).

For animals with sperm absent in the baseline semen sample,terminal age, body weight, and study duration were lowest/short-est in those animals with immature spermatogenesis and highest/longest in animals with mature spermatogenesis (p < 0.05, Table 4).This was particularly evident for the immature spermatogenesis

diagnosis with an average duration of 3.0 vs 9.6 months for the ma-ture spermatogenesis diagnosis. Duration for the adolescent–immature diagnosis was 5.8 months, on average (p < 0.05 vs imma-ture diagnosis). Also, terminal age and body weight were signifi-cantly larger (p < 0.05) for animals with sperm present andmature spermatogenesis diagnosis compared to animals withsperm absent but also mature spermatogenesis diagnosis albeitstudy duration was shorter (p < 0.05) (Table 4).

The weights of testes and seminal vesicles were largest in ani-mals that had sperm present in the baseline semen sample(p < 0.05, Table 4). Among animals without sperm in the baselinesemen sample, reproductive organ weights correlated with testic-ular histology diagnosis with largest weights associated withmature spermatogenesis and lowest weights with immature sper-matogenesis (p < 0.05). For animals with mature spermatogenesisdiagnosis, testis and seminal vesicle weights were larger(p < 0.05) in animals with sperm present compared to animals withsperm absent in the baseline semen sample (Table 4).

3.4. Testes volume

Baseline testes volume was available for 667 animals withsperm present in the baseline semen samples but only for 16 ani-mals with sperm absent in the baseline semen sample (Fig. 4). Forthe presence of sperm, testis volumes were highly variable andranged from 4.9–68.0 mL. For the absence of sperm, testis volumeswere below 4 mL in the three animals and within the above rangefor the remaining 13 animals. Testis volumes were slightly but sig-nificantly larger (p < 0.05) in Mauritian animals (26.2 ± 10.6 mL)compared to Asian animals (22.9 ± 12.1 mL) whilst Mauritian ani-mals were younger than Asian animals (4.6 ± 0.6 years vs6.5 ± 1.3 years, p < 0.05). Regression analysis indicated that testesvolume increases by 5.7 mL/year in Mauritian animals and by3.6 mL/year in Asian animals (based upon Fig. 4).

For 14 animals, baseline and terminal testes volume, durationand testicular histology diagnosis were available (Table 5). Theseanimals had no sperm in the baseline semen sample but were diag-nosed with mature spermatogenesis at termination. Duration frombaseline to termination ranged from 3.2–16.6 months. The changesin testes volume from baseline to termination were between �2.2–54.2 mL and, generally, longer duration was associated with agreater testes volume increase. These limited data set indicates,that for animals that provide a semen sample – albeit withoutsperm – spermatogenesis is already mature or ongoing and will at-tain maturity with in a period of > 3 months.

3.5. Baseline prediction of male sexual maturity

Neither baseline body weight nor baseline age correlated withthe presence or absence of sperm in the baseline semen sampleon an individual animal basis but instead displayed great variabil-ity (Figs. 1 and 2). The age of the youngest animal with sperm pres-ent in the baseline semen sample was 2.8 years and the age of theoldest animal without sperm in the baseline semen sample was7.7 years. Similarly, despite a large variation of baseline testicularvolumes from 4.9–68.0 mL determined from 667 animals, each ofthese animals had sperm present in the baseline semen sampleand upon termination was diagnosed with mature spermatogene-sis (Fig. 4). The youngest animal with mature spermatogenesisdiagnosis was 3.4 years and the oldest animal with immature sper-matogenesis diagnosis was 5.1 years. It appears that in the approx-imate range of 3.0–5.5 years the testis can be mature, adolescent–immature or immature (Fig. 3). The lowest combined testicularvolume associated with sperm in the baseline semen sample wasas low as 4.9 mL. These observations demonstrate that neitherbaseline body weight, age nor testes volume size are unequivocally

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predictive of mature spermatogenesis on an individual animal ba-sis. In contrast, the presence of sperm in a semen sample was asso-ciated with mature spermatogenesis in every single animal. Thispredictivity was not related to the time interval between baselineand terminal evaluation which ranged from 1.0–43.4 months (Ta-ble 2 and Fig. 3) indicating that the presence of sperm in a semensample directly reflects mature spermatogenesis.

4. Discussion

The long-tailed macaque (cynomolgus monkey) currently is afrequently used NHP model in safety assessment, and recent guide-line revisions have markedly increased the need for sexually ma-ture NHPs. Current approaches towards identifying sexualmaturity in the male NHP comprise consideration of age, bodyweight and sometimes testis size and hormone levels. However,none of these parameters provides a definitive criterion on an indi-vidual animal basis and, equally important, if these criteria areused to develop minimal cut-off criteria – many animals alreadymature but below these cut-off levels, will be missed. The presentwork investigates whether the sole presence of sperm in a semensample can be used to unequivocally predict sexual maturity forevery male long-tailed macaque. In the context of this paper, malesexual maturity is defined via a functional parameter, i.e. the com-pletion of gametogenesis as evidenced by the presence of sperm ina semen sample (and the ability to produce a semen sample).

A pivotal question addressed here is whether the sole presenceof sperm in a semen sample is compatible with mature spermato-

genesis or whether – in spite of ejaculated sperm – it is possiblethat spermatogenesis is not yet completed throughout the entiretestis. Our data from 197 animals demonstrated complete sper-matogenesis by standard histopathology analysis of an entire testiscross-section when sperm were present in a baseline semen sam-ple in these animals. It could be argued that at the time of collec-tion of the baseline semen sample, spermatogenesis had only beenestablished in some testicular areas, and that the subsequent timeperiod until study termination had been sufficient to allow com-pletion of spermatogenesis throughout the entire testis. This isconsidered very unlikely since (1) the duration between baselineand termination was not related to the histological diagnosis(Fig. 3 for details), (2) in many animals the duration was as shortas 1–3 months, and (3) the average duration between baselineand termination was shorter in animals with sperm present vssperm absent at baseline (6.9 vs 8.3/9.6 months, p < 0.05, Tables2 and 4 for details). Thus, in all likelihood, the sole presence ofsperm in a semen sample is associated with mature spermatogen-esis in the male long-tailed macaque.

Surprisingly, among those animals with sperm absent in thebaseline sample, mature spermatogenesis was diagnosed at termi-nation at a rate of approx. 75% (95/125). In these animals, the aver-age duration between baseline and termination was 9.6 monthsbut varied widely (1.0–18.9 months). Previous histological studieson the pubertal establishment of spermatogenesis in the long-tailed macaque concluded that there is no privileged region forspermatogenic activity (Dang and Meusy-Dessolle, 1984). In fact,spermatogenic progress was encountered homogenously irrespec-tive of the part of test examined or of arterial blood supply. Based

Table 2Baseline and terminal age and body weight of 322 long-tailed macaques with or without the presence of sperm in the baseline semen sample and available testicular histologydiagnosis.

Terminal data All animals Mauritian Asian

Sperm in the ejaculate Sperm absent Sperm present Sperm absent Sperm present Sperm absent Sperm present

Total number of animals 125 197 103 131 22 66

Baseline age [years]Mean ± SD 4.0 ± 1.0* 5.2 ± 1.1 3.7 ± 0.6*,** 4.7 ± 0.6** 5.4 ± 1.2* 6.2 ± 1.1Range 3.1–7.6 3.1–9.2 3.1–5.8 3.1–6.1 3.2–7.6 4.6–9.2Median 3.7 5.1 3.6 4.7 5.3 6.2Number of animals 125 197 103 131 22 66

Terminal age [years]Mean ± SD 4.7 ± 0.9*,*** 5.7 ± 1.1*** 4.5 ± 0.6*,**,*** 5.2 ± 0.7**,*** 6.0 ± 1.2* 6.7 ± 1.1***

Range 3.5–8.4 3.4–9.6 3.5–6.6 3.4–6.5 4.1–8.4 4.9–9.6Median 4.6 5.5 4.5 5.3 6 6.6Number of animals 125 197 103 131 22 66

Duration [months]Mean ± SD 8.3 ± 5.1* 6.9 ± 4.9 8.5 ± 5.4* 6.6 ± 3.7 7.5 ± 3.2 7.3 ± 6.6Range 1.0–18.9 1.0–43.4 1.0–18.9 1.6–14.2 3.0–17.3 1.0–43.4Median 7.0 5.5 6.7 5.9 8.1 5.5Number of animals 125 197 103 131 22 66

Baseline body weight [kg]Mean ± SD 4.5 ± 1.0* 5.8 ± 1.1 4.5 ± 1.0* 5.7 ± 0.9 4.6 ± 0.9* 6.0 ± 1.3Range 3.0–7.4 3.7–11.0 3.0–7.4 4.0–8.6 3.1–6.5 3.7–11.0Median 4.3 5.7 4.3 5.6 4.3 6.0Number of animals 122 197 100 131 22 66

Terminal body weight [kg]Mean ± SD 5.2 ± 1.1*,*** 6.0 ± 1.1 5.2 ± 1.1*,*** 5.8 ± 1.0 5.3 ± 1.1*,*** 6.3 ± 1.2Range 3.2–8.5 3.2–9.8 3.2–8.5 3.2–9.2 3.4–7.6 4.3–9.8Median 5.1 5.9 5.1 5.8 5.3 6.3Number of animals 125 191 103 128 22 63

Body weight ratioBaseline/terminal (median based) 0.84 0.97 0.84 0.97 0.81 0.95

Animals of Asian origin were from Chinese or Vietnamese breeding facilities.Testicular histology diagnosis is presented in Tables 3 and 4.* p < 0.05 sperm present vs sperm absent.** p < 0.05 Mauritian vs Asian.*** p < 0.05 terminal vs baseline.

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upon serial testicular biopsies it was concluded that between 2 and5 months were needed between meiosis (presence of spermato-cytes) and the appearance of elongated spermatids, e.g. testicularspermatozoa. For the long-tailed macaque, spermatogenic cycleduration is around 10 days (Aslam et al., 1999) and the entire sper-matogenic process lasts around 40 days. For the Cebus monkey, itwas concluded that 30 complete spermatogenic processes areneeded to establish spermatogenesis throughout the testis (Reyet al., 1993). Since these processes run in parallel or synchronouslyin the various seminiferous tubules, the onset of complete sper-matogenesis throughout the testis is achieved rapidly once the firstelongated spermatids are present. Human studies indicated – fol-lowing the first ejaculation - azoospermia for 5 months and nor-mospermia after 21st months (Janczewski and Bablok, 1985).Using the age conversions factor 4 between human and macaques(Morford et al., 2011), the corresponding timing would be1.2 months until sperm appearance and 5–6 months until fullsperm count in the long-tailed monkey, respectively. These consid-erations would explain why histological evaluation consistently re-vealed mature spermatogenesis in the present study even inanimals with sperm absent in the baseline semen sample.

It is also pertinent to mention that the ability to transportsperm from the testis throughout the reproductive tract requiresthat the seminiferous tubules, rete testis, efferent ducts, epididy-midal duct and vas deferens are patent and that seminal vesicles

and prostate are functional in terms of producing seminal fluid. Fi-nally, a certain degree of maturation of the external genitalia ismandatory. Therefore, the ability to provide a semen sample im-plies a substantial degree of maturation along the entire malereproductive tract that is compatible with sperm transport andproduction of seminal fluid. From a clinical viewpoint, spermarche– defined as the onset of release of spermatozoa – has been consid-ered a hallmark of pubertal development in boys (Laron, 2010),since the ability to emit a semen samples implies and necessitatessufficient maturation of the male reproductive system. In the long-tailed macaque, the ejaculatory response to electro-stimulation isessentially androgen-dependent (Weinbauer et al., 1994). Sincethe testis are the source of androgens and since androgens can ini-tiate spermatogenesis in macaques (Marshall et al., 1986; Marshallet al., 1995), it is entirely plausible that at the time point of periph-eral androgen-dependent reproductive organ maturation, testicu-lar gametogenesis is already ongoing because of the exposure tohigh local androgen levels. At initiation of pubertal developmentin boys, seminiferous tubules receive a high androgen load at atime when peripheral androgen-dependent organs are hardlydeveloped (Rivarola et al., 1983). In fact, it appears that some tes-ticular sperm production may even start prior to the ability to pro-duce an ejaculate – cases have been reported that spermaturia wasmore common in early-to-mid-pubertal boys versus late-pubertalboys (Pedersen et al., 1993). It has also been reported that sperm-arche in boys is an early pubertal event that can occur even whentestis have only grown slightly (Nielsen et al., 1986). On the otherhand, a recent study reported a mean age of pubertal onset (de-fined by testis size increase) of 10.55 years versus an age of12.78 years for spermarche (Ma, 2010). In any case, the onset ofspermatogenic activity of the primate testis may well precedethe external maturation of the reproductive system.

The present work demonstrated a very large individual varia-tion for age, body weight, and testis associated with mature sper-matogenesis and ejaculatory ability (see section 3.5 for details).These observations generally support previous work in that noneof these parameters will provide 100% confidence for identifyingNHPs with mature spermatogenesis. Ku et al. (Ku et al., 2010) re-ported an animal weighing 2.3 kg that already had a fully maturetesticular histology. Others observed full spermatogenesis at agesbetween 3 years 8 months and 4 years 4 months at a body weight

Table 4Baseline and terminal age, body weight and reproductive organ weights relative to testicular histology diagnosis in 322 long-tailed macaques with absence/presence of sperm inthe baseline semen sample.

Sperm absent/present No of animals Age (years) Body weight (kg) Duration (months) Organ weight (gram)

Histology diagnosis Baseline Terminal Baseline Terminal Duration Range Testes Seminal vesicles

Sperm absentMature 95 4.2 ± 1.1 5.0 ± 1.0** 4.6 ± 1.1** 5.5 ± 1.1**,*** 9.6 ± 5.1** 1.0–18.9 29.4 ± 11.5**,*** (93) 5.7 ± 3.5**,*** (61)Adolescent-immature 12 3.7 ± 0.5 4.2 ± 0.5 4.3 ± 0.9 4.5 ± 0.8 5.8 ± 2.1**** 1.6–8.7 7.8 ± 3.4 2.2 ± 0.8 (9)Immature 18 3.7 ± 0.4 3.9 ± 0.4 3.8 ± 0.7 (17) 4.1 ± 0.5 3.0 ± 1.7 1.6 –7.5 4.0 ± 3.1 1.6 ± 1.3 (7)

Sperm presentMaturea 197 5.2 ± 1.1* 5.7 ± 1.1* 5.8 ± 1.1* 6.0 ± 1.1* (191) 6.9 ± 4.9* 1.0–43.4 36.2 ± 12.3* (193) 9.5 ± 5.6* (158)

Date are mean ± SD.Numbers in brackets represent animals number different from column 2.Data represent 322 long-tailed macaques (cynomolgus monkeys).Duration represents the time period between collection of the baseline semen sample and study termination.Terminal data for age, body weight and duration for the animals with sperm present are the same as those presented in Table 2.Spermatogenesis was histologically classified as mature (complete spermatogenesis), adolescent immature (a mixture of seminiferous tubules with complete and incompletespermatogenesis) and immature (spermatogenesis incomplete in all seminiferous tubules).

a Only mature spermatogenesis was diagnosed in all animals with sperm present in the baseline semen sample.* p < 0.05 present vs absent.** p < 0.05 mature vs immature (absent).*** p < 0.05 mature vs immature adolescent (absent).**** p < 0.05 immature adolescent vs immature (absent).

Table 3Testicular histology diagnosis in 322 long-tailed macaques with or without thepresence of sperm in the baseline semen sample.

Histology diagnosis Spermabsent

Spermpresent

Combined animalnumber

Mature 95 (76.0) 197 (100) 292 (90.7)Adolescent-

immature12 (9.6) 0 12 (3.7)

Immature 18 (14.4) 0 18 (5.6)Total number of

animals125 197 322 (100)

Numbers in brackets are percentages.Spermatogenesis was histologically classified as mature (complete spermatogene-sis), adolescent immature (a mixture of seminiferous tubules with complete andincomplete spermatogenesis) and immature (spermatogenesis incomplete in allseminiferous tubules).

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of 3.5–3.8 kg (Dang and Meusy-Dessolle, 1984). Based upon semenanalysis, some long-tailed macaques at 3 years of age (7/194 ani-mals) and at 4 years of age (58/194 animals) already producedsperm (Zühlke et al., 2006). Also for rhesus monkey, testis size

around 3 mL was accompanied by presence of tubules with com-plete spermatogenesis (Schlatt et al., 2009). It is of interest to notethat in boys, spermarche also can be an early-to-mid pubertalevent, e.g. preceding the pubertal growth spurt (Kulin et al.,

Fig. 1. Relation between baseline age and baseline body weight of long-tailed macaques relative to their origin (Mauritian or Asian) and relative to the presence or absence ofsperm in the baseline semen sample. Animals with sperm present tend to be older and heavier compared to animals with sperm absent. Also, Mauritian animals tend to beyounger relative to body weight compared to Asian animals. Finally, neither age nor body weight – on an individual animal basis – correlate with the presence or absence ofsperm in the baseline semen sample. Animals numbers: N = 505 for Mauritian sperm present, n = 166 for Mauritian sperm absent, n = 221 for Asian sperm present and n = 56for Asian sperm absent. Descriptive statistics are provided in Table 1.

Fig. 2. Relation between baseline age and baseline body weight of long-tailed macaques relative to their testicular histology diagnosis at termination. Animals with spermabsent in the baseline semen sample tend to be younger and lighter compared to animals with sperm present in the baseline semen sample. Note that there is clear overlap inbody weight and age irrespective of testicular histology diagnosis. Codes and animal numbers: ‘‘Sperm present mature’’ means the presence of sperm in the baseline semensample and histological mature spermatogenesis at termination (n = 197); ‘‘sperm absent mature’’ means the absence of sperm in the baseline semen sample but histologicalmature spermatogenesis at termination (n = 95). ‘‘Sperm absent adolescent-immature’’ (n = 12) and ‘‘Sperm absent immature’’ (n = 18). Descriptive statistics are provided inTable 4.

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1989; Nielsen et al., 1986; Pedersen et al., 1993; Schaefer et al.,1990).

Unlike some other macaque species (e.g. rhesus monkey, bon-net monkey, Japanese macaque), the reproductive physiology of

long-tailed macaques appears not to be dependant on seasonalinfluences. This observation has been made for both female ani-mals (Dang, 1977; Kavanagh and Laursen, 1984) and for male ani-mals (Dang and Meusy-Desolle, 1984; Kluin et al., 1983) using a

Fig. 3. Relation between baseline age and terminal age of long-tailed macaques relative to their testicular histology diagnosis at termination. The youngest animal withmature spermatogenesis diagnosis was 3.4 years, and the oldest animal with immature spermatogenesis diagnosis was 5.1 years. Note that in the approximate 3–5.5 yearsterminal age range (marked with an arrow) the testis can either be mature, adolescent–immature or immature. Note that there is clear overlap in body weight and ageirrespective of testicular histology diagnosis. Codes and animal numbers: ‘‘Sperm present mature’’ means the presence of sperm in the baseline semen sample and histologicalmature spermatogenesis at termination (n = 197); ‘‘sperm absent mature’’ means the absence of sperm in the baseline semen sample but histological mature spermatogenesisat termination (n = 95). ‘‘Sperm absent adolescent-immature’’ (n = 12) and ‘‘Sperm absent immature’’ (n = 18). Descriptive statistics are provided in Table 4 Descriptivestatistics are provided in Table 4.

Fig. 4. Relation between baseline age and baseline testes volume of long-tailed macaques relative to their origin (Mauritian or Asian) and relative to the presence or absenceof sperm in the baseline semen sample. Animals numbers: N = 449 for Mauritian sperm present, n = 16 for Mauritian sperm absent, and n = 218 for Asian sperm present. Notestes volume data were available for Asian animals without sperm in the baseline semen sample.

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variety of reproductive endpoints such as ovarian cyclicity, gonadalhistology, reproductive hormone levels and fertility. Our ownexperience with long-tailed macaque reproduction also supportsthis view for female animals (Weinbauer et al., 2008) and for maleanimals. Male animals successfully impregnate female animalsthroughout the entire year in our colony (unpublished observa-tions). For these various reasons, it is considered unlikely that sea-sonal variations of male reproductive functions were relevant inthe present study.

In this work we observed a clear difference in terms of bodyweight development and testicular maturation between animalsof Mauritian and Asian mainland origin. Mauritian animals grewby 1.7 kg/year whilst Asian mainland animals gained by 0.9 kg/year, and for testicular size, volume increased by 5.7 mL/year inMauritian animals versus by 3.6 mL/year in Asian mainland ani-mals. Overall, testicular maturation apparently was complete ap-prox. 2 years earlier in Mauritian compared to Asian mainlandorigin animals. These origin-related differences bear some rele-vance with regard to study planning and animal supply if sexuallymature animals are required in such investigations. The reasons(s)underlying the observed differences are not entirely clear. Long-tailed macaques are not endemic in Mauritius and mitochondrialDNA analysis supports the hypothesis that a few original founderanimals came from Indonesia (Lawler et al., 1995) a few hundredyears ago. Mauritian cynomolgus monkeys have a lower degreeof genetic variability (Leuchte et al., 2004), can respond differentlyto immune system challenges compared to animals of other origins(Menninger et al., 2002; Migot-Nabias et al., 1999) and have somemajor differences regarding clinical pathology parameters (Zühlkeet al., 2006). It would be interesting to know whether Asian islandanimals, e.g. from Indonesia, also exhibit earlier sexual maturationcompared to Asian mainland animals – if so, this could serve as anexplanation for our own observations on the difference betweenMauritian and Asian mainland animals.

It is important to emphasize that sexual maturity – in the con-text of this paper – is not meant to reflect reproductive maturity,i.e. fertility. Clinically there is neither a single parameter nor acombination of parameters that allows prediction of fertility withcertainty. Meyer et al. (Meyer et al., 2006) compared age, bodyweight, testis size, semen parameters, and testosterone levels inlong-tailed macaques with proven fertility and in animals thatfailed to impregnate and, interestingly, were unable to detect dif-ferences in these parameters. This confirms clinical observations.In another study, African green monkeys with confirmed fertilityhad sperm concentrations between 1–390 mill/mL (Brady et al.,

1985). Fertility proof in NHP models could – in principle – beachieved by a pre-study mating test. However, for a variety of rea-sons including less than 100% mating success, timing and animalwelfare considerations, this approach is not considered feasiblenor is it recommended (Chellman et al., 2009; Martin et al.,2009). If information of reproductive maturity is desired, it is rec-ommended to assess whether the available (reproductive) param-eters are within the normal and age-related reference range of aparticular laboratory – if so, reproductive maturity is likely.

The presence of sperm in the baseline semen samples was con-sistently associated with older and larger animals compared tothose animals with sperm absent from the baseline semen sample(Tables 1 and 2). Interestingly, at termination, testis and seminalvesicle weights of animals with sperm present at baseline werealso higher compared to those animals with sperm absent (Table 4).This observation suggests that the presence/absence of sperm in asemen sample is apparently associated with a different degree ofmaturation of the reproductive tract and underlines the relevanceof the ability to obtain semen samples. Similar observations werereported for the African Green monkey (Chlorocebus aethiops (Bra-dy et al., 1985). In this study, in addition to body weight, animalswithout sperm in a semen sample also had lower testis size andtestosterone levels. Interestingly, when sperm were present in asemen sample, testis size, and testosterone levels were in the rangeof adult animals. Body weight differences were also reported forpre- and post-spermarchial boys (Ji, 2001).

This work demonstrates that the sole presence of sperm in along-tailed macaque semen sample provided unequivocal evidencefor sexual maturity. This is considered important since this obser-vation eliminates the need for complete semen analysis includingejaculate volume, sperm number, sperm motility, and sperm mor-phology. Quantitative semen analysis is challenging, can be highlyvariable within and between laboratories and mandates a qualitycontrol program (Cooper et al., 2002). Whereas collection of semensamples from macaques has not been a common approach previ-ously, it appears to be more widespread recently. Hence, obtaininga semen sample and checking for presence of sperm by simple lightmicroscopy should enable a larger scale animal screening and mayalso be feasible for NHP breeding facilities. In the present work adifference in body and testicular growth rate between animals ofMauritian and Asian origin was encountered (Figs. 1 and 4 for de-tails). Overall, these baseline data suggest that Mauritian animalsapparently grow faster and complete male reproductive tract mat-uration earlier than Asian animals. For the purpose of identifyingsexually mature animals based upon age, body weight or testessize, the animal origin would be relevant. However, using the pres-ence of sperm in a semen sample as a functional endpoint wouldeliminate the need to consider the male long-tailed macaqueorigin.

5. Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

The authors wish to specifically acknowledge the dedicated andcompetent work of our scientific and laboratory technical staff.

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