American Journal of Primatology 47:105–115 (1999)

© 1999 Wiley-Liss, Inc.

Morphological Characterization of Ejaculated CynomolgusMonkey (Macaca fascicularis) Sperm

C. GAGO,1 F. PÉREZ-SÁNCHEZ,2 C.H. YEUNG,3 L. TABLADO,1 T.G. COOPER,3 ANDC. SOLER1*1Departament de Biologia Animal, Facultat de Biologia, Universitat de València, PaísValencià, Spain

2Departament de Biologia Cel.lular, Facultat de Biologia, Universitat de València, PaísValencià, Spain

3Institut für Reproduktionsmedizin der Universität, Münster, Germany

The aim of this study was to give reference values for the frequency ofmorphological sperm abnormalities present in the semen from non-experimental cynomolgus monkeys as well as for the dimensions of spermheads. Spermatozoa from the liquid portion of electroejaculates from 14cynomolgus monkeys were air-dried as smears, fixed, and stained withHarris’s Haematoxylin and subjected to visual analysis of morphologyand computer-aided analysis of ten morphometric variables. The major-ity (83%) of sperm were morphologically normal. Tail defects were themost common (11%), and showed the highest variation between individu-als, the values ranging between 4 and 23%. Head abnormalities consistedof large, tapering, and amorphous forms but were not frequent (0.4%),the values ranging between 0 and 1.3%. Midpiece imperfections werefound in all the individuals; the mean percentage was 5%, and the rangevaried between 3 and 9%. Tail plus midpiece was the only multiple ab-normality observed, with a mean value of 1.5% and a range between 0and 8%. The majority of these double defects consisted of a coiled tailtogether with a coiled midpiece. Mean values for the morphometric pa-rameters characterizing sperm heads were as follows: area 17.2 µm2, pe-rimeter 15.2 µm, length 5.8 µm, width 4.0 µm, L/W ratio 1.5, gray-level98, ellipticity 0.2, first shape factor 0.9, second shape factor 1.4, and thirdshape factor 1.1. Overall coefficients of variation for the majority of pa-rameters were below 7%, showing the great homogeneity in the dimen-sions of cynomolgus sperm heads. Most useful parameters for spermcharacterization, according to their low variability, were perimeter, length,width, L/W ratio, and shape factors. Differences in these parameters were,however, observed between monkeys. Am. J. Primatol. 47:105–115,1999. © 1999 Wiley-Liss, Inc.

Key words: sperm head morphology; morphometry; automated spermmorphometry analysis; monkey; cynomolgus

Contract grant sponsor: University of València; Contract grant sponsor: Generalitat Valenciana

*Correspondence to: Carles Soler, Departament de Biologia Animal, Facultat de Biologia, Universitatde València, C/. Dr Moliner 50, 46100 Burjassot, País Valencià, Spain. E-mail: pacops@uv.es

Received 6 October 1997; revision accepted 26 May 1998

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INTRODUCTIONSperm morphology has long been considered one of the most important indica-

tors of sperm quality, in addition to sperm concentration, motility, viability, mucus-penetrating ability, capacity to bind to oocyte investments, and other physical andbiochemical properties [World Health Organization, 1992; Comhaire & Vermeulen,1995]. The concept and evaluation of the normal and abnormal morphology of sper-matozoa have received most attention, with sperm abnormalities being taken as indi-cators of biological stress [Kasa & Thwaits, 1992] or of exposure to toxicants [Foote etal., 1986]. Recent studies suggest that aberrant sperm heads are associated withchromosomal defects [Lee et al., 1996]; furthermore, the relationship between thefrequency of morphologically abnormal spermatozoa and fertility has been reportedboth in man and animals [Karabinus et al., 1990; Leroy-Martin et al., 1994].

Morphological analysis is therefore of great significance in the study of sperm.Various techniques have been described for such analysis, but the study of spermmorphology is difficult, owing to inter- and intraobserver variability [World HealthOrganization, 1992]. Some work has addressed the standardization of semen analysisin man [Davis & Gravance, 1993] and in animals [Ball & Mohammed, 1995; Gravance& Davis, 1995; Gravance et al., 1995, 1996], but in species with a large proportion ofabnormal morphological forms, such as man [World Health Organization, 1992], theanalysis of spermatozoa has presented many difficulties since there is no standardmethodology. This lack of standardization adds to the subjectivity of the methods andmay explain the widely differing conclusions that have been reached concerning theimportance of morphological assessment.

Williams and Savage [1925] were the first to highlight the importance of spermhead morphometry to the reproductive potential of spermatozoa in bulls. Subsequently,numerous studies have investigated the morphometric characteristics of spermato-zoa, both in humans and in other animals [for review see Cummins & Woodwall,1985]. In recent years, automated sperm morphometry analysis (ASMA) systems havebeen developed, and some studies have stressed the importance and value of thesesystems in providing more objective and accurate information than visual measure-ments. ASMA technology employs techniques of computer image analysis, reducingthe element of uncertainty in the estimates of sperm morphology, and has become thefavored technique where subtle differences between samples are to be detected.

In most nonhuman species, the need to assess sperm quality is initiated by fi-nancial concerns and is based on maximizing reproductive efficiency. The monkey,because of its close evolutionary relationship to the human, is of special interest instudies of the reproductive function [Naz & Wolf, 1994; Cooper & Yeung, 1998]. Rela-tively little is known, however, about ejaculated sperm from adult male monkeys[Martin et al., 1975; Yeung et al., 1989; Gould et al., 1993]. Moreover, sperm cells arelikely to be under intense selective pressure given their crucial role in the reproduc-tion of individuals. The evolution of features such as sperm size and shape is prob-ably the result of two major selective forces: female reproductive physiology and spermcompetition [Roldan et al., 1991], but the information available on these two charac-ters is scarce and scattered [Cummins & Woodwall, 1985]. Therefore, the aim of thepresent investigation is to study the morphology of cynomolgus monkey sperm and toprovide reference values for their head dimensions using an ASMA system.

METHODSFourteen cynomolgus monkeys (Macaca fascicularis) were used, and ejacu-

lates were obtained as previously described [Yeung et al., 1989]. Semen sampleswere taken to the laboratory from the animal house in a 37°C incubator and

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maintained for 30 min at this temperature before being smeared as for humanspecimens [World Health Organization, 1992]. All animals were untreated andfully adult, but their ages were unknown since they were caught in the wild.Body weights are summarized in Table I.

Slides were fixed in a mixture of equal volumes of 96% ethanol and diethylether and were stained with Harris’s Haematoxylin (Papanicolaou’s solution 1;Merck (catalog number 9253), Darmstadt, Germany) by leaving them in the stainfor 1 h. All slides were then permanently sealed with mounting medium (Eukitt;O. Kindler GmbH & Co., Freiburg, Germany) and a coverslip.

Morphological analysis was carried out with an Olympus BH-2 microscopewith a bright-field ×100 oil immersion objective and a ×10 ocular. For each sample,300 spermatozoa were selected at random and evaluated visually (by C. Gago) torecord the morphological abnormalities. The morphological categories into whichthe spermatozoa were classified were determined following a preliminary studyof the samples and are described in Results.

Morphometric analysis was performed using the morphometric module of theSperm Class Analyzer® (SCA®) computer-aided sperm analysis system (Microp-tic, Barcelona, Spain). The equipment used consisted of an Olympus BH-2 micro-scope with a bright-field ×100 oil immersion objective (plan apochromatic; N.A.1.3), on which was mounted a Sony CCD AVC-D7CE video camera (Sony Corpo-ration, Tokyo, Japan) with a ×3.3 photo-ocular. The SCA® configuration includesa 486 PC with a PIP-1024 B video digitizer board (Matrox Electronic SystemsLtd., Quebec, Canada), an Intel 487 DX math coprocessor, and the sperm imageanalysis software. Final magnification on a Sony Trinitron PVM-1443MD moni-tor after zooming was 7000, giving a resolution of 0.15 µm and 0.11 µm per pixelin the horizontal and vertical axes, respectively. A minimum of 100 spermatozoafrom each slide was analyzed, a sample size which has been demonstrated togive values statistically representative of the total population for this species[Gago et al., 1998]. The sperm heads were captured at random, only those thatoverlapped other cells or debris being rejected. This process was performed manu-

TABLE I. Body Weight and Frequency of Abnormalities Appearing in CynomolgusMonkey Sperm*

Weight % normal %Abnormal spermatozoaAnimal (kg) spermatozoa Head Midpiece Tail Midpiece + tail

1 6.0 87.0 0.0 4.0 8.3 0.72 7.3 89.0 0.0 5.0 5.7 0.33 4.9 88.0 0.0 7.7 3.7 0.74 4.1 78.7 0.3 3.0 17.0 1.05 7.0 85.3 0.3 9.0 4.7 0.76 5.9 63.3 0.3 9.0 19.7 7.77 5.1 91.3 0.0 4.3 4.0 0.38 5.1 72.7 0.0 2.0 23.3 2.09 6.1 85.0 1.0 3.7 10.3 0.0

10 6.3 90.0 1.3 2.0 5.0 1.711 5.4 82.3 0.7 4.0 11.3 1.712 6.6 75.0 0.7 4.0 19.0 1.313 5.3 87.3 0.3 4.3 7.0 1.014 6.2 80.3 0.3 6.7 11.7 1.0Mean 5.8 ± 0.9 82.5 ± 7.9 0.4 ± 0.4 4.9 ± 2.3 10.8 ± 6.6 1.4 ± 1.9

*Values are mean ± SD.

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ally in order to avoid the inclusion of particles that might put obstacles in theway of subsequent image processing. A predetermined range of gray-level valueswas then introduced for the segmentation of sperm heads. Erroneous definitionof the head boundary was corrected with an editing facility incorporated into thesystem. The metric measurements obtained for area, perimeter, length, width,gray-level, length/width ratio, ellipticity, and three shape factors from each prop-erly digitized sperm head were saved on the hard disk of the computer for fur-ther statistical analysis (Fig. 1).

StatisticsStatistical analysis was performed with version 6.1.2 SPSS for Windows.

Kolmogorov-Smirnov and Levene tests were applied to analyze the data normal-ity and homogeneity of variance, respectively. If data for one variable were nor-mally distributed and their variance was homogeneous, ANOVA followed by theTukey-HSD test for multiple comparison was performed (for the second shapefactor). When data showed a normal distribution but did not satisfy the assump-

Fig. 1. Schematic representation of a spermatozoon withdefinition of parameters assessed for the morphometric char-acterization of sperm heads from cynomolgus monkey ejacu-lates. Area (A) is the number of pixels contained within theboundary multiplied by the area occupied by each individualpixel. Length (L) is the largest value of the feret diameters,and width (W) is the smallest value of the feret diameters;feret diameters were measured at angles of 0, 30, 60, 90,120 and 150°, and W is not necessarily orthogonal to L. Pe-rimeter (P) is the sum of external boundary pixels. Gray levelis the mean object gray level; values range from 0 (black) to255 (white). Derived parameters were calculated followingthe expressions: L/W ratio = L/W; ellipticity = (L - W)/(L +W); shape 1 factor (S1) = 4 π A/P2; shape 2 factor = S1 L/W;shape 3 factor = π L W/(4 A).

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tion of the homogeneity of variance, a conservative F proof was carried out be-fore the a posteriori test for group comparison (for area, perimeter, length, ellip-ticity, and ratio). Finally, where data did not adjust to a normal distribution, theKruskall-Wallis nonparametric test followed by the Mann-Whitney U test wasperformed (for width, gray level, first shape factor, and third shape factor). Inaddition, in order to characterize cynomolgus sperm heads, a descriptive statisti-cal analysis of the 14 individuals was performed. Estimates of location (mean),dispersion (standard deviation and coefficient of variation), and shape (skewnessand kurtosis) were obtained for each variable. Lastly, once the data variationhad been established, power analysis was performed in order to establish theminimum number of animals to be used in future studies (SOLO Power Analy-sis; BMDP Statistical Software Inc., Los Angeles, CA).

RESULTSFollowing the analysis of the sperm morphology, it was possible to determine

the following sperm types: 1) normal spermatozoa, characterized by an ovoid, well-delineated head shape with an apical half representing the acrosome less stainedthan the basal part and a flagellum composed of the midpiece and tail portionswith no bending, coiling, breakage, or thickening along its length (Fig. 2a), 2)spermatozoa with an abnormal head, in which size or shape deviations from anormal spermatozoon could be distinguished (Fig. 2b), 3) spermatozoa with a de-fective midpiece (Fig. 2c) or tail (Fig. 2d), and 4) spermatozoa having any combi-nation of the above mentioned morphological defects, with spermatozoa presentinga coiling of the midpiece and tail being the most frequent (Fig. 2e).

In the morphological analysis, 82.5% normal spermatozoa and 17.5% ab-normal spermatozoa were found. The percentages of head, midpiece, tail, andmultiple abnormalities from each individual and globally are shown in TableI. Head abnormalities were not frequent (0.4%), the values ranging between 0and 1.3%. Large (macrocephalic), tapering (narrowed at the basal part), andamorphous forms were found. Midpiece imperfections were found in all indi-viduals; the mean percentage was 4.9%, and the range varied between 2 and9%. Tail defects were the most common (10.8%) and showed the highest varia-tion between individuals, the values ranging between 3.7 and 23.3%. Tail de-fects corresponded to coiled or folded tails. Tail plus midpiece was the onlymultiple abnormality observed, with a mean value of 1.4% and a range be-tween 0 and 7.7%. The majority of these double defects consisted of a coiledtail together with a coiled midpiece.

Statistical description of the morphometric values of morphologically normalsperm heads for the 14 individuals was made (the values are presented in TableII). Higher CVs were obtained for area, ellipticity, and gray level than for theremaining parameters (perimeter, length, width, L/W ratio, and the three shapefactors), for which CVs were below 7%. Values of skewness and kurtosis wereclose to zero, which corresponds to a normal distribution.

Additionally, statistical analysis of morphometric values denoted differencesbetween individuals for all the parameters under consideration. Figure 3 pre-sents graphs of each parameter for the 14 individuals. A power value >0.8 wasobtained with a sample size of eight animals and a value of 1 for 14 animals,which demonstrates that differences between animals do exist. Even though thevalues obtained for all the animals are similar, the fact that closely ranged val-ues for each morphometric variable are found within individual animals is re-sponsible for the statistically significant differences observed.

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DISCUSSIONThe standardization of semen processing procedures has been recognized

as an important step towards reducing the subjectivity of the assessment ofsperm head morphometry, as has the description of the optimum operationalconditions of preparation, fixation, and staining methods [Davis & Gravance,1993; Gago et al., 1998]. In the present study, the 14 samples were stainedwith Harris's Haematoxylin. In a previous work, research was done into themost suitable staining method for cynomolgus sperm using the SCA®, andHaematoxylin was selected because the definition of the sperm head boundarywas more accurate and precise when compared to Diff-Quik and Hemacolor[Gago et al., 1998].

Mammals, except humans, gorillas, and certain endangered species, usu-ally have a high percentage of morphologically normal spermatozoa. In thepresent study, the percentage of abnormalities present in cynomolgus spermwas 17.5%, the majority being tail abnormalities (10.8%). This value is lowwhen compared to percentages reported for the same species (30–36%) and for

Fig. 2. Light micrographs of Haematoxylin-stained cynomolgus monkey ejaculated spermatozoa showingnormal and abnormal morphologies. a: Normal spermatozoon. b: Head defect. c: Midpiece defect. d: Taildefect. e: Midpiece plus tail defects. Scale bar = 10 µm.

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other species of the genus Macaca: values from 22–47% in a number of studiesof rhesus monkeys and 32% for M. radiata [Wildt, 1986]. Different values mayarise due to differences in sampling and preparation methods as well as tointeranimal variations and to the variability inherent in the subjective assess-ment of morphology.

The cause of abnormal morphology of the head, midpiece, and tail is notclear. It has been suggested that this may occur during spermatogenesis (pri-mary defects) or that morphological defects arise during posttesticular matura-tion, thus being considered secondary defects [Barth & Oko, 1989]. However,abnormalities can also be caused during the manipulation of the slides. Footeet al. [1992] observed that in bulls 1% of spermatozoa had no tail, and theywere of the opinion that this fact was probably due to the breaking off of anoccasional tail in preparing semen smears. In the monkey, as in man, the per-centage of normal forms increases on passage from the caput to the cauda ofthe epididymis [Yeung et al., 1997]. The majority of defective cells from theproximal epididymis is in the form of swollen or exploded heads which occurwhen making air-dried smears of nonfixed immature sperm. This sensitivity toair-drying shown by immature sperm is lost upon maturation and is not foundin the more mature sperm from the epididymal cauda and ejaculate. The mor-phological defects of sperm heads found in the present study do not seem tocorrespond to this type of abnormality and probably constitute primary defects.The primary or secondary origin of midpiece and tail defects found in cynomol-gus sperm is unclear, and further studies must be carried out before a definiteconclusion can be reached.

Numerous studies have been done into the morphological and morphometriccharacteristics of semen of several species, but, while significant differences inmorphometry were detected in mean group values, the criteria by which indi-vidual spermatozoa may be classified as normal or abnormal and what their fer-tility potential is are still under discussion. Many studies have described acorrelation between the percentage of normal morphology and in vitro fertility[see Ombelet et al., 1995], but there is virtually no information on the role thatspecific types of sperm morphological defects play in fertility in vivo. In a previ-ous study in man, Carrell et al. [1994] showed that spermatozoa having taperedheads had a viability, as determined by the triple-stain technique of Talbot andChacon [1981], that was not significantly different from that of normal sperm.However, for all the other categories of sperm abnormalities considered in theirstudy, viability was significantly lower. Moreover, Lee et al. [1996] suggestedthat the abnormal size of human spermatozoa is not associated with chromo-some defects but merely with aberrant head development (amorphous, round,and elongated). Further accumulation of experimental data is needed, however,

TABLE II. Morphometric Characterization of Cynomolgus Monkey Sperm Heads*

L/W Gray Shape Shape ShapeArea Perimeter Length Width ratio level Ellipticity 1 2 3

Mean 17.17 15.24 5.81 4.01 1.45 98.30 0.18 0.93 1.35 1.06SD 1.44 0.66 0.31 0.26 0.09 15.72 0.03 0.03 0.07 0.05CV (%) 8.44 4.15 5.26 6.37 6.34 15.99 16.39 2.90 5.57 5.00Skewness 0.26 –0.01 0.08 0.14 0.28 0.21 0.06 –0.37 0.05 –0.13Kurtosis 0.26 0.05 –0.03 0.06 0.07 –0.88 –0.08 –0.03 0.13 –0.92

*Values of mean and SD are given in microns (perimeter, length, and width), squared microns (area), andvalues from 0–255 (gray level); shape factors and ellipticity are nondimensional.

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Fig. 3. Box-and-whisker plots showing sperm head morphometric values corresponding to the 14 cynomol-gus monkeys. Units are given in microns (perimeter, length, and width), squared microns (area) and valuesfrom 0–255 (gray level); shape factors and ellipticity are nondimensional. Each box encloses the 25 and 75percentiles; the line in the middle is the median, and the whiskers extend to the 5 and 95 percentiles of themean values. A minimum of 100 spermatozoa from each animal was analyzed. Boxes labelled with differentsuperscripts are statistically different from each other (P < 0.05).

Morphology of Cynomolgus Sperm / 113

before any definitive conclusions can be drawn about the implications that thepresence of specific sperm morphological abnormalities may have on the abilityof a semen sample to fertilize.

The morphometric results obtained here are slightly higher than those de-scribed by Martin et al. [1975] after scanning electron microscopy (i.e., 5.53–5.80µm for head length and 3.45-4.01 µm for width), but their technique was differ-ent and when employed on human sperm provided higher values; thus, the headlength obtained by Martin and coworkers for human spermatozoa was 6.11 µm,while the World Health Organization (1992) reference value is 4.0–5.5 µm.

When the cynomolgus monkey sperm were compared with other species ofthe order Primates, it was observed that the morphometric values were conser-vative [Cummins & Woodwall, 1985], although it is necessary to point out thatthese data have been obtained using different methodological approaches. As aconsequence, more work is needed before any serious evolutionary conclusionscan be drawn, as is the case with other biological groups [Roldan et al., 1991].

It is fundamental to establish which are the most representative parametersfor each particular species. In rabbit ejaculate, the sperm head width/length ra-tio seems to be sufficient for the discrimination between animals [Gravance &Davis, 1995]. In the present work, among the most useful parameters for spermcharacterization, according to their low variability, were perimeter, length, width,L/W ratio, and the three shape factors, all showing the lowest values in coeffi-cient of variation (%: perimeter, 4.2; length, 5.3; width, 6.4; L/W ratio, 6.3; firstshape factor, 2.9; second shape factor, 5.6; third shape factor, 5.0). Therefore, theseparameters could be the most important in posterior studies of cynomolgus mon-key sperm.

In addition to the general study, an appraisal of individuals was made. Themorphometric analysis of sperm head dimensions revealed significant differencesbetween individuals in all the parameters. In Figure 3, a distinctive distributionof values is clear for each individual. The determination of significant differencesin the morphometric dimensions between individuals is probably due to the ac-curate and objective measurements obtained by the SCA® system, which has beenput to the test in humans with good results [De Monserrat et al., 1995] and hasdemonstrated its accuracy and sensitivity in other species [Sancho et al., 1996;Gago et al., 1998]. Individual variation is an important factor, and it must betaken into account in any sperm research. The morphometric differences observedcould indicate the presence of spermatozoa with a normal aspect but of differentsize. Thus, two animals with the same percentage of normal spermatozoa couldhave different morphometric values. With ASMA systems, subtle differences be-tween samples can be detected, as has been observed in man and other animalsunder both normal [Jagoe et al., 1986; Gravance & Davis, 1995] and experimen-tal [Young et al., 1982; Davis et al., 1994] conditions. Subpopulations of “normalspermatozoa” with possible different fertility profiles might exist, and thus se-men analyses would need to be performed in order to identify these subpopula-tions separately from the average semen population values [Pérez-Sánchez etal., 1994; Gravance and Davis, 1995].

The results of the present study, with the accuracy and precision of the SCA®

system, provide a definition of morphometric parameters of normal sperm headsfrom cynomolgus monkeys that helps in the establishment of a nonhuman pri-mate model for the study of reproductive toxicology, contraception, and fertility.The data presented might also be considered the beginning of a systematic mor-phometric characterization of primate sperm which could provide new ways ofunderstanding the evolutionary process of reproduction of this Order.

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CONCLUSIONS1. The Sperm Class Analyzer® instrument constitutes a useful tool for the

definition of the dimensional features of monkey sperm heads.2. Normal cynomolgus monkey spermatozoa are defined as having no defects

in the tail or midpiece and a head with a regular ovoid shape with a length of5.5–6.1 µm, a width of 3.7–4.3 µm, and a length/width ratio of 1.4–1.5.

3. In cynomolgus monkeys, 82.5% of ejaculated spermatozoa are morphologi-cally normal (range 63–91%), although differences in sperm head morphometrybetween animals are noticeable. Parameters showing the least variation are pe-rimeter, length, width, L/W ratio, and shape factors of the sperm head.

ACKNOWLEDGMENTSWe thank Dr. G.F. Weinbauer (IRM Münster) for helping with the provision

of monkey ejaculates, Mr. I.V. Costello for his assistance in the preparation ofthe manuscript, and P.M. Medina and V. Girbes for data analysis advice. Thisstudy was partially conducted during C.S.’s stay at the Institute of ReproductiveMedicine of the University, Münster, and T.G.C.’s stay at the University of Valènciaand was supported by travel grants from the University of València and theGeneralitat Valenciana.

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