Table of Contents  
Year : 2016  |  Volume : 18  |  Issue : 6  |  Page : 863-870

Current status and potential of morphometric sperm analysis

1 SaBio IREC (CSIC - UCLM - JCCM), Albacete, Spain
2 Biomedical Center, Medical Faculty in Pilsen, Charles University in Prague, Pilsen, Czech Republic
3 Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, Valdepeñas, Spain
4 Institute for Animal Health and Cattle Development, University of León, León, Spain
5 Department of Molecular Biology, University of León, León, Spain
6 Faculty of Pharmacy (UCLM), Albacete, Spain

Date of Web Publication27-Sep-2016

Correspondence Address:
Dr. José Julián Garde
SaBio IREC (CSIC - UCLM - JCCM), Albacete
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1008-682X.187581

Rights and Permissions

The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment.

Keywords: computer-assisted sperm morphometric analysis; mammals; sperm function; sperm morphometry

How to cite this article:
Maroto-Morales A, García-Álvarez O, Ramón M, Martínez-Pastor F, Fernández-Santos M R, Soler A J, Garde JJ. Current status and potential of morphometric sperm analysis. Asian J Androl 2016;18:863-70

How to cite this URL:
Maroto-Morales A, García-Álvarez O, Ramón M, Martínez-Pastor F, Fernández-Santos M R, Soler A J, Garde JJ. Current status and potential of morphometric sperm analysis. Asian J Androl [serial online] 2016 [cited 2021 Aug 1];18:863-70. Available from: - DOI: 10.4103/1008-682X.187581

  Introduction Top

Sperm research has received increased attention in recent decades, given the peculiarities of this cell: it develops part of its lifespan outside the male body, often in a hostile environment, and it carries the genetic material from the male to the oocyte. The differences in sperm morphology and physiology, even between related species, and the presence of highly specialized structures, have led to questions on the reasons for this diversity and specialization. Considering different sperm features, the study of sperm morphology has been considered an essential part of sperm research. Two main questions arise: how did the diversity in sperm morphology arise during speciation (a backward perspective) and what role do the different specializations play in sperm function (a forward perspective)?

Several studies have addressed the first question, many of them from an evolutionary point of view, and the majority being descriptive.[1],[2],[3] These studies have put considerable effort into finding the ultimate cause of this sperm diversification, and how within- and between-male variation and within- and between-species variation contribute to sperm performance and behavior.[4],[5] Adaptation to specific fertilization environments and the fertilization process itself have been proposed as the main selection forces.[4],[6],[7] With these forces, sperm competition seems to play a major role, influencing not only sperm morphology but also sperm length and sperm numbers.[8],[9]

Considering the forward perspective, most studies have tried to establish functional relationships between sperm traits during the fertilization process and their performance in assisted reproductive techniques (ARTs), with regard to sperm resilience or fertility. Typically, traits such as motility, viability (either plasma-lemma integrity or the hyper-osmotic shock test [HOST] responsiveness), acrosomal integrity, or absence of abnormalities have been used as endpoints for predicting sperm fertility or, rather, discarding potentially low-fertility semen doses.[10],[11],[12],[13] Availability of advanced techniques and hardware such as computer-assisted sperm analysis (CASA),[14],[15],[16] fluorescence probes and ultimately flow cytometry,[17],[18] and new endpoints, e.g. capacitation and chromatin assessment,[19],[20] have allowed more objective and faster analysis, but the predictive power of laboratory sperm assessment still needs improvement.[21] The routine evaluation of semen has traditionally included the assessment of normal sperm morphology, but the important subjective component has limited its practical use.[22] The development of automatic image-processing systems has displaced classical methods and is a major advance in sperm analysis. Computer-assisted sperm morphometric analysis (CASA-Morph) systems have been successfully used to determine the relationships between sperm shape and fertility of males[23],[24] or sperm freezability.[25],[26]

There is a need to develop new analytical tools to capture sperm diversity better, and to improve data analysis methods. New equipment should take advantage of high-resolution optics and image analysis programs to increase accuracy and precision of laboratory tests; the implementation of this new equipment should allow the simultaneous assessment of morphological and functional sperm features. Given the high amount of information that is expected in the mid-term future, the development of new statistical methods is also necessary for the joint analysis of all these sources of information, which will allow a better assessment of relationships among sperm features and their functional meaning.

  Morphological Sperm Diversity Top

The spermatozoon is the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which the cells function, as a way to ensure their survival in different fertilization environments and to maximize their fertilizing capacity.[4],[6] The spermatozoon of thousands of species has been described including insects,[27] crustaceans,[28] fish,[29] birds,[30] mammals,[31],[32] and many other groups. The existence of morphological diversity among species is widely recognized, with a high degree of variation in size, shape, and behavior,[3],[32],[33] reflected in main structures (i.e., head, midpiece, and principal piece) and overall size. Different specializations have been observed, such as the absence of a flagellum or multiple flagella, gigantism, the presence of an apical hook, bristles or brushtail, etc. In some species, the shape of the spermatozoon allows it to cooperate with others, as in the case of sperm conjugation, a phenomenon in which two or more spermatozoa are physically united during transport through female reproductive tract.

A first attempt to explain this diversity was conducted by Franzén[4],[6] who proposed that sperm modifications are adaptations to their specific fertilization environment. Thus, pre- and post-copulatory selection such as mate choice, mode of fertilization, cryptic female choice, sexual conflict, and sperm competition may influence the evolution of sperm traits.[5] Two of these traits are likely to play an important role as selective forces: female selection and sperm competition.[9] Numerous studies have focused on the correlated evolution of sperm length and female reproductive tract design,[34],[35],[36],[37],[38] suggesting a sexual coevolution of the fertilization system.[39] Selection would favor very different sperm traits depending on whether spermatozoa are released into open water (e.g., external fertilizers), have to remain for longer periods of time in female storage organs (e.g., birds, bats and insects), or have a short time window to fertilize after being transferred to the female tract (e.g., mammals).

Sperm competition has also been found to be a significant source of sperm variability.[40],[41],[42],[43],[44] High levels of sperm competition are associated with increases in testicular mass relative to body size[5],[41],[45],[46] and with high relative sperm numbers[45],[47],[48] in many taxa. While initial hypotheses were that an energy trade-off exists between sperm numbers and sperm size,[49] and consequently sperm competition would result in a reduction in sperm size, later studies demonstrated that higher sperm size represented an advantage by increasing sperm velocity[43],[44],[48],[50],[51],[52] and that sperm competition, therefore, resulted in an increase of sperm size.[41],[53],[54]

Evolutionary forces

Morphological variation has been driven by evolutionary forces. However, the development of assisted reproductive techniques (ARTs) has introduced a new - artificial - source of variation in sperm morphology.[55] The in vitro fertilization process includes stages outside the male and female reproductive tracts, during which spermatozoa are subjected to procedures aimed at maximizing reproductive success. The sperm characteristics that determine which are the best for ART may not be the same as those determined from a physiological assessment. Therefore, it may be expected that those males having a higher proportion of the relevant kind of spermatozoa would produce more offspring, ultimately leading to a selection toward more favorable sperm design for ART.

A number of studies have reported that spermatozoa with smaller heads withstand the cryopreservation process better[56],[57] and that the sperm head morphometry-cryoresistance relationship is in part genetically determined.[26] The use of selection media before AI could also select cells of a specific morphology[58] and could favor a certain shape although these methods are expected to work in a similar manner to the selection processes occurring in the female reproductive tract.[59],[60] It is too soon to assess the role that ART plays in the evolution of sperm morphology, but it might be advisable to follow up morphological changes derived from the use of these techniques, and their consequences.

  Assessment of Sperm Morphometry I - Technological Aspects Top

Many techniques can assess sperm morphometry[61],[62] but CASA-Morph has become the main choice, because it provides increased reliability and repeatability, and reduced subjectivity.[62],[63],[64] Studies that assess the different sources of variation affecting CASA-Morph are critical for guaranteeing its repeatability and consistency among laboratories. The main sources of variation of CASA-Morph, other than the software and data analysis, are the sample preparation, fixation method, staining method, microscopic system (optics and camera), and technician. All these steps can affect not only the repeatability of the experiments but also the reproducibility and the comparison of results among laboratories, which are necessary for the practical use of sperm morphometric analyses. These aspects have been studied by several authors,[64],[65],[66],[67],[68],[69],[70],[71] and they have not yet been completely resolved.

Sample preparation

The preparation of the sample, its concentration, and the fixation procedure are the first steps to consider in a CASA-Morph protocol. Varying the sperm concentration may affect CASA-Morph performance.[66],[72],[73],[74] Fixation, together with drying of the sample, has received little attention, but they are both critical steps, and it has been demonstrated that they affect CASA-Morph results.[75],[76] During slide preparation, it is advisable to make at least one replicate so that inter-slide variability can be assessed,[77],[78] and that replicates that fall outside certain thresholds can be rejected, because of unacceptable variability in slide preparation.

The choice of staining protocol is the issue on which most authors have conducted their studies[63],[65],[66],[68],[70],[71],[72],[73],[79],[80] since it influences background noise, sperm contrast, and the identification of different areas within the cell. To prevent these problems during morphometric sperm analysis, fluorescence-based methods in combination with a CASA-Morph system have been developed for a more precise measurement of the nucleus, acrosome, and sperm head, yielding promising results.[64],[81] In addition, a new system has been developed (Trumorph® , Proiser R+D, Paterna, Spain) that avoids fixing and staining of the sample, and in combination with phase-contrast microscopy, allows assessment of sperm size and shape in wet-mount preparations.[82],[83]

The higher the quality of the hardware (microscope, lenses, and camera), the more reliable the analysis, at least for experimental work. At high magnification, lenses capable of providing a sharp, aberration-free and bright image, and a camera capable of high resolution will reduce the errors of the CASA-Morph software and will allow a more reliable analysis.[69],[74] However, widespread use of CASA-Morph systems in clinical or production environments may require the use of cheaper equipment. In such cases, efforts should be directed at improving staining results and optimizing software to cope with the limitations due to the use of basic equipment.

Regarding software, automatic methods should be preferred, to save analysis time and to reduce operator errors. Goulart et al.[69] suggested using semi-automatic methods, with some operator intervention. When the interaction of the technician with the software is limited to removing misdigitized spermatozoa, the effect of the technician in the analysis is relatively low, compared with subjective morphology assessment.[25],[78],[84]

Replication and statistical procedures

Some authors have tried to establish the minimum number of spermatozoa necessary for obtaining reliable and stable morphometry parameter values. A general recommendation is that at least 200 spermatozoa should be analyzed per sample[85] although some authors have suggested that lower numbers could be adequate for some species and experiments.[25],[68],[70],[72],[84],[86] If the aim of the analysis is to obtain a proportion, usually the percentage of normal spermatozoa, the confidence intervals vary with the value of that proportion. If the proportion of abnormal forms is close to the extremes of the range (0%-100%), a situation that is fairly common in animal reproduction, the coefficient of variation (CV%) increases considerably.[85],[87] Currently, the capabilities of computers, sophistication of CASA-Morph software, and availability of automatic acquisition systems (e.g., microscopes with motorized stages and autofocus) allow the acquisition of large amounts of data with little operator time. Moreover, if the aim of CASA-Morph is subpopulation analysis, the number of spermatozoa analyzed needs to be high, because the total number analyzed (the sample size) must be divided among the subpopulations, thereby decreasing the statistical confidence of the statistics defining each population. This would be aggravated if there were subpopulations of relatively low size (thus receiving a small percentage of the total number of spermatozoa analyzed).

In addition, it is also advisable to assess a high number of individuals, to have an adequate representation of the species, allowing conclusive results to be obtained. Thus, the main problem of most CASA-Morph studies so far is the use of a low sample size.[23],[26],[88],[89],[90],[91] Only a few authors[24],[92],[93],[94] have conducted large-scale studies to assess, in the same species, the sperm head morphometry and also to study its relationship with sperm function.

It must be pointed out that most studies have used incorrect statistical techniques to compare protocols, not only regarding the number of spermatozoa analyzed but also technician effect, stains, etc. These statistics, generally based on a means comparison or correlations/regressions, are not appropriate for assessing differences between methods.[95],[96] Thus, the conclusions are usually limited and should be reevaluated using the appropriate methodology. Only a few studies[84] have applied correct statistical methods (e.g. Bland and Altman agreement coefficients[97]).

Regarding the consistency and repeatability of analyses, laboratories should set up a quality assurance system for CASA-Morph. The use of latex beads of a determined size[69] or standard sperm doses could be combined in a quality assurance protocol, which could also be used for assessing inter-laboratory variability.

As a final point, CASA-Morph protocols and software should be validated, a process that is not always straightforward. In general, protocols have been validated for reduced intra-slide and between-slide (for the same sample) variability, while enhancing between-male or between-treatment variability and reducing digitization or analytical errors.[66],[68],[78] Some studies have compared different protocols, describing their respective strengths and weaknesses. However, most validations lack the definition of a "gold standard" that would allow a broader comparison among studies and protocols. Examples of such a "gold standard" would be morphometric data obtained from electron microscopy (much more resolution and thus more reliable, although not fit for routine use) or a previously validated technique. Some authors have used other methods, such as measuring the heads directly on screen with calipers, and comparing these measurements with those provided by the CASA-Morph software.[70]

  Assessment of Sperm Morphometry II - Functional Aspects Top

Studies on the relationships between sperm design and sperm function have often yielded contradictory results.[24],[44],[89],[94],[98],[99],[100] Most of the research made in this aspect has usually been conducted at an interspecific level[44],[101] since finding differences between species is easier than intraspecific or intra-male levels. Studies at the intraspecific level are quite limited and most of them have used a low sample size (fewer than 25 individuals)[23],[88],[89],[91] making robust conclusions difficult to obtain.

Recently, some authors have reported that some sperm characteristics are genetically determined,[26],[102] sperm morphometry being one of them. Thus, it is expected that sperm morphometry reflects differences in sperm function. Indeed, different sperm morphometric features have been identified between breeds[103] but also between animals from the same breed belonging to different herds (i.e., reflecting their origin).[92]

Ignoring the differences between the morphometric dimensions of X- and Y-bearing spermatozoa due to their DNA content,[104] most differences detected between spermatozoa are probably caused by changes in the media in which spermatozoa are suspended, which could modify the sperm volume. Some authors have explored the morphological response to diluting or washing the sperm sample,[105],[106] to capacitation[107] or to cryopreservation.[26],[108],[109],[110]

Sperm cryopreservation and morphology

The effects of cryopreservation on sperm head morphometry have been studied in numerous species: humans,[111] bulls,[105],[112] boars,[113],[114] rams,[26] goats,[115],[116] stallions,[117] dogs,[118] bears,[119] and red deer.[56],[57] All these studies have reported a significant reduction in sperm head dimensions by cryopreservation of freshly extended samples. This reduction in sperm head dimension is reflected not only in the size of sperm head but also in its shape. Different hypotheses have been proposed to explain the reasons for the sperm head dimension decrease after cryopreservation, including osmotic changes,[117],[120] alterations of some cell compartments,[25],[117] damage or loss of the sperm acrosome,[121],[122] and over-condensation of sperm nuclear chromatin.[74],[119]

The spermatozoa from different individuals may exhibit significantly different responses to the same freezing treatment.[123],[124],[125] Thus, males can be classified as "good" or "bad" freezers on the basis of their sperm cells' resistance to the cryopreservation process, and sperm morphometry is a useful tool to this end. For example, Hidalgo et al.[115] observed that in the goat, sperm morphometric changes after cryopreservation were lower in semen samples showing better quality in fresh semen, while a further reduction in sperm dimensions was observed for those semen samples with initial poor semen quality. Esteso et al.[126] observed that deer with better cryoresistant spermatozoa were characterized by a low sperm head area and a large sperm head shape factor (defined as the length/width ratio). These authors also observed that semen samples with lower intravariability for sperm morphometric measurements showed smaller changes in their morphometry.[56] Moreover, they reported that those males with no or small changes in sperm dimensions after freezing-thawing showed a low sperm head dimension in the extended samples. These authors thus suggested that in semen samples of better quality the sperm cryodamage was less and the effect of cryopreservation on the sperm head morphometry was also less. Ramon et al.[26] have gone a little further on the prediction of sperm cryoresistance. Whereas a general study of the morphometric characteristics of the ram sperm head at each stage of the freezing process did not allow an adequate identification of the males with better sperm cryopreservation, the study of patterns of change throughout the cryopreservation process led to the identification of differently defined patterns clearly related to cryopreservation ability. Furthermore, these authors showed that each male retained its pattern of response for all ejaculates examined, and that those males sharing the same pattern of response were more closely related, suggesting the possibility of a genetic control of the response.

Therefore, the study of the morphological changes in response to cryopreservation may be presented as an opportunity to improve the reproductive ability of individuals. First, as a way to indicate what changes happen and how they occur, and second, as a tool to develop better ART methodologies to prevent undesirable response patterns, or for designing selection programs toward fitter sperm designs.

Despite most of the studies on the effect of cryopreservation on sperm morphology have been focused on the evaluation of sperm head features, Ros-Santaella et al.[109] recently reported that stags not differing in sperm head dimensions showed significant differences in sperm cryoresistance that were strongly related to the volume of the sperm principal piece. This study indicates a key role of the sperm flagellum during cryopreservation and suggests new approaches for the characterization of those spermatozoa with good cryoresistance.

Sperm physiology and morphology

Other aspects of sperm physiology and morphology may be considered; spermatozoa actively migrate through the female genital tract. In many species, the first barrier is cervical mucus, which only allows the advance to the uterus of progressively motile sperm with normal morphology and through which they migrate (with the aid of myometrial contractions) to the oviduct, where fertilization will take place.[127] In attempts to mimic this process of in vivo selection, different sperm selection methods are routinely used in in vitro protocols, such as in vitro fertilization (IVF) or sperm sorting, to enrich the sample with morphologically normal and motile spermatozoa.[128] During this process, not only morphologically normal and progressively motile spermatozoa are selected, but also the male germ cells undergo physiological changes, termed capacitation, which are fundamental prerequisites for the acquisition of functional sperm competence to undergo the acrosome reaction and hence fertilize the oocyte.[129] However, it is not yet known in detail how these processes affect sperm morphology; more importantly, the morphometric characteristics of the cells that eventually fertilize the oocyte are unknown. Recently, García-Vázquez et al.[130] reported how boar spermatozoa in the female reproductive tract are selected on the basis of their size and shape, with those with a larger head and longer tail being those reaching the fertilization site. For sperm morphometric changes during capacitation, García-Herreros and Leal[107] reported that the induction of in vitro capacitation in bovine spermatozoa modified sperm head morphometry. As capacitated spermatozoa showed a decrease in all sperm head size and shape parameters, the authors concluded that sperm head morphometry is an objective diagnostic tool for sperm assessment during capacitation.

During the migration of spermatozoa through the female genital tract toward the fertilization site, sperm motility is essential.[131] Several studies have addressed how sperm morphology and sperm velocity may be related,[23],[44],[51],[99],[109],[132] and their impact on reproductive performance.[23],[98],[133] However, results are contradictory on how sperm diversity translates into variation in sperm velocity.[43],[99],[132] Only a few studies have been made in the same species to study directly sperm design and motility. Ramon et al.[23] reported, for the first time, the relationships between stag sperm design and velocity (in the same sample) in a species with internal fertilization, and the role that both may play in male fertility. These researchers observed that males with ejaculates containing a high percentage of spermatozoa with fast and linear motility also had small and elongated heads and yielded higher fertility rates. These relationships were also reported by Fitzpatrick et al.[134] in externally fertilizing species (fish). Sperm head elongation may play an important role by making sperm more hydrodynamically efficient, which, in turn, may influence sperm fertilizing ability. Indeed, other authors have reported that spermatozoa with elongated heads may be faster[23],[44],[98] because of lower resistance to forward progression. This could compensate evolutionary constraints to increases in sperm length by allowing increased swimming efficiency, or for the same reason, it might increase sperm lifespan if energy reserves last longer or are used more efficiently, which would result in more spermatozoa reaching the fertilization site.[43]

In addition, because most of the sperm head is occupied by the nucleus, its compactness can influence sperm head shape. Some authors have presented evidence supporting the involvement of protamines in sperm head shaping, leading to smaller and more elongated sperm heads when they are present in high proportions.[135],[136] Similar results have been reported by Gömez Montoto et al.[137] who observed in rodents that an increase in sperm swimming speed is also associated with elongated sperm heads. On the other hand, they also found that an increase in total sperm size maximized the chances that sperm cells would reach the ova in a sperm competition context. On whether sperm shape and dimensions reflect defects in the structure and integrity of the sperm chromatin, Sailer et al. (1996) reported that variations in sperm head morphometry were related to abnormal chromatin structure in the bull. However, many researchers have not found these relationships in other species.[103],[138],[139]

There are few intraspecific studies on the role of sperm morphometry and reproductive performance, and they have provided contradictory results.[23],[24],[94],[100],[112] For this reason, it is advisable to settle these questions using more sophisticated techniques that define which sperm morphometric parameters are crucial for breeding success.

  Perspectives on the Future of Sperm Morphometric Studies Top

Normal sperm morphology is a major criterion in sperm quality evaluation. However, although there is evidence of relationships between sperm morphometric characteristics and fertility, results vary widely and are sometimes contradictory. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, with emphasis on intraspecific variability. In addition, more attention should be focused on other sperm structures besides the head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function. Such technology could combine high-resolution optics and image analysis programs to increase the accuracy and precision of laboratory tests, and these should go hand-in-hand with the advance in new statistical methods that allow the analysis of large amounts of data given for these novel technologies.

Statistical approaches

Since the importance of sperm flagellar dimensions on sperm function has been demonstrated,[98],[109] CASA-Morph systems should be modified to incorporate new tools that allow an automatic and accurate evaluation of these sperm structures. These advances will allow the undertaking of basic studies of sperm morphometry since most of the research done so far has focused only on the sperm head. Therefore, future studies of sperm morphometry should not overlook the role of the flagellum as a modulator of sperm function.

Regarding the accuracy of the evaluation of the sperm shape, CASA-Morph systems only provide an approximation of head shape on the basis of sperm head linear dimensions.[26],[55],[73],[92] Elliptic Fourier analysis is one method, based on the use of successive points located by a coordinate system that fits the cell perimeter to a Fourier function, and that can identify more features of morphological variation in spermatozoa than manual methods.[140],[141] However, it has not been used by many researchers. Moreover, geometric morphometrics has been developed to avoid the shortcomings of CASA-Morph morphometric parameters and was recently used by Varea Sánchez et al.[142] to evaluate sperm head morphometry in rodents. These authors obtained a better characterization of sperm shape, finding some regions in the sperm head that were not characterized by the linear descriptors, and which were nevertheless susceptible to change. Furthermore, geometric morphometrics should allow the assessment of size and shape separately, and the exact definition of where the main shape differences are located in the sperm head. Nevertheless, that study dealt with different mouse species, whose sperm heads have convoluted shapes and vary noticeably between species. However, it is possible that in species with spermatozoa of more simple shape (ungulates, primates, and humans), classical CASA-Morph systems can provide enough information to define sperm head shape adequately.

Sperm preparation

Other new methods have been developed to evaluate sperm morphometry without the need for sperm staining.[143] These methods are focused on humans and allow the use of the observed spermatozoon to be used in assisted reproductive techniques such as ICSI after the measurement. However, since chromatin and DNA integrity are not always related to sperm head size or shape,[103],[138],[139] further studies should be done to find phenotypic sperm parameters providing accurate information about sperm function, allowing the selection of spermatozoa which will generate healthy offspring. Along this line, the study of proteins associated with sperm head shape[144] would offer an important new tool to deepen knowledge of sperm shape and sperm function.

The combination of flow cytometry with single quantitative image analysis will provide new and interesting capabilities. This type of analysis will couple the collection of high-throughput data with streamlined image analysis. Sperm features such as size and shape, granularity, intensity, radial distribution, and texture could be obtained[145] in a large sperm population. In addition, the main advantage of this technique, which makes it unique, is the ability simultaneously to evaluate the morphometric and physiological parameters in the same sperm cell.

  Author Contributions Top

AMM, OGA, MR, FMP, MRFS, AJS, and JJG conceived the study, wrote the manuscript, and reviewed drafts of the manuscript. All authors read and approved the final version of the manuscript, and agree with the order of presentation of the authors.

  Competing Interests Top

The authors declared no competing interest.

  Acknowledgments Top

This study was sponsored by Grant PEII-2014-032-P from Junta de Comunidades de Castilla-La Mancha. AMM was supported by the Postdoctoral Fellowship from JCCM. OGA was supported by the Postdoctoral Fellowship from CYTEMA-UCLM. MR was supported by the DOC-INIA program.

  References Top

Jamieson BG. Reproductive biology of invertebrates. In: Progress in Male Gamete Ultrastructure and Phylogeny. Part A. Vol. IX. New York: John Wiley and Sons, Ltd.; 1999a.  Back to cited text no. 1
Jamieson BG, editor. Spermatozoal phylogeny of the vertebrata. In: The Male Gamete: From Basic Science to Clinical Applications. Vienna: Cache River Press; 1999b. p. 301-31.  Back to cited text no. 2
Pitnick S, Hosken DJ, Birkhead TR, editors. Sperm morphology diversity. In: Sperm Biology and Evolutionary Perspective. Oxford, UK: Academic Press; 2009. p. 69-149.  Back to cited text no. 3
Franzén JR. Sperm structure with regard to fertilization biology and phylogenetics. Verh Dtsch Zool Ges 1977; 70: 123-38.  Back to cited text no. 4
Birkhead TR, Hosken DJ, Pitnick S, editors. Sperm Biology: An Evolutionary Perspective. London: Academic Press; 2009. p. 674.  Back to cited text no. 5
Franzén A. On spermiogenesis, morphology of the spermatozoon and biology of fertilization among invertebrates. Zool Bidr Upps 1956; 31: 355-482.  Back to cited text no. 6
Parker GA. Sperm competition and its evolutionary consequences in insects. Biol Rev 1970; 45: 525-67.  Back to cited text no. 7
Roldan ER. Sperm shape and size: Evolutionary processes in mammals. In: Baccetti B, editor. Comparative Spermatology, 20 Years After. New York: Raven Press; 1991. p. 1001-10.  Back to cited text no. 8
Roldan ER, Gomendio M, Vitullo AD. The evolution of eutherian spermatozoa and underlying selective forces: female selection and sperm competition. Biol Rev 1992; 67: 551-93.  Back to cited text no. 9
Sánchez-Partida LG, Windsor DP, Eppleston J, Setchell BP, Maxwell WM. Fertility and its relationship to motility characteristics of spermatozoa in ewes after cervical, transcervical, and intrauterine insemination with frozen-thawed ram semen. J Androl 1999; 20: 280-8.  Back to cited text no. 10
Foote RH. Fertility estimation: a review of past experience and future prospects. Anim Reprod Sci 2003; 75: 119-39.  Back to cited text no. 11
Love CC. Relationship between sperm motility, morphology and the fertility of stallions. Theriogenology 2011; 76: 547-57.  Back to cited text no. 12
David I, Kohnke P, Lagriffoul G, Praud O, Plouarboué F, et al. Mass sperm motility is associated with fertility in sheep. Anim Reprod Sci 2015; 161: 75-81.  Back to cited text no. 13
Holt C, Holt WV, Moore HD, Reed HC, Curnock RM. Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials. J Androl 1997; 18: 312-23.  Back to cited text no. 14
Martínez-Pastor F, Tizado EJ, Garde JJ, Anel L, de Paz P. Statistical series: opportunities and challenges of sperm motility subpopulation analysis. Theriogenology 2011; 75: 783-95.  Back to cited text no. 15
Amann RP, Waberski D. Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology 2014; 81: 5-17.  Back to cited text no. 16
Cordelli E, Eleuteri P, Leter G, Rescia M, Spanö M. Flow cytometry applications in the evaluation of sperm quality: semen analysis, sperm function and DNA integrity. Contraception 2005; 72: 273-9.  Back to cited text no. 17
Martínez-Pastor F, Mata-Campuzano M, Álvarez-Rodriguez M, Alvarez M, Anel L, et al. Probes and techniques for sperm evaluation by flow cytometry. Reprod Domest Anim 2010; 45: 67-78.  Back to cited text no. 18
Hallap T, Nagy S, Haard M, Jaakma U, Johannisson A, et al. Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls. Theriogenology 2005; 63: 1752-63.  Back to cited text no. 19
García-Macías V, Martinez-Pastor F, Alvarez M, Borragan S, Chamorro CA, et al. Seasonal changes in sperm chromatin condensation in ram (Ovis aries), Iberian red deer (Cervus elaphus hispanicus), and brown bear (Ursus arctos). J Androl 2006; 27: 837-46.  Back to cited text no. 20
Kummer AB, Gaggini TS, Bernardi ML, McManus C, Gonçales EM, et al. Multivariate analyses for determining the association of field porcine fertility with sperm motion traits analysed by computer-assisted semen analysis and with sperm morphology. Reprod Domest Anim 2013; 48: 747-54.  Back to cited text no. 21
Rodriguez-Martinez H. Can we increase the estimated value of semen assessment? Reprod Domest Anim 2006; 41: 2-10.  Back to cited text no. 22
Ramon M, Soler AJ, Ortiz JA, García-Álvarez O, Maroto-Morales A, et al. Sperm population structure and male fertility: an intraspecific study of sperm design and velocity in red deer. Biol Reprod 2013; 89: 110-4.  Back to cited text no. 23
Maroto-Morales A, Ramon M, García-Álvarez O, Montoro V, Soler AJ, et al. Sperm head phenotype and male fertility in ram semen. Theriogenology 2015; 84: 1536-41.  Back to cited text no. 24
Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl 1998; 19: 704-9.  Back to cited text no. 25
Ramon M, Pérez-Guzmán MD, Jiménez-Rabadán P, Esteso MC, García-Álvarez O, et al. Sperm cell population dynamics in ram semen during the cryopreservation process. PLoS One 2013b; 8: e59189.  Back to cited text no. 26
Jamieson BG. The Ultrastructure and Phylogeny of Insect Spermatozoa. Cambridge: Cambridge University Press; 1987.  Back to cited text no. 27
Jamieson BG. Ultrastructure and phylogeny of crustacean spermatozoa. Mem Qld Mus 1991; 31: 109-42.  Back to cited text no. 28
Jamieson BG. Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge: Cambridge University Press; 1991.  Back to cited text no. 29
Jamieson BG, editor. Avian spermatozoa: Structure and phylogeny. In: Reproductive Biology and Phylogeny of Birds. Part A. Phylogeny, Morphology and Fertilization. Enfield: Science Publishers, Inc.; 2007. p. 349-511.  Back to cited text no. 30
Bedford JM, Hoskins DD. The mammalian spermatozoon: Morphology, biochemistry and physiology. In: Lamming GE, editor. Marshall′s Physiology of Reproduction. II. Reproduction in the Male. UK: Longman; 1990. p. 379-568.  Back to cited text no. 31
Gage MJ. Mammalian sperm morphometry. Proc R Soc Lond B 1998; 265: 97-103.  Back to cited text no. 32
Cohen J. Reproduction. London: Butterworths; 1977.  Back to cited text no. 33
Briskie JV, Montgomerie R. Sperm size and sperm competition in birds. Proc Roy Soc B 1992; 247: 89-95.  Back to cited text no. 34
Pitnick S, Miller GT, Schneider K, Markow TA. Ejaculate-female coevolution in Drosophila mojavensis. Proc Roy Soc B 2003; 270: 1507-12.  Back to cited text no. 35
Morrow EH, Gage MJ. The evolution of sperm length in moths. Proc Roy Soc B 2000; 267: 307-13.  Back to cited text no. 36
Anderson MJ, Dixon AS, Dixon AF. Mammalain sperm and oviducts are sexually selected: evidence for co_evolution. J Zool 2006; 270: 682-6.  Back to cited text no. 37
Higginson DM, Miller KB, Segraves KA, Pitnick S. Female reproductive tract form drives the evolution of complex sperm morphology. Proc Natl Acad Sci U S A 2012; 109: 4538-43.  Back to cited text no. 38
Pitnick S, Miller GT, Schneider K, Markow TA. Ejaculate-female coevolution in Drosophila mojavensis. Proc Roy Soc B 2003; 270: 1507-12.  Back to cited text no. 39
Gomendio M, Roldan ER. Sperm competition influences sperm size in mammals. Proc Roy Soc B 1991; 243: 181-5.  Back to cited text no. 40
Gage MJ, Cook PA. Sperm size or numbers? Effects of nutritional stress upon eupyrene and apyrene sperm production strategies in the moth Plodia interpunctella (Lepidoptera: pytalidea). Funct Ecol 1994; 8: 594-9.  Back to cited text no. 41
Byrne PG, Simmons LW, Roberts JD. Sperm competition and the evolution of gamete morphology in frogs. Proc Roy Soc B 2003; 270: 2079-86.  Back to cited text no. 42
Gomendio M, Roldan ER. Implications of diversity in sperm size and function for sperm competition and fertility. Int J Dev Biol 2008; 52: 439-47.  Back to cited text no. 43
Tourmente M, Gomendio M, Roldan ER. Sperm competition and the evolution of sperm design in mammals. BMC Evol Biol 2011; 11: 12.  Back to cited text no. 44
Birkhead TR, Møller AP. Sperm Competition and Sexual Selection. San Diego, CA, USA: Academic Press; 1998.  Back to cited text no. 45
Simmons LW. Sperm Competition and its Evolutionary Consequences in Insects. Princeton, NJ, USA: Princeton University Press; 2001.  Back to cited text no. 46
Parker GA, Pizzari T. Sperm competition and ejaculate economics. Biol Rev Camb Philos Soc 2010; 85: 897-934.  Back to cited text no. 47
Gomez Montoto L, Magana C, Tourmente M, Martin-Coello J, Crespo C, et al. Sperm competition, sperm numbers and sperm quality in muroid rodents. PLoS One 2011; 6: e18173.  Back to cited text no. 48
Parker GA. Why are there so many tiny sperm? Sperm competition and the maintenance of two sexes. J Theor Biol 1982; 96: 281-94.  Back to cited text no. 49
Fitzpatrick JL, Montgomerie R, Desjardins JK, Stiver KA, Kolm N, et al. Female promiscuity promotes the evolution of faster sperm in cichlid fishes. Proc Natl Acad Sci U S A 2009; 106: 1128-32.  Back to cited text no. 50
Lüpold S, Calhim S, Immler S, Birkhead TR. Sperm morphology and sperm velocity in passerine birds. Proc Roy Soc B 2009; 276: 1175-81.  Back to cited text no. 51
Tourmente M, Delbarco-Trillo J, Roldan ER. No evidence of tradeoffs in the evolution of sperm numbers and sperm size in mammals. J Evol Biol 2015; 28: 1816-27.  Back to cited text no. 52
Tourmente M, Gomendio M, Roldan ER, Giojalas LC, Chiaraviglio M. Sperm competition and reproductive mode influence sperm dimensions and structure among snakes. Evolution 2009; 63: 2513-24.  Back to cited text no. 53
Tourmente M, Gomendio M, Roldan ER. Mass-specific metabolic rate and sperm competition determine sperm size in marsupial mammals. PLoS One 2011; 6: e21244.  Back to cited text no. 54
Ramon M, Jiménez-Rabadán P, García-Álvarez O, Maroto-Morales A, Soler AJ, et al. Understanding sperm heterogeneity: biological and practical implications. Reprod Domest Anim 2014; 49: 30-6.  Back to cited text no. 55
Esteso MC, Soler AJ, Fernández-Santos MR, Quintero-Moreno AA, Garde JJ. Functional significance of the sperm head morphometric size and shape for determining freezability in Iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples. J Androl 2006; 27: 662-70.  Back to cited text no. 56
Esteso MC, Fernández-Santos MR, Soler AJ, Montoro V, Martínez-Pastor F, et al. Identification of sperm-head morphometric subpopulations in Iberian red deer epididymal sperm samples. Reprod Domest Anim 2009; 44: 206-11.  Back to cited text no. 57
Morrell J, Rodriguez-Martinez H. Practical applications of sperm selection techniques as a tool for improving reproductive efficiency. Vet Med Int 2010; 2011. [pii: 894767].  Back to cited text no. 58
Morrell JM, Rodriguez_Martinez H. Biomimetic techniques for improving sperm quality in animal breeding: a review. Open Androl J 2009; 1: 1-9.  Back to cited text no. 59
García_Álvarez O, Maroto_Morales A, Ramön M, del Olmo E, Montoro V, et al. Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa. Theriogenology 2010; 74: 979-88.   Back to cited text no. 60
Ombelet W, Menkveld R, Kruger TF, Steeno O. Sperm morphology assessment: historical review in relation to fertility. Hum Reprod Update 1995; 1: 543-57.  Back to cited text no. 61
Kruger TF, Coetzee K. The role of sperm morphology in assisted reproduction. Hum Reprod Update 1999; 5: 172-8.  Back to cited text no. 62
Gago C, Perez-Sanchez F, Yeung C, Tablado L, Cooper T, et al. Standardization of sampling and staining methods for the morphometric evaluation of sperm heads in the cynomolgus monkey (Macaca fascicularis) using computer-assisted image analysis. Int J Androl 1998; 21: 169-76.  Back to cited text no. 63
Yániz JL, Soler C, Santolaria P. Computer assisted sperm morphometry in mammals: a review. Anim Reprod Sci 2015; 156: 1-12.  Back to cited text no. 64
Banaszewska D, Andraszek K, Czubaszek M, Biesiada-Drzazga B. The effect of selected staining techniques on bull sperm morphometry. Anim Reprod Sci 2015; 159: 17-24.  Back to cited text no. 65
Davis RO, Gravance CG, Casey P. Automated morphometric analysis of stallion spermatozoa. Am J Vet Res 1993; 54: 1808-11.  Back to cited text no. 66
Esteso MC, Rodríguez E, Toledano-Díaz A, Castaño C, Pradiee J, et al. Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): optimum sampling procedure and staining methods using Sperm-Class Analyzer ® . Anim Reprod Sci 2015; 155: 42-9.  Back to cited text no. 67
García-Herreros M, Aparicio IM, Barön FJ, García-Marín LJ, Gil MC. Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa. Int J Androl 2006; 29: 553-63.  Back to cited text no. 68
Goulart AR, de Alencar Hausen M, Monteiro-Leal LH. Comparison of three computer methods of sperm head analysis. Fertil Steril 2003; 80: 625-30.  Back to cited text no. 69
Maree L, du Plessis SS, Menkveld R, van der Horst G. Morphometric dimensions of the human sperm head depend on the staining method used. Hum Reprod 2010; 25: 1369-82.  Back to cited text no. 70
Villaverde-Morcillo S, Esteso MC, Castaño C, Toledano Díaz A, Löpez-Sebastián A, et al. Influence of staining method on the values of avian sperm head morphometric variables. Reprod Domest Anim 2015; 50: 750-5.  Back to cited text no. 71
Gravance CG, Davis RO. Automated sperm morphometry analysis (ASMA) in the rabbit. J Androl 1995; 16: 88-93.  Back to cited text no. 72
Gravance CG, Vishwanath R, Pitt C, Casey PJ. Computer automated morphometric analysis of bull sperm heads. Theriogenology 1996; 46: 1205-15.  Back to cited text no. 73
Rijsselaere T, Van Soom A, Hoflack G, Maes D, de Kruif A. Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser. Theriogenology 2004; 62: 1292-306.  Back to cited text no. 74
Sancho M, Perez-Sanchez F, Tablado L, de Monserrat J, Soler C. Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques. Theriogenology 1998; 50: 27-37.  Back to cited text no. 75
Yániz JL, Vicente-Fiel S, Capiströs S, Palacín I, Santolaria P. Automatic evaluation of ram sperm morphometry. Theriogenology 2012; 77: 1343-50.  Back to cited text no. 76
Coetzee K, Kruger TF, Lombard CJ. Repeatability and variance analysis on multiple computer-assisted (IVOS) sperm morphology readings. Andrologia 1999; 31: 163-8.  Back to cited text no. 77
Gravance CG, Garner DL, Pitt C, Vishwanath R, Sax-Gravance SK, et al. Replicate and technician variation associated with computer aided bull sperm head morphometry analysis (ASMA). Int J Androl 1999; 22: 77-82.  Back to cited text no. 78
Garrett C, Baker HW. A new fully automated system for the morphometric analysis of human sperm heads. Fertil Steril 1995; 63: 1306-17.  Back to cited text no. 79
Soler C, Gadea B, Soler A, Fernández_Santos M, Esteso M, et al. Comparison of three different staining methods for the assessment of epididymal red deer sperm morphometry by computerized analysis with ISAS. Theriogenology 2005; 64: 1236-43.  Back to cited text no. 80
Yániz JL, Capiströs S, Vicente_Fiel S, Soler C, Nuñez de Murga J, et al. Study of nuclear and acrosomal sperm morphometry in ram using a computer_assisted sperm morphometry analysis fluorescence (CASMA_F) method. Theriogenology 2014; 82: 921-4.  Back to cited text no. 81
Soler C, García A, Sancho M, Contell J, Nuñez M, et al. A new technique for human sperm morphology analysis in unstained cells from raw semen. Reprod Fertil Dev 2016; 28: 428-33.  Back to cited text no. 82
Soler C, García A, Contell J, Silvestre MA, Sancho M. The Trumorph® system: the new universal technique for the observation and analysis of the morphology of living sperm. Anim Reprod Sci 2015; 158: 1-10.  Back to cited text no. 83
Gravance CG, Champion ZJ, Casey PJ. Computer_assisted sperm head morphometry analysis (ASMA) of cryopreserved ram spermatozoa. Theriogenology 1998; 49: 1219-30.  Back to cited text no. 84
Davis RO, Gravance CG. Standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis. Fertil Steril 1993; 59: 412-7.  Back to cited text no. 85
Buendia P, Soler C, Paolicchi F, Gago G, Urquieta B, et al. Morphometric characterization and classification of alpaca sperm heads using the Sperm-Class Analyzer (R) computer-assisted system. Theriogenology 2002; 57: 1207-18.  Back to cited text no. 86
Verstegen J, Iguer-Ouada M, Onclin K. Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002; 57: 149-79.  Back to cited text no. 87
de Paz P, Mata-Campuzano M, Tizado EJ, Alvarez M, Alvarez-Rodríguez M, et al. The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used. Theriogenology 2011; 76: 1313-25.  Back to cited text no. 88
Vicente_Fiel S, Palacín I, Santolaria P, Fantova E, Quintín_Casorrán FJ, et al. In vitro assessment of sperm quality from rams of high and low field fertility. Anim Reprod Sci 2014; 146: 15-20.  Back to cited text no. 89
Ros_Santaella JL, Pintus E, Garde JJ. Intramale variation in sperm size: functional significance in a polygynous mammal. PeerJ 2015; 3: e1478.  Back to cited text no. 90
Yániz JL, Palacín I, Vicente-Fiel S, Sánchez Nadal JA, Santolaria P. Sperm population structure in high and low field fertility rams. Anim Reprod Sci 2015; 154: 128-34.  Back to cited text no. 91
Maroto-Morales A, Ramön M, García-Alvarez O, Soler AJ, Esteso MC, et al. Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer. Theriogenology 2010; 73: 437-48.  Back to cited text no. 92
Maroto-Morales A, Ramön M, García-Álvarez O, Soler AJ, Fernández-Santos MR, et al. Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: intra- and inter-individual variation. Theriogenology 2012; 77: 1529-39.  Back to cited text no. 93
Santolaria P, Vicente-Fiel S, Palacín I, Fantova E, Blasco ME, et al. Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep. Anim Reprod Sci 2015; 163: 82-8.  Back to cited text no. 94
Martinez-Pastor F. Use of misleading statistics in method comparison analyses. J Androl 2010; 31: 323.  Back to cited text no. 95
Watson PF, Petrie A. Method agreement analysis: a review of correct methodology. Theriogenology 2010; 73: 1167-79.  Back to cited text no. 96
Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; 1: 307-10.  Back to cited text no. 97
Malo AF, Garde JJ, Soler AJ, Garcia AJ, Gomendio M, et al. Male fertility in natural population of red deer is determined by sperm velocity and the proportion of normal spermatozoa. Biol Reprod 2005; 72: 822-9.  Back to cited text no. 98
Malo AF, Gomendio M, Garde J, Lang_Lenton B, Soler AJ. Sperm design and sperm function. Biol Lett 2006; 2: 246-9.  Back to cited text no. 99
Marco_Jiménez F, Vicente JS, Lavara R, Balasch S, Viudes_de_Castro MP. Poor prediction value of sperm head morphometry for fertility and litter size in rabbit. Reprod Domest Anim 2010; 45: e118-23.  Back to cited text no. 100
Vicente-Fiel S, Palacín I, Santolaria P, Hidalgo CO, Silvestre MA, et al. A comparative study of the sperm nuclear morphometry in cattle, goat, sheep, and pigs using a new computer-assisted method (CASMA-F). Theriogenology 2013; 79: 436-42.  Back to cited text no. 101
Lavara R, Vicente JS, Baselga M. Genetic variation in head morphometry of rabbit sperm. Theriogenology 2013; 80: 313-8.  Back to cited text no. 102
Saravia F, Núñez-Martínez I, Morán JM, Soler C, Muriel A, et al. Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity. Theriogenology 2007; 68: 196-203.  Back to cited text no. 103
Carvalho JO, Silva LP, Sartori R, Dode MA. Nanoscale differences in the shape and size of X and Y chromosome-bearing bovine sperm heads assessed by atomic force microscopy. PLoS One 2013; 8: e59387.  Back to cited text no. 104
Petrunkina AM, Topfer_Petersen E. Heterogeneous osmotic behaviour in boar sperm populations and its relevance for detection of changes in plasma membrane. Reprod Fertil Dev 2000; 12: 297-305.  Back to cited text no. 105
García_Herreros M, Leal CL. Comparative study of sperm washing and selection methods after cryopreservation and its influence on sperm subpopulational structure in a bovine model. Syst Biol Reprod Med 2014; 60: 338-47.  Back to cited text no. 106
García-Herreros M, Leal CL. Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa. Animal 2015; 9: 1016-24.  Back to cited text no. 107
Blässe AK, Oldenhof H, Ekhlasi-Hundrieser M, Wolkers WF, Sieme H, et al. Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation. Theriogenology 2012; 78: 1312-20.  Back to cited text no. 108
Ros-Santaella JL, Domínguez-Rebolledo AE, Garde JJ. Sperm flagellum volume determines freezability in red deer spermatozoa. PLoS One 2014; 9: e112382.  Back to cited text no. 109
Santiago-Moreno J, Esteso MC, Pradiee J, Castaño C, Toledano-Díaz A, et al. Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing. Andrologia 2016; 48: 470-4.  Back to cited text no. 110
Thompson LA, Brook PF, Warren M, Barrat C, Cooke I. A morphometric comparison of the nuclear morphology of fresh and frozen-thawed human zona-bound and unbound sperm. J Androl 1994; 15: 337-42.  Back to cited text no. 111
Gravance CG, Casey ME, Case PJ. Pre-freeze bull sperm head morphometry related with post-thaw fertility. Anim Reprod Sci 2009; 114: 81-8.  Back to cited text no. 112
Peña FJ, Saravia F, García_Herreros M, Núñez_martínez I, Tapia JA, et al. Identification of sperm morphometric subpopulations in two different portions of the boar ejaculate and its relation to post_thaw quality. J Androl 2005; 26: 716-23.  Back to cited text no. 113
García_Herreros M, Barön FJ, Aparicio IM, Santos AJ, García_Marín LJ, et al. Morphometric changes in boar spermatozoa induced by cryopreservation. Int J Androl 2008; 31: 490-8.  Back to cited text no. 114
Hidalgo M, Rodríguez I, Dorado JM. The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability. Anim Reprod Sci 2001; 100: 61-72.  Back to cited text no. 115
Marco-Jiménez F, Viudes-de-Castro MP, Balasch S, Mocé E, Silvestre MA, et al. Morphometric changes in goat sperm heads induced by cryopreservation. Cryobiology 2006; 52: 295-304.  Back to cited text no. 116
Arruda RP, Ball BA, Gravance CG, Garcia RP, Liu IK. Effects of extenders and cryoprotectants on stallion sperm head morphometry. Theriogenology 2002; 58: 252-6.  Back to cited text no. 117
Rijsselaere T, Van Soom A, Maes D, Nizanski W. Computer-assisted sperm analysis in dogs and cats: an update after 20 years. Reprod Domest Anim 2012; 47 Suppl 6: 204-7.  Back to cited text no. 118
Alvarez M, Garcia-Macias V, Martinez-Pastor F, Martinez F, Borragan S, et al. Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa. Theriogenology 2008; 70: 1498-506.  Back to cited text no. 119
Curry MR, Kleinhans FW, Watson PF. Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore. Cryobiology 2000; 41: 167-73.  Back to cited text no. 120
Peña AI, Lugilde LL, Barrio M, Herradon PG, Quintela LA. Effects of Equex from different sources on post_thaw survival, long_ evity and intracelular Ca2+concentration of dog spermatozoa. Theriogenology 2003; 59: 1725-39.  Back to cited text no. 121
Thomas C, Garner D, DeJarnette J, Marshall C. Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa. Biol Reprod 1997; 56: 991-8.  Back to cited text no. 122
Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000; 53: 47-58.  Back to cited text no. 123
Thurston LM, Watson PF, Holt WV. Sperm cryopreservation: a genetic explanation for species and individual variation. Cryo Letters 2002; 23: 255-62.  Back to cited text no. 124
Soler AJ, García AJ, Fernández-Santos MR, Esteso MC, Garde JJ. Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at −196°C. J Androl 2003; 24: 746-56.  Back to cited text no. 125
Esteso MC, Fernández-Santos MR, Soler AJ, Montoro V, Quintero-Moreno A, et al. The effects of cryopreservation on the morphometric dimensions of Iberian red deer (Cervus elaphus hispanicus) epididymal sperm heads. Reprod Domest Anim 2006; 41: 241-6.  Back to cited text no. 126
Druart X. Sperm interaction with the female reproductive tract. Reprod Domest Anim 2012; 4: 348-52.  Back to cited text no. 127
Mortom KM, Rowe AM, Maxwell WM, Evans G. In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen thawed ram spermatozoa. Theriogenology 2006; 65: 1333-45.  Back to cited text no. 128
Bedford JM. Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol Reprod 1983; 28: 108-20.  Back to cited text no. 129
García-Vázquez FA, Hernández-Caravaca I, Yánez-Quintana W, Matás C, Soriano-Úbeda C, et al. Morphometry of boar sperm head and flagellum in semen backflow after insemination. Theriogenology 2015; 84: 566-74.  Back to cited text no. 130
Hernández-Caravaca I, Soriano-Úbeda C, Matás C, Izquierdo-Rico MJ, García-Vázquez FA. Boar sperm with defective motility are discriminated in the backflow moments after insemination. Theriogenology 2015; 83: 655-61.  Back to cited text no. 131
Humphries ST, Evans JP, Simmons LW. Sperm competition: linking form to function. BMC Evol Biol 2008; 8: 319-29.  Back to cited text no. 132
Gomendio M, Malo AF, Garde J, Roldan ER. Sperm traits and male fertility in natural populations. Reproduction 2007; 134: 19-29.  Back to cited text no. 133
Fitzpatrick JL, Garcia-Gonzalez F, Evans JP. Linking sperm length and velocity: the importance of intramale variation. Biol Lett 2010; 6: 797-9.  Back to cited text no. 134
Martin_Coello J, Dopazo H, Arbiza L, Ausiö J, Roldan ER, et al. Sexual selection drives weak positive selection in protamine genes and high promoter divergence, enhancing sperm competitiveness. Proc Biol Sci 2009; 27: 2427-36.  Back to cited text no. 135
Lüke L, Vicens A, Tourmente M, Roldan ER. Evolution of protamine genes and changes in sperm head phenotype in rodents. Biol Reprod 2014; 90: 67.  Back to cited text no. 136
Gömez Montoto L, Varea Sánchez M, Tourmente M, Martín-Coello J, Luque-Larena JJ, et al. Sperm competition differentially affects swimming velocity and size of spermatozoa from closely related muroid rodents: head first. Reproduction 2011; 142: 819-30.  Back to cited text no. 137
Vernocchi V, Morselli MG, Lange Consiglio A, Faustini M, Luvoni GC. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa. Theriogenology 2014; 82: 982-7.  Back to cited text no. 138
Larson KL, Brannian JD, Singh NP, Burbach JA, Jost LK, et al. Chromatin structure in globozoospermia: a case report. J Androl 2001; 22: 424-31.  Back to cited text no. 139
Thurston LM, Watson PF, Mileham AJ, Holt WV. Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation. J Androl 2001; 22: 382-94.  Back to cited text no. 140
Severa L, Machal L, Svabova L, Mamica O. Evaluation of shape variability of stallion sperm heads by means of image analysis and Fourier descriptors. Anim Reprod Sci 2010; 119: 50-5.  Back to cited text no. 141
Varea Sánchez M, Bastir M, Roldan ER. Geometric morphometrics of rodent sperm head shape. PLoS One 2013; 8: e80607.  Back to cited text no. 142
Ghasemian F, Mirroshandel SA, Monji-Azad S, Azarnia M, Zahiri Z. An efficient method for automatic morphological abnormality detection from human sperm images. Comput Methods Programs Biomed 2015; 122: 409-20.  Back to cited text no. 143
Shojaei Saadi HA, van Riemsdijk E, Dance AL, Rajamanickam GD, Kastelic JP, et al. Proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulations. J Proteomics 2013; 82: 64-80.  Back to cited text no. 144
Blasi T, Hennig H, Summers HD, Theis FJ, Cerveira J, et al. Label-free cell cycle analysis for high-throughput imaging flow cytometry. Nat Commun 2016; 7: 10256.  Back to cited text no. 145

This article has been cited by
1 Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits
María del Carmen Fuentes-Albero,Silvia Abril Sánchez,José Luis Ros-Santaella,Eliana Pintus,Chiara Luongo,Sara Ruiz Díaz,Carlos Barros García,María Jesús Sánchez Calabuig,Daniel García Párraga,Francisco Alberto García Vázquez
Biology. 2021; 10(5): 355
[Pubmed] | [DOI]
2 Semen evaluation: methodological advancements in sperm quality-specific fertility assessment — A review
Bereket Molla Tanga,Ahmad Yar Qamar,Sanan Raza,Seonggyu Bang,Xun Fang,Kiyoung Yoon,Jongki Cho
Animal Bioscience. 2021; 34(8): 1253
[Pubmed] | [DOI]
3 Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination
Vinicio Barquero,Eduardo R. S. Roldan,Carles Soler,Jesús L. Yániz,Marlen Camacho,Anthony Valverde
Animals. 2021; 11(4): 920
[Pubmed] | [DOI]
4 Evaluation of the Morphometry of Sperm from the Epididymides of Dogs Using Different Staining Methods
Anna Wysokinska,Ewa Wójcik,Angelika Chlopik
Animals. 2021; 11(1): 227
[Pubmed] | [DOI]
5 Crioconservación seminal en peces de agua dulce: aspectos biotecnológicos, celulares y bioquímicos
Víctor M. Medina-Robles,Astrid S. Duarte-Trujillo,Pablo E. Cruz-Casallas
Orinoquia. 2020; 24(2): 51
[Pubmed] | [DOI]
6 Comparison of three different staining methods for the morphometric characterization of Alpaca (Vicugna pacos) sperm, using ISAS® CASA-Morph system
Hernán Cucho,Yolvi López,Carina Caldeira,Anthony Valverde,César Ordóñez,Carles Soler
Nova Biologica Reperta. 2019; 6(3): 284
[Pubmed] | [DOI]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Morphological Sp...
Assessment of Sp...
Assessment of Sp...
Perspectives on ...
Author Contributions
Competing Interests

 Article Access Statistics
    PDF Downloaded586    
    Comments [Add]    
    Cited by others 6    

Recommend this journal