Asian Journal of Andrology (2016) 18, 863–870
© 2016 AJA, SIMM & SJTU. All rights reserved 1008-682X
www.asiaandro.com; www.ajandrology.com
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.
10–13
Availability of advanced techniques and hardware such as
computer-assisted sperm analysis(CASA),
14–16
uorescence probes
and ultimately ow 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
e 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
ere is a need to develop new analytical tools to capture sperm
diversity better, and to improve data analysis methods. New equipment
INTRODUCTION
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, oen in a hostile environment, and it carries
the genetic material from the male to the oocyte. e dierences 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 dierent
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 dierent 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.
1–3
ese studies have put considerable eort into nding
the ultimate cause of this sperm diversication, and how within-and
between-male variation and within-and between-species variation
contribute to sperm performance and behavior.
4,5
Adaptation to specic
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, inuencing not only sperm
morphology but also sperm length and sperm numbers.
8,9
Current status and potential of morphometric sperm
analysis
Alejandro Maroto‑Morales
1
, Olga García‑Álvarez
1,2
, Manuel Ramón
3
, Felipe Martínez‑Pastor
4,5
,
M Rocío Fernández‑Santos
1,6
, A Josefa Soler
1
, José Julián Garde
1
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.
Asian Journal of Andrology (2016) 18, 863–870; doi: 10.4103/1008-682X.187581; published online: 27 September 2016
Keywords: computer‑assisted sperm morphometric analysis; mammals; sperm function; sperm morphometry
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.
Correspondence: Dr. JJ Garde (Julian.garde@uclm.es)
Semen Analysis
INVITED REVIEW
Open Access
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Asian Journal of Andrology
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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
e spermatozoon is the most diverse cell type known. is diversity
is thought to reect adaptation to conditions under which the cells
function, as a way to ensure their survival in dierent fertilization
environments and to maximize their fertilizing capacity.
4,6
The
spermatozoon of thousands of species has been described including
insects,
27
crustaceans,
28
sh,
29
birds,
30
mammals,
31,32
and many other
groups. e existence of morphological diversity among species is
widely recognized, with a high degree of variation in size, shape,
and behavior,
3,32,33
reected in main structures(i.e.,head, midpiece,
and principal piece) and overall size. Dierent specializations have
been observed, such as the absence of a agellum or multiple agella,
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 rst attempt to explain this diversity was conducted by Franzén
4,6
who proposed that sperm modications are adaptations to their specic
fertilization environment. us, pre-and post-copulatory selection
such as mate choice, mode of fertilization, cryptic female choice,
sexual conict, and sperm competition may inuence 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,
34–38
suggesting a sexual coevolution
of the fertilization system.
39
Selection would favor very dierent 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 aer being transferred to the female
tract(e.g.,mammals).
Sperm competition has also been found to be a significant
source of sperm variability.
40–44
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-o 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
43,44,48,50–52
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–articial– source of variation in sperm
morphology.
55
e invitro 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. erefore, it may be expected that those males having a
higher proportion of the relevant kind of spermatozoa would produce
more ospring, 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
e use of selection media before
AI could also select cells of a specic 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
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.
62–64
Studies that assess the
dierent sources of variation aecting CASA-Morph are critical for
guaranteeing its repeatability and consistency among laboratories. e
main sources of variation of CASA-Morph, other than the soware and
data analysis, are the sample preparation, xation method, staining
method, microscopic system(optics and camera), and technician.
All these steps can aect 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. ese aspects have been studied by several
authors,
64–71
and they have not yet been completely resolved.
Sample preparation
e preparation of the sample, its concentration, and the xation
procedure are the rst steps to consider in a CASA-Morph protocol.
Varying the sperm concentration may affect CASA-Morph
performance.
66,72–74
Fixation, together with drying of the sample, has
received little attention, but they are both critical steps, and it has
been demonstrated that they aect 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
63,65,66,68,70–73,79,80
since it inuences
background noise, sperm contrast, and the identification of
dierent 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 xing
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
e higher the quality of the hardware(microscope, lenses, and
camera), the more reliable the analysis, at least for experimental
work. At high magnication, lenses capable of providing a sharp,
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aberration-free and bright image, and a camera capable of high
resolution will reduce the errors of the CASA-Morph soware 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, eorts should be
directed at improving staining results and optimizing soware to cope
with the limitations due to the use of basic equipment.
Regarding soware, automatic methods should be preferred,
to save analysis time and to reduce operator errors. Goulart etal.
69
suggested using semi-automatic methods, with some operator
intervention. When the interaction of the technician with the soware
is limited to removing misdigitized spermatozoa, the eect 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. Ageneral 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 condence 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 coecient of variation(CV%) increases
considerably.
85,87
Currently, the capabilities of computers, sophistication
of CASA-Morph soware, 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 condence of the
statistics dening each population. is 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. us, the main problem of most
CASA-Morph studies so far is the use of a low sample size.
23,26,88–91
Only a few authors
24,92–94
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 eect, stains,
etc. These statistics, generally based on a means comparison or
correlations/regressions, are not appropriate for assessing dierences
between methods.
95,96
us, 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 coecients
97
).
Regarding the consistency and repeatability of analyses,
laboratories should set up a quality assurance system for CASA-Morph.
e 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 nal point, CASA-Morph protocols and soware 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 denition 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 t 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
soware.
70
ASSESSMENT OF SPERM MORPHOMETRY II – FUNCTIONAL
ASPECTS
Studies on the relationships between sperm design and sperm
function have oen yielded contradictory results.
24,44,89,94,98–100
Most
of the research made in this aspect has usually been conducted at an
interspecic level
44,101
since nding dierences between species is easier
than intraspecic or intra-male levels. Studies at the intraspecic 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 dicult to
obtain.
Recently, some authors have reported that some sperm
characteristics are genetically determined,
26,102
sperm morphometry
being one of them. us, it is expected that sperm morphometry
reflects differences in sperm function. Indeed, different sperm
morphometric features have been identied between breeds
103
but
also between animals from the same breed belonging to dierent
herds(i.e.,reecting their origin).
92
Ignoring the dierences between the morphometric dimensions
of X-and Y-bearing spermatozoa due to their DNA content,
104
most
dierences 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.
26,108–110
Sperm cryopreservation and morphology
e eects 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 signicant reduction in sperm head dimensions
by cryopreservation of freshly extended samples. This reduction
in sperm head dimension is reected not only in the size of sperm
head but also in its shape. Dierent hypotheses have been proposed
to explain the reasons for the sperm head dimension decrease aer
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
signicantly dierent responses to the same freezing treatment.
123–125
us, males can be classied 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
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Asian Journal of Andrology
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et al.
115
observed that in the goat, sperm morphometric changes
aer 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(dened as the length/width ratio). ese
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 aer freezing-thawing
showed a low sperm head dimension in the extended samples. ese
authors thus suggested that in semen samples of better quality the
sperm cryodamage was less and the eect of cryopreservation on the
sperm head morphometry was also less. Ramon etal.
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
identication of the males with better sperm cryopreservation, the
study of patterns of change throughout the cryopreservation process
led to the identication of dierently dened 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.
erefore, 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 tter sperm designs.
Despite most of the studies on the eect of cryopreservation on
sperm morphology have been focused on the evaluation of sperm
head features, Ros-Santaella etal.
109
recently reported that stags not
diering in sperm head dimensions showed signicant dierences in
sperm cryoresistance that were strongly related to the volume of the
sperm principal piece. is study indicates a key role of the sperm
agellum 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 rst 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 invivo selection, dierent
sperm selection methods are routinely used in invitro protocols, such
as invitro 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 aect sperm
morphology; more importantly, the morphometric characteristics of
the cells that eventually fertilize the oocyte are unknown. Recently,
García-Vázquez etal.
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 invitro capacitation in bovine spermatozoa modied 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 rst 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. ese 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. ese relationships were also reported by Fitzpatrick
etal.
134
in externally fertilizing species(sh). Sperm head elongation
may play an important role by making sperm more hydrodynamically
ecient, which, in turn, may inuence 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.
is could compensate evolutionary constraints to increases in sperm
length by allowing increased swimming eciency, or for the same
reason, it might increase sperm lifespan if energy reserves last longer
or are used more eciently, 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 inuence 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 Gómez 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 reect defects in the structure and integrity of the
sperm chromatin, Sailer etal.(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 dene which
sperm morphometric parameters are crucial for breeding success.
PERSPECTIVES ON THE FUTURE OF SPERM
MORPHOMETRIC STUDIES
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
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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. e implementation of scientic and
technological advances could benet 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 agellar dimensions on sperm function
has been demonstrated,
98,109
CASA-Morph systems should be modied
to incorporate new tools that allow an automatic and accurate
evaluation of these sperm structures. ese 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. erefore,
future studies of sperm morphometry should not overlook the role of
the agellum 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 ts 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 etal.
142
to evaluate
sperm head morphometry in rodents. ese authors obtained a better
characterization of sperm shape, nding 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 denition of where the main shape dierences
are located in the sperm head. Nevertheless, that study dealt with
dierent 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 dene sperm head shape adequately.
Sperm preparation
Other new methods have been developed to evaluate sperm
morphometry without the need for sperm staining.
143
ese methods
are focused on humans and allow the use of the observed spermatozoon
to be used in assisted reproductive techniques such as ICSI aer 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 ospring. Along this line, the
study of proteins associated with sperm head shape
144
would oer 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. is
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
AMM, OGA, MR, FMP, MRFS, AJS, and JJG conceived the study, wrote
the manuscript, and reviewed dras of the manuscript. All authors read
and approved the nal version of the manuscript, and agree with the
order of presentation of the authors.
COMPETING INTERESTS
e authors declared no competing interest.
ACKNOWLEDGMENTS
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.
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