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Syst Biol Reprod Med, Early Online: 1–7
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2013.869273

REVIEW AND HYPOTHESIS

Motility, viability, and calcium in the sperm cells
Jorge Parodi
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Laboratorio de Fisiologıa de la Reproduccion, Escuela de Medicina Veterinaria, Nucleo de Investigacion en Produccion Alimentaria, Facultad de
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Recursos Naturales, Universidad Catolica de Temuco, Temuco, Chile

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Abstract

Keywords

Sperm cells are complicated in vitro models. Their viability is limited, and physiology is
complex. The study of their properties is of great application in the animal production as
viable and functional gametes are essential. It has been shown that the decrease of sperm
cell viability parallels an increase of the reactive oxygen species (ROS). Reactive oxygen
species is secondary to normal metabolic processes of the cell-like flagellar movement.
There is evidence of strategies that reduce ROS levels by using exogenous or endogenous
antioxidants with the intention that seminal plasma protects the sperm cells and
increases viability. Perhaps viability can increase by reducing that flagellar movement
which is regulated by calcium. The phenomenon has not been fully characterized, but it is
established that in certain mammalian models, the entrance of calcium via specific channels
such as CATsper or voltage-dependent channels, signals flagellar movement. Previous
reports have indicated that a change in the concentration of calcium or if the temperature
is altered, the function of mammal sperm cells is reduced or blocked and viability
prolonged. Fish sperm can remain immobile for several weeks but when activated the
number of mobile and viable sperm is reduced at a faster rate. However, if the cells are not
mobilized the semen can be preserved for longer periods. As presented in this paper, this
supports the notion that by modulating calcium channels to reduce motility the viability of
these cells can increase.

Calcium, motility, sperm
History
Received 4 July 2013
Revised 2 October 2013
Accepted 4 October 2013
Published online 13 December 2013

Abbreviations: ROS: reactive oxygen species; ZP: zona pellucid; AR: acrosome reaction; DF:
disinhibit factor; TEA: tetraethylammonium chloride; CAVs: calcium voltage channels; CatSper:
cationic sperm

Sperm capacitation
Fertilization is a unique and amazing process involving
two morphologically distinct cells, the sperm and the oocyte,
which are recognized and fused together. This process begins
when the sperm starts to penetrate the oocyte envelope and
plasma membrane and ends in the exchange of maternal and
paternal chromosomes, forming the zygote [Patrat et al.
2006]. The sperm must undergo functional changes following
its genesis and subsequent maturation in the epididymis.
Only sperm that have become capacitated can recognize and
bind to the zona pellucida (ZP). The interaction between the
sperm and the ZP initiates a signal transduction process
resulting in exocytosis of the acrosomal contents during the
acrosome reaction (AR) [Breitbart 2003; Rossato et al. 2001].

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Address correspondence to Jorge Parodi, Laboratorio de Fisiologıa de la
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Reproduccion, Escuela de Medicina Veterinaria, Nucleo de Investigacion
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en Produccion Alimentaria, Facultad de Recursos Naturales, Universidad
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Catolica de Temuco, Temuco, Chile. Tel: þ56-45-2205564. E-mail:
jparodi@uct.cl

However, this is only a general picture of the AR phenomenon, and some reports have suggested that an intact ZP is
not sufficient to induce acrosomal exocytosis [Baibakov
et al. 2007]. Furthermore, according to the work of
Dr. Yanagimachi’s group, some mouse sperm passing through
the cumulus layers are already undergoing or have completed
the acrosome reaction [Knobil and Neill 1994]. In shrews, the
acrosome reaction is induced by cumulus cells, but not by the
ZP [Bedford et al. 2004]. The available evidence suggests a
general but not unique mechanism of penetration, and it is
important to consider particular species adaptations when
manipulating different samples in vitro. The sperm must
penetrate physical barriers imposed by the oocyte, including
cumulus oophorus cells, the plasma membrane, and the ZP,
for which hydrolytic enzymes such as glycohydrolases and
proteinases are necessary. During capacitation, the sperm
undergoes functional biochemical and biophysical modifications, including changes in the activity of membrane enzymes
and motility patterns, enabling it to undergo the AR prior to
fertilization. These modifications include the removal of
roadblocks to capacitation factors from the sperm surface and
increased membrane fluidity, cholesterol efflux, intracellular

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J. Parodi



Plasma membrane and ion channels

Figure 1. Calcium wave. The upper panel shows a bovine sperm with a
calcium probe exposed to a high potassium concentration, while the
lower panel shows a graphic representation of the fluorescence intensity,
both in control conditions and when sperm are exposed to potassium.
The figure indicates that there is a wave from the middle piece to the
head when the sperm are depolarized. Modified figure from Navarrete
et al. 2010.

calcium, cAMP, and protein tyrosine phosphorylation
[Aitken and McLaughlin 2007]. All of these processes
are regulated by the entry of calcium into the cells, but
another key factor is the motility of the sperm. The complete
process had previously been described as a single step:
the entry of calcium increases motility and the AR.
However, recently, the calcium wave concept has been
incorporated (see Figure 1; [Navarrete et al. 2010]),
indicating that in sperm cells, the first part of the calcium
wave generates an increase in motility, while the second
part induces the AR. Furthermore, this process can be
manipulated while the cells maintain a healthy state [Darszon
et al. 2011].

Capacitation factors
The reduction of factors inhibiting capacitation (disinhibit
factor, DF) due to the seminal flow involves a gradual
release of these factors, from the sperm surface. Their release
results in a transient state of sperm DF capacitation. This
ensures the maximum capacity of fertilization at the appropriate location [Acott and Carr 1984; Zhong et al. 1993].
Once the DF binds to the sperm surface, it activates a calcium
ATPase, thus maintaining a low calcium concentration.
When the DF is released from the sperm surface, an increase
in intracellular calcium levels is initiated. In vitro studies in
which the calcium ATPase was inhibited revealed acceleration of capacitation [Perry et al. 1997].

The plasma membrane is a lipoprotein interface that acts as a
permeability barrier allowing the cell to maintain a different
composition in the intracellular in comparison to the extracellular medium. The most abundant components of the
plasma membrane are phospholipids and proteins, which
together form the fluid mosaic pattern [Hasdemir 2007].
The resting potential is a particular state of the membrane
potential in which the sum of ion currents through the
membrane is zero. This is due to the presence of transmembrane electrochemical gradients resulting from selective
permeability to ions and secondary various structures such
as transmembrane channels, pumps, and ion exchangers.
From the resting potential, cell excitation can generate an
action potential that allows the cell to respond to different
stimuli. During this process, each ion tends to draw the
membrane potential towards its own electrochemical equilibrium potential (Nernst equation) [Hille 1992]. Ionic currents
through channels determine transmembrane bioelectric
phenomena related to the membrane potential in addition
to modulating enzyme activity, metabolism, and cellular
genetics activity. Specifically, in sperm cells, the transmembrane ionic currents and their potential, among other factors,
regulate the intracellular concentration of calcium and the
genesis of second messengers. These factors are essential for
fertilization-associated processes, such as sperm motility,
capacitation, and the AR. Therefore, the study of ion channels
is extremely valuable for understanding the electrophysiological processes and biological responses of both excitable
cells and isolated cells. In particular, determining the roles of
these channels in the mammalian sperm membrane is
essential to understand the processes involved in fertilization.
The main tool for investigating the characteristics and
distribution of ion channels in the plasma membrane is the
patch-clamp technique [Neher and Sakmann 1976; Neher
et al. 1978], which is a high-resolution method that is
currently used to determine the electrophysiological and
pharmacological properties of the cell structure.

Sperm cell viability and function
One must be careful during the various procedures in which
sperm are manipulated as alterations can cause premature
sperm capacitation [Gomez et al. 1997]. This leads to the
AR impacting the longevity of the sperm. A decrease in
fertilization capacity can result from the presence of large
amounts of ROS following ejaculation. Kirchhoff and associates [1998] and Alvarez and Agarwal [2006] indicated
that sperm produce and export ROS to the extracellular
environment, most of which are generated by the mitochondria, secondary to the flagellar activity of the cells. The loss
of sperm function, i.e., the fertilization capacity, results from
the presence of high levels of ROS, either following
ejaculation or secondarily to high levels of motility. These
studies have indicated that the sperm produced and exported
ROS to the extracellular environment are the product of the
monovalent reduction of molecular oxygen during oxidative
phosphorylation [Alvarez and Agarwal 2006; Kirchhoff et al.
1998]. Observations made in the laboratory in models
of immobile sperm cells (salmon or trout) have suggested


DOI: 10.3109/19396368.2013.869273

the existence of long-term viability, lasting for days or weeks,
while retaining a high rate of fertilization. The common
feature of such models is the inactive state of the cells,
without metabolic changes, i.e., ROS production. In mammalian sperm cells, we have observed only a one-hour period
of viability and function, and these cells show a high motility
and metabolism. Temperature is important for the regulation
of cell function, and the preservation and the quality of sperm.
For example, fish semen display extreme temperature control
[Alavi and Cosson 2005], and in porcine sperm, temperature
conservation increases the time of preservation of a sample
[Althouse et al. 1998]. Furthermore, in fowl, temperature
regulates calcium influx [Thomson and Wishart 1991].
These lines of evidence suggest the importance of temperature control during the in vitro manipulation of sperm cells,
which is correlated with changes during travel in the
oviduct or in fresh water, in the case of the aquatic species.
In particular, there may be temperature gradients within the
oviduct of animals in estrous [Bahat and Eisenbach 2006;
Hunter and Nichol 1986]. Values presented in the literature
suggest that these gradients can be on the order of 1–2 C or
more between the caudal portion of the isthmus and the
cranial portion of the ampulla in the hours before ovulation.
This has been proposed to contribute to reducing sperm
motility and the sperm storage function of the caudal isthmus
[Hunter and Nichol 1986]. The magnitude of the temperature
gradient may change according to the stage of the cycle and,
especially, according to the time of ovulation [Hunter 2012].
Therefore, there is a possible influence of temperature on the
viscosity and viscoelasticity of female tract fluids and on the
ZP, as in other cell models, membrane viscosity is affected
by temperature [Stokke et al. 1985]. This factor must be
considered, and it might be most significant at the time when
viable spermatozoa are expected to be found in the oviduct
[Coy et al. 2009]. Temperature is a key factor in the function
of sperm cells, and we can control it in vitro. Thus, we
observed natural changes in the oviduct when the sperm cells
are swimming towards the oocyte. Moreover, in aquatic
species, environmental conditions are vital to fecundity.

Kv currents identified in sperm
A previous study revealed the presence of different types
and differentially localized potassium channels [Darszon et al.
2006; Hagiwara and Kawa 1984]. An example is the delayed
rectifier Kþ type channel found in rat spermatogenic cells,
which shows a trend that is independent of extracellular
calcium and is blocked by tetraethylammonium chloride
(TEA) [Hagiwara and Kawa 1984]. Based on these characteristics, we identified an inward rectifier Kþ channel referred
to as Kir [Munoz-Garay et al. 2001]. This channel is also
regulated by the intracellular pH, with an acidic intracellular
pH (6.3) inhibiting the current in spermatogenic cells, while a
rising intracellular pH (7.4) significantly increases conductance in these cells. We further identified a third type of Kþ
channel, designated mSlo3, which was cloned in rat
spermatogenic cells and has been expressed in Xenopus
laevis oocytes for biophysical analyses. Recent studies using
electrophysiological methods allowed an output current from
the sperm midpiece that is sensitive to TEA to be detected

Sperm cells viability

3

[Marconi et al. 2008], and depolarization regulating calcium
entry was described.

Regulation of calcium voltage channels (CAVs)
during capacitation
During capacitation, ionic channels are susceptible to being
activated when a change in the configuration of these
channels occurs and are mediated by a change in the
membrane potential. In rat and bovine sperm, the membrane
potential is between À10 and À50 mV [Clapham et al. 2003;
Darszon et al. 2005]. Low voltage calcium is inactivated at
these voltages and therefore does not respond to depolarizing
stimuli. Analysis of the membrane potential of rat spermatozoa showed that only cells that maintain hyperpolarization
are able to generate an increased flow of calcium secondary
to contact with the ZP (likely secondary CAVs) and carry
out the RA [Arnoult et al. 1999]. Capacitation, resulting in
hyperpolarization, changes the configuration of the CAV in a
manner that is open to the agonist-mediated ion flow only at
a specific stage, thus avoiding early RA. Studies in sperm
conducted using electrophysiological methods have demonstrated the role of calcium channel functional are keys in
capacitation, which are dependent on the membrane potential
[Darszon et al. 2005; Wennemuth et al. 2000]. However, the
complete mechanism underlying this phenomenon and its
regulation via calcium entry is not completely understood.
In this context, it was recently suggested that calcium entry
occurs via depolarization and the regulation of motility, with
a second entry event occurring due to pH regulation and
depolarization, and this second calcium influx is mediated by
the AR [Escoffier et al. 2007]. These findings have led to new
models in which not only the type of CatSper channel is
responsible for this phenomenon [Xia et al. 2007] but have
further allowed the electrophysiological investigation of new
phenomena, such as depolarization, that are also involved in
the regulation of these voltage-dependent calcium channels.

Cationic sperm (CatSper) channels
Four members of the CatSper channels have been described
(CatSper1-4) in murine sperm [Quill et al. 2001; Ren et al.
2001]. These channels consist of 6 transmembrane domains
(6TM1) that are voltage-dependent and calcium-permeable
and appear to be found only in sperm cells. CatSper1 and
2 channels have been reported to be essential for sperm
hyperactivation and fertility. However, reports concerning
these channels still mainly result from studies of humans and
mice [Clapham and Garbers 2005].
Functional features of the plasma membrane of the sperm
tail have been described [Ren et al. 2001]. Other reports have
localized these proteins to the principal piece of the flagellum
[Kirichok et al. 2006; Qi et al. 2007]. Additional evidence
regarding the distribution of CatSper in different species and
its localization in sperm cells is being obtained through
ongoing investigations, which is important for designing
solutions for the manipulation of samples. Studies in which
the expression of this protein has been manipulated have led
to the generation of a male sterile phenotype in a normal
mouse model. While the mating behavior, sperm counts, and
sperm cell morphology of these mutant mice are


4

J. Parodi

indistinguishable from those of wild type mice, the CatSper1
mutant sperm cells are slow, exhibit a reduced basal rate, and
have no effect on the bathing or the bending of the tail region.
The mutant sperm cells cannot fertilize the eggs with an intact
zone pellucid but can fertilize eggs when the outer layers have
been enzymatically removed [Ren et al. 2001], suggesting
changes in some cell functions. Male mice lacking CatSper2
are also infertile due to a lack of the hyperactivated motility
required for penetration of the extracellular matrix of the
egg [Quill et al. 2003]. In a study in humans, subfertile men
with deficient sperm motility showed significantly reduced
expression of CatSper1 [Nikpoor et al. 2004]. Little is known
about CatSper3 and CatSper4, but they appear to be involved
in supporting cell functions in the sperm [Clapham and
Garbers 2005].
The above leads to two questions: (1) can these channels
explain all of the phenomena observed in the sperm cells?
(2) is there sufficient evidence to support the idea that
CatSper channels explain the entire model of the sperm
activity? It is accepted that CatSper channels and their various
isoforms are responsible for cellular functions in sperm.
Additionally, the relationship between CatSper and progesterone has been described, and the authors indicated the effect
of progesterone on increasing intracellular calcium levels
[Blackmore 1993; Turner and Meizel 1995]. While the
relationship between progesterone and CatSper has been
described [Lishko et al. 2011; Strunker et al. 2011], the
mechanism underlying the regulation of CatSper function
by progesterone is not completely understood, although the
intracellular PI3K-AKT signaling pathway was recently
implicated in this process. However, progesterone may be
associated with other receptors in sperm cells, such as
GABAa [Shi and Roldan 1995], or in the regulation of another
channel, such as potassium [Kumar et al. 2000], or voltagedependent calcium channels [Bonaccorsi et al. 2001].
Progesterone has been described to play a role in the specific
functions of sperm cell channels [Sagare-Patil et al. 2013].
Additionally, CatSper is modulated by pH [Fraire-Zamora and
Gonzalez-Martinez 2004] and bicarbonate [Wennemuth et al.
2003]. Nevertheless, additional events must be coordinated
for fecundation to occur successfully, including the AR, the
regulation of membrane stability, calcium signaling, and
mitochondrial function, among others, beyond Catsper modulation. However, these events are not described in all models,
and other electrical phenomena can cooperate in the cellular
events described in sperm. A complete table of ion channels,
indicating the presence of voltage-dependent calcium channels and CatSper, in humans and mice is available [Darszon
et al. 2011]. This review indicates that we lack a complete
understanding of the localization of these channels, and there
are other mechanisms that may alter intracellular calcium.

Changes observed in the membrane potential
of sperm cells
An increase in the membrane potential, described as
hyperpolarization, occurs during capacitation in rat, bovine,
and human spermatozoa [Arnoult et al. 1996; Brewis et al.
2001; Zeng et al. 1996]. In rat sperm, hyperpolarization is the
result of increased permeability to Kþ [Zeng et al. 1995],


leading to a change in the membrane potential. During
capacitation, there is an increase in the pHi of more than
0.2 units [Zeng et al. 1996], which is sufficient to induce an
increase of 0.5 to 3 times in the probability of the opening
of Kir channels found in other tissues [Gutman et al. 2003].
Thus, under physiological conditions, an increase in pHi
activates Kir channels. It has been suggested that this process
hyperpolarizes the sperm membrane [Krasznai et al. 2000].
Furthermore, Kv-activated intracellular calcium is modulated
by the increase in the concentration of intracellular calcium
that occurs during capacitation, thus contributing to hyperpolarization [Jagannathan et al. 2002]. Together these observations confirm the role of Kþ currents in the hyperpolarization
of the sperm membrane and its effect on capacitation and the
subsequent AR. However, in other cell models, the mechanism reflects blocking the Kþ channel shaft, depolarization,
and calcium channel opening [Baker et al. 1973; Wellman
et al. 2001]. In sperm models, it is accepted that Kir channels
are able to hyperpolarize the membrane, but these channels
are controlled by physiological phenomena, leading to changes
in the membrane potential and correcting this potential,
allowing positive charges to be relocated to restore balance
and maintain a physiological membrane potential [Gutman
et al. 2003]. Kv-type channels are present in sperm [Marconi
et al. 2008], and their current is modulated by peptides,
suggesting a means to modulate currents in sperm [Parodi
et al. 2010]. This model is sensitive to ASD and can be applied
to generate depolarization in other cell models, leading to an
increase in intracellular calcium levels and consequent cellular
changes [Navarrete et al. 2010]. Some evidence suggests that
this mechanism is part of a complex mechanism of regulation
that also includes the hyperpolarization and depolarization
described in sperm [Fraire-Zamora and Gonzalez-Martinez
2004; Gonzalez-Martinez 2003; Neri-Vidaurri Pdel et al.
2006], which can generate changes in the membrane potential,
causing an influx of calcium and alterations in the physiology
of sperm [Babcock and Pfeiffer 1987; Linares-Hernandez et al.
1998]. It is not hyperpolarization alone that mediates this
phenomenon. The control of the membrane potential of sperm
cells can block calcium entry and the associated secondary
signaling. Many drugs can block changes in the membrane
potential; could these drugs be used as potential regulators of
sperm motility? A high concentration of potassium can induce
changes in intracellular calcium levels, in the form of a wave
from the middle piece to the head of the sperm. Figure 1 shows
the effect of high potassium on intracellular calcium levels in
bovine sperm (from [Navarrete et al. 2010]).

Calcium as a second messenger
The processes that generate second messengers that regulate
cellular physiology have been studied for several years.
Calcium is important for the regulation of kinase activity,
phosphatases, gene activation, and protein translation. It is
required at high concentrations for short periods of time,
and cells display various mechanisms for finely regulating its
intracellular concentration and maintaining a physiological
calcium gradient [Hurwitz 1996; Stewart 1985]. Thus, various
signals transiently increase intracellular calcium, which is
indicative of activation of cellular processes, whereas a



DOI: 10.3109/19396368.2013.869273

sustained increase in the cellular concentration indicates cell
toxicity [Pounds 1984]. These changes in intracellular
calcium concentrations may vary depending on cell type
and the type of stimulation involved. Furthermore, they have
been correlated with the process of vesicular exocytosis
based on observations made through different techniques for
measuring calcium currents and cell capacitance [Trifaro
et al. 2000], including fluorometric measurements of calcium
levels and amperometric records [Elhamdani et al. 1994].
For example, the normal process of vesicle release is
highly dependent on calcium entry, which is crucial for the
propagation of nerve impulses and the establishment of neural
connections responsible for cognitive brain functions. In
sperm cells, the intracellular activation of vesicles in the
AR is similar to what is observed in other types of somatic
cells and depends on changes in calcium levels. These
findings suggest that different pathways leading to changes
in calcium levels play a role in the development of different
models of cell physiology.

Animal species of industrial interest
Understanding the influence of reproduction in food production is important in relation to increasing output and yield
as well as maintaining and preserving genetic markers to
improve productivity. Regarding the production of meat for
consumption, cows, goats, pigs, and fish have been instrumental in the development of this industry. In recent years,
assisted reproduction has begun to be applied in these species
by preserving oocytes and sperm for later use in artificial
insemination. The main reference models studied have been
mice and humans, and similar techniques have been implemented in cows. Work aimed at the cryopreservation of sperm
from salmon and other species was recently initiated, with
sperm being frozen for transport, storage, and handling. There
is high national and international demand for animal reproduction, as the meat market is steadily increasing, and the
requirements for animal protein for human populations are
also increasing [Food and Agriculture Organization of the
United Nations, 2003]. The world population in 2030 will
consume more and better food, with 3050 kilocalories (kcal)
being available per person, compared to 2360 kcal per person/
day in the mid-1960s and the 2800 kcal available currently.
This change reflects the increase in consumption in many
developing countries, whose average daily intake will be
approximately 3000 kcal in 2030. For example, it has been
reported that the domestic consumption of pork per person
has increased [Oficina de Estudios y Politicas Agrarias,
2011], reaching values of 23 kg/capita in recent years. Thus,
pork has become the second most commonly consumed meat,
while poultry consumption decreased from 2000 to 2006 and
has remained even at levels of 18 kg/capita over the last 4
years. The economic returns from the exploitation of animal
flesh under current market conditions are based on the
management of their genes and the use of high-genetic value
players together with the best production techniques to obtain
high-quality meat products at competitive cost. Reproduction
is one of the most important aspects of the animal resource, as
it allows the continuity of the species to be maintained.
Additionally, the economic importance of reproductive

5

behavior in cattle is well-known. Ingvartsen and Moyes
[2013] summarized that essential studies examining the
factors that affect the same traits will increase productivity
in females. Thus, techniques including the control of insemination have begun to be viewed as an alternative for
improving production, and the discussion regarding phenotypic traits of importance to the industry is increasing.
How do we maintain these gametes, increase cell function,
and apply these techniques under various industrial conditions? This is not an easy question to answer, but the cellular
functions of sperm related to generating such compounds as
well as protocols and conditions applicable in this industry
should be determined.
Mature sperm cells are complex cellular machines that
through a series of steps and environments reach their target,
the oocyte, and fulfill the purpose of delivering their genetic
material via fertilization. In this review, we have highlighted
flagellar motility and capacitation, which is characterized
by the AR. In recent years, the function of CatSper channels
as regulatory elements has shown to be indirectly involved in
modulating the motility and fertilization capacity of sperm as
well as calcium entry. A recent study has now demonstrated
that a CatSper channel is involved in the motility but not in
the AR [Sagare-Patil et al. 2013]. Flagellar movement
generates various changes, including the production of ROS,
and these increases can explain the reduction of cell viability.
Moreover, some sperm cell models can remain immobile for a
period of time. These sperm cells show a long period of
viability and maintain their cellular functions for days. When
activated, the cells become motile upon external signaling
(i.e., osmotic changes). Calcium regulation is important for
the general function of cells. In mammalian sperm cells, a
recent study has suggested that there are two steps regulated
by calcium entry: first, the motility of sperm cells, and
second, the AR. Since motility generates ROS it is
hypothesized here that regulation by calcium reduces the
motility and the general metabolic state of the cells, leading to
a reduction of cell mortality. All of these regulatory
mechanisms are important for the conservation and manipulation of sperm cells. Because food production, and especially
that of animal protein, has increased in recent decades,
reproductive processes must be understood to provide an
efficient means of control. It is vital for the development of
the food industry to study these processes, yet little is known
about the cells involved and the conditions that must occur.
Thus, we should study other species as a reference for the
development and maintenance of sperm as a function of
process.

Declaration of interest
The author reports no conflicts of interest. The author alone
is responsible for the content and writing of the paper.

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