Download The impact of sperm metabolism during in vitro storage: the stallion

The impact of sperm metabolism during in
vitro storage: the stallion as a model
Zamira Gibb, PhD
The University of Newcastle
Background on cellular respiration
Adenosine triphosphate (ATP) is the energy molecule used by all living cells. It is an organic
molecule containing high phosphate bonds. Energy is harvests from ATP through the breaking of these
phosphate bonds by the ATPase enzyme, leaving behind adenosine diphosphate (ADP) which is
rephosphorylated by ATP synthtase during glycolysis and the citric acid cycle (aka Krebs cycle or
tricarboxylic acid cycle; TCA). Carbohydrates, such as glucose, are broken into smaller molecules in the
first step of cellular respiration; glycolysis. During this process, some ATP is produced (4 molecules), but
ATP is also consumed (2 molecules), so that overall there are only 2 molecules of ATP produced during
glycolysis. Glucose is broken down into pyruvate which, under anaerobic conditions is converted to lactate
but under aerobic conditions (in the presence of oxygen) is converted to acyl-CoA which, with the help of
carnitine enters the mitochondria, is oxidised to acetyl-CoA and enters the citric acid cycle. The citric acid
cycle produces some ATP, but it also produces the NADH and FADH that is essential for driving the electron
transport chain, located on the inner mitochondrial membrane. The electron transport chain results in a high
concentration of hydrogen ions (or ‘protons’) in the mitochondrial inner membrane space. This gradient
generates energy through the movement of hydrogen ions back in to the mitochondrial matrix via ATP
synthase, a membrane bound enzyme which uses this energy to add a phosphate group to ADP (oxidative
phosphorylation; OXPHOS), in turn producing ATP. A graphical overview of ATP production via aerobic
respiration is presented in Figure 1.
Figure 1. Overview of ATP production via aerobic cellular respiration
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History
Energy production by mammalian spermatozoa was first recognised during the two decades
following the 1919 publication of Frank Lillie’s famous book in which mitochondria in the sperm midpiece
were first identified [1]. Much of this early research was conducted on bulls by virtue of their availability
and the increased use of artificial insemination by the dairy industry. In 1933 the need for ‘outside
nourishment’ of bull spermatozoa in the absence of oxygen was first reported [2] with the observation that
washed spermatozoa (free of seminal plasma) maintained motility in the presence of O2, but required glucose
or other sugars to survive anaerobically. In the 1940s several studies supporting these observations were
performed at the University of Wisconsin [3-5] and it was concluded that bull spermatozoa could exhibit
both oxidative and glycolytic metabolism. Zittle and Zitin [6] further investigated the relative importance of
glycolysis and OXPHOS, concluding that although there was no close correlation between O2 consumption
and motility, if mitochondrial activity was inhibited with cyanide, even in the presence of exogenous sugars,
motility was impeded. From a very early time in animal andrology research the ability of sperm cells to
utilise both oxidative and glycolytic pathways has been well accepted. However, the importance of OXPHOS
has been somewhat overlooked in recent years, particularly with respect to inter-species differences and the
production of reactive oxygen species (ROS).
Physiology of energy production by spermatozoa
Spermatozoa are among the most highly specialised mammalian cells, with the roles of delivering
paternal DNA and triggering activation of the oocyte. Given that the site of sperm deposition is somewhat
removed from the site of fertilisation, the ability to generate energy in the form of ATP for the function of
motility is essential. In addition, the process of capacitation whereby spermatozoa develop the ability to
fertilise the oocyte, is a highly energy dependant process involving numerous modifications to motility
patterns and plasma membrane structure [7].
There are two main metabolic pathways of energy production by spermatozoa; glycolysis and
oxidative phosphorylation (OXPHOS). The enzymes associated with glycolysis are located in the principal
part of the tail, while OXPHOS occurs in the mitochondria which are arranged in a sheath around the sperm
midpiece. Despite the fact that OXPHOS is significantly more efficient at producing ATP than glycolysis,
spermatozoa from most species depend predominantly on glycolysis for ATP production [8]. During
spermatogenesis, DNA is condensed to a crystalline structure which provides not only mechanical protection
from ROS damage, but also allows the spermatozoa to become streamlined for ease of movement [9]. During
this process, the majority of the cytoplasm is removed from the cell and as such, spermatozoa lose the specific
locations directed to energy accumulation and are almost entirely dependent on an external supply of sugars
for glycolysis [10].
There has been considerable attention focussed on the role of glycolysis in sperm motility over a
number of species. While glucose may passively diffuse across the bilipid layer, this process is inefficient
and the more rapid uptake of glucose is facilitated by glucose transporters (GLUTs), which were first
described by Kasahara and Hinkle in 1977 [11]. A number of GLUTs have been identified, but they are
largely characterised by their ability to transport hexoses (glucose, fructose, mannitol), vitamins and amino
sugars [12]. GLUT 1 is ubiquitous in mammalian cells, and its role is to provide basal energy uptake [13].
GLUTs 1, 2, 3, 4 and 5 have been identified on stallion spermatozoa, with the location of GLUTs 1, 2 and 5
being the tail and the acrosome, GLUT 3 being located on the subequatorial region and GLUT 4 having no
specific immunolabelling pattern, but its presence has been identified using Western blotting, with a weak
band at 70/80 kDa in the horse [10; Fig 2]. These patterns of distribution suggest that glycolytic processes
are involved in generating energy for the membrane modifications required for capacitation and the
acrosome reaction. Should this be the case, then the distribution of GLUTs would be expected to change
with the functional status of the cell (i.e. between non-capacitated and capacitated states), a phenomenon
which has been reported in the dog, but does not occur in the horse [14]. At this stage, the significance of
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glycolysis to stallion sperm ATP production for either motility or capacitation and acrosome reaction is
poorly understood, and further investigations are necessary to elucidate its importance.
As previously stated, while spermatozoa from the majority of species rely almost completely on
glycolysis for ATP generation, there is emerging evidence to suggest that stallion spermatozoa are almost
entirely dependent on OXPHOS for the generation of ATP for motility. In the presence of mitochondrial
inhibitors, stallion spermatozoa rapidly lose velocity and ATP levels decline dramatically, while human
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spermatozoa display no significant decrease in motility parameters or ATP levels following treatment with
mitochondrial inhibitors. Although studies such as these have highlighted the relative contribution of
OXPHOS to energy production in these species, deciphering the relative importance of glycolysis to stallion
sperm function is somewhat more problematic. Many studies have attempted to quantify the relative
contribution of glycolysis by inhibition of the process using the biologically unavailable isomer 2-DeoxyD-glucose. While this method does indeed result in a decrease in ATP, it is a somewhat ‘blunt’ instrument
in that the depletion of ATP is due to both the absence of usable hexoses as well as ATP exhaustion through
a lack of negative feedback (as the cell does not recognise the presence of glucose) and the resulting futile
phosphorylation of the unusable 2-Deoxy-D-glucose by hexokinases; a highly energy dependant activity
[15]. A more direct approach to the quantification of glycolytic processes would be to perform flux and
product distribution measurements using 14C-labelled glucose and evaluating the production of 14C-labelled
pyruvate and lactate in the presence and absence of mitochondrial inhibition.
The hypothesis that stallion spermatozoa depend predominantly on OXPHOS for energy generation
has been developing for some time. This idea arose from observations of a non-conventional relationship
between ROS and fertility in the stallion [16, 17]. The source of ROS under these conditions is the electron
transport chain, in which about 1-3% of O2 reduced in the mitochondria during OXPHOS forms superoxide
[18]. While the finding that stallion spermatozoa generate ROS is not novel, the general dogma has always
been that it is the non-viable or poor quality spermatozoa that generate the most ROS [19]. However an
alternative explanation is that rapidly metabolizing spermatozoa from highly fertile stallions exhibit higher
levels of OXPHOS activity and therefore present with elevated levels of ROS generation and lipid
peroxidation coupled with impaired vitality.
The significance of understanding the modes by which stallion spermatozoa generate energy are
numerous. Sperm motility is not only lost as a consequence of lipid peroxidation due to ROS attack [20], but
also due to the depletion of ATP [21]. With respect to the storage of stallion spermatozoa for AI, a better
understanding of metabolic processes will allow tailoring of the media constituents to provide the correct
energy sources (glucose vs. pyruvate) and carrier molecules to allow the unimpeded generation of ATP for
optimal sperm motility, functionality and fertility. Given that mitochondrial metabolism is the source of the
majority of ROS, a mitochondrial antioxidant that can effectively scavenge leaked superoxide may present
the best option to reduce the downstream effects of ROS on sperm functionality and DNA integrity. In
addition, the relevance of oxidative stress to stallion fertility must be re-assessed. While the symptoms of
oxidative stress are never going to be beneficial, they may be indicative of an extremely fertile sample while
remaining at sub-clinical levels. A major implication of this phenomenon is that semen assessments must be
performed as close as possible to the time of collection to avoid ROS-induced artefacts.
In vitro sperm storage: the horse
Horses are selected for breeding on the basis of pedigree and athletic performance as opposed to
reproductive traits and therefore, like humans, are not subjected to selection pressure for fertility.
Reproductive fitness traits are heritable [22], and the practice of circumventing subfertility through the use
of assisted reproductive technologies (ART), because it places no importance on reproductive fitness in the
selection of breeding animals or partners, has resulted in equine and human populations with significantly
lower per cycle conception rates than other species [22-24]. As artificial Insemination (AI) is a widely
utilised tool in modern horse reproduction [25], with around 90% of Standardbred and Hanoverian foals
being produced via AI of chilled or cryopreserved stallion spermatozoa [23, 26], this animal model provides
an excellent source of information about the influence of cell metabolism on the storage of male gametes.
For its part, the use of AI brings a number of advantages, such as the prevention and control of disease
through the eradication of direct male to female contact [27], an increased rate of genetic gain through the
importation of new genetics and the preservation of spermatozoa for later use in case of death or infertility.
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Sperm metabolism
Spermatozoa are highly specialised mammalian cells, playing the vital roles of paternal DNA
delivery and activation of the oocyte following fertilisation. The site of sperm deposition (in the vagina for
the human and the uterus for the horse) is physically removed from the site of fertilisation (the oviduct).
While a proportion of sperm transport is facilitated by uterine contractions, the spermatozoa must in
themselves be sufficiently motile to traverse the uterotubal junction prior to oviduct binding, and to locate
the egg following ovulation. In addition, spermatozoa must undergo a process called capacitation for the
final maturational changes that are required to allow them to fertilise the oocyte. This process involves
extreme membrane remodelling and the hyperactivation of motility and, as such, is a highly energydependent process [7].
The process of spermatogenesis requires extensive remodelling of a conventional spherical cell to
become one the most highly specialised and morphologically distinct cells in the body. During this
transformation, the DNA in the sperm nucleus reaches the physical limits of compaction to achieve a
quasicrystalline state [9]. This extreme compaction requires the removal or resorption of most of the
cytoplasm, at the same time removing the majority of the organelles (such as the endoplasmic reticulum,
ribosomes and Golgi apparatus) that are responsible for the regulation of metabolism in somatic cells. The
result of this drastic modification is that spermatozoa are left both translationally silent and depleted of
intracellular enzymes and energy reserves such as fat droplets, yolk granules and glycogen. For this reason,
spermatozoa are highly dependent on their immediate extracellular environment for both the enzymatic
activities that would normally be conducted intracellularly and the supply of energy substrates [10]. In
somatic cells, the array of enzymes involved in protecting spermatozoa against oxidative stress would also
be housed intracellularly within the cytoplasm. Spermatozoa, on the other hand, depend upon epididymal
and seminal fluids to provide the richest and most diverse combination of antioxidants in the body, including
several that are unique to the male reproductive tract [28, 29].
As with somatic cells, the predominant metabolic pathways that spermatozoa use to produce ATP
are glycolysis and oxidative phosphorylation (OXPHOS) [8]. The enzymes necessary for glycolysis are
primarily associated with the fibrous sheath located in the principal piece of the tail. In contrast, OXPHOS
occurs in the mitochondrial gyres located in the midpiece. OXPHOS is a significantly more efficient method
of ATP production than glycolysis. Despite this, spermatozoa from most heavily researched species,
including the human and laboratory rodents, depend predominantly on glycolysis for ATP production [8].
The role of glycolysis in driving the production of ATP for motility has been well researched due
to its relative importance in human and laboratory species. Large polar molecules such as glucose cannot
diffuse across membranes, and their transport is facilitated by membrane bound proteins called glucose
transporters (GLUTs) [11]. GLUTs are categorised according to their relative ability to transport hexoses
(such as glucose, mannitol and fructose), amino sugars or vitamins [12]. Since the discovery of the glucose
transporter GLUT 1, a great many additional GLUTs have been characterised [13, 14]. In spermatozoa of
the stallion, GLUTs are localised to the tail and acrosome, suggesting that glycolytic processes are involved
in generating energy for the membrane modifications required for capacitation and the acrosome reaction
[14]. In glycolytic spermatozoa, the distribution of GLUTs changes along with the capacitation status of the
cell (i.e. between noncapacitated and capacitated states) to provide energy at the sites requiring membrane
modifications or hyperactivation of motility [14]. In contrast, the distribution of GLUTs on stallion
spermatozoa does not change with the capacitation status of the cell [14], indicating that in species who rely
on OXPHOS, glycolysis is not required to support ATP production for motility, capacitation or the acrosome
reaction.
Despite the well characterised presence of GLUTs on equine spermatozoa, it has become abundantly
evident that stallion spermatozoa differ from that of other well-studied mammalian species, in that their
energy demands are met not by glycolytic pathways, but by using OXPHOS [17, 30, 31], and in the presence
of mitochondrial inhibitors, they suffer a rapid loss of velocity and a dramatic decline in ATP content [30;
Fig 3]. This dependence results in a nonconventional relationship between ROS production and fertility in
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the stallion [17, 30, 31], with the source of ROS being the mitochondrial electron transport chain, in which
about 1–3% of O2 reduced in the mitochondria during OXPHOS forms superoxide [18].
Figure 3. Effect of OXPHOS inhibition (using diphenyleneiodonium at 10 µM) on A) equine and human
sperm ATP content and B) equine and human sperm average path velocity (VAP). *P ≤ 0.05, **P ≤ 0.01.
There is a long-standing paradigm that it is the nonviable or poor quality spermatozoa that generate
the most ROS [19]. An alternative explanation is that rapidly metabolising spermatozoa from highly fertile
stallions exhibit higher levels of OXPHOS activity, and following in vitro storage prior to AI present with
elevated levels of ROS generation and lipid peroxidation. Thus, while human clinical data steadily report
negative correlations between male fertility and sperm oxidative stress [32, 33], a recent study has revealed
a paradoxical inverse relationship between fertility and the percentage of live cells without oxidative damage
in the stallion[30]. In addition, more fertile ejaculates (those which resulted in a pregnancy following
insemination) had lower vitality and a higher percentage of cells displaying ROS-induced damage following
in vitro storage than less fertile ejaculates which did not result in a pregnancy [30; Fig 4].
Figure 4. Comparison of A) vitality, B) acrosome integrity (vital cells), C) sperm concentration, D) ROS
production (DHE), E, F and G) oxidative DNA damage (8OHdG) between equine sperm samples that did
(closed bar) or did not (open bar) result in a pregnancy at day 14 following mating. Values shown are
mean + SEM. *P ≤ 0.05, **P ≤ 0.01
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From these results, it was hypothesised that during in vitro storage spermatozoa from the more
fertile stallions (assumed to be more metabolically active) were becoming exhausted more rapidly, such that
by the time that the assays were performed in the laboratory, these cells had suffered an accelerated demise
due to the accumulation of metabolic by-products, such as ROS and cytotoxic lipid aldehydes in a ‘live fastdie young’ paradigm. Another interesting observation was that the greater efficiency of OXPHOS mediated
ATP production by equine spermatozoa supported a higher velocity, with stallion spermatozoa being around
60% faster than human spermatozoa (Fig 3). Ultimately, high ROS production by stallion spermatozoa
appears to be a physiologically normal scenario brought about by superoxide leakage from the mitochondrial
electron transport chain during OXPHOS [31], with a positive relationship between mitochondrial ROS
production and sperm velocity, leading to increased rates of lipid peroxidation [30; Fig 5] and, following
prolonged storage, a loss of motility and vitality [20]. This phenomenon introduces a number of implications
for the in vitro storage of stallion spermatozoa, since the prolonged generation of ROS in the absence of
extracellular free radical and lipid aldehyde scavengers will lead to irreversible oxidative damage, impairing
DNA integrity and sperm functionality (Fig 6).
Figure 5. Significant correlations between equine sperm motility parameters (total motility, rapid motility,
VAP and VCL) and mitochondrial ROS production or lipid peroxidation in live cells (n = 28, P ≤ 0.001).
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Figure 6. Implications of oxidative phosphorylation (OXPHOS) on sperm storage in vitro. Mitochondrial
superoxide (O2·˗) leakage causes lipid peroxidation and reactive electrophilic aldehyde production. These
aldehydes adduct to functional proteins resulting in motility loss and mitochondrial damage, which may
trigger apoptosis and DNA damage. If this damage does not result in cell death, then germ line mutations
may cause embryonic failure and abortion, or should the mutations not be lethal, result in poor health in the
resulting offspring. The potential outcomes of oxidative damage as a result of OXPHOS are highlighted.
In vitro storage of spermatozoa
In the horse, the most common reason for sperm storage prior to AI is the asynchronous nature of
ovulation in the mare. This makes it difficult to predict the precise time of ovulation [34] and means that
stored spermatozoa must retain their functionality and longevity for extended periods to allow for the
possibility of a delayed ovulation. The long term storage of spermatozoa is useful so that AI may be
performed when ovulation is deemed imminent (based on follicle size determined via transrectal
ultrasonography). If AI is to be performed within 12 h of semen collection, spermatozoa are generally left at
room temperature (‘fresh’). If sperm longevity must be maintained for longer periods, spermatozoa are either
chilled (up to 72 h) or cryopreserved (indefinite) to restrict the metabolic rate of the spermatozoa. This
temperature-induced metabolic restriction reduces the rates of both reactive oxygen species (ROS)
production and acidification of the storage medium through the accumulation of lactic acid and CO2 from
glycolysis and OXPHOS respectively. However, the spermatozoa of many stallions, and indeed men, do not
tolerate the stressors associated with chilling or cryopreservation [35-38]. Therefore, there is a need to
develop a medium which will extend the longevity of spermatozoa without the need to chill or cryopreserve.
Sperm cryopreservation
Cryopreservation is presently the only viable method of in vitro storage of spermatozoa for periods
exceeding 72 h. However, the process of cryopreservation and thawing reduces the acrosomal integrity,
viability and motility of spermatozoa in all species examined including the horse [39-41] while for human
spermatozoa there is evidence that cryopreservation results in the formation of DNA lesions on genes that
are essential for fertilisation and normal embryonic development [42]. Many of the deleterious effects
induced by cryopreservation may be attributed to osmotic stress. During cooling below 0°C, extracellular
ice crystals begin to form. This phase change causes a large increase in the osmolarity of any remaining
liquid to which the spermatozoa are exposed, placing cells under extreme osmotic stress [43, 44].
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Additionally, the cryoprotectants make the cryodiluent hyperosmotic, which causes dehydration of the cells
through osmosis [45]. While this dehydration is essential for post-thaw viability to be maintained, the
extreme hyperosmolarity induces cellular stress as water rushes across the sperm membrane via water
channels in an attempt to balance the osmolarity [46]. The result of these osmotic stressors include membrane
damage [47], DNA damage [48, 49] and the production of ROS which causes premature capacitation-like
changes [50].
Chilling
The Standardbred, Sporthorse and Polo pony industries, along with the cattle and pig breeding
industries, are almost entirely dependent on AI for breeding purposes. Sperm chilling is the most widely
utilised technique for the transport and storage of stallion spermatozoa. ‘Chilling’ is most commonly
achieved using commercial passive cooling devices which slowly cool extended semen to a temperature of
between 4 and 10°C, an adequately low enough temperature to restrict metabolism sufficiently to maintain
acceptable sperm functionality for up to 72 h. However, stallion spermatozoa are significantly more
susceptible to cold shock than those of other species, probably due to a lower ratio of cholesterol to
phospholipid in the sperm membranes [51] and, as a result, the insemination of chilled semen is associated
with success rates as low as 44% per cycle [24]. As with cryopreservation, there are significant unexplained
differences between stallions in the suitability of their semen for low temperature storage [52], a phenomenon
which reduces the commercial viability of such animals due to sperm damage following chilling [53].
Additionally, animal derived compounds, such as milk and egg yolk, are routinely incorporated into media
for chilled and frozen semen due to their membrane-stabilising effects [54]. This presents a major biosecurity
concern for customs authorities and, as such, is a chief limiting factor for the genetic improvement of herds
in geographically isolated countries such as Australia.
Ambient temperature storage
The development of a medium that allows spermatozoa to be stored at ambient temperatures for at
least one week would permit the importation of new genetics into geographically isolated areas, while
avoiding the loss of fertility that occurs following semen chilling and cryopreservation. Moreover, as ambient
temperature storage does not require the addition of animal-derived products, such as egg yolk and skim
milk for membrane stabilisation, the biosecurity risks associated with importing spermatozoa will be
considerably reduced.
There are several implications that arise when higher temperatures are utilised for the in vitro storage
of stallion spermatozoa. The first of these is the growth of bacteria in the nutrient rich semen extender during
storage. Many microbes are present on the penis of the stallion; these include normal commensal bacteria
along with microbes from soil, water and faeces which may contaminate the penis when the stallion gallops,
rolls or lies down in the paddock. Through the process of semen collection using an artificial vagina, these
bacteria will inevitably contaminate the ejaculate [55]. Several of these strains have been shown to be
deleterious to sperm motility and vitality, even during chilled storage at 4°C [56] and following
cryopreservation [57]. However, several antibiotic formulations have been investigated for their effects on
curtailing bacterial growth in extended stallion semen [56, 58], and based on these studies, further work in
our laboratory has revealed that a storage medium containing 0.25 mg/mL gentamicin, 50 µg/mL
streptomycin and 50 IU/mL penicillin is able to suppress the growth of bacteria for up to one week at room
temperature (Gibb et al. unpublished data).
If sperm metabolism is not restricted by temperature reduction, OXPHOS will produce significant
quantities of ROS [18], which will compromise sperm function [20, 29]. The majority of attempts to assuage
the damaging effects of ROS on stallion spermatozoa through antioxidant supplementation have produced
either marginal improvements [59-63] or have had detrimental effects [64, 65]. This is in contrast to the
positive effects seen in human spermatozoa [66, 67], and may be due in part to their alternative mode of
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ATP production. More recently, the antioxidant properties of carnitine have come into the spotlight [68-72].
L-carnitine supplementation of stallion spermatozoa during in vitro storage significantly reduces both
mitochondrial free radical production and lipid peroxidation [72], suggesting that the beneficial effects
observed by others may well be attributed to L-carnitine’s antioxidant properties. However, supplementation
with L-carnitine alone does not completely abolish ROS-induced damage, indicating that it is insufficient
for the complete scavenging of ROS [72]. Clearly, further refinement of the antioxidants that might be used
to facilitate the long-term storage of stallion spermatozoa is required. Given that mitochondrial metabolism
is the source of the majority of ROS, a mitochondrial antioxidant that will also act as a regulator of
mitochondrial bioenergetic functions may present the best option to reduce the downstream effects of ROS
on sperm function and DNA integrity. While L-carnitine meets these requirements, it is possible that at 50
mM we have reached its beneficial limits, and that supplementation with additional antioxidants capable of
performing alternative roles in mitochondrial energy production homeostasis may be necessary. Possible
candidates for this role are co-enzyme Q10, an integral component of the electron transport chain and an
antioxidant capable of counteracting the ROS-induced peroxidation of mitochondrial phospholipids [73],
and melatonin, a free radical scavenger which reduces nitric oxide generation within mitochondria while
performing bioenergetic functions by regulating respiratory complex activities, Ca2+ influx and
mitochondrial permeability transition pore opening [74].
Sperm motility is not only lost as a consequence of lipid peroxidation due to ROS attack [75], but
also due to the concomitant depletion of ATP [21] which compromises myriad ATP-dependent functions,
disrupting homeostasis and hastening cell death [76]. If spermatozoa are to be stored at ambient
temperatures, it is vital to support mitochondrial energy production while reducing avoidable ATP depletion
which results when pressure is placed on ATP–dependent pathways such as the regulation of ionic flux [77].
By utilizing non-ionic, organic osmolytes, such as betaines, carbohydrates and amino acids, in place of
sodium chloride, pressure on the ATP-dependent Na+/K+ pumps is alleviated, deducing the rate of ATP
depletion [77]. Recent research has revealed that supplementing media with pyruvate, the primary energy
source utilized for OXPHOS, and L-carnitine, the biologically active free form of carnitine which plays an
essential role in mitochondrial ATP synthesis while being a powerful antioxidant [78] and an organic, nonionic osmolyte [79] results in the maintenance of motility and viability at room temperature akin to that of
chilled semen for up to 72 h [72]. Furthermore, stallion spermatozoa contain a number of proteins involved
in beta-oxidation of mitochondrial fatty acids and inhibition of this metabolic pathway leads to reduced
motility, indicating its significance in fertility [80]. As L-carnitine plays an essential role in beta-oxidation,
in addition to its role as an antioxidant and osmolyte, it boosts mitochondrial ATP production through the
transportation of acetyl groups from pyruvate into the mitochondrial matrix and through the buffering of free
CoA. The acetylation of carnitine (acetyl-L-carnitine; ALCAR) by spermatozoa occurs across the outer
mitochondrial membrane to facilitate the provision of acetyl groups for β-oxidation and entry into the citric
acid cycle for ATP production. The in vivo importance of L-carnitine in sperm quality is well recognized
[81-86]. Androgen regulated epithelial cells actively secrete L-carnitine into the epididymal lumen [87, 88]
resulting in concentrations of up to 2000 fold higher than that of blood, with spermatozoa containing the
highest intracellular concentrations of L-carnitine in the body [84], suggesting that this molecule is of
extreme importance in fertility. In addition, oral supplementation of L-carnitine results in increased uptake
of pyruvate by spermatozoa [89], demonstrating an important interactive role between these compounds in
the support of sperm metabolism.
Conclusion
As the major implication for ambient temperature storage of equine spermatzoa is the production of
ROS as a consequence of OXPHOS, future research should concentrate on reducing the deleterious effects
of this pathway either through the redirection of metabolism towards the less deleterious glycolysis, or
through elucidating the mechanisms behind the reversible mitochondrial uncoupling which induces a
quiescent state during the in vivo storage of spermatozoa in the epididymis. Work in our laboratory has
revealed that mouse epididymal fluid can reversibly uncouple sperm mitochondria [90], a phenomenon
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which we have also observed in the horse. Once this factor has been identified, there is the potential to exploit
it to induce sperm quiescence during in vitro storage akin to that in the epididymis. In addition, we have
found that supplementation of sperm storage medium media with rosiglitazone, a member of the
thiazolidinedione family of compounds, significantly enhances sperm longevity during storage at ambient
temperature. We hypothesize that rosiglitazone is redirecting stallion sperm metabolism from OXPHOS to
glycolysis by increasing the efficiency of glucose uptake through GLUT1 [91] and the preferential utilization
of both aerobic [92] and anaerobic glycolysis [93]. While currently the only feasible method for the indefinite
storage of spermatozoa is cryopreservation, methods for the storage of spermatozoa at room temperature for
at least one week, a sufficient period of time to account for the logistical constraints surrounding
insemination and IVF protocols, are in the final stages of optimization. This development will make the need
to chill spermatozoa obsolete and in many cases will also negate the need to cryopreserve for gamete
importation purposes, resulting in improved per-cycle fertility and embryo development rates.
References
[1] Lillie FR. Problems of Fertilization. Chicago, IL: University of Chicago Press; 1919.
[2] Redenz E. Über den spaltingsstoffwechsel der säugertierspermatozoen in zusammenhang mit der
beweglichkeit. Biochem Z. 1933;257:234-41.
[3] Lardy HA, Phillips PH. The effect of certain inhibitors and activators on sperm metabolism. J Biol
Chem. 1941;138:195-202.
[4] Lardy HA, Phillips PH. The interrelation of oxidative and glycolytic processes as sources of energy for
bull spermatozoa. Am J Physiol. 1941;133:602-9.
[5] Lardy HA, Phillips PH. Phospholipids as a source of energy for motility of bull spermatozoa. Am J
Physiol. 1941;134:542-8.
[6] Zittle CA, Zitin B. The amount and distribution of cytochrome oxidase in bull spermatozoa. J Biol
Chem. 1942;144:99-104.
[7] Flesch FM, Gadella BM. Dynamics of the mammalian sperm plasma membrane in the process of
fertilization. Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes. 2000;1469:197-235.
[8] Storey BT. Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int J Dev Biol.
2008;52:427-37.
[9] Miller D, Brinkworth M, Iles D. Paternal DNA packaging in spermatozoa: more than the sum of its
parts? DNA, histones, protamines and epigenetics. Reproduction. 2010;139:287-301.
[10] Bucci D, Rodriguez-Gil JE, Vallorani C, Spinaci M, Galeati G, Tamanini C. GLUTs and mammalian
sperm metabolism. J Androl. 2011;32:348-55.
[11] Kasahara M, Hinkle PC. Reconstitution and purification of the D-glucose transporter from human
erythrocytes. J Biol Chem. 1977;252:7384-90.
[12] Angulo C, Rauch MC, Droppelmann A, Reyes AM, Slebe JC, Delgado-López F, et al. Hexose
transporter expression and function in mammalian spermatozoa: Cellular localization and transport of
hexoses and vitamin C. J Cell Biochem. 1998;71:189-203.
[13] Mueckler M, Caruso C, Baldwin SA, Panico M, Blench I, Morris HR, et al. Sequence and structure of
a human glucose transporter. Science. 1985;229:941-5.
[14] Bucci D, Isani G, Spinaci M, Tamanini C, Mari G, Zambelli D, et al. Comparative
immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa. Reprod Dom Anim.
2010;45:315-22.
[15] Ford WC. Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? Hum
Reprod Update. 2006;12:269-74.
[16] Ferrusola CO, Fernández LGl, Sandoval CS, GarcÃa BM, MartÃnez HR, Tapia JA, et al. Inhibition
of the mitochondrial permeability transition pore reduces “apoptosis like” changes during
cryopreservation of stallion spermatozoa. Theriogenology. 2010;74:458-65.
15
Proceedings of the Australian Reproduction Veterinarians (ARV)
Muswellbrook, NSW 2015
[17] Morrell JM, Johannisson A, Dalin AM, Hammar L, Sandebert T, Rodriguez-Martinez H. Sperm
morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy
rates. Acta Vet Scand. 2008;50:(07 January 2008).
[18] Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine (3rd Ed.). Third ed. Oxford,
UK: Oxford University Press; 2003.
[19] Ball BA, Vo AT, Baumber J. Generation of reactive oxygen species by equine spermatozoa. Am J Vet
Res. 2001;62:508-15.
[20] Aitken RJ, Gibb Z, Mitchell LA, Lambourne SR, Connaughton HS, De Iuliis GN. Sperm motility is
lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic
aldehydes but can be significantly rescued by the presence of nucleophilic thiols. Biol Reprod.
2012;87:110, 1-11.
[21] de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm
physiology. Rev Reprod. 1997;2:48-54.
[22] Kosova G, Abney M, Ober C. Heritability of reproductive fitness traits in a human population.
Proceedings of the National Academy of Sciences. 2010;107:1772-8.
[23] Nath LC, Anderson GA, McKinnon AO. Reproductive efficiency of Thoroughbred and Standardbred
horses in north-east Victoria. Aust Vet J. 2010;88:169-75.
[24] Squires E, Barbacini S, Matthews P, Byers W, Schwenzer K, Steiner J, et al. Retrospective study of
factors affecting fertility of fresh, cooled and frozen semen. Equine Veterinary Education. 2006;18:96-9.
[25] Pagl R, Aurich JE, Schlosser FM, Kankofer M, Aurich C. Comparison of an extender containing
defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5°C.
Theriogenology. 2006;66:1115-22.
[26] Aurich J, Aurich C. Developments in European Horse Breeding and Consequences for Veterinarians
in Equine Reproduction. Reproduction in Domestic Animals. 2006;41:275-9.
[27] Jane MM. Artificial Insemination: Current and Future Trends. Artificial Insemination in Farm
Animals. 2011;1:1-13.
[28] Vernet P, Aitken RJ, Drevet JR. Antioxidant strategies in the epididymis. Mol Cell Endocrinol.
2004;216:31-9.
[29] Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the physiological control of
sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxid Redox Signal.
2011;14:367-81.
[30] Gibb Z, Lambourne SR, Aitken RJ. The paradoxical relationship between stallion fertility and
oxidative stress. Biol Reprod. 2014;91(3):1-10.
[31] Ferrusola CO, Fernández LG, Sandoval CS, García BM, Martínez HR, Tapia JA, et al. Inhibition of
the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of
stallion spermatozoa. Theriogenology. 2010;74:458-65.
[32] Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis
of reactive oxygen species by human spermatozoa. J Reprod Fertil. 1987;81:459-69.
[33] Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids in human spermatozoa,
spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril.
1979;31:531-7.
[34] Ginther OJ, Whitmore HL, Squires EL. Characteristics of estrus, diestrus and ovulation in mares and
effects of season and nursing. Am J Vet Res. 1972;33:1935-39.
[35] Brinsko SP, Crockett EC, Squires EL. Effect of centrifugation and partial removal of seminal plasma
on equine spermatozoal motility after cooling and storage. Theriogenology. 2000;54:129-36.
[36] Batellier F, Vidament M, Fauquant J, Duchamp G, Arnaud G, Yvon JM, et al. Advances in cooled
semen technology. Anim Reprod Sci. 2001;68:181-90.
16
Proceedings of the Australian Reproduction Veterinarians (ARV)
Muswellbrook, NSW 2015
[37] Brinsko SP, Van Wagner GS, Graham JK, Squires EL. Motility, morphology and triple stain analysis
of fresh, cooled and frozen-thawed stallion spermatozoa. J Reprod Fertil Suppl. 2000:111-20.
[38] Nallella KP, Sharma RK, Said TM, Agarwal A. Inter-sample variability in post-thaw human
spermatozoa. Cryobiology. 2004;49:195-9.
[39] Bedford SJ, Varner DD, Meyers SA. Effects of cryopreservation on the acrosomal status of stallion
spermatozoa. J Reprod Fertil Suppl. 2000;56:133-40.
[40] Brum AM, Sabeur K, Ball BA. Apoptotic-like changes in equine spermatozoa separated by densitygradient centrifugation or after cryopreservation. Theriogenology. 2008;69:1041-55.
[41] Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Aguero A. Membrane changes
during different stages of a freeze-thaw protocol for equine semen cryopreservation. Theriogenology.
2003;59:1693-705.
[42] Valcarce DG, Cartón-García F, Riesco MF, Herráez MP, Robles V. Analysis of DNA damage after
human sperm cryopreservation in genes crucial for fertilization and early embryo development.
Andrology. 2013;1:723-30.
[43] Mazur P, Cole KW. Roles of unfrozen fraction, salt concentration and change in cell volumes in the
survival of frozen human erythrocytes. Cryobiology. 1989;26:1-29.
[44] Pegg DT, Diaper MD. The unfrozen fraction hypothesis of freezing injury to human erythrocytes: A
critical examination of the evidence. Cryobiology. 1989;26:30-43.
[45] Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2007;368:39-57.
[46] Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: what we ask them
to survive. J Androl. 1990;11:73-88.
[47] Morris GJ, Faszer K, Green JE, Draper D, Grout BWW, Fonseca F. Rapidly cooled horse
spermatozoa: Loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation.
Theriogenology. 2007;68:804-12.
[48] Sardoy MC, Carretero MI, Neild DM. Evaluation of stallion sperm DNA alterations during
cryopreservation using toluidine blue. Anim Reprod Sci. 2008;107:349-50.
[49] Klewitz J, Hagen C, Behrendt D, Martinsson G, Sieme H. Effect of multiple freezing of stallion
semen on sperm quality and fertility. Anim Reprod Sci. 2008;107:327-8.
[50] Burnaugh L, Ball BA, Sabeur K, Thomas AD, Meyers SA. Osmotic stress stimulates generation of
superoxide anion by spermatozoa in horses. Anim Reprod Sci. 2010;117:249-60.
[51] Darin-Bennett A, White IG. Influence of the cholesterol content of mammalian spermatozoa on
susceptibility to cold-shock. Cryobiology. 1977;14:466-70.
[52] Colenbrander B, Gadella BM, Stout TA. The predictive value of semen analysis in the evaluation of
stallion fertility. Reprod Domest Anim. 2003;38:305-11.
[53] Bosh KA, Powell D, Shelton B, Zent W. Reproductive performance measures among Thoroughbred
mares in central Kentucky, during the 2004 mating season. Equine Vet J. 2009;41:883-8.
[54] Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci. 2000;62:3-22.
[55] Rota A, Calicchio E, Nardoni S, Fratini F, Ebani VV, Sgorbini M, et al. Presence and distribution of
fungi and bacteria in the reproductive tract of healthy stallions. Theriogenology. 2011;76:464-70.
[56] Aurich C, Spergser J. Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa.
Theriogenology. 2007;67:912-8.
[57] Ortega-Ferrusola C, Gonzalez-Fernandez L, Muriel A, Macias-Garcia B, Rodriguez-Martinez H,
Tapia JA, et al. Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? Reprod
Domest Anim. 2009;44:518-22.
[58] Varner DD, Scanlan CM, Thompson JA, Brumbaugh GW, Blanchard TL, Carlton CM, et al.
Bacteriology of preserved stallion semen and antibiotics in semen extenders. Theriogenology.
1998;50:559-73.
17
Proceedings of the Australian Reproduction Veterinarians (ARV)
Muswellbrook, NSW 2015
[59] Gibb Z, Butler TJ, Morris LHA, Maxwell WMC, Grupen CG. Quercetin improves the postthaw
characteristics of cryopreserved sex-sorted and nonsorted stallion sperm. Theriogenology. 2013;79:1001-9.
[60] de Oliveira RA, Wolf CA, de Oliveira Viu MA, Gambarini ML. Addition of Glutathione to an
Extender for Frozen Equine Semen. J Equine Vet Sci. 2013;33:1148-52.
[61] Franco JSV, Chaveiro A, da Silva FM. Effect of Freezing Rates and Supplementation of alphaTocopherol in the Freezing Extender in Equine Sperm Cryosurvival. J Equine Vet Sci. 2014;34:992-7.
[62] de Oliveira RA, Wolf CA, Viu MAD, Gambarini ML. Cooling of equine semen at 16 degrees C for 36
h with the addition of cysteine in different concentrations. Pferdeheilkunde. 2015;31:27-32.
[63] da Silva CMB, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A,
Ortega-Ferrusola C, et al. Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion
spermatozoa. J Pineal Res. 2011;51:172-9.
[64] Giaretta E, Bucci D, Mari G, Galeati G, Love CC, Tamanini C, et al. Is Resveratrol Effective in
Protecting Stallion Cooled Semen? J Equine Vet Sci. 2014;34:1307-12.
[65] Baumber J, Ball BA, Linfor JJ. Assessment of the cryopreservation of equine spermatozoa in the
presence of enzyme scavengers and antioxidants. Am J Vet Res. 2005;66:772-9.
[66] Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservationinduced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.
Hum Reprod. 2009;24:2061-70.
[67] Moubasher AE, El Din AM, Ali ME, El-sherif WT, Gaber HD. Catalase improves motility, vitality
and DNA integrity of cryopreserved human spermatozoa. Andrologia. 2013;45:135-9.
[68] Banihani S, Agarwal A, Sharma R, Bayachou M. Cryoprotective effect of l-carnitine on motility,
vitality and DNA oxidation of human spermatozoa. Andrologia. 2014;46:637-41.
[69] Lisboa FL, Hartwig FP, Freitas-Dell'Aqua CP, Hartwig FP, Papa FO, Dell’aqua JA. Improvement of
cooled equine semen by addition of carnitines. J Equine Vet Sci. 2014;34:48.
[70] Lisboa FL, Hartwig FP, Maziero RRD, Monteiro GA, Papa FO, Dell'aqua JA. Use of L-carnitine and
acetyl-L-carnitine in cooled-stored stallion semen. J Equine Vet Sci. 2012;32:493-4.
[71] Banihani S, Sharma R, Bayachou M, Sabanegh E, Agarwal A. Human sperm DNA oxidation, motility
and viability in the presence of l-carnitine during in vitro incubation and centrifugation. Andrologia.
2012;44:505-12.
[72] Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ. L-Carnitine and pyruvate are prosurvival
factors during the storage of stallion spermatozoa at room temperature. Biol Reprod. 2015;(In Press).
[73] Littarru G, Tiano L. Bioenergetic and Antioxidant Properties of Coenzyme Q10: Recent
Developments. Mol Biotechnol. 2007;37:31-7.
[74] Srinivasan V, Spence DW, Pandi-Perumal SR, Brown GM, Cardinali DP. Melatonin in Mitochondrial
Dysfunction and Related Disorders. International Journal of Alzheimer's Disease. 2011;2011:326320.
[75] Shimizu T, Johnson KA. Kinetic Evidence for Multiple Dynein ATPase Sites. J Biol Chem.
1983;258:3841-6.
[76] Kamp G, Büsselmann G, Lauterwein J. Spermatozoa: models for studying regulatory aspects of
energy metabolism. Cell Mol Life Sci. 1996;52:487-94.
[77] Silver IA, Erecińska M. Energetic demands of the Na+/K+ ATPase in mammalian astrocytes. Glia.
1997;21:35-45.
[78] Gülçin İ. Antioxidant and antiradical activities of l-carnitine. Life Sci. 2006;78:803-11.
[79] Peluso G, Barbarisi A, Savica V, Reda E, Nicolai R, Benatti P, et al. Carnitine: An osmolyte that
plays a metabolic role. J Cell Biochem. 2001;80:1-10.
[80] Swegen A, Curry BJ, Gibb Z, Lambourne SR, Smith ND, Aitken RJ. Investigation of the stallion
sperm proteome by mass spectrometry. Reproduction. 2015;149:235-44.
18
Proceedings of the Australian Reproduction Veterinarians (ARV)
Muswellbrook, NSW 2015
[81] Li K, Li W, Huang YF, Shang XJ. [Level of free L-carnitine in human seminal plasma and its
correlation with semen quality]. Zhonghua nan ke xue = National journal of andrology. 2007;13:143-6.
[82] Matalliotakis I, Koumantaki Y, Evageliou A, Matalliotakis G, Goumenou A, Koumantakis E. Lcarnitine levels in the seminal plasma of fertile and infertile men: correlation with sperm quality. Int J
Fertil Womens Med. 2000;45:236-40.
[83] Golan R, Weissenberg R, Lewin LM. Carnitine and acetylcarnitine in motile and immotile human
spermatozoa. Int J Androl. 1984;7:484-94.
[84] Jeulin C, Lewin L. Role of free L-carnitine and acetyl-L-carnitine in post-gonadal maturation of
mammalian spermatozoa. Hum Reprod Update. 1996;2:87-102.
[85] Lewin LM, Shalev DP, Weissenberg R, Soffer Y. Carnitine and acylcarnitines in semen from
azoospermic patients. Fertil Steril. 1981;36:214-8.
[86] Stradaioli G, Sylla L, Zelli R, Verini Supplizi A, Chiodi P, Arduini A, et al. Seminal carnitine and
acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions. Anim
Reprod Sci. 2000;64:233-45.
[87] Brooks DE. Carnitine in the male reproductive tract and its relation to the metabolism of the
epididymis and spermatozoa. In: McGarry JD, Frenkel RA, editors. Carnitine biosynthesis metabolism and
function. New York: Academic Press; 1980. p. 219-35.
[88] Hinton BT, Setchell BP. Concentration and uptake of carnitine in the rat epididymis. A micropuncture
study. In: Frenkel RA, McGarry JD, editors. Carnitine Biosynthesis, Metabolism and Function. New York:
Academic Press; 1980. p. 237.
[89] Stradaioli G, Sylla L, Zelli R, Chiodi P, Monaci M. Effect of L-carnitine administration on the
seminal characteristics of oligoasthenospermic stallions. Theriogenology. 2004;62:761-77.
[90] Lee YH. Functional Maturation of Mouse Epididymal Spermatozoa [PhD]. Callaghan, NSW,
Australia: The University of Newcastle; 2008.
[91] Dello Russo C, Gavrilyuk V, Weinberg G, Almeida A, Bolanos JP, Palmer J, et al. Peroxisome
proliferator-activated receptor gamma thiazolidinedione agonists increase glucose metabolism in
astrocytes. J Biol Chem. 2003;278:5828-36.
[92] Vazquez A, Liu JX, Zhou Y, Oltvai ZN. Catabolic efficiency of aerobic glycolysis: The Warburg
effect revisited. Bmc Syst Biol. 2010;4.
[93] Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, et al. Thiazolidinediones, like
metformin, inhibit respiratory complex I - A common mechanism contributing to their antidiabetic
actions? Diabetes. 2004;53:1052-9.
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Proceedings of the Australian Reproduction Veterinarians (ARV)
Muswellbrook, NSW 2015