BioMed Research International
Volume 2016 (2016), Article ID 9380609, 8 pages
http://dx.doi.org/10.1155/2016/9380609

Review Article

The Impact of Sperm Metabolism during In Vitro Storage: The Stallion as a Model

Zamira Gibb and Robert J. Aitken

Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia

Received 28 October 2015; Accepted 2 December 2015

Academic Editor: Elisabetta Baldi

Copyright © 2016 Zamira Gibb and Robert J. Aitken. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

In vitro sperm storage is a necessary part of many artificial insemination or in vitro fertilization regimes for many species, including the human and the horse. In many situations spermatozoa are chilled to temperatures between 4 and 10°C for the purpose of restricting the metabolic rate during storage, in turn, reducing the depletion of ATP and the production of detrimental by-products such as reactive oxygen species (ROS). Another result of lowering the temperature is that spermatozoa may be “cold shocked” due to lipid membrane phase separation, resulting in reduced fertility. To overcome this, a method of sperm storage must be developed that will preclude the need to chill spermatozoa. If a thermally induced restriction-of-metabolic-rate strategy is not employed, ATP production must be supported while ameliorating the deleterious effects of ROS. To achieve this end, an understanding of the nature of energy production by the spermatozoa of the species of interest is essential. Human spermatozoa depend predominantly on glycolytic ATP production, producing significantly less ROS than oxidative phosphorylation, with the more efficient pathway predominantly employed by stallion spermatozoa. This review provides an overview of the implications of sperm metabolism for in vitro sperm storage, with a focus on ambient temperature storage in the stallion.

1. Introduction

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 [1], 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 [1–3]. As artificial insemination (AI) is a widely utilised tool in modern horse reproduction [4], with around 90% of Standardbred and Hanoverian foals being produced via AI of chilled or cryopreserved stallion spermatozoa [2, 5], 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 [6], 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.

2. 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 energy-dependent process [7].

The process of spermatogenesis requires extensive remodelling of a conventional spherical cell to become one of 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 [8]. 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 [9]. 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 antioxidants that are unique to the male reproductive tract [10, 11].

As with somatic cells, the predominant metabolic pathways that spermatozoa use to produce ATP are glycolysis and oxidative phosphorylation (OXPHOS) [12]. 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 [12].

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) [13]. GLUTs are categorised according to their relative ability to transport hexoses (such as glucose, mannitol, and fructose), amino sugars, or vitamins [14]. Since the discovery of the glucose transporter GLUT1, many additional GLUTs have been characterised [15, 16]. 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 [16]. 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 [16]. In contrast, the distribution of GLUTs on stallion spermatozoa does not change with the capacitation status of the cell [16], 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 sperm, 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 [17–19], and in the presence of mitochondrial inhibitors, they suffer a rapid loss of velocity and a dramatic decline in ATP content [17]. This dependence results in a nonconventional relationship between ROS production and fertility in the stallion [17–19], 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 [20].

There is a long-standing paradigm that it is the nonviable or poor quality spermatozoa that generate the most ROS [21]. An alternative explanation is that rapidly metabolising spermatozoa from highly fertile stallions exhibit higher levels of OXPHOS activity, 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 [22, 23], a recent study has revealed a paradoxical inverse relationship between fertility and the percentage of live cells without oxidative damage in the stallion [17]. 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 compared to ejaculates which did not result in a pregnancy [17]. 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 fast-die 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. 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 [18], with a positive relationship between mitochondrial ROS production and sperm velocity, leading to increased rates of lipid peroxidation [17] and, following prolonged storage, a loss of motility and vitality [24]. 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.

3. 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 [25] 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 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 [26–29]. Therefore, there is a need to develop a medium which will extend the longevity of spermatozoa without the need to chill or be cryopreserved.

4. 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 [30–32] 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 [33]. 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 [34, 35]. Additionally, the cryoprotectants make the cryodiluent hyperosmotic, which causes dehydration of the cells through osmosis [36]. While this dehydration is essential for postthaw 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 [37]. The result of these osmotic stressors includes membrane damage [38], DNA damage [39, 40], and the production of ROS which causes premature capacitation-like changes [41].

5. Chilling

The Standardbred, Sport Horse, and Polo Pony 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 [42] and, as a result, the insemination of chilled semen is associated with success rates as low as 44% per cycle [3]. As with cryopreservation, there are significant unexplained differences between stallions in the suitability of their semen for low temperature storage [43], a phenomenon which reduces the commercial viability of such animals due to sperm damage following chilling [44]. 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 [45]. 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.

6. 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 [46]. Several of these strains have been shown to be deleterious to sperm motility and vitality, even during chilled storage at 4°C [47] and following cryopreservation [48]. However, several antibiotic formulations have been investigated for their effects on curtailing bacterial growth in extended stallion semen [47, 49], 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 [20], which will compromise sperm function [11, 24] (Figure 1). The majority of attempts to assuage the damaging effects of ROS on stallion spermatozoa through antioxidant supplementation either have produced marginal improvements [50–54] or have had detrimental effects [55, 56]. This is in contrast to the positive effects seen in human spermatozoa [57, 58] and may be due in part to their alternative mode of ATP production. More recently, the antioxidant properties of carnitine have come into the spotlight [59–63]. L-Carnitine supplementation of stallion spermatozoa during in vitro storage significantly reduces both mitochondrial free radical production and lipid peroxidation [63], 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 [63]. 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 coenzyme Q₁₀, an integral component of the electron transport chain and an antioxidant capable of counteracting the ROS-induced peroxidation of mitochondrial phospholipids [64], 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 [65].

Figure 1: Implications of oxidative phosphorylation (OXPHOS) on sperm storage in vitro. Mitochondrial superoxide () 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 oxidative 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.

Sperm motility is lost as a consequence of lipid peroxidation not only due to ROS attack [66], but also due to the concomitant depletion of ATP [67] which compromises myriad ATP-dependent functions, disrupting homeostasis and hastening cell death [68]. 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 [69]. By utilising nonionic, 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 [69]. Recent research has revealed that supplementing media with pyruvate, the primary energy source utilise 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 [70] and an organic, nonionic osmolyte [71], results in the maintenance of motility and viability at room temperature akin to that of chilled semen for up to 72 h [63]. 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 [72]. 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 [73–78]. Androgen regulated epithelial cells actively secrete L-carnitine into the epididymal lumen [79, 80] 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 [76], 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 [81], demonstrating an important interactive role between these compounds in the support of sperm metabolism.

7. Conclusion

As the major implication for ambient temperature storage of equine spermatozoa 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 [82], a phenomenon 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 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 [83] and the preferential utilisation of both aerobic [84] and anaerobic glycolysis [85]. 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.

Conflict of Interests

The authors declare no conflict of interests.

Acknowledgments

The authors would like to thank the Hunter Valley Equine Research Centre, Harness Racing Australia, EquiBreed NZ Ltd., and Alabar Farms for supporting this research. This work is supported by ARC Linkage Grant, the Hunter Valley Equine Research Centre, and Harness Racing Australia.

References

G. Kosova, M. Abney, and C. Ober, “Heritability of reproductive fitness traits in a human population,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 1, pp. 1772–1778, 2010.

L. C. Nath, G. A. Anderson, and A. O. McKinnon, “Reproductive efficiency of Thoroughbred and Standardbred horses in north-east Victoria,” Australian Veterinary Journal, vol. 88, no. 5, pp. 169–175, 2010.

E. Squires, S. Barbacini, P. Matthews et al., “Retrospective study of factors affecting fertility of fresh, cooled and frozen semen,” Equine Veterinary Education, vol. 18, no. 2, pp. 96–99, 2006.

R. Pagl, J. E. Aurich, F. Müller-Schlösser, M. Kankofer, and C. Aurich, “Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5°C,” Theriogenology, vol. 66, no. 5, pp. 1115–1122, 2006.

J. Aurich and C. Aurich, “Developments in European horse breeding and consequences for veterinarians in equine reproduction,” Reproduction in Domestic Animals, vol. 41, no. 4, pp. 275–279, 2006.

M. M. Jane, “Artificial insemination: current and future trends,” Artificial Insemination in Farm Animals, vol. 1, pp. 1–13, 2011.

F. M. Flesch and B. M. Gadella, “Dynamics of the mammalian sperm plasma membrane in the process of fertilization,” Biochimica et Biophysica Acta (BBA)—Reviews on Biomembranes, vol. 1469, no. 3, pp. 197–235, 2000.

D. Miller, M. Brinkworth, and D. Iles, “Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics,” Reproduction, vol. 139, no. 2, pp. 287–301, 2010.

D. Bucci, J. E. Rodriguez-Gil, C. Vallorani, M. Spinaci, G. Galeati, and C. Tamanini, “GLUTs and mammalian sperm metabolism,” Journal of Andrology, vol. 32, no. 4, pp. 348–355, 2011.

P. Vernet, R. J. Aitken, and J. R. Drevet, “Antioxidant strategies in the epididymis,” Molecular and Cellular Endocrinology, vol. 216, no. 1, pp. 31–39, 2004.

R. J. Aitken and B. J. Curry, “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,” Antioxidants and Redox Signaling, vol. 14, no. 3, pp. 367–381, 2011.

B. T. Storey, “Mammalian sperm metabolism: oxygen and sugar, friend and foe,” International Journal of Developmental Biology, vol. 52, no. 5-6, pp. 427–437, 2008.

M. Kasahara and P. C. Hinkle, “Reconstitution and purification of the D-glucose transporter from human erythrocytes,” The Journal of Biological Chemistry, vol. 252, no. 20, pp. 7384–7390, 1977.

C. Angulo, M. C. Rauch, A. Droppelmann et al., “Hexose transporter expression and function in mammalian spermatozoa: cellular localization and transport of hexoses and vitamin C,” Journal of Cellular Biochemistry, vol. 71, no. 2, pp. 189–203, 1998.

M. Mueckler, C. Caruso, S. A. Baldwin et al., “Sequence and structure of a human glucose transporter,” Science, vol. 229, no. 4717, pp. 941–945, 1985.

D. Bucci, G. Isani, M. Spinaci et al., “Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa,” Reproduction in Domestic Animals, vol. 45, no. 2, pp. 315–322, 2010.

Z. Gibb, S. R. Lambourne, and R. J. Aitken, “The paradoxical relationship between stallion fertility and oxidative stress,” Biology of Reproduction, vol. 91, no. 3, article 77, 2014.

C. O. Ferrusola, L. G. Fernández, C. S. Sandoval et al., “Inhibition of the mitochondrial permeability transition pore reduces ‘apoptosis like’ changes during cryopreservation of stallion spermatozoa,” Theriogenology, vol. 74, no. 3, pp. 458–465, 2010.

J. M. Morrell, A. Johannisson, A.-M. Dalin, L. Hammar, T. Sandebert, and H. Rodriguez-Martinez, “Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates,” Acta Veterinaria Scandinavica, vol. 50, article 2, 2008.

B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, Oxford, UK, 3rd edition, 2003.

B. A. Ball, A. T. Vo, and J. Baumber, “Generation of reactive oxygen species by equine spermatozoa,” American Journal of Veterinary Research, vol. 62, no. 4, pp. 508–515, 2001.

R. J. Aitken and J. S. Clarkson, “Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa,” Journal of Reproduction and Fertility, vol. 81, no. 2, pp. 459–469, 1987.

R. Jones, T. Mann, and R. Sherins, “Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma,” Fertility and Sterility, vol. 31, no. 5, pp. 531–537, 1979.

R. J. Aitken, Z. Gibb, L. A. Mitchell, S. R. Lambourne, H. S. Connaughton, and G. N. De Iuliis, “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,” Biology of Reproduction, vol. 87, no. 5, article 110, Article ID Article 110, 2012.

O. J. Ginther, H. L. Whitmore, and E. L. Squires, “Characteristics of estrus, diestrus, and ovulation in mares and effects of season and nursing,” American Journal of Veterinary Research, vol. 33, no. 10, pp. 1935–1939, 1972.

S. P. Brinsko, E. C. Crockett, and E. L. Squires, “Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage,” Theriogenology, vol. 54, no. 1, pp. 129–136, 2000.

F. Batellier, M. Vidament, J. Fauquant et al., “Advances in cooled semen technology,” Animal Reproduction Science, vol. 68, no. 3-4, pp. 181–190, 2001.

S. P. Brinsko, G. S. Van Wagner, J. K. Graham, and E. L. Squires, “Motility, morphology and triple stain analysis of fresh, cooled and frozen-thawed stallion spermatozoa,” Journal of Reproduction and Fertility. Supplement, no. 56, pp. 111–120, 2000.

K. P. Nallella, R. K. Sharma, T. M. Said, and A. Agarwal, “Inter-sample variability in post-thaw human spermatozoa,” Cryobiology, vol. 49, no. 2, pp. 195–199, 2004.

S. J. Bedford, D. D. Varner, and S. A. Meyers, “Effects of cryopreservation on the acrosomal status of stallion spermatozoa,” Journal of Reproduction and Fertility Supplement, vol. 56, pp. 133–140, 2000.

A. M. Brum, K. Sabeur, and B. A. Ball, “Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation,” Theriogenology, vol. 69, no. 9, pp. 1041–1055, 2008.

D. M. Neild, B. M. Gadella, M. G. Chaves, M. H. Miragaya, B. Colenbrander, and A. Agüero, “Membrane changes during different stages of a freeze-thaw protocol for equine semen cryopreservation,” Theriogenology, vol. 59, no. 8, pp. 1693–1705, 2003.

D. G. Valcarce, F. Cartón-García, M. F. Riesco, M. P. Herráez, and V. Robles, “Analysis of DNA damage after human sperm cryopreservation in genes crucial for fertilization and early embryo development,” Andrology, vol. 1, no. 5, pp. 723–730, 2013.

P. Mazur and K. W. Cole, “Roles of unfrozen fraction, salt concentration, and changes in cell volume in the survival of frozen human erythrocytes,” Cryobiology, vol. 26, no. 1, pp. 1–29, 1989.

D. E. Pegg and M. P. Diaper, “The ‘unfrozen fraction’ hypothesis of freezing injury to human erythrocytes: a critical examination of the evidence,” Cryobiology, vol. 26, no. 1, pp. 30–43, 1989.

D. E. Pegg, “Principles of cryopreservation,” Methods in Molecular Biology, vol. 368, pp. 39–57, 2007.

R. H. Hammerstedt, J. K. Graham, and J. P. Nolan, “Cryopreservation of mammalian sperm: what we ask them to survive,” Journal of Andrology, vol. 11, no. 1, pp. 73–88, 1990.

G. J. Morris, K. Faszer, J. E. Green, D. Draper, B. W. W. Grout, and F. Fonseca, “Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation,” Theriogenology, vol. 68, no. 5, pp. 804–812, 2007.

M. C. Sardoy, M. I. Carretero, and D. M. Neild, “Evaluation of stallion sperm DNA alterations during cryopreservation using toluidine blue,” Animal Reproduction Science, vol. 107, no. 3-4, pp. 349–350, 2008.

J. Klewitz, C. Hagen, D. Behrendt, G. Martinsson, and H. Sieme, “Effect of multiple freezing of stallion semen on sperm quality and fertility,” Animal Reproduction Science, vol. 107, no. 3-4, pp. 327–328, 2008.

L. Burnaugh, B. A. Ball, K. Sabeur, A. D. Thomas, and S. A. Meyers, “Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses,” Animal Reproduction Science, vol. 117, no. 3-4, pp. 249–260, 2010.

A. Darin-Bennett and I. G. White, “Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock,” Cryobiology, vol. 14, no. 4, pp. 466–470, 1977.

B. Colenbrander, B. M. Gadella, and T. A. E. Stout, “The predictive value of semen analysis in the evaluation of stallion fertility,” Reproduction in Domestic Animals, vol. 38, no. 4, pp. 305–311, 2003.

K. A. Bosh, D. Powell, B. Shelton, and W. Zent, “Reproductive performance measures among Thoroughbred mares in central Kentucky, during the 2004 mating season,” Equine Veterinary Journal, vol. 41, no. 9, pp. 883–888, 2009.

W. V. Holt, “Basic aspects of frozen storage of semen,” Animal Reproduction Science, vol. 62, no. 1–3, pp. 3–22, 2000.

A. Rota, E. Calicchio, S. Nardoni et al., “Presence and distribution of fungi and bacteria in the reproductive tract of healthy stallions,” Theriogenology, vol. 76, no. 3, pp. 464–470, 2011.

C. Aurich and J. Spergser, “Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa,” Theriogenology, vol. 67, no. 5, pp. 912–918, 2007.

C. Ortega-Ferrusola, L. González-Fernández, A. Muriel et al., “Does the microbial flora in the ejaculate affect the freezeability of stallion sperm?” Reproduction in Domestic Animals, vol. 44, no. 3, pp. 518–522, 2009.

D. D. Varner, C. M. Scanlan, J. A. Thompson et al., “Bacteriology of preserved stallion semen and antibiotics in semen extenders,” Theriogenology, vol. 50, no. 4, pp. 559–573, 1998.

Z. Gibb, T. J. Butler, L. H. A. Morris, W. M. C. Maxwell, and C. G. Grupen, “Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm,” Theriogenology, vol. 79, no. 6, pp. 1001–1009, 2013.

R. A. de Oliveira, C. A. Wolf, M. A. de Oliveira Viu, and M. L. Gambarini, “Addition of glutathione to an extender for frozen equine semen,” Journal of Equine Veterinary Science, vol. 33, no. 12, pp. 1148–1152, 2013.

J. S. V. Franco, A. Chaveiro, and F. M. da Silva, “Effect of freezing rates and supplementation of α-tocopherol in the freezing extender in equine sperm cryosurvival,” Journal of Equine Veterinary Science, vol. 34, no. 8, pp. 992–997, 2014.

R. A. de Oliveira, C. A. Wolf, M. A. D. Viu, and M. L. Gambarini, “Cooling of equine semen at 16 degrees C for 36 h with the addition of cysteine in different concentrations,” Pferdeheilkunde, vol. 31, pp. 27–32, 2015.

C. M. B. da Silva, B. MacÍas-García, A. Miró-Morán et al., “Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa,” Journal of Pineal Research, vol. 51, no. 2, pp. 172–179, 2011.

E. Giaretta, D. Bucci, G. Mari et al., “Is resveratrol effective in protecting stallion cooled semen?” Journal of Equine Veterinary Science, vol. 34, no. 11-12, pp. 1307–1312, 2014.

J. Baumber, B. A. Ball, and J. J. Linfor, “Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants,” American Journal of Veterinary Research, vol. 66, no. 5, pp. 772–779, 2005.

L. K. Thomson, S. D. Fleming, R. J. Aitken, G. N. De Iuliis, J.-A. Zieschang, and A. M. Clark, “Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis,” Human Reproduction, vol. 24, no. 9, pp. 2061–2070, 2009.

A. E. Moubasher, A. M. E. El Din, M. E. Ali, W. T. El-Sherif, and H. D. Gaber, “Catalase improves motility, vitality and DNA integrity of cryopreserved human spermatozoa,” Andrologia, vol. 45, no. 2, pp. 135–139, 2013.

S. Banihani, A. Agarwal, R. Sharma, and M. Bayachou, “Cryoprotective effect of l-carnitine on motility, vitality and DNA oxidation of human spermatozoa,” Andrologia, vol. 46, no. 6, pp. 637–641, 2014.

F. Lisboa, F. Hartwig, C. Freitas-Dell'Aqua, F. Hartwig, F. Papa, and J. Dell’aqua, “Improvement of cooled equine semen by addition of carnitines,” Journal of Equine Veterinary Science, vol. 34, no. 1, p. 48, 2014.

F. L. Lisboa, F. P. Hartwig, R. R. D. Maziero, G. A. Monteiro, F. O. Papa, and J. A. Dell'aqua, “Use of L-carnitine and acetyl-L-carnitine in cooled-stored stallion semen,” Journal of Equine Veterinary Science, vol. 32, no. 8, pp. 493–494, 2012.

S. Banihani, R. Sharma, M. Bayachou, E. Sabanegh, and A. Agarwal, “Human sperm DNA oxidation, motility and viability in the presence of l-carnitine during in vitro incubation and centrifugation,” Andrologia, vol. 44, no. 1, pp. 505–512, 2012.

Z. Gibb, S. R. Lambourne, J. Quadrelli, N. D. Smith, and R. J. Aitken, “L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature,” Biology of Reproduction, vol. 93, no. 4, article 104, 2015.

G. P. Littarru and L. Tiano, “Bioenergetic and antioxidant properties of coenzyme Q10: recent developments,” Molecular Biotechnology, vol. 37, no. 1, pp. 31–37, 2007.

V. Srinivasan, D. W. Spence, S. R. Pandi-Perumal, G. M. Brown, and D. P. Cardinali, “Melatonin in mitochondrial dysfunction and related disorders,” International Journal of Alzheimer's Disease, vol. 2011, Article ID 326320, 16 pages, 2011.

T. Shimizu and K. A. Johnson, “Kinetic evidence for multiple dynein ATPase sites,” The Journal of Biological Chemistry, vol. 258, no. 22, pp. 3841–3846, 1983.

E. de Lamirande, H. Jiang, A. Zini, H. Kodama, and C. Gagnon, “Reactive oxygen species and sperm physiology,” Reviews of Reproduction, vol. 2, no. 1, pp. 48–54, 1997.

G. Kamp, G. Büsselmann, and J. Lauterwein, “Spermatozoa: models for studying regulatory aspects of energy metabolism,” Cellular and Molecular Life Sciences, vol. 52, pp. 487–494, 1996.

I. A. Silver and M. Erecińska, “Energetic demands of the Na⁺/K⁺ ATPase in mammalian astrocytes,” Glia, vol. 21, no. 1, pp. 35–45, 1997.

I. Gülçin, “Antioxidant and antiradical activities of L-carnitine,” Life Sciences, vol. 78, no. 8, pp. 803–811, 2006.

G. Peluso, A. Barbarisi, V. Savica et al., “Carnitine: an osmolyte that plays a metabolic role,” Journal of Cellular Biochemistry, vol. 80, no. 1, pp. 1–10, 2000.

A. Swegen, B. J. Curry, Z. Gibb, S. R. Lambourne, N. D. Smith, and R. J. Aitken, “Investigation of the stallion sperm proteome by mass spectrometry,” Reproduction, vol. 149, no. 3, pp. 235–244, 2015.

K. Li, W. Li, Y.-F. Huang, and X.-J. Shang, “Level of free L-carnitine in human seminal plasma and its correlation with semen quality,” National Journal of Andrology, vol. 13, no. 2, pp. 143–146, 2007.

I. Matalliotakis, Y. Koumantaki, A. Evageliou, G. Matalliotakis, A. Goumenou, and E. Koumantakis, “L-carnitine levels in the seminal plasma of fertile and infertile men: correlation with sperm quality,” International Journal of Fertility and Women's Medicine, vol. 45, no. 3, pp. 236–240, 2000.

R. Golan, R. Weissenberg, and L. M. Lewin, “Carnitine and acetylcarnitine in motile and immotile human spermatozoa,” International Journal of Andrology, vol. 7, no. 6, pp. 484–494, 1984.

C. Jeulin and L. M. Lewin, “Role of free L-carnitine and acetyl-L-carnitine in post-gonadal maturation of mammalian spermatozoa,” Human Reproduction Update, vol. 2, no. 2, pp. 87–102, 1996.

L. M. Lewin, D. P. Shalev, R. Weissenberg, and Y. Soffer, “Carnitine and acylcarnitines in semen from azoospermic patients,” Fertility and Sterility, vol. 36, no. 2, pp. 214–218, 1981.

G. Stradaioli, L. Sylla, R. Zelli et al., “Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions,” Animal Reproduction Science, vol. 64, no. 3-4, pp. 233–245, 2000.

D. E. Brooks, “Carnitine in the male reproductive tract and its relation to the metabolism of the epididymis and spermatozoa,” in Carnitine Biosynthesis Metabolism and Function, J. D. McGarry and R. A. Frenkel, Eds., pp. 219–235, Academic Press, New York, NY, USA, 1980.

B. T. Hinton and B. P. Setchell, “Concentration and uptake of carnitine in the rat epididymis. A micropuncture study,” in Carnitine Biosynthesis, Metabolism and Function, R. A. Frenkel and J. D. McGarry, Eds., p. 237, Academic Press, New York, NY, USA, 1980.

G. Stradaioli, L. Sylla, R. Zelli, P. Chiodi, and M. Monaci, “Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions,” Theriogenology, vol. 62, no. 3-4, pp. 761–777, 2004.

Y. H. Lee, Functional maturation of mouse epididymal spermatozoa [Ph.D. thesis], The University of Newcastle, Callaghan, Australia, 2008.

C. Dello Russo, V. Gavrilyuk, G. Weinberg et al., “Peroxisome proliferator-activated receptor gamma thiazolidinedione agonists increase glucose metabolism in astrocytes,” Journal of Biological Chemistry, vol. 278, no. 8, pp. 5828–5836, 2003.

A. Vazquez, J. Liu, Y. Zhou, and Z. N. Oltvai, “Catabolic efficiency of aerobic glycolysis: the Warburg effect revisited,” BMC Systems Biology, vol. 4, article 58, 2010.

B. Brunmair, K. Staniek, F. Gras et al., “Thiazolidinediones, like metformin, inhibit respiratory complex I—a common mechanism contributing to their antidiabetic actions?” Diabetes, vol. 53, no. 4, pp. 1052–1059, 2004.