Proteomics is advancing the understanding of stallion sperm biology.

The mammalian ejaculate is very well suited to proteomics studies. As such, research concerning sperm proteomics is offering a huge amount of new information on the biology of spermatozoa. Among domestic animals, horses represent a species of special interest, in which reproductive technologies and a sizeable market of genetic material have grown exponentially in the last decade. Studies using proteomic approaches have been conducted in recent years, showing that proteomics is a potent tool to dig into the biology of the stallion spermatozoa. The aim of this review is to present an overview of the research conducted, and how these studies have improved our knowledge of stallion sperm biology. The main outcomes of the research conducted so far have been an improved knowledge of metabolism, and its importance in sperm functions, the impact of different technologies on the sperm proteome, and the identification of potential biomarkers. Moreover, proteomics of seminal plasma and phosphoproteomics are identified as areas of major interest.

The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

In brief: Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis. Abstract: Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract, exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress), stallion spermatozoa were incubated under different concentrations of glucose, 67 mM (HG) or 1 mM plus 10 mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa. Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P < 0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P < 0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67 mM glucose but not in media containing 1 mM glucose and 10 mM pyruvate. The composition of the media, especially the concentration of glucose, exerts a major impact on the functionality and life span of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.

Endometrial area of the blood flow as a marker of endometritis in equine.

In this study, uterine blood flow area (BFA) has been evaluated for the first time using power Doppler ultrasound (PD) as a marker of endometritis in mares and jennies. The uterine BFA in healthy mares was greater in oestrus than in diestrus (p < .001). However, differences in endometrial blood flow between oestrus and diestrus were not observed in mares with endometritis. The uterine blood flow in healthy jennies is not affected by the oestrus cycle. Both species showed an increase in endometrial BFA in pathological uterine conditions compared to controls. BFA was a good marker of endometritis with an area under curve (AUC) (estrus:0.94 (p < .001) diestrus:0.98 (p < .001) in mares and AUC (0.91 (p < .0001) in jennies. The results of this preliminary study suggest that PD ultrasound in combination with computerized image analysis has the potential to be a very useful tool in the diagnosis of endometritis.

The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism.

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.

The calcium/CaMKKalpha/beta and the cAMP/PKA pathways are essential upstream regulators of AMPK activity in boar spermatozoa.

Spermatozoa successfully fertilize oocytes depending on cell energy-sensitive processes. We recently showed that the cell energy sensor, the AMP-activated protein kinase (AMPK), plays a relevant role in spermatozoa by regulating motility as well as plasma membrane organization and acrosomal integrity, and contributes to the maintenance of mitochondrial membrane potential. As the signaling pathways that control AMPK activity have been studied exclusively in somatic cells, our aim is to investigate the intracellular pathways that regulate AMPK phosphorylation at Thr(172) (activity) in male germ cells. Boar spermatozoa were incubated under different conditions in the presence or absence of Ca(2+), 8Br-cAMP, IBMX, PMA, the AMPK activator A769662, or inhibitors of PKA, PKC, or CaMKKalpha/beta. AMPK phosphorylation was evaluated by Western blot using anti-phospho-Thr(172)-AMPK antibody. Data show that AMPK phosphorylation in spermatozoa is potently stimulated by an elevation of cAMP levels through the activation of PKA, as the PKA inhibitor H89 blocks phospho-Thr(172)-AMPK. Another mechanism to potently activate AMPK is Ca(2+) that acts through two pathways, PKA (blocked by H89) and CaMKKalpha/beta (blocked by STO-609). Moreover, phospho-Thr(172)-AMPK levels greatly increased upon PKC activation induced by PMA, and the PKC inhibitor Ro-32-0432 inhibits TCM-induced AMPK activation. Different stimuli considered as cell stresses (rotenone, cyanide, sorbitol, and complete absence of intracellular Ca(2+) by BAPTA-AM) also cause AMPK phosphorylation in spermatozoa. In summary, AMPK activity in boar spermatozoa is regulated upstream by different kinases, such as PKA, CaMKKalpha/beta, and PKC, as well as by the essential intracellular messengers for spermatozoan function, Ca(2+) and cAMP levels.

The future of equine semen analysis.

We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput 'omics' techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors' perspective, using the stallion as a model. We aim to portray a brief overview of the changes occurring in the assessment of sperm motility and kinematics, advances in flow cytometry, implementation of 'omics' technologies, and the use of artificial intelligence/self-learning in data analysis. We also briefly discuss how some of the advances can be readily available to the practitioner, through the implementation of 'on-farm' devices and telemedicine.

Artificial intelligence in Andrological flow cytometry: The next step?

Since its introduction in animal andrology, flow cytometry (FC) has dramatically evolved. Nowadays, many compartments and functions of the spermatozoa can be analyzed in thousands of spermatozoa, including, but not limited to DNA, acrosome, membrane integrity, membrane symmetry, permeability, and polarity; mitochondrial mass and mitochondrial membrane potential, identification of reactive oxygen species, ion dynamics, and cellular signaling among many others. Improved machines, many more probes, and new software are greatly expanding the amount of information that can be obtained from each flow cytometry analysis. Modern flow cytometers permit the simultaneous investigation of many different sperm compartments and functions and their interactions, allowing the identification of sperm phenotypes, helping to disclose different sperm populations within the ejaculate. Complex flow cytometry panels require a careful design of the experiment, including selecting probes (fully understanding the characteristics and properties of them) and adequate controls (technical and biological). Ideally, compensation and management of data ("cleaning", transformations, the establishment of gates) are better performed post-acquisition using specific software. Data can be expressed as a percentage of positive cells (typically viability assays), intensity of fluorescence (arbitrary fluorescence units, i.e. changes in intracellular Ca2+) or dim and bright populations (typically assays of membrane permeability or antigen expression). Furthermore, artificial intelligence/self-learning algorithms are improving visualization and management of data generated by modern flow cytometers. In this paper, recent developments in flow cytometry for animal andrology will be briefly reviewed; moreover, a small flow cytometry experiment will be used to illustrate how these techniques can improve data analysis.

Pyruvate enhances stallion sperm function in high glucose media improving overall metabolic efficiency.

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis.

In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism.

BACKGROUND: Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. MATERIAL AND METHODS: Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca2+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. RESULTS: After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca2+ increased in all treatment groups compared to time 0h. DISCUSSION AND CONCLUSION: Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.

Reimagining stallion sperm conservation: Combating carbotoxicity through pyruvate-induced Warburg effect to enhance sperm longevity and function.

Although stallion spermatozoa are now recognized as highly dependent on oxidative phosphorylation for ATP production in the mitochondria, most extenders in use contain supraphysiological concentrations of glucose as the main energy source. While the toxicity of cryoprotectants has been well documented in the literature, the potential toxicity of excessive glucose in extenders is largely ignored. However, the toxicity of excess glucose, known as "carbotoxicity", is well-established in many areas of medicine. In this paper, we review the basic aspects of stallion spermatozoa metabolism, focusing on factors that significantly impact the lifespan and functionality of spermatozoa during conservation.

Aging of stallion spermatozoa stored in vitro is delayed at 22°C using a 67 mm glucose-10 mm pyruvate-based media.

BACKGROUND: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C. OBJECTIVES: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C. MATERIAL AND METHODS: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode's (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 µM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode's 67 mm glucose-10 mm pyruvate and modified Tyrode's 67 mm glucose, and metabolic analysis conducted. RESULTS: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose-10 mm pyruvate/10 µm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+ , pyruvate, lactate, and ATP. DISCUSSION AND CONCLUSIONS: Aliquots stored in a 67 mm glucose-10 mm pyruvate-based medium supplemented with 10 µM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+ .