This was seen for sperm progressive motility, sperm concentration, total sperm count and total progressively motile sperm. No differences occurred in sperm progressive motility and sperm concentration between higher and elite levels. The majority of stallions included (93.6%) had multiple sample collections within the data, however as environmental conditions, management and competition schedules have previously been shown to impact semen quality 19,20, each collection was treated as a unique and individual data point. 4.6. Model B6: Effect of 15 Factors on Total Progressively Motile Sperm Count The kinematic parameters, total motile spermatozoa (MT%), progressive motility (MP%), rectilinear velocity (VSL μm/s), trajectory velocity (VAP μm/s) and curvilinear velocity (VCL μm/s) of the stallions improved (p 1). On the basis of sperm concentration and semen kinematic parameters, total motility (MT%), progressive motility (MP%), rectilinear velocity (VSL μm/s), trajectory velocity (VAP μm/s) and curvilinear velocity (VCL μm/s). There are objective parameters that assess sperm quality, such as concentration, total motility, or progressive motility 1,2,3. In the present study, non-competing stallion sperm concentration, total sperm count and total progressively motile sperm values were increased compared to competing stallions. Elite stallions were found to have significantly higher values in total sperm count and total progressively motile sperm compared to all other competition levels for the actively competing stallions. Each region of the membrane can be characterized by a phospholipid bilayer of heterogeneously expressed lipids, proteins, carbohydrates, and cholesterol that is primarily established during spermatogenesis 33,41,42,43. Although parameters of a morphologically normal sperm may vary significantly on an individual basis, abnormalities in the sperm anatomy may be an indication of subfertility or a problem with spermatogenesis . The mitochondrial helix is critical for supplying energy to the sperm tail, allowing for the motility that is necessary in fertilization events. The cell surface is additionally covered by a glycocalyx, a network of glycoproteins and glycolipids attached to a matrix of oligosaccharides and polysaccharides, that is known to aid in the proper function of sperm, as well as survival as it passes through the female reproductive tract 43,44. The outer plasma membrane can be partitioned into the acrosomal, post-acrosomal, neck, midpiece, and principal piece domains . The end of the midpiece is defined as the caudal end of the mitochondrial sheath where the annulus, a ring of dense filaments, is located to separate the mitochondria and the sperm tail . Disruption of mitochondrial integrity, including the depolarization of the membrane, can disrupt ATP production and cause a sperm cell to become immotile and non-functional 6,90. Therefore, it is of interest to remove the prematurely acrosome reacted spermatozoa during selection procedures. This results in the entrance of the sperm into the perivitelline space and the fusion of the inner acrosomal membrane and the equatorial region of the sperm head with the oolemma 133,134,135. These enzymes aid in the digestion of the zona pellucida so the sperm can penetrate the zona pellucida using hyperactivated motility acquired during capacitation. Subsequently, the acrosome reaction, or the fusion of the outer acrosomal membrane with the sperm plasma membrane, is marked by the exocytosis of proteolytic and hydrolytic enzymes from the acrosomal compartment 132,133. However, when there is an imbalance in ROS due to oxidative stress (Del Prete et al., 2018; Halliwell & Whiteman, 2004), it produces alterations that modify semen homeostasis (El Sisy et al., 2016), affecting fertility (Freitas & De Oliveira, 2018; Lançoni et al., 2018).Sperm evaluation for morphological defects could assist with detecting the time frame in which damage to the sperm occurs.The data that support the findings of this study are available from the corresponding author upon reasonable request.This study aimed to evaluate the effect of nutraceutical supplementation on semen quality in Quarter Horse stallions.Assessment of sperm viability can also be indicative of early apoptotic changes, which could also be correlated with other severe sperm abnormalities or infertilities.Sperm viability is a generalized term that can be used to describe a number of traits, including an intact membrane, metabolic activity, and overall physiological health of the cell .Only DEFRA approved stud farms were used within this research, anonymity was maintained throughout and data were collected and maintained in accordance with the Data Protection Act 1988 . Among this population of sperm there is a wide array of sperm “quality”, which represents the ability of the sperm to fertilize an oocyte and produce viable offspring . During natural breeding, a stallion will deposit millions of sperm within the intra-uterine environment of the mare . Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed. Modulating reproductive activity in stallions: a review The model explained 87.9% (Nagelkerke R2) of the variance in total volume and correctly classified 99.4% of cases. The model explained 83.6% (Nagelkerke R2) of the variance in total volume and correctly classified 96.4% of cases. The model explained 82.7% (Nagelkerke R2) of the variance in total progressively motile sperm and correctly classified 98.1% of cases. Authors These changes in seminal flora may contribute to the idiopathically poorer sperm quality in certain animals. This suggests that the management for optimal performance may attenuate the negative impacts on semen quality which can occur with exercise. The results seen within this study could be resultant of similar management protocol between the two competition levels. Its predominance has also been linked to low motility . Stallion semen is not abundant in the Lactobacillus genus 10,11, but there are related bacterial families. Four common phyla were detected in samples and a total of 74 families. Quality control was performed by submitting the ZymoBIOMICS Microbial Community Standard® to the same extraction and analysis process as the rest of the samples. In human in vitro experiments, a premature acrosome reaction precluded the binding of sperm to the oocyte, and sperm that were able to bind were less successful in penetration 131,138. However, if a sperm cell undergoes the acrosome reaction prematurely, which can occur during cryopreservation or in vitro processing, it loses its ability to penetrate the cumulus oophorus and zona pellucida for fertilization 136,137. In the context of fertilization, capacitation involves calcium oscillations that trigger a complex cascade of intracellular events leading to the binding of specific zona ligands on the outer plasma membrane with the zona pellucida of the oocyte 130,131. Capacitation can be generally characterized by the acquisition of both hyperactive motility and the ability to undergo the acrosome reaction through various molecular pathways and protein phosphorylation cascades 51,121,122. Table 6. They were evaluated for 60 days before supplementation; then, the same stallions were re‐evaluated for 60 days with nutraceutical supplementation (30 g/day). The use of reproductive biotechnologies in equine practice has shown that some stallions are subfertile, so ways to improve fertility have been sought. Furthermore, saliva, as well as seminal plasma samples were taken, and the concentration of cortisol therein was determined. Two ejaculates were collected at a one-week interval and analysed with an extended spectrum of spermatological methods. Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Adequate production of high-quality sperm by the male is critical to both natural and artificial reproductive processes. Studies of equine sperm fertility encompass sperm biogenesis 3,4, motility and metabolism 5,6, morphology , sperm ultrastructure , and biochemical elements of sperm function 9,10,11,12, including sperm interactions with accessory sex gland secretions 13,14,15. Thus, it becomes necessary to understand which physiological and functional parameters are the biggest contributors to sperm fertility. Although some variation in sperm morphology and physiology between either individuals of the same species or within an ejaculate will not affect fertilization and embryo development outcomes, some parameters are correlated with fertilization, embryo development, and pregnancy outcomes. In contrast to previous research, stallions aged 20 years and over presented with increased semen characteristic values.Fertility is not considered to be directly affected by semen volume ; therefore, the lower values seen within non-competing stallions should not be considered a negative impact on semen quality, unless they fall below industry standards or what would be classed as “normal” for individual stallions.Although proportions vary among individuals, sperm quality values are similar.Semen from six Quarter Horse stallions was assessed for 4 months every 20 days using the computer‐assisted semen analysis system.AI allows for eliminating geographic barriers and allowing sport stallions within breeding programmes to breed and compete simultaneously (Alvarenga et al., 2016; Wilson & Flesner, 2017).Therefore, it is of interest to remove the prematurely acrosome reacted spermatozoa during selection procedures.However, it is necessary to perform further experiments involving a larger number of animals and evaluate the semen during the cooling and freezing process of the spermatozoa. Fertility is not considered to be directly affected by semen volume ; therefore, the lower values seen within non-competing stallions should not be considered a negative impact on semen quality, unless they fall below industry standards or what would be classed as “normal” for individual stallions. In addition, sperm progressive motility and sperm concentration did not differ between lower and elite levels of competition. Stallions at elite levels of competition recorded higher total sperm count and progressive motility compared to those competing at lower levels of competition. Using statistical analysis, we found that competing stallions have lower quality semen than non-competing stallions and competitive activity may have a greater negative impact on spermatogenesis than age. This need for constant genetic progression means that a successful career in competition is considered vital to increase a horse’s potential as a valuable breeding stallion . The exercising of stallions with regards to training regimes during the breeding season is a controversial subject. Most stallions within breeding programmes are expected to breed and compete concurrently. This paper reviews the major pharmacological agents reported to usefully modify reproductive activity in stallions, and outlines their pros and cons when compared to training, management or surgical alternatives. And J.L.V.-P.; formal analysis, C.Q.-P.; resources, A.M.; writing—original draft preparation, C.Q.-P.; writing—review and editing, A.M., F.C., I.O., and J.L.V.-P.; visualization, C.Q.-P. However, the data process may be more complicated . Our study wanted to show that there may be certain bacterial families that harmoniously dwell in semen. In equine spermatozoa, the precursor to the acrosome reaction is sperm activation, or capacitation, which occurs in the female reproductive tract as the spermatozoa approaches the oocyte. Fluorescent dyes are a common method of sperm quality assessment and a more extensive discussion of their use in ARTs can be found in Section II of this review. However, stallion fertility may be poor even with a highly motile population , and, thus, it is critical to understand other common sperm abnormalities. Sperm motility is essential for in vivo fertilization and in vitro fertilization (IVF), and is not necessarily required for ICSI where the sperm is manually injected 94,95,96,97. Although parameters of a morphologically normal sperm may vary significantly on an individual basis, abnormalities in the sperm anatomy may be an indication of subfertility or a problem with spermatogenesis .A dominant family in fertile stallions is Porphyromonadaceae .Although exact mechanisms of cryoinjury to equine sperm are poorly understood, potential targets include disrupted plasma and mitochondrial membranes, increased ROS production, and generation of apoptotic factors 6,93,140.The charge may also change significantly as a sperm changes environments, or when it undergoes capacitation or acrosome reaction 41,79.The sperm head is an elongated, oval shape that is also relatively flat, with some variation on an individual basis 7,36,37.Starting with Firmicutes phylum, the literature contains divergent findings regarding the effect of bacterial families on sperm quality.Pairwise comparisons to identify where differences in semen characteristics occur among stallion competition levels.No difference between BS and BSC could be found in regard to the sperm quality parameters and the cortisol concentration in seminal plasma samples.The thermal effect of higher intensity exercise could also impact on semen characteristics . Aaligned spermatogonia then undergo differentiation into committed A1-spermatogonia, which also reside in the basal compartment . However, A-spermatogonia exist both to replenish the population of gametic stem cells and differentiate for continuation of spermatogenesis, and, therefore, a subpopulation of A-spermatogonia become committed to differentiation 27,30. A non-committed store of A-spermatogonial cells exists at the basal layer and remains undifferentiated due to the expression of the Neurogenin 3 (NGN3) gene 27,28,29. The sperm head is overlaid by a plasma membrane, and an acrosomal compartment containing enzymes to aid in fertilization. Species specific traits of the stallion sperm head include a characteristic asymmetrical head, a paraxially inserted tail, and a small acrosome relevant to other species . However, the remainder of this review will be focusing on the mature ejaculated spermatozoa and its relation to IVP, a process during which seminal plasma is largely removed, and, thus, we will not be elaborating on the significance of accessory sex glands and their secretions. Where these tests found significant differences, post-hoc analysis with Mann–Whitney U tests determined where differences occurred among groups. The frequency of collections for age, discipline and competition level were also recorded. Only DEFRA approved stud farms were used within this research, anonymity was maintained throughout and data were collected and maintained in accordance with the Data Protection Act 1988 . All samples were collected and assessed by Department for Environment, Food and Rural Affairs (DEFRA) qualified Artificial Insemination (AI) technicians and both stud farms used the same techniques for semen evaluation. Stallion breed was controlled to include only warmbloods and competition level was controlled in accordance with previous research 15,16,17,18. Sperm viability is a generalized term that can be used to describe a number of traits, including an intact membrane, metabolic activity, and overall physiological health of the cell . CASA can also analyze more complicated motion parameters including the amplitude of lateral head displacement, average path velocity, straight line velocity, curvilinear velocity, linearity of the curvilinear path, and beat-cross frequency . Common issues in patients with male factor infertility can be either obvious or indiscernible to the human eye, and thus the depth of analysis by a technician depends on the technology immediately available to them. Sperm analysis is a significant method of infertility diagnoses and is critical in order to maximize IVP outcomes. Lesser amounts of ATP may be produced by glycolysis under oxygen depleted conditions for maintenance of high sperm velocity 91,92. Interestingly, sperm from stallions classified as fertile based on their breeding history are more likely to undergo the acrosome reaction in vitro when incubated with progesterone than sperm from subfertile stallions .Model B6 was performed to ascertain the effects of 15 independent variables (Table S13) on the likelihood that total semen volume was above industry standards (Table 7).This can cause testicular hyperthermia and thermal insult could negatively impact on spermatogenesis 31,32,33.This does not concur with findings from previous studies 4,14,28, which found that higher intensity exercise have detrimental effects on semen characteristics.It is therefore likely that changes also occur within equine spermatogenesis and semen quality in response to exercise loads and training regimes .Whilst thermal stress could explain some of the negative effects seen in semen characteristics among stallion disciplines, this is still a debated topic area within equine research 10,31,32,34; and no definitive conclusions have been drawn.Following exercise, stallion internal scrotal temperature has been recorded to rise over 2 °C .The sperm head is overlaid by a plasma membrane, and an acrosomal compartment containing enzymes to aid in fertilization. As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. Male fertility is often estimated by simple sperm assessment, and therefore, it is crucial to establish species-specific baselines for normal sperm parameters. In a study carried out on Mangalarga Marchador stallions, they reported that there was no statistical difference in rectilinear speed (Rodrigues et al., 2017). Six clinically healthy stallions with proven fertility and an age range of 9–12 years were used. The diet of the six stallions was based on alfalfa with an intake of 20% MS and 1 kg concentrate containing 14% crude protein, which was mixed with 30 g of nutraceutical. The stallions were exercised thrice weekly with forced exercise and electric treadmills for approximately 30 min. Six Quarter Horse stallions, aged 9–12 years and with a body condition of 5 (scale 1–9) according to the classification proposed by Carroll and Huntington (1988), were used for the experiment. Several studies have focused on investigating the effects of different dietary compounds on semen quality. Model A6 was performed to ascertain the effects of 18 independent variables (Table S7) on the likelihood that total progressively motile sperm was above industry standards (Table 7). The model explained 88.3% (Nagelkerke R2) of the variance in total volume and correctly classified 95.9% of cases. Pairwise comparisons to identify where differences in semen characteristics occur within stallion disciplines. Results (probability values) of pairwise comparisons to identify where differences in semen characteristics occur within stallion age categories. The mitochondrial membrane potential must be maintained, as reduced polarization can lead to an ATP shortage and cellular damage and hyperpolarization may produce an over-abundance of reactive oxygen species (ROS) and cause lipid peroxidation, which can be detrimental to overall cell integrity 6,90. ATP production occurs on the inner mitochondrial membrane within the intermembrane space between inner and outer membranes 6,86. Thus, membrane charge is both a revealing and complex trait to accurately measure and interpret. 3. Difference in Semen Characteristics among Stallion Disciplines This optimised management may attenuate the negative impact of exercise on semen quality within the elite level stallions. Further studies are required to investigate this effect and would benefit from the inclusion of stallions within the competitive environment and the classification of exercise intensity, as well as measuring both subcutaneous testicular and rectal temperatures concurrently with the evaluation of semen characteristics. Within this study, competing stallions had significantly reduced semen characteristic values compared to non-competing stallions. To the authors’ knowledge, no previous equine studies have evaluated the effect of stallion competitive discipline participation on the quality of semen characteristics. Whilst previous research states that stallion fertility is optimal at 5–9 years 15,26, the current study observed an increase of 23% in the total progressively motile sperm of stallions aged ≥20 years when compared to stallions aged 5–9 years. An artificial vagina (VA), the Botucatu model, was used for semen collection. To assess the actual quality of the semen, semen was collected three times a day, every 2 days, on two occasions. The stallions were evaluated before and after supplementation with nutraceutical. The outer plasma membrane can be partitioned into the acrosomal, post-acrosomal, neck, midpiece, and principal piece domains .Therefore, this study hypothesized that supplementation with various antioxidants of nutraceutical origin would improve semen quality in Quarter Horse stallions.In the context of fertilization, capacitation involves calcium oscillations that trigger a complex cascade of intracellular events leading to the binding of specific zona ligands on the outer plasma membrane with the zona pellucida of the oocyte 130,131.The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates.However, A-spermatogonia exist both to replenish the population of gametic stem cells and differentiate for continuation of spermatogenesis, and, therefore, a subpopulation of A-spermatogonia become committed to differentiation 27,30.To be included in the study, stallions were required to be warmbloods, and to participate in show jumping, dressage or eventing, or be non-competing.Parameters were evaluated using custom configurations for equine sperm.Within all models many of the confidence intervals (95% CI) presented values above 1, suggesting high variability in the data, therefore caution must be applied when interpreting the outcomes.Poor DNA integrity of sperm, or sperm with increased DNA fragmentation, can, thus, have detrimental effects on reproductive outcomes. It is also noteworthy that oxidative phosphorylation (the primary method of ATP generation in stallion sperm) coupled with mild oxidative stress is beneficial to sperm functional pathways such as hyperactivation, capacitation, acrosome reaction, and fertilization . In fact, mitochondrial function, which can be approximated by mitochondrial membrane potential and electron transport chain (ETC) activity, are known to be positively correlated with overall sperm function 82,83,84,85. As previously mentioned, the mitochondrial helix is the primary grouping of organelles responsible for active motility and metabolism in the sperm cell. Therefore, it could be postulated that dressage competition has a reduced impact on stallion endocrinology, due to the lower competition intensities and energy demands . Within all models many of the confidence intervals (95% CI) presented values above 1, suggesting high variability in the data, therefore caution must be applied when interpreting the outcomes. However, within this model, many of the confidence intervals (95% CI) presented values above 1, suggesting high variability in the data. The difference in semen quality among competing and non-competing stallions, as well as the difference among disciplines suggests endocrinological and physiological changes occur in relation to training intensity and competition. It can be concluded that while active participation in competitions represents a stress factor, the dual use of stallions in breeding programs and sports competitions is possible without negative effects on their sperm quality. Our results show that there might be a correlation between some sperm quality parameters and the seminal flora composition of healthy, fertile stallions, in particular, Firmicutes phylum. Concentration, number of spermatozoa, total and progressive sperm motility parameters, and sperm DNA fragmentation were included. Due to the ease of analysis, sperm motility, viability, and morphology are the most common sperm assessments. In the stallion, remodeling of the plasma membrane has been partially described through the domain-specific patterning of filipin–sterol complexes acquired during epididymal maturation as well as changes in intermembrane proteins . In the stallion, it has been shown that seasonal fertility is partially attributed to changes in the number of Sertoli cells in the testis, which is directly correlated with the numbers of spermatozoa ultimately produced 25,26. Cross sections of the seminiferous tubules reveal adjacent cellular associations that produce sperm in a cyclic manner, repeating approximately every 12 days in the stallion for the constant production of spermatozoa 3,16,17,18. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. To be included in the study, stallions were required to be warmbloods, and to participate in show jumping, dressage or eventing, or be non-competing. It is therefore likely that changes also occur within equine spermatogenesis and semen quality in response to exercise loads and training regimes . Consequently, many sports horse stallions have dual careers and often need to fit breeding schedules around competition dates. This was a retrospective study that evaluated the seminal data of 1130 stallion semen collections from two UK based stud farms between 2009 and 2016. Peptoniphilaceae correlated positively with total sperm motility, whereas Clostridiales Incertae Sedis XI correlated negatively with progressive motility. Despite the advances in reproductive technology, there is still a considerable number of low sperm quality cases in stallions. Due to the retrospective nature of the data within the current study, this information was not available, however future work integrating sperm morphology data to gain a greater understanding on the stallions testicular functioning is warranted. Alternatively, it could be suggested that competition stallions should have semen collections scheduled outside of competition seasons and semen frozen for use within the consecutive competition season. Some researchers have suggested that long periods of intratesticular hypoxia could be part of the aetiology of certain spermatogenic failures (Sharma et al., 2021). Sperm concentration was lower (p 6) than after supplementation (339.4 ± 17.5x6). Parameters were evaluated using custom configurations for equine sperm. Immediately after collection, it was placed in a water bath at 37°C; the gel fraction was removed by filtration using an equine semen filter (Animal Reproduction Systems). Prior to fertilization, the acrosome undergoes a calcium-dependent exocytotic reaction (acrosome reaction) as a result of sperm-oocyte binding that is essential for the subsequent penetration of the oocyte . Rather than, or in addition to, a viability stain with a permeable cell marker, another fluorescent dye may be added to expand upon the assessment of sperm integrity or function. Assessment of sperm viability can also be indicative of early apoptotic changes, which could also be correlated with other severe sperm abnormalities or infertilities. Another double staining fluorescent method for viability used in the equine industry is SYBR-14 and PI for viability 103,105. Although Hoechst is permeable with all cells, PI is only able to penetrate cells with disrupted plasma membranes. Sperm evaluation for morphological defects could assist with detecting the time frame in which damage to the sperm occurs. It has been observed that horses competing within non-elite level dressage train for a duration of approximately one hour, three to four times per week, suggesting similar exercise regimes . This can cause testicular hyperthermia and thermal insult could negatively impact on spermatogenesis 31,32,33. Following exercise, stallion internal scrotal temperature has been recorded to rise over 2 °C . In the human species, however, some authors have already pointed out the influence of bacteria on semen quality 12,13,14,15,16. Factors affecting these parameters have been subject to large-scale analysis in horse reproductive science . These outcomes depend on a variety of factors, such as sperm quality. In conclusion, the seminal microbiome may affect spermatozoa activity. Recent studies in humans have detected several seminal microflora–spermatozoa associations behind some idiopathic infertility cases. Extrapolating from these data, selecting sperm with a greater net negative zeta potential will theoretically select for mature, functional, and viable spermatozoa. Interestingly, sperm from stallions classified as fertile based on their breeding history are more likely to undergo the acrosome reaction in vitro when incubated with progesterone than sperm from subfertile stallions . Generally, in sperm analysis, viability of the population is estimated by determining the percent of intact membranes using fluorescent dyes such as propidium iodide (PI) and Hoechst 103,104. Associated Data This could also explain why disciplines anecdotally considered less physiologically demanding had higher semen values. The models explained 100% (Nagelkerke R2) of the variance in the dependant variables and correctly classified 100% of cases. Only five variables made a unique statistically significant contribution to the model. Stallions at elite level of competition recorded higher total sperm count and sperm progressive motility compared to stallions active within lower competition levels. Dressage stallions had significantly higher sperm progressive motility, sperm concentration, total sperm count and total progressively motile sperm measurements compared to show jumping stallions. Volume of semen sample (mL), volume of gel-free sample (mL), sperm progressive motility (percentage), sperm concentration (million/mL), total sperm count (billion), total progressively motile sperm (billion) and collection date were recorded. In conclusion, semen parameters such as sperm concentration, morphology and motility were significantly improved in the stallions after supplementation with the nutraceutical. However, spermatozoa in several species, including the ram, bull, rat, boar, buck, man, and stallion have been documented to undergo significant remodeling to the lipid and protein compositions during epididymal maturation 41,45,46,47,48,49,50. When sperm morphology was evaluated, it was observed that abnormalities decreased after supplementation concerning the data obtained before being supplemented (14.3 ± 0.6% vs. 19.1 ± 0.4%, p 2008) and Andalusian stallions (Ruiz et al., 2021). The results of this study show the importance of nutraceutical in the production and function of spermatozoa in stallions. Mean ± standard error of the mean (SEM) of volume, concentration and abnormal morphology evaluated every 20 days in Quarter Horse stallions without and with nutraceutical. Mean ± standard error of the mean (SEM) of volume, concentration and abnormal morphology evaluated in Quarter Horse stallions without and with nutraceutical. These proteins, as well as the total glycoproteic population in the plasma membrane, undergo compositional changes throughout maturation, capacitation, and acrosome reaction, and are thought to play a physiological role in these processes as well as in fertilization 41,77,79. The source of the net negative charge is due to the addition of negatively charged sialoglycoproteins to the glycocalyx, such as the bipolar glycopeptide CD52, that appear in the sperm membrane during epididymal maturation 43,69,77,78. Through freeze-fracture analysis, altered quantities and distributions of various intramembrane particles were observed over the course of epididymal transit in the equine testis, which is hypothesized to play a role in the establishment of various functional domains 50,67. Factors that affect spermatogenesis, such as thermal stress, hormones and oxidative stress, have the potential to negatively impact stallions’ fertility through detrimental effects on semen characteristics. To calculate if competition does affect semen quality, this study investigated the effect that equestrian discipline and timing of competition had on a range of stallion semen characteristics. Despite the increased demand for breeding stallions to be performing at elite levels of competition, little research has been conducted into how equestrian disciplines and competition level affect seminal quality. Using of antioxidants directly in semen or antioxidant supplementation of stallions has improved sperm quality (Brinsko et al., 2005; Campos et al., 2020; Deichsel et al., 2008; Freitas et al., 2016). Then, semen was diluted until reaching an approximate concentration of 25 × 106 sperm/mL in milk-based extender (INRA 96®, IMV Technologies, L’Aigle, France) and placed in a 37 °C water bath. Only two families appeared to have a correlation with two sperm quality parameters. Our study detected a correlation between the presence of the Peptoniphilaceae family and higher total motility and the presence of Clostridiales Incertae Sedis XI and lower progressive motility. In contrast to previous research, stallions aged 20 years and over presented with increased semen characteristic values. Since differences among semen characteristics were found between competing level and discipline, this suggests that confounding management factors which were not recorded could be influential. We suggest that appropriate management of the competition stallion may reduce the negative impacts which exercise can induce on semen quality.Following spermatocytogenesis, the final stage of spermatogenesis is the morphological shift denoted as spermiogenesis.Descriptive analysis of data was performed to establish mean ± standard deviation (sd), medians, ranges and interquartile ranges for the semen characteristics recorded.Pairwise comparisons to identify where differences in semen characteristics occur within stallion disciplines.This significant diversity in sperm fertility within an ejaculate becomes more pertinent when applied to in vitro embryo production (IVP), during which a smaller number of sperm are generally selected for either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).Within the 5–9 years category, 75% of stallions were competing within various disciplines, 67% of which were at higher or elite competition levels.It was evaluated by extracting a sample of approximately 2.7 μL of semen and placing it on a slide, after which semen motility was analysed with respect to five digital images of various fields at a temperature of 37°C in a computer‐aided semen analyser (HC‐B028V).Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed. Zeta potential of sperm cells has been investigated in men, rats, bulls, rabbits, golden hamsters, guinea pigs, and mongoose 69,73,74,75,76. One of the physiological outcomes of membrane protein modifications is the overall change in net surface charge. It is hypothesized that functional domains assist the sperm cell in adapting to new conditions in the seminal plasma and female reproductive tract . Due to the compaction of the sperm genome and the reduction in transcription, significant changes in protein, lipid, and sugar contents are thought to be a result of the uptake of epididymal epithelial secretions . By the same token, a breeding stallion that displays little interest in mounting a mare/phantom, or is slow to achieve erection and/or ejaculation, can be extremely frustrating. However, the most common reason for wanting to modulate reproductive activity in a stallion is to alter the expression of sexual behaviour. For a review of current and prospective sperm selection technologies, please refer to Section II of this review. This study aimed to evaluate the effect of nutraceutical supplementation on improving semen quality in Quarter Horse stallions. This corresponds with lower freezability of stallion spermatozoa, up to this age. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. However, this family has been regularly found in the semen of fertile stallions 46,48. Following spermatogenesis, spermatozoa are exposed to a variety of proteinic and non-proteinic substances secreted by the accessory sex glands which aid in the acquisition of mature male fertility and sperm survival during transportation through the male tract and into the female tract 13,14,15,34. However, during spermiogenesis, somatic histones are replaced by protamines, the dominant nuclear proteins of mature spermatozoa . The process by which mature spermatozoa are generated is a highly regulated process spanning across multiple domains of the testis. Facilitated by the nurturing Sertoli cells, basal spermatogonia replicate and differentiate into primary spermatocytes, and sequentially develop into secondary spermatocytes, spermatids, and the morphologically distinct spermatozoa during spermatogenesis. A greater net negative zeta potential, a parameter determined by surface composition as described previously, is acquired during epididymal maturation through extensive membrane remodeling and has been correlated with sperm quality in men 154,155. In the stallion, progressive motility is used as a general estimate of fertility, with less than 50% progressively motile in raw semen or less than 10% progressively motile two hours post collection being an indicator of potential subfertility . Sperm motility measures are widely considered to be indicative of fertility based on obvious biological functions, despite variable correlations with other sperm quality parameters 97,99. Additionally, research in stallions has shown correlations between ROS and motility, viability, and mitochondrial function 87,91,93, and, thus, it is highly beneficial to understand mitochondrial mechanisms as they relate to sperm fertility. The number of mitochondrial gyres in the midpiece of the equine spermatozoa varies between 40 and 50, and their organization, or more specifically a disrupted organization, has been shown to play a role in the fertility of stallions through localized ATP production for sperm flagellar movement 80,81,82. Stallion infertility is a major cause of concern in the horse industry. (Jane Williams) and V.T.M.; investigation, M.W.; resources, M.W.; data curation, M.W., J.W. We would like to thank Alison Wills for her support with statistical modelling and the participating studs for their support this project. This may be related to their non-competitive status, suggesting that competitive activity may have a greater negative impact on spermatogenesis than age. Thorough interpretation of sperm physiology, despite its complexity, is the best method for assessing male fertility. Externalization of sialoglycans by the sperm has been shown to play a role in avoidance of the uterine immune systems, as well as playing roles in capacitation and being an important component of sperm-zona pellucida binding, and, therefore, fertilization. The charge may also change significantly as a sperm changes environments, or when it undergoes capacitation or acrosome reaction 41,79. The acquisition of a net negative charge is primarily based on the extrusion of sialic acid (sialoglycoproteins) and other charged proteins to the outer membrane of the head region during epididymal maturation 44,69,77,156. Kinematic parameters reflect the ability of sperm to migrate through the mare's reproductive system for fertilization (Alvarenga et al., 2016). A Student's t test analysis was used with significance levels of p ≤ 0.05. Experimental endpoints were analysed using an ANOVA to compare the values of the stallions fed their control vs. the same stallions fed the nutraceutical‐enriched diet. It was evaluated by extracting a sample of approximately 2.7 μL of semen and placing it on a slide, after which semen motility was analysed with respect to five digital images of various fields at a temperature of 37°C in a computer‐aided semen analyser (HC‐B028V). Starting with Firmicutes phylum, the literature contains divergent findings regarding the effect of bacterial families on sperm quality. Total (TM, %) and progressive sperm motility (PM, %) were evaluated as described by . Samples were collected from 12 clinically stallions (7 Andalusians, 4 Arabs, and 1 Anglo-Arab) in Écija (Seville, Spain) during the breeding season (March–June). In the case of intact stallions used for competitive or recreational purposes, the overt expression of sexual or aggressive behaviour can be distracting for both animal and owner and, in some cases, dangerous to all concerned. In conclusion, sperm physiological assessment is an invaluable tool for the equine breeding industry and merits continued consideration in clinical and research settings. In addition, characterization of new biophysical properties, such as zeta potential, will not only aid in our understanding of what makes a fertile sperm, but will also allow for the development of new semen selection technologies. No difference between BS and BSC could be found in regard to the sperm quality parameters and the cortisol concentration in seminal plasma samples. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. Further studies including a larger number of animals, including subfertile stallions with low sperm quality, are needed in order to find the possible relationship between the seminal microbiome and sperm quality. In this sense, it has been observed that supplementation based on nutraceuticals containing antioxidants improves semen quality (Freitas et al., 2016). Several recent studies have mentioned that semen quality efficiency is improved by reducing oxidative stress damage (Aurich et al., 2020; Aurich, 2008). AI allows for eliminating geographic barriers and allowing sport stallions within breeding programmes to breed and compete simultaneously (Alvarenga et al., 2016; Wilson & Flesner, 2017). Sperm concentration (10x6) was significantly higher with supplementation (339.4 ± 17.5 sperm/mL) than without supplementation (224.6 ± 19.9). This section collects any data citations, data availability statements, or supplementary materials included in this article. The data that support the findings of this study are available from the corresponding author upon reasonable request. However, in other studies, the kinematic parameters did not increase after nutraceutical supplementation (Alamaary et al., 2019; Brinsko et al., 2005; Ravi et al., 2016). Other studies have observed an increase in the percentage of abnormal sperm in conditions of hypoxia, where the predominant anomalies are head malformations, unlike tail anomalies that are more frequent in normal conditions (Dehdari et al., 2023). Apoptosis of primary spermatocytes and spermatogonia, induced by hypoxia, may contribute to the loss in this population of cells due to the large distance of oxygen diffusion from the blood vessels to the luminal region of the seminiferous tubules (Govindaraj et al., 2017). Leydig cells, which are stimulated by Luteinizing Hormone (LH) to produce sex hormones, including testosterone, are key for regulating spermatogenesis as well as being responsible for the male phenotype 20,21. Spermatozoa are released into the lumen of the seminiferous tubule of the testis during spermiation. This process, known as spermatogenesis, occurs in the germinal epithelium of the seminiferous tubules of the testis, and is initiated during puberty (Figure 1) . The wholistic picture of sperm fertility is integral to the maximization of IVP outcomes, and, therefore, in Section I of this review we will focus on equine spermatogenesis, sperm morphology, and common sperm abnormalities leading to infertility. This significant diversity in sperm fertility within an ejaculate becomes more pertinent when applied to in vitro embryo production (IVP), during which a smaller number of sperm are generally selected for either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). To calculate if competition affects semen quality, this study investigated the effect that equestrian discipline, competition level and timing of competition have on a range of stallion semen characteristics (total volume, gel-free volume, sperm concentration, sperm progressive motility, total sperm count and total number of progressively motile sperm). Model B2–B5 were performed to establish the effects of 15 independent variables (Tables S9–S12) on the likelihood that gel-free semen volume (MB2), sperm progressive motility (MB3), sperm concentration (MB4) and total sperm count (MB5) were above industry standards. Model A2–A5 were performed to ascertain the effects of 18 independent variables (Tables S3–S6) on the likelihood that gel-free semen volume (MA2), sperm progressive motility (MA3), sperm concentration (MA4) and total sperm count (MA5) were above industry standards. These stressors, as well as abnormal morphology, can initiate a variety of negative effects such as membrane and mitochondrial damage, plasma membrane restructuring (including the externalization of proteins such as phosphatidylserine), generation of ROS, and subsequent DNA damage 10,12,141,142,143. Apoptosis is also a common issue seen in sperm samples, especially those that undergo thermal, oxidative, or osmotic stressors from extending, cooling, or cryopreservation 141,142. Alternatively, hyperpolarization of the mitochondrial membrane will lead to lipid peroxidation and an over-abundance of ROS, leading to cellular damage 6,90. Add to Collections No significant differences were found in semen characteristics between lower and higher levels of competition. Medians, ranges and interquartile ranges for each semen characteristic among age categories of competing and non-competing stallions. Differences in total volume and gel-free volume were also found between stallions aged 5–9 years and 10–14 years to stallions aged 20 years and over. Descriptive analysis of data was performed to establish mean ± standard deviation (sd), medians, ranges and interquartile ranges for the semen characteristics recorded. The purpose of this study was to evaluate the effect of nutraceutical supplementation with β‐carotene, lutein, lycopene, casein, selenium, docosahexaenoic acid (DHA, omega‐3), l‐carnitine, vitamin A, vitamin C and vitamin E on semen quality in Quarter Horse stallions. Therefore, this study hypothesized that supplementation with various antioxidants of nutraceutical origin would improve semen quality in Quarter Horse stallions. This study aimed to evaluate the effect of nutraceutical supplementation on semen quality in Quarter Horse stallions. Based on the results, it is concluded that nutraceutical supplementation improved semen parameters in Quarter Horse stallions. Semen from six Quarter Horse stallions was assessed for 4 months every 20 days using the computer‐assisted semen analysis system. However, there is little research evaluating the effects of exercise in competitively active stallions, despite the increasing popularity of dual purpose (breeding and competition) stallions in the industry. Daily exercise at low intensities is important for the mental and reproductive well-being of the stallion, however higher intensities of exercise, as seen in competing stallions, may have detrimental effects on seminal quality. We suggest that appropriate management of the competition stallion may reduce the negative impacts which exercise can induce on semen quality. In addition, dressage stallions recorded improved semen characteristics when compared to show jumping and eventing stallions. Statistical analysis was performed using Microsoft Excel® 2013. Next-generation analysis was performed using Ion semiconductor sequencing following the protocol described by Quiñones . Briefly, 100 μL of the raw semen sample was broken down for 1 h with 10 mg/mL lysozyme, 4000 U/mL lysophosphatin, and 25,000 U/mL mutanolysin. Sperm concentration was measured using a spectrophotometer (Spermacue®, Minitüb, Tiefenbach, Germany). In order to evaluate the extraction and amplification quality, the pattern sample ZymoBIOMICS Microbial Community Standard® (Zymo Research, Irvine, CA, USA) was included during DNA extraction. In stallions, the presence of the Enterobacteriaceae family typically has a fecal origin and worsens various seminal parameters 22,39,40. Additionally, the positive effects of lactobacilli supplementation on sperm quality parameters have also been described in humans 19,20, mice , and broilers . Then, samples were submitted to a sperm quality analysis. Extended semen was only used for sperm parameters evaluation. 1.3. Multivariable Modelling: Effect of Related Factors on Semen Characteristics Previous research states that disruptions within spermatogenesis can take up to 57 days for semen characteristics to return to basal values . Therefore, whilst it may be possible to optimise the management of dual career stallions for competition performance, it may be difficult to also improve breeding performance concurrently. Interestingly, no significant differences were observed between lower and higher competition level horses for any of the semen characteristics. Future research should explore the management practices of competing horses and breeding stallions with the goal of creating a successful management protocol for dual career stallions to enhance performance and breeding. This does not concur with findings from previous studies 4,14,28, which found that higher intensity exercise have detrimental effects on semen characteristics. Interestingly, a reduced total sperm count was found in eventing horses compared to other disciplines. The thermal effect of higher intensity exercise could also impact on semen characteristics . Thus, DNA integrity of semen can be a good indication of fertilization potential and the potential effects on embryo development and offspring characteristics. Causes of DNA fragmentation may include the mispackaging of chromatin during spermatogenesis , apoptosis , excessive ROS 146,148, and other environmental factors . Poor DNA integrity of sperm, or sperm with increased DNA fragmentation, can, thus, have detrimental effects on reproductive outcomes. DNA integrity assessment is one of the most valuable assessments of sperm fertilization potential due to the strong correlation with sperm reproductive competence; in fertilization as well as in subsequent embryo development and offspring phenotype . Physiological processes connected with sexual maturation of stallions were observed on 10 half-breed Anglo-Arab stallions beginning from 8 months of age, until 4.5 years of age. Further studies are needed to better understand the interactions between seminal flora and sperm quality. Although proportions vary among individuals, sperm quality values are similar. Regarding Pseudomonadaceae, this family has been related to lower values of motility and integrity parameters 23,46,47, while other authors agree to consider it an opportunistic pathogen . The other highly dominant family in stallion semen is Corynebacteriaceae 10,11, which has regularly been found in fertile individuals 10,23,37,38,39,40. Medians, ranges and interquartile ranges for each semen characteristic among varying stallion disciplines. The predictive abilities of the final models were investigated using receiver operating characteristic (ROC) curve analysis . For each stallion, age, breed, discipline, competition level and dates competed were recorded alongside key seminal characteristics identified from previous research . Data were collected for 142 resident sports horse stallions aged between two and twenty-five years (9.21 ± 4.69 years) used within artificial insemination breeding programmes. Retrospective data for six semen performance determinants were collected from two UK based stud farms for a seven-year period (2009–2016). To the best of our knowledge, there are no studies evaluating sperm quality and the seminal microbiome in this species. Regarding stallions, papers usually focus on pathogenic bacteria or on their effect on reproductive technologies 22,23. In fact, these studies have opened the door to a potential therapeutic tool in infertility cases, and some authors have already published some positive effects of prebiotics in improving sperm quality 17,18,19,20. Differences were found in the average and median values for the semen factors investigated which related to the age of the stallions that semen was collected from (Table 1). Number of semen collections from each age category within stallion discipline and competition level. The majority of stallions were aged between five and nine years and the level horses were competing at varied across the discipline (Figure 1). Age was also included in all models as previous studies highlight this as a biologically plausible factor related to semen quality 15,16. Even though exercise modality and intensity has been frequently researched within human science, this is not the case for the equine industry. Physiological and endocrinological responses to exercise can affect spermatogenesis 4,5,6 and therefore produce fluctuations in seminal characteristics 7,8,9,10. The aim within British sport horse breeding is to produce offspring that will, in the future, perform successfully within international competitions 1,2. These results differ from previous research within commercial breeding stallions 16,25,26, where declines in semen quality by the age of 10 years are consistently reported. Model B6 was performed to ascertain the effects of 15 independent variables (Table S13) on the likelihood that total semen volume was above industry standards (Table 7). Model B1 was performed to ascertain the effects of 15 independent variables (Table S8) on the likelihood that total semen volume was above industry standards (Table 7). Model A1 was performed to ascertain the effects of 18 independent variables (Table S2) on the 6likelihood that total semen volume was above industry standards (Table 7). Medians, ranges and interquartile ranges for each semen characteristic among stallion competition levels. The total number of spermatozoa was calculated by multiplying the concentration and volume. Competing stallions within dressage, show jumping and eventing had significantly lower quality semen than non-competing stallions. We found stallion competition level and discipline did not significantly affect whether the semen characteristics investigated were above or below the current industry standards for artificial insemination. Our results suggest that both competition level and discipline have significant negative effects on semen characteristics and we would recommend that competition programs for dual career stallions should be managed to ensure sufficient time in light-moderate exercise and that stallions be removed from active competition before collection takes place. One of the physiological outcomes of membrane protein modifications is the overall change in net surface charge.Our study wanted to show that there may be certain bacterial families that harmoniously dwell in semen.Due to the ease of analysis, sperm motility, viability, and morphology are the most common sperm assessments.Aaligned spermatogonia then undergo differentiation into committed A1-spermatogonia, which also reside in the basal compartment .At the beginning of the breeding season, the penis and prepuce of the stallion were gently washed with warm water to remove smegma excess.This suggests that the management for optimal performance may attenuate the negative impacts on semen quality which can occur with exercise.In fact, mitochondrial function, which can be approximated by mitochondrial membrane potential and electron transport chain (ETC) activity, are known to be positively correlated with overall sperm function 82,83,84,85.Therefore, it could be postulated that dressage competition has a reduced impact on stallion endocrinology, due to the lower competition intensities and energy demands .Despite the increased demand for breeding stallions to be performing at elite levels of competition, little research has been conducted into how equestrian disciplines and competition level affect seminal quality. Horses competing at elite levels are managed for the highest level of athletic performance, which may equate to less demanding training regimes and stringently managed competition schedules. Whilst thermal stress could explain some of the negative effects seen in semen characteristics among stallion disciplines, this is still a debated topic area within equine research 10,31,32,34; and no definitive conclusions have been drawn. Horses are shown to have reduced heart rates (46 bpm vs. 81.9 bpm) and cortisol levels (3.5 nmol/L vs. 5.01 nmol/L) when comparing dressage and show jumping stallions, respectively 29,30, most likely reflecting differences in discipline workload related to exercise frequency, duration and intensity . Therefore, the declines observed here in semen quality could be an indicator that as competition performance peaks, reproductive performance is negatively affected. This decline is postulated to be due to aging stallions being more susceptible to substandard spermatogenesis and testicular degeneration 16,26. At the beginning of the breeding season, the penis and prepuce of the stallion were gently washed with warm water to remove smegma excess. Semen collection was performed using a phantom for stallion support, with a mare in estrus to stimulate sexual behavior. The success of the equine industry greatly depends on good reproductive outcomes. No significant differences were found for the rest of the parameters. However, no studies are available on horses, and there is limited information on the microflora present in stallion ejaculates. However, when there is an imbalance in ROS due to oxidative stress (Del Prete et al., 2018; Halliwell & Whiteman, 2004), it produces alterations that modify semen homeostasis (El Sisy et al., 2016), affecting fertility (Freitas & De Oliveira, 2018; Lançoni et al., 2018). It has been shown that sperm can produce reactive oxygen species (ROS) that are involved in various physiological events, such as sperm capacitation, if they are controlled (Aitken & Baker, 2004). The neck piece connects the sperm head to the tail and is made up of the connecting piece, the proximal centriole, and mitochondria. The tail points distally, is also covered by a plasma membrane, surrounding the structural axoneme. The acrosome covers the anterior portion of the sperm head and contains hydrolytic enzymes which are released in order for the sperm to penetrate an oocyte . The sperm head is an elongated, oval shape that is also relatively flat, with some variation on an individual basis 7,36,37. This final form produced via spermiogenesis is known as a spermatozoon and is released into the lumen of the seminiferous tubule during the event of spermiation 4,32. A two-way ANOVA with log-transformation assessed if interactions occurred between age and discipline, and age and competition level for the semen characteristics assessed. Differences in semen collection technique (number of mounts, teasing time, and the artificial vagina used) were not factored into the model as these data were unknown. Sperm concentration was analysed using an automated sperm cell counter (NucleoCounter SP-100) and motility was assessed via microscopic evaluation. Here, the sperm cell acquires its characteristic shape, including a species-specific streamlined head containing penetrative enzymes, a structured midpiece, a propelling tail, and the condensation of the male genome . Following spermatocytogenesis, the final stage of spermatogenesis is the morphological shift denoted as spermiogenesis. These primary spermatocytes then enter the meiosis phase, where they pass into the adluminal compartment and participate in two meiotic divisions, first becoming haploid secondary spermatocytes and, ultimately, producing haploid spermatids 3,4. This period of cell replication is known as spermatocytogenesis and, ultimately, produces preleptotene primary spermatocytes . A dominant family in fertile stallions is Porphyromonadaceae . In the literature, we found Anaerococcus, a Clostridiales genus, to have a detrimental effect on sperm quality 15,33. However, other authors have found some Firmicutes to have a detrimental effect on sperm parameters. In this regard, Lactobacillus gender has been proven to have a protective effect on spermatozoa 13,30,31,32. Some authors have highlighted the positive correlation between specific Firmicutes families and good sperm quality. In this paper, sperm physiology, function, and common abnormalities in stallions will be reviewed. However, it is necessary to perform further experiments involving a larger number of animals and evaluate the semen during the cooling and freezing process of the spermatozoa. This may be mainly because some antioxidants in high concentrations in the epididymis provide energy to the sperm by helping mitochondrial β‐oxidation and increasing sperm concentration (Mongioi et al., 2016). Brinsko et al. (2000) suggested the term ‘bad coolers’ or ‘bad freezers’ as appropriate for stallions that exhibit a sudden decline in motility or fertility. It was found that there is full somatic and sexual development in the stallion reached around the age of 3.5 years, and the sperm morphology stabilized in the range of the physiological norm around 3.0 years of age. Specifically, membrane charge increases, or becomes less negative, when the sperm undergoes capacitation . Surface composition and the resulting membrane charge are also of interest in sperm fertility studies. Although exact mechanisms of cryoinjury to equine sperm are poorly understood, potential targets include disrupted plasma and mitochondrial membranes, increased ROS production, and generation of apoptotic factors 6,93,140. Significantly higher semen volumes were found within competing stallions, with the lowest volumes within non-competing stallions. Within the 5–9 years category, 75% of stallions were competing within various disciplines, 67% of which were at higher or elite competition levels. Pairwise comparisons to identify where differences in semen characteristics occur among stallion competition levels. For the purpose of this research, 12 predictive multivariable binary logistic regression models were produced; data from all stallions (Model A 1–6) and data from competing stallions only (Model B 1–6) (Table S1).