Reproductive ageing influences fertility and offspring fitness across mammals. In humans and model organisms, paternal age has been associated with changes in sperm quality and genomic integrity, including oxidative stress, DNA damage, and epigenetic alterations; however, these mechanisms have not been directly examined in large equine populations. Thoroughbred racehorses provide a system to investigate paternal age effects at scale using detailed breeding records. In this retrospective observational study, with data from 35 retired stallions and 33,546 progeny, we assessed associations between sire age at conception, reproductive outcomes and progeny racing performance using generalized linear mixed-effects models. Progeny conceived when stallions were ≤ 8 years were more likely to race (odds ratio [OR] = 2.21, 95% CI 1.93-2.52), place in a stakes race (OR = 3.71, 95% CI 2.74-5.04), and win a stakes race (OR = 3.58, 95% CI 2.43-5.29) than those conceived at ≥ 19 years. These findings demonstrate population-level associations between paternal age and reproductive outcomes.

Ambient heat exposure reduces male fertility in mammals with scrotal testes. Our previous work has demonstrated that some stallions are more susceptible to ambient heat-related subfertility than others, yet the mechanism for heat-induced subfertility remains uncertain, limiting both diagnosis and preventative measures. This study sought to define how the phenotype of stallions susceptible to heat-induced subfertility differs from that of more resilient animals, by measuring the systemic (blood plasma) and localized (reproductive tract) inflammatory and oxidative stress markers of sperm concentration, sperm motility assessments, total antioxidant capacity (TAC; in blood and seminal plasma), malondialdehyde (MDA; in blood and seminal plasma), oxidized guanine species (8-OH-2dG; in blood plasma and spermatozoa DNA), sperm DNA damage (assessed via Halo, SCSA (Sperm Chromatin Structure Assay) and CMA3 (Chromomycin A3)), and c-reactive protein (CRP; in blood plasma). Post-breeding dismount semen samples (n = 357) and blood plasma samples (n = 97) were collected from 31 stallions at commercial thoroughbred studs throughout one breeding season (NSW, Australia). A subset of stallions (16%) was deemed heat-induced subfertility-susceptible (HISS) stallions. These animals showed reduced seminal plasma antioxidant capacity, increased systemic and localized lipid peroxidation, and distinct systemic inflammatory response. Seminal antioxidant capacity was found to be strongly associated with impaired sperm motility (r = 0.739 * vs. r = -0.059). The plasma c-reactive protein of heat-susceptible stallions correlated to heat exposure (r = 0.597 *) and affected sperm motilities (r = -0.527 **, r = -0.434 *). Systemic oxidative DNA damage (8-OH-2dG) also increased following heat events (r = 0.862 ***) and correlated with fertility losses (FCP: r = -0.740 **, PCP: r = -0.603 *). Non-HISS stallions displayed greater variability in systemic antioxidant status and robust response following heat exposure (r = 0.307 *) and localized antioxidant capacity was more strongly correlated to systemic antioxidant capacity than in the heat-susceptible group (r = 0.897 *** vs. r = 0.482 **). We demonstrate that impaired antioxidant responses, altered redox balance and suppressed acute-phase inflammatory signalling are key features associated with heat-induced subfertility in stallions and highlight biomarkers that could be used to identify animals with heat-susceptible fertility.

Exposure to systemic heat stress in male mammals adversely affects sperm production, fertility, and DNA integrity. To date, few studies have investigated this phenomenon in horses, particularly in industry-relevant environments. Therefore, this study examined the relationship between ambient climatic conditions and fertility within a population of commercially fertile stallions. Post-coital semen samples (n = 804) were collected weekly from 46 Thoroughbred stallions during two successive breeding seasons (NSW, Australia; 22 weeks total). Semen samples were processed via single-layer colloidal centrifugation on-site to remove contaminants, seminal plasma and poor-quality sperm cells. The remaining sperm fraction was resuspended in Biggers, Whitten and Whittingham (BWW) medium for analyses, including sperm concentration, motility, and DNA damage. Fertility data (first cycle and per-cycle pregnancy rates) were collected from farms. Loggers were placed in the stables and paddocks of stallions to record ambient temperature and humidity, on 3-min cycles. Our results indicate that current management regimens, involving the stabling of stallions at night, with paddock access during the day, expose stallions to highest ambient climatic conditions. Polynomial distributed lag modelling identified 18 stallions whose fertility was adversely affected by heat stress, based on correlations between fertility and ambient climatic conditions (p ≤ 0.05). Of these 18 candidate stallions, six (13

AbstractThe sperm plasma membrane is of the utmost importance to the structural integrity and fertilising ability of the spermatozoon. Without a functional membrane, a spermatozoon cannot be expected to survive the arduous journey from testes to oviduct, particularly when its journey is disrupted by assisted reproductive technology (ART). In this study, we endeavoured to characterise a more complete bull sperm membrane-associated proteome through reanalysing publicly available RAW mass spectrometer data files from three previously published studies. Individually, these studies have utilised differing software platforms and inclusion criteria for their identification of membrane-associated proteins. The reanalysis of these datasets with standardised search criteria against up-to-date databases has identified 742 proteins. Amongst the proteins identified, 378 overlap with those reported in earlier studies, whereas we describe 364 previously unreported bovine sperm membrane-associated proteins linked to key functions, including zona pellucida binding. Next, the bovine proteome was mapped to human homologues (humanised), with a 96.8% conversion rate, allowing us to perform a more advanced functional analysis of the identified membrane-associated proteins, highlighting roles in energy metabolism, sperm motility, actin polymerisation, and protein ubiquitination. This deeper understanding of the bull sperm membrane-associated proteome will inform biomarker discovery for evaluating bull fertility and guide strategies to preserve sperm integrity during storage and ART procedures. Lay summaryIn this study, we combined and reanalysed publicly available data files from three previously published studies to generate an updated list of the proteins associated with the membrane of bull sperm cells. We identified 742 proteins in total, of which 364 were not reported previously in the original studies. Membrane proteins are important because they sit on the surface of the sperm and control how it recognises and communicates with the egg. Our functional analysis of these proteins revealed roles in sperm motility, energy production, and key fertilisation events, such as binding to the egg. This study highlights the value of depositing data in public repositories so that older datasets can be reanalysed as analytical tools improve. A greater understanding of sperm membrane-associated proteins will improve knowledge of bull fertility with important implications for breeding and livestock production industries.

Over the past half-century, the world has witnessed a dramatic decline in human fertility, which began in the 1960s and has continued unabated ever since [...]

Heavy use of antioxidants in equine in vitro fertilisation (IVF) media may suppress the reactive oxygen species (ROS) signals stallion sperm need for capacitation, which depends on high mitochondrial activity and a well-maintained endogenous redox homeostasis. A species-specific approach balancing oxidative protection with controlled ROS signalling could improve fertilisation efficiency and reduce prolonged incubation times.

CONTEXT:Intracytoplasmic sperm injection (ICSI) is the primary method for equine in vitro embryo production, yet outcomes may be influenced by sperm selection techniques. AIMS:This study compared four isolation methods: EquiPure™, Vetmotl™, Felix™, and Samson™, to assess effects on sperm quality and embryo development. METHODS:Semen was collected from four fertile stallions, processed and evaluated for sperm motility, viability, DNA integrity, and yield of high-quality cells relative to input. Isolated samples were compared to non-isolated (centrifuged) samples. Spermatozoa were used for ICSI with in vitro-matured oocytes, and cleavage and blastocyst rates were recorded. KEY RESULTS:Compared with unfractionated samples, sperm quality (motility and viability) was improved in samples recovered with VetMotl, Felix and Samson while DNA integrity was improved in Vetmotl-isolated samples. When concentration of recovered cells was considered, Samson, EquiPure, and Felix yielded significantly more motile, viable, and DNA-intact spermatozoa than Vetmotl, showing the lowest recovery. Samson achieved the greatest recovery of progressively motile cells. Isolation with all methods supported in vitro embryo production; cleavage (51.2-69.4%) and blastocyst rates (20.5-24.5%) did not differ significantly between methods. CONCLUSIONS:Several established and emerging methods for sperm isolation are effective in isolating high-quality spermatozoa suitable for use in embryo production by ICSI. VetMotl, Felix, and Samson performed comparably to conventional methods, supporting their clinical utility. IMPLICATIONS:Isolation methods vary in efficiency, labour intensiveness and sperm selection stringency, with potential implications for sperm storage, low dose insemination and IVF. Further studies incorporating multiple stallions and embryo transfer outcomes are needed to confirm reproductive relevance.

Oxidative stress plays a critical role in regulating sperm function, yet species-specific antioxidant mechanisms remain poorly understood. This study compared hydrogen peroxide (H2O2) tolerance in horse and human sperm and investigated the roles of catalase and glutathione peroxidase (GPx) in horses. A H2O2 dose-response assay (0-2000 µM) showed that horse sperm were significantly more resistant to oxidative damage, with an IC50 for progressive motility over 14-fold higher than that of human sperm (391.6 µM vs. 27.3 µM). Horse sperm also accumulated more intracellular H2O2 without loss of motility or viability. DNA damage assays (Halo and SCSA) revealed H2O2-induced fragmentation in human but not horse sperm. Enzyme inhibition experiments in horse sperm using 3-amino-1,2,4-triazole (catalase inhibitor) and (1S,3R)-RSL3 (GPx inhibitor) at 250 µM H2O2 showed that catalase inhibition severely impaired motility and increased intracellular H2O2 > 100-fold, while GPx inhibition had a milder effect (~5-fold increase). Immunocytochemistry localized catalase to the sperm head, particularly the post-acrosomal region, challenging the notion that sperm lack peroxisomes. The dependence of horse sperm on oxidative phosphorylation may drive the need for enhanced antioxidant defenses. These findings reveal species-specific oxidative stress adaptations and highlight catalase as a key antioxidant in equine sperm.

Oxidative stress, characterized by an imbalance between prooxidants and antioxidants in favor of the former, can lead to cellular damage due to the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In spermatozoa, oxidative stress plays a crucial role in signaling pathways associated with capacitation, a process essential for fertilization. However, the limited capacity of spermatozoa to buffer ROS makes them susceptible to oxidative stress. Measurement of oxidative stress in spermatozoa is important as it precedes or directly causes the loss of basic sperm functions-such as motility and the membrane changes required for zona binding and gamete fusion-associated with fertility. This chapter aims to describe protocols for assessing oxidative damage in spermatozoa, encompassing the generation of ROS in mitochondria to the manifestation of oxidative damage in DNA, proteins, and lipids. In addition to the detailed instructions provided within this chapter, we have sought to provide background information on the development and significance of the assays which we most commonly utilize in our laboratory.

(1) Background: The RoXstaTM system has been developed as a rapid, effective means of profiling different types of antioxidant activity. The purpose of this study was to examine its performance utilizing a diverse array of biological fluids including semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts. (2) Methods: The RoXstaTM system was used to assess the ability of different fluids to suppress free radical formation as well as scavenge a variety of toxic oxygen metabolites including free radicals and both hydrogen and organic peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested (10–14 mM vitamin C equivalent compared with 1–2 mM for blood serum or plasma), while urine was particularly effective in scavenging free radicals and preventing free radical formation (p < 0.001). The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: The RoXstaTM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen and organic peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage.

PURPOSE:Embryo-maternal signaling during the establishment of pregnancy in horses remains one of the biggest mysteries in large animal physiology. Early pregnancy loss represents a major source of economic loss to the breeding industry. This study aimed to investigate the systemic changes associated with early pregnancy by mapping the proteome of blood plasma at 14 days in pregnant and non-pregnant mares. EXPERIMENTAL DESIGN:Plasma proteomes were analysed in commercially bred pregnant (n = 17) and non-pregnant (n = 17) Thoroughbred mares at 14 days after ovulation, using high-resolution mass spectrometry. Day 14 histotroph and yolk sac fluid were also profiled and datasets were integrated through pathway analysis. RESULTS:We identified 229 total protein IDs, with 12 increased and 10 decreased significantly in pregnant versus non-pregnant plasma. To gain functional insight, these data were aligned with proteomes of 14-day pregnant mare uterine fluid (n = 4; 1358 IDs) and conceptus fluid (soluble proteins within the yolk sac fluid; n = 4; 1152 IDs), and further interrogated using gene ontology databases and pathway analysis. CONCLUSIONS AND CLINICAL RELEVANCE:These analyses identified consistent systemic changes in the mare's proteome that indicate a profound and specific immune response to early pregnancy, which appears to precede the systemic endocrine response to pregnancy. Integrated pathway analysis suggests that embryo-maternal interactions in early pregnancy may mimic elements of the virus-host interaction to modulate the maternal immune response. Transthyretin (TTR) and uteroglobin (SCGB1A1) were respectively down- and upregulated in plasma while also present in uterine fluid, and are proposed to be key proteins in early pregnancy establishment. These findings contribute significantly to our knowledge of early pregnancy in the mare and identify potential new avenues for developing clinical approaches to reduce early embryo loss.

Context Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances. Aims This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproductive success in equine breeding. Methods Traditional sperm isolation methods, including sperm washing and single-layer centrifugation (SLC), are compared with emerging techniques such as microfluidic-based technologies and electrophoretic separation to assess their ability to improve sperm quality while minimising DNA damage. Key results While conventional methods are widely used, they present limitations, such as reduced motility, cost, and potential DNA damage. Novel approaches, including the VetMotl™ and Samson™, replicate natural sperm selection to enhance motility while preserving DNA integrity, and the electrophoretic sperm isolation device, Felix™, separates sperm based on surface charge and motility, benefiting cryopreserved samples. These innovations offer promising improvements in ART outcomes, though challenges remain, including high costs and limited sperm yields. Conclusions Emerging sperm isolation techniques have the potential to improve ART success, but further research is required to optimise these methods and validate their efficacy in fertility trials. Implications Advancements in sperm isolation could modernise equine reproductive practices by improving sperm quality and fertility outcomes, though accessibility and practical applications require continued investigation.

The lipidomic secretions of embryos provide a unique opportunity to examine the cellular processes of the early conceptus. In this study we profiled lipids released by the early equine conceptus, using high-resolution mass spectrometry to detect individual lipid species. This study examined the lipidomic profile in embryo-conditioned media from in vivo-produced, 8-9 day-old equine embryos (n = 3) cultured in vitro for 36 h, analyzed over 3 timepoints. A total of 1,077 lipid IDs were recorded across all samples, containing predominantly glycerolipids. Seventy-nine of these were significantly altered in embryo conditioned-media versus media only control (p < 0.05, fold-change >2 or < 0.5). Fifty-five lipids were found to be released into the embryo-conditioned media, of which 54.5% were triacylglycerols and 23.6% were ceramides. The sterol lipid, cholesterol, was also identified and secreted in significant amounts as embryos developed. Further, 24 lipids were found to be depleted from the media during culture, of which 70.8% were diacylglycerols, 16.7% were triacylglycerols and 12.5% were ceramides. As lipid-free media contained consistently detectable lipid peaks, a further profile analysis of the various components of non-embryo-conditioned media consistently showed the presence of 137 lipids. Lipid peaks in non-embryo-conditioned media increased in response to incubation under mineral oil, and contained ceramides, diacylglycerols and triacylglycerols. These results emphasize the importance of a defined embryo culture medium and a need to identify the lipid requirements of the embryo precisely. This study sheds light on early embryo lipid metabolism and the transfer of lipids during in vitro culture.

(1) Background: The RoXstaTM system has been developed as a potential diagnostic tool to profile the antioxidant activity of complex biological fluids. (2) Methods: This methodology was used to assess the ability of different biofluids (semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts) to suppress free radical formation, as well as scavenge a variety of toxic oxygen metabolites including free radicals and peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested, while urine was particularly effective in scavenging free radicals and preventing free radical formation. The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: the RoXstaTM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen-, and organic- peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage.

CONTEXT:Little is known about the microbial composition of stallion semen.AIMS:To describe the microbiota detected in equine semen of healthy miniature pony stallions.METHODS:Semen specimens were collected using a Missouri artificial vagina at a single time point. PacBio (Pacific Biosciences) genomic DNA sequencing of the 16S rRNA gene was performed on these specimens, following which next-generation microbiome bioinformatics platform QIIME2 was used to process fastq files and analyse the amplicon data. The data were categorised into genus, family, class, order and phylum.KEY RESULTS:Firmicutes and Bacteroidetes phyla predominated (76%), followed by Proteobacteria (15%). Bacteroidales, Clostridiales and Cardiobacteriales predominated the microbial rank of order (86%). Class was mainly composed of Bacteroidia, Clostridia and Gammaproteobacteria (87%), while family was mainly composed of Porphyromonadaceae , Family_XI and Cardiobacteriaceae (62%). At the level of genus, 80% of the abundance was composed of seven genera, namely Porphyromonas, Suttonella, Peptoniphilus, Fastidiosipila, Ezakiella, Petrimonas and an unknown taxon.CONCLUSIONS:The findings indicate that specific microbiota may be characteristic of healthy miniature pony stallions' semen with some inter-individual variations observed.IMPLICATIONS:Larger equine studies involving fertile and infertile subjects could be informed by this study and could explore the relationship of the semen microbiome to male fertility.

Probiotic and prebiotic effects on equine semen and gastrointestinal microbiome composition and sperm quality are unknown. This study aimed to evaluate the effects of pre-, pro- or synbiotic supplementation on fecal and semen microbiome composition and sperm quality parameters of stallions. This Latin square crossover trial involved four miniature pony stallions receiving control diet only, or addition of a pro-, pre- or synbiotic formulation. Full-length 16S rRNA gene amplicon sequencing was used to measure diversity of semen and fecal microbiomes. Total sperm count, total motility, progressive motility, DNA integrity, lipid peroxidation and mitochondrial oxidative stress, biomarkers of sperm quality, were measured after each intervention. A general linear model was employed to analyse and compare microbiome diversity measures and sperm quality data across four time points. Shannon's diversity index (alpha-diversity), and evenness of semen and gastrointestinal microbiomes were significantly different (p<0.001). A trend was observed for prebiotic effects on the diversity indices of the GI microbiome (p= 0.07). No effects of treatments were observed on either semen microbiome or sperm quality. Pre-, pro- and synbiotic supplements showed no negative effect on sperm quality parameters observed. This proof of concept provides preliminary data to inform future studies exploring the relationship between microbiomes and fertility.

Graphical abstract Abstract In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process in order to generate the highest possible chance of oocyte fertilization following artificial insemination. Numerous methods have been developed to isolate functional spermatozoa for artificial insemination, yet only one method, single-layer centrifugation using commercial preparations like EquiPure, has been widely employed. In this study, we have introduced a novel approach for isolating spermatozoa and compared their quality to those isolated using EquiPure. The AI port system (Memphasys, Ltd. in Sydney, Australia) features a disposable cartridge with an inoculation chamber for depositing extended semen and a harvest chamber for extracting isolated spermatozoa. These chambers are separated by a 5 µm polyethylene terephthalate (PETE) membrane, allowing highly motile spermatozoa to migrate from the inoculation chamber to the harvest chamber over a 20-minute period. This migration effectively leaves behind seminal plasma and other cell types, such as leukocytes. Comparative analyses between spermatozoa isolated with the AI port and EquiPure demonstrated that, across all measured sperm parameters, including yield, vitality, motility, morphology, DNA fragmentation, and mitochondrial superoxide generation, the AI port-isolated cells exhibited comparable or superior performance, particularly in terms of DNA fragmentation. In summary, the AI port system demonstrates the potential to efficiently isolate high-quality spermatozoa, possibly offering a cost-effective and user-friendly alternative that may enhance the success rates of artificial insemination in breeding programs. Lay summary This study aimed to create a new method for refining stallion semen to increase the likelihood of a successful pregnancy through artificial insemination. While there are existing techniques for isolating high-quality sperm, the most common involves a complicated process using a centrifuge, which spins the semen to separate it. This research introduces a new approach called the AI port system that uses a disposable cartridge with two separate chambers for putting in semen and getting out isolated sperm. A membrane between the chambers acts like a filter, letting highly motile sperm swim across, leaving behind unwanted substances like bacteria and blood cells. Compared to the centrifugation method, the AI port system effectively produces sperm with comparable or better quality in various aspects, including vitality, movement, shape, DNA integrity, and energy production. In summary, the AI port system is an easy-to-use alternative with the potential to improve the success of artificial insemination in horse breeding programs.

Understanding the systemic biochemistry of early pregnancy in the mare is essential for developing new diagnostics and identifying causes for pregnancy loss. This study aimed to elucidate the dynamic lipidomic changes occurring during the initial stages of equine pregnancy, with a specific focus on days 7 and 14 post-ovulation. By analysing and comparing the plasma lipid profiles of pregnant and non-pregnant mares, the objective of this study was to identify potential biomarkers for pregnancy and gain insights into the biochemical adaptations essential for supporting maternal recognition of pregnancy and early embryonic development. Employing discovery lipidomics, we analysed plasma samples from pregnant and non-pregnant mares on days 7 and 14 post-conception using the SCIEX ZenoTOF 7600 system. This high-resolution mass spectrometry approach enabled us to comprehensively profile and compare the lipidomes across these critical early gestational timepoints. Our analysis revealed significant lipidomic alterations between pregnant and non-pregnant mares and between days 7 and 14 of pregnancy. Key findings include the upregulation of bile acids, sphingomyelins, phosphatidylinositols, and triglycerides in pregnant mares. These changes suggest enhanced lipid synthesis and mobilization, likely associated with the embryo's nutritional requirements and the establishment of embryo-maternal interactions. There were significant differences in lipid metabolism between pregnant and non-pregnant mares, with a notable increase in the sterol lipid BA 24:1;O5 in pregnant mares as early as day 7 of gestation, suggesting it as a sensitive biomarker for early pregnancy detection. Notably, the transition from day 7 to day 14 in pregnant mares is characterized by a shift towards lipids indicative of membrane biosynthesis, signalling activity, and preparation for implantation. The study demonstrates the profound lipidomic shifts that occur in early equine pregnancy, highlighting the critical role of lipid metabolism in supporting embryonic development. These findings provide valuable insights into the metabolic adaptations during these period and potential biomarkers for early pregnancy detection in mares.