The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca²⁺ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca²⁺ to form the mPTP. However, chelation of endogenous Ca²⁺ prevented mPTP formation, indicating a role for intracellular Ca²⁺ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca²⁺ stores to form the mPTP in response to low Ca²⁺ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca²⁺, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca²⁺ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

A deficiency in NAD⁺ has previously been linked with increased occurrences of congenital abnormalities and embryonic death in humans and mice. Early embryonic death is a major factor involved in pregnancy loss in mares, and very little is known regarding the NAD⁺ requirements for optimum reproductive function in horses. The aim of this study was to determine the effect of supplementing the diet of mares with nicotinic acid (NA) on the composition of NAD⁺ metabolites in the blood and follicular fluid. Vehicle alone or NA (3 g per os) were administered to seven mares over a minimum of 3 consecutive days during the follicular phase of the oestrous cycle. Blood samples were collected immediately prior to supplemental feeding and follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound-guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was 4-fold greater than that in the corresponding plasma and 10-fold greater than that in the follicular fluid of vehicle-fed mares. The concentrations of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA-supplemented mares than in the corresponding plasma. The results show that NA supplementation increased the bioavailability of NAD⁺ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.

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.

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% of the overall population) exhibited repeated positive relationships between ambient climatic conditions and sperm DNA damage, and repeated negative relationships between fertility and sperm DNA damage ( r ≥ 0.50; p ≤ 0.05). These results suggest that heat-induced subfertility was most likely mediated via oxidative damage to the sperm DNA, and that current management strategies should be assessed to prevent exposure of stallions to maximal climatic conditions.