For successful fertilisation to occur, spermatozoa need to successfully migrate through the female reproductive tract and penetrate the oocyte. Predictably, poor sperm motility has been associated with low rates of fertilisation in many mammalian species, including the ram. As such, motility is one of the most important parameters used for in vitro evaluation of ram sperm quality and function. This review aims to outline the mechanical and energetic processes which underpin sperm motility, describe changes in motility which occur as a result of differences in sperm structure and the surrounding microenvironment, and assess the effectiveness of the various methods used to assess sperm motility in rams. Methods of subjective motility estimation are convenient, inexpensive methods widely used in the livestock industries, however, the subjective nature of these methods can make them unreliable. Computer-assisted sperm analysis (CASA) technology accurately and objectively measures sperm motility via two-dimensional tracing of sperm head motion, making it a popular method for sperm quality assurance in domesticated animal production laboratories. Newly developed methods of motility assessment including flagellar tracing, three-dimensional sperm tracing, in vivo motility assessment, and molecular assays which quantify motility-associated biomarkers, enable analysis of a new range of sperm motion parameters with the potential to reveal new mechanistic insights and improve ram semen assessment. Experimental application of these technologies is required to fully understand their potential to improve semen quality assessment and prediction of reproductive success in ovine artificial breeding programs.

Reproductive success in mammals hinges on the ability of sperm to generate sufficient energy through cellular metabolism to perform the energy-intensive processes required for fertilisation, including motility, maturation, and oocyte interactions. It is now widely accepted that sperm exhibit metabolic flexibility, utilising a combination of glycolysis and oxidative phosphorylation (supported by the Krebs cycle and other complementary pathways) to meet their energy demands. However, the preferred pathway for energy production varies significantly among species, making it challenging to map species-specific metabolic strategies, particularly in species with high metabolic flexibility, like the ram. Additionally, differences in methodologies used to measure metabolism have led to biased interpretations of species' metabolic strategies, complicating the development of liquid storage methods aimed at preserving spermatozoa by manipulating energy generation based on species-specific requirements. This review examines sperm energy requirements, current methods for assessing metabolic capacity, and the current research on species-specific metabolism. Future research should focus on establishing a standardised approach for determining metabolic preferences to accurately map species-specific strategies, a critical step before developing effective liquid preservation methods. By identifying species-specific regulatory points, strategies can be designed to temporarily inhibit metabolic pathways, conserving resources and reducing the accumulation of metabolic by-products. Alternatively, supplementation with depleted metabolites can be guided by understanding areas of excessive consumption during prolonged metabolism. Applying this knowledge to develop tailored preservation techniques will help minimise sperm damage and improve survival during in vitro processing and liquid storage, ultimately enhancing the success of artificial breeding programs.

Context: Mass motility assessment is widely used to evaluate semen quality in the sheep breeding industry, yet the biological factors influencing motility duration remain poorly characterised. At ejaculation, spermatozoa are combined with seminal plasma (SP), which initially supports motility and function but may compromise long-term metabolic endurance. Aims: This study aimed to investigate the effects of SP on mass motility duration and pH regulation of epididymal ram spermatozoa. Methods: Epididymal sperm were incubated with whole SP, saline, or left undiluted to assess motility duration. Subsequently, sperm were incubated with varying SP concentrations and molecular weight-separated fractions (protein-free 3 kDa) and assessed for motility and pH over 24 h. Metabolomic and lipidomic profiling via targeted mass spectrometry characterised the biochemical composition of each SP fraction. Key results: SP exposure significantly reduced mass motility duration (195 ± 11 min) compared with undiluted sperm (1797 ± 185 min; P = 0.0123) and saline dilution (2667 ± 134 min; P < 0.0001). All SP-treated groups exhibited a progressive pH decline, consistent with increased glycolytic flux, whereas SP-naïve sperm showed stable or increasing pH, indicating a more regulated metabolic state and greater metabolic flexibility. The protein-free SP fraction was most detrimental to motility, which was likely to be the result of the depletion of key proteins, lipids, and metabolites associated with energy production. Conclusions: Prolonged exposure to SP impairs ram sperm motility longevity by promoting a glycolysis-biased metabolic state and reducing metabolic endurance. Implications: Understanding how seminal plasma composition influences sperm metabolic regulation may inform strategies to better preserve sperm motility during semen handling and storage.

Context: Motility longevity offers valuable insights into sperm function and metabolic resilience; however, the extent of inter-individual variation and its mechanistic underpinnings remain poorly defined. Aims: This study aimed to characterise variation in mass motility duration in rams and determine whether differences in motility longevity are associated with distinct metabolic profiles. Methods: Successive ejaculates (n = 314) were collected and assessed from 23 Merino rams. Seminal plasma from ejaculates exhibiting the shortest and longest motility duration was analysed immediately post-collection and at motility cessation using targeted mass spectrometry to characterise changes in metabolite abundance over time. Key results: Mass motility duration varied markedly between rams, ranging from 11 min to over 20 h. Although mass motility duration, ejaculate volume, and sperm concentration were influenced by sequential ejaculate collection, mass motility duration was not strongly correlated with ejaculate volume or sperm concentration, indicating that variation in motility longevity is not simply explained by these traits. Rather, distinct metabolic strategies appear to drive variation among phenotypes. Short-lasting ejaculates showed rapid depletion of fructose and marked lactate accumulation, consistent with heavy reliance on glycolysis and limited mitochondrial engagement. In contrast, long-lasting ejaculates maintained stable fructose levels while exhibiting increased hexose phosphate abundance and substantial pyruvate utilisation, suggesting more controlled glycolytic flux and sustained mitochondrial oxidation. Accumulation of α-ketoglutarate and 2-hydroxyglutarate was exclusive to short-lasting ejaculates, indicating mitochondrial bottlenecks and redox imbalance. Conclusions: These findings highlight metabolic regulation, rather than substrate availability alone, as a key determinant of motility longevity in ram spermatozoa. Implications: Understanding how long-lasting ejaculates maintain coordinated glycolytic and mitochondrial activity provides a framework for developing metabolically informed strategies to prolong sperm survival during processing and storage.