Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions

Simple Summary Stallion breeding soundness is indicated for infertility or to evaluate the potential to mate or to produce and ship doses of fresh, refrigerated, or frozen semen. This review describes the methods used to evaluate equine semen and their relation to semen production pathways and fertility. Clinical examination is useful for detecting genital tract pathologies, but also for predicting the expected spermatozoa production. Basic semen evaluation in the laboratory includes analysis of the volume, spermatozoa concentration, motility, and morphology. These factors can be used to evaluate the spermatozoa production and its quality, and predict the efficiency of natural mating and the potential fresh or refrigerated semen dose production. The post-thaw quality of some stallions is below standard, despite good fresh semen quality, requiring methods for predicting this outcome. To overcome this concern and the unresolved infertility cases, biotechnologies offer new opportunities. The potential toxicity of seminal plasma can be investigated, and damaged parts of the spermatozoa can be imaged with specific staining and fluorescent microscopy or flow cytometry. These data will deepen the understanding of stallion infertility and enhance basic semen evaluation. Abstract Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners’ concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation.