Aims: Improving fertilisation efficiency in equine in vitro fertilisation (IVF) depends heavily on effective sperm capacitation. We explored how capacitating agents individually influence stallion sperm function, aiming to identify the most effective doses for IVF regimens. Methods: Semen from six normospermic Shetland and miniature crossbred pony stallions were collected via artificial vagina. The samples were immediately diluted with semen extender and processed via single-layer colloidal centrifugation. After centrifugation sperm pellets were resuspended in Biggers, Whitten, and Whittingham (BWW) medium containing different doses of each of the following elements: heparin, bovine serum albumin (BSA), D-penicillamine, epinephrine, hypotaurine, and sodium metabisulfite (SMB); and incubated for 22 h (37˚C, 5% CO2). Sperm motility was assessed using CASA. Viability and acrosome status were evaluated with LIVE/DEAD™ Far Red and FITC-PNA staining, respectively. Capacitation-associated tyrosine phosphorylation was detected via immunofluorescence using anti-phosphotyrosine antibodies and fluorescent secondary labelling, followed by flowcytometric evaluation. Results: Increasing concentrations of epinephrine and D-penicillamine significantly improved sperm viability (P≤0.05). Treatment with SMB increased the percentage of tyrosine phosphorylated spermatozoa at 20, 100, and 200 μM compared to the control (20 μM 28.06 ± 3.52, 100 μM 32.44 ± 6.15, and 200 μM 29.27 ± 3.01 vs Control μM 17.21 ± 2.22; respectively, P≤0.05). Hypotaurine and heparin did not affect sperm motility or capacitation. Notably, while BSA enhanced sperm viability, it also caused a significant decline in total and progressive motility. Conclusion: These results suggest that epinephrine, D-penicillamine, and SMB can improve specific aspects of stallion sperm fertilising capacity. Further research will be essential to optimise a capacitation protocol that can reliably support successful IVF outcomes in horses.