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Summary: Background: In the United States, the carrying capacity of the land has been overwhelmed with the wild horse and burro populations. Identification of a long‐term contraception method is needed to control growing populations as current methods cannot quell these growing numbers. Sperm acrosome‐associated 3 protein (SPACA3) is a protein expressed in both male and female reproductive systems. SPACA3 may prove to be a viable contraception target in horses in immunosterilant research. Objectives: The objective of the current study was to evaluate and compare ovarian immunoexpression of SPACA3 between domesticated and free‐roaming mares. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations. Study design: Non‐randomised comparative study design. Methods: Routine immunohistochemistry was performed on ovarian sections from domesticated (n = 8) and free‐roaming mares (n = 8). At least three representative images of each follicle stage (primordial, primary, secondary, tertiary) from each ovary were digitally captured and analysed using Image J software. Expression of SPACA3 was quantified in cellular locations (granulosa or theca) specific to each follicle stage: primordial, primary, secondary, and tertiary. Results: Domesticated horses had higher SPACA3 expression compared to free‐roaming mares in granulosa cells of primordial (p = 0.0022), primary (p < 0.001), secondary (p = 0.025), and tertiary follicles (p = 0.0078). In theca cells, domesticated horses had higher SPACA3 expression in tertiary follicles (p = 0.022), but not secondary follicles (p = 0.089). The highest SPACA3 expression was observed in the granulosa cells of tertiary follicles (p = 0.022). Main limitations: Varying background colours of representative images taken of ovaries, leading to slight technical issues in Image J software. Conclusions: This is the first report quantifying SPACA3 immunoexpression during folliculogenesis and describing the spatial and temporal expression of SPACA3 in the equine ovary. This information may lead to the development of a SPACA3 immunosterilant to reduce free‐roaming horse and burro populations. [ABSTRACT FROM AUTHOR]