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Heparin is a glycosaminoglycan polymer that is commonly used as an anticoagulant. Heparin also induces in vitro capacitation in spermatozoa, although its molecular mechanism is elusive. This study investigated the effect of heparin on in vitro capacitation and spermatozoal RNA (spRNA) population in goats. Goat spermatozoa were treated with 20 μM heparin for 0-6 h and evaluated for motility, capacitation, acrosome reaction, and spRNA population by RNA sequencing (RNA-seq). It was observed that heparin enhanced sperm motility up to 6 h of incubation (p < 0.05). Heparin also induced capacitation and acrosome reaction within 4 h. RNA-seq identified 1254 differentially expressed genes (DEGs) between heparin-treated and control spermatozoa. Most DEGs (1251 nos.) were upregulated and included 1090 protein-coding genes. A few genes (PRND, ITPR1, LLCFC1, and CHRM2) showed >5-fold increased expression in heparin-treated spermatozoa compared to the control. The upregulated genes were found to be involved in cAMP-PKA, PI3-Akt, calcium, MAPK signaling, and oxidative stress pathways. DCFDA staining confirmed the increased oxidative stress in heparin-treated spermatozoa compared to the control (p < 0.05). In conclusion, the results of the present study suggest that heparin enhances sperm motility and induces capacitation by upregulation of the spRNA population and oxidative stress pathway., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)