Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 19 1 2024 03 01 75 85 Gholamreza Habibi Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricul-ture Research, Education and Extension Organization, Karaj, Iran Asghar Afshari Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricul-ture Research, Education and Extension Organization, Karaj, Iran Amin Shahedi Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricul-ture Research, Education and Extension Organization, Karaj, Iran Mousa Hashemlo Department of Veterinary Biological Products Approval Group of Razi Vaccine and Serum Research, Agricultural Research, Education and Extension Organization, Karaj, Iran Ali Es-Haghi Department of Physico Chemistry, Razi Vaccine & Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran Mohamad Hosein Fallah Mehrabadi Department of Epidemiology, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran Saeid Fathi Department of Parasite Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization, Karaj, Iran 2023 05 13 2024 02 17 Background: The production of bovine theileriosis vaccine involves in vitro cultivation of Theileria annulata schizont-infected cell lines. Fetal bovine serum (FBS) is commonly used in animal cell culture, including the Theileria cell line. However, we aimed to reduce the amount of serum needed for cell culture by modifying the Stoker culture medium with supplements such as excretion factor and serum substitutes. Methods: To evaluate the effectiveness of these modifications, techniques such as cell counting, cell viability assays, and genomic analysis were employed in the Parasitic Vaccines Production Department of Razi institute of Iran, from 2020 to 2022.  Statistical analysis was used to compare the results of different experimental conditions.  Results: The three experimental media were as effective as the commonly used 10% Stoker medium in supporting the growth and viability of cells. Conclusion: The significant reduction in the required amount of serum and the remarkable cell growth achieved by using defined serum replacements for the production of cell culture media is a significant step towards the preparation of a proper cell culture medium for the production of bovine Theileriosis vaccine. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3949 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3949/1343