Abstract：Mycoplasma synoviae (MS) is a significant pathogen in poultry, its infection frequently causes symptoms such as arthritis, synovitis, and eggshell apex abnormalities. Infection in laying hens (Gallus gallus domesticus) leads to a decreased egg production rate, which seriously affects the economic benefits of the poultry industry. Follicular granulosa cells are crucial in the egg-laying process, primarily through their role in hormone secretion, which is essential for maintaining normal follicular development and ovulation. This study aimed to establish a stable in vitro model of MS-infected chicken follicular granulosa cells and to investigate the regulatory effects of MS on the proliferation and apoptosis of chicken follicular granulosa cells. The optimal multiplicity of infection (MOI) and infection duration for MS were determined through colony-forming unit (CFU) counting, CCK-8 assays, and observation of cytopathic effects (CPE) in cells. Immunofluorescence was employed to observe the localization of MS in infected cells, while changeed in progesterone secretion post-MS infection were assessed using ELISA. The results indicated that when MS was cultured for 32 h, infection at a multiplicity of infection (MOI) of 1 000 for 12 h facilitated the successful adhesion of MS to the cell surface, resulting in a significant reduction in progesterone secretion by chicken follicular granulosa cells. Furthermore, qPCR analysis showed an upregulation of apoptosis-related genes, including cysteine-aspartic protease 3 (Caspase-3), tumor protein p53 (p53) and Bcl-2-associated X protein (Bax). These results suggested that MS could infect chicken follicular granulosa cells and affect follicular function by promoting apoptosis and inhibiting progesterone secretion. This study provides a reference for further elucidating the pathogenic mechanisms associated with MS.
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