Tehran University of Medical Sciences Acta Biochimica Iranica 0001-5261 3 3 2025 09 22 180 188 Mahla Ghanbari Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran. Siavash Amiri Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran Mitra Motallebi Infectious Disease Research Center, Kashan University of Medical Sciences, Kashan, Iran. Merat Karimi Institute of Nanoscience and Nanotechnology, University of Kashan, Kashan, Iran Esmat Aghadavood Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran Maryam Akhavan Taheri Anatomical Sciences Research Center, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran. Mohamad Reza Karbalaei Hashemian Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran. Atefe Saber Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran Sahar Ahmadi Department of Clinical Biochemistry, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran Hamed Mirzaei Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran Mohamad Esmaeil Shahabodin 2Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran 2025 10 02 2025 11 02 Objective: Probiotics such as Bacillus subtilis exhibit antioxidant and anti-inflammatory activities. However, their viability may be reduced in the gastrointestinal tract (GIT) due to acidity and enzymatic degradation. Encapsulation can improve probiotic survival and bioactivity. Probiotics have also been implicated in mitigating oxidative stress–induced reproductive toxicity. This study evaluated the effects of encapsulated B. subtilis on oxidative balance, sperm quality, and testicular histology in an experimental model of liver cholestasis. Methods: Forty-eight male Wistar rats were randomly assigned to four groups: control, probiotic, free capsule, and encapsulated probiotic. Encapsulated B. subtilis (3 × 109 CFU/day) was administered orally for one week prior to bile duct ligation (BDL) and for three weeks afterward. At the end of the experiment, rats were euthanized, and blood and testes were collected for biochemical, hormonal, and histological analyses. Results: BDL markedly reduced sperm concentration and viability while increasing morphological abnormalities. Encapsulated B. subtilis significantly improved sperm parameters compared to free probiotics. Testicular oxidative stress, evidenced by increased protein carbonyls and total oxidant status with decreased reduced glutathione, was attenuated by encapsulated probiotics. Histological analysis revealed disrupted testicular architecture and decreased Johnson’s scores after BDL, whereas encapsulated B. subtilis restored seminiferous tubule integrity and spermatogenesis. Conclusion: Encapsulated B. subtilis enhanced probiotic efficacy, improving sperm quality, antioxidant status, and testicular structure in cholestatic rats. These findings suggest a protective role for encapsulated probiotics in male infertility associated with oxidative stress and liver disease. https://abi.tums.ac.ir/index.php/abi/article/view/163 https://abi.tums.ac.ir/index.php/abi/article/download/163/119