Protective Effects of Encapsulated Bacillus subtilis on Oxidative Stress, Spermatogenesis, and Fertility Outcomes in Experimental Cholestasis
Microencapsulation Strategy to Improve Male Fertility in Liver Disease
-
Mahla Ghanbari
,
Siavash Amiri
,
Mitra Motallebi
,
Merat Karimi
,
Esmat Aghadavood
,
Maryam Akhavan Taheri
,
Mohamad Reza Karbalaei Hashemian
,
Atefe Saber
,
Sahar Ahmadi
,
Hamed Mirzaei
,
Mohamad Esmaeil Shahabodin
Abstract
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.
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13. Machado AR, Silva PMP, Vicente AA, Souza-Soares LA, Pinheiro AC, Cerqueira MA. Alginate Particles for Encapsulation of Phenolic Extract from Spirulina sp. LEB-18: Physicochemical Characterization and Assessment of In Vitro Gastrointestinal Behavior. Polymers. 2022;14(21):4759. https://doi.org/10.3390/polym14214759
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15. Ghanbari M, Motallebi M, Karimi M, Aghadavood E, Akhavan Taheri M, Ahmadi Asouri S, et al. Alginate/Arabic gum-chitosan-encapsulated Bacillus subtilis enhances probiotic viability and alleviates liver injury and fibrosis in cholestatic rats. Naunyn Schmiedebergs Arch Pharmacol. 2025. https://doi.org/10.1007/s00210-025-04452-w.
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17. Cadamuro J, Mrazek C, Leichtle AB, Kipman U, Felder TK, Wiedemann H, et al. Influence of centrifugation conditions on the results of 77 routine clinical chemistry analytes using standard vacuum blood collection tubes and the new BD-Barricor tubes. Biochem Med (Zagreb). 2018;28(1):010704. https://doi.org/10.11613/bm.2018.010704.
18. Cao XW, Lin K, Li CY, Yuan CW. [A review of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition)]. Zhonghua Nan Ke Xue. 2011;17(12):1059-63.
19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. https://doi.org/10.1006/abio.1976.9999.
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21. Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005;38(12):1103-11. https://doi.org/10.1016/j.clinbiochem.2005.08.008.
22. Hu ML. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol. 1994;233:380-5. https://doi.org/10.1016/s0076-6879(94)33044-1.
23. PROMEGA GRS, Instructions for Use of Product G2930. Promega Madison, WI, USA.
24. Hajian H, Motallebi M, Akhavan Taheri M, Kheiripour N, Aghadavod E, Shahaboddin ME. The preventive effect of heat-killed Lactobacillus plantarum on male reproductive toxicity induced by cholestasis in rats. Food Chem Toxicol. 2024;186:114571. https://doi.org/10.1016/j.fct.2024.114571.
25. Liu X, Chen X, Wang C, Song J, Xu J, Gao Z, et al. Mechanisms of probiotic modulation of ovarian sex hormone production and metabolism: a review. Food Funct 2024;15(6):2860-78. https://doi.org/10.1039/D3FO04345B.
26. Zou S, Yang X, Li N, Wang H, Gui J, Li J. Association of probiotic ingestion with serum sex steroid hormones among pre- and postmenopausal women from the NHANES, 2013-2016. PLoS One. 2023;18(11):e0294436. https://doi.org/10.1371/journal.pone.0294436.
27. Wang L, Li A, Zhang X, Iqbal M, Aabdin ZU, Xu M, et al. Effect of Bacillus subtilis isolated from yaks on D-galactose-induced oxidative stress and hepatic damage in mice. Front Microbiol. 2025;Volume 16 - 2025. https://doi.org/10.3389/fmicb.2025.1550556.
28. Arias Padró MD, Caboni E, Salazar Morin KA, Meraz Mercado MA, Olalde-Portugal V. Effect of Bacillus subtilis on antioxidant enzyme activities in tomato grafting. PeerJ. 2021;9:e10984. https://doi.org/10.7717/peerj.10984.
29. Cömert M, Tekin IO, Acikgöz S, Ustündağ Y, Uçan BH, Acun Z, et al. Experimental bile-duct ligation resulted in accumulation of oxidized low-density lipoproteins in BALB/c mice liver. J Gastroenterol Hepatol. 2004;19(9):1052-7. https://doi.org/10.1111/j.1440-1746.2004.03400.x.
30. Hasani Fard AH, Mohseni Kouchesfehani H, Jalali H. Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats. Andrologia. 2020;52(9):e13660. https://doi.org/10.1111/and.13660.
31. Mavaddat H, Ale-Ebrahim M, Aghdam DS. Effects of Saponin on Testes Oxidative Stress, Apoptosis, and Steroid Hormone Synthesis Pathway in Bile Duct Ligation Model of Obstructive Cholestasis in male Wistar rats. J Nut Biochem. 2025:110020. https://doi.org/10.1016/j.jnutbio.2025.110020.
32. Wu Y, Wang B, Tang L, Zhou Y, Wang Q, Gong L, et al. Probiotic Bacillus Alleviates Oxidative Stress-Induced Liver Injury by Modulating Gut-Liver Axis in a Rat Model. Antioxidants (Basel). 2022;11(2). https://doi.org/10.3390/antiox11020291.
33. Li MW, Mruk DD, Lee WM, Cheng CY. Disruption of the blood-testis barrier integrity by bisphenol A in vitro: is this a suitable model for studying blood-testis barrier dynamics? Int J Biochem Cell Biol. 2009;41(11):2302-14. https://doi.org/10.1016/j.biocel.2009.05.016.
34. Dutta S, Sengupta P, Slama P, Roychoudhury S. Oxidative Stress, Testicular Inflammatory Pathways, and Male Reproduction. Int Mol Sci. 2021;22(18):10043.
35. Dowds BC. The oxidative stress response in Bacillus subtilis. FEMS Microbiol Lett. 1994;124(3):255-63. https://doi.org/10.1111/j.1574-6968.1994.tb07294.x.
2. Faddladdeen K. Effect of liver fibrotic changes on testicular histological structure: An updated review. Ann Clin Anal Med. 2019;10. https://doi.org/10.4328/ACAM.6102.
3. Liedtke C, Luedde T, Sauerbruch T, Scholten D, Streetz K, Tacke F, et al. Experimental liver fibrosis research: update on animal models, legal issues and translational aspects. Fibrogenesis Tissue Repair. 2013;6(1):19. https://doi.org/10.1186/1755-1536-6-19.
4. Ljubuncic P, Tanne Z, Bomzon A. Evidence of a systemic phenomenon for oxidative stress in cholestatic liver disease. Gut. 2000;47(5):710-6. https://doi.org/10.1136/gut.47.5.710.
5. Ommati MM, Farshad O, Niknahad H, Arabnezhad MR, Azarpira N, Mohammadi HR, et al. Cholestasis-associated reproductive toxicity in male and female rats: The fundamental role of mitochondrial impairment and oxidative stress. Toxicol Lett. 2019;316:60-72. https://doi.org/10.1016/j.toxlet.2019.09.009.
6. Poli G. Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med. 2000;21(3):49-98. https://doi.org/10.1016/s0098-2997(00)00004-2.
7. Perez MJ, Briz O. Bile-acid-induced cell injury and protection. World J Gastroenterol. 2009;15(14):1677-89. https://doi.org/10.3748/wjg.15.1677.
8. Azenabor A, Ekun A, Akinloye O. Impact of Inflammation on Male Reproductive Tract. J Reprod Infertil. 2015;16(3):123-30.
9. Tomaiuolo R, Veneruso I, Cariati F, D'Argenio V. Microbiota and Human Reproduction: The Case of Male Infertility. High Throughput. 2020;9(2). https://doi.org/10.3390/ht9020010.
10. Markowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017;9(9). https://doi.org/10.3390/nu9091021.
11. Wang X, Gao S, Yun S, Zhang M, Peng L, Li Y, Zhou Y. Microencapsulating Alginate-Based Polymers for Probiotics Delivery Systems and Their Application. Pharmaceuticals (Basel). 2022;15(5). https://doi.org/10.3390/ph15050644.
12. Jiménez-Villeda BE, Falfán-Cortés RN, Rangel-Vargas E, Santos-López EM, Gómez-Aldapa CA, Torres-Vitela MR, et al. Synbiotic Encapsulation: A Trend towards Increasing Viability and Probiotic Effect. J Food Process Preserv. 2023;2023(1):7057462. https://doi.org/10.1155/2023/7057462.
13. Machado AR, Silva PMP, Vicente AA, Souza-Soares LA, Pinheiro AC, Cerqueira MA. Alginate Particles for Encapsulation of Phenolic Extract from Spirulina sp. LEB-18: Physicochemical Characterization and Assessment of In Vitro Gastrointestinal Behavior. Polymers. 2022;14(21):4759. https://doi.org/10.3390/polym14214759
14. Pournaki SK, Aleman RS, Hasani-Azhdari M, Marcia J, Yadav A, Moncada M. Current Review: Alginate in the Food Applications. J. 2024;7(3):281-301. https://doi.org/10.3390/j7030016
15. Ghanbari M, Motallebi M, Karimi M, Aghadavood E, Akhavan Taheri M, Ahmadi Asouri S, et al. Alginate/Arabic gum-chitosan-encapsulated Bacillus subtilis enhances probiotic viability and alleviates liver injury and fibrosis in cholestatic rats. Naunyn Schmiedebergs Arch Pharmacol. 2025. https://doi.org/10.1007/s00210-025-04452-w.
16. Tang W, Xing Z, Li C, Wang J, Wang Y. Molecular mechanisms and in vitro antioxidant effects of Lactobacillus plantarum MA2. Food Chem. 2017;221:1642-9. https://doi.org/10.1016/j.foodchem.2016.10.124.
17. Cadamuro J, Mrazek C, Leichtle AB, Kipman U, Felder TK, Wiedemann H, et al. Influence of centrifugation conditions on the results of 77 routine clinical chemistry analytes using standard vacuum blood collection tubes and the new BD-Barricor tubes. Biochem Med (Zagreb). 2018;28(1):010704. https://doi.org/10.11613/bm.2018.010704.
18. Cao XW, Lin K, Li CY, Yuan CW. [A review of WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition)]. Zhonghua Nan Ke Xue. 2011;17(12):1059-63.
19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. https://doi.org/10.1006/abio.1976.9999.
20. Lenz AG, Costabel U, Shaltiel S, Levine RL. Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride. Anal Biochem. 1989;177(2):419-25. https://doi.org/10.1016/0003-2697(89)90077-8.
21. Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005;38(12):1103-11. https://doi.org/10.1016/j.clinbiochem.2005.08.008.
22. Hu ML. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol. 1994;233:380-5. https://doi.org/10.1016/s0076-6879(94)33044-1.
23. PROMEGA GRS, Instructions for Use of Product G2930. Promega Madison, WI, USA.
24. Hajian H, Motallebi M, Akhavan Taheri M, Kheiripour N, Aghadavod E, Shahaboddin ME. The preventive effect of heat-killed Lactobacillus plantarum on male reproductive toxicity induced by cholestasis in rats. Food Chem Toxicol. 2024;186:114571. https://doi.org/10.1016/j.fct.2024.114571.
25. Liu X, Chen X, Wang C, Song J, Xu J, Gao Z, et al. Mechanisms of probiotic modulation of ovarian sex hormone production and metabolism: a review. Food Funct 2024;15(6):2860-78. https://doi.org/10.1039/D3FO04345B.
26. Zou S, Yang X, Li N, Wang H, Gui J, Li J. Association of probiotic ingestion with serum sex steroid hormones among pre- and postmenopausal women from the NHANES, 2013-2016. PLoS One. 2023;18(11):e0294436. https://doi.org/10.1371/journal.pone.0294436.
27. Wang L, Li A, Zhang X, Iqbal M, Aabdin ZU, Xu M, et al. Effect of Bacillus subtilis isolated from yaks on D-galactose-induced oxidative stress and hepatic damage in mice. Front Microbiol. 2025;Volume 16 - 2025. https://doi.org/10.3389/fmicb.2025.1550556.
28. Arias Padró MD, Caboni E, Salazar Morin KA, Meraz Mercado MA, Olalde-Portugal V. Effect of Bacillus subtilis on antioxidant enzyme activities in tomato grafting. PeerJ. 2021;9:e10984. https://doi.org/10.7717/peerj.10984.
29. Cömert M, Tekin IO, Acikgöz S, Ustündağ Y, Uçan BH, Acun Z, et al. Experimental bile-duct ligation resulted in accumulation of oxidized low-density lipoproteins in BALB/c mice liver. J Gastroenterol Hepatol. 2004;19(9):1052-7. https://doi.org/10.1111/j.1440-1746.2004.03400.x.
30. Hasani Fard AH, Mohseni Kouchesfehani H, Jalali H. Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats. Andrologia. 2020;52(9):e13660. https://doi.org/10.1111/and.13660.
31. Mavaddat H, Ale-Ebrahim M, Aghdam DS. Effects of Saponin on Testes Oxidative Stress, Apoptosis, and Steroid Hormone Synthesis Pathway in Bile Duct Ligation Model of Obstructive Cholestasis in male Wistar rats. J Nut Biochem. 2025:110020. https://doi.org/10.1016/j.jnutbio.2025.110020.
32. Wu Y, Wang B, Tang L, Zhou Y, Wang Q, Gong L, et al. Probiotic Bacillus Alleviates Oxidative Stress-Induced Liver Injury by Modulating Gut-Liver Axis in a Rat Model. Antioxidants (Basel). 2022;11(2). https://doi.org/10.3390/antiox11020291.
33. Li MW, Mruk DD, Lee WM, Cheng CY. Disruption of the blood-testis barrier integrity by bisphenol A in vitro: is this a suitable model for studying blood-testis barrier dynamics? Int J Biochem Cell Biol. 2009;41(11):2302-14. https://doi.org/10.1016/j.biocel.2009.05.016.
34. Dutta S, Sengupta P, Slama P, Roychoudhury S. Oxidative Stress, Testicular Inflammatory Pathways, and Male Reproduction. Int Mol Sci. 2021;22(18):10043.
35. Dowds BC. The oxidative stress response in Bacillus subtilis. FEMS Microbiol Lett. 1994;124(3):255-63. https://doi.org/10.1111/j.1574-6968.1994.tb07294.x.
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| Issue | Vol 3 No 3 (2025) | |
| Section | Original Articles | |
| Keywords | ||
| Probiotics; Bacillus subtilis; Encapsulation; Cholestasis; Male infertility | ||
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How to Cite
1.
Ghanbari M, Amiri S, Motallebi M, Karimi M, Aghadavood E, Akhavan Taheri M, Karbalaei Hashemian MR, Saber A, Ahmadi S, Mirzaei H, Shahabodin ME. Protective Effects of Encapsulated Bacillus subtilis on Oxidative Stress, Spermatogenesis, and Fertility Outcomes in Experimental Cholestasis. ABI. 2025;3(3):180-188.
