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The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)\/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.<\/jats:p>","DOI":"10.1093\/molehr\/gaad031","type":"journal-article","created":{"date-parts":[[2023,9,1]],"date-time":"2023-09-01T19:55:39Z","timestamp":1693598139000},"source":"Crossref","is-referenced-by-count":5,"title":["Treatment with cholesterol just after thawing maintains the fertility of bull sperm"],"prefix":"10.1093","volume":"29","author":[{"given":"Md Mazharul","family":"Islam","sequence":"first","affiliation":[{"name":"Laboratory of Reproductive Endocrinology, Department of Bioresource Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan"},{"name":"Department of Animal Breeding and Genetics, Bangabandhu Sheikh Mujibur Rahman Agricultural University , Gazipur, Bangladesh"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3897-9739","authenticated-orcid":false,"given":"Takashi","family":"Umehara","sequence":"additional","affiliation":[{"name":"Laboratory of Reproductive Endocrinology, Department of Bioresource Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan"},{"name":"Graduate School of Integrated Sciences for Life, Hiroshima University , Higashi-Hiroshima, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natsumi","family":"Tsujita","sequence":"additional","affiliation":[{"name":"Graduate School of Integrated Sciences for Life, Hiroshima University , Higashi-Hiroshima, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masanori","family":"Koyago","sequence":"additional","affiliation":[{"name":"Graduate School of Integrated Sciences for Life, Hiroshima University , Higashi-Hiroshima, Hiroshima, Japan"},{"name":"Livestock Improvement Association of Japan Inc. , Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6500-2088","authenticated-orcid":false,"given":"Masayuki","family":"Shimada","sequence":"additional","affiliation":[{"name":"Laboratory of Reproductive Endocrinology, Department of Bioresource Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan"},{"name":"Graduate School of Integrated Sciences for Life, Hiroshima University , Higashi-Hiroshima, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,9,1]]},"reference":[{"key":"2023091505021417400_gaad031-B1","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1006\/abbi.2000.1880","article-title":"Mammalian sperm acrosome: formation, contents, and function","volume":"379","author":"Abou-Haila","year":"2000","journal-title":"Arch Biochem Biophys"},{"key":"2023091505021417400_gaad031-B2","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1530\/REP-19-0414","article-title":"Sperm enter glands of preovulatory bovine endometrial explants and initiate inflammation","volume":"159","author":"Akthar","year":"2020","journal-title":"Reproduction"},{"key":"2023091505021417400_gaad031-B3","doi-asserted-by":"crossref","first-page":"3555","DOI":"10.3390\/ijms22073555","article-title":"Molecular determinants of seminal plasma on sperm biology and fertility","volume":"22","author":"\u00c1lvarez-Rodr\u00edguez","year":"2021","journal-title":"Int J Mol Sci"},{"key":"2023091505021417400_gaad031-B4","doi-asserted-by":"crossref","first-page":"18797","DOI":"10.1016\/S0021-9258(17)30583-5","article-title":"Mitochondrial contact sites. 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