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Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 \u00b0C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 \u00b5M Y-27632 significantly increased plasma membrane integrity (p &lt; 0.05), and acrosome integrity (p &lt; 0.05) and sperm motility (p &lt; 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p &lt; 0.01), increased total antioxidant capacity (T-AOC) (p &lt; 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p &lt; 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p &lt; 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 \u03bcM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.<\/jats:p>","DOI":"10.3390\/ani14101441","type":"journal-article","created":{"date-parts":[[2024,5,13]],"date-time":"2024-05-13T03:34:01Z","timestamp":1715571241000},"page":"1441","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-2646-878X","authenticated-orcid":false,"given":"Yongjie","family":"Xu","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-8669-6006","authenticated-orcid":false,"given":"Shixin","family":"Sun","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3829-4628","authenticated-orcid":false,"given":"Yu","family":"Fu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-8732-9129","authenticated-orcid":false,"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1704-059X","authenticated-orcid":false,"given":"Chunhuan","family":"Ren","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"},{"name":"Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0127-0275","authenticated-orcid":false,"given":"Yinghui","family":"Ling","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"},{"name":"Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4292-4709","authenticated-orcid":false,"given":"Zijun","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"},{"name":"Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7598-6682","authenticated-orcid":false,"given":"Hongguo","family":"Cao","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China"},{"name":"Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Anhui Agricultural University, Hefei 230036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chen, Z., He, W., Leung, T.C.N., and Chung, H.Y. 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