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Lipids were extracted from cryopreserved semen samples of Alpine (n = 7) and Spanish\u2013Creole (n = 4) mature bucks and subsequently analyzed by gas chromatography\u2013mass spectrometry (GC-MS), with 21 fatty acids identified within the two breeds. Eight of these fatty acids, namely 13:0, 16:0, 18:0, 24:0, 14:1, 18:1 (cis-9), 24:1, and 18:2 showed significant differences (p &lt; 0.05). The levels of 16:0, 18:0, 24:0, 18:1 (cis-9), 18:1, and 18:2 were higher in the Alpine breed, whereas the levels of 13:0, 14:1, and 24:1 were higher in the Spanish\u2013Creole breed (p &lt; 0.05). Of those, 16:0, 18:1 (cis-9), and 18:2 were both statistically and biologically significant (p &lt; 0.05, FC &gt; 2). Concentrations of the total fatty acids, total saturated fatty acids (Total-SFA), and total polyunsaturated fatty acids (Total-PUFA) were significantly higher in the Alpine breed, whereas the concentrations of the total cis-monounsaturated fatty acid (Total cis-MUFA) were significantly higher in the Spanish\u2013Creole breed (p &lt; 0.05). Network and pathway analyses revealed that 16:0, 18:1 (cis-9), and 18:2 contributed to the most central nodes of the lipidomic network, which may support membrane stability and cryotolerance. The lipidomic differences observed between breeds may be attributed to both genetic and environmental factors and may provide valuable tools for enhancing breeding strategies, artificial insemination programs, and sperm cryopreservation techniques.<\/jats:p>","DOI":"10.3390\/ani15131897","type":"journal-article","created":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T05:29:58Z","timestamp":1751002198000},"page":"1897","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Lipidomic Landscapes of Cryopreserved Sperm from Alpine and Spanish\u2013Creole Bucks"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8432-7347","authenticated-orcid":false,"given":"Mustafa","family":"Bodu","sequence":"first","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"},{"name":"Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Sel\u00e7uk University, Konya 42075, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mustafa","family":"Hitit","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Selamawit","family":"Woldesenbet","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammet Ra\u015fit","family":"U\u011fur","sequence":"additional","affiliation":[{"name":"IVF Michigan Fertility Centers, 37000 Woodward Ave #350, Bloomfield Hills, MI 48304, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-8516-2740","authenticated-orcid":false,"given":"Zeynep","family":"Erdo\u011fan","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olivia Chika","family":"Greenwood","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9368-7262","authenticated-orcid":false,"given":"Raheem Davian","family":"Murray","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andres Pech","family":"Cervantes","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erdo\u011fan","family":"Memili","sequence":"additional","affiliation":[{"name":"Cooperative Agricultural Research Center, College of Agriculture, Food, and Natural Resources, Prairie View A&M University, Prairie View, TX 77446, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"107643","DOI":"10.1016\/j.anireprosci.2024.107643","article-title":"Harnessing the value of fertility biomarkers in bull sperm for buck sperm","volume":"272","author":"Bodu","year":"2025","journal-title":"Anim. 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