RT Journal Article T1 Variation of existence and location of aquaporin 3 in relation to cryoresistance of ram spermatozoa A1 Pequeño, Belén A1 Castaño, Cristina A1 Alvarez-Rodriguez, Manuel A1 Bóveda, Paula A1 Millán de la Blanca, María Gema A1 Toledano-Díaz, Adolfo A1 Galarza, Diego Andrés A1 Rodriguez-Martinez, Heriberto A1 Martínez Madrid, Carmen Belén A1 Santiago-Moreno, Julián A2 Ortega, M. Sofia AB Introduction and objective: Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa.Methods: Thirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE (n = 13) or PFE (n = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219).Results and discussion: Although AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawed samples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater (P < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance.Conclusion: A greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes. PB Frontiers Media YR 2023 FD 2023-03-28 LK https://hdl.handle.net/20.500.14352/94180 UL https://hdl.handle.net/20.500.14352/94180 LA eng NO Pequeño B, Castaño C, Alvarez-Rodriguez M, Bóveda P, Millán de la Blanca MG, Toledano-Díaz A, Galarza DA, Rodriguez-Martinez H, Martínez-Madrid B, Santiago-Moreno J. Variation of existence and location of aquaporin 3 in relation to cryoresistance of ram spermatozoa. Front Vet Sci. 2023 Mar 28;10:1167832 NO Belén Pequeño recibió una beca predoctoral financiada por la AEI (PRE2018-085637).Author contributionsBP performed the experiments, analyzed generated data, and wrote the first draft of the manuscript. MA-R, HR-M, and BM-M performed study design, data analysis, and reviewed manuscript. CC, PB, MM, AT-D, and DG contributed to the investigation and methodology. JS-M designed the experiment, data analysis, and corrected the manuscript. All authors contributed to the article and approved the submitted version. NO Agencia Estatal de Investigación (AEI) DS Docta Complutense RD 11 abr 2025