Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm
[EN] The Cantabrian brown bear subpopulation is currently endangered, and a better understanding of its sperm metabolome may contribute to improvements in semen cryopreservation. Nevertheless, diluent residues present a problem for sample processing in metabolomic analysis. This study aims to optimi...
| Autores: | , , , , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2026 |
| País: | España |
| Institución: | Universidad de León |
| Repositorio: | BULERIA. Repositorio Institucional de la Universidad de León |
| OAI Identifier: | oai:buleria.unileon.es:10612/27559 |
| Acceso en línea: | https://hdl.handle.net/10612/27559 |
| Access Level: | acceso abierto |
| Palabra clave: | Veterinaria Brown bear Metabolomic Semen Sperm quality Extender Washing protocol 3104.11 Reproducción |
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Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear spermPalacín Martínez, CristinaMontes Garrido, RafaelÁlvarez García, MercedesNeila Montero, MartaRamírez González, DavidDíez Zavala, VictoriaSoriano Úbeda, Cristina de las MercedesAnel López, LuisBarragán, SantiagoManrique Revuelta, PatriciaPaz Cabello, Paulino deAnel Rodríguez, LuisFernández Riesco, MartaVeterinariaBrown bearMetabolomicSemenSperm qualityExtenderWashing protocol3104.11 Reproducción[EN] The Cantabrian brown bear subpopulation is currently endangered, and a better understanding of its sperm metabolome may contribute to improvements in semen cryopreservation. Nevertheless, diluent residues present a problem for sample processing in metabolomic analysis. This study aims to optimize sperm washing protocols. Five thawed ejaculates from brown bears were used. Two centrifugal forces (1200 × g and 10,000 × g) and two washing volumes (0.5 and 1 mL) (Experiment 1), and different washing steps at 1200 × g (Experiment 2) were tested, accompanied by a non-diluent contact group (Negative control). Sperm quality was assessed by motility and functionality parameters, including viability, apoptosis, mitochondrial functionality, and acrosome status. Metabolomic evaluation comprised lipidomic analysis and the determination of polar metabolites. In Experiment 1, centrifugation at 1200 × g with a washing volume of 0.5 mL resulted in the best preservation of semen quality. Nevertheless, metabolomic analyses were compromised by the presence of egg-yolk residues. To address this limitation, Experiment 2 was conducted, selecting this experimental group and performing consecutive washing step. Principal component analysis revealed that samples subjected to three washes at 1200 × g clustered closest to the negative control group, indicating effective removal of diluent residues without inducing sperm damage (only minor reduction in sperm quality). In conclusion, the ideal washing method for metabolomic analyses consists of three washing steps at 1200 × g for 6 min, using a 0.5 mL PBS dilution at 4 °C.SIThis work is part of the project TED2021-130774B-I00, supported by the MCIN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU”/PRTR. Moreover, it is partially financed by Cantur S.A. and the University of León.ElsevierMedicina y Cirugia AnimalFacultad de Veterinaria2026info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://hdl.handle.net/10612/27559reponame:BULERIA. Repositorio Institucional de la Universidad de Leóninstname:Universidad de LeónIngléshttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:buleria.unileon.es:10612/275592026-06-24T12:43:27Z |
| dc.title.none.fl_str_mv |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| title |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| spellingShingle |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm Palacín Martínez, Cristina Veterinaria Brown bear Metabolomic Semen Sperm quality Extender Washing protocol 3104.11 Reproducción |
| title_short |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| title_full |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| title_fullStr |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| title_full_unstemmed |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| title_sort |
Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm |
| dc.creator.none.fl_str_mv |
Palacín Martínez, Cristina Montes Garrido, Rafael Álvarez García, Mercedes Neila Montero, Marta Ramírez González, David Díez Zavala, Victoria Soriano Úbeda, Cristina de las Mercedes Anel López, Luis Barragán, Santiago Manrique Revuelta, Patricia Paz Cabello, Paulino de Anel Rodríguez, Luis Fernández Riesco, Marta |
| author |
Palacín Martínez, Cristina |
| author_facet |
Palacín Martínez, Cristina Montes Garrido, Rafael Álvarez García, Mercedes Neila Montero, Marta Ramírez González, David Díez Zavala, Victoria Soriano Úbeda, Cristina de las Mercedes Anel López, Luis Barragán, Santiago Manrique Revuelta, Patricia Paz Cabello, Paulino de Anel Rodríguez, Luis Fernández Riesco, Marta |
| author_role |
author |
| author2 |
Montes Garrido, Rafael Álvarez García, Mercedes Neila Montero, Marta Ramírez González, David Díez Zavala, Victoria Soriano Úbeda, Cristina de las Mercedes Anel López, Luis Barragán, Santiago Manrique Revuelta, Patricia Paz Cabello, Paulino de Anel Rodríguez, Luis Fernández Riesco, Marta |
| author2_role |
author author author author author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Medicina y Cirugia Animal Facultad de Veterinaria |
| dc.subject.none.fl_str_mv |
Veterinaria Brown bear Metabolomic Semen Sperm quality Extender Washing protocol 3104.11 Reproducción |
| topic |
Veterinaria Brown bear Metabolomic Semen Sperm quality Extender Washing protocol 3104.11 Reproducción |
| description |
[EN] The Cantabrian brown bear subpopulation is currently endangered, and a better understanding of its sperm metabolome may contribute to improvements in semen cryopreservation. Nevertheless, diluent residues present a problem for sample processing in metabolomic analysis. This study aims to optimize sperm washing protocols. Five thawed ejaculates from brown bears were used. Two centrifugal forces (1200 × g and 10,000 × g) and two washing volumes (0.5 and 1 mL) (Experiment 1), and different washing steps at 1200 × g (Experiment 2) were tested, accompanied by a non-diluent contact group (Negative control). Sperm quality was assessed by motility and functionality parameters, including viability, apoptosis, mitochondrial functionality, and acrosome status. Metabolomic evaluation comprised lipidomic analysis and the determination of polar metabolites. In Experiment 1, centrifugation at 1200 × g with a washing volume of 0.5 mL resulted in the best preservation of semen quality. Nevertheless, metabolomic analyses were compromised by the presence of egg-yolk residues. To address this limitation, Experiment 2 was conducted, selecting this experimental group and performing consecutive washing step. Principal component analysis revealed that samples subjected to three washes at 1200 × g clustered closest to the negative control group, indicating effective removal of diluent residues without inducing sperm damage (only minor reduction in sperm quality). In conclusion, the ideal washing method for metabolomic analyses consists of three washing steps at 1200 × g for 6 min, using a 0.5 mL PBS dilution at 4 °C. |
| publishDate |
2026 |
| dc.date.none.fl_str_mv |
2026 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
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article |
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publishedVersion |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/10612/27559 |
| url |
https://hdl.handle.net/10612/27559 |
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Inglés |
| language_invalid_str_mv |
Inglés |
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http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
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http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| eu_rights_str_mv |
openAccess |
| dc.publisher.none.fl_str_mv |
Elsevier |
| publisher.none.fl_str_mv |
Elsevier |
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reponame:BULERIA. Repositorio Institucional de la Universidad de León instname:Universidad de León |
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Universidad de León |
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BULERIA. Repositorio Institucional de la Universidad de León |
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BULERIA. Repositorio Institucional de la Universidad de León |
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15,812429 |