Czech J. Anim. Sci., 2025, 70(11):489-497 | DOI: 10.17221/103/2025-CJAS

Sperm quality improvement of cryopreserved boar semen through colloidal centrifugation gradientOriginal Paper

Francisco Sevilla ORCID...1,2, Ignacio Araya-Zúñiga ORCID...2, Patricia Cervantes-Acosta ORCID...3, Antonio Hernández-Beltrán ORCID...3, Manuel Barrientos ORCID...3, Belisario Domínguez ORCID...3, Anthony Valverde ORCID...2
1 Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica, Universidad Nacional, Universidad Estatal a Distancia, Alajuela, Costa Rica
2 Laboratory of Animal Reproduction, School of Agronomy, Research and Development Center for Sustainable Agriculture in the Humid Tropics, Costa Rica Institute of Technology, San Carlos Campus, Alajuela, Costa Rica
3 School of Veterinary Medicine and Animal Husbandry, Master’s in Animal Science Reproduction Biology, Biotechnology for Animal Health and Reproduction, Veracruz University, Veracruz, México

Boar semen cryopreservation is limited due to the lower thermal shock resistance of sperm cells. A colloidal centrifugation gradient is an enhancement to cryopreservation that could improve the frozen boar semen quality. Cryopreserved boar semen quality was evaluated using a commercial colloidal centrifugation gradient. A total of 15 ejaculates from 5 boars were evaluated across two treatments: a control without colloidal centrifugation and a treatment with commercial colloidal centrifugation. A manual freezing method used two freezing curves in liquid nitrogen. Sperm motility was assessed by Computer-Assisted Semen Analysis, as well as sperm capacitation-like membrane destabilisation at 30 min and 150 min after thawing using Merocyanine 540 (M540) for samples incubated at 37 °C. Spectrophotometry measured lipid peroxidation indirectly by the amount of malondialdehyde; reactive oxygen species production was also determined for sperm samples incubated for 30 minutes. The gradient centrifugation treatment improved ejaculate sperm motility and membrane destabilisation. The proportion of morphologically normal sperm was higher in the gradient than in the control. A total of 74% of spermatozoa retained normal morphology. Lipid peroxidation was lower in the colloidal centrifugation treatment. MDA was lower with gradient (16.4 ± 2.5 vs 22.3 ± 2.5 µmol/30 × 106 sperm; P < 0.05). Intracellular reactive oxygen species (ROS) and M540 positivity did not differ significantly between the treatments at either time point. The centrifugation process improved the quality parameters of frozen boar semen after thawing.

Keywords: animal science; cryopreservation; frozen-thawed sperm; oxidative stress; reproduction

Received: July 17, 2025; Accepted: November 3, 2025; Published: November 28, 2025  Show citation

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Sevilla F, Araya-Zúñiga I, Cervantes-Acosta P, Hernández-Beltrán A, Barrientos M, Domínguez B, Valverde A. Sperm quality improvement of cryopreserved boar semen through colloidal centrifugation gradient. Czech J. Anim. Sci. 2025;70(11):489-497. doi: 10.17221/103/2025-CJAS.
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