Refining freezing techniques to enhance post-thaw sperm functionality in bovine semen

Laura Murillo; Francisco Sevilla; Ignacio  Araya-Zúñiga; Miguel A Silvestre; Anthony Valverde

Laura Murillo Costa Rica Institute of Technology https://orcid.org/0000-0002-8628-4651
Francisco Sevilla Costa Rica Institute of Technology
Ignacio Araya-Zúñiga Costa Rica Institute of Technology
Miguel A Silvestre University of Valencia
Anthony Valverde Costa Rica Institute of Technology https://orcid.org/0000-0002-3191-6965

Keywords: spermatozoa, semen analysis, animal reproduction, cryopreservation, beef cattle

Abstract

Animal breeding plays a pivotal role in enhancing livestock production and adaptability to various challenges, including climate change and disease. In Latin America, genetic improvement of the Brahman breed is critical for increasing meat production and optimizing farming practices, thereby contributing to sustainable and efficient livestock development. This study evaluates two protocols for bovine semen cryopreservation by analyzing post-thaw sperm quality using Computer-Assisted Semen Analysis (CASA) technology. Six bulls, with an average age of 48.6 ± 11.5 months, were used. Two ejaculates were collected from each bull, and semen was diluted with three extenders: Andromed^®, BioXcell^®, and OptiXcell^®. The samples were equilibrated for either four or six hours and frozen using static/manual or controlled programable methods. Results showed significant differences (p<0.05) in sperm motility and kinematics based on the extender used. The highest sperm motility was obtained with OptiXcell^® (31.61 ± 0.61%). Semen diluted with BioXcell^® exhibited a more linear and progressive kinematic pattern, whereas Andromed^® resulted in the lowest motility and kinematic values. Bull age had a significant effect (p<0.05) on the percentage of fast and medium sperm. Bulls over 48 months showed higher progressive motility, while bulls under 48 months had higher curvilinear velocity (VCL = 80.15 ± 0.43 µm·s^-1). Cooling time did not affect motility variables (p>0.05), but significant differences (p<0.05) were observed in progressive motility variables. No differences were found in total motility rate between freezing methods, but significant differences (p < 0.05) were noted in sperm kinematic variables. Livestock production has significantly enhanced with the more efficient use of genetically superior bulls for AI. These cryopreservation-induced damages can compromise sperm viability, reducing fertility and productivity. The findings suggest that male age, extender type, cooling time, and freezing method significantly influence post-thaw sperm quality in the Brahman breed, with implications for optimizing cryopreservation protocols.

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