Effect of Different Thawing Methods on Frozen Semen Characteristics and DNA Damage of Indonesian Simmental Bull

The thawing method is a crucial factor affecting frozen semen quality.This study evaluated the effect of different thawing methods on the post-thaw quality of frozen Simmental semen. The study was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, and the Integrated Research Laboratory, Faculty of Medicine, Universitas Gadjah Mada. This study used frozen semen straws from a superior bull with same production date, produced by the Center for Artificial Insemination (BBIB) Singosari. A total of ninety straws were divided into three groups with different thawing methods such as; 28°C for 30 seconds (T1), 28°C for 45 seconds (T2), and 37°C for 15 seconds (T3). Motility was evaluated by observing spermatozoa movement. Viability and abnormalities were analyzed using eosin-nigrosin staining. Plasma membrane integrity was assessed using the hypoosmotic swelling test (HOS-test), and DNA damage was evaluated using the Halomax® kit. The results showed that the thawing method significantly affected motility, abnormalities, and plasma membrane integrity (p<0.05). The average motility in T1, T2, and T3 was 44.03±3.47, 42.13±2.99, and 42.30±2.56, respectively. Abnormalities were 10.30±2.16, 12.25±2.85, and 12.77±3.14, respectively. Therefore, plasma membrane integrity was 75.70±3.23, 73.98±4.30, and 70.40±3.87, respectively. However, the thawing method did not significantly affect viability and DNA damage (p>0.05). It can be concluded that, the thawing method affects the post-thaw quality of Simmental spermatozoa, specifically motility, abnormalities, and plasma membrane integrity. Among the methods tested, 28°C tap water for 30 seconds was the best thawing method under field conditions. Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).