Work

Influence of extender type, freezing temperature, and sperm concentration on the cryosurvival of Santa Inês ram spermatozoa

Year: 2026

Type: article

Abstract: This study evaluated the effects of three extenders (Tris, HEPES, and skim milk), three freezing temperatures (-79, -90, and − 120 °C), and three final sperm concentrations (600, 800, and 1000 × 10⁶ spermatozoa/mL) on the post-thaw quality of Santa Inês ram semen. Semen was collected weekly from five rams during six consecutive weeks, pooled, and subjected to a 3 × 3 × 3 factorial arrangement. Samples were cryopreserved using a programmable freezing system (TK3000 ® ) and stored in liquid nitrogen. After thawing, sperm quality was assessed by computer-assisted sperm analysis (CASA) and sperm morphology evaluation. Data were analyzed using mixed models, considering extender, freezing temperature, sperm concentration, and their interactions as fixed effects and collection week as a random effect. Tris consistently resulted in greater ( P < 0.05) total motility, progressive motility, and percentage of rapid spermatozoa compared with HEPES and skim milk extenders. Semen frozen at 600 × 10⁶ spermatozoa/mL showed superior post-thaw motility characteristics relative to higher sperm concentrations. Freezing temperature affected sperm kinetics, particularly in skim milk and HEPES extenders, whereas Tris maintained stable performance across the evaluated temperatures. Total morphological abnormalities were minimally influenced by the treatments. Overall, under the experimental conditions evaluated, the combination of Tris extender, a freezing temperature of -79 °C, and a sperm concentration of 600 × 10⁶ spermatozoa/mL provided the most favorable post-thaw sperm quality among the treatments tested. However, progressive motility remained relatively low, indicating that further optimization and fertility validation are required before this combination can be recommended for practical application.

Source: Tropical Animal Health and Production

DOI: 10.1007/s11250-026-05435-6