Title : Effect of pre freezing and thawing on the sperm quality of climbing perch (Anabas testudineus) sperm post cryopreservation
Abstract:
Cryopreservation of climbing perch sperm Anabas testudineus is developed by several methods. However, the quality of post-cryopreservation remains suboptimal and requires improvement through optimization of pre-freezing and thawing methods. Therefore, this study aimed to determine the best pre-freezing and thawing method during cryopreservation of climbing perch sperm. The experiment was conducted from March to April 2025 at the Fish Hatchery and Breeding Laboratory, Faculty of Marine Affairs and Fisheries, Syiah Kuala University. The method used was a non-factorial Completely Randomized Design, conducted in two stages of experiment. The first stage included testing of several gradual pre-freezing temperature, namely P1: 4°C >> 0°C >> -79°C >> -196°C (pre-freezing time 15 minutes), P2: 4°C >> 0°C >> -196°C (pre-freezing time 10 minutes), P3: 4°C >> 79°C, >> -196°C (pre-freezing time 10 minutes), and P4: 4°C >> -196°C (pre-freezing time 5 minutes). The second stage was testing of several levels of temperature and thawing time, including 25°C, 30°C, and 35°C, with immersion times of 5, 10, and 15 minutes, respectively. The result of the first stage showed that pre-freezing temperature significantly affected the motility, viability, sperm abnormalities, and fertility of climbing perch eggs (P<0.05), without a substantial effect on life span (P>0.05). The best post-cryopreservation sperm quality was produced in P1: 4°C >> 0°C >> -79°C >> -196°C (pre-freezing time 15 minutes) with motility of 78.50 ± 1.70%, viability 77.50 ± 1.91%, abnormalities 1.50 ± 0.57%, fertilization 62.50 ± 8.18%, and life span of 9.82 ± 1.58 minutes. In the second experiment, thawing method significantly affected motility, viability, abnormalities, fertility, and life expectancy of sperm after freezing (P<0.05). The best quality was found in treatment T8, which included thawing temperature of 35°C with a 10-minute soaking, leading to motility 80.25%, viability 81.75%, abnormalities 3.25%, fertilization 62.25%, and a duration of lifespan of 10.11 minutes. The DNA laddering analysis using gel electrophoresis method showed intact bands without fragments, showing no damage across all samples. This suggested that sperm quality improved through optimization of pre-freezing and thawing. In line with the analysis, the recommended pre-freezing was 4°C >> 0°C>> -79°C, and >> -196°C, while the best thawing was 35°C for 10 minutes.
