Post-maceration storage temperature affects sperm quality and fertilization in sex-reversed rainbow trout

Optimizing sperm quality is essential in aquaculture, especially for producing all-female Oncorhynchus mykiss populations using sex-reversed males. Because sex-reversed males lack sperm ducts, semen is collected by gonadal maceration, a step that may expose spermatozoa to thermal and mechanical stress. We evaluated how post-maceration storage temperature (2, 4, 12 °C for 24 h) is associated with sperm performance in a within-male design, using an immediate post-extraction control (T0, 0 h) as a descriptive baseline for comparison. Sperm kinematics were quantified by computer-assisted sperm analysis, and cellular readouts were assessed by flow cytometry for viability (% SYTOX-negative), mitochondrial membrane potential (ΔΨm/% JC-1 red), superoxide anion production (DHE/% DHE-high), and DNA fragmentation (% TUNEL-positive). After 24 h, the warmer condition (12 °C) was associated with lower total and progressive motility, lower mitochondrial membrane potential and viability, and higher superoxide levels, relative to cold conditions (2-4 °C). Fertilization was analyzed as binomial data (k/n); proportions were lower at 12 °C, but should be interpreted with caution given the small number of eggs assessed per replicate. Correlations between cellular biomarkers and motility/fertilization were generally weak, although a moderate negative association was observed between mitochondrial status and superoxide anion production. Overall, these results suggest that sperm from sex-reversed males is sensitive to post-maceration handling temperature, supporting the practical approach of maintaining tissues, tools and homogenates at ~ 0-4 °C throughout the collection and storage workflow.