Determining Reference Intervals of Serum Biochemical Parameters in Juvenile Hybrid Snakehead Channa argus & C. maculata in Mesocosm

Hybrid snakehead (Channa argus × Channa maculata) is a major cultured freshwater fish in China, but standardized health monitoring using serum biochemistry is limited by the lack of species-specific reference intervals. This study established reference intervals for 20 serum biochemical parameters in hybrid snakehead reared under 27 °C for 90 days. The body weights of the sampled fish ranged from 50 g to 160 g and were exempted from diseases by health check. All parameters were measured using an automated analyzer with commercial reagent kits. Most parameters exhibited non-normal, right-skewed distributions, and only total protein (TP) was normally distributed. Smoothed bootstrap resampling and kernel density estimation were applied to extract the main peak distribution and reduce bias from outliers and long tails. Species-specific reference intervals were established based on the main peak data, providing more reliable physiological baselines than conventional percentiles. Correlation analysis revealed coordinated changes among liver function, nutrient metabolism, tissue damage, and digestive enzymes. These results provide a standardized tool for health assessment, subclinical disease diagnosis, and comparative analysis in juvenile hybrid snakehead maintained at an optimal temperature in indoor mesocosm systems.