Standardization of the Dye Concentration and Volume for Sperm Viability Assay in a Ladybird Beetle, <i>Cheilomenes sexmaculata</i> (Coccinellidae: Coleoptera)

ABSTRACT Sperm viability is a key determinant of male reproductive fitness and is widely used in studies assessing environmental, physiological, or behavioral impacts on reproduction. However, assessing viability depends on the effectiveness of staining techniques that accurately differentiate live and dead sperm cells. This study aimed to standardize the concentration and volume of sperm viability dye (SYBR‐14 and PI) for optimal visualization of sperm viability in a commonly studied ladybird beetle species, Cheilomenes sexmaculata . Different concentrations of SYBR‐14 (1:10, 1:20, 1:30, 1:40, and 1:50 ratio) (SYBR‐14: DMSO) were tested with a range of volumes (2 μL, 4 μL, 6 μL, 8 μL, and 10 μL per 10 μL sample (volume of sperm containing sample)) and PI (2 μL per 10 μL sample) for the fluorescence. The results demonstrated that 1:50 diluted 3 μL SYBR‐14 and 2 μL PI per 10 μL sample yielded the best contrast with live sperm fluorescing green and dead sperm fluorescing red. This standardized protocol will improve reproducibility and accuracy in future studies evaluating male reproductive performance in ladybird beetle, C. sexmaculata .