Nota de investigación: Efecto del tipo de muestra, la concentración de espermatozoides y las condiciones de...

Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation and oxidative stress in avian semen. However, inconsistencies in sample fraction, sperm concentration, and storage conditions complicate comparisons across studies. This study systematically evaluated how these factors influence MDA in rooster semen. In Experiment 1, pooled semen was diluted to 100, 150, 200, and 250 × 10⁶ spz/mL and fractionated into whole semen, seminal plasma, and sperm pellet before MDA quantification. In Experiment 2, the three fractions were stored at 4°C or -20°C and analyzed on Days 1, 3, and 7 (Day 0 baseline). In Experiment 1, both sperm concentration and sample fraction affected MDA (P < 0.05), with the highest values in sperm pellets, moderate in whole semen, and the lowest in seminal plasma. In Experiment 2, whole semen exhibited time-dependent increases at both temperatures, whereas seminal plasma and sperm pellets were comparatively stable. These findings indicate that sample origin and sperm density materially influence MDA readouts, and that storage particularly impacts whole semen. We recommend standardizing semen fraction and sperm concentration, and reporting storage conditions, to improve reproducibility of oxidative stress assessments in poultry reproduction.