Choosing the best washing condition could be crucial in metabolomic analyses of frozen-thawed brown bear sperm

The Cantabrian brown bear subpopulation is currently endangered, and a better understanding of its sperm metabolome may contribute to improvements in semen cryopreservation. Nevertheless, diluent residues present a problem for sample processing in metabolomic analysis. This study aims to optimize sperm washing protocols. Five thawed ejaculates from brown bears were used. Two centrifugal forces (1200 × g and 10000 × g) and two washing volumes (0.5 and 1 mL) ( Experiment 1 ), and different washing steps at 1200 × g ( Experiment 2 ) were tested, accompanied by a non-diluent contact group (Negative control). Sperm quality was assessed by motility and functionality parameters, including viability, apoptosis, mitochondrial functionality, and acrosome status. Metabolomic evaluation comprised lipidomic analysis and the determination of polar metabolites. In Experiment 1, centrifugation at 1200 × g with a washing volume of 0.5 mL resulted in the best preservation of semen quality. Nevertheless, metabolomic analyses were compromised by the presence of egg-yolk residues. To address this limitation, Experiment 2 was conducted, selecting this experimental group and performing consecutive washing step. Principal component analysis revealed that samples subjected to three washes at 1200 x g clustered closest to the negative control group, indicating effective removal of diluent residues without inducing sperm damage (only minor reduction in sperm quality). In conclusion, the ideal washing method for metabolomic analyses consists of three washing steps at 1200 x g for 6 minutes, using a 0.5 mL PBS dilution at 4 °C. Graphic scheme of the experimental design (created with BioRender.com). • A single washing step does not efficiently remove diluent residues in thawed sperm. • High centrifugal forces decrease semen quality in thawed brown bear samples. • Different washing steps negatively affect sperm functionality in brown bears. • The ideal washing protocol for thawed brown bear semen is three steps at 1,200 × g.