Efficacy of glyoxal acid-free (GAF®) as a non-toxic fixative for fish gonads, gametes and larvae: widespread implications for laboratory studies, field sampling and applied aquaculture

Context: Traditional fixatives such as buffered formaldehyde (BF) and paraformaldehyde (PFA) are widely used but raise health concerns because of their toxicity. Their use is increasingly subject to regulatory restrictions, calling for the development of non-toxic alternatives. Glyoxal acid-free (GAF®) has emerged as a potential substitute. Aims: This study evaluated GAF® for preserving gonadal tissues, oocyte, sperm, and larvae of the European seabass (Dicentrarchus labrax). Methods: GAF formulations – standard and an optimized, marine-adapted formulation – were compared to conventional fixatives or fresh samples. Key results: The standard GAF® led to tissue shrinkage and poor gamete preservation, whereas the optimized version effectively preserved oocyte, sperm, gonadal tissue, and delicate larval structures. Although 10% formalin still outperformed GAF® for certain larval tissues, extended fixation in optimized GAF® improved results. Conclusions: Overall, optimized GAF® represents a promising non-toxic alternative for histological applications in marine fish, although further refinement is needed for certain tissues and for the use in the oocyte cytoplasmic clarification applied in aquaculture broodstock selection. Implications: Non-toxic fixatives are fundamental to ensure operator safety, and identifying solutions that effectively preserve samples is essential; therefore, studies such as this one have important implications for both field and laboratory settings.