Aquatic Advancements: Refining and Validating a Zebrafish Survival Blood Collection Technique

The zebrafish (Danio rerio) has become a cornerstone model organism for studying vertebrate development, human diseases, drug discovery, and toxicology. A key challenge in leveraging this model lies in the need for repeatable, nonlethal blood sampling, essential for longitudinal studies. Due to the aquatic environment, lack of easily accessible vasculature, small size, and blood volume of this species, blood sampling of zebrafish presents a unique set of biologic, technical, and logistical challenges. Despite advancements, standardized survival blood collection protocols for zebrafish remain underdeveloped. We refined and validated a survival blood collection methodology using the dorsal aorta as an accessible vascular site. We used anesthetized fish, precise glass pipette creation for microscale blood draws, and the adoption of safe capillary collection techniques, enabling the ability to perform blood collection weekly for up to 4 weeks. Both male and female zebrafish were evaluated to account for potential sex-based differences, enhancing reproducibility and translational relevance. Hemoglobin (Hgb) levels were monitored as a biomarker of anemia, alongside behavioral and clinical assessments. Histopathological analyses showed no significant differences in tissue response between 1 and up to 4 blood draws. Hgb values decreased proportionately to increasing blood collection volumes, while behavioral and clinical assessments revealed no observable detriment even at higher blood volumes. This study presents a validated and standardized application of previously described survival blood collection techniques, demonstrating the feasibility of repeated sampling over 4 weeks while incorporating behavioral and histopathologic outcome measures relevant to clinical tolerance and longitudinal study design, thus continually broadening zebrafish utility in preclinical research.