ZebraPace: An Open-Source Method for Cardiac-Rhythm Estimation in Untethered Zebrafish Larvae.

For the assessment of cardiac function, heartbeat represents one key parameter. Current methods of heartbeat measurements in the zebrafish larvae usually require larval immobilization, fluorescent transgenic strains and a confocal microscope, costly commercial software for analysis, or strong programming skills if the software is open-source. Here, we present a simple yet powerful method of heartbeat analysis using untethered, unlabeled zebrafish larva using ImageJ (open-source software), which does not require programming skills. We named it as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. ZebraPace works directly with AVI videos and requires no image processing steps. ZebraPace uses pixel intensity change in a grayscale video to count the number of beats. We have validated the ZebraPace method by pharmacological alterations of the heartbeat in zebrafish larvae of 48 and 72 hpf stages. We have also determined beat-to-beat interval, which relates to rhythmicity of heartbeat. The results obtained by using ZebraPace corroborates well with the heartbeat values previously reported for similarly aged larvae as determined by using specialized software. We believe that the ZebraPace method is simple, cost-effective, and easy to grasp as it involves fewer steps. It not only reduces the manual workload but also eliminates sample preparation time and researcher subjectivity.