The heart is the most important muscular organ of the cardiovascular system, which pumps blood and circulates, supplying oxygen and nutrients to peripheral tissues. Zebrafish have been widely explored in cardiotoxicity research. For example, the zebrafish embryo has been used as a human heart model due to its body transparency, surviving several days without circulation, and facilitating mutant identification to recapitulate human diseases. On the other hand, adult zebrafish can exhibit the amazing regenerative heart muscle capacity, while adult mammalian hearts lack this potential. This review paper offers a brief description of the major methodologies used to detect zebrafish cardiac rhythm at both embryonic and adult stages. The dynamic pixel change method was mostly performed for the embryonic stage. Other techniques, such as kymography, laser confocal microscopy, artificial intelligence, and electrocardiography (ECG) have also been applied to study heartbeat in zebrafish embryos. Nevertheless, ECG is widely used for heartbeat detection in adult zebrafish since ECG waveforms’ similarity between zebrafish and humans is prominent. High-frequency ultrasound imaging (echocardiography) and modern electronic sensor tag also have been proposed. Despite the fact that each method has its benefits and limitations, it is proved that zebrafish have become a promising animal model for human cardiovascular disease, drug pharmaceutical, and toxicological research. Using those tools, we conclude that zebrafish behaviors as an excellent small animal model to perform real-time monitoring for the developmental heart process with transparent body appearance, to conduct the in vivo cardiovascular performance and gene function assays, as well as to perform high-throughput/high content drug screening.

Cardiovascular disease (CVD) is the number one cause of death worldwide. This condition resulted in huge research on CVD increasing the need for animal models suitable for in vivo research. Daphnia and zebrafish are good animal models for cardiovascular research due to their relative body transparency and easy culture property. Several methods have been developed to conduct cardiac performance measurement in Daphnia and zebrafish. However, most of the methods are only able to obtain heartbeat rate. The other important cardiac endpoints like stroke volume, ejection fraction, fraction shortening, cardiac output, and heartbeat regularity must use other programs for measurement. To overcome this limitation, in this study, we successfully developed a one-stop ImageJ-based method using kymograph macros language that is able to obtain multiple cardiac performance endpoints simultaneously for the first time. To validate its utility, we incubated Daphnia magna at different ambient temperatures and exposed zebrafish with astemizole to detect the corresponding cardiac performance alterations. In summary, the kymograph method reported in this study provides a new, easy to use, and inexpensive one-stop method obtaining multiple cardiac performance endpoints with high accuracy and convenience.

As a nicotinoid neurotoxic insecticide, imidacloprid (IMI) works by disrupting nerve transmission via nicotinic acetylcholine receptor (nAChR). Although IMI is specifically targeting insects, nontarget animals such as the freshwater shrimp, Neocaridina denticulata, could also be affected, thus causing adverse effects on the aquatic environment. To investigate IMI toxicity on nontarget organisms like N. denticulata, their physiology (locomotor activity, heartbeat, and gill ventilation) and biochemical factors (oxidative stress, energy metabolism) after IMI exposure were examined. IMI exposure at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 ppm) to shrimp after 24, 48, 72 h led to dramatic reduction of locomotor activity even at low concentrations. Meanwhile, IMI exposure after 92 h caused reduced heartbeat and gill ventilation at high concentrations. Biochemical assays were performed to investigate oxidative stress and energy metabolism. Interestingly, locomotion immobilization and cardiac activity were rescued after acetylcholine administration. Through molecular docking, IMI demonstrated high binding affinity to nAChR. Thus, locomotor activity and heartbeat in shrimp after IMI exposure may be caused by nAChR blockade and not alterations caused by oxidative stress and energy metabolism. To summarize, N. denticulata serves as an excellent and sensitive aquatic invertebrate model to conduct pesticide toxicity assays that encompass physiologic and biochemical examinations.

Previous methods to measure protozoan numbers mostly rely on manual counting, which suffers from high variation and poor efficiency. Although advanced counting devices are available, the specialized and usually expensive machinery precludes their prevalent utilization in the regular laboratory routine. In this study, we established the ImageJ-based workflow to quantify ciliate numbers in a high-throughput manner. We conducted Tetrahymena number measurement using five different methods: particle analyzer method (PAM), find maxima method (FMM), trainable WEKA segmentation method (TWS), watershed segmentation method (WSM) and StarDist method (SDM), and compared their results with the data obtained from the manual counting. Among the five methods tested, all of them could yield decent results, but the deep-learning-based SDM displayed the best performance for Tetrahymena cell counting. The optimized methods reported in this paper provide scientists with a convenient tool to perform cell counting for Tetrahymena ecotoxicity assessment.

Tail coiling is a reflection response in fish embryos that can be used as a model for neurotoxic analysis. The previous method to analyze fish tail coiling is largely based on third-party software. In this study, we aim to develop a simple and cost-effective method called TCMacro by using ImageJ macro to reduce the operational complexity. The basic principle of the current method is based on the dynamic change of pixel intensity in the region of interest (ROI). When the fish tail is moving, the average intensity is increasing. In time when the fish freeze, the peak of mean intensity is maintaining at a relatively low level. By using the optimized macro settings and excel VBA scripts, all the tail coiling measurement processes can be archived with few operation steps with high precision. Three major endpoints of tail coiling counts, tail coiling duration and tail coiling intervals can be obtained in batch. To validate this established method, we tested the potential neurotoxic activity of Tricaine (methanesulfonate, MS-222) and psychoactive compound of caffeine. Zebrafish embryos after Tricaine exposure displayed significantly less tail coiling activity in a dose-dependent manner, and were comparable to manual counting through the Wilcoxon test and Pearson correlation double validation. Zebrafish embryos after caffeine exposure displayed significantly high tail coiling activity. In conclusion, the TCMacro method presented in this study provides a simple and robust method that is able to measure the relative tail coiling activities in zebrafish embryos in a high-throughput manner.