Light cardiogram, a simple technique to determine heart rate in adult zebrafish, Danio rerio (2023)

part 246,

august 2020

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Current techniques for determining heart rate in adult zebrafish require experience and are often invasive, technically demanding and not easily transferable to other laboratories for routine testing. Here we present a simple, non-invasive and inexpensive lightcardiogram technique to assess heart rate and heart rate in adult zebrafish. Brightfield microscope in combination with a high resolution camera and ImageJ (an open source software) were used as central acquisition and processing platforms, respectively. The heart was visualized and located ventrally by juxtaposing an isosceles triangle between the operculae as a reference to analyze the pixel intensity fluctuations generated by each cardiac cycle to derive heart rate and rate. Compared to transparent embryos, the cardiograms generated inverse oscillations of the light signal, with contraction and relaxation of the heart (ventricle) corresponding to decrease and increase in pixel intensity, respectively. Heart rates (♂ 122.58 ± 2.15 and ♀ 121.37 ± 2.63 beats/min) and mean dominant frequency (♂ 2.04 ± 0.035 and ♀ 2.05 ± 0.048 Hz) between genders did not were significant (P>.05) different at 28°C. However, theDThe amplitudes between men (0.26 ± 0.03) and women (0.45 ± 0.05) were significantly different (P<0.05) indicates gender-specific diastolic cardiac output. Overall, the technique can be used to measure heart rates, but it can also be easily adapted to record relative cardiac output and compare differences between physiological states (eg, gender). Furthermore, the approach can be automated and applicable to other fish species, giving researchers the flexibility to measure these and other critical heart health parameters with relative ease.


from zebravis (denmark rerio) has emerged as an excellent vertebrate model for cardiovascular research. Despite having only one atrium and one ventricle (i.e. anatomically distinct), the electrophysiology of the zebrafish heart is remarkably conserved (Liu et al., 2016) with many key functions similar to those of the human heart (Nemtsas et al. ., 2010; Zhang et al., 2018). For example, spontaneous heartbeats are comparable to those of humans (Stainier, 2001).

Optically transparent zebrafish embryos are used to study cardiac development (Bakkers, 2011; Brown et al., 2016), physiology (Hoage et al., 2012; Yalcin et al., 2017) and associated human congenital diseases (Asnani and Peterson, 2014). ; Giardoglou and Beis, 2019). Typically, brightfield video digital motion analysis is used to determine cardiac functions such as heart rate and rate (Hoage et al., 2012; Gieren et al., 2020). As a result, researchers can now record and process thousands of videos simultaneously to analyze fluctuations in pixel intensity generated by each cardiac cycle of zebrafish embryos (Martin et al., 2019; Gieren et al., 2020). However, embryonic zebrafish transparency is gradually lost with age and effective tools for cardiac phenotyping are lacking for later stages of development, particularly in juvenile and adult animals (Zhang et al., 2018). An exception to this is a transgenic zebrafish strain that expresses red fluorescence in vessel walls (Littleton et al., 2013), transparent adult zebrafish (White et al., 2008), and the use of Eulerian video magnification. (Lauridsen et al., 2019). ). However, these are limited in terms of payload, relatively expensive and require specialist knowledge.

In mammalian models, cardiac function is usually assessed by echocardiography (Locatelli et al., 2011; Abduch et al., 2014). However, due to the small size of juvenile and adult zebrafish hearts (~0.25 and 1 mm in diameter, respectively), the resolution of classical low-frequency ultrasound is not satisfactory (Hoage et al., 2012; Wang et al., 2017). Recently, high-frequency echocardiography (HFE) allowed the study of heart rate in adult zebrafish (≥20 mm in length) (Liu et al., 2016; Wang et al., 2017; Zhang et al., 2018; Evangelisti et al., 2017; al., 2020) with acceptable image quality only for larger and older fish (6-9 months) (Wang et al., 2017). Furthermore, the HFE image quality of female zebrafish was impaired compared to males due to female attraction (Wang et al., 2017).

A simplified electrocardiogram (ECG) has also been used to study heart rate in zebrafish adults (Milan et al., 2006; Yu et al., 2010; Lin et al., 2018) and larvae (Dhillon et al., 2013 ) with an ECG profile fundamentally similar to humans (Milan et al., 2006; Liu et al., 2016). However, HFE and ECG techniques require expertise (Lin et al., 2018) and are often invasive. For example, ECG requires the implantation of an array of microelectrodes in direct contact with the epicardium (Cao et al., 2014; Liu et al., 2016; Lin et al., 2018), using an in vitro recording of the explanted heart (Yu et al., 2010; Tsai et al., 2011; Zhang et al., 2018) or the placement of specialized electrodes (Marchant and Farrell, 2019), all of which require specialized and often complicated micromanipulation. The ECG can also be affected by various sources of noise, such as: B. Power cord artifacts, electrode contact noise and muscle movements (Liu et al., 2016). ECG and HFE also require a complex and expensive software-hardware combination that is unusual, well-integrated, and easily transferable to other laboratories (Liu et al., 2016; Zhang et al., 2018; Martin et al., 2019).

Until now, brightfield imaging to study heart rate in zebrafish has been limited to optically transparent embryos and larvae. However, based on visual (ventral) observations, we hypothesize that the heart may emit fluctuating pixel intensities on the ventral surface of the fish skin that are synchronized with its beats. Therefore, this study aimed to develop a simple light imaging method to assess heart rate and heart rate in adult zebrafish.

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All animal testing has been approved by the University of Tasmania Animal Ethics Committee (A12787). Adult zebrafish were maintained in a continuous flow system with a light:dark photoperiod of 12:12 hours at 28°C (7 fish per 30 liters) and fed twice a day. Feeding was interrupted six hours before the experiments.

Determining the optimal anesthesia

A preliminary test was carried out to determine the optimal duration and concentration of the AQUI-S® anesthetic. Adult (N= 10/treatment; CT = 35.5 ± 1.5 mm; eight months).

A low dose of AQUI-S® maintains a stable heart rate

Adequate sedation to immobilize the zebrafish is required to record cardiovascular data (Liu et al., 2016; Lin et al., 2018). Prior to the LCG assessment, we tested AQUI-S® as an alternative sedative to tricaine (MS-222) due to the latter's negative chronotropic effects (Huang et al., 2010; Liu et al., 2016). There were progressive cuts (P> 0.05) in HR from 1 to 6 min of exposure to 6 ppm AQUI-S® (Table 1). However, at 3 ppm there was no significant reduction in HR or HF compared to

(Video) Fishing for Insights from Single-Lead and Multi-Lead ECG of Live Adult Zebrafish


We developed a semi-automated, simple and non-invasive approach for CF and FDof adult zebrafish with brightfield images that can be easily adopted by any laboratory. This study offers the opportunity to comparatively examine cardiovascular function across all life stages of zebrafish and can be easily adapted to other fish species and for automation.


This work was supported byAustralian Research Council(THE BOOKLP140100428).

Declaration of competitive interest

The authors declare no conflicts of interest.


Comments on earlier drafts of the manuscript by Nicholas Perkins (IMAS, University of Tasmania) and two anonymous reviewers significantly improved the manuscript.


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    What is the normal heart rate of a zebra fish? ›

    In zebrafish, the normal embryonic heart rate is much closer to that of humans, at 120–180 beats per minute32.

    What is the cardiac output of zebrafish? ›

    Key findings: Cardiac output (CO) in PHZ-treated zebrafish was significantly higher than that in control zebrafish (151 ± 67 vs. 84 ± 37 μl/min, P = 0.004), whereas ejection fraction (EF) was lower (36.3 ± 10.9 vs.

    Do zebrafish have a heart? ›

    The zebrafish adult heart has one atrium and one ventricle; it is smaller and simpler than the mammalian heart but the histological and structural composition is very similar to that of other vertebrates.

    How many times does a zebrafish heart beat per minute? ›

    Similarly, after onset of heartbeat in zebrafish, heart rate increased and plateaued at around 210 bpm (28 °C, 72 hpf; Fig. 4b). Zebrafish heart rate did not exhibit a day-night dependent fluctuation during the period of analysis (22–62 hpf). Heart rates of medaka and zebrafish during embryonic development.

    How many times does a zebra heart beat per minute? ›

    Evaluation of ECGs from 19 zebras revealed sinus rhythm with a predominantly negative QRS complex and a mean +/- SD heart rate of 67 +/- 10 beats/min.

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    The zebrafish as a model organism

    Unlike humans, zebrafish can regenerate new cardiac tissue after an injury such as a heart attack, which makes them a great model to study the cellular and molecular mechanisms involved in cardiac regeneration.

    What does a zebrafish heart have in common with a human heart? ›

    The Zebrafish adult heart is not the same as the human heart as it has a simpler structure, but it is similar enough to replicate cardiovascular function. It only has a single atrium and ventricle (which contains two kinds of cardiac muscle), but these work in an equivalent way to those of a human heart.

    Why might cardiologists find it useful to study zebrafish? ›

    Zebrafish embryos are transparent, making it easy to spot abnormalities. Particularly helpful for cardiology research: mutant embryos lacking active circulation are capable of surviving up to 5 days post fertilization. And zebrafish hearts are simpler than mammals': theirs have just two chambers, while ours have four.

    What is unique about zebrafish? ›

    Because of its fully sequenced genome, easy genetic manipulation, high fecundity, external fertilization and rapid development, and nearly transparent embryo, zebrafish are a unique model animal for biomedical research, including studies of biological processes and human diseases.

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    One important advantage of zebrafish is that the adults are small and prefer to be housed in large groups, or “shoals”. As a result, they require much less space and are cheaper to maintain than mice. The NIH Zebrafish Core houses hundreds of thousands of zebrafish in a state-of-the-art facility.

    What is the structure of the zebrafish heart? ›

    Zebrafish have a prototypic heart with only one ventricle and one atrium.

    How do you calculate animal heart rate? ›

    Your pet should be calm and quiet. Place your hand over this area of the chest and feel for a heartbeat. You can also use a stethoscope if you have one. Count the number of heartbeats for 15 seconds and multiply that number by 4.

    What species has the fastest heartbeat? ›

    The heart of the Etruscan shrew, one of the world's smallest mammals, beats incredibly fast — up to 1,500 times per minute, or 25 times per second. The human heart, in comparison, is sluggish, beating only 60 to 100 times a minute. Then there's the heart of the blue whale, the largest animal ever to have lived.

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    How fast does a zebra run per hour? ›

    Zebras are equids, members of the horse family. They have excellent hearing and eyesight and can run at speeds of up to 35 miles per hour (56 kilometers per hour).

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    An adult human resting heart rate is normally 60 to 100 beats per minute, while shrews clock in at “over 1,000 beats per minute—that's over 16 times a second,” Mark Oyama, cardiology professor at the University of Pennsylvania School of Veterinary Medicine, says via email.

    What animal has the lowest heart rate per minute? ›

    The largest mammal, the blue whale, has a heart the size of a sofa, and their heart beats have been recorded as low as two per minute. That's the slowest heartbeat of any warm blooded mammal.

    What is the zebrafish in the study of early cardiac development? ›

    The zebrafish has emerged as a powerful model system to unravel the basic genetic, molecular, and cellular mechanisms of cardiac development and function. We summarize and discuss recent discoveries on early cardiac specification and the identification of the second heart field in zebrafish.

    What do scientists use zebrafish to study? ›

    Since the 1970s, zebrafish have been used to study a variety of human diseases, including cancer, cardiovascular disease, and diabetes.

    What is the main advantage of using the zebrafish embryos for this type of study? ›

    The zebrafish model has significantly improved our ability to study vertebrate developmental biology. The strengths of this model system lie in its external, visually accessible development, ease of experimental manipulation, and common genetic underpinnings with other vertebrates including humans.

    Do zebrafish have a four chambered heart? ›

    Unlike mammals, which have four chambers, the zebrafish heart consists of only two: a single ventricle (left) and a single atrium (right). Despite the difference in the number of chambers, the heart is the first organ to form in both mammals and zebrafish.

    Why is the fish heart different from the human heart? ›

    Unlike humans, they have a single circulatory pattern. Fish have a simple circulatory system, which consists of a two-chambered heart, blood, and blood vessels. Unlike humans, they have a single circulatory pattern.

    How does the zebrafish heart regenerate? ›

    Regeneration of the zebrafish heart

    By 7dpa, the wound is sealed by fibrin and is replaced by cardiac muscle by 30 dpa. (b) Proliferation, based on BrdU incorporation, is activated in cardiomyocytes by 7 dpa. The ventricular wall is restored by proliferation at the leading edge of the regenerating tissue.

    Why is zebra Danio used in scientific research? ›

    Because of its fully sequenced genome, easy genetic manipulation, high fecundity, external fertilization and rapid development, and nearly transparent embryo, zebrafish are a unique model animal for biomedical research, including studies of biological processes and human diseases.

    How zebrafish research has helped in understanding thyroid diseases? ›

    Zebrafish and mammals possess the same molecular mechanism of thyroid organogenesis and development. Thus, thyroid hormone signaling, embryonic development, thyroid-related disorders, and novel genes involved in early thyroid development can all be studied using zebrafish as a model.

    Why are zebrafish good models to use to study human disease? ›

    As zebrafish eggs are fertilised and develop outside the mother's body it is an ideal model organism for studying early development. Zebrafish have a similar genetic structure to humans. They share 70 per cent of genes with us. 84 per cent of genes known to be associated with human disease have a zebrafish counterpart.

    What is one advantage of using the zebrafish as a research animal? ›

    Similarity to humans

    This tiny tropical fish contains 70% of the human genome code, meaning its organs and cells are very similar to those of humans. Moreover, zebrafish have orthologs in 82% of human disease-associated genes, making them especially translatable for genetic research.

    What are the disadvantages of using zebrafish for research? ›

    Disadvantages: They require water systems to maintain them. They are not mammals and are not as closely related to humans as a mouse is. Reverse genetics has not been worked out for zebrafish as it has in the mouse.

    What are some interesting facts about zebra Danios? ›

    The scientific name of zebrafish is Danio rerio and it belongs to the minnow family, Cyprinidae. The fish got its common name from the presence of five uniform and pigmented horizontal stripes on the side of its body, which resemble the stripes of a zebra.

    What color do zebrafish prefer? ›

    Results showed that zebrafish preferred green over blue and domesticated fish chose green more than blue when there was a reward attached. Zebrafish also preferred red over green. Fish from one wild population learned with both colors and reversed learning only from green to red and not vice-versa.

    What do zebrafish need? ›

    To maintain zebrafish in a healthy condition, it is important to provide them with a clean environment in a properly functioning aquarium system. An important part of this is changing system filters regularly so that all the tanks receive proper water flow and clean water.

    What anesthetic is most commonly used for zebrafish? ›

    MS-222 (tricaine methanesulfonate) is the anaesthetic that has been the most largely used by the scientific community [18].

    What is the structure and function of the developing zebrafish heart? ›

    The zebrafish heart consists of four chambers (sinus venosus, atrium, ventricle, and bulbus arteriosus) connected in series. It pumps desaturated venous blood to the ventral aorta leading to the gill arches where oxygenation occurs, and from where it is distributed to the rest of the body.

    What are the cells of zebrafish heart? ›

    The zebrafish heart contains the same types of cells compared to the mammalian heart, including cardiomyocytes, epicardial cells, endocardial cells, and vascular endothelial cells.

    What are the features of fish heart? ›

    The systemic heart of fishes consists of four chambers in series, the sinus venosus, atrium, ventricle, and conus or bulbus. Valves between the chambers and contraction of all chambers except the bulbus maintain a unidirectional blood flow through the heart.

    What is the respiratory rate of zebrafish? ›

    A typical resting ventilatory rate for adult zebrafish is approximately 160 breaths min1; whereas hypoxia ( P O 2 of 35–40 mmHg) may elevate ventilation frequency to above 300 breaths min1 (Jonz and Nurse, 2005, Vulesevic et al., 2006).

    How many hearts does a zebrafish have? ›

    Zebrafish have a prototypic heart with only one ventricle and one atrium.

    What is the size of zebra fish heart? ›

    Due to the small size of the adult zebrafish heart (about 1 mm in diameter), the resolution of classic ultrasound-based technology is not satisfactory for reliable measurements of shortening fraction.

    What is the normal heart rate of a shark? ›

    In small, relatively inactive sharks such as the Spiny Dogfish, Lesser and Greater Spotted (Scyliorhinus stellaris) Catsharks. and the Leopard Shark (Triakis semifasciata), heart rate measures about 19 to 48 beats per minute. No one has yet managed to measure the heart rate of a White Shark.

    How do you take a fish's respiratory rate? ›

    The mouth working in unison with the gills, moves water from the mouth over the gills to remove oxygen from the water. The respiration rate of a goldfish can be measured by counting the number of times each minute the gills and their coverings move.

    What is the heart rate of zebrafish with caffeine? ›

    Exposure to high concentrations of caffeine significantly decreases the heart rate32 and increases stress-sensitive cortisol levels31. Hence, given that caffeine affects the behavior and physiological state of zebrafish, it may also affect ventilation.

    What is the normal rate for respiratory rate? ›

    Respiration rates may increase with fever, illness, and other medical conditions. When checking respiration, it is important to also note whether a person has any difficulty breathing. Normal respiration rates for an adult person at rest range from 12 to 16 breaths per minute.

    Why might cardiologist find it useful to study zebrafish? ›

    Zebrafish embryos are transparent, making it easy to spot abnormalities. Particularly helpful for cardiology research: mutant embryos lacking active circulation are capable of surviving up to 5 days post fertilization. And zebrafish hearts are simpler than mammals': theirs have just two chambers, while ours have four.

    How is a zebrafish heart similar to a human heart? ›

    The Zebrafish adult heart is not the same as the human heart as it has a simpler structure, but it is similar enough to replicate cardiovascular function. It only has a single atrium and ventricle (which contains two kinds of cardiac muscle), but these work in an equivalent way to those of a human heart.

    Why might cardiologists heart doctors find it useful to study zebrafish? ›

    In humans, the heart cannot repair itself. But zebrafish can repair their hearts - heart muscle cells near the damaged area lose their muscle properties and revert back to stem cells, which can repair the heart tissue.

    What is unique about the fish heart? ›

    Fish do not have a very powerful heart. It's a simple, four-chambered pump with two valves that circulates blood slowly throughout the body, which in turn slows the movement of oxygen and food in the body.

    Which bird has the fastest heart rate? ›

    An active hummingbird's heart pumps at 1,200 beats per minute; a flying pigeon's heart beats at 600. But a human athlete during exercise builds up a heart rate to around only 150 beats, a mere fraction of the hummingbird's heart rate. Exercise as hard as you like.

    Which animal has the fastest heartbeat rate? ›

    The heart of the Etruscan shrew, one of the world's smallest mammals, beats incredibly fast — up to 1,500 times per minute, or 25 times per second. The human heart, in comparison, is sluggish, beating only 60 to 100 times a minute. Then there's the heart of the blue whale, the largest animal ever to have lived.


    1. Imaging a Beating Zebrafish Heart - MRI in the Heart & Lung Research
    (Bruker Corporation)
    2. IMPART Research Reels - Dr. Matthew Stoyek (The Zebrafish Model in Cardiac Research)
    (Research IMPART)
    3. Science Lite -- Using Zebrafish to Study and Model Cancer
    (Huntsman Cancer Institute)
    4. Zebrafish Heart Imaging - In Vivo Preclinical Imaging
    (Bruker Corporation)
    5. A slide of zebra fish thesis from professor Xiao. Use our RvVisualPulse doing analysis.
    (Daniel Lu)
    6. Can neonatologists fix the heart for life_ Stories of the preterm heart
    (Salta Neo)


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