The use of zebrafish (Danio rerio) as biomedical models

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. Zebrafish have all the main organs involved in the process of metabolism and can be used to study several human metabolic disorders such as nonalcoholic fatty liver disease, type 2 diabetes mellitus, dyslipidemia, and other hepatic diseases. With innovation and improvement of molecular techniques, zebrafish will continue to be an important biomedical model in the future. Various animal species have important roles as experimental models to advance biomedical research. Animal models provide consistency and validity of research results from in vitro studies or studies with rodents. Zebrafish has become a popular animal model for biomedical research. As shown in Figure 1, the number of publications per year on zebrafish as a model for biomedical research has been significantly increasing in recent years. One reason that zebrafish are an important biomedical model is because zebrafish embryos are transparent and they develop outside of the uterus. This unique developmental process allows scientists to study the details of development starting from fertilization and continuing throughout development. Innovation and development of molecular techniques in the later 20th century allowed zebrafish to be used as a model organism in almost all aspects of biology throughout the world. This review focuses on the use of zebrafish as a biomedical model in areas mainly related to diet-induced diseases, metabolic disorders, liver diseases, and intestinal diseases in humans. The number of publications in PubMed per year when searching with the keywords “zebrafish” and “Biomedical.” For more than 200 years, scientists used fish as model species with goldfish (Carassius auratus) the oldest model species. Goldfish were primarily used for applied studies of aquatic toxicology. Additional fish species have also been used, including zebrafish (Danio rerio), goldfish (Carassius auratus), medaka (Oryzias latipes), roach (Rutilus rutilus), three-spined stickleback (Gasterosteus aculeatus), pufferfish (Takifugu rubripes), and the swordtail (Xiphophorus hellerii) (Ribas and Piferrer, 2014). Every fish species has its unique advantages and disadvantages. For instance, goldfish have been used to study growth, stress, immunology, and reproduction. Medaka fish were the most popular species of fish used to study genetics, reproduction, and development. In recent years, the popularity of zebrafish as a model has increased due to its suitable features for many research areas. Danio rerio the Latin name for zebrafish formerly called Brachydanio rerio is a small tropical freshwater fish originating in the Ganges River and its tributaries in northern India (Tavares and Santos Lopes, 2013). In the natural habitat, zebrafish are usually found near the bottom of the water to minimize attack by predators. The morphology of male and female zebrafish is shown in Figure 2. Adult male and female AB strain of zebrafish, adapted from https://www.asianscientist.com/2014/12/in-the-lab/zebrafish-switch-sex/ with minor modification. Currently, zebrafish are considered as a suitable model to investigate development, genetics, immunity, behavior, physiology, and nutrition. According to its feeding habits, zebrafish are classified as omnivores and they eat a variety of foods (euryphagous). During experimental trials, scientists use different types and levels of dietary feeds. The same amounts of ingredients are used for adult and larvae zebrafish. Moreover, the feeds and feeding regimes implemented by some laboratories for rearing zebrafish are varied and, in some cases, are implemented without formal evaluation (Castranova et al., 2011; Gonzales and Law, 2013). In the laboratory, to get reasonable research results, zebrafish should receive the appropriate type and level of dietary nutrients. Most of the time researchers use different commercial diets for zebrafish, but several commercial diets have undefined nutritional composition and may have an effect on experimental results (Gonzales and Law, 2013). In addition, the dietary requirement for larvae and adults are different in the amount and composition of ingredients. In research studies, it is important to use a standard diet with adequate nutritional composition and known ingredients, which promote optimum growth and physiological status of the fish and to minimize the contribution of unintended nutritional effects on experimental results. The following diet formulas (Tables 1 and 2) were developed in our laboratory and give consistent experimental results with zebrafish. We recommend that researchers use these dietary formulas in their studies with zebrafish. Dietary formula for zebrafish larvae (5 to 29 d post fertilization) 1Vitamin premix (g/kg): thiamine, 0.438; riboflavin, 0.632; pyridoxine·HCl, 0.908; d-pantothenic acid, 1.724; nicotinic acid, 4.583; biotin, 0.211; folic acid, 0.549; vitamin B12, 0.001; inositol, 21.053; menadione sodium bisulfite, 0.889; retinyl acetate, 0.677; cholecalciferol, 0.116; dl-α-tocopherol-acetate, 12.632. 2Mineral premix (g/kg): CoCl2·6H2O, 0.074; CuSO4·5H2O, 2.5; FeSO4·7H2O, 73.2; NaCl, 40.0; MgSO4·7H2O, 284.0; MnSO4·H2O, 6.50; KI, 0.68; Na2SeO3, 0.10; ZnSO4·7H2O, 131.93; cellulose, 501.09. (Unpublished data; formulated in our zebrafish laboratory.) Dietary formula for zebrafish larvae (5 to 29 d post fertilization) 1Vitamin premix (g/kg): thiamine, 0.438; riboflavin, 0.632; pyridoxine·HCl, 0.908; d-pantothenic acid, 1.724; nicotinic acid, 4.583; biotin, 0.211; folic acid, 0.549; vitamin B12, 0.001; inositol, 21.053; menadione sodium bisulfite, 0.889; retinyl acetate, 0.677; cholecalciferol, 0.116; dl-α-tocopherol-acetate, 12.632. 2Mineral premix (g/kg): CoCl2·6H2O, 0.074; CuSO4·5H2O, 2.5; FeSO4·7H2O, 73.2; NaCl, 40.0; MgSO4·7H2O, 284.0; MnSO4·H2O, 6.50; KI, 0.68; Na2SeO3, 0.10; ZnSO4·7H2O, 131.93; cellulose, 501.09. (Unpublished data; formulated in our zebrafish laboratory.) Dietary formula for zebrafish (1 to 3 mo of age) 1Vitamin premix (g/kg): thiamine, 0.438; riboflavin, 0.632; pyridoxine·HCl, 0.908; d-pantothenic acid, 1.724; nicotinic acid, 4.583; biotin, 0.211; folic acid, 0.549; vitamin B12, 0.001; inositol, 21.053; menadione sodium bisulfite, 0.889; retinyl acetate, 0.677; cholecalciferol, 0.116; dl-α-tocopherol-acetate, 12.632. 2Mineral premix (g/kg): CoCl2·6H2O, 0.074; CuSO4·5H2O, 2.5; FeSO4·7H2O, 73.2; NaCl, 40.0; MgSO4·7H2O, 284.0; MnSO4·H2O, 6.50; KI, 0.68; Na2SeO3, 0.10; ZnSO4·7H2O, 131.93; cellulose, 501.09. (Unpublished data; formulated in our zebrafish laboratory.) Dietary formula for zebrafish (1 to 3 mo of age) 1Vitamin premix (g/kg): thiamine, 0.438; riboflavin, 0.632; pyridoxine·HCl, 0.908; d-pantothenic acid, 1.724; nicotinic acid, 4.583; biotin, 0.211; folic acid, 0.549; vitamin B12, 0.001; inositol, 21.053; menadione sodium bisulfite, 0.889; retinyl acetate, 0.677; cholecalciferol, 0.116; dl-α-tocopherol-acetate, 12.632. 2Mineral premix (g/kg): CoCl2·6H2O, 0.074; CuSO4·5H2O, 2.5; FeSO4·7H2O, 73.2; NaCl, 40.0; MgSO4·7H2O, 284.0; MnSO4·H2O, 6.50; KI, 0.68; Na2SeO3, 0.10; ZnSO4·7H2O, 131.93; cellulose, 501.09. (Unpublished data; formulated in our zebrafish laboratory.) The amount of feed varies across the different growth stages of the fish and is dependent on the stage of growth. From 5 d post fertilization, zebrafish larvae are mostly fed zooplanktons such as paramecium and rotifers and young larvae can be fed with artificial food up to 100 μm in size or live feed. For adult fish, the size of the dry food can range from 300 to 400 μm (Avdesh et al., 2012). The size of the dry food can increase with increasing size of the fish. The commonly practiced feeding ratio of zebrafish is about 4% of its bodyweight. Overfeeding may increase the concentration of nitrate in the water and affect the physiology of the fish. In addition, overeating may cause death of the fish. The criteria to select animal models for biomedical research are directly related to the final goal of the research. The use of zebrafish as a biomedical model was suggested by George Streisinger and colleagues at the University of Oregon, who launched the modern era for zebrafish in the field of biomedical research (Clark and Ekker, 2015). Zebrafish are popular animal models because they have numerous advantages over other species. The most advantageous features of zebrafish are a fully sequenced genome, easy manipulation of its genome, high fecundity, short generation time (about 3 mo), rapid embryonic development (24 hr), and external fertilization. The translucent zebrafish embryo allows study of the different developmental stages starting from the early stage of embryogenesis. In addition, zebrafish embryos form complete organ systems, including heart, intestine and blood vessels within 48 hr after fertilization. More than 10,000 mutants in protein-coding genes have been generated (Howe et al., 2013) and several transgenic lines of zebrafish have been made to study human diseases. The availability of multiple strains of zebrafish is another important advantage of this species. In addition, it is also very affordable to maintain a large number of zebrafish in a relatively small amount of laboratory space. Although zebrafish require relatively easy management, special attention must be paid to ensuring a healthy diet and adequate water quality to optimize fish health and growth. While there are several strains of zebrafish in the world, the most widely used strains in biomedical research are AB, Casper, Ekkwill, Nadia, Wild Indian Karyotype, wild-caught, and Tubingen. According to the ZFIN website, more than 800 biological laboratories around the world conduct basic and applied research with zebrafish (https://zfin.org/search?q=Zebrafish+laboratories&category). Many of these laboratories use zebrafish to study human diseases, including neural disorders, cancer, infectious diseases, cardiovascular diseases, kidney diseases, diabetes, blindness, deafness, digestive diseases, hematopoiesis, and muscle disorders. Mutant zebrafish have been established by knocking out or knocking in specific genes. These alterations create novel biomedical models. For example, if the patient has a disease related to metabolism, different mutations in zebrafish genes related to metabolism can be made and then changes in gene expression can be monitored using different molecular techniques. The short generation time of zebrafish makes it difficult to produce stable transgenic adults or homozygous mutant embryos, which usually requires about 4 months. Recently, scientists have developed many technologies to expedite the transgenic process (Burger et al., 2016). The presence or absence of genomic duplication events in zebrafish makes it complicated to study some human diseases such as diabetes mellitus. Zebrafish are also important for developing new therapies or screening novel drugs to treat or prevent human diseases. Even though zebrafish are an important biomedical model, they have some limitations, including the dissimilarity of some organs like the respiratory system and the reproductive system. Thus, it is difficult to use zebrafish as a model for respiration or reproduction in humans. In addition, because zebrafish live in an aquatic habitat, screening of some water soluble drugs in zebrafish is another limitation. There are several examples of human diseases that have been successfully modeled in zebrafish such as Duchenne muscular dystrophy, human melanoma, acute lymphoblastic leukemia, polycystic kidney disease, nephronophthisis, acute kidney injury, Parkinson’s disease, Huntington’s disease, Alzheimer disease, myocardial infarction, and some metabolic diseases. As shown in Figure 3, in addition to genomic similarity, the presence of conserved organs and organ systems between human and zebrafish contributes to development of a number of successful models of human diseases. Some of the conserved organ systems between zebrafish and humans (adapted from http://www.intl.upm.edu.my/article/zebrafish_replace_lab_rat-30977 with minor modification). We will focus on the common human metabolic diseases successfully modeled in zebrafish, including obesity, type 2 diabetes mellitus, nonalcoholic steatohepatitis, and atherosclerosis. Disturbance of the normal process of converting food to energy in the cell results in different metabolic disorders. Even though zebrafish and humans have differences in basic nutrient requirements, different metabolic mechanisms may not be needed. To keep the balance between the production and utilization of energy several organs are involved, including the brain, intestines, liver, skeletal muscle, and adipose tissue. Whole animal models are needed to study the entire process of metabolism. Zebrafish are an appropriate model to study metabolic dysfunction because they have all the organs involved in energy homeostasis and metabolism including appetite and insulin regulation and a lipid storage system which is conserved with that found in humans (Nishio et al., 2012). A report from World Health Organization indicated that, of the metabolism-related human diseases, cardiovascular disease is currently the most predominant fatal disease (Lozano et al., 2012). Obesity (Ng et al., 2014), type 2 diabetes mellitus, and nonalcoholic fatty liver disease (LaBrecque et al., 2014) increase the risk of cardiovascular disease. Because zebrafish and humans have similar metabolic organs (including the digestive organs, adipose tissue, and muscle), zebrafish are a popular model to study metabolic disorders. In addition, the availability of several new tools and approaches such as talens, CRISPR/Cas9 (Wu et al., 2018), compound treatment (Poureetezadi et al., 2016), mass spectrometry-based polar metabolomics and lipidomics (Zhang et al., 2018), and in vivo imaging of fluorescent dyes (Minchin et al., 2018) make it possible to investigate the molecular mechanisms of metabolic processes in zebrafish. Researchers have also used zebrafish as a model organism to study different types of metabolic diseases such as congenital errors of metabolism, hyper- and hypothyroidism, disorders of the hypothalamus–pituitary–adrenal axis, dysregulation of the circadian clock, and cancer metabolism (Gut et al., 2017). In this review, our emphasis will be on diet-induced metabolic disorders. Utilization of zebrafish in diet-induced obesity studies was first developed by Oka et al. (2010) by feeding adult zebrafish Artemia nauplii. In these studies, the fish showed increased body mass index, developed hepatic steatosis, hypertriglyceridemia, and dysregulation of some lipid metabolism genes. Chen et al. fed zebrafish a diet of high which in increased body increased and lipid in the of zebrafish with high from different or also to and lipid increased body increased adipose tissue, cardiovascular and et al., 2015). et al. used zebrafish to the of with normal and diets on obesity development. that diets showed an increase in adipose and the fish fed the normal diet developed obesity, but these fish were The other fish fed a diet were with the in our laboratory, also found that larvae and adult zebrafish fed a diet developed hepatic as shown in Figure In zebrafish, diet-induced obesity is also used to the type of food and effect of nutrient on development, and different drugs to prevent or treat obesity and by metabolism. The diet-induced obesity zebrafish model with Artemia common with obesity and can be used to of human obesity et al., the diet-induced obesity allows to the disease in the of obesity, the most common process in humans by this diets hepatic in adult and zebrafish. Adult zebrafish (1 mo and zebrafish (5 d post liver by and and of adult zebrafish fed with a diet or for 4 The is intestinal by and of adult zebrafish fed with a diet or for 4 The is 100 of in zebrafish larvae fed diet and for The is 200 (Unpublished from our zebrafish laboratory.) The main cause for development of diabetes is the of to produce which to insulin These and processes are conserved between zebrafish and humans. 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These results that zebrafish are a suitable model to study human disease. et al. also developed a zebrafish model for by human insulin in zebrafish These studies of the type in research results of et al. showed that mutant zebrafish with a in insulin a and genes when fed a diet showed growth increased and fatty liver development, which are similar to the human disease. The level in zebrafish can be using for use in humans with et al., for and can be There are several of insulin levels in zebrafish, including the insulin expression level by et al., 2016), insulin for et al., or et al., 2012). can also be by of insulin in zebrafish et al., 2014). the level of or in dyslipidemia, and in to development of atherosclerosis. the nutritional of zebrafish are several researchers established different models by the standard diet as feeding zebrafish a diet to develop obesity, and to metabolic on the fish. 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