This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

All animal procedures were approved by the Ethics Committee of Mie University, and were performed according to Japanese animal welfare regulation &#8216;Act on Welfare and Management of Animals&#8217; (Ministry of Environment of Japan) and complied with international guidelines.
1. Preparation of the Needle &#160;
NOTE: All experiments were performed under anesthesia, and all efforts were made to minimize suffering. For euthanasia, fish were immersed in an ice&#8211;water bath (5 parts ice/1 part water at &#8804;4 &#176;C) for &#8805;20 min.
<ol>
	<li>Prepare the glass microcapillary needles by pulling a 1.0-mm-outer-diameter glass capillary with a needle puller (Figure 1A).</li>
	<li>Cut the tips of the needles obliquely using fine scissors (Figure 1B). The ideal tip diameter should be approximately 100 &#8211; 200 &#181;m (Figure 1C). If the tip diameter is too narrow, blood will not enter the needle.</li>
	<li>Dissolve heparin in saline to a concentration of 5 mg/ml.</li>
	<li>Place a precut needle in the nosepiece end of an aspirator tube assembly and hold the mouthpiece in the mouth, or connect the needle to a bulb dispenser. Immerse the needle tip into the heparin solution, and heparinize the needle by suction and blowing through the solution (Figure 1D and 1E). 
	Note: The aspirator tube assembly has been used for zebrafish sperm cryopreservation16. Long rubber tube can stop any blood flying into mouth. Setting a filter in the midway of the tube can avoid the hazards.</li>
	<li>Store the heparinized needles in a 10-cm Petri dish and air-dry for at least 1 hr. A large number of needles can be prepared in advance.</li>
</ol>
2. Anesthesia
<ol>
	<li>Prepare the anesthetic solution in a small plastic case by mixing 200 ml of fish water with 100 &#181;l of 2-phenoxyethanol (2-PE). Final concentration of the anesthetics is 500 ppm. 
	CAUTION! 2-PE has a rapid effect.</li>
	<li>Remove desired number of fish (AB strain) from the circulation system.</li>
	<li>Use a net to transfer the fish into the anesthetics for 1 &#8211; 2 min (Figure 1F). Observe the fish gradually swim, spread the pectoral fins horizontally, gasp, and have rapid operculum movements within 1 min.</li>
	<li>As the time goes on, observe the fish lay on the bottom of the case and finally stop swimming. The surgical plane of anesthesia will reach when the fish stops to gasp and the operculum movements are slow. At this point, fish is ready for blood collection.</li>
	<li>Using a skimmer lift the anesthetized fish from the 2-PE and gently place it on a paper towel soaked with the anesthetics ( Figure 1G). Cover the fish&#8217;s head with soft tissue paper also soaked with 2-PE solution to prevent eye dryness and use another dry soft tissue paper to gently dry off the body surface.</li>
</ol>
3. Blood Collection
<ol>
	<li>Place a heparinized needle in the nosepiece end of the aspirator tube assembly (or the bulb dispenser) and hold the mouthpiece end of the aspirator tube assembly in mouth.</li>
	<li>Grasp the nosepiece end and the needle together, and carefully remove the interfering scales with the needle tip. Insert the needle at a 30 &#8211; 45&#176; angle into the blood collection site. Avoid puncture of the gastrointestinal tract (Figure 1H). In case of using bulb dispenser, press the bulb with thumb and middle finger, and block the hole at the tip of the bulb with first finger, then insert the needle as described above. 
	Note: The site for blood collection is along the body axis and posterior to the anus in the region of the dorsal aorta. The dorsal aorta (DA) and the posterior cardinal vein (PCV) are just ventral to the spine (Figure 2).</li>
	<li>Begin to suck the mouthpiece end of the aspiration tube assembly when the needle is felt touching the spine. If blood does not rise, move the needle tip subtly by hand to encourage blood flow. Note that once blood is rising into the needle, immediately stop shaking and suck gently (Figure 1I). In case of using bulb dispenser, release the pressure of bulb to aspirate the blood.</li>
	<li>Observe the blood will slowly rise into the needle in a pulsatile manner without suction, which is likely because of the arterial blood pressure. Thus, it is not necessary to suck if the needle correctly penetrates the artery.</li>
	<li>Stop suction after the appropriate volume of blood is collected. Remove the needle from the fish and press the puncture site using soft tissue paper to stop any bleeding. Observe the bleeding stop after approximately 10 &#8211; 20 sec&#160;of finger pressure (Figure 1J).</li>
	<li>After the bleeding is stopped, immediately transfer the fish back to a clean warm&#8211;water (~ 28 &#176;C) tank. Help the fish to recover by gently swirling water towards the gills until it begins to swim. 
	Note: Keep up to 5 postsampling fish in a 2 L tank and connect the tank to the circulation system to oxygenate the fish. Addition of antibiotics to the fish water is not necessary. Maintain the fish with normal housing and feeding.</li>
	<li>Expel the blood from the needle onto a clean area of a piece of parafilm (Figure 1K).</li>
	<li>Measure blood glucose using any commercial handheld glucometer (Figure 1L). 
	Note: The glucometer uses a glucose dehydrogenase&#8211;flavin adenine dinucleotide electrode and requires a sample volume of 0.6 &#181;l.
	<ol>
		<li>Insert a test strip completely into the meter and directly touch the blood drop. Observe the blood draw into the test strip automatically and obtain the blood glucose result 5 sec on the display area. Record the result and discard the test strip. Use a new test strip for each measurement.</li>
	</ol>
	</li>
	<li>(optional step) Take accurate quantities of blood by a pipetting and transfer the blood to a microcentrifuge tube for further analyses (hemoglobin, triacylglycerol (TG), total cholesterol, etc.). If necessary, dilute the whole blood from one fish with saline. Centrifuge the blood samples for 3 min at 680 x g at RT and harvest the plasma. Transfer the plasma into a new tube. At this point, it is ready to be used in biochemical analyses.</li>
</ol>

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The authors have nothing to disclose.

We present here a detailed protocol for serially obtaining blood from adult zebrafish. This method is straightforward to carry out and we use it in lab on a daily basis. This blood collection method is based on inserting a glass capillary needle into the zebrafish&#8217;s dorsal aorta. During this procedure, it is critical to be careful to not ablate the spine because it is the criterion for searching for the dorsal aorta. Reducing the spine injury will improve the survival rate. Although this technique is simple and easy to master, there are best practices that can ensure high success and survival rates. A skillful researcher will take 1&#8211;2 min to carry out the blood collection procedure (Protocol 2.3 to 3.6, which is the time that the fish is out of water). To save time in large-scale experiments, a double-team approach is recommended to perform blood collection. For example, one researcher could perform the blood sampling, while a second could handle the fish, perform the anesthesia and analyze the collected blood (measuring blood glucose, recording, or moving the blood to microcentrifuge tube, etc.).
Using this method, we demonstrated that the maximum blood sample volume that could be collected from individual fish is approximately 2% of body weight regardless of sex15, suggesting that the total circulating blood volume of zebrafish is greater than 2% of body weight. Previous studies demonstrated that teleost fish (Osteichthyes spp. and Salmogairdnerigairdneri) possess a total blood volume ranging between 1.8&#8211;3.8% of body weight17,18, thus we predict the total circulating blood volume of zebrafish to be 2&#8211;3.8% of body weight in zebrafish. For a single blood collection, we strongly recommend that blood sampling should be performed from 3 months old or &#62;0.3 g body weight zebrafish to obtain &#8805;5 &#181;l of blood. It is noteworthy that approximately half of the zebrafish survived even though 2% of body weight of their blood was removed, which revealed that zebrafish may cope well with blood loss.
The most significant advantage of this method is that it enables repeated blood sampling from the same individual. We determined the optimal volume and frequency of blood sampling by measuring the changes in hemoglobin levels (Figure 4). We recommend that the volume and interval for repeated blood sampling be &#8804;0.4% of body weight per week and &#8804;1% of body weight per 2 weeks to avoid blood loss anemia and hemorrhagic death. This conclusion is consistent with the practice guidelines for repeated blood sampling of rodent model animals19,20.
If the experiment allows the sacrifice of zebrafish, the needle can be inserted into a position along the body axis, posterior to the gill in the region of the dorsal aorta as a troubleshooting or an alternative method. This site is near the heart and aorta is relatively big, which can make the blood collection procedure easier.
Zebrafish have been successfully used in modeling the interrelated conditions of metabolic syndromes, including diabetes21,22, obesity23,24, fatty liver disease25 and atherosclerosis26. We further applied this technique to observe glucose metabolism in diet-induced obesity (Figure 5). Similar to mammals, zebrafish also developed abnormal glycolipid metabolism when fed a high-fat diet. The changes in fasting blood glucose of each individual showed individual differences in the response to overfeeding similar to that in humans15,27. This result indicates that an investigation of the temporal changes in blood biochemical parameters of individual fish will provide a good opportunity to determine the individual differences in metabolic disorders such as obesity and type 2 diabetes mellitus, thus further confirming its value as an animal model for human disease.
Overall, we developed a novel method for repeated blood collection from adult zebrafish. This method is essential for zebrafish research requiring repeated blood samples, such as studies of toxicokinetics, pharmacokinetics, and hematology. Furthermore, this repeated blood sampling method can also be applied to other small aquarium fish in biomedical research, for example, medaka (Oryzias latipes) or Xiphophorus (Xiphophorushelleri).

Zebrafish are gaining increasing popularity as a valuable model of human diseases because their organs and genetics are similar to those of humans1,2. In the field of developmental biology, many studies have demonstrated that zebrafish and human show marked similarity in hematopoiesis3, hemostasis4,5, and myelopoiesis6. Adult zebrafish are also used for studying immunological7, neurodegenerative8 and obesity-related diseases9 because this model organism shares common pathways with those disrupted in human diseases. For obesity and obesity-related diseases (diabetes, hepatic steatosis and nonalcoholic steatohepatitis and atherosclerosis), zebrafish blood glucose and lipids levels have been thoroughly investigated in several transgenic and diet-induced obesity models10-13.
Repeated blood sampling from individual animals will reduce animal use and decrease interindividual differences. However, repeated sample collection is technically difficult in small animals such as zebrafish because of their relatively small blood volume and the lack of easily accessible vessels. Several methods for one-time blood collection from zebrafish have been developed, although these methods have their own drawbacks, including lethality, associated tissue damage and limited blood volume. For example, 1&#8211;5 &#181;l blood can be harvested from a lateral incision of approximately 0.3 cm in length in the region of the dorsal aorta5. Decapitation with scissors by cutting through the pectoral girdle can collect 5&#8211;10 &#181;l blood10. Another convenient blood sampling method is tail ablation14. Cardiac puncture is one potential alternative method for repeated blood collection from the same fish, but the very small amount obtained (approximately 50 nl) with this procedure limits the number of analyses that can be performed11. Accordingly, a new protocol is needed to enable repeated non-lethal blood sampling, which would be a critical advance necessary for this organism to be a standard model organism for human diseases. This technique would allow for testing pharmacological response, discovery of molecular biomarkers for diagnosis, determination of prognosis, and the monitoring of various diseases, such as metabolic diseases, degenerative diseases and several types of malignancies.
We therefore developed a minimally invasive method for obtaining blood from zebrafish serially15. Here we demonstrate the procedure visually and provide a detailed protocol for this technique. Using this method, the normal value based on various parameters, including hemoglobin, fasting blood glucose, and lipids in the blood of healthy adult zebrafish were evaluated. Additionally, we also evaluated whether this method is suitable for studies that require serial samples by monitoring the temporal changes in blood glucose levels during overfeeding experiments.

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

This blood collection method causes minimal injury to zebrafish (a &#60;1 mm puncture; Figure 1J) and yields a very low mortality rate of 2.3%. We examined the maximum volume of blood that could be collected from a single fish and evaluated the relationship to its body weight (Figure 3). We found that the maximum blood volume collected was linearly correlated with body weight (R = 0.813). The largest volume of blood collected from an individual fish (body weight = 1.071 g) was 25 &#181;l, and the smallest volume was 1.3 &#181;l from a fish weighing 0.115 g. This suggests that the maximum volume of blood collected depends on the body weight of the zebrafish.
Biochemical analysis of hemoglobin, blood glucose, TG and total cholesterol were performed after blood collection (Table 1). Male and female healthy adult zebrafish (4&#8211;6 months old) were fasted for 18 hr before blood collection. The biochemical analysis revealed that the normal value of hemoglobin (male 9.91 &#177; 0.49 g/dl and female 10.02 &#177; 0.48 g/dl) and TG (male 417 &#177; 45 mg/dl and female 404 &#177; 35 mg/dl) did not differ significantly between the two groups. However, the fasting blood glucose and total cholesterol levels of the male group (44 &#177; 3 mg/dl and 365 &#177; 18 mg/dl, respectively) were significantly lower (p &#60;0.05) than the female group (69 &#177; 3mg/dl and 511 &#177; 52 mg/dl, respectively).
While the minimal trauma to zebrafish using the present method enables repeated blood sampling from the same individual, the effects of repeated blood drawing have not been evaluated. We investigated these effects using measurements of blood hemoglobin level (Figure 4). As we have shown in a previous publication15, adult male fish weighing approximately 0.5 g each were assigned to four groups. Repeated blood sampling (2 &#181;l each time) of the same individual fish once daily for 7 days (for a total of seven blood samples) resulted in significant decrease (p &#60;0.01) in hemoglobin levels from 10.82 &#177; 0.78 g/dl to 2.38 &#177; 0.8 g/dl. Removal of 2 &#181;l of blood every 2 days or a single collection of 5 &#181;l per week also yielded a significant decrease (p &#60;0.05) in hemoglobin levels. In addition, one week after a single collection of a 2 &#181;l blood sample, hemoglobin levels were slightly below normal (from 8.11 &#177; 1.15 g/dl to 7.15 &#177; 1.17 g/dl). The hemoglobin levels had no effects after a single collection of a 2 or 5 &#181;l blood sample for a 2-week recovery period. Thus, we concluded that repeated collection of 2 &#181;l of blood (0.4% of body weight) per week or 2&#8211;5 &#181;l (0.4&#8211;1% of body weight) per 2 weeks from individual fish can avoid blood loss anemia.
We further applied this method to the study of glucose metabolism. The changes in blood glucose levels of each individual in the normal diet group (once daily feeding) and overfeeding group (five daily feedings) were monitored over a 5-week period. Normal diet-fed zebrafish (Fish A, B, C) exhibited stable blood sugar levels all the time, while the overfed zebrafish (Fish D, E, F) experienced high blood glucose levels as early as week 1, and maintained this hyperglycemia condition throughout the 5-week study period (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/53272/53272fig1.jpg" /> 
Figure 1: Procedure for Blood Collection From Adult Zebrafish. (A) Glass needles prepared using a needle puller. (B) Cutting the tip of the needle obliquely using a fine scissors. (C) A precut needle with a tip diameter of approximately 135 &#181;m. Scale bar = 1 mm. (D) Blood collection devices: an aspirator tube assembly (left) and a bulb dispenser (right). Arrows indicate the nosepiece to hold the microcapillary needle. The arrowhead shows the mouthpiece of the aspirator tube assembly. The needle is positioned in the end of the nosepiece before sample collection. (E) Heparinizing the needle. (F) An anesthetized fish. (G) Place the fish on a paper towel soaked with the anesthetics. (H) Insert the needle at a 30&#8211;45&#176; angle into the blood collection site. (I) Blood rising into the needle. (J) Bleeding has stopped and a &#60;650 &#181;m puncture is circled and shown in a high magnification. (K) Expelling the blood from the needle onto a piece of parafilm. (L) Measurement of blood glucose using a glucose meter.
<img alt="Figure 2" src="/files/ftp_upload/53272/53272fig2.jpg" /> 
Figure 2: Schematic Representation of the Anatomic Landmarks for Blood Collection from the Adult Zebrafish. (A) The white line shows the puncture site for blood collection, which is along the body axis and posterior to the anus in the region of the dorsal aorta. (B) The primary vessels are the dorsal aorta and posterior cardinal vein; these are located ventral to the spine. S, spine; DA, dorsal aorta; PCV, posterior cardinal vein.
<img alt="Figure 3" src="/files/ftp_upload/53272/53272fig3.jpg" /> 
Figure 3: The Relationship of Maximum Volume of Blood Sampling and Body Weight. A total of 83 zebrafish (2&#8211;6 months old, 42 male and 41 female) underwent maximal blood collection.
<img alt="Figure 4" src="/files/ftp_upload/53272/53272fig4.jpg" /> 
Figure 4: Changes in Hemoglobin Levels Over a 1-week Period with Repeated Blood Sampling.A 2&#160;&#181;l sample of blood was collected from the same individual fish daily, once in 2 days, once weekly or 5 &#181;l once weekly (n = 5). The hemoglobin levels of each group before blood collection (day 0, white bar) and after repeated blood sampling (day 7, gray bar) are shown. Values are means &#177; standard error of the mean (SEM). *p&#160;&#60;0.05, **p&#160;&#60;0.01 vs. day 0. Adapted from ref.15.
<img alt="Figure 5" src="/files/ftp_upload/53272/53272fig5.jpg" /> 
Figure 5: Changes in the Fasting Blood Glucose Concentrations of Six Individual Male Fish over a 5-week Period. Fish A, B and C were the normal diet group. Fish D, E and F were the overfed group.
<img alt="Table 1" src="/files/ftp_upload/53272/53272table1.jpg" /> 
Table 1:&#160;Hemoglobin, Blood Glucose, TG and Total Cholesterol Levels for Male and Female Zebrafish 4&#8211;6 Months of Age.

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Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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Nanyang Technological University

Research

JoVE Journal

Biology

35.0K Views.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Video: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetics

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Developmental Biology

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Cancer Research

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

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