A Custom-Built Recirculating Zebrafish Housing System

Summary

Here, we present a novel, easy-to-construct, and low-cost protocol for recirculating zebrafish housing.

Abstract

Zebrafish are a popular model organism for performing in vivo studies, partly due to being considered inexpensive to house and maintain. Yet, commercial housing systems are typically cost-prohibitive to smaller research groups. Multiple custom-built zebrafish housing systems exist, but have issues with inconsistent water conditions, poor water cycling rates, and fragile housing tanks. These design issues reduce the viability of these systems for long-term research programs. In this article, we present a custom-built recirculating zebrafish housing rack, which is novel, affordable, and easy to construct. This system, when paired with our previously published housing tank design, allows for the housing of zebrafish at any stage of development. There are multiple levels of filtration and sterilization, which establish stable water conditions, allowing for the establishment of long-term usage of zebrafish for research and teaching. The system described below has been used by our laboratory to house zebrafish for 9 years, representing multiple generations of housed fish and hundreds of experiments.

Introduction

Zebrafish are a common model organism used in biological research. They are tolerant of many environmental conditions and breed easily with high numbers of offspring¹. They make a great model for a wide range of biological research, including human health, inflammation, genetics, and toxicology^{1,2,3,4,5,6,7}. They also offer a cost-effective option with relatively low maintenance requirements. However, creating a zebrafish housing system can be intimidating, especially for those with less experience - many factors should be kept in mind, such as recirculation rates, water quality, and the challenges of housing zebrafish at multiple stages of their development.

Commercial zebrafish housing systems are often expensive, which can pose a significant barrier for smaller laboratories or those with limited funding. These systems can cost tens of thousands of dollars, especially with additions such as automatic feeders and autocleaning, but these additions are unnecessary for budget-conscious laboratories, as well as those hoping to utilize the zebrafish in a classroom setting. For these types of facilities, constructing a zebrafish housing system is much more reasonable. There have been other articles published with custom construction plans, but each has its own disadvantages. Kim et al. demonstrated a method to create an easy-to-construct, 80-tank system⁸. At the time of publication, it was around $500 to build, but in the present time, it would be much more expensive. Paige et al. created a plan meant as an improvement to the previously mentioned method, but the cost to construct was $1500⁹. Additionally, maintenance of a 150 fish colony was estimated to be $1000 every 3 months⁹.

In this article, we have established a method of constructing a recirculating zebrafish housing and filtration system, which, when paired with the previously published housing tank design¹⁰, is significantly less expensive than commercially available systems. In addition, it is simple to construct with a few common tools. The system, when utilizing 30 individual tanks (~70% capacity), is also relatively quiet, producing roughly 60 dBA of sound, the equivalent of a normal conversation or a television¹¹. With a couple of simple modifications or by running it with fewer tanks, we believe the system could be used directly in a classroom setting without being a distraction to students. The overall design of the recirculating system is as follows: water that has been heated and conditioned is pumped from a sump basin through a main water supply line, which carries it up through the center of the housing rack. Ball valves off the main supply line provide water to a baffle, which supplies water at adjustable flow rates to the individual tanks at each level. To prevent excess buildup of pressure within the main supply line, the system utilizes a pressure release line with an adjustable ball valve to return excess water back to the sump. Water entering the tanks from the baffle recirculates within the tanks, with "overflow" captured by a slightly angled trough that runs through the center of the housing rack at each level. Water from the trough then flows into a vertical return pipe, which returns it to the sump basin, where it is cleaned by a multistage canister filter and sterilized with a UV light.

Protocol

The protocol described herein has been previously approved for use by the Institutional Animal Care and Use Committees at both Northern Michigan University and Shenandoah University.

NOTES: A saw will be required to cut the PVC piping for the water return portion of the system. This protocol used a rotary tool, but other types of saws will be sufficient for most cuts besides those needed for the tank return pipes. To cut the clear vinyl tubing for the water supply lines, a utility knife was used. To tighten hose clamps, a screwdriver is required. To attach the PVC tank returns to the PVC return holder, a drill with a 3/16" (0.47 cm) bit (slightly larger/smaller bits will also work) will be required. To avoid injury, care must be taken when using power tools. The following protocol describes the production of a housing rack that will pair with the previously published tank design¹⁰. If an alternative tank will be used, alterations to the tank water supply and water return instructions will need to be made to fit the tank specifications.

1. Assembling the housing rack (Figure 1A)

1. Assemble the support brackets. Each support bracket must be 21-1/4" (54 cm), 37" (94 cm), and 52-3/4" (134 cm) off the ground, with the fourth level being at 68-1/8" (173 cm) from the ground. Place each shelf onto the supports. These numbers represent the exact heights of the design presented here.
2. If the system will utilize a different rack due to cost considerations or availability, adjust all other measurements accordingly. The same is true if the tanks used are not those described in Lipscomb et al.¹⁰.

2. Support bracket for tank return system (Figure 1A)

1. Cut a 3/4" (1.9 cm) diameter polyvinyl chloride (PVC) pipe to 5' (152.4 cm). This will be the support for the tank return lines.
2. Slide the support onto the system. Position the support vertically running through the four layers of shelving. Place it at the end of the rack (lengthwise) and in the middle width). Insert a small (2" x 2" / 5.08 cm x 5.08 cm) piece of Styrofoam (or anything that can endure moisture) under the return support to keep it from falling through the bottom shelf of the rack.
3. Drill holes in the support, one hole directly through the support at each shelving level. Drill the holes at 7" (17.78 cm) above the surface of each shelf. Then, thread a 10" (25.4 cm) piece of wire or string through the hole. Use this piece later to secure the PVC water return pipes.

3. Assembling the water return system (Figure 1B-D)

NOTE: The water return pipes receive water from the individual tanks and carry it to the vertical return pipe that transports water to the sump, where it is filtered and ultimately recirculates back into the system.

1. Assemble the vertical return pipe (Figure 1B). The vertical return pipe guides water from the water return pipes to the sump basin.
   NOTE: Figure 1 illustrates the assembly of only two of the three layers of the return pipe.
   1. Cut a 2" (5.08 cm) diameter PVC pipe into four pieces of the corresponding length: 4" (10.16 cm), 12-3/4" (32.39 cm), 11-1/2" (29.21 cm), and 3" (7.62 cm).
   2. Take the 4" (10.16 cm) piece and mark the top and bottom sides. Attach a PVC T joint to the bottom side (attach the T joint so that its 45° angle is facing down).
   3. Connect the 12-3/4" (32.39 cm) PVC piece to the opposite end of the T joint from step 3.1.2.
   4. Connect another PVC T joint as before to the available end of the PVC from step
   5. Connect the 11-1/2" (29.21 cm) piece of PVC to the opposite end of the T joint from step 3.1.4.
   6. Connect another PVC T joint as before to the available end of the PVC from step 3.1.5.
   7. Connect the 3" (7.62 cm) piece of PVC to the opposite end of the T joint from step 3.1.6.
   8. Attach the 2" (5.08 cm) PVC cap onto the available end of the PVC from step 3.1.7.
   9. Attach the 2" (5.08 cm) PVC 45° elbow to the end of the 3" (7.62 cm) piece of PVC pipe from step 3.1.8
   10. Use the fastening straps to fasten the vertical return to the stand.
       1. Install the vertical return in the center of the stand. Face the open end of each T joint inwards towards the housing rack and locate about 1-2" (2.5-5.08 cm) above the shelf directly below it.
       2. Place the bottom 45° elbow just above the sump so that water will drain directly into the basin upon completion.
2. Assemble the water return pipes (Figure 1C-D). The PVC pipes transport water from the housing tanks back into the vertical return from step 3.1.
   1. Cut the 1-1/2" (3.81 cm) PVC pipe into three pieces of 4' (121.92 cm) length.
   2. Cut the 1-1/2" (3.81 cm) PVC pipe lengthwise to establish a U-shaped trough. This step requires two cuts along the pipe length to create a trough.
   3. Place the cut PVC pipes from step two onto each shelf so that one end is inserted into the T joint of the main vertical return. Using the string/wire previously inserted through the hole in the support bracket, secure the other end of the cut PVC pipes so that it slopes gently down towards the T joint of the vertical return. Aim for a 1.5 to 2° slope down to the T joint of the main vertical return.
   4. Optional: Put a cap onto the cut 1-1/2" (3.81 cm) tank return PVC pipes to prevent accidental backflows from coming out of the system.

4. Assembling the sump basin

NOTE: The sump basin limits fluctuations in conductivity, pH, nitrate/nitrite levels, and temperature by acting as a water reserve (Figure 1A). Multiple stages of filtration also occur at the sump basin.

1. Position sump basin under the assembled rack. Ensure an allowance of about 5-5/8" (14.29 cm) above the basin and below the first shelf. Ensure that the sump basin protrudes slightly from the shelf so that the vertical return pipe overhangs it to bring tank water back to the sump basin.
2. Place two foam filters into the sump basin and attach a 2-1/2' (76.2 cm) length of the 0.17" (0.43 cm) clear vinyl tubing. Later, connect the tubing's free end to the air pump.
3. Place the assembled canister filter intake tube against one of the legs that is adjacent to the sump basin.
4. Place the canister filter out port against the other leg of the system that is adjacent to the sump basin.
5. To install the ultraviolet (UV) light sterilizer, feed the out-port line of the canister filter into the UV light sterilizer to force particulate-free water through it.
6. If the ambient room temperature is not maintained at a constant 28.5 °C, add a submersible heater to the sump basin. Add a second heater depending on the ambient temperature of the room, and to act as a failsafe against the failure of a single unit.
7. Position the water pump into the sump basin so that the pump aligns with the support bracket of the tank return system.

5. Assembling the main water supply line (Figure 1A,F)

NOTE: The main supply line can be built and broken down quickly for cleaning due to a lack of adhesives. To avoid leakage, all vinyl tubing can have hose clamps attached. These can be added after starting up the system for the first time and assessing for leaks.

1. Cut a 3/4" (1.905 cm) vinyl tubing to 27.5" (69.85 cm) length and connect it to the pump.
2. Join a T coupler to the tubing from step 5.1. Place this T coupler slightly below the second shelf and oriented to the far side of the sump basin.
3. Cut a 3/4" (1.905 cm) vinyl tubing to 14-1/2" (36.195 cm) length and fasten it directly to the other end of the T coupler from step 5.2.
4. Connect another T coupler to the tubing. Place the T coupler slightly below the second shelf and oriented to the far side of the sump basin.
5. Cut a 3/4" (1.905 cm) vinyl tubing to 14-1/2" (36.195 cm) length and fasten it directly to the other end of the T coupler from step 5.4.
6. Connect another T coupler to the tubing. Place the T coupler slightly below the second shelf and oriented to the far side of the sump basin.
7. Cut a 3/4" (1.905 cm) vinyl tubing to 14-1/2" (36.83 cm) length and fasten it directly to the other end of the T coupler from step 5.6.
8. Connect the 3/4" (1.905 cm) to 1/2" (1.27 cm) reducing elbow with 1/2" (1.27 cm) male adaptor to the tubing. Seat the reducing elbow and male adaptor on the top surface of the fourth shelf. The elbow and adaptor will connect to the vinyl pressure release line, which runs back down to the sump.

6. Assembling the pressure release line (Figure 1A, Left panel)

1. Cut a 5/8" (1.5875 cm) vinyl tubing to a 3" (7.62 cm) length. Connect it to the 1/2" (1.27 cm) male adaptor at the top of the shelf.
2. Connect one of the ball valves to the other end of the tubing from step 6.1.
3. Cut a 5/8" (1.5875 cm) vinyl tubing to a 7' (213.36 cm) length and connect to the free fitting from step 6.2.
4. Thread tubing across the top of the rack and then back down into the sump basin along the leg of the housing rack.
5. To hold the tubing in place along the leg of the housing rack, use fastening straps.

7. Assembling the tank water supply lines (Figure 1E)

NOTE: The tank water supply lines carry water from the main supply line to the individual tanks.

1. Cut three pieces of 5/8" (1.575 cm) vinyl tubing to 2.5" (6.35 cm) length. Connect each to one of the three free T couplers coming off the main water supply line.
2. Connect a ball joint to each of the three free ends from step 7.1.
3. Cut three pieces of 5/8" (1.575 cm) vinyl tubing to 2.5" (6.35 cm) length and attach to the free end of each of the three ball joints.
4. Connect a 5/8" to 3/8" (1.5875 cm to 0.9525 cm) reducer to the 5/8" (1.5875 cm) tubing from step 7.3.
5. Cut three pieces of 3/8" (0.9525 cm) tubing to 2-1/4" (5.715 cm) length. Connect to the reducer from step 7.4.
6. Connect a 3/8" (0.9525 cm) nylon T coupler to each tube from step 7.5.
7. Cut six pieces of 3/8" (0.9525 cm) vinyl tubing to 2" (5.08 cm) length. Connect to the T joints from step 7.6.
8. Attach a hose clamp to each of the six pieces from step 7.7.
9. Slip a steel aquarium valve onto each of the available ends from step 7.7. Tighten hose clamps onto the steel aquarium valve.
10. Secure the steel aquarium valve to the shelf above it with fastening straps.
11. Cut 0.17" (0.4318 cm) vinyl tubing to the appropriate length to reach each of the tanks.

8. Preparing the system for operation

NOTE: The subsequent steps must be taken to ensure the health and survival of fish to be housed in the system. This process must occur at least a week in advance of adding fish to the system.

1. Prepare the system water as has been well described⁹.
2. Join the two bio sponge filters through the two free ends of the tubing to the air pump.
3. Make sure that all of the ball valves are in a suitable orientation. Close all of the ball valves except for the pressure release valve at the top of the system. To prevent significant pressure accumulation, keep this valve currently open.
   Contact the institution's IACUC representative to ensure electrical compliance prior to proceeding. Some electrical connection considerations are as follows:
   1. Use ground fault circuit interrupter (GFCI) outlets for all electrical components.
   2. Use power strips to prevent damage to equipment in the case of power surge.
   3. Ensure that power strips are elevated above the highest point of the system to minimize the chance of accidental introduction of system water.
   4. Utilize drip loops for all cords to minimize the risk of water traveling along cords to the power sources.
4. Turn on the pump and check the system for leaks.
5. If leaks occur, add hose clamps to each leaky junction.
6. Turn on the canister filter and ensure water flow is occurring. Be sure that the water intake for the canister filter is at the bottom of the sump.
   NOTE: In case of a serious system leak, the pump will automatically turn off before the entire system empties. By having the filter intake at the bottom, water will continue to circulate through the pump, saving it from damage.
7. Turn on the UV sterilizer only after the canister filter is running.

9. Routine maintenance

1. For optimal housing, check and adjust each of the following items daily:
   1. Keep conductivity between 600 and 800 µS/cm.
   2. Keep pH between 7-8.
   3. Maintain water temperature of 28.5 °C.
   4. Ensure that ammonia levels equal zero.
   5. Ensure chlorine is not present.
   6. Keep nitrate levels below 50 mg/L.
   7. Ensure water is moving through the pressure release line.
   8. Ensure water is flowing into and out of each tank on the system.
   9. Ensure water is flowing through the filter system.
   10. Be sure that the UV sterilizer bulb is on.
   11. Maintain water levels. If no matings or water exchanges are taking place, the system will need roughly 10 gallons of water added weekly due to evaporation. This volume will vary based on ambient room temperature and mating frequency.
2. Monitor each of the following items weekly:
   1. Check the flow of water through the tank water supply lines. Salt can build up within the steel aquarium valves, reducing water flow. Insert a pipe cleaner into the blocked valve to clean the salt buildup.
   2. Address water levels and water exchanges. If water is not being removed from the system routinely (such as during matings), exchange roughly 30% of the system water if ammonia levels rise or nitrate levels become unsafe.
      1. Fill a bucket or other large container with system water using the water exiting the pressure release line. Remove the straps holding it against the leg of the housing rack.
      2. Dump the collected water into a sink drain and repeat the process until roughly 30% of the water in the system (tanks plus sump) has been removed.
      3. Add new reverse osmosis (RO) or dechlorinated tap water to the system, bringing the water level up to a couple of centimeters from the top of the sump.
      4. Adjust conductivity and pH. Formulate a standard addition of salts/pH modifiers per liter of water added to the system, based on the water used within the facility.
   3. Remove mulm using a standard turkey baster weekly.
3. Check each of the following items monthly:
   1. Clean the canister filter.
      1. Unplug the canister filter.
      2. Unplug the UV sterilizer. Turn off the UV sterilizer when the filter is not running to prevent overheating of the UV light.
      3. Replace system filter pads and charcoal every 3 months when fully stocked with fish.
      4. Manually clean the canister filter components using tap water and clean sponges, with the exception of the bio beads, which are soaked in RO water (or dechlorinated tap water).
      5. Refill the canister filter with RO (or dechlorinated tap water) water after manual cleaning and filter replacements, then reconnect to the system.
      6. Ensure consistent flow through the canister filter is occurring before turning on the UV sterilizer or leaving the facility.
         NOTE: Small air bubbles can be introduced during the cleaning process. These bubbles stop the flow through the filter even after a steady flow has been initially established. If after 5 min of filter flow, no disruption has occurred, it is safe to assume the filter is running properly.
   2. Check the bio sponge every month. Replace every 4 to 6 months, depending on the density of adult fish in the system.
4. Perform each of the following maintenance items yearly.
   1. Full system cleaning: Once per year, turn off and disassemble the entire system. Manually scrub all components using standard laboratory brushes.
      NOTE: Soap is never to be used on any component.
   2. Sump cleaning: Use a turkey baster to remove any accumulated mulm from the bottom of the sump. Use a brush to gently remove buildup from the sides and bottom of the sump. Then, remove the "sediment" using the baster.

10. Common troubleshooting

1. If small leaks are observed along the tank water supply lines, utilize ring clamps at the leaking junction.
2. If water flow through the tank supply lines is reduced, use a pipe cleaner to remove salt buildup within the steel aquarium valve. Leave the valve open during cleaning.
3. Mulm can accumulate on the bottom of tanks due to overfeeding or a lack of water circulation. Use a turkey baster to remove excess buildup from the bottom of all tanks and reduce the amount of food per feeding.
4. Rapid or excessive algae buildup within the system may be observed. Placement of lights and excess food can both contribute to algae growth. Ensure overfeeding is not occurring and adjust lighting brightness to slow algae growth.
5. Water is not flowing through the system. Following power surges, the magnetic motor on the pump can become misaligned, causing water flow to cease. Unplug the pump from power, remove the magnetic motor, and reinsert it, making sure to realign it.
6. If the canister filter is not moving water after filter pad changes, manually prime the pump until water flows freely again.
7. If fish are not mating efficiently, ensure that both feeding and water quality are optimized. Dips in the pH of the water cause mating efficiencies to drop dramatically.
8. Water quality fluctuates greatly during water exchanges. This can occur when large volumes of fresh water (RO or dechlorinated) are added all at one time. To minimize fluctuations, add fresh water first to a dedicated 5-gallon (18.9 L) container and adjust the pH prior to being introduced into the sump. Do not, however, adjust conductivity prior to sump addition.
9. The pump has died, and water is not moving through the system. If water quality has been maintained appropriately, the fish will have no issues without water circulation for 48 h or more. However, reduce feedings to once a day during this time. If a lack of water circulation lasts for more than 48 h, manually remove a volume of 1/3 of the tank volume and replace it with water from the sump.
10. The canister filter has died, and filtration is not occurring. Assuming water quality has been maintained appropriately, the fish will have no issues without filtration for up to a week. Reduce feedings to once a day, and testing water quality every day until filtration resumes. If water quality drops, perform a water exchange.

11. Additional considerations

1. As with any recirculating housing system, the health of the fish is dependent upon consistent filtering of system water and recirculation of the water throughout the tanks. As component failure can compromise both filtration and recirculation, always have a backup water pump, canister filter (or a second motor for the canister filter), and a submersible water heater on hand.

Results

The protocol presented here will create a three-level recirculating zebrafish housing system (Figure 1). When paired with the previously published modular zebrafish tank design¹⁰, the system can easily accommodate 30-36 individual tanks, with each large tank capable of holding ~40 fish at a density under 10 adult fish/liter¹. In total, the housing system can hold over 1,200 adult zebrafish with a small overall footprint (~5' x 7') (~1.52 m x 2....

Discussion

The recirculating housing system described herein can be constructed in total for under $1500. The yearly cost is estimated to only be $75, assuming no major component replacements are needed, with all components available through local and online retailers. No special tools or skills are needed in order to complete the construction of this project, which makes it accessible for most to assemble, though it is essential to modify the lengths of materials if not using the exact metal stand utilized in the protocol describe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.17" inner diameter clear vinyl tubing 20 ft	Lowes	814315
1-1/2" PVC pipe cap x3	Lowes	23899
2" PVC pipe cap x1	Lowes	23900
3/4" inner diameter clear vinyl tubing 5 ft	Lowes	879267
3/4" to 1/2" threaded elbow and 1/2" adaptor	Lowes	23931
3/4" x 3/4" x 1/2" Tx4	Lowes	3311539
3/8" hose barb nylon T coupler x3	Lowes	877110
3/8" inner diameter clear vinyl tubing 5 ft	Lowes	879263
40 gallon sump basin	Tractor Supply	2229846
5/8" inner diameter clear vinyl tubing 10 ft	Lowes	814320
AAwipes 8-in-1 Aquarium Test Kit	Amazon	B0D2RG3LXR
Attmu 50 pcs reusable fastening straps	Attmu	8350503624091
Bacto-sure high-density foam filter (2-pack)	Pets Warehouse	8729780257055
Cascade CCF3UL Canister Filter, 100 Gallon, 265sph	Aquatic Warehouse	30172015946	Purchased from Amazon
Charlotte pipe 10 ft PVC pipe 1-1/2" diameter x2	Lowes	23830
Charlotte pipe 2-in x 2-in PVC DWV sanitary T	Lowes	23395
Charlotte pipe 5 ft PVC pipe 1-1/4" diameter	Lowes	23982
Charlotte pipe 5 ft PVC pipe 2" diameter	Lowes	23832
Charlotte pipe 5 ft PVC pipe 3/4" diameter	Lowes	23990
Honeforest TDS meter	Walmart	B073713G5F
Industrial strength rack 48 x 24 x 72	Lowes	23395
Ispinner 28 pcs worm gear hose clamps	Home Depot	313206783
JoyTube plastic hose barb reducer pipe fittings 5/8" to 3/8" pack of 3	Amazon	B08DNJY57G
LASCO 2-in x 2-in x 2-in dia Elbow PVC fitting	Lowes	23910
Pond pump submersible (2100 GPH)	Amazon	B004DFMZME
Qmseller stainless steel aquarium valve x6	Amazon	B07PDSVH3N
Submersible aquarium heater	Amazon	B0DLG8VSL8
Tetra whisper easy to use air pump for aquariums	Amazon	B0009YF4FI
UV light sterilizer Coralife Turbo Twist UV sterilizer, 6x	Coralife	96316156005	Purchased from Amazon
Uxcell drip irrigation barbed value for 5/8" double male barbed valve aquarium water flow control - 3 pcs x2	Harfington	1003460	Purchased from Amazon