An Affordable and Easy-to-Construct Zebrafish Housing System for Stable Long-Term Laboratory Research

Abstract

Zebrafish have become a go-to model organism for in vivo studies, in part because of their reputation as being inexpensive to rear and house. Multiple do-it-yourself designs are currently available that provide laboratories with cost-effective housing systems. Unfortunately, these designs suffer from a range of issues ranging from poor water cycling rates and fragile housing tanks to inconsistent water conditions and designs that are prohibitively expensive for smaller laboratories to construct and maintain. These issues cause many of these housing systems to fall far short of the quality of commercially available zebrafish housing facilities. In this article, we present a novel, affordable, and easy-to-construct zebrafish housing system that improves upon previously published systems. The system utilizes three-dimensional printing technology to construct adaptable zebrafish tanks allowing for the housing of zebrafish at any stage of development. In addition, the water recirculation system utilizes multiple layers of filtration and no chemical adhesives, which allows for stable, long-term, housing of zebrafish in conditions suitable for research and teaching laboratories. The build described herein has been used by our laboratory to house zebrafish for over 3 years, representing multiple generations of housed fish.

Introduction

Zebrafish (Danio rerio) are a widely used animal model in biology.^1–9 A chief reason that zebrafish has grown in popularity is that they are generally considered to be easy and “inexpensive” to house and maintain.¹⁰ However, even entry-level commercial systems start in the thousands of dollars and escalate in price for larger units. While zebrafish are more affordable to maintain than other species, there is still an unmet need for quality housing systems that are accessible to budget-conscious laboratories.

To address this hurdle, Kim et al. published plans for an 80-tank zebrafish housing facility that could be built, at the time of publication, for ∼$500.¹¹ This system represented a significant first step toward a low-cost zebrafish facility. However, the system would cost substantially more to construct at the present time. Paige et al. modified and improved upon the design, but their system still required an initial $1500 investment and another $1000 in maintenance costs every 3 months.¹² Since these initial publications, numerous other do-it-yourself (DIY) systems have been designed. A recent review of these DIY systems provides an overview of the benefits and pitfalls of each.¹³

In this article we present a housing system that is significantly cheaper than commercial systems and has a simpler, more robust design than other DIY housing options. We describe an inexpensive, easy-to-construct water recirculation and filtration system sufficient to maintain 750 adult fish housed in 26 tanks (system can hold 48 tanks at full capacity). Additionally, we describe a novel tank design that can house either larval or adult fish. Over the course of 3 years of constant use, we have found that zebrafish reared and housed on this system display normal development and maintain reproductive rates sufficient for research-scale experimentation.

Materials and Methods

System assembly

Before proceeding, obtain the components listed in Tables 1 and 2 and Supplementary Figure S1.

Table 1.

List of Required Materials to Build the Housing System

Item	Total cost	Retailer
Tetra whisper easy to use air pump for aquariums (Fig. 1A)	17.89	Amazon.com
LASCO 2-in × 2-in × 2-in dia Elbow PVC Fitting (Fig. 1B)	4.09	Lowes
Cascade CCF3UL Canister Filter, 100 Gallon, 265 gph (Fig. 1C)	134.99	Amazon.com
Optional: UV light sterilizer Coralife Turbo Twist UV Sterilizer, 6X—price not included in total (Figure D)	175.88	Amazon.com
Uxcell drip irrigation barbed valve for 5/8′′ double male barbed valve aquarium water flow control—3 pcs × 2 (Fig. 1E)	26.98	Amazon.com
3/8′′ hose barb nylon T coupler × 3 (Fig. 1F)	11.34	Lowes
3/4′′ × 3/4′′ × 1/2′′ T × 4 (Fig. 1G)	9.92	Lowes
1-1/2′′ PVC pipe cap × 3 (Fig. 1H)	6.33	Lowes
JoyTube plastic hose barb reducer pipe fittings 5/8′′ to 3/8′′ pack of 3 (Fig. 1I)	5.99	Amazon.com
QMseller stainless steel aquarium valve × 6 (Fig. 1J)	77.94	Amazon.com
Pond pump submersible (2100 GPH) (Fig. 1K)	65.99	Amazon.com
3/4′′ inner diameter clear vinyl tubing 5 ft (Fig. 1 L)	11.89	Amazon.com
5/8′′ inner diameter clear vinyl tubing 10 ft (Fig. 1 M)	14.89	Amazon.com
Ispinner 28 pcs worm gear hose clamps (Fig. 1N)	16.99	Amazon.com
3/8′′ inner diameter clear vinyl tubing 5 ft (Fig. 1O)	6.99	Amazon.com
0.17′′ inner diameter clear vinyl tubing 20 ft (Fig. 1P)	5.98	Lowes
Charlotte pipe 5-ft PVC Pipe 2′′ diameter (Fig. 1Q)	15.08	Lowes
Charlotte pipe 10-ft PVC Pipe 1-1/2′′ diameter × 2 (Fig. 1Q)	12.78	Lowes
Charlotte pipe 5-ft PVC Pipe 3/4′′ diameter (Fig. 1Q)	5.35	Lowes
Charlotte pipe 5-ft PVC Pipe 1-1/4′′ diameter (Fig. 1Q)	10.05	Lowes
40 gallon sump basin (Fig. 1R)	59.99	Tractor supply
Submersible aquarium heater (Figure S1)	25.99	Amazon.com
3/4′′ inner diameter clear vinyl tubing 5 ft (Fig. 1L)	11.89	Amazon.com
Bacto-sure high-density foam filter (2-pack) (Fig. 1T)	29.98	Amazon.com
3/4′′ to 1/2′′ threaded elbow and 1/2′′ adaptor (Fig. 1U)	1.94	Lowes
Kingspan insulation R 5 unfaced polystyrene foam board insulation—1′′ thick (Fig. 1V)	24.97	Lowes
2′′ PVC pipe cap × 1 (Fig. 1W)	2.6	Lowes
Attmu 50 PCS reusable fastening straps (Fig. 1X)	7.99	Amazon.com
Charlotte pipe 10-ft PVC Pipe 1-1/2′′ diameter × 2 (Fig. 1Q)	12.78	Lowes
Charlotte pipe 2-in × 2-in PVC DWV Sanitary T	13.47	Lowes.com
Industrial strength rack 48 × 24 × 72 (Fig. 1Z)	169.99	Lowes
3D printed return pipe support brace (Figure Z’) x3	21.48	AutotivMFG
3D printed water port support brace (Figure Z’’) x6	14.52	AutotivMFG
Total	1010.24

Photos of the individual components are shown in Supplementary Figure S1 for clarity. The total cost column is the sum of the cost for all the parts in a given row, not the cost per item. The retailer that was used is also indicated, but other purchasing options may be possible.

3D, three-dimensional; UV, ultraviolet.

Table 2.

List of Tools Required to Assemble the Housing System

Tools	Retailer
Rotary tool with circular blade	Lowes
Utility knife	Lowes
Screw driver	Lowes

A saw is required to cut the PVC piping for the water return portion of the system. We utilized a rotary tool, although other types of saws would suffice for all cuts other than those needed for the support brackets and tank return pipes. Construction also requires the cutting of the foam board and clear vinyl tubing. We used a utility knife for these cuts. The screwdriver is used to tighten the hose clams.

Briefly, heated and conditioned water is pumped from a sump basin. The main water supply line carries the water up through the center of the rack. Ball valves located off the main supply line allow water to be directed to tanks at each level (Figs. 1A and 2A), to a pressure release line (Fig. 1A, F), or out of the system to accommodate large-volume water exchanges (Fig. 1A, E). At each level, water flows to a pair of stainless steel eight-port valves (Fig. 2B) that control the delivery of water to the tanks. Overflow from each tank drips into a slightly angled trough that runs through the center of the housing rack (Fig. 2J, K). Water in the trough flows into the vertical return pipe (Fig. 2D, H) and back to the sump basin (Fig. 2I) where it is filtered.

FIG. 1.

Assembly of the housing system. (A) The illustration on the left shows the overall design of the system. The areas indicated by the dashed box are shown in greater detail. Each box has a number (indicating the figure number) and letter (indicating the panel within the figure). The photo on the right displays the fully assembled system. (B) Indicates the distance from the ground (inches) that the housing racks are positioned. (C) Top down view of the positions of the main water supply line support and the return support. The gray box indicates the position through which the water line support brace should run. The return support should be positioned through the location with the star. (D) The two gray circles indicate the foam filters and the star indicates the position of the pump. The 1 indicates where the water return should overhang the sump basin to ensure proper water return. The 2 and 3 are the suggested locations for the water intake and water output ports of the canister filter. (E) Shows a T coming off of the main water supply line (in the white dashed box). Also shown is the connection off the T to a barbed ball valve. (F) Image taken from above the top shelf of the housing rack looking down on the top of the rack. Image indicates the elbow at the top of the housing system (dashed black box). The elbow connects to a barbed ball valve, which acts as the pressure release for the main water supply line. The arrow indicates the tubing that runs back down to the sump basin. (G) The arrow is pointing to the tubing from part E that is fastened to the rack leg and runs back to the sump basin. It is the final portion of the pressure release for the main water supply line. (H) Shown is the main water supply line running up slightly off of center of the housing system. The dashed box shows fastener strap that are attaching the water supply line to the water supply brace.

FIG. 2.

Assembly of the water circulation system. The water circulation system takes water from the main water supply line and brings it to each individual tank and takes water from each tank and brings it back to the sump. (A) A T-coupler coming off of the main water supply line connects to 1/2′′ tubing (1) and then to a barbed ball valve. The other end connects to another 1/2′′ piece of tubing (2), then a 1/2′′ to 3/8′′ reducer (3), then to a 3/8′′ tubing (4) and finally to a 3/8′′ T (5). (B) The same components from part A are shown, but with the addition of 3/8′′ tubing (6) coming off each end of the T-couplers (5) and attaches to the steel 8 port valves. This connection often requires a hose clamp. (C) 3D printed port supports hold the eight port valves onto the shelf above it. Fastening straps can also be used. (D) Shows the vertical water return system. (E) The lowest portion of the vertical return. (F) The middle section of the vertical return. This portion collects water from the lowest and middle levels of tanks. (G) The top of the vertical return. (H) The top of the vertical return placed onto the system. Note the water return pipe is inserted into the T joint. (I) The lowest portion of the vertical return is placed onto the system. Note the 45° PVC elbow is positioned to return water to the sump. (J) End on view of the water return pipe that has been cut to create a trough. (K) The troughed water return pipe placed onto the 3D printed tank return brackets. (L) Foam board cover for the sump. 3D, three-dimensional.

Assembling the housing rack

(1)

Assemble the housing rack (Supplementary Fig. S1Z) such that the bottom attachment for each support bracket for the three housing shelves are; 21-1/4′′, 37′′, and 52-3/4′′ off the ground (Fig. 1B). Place the shelves onto the supports.

(2)

Place the shelf supports for the fourth level at 68-1/8′′ from the ground (Fig. 1B).

Support brackets for main water supply line and tank return system

To reduce flow turbulence through the system, the flexible tubing of the main supply line is supported by a bracket that runs through the center of the rack.

(1)

Cut a 1-1/4′′ (Supplementary Fig. S1Q) PVC pipe to 4-1/2′ length.

(2)

Cut the PVC pipe in half lengthwise creating a trough. This is the main water line support brace.

(3)

Insert the support brace through the shelves as shown (Fig. 1C, gray box). The main water supply line will run along the path of the support brace.

(4)

Cut a 3/4′′ diameter PVC pipe to a length of 5′. This is the support for the tank return lines (Fig. 1A, C).

(5)

Fasten support brackets (Supplementary Fig. S1Z’) onto the support from step 4.

(6)

Slide the return support down through the top shelf in the correct location (Fig. 1C, star). The three-dimensional (3D) printed return holders are too large to slide through the holes in the grates of each shelf and must be added one at a time as the pipe slides through each level.

(7)

Place a small (2′′ × 2′′) piece Styrofoam (anything that can withstand moisture) under the return support to keep it from falling through the bottom shelf of the rack (Fig. 1A, right).

(8)

Place the fourth shelf onto the housing rack.

Assembling the sump basin

The sump basin acts as a water reserve, which prevents large swings in conductivity, pH, nitrate/nitrite levels, and temperature. The sump basin is also the location where multiple stages of filtration occurs.

(1)

Position the sump basin under the rack as shown (Fig. 1A, D). There should be a clearance of ∼5–5/8′′ between the top of the basin and the bottom of the first shelf if the shelf is in the correct position. The sump basin should stick out slightly from the shelf to give the system's vertical return pipe enough room to bring tank water to the sump basin (Fig. 1D arrow 1, 2H).

(2)

Place the two foam filters (Supplementary Fig. S1T) into the sump basin and attach a 2-1/2′ length of the 0.17 clear vinyl tubing (Supplementary Fig. S1P). The free end of the tubing will be attached to the air pump later.

(3)

Position the assembled canister filter (Supplementary Fig. S1C) intake tube along one of the legs that rests against the sump basin (Fig. 1D, arrow 2).

(4)

Position the canister filter out port along the other leg of the system that rests against the sump basin (Fig. 1D, arrow 3).

(5)

Optional: To install an ultraviolet (UV) light sterilizer (Supplementary Fig. S1D), the out-port line of the canister filter will need to feed into the UV light sterilizer. This will prevent the buildup of sediments in the UV sterilizer.

(6)

Add a submersible heater (Supplementary Fig. S1) to the sump basin if needed.

(7)

Place the water pump (Supplementary Fig. S1K) into the sump basin such that the pump lines up with the support brace (Fig. 1C gray box, Fig. 1D star).

Assembling the main water supply line

The main supply line does not require any adhesives, which allows for quick construction (Supplementary Movie S1) and breakdown for cleaning. Hose clamps can be added to any/all vinyl tubing connections to prevent leaks.

(1)

Cut a 10′′ length of 3/4′′ vinyl tubing (Supplementary Fig. S1L) and attach it to the pump.

(2)

Attach a 3/4′′ × 3/4′′ × 1/2′′ T coupler (Supplementary Fig. S1G) to the tubing. The T coupler should be situated below the first shelf but above the lid of the sump basin (Fig. 1E, dashed box).

(3)

Cut a 14-1/4′′ length of 3/4′′ vinyl tubing and attach to the T from step 2. The tubing should be running directly next to the main water supply line support bracket (Fig. 1C gray box).

(4)

Attach another T coupler to the tubing from step 3. The T coupler should be positioned slightly below the second shelf and the oriented toward the far side of the sump basin (toward number 1 in Fig. 1D).

(5)

Cut a 14-1/2′′ length of 3/4′′ vinyl tubing and attach it directly to the other end of the T coupler from step 4.

(6)

Attach another T coupler to the tubing. The T coupler should be positioned slightly below the second shelf and the oriented toward the far side of the sump basin.

(7)

Cut a 14-1/4′′ length of 3/4′′ vinyl tubing and attach it directly to the other end of the T coupler from step 6.

(8)

Attach another T coupler to the tubing. The T coupler should be positioned slightly below the second shelf and oriented toward the far side of the sump basin.

(9)

Cut a 2-3/4′′ length of 3/4′′ vinyl tubing and attach it directly to the other end of the T coupler from step 8.

(10)

Attach the 3/4′′ to 1/2′′ reducing elbow with 1/2′′ male adaptor to the tubing (Supplementary Fig. S1U). The reducing elbow and male adaptor should sit on the top surface of the fourth shelf (Fig. 1F, dashed box). The elbow and adaptor will connect to the vinyl pressure release line, which runs back down to the sump.

(11)

Fasten the support brace to the main water supply line using the Velcro fasteners (Supplementary Fig. S1X) (Fig. 1H, dashed box).

Assembling the pressure release line

(1)

Cut a 3′′ length of 5/8′′ vinyl tubing (Supplementary Fig. S1M). Attach to the 1/2′′ male adaptor at the top of the shelf (Supplementary Fig. S1U).

(2)

Attach one of the ball valves to the free end of the tubing from step 1 (Supplementary Fig. S1E).

(3)

Cut a 7′ length of 5/8′′ vinyl tubing and attach to the free fitting from step 2.

(4)

Feed the tubing across the top of the rack and then back down into the sump basin along the leg of the housing rack (Fig. 1F arrow, and G).

(5)

Use fastening straps to keep the tubing in place along the leg of the housing rack (Fig. 1G, arrow).

Assembling the water exchange/breeding tank filling line

This line allows for quick water exchanges (Supplementary Movie S2).

(1)

Cut a 2.5′′ length of 5/8′′ diameter vinyl tubing (Supplementary Fig. S1M). Attach to the free T coupler (Supplementary Fig. S1G) that comes off of the main supply line below the first level of the rack (Fig. 1E, dashed box).

(2)

Attach one of the ball valves to the free end of the tubing from step 1 (Fig. 1E).

(3)

Cut a 1.5′ length of 5/8′′ diameter vinyl tubing. Attach one end to the free fitting from step 2.

Assembling the tank water supply lines

The tank water supply lines take water from the main supply line and deliver it to individual tanks (Supplementary Movie S3).

(1)

Cut three pieces of 2.5′′ length of 5/8′′ diameter vinyl tubing (Supplementary Fig. S1M). Attach each to one of the three free T couplers (Supplementary Fig. S1G) coming off of the main water supply line [Fig. 2A, (1)].

(2)

Attach a ball joint (Supplementary Fig. S1E) to each of the three free ends from step 1.

(3)

Cut three pieces of 2.5′′ length of 5/8′′ diameter vinyl tubing. Attach to the free end of each of the three ball joints [Fig. 2A, (2)].

(4)

Attach a 5/8′′ to 3/8′′ reducer (Supplementary Fig. S1I) to the 5/8′′ tubing from step 3 [Fig. 2A, (3)].

(5)

Cut three pieces of 2-1/4′′ length of 3/8′′ diameter vinyl tubing (Supplementary Fig. S1O). Attach to the reducer from step 4 [Fig. 2A, (4)].

(6)

Attach a 3/8′′ nylon T coupler to each tube from step 5 [Fig. 2A and F, (5)].

(7)

Cut six pieces of 2′′ length of 3/8′′ diameter vinyl tubing. Attach to the T joints from step 6 [Fig. 2B, (6)].

(8)

A hose clamp (Supplementary Fig. S1N) can be added to each of the six pieces from step 7.

(9)

Slide a steel water port onto each of the free ends from step 7 (Fig. 2B). Tighten the hose clamps onto the steel water ports.

(10)

Support the steel water ports (Supplementary Fig. S1J) by securing them to the shelf above it with 3D printed support couplers (Fig. 2C)

(11)

Cut lengths of 0.17′′ (Supplementary Fig. S1P) diameter clear vinyl tubing to length to reach each of the tanks.

Assembling the water return system

The water return pipes take water from the individual tanks to the vertical return pipe that carries water to the sump where it is filtered and eventually recirculated back to the system.

Part 1: Assembling the vertical return

The vertical return pipe directs water coming from the water return pipes and to the sump basin (Supplementary Movie S4).

(1)

Cut a 2′′ diameter PVC pipe (Supplementary Fig. S1Q) into four pieces of the following lengths: 4′′, 12-3/4′′, 11-1/2′′, and 3′′.

(2)

Take your 4′′ piece and mark a top and bottom side. Attach a PVC T joint (Supplementary Fig. S1Y) to the bottom side of the 4′′ piece (be sure the T joint is positioned with the 45 degree angle facing down (Fig. 2D, G).

(3)

Attach the 12-3/4′′ Length PVC piece to the opposite end of the T joint from step 2 (Fig. 2D, G).

(4)

Attach another PVC T joint as before to the free end of PVC from step 3 (Fig. 2D, F).

(5)

Attach the 11-1/2′′ length piece of PVC to the opposite end of the T joint from step 4 (Fig. 2D, F).

(6)

Attach another PVC T joint as before to the free end of the PVC from step 5 (Fig. 2D, G, H).

(7)

Attach the 3′′ length piece of PVC to the opposite end of the T joint from step 6 (Fig. 2D, E).

(8)

Attach the 2′′ PVC cap (Supplementary Fig. S1H) onto free end of the PVC from step 2 (Fig. 2D, G, H).

(9)

Attach the 2′′ PVC 45° (Supplementary Fig. S1B) elbow to the bottom end of the 3′′ piece of PVC pipe from step 7 (Fig. 2D, E, I).

(10)

Use the fastening straps (Supplementary Fig. S1X) to secure the assembled vertical return to the stand (Fig. 2D, H, I). The vertical return should be positioned in the center of the stand. The open end of each T joint should be facing inwards toward the housing rack and positioned roughly (1-2′′) above the shelf below it (Fig. 2D, H). The bottom 45° elbow should be just above the sump basin (Fig. 2I) when finished.

Part 2: Assembling the water return pipes

These PVC pipes carry water directly from the housing tanks back into the vertical return described in part 1.

(1)

Cut the 1-1/2′′ PVC (Supplementary Fig. S1Q) pipe into three pieces of 4′ length.

(2)

Remove 1/3 of the 1-1/2′′ PVC pipe lengthwise to create a U-shaped trough (Fig. 2J). This step will require two cuts lengthwise along the pipe to create a trough.

(3)

Position the 3D printed tank return brackets such that they are ∼3–4′′ above the shelf immediately below them (Fig. 2K).

(4)

Slide the cut 1-1/2′′ PVC pipe from step 2 onto the tank return holders (Fig. 2K). One end should be on the holder, the other end should be fully inserted into the T joint of the main vertical return (Fig. 2H)

(5)

Adjust the tank return holder height (by sliding it up or down) to achieve a 1.5 to 2-degree slope down to the T joint of the main vertical return.

(6)

Place a cap (Supplementary Fig. S1W) onto the 1-1/2′′ tank return PVC pipes.

Preparing the system for operation

The following steps must be taken to ensure that the health and survival of fish housed on the system is not jeopardized.

(1)

Prepare the system water as has been well described (12)

(2)

Attach the two bio sponge filters (Supplementary Fig. S1L) through the two free ends of the tubing to the air pump [Supplementary Fig. S1A)].

(3)

Ensure that all of the ball valves are in the appropriate orientation. At this time, all of the ball valves should be in the closed orientation, except for the pressure release valve at the top of the system. This valve must be open to prevent substantial pressure buildup.

(4)

Turn on the pump and check system for leaks.

(5)

Additional hose clamps can be added to any junction that leaks.

(6)

Cut the Styrofoam board to cover the sump basin (Fig. 2L). We cut to fit and then cut in half. This allows for easier removal during water exchanges.

(7)

Just before adding fish to the system, turn on the UV sterilizer.

Housing tank construction

To assemble the tanks described herein, be sure to obtain all tools and products (Tables 3 and 4). The 3D printed items can be manufactured in-house, or purchased through online retailers. The provided instructions yield 26 total tanks: 12 large and 14 small tanks.

Table 3.

List of Tools Required to Assemble the Housing Tanks

Tools	Retailer
Utility knife	Lowes
Power drill	Lowes
5/8′′ boring-style drill bit	Lowes
1/16′′ Pilot drill bit	Lowes

The power drill and bits are required to create the guide hole and the larger hole in which the elbow setup is constructed. The utility tool is required to create the foam lids.

Table 4.

Product List for the Construction of 26 Zebrafish Housing Tanks

Item	Total cost	Retailor
Kritter keepers—medium × 12	144	kensfish.com
Kritter keepers—small × 14	101.9	Petmountain.com
Kingspan insulation R 5 unfaced polystyrene foam board insulation	24.97	Lowes
Downports × 26	21.84	Hosewarehouse.com
Aqueon silicone aquarium sealant	3.97	Amazon.com
FunLavie aquarium filter media bag nylon mesh bag net bag zipper	9.35	Amazon.com
Phifer BetterVue 3 ft × 7 ft black fiberglass screen mesh	20.34	Amazon.com
Uxcell acrylic round rod, 2 mm 5/64 inch	8.99	Amazon.com
Total	335.36

Items to 3D print	Cost to purchase from retailer ^a	Retailor
Inner elbow adaptor × 26	26.78	AutotivMFG
Outer elbow adaptor × 26	21.12	AutotivMFG
Adult screen body × 26	28.6	AutotivMFG
Adult screen cap × 26	16.9	AutotivMFG
Larval screen body × 26	39.78	AutotivMFG
Larval screen cap × 26	27.3	AutotivMFG
Total	160.48

The costs are assuming that the items are purchased via an online retailer. If a 3D printer is already available, there will be no need to purchase these items.

The list is complete for the construction of 26 housing tanks. Included are the prices for the production of the 3D printed items from commercial vendors, although we suggest purchasing a 3D printer to save on costs in the long term.

3D printed items

3D printing of quick connect filters

All models were designed using the free, web-based CAD program Tinkercad (https://www.tinkercad.com/). Models were saved as stl files and imported into Ultimaker Cura (https://ultimaker.com/software/ultimaker-cura) for conversion into gcode, a printable file format. Files were printed on an Ender 3 using a 0.2 mm layer height with 100% infill. Models were printed in polylactic acid (PLA). Those with more printing experience may wish to use PETG, which is more stable than PLA and is available in explicitly food-safe varieties.

Stl files used in this article can be found on NIH 3D (https://3d.nih.gov/discover?sort=relevant&createdby=blipscom).

Preparing the housing tanks

The measurements given below for the exit port location have been optimized for our system but can be modified to suit your tanks (Supplementary Movie S5).

(1)

Drill a guide hole in one of the short sides of each of the tanks. The hole should be positioned at 3-1/4′′ from the edge and 1-1/2′′ from the top of the tank (Fig. 3A). Be careful to not exert too much pressure or the tanks can crack.

(2)

Use a 5/8′′ boring-style drill bit to expand the opening (Fig. 3B). Again, work slowly so as to not crack the tanks.

(3)

Insert and seal an inner elbow adaptor (Fig. 3C) to each of the holes created in steps 1 and 2. The inner elbow adaptor adheres to the inside of the tank. Liberally apply the silicone aquarium sealant to the adaptor and press firmly into the 5/8′′ hole. There should be excess sealant pushed out around the entire circumference of the adaptor. Note, the adaptor should be positioned with the locking mechanism side up.

(4)

Attach the outer elbow adaptor (Fig. 3D). Liberally apply the silicone aquarium sealant to the outer adaptor before inserting it onto the inner elbow adaptor. There should be excess sealant pushed out around the entire circumference of the adaptor.

(5)

Apply a thin layer of silicone aquarium seal to the threaded end of the elbow insert (Fig. 3E) and slide it into the inner elbow adaptor. It is important that the nonthreaded end be situated outside the tank and positioned down.

(6)

Leave to cure for at least 48 h.

(7)

Use a utility knife to remove excess silicone from around the inner elbow adaptor. Excess silicone interferes with the filter screen attachment.

FIG. 3.

Construction of Larval and Adult Housing Tanks. (A) Top shows the positioning for proper circulation. Bottom shows the drilling of the small pilot hole. (B) Top, the pilot hole is next expanded to the final size. The bottom shows the end result. (C) Top, inner elbow adaptor with silicone added. Bottom is a side view of the adaptor pushed through the drilled tank. (D) Top, outer elbow adaptor. Bottom, outer elbow adaptor placed onto opposite side of the tank with silicone. (E) Top, threaded elbow. Bottom, threaded elbow inserted into the openings of the inner and outer elbow adaptors and sealed with silicone. (F) Top, adult screen cap and adult screen body. Adult screen body has acrylic rod inserted. Bottom shows the cap and body connected with layer of screen sandwiched between and sealed with silicone. (G) Top is a side view of the larval screen. Bottom is the same screen from the side. (H) Top shows the alignment of the adult screen onto the inner elbow adaptor within the tank. Bottom shows the completed water exit port with adult screen in place. (I) Dimensions of a large tank Lid with positioning of holes for water delivery. (J) Dimensions of a small tank lid with positioning of holes for water delivery. (K) Fully assembled large tank and a small tank before attachment of the outer elbow adaptor and elbow insert. (L) Fully assembled tanks with proper orientation above the water return pipe.

Preparing the filter screens

Before starting, obtain all components in Tables 3 and 4. The following instructions will produce 26 of both the adult and larval screens.

Adult screen preparation

(1)

To prepare the screens for the adult tanks, cut 26 pieces of 3′′ × 3′′ fiberglass screen.

(2)

Cut the acrylic rod into 1 cm lengths (one for each screen).

(3)

Insert the rod into the hole in the adult screen body (Fig. 3F top, right). The rod should fit snuggly and sealed in place with silicone aquarium seal.

(4)

Position the adult screen body piece with prong-sides facing up.

(5)

Center the fiberglass screen onto the adult screen body.

(6)

Apply a thin layer of silicone aquarium seal onto an assembled adult screen cap (Fig. 3F, top left) and squeeze it tightly onto the adult screen body. Both the adult screen body and cap have a small furrow that should line up to ensure a proper fit. Cure for 48 h before proceeding.

(7)

Trim away excess screen.

(8)

Insert a prepared adult screen onto the locking mechanism on the inner elbow adaptor inside the tank. Spin the attached screen to lock it into place (Fig. 3H).

Larval screen preparation

(1) Prepare 26 larval screens using the same procedure as above.

The larval screen is considerably larger than the adult screen to allow for adequate water flow through a much finer mesh (Fig. 3G). Larval screens should be checked weekly for debris buildup to reduce the likelihood of tank overflow.

Lid preparation

The lids create shaded regions within the tanks that provide “cover” for the fish. They also prevent fish from escaping out the top of the tank and position the water supply lines to drive flow toward the exit port. The dimensions below are specific to the tanks in the product list.

(1)

Cut the tank lids with a utility knife:

(a) Adult tanks—Cut pieces of foamboard to the following dimension—7.5′′ × 11.5′′ (Fig. 3I)

(b) Larval tanks—Cut pieces of foamboard to the following dimension—5 11/16′′ × 9′′ (Fig. 3J)

(2)

Using the utility knife, carefully trim back the inner perimeter of the foam pieces by 1/2′′ to create a lip around each lid. This keeps the lid from accidently shifting off the tank.

(3)

Drill three holes in the large lids and one hole in the small lids. Having three holes allows the lid to be rotated and allows for the introduction of a glass thermometer.

(a) Drill a (1/4′′) hole in the center of all lids (Fig. 3J).

(b) Drill a (1/4′′) hole 1.5′′ from the edge on both ends of the larger lids (Fig. 3I).

Constructed tanks and proper orientation relative to the return trough are shown in Figure 3K L.

Results/Discussion

We have described a recirculating housing system that is accessible to even the most budget-conscious laboratory. The system, complete with the optional UV sterilizer and 26 interchangeable tanks can be assembled for under $1500, with all components readily available through local and online retailers. Using the filtering described, we estimate the yearly cost to be roughly $75. The UV bulb is not replaced yearly, but when replaced will cost another $60.

We designed the system with an emphasis on simplicity of construction, ensuring that no specialized tools or construction skills would be required. Furthermore, no harmful chemical adhesives are used, eliminating the fear of introducing mutagens to the system water. This is a significant advantage over other published DIY housing systems. The lack of adhesives greatly simplifies routine maintenance and cleaning as the system described in this study can be disassembled, cleaned, and reassembled in under an hour (Supplementary Movie S6). The sump is also significantly larger than those used in other DIY designs. A large sump makes it easier to maintain water quality by buffering against changes in temperature, salt content, and pH that result from adding Reverse Osmosis (RO) water during water exchanges or from normal water evaporation.

Obtaining adequate water recirculation was a critical concern. Other publications describe recirculating systems that use gravity to bring fresh system water into each tank.^11,12 Some models even allow the flow rate to be adjusted to increase or decrease the delivery of fresh system water.¹¹ While effective in delivering clean water, low pressure systems fail to circulate existing tank water resulting in the accumulation of uneaten food and waste in the tank. This can lead to toxic levels of ammonia and nitrate/nitrite.¹⁴ To address this issue, we created a water distribution system that maximizes recirculation to each tank. Even when fully stocked (40 tanks), the pump recirculates ∼20 L/h through each tank. This equates to 4 hourly water exchanges in large tanks and nearly 9 hourly water exchanges in small tanks. This volume of recirculation has, in our hands, been sufficient to effectively eliminate excess food and waste accumulation.

For our tank design we wanted to emphasize the ability to use the same tanks with different-sized fish as well as be compatible with a variety of tanks. We settled on a common exit port that interlocks with various filters that prevent fish from escaping the tank. The two types of filters developed for the system allow the same tank to be used to house either adult or larval/juvenile zebrafish. The common exit port we developed could be used on a variety of plastic containers, besides the one we used. Our tank parts are produced with polylactic acid (PLA). PLA is the most widely used fused deposition modeling (FDM) printing material, but it does degrade with exposure to heat and UV radiation. On our system, PLA parts start to become brittle after about a year. Accordingly, we routinely replace older parts during tank maintenance.

In conclusion, the housing system described in this article is inexpensive, easy to construct, and easy to maintain. Because the system is relatively simple, it is very easy to move, reconfigure, or expand as a laboratory's circumstances change. Additionally, the disassembled system can be stored in a small space and reassembled as needed. The ability to have a “pop-up” colony should be useful for departments who wish to occasionally use zebrafish for a laboratory course but do not have the personnel or desire to continuously maintain a colony.

Footnotes

Acknowledgments

The authors would like to thank the following individuals for their editing of this article: Sam Grzesik and Jason Morgan. They would also like to thank the many students who have helped maintain and modify the housing system over the past 3 years.

Authors' Contributions

B.L.—Conceptualization, Methodology, Software, Resources, Writing—Review and Editing, and Supervision. J.L.—Investigation, and Writing—Review and Editing. N.S.—Investigation, and Writing—Review and Editing. D.L.—Conceptualization, Methodology, Investigation, Resources, Writing—Original Draft, Writing—Review and Editing, Visualization, and Supervision.

Disclosure Statement

No competing financial interests exist.

Funding Information

Funding was provided through internal research support funds from Shenandoah University and Northern Michigan University.

Supplementary Material

References

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Supplementary Material

Please find the following supplemental material available below.

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