Construction of an Affordable and Easy-to-Build Zebrafish Facility

Summary

A Zebrafish facility suitable for breeding and in vivo research is built in an easy and affordable manner. Maintenance is minimal. This facility is ideal for student involvement and independent research.

Abstract

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.

Introduction

In vivo models are essential in conducting comprehensive biomedical research since they allow in vitro research to be translated at the system and organismal level. For example, research with small mammals, namely rodents, has been proven necessary in the development of drugs and in the improvement of available drugs. However, the complexity (financially and logistically) might make it virtually impossible to build and maintain rodent facilities for experimentation proposes at small research academic institutions. Nonetheless, in vivo research can be performed using other animal species that require less elaborate logistics. Zebrafish (Danio rerio) have been successfully used as an in vivo model to study genetics, development, pathophysiology, and pharmacology^1,2,10,11,12. The facilities required to house zebrafish for experimentation are relatively simple and significantly less expensive when compared to rodent facilities. Kim et al.³ have recently described in detail an 80 tank zebrafish housing system that can be built for approximately $1,500, which includes both parts and labor costs. This stands in stark contrast to the nearly $8,500 that would be required to purchase a ready-made, 80 tank system from any number of commercial suppliers. However, the description given by Kim et al. may be complicated and difficult to follow for novices of fish research. We believe that a zebrafish facility can be constructed in simplified manner for an investment of $1,500.

The establishment of an in vivo facility at small academic institutions provides their faculties an additional tool with which to produce more powerful and thorough research. In vitro data supported by in vivo results generally produce more comprehensive biomedical research than data obtained using only in vitro approaches. Therefore, the implementation of an in vivo research facility will significantly enhance the institution success in its overall research endeavors (student exposure to research, publications, communications, pursuit of external funding, etc.).

The construction of a Zebrafish facility like the one described in this study is ideal for research academic institutions with limited facilities (or institutions without in vivo research facilities) during constricting financial times.

Protocol

NOTE: These protocols follow the guidelines of IACUC of Presbyterian College.

1. Acquire Materials Listed in Table 1.

2. Assemble the 5 Shelf Chrome According to the Manufacturer’s Instructions. This will be used to House the Facility.

3. Construct the water reservoir and filtration system (Figures 1A and 2).

1. Using Figure 1A as a guide, layout the 18 gallon bucket, pump, filters and UV lamp to determine final installation location and size.
2. Once parts are laid out, measure and cut the ¾” PVC pipe to the length needed for assembly. Vary the lengths of all pipes according to specific space needs and limitations.
3. Assemble the pipes and fittings as laid out previously using the PVC primer and cement, see below (3.3.2 and 3.3.3).
   1. Sand the cut edges of the pipe with sandpaper until they are smooth to the touch and there is no debris.
   2. Prime the outside of the pipe and the inside of the fitting with the purchased PVC primer with the included swab from the PVC primer.
   3. Cement the pipe and fitting together by applying the PVC cement to the outside of the pipe using the included swab. Repeat for all steps that need PVC cementing.
   4. Place the 18 gallon bucket to one side of the shelf construction.
   5. Install a bulkhead connector in the side of the reservoir tank.
   6. Attach a ¾” PVC pipe to the bulkhead.
   7. Attach the other end of the ¾” pipe to the pump inlet using a ¾” threaded make fitting.
   8. Connect the filters to the pump using two 90 degree elbows and ¾” PVC pipe.
      1. Use one paper filter followed by one activated carbon filters in order to filter out debris and reduce chlorine and chemical levels in the water.
   9. Connect the UV lamp to the filters according to the manufacturer’s instructions.
4. Attach the pump and filter system to the top side of the first shelf of the rack using wire ties. Vary the order of the assembly and attachment according to the size and layout of the shelf/rack chosen.
5. Add 1 bioball per gallon of water to the water reservoir once the pump and filter system are assembled and attached to the shelf.

4. Construct the main supply line (Figures 1B and 2).

1. Measure and cut a piece of ¾” PVC pipe to connect the UV lamp to the main supply
2. Use a ¾” PVC tee to start the main supply line by connecting the tee to the ¾” PVC exhaust coming from the UV filter. .
   1. Add an on-off valve to the bottom of the PVC tee to serve as a drain for maintenance flushing of the system.
   2. Start the main supply line from the top of the ¾” PVC tee by measuring and cutting ¾” PVC pipe to desired length to provide feed pipes for each of the shelves on the rack.
   3. Install ¾” PVC tees for the horizontal lines that will enter each shelf of the rack.
   4. Optionally (and suggested), add a ¾” on-off valve for each shelf supply line to allow for isolation of each shelf and a modular design of the system.
3. Use a ¾” PVC pipe as a pressure reducing line on the top shelf. If this line runs on the back of the shelf, use this shelf for storage. End the pressure reducing line in the main exhaust line (Figure 1B) with a ¾” on-off valve.
4. Once the above steps have been completed, prime and cement the PVC pipe together as previously done when constructing the reservoir and filtration system.

5. Construct the main exhaust line (Figures 1C and 2).

1. Run the vertical length of the main exhaust line to the other side of the rack from the main supply line. Use a 2” PVC pipe for this vertical line to receive the main supply line at the top and drain lines from the fish tanks from each shelf.
2. Cut the pieces of 2” PVC pipe for the vertical main exhaust line and use 2” PVC tees to receive the fish tank drain lines from each shelf. End the main exhaust line in the stock tank to expel the recycled water over a sponge filter.
3. Cut a one inch opening the entire length of the 2” PVC pipe pieces and attach to vertical main exhaust line with PVC tees for the horizontal drain lines from the fish tanks.
4. Cap the 2” horizontal drain lines with a 2” PVC cap.
5. Prime and cement the pieces using the necessary fittings (connectors, nipples or elbows).

6. Construct the fish tanks (Figure 3).

1. With the drill, cut a 1 1/8” hole at the back of the fish tank 1” below the upper edge with a hole saw.
2. Slide a threaded ¾” PVC male adapter (with a #18 O ring attached) through the hole (from the inside of the tank to the outside).
3. Attach and tighten a 90 degree 3/4' threaded PVC elbow to the male adapter to create the fish tank exhaust pipe. Add a piece of fine mesh before screwing the holding nut to prevent zebrafish going through the exhaust system.
4. Place the fish tank with its exhaust pipe ending into the open space of the draining line behind the tanks

7. Starting the water system

1. In several containers, such as the one used for the reservoir, measure three times the amount of water contained within the system. Let this water stand at room temperature for conditioning for 48 hr.
2. Add water to the system and let it circulate for a minimum of 3 days.
   1. Change the water in the system vigorously throughout the initial 3 days of circulation in order to rid the system of contaminants that reside within the pipes.
3. Add starter zebrafish and allow the nitrogen cycle to get established. Normalize pH to a consistent 7. Keep seven zebrafish per 1.8 L, and feed them twice daily during week days and once daily during weekends as suggested by Linbo⁴ and McNabb⁶.
4. Monitor the nitrate (<75 mg/L), nitrite (<10 mg/L), and ammonium (0 mg/L) daily during the first week after adding fish, and twice weekly once desired levels have been maintained with regular water changes.

Results

Construction of the Pump and Flters System
Once the rack is assembled and set up the first step is to build the pump and filter system. The filters are connected to the system pump and are located above the bottom level of the first shelf, which allows for easy filter maintenance and replacement. Similarly, the UV lamp is connected with PVC pipe to the filter and held in place with plastic wire ties to allow for easy replacement of the UV lamp as needed (Figure 4A). Immediately following the...

Discussion

The main purpose of this article is to demonstrate that the construction of a functional zebrafish facility is not complicated and can be built by novice fish researchers with minimal construction knowledge. Furthermore, this article shows that the materials for this functional zebrafish facility can be acquired readily and affordably from local pet supply stores or hardware stores. Once all the materials and tools have been acquired the construction of the system and the fish tanks takes about one working day. The 72 hr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Item	Brand	Quantity
74-in H x 48-in W x 18-in D 5-shelf Steel Freestanding Shelving Unit	Style Selections	1
18-Gallon General Bucket/Tote	Centrex Plastics: LLC Rugged Tote	1
6-Outlet Power Strip With 15-Ft. Cord	Master Electrician	1
9-Watt UV Water Garden Clarifier FiIter and UV lamp	Smartpond	1
Whole House Water Replacement Filter	Whirlpool	6
Opaque Whole-House Pre-Filtration Housing	Whirlpool	2
Thread Seal Tape	William H Harvey	1
8-fl oz LO-VOC PVC Cement	Oatey	1
8-fl oz LO-VOC Primer	Oatey	1
1/4-in x 100-ft Vinyl Drip Irrigation Distribution Tubing	Mister Landscaper	1
1/4-in Barbed Drip Irrigation On/Off Valve	Mister Landscaper	33
9-Port NPT Irrigation Manifold with Filter	Mister Landscaper	6
Drop-In Filter with House	Whirlpool	6
2-in Dia 90-Degree PVC Elbow	LASCO	3
2-in Dia 90-Degree Tee	LASCO	3
3/4-in Dia 90-Degree Slip Elbow	LASCO	15
3/4-in Dia PVC Adapter	LASCO	7
3/4-in Dia PVC Tee	LASCO	12
2-in Dia PVC Cap	LASCO	3
1-in Dia x 3/4-in Dia PVC Adapter	LASCO	2
3/4-in PVC Socket In-Line Ball Valve	American Valve	5
1/2-in Dia x 3/4-in Dia PVC Adapter	Lasco	8
3/4-in Dia PVC Cap	Lasco	1
2-In x 5-Ft Pvc Pipe	Charlotte Pipe	4
¾-In x 10-Ft Pvc pipe	Charlotte Pipe	3
11/16x1-7/80 PVC ring	SXT	10
½” PVC male adapter	SXT	1
¾” PVC coupling	SXT	1
½” PVC female adapter	SXT	1
link
1.8 L Dual Beta Keeper	Top Fin	27
¾” PVC male adapter (w/ #18 O-ring)	SXT	28
90-degree ¾” threaded PVC elbow	SXT	27
Fine Mesh	N/A	1 yard
Bulkhead Connector	N/A	1
Hacksaw	N/A	1
Drill	N/A	1
Hole saw	N/A	1
Adjustable Wrench	N/A	1
Pliers	N/A	1
Tape Measure	N/A	1
Safety Glasses	N/A	1
Work Gloves	N/A	1