One of the characteristics that make zebrafish a fantastic model for research is that they spawn…a lot. A healthy female zebrafish can lay upwards of 1,000 eggs each week. By comparison, the mouse, another species widely used in research, may have a single 12-pup litter each month.
Sometimes, though, that isn’t enough. A researcher screening a library of chemicals for potential drugs, for instance, might need tens of thousands of zebrafish embryos, all at the same developmental stage, to have statistically meaningful results.
That researcher could really use the iSpawn.
Developed by Christian Lawrence and Isaac Adatto of Children’s Aquatic Resources Program and the laboratory of Leonard Zon, the iSpawn is a specialized breeding tank designed to take advantage of the zebrafish’s natural preference for breeding in shallow water. “Zebrafish are native to South Asia,” explains Lawrence, who manages the zebrafish facilities at Children’s Hospital Boston. “When the rainy season comes, you find them spawning in the shallow margins of rivers, ponds, rice paddies, any body of fresh water.
“What we’ve done,” he continues, “is take that knowledge of how the fish behave in the wild and applied to the laboratory setting.”
Building a better breeding tank
Typically when you want to breed zebrafish, you take males and females out of a housing tank and put them in a breeding tank overnight, separated by a mesh barrier. “Zebrafish prefer to spawn in the morning,” Lawrence says. “And you need to give them some time in the same tank to respond to each other’s pheromones. So we set the experiments up the night before.”
The next morning, you remove the barrier; the fish do the rest.
The problem with this 30-odd-year-old method is that getting enough zebrafish embryos to conduct a large-scale experiment can take dozens of tanks, several man-hours of labor to transfer the fish into the tanks, and – if you need developmentally synchronized embryos – a couple dozen people to remove the barriers nearly simultaneously in the morning.
Aside from its greater volume (the most recent version holds 100 liters of water and up to 250 fish at a time), what really sets the iSpawn apart is its sliding bottom insert. The insert has an undulating mesh floor reminiscent of the contours of a riverbank. You set experiments up much as you would in a standard tank, but in the morning you raise the insert to its shallowest setting, nearly beaching the fish in about a half-inch of water. “Almost immediately, the fish start spawning, and you can see embryos floating down to the bottom of the tank,” says Adatto. “It almost looks like it’s snowing.”
He’s not kidding. On a recent morning, Adatto and Lawrence treated me to a demonstration of the iSpawn. Sure enough, almost as soon as Adatto removed the separator and raised the insert, the tank started to resemble the inside of a snow globe.
The system holds much promise for genetic and stem cell researchers who use zebrafish. In a paper in PLoS ONE, Adatto, Lawrence, and Zon, director of Children’s Stem Cell Research Program and a leading authority on zebrafish as a research model, directly compared the iSpawn’s performance with that of the standard breeding practice, looking at parameters like the time needed to conduct a large-scale collection and the numbers of embryos collected. The iSpawn won hands down, allowing the team to collect double the number of fish embryos (8,400 versus 4,200) in a quarter of the time and less than one-fifth the amount of lab space.
I asked Adatto to tell me the highest number of zebrafish embryos they’ve collected in one breeding session with the iSpawn. His answer: 10,000 in 10 minutes.
“The iSpawn is going to have a major impact on how researchers work with zebrafish, not only at Children’s but internationally,” Zon states. “The potential savings in time and efficiency is really significant and could speed up our ability to search for therapies through chemical screening.”
An evolving concept
The apparatus has come a long way from its humble beginnings. “A former Zon lab technician had noticed a spawning preference when she simply tilted a breeding tank to make one side more shallow,” Adatto relates. “I thought I could create something that would work for more than two or three fish. So I built a tank with a sliding insert using a five-gallon bucket, some mesh, hot glue, and a hacksaw.” It worked well enough that Adatto and Lawrence decided to try to scale it up into something that would be efficient enough for lab use.
Adatto and Lawrence worked with a plastics fabricator to develop several large-scale prototypes, and then with the hospital’s Technology and Innovation Development Office (TIDO) to license the design to Tecniplast, a manufacturer of animal care equipment for research. The company, which will further develop and market the iSpawn, unveiled its latest design at the recent European Zebrafish Meeting in Scotland.
Lawrence readily acknowledges that they’re not done learning about the principles of zebrafish behavior. “There is a staggering number of things about this fish and the way it behaves that we don’t yet understand,” he says. “So while the iSpawn has answered many questions, it’s actually opened up many, many more. There is a plethora of things that we could learn about their reproductive behavior that will hopefully help us make the iSpawn even more efficient in the future.”