Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus

So as the Astyanax mexicanas is a small fish. It is indigenous to Northeastern Mexico and Southern Texas. The important thing about it is it exists in two forms.

One form is a surface dwelling fish that has large eyes and pigmentation, and the other form is a cave-dwelling fish that has lost its eyes and lost its pigmentation. They're still capable of spawning, of hybridizing with each other, so they can be used in a forward genetic analysis to study genetics and development. Also have large embryos that are transparent, and they can be used to study comparative developmental biology.

So they've been used for many years now as a system to understand the evolution of development. Several days before the spawning is desired, we begin to feed the fish better. We also increase the temperature incrementally such that within the time we want them to spawn, they will then undergo about two or three days of excessive spawning, producing large numbers of fertilized embryos that develop well and through the larval stages to adults.

After feeding the fish at 10:00 a.m. with a pinch of Tetra flakes, check the incubator that will be used to house finger bowls of developing embryos and change the water if necessary. Check the water level in all of the reservoirs and add system water to any reservoirs that are running low.

Then set the incubator to 23 degrees Celsius At least 30 minutes after the first feeding, feed the fish in the breeding tanks with a pinch of egg yolk flakes, enough black worm clusters to allow each fish in the tank to consume about five to 10 worms or both. At least one hour after the second feeding, scrub the breeding tanks as necessary and carefully set up breeding nets without interfering with the tank air supply. The next morning, remove the breeding nets from the bottoms of the adult system tanks between 9:00 and 10:00 a.m.

and use the hose attached to the to gently rinse the embryos into a handheld net. Invert the net into a finger bowl of clean system water and transfer about 100 embryos per finger bowl into 200 milliliters of clean system water supplemented with 0.00003%methylene blue. Stage the embryos under a light microscope to estimate the time of fertilization according to the A-mexicanas developmental timetable.

After staging, place the finger bowls into an incubator for five to seven days and feed the cultures with living brine shrimp. For every tank that dropped embryos, record the date and tank number and record the number and quality of embryos dropped. Record the average time of spawn according to staging table and record the temperature of the system at the time when the fish spawned.

Monitor the finger bowls of embryos throughout the day, using a pasture pipette to remove any dead or deformed embryos and debris, such as uneaten food or feces, and changing the blue water as necessary. At 1:00 p.m. feed the fish in the breeding tanks as demonstrated and set the water temperature to 24 degrees Celsius.

Then remove any excess food and debris from the tanks and scrub the tanks before resetting the nets. On Wednesday and Thursday, repeat the embryo collection and evaluation as demonstrated for Tuesday. Feed the fish and set the water to the appropriate temperature.

Check the embryos in the incubator and replace the finger bowl water with fresh methylene blue treated system water. Then clean the breeding tanks as needed and reset the nets. On Friday, after feeding the fish, set the water temperature to 23 degrees Celsius and check the embryos in the incubator.

On Saturday and Sunday, feed the fish. In this figure, the breeding data from July 2017 through March 2020 for the Rio Choy and Texas Surface Fish and the Los Sabinos, Tinaha, and Pachon cavefish are shown. The data were analyzed by breeding week and the average number of embryos collected per day during a single breeding week.

The data indicates that breeding was continuous throughout the year in Rio Choy and Texas Surface fish and in Pachon cavefish. The quantity and quality of most Rio Choy surface fish was between low and high, while the quantity and quality of most Texas surface fish and Pachon cavefish was between low and medium. The occurrence of spawning was not continuous in Tinaja or Los Sabinos cave fish, with spawning being low or non-existent during the late summer to autumn in these fish.

Although the lowest levels of spawning were recorded for Los Sabinos cave fish, the quality of embryos was the best. Overall, this surface fish exhibited a better spawning quantity and quality than cavefish. Using this system, we can spawn hundreds, actually thousands of eggs bi-monthly, and these eggs are then used, these embryos are then used for many different purposes.