Couples Counselling for Zebrafish: How to Optimize Breeding Efficiency

It’s Sunday morning, the sun has just begun to rise, and you find yourself on the way to the lab (again!), sipping hot coffee and melancholically thinking of your abandoned bed. But something is different this time. Today, the freezing-cold wind blowing from behind is not the only motivation pushing you to sacrifice another weekend in the name of science. Today, you will perform the experiment that will boost your scientific career—you can bet on that!  And then lose. Only an hour later, indeed, a way less motivated version of yourself is staring hopelessly at a bunch of zebrafish couples lazily swimming around, with no intention of spawning a single egg for your “Nature” cover.

If you work with zebrafish (Danio rerio) as a model system, I am sure that sooner or later this has happened to you.

But why do zebrafish couples refuse to mate sometimes? And most important, is there anything you can do to make them spawn and rescue your experiment? More often than not, the answer is yes! Let’s see together how to optimize zebrafish breeding efficiency in a laboratory setting.

Major Factors Affecting Spawning

One of the advantages of zebrafish as a model system includes high fecundity, with up to several hundred embryos produced per spawning session. However, these optimal results are a factor of animal health, which in turn depends on different parameters. To maximize breeding efficiency in captivity, here are some of the most important aspects you must consider.

1. Light Cycle

Zebrafish reproduction depends strongly on light cycle. The photoperiod commonly used is 14-hour light and 10-hour dark, which mimics the light cycle during peak breeding season in the native environment.

Disrupting the photoperiod can have devastating effects on breeding efficiency. Therefore, always make sure the light cycle timers work properly and never open the door or switch on any light during the dark period.

2. Diet and Physical Appearance

Since a male fish can’t take his lady out to a fancy restaurant the night before mating, it is your responsibility to ensure that breeders are properly fed. In my experience, the best feeding strategy is a combination of brine shrimp and a flake diet—occasionally supplemented with high protein food (for example, spirulina) if needed—provided twice a day.

To make sure fish are supplied with a balanced diet and matings are going be successful, you should observe every aspect of the fish. Although it might be true that for real love physical appearance does not matter, body shape is very informative when it comes to fish fecundity.

Females that do not show a nicely round belly have very low chances to produce a good amount of eggs. If this is the case, provide the fish with additional food for some days before mating them. On the other hand, females that appear too chubby probably will not lay if bred. Indeed, if fish are not spawned often, necrotic cloned eggs might clog the oviduct. To prevent fish from becoming ‘eggbound’, breed them at regular intervals even if eggs are not required.

3. Rest

Mating is an energy-demanding process, especially for female fish. Therefore, fish must rest for an adequate period of time before they are used for the next spawning. I personally let females rest for at least a week and males for a minimum of two days.

To ensure fish are not bred too often, a good strategy is to record the mating date and the number of embryos laid by fish from each tank.

4. Age

Just like for humans, age is a key factor in fish fertility, with fecundity beginning to drop with fish older than 1,5 years. I usually rotate animals off the system by 18 months when possible, and never rely on fish older than 2 years to perform experiments.

It is important to consider that young fish which just reached sexual maturity (3 months) may produce eggs that are of lower quality and quantity at the beginning.

5. Inbreeding

Like for other animals, repeated sibling-crossing of zebrafish leads to reduced fertility and quality of offspring due to inbreeding. Therefore, I strongly recommend to propagate fish stocks by out-crossing.

How to Deal with a Non-Cooperative Fish Couple

Let’s assume you have taken care of all the aspects discussed above but the young, non in-bred, properly fed, and well-rested fish couples that you have set up still refuse to mate.

What to do now (hint: staring helpless at the breeders it’s not the answer. I suspect they might prefer some privacy, you know)? All is not lost! Don’t let your motivation circle the drain and try out these tricks instead.

• Water quality is an essential parameter not only for fish health, but also in the mating environment. Since breeding are usually set up in the late afternoon and fish start mating the next morning, the water can become foul overnight. Placing mating fish in fresh water can frequently initiate spawning.
• If simply freshening the water does not help, you can try to exchange it with water at two degrees lower. I must admit I never tried this myself, but it is known to serve as a trigger for the breeders to spawn.
• The zebrafish like to spawn in shallow water. To mimic this mating behavior, I reduce the amount of water inside the breeding tank or tilt the mating tank inserts so that the grating lies at an angle.
• Like their human counterpart, male zebrafish prefer to change their partner. This promiscuous – no judgments here – sexual behavior suggests that using more females than males in a breeding set-up can improve embryo production. If possible, you should then consider using group crosses, which allow you to control gender ratios. Alternatively, if you are like me and still prefer single-pair matings, you can transfer one of the female into another tank (to obtain a ratio of two females per one male) directly on the breeding day. This can have an additional positive effect on spawning because mate choice also depends on olfactory cues and visual stimuli. Therefore, the same fish may mate with one partner but not with another.
• Put on your romantic playlist, be patient and let the fish mate for as long as needed. This is not a real trick, I know. But you have exhausted all your options and you have nothing to lose. Although spawning typically takes place shortly after mating is initiated, sometimes it can happen even after several hours. And even if the romantic music will not make fish spawn, it can help you relax and overcome frustration. Well, unless you are getting over a breakup. In that case, I highly recommend you skip this step.