Zebrafish are social fish, used in research notably for their ability to produce large numbers of offspring. At the end of this month under the sign of love, we are going to present to you the mating process of zebrafish and how laboratories and fish facilities manipulate this natural process to obtain many eggs at the right time. In order to ensure the quantity of eggs needed for the various experiments, it is necessary to understand and have solid knowledge about these mechanisms. Major protocols used are highlighted in this article.
From flirting to childs
Zebrafish are social fish, living in a shoal. In the wild, the reproduction season is correlated with the arrival of the monsoon. But reproduction is not only linked with the abundance of rainfall but also with the increase of prey and the food availability (1). In laboratories, several methods are used to mimic these conditions and are described in the next chapter.
The mate choice is depending on olfactory cues, visual stimuli and social interactions (2). Females seem to have more exigent physical criteria to select a male. In fact, they preferred larger male with certain pigmentation patterns (2). Furthermore, their selection doesn’t stop at these criteria. Females also look to breed with males defending a more favorable territory. In fact, the optimal spot to spawn their eggs contain gravels and vegetation (1). In order to be noticed by the females, the males have developed two strategies: territorial defense and active pursuit of females (2).
Once the female has set her sights on a male, a courtship behavior is observed. It consists of a kind of dance, where males stimulate the release of eggs. All the steps of this particular behavior are described in our article “Do zebrafish fall in love?”. During the courtship, an important chemical process occurs: pheromones are released to synchronize the reproductive function between males and females. Male’s pheromones stimulate ovulation in females and then, females release pheromones to attract males and to stimulate courtship behavior. Pheromones coming from dominant females have also an inhibitory effect on ovulation of subordinate females (2).
At the end of the courtship behavior, the female releases the eggs and simultaneously, male releases sperm in the environment. In fact, zebrafish have an external fertilization (2). Fertilized and unfertilized eggs fall on gravel bottom and after 2 to 3 days, fertilized eggs hatch into free swimming larvae (1). Zebrafish don’t provide parental care; so male and female are fertile again some hours after the spawning (1,2).
Mating protocols in laboratories
For all laboratories working with animal models, one of the objectives is to replicate the wild conditions (3). Although in the wild zebrafish spawn in groups with multiple males and females, it is also possible to form spawning pairs.
- Advantages of group spawning: follow the natural behavior, ensure genetic variability (3).
- Advantages of pairs spawning: to obtain a particular mutant or transgenic line, to perform mutagenesis screen (3).
In both cases, mating happened normally at the onset of light. But if eggs are required later during the day, isolation cabinets can be used, with a shift in dark and light phases. Another possibility is to perform in vitro fertilization (2). To realize this artificial spawning methods, Brand et al. (2002) have developed an in vitro fertilization protocol, using frozen sperm. In fact, eggs can be collected on anesthetized females by hand pressure on the ventral side of the fish. Sperm can also be collected from anesthetized fishes or from dissected testes (4).
Zebrafish mating is influenced by a lot of factors, including environmental conditions. Zebrafish are able to tolerate a wide range of conditions but operating outside their optimum range involves an increase in energy costs and as a direct consequence, a decrease in reproductive activities (1).
Another factor influencing the mating is also the fish’s age. Zebrafish reach sexual maturity at three months (2). The maximal breeding performances happen between 6 months and one year (2). After that, zebrafish are less efficient (5).
The breeding frequency also influences breeding efficiency. Females not exposed to males for a prolonged period of time develop a plug that clogs the oviduct and prevents oviposition. In contrast, females exposed to males can develop malformed eggs that reduce the embryo’s survival (2). It has been reported that the optimal breeding frequency is every 10 days (2).
Then, when a laboratory has met all these conditions, zebrafish can be placed in the evening in a mating tank with high hopes of obtaining eggs. Laboratories have developed many techniques but the most basic breeding techniques require placing marbles at the bottom of the mating tank (1). This simple technique mimics the gravel substrate present in the wild and also prevents egg cannibalism by the adult. But the collection of eggs in the space between the marble isn’t easy and some eggs can be damaged by being crushed between or under the marbles.
To facilitate the egg collection, two other major techniques have emerged. The first consists in a breeding tank where the “floor” is meshed, placed in a larger tank. This method allows the eggs to fall through the mesh to prevent cannibalism by adults (1,4). Although this method has the major advantage that all the eggs spawn can be collected, zebrafish have to be manipulated (inside and outside the larger tank) and can generate some stress.
Another method consists of carefully emerging a container inside the main tank. The container has a grilled top through which eggs can fall (5). The advantage of this method is the ease of collecting the eggs just by removing the container. But another consideration is that zebrafish have to spawn above it and sometimes it is not the case. To increase the probability that they spawn just above it, plants and marbles can be placed on the grill.
If, despite these efforts, spawning does not take place, it is still possible to play on the breeding tank size or the male:female ratio. A larger breeding tank can allow swimming in bigger space and it is easier to control the physico-chemical water parameters. Concerning the ratio between male and female, it plays a bigger role in case of group spawning, where placing less male than female ensures aggressivity avoidance (3,5).
Conclusion
Zebrafish mating depends on a lot of biotic and abiotic factors. From the age of the fish, the male:female ratio to the temperature and the hardness of water, all factors have to be in optimal range to ensure a successful breeding.
The lack of eggs can reflect sub-optimal husbandry conditions or also be a sign of parasitic disease.
In research, replicability is essential and the zebrafish breeding doesn’t make the task obvious. To address replicability issues, it is essential to identify all factors influencing our results, to control them and to standardize protocols.
References
- Lawrence C. The husbandry of zebrafish (Danio rerio): A review. Aquaculture. 2007;269.
- Nasiadka A, Clark MD. Zebrafish Breeding in the Laboratory Environment. Inst Lab Anim Res J. 2012;53(2).
- Tsang B, Zahid H, Ansari R, Lee RCY, Partap A, Gerlai R. Breeding Zebrafish: A Review of Different Methods and a Discussion on Standardization. Zebrafish. 2017;14(6).
- Brand M, Granato M, Nüsslein-Volhard C. Keeping and Raising zebrafish. Zebrafish. 2002;
- Avdesh A, Chen M, Martin-Iverson MT, Mondal A, Ong D, Rainey-Smith S, et al. Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction. J Vis Exp. 2012;69.
First image: zebrafish embryo. Picture taken by the EggSorter