The use of zebrafish as a laboratory animal has been increasing in recent years amongst Malaysian researchers. The popularity of the zebrafish lies in its pliability as an adult as well as embryonic model for research in fields ranging from genetics to disease modelling to toxicity screening and drug discovery. Zebrafish are generally less costly compared to rodents, and their high fecundity is not dependent on seasonal changes thus ensuring that embryos are available all year long.

 

Despite the ease of use of the model, regular zebrafish users would be familiar with one distressing complication – zebrafish adults either failing or refusing to spawn, thus disrupting schedules and planned experiments. This article will address some common issues that might lead to failure to spawn, and offers a few suggestions to resolve the problem.

 

1. Water quality parameter. Water quality must be maintained to be stable to ensure efficient zebrafish homeostasis. Inconsistent water quality results in zebrafish using up their body’s energy and resources to maintain homeostasis, and as a consequence, the fish will not spawn. Zebrafish can reasonably tolerate a pH range of 6.8 – 7.5, and the zebrafish system’s pH must be checked daily. The exact pH chosen for lab is not as important as keeping pH stable. The breakdown of organic waste products can result in the water becoming acidic and sodium bicarbonate can be used to gradually increase the pH to its predetermined value.

 

 

 

Zebrafish are stable at temperatures within 24 – 30°C, but again once a temperature is fixed it should remain stable. Water temperature should also be monitored daily and abrupt changes to temperature should be avoided in order to avert stress. Zebrafish are particularly affected by higher temperatures because oxygen concentration in water drops quite quickly with increasing temperature. The zebrafish’s metabolism and oxygen requirements increase by 50% for every 5° increase in temperature.

 

 

 

Salinity levels in a zebrafish system can vary whenever fresh water is added or when water is lost to evaporation. Since zebrafish are freshwater fish, osmoregulation is required to balance internal body salts and water against the salinity of the water environment. Osmoregulation saps energy, but energy loss can be minimized by keeping water conductivity stable (the recommended range is between 300 and 1,500 μS) as this would reduce the energy the zebrafish needs to maintain body salts, which it turn would maximize energy dedicated to breeding purposes.

 

Ideally, about 10% of the system’s water should be refreshed daily to maintain good water quality. Overfeeding should be avoided in order to minimize waste in the system and mechanical filters should be changed regularly to avoid clogging and interruptions to water flow. Very importantly, there should be zero levels of ammonia in the water as ammonia is extremely toxic to fish.

 

Another significant water concern is that zebrafish are strongly reliant on olfactory inputs. Spawning and social cues are mediated by olfactory response; a zebrafish overwhelmed by excess pheromones in the water is unlikely to breed. Thus, it is important to ensure pheromones and other wastes generated by metabolic processes are diluted or adsorbed effectively. This can be accomplished by changing the carbon filter regularly. Activated carbon will bind to and remove organic compounds that contribute to colour and odours.

                

 

2. Breeding methods. Zebrafish will spawn at the onset of light, which can be simulated by lights in the room or lab turning on at the same time, daily. Some labs might rely on natural lighting through a window if the room is not equipped with lights on a timer. Adults can be set up for spawning through in-tank breeding or pairwise breeding. In-tank breeding is more labour-efficient whereby a container is filled marbles and dropped into the fish tank at the moment when there is light. Alternatively, the in-tank breeder container can be left overnight in the fish tank. Bear in mind that adults consume their own eggs, so the in-tank breeder container should be one that prevents the fish from doing so. Having marbles in the container encourages the females to lay over the marbles (mimicking their behaviour in the wild where they prefer to spawn over gravel); the eggs then sink past the marbles to the bottom of the container and the adult fish are unable to squeeze past the marbles to suck up the eggs. The addition of environmental enrichment in the form of artificial greens/leaves can also encourage spawning behaviour.

 

Pairwise breeding is performed by taking the adults out of their tanks and placing them in breeding tanks. The water used for the breeding tanks should be of the same quality as the water the fish live in. Pairwise breeding should be set up late in the afternoon to minimize the time spent in the breeding tanks. Avoid feeding the fish once they are in their breeding tanks, as the leftover food and waste excreted can result in a dangerous buildup of ammonia levels. There should also not be too many fish in one breeding tank and the fish should not be left in the breeding tank for too long the next day, as waste and ammonia will accumulate the longer the fish remain in the tank. Meanwhile, changing the water in the breeding tanks at first light the next morning might encourage the fish to spawn, and this can be done by carefully transferring the internal breeding tank into another tank filled with fresh water.

 

Lastly, there is sufficient evidence to suggest that zebrafish prefer to spawn where the water is shallow, as opposed to laying their eggs in deep water. Having an incline in the breeding tank, or positioning an in-tank laying tray nearer to the surface of the water should encourage the fish to spawn with greater enthusiasm.

 

3. The right gender? The right mates? The selection of males and females to set up for spawning requires skill and patience to differentiate between the genders, especially as the zebrafish never stay still! Males have a more streamlined body and are typically described as being more golden, with darker blue stripes. Females appear more silver or bluish-white, and have white protruding bellies laden with eggs. However, individual colouring can be difficult to discern while the fish are darting around their tank to evade the nets that are trying to scoop them up. The protruding belly is the easier distinguishing factor, but there remains the possibility that a fat male might be mistaken for a female. For definitive evidence that a fish is female, look at her urogenital area for the ovipositor, a small triangular flap positioned anterior to the anal fin and posterior to the pelvic fin.

 

Zebrafish are generally sexually mature by 3 months of age and can continue breeding till they are 18-months-old. However, the best breeders are typically between 6 – 12-monthsold. Males are likely to be able to spawn even past the age of 2 years. There must be sufficient nutrition provided to fish that are spawning regularly, and regular spawning is to be encouraged. Females that do not lay frequently are at risk of becoming egg-bound where their ovarian ducts become blocked. Housing zebrafish according to gender, without setting them up to spawn at regular intervals, can increase the risk of the females becoming eggbound so it is important to ensure all fish of breeding age are allowed to spawn regularly.

 

The exact ratio of male to female fish for successful spawning is worth debating. There are anecdotes of some labs having success with a higher male to female ratio, or even having a young male alongside an older male so as to promote competition amongst the males to engage in spawning activity.

 

Revisiting the point about zebrafish responding to olfactory cues, it is now known that female zebrafish are selective about the males they would respond to wherein females prefer not to mate with males they are related to. Many zebrafish labs have inbred fish colonies, and this does adversely affect spawning rates. Avoid genetic bottlenecks to avert this problem. 

 

4. Environmental disruptions. Zebrafish have a fixed photoperiod and any disruption to the light cycle can result in the zebrafish not spawning. It is worth checking whether lights are unexpectedly coming on at the wrong time (having lights on when the zebrafish should be in the dark), or if there are lights being left on overnight.

 

Noise and vibration can have an adverse effect on the fish, inducing stress. Sudden loud noises from a room next door, persistent noises within the lab, vibrations from other equipment near the zebrafish system – these can all be disruptive. Zebrafish can also sense vibrations from renovation works that may be happening in other parts of the building, and over time, the stress arising from such vibrations will result in poor spawning.

 

5. Health. In addition to stress, fish can be affected by the disease and if the fish are unwell, they will not spawn. Practice strict quarantine protocols when bringing new fish into the lab. There are helpful resources online that provide lists as well as reference photos of pathologies that can affect zebrafish (one such online manual is listed in the References below). Being aware of these conditions can enhance the daily monitoring and observation of the zebrafish for signs of ill health. Zebrafish that are emaciated, diseased and egg-bound will never spawn. Commonly, these sick fish are to be immediately and humanely culled, which will also effectively reduce the risk of a disease spreading through a system.

 

By methodically assessing the issues outlined above, it is possible to begin to troubleshoot what may be causing poor spawning incidents. For additional information, a video lecture by Mr. Christian Lawrence on “The Art of Breeding Laboratory Zebrafish” (the URL is provided in the References below) is exceptionally valuable – parts of this article were sourced from Mr. Lawrence’s presentation – and I would recommend for all zebrafish users whether or not they are presently having problems. In conclusion, the key to successful spawning everytime is to ensure that the zebrafish are of good genetic quality (avoid in-breeding), healthy, and living in a stable environmental free of stress-inducing factors. In other words: keep the fish happy and the fish will provide!

 

Avdesh, et al. Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction. J Vis Exp. 2012; (69): 4196. Accessible at

 

Guide for the Care and Use of Laboratory Animals, 8th edition. National Academies Press (US); 2011. Accessible at https://www.ncbi.nlm.nih.gov/books/NBK54050/

 

Kent, et al. Diseases of Zebrafish in Research Facilities. Accessible at

 

Lawrence. The Art of Breeding Laboratory Zebrafish. 2015. Accessible at