How to produce millions of high quality monosex tilapia fry
by Dr Ram C Bhujel, Director, Aqua-Centre
Historical evidence has shown that tilapia formed part of a nomadic lifestyle and that they were captured from the river Nile in Egypt or as some evidence suggests, might have even been cultured in ponds as far back as 3,000 years ago.
Since then, tilapia have been an important fish from the point of view of food security. However, documented culture of tilapia started since the 1950s or so and its culture was heavily constrained by the limited availability of quality seed.
This is mainly due to its intrinsic characteristics of producing a few eggs at a time asynchronously. Breeding occurs at different times and days among the females within the population, even in the same age group.
Farming of tilapia, especially the Nile tilapia (Oreochromis niloticus), has become increasingly popular during the last two decades due mainly to the availability of high quality monosex tilapia fry and its production technology which has become common.
As a result, mass-scale production of fry technology has been well-taken up by the private sector in Asia and around the globe. There are hundreds of hatcheries in Thailand, with some of them produce over 20 million monosex fry per month. They supply both monosex and brood fry around the globe contributing to the expansion of tilapia industry globally.
As a result of the successes in mass-scale production of monosex fry, tilapia culture in ponds, cages and tanks is expanding all over the world and become the most common species globally. It is now grown in about 150 countries and its products are traded worldwide due to the taste of its mild flavoured, firm textured and boneless meat.
Their fast growth, short generation and frequent breeding behavior means that they are favoured by fish farmers. In accordance with this, global tilapia production has doubled each decade during the last thirty years, with its farming enjoying steady and sustainable growth.
Annual global production of Nile tilapia, including its hybrids, surpassed seven million metric tons in 2020, which is arguably the highest among the individual species. Prior to this, grass carp and silver carp were the two most produced fish with the production of less than six million tons each, according to FAO statistics.
This article will highlight the salient features of mass seed production technology, which has performed a key role in taking it to the top.
Tilapia reproductive behaviour happens naturally
The breeding method of tilapia is unique as they breed naturally and easily if mature males and females are stocked together in ponds, cages or tanks. There is no need to inject hormone or make any arrangements of water flow, kakabans or any shelter.
However, as it is tropical fish, it will need environment especially suitable water temperature range of 24-32°C. Nile tilapia show a high degree of parental care to their eggs and fry as they only release a small number of egg clutches at a time, which ranges from a few hundred to a maximum of 1,500 eggs.
Therefore, thousands of females and males are needed to produce monosexed fry on a mass scale. Another problem is that they spawn at different times and days. Synchronisation has not been possible as they do not respond to hormone, neither can their eggs can be easily stripped. Nevertheless, they can spawn 10-12 times per year or almost once a month - which helps to some extent.
Nile tilapia is a maternal mouth breeder and a female releases eggs in the presence of a mature male which releases sperms to fertilise those eggs, after fertilisation, the female takes back into her mouth and incubates the fertilised eggs in her mouth until the young can swim independently, which is also referred to as swim-ups.
Partial harvesting does not yield good results
Some farmers collect free swimming fry from the edges of breeding ponds, hapas or tanks either on a daily basis, or at an interval of a few days using scoop nets where broods are stocked maintaining approximately 1:1 male:female ratio.
This was the traditional system, and it is still followed in some countries as it is cheap and easy. But net fry production per unit area of space is very low because it is not possible to collect all of the fry from the system, which is why it is also called 'partial harvesting method'.
Survival is also low due to predation and adverse environmental conditions especially in ponds. Furthermore, fry vary in size and age. Therefore, if sex-reversal technique is to follow, partial harvesting does not yield good results because most of the fry are older than 15 days.
Hormone treatment works only for the fry which are sexually undifferentiated, normally before the fifteenth day post-hatch, for example. Some of the hatcheries, harvest fry more frequently two times a day (morning and evening) to get more uniform fry by age and size. However, results are still not of high standard. Those hatcheries grade the fry to maintain uniformity in size but still the age of fry matters. Some fry may remain small if they get less chance to access food. Therefore, egg collection method has become more common practice.
The mouths of the females are small to keep all the eggs for long, which they may lose gradually over the period. Water quality of the breeding ponds or tanks is also difficult to control and is normally sub-optimal and often may contain pathogens and predatory animals such as insects, which can infect fertilised eggs or hatchlings during the incubation in the mouths of the female, or even attack when they come out of the mother's mouth.
Therefore, collecting eggs as soon as they are fertilised and incubating them in good quality water maximises the egg number, thereby the seed output. For this reason, large scale hatcheries have adopted the collection of fertilised eggs or yolk-sac larvae (seed) and their artificial incubation and larval rearing techniques (Fig. 1). The harvesting of the eggs is done once a week or at 5-day intervals.
This method becomes most efficient and cost-effective, if the broodfish are in hapas or ponds. Seed harvest can be even easier in tanks but initial investment to installation is too high for the construction of large or sufficient number of tanks as over 100,000 broods need to be maintained and managed for the production of millions of seed each month.
The same broods can be utilised for one-to-two years. They grow bigger in size and vary in size so much therefore hatcheries prepare new batch batches at least six months in advance to replace the old batch. Maintaining a large number of broods is done in hapa-in pond systems by most of the commercial hatcheries. If the broods are in hapas, it is easy to collect seed by gathering them at a corner on a side of the pond of tank.
Collection of eggs is done using a pair of hand nets with large mesh and small mesh (Fig. 1, left). The large mesh net is for scooping up the broodfish and to check eggs in the mouth, and the small mess net is to collect the seed.
A fore finger is placed in the mouth of a brooding female after catching it making head down and shaking is done to release eggs or yolk-sac fry. Eggs or yolk-sac fry are then transferred to a plastic bowl half-full of water to keeping them submerged. During harvesting, seeds are separated by stages described in Macintosh and Little, (1995) and Little et al., (1997) as follows:
Stage 1 – just fertilised, yellow in color without any spots
Stage 2 – with two eyespots,
Stage 3 – darker in color and with small tail and protruding eyes,
Stage 4 – with longer tail and head,
Stage 5 – swim up fry
These are arbitrary stages based on the embryonic developmental phase which can be observed by the naked eye and kept separately. However, the seeds of a stage from many females are pooled together in a single bowl and are incubated in a single jar. Fortunately, a female lays the same stage eggs at a time, so they don't need to be separated. Normally, each stage takes roughly one day for the embryonic development.
The various stages of egg incubation and larval rearing
The harvested eggs and yolk-sac larvae are washed with clean tap water and passed through a fine net (500 micron), disinfected with 40 percent formalin (@200 ppm) for one-to-three minutes and acriflavine (1g/200 L water) solutions followed by rinsing in clear water each treatment, weighed and then placed into down-welling incubator jars.
The jars are supplied with slow sand filtered water with a constant pressure from a header tank. The flow rate of the water entering into the jars and trays can be adjusted in such a way that all the eggs in incubator jars and yolk-sac larvae in trays are gently churned constantly throughout the day and night.
Therefore, it requires continuous electricity supply. In a country where electricity supply is not regular, a back-up generator or batteries need to be standby.
Freshly laid eggs are normally yellow in colour and it takes about four days to hatch, depending upon the temperature. Each subsequent stage takes approximately one day less to hatch. After hatching takes place, yolk-sac fry are transferred into shallow trays filled with re-circulated clear water.
If the stages 4 and 5 seed and swim-up fry are harvested, they are directly transferred to the tray system for larval rearing for about a week. They become free swimming fry often called 'swim-up fry' are then transferred to the sex-reversal system (hapa-in-pond or tanks). Until then, fry are not fed at all.
By the time, they are transferred to sex-reversal system, feeding of hormone (17a-methyltestosterone or MT) mixed with high quality feed starts. Post-hatch, these fry are approximately 9-10 day old. It is essential to start MT feeding from this age as the sex determination takes place during 12-15th post-hatch.
When they start taking MT feed, the level of hormone in their body is raised which directs the sex towards males. If MT feeding begins late, or when they are older than 15 days, sex reversal won't be possible. This is one of the fundamental principles that the hatchery operators should keep in mind while producing monosex tilapia fry.
Hormonal sex-reversal technique is the most reliable method
Although there are a number of other methods of producing all-male tilapia fry have been attempted, hormonal sex-reversal technique has been the most reliable and common method. The period of sex-reversal can be divided into two phases; 21-day hormone treatment phase, which is followed by the subsequent nursing phase that can range from a week to several weeks.
Feeding young fry with a male hormone (17α-methyltestosterone) produces phenotypically all-male fry (not genetically). It stops breeding in grow-out systems and males grow about 50 percent faster than females, which can be seen specially when fish are bigger in size.
For sex-reversal, 60 mg of 17a-methyl-testosterone (MT) is mixed with 1 kg of fish meal together with 10-15 g of vitamin C or vitamin mixture. The MT is insoluble in water, an organic solvent such as ethanol (95 percent) is necessary to dissolve it. The alcohol then helps spread the hormone evenly in each particle of the feed then it can be evaporated off easily and quickly under shade at ambient temperature.
While making stock solution, normally 5g of MT hormone is dissolved in one litre of ethanol using a magnetic stirrer. The volume is then made up 10 litres (by adding 9 litres), which serves as stock solution and can be stored for about 6 months in a refrigerator at about 4-7ºC. The stock solution contains 0.5 mg of MT hormone per ml of alcohol, that means that 120 ml of stock solution is required to raise the required dose of 60 mg/kg feed.
Some people are concerned about the use of MT in fish food but the amount is so tiny that 300 mg MT is enough to convert 30,000 fish. More importantly, MT is fed during the first month after hatch or when they are very small and fish are grown at least 4-5 months before they are deemed ready to consume it. Research has showed that the MT level returns to normal in the fish after a week of stoppage of the hormone feeding.
Hormone is mixed with feed in batches and of fishmeal is poured into a mixer, with large scale hatcheries typically preparing about 10-20 kg at a time. For five kilograms of feed, 50g of vitamin C or 100 and mineral mixture is gradually added.
Gradual pouring of 300 ml of stock solution is done while mixing and then the same amount of fresh ethanol (300 ml) is also added. This process is repeated once more and churned for about 10 minutes. The ethanol is then evaporated by spreading the mixed feed under shade for over a night or day or about an hour if under sun in the early morning or late afternoon.
After drying, the MT feed should be packed in a plastic bag or kept in a container with an air-tight lid and stored in a room at low temperature or in a refrigerator (approx. 4-7°C). When the hatchery operation gets bigger or expands, 10-20 kg is prepared at a time, doubling all the ingredients.
Normally 20,000-30,000 fry are stocked in 3m x 2m hapa and are fed with the MT mixed feed at 75, 150, 250 and 420 g each day for the first five days. On days 6 - 10, days 11-15 and days 16-21 respectively, the feed is divided into five equal portions or meals for each day to feed 5 times daily. The size of the hapas during the 21 days of hormone treatment for sex-reversal depends on the scale of production. If the fry are not going to be sex-reversed, then they can be fed on a mixture of untreated rice bran and fish meal (2:1) using the same routine.
Uniformity in size is an indicator of good quality
Although each batch of fry coming out from sex-reversal are from the same age group, their size starts getting high variation over the period. Uniformity in size is one of the most important indicators of quality perceived by the farmers. Therefore, grading of fry is an important part of the fry production.
It minimizes mortality due to cannibalism and social dominance, as tilapia fry are aggressive in nature when they are small. The first grading is done the following day of end of sex-reversal on the 22nd day, followed by second grading after a week and most importantly, grading a day before sale is important.
Fry are grouped into three-to-four categories or more such as small, medium, big and very big, depending upon the size. During nursing fry are kept at a density of 1,000 – 2,000 fry.m-2 of the hapa space. The size of the nursing hapa can vary from 20-120 m2 depending upon the scale of production. Fry are then fed on a mixture of rice bran and fish meal (2:1) about four times a day at about 25-50 g.m-2 per day.
The first month after sex-reversal is very critical
Small fry (less than one inch) are susceptible to predation by carnivorous fish, insects, amphibians, reptiles and birds. Small fry are also less tolerant to poor water quality, which is a common feature of ponds with organic manures.
The first month after sex-reversal is very critical and high mortality may occur if they are directly stocked into the grow-out ponds. Therefore, if they are nursed for at least a month before stocking, this can increase survival considerably. Traditional nursing was done in small ponds but nowadays, nursing in hapas is popular as it allows better care including covering by bird nets.
Fry are stocked at 500-1,000 fry m-2 of hapa space and for those who want to nurse in ponds, stocking density can be around 100-200 fry m-2 of pond area. Whether fry are raised in hapas or directly in the pond, water should be drained, dried and limed (300 kg/ha) and fertilised with urea (30 kg/ha/wk) and triple superphosphate (30 kg/ha/wk) fertilisers. Fry are fed with floating pellets or with a mixture of rice bran and fish meal (2:1), 2-4 times a day at about 5-10 percent of biomass or to satiation.
The demand for seed is unpredictable and it differs with season and year to year. It is common to have a large number of fry left unsold during the dry season. These fry can be kept at high densities when unsold and also during the cold season (Overwintering), so that they can be sold early in the next warm season at higher prices.
During this period, fry may not grow due to restricted feeding, (less than one percent) or even less in alternate day and high densities (2,000/m2), but they are old and when they are stocked in the grow-out pond, they can grow fast, meeting their growth potential.
These fry may fetch higher prices because they will be bigger, stronger and show faster growth. This is a common practice in the countries where winter temperatures are generally below 20°C, in which fish do not eat and grow normally. The main objective of this act is to save the fry during these cold season periods.
Overwintering of fry/fingerlings can be done in several ways: using warm water from a heating facility or geothermal water, green houses or plastic coverings for insulation, and stocking in deep ponds, tanks and deep hapas in ponds. The former methods are cost intensive though survival is higher. The most popular overwintering method used by farmers in Northern Vietnam (where winter temperatures can be around 12°C) is stocking seed in deep ponds.
Conditioning before sale
Normally fry are transported using plastic bags. Although they might also be transported using tanks with oxygen in some cases, the use of plastic bags is still the most common method. Fry need to be prepared for transport before they are packed and it is essential to condition the fry/fingerlings by starving overnight so that they empty their stomach and intestines.
This reduces the production of excreta in plastic bags/transporting containers and it also avoids mortality when long transportation is involved. The main problems during fish transportation are shortage of oxygen, high ammonia production from excreta, high temperatures (in hot seasons or areas), as well as the stress due to handling. Fry harvesting should be done at least one day before selling and they should also be graded before harvest and kept in separate hapas based on their size.
After grading, fry are counted and transferred to containers or tanks filled with the water from conditioning tanks. They should be transported to conditioning tanks immediately. The conditioning tanks are supplied with clear water using sprinklers or with air-stones and the fry can be kept at high density over night or more.
Before putting fry into the conditioning tanks, they are dipped in a formalin bath (400 ml/100 L water) for 5-10 second to make sure parasites or other disease-causing organisms are not transferred to the conditioning tanks and to the farmers field.
Quality assessment and certification
Quality of monosex tilapia fry is expressed mainly as percentage of male achieved through sex-reversal process because if all fry are males, they will not breed in the grow-out systems, grow faster and bigger. The best quality means achieving higher than 99 percent male in the fry population.
However, some of the hatcheries can achieve hardly 90 percent or so while others may not even that. If 10 females are present in a grow-out pond, it can create problems. Therefore, there are some issues about the quality produced by some hatcheries. Quality monitoring and control system is currently lacking. For checking the percentage of males, three random and representative samples from the batch sold to the customers are further nursed in isolation for about two months or until they get five gram in weight.
They are dissected to see their gonadal tissues under microscope. The method has been described as Gonad Squash Method by Guerrero and Shelton, (1974). Fry quality can be assessed by observing the movement, colour, shape, size and responses to feed and strangers. Another method is to test their strength using a salinity challenge using 24 ppt salt solution for about two hours and to see the survival.
Nile tilapia is the aquatic chicken
Methods developed through a series of collaborative research on monosex tilapia fry production technology as described above has helped Nile tilapia to become the most widely cultured species in the world.
However, dependence on fishmeal, alcohol and steroid hormones have some issues. More research is needed to minimise or avoid them. Tilapia farming is spreading rapidly all over the world and rapid expansion has made it difficult to keep up-to-date data regarding production and the number of people engaged in tilapia industry.
It has expanded across the wide range of culture systems; from small ditches to large ponds, tanks, and cages in rivers, canals, lakes, and reservoirs, in fresh and seawaters within peri-urban to rural areas.
Tilapia is playing a significant role in food and nutrition security as it is consumed by the poor and middle class especially those residing in the rural areas. Tilapia is also consumed by the urban elites too, who go to restaurants frequently and buy their foods from supermarkets. Therefore, tilapia has become 'everyone's fish'.
Although it was originally from Africa, it has been introduced to many countries of the world, where it has great contribution to the income and employment of the people. Tilapia has a potential to emerge or stand out from among the hundreds of farmed aquatic species, like that of chicken among the fowls, to play much greater role to contribute to the food and nutrition security specially to reduce protein malnutrition and mineral deficiencies.
It will then become the true aquatic chicken. However, more partnerships and cooperations among the academia, private sector, policy makers and the practitioners are needed to carry out further research and promote the fry production technology further. This will play a key role in expanding the farming of tilapia wider for the benefit of more people and the communities.