by

Jan Vos
FAO Associate Expert
(Culture of Food Organisms)
National Freshwater Prawn Research and Training Center
Freshwater Fisheries Division, Department of Fisheries
Ministry of Agriculture and Cooperatives
(FAO/UNDP:THA/75/008)

Bangpakong, Chachoengsao
Thailand, February 1979

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CONTENTS

1. INTRODUCTION

2. BIOLOGY OF ARTEMIA IN CONNEXION WITH CYST PRODUCTION

2.1 Systematic classification
2.2 Different strains
2.3 Reproduction

2.3.1 Oviparous reproduction
2.3.2 Ovoviviparous reproduction

2.4 Cysts
2.5 Growth and feeding
2.6 Tolerance to environmental factors

2.6.1 Nauplii
2.6.2 Adults

2.7 Phototactism and distribution patterns

3. STRAIN SELECTION

4. POND SELECTION AND PREPARATION

4.1 Suitable ponds
4.2 Design of the ponds
4.3 Multipurpose ponds

5. PREPARATION OF THE INOCULUM

5.1 Nauplii or adults
5.2 Inoculum preparation

5.2.1 Nauplii
5.2.2 Adults

5.3 Transportation

6. INOCULATION

6.1 Condition of the pond
6.2 Inoculation

7. OBSERVATIONS AND ACTIVITIES DURING FARMING

7.1 General method
7.2 Specific problems
7.3 Observations

7.3.1 Daily observations
7.3.2 Weekly observations

8. CYST AND ADULT HARVESTING

9. PROCESSING OF PRODUCED CYSTS AND ADULTS

9.1 Cleaning
9.2 Dehydration
9.3 Packaging
9.4 Quality control

10. PREVIOUS INOCULATIONS

10.1 Macau (Rio Grande del Norte, Brazil)
10.2 Iloilo (Barotac Nuevo, Philippines)

BRINE SHRIMP (ARTEMIA SALINA) INOCULATION IN TROPICAL SALT PONDS:
A PRELIMINARY GUIDE FOR USE IN THAILAND

by

Jan Vos
FAO Associate Expert
(Culture of Food Organisms)

1. INTRODUCTION

This work paper is prepared in view of the wish of the Thai Department of Fisheries to inoculate salt ponds with Artemia salina to produce cysts.

Artemia cysts are used to obtain live food in many aquacultural operations, but especially in the hatchery of the giant freshwater prawn, Macrobrachium rosenbergii. These inoculations, if successful, could encourage the Thai Government in the future to conduct experiments in large-scale cyst production. Indeed, up to this date, Thailand completely depends on import of expensive Artemia cysts from abroad. The yearly expenditure in cysts by the Department of Fisheries for the national fisheries stations, is estimated to be more than US$50 000.

The inoculation ponds considered are: (i) ponds in the National Fishery Station at Samut Sakhorn (still to be completed), and (ii) privately-owned salt ponds of salt farmers in the vicinity of the National Fishery Station at Chachoengsao.

Although previously, several inoculations in other parts of the world have proven to be successful, no written guides are yet available. Trial inoculations in Thailand being considered important, the necessity was felt to gather all available information on Artemia inoculations and cyst production into one working paper to serve as guide on improving the chances of a successful experiment.

The purposes of this paper are:

1. To give fundamental scientific background for an inoculation work programme
2. To give information about practical knowledge obtained from previous inoculations
3. To recommend inoculation and pond management procedures
4. To point out the contribution and possibilities of new inoculations in the growth of fundamental science

This paper may also mean to serve as a first basis for a future and more elaborate inoculation manual. Therefore, no references are given for particular findings, although they have been checked before use on their scientific credibility.

We are greatly indebted to Dr. P. Sorgeloos of the Artemia Reference Centre, University of Ghent, Belgium, who furnished a great part of the information used.

2. BIOLOGY OF ARTEMIA IN CONNEXION WITH CYST PRODUCTION

2.1 Systematic classification

Class-Cruatacea, Subclass-Branchiopoda, Order-Anostraca, Family- Genus-Artemia, Species-Artemia salina Linnaeus.

2.2 Different strains

Many different geographical strains of Artemia salina L. exist. Over 50 strains from all continents have been registered: Algeria, Kenya, Tunisia, Argentina, Brazil, Canada, Mexico, Peru, Puerto Rico, U.S.A., Venezuela, India, Iran, Iraq, Israel, Japan, China, Australia, Bulgaria, France, Italy, Spain, U.S.S.R. These wild populations reveal important differences in specific characteristics such as hatching rate, nauplii size, viability, and optimal temperature and salinity range requirements. A comparative study in Europe and America is underway. Up to now, about 11 commercial harvester-distributors, selling brands with different qualities, exist.

2.3 Reproduction

Two main modes of reproduction occur in Artemia. These are described as follows:

2.3.1 Oviparous reproduction

The fertilized egg develops into a gastrula stage. This gastrula is dehydrated, resulting in metabolic dormancy, and becomes encysted. A brown shell, containing haematine, lipoproteins and chitin is produced by the shell glands and surrounds the dormant gastrula.

The wintering eggs or cysts collect in the broodpouch of the female Artemia and are released in the water. The cysts deposited in this way will not hatch into nauplii. First, they have to go through a dehydration process. Only when this initial dehydration has taken place will the cysts hatch into free-swimming nauplii when hydrated again. In case of an oviparous reproductive cycle, the shell glands have a brown colour.

2.3.2 Ovoviviparous reproduction

The fertilized egg develops to the gastrula stage but instead of being encysted, the gastrula further differentiates in a nauplius in the brood pouch of the female Artemia. Thus, in the ovoviviparous reproduction mode, the female releases directly free-swimming nauplii, which have hatched out of the eggs in the brood pouch. The eggs are not surrounded by a shell and in the shell glands have a whitish colour.

The mode of reproduction is controlled by environmental factors, the most important one being the oxygen content and/or fluctuations in oxygen concentration in the water. Also, the type of food seems to have an important influence.

The control of the mode of reproduction is also correlated with the haemoglobin content of the blood of Artemia (red Artemia), which in turn is correlated with the oxygen concentration of the water. In low oxygen concentration, extra haemoglobin production is induced. Since with the increasing salinity the oxygen concentration of the water decreases, the salinity also has a strong influence on the mode of reproduction. Ovoviviparous reproduction occurs at salinities lower than 150 ppt, while reproduction is predominantly oviparous at salinities between 150–200 ppt (lower oxygen concentration). An oxygen-controlled reproduction theory has been put forward by Dr. P. Sorgeloos, which gives an ecological explanation for the variation of the mode of reproduction of Artemia in nature.

In the San Francisco Bay area, highest cyst production, and accordingly best harvest occurs in periods of primary production blooms. In these periods oxygen concentration fluctuations (night-day) are greatest.

Apart from oviparity and ovoviviparity, other reproduction types exist such as subitan eggs, i.e. “cysts” which are deposited and hatched immediately and abortive cysts, i.e. cysts produced by a non-fertilized female. However, these types are rather rare. Within one reproductive cycle, only one mode of reproduction may be favoured. The shortest period registered between two consecutive reproductive cycles is four days.

The number of offspring, cysts or nauplii per female per reproductive cycle is 50–200, but in oviparous reproduction, the number of offspring is generally lower than in ovoviviparous reproduction. In very high salinities (more than 180–200 ppt), the oviparous-produced offspring tends to decrease.

Although most Artemia strains are zygogenetic, some are parthenogenetic.

2.4 Cysts

The colour of the cysts is correlated (due to haemoglobin-haematine) with the Fe++-concentration in the natural environment. The paler the colour, the lower the viability of the cysts. Cyst size seems to be a strain characteristic. Most dehydrated cysts measure between 200–270 microns and the weight/ cyst averages at 3.5 micrograms.

Dry cysts are very resistant to extreme conditions. Up to 80°C they show no reduction of the hatching efficiency. Only at temperatures of 90–100°C the hatching efficiency decreases. Cysts are dehydrated dormant eggs. The minimum water content is 0.02 g H₂O/g cyst or 2% (residual water). When stored with water-content lower than 0.09 g H₂O/g cyst or 10%, viability and hatching efficiency will not decrease for many years, provided they are kept in oxygen free conditions. With oxygen, oxygen-free radicals are formed which are very harmful for the embryos. Moreover, since cysts are very hygroscopic, they should be stored in perfectly dry conditions. At 0.3 g H₂O/g cyst or 25%, the carbohydrate metabolism of the dormant cyst starts.

To hatch into a free-swimming nauplius, the cyst needs water (hydration) and consumes oxygen to initiate and to complete the carbohydrate metabolism. Complete hydration of the cyst takes about one hour. At salinities higher than 70 ppt, hydration is not completed, the metabolism does not start and consequently the cyst will not hatch. In salinities lower than 5 ppt, the cyst may hatch but the resulting nauplii will die very quickly.

Before actually hatching, cysts go through a breaking period, during which the cyst shell bursts open. This is activated by glycerol production in the cyst to form an osmotic pressure higher than the osmotic pressure of the NaCl solution outside, and high enough to break the shell. Therefore, hatching should be performed in salinities as low as possible. Once the cyst is hydrated, it becomes very sensitive for specific conditions. At temperatures lower than 40°C and higher than 34–40°C and in anaerobic conditions, metabolism shows a reversible stop. Temperatures higher than 40°C or lower than 0°C kill the hydrated cyst. Within 10–30°C, the hatching efficiency remains constant but the hatching rate increases with increasing temperature. Light triggering is needed at the start of the carbohydrate metabolism.

The hatching time needed is cumulative. A cyst which after hydration is dehydrated (reversible stop) and hereafter rehydrated again will continue its metabolism at the point (time) where it was stopped by the dehydration; this is for short reversible stops. For long reversible stops (especially at elevated temperatures), the incubation time until hatching becomes progressively longer. Thus, a cyst can undergo a series of hydration-dehydration-rehydration cycles. However, the more cycles are administered the lower the hatching efficiency since “energy reserves” are used up. The reversible stop has to be considered as an ecological adaptation to varying climatological conditions, i.e. wash-ashore, dehydration by sun, hydration by rainfall, dehydration by sun, etc. In freshwater dehydrated cyst sink to the bottom whereas in brine (saturated salt solution = 300 ppt) they float at the surface.

2.5 Growth and feeding

Adult Artemia of most strains measure 1–1.2 cm. Some strains have been recorded to measure about 2 cm such as the parthenogenetic Italian Margaritha di Savoia strain.

In optimal conditions, Artemia becomes adult in seven days. Males and females in zygogenetic strains are easily discriminated. Males have huge claspers in front of the head, which are used to grasp the female during the riding position, previous to copulation. Only females have brood pouches on the ventral side of the body. During growth, the thoracopods develop and finally have a triple function: swimming, feeding (filtration) and respiration. From nauplius to adult, the length increases 20 times and the weight 500 times (2 micrograms to 1 mg).

Artemia is an obligatory non-selective filter feeder and removes suspended particles smaller than 40–60 microns down to a few microns from the water with great effectiveness. These food particles may consist of algal cells (non-filamentous algae), Protozoa and detritus.

Only from the second larval stage (Instar II) food is taken up. Instar stages I, II and III have a yolk reserve. Although a suspension feeder, Artemia is found browsing on submerged stones, clumps of blue-green algae and detritus. In most places where Artemia is found no other plankton feeder is present The only food competition will be among the Artemia themselves. Artemia is a very efficient protein converter. The adult has a protein content of 60% of the dry weight. Nauplii, Instar I, have 40% protein content.

2.6 Tolerance to environmental factors

2.6.1 Nauplii

Growth is optimal at 28°C and 35 ppt and drops at pH values below 7. The respiratory metabolism shows a reversible stop at 5–8°C and death occurs at 0°C and 37–38°C. Salinity changes can be administered very abruptly. No mortality occurs when Instar I larvae, hatched in 30 ppt are transferred to 90–110 ppt immediately. Nauplii can also, without harm, be transferred from 30°C to 4°C. Activity stops suddenly but can be reactivated by increasing the temperature again.

2.6.2 Adults

When put in freshwater, adults survive for 2–3 hours. For most strains maximum salinity tolerance seems to be at 200 ppt. Increasing salinity with 10 ppt changes in 4 hour intervals is very well tolerated. Morphology and general appearance of adults change within different salinities. The higher the salinity the smaller the claspers of the male Artemia. Also in high salinities, the body becomes longer and thinner than in low salinities.

At pH lower than 7, the general appearance of Artemia grows worse; pH 8–8.5 seems to be optimal. At 28°C growth is optimal and temperature higher than 36–37°C are lethal. Nauplii as well as adults survive a sudden fluctuation from 28–30°C to 0–4°C and vice versa. At temperatures of 0–4°C most motion activity stops. Low oxygen concentrations are more harmful for nauplii in the first stages than for later stages and adults, since then gills are formed.

2.7 Phototactism and distribution patterns

Freshly hatched nauplii (Instar I, II and III) are positively phototactic. On the other hand, adults are negatively phototactic. This phototactism, among other phenomena, probably lies at the basis of the cloud forming tendency in Artemia populations. Nauplii as well as adults always tend to gather in “clouds” so that population and density determinations in big water systems are extremely difficult. On the other hand, collection of nauplii, sub-adults and adults is facilitated by good knowledge of this behavioural pattern. Distribution patterns during night and day are completely different.

When the oxygen concentration in the water is very low, Artemia swims on its back, at the surface. This is very often the case in salt lakes with high phytoblooms. During night time, the phytoblooms cause serious oxygen depletion so that in the early mornings, great part of Artemia populations may be found floating at the surface.

3. STRAIN SELECTION

In fact, cysts of all the different strains can be used for inoculation. Nonetheless, some criteria have to be considered. Firstly, preference should be given to a strain with good hatching efficiency. In some cases, however, starting from a low hatching efficiency strain, better cyst may be produced, since low hatching efficiency is mostly due to a bad cyst processing.

Secondly, in the absence of available information on the temperature tolerance of a specific strain, a similarity of the climatic conditions between the wild population habitat and the new inoculation spot should be favoured. Attention has to be paid to degrees of latitude, average, minimum and maximum air- and water temperatures, length of dry season, etc.

Thirdly, the chemical nature of the salt lake the strain is coming from is important. Some salt lakes with natural Artemia populations are sulphate lakes instead of chloride lakes. Inoculation of a sulphate strain in a chloride pond should be avoided since this might cause serious deviation in the ionic balance which will affect the reproduction rate or hatchability of the produced cysts.

Up to now, no research has been performed on the above points. In most Southeast Asian countries, inoculation will have to be renewed every year as a consequence of the rainy season. During the rainy season, the established Artemia population will completely die off and/or be finished by predators. Therefore, every year another strain should be selected for inoculation. Further studies on the determination of the ecological characteristics as well as the nutritional value of the different strains, should lead to the selection of the strains best suited for a particular region. For instance, the selection of high temperature resistant strains, could imply that the existing shallow salt ponds (with very high maximum water temperatures) are fit for use without any adjustment. Also, the use of parthenogenetic strains for inoculation would be a very beneficial improvement.

4. POND SELECTION AND PREPARATION

4.1 Suitable ponds

The construction of new salt ponds especially for Artemia inoculation is not indispensable. Also, existing salt ponds strictly used for salt production are very well suited, provided some minor adjustments are made. Concrete as well as earthen and plastic lined ponds can be used, although preference should be given to earthen ponds since they provide a beneficial nutrient exchange with the water. Warning has to be given for chemically non-stabilized pond bottoms after excavation.

The source of water intake in the pond system has a major influence on the success of the inoculation. In order to sustain a large Artemia population, suspended food particles and/or high nutrient concentrations should be provided by the intake water. In this respect, salt ponds situated in mangrove areas, being one of the most productive ecosystems, are much in favour. In natural waters, no Artemia will be found when the water is very turbid, whether the turbidity is caused by algae, organic matter or by inorganic salts. Salt ponds with water intake directly from the sea are not preferred.

4.2 Design of the ponds

Inoculation ponds for mass cyst production should have the biggest possible surface, since the same labour will be involved for small ponds as for large ponds. However, for initial experimental inoculation, a series of small ponds (0.2–1.0 ha) should be preferred above one large pond with the same surface. The inoculation ponds need good water intake and drainage facilities since after the inoculation, salinity has to be completely controllable. Instead of gates, eventually portable water pumps could be used.

The necessary depth of the ponds depends on the maximum water temperature, which should not exceed 35–36°C. In Thailand, a depth of 40–45 cm has to be considered to keep the temperature within safe margins. Although existing salt ponds could be excavated, preference has to be given to raising of the earthen dikes, because of the lower costs involved and the eventual risks of affecting the water chemistry through the excavated new pond bottom. Seepage in earthen ponds has to be prevented so that enough high salinities can be reached. To facilitate cyst collection, the ponds have to be orientated in such a way that the cysts will be gathered by the main wind direction in one corner of the pond.

4.3 Multipurpose ponds

Many salt farmers are afraid that their salt production will be threatened by the presence of Artemia population. This is not true. Firstly, only the specific suited part of the salt farm can be reserved for cyst production. Secondly, the water will be swept clear (filter feeding) by the Artemia, resulting in a better quality salt production. As a matter of fact, in the Antillean islands, Artemia is called “clearer worms” by the salt farmers since they consider the presence of Artemia to be an obligatory condition for cleaning brine with gypsum (CaSO₄) suspension. Screens can be used to keep the brine shrimps in the restricted area.

During the rainy season, the Artemia population will die off and the ponds can then be used for other purposes such as shrimp and fish growing.

5. PREPARATION OF THE INOCULUM

5.1 Nauplii or adults

Nauplii as well as sub-adults can be inoculated. Inoculated nauplii will first grow to the adult size in the ponds. Hereafter, a specific management for population increase can be started. In our opinion, inoculation with nauplii

should be the rule. However, if large amounts of adult Artemia are available from, for instance, other already productive ponds or from routine high density culturing units, preference might be given to adult inoculation.

Cyst inoculation has to be discarded since cysts will not hatch in high salinity water and if inoculation is started in lower salinities than 100 ppt, predators which will decimate the population cannot be prevented.

5.2 Inoculum preparation

5.2.1 Nauplii

After determination of the number of nauplii needed, the corresponding amount of cysts can be calculated knowing the hatching efficiency of a particular batch of cysts. The hatching efficiency is the weight of cyst (grams) needed to produce one million nauplii.

Previously to the hatching, the cysts can be decapsulated, during which the outer cyst shell is dissolved in a hypochlorite solution. Decapsulation eliminates the cumbersome shell separation after the hatching. Hatching has to be performed in salinity as low as possible and can be carried out in densities up to 10 g/liter, provided that funnel-shaped hatching containers with strong aeration at the bottom instead of flat-bottomed tanks are used. Hereafter, the nauplii after being sieved off can be transferred directly to the same salinity as the inoculation pond, which initially must not be higher than 100– 110 ppt.

5.2.2 Adults

Adults and sub-adults used for inoculation should be in good health. After being collected, they have to be adapted gradually to the salinity of the inoculation pond. For the moment, 10 ppt changes in 4-hour intervals are recommended.

5.3 Transportation

If the inoculation pond is near (within 15 minutes) walking distance to the hatching site, no special transportation methods have to be used. As long as the temperature is kept below 30°C and oxygen is provided by means of aeration, 100% survival is guaranteed. A useful density for nauplii is half the hatching density, for adults 2000–4000 per liter. However, if long distances have to be covered and/or large quantities of nauplii or adults have to be transported, the following method can be used.

After collection and sieving of nauplii or adults and transferring to the appropriate salinity, they can be rinsed with 0–4°C salt water. This temperature stress causes a metabolic stoppage and motion activity is strongly reduced. When kept in the appropriate salinity at such low temperatures, nauplii as well as adults can be stored in much higher densities for many hours. For nauplii a density of 10.16⁶/20 liter (equivalent to 50 g cysts/20 liters) is recommended, while adults can be transported at densities up to 20 000 per liter. Slow aeration has to be provided to keep the animals in suspension.

6. INOCULATION

6.1 Condition of the pond

Before actually inoculating a pond, the following points have to be checked. First, there should be absence of predators in the water. To eliminate predators, screens at the intake can be used, although this does not give complete guarantee, and flying insects such as Corixidae can still prey on Artemia.

A better method is to work at a salinity too high to allow survival of any predatory organisms. Tilapia and gobies tolerate salinities up to 100 ppt. Therefore, the inoculation ponds should have an initial salinity of 100– 110 ppt. Presence of predators can be checked by walking through the pond.

Secondly, the water of the pond should have productivity high enough to sustain the inoculated population. A medium turbidity or presence of slight phytobloom is desirable. In case the water is very clear, food particles can be brought in the water by stirring up the bottom with a stick or by walking through the pond once a day. Eventually, ricebran feeding, sieved over a 60 micron screen, can be considered.

Thirdly, the pond should be free of seepage and the water depth has to be adjusted to 30–40 cm. Knowledge of prevailing water temperature readings and pH measurements are useful.

6.2 Inoculation

The best time of the day to inoculate is late evening since at this time, temperatures are low and will continue to drop till the early morning. In this case, the inoculated organisms, nauplii or adults, have the time to adapt to higher afternoon temperatures. Nauplii as well as adults can be transferred directly from their low temperature transportation containers to the inoculation pond. No absolute guidance for inoculation densities can be given as yet. This depends very much on the productivity of the pond. Previous inoculations with initial densities of 20.10⁶ nauplii per ha (equivalent to 100 g cysts) have been proven to be successful. In the end, too dense inoculation will be regulated down to a sustainable population anyhow. On the other hand, a high initial density implies higher cyst expenses, while the main idea of an inoculation is that the population will increase to a sustainable level by itself, with an initial input as low as possible. The most economic input/output ratios should be determined.

7. OBSERVATIONS AND ACTIVITIES DURING FARMING

7.1 General method

In most salt farms, water of 30–35 ppt is taken into large shallow evaporation ponds. When 100–120 ppt is reached, the water is lead into condensation ponds. Finally, when about 180 ppt is reached, the brine is taken into the crystalization ponds (pans). Salt crystalizes at 280–300 ppt. Under optimal insolation, the evaporation rate is about 5 ppt per day. The run-off of the crystalization pans contains high concentrations of CaSO₄ (gypsum). The salinity range used for the Artemia cyst production is 100–200 ppt. Therefore, the Artemia population will most be located in the evaporation ponds and condensation pans, although for some strains Artemia has been reported to thrive even at salinities of 300 ppt.

In another method, specific ponds are reserved for Artemia and have to be considered as a separate production unit, using water from the salt farm unit. In the last case, a specific Artemia management distinct from salt production, can be followed in these ponds.

Based on previous inoculations, the following management is recommended:

1. Intake of 35 ppt in Artemia pond up to 10 cm. Let salinity rise and keep the depth at 10 cm by regular water intake.
2. Fill up to 30–40 cm with 100–110 ppt from evaporation ponds.
3. Check for predators.
4. Inoculate and keep for one week at 30–40 cm, 100 ppt.
5. Let salinity increase up to 150 ppt. Keep water level at 30–40 cm by regular water intake.
6. Decrease salinity abruptly to 110 ppt by drainage and intake of 35 ppt water. Adjust water level at 45 cm.
7. Let salinity increase again up to 150 ppt. Allow water level not to drop lower than 30–40 cm.
8. Repeat several cycles of procedure items (f) and (g).
9. Let salinity rise to 180–200 ppt and do not allow salinity to exceed this limit by regular water intake. Keep water level at 40 cm.

Explanation:

Procedure (d) will allow nauplii to grow out to adult size. Procedures (e), (f), (g) and (h) will increase population through ovoviviparity and regular food intake (e.g. use of mangrove water) On using procedure (i) oviparity will become the dominant reproduction method.

Predators present in the low salinity water intake, will be killed directly in the Artemia ponds as a consequence of salinity shock. When the inoculation pond is drained, the adult Artemia can be recovered by use of a funnel-shaped net at the outlet.

7.2 Specific problems

7.2.1 Since the run-off of crystalization pans contains a high CaSO₄ concentration, it is not recommended to use this brine to increase the salinity in the inoculation pond. This run-off is poisonous for Artemia, probably also because the equilibrium between calcium and magnesium has been changed too much. After dilution with sea water, however, the equilibrium of ions will be partly restored and the brine might become suitable again for maintaining Artemia.

7.2.2 As a consequence of the specific gravity, mixing salt water of different salinities in large systems is difficult. When a layer of freshwater covers a concentrated brine (freshwater lens), the temperature in the brine may rise to high and lethal temperature levels. This effect is intensified, the darker the colour of the bottom and the shallower the water. Temperatures of 70°C have been recorded. Therefore, care should be taken to mix thoroughly the low salinity intake water with the high salinity pond water. Again, stirring the pond bottom such as walking through the pond could be the most convenient solution.

7.2.3 Lethal oxygen depletion during the night as a consequence of a phytoblooms should be anticipated and possibly prevented. For some reason or another, heavy rainfall at the end of the dry season seems to cause high mortality in the Artemia population, even if salinity remains at sufficiently high levels and temperatures kept within tolerance ranges.

7.3 Observations

It is of extreme importance to monitor the fluctuations of specific parameters during cyst farming, since these environmental factors determine the success of an inoculation. Furthermore, still many questions are unresolved and these data, in the future, could lead to an improvement of farming procedures and cyst production.

Some observations are recommended to be carried out daily, others weekly. The daily observations consist only of easy short time measurements, which could be performed by the salt farmer himself. The weekly observations should be carried out by a fishery biologist.

7.3.1 Daily observations

Depth of water, salinity by optical refractometer or densitometer, temperature (minimum and maximum air and water temperature) and weather conditions (cloudiness, wind and amount and occurrence of rainfall).

7.3.2 Weekly observations

Productivity of the water, population characteristics (size frequency, density-multiple at random sampling), reproduction mode, fertility, state of health, pH and dissolved oxygen.

8. CYST AND ADULT HARVESTING

The cyst deposited by the females will float at the surface. They should be prevented to wash ashore since if this happens collection of the cysts will be made difficult, the cleaning procedures will be complicated and the cysts may be blown away once they are dry. Cysts washed ashore may also be subject to several dehydration-hydration cycles decreasing the cyst quality. It is recommended to collect the cysts while still floating in the water.

Cysts gathered by the wind in one corner of the pond are easily collected with dipnets. Eventually, floating windshields made of plastic sheets and bamboo can be used. Cyst collection should be performed as regularly as possible, preferably daily. At the end of the dry season, when the pond is completely drain the adult Artemia population can be harvested also.

9. PROCESSING OF PRODUCED CYSTS AND ADULTS

The processing of the cysts is of crucial importance with regard to the quality of the product. Not only have the cysts to be dehydrated to a sufficient low level (lower than 10%) but the quality of the product (hatching efficiency) is also determined by the effective removal of dirt from the cysts. Although many different processing procedures exist, all of them go through the following three steps.

9.1 Cleaning

The so-called biphase flotation method is recommended for its simplicity, low cost and good results. First, the cyst material is suspended in brine. In this solution, the cysts and light debris will float while the heavy particles such as sand, shells, etc. will sink to the bottom. Intermittent aeration from an air tube at a certain distance of the bottom or regular mixing with a stick, improves the separation of the cysts and heavy debris. This brine separation should be continued for at least 24 hours. The layer of floating cysts is scopped off and the cysts are throughly washed with tap water on a 200 micron screen.

Second, the separation of the light debris is carried out in tap water and should take only about half an hour. The full cysts (viable cysts) will sink to the bottom, whereas the empty cyst shells, plumes, etc. float at the surface. The cleaned cysts are then siphoned into a cloth bag and water is removed as much as possible.

9.2 Dehydration

After the cleaning process, the cysts have to be dehydrated as soon and as possible very quickly. The easiest method uses submersion in brine (300 ppt). First, the cysts are dehydrated down to 25% water so that metabolism stops. Second, dehydration is continued till water content lower than 10% is reached. The complete dehydration process takes about 3–5 hours. During this treatment, the cysts should be kept in continuous suspension. At intervals, brine has to be changed during the process since the brine solution is diluted by the extraction of water from the cysts.

9.3 Packaging

After the brine dehydration, the cysts have to be washed with tap water to remove salt after which most water must be eliminated by a cloth bag. This procedure should be carried out as quickly as possible and preferably with cold water, to prevent initiation of metabolism. Hereafter, they can be dried on shelves, not in direct sunlight.

There are three equally good procedures for packaging that exist. First, the cysts can be packed in vacuum sealed or nitrogen flushed cans. This is the classic method used for all commercial brands. Another recently developed method consists of decapsulation of the cysts in a technical hypochlorite solution. The embryos, only surrounded by their embryonic membrane, can then be dehydrated in brine, resulting in a coffee-bean shaped embryo. These dehydrated decapsulated cysts can then be stored in brine and packed in non-transparent containers to prevent harmful effects of sunlight. Third, cysts dehydrated but not decapsulated can also be packed in cans or plastic bags containing brine. Generally speaking, any method can be used which guarantees (i) the absence of oxygen, and (ii) absence of water absorption by the cysts.

9.4 Quality control

The processed product should be checked for its quality. Quality control is easily performed by routine determination of hatching efficiency and hatching rate. It has been proven to be very useful, to include determination of the nutritional value in the quality control procedure. However, the routine determination of this quality criterion is still in the experimental stage.

10 PREVIOUS INOCULATIONS

Previous successful inoculations have been recently performed by Sorgeloos (1978) in Brazil and the Philippines. Information on these inoculations can be used to have an idea of the possible production of cysts in Thailand.

10.1 Macau (Rio Grande del Norte, Brazil)

In April 1977, a 10-ha salt pond (100 ppt) was inoculated with nauplii of the San Francisco Bay Brand strain of brine shrimp hatched out of 250 g of cysts. In June of the same year, the first batch of cysts was harvested and the Artemia population spread out over the entire salt pond system (1000 ha). In December 1977, over 3000 kg of good quality cyst were harvested.

10.2 Iloilo (Barotac Nuevo, Philippines)

In February 1978, 125 000 pre-adults and nauplii hatched out of 80 g of cysts of the San Francisco Bay Brand strain were inoculated in a 300 m² concrete tank (90 ppt). The Artemia were additionally fed with ricebran. After 14 days, they were transferred from the concrete tank to two ponds (140 ppt), one of about 3000 m² and the other of 3500 m². Creek water was taken in at weekly intervals to let salinity fluctuate between 100–150 ppt and to take in food. This resulted in a population increase. At the end of May 1978, a total of 35 kg of good quality cysts were harvested plus 30–40 kg wet weight of adults.

Caution has to be given for rash extrapolations of these results, since the production of cysts depends on many factors as outlined in this paper. Moreover, many questions on pond management, strain selection and optimal inoculation density still have to be answered.

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