ANNEX A
ITINERARY AND ACTIVITIES DURING THE TRIPS OF DR. PATRICK SORGELOOS
TO THAILAND FOR THE FAO-UNDP THAILAND PRAWN PROJECT (THA/75/008)
30 March - 15 April 1978 and 22 October - 1 November 1978

ANNEX B
CONCENSUS OF THE INFORMAL SESSION ON ARTEMIA SALINA HELD ON THE OCCASION
OF THE FAO TECHNICAL CONFERENCE ON AQUACULTURE
Kyoto, Japan, 26 May - 2 June 1976

Whereas there is a present and potential future shortage of good quality Artemia cysts, and considering the widespread need of Artemia cysts in aquaculture research and development in many countries of the world, the following recommendations are proposed:

1. Research on the biology and ecology of Artemia should be encouraged to provide needed background data for more efficient production of Artemia.

2. More efficient procedures for hatching and utilization of Artemia should be encouraged based upon available information.

3. Research on automated production systems for the mass production of Artemia cysts should be encouraged.

4. Investigation should be conducted to develop substitutes for Artemia.

5. A committee should be appointed by FAO to collect and disseminate statistics on:

   1. the current demand and supply situations for Artemia cysts, and

   2. projected future demand for Artemia cysts

6. This committee should send a questionnaire to likely countries to establish information on under-utilized sources of Artemia cysts.

ANNEX C
PROCEDURE FOR TESTING THE HATCHABILITY OF ARTEMIA CYSTS

In literature, the hatchability of a given sample of cysts mostly has been expressed as hatching percentage, namely the number of live nauplii hatching out of 100 cysts. This criterion, however, does not take into account the degree of purity of the product; in other words it does not consider the quantity of debris included in the batch of cysts. In this regard, the concept of hatching percentage is misleading since a figure of 90% hatching may indeed be correct, despite the fact that the product may carry a significant quantity of debris.

Inasmuch as Artemia cysts are always sold on a weight basis, the criterion of first importance to the customer is of course the number of live nauplii which he will get from the total quantity of product purchased.

The following simple techniques can be applied to determine the weight of product, which under standard conditions of incubation, will yield 1 million nauplii:

• 3 samples of 250 mg are taken at random from the batch of cysts to be analyzed

• the cysts are hydrated in 100 ml graduated cylinders in 80 ml natural seawater at 30°C and are kept in suspension by a continuous aeration from the bottom of the tubes

• after 1 hour, the water volumes are adjusted to 100 ml with seawater and 5 subsamples of 0.250 ml each are taken from each tube with a glass pipette or preferentially with an automatic micropipette

• each subsample is pipetted in a small plastic tube (5 ml content), the pipet is cleaned with seawater in the same tube and the water level in the tube is finally brought to 4 ml with seawater

• since at the end of the hatching period a total count will be performed on each tube, the volume of seawater in the tubes may vary in function of the type of tube used (minimum 4 ml)

• the tubes are closed with a cap and clamped into a rotating axle at 5 rpm; the whole set up is incubated at 30°C in continuous light conditions

• after 48 hours, the content of each tube is fixed by addition of a few drops of Lugol's solution¹ (stains the nauplii dark) or with another fixative.

• The total number of nauplii hatched in each tube is determined by filtering the suspension on a small gauze filter, placing the filter in a petri dish and counting the nauplii under a dissection microscope. The average number of nauplii produced per gram of cyst product is then calculated. This number can also be expressed more practically as the quantity of product that has to be incubated to produce 1 million nauplii.

• Summarized:

  • 250 mg product in 100 ml seawater

  • 5 sub-samples of 0.250 ml giving n₁ n₂ n₃ n₄ n₅ nauplii or average n nauplii in 0.250 ml

  • number of larvae per gram product: n × 4 × 100 × 4

  • weight of product needed for the production of 1 million nauplii

    

¹ Preparation of Lugol's solution:

1. Dissolve 10 g neutral KI and 5 g I₂ sublimate in 50 ml boiling distilled water
2. Dissolve 5 g Na-acetate in 50 ml distilled water
3. Mix solutions (a) and (b)

ANNEX D
DECAPSULATION METHOD FOR ARTEMIA CYSTS

The reasons for applying the cyst decapsulation technique in the aquaculture hatcheries are discussed in detail in the SEAFDEC consultancy reports (see above) and in the following papers: Sorgeloos, P. et al. (1977b), Bruggeman, E. et al. (1977), and Sorgeloos and Bruggeman (1978).

In view of the local price difference between the two forms of hypochlorite, the decapsulation of Artemia cysts in Thailand should preferentially be done with technical bleaching power (e.g. calcium hypochlorite, for example, Nielon-60-P power containing 60% active product imported from Nissin Denka Co., Ltd., Japan. Local price: ± 38 Baht per kg).

Principles

1. Ratio cysts/active product: 2–3 g cysts for 1 g active product (take into account the percentage of inert ingredients when calculating the quantity of product needed).

2. Ratio cysts/sodium carbonate: 7 g technical Na₂CO₃ per 10 g cysts. For economy reasons, Mr. Anand Tunsutapanich (Chachoengsao Macrobrachium Research Center) is using technical calcium oxide instead of sodium carbonate at a rate of 3 g CaO per 10 g cysts. The use of CaO instead of Na₂CO₃ does not affect the efficiency of decapsulation, however, no information is available yet of its impact on the hatching efficiency.

3. Total volume of decapsulation solution (seawater + bleaching powder + sodium carbonate): 200 ml per g cysts. Preparation of the decapsulation solution - first dissolve the bleaching power (e.g. 10 minutes of aeration) then add the sodium carbonate. If a small particulate precipitate is formed, let it sediment (eventually overnight) and only use the supernatant.

4. Ratio desactivation solution/cysts: minimum of 0.5 ml of a 1% solution of technical sodium thiosulphate (Na₂S₂O₃.5H₂O) or sodium sulphite (Na₂SO₃) per 10 g cysts.

Working procedures:

1. Hydrate the Artemia cysts in seawater or tapwater (the aeration should keep all the cysts in suspension).

2. After 1 hour filter off the cysts on a 150–200 micron screen and wash them with seawater or tapwater.

3. Resuspend the cysts in a bucket with the decapsulation solution which has been cooled to a temperature of 15–20°C (eventually add ice).

4. Stirr the suspension continuously with a stick in order to keep all the cysts in suspension. Check the temperature and eventually add ice to avoid a temperature increase above 40°C.

5. Depending on the temperature, it takes from 5–10 minutes before the decapsulation process is finished (orange-like colour of the suspension). Filter off the decapsulated cysts on a 150–200 micron screen and thoroughly wash them with seawater or tapwater until there is no more smell of chlorine.

6. Resuspend the decapsulated cysts in seawater or freshwater (1 kg of cysts in about 5–10 liters water). Add the desactivation solution and agitate the suspension during a few minutes.

7. After sedimentation of the decapsulated cysts to the bottom of the container, remove the floating materials (non-decapsulated cysts, transparent membranes, plumes, etc.). If a large portion of non-decapsulated cysts (which means that the products used do not have the exact activity or that the procedure has not been followed properly): harvest the floating non-decapsulated cysts and resuspend in saturated brine solution (300 g technical salt per liter), change the brine solution after 2 hours and again after 4–12 hours. These cysts can now be stored until a new batch of cysts will be decapsulated: filter off the brine and add the cysts to the new batch of cysts which will be decapsulated - see working procedure 1 and onwards.

8. The further treatement of the decapsulated cysts which settled out in the desactivation solution can be either one of the following methods:

   1. direct distribution to the cultured species after washing out the sodium-thiosulphate or sulphite.

   2. incubation for hatching:

      • washing out of the sodium-thiosulphate or sulphite
      • incubation in optimal hatching conditions

      • upon completion of the hatching process, harvest of the nauplii on a 150–200 micron screen; thorough washing (removal of all dissolved organics, mainly glycerol); distribution to the cultured species.

   3. dehydration in brine and storage until later use:

      • suspend the wet dry cysts in a saturated brine solution (300 g technical salt per liter; brine can be made up automatically with a “brinomat” (Spotte, S.H., 1970, Fish and Invertebrate Culture, Wiley, New York)
      • aerate the suspension for about 5 minutes
      • stop aeration: the decapsulated cysts float while the heavy debris (e.g. sand) sink and can be drained or siphoned off
      • aerate the suspension for about 2 hours; filter off and resuspend the cysts in fresh brine

      • aerate the suspension for another 2 hours (eventually overnight) store in fresh brine until use; see above (a) and (b)

9. Do not expose the decapsulated cysts (hydrated or dehydrated) to direct sunlight (UV-rays kill the embryos).

ANNEX E
LIST OF COMMERCIAL HARVESTERS-DISTRIBUTORS OF ARTEMIA CYSTS FROM DIFFERENT GEOGRAPHICAL ORIGIN

Whereas for years the demand for Artemia cysts has exceeded by far the offer resulting in a continuous increase of the price of this product, and creating a serious bottleneck for the further expansion of the aquaculture hatcheries (Sorgeloos, 1976), this situation was reversed recently by the commercial exploitation of several new natural resources.

At the recent FAO/EIFAC Symposium on Finfish Nutrition and Feed Technology (Hamburg, Germany, June 20–23, 1978), it appeared however that there is still an important lack of information on the commercial sources of Artemia cysts.

We hope to alleviate this gap by the list given below. We would like to emphasize that the latter list has been worked out without any consideration on the potential quality of the respective cyst-products (Sorgeloos et al., 1978).

Informations gained from various sources indicate that the present commercial supply of cysts averages about 100 metric tons per year.

ANNEX F
AIDE MEMOIRE

Meeting with Mr. Somsuk Singholka (Director, Chachoengsao Macrobrachium Research Center), Mr. Sombhong Suwannatous and Mr. Anand Tunsutapanich (Officers, Chachoengsao Macrobrachium Research Center) and Mr. Jan Vos (FAO Associate Expert - Growing Food Organisms, THA/75/008)

1. Mr. Jan Vos, as necessary, to provide with a full-time technical aid for better execution of his Artemia activities.

2. Mr. Anand Tunsutapanich and Mr. Jan Vos will alternately have weekend responsibility with regard to the supervision of the running Artemia activities. Their mutual arrangements will be communicated to the Station Director.

3. Short weekly meetings will be called by the Station Director with Mr. Anand Tunsutapanich and Mr. Jan Vos for reporting on their respective progress and problems with regard to Artemia matters in order to stimulate an efficient exchange of information.

ANNEX G
NOTES ON THE PROCESSING OF ARTEMIA SALINA CYSTS

1.    Separation of the heavy debris (sand, skelets, etc.) in a saturated brine solution: poor the cysts in brine, either agitate (aerate) continuously or discontinuously in order to detach all clumps of cysts and have all dirt settled on the bottom of the container. There is no time limitation for this process.

2.    Harvest the floating cysts from the surface of the brine solution; wash the cysts in freshwater or seawater (the cooler the better-cf. start cyst metabolism)

3.    Pour the cysts in freshwater or seawater and let them settle at the bottom: the light debris (empty shells, plumes, etc.) will float at the surface (for some batches agitation of the surface layer might be needed to detach the cyst-clumps. This process should last not longer than 15 minutes.

4.    Further processing can be done following various ways:

1. - decapsulation - dehydration and storage in brine
2. - dehydration and storage in brine
3. - air-drying and storage under vacuum or nitrogen.

4c.     Transfer the cysts to a cloth sack and remove the excess water by gently squeezing the sack and cyst-mass.

5c.    5c. Distribute the cysts on a drying surface consisting of a table or trays made of wire screen covered with cotton muslin (tight woven grade; will absorb water from the cysts). In order to spread the cysts in thin layers (maximum a few mm thick) use a woven wire basket (5 mm mesh).
By properly shaking this basket containing the cysts, the cysts will be sifted through the mesh and be evenly distributed over the drying surface.

6c.    Drying should take place in a housing designed as a drying room. Temperatures as high as 40°C will not harm the cysts. Air exchange is required in order to maintain a minimal humidity (drying the cysts in direct sunlight might result in a decrease of the viability - due to heat-absorption the temperature inside the cyst shell might exceed 40°C which is lethal for hydrated cysts).

7c.    Stop the drying process once the cysts are dry (the water content should be below 9%; preferably between 2 and 5%). Cool the drying room in order to eliminate or reduce the static charges that can be generated when hot dry cysts are brushed off the drying tables. Screen the cysts through a 300 to 500 micron screen.

8c.    The viability of the dried cysts is guaranteed when packed under vacuum or under nitrogen atmosphere (away from oxygen and otherwise free radicals can be produced and these irreversibly damage the embryos).
The nitrogen packaging process has to be performed in 2 steps:

• flushing of the cysts with nitrogen; overnight storage under nitrogen (description of oxygen from the alveolar layer);

• flushing of the cysts with nitrogen followed by packaging under nitrogen atmosphere.

LITERATURES RELATED TO THIS SUBJECT

Benijts, F., G. Vandeputte, P. Sorgeloos, 1977 Energetic aspects of the metabolism of hydrated Artemia cysts: 79–87. In Fundamental and Applied Research on the Brine Shrimp, Artemia salina (L.) in Belgium, Special Publication No. 2, European Mariculture Society, Ed. Jaspers, E., Institute for Marine Scientific Research, Bredene, Belgium: 110p.

Clegg, J.S., 1976 Interrelationships between water and metabolism in Artemia cysts. II. Carbohydrates. Comp. Biochem. Physiol., 53A: 83–87

Clegg, J.S., J. Cavagnaro, 1976 Interrelationships between water and cellular metabolism in Artemia cysts. IV. Adenosine 5'-triphosphate and cyst hydration. J. Cell. Physiol., 88: 159–166

Crowe, J.H., 1971 Anhydrobiosis: an unsolved problem. Amer. Naturalist, 105: 563–574

Crowe, J.H., J.S. Clegg, 1973 Anhydrobiosis. Dowden, Hutchinson and Ross, Inc. Stroudsburg: 477p.

Hellfrich, P., 1973 The feasibility of brine shrimp production on Christmas Island. Sea Grant Technical Report UNIHI-SEA GRANT-TR-73-02: 173p.

Rakowicz, M., 1975 Notes on Artemia salina. Manuxcript: 13p.

Sorgeloos, P., 1976 The brine shrimp Artemia salina L.: a bottleneck in mariculture? FAO Technical Conference on Aquaculture, Kyoto, Japan, 26 May - 2 June 1976, Experience Paper 77: 12p.

Sorgeloos, P., 1977 Automatic washing and sieving of Artemia cysts. Manuscript: 3p.

Sorgeloos, P., 1977 et al., The use of brine shrimp, Artemia salina in aquaculture Proceedings of the 3rd Meeting of the ICES Working Group on Mariculture, Brest, France, May 10–13, 1977. Actes de Colloques du C.N.E.X.O., 4: 21–25