AN IMPROVED TECHNIQUE FOR DECAPSULATION AND PRESERVATION
OF ARTEMIA CYSTS (BRINE SHRIMP EGGS)
Decapsulation of brine shrimp eggs was originally carried out with sodium hydroxide and sodium hypochlorite at the Chacheongsao Fisheries Station. The cost of decapsulation by that technique was estimated at B300/kg. of brine shrimp eggs. A modified technique has been developed whereby decapsulation is conducted by using calcium oxide and calcium hypochlorite as stimulating agents. Promising results were achieved with the same degree of success as with the former technique. However, the latest technique is better because the cost of decapsulation can be reduced from B300/kg. to only B18/kg. of brine shrimp eggs.
Nowadays, in the culturing of aquatic animals, artemia (Aremia salina), commonly called brine shrimp, is widely used in feeding larval or young fish or crustacea. The artemia is a very tiny animal which has no shell. Its body is enclosed in thin tissues. The eggs of artemia do however have a shell which is dark brown in color. The size of the egg is approximately 200–270 microns. The fact that the eggs can be preserved for a long time makes it convenient to hatch them whenever necessary. Lately the techniques for hatching have been improved. Decapsulation of brine shrimp eggs is carried out before hatching. Decapsulation is important because the young aquatic animals do not eat artemia egg shells. Additionally, the shells tend to cause water pollution and act as a carrier of disease. Decapsulation of the brine shrimp eggs solves these problems. Brine shrimp eggs which have been decapsulated are in a better condition for hatching than those which are not decapsulated (Table 1). We can therefore use artemia eggs, which are very costly, in a more efficient way.
The Chacheongsao Fisheries Station, which is responsible for this project experiment, carries out research, training and technological development in the field of fresh and brackishwater prawn farming. This involves the need for a large quantity of brine shrimp eggs. In April 1978, an experiment was conducted which decapsulated brine shrimp eggs with sodium hydroxide (caustic soda) and sodium hypochlorite, following the procedures of Bruggeman et al. (1977) and Sorgeloos et al. (1978). This experiment, which was successful, showed that the cost of this treatment in Thailand was high, at B300/kg. of brine shrimp eggs.
a Translated from the Thai Fisheries Gazette, 1979, 32(2), 181–186.
b Fisheries Biologist, Chacheongsao Fisheries Station, Bangpagong, Chacheongsao, Thailand
The Chacheongsao Fisheries Station, considering the excessive cost of decapsulating artemia eggs by this method, supported a new experiment in this field. This experiment introduced new chemical substances for use in the decapsulation process and investigated ways and means of improving and modifying the technique at different stages. The result of this experiment proved that chemical substances that are cheaper and more readily available in the Thai market, such as lime (CaO) and bleaching powder (calcium hypochlorite), can be used as stimulating agentsin the decapsulation of artemia eggs. These can be used by modifying the technique at various stages. The volume of water and the quantity of chemicals used in this modification are also decreased enormously thus reducing the cost of decapsulation to about B18/kg. of brine shrimp eggs.
Additionally the technique for the preservation of decapsulated artemia eggs has been improved by a more efficient method. Instead of preserving decapsulated artemia eggs in strong salt solution (brine), they can be kept directly in refined salt.
Hatching Environment for sell and shell-less sucess
Follow these guidelines for the best results:
25 parts per thousand (ppt) salt solution, or approximately 1 and 2/3 tablespoons of salt per quart (or liter) of water. This equates to around 1.018 specific gravity as measured with a hydrometer. Be sure to use marine salt or solar salt.
Proper pH is important in hatching brine shrimp. A starting pH of 8.0 or higher is recommended. In areas where the water pH is below 7, Epson salt or magnesium sulfate can be added at the rate of 1/2 teaspoon per quart of solution to buffer the hatching solution.
Optimum water temperature for a 24-hour complete hatch is 80-82°F or 26-28°C. Lowering the temperature would result in a longer hatching time. Do not exceed 30°C.
Illumination is necessary to trigger the hatching mechanism within the embryo during the first few hours of incubation. Maintaining a light source during the entire incubation period is recommended to obtain optimum hatch results and for temperature control.
Constant aeration is necessary to keep cysts in suspension and to provide sufficient oxygen levels for the cysts to hatch. A minimum of 3 parts per million dissolved oxygen during the incubation is recommended. Strong aeration should not damage or hurt the brine shrimp cysts or nauplii.
- Stocking Density:
1 gram per liter or quart or approximately 1/2 level teaspoon of cysts per quart is recommended. A higher stocking density will result in a lower hatch percentage.
- Hatching Cone:
Flat-bottom hatching vessels should be avoided. Cone or “V” bottomed containers are best to insure that the cysts remain in suspension during hatching. Be sure to thoroughly wash the hatching cone with a light chlorine solution, rinse, and allow to air-dry between uses. Avoid soap. Soap will leave a slight residue which will foam from aeration during hatching and leave cysts stranded above the water level.
- Incubation Period:
Generally, the optimum incubation time is 24 hours and 12 hours for shell-less eggs. Egg which has been properly stored for more than 2-3 months may require additional incubation time — up to 30-36 hours. Oftentimes, eggs will hatch in as few as 18 hours. If a smaller size nauplii (Instar I) is desired, a harvest time of 18 hours is recommended.
Brine shrimp egg is sometimes very buoyant. In order to maximize the hatching percentage, it is sometimes helpful to swirl the water inside the hatching container with your finger once or twice at intervals in the first 4 to 6 hours of incubation in order to knock down eggs that have been stranded on the side of the container above the water-line. After about 6 hours, the eggs are usually well-hydrated and will stay in the water column.
2 TECHNICAL PROCEDURES
Introduce the artemia eggs into plastic containers. Add a small amount of water. Aeration of both water and eggs must be continuous to re-hydrate the eggs fully. (Dry artemia eggs are deflated like bean seeds and change into a round shape when completely hydrated). Leave the artemia eggs at this stage for 45–60 minutes. Then put them into a filter bag and wash them until they are completely clean.
After cleaning transfer the eggs back into the plastic containers. Add some water and ice until it contains a volume of 700 ml per 100 g of eggs and the temperature is maintained at 20°C.
Add lime (CaO) at the rate of 12.5% of the weight of eggs. Stir until mixed thoroughly. Then add bleaching powder at the rate of 27.5% of the weight of artemia eggs and keep on stirring until the substances are mixed thoroughly; this stimulates the reaction of the bleaching powder in removing the shell of the eggs. The procedure must take place at a controlled temperature of 40°C (do not let the temperature exceed 40°C; it can be maintained by adding more ice). Stir for 5–8 minutes until the temperature stays steady; this shows that the reaction of the first step of decapsulation has been completed.
Add some more ice to decrease the temperature to 30°C and then continue with a second decapsulation stage, in the same manner as the first stage, adding lime and bleaching powder in the same amounts. Stir for 5–8 minutes until the decapsulation reaction is complete. The colour of the eggs will change from dark brown to white. Leave them at that stage until the white color gradually becomes orange. The whole procedure, including both stage will now have taken 10–16 minutes.
After the eggs have been decapsulated, transfer them into a filter bag. Wash them with several changes of clean water to remove the smell and presence of dissolved chemicals. Then put the eggs in a solution of sodium thiosulphate (Na2S2O3.5H2O) prepared to provide a strength of not less than 0.05 g per 100 g of artemia eggs. Add 100 ml of water and stir for 2–5 minutes until mixed thoroughly (this will neutralise the chlorine which still remains on the artemia eggs). At this stage, decapsulated eggs will have sunk to the bottom of the plastic container. However, the eggs which are not completely bleached as well as some dirt will be floating on the surface. Siphon all these eggs out and discard (if the quantity of incompletely decapsulated eggs is large however, they can be stored in refined salt until the next decapsulation process). Wash the eggs in the filter bag with water once more. They are then ready for hatching or storage in refined salt until required.
Store the eggs in refined salt in the ration of 30g NaC1/100g of brine shrimp eggs. The salt will dehydrate the eggs. Pour off the water produced and store the eggs in shade at normal room temperatures. Artemia eggs can be preserved in this manner for a long time. From a comparison of eggs preserved for 7 weeks in brine of about 300 ppt and those preserved in refined salt, it has been found that the hatching percentage of the latter is higher than in the former (Table 2).
Comparisons of hatching efficiency between non-decapsulated eggs and those decapsulated either with sodium hypochlorite or calcium hypochlorite (bleaching powder) show the decapsulated artemia eggs to have better hatch-ability. Eggs decapsulated by either process give the same result.
Artemia eggs which have been decapsulated have a higher hatching efficiency than those which are not decapsulated.
Artemia eggs which have been decapsulated with bleaching powder have the same hatching efficiency as those decapsulated by sodium hypochlorite; the process is however reduced in cost by the use of bleaching powder from B300/kg. to B18/kg. of artemia eggs.
Preservation of decapsulated artemia eggs in refined salt is more efficient than preservation in brine.
Bruggeman, E., Baeza-Mesa, M., Bossuyt, E. and Sorgeloos, P., 1977. Improvement in the decapsulation of Artemia cysts. Proceedings of the Conference on Aquaculture “Cultivation of fish fry and its live food,” Szymbark, Poland, September 23–28, 1977 (in press). Sorgeloos, P., Persoone, G., Baeza-Mesa, M., Bossuyt, E. and Bruggeman, E., 1978. The use of Artemia cysts in Aquaculture. The concept ‘hatching efficiency’ and description of a new method for cyst processing. Proceedings of the World Mariculture Society, 9: 715–721.
Table 1. Comparison of the hatching efficiency of non-decapsulated artemia eggs, eggs decapsulated by sodium hypochlorite and those decapsulated by calcium hypochlorite (bleaching powder).
|24 hrs||36 hrs||24 hrs||36 hrs||24 hrs||36 hrs|
a Number of eggs
b Number of nauplii hatched
Table 2. Average % hatched after 36 hours at 8 ppt following different storage techniques and periods for eggs of Artemia salina.a
|Number of weeks stored||% Hatchability of decapsulated brine shrimp eggsb|
|Preserved in refined salt||Preserved in strong brinec|
a Decapsulated eggs were preserved either in refined salt or in brine at ambient room temperatures (28–30°C).
b In accordance with the advice of other scientists working on brine shrimp eggs, the incubation period lasted less than 48 hours.
c Decapsulated artemia eggs preserved in brine for more than 3 weeks smelt rotten and their colour gradually changed to pink; the colour of the brine solution turned black. Decapsulated eggs preserved in refined salt for more than 7 weeks retained normal smell and colour.
****The Super Simple Way!
Ok… so here’s what you REALLY came to this page for. Here are instructions for you to decapsulate your own brine shrimp eggs and chances are it is more easy than you first thought.
You will need :
- Brine Shrimp Cysts (duh!)
- Your normal 2 liter brine shrimp hatcher* + airpump
- Bleach – Sodium hypochlorite solution. (Ol’fashioned Bleach. NONE of that flowery smelling, color safe stuff).
- Table Salt. You can use aquarium salt, but table salt is cheaper and is less likely to leave white precipitate.
- Brine Shrimp Net or rotifer sieve
- Dechlorinator (I like “Prime”)
- Small saucepan or microwaveable container (to heat water)
- Water (tap water is fine)
- Measuring spoon (1 cup)
Step 1 – Rehydrate The Dehydrated Cysts
If you look at the dried cysts under a microscope, you will notice that it is not perfectly spherical. In fact, it looks kinda scrunched up and bumpy. Kinda like those dried prunes that grandma made you eat when you were *ahem* clogged up. This uneven surfaces makes it difficult to uniformly remove the protective shell. When re-hydrated, the cyst sucks up water and balloons into a something that looks more spherical, which will help when we are trying to strip the surface shell later.
Hook up your air pump to your hatchery and fill with a cup or 2 of tap water (no salt yet!!). Grab a tablespoon of brine shrimp eggs and add it to the water. Let it it bubble for 1 to 2 hrs depending on the brand of eggs that you have. If you have no idea where your eggs came from or just want to play it safe, then let it rehydrate for 1.5 hrs.
The only accurate way to determine how long to rehydrate is to view cysts under a microscope to determine if they have fully formed into spheres. Too short and your cysts would not have ballooned enough. Too long and the embryo insides starts developing and will definitely be killed later when we dehydrate it again for storage.
Step 2 – Prepare Saturated Brine (Salt) Solution.
This step should be performed while waiting for step 1 to finish. A saturated salt solution will later be used to dehydrate the eggs for storage after decapsulation. The only way to ensure a super saturated solution is to dissolve salt into warm or hot water. This can be done by heating a bowl of water in the microwave and then dissolve as much salt as will dissolve before the liquid cools down. You will know the solution is saturated when the liquid is still warm and no more salt will dissolve in it.
Alternatively you can use a saucepan and heat water on the stove top while stirring in salt. When the liquid is saturated (no more salt will dissolve), you can take it off the heat and allow to cool. In this pic you can see what happens after cooling for an hour. The salt precipitates out of the saturated solution, forming a ring of salt on the surface and a mound of salt at the bottom.
Now that the cysts are adequately re-hydrated and the salt solution is prepared, we are now ready to start decapsulating. The next few steps happen very quickly, so make sure you read the next few steps and understand them thoroughly before you start.
Step 3 – Decapsulating
After an 1 to 2 hrs of rehydrating, prepare 1 to 2 times as much of clorox bleach compared to the amount of water you used to re-hydrate the eggs. Again the amount of bleach required is dependant on the brand of cysts that you have. The first time you do this it is pretty much trial and error, but after a couple of times, you will be able to get a feel of how much bleach to use. In my case, I find that it requires twice as much bleach as water i.e. 1:2 ratio. (If I used 1 cup of water to rehydrate the brine shrimp before, now I will need 2 more cups of bleach). Pour the bleach into the hatcher with the cysts already bubbling inside.
Now things become crucial. DON’T TAKE YOUR EYES OFF THE CYSTS FOR THE NEXT 5 – 10 MIN!!!!!!! Depending on the brand of cysts and how much bleach you use, the decapsulation process will take anywhere from 5 to 10 min. If you find that it takes more than 10 min, then you haven’t added enough bleach. Adjust your ratio for the next time round.
You are watching for several color changes…. The cysts start out golden brown at first (before adding bleach)…..
The next color change you are looking out for is WHITE. They cysts will turn white as the brown outer shell gets stripped off.
Then it will slowly turn orangey brown again as the white layer gets removed.
Lastly, it should turn completely ORANGE as the shell is completely stripped off, revealing the orange embryo inside. And no… Do NOT add soy sauce and olive oil !!
Step 4 – Rinse Decapsulated Eggs
The moment the eggs turn orange IT IS DONE!!! Now we need to immediately stop the decapsulation process before the bleach eats through the remaining membrane and kills the embryo inside.
Pour the contents of the hatcher into a brine shrimp net (or plankton sieve) to drain away all the bleach solution and immediately run it under the tap for a few minutes. Rinse it until you can barely smell any chlorine in the net.
Step 5 – Dehydrate Using Super Saturated Brine
Once you are satisfied that you have washed away most of the bleach, turn your attention to the hatchery. Rinse it thoroughly until most of the bleach is gone. Then return the rinsed brine shrimp eggs to the hatchery. Make sure that your super saturated brine solution has cooled to room temperature and pour into the hatchery with the decapsulated eggs.
Step 6 – Allow to dehydrate for at least 12 hrs
I usually add a partial cupful of prime in there to make sure that all the chlorine has been removed.
Allow the solution to bubble for about 12 hrs. As the eggs dehydrate due to osmotic pressure, the water coming out of the eggs will start diluting the once super saturated brine solution. After the first 5 hours or so, you may want to add a little bit more super saturated brine or a couple pinches of salt to keep the brine saturated enough to keep pulling water out of the eggs. After 12 hrs or so of dehydrating, the eggs should be ready for medium – term storage
Storing Decapsulated Eggs
There has been a lot of debate about storing decapsulated brine shrimp eggs and for how long you can store them for. Some places tell you to simply store them in a tupperware in super saturated brine in the fridge, some places tell you to store them dry in 100% salt. While the latter actually preserves the decapsulated cysts much better and ensures a much better hatch rate, good luck trying to separate eggs and salt when it comes time to hatch them. It’s going to be a nightmare to ensure that you have the correct salinity to hatch the eggs as it is unavoidable that you will pick up some salt along with the eggs. The first method is ok, but as eggs continue to dry out and the storage solution becomes less and less salty, there is always a risk that the brine solution may become dilute enough such that fungus may start to grow on the eggs. I found the best way is to combine the above two methods.
After allowing 12 hrs for dehydration, pour the eggs through a brine shrimp net again to drain away all the water. Transfer the eggs to a little tupperware (I like these tiny ones). Pour enough fresh super saturated brine solution over the eggs to cover them by at least 1/2″. On top of that, add a pinch of granulated salt into the solution so that it forms a very thin layer of solid salt granules. Mix everything up.
Harvesting is easy, have a little pipette or syringe ready. Give the container a good shake. All the salt granules will settle to the bottom first. Next the eggs will settle on top of the salt layer. Harvesting is as simple as sucking up the top layer of eggs with the pipette. When you get real good at it, you will be able to notice that if you tilt the container at certain times while the layers are settling, you will be able to get a good thick layer of eggs at the corners which you can very easily suck up.
In order to keep the eggs in tip top shape, make sure that there is always a certain amount of salt granules with the eggs. In the even that you no longer see any salt granules, it means that the solution has become diluted and hence caused the salt to dissolve. If that happens, simply add a couple pinches or salt to the container.
Hatching After Decapsulation
Hatching is exactly the same as hatching cysts that are not decapsulated. 1 quart of water + 1 tablespoon of salt + bring shrimp cysts. The only difference is that you will notice that BBS will start to hatch much earlier. Check the hatchery at about 16 hrs and you should be able to partially harvest the ones that have already hatched. Unhatched eggs will sink to the bottom so all you need to do is skim the surface with a brine shrimp net to pick up any ones that have already hatched. Keep areating unhatched eggs for a couple more hours and you will be able to do a second harvest 4 – 5 hrs later.