8 replies to this topic

[littlen]

To all interested parties:

There has been recent discussion on the decapsulation process, or the removal of the outer cyst/shell of Artemia nauplii, of [baby] brine shrimp eggs. “BBS” will be used in future references as it is an in-house term frequently used by the aquatic industry. Below, I will touch upon the methods I used to process large quantities of eggs over the years. Please keep in mind that this technique is not by any means the only one and there are many variations of it. There is definitely an art to decapsulating the perfect batch and it can take several attempts before you finally succeed. I will point out some of the reasons batches would fail from time to time in addition to the step-by-step process from hatching to harvesting. The following denotes how this technique was used to produce decasulated BBS for feeding Moon jellies, corals, and various aquatic larvae.

First, there are a few reasons to decapsulate BBS eggs. If done correctly, you can expect a higher hatch rate. The process dissolves the outer shell making the harvesting and feeding steps much simpler. As some may know, BBS eggs can cause an animal (fish fry in particular) to become impacted if enough are ingested. Artemia nauplii are a cheap, effective, and nutritional food item to culture to feed your aquatic species. They are the most nutritional immediately after hatching and are the smallest at this time too. They quickly grow and lose their nutritional value exponentially 12-24 hours after hatching.


Decapsulating:

The first step in the process starts with selecting a quality brand of freeze-dried eggs. Like almost everything offered for sale, there are some brands that are better than others. We used 425g cans of Bio-Marine as they proclaim, “90% Min. hatch” on the can, which I am certain does not pertain to decapsulating the eggs first. In any event, 200g of eggs (dry weight) were weighed out and poured into a 3 gallon, clear, plastic container filled with roughly 1 gallon of filtered freshwater [FFW]. The container was placed on top of a large, magnetic stir-plate and a 2” stir bar was added. It was set on high so that most of the eggs would become suspended to ensure proper hydration. Rigid air lines attached to air pumps were also added to opposite corners due to settlement, or dead-zones where the agitation caused by the stir bar would have no effect on churning the eggs. This is why a clear container was used. It allows you to see any dead-zones. The eggs were allowed to hydrate for 2 hours at room temperature.

After 2 hours, the eggs were strained through a large sieve with a 100 micron screen at the bottom. They were then removed using a rubber spatula and placed into a second, clear, 1 gallon container. The next couple steps are the most important and are likely where many of you have unknowing gone wrong. Before the actual decapsulation takes place, the original, 3 gallon container was rinsed and refilled with another gallon of FFW along with 20 grams of sodium thiosulfate crystals. Note: this set up is important in order to immediately stop the chemical process of dissolving the outer cyst. (Much the same as penaeid shrimp are added to a bowl of ice to arrest the cooking process after steaming). With the now rehydrated BBS eggs in the 1 gallon container, 2000mL of sodium hypochlorite (bleach) and 100mL of sodium hydroxide were measured out in a large measuring cup and graduated cylinder respectively. A 20:1 ratio of the two ingredients can be used for smaller batches. Note: both of these chemicals were stored in the refrigerator which was just above freezing (~35F). This is important as mixing these two chemicals together causes an exothermic reaction and heat is generated. It is significant that your BBS eggs do not get burned, thermally speaking.

{Side note: different brands of bleach contain varying concentrations of the active ingredient sodium hypochlorite. “Chlorox” was preferred as it generally had a higher concentrations than the generic, store brands. The MSDS sheet for Chlorox Bleach lists the concentration between 5-10%. Even using the same brand each time, the concentration could still vary and the “cooking time” (chemically speaking) would change. More on that process shortly}.

Next, the chemical cocktail was added to the strained eggs in the 1 gallon container. The solution was thoroughly mixed on the stir plate and by hand for 3 to 5 minutes, depending on the color change. This would have to be the most important step in the entire process where a trained eye is required to notice the color change from brown to pale yellow, to a faint orange.

**Please be aware that a full face respirator was used in addition to being in a room with a ventilation fan. Chlorine can cause serious chemical burns to the skin, lungs, and eyes. Please wear proper protection in addition to a smock or apron to protect your clothing from accidental splashes.**

Watching for the color change can be challenging. Nothing happens for the first few minutes, followed by the subtle formation of some foam. Finally, the color change from brown to yellow to orange happens quickly somewhere between the 3 and 5 minute mark. At this point you have to immediately halt the process, (hence the other container with FFW and sodium thiosulfate). Please understand that it is very hard to give an exact amount of time required to remove the outer shell in the bleach/sodium hydroxide solution. If not enough time is allowed then the shells will not completely dissolve. While you will still have a good hatch rate, there will be shells present when you hatch your next batch of ‘eggs’—effectively defeating the purpose of decapsulating in the first place. Too long, and you’ll burn all the way through the cyst and kill the Artemia nauplii inside. If trying your hand at decapulating BBS eggs for the first time, err on the side of too little time in the chemical bath as opposed to too long. For the record, even if you keep the eggs in the bath for too long and they die, the nauplii are still viable, aka, nutritional and safe to feed to your fish (after rinsing). Although if you require live nauplii to trigger your fish to feed, then these will be useless.

After the 3-5 minute bath, the eggs are quickly strained through the sieve and scraped into the original container now holding a gallon of FFW along with the sodium thiosulfate. The eggs will spend one minute suspended in this second bath by means of manual stirring in order to neutralize the chlorine. Finally, strain the eggs one last time into the sieve. At this point some residual chlorine will still persist so I would rinse them for an additional 15-20 minutes with FFW. Leave the room for a few minutes to clear your sense of smell or have someone else come in and smell the eggs. If you/they can still detect a faint hint of chlorine on the eggs, continue to rinse. 15-20 minutes should be thorough enough with a steady flow of FFW although you may not have access to that much. I do not know if using a (chlorinated) tap water rinse, followed by a final rinse with FFW will have a detrimental effect on your eggs. I suspect it would, but have no test results to prove this. The extra rinsing is essential and certainly doesn’t hurt.

The final step before storage is to allow them to dry for about a half day in the fridge. I would blot the bottom of the sieve several times using new paper towels and keep a towel under the screen to absorb as much water off of the eggs as possible. I again to not have any test results showing the effects of remaining moisture content of the eggs to hatch rate or shelf life in the fridge. I speculate the drier the better. They could probably last longer than 2 weeks, but that theory was never tested. I would look for the eggs to clump together like moist sand to determine if they were dry enough. If a large clump is poked, it should easily break apart. Afterwards, 15g worth would be weighed at a time and put into separate test tubes. This provided us with over 2 weeks worth of decapsulated eggs. This is likely way more than the home hobbyist will use in an entire year, or possibly even a lifetime. After hatching, there was enough to densely fill a 5 gallon bucket such that the visibility was less than 3” when the bucket was filled. In case it isn’t clear, the now decapsulated eggs are being stored dry—and not in a brine solution—in the refrigerator. Other techniques call for storage in an extremely saline solution before hatching, but not ours at any rate.


Hatching/Culturing:

I will quickly give an overview on how the nauplii were cultured as the inquiries are mainly on the decapsulation process. We had two, 5-gallon hatching cones that we used in our BBS rotation. They were inverted, acrylic, isosceles pyramids with a long piece of rigid airline running down the center. This provided sufficient aeration and turnover for the eggs. The first cone would be filled with a brine solution somewhere between 20-25ppt. We opted for a lower salinity to speed up the time required to hatch. We would also keep the cone at 80-85F for this same reason. A test tube, or ~15 grams of decapsulated eggs would be added and allowed to hatch for 24 hours. On day 2, we added enrichment, (‘Selco’ for those interested) to cone 1, and cone 2 would be set up exactly the same as the first cone on day 1. On day 3, the first cone would be ready to harvest after allowing the nauplii to feed on the enrichment for 24 hours. Before harvesting, the rigid airline was removed and the cone was allowed to settle for several minutes. This allows any dead nauplii, remaining or unhatched eggs to settle to the bottom while a light placed at the top draws the swimming nauplii towards the surface. A small valve at the bottom of the cone can be partially opened to allow the unwanted sludge at the bottom to drain out. A 5 gallon collection bucket is then used to harvest the rest of the viable culture. Cone 2 then receives its enrichment and cone 1 is rinsed and set up all over again.

[littlen]

I apologize for the long post and should have just offered it as a Word document. If anyone is interested, PM me and I can email you a copy.

Also, I'd be happy to elaborate on any of the material if it isn't clear enough. Please know that I am not the leading expert in the brine shrimp decapsulation process and do not understand every principle involved. This is just the way I learned. There are many great resources out there but trial and error will play a significant role regardless.

Good luck!

[mattknepley]

Very thorough and very interesting. Thanks for taking the time to write that up!

[littlen]

We are no longer producing any decapsulated brine as we're in the final stages of closing the aquarium and do not have a need for it. I really have no idea how long the shelf life would be even if kept refrigerated as I mentioned in the original post. My guess would be that you would see a significant drop in the hatch rate after 3 to 4 weeks.

Matt is spot on with the thio crystals. We order a 5lb. bucket at a time. It's pretty inexpensive and useful for folks with large systems who need to dechlorinate a lot of water. Certainly cheaper than the bottled solutions out there.

[littlen]

labgirl, as Gerald points out--larger fry rarely have any problems if a few eggs are consumed. Again, they seem to be able to know nauplii from egg. Decapsulating isn't necessary if you're hatching small batches and siphon off the free-swimming nauplii as you are. Some people can be careless and net out as many unhatched eggs/shells to feed to their fry as they do BBS. Seems obvious that you'd want to avoid this if possible but it isn't something everyone thinks about.


Gerald, the 2100mL bleach/NaOH mixture is poured into a container with only the re-hydrated eggs---no water as you indicated. (1)

My understanding of using the NaOH is that it works in conjunction with the bleach and is more efficient at removing the cyst as well as speeding up the process. I have heard of many methods of just using bleach too. I'm sure the problem is still the same that it is hard to tell when they are "done cooking" due to the inconsistency of the concentration. (2)

I am also under the impression that they do not begin feeding until after the first molt as well. Most of the commercial enrichment's particle sizes are too large for newly hatched nauplii to feed on. They must molt in order to begin feeding. There are some who state that the enrichment will adhere to the exoskeleton, still providing nutrition to whatever feeds on it. I cannot say for sure if they become more nutritional after the first molt--once they start feeding on the enrichment. Our needs didn't require that we feed out newly hatched shrimp. Technically, they were over 48 hours old before they were fed out. Without enrichment, they would be significantly less nutritional. So we gut load them for 24 hours to replenish what was lost the first day.