From http://dianawalstad.com HATCHING and GROWING BRINE SHRIMP By Diana Walstad 1 February 2018 The usual method for providing brine shrimp (Artemia franciscana) as live food for fish involves making new batches every day in freshly prepared saltwater. This means mixing up lots of saltwater. If the shrimp don t all hatch the first day, which is often the case, it wastes expensive eggs. More than one fish breeder has decided that feeding live brine shrimp just isn t worth the trouble. My method involves raising the shrimp for 3-4 days before feeding them to the fish. I get maximal growth of the shrimp before the hatching bottle gets overpopulated. 2 The shrimp are still small enough for newborne livebearers, but now, having grown 3-4 times their hatched size, they are still attractive to juveniles and even adults (Fig). But wait.if my method was easy or intuitive, aquarium hobbyists would have been using it long ago. Yes, I can get a good hatch in clean, freshly prepared salt water, but the brine shrimp quickly lose their food value and die within a few days. Guppies Going After 4-Day-Old Brine Shrimp The shrimp are big enough to attract all ages from newborne fry to adults. The juvenile guppies in this photo are less than a month old and growing fast. They get fed live brine shrimp 1-2 times per day along with other foods. If their enthusiasm for hunting down live brine shrimp is any indication of nutritional value, then live brine shrimp is good food indeed! 1 I would like to thank Gerald Pottern, charter member of the Raleigh Aquarium Society and an authority on live foods, for his assistance on this article. 2 The maximum density of adult brine shrimp under the best natural growth conditions (summertime in Mono Lake, Calif.) is 6-8 adults per liter (~quart). Peak densities in the Great Salt Lake are only ~3 per liter [1].
2 I assume that most readers know the basics of hatching brine shrimp. I ve found that there is plenty of leeway in terms of temperature, salinity, egg source, etc. For example, if the water temperature is 65ºF instead of 80ºF, the eggs will take longer to hatch, but they will still hatch. GETTING STARTED I recommend that hobbyists start out small just to see for themselves how the hatching bottle with patience can be turned into a brine shrimp nursery. Try hatching out brine shrimp as usual but add a pinch of food to encourage bacterial growth. Harvest the nauplii (i.e., newly hatched brine shrimp) the next day, but instead of discarding the hatch water, filter out the egg shells (using the brine shrimp net) and pour the water back into the same bottle. No need to clean the bottle. Start another hatch in the bottle with the filtered saltwater. See how it does. If the water is completely clear, you could add more food. You want the water to be slightly cloudy. And it shouldn t smell. Keep using the bottle and old saltwater for a few weeks. Ideally, you should be getting the same good hatch rate that you got when you first set up the bottle. Over time, the bottle will become seasoned colonized by bacteria and microalgae that will sustain brine shrimp growth. If you are encouraged, try using the seasoned bottle to grow out shrimp for a few days before harvesting. You can set up a new bottle for just providing nauplii. In this way, I got a feel for what works to grow out the shrimp, plus gradually added new bottles to my system. Now, I have 4 bottles going at a time, enough to feed 7 tanks of guppies and their babies. I use clothespins marked A,B,C, and D to more easily identify which bottles have the oldest cultures and which ones have the youngest. Generally, I start each culture on a different day, but the system is flexible. The brine shrimp may be 3-5 days old depending on when I get to the bottle. HATCHING FACTORS Brine Shrimp nauplii These newly hatched shrimp are the same size as the eggs (dark dots). 3-Day-Old Brine Shrimp After hatching, I let the brine shrimp grow for 3 days in their hatching bottle. Note the increased size compared to the eggs. Temperature was ~74 F. Eggs: I ve gotten essentially the same results using eggs from either San Francisco Bay or the Great Salt Lake. I keep the eggs in a sealed container in the refrigerator and add 1/8 level teaspoon to about 2 quarts of saltwater. By always starting with the same concentration of eggs, I can better gauge how successful the hatch is. I have found that brine shrimp cultures started with more eggs sometimes do well for a day or two, but usually won t last 3-4 days. Brine shrimp eggs that I bought in 1990 and stored in the freezer, are still hatching after 27 years. 2
3 Food: In nature, brine shrimp feed off of microalgae. For artificial cultivation, the following foods have been used: Spirulina algae, fry food, yeast, boiled egg yolk, and rice bran. Foods high in lipid (i.e., fats) and protein, are recommended for young brine shrimp [2]. Yeast is actually a relatively poor food, because it has cell wall proteins that are hard for the shrimp to digest [2]. I like to feed my brine shrimp Spirulina algae, because it contains high percentages of both proteins and fat. I purchase the Spirulina algae either as a powder in gelatin capsules or as pills from the nutrition centers at Whole Foods, GNC, etc. (A bottle of Spirulina pills or capsules stored in the freezer will last for years.) For the pill form, I use a mortar and pestle to first make a powder. Then, I prepare a water slurry from the powder. (Because brine shrimp are filter feeders, they cannot ingest large particles.) I squirt in enough of the Spirulina algae slurry to the shrimp bottles to turn the water somewhat green. I add a fair amount of food the first week or two, mainly to get the culture going. After Brine Shrimp Factory The four bottles shown here share a clamp light (23 watt CFL bulb of 6,500K) with a planted 5 gal tank nearby. The overhead light stimulates the growth of beneficial microalgae in the water and attached algae on the sides. Water circulation via gentle air bubbling in the bottles constantly brings microalgae to the surface where it harvests light efficiently. Brine shrimp feed on the algae, plus the algae maintains good water quality. that, I add more Spirulina algae every week or two. As the bottles become seasoned, the sides become coated with algae that the shrimp feed on. Lighting: I ve gotten much better shrimp harvests since I added good lighting (Fig). Although some fish breeders use continuous light for their hatching bottles, it may not be necessary. One recent investigation [3] showed that there was no significant difference in hatching efficiency using either a 2, 12, or 24 hour photoperiod. My bottles get 12 hours of light per day. Good lighting stimulates the growth of microalgae in the bottles. Microalgae is not only a natural food source for the shrimp, but these tiny factories of photosynthesis consume ammonia and CO 2, keep the ph up, and produce oxygen. With good lighting, I can add much more food to the bottles without the water fouling. And the shrimp do better with the extra food. Aeration: Aeration can be gentle in a bottle ecosystem where algae is flooding the water with oxygen. Brine shrimp like newborn fish will lose energy and die if they are constantly fighting a strong current. While nauplii may survive vigorous aeration, it is not ideal. I keep the bubbling gentle such that the water surface is barely disturbed and no foam is generated. Bubbles are released at a rate of about 1-2 per minute from a glass tube (5 mm O.D. X 12 inches long) attached to airline tubing. I keep pieces of saran wrap on top of the bottles to minimize water evaporation. 3
4 Temperature: Eggs hatch overnight at 80-85 F. At 65-75ºF, it takes 1-2 days for all the eggs to hatch. (I wonder how many hobbyists have thrown out perfectly good eggs when they didn t all hatch the next day.) With my method, temperature doesn t matter, because I am not depending on an overnight hatch. I give every egg time to hatch. Saltwater Mix: I add 1/3 cup of either pure aquarium salt or non-iodized 3 table salt (NaCl) and ¼ teaspoon of baking soda (NaHCO 3 ) to a gallon jug and fill it with tapwater. The density works out to 1.020 g/cc and a salinity of 27 (parts per thousand). The baking soda insures that the water has an alkaline ph. (The oyster shells that I add to the bottles provide some calcium.) Hobbyists with softwater (GH of 4 or less) may need to use marine salts for preparing their saltwater. Marine salts automatically adjust the ph to seawater s ~8.3 ph, and they contain more minerals (e.g., potassium, magnesium, calcium, etc) than just table salt. For a Harvesting To collect the brine shrimp, I use a 2 quart pitcher, brine shrimp net, airline tubing (attached to glass tubing), and gravity. time I used marine salts, but I found that table salt worked just as well. (My hard tapwater and the oyster shells apparently provide enough minerals for the shrimp.) Brine shrimp can be hatched at 5 to 85 [4]. Salinities higher than seawater s 35 were found to increasingly inhibit hatching [5]. One investigator [6] got his brine shrimp to grow and reproduce just fine at 20. My 27 salinity represents an arbitrary compromise. Ecosystem in a Bottle The sides are covered with algae and bacteria that the brine shrimp can feed on. Water Conditioner: After the salts dissolve, I have to add an aquarium water conditioner that eliminates heavy metals. Otherwise, the eggs will not hatch in my tapwater, because it contains zinc. Hobbyists who continuously get suboptimal hatches should try using a water conditioner. Many conditioners contain EDTA, which chelates heavy metals such as copper, aluminum, and zinc, thereby rendering the metals non-toxic to the eggs and young shrimp. 3 I have used iodized salt at times without obvious problem. Hobbyists should probably avoid table salts that contain anti-caking agents such as aluminum silicate, which can be toxic to shrimp. I use Morton s Salt, which contains calcium silicate, a more desirable anti-caking agent. 4
5 Bottles: To ordinary plastic bottles of about 2 quarts, I add a little aquarium gravel, along with sea shells and oyster grit to maintain water calcium levels. This added material increases the surface area for bacteria and algae, many of which live attached to surfaces. Added material is optional and should not be so deep that it creates anaerobic pockets at the bottom. Harvesting: I siphon out as much brine shrimp as I need. Light will encourage them to collect in a certain area of the bottle for partial harvests. To collect an entire batch, I siphon down to the bottom including some of the dregs. (A goodly number of shrimp will congregate at the bottom of the bottle.) After collecting the shrimp in a brine shrimp net and the saltwater in a pitcher, I rinse the net contents with tapwater before adding the shrimp to tapwater in a small cup. From this cup, I feed the fish. Using a 10 ml pipette, I can easily distribute portions between several tanks. I don t mind if debris and some egg casings come along with the shrimp. (I trust my fish to know the difference between egg shells and live brine shrimp!) Setting up a New Hatch: After getting the shrimp into the feeding cup, I transfer some of the old saltwater back to the bottle, swirl the water to suspend the empty shells and then quickly pour the saltwater back through the brine shrimp net into my collection pitcher. I discard the dregs caught in the net and then pour the old saltwater (from the pitcher) back into the bottle. I add the eggs and if the water volume has declined a bit of replacement water. DISCUSSION Live brine shrimp have long been recognized as a superior food for young aquarium fish. Indeed, aquaculturists the world over have not yet found a better food source than brine shrimp nauplii for raising their farmed lobsters, scallops, crabs, tiger shrimp, and aquarium fish. Less recognized is that adult and juvenile shrimp are better nutritionally than nauplii. Aquarium hobbyists are instructed to use the shrimp soon after hatching. Within 6-8 hours (at 80ºF), the nauplii reach the Instar II stage of development [7]. They cannot eat during this time and have lost energy breaking out of their shells and swimming around. 4 This explains why decapsulated eggs (i.e., the brine shrimp egg minus its shell) fed directly to fish has been shown experimentally and repeatedly to support faster fish growth and greater egg production than nauplii [8,9,10]. 5 Since brine shrimp eggs never hatch all at once, it is virtually impossible to get an optimal harvest with the conventional method. If one harvests too soon, many of the eggs haven t hatched and are wasted; if one waits for a more complete hatch, many of the nauplii will have lost their food value. What to do? Culturing the shrimp for a few days after hatching solves the harvest timing problem. Moreover, the overall nutritional value of brine shrimp increases once the brine shrimp start feeding. For example, the average protein concentration increases from 42% in nauplii to 60% in adults [4]. Critical amino acids (histamine, methionine, etc.) absent in nauplii are present in adults [11]. 4 The Instar II stage of the San Francisco Bay strain was found to contain 27% less energy than Instar I [7]. 5 The high concentration of HUFA (highly unsaturated fatty acids) in the eggs is believed to be responsible for the superiority of decapsulated eggs as a fishfood. Other advantages are lower production costs and the smaller size (0.2 mm for the eggs as opposed to 0.4 mm for the nauplii) such that the small fry of egg layers can eat them [11]. That said, when the experimental fish in one study [8] received nauplii plus a commercial pelleted food, they did just as well (weight gain and egg production) as fish receiving only decapsulated eggs. 5
6 Some hobbyists describe old saltwater as liable to cause disease in fish. However, because bacteria have trouble crossing the osmotic divide from saltwater to freshwater, brine shrimp are much less likely to cause disease in fish than freshwater live foods. Years earlier, when I had less fish to feed, I kept a shrimp batch going 5-7 days in a seasoned bottle with old saltwater. I harvested only a small portion of the shrimp each day and allowed the rest to continue growing. This system worked very well. The daily removal of some shrimp kept the bottle from becoming overpopulated so that on Day 5 or Day 6 I still recovered a fair number of shrimp. Now that I am raising a large number of guppy fry, I use up a bottle about every day. The brine shrimp are 3 to 4 days-old, approaching the sweet spot for maximal growth and survival. Investigators [12] showed that brine shrimp, even when well-fed on a good food source (rice bran), began to decrease in numbers at 4-5 days (See Graph). The decline in survival at 4-5 days for all parameters coincides with the shrimp s complete absorption of its yolk sac. The investigators obtained interesting results using bacteria as the only food source. Apparently, not just any old bacterium will do. The graph shows that shrimp grew much faster when fed Pseudomonads than Vibrio bacteria. At 4 days, shrimp fed on Pseudomonad bacteria did as well as those fed rice bran over 90% survival In contrast, unfed shrimp or those fed Vibrio bacteria were dead at 4.5 days. (Brine shrimp probably do best feeding off a combination of food sources.) The same investigators [12] also showed that it took time to colonize desirable bacteria in the shrimps environment. On Day 1, the population of tasty Pseudomonads was insignificant. After one week of brine shrimp culturing, though, the Pseudomonads in the bacteria microflora had increased to 20%; after two weeks, 90%. This means that one may have to wait for the optimal colonization of microorganisms. I ve found that new bottles are much less productive than older, seasoned bottles. Colonization with desirable bacteria takes time perhaps analogous to waiting 3-4 weeks for the development of nitrifying bacteria in the bio-filter of new aquarium setups. Microalgae such as species of Dunaliella, which make up 95% of the phytoplankton in the Great Salt Lake [1], would similarly require time to colonize the bottles. Food Type and Survival [12] Graph shows the effect of no food and 3 food sources rice bran, Pseudomonas bacteria, or Vibrio bacteria over an 8 day culture period. Except for the two conditions where investigators added bacteria, brine shrimp were grown under sterile conditions. Bacteria and microalgae are introduced automatically into the bottle by hitching a ride on the brine shrimp eggs. These microorganisms are part of the shrimps natural habitat. Investigators showed significantly greater survival for shrimp cultured in old shrimp water than in seawater seeded with biofilter bacteria [13]. 6
7 Nauplii can tolerate low oxygen (2 ppm [14]) and seem almost impervious to ammonia. The lethal concentration (LC 50 at 24 hours) for total ammonia and ammonium is over 1,000 ppm [15]. However, brine shrimp are exquisitely sensitive to heavy metals. I suspect that many poor brine shrimp hatches may be due to metal toxicity, not bad eggs. Metal toxicity probably explains why some hobbyists can never get a decent hatch in freshly prepared saltwater. [My reused saltwater has enough DOC (dissolved organic carbon) to chelate the metals, and therefore, prevent metal toxicity.] Graph shows the effect of various concentrations of zinc on brine shrimp hatching. The zinc concentration blocking 50% of hatching was determined to be about 1 um, which is 0.065 ppm. My well water contains 0.8 ppm zinc over 10 times more; brine shrimp will not hatch in saltwater prepared from it unless I add a water conditioner that contains a metal Effect of Zinc on Brine Shrimp Hatching [16] Experiments were conducted at 28 C (82 F). Zinc levels in graph (0, 0.1, 1, 5 and 10) represent µm (micromolar) concentrations with 1 µm equal to 0.065 ppm zinc. In separate experiments, the investigators showed that copper was 10 to 100 times more toxic than zinc. chelator (e.g., EDTA). Other hobbyists have reported traces of copper in their municipal tapwater, often enough to keep brine shrimp from hatching. My bottles can go for many months producing abundant brine shrimp. I ve stored old saltwater for a year or more before putting it back to use. I cannot think of any reason to throw it away. The Balanced Shrimp Bottle may be analogous to the Balanced Aquarium. Nutrient input (food, eggs) is balanced by nutrient removal (harvested shrimp and debris removal). Algae, fueled by adequate lighting, further stabilize the bottle ecosystem by removing ammonia, consuming CO 2, keeping the ph up, and producing oxygen. The added oxygen helps keep a nutrient-rich system like this from going anaerobic. The fact that microalgae is also good food for the shrimp is an added bonus. It prompted me to get my bottles under some good overhead lighting for 12 hours per day. Like plants in an aquarium, microalgae contribute to a healthier environment. Readers should not expect to get a perfect harvest every time. Once in awhile, I get a lousy harvest, but the next harvest from that same bottle might be spectacular. Sometimes, it just prompts me to add more Spirulina algae to the bottles. The bottles contain bacteria and microalgae with population doubling times of minutes and hours. Bottles can go downhill fast, but they can recover just as quickly. Because of their intrinsic value as a source of food for farmed fish and marine invertebrates, the demand (and high price) for brine shrimp eggs is predicted to continue [17]. Aquarium hobbyists can help themselves by learning how to use brine shrimp eggs frugally and optimizing the hatching conditions. Allowing brine shrimp to grow for 3-4 days before using them enhances brine shrimp as a live food for aquarium fish. 7
8 REFERENCES 1. Mohebbi F. 2010. The brine shrimp Artemia and hypersaline environments microalgal composition: a mutual interaction (Review). Int. J. Aquat. Sci. 1: 19-27. 2. Marques A et al. 2005. Effects of bacteria on Artemia franciscana cultured in different gnotobiotic environments. Appl. Environ. Microbiol. 71: 4307-4317. 3. Asil SM et al. 2012. The influence of light (intensity and duration) on the cysts hatching parameters and nauplii growth of Artemia urmiana (Günther 1890). World J. Zoology 1:60-64. 4. Persoone G and Sorgeloos P. 1980. General aspects of the ecology and biogeography of Artemia. In: Persoone G et al (Eds). The Brine Shrimp Artemia (Vol 3). Universa Press (Wetteren, Belguim), p 36. 5. Dwivedi SN et al. 1980. Mass culture of brine shrimp under controlled conditions in cement pools at Bombay, India. In: Persoone G et al (Eds). The Brine Shrimp Artemia (Vol 3). Universa Press (Wetteren, Belguim), pp. 175-183. 6. Nambu Z, Tanaka S and Nambu F. 2004. Influence of photoperiod and temperature on reproductive mode in the brine shrimp, Artemia franciscana. J. Expt. Zoology 301A:542-46. 7. Dhert P et al. 1997. Possible applications of modern fish larviculture technology to ornamental fish production. Aquarium Sciences and Conservation 1: 119-128. 8. Tye M et al. 2014. Nonhatching decapsulated Artemia cysts as a replacement to Artemia nauplii in juvenile and adult zebrafish culture. Zebrafish DOI: 0.1089 9. Lim IC et al. 2002. Use of decapsulated Artemia cysts in ornamental fish culture. Aquaculture Research 33: 575-589. 10. Adewolu MA et al. 2009. Growth performance and survival of hybrid African catfish larvae (Clarias gariepinus X Heterobranchus bidorsalis) fed on different diets. The Zoologist 7: 45-51. 11. Malla S and Banik S. 2015 Production and application of live food organisms for freshwater ornamental fish larviculture. Adv. Biores. 6: 159-167. 12. Gorospe J and Nakamura K. 1996. Associated bacterial microflora in Artemia-rice bran culture. Israeli J. Aquaculture 48: 99-107. 13. Verschuere L et al. 1999. Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. Appl. Environ. Microbiol. 65: 2527-33. 14. Bossuyt E and Sorgeloos P. 1980. Technological aspects of the batch culturing of Artemia in high density. In: Persoone G et al (Eds). The Brine Shrimp Artemia (Vol 2). Universa Press (Wetteren, Belguim), pp 55-82. 15. Chen, J-C, Chen K-J, and Liao J-M. 1989. Joint action of ammonia and nitrite on Artemia nauplii. Aquaculture 77: 329-36. 16. MacRae TH and Pandey AS. 1991. Effects of metals on the early life stages of the brine shrimp, Artemia: A developmental toxicity assay. Arch. Environ. Contam. Toxicol. 20: 247-52. 17. Lavens P and Sorgeloos P. 2000. The history, present status and prospects of the availability of Artemia cysts for aquaculture. Aquaculture 181: 397-40 Diana Walstad is the author of Ecology of the Planted Aquarium (2013). For more information about her books, see: http://dianawalstad.com. 8