Paper:

Preliminary Evaluation of Selected Nutrient Composition of Two Life Stages of Artemia salina Before and After Feeding an Enriched Torula Yeast Product

Submitted by Frank M. Greco

By Frank M. Greco1,3, Marianne P. Fitzpatrick, M.S.2, Wendy S. Graffam, Ph.D.2, Ellen S. Dierenfeld, Ph.D., C.N.S.2, and Dennis A. Thoney, Ph.D.1

1 Aquarium for Wildlife Conservation, Wildlife Conservation Society, Boardwalk and West 8th St., Brooklyn, NY 11224

2 Wildlife Health Sciences-Nutrition Department, Wildlife Conservation Society, 2300 Southern Blvd., Bronx, NY 10460

3Contact: phrank2139@netzero.net

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Abstract

Aquarists commonly feed brine shrimp, Artemia salina-variation San Francisco, to invertebrates and both larval and adult fishes. We analyzed both larval and adult stages of Artemia, before and after being fed an enriched torula yeast diet (Microfeast Plus® L-10 Larval Diet). Moisture, ash, crude fat, crude protein, water soluble carbohydrates, neutral and acid detergent fiber, lignin, thiamin, ascorbic acid, choline, vitamin E and A activity, carotenoids and 12 minerals (Ca, K, Mg, Na, P, Cr, Co, Cu, Fe, Mn, Mo, and Zn) were analyzed. There were 5 groups of Artemia each with n=2:

1. newly hatched unfed nauplii;
2. 24-hour post-hatch unfed nauplii;
3. 72-hour post-hatch fed nauplii
4. unfed adults; and
5. fed adults.

Artemia fed the yeast diet had increased vitamin E activity (nauplii: 418.1 versus 618.6 IU/kg, adults: 75.9 vs. 155.6 IU/kg; unfed vs. fed, respectively), increased ash in adults (16.1 vs. 27.5%; unfed vs. fed, respectively), and decreased fiber fractions in nauplii. Results of water-soluble vitamin and mineral analyses were inconsistent, making it difficult to determine trends. Vitamin A and carotenoids were detected in only adult life stages of Artemia. While literature suggests that yeast enrichment may be useful for altering fatty acids, amino acids, and many water-soluble vitamins, we could find few references to the nutrients assayed in our study. Overall, nauplii and adult Artemia (both enriched and not) met the 1993 NRC recommended nutritional requirements of fishes for crude protein and crude fat, vitamin E, and all minerals measured, with the exception of Ca. Based on these preliminary data, we cannot determine whether feeding Artemia with Microfeast Plus® L-10 enhances its nutritional value.

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Introduction

Appropriate nutrition is a major factor in improving the longevity and fecundity of fish and invertebrate species in captivity. Deficiencies or excesses of individual nutrients can lead to circumstances where the life of the fish is severely shortened or its ability to reproduce compromised (Halver, 1972; Post, 1987; Noga, 1996). In fish fry or larvae, nutrition plays an even more important role, and improper or inadequate diets during the early stages of development can lead to morphological malformations and/or an inability to successfully reproduce later in life (Halver, 1972; Post, 1987; Verreth, et al., 1987; Noga, 1996). We have observed that fishes of the genera Hippocampus (sea horses) and Syngnathus (pipefish), commonly kept at the New york Aquarium and fed Artemia salina (brine shrimp) as a major portion of their diet, exhibit shortened lifespans, decalcification of their exoskeleton, and poor survival rate amongst their fry.

As public aquariums become more involved with the captive breeding and the long-term husbandry of endangered or threatened fish and invertebrate species, it is imperative that a better understanding of the nutritional needs of these animals be utilized when choosing diets. Artemia salina (brine shrimp) are a common food source for invertebrates and both larval and adult fishes such as juvenile butterfly fish and larval anemonefish. The ease of hatching cysts and the commercial availability of the adult stage makes it a reliable food source. The quick recognition of Artemia as a food organism by both larval and adult fishes and invertebrates also makes it an ideal food choice. Previous studies have shown that enriching the diets of Artemia can increase their nutritional value as a food item (Wantanabe, et al., 1982; Clawson and Lovell, 1992; Ako, et al., 1994), but most studies concentrate on the fatty acid composition of Artemia. Few studies report the proximate, vitamin and mineral levels of Artemia life stages (Wantanabe, et al., 1983; Verreth, et al., 1987; Webster and Lovell, 1990).

Therefore, this study was performed to determine whether juvenile and adult Artemia were a nutritious food source with regard to proximate, vitamin and mineral components both before and after being fed a torula yeast product. Five groups of Artemia n=2:

1. newly hatched unfed nauplii;
2. 24-hour post-hatch unfed nauplii;
3. 72-hour post-hatch fed nauplii;
4. unfed adults; and
5. fed adults

were analyzed for moisture, ash, fat, crude protein, water-soluble carbohydrates, neutral (NDF) and acid (ADF) detergent fiber, lignin, thiamin, ascorbic acid, choline, vitamin E activity, retinol, and eleven minerals (Ca, Co, Mo, K, Mg, Na, P, Cu, Fe, Mn, Zn). Sample sizes were limited due to the cost of analysis.

Artemia Life History

The brine shrimp (Artemia salina) is in the phylum Arthropoda, class Crustacean and is closely related to zooplankton like copepods and Daphnia. Artemia life cycle begins by the hatching of dormant cysts which are encased embryos that are metabolically inactive. The cysts can remain dormant for many years as long as they are kept dry. When the cysts are placed into salt water, they are re-hydrated and resume their development.

After 15 to 20 hours at 77°F (25°C) the cyst bursts and the embryo leaves the shell. For the first few hours, the embryo hangs beneath the cyst shell, still enclosed in a hatching membrane. This is called the umbrella stage, and it is during this stage the nauplius completes its development and emerges as a free swimming nauplii. In the first larval stage, the nauplii is a brownish orange color because of its yolk reserves, and does not feed because its mouth and anus are not fully developed. Approximately 12 hours after hatch they molt into the second larval stage and they start filter feeding on various microalgae, bacteria, and detritus. The nauplii will grow and progress through 15 molts before reaching adulthood in about 8 days. Adult Artemia average about 0.32" (8mm) long, but can reach lengths up to 0.78" (20mm). An adult is a 20 times increase in length, and a 500 times increase in biomass from the nauplii stage.

In low salinity and optimal food levels, fertilized females usually produce free swimming nauplii at a rate of up to 75 nauplii per day. They will produce 10-11 broods over an average life cycle of 50 days. Under super ideal conditions, adult Artemia can live as long as three months and produce up to 300 nauplii or cysts every 4 days. Cyst production is induced by conditions of high salinity, and chronic food shortages with high oxygen fluctuations between day and night.

Adults can tolerate brief exposures to temperatures as extreme as 0° to 104°F (-18° to 40°C). Optimal temperature for cyst hatching and adult grow out is 77° to 86°F (25° to 30°C), but there are differences between strains. Artemia prefer a salinity of 30-35 ppt (1.0222-1.0260 density) and can live in fresh water for about 5 hours before they die.

Materials and Methods

Enrichment media

Provesta Micro-Feast® L-10 larval diet, an enriched dehydrated torula yeast, was used in this study. This diet claims to have benefits over other enrichment media for several reasons:

1. It contains fatty acids, various macro- and micro- minerals, amino acids and vitamins.
2. Whereas other supplements tend to have a usable life of seven to ten days, Micro-Feast L-10 tends to have a shelf life measured in weeks, longer if kept frozen.
3. It tends not to foul the water as quickly as many other supplements, thereby reducing the mortality of Artemia due to degrading water quality.
4. It does not have to be cultured by the end user.
5. The nutritional makeup of the diet can be augmented to meet the specific need of the culturist.

Obtaining Artemia salina var. San Francisco nauplii

A 132.5 l Cal-Wal container was filled with 94.6 l of filtered seawater buffered with calcium hydroxide to pH 8.2, and heated to a temperature of approximately 27.7°C utilizing a 250 watt Visi-Therm aquarium heater. Artemia salina var. San Francisco cysts (200 g) were added and aeration sufficient to fully suspend the cysts was provided by three 4.5 mm air lines. Full spectrum illumination (20 watt DuroTest Vita-lite!), placed directly next to the hatching container, was provided constantly to enhance hatching (Hoff and Snell, 1987).

After 22 hours, the heater was removed from the container, and aeration stopped, allowing the newly hatched nauplii to separate from the cysts. After 30 minutes, the nauplii were run through a 200 micron mesh to further separate them from any remaining cysts. Nauplii were subjected to one of three treatments:

1. The first batch of nauplii (newly hatched), were collected and concentrated using a cheesecloth net, and rinsed with reverse osmosis (RO) water to remove any traces of salt water. They were then placed in clean high density polyethylene (HDPE) containers and frozen to -20°C.
2. The second batch of nauplii (24 hour post-hatch) were collected and concentrated using a cheesecloth net, then divided between two containers of approximately 75.7 l each. The nauplii were allowed to remain in these containers for an additional 24 hours before being harvested, rinsed in RO water, and frozen at -20°C.
3. The third batch of nauplii (fed nauplii), were collected and concentrated using a cheesecloth net, and were divided between two containers of approximately 75.7 l, each filled with 73.8 l of filtered seawater. Water temperature was kept at 23.9°C, and aeration was moderate. The nauplii remained unfed for the next 24 hours. In a Waring blender, 1.89 l of filtered seawater and 10 grams of Microfeast® L-10 Larval Diet were added. The enriched yeast was blended on high speed for 30 seconds, and the resulting suspension was added to each container, bringing the total volume to 75.7 l. The nauplii were then allowed to feed for 24 hours before they were harvested, rinsed in RO water, placed in clean HDPE containers and frozen at -20°C.

Obtaining Artemia salina var. San Francisco adult stage

Adult Artemia salina var. San Francisco were obtained through Zimmer and Son, Brooklyn, NY. Due to unfavorable harvesting conditions in San Francisco Bay brought on by El Nino, it was impossible to obtain sufficient amounts adult Artemia in one shipment. The resulting data is based upon five separate shipments, all originating from the same collection locale. The first batch of adult Artemia (unfed adult) was collected and concentrated using a cheesecloth net, rinsed in RO water, and frozen in clean HDPE jars at -20°C. The second batch (fed adult) was collected in the same way, and then divided between two containers of approximately 75.7 l each filled with 73.8 l of filtered seawater. Temperature was kept at 23.9°C, and aeration was moderate. The adult Artemia remained unfed for the next 24 hours, then allowed to feed for 24 hours on the same dietary suspension fed to the nauplii before they were harvested, rinsed in RO water, placed in clean HDPE containers and frozen at -20°F.

Analyses

Upon arrival at the Wildlife Conservation Society Nutrition Lab, Bronx, N.Y., all samples were thawed overnight in a refrigerator at 4°C. Samples were mixed thoroughly and sub-sampled. Vitamin A and E assays were performed immediately. An 80 g sub-sample was frozen at -30°C for choline, thiamin and vitamin C assays, and the remaining sample was freeze-dried and ground using a laboratory grinder prior to fat, protein, fiber, carbohydrate and mineral analyses. Extracts from the vitamin analyses were frozen and analyzed for carotenoids.

Proximate Composition

Percent moisture, ash, crude fat, and crude protein were obtained for all samples using AOAC methodology (Ellis, 1984; AOAC,1996). Duplicate samples (> 0.5 g) were weighed, then freeze-dried, and percent moisture calculated. These samples were then incinerated in a Thermolyne muffle furnace at 550°C overnight and total ash was calculated. Crude fat was determined by extraction with petroleum ether using AOAC Official method 991.36 (1996). Crude protein was determined using a macro-Kjeldahl method with a copper catalyst. Water soluble carbohydrates were obtained using a phenol/sulfuric acid colorimetric assay of Dubois et al. (1956) as modified by Strickland and Parsons (1972) and using sucrose as a standard. Fiber fractions (NDF, ADF, and lignin) were obtained using the methods of Van Soest (1994).

Vitamins

Vitamins A and E were analyzed using a modification of the methods of Taylor et al. (1976) as detailed in Barker, et al. (1998). Vitamin E activity was calculated as 1 mg a-tocopherol = 1.49 IU; 1 mg g-tocopherol = 0.15 IU; 1 mg d-tocopherol = 0.05 IU (Horwitt, 1993). Vitamin A activity was calculated as 0.3 mg retinol = 1 IU; 0.55 µg retinyl palmitate = 1 IU (Olson, 1984). Carotenoids were analyzed at Our Lady of Mercy Research Facility (Bronx, New York) using HPLC methodology. Choline, thiamin and vitamin C levels were run by Woodson Tenet Laboratories (Memphis, TN).

Elemental Composition

All mineral values were obtained by inductively coupled plasma-atomic emission spectroscopy at the Laboratory of Large Animal Pathology and Toxicology at the University of Pennsylvania.

LIPIDS
Lipids, in the form of fatty acids, appear to be essential to the proper growth and development of both marine and freshwater fishes. However, there is a differing need in these acids between the two, with marine species requiring eicosapentaenoic (EPA, 20: 5n-3) and docosahexaenoic (DHA, 22:6n-3) acids and freshwater species requiring fatty acids more along the line of the n-3 unsaturated fatty acids. Artemia, depending on which variety it is, may contain EPA in the nauplii stage thus making it suitable for marine species (marine-type Artemia), or n-3 unsaturated fatty acids such as 18: 3n-3 (but lacking EPA), making it suitable for freshwater organisms (freshwater-type Artemia) (Navarro and Amat, 1992; Navarro, Amat, and Sargent, 1993). Some Artemia strains contain the 20:5n-3 fatty acids, while none contain the 22:6n-3 fatty acids (Navarro, amat, and Sargent, 1993). The cause for this variability in fatty acids is unknown, but it may well be that the types of fatty acids found in certain strains of Artemia is influenced by the type and quality of food items (Navarro and Amat, 1992; Navarro, Amat and Sargent, 1992). Artemia salina var. San Francisco tend to vary greatly in their fatty acid profile between batches and location of harvest. Since this variety is most commonly utilized within the USA for a wide range of aquaculture organisms, and since fatty acid composition of this strain is so uncertain, enrichment appears to be key here.

Results and Discussion

Proximate Composition

The results of the proximate analyses are found in Table 1. Feeding had little effect on the proximate nutrient composition of the Artemia tested. Percent moisture was similar across all life stages of Artemia. Nauplii ash (mineral) content (X=10.4%) was comparable to those previously reported (X=10.07%, Wantanabe, et al., 1983; 4.5% Webster and Lovell, 1990). Feeding increased ash content only in adults. This may be attributed to the ingestion of the diet, which contained 12.01% DM ash as well as the presence of the exoskeleton in the adult shrimp. However, we cannot discount the contribution of minerals from the water as a potential source of inorganic material; the water mineral content was not analyzed in this study.

Crude protein content of Artemia was similar across all life stages (nauplii X=58.4%, adults X=60.0%, DM basis) and was higher than the level present in the diet (42.7%). The values reported here are like those of nauplii reported by Wantanabe et al. (1983) X=61.6% DM and Webster and Lovell (1990) 55.6% DM. Crude fat was almost two times higher in the nauplii stage than in adults. It appears that the crude fat content of the diet (20.22% DM) did not contribute to the crude fat content of Artemia in any stage, or the level present was not adequate for the needs of Artemia. Fat values reported earlier for nauplii (X=19.4% DM, Wantanabe et al., 1983; 20.1% DM, Webster and Lovell, 1990) are comparable to the nauplii values from this study (X=13.2% DM).

Soluble carbohydrates were higher in nauplii than in adults, and lower in all life stages compared to the diet. Either the diet is not a significant source of carbohydrate, or the level present in the diet is not adequate for the needs of Artemia in any life stage. Fiber constituents (NDF, ADF, and lignin) were diluted by feeding in both life stages, perhaps due to the lack of any fiber in the yeast diet. It should be noted that in the 24-hour post-hatch and the unfed adult Artemia, data indicated a downward trend for all nutrients tested. It is therefore essential that Artemia be utilized as quickly as possible in order to avoid nutrient degradation.

Vitamin composition

There was no straight-forward relationship between dietary concentrations of any of the vitamins measured and their presence in Artemia (Table 2). Although the fish choline requirement (NRC, 1993) was met by all nauplii and adult Artemia, levels decreased over time for both life stages despite the high dietary concentration of this nutrient (268.7 mg/100g). This is perhaps due to inadequate uptake of the diet, or the use of choline directly by the Artemia. Feeding increased thiamin levels in both fed nauplii and adult Artemia, although only nauplii stages met the 1993 NRC requirement of fish (NRC, 1993). Feeding did not increase vitamin C levels in any life stage, and was below detectable levels in the adult stage. The level of this nutrient may be low since the dietary concentration (11.3 mg/100g) did not meet the dietary recommendation for penaied shrimp (100-1000 mg/100g; Lim and Persyn, 1989). If this recommendation holds true for Artemia, it may explain the steady decrease of vitamin C in the nauplii stage and the non-detectable levels in all adults. Only newly hatched nauplii met the fish requirement for vitamin C (NRC, 1993). Vitamin A activity and carotenoids were detected only in the adult Artemia regardless of feeding. Therefore, if feeding carotenoids for fish coloration is important, only adult Artemia will meet those needs. Vitamin E activity increased in both fed nauplii and adults. However, even the unfed life stages met the vitamin E requirement for fishes (NRC, 1993).

Mineral composition

With the exception of calcium, all mineral requirements of fishes were met or greatly exceeded by all stages of Artemia analyzed in this study (Table 3). Besides the low Ca level, the Ca:P ratio provided by Artemia in all life stages was less than the recommended optimal 2:1. This may be why some species, notably seahorses and pipefish, come down with a "soft plate" condition after being fed a diet consisting solely of Artemia. However, it is not known how important dietary mineral concentrations are since minerals can be obtained by fish directly from the water. Future studies should determine how to improve the Ca:P ratio in Artemia. Calcium values of nauplii (0.26 ± 0.06% DM) reported by Wantanabe, et al. (1983) were double those reported here (0.08 ± 0.02% DM).

Iron concentrations were exceedingly high in 24-hour post-hatch nauplii, fed nauplii, and both stages of adults. While the iron level in the diet was significant (221.84 ppm), this alone does not appear to be contributing to increasing iron concentrations. It is possible that Artemia may be concentrating iron found in the water. The high level found in the above mentioned life stages are of interest since iron toxicity has been reported in rainbow trout fed diets containing >1380 mg Fe/kg (Desjardins et al., 1987 in NRC, 1993). Wantanabe, et al. (1983) also reported high levels of iron from Artemia nauplii from South America (3237 mg/kg DM) and Canada (2434.8 mg/kg DM), but not from San Francisco (n=4, X=365.4 ± 113.4 mg/kg DM). All other minerals reported here were similar to previously reported values for nauplii regardless of their geographic origin (Wantanabe, et al. 1983).

Conclusions

Based on these preliminary data, we cannot determine whether feeding Artemia Microfeast Plus® L-10 enhances their nutritional value. Feeding had little effect on the proximate nutrient composition of the various life stages. Ash content increased for the adult Artemia, but water could not be discounted as a potential source of minerals. There was also no straightforward relationship between dietary vitamin levels and their presence in the Artemia. One cannot assume that increasing vitamin levels in the diet will lead to higher concentrations in the feed organisms.

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Acknowledgments

We would like to thank the Species Survival Fund for providing the funding for this project.

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