Improved Enrichment of Artemia Using a Novel Algal Derived Material

Ingrid Lein1, Y. Barr1, Moti Harel3, P. Behrens3, A. Place2, G.M. Berge1

1AKVAFORSK (Institute of Aquaculture Research), N-6600 Sunndalsora, Norway
2Center of Marine Biotechnology, University of Maryland, Biotechnology Institute,
701 E. Pratt St., Baltimore, MD 21202 USA
3Martek Biosciences Corporation, 6480 Dobbin Road, Columbia, MD 21045 USA

Source: "Cultivation of Cold Water Species: Production, Technology and Diversification," Aquaculture Trondheim '97, Trondheim, Norway

Introduction

Enrichment techniques for improving the nutritional value of live food is widely used in marine fish hatcheries. Recent studies indicate the importance of the essential fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and also the DHA/EPA-ratio, for normal pigmentation, metamorphosis, and stress tolerance in marine fishes. Larvae of cold water species, e.g. Atlantic halibut (Hippoglossus hippoglossus L.) contain high levels of DHA (25-30% of total lipids), and also have a high DHA/EPA-ratio (about 2.5) at onset of exogenous feeding, indicating that these larvae need high amounts of DHA and DHA/EPA ratios in their feed. However, such high levels in feeds have not been achieved when using traditional enrichment products based on emulsified fish oils. The present work was carried out in order to test enrichment products which facilitate high contents of DHA and DHA/EPA-ratios in Artemia.

Material and methods

A novel algal derived material was used as experimental enrichment material. This material is high in DHA (>48% with no EPA), and constitutes spray dried phospholipid extract of algal biomasss. The effect of dosage and duration of the enrichment was tested with brine shrimp Artemia containing 1% DHA of the total fatty acids (premium grade, Sanders Brine Shrimp Co., Ogden, Utah, USA). In the first set of experiments, 2-3 portions of 0.1, 0.2 or 0.3 1-1 were added to artificial sea water (20ppt, 28ºC) to instar-II-stage Artemia nauplii (200,0001-1) after 8, 12 or 16h. Survival, growth, lipid content, lipid composition and fatty acid profiles were measured during 24h enrichment period.

In the second set of experiments, comparisons with four other commercial enrichment products which are all used to gain high DHA-contents and high DHA/EPA-ratios in the live food were carried out with instar-II Artemia and 60h old Artemia. Duration of enrichment was 16h with the DHA phospholipid, and 24h with the four commercial products, in accordance to producers' recommendations. Artemia survival, lipid content, lipid composition and fatty acid profile were measured during the enrichment period. All experimental treatments were carried out in triplicates.

Results

In the first set of the experiments, Artemia survival after 16h enrichment was over 80% at the highest portion of 0.3g/liter, with a significant drop to 50% after 24h. Size of Artemia nauplii increased from 620µm in length at instar-II up to a maximal size of 900µm after 16h. Lipid content increased from 16.3% DW at instar-II to 23.8 and 25.3% DW at 0.2 and 0.3g/liter portions (fed at 0 and 8h), respectively after 24h enrichment. Triglycerids/phospholipids ratio increased only at the higher portion of 0.3g/liter from an initial ratio of 3.18 at instar-II to 3.83 after 16h enrichment. Maximal DHA content of 17.2% of the total fatty acids was achieved after 16h enrichment at the highest portion of 0.3g/liter, whereas EPA content slightly increased from an initial content of 5% to 6.2%. In the second set of experiments, the survival of Artemia enriched with DHA phospholipid (0.3g/liter fed at 0 and 12h) and with the four commercial diets was not significantly different (75-100%) at the end of the enrichment period. On the other hand, the DHA phospholipid material resulted in a high content of DHA and a DHA/EPA-ratio significantly higher than in any of the commercial diets (Table I). There was a large variation in total lipid content of the Artemia fed on the different diets (ranging from 16.0-34.3%, Table I).

Table I. Relative content (% of fatty acids) of DHA and EPA DHA/EPA ratio and total lipid in starved Artemia nauplii and 16h enrichment with the DHA-rich phospholipid diet, or for 24 hours with four commercial enrichment products.

Conclusion

The results from the present study show that the DHA spray dried phospholipid diet resulted in the highest DHA content and the highest DHA/EPA-ratio reported so far. The DHA content of the enriched Artemia was 17.2% of the total fatty acids, which is almost similar top that present in halibut larvae at onset of exogenous feeding (20-25%). Maximal DHA/EPA-ratio in Artemia enriched with DHA phospholipid diet, in both sets of experiments, was ranging from 2.78-3.8, which is even higher than that found in halibut larvae (2.5). These results provides the possibility to test the hypothesis that cold water species and flatfishes need high levels of DHA and high DHA/EPA-ratios in the feed. Artemia enriched with the DHA-rich phospholipid extract are currently being tested in first feeding of halibut larvae.

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Enhanced Absorption of Docosahexaenoic Acid (DHA, 22:6n-3) in Artemia nauplii Using a Dietary Combination of DHA-Rich Phospholipids and DHA-Sodium Salts

Authors: Moti Harel1, Sureyya Ozkizilcik1, Eric Lund1, Paul Behrens2, and Allen R. Place1

1Center of Marine Biotechnology, University of Maryland, 701 East, Pratt St., Baltimore, MD 21202, USA

2Martek Biosciences Corporation, 6480 Dobbin Road, Columbia, MD 21045, USA

Source: Comparative Biochemistry and Physiology, Part B, Vol. 124 (2) (1999) 169-76

Abstract

The effects of surface-active agents such as phospholipids (PL) and salts of long chain fatty acids in enrichment lipids on emulsification, and hence digestion and absorption of essential fatty acids (EFA) were studied. The advantages obtained by using a mixture of docosahexaenoic acid (DHA, 22:6n-3)-rich phospholipids (DHA-PL) and DHA-salts in the process of live feed enrichment are reported on. Enrichment diets at equal levels of DHA, but with varying proportions of DHA-rich triacylglycerols (DHA-TAG), DHA-PL and DHA-sodium salt (DHA-Na) isolated from heterotrophically grown Crypthecodinium sp. algae were fed to instar-II stage Artemia nauplii. Artemia nauplii survival and lipid content after 16h of enrichment were 74.0 ± 17.8% and 24.3 ± 0.6% (dry weight) respectively, and not significantly affected (P >0.05) with increasing dietary quantities of PL and DHA-salts (up to 40 and 30%, respectively). Artemia lipid class composition was independent of dietary phospholipid level. However, higher proportions of polar lipid fraction was evident in Artemia lipids with the addition of DHA-Na. Dietary inclusion of 20% PL or a mixture of 10% PL and 10% DHA-Na resulted in maximal (P <0.05) absorption of dietary DHA by the Artemia (27.5 ± 2.6 mg/g dry weight) with a DHA to eicosapentaenoic acid (EPA, 20:5n-3) ratio greater than 3. Furthermore, PL was the most efficient dietary fraction to deliver DHA to Artemia nauplii as compared to TAG or unesterified delivery forms.

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