Optimizing Growth and Ensuring Safety: A Research-Based Guide to Artemia salina Supplements

A. Significance of Artemia salina in Aquaculture

Artemia, commonly known as brine shrimp, holds a pivotal position as a live feed in aquaculture due to its exceptional convenience and minimal labor requirements. These small crustaceans are widely utilized as a nutritious live food source for the larval stages of a diverse array of marine and freshwater organisms, including various fish and crustacean species. Their widespread adoption is largely attributed to the remarkable ability of their cysts (dormant eggs) to be stored in a dry, metabolically inactive state for extended periods, often years, in vacuum-packed tins. This allows for the on-demand hatching of free-swimming nauplii (larvae) in precise quantities as needed, eliminating the logistical complexities and infrastructure typically associated with maintaining continuous live food cultures.

Freshly hatched Artemia nauplii, particularly at the Instar I (first larval) stage, boast a high protein content, reaching up to 40% of their dry weight. Their overall nutritional profile is recognized for its richness in proteins, essential fatty acids, and vitamins, making them an ideal dietary component for developing fish larvae. This combination of practical utility and inherent nutritional value underscores their foundational role in modern aquaculture practices.

B. The Rationale for Nutritional Supplementation

Despite the inherent advantages of Artemia as a live feed, their nutritional composition is not consistently optimal across all strains or batches, which can lead to variable and sometimes unreliable outcomes in marine larviculture. A significant limitation is the naturally low concentration of certain essential fatty acids, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These highly unsaturated fatty acids (HUFAs) are indispensable for the robust growth, proper development, and high survival rates of many marine fish and crustacean larvae. Without adequate levels of these critical nutrients, marine fish larvae fed solely on Artemia can experience high mortality rates.

To overcome these nutritional shortcomings, a process known as "bio-encapsulation" or "enrichment" is routinely employed. This technique involves feeding Artemia specific particulate or emulsified products that are rich in the desired nutrients. By ingesting these supplements, Artemia effectively become living "feed transmitters," delivering concentrated essential elements directly to the predatory larvae. This approach transforms Artemia from a merely convenient food source into a precisely tailored nutritional delivery system.

A. Artemia as a Filter Feeder

Artemia are characterized as non-selective filter feeders, meaning they consume a broad spectrum of suspended organic matter, including microscopic algae and bacteria, from their aquatic environment. This non-selective feeding behavior is fundamental to their utility in bio-encapsulation, as it allows them to readily ingest and incorporate various supplements suspended in their culture water.

The size of food particles is a critical factor in Artemia feeding efficiency. They are capable of filtering particles ranging from 1 to 50 micrometers (µm). However, research indicates that Artemia exhibit a distinct preference for specific particle sizes, with an optimal ingestion range between 6.8 and 27.5 µm, and a peak preference around 16.0 µm. The preferred particle size can also vary with the Artemia's developmental stage; for instance, larger individuals (e.g., 3-3.99 mm in length) can ingest particles up to 29 µm, while smaller ones (1-1.99 mm) show a preference for particles between 4.1 and 17.7 µm.

Important: Freshly hatched Instar I Artemia larvae do not actively feed, as they rely on their internal yolk reserves for initial development. Active feeding, and thus the ability to ingest external supplements, commences only after their first molt into the Instar II stage. This developmental milestone is crucial for timing enrichment strategies effectively.

B. Optimal Environmental Conditions for Culture

Successful Artemia culture and effective supplementation are highly dependent on maintaining precise environmental parameters:

• Temperature: Optimal growth rates for Artemia are observed within a temperature range of 25-30°C. Within this optimal range, higher temperatures generally accelerate metabolic rates, leading to faster growth and development. Conversely, temperatures below this range will slow these processes. Extreme temperatures can be lethal; adults may perish at temperatures exceeding 36-37°C, and cyst metabolism ceases above 33°C.
• Salinity: Artemia thrive in saline environments, with optimal growth achieved at salinities between 28 and 35 parts per thousand (ppt). While Artemia are remarkably tolerant of a wide range of salinities, capable of surviving from 5 to 150 ppt for reproduction and up to 200 ppt for survival, extreme physiological stress and mortality occur at salinities approaching or exceeding NaCl saturation (250 g/L and higher).
• pH: An optimal pH range of 8-9 is conducive to Artemia health and growth, with a more precise optimum around 8.4. They can, however, survive in a broader pH range of 5-10.
• Aeration and Oxygen: A consistent and adequate supply of oxygen is paramount for Artemia culture. Proper aeration not only maintains sufficient dissolved oxygen levels (preferably 5 mg/L) but also serves to keep food particles suspended uniformly throughout the culture volume, ensuring efficient feeding.
• Water Quality Management: Regular removal of metabolic waste products is essential to prevent water quality degradation. Water transparency can serve as a practical indicator for feeding amounts; maintaining transparency between 15-20 cm initially, and 20-25 cm in later weeks, helps prevent overfeeding.
• Light: Strong illumination, approximately 2000 Lux at the water surface, is crucial for maximizing the hatching rate of Artemia cysts, particularly during the initial hours following complete hydration.

Organic supplements play a vital role in enhancing Artemia growth and nutritional value, often serving as primary feed sources or complementary additives.

A. Microalgae-Based Feeds

Microalgae are the natural and foundational food source for Artemia, forming the base of their food chain. These microscopic green plants are rich in essential macronutrients—proteins, carbohydrates, and lipids—as well as various vitamins.

• Spirulina (Spirulina maxima): This blue-green alga is highly regarded for its exceptional protein content, typically comprising around 60% of its dry weight. Research indicates that a combination of spirulina and rice bran constitutes the most effective supplementary diet for Artemia in aquarium settings, resulting in superior average length (8.7 ± 0.21mm) and biomass concentration (66.7 ± 0.17). Artemia enriched with Spirulina maxima consistently demonstrate the highest protein concentrations, reaching up to 1.8 ± 0.7 mg/g after 24 hours of enrichment.
• Nannochloropsis (N. granulata / N. salina): Nannochloropsis species are particularly effective for lipid enrichment in Artemia. Artemia enriched with N. granulata for 24 hours showed the highest lipid concentration (2.08 ± 0.42 mg/g) among tested enrichment processes. Nannochloropsis salina has been shown to significantly elevate essential fatty acid levels in Artemia nauplii, including arachidonic acid (AA, 9.50%), eicosapentaenoic acid (EPA, 25.80%), and docosahexaenoic acid (DHA, 4.18%).
• Chlorella (C. vulgaris / C. salina): Similar to Nannochloropsis, Chlorella vulgaris enrichment leads to high lipid content in Artemia. However, it's important to note that Artemia enriched with Chlorella salina have shown a reduction in overall PUFA content even after just 6 hours of enrichment. A significant limitation with some unicellular algae, such as Chlorella and Stichococcus, is their thick cell walls, which Artemia cannot effectively digest.
• Chaetoceros (C. calcitrans): This microalga is specifically recommended as a feed source for Macrobrachium americanum larvae when provided to Artemia, leading to improvements in growth, growth rates, and survivability of the consuming larvae.

B. Yeast-Based Feeds

Baker's Yeast (Saccharomyces cerevisiae): While fresh baker's yeast is inexpensive and widely available, its direct use as a sole feed for Artemia typically results in poor growth and low survival rates. This inefficiency is primarily due to the low digestibility of its robust cell wall. To overcome this barrier, the nutritional value of baker's yeast can be significantly improved through pre-treatment involving either complete enzymatic removal of the yeast cell wall or chemical treatment that renders the cell wall permeable to Artemia's digestive enzymes.

Specialized Yeasts: Seleno-yeast, such as Sel-Plex, is an effective means of enriching Artemia with selenium (Se). Artemia nauplii can be enhanced by exposure to 12 mg of Sel-Plex per liter for 4 hours before being fed to fish larvae. Saccharomyces boulardii, a probiotic yeast, whose bio-encapsulation in Artemia nauplii has been shown to enhance the brine shrimp's resistance against pathogens like Vibrio harveyi.

C. Other Natural By-products

Various agricultural and animal by-products can serve as economical organic supplements:

• Rice Bran: This common agricultural by-product is valuable when combined with spirulina, contributing to the best observed growth performance for Artemia.
• Soybean Meal: Often included with rice bran, contributes to good Artemia growth performance in supplement diets.
• Mustard Oilcake: Another component frequently utilized alongside rice bran in Artemia feed formulations.
• Egg Yolk: Can be used as a component in enriching Artemia cultures, providing additional nutrients.
• Chicken Feed/Dung and Minced Fish: These materials can be fed directly to Artemia and also act as fertilizers, promoting the growth of natural food sources.

Synthetic and enriched supplements are crucial for precisely tailoring the nutritional profile of Artemia, particularly for addressing specific deficiencies and enhancing the health of the organisms that consume them.

A. Highly Unsaturated Fatty Acids (HUFAs)

HUFAs, especially EPA and DHA, are critically important for the development and survival of marine fish and crustacean larvae, yet these are often present in insufficient quantities in Artemia.

• Enrichment Strategies: Commercial lipid emulsions, such as A1 DHA SELCO, Selco, Algamac-2000, and Sander's Rich, are widely adopted for HUFA enrichment. These emulsions are formulated with varying HUFA concentrations, typically ranging from 30% to 50%. DHA powder is another effective option, with studies showing that concentrations of 3 g/l can yield the highest DHA content in Artemia (3.32% of total fatty acids).
• Dosages and Enrichment Times: Typical enrichment doses for lipid emulsions range from 0.05 to 0.4 g/l, or 0.25 ml/liter (250 ppm). The duration of enrichment is also critical, commonly ranging from 2 to 24 hours. Optimal PUFA content with Nannochloropsis salina was achieved after 8 hours of enrichment, with a notable reduction observed at 24 hours.
• Safety and Adverse Effects: While HUFA enrichment generally leads to improved performance in predators, commercial enrichment diets with high PUFA content can generate harmful trans fatty acids when exposed to light, high temperatures, and air. Furthermore, elevated bacterial loads and accelerated Artemia metabolism at higher temperatures during enrichment can increase naupliar mortality.

B. Vitamins and Amino Acids

• Vitamins: Enrichment with various vitamins, including Vitamin C, A, D3, and E, is crucial for both Artemia and their predators. Vitamin C enrichment has been shown to boost the growth performance and survival rates of Sepia pharaonis. The synergistic effect of essential fatty acids and Vitamin C has also been linked to lower mortality rates and increased stress resistance in rainbow trout larvae.
• Amino Acids: The natural content of free amino acids can vary among different Artemia strains. Enrichment with specific free amino acids, such as methionine, is achievable either by direct dissolution in the culture water or by encapsulation within liposomes.
• Safety: Studies confirm that vitamin and amino acid complex use is considered "absolutely safe," with detected levels of toxic elements such as cadmium (Cd), arsenic (As), mercury (Hg), and lead (Pb) remaining low and well within permissible concentrations.

C. Probiotics

Probiotic supplementation represents a significant advancement in Artemia enrichment, offering benefits that extend beyond basic nutrition to include improved disease resistance and gut health.

• Types Used: Bacterial species such as Lactobacillus johnsonii, Bifidobacterium animalis, Bacillus subtilis, Enterococcus durans, Bacillus clausii, and Bacillus pumilus, as well as probiotic yeast like Saccharomyces boulardii.
• Impact on Artemia: Probiotic bio-encapsulation can enhance the survival of Artemia and increase their maximum population density and growth rate. Optimal loading of Artemia metanauplii with L. johnsonii and B. animalis has been achieved within 40 minutes at a concentration of 2.0 × 10³ CFU mL⁻¹ of viable cells.
• Impact on Predators: Benefits include promotion of growth, increases in weight and specific growth rate, balancing of gut microbiota, improvements in intestinal morphology, enhancement of immune system function, and increased resistance to common pathogens like Vibrio sp.

D. Emerging Supplements (Poly-beta-hydroxybutyrate - PHB)

PHB is a natural biopolymer that undergoes depolymerization into water-soluble short-chain fatty acid monomers. When supplemented to Artemia (e.g., 1 g/L for Instar II nauplii), PHB has been shown to increase the survival of Penaeus monodon postlarvae following ammonia exposure and enhance their resistance to pathogenic Vibrio campbelli.

A. General Principles of Toxicity Assessment in Artemia

Artemia salina is extensively utilized as a cost-effective and straightforward model organism for toxicity screening and preliminary cytotoxicity testing. Its ease of handling, rapid developmental cycle, and adaptability to laboratory conditions make it a convenient tool for such assessments. Toxicity in Artemia can manifest through various observable effects, including increased mortality rates, delays in development, reduced feeding activity, and physical damage to the gastrointestinal tract.

B. Minimizing Risks from Contaminants

• Heavy Metals and Pesticides: The nutritional quality of Artemia can be compromised by contamination with various chemicals, including pesticides and heavy metals, which can vary significantly among different geographical strains. Although Artemia is generally tolerant to high salinities and is often considered not highly sensitive to contaminants, this robustness raises concerns about the potential for trophic transfer of accumulated pollutants to the consuming organisms.
• Microplastics (MPs): Due to their non-selective filter-feeding nature, Artemia can readily ingest MPs, particularly when other food sources are scarce. The presence of MPs can lead to adverse effects including developmental delays, reduced body length, impaired microalgal feeding, alterations in gut microbiota, physical damage to the digestive system, and even increased mortality.
• Mycotoxins: Artemia salina larvae have demonstrated susceptibility to mycotoxins, such as fumonisins and zearalenone. This sensitivity makes them valuable biological indicators for assessing the toxicity of such contaminants in food and feed products.

C. Maintaining Optimal Water Quality During Culture and Enrichment

• Aeration: Continuous and vigorous aeration is indispensable. It maintains adequate dissolved oxygen levels (ideally 5 mg/L) and ensures that food particles remain uniformly suspended throughout the culture medium.
• pH and Salinity Control: Regular monitoring and precise adjustment of pH (optimal range 8-9) and salinity (optimal range 28-35 ppt) are crucial for maintaining a healthy culture environment.
• Waste Removal: Prompt and regular removal of Artemia waste products is recommended to prevent the accumulation of harmful substances. Overfeeding can lead to decreased water transparency and water quality deterioration.
• Chlorine Neutralization: Water that has been treated with chlorine must be thoroughly neutralized using agents like rock salt or sodium thiosulfate before Artemia are introduced.

D. Practical Feeding Guidelines

• Timing: Feed Artemia as soon as possible after harvesting, ideally within 12 hours, as they expend energy and nutrients rapidly after hatching.
• Frequency: Generally, Artemia can be fed twice a day or more frequently. Since Artemia are continuous filter-feeders, distributing food as frequently as possible promotes the highest growth rates.
• Quantity: Feeding should commence with lower amounts, gradually increasing as the nauplii grow. Water transparency serves as a reliable indicator of appropriate feeding levels.
• Particle Size: Feed particles must be appropriately sized for ingestion by Artemia, ranging from 1 to 50 µm, with optimal range between 6.8 and 27.5 µm.

E. Importance of Cyst Decapsulation and Hygiene

• Decapsulation: The process of decapsulation, which involves removing the tough outer shell of Artemia cysts, significantly improves hatching efficiency and reduces the potential introduction of contaminants.
• Disinfection: Routine disinfection of Artemia cysts and all culture apparatus with solutions like hypochlorite is crucial to prevent outbreaks of common bacterial infections, such as filamentous Leucothrix and Vibrio sp.

A. Summary of Effective and Safe Growth Supplements

The research comprehensively demonstrates that while Artemia salina is an invaluable live feed in aquaculture due to its convenience, its inherent nutritional limitations necessitate strategic supplementation for optimal growth and effective nutrient transfer to target organisms.

• Microalgae: Spirulina maxima, Nannochloropsis granulata, and Chaetoceros calcitrans emerge as highly recommended organic supplements. Spirulina is particularly effective for its high protein content and overall growth promotion, while Nannochloropsis excels in providing essential fatty acids like EPA and DHA.
• Treated Yeast: Although raw baker's yeast is poorly digestible, chemical or enzymatic pre-treatment significantly improves its nutritional value, rendering it a viable and cost-effective protein source.
• HUFAs: For marine aquaculture, enrichment with lipid emulsions or DHA powder is critical. These supplements effectively boost EPA and DHA levels in Artemia, which are vital for the robust growth, development, and survival of fish and crustacean larvae.
• Vitamins and Amino Acids: Supplementation with a complex of vitamins (e.g., Vitamin C) and amino acids significantly enhances Artemia's nutritional, biological, and energy value.
• Probiotics: Bio-encapsulation with beneficial microorganisms improves Artemia survival, growth, and gut health, with these benefits effectively transferring to the consuming larvae.
• PHB: As an emerging prebiotic, PHB enhances Artemia's resistance to pathogens and improves growth in the consuming organisms.

B. Integrated Recommendations for Optimal Artemia Culture

• Environmental Control: Consistently maintain optimal environmental parameters: temperature between 25-30°C, salinity at 28-35 ppt, and pH within 8-9. Ensure continuous and adequate aeration to maintain dissolved oxygen levels and keep feed particles suspended.
• Hygiene and Water Quality: Implement rigorous hygiene protocols, including the decapsulation of cysts and regular disinfection of all culture equipment. Neutralize chlorine in water used for culture. Monitor water transparency diligently to prevent overfeeding.
• Feeding Strategy: Initiate feeding only when Artemia reach the Instar II stage. Select supplements based on the specific nutritional requirements of the target organisms. Favor microalgae as primary organic feeds due to their comprehensive nutritional profiles and inherent safety.
• Safety First: Focus on maintaining Artemia's physiological health and nutritional integrity as a feed organism. Be aware of the potential for contaminant transfer, such as microplastics or heavy metals, from water sources or impure supplements. Proactive contaminant prevention and understanding of sub-lethal effects are crucial to ensure that Artemia are truly "safe to use" and maximally beneficial for the organisms they are intended to nourish.