I. Core Function Overview
Trimethylamine N-oxide dihydrate (TMAO·2H₂O) is a very important multifunctional feed additive in aquaculture. It was initially discovered as a key feeding attractant in fishmeal. However, with in-depth research, more significant physiological functions have been revealed, making it a crucial tool for improving the health and growth performance of aquatic animals.

II. Main Applications and Mechanisms of Action

1. Potent Feeding Attractant
This is the most classic and well-known role of TMAO.

• Mechanism: Many aquatic products, especially marine fish, naturally contain high concentrations of TMAO, which is a key source of the characteristic "umami" flavor of marine fish. The olfactory and taste systems of aquatic animals are highly sensitive to TMAO, recognizing it as a "food signal".
• Effects:
  • Increased Feed Intake: Adding TMAO to feed can significantly stimulate the appetite of fish and shrimp, especially during initial feeding stages or for picky species, quickly attracting them to feed.
  • Reduced Feeding Time: Shortens the time feed remains in the water, reducing feed loss and water pollution.
  • Applicability in Alternative Feeds: When plant protein sources (e.g., soybean meal) are used to replace fishmeal, adding TMAO can compensate for the lack of flavor and improve feed palatability.

2. Osmolyte (Osmotic Pressure Regulator)
This is a vital physiological function of TMAO for marine fish and diadromous fish.

• Mechanism: Seawater is a hyperosmotic environment, causing water inside the fish's body to constantly be lost to the sea. To maintain internal water balance, marine fish drink seawater and accumulate high concentrations of inorganic ions (e.g., Na⁺, Cl⁻). TMAO acts as a "compatible solute" that can counteract the disruptive effects of high ion concentrations on protein structure, helping to stabilize intracellular protein function.
• Effects:
  • Reduced Osmoregulatory Energy Expenditure: Supplementing with TMAO helps marine fish regulate osmotic pressure more efficiently, thereby directing more energy from "maintaining life" towards "growth and reproduction".
  • Improved Stress Tolerance: Under conditions of salinity fluctuation or environmental stress, TMAO supplementation helps maintain organismal homeostasis and improve survival rates.

3. Protein Stabilizer
TMAO has the unique ability to protect the three-dimensional structure of proteins.

• Mechanism: Under stress conditions (e.g., high temperature, dehydration, high pressure), proteins are prone to denaturation and inactivation. TMAO can indirectly interact with protein molecules, being preferentially excluded from the protein's hydration sphere, thereby thermodynamically stabilizing the native folded state of the protein and preventing denaturation.
• Effects:
  • Protects Gut Health: During digestion, intestinal enzymes need to remain active. TMAO can stabilize these digestive enzymes, improving feed digestibility and utilization.
  • Enhances Stress Resistance: During high-temperature seasons or transportation, when aquatic animals face heat stress, TMAO helps protect the stability of various functional proteins (e.g., enzymes, structural proteins) in the body, reducing stress-related damage.

4. Improves Intestinal Health and Morphology

• Mechanism: The osmoregulatory and protein-stabilizing effects of TMAO collectively provide a more stable microenvironment for intestinal cells. It may promote the development of intestinal villi, increasing the absorptive surface area.
• Effects:
  • Promotes Nutrient Absorption: A healthier intestinal morphology means better nutrient absorption capacity, which is key to improving feed conversion ratio.
  • Enhances Intestinal Barrier Function: May help maintain the integrity of the intestinal mucosa, reducing the invasion of pathogens and toxins.

5. Methyl Donor
TMAO can participate in metabolism within the body, acting as a methyl donor.

• Mechanism: During metabolism, TMAO can provide active methyl groups, participating in various important biochemical reactions, such as the synthesis of phospholipids, creatine, and neurotransmitters.
• Effect: Promotes growth, especially during rapid growth phases where the demand for methyl groups increases; TMAO supplementation can help meet this demand.

III. Application Targets and Considerations

• Primary Application Targets:
  • Marine Fish: Such as turbot, grouper, large yellow croaker, sea bass, etc. Their requirement for TMAO is most significant because its osmoregulatory function is indispensable.
  • Diadromous Fish: Such as salmonids (salmon), which also require it during the marine farming phase.
  • Crustaceans: Such as prawns/shrimp and crabs. Studies also show that TMAO has good attractant and growth-promoting effects.
  • Freshwater Fish: Although freshwater fish do not synthesize TMAO themselves, their olfactory systems can still detect it, making it effective as a feeding attractant. However, the osmoregulatory function is not operative in freshwater.
• Dosage and Considerations:
  • Dosage: The typical addition level in feed is usually 0.1% to 0.3% (i.e., 1-3 kg per ton of feed). The specific dosage should be determined based on trials considering the cultured species, growth stage, feed formulation, and water environmental conditions.
  • Relationship with Choline and Betaine: Choline and betaine are precursors to TMAO and can be converted to TMAO in the body. However, they cannot fully replace TMAO due to limited conversion efficiency and TMAO's unique attractant and protein-stabilizing functions. In practice, they are often used synergistically.
  • Overdosing Issues: Excessive addition (far above recommended doses) may lead to cost waste and potentially have negative effects on certain species, but it is currently considered safe at conventional addition levels.

IV. Summary
Trimethylamine N-oxide dihydrate (TMAO·2H₂O) is a highly efficient, multifunctional feed additive in aquaculture that integrates the functions of feeding attraction, osmotic pressure regulation, protein stabilization, and intestinal health improvement.

Its application not only directly increases the feed intake rate and growth speed of aquatic animals but also indirectly enhances feed utilization efficiency and organism health by reducing physiological energy expenditure and strengthening stress resistance. Ultimately, it provides powerful technical support for increasing production, efficiency, and the sustainable development of aquaculture. In modern aquatic feed, especially high-end marine fish feed, it has become an indispensable key component.