All living organisms, including fish and crustaceans, do not require protein per se, but amino acids (AA), the building blocks of proteins. All AA structurally contain three common parts: a central carbon bond to a hydrogen, a nitrogen containing amino group and a carboxylic group. Proteins, consisted of few up to thousands of AA, having numerous structural and metabolic functions. In animal production, the most direct result of AA deficiencies translates to reduced growth. Traditionally, it was the economic incentive, which resulted in the use of supplemental AA in diet formulation. However, there has been a gradual evolution with more emphasis directed towards sustainability and total nutrient supply.

In aquaculture feed formulations, Methionine (Met) is usually the first limiting essential amino acid (EAA) especially in low fish meal (FM) diets. It is therefore, required to include a supplemental source of Met in order to meet the feeds" specification, targeting the animal"s requirement for this particular EAA. While evaluating supplemental nutrients or additives for use in feed formulation, three parameters needs to be considered: (i) nutritional value (biological effectiveness) of the supplemental nutrient, (ii) stability, homogeneity etc. during the feed production process and (iii) physical properties of the nutrient source during feeding practice.

There are several commercially available Met sources in the market like DL-Met (DL-Methionine for Aquaculture), DL-Methionyl-DL-Methionine (AQUAVI® Met-Met), L-Met (L-Methionine), Methionine Hydroxy Analogue-free acid (MHA-FA or liquid MHA) and Methionine Hydroxy Analogue calcium salt (MHA-Ca). Both terrestrial and aquatic animals can utilise crystalline AA such as Methionine; however, the biological availability of the different methionine sources differs greatly. The differences in biological availability are a reflection of differences in product matrix, digestibility, transport mechanism and metabolic conversion requirements.

DL-Met, as well as the dipeptide DL-Methionyl-DL-Methionine (Met-Met), are the racemic mixture of D- and L-isomer of Methionine and are commercially available as feed additive, with 99 percent DL-Met and 95% Met-Met (95% DL-Methionyl-DL-Methionine and 2% DL-Met) purity, respectively. As only the L-isomer can be utilized for the protein synthesis by the animals" body, D-isomer is metabolically converted to L-isomer first through oxidation to keto-Methionine by the enzyme D-amino oxidase and then transaminated by transaminase enzyme to L-Met. The enzymes required in the conversion of D-form to L-form are not at a rate-limiting factor in fish and shrimp alike poultry and swine.

On the other hand, MHA-Ca and MHA-FA are also racemic mixtures of its D- and L-isomer. MHA-Ca consists of about 84 percent MHA monomer, 12 percent calcium and four percent water and MHA-FA, 65 percent monomer, 23 percent dimers/trimers and the remaining 12 percent water.

Chemically, both MHA-Ca and MHA-FA cannot be classified as an AA. AA contains both a carboxyl (COOH) and amino group (NH2), however, in Methionine Hydroxy Analogue, the NH2 group is replaced by a hydroxyl (OH) group and for that reason cannot be classified as an amino acid (Dibner 2003). Methionine Hydroxy Analogue has to undergo a series of metabolic transformation in order to be utilised by the animals. Through a dehydrogenase reaction, it is first converted to a-keto analogue of methionine and then to the utilisable L-methionine via a transaminase reaction.

Finally L-Met is also commercially available as a feed additive with 99 percent purity and do not require any conversion as the L form can be utilised by the body. It is however clearly stated by the National Research Council based on nutritional studies (NRC, 2011), fish and shrimp can use D-Met to replace L-Met on an equimolar basis.

Evonik Animal Nutrition compiled a recent critical review, updating the original publication entitled "Relative bioavailability of methionine sources in fish" (Lemme, 2010), with all the latest scientific publications and industry trial data on both fish and shrimp.

Several studies comparing the nutritional value of MHA products with DL-Met conducted in fish concluded that both MHA-FA and MHA-Ca are significantly less available than DL-Met (Lemme 2010; Lemme et al. 2012; Figueiredo-Silva et al. 2014; Powell et al. 2017). By applying regression analysis and comparing the slopes for weight gain between Met sources, revealed nutritional value of MHA-Ca relative to DL-Met varied between 22 percent in Nile tilapia and channel catfish to 62 percent in red drum on weight to weight basis (wt/wt).

At this point it is worth explaining the meaning of equimolar and wt/wt basis with a simple example. According to NRC (2011), "on the basis of available experimental evidences, the committee considers it reasonable to assume that the biological efficacy of HMB (2-hydroxy-4-(methylthio)butanoic acid known as MHA) for fish is about 75 to 80% that of DL-Met on a equimolar basis". The equimolar ratio, results from in vivo scientific experiments determining the relative bioavailability of the tested nutrient sources through the analysis of key growth performance parameters of a dose response trial using regression analysis. The translation to the wt/wt basis is by multiplying the equimolar ratio of the two Met sources with the active ingredient of the product. As mentioned above DL-Met has >99 percent Methionine purity and MHA-Ca 84 percent 2-hydroxy-4-(methylthio) butanoic acid hence, with an equimolar basis of 77 percent, MHA-Ca has around 65 percent biological efficacy on a wt/wt basis compared to DL-Met [77 (equimolar) x 0.84 (MHA content in the MHA-Ca product) = 65%].

Further studies comparing DL-Met and L-Met showed a non-statistically significant but slightly lower bioavailability of L-Met (82-83%) relative to DL-Met in salmonids. This requires further investigation, but agrees with earlier data obtained in salmon (Sveier et al. 2001), rainbow trout (Kim et al. 1992) and in hybrid striped bass (Keembiyehetty and Gatlin III, 1995), showing that D- and/or DL-Met are at least as effective as L-Met. The simultaneous regression analysis from a recent study in Indonesia in L. vannamei revealed that the nutritional efficiency of Met-Met relative to L-Methionine is 194 percent based on biomass gain, 190 percent for SGR and 212 percent based on FCR; Facts and Figure 1634). Further studies in L. vannamei with Met-Met show a higher bioavailability ranging from 178 percent up to 298 percent compared with DL-Met establishing an average minimum of 200 percent bioavailability of Met-Met relative to DL-Met as well as, on average a 65 percent bioavailability for MHA-CA compared with DL-Met as it was validated in the Thailand trial.

It is not only the nutritional value of the different Met sources, but the physical characteristics of the products are equally important. Feed milling calls for extra attention looking at the caking tendency and flowability of the different Met sources as well as the mixability and homogenous distribution of the critical and costly nutrients in the feeds. Hence, the average size of the particles is considered as one of the main factors for the mixing homogeneity in the feeds.

Finally, a critical parameter in aquaculture feeds is the solubility and leaching of the supplemental nutrients, like Met, especially since the feeding takes place exclusively in salt or fresh water. In vitro tests proved that the water solubility levels of each of the commercially available Met sources also vary significantly. The dipeptide (Met-Met) being five to 10 times less water soluble compared to other commercially available Met sources. Since the nutrients" leaching from the pellets is strongly related to the feeding behavior of the targeted aquaculture species. Thus, Met-Met is more suitable to crustaceans where the feed stays for a prolonged time in the water before it is fully consumed.

By Dr Alexandros Samartzis Senior Technical Service Manager, Evonik

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