Alpha Ketoglutarate – A promising supplement in aquafeed

Replacing fish meal in aquafeed is one of the biggest challenges in aquaculture. Till now no feed ingredients was able to effectively replace fish meal especially in terms of its amino acid profile and its unknown growth factors. Alpha ketoglutarate (AKG) is known for its nutritional modulation, where a low protein and inadequate amino acid profile feeds supplemented with AKG showing promising growth & immune performances.

INTRODUCTION
Globally, aquaculture is the fastest growing food-producing sector with an annual growth rate of 5.8% and provides about half of the global fish supply. It is diversifying, expanding and intensifying in almost all regions of the world. Rapid growing of human population has led to increasing demand of aquaculture production. Sustaining fish supplies from capture fisheries will, therefore, not be able to meet the growing global demand for aquatic food. Aquaculture can bridge the supply and demand gap of aquatic food in most regions of the world. Aquaculture production is heavily reliant on artificial feeds, which account for 50-60% of overall production costs, making it the most expensive variable in the industry. It’s due to the rising cost of protein components in feed formulations like fish meal and fish oil. The most efficient use of nutrients from feed by the fish for increased development is the primary purpose of feed formulation, which can lower production costs. Fish bodies contain 70% protein by dry weight basis, making protein the most significant and expensive portion of the fish diet. The entire utilisation of protein for tissue building is prioritised over its use for energy. The feed with inadequate protein level or low protein: energy ratio does not contribute to optimum growth performance of fish. The high protein content diet has also some disadvantages as it is expensive and on utilisation produces excess ammonia in the culture environment which is detrimental to fish. Adequate protein content with suitable amino acid profile and high digestibility increases the protein retention in fish and enhances growth. Maximum utilization of lipid is also highly beneficial for fish which leads to protein sparing for growth and less fat get stored in the fish muscle. Low protein and adequate energy could be a nutritional strategy for fish in the presence of protein synthesizing compound. Nutritional modulation is an efficient way to enhance growth and body composition however, the underlying mechanisms are still largely unknown. Certain substances, such as alpha ketoglutarate (AKG), have been shown to improve protein synthesis and fat utilisation when fed to animals.

ALPHA KETOGLUTARATE
Glutamine (Gln), and its derivatives, is considered as one of the crucial molecules in protein metabolism, amino acids transport across membranes, gene and cellular redox regulation. However, because of the low solubility and poor stability of glutamine, its precursor α-ketoglutarate (AKG) appears to be more preferable for supplementation.

AKG is comprising of two carboxyl groups and a ketone body (Figure 1), also called as 2-ketoglutaric acid, 2-oxoglutamate, 2-oxoglutaric acid, oxo glutaric acid and 2-oxopentanedioic acid, is a rate-determining intermediate in the tricarboxylic acid (TCA) and has a crucial role in cellular energy metabolism. AKG is produced and degraded by a number of different metabolic routes throughout cellular metabolism. In the TCA cycle, AKG is decarboxylated to succinyl CoA and CO₂ by AKG dehydrogenase, a key control point of the TCA cycle. Otherwise, AKG can be generated from isocitrate by oxidative decarboxylation catalysed by isocitrate dehydrogenase.

Also, AKG can be produced anaplerotically from glutamate (Glu) by oxidative deamination using glutamate dehydrogenase, and as a product of pyridoxal phosphate-dependent trans-amination reactions in which glutamate is a common amino donor. AKG can dissolve well in water, does not show toxic properties and its water solutions characterize has high stability. AKG is the precursor of glutamine and glutamate (Figure 2) and is known to have beneficial effects on protein synthesis, prevents protein degradation in muscles and constitutes an important fuel for gastrointestinal tract cells when provided as a supplement. In the cellular metabolism, it is impossible to utilize AKG from the TCA cycle in the synthesis of amino acids, for this to occur, one must provide AKG as a pure dietary supplement.

AKG AND IT’S FUNCTIONS IN AQUACULTURE
In several livestock industries, including pig, poultry, and turkey culture, alpha ketoglutarate has been used as a significant feed supplement. However, researchers have been employing it in aquaculture from the last decade (Table 1). The common functions of AKG in aquaculture are,
• Growth promotor
• Protein synthesis
• Improves antioxidant capacity
• Improves digestive enzyme activity
• Reduces ammonia toxicity
• Bone mineralization
• Amino acid synthesis
• Fat metabolism
• Gene expression
• Improves fillet quality

1. On growth performances and protein synthesis
AKG serves as a crucial supply of glutamine and glutamate for cellular metabolism, which promotes protein synthesis, prevents muscle protein breakdown, and serves as a crucial metabolic fuel for gastrointestinal tract cells. Gln, which makes up more than 60% of the entire amino acid pool, provides an energy source for all types of cells in the organism. As a way, AKG, which is a precursor to Gln, serves as both an important source of energy for intestinal cells and a favoured substrate for enterocytes and other rapidly proliferating cells. Moreover, the transformation of Gln and Glu into ornithine and later arginine, stimulates the release of growth hormone and insulin-like growth factor I. The animal will grow more effectively with these kinds of molecular modifications. Many studies have shown that dietary AKG decreases nitrogen excretion from the body and enhances nitrogen digestion, deposition, and retention, which promotes protein synthesis and helps in animal growth. By increasing the intestinal functions in animals, AKG enhances better digestion and absorption of nutrients. Wang et al., (2016) reported that the development of the intestine was promoted by the AKG supplementation in hybrid sturgeon. Thus, the growth performance was increased by the addition of AKG. In a recent study, 1% AKG supplementation in Juvenile mirror carp (Cyprinus carpio) feed promoted the growth performance, final body weight, weight gain rate, feed intake and phosphorus intake. Pradhan et al., (2021) reported that the combination of L-carnitine and AKG could enhance the growth performance, and nutrient utilization in tilapia.

2. On digestive enzymes
The levels of activities of digestive enzymes are used as comparative indicators of growth rate and digestive capacity of the fish. It is speculated that AKG, as an intermediate of TCA, also an intermediate of Glu and Gln, may contribute to ATP homeostasis in the small intestine. By interacting with the glutamine metabolic pathway, AKG can produce glutamine (Gln) which increases the functional surface area of the intestinal mucosa and improve the nutrition absorption. Feeding the 0.75 % AKG diet increased significantly amylase, protease, and lipase in the foregut of grass, when compared with the basal diet without AKG supplementation. A study conducted by Pradhan et al. (2021), showed that when AKG and L-carnitine combination was fed in the diet increased activity of digestive enzymes in tilapia. Increased digestive enzyme activity will improve the digestion and absorption of nutrients, thus enhance growth performance.

3. As an antioxidant
Antioxidant capacity is an important indicator to reflect the health status of organisms. The antioxidant system includes the enzyme system (Superoxide dismutase, Catalase, Glutathione) and the coenzyme system, which will be affected by the breeding environment and the feed composition. AKG may indirectly regulate an organism’s redox status as it is a highly ammonia and phosphate-binding factor. The antioxidant capacity of alpha-ketoglutarate helps in scavenging reactive oxygen species (ROS) in organisms. AKG plays an important role in antioxidant mechanism as precursors of Glu, Gln, and proline. Gln plays a role in eliminating free radicals because it acts as a precursor for synthesis of the antioxidant glutathione. Specially, proline, as conditionally essential amino acids in fish, can be produced from AKG and plays a key role in protein synthesis, wound healing, and antioxidative reaction. These may be the reasons for effective functions of AKG in antioxidant mechanism. Studies report that dietary AKG supplementation in fishes resulted in inhibiting lipid peroxidation by enhancing the activities of antioxidant enzymes like SOD, CAT, Glutathione peroxidase (GSHPx). The process of non-enzymatic oxidative decarboxylation of AKG in hydrogen peroxide decomposition reduces the level of hydrogen peroxide in the cells, resulting in the conversion of AKG to succinate with release of carbon dioxide and water. Recent studies on AKG supplementation proved that the rise in contents of CAT and total antioxidant capacity (T-AOC) are paralleled by a decrease in the content of malondialdehyde (MDA) in the intestine of Songpu mirror carp, and significantly increased SOD, GSHPx, and CAT activities in grass carp. This is indicating that AKG supplementation could reduce oxidative damage and increase antioxidant capacity, as well as promote the radical scavenging ability when supplemented in aquafeed.

4. As an immunomodulatory agent
AKG is also known as the immune nutrition factor and therefore crucial for the function of the immune system. The importance of AKG as a source of Gln and Glu is already well documented, i.e., AKG as a glutamine homologue has immuno-enhancing properties, can maintain a gut barrier, increase immune cells and the activity of neutrophils and phagocytosis, reduce bacterial translocation in vivo derivative. Glutamine is one of the crucial fuels for lymphocytes and macrophages. Macrophages and neutrophils are involved in the early, nonspecific host-defence responses and play an important role in the pathophysiology and/or protection against sepsis. Supplementation of dietary AKG may activate the NF-kB, thereby upregulating the expression of pro-inflammatory cytokines and downregulating the expression of anti-inflammatory cytokines. AKG is also capable of reducing the expression of tumour necrosis factor-α. AKG and herbs together may provide a novel, alluring way to improve gut immunity. For example, the administration of AKG along with Chinese herbal medicine can enhance the size of intestinal epithelial lymph nodes and the amount of intestinal Immunoglobulin A (IgA) secreted, indicating that AKG has a positive impact on the immunological function. Wei et al., (2021) reported that AKG supplementation increased the alkaline phosphate which could improve intestinal injury in songpu mirror carp after A. hydrophila injection. Also, Xu et al., (2017) stated that dietary AKG had positive effect on the lysozyme and neutrophil oxidative radical production. These findings suggest the use of AKG as an immunotherapeutic drug with potential advantages in the ability to fend against infection by harmful microorganisms in aquaculture.

5. Ammonia toxicity to amino acids
Ammonia is a natural metabolite and plays an important role in maintaining the acid–base metabolic balance in the body. But ammonia toxicity has become one of the most common problems for aquatic animals, especially fish. As a final product of protein catabolism in most teleosts, ammonia is produced and excreted into the surrounding environment through the gills. Ammonium levels can rapidly rise to unsafe levels in aquatic systems unless the substance is continuously removed, usually by biological filtration or periodical water change. AKG is considered as a crucial molecule in transmembrane amino acid transport, protein metabolism, and cellular redox regulation. Dietary supplementation of AKG will increase the activity of glutamine synthetase (GS) enzyme which plays an important role in nitrogen metabolism (amino acid production) and reduces the toxicity of ammonium in the fish body. As the numbers of studies on Rh genes in animals have increased, the important roles of Rh glycoproteins in ammonia transport processes of aquatic animals have been confirmed. AKG up-regulates the expressions of Rhag and Rhbg genes, which promote ammonia excretion. In Atlantic salmon a 5.0 % AKG supplement reduced the nitrogen excretion without any negative effects on growth or vitellogenin synthesis. A study of Wang et al., (2016) showed that the concentrations of arginine, aspartate, glutamate, glutamine, and the total amino acids were increased by the addition of AKG, which are important amino acids for animals. These amino acids can also improve the growth of fish.

CONCLUSION
The primary goal of aquaculture scientists nowadays is to discover a vital supplement for improving the growth performance of fish. AKG has been a promising ingredient in aquaculture due to its multiple crucial functions even in molecular level. Adding an appropriate amount of AKG to the feed could increase the growth & immune performances of fish by reducing the feed conversion ratio, feed coefficient, promoting the metabolism of protein and fat in the fish, antioxidant capacity and digestive enzyme activity. The supplementation level of AKG shows species specific and feed protein level variations. But as far as the latest research findings, 0.75% and 1.0% of AKG supplementation showed promising results in aquaculture. There is still more to explore the molecular level activities of AKG and its effect in fish nutrition and physiology.

About S. R. Gokulnath
S R. Gokulnath is a fisheries science graduate (B.F.Sc) of Tamil Nadu Dr J Jayalalithaa Fisheries University (TNJFU), Nagapattinam and have completed his Master’s degree program in Fish Nutrition and Feed Technology discipline at Kerala University for Fisheries and Ocean Studies, Kerala. His masters’s work was ‘Effects of alpha ketoglutarate on growth, antioxidant capacity, gene expression and amino acid profile in Nile tilapia (Oreochromis niloticus) fed with different level of protein diets’. He is an awardee of ‘National Talent Scholarship’ (NTS) of ICAR.

About Phibi Philip Naduvathu
Phibi Philip Naduvathu is a fisheries science graduate (B.F.Sc) of Tamil Nadu Dr J Jayalalithaa Fisheries University, Nagapattinam and Post-Graduate (M.F.Sc) of Central Institute of Fisheries Education (CIFE), Mumbai. His expertise is in fish genetics & biotechnology, aquaculture, fisheries & computational biology. Fish nutrition, aquatic ecology & conservation, climate change & AI are his extended interests. He is an awardee of prestigious ‘Commonwealth Scholarship’ by the UK government, ‘National Talent Scholarship’ (NTS) of ICAR and Institutional Scholarship of CIFE.

About Dr. Chiranjiv Pradhan
Dr. Chiranjiv Pradhan is a Bio-chemistry graduate with a doctoral degree in Fish Nutrition. His expertise is in bio-chemistry, fish nutrition, feed formulation, aquaculture, and animal health management.