Phosphorus and calcium

Despite the importance of Ca in bone formation and maintenance of skeletal structure, it is widely distributed in all soft tissues, having an integral role in muscle contraction, blood clot formation, transmission of nerve impulses across synapses, maintenance of cell membrane integrity and activation of several important enzymes. Furthermore, Ca in the cell membrane

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is closely bound to phospholipids, regulating membrane permeability and nutrient uptake by the cells (Lall, 2003). Moreover, Ca and P are both involved in the maintenance of acid-base equilibrium (Lall, 2003).

Phosphate serves as a structural cell component, a factor in intermediate metabolism and a component of genetic material (Lall, 2003). Phospholipids constitute the major component of the cell membranes and intracellular organelles, while phosphate is also an integral constituent of nucleic acids (DNA and RNA) (Lall, 2003). Moreover, phosphorus is part of high energy phosphate esters such as adenosine phosphate (ATP), the hydrolysis of which can release high energy for metabolic processes and muscle contraction. In addition, phosphate is essential for carbohydrate, lipid and amino acid metabolism and in muscle and nervous tissue metabolism.

As mentioned previously, Ca and P are closely connected to the normal bone formation and mineralisation, while the stability of the vertebral bone is maintained by a solid form of calcium phosphate (Lall, 2003; Lall and Lewis-McCrea, 2007). Fish can derive Ca and P from the surrounding aquatic environment, while Ca requirements can be met by their ability to sequester this mineral from the water. On the contrary, the concentration of P in the water is very low making the dietary supply of this element the main source of P. In fish, gills are the major site of Ca regulation (Lall, 2003). Other sites of significant Ca regulation include the fins and oral epithelia and tissues (Lall, 2003). The absorbed Ca is then deposited in the bones, scales and skin. While scale chemical composition is similar to that of other bony tissues, Ca metabolism in scales is physiologically different compared to the other skeletal structures. Generally, the regulation of P is considered to be more crucial than that of Ca, since fish have to absorb, store, mobilise and conserve P in both fresh and saline aquatic environments (Lall, 2003; Lall and Lewis-McCrea, 2007). Absorbed P is mainly

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accumulated in soft tissues (heart, liver, muscle, blood etc) and to a lesser degree in skeletal tissues.

In general, chemical analysis of the diets cannot be used to define the biological effectiveness of a nutrient. It has been shown that the actual bioavailability of an element can vary considerably when it is provided from different feed ingredients or even within the same feed component in different diets (Lall, 2003). Moreover, it has been reported that elemental bioavailability can be influenced by a wide range of factors. These include levels of mineral, the particle size, interactions with other dietary components (chelators or inhibitors) or nutrients, the physiological and pathological status of the animal and the type of feed processing (Lall, 2003). Dietary mineral bioavailability can differ depending on the molecular form of the element, its valence state and the number of ligands in the ingested dietary element (Lall, 2003). Furthermore, several mechanisms that are associated with the formation of insoluble and non-absorbable substances in the intestinal tract may inhibit or promote the mucosal absorption, transport and metabolism of an element in the body (Lall, 2003). Feedstuffs of animal origin, such as FM and meat meals, demonstrate the highest levels of Ca and P with P ranging between 1.5-3.2 % and 3.5-5.5 % in these ingredients respectively. In these feedstuffs, the bony tissues contribute significantly to the P content, where the major proportion is present as inorganic phosphates, whereas the remainder is found as organic phosphate complexes, which increase the availability of P to fish compared to the P forms found in plant feedstuffs.

Phosphorus in cereal grains and plant protein concentrates varies from 0.3-0.4 % and 0.5-1.4 % respectively. However, oilseeds including soybean store P as phytates, i.e., salts of phytic acid (inositol hexaphosphoric acid). The average amount of phytic acid in cereal and oilseed feedstuffs is 1-2% by weight, however certain varieties may contain higher levels of this compound (3-7%) (Lall, 2003). In soy products phytic acid, phosphatides and

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inorganic P constitute approximately 75, 12 and 4% of the total P, respectively. Phytate is not digestible by fish, due to the lack of an endogenous enzyme (phytase) in the gut, which is able to break down the phytic acid to its moieties. Due to the high volume of negatively charged phosphate groups, phytate can reduce the availability of several positively charged ions, including Ca, Mg and Zn at high pH, by binding them and forming insoluble salts within the intestine (Francis et al., 2001). As mentioned before many dietary minerals are found as organic salt complexes, which have to be broken down by intestinal enzymes to be released. Phytate has also been shown to inhibit the function of intestinal enzymes, especially the ones involved in proteolysis (Francis et al., 2001; Denstadli et al., 2006), while inhibition of lipid digestibility has also been reported in rainbow trout (Mambrini et al., 1999).

Furthermore, phytate has been proven to form insoluble complexes with proteins reducing their digestibility and availability (Francis et al., 2001). Lastly, non-starch polysaccharides (NSPs) and oligosaccharides in plant derived protein ingredients have been linked with reduced nutrient availability (including minerals) due to the formation of gelatinized solutions within the gut, entrapping nutrients and decreasing their availability, reducing the distribution of digestive enzymes and the flow on the mucosal layer (Storebakken et al., 2000, 1998b).

Dietary available P requirements for Atlantic salmon range between 6 to 10 g × kg^-1 (Åsgard and Shearer, 1997). Many studies with mono-gastric animals have reported that an optimum dietary Ca:P ratio is important and that by increasing it, the absorption of P could be adversely affected, whereas a high P: Ca ratio may cause reductions in Ca absorption.

Commercial feed trials have shown that diets with an adverse Ca: P ratio (≤ 1) are preferred for salmonid nutrition due to their higher digestibility and increased availability of P (Aliphos, 2012).

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Calcium deficiency is not very common in fish since this mineral can be obtained straight from the aquatic environment. However, P deficiencies can lead to reduced growth, decreased feed efficiency, reduced bone mineralisation, skeletal deformities and increased mortalities (Lall, 2003). Atlantic salmon fed on low dietary P possessed abnormally soft bones; wrinkly ribs and scoliotic spines (Baeverfjord et al., 1998). In general, P and other elemental deficiencies have been proposed as the main cause of vertebral deformities in farmed Atlantic salmon (Witten et al., 2005; Fjelldal et al., 2007; 2009a, 2009b). The reason for this, is the production of fish with under-mineralised bones which are soft, brittle and more prone to the effects of muscular contructions (Lall and Lewis-McCrea, 2007).

Vertebral fusions and compressions are the most commonly occurring types of skeletal deformities (Witten et al., 2005, 2006, 2009), however, curvatures have been observed in farmed Atlantic salmon (Waagbø et al., 2000; Silverstone and Hammell, 2002; Fjelldal et al., 2004; Waagbø, 2006, 2008; Gil Martens et al., 2012). Moreover, a condition termed as hyper-dense vertebrae, caused by the replacement of the adipose tissue with ectopic cartilage within the trabecular network, giving vertebrae a denser appearance in radiographic images, has been reported in Atlantic salmon parr (Helland et al., 2006). The number of fish with this condition was found to increase when salmon parr were fed with increasing amounts of dietary phytate (Helland et al., 2006).

In P deficient fish exhibiting bone deformities, a decrease in bone mineralisation has been described, due to an increment in the amount of osteoid tissue and osteoclast numbers combined with a subsequent reduction in the number of osteoblasts (Takagi and Yamada, 1991; Roy, 2002). Hence, normal development and growth of bones are dependent on the amounts of dietary P, and even most importantly the levels of dietary available P. A deficiency or excessive intake of P can result in the formation of skeletal anomalies (Lall and Lewis-McCrea, 2007).

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