D functions in calcium homeostasis and is important for the development of skeletal tissues and cellular differentiation (NRC 2011). Vitamin D can be sourced from plants (ergocalciferol or vitamin D2) and animals (cholecalciferol or vitamin D3). Of these, D3 is significantly more effective than D2. Larval requirements for vitamin D seem to be significantly higher than those of juvenile fish. Toxicity can result in vertebral deformities, so further research is warranted before recommendations of high intakes for larval fish are made (Hamre et al. 2013).
Within the family of vitamin E molecules, α‐tocopherol is the most biologically active form of the tocopherols and tocotrienols. These serve as antioxidants, stabilize membranes, affect eicosanoid signaling and cellular proliferation, and modulate immune responses. Efficacy of vitamin E as an antioxidant is dependent on levels of other antioxidants such as vitamin C and selenium‐based glutathione systems. When the latter are low, vitamin E can become a pro‐oxidant (Hamre 2011). As an antioxidant, vitamin E protects PUFAs at an approximate ratio of 1 molecule vitamin E:1000 molecules PUFA, with higher levels of unsaturation requiring more protection (Schwarz et al. 1988; Hamre 2011). This is why nutrient requirements for vitamin E are often described in relation to the PUFA content of the diet. This is particularly critical for fish, where diets are often rich in PUFA. Vitamin E requirements in most experiments are determined based on maximal growth rates. When other parameters are used (e.g. red blood cell fragility, immune responses), higher requirements are predicted. For example, Malabar grouper (Epinephelus malabaricus) required 60–100 mg D,L‐α‐tocopheryl acetate/kg diet for growth and up to 800 mg/kg for better nonspecific immune parameters (Lin and Shiau 2005). Requirements may increase further during disease (Durve and Lovell 1982). Commercially prepared larval fish feeds may require higher vitamin E due to the large surface area, higher risk of lipid oxidation, and high level of omega‐3 PUFAs, particularly for marine fish (Hamre 2011). Very high levels of dietary vitamin E (5000–10 000 IU/kg) have been shown to promote oxidation and damage erythrocytes (NRC 2011).
Little work has been conducted on vitamin K in fish. Existing research has been inconsistent in showing a dietary requirement or GI microbial synthesis of vitamin K in fish (NRC 2011).
Water‐soluble vitamins pose a lower risk of toxicity and are frequently supplemented at high levels to ensure optimal levels are met. Research has focused primarily on defining minimal requirements. Of the water‐soluble vitamins, vitamin C receives the most attention. It is considered an essential or semi‐essential dietary nutrient for teleosts, as there is evidence that some cartilaginous fish can synthesize ascorbic acid de novo (Cho et al. 2007). However, it is unclear if de novo synthesis is adequate to meet needs in all life stages and environmental conditions, and it has been demonstrated that supplemental vitamin C improves immune responses in some species with de novo synthesis (Xie et al. 2006). So it is advisable to supplement vitamin C in the diet of all fish species. Signs of vitamin C deficiency include lordosis, scoliosis, cartilage and collagen defects, petechial hemorrhaging, and fractures of the spine (Halver et al. 1975; Sandnes et al. 1992). These have been reported within weeks of feeding a vitamin C‐deficient diet (Halver et al. 1975; Sandnes et al. 1992). Minimum requirements for growth are often much lower than the requirements for optimal immune function (Ai et al. 2004). While minimum requirements for fish are ~50 mg/kg diet, much higher doses (190–1000 mg/kg diet) often result in significant improvements in immune function, notably antibody production, complement activity, and survival after disease challenge (Navarre and Halver 1989; NRC 2011; Hamre et al. 2016). Vitamin C needs are increased during reproduction and larval fish growth (NRC 2011). Since vitamin C is heat labile, stabilized forms should be used for supplementation, e.g. L‐ascorbyl‐2‐polyphosphate. The requirement for vitamin C is related to the level of vitamin E in the diet, as vitamin C or vitamin E can counteract the signs of deficiency of the other nutrient to some extent (Wahli et al. 1998; Sealey and Gatlin 2002).
Work on B vitamins in fish is limited. Thiamine (B1) is of concern for any fish‐eating species, as thiaminases in fish tissues are activated upon death and rapidly destroy available thiamine. Thiaminase activity is particularly high in clupeids such as alewives (Alosa pseudoharengus) and capelin (Mallotus villosus), and cyprinids such as feeder goldfish (Carassius auratus). Any frozen/thawed fish should be supplemented with thiamine (Fitzsimons et al. 2005). Thiaminase activity has also been demonstrated in insects, blue‐green algae (Microcystis aeruginosa) and bacteria (Honeyfield et al. 2002). Pyridoxine (B6) may be needed in the diet during larval development, as levels in the yolk sac are depleted rapidly (Hamre et al. 2013; Saha et al. 2016).
Choline, a vitamin‐like nutrient, is an important component of phospholipids and a precursor of the neurotransmitter acetylcholine. Most fish cannot synthesize sufficient choline and it is a common supplement in cultured fish diets, usually as choline chloride (Wilson and Poe 1988). Additionally, choline may reduce excessive lipid accumulation and the development of fatty liver, although studies have produced mixed results (Halver 2002). Some species have shown a negative correlation between dietary choline and liver lipid levels (channel catfish Ictalurus punctatus, hybrid striped bass Morone saxatilis x M. chrysops, barred knifejaw Oplegnathus fasciatus), while others have shown a positive correlation (hybrid tilapia) or no correlation (rainbow trout, yellow perch Perca flavescens, Siberian sturgeon Acipenser baerii) (Wilson and Poe 1988; Rumsey 1991; Griffin et al. 1994; Shiau and Lo 2000; Twibell and Brown 2000; Yazdani‐Sadati et al. 2014; Khosravi et al. 2015). Further studies are needed to understand the potential benefits of supplementing dietary choline.
Minerals
Minerals in the diet are often subdivided into:
Macrominerals (calcium [Ca], phosphorus [P], sodium [Na], potassium [K], magnesium [Mg]).
Trace minerals or microminerals (copper [Cu], zinc [Zn], iron [Fe], manganese [Mn], selenium [Se]).
Ultra‐trace minerals (iodine [I], cobalt [Co], chromium [Cr], molybdenum [Mo]).
Some minerals can be acquired by fish from the water, including Ca, Cu, Fe, K, Mg, Na, Se, and Zn (Terech‐Majewska et al. 2016). It is generally assumed that Ca, K, Mg, and Na requirements can be met by the water, particularly seawater, although low pH does restrict absorption (Terech‐Majewska et al. 2016). However, most commercial fish diets also meet minimum requirements. As with other nutrients, these recommended levels are generally determined for optimal performance of aquaculture species and should be used as a guide only (Table A4.4). Finally, many trace minerals interact with each other and with vitamins or other elements in the water, making it hard to determine their bioavailability to fish.
Phosphorus is likely the most discussed mineral in fish as dietary sources are required, and because of the variability in bioavailability and the environmental impact of excreted phosphorus. Phosphorus may be present in the diet from plant, animal, or chemical sources. Plant‐based forms are often complexed with phytic acid (e.g. phytate‐P) which generally has very low bioavailability to fish. A notable exception is Nile tilapia which can digest some phytate‐P (Kumar et al. 2012). Undigested phytate‐P can form complexes with other nutrients (e.g. Zn), reducing their bioavailability, and can contribute to algal blooms when excreted. Efforts to improve phytate‐P digestibility have been a focus of extensive research in fish nutrition. Exogenous phytase enzymes may be provided in the diet to improve phytate‐P digestibility, although efficacy varies. For example, agastric species such as the common carp (Cyprinus carpio) have a higher GI pH and most phytase enzymes have little effect unless dietary acidifiers or neutral pH‐active phytase enzymes are used (Kumar et al. 2012; Lemos and Tacon 2016).
Selenium is a trace mineral involved in antioxidant and housekeeping functions. Deficiencies of selenium reduce antioxidant function and can result in vitamin E or vitamin C based pro‐oxidation (Hamre et al. 2016). Deficiencies may be seen in captive‐bred larval fish, as rotifers and other common aquarium food items have much lower levels than wild food items (Hamre et al. 2008; Penglase et al. 2010). Selenium can also be toxic; excess can reduce growth rates and increase mortality and has been associated