Aquaculture: Vitamin E

Table 1 lists Vitamin E requirements for various species.
Vitamin E is insoluble in water and is typically supplied in the form of dl-alpha-tocopherol acetate, either as a spray-dried or an absorbed product. Vitamin E, as dl-alpha-tocopherol acetate, is moderately stable in dry multivitamin premixes if stored below room temperature. However, the vitamin is prone to oxidation on storage in the presence of oxidation products such as rancid oils or at high ambient temperatures.

The dietary requirement of alpha-tocopherol is dependent on dietary levels of PUFA, oxidizing agents, vitamin A, vitamin C, carotenoids, and trace minerals and decreased with increasing levels of fat-soluble antioxidants, sulfur amino acids, and selenium (Cowey, 1986; Hamre et al., 1997). Limited work suggests a connection between water temperature and vitamin E requirement; the vitamin E requirement may be increased as water temperature decreases (Cowey et al., 1984). A detailed experiment to look at this question remains yet to be completed (Cowey and Cho, 1993).

Fish can be placed into three categories based on their requirements for essential fatty acids: those that require n-6 only, those that require n-6 and n-3, and those that require n-3 only (Takeuchi, 1997; Sargent et al., 1999). Juvenile marine fish generally require around 0.5% to 1% of the dietary dry matter as n-3 PUFA (Izquierdo, 1996; Sargent et al., 1997). Recent work in fish has shown the importance of fatty acids as precursors for eicosanoids and their importance as paracrine messengers and their effect on the immune system (Kiron et al., 1995; Bell et al., 1996).

The nature of PUFAs makes them more prone to oxidation, and thus they have the potential to increase the need for vitamin E. Tacon (1996) has summarized the pathologic effects of feeding oxidized oil to fish.

Numerous studies have been completed on the effects of oxidized oil on vitamin E requirement of fishes. Studies with trout found that the natural level of vitamin E in steam-pelleted diets was adequate to meet the vitamin E requirements when fresh oil was fed, but not when oxidized oil was fed (Hung et al., 1980; Hung et al., 1981). Other have found that oxidized oils in the diet increase the level of supplemental vitamin E required (Kubota et al., 1981; Moccia et al., 1984; Cowey et al., 1984; Oberbach et al., 1989; Sakai et al., 1992; Stéphan et al., 1993; Stéphan et al., 1995; Baker and Davies, 1996a, b; Baker, 1997; Baker and Davies, 1997a, c).

Still, other have found only minimal vitamin E levels are required to protect fish from the effects of oxidized oil in the diet (Forster et al., 1988).

Jaundice of cultured yellowtail (Seriola quinqueradiata) and hybrid catfish (Clarias macrocephalus x C. gariepinus) have been linked with the feeding of rancid oil (Sakai et al., 1989; Pearson et al., 1994).

Various workers have also investigated the impact of dietary vitamin E supplementation on the fatty acid profile of fishes. Depending on the amount of PUFA in various oils used, vitamin E was seen to influence the fatty acid profile of fish tissue (Runge et al., 1987; Schwarz et al., 1988; Runge et al., 1992; Schwarz, 1996; Baker and Davies, 1997c). In other studies with rainbow trout or Atlantic salmon, no impact of vitamin E was seen on fatty acid profiles (Boggio et al., 1985; Waagbø et al., 1993d; Sigurgisladottir et al., 1994b).

Elevation of the dietary lipid levels from 5% to 15% in the diet containing 50 IU of alpha-tocopherol reduced the concentration of alpha-tocopherol in the whole body of tilapia (Oreochromis niloticus) (Satoh et al., 1987). Increasing the PUFA level in the diet of Atlantic salmon (Salmo salar) reduced the liver vitamin E concentration (Figure 1) (Waagbø et al., 1991b). Increasing the dietay lipid level from 5% to 20% increased the severity of vitamin E deficiency signs in carp (Watanabe et al., 1981a).

Fish has the shortest shelf life of all meats, even when stored optimally. A number of factors influence shelf life: fish species, fish genetics, handling during harvest and processing, storage temperature and storage conditions, to name a few.

Genetics have been suggested to play a role in the oxidative stability of fish muscle. Studies by Erickson found that different strains of catfish (Ictalurus punctatus) (Erickson, 1992b), different varieties of tilapia (Tilapia nilotica–black skin coloration and red skin coloration) (Erickson, 1992c), and striped bass (Morone saxatilis) and hybrid striped bass (Morone chrysops x M. saxatilis) (Erickson, 1992a) reared under identical conditions had different muscle lipid compositions, fatty acid profiles, and alpha-tocopherol levels. It was suggested that this could have an impact on the rate of lipid oxidation and thus shelf life.

Antioxidants have been found to help increase certain aspects of the shelf life of fish. Muscle tissue contains several lipid oxidation catalysts, including enzyme NADPH- or NADH-dependent redox cycling of iron in microsomal fractions and sacroplasmic reticulum; non-enzymatic iron-redox cycling system utilizing ascorbate, superoxide, and cysteine; non-enzymatic lipid oxidation catalyzed by H2O2-activated metmyoglobin; and lipoxygenase. In addition, skeletal muscle contains high concentrations of unsaturated fatty acids, especially in cellular membranes such as mitochondria, sacroplasmic reticulum and microsomes (Chan and Decker, 1994). Vitamin E was found to protect shrimp (Penaeus vannamei) tail muscle, stored at -60†C, from oxidation (Figure 2) (He and Lawrence, 1993b).

The dietary vitamin E level significantly influenced lipid oxidation rate in turbot (Psetta maxima) muscle frozen at -20†C for six months (Figure 3) (Stéphan et al., 1995).

The dietary level of vitamin E significantly affects fillet vitamin E content in trout (Figure 4) (Hung and Slinger, 1982; Boggio et al., 1985; Frigg et al., 1990; Gessl et al., 1995; Jensen et al., 1998; Akhtar et al., 1999). Also, the fillet alpha-tocopherol concentration influences the oxidative stability of trout fillets (Figure 5) (Frigg et al., 1990; Gessl et al., 1995). In organoleptic tests all significant preferences were for fillets with higher vitamin E levels (Frigg et al., 1990). Hung and Slinger (1982) were unable to show in small trout (15 g) fed low lipid diets (7.5%) any benefit of feeding high levels of vitamin E (770 IU) on muscle storage stability. Jensen et al. (1998) found that astaxanthin and alpha-tocopherol helped protect frozen trout fillets from oxidation.

The fillet alpha-tocopherol concentration was found to improve the oxidative stability of Atlantic salmon (Salmo salar) fillets (Sigurgisladottir et al., 1994b). In Atlantic salmon fed different combinations of n-3 PUFA levels (1.0%, 2.5% or 5.0% of the diet at 17% total lipid) and each with two vitamin E levels (45 to 70 IU or 300 IU vitamin E), there was a significant interaction between PUFA level and vitamin E level on organoleptic parameters (Waagbø et al., 1993d). The drip loss of Atlantic salmon fillets after storage at 4°C for 12 days was significantly improved by having fed diets that contained 29% lipid and that were supplemented with 521 mg or 786 mg of alpha-tocopherol per kg (Weber, 1996). Refsgaard et al. (1998) was unable to find any correlation between lipid hydroperoxides and the oil content, the concentrations of tocopherols, astaxanthin, or canthaxanthin; the fatty acid composition; or the content of free fatty acids measured in fresh Atlantic salmon, that would predict the sensory quality during storage.

Feeding catfish (Ictalurus punctatus) 240 IU of vitamin E reduced the 2-thiobarbituric acid value compared to fish fed 60 IU of vitamin E (Gatlin et al., 1992). In a vitamin E muscle titration study with small catfish (36 g), Bai and Gatlin (1993) were able to reach approximately 25 µg of alpha-tocopherol per g of fillet in two weeks or six weeks, by feeding 1,000 or 240 IU of vitamin E per kg of diet at 3% of total body weight per day.

High doses of vitamin E in catfish (Ictalurus punctatus) diets failed to promote increased frozen shelf life (Silva et al., 1994). In a study with African catfish (Clarias gariepinus), fish fed a diet containing 100 IU of vitamin E per kg had reduced drop loss after thawing, especially when an oxidized oil was fed (Baker, 1997).

Very limited work has been directed at this area. Dietary PUFA level (1.9%, 3.5% or 6.0% of diet), obtained by using soybean, capelin or sardine oil, respectively, and dietary vitamin E level (45 IU to 70 IU vs. 250 IU to 300 IU) affected fillet whiteness, color tone, and color intensity in Atlantic salmon (Salmo salar) (Waagbø et al., 1993d). Increasing the PUFA improved color and increasing the vitamin E seemed to decrease color (Waagbø et al., 1993d). Dietary vitamin E (400 IU vs. 15 IU) did not improve astaxanthin deposition in Atlantic salmon (Salmo salar) (Sigurgisladottir et al., 1994b). Jensen et al. (1998) found the same effect in trout (Oncorhynchus mykiss).

Yet, there is an interation between dietary astaxanthin and vitamin E. It was found that dietary astaxanthin enhanced fish growth, muscle (retinol and alpha-tocopherol) and liver (retinol, alpha-tocopherol and ascorbic acid) levels of antioxidants, and resistance to challenge with Aeromonas salmonicida (Christiansen et al., 1995b). High levels of vitamin C and vitamin E were found to reduce astaxanthin oxidation in Atlantic salmon (Salmo salar) fillets stored in the dark on ice for 12 days (Figure 6) (Robb et al., 1997). Pozo et al. (1988) saw only a small effect (P < 0.1) of dietary alpha-tocopherol level (26 IU vs. 526 IU) on canthaxanthin stability in trout (Oncorhynchus mykiss) fillets frozen at -12°C.

Our knowledge of the fishes’ immune system and non-specific disease resistance factors has increased, and so has the methodology for examining mechanisms of diet-induced effects on infectious disease. It must be realized that feeds producing the fastest growth may not provide for the best disease resistance. A number of articles are available that review general and specific roles of various nutrients on the immune system in fish (Blazer and Wolke, 1984a; Lall et al., 1988; Landolt, 1989; Blazer, 1992; Lall and Olivier, 1993; Waagbø, 1994; Kiron et al., 1995; Olivier, 1997; Bell et al., 1996; Bowers, 1997). Vitamin E has been shown to act as a immunopotentiator in homeotherms, when fed at levels above the daily requirement (Kelleher, 1991; Blazer, 1992; Packer, 1992).

Blazer (1982) and Blazer and Wolke (1984b) found that vitamin E deficiency depressed all aspects of humoral and cellular immunity as well as phagocytosis, although no clinical histopathologic deficiency signs were observed in the rainbow trout (Oncorhynchus mykiss).

Early diagnosis of cold-water vibriosis—"Hitra disease" (Vibro salmonicida) — was thought to be due to vitamin E and selenium deficiencies (Fjølstad and Heyeraas, 1985) as muscle and endothelium degeneration and anemia followed the infection. However, further investigation into this problem showed the administration of selenium or vitamin E to infected fish did not prevent mortalities (Salte et al., 1988). Again, before the true etiology was ascertained, pancreas disease was also associated with reduced plasma and tissue vitamin E and selenium levels (Ferguson et al., 1986).

In one case, further work found that pancreas disease could be influenced by modulating the dietary vitamin E and PUFA levels (Raynard et al., 1991). In another study (McCoy et al., 1994), vitamin E did not influence the outbreak of pancreas disease.

Lall et al. (1988) did not see any effect of vitamin E (up to 240 IU) on resistance to furunculosis (Aeromonas salmonicida) in Atlantic salmon (Salmo salar). In a study with coho salmon (Oncorhynchus kisutch), Forster et al. (1988) did not see any benefit of low (30 IU) or high (1,030 IU) vitamin E supplementation to diets containing oxidized herring oil on the immune function or disease resistance. In studies with chinook salmon (Oncorhynchus tshawytscha) fed diets containing increasing amounts (10 to 2,800 IU) of vitamin E, no benefit on immune function or disease resistance was seen (Leith et al., 1989).

Atlantic salmon fed diets containing 7 IU or 326 IU of vitamin E per kg had significantly different latency periods and survival following a bath challenge with Aeromonas salmonicida (Hardie et al., 1990). Some non-specific immune parameters seemed to be influenced as well in this study (Hardie et al., 1990). Feeding rainbow trout (Oncorhynchus mykiss) diets containing 806 mg vitamin E significantly improved survival after challenge with Yersinia ruckeri (Furones et al., 1992). Increasing the dietary vitamin E concentration was found to have a modulatory effect on specific immunity (Verlhac et al., 1993). Various combinations of vitamins C and E were found to influence various aspects of the immune response and disease resistance in rainbow trout (Oncorhynchus mykiss) (Wahli et al., 1998), but not always in a consistent fashion.

By increasing the oxidative stress by feeding turbot (Psetta maxima) a diet low in antioxidants and with oxidized oil, the immune function and disease resistance were lowered (Obach and Baudin Laurencin, 1992). The activity of the alternative complement pathway in gilthead sea bream (Sparus auratus) was reduced in fish fed diets deficient in vitamin E or n-3 PUFA (Montero et al., 1998). Vitamin E was found to help mediate the negative effects of stocking density on serum lysozyme and the alternative complement pathway in gilthead sea bream (Sparus auratus) (Montero et al., 1999).

In sea bass (Dicentrarchus labrax), the combination of vitamin E (up to 300 IU) with fresh or oxidized oil did influence various immune parameters (Obach et al., 1993), with low vitamin E levels and oxidized oil being detrimental.

Vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent Edwardsiella ictaluri (Figure 7) (Wise et al., 1993).

Beneficial effects of vitamin E (272 IU versus 57 IU) were seen in Atlantic salmon challenged with Vibrio salmonicida. In the same studies, dietary fatty acid composition and water temperature had an influence on the effects (Waagbø et al., 1993b, c). Feeding rainbow trout 600 IU of vitamin E for four months improved non-specific (Figure 8) and specific immune functions (Figure 9) (Verlhac et al., 1997).

The survival of chinook salmon (Oncrohynchus tshawytscha) with a natural Renibacterium salmoninarium infection was higher when fed diets containing 300 IU vitamin E and 2.49 mg selenium per kg of dry diet when compared to diets with lower levels of these nutrients (Thorarinsson et al., 1994).

Dietary astaxanthin was found to enhance fish growth, muscle (retinol and alpha-tocopherol) and liver (retinol, alpha-tocopherol and ascorbic acid) levels of antioxidants and resistance to challenge with Aeromonas salmonicida (Christiansen et al., 1995b).

Alpha-tocopherol may exert indirect immunomodulatory effects by influencing other antioxidants as retinol, ascorbic acid and ubiquinones as may those antioxidants impact on alpha-tocopherol levels (Cowey, 1986; Parker, 1989; Packer , 1992; Chan and Decker, 1994). In view of the temperature-related PUFA content in cell membranes and changes in vitamin E requirement, it is possible that antioxidant interactions may vary with temperature as well as other environmental variable (Cowey et al., 1984; Hilton, 1989). Depending on water temperature and dietary lipid fatty acid composition, supplementation of vitamin E above the requirement for growth reduces negative effects of oxidized lipid, reduces mortality as demonstrated by bacterial challenge experiments, and modifies non-specific humoral immune factors as well as cellular immunity (B-cells, T-Cells, macrophages). These effects seem to be related to the antioxidant properties and membrane stabilizing effect of vitamin E and the influence of vitamin E in eicosanoid synthesis.

During vetellogenesis in Atlantic salmon (Salmo salar), muscle alpha-tocopherol levels decrease as ovary levels increase (Lie et al., 1994). A higher vitamin E requirement (190 versus 90) was suggested for mullet (Mugil cephalus) for optimal ovary development and alpha-tocopherol levels (Fung Shyu and Sun Pan, 1993). Ayu (Plecoglossus altivelis) broodstock were found to require 34 IU of vitamin E per kg of diet to promote good egg survival and hatching rate (Takeuchi et al., 1981).

Vitamin C protected Atlantic salmon fry (Salmo salar) against vitamin E deficiency in a dose-dependent manner. Vitamin C did not influence the tissue levels of vitamin E, except in vitamin C deficiency, which induced a large drop in liver vitamin E concentration (Hamre et al., 1997).
In a study to evaluate the interaction of vitamin C and vitamin E, a megadose (2,000 mg) of vitamin C prevented myodegeneration occurring as a consequence of vitamin E deficiency in rainbow trout (Oncorhynchus mykiss) (Frischknecht et al., 1994). Vitamin E prevented negative effects of vitamin C deficiency on blood parameters (Frischknecht et al., 1994).
Results indicated that stocking density and vitamin C status did not affect the severity of vitamin E deficiency in channel catfish (Gatlin et al., 1986b).

In various studies, the inclusion of ethoxyquin, BHT (butylated hydroxytoluene), or BHA (butylated hydroxyanisole) has been shown to influence the requirement of vitamin E. Murai and Andrews (1974) recommended 25 IU of vitamin E when the diet contained 125 mg of ethoxyquin per kg for fingerling catfish. If the diet did not contain ethoxyquin, they recommended the diet to be supplemented with 100 IU of vitamin per kg. Yet, ethoxyquin does not completely replace the need for vitamin E in catfish diets (Lovell et al., 1984). Nor did BHT completely replace the need for vitamin E in shrimp (Penaeus vannamei) diets (He and Lawrence, 1993b).

Roem et al. (1990a) found the vitamin E requirement of tilapia (Oreochromis aureus) to be 10 IU or 25 IU per kg of a diet containing 3% or 6% of corn oil, respectively. The diets also contained 120 mg of BHA.

Gatlin et al. (1992) found that neither ethoxyquin, BHT, BHA, nor Endox™ improved fish growth or helped to reduce oxidation in frozen catfish fillets, when adequate dietary vitamin E levels were present.

During smoltification, Atlantic salmon (Salmo salar) kidney and adipose tissue showed a clear reduction in alpha-tocopherol concentration during the parr-smolt transformation (Hamre and Lie, 1995b). In another study, Roy et al. (1995) found the liver vitamin E concentrations in Atlantic salmon smolts increased two to six weeks after saltwater entry.

During a clinical investigation of increased mortalities suggestive of vitamin E deficiency signs, rainbow trout (Oncorhynchus mykiss) fry, reared under commercial conditions, showed apparent vitamin E deficiency signs when the diet contained 239 IU vitamin E and 31,000 IU of vitamin A. Fish fed diets supplying 532 IU of vitamin E and 20,000 of vitamin A did not show any vitamin E deficiency signs (McLoughlin et al., 1992). It has been shown that high levels of vitamin A (50,000 IU per kg) reduce the availability of vitamin E in the chick (Frigg and Broz, 1984). This interaction has not been specifically investigated in fish.

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