58 MARCH 2015 • WORLD AQUACULTURE • WWW.WAS.ORG pigments, such as b-carotene, zeaxanthin and canthaxanthin, can be converted to astaxanthin (Tanaka et al. 1976). Astaxanthin attracted considerable interest because of its potent antioxidant activity and for its economic value as a pigment source in the aquaculture and food industries. Changes in the levels or activities of the main antioxidants have been proposed as biomarkers of health status in marine organisms. Electron paramagnetic spin resonance (EPR) is a potent tool for determining the level of oxidative stress in vitro and in vivo (Díaz et al. 2004, Buico et al. 2008). The antioxidant activity of a molecule is measured by evaluating its ability to scavenge radicals. Because of their short lifetime, radicals cannot be detected directly by EPR in aqueous solution at room temperature, and they can only be measured using a spin-trapping agent. In this study, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was employed to measure antioxidant capacity. Study Methods Unilaterally-ablated immature females with an initial mean weight of 18.5 g were held in aerated circular tanks of 3 m diameter (33 g/L salinity, temperature 20-21°C, pH 7, 12:12 h photoperiod). Shrimp were maintained at 10/m2. Two feeds with the same protein and lipid content were formulated and supplemented with the carotenoids astaxanthin and β-carotene, each at 300 mg/kg diet. A formulated diet without carotenoid supplementation served as control (Table 1). After 60 days of experimentation, shrimp were dissected and ovaries, integument and midgut gland were extracted and lyophilized. Female specimens after one day (wild) and 7 days of acclimatization (initial) were also sampled as controls. Samples were homogenized under an argon atmosphere in darkness. Carotenoids were extracted and separated following the procedure of Schiedt et al. (1993). Non-polar carotenoids were extracted with hexane and polar carotenoids with dimethyl sulfoxide/acetone. Absorption peaks of different carotenoids were identified by scanning the spectrum between 200 and 750 nm using an UVvisible spectrophotometer. Antioxidant activity of the midgut gland was investigated on the basis of the scavenging activity on the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH) free radical using spectroscopy of the EPR technique (Fig.3) (Díaz et al. 2004). Broodstock Performance Food is a critical factor to promote maturation in females of Argentine red shrimp under culture conditions, so a diversified diet covers most nutritional requirements. Mixed diets consisting of different natural fresh-frozen food and dried formulated feed are more efficient than a single food. This feeding regime has a synergetic effect on ablated specimens, improving female maturation in this species, with 50 percent success (Fig. 4; Díaz and Fenucci 2004). Addition of carotene (as astaxanthin or β-carotene) increased TABLE 1. Ingredient composition of reference diet. Ingredient g/100 g Fish meal 41.5 Squid meal 25.0 Clam meal 4.0 Soybean meal 4.5 Manioc starch 15.0 Fish oil 3.0 Fish soluble 3.0 Soy lecithin 1.0 Cholesterol 1.0 Vitamins 0.5 Na alginate 1.5 Proximate composition % dry matter Crude protein 54.5 Lipid 13.7 Ash 7.1 Moisture 7.0 TOP AND BOTTOM, FIGURE 3. UV-visible spectrophotometer (top) and EPR spectra of DPPH free radical (bottom).
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