Liquid chromatographic determination of efficacy of incorporation of trimethoprim and sulfamethoxazole in brine shrimp (<i>Artemia</i> spp.) used for prophylactic chemotherapy of fish

The brine shrimp Artemia , an excellent live food source in aquaculture, has been studied as a carrier to deliver selected chemotherapeutic agents to fish for prophylactic treatment of infectious diseases. To monitor the efficiency of incorporation of trimethoprim and sulfamethoxazole in Artemia franciscana, a sensitive and specific analytical method was developed. It is based on homogenization of Artemia nauplii in methanol, extraction of lipids with hexane, solid-phase cleanup on C18 cartridges, and reversed-phase liquid chromatography with detection at 210 nm. The method is sensitive (detection limit, on the order of 3 µg/g with a sample quantity of 30 mg [dry weight]) and reproducible (coefficients of variation, 2.2 and 1.8% for trimethoprim and sulfamethoxazole at levels of 79.6 and 257 µg/g of body weight, respectively). Preliminary quantitative data indicated excellent uptake and persistence of both therapeutic agents in A. franciscana, with levels of 115 µg/g for trimethoprim and 277 µg/g for sulfamethoxazole

The content of ascorbic acid and tocopherol in the tissues and eggs of wild <i>Macrobrachium rosenbergii</i> during maturation

Variations in the concentrations of ascorbic acid (AA) and tocopherols in association with the gonadal development of the freshwater prawn Macrobrachium rosenbergii were investigated in females captured in the Mae Klong River, Thailand. Mean ovarian AA levels ranged from 210 to 540 µg/g dry weight (dw) and were at least 11-fold higher than midgut gland (MG) levels. Variations in ovarian AA levels are believed to be related to the biosynthesis of steroid hormones, the formation of collagen, and the deposition of egg yolk compounds. alpha-Tocopherol (alpha-T) was the predominant form of vitamin E in prawn tissues and eggs. The level of alpha-T in the MG was constant, whereas in the ovaries, it ranged from 143 to 425 µg/g dw. The incorporation of a-T into the ovary was highly correlated (r² = 0.87) to ovarian lipid levels, which probably reflects the role of this vitamin as a major antioxidant agent. The present results provide further evidence of the essentiality of these vitamins in crustacean reproduction

Effect of vitamin C incorporation in live food on the larviculture success of aquaculture species

A study was undertaken to upgrade the levels of vitamin C in Artemia and Brachionus through bioencapsulation and to investigate in this way the requirements for ascorbic acid (AA) during larviculture of fish and prawn. Ascorbyl palmitate (AP) was used as the vit C source because of its stable and lipophylic characteristics which allowed its incorporation in booster emulsions and its readily conversion into AA by Brachionus and Artemia . Under standard conditions very high levels of AA could be incorporated into the Artemia nauplii: supplementation of the enrichment emulsion with 20% AP (w/w) increased the AA-content up to 2000 ppm after 24h enrichment. Also in Brachionus the AA-level changed in function of the concentration of AP applied. 20% AP in the diet enhanced the AA-content in the rotifers 10-fold over 3 days of culture. Using three different enrichment levels in the live food (0%, 10% and 20%), no differences on growth nor survival could be observed for Macrobrachium rosenbergii under standard culture conditions. However, a significantly positive effect on the psychological condition of the postlarvae could be demonstrated when the vit C- boosted live food was administrated. For Clarias gariepinus larvae fed vit C- boosted Artemia , supplemental dietary ascorbate resulted in a significantly positive effect on growth: the 20% AP group weighed 30% more than the control (0% AP) on the final day of the experiment. Evaluation of the physiological condition showed a significantly higher resistance of the larvae according to the dietary vit C level.Dicentrarchus labrax larvae were successively fed rotifers (day 4-12) and enriched Artemia nauplii (day 13-46), supplemented with the same three vitamin C enrichment levels. No significant differences in production characteristics nor in stress resistance of the fish larvae could be observed, however, for all salinity stress tests the 20% AP group performed better.Comparing the results for the two aquaculture fish species, with those reported earlier for the larvae of the prawn Macrobrachium, it appears that requirements for vit C are species specific, and might even differ according to the culture conditions

Solid-phase extraction of ascorbic acid 2-sulfate from cysts of the brine shrimp <i>Artemia fransiscana</i>

Two producers are described for the solid-phase extraction of ascorbic acid 2-sulfate from cysts of the brine shrimp Artemia . The first one is based on reversed-phase ion pair extraction on an octadecylsilica cartridge using tetrabutylammonium as the counterion, followed by elution with methanol. In the second procedure ascorbic acid 2-sulfate is retained on a DEAE silica cartridge and eluted with 0.1M sodium salicylate. Both approaches were used as part of a quantitative reversed-phase ion pair liquid chromatographic method with detection at 254nm. The anion exchange extraction procedure is superior to its ion pair counterpart in terms of recovery (88.7±3,5% versus 63.0±12.7%), reproducibility (CVs of 3.6-7.7% versus 6.3-18.7%), and simplicity

Determination of vitamin E in aquatic organisms by high-performance liquid chromatography with fluorescence detection

A liquid chromatographic (HPLC) method has been developed for the quantitative determination of different forms of vitamin E (alpha-, gamma-, and delta-tocopherol) in aquatic organisms. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed phase chromatography with fluorescence detection. The efficiency of the extraction method was equivalent or superior to that of more complex approaches for the isolation of tocopherols. Linearity has been achieved over the range of 0.02 to 3 µg/ml for alpha-tocopherol and within-run and between-run coefficients of variation at three levels were 0.7-2.9 and 1.2-3.7%, respectively. The recovery at three concentrations ranged from 73.8 to 96.6% and the minimal quantity that could be detected was 0.6 ng. Comparable figures were obtained for gamma- and delta-tocopherol. This method has been routinely applied to determine vitamin E in Artemia , rotifers, turbot and sea bass larvae, and shrimp postlarvae

Simultaneous determination of a-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, alpha-tocopheryl acetate (alpha-TA) is the main source of vitamin E used to fortify fish feed. Alpha-TA in fish is often determined indirectly, i.e. by alkaline hydrolysis, followed by quantitation of ‘total alpha-tocopherol’ (alpha-T) and substraction of the natively present alpha-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of alpha-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of alpha-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 µg alpha-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 µg/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 µg/g dry mass. The recovery of alpha-TA ranged from 97.7 to 100.8% (concentration=2.1 and 20.5 µg/ml, n=6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 µg/ml. This method was routinely applied to determine alpha-TA and alpha-, gamma- and delta-tocopherol (alpha-T, gamma-T, delta-T) simultaneously in Artemia , fish feed, shrimp eggs and various other aquatic organisms

<i>Cis</i>- and <i>trans</i>-canthaxanthin levels in <i>Artemia</i> cysts of different geographical origin

The carotenoid composition of Artemia cysts from 17 geographical strains was studied. All-trans-canthaxanthins and cis-canthaxanthins were quantitatively determined using a non-aqueous reversed phase chromatographic method. In dehydrated cysts, the average cis- and trans-canthaxanthin levels (+/- SD) were 152 +/- 52 and 134 +/- 42 µg/g, respectively (range 38-227 and 67-201µg/g). The corresponding concentrations in hydrated cysts were (cis) 177 +/- 60 µg/g (range 47-271 µg/g) and (trans) 112 +/- 35 µg/g (range 52-169 µg/g). The cis/trans concentration ratios averaged 1.13 (range 0.57-1.47) and 1.60 (range 0.84-2.54) for dehydrated and hydrated cysts, respectively. Differences in total canthaxanthin content in Artemia cysts may partly reflect environmental variability (e.g. algal supply), whereas the divergence in the relative abundance of cis- and trans-canthaxanthin may be associated with differences in some physical and biological characteristics of the cysts