nutrient
Requirements
and Interactions
HOÃœHG-YÃœHGCHEN2 AND GLORIA HWANG Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung, Taiwan 804, Republic of China growth (Halver 1985 and 1989). Riboflavin deficiency in shrimp species is unknown. The requirements for riboflavin have been determined only for larval Penaeus japonicus, based on survival results (Kanazawa 1985). A requirement of 80 mg/kg diet was suggested. The riboflavin requirements of coldwater salmonids (Halver 1985 and 1989) and warmwater channel catfish (Murai and Andrews 1978) have been reported to be 20-30 and 9 mg/kg dry diet, respec tively. The allowance for riboflavin recommended by the National Research Council (1977) in complete diets for warmwater herbivorous fish is 20 mg/kg dry diet; that required for coldwater fish is 0.15 to 0.20 mg/kg body wt per day (NRC 1981). These values are very low when compared with the only reported re quirement level (80 mg/kg diet) for larval P. japonicus (Kanazawa 1985). Cowey (1976) suggested that the nutrient require ments of cultured fish be established by biochemical measurements, such as a particular enzyme's activity. The measurements of specific nutrient-dependent bio chemical functions are superior to growth perfor mance or simple measurement of tissue nutrient level for discerning the true requirement. Hughes and Rumsey (1981) found that the measurement of the activation coefficient (ratio of activity following preincubation with FAD to basal activity) of erythrocyte glutathione reducÃ-ase (EC 1.6.4.2) is a sensitive and specific indicator of the riboflavin status for rainbow trout as shown in rats (Bamji and Sharada 1972, Glatzle et al. 1968). Because no quantitative riboflavin requirement has been reported for P. monodon (the most widely cul tured shrimp species), the following study was
ABSTRACT The nboflavin requirements of marine shrimp (Penaeus monodon) were evaluated in a 15-wk feeding trial. Juvenile shrimp (initial mean weight, 0.13 ±0.05 g) were fed purified diets containing seven levels (0, 8, 12, 16, 20, 40 and 80 mg/kg diet) of supplemental nboflavin. There were no significant differ ences in weight gains, feed efficiency ratios and survival of shrimp over the dietary riboflavin range. The riboflavin concentrations in shrimp bodies increased with the in creasing vitamin supplementation. Hemolymph (blood) glutathione reducÃ-ase activity coefficient was not a sen sitive and specific indicator of riboflavin status of the shrimp. The dietary riboflavin level required for P. monodon was found to be 22.3 mg/kg diet, based on the broken-line model analysis of body riboflavin concen trations. Shrimp fed unsupplemented diet (riboflavin concentration of 0.48 mg/kg diet) for 15 wk showed signs of deficiency: light coloration, irritability, pro tuberant cuticle at intersomites and short-head dwarfism. J. Nutr. 122: 2474-2478, 1992. INDEXING KEY WORDS:
•riboflavin requirement •riboflauin deficiency •glutathione reducÃ-ase
penaeid shrimp
Riboflavin is a precursor of FAD and FMN, which are vital nutrients for all animals, including crusta ceans (DalÃ-and Moriarty 1983). FAD and FMN are cofactors in many enzyme systems involved in oxi dation-reduction reactions, tissue respiration and hydrogen transport. Riboflavin plays an important role in the respiration of poorly vascularized tissues and in the retinal pigment during light adaptation (Pike and Brown 1984). Many deficiency symptoms of this vitamin, such as changes in the skin, eye and nervous system, have been reported in humans (Pike and Brown 1984), farm animals (Maynard et al. 1979) and fishes (Halver 1985). The deficiency signs in fishes are usually species specific, and the only common signs among species are anorexia and poor 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
1Supported by a grant (NSC80-0409-B110-01) from the National Science Council of the Republic of China. •^To whom correspondence should be addressed.
Received 19 May 1992. Accepted 6 August 1992. 2474
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Estimation of the Dietary Riboflavin Required to Maximize Tissue Riboflavin Concentration in Juvenile Shrimp (Penaeus monodori)1
RIBOFLAVIN REQUIREMENT
MATERIALS AND METHODS Juvenile P. monodon from a single spawner were obtained from a commercial hatchery (Won-Zai Hatchery, Lin-Yuan, Kaohsiung County, Taiwan). They were acclimated to experimental conditions and semipurified diets for 3 wk before the experiment. A total of 168 apparently healthy shrimp (initial mean weight, 0.13 ±0.05 g) were divided into seven ex perimental groups with three replicates of eight shrimp each. Each of the 21 replicates was randomly assigned to a 60 x 60 x 46.6 cm rectangular glass aquarium fitted with under-gravel filters. The filter bed consisted of crushed oyster shell and sand. Each aquarium was divided into eight equal compartments by clear plastic plates. Holes with a diameter of 4 mm allowed exchange of water among neighboring com partments. Each shrimp was individually housed in one of the compartments. Aquariums were placed under fluorescent light with a light cycle of 12 h light (0700-1900 h):12 h dark. Natural sea water drawn through a beach sand filter was first passed through a polyester cartridge and activated carbon filter and then through a quartz UV sterilizer (Sanitron, Atlantic Ultraviolet Co., Bayshore, NY). Deionized water was used to adjust salinity to be approximately 28-29 g/L. Approxi mately one tenth of the sea water in each aquarium was replaced every other day. Temperature was not controlled but monitored daily. The water temper ature, dissolved oxygen, pH and ammonia were 25-29°C, 7.5-8.0 mg/L, 7.5-8.0 and 0-0.5 mg/L, respectively, during the trial period. These environ mental factors are considered to be not detrimental to shrimp growth. The shrimp were fed three times daily (0800, 1700 and 2300 h) for 15 wk. Feeding level was 10% of shrimp body weight during the first 3 wk and was adjusted thereafter according to the feeding response of the shrimp. Uneaten food and exuviae were re moved each morning before feeding. The shrimp were observed daily for mortality and unusual behavior or morphological changes. All shrimp were weighed every 3 wk in morning hours before the first feeding. Shrimp were netted, blotted with dry gauze cloth and weighed individually to the nearest 0.01 g. Each shrimp was placed between folded gauze during weighing to prevent sudden jumping. No anesthesia was used.
Seven purified experimental diets were formulated to provide graded levels of supplemental riboflavin. The basic vitamin-free diet (Table 1) was based on a formula for penaeid shrimp (Chen et al. 1991). The basal diet contained 15.08 MJ/kg diet of gross energy and 40% crude protein and had a protein:energy ratio of 26.53 mg protein per kj. Riboflavin was added to the test diets at the expense of small amounts of cellulose to provide concentrations of 0, 8, 12, 16, 20, 40 and 80 mg/kg. All vitamins used in the study were supplied by Hoffmann La Roche (Basel, Switzerland). The diets were prepared under reduced-light condi tions because of the light sensitivity of riboflavin. The diets were processed into pellets (3 mm in di ameter by 3 mm in length) and were freeze-dried. Each diet was sealed in opaque plastic bags and stored at -20°C until fed. The riboflavin concentrations of the seven diets were chemically determined (AOAC 1984) to be 0.48 (unsupplemented), 7.39 (8 mg/kg), 11.35 (12 mg/kg), 14.64 (16 mg/kg), 19.08 (20 mg/kg), 40.00 (40 mg/kg) and 76.96 (80 mg/kg) mg/kg diet, respectively. At the end of the feeding trial, blood (hemolymph) samples were withdrawn from the ostium of the heart of each shrimp by a 1-mL syringe rinsed with 80 mmol/L dipotassium EDTA solution. Blood samples from within the same dietary group were pooled into two samples because of their small volumes and frozen in liquid nitrogen until the assay of glutathione reducÃ-ase was conducted. The remaining shrimp bodies were pooled into ihree or four samples within each treatment and were freeze-dried, pul verized and used to determine the riboflavin concen tration according to the method of AOAC (1984), using a fluorospectrophotometer (F-3000, Hitachi In struments, Tokyo, Japan). Before the shrimp were frozen, body and carapace lengths of each shrimp were measured to the nearest 1 mm. Body length is the length between the base of the rostrum (rostral spine) and the end of tail fan (uropods). Carapace length represents the head length of a shrimp and is the distance between the base of rostrum and the end of carapace. The assay and calculation procedures of hemo lymph glutathione reducÃ-ase followed the method of Glalzle el al. (1970) and Nichoalds (1974). Because ihe cell volume of shrimp hemolymph is much smaller than mammalian blood, whole hemolymph was used in assay. Before analysis, the frozen blood samples were thawed and then ultra-sonicated for 5 min to erupt blood cell membranes. The supernatants de rived from a 15-min centrifugation (Model Himac, RPR20-3 rotor, Hitachi Instruments) at 0°C and 14,500 x g were used as the enzyme solution. Glutathione reducÃ-ase activity was measured in the presence and absence of in vitro FAD by moniioring ihe oxidation of NADPH at 340 nm using a thermoslaÃ--conÃ-rolled (37°C)specÃ-rophoÃ-omeler (U-2000, Hilachi
Inslrumenls).
The
sialus
of
riboflavin
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designed to estimate the requirement for juvenile P. monodon. Weight gains, feed efficiency ratios, sur vival rates and biochemical measurements (including body riboflavin concentration and hemolymph glutathione reducÃ-ase activity coefficient) were used simultaneously to assess the riboflavin status and optimal requirement of the shrimp.
2475
OF PENAEID SHRIMP
2476
CHEN AND HWANG
TABLE 2
Composition of the basal diet
Weight gain, feed efficiency and survival rates of juvenile Penaeus monodoa fed setnipurified diets containing graded levels of riboflavin for 15 wk1
IngredientCasein
diet4573020050SO58338627462510 Supplemental free)1Amino (vitamin mixture2Potato acid starch1Fish oil3Soybean lecithin4Cholesterol1Glucosamine
riboflavin efficiency diet)081216204080Weight (mg/kg gaing/shiimp.83 ratiog
feed0.61 gain/g 0.82.42 ± 0.33.56 ± 0.24.36 ± 0.38.71 ± 0.14.91 ± 0.50.92 ± ±0.43Feed
HC11Sodium Buccinate5Sodium
citrate5Mineral mixture6Vitamin mixtureCellulose1Sodium alginate1Sodium hexametaphosphate8Amountg/kg 1Sigma Chemical, St. Louis, MO. 2Served as attractant and contained alanine-glycine-glutamic acid-betaine (1:1:1:2) (Sigma Chemical). 3Hanaqua Feed Co., Kaohsiung, Taiwan. 4Great Wall Enterprise Co., Tainan, Taiwan. 5Merck, Darmstadt, Germany. 6Mineral mixture (Merck) supplied the following minerals (g/kg diet): K2HPO4, 20; Ca3|PO4)2, 27.2; MgSO4-7H2O, 30.4; NaH2PO4-2H2O, 7.9. 7Provided the following amounts of vitamins (mg/kg diet): pamino benzoic acid, 100; biotin, 4; inositol, 4000,- nicotinic acid, 400; Ca-pantothenate, 600; thiamin HC1, 40; pyridoxine-HCl, 120; menadione, 40; all-rac-cc-tocopherol, 200; ß-carotene,96; vitamin B12, 0.8; cholecalciferol, 12; sodium ascorbate, 20000; folie acid, 8; choline chloride, 1200 (Hoffmann La Roche, Basel, Switzerland). 8Yakuri Chemicals, Osaka, Japan.
nutriture was indicated by the extent of the stimu lated glutathione reducÃ-ase activity caused by the addition of FAD in vitro and was expressed as an activity coefficient. The coefficient is defined as the diminution of absorbance of NADPH in the presence of FAD divided by the diminution of absorbance of NADPH without added FAD during 10 min (Nichoalds 1974). Growth (expressed as absolute weight gain per shrimp), survival rates, feed efficiency ratios (the ratio of the weight gain to amount of feed fed in each replicate), shrimp body riboflavin concentrations and ratios of carapace length and body length were ana lyzed for statistical significance (P < 0.05) by ANOVA. If collective statistical significance was indicated, in dividual differences between treatments were deter mined by the Duncan's new multiple range test (Snedecor and Cochran 1978). Simple regression analysis was used to examine the effects of riboflavin supplementation on hemolymph glutathione reducÃ-ase activity coefficients (Snedecor and Cochran 1978). The broken-line analysis technique (Robbins 1986) was used to estimate the riboflavin requirement based on body riboflavin concentrations.
rate%58.3
'initial
0.290.42 ± 15.666.7 ± 0.100.47 ± 5.945.8 ± 0.090.42 ± 21.245.8 ± 0.130.51 ± 15.662.5 ± 0.040.65 ± 0.045.8 ± 0.110.62 ± 6.354.2 ± ±0.12Survival ± 5.9
average weight was approximately
0.13 ±0.05 g. Values
are means (three replicates) ±SEM. ANOVA results indicate no significant differences (P > 0.05) in weight gain, feed efficiency ratio and survival of the shrimp over the dietary range.
RESULTS The groups of juvenile shrimp fed the experimental diets grew well (to at least 10-fold their initial weights) throughout the 15-wk feeding trial, although average survival was not satisfactory. After 15 wk, no significant differences (P > 0.05) in weight gain, feed efficiency and survival of shrimp were observed (Table 2) over the experimental range. Gross defi ciency symptoms were observed in the shrimp fed the unsupplemented diet (residual riboflavin level of 0.48 mg/kg diet). These symptoms were light coloration, irritability and protuberant cuticle at the joints of abdominal somites. The same deficiency signs were also observed, but with a much lower frequency, in shrimp fed diets supplemented with the lowest level (8 mg/kg diet) of riboflavin. The body riboflavin concentration, but not hemo lymph glutathione reducÃ-ase activily coefficienl, was shown io be a sensilive and specific indicator of ribo flavin staius of ihe shrimp (Table 3). Increased levels of supplemenied riboflavin yielded posiiive effeels (P < 0.05) on riboflavin relenlion in shrimp. The shrimp fed ihe unsupplemenied diet showed significantly lower concentrations (11.0 ±0.5 umol/g tissue) of body riboflavin than all riboflavin-supplemented groups. Body riboflavin concentration (13.0 ±0.3 umol/g tissue) was significantly greater in the group that was fed the diet supplemented with the lowest level of riboflavin (8 mg/kg diet). In a preliminary study, we found the body riboflavin concentrations of farm-raised subadult P. monodon to be generally be tween 12.7 and 13.2 umol/g tissue. The group fed the unsupplemented diet was the only group with a body riboflavin concentration lower than that range.
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TABLE 1
RIBOFLAVIN REQUIREMENT
OF PENAEID SHRIMP
2477
TABLE 3
Supplemental riboflavin (mg/kg diet)0
8 12 16 20 40 80Body
riboflavin1\unol/g tissue10.95 ±0.51a 12.99 ±0.32b 13.28 ±0.37b 13.42 ±0.24b 13.74 ±0.29bc 14.64 ±0.74cd 14.80 ±0.98d
(4) (4) (4) (3) (3) (3) (3)AC2_3
ratio1mm/mm0.372
1.24, 1.20, 1.25, 1.07, 1.00, 0.90,
1.48 1.60 1.33 1.39 1.50 1.07Head:body
0.385 0.394 0.394 0.394 0.398 0.395
±0.004a ±0.015b ±0.006b ±0.009b ±0.006b ±0.003b ±0.008b
(14) (16) (11) (11) (15) (11) (13)
Values are means ±SEM.Numbers in parentheses indicate number of pooled samples in body riboflavin measurement and number of shrimp for headibody ratio calculation. Values with different superscript are significantly different (P < 0.05, Duncan's new multiple range test). 2AC represents hemolymph glutathione reducÃ-aseactivity coefficient. Regression analysis indicated a significant linear relation between riboflavin supplements and AC (r2 = 0.367, P < 0.05). 3Unable to measure.
The hemolymph glutathione reductase activity coefficient decreased linearly (r2 = 0.37, P < 0.05) with increased riboflavin supplements (Table 3). The ac tivity coefficient continued to decrease with each increment in the dietary level of riboflavin, up to the maximum (80 mg/kg diet). This suggests that either the sensitivity of the coefficient with respect to ribo flavin status is poor or that the maximum dietary riboflavin level needed to obtain a normal activity coefficient was not employed. In either case, the results indicate that hemolymph glutathione reductase activity coefficient is not a good test for riboflavin status in shrimp. The activity coefficient in the group fed unsupplemented diet was not obtained (Table 3) because of unusual responses during the assay. In general, the glutathione reductase activity is measured by the de crease in absorbance of NADPH in the stoichiometric reaction. In the present study, however, the absor bance reading showed an increment instead of a de crease. Moreover, this phenomenon was observed in repeated tests in other riboflavin-deficient shrimp in other studies (data not shown). Thus no value was obtained for this dietary group. Riboflavin deficiency resulted in short-head dwarfism in the shrimp. The ratios of head (carapace) length to body length of the shrimp were significantly lower (P < 0.05) in the unsupplemented group than in all supplemented groups (Table 3). The deficient shrimp continued to grow in spite of the dwarfism. Their growth and survival were not significantly different from those of the supplemented groups in the 15-wk period (Table 2). Body storage of riboflavin was affected by the di etary level of this vitamin (Table 3). Based on the
broken-line analyses of the residual riboflavin concen trations in shrimp bodies, the dietary riboflavin con centration needed for juvenile P. monodon to max imize body riboflavin concentration was estimated to be 22.3 mg/kg dry diet. The regression line that fits the residual riboflavin level has a breakpoint at 22.3 mg riboflavin/kg diet. The relationship between residual riboflavin level (Y) and dietary riboflavin level IX] is Y = 4.25 + 0.056X for X < 22.3 and Y = 5.50 for X > 22.3. This analysis suggested that the body riboflavin concentration reached saturation when the dietary riboflavin level was >22.3 mg/kg.
DISCUSSION The minimum physiological requirement of ribo flavin by P. monodon for either growth or prevention of deficiency symptoms is likely to be lower than 22.3 mg/kg diet, the level that maximized tissue riboflavin concentration in the present study. Studies of ribo flavin requirement with rainbow trout indicated a growth requirement (dietary level needed to achieve maximal growth) of 3.6 mg/kg diet, a requirement of 4.6 mg/kg for liver flavin saturation and a re quirement of 6.6 mg/kg for saturation of spleen and head kidney with flavin compounds (Woodward 1985). Similarly, the riboflavin requirement of chicks is between 0.17 and 0.19 mg/MJ dietary gross energy for maximal growth and between 0.19 and 0.29 mg/ MJ for liver flavin saturation (Bro-Rasmussen 1958). In analogy with the above animal models, the growth requirement of P. monodon should be substantially less than 22.3 mg/kg diet. The observations that defi ciency symptoms occurred only in the groups fed
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Body riboflavin concentrations, hemolymph glutathione reducÃ-aseactivity coefficients and ratios of head (carapace) length and body length of juvenile Penaeus monodon fed semipurified diets containing graded levels of riboflavin for 15 wk
2478
CHEN AND HWANG
LITERATURE Amezaga, M. R. & Knox, growing rainbow trout, 87-98. Association of Official Methods of Analysis, Bamji, M. S. & Sharada,
CITED
D. (1990] Riboflavin requirements in onOncorhynchus mykiss. Aquaculture 88: Analytical Chemists (1984) Official 14th ed. AOAC, Washington, DC. D. (1972| Hepatic glutathione reductase
and riboflavin concentrations in experimental deficiency of thiamin and riboflavin in rats. J. Nutr. 102: 443-448. Bro-Rasmussen, F. J1958] The riboflavin requirement of animals and man and associated metabolic relations. Part II: Relation of requirement to the metabolism of protein and energy. Nutr. Abstr. Rev. 28: 369-386. Chen, H. Y., Wu, F. C. & Tang, S. Y. (1991] Thiamin requirement of juvenile shrimp (Penaeus monodon}. ]. Nutr. 121: 1984-1989. Cooperman, J. M. & Lopez, R. (1984) Riboflavin. In: Handbook of Vitamins (Machlin, L. J., éd.), pp. 299-327. Marcel Dekker, New York, NY. Cowey, C. B. (1976) Use of synthetic diets and biochemical criteria in the assessment of nutrient requirement of fish. J. Fish. Res. Board Can. 33: 1040-1045. Dali, W. & Moriarty, D.J.W. (1983) Functional aspects of nutrition and digestion. In: The Biology of Crustacea, Vol. 5 (Mantel, L. H., éd.),pp. 215-261. Academic Press, New York, NY. Glatzle, D., Korner, W. F., Christeller, S. & Wiss, O. (1970) Method for the detection of a biochemical riboflavin deficiency stimu lation of NADPHj-dependent glutathione reductase from human erythrocytes by FAD in vitro investigations on the vitamin 62 status in healthy people and geriatric patients. Int. J. Vitam. Nutr. Res. 40: 166-183. Glatzle, D., Weber, F. & Wiss, O. (1968) Enzymatic test for the detection of a riboflavin deficiency. Experimentia (Basel) 24: 11-22. Halver, J. E. (1985) Recent advances in vitamin nutrition and metabolism in fish. In: Nutrition and Feeding in Fish (Cowey, C. B., Mackie, A. M. & Bell, J. B., eds.), pp. 415-430. Academic Press, New York, NY. Halver, ]. E. (1989) The vitamins. In: Fish Nutrition (Halver, J. E., ed.), pp. 31-109. Academic Press, San Diego, CA. Hughes, S. G. & Rumsey, G. L. (1981) Riboflavin requirement of fingerling rainbow trout. Prog. Fish-Cult. 43: 167-172. Kanazawa, A. (1985) Prawn nutrition and microparticulated feeds. In: Prawn Feeds, pp. 1-51. American Soybean Association, Taipei, Taiwan. Lightner, D., Colvin, L., Brand, C. & Nonald, D. (1977) Black death, a disease syndrome of penaeid shrimp related to a dietary defi ciency of ascorbic acid. Proc. World Maricult. Soc. 8: 611-623. Maynard, L. A., Loosli, J. K., Hintz, H. F. & Warner, R. G. (1979) Animal Nutrition, pp. 283^55. McGraw-Hill, New York, NY. Murai, T. & Andrews, J. W. (1978) Riboflavin requirement of channel catfish fingerlings. J. Nutr. 108: 1512-1517. National Research Council (1977) Nutrient Requirements of Warmwater Fishes. National Academy of Sciences, Washington, DC. National Research Council (1981) Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC. Nichoalds, G. E. (1974) Assessment of status of riboflavin nutriture by assay of erythrocyte glutathione reductase activity. Clin. Chem. 20: 624-628. Pike, R. H. & Brown, M. L. (1984) Nutrition: An Integrated Ap proach, 3rd éd.,pp. 84-136. lohn Wiley and Sons, New York, NY. Robbins, K. R. (1986) A Method, SAS Program, and Example for Fitting the Broken-Line to Growth Data. University of Ten nessee Agricultural Experiment Station Research Report, University of Tennessee, Knoxville, TN. Snedecor, G. W. & Cochran, W. G. (1978) Statistical Methods, pp. 172-257. Iowa State University Press, Ames, IA. Woodward, B. (1983) Sensitivity of hepatic D-amino acid oxidase and glutathione reductase to the riboflavin status of the rainbow trout (Salmo gairdneri). Aquaculture 34: 193-201. Woodward, B. (1985) Riboflavin requirement for growth, tissue saturation and maximal flavin-dependent enzyme activity in young rainbow trout (Salmo gairdneri} at two temperatures. J. Nutr. 115: 78-84.
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diets with supplemental riboflavin levels of 1.3 represent a high risk (Cooperman and Lopez 1984). In the present study, shrimp fed either the unsupplemented or the lowest supplemented diet (8 mg/kg diet) showed signs of deficiency, including light color ation, irritability, protuberant intersomite cuticle and short-head dwarfism. Riboflavin deficiency syn dromes in fish include many abnormal signs related to vision, dark coloration, incoordination, striated constriction of abdominal wall, poor appetite anemia and poor growth (Halver 1989). It is very difficult to speculate on possible reasons for the deficiency signs in the shrimp, because no histological examinations on the shrimp were conducted. Most of the vitaminrelated studies on shrimp concern the effects of sup plementation on growth, feed efficiency, survival and biochemical indices. Information on vitamin defi ciencies in shrimp is scarce. The only welldocumented vitamin deficiency in shrimp species is the black death syndrome related to vitamin C defi ciency in penaeid shrimp (Lightner et al. 1977).
Requirements
and Interactions
HOÃœHG-YÃœHGCHEN2 AND GLORIA HWANG Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung, Taiwan 804, Republic of China growth (Halver 1985 and 1989). Riboflavin deficiency in shrimp species is unknown. The requirements for riboflavin have been determined only for larval Penaeus japonicus, based on survival results (Kanazawa 1985). A requirement of 80 mg/kg diet was suggested. The riboflavin requirements of coldwater salmonids (Halver 1985 and 1989) and warmwater channel catfish (Murai and Andrews 1978) have been reported to be 20-30 and 9 mg/kg dry diet, respec tively. The allowance for riboflavin recommended by the National Research Council (1977) in complete diets for warmwater herbivorous fish is 20 mg/kg dry diet; that required for coldwater fish is 0.15 to 0.20 mg/kg body wt per day (NRC 1981). These values are very low when compared with the only reported re quirement level (80 mg/kg diet) for larval P. japonicus (Kanazawa 1985). Cowey (1976) suggested that the nutrient require ments of cultured fish be established by biochemical measurements, such as a particular enzyme's activity. The measurements of specific nutrient-dependent bio chemical functions are superior to growth perfor mance or simple measurement of tissue nutrient level for discerning the true requirement. Hughes and Rumsey (1981) found that the measurement of the activation coefficient (ratio of activity following preincubation with FAD to basal activity) of erythrocyte glutathione reducÃ-ase (EC 1.6.4.2) is a sensitive and specific indicator of the riboflavin status for rainbow trout as shown in rats (Bamji and Sharada 1972, Glatzle et al. 1968). Because no quantitative riboflavin requirement has been reported for P. monodon (the most widely cul tured shrimp species), the following study was
ABSTRACT The nboflavin requirements of marine shrimp (Penaeus monodon) were evaluated in a 15-wk feeding trial. Juvenile shrimp (initial mean weight, 0.13 ±0.05 g) were fed purified diets containing seven levels (0, 8, 12, 16, 20, 40 and 80 mg/kg diet) of supplemental nboflavin. There were no significant differ ences in weight gains, feed efficiency ratios and survival of shrimp over the dietary riboflavin range. The riboflavin concentrations in shrimp bodies increased with the in creasing vitamin supplementation. Hemolymph (blood) glutathione reducÃ-ase activity coefficient was not a sen sitive and specific indicator of riboflavin status of the shrimp. The dietary riboflavin level required for P. monodon was found to be 22.3 mg/kg diet, based on the broken-line model analysis of body riboflavin concen trations. Shrimp fed unsupplemented diet (riboflavin concentration of 0.48 mg/kg diet) for 15 wk showed signs of deficiency: light coloration, irritability, pro tuberant cuticle at intersomites and short-head dwarfism. J. Nutr. 122: 2474-2478, 1992. INDEXING KEY WORDS:
•riboflavin requirement •riboflauin deficiency •glutathione reducÃ-ase
penaeid shrimp
Riboflavin is a precursor of FAD and FMN, which are vital nutrients for all animals, including crusta ceans (DalÃ-and Moriarty 1983). FAD and FMN are cofactors in many enzyme systems involved in oxi dation-reduction reactions, tissue respiration and hydrogen transport. Riboflavin plays an important role in the respiration of poorly vascularized tissues and in the retinal pigment during light adaptation (Pike and Brown 1984). Many deficiency symptoms of this vitamin, such as changes in the skin, eye and nervous system, have been reported in humans (Pike and Brown 1984), farm animals (Maynard et al. 1979) and fishes (Halver 1985). The deficiency signs in fishes are usually species specific, and the only common signs among species are anorexia and poor 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
1Supported by a grant (NSC80-0409-B110-01) from the National Science Council of the Republic of China. •^To whom correspondence should be addressed.
Received 19 May 1992. Accepted 6 August 1992. 2474
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Estimation of the Dietary Riboflavin Required to Maximize Tissue Riboflavin Concentration in Juvenile Shrimp (Penaeus monodori)1
RIBOFLAVIN REQUIREMENT
MATERIALS AND METHODS Juvenile P. monodon from a single spawner were obtained from a commercial hatchery (Won-Zai Hatchery, Lin-Yuan, Kaohsiung County, Taiwan). They were acclimated to experimental conditions and semipurified diets for 3 wk before the experiment. A total of 168 apparently healthy shrimp (initial mean weight, 0.13 ±0.05 g) were divided into seven ex perimental groups with three replicates of eight shrimp each. Each of the 21 replicates was randomly assigned to a 60 x 60 x 46.6 cm rectangular glass aquarium fitted with under-gravel filters. The filter bed consisted of crushed oyster shell and sand. Each aquarium was divided into eight equal compartments by clear plastic plates. Holes with a diameter of 4 mm allowed exchange of water among neighboring com partments. Each shrimp was individually housed in one of the compartments. Aquariums were placed under fluorescent light with a light cycle of 12 h light (0700-1900 h):12 h dark. Natural sea water drawn through a beach sand filter was first passed through a polyester cartridge and activated carbon filter and then through a quartz UV sterilizer (Sanitron, Atlantic Ultraviolet Co., Bayshore, NY). Deionized water was used to adjust salinity to be approximately 28-29 g/L. Approxi mately one tenth of the sea water in each aquarium was replaced every other day. Temperature was not controlled but monitored daily. The water temper ature, dissolved oxygen, pH and ammonia were 25-29°C, 7.5-8.0 mg/L, 7.5-8.0 and 0-0.5 mg/L, respectively, during the trial period. These environ mental factors are considered to be not detrimental to shrimp growth. The shrimp were fed three times daily (0800, 1700 and 2300 h) for 15 wk. Feeding level was 10% of shrimp body weight during the first 3 wk and was adjusted thereafter according to the feeding response of the shrimp. Uneaten food and exuviae were re moved each morning before feeding. The shrimp were observed daily for mortality and unusual behavior or morphological changes. All shrimp were weighed every 3 wk in morning hours before the first feeding. Shrimp were netted, blotted with dry gauze cloth and weighed individually to the nearest 0.01 g. Each shrimp was placed between folded gauze during weighing to prevent sudden jumping. No anesthesia was used.
Seven purified experimental diets were formulated to provide graded levels of supplemental riboflavin. The basic vitamin-free diet (Table 1) was based on a formula for penaeid shrimp (Chen et al. 1991). The basal diet contained 15.08 MJ/kg diet of gross energy and 40% crude protein and had a protein:energy ratio of 26.53 mg protein per kj. Riboflavin was added to the test diets at the expense of small amounts of cellulose to provide concentrations of 0, 8, 12, 16, 20, 40 and 80 mg/kg. All vitamins used in the study were supplied by Hoffmann La Roche (Basel, Switzerland). The diets were prepared under reduced-light condi tions because of the light sensitivity of riboflavin. The diets were processed into pellets (3 mm in di ameter by 3 mm in length) and were freeze-dried. Each diet was sealed in opaque plastic bags and stored at -20°C until fed. The riboflavin concentrations of the seven diets were chemically determined (AOAC 1984) to be 0.48 (unsupplemented), 7.39 (8 mg/kg), 11.35 (12 mg/kg), 14.64 (16 mg/kg), 19.08 (20 mg/kg), 40.00 (40 mg/kg) and 76.96 (80 mg/kg) mg/kg diet, respectively. At the end of the feeding trial, blood (hemolymph) samples were withdrawn from the ostium of the heart of each shrimp by a 1-mL syringe rinsed with 80 mmol/L dipotassium EDTA solution. Blood samples from within the same dietary group were pooled into two samples because of their small volumes and frozen in liquid nitrogen until the assay of glutathione reducÃ-ase was conducted. The remaining shrimp bodies were pooled into ihree or four samples within each treatment and were freeze-dried, pul verized and used to determine the riboflavin concen tration according to the method of AOAC (1984), using a fluorospectrophotometer (F-3000, Hitachi In struments, Tokyo, Japan). Before the shrimp were frozen, body and carapace lengths of each shrimp were measured to the nearest 1 mm. Body length is the length between the base of the rostrum (rostral spine) and the end of tail fan (uropods). Carapace length represents the head length of a shrimp and is the distance between the base of rostrum and the end of carapace. The assay and calculation procedures of hemo lymph glutathione reducÃ-ase followed the method of Glalzle el al. (1970) and Nichoalds (1974). Because ihe cell volume of shrimp hemolymph is much smaller than mammalian blood, whole hemolymph was used in assay. Before analysis, the frozen blood samples were thawed and then ultra-sonicated for 5 min to erupt blood cell membranes. The supernatants de rived from a 15-min centrifugation (Model Himac, RPR20-3 rotor, Hitachi Instruments) at 0°C and 14,500 x g were used as the enzyme solution. Glutathione reducÃ-ase activity was measured in the presence and absence of in vitro FAD by moniioring ihe oxidation of NADPH at 340 nm using a thermoslaÃ--conÃ-rolled (37°C)specÃ-rophoÃ-omeler (U-2000, Hilachi
Inslrumenls).
The
sialus
of
riboflavin
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designed to estimate the requirement for juvenile P. monodon. Weight gains, feed efficiency ratios, sur vival rates and biochemical measurements (including body riboflavin concentration and hemolymph glutathione reducÃ-ase activity coefficient) were used simultaneously to assess the riboflavin status and optimal requirement of the shrimp.
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OF PENAEID SHRIMP
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CHEN AND HWANG
TABLE 2
Composition of the basal diet
Weight gain, feed efficiency and survival rates of juvenile Penaeus monodoa fed setnipurified diets containing graded levels of riboflavin for 15 wk1
IngredientCasein
diet4573020050SO58338627462510 Supplemental free)1Amino (vitamin mixture2Potato acid starch1Fish oil3Soybean lecithin4Cholesterol1Glucosamine
riboflavin efficiency diet)081216204080Weight (mg/kg gaing/shiimp.83 ratiog
feed0.61 gain/g 0.82.42 ± 0.33.56 ± 0.24.36 ± 0.38.71 ± 0.14.91 ± 0.50.92 ± ±0.43Feed
HC11Sodium Buccinate5Sodium
citrate5Mineral mixture6Vitamin mixtureCellulose1Sodium alginate1Sodium hexametaphosphate8Amountg/kg 1Sigma Chemical, St. Louis, MO. 2Served as attractant and contained alanine-glycine-glutamic acid-betaine (1:1:1:2) (Sigma Chemical). 3Hanaqua Feed Co., Kaohsiung, Taiwan. 4Great Wall Enterprise Co., Tainan, Taiwan. 5Merck, Darmstadt, Germany. 6Mineral mixture (Merck) supplied the following minerals (g/kg diet): K2HPO4, 20; Ca3|PO4)2, 27.2; MgSO4-7H2O, 30.4; NaH2PO4-2H2O, 7.9. 7Provided the following amounts of vitamins (mg/kg diet): pamino benzoic acid, 100; biotin, 4; inositol, 4000,- nicotinic acid, 400; Ca-pantothenate, 600; thiamin HC1, 40; pyridoxine-HCl, 120; menadione, 40; all-rac-cc-tocopherol, 200; ß-carotene,96; vitamin B12, 0.8; cholecalciferol, 12; sodium ascorbate, 20000; folie acid, 8; choline chloride, 1200 (Hoffmann La Roche, Basel, Switzerland). 8Yakuri Chemicals, Osaka, Japan.
nutriture was indicated by the extent of the stimu lated glutathione reducÃ-ase activity caused by the addition of FAD in vitro and was expressed as an activity coefficient. The coefficient is defined as the diminution of absorbance of NADPH in the presence of FAD divided by the diminution of absorbance of NADPH without added FAD during 10 min (Nichoalds 1974). Growth (expressed as absolute weight gain per shrimp), survival rates, feed efficiency ratios (the ratio of the weight gain to amount of feed fed in each replicate), shrimp body riboflavin concentrations and ratios of carapace length and body length were ana lyzed for statistical significance (P < 0.05) by ANOVA. If collective statistical significance was indicated, in dividual differences between treatments were deter mined by the Duncan's new multiple range test (Snedecor and Cochran 1978). Simple regression analysis was used to examine the effects of riboflavin supplementation on hemolymph glutathione reducÃ-ase activity coefficients (Snedecor and Cochran 1978). The broken-line analysis technique (Robbins 1986) was used to estimate the riboflavin requirement based on body riboflavin concentrations.
rate%58.3
'initial
0.290.42 ± 15.666.7 ± 0.100.47 ± 5.945.8 ± 0.090.42 ± 21.245.8 ± 0.130.51 ± 15.662.5 ± 0.040.65 ± 0.045.8 ± 0.110.62 ± 6.354.2 ± ±0.12Survival ± 5.9
average weight was approximately
0.13 ±0.05 g. Values
are means (three replicates) ±SEM. ANOVA results indicate no significant differences (P > 0.05) in weight gain, feed efficiency ratio and survival of the shrimp over the dietary range.
RESULTS The groups of juvenile shrimp fed the experimental diets grew well (to at least 10-fold their initial weights) throughout the 15-wk feeding trial, although average survival was not satisfactory. After 15 wk, no significant differences (P > 0.05) in weight gain, feed efficiency and survival of shrimp were observed (Table 2) over the experimental range. Gross defi ciency symptoms were observed in the shrimp fed the unsupplemented diet (residual riboflavin level of 0.48 mg/kg diet). These symptoms were light coloration, irritability and protuberant cuticle at the joints of abdominal somites. The same deficiency signs were also observed, but with a much lower frequency, in shrimp fed diets supplemented with the lowest level (8 mg/kg diet) of riboflavin. The body riboflavin concentration, but not hemo lymph glutathione reducÃ-ase activily coefficienl, was shown io be a sensilive and specific indicator of ribo flavin staius of ihe shrimp (Table 3). Increased levels of supplemenied riboflavin yielded posiiive effeels (P < 0.05) on riboflavin relenlion in shrimp. The shrimp fed ihe unsupplemenied diet showed significantly lower concentrations (11.0 ±0.5 umol/g tissue) of body riboflavin than all riboflavin-supplemented groups. Body riboflavin concentration (13.0 ±0.3 umol/g tissue) was significantly greater in the group that was fed the diet supplemented with the lowest level of riboflavin (8 mg/kg diet). In a preliminary study, we found the body riboflavin concentrations of farm-raised subadult P. monodon to be generally be tween 12.7 and 13.2 umol/g tissue. The group fed the unsupplemented diet was the only group with a body riboflavin concentration lower than that range.
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TABLE 1
RIBOFLAVIN REQUIREMENT
OF PENAEID SHRIMP
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TABLE 3
Supplemental riboflavin (mg/kg diet)0
8 12 16 20 40 80Body
riboflavin1\unol/g tissue10.95 ±0.51a 12.99 ±0.32b 13.28 ±0.37b 13.42 ±0.24b 13.74 ±0.29bc 14.64 ±0.74cd 14.80 ±0.98d
(4) (4) (4) (3) (3) (3) (3)AC2_3
ratio1mm/mm0.372
1.24, 1.20, 1.25, 1.07, 1.00, 0.90,
1.48 1.60 1.33 1.39 1.50 1.07Head:body
0.385 0.394 0.394 0.394 0.398 0.395
±0.004a ±0.015b ±0.006b ±0.009b ±0.006b ±0.003b ±0.008b
(14) (16) (11) (11) (15) (11) (13)
Values are means ±SEM.Numbers in parentheses indicate number of pooled samples in body riboflavin measurement and number of shrimp for headibody ratio calculation. Values with different superscript are significantly different (P < 0.05, Duncan's new multiple range test). 2AC represents hemolymph glutathione reducÃ-aseactivity coefficient. Regression analysis indicated a significant linear relation between riboflavin supplements and AC (r2 = 0.367, P < 0.05). 3Unable to measure.
The hemolymph glutathione reductase activity coefficient decreased linearly (r2 = 0.37, P < 0.05) with increased riboflavin supplements (Table 3). The ac tivity coefficient continued to decrease with each increment in the dietary level of riboflavin, up to the maximum (80 mg/kg diet). This suggests that either the sensitivity of the coefficient with respect to ribo flavin status is poor or that the maximum dietary riboflavin level needed to obtain a normal activity coefficient was not employed. In either case, the results indicate that hemolymph glutathione reductase activity coefficient is not a good test for riboflavin status in shrimp. The activity coefficient in the group fed unsupplemented diet was not obtained (Table 3) because of unusual responses during the assay. In general, the glutathione reductase activity is measured by the de crease in absorbance of NADPH in the stoichiometric reaction. In the present study, however, the absor bance reading showed an increment instead of a de crease. Moreover, this phenomenon was observed in repeated tests in other riboflavin-deficient shrimp in other studies (data not shown). Thus no value was obtained for this dietary group. Riboflavin deficiency resulted in short-head dwarfism in the shrimp. The ratios of head (carapace) length to body length of the shrimp were significantly lower (P < 0.05) in the unsupplemented group than in all supplemented groups (Table 3). The deficient shrimp continued to grow in spite of the dwarfism. Their growth and survival were not significantly different from those of the supplemented groups in the 15-wk period (Table 2). Body storage of riboflavin was affected by the di etary level of this vitamin (Table 3). Based on the
broken-line analyses of the residual riboflavin concen trations in shrimp bodies, the dietary riboflavin con centration needed for juvenile P. monodon to max imize body riboflavin concentration was estimated to be 22.3 mg/kg dry diet. The regression line that fits the residual riboflavin level has a breakpoint at 22.3 mg riboflavin/kg diet. The relationship between residual riboflavin level (Y) and dietary riboflavin level IX] is Y = 4.25 + 0.056X for X < 22.3 and Y = 5.50 for X > 22.3. This analysis suggested that the body riboflavin concentration reached saturation when the dietary riboflavin level was >22.3 mg/kg.
DISCUSSION The minimum physiological requirement of ribo flavin by P. monodon for either growth or prevention of deficiency symptoms is likely to be lower than 22.3 mg/kg diet, the level that maximized tissue riboflavin concentration in the present study. Studies of ribo flavin requirement with rainbow trout indicated a growth requirement (dietary level needed to achieve maximal growth) of 3.6 mg/kg diet, a requirement of 4.6 mg/kg for liver flavin saturation and a re quirement of 6.6 mg/kg for saturation of spleen and head kidney with flavin compounds (Woodward 1985). Similarly, the riboflavin requirement of chicks is between 0.17 and 0.19 mg/MJ dietary gross energy for maximal growth and between 0.19 and 0.29 mg/ MJ for liver flavin saturation (Bro-Rasmussen 1958). In analogy with the above animal models, the growth requirement of P. monodon should be substantially less than 22.3 mg/kg diet. The observations that defi ciency symptoms occurred only in the groups fed
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Body riboflavin concentrations, hemolymph glutathione reducÃ-aseactivity coefficients and ratios of head (carapace) length and body length of juvenile Penaeus monodon fed semipurified diets containing graded levels of riboflavin for 15 wk
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LITERATURE Amezaga, M. R. & Knox, growing rainbow trout, 87-98. Association of Official Methods of Analysis, Bamji, M. S. & Sharada,
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D. (1990] Riboflavin requirements in onOncorhynchus mykiss. Aquaculture 88: Analytical Chemists (1984) Official 14th ed. AOAC, Washington, DC. D. (1972| Hepatic glutathione reductase
and riboflavin concentrations in experimental deficiency of thiamin and riboflavin in rats. J. Nutr. 102: 443-448. Bro-Rasmussen, F. J1958] The riboflavin requirement of animals and man and associated metabolic relations. Part II: Relation of requirement to the metabolism of protein and energy. Nutr. Abstr. Rev. 28: 369-386. Chen, H. Y., Wu, F. C. & Tang, S. Y. (1991] Thiamin requirement of juvenile shrimp (Penaeus monodon}. ]. Nutr. 121: 1984-1989. Cooperman, J. M. & Lopez, R. (1984) Riboflavin. In: Handbook of Vitamins (Machlin, L. J., éd.), pp. 299-327. Marcel Dekker, New York, NY. Cowey, C. B. (1976) Use of synthetic diets and biochemical criteria in the assessment of nutrient requirement of fish. J. Fish. Res. Board Can. 33: 1040-1045. Dali, W. & Moriarty, D.J.W. (1983) Functional aspects of nutrition and digestion. In: The Biology of Crustacea, Vol. 5 (Mantel, L. H., éd.),pp. 215-261. Academic Press, New York, NY. Glatzle, D., Korner, W. F., Christeller, S. & Wiss, O. (1970) Method for the detection of a biochemical riboflavin deficiency stimu lation of NADPHj-dependent glutathione reductase from human erythrocytes by FAD in vitro investigations on the vitamin 62 status in healthy people and geriatric patients. Int. J. Vitam. Nutr. Res. 40: 166-183. Glatzle, D., Weber, F. & Wiss, O. (1968) Enzymatic test for the detection of a riboflavin deficiency. Experimentia (Basel) 24: 11-22. Halver, J. E. (1985) Recent advances in vitamin nutrition and metabolism in fish. In: Nutrition and Feeding in Fish (Cowey, C. B., Mackie, A. M. & Bell, J. B., eds.), pp. 415-430. Academic Press, New York, NY. Halver, ]. E. (1989) The vitamins. In: Fish Nutrition (Halver, J. E., ed.), pp. 31-109. Academic Press, San Diego, CA. Hughes, S. G. & Rumsey, G. L. (1981) Riboflavin requirement of fingerling rainbow trout. Prog. Fish-Cult. 43: 167-172. Kanazawa, A. (1985) Prawn nutrition and microparticulated feeds. In: Prawn Feeds, pp. 1-51. American Soybean Association, Taipei, Taiwan. Lightner, D., Colvin, L., Brand, C. & Nonald, D. (1977) Black death, a disease syndrome of penaeid shrimp related to a dietary defi ciency of ascorbic acid. Proc. World Maricult. Soc. 8: 611-623. Maynard, L. A., Loosli, J. K., Hintz, H. F. & Warner, R. G. (1979) Animal Nutrition, pp. 283^55. McGraw-Hill, New York, NY. Murai, T. & Andrews, J. W. (1978) Riboflavin requirement of channel catfish fingerlings. J. Nutr. 108: 1512-1517. National Research Council (1977) Nutrient Requirements of Warmwater Fishes. National Academy of Sciences, Washington, DC. National Research Council (1981) Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC. Nichoalds, G. E. (1974) Assessment of status of riboflavin nutriture by assay of erythrocyte glutathione reductase activity. Clin. Chem. 20: 624-628. Pike, R. H. & Brown, M. L. (1984) Nutrition: An Integrated Ap proach, 3rd éd.,pp. 84-136. lohn Wiley and Sons, New York, NY. Robbins, K. R. (1986) A Method, SAS Program, and Example for Fitting the Broken-Line to Growth Data. University of Ten nessee Agricultural Experiment Station Research Report, University of Tennessee, Knoxville, TN. Snedecor, G. W. & Cochran, W. G. (1978) Statistical Methods, pp. 172-257. Iowa State University Press, Ames, IA. Woodward, B. (1983) Sensitivity of hepatic D-amino acid oxidase and glutathione reductase to the riboflavin status of the rainbow trout (Salmo gairdneri). Aquaculture 34: 193-201. Woodward, B. (1985) Riboflavin requirement for growth, tissue saturation and maximal flavin-dependent enzyme activity in young rainbow trout (Salmo gairdneri} at two temperatures. J. Nutr. 115: 78-84.
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diets with supplemental riboflavin levels of 1.3 represent a high risk (Cooperman and Lopez 1984). In the present study, shrimp fed either the unsupplemented or the lowest supplemented diet (8 mg/kg diet) showed signs of deficiency, including light color ation, irritability, protuberant intersomite cuticle and short-head dwarfism. Riboflavin deficiency syn dromes in fish include many abnormal signs related to vision, dark coloration, incoordination, striated constriction of abdominal wall, poor appetite anemia and poor growth (Halver 1989). It is very difficult to speculate on possible reasons for the deficiency signs in the shrimp, because no histological examinations on the shrimp were conducted. Most of the vitaminrelated studies on shrimp concern the effects of sup plementation on growth, feed efficiency, survival and biochemical indices. Information on vitamin defi ciencies in shrimp is scarce. The only welldocumented vitamin deficiency in shrimp species is the black death syndrome related to vitamin C defi ciency in penaeid shrimp (Lightner et al. 1977).
