Nutrient Requirements

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1 Nutrient Requirements Estimation of the Dietary Requirement of Juvenile Grass Shrimp, Penaeus monodon 1 Shi-Yen Shiau 2 and Ying Chen Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China ABSTRACT Two growth experiments were conducted to estimate the minimal dietary vitamin A requirement for juvenile grass shrimp, Penaeus monodon. In expt. 1, purified diets containing 0, 1,500, 3,000, 15,000, 30,000, 45,000 and 60,000 retinol equivalent (RE)/kg (i.e., 0, 5,000, 10,000, 50,000, 100,000, 150,000, 200,000 IU/kg) of supplemental vitamin A (retinyl acetate) were fed to P. monodon (mean initial weight g) for 8 wk. In expt. 2, diets with 0, 600, 1,200, 1,800, 2,400, 3,000, 3,600, and 4,500 (i.e., 0, 2,000, 4,000, 6,000, 8,000, 10,000, 12,000, 15,000 IU/kg) of supplemental vitamin A were fed to the shrimp (mean weight g) for 6 wk. The basal unsupplemented diet contained 54 RE vitamin A/kg, and supplemental levels were confirmed by analysis. Each diet was fed to three replicate groups of shrimp. In expt. 1, shrimp fed diets supplemented with 300 RE vitamin A/kg had significantly greater weight gain (P 0.05) than those fed the unsupplemented control diet and diets supplemented with 30,000 RE vitamin A/kg. Survival rate was higher in shrimp fed diets supplemented with 1,500 30,000 RE vitamin A/kg than shrimp fed the control diet. Highest blood triglyceride concentration and body lipid concentration were in shrimp fed diets supplemented with 45,000 and 60,000 RE vitamin A/kg, respectively. Eye vitamin A concentration and hepatopancreatic total lipid concentration in shrimp generally increased as dietary vitamin A supplementation increased. In expt. 2, feed efficiency was highest in shrimp fed diets supplemented with 2,400, 3,000, 3,600 and 4,500 RE vitamin A/kg, followed by shrimp fed diets with 600 and 1,200 RE vitamin A/kg and finally the unsupplemented control group. Shrimp fed diets supplemented with vitamin A had significantly higher survival percentages than those fed the unsupplemented control diet. Weight gain percentage of the shrimp analyzed by broken-line regression indicated that the minimal dietary vitamin A concentration in growing P. monodon is 2,511 ( 8,400 IU/kg). J. Nutr. 130: 90 94, KEY WORDS: vitamin A shrimp Penaeus monodon is important in a number of physiological processes such as vision, reproduction and in the maintenance of differentiated epithelia in warm-blood vertebrates. The dietary vitamin A requirement for land animals has been studied extensively. Aquatic organisms are exposed to environmental factors different from those that affect the land animals, namely temperature, pressure, salt concentration, availability of oxygen and the presence and concentrations of pollutants. Therefore, nutrient requirements of aquatic species may differ from those of the land animals. The quantitative requirement for growth has been studied in only a few species of fish. For example, 2,500 3,500, 2,000 4,000 and 4,000 20,000 IU 3 vitamin A/kg diet were reported as requirements for rainbow trout (Kitamura et al. 1967), guppy (Shim and Tan 1990) and common carp (Aoe et al. 1968, Suhenda and Djajadiredja 1985), respectively. These values for teleost species are somewhat different from those established for homeotherms such as chickens and pigs in which a requirement of 1,500 and 2,000 IU/kg, respectively, were reported (NRC 1984 and 1988). 1 Supported by a grant from the National Science Council of the Republic of China, grant number NSC B To whom correspondence and reprint request should be addressed. 3 Abbreviations used: FE, feed efficiency; IU, international unit; PER, protein efficiency ratio; RE, retinol equivalent. For crustaceans, only limited qualitative data concerning vitamin requirements are available. He et al. (1992) and Chen and Li (1994) reported that penaeid shrimp, Penaeus vannamei and P. chinensis, required dietary vitamin A for optimal growth. However, quantitative information on the requirements of vitamin A for penaeid shrimp is lacking. The purpose of this study was to define the minimal dietary vitamin A requirement of juvenile grass shrimp P. monodon, the most widely cultured shrimp worldwide. MATERIALS AND METHODS Diet preparation. Experimental diet formulation is given in Table 1. The formulation is similar to that of Shiau and Hwang (1994), which has been shown to be adequate for P. monodon. Vitamin-free casein (Sigma Chemical, St. Louis, MO), cod liver oil (Scott and Bowne, London, United Kingdom) and corn starch (Sigma Chemical) were used as dietary protein, lipid and carbohydrate sources, respectively. A mixture of amino acids [including glycine, L-alanine, L-glutamate and betaine (Sigma Chemical)] was included in the diets as an attractant (Shiau and Chin 1998). Sodium alginate (Hayashi Pure Chemical, Osaka, Japan) was used as a binder. This constituent is not digested by the shrimp, and it is essential for shrimp diet as it holds the ingredients in a stable pellet form in water. The vitamin mixture was similar to that used by Shiau and Hsu (1999), except that it did not contain vitamin A. In expt. 1, vitamin A /00 $ American Society for Nutritional Sciences. Manuscript received 30 August Initial review completed 10 September Revision accepted 16 September

2 VITAMIN A REQUIREMENT OF PENAEID SHRIMP 91 Ingredient TABLE 1 Composition of the basal diet1 Amount g/kg diet Vitamin-free casein (89% crude protein) 457 Corn starch 200 Cod liver oil 50 Amino acid mixture2 30 Glucosamine HCl 8 Sodium citrate 3 Sodium succinate 3 Sodium alginate 25 Sodium hexametaphosphate 10 Cholesterol 5 Vitamin mix3 27 Mineral mix4 86 -Cellulose 96 1 Proximate analysis of basal diet: moisture, 10.84%; ash, 7.41%; crude protein, 41.65%; ether extract, 5.17%; crude fiber, 9.37%. 2 Amino acid mixture contained the following (g/kg diet): glycine, 6; L-alanine, 6; L-glutamic acid, 6; betaine, Vitamin mixture supplied the following (mg/g mixture): myo-inositol, 100; pyridoxine-hcl, 1.5; riboflavin, ; nicotinic acid, ; thiamin-hcl, ; menadione, ; all-rac- -tocopherol, 10; vitamin B-12, ; cholecaliferol, ; folic acid, ; choline HCl, 200; ascorbic acid (L-ascorbyl-2-monophosphate-Mg), ; biotin, ; d-ca pantothenate, All ingredients were diluted with -cellulose to 1 g. 4 Mineral mixture supplied the following (mg/kg diet): K 2 HPO 4, 100; NaH 2 PO 4, 215; Ca(H 2 PO 4 ) 2 H 2 O, 265; CaCO 3, 105; Ca-lactate, 165; KCl, 28; KI, 0.23; MgSO 4 7H 2 O, 100; Fe-citrate, 10; CuCl 2 2H 2 O, 0.15; AlCl 3 6H 2 O, 0.24; CoCl 2 6H 2 O, 1.4; ZnSO 4 7H 2 O, 4.76; MnSO 4 H 2 O, 1.07; -cellulose, (retinyl acetate) (Sigma Chemical) was added to the test diets at the expense of small amounts of cellulose to provide concentrations of 0, 1,500, 3,000, 15,000, 30,000, 45,000 and 60,000 retinol equivalent (RE)/kg diet (0, 5,000, 10,000, 50,000, 100,000, 150,000, 200,000 IU/kg). In expt. 2, retinyl acetate was added at 0, 600, 1,200, 1,800, 2,400, 3,000, 3,600 and 4,500 diet (0, 2,000, 4,000, 6,000, 8,000, 10,000, 12,000, 15,000 IU/kg). The vitamin A concentrations of the experimental diets were determined by HPLC (Larry et al. 1991) to be 54 (unsupplemented control), 1,380 (1,500 ), 2,856 (3,000 ), 13,466 (15,000 ), 28,246 (30,000 ), 39,653 (45,000 ) and 55,978 (60,000 ) in expt. 1 and 54 (unsupplemented), 572 (600 ), 1,166 (1,200 ), 1,747 (1,800 ), 2,338 (2,400 ), 2,909 (3,000 ), 3,521 (3,600 ) and 4,346 (4,500 ) in expt. 2. The diets were prepared and stored as previously described (Shiau and Yu 1998). Experimental procedure. Juvenile P. monodon were supplied by the Tungkang Marine Laboratory (Tungkang, Pingtung, Taiwan). Upon arrival, they were acclimated to laboratory conditions for 2 wk in a plastic tank [74 cm (w) 95 cm (l) 45 cm (h), 290 L] and fed a commercial diet (Yung-Hsien, Taipei, Taiwan). The laboratory conditions during the acclimation period were similar to those at the initiation of the experiment. The proximate composition (g/100 g) of the commercial diet was as follows: moisture, 10.74; crude protein (N 6.25), 40.90; lipid, 7.19; and ash, Shrimp were then fed the basal diet without vitamin A supplementation for 7 d prior to the start of the growth trial. At the beginning of the experiment, 16 shrimp [mean weight: g (SD)] in expt. 1 and 20 shrimp [mean weight: g (SD)] in expt. 2 were stocked in each experimental aquarium ( cm 3, 125 L). Each experimental diet was fed to three groups of shrimp. Each aquarium received continuous aeration. In each aquarium, impurities from uneaten feed and fecal pellets in the water were removed by siphoning every day, and 75% of the water was exchanged at 2, 4 and 6 wk in expt. 1 and 2, and 4 wk in expt. 2 to maintain water quality. Dissolved oxygen concentration was monitored weekly and maintained at 7.5 mg of O 2 /L throughout the experimental period. Water temperature ranged from 26 to 28 C, ph of , and salinity of g/kg. The ammonia level was about 1.5 mg/kg. The water quality variables were recorded weekly. A photoperiod of 12 h light, 12 h dark ( h) was used. Shrimp were fed their respective diets at a rate of 8 g/100 g body weight. This daily ration was subdivided into two equal feedings given at 0830 and 1730 h. Shrimp were weighed biweekly, and the daily ration was adjusted accordingly. Any dead shrimp were not replaced during the experiment. The shrimp were fed the test diets for 8 wk in expt. 1, and 6 wk in expt. 2. Throughout the experiment, the shrimp were maintained and used under humane conditions. At the end of the feeding trial, the shrimp were weighed. Growth was measured by the percentage of body weight gain [100(final body wt-initial body wt)/initial body wt], feed efficiency (FE) and protein efficiency ratio (PER) were calculated as described previously (Shiau and Chou 1991, Shiau and Liu 1994). After the final weighing, three shrimp were randomly removed from each aquarium, blood samples were collected from the ostium of each shrimp and pooled for blood triglyceride and cholesterol concentrations estimation (Carson and Goldfard 1979). Hepatopancreata and eyes were removed and pooled for total lipid determination (Folch et al. 1957) and vitamin A (retinol) determination (Alava et al. 1993), respectively. Three other shrimp were then taken randomly from each aquarium and pooled for body composition analysis (AOAC 1995). Statistical analysis. Each experimental diet was fed to three groups of shrimp. Growth data were means of three groups of shrimp, with 16 shrimp per group (n 3) in expt. 1 and 20 shrimp per group (n 3) in expt. 2. Blood triglyceride concentration, hepatopancreatic total lipid concentration and eye vitamin A concentration were means of the three groups of shrimp, with three shrimp randomly selected from each group and pooled (n 3). Results were analyzed by one-way ANOVA. When the ANOVA identified differences among groups, multiple comparisons among means were made with Duncan s new multiple range test. Statistical significance of difference was determined by setting the aggregate type I error at 5% (P 0.05) for each set of comparisons. Dietary vitamin A requirements for juvenile P. monodon were estimated by the broken-line method (Robbins 1986). RESULTS Experiment 1. The weight gains were significantly higher (P 0.05) in shrimp fed the diet supplemented with 3,000 RE vitamin A/kg than shrimp fed the control group and shrimp fed diets supplemented with 30,000 RE vitamin A/kg (Table 2). Patterns of difference in FE and PER were similar to those of the weight gain. Survival of shrimp fed the control diet was significantly lower (P 0.05) than shrimp fed diets supplemented with 1,500 30,000 RE vitamin A/kg. Blood triglyceride concentrations were highest in shrimp fed diets with 15,000 RE vitamin A/kg, followed by shrimp fed diet with 30,000 RE vitamin A/kg and lowest in shrimp fed diets with 45,000 RE vitamin A/kg. The differences between the highest groups and the lowest groups were significant (Table 3). Hepatopancreatic total lipid concentration was highest for shrimp fed diets supplemented with 45,000 RE vitamin A/kg, intermediate for shrimp fed the diet supplemented with 15,000 and 30,000 RE vitamin A/kg, and lowest for shrimp fed the diet supplemented with 3,000 RE vitamin A/kg. The differences between the three groups were all significantly different from one another. Eye vitamin A concentration was highest in shrimp fed the diet supplemented with 60,000 RE vitamin A/kg, followed by shrimp fed the diets supplemented with 15,000, 30,000 and 45,000 RE vitamin A/kg and lowest in shrimp fed the diets supplemented with 3,000 RE vitamin A/kg. The differences among the three groups were significant. The lipid concentration of shrimp was highest in shrimp fed

3 92 SHIAU AND CHEN TABLE 2 Weight gain, feed efficiency (FE), protein efficiency ratio (PER) and survival of Penaeus monodon-fed diets containing various levels of vitamin A (Experiment 1)1 supplementation levels Initial weight Final weight Weight gain FE PER Survival g/shrimp g/100 g body weight g gain/g feed consumed g gain/g protein consumed % ab 0.26a 0.64ab 44.4a 1, bc 0.37b 0.95d 66.3b 3, c 0.45c 1.13e 71.1b 15, bc 0.36b 0.90cd 67.8b 30, b 0.35b 0.87cd 66.3b 45, ab 0.33b 0.77bc 55.6ab 60, a 0.22a 0.49a 57.8ab Pooled SEM Values are means of three groups of shrimp (n 3), with 16 shrimp per group. Within a column, values with no common superscripts differ, P the diet supplemented with 60,000 RE vitamin A/kg, followed by shrimp fed the diets supplemented with 30,000 and 45,000 RE vitamin A/kg, and lowest in shrimp fed the diets supplemented with 15,000 RE vitamin A/kg. The differences between the highest groups and the lowest groups were significant (Table 4). The moisture, crude protein and ash concentrations of shrimp were not affected by the dietary treatments. Experiment 2. FE was highest in shrimp fed the diets supplemented with 2,400, 3,000, 3,600 and 4,500 RE vitamin A/kg diet, followed by the groups fed 600 and 1,200 RE vitamin A/kg, and finally the unsupplemented control group (Table 5, P 0.05). Patterns of difference in PER were similar to those of the FE ratio. The survival percentage was significantly higher in shrimp fed diets supplemented with vitamin A than in shrimp fed the unsupplemented control diet. Broken-line analysis was used for estimating the minimal requirements of vitamin A (as shown in Fig. 1). Based on the TABLE 3 Blood triglyceride concentration, hepatopancreatic total lipid concentration and eye vitamin A concentration of Penaeus monodon-fed diets containing various levels of vitamin A (Experiment 1)1 supplementation levels Blood Hepatopancreas Eye Triglyceride Total lipid mmol/l mg/g tissue nmol retinol/g tissue b 50.60a 1.22a 1, b 52.10a 1.33a 3, b 52.33a 1.26a 15, b 71.96b 1.95b 30, ab 73.05b 2.02b 45, a 82.00c 2.06b 60, a 82.80c 3.56c Pooled SEM Values are means of three groups of shrimp (n 3), with three shrimp per group. Within a column, values with different superscripts are significantly different (P 0.05). weight gain percentage, the regression equations were Y 0.055X (r 0.98) and Y X (r 0.73). Because the point of intersection between the two lines is 2,511, the adequate amount of dietary vitamin A for juvenile P. monodon was estimated to be 2,511 (about 8,400 IU/kg). DISCUSSION The essentiality of vitamin A for normal growth of P. monodon is clearly demonstrated in the present study. After 8 wk, the shrimp with the greatest growth rates had nearly quadrupled their weight. These growth performances are comparable to those of our previous study with P. monodon fed a complete purified diet which corresponds to the 3,000 RE vitamin A/kg diet of the present study (Shiau and Liu 1994). Retinoids, or their precursors, are essential in the diet of vertebrates (Blomhoff et al. 1991,Wolf 1984). Their function as a component of retinal pigment in the eye has long been known. Recent research showed however, that they have a more fundamental function in gene transcription and thus are TABLE 4 Body composition of Penaeus monodon-fed diets containing various levels of vitamin A (Experiment 1)1 supplementation levels Moisture Crude protein Ash Crude lipid g/100 g a 1, a 3, a 15, a 30, ab 45, ab 60, b Pooled SEM Values are means of three groups of shrimp (n 3), with three shrimp per group. Within a column, values with different superscripts are significantly different (P 0.05).

4 VITAMIN A REQUIREMENT OF PENAEID SHRIMP 93 TABLE 5 Feed efficiency (FE), protein efficiency ratio (PER) and survival of Penaeus monodon-fed diets containing various levels of vitamin A (Experiment 2)1 supplementation levels Initial weight FE PER Survival g/shrimp g gain/g feed consumed g gain/g protein consumed % a 0.65a 54.3a b 0.76ab 66.6b 1, b 0.84b 71.3b 1, bc 0.97c 76.4b 2, c 1.20d 77.7b 3, c 1.14d 76.4b 3, c 1.00cd 75.2b 4, c 0.99cd 74.4b Pooled SEM Values are means of three group of shrimp (n 3), with 20 shrimp per group. Within a column, values with different superscripts are significantly different (P 0.05). essential in cell division and differentiation (Blomhoff et al. 1991, Ross 1993,Wolf 1984). Hence, retinoids are of particular importance in young, rapidly growing vertebrates. Since invertebrates cannot synthesize carotenoids, and thus cannot synthesize retinoids, it would be expected that retinoids are essential to their metabolism, but in crustaceans the situation is unclear. Fisher et al. (1952) found very large quantities of retinoids in the eyes of euphausiid species but only small amounts in the rest of the body. In various other crustacean species, retinoids were either absent or constituted 1 g/g wet mass. In eight species of Penaeidea, some pelagic or deep-sea species had no retinoids in the eyes and only trace amounts or none in the body. Shallow-water species such as Penaeus aztecus had none in the body, but 4 g/g in the eyes (Fisher et al. 1957). A similar situation was found in the freshwater crayfish Orconectes rusticus (Wolfe and Cornwell 1965). Thus, retinoids if present at all in crustaceans, are concentrated in the eyes suggesting that their principal function is in vision. It was also suggested that vitamin A supplementation is unnecessary when fish oils and carotenoids are FIGURE 1 The effect of dietary vitamin A on relative weight gain (g/100 g initial body weight) of Penaeus monodon. Each point represents the mean of three groups of shrimp (n 3), with 20 shrimp per group. Requirements derived with the broken-line method for weight gain is 2,511 diet. added to diets for nonfeeding stage of P. semisulcatus (Dall 1995). In the present study 5% fish oil was used in the basal diet to meet the requirement of the shrimp (Sheen et al. 1994). Nevertheless, vitamin A supplementation is needed. He et al. (1992) used 4,800 IU of vitamin A/kg in their investigation with P. vannamei and qualitatively evaluated the dietary essentiality of fat-soluble vitamins, A, D, E and K. There was only one dietary vitamin A inclusion level in their study (i.e., 4,800 IU/kg), and they did not provide formal estimates of dietary requirement. Shrimp fed the vitamin A-unsupplemented diet showed signs of deficiency including light coloration beginning at wk 4 and increasing mortality at wk 6 of the experiment. We cannot speculate about possible reasons for the deficiency signs in the shrimp because no histological examinations on the shrimp were conducted. Information on vitamin deficiencies in shrimp is scarce. The only well-documented vitamin deficiency in a shrimp species is the Black Death syndrome related to vitamin C deficiency in penaeid shrimp (Lightner et al. 1977). The most marked effect of retinoid feeding seen in animal studies is that of hypertriglyceridemia. In rats, oral all-transretinoic acid, 13-cis-retinoic acid and natural vitamin A resulted in hypertriglyceridemia (Gerber and Erdman 1982). Singh et al. (1969) found elevated plasma-free fatty acids and increased liver lipids in vitamin A-fed rats and postulated that these changes were caused by a mobilization of fatty acids from adipose tissue. Ramachandran et al. (1986) indicated that fatty acid homeostasis in rats can be greatly influenced by their vitamin A status in which hypervitaminosis A caused a decrease in the fatty acid release from adipose. On the other hand, adipose tissue from vitamin A-deficient animals showed an increased lipolytic rate as compared to that of the controls. A similar phenomenon was also reported in fish. The reduced percentage of body fat in the trout fed excess vitamin A may have resulted from a similar mobilization of fatty acids in body fat (Poston 1970). High body content of crude fat was also found in vitamin A-deficient guppies (Shim and Tan 1990). However, in the present study, decreased blood triglyceride concentrations in shrimp were associated with higher vitamin A in their diets (Table 3) and high body fat content was found in shrimp fed high vitamin A diets (Table 4). Generally speaking, body fat content in crustaceans is in the range of

5 94 SHIAU AND CHEN 1 2% which is much lower than other animals. The extent to which the low body lipid affects fatty acid mobilization is not known. The mechanism of dietary vitamin A concentration on lipid nutrition in crustaceans requires further investigation. We could not measure the vitamin A concentration in the hepatopancreas of shrimp. concentration in the eyes of shrimp fed vitamin A supplemented diets generally increased as the vitamin A supplementation level increased (Table 3), indicating that the tissue vitamin A concentration of shrimp did not plateau. It was reported that higher levels of nutrients are needed to maximize tissue concentration. For example, studies of the riboflavin requirement of rainbow trout indicated a growth requirement (dietary level needed to achieve maximal growth) of 3.6 mg/kg, a requirement of 4.6 mg/kg for liver flavin saturation and a requirement of 6.6 mg/kg for saturation of spleen and head kidney with flavin compounds (Woodward 1985). 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