Vitamin C requirements of the shrimp Penaeus vannamei

Aquaculture, 114 (1993) 305-316 Elsevier Science Publishers

305

B.V., Amsterdam

AQUA 50080

Vitamin C requirements of the shrimp Penaeus vannamei Haiqi He and Addison L. Lawrence Shrimp Mariculture Project, Texas A&M University System, Port Aransas, TX, USA (Accepted

11 January 1993)

ABSTRACT Two experiments were conducted to investigate vitamin C requirements of the shrimp Penaeus vannamei using Lascorbyl-2-polyphosphate (APP) in semi-purified diets. Shrimp survival but not growth was significantly (PcO.05) reduced by feeding vitamin C-deficient diets. Whole-body ascorbic acid (WBAA) content in shrimp increased as dietary vitamin C increased. After shrimp were fed a vitamin C-free diet for 3 weeks, the depletion levels for WBAA of shrimp were found to be 2.6 and 4.8 pgg/g for shrimp with an initial weight of 0.1 and 0.5 g, respectively. Minimum dietary vitamin C levels required for normal survival of P. vannamei were estimated to be 120 mg ascorbic acid-equivalent (AAE)/kg diet for shrimp with an initial weight of 0.1 g and 41 mg AAE/kg diet for shrimp with an initial weight of 0.5 g. Dietary vitamin C levels of 120 and 90 mg AAE/kg were required for maintaining a normal WBAA content in shrimp with an initial weight of 0.1 and 0.5 g, respectively. These data indicated that dietary vitamin C requirement of P. vannamei decreased with increased size.

INTRODUCTION

Dietary vitamin C is an essential nutrient for penaeid shrimp. Vitamin C deficiency in penaeid shrimp has been characterized by poor growth, poor feed conversion, reduced molting frequency or incomplete molting, decreased resistance to stress, impaired collagen synthesis and wound healing, melanized lesions underneath the exoskeleton, and high mortality (Kitabayashi et al., 197 1; Deshimaru and Kuroki, 1976; Guary et al., 1976; Lightner, 1977; Lightner et al., 1977, 1979; Magarelli and Colvin, 1978; Hunter et al., 1979; Magarelli et al., 1979; Shigueno and Itoh, 1988). Reported dietary vitamin C requirements for P. juponicus range from 3000 to 10 000 mg/kg when L-ascorbic acid was used as the vitamin C source (Deshimaru and Kuroki, 1976; Guary et al., 1976) and 100 mg/kg when Mg-Lascorbyl-2-phosphate Correspondence to: H. He, Shrimp Mariculture Drawer Q, Port Aransas, TX 78373, USA.

0044-8486/93/$06.00

Project,

Texas A&M University

0 1993 Elsevier Science Publishers

System, P.O.

B.V. All rights reserved.

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was used (Shigueno and Itoh, 1988). However, the vitamin C requirement has not been determined for most farmed species of penaeid shrimp. Ascorbic acid is the most sensitive vitamin to degradation during feed processing and storage. As much as 80 to 100% of the initial amount of supplemental L-ascorbic acid in commercially manufactured fish feeds can be lost during processing and subsequent short-term storage at ambient temperature (Eva et al., 1976; Hilton et al., 1977; Love11 and Lim, 1978; Slinger et al., 1979; Soliman et al., 1987; Grant et al., 1989). Attempts have been made to increase retention of ascorbic acid activity in fish feeds by using alternate forms of vitamin C, such as coated L-ascorbic acid (Hilton et al., 1977; Adams, 1978; Murai et al., 1978), ascorbate-2-sulfate (Halver et al., 1975; Brandt et al., 1985; Tucker and Halver, 1986; Dabrowski et al., 1990; Maage et al., 1990), ascorbate-2-phosphate and ascorbate-6-palmitate (Brandt et al., 1985; Albrektsen et al., 1988; Robinson et al., 1989; El Naggar and Lovell, 1991), and L-ascorbyl-2-polyphosphate (Grant et al., 1989; Wilson et al., 1989). L-ascorbyl-2-polyphosphate ( APP ) is relatively resistant to oxidation (Grant et al., 1989) and has confirmed vitamin C activity in channel catfish (Wilson et al., 1989), trout and fathead minnow (Grant et al., 1989). Information concerning the use of APP in shrimp feeds is not available. The objective of this study was to determine the dietary vitamin C requirement of P. vannamei using APP as the vitamin source. MATERIALS AND METHODS

Feeding trials were conducted at the Nutrition Laboratory of Shrimp Mariculture Project, Texas A&M University System, Port Aransas, Texas. Natural seawater, supplied by an 86-ton, semi-closed, recirculating water system with a 6% daily replacement, was filtered through a 50 pm microfilter and distributed into each rearing tank ( 19 1) at a rate of 450-500 ml/min. A 12h light 12-h dark photoperiod was maintained throughout the experiment. Water temperature, salinity, and dissolved oxygen were measured daily and ammonia and nitrite nitrogen were measured weekly. During the experiments, water temperature and dissolved oxygen were maintained at about 28 “C and 6.3 ppm, respectively. Salinity ranged from 26 to 3 1 ppt. Ammonia nitrogen and nitrite nitrogen levels in the system were less than 0.1 ppm. Experiment one The feeding trial consisted of five dietary treatments containing APP (containing 11.61% ascorbic acid activity) equivalent to 0, 25, 50, 75, 100 mg ascorbic acid-equivalent (AAE) /kg diet and a control diet ( 1000 mg AAE/ kg diet). Ingredients (Table 1) were mixed with approximately 50% (v/w) warm distilled water (60’ C) and cold extruded through a 2-mm orifice die. Extruded diets were dried in an oven by circulated warm air ( 55 oC) for 3 h,

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C REQUIREMENTS

OF PENAEUS

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TABLE 1 Ingredient composition

(g/kg diet) of semi-purified

Ingredient

Conditioning

Casein, vitamin-free’ Gelatin’ Wheat starch’ Menhaden fish oil* Lecithin, purified’ Cholesterol’ Mineral premix3 Vitamin premix4 Lu-Cellulose’ Carboxymethylcellulose’

450.0 80.0 200.0 70.0 10.0 5.0 120.0 0.0 45.0 20.0

diet

diet Test diet 400.0 80.0 250.0 70.0 10.0 5.0 120.0 45.0 0.0 20.0

‘ICN Biochemicals Inc., Cleveland, OH, USA. ‘Zapata Haynie Corp., Reedville, VA, USA. 3AIN mineral mixture 76. ICN Biochemicals Inc., Cleveland, OH, USA. 4Vitamin premix without vitamin C (mg/kg diet): vitamin A acetate (20 IU/mg), 240; cholecalciferal (400 IU/mg), 20; vitamin E acetate (250 IU/mg), 1600; menadione, 20;p-aminobenzoic acid, 100; thiamine HCl, 150; riboflavin, 100; pyridoxine HCl, 100; calcium d-pantothenate, 200; nicotinic acid, 200; biotin, 20; folic acid, 80; Br2, 20; choline chloride, 2000; inositol, 1000; or-cellulose, 39 150. Replacing ol-cellulose with appropriate amounts of APP in vitamin premix to give different levels of dietary vitamin C.

crumbled with a blender, and sifted through sieves to give particle sizes suitable for shrimp. Prepared diets were stored at - 20’ C in sealed plastic bags during the experiment. Penaeus vunnamei postlarvae (PL5-6)) obtained from a commercial shrimp hatchery in Texas, were fed a combination of Artemia nauplii and a commercial shrimp feed for 4 weeks. Prior to initiation of the feeding trial, a conditioning diet (Table 1) that contained no vitamin premix was fed to shrimp for 6 days. The conditioning period was used to deplete the vitamin reserves in shrimp tissue to increase the level of response to the experimental diets and to acclimate shrimp to the experimental semi-purified diet. After the conditioning feeding period, shrimp (0.23 2 0.07 g, y1=40) were randomly distributed into 42 tanks at a density of 8 shrimp per tank. Seven replicate tanks were assigned to each treatment. During the feeding trial, shrimp were fed continuously and in slight excess using automatic feeders and feeding rates were adjusted daily based on feed consumption. Feces and the uneaten feed were siphoned out and mortality recorded daily. At the end of the second week, treatments containing 0 and 25 mg AAE/kg diet were terminated due to high mortality. For the remaining treatments, density was reduced to 4 shrimp per tank to eliminate a potential effect of biomass on individual shrimp growth and the trial was continued for an additional 4 weeks. Shrimp in each

308

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tank were weighed and counted at the end of the second week and at the end of the experiment to determine weight gain and survival. Experiment two The feeding trial consisted of 8 dietary treatments containing APP equivalent to 0, 30, 60, 90, 120, 150, 200, and 400 mg AAE/kg diet. To investigate if shrimp size would affect the vitamin C requirement, 4 replicates of large size shrimp (0.50 + 0.06 g, n = 20)) designated as 0.5 g shrimp, and 4 replicates of small size shrimp (0.10 & 0.03 g, IZ= 20)) designated as 0.1 g shrimp, were assigned to each treatment. Stocking density was 7 per tank for 0.5 g shrimp and 10 per tank for 0.1 g shrimp. Diets were prepared as described in experiment one. All experimental conditions were identical to experiment one. The length of the feeding trial was 3 weeks. At the termination of feeding trial, shrimp were weighed and then frozen immediately at - 80°C for whole-body tissue ascorbic acid analysis. Samples were prepared by homogenizing a whole shrimp in an appropriate amount of ice-cold 5% trichloroacetic acid (TCA) and the homogenates were centrifuged for 20 min at 3000 rpm. Ascorbic acid was analyzed using the DNPH method described by Omaye et al. ( 1979). Briefly, 0.5 ml of supernatant was mixed with 0.1 ml of reaction solution (0.4 g thiourea, 0.05 g CuS04-5H20, and 3.0 g 2,4_dinitrophenylhydrazine (DNPH) dissolved in 9 NH&SO, and brought to 100 ml) and incubated for 3 h at 37°C. Then, 0.75 ml of ice-cold 65% H2S04 was added and mixed well. The mixtures were allowed to stand at room temperature for an additional 30 min. Absorbances were determined at 520 nm. Growth, survival, and WBAA content of shrimp fed the different levels of vitamin C were statistically evaluated by analysis of variance (ANOVA). The Student-Newman-Keuls (SNK) multiple range test was used to determine significant differences (PcO.05) between means (SAS Institute Inc., 1988). Vitamin C requirements were estimated by Iitting the broken-line model to the survival data (Robbins et al., 1979) using SAS nonlinear regression procedure (SAS Institute Inc., 1988). RESULTS

Experiment one Weight gain and survival of shrimp fed diets containing different levels of vitamin C are presented in Table 2. After 14 days, survival of shrimp fed diets containing 0,25, 50,75, and 100 mg AAE/kg diet was significantly (PcO.05) lower than that of shrimp fed the control diet containing 1000 mg AAE/kg diet. Within the range of dietary vitamin C levels tested, survival of shrimp increased from 23 to 9 1% as dietary levels of vitamin C increased from 0 to 1000 mg AAE/kg diet.

VITAMIN C REQUIREMENTS OF PENAEUS

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VANNAMEI

TABLE 2 Weight gain and survival of shrimp fed graded levels of vitamin C in experiment one. Values represent mean k standard error for 7 replicate tanks’ Vitamin C (mg AAE/kg diet)

0

25 50 75 100 1000

Survival (% )

Weight gain (g)

Day 1-14

Day 15-42

14 days

42 days

23.2L4.7” 35.7?3.0b 50.0+ 7.2’ 51.8k3.0” 62.5 *4.4a 91.1 k3.3”

39.354.7” 57.1* 8.3b 89.3 z!z3.6” 96.4? 3.3”

0.62kO.08 0.56+0.05 0.51 kO.08 0.60f0.06 0.58 z!I0.07 0.59 + 0.05

4.00+0.53 3.97LO.34 3.96kO.31 4.13iO.20

‘Means in the same column with different superscript letters are significantly different. 30 7

1

st week

2nd week

3rd week

4th week

5th week

61h week

Time

Fig. 1. Weekly mortality experiment one.

(number of deaths) of shrimp fed different levels of dietary vitamin C in

After continuing the feeding trial an additional 4 weeks, shrimp fed diets containing 50 and 75 mg AAE/kg diet had significantly (PC 0.05) lower survival than shrimp fed the control diet and the diet containing 100 mg AAE/ kg diet. Survival of shrimp fed diets containing 100 and 1000 mg AAE/kg diet was not different. Most mortality occurred in shrimp fed vitamin C-deficient diets during the second week and third week after initiation of the feeding trial and mortality was reduced thereafter (Fig. 1) . There was no difference in weight gain of shrimp fed diets containing different levels of vitamin C at the end of the second and sixth week (Table 2).

N. HE AND

310

A.L. LAWRENCE

Experiment two Since no level of dietary vitamin C between 100 and 1000 mg/kg diet was tested in the first experiment, a second experiment was conducted to more accurately examine the vitamin C requirement of P. vannamei. Two-way ANOVA, with shrimp size and diet as class variables, indicated that there was a significant interaction (PC 0.02 1) on survival of shrimp between shrimp size and dietary treatment. For 0.5 g shrimp, SNK-test indicated that survival of shrimp fed vitamin C-free diet was significantly (PC 0.05) lower than that of shrimp fed other diets (Table 3). For 0.1 g shrimp, SNK-test indicated that survival of shrimp fed diets containing vitamin C from 0 to 90 mg AAE/kg diet increased significantly from 35.0% to 77.5% and there was no difference in survival when shrimp were fed diets containing vitamin C from 90 to 400 mg AAE/kg diet (Table 3). Vitamin C requirements, estimated by fitting a broken-line model to the survival data, were 4 1 mg AAE/kg diet for 0.5 g shrimp and 120 mg AAE/kg diet for 0.1 g shrimp (Fig. 2 ) . Whole-body ascorbic acid (WBAA) contents of shrimp fed different levels of dietary vitamin C are presented in Table 4. WBAA contents of 0.1 g shrimp fed diets containing vitamin C from 0 to 90 mg AAE/kg diet were significantly lower than those of shrimp fed diets containing vitamin C from 120 to 400 mg AAE/kg diet. Similarly, 0.5 g shrimp fed diets containing vitamin C from 0 to 60 mg AAEjkg diet had significantly lower WBAA contents than those fed diets cont~ning vitamin C from 90 to 400 mg AAE/kg diet. There was no-difference in weight gain of shrimp fed different diets containing vitamin C from 0 to 400 mg AAE/kg diet after the 3 week feeding trial. For both experiments, the “black death” syndrome and incomplete moltTABLE 3 Weight gain and survival of shrimp with different initial weight fed graded levels of vitamin C in experiment two. Values represent mean + standard error for 4 replicate tanks’ Vitamin C (mg AAE/kg diet)

0

30 60 90 120 150 200 400

0.5 g Shrimp

0.1 g Shrimp Survival (%)

Weight gain (g )

Survival (%)

Weight gain (g )

35.0,6.5* 50.0+ 8.3ab 65.05 5.gbC 77.5 f 7.5Cd 92.5 + 2.5* 90.0 + O,Od 92.5 f 7.Sd 95.02 2.9”

0.87tO.10 0.93 + 0.06 0.88+0.10 0.96 + 0.06 0.97 ?I 0.04 0.94 + 0.04 1.02+0.09 1.06+0.03

28.6+ 5.8” 75.01f: 10.7b 82.1 ?c 3.6bc 89.3 IfI 3.6bC lOO.O? 0.0” 96.41 3.P 92.9+ 4.l& 92.91 4.1bC

2.00+0.17 1.78kO.06 2.14kO.18 1.97+0.07 2.00+ 0.07 2.07kO.13 2.042 0.08 1.97kO.13

‘Means in the same column with different superscript letters are significantly different.

VITAMIN C REQUIREMENTS

0

OF PENAEUS

100

200

Dietary

vitamin

311

VANNAMEI

300

400

C (mglkg)

Fig. 2. Plot of predicted survival of shrimp to graded levels of dietary vitamin C in experiment two by broken-line regression model. Requirement for vitamin C defined at points where the relationship breaks horizontal. 0.1 gshrimp: Y=35.5+0.5X, Y=92.5.0.5 g shrimp: Y=28.6+ 1.5X, Y=92.3. TABLE 4 Whole-body ascorbic acid contents of shrimp fed graded levels of vitamin C at the termination experiment two. Values represent mean + standard error for 5 replicate shrimp’ Vitamin C (mg AAE/kg diet)

0 30 60 90 120 150 200 400

of

Whole-body ascorbic acid contents @g/g) 0.1 g shrimp

0.5 g shrimp

2.650.2” 2.8f0.2a 2.5kO.l” 3.OkO.3” 10.1 +O.gb 13.02 1.2’ 13.0*0.5= 20.4+ 1.4d

4.8 50.4” 5.5 10.5” 6.6jIO.3” 10.52 l.Ob 10.0?0.4b 11.9?1.3b 12.6+0.6b 21.5kO.5’

‘Means in the same column with different superscript letters are significantly different.

ing were observed on shrimp which died from vitamin C deficiency. A few shrimp fed vitamin C-deficient diets showed abnormal coloration and swollen hepatopancreata. Those vitamin C-deficient shrimp appeared motionless and were unresponsive to disturbances. DISCUSSION

Vitamin C is required for hydroxylation of proline and lysine in the formation ‘of collagen. Many of the vitamin C deficiency signs previously de-

312

H. HE AND AL. LAWRENCE

scribed for penaeid shrimp, such as the “black death” syndrome, are related to the malfunction of collagen synthesis (Lightner et al., 1979). In the present study, the importance of dietary vitamin C for normal survival of juvenile P. vunnamei has been demonstrated. High mortality occurred in those shrimp fed vitamin C-deficient diets in less than 2 weeks. This indicates a great sensitivity of juvenile P. vannamei to dietary vitamin C deficiency. In fish culture, inclusion of dietary vitamin C has been demonstrated to enhance tolerance to environmental contaminants and stressors and immunity against pathogens (Mayer et al., 1978; Durve and Lovell, 1982; Halver, 1985; Li and Lovell, 1985; Liu et al., 1989; Navarre and Halver, 1989). The same functions may also be present in penaeid shrimp. In the present study, besides the “black death” syndrome, a few shrimp in the vitamin C-deficient treatments showed abnormal coloration and swollen hepatopancreata. Vitamin C-deficient shrimp appeared motionless and were unresponsive to disturbances. This suggests that death may have been due to reduced resistance to stress such as bacterial infection. Results of this study indicate that dietary vitamin C requirements of P. vannamei are size-dependent and decreased with age. In the first experiment, dietary vitamin C at 100 mg AAE/kg diet was apparently sufficient to prevent vitamin C deficiency-related high mortality for shrimp larger than 0.8 g (during the second period of the feeding trial), whereas 1000 mg AAE/kg diet was needed for shrimp with an initial weight of 0.23 g. In addition, the obvious reduction of shrimp mortality in vitamin C-deficient treatments after the third week of the first feeding trial suggests that there was a decrease in vitamin C demand by shrimp. In the second experiment, a significant interaction (P~O.021) between shrimp size and dietary vitamin C level confirmed the conclusion that vitamin C requirement of P. vannamei was size-dependent. Shrimp with an initial weight of 0.1 g were found to require nearly 3 times as much dietary vitamin C as required by shrimp with initial weight of 0.5 g in order to maintain normal survival ( 120 vs 4 1 mg AAE/kg diet ) . Decreased vitamin C requirement with size has also been reported in fish (Sato et al., 1978; Li and Lovell, 1985). Size-dependent vitamin C requirements may be the result of different percent changes in weight per unit time. A smaller or a younger animal has a higher percent change in weight per unit time and consequently may require more vitamin C to meet its metabolic needs. In the present study, 0.1 g shrimp had a much higher percent weight gain (91311 11%) than 0.5 g shrimp (374-449%) after the 3-week feeding trial. For penaeid shrimp, however, a lower requirement for vitamin C of a larger animal may also be due to development of the ability to biosynthesize vitamin C de nova Lightner et al. ( 1979) suggested that shrimp have a limited ability to synthesize vitamin C, which was sufficient to meet the low requirements of large shrimp ( 2 12 g ) but insufficient in young shrimp. WBAA contents of shrimp were significantly reduced by feeding vitamin

VITAMIN C REQUIREMENTS OF PENAEUS

VANNAMEI

313

C-deficient diets. For shrimp having an initial weight of 0.1 g, WBAA contents from 10.1 to 20.4 pg/g were observed for shrimp fed diets containing vitamin C from 120 to 400 mg AAE/kg diet. Normal survival of shrimp fed these dietary treatments suggests that WBAA content of about 10 ,ug/g was required to prevent the occurrence of high mortality and that values below this level may indicate vitamin C deficiency in the shrimp, P. vannamei. Relatively low depletion levels for WBAA from 2.5 to 3.0 pg/g were observed on these shrimp fed diets containing vitamin C from 0 to 90 mg AAE/kg diet. These results indicate that P. vannamei with an initial weight of 0.1 g appeared to require a minimum level of dietary vitamin C of 120 mg AAE/kg diet in order to maintain a normal tissue ascorbic acid content. This value is in good agreement with that required for normal survival of shrimp. For shrimp having an initial weight of 0.5 g, WBAA contents from 10.0 to 21.5 ,ug/g were observed on shrimp fed diets containing vitamin C from 90 to 400 mg AAE/kg diet. The depletion level for WBAA of shrimp fed a vitamin Cfree diet was 4.8 ,ug/g. The WBAA content of shrimp fed the diet containing 60 mg AAE/kg diet was, however, significantly lower than that of shrimp fed diets containing vitamin C from 90 to 400 mg AAE/kg diet but not different from that of shrimp fed the vitamin C-free diet. These results indicate that a dietary vitamin C level of 90 mg/kg diet was required for maintaining normal tissue ascorbic acid level by P. vannamei with an initial weight of 0.5 g. The disagreement between the value required for normal survival and the value required for normal tissue ascorbic acid level in 0.5 g shrimp suggests the vitamin C requirement may vary as different criteria were used. In the present study, the lower depletion level for WBAA with small shrimp vs larger shrimp may either be due to limited ability to synthesize vitamin C and/or the length of experiment. The lack of any differences in weight gain in response to different levels of vitamin C was observed in both experiments. Similarly, greatest growth was obtained for P. ja~u~ic~s fed a diet without vitamin C supplementation even though. a dietary level of at least 3000 mg ascorbic acid/kg diet was required for preventing mortalities (Deshimaru and Kuroki, 1976). APP is a stable form of vitamin C and has been used in fish and shrimp feeds (Grant et al., 1989). Stability of APP in pelleted feeds was reported up to 83 times or 45 times greater than that of ascorbic acid at 25 “C or 40’ C and the retention of vitamin C activity was 84% in the finished feed after steampelleting and 65% after 60-90 day storage at 40’ C (Grant et al., 1989 ) . In the present study, since diets were prepared under much milder conditions and stored at - 20’ C during the feeding trial and shrimp were fed continuously, the loss of vitamin C activity due to feed processing, storage, and water leaching was minimized. The great improvement of shrimp survival by adding relatively small amounts of APP in diets indicates that APP is an effective dietary vitamin C source and can be utilized by P. vannamei to prevent the

314

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vitamin C de~ciency-related mortality. The requirement of P. v~~~ff~e~ for vitamin C estimated in the present study was much less than the reported requirement of P.japonicus (3000- 10000 mg vitamin C/kg diet) determined by using L-ascorbic acid (Deshimaru and Kuroki, 1976; Guary et al., 1976) and was close to the value (100 mg vitamin C-equivalent/kg diet) determined by using Mg-L-ascorbyl-2-phosphate (Shigueno and Itoh, 1988), ACKNOWLEDGMENTS

The authors wish to thank Dr. Allen Davis, Dr. Delbert Gatlin, Dr. Edwin H. Robinson, Dr. Frank Castille, and Dr. Louis R. D’Abramo for reviewing a draft of the m~uscript and Mrs. Karen Hall for technical assistance during the experiment. We also thank Rangen Feeds, Inc. for providing L-ascorbyl2-polyphosphate. This research was funded in part under Grant No. H-8 158 from the Texas Agriculture Experiment Station, Texas A&M University System and U.S. Department of Agriculture, Cooperative, State Research Service, Grant No. 88-38808-3319.

REFERENCES Adams, CR., 1978. Vitamin product forms for animal feeds. In: Proceedings of the Roche Vitamin Nutrition Update Meeting, Arkansas Nut~tion Conference, RCD 5483/ 1078 Hoffman-La Roche Inc., Nutley, NJ, USA, pp. 54-60. Albrektsen, S., Lie, 0. and Sandnes, K., 1988. Ascorbyl palmitate as a dietary vitamin C source for rainbow trout (Salmo gairdneri). Aquaculture, 7 1: 359-368. Brandt, T.M., Deyoe, C.W. and Seib, P.A., 1985. Alternate source of vitamin C for channel catfish. Prog. Fish-Cult., 47: 55-59. Dabrows~, K., El-Fiky, N., K&k, G., Frigg, M. and Wieser, W., 1990. Requirement and utilization of ascorbic acid and ascorbic sulfate in juvenile rainbow trout. Aquaculture, 9 1: 3 17337. Deshimaru, 0. and Kuroki, K., 1976. Studies on a purified diet for prawn. VII. Adequate dietary levels of ascorbic acid and inositol. Bull. Jpn. Sot. Sci. Fish., 42: 57 l-576. Durve, V.S. and Lovell, R.T., 1982. Vitamin C and disease resistance in channel catfish (&al~r~~~~ctat~). Can. J. Fish. Aquat. Sci., 39: 948-951. El Naggar, G.O. and LoveIl, R.T., 199 1. ~ascorbyl-Z-monophosphate has equal antiscorbutic activity as L-ascorbic acid for channel catfish. J. Nutr., 12 1: 1622- 1626. Eva, J.K., Fifiled, R. and Rickett, M., 1976. Decomposition of supplementary vitamin C in diets compounded for laboratory animals. Lab. Anim., 10: 157-l 59. Grant, B.F., Seib, P.A., Liao, M. and Corpron, K.E., 1989. Polyphosphorylated L-ascorbic acid: a stable form of vitamin C for aquaculture feeds. J. World Aquacult. Sot., 20: 143-l 57. Guary, M., Kanazawa, A., Tanaka, N. and Ceccaldi, H.L., 1976. Notational requirements of prawn. VI. Requirement for ascorbic acid. Mem. Fat. Fish. Kagoshima Univ., 25: 53-57. Halver, J.E., 1985. Recent advances in vitamin nutrition and metabolism in fish. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press Inc., London, pp. 4 15-429.

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Halver, J.E., Smith, R.R., Tolbert, B.M. and Baker, E.M., 1975. Utilization of ascorbic acid in fish-Ann. N.Y. Acad. Sci., 258: 81-101. Hilton, J.W., Cho, C.Y. and Slinger, S-J., 1977. Factors affecting and stability ofsupplem~ntal ascorbic acid in practical trout diets. J. Fish. Res. Board Can., 34: 683-687. Hunter, B., Magrelli, P.C. Jr., Lightner, D.V. and Colvin, L.B., 1979. Ascorbic acid-dependent collagen formation in penaeid shrimp. Comp. Biochem. Physiol., 64B: 38 l-385. Kitabayashi, K., Shudo, K., Nakamura, K. and Ishikawa, S., 197 1. Studies on formuIa feed for Kuruma prawn. II. On the utilization values of glucose. Bull. Tokai Reg. Fish. Res. Lab., 65: 109-118. Li, L. and Love& R.T., 1985. Elevated levels of ascorbic acid increase immune responses in channel catfish. J. Nutr., 115: 123- 13 1. Lightner, D.V., 1977. Black death disease of shrimps. In: C.J. Sindermann (Editor), Shrimp Diseases: Disease Diagnosis and Control in North American Marine Aquaculture. Elsevier Sci. Publ. Comp. New York, pp. 65-68. Lightner, D.V., Colvin, LB., Brand, C. and Danald, D.A., 1977. Black death, a disease syndrome of penaeid shrimp related to a dietary deficiency of ascorbic acid. Proc. World Maricult. Sot., 8: 61 l-623. Lightner, D.V., Hunter, B., Magarelli, P.C., Jr. and Colvin, L.B., 1979. Ascorbic acid: nutritional requirement and role in wound repair in penaeid shrimp. Proc. World Maricult. Sot., 10: 513-528. Liu, P.R., Plumb, J.A., Guerin, M. and Love& R.T., 1989. Effect of megalevels of dietary vitamin C on the immune response of channel catfish I~t~iu~ punctat~ in ponds. Dis. Aquat. Org., 7: 191-194. Lovell, R.T. and Lim, C., 1978. Vitamin C in pond diets for channel catfish. Trans. Am. Fish. Sot., 107: 321-325. Maage, A., Waagbe, R., Olsson, P.E., Julshamn, K. and Sandnes, K., 1990. Ascorbate-2-sulfate as a dietary vitamin C source for Atlantic salmon (Saho salar). 2. Effects of dietary levels and immunization on the metabolism of trace elements. Fish Physiol. Biochem., 8: 429-436. Magarelli, PC, Jr. and Calvin, L.B., 1978. Depletion/repletion dynamics of ascorbic acid in two species of penaeid: Penaeuf cahfornieasis and Penaeus stybmiris. Proc. World Aquacult. Sot., 9: 235-241. Magarelli, P.C., Jr., Hunter, B., Lightner, D.V. and Colvin, L.B., 1979. Black death: an ascorbic acid deficiency disease in penaeid shrimp. Comp. Biochem. Physiol., 63A: !03- 108. Mayer, F.L., Mehrle, P.M. and Crutcher, P.L., 1978. Interactions of toxaphene and vitamin C in channel catfish. Trans. Am. Fish. Sot., 107: 326-333. Murai, T., Andrews, J.W. and Bauemfeind, J.C., 1978. Use of L-ascorbic acid, ethocel coated ascorbic acid and ascorbate 2-sulfate in diets for channel catfish, Ictalurus punctatus. J. Nutr., 108: 1761-1766. Navarre, 0. and Halver, J.E., 1989. Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C. Aquaculture, 79: 207-22 1. Omaye, S.T., Tumbull, L.D. and Sauberlich, H.E., 1979. Selected methods for the determination of ascorbic. acid in animal cells, tissues, and fluids. In: D.B. McCo~ick and L.D. Wright (Editors), Methods in Enzymology, Vol. 62. Academic Press, Inc., New York, pp. 3-l 1. Robbins, K.R., Norton, H.W. and Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J. Nutr., 109: 1710-1714. Robinson, E.H., Brent, J.R. and Crabtree, J.T., 1989. Ascorbyl-2-phosphate, an ascorbic acid source that resists oxidation in fish feed. Feedstuffs, 6 1: 64-66. SAS Institute Inc., 1988. SAS/STAT User’s Guide, Release 6.03 Edition. Gary, NC, USA, 1028 PP. Sato, RI., Yoshinaka, R. and Ikeda, S., 1978. Dietary ascorbic acid requirement of rainbow trout for growth and collagen formation. Bull. Jpn. Sot. Sci. Fish., 44: 1029-l 035.

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LAWRENCE

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