Dietary pantothenic acid requirement of juvenile abalone, Haliotis discus hannai Ino.

ABSTRACT Juvenile abalone, Haliotis discus hannai Ino, of 0.55 [+ or -] 0.02 g in body weight, and 15.48 [+ or -] 0.31 mm in shell length, were fed with six purified diets containing 0, 9.8, 19.8, 39.7, 79.4, 158.5 mg Ca-pantothenic acid/kg diet for 16 wk. The Ca-pantothenic acid was microbound with sodium alginate, and other water-soluble vitamins were encapsulated with sodium alginate, by an emulsion coacervation process, prior to supplementation in experimental diets. The dietary Ca-pantothenic acid concentration did not affect the body composition of abalone. Weight gain (WG), daily increment in shell length (DISL), and tissue pantothenic acid were significantly affected by dietary pantothenic acid. According to broken-line regression analysis, 21 mg Ca-pantothenic acid/kg diet was found to satisfy the maximum growth of abalone, whereas on the basis of the pantothenic acid concentration in the viscera and muscle of the experimental abalone, 24 and 26 mg Ca-pantothenic acid/kg diet were required, respectively. Hence, the optimum dietary Ca-pantothenic acid requirement for H. discus hannai Ino is estimated to be 26 mg/kg diet.

KEY WORDS: abalone, Haliotis discus hannai Ino, Ca-pantothenic acid, requirement, feeding and nutrition, molluscs

INTRODUCTION

Pantothenic acid (PA) has crucial roles in intermediary metabolism as a constituent of coenzyme A (CoA) and phosphopantotheine. As a part of coenzyme A, pantothenic acid transfers acyl groups in enzymatic reaction such as oxidation of pyruvate and fatty acids (NRC 1983).

Pantothenic acid deficiency signs in fish are usually growth retardation, anorexia, abnormal swimming behavior, and death (Halver 1989). The most common signs of PA deficiency are clubbed gill and fusion of gill filaments (NRC 1981, Wilson et al. 1983, Butthep et al. 1985, Masumoto et al. 1994). Dietary PA requirement for maximum growth and prevention of deficiency signs have been reported to be 10-20 mg/kg diet for salmonids (NRC 1981), 10 mg/kg of diet for channel catfish (Mural & Andrews 1979, Wilson et al. 1983) and blue tilapia (Roem et al. 1990, Soliman & Wilson 1992), and 30-50 mg/kg for common carp (Ogino 1967).

Masumoto et al. (1994) suggested that free PA tissue concentrations measured by radioimmunoassay (RIA) are a more sensitive indicator of pantothenic acid intake in rainbow trout than reduction in appetite or reduced growth.

Abalone are large algivorous marine mollusks of the genus, Haliotis (Gastropoda, Prosobranchia, Archaeogastropoda, Haliotidae). They are the most commercially important gastropods in aquaculture. Water-soluble vitamin requirements of the abalone, H. discus hannai, were recently reported to include; vitamin C, vitamin [B.sub.1], inositol, and biotin (Mai 1998, Mai et al. 2001, Wu et al. 2002, Zhu et al. 2002). The objective of this study is to investigate the effects of dietary PA on survival, growth, and tissue PA concentration and further to estimate the optimum level of dietary PA for the abalone, H. discus hannai.

MATERIALS AND METHODS

Experimental Diet

Formulations of the experimental diets and their proximate composition are shown in Table 1. Crude protein level of the experimental diets was 28.2%, which is considered to be sufficient to maintain optimum growth for H. discus hannai (Mai et al. 1995b). Soybean oil and menhaden fish oil (1:1) was used as the basal lipid source. Dietary lipid level was 3.75%, which was sufficient to support optimum growth and provide enough EFA for the abalone (Mai et al. 1995a). The vitamin (except for Ca-PA) and mineral mixture were slightly modified from those used by Uki et al. (1986). Six experimental diets were formulated from purified ingredients to provide graded levels of Ca-pantothenic acid ranging from 0-158.5 mg/kg diet.

Ca-PA was microbound with sodium alginate prior to supplementation to the experimental diet. One gram Ca-PA was mixed well with l-g sodium alginate; appropriate water was added and mixed. The paste was lyophilized for 24 h, and then ground to a powder (passed through a sieve with 125-[micro]m pore size). Other water-soluble vitamins were microencapsulated with sodium alginate by an emulsion coacervation process prior to supplementation to the experimental diets. The method of microencapsulation was modified from that described by Bodmeier and Wang (1993). A portion of sodium alginate solution (2%, w/w) containing 2% (w/ w) of the vitamins to be microencapsulated, was mixed with another portion of oil containing 6.74 g/L of Span 80, stirred at 400 rpm for 15 min. The emulsified solution was slowly poured into 1% (w/w) Ca[Cl.sub.2] solution, with continuous stirring for 1 min, and then filtered with a vacuum filter. The harvested microcapsules were washed with cyclohexane and non-water alcohol in turn, to remove oil and water, dried at room temperature, and then kept at -20[degrees] until use. The actual quantities of Ca-pantothenic acid in diets were measured by HPLC (Table 2).

Procedures for diet preparation were based on the method of Mai et al. (1995a). All the ingredients (passed through a sieve with 125 [micro]m pore size) were mixed thoroughly and were made to a paste by gradually adding water (about 120%, v/w). The paste was shaped into 0.5-mm thick sheets, which were cut into 1-[cm.sup.2] flakes. The flakes were dipped in an aqueous solution of Ca[Cl.sub.2] (5%, w/v) for about 1 min, and then the surplus solution was drained naturally. The diet flakes were sealed in a sample bag and stored at -20[degrees] until use.

Experimental Procedures

Abalone juveniles (H. discus hannai), of 0.55 [+ or -] 0.02 g average weight and 15.48 [+ or -] 0.31 mm mean shell length, used in this experiment, were from a spawning at Mashan Fisheries Co., Shan dong Province of China. Thirty abalone were placed into each rearing unit, a plastic cage (20cm x 20 cmx 15 cm) with a corrugated plastic plate as a shelter. There were three replicates for each of the six dietary treatments. All the cages were distributed randomly in a rectangle pool (1 m x 1 m x 6 m). Fresh seawater, flowing through 30-[micro]m primary sand filters, then through 10-[micro]m secondary composite sand filters, was continuously supplied to the pool. The flow rate was about 0.5 L per rain per cage. Cages were kept in dim light by screening a large drape above. Before the experiment, the abalone underwent a week conditioning period. The animals were fed with test diets once a day at 17:00 at a satiation level with a little leftover (5% to 10% of abalone wet weight per day). The plastic cages were cleaned at 8:00 o'clock every morning to remove the uneaten feed and feces during the experimental period. The temperature of the water was 15 [degrees]C to 25[degrees]C, salinity 30[per thousand] to 34[per thousand]., pH 7.6 to 7.9. Dissolved oxygen was not less than 7 mg/L, and there were negligible levels of free ammonia and nitrite. The Ca-pantothenic acid in the seawater was below the level of detection. The whole feeding experiment was conducted for 16 wk.

Sample Collection and Analysis

At the termination of the experiment, all abalone from each replicate, were fasted for 3 days, then removed from the cages, counted, weighed, measured, and then frozen in -70[degrees] for subsequent chemical analysis. Growth performance is expressed as weight gain (WG, %) and daily increment in shell length (DISL, [micro]m/day). The calculation formulae are as follows:

WG (%) = [(Wt-Wi) / Wi] x 100

DISL ([micro]m/day) = [(SLt-SLi) / t] x 1000

where, Wt, Wi are final and initial mean weight (g), SLt, SLi are final and initial mean length (mm), respectively: and t is the feeding experiment period (days).

Proximate analyses to determine protein, lipid, and moisture levels in diets and abalone carcass were conducted using standard methods (AOAC 1995). Moisture content was calculated following drying in an oven at 105[degrees] for 24 h. Crude protein was estimated by the Kjedahl method. Crude lipid was determined after ether extraction by the Soxhlet method.

Water Stability of Ca-Pantothenie Acid in Experimental Diets

The leaching measurement of dietary Ca-PA was carried out with the modified method of Zhu et al. (2002). The experimental feeds, together with a control feed supplemented with 200 mg/kg crystalline Ca-pantothenic acid, were put into packets of 100-mm screens and placed onto the bottom of experimental cages without abalone. Temperature and flow rate were 20 [+ or -] 0.8[degrees]C and about 0.5 L per rain per cage that matched those of the experiment respectively. At the end of allotted times (0, 1, 2, 6, 12 h, respectively), the remaining diets were removed from the cages and freeze dried for 24 h. Then the Ca-PA concentrations were measured by HPLC.

The Ca-PA levels in abalone tissues and diets were analyzed with HPLC by a method modified from the HP operation manual (HP 1999). Weighted viscera and muscle tissues were homogenized with 3 mL of Millipore water. Homogenates were deproteinized with saturated barium hydroxide and 10% zinc sulfate. Samples were then centrifuged (6000g for 10 min.) and the supernatants were filtered through a 0.45-[micro]m filter prior to being analyzed by HPLC with an ODS hyzersil column (4 x 250 mm). The mobile phase A was 15 mM phosphate buffer (pH 7.5) and mobile phase B was methanol. A: B = 70:30 was used as eluant. The flow rate was 0.8 mL/min. Pantothenic acid was detected with a diode array detector (DAD) and detecting wavelength was 245 nm. The Ca-PA (from Sigma Co.) was solved in Millipore water as a standard.

Statistical Analysis

All percentage data were square-root arcsine transformed prior to analysis, Data from each treatment were subjected to one way ANOVA. When overall differences were significant at less then 5%, Tukey HSD test was used to compare the mean values between individual treatments. Ca-PA requirements of the juvenile abalone were estimated from weight gain (WG), daily increment in shell length (DISL), PA concentrations in the viscera and muscle using broken-line analysis (Robbins et al. 1979). Statistical analyses were performed using the STATISTICA[TM] package.

RESULTS

Leaching

The leaching of microbound Ca-PA in the experimental diets was significantly lower than that of crystalline Ca-PA (Table 3). The retentions of microbound Ca-PA in the diets were about 80% after 2-h immersion in seawater and about 40% after 12-h. Meanwhile, the corresponding retentions of crystalline Ca-PA in the control diet were 53.5% and 28.5% respectively. There was no significant difference in water stability among the diets containing different levels of microbound Ca-PA at each sampling time.

Survival, Weight Gain and Daily Increment of Shell Length

There were no significant differences in survival of the abalone among the dietary treatments (Table 4), which ranged from 90% to 97.8%. The weight gain (WG) was significantly affected by dietary Ca-PA after the 16-wk rearing period. The WG was lowest in the group fed Ca-PA free diet and increased from 92.0% to 147.8% as the dietary PA increased from 0 to 19.8 mg/kg and leveled off when dietary PA further increased. The change of the values of DISL was similar to WG (Table 4). It increased from 45.5 to 64.0 [micro]m/day when dietary Ca-PA increased from 0 to 19.8 mg/kg and reached a plateau when dietary Ca-PA further increased. Based on broken line analysis, the relationship between WG and dietary PA level (X) is Y = 150.24 - 2.787 (21- X), and that of DISL is Y = 63.88 - 0.964 (20 - X).

Carcass Composition

The results of carcass composition are shown in Table 5. There were no significant differences in soft body protein, lipid or moisture among all the treatments (P > 0.05) after feeding with the feeds containing graded levels of Ca-PA for 16 wk. Soft body protein, lipid, and moisture of the experimental abalone ranged from 52.7% to 55.0%, 7.5% to 8.1%, and 75.7% to 79.5%, respectively.

Tissue Pantothenic Acid Concentration

Visceral and muscle concentrations of PA are shown in Table 6. They were significantly affected by the dietary Ca-PA level. PA content in the viscera increased from 5.82 to 10.74 [micro]g/g wet tissue when dietary PA increased from 0 to 39.7 mg/kg diet, then leveled off when dietary PA further increased. Changes in the PA contents in muscle were similar to those in the viscera, but were much lower than those in viscera, ranging from 1.25-4.71 [micro]g/g. Both of them responded in a broken-line model to dietary Ca-PA levels, the breakpoints were 24 and 26 mg/kg respectively.

DISCUSSION

Leaching of vitamins from fish feed is a serious problem in aquaculture: immersion of feed, especially if prolonged, may lead to very high rates of vitamin depletion and to insufficient or unbalanced supplies of these micronutrients to farmed animals (Marchetti et al. 1999). In the present study, to reduce leaching of dietary water-soluble vitamins during the period before being consumed, Ca-pantothenic acid was treated with microbinding with sodium alginate, and other water soluble vitamins were microencapsulated with sodium alginate by an emulsion coacervation process before supplementation. A leaching test showed that the leaching rates for the first 2 h of dietary Ca-PA in seawater was significantly reduced from 46.5% to 21.1% by the above mentioned processes (Table 3). According to previous studies, it is reliable to determine the effect of dietary pantothenic acid on abalone using these experimental feeds (Zhu et al. 2002).

The optimum PA requirement is 26 mg/kg diet according to the results of this study, which is higher than 10-20 mg/kg for salmonids (NRC 1981), 10 mg/kg of diet for channel catfish (Murai & Andrews 1979, Wilson et al. 1983), and blue tilapia (Roem et al. 1990, Soliman & Wilson 1992) but lower than 30-50 mg/kg for common carp (Ogino 1967). Abalone are slow feeders. The extended period before consuming food increases the leaching of nutrients, including water-soluble vitamins like Ca-PA. In the present study, leaching of the vitamin still exists although the microbinding technique was used. If the water stability of dietary Ca-PA is further improved, the requirement of dietary Ca-PA for this species will be further reduced.

Masumoto et al. (1994) pointed out that free PA in the gill, heart, and liver were 15.2-81.5, 24.4-48.1, 68.6-83.7 nmol/g tissue of rainbow trout, and suggested that free PA in the gill and heart are sensitive indicators. In the present study, the free PA in the viscera and muscle of the abalone measured by HPLC were 5.82-11.02 [micro]g/g viscera and 1.25-4.71 [micro]g/g muscle respectively, and they were also significantly affected by dietary PA, indicating that they were all sensitive to PA status of the abalone.

In some fish, PA deficiency can result in various deficiency signs (NRC 1981, Wilson et al. 1983, Butthep et al. 1985, Masumoto et al. 1994). In this study, there was no other overt deficiency sign found except for poor growth. This is perhaps partly due to the bottom-living habit and slow growth of abalone (Chert et al. 1991, Wu et al. 2002, Zhu et al. 2002).

TABLE 1.

Ingredient and proximate composition of the basal diet (% on dry
weight basis).

                                                        Content
                   Ingredients                       (g/100g Diet)

Casein, vitamin free (Sigma Chemical, St. Louis,         25
  MO, USA)
Gelatin (Sigma)                                           6
Dextrin (Shanghai Chemical Co., Shanghai, China)         34
CM-cellulose (Shanghai Chemical Co.)                      5
Sodium alginate (Shanghai Chemical Co.)                  30
Vitamin mix. (a),  Ca-PA free
Mineral mix. (b)                                          4
Choline chloride (Shanghai Chemical Co.)                  0.5
SO/MFO (c)                                                3.5
Proximate analysis (means of triplicates)
  Protein (%)                                            28.5
  Lipid (%)                                               3.80
  Ash (%)                                                 8.37

(a) See Table 2.

(b) Mineral mix: each 1000 g of diet contained NaCl, 0.4 g;
MgS[O.sub.4-] 7[H.sub.2]O, 6.0 g: Na[H.sub.2]P[O.sub.4-]2[H.sub.2]O,
10 g; K[H.sub.2]P[O.sub.4], 12.8 g: Ca]([H.sub.2]P[O.sub.4]).sub.2-]
[H.sub.2]O, 8 g: Fe citrate, 1.0 g: Ca lactate, 1.4 g; ZnS[O.sub.4-]
7[H.sub.2]O, 141.4 mg: MnS[O.sub.4-] 4[H.sub.2]O, 64 mg:
CuS[O.sub.4-]5[H.sub.2]O, 12.4 mg; Co[Cl.sub.2-] 6[H.sub.2]O, 0.4 mg;
KI[O.sub.3], 1.2 mg.

(c) Soybean oil and menhaden fish oil (1:1) with 0.001% ethoxyquin.

TABLE 2.

Composition of vitamin mix and Ca-pantothenic acid (Ca-PA) in the
diet (on dry weight basis).

                                     Actual Ca-PA Content in
Vitamin Mix (Without Ca-PA) (a)      Test Diet (mg/kg Diet)

  Vitamin         1000 g Diet      Diet           Ca-PA

Thiamin HCl          120 mg       D0                0
Riboflavin           100 mg       D10        9.4 [+ or -] 0.87
Folic acid            30 mg       D20       19.8 [+ or -] 2.35
PABA                 400 mg       D40       39.7 [+ or -] 3.74
Pyridoxine HCl        40 mg       D80       79.4 [+ or -] 6.88
Niacin               200 mg       D160     158.5 [+ or -] 10.73
Inositol            4000 mg
Biotin                12 mg
Vitamin E            450 mg
Menadione            890 mg
V[B.sub.12]         0.18 mg
Ascorbic acid       4000 mg
Retinol acetate   I00000 IU
Cholecalciferol     2000 1U

(a) 400 mg ethoxyquin was used as an antioxidant.

TABLE 3.

Retention of Ca-pantothenic acid (%) of the experimental diets
immersed in sea water for different times.

                              Immersion Time
  Dietary Ca-PA
  (mg/kg Diet)          0                    1

        0            -- (a)                 --
       9.8           100        90.7 [+ or -] [3.8.sup.a] (2)
      19.8           100        90.3 [+ or -] [3.0.sup.a]
      39.7           100        89.5 [+ or -] [3.6.sup.a]
      79.4           100        88.2 [+ or -] [3.2.sup.a]
      158.5          100        87.0 [+ or -] [2.7.sup.a]
200 (crystalline)    100        65.5 [+ or -] [3.9.sup.b]
      ANOVA
     F value                            15.421
     P value                             0.003

                             Immersion Time
  Dietary Ca-PA
  (mg/kg Diet)                     2
        0
       9.8             84.2 [+ or -] [2.1.sup.a]
      19.8             83.0 [+ or -] [3.4.sup.a]
      39.7             82.2 [+ or -] [4.2.sup.a]
      79.4             81.3 [+ or -] [3.5.sup.a]
      158.5            78.9 [+ or -] [2.6.sup.a]'
200 (crystalline)      53.5 [+ or -] [3.7.sup.b]
      ANOVA
     F value                    20.522
     P value                     0.001

                             Immersion Time
  Dietary Ca-PA
  (mg/kg Diet)                     6
        0
       9.8             64.4 [+ or -] [4.1.sup.a]
      19.8             63.0 [+ or -] [3.7.sup.a]
      39.7             62.2 [+ or -] [2.5.sup.a]
      79.4             61.7 [+ or -] [4.2.sup.a]
      158.5            59.4 [+ or -] [3.3.sup.a]
200 (crystalline)      38.5 [+ or -] [2.6.sup.b]
      ANOVA
     F value                    22.384
     P value                     0.001

                             Immersion Time
  Dietary Ca-PA
  (mg/kg Diet)                     12
        0
       9.8             44.2 [+ or -] [3.2.sup.a]
      19.8             43.4 [+ or -] [3.4.sup.a]
      39.7             42.4 [+ or -] [3.0.sup.a]
      79.4             41.1 [+ or -] [1.9.sup.a]
      158.5            40.5 [+ or -] [2.4.sup.a]
200 (crystalline)      28.5 [+ or -] [1.7.sup.b]
      ANOVA
     F value                    28.671
     P value                     0.000

(a) Not detectable.

(2) Means in each column not sharing a common superscript are
significantly different according to Tukey test (P < 0.05).
Mean [+ or -] SE, n = 3.

TABLE 4.

Growth and survival of abalone, Haliotis discus hannai, fed diets
containing different levels of pantothenic acid for 16 weeks.

Dietary PA          Initial Weight        Initial Shell
(mg/kg Diet)             (mg)              Length (mm)

     0           543.3 [+ or -] 32.1    15.49 [+ or -] 0.37
     9.8         547.8 [+ or -] 10.7    15.57 [+ or -] 0.15
    19.8         596.7 [+ or -] 12.0    15.78 [+ or -] 0.39
    39.7         533.3 [+ or -] 14.5    15.26 [+ or -] 0.39
    79.4         555.6 [+ or -] 28.3    15.44 [+ or -] 0.23
   158.5         543.1 [+ or -] 63.8    15.36 [+ or -] 0.65
   ANOVA
  F value               1.417                  0.636
  P value               0.287                  0.677

Dietary PA               Final Weight
(mg/kg Diet)                 (mg)

     0          1044.7 [+ or -] [94.9.sup.a] (3)
     9.8        1202.3 [+ or -] [65.7.sup.ab]
    19.8        1477.4 [+ or -] [80.9.sup.b]
    39.7        1317.2 [+ or -] [76.9.sup.ab]
    79.4        1440.4 [+ or -] [104.4.sup.b]
   158.5        1321.2 [+ or -] [78.4.sup.ab]
   ANOVA
  F value                    7.120
  P value                    0.003

Dietary PA                Final Shell
(mg/kg Diet)              Length (mm)

     0           20.58 [+ or -] [0.02.sup.a]
     9.8         21.48 [+ or -] [0.48.sup.ab]
    19.8         22.95 [+ or -] [0.53.sup.b]
    39.7         22.17 [+ or -] [0.63.sup.ab]
    79.4         22.72 [+ or -] [0.56.sup.b]
   158.5         22.59 [+ or -] [0.86.sup.b]
   ANOVA
  F value                    4.998
  P value                    0.010

Dietary PA
(mg/kg Diet)              WG (1) (%)

     0           92.0 [+ or -] [13.4.sup.a]
     9.8        119.4 [+ or -] [10.7.sup.a]
    19.8        147.8 [+ or -] [19.2.sup.ab]
    39.7        146.8 [+ or -] [11.3.sup.ab]
    79.4        158.9 [+ or -] [9.99.sup.b]
   158.5       143.97 [+ or -] [10.6.sup.ab]
   ANOVA
  F value                   10.960
  P value                    0.000

Dietary PA                DISL (2)
(mg/kg Diet)          ([micro] m/day)

     0          45.5 [+ or -] [2.74.sup.a]
     9.8        52.7 [+ or -] [4.24.sup.ab]
    19.8        64.0 [+ or -] [2.79.sup.b]
    39.7        62.3 [+ or -] [3.73.sup.b]
    79.4        65.1 [+ or -] [4.52.sup.b]
   158.5        64.6 [+ or -] [5.34.sup.b]
   ANOVA
  F value                8.138
  P value                0.001

Dietary PA               Survival
(mg/kg Diet)                (%)

     0              96.7 [+ or -] 2.74
     9.8            97.8 [+ or -] 3.16
    19.8            90.0 [+ or -] 0.00
    39.7            95.5 [+ or -] 5.63
    79.4            97.8 [+ or -] 3.16
   158.5            97.8 [+ or -] 3.16
   ANOVA
  F value                  1.062
  P value                  0.429

(1) Weight gain.

(2) Daily increment of shell length.

(3) Means in each column not sharing a common superscript are
significantly different according to Tukey's test (p < 0.05).
Means [+ or -] S.E., n = 3.

TABLE 5.

Soft body composition of juvenile abalone, H. discus hannai fed diets
with graded levels of Ca-Pantothenic acid for 16 weeks.

Number         Dietary Ca-PA          Soft Body
of Diets        (mg/kg Diet)       Protein (%) (1)

D0                  0             54.9 [+ or -] 4.1
D10                 9.8           53.3 [+ or -] 3.4
D20                19.8           52.8 [+ or -] 1.7
D40                39.7           55.0 [+ or -] 2.5
D80                79.4           52.7 [+ or -] 4.6
D160              158.5           53.5 [+ or -] 3.2
ANOVA
  F value                               0.469
  P value                               0.793

                                         Soft Body
Number            Soft Body               Moisture
of Diets      Lipid [(%).sup.a]             (%)

D0            7.5 [+ or -] 0.14      75.7 [+ or -] 3.4
D10           7.9 [+ or -] 0.42      79.5 [+ or -] 5.7
D20           7.6 [+ or -] 0.23      76.3 [+ or -] 2.8
D40           7.5 [+ or -] 0.30      78.5 [+ or -] 3.6
D80           8.1 [+ or -] 0.27      77.4 [+ or -] 2.9
D160          7.8 [+ or -] 0.51      79.4 [+ or -] 4.6
ANOVA
  F value           1.982                  0.582
  P value           0.143                  0.824

(1) On dry weight basis.

Means [+ or -] S.E., n = 3.

TABLE 6.

Pantothenic acid (PA) in viscera and muscle of juvenile abalone,
Haliotis discus hannai fed diets with graded levels of PA for
16 weeks.

Dietary       Dietary PA            Visceral PA
Number         (mg/kg)              ([micro]g/g)

0                0          5.82 [+ or -] [0.65.sup.a] (1)
1                9.8        8.03 [+ or -] [0.87.sup.ab]
2               19.8        9.75 [+ or -] [1.23.sup.b]
3               39.7       10.74 [+ or -] [1.25.sup.b]
4               79.4       10.48 [+ or -] [1.29.sup.b]
5              158.5       11.02 [+ or -] [1.10.sup.b]
ANOVA
  F value                             6.859
  P value                             0.003

Dietary            Muscle PA
Number            ([micro]g/g)

0              1.25 [+ or -] [0.19.sup.a]
1              2.52 [+ or -] [0.20.sup.b]
2              3.76 [+ or -] [0.21.sup.c]
3              4.29 [+ or -] [0.28.sup.cd]
4              4.71 [+ or -] [0.29.sup.d]
5              4.53 [+ or -] [0.29.sup.cd]
ANOVA
  F value                 59.65
  P value                  0.000

(1) Means in each column not sharing a common superscript are
significantly different according to Tukey test (p < 0.05).
Means [+ or -] SE, n = 3.

ACKNOWLEDGMENTS

The authors thank Mr. Hongming Ma and Ms. Wei Xu for their kind assistance in the biochemistry analysis and Mr. Shaobo Du for their assistance in the feeding experiment. This work is supported by grants from the National Science Fund for Distinguished Young Scholars (NSFC, No.39925029), and from the Excellent Young Teachers Program of MOE, P. R. China.

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WEI ZHU, KANGSEN MAI, * BEIPING TAN, AND GETIAN WU

The Key Lab of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, People's Republic of China

* Corresponding author. Fax: [phone]; E-mail: [e-mail]