Influence of dietary lipid sources on growth and fatty acid composition of juvenile abalone, Haliotis discus hannai Ino.

A study was conducted to evaluate the effects of dietary lipid sources on the growth and fatty acid composition of juvenile abalone Haliotis discus hannai Ino. Four triplicate groups of juvenile abalone (initial weight: 0.13 [+ or -] 0.04 g; initial shell length: 10.23 [+ or -] 1.48 mm) were led with one of four semipurified diets containing 3.5% of dietary lipid from either tripalmitin (TP), soybean oil (SO), linseed oil (LO), or 20:5n-3-enriched fish oil (FO), respectively, and were reared for 120 days in a recirculation water system. Results showed that the growth rate of abalone was significantly affected by dietary lipid sources (P < 0.05). The dietary lipid, FO produced the highest weight gain rate (WGR, 413.2%), closely followed by LO (389.0%) and SO (382.4%). These three WGR values were not significantly different to each other. However, abalone fed the TP diet showed a significantly lower WGR (267.3%). Carcass moisture and protein were independent of dietary treatment, but the TP diet resulted in significantly lower carcass lipid. The fatty acid profile in abalone carcass reflected that of dietary lipids, especially the unsaturated fatty acids. It appears that H. discus hannai have a capacity to synthesize 20:5n-3, 20:4n-6 and 22:6n-3 from 18-carbon PUFAs and even from 16:0 through elongation and desaturation. However, the capacity seems to be insufficient to support the maximum growth of abalone.

KEY WORDS: Haliotis discus hannai, lipid sources, fatty acid composition, growth, feeding and nutrition mollusk 

INTRODUCTION

Dietary lipid plays important roles in providing concentrated energy, essential fatty acids (EFA) and some other nonfat nutrients to organisms. The qualitative and quantitative requirements of fish and crustaceans have been extensively studied (NRC 1993, Sargent et al. 2002). Previous studies have demonstrated that dietary lipid sources may influence the growth, survival, feed efficiency, and fatty acid composition of aquatic animals including fish and shrimp, and that the qualitative and quantitative requirements of fish and crustaceans for lipids considerably differ with different species (NRC 1993, D'Abramo 1997, Sargent et al. 2002). Results suggest that many marine fish and prawns require n-3 highly unsaturated fatty acids (HUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to maintain normal physiology and growth. For some marine species, it is believed that DHA appears to be superior to EPA (Watanabe 1993, Mourente & Toche 1993, Xu et al. 1994, Rodriguez et al. 1997).

For mollusks, however, less is known about the nutritional value of different lipid sources. According to available information, the polyunsaturated fatty acids (PUFA) of n-3 and n-6 families are able to enhance the growth of Haliotis discus hannai and H. tuberculata. Especially, 20:5n-3 played a more prominent role in abalone nutrition (Uki et al. 1986, Mai et al. 1996a, Mai et al. 1996b). It is suggested that with different EFA composition, lipids from different sources have different nutritional values for abalone. However, no significant differences were found in the growth of H. fulgens fed the different oil types, olive, corn, linseed, and cod liver oils (Durazo-Beltran et al. 2003). There is no information available about the effects of different lipid sources on the growth of H. discus hannai. This study was conducted to evaluate the effects of different dietary lipid sources on the growth, survival, and carcass composition of juvenile abalone H. discus hannai, which would provide useful information for abalone aquaculture.

MATERIALS AND METHODS

Experimental Diets and Design

Experimental basal diet was formulated to provide 28% crude protein from casein and gelatin (Table 1), which is considered to be sufficient to maintain optimum growth for H. discus hannai (Mai et al. 1995a). Tripalmitin (TP), soybean oil (SO), linseed oil (LO) or fish oil (FO) was individually added to the basal diet to produce four experimental diets, respectively. Dietary lipid level was maintained at 3.5%, which was considered to be an optimal level for this abalone (Mai et al. 1995b). The main fatty acid compositions of SO, LO and FO were determined by GC, and the results are given in Table 2. Procedures for diet preparation and storage were the same as those previously described by Mai et al. (1995a).

Animal Rearing

Abalone (initial weight: 0.13 [+ or -] 0.04 g; initial shell length: 10.23 [+ or -] 1.48 mm) were derived from a spawning at Mashan Fisheries Co. Shandong, China. Shell length was measured with calipers to the nearest 0.02 mm. The individual abalone was superficially dried with tissues and then weighed to 0.01 g using an electronic balance. Growth trial was conducted in a recirculation water system. Twenty-five juvenile abalone were stocked in a rearing unit, a plastic basket (20 cm x 20 cm x 10 cm) placed in a glass aquarium (45 cm x 25 cm x 35 cm), as one replicate. There were three replicates for each of the four dietary treatments.

Prior to initiation of the experiment, abalone were placed in glass aquariums and conditioned with the basal diet for 1 wk. Animals were hand-fed with the test diets at a rate equaling 5% to 10% of wet body weight once daily at 17:00. Every morning, feces and excess feed were removed to maintain water quality. The feeding trial was run for 120 days. During the 120-day experiment, water temperature was maintained at 18[degrees]C to 25[degress]C, salinity 31 to 35, pH 7.6 to 8.0. Dissolved oxygen was not less than 6 mg/L, and there were negligible levels of free ammonia and nitrite.

Sample Collection and Analysis

At the termination of the experiment, animals were not fed for 3 days. All abalone were collected from the cages, counted, weighed, and measured then immediately frozen (-70[degrees]C) for subsequent analyses. Growth was expressed as weight gain rate (WGR, %) and daily increment in shell length (DISL, [micro]m/d). The calculation formulae are as follows:

WGR (%) = (Wt-Wi)/Wi x 100

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

Where, Wt and Wi are final and initial mean weight (g), respectively; SLt and SLi are final and initial mean shell length (mm), respectively.

The procedures for analysis of the fatty acid profiles in lipid sources and abalone carcass were modified from those described by Metcalfe et al. (1966). Briefly, fatty acid methyl esters were separated and quantified by the gas chromatograph equipment (HP5890) with a fused silica capillary column (007-CW, HP) and a flame ionization detector. The column temperature was programmed to rise from 150[degrees] up to 200[degrees] at a rate of 15[degrees] [min.sup.-1], from 200[degrees] to 250[degrees] at a rate of 2[degrees] [min.sup.-1].

Proximate analyses of the diets and abalone soft-body samples to determine protein, lipid, ash, and moisture levels were conducted using standard procedures (AOAC 1995).

Statistical Analysis

All percentage data were square-root arcsine transformed prior to analysis. Data were submitted to 1-way analysis of variance using the STATISTICA[TM] package. When significant differences (P < 0.05) were found, means were compared using the Tukey's test.

RESULTS

Survival and Growth

The data of abalone survival and growth performances are presented in Table 3. Analysis of variance showed that there was no significant difference in survival, which ranged from 90% to 96%, among the dietary treatments. However, weight gain rate (WGR) of the animals was significantly affected by the different lipid sources. Abalone fed tripalmitin (TP) diet had a significantly lower WGR (267.32%). Meanwhile, those fed the diet with EPA-enriched fish oil (FO) showed the highest WGR (413.19%). Soybean oil (SO) and linseed oil (LO) resulted in the WGRs of 382.43% and 389.04%, respectively. These later three WGR values were not significantly different from each other. The trend of daily increment in shell length (DISL) changing with different dietary lipids was similar to that of WGR. However, the effects of dietary lipids on DISL were not significant.

Fatty acid Composition in Carcass

Fatty acid composition in abalone carcass is shown in Table 4. Different dietary lipids significantly influenced the fatty acid patterns in carcass, particularly the polyunsaturated fatty acids (PUFA). Abalone fed TP accumulated a higher level of palmitic acid (16:0) in tissues. There were significantly higher levels of EPA (20:5n-3, 12.0%) and DHA (22:6n-3, 2.6%) in the abalone fed diet with FO than those in all other treatments. The highest values of linoleic acid (18:2n-6) and arachidonic acid (20:4n-6, ARA) were found to be 1.64% and 6.03%, respectively, in abalone fed the diet with SO. Abalone fed LO had the highest values of linolenic acid (18:3n-3, 2.97%), and had a significantly higher level of EPA (7.86%) than those fed the diet with TP (5.87%) or with SO (4.61%), but it was significantly lower than that resulted from feeding FO diet (12.0%). Contents of carcass EPA in all experimental treatments were generally higher than those of DHA.

Carcass Proximate Composition

Data on abalone carcass moisture, protein, and lipid contents are given in Table 5. Among the dietary treatments, there were no significant differences in abalone carcass moisture ranging from 77.1% to 78.1%, and in abalone carcass protein ranging from 64.8% to 66.2%. However, the significant effect of the dietary treatments on abalone carcass lipid contents was observed. The diet containing TP resulted in significantly lower carcass lipid (4.7%) than those containing either SO (6.1%), LO (6.0%) or FO (5.6%).

DISCUSSION

Previous studies on the requirement of H. discus hannai for essential fatty acids using purified diets or macroalgae demonstrated that EPA (20:5n-3) played a prominent role in the nutrition of abalone, and linoleic acid together with other n-3 and n-6 PUFA also contributed to the faster growth of 14. discus hannai (Uki et al. 1986, Mai et al. 1996a, Mai et al. 1996b). During the 120-day feeding trial of the present study, the mean body weights of abalone in all the four treatments increased by high percentages, from 267.3% to 413.1%. This means that, on one hand, the carcass storage of essential fatty acids are probably able to support abalone growth for a long period. On the other hand, abalone is probably able to biosynthesize essential fatty acids, to a certain extent, from different lipid sources. It can be seen from Table 4 that the carcass of abalone fed the TP diet showed fairly high levels of 18:2n-6, 20:4n-6 and 20:5n-3, indicating that active elongation and desaturation of palmitic acid take place in H. discus hannai. Similarly, the highest level of 20:4n-6 in carcass was found in the treatment with dietary SO that contains the highest level of 18:2n-6 (50.6%), and significantly higher carcass level of 20:5n-3 was observed in abalone fed the diet with LO, which contains the highest level of 18:3n-3 (49.0%), than those fed diets with TP or SO. It is suggested that H. discus hannai have the ability to bio-convert 18:2n-6 to 20:4n-6, and 18:3n-3 to 20:5n-3. Similar findings have been reported in H. laevigam (Dunstan et al. 1996) and H. fulgens (Durazo-Beltran et al. 2003).

Abalone is probably able to biosynthesize highly unsaturated fatty acids (HUFA), to a certain extent, by elongation and desaturation of short-chain fatty acids, however, the differences in weight gain rate were observed among the dietary treatments of different lipid sources (Table 3). This is probably due to the fact that the capacity of abalone to biosynthesize essential fatty acids is insufficient to support normal growth, and that biosynthesis of essential fatty acids is an energy-consuming process that could suppress the growth of abalone fed diets with short-chain fatty acids. The present study and previous ones have demonstrated that HUFAs are superior to short-chain PUFAs in enhancing abalone growth (Uki et al. 1986, Mat et al. 1996a, Mat et al. 1996b). Lipid sources, rich in HUFA, mainly come from marine origins, such as fish oils. However, fish oils are increasingly in short supply globally, and intensive studies are underway on fish oil substitution in the aquaculture feed industry. Hence, further studies should be conducted to determine the optimal ratio of fish oils to plant oils in diets for abalone. Durazo-Beltran et al. (2003) did not find significant differences in the growth of H. fulgens fed the different oil types (olive, corn, linseed, and cod liver oils). The authors assumed that the lack of a growth response to the different lipid sources and different levels of HUFA may be due to insufficient duration (75 days) of the experiment to achieve an essential fatty acid deficiency.

Fatty acid biosynthesis in fish and crustaceans has been extensively studied and it has been shown that the overall conversion of 18-carbon PUFA to HUFA occurs poorly in the marine species studied so far (Ito & Simpson 1996, D'Abramo 1997, Sargent et al. 2002). For abalone, however, there is still no direct evidence on the pathways of fatty acid biosynthesis. Further investigations are needed to elucidate detailed pathways of fatty acid biosynthesis and their regulation in abalone and other mollusks.

TABLE 1.

Ingredient and proximate analysis of the experimental diets (g/100 g
dry wt.).

    Ingredients         TP       SO       LO       FO

Casein                 25.00    25.00    25.00    25.00
Gelatin                 6.00     6.00     6.00     6.00
Dextrin                33.00    33.00    33.00    33.00
Na alginate            20.00    20.00    20.00    20.00
Choline chloride        0.50     0.50     0.50     0.50
CM-cellulose            5.00     5.00     5.00     5.00
vitamin mix (a)         2.00     2.00     2.00     2.00
Mineral mix (b)         5.00     5.00     5.00     5.00
Tripalmitin             3.50     1.50     1.50     1.50
Soybean oil             0.00     2.00     0.00     0.00
Linseed oil             0.00     0.00     2.00     0.00
Fish oil                0.00     0.00     0.00     2.00
Proximate analysis
  Protein              28.71    28.22    28.28    28.93
  Lipid                 3.54     3.28     3.53     3.43
  Ash                   9.40     9.24     9.03     8.93

(a) Vitamin mix: each 1000 g of diet contained thiamin HCl, 120 g;
riboflavin, 100 mg; folic acid 30 mg; PABA, 400 g; niacin, 200 mg;
Ca pantothenate, 200 mg; inositol, 4000 mg; biotin, 12 mg;
vitamin E, 450 mg, menadione, 890 mg; V[B.sub.12], 0.18 mg;
ascorbic acid, 4000 mg; retinel acetate, 100000 IU; cholecalciferol,
2000 IU; ethoxquin, 400 mg.

(b) Mineral mix: each 1000 g of diet contained NaCl, 0.4 g;
MgS[O.sub.4] x 7[H.sub.2]O, 6.0 g; Na[H.sub.2]P[O.sub.4] x
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]) x [H.sub.2]O, 8 g; Fecitrate, 1.0 g;
Ca-lactate 1.4 g; ZnS[O.sub.4] x 7[H.sub.2]O, 141.4 mg;
MnS[0.sub.4] x 4[H.sub.2]O, 64 mg; CuS[O.sub.4] x 5[H.sub.2]O, 12.4 mg;
Co[Cl.sub.2] x 6[H.sub.2]O, 0.4 mg; KI[O.sub.3], 1.2 mg.

TABLE 2.

Fatty acid composition of dietary lipid sources (%area).

                     Lipid Source (b)

Fatty Acid       SO        LO        FO

14:0            0.51      0.22       ND
16:0            9.42      6.41       ND
16:1            0.24      0.16       ND
18:0            3.82      3.58      2.02
18:1n-9        21.81     21.31      3.43
18:1n-7         0.90      1.20      1.05
18:2n-6        50.60     15.49      0.82
18:3n-3         5.70     49.03      3.11
20:4n-6          ND        ND       1.09
20:5n-3          ND        ND      49.77
22:6n-3          ND        ND      21.60

ND, undetectable (<0.01 g/100 g fatty acids): SO, soybean oil: LO,
linseed oil; FO, fish oil.

TABLE 3.

Effects of the experimental diets supplemented with different lipid
sources on growth, survival of juvenile abalone, Haliotis discus
hannai.

             Initial Weight               Final Weight
Diets            (mg)                         (mg)

TP        132.00 [+ or -] 8.51    508.83 [+ or -] [12.27.sup.b]
SO        128.70 [+ or -] 8.60    620.12 [+ or -] [30.49.sup.a]
LO        125.33 [+ or -] 8.20    610.53 [+ or -] [25.86.sup.a]
FO        131.33 [+ or -] 7.57    671.50 [+ or -] [34.30.sup.a]
ANOVA
  F               0.41                        19.01
  P               0.75                         0.00

                   Weight Gain                Initial Shell
Diets               Rate (%)                   Length (mm)

TP       267.32 [+ or -] [7.89.sup.b]     10.35 [+ or -] 0.11
SO       382.43 [+ or -] [19.36.sup.a]    10.15 [+ or -] 0.06
LO       389.04 [+ or -] [46.82.sup.a]    10.20 [+ or -] 0.06
FO       413.19 [+ or -] [53.09.sup.a]    10.21 [+ or -] 0.09
ANOVA
  F                  9.22                        3.00
  P                  0.01                        0.10

                   Final Shell                    DISL
Diets              Length (mm)                ([micro]m/day)

TP             17.35 [+ or -] 0.08        58.26 [+ or -] 1.36
SO             17.80 [+ or -] 0.57        63.28 [+ or -] 5.13
LO             18.02 [+ or -] 0.36        65.47 [+ or -] 3.21
FO             18.30 [+ or -] 0.42        67.14 [+ or -] 4.88
ANOVA
  F                   3.04                        2.86
  P                   0.09                        0.10

                    Survival

Diets               Rate (%)

TP              92 [+ or -] 6.93
SO              92 [+ or -] 4.00
LO              90 [+ or -] 9.24
FO              96 [+ or -] 0.00
ANOVA
  F                   0.43
  P                   0.74

DISL, daily increment in shell length.

TP, Tripalmitin; SO, Soybean oil; LO, linseed oil: FO, fish oil.

[sup.a-b] Means in the same column sharing a common superscript letter
were not significantly different (P > 0.05) as determined by
Tukey's test. Mean [+ or -] SD, n = 3.

TABLE 4.

Effects of the experimental diets supplemented with different lipid
sources on fatty acid compositions in the carcass of juvenile abalone,
Haliotis discus hannai.

Fatty Acid             TP                             SO

14:0          2.65 [+ or -] 0.09             2.08 [+ or -] 0.30
16:0         18.99 [+ or -] 1.99            15.57 [+ or -] 1.19
16:1          1.15 [+ or -] 0.06             0.97 [+ or -] 0.02
18:0          5.29 [+ or -] 1.22             5.85 [+ or -] 0.38
18:1n-9       7.43 [+ or -] 0.24             6.71 [+ or -] 0.41
18:1n-7       5.55 [+ or -] 0.41             4.48 [+ or -] 0.28
18:2n-6       1.29 [+ or -] [0.09.sup.ab]    1.64 [+ or -] [0.07.sup.a]
18:3n-3       0.49 [+ or -] [0.02.sup.b]     0.39 [+ or -] [0.02.sup.b]
20:4n-6       4.78 [+ or -] [0.90.sup.ab]    6.03 [+ or -] [0.52.sup.a]
20:5n-3       5.87 [+ or -] [0.64.sup.c]     4.61 [+ or -] [0.01.sup.c]
22:6n-3       0.89 [+ or -] [0.02.sup.b]     0.93 [+ or -] [0.03.sup.b]

Fatty Acid           LO                              FO

14:0          2.57 [+ or -] 0.37             2.90 [+ or -] 0.15
16:0         15.64 [+ or -] 0.42            15.29 [+ or -] 2.05
16:1         1 .05 [+ or -] 0.08             1.05 [+ or -] 0.08
18:0          5.69 [+ or -] 0.32             5.39 [+ or -] 0.64
18:1n-9       6.51 [+ or -] 0.58             5.95 [+ or -] 0.17
18:1n-7       5.34 [+ or -] 0.52             4.36 [+ or -] 0.37
18:2n-6       0.65 [+ or -] [0.04.sup.ab]    0.28 [+ or -] [0.01.sup.b]
18:3n-3       2.97 [+ or -] [0.49.sup.a]     0.58 [+ or -] [0.03.sup.b]
20:4n-6       4.03 [+ or -] [0.34.sup.b]     3.55 [+ or -] [0.46.sup.b]
20:5n-3       7.86 [+ or -] [0.36.sup.b]    12.00 [+ or -] [1.15.sup.a]
22:6n-3       1.28 [+ or -] [0.13.sup.b]     2.59 [+ or -] [0.10.sup.a]

                  ANOVA

Fatty Acid      F         P

14:0          1.02     0.43
16:0          3.77     0.06
16:1          0.15     0.92
18:0          0.17     0.91
18:1n-9       0.37     0.77
18:1n-7       1.74     0.23
18:2n-6       4.91     0.03
18:3n-3      54.95     0.01
20:4n-6       9.70     0.01
20:5n-3      53.48     0.00
22:6n-3      48.19     0.00

TP, Tripalmitin: SO, soybean oil; LO, linseed oil: FO, fish oil.

[sup.a-c] Means in the same line sharing a common superscript letter
were not significantly different (P > 0.05) as determined by Tukey's
test. Mean [+ or -] SD. n = 3.

TABLE 5.

Effects of different lipid sources on the carcass chemical
composition of juvenile abalone, Haliotis discus hannai.

Diet           Moisture               Protein

TP        77.81 [+ or -] 0.66    65.35 [+ or -] 0.42
SO        77.19 [+ or -] 0.42    64.83 [+ or -] 0.41
LO        77.50 [+ or -] 1.08    65.18 [+ or -] 0.86
FO        77.14 [+ or -] 0.85    66.17 [+ or -] 0.32
ANOVA
  F              0.731                  3.370
  P              0.591                  0.080

Diet                 Crude Lipid

TP            4.74 [+ or -] [0.11.sup.c]
SO            6.11 [+ or -] [0.25.sup.a]
LO            6.00 [+ or -] [0.18.sup.ab]
FO            5.56 [+ or -] [0.20.sup.b]
ANOVA
  F                  18.551
  P                   0.000

TP, Tripalmitin: SO, soybean oil: LO, linseed oil: FO, fish oil.

[sup.a-c] Means in the same column sharing a common superscript letter
were not significantly different (P > 0.05) as determined by Tukey
test. Mean [+ or -] SD, n = 3.

ACKNOWLEDGMENTS

This study was financially supported by grant No. 30200215 from the National Natural Science Foundation of China (NNSFC) and grant No. 39925029 from National Science Fund for Distinguished Young Scholars (NNSFC).

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WEI XU, KANGSEN MAI, * WENBING ZHANG, ZHIGUO LIUFU, BEIPING TAN, HONGMING MA, AND QINGHUI AI

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

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