B . Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
233
DISL 5 [SL 2 SL t] 3 1000
t i
Where, W , W are final and initial mean weight g, respectively; SL , SL are final and
t i
t i
initial mean shell length mm, respectively; t is the feeding trial period days. Proximate analyses of the samples to determine moisture, protein, lipid, ash and
calcium content were conducted using the standard procedures AOAC, 1984. The samples of abalone were slightly thawed, and shell and soft-body were separated.
The soft-body to shell ratio w w was calculated to provide an index of nutritional status for abalone Mai et al., 1995a. An aliquot of soft body tissue from each sample
was defatted and dried by a chloroform–methanol–ether process and the dried fat-free tissue DFFT was stored 2208C for subsequent nucleic acid analysis Bulow, 1970,
1971; Bulow et al., 1981. Nucleic acid was extracted from DFFT by the methods of Webb and Levy 1955. Analysis of RNA content basically followed the orcinol method
described by Schneider 1957 and DNA was determined by the Burton modification of the diphenylamine reaction Burton, 1956.
The rest of the soft body samples was used for measuring the content of phylloquinone PK and menaquinone-4 MK-4 with a reversed-phase HPLC method
modified from that described by Udagawa et al. 1993. In brief, the muscle and viscera was separated. About 5 g of the wet tissue was homogenized with 20 ml methanol.
Then, 30 ml n-hexane was added and the mixture was shaken for 5 min, followed by centrifugation at 8003g for 8 min. The upper layer was evaporated in a rotatory
evaporator 608C, followed by drying under nitrogen at 258C. The residue was dissolved in 0.2 ml methanol and store at 2208C for analysis. The HPLC system
consisted of an ultrasphere ODS C
column I.D. 5 mm, 4.63250 mm. The mobile
18 21
phase was methanol containing 2 n-hexane. The flow-rate was 1.0 ml min . K
vitamins were detected at 254 nm by UV spectrophotometer. Standard PK, MK-4 were purchased from Sigma Chemical Co. The n-hexane and methanol were of HPLC grade.
Vitamin K concentrations of the samples are expressed on a wet weight basis. All study was conducted in the absence of daylight by using brown glass.
2.4. Statistical analysis Data from each treatment were subject to one-way ANOVA. When overall differences
were significant at less than 5 level, Tukey’s test was used to compare the mean values between individual treatments. Statistical analysis was performed using the
STATISTICA
E package.
3. Results
3.1. Survival and growth Survival and growth data are presented in Table 3. During the 120-day experimental
period, the mean survival, ranging from 75.0 to 83.3, was relatively low, and not related to dietary levels of vitamin K ANOVA, P .0.05. No significant difference
234 B
. Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239 Table 3
Effect of dietary vitamin K on survival and growth of the abalone, Haliotis discus hannai fed diets containing different levels of vitamin K for 120 days means6S.D., n 53
Diet WGR
DISL SB S
RNA DNA Survival
21
mm day ratio
ratio
b ab
D0 54.6166.0
43.6266.7 1.6360.1
3.9960.1 76.6765.8
a a
DA-0 46.9664.2
38.6662.3 1.5860.1
3.1060.1 76.6765.8
b b
D10 57.6165.9
45.0666.8 1.6460.2
4.0560.1 81.6762.9
b b
D20 59.6067.2
46.6267.2 1.6660.1
4.0760.1 75.0060.0
b b
D40 56.8565.4
44.4764.1 1.6060.1
4.1460.1 75.00610.0
b b
D80 56.8265.0
47.7761.1 1.6460.1
4.0960.2 75.0065.0
ab ab
D160 52.7364.4
42.6762.9 1.6160.1
3.8960.1 75.00610.0
b b
D320 58.4865.8
43.1763.2 1.6960.1
4.1360.1 83.3362.9
b b
L . japonica
55.5165.2 42.1063.8
1.5960.1 4.1260.1
75.00610.0 ANOVA
F value 3.3912
2.0599 1.5520
4.1741 0.4963
P value 0.0175
0.1008 0.2627
0.0159 0.8432
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. WGR, weight gain rate; DISL, daily increment in shell length; SB S, soft body
to shell ratio w w; RNA DNA, muscle RNA to DNA ratio w w.
P .0.05 was observed in SB S ratio for abalone fed the diets containing different levels of vitamin K. The mean weight gain rate WGR, daily increment in shell length
DISL and muscle RNA DNA ratio of the abalone were not significantly P .0.05 affected by the varying levels of dietary vitamin K except that the abalone fed the diet
without MSB but with sulphaguanidine supplementation DA-0, showed a slightly lower WGR and RNA DNA ratio. The WGR, DISL and RNA DNA ratio ranged from
21
46.96 to 59.60, 38.66 to 47.77 mm day and 3.10 to 4.14, respectively.
3.2. Carcass composition Data on carcass composition are shown in Table 4. There were no significant
differences P .0.05 in soft body moisture , protein , dry weight basis, lipid , dry weight basis or ash , dry weight basis of the abalone fed diets containing
different levels of vitamin K. Soft body moisture ranged from 76.88 to 78.05. Soft body protein, lipid and ash ranged from 51.14 to 53.04, 7.17 to 7.62, and 11.07 to
11.76, respectively.
No significant difference P .0.05 in soft-body or shell calcium deposition was observed among dietary treatments Table 4.
3.3. Tissue concentrations of MK-4 and PK Tissue concentrations of menaquinone-4 MK-4 and phylloquinone PK of abalone
are shown in Table 5. No PK was detected either in muscle or in viscera of the abalone fed the various levels of MSB, whereas the abalone fed the fresh brown alga, Laminaria
B . Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
235 Table 4
Carcass composition of the abalone fed the diets containing different levels of vitamin K for 120 days means6S.D., n 53
a a
a a
Diet Moisture
Protein Lipid
Ash Ca deposition
Soft-body Shell
D-0 77.960.20
52.8260.50 7.4060.15
11.0760.12 0.6260.07
35.2860.22 D-A0
76.9760.18 51.7960.54
7.1760.20 11.6860.15
0.5860.04 36.6660.14
D-10 77.1560.12
52.6260.50 7.6260.12
11.5460.10 0.5660.09
35.7760.35 D-20
76.8860.15 51.8860.62
7.5360.10 11.7260.12
0.6160.07 34.4860.20
D-40 77.5060.10
51.1460.45 7.2860.15
11.3860.36 0.5560.07
36.2660.24 D-80
77.1060.16 53.0460.63
7.7460.11 11.5060.24
0.5260.04 36.5360.31
D-160 77.5860.46
52.5860.50 7.2560.10
11.4660.12 0.5660.06
35.9160.28 D-320
78.0560.14 51.7960.47
7.6260.16 11.7660.16
0.6360.10 36.4860.16
L . japonica
77.5860.21 52.4660.33
7.3860.12 11.4060.14
0.5960.05 36.9660.30
ANOVA F value
1.8568 1.1584
0.9976 1.6282
2.0956 1.0825
P value 0.2496
0.5042 0.5726
0.2633 0.1824
0.4819
a
Dry weight basis.
japonica, showed a relatively high levels of PK both in muscle and in viscera, the values
21
being 25.42 and 32.16 ng g , respectively.
Menaquinones as MK-4 were detected in muscle and viscera of abalone fed diets with MSB or the fresh brown alga. The level of MK-4 in muscle and viscera increased with
21
increasing dietary MSB up to 10 mg kg . The maximum level of MK-4 in muscle was
21 21
20.79 ng g for animals fed diet with 80 mg MSB kg
, whereas this value in viscera
21 21
was 35.13 ng g for animals fed 160 mg MSB kg
. The brown alga, Laminaria
Table 5 Concentrations of phylloquinone PK and menaquinone-4 MK-4 in muscle and viscera of the abalone fed
the diets containing different levels of vitamin K for 120 days means6S.D., n 53
21 21
Diet Muscle ng g
wet weight Viscera ng g
wet weight PK
MK-4 PK
MK-4
a a
D0 ND
10.4461.49 ND
16.160.94
a a
DA-0 ND
10.9462.38 ND
14.161.63
b b
D10 ND
16.2963.20 ND
27.2362.01
b b
D20 ND
18.7661.96 ND
34.4961.82
b b
D40 ND
18.5164.77 ND
33.3064.07
b b
D80 ND
20.7962.64 ND
31.7264.62
b b
D160 ND
18.4962.65 ND
35.1362.99
b b
D320 ND
19.4762.84 ND
33.4562.38
ab a
L . japonica
25.4264.74 14.3262.07
32.1663.04 16.3661.87
ANOVA F value
– 3.5744
– 19.3142
P value –
0.0356 –
0.0001
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. ND, not detected.
236 B
. Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
21
japonica, produced a relatively lower MK-4 content 14.32 ng g in muscle than those
21
resulting from feeding diets containing 10 mg MSB kg diet, but the difference was
not significant P .0.05. However, the abalone fed L . Japonica showed a significantly
21
P ,0.01 lower MK-4 content 16.36 ng g in viscera than those fed diets containing
21
10 mg MSB kg diet.
4. Discussion
