230 B
. Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
1. Introduction
Vitamin K is essential for the synthesis of clotting factors in the livers of animals. Its metabolic role has been more clearly defined than that of the other three fat-soluble
vitamins in terrestrial animals even though it was the last of the fat-soluble vitamins to be discovered Suttie, 1980; Kormann and Weiser, 1983; Udagawa et al., 1993. The
classical role of vitamin K has been in the maintenance of normal hemostasis. Many types of compounds, such as phylloquinone PK , menaquinones VK and menadione
1 2
VK exhibit vitamin activity Udagawa et al., 1993. Phylloquinone vitamin K is
3 1
synthesised by plants and algae, whereas the menaquinone family MK vitamin K
2n 2
are products of bacterial biosynthesis. Water-soluble salts of the synthetic menadione are used in animal diets. Fish diets are commonly supplemented with menadione sodium
bisulfite MSB. Menadione and its salts are usually converted to MK-4 in animal tissues Nestor and Conrad, 1990; Udagawa et al., 1993; Grahl-Madsen and Lie, 1997.
In mammals, the requirement for vitamin K is met by a combination of dietary intake and microbiological synthesis in the intestine, but composition of diets and antibiotics
affect intestinal production Mathers et al., 1990. The effect of dietary vitamin K on physiological role and requirement has been
studied in several species of fishes and crustaceans. Vitamin K deficiency results in anemia and prolonged coagulation time in fish Halver, 1989; NRC, 1993. Menadione
is highly effective in preventing the molinate-induced anemia in common carp Kawatsu and Ikeda, 1988; Kawatsu et al., 1989. Menadione has been reported to be required for
larval kuruma shrimp, Penaeus japonicus Kanazawa, 1985, P
. monodon Shiau and Liu, 1994a and P
. chinensis Shiau and Liu, 1994b. However, the significance of intestinal production of vitamin K in fishes or crustaceans has not been established. In
21
salmonids, the vitamin K requirement for growth is suggested to be 10 mg kg dry diet
Halver, 1989. Increased blood clotting times, anaemia and hemorrhages in gills, eyes and vascular tissues have been reported in salmonids fed diets low in vitamin K or diets
supplemented with antibiotics Phillips et al., 1963; Kitamura et al., 1967; Halver, 1989. On the other hand, deficiency signs were not induced in channel catfish fed
antibiotics Murai and Andrews, 1977.
For mollusks, however, direct evidence is lacking in either the significance of intestinal production of vitamin K or dietary requirement for this vitamin. Haliotis discus
hannai is one of the most commercially important gastropods in aquaculture. At present, no information has been reported as to the essentiality or quantitative requirements of
vitamin K for this species. Therefore, the objective of this study was to investigate the effect of dietary vitamin K on survival, growth and tissue concentrations of
phylloquinone and or menaquinone-4 in the juvenile abalone, Haliotis discus hannai.
2. Materials and methods
2.1. Feed formulation and manufacture The basal diet formulation was given in Table 1. Casein and gelatin were used as
B . Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
231 Table 1
Ingredient and proximate composition of the basal diet Ingredient
Percent in diet Casein, vitamin-free Sigma Chemical, St. Louis, MO, USA
25.00 Gelatin Sigma Chemical, St. Louis, MO, USA
6.00 Dextrin Shanghai Chemical Co., Shanghai, China
34.00 Carboxymethylcellulose Shanghai Chemical Co., Shanghai, China
5.00 Sodium alginate Shanghai Chemical Co., Shanghai, China
20.00
a
Vitamin mix 2.00
b
Mineral mix 4.00
c
SO MFO food grade 3.50
Choline chloride Shanghai Chemical Co., Shanghai, China 0.50
Proximate composition means of triplicate Crude protein
28.63 Crude lipid
3.60 Ash
8.88
a
Vitamin mix, see Table 2.
b
Mineral mix, each 1000 g of diet contained: NaCl, 0.4 g; MgSO ?7H O, 6.0 g; NaH PO ?2H O, 10.0 g;
4 2
2 4
2
KH PO , 12.8 g; CaH PO ?H O, 8.0 g; Fe-citrate, 1.0 g; Ca-lactate, 1.4 g; ZnSO ?7H O, 141.2 mg;
2 4
2 4 2
2 4
2
MnSO ?H O, 64.8 mg; CuSO ?5H O, 12.4 mg; CoCl ?6H O, 0.4 mg; KIO , 1.2 mg.
4 2
4 2
2 2
3 c
Soybean oil and menhaden fish oil 1:1 with 0.01 ethoxyquin.
protein sources. Crude protein level of the experimental diets was about 30, which is considered to be sufficient to maintain optimum growth for Haliotis discus hannai Mai
et al., 1995b. Soybean oil and menhaden fish oil 1:1 was used as the basal lipid sources. Dietary lipid level was about 3.5, which was sufficient to support optimum
growth and provide enough essential fatty acids EFA for abalone Mai et al., 1995a. Dextrin was the major carbohydrate source. The compositions of vitamin mixture were
similar to those used by Uki et al. 1985, except that it did not contain vitamin K. Menadione sodium bisulfite MSB Sigma Chemicals was added to the test diets at the
expense of small amounts of dextrin to provide concentrations of 0, A-0 without MSB but supplemented with sulphaguanidine, Sigma Chemical, 10, 20, 40, 80, 160 and 320
21
mg kg diet.
Procedures for diet preparation were similar to those described by Mai et al. 1995a,b.
2.2. Animal rearing Juvenile abalone, Haliotis discus hannai used in this experiment were derived from a
spawning in June 1997, at Xunshan Fisheries Co., Shandong, China. Before trial, shell length was measured with calipers to the nearest 0.02 mm and the animals were weighed
to the nearest 0.01-g using an electronic balance. Animals were kept in acrylic square cages 20 3 20 3 20 cm. Each rearing unit was
stocked with 30 abalone juveniles mean weight 1.1860.04 g; mean shell length 18.6560.18 mm. Cages were assigned to a rectangle cement tank 5 3 2 m using a
completely randomized design with nine treatments and three replicates per treatment.
232 B
. Tan, K. Mai J. Exp. Mar. Biol. Ecol. 256 2001 229 –239
Seawater was pumped into a precipitating tank, then filtered to 30-mm by primary sand filters, followed to 10-mm by secondary composite sand filters. The system was
flow-through. The flow-rate was about 0.5-l per min per cage. During the experimental period, water temperature ranged from 8.6 to 26.48C, salinity 30–34‰, pH 7.6–7.9.
21
Dissolved oxygen was not less than 7 mg l , and there were negligible levels of free
ammonia and nitrite. Prior to initiation of the experiment, the abalone underwent a 2-week conditioning
period during which they readily adjusted to a vitamin K-depleted basal diet Table 1 and standardized environmental conditions. The feeding trial was run for 120 days.
Abalone were hand-fed with the experimental diets at a rate equaling 5–10 of wet body weight per day, once daily at 17:00. Every morning, uneaten feed and feces were
cleaned to maintain water quality.
2.3. Sample collection and analyses At the termination of the experiment, animals were not fed for 3 days, then all abalone
were removed from the cage, weighed, measured and counted. Then, 15 abalone from each replicate were frozen 2708C for subsequent analyses. Growth was expressed as
21
weight gain rate WGR, and daily increment in shell length DISL, mm day . The
calculation formulae are as follows: WGR 5 [W 2 W W ] 3 100
t i
i
Table 2 Composition of vitamin mix and vitamin K in the experimental diets on dry weight basis
a 21
Vitamin mix without vitamin K Vitamin K in test diets mg MSB kg
diet Vitamin
100 g mix 1000 g diet
Diet Sulphaguanidine
MSB Thiamin HCl
0.6 g 120.0 mg
D0 Riboflavin
0.5 g 100.0 mg
DA-0 10 000
Folic acid 0.15 g
30.0 mg D10
10.0 PABA
2.0 g 400.0 mg
D20 20.0
Pyridoxine HCl 0.2 g
40 mg D40
40.0 Niacin
4.0 g 800.0 mg
D80 80.0
Ca pantothenate 1.0 g
200.0 mg D160
160.0 Inositol
20.0 g 4000.0 mg
D320 320.0
b
Biotin 60.0 mg
12.0 mg L
. japonica 0.66
Vitamin E 2.25 g
450.0 mg Ascorbic acid
20.0 g 4000.0 mg
B12 900.0 mg
180.0 mg Retinol acetate
500 000 IU 100 000 IU
Cholecalciferol 10 000 IU
2000 IU
a
Dextrin was used as a filler to prepare 100 g vitamin mix, in which 2.0 g of ethoxyquin was used as an antioxidant.
b
Presented in the form of phylloquinone PK as determined by the HPLC method modified from that described by Udagawa et al. 1993.
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
