256 (2001) 229–239
www.elsevier.nl / locate / jembe
Effects of dietary vitamin K on survival, growth, and tissue
concentrations of phylloquinone (PK) and menaquinone-4
(MK-4) for juvenile abalone, Haliotis discus hannai Ino
* Beiping Tan, Kangsen Mai
Aquaculture Research Laboratory, College of Fisheries, Ocean University of Qingdao, Qingdao 266003, P.R. China
Received 14 June 2000; received in revised form 7 September 2000; accepted 26 October 2000
Abstract
The experiment was conducted to investigate the effects of dietary vitamin K on survival, growth and tissue vitamin K concentrations of abalone, Haliotis discus hannai. Eight purified diets were formulated to provide a series of graded levels of menadione sodium bisulfite (MSB) (0–320
21
mg kg diet). The brown alga, Laminaria japonica, was used as a control diet. Abalone juveniles of similar size (mean weight 1.1860.04 g; mean shell length 18.6560.18 mm) were distributed in a flow-through system using a completely randomized design with nine treatments and three replicate groups per treatment. Animals were hand-fed the appropriate diets once daily at 17:00 for a 120-day period. Survival and carcass composition were not significantly affected by the dietary treatments (P.0.05). Significantly lower growth rate was found in abalone fed diet supplemented with antibiotic (suphaguanidine) than in those fed the other diets, but no significant difference in growth rate related to dietary level of MSB was observed. The concentrations of menaquinone-4
21
(MK-4) in muscle and viscera increased with increasing dietary MSB up to 10 mg kg . No phylloquinone (PK) was detected in tissues except that the abalone fed the control diet, Laminaria
21
japonica, produced a relatively high level of PK. It seemed to be that the 10-mg MSB kg diet is sufficient to that allowing the maintenance of ‘steady-state’ tissue concentration in juvenile abalone. Dietary MSB was converted to MK-4 in abalone. Either MK-4 or PK deposited in the body is derived from food. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Haliotis discus hannai; Menaquinone-4; Nutrition; Phylloquinone; Vitamin K
*Corresponding author. Tel.:186-532-203-2282; fax:186-532-203-2799. E-mail address: kmai@mail.ouqd.edu.cn (K. Mai).
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 menadione1 2 (VK ) exhibit vitamin activity (Udagawa et al., 1993). Phylloquinone (vitamin K ) is3 1 synthesised by plants and algae, whereas the menaquinone family (MK2n) (vitamin K )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
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
Choline chloride (Shanghai Chemical Co., Shanghai, China) 0.50 Proximate composition (means of triplicate)
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.
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[(Wt2W ) /W ]i i 3100
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 0 0
Riboflavin 0.5 g 100.0 mg DA-0 10 000 0
Folic acid 0.15 g 30.0 mg D10 0 10.0
PABA 2.0 g 400.0 mg D20 0 20.0
Pyridoxine HCl 0.2 g 40 mg D40 0 40.0
Niacin 4.0 g 800.0 mg D80 0 80.0
Ca pantothenate 1.0 g 200.0 mg D160 0 160.0
Inositol 20.0 g 4000.0 mg D320 0 320.0
b
Biotin 60.0 mg 12.0 mg L. japonica 0 0.66
Vitamin E 2.25 g 450.0 mg Ascorbic acid 20.0 g 4000.0 mg
B12 900.0mg 180.0mg
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
DISL5[(SLt2SL ) /t]i 31000
Where, W , W are final and initial mean weight (g), respectively; SL , SL are final andt 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 C18 column (I.D. 5 mm, 4.63250 mm). The mobile
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 theSTATISTICAE package.
3. Results
3.1. Survival and growth
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., n53)
Diet WGR DISL SB / S RNA / DNA Survival
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
Table 4
Carcass composition of the abalone fed the diets containing different levels of vitamin K for 120 days (means6S.D., n53)
a a a a
Diet Moisture (%) Protein (%) Lipid (%) Ash (%) Ca deposition (%) Soft-body Shell
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., n53)
21 21
Diet Muscle (ng g wet weight) Viscera (ng g wet weight)
PK MK-4 PK MK-4
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
The growth rates achieved in our experiment were relatively low, mainly due to the greater variation of water temperature during the rearing period, ranging from 9.8 to 26.48C, in comparison to those reported by other authors (Uki et al., 1985; Uki and Watanabe, 1992; Mai et al., 1995a,b; Mai, 1998).
Information on the physiological role of vitamin K in aquatic animals is limited, and its necessity for survival and growth has not yet been established (Murai and Andrews, 1977; Kawatsu and Ikeda, 1988; Kawatsu et al., 1989; He et al., 1992; Shiau and Liu, 1994a,b; Grahl-Madsen and Lie, 1997). No study has been carried out on the synthesis, dietary requirements, and tissue concentrations of vitamin K in molluscs. This is the first report on the effects of dietary vitamin K on survival, growth and tissue vitamin K concentrations of abalone. No significant effects of dietary vitamin K on survival and growth of abalone were observed in the 120-day experiment except that the abalone fed antibiotic (DA-0) showed a reduced growth rate (Table 3). Toxic effects of high doses of menadione and its analogues have been observed in mammals (Suttie, 1991). However, Poston (1971) did not observe any adverse effects on growth rates of brook
21
trout fed high dietary levels (100–2400 mg MSB kg diet). In the present study, no detrimental effect of high doses of vitamin K was observed. From these results, it cannot be concluded that dietary vitamin K is essential for this mollusc species. Due to the relatively slow growth of abalone, a longer experimental duration and younger abalone should be used for further evaluate the nutritional functions of vitamin K in abalone.
In this study, only PK and MK-4 were measured because no rich source of typical vitamin K-synthesizing microorganisms has been described in the intestines of cultured fishes (Margolis, 1953). Will et al. (1992) have also reported that long-chain mena-quinones (MK-5 to MK-13) of gut origin makes a relatively minor contribution to the hepatic stores of vitamin K in rats and chicks.
The tissue concentrations of MK-4 increased with increasing dietary MSB up to 10
21 21
mg kg . This suggested that the 10-mg MSB kg diet is sufficient to that allowing the maintenance of ‘steady-state’ tissue concentration in juvenile abalone, Haliotis discus
21
hannai. If the 10-mg MSB kg diet is taken as the optimal dietary concentration for abalone, this requirement is similar to that reported for salmonids (Halver, 1989), but much lower than the values reported for shrimps (Shiau and Liu, 1994a,b).
The fact that MK-4, but not menadione, was detected in the tissue of abalone fed the experimental diets indicated that dietary MSB converted to MK-4 in the body of abalone, as has been known to take place in mammals (Billeter et al., 1964), cultured sardine (Udagawa et al., 1993) and cod (Grahl-Madsen and Lie, 1997).
produced a relatively high level of PK in tissues. Comparing the dietary levels of vitamin K (PK or MK-4) and their corresponding tissue concentrations (Table 5), it can be said that the bioavailability of PK in L. Japonica is probably much higher than that in the prepared diets. On the other hand, the leaching of dietary MSB should also be considered.
Studies on the haematology of abalone indicated that abalone have no blood coagulation factor (Meyer, 1967), therefore, it is impossible to evaluate dietary vitamin K requirements of abalone on the basis of the classical haematological criteria, such as prothrombin time and prothrombin content.
Osteocalcin is an abundant calcium-binding protein of bone containing three residues of vitamin K-dependent g-carboxyglutamic acid (Gla) among its amino acids. The Gla side chains participate directly in the binding of calcium ions and the absorption of osteocalcin to hydroxyapatite surfaces in vivo and in vitro (Hauschka and Carr, 1982). The importance ofg-carboxyglutamic acid (Gla) for calcium binding and its essentiality for biological activity of vitamin K-dependent protein have been demonstrated in vertebrates (Hauschka and Carr, 1982; Suttie, 1991; Will et al., 1992), whereas King (1978) reported that matrix protein-bound g-carboxyglutamic acid (Gla) is not
obligat-21
ory (the amount is ,0.1 nmol g tissue) for the calcification process in invertebrates. Shiau and Liu (1994a) reported that increased vitamin K resulted in increasing calcium deposition in juvenile P. monodon. However, the calcium deposition in soft body or shell of the abalone was maintained relatively constant regardless of dietary vitamin K content in the present study. It may be interesting to clarify the role of vitamin K in the mineralization of shell in abalone.
Acknowledgements
The authors are grateful for financial support by grant No. 39670572 from the National Natural Science Foundation of China (NNSFC). We also thank Hongming Ma and Wei Xu of Ocean University of Qingdao for assistance in vitamin K analyses. [SS]
References
AOAC, 1984. In: Method of Analysis. Association of the Official Analytical Chemists, Washington, DC, 1141 pp.
Billeter, M., Bollinger, W., Martius, C., 1964. Untersuchungen uber die Unwandlung von Verfutterten K-vitaminen durch Austausch der Seitenkette und die Rolle der Darmbarkterien Heirbei. Biochem. Z. 340, 290–303.
Bulow, F.J., 1970. RNA–DNA ratios as indicators of recent growth rates of a fish. J. Fish. Res. Board Can. 27, 2343–2349.
Bulow, F.J., 1971. Selection of suitable tissues for use in the RNA–DNA ratio technique of assessing recent growth rate of a fish. Iowa State J. Sci. 46, 71–78.
Burton, K., 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315–323.
Grahl-Madsen, E., Lie, Ø., 1997. Effects of different levels of vitamin K in diets for cod (Gadus morhua). Aquaculture 151, 269–274.
Halver, J.E., 1989. The vitamins. In: Halver, J.E. (Ed.), Fish Nutrition. Academic Press, New York, pp. 31–109.
Hauschka, P.V., Carr, S.A., 1982. Calcium-dependent alfa-helical structure in osteocalcin. Biochemistry 21, 2539–2547.
He, J., Lawrence, A., Liu, R., 1992. Evaluation of dietary essentiality of fat-soluble vitamins A, D, E and K for penaeid shrimp (Penaeus vannamei ). Aquaculture 103, 177–185.
Kanazawa, A., 1985. Nutrition of Penaeid Prawn and Shrimp. In: Taki, Y., Primavera, L.H. and Lobrera, J.A. (Eds.), Proceedings of the First International Conferences of Culture of Penaeid Prawns / Shrimps. Aquacult. Dept. Southeast Asian Fish. Dev. Center, Iloilo, Philippines, pp. 123–130.
Kawatsu, H., Ikeda, T., 1988. Anti-anemic effect of anemia in common carp. Bull. Jpn. Soc. Sci. Fish. 54, 1731–1736.
Kawatsu, H., Kondo, K., Kubono, K., 1989. Hemostatic disorder in common carp induced by exposure to the herbicide molinate. Bull. Jpn. Soc. Sci. Fish. 55, 779–783.
King, K., 1978. Distribution ofg-carboxyglutamic acid in calcified tissues. Biochem. Biophys. Acta 542, 542–546.
Kitamura, S., Suwa, T., Ohara, S., Nakagawa, K., 1967. Studies on vitamin requirements of rainbow trout. II. The deficiency symptoms of fourteen kinds of vitamin. Bull. Jpn. Soc. Sci. Fish. 33, 1120–1125. Kormann, A.W., Weiser, H., 1983. Protective functions of fat-soluble vitamins. In: Proceedings of the 37th
Nottingham Feed Manufacturer’s Conference, Nottingham, UK.
Mai, K., 1998. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and H. discus hannai Ino. VII. Effects of dietary vitamin C on survival, growth and tissue concentration of ascorbic acid. Aquaculture 161, 383–392.
Mai, K., Mercer, J.P., Donlon, J., 1995a. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and H. discus hannai Ino. III. Response of abalone to various levels of dietary lipids. Aquaculture 134, 65–80.
Mai, K., Mercer, J.P., Donlon, J., 1995b. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and H. discus hannai Ino. IV. Optimum dietary protein level for growth. Aquaculture 136, 165–180.
Margolis, L., 1953. The effect of fasting on the bacterial flora of the intestine of fish. J. Fish. Res. Board Can. 10, 62–63.
Mathers, J.C., Fernandez, F., Hill, M.J., McCarthy, P.T., Shearer, M.J., Oxley, A., 1990. Dietary modification of potential vitamin K supply from enteric bacterial menaquinones in rats. Br. J. Nutr. 63, 639–652. Meyer, R.J., 1967. Hemocyanins and the systematic of California Haliotis. PhD thesis, Stanford University. Murai, T., Andrews, J.W., 1977. Vitamin K and anticoagulant relationships in catfish diets. Bull. Jpn. Soc. Sci.
Fish. 43, 785–794.
Nestor, K.E., Conrad, H.R., 1990. Metabolism of vitamin K and influence on prothrombin time in milk-fed preruminant calves. J. Dairy Sci. 73, 3291–3296.
NRC (National Research Council), 1993. In: Nutrient Requirements of Fish. National Academy Press, Washington, DC, 114 pp.
Phillips, A.M., Podiliak, H.A., Poston, H.A., Livingston, D.L., 1963. Effect of dietary vitamin K and sulfaguanidine on blood coagulation time, microhematocrit, and growth of immature brook trout. Fish. Res. Bull. NY 26, 15.
Poston, H.A., 1971. Effect of excess vitamin K on growth, coagulation time and hematocrit values of brook trout fingerlings. Fish. Res. Bull. NY 26, 15.
Schneider, W.C., 1957. Determination of nucleic acids in tissues by pentose analysis. In: Colowick, S.P., Kaplan, N.O. (Eds.). Methods in Enzymology, Vol. 3. Academic Press, New York, pp. 680–684. Shiau, S.Y., Liu, J.S., 1994a. Quantifying the vitamin K requirement of juvenile marine shrimp (Penaeus
monodon) with menadion. J. Nutr. 124, 277–282.
Suttie, J.W., 1980. The metabolic role of vitamin K. Fed. Proc. Fed. Am. Soc. Exp. Biol. 39, 2730. Suttie, J.W., 1991. Vitamin K. In: Machlin, L.J. (Ed.), Handbook of Vitamins. Marcel Dekker, New York, pp.
154–194.
Udagawa, M., Nakazoe, J.-I., Murai, T., 1993. Tissue distribution of phylloquinone and menaquinone-4 in sardine (Sardinops melanostictus). Comp. Biochem. Physiol. B 106, 297–301.
Uki, N., Kemuyama, A., Watanabe, T., 1985. Development of semipurified test diets for abalone. Bull. Jpn. Soc. Sci. Fish 51, 1825–1833, in Japanese, with English abstract.
Uki, N., Watanabe, T., 1992. Review of the nutritional requirements of abalone (Haliotis spp.) and
´ ´
development of more efficient artificial diets. In: Shepherd, S.A., Tegner, M.J., Guzman del Proo, S.A. (Eds.), Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 504–517. Webb, J.M., Levy, H.B., 1955. A sensitive method for the determination of deoxyribonucleic acid in tissues
and microorganisms. J. Biol. Chem. 213, 107–117.
