Interaction between vitamins A and D on growth and metabolic responses of abalone Haliotis discus hannai, Ino.

ABSTRACT A 152-day growth experiment was conducted in a recirculated water system to investigate the interaction between vitamins A (retinol) and D (cholecalciferol) on growth and metabolic responses in abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 0.35 [+ or -] 0.03 g; initial shell length: 11.31 [+ or -] 0.25 mm) were fed to satiation one of 16 semipurified diets containing 0, 1 x [10.sup.3], 1 x [10.sup.5], 1 x [10.sup.6] IU/kg vitamin A and 0, 500, 1 x [10.sup.3], 5 x [10.sup.3] IU/kg vitamin D in a 4 x 4 factorial design. Abalone were weighed and shell-length measured on the 76th day and the 152nd day, respectively. The total specific growth rate (SGR) during the 152 days, neither the SGR in the first 76 days nor in the second 76 days, was significantly influenced by the interaction between vitamins A and D. Dietary vitamins A and D significantly stimulated viscera 25-hydroxyvitamin [D.sub.3] [25(OH)[D.sub.3]] and 1[alpha],25-dihydroxyvitamin [D.sub.3] [1[alpha],25[(OH).sub.2][D.sub.3]] contents in a cooperative fashion. Dietary vitamin A generally increased the alkaline phosphatase (AKP) activity in viscera except the excessive supplement(1 x [10.sup.3] IU/kg), which significantly decreased AKP activity. Dietary vitamin D significantly increased AKP activity. Contents of P, not Ca and Mg, in soft body increased with dietary vitamin D supplement. Dietary vitamin A significantly improved contents of lipid and retinol in soft body and viscera, respectively. Meanwhile, dietary vitamin D significantly increased contents of ash and cholecalciferol in soft body and viscera, respectively. Based on these results, interaction between vitamins A and D was expressed in various manners as different indicators were considered, though there was potential antagonism mechanism at molecular level between the two fat-soluble vitamins.

KEY WORDS: Haliotis discus hannai, retinol, cholecalciferol, growth, metabolism, abalone

INTRODUCTION

Vitamin A (retinol) has many functions: it is required for vision, reproduction, and the development and maintenance of differentiated tissues (Sporn & Roberts 1984). It has a number of significant effects on innate and specific immune responses in homeotherms (Dhur et al. 1991) and fish (Thompson et al. 1994). Furthermore, it induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix (Iwamoto et al. 1993). Retinol is sequentially metabolized to retinaldehyde, retinoic acid for its functions (Hofman & Eichele 1994, Saari 1994). All-trans-retinoic acid (ATRA) acts through a series of receptors, termed retinoic acid receptors (RAR) [alpha], [beta], and [gamma]. Meanwhile, 9-cis-retinoic acid (9-cis-RA) can bind another series of retinoid receptors, termed retinoid X receptors (RXR), and initiate transcription of RXR responsive genes in a ligand-dependent fashion (discussed by Rohde et al. 1999).

Vitamin D (cholecalciferol) is essential to animals. Cholecalciferol is a kind of prohormone with little biological activity. It must be metabolized to 25-hydroxyvitamin [D.sub.3] [25(OH)[D.sub.3]] and subsequently hydroxylated to 1[alpha],25-dihydroxyvitamin [D.sub.3] [1[alpha], 25(OH)2[D.sub.3]] to exert its functions. The classical actions of 1[alpha], 25[(OH).sub.2][D.sub.3] are to regulate calcium and phosphate homeostasis and to promote the mineralization of bone (Feldman et al. 1996). Furthermore, 1[alpha],25[(OH).sub.2][D.sub.3] involves in many other biological processes, such as cell differentiation, immunological responses, gene expression (Bouillon et al. 1995). Most of the known biological effects occur through the direct transcriptional regulation of specific target genes by binding to the vitamin D receptor (VDR), which binds to its response element in target genes as a heterodimer with the RXR (Glass 1994).

In respect of RAR to influence the activation or expression of appropriate target genes, the preferred receptor partner for heterodimerisation is RXR. Therefore, there is potential for interaction between retinoic acid and vitamin D signaling pathways (Glass 1994, Carlberg 1995). Based on the researches at the cellular and molecular levels, results of interaction between vitamins A and D lie in two possibilities: passive effects and positive effects. For the former, there are many available studies (e.g., Folgueira et al. 1998, Hida et al. 1998, Thompson et al. 1998). In contrast, up to now there is less available data to demonstrate the positive interaction. Cellular studies have revealed that combinations of vitamin D derivatives and retinoids, such as ATRA and 9-cis-RA, exhibit positive effects on differentiation in established leukemia cell lines such as HL-60, U937, and NB4 (James et al. 1999). There is little data available on changes of physiological and biochemical properties in animal caused by the interaction between vitamins A and D, partly because reasons leading to such changes are complicated. Rohde et al. (1999) demonstrated that vitamin A antagonized the action of vitamin D on intestine and bone in rats, and ascribed the antagonisms to domination or utilization much of the RXR proteins by retinoids. However, vitamin A could interfere with the absorption of vitamin D, transport of vitamin D, conversion of vitamin D to its active form, or vitamin A could stimulate the metabolic degradation of vitamin D (Rohde et al. 1999). The mechanism of antagonism of vitamin A to vitamin D is still left obscure.

All the current information on interaction between vitamins A and D were from researches in vertebrates. At this time point, there is no report on such interaction in invertebrates. Requirements of vitamins A and D of abalone Haliotis discus hannai Ino of the archaeogastropod genus of mollusca were established in previous studies as 1,000 IU vitamin A/kg diet and 1000 IU vitamin D/kg diet, respectively (Zhou et al. 2000, Zhou & Mai 2004). The aim of this study is to preliminarily investigate the interaction between vitamins A and D on survival, growth and metabolism in H. discus hannai.

MATERIALS AND METHODS

Experimental Diets and Design

Experimental diets were formulated with purified ingredients (Table 1) to provide 29% crude protein from casein and gelatin, which is considered to be sufficient to maintain optimum growth for H. discus hannai (Mai et al. 1995a). A mixture of soybean oil and menhaden fish oil (1:1) was used as the basal lipid sources. Dietary lipid level was 3.3%, which was sufficient to support optimum growth and provide adequate HUFA (about 1.0%) for abalone (Mai et al. 1995b). Except vitamins A and D, all vitamins and minerals were added in premixes. The basal diet was analyzed by high-performance liquid chromatography (HPLC) to contain 32.8 IU vitamin A/kg diet and 25.6 IU vitamin D/kg diet. Diets supplemented with four levels of vitamin A (0, 1 x [10.sup.3], 1 x [10.sup.5] and 1 x [10.sup.6] IU/kg) and four levels of vitamin D (0, 500, 1 x [10.sup.3] and 5 x [10.sup.3] IU/kg) were prepared by adding appropriate quantifies of all-trans-retinol acetate and cholecalciferol to the basal diet in a 4 x 4 factorial arrangement. Vitamins A and D were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Procedures for diet preparation and storage were as previously described (Mai et al. 1995a).

Animal Rearing

Abalone (initial weight: 0.35 [+ or -] 0.03 g; initial shell length: 11.31 [+ or -] 0.25 mm) were derived from a spawning at Huaxin Fisheries Co., Shandong, China. Shell length was measured with calipers to the nearest 0.02 mm. The shell of each abalone was then superficially dried and the animal was weighed to 0.01 g using an electronic balance.

Growth experiment was conducted in a recirculated water system. Abalone were stocked at 30 animals for each rearing unit in a plastic basket (20 cm x 20 cm x 10 cm) per glass aquarium (45 cm x 25 cm x 35 cm). There were 16 treatments, and each treatment was conducted in three replicates. Prior to initiation of the experiment, abalone were placed in glass aquariums and conditioned with the basal diet for 1 wk. Abalone 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 152 days. During the 152-day experiment, water temperature was maintained at 18 [+ or -] 1[degrees]C, salinity 31-35, pH 7.8-8.0. Dissolved oxygen was not less than 6 mg/L, and there were negligible levels of free ammonia and nitrite.

Sample Collection and Analyses

At the termination of a sampling period (i.e., on the 76th and 152nd day, respectively), animals were not fed for 3 days. All the abalone were removed from the baskets, weighed, measured and counted. When the feeding trial finished, abalone from each replicate were immediately frozen (-70[degrees]C) for subsequent analyses. Growth was expressed as the specific growth rate (SGR, %) and daily increment in shell length (DISL, [micro]m/d). The calculation formulae are as follows:

SGR (%) = [(LnWt - LnWi)/t] x 100

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

Where, Wt, Wi are final and initial mean weight (g) during a sampling period, respectively; SLt, SLi are final and initial mean shell length (mm) during a sampling period, respectively; t is the period (day) between the two times of sampling to obtain the data of Wt (or SLt) and Wi (or SLi).

Contents of 25(OH)[D.sub.3] in viscera were determined by radioimmunoassay. The MEDGENIX 25OH-VIT.[D.sub.3]-RIA-CT kit was purchased from BioSource (BioSource Europe S.A. Zoning Industriel B-1400 Nivelles Belgium). [I.sup.125] 25(OH)[D.sub.3] (280 kBq) (BioSource Europe S.A.) was used as the tracer. All steps were followed with the methods described in the Instruction Manual (BioSource Europe S.A.) with some modifications. Briefly, lipid extract of viscera was placed in glass tubes (12 x 75 mm), then acetonitrile (0.5 mL) was added and the mixture was stirred, followed by centrifugation at x800 g at room temperature for 5 min. Supernatant (0.1 mL) was transferred to another tube. Subsequently, incubation buffer (0.4 mL) and 0.05 mL tracer (BioSource Europe S.A.) were added into the tube. The mixture was stirred, and then stored at room temperature for 2 h. The content in tube was aspirated, and the tube was washed twice with 2 mL wash solution (BioSource Europe S.A.). After the washing, the remaining drop of liquid in tube was aspirated. Then the tube was applied to a gamma radioimmunoassay counter (GC-911, Science & Technology Development Co., USTC, PRC).

Contents of 1[alpha],25[(OH).sub.2][D.sub.3] in viscera were also determined by radioimmunoassay. The BIOSOURCE 1,25[(OH).sub.2]-VIT.D-RIA-CT kit was purchased from BioSource (BioSource Europe S.A. Rue de l'Industrie, 8 B-1400 Nivelles Belgium). [I.sup.125] 1[alpha],25[(OH).sub.2][D.sub.3] (75 kBq) (BioSource Europe S.A.) was used as the tracer. All steps were followed with the methods described in the Instruction Manual (BioSource Europe S.A.) with some modifications. In brief, lipid extract of viscera was extracted by extraction solution and extraction solvent (BioSource Europe S.A.). Separation step was taken on bond elute silica cartridges (BioSource Europe S.A.). After elution step, the sample (0.2 mL) and the tracer (0.5 mL) were incubated together in tubes coated with anti1[alpha],25[(OH).sub.2][D.sub.3] (BioSource Europe S.A.) overnight at room temperature. After washed with the washing solution (BioSource Europe S.A.) and aspirated, the tubes were applied to a gamma radioimmunoassay counter (GC-911, Science & Technology Development Co., USTC, PRC).

For assay of alkaline phosphatase (AKP) activity in viscera, samples were homogenized in cold (4[degrees]C) 0.01 M Tris buffer (pH 7.5) at a ratio of 1:10 (w/v). A crude extract was obtained by centrifuging the homogenate at x13,500 g for 20 min at 4[degrees]C. Activity of AKP in the crude extract was determined spectrophotometrically using a [rho]-nitrophenyl-phosphate substrate assay (Tietz 1986). Protein was estimated by a modification of the Lowry procedure (Hartee 1972). Activity of AKP was expressed as specific activity (U/g protein), where one unit (U) is equal to the amount of enzyme necessary to produce 1 [micro]mol of nitrophenol (from [rho]-nitrophenyl-phosphate) per min at 37[degrees]C.

Elemental analyses of the soft body were performed by the method of Shearer (1984). The soft body samples were digested in perchloric acid (HCl[O.sub.4], 70%, ACS Reagent) at a ratio of 1:20 (w/v). Concentrations of elements in digests were determined by inductively coupled plasma-atomic emission spectrophotometer (ICP-OES; VISTA-MPX, VARIAN). Elemental concentrations of the soft body samples were expressed on a wet-weight basis.

In case of retinol and cholecalciferol contents in diet and viscera of abalone, a method for simultaneous determination of fat-soluble vitamins by HPLC was used. Saponification of samples and extraction of fat-soluble vitamins were followed the procedures described elsewhere (Salo-Vaananen et al. 2000) with some modifications. In brief, homogenized viscera sample (2 g, wt.) was mixed with 10 mL of 2% ascorbic acid, 20 mL of ethanol and 8 mL of 100% KOH (w/v) into glass flask. The flask was stirred and flushed with nitrogen, and then transferred into a boiling water bath for 20 min. After boiling water bath, the flask was cooled immediately in an ice-water bath for 10 min. To avoid emulsion formation, 20 mL of 10% NaC1 was added. Extraction of the saponified mixture was performed with 3 x 30 mL n-hexane in a separating funnel. The extracts were washed with 2 x 20 mL of 5% NaCl. The supernatants were evaporated to near dryness in a rotary evaporator in the dark at 30[degrees]C, and the remaining solvent was removed using a stream of nitrogen. The residue was dissolved in 1.0 mL methanol for HPLC analysis. The HPLC equipment consisted of a Hewlett Packard Model HP 1100 system with ODS (octadecylsilane) Hypersil column (HP; 250 x 4 mm; 5 [micro]m). UV detect was operated at wavelengths of 325 and 264 nm for retinol and cholecalciferol, respectively. The mobile phase was methanol with a flow rate of 0.5 mL/min. For the diet, fine powder of lyophilized sample was analyzed. Correspondingly, some adjustments of sample size and solvent volumes for saponification and extraction were needed.

Proximate analyses of soft-body samples to determine protein, lipid, ash and moisture levels were conducted using standard procedures (AOAC 1984).

Statistical Analysis

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

RESULTS

Survival and Growth

Survival and growth performances are presented in Table 2 and Table 3. Analysis of variance showed that dietary vitamin A, not vitamin D, significantly influenced the 76-day survival and the 152-day survival. Generally, excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) resulted in higher mortality. Weight of abalone on the 76th day and the 152nd day were significantly influenced by interaction between vitamins A and D. however, such interaction was not significant on shell length and survival of abalone.

Statistical analyses of these data suggested that vitamin A (0-1 x [10.sup.3] IU/kg) increased the specific growth rates in the first 76 days (SG[R.sub.1]), in the second 76 days (SG[R.sub.2]) and during the 152-day growth experiment (SG[R.sub.t]). Nevertheless, higher vitamin A levels decreased the SGRs (SG[R.sub.1], SG[R.sub.2], and SG[R.sub.t]). Furthermore such decrease was significant when dietary vitamin A supplement reached to 1 x [10.sup.6] IU/kg. Values of SG[R.sub.2] were generally lower than those of SG[R.sub.1], and excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) enlarged such difference. Values of SG[R.sub.t] of abalone fed the diet with 1 x [10.sup.3] IU/kg vitamin A and 5 x [10.sup.3] IU/kg vitamin D were higher than those of all other treatments. Vitamin D generally increased the SGRs, regardless of dietary vitamin A supplement. Interaction between vitamins A and D on SG[R.sub.1] and SG[R.sub.2] was not significant. However, such interaction significantly influenced SG[R.sub.t].

The daily increments in shell length in the first 76 days, in the second 76 days, and during the 152-day growth experiment were expressed as DIS[L.sub.1], DIS[L.sub.2], and DIS[L.sub.t], respectively. Trends of these DISLs changing with the different dietary vitamins A and D supplements were similar to those of the SGRs. Values of DIS[L.sub.2] were generally lower than those of DIS[L.sub.1], and excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) obviously enlarged such difference. The highest value for DIS[L.sup.t] was found as 84.19 [micro]m/d in treatment with 1 x [10.sup.3] IU/kg vitamin A and 5 x [10.sup.3] IU/kg vitamin D supplements. Interaction between vitamins A and D on the DISLs was not significant.

Contents of Cholecalciferol Metabolites and Activities of AKP

Contents of 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3], activities of AKP in viscera are presented in Table 4. The two vitamin D metabolites, 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] were detected in the viscera of abalone. Dietary vitamins A and D significantly increased 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] contents in a cooperative fashion. The highest values for 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] contents were found as 43.02 ng/g and 62.21 pg/g, respectively, in treatment with 1 x [10.sup.6] IU/kg vitamin A and 5 x [10.sup.3] IU/kg vitamin D supplements.

Activities of AKP significantly increased with dietary vitamin A supplements, which ranged from 0-1 x [10.sup.5] IU/kg. Excessive vitamin A supplement (1 x [10.sup.6] IU/kg) significantly depressed AKP activities at the all studied dietary vitamin D levels. Dietary vitamin D significantly increased AKP activities, regardless of the dietary vitamin A level. When dietary vitamin A supplements ranged from 0-1 x [10.sup.5] IU/kg, interaction between vitamins A and D significantly elevated AKP activity.

Mineral Concentrations

Concentrations of Ca, P, Mg, Na, K, and Zn in soft body are given in Table 5. Excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) significantly decreased the soft body Ca concentration, regardless of dietary vitamin D level. Vitamin D had no significant effect on the soft body Ca concentration. When dietary vitamin A supplements ranged from 0-1 x [10.sup.5] IU/kg, the soft body P concentration significantly increased with dietary vitamin D supplement. The highest value of P concentrations was found as 1.87 mg/g in the treatment with 0 IU/kg vitamin A and 5 x [10.sup.3] IU/kg vitamin D. Concentrations of Zn in the soft body significantly increased with either dietary vitamin A or dietary vitamin D levels. The highest value of Zn concentrations in the soft body was shown as 40.23 [micro]g/g in the treatment with 1 x [10.sup.6] IU/kg vitamin A and 5 x [10.sup.3] IU/kg vitamin D supplements. There was no significant interaction between vitamins A and D on contents of Ca, P, Mg, Na, K, and Zn in the soft body of H. discus hannai.

Soft Body Compositions, Retinol, and Cholecalciferol Contents

The soft body compositions, contents of retinol and cholecalciferol in viscera were listed in Table 6. Both vitamins A and D had no significant effects on the moisture and protein contents in the soft body. Lipid contents in the soft body were significantly increased with dietary vitamin A supplement, regardless of dietary vitamin D levels. The highest value of the soft body lipid content was found as 9.15% in the treatment with 1 x 106 IU/kg dietary vitamin A and 1 x [10.sup.3] IU/kg dietary vitamin D supplement. Higher dietary vitamin D supplement significantly lead to higher soft body ash content. Retinol contents in viscera significantly increased with dietary vitamin A levels, regardless of the dietary vitamin D levels. Cholecalciferol content significantly increased with dietary vitamin D supplements. Interaction between vitamins A and D had no significant effects on the soft body compositions, contents of retinol, and cholecalciferol in viscera.

DISCUSSION

It is demonstrated in present study that interaction between vitamins A and D on SGR either in the first 76 days (SG[R.sub.1]) or in the second 76 days (SG[R.sub.2]) is not significant. However, effect of such interaction on SG[R.sub.t] is found significant. That is to say, enough time is needed for such interaction to exert its function. Comparing the values of SG[R.sub.2] to SG[R.sub.1] and DIS[L.sub.2] to DIS[L.sub.1] (Table 3), it is shown that the growth rate of weight and shell length in the first 76 days was slowed down in the second 76 days. In this study, it is also found that excessive dietary vitamin A supplement accelerate the decreasing rate, regardless of the dietary vitamin D supplement. The reasons resulted in slowing down of growth rate could be ascribed to the antagonism of vitamin A to vitamin D.

Antagonism of vitamin A to vitamin D had been demonstrated in other studies. Rohde et al. (1999) pointed out that vitamin A antagonized the action of vitamin D in rats, when weight gain and minerals (Ca and P) concentrations were considered as the indicators. In fact, interaction between vitamins A and D has its molecular mechanisms. The biological active form of vitamin D, 1[alpha],25[(OH).sub.2][D.sub.3], mediates most of its actions through the intracellular vitamin D receptor (VDR), which binds to vitamin D responsive dements (VDREs) in the promoter region of responsive genes and regulates transcription. Usually the VDREs consist of a direct repeat of two hexanucleotides spaced by three nucleotides (DR-3), to which VDR preferentially binds as a heterodimer with the retinoid X receptor (RXR) (MacDonald et al. 1995, Darwish & DeLuca 1996). RXR is also the preferred receptor partner of retionic acid for homodimerisation. It is suggested that vitamin A competes with vitamin D for RXR. Furthermore, retinoic acid decreases VDR protein expression (Folgueira et al. 1998). Higher concentrations of 9-cis-RA inhibit the interaction between 1[alpha],25[(OH).sub.2][D.sub.3] and heterodimer-VDREs (Thompson et al. 1998). In contrast, 1[alpha],25[(OH).sub.2][D.sub.3] increased VDR protein expression (Jensen et al. 1998), and VDR-RXR heterodimer is resistant to dissociation and diversion to other pathways by 9-cis-RA if VDR is occupied by 1[alpha],25[(OH).sub.2][D.sub.3] prior to complexing with RXR (Thompson et al. 1998). Therefore, increasing 1[alpha],25[(OH).sub.2][D.sub.3] levels could soothe the antagonism of vitamin A to the action of vitamin D. In this study, contents of 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] in viscera of H. discus hannai increased with dietary vitamin D as well as dietary vitamin A. The highest values of 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] contents (43.02 ng/g, 62.21 pg/g, respectively) were found in the treatment with 1 x [10.sup.6] IU/kg dietary vitamin A and 5 x [10.sup.3] IU/kg dietary vitamin D supplement. That is to say, interaction between vitamins A and D had a positive effect on contents of 25(OH)[D.sup.3] and 1[alpha],25[(OH).sub.2][D.sub.3]. It is implied that accelerating metabolism of vitamin D (cholecalciferol) to 25(OH)[D.sub.3], subsequently to 1[alpha],25[(OH).sub.2][D.sub.3] is the strategy of H. discus hannai to overcome the antagonism of higher vitamin A. Meanwhile, it is interesting in this study that increasing contents of 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] is not able to significantly improve the growth of abalone any more, when dietary vitamin A supplement is up to 1 x [10.sup.5] IU/kg. Furthermore, excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) significantly decreased the growth of H. discus hannai. It is suggested that excessive vitamin A supplement antagonizes actions of vitamin D, and such antagonism cannot be overcome by the limited increases of 1[alpha],25[(OH).sub.2][D.sub.3] content.

In addition to contents of vitamin D metabolites, tissue mineral contents, particularly Ca and P, have also been used as indicators of vitamin D status in vertebrates (Brown & Robinson 1992). In this study, excessive dietary vitamin A supplement (1 x [10.sup.6] IU/kg) decreased the soft body Ca content, regardless of dietary vitamin D supplement. Dietary vitamin D increased the soft body P content, but had no significant effect on the soft body Ca content. It is evident that trends of soft body Ca and P contents changing with various dietary vitamins A and D levels differed from those of viscera 25(OH)[D.sup.3] and 1[alpha],25[(OH).sub.2][D.sub.3] contents. That is to say vitamin D status reflected by Ca and P contents differed from that by 25(OH)[D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3] contents. Retention of Ca and P in body is related to absorption, transportation, and excretion of the two minerals. It is abundantly clear that 1[alpha],25[(OH).sub.2][D.sub.3] plays a pivotal role in maintaining Ca and P homeostasis (Tanaka & DeLuca 1974, Minghetti & Norman 1988). Many steps in Ca and P metabolism process are tightly regulated by 1[alpha],25[(OH).sub.2][D.sub.3]. However, it is a complicated process that 1[alpha],25[(OH).sub.2][D.sub.3] exerts its functions. Any change that occurred in this process may lead to different results. Interaction between vitamins A and D was proved to be significant on growth, contents of 25(OH)[D.sub.3], and 1[alpha],25[(OH).sub.2][D.sub.3] in viscera of abalone. It is demonstrated that vitamin A has effect on the actions of vitamin D, which could be used to explain the differences between vitamin D status deduced from contents of vitamin D metabolites (25[OH][D.sub.3] and 1[alpha],25[(OH).sub.2][D.sub.3]) and that from body Ca and P contents.

The involvement of alkaline phosphatase (AKP) in metabolism of Ca and P has been proposed (Norman et al. 1970, Birge & Avioli 1981). Activity of AKP is also used as an important physiological parameter in research on animal immunities (Cheng & Rodrick 1975). It has been demonstrated that AKP activity is a responsive indicator in responses of H. discus hannai to many nutrients. Dietary Ca, P, Zn, and Fe had significant effect on AKP activity in H. discus hannai (Mai & Tan 2000, Tan & Mai 2001, Tan et al. 2001). In this study, it is demonstrated that AKP activity in viscera of H. discus hannai generally increased with dietary vitamins A and D supplements. Cellular study showed that vitamin A (retinoic acid) increased AKP activity by improving the expression of AKP gene (Iwamoto et al. 1993, Orimo & Shimada 2005). With respect to the relationship between vitamin D and AKP activity, 1[alpha],25[(OH).sub.2][D.sub.3] may play an important role. It was suggested that 1[alpha],25[(OH).sub.2][D.sub.3] stimulated the gene expression of AKP in HOS 58 human osteosarcoma cells (Siggelkow et al. 2002). Activity of AKP, in this study, is positively related to 1[alpha],25[(OH).sub.2][D.sub.3] contents. In vertebrates, many different genes playing a direct role in Ca endocrinology or bone mineralization have been shown to be responsive to 1[alpha],25[(OH).sub.2][D.sub.3]. Examples of these include osteocalcin (Demay et al. 1990), osteopontin (Noda et al. 1990), PTH (Okazaki et al. 1988), the hydroxylases CYP1[alpha] (Shinki et al. 1998) and the calbindin genes (Christakos et al. 1992). At this time point, it is not clear if vitamins A and D regulate the gene expression of AKP or subsequently have effects on the Ca and P metabolism in H. discus hannai. Further work is required to elucidate the mechanism of effects of vitamins A and D on AKP activity in abalone.

ACKNOWLEDGMENTS

This study was financially supported by grant No. 30200215 from the National Natural Science Foundation of China (NNSFC).

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TABLE 1.

Composition of the basal diet.

Ingredient                                          g/100 g dry wt.

Casein (vitamin-free, Sigma Chemical,
  St. Louis, MO, USA)                                    25.0
Gelatin (Sigma Chemical, St. Louis,
  MO, USA)                                                6.0
Dextrin (Shanghai Chemical Co.,
  Shanghai, China)                                       33.5
Sodium alginate (Shanghai Chemical Co.,
  Shanghai, China)                                       20.0
SO/MFO (Food grade) (a)                                   3.5
Choline chloride (Shanghai Chemical Co.,
  Shanghai, China)                                        0.5
Carboxymethylcellulose (Shanghai Chemical Co.,
  Shanghai, China)                                        5.0
Mineral mix (b)                                           4.5
Cholecalicferol and retinol-free vitamin mix (c)          2.0

Proximate analysis (means of triplicate)
Crude protein (%)                                        29.0
Crude lipid (%)                                           3.3
Ash (%)                                                   9.6
Gross energy (kJ/g) (d)                                  18.8
Retinol (IU/kg)                                          32.8
Cholecalciferol (IU/kg)                                  25.6

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

(b) Mineral mix, each 1,000 g of diet contained: NaCl, 0.4g;
MgS[O.sub.4] x 7[H.sub.2]O, 6.0g; Na[H.sub.2]P[O.sub.4] x 2[Hsub.2]O,
10.0g; K[H.sub.2]P[O.sub.4], 12.8g; Ca[([H.sub.2]-P[O.sub4]).sub.2] x
[H.sub.2]O, 8.0g; Fe-citrate, 1.0g; Ca-lactate, 1.4g; ZnS[O.sub.4] x
7[H.sub.2]O, 141.2 mg; MnS[O.sub.4]-[H.sub.2]O, 64.8mg;
CUS[O.sub.4]-5[H.sub.2]O, 12.4mg; Co[Cl.sub.2]-6[H.sub.2]O, 0.4mg;
KI[O.sub.3], 1.2 mg; [Na.sub.2]Se[O.sub.3], 0.4mg.

(c) Cholecalciferol and retinol-free vitamin mix, each 1,000 g of diet
contained: thiamin HCl, 120 mg; riboflavin, 100 mg; folic acid, 30 mg;
PABA, 400 mg; pyridoxine HCl, 40 mg; niacin, 800 mg; Ca pantothenate,
200 mg; inositol, 4,000 mg; ascorbic acid, 4000 mg; biotin, 12 mg;
[B.sub.12],0.18 mg; menadione, 80 mg; vitamin E, 450 mg; ethoxyquin,
400 mg.

(d) Estimated with an XYR-1 bomb calorimeter.

TABLE 2.

Weight, shell length and survival rate of Haliotis discus hannai fed
diets with different vitamins A (A) and D (D) levels for 152 days.

   Dietary Supplement

 A (IU/kg)     D (IU/kg)    [W.sub.1] (g)    [W.sub.2] (g)

         0           0       0.78 (abc)        1.50 (e)
                   500       0.72 (bc)         1.53 (e)
                 1,000       0.80 (abc)        1.85 (c)
                 5,000       0.86 (ab)         1.97 (bc)
     1,000           0       0.83 (abc)        1.80 (cd)
                   500       0.84 (abc)        1.93 (c)
                 1,000       0.90 (a)          2.33 (a)
                 5,000       0.88 (a)          2.31 (a)
   100,000           0       0.77 (abc)        1.58 (de)
                   500       0.82 (abc)        1.85 (c)
                 1,000       0.89 (a)          2.18 (ab)
                 5,000       0.84 (abc)        2.07 (b)
 1,000,000           0       0.76 (abc)        1.35 (e)
                   500       0.80 (abc)        1.55 (e)
                 1,000       0.79 (abc)        1.54 (e)
                 5,000       0.71 (c)          1.40 (e)
ANOVA
A                            0.000            0.000
D                            0.023            0.000
A x D                        0.039            0.000
Pooled s.e.                  0.068            0.317

   Dietary Supplement

 A (IU/kg)     D (IU/kg)    S[L.sub.1] (mm)      SLZ (mm)

         0           0         16.03 (ab)      20.29 (e)
                   500         16.59 (ab)      21.87 (bcde)
                 1,000         17.34 (ab)      22.78 (abcd)
                 5,000         17.22 (ab)      23.07 (abcd)
     1,000           0         16.41 (ab)      21.09 (cde)
                   500         17.47 (ab)      23.35 (abc)
                 1,000         17.36 (ab)      23.55 (abc)
                 5,000         17.92 (a)       24.20 (a)
   100,000           0         16.87 (ab)      21.70 (bcde)
                   500         17.10 (ab)      22.87 (abed)
                 1,000         17.76 (ab)      23.82 (ab)
                 5,000         17.44 (ab)      23.65 (abc)
 1,000,000           0         15.84 (b)       20.26 (e)
                   500         16.68 (ab)      21.58 (cde)
                 1,000         17.03 (ab)      22.17 (abcde)
                 5,000         16.81 (ab)      21.95 (bcde)
ANOVA
A                               0.024           0.000
D                               0.000           0.000
A x D                           0.925           0.831
Pooled s.e.                     0.783           1.338

   Dietary Supplement           Survival Rate (%)

 A (IU/kg)     D (IU/kg)      76-day       152-day

         0           0      90.18 (ab)    89.42 (ab)
                   500      91.26 (ab)    89.36 (ab)
                 1,000      90.36 (ab)    90.36 (ab)
                 5,000      89.47 (ab)    88.87 (ab)
     1,000           0      90.04 (ab)    89.41 (ab)
                   500      92.17 (ab)    89.77 (ab)
                 1,000      90.29 (ab)    90.29 (ab)
                 5,000      93.15 9a)     92.41 (a)
   100,000           0      91.87(ab)     91.87 (a)
                   500      91.33 (ab)    91.33 (ab)
                 1,000      92.93 (a)     88.33 (ab)
                 5,000      92.58 (ab)    92.58 (a)
 1,000,000           0      90.23 (ab)    88.95 (ab)
                   500      90.25 (ab)    90.25 (ab)
                 1,000      90.09 (ab)    88.09 (ab)
                 5,000      88.46 (b)     84.22 (b)
ANOVA
A                            0.001         0.017
D                            0.743         0.799
A x D                        0.122         0.068
Pooled s.e.                  1.777         2.789

[W.sub.1], [W.sub.2]: Mean weight of abalone on the 76th day and the
152nd day, respectively.

S[L.sub.1], S[L.sub.2]: Mean shell length of abalone on the 76th day
and the 152nd day, respectively.

(a-e) Means in the same column sharing a common superscript letter were
not significantly different (P > 0.05) as determined by Tukey test.

TABLE 3.

Specific growth rate (SGR) and daily increment in shell length (DISL)
of Haliotis discus hannai fed diets with different vitamins A (A) and
D (D) levels for 152 days.

   Dietary Supplement

A (IU/kg)     D (IU/kg)     SGRI (%)     SG[R.sub.2] (%)

        0           0      1.00 (bc)        0.87 (ef)
                  500      1.08 (abc)       0.99 (cde)
                1,000      1.17 (abc)       1.11 (bc)
                5,000      1.18 (abc)       1.09 (cd)
    1,000           0      1.08 (abc)       1.02 (cde)
                  500      1.12 (abc)       1.10 (c)
                1,000      1.27 (a)         1.25 (ab)
                5,000      1.29 (a)         1.27 (a)
  100,000           0      1.Ol (bc)        0.94 (de)
                  500      1.1l (abc)       1.07 (cd)
                1,000      1.23 (ab)        1.19 (abc)
                5,000      1.28 (a)         1.19 (abc)
1,000,000           0      0.94 (c)         0.76 (f)
                  500      1.08 (abc)       0.87 (ef)
                1,000      1.08 (abc)       0.89 (ef)
                5,000      1.07 (abc)       0.89 (ef)
ANOVA
A                          0.000            0.000
D                          0.000            0.000
A x D                      0.743            0.253
Pooled s.e.                0.122            0.155

   Dietary Supplement

A (IU/kg)     D (IU/kg)    SG[R.sub.1] (%)    DIS[L.sub.1] ([micro]m/d)

        0           0        0.93 (gh)               63.55 (bc)
                  500        1.04 (cdef)             70.63 (abc)
                1,000        1.14 (b)                78.51 (abc)
                5,000        1.13 (bc)               79.65 (abc)
    1,000           0        1.05 (cde)              69.61 (abc)
                  500        1.11 (c)                77.72 (abc)
                1,000        1.26 (a)                81.27 (abc)
                5,000        1.28 (a)                85.83 (a)
  100,000           0        0.98 (efg)              72.11 (abc)
                  500        1.09 (cd)               76.10 (abc)
                1,000        1.21 (ab)               82.72 (ab)
                5,000        1.24 (a)                81.97 (ab)
1,000,000           0        0.85 (h)                61.18 (c)
                  500        0.97 (efg)              69.69 (abc)
                1,000        0.98 (efg)              73.90 (abc)
                5,000        0.98 (efg)              72.76 (abc)
ANOVA
A                             0.000                   0.006
D                             0.000                   0.000
A x D                         0.004                   0.993
Pooled s.e.                   0.127                   8.803

   Dietary Supplement
                            DIS[L.sub.2]
A (IU/kg)     D (IU/kg)     ([micro]m/d)     DIS[L.sub.t] ([micro]m/d)

        0           0      56.14 (h)               59.85 (e)
                  500      69.77 (bcdefg)          70.00 (bcde)
                1,000      71.62 (abcdef)          75.07 (abcd)
                5,000      76.97 (abcd)            78.31 (abc)
    1,000           0      61.54 (fg)              65.57 (de)
                  500      77.37 (abc)             77.54 (abcd)
                1,000      81.40 (ab)              81.34 (ab)
                5,000      82.54 (a)               84.19 (a)
  100,000           0      63.54 (efg)             67.82 (cde)
                  500      75.92 (abcde)           76.01 (abcd)
                1,000      79.74 (abc)             81.23 (ab)
                5,000      81.71 (ab)              81.84 (ab)
1,000,000           0      58.11 (gh)              59.65 (e)
                  500      64.47 (defg)            67.08 (cde)
                1,000      67.63 (cdefg)           70.77 (bcde)
                5,000      67.59 (cdefg)           70.18 (bcde)
ANOVA
A                           0.000                   0.000
D                           0.000                   0.000
A x D                       0.408                   0.869
Pooled s.e.                 9.164                   8.260

SG[R.sub.1], SG[R.sub.2], SG[R.sub.t]: Mean specific growth rate of
abalone in the first 76 days, in the second 76 days and during the
152-day growth experiment, respectively.

DIS[L.sub.1], DIS[L.sub.2], DIS[L.sub.t]: Mean daily increment in shell
length of abalone in the first 76 days, in the second 76 days and
during the 152-day growth experiment, respectively.

(a-h) Means in the same column sharing a common superscript letter were
not significantly different (P > 0.05) as determined by Tukey test.

TABLE 4.

Contents of 25-hydroxyvitamin [D.sub.3] [25(OH)[D.sub.3]] and 1[alpha],
25-dihydroxyvitamin [D.sub.3] [1,25[(OH).sub.2][D.sub.3]], activities
of alkaline phosphatase (AKP) in viscera of Haliotis discus hannai fed
diets with different vitamins A (A) and D (D) levels for 152 days.

   Dietary Supplement                               1[alpha],25
                            25(OH)[D.sub.3]    [(OH).sub.2][D.sub.3]
A (IU/kg)      D (IU/kg)        (ng/g)                (pg/g)

         0           0        21.88 (g)            27.13 (h)
                   500        22.62 (fg)           31.55 (fgh)
                 1,000        25.54 (def)          33.01 (efgh)
                 5,000        28.26 (cd)           36.92 (efg)
     1,000           0        22.53 (fg)           28.77 (gh)
                   500        24.18 (efg)          35.21 (efgh)
                 1,000        28.76 (c)            31.13 (fgh)
                 5,000        34.92 (b)            40.34 (de)
   100,000           0        23.19 (efg)          28.01 (h)
                   500        26.06 (cde)          36.92 (efg)
                 1,000        33.92 (b)            48.68 (cd)
                 5,000        41.60 (a)            58.57 (ab)
 1,000,000           0        23.90 (efg)          30.94 (fgh)
                   500        28.92 (c)            38.76 (ef)
                 1,000        36.39 (b)            50.52 (bc)
                 5,000        43.02 (a)            62.21 (a)
ANOVA
A                              0.006                0.001
D                              0.000                0.000
A x D                          0.008                0.003
Pooled s.e.                    6.766               10.844

   Dietary Supplement         AKP (U/g
                              protein)
A (IU/kg)      D (IU/kg)

         0           0      52.43 (fg)
                   500      63.97 (def)
                 1,000      69.87 (bcde)
                 5,000      71.46 (bcde)
     1,000           0      55.18 (fg)
                   500      63.84 (def)
                 1,000      76.37 (abcd)
                 5,000      78.25 (abc)
   100,000           0      50.08 (g)
                   500      64.60 (cdef)
                 1,000      78.15 (abc)
                 5,000      85.57 (a)
 1,000,000           0      35.25 (h)
                   500      35.42 (h)
                 1,000      58.72 (efg)
                 5,000      81.62 (ab)
ANOVA
A                            0.000
D                            0.000
A x D                        0.000
Pooled s.e.                 15.405

(a-h) Means in the same column sharing a common superscript letter were
not significantly different (P > 0.05) as determined by Tukey test.

TABLE 5.

Concentrations of the selected minerals in the soft body of Haliotis
discus hannai fed diets with different vitamins A (A) and D (D) levels
for 152 days.

   Dietary Supplement

A (IU/kg)      D (IU/kg)    Ca (mg/g)      P (mg/g)     Mg (mg/g)

         0           0      4.31 (abc)    1.45 (c)        1.37
                   500      4.48 (ab)     1.52 (bc)       1.30
                 1,000      4.45 (ab)     1.68 (abc)      1.28
                 5,000      4.66 (a)      1.87 (a)        1.28
     1,000           0      4.26 (abc)    1.38 (c)        1.22
                   500      4.35 (abc)    1.41 (c)        1.20
                 1,000      4.36 (abc)    1.52 (bc)       1.10
                 5,000      4.49 (ab)     1.68 (abc)      1.14
   100,000           0      4.05 (abc)    1.38 (c)        1.16
                   500      4.09 (abc)    1.44 (c)        1.16
                 1,000      4.07 (abc)    1.56 (abc)      1.19
                 5,000      4.03 (abc)    1.85 (ab)       1.08
 1,000,000           0      3.62 (bc)     1.36 (c)        1.18
                   500      3.52 (c)      1.59 (abc)      1.19
                 1,000      3.55 (c)      1.52 (bc)       1.24
                 5,000      3.67 (bc)     1.52 (bc)       1.38
ANOVA
A                           0.000         0.240           0.058
D                           0.610         0.001           0.969
A x D                       0.990         0.604           0.738
Pooled s.e.                 0.428         0.212           0.152

   Dietary Supplement

A (IU/kg)      D (IU/kg)    Na (mg/g)    K (mg/g)      Zn (FLg/g)

         0           0        12.71       2.65       31.08 (f)
                   500        12.85       2.56       31.52 (f)
                 1,000        12.50       2.58       32.39 (ef)
                 5,000        12.58       2.62       34.65 (bcdef)
     1,000           0        12.46       2.66       32.44 (ef)
                   500        12.78       2.50       32.85 (def)
                 1,000        13.02       2.63       33.65 (cdef)
                 5,000        12.01       2.49       34.03 (bcdef)
   100,000           0        12.63       2.55       35.31 (abcdef)
                   500        12.46       2.56       36.90 (abcde)
                 1,000        12.23       2.66       37.59 (abcde)
                 5,000        12.62       2.54       38.85 (abc)
 1,000,000           0        12.86       2.66       37.21 (abcde)
                   500        12.72       2.60       38.02 (abcd)
                 1,000        13.79       2.48       39.28 (ab)
                 5,000        14.52       2.43       40.23 (a)
ANOVA
A                              0.171      0.914       0.000
D                              0.929      0.651       0.002
A x D                          0.760      0.959       0.988
Pooled s.e.                    1.139      0.187       3.234

(a-f) Means in the same column sharing a common superscript letter were
not significantly different (P > 0.05) as determined by Tukey test.

TABLE 6.

Soft body compositions and contents of retinol and cholecalciferol in
viscera of Haliotis discus hannai fed diets with different vitamins A
(A) and D (D) levels for 152 days.

   Dietary Supplement

               [D.sub.3]
A (IU/kg)       (IU/kg)     Moisture (%)    Protein (%)    Lipid (%)

         0            0        79.44          56.98        7.29 (b)
                    500        79.61          56.61        7.35 (b)
                  1,000        79.40          57.20        7.34 (b)
                  5,000        77.46          56.76        7.49 (b)
     1,000            0        78.28          57.09        7.32 (b)
                    500        79.47          56.22        7.31 (b)
                  1,000        78.52          57.16        7.75 (b)
                  5,000        78.62          57.02        7.62 (b)
   100,000            0        77.79          56.71        7.94 (b)
                    500        78.53          57.59        7.90 (b)
                  1,000        77.51          56.45        7.89 (b)
                  5,000        77.65          56.98        8.06 (b)
 1,000,000            0        79.34          57.34        8.85 (a)
                    500        77.47          56.67        8.97 (a)
                  1,000        79.22          56.49        9.15 (b)
                  5,000        79.29          56.52        9.06 (b)
ANO VA
A                               0.481          0.900       0.000
D                               0.905          0.737       0.137
A x D                           0.790          0.142       0.855
Pooled s.e.                     1.745          0.617       0.695

   Dietary Supplement

               [D.sub.3]
A (IU/kg)       (IU/kg)       Ash (%)      Refinol ([micro]g/g)

         0            0     10.65 (e)           0.03 (d)
                    500     11.59 (c)           0.04 (d)
                  1,000     11.70 (bc)          0.07 (d)
                  5,000     12.46 (ab)          0.06 (d)
     1,000            0     10.74 (de)          0.18 (d)
                    500     11.23 (cde)         0.12 (d)
                  1,000     11.68 (bc)          0.23 (d)
                  5,000     12.72 (a)           0.24 (d)
   100,000            0     10.63 (e)           0.64 (c)
                    500     11.45 (cd)          0.58 (c)
                  1,000     11.42 (cde)         0.59 (c)
                  5,000     12.61 (a)           0.67 (c)
 1,000,000            0     11.01 (cde)         1.28 (ab)
                    500     11.19 (cde)         1.31 (a)
                  1,000     11.45 (cd)          0.98 (b)
                  5,000     12.61 (a)           1.26 (ab)
ANO VA
A                            0.913              0.000
D                            0.000              0.167
A x D                        0.239              0.064
Pooled s.e.                  0.719              0.469

   Dietary Supplement

               [D.sub.3]
A (IU/kg)       (IU/kg)     Cholecalciferol ([micro]g/g)

         0            0              0.06 (f)
                    500              0.12 (ef)
                  1,000              0.23 (def)
                  5,000              0.48 (abc)
     1,000            0              0.06 (f)
                    500              0.13 (ef)
                  1,000              0.26 (def)
                  5,000              0.53 (ab)
   100,000            0              0.08 (f)
                    500              0.18 (def)
                  1,000              0.30 (cde)
                  5,000              0.62 (a)
 1,000,000            0              0.06 (f)
                    500              0.15 (def)
                  1,000              0.35 (bcd)
                  5,000              0.60 (a)
ANO VA
A                                    0.032
D                                    0.000
A x D                                0.828
Pooled s.e.                          0.021

(a-f) Means in the same column sharing a common superscript letter were
not significantly different (P > 0.05) as determined by Tukey test.

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

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

* Corresponding author. E-mail: [e-mail]