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Estimation of dietary pantothenic acid requirement of grouper, Epinephelus malabaricus according to physiological and biochemical parameters
Aquaculture 324–325 (2012) 92–96
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Estimation of dietary pantothenic acid requirement of grouper, Epinephelus malabaricus according to physiological and biochemical parameters Yu-Hung Lin a, Hui-You Lin b, Shi-Yen Shiau b, c,⁎ a b c
Department of Aquaculture, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 912, Taiwan, ROC Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 202, Taiwan, ROC Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 5 August 2011 Received in revised form 19 October 2011 Accepted 19 October 2011 Available online 26 October 2011 Keywords: grouper pantothenic acid nutritional requirements deficiency
a b s t r a c t To quantify the dietary pantothenic acid (PA) requirements of grouper, Epinephelus malabaricus, calcium d-PA was added to basal diet at 0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet. Triplicate groups of fish (initial body weight: 15.3 ± 0.3 g) were fed with each experimental diet for 8 weeks in a recirculating seawater rearing system. After feeding trial, besides growth and survival, hematological index, hepatic PA, CoA and total lipid concentrations in fish were also monitored. Pantothenic acid deficiency signs including clubbed gills, anemia, high mortality, anorexia, sluggishness, and poor growth were observed in fish fed the PA-free control diet. Fish fed diets supplemented with ≥ 10 mg PA/kg diet had significantly (P ≤ 0.05.) higher weight gain (WG) and feed efficiency than fish fed the PA-free control diet. Fish fed diets supplemented with ≥ 5 mg PA/kg diet had higher survival, red blood cell count, hematocrit and hemoglobin concentrations, but lower hepatic total lipid concentration than fish fed the control diet. Hepatic PA concentration was highest in fish fed diets with ≥10 mg PA/kg diet, followed by 5 mg PA/kg diet group, and lowest in fish fed the control diet. Fish fed diets with 10–40 and ≥ 100 mg PA/kg diet had higher hepatic CoA concentration than fish fed diets with ≤5 mg PA/kg diet. Analysis of WG, hepatic PA and CoA concentrations by broken-line regression indicated that the optimum dietary PA requirement of growing grouper is about 11 mg PA/kg diet. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Pantothenic acid (PA) is a component of coenzyme A (CoA), acyl CoA synthetase, and acyl carrier protein. The coenzyme form of the vitamin is therefore responsible for acyl group transfer reactions. Coenzyme A is required in reactions in which the carbon skeletons of glucose, fatty acids, and amino acids enter into the energyyielding tricarboxylic acid cycle (NRC, 2011). This coenzyme is also involved in acetylation of choline to form the neurotransmitter acetylcholine and biosynthesis of cholesterol. Acyl carrier protein is required for fatty acid synthesis. A dietary PA deficiency impairs normal metabolism in cells undergoing rapid mitosis and high energy expenditure (Roem et al., 1991). Many of the effects of PA deficiency could result from lower tissue levels of CoA in animals (Reibel et al., 1982). The dietary PA requirement has been reported in several fish species, including rainbow trout (Oncorhynchus mykiss) (Cho and Woodward, 1990), common carp (Cyprinus carpio) (Ogino, 1967),
⁎ Corresponding author at: Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC. Fax: + 886 2 2462 1684. E-mail address: [email protected] (S.-Y. Shiau). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.020
channel catfish (Ictalurus punctatus) (Murai and Andrews, 1979; Wilson et al., 1983), blue tilapia (Oreochromis aureus) (Roem et al., 1991; Soliman and Wilson, 1992), Jian carp (Cyprinus carpio var. Jian) (Wen et al., 2009) and yellowtail (Seriola quinqueradiata) (Shimeno, 1991), ranging from 10 to 45 mg PA/kg diet. Groupers are popular and high value marine fish in many parts of the world such as in Kuwait, Indonesia, Malaysia, Thailand, the Philippines, Hong Kong, Taiwan, China, Mexico and Japan. They are also good candidates for intensive aquaculture because of their desirable taste, hardiness and rapid growth (Chen and Tsai, 1994; Millamena, 2002). Global grouper production increased dramatically in recent years, recorded as 60,774, 99,498, 163,328 and 204,284 MT in 1990, 2000, 2005, and 2009 respectively (FAO Yearbooks of Fisheries Statistics, 2011). Because no quantitative PA requirement has been reported for grouper, the following study was designed to estimate the dietary PA requirements for juvenile grouper, Epinephelus malabaricus. The PA levels in assayed diet were based on published PA requirement results of other fish species (10 to 45 mg PA/kg diet) plus lower (5 mg PA/kg diet) and excessive (60, 100 and 200 mg PA/kg diet). Other than growth performance, histological assay for gill, hematological index and hepatic PA, CoA and total lipid concentrations in fish were also monitored.
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2. Materials and methods
2.2. Experimental procedure
2.1. Experimental diets
Juveniles E. malabaricus obtained from a local hatchery (Pingtung, Taiwan) were used in the study. Upon arrival, they were acclimated to laboratory conditions for 4 weeks in a 1000 L plastic tank and fed a commercial diet (Uni-President Enterprise Corp., Tainan, Taiwan): moisture, 11.7%; crude protein, 43.3%; lipid, 8.8%; ash, 9.3%. At the beginning of the experiment, 10 fish (mean initial weight ± SD: 15.3 ± 0.3 g) were stocked in each of 24 aquariums (width × length × height: 0.305 × 0.610 × 0.360 m 3). Each experimental diet was assayed in triplicate (8 experimental diets × 3 tanks). Diets were assigned to groups of fish randomly. Each aquarium was part of a closed recirculating system with a common reservoir of seawater at 29 to 32‰ salinity. The seawater was obtained from the coast near the National Taiwan Ocean University (Keelung, Taiwan). Water temperature of the rearing system was 29 ± 1 °C. The flow water was 2 L/min through two separate biofilters to remove impurities and reduce ammonia concentrations. Half of the water in the system was exchanged daily. Fish were fed 3% of their body weight per day. This amount was close to the maximal daily ration for grouper according to feed consumption during the acclimation period of the study. The daily ration was divided into two equal meals (08:30 and 16:30 h). Fish were weighed once every 2 weeks and the daily ration adjusted accordingly. The remaining feed and feces were removed by a siphon immediately after feeding. A photoperiod of 12 h light (08:00 to 20:00 h), 12 h dark was used. Dead fish were removed and not replaced during the experiment. Fish were fed the test diets for an 8-week period.
Eight experimental diets were formulated according to different levels of PA (0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet). Vitamin-free casein (Sigma Chemical Co., St. Louis, MO), fish oil (Semi-refined fish oil, Oleaginosa Victoria S.A., Peru) and corn oil (Tai-Tang Industrial, Taiwan), and corn starch (Sigma Chemical) were used as dietary protein, lipid and carbohydrate sources, respectively. Dietary protein and lipid supplementation levels were recommended by Chen and Tsai (1994) and Lin and Shiau (2003), respectively. All experimental diets were adjusted to be isoenergetic at 15.05 KJ/g diet. An attractant that had a similar chemical composition to squid mantle tissue (Mackie and Mitchell, 1985) was added at 6% to all diets to increase palatability and diet acceptance. Basal diet was supplemented with calcium d-PA (92% PA, Sigma Chemical) at 0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet. Pantothenic acid-free basal diet formulation and proximate composition analysis (AOAC, 1995) are shown in Table 1. Dietary PA concentrations of the eight experimental diets were determined with a microbiological assay using Lactobacillus plantarum (BCRC 10357) from the Bioresource Collection and Research Center (Hsinchu, Taiwan) according to the procedure of AOAC (1995). The growth of L. plantarum was dependent on the PA levels. The determined values were 0, 4.6, 9.9, 19.4, 34.3, 50.2, 98.9, and 192.8 mg PA/kg diet. All ingredients of the experimental diet were mixed, and then enough cold water was added to form a stiff dough. This was then passed through a mincer with die, and the resulting strands were dried using an electrical fan at 20 °C. After drying, the diets were broken up and sieved into pellets (1.2 mm in diameter) and stored at −20 °C until used.
Table 1 Formulation and proximate composition of the basal diet. % (wet matter) Ingredient Casein Fish oil Corn oil Corn starch Vitamin mixturea Mineral mixtureb Attractantc Carboxylmethylcellulose Alpha-cellulose Proximate composition Moisture Ash Crude protein Ether extract
51 4.5 4.5 16.7 2 4 6 3 8.3 10.46 3.17 47.61 8.78
a Vitamin mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboflavin, 10; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; L-ascorbyl2-monophosphate Mg, 5; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025; biotin, 0.25. All ingredients were diluted with alpha-cellulose to 1 g. b Mineral mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4.2H2O, 135.8; MgSO4.7H2O, 132; Na2HPO4.2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4.7H2O, 3; MnSO4.H2O, 0.8; CoCl2.6H2O, 1; KI, 0.15; CuSO4, 0.15; AlCl3.6H2O, 0.15; selenomethionine, 0.02. All ingredients were diluted with alpha-cellulose to 1 g. c As mg/100 g diet: L-aspartic acid, 18; L-threonine, 44; L-serine, 33; L-glutamic acid, 53; Lvaline, 36; L-methionine, 36; L-isoleucine, 29; L-leucine, 55; L-tyrosine, 22; L-phenylalanine, 29; L-lysine-HCl, 29; L-histidine-HCl, 15; L-proline, 1456; L-alanine, 273; L-arginine, 228; taurine, 337; glycine, 892; betain-HCl, 910; trimethylamine-HCl, 91; trimethylamine n-oxide HCl, 1138; hypoxanthine, 47; inosine, 25; adenosine-5′-monophosphate, 40; L-(+)-lactic acid, 91; alpha-cellulose, 80 (Mackie and Mitchell, 1985).
2.3. Growth performance and assay methods At the end of the feeding trial, percentage of body weight gain (WG) in each aquarium [100 × (final body weight–initial body weight)/initial body weight], feed efficiency (FE) [(final body weight–initial body weight)/feed intake], and survival [100 × (final fish number/initial fish number] were calculated. After the final weight was noted, blood was collected using heparinized syringes from the caudal vein from six ice-anesthetized fish selected randomly per aquarium and pooled. The red blood cell (RBC) count, hematocrit (Hct), and hemoglobin (Hb) concentrations were measured by auto hematological analyzer (KX 21N, Sysmex Co., Japan). After blood collecting, the fish were sacrificed. The livers of six fish from a same tank were removed and pooled. Liver samples were stored at −80 °C freezer until analysis. Hepatic PA concentration was determined with the same method of feed analysis (AOAC, 1995). Hepatic CoA was measured according to the method described by Tubbs and Garland (1969). Briefly, hepatic CoA was extracted by perchloric acid (8%, containing 15 mM dithiothreitol). Then the extractions were reacted with acyl-CoA synthetase and absorbance was recorded by the spectrophotometer to calculate CoA concentration (Hitachi U-2000, Tokyo, Japan). Total lipid concentration in liver was determined by Folch method (Folch et al., 1957) using chloroform/methanol (2:1, v/v) to extract the total lipid from liver sample. In addition, gills from the same fish were dissected, fixed in 10% formalin, dehydrated by ethanol (100%, Merch Co., Germany), embedded in Paraplast (Sigma Chemical), sectioned at 6 μm and stained with hematoxylin and eosin (Sigma Chemical) (Wilson et al., 1983). The gill sections were examined microscopically for histological changes. 2.4. Statistical analysis Results are expressed as mean ± SD. Data were analyzed by oneway analysis of variance (ANOVA), and significance was set at
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P ≤ 0.05. Multiple comparisons among means were made with Duncan's new multiple range test (Duncan, 1955). Dietary PA requirements for juvenile grouper were estimated by the brokenline model with the weight gain, hepatic PA and CoA concentrations. Data analyses were performed with using the SAS/PC statistical software (SAS Inst. Inc., Cary, NC). 3. Results Fish fed the PA-free control diet began to exhibit deficiency signs including anorexia, sluggishness, poor growth and increased mortality within 4 weeks of the trial. After 8 weeks, clubbed gills with marked interlamellar proliferative lesions were observed (Fig. 1). No lesions were found in gills from the fish fed PA-supplemented diets. Red blood cell (RBC) count, hematocrit (Hct) and hemoglobin (Hb) concentrations were significantly higher in fish fed diets with ≥5 mg PA/kg diet than fish fed the control diet (Table 2). Fish fed diets supplemented with ≥10 mg PA/kg diet had significantly (P ≤ 0.05) higher weight gain (WG) and feed efficiency (FE) than fish fed the PA-free control diet (Table 3). Survival was significantly higher in fish fed diets with ≥ 5 mg PA/kg diet than those fed the control diet. Hepatic PA concentration was the highest in fish fed diets with ≥ 10 mg PA/kg diet, followed by 5 mg PA/kg diet, and lowest in fish fed the control diet (Table 4). Fish fed diets with 10–40 and ≥100 mg PA/kg diet had higher hepatic CoA concentration than fish fed diets
Table 2 Red blood cell (RBC) count, hematocrit (Hct) and hemoglobin (Hb) concentrations of grouper fed different diets for 8 weeks1. Experimental diets
RBC
Hct
Hb
mg PA/kg diet
106/μL
%
g/dL
0 5 10 20 40 50 100 200
1.58 ± 0.01a 2.27 ± 0.21b 2.32 ± 0.27b 2.26 ± 0.07b 2.26 ± 0.07b 2.19 ± 0.23b 2.14 ± 0.31b 2.14 ± 0.32b
27.15 ± 3.75a 32.40 ± 0.72b 33.80 ± 2.91b 32.50 ± 2.97b 33.98 ± 2.03b 33.62 ± 2.57b 36.15 ± 0.92b 36.40 ± 1.70b
6.70 ± 0.28a 7.57 ± 0.06b 8.27 ± 0.55b 7.80 ± 0.28b 8.07 ± 0.31b 7.67 ± 0.74b 7.05 ± 0.71b 7.80 ± 0.42b
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 6 fish per group.
with ≤5 mg PA/kg diet. Hepatic total lipid concentration was lower in fish fed diets with ≥4.6 mg PA/kg diet than that in fish fed the control diet. Analysis of WG, hepatic PA and CoA concentrations by broken-line regression analysis indicated that the optimum dietary PA requirements of growing grouper were 10.7, 11.9 and 10.4 mg PA/kg diet, respectively (Fig. 2).
4. Discussion The essentiality of dietary pantothenic acid for grouper is clearly demonstrated in the present study. Fish fed the PA-free diet started floating on water surface with short breath within 4 weeks in the trial. After 8 weeks, significant lower survival (~ 66%) was recorded in fish fed the PA-free control diet compared to fish fed diet with PA supplementation (≥ 90%) (Table 3). Gill lesions (clubbed gill) were observed in fish fed the PA-free control diet. According to this, similar deficiency sign was also reported in blue tilapia (Soliman and Wilson, 1992), channel catfish (Murai and Andrews, 1979; Wilson et al., 1983) and rainbow trout (Karges and Woodward, 1984) when these fish fed PA-free diet. This gill deficiency sign is characterized by an interlamellar tissue proliferation, which is most marked at the distal end of the gill filaments (Wilson et al., 1983). This gill lesion may result in short breath of the grouper fed the PA-free diet, consequently with high mortality (Table 3). Anemia is another PA deficiency sign in fish (NRC, 2011), which is considered to be associated with anorexia in fish fed with PAdeficient diet (Murai and Andrews, 1979; Soliman and Wilson, 1992). Low hematological parameters, including RBC, Hct and Hb Table 3 Weight gain, feed efficiency (FE) and survival of grouper fed different diets for 8 weeks1.
Fig. 1. Histological changes (×200) of gills in grouper fed diets with 0 and 10 mg PA/kg diet for 8 weeks. (a) Clubbed gills with marked interlamellar proliferative lesions were observed in fish fed the diet with 0 mg PA/kg diet; (b) no lesions were found in gills in fish fed the diet with 10 mg PA/kg diet.
Experimental diets
Weight gain
mg PA/kg diet
%
0 5 10 20 40 50 100 200
175.8 ± 35.3a 232.6 ± 42.9ab 260.7 ± 16. 8b 269.6 ± 14.7b 277.1 ± 29.4b 280.0 ± 16.7b 265.4 ± 24.6b 256.7 ± 21.3b
FE
Survival %
0.57 ± 0.24a 0.87 ± 0.17ab 0.91 ± 0.09b 0.98 ± 0.09b 0.96 ± 0.12b 0.98 ± 0.05b 0.90 ± 0.16b 0.89 ± 0.03b
66.7 ± 25.2a 90.0 ± 10.0b 96.7 ± 5.8b 93.3 ± 11.6b 90.0 ± 10.0b 96.7 ± 5.8b 96.7 ± 5.8b 93.3 ± 5.8b
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 10 fish per group.
Y.-H. Lin et al. / Aquaculture 324–325 (2012) 92–96 Table 4 Hepatic pantothenic acid (PA), coenzyme A (CoA) and total lipid concentration of grouper fed different diets for 8 weeks1. Experimental diets
PA
CoA
Total lipid
mg PA/kg diet
μg/g tissue
μg/g tissue
%
0 5 10 20 40 50 100 200
23.58 ± 1.06a 30.34 ± 3.56b 42.05 ± 1.69c 44.70 ± 0.67c 45.44 ± 4.20c 44.91 ± 0.55c 46.38 ± 3.15c 47.80 ± 3.18c
9.34 ± 0.21a 12.64 ± 3.59ab 20.41 ± 1.36c 20.65 ± 0.33c 21.25 ± 0.45c 18.38 ± 4.34bc 19.28 ± 3.96c 22.89 ± 2.43c
22.05 ± 2.63b 13.78 ± 0.34a 11.57 ± 0.40a 13.38 ± 0.58a 14.54 ± 1.97a 14.88 ± 2.63a 11.43 ± 1.38a 12.24 ± 0.09a
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 6 fish per group.
are characterized as anemia sign in fish. These were observed in grouper fed the PA-free control diet (Table 2). Hemoglobin synthesis in animal starts combining succinyl CoA and glycine as its first step (Shemin and Russell, 1953). Deficiency in PA depletes tissue CoA concentration which in turn depresses the conversion of succinate to succinyl CoA. In the present study, grouper fed the PA-free diet exhibits the lowest hepatic CoA and blood Hb concentrations (Tables 2 and 4). Liver is major PA storage site in animals. In the literature, besides WG hepatic PA concentration has also been used as a parameter to quantify PA requirements for common carp (Ogino, 1967) and
300
Ymax=272.8
200 150
Y=8 (R 2 .49X+ =0. 1 89) 82.0
250 10.7
50 Ymax=45.21
Y=1.8 7 (R 2=0 +22.93 .98)
40
30
11.9
25 Ymax=20.48
20 15 10 5
Y=1 .1 (R 2= 3X+8.6 0.96 8 )
Hepatic CoA concentration Hepatic PA concentration (µg/g tissue) (µg/g tissue)
Weight gain (%)
350
10.4
0 0
50
100
150
200
Analyzed dietary PA concentration (mg PA/kg diet) Fig. 2. Broken-line analysis of weight gain, hepatic pantothenic acid (PA) and coenzyme A (CoA) concentrations on analyzed dietary PA concentration indicates that the optimal pantothenic acid levels for of juvenile grouper are 10.7, 11.9 and 10.4 mg PA/kg diet, respectively. Each point represents the mean ± SD of three aquaria within a treatment with 10 fish per aquarium for weight gain and with 6 fish per aquarium for hepatic PA and CoA concentrations, respectively.
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yellowtail (Shimeno, 1991). Higher requirement values were obtained from hepatic PA concentration data (45 and 35.9 mg PA/kg diet for common carp and yellowtail, respectively) than those obtained with WG data (30 and 13.5 mg PA/kg diet for common carp and yellowtail, respectively). This difference in PA requirements obtained from the two parameters was explained as hepatic PA concentration responds more rapidly to dietary vitamin intake than weight gain (Shimeno, 1991). It is noted, however, that the two studies were conducted for 6 weeks (common carp) and 30 days (yellowtail). It has been suggested that the duration of feeding normally encompass at least 8 to 12 weeks, but it is not appropriate to arbitrarily assign a specific time period (Cowey, 1992). The important point is that if a dose–response experiment is conducted, then a sufficient increase in weight will be required to observe recognizable and perhaps significant dose–response relationships (NRC, 2011). This means that using WG as a response variable, care needs to be exercised to ensure that the magnitude of the response achieves a level that is sufficiently great for comparison among treatments before an experiment is terminated. A standard often used is a 300% increase in body weight (which represents the twice doubling of the initial weight) (NRC, 2011). In our study, the highest WG of grouper obtained (280%, Table 3) compares favorably with those of previous studies in our laboratory (Lin and Shiau, 2003, 2007; Lin et al., 2008), in which about 300% WG was observed in similar sized fish fed a nutritionally adequate diet for 8 weeks. Whereas, WG of the common carp was only 114–171% (Ogino, 1967). Other parameters, such as survival, RBC, Hct, Hb and hepatic lipid concentrations have been analyzed by the broken-line model. However, these parameters were not fit the model well. Hence, only the three parameters, i.e. WG, hepatic PA and CoA concentrations, were presented in our study. Broken-line model is a widely used method of evaluating dose response data in nutrient requirement studies for aquatic species. This technique involves using two straight lines to model the dose–response relationship (Robbins et al., 1979). This linear model assumes that the response of an animal to increasing dietary intake of a limiting indispensable nutrient will increase linearly until the requirement is met, after which no increase in response, represented by a horizontal line (slope = 0), or a negative response will be observed. The break point corresponds to the nutrient requirement or maximum nutrient level that will produce the maximum response (NRC, 2011). However, the dose–response relationship, in some cases, is curvilinear as the requirement is approached, thus broken-line model may underestimate the requirement. For data such as these, an alternate model that includes a quadratic component (polynomial regression) is required (Robbins et al., 2006). In the present study, when the polynomial (cubic) regression model was employed with WG data, a peak of 54.0 mg PA/kg diet was obtained (Y = 0.00015X 3–0.048X 2 + 3.82X + 201.97, R 2 = 0.82). Judging from the ANOVA analysis (Table 3), fish fed the diet supplemented with 10 mg PA/kg diet reached a plateau, and remained unchanged statistically thereafter. It is unlikely that 54.0 mg PA/kg diet would be the optimum requirement level for the fish. Thus, the broken-line model is more appropriate for quantifying PA requirements of grouper in which values of 10.4–11.9 were obtained (Fig. 2). Both hepatic PA and CoA concentrations of grouper were also used as parameter to quantify PA requirements, the values (11.9 and 10.4 mg PA/kg diet, Fig. 2) agree with the obtained from WG data (10.7 mg PA/kg diet). High hepatic lipid concentration (fatty liver) was observed in grouper fed the PA-free diet (Table 4). Fatty liver was also reported in lake trout (Salvelinus namaycush) (Poston and Page, 1982), Mexican cichlid (Cichlasoma urophthalmus) (Martinez et al., 1990), and blue tilapia (Roem et al., 1991) fed with the PA-free diet. In the present study, low hepatic CoA concentration was also observed in grouper fed the PA-free diet (Table 4). Coenzyme A and phosphopantotheine are the recognized coenzyme forms of PA which involved in
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many reactions in intermediary metabolism. As the universal carriers of acyl groups, they are particularly important in fatty acid metabolism. Fatty acids must also be activated by CoA before they can be synthesized into triglycerides (NRC, 2011). The low hepatic CoA concentration in fish fed the PA-free diet may explain fatty liver in the PA-deficient grouper. In conclusion, grouper fed diet deficient in PA showed deficiency signs including clubbed gills, anemia, high mortality, anorexia, sluggishness, poor growth, and high lipid but low PA and CoA concentrations in liver. An adequate level of 11 mg PA/kg diet is recommended for grouper diet to improve performance of the fish. Acknowledgments This work was supported by a grant from the National Science Council of the Republic of China, grant number NSC 98-2321-B-126001-MY3. References Association of Official Analytical Chemists (AOAC), 1995. Official Methods of Analysis, 16th ed. AOAC, Arlington, VA. Chen, H.Y., Tsai, J.C., 1994. Optimum dietary protein level for the growth of juvenile grouper, Epinephelus malabaricus, fed semipurified diets. Aquaculture 119, 265–271. Cho, C.Y., Woodward, B., 1990. Dietary pantothenic acid requirements of young rainbow trout (Oncorhynchus mykiss). The FASEB Journal 4, 3747. Cowey, C.B., 1992. Nutrition: estimating requirements of rainbow trout. Aquaculture 100, 177–189. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics 11, 1–42. FAO (Food Agriculture Organization, 2011. Yearbooks of Fisheries Statistics. Food Agriculture Organization of the United Nations, Rome. Folch, J., Lees, M., Solane Stanley, G.M., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509. Karges, R.G., Woodward, B., 1984. Development of lamellar epithelial hyperplasia in gills of pantothenic acid-deficient rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology 25, 57–62. Lin, Y.H., Shiau, S.Y., 2003. Dietary lipid requirement of grouper, Epinephelus malabaricus, and effects on immune response. Aquaculture 225, 243–250.
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Estimation of dietary pantothenic acid requirement of grouper, Epinephelus malabaricus according to physiological and biochemical parameters Yu-Hung Lin a, Hui-You Lin b, Shi-Yen Shiau b, c,⁎ a b c
Department of Aquaculture, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 912, Taiwan, ROC Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 202, Taiwan, ROC Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC
a r t i c l e
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Article history: Received 5 August 2011 Received in revised form 19 October 2011 Accepted 19 October 2011 Available online 26 October 2011 Keywords: grouper pantothenic acid nutritional requirements deficiency
a b s t r a c t To quantify the dietary pantothenic acid (PA) requirements of grouper, Epinephelus malabaricus, calcium d-PA was added to basal diet at 0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet. Triplicate groups of fish (initial body weight: 15.3 ± 0.3 g) were fed with each experimental diet for 8 weeks in a recirculating seawater rearing system. After feeding trial, besides growth and survival, hematological index, hepatic PA, CoA and total lipid concentrations in fish were also monitored. Pantothenic acid deficiency signs including clubbed gills, anemia, high mortality, anorexia, sluggishness, and poor growth were observed in fish fed the PA-free control diet. Fish fed diets supplemented with ≥ 10 mg PA/kg diet had significantly (P ≤ 0.05.) higher weight gain (WG) and feed efficiency than fish fed the PA-free control diet. Fish fed diets supplemented with ≥ 5 mg PA/kg diet had higher survival, red blood cell count, hematocrit and hemoglobin concentrations, but lower hepatic total lipid concentration than fish fed the control diet. Hepatic PA concentration was highest in fish fed diets with ≥10 mg PA/kg diet, followed by 5 mg PA/kg diet group, and lowest in fish fed the control diet. Fish fed diets with 10–40 and ≥ 100 mg PA/kg diet had higher hepatic CoA concentration than fish fed diets with ≤5 mg PA/kg diet. Analysis of WG, hepatic PA and CoA concentrations by broken-line regression indicated that the optimum dietary PA requirement of growing grouper is about 11 mg PA/kg diet. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Pantothenic acid (PA) is a component of coenzyme A (CoA), acyl CoA synthetase, and acyl carrier protein. The coenzyme form of the vitamin is therefore responsible for acyl group transfer reactions. Coenzyme A is required in reactions in which the carbon skeletons of glucose, fatty acids, and amino acids enter into the energyyielding tricarboxylic acid cycle (NRC, 2011). This coenzyme is also involved in acetylation of choline to form the neurotransmitter acetylcholine and biosynthesis of cholesterol. Acyl carrier protein is required for fatty acid synthesis. A dietary PA deficiency impairs normal metabolism in cells undergoing rapid mitosis and high energy expenditure (Roem et al., 1991). Many of the effects of PA deficiency could result from lower tissue levels of CoA in animals (Reibel et al., 1982). The dietary PA requirement has been reported in several fish species, including rainbow trout (Oncorhynchus mykiss) (Cho and Woodward, 1990), common carp (Cyprinus carpio) (Ogino, 1967),
⁎ Corresponding author at: Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC. Fax: + 886 2 2462 1684. E-mail address: [email protected] (S.-Y. Shiau). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.020
channel catfish (Ictalurus punctatus) (Murai and Andrews, 1979; Wilson et al., 1983), blue tilapia (Oreochromis aureus) (Roem et al., 1991; Soliman and Wilson, 1992), Jian carp (Cyprinus carpio var. Jian) (Wen et al., 2009) and yellowtail (Seriola quinqueradiata) (Shimeno, 1991), ranging from 10 to 45 mg PA/kg diet. Groupers are popular and high value marine fish in many parts of the world such as in Kuwait, Indonesia, Malaysia, Thailand, the Philippines, Hong Kong, Taiwan, China, Mexico and Japan. They are also good candidates for intensive aquaculture because of their desirable taste, hardiness and rapid growth (Chen and Tsai, 1994; Millamena, 2002). Global grouper production increased dramatically in recent years, recorded as 60,774, 99,498, 163,328 and 204,284 MT in 1990, 2000, 2005, and 2009 respectively (FAO Yearbooks of Fisheries Statistics, 2011). Because no quantitative PA requirement has been reported for grouper, the following study was designed to estimate the dietary PA requirements for juvenile grouper, Epinephelus malabaricus. The PA levels in assayed diet were based on published PA requirement results of other fish species (10 to 45 mg PA/kg diet) plus lower (5 mg PA/kg diet) and excessive (60, 100 and 200 mg PA/kg diet). Other than growth performance, histological assay for gill, hematological index and hepatic PA, CoA and total lipid concentrations in fish were also monitored.
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2. Materials and methods
2.2. Experimental procedure
2.1. Experimental diets
Juveniles E. malabaricus obtained from a local hatchery (Pingtung, Taiwan) were used in the study. Upon arrival, they were acclimated to laboratory conditions for 4 weeks in a 1000 L plastic tank and fed a commercial diet (Uni-President Enterprise Corp., Tainan, Taiwan): moisture, 11.7%; crude protein, 43.3%; lipid, 8.8%; ash, 9.3%. At the beginning of the experiment, 10 fish (mean initial weight ± SD: 15.3 ± 0.3 g) were stocked in each of 24 aquariums (width × length × height: 0.305 × 0.610 × 0.360 m 3). Each experimental diet was assayed in triplicate (8 experimental diets × 3 tanks). Diets were assigned to groups of fish randomly. Each aquarium was part of a closed recirculating system with a common reservoir of seawater at 29 to 32‰ salinity. The seawater was obtained from the coast near the National Taiwan Ocean University (Keelung, Taiwan). Water temperature of the rearing system was 29 ± 1 °C. The flow water was 2 L/min through two separate biofilters to remove impurities and reduce ammonia concentrations. Half of the water in the system was exchanged daily. Fish were fed 3% of their body weight per day. This amount was close to the maximal daily ration for grouper according to feed consumption during the acclimation period of the study. The daily ration was divided into two equal meals (08:30 and 16:30 h). Fish were weighed once every 2 weeks and the daily ration adjusted accordingly. The remaining feed and feces were removed by a siphon immediately after feeding. A photoperiod of 12 h light (08:00 to 20:00 h), 12 h dark was used. Dead fish were removed and not replaced during the experiment. Fish were fed the test diets for an 8-week period.
Eight experimental diets were formulated according to different levels of PA (0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet). Vitamin-free casein (Sigma Chemical Co., St. Louis, MO), fish oil (Semi-refined fish oil, Oleaginosa Victoria S.A., Peru) and corn oil (Tai-Tang Industrial, Taiwan), and corn starch (Sigma Chemical) were used as dietary protein, lipid and carbohydrate sources, respectively. Dietary protein and lipid supplementation levels were recommended by Chen and Tsai (1994) and Lin and Shiau (2003), respectively. All experimental diets were adjusted to be isoenergetic at 15.05 KJ/g diet. An attractant that had a similar chemical composition to squid mantle tissue (Mackie and Mitchell, 1985) was added at 6% to all diets to increase palatability and diet acceptance. Basal diet was supplemented with calcium d-PA (92% PA, Sigma Chemical) at 0, 5, 10, 20, 40, 60, 100 and 200 mg PA/kg diet. Pantothenic acid-free basal diet formulation and proximate composition analysis (AOAC, 1995) are shown in Table 1. Dietary PA concentrations of the eight experimental diets were determined with a microbiological assay using Lactobacillus plantarum (BCRC 10357) from the Bioresource Collection and Research Center (Hsinchu, Taiwan) according to the procedure of AOAC (1995). The growth of L. plantarum was dependent on the PA levels. The determined values were 0, 4.6, 9.9, 19.4, 34.3, 50.2, 98.9, and 192.8 mg PA/kg diet. All ingredients of the experimental diet were mixed, and then enough cold water was added to form a stiff dough. This was then passed through a mincer with die, and the resulting strands were dried using an electrical fan at 20 °C. After drying, the diets were broken up and sieved into pellets (1.2 mm in diameter) and stored at −20 °C until used.
Table 1 Formulation and proximate composition of the basal diet. % (wet matter) Ingredient Casein Fish oil Corn oil Corn starch Vitamin mixturea Mineral mixtureb Attractantc Carboxylmethylcellulose Alpha-cellulose Proximate composition Moisture Ash Crude protein Ether extract
51 4.5 4.5 16.7 2 4 6 3 8.3 10.46 3.17 47.61 8.78
a Vitamin mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboflavin, 10; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; L-ascorbyl2-monophosphate Mg, 5; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025; biotin, 0.25. All ingredients were diluted with alpha-cellulose to 1 g. b Mineral mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4.2H2O, 135.8; MgSO4.7H2O, 132; Na2HPO4.2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4.7H2O, 3; MnSO4.H2O, 0.8; CoCl2.6H2O, 1; KI, 0.15; CuSO4, 0.15; AlCl3.6H2O, 0.15; selenomethionine, 0.02. All ingredients were diluted with alpha-cellulose to 1 g. c As mg/100 g diet: L-aspartic acid, 18; L-threonine, 44; L-serine, 33; L-glutamic acid, 53; Lvaline, 36; L-methionine, 36; L-isoleucine, 29; L-leucine, 55; L-tyrosine, 22; L-phenylalanine, 29; L-lysine-HCl, 29; L-histidine-HCl, 15; L-proline, 1456; L-alanine, 273; L-arginine, 228; taurine, 337; glycine, 892; betain-HCl, 910; trimethylamine-HCl, 91; trimethylamine n-oxide HCl, 1138; hypoxanthine, 47; inosine, 25; adenosine-5′-monophosphate, 40; L-(+)-lactic acid, 91; alpha-cellulose, 80 (Mackie and Mitchell, 1985).
2.3. Growth performance and assay methods At the end of the feeding trial, percentage of body weight gain (WG) in each aquarium [100 × (final body weight–initial body weight)/initial body weight], feed efficiency (FE) [(final body weight–initial body weight)/feed intake], and survival [100 × (final fish number/initial fish number] were calculated. After the final weight was noted, blood was collected using heparinized syringes from the caudal vein from six ice-anesthetized fish selected randomly per aquarium and pooled. The red blood cell (RBC) count, hematocrit (Hct), and hemoglobin (Hb) concentrations were measured by auto hematological analyzer (KX 21N, Sysmex Co., Japan). After blood collecting, the fish were sacrificed. The livers of six fish from a same tank were removed and pooled. Liver samples were stored at −80 °C freezer until analysis. Hepatic PA concentration was determined with the same method of feed analysis (AOAC, 1995). Hepatic CoA was measured according to the method described by Tubbs and Garland (1969). Briefly, hepatic CoA was extracted by perchloric acid (8%, containing 15 mM dithiothreitol). Then the extractions were reacted with acyl-CoA synthetase and absorbance was recorded by the spectrophotometer to calculate CoA concentration (Hitachi U-2000, Tokyo, Japan). Total lipid concentration in liver was determined by Folch method (Folch et al., 1957) using chloroform/methanol (2:1, v/v) to extract the total lipid from liver sample. In addition, gills from the same fish were dissected, fixed in 10% formalin, dehydrated by ethanol (100%, Merch Co., Germany), embedded in Paraplast (Sigma Chemical), sectioned at 6 μm and stained with hematoxylin and eosin (Sigma Chemical) (Wilson et al., 1983). The gill sections were examined microscopically for histological changes. 2.4. Statistical analysis Results are expressed as mean ± SD. Data were analyzed by oneway analysis of variance (ANOVA), and significance was set at
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P ≤ 0.05. Multiple comparisons among means were made with Duncan's new multiple range test (Duncan, 1955). Dietary PA requirements for juvenile grouper were estimated by the brokenline model with the weight gain, hepatic PA and CoA concentrations. Data analyses were performed with using the SAS/PC statistical software (SAS Inst. Inc., Cary, NC). 3. Results Fish fed the PA-free control diet began to exhibit deficiency signs including anorexia, sluggishness, poor growth and increased mortality within 4 weeks of the trial. After 8 weeks, clubbed gills with marked interlamellar proliferative lesions were observed (Fig. 1). No lesions were found in gills from the fish fed PA-supplemented diets. Red blood cell (RBC) count, hematocrit (Hct) and hemoglobin (Hb) concentrations were significantly higher in fish fed diets with ≥5 mg PA/kg diet than fish fed the control diet (Table 2). Fish fed diets supplemented with ≥10 mg PA/kg diet had significantly (P ≤ 0.05) higher weight gain (WG) and feed efficiency (FE) than fish fed the PA-free control diet (Table 3). Survival was significantly higher in fish fed diets with ≥ 5 mg PA/kg diet than those fed the control diet. Hepatic PA concentration was the highest in fish fed diets with ≥ 10 mg PA/kg diet, followed by 5 mg PA/kg diet, and lowest in fish fed the control diet (Table 4). Fish fed diets with 10–40 and ≥100 mg PA/kg diet had higher hepatic CoA concentration than fish fed diets
Table 2 Red blood cell (RBC) count, hematocrit (Hct) and hemoglobin (Hb) concentrations of grouper fed different diets for 8 weeks1. Experimental diets
RBC
Hct
Hb
mg PA/kg diet
106/μL
%
g/dL
0 5 10 20 40 50 100 200
1.58 ± 0.01a 2.27 ± 0.21b 2.32 ± 0.27b 2.26 ± 0.07b 2.26 ± 0.07b 2.19 ± 0.23b 2.14 ± 0.31b 2.14 ± 0.32b
27.15 ± 3.75a 32.40 ± 0.72b 33.80 ± 2.91b 32.50 ± 2.97b 33.98 ± 2.03b 33.62 ± 2.57b 36.15 ± 0.92b 36.40 ± 1.70b
6.70 ± 0.28a 7.57 ± 0.06b 8.27 ± 0.55b 7.80 ± 0.28b 8.07 ± 0.31b 7.67 ± 0.74b 7.05 ± 0.71b 7.80 ± 0.42b
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 6 fish per group.
with ≤5 mg PA/kg diet. Hepatic total lipid concentration was lower in fish fed diets with ≥4.6 mg PA/kg diet than that in fish fed the control diet. Analysis of WG, hepatic PA and CoA concentrations by broken-line regression analysis indicated that the optimum dietary PA requirements of growing grouper were 10.7, 11.9 and 10.4 mg PA/kg diet, respectively (Fig. 2).
4. Discussion The essentiality of dietary pantothenic acid for grouper is clearly demonstrated in the present study. Fish fed the PA-free diet started floating on water surface with short breath within 4 weeks in the trial. After 8 weeks, significant lower survival (~ 66%) was recorded in fish fed the PA-free control diet compared to fish fed diet with PA supplementation (≥ 90%) (Table 3). Gill lesions (clubbed gill) were observed in fish fed the PA-free control diet. According to this, similar deficiency sign was also reported in blue tilapia (Soliman and Wilson, 1992), channel catfish (Murai and Andrews, 1979; Wilson et al., 1983) and rainbow trout (Karges and Woodward, 1984) when these fish fed PA-free diet. This gill deficiency sign is characterized by an interlamellar tissue proliferation, which is most marked at the distal end of the gill filaments (Wilson et al., 1983). This gill lesion may result in short breath of the grouper fed the PA-free diet, consequently with high mortality (Table 3). Anemia is another PA deficiency sign in fish (NRC, 2011), which is considered to be associated with anorexia in fish fed with PAdeficient diet (Murai and Andrews, 1979; Soliman and Wilson, 1992). Low hematological parameters, including RBC, Hct and Hb Table 3 Weight gain, feed efficiency (FE) and survival of grouper fed different diets for 8 weeks1.
Fig. 1. Histological changes (×200) of gills in grouper fed diets with 0 and 10 mg PA/kg diet for 8 weeks. (a) Clubbed gills with marked interlamellar proliferative lesions were observed in fish fed the diet with 0 mg PA/kg diet; (b) no lesions were found in gills in fish fed the diet with 10 mg PA/kg diet.
Experimental diets
Weight gain
mg PA/kg diet
%
0 5 10 20 40 50 100 200
175.8 ± 35.3a 232.6 ± 42.9ab 260.7 ± 16. 8b 269.6 ± 14.7b 277.1 ± 29.4b 280.0 ± 16.7b 265.4 ± 24.6b 256.7 ± 21.3b
FE
Survival %
0.57 ± 0.24a 0.87 ± 0.17ab 0.91 ± 0.09b 0.98 ± 0.09b 0.96 ± 0.12b 0.98 ± 0.05b 0.90 ± 0.16b 0.89 ± 0.03b
66.7 ± 25.2a 90.0 ± 10.0b 96.7 ± 5.8b 93.3 ± 11.6b 90.0 ± 10.0b 96.7 ± 5.8b 96.7 ± 5.8b 93.3 ± 5.8b
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 10 fish per group.
Y.-H. Lin et al. / Aquaculture 324–325 (2012) 92–96 Table 4 Hepatic pantothenic acid (PA), coenzyme A (CoA) and total lipid concentration of grouper fed different diets for 8 weeks1. Experimental diets
PA
CoA
Total lipid
mg PA/kg diet
μg/g tissue
μg/g tissue
%
0 5 10 20 40 50 100 200
23.58 ± 1.06a 30.34 ± 3.56b 42.05 ± 1.69c 44.70 ± 0.67c 45.44 ± 4.20c 44.91 ± 0.55c 46.38 ± 3.15c 47.80 ± 3.18c
9.34 ± 0.21a 12.64 ± 3.59ab 20.41 ± 1.36c 20.65 ± 0.33c 21.25 ± 0.45c 18.38 ± 4.34bc 19.28 ± 3.96c 22.89 ± 2.43c
22.05 ± 2.63b 13.78 ± 0.34a 11.57 ± 0.40a 13.38 ± 0.58a 14.54 ± 1.97a 14.88 ± 2.63a 11.43 ± 1.38a 12.24 ± 0.09a
Different superscripts in the column indicate significant (P ≤ 0.05) difference between different dietary treatments. 1 Values are means ± SD from three groups of fish fed on a same experimental diet (n = 3 tanks) with 6 fish per group.
are characterized as anemia sign in fish. These were observed in grouper fed the PA-free control diet (Table 2). Hemoglobin synthesis in animal starts combining succinyl CoA and glycine as its first step (Shemin and Russell, 1953). Deficiency in PA depletes tissue CoA concentration which in turn depresses the conversion of succinate to succinyl CoA. In the present study, grouper fed the PA-free diet exhibits the lowest hepatic CoA and blood Hb concentrations (Tables 2 and 4). Liver is major PA storage site in animals. In the literature, besides WG hepatic PA concentration has also been used as a parameter to quantify PA requirements for common carp (Ogino, 1967) and
300
Ymax=272.8
200 150
Y=8 (R 2 .49X+ =0. 1 89) 82.0
250 10.7
50 Ymax=45.21
Y=1.8 7 (R 2=0 +22.93 .98)
40
30
11.9
25 Ymax=20.48
20 15 10 5
Y=1 .1 (R 2= 3X+8.6 0.96 8 )
Hepatic CoA concentration Hepatic PA concentration (µg/g tissue) (µg/g tissue)
Weight gain (%)
350
10.4
0 0
50
100
150
200
Analyzed dietary PA concentration (mg PA/kg diet) Fig. 2. Broken-line analysis of weight gain, hepatic pantothenic acid (PA) and coenzyme A (CoA) concentrations on analyzed dietary PA concentration indicates that the optimal pantothenic acid levels for of juvenile grouper are 10.7, 11.9 and 10.4 mg PA/kg diet, respectively. Each point represents the mean ± SD of three aquaria within a treatment with 10 fish per aquarium for weight gain and with 6 fish per aquarium for hepatic PA and CoA concentrations, respectively.
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yellowtail (Shimeno, 1991). Higher requirement values were obtained from hepatic PA concentration data (45 and 35.9 mg PA/kg diet for common carp and yellowtail, respectively) than those obtained with WG data (30 and 13.5 mg PA/kg diet for common carp and yellowtail, respectively). This difference in PA requirements obtained from the two parameters was explained as hepatic PA concentration responds more rapidly to dietary vitamin intake than weight gain (Shimeno, 1991). It is noted, however, that the two studies were conducted for 6 weeks (common carp) and 30 days (yellowtail). It has been suggested that the duration of feeding normally encompass at least 8 to 12 weeks, but it is not appropriate to arbitrarily assign a specific time period (Cowey, 1992). The important point is that if a dose–response experiment is conducted, then a sufficient increase in weight will be required to observe recognizable and perhaps significant dose–response relationships (NRC, 2011). This means that using WG as a response variable, care needs to be exercised to ensure that the magnitude of the response achieves a level that is sufficiently great for comparison among treatments before an experiment is terminated. A standard often used is a 300% increase in body weight (which represents the twice doubling of the initial weight) (NRC, 2011). In our study, the highest WG of grouper obtained (280%, Table 3) compares favorably with those of previous studies in our laboratory (Lin and Shiau, 2003, 2007; Lin et al., 2008), in which about 300% WG was observed in similar sized fish fed a nutritionally adequate diet for 8 weeks. Whereas, WG of the common carp was only 114–171% (Ogino, 1967). Other parameters, such as survival, RBC, Hct, Hb and hepatic lipid concentrations have been analyzed by the broken-line model. However, these parameters were not fit the model well. Hence, only the three parameters, i.e. WG, hepatic PA and CoA concentrations, were presented in our study. Broken-line model is a widely used method of evaluating dose response data in nutrient requirement studies for aquatic species. This technique involves using two straight lines to model the dose–response relationship (Robbins et al., 1979). This linear model assumes that the response of an animal to increasing dietary intake of a limiting indispensable nutrient will increase linearly until the requirement is met, after which no increase in response, represented by a horizontal line (slope = 0), or a negative response will be observed. The break point corresponds to the nutrient requirement or maximum nutrient level that will produce the maximum response (NRC, 2011). However, the dose–response relationship, in some cases, is curvilinear as the requirement is approached, thus broken-line model may underestimate the requirement. For data such as these, an alternate model that includes a quadratic component (polynomial regression) is required (Robbins et al., 2006). In the present study, when the polynomial (cubic) regression model was employed with WG data, a peak of 54.0 mg PA/kg diet was obtained (Y = 0.00015X 3–0.048X 2 + 3.82X + 201.97, R 2 = 0.82). Judging from the ANOVA analysis (Table 3), fish fed the diet supplemented with 10 mg PA/kg diet reached a plateau, and remained unchanged statistically thereafter. It is unlikely that 54.0 mg PA/kg diet would be the optimum requirement level for the fish. Thus, the broken-line model is more appropriate for quantifying PA requirements of grouper in which values of 10.4–11.9 were obtained (Fig. 2). Both hepatic PA and CoA concentrations of grouper were also used as parameter to quantify PA requirements, the values (11.9 and 10.4 mg PA/kg diet, Fig. 2) agree with the obtained from WG data (10.7 mg PA/kg diet). High hepatic lipid concentration (fatty liver) was observed in grouper fed the PA-free diet (Table 4). Fatty liver was also reported in lake trout (Salvelinus namaycush) (Poston and Page, 1982), Mexican cichlid (Cichlasoma urophthalmus) (Martinez et al., 1990), and blue tilapia (Roem et al., 1991) fed with the PA-free diet. In the present study, low hepatic CoA concentration was also observed in grouper fed the PA-free diet (Table 4). Coenzyme A and phosphopantotheine are the recognized coenzyme forms of PA which involved in
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many reactions in intermediary metabolism. As the universal carriers of acyl groups, they are particularly important in fatty acid metabolism. Fatty acids must also be activated by CoA before they can be synthesized into triglycerides (NRC, 2011). The low hepatic CoA concentration in fish fed the PA-free diet may explain fatty liver in the PA-deficient grouper. In conclusion, grouper fed diet deficient in PA showed deficiency signs including clubbed gills, anemia, high mortality, anorexia, sluggishness, poor growth, and high lipid but low PA and CoA concentrations in liver. An adequate level of 11 mg PA/kg diet is recommended for grouper diet to improve performance of the fish. Acknowledgments This work was supported by a grant from the National Science Council of the Republic of China, grant number NSC 98-2321-B-126001-MY3. References Association of Official Analytical Chemists (AOAC), 1995. Official Methods of Analysis, 16th ed. AOAC, Arlington, VA. Chen, H.Y., Tsai, J.C., 1994. Optimum dietary protein level for the growth of juvenile grouper, Epinephelus malabaricus, fed semipurified diets. Aquaculture 119, 265–271. Cho, C.Y., Woodward, B., 1990. Dietary pantothenic acid requirements of young rainbow trout (Oncorhynchus mykiss). The FASEB Journal 4, 3747. Cowey, C.B., 1992. Nutrition: estimating requirements of rainbow trout. Aquaculture 100, 177–189. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics 11, 1–42. FAO (Food Agriculture Organization, 2011. Yearbooks of Fisheries Statistics. Food Agriculture Organization of the United Nations, Rome. Folch, J., Lees, M., Solane Stanley, G.M., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509. Karges, R.G., Woodward, B., 1984. Development of lamellar epithelial hyperplasia in gills of pantothenic acid-deficient rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology 25, 57–62. Lin, Y.H., Shiau, S.Y., 2003. Dietary lipid requirement of grouper, Epinephelus malabaricus, and effects on immune response. Aquaculture 225, 243–250.
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