Fish Sci (2009) 75:665–672 DOI 10.1007/s12562-009-0058-4
ORIGINAL ARTICLE
Aquaculture
Effect of dietary medicinal herbs on lipid metabolism and stress recovery in red sea bream Pagrus major Seung-Cheol Ji Æ Osamu Takaoka Æ Si-Woo Lee Æ Jae-Ho Hwang Æ Yang-Su Kim Æ Katsuya Ishimaru Æ Manabu Seoka Æ Gwan-Sik Jeong Æ Kenji Takii
Received: 28 March 2008 / Accepted: 2 September 2008 / Published online: 27 March 2009 Ó The Japanese Society of Fisheries Science 2009
Abstract The effect of dietary medicinal herbs on lipid metabolism and stress recovery was investigated in red sea bream Pagrus major. Fish (mean body weight 24.0 ± 0.2 g) were fed on test fish meal diets supplemented with either Massa Medicata (Mm), Crataegi Fructus (Cf), Artemisia capillaries (Ac), or Cnidium officinale (Co), or with a mixture of the four herbs (HM) for 12 weeks. A control group was fed a diet without herbs in the same manner. A high survival rate was observed in the herbal diet groups. The final mean body weight, feed efficiency, protein efficiency ratio, and apparent protein and lipid retention in the Ac, Co, and HM groups were higher than those in the control and Mm groups. Final carcass, hepatic lipid and triglyceride contents, and plasma triglyceride and nonesterified fatty acid levels were lower in the Ac, Co, and HM groups compared to those of control and Mm groups. However, final hepatic phospholipid, plasma phospholipid, and high density lipoprotein cholesterol levels were higher in the Ac, Co, and HM diets groups than in the control and Mm groups. The Cf, Co, Ac, and HM groups showed faster
S.-C. Ji (&) Jeju Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Jeju 690-192, Korea e-mail:
[email protected] O. Takaoka Y.-S. Kim M. Seoka K. Takii Fisheries Laboratory, Kinki University, Uragami, Wakayama 649-5145, Japan S.-W. Lee J.-H. Hwang G.-S. Jeong College of Fisheries and Ocean Sciences, Chonnam National University, Yeosu 550-749, Korea K. Ishimaru Fisheries Laboratory, Kinki University, Shirahama, Wakayama 649-2211, Japan
recovery time in the 2-phenoxyethanol anesthesia test and a higher recovery rate in the 10-min air exposure test than those of the control and Mm groups. Moreover, the Cf, Ac, Co, and HM diet groups had a significantly lower plasma cortisol level than the control and Mm diet group, but the glucose level in the herbal diet groups was higher than that in the control group after a 1-h air exposure. These results indicate that the addition of medicinal herbs to the fish diet improved lipid utilization and stress recovery in red sea bream. Keywords Air exposure Anesthesia Lipid metabolism Medicinal herbs Red sea bream
Introduction Red sea bream Pagrus major is one of the important cultured fish species and is in high demand by consumers in Japan and Korea. Reared fish can often be subjected to various stresses, including handling, transportation, and unsuitable rearing conditions, which can increase their susceptibility to infectious disease outbreaks [1]. As a result, the use of antibacterial agents has become standard practice in disease management. However, stricter rules and regulations on the use of antibacterial agents in combination with consumer concern over their long-term health effects is causing fish farmers to search for alternative disease management measures. The development of safe and functional fish feed that can enhance immune system activity and promote somatic growth is increasingly becoming a priority. Herbal remedies have a long history in Asian countries, where their use in preventing or curing diseases is highly popular among the general population. This popularity has
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been established on the belief that herbal remedies provide an organic and side-effect-free alternative to standard medicine for treating illnesses. Given this context, the use of herbal extracts in fish feed is increasingly being viewed as a practical and safe alternative to synthetic pharmaceutical agents. There are various reports of herbal fish diets promoting growth performance [2], increasing stress tolerance [3], and enhancing immune system efficiency [4]. The medicinal herbs Massa Medicate (Mm), Crataegi Fructus (Cf), Artemisia capillaries (Ac), and Cnidium officinale (Co) have been used for many purposes. Mm has been found to promote digestion by stimulating the secretion of digestive enzyme [5]. Cf, Ac, and Co have been reported to be anti-oxidants [6] and to improve lipid metabolism, enhance the immune system [7], and have an antibacterial function [8] in land mammals. It has recently been reported that a 0.5% addition of Mm, Cf, Ac, Co, or a mixture of the four herbs (HM) to the diet of juvenile red sea bream resulted in increased survival, weight gain, feed efficiency, and immune activity [9]. A 0.5% dietary supplement of HM has also been reported to induce better growth performance, improve fatty acid utilization, and increase stress recovery in cultured Japanese flounder [10]. However, there is a paucity of data on herbal supplemented feed and their effects on biological parameters. We report here our study on the effects of supplementing fish feed with Mm, Cf, Ac, or Co, either singularly and in combination, on lipid metabolic response, and stress recovery to an anesthetic and air exposure in red sea bream. Our aim was to add to the knowledge base of the effect of medicinal herbs on fish physiological parameters.
Fish Sci (2009) 75:665–672 Table 1 Formulation and chemical proximate composition of basal diet (% of dry matter) Ingredients
Basal diet
Brown fish meal
55
Soybean meal
10
Wheat flour
10
Fish oila
10
a-Potato starch
5
Vitamin mixtureb
3
c
3 4
Mineral mixture a-Cellulose
Proximate analysis (%) Crude protein
47.0
Crude lipid
14.7
Crude sugar
17.9
Crude ash
12.2
a
Pollack liver oil containing 10% of DHA oil
b
Vitamin mixture (mg/g mixture): thiamine hydrochloride, 2.0 mg; riboflavin, 6.7 mg; pyridoxine hydrochloride, 1.34 mg; nicotine acid, 26.7 mg; calcium pantothenate, 9.4 mg; inositol, 133.6 mg; biotin, 0.2 mg; folic acid, 0.5 mg; p-aminobenzoic acid, 13.4 mg; choline chloride, 267.2 mg; vitamin B12, 0.02 mg; vitamin K3, 3.0 mg; L-ascorbic acid, 66.8 mg; alpha-tocopherol, 13.4 mg; cholecalciferol, 0.0015 mg; retinol acetate, 0.23 mg. All ingredients were diluted with alpha-cellulose to 1 g c
Mineral mixture (mg/g mixture): KH2PO4, 206.0 mg; zinc sulfate, 357 mg; calcium lactate, 141.0 mg; iron proteinate, 83.0 mg; manganous sulfate, 80 mg; Ca2H2PO4, 309 mg; MgSO4, 12.5 mg; CoCl2, 0.1 mg; CuSO4, 4.0 mg; KIO3, 0.3 mg. All ingredients were diluted with alpha-cellulose to 1 g
water and then added to 100 g of basal diet. All diets, including the control diet, which contained no herb, were manufactured using a pellet machine (diameter 5 mm) and stored at -20°C before use.
Materials and methods Feeding trial Medicinal herb and test diet The basal composition of the test diet has been described in detail in previous studies [11, 12]; the dietary formula and proximate composition of the feed used in this study are presented in Table 1. We prepared four kinds of herbal supplements: powdered fruit of Massa Medicata (Kamikouji in Japanese) yeast-leavened wheat, fruit of Crataegi Fructus (Sanzashi in Japanese), leaves of Artemisia capillaries (Kawarayomogi in Japanese), and the root of Cnidium officinale (Senkyu in Japanese). These four types of powdered herbs were purchased from Korean local commercial markets. We also prepared a mixture of the four herbs (HM) which contained two parts of Mm and CF and 1 part of Ac and Co in dry weight ratio (2:2:1:1). In accordance with previous studies [9, 10], 0.5 g herb was mixed with 30-ml tap
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Juvenile red sea breams (mean body weight 24.0 ± 0.2 g) were obtained from the Fish Nursery Center, Kinki University, Uragami. Groups of 25 fish were introduced into 18 indoor 300-l rectangular tanks after being acclimatized to the rearing conditions, which included feeding on the control diet for 2 weeks. Rearing trials were performed in triplicate for each diet. Fish were fed twice daily (1000 and 1500 hours) for 12 weeks. The photoperiod was maintained at a 12/12-h [light (0600–1800 hours)/dark] cycle. Flow rate of sand-filtered seawater in each tank was maintained at 3 l/min, while the water temperature, salinity, and dissolved oxygen (DO) were 22.0 ± 4.6°C, 33.0 ± 1.7 %, and 7.5 ± 0.3 mg/l, respectively. Body weight and body length were measured at the start and end of each feeding trial.
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Sampling and assay At the end of the feeding trials, fish were randomly chosen and anaesthetized with ice seawater. The abdominal fat of six fish from each group was weighed and used for calculating the mean abdominal fat weight ratio (AFR). A further six fish from each group were extracted for proximate lipid content of the carcass and stored at -25°C. Livers were subsequently removed for proximate lipid content and lipid class analysis, and stored at -80°C. Proximate compositions of diets, carcass, and liver were assayed according to the AOAC method [13]. Liver fats were extracted following the method of Folch et al. [14], while the amount of different lipid classes was determined using a commercial kit (Wako Purechem, Osaka, Japan) according to Røsjø et al. [15]. Using a heparinized syringe, we collected blood samples from the caudal vein of six fish from each dietary group. After centrifugation (20,000 g, 10 min) at 4°C, plasma was sampled for assaying of total triglyceride, total cholesterol, phospolipid, high-density lipoprotein-cholesterol (HDLCHO), and nonesterified fatty acid (NEFA) levels using commercial kits (Wako Purechem). Anesthesia test Ten fish from each dietary group were subjected to 200, 400, and 800 ppm 2-phenoxyethanol (Wako Purechem) in solution for 2 min. The procedure was conducted twice. The fish were then put back into seawater, and recovery time was recorded with a chronometer. Recovery was established when the fish exhibited normal equilibrium and swimming behavior [16]. Air exposure test and analysis of plasma glucose and cortisol The air exposure test was conducted at the completion of feeding trials, as described by Yokoyama et al. [17]. Ten fish were randomly captured from each group, placed on a dry nylon net, and exposed to air for 5 min. This process was replicated with a second group at a 10-min exposure time. Following air exposure, the fish were placed back into the recovery tank, and the recovery rate was monitored for 6 h. After a 5-min air exposure, three fish were randomly selected from the recovery group at 0, 1, 2, 4 and 6 h postexposure to be used for blood glucose and cortisol level analysis. Blood was extracted from the caudal vein using a 1-ml heparinized disposable syringe. Plasma was obtained from blood samples by centrifugation (20,000 g, 4°C), from which glucose was measured by an analytical kit (Wako, Japan), while the enzyme-linked immunosorbent
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(ELISA) method was applied for measuring the cortisol level following the manufacturer’s instructions (Oxford Biomedical Research, Oxford, MI). Statistical analysis The SPSS statistical package (SPSS, Chicago, IL) was used to analyze data by one-way analysis of variance (ANOVA). When differences were found among dietary treatments, Tukey’s test was used to compare mean differences. Differences were considered significant at P \ 0.05.
Results Growth performance and lipid contents of carcass, liver and plasma As shown in Table 2, the survival of fish fed herbal diets was higher than that of the control group. Final mean body weight of juvenile red sea bream fed diets containing Cf, Ac, Co, or HM was significantly higher than those of the control and Mm groups (P \ 0.05). The feed efficiency and protein efficiency ratio (PER) of the Ac, Co, and HM diet groups were significantly higher than those of the control diet group. The AFR in the control and Mm diet groups were between 1.12–1.26%, while that for the Cf, Ac, and Co diet groups was between 1.03 and 0.93%. Apparent lipid retention of fish in the Ac, Co, and HM diet groups was lower than that of fish in the control, Mm, and Cf groups. Apparent protein retention in the Hm diet group was significantly higher than that of the control diet group. Diets containing Cf, Ac, Co, and HM led to lower final carcass and hepatic lipid contents compared to the control and Mm diets, although the differences were not significant (Table 3). Diets containing Ac, Co, and HM led to lower hepatic triglyceride contents than those found in the control and Cf groups. Phospholipid content was higher in fish fed diets of Cf, Ac, Co, or HM than in those of the control and Mm groups, but no significant differences in total cholesterol content was observed among all dietary groups. There were no significant differences in final plasma total cholesterol and phospholipid levels among dietary treatments (Table 4). The fish of the HM diet group showed significantly lower plasma triglyceride levels than those of the control and Mm diet groups (P \ 0.05). The Ac, Co, and HM diet groups had higher plasma HDL-CHO levels than those of control and Mm diet groups. Significantly lower nonesterified fatty acid (NEFA) levels were induced in the Ac, Co, and HM diet groups than in the control and Cf diet groups.
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Table 2 Growth performance and apparent protein and lipid retention in red sea bream fed diets containing different medicinal herbs for 12 weeks Physiological parameters
Dietary groups Control
Mm
Cf
Ac
Co
HM
Initial body weight (g)
24.1 ± 0.2
24.2 ± 0.1
24.2 ± 0.1
24.3 ± 0.3
23.8 ± 0.1
23.9 ± 0.2
Final body weight (g)
60.4 ± 2.2 a
63.2 ± 1.8 a
70.9 ± 2.8 b
72.8 ± 1.4 b
72.9 ± 3.0 b
81.4 ± 4.7 c
Feed efficiency (%) PERa
58.7 ± 3.2 a 1.23 ± 0.1 a
64.8 ± 1.6 ab 1.37 ± 0.0 a
60.5 ± 3.3 a 1.28 ± 0.1 a
71.2 ± 1.4 bc 1.50 ± 0.0 b
70.0 ± 3.3 bc 1.47 ± 0.1 b
74.4 ± 2.9 c 1.57 ± 0.1 b
Survival rate (%)
84.0 ± 5.3 a
96.0 ± 2.7 b
96.0 ± 2.7 b
100 ± 0.0 b
93.3 ± 3.6 b
97.3 ± 3.6 b 0.93 ± 0.6
AFR (%)
b
1.12 ± 0.4
1.26 ± 1.2
1.03 ± 0.4
0.97 ± 0.0
1.02 ± 0.4
Apparent protein retentionc
25.6 ± 2.4 a
26.0 ± 0.6 a
24.7 ± 1.3 a
26.8 ± 2.5 ab
28.4 ± 5.0 ab
30.8 ± 2.1 b
Apparent lipid retentionc
52.1 ± 9.6
52.0 ± 6.7
53.5 ± 11.9
49.6 ± 3.4
46.1 ± 9.7
46.6 ± 5.5
Mm, Massa Medicate; Cf, Crataegi Fructus; Ac, Artemisia capillaries; Co, Cnidium officinale; HM, mixture of four herbs Values are given as the mean ± standard deviation of three groups of fish (n = 6). Values within a column followed by different letters are significantly different (P \ 0.05) a Protein efficiency ratio = (final body weight - initial body weight)/protein intake b
Abdominal fat ratio = (abdominal fat weight/body weight) 9 100
c
(Final body protein and lipid content - initial body protein and lipid content)/protein and lipid intake
Table 3 Final lipid content of carcass and liver, and lipid class of liver in red sea bream fed diets containing different medicinal herbs for 12 weeks Lipid content
Dietary groups Control
Mm
Cf
Ac
Co
HM
Crude lipid contents (% of dry matter basis) 30.9 ± 4.3
30.7 ± 1.9
29.5 ± 9.6
28.8 ± 1.1
27.8 ± 3.0
27.6 ± 3.5
Liver 19.6 ± 3.8 Lipid class of liver (mg/g tissue)
Whole carcass
19.5 ± 3.0
15.6 ± 1.6
15.0 ± 7.2
17.7 ± 6.0
15.1 ± 3.3
40.7 ± 8.6 a
Triglyceride
54.3 ± 11.7 ab
48.8 ± 8.9 ab
55.1 ± 1.6 b
Phospholipid
8.2 ± 0.5 a
8.6 ± 0.8 a
10.4 ± 0.3 b
Total cholesterol
3.8 ± 0.2
3.4 ± 0.4
4.3 ± 0.3
9.0 ± 0.6 ab 3.3 ± 0.9
44.4 ± 14.0 ab
42.9 ± 1.5 a
11.2 ± 0.8 b
10.2 ± 0.3 b
3.9 ± 0.6
3.5 ± 0.3
Values are given as the mean ± standard deviation of three groups of fish (n = 6). Values within a column followed by different letters are significantly different (P \ 0.05)
Table 4 Hematological values of red sea bream fed diets containing different medicinal herbs for 12 weeks Hematological values
Dietary groups Control
Mm
Cf
Ac
Co
HM
Triglyceride (mg/dl)
321.1 ± 32.7 bc
493.3 ± 126.5 c
367.3 ± 76.8 bc
292.3 ± 96.9 ab
276.9 ± 17.3 ab
194.2 ± 48.0 a
Total cholesterol (mg/dl)
250.9 ± 49.8
245.2 ± 15.6
298.8 ± 40.1
223.6 ± 113.7
306.6 ± 37.4
259.4 ± 69.3
HDL-cholesterol (mg/dl)
143.0 ± 28.7 ab
116.3 ± 1.9 a
158.2 ± 3.7 bc
170.4 ± 35.0 bc
192.0 ± 15.9 cd
218.0 ± 12.8 d
Phospholipid (mg/dl)
841.5 ± 96.2
924.2 ± 34.6
858.2 ± 59.0
854.7 ± 40.2
998.7 ± 89.5
954.1 ± 53.6
Free fatty acid (lEq/l)
215.3 ± 28.5 ab
312.7 ± 138.3 b
275.3 ± 52.1 ab
160.1 ± 21.0 ab
122.7 ± 38.6 a
152.7 ± 28.0 a
HDL, High-density lipoprotein Values are given as the mean ± standard deviation of three groups of fish (n = 6). Values within a column followed by different letters are significantly different (P \ 0.05)
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669 120
As shown in Fig. 1, no significant difference was detected in recovery time at 200 ppm and 400 ppm 2-phenoxyethanol concentrations. However, recovery time in the Cf, Co, and HM diet groups at the 800 ppm 2-phenoxyethanol concentrations were significantly faster than those of the control and Mm diet groups (P \ 0.05). The recovery rate of fishes exposed to 5 min of air was not significantly different among the dietary groups (Fig. 2). However, the recovery rate after a 10 min exposure to air was significantly higher in all fish on diets supplemented with herbs compared to that of control group. Cortisol level changes after 5 min of air exposure are shown in Fig. 3. The post-stress cortisol response reached a peak after 1 h. The cortisol level of the control diet group (60.0 ng/ml) was significantly higher than that of the herbal diet groups (15.3–35.0 ng/ml). After 2 h of recovery time, the cortisol level returned back to normal levels, and there was no significant difference among dietary treatments. Changes in the plasma glucose level after 5 min of air exposure are shown in Fig. 4. The glucose level of the control group (172.1 mg/100 ml) was lower than those of herbal diet groups (210–255.2 mg/100 ml) after 1 h. However, the lowest glucose level was detected in the HM diet group (165.0 mg/100 ml), whereas the control and Cf groups showed 217.8 and 275.1 mg/100 ml, respectively, after 2 h. After 4 h, the Cf and HM diet groups showed
100
Recovery rate (%)
Recovery from the stress
5 min 10 min
80
c c
60
bc
40
bc
b a
20 0 Control
Mm
Cf
Ac
Co
HM
Dietary groups Fig. 2 Changes in recovery rate of red sea bream during the first 6 h following the air exposure test. Duplicate tests for 5 and 10 min each was carried out on ten fish from each dietary group. Bar Mean and standard deviation (n = 2). Bars with different letters are significantly different (P \ 0.05) 70
Cortisol (ng/mL)
60
c Control Mm Cf Ac Co HM
50 40 b
30 20
)a 10 7
Recovery time (min)
6
a
a
200 ppm 400 ppm 800 ppm
ab
0 0
2
4
6
Recovery time (h)
5
Fig. 3 Changes in plasma cortisol level of red sea bream after the 5-min air exposure test. Different letters indicate significant differences (P \ 0.05)
bc
4
1
b 3
c
2
lower glucose levels (121.4 and 109.8 mg/100 ml, respectively) than the control group (174.7 mg/100 ml).
1
Discussion
0 Control
Mm
Cf
Ac
Co
HM
Dietary groups Fig. 1 Recovery time of red sea bream after the anesthesia test. Duplicate treatments with 200, 400, or 800 ppm 2-phenoxyethanol solution were given to ten fish from each dietary group for 1 min each time. Bar Mean and standard deviation (n = 2). Bars with different letters are significantly different (P \ 0.05). Mm Massa Medicate, Cf Crataegi Fructus, Ac Artemisia capillaries, Co Cnidium officinale, HM mixture of four herbs
Our results indicate that medicinal herbs improved lipid metabolism and stress tolerance in red sea bream. The involvement of Ac, Cf, and Co in improving lipid metabolism and immunity in land animals has already been reported [7, 18]. The AFR and the crude lipid content of the carcass and liver tended to be lower in the Ac, Co, and HM diet groups
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Fish Sci (2009) 75:665–672 300 Control Mm Cf Ac Co HM
b b
Glucose (mg/100 mL)
250 ab
(
)ab
200 a
b a ab
150
a
) 100
50
0 0
1
2
4
6
Recovery time (h) Fig. 4 Changes in plasma glucose level of red sea bream after the 5-min air exposure test. Different letters indicate significant differences (P \ 0.05)
than in the control group. We also observed that serum NEFA level and triglyceride concentration of the liver in the Ac, Co, and HM diet groups were lower than those of the control group. The dissolution of triglyceride occurs continuously in fat cells around the abdomen, which increases the density of free fatty acid in the blood. Free fatty acid is then absorbed into fat cells and accumulated in fat tissues as triglycerides [18]. The involvement of Ac in decreasing the abdominal lipid content [19] and Co in repressing of lipid accumulation [20] have already been reported in rats. Here, we have demonstrated that Ac, Co, and HM reduced abdominal lipid accumulation and promoted lipid metabolism in the red sea bream. In contrast, serum phospholipid, HDL-CHO level, and liver phospholipid concentration showed a tendency to increase in the herbal diet groups. Phospholipid plays an important role as the transporter of triglyceride and cholesterol. It has been reported that HDL–CHO plays an important role in transporting lipid and enhancing immunity in shrimp [21], crayfish [22], and Characidae pacu Piaractus mesopotamicus [23]. Other studies have found that Ac and Co have a high effectiveness for the improvement of lipid metabolism by increasing the phospholipid and HDL-CHO levels in the liver or plasma in rat [19, 24]. As such herb supplementation helps transport fatty acid to diverse tissues and cells and improves lipid metabolism. In our study, this promotion of lipid metabolism by herb supplementation facilitated growth directly by increasing protein utilization and also caused high feed efficiency, PER, and protein retention (Table 2). The cortisol level increases in fish subjected to handling or transportation stress [25]. In this study, the plasma
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cortisol level at 1 h post-air exposure had increased rapidly in the control and Mm groups, and these values were significantly higher than those of herbal diet groups. However, an opposite trend was identified for the glucose level. At the 1-h recovery, glucose levels in the herbal diet groups were higher than those of the control group, but the levels decreased rapidly in the herbal diet groups. Iversen et al. [26] also reported opposite cortisol and glucose level trends in Atlantic salmon. The transport stress reaction was interpreted as a fast response reaction of glucose metabolism against cortisol secretion [27]. This result also indicates that herbs promote glucose metabolism, which is a secondary stress response, and that the rapid and strong response to physiological stressor contributes to the constraining of cortisol increase as well as the rapid recovery against stress. The herbs Crataegi Fructus, Artemisia capillaries, and Cnidium officinale used in this study contain various antioxidants compounds that include carotinoids, flavonoides, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, and tocotorienols [23, 28–30]. It is generally accepted that endogenous oxidative damage leads to ageing, dysfunction, and disease. The mammalian disease and lifespan correlates directly with the resistance of cellular structure potency to oxidative challenge [31]. The effectiveness of herbal diet groups was confirmed by the rapid recovery after anesthetic test and high survival rate after the air exposure test. Mortality was frequent in the control group due to Flexibacter columnaris infection from the second to seventh week, resulting in significantly low overall survival rate. Our results suggest that the herbal diet can be used effectively in aquaculture in red sea bream. The increase in cortisol levels leads to the consumption of stored energy, which reduces growth due to increasing gluconeogenesis and aminotransferase activity in the liver as well as protein catabolism [32]. Therefore, when recovery from stress is delayed, a decrease in growth is accelerated by increases in the metabolic rate and continuous energy consumption [27]. In this study, the high growth rate observed in the herbal diet groups was attributed to rapid stress recovery, which decreased the consumption of stored energy. In summary, medicinal herbs Cf, Ac, Co, and HM contributed to good growth performance and survival rate by promoting lipid metabolism and stress recovery in red sea bream. In particular, the HM diet group was superior in terms of growth performance and stress recovery relative to the other herbal diet groups. Medicinal herbs are commonly used in combined form, and their supplementary effects in mariculture feeds for flounder [33], parrotfish [34], and yellow croaker [3] have been reported. In the red sea bream, HM rather than a single herb supplement has been reported to be highly effective for improved growth
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and immunity [9]. Future research initiatives should focus on establishing optimum HM mixture concentrations and applications during seedling production in mariculture. Acknowledgments The authors would like to thank Prof. Hidemi Kumai, the director of the Fisheries Laboratory, Kinki University, and the staff of the Fisheries Laboratory for their kind advice and assistance. This study was partially supported by the 21st Century COE Program of the Ministry of Education, Science, Sports and Culture of Japan. This work is also funded by a grant from the National Fisheries Research and Development Institute (NFRDI, RP-2008-AQ-041), Korea.
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