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Effects of dietary ethoxyquin on growth performance and body composition of large yellow croaker Pseudosciaena crocea
Aquaculture 306 (2010) 80–84
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Effects of dietary ethoxyquin on growth performance and body composition of large yellow croaker Pseudosciaena crocea Jun Wang, Qinghui Ai ⁎, Kangsen Mai, Wei Xu, Houguo Xu, Wenbing Zhang, Xiaojie Wang, Zhiguo Liufu Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, PR China
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Article history: Received 21 March 2010 Received in revised form 15 June 2010 Accepted 17 June 2010 Keywords: Large yellow croaker Pseudosciaena crocea Ethoxyquin Growth performance Body composition Aquaculture
a b s t r a c t Ethoxyquin (6-ethoxy-2, 2, 4-trimethyl-1, 2-dihydroquinoline (EQ)) is the most common synthetic antioxidant in fish feed. However, there was little literature related to the effects of dietary EQ on growth performance of fish. The present study was conducted to investigate the effects of EQ on survival, growth, feed utilization, and body composition of large yellow croaker Pseudosciaena crocea. Five experimental diets were formulated to contain graded levels of EQ (0, 50, 150, 450 and 1350 mg kg− 1). Each diet was randomly fed to three sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (with an initial body weight of 7.82 ± 0.68 g). Survival was more than 93%, and irrespective of dietary EQ levels. Specific growth rate (SGR) decreased with the increase of dietary EQ. SGR of fish fed the diet with 1350 mg kg− 1 EQ was significantly lower than the other treatments. There were no significant differences in SGR among fish fed diets with or less than 450 mg kg− 1 EQ. No significant difference in feed intake (FI) was found among dietary treatments. Feed efficiency ratio (FER) first increased, and then decreased with increasing dietary EQ, but no significant differences were observed among dietary treatments. No significant differences were found in moisture, protein, and ash content among dietary treatments. The lipid content, however, in fish fed the diets with EQ was higher than those fed the diet without EQ. The hepatosomatic index (HSI) and condition factor (CF) generally decreased with increasing dietary EQ level. The CF in fish fed the diet containing 1350 mg kg− 1 EQ was significantly lower than the others. The viscerosomatic index (VSI) of fish firstly decreased with the increase of dietary EQ, the lowest value was recorded in fish fed diet with 150 mg kg− 1 EQ, then increased with increasing dietary EQ. Results of the present study showed that EQ level in the diet of large yellow croaker should not exceed 50 mg kg− 1. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ethoxyquin (6-ethoxy-2, 2, 4-trimethyl-1, 2-dihydroquinoline (EQ)), a synthetic antioxidant, has been used as a preservative since the 1950s (Coelho, 1995). Because of its antioxidant properties, EQ has been widely used in animal foodstuffs to prevent rancidity (Olcott, 1958; Weil et al., 1968; FAO/PL: 1969/M/17/1, WHO/FOOD./70.38; Thorisson et al., 1992; Drewhurst, 1998) and preserve a variety of auto-oxidation-prone materials which are rich in unsaturated hydrocarbons, such as lipids, carotenes, vitamin A and vitamin E. Some previous studies demonstrated that supplementation of EQ in diets can cause increases in feed intake, growth, feed efficiency and digestibility in cattle and Eurasian perch Perca fluviatilis (Krumsiek and Owens, 1998; McBribe, 2000; Kestemont et al., 2001) and decreases in mortality and morbidity in calves (Kegley et al., 1999). However, other investigations also indicated that EQ may also cause a number of undesirable side effects, including physiological stress such
⁎ Corresponding author. Tel.: +86 532 82031943. E-mail address: [email protected] (Q. Ai). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.06.024
as inhibit energy metabolism and ATP production (Reyes et al., 1995; Saxena et al., 2000; Błaszczyk and Skolimowski, 2005). Given the potential for these negative effects, the dietary concentration of EQ should be closely monitored. At present, most of the studies in EQ was performed on land animals, such as beef cattle (Kegley et al., 1999), dog (Dzanis, 1991), and chicks (Rubel and Freeman, 1989; Ohshima et al., 1996). Although EQ is the most common synthetic antioxidant in fish feed, there was little literature related to the effects of dietary EQ on fish growth performance (Saxena et al., 2000; Kestemont et al., 2001; Bohne et al., 2008). The information on optimal concentration of EQ in fish diet was very limited. Till now, the only available information was that the European Council recommended that EQ concentrations in fish feed should be below 150 mg kg− 1 when used alone or in combination with other legislated synthetic antioxidants (European Council, 1970). Therefore, it is urgent to investigate the effects of EQ supplementation on growth performance, feed utilization and healthy status of fish, which is necessary for determining the optimum EQ concentration in fish diets. Large yellow croaker Pseudosciaena crocea is a carnivorous species that is widely cultured in China. There are many studies on the
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nutrition requirement (Duan et al., 2002; Ma et al., 2005; Ai et al., 2006a,b; Mai et al., 2006a, b; Li et al., 2009) and immunity (Jian and Wu, 2003; Ai et al., 2006b, 2007) of large yellow croaker. Protein and lipid levels in diets of carnivorous fish are relatively high (Ai et al., 2006a). Therefore, the formulated diets are susceptible to oxidation during the summer season, particularly in South China, where the temperature and humidity are very high. To prevent oxidation, high quantities of EQ are used, e.g. in some fishmeal products the content of EQ was as high as 400 mg kg− 1 (Saxena et al., 2000). However, it is unclear whether such high level of EQ was necessary or harmful to the fish. The purpose of this study was to evaluate the effects of EQ on the survival and growth performance of P. crocea, which is useful for the development of commercial feeds for the culture of this fish.
At the end of the acclimation, the fish were starved for 24 h, anesthetized with eugenol (1:10,000) (Shanghai Reagent, China), and weighed. Fish with similar sizes (7.82 ± 0.68 g) were randomly distributed into 15 sea cages (n = 60 fish per cage: 1.0 × 1.0 × 1.5 m). The fifteen tanks were then divided randomly into five treatments (n = 3 tanks per treatment). The fish were hand-fed to apparent satiation twice daily (04:30 and 16:30). Fish were considered satiated when they did not exhibit a feeding behavior towards the pellets. Smaller pellets (1.5 mm) were fed to fish during weeks 1–4 and the larger pellets (2.5 mm) during weeks 5–10. The consumption of food in each cage was recorded. The feeding trial lasted for 10 weeks. During the trial period, water temperature ranged from 26.5 to 29.5 °C, the salinity was about 28‰, and dissolved oxygen content was approximately 7 mg l− 1.
2. Materials and methods
2.3. Analysis and measurement
2.1. Experimental diets
The fish were starved for 24 h at the end of the feeding trail before sampling. Total number and mean body weight of the fish in each cage were measured. Ten fish were randomly sampled from each cage for individual proximate composition analysis. Proximate composition analysis on feed ingredients, experimental diets and fish body were performed by the standard methods of AOAC (1995). Samples of the diets and fish were dried to a constant weight at 105 °C to determine moisture content. Protein was determined by measuring nitrogen (N × 6.25) using the Kjeldahl method. Lipid levels were quantified by ether extraction using Soxhlet. The level of ash was measured by combustion at 550 °C.
EQ-free white fish meal and soybean meal were used as protein sources. EQ-free menhaden fish oil was used as the lipid source. Five diets were prepared (Control, Diet 1, Diet 2, Diet 3, and Diet 4) to contain graded levels of EQ (0, 50, 150, 450, and 1350 mg kg− 1, respectively) (Table 1). The ingredients were first ground into a fine powder through a 320 μm mesh. All the ingredients were then thoroughly mixed with fish oil, and water was added to produce a stiff dough. The dough was then pelletized using an experimental feed mill (F-26 (II), South China University of Technology, China) and dried for ∼ 24 h in a ventilated oven at 38 °C. After drying, the diets were broken up and sieved into two size classes (1.5 × 3.0 mm and 2.5 × 5.0 mm) (at a ratio of 1:2).
2.4. Calculations and statistical analysis The following variables were calculated:
2.2. Experimental procedures
−1 Specific growth rate SGR: %day = 100 × ðLnWt −LnW0 Þ = t
The experiment was conducted in Xiangshan Bay, Zhejiang province, southern China. Juvenile large yellow croaker was obtained from a local commercial hatchery farm. Prior to the start of the experiment, the juvenile fish were reared in floating sea cages (3.0 × 3.0 × 3.0 m) and were fed the control diet for 2 weeks to acclimate to the experimental diet and culture conditions.
Survivalð%Þ = 100 × Nt = N0 Feed intakeðFIÞ = Df = ððW t + W0 Þ = 2 × t Þ Feed efficiency ratioðFERÞ = ðW t −W0 Þ = Df (Hardy and Barrows, 2002)
Table 1 Formulation and proximate composition of the experimental diets (% dry matter). Ingredients
EQ-free fishmeal1 Soybean meal EQ-free oil2 Beer yeast Wheat meal Lecithin Attractants Potassium chloride Mold inhibitor3 Vitamin mix4 Mineral mix4 Ethoxyquin5 Chemical analyses (%) Dry matter Crude protein Crude lipid Ash 1
Diet no. (EQ supplementation level, mg kg− 1 diet) Control (0)
Diet 1 (50)
Diet 2 (150)
Diet 3 (450)
Diet 4 (1350)
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0.005
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0.015
43.0 15.0 7.0 3.0 25.24 2.5 0.3 0.2 0.1 1.8 0.8 0.045
43.0 15.0 7.0 3.0 25.15 2.5 0.3 0.2 0.1 1.8 0.8 0.135
92.22 45.58 12.18 13.18
91.49 45.38 12.30 13.20
91.34 45.39 12.21 13.14
91.51 45.37 12.35 13.79
90.71 45.29 12.38 13.19
EQ free fishmeal, obtained from AKROS Fishing Co., Ltd (Russia). EQ free oil obtained from Shengda Fish Meal & Oil Co., Ltd, (Shandong, China). Mold inhibitor: 50% calcium propionic acid and 50% fumaric acid. 4 Vitamin premix and mineral premix as described by Ai et al. (2006a). 5 Ethoxyquin obtained from Sigma Company (USA). 2
Hepatosomatic indexðHSI; %Þ = 100 × liver wet weight = Wt Viscerosomatic indexðVSI; %Þ = 100 × viscera wet weight = Wt 3
Condition factorðCF; %Þ = 100 × Wt = L
where Wt is the final body weight (g), W0 is the initial body weight (g), and t is the experimental duration in day, respectively. N0 and Nt represent the initial and final numbers of fish in each cage, respectively. Df is the dry diet intake (g). L is the body length of fish (cm). One-way analysis of variance (ANOVA) was conducted to compare differences among dietary treatments. When overall differences were significant (P b 0.05), Duncan's multiple range test was used to compare the mean values between individual treatment groups. All tests were performed in Statistica 6.0 (Statsoft, USA). 3. Results 3.1. Survival and growth performance
3
Survival ranged from 93.9% to 99.4%, and was independent of dietary treatments (P N 0.05) (Table 2). SGR significantly decreased
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Table 2 Survival and growth performance of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1. SGR2 (%·day− 1)
Diet no. (EQ level)
Final body weight (g)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.5 ANOVA6 F value P value
32.50a 33.43a 33.16a 31.66a 28.65b 0.55
2.03a 2.08a 2.06a 2.00a 1.85b 0.02
11.510 N0.000
11.970 N0.000
Survival (%)
FI3 (% body weight day− 1)
FER4
98.9 95.0 99.4 93.9 99.4 2.0
1.45 1.38 1.36 1.30 1.28 0.05
1.23 1.29 1.30 1.32 1.27 0.05
1.086 0.414
0.360 0.832
1.637 0.240
1
Data represent the mean of triplicate groups. Values in the same row with the same letters are not significantly different (P N 0.05, Duncan's test). 2 SGR: specific growth rate. 3 FI: feed intake. 4 FER: feed efficiency ratio. 5 S.E.M.: standard error of means. 6 ANOVA: one-way analysis of variance.
with the increasing of dietary EQ. Fish fed the diet with 50 mg kg− 1 EQ showed the highest growth rate. However, SGR of fish fed the diet containing 1350 mg kg− 1 EQ was significantly lower than the other treatments (P b 0.05). There was no significant difference in SGR among fish fed diets with or lower than 450 mg kg− 1 EQ (P N 0.05). No significant difference in feed intake (FI) was found among dietary treatments, although there was a decreasing trend with the increase of dietary EQ (P N 0.05) (Table 2). Feed efficiency ratio (FER) first increased, and decreased with increasing dietary EQ, but no significant differences were observed among dietary treatments (P N 0.05). 3.2. Body composition There was no significant difference in body composition (P N 0.05), except the lipid content (P b 0.05) among dietary treatments with graded supplementation of EQ (Table 3). The lipid content of fish fed diets with 50 and 150 mg kg− 1 EQ was significantly higher than that without EQ supplementation. The lipid content peaked in fish fed the diet with 150 mg kg− 1 EQ, significantly higher than the other treatments, followed by the group with 50 mg kg− 1 EQ. 3.3. Hepatosomatic, viscerosomatic index and condition factor The HSI and CF generally decreased with the increasing of dietary EQ level, and the lowest values were recorded in fish that were fed 1350 mg kg− 1 EQ (Table 4). The HSI was significantly lower in fish fed
Table 3 Body composition of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1.
1
Diet no. (EQ level)
Moisture (%)
Crude protein (% w.w.2)
Crude lipid (% w.w.)
Crude ash (% w.w.)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.3 ANOVAd F value P value
77.5 77.2 76.6 77.0 77.3 0.8
14.9 14.6 14.7 14.7 14.8 0.2
4.6a 5.2b 5.7c 5.0ab 4.9ab 0.2
3.8 3.5 3.6 3.6 3.7 0.1
6.132 0.001
0.196 0.935
0.600 0.641
0.190 0.941
Data represent the mean of triplicate groups. w.w.: wet weight. 3 S.E.M.: standard error of means. 4 ANOVA: one-way analysis of variance. 2
Table 4 Hepatosomatic (HSI), viscerosomatic (VSI) index and condition factor (CF) of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1. Diet no. (EQ level)
HSI (%)
VSI (%)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.2 ANOVA3 F value P value
1.30ab 1.32b 1.17abc 1.08c 1.00c 0.07
8.18 7.93 7.65 7.85 7.90 0.21
CF (%) 1.67a 1.64a 1.63a 1.67a 1.59b 0.01
3.354 0.013
1.021 0.401
6.410 b 0.001
1
Data represent the mean of triplicate groups. Values in the same row with same letters are not significantly different (P N 0.05, Duncan's test). S.E.M.: standard error of means. 3 ANOVA: one-way analysis of variance. 2
the diet with 1350 mg kg− 1 EQ than in those fed the diets with 50 mg kg− 1 or less EQ (P b 0.05). Similarly, the CF of fish fed 1350 mg kg− 1 EQ was significantly lower than the other groups (P b 0.05). The VSI firstly decreased with the increase of dietary EQ, the lowest value recorded in fish fed the diet containing 150 mg kg− 1 EQ, and then increased (Table 4).
4. Discussion In the present study, the growth of large yellow croaker was slightly affected by EQ supplementation. Since the experimental fish were from the same stock and had been reared under identical conditions throughout the experiment, the differences in growth performance were most likely a result of changes in dietary EQ levels. Inconsistent findings of the effects of EQ on the growth response have been reported. Improved growth has been reported in animals by many studies (Dibner et al., 1996; Krumsiek and Owens, 1998; McBribe, 2000; Kestemont et al., 2001; Han et al., 2002). This was probably due to the protective effects of EQ on the nutritive value of the diet, especially preventing or slowing lipid or fatty acid oxidation process (Kestemont et al., 2001). However, Bohne et al. (2008) reported that the growth of Atlantic salmon Salmo salar was not significantly affected by dietary EQ supplementation that ranged from 0 to 1500 mg kg− 1. In the present study, the growth rate (SGR) of large yellow croaker was improved when dietary EQ was equal to or less than 150 mg kg− 1. However, as the dietary EQ concentration continued to increase, the growth of croaker decreased. Especially, when dietary EQ was 1350 mg kg− 1, the growth was significantly decreased. The finding of the present study was consistent with that reported by Ohshima et al. (1996), who found that weight gain of chick was highest when fed the diet containing 50 and 125 mg kg− 1 EQ, and the growth rate significantly decreased when dietary EQ was higher than 350 mg kg− 1. The difference in growth response could have resulted from the effects of EQ on feed intake. Kestemont et al. (2001) reported that the feed intake increased in the Eurasian perch that received diets supplemented with EQ (500 mg kg− 1), while the fish fed diets without EQ displayed a significantly lower feed intake, particularly at high lipid content. However, in the present study, there was a decreased trend in feed intake with increasing dietary EQ, although no significant differences were observed (Table 2). It was reasonable and easy to understand that the higher lipid content in the diet, the more EQ should be supplemented to protect the dietary lipid from oxidization. This was somewhat confirmed by the finding of Kestemont et al. (2001), who found that the growth of Eurasian perch P. fluviatilis fed diet with higher lipid level (18%) was more improved by EQ compared to that fed with lower dietary lipid (6% and 12%). The lipid levels of the diets in the present experiment were
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about 12.35%. Too high EQ concentration was unnecessary and resulted in reduced feed intake and feeding behavior (Laohabanjong et al., 2009). This was confirmed by the findings of Bohne et al. (2008), who found that the diet with high EQ concentration was rejected by Atlantic salmon. Another possible reason was the species-difference in the upper tolerance limit to EQ. For the large yellow croaker, the concentration of 50 mg kg− 1 EQ was enough to cause some negative effects on feed intake. Saxena et al. (2000) reported that the turbot Scophthalmus maximus fed the diet with 400 mg kg− 1 displayed a substantial increase in liver weight. This was also confirmed by the findings of Bailey et al. (1996), who observed that the relative liver weight of leghorn received the diet supplemented with 1000 mg kg− 1 EQ was significantly higher than those with lower EQ. This could have resulted from an increase in hepatic cell proliferation or cellular water content caused by EQ (Dibner et al., 1996). However, Ohshima et al. (1996) reported that the liver weight of chick was not significantly affected by dietary EQ supplementation. In contrast to these findings, in the present study, the hepatosomatic and viscerosomatic indices of croaker were generally lower for those fed the diet with higher concentrations of EQ than those with lower EQ levels (Table 4). According to the finding of Kestemont et al. (2001), the effects of EQ on HSI and VSI were influenced by the dietary lipid level. They found that when the lipid content in the diet was lower (6%), the HSI and VSI of Eurasian perch were decreased by the addition of EQ, while when the lipid level was higher (12% and 18%), the HSI and VSI of fish fed diets with EQ addition were increased. The different responses probably also were caused by species-specific differences in the metabolic breakdown products of EQ (Burka et al., 1996) and/or the different culture condition and feed. It was well demonstrated that the carcass composition of lipid and fatty acids could be influenced by the dietary lipid profile (Estévez et al., 1997; Kestemont et al., 2001; Lewis-McCrea and Lall, 2007; López et al., 2009). As an antioxidant, EQ can protect lipid from oxidation, therefore to some extent cause changing of dietary lipid profile (Reyes et al., 1995). In the present study, the whole body lipid content of large yellow croaker fed the diet with EQ supplementation was higher compared with the control group (Table 3). This was probably due to the facts that dietary lipid was protected by EQ addition, and higher body lipid content was accumulated. This was in agreement with the finding of Kestemont et al. (2001), who found that the total lipid concentration significantly increased with increasing dietary lipid content of fish fed diets containing EQ. While, higher EQ can completely inhibit lipid peroxidation activity (Eun et al., 1993) and retard normal lipid metabolism, resulting in decreased body lipid accumulation. This was a possible explanation for the decreasing of lipid content in fish fed diet with or more than 450 mg kg− 1. However, lipid metabolism and oxidation were complex processes and not clearly understood (Gutteridge and Halliwell, 1990). Further studies are needed to investigate the effects of EQ on lipid metabolism in this fish. According to the results, the EQ in the diets up to 1350 mg kg− 1 did not show significant benefits in terms of survival, growth and feed utilization, moreover higher EQ concentrations (1350 mg kg− 1) decreased the growth of large yellow croaker. It is recommended that the concentration of EQ in the feed for large yellow croaker should not exceed 50 mg kg− 1.
Acknowledgements The study was supported by the National Key Technologies R&D Program for the 11th Five-year Plan of China (grant no. 2006BAD03B03). We thank W.W. Dai, A. Hiskia and R.T. Zuo for their help in diet production. We are also grateful to J.K. Shentu and F.P. Tan for their assistance during the study.
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Effects of dietary ethoxyquin on growth performance and body composition of large yellow croaker Pseudosciaena crocea Jun Wang, Qinghui Ai ⁎, Kangsen Mai, Wei Xu, Houguo Xu, Wenbing Zhang, Xiaojie Wang, Zhiguo Liufu Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, PR China
a r t i c l e
i n f o
Article history: Received 21 March 2010 Received in revised form 15 June 2010 Accepted 17 June 2010 Keywords: Large yellow croaker Pseudosciaena crocea Ethoxyquin Growth performance Body composition Aquaculture
a b s t r a c t Ethoxyquin (6-ethoxy-2, 2, 4-trimethyl-1, 2-dihydroquinoline (EQ)) is the most common synthetic antioxidant in fish feed. However, there was little literature related to the effects of dietary EQ on growth performance of fish. The present study was conducted to investigate the effects of EQ on survival, growth, feed utilization, and body composition of large yellow croaker Pseudosciaena crocea. Five experimental diets were formulated to contain graded levels of EQ (0, 50, 150, 450 and 1350 mg kg− 1). Each diet was randomly fed to three sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (with an initial body weight of 7.82 ± 0.68 g). Survival was more than 93%, and irrespective of dietary EQ levels. Specific growth rate (SGR) decreased with the increase of dietary EQ. SGR of fish fed the diet with 1350 mg kg− 1 EQ was significantly lower than the other treatments. There were no significant differences in SGR among fish fed diets with or less than 450 mg kg− 1 EQ. No significant difference in feed intake (FI) was found among dietary treatments. Feed efficiency ratio (FER) first increased, and then decreased with increasing dietary EQ, but no significant differences were observed among dietary treatments. No significant differences were found in moisture, protein, and ash content among dietary treatments. The lipid content, however, in fish fed the diets with EQ was higher than those fed the diet without EQ. The hepatosomatic index (HSI) and condition factor (CF) generally decreased with increasing dietary EQ level. The CF in fish fed the diet containing 1350 mg kg− 1 EQ was significantly lower than the others. The viscerosomatic index (VSI) of fish firstly decreased with the increase of dietary EQ, the lowest value was recorded in fish fed diet with 150 mg kg− 1 EQ, then increased with increasing dietary EQ. Results of the present study showed that EQ level in the diet of large yellow croaker should not exceed 50 mg kg− 1. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ethoxyquin (6-ethoxy-2, 2, 4-trimethyl-1, 2-dihydroquinoline (EQ)), a synthetic antioxidant, has been used as a preservative since the 1950s (Coelho, 1995). Because of its antioxidant properties, EQ has been widely used in animal foodstuffs to prevent rancidity (Olcott, 1958; Weil et al., 1968; FAO/PL: 1969/M/17/1, WHO/FOOD./70.38; Thorisson et al., 1992; Drewhurst, 1998) and preserve a variety of auto-oxidation-prone materials which are rich in unsaturated hydrocarbons, such as lipids, carotenes, vitamin A and vitamin E. Some previous studies demonstrated that supplementation of EQ in diets can cause increases in feed intake, growth, feed efficiency and digestibility in cattle and Eurasian perch Perca fluviatilis (Krumsiek and Owens, 1998; McBribe, 2000; Kestemont et al., 2001) and decreases in mortality and morbidity in calves (Kegley et al., 1999). However, other investigations also indicated that EQ may also cause a number of undesirable side effects, including physiological stress such
⁎ Corresponding author. Tel.: +86 532 82031943. E-mail address: [email protected] (Q. Ai). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.06.024
as inhibit energy metabolism and ATP production (Reyes et al., 1995; Saxena et al., 2000; Błaszczyk and Skolimowski, 2005). Given the potential for these negative effects, the dietary concentration of EQ should be closely monitored. At present, most of the studies in EQ was performed on land animals, such as beef cattle (Kegley et al., 1999), dog (Dzanis, 1991), and chicks (Rubel and Freeman, 1989; Ohshima et al., 1996). Although EQ is the most common synthetic antioxidant in fish feed, there was little literature related to the effects of dietary EQ on fish growth performance (Saxena et al., 2000; Kestemont et al., 2001; Bohne et al., 2008). The information on optimal concentration of EQ in fish diet was very limited. Till now, the only available information was that the European Council recommended that EQ concentrations in fish feed should be below 150 mg kg− 1 when used alone or in combination with other legislated synthetic antioxidants (European Council, 1970). Therefore, it is urgent to investigate the effects of EQ supplementation on growth performance, feed utilization and healthy status of fish, which is necessary for determining the optimum EQ concentration in fish diets. Large yellow croaker Pseudosciaena crocea is a carnivorous species that is widely cultured in China. There are many studies on the
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nutrition requirement (Duan et al., 2002; Ma et al., 2005; Ai et al., 2006a,b; Mai et al., 2006a, b; Li et al., 2009) and immunity (Jian and Wu, 2003; Ai et al., 2006b, 2007) of large yellow croaker. Protein and lipid levels in diets of carnivorous fish are relatively high (Ai et al., 2006a). Therefore, the formulated diets are susceptible to oxidation during the summer season, particularly in South China, where the temperature and humidity are very high. To prevent oxidation, high quantities of EQ are used, e.g. in some fishmeal products the content of EQ was as high as 400 mg kg− 1 (Saxena et al., 2000). However, it is unclear whether such high level of EQ was necessary or harmful to the fish. The purpose of this study was to evaluate the effects of EQ on the survival and growth performance of P. crocea, which is useful for the development of commercial feeds for the culture of this fish.
At the end of the acclimation, the fish were starved for 24 h, anesthetized with eugenol (1:10,000) (Shanghai Reagent, China), and weighed. Fish with similar sizes (7.82 ± 0.68 g) were randomly distributed into 15 sea cages (n = 60 fish per cage: 1.0 × 1.0 × 1.5 m). The fifteen tanks were then divided randomly into five treatments (n = 3 tanks per treatment). The fish were hand-fed to apparent satiation twice daily (04:30 and 16:30). Fish were considered satiated when they did not exhibit a feeding behavior towards the pellets. Smaller pellets (1.5 mm) were fed to fish during weeks 1–4 and the larger pellets (2.5 mm) during weeks 5–10. The consumption of food in each cage was recorded. The feeding trial lasted for 10 weeks. During the trial period, water temperature ranged from 26.5 to 29.5 °C, the salinity was about 28‰, and dissolved oxygen content was approximately 7 mg l− 1.
2. Materials and methods
2.3. Analysis and measurement
2.1. Experimental diets
The fish were starved for 24 h at the end of the feeding trail before sampling. Total number and mean body weight of the fish in each cage were measured. Ten fish were randomly sampled from each cage for individual proximate composition analysis. Proximate composition analysis on feed ingredients, experimental diets and fish body were performed by the standard methods of AOAC (1995). Samples of the diets and fish were dried to a constant weight at 105 °C to determine moisture content. Protein was determined by measuring nitrogen (N × 6.25) using the Kjeldahl method. Lipid levels were quantified by ether extraction using Soxhlet. The level of ash was measured by combustion at 550 °C.
EQ-free white fish meal and soybean meal were used as protein sources. EQ-free menhaden fish oil was used as the lipid source. Five diets were prepared (Control, Diet 1, Diet 2, Diet 3, and Diet 4) to contain graded levels of EQ (0, 50, 150, 450, and 1350 mg kg− 1, respectively) (Table 1). The ingredients were first ground into a fine powder through a 320 μm mesh. All the ingredients were then thoroughly mixed with fish oil, and water was added to produce a stiff dough. The dough was then pelletized using an experimental feed mill (F-26 (II), South China University of Technology, China) and dried for ∼ 24 h in a ventilated oven at 38 °C. After drying, the diets were broken up and sieved into two size classes (1.5 × 3.0 mm and 2.5 × 5.0 mm) (at a ratio of 1:2).
2.4. Calculations and statistical analysis The following variables were calculated:
2.2. Experimental procedures
−1 Specific growth rate SGR: %day = 100 × ðLnWt −LnW0 Þ = t
The experiment was conducted in Xiangshan Bay, Zhejiang province, southern China. Juvenile large yellow croaker was obtained from a local commercial hatchery farm. Prior to the start of the experiment, the juvenile fish were reared in floating sea cages (3.0 × 3.0 × 3.0 m) and were fed the control diet for 2 weeks to acclimate to the experimental diet and culture conditions.
Survivalð%Þ = 100 × Nt = N0 Feed intakeðFIÞ = Df = ððW t + W0 Þ = 2 × t Þ Feed efficiency ratioðFERÞ = ðW t −W0 Þ = Df (Hardy and Barrows, 2002)
Table 1 Formulation and proximate composition of the experimental diets (% dry matter). Ingredients
EQ-free fishmeal1 Soybean meal EQ-free oil2 Beer yeast Wheat meal Lecithin Attractants Potassium chloride Mold inhibitor3 Vitamin mix4 Mineral mix4 Ethoxyquin5 Chemical analyses (%) Dry matter Crude protein Crude lipid Ash 1
Diet no. (EQ supplementation level, mg kg− 1 diet) Control (0)
Diet 1 (50)
Diet 2 (150)
Diet 3 (450)
Diet 4 (1350)
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0.005
43.0 15.0 7.0 3.0 25.28 2.5 0.3 0.2 0.1 1.8 0.8 0.015
43.0 15.0 7.0 3.0 25.24 2.5 0.3 0.2 0.1 1.8 0.8 0.045
43.0 15.0 7.0 3.0 25.15 2.5 0.3 0.2 0.1 1.8 0.8 0.135
92.22 45.58 12.18 13.18
91.49 45.38 12.30 13.20
91.34 45.39 12.21 13.14
91.51 45.37 12.35 13.79
90.71 45.29 12.38 13.19
EQ free fishmeal, obtained from AKROS Fishing Co., Ltd (Russia). EQ free oil obtained from Shengda Fish Meal & Oil Co., Ltd, (Shandong, China). Mold inhibitor: 50% calcium propionic acid and 50% fumaric acid. 4 Vitamin premix and mineral premix as described by Ai et al. (2006a). 5 Ethoxyquin obtained from Sigma Company (USA). 2
Hepatosomatic indexðHSI; %Þ = 100 × liver wet weight = Wt Viscerosomatic indexðVSI; %Þ = 100 × viscera wet weight = Wt 3
Condition factorðCF; %Þ = 100 × Wt = L
where Wt is the final body weight (g), W0 is the initial body weight (g), and t is the experimental duration in day, respectively. N0 and Nt represent the initial and final numbers of fish in each cage, respectively. Df is the dry diet intake (g). L is the body length of fish (cm). One-way analysis of variance (ANOVA) was conducted to compare differences among dietary treatments. When overall differences were significant (P b 0.05), Duncan's multiple range test was used to compare the mean values between individual treatment groups. All tests were performed in Statistica 6.0 (Statsoft, USA). 3. Results 3.1. Survival and growth performance
3
Survival ranged from 93.9% to 99.4%, and was independent of dietary treatments (P N 0.05) (Table 2). SGR significantly decreased
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Table 2 Survival and growth performance of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1. SGR2 (%·day− 1)
Diet no. (EQ level)
Final body weight (g)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.5 ANOVA6 F value P value
32.50a 33.43a 33.16a 31.66a 28.65b 0.55
2.03a 2.08a 2.06a 2.00a 1.85b 0.02
11.510 N0.000
11.970 N0.000
Survival (%)
FI3 (% body weight day− 1)
FER4
98.9 95.0 99.4 93.9 99.4 2.0
1.45 1.38 1.36 1.30 1.28 0.05
1.23 1.29 1.30 1.32 1.27 0.05
1.086 0.414
0.360 0.832
1.637 0.240
1
Data represent the mean of triplicate groups. Values in the same row with the same letters are not significantly different (P N 0.05, Duncan's test). 2 SGR: specific growth rate. 3 FI: feed intake. 4 FER: feed efficiency ratio. 5 S.E.M.: standard error of means. 6 ANOVA: one-way analysis of variance.
with the increasing of dietary EQ. Fish fed the diet with 50 mg kg− 1 EQ showed the highest growth rate. However, SGR of fish fed the diet containing 1350 mg kg− 1 EQ was significantly lower than the other treatments (P b 0.05). There was no significant difference in SGR among fish fed diets with or lower than 450 mg kg− 1 EQ (P N 0.05). No significant difference in feed intake (FI) was found among dietary treatments, although there was a decreasing trend with the increase of dietary EQ (P N 0.05) (Table 2). Feed efficiency ratio (FER) first increased, and decreased with increasing dietary EQ, but no significant differences were observed among dietary treatments (P N 0.05). 3.2. Body composition There was no significant difference in body composition (P N 0.05), except the lipid content (P b 0.05) among dietary treatments with graded supplementation of EQ (Table 3). The lipid content of fish fed diets with 50 and 150 mg kg− 1 EQ was significantly higher than that without EQ supplementation. The lipid content peaked in fish fed the diet with 150 mg kg− 1 EQ, significantly higher than the other treatments, followed by the group with 50 mg kg− 1 EQ. 3.3. Hepatosomatic, viscerosomatic index and condition factor The HSI and CF generally decreased with the increasing of dietary EQ level, and the lowest values were recorded in fish that were fed 1350 mg kg− 1 EQ (Table 4). The HSI was significantly lower in fish fed
Table 3 Body composition of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1.
1
Diet no. (EQ level)
Moisture (%)
Crude protein (% w.w.2)
Crude lipid (% w.w.)
Crude ash (% w.w.)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.3 ANOVAd F value P value
77.5 77.2 76.6 77.0 77.3 0.8
14.9 14.6 14.7 14.7 14.8 0.2
4.6a 5.2b 5.7c 5.0ab 4.9ab 0.2
3.8 3.5 3.6 3.6 3.7 0.1
6.132 0.001
0.196 0.935
0.600 0.641
0.190 0.941
Data represent the mean of triplicate groups. w.w.: wet weight. 3 S.E.M.: standard error of means. 4 ANOVA: one-way analysis of variance. 2
Table 4 Hepatosomatic (HSI), viscerosomatic (VSI) index and condition factor (CF) of large yellow croaker fed diets containing different concentrations of EQ for 10 weeks1. Diet no. (EQ level)
HSI (%)
VSI (%)
Control (0) Diet 1 (50) Diet 2 (150) Diet 3 (450) Diet 4 (1350) Pooled S.E.M.2 ANOVA3 F value P value
1.30ab 1.32b 1.17abc 1.08c 1.00c 0.07
8.18 7.93 7.65 7.85 7.90 0.21
CF (%) 1.67a 1.64a 1.63a 1.67a 1.59b 0.01
3.354 0.013
1.021 0.401
6.410 b 0.001
1
Data represent the mean of triplicate groups. Values in the same row with same letters are not significantly different (P N 0.05, Duncan's test). S.E.M.: standard error of means. 3 ANOVA: one-way analysis of variance. 2
the diet with 1350 mg kg− 1 EQ than in those fed the diets with 50 mg kg− 1 or less EQ (P b 0.05). Similarly, the CF of fish fed 1350 mg kg− 1 EQ was significantly lower than the other groups (P b 0.05). The VSI firstly decreased with the increase of dietary EQ, the lowest value recorded in fish fed the diet containing 150 mg kg− 1 EQ, and then increased (Table 4).
4. Discussion In the present study, the growth of large yellow croaker was slightly affected by EQ supplementation. Since the experimental fish were from the same stock and had been reared under identical conditions throughout the experiment, the differences in growth performance were most likely a result of changes in dietary EQ levels. Inconsistent findings of the effects of EQ on the growth response have been reported. Improved growth has been reported in animals by many studies (Dibner et al., 1996; Krumsiek and Owens, 1998; McBribe, 2000; Kestemont et al., 2001; Han et al., 2002). This was probably due to the protective effects of EQ on the nutritive value of the diet, especially preventing or slowing lipid or fatty acid oxidation process (Kestemont et al., 2001). However, Bohne et al. (2008) reported that the growth of Atlantic salmon Salmo salar was not significantly affected by dietary EQ supplementation that ranged from 0 to 1500 mg kg− 1. In the present study, the growth rate (SGR) of large yellow croaker was improved when dietary EQ was equal to or less than 150 mg kg− 1. However, as the dietary EQ concentration continued to increase, the growth of croaker decreased. Especially, when dietary EQ was 1350 mg kg− 1, the growth was significantly decreased. The finding of the present study was consistent with that reported by Ohshima et al. (1996), who found that weight gain of chick was highest when fed the diet containing 50 and 125 mg kg− 1 EQ, and the growth rate significantly decreased when dietary EQ was higher than 350 mg kg− 1. The difference in growth response could have resulted from the effects of EQ on feed intake. Kestemont et al. (2001) reported that the feed intake increased in the Eurasian perch that received diets supplemented with EQ (500 mg kg− 1), while the fish fed diets without EQ displayed a significantly lower feed intake, particularly at high lipid content. However, in the present study, there was a decreased trend in feed intake with increasing dietary EQ, although no significant differences were observed (Table 2). It was reasonable and easy to understand that the higher lipid content in the diet, the more EQ should be supplemented to protect the dietary lipid from oxidization. This was somewhat confirmed by the finding of Kestemont et al. (2001), who found that the growth of Eurasian perch P. fluviatilis fed diet with higher lipid level (18%) was more improved by EQ compared to that fed with lower dietary lipid (6% and 12%). The lipid levels of the diets in the present experiment were
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about 12.35%. Too high EQ concentration was unnecessary and resulted in reduced feed intake and feeding behavior (Laohabanjong et al., 2009). This was confirmed by the findings of Bohne et al. (2008), who found that the diet with high EQ concentration was rejected by Atlantic salmon. Another possible reason was the species-difference in the upper tolerance limit to EQ. For the large yellow croaker, the concentration of 50 mg kg− 1 EQ was enough to cause some negative effects on feed intake. Saxena et al. (2000) reported that the turbot Scophthalmus maximus fed the diet with 400 mg kg− 1 displayed a substantial increase in liver weight. This was also confirmed by the findings of Bailey et al. (1996), who observed that the relative liver weight of leghorn received the diet supplemented with 1000 mg kg− 1 EQ was significantly higher than those with lower EQ. This could have resulted from an increase in hepatic cell proliferation or cellular water content caused by EQ (Dibner et al., 1996). However, Ohshima et al. (1996) reported that the liver weight of chick was not significantly affected by dietary EQ supplementation. In contrast to these findings, in the present study, the hepatosomatic and viscerosomatic indices of croaker were generally lower for those fed the diet with higher concentrations of EQ than those with lower EQ levels (Table 4). According to the finding of Kestemont et al. (2001), the effects of EQ on HSI and VSI were influenced by the dietary lipid level. They found that when the lipid content in the diet was lower (6%), the HSI and VSI of Eurasian perch were decreased by the addition of EQ, while when the lipid level was higher (12% and 18%), the HSI and VSI of fish fed diets with EQ addition were increased. The different responses probably also were caused by species-specific differences in the metabolic breakdown products of EQ (Burka et al., 1996) and/or the different culture condition and feed. It was well demonstrated that the carcass composition of lipid and fatty acids could be influenced by the dietary lipid profile (Estévez et al., 1997; Kestemont et al., 2001; Lewis-McCrea and Lall, 2007; López et al., 2009). As an antioxidant, EQ can protect lipid from oxidation, therefore to some extent cause changing of dietary lipid profile (Reyes et al., 1995). In the present study, the whole body lipid content of large yellow croaker fed the diet with EQ supplementation was higher compared with the control group (Table 3). This was probably due to the facts that dietary lipid was protected by EQ addition, and higher body lipid content was accumulated. This was in agreement with the finding of Kestemont et al. (2001), who found that the total lipid concentration significantly increased with increasing dietary lipid content of fish fed diets containing EQ. While, higher EQ can completely inhibit lipid peroxidation activity (Eun et al., 1993) and retard normal lipid metabolism, resulting in decreased body lipid accumulation. This was a possible explanation for the decreasing of lipid content in fish fed diet with or more than 450 mg kg− 1. However, lipid metabolism and oxidation were complex processes and not clearly understood (Gutteridge and Halliwell, 1990). Further studies are needed to investigate the effects of EQ on lipid metabolism in this fish. According to the results, the EQ in the diets up to 1350 mg kg− 1 did not show significant benefits in terms of survival, growth and feed utilization, moreover higher EQ concentrations (1350 mg kg− 1) decreased the growth of large yellow croaker. It is recommended that the concentration of EQ in the feed for large yellow croaker should not exceed 50 mg kg− 1.
Acknowledgements The study was supported by the National Key Technologies R&D Program for the 11th Five-year Plan of China (grant no. 2006BAD03B03). We thank W.W. Dai, A. Hiskia and R.T. Zuo for their help in diet production. We are also grateful to J.K. Shentu and F.P. Tan for their assistance during the study.
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