Fasted and postprandial response of serum physiological response , hepatic antioxidant abilities and HSP 70 expression in Wuchang bream ( Megalobrama amblycephala ) fed different dietary carbohydrate levels

The effect of dietary carbohydrate (CHO) level on serum physiological response, hepatic antioxidant abilities and heat shock protein 70 (HSP70) expression of Wuchang bream (Megalobrama amblycephala) was studied. Two isonitrogenous (28.56% crude protein) and isolipidic (5.28% crude lipid) diets were formulated to contain 30% or 53% wheat starch. Diets were fed for 90 days to fish in triplicate tanks (28 fish per tank). At the end of feeding trial, significantly higher serum triglyceride level, insulin level, cortisol level, and malondialdehyde (MDA) content were observed in fish fed the 53% CHO diet, while significantly lower serum total protein content, alkaline phosphatase activity, superoxide dismutase activity and total antioxidative capacity were found in fish fed the 53% CHO diet compared with those fed the 30% diet. The relative level of hepatic heat shock protein 70 mRNA was significantly higher in the 53% CHO group than that in the 30% CHO at 6, 12 and 48 h after feeding. Ingestion of 53% dietary CHO impacts the nonspecific immune ability and causes metabolic stress in Megalobrama amblycephala.


Introduction
Fish are known to have a limited ability for digesting and metabolizing carbohydrates, and excessive intake of this nutrient may result in nutritional problems (Jauncey, 1982;Roberts and Bullock, 1989;Roberts, 1989;Lall, 1991).The carbohydrate utilization by fish varies among fish species and carbohydrate sources (Wilson, 1994;Krogdahl et al., 2005).In general, herbivorous fish are more capable of utilizing dietary carbohydrate than carnivorous and omnivorous fish (Hemre et al., 2002).Fish with excess glucose are assumed to be under constant metabolic stress (Pieper and Pfeffer, 1980;Fletcher, 1981), which may lead to suppressed immune functions (Erfanullah, 1995;Fu and Xie, 2005).Thus, high dietary carbohydrate intake may affect the antioxidation ability and increase the incidence of diseases in fish.
Wuchang bream (Megalobrama amblycephala), a freshwater herbivorous species, was originally found in Newshan Lake and Yuli Lake (Ke, 1975).Its main distribution is in the mid reach of the Yangtze River, China (Zhu, 1995).Due to the merits of this species including tender flesh, fast growth, feeding on natural foods, high disease resistance, economic profitability, and cultural values (Ke, 1986;Zhou et al., 2008), Wuchang bream is widely cultured in China with the output of 625,789 t in 2009 -an increase of 31.50% in the past decade.Shi et al. (1988) reported that Wuchang bream requires 27.04-30.39%protein for optimal growth when the water temperature is about 20 ºC, and the optimum protein requirement ranges from 25.58% to 41.40% when water temperature varies from 25 °C to 30 ºC.Zou et al. (1987) reported that Wuchang bream required a diet containing 21.05-30.83%,using methods of linear and polynomial regression analysis.According to the previous reports (Yang et al., 1989;Liu et al., 1992;Zhou et al., 1997;Li et al., 2010), the carbohydrate requirement was reported to be between 25% and 30%.However, little research has been done on the health implications of high dietary carbohydrate on Wuchang bream.
Therefore, the objectives of the present study were to investigate the effect of dietary carbohydrate (CHO) on serum physiological response, hepatic antioxidative enzyme activities and HSP70 expression of M. amblycephala to gain a better understanding of the health status of M. amblycephala fed high CHO diets.

Material and Methods
Wuchang bream juveniles were obtained from a fish farm at the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, in China and reared in six 300-L cylindrical fiberglass tanks.Fish were allocated to each tank and acclimated for 15 days before the feeding trial.Thereafter, the fish in the control group and the high-CHO group in triplicate (three tanks, 28 fish per tank, 34.73±0.67g initial body weight) were fed 30 and 53% CHO, respectively (Table 1).
Two isonitrogenous (28.6% crude protein) and isolipidic (5.3% crude lipid) diets were formulated to contain 30 and 53% CHO, respectively.The dietary crude protein and lipid contents have been evidenced for optimal growth of Wuchang bream (Shi et al., 1988;Yang et al., 1989;Liu et al., 1992).Dry ingredients were well ground and mixed thoroughly in a mixer, and then water was added.The 2-mmdiameter pellets were wet-extruded.The diets were dried in a forced air oven at 40 °C to a moisture content of 10% and stored at −20 °C until use.
Wuchang bream juveniles were acclimated in cylindrical fiberglass tanks for 15 days and then fed the trial diet in a quantity equivalent to about 2.0-4.0%BW three times a day at 06. 00-06.30, 11.00-11.30, and 16.00-16.30h, provided with a continuous flow of sand-filtered freshwater (2 L/min) with continuous aeration.Tanks were thoroughly cleaned biweekly.Water quality parameters were monitored daily.Feeding trials lasted 90 days.During the test period, the water quality on average was as follows: water temperature 26.0±1.5 °C, dissolved oxygen (DO) >5 mg L -1 , NH 3 <0.05mg L -1 , H 2 S<0.1 mg L -1 , and pH 7.8-8.0.The feeding amount was increased every other week.
At the end of the trial, the fish were starved for 48 h and then fed once.Nine fish from each group (three tanks, three fish per tank) were anesthetized with MS-222 and sampled at 0, 6, 12, 24, and 48 h after the meal.Gut contents were systematically checked to make sure that the fish had actually consumed the test diets.Blood was sampled from the caudal vein, centrifuged for 10 min (4 °C, 3,000 × g), and kept frozen until analysis.The liver was excised, frozen in liquid nitrogen, and stored at −80 °C until analysis.
Serum total protein and albumin contents were estimated by the Biuret and BCG dye binding method and the bromocresol green binding method (Reinhold, 1953), respectively.Serum total protein content was measured using the biuret method (kit purchased from Shanghai Fudan Zhangjiang Biopharmaceutical Co., Ltd., China) in a Beckman Cx-4 type auto biochemical analyzer (Beckman Coulter, USA).Bovine serum albumin was used as standard (BSA: 66 kDa; Nanjing Jiancheng Biological Engineering Research Institute of China).Serum globulin was determined by subtracting serum albumin from total protein.Activity of serum aspartate aminotransferase (GOT) was estimated according to the methods described by the study of Krajnović-Ozretić and Ozretić (1978).Serum GOT activity was determined using a colorimetric kit (Shanghai Fudan Zhangjiang Bio Medical Co., Ltd., China) in a Beckman Cx-4 type Auto Bio-chemical Analyzer (Beckman Coulter, USA).
Serum insulin was measured by radioimmunoassay (RIA) using a test kit (Beijing Beifang Biotech Research Institute, China) and following the method described by Clark and Hales (Clark and Hales, 1994;Li et al., 2012a).Serum cortisol was measured by RIA using a test kit (Beijing Beifang Biotech Research Institute, China) and following the method described by Pickering and Pottinger (Liu et al., 2012a).Serum alkaline phosphatase (AKP) activity was measured according to the procedure described  4 Carbohydrate contents of feed were analyzed by the 3,5-dinitro salicylic acid method (Yu et al., 1997). 5Gross energy (kJ/g) was calculated using energy equivalents 23.64 kJ/g, 39.54 kJ/g, and 17.15 kJ/g for protein, lipid and digestible carbohydrate, respectively.
by Dabrowski (1990) using kits from Junshi Biotechnology (Shanghai) Co., Ltd., China.Hepatic superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and total antioxidation capacity (T-AOC) were determined by following the methods described in the previous studies (Marklund and Marklund, 1974;Drape et al., 1993;Rice-Evans and Miller, 1994).Test kits for these assays were purchased from Nanjing Jiancheng Biological Engineering Research Institute in China.
Real-time PCR was used to determine mRNA levels with an SYBR GreenⅠfluorescence kit (Ming et al., 2010).Real-time PCR was performed in a Mini Opticon Real-Time Detector (Bio-Rad, USA).The fluorescent quantitative PCR reaction solution consisted of 12.5 μL SYBRR premix Ex Taq TM (2×), 0.5 μL PCR Forward Primer (10 μM), 0.5 μL PCR Reverse Primer (10 μM), 2.0 μL RT reaction mix (cDNA solution), and 9.5 μL dH 2 O.The reaction conditions were as follows: 95 ºC for 2 min, followed by 44 cycles consisting of 95 ºC for 10 s, 59 ºC for 20 s, and 72 ºC for 20 s.The florescent flux was then recorded and the reaction continued at 72 ºC for 3 min.Dissolution rate was measured between 65 and 92 ºC.Each increase of 0.2 ºC was maintained for 1 s and the fluorescent flux was recorded.The relative quantification of the target gene transcript (HSP70) was calculated with a chosen reference gene transcript (β-actin) using the 2 −ΔΔCT method (Livak and Schmittgen, 2001).This mathematical algorithm, which does not require a calibration curve, computes an expression ratio based on real-time PCR efficiency and the crossing point deviation of the sample versus a control.The

Results and Discussion
There was no significant change in serum total protein content in the control group (Figure 1A).Serum total protein content at 12 h after feeding was significantly lower than that at 0 h in the 53% CHO group (P<0.05)(Figure 1A).Serum total protein content was significantly lower in the 53% CHO group than that in the control group at 12 h (P<0.05)(Figure 1A).There was no significant change in serum globulin content in both groups (Figure 1B).No significant change in serum GOT activity was observed in the control group (Figure 1C).Serum GOT activity at 12 h after feeding was significantly lower than that at 0 h in the 53% CHO group, but serum GOT activity at 48 h after feeding was significantly higher than that at 0 h in the 53% CHO group (P<0.05)(Figure 1C).Serum GOT activity was significantly lower in the 53% CHO group than that in the control group at 12 h, but higher at 48 h (P<0.05)(Figure 1C).
Proteins are the most important compounds in the serum.The serum protein content is used as an immune parameter that can indicate whether or not a fish is healthy (De Smet and Blust, 2001).Excess dietary CHO can impact serum metabolites such as serum total protein and globulin (Kumar et al., 2005).In the present study, the serum total protein content was significantly lower in the 53% CHO group than that in the control group at 12 h.It indicated that the excess dietary CHO have an immuno-suppressive effect on Wuchang bream.The results were in agreement with the previous study in fish in which the serum total protein content was negatively correlated with dietary carbohydrate (Hemre et al., 1995).Serum aspartate aminotransferase, ubiquitous aminotransferase located in the mitochondria of fish, is an important index for the diagnosis of liver function and damage (Cho et al., 1994;Liu et al., 2010).In this study, the serum GOT activity was significantly higher in the 53% CHO group than that in the control group at 48 h, which indicated that, to some extent, the excess dietary carbohydrate caused damage to the liver function of M. amblycephala.Similar findings were obtained in top mouth culter (Erythroculter ilishaeformis Bleeker) (Liu et al., 2012b).However, in this study, the serum GOT activity was significantly lower in the 53% CHO group than that in the control group at 12 h.Miao et al. (2011) also found that Carassius auratus gibelio fed higher levels (50%) of CHO had lower GOT activity.
There was no significant change in serum glucose concentration in both groups (Figure 2A).Serum cholesterol content at 12 h after feeding was significantly lower than that at 0 h in the control group (P<0.05)(Figure 2B).Serum cholesterol content at 48 h after feeding was significantly lower than that at 0 h in the 53% CHO group (P<0.05)(Figure 2B).Serum triglyceride content at 6 h after feeding was significantly higher than those at 0 h in the 53% CHO group and then serum triglyceride content in the 53% CHO group returned to the level at 0 h (P<0.05)(Figure 2C).There was no significant change in serum triglyceride content in the control group (Figure 2C).Serum triglyceride content was significantly higher in the 53% CHO group than those in the control group at 6 h (P<0.05)(Figure 2C).
Serum insulin at 6 and 12 h after feeding was significantly higher than that at 0 h in the 53% CHO group and then serum insulin in the 53% CHO group at 24 and 48 h after feeding returned to the level at 0 h.Serum insulin at 6, 12, 24 and 48 h after feeding was significantly higher than that at 0 h in the control group (Figure 3A).Serum insulin was significantly higher in the 53% CHO group than that in the control group at 0 h, 24 h and 48 h (P<0.05)(Figure 3A).Serum cortisol levels at 6, 12, 24 and 48 h after feeding were significantly lower than those at 0 h in    both groups (P<0.05)(Figure 3B).Serum cortisol levels were significantly higher in the 53% CHO group than those in the control group at 0 h, 12 h, 24 h and 48 h (P<0.05)(Figure 3B).Serum alkaline phosphatase at 6 and 12 h after feeding was significantly higher than that at 0 h in the control group, but significantly lower at 24 and 48 h after the meal than those at 0 h in the control group (P<0.05)(Figure 3C).Serum alkaline phosphatase at 6, 12, 24 and 48 h after feeding was significantly lower than that at 0 h in the 53% CHO group (Figure 3C).Serum alkaline phosphatase was significantly lower in the 53% CHO group than that in the control group at 6, 12 and 24 h (P<0.05)(Figure 3C).Insulin is very effective in stimulating glucose and amino acid uptake in fish (Castillo et al., 2004).In this study, the serum insulin was significantly higher in the 53% CHO group than that in the control group at 0 h, 24 h and 48 h.However, the results obtained here were quite preliminary.Further studies concerning effects of dietary CHO in this species are needed.Stress results in adrenergic and hypothalamus-pituitary-interrenal (HPI) axis responses, and the HPI response culminates in an increase in the plasma cortisol level.Increase in the blood cortisol level is an indication that a fish is under stress (Hsieh et al., 2003).The plasma cortisol levels increased significantly as the dietary CHO:L ratios decreased from 5.64 to 1.62 in M. amblycephala (Li et al., 2012b).The dietary intake of high CHO was shown to improve the serum cortisol level (Waagbø et al., 1994), whereas a high-CHO diet did not significantly affect the serum cortisol level in rainbow trout (Oncorhynchus mykiss) (Hilton et al., 1987) or cod (Gadus morhua) (Hemre et al., 1991).In the present study, the serum cortisol levels at 6, 12, 24 and 48 h after feeding were significantly lower than those at 0 h in both groups.Serum cortisol levels were significantly higher in the 53% CHO group than those in the control group at 0 h, 12 h, 24 h and 48 h.These results indicate that a diet containing higher CHO would lead to a stress response in M. amblycephala.Alkaline phosphatase (AKP) is an important enzyme that regulates a number of essential functions in all living organisms.In Megalobrama amblycephala, supplementation with 0.1% anthraquinone extract significantly increased serum AKP activity at 24 h after infection (Liu et al., 2012a).The AKP activity increased dramatically in Labeo rohita that were fed Achyranthes aspera (Rao et al., 2006).The AKP activity in the highglucose group (40% CHO) was significantly lower than that in the low-glucose group (20% CHO) (Cai, 2004).The AKP activity decreased in allogynogenetic crucian carp (Carassius auratus gibelio) fed high-CHO diet (Miao et al., 2011).In our study, serum AKP activities at 6, 12, 24 and 48 h after feeding were significantly lower than those at 0 h in the 53% CHO group.Serum AKP activities were significantly lower in the 53% CHO group than those in the control group at 6, 12 and 24 h (P<0.05).In the present study, the results suggested that supplementation with higher dietary CHO decreased the levels of serum AKP.
Hepatic superoxide dismutase activity at 6, 12 and 24 h after feeding was significantly higher than at 0 h in both groups, but significantly lower at 48 h after the meal than at 0 h in both groups (P<0.05)(Figure 4A).Hepatic SOD activity was significantly lower in the 53% CHO group than in the control group at 0 h, 12 h, 24 h and 48 h (P<0.05)(Figure 4A).Hepatic MDA contents at 12, 24 and 48 h after feeding were significantly higher than those at 0 h in the control group, and significantly higher at 6, 12, 24 and 48 h after the meal than those at 0 h in the 53% CHO group (P<0.05)(Figure 4B).Hepatic MDA content was significantly higher in the 53% CHO group than in the control group at 6 h (P<0.05)(Figure 4B).Hepatic T-AOC at 12 h after feeding was significantly higher than at 0 h in the control group, but significantly lower at 24 and 48 h after feeding than those at 0 h in both groups (P<0.05)(Figure 4C).Hepatic T-AOC was significantly lower in the 53% CHO group than in the control group at 6 h, 12 h and 48 h (P<0.05)(Figure 4C).
The nonspecific defense mechanisms of fish include neutrophil activation, production of peroxidase and oxidative free radicals, and initiation of other inflammatory factors (Ainsworth et al., 1991).The stress response might also impact factors such as total antioxidation capacity and levels of SOD, MDA and various peroxidases (Itou et al., 1996;Liu et al., 2010).In this study, the hepatic SOD activities were significantly lower in the 53% CHO group than those in the control group at 0 h, 12 h, 24 h and 48 h.Hepatic MDA content was significantly higher in the 53% CHO group than in the control group at 6 h.Hepatic T-AOC was significantly lower in the 53% CHO group than in the control group at 6 h, 12 h and 48 h.These results indicated that the antioxidant ability of Wuchang bream fed high-CHO diets decreased.Similar results were obtained in previous studies on Wuchang bream (Megalobrama amblycephala) (Zhou et al., 2013), top-mouth culter (Erythroculter ilishaeformis Bleeker) (Liu et al., 2012b) and black carp (Mylopharyngodon piceus) (Cai et al., 2009).
The relative levels of hepatic HSP70 mRNA at 6 h and 12 h after the meal were significantly higher than those at 0 h in both groups (P<0.05)(Figure 5).However, levels in both groups at 24 and 48 h were significantly lower than those at 0 h (P<0.05)(Figure 5).Furthermore, the relative level of hepatic HSP70 mRNA was significantly higher in the 53% CHO group than in the control group at 6, 12 and 48 h after feeding (P<0.05)(Figure 5).
Heat shock proteins (HSP) are one of the most conserved and important protein families, present in all organisms including fish (Basu et al., 2002).HSP70 is mainly involved in stress protection, improving cell survival and raising tolerance to environmental stressors or harm (Basu et al., 2002).Thus, HSP70 has been most widely used as a bioindicator of stress.HSP70 is induced by heat and chemical shocks in fish, like in mammals (Gornati et al., 2004).Gornati et al. (2004) observed that HSP70 was also shown to be inducible by increasing stocking density in sea bass.Enes et al. (2006) reported that HSP70 gene expression was affected by water temperature and dietary carbohydrate.In this study, the relative level of hepatic HSP70 mRNA was significantly higher in the 53% CHO group than in the control group at 6, 12 and 48 h after feeding.The results of this study indicated that high dietary CHO may lead to metabolic stress in M. amblycephala.

Conclusions
High dietary carbohydrate intake impacts the nonspecific immunity of M. amblycephala to some extent and also causes metabolic stress.The underlying mechanisms of carbohydrate metabolism are far from being understood in M. amblycephala, and the effect of dietary carbohydrates on the health status in M. amblycephala requires further study.
PCR efficiency was measured by constructing a standard curve using a serial dilution of cDNA; ΔΔC T = (C T , Target − C T, β-actin ) time x − (C T , Target − C T, β-actin )time 0. The SPSS (version 16.0) software was used to perform Duncan's multiple range tests and Independent-Samples t-tests to determine differences among treatments.Different little letters above histogram bars denote statistically significant differences (P<0.05) in different CHO groups of each sampling point in Duncan's multiple range tests.Significant differences (P<0.05) between values obtained from normal and high CHO groups are indicated by asterisks above histogram bars in Independent-Samples t-tests.All the results were expressed as means ± standard error ( ±SE).
Data are expressed as the mean of triplication of each group ± standard error (n = 9).Significant differences (P<0.05) between values obtained in control group and high CHO group are marked by asterisk by t-tests.Different letters indicate significant differences (P<0.05) in different sampling points by Duncan's multiple range tests.

Figure 2 -
Figure 2 -Fasted and postprandial patterns of serum glucose (a), cholesterol (b) and triglyceride (c) in M. amblycephala fed the different-carbohydrate-level diets for 10 weeks.

Figure 3 -
Figure 3 -Fasted and postprandial patterns of serum insulin (a), cortisol (b) and alkaline phosphatase (c) in M. amblycephala fed the different-carbohydrate-level diets for 10 weeks.
Data are expressed as the mean of triplication of each group ± standard error (n = 9).Significant differences (P<0.05) between values obtained in control group and high CHO group are marked by asterisk by t-tests.Different letters indicate significant differences (P<0.05) in different sampling points by Duncan's multiple range tests.

Figure 4 -
Figure 4 -Fasted and postprandial patterns of heaptic superoxide dismutase (a) malondialdehyde (b), and total antioxidative capacity (c) in M. amblycephala fed the different-carbohydrate-level diets for 10 weeks.

Figure 5 -
Figure 5 -Fasted and postprandial patterns of the relative level of hepatic HSP70 mRNA in M. amblycephala fed the different-carbohydrate-level diets for 10 weeks.