Chinese Journal of Oceanology and Limnology   2017, Vol. 35 issue(2): 251-257     PDF       
http://dx.doi.org/10.1007/s00343-016-5332-6
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

CHENG Zhenyan(程镇燕), LI Jinghui(李静辉), ZHANG Baolong(张宝龙), FANG Zhenzhen(方珍珍), SUN Jinhui(孙金辉), BAI Dongqing(白东清), SUN Jinsheng(孙金生), QIAO Xiuting(乔秀亭)
Verification of protein sparing by feeding carbohydrate to common carp Cyprinus carpio
Chinese Journal of Oceanology and Limnology, 35(2): 251-257
http://dx.doi.org/10.1007/s00343-016-5332-6

Article History

Received Nov. 27, 2015
accepted in principle Feb. 1, 2016
accepted for publication Feb. 29, 2016
Verification of protein sparing by feeding carbohydrate to common carp Cyprinus carpio
CHENG Zhenyan(程镇燕)1,2, LI Jinghui(李静辉)2, ZHANG Baolong(张宝龙)2, FANG Zhenzhen(方珍珍)2, SUN Jinhui(孙金辉)2, BAI Dongqing(白东清)2, SUN Jinsheng(孙金生)1, QIAO Xiuting(乔秀亭)2        
1 Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University, Tianjin 300387, China;
2 Tianjin Key Lab of Aqua-Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural University, Tianjin 300384, China
ABSTRACT: A 9-week feeding trial in floating freshwater cages (1.0 m×1.0 m×2.0 m) was conducted to study the effects of different dietary levels of protein and starch on growth, body composition, and gene expression of enzymes in common carp, Cyprinus carpio (mean body weight, 36.12±1.18 g) to evaluate the protein-sparing effect of dietary carbohydrate. Four diets were formulated with corn starch as the carbohydrate source to obtain corn starch levels of 6.5%, 13%, 19.5%, or 26% and protein levels of 30.5%, 28.2%, 26.4%, and 24.2%. The results showed no differences in growth performance of fish fed the diets with different protein and corn starch levels, but body composition and glucose metabolic enzyme activity of carp were significantly affected by the different diets (P < 0.05). Weight gain, specific growth rate, and the feed conversion ratio were not different in fish fed the different dietary treatments. Protein efficiency ratio increased significantly as corn starch level increased (P < 0.05). Whole-body crude lipid composition increased with increasing dietary corn starch level (P < 0.05). Glucokinase (GK), hexokinase, and pyruvate kinase (PK) activities increased significantly with increasing dietary corn starch level (P < 0.05), whereas glucose-6-phosphate (G6Pase) activity decreased with increasing dietary corn starch level (P < 0.05). GK gene expression was significantly higher in fish fed the high-corn starch diet than those fed the low-corn starch diet (P < 0.05). G6pase gene expression tended to decrease with increasing starch level (P>0.05). In summary, the results indicate a protein-sparing effect by substituting carbohydrate in the diet of common carp.
Key words: common carp     protein sparing     corn starch     growth performance     glucose metabolism    
1 INTRODUCTION

Protein is an essential but the most expensive dietary component for normal animal growth and development. However, excess protein is consumed to provide energy. Fishmeal shortages have started to occur in recent years with further development of aquaculture and deterioration of the environment. Thus, efficient transformation of protein into tissue protein for growth has become increasingly more important (Shiau, 1997; Orire and Sadiku, 2011). Dietary carbohydrate and fat act as a stimulus for protein accretion in growing animals, which is a protein-sparing effect.

Carbohydrates are the most economical source of dietary energy, compared with protein and lipid, and much effort has been invested in studying the ability of fish to use energy from starch (Shiau and Lin, 2001; Hemre et al., 2002), which also helps reduce the accumulation of total nitrogen and total phosphorus in culture water. However, the ability of fish to use carbohydrate is limited and varies among species (NRC, 2011). Utilization of dietary carbohydrate by fish appears to be related to how their digestive and metabolic systems adapt to different aquatic environments and to dietary carbohydrate level and complexity. In general, utilization of complex macromolecular-structured carbohydrates is better than that of smaller structured carbohydrates. Starch is composed of glucose and is a potential energy source (Stone et al., 2003; Krogdahl et al., 2005). Furthermore, more corn starch is produced than any other type of starch. Therefore, starch is an extensive resource that is unmatched by other carbohydrates.

Carp remain the most abundantly cultured fish in China; however, profits for farmers are low because of increasing material costs. Extensive research has been performed on the protein-sparing effects of carbohydrate and lipid in the carp diet, most of which has focused on the effects of carbohydrate level on growth performance and digestive enzyme activities, but investigations into the protein-sparing effect of carbohydrate in practical diets for carp are limited. Moreover, data on the effects of dietary carbohydrate level on carbohydrate metabolic gene expression are rare, particularly in fish fed different levels of protein. The purpose of this study was to evaluate the effects of different combinations of protein and corn starch on growth performance, whole-body and muscle composition, glucose metabolism, enzyme activities, and gene expression to provide useful information for aquaculture and future research.

2 MATERIAL AND METHOD 2.1 Experimental animals

A total of 720 carp (Cyprinus carpio) were obtained from Tianjin Huanxin Aquatic Breeding Farm. The fish were acclimated for 1 week during which they were fed a basal diet twice daily to satiation. At the start of the experiment, the fish were fasted for 24 h and then weighed. Sixty fish (mean weight, 36.12±1.18 g) were distributed randomly into each of 12 floating freshwater cages (1.0 m×1.0 m×2 m). Each diet was assigned randomly to three cages. Fish were fed by hand ad libitum (until uneaten feed was visible) twice daily (09:00 and 15:30) for 9 weeks, and daily feed intake was recorded. Water temperature was 22-29°C during the experiment.

2.2 Experimental diets

Soybean meal, peanut meal, cottonseed meal, and rapeseed meal were used as vegetable protein sources, and fish meal was the animal protein source. Raw corn starch was used as the carbohydrate source. The diets contained four protein levels of 30.5%, 28.2%, 26.4%, and 24.2%, in combination with starch levels of 6.5%, 13%, 19.5%, and 26%. Four experimental diets were marked diet 1 (30.5P/6.5C), diet 2 (28.2P/13C), diet 3 (26.4P/19.5C), and diet 4 (24.2P/26C). The ingredients and proximate composition varied (Table 1).

Table 1 Formulations and proximate compositions of the common carp diets
2.3 Sampling

The fish were fasted for 48 h at the end of the feeding experiment. All fish from each cage were collected and weighed to determine their final biomass. Seven fish per cage were stored frozen at -20°C to determine their proximate composition. Three fish per cage were anesthetized with MS-222, and blood was removed from the caudal vein into ammonium-heparinized syringes and centrifuged; the plasma was recovered, immediately frozen, and stored at -20°C until analyses. The hepatopancreas, intestine, kidney, and spleen were removed and weighed, frozen in liquid nitrogen, and stored at -80°C until assay. A fasting-refeeding trial was carried out to measure gene expression. The remaining fish were refed after the 48-h fast. Then, the hepatopancreas, intestine, and kidney of three fish per cage were collected 0, 3, 6, 12, 24, and 48 h after refeeding and stored at -80°C for later analyses.

2.4 Analysis of diets and fish body composition

The chemical composition of the diets and proximate fish composition were analyzed using the following procedures. Dry matter was determined after drying the material to constant weight at 105°C; ash was determined by incineration in a muffle furnace at 550°C until constant weight was achieved; protein content (N×6.25) was determined by the Kjeldahl method, lipids were evaluated by ether extraction using the Soxhlet method, and starch was determined according to Thivend et al. (1972).

2.5 Glucose metabolic enzyme activity

Glucose-6-phosphate (G6Pase) and glucokinase (GK) activities were determined using commercial kits (Jiang Lai Biotechnology Shanghai Co. Ltd., Shanghai, China).

2.6 Glucose metabolic gene expression

Tissue levels of the G6Pase and GK genes were determined by semi-quantitative reverse transcriptionpolymerase chain reaction analysis. The primers (Table 2) were designed with Primer 5.0. Relative target gene transcript levels were quantified using β-actin gene expression as a reference.

Table 2 Sequences of the primer pairs
2.7 Calculations

Weight gain percentage (WG, %)=(Wt-W0)/W0×100; Specific growth rate (SGR, %/d)=100×(ln (Wt)-ln (W0))/t;

Feed conversion ratio (FCR, %)=100×Id/(Wt-W0); Protein efficiency ratio (PER, %)=(Wt-W0)/(Id×Pd)×100;

Survival=Nt/N0×100;

Hepatosomatic index (HSI, %)=hepatopancreas weight/Wt×100;

Condition factor (CF, %)=Wt(g)/B3(cm)×100, where Wt is final mean weight, W0 is initial mean weight, Nt and N0 are final and initial fish number, respectively, t is number of experimental days; B is fork length (cm), Id is food intake, and Pd is protein intake.

2.8 Statistical analysis

All data are expressed as means±standard error and were subjected to analysis of variance using SPSS 16.0 for Windows software (SPSS Inc., Chicago, IL, USA). Differences between means were detected by Duncan’s multiple range test, and a P-value < 0.05 was considered significant.

3 RESULT 3.1 Effects of different protein and corn starch levels on growth performance of carp

Growth and feed utilization by carp are shown in Table 3. No differences in WG, SGR, or FCR were observed between treatments. Survival rate was more than 95% during all the groups. PER increased significantly when dietary corn starch level was increased and reached a peak in fish fed diet 4 (24.2P/26C) (P < 0.05).

Table 3 Growth and feed utilization of carp fed different protein and starch levels
3.2 Effects of different protein and corn starch levels on body composition of carp

Whole-body carp composition is shown in Table 4. No differences in whole-body moisture or crude protein contents were detected, but crude lipid increased significantly with increasing dietary corn starch level (P < 0.05). Ash content increased significantly with increasing dietary corn starch level (P < 0.05). The HSI of fish fed the diet supplemented with high-corn starch increased significantly compared with that of fish fed the diet with low-corn starch and reached a peak in fish fed diet 4 (24.2P/26C). No difference in CF was observed among the groups.

Table 4 Whole-body composition, HSI, and CF ratio of carp fed different protein and corn starch levels
3.3 Effects of different protein and corn starch levels on metabolic enzyme activities of carp

Higher carbohydrate levels enhanced glycogen and glucose concentrations. Hexokinase (HK), pyruvate kinase (PK), and GK activities increased significantly with increasing dietary corn starch level, but G6Pase activity decreased significantly in the blood and hepatopancreas (P < 0.05) (Table 5).

Table 5 Hepatopancreas, blood glucose, and metabolic enzyme activities of carp fed different protein and corn starch levels (mean±standard error, n=3)
3.4 Effects of different protein and corn starch levels on gene expression of carp

GK and G6Pase expression was high in the hepatopancreas, intestine, and kidney (Figs. 1 and 2). Dietary corn starch level significantly affected GK and G6Pase gene expression in fish sampled at different refeeding times after the 48-h fast (Table 6). G6Pase gene expression of fish fed diet 4 (high-corn starch group) tended to be lower than that of fish fed diet 1 (low-corn starch group) (P>0.05). G6Pase gene expression in the hepatopancreas peaked at 3 h, and no difference was detected between 3 and 6 h, but the levels were significantly higher (P < 0.05) than those in the low dietary corn starch group. No differences in kidney G6Pase gene expression were observed among the different sampling times, regardless of dietary starch level. Intestinal G6pase gene expression peaked at 6 h in the low and high dietary corn starch groups. GK gene expression levels in the hepatopancreas were significantly higher in the highcorn starch group than those in the lower corn starch group at 3, 12, and 24 h (P < 0.05). GK gene expression increased sharply after 12 h in the low-corn starch group 3-6 h after refeeding and then decreased sharply. GK gene expression levels at 6 and 12 h were significantly higher than those at other times (P < 0.05). GK gene expression in the high-corn starch group increased sharply for 24 h at 3-6 h after refeeding intake but then decreased sharply. GK gene expression levels at 6, 12, and 24 h were significantly higher than those at other times (P < 0.05). GK gene expression levels in the kidney and intestine of the high-corn starch group tended to be higher than those in the low-corn starch group (P>0.05). GK gene expression reached the maximum at 12 h, regardless of dietary corn starch level. GK gene expression was significantly higher in the kidney from 6 to 24 h after refeeding than that during fasting in the high-corn starch group.

Figure 1 Reverse transcription-polymerase chain reaction glucokinase (GK) and glucose-6-phosphatase (G6Pase) amplification products in various carp tissues M: DL 2000 marker; lanes 1-6 are hepatopancreas, intestine, kidney, spleen, brain, and heart.
Figure 2 Reverse transcription-polymerase chain reaction glucokinase (GK) and glucose-6-phosphatase (G6Pase) amplification products in various carp tissues
Table 6 Effects of fasting and refeeding on G6Pase and GK gene expression in carp fed different diets
4 DISCUSSION

Carbohydrate is the cheapest source of energy in food. Carbohydrate can be used by fish but it can inhibit growth and enhance mortality if fed in excess (Miao et al., 2013). In this experiment, WG, SGR, and FCR were not affected by the different diets, but fish fed diet 2 (28.2P/13C) obtained maximum growth. These results are in accordance with previous findings in common carp (Keshavanath et al., 2002). The PER was higher in fish given the high-corn starch feeds, suggesting a protein-sparing effect. Results from a previous study on the efficiency of dietary carbohydrate for protein sparing varied, even within a single species (Peres and Oliva-Teles, 2002). In our previous study, incorporating dextrin and reducing dietary protein improved growth and feed efficiency of carp (Guo et al., 2013), indicating protein sparing by carbohydrate at optimal levels in common carp.

In the present study, the whole-body composition results demonstrated that crude lipid contents increased as dietary corn starch level was increased. Similar results were found in Carassius auratus (Cai et al., 2006) and Labeo rohita fry (Mohapatra et al., 2003), indicating that excess dietary carbohydrate is deposited as lipid in carp. Incorporating carbohydrate may involve reducing the rate of gluconeogenesis from amino acids. In the present study, whole-body protein content was not affected by decreasing dietary protein or increasing dietary corn starch, as reported previously for carp (Keshavanath et al., 2002), suggesting a protein-sparing effect to some extent. The liver is an important metabolic organ in fish. Dietary carbohydrate can be converted to fat, which accumulates in the hepatopancreas and mesentery (Tian et al., 2002). Some studies have shown that the visceral organs are affected after fish consume a highcarbohydrate diet (Panserat et al., 2000a).

In the present study, liver-glycogen content and the HSI increased significantly as dietary corn starch level was increased. Microscopic observations of the hepatopancreas for this study, which will be published in another article, showed that hepatic steatosis was aggravated as dietary corn starch level increased, suggesting that lipid deposition and glycogen synthesis increased in the hepatopancreas as dietary corn starch level was increased. The increase in liverglycogen content reflects the relative inability of carp to metabolize glucose through the glycolytic pathway.

Early studies showed that fish lack GK or its activity is low (Tranulis et al., 1991), which was considered one of the reasons for the limited utilization of carbohydrate in carp. In our study, HK and GK activities increased significantly along with serum glucose level as dietary corn starch level was increased. This finding indicates that higher carbohydrate levels could enhance carbohydraterelated metabolism in carp. Similar results were reported in perch (Borrebaek and Christophersen, 2000), salmon, rainbow trout, and carp (Panserat et al., 2000a; Capilla et al., 2003). GK activity was in accordance with its gene expression. GK gene expression in the hepatopancreas of the high-corn starch diet group was significantly higher than that in the low-corn starch diet group and reached the maximum 6-24 h after refeeding, regardless of dietary starch level, suggesting that dietary carbohydrate was fully digested and absorbed during this period. Similar results have been reported previously in carp (Panserat et al., 2000b, 2001) and rainbow trout (Polakof et al., 2012). Thus, this study demonstrated that GK expression could be induced in carp fed a high-corn starch diet. PK activity increased with increasing dietary corn starch level, which was in accordance with Enes et al. (2006) and Cowey et al. (1977). G6Pase activity decreased with increasing corn starch level, which was in accordance with Panserat et al. (2000b). In summary, glycolytic activity was enhanced when a suitable range of carbohydrate was consumed. G6Pase gene expression tended to be lower in fish fed the high-corn starch diet than that in fish fed the low-corn starch diet. Similar results were reported in gilthead seabream (Caseras et al., 2002; Metón et al., 2004) and carp (Panserat et al., 2002) fed different dietary carbohydrate levels, indicating that supplementing dietary carbohydrate does not significantly inhibit gluconeogenesis. Although glycolytic enzyme activities were enhanced to some extent, the carp were unable to metabolize excessive glucose, which would be stored in serum, the hepatopancreas, or converted to body lipid, and cause metabolic problems.

5 CONCLUSION

The present results indicate that carp were able to use a higher carbohydrate level, but it had a negative effect on growth and lowered the capacity to metabolize glucose, which may have decreased WG and resulted in the accumulation of glucose in the blood or hepatopancreas. The higher carbohydrate level enhanced the activities and expression of some glucose metabolic enzymes, such as HK, GK, and PK. The results of our study indicate that protein sparing with carbohydrate is feasible in common carp. Thus, replacing dietary protein with carbohydrate could reduce feed costs for common carp.

6 ACKNOWLEDGEMENT

The authors would like to thank Tianjin Tianxiang Aquatic Co. Ltd. for their assistance preparing the farming equipment and supplements.

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