Effects of Dietary Protein and Temperature on Growth and Flesh Quality of Songpu Mirror Carp

Wang Chang-an, Xu Qi-you, Zhao Zhi-gang Li Jin-nan Wang Lian-sheng and Luo Liang

1Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China

2College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China

Effects of Dietary Protein and Temperature on Growth and Flesh Quality of Songpu Mirror Carp

Wang Chang-an1,2, Xu Qi-you1*, Zhao Zhi-gang1, Li Jin-nan1, Wang Lian-sheng1, and Luo Liang1

1Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China

2College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China

The study was conducted to demonstrate the effects of dietary protein and water temperature on growth and flesh quality of Songpu mirror carp (Cyprinus carpio) at an initial weight (165.24±5.08) g. Practical diets were formulated to contain five protein levels (29.12%, 31.46%, 34.49%, 38.17%, and 40.13%), and each diet was randomly assigned triplicate groups of 15 fishes at three temperatures (18℃, 22℃, and 26℃) in the recirculation system. Fishes were fed twice daily to apparent satiation for 56 days. Results indicated that fishes had higher weight gain rate at 22℃ and 26℃ than that at 18℃ (P＜0.05), but there were no significant differences between 22℃ and 26℃ (P＞0.05). Based on the weight gain rate, dietary protein level 29.12% could meet the requirement of the body at 18℃, 22℃, or 26℃. Crude protein, crude lipid, moisture and ash of muscle had no significant differences among those treatments (P＞0.05). pH (after 24 h) of muscle was the highest at 18℃ and the lowest at 22℃ (P＜0.05), but no differences were observed among different protein level groups at each temperature (P＞0.05). No significant differences on shear force, water holding capacity, collagen, glycogen and lactate among all the treatments were found (P＞0.05). It was concluded that when C. carpio fed to apparent satiation, the growth mainly depended upon temperature. Dietary protein could not significantly affect flesh quality, but temperature significantly affected pH of muscles. A dietary protein level 29.12% could meet the requirement of the body at 18℃, 22℃, or 26℃.

Songpu mirror carp, flesh quality, growth, protein, temperature

Introduction

Water temperature and dietary protein are two of the most important factors for fish growth. Nonoptimal water temperature, insufficient food and low dietary protein have been found to inhibit fish growth (Danzmann et al., 1990, Hertz et al., 1992). Protein accounts for the major ingredient cost in aquaculture feed (Cuzon et al., 1994). As a result, it is essential to explore optimal protein at different temperatures to develop a cost-effective strategy.

The dietary protein requirement is dependent on a variety of factors like fish size, water temperature, feeding frequency, non-protein dietary energy and dietary protein quality (NRC, 1983). It is reported that the protein requirement of fish varies with temperatures. The protein requirement of Morne saxatilis fingerling is 55% when water temperature is 24.5℃, and protein requirement is 47% at 20.5℃ (Millikin, 1982). While other reports showed that the protein requirements of fish remain constant even if thetemperature fluctuates. Diet with 36% dietary protein is observed to be the best diet for Cirrhinus mrigala fry at 28-32.1℃ (Singh et al., 2008).

Diet characteristics are known to modify the textural properties of fish muscle. Some authors have reported increases in lipid concentration in whole body and muscle when fed different protein concentrations (isocaloric diets) (Kaushik, 2004). The temperature may significantly influence the muscle growth patterns of fish by a modulation of the rates of hypertrophy and hyperplasia of muscle fibers (Johnston et al., 2003). However, flesh quality of sea bass does not vary with temperatures (Octavio et al., 2008). Therefore, it is necessary to investigate the effects of the water temperature and protein on flesh quality of fish as fish nutritional value is the essential characteristic for consumers.

C. carpio is a commercially important farmed species in the north of China, which has favorable growing characteristics, resistance to diseases and high commercial value. It has been exported to many regions, such as Korea, Japan and Vietnam. However, nutritional requirements of C. carpio have been not totally understood. Limited studies related to the nutritional requirements of C. carpio are available (Zhang et al., 2011, Wang et al., 2011). Recently, C. carpio has been commercially cultured at optimum temperatures from 18℃ to 26℃ in the north of China for 150-230 days, and no knowledge has been known about the optimal protein requirement at different temperatures. Therefore, the present experiment was conducted to identify the suitable dietary protein level at different temperatures to lower the feed cost and throw light on effects of temperatures and dietary proteins on the growth and flesh quality of C. carpio.

Materials and Methods

Experimental animals

C. carpio (165.24±5.08 g) was obtained from the Aquatic Farm of Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences. Every three aquaria was connected to a recirculation system (total volume 1 000 L) and supplied with aerated water and filtered through zeolite, corallite and activated carbon. Water quality parameters were monitored daily between 8:00 a.m. and 4:00 p.m. During the experiment period, pH from 7.8 to 8.0, am monia nitrogen was lower than 0.02 mg ? L-1and dissolved oxygen (JPB-607determinator, Shanghai, China, precision±0.03 mg ? L-1) was higher than 6.0 mg ? L-1. Fish were fed twice daily (8:00 a.m. and 4:00 p.m.) to apparent satiation for 56 days. The water exchanged approximately 30% of the capacity each day.

Diets preparation

Diet formulation contained wheat protein hydrolysates, soybean meal, wheat meal, white fish meal, rapeseed meal, cottonseed meal, corn gluten meal, soy oil, fish oil, calcium phosphate, α-cellulose, choline, ethoxyquin, magnesium sulfate, antioxidant, zeolite, mineral and vitamin premix (Table 1). Dry ingredients were sieved through a 60 mesh screen and homogenized by blending thoroughly in a feed mixer. The required amount of fish oil and soy oil were added to the ingredients and the appropriate amount of water prepared dough that allowed pelleting (diameter 2.0 mm). After pelleting, the pellet air-dried in a workshop until 8%-10% moisture reached were stored in airtight plastic bottles, and then refrigerated at –20℃ until fed to fish.

Experimental design

Using two-factor design, there were 15 treatments with five protein levels (29.12%, 31.46%, 34.49%, 38.17%, and 40.13%) and three temperatures (18℃, 22℃, and 26℃). Fishes were randomly stocked in each aquarium with triplicates per treatment and fishes were acclimated to experimental conditions for two weeks before the experiment. At the end of the experiment, fishes were measured for the analyses of growth performance. Weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), and feed conversation ratio (FCR) were calculated as follows:

WGR (%)=100×(final weight–initial weight)/initial weight

SGR (% day-1)=100×(ln average final weight– ln average initial weight)/days

CF=100×(body weight/fork length3)

FCR=feed intake/body weight gain

Biochemical analysis

After fed for 56 days, five fishes fasted for 24 h from each replicate were collected, and the muscle was removed for analyses of proximate composition. Proximate compositions of muscle and experimental diets were determined as described in AOAC (1995). Moisture was desiccated in an oven at 105℃ for 24 h, ash by incinerated at 550℃ for 12 h, crude protein by Kjeldhal's method (crude protein=N×6.25), and crude lipid by Soxhlet method. Shear force was measured by using a Tenderness Analyzer (C-LM 3B). The fillet was placed under the probe and the probe moved downwards at a constant speed of 2.0 mm-1. When the probe first came in contact with the fillet, the force of the fillet was automatically recorded by the analyzer. The probe continued downwards and cut off the fillet. During the test, the force of the fillet to compress was recorded every 0.01 s. Modified original method was used to analyze the water holding capacity (WHC) and pH of muscle (Suárez et al., 2009). Muscle samples were placed in a tube inside of which another tubewas fitted in such a way that the muscle sample was never in contact with the water released, and then were slightly centrifuged (630×g, 30 min, 10℃). The amount of water lost from the muscle portion after centrifugation was expressed as a percentage of the initial fresh weight. WHC was calculated as the difference between the initial percentage of water in the muscle (determined by dehydration at 105℃ until constant weight) and the percentage of water released by centrifugation. pH of the flesh was determined by using a METTLER TOLEDO penetration electrode (model FE, 0.01 pH unit accuracy) after carrying out a lateral incision at the dorsal muscle in order to place the tip of the electrode deep in the muscle mass (approx. 5 mm depth). Collagen was determined according to the method of Palka (1999), based on the measurement of the hydroxyproline (OH-Pro) content in a muscle sample. This procedure is based on the oxidation of OH-Pro by chloramine-T, followed by the addition of 4-dimethyl-aminobenzaldehyde, which generates a colored complex quantifiable spectrophotometrically at 560 nm. A factor of 11.42 was used to transform the amount of OH-Pro to collagen. Glycogen in muscle samples was measured by the amyloglucosidase method (Keppler and Decker 1974). Muscle samples were homogenized (0.1 g ? mL-1) in cold 0.6 N perchloric acid by using a mechanical homogenizer (FJ-200CL, China). Homogenates were centrifuged (3 000×g); 0.2 mL of the supernates were incubated with 1 mL of a solution of amyloglucosidase (15 U ? mL-1) at 60℃ for 2 h, then pH was neutralized with 1 mol ? L-1Na2CO3. Lactate in homogenates was determined spectrophotometrically (365 nm) by means of a commercial kit (Lactate, from Nanjing jiancheng Co., Ltd., China), which is based on the reactions catalyzed by lactate oxidase and peroxidase.

Data analysis and statistics

Experimental data was subjected to statistical analyses using the specific software which was SPSS 19.0 for Windows. Means of each variable were analyzed using two-way ANOVA to find effects of temperature, dietary protein and interaction of temperature and dietary protein. Sample means of each variable were compared using the Duncan's means procedure. A significance level of 95% was considered to indicate statistical differences (P＜0.05).

Results

Effects on growth performance

Effects of dietary protein and water temperature on growth performances of C. carpio are presented in Table 2. Weight gain rate (50.11%) was the highest in 31.46% dietary protein at 26℃ and the lowest in diet 2 (31.46% protein level) at 18℃. Based on the weight gain rate and specific growth rate, dietary protein level 29.12% could meet the requirement of the body at 18℃, 22℃, or 26℃. Feed conversion ratio had no significant differences among all the treatments (P＞0.05). At 18℃, daily feed intake was the highest (4.04 g) in 38.17% dietary protein level compared to other dietary protein levels. At 18℃, the highest (2.98 g) daily feed intake was observed in 29.12% dietary protein level. At 26℃, daily feed intake was the highest (2.85 g) in 31.46% dietary protein level compared to other dietary protein levels. Significant difference (P＜0.05) in feed intake was observed at all temperatures. Condition factor was the highest (2.25) in 31.46% dietary protein level at 18℃ and the lowest (1.48) in 38.17% dietary protein level at 22℃ compared to other groups. Condition factor was significantly higher at 18℃ than that of 22℃ or 26℃ (P＜0.05).

Two-way ANOVA analysis of growth performance (Table 3) indicated significant effects of temperature on weight gain rate, specific growth rate, feed intake, and condition factor (P＜0.05). Fishes had the higher weight gain rate at 22℃ and 26℃ than 18℃ (P＜0.05), but there were no significant differences between 22℃ and 26℃ (P＞0.05). Fishes had the higher feed intake and condition factor at 18℃ than 22℃ or 26℃(P＜0.05), but there were no significant differences between 22℃ and 26℃ (P＞0.05).

Table 2 Growth performance of C. carpio with different dietary proteins at different temperatures

Table 3 Analysis of growth performance of C. carpio with different dietary proteins at different temperatures

Effects on proximate composition

The whole proximate composition of C. carpio muscle is presented in Table 4. Two-way ANOVA analyses (Table 5) indicated that temperature and dietary protein level had no significant effects (P＞0.05) on muscle protein, lipid, moisture and ash. Interaction between diet and temperature could not modify the influence on muscle composition, as confirmed by one-way variance analysis (P＞0.05).

The highest protein concentration (19.10%) was obtained at 26℃ in diet 1 (29.12% dietary protein level) and the lowest protein concentration (17.87%) was obtained at 22℃ in diet 4 (38.17% dietary protein level). The highest lipid concentration (2.25%) was obtained at 26℃ in diet 3 (34.49% dietary proteinlevel) and at 22℃ in diet 3 (34.49% dietary protein level). The lowest lipid concentration (2.05%) was obtained at 18℃ in diet 3 (39.49% dietary protein level). The highest protein concentration (19.10%) was obtained at 26℃ in diet 1 (29.12% dietary protein level) and the lowest protein concentration (17.87%) was obtained at 22℃ in diet 4 (38.17% dietary protein level). The highest ash concentration (1.93%) was obtained at 22℃ in diet 3 (39.49% dietary protein level) and the lowest ash concentration (1.47%) was obtained at 18℃ in diet 5 (40.13% dietary protein level).

Table 4 Proximate composition of muscle of C. carpio with different dietary proteins at different temperatures

Table 5 Analysis of proximate composition of muscle of C. carpio with different dietary proteins at different temperatures

Effects on flesh quality

Shear force, water holding capacity, collagen, glycogen, lactate and pH of muscle for C. carpio are presented in Table 6. No significant differences were observed on shear force, water holding capacity, collagen, glycogen and lactate among all the treatments (P＞0.05). pH (after 24 h) of muscle (Table 7) was the highest at 18℃and the lowest at 22℃ (P＜0.05), but there were no significant differences among dietary protein groups at each temperature. There were no effects of interaction between temperature and protein on pH (after 24 h) of muscle (P＞0.05). There were no significant differences in pH of muscle among all the groups (P＞0.05).

Table 6 Flesh quality of C. carpio with different dietary proteins at different temperatures

Table 7 Analysis of flesh quality of C. carpio with different dietary proteins at different temperatures

Discussion

It has been extensively demonstrated that temperature had an intense effect on fish development and growth (Blaxter, 1988). This study also showed that water temperature significantly improved growth performances of C. carpio. Similar results were obtained from other species (Hidalgo et al., 1987, 1988; Helena et al., 1999). Fishes are ectothermic animals and their growth and metabolic rates are affected by water temperature. The growth improvement of C. carpio could be explained by metabolic rate increasement. Higher feed intake of C. carpio resulted in poor growth performance at 18℃. This indicated that growth improvement of C. carpio was not only affected by an increase of food intake. Although no significant differences were observed in feed conversion ratio at each temperature, and was higher at lower temperature. Feed efficiency of C. carpio might be lower at lower temperature and this needed more researches to confirm. In the present study, irrespective of water temperature, lower protein level could meet protein requirement of C. carpio. Some reports indicated that when fishdiet contained lower levels of protein, the amino acids were utilized effectively to meet the protein requirements (Oliva-Teles, 2012); once requirements were met, additional protein allowed protein to be excrete nitrogenous wastes in the form of ammonia. Thus, lower dietary protein (29.12%) may be sufficient for growth and physiological function. Similarly, Hidalgo and Alliot (1988) reported that at 15℃ and 20℃, optimal protein concentrations were similar and lower dietary protein could be met for growth.

Some reports indicated that proximate composition could be slightly modified by rearing conditions and lifestyles (Haard et al., 1992). It was reported that temperature affects (P＜0.05) body protein, lipid, moisture and ash (Singh et al., 2008). No changes in proximate composition among all the treatments in this study were found as C. carpio of bigger size might be not sensitive to temperature ranged from 18℃ to 26℃. Thus, the longer growth period needs to be set in the future researches. On the other hand, fish are poikilotherms and live in an environment where temperatures can fluctuate a great deal and they must be capable of physiological responses that will enable them to maintain the homeostatic conditions necessary for life. Regardless of temperature, proximate composition varies with the change of dietary ingredients. An increase of moisture and protein contents of sea bass as the dietary protein level increased (Ballestrazzi et al., 1994). Body protein contents increased with increasing dietary protein levels in fishes such as grouper and flounder (Shiau et al., 1996; Kim et al., 2002). However, this study indicated that proximate composition was found to be almost constant, as C. carpio was able to regulate their feed intake to meet their protein and metabolic energy requirements (Boujard, 2001) and made the proximate composition of muscle remain stable.

Temperature did not affect shear force, juiciness, fattiness, chewiness, flavor and odor of the flesh of sea bass (Octavio et al., 2008). Similarly, flesh quality was not affected by temperature except pH of muscle (after 24 h) which changed with temperature in this study. This effect has also been reported in other species, such as Atlantic salmon (Hultman and Rustad, 2002), and likely the origin is the production of acidic substances from the hydrolysis of glycogen in muscle tissue. It would therefore be interesting to study the effect of rearing temperature on pH of C. carpio and investigate how this correlates with texture. For the pH of muscle (after 24 h) in this study, muscle storage property of C. carpio cultured at lower temperature might be better than that at higher temperature.

Fish texture characteristics of muscles mainly depend on lipid and collagen (Thakur et al., 2003; Regost et al., 2001), higher levels of dietary lipid affected textural characteristics of the fish fillet. Interestingly, some reports showed dietary protein could affect the flesh quality of fish. It was found that Dentex (Dentex dentex) fed a lower protein diet (38%) displayed increased firmness as well as increased water holding capacity when compared to fish fed on higher protein diets (Suárez et al., 2009). In this study, flesh quality of C. carpio had no significant effect on shear force, water holding capacity, collagen, glycogen, pH, and lactate of muscle by dietary protein levels. When dietary protein requirement met C. carpio, flesh quality might be not affected by excessive dietary protein.

Conclusions

The results of the present study indicated that growth of C. carpio mainly depended upon temperature when fishes fed to apparent satiation. Dietary protein could not significantly affect flesh quality, but temperature significantly affected pH of muscle (after 24 h). Dietary protein level 29.12% could meet the requirement of body at 18℃, 22℃, or 26℃.

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S965.116; S963

A

1006-8104(2014)-02-0053-09

Received 22 November 2013

Supported by the Special Fund for Agro-scientific Research in Public Interest (CARS-46); the National Key Technology Research and Development Program in 12th Five-year Plan of China (2012BAD25B10)

Wang Chang-an (1981-), male, researcher associate, Ph. D, engaged in the research of aquatic animal nutrition. E-mail: gordoncase@126.com

* Corresponding author. Xu Qi-you, professor, Ph. D, engaged in the research of animal nutrition. E-mail: xuqiyou@sina.com