In uence of dietary composition on growth and energy reserves in tench (Tinca tinca)

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1 J. Appl. Ichthyol. 17 (2001), 25±29 # 2001 Blackwell Wissenschafts-Verlag, Berlin ISSN 0175±8659 Received: December 18, 1998 Accepted: May 23, 2000 In uence of dietary composition on growth and energy reserves in tench (Tinca tinca) By N. De Pedro, A. I. Guijarro, M. J. Delgado, M. A. Lo pez-patinä o, M. L. Pinillos and M. Alonso-Bedate Departamento BiologõÂa Animal II, Facultad de BiologõÂa, Universidad Complutense, Madrid, Spain Summary The e ects of di erent protein, lipid and carbohydrate diets on growth and energy storage in tench, Tinca tinca L., were studied. Over a 2-month period sh were fed four di erent diets: control, protein-enriched, carbohydrate-enriched and lipidenriched. The best growth rates were obtained with the control and protein-enriched diets; the carbohydrate diet produced the worst results (lowest speci c growth rate, weight gain, nutritional index and hepatosomatic index). These results suggest that it is not advisable to reduce dietary sh protein below 35%, and that it is not possible to obtain a protein-sparing e ect of either lipids or carbohydrates, at least in our experimental conditions. The high-protein diet resulted in the storage of energy excess as muscle proteins and hepatic glycogen. Tench fed the high-carbohydrate diet stored carbohydrates as muscle glycogen and reduced plasma triglycerides. Finally, both liver and muscle lipid content were in positive correlation to dietary lipid. Introduction Tench (Tinca tinca L.) is a cyprinid species of particular interest in European pond sh farming (Pe rez-regadera 1997). However, manufactured feed development is restricted by the lack of knowledge on the feeding and nutrition requirements of this species; thus, a main objective in this study was to optimise the feed composition and feeding strategies of this species. An important prerequisite for optimum nutrition and growth is an appropriate supply of dietary macronutrients. This optimal nutrient composition varies among sh species. Generally, sh appear to require 30±50% dietary protein to achieve maximum growth rates (Wilson and Halver 1986; Cowey 1992). Lipids also play an important role as dietary energy sources for both carnivorous and herbivorous sh (Takeuchi et al. 1978). In relation to carbohydrates (CH), herbivorous and omnivorous sh accept more than 25% CH in their diet, while carnivorous sh show an optimum below 20% (Wilson 1994). Feed represents one of the major costs in sh farming, proteins being the most expensive component. A cost reduction can be obtained by replacing part of the protein energy with energy from lipids or carbohydrates. The protein-sparing e ect of lipids has been established in sh (Takeuchi et al. 1978; De Silva et al. 1991; Bjornson et al. 1992; Garcõ a-gallego et al. 1993; Grisdale-Helland and Helland 1997). CH are a cheaper source of energy than proteins and lipids, but carnivorous sh have limitations in their absorption and metabolism of CH; an excessive intake can result in pathological conditions (Lall 1988; Roberts and Bullock 1989). Nevertheless, recent studies have indicated that high levels of CH are well tolerated by Salmo salar L. (Hemre et al. 1994), and a protein-sparing e ect of CH in Oreochromis niloticus L. (Anderson et al. 1984) and Anguilla anguilla L. (Hidalgo et al. 1993; Sanz et al. 1993) has been shown. It is generally accepted that the main energy reserves in sh are stored in the liver and muscle (Lie et al. 1988; Berge and Storebakken 1991; Hemre et al. 1992). CH are stored as glycogen in these tissues in all vertebrates so far studied. Moreover, sh store their surplus energy as lipids and proteins in muscle and the liver. However, the in uence of diet composition on such energy storage in the tench is unknown. Taking this into account, the objective of the present study was to evaluate the e ects in the tench of diets with di erent protein, lipid and carbohydrate contents on their growth rate and energy storage. Materials and Methods Fish and diets Experiments were carried out on tench, T. tinca (L.) ( g body weight) provided by the Ipescon sh farm (Salamanca, Spain). The tench were kept at the laboratory in glass aquaria (50±100 L), with walls painted black to reduce the stress during the acclimatization period. The sh were maintained in owing and aerated tap water under 12 L:12 D photoperiod and C water temperature. All sh were fed Sera Biogran (Heinsberg, Germany) pellets at a daily ratio of 1% body weight (bw). Experimental procedure Following a 30-day period of adaptation to laboratory conditions, the tench were divided into four groups (n ˆ 7±9/group) and fed with the respective experimental diet for 2 months, at a daily ratio of 2% bw. Four experimental diets were formulated (Table 1) varying in protein, lipid and carbohydrate content: control diet (C), and diets enriched with proteins (P), carbohydrates (CH) or lipids (L), respectively. All experiments were carried out in duplicate. In order to obtain individual data, each tench was marked with a visible implant ( uorescent elastomer tags, Northwest Marine Technology, ICN, Washington, USA) using a special biocompatible elastomer material containing uorescent colouring which provided internal marks that were externally visible. The best place to locate and identify the tags was tested in a previous experiment whereby the tench were marked with yellow and red elastomers on di erent body locations: top of the head (n ˆ 60), the base of both dorsal and pectoral ns, and between the caudal n rays (n ˆ 60). Retention of tags was monitored weekly for 6 weeks. Fish were individually weighed once each week throughout the 2-month experimental period. Daily rations were adjusted U.S. Copyright Clearance Center Code Statement: 0175±8659/2001/1701±0025$15.00/0

2 26 N. De Pedro et al. Table 1 General composition of the experimental diets (g/100 g dry diet) Diets Dietary composition C P CH L Fish meal Casein Fish oil 1.95 ± Sun ower oil Dextrine Betaine Vitamin mixture Mineral mixture Cellulose Carboxymetilcellulose Total protein (%){ Total fat (%){ Total carbohydrate (%){ Energy (MJ/100 g)* C: control diet; P: protein-enriched diet; CH: carbohydrate-enriched diet; L: lipid-enriched diet. *1 g protein: 19.6 KJ; 1 g fat: 39.5 KJ; 1 g carbohydrate: 17.2 KJ. {% according to formula and raw material composition. weekly on the basis of bw. At the end of the 2-month growth trial the sh were decapitated after 24 h food deprivation. Blood samples were collected using heparinized capillary tubes and centrifuged (6000 r.p.m. for 3 min) to obtain plasma samples. The livers and muscle were immediately removed and frozen on solid CO 2. All plasma and tissue samples were stored at 80 C until biochemical analyses were performed. Calculations The weight increase (body weight gain) and speci c growth rate (SGR) were determined. The SGR was calculated as a percentage of bw per day according to the formula: SGR ˆ [(ln Wf ln Wi)/t] 100, where Wf is the weight of the sh at the end of experiment, Wi is the weight at the start of the experiment, and t is the duration of the growth period in days. Two biometric indices were also calculated: the nutritional index, NI ˆ nal body weight 100/[length (cm)] 3, and the hepatosomatic index, HSI ˆ liver weight 100/body weight. Biochemical analysis Plasma glucose levels were determined by the glucose-oxidase method using a commercial kit (Peridochrom Glucose, Boehringer Mannheim, Barcelona, Spain). Plasma levels of triglycerides (TG) were quanti ed by the GOP-PAP method (enzymatic hydrolysis of TG with subsequent determination of liberated glycerol by colourimetry) using a commercial kit (Peridochrom Triglycerides, Boehringer Mannheim). Hepatic and muscle glycogen were measured by spectrophotometry (Dubois et al. 1956) after extraction with ethanol and previous digestion with KOH (Cifonelli et al. 1956; Montgomery 1957). Protein content in liver and muscle homogenates was determined by applying the method of Lowry et al. (1951) using bovine serum albumin as a standard. Total lipids from liver and muscle samples were extracted with chloroform/methanol (Folch et al. 1957) and determined by turbidimetry. Statistics A one-way ANOVA was performed to ascertain statistical di erences between duplicate tanks exposed to the same treatment (diet). When no statistical di erences were found, we considered both tanks per diet as two homogeneous populations, and the individual data were used to calculate the mean and SEM of the di erent groups. A Student's t-test was performed to examine statistical di erences between the control sh and the sh fed the protein-, carbohydrate- and lipid-enriched diets. A probability level of P <0.05 was considered statistically signi cant. Results Retainment of n elastomer tags was 95% after 1 week and 83.3% after 6 weeks (Fig. 1). At the end of the experiment there was an 88.9% loss of tags placed in the head region, indicating that the elastomer n tag locations are more reliable and probably better detectable, and therefore a useful marking system for T. tinca, at least for 6 weeks. Fish that lost their markings were retagged each week. Weight gain, SGR and both NI and HSI obtained at the end of the 2-month experiment are shown in Table 2. Fish fed the carbohydrate-enriched diet showed signi cantly reduced weight gains, SGR, NI and HSI. No signi cant di erences in these indices were observed when dietary proteins were increased. Weight gain and SGR were reduced (P <0.05), compared to the control diet, when dietary lipids were increased. Plasma glucose levels did not vary among tench fed di erent diet compositions (Fig. 2a). However, the carbohydrateenriched diet signi cantly (P <0.001) decreased plasma triglycerides levels. There were no signi cant di erences in these values in the other diets (Fig. 2b). The hepatic contents of glycogen, proteins and lipids after 2 months of feeding the di erent diets are shown in Fig. 3. Fish fed the high-protein diet contained more hepatic glycogen than did the other groups (Fig. 3a). The di erent diets caused no signi cant changes in liver proteins (Fig. 3b). An increase in liver lipid content was observed in the tench fed the lipid-enriched diet (Fig. 3c). Figure 4 summarizes the results for muscle content of glycogen, proteins and lipids. Glycogen increased in the sh fed the protein-, lipid- and carbohydrate-enriched diets compared to the control diet; this increase was statistically signi cant for the

3 Growth and energy reserves in tench 27 Fig. 1. Retention rate of elastomer marks in ns (n ˆ 60) or head (n ˆ 60) of tench through 6 weeks. Data are expressed as mean +SEM. ***P <0.001 compared to n marks. CH and L diets. The muscle protein content signi cantly (P <0.05) increased in both the protein- and lipid-enriched diets. Finally, an increase in muscle lipids was found in the tench fed the lipid-enriched diet. Discussion In our study, the best growth rate was obtained in tench fed the control and protein-enriched diets; the carbohydrate diet produced the lowest SGR, body weight gain, NI and HSI. The negative results obtained with the CH-enriched diet might indicate that the tench did not tolerate large amounts of CH. In fact, the natural diet for tench contains little CH and it could be expected that this species would not be able to digest or metabolize large amounts thereof. This hypothesis is in accordance with results obtained for other carnivorous teleosts, which tolerate approximately 10% dietary starch, but they have signi cant problems in e ciently metabolizing higher starch amounts (Hemre et al. 1992, 1993). Similarly, when the dietary CH content exceeded 22%, feed utilization was reduced in S. salar (Hemre et al. 1995). The present tench growth results suggest that, in this species, reduction of dietary sh protein Fig. 2. Plasma levels of (a) glucose and (b) triglycerides in tench fed diets of di erent composition: C, control diet (n ˆ 10); P, proteinenriched diet (n ˆ 10); CH, carbohydrate-enriched diet (n ˆ 8) and L, lipid-enriched diet (n ˆ 7). Data refer to samples taken at the end of the experiment and are expressed as mean +SEM. ***P <0.001 compared to control diet. below 35% is not recommendable. On the other hand, an increase in dietary protein from 35 to 50% did not result in further improvements in tench growth. These results are in agreement with previous data for halibut (Hippoglossus hippoglossus L.), by Aksnes et al. (1996). Table 2 In uence of diet composition on weight gain, SGR and biometrical indices in Tinca tinca (L.) Diets C High P High CH High L Final body weight gain (%) { { Standard growth rate (% day 1 ) { * Initial length (cm) Final length (cm) Nutritional index (g cm 3 ) { Hepatosomatic index (%) { Data are presented as mean +SEM. *P <0.05; {P <0.01; {P <0.005 compared to control group. C, control (n = 15); P, protein (n = 18); CH, carbohydrate (n = 19); L, lipid (n = 7).

4 28 N. De Pedro et al. Fig. 3. Hepatic content of (a) glycogen (b) proteins and (c) lipids in tench fed diets of di erent composition: C, control diet (n ˆ 8); P, protein-enriched diet (n ˆ 8); CH, carbohydrate-enriched diet (n ˆ 8) and L, lipid-enriched diet (n ˆ 7). Data refer to samples taken at the end of the experiment and are expressed as mean +SEM. **P <0.01, ***P <0.001 compared to control diet. Fig. 4. Muscle content of (a) glycogen (b) proteins and (c) lipids in tench fed diets of di erent composition: C, control diet (n ˆ 8); P, protein-enriched diet (n ˆ 8); CH, carbohydrate-enriched diet (n ˆ 8) and L, lipid-enriched diet (n ˆ 7). Data refer to samples taken at the end of the experiment and are expressed as mean +SEM. *P <0.05 compared to control diet. The storage of energy as muscle protein in tench fed an enriched protein diet is in accord with the maintenance of highprotein deposition, one of the roles of the muscle. Thus, it was shown that protein-synthesis rates in the muscle of cod (Gadus morhua L.) fed with di erent protein level diets increased when the metabolizable energy in the form of protein was increased from 10 to 48% (Lied and Braaten 1984). Perago n et al. (1994) reported that an enhancement of dietary protein caused a signi cant increase in both protein-accumulation and proteinsynthesis rates in the muscle of the rainbow trout. Despite a poor utilization of dietary CH, sh require glucose as the main fuel for certain tissues, such as brain and active gonads. Gluconeogenesis from amino acids is an important metabolic pathway in salmonids (Walton and Cowey 1982), representing a certain waste of dietary protein. This could explain the increase in hepatic glycogen observed in tench fed with a high-protein diet in the present study, probably due to the activation of gluconeogenesis from amino acids, as suggested by Hemre et al. (1993) in G. morhua. The tench fed with the high-carbohydrate diet stored CH as muscle glycogen, without correlation between dietary CH and hepatic glycogen. Similarly, no correlation between dietary starch and liver glycogen was found in Atlantic salmon, S. salar (Hemre et al. 1996). In this species the liver was not the main target organ for plasma glucose, and muscle could reserve more glucose than the liver, as reported by Garcõ a-riera and

5 Growth and energy reserves in tench Hemre (1996). On the other hand, it was reported extensively that feeding (Walton and Cowey 1982) or dietary CH (Hemre et al. 1996) increased plasma glucose levels. However, it was found that the plasma glucose response after feeding or a glucose load showed an initial increase but returned to basal levels within 24 h (Garcõ a-riera and Hemre 1996). Thus, the lack of glucemia variation in tench fed a high CH diet could be explained by the previous 24 h fasting carried out to obtain plasma samples. Earlier studies have shown that glucose stimulates triglyceride hydrolysis in sh, probably mediated by glucagon (Harmon and Sheridan 1992). A similar mechanism could be functioning in tench since there was a signi cant drop of plasma triglyceride levels when they were fed a high CH diet. Accordingly, recent studies on glucose tolerance in turbot, Scophthalmus maximus L., demonstrated that a glucose load decreased plasma TG levels (Garcõ a-riera and Hemre 1996). The increase in lipid storage found in the liver and muscle of the tench fed with high dietary lipid is in accord with previous reports (Garcõ a-gallego et al. 1993; Aksnes et al. 1996; Koskela et al. 1998). The interdependence between dietary lipids levels and storage of surplus energy as lipid depots has been corroborated in di erent species and tissues. Given the present results, two principal conclusions can be drawn. On the one hand, because of the negative results in the tench, at least under our experimental conditions, reduction of dietary sh protein below 35% is not recommended. On the other hand, neither lipid nor CH had a pronounced proteinsparing e ect. Finally, dietary composition plays an important role in the regulation of protein, carbohydrate and lipid reserves in tench, as is the case with other sh. Acknowledgements We are grateful to Dr M. De la Higuera for the supply of diets. This work was supported by CAM project 7B/13/97. References Aksnes, A.; Hjertnes, T.; Opstvedt. J., 1996: E ect of dietary protein level on growth and carcass composition in Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture 145, 225±233. Anderson, J.; Jackson, J. A.; Matty, A. J.; Capper, B. S., 1984: E ects of dietary carbohydrate and ber on the Tilapia (Oreochromis niloticus). Aquaculture 37, 303±314. Berge, G. M.; Storebakken, T., 1991: E ect of dietary fat level on weight gain, digestibility, and llet composition of Atlantic halibut. Aquaculture 99, 331±338. Bjornson, B.; Sigurthorsson, G.; Hemre, G.-I.; Lie, O., 1992: Growth rate and feed conversion factor of young halibut (Hippoglossus hippoglossus L.) fed six di erent diets. Fisk. Dir. 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