Firat University, Faculty of Fisheries, Elazig
A growth trial was conducted for 90 days to evaluate the effects of dietary protein and lipid levels on growth performance, feed utilization, and whole body chemical composition of juve-nile grass carp. Six practical diets containing two dietary digestible protein levels (P1: 33% and P2: 37%) and three lipid levels (L1: 4%, L2: 6% and L3: 8%) were prepared. Fish were fed to satiation three times a day. There were significant differences between the weight gain (387-594%), specific growth rate (1.7-2.1%), feed intake (46.1-51.4 g fish-1), feed conversion ratio (FCR, 1.2-1.7), protein efficiency ratio (PER, 1.6-2.4), hepatosomatic index (3-3.2%), digesti-ble protein (87.4-93.5%), lipid (91.1-97.6%) and energy (87.6-93.5%) values and the whole body fat content (16.6-18.6%) for fish fed on the experimental diets. Growth-performance, FCR, PER, feed intake, digestible protein, lipid and energy values were found higher for P1L2 diet group compared to all other experimental diets (P
Diet, Digestibility, Grass carp, Growth, Lipid, Protein
It was well known that fish utilize protein preferentially to lipid or carbohydrate as an en-ergy source. Therefore, from the nutritional, envi-ronmental and economical points of view, it is important to improve protein utilization for tissue synthesis rather than for energy purpose. Within certain limits, increasing dietary lipid levels im-prove diet utilization (Sargent et al., 1999).
Dietary lipid was also reported to bring pro-tein sparing effect, replacing protein, which may otherwise be used to provide energy (NRC, 1992; Vargara et al., 1996), to reduce organic matter and nitrogen losses (Du et al., 2005). But, some authors have observed no protein sparing effect of lipid in some fish species (Danielssen and Hjertnes, 1993). In salmon aquaculture today, high fat diet has normally been used. However, excessive dietary lipid negatively affected growth performance and reduced feed consumptions and utilization of other nutrients, which led to fat deposition on viscera or the carcass (Shiau and Huang, 1990). Excessive lipid in the diet not only suppresses fatty acid synthesis, but also reduces the ability of fish to digest and assimilate lipids (Sargent et al., 1999). Therefore, a proper ratio of digestible protein to digestible lipid is necessary to achieve high growth rate and feed conversion efficiency (Takeuchi et al., 1978).
Grass carp (Ctenopharyngodon idella) is a typical herbivorous finfish without stomach. In its natural environment, it consumes water plants. It has a long history in aquaculture and is one of the most important species cultured in inland water bodies in China. Although studies were in-itiated as far back as the late 1970s on the nutri-tion of grass carp (Dabrowski, 1977; Law, 1986; Cai et al., 2005), there is no adequate knowledge on the suitable protein and lipid levels in juvenile diet of grass carp. Only protein (Dabrowski, 1977) and lipid (Du et al., 2005) and energy (Carter and Brafield, 1991; Cui et al., 1992) have been studied. It is necessary to determine the di-etary protein and lipid requirements of grass carp to achive optimum growth rates in aquaculture. Since the protein content in finfish diets usually constitutes the largest single cost factor in feeds, the objective is to minimize protein while opti-mizing growth and survival.
In the present study, the effects of dietary protein and lipid levels on the growth perfor-mance, feed utilization, body composition and digestibility of dietary nutrient and energy in ju-venile grass carp were investigated. Responses to these parameters of the fish were used to derive an optimum dietary protein and lipid levels.
This study was conducted at the Fish Nutri-tion Unit of Fisheries Faculty of Firat University, Elazig, Turkey. Each treatment had three repli-cates, 30 juvenile grass carp per replicate, with mean initial fish weights of 7.1 ± 0.1 g. Each treatment combination was randomly assigned to three conic glass aquaria (50 L). Fish were ex-posed to natural daily light regime. During the experimental periods, the aquaria continuously supplied with 250 mL min-1 of freshwater with 26 ± 2 oC temperatures, 7.0 ±0.4 mg L-1 dissolved oxygen and 7.2 ± 0.2 pH.
Feed ingredients were obtained from a local supplier (Oz Ugur-Feed Manufacture Company, Elazig, Turkey). Chromic oxide (marker) was donated by Merck Products Ltd. Vitamin and mineral premixes were obtained from Roche Products Ltd. All test ingredients were grounded until achiving a diameter of 500 μm. Diets were mechanically mixed with distilled water, and pelleted using a hand mincer fitted with a 3-mm die. The resultant moist pellets were then oven dried at 70 °C for approximately 12-h and then allowed to cool to ambient temperature in the oven and stored in polythene bags at 4 oC until used (Cai et al., 2005).
In the present experiment, a feeding trial was performed, at juvenile stage, to identify suitable protein and lipid levels and optimum protein to energy ratio to be used in formulating dry diets for this species. In the study, six different treat-ments were conducted. Triplicate groups were fed to satiation one of six practical diets (P1L1, P1L2, P1L3, P2L1, P2L2 and P2L3) with in-creasing levels of digestible protein (P1: 33% and P2: 37%) and three lipid levels (L1: 4%, L2: 6% and L3: 8%) within each protein level (Table 1).
All fish were fed three times at 4-h intervals from 09:00 to 18:00-h during 90 days. On termi-nation of the experiment, fishes were weighed individually and mean weight for each aquaria was calculated. During acclimatization period, fish were fed with experimental diets for 7 days prior to the beginning of fecal collection. After that first fecal collection was carried out. One hour after the feed was administered; any feed and feces present in the aquaria were removed. Fresh feces produced by the fish after this period and before the second feed was given, were si-phoned out. At least two siphonings were done from each aquaria during each period to minimise nutrient leaching from the feces. Fecal samples collected by siphoning were filtered onto filter paper and dried at 105 oC for 12-h. The faecal samples from each treatment replicate were sepa-rately analysed for the determination of nutrients and energy digestibility (Windell et al., 1978).
Whole body composition was determined af-ter homogenization of individual fish. The dry matter, protein, fat, fiber and ash in all samples were determined by standard methods (AOAC, 2000). The chromic oxide content of the experi-mental diets and fecal samples was determined by the acid digestion method of Furukawa and Tsukahara (1966). These samples were analysed for dry matter at 65 oC for 24-h in a vacuum oven. Crude protein (%N x 6.25) was determined by the micro-Kjeldahl method using an Auto Kjeldahl System and lipid by ether extraction. Nitrogen-free extract (NFE) was calculated by subtracting the percentages calculated for crude protein, fat, ash and fiber from 100. Gross energy content was determined in an Adiabatic Calori-metric Bomb.
Apparent digestibility coefficients for control and test diets were calculated according to the following equation described by Cho and Slinger (1979).
ADC = 100 x [1 - (F/D) x (Di/Fi)]
where ADC = % apparent digestibility coeffi-cient; F = % nutrient or energy of feces; D = % nutrient or energy of diet; Di = % marker in diet; Fi = % marker in feces.
The trial consisted of six treatments and three replications arranged in a completely randomized design. The data were expressed as mean ± SE of three replicate groups. The data of different treatments were subjected to ANOVA. When significant (P<0.05) difference was found, a Duncan’s multiple range test was used to esti-mate the difference. Correlation analysis was per-formed to compare differences between dietary lipid level and whole body fat content of fish. All statistical analyses were made using the SPSS 10.1 computer program for Windows (SPSS Inc. Chicago, Illinois, USA).
All the experimental diets were well accepted by juvenile grass carp, which actively fed on them, confirming the voracious feeding nature of this species. During the experimental period, a 100% survival rate was observed in all treat-ments; no external pathological signs were ob-served during the experiment.
The specific growth rates (SGR) determined in all treatments showed mean values higher than 1.7% of body weight per day. The grass carp fed the P1L1 diet group had the lowest weight gain (387%) and SGR (1.7%), which was significantly lower than those fed other experimental diets. But, when the lipid levels of diet containing 33% digestible protein was increased, the weight gain and SGR significantly increased. The feed con-version ratio (FCR) ranged from 1.7 to 1.2. Thus, in the P1L2 diet (containing 33% digestible pro-tein, 6% lipid and 10.7 kJ g-1 digestible energy) group, FCR was near to 1 and it was higher than those fed other diets (P<0.05). The protein effi-ciency ratio (PER) followed the same general pattern as FCR (Table 2).
No significant differences were found in whole body protein, fiber, ash or gross energy contents of fish fed diets with different protein and lipid levels (Table 3). On the other hand, body fat level increased with the increase from 4% to 8% in dietary lipid levels (P<0.05). Fur-thermore, the results showed a positive correla-tion (R = 0.926, P<0.01) between dietary lipid level and body fat content.
In addition, the P1L2 diet group had the high-est ADC values for protein, lipid and energy, and the P2L3 diet group the lowest (P<0.05). But, there were no significant differences between the digestibility values of dry matter, fiber, ash and NFE (carbohydrate) in the experimental diets (Table 4).
The SGR of fish fed the test diets in experi-ment showed high mean values (> 1.7%). In-creasing dietary lipid from 4% to 6% allowed di-gestible protein level to be decreased from 37% (P2L1 diet) to 33% (P1L2 diet) without negative affects on the SGR and FCR. Likewise, when comparing diets contained 33% digestible pro-tein, the increase in dietary lipid led to an im-provement in growth performance and feed utili-zation, suggesting that protein may be utilized for growth rather than for energy and, therefore, en-ergy from lipid spares protein in juvenile grass carp. This fact has been demonstrated in a num-ber of freshwater fish and marine carnivorous tel-eosts (Vergara et al., 1996).
A significant improvement in growth perfor-mance due to the sparing effect of lipid on dietary digestible protein has been reported for the gilt-head seabream, Sparus aurata (Vergara et al., 1996). This conclusion is supported by the PERs found for grass carp in the present study. Thus, in this work, a repeated trend was observed towards a higher PER as dietary lipid increased. This fact was evident when comparing, once again, diets with the same protein contents but different en-ergy levels. This phenomenon has been observed in rainbow trout, Oncorhynchus mykiss (Takuechi et al., 1978). However, some reports showed no effect of dietary lipid on body weight gain in juvenile turbot, Scophthalmus maximus (Danielssen and Hjertnes, 1993) and grass carp, C. idella (Du et al., 2005). Peres and Oliva-Teles (1999) believed this lack of protein sparing effect by dietary lipid may be related to the high protein level of the diet and according to Dias et al. (1998), the beneficial effects of an increase of the lipid level from 10 to 18% in sea bass (Dicen-trarchus labrax) diets were significant only with a low protein diet, but not with a high protein diet. Similarly in the present study, the dietary protein content was relatively low, when lipid level was below 6%, the protein utilization in-creased with the lipid level. This suggests, in low protein diets, the protein sparing effect by lipid is possible within a high upper limit.
The results of whole body composition showed positive correlation between dietary lipid level and body fat content, as reported in most other fish species investigated to date (Vergara et al., 1996). The body lipid content of grass carp increased with increasing dietary lipid levels, in-dicating that this fish could deposit lipid in the muscle. These results agreed with the report of gilthead sea bream (Vergara et al., 1996) and grass carp (Du et al., 2005). Thus in many fish species, the increase of dietary lipid levels should be evaluated carefully for it may lead to in-creased fat deposition in fish.
Digestible protein, lipid and energy values de-creased significantly with an increase in dietary lipid level from 4% to 8% (except P1L2 diet), which is probably one of the causes of the low-ered growth performance described above. Her-nandez et al., (2001) reported that sharp snout seabream (Diplodus puntazzo) showed no de-crease in protein digestibility and an increase of lipid digestibility with an increase in dietary lipid level. The digestibilities of protein and lipid for a diet with high lipid might differ among fish spe-cies. The present study shows that protein di-gestibility values (93 -93.5%) in diet P1L1, P1L2 and P2L1 for juvenile grass carp was found higher than the value (90%) reported by Law (1986) who fed commercial feed containing 38% of protein to grass carp. But, the digestible lipid (91.1-97.6%) and energy (87.6-93.5%) values for all diets used in this study are lower than the val-ues (lipid: 100% and energy: 98%) determined by Law (1986).
In conclusion, the maximum growth perfor-mance, feed intake, FCR, PER, digestible protein, lipid and energy values were obtained from the P1L2 diet group. These results suggest that P1L2 diet appears to be more adequate for growth of fish. However, a high-lipid diet (P2L3 diet) con-taining 37% digestible protein negatively affected growth performance and protein digestibility in juvenile grass carp. Thus in low protein diets, the protein sparing affect by lipid is possible within a high upper limit. Dietary levels of 33% digestible protein, 6% lipid and 10.7 kJ g-1 digestible energy could be assumed to be adequate levels in for-mulating practical diets for juvenile grass carp.
AOAC, (2000). Official Methods of Analysis. 17th edn., Association of Official Analytical Chemists, Washington, DC, USA
Cai, X., Luo, L., Xue, M., Wu, X., Zhan, W., (2005). Growth performance, body compo-sition and phosphorus availability of juve-nile grass carp (Ctenopharyngodonidellus) as affected by diet processing and replace-ment of fishmeal by detoxified castor bean meal, Aquaculture Nutrition, 11: 293-299. doi: 10.1111/j.1365-2095.2005.00354.x
Carter, C.G., Brafield, A.R., (1991). The bioen-ergetics of grass carp Ctenopharyngodonidella(Val.): energy allocation at different planes of nutrition, Journal of Fish Biology, 39: 873-887. doi: 10.1111/j.1095-8649.1991.tb04416.x
Cho, C.Y., Slinger, S.J., (1979). Apparent di-gestibility measurement in feedstuffs for rainbow trout. In: Halver, J., Tiews, K. (Eds.), Proc. World Symposium on Finfish Nutrition and Fishfeed Technology. Vol. II. Heenemann, Berlin, pp. 239-247.
Cui, Y., Liu, X.F., Wang, S.M., Chen, S.L., (1992). Growth and energy budget in young grass carp CtenopharyngodonidellaVal. fed plant and animal diets, Journal of Fish Biol-ogy, 41: 231-238. doi:10.1111/j.1095-8649.1992.tb02653.x
Dabrowski, K., (1977). Protein requirements of grass carp fry (Ctenopharyngodonidella), Aquaculture, 12: 63-73. doi: 10.1016/0044-8486(77)90047-3
Danielssen, D.S., Hjertnes. T., (1993). Effect of dietary protein levels in diets for turbot (Scophthalmusmaximus) to market size. In: Kaushik, S.J., Luquet, P. (Eds.), Fish Nutri-tion in Practice. INRA Editions, Les Collo-ques no. 61, pp. 89-96.
Dias, J., Alvarez, M.J, Diez, A., Arzel, J., Cor-raze, G., Bautista, J.M., Kaushik, S.J., (1998). Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchuslabrax), Aquacul-ture, 161: 169-186. Doi: 10.1016/S0044-8486(97)00268-8
Du, Z.Y., Liu, Y.J., Tian, L.X., Wang, J.T., Wang, Y., Liang, G.Y., (2005). Effect of di-etary lipid level on growth, feed utilization and body composition by juvenile grass carp (Ctenopharyngodonidella), Aquaculture Nutrition, 11: 139-146. doi: 10.1111/j.1365-2095.2004.00333.x
Furukawa, A., Tsukahara, H., (1966). On the acid digestion of chromic oxide as an index sub-stance in the study of digestibility of fish feed, Bulletin of the Japanese Society of Sci-entific Fisheries, 32: 502-506. doi: 10.2331/suisan.32.502
Hernandez, M.D., Egea, M.A., Rueda, F.M., Aguado, F., Martinez, F.J., Garcia, B., (2001). Effects of commercial diets with dif-ferent P/E ratios on sharp snout seabream (Diploduspuntazzo) growth and nutrient utilization, Aquaculture, 195: 321-329. doi: 10.1016/S0044-8486(00)00564-0
Law, A.T., (1986). Digestibility of low-cost in-gredients in peletted feed by grass carp (CtenopharyngodonidellaC. et V.), Aqua-culture, 51: 97-103. doi: 10.1016/0044-8486(86)90131-6
NRC, (1992). Nutrient Requirement of Warm-water Fishes and Shellfishes. Revised edn., National Academy Press, National Research Council, Washington DC.
Peres, H., Oliva-Teles, A., (1999). Effect of die-tary lipid level on growth performance and feed utilization by European sea bass juve-nile (Dicentrarchuslabrax), Aquaculture, 179: 325-334.
Sargent, J., Bell, G., McEvoy, L., Tocher, D., Estevez, A., (1999). Recent developments in the essential fatty acid nutrition of fish, Aq-uaculture, 177: 191-199. doi: 10.1016/S0044-8486(99)00083-6
Shiau, S.Y., Huang, S.L., (1990). Influence of varying energy levels with two protein con-centrations in diets for hybrid tilapia (Oreo-chromisniloticus x O. aureus) reared in sea water, Aquaculture, 91: 143-152. doi: 10.1016/0044-8486(90)90183-N
Takuechi, T., Yokoyama, H., Watanabe, T., Ogino, C., (1978). Optimum ratio of dietary energy to protein for rainbow trout, Nippon Suisan Gakkaishi, 44: 729-732. doi: 10.2331/suisan.44.729
Vergara, J.M., Robaina, I., Izquierdo, M., de La Higuera, M., (1996). Protein sparing effect of lipids in diets for fingerlings of gilthead sea bream, Fisheries Science, 62: 624-628.
Windell, J.T., Foltz, J.W., Sarokon, J.A., (1978). Methods of fecal collection and nutrient leaching in digestibility studies, Progressive Fish-Culturist, 40(2): 51-55.doi: 10.1577/1548-8659(1978)40[51:MOFCAN]2.0.CO;2