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J. B. Ulloa Rojas1 and Hans van Weerd2. 1 Escuela de Ciencias Biol;gicas, Universidad Nacional, Heredia 863000, Costa Rica. 2 Dept. Fish Culture and Fisheries, Wageningen Agricultural University. P.O Box 3386700 AH Wageningen, The Netherlands. Summary. An experiment was conducted in one recirculation system with 16 aquaria of 45 L each over an eight weeks period. The objective was to evaluate the effect of different dietary inclusion levels of NaOHtreated coffee pulp  7r (CoP) on growth and feed utilization of tilapia fingerlings (Oreochromis  7< aureus). The highest relative metabolic growth rate (RmGR) was found with control diet (D4: 17.2% g/kg0.8/d, no CoP), followed by diets with 13% CoP (D1a: 11.8% and D1: 12.5% g/kg0.8/d), 26% CoP (D2: 5.6% g/kg0.8/d) and 39% CoP (D3: 0.9% g/kg0.8/d) (P<0.05). Body weight gain also decreased as CoP inclusion levels increased (P<0.05). The protein efficiency ratio (PER) was higher with D4 but not different from diets D1, D2 and D1a. The lowest PER was obtained with diet D3 (39% CoP). The feed conversion (FC) of control diet and diets containing 1326% CoP were similar (P>0.05), being significantly higher for diet with 39% CoP. The highest apparent net protein utilization (NPUa) values were found in fish fed diet D4 and diets with 13% CoP (30.633.4%). The growth, feed utilization and the survival were better with fish fed diet D4. No differences were found for these parameters between fish fed diets with treated/washed (D1) and treated/unwashed (D1a) CoP. A decreasing dry matter and carbohydrate digestibilities were found at increasing dietary treatedCoP (P<0.05). However, the protein digestibility was not unpaired. Fish body ash, moisture and body protein increased with dietary inclusion of CoP whereas body fat diminished. The'0*(( allometric analysis showed that, body protein and body lipid (R2:0.9973 and R2:0.9652, respectively) were positively correlated with body weight (BW). The body ash and BW were inversely related (R2:0.9609).  7  It is concluded that juvenile Oreochromis aureus can tolerate the inclusion of CoP in the feed, however, it retarded the growth and feed utilization indices. The low feed intake could be a consequence of the increasing dietary fiber content at higher CoP inclusion levels. This together with the low feed palatability observed could explain the reduction on growth and feed utilization indices at increasing dietary CoP. Introduction In the tropics there are a great variety of agricultural residues scarcely used. Because of their enormous volume and potential feed value, these residues should be investigated as alternate foodstuffs. The greatest amounts of these wastes come from the processing of coffee, bananas, sugarcane, cocoa and fruits. If they could be incorporated into animal feeds, a great reduction of the environmental impact could be achieved. Coffee wastes, especially coffee pulp (CoP), have been used in animal  72 feeds with variable results (Bressani and Braham 1980). In fish, Bayne et  7 al. (1976) and Garc1a and Bayne (1974) found that 30% CoP in supplementary foods gave no adverse effects on the growth of tilapia, while Christensen (1981) and Fagbenro and Arowosoge (1991) found that inclusion of CoP in diets for carp and catfish reduced growth significantly. The adverse effects of CoP may be attributed to the presence of antinutritional factors (ANF's, e.g. caffeine, polyphenols and tannins (Bressani 1979,  7 V)lez et al. 1985). Several chemical, physical and biological treatments have been applied to CoP and other wastes to reduce the content of these components and to improve their nutritional value (Braham and Bressani 1979). It has been found previously that the composition of CoP treated with NaOH improved significantly, e.g. reducing caffeine, tannin and polyphenol contengw (Ulloa, in preparation). The purpose of this work is to assess the effect of NaOHtreated and ovendried CoP at different dietary levels  7 on the growth and feed utilization of Oreochromis aureus fingerlings. Materials and methods. Diet preparation and analysis: Fresh CoP was collected from a processing plant and processed as follows: It was treated with 5% NaOH for 24 h, either washed or not five times with tapwater, ovendried at 50 $C for 24 h, and milled to 1 mm. Five diets were formulated: D1, D2 and D3 containing 13%, 26% and 39% of treated and washed CoP, respectively, D1a with 13% treated and unwashed CoP and D4 with 0% CoP (control). Diets were formulated to be isoproteinous, but differences in feedstuff compositions produced an imbalance in dietary crude protein content (tables 1 and 2). Dietary aminoacid and essential fatty acid contents followed the'0*(( requirements for tilapia (Tacon 1990, Ulloa 1995b). Diet preparation was realized as previously described by Ulloa and Verdegem (1994) but including 1% Cr2O3 as a marker for digestibility measurements. Diets were analysed for moisture, protein, lipid, fibre and ash by standard methods (AOAC 1980). The "Total Utilizable Carbohydrate" was determined by the anthrone method (Osborne and Voogt 1986). Table 1. Ingredient composition (%) of the diets used in the feeding trial  7 with O. aureus fingerlings. c ddx !ddJZ? c  R R DietsR (% CoP)R R ` Ingredient D1(13) D2(26) D3(39) D4(0) D1a (13) `R  Fish meal Blood meal Soybean meal Coffee pulp1 Cassava starch Soybean oil Vitamins2 Sodium alginate3 Chromium oxide 43.0 5.0 12.0 13.0 22.0 2.0 2.0 1.0 43.0 6.0 7.0 26.0 13.0 2.0 2.0 1.0 45.0 6.0 0.0 39.0 3.0 2.0 2.0 2.0 1.0 42.0 5.0 17.0 0.0 31.0 2.0 2.0 1.0 43.0 5.0 12.0 13.0 22.0 2.0 2.0 1.0  1 D1a was similar to D1 but with treated and unwashed CoP. 2 (amount/kg premix): 800000 IU vitamin A; 200000 IU vitamin D; 10 g vitamin E; 1 g vitamin K; 2 g Thiamine; 3 g Riboflavin; 15 g Pantothenate; 2 g Pyridoxine; 2 mg B12; 20 g Niacinamide; 0.5 g Biotin; 200 g Ascorbic acid; 1 g Folic acid; 100 g Choline. 3 Used as binder. Experimental procedure: The experiment was run in a recirculating unit consisting of 16 aquaria (30 x 50 x 30 cm), a biofilter and a sedimentation tank for a period of eight weeks. Water quality was checked in the outflow of the aquaria. Water temperature and dissolved oxygen were measured twice daily (8 and 17 h). The pH, nitrite and ammonium levels were measured every week. By adjusting the water flow, nitrite and total ammonia levels were maintained below 0.20 and 3.4 ppm, respectively, and oxygen above 3.5 ppm. The photoperiod was 13 hours light per day. Fries were taken from an all male sexreversed population (17methyltestosterone) and acclimatized in the experimental unit for two weeks. At the beginning of the experiment each aquarium was stocked with 10 fish weighing between 410 g. The five diets were assigned randomly in triplicate over the system. At the beginning and at the end of the study all fish were weighed individually. Fish were fed "ad libitum" twice daily (10 and 15 h) for eight weeks. Faeces were collected after four weeks by pipetting them from the aquarium'0*(( bottom immediately after deposition, then dried and frozen for further analysis. Fish samples were taken from the initial population and, at the end of the feeding trial, from the treatment replicates (46 fish) for body composition analysis (AOAC 1980).  7 Table 2. Proximate composition, cell wall fractions 1 and other compounds 1 (% as drymatter) of CoP and  7x diets used to feed O. aureus fingerlings. | !ddJZ? AddBer | &` &      Diet (%CoP) "``B" Components WT CoP 1 UWT CoP  D1 (13)   D2 (26)   D3 (39)  D4 (0)   D1a (13)$`$ Moisture Lipids Fibre Ash Crude protein Carbohydrate" 10.5 0.24 56.3 14.4 8.8 20.3"  5.7 1.3 38.6 39.4 8.5 12.2"  6.7 8.6 11.0 12.6 37.1 30.6"  6.0 9.8 16.7 13.6 36.9 23.0"  4.7 9.8 27.8 15.5 34.8 12.1"  4.7 9.6 3.9 11.6 39.3 35.6"  5.3 8.0 9.6 14.8 37.2 30.4" " Cell walls 2 NDF ADF Cellulose Hemicellulose Caffeine Polyphenols TanninsF  68.6 48.8 20.8 19.8 0.22 0.14 0.05F  35.1 29.1 28.2 6.0 0.21 0.28 0.13F  8.9 6.3 2.7 2.6 0.03 0.02 0.006F  17.8 12.7 5.4 5.2 0.06 0.04 0.01F  26.8 19.0 8.1 7.7 0.09 0.06 0.02F  ND ND ND ND ND ND NDF  4.6 3.8 3.7 0.78 0.03 0.04 0.02(1) WT CoP: treated/washed CoP, UWT CoP: treated/unwashed CoP. (2) Cell walls and other component contents in diets were calculated from the CoP composition data.  7z ND: Not Determined. Data analysis: The following growth and feed utilization parameters were calculated: Relative growth rate of metabolic weight (RGRm): (WfĩWi/t)/BW0.8 (g/kg0.8/d) Weight gain (WG): (WfĩWi)/t (g/fish/d) J'0*((ԌFeed conversion (FC): Feed given (dry matter, g)/wet weight gain (g) Protein efficiency ratio (PER): (body growth, g)/(protein fed, g) Apparent net protein utilization (NPUa): (protein growth, g)/(protein fed, g) x 100 (%) Survival (S): NiĩNf/Nix100 (%), Where W= mean weight (g); N= number of fish; t= number of days; BW0.8:(exp((lnWi+lnWf)/2)/1000)0.8= geometric mean metabolic body weight; i= initial and f= final. Chromic oxide in faeces and feed was measured in duplicate by the dry ash  7 method of Mink et al. (1969) adapted for small faeces samples. The apparent digestibility coefficients (ADC) were calculated using the equations: ADC of dry matter: 100x(1Ii/If) (%) ADC of nutrients: 100x[1(IixNf)/(IfxNi)] (%), Where: I= indicator concentration, N= nutrient concentration, f= faeces and i= ingesta or feed. Data analysis was done using the ANOVA and the initial weight as a covariate. Treatment means were compared by the HSD Tukey test with 95% confidence intervals. Results. The water quality parameters remained within the ranges for tilapia culture (pH: 5.97.6, NO2é:0.040.2 ppm, total NH4+: 0.283.36 ppm, temperature: 28.632.5 $C and oxygen: 3.46.6 ppm). The survival ranged from 92.5 to 100% and there were no differences between treatments. Fish fed diet D4 (0% CoP) showed the highest final body weight (45 g), WG (0.7 g/fish/d) and RGRm (17.2 g/kg0.8/d) (P<0.05). As dietary CoP level increased (13 to 39%), growth decreased. Diet D1 (13% treatedwashed CoP) showed better growth and feed utilization vaalues than D1a (13% treatedunwashed CoP) but differences were not significant (table 3). The feed utilization decreased with increasing CoP levels in the diet: increased FC and decreasing PER and NPUa values. Feed and nutrient intakes increased as dietary CoP inclusion level decreased. In general, dry matter and carbohydrate digestibility coefficients decreased with increasing CoP level in the diet. The digestibility of protein was, however, not unpaired (Table 3). '0*((Ԍ 7 Table 3. Growth and feed utilization values of O. aureus fingerlings fed diets with different CoP levels. | AddBer addxt@ |    J Z DietsZ (% CoP)Z Z  p  Parameters D1 (13) D2 (26) D3 (36) D4 (0) D1a (13) pZ  Initial mean body weight (g) Final mean body weight (g) WG (g/fish/d) RGRm (g/kg0.8/day) Feed conversion PER NPUa (%)   7.0 a 31.3 b 0.43 b 12.5 b 1.46 c 1.94 d 30.6 d   7.0 ab 15.6 c 0.15 c 5.6 c 2.15 c 1.34 c 21.1 c   7.2 a 7.4 d 0.01 c 0.9 d 7.68 a 0.40 a 6.1 a   7.2 a 45.0 a 0.68 a 17.2 a 1.24 c 2.09 d 33.4 d   7.0 ab 28.8 b 0.39 b 11.8 b 1.40 c 1.98 d 32.6 d Feed intake (1) Protein intake Lipid intake Carbohydrate intake  33.0 c 12.3 c 2.9 d 10.1 d  16.6 b 6.1 b 1.6 bc 3.8 c   6.4 a 2.2 a 0.6 a 0.7 ab  46.3 d 18.2 d 4.4 e 16.5 e  29.2 c 10.9 c 2.3 cd 8.9 d  Digestibility Dry matter Protein Carbohydrater  68.9 bcd 83.4 ab 81.7 bcr  64.5 abc 85.4 b 73.3 abcr  59.4 a 78.5 a 55.0 ar  74.0 d 82.7 ab 87.4 cr  70.3 cd 82.9 ab 88.8 c abcd value in the same row with letter in common are not statistically different (P<0.05).  7: (1) Feed and nutrient intakes: g/fish/feeding period.  7 Table 4. Proximal body composition (% wet basis) of fingerlings O. aureus fed diets with different CoP levels. w addxt@ ddd0$A w  d  Diet(%CoP)/ Components Initial fish D1 (13) D2 (26) D3 (39) D4 (0) D1a (13) d\$  Moisture (%) 71.8 a 73.4 a 75.5 a 75.7 a 71.3 a 72.0 a \\  Ash (%) @  4.2 a@ 4.7 a@  5.1 a@  6.7 c@ 4.5 a@  5.0 a \\  Lipid (%) 9.2 a 6.2 c 4.3 deQQQ  3.0 e  8.0 ab  6.8 bc \@  Protein (%)( 14.1 a( 15.4 a( 14.9 a( 14.2 a( 15.7 a( 15.7 a 7(  abcde Values in the same row with no letter in common are not statistically different (P<0.05). At higher dietary CoP levels, trends for higher body ash and lower fat contents were found (Table 4). The initial fish and fish fed diet D4 showed the highest body fat, but also the lowest body ash, protein and moisture contents. Allometric analysis showed positive correlation between body protein vs. fish weight (R 2:0.9973, log protein: 0.876 + 1.0451x log fish weight) and, body lipid vs. fish weight (R2:0.9652, log lipid: 0.0798 + 0.6002x log fish weight). The body ash and fish weight showed an inverse relationship (R 2:0.9609, log ash: 39.4524 14.5846x log fish weight). An inverse correlation between moisture and body lipid was also'0*(( observed (R 2:0.7209, moisture(%): 67.2549 0.8399x lipid(%)). Discussion.  7  The growth and feed utilization of O. aureus decreased with the increasing inclusion of CoP in diets (0% to 39%). This was also observed with common  7 carp (Cyprinus carpio), catfish (Clarias mossambicus) and Clarias  7| isheriensis (Christensen 1981, Fagbenro and Arowosoge 1991). These findings also agree with those found by Ulloa (1995a) for tilapia fed diets containing untreated CoP, but these values appear worse in the present study. On the contrary, Garc1a and Bayne (1974) found higher weight gain (WG) values with tilapia fed a 30% CoP diet than those raised  7f in ponds treated with chicken manure. Bayne et al. (1976) found similar WG's using a diet with 30% CoP and a control diet. Natural food present in ponds used in those researches could partially support the tilapia growth and minimize the possible negative effect of CoP on the fish. The FC values for fish fed up to 26% CoP (1.32.15) seem acceptable. They  7 suggest that despite the high fibre dietary contents, O. aureus may use efficiently diets containing CoP. Such observation concurs with Fagbenro and Arowosoge (1991) and Ulloa (1995a). Despite the great reduction on ANF's (e.g. polyphenols, tannins and caffeine) in NaOH treatedCoP diets, the growth and feed utilization were still lower than the control diet. The remaining levels of these ANF's in the diets appears too low to still exert a negative effect on the fish response. However, the high fibre levels in diets containing CoP would explain the decreasing tilapia response. Their values were above the least  7 recommended level for O. niloticus x O. aureus and O. mossambicus (Shiau  7 et al. 1989, Dioundick and Stom 1990, De Silva and Anderson 1995). The reduction in the feed intake, dry matter and carbohydrate digestibilities as dietary CoP level increased could be caused by a deleterious effect of the dietary fibre excess and the remaining ANF's (Tacon 1990, De Silva and Anderson 1995). This also coincided with the low feed palatability observed. The high fibre levels could result in dilution of nutrients producing poor fish growth, especially in diets containing 26 and 39% CoP. The high protein digestibility coefficients for all diets containing CoP  7N (78.5% to 85.4%) could suggest that O. aureus digest well the dietary protein. These values are higher than those reported by Popma (1982) for  7 O. niloticus and Fagbenro and Arowosoge (1991) for Clarias isheriensis. Differences could be caused by (a) variation on diet composition (e.g. CoP alone, diets with low and high fish meal levels), (b) species differences (e.g. selective food consumption) or, (c) variation in the faeces collection methods used which could result in differences in leaching of faecal nutrients. The short collection intervals used in this study (30 minutes) could reduce nutrient leaching. The maximum inclusion level of CoP recommended in rations for ruminants"'0*((  7 (up to 30%, Bressani et al. 1975) and for swine (up to 16%18%, Jarqu1n et  7 al. 1977) is higher than the CoP level (13%) found in this study causing adverse effects. However, it concurs with the maximal level for rats and  7\ poultry (10% CoP addition) (Bressani et al. 1973). The differences may be explained by the better capability of ruminants and, possibly of swine to digest higher fibre levels in the diet. The high survival rate (above 92%) at any CoP inclusion level suggests  7F that Oreochromis aureus juveniles can tolerate high dietary levels of CoP.  7 This finding agrees with Bayne et al. (1976) and Fagbenro and Arowosoge (1991). However, it is opposite to findings with poultry and, other  7 monogastric animals as rats and turkeys (Bressani et al. 1973, G;mez 7l Brenes et al. 1985, Donkoh et al. 1988). It could be explained by the relatively low levels of ANF's in the diets with treated CoP. The depressed feed intake at higher CoP dietary levels could lead fish to use body energy reserves that may produce the reduction on body fat and the relative increase in body ash agreeing with Ulloa (1995a). The allometric analysis showed that body protein, body fat and body ash were dependant of fish weight, but, body ash showed a negative correlation. The protein growth coefficients found for tilapia (a:0.88 and b:1.04) agreed with those cited by Shearer (1994). The results showed that diets with 13% CoP (or higher) fed to fingerlings  7 of O. aureus produced adverse effects on growth and feed utilization. As CoP increased in the diets (26 or 39%), the reduction in fish growth was much bigger. This reduction may be associated to the high level of fibre in the CoP diets. Acknowledgements. This research was supported by the Programme UNALUW, Universidad Nacional, Costa Rica and Wageningen Agricultural University, The Netherlands. Thanks to the CICAFE (Costa Rican Coffee Research Centre) for doing the caffeine analysis. References.  7P AOAC (Association of Official Analytical Chemists). Official Methods of  7 Analysis. 12th edition. Washington DC, USA: AOAC, 1980. Bayne D., Dunseth D. and C. Garc1a. "Supplemental feeds containing coffee  7t" pulp for rearing Tilapia in Central America". 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San Jos), Costa Rica: EUNA, 1995a.  7 Ulloa, J. 1995b. "Nutrici;n de tilapia". p. 3353. In: 1er. Simposio  7 Centroamericano sobre el cultivo de tilapia. San Jos), Costa Rica: EUNA, 1995b. Ulloa, J. and M. Verdegem. "Effects of the protein: energy ratio in isocaloric diets on the growth of Cichlasoma managuense (GGnther 18969)".  7 Aquaculture and Fisheries Management 25 (1994): 631637.  7> V)lez, A., Garc1a, L. y M. de Rozo. 1985. "Interacci;n in vitro entre los  7 polifenoles de la pulpa de caf) y algunas prote1nas". Archivos  7 Latinoamericanos de Nutrici;n 35 (1985): 297301.