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Determination of digestibility of commercial salmon feeds
Aquaculture 179 Ž1999. 81–94
Determination of digestibility of commercial salmon feeds ˚ ˚ ) , Gerd Marit Berge Marie Hillestad, Torbjørn Asgard AKVAFORSK, Institute of Aquaculture Research AS N-6600 Sunndalsøra, Norway
Abstract The aim of the study was to develop an applicable method for determination of the digestibility of nutrients in commercial feeds. Leaching of markers and dry matter ŽDM. were compared between a commercial extruded feed containing La 2 O 3 , the same commercial feed coated with oil and other additional markers, and the commercial feed that had been ground and re-pelleted with water, marker and a crude alginate binder. Two markers, Y2 O 3 and Cr2 O 3 , were added in both the coated and the moist feeds. The optimal inclusion level of binder and water to minimize the leaching of DM from the moist pellet was determined in a preliminary study. The lowest leaching of DM was achieved with the inclusion of 2% alginate and 35% water. Added markers tended to be less homogeneously included in the coated feed than in the moist feed, and the markers leached relatively more than the DM from the coated feed. Apparent digestibility coefficients ŽADCs. of DM, fat, nitrogen ŽN., energy, organic matter ŽOM., and phosphorus ŽP. in the commercial dry feed and the moist feed were compared in two experiments with Atlantic salmon. Differences between the ADCs in the two experiments were observed for N and P. The ADC estimates of the moist feed were slightly lower than those of the dry feed for all nutrients. Similar ADC estimates for each nutrient were obtained for all markers, except for Y2 O 3 which produced a slightly higher ADC for N than did La 2 O 3. By using the moist feed method recommended from this study, a difference of about 1.4% units in ADC of energy and of 0.8% units for N between the original and the modified test feed was demonstrated. This means the error of ADC estimates is low compared to using fixed digestibility coefficients for each of protein, fat and carbohydrate, a method that has been employed for declaration of commercial fish feed. Further development of the method is discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Digestibility; Nitrogen; Energy; Phosphorus; Marker; Commercial feeds; Effluents
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Corresponding author: Tel.: q47-71-69-53-00; fax: q47-71-695301; E-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 5 4 - 4
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1. Introduction Commercial fish feeds are usually provided with a declaration of total digestible energy. Many feed manufacturers base the declaration on measured proximate composition and fixed digestibility coefficients for protein, fat and starch. Coefficients commonly employed in Norway have been 87, 90, and 65% for protein, fat and carbohydrate, respectively Žconsensus in the Norwegian Fish Feed Producers Association.. Because the digestibility may vary considerably, depending on feed ingredients, inclusion levels, processing conditions and nutrient interactions, the use of fixed digestibility values can be misleading. Feeds that are equal in proximate composition may have up to 10% units difference in energy digestibility due to the use of different fat sources ŽAustreng et al., 1979., different protein sources ŽMorales et al., 1994. or different carbohydrate sources ŽStorebakken et al., 1998a.. Carbohydrate levels in otherwise equal feeds can result in digestibility differences of the same extent ŽBergot and Breque, 1983; Aksnes, 1995.. However, no standard method for determination, verification or invalidation of the digestibility of the nutrients in commercial feeds is available. The digestibility level of the feed may have both economical and ecological consequences. The nutrients of environmental concern are nitrogen ŽN., phosphorus ŽP. and organic matter ŽOM.. The oxygen consumption required for degradation of OM depends largely on its energy content. With respect to the digestibility differences mentioned above, the energy load, through faeces, from a feed with an energy digestibility of 80% will be twice that of a feed with an energy digestibility of 90%. Correspondingly, knowledge of the P and N digestibility of the feeds is of crucial importance when modelling discharge to the environment. Due to the problems described above the nutrient digestibility of commercial feeds should be declared based on measurements. To do so, we need reproducible methods and standardized conditions. The direct method for determination of digestibility by exact measurement of feed intake and complete collection of excreta has been suggested by Choubert et al. Ž1979.. But there are important limitations with the faeces collection in water for nutrients easily dissolving. Careful stripping produces digestibility estimates close to the dissection method ŽStorebakken et al., 1998b., and stripping can also be employed in field studies or in tanks without special collection equipment, in contrast to other methods where the faecal matter is collected from the water. However, the stripping method requires indigestible markers in the feed for estimation of the digestibility. Naturally occurring indigestible fractions such as acid insoluble ash ŽBowen, 1981; Atkinson et al., 1984., fibre ŽTacon and Rodrigues, 1984., cellulose ŽBuddington, 1980. and hydrolysis resistant matter ŽBuddington, 1980; De Silva and Perera, 1983. have been evaluated as markers for digestibility determination. These internal markers have been of particular interest when the aim was to test a feed with no markers added, as e.g., natural food or commercial feeds. The naturally occurring level of these markers is, however, low in commercial fish feeds, relative to the amount required for analysis, thus making them unsuitable as test markers. The indirect method using Cr2 O 3 as an inert marker in salmonid feed ŽAustreng, 1978. has been commonly used. However, analytical variability and poor recovery of the Cr2 O 3 have been reported ŽKotb and Luckey, 1972; Dabrowski and Dabrowska, 1981; Blincoe et al., 1987., and several
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studies reveal digestive or metabolic effects of Cr2 O 3 , which in turn might affect the nutrient digestibility measurements ŽBowen, 1978; Dabrowski and Dabrowska, 1981; Ringø, 1993; Shiau and Liang, 1995; Sugiura et al., 1998a.. Further, Cr2 O 3 changes the appearance and possibly the immediate acceptability of the feed. Concerns have therefore been raised about the use of Cr2 O 3 in digestibility studies ŽRingø, 1993; Riche et al., 1995.. Ng and Wilson Ž1997. studied some of the these problems and did not find negative effects of Cr2 O 3 . Alternatives to Cr2 O 3 as added inert marker have successfully been used in digestibility studies of fish feeds, like oxides of Y ŽBjerkeng et al., 1997; Hung et al., 1997; Refstie et al., 1997; Sugiura et al., 1998a,b; Storebakken et al., 1998a., Yb ŽRefstie et al., 1998., Dy, Sc, La ŽHillestad and Olafsen, 1990; Meland, 1991; Winther, 1991. and Ti ŽWeatherup and McCracken, 1998.. Recently, the microtracer Ferro-Nickel was reported a promising marker by Kabir et al. Ž1998.. The aim of the present study was to find an applicable method for digestibility assessment of commercial salmon feeds. The first step was to select a method of adding marker to the feed. Two methods were tested: either by grinding the feed and mixing it with the marker before preparing a moist feed, or by coating the intact feed with the marker. Homogeneity of markers in the feeds and leaching to the water of markers and nutrients were compared. Inclusion of binder and water in the moist feed was optimized in a preliminary study. Finally, the apparent digestibility of the macronutrients and P in the intact commercial feed and the moist feed were compared in an experiment with Atlantic salmon. 2. Materials and methods 2.1. Sinking speed and loss of dry matter (DM), preliminary studies Moist feed prepared from a commercial dry feed was optimized for low DM leaching in a preliminary study. A feed produced according to the process of commercial extruded feed production, containing 46% protein and 30% fat, was used for the experiment. Nine experimental moist feeds were produced by adding 30, 35 or 40% water to each of 3 batches Žeach 5 kg. of ground extruded basic feed containing either 1, 2 or 3% crude alginate ŽAlgibinde Algea AS, Oslo, Norway. as a binder, in a 25-kg blender. After mixing for 5 min, each feed was pressed through a mincer equipped with a 5 mm matrix. The feeds were then frozen Žy208C. and later broken into pellets. Sinking speed of the basic feed pellets and pellets of the nine moist feeds were measured by using a 200 cm high = 30 cm diameter cylinder filled with 108C freshwater. Timing started 10 cm below the water surface and lasted during the next 150 cm of sinking. Leaching of DM was measured by dipping 100 g of each thawed feed in a sieve for 10 min in 208C seawater with a salinity of 33 g ly1 . Each sample was analyzed for DM immediately, and results were not corrected for uptake of salt from the water. 2.2. Experimental feeds The 6 mm extruded basic feed ŽBasic. was produced by Stormøllen ŽVaksdal, Norway.. The feed contained Norse-LT 94TM and NorSeaMinkTM fish meal, fish oil,
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wheat, fish protein concentrate, shrimp peel meal and a vitaminrmineral premix. The feed contained 24, 282, 490, 103 and 15.3 g kgy1 of water, fat, protein, ash and P, respectively, and 23.9 MJ energy kgy1 . An even distribution of the marker, La 2 O 3 , was ensured by manually mixing 125 g La 2 O 3 with 3.5 kg of the dry premix before the batch Ž750 kg. was blended Ž5 min. and further dry-extruded. A feed coated with marker ŽCoated. was made from a 10-kg batch of the basic feed by coating with 10,000 mg Cr2 O 3 kgy1 , 100 mg Y2 O 3 kgy1 , and 2% fish oil ŽTable 1.. The Y2 O 3 was mixed with half of the oil at 258C and added to the mixture in a narrow stream into a bakers blender during a 1 min run. The Cr2 O 3 was sieved into the mixture and the blending continued for 2 min. Then the rest of the oil was added and the mixture blended for another 2 min. A moist feed ŽMoist. was made by grinding 10 kg of the basic feed in a coffee grinder and adding 2% AlgibindTM , 10,000 mg Cr2 O 3 kgy1 , and 100 mg Y2 O 3 kgy1 . An even distribution of the markers was ensured by first mixing the Y2 O 3 with a small amount of the ground feed, then with a larger amount before it was added to the total batch. The Cr2 O 3 was diluted in the same way, but only in one step, before being added to the total batch. Then 3.5 l of water was added and the blending was proceeded for 5 min before running the batch through a mincer with a 5 mm matrix. Three samples Ž15–20 g. from each of the experimental feeds were drawn and stored at y208C. Moist feed was freeze dried and all feeds were analyzed for marker, P content and proximate composition. All experimental feeds were stored at y208C. 2.3. Leaching of markers and DM A square tank Ž1 m2 . with 1.2 m water depth Žsalinity 33 g ly1 . and a water exchange of 15 l per min was employed for the leaching test. A pre-weighed amount of thawed feed was dropped into the water and collected from the outlet in a wire mesh collector ŽHelland et al., 1996.. The average time spent in water was calculated as the time that elapsed from when pellets were dropped to the average between when the first and the last pellet were collected from the outlet. Samples were frozen Žy208C. before
Table 1 Ingredients addeda to feeds used in digestibility studies Feed
Basic
Coated
Moist
Experiment
1 and 2
1
1
2
La 2 O 3 Žmg kg y1 . Y2 O 3 Žmg kg y1 . Cr2 O 3 Žmg kg y1 . Water Ž%. Alginateb Ž%. Capelin oil Ž%.
166 – – – – –
161 100 10,000 – – 2.0
164 100 10,000 35 2 –
164 100 – 35 2 –
a
Added per kg of basic feed. Algibinde ŽAlgea, Oslo, Norway..
b
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analysis of DM and markers. DM results were not corrected for uptake of salt from the water. 2.4. Apparent digestibility Two digestibility trials were conducted with the basic feed and the moist feed. The coated feed was excluded based on the results from the preliminary studies on leaching. In Experiment 1 ŽExp. 1. a moist feed with both Y2 O 3 and Cr2 O 3 was used, while Cr2 O 3 was excluded from the moist feed in Experiment 2 ŽExp. 2., due to concerns about confounding effects on the digestibility of adding Cr2 O 3 and alginate to the diet. 2.4.1. Exp. 1 Thirty Atlantic salmon Ž Salmo salar L.., with an initial average weight of 540 g, were in August 1996 randomly distributed to each of six tanks Ž1 m2 . supplied with 15 l per min of seawater Ž8.1–8.98C and salinity 33 g ly1 .. The two experimental feeds, Basic and Moist, were thawed and randomly fed to fish in three tanks each. Feed ration was kept approximately 10% above satiation. Feed intake was monitored by collecting all feed waste from the tanks as described by Helland et al. Ž1996.. Faeces were stripped from all fish after 8 and 15 days, and samples were pooled within tank and frozen at y208C. The amount of faecal samples from day 15 was sufficient for all chemical analyses which were carried out after freeze drying. 2.4.2. Exp. 2 Twenty Atlantic salmon with an initial average weight of 1.0 kg were kept in the same experimental set-up as in Exp. 1 ŽJune 1997., with water temperature 6.8–7.08C. Faeces were collected by stripping after 7 and 12 days. As in Exp. 1, samples from the second stripping were used for chemical analyses. 2.5. Analyses and calculations Feeds and faeces were analyzed for DM Ž1058C, 16–18 h., ash Žcombusted at 5508C, 16–18 h., nitrogen Žsemi-micro-Kjeldahl, Kjeltec-Auto System, Tecator, Hoganes, ¨ ¨ Sweden., fat Ždiethyl ether extraction in a Fosstec ŽTecator. analyser after HCl-hydrolysis. and gross energy ŽParr 1271 Bomb calorimeter, Parr, Moline, IL, USA.. Y2 O 3 , La 2 O 3 and P were analyzed according to Refstie et al. Ž1997.. Cr2 O 3 was analyzed according to Williams et al. Ž1962.. Organic matter ŽOM. was calculated as the DM y ash difference. ADCs were calculated as ADC n s 100 y Ž100 = feed i Ž%.rfaeces i Ž%. = faeces nŽ%.rfeed nŽ%.., where subscript i means indicator Žmarker. and n means nutrient concentrations. ADCs of DM and ash were calculated from the original content in feed and faeces exclusive of the added marker. ADC estimates were corrected for content of alginate in feed and faeces, assuming the alginate is completely indigestible and containing 89.0, 0.8, 35.0 and 0.0% of DM, N, ash and fat, respectively ŽAlgea, Oslo, Norway..
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Two-way ANOVA was employed for testing the effect of the inclusion level of water and alginate and their interaction on sinking speed and DM loss of the moist feed in the preliminary study. One way ANOVA and Duncan’s multiple rank test was used for testing the difference between means of leaching of DM and markers from the basic, coated and moist feeds. Possible differences in ADCs obtained by Cr2 O 3 and La 2 O 3 were tested in Exp. 1 in a One-way ANOVA with digestibility data from the moist feed. After exclusion of the Cr2 O 3 measurements, the ADC data of moist feed from both experiments were merged in a two-way ANOVA for testing the possible differences obtained by La 2 O 3 and Y2 O 3 . Effect of feed and experiment and their interaction were tested in a two-way ANOVA by including only the ADCs measured by La 2 O 3 in the two feeds. In the preliminary study three observed DM analysis values made no sense and fell out of the 99% confidence interval. They were removed from the data set before statistical analyses. Statistical power was calculated according to Searcy-Bernal Ž1994., and minimum detectable difference between means were based on a power of at least 0.80. Statistical differences were indicated at a 5% level for all analyses.
3. Results 3.1. Sinking speed and loss of DM, preliminary study The dry feed sank approximately twice as fast Ž14.4 " 0.2 cm sy1 Žmean " S.E.M... as the moist feed Ž7.8 " 0.1.. Sinking speed of the moist feed decreased with increasing content of alginate and increased with increasing inclusion of water. A significant interaction between the two main effects on sinking speed was observed. Loss of DM was lower with the feeds containing 2% alginate compared to the feeds containing 3% alginate, and lowest for the feeds containing 35% water compared to the others Ž1.1% " 0.1.. If corrected for assumed uptake of salt Ž33 g ly1 water. the estimated losses of DM would have been approximately 0.4% units higher. 3.2. Marker homogeneity and leaching of markers and DM The coefficient of variation ŽCV. for marker content between feed samples ranged from 1 to 7 ŽTable 2.. The CV of the added markers tended to be higher in the coated than in dry and moist feed. The average recovery of La 2 O 3 , measured as the analyzed amount in percentage of the formulated, in all feeds was 86%. Recovery of Y2 O 3 in coated feed was 79% and in moist feed 84 and 93% in Exp. 1 and Exp. 2, respectively. Recovery of Cr2 O 3 was 84% in coated feed and 88% in moist feed. The leaching of the added markers were higher from the coated feed than from the moist feed Ž P s 0.01, 0.07 and 0.08, respectively, for Cr2 O 3 , Y2 O 3 and La 2 O 3 .. From the coated feed the leaching of added markers were 10-fold higher than the leaching of DM ŽTable 3.. If corrected for assumed uptake of salt the estimated leaching of DM would have been approximately 0.6% units higher. Time elapsed from when the feed left the automatic
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Table 2 Marker content of extruded basic feed, feed coated with oil and marker, and freeze dried moist feed Ž ns 3. in Experiment 1 Feed
Basic
Coated
Marker
Average
CV
La 2 O 3 Žmg kgy1 . Y2 O 3 Žmg kgy1 . Cr2 O 3 Žmg kgy1 .
141 – –
2.3 – –
a
a
Moist
Average
CV
Average
CV
138 76 8,069
1.1 6.8 5.1
136 81 8,500
2.5 3.8 3.5
CVs coefficient of variance between 3 samples.
feeder until it was collected in the outlet tended to be longer for moist feed than for the basic and coated feeds Ž P s 0.11.. 3.3. ADC Feed intake by the end of both experiments was acceptable Žin average 8 g kgy1 fish dayy1 .. Mean ADCs are presented in Table 4, while the differences between means and their significance within each model are presented in Table 5. The ADCs of dry feed were significantly higher than those of moist feed for all parameters measured. The greatest difference between feeds was measured in ADCs of ash Ž10.3% units. and P Ž7.6% units.. The differences in ADCs between dry and moist feed were at the same level in Exp. 1 and Exp. 2. If corrected for content of alginate in feed and faeces, assuming the alginate is completely indigestible, the mean ADC estimates of DM, energy and N in the Moist feed obtained by Y2 O 3 would be 1.4, 0.8 and 0.2% units higher than the ADCs shown in Table 4, as an average for both experiments. Significant differences between the two experiments were revealed only for the ADCs of N and P, with 1.3 and 7.6% units lower ADCs, respectively, in Exp.1 than in Exp. 2. No significant differences between ADCs measured by the different markers were observed, except for the difference between ADC of N measured by La 2 O 3 and Y2 O 3 Ž P s 0.05.. Except for ash, the differences between ADCs measured by different markers were small Ž- 2.1% units.. The power of the test to unveil differences in ADCs
Table 3 Leaching of dry matter and markers Ž%. from different feeds during the time spent from automatic feeder to collection in effluent water Žmean"S.E.M., ns 3. Feed Dry matter La 2 O 3 Y2 O 3 Cr2 O 3 , Time elapsed Žs. a
Basic 0.5"0.6 y0.3"0.3 Aa – – 23.8"0.9
Coated
Moist a
1.4"0.3 a 5.1"2.3 Ba 18.2"6.4 b 24.4"3.7 Ab 26.2"2.4
0.9"0.2 1.9"0.7 AB 2.3"0.4 2.5"3.0 B 32.5"3.5
Different letters in the same row ŽAB. or in the same column Žab. denote significant difference Ž P - 0.05. between treatment means.
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Table 4 Apparent digestibility Ž"S.E.M.. of dry and moist feed in Atlantic salmon measured by different markers and repeated experiments Ž ns 3. Feed
Dry
Moist
Marker
La 2 O 3
La 2 O 3
Y2 O 3
Cr2 O 3
Experiment 1 Dry matter Fat Nitrogen Energy Organic matter Asha Phosphorus
69.5"0.5 94.6"0.5 83.9"0.2 84.2"0.5 77.5"0.4 2.3"1.2 25.7"0.2
65.8"0.5 89.7"2.0 82.7"0.4 81.0"0.7 74.7"0.6 y8.0"1.3 19.7"0.6
66.3"1.1 89.7"2.3 82.9"0.2 81.2"1.2 75.0"1.1 y5.0"1.6 20.8"1.6
66.7"0.2 90.0"1.9 83.1"0.3 81.5"0.7 75.3"0.3 y5.3"2.1 21.7"1.3
Experiment 2 Dry matter Fat Nitrogen Energy Organic matter Asha Phosphorus
70.3"0.4 92.0"0.9 85.5"0.4 83.9"0.2 77.8"0.4 3.0"0.7 34.9"0.5
65.7"0.9 90.6"0.9 83.7"0.1 81.4"0.8 74.4"0.8 y7.3"2.8 25.7"1.8
67.7"0.6 91.2"0.7 84.7"0.2 82.5"0.5 75.9"0.4 y0.8"2.7 30.2"1.2
a
Measured ash ADCs are affected by uptake of minerals from seawater.
between experiments was approximately 0.20 for other parameters than N and P. To unveil differences between La 2 O 3 and Y2 O 3 the power was between 0.70 and 0.90 for ash and P, and in the range 0.20–0.50 for the other nutrients. To unveil differences between Cr2 O 3 and Y2 O 3 the power was below 0.35.
Table 5 Differences Ž% units. between means of apparent digestibility of dry and moist feed in Atlantic salmon measured by different markers and repeated experiments
Dry matter Fat Nitrogen Energy Organic matter Ash Phosphorus a
Feed a
Experimentsa
Markers
Dry–moist feed
2–1
Y2 O 3 –La 2 O 3b
Cr2 O 3 –La 2 O 3c
4.2U 3.1U 1.5U 2.9U 3.1U 10.3U 7.6U
0.3 y0.8 1.3U 0.1 0.1 0.7 7.6U
1.3 0.3 0.6U 0.7 0.9 4.0 2.8
0.9 0.3 0.5 0.5 0.7 2.8 2.1
Data obtained by La were included in the analysis when comparing feeds and experiments Ž N s12.. Data from moist feed were included in the analysis when comparing Y and La Ž N s12.. c Data from moist feed in Exp. 1 were included in the analysis when comparing Cr and La Ž N s6.. U The contrasts with asterisk are significantly different Ž P - 0.05.. b
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4. Discussion In the preliminary study with moist feeds, the intermediate levels of alginate Ž2%. and water Ž35%. resulted in the lower loss of DM and the best feed consistency. Therefore, these levels of alginate and water were used in the later experiments. The leaching measured by immersion of pellets in a sieve in water for 10 min or by dropping the pellet into a tank and collecting it in the outlet, both gave a DM loss of about 1%, which is lower than Fagbenro and Jauncey Ž1995. found with extruded fish silage feeds. The thawing did not seem to cause any visible leaching or crumbling of the feed before feeding. The residence time in water, before pellets are eaten by Atlantic salmon in tanks, is most likely less than measured in the drop-and-collect experiment. Thus, the leaching before the pellets are eaten is anticipated to be less than 1%. The low sinking speed of the moist feed makes it well suited for experiments in shallow tanks, as the pellet will remain in the water column for a longer period, and easily be caught by the fish. On the other hand, when the residence time of the feed in water increases Žnot significantly different in our experiment. the leaching of nutrients and markers may also increase. However, if markers and nutrients are leaching at the same rate, the digestibility measurements will not be affected. The coated feed leached relatively more markers than DM, thus lowering the digestibility estimates. Our results also indicate that it is more difficult to add markers homogeneously by coating them on the pellet surface than by including them into a moist feed ŽTable 3.. Thus, adding an external marker into a commercial feed by mixing it into the feed seems to be advisable rather than coating it on. Based on these results, the coated feed was excluded from the digestibility experiment. Unfortunately, insufficient amounts of faeces were available to run replicate nutrient analyses. The accordance between the fat analyses of faeces from the three tanks within each treatment was low ŽCV s 16–33., while fat analyses of feed had high accuracy ŽCV s 0.3–1.6.. Based on the present data it cannot be concluded whether the high variation in fat content of faeces is a general picture of the variation of ADC of fat, or if we might have some analytical problems. Recovery of markers, the analyzed amount in percentage of the formulated, may be an indication of accuracy in the inclusion of markers in the feed or in the analysis of the markers. Recoveries of the markers less than 100% might be caused by the hygroscopic properties of the oxides. General recovery of markers were lower in the coated feed than in the moist feed. This may be due to the fact that coated fat and markers had a tendency to stick to the mixer and holding containers. The different markers used in the experiments produced similar ADC estimates. Concerns about the suitability of Cr2 O 3 as a marker for digestibility studies have, however, been raised ŽRingø, 1993; Riche et al., 1995.. La 2 O 3 and Y2 O 3 can be homogeneously included and accurately detected at levels that are about 1r100 of the level of Cr2 O 3 commonly applied, and they do not colour the feed. We therefore recommend these markers in digestibility studies instead of the more commonly used Cr2 O 3 , which still was recommended by Ng and Wilson Ž1997.. The ADCs in our study ŽTable 4. seem to be in the same range as reported for Atlantic salmon fed comparable feeds and faecal matter collected by stripping ŽJohnsen
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et al., 1993; Arnesen et al., 1995; Einen and Roem, 1997; Storebakken et al., 1998a., though somewhat lower than the ADC of N presented by Storebakken et al. Ž1998b.. The ADCs of P Ž26–35%. are in the lower range of what was found in other studies with fish meal based feeds for salmonids ŽLovell, 1989; Riche and Brown, 1996. and demonstrate that the digestibility of P from fish meal based modern feeds may be low. Due to similar conditions in the two experiments the ADC results were expected to be similar. The difference in body size from 0.5 kg in Exp. 1 to 1.0 kg in Exp. 2 was not presumed to affect the ADC, as other experiments have shown no differences in ADC of macronutrients between body sizes ŽCho and Slinger, 1979; Torrissen and Shearer, 1992.. Nevertheless, both the N and the P estimates of ADC were higher in Exp. 2 than in Exp. 1 ŽTable 5.. The average differences between moist and dry feed were at the same level in Exp. 1 as in Exp. 2, indicating that the Cr2 O 3 itself did not affect the digestibility. The slightly lowered ADCs in the moist feed might have been affected by the inclusion of alginate. Storebakken and Austreng Ž1987. reported lower ADCs of N, fat and ash when 5% alginate was added to rainbow trout feeds, and results reported by Storebakken Ž1985. indicate that alginate levels as low as 2% may depress the digestibility of fat and nitrogen. On the other hand, Ravndal and Austreng Ž1992. did not find any adverse effects on digestibility of N and fat in Atlantic salmon, by using alginate inclusion levels up to 9%. However, the power of their test was low, as only two replicates were employed. Process technology, water inclusion level and feed consistency may have an effect on the ADC measurements, and these factors should be further investigated. Correction for assumed indigestible alginate produced slightly higher ADC estimates of the moist feed, making the difference in ADC between dry feed and moist feed minor. The digestibility of carbohydrate, fat and protein in practical feeds may vary considerably. Starch digestibility in salmonids is affected by carbohydrate source ŽArnesen et al., 1995; Storebakken et al., 1998a. and inclusion level ŽAksnes, 1995; Arnesen et al., 1995; Hemre et al., 1995; Grisdale-Helland and Helland, 1997.. Native starches are associated with low starch ADCs Ž10–40%., while heat treated starches can be highly digestible. Hemre et al. Ž1995. found a starch ADC above 90% in Atlantic salmon fed a diet with 9% extruded wheat. Fat digestibility is also known to vary in a broad range, fat source being one of the influencing factors. Fat ADC down to 46% was reported by Austreng et al. Ž1979. in rainbow trout fed a fish meal feed with hydrogenatedrhigh melting point capelin oil, while ADCs as high as 98% were reported by Grisdale-Helland and Helland Ž1997. in Atlantic salmon fed fish meal based feeds with capelin oil Žimmediate faeces collection from outlet water.. ADC of fat is also affected by the inclusion of other ingredients in the feed, such as the carbohydrate source and level ŽBaeverfjord, 1992; Aksnes, 1995; Hemre et al., 1995; Grisdale-Helland and Helland, 1997; Storebakken et al., 1998a.. Concerning the variation in protein digestibility, Baeverfjord Ž1992. found a protein ADC as low as 47% in rainbow trout fed a fish meal based feed with insoluble and soluble fibres Ž20% a-cellulose and 5% guargum., while Hemre et al. Ž1995. measured protein ADCs of 93–95% when Atlantic salmon were fed a 10:1 mix of cusk fillet and squid. The current declaration system is suitable for high energy feeds based on high quality fish meal, as the feed used in the present experiment, and most of the commercial
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salmon feeds available on the market today. However, when other ingredients and feed formula are employed, the fixed digestibility coefficients will give erroneous digestibility estimates. This can be illustrated using the results from Aksnes Ž1995., in which two fish meal based feeds were fed to Atlantic salmon. The ADC of energy was measured to 93% in a low carbohydrate feed Ž2.4% inclusion. and 81% in a high carbohydrate feed Ž23% inclusion.. With fixed coefficients of 87, 90, and 65% for protein, fat and carbohydrate, respectively, the ADC of energy would be estimated to 88 and 85% in the low and high carbohydrate feed, respectively. This means a calculated difference of only 3% units between the two feeds, while the measured difference was 12% units. The fish farmers may experience corresponding effects on feed conversion and economy. Concerning possible impacts from fish farming on the environment, the outlet of energy is almost three times higher from the high carbohydrate feed, thus three times higher oxygen demand for degrading the OM in the recipient is required. The low and high carbohydrate feeds would liberate 175 g kgy1 and 313 g kgy1 faecal DM to the effluent water, respectively. Therefore, the high carbohydrate feed would result in the release of twice the amount of faecal DM per kg feed eaten. These great differences are not detected when the fixed coefficients are used. A test of a commercial feed should be conducted using the same procedure as in Exp. 2, as the precision of this method appears satisfactory. The Ferro-Nickel and the Cr2 O 3 used by Kabir et al. Ž1998. in feeds for rainbow trout, the Cr2 O 3 used by Fernandez et ´ al. Ž1998. in feeds for gilthead sea bream, and the Y2 O 3 used in feeds for rainbow trout by Sugiura et al. Ž1998a., produced ADC estimates with higher standard error of mean ŽS.E.M.. values than in the present study with Y2 O 3 . In studies with rainbow trout ŽAksnes et al., 1996. and Atlantic salmon ŽAksnes, 1995. similar or slightly lower S.E.M. were reported. Considering the same variance of the estimated ADCs as in Exp. 2 and a test power of 0.80, the minimum detectable differences between ADCs of N and energy would be 1.3 and 2.5% units, respectively. Even as small differences as these can represent large values for the fish farming industry, and efforts should be made to improve and fine-tune the method, either by increasing the number of replicates, or by decreasing the variation. In conclusion, the recommended moist feed method produced parallel but slightly lower ADCs Ž1.4% units for energy and 0.8% units for N. compared to the ADCs measured directly in the intact commercial feed with marker included. The method can be successfully used as a tool for improved declaration and re-examination of digestibility of commercial feeds. However, the method needs further development, and the factors causing the difference between ADC of dry and moist feed should be identified and eliminated. Further, a more accurate ADC determination of P is required. Acknowledgements The project was funded by the Norwegian Research Council as a Dr. Scient. grant ŽNo.103005r120. to Marie Hillestad, and by Institute of Aquaculture Research ŽAKVAFORSK.. Thanks to Asbjørn Bakken for technical assistance, and to Brit Seljebø for laboratory guidance. The authors are also grateful to Stormøllen AS for production of experimental feeds.
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References Aksnes, A., 1995. Growth, feed efficiency and slaughter quality of salmon, Salmo salar L., given feeds with different ratios of carbohydrate and protein. Aquacult. Nutr. 1, 241–248. Aksnes, A., Hjertnes, T., Opstvedt, J., 1996. Comparison of two assay methods for determination of nutrient and energy digestibility in fish. Aquaculture 140, 343–359. ˚ Sundby, A., 1995. Nutrient digestibilities, weight gain and plasma liver levels of Arnesen, P., Krogdahl, A., carbohydrate in Atlantic salmon Ž Salmo salar, L.. fed diets containing oats and maize. Aquacult. Nutr. 1, 151–158. Atkinson, J.L., Hilton, J.W., Slinger, S.J., 1984. Evaluation of acid-insoluble ash as an indicator of feed digestibility in rainbow trout Ž Salmo gairdneri .. Can. J. Fish. Aquat. Sci. 41, 1384–1386. Austreng, E., 1978. Digestibility determination in fish using chromium oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265–272. ˚ 1979. Effect of dietary fat source on the digestibility of fat and fatty Austreng, E., Skrede, A., Eldegard, A., acids in rainbow trout and mink. Acta Agric. Scand. 29, 119–126. Baeverfjord, G., 1992. Digestible and indigestible carbohydrates in rainbow trout diets: Effects on growth, digestibility, pancreatic hormones, liver glycogen deposition and liver morphology. Dr. Scient.-thesis, Norwegian College of Veterinary Medicine, Oslo, Norway, p. 117. Bergot, F., Breque, J., 1983. Digestibility of starch by rainbow trout: effects of the physical state and of the intake level. Aquaculture 34, 203–212. Bjerkeng, B., Følling, M., Lagocki, S., Storebakken, T., Olli, J.J., Alsted, N., 1997. Bioavailability of all E-astaxanthin and Z-isomers of astaxanthin in rainbow trout Ž Oncorhynchus mykiss .. Aquaculture 157, 63–82. Blincoe, C., Theisen, M.O., Stoddard-Gilbert, K., 1987. Sample oxidation procedures for the determination of chromium and nickel in biological material. Comm. Soil. Sci. Plant. Anal. 18, 687–692. Bowen, S.H., 1978. Chromic oxide in assimilation studies — a caution. Trans. Am. Fish. Soc. 107, 755–756. Bowen, S.H., 1981. Digestion and assimilation of periphytical detrital aggregate by Tilapia mossambica. Trans. Am. Fish. Soc. 110, 239–245. Buddington, R.K., 1980. Hydrolysis-resistant organic matter as a reference for measurement of fish digestive efficiency. Trans. Am. Fish. Soc. 109, 653–656. Cho, C.Y., Slinger, S.J., 1979. Apparent digestibility measurement in feedstuffs for rainbow trout. In: Halver, J.H., Tiews, K. ŽEds.., Finfish Nutrition and Fishfeed Technology, Vol. II. Heeneman, Berlin, pp. 239–247. Choubert, G., De la Noue, ¨ J., Luquet, P., 1979. Continuous quantitative automatic collector for fish feces. Prog Fish-Cult. 41, 64–67. Dabrowski, K., Dabrowska, H., 1981. Digestion of protein by rainbow trout Ž Salmo gairdneri Rich.. and absorption of amino acids within the alimentary tract. Comp. Biochem. Physiol. 69A, 99–111. De Silva, S.S., Perera, M.K., 1983. Digestibility of an aquatic macrophyte by the cicchlid Etroplus suratersis ŽBloch. with observations on the relative merits of three indigenous markers and daily changes in protein digestibility. J. Fish Biol. 23, 675–684. Einen, O., Roem, A.J., 1997. Dietary proteinrenergy ratios for Atlantic salmon in relation to fish size: growth, feed utilization and slaughter quality. Aquacult. Nutr. 3, 115–126. Fagbenro, O., Jauncey, K., 1995. Water stability, nutrient leaching and nutritional properties of moist fermented fish silage diets. Aquacult. Eng. 14, 143–153. Fernandez, F., Miguel, A.G., Guinea, J., Martınez, R., 1998. Digestion and digestibility in gilthead sea bream ´ ´ Ž Sparus aurata.: the effect of diet composition and ration size. Aquaculture 166, 67–84. Grisdale-Helland, B., Helland, S.J., 1997. Replacement of protein by fat and carbohydrate in diets for Atlantic salmon Ž Salmo salar . at the end of the freshwater stage. Aquaculture 152, 167–180. Helland, S.J., Grisdale-Helland, B., Nerland, S., 1996. A simple method for the measurement of daily feed intake of groups of fish in tanks. Aquaculture 139, 157–163. Hemre, G.-I., Sandnes, K., Lie, Ø., Torrissen, O., Waagbø, R., 1995. Carbohydrate nutrition in Atlantic salmon, Salmo salar L.: growth and feed utilization. Aquacult. Res. 26, 149–154. Hillestad, M., Olafsen, T., 1990. Nar ˚ spiser syk fisk? Opptak og effekt av medisinfor ˆ hos vibriosesmittet regnburørret ŽWhen do diseased fish eat? Uptake and effect of medical feed by vibriosis infected rainbow
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trout.. Cand. Agric. Thesis. AKVAFORSK, Dept. Animal Sci., Norw. Univ. Agric., Norway, 62 pp. ŽIn Norwegian.. Hung, S.S.O., Berge, G.M., Storebakken, T., 1997. Growth and digestibility effects of soya lecithin and choline chloride on juvenile Atlantic salmon. Aquacult. Nutr. 3, 141–144. Johnsen, F., Hillestad, M., Austreng, E., 1993. High energy diets for Atlantic salmon, effects on pollution. In: Kaushik, S.J., Luquet, P. ŽEds.., Fish Nutrition in Practice. Proceedings from the Fourth International Symposium on Fish Nutrition and Feeding. INRA, St. Pee-sur-Neville, France, pp. 391–401. Kabir, N.M.J., Wee, K. L, Maguire, G., 1998. Estimation of apparent digestibility coefficients in rainbow trout Ž Oncorhynchus mykiss . using different markers: 1. Validation of microtracer F-Ni as marker. Aquaculture 167, 259–272. Kotb, A.R., Luckey, T.D., 1972. Markers in nutrition. Nutr. Abstr. Rev. 42, 814–845. Lovell, T., 1989. Nutrition and Feeding of Fish. AVI, New York, NY. Meland, A.K., 1991. Fargepreferanse pa˚ for: ˆ laks og regnbueørret tilbudt for ˆ med forskjellig farge ŽColour preference in diets: Atlantic salmon and rainbow trout offered diets with different colour.. Cand. Agric. AKVAFORSK, Thesis. Dept. Animal Sci., Norw. Univ. Agric., Norway, 41 pp. Žin Norwegian.. Morales, A.E., Cardenete, G., De la Higuera, M., Sanz, A., 1994. Effects of dietary protein source on growth, feed conversion and energy utilization in rainbow trout, Oncorhynchus mykiss. Aquaculture 124, 117–126. Ng, W.-K., Wilson, R.P., 1997. Chromic oxide inclusion in the diet does not affect glucose utilization or chromium retention by channel catfish, Ictalurus punctatus. J. Nutr. 127, 2357–2362. Ravndal, J., Austreng, E., 1992. Rubin-for ˆ til oppdrettsfisk-delrapport I. ŽRubin feed for farmed fish. Report no. 302r8. Rubin, Trondheim, Norway, 6 pp. Žin Norwegian.. Refstie, S., Helland, S.J., Storebakken, T., 1997. Adaption to soybean meal diets for rainbow trout, Oncorhynchus mykiss. Aquaculture 153, 263–272. Refstie, S., Storebakken, T., Roem, A.J., 1998. Feed consumption and conversion in Atlantic salmon Ž Salmo salar . fed diets with fish meal, extracted soybean meal or soybean meal with reduced content of oligosaccharides, trypsin inhibitors, lectins and soya antigens. Aquaculture 162, 301–312. Riche, M., Brown, P.B., 1996. Availability of phosphorus from feedstuffs fed to rainbow trout, Oncorhynchus mykiss. Aquaculture 142, 269–282. Riche, M., White, M.R., Brown, P.B., 1995. Barium carbonate as an alternative indicator to chromic oxide for use in digestibility experiments with rainbow trout. Nutr. Res. 15, 1323–1331. Ringø, E., 1993. Does chromic oxide ŽCr2 O 3 . affect faecal lipid and intestinal bacterial flora in Arctic charr, SalÕelinus alpinus ŽL..?. Aquacult. Fish. Manage. 24, 767–776. Searcy-Bernal, R., 1994. Statistical power and aquaculture research. Aquaculture 127, 371–388. Shiau, S.-Y., Liang, H.-S., 1995. Carbohydrate utilization and digestibility by Tilapia, Oreochromis niloticus = O. aureus, are affected by chromic oxide inclusion in the diet. J. Nutr. 125, 976–983. Storebakken, T., 1985. Binders in fish feeds: I. Effect of alginate and guargum on growth, digestibility, feed intake and passage through the gastrointestinal tract of rainbow trout. Aquaculture 47, 11–26. Storebakken, T., Austreng, E., 1987. Binders in fish feeds: II. Effect of different alginates on the digestibility of macronutrients in rainbow trout. Aquaculture 60, 121–131. Storebakken, T., Shearer, K.D., Refstie, S., Lagocki, S., McCool, J., 1998a. Interactions between salinity, dietary carbohydrate source and carbohydrate concentration on the digestibility of macronutrients and energy in rainbow trout Ž Oncorhynchus mykiss .. Aquaculture 163, 347–359. Storebakken, T., Kvien, I.S., Shearer, K.D., Grisdale-Helland, B., Helland, S., Berge, G.M., 1998b. The apparent digestibility of diets containing fish meal, soybean meal or bacterial meal fed to Atlantic salmon Ž Salmo salar .: evaluation of different faecal collection methods. Aquaculture 169, 195–210. Sugiura, S.H., Dong, F.M., Hardy, R.W., 1998a. Effects of dietary supplements on the availability of minerals in fish meal; preliminary observations. Aquaculture 160, 283–303. Sugiura, S.H., Dong, F.M., Rathbone, C.K., Hardy, R.W., 1998b. Apparent protein digestibility and mineral availability in various feed ingredients for salmonid feed. Aquaculture 159, 177–202. Tacon, A.G.J., Rodrigues, A.M.P., 1984. Comparison of chromic oxide, crude fiber, polyethylene and acid-insoluble ash as dietary markers for the estimation of apparent digestibility coefficients in rainbow trout. Aquaculture 43, 391–399. Torrissen, K.R., Shearer, K.D., 1992. Protein digestion, growth and food conversion in Atlantic salmon and Arctic charr with different trypsin-like isozyme patterns. J. Fish Biol. 41, 409–415.
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Weatherup, R.N., McCracken, K.J., 1998. Comparison of estimates of digestibility of two diets for rainbow trout, Oncorhynchus mykiss ŽWalbaum., using two markers and two methods of faeces collection. Aquacult. Res. 29, 527–533. Williams, C.H., Davis, D.J., Iisma, O., 1962. The determination of chromic oxide in faeces samples by atomic absorbtion spectrophotometry. J. Agric. Sci. 59, 381–386. Winther, H., 1991. Nye indikatorer til bruk i fordøyelighets-, appetitt-og preferanseforsøk hos fisk ŽNew markers for experiments on digestibility, appetite and preference in fish.. Cand. Agric. Thesis. AKVAFORSK, Dept. Animal Sci., Norw. Univ. Agric., Norway, 50 pp. Žin Norwegian..
Determination of digestibility of commercial salmon feeds ˚ ˚ ) , Gerd Marit Berge Marie Hillestad, Torbjørn Asgard AKVAFORSK, Institute of Aquaculture Research AS N-6600 Sunndalsøra, Norway
Abstract The aim of the study was to develop an applicable method for determination of the digestibility of nutrients in commercial feeds. Leaching of markers and dry matter ŽDM. were compared between a commercial extruded feed containing La 2 O 3 , the same commercial feed coated with oil and other additional markers, and the commercial feed that had been ground and re-pelleted with water, marker and a crude alginate binder. Two markers, Y2 O 3 and Cr2 O 3 , were added in both the coated and the moist feeds. The optimal inclusion level of binder and water to minimize the leaching of DM from the moist pellet was determined in a preliminary study. The lowest leaching of DM was achieved with the inclusion of 2% alginate and 35% water. Added markers tended to be less homogeneously included in the coated feed than in the moist feed, and the markers leached relatively more than the DM from the coated feed. Apparent digestibility coefficients ŽADCs. of DM, fat, nitrogen ŽN., energy, organic matter ŽOM., and phosphorus ŽP. in the commercial dry feed and the moist feed were compared in two experiments with Atlantic salmon. Differences between the ADCs in the two experiments were observed for N and P. The ADC estimates of the moist feed were slightly lower than those of the dry feed for all nutrients. Similar ADC estimates for each nutrient were obtained for all markers, except for Y2 O 3 which produced a slightly higher ADC for N than did La 2 O 3. By using the moist feed method recommended from this study, a difference of about 1.4% units in ADC of energy and of 0.8% units for N between the original and the modified test feed was demonstrated. This means the error of ADC estimates is low compared to using fixed digestibility coefficients for each of protein, fat and carbohydrate, a method that has been employed for declaration of commercial fish feed. Further development of the method is discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Digestibility; Nitrogen; Energy; Phosphorus; Marker; Commercial feeds; Effluents
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Corresponding author: Tel.: q47-71-69-53-00; fax: q47-71-695301; E-mail: [email protected] 0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 1 5 4 - 4
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1. Introduction Commercial fish feeds are usually provided with a declaration of total digestible energy. Many feed manufacturers base the declaration on measured proximate composition and fixed digestibility coefficients for protein, fat and starch. Coefficients commonly employed in Norway have been 87, 90, and 65% for protein, fat and carbohydrate, respectively Žconsensus in the Norwegian Fish Feed Producers Association.. Because the digestibility may vary considerably, depending on feed ingredients, inclusion levels, processing conditions and nutrient interactions, the use of fixed digestibility values can be misleading. Feeds that are equal in proximate composition may have up to 10% units difference in energy digestibility due to the use of different fat sources ŽAustreng et al., 1979., different protein sources ŽMorales et al., 1994. or different carbohydrate sources ŽStorebakken et al., 1998a.. Carbohydrate levels in otherwise equal feeds can result in digestibility differences of the same extent ŽBergot and Breque, 1983; Aksnes, 1995.. However, no standard method for determination, verification or invalidation of the digestibility of the nutrients in commercial feeds is available. The digestibility level of the feed may have both economical and ecological consequences. The nutrients of environmental concern are nitrogen ŽN., phosphorus ŽP. and organic matter ŽOM.. The oxygen consumption required for degradation of OM depends largely on its energy content. With respect to the digestibility differences mentioned above, the energy load, through faeces, from a feed with an energy digestibility of 80% will be twice that of a feed with an energy digestibility of 90%. Correspondingly, knowledge of the P and N digestibility of the feeds is of crucial importance when modelling discharge to the environment. Due to the problems described above the nutrient digestibility of commercial feeds should be declared based on measurements. To do so, we need reproducible methods and standardized conditions. The direct method for determination of digestibility by exact measurement of feed intake and complete collection of excreta has been suggested by Choubert et al. Ž1979.. But there are important limitations with the faeces collection in water for nutrients easily dissolving. Careful stripping produces digestibility estimates close to the dissection method ŽStorebakken et al., 1998b., and stripping can also be employed in field studies or in tanks without special collection equipment, in contrast to other methods where the faecal matter is collected from the water. However, the stripping method requires indigestible markers in the feed for estimation of the digestibility. Naturally occurring indigestible fractions such as acid insoluble ash ŽBowen, 1981; Atkinson et al., 1984., fibre ŽTacon and Rodrigues, 1984., cellulose ŽBuddington, 1980. and hydrolysis resistant matter ŽBuddington, 1980; De Silva and Perera, 1983. have been evaluated as markers for digestibility determination. These internal markers have been of particular interest when the aim was to test a feed with no markers added, as e.g., natural food or commercial feeds. The naturally occurring level of these markers is, however, low in commercial fish feeds, relative to the amount required for analysis, thus making them unsuitable as test markers. The indirect method using Cr2 O 3 as an inert marker in salmonid feed ŽAustreng, 1978. has been commonly used. However, analytical variability and poor recovery of the Cr2 O 3 have been reported ŽKotb and Luckey, 1972; Dabrowski and Dabrowska, 1981; Blincoe et al., 1987., and several
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studies reveal digestive or metabolic effects of Cr2 O 3 , which in turn might affect the nutrient digestibility measurements ŽBowen, 1978; Dabrowski and Dabrowska, 1981; Ringø, 1993; Shiau and Liang, 1995; Sugiura et al., 1998a.. Further, Cr2 O 3 changes the appearance and possibly the immediate acceptability of the feed. Concerns have therefore been raised about the use of Cr2 O 3 in digestibility studies ŽRingø, 1993; Riche et al., 1995.. Ng and Wilson Ž1997. studied some of the these problems and did not find negative effects of Cr2 O 3 . Alternatives to Cr2 O 3 as added inert marker have successfully been used in digestibility studies of fish feeds, like oxides of Y ŽBjerkeng et al., 1997; Hung et al., 1997; Refstie et al., 1997; Sugiura et al., 1998a,b; Storebakken et al., 1998a., Yb ŽRefstie et al., 1998., Dy, Sc, La ŽHillestad and Olafsen, 1990; Meland, 1991; Winther, 1991. and Ti ŽWeatherup and McCracken, 1998.. Recently, the microtracer Ferro-Nickel was reported a promising marker by Kabir et al. Ž1998.. The aim of the present study was to find an applicable method for digestibility assessment of commercial salmon feeds. The first step was to select a method of adding marker to the feed. Two methods were tested: either by grinding the feed and mixing it with the marker before preparing a moist feed, or by coating the intact feed with the marker. Homogeneity of markers in the feeds and leaching to the water of markers and nutrients were compared. Inclusion of binder and water in the moist feed was optimized in a preliminary study. Finally, the apparent digestibility of the macronutrients and P in the intact commercial feed and the moist feed were compared in an experiment with Atlantic salmon. 2. Materials and methods 2.1. Sinking speed and loss of dry matter (DM), preliminary studies Moist feed prepared from a commercial dry feed was optimized for low DM leaching in a preliminary study. A feed produced according to the process of commercial extruded feed production, containing 46% protein and 30% fat, was used for the experiment. Nine experimental moist feeds were produced by adding 30, 35 or 40% water to each of 3 batches Žeach 5 kg. of ground extruded basic feed containing either 1, 2 or 3% crude alginate ŽAlgibinde Algea AS, Oslo, Norway. as a binder, in a 25-kg blender. After mixing for 5 min, each feed was pressed through a mincer equipped with a 5 mm matrix. The feeds were then frozen Žy208C. and later broken into pellets. Sinking speed of the basic feed pellets and pellets of the nine moist feeds were measured by using a 200 cm high = 30 cm diameter cylinder filled with 108C freshwater. Timing started 10 cm below the water surface and lasted during the next 150 cm of sinking. Leaching of DM was measured by dipping 100 g of each thawed feed in a sieve for 10 min in 208C seawater with a salinity of 33 g ly1 . Each sample was analyzed for DM immediately, and results were not corrected for uptake of salt from the water. 2.2. Experimental feeds The 6 mm extruded basic feed ŽBasic. was produced by Stormøllen ŽVaksdal, Norway.. The feed contained Norse-LT 94TM and NorSeaMinkTM fish meal, fish oil,
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wheat, fish protein concentrate, shrimp peel meal and a vitaminrmineral premix. The feed contained 24, 282, 490, 103 and 15.3 g kgy1 of water, fat, protein, ash and P, respectively, and 23.9 MJ energy kgy1 . An even distribution of the marker, La 2 O 3 , was ensured by manually mixing 125 g La 2 O 3 with 3.5 kg of the dry premix before the batch Ž750 kg. was blended Ž5 min. and further dry-extruded. A feed coated with marker ŽCoated. was made from a 10-kg batch of the basic feed by coating with 10,000 mg Cr2 O 3 kgy1 , 100 mg Y2 O 3 kgy1 , and 2% fish oil ŽTable 1.. The Y2 O 3 was mixed with half of the oil at 258C and added to the mixture in a narrow stream into a bakers blender during a 1 min run. The Cr2 O 3 was sieved into the mixture and the blending continued for 2 min. Then the rest of the oil was added and the mixture blended for another 2 min. A moist feed ŽMoist. was made by grinding 10 kg of the basic feed in a coffee grinder and adding 2% AlgibindTM , 10,000 mg Cr2 O 3 kgy1 , and 100 mg Y2 O 3 kgy1 . An even distribution of the markers was ensured by first mixing the Y2 O 3 with a small amount of the ground feed, then with a larger amount before it was added to the total batch. The Cr2 O 3 was diluted in the same way, but only in one step, before being added to the total batch. Then 3.5 l of water was added and the blending was proceeded for 5 min before running the batch through a mincer with a 5 mm matrix. Three samples Ž15–20 g. from each of the experimental feeds were drawn and stored at y208C. Moist feed was freeze dried and all feeds were analyzed for marker, P content and proximate composition. All experimental feeds were stored at y208C. 2.3. Leaching of markers and DM A square tank Ž1 m2 . with 1.2 m water depth Žsalinity 33 g ly1 . and a water exchange of 15 l per min was employed for the leaching test. A pre-weighed amount of thawed feed was dropped into the water and collected from the outlet in a wire mesh collector ŽHelland et al., 1996.. The average time spent in water was calculated as the time that elapsed from when pellets were dropped to the average between when the first and the last pellet were collected from the outlet. Samples were frozen Žy208C. before
Table 1 Ingredients addeda to feeds used in digestibility studies Feed
Basic
Coated
Moist
Experiment
1 and 2
1
1
2
La 2 O 3 Žmg kg y1 . Y2 O 3 Žmg kg y1 . Cr2 O 3 Žmg kg y1 . Water Ž%. Alginateb Ž%. Capelin oil Ž%.
166 – – – – –
161 100 10,000 – – 2.0
164 100 10,000 35 2 –
164 100 – 35 2 –
a
Added per kg of basic feed. Algibinde ŽAlgea, Oslo, Norway..
b
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analysis of DM and markers. DM results were not corrected for uptake of salt from the water. 2.4. Apparent digestibility Two digestibility trials were conducted with the basic feed and the moist feed. The coated feed was excluded based on the results from the preliminary studies on leaching. In Experiment 1 ŽExp. 1. a moist feed with both Y2 O 3 and Cr2 O 3 was used, while Cr2 O 3 was excluded from the moist feed in Experiment 2 ŽExp. 2., due to concerns about confounding effects on the digestibility of adding Cr2 O 3 and alginate to the diet. 2.4.1. Exp. 1 Thirty Atlantic salmon Ž Salmo salar L.., with an initial average weight of 540 g, were in August 1996 randomly distributed to each of six tanks Ž1 m2 . supplied with 15 l per min of seawater Ž8.1–8.98C and salinity 33 g ly1 .. The two experimental feeds, Basic and Moist, were thawed and randomly fed to fish in three tanks each. Feed ration was kept approximately 10% above satiation. Feed intake was monitored by collecting all feed waste from the tanks as described by Helland et al. Ž1996.. Faeces were stripped from all fish after 8 and 15 days, and samples were pooled within tank and frozen at y208C. The amount of faecal samples from day 15 was sufficient for all chemical analyses which were carried out after freeze drying. 2.4.2. Exp. 2 Twenty Atlantic salmon with an initial average weight of 1.0 kg were kept in the same experimental set-up as in Exp. 1 ŽJune 1997., with water temperature 6.8–7.08C. Faeces were collected by stripping after 7 and 12 days. As in Exp. 1, samples from the second stripping were used for chemical analyses. 2.5. Analyses and calculations Feeds and faeces were analyzed for DM Ž1058C, 16–18 h., ash Žcombusted at 5508C, 16–18 h., nitrogen Žsemi-micro-Kjeldahl, Kjeltec-Auto System, Tecator, Hoganes, ¨ ¨ Sweden., fat Ždiethyl ether extraction in a Fosstec ŽTecator. analyser after HCl-hydrolysis. and gross energy ŽParr 1271 Bomb calorimeter, Parr, Moline, IL, USA.. Y2 O 3 , La 2 O 3 and P were analyzed according to Refstie et al. Ž1997.. Cr2 O 3 was analyzed according to Williams et al. Ž1962.. Organic matter ŽOM. was calculated as the DM y ash difference. ADCs were calculated as ADC n s 100 y Ž100 = feed i Ž%.rfaeces i Ž%. = faeces nŽ%.rfeed nŽ%.., where subscript i means indicator Žmarker. and n means nutrient concentrations. ADCs of DM and ash were calculated from the original content in feed and faeces exclusive of the added marker. ADC estimates were corrected for content of alginate in feed and faeces, assuming the alginate is completely indigestible and containing 89.0, 0.8, 35.0 and 0.0% of DM, N, ash and fat, respectively ŽAlgea, Oslo, Norway..
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Two-way ANOVA was employed for testing the effect of the inclusion level of water and alginate and their interaction on sinking speed and DM loss of the moist feed in the preliminary study. One way ANOVA and Duncan’s multiple rank test was used for testing the difference between means of leaching of DM and markers from the basic, coated and moist feeds. Possible differences in ADCs obtained by Cr2 O 3 and La 2 O 3 were tested in Exp. 1 in a One-way ANOVA with digestibility data from the moist feed. After exclusion of the Cr2 O 3 measurements, the ADC data of moist feed from both experiments were merged in a two-way ANOVA for testing the possible differences obtained by La 2 O 3 and Y2 O 3 . Effect of feed and experiment and their interaction were tested in a two-way ANOVA by including only the ADCs measured by La 2 O 3 in the two feeds. In the preliminary study three observed DM analysis values made no sense and fell out of the 99% confidence interval. They were removed from the data set before statistical analyses. Statistical power was calculated according to Searcy-Bernal Ž1994., and minimum detectable difference between means were based on a power of at least 0.80. Statistical differences were indicated at a 5% level for all analyses.
3. Results 3.1. Sinking speed and loss of DM, preliminary study The dry feed sank approximately twice as fast Ž14.4 " 0.2 cm sy1 Žmean " S.E.M... as the moist feed Ž7.8 " 0.1.. Sinking speed of the moist feed decreased with increasing content of alginate and increased with increasing inclusion of water. A significant interaction between the two main effects on sinking speed was observed. Loss of DM was lower with the feeds containing 2% alginate compared to the feeds containing 3% alginate, and lowest for the feeds containing 35% water compared to the others Ž1.1% " 0.1.. If corrected for assumed uptake of salt Ž33 g ly1 water. the estimated losses of DM would have been approximately 0.4% units higher. 3.2. Marker homogeneity and leaching of markers and DM The coefficient of variation ŽCV. for marker content between feed samples ranged from 1 to 7 ŽTable 2.. The CV of the added markers tended to be higher in the coated than in dry and moist feed. The average recovery of La 2 O 3 , measured as the analyzed amount in percentage of the formulated, in all feeds was 86%. Recovery of Y2 O 3 in coated feed was 79% and in moist feed 84 and 93% in Exp. 1 and Exp. 2, respectively. Recovery of Cr2 O 3 was 84% in coated feed and 88% in moist feed. The leaching of the added markers were higher from the coated feed than from the moist feed Ž P s 0.01, 0.07 and 0.08, respectively, for Cr2 O 3 , Y2 O 3 and La 2 O 3 .. From the coated feed the leaching of added markers were 10-fold higher than the leaching of DM ŽTable 3.. If corrected for assumed uptake of salt the estimated leaching of DM would have been approximately 0.6% units higher. Time elapsed from when the feed left the automatic
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Table 2 Marker content of extruded basic feed, feed coated with oil and marker, and freeze dried moist feed Ž ns 3. in Experiment 1 Feed
Basic
Coated
Marker
Average
CV
La 2 O 3 Žmg kgy1 . Y2 O 3 Žmg kgy1 . Cr2 O 3 Žmg kgy1 .
141 – –
2.3 – –
a
a
Moist
Average
CV
Average
CV
138 76 8,069
1.1 6.8 5.1
136 81 8,500
2.5 3.8 3.5
CVs coefficient of variance between 3 samples.
feeder until it was collected in the outlet tended to be longer for moist feed than for the basic and coated feeds Ž P s 0.11.. 3.3. ADC Feed intake by the end of both experiments was acceptable Žin average 8 g kgy1 fish dayy1 .. Mean ADCs are presented in Table 4, while the differences between means and their significance within each model are presented in Table 5. The ADCs of dry feed were significantly higher than those of moist feed for all parameters measured. The greatest difference between feeds was measured in ADCs of ash Ž10.3% units. and P Ž7.6% units.. The differences in ADCs between dry and moist feed were at the same level in Exp. 1 and Exp. 2. If corrected for content of alginate in feed and faeces, assuming the alginate is completely indigestible, the mean ADC estimates of DM, energy and N in the Moist feed obtained by Y2 O 3 would be 1.4, 0.8 and 0.2% units higher than the ADCs shown in Table 4, as an average for both experiments. Significant differences between the two experiments were revealed only for the ADCs of N and P, with 1.3 and 7.6% units lower ADCs, respectively, in Exp.1 than in Exp. 2. No significant differences between ADCs measured by the different markers were observed, except for the difference between ADC of N measured by La 2 O 3 and Y2 O 3 Ž P s 0.05.. Except for ash, the differences between ADCs measured by different markers were small Ž- 2.1% units.. The power of the test to unveil differences in ADCs
Table 3 Leaching of dry matter and markers Ž%. from different feeds during the time spent from automatic feeder to collection in effluent water Žmean"S.E.M., ns 3. Feed Dry matter La 2 O 3 Y2 O 3 Cr2 O 3 , Time elapsed Žs. a
Basic 0.5"0.6 y0.3"0.3 Aa – – 23.8"0.9
Coated
Moist a
1.4"0.3 a 5.1"2.3 Ba 18.2"6.4 b 24.4"3.7 Ab 26.2"2.4
0.9"0.2 1.9"0.7 AB 2.3"0.4 2.5"3.0 B 32.5"3.5
Different letters in the same row ŽAB. or in the same column Žab. denote significant difference Ž P - 0.05. between treatment means.
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Table 4 Apparent digestibility Ž"S.E.M.. of dry and moist feed in Atlantic salmon measured by different markers and repeated experiments Ž ns 3. Feed
Dry
Moist
Marker
La 2 O 3
La 2 O 3
Y2 O 3
Cr2 O 3
Experiment 1 Dry matter Fat Nitrogen Energy Organic matter Asha Phosphorus
69.5"0.5 94.6"0.5 83.9"0.2 84.2"0.5 77.5"0.4 2.3"1.2 25.7"0.2
65.8"0.5 89.7"2.0 82.7"0.4 81.0"0.7 74.7"0.6 y8.0"1.3 19.7"0.6
66.3"1.1 89.7"2.3 82.9"0.2 81.2"1.2 75.0"1.1 y5.0"1.6 20.8"1.6
66.7"0.2 90.0"1.9 83.1"0.3 81.5"0.7 75.3"0.3 y5.3"2.1 21.7"1.3
Experiment 2 Dry matter Fat Nitrogen Energy Organic matter Asha Phosphorus
70.3"0.4 92.0"0.9 85.5"0.4 83.9"0.2 77.8"0.4 3.0"0.7 34.9"0.5
65.7"0.9 90.6"0.9 83.7"0.1 81.4"0.8 74.4"0.8 y7.3"2.8 25.7"1.8
67.7"0.6 91.2"0.7 84.7"0.2 82.5"0.5 75.9"0.4 y0.8"2.7 30.2"1.2
a
Measured ash ADCs are affected by uptake of minerals from seawater.
between experiments was approximately 0.20 for other parameters than N and P. To unveil differences between La 2 O 3 and Y2 O 3 the power was between 0.70 and 0.90 for ash and P, and in the range 0.20–0.50 for the other nutrients. To unveil differences between Cr2 O 3 and Y2 O 3 the power was below 0.35.
Table 5 Differences Ž% units. between means of apparent digestibility of dry and moist feed in Atlantic salmon measured by different markers and repeated experiments
Dry matter Fat Nitrogen Energy Organic matter Ash Phosphorus a
Feed a
Experimentsa
Markers
Dry–moist feed
2–1
Y2 O 3 –La 2 O 3b
Cr2 O 3 –La 2 O 3c
4.2U 3.1U 1.5U 2.9U 3.1U 10.3U 7.6U
0.3 y0.8 1.3U 0.1 0.1 0.7 7.6U
1.3 0.3 0.6U 0.7 0.9 4.0 2.8
0.9 0.3 0.5 0.5 0.7 2.8 2.1
Data obtained by La were included in the analysis when comparing feeds and experiments Ž N s12.. Data from moist feed were included in the analysis when comparing Y and La Ž N s12.. c Data from moist feed in Exp. 1 were included in the analysis when comparing Cr and La Ž N s6.. U The contrasts with asterisk are significantly different Ž P - 0.05.. b
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4. Discussion In the preliminary study with moist feeds, the intermediate levels of alginate Ž2%. and water Ž35%. resulted in the lower loss of DM and the best feed consistency. Therefore, these levels of alginate and water were used in the later experiments. The leaching measured by immersion of pellets in a sieve in water for 10 min or by dropping the pellet into a tank and collecting it in the outlet, both gave a DM loss of about 1%, which is lower than Fagbenro and Jauncey Ž1995. found with extruded fish silage feeds. The thawing did not seem to cause any visible leaching or crumbling of the feed before feeding. The residence time in water, before pellets are eaten by Atlantic salmon in tanks, is most likely less than measured in the drop-and-collect experiment. Thus, the leaching before the pellets are eaten is anticipated to be less than 1%. The low sinking speed of the moist feed makes it well suited for experiments in shallow tanks, as the pellet will remain in the water column for a longer period, and easily be caught by the fish. On the other hand, when the residence time of the feed in water increases Žnot significantly different in our experiment. the leaching of nutrients and markers may also increase. However, if markers and nutrients are leaching at the same rate, the digestibility measurements will not be affected. The coated feed leached relatively more markers than DM, thus lowering the digestibility estimates. Our results also indicate that it is more difficult to add markers homogeneously by coating them on the pellet surface than by including them into a moist feed ŽTable 3.. Thus, adding an external marker into a commercial feed by mixing it into the feed seems to be advisable rather than coating it on. Based on these results, the coated feed was excluded from the digestibility experiment. Unfortunately, insufficient amounts of faeces were available to run replicate nutrient analyses. The accordance between the fat analyses of faeces from the three tanks within each treatment was low ŽCV s 16–33., while fat analyses of feed had high accuracy ŽCV s 0.3–1.6.. Based on the present data it cannot be concluded whether the high variation in fat content of faeces is a general picture of the variation of ADC of fat, or if we might have some analytical problems. Recovery of markers, the analyzed amount in percentage of the formulated, may be an indication of accuracy in the inclusion of markers in the feed or in the analysis of the markers. Recoveries of the markers less than 100% might be caused by the hygroscopic properties of the oxides. General recovery of markers were lower in the coated feed than in the moist feed. This may be due to the fact that coated fat and markers had a tendency to stick to the mixer and holding containers. The different markers used in the experiments produced similar ADC estimates. Concerns about the suitability of Cr2 O 3 as a marker for digestibility studies have, however, been raised ŽRingø, 1993; Riche et al., 1995.. La 2 O 3 and Y2 O 3 can be homogeneously included and accurately detected at levels that are about 1r100 of the level of Cr2 O 3 commonly applied, and they do not colour the feed. We therefore recommend these markers in digestibility studies instead of the more commonly used Cr2 O 3 , which still was recommended by Ng and Wilson Ž1997.. The ADCs in our study ŽTable 4. seem to be in the same range as reported for Atlantic salmon fed comparable feeds and faecal matter collected by stripping ŽJohnsen
90
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et al., 1993; Arnesen et al., 1995; Einen and Roem, 1997; Storebakken et al., 1998a., though somewhat lower than the ADC of N presented by Storebakken et al. Ž1998b.. The ADCs of P Ž26–35%. are in the lower range of what was found in other studies with fish meal based feeds for salmonids ŽLovell, 1989; Riche and Brown, 1996. and demonstrate that the digestibility of P from fish meal based modern feeds may be low. Due to similar conditions in the two experiments the ADC results were expected to be similar. The difference in body size from 0.5 kg in Exp. 1 to 1.0 kg in Exp. 2 was not presumed to affect the ADC, as other experiments have shown no differences in ADC of macronutrients between body sizes ŽCho and Slinger, 1979; Torrissen and Shearer, 1992.. Nevertheless, both the N and the P estimates of ADC were higher in Exp. 2 than in Exp. 1 ŽTable 5.. The average differences between moist and dry feed were at the same level in Exp. 1 as in Exp. 2, indicating that the Cr2 O 3 itself did not affect the digestibility. The slightly lowered ADCs in the moist feed might have been affected by the inclusion of alginate. Storebakken and Austreng Ž1987. reported lower ADCs of N, fat and ash when 5% alginate was added to rainbow trout feeds, and results reported by Storebakken Ž1985. indicate that alginate levels as low as 2% may depress the digestibility of fat and nitrogen. On the other hand, Ravndal and Austreng Ž1992. did not find any adverse effects on digestibility of N and fat in Atlantic salmon, by using alginate inclusion levels up to 9%. However, the power of their test was low, as only two replicates were employed. Process technology, water inclusion level and feed consistency may have an effect on the ADC measurements, and these factors should be further investigated. Correction for assumed indigestible alginate produced slightly higher ADC estimates of the moist feed, making the difference in ADC between dry feed and moist feed minor. The digestibility of carbohydrate, fat and protein in practical feeds may vary considerably. Starch digestibility in salmonids is affected by carbohydrate source ŽArnesen et al., 1995; Storebakken et al., 1998a. and inclusion level ŽAksnes, 1995; Arnesen et al., 1995; Hemre et al., 1995; Grisdale-Helland and Helland, 1997.. Native starches are associated with low starch ADCs Ž10–40%., while heat treated starches can be highly digestible. Hemre et al. Ž1995. found a starch ADC above 90% in Atlantic salmon fed a diet with 9% extruded wheat. Fat digestibility is also known to vary in a broad range, fat source being one of the influencing factors. Fat ADC down to 46% was reported by Austreng et al. Ž1979. in rainbow trout fed a fish meal feed with hydrogenatedrhigh melting point capelin oil, while ADCs as high as 98% were reported by Grisdale-Helland and Helland Ž1997. in Atlantic salmon fed fish meal based feeds with capelin oil Žimmediate faeces collection from outlet water.. ADC of fat is also affected by the inclusion of other ingredients in the feed, such as the carbohydrate source and level ŽBaeverfjord, 1992; Aksnes, 1995; Hemre et al., 1995; Grisdale-Helland and Helland, 1997; Storebakken et al., 1998a.. Concerning the variation in protein digestibility, Baeverfjord Ž1992. found a protein ADC as low as 47% in rainbow trout fed a fish meal based feed with insoluble and soluble fibres Ž20% a-cellulose and 5% guargum., while Hemre et al. Ž1995. measured protein ADCs of 93–95% when Atlantic salmon were fed a 10:1 mix of cusk fillet and squid. The current declaration system is suitable for high energy feeds based on high quality fish meal, as the feed used in the present experiment, and most of the commercial
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salmon feeds available on the market today. However, when other ingredients and feed formula are employed, the fixed digestibility coefficients will give erroneous digestibility estimates. This can be illustrated using the results from Aksnes Ž1995., in which two fish meal based feeds were fed to Atlantic salmon. The ADC of energy was measured to 93% in a low carbohydrate feed Ž2.4% inclusion. and 81% in a high carbohydrate feed Ž23% inclusion.. With fixed coefficients of 87, 90, and 65% for protein, fat and carbohydrate, respectively, the ADC of energy would be estimated to 88 and 85% in the low and high carbohydrate feed, respectively. This means a calculated difference of only 3% units between the two feeds, while the measured difference was 12% units. The fish farmers may experience corresponding effects on feed conversion and economy. Concerning possible impacts from fish farming on the environment, the outlet of energy is almost three times higher from the high carbohydrate feed, thus three times higher oxygen demand for degrading the OM in the recipient is required. The low and high carbohydrate feeds would liberate 175 g kgy1 and 313 g kgy1 faecal DM to the effluent water, respectively. Therefore, the high carbohydrate feed would result in the release of twice the amount of faecal DM per kg feed eaten. These great differences are not detected when the fixed coefficients are used. A test of a commercial feed should be conducted using the same procedure as in Exp. 2, as the precision of this method appears satisfactory. The Ferro-Nickel and the Cr2 O 3 used by Kabir et al. Ž1998. in feeds for rainbow trout, the Cr2 O 3 used by Fernandez et ´ al. Ž1998. in feeds for gilthead sea bream, and the Y2 O 3 used in feeds for rainbow trout by Sugiura et al. Ž1998a., produced ADC estimates with higher standard error of mean ŽS.E.M.. values than in the present study with Y2 O 3 . In studies with rainbow trout ŽAksnes et al., 1996. and Atlantic salmon ŽAksnes, 1995. similar or slightly lower S.E.M. were reported. Considering the same variance of the estimated ADCs as in Exp. 2 and a test power of 0.80, the minimum detectable differences between ADCs of N and energy would be 1.3 and 2.5% units, respectively. Even as small differences as these can represent large values for the fish farming industry, and efforts should be made to improve and fine-tune the method, either by increasing the number of replicates, or by decreasing the variation. In conclusion, the recommended moist feed method produced parallel but slightly lower ADCs Ž1.4% units for energy and 0.8% units for N. compared to the ADCs measured directly in the intact commercial feed with marker included. The method can be successfully used as a tool for improved declaration and re-examination of digestibility of commercial feeds. However, the method needs further development, and the factors causing the difference between ADC of dry and moist feed should be identified and eliminated. Further, a more accurate ADC determination of P is required. Acknowledgements The project was funded by the Norwegian Research Council as a Dr. Scient. grant ŽNo.103005r120. to Marie Hillestad, and by Institute of Aquaculture Research ŽAKVAFORSK.. Thanks to Asbjørn Bakken for technical assistance, and to Brit Seljebø for laboratory guidance. The authors are also grateful to Stormøllen AS for production of experimental feeds.
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Weatherup, R.N., McCracken, K.J., 1998. Comparison of estimates of digestibility of two diets for rainbow trout, Oncorhynchus mykiss ŽWalbaum., using two markers and two methods of faeces collection. Aquacult. Res. 29, 527–533. Williams, C.H., Davis, D.J., Iisma, O., 1962. The determination of chromic oxide in faeces samples by atomic absorbtion spectrophotometry. J. Agric. Sci. 59, 381–386. Winther, H., 1991. Nye indikatorer til bruk i fordøyelighets-, appetitt-og preferanseforsøk hos fisk ŽNew markers for experiments on digestibility, appetite and preference in fish.. Cand. Agric. Thesis. AKVAFORSK, Dept. Animal Sci., Norw. Univ. Agric., Norway, 50 pp. Žin Norwegian..