Aquaeultural Engineering 4 (1985) 33-49

Methodical Studies on Digestion in Trout. 1. Reliability of Digestion Coefficients in Relation to Methods for Faeces Collection B. Vens-Cappell*
Teichwirtschaftlicher Beispielsbetrieb, 8482 W611ershof, Bavaria, West Germany

ABSTRA CT Two methods or faeces collection were compared when studying digestion in trout (78-200g individual weight; 12-15C): (a) Faeces stripped from the lower intestine; (b) faecal pellets filtered continuously from water using an automated faeces collector. This apparatus consisted of a cylindrical tank with a funnel shaped bottom, from which the effluent water was drained over a rotating cloth filter, separating the faeeal pellets immediately from water. The digestion coefficients for energy and protein of a commercial feed and an experimental diet were determined applying both methods of faeces collection. Digestion coefficients for protein of the experimentally composed diet were 1-2.3% higher for the filtered faeces than for the stripped faeces (88.6-87.3%). The protein digestion coefficients for the commercial feed were about 5% higher when faeces were sampled by continued filtration. Results obtained with the stripp#zg method averaged 79.5%. The energy digestion coefficients followed a similar trend. It was concluded that the stripping method gives more reliable digestion coefficients.

1. INTRODUCTION Since the introduction of the chromium indicator m e t h o d into fish nutrition research (Nose, 1960) the problem of collecting representative unadulterated faeces samples has not yet been solved. A critical review 33 * Present address: Dorpsfelde 4, 2000 Hamburg, West Germany. Aquacultural Engineering 0144-8609/85/$03.30 Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain

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of methods for faeces collection has been given by Smith (1979). A fundamental difference exists between methods for faeces collection from water and those for sampling faeces directly from the lower intestine. Windell et al. (1978) removed faeces, which had settled in the tank, simply by using a hand net in intervals of 1-4 h. In the 'Guelph System' (Cho and Slinger, 1979) the effluent of the experimental fish tank passes a settling column, from which faeces are easily and quantitatively removed. French authors have tried to construct automatic faeces samplers with the purpose of minimizing the contact of released faeces with water (Possompes, 1973; Bergot, 1979; Choubert et al., 1982). However, leaching of faecal material into the water seems to be inevitable with all of the methods mentioned above, resulting in high digestion coefficients. This is the major reason why numerous authors have tried to avoid the contact of faeces with water by developing sophisticated methods. Among these, the most simple one is to strip the content of the lower intestine before its natural defaecation (Inaba et al., 1962; Nose, 1967; Possompes, 1973; Austreng, 1978; Windell et al., 1978; Cho and Slinger, 1979). Faeces collected by stripping may be contaminated by body slime, urine or sexual products. Therefore, faeces were also sampled by dissecting the fish and removing the faeces from a certain section of the intestine (Austreng, 1978; Windell et al., 1978) or by sucking the lower intestine (Windell et al., 1978). Objections to all these methods of direct faeces sampling from intestine relate to the fact that faecal material may be removed prior to the completion of the natural retention time, thereby reducing the digestion and absorption capacity and simulating poor digestibility. Hence, it follows that there is presently no method for faeces collection without any inherent errors. Digestion coefficients obtained with faeces collected from water have been consistently higher as compared with those stripped or dissected from the intestine. Differences between these two methods may exceed 15% of the observed digestibility (Windell et al., 1978). The question still remains unsolved which of the methods of faeces collection allows the least systematic error. The motivation to deal with the problems of faeces collection in this study was not at all a sense of methodical purism. The standardization of methods in digestion studies with fish is an indispensable precondition for the compilation of feed value tables with respect to the formulation of least cost diets using linear programming techniques (Vens-Cappell, 1983).

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The objective o f the study presented here was a new approach to the problem. For this reason, digestion coefficients for protein, energy and amino acids of different types of feeds were compared applying two methods of faeces collection: (a) stripping faeces from the lower intestine; (b) filtering faeces continually from water using a faeces sampling apparatus similar to that described by Bergot (1979). In addition the influence o f time of faeces collection on digestion coefficients was studied.

2. METHODS AND MATERIALS 2.1. Experimental

procedures

2.1.1. Faeces collection 2.1.1.1. S t r i p p i n g m e t h o d . Fish were anaesthetized with 0.5 g 1, 1, ltrichlorine-2-methyl-2-propanol litre -1 water, and adherent water was removed with a towel. While the right hand kept the head, forefinger and thumb of the left hand, embracing the body, were gently moved along the lateral line system towards the anus. By this method some urine could be pressed out, reducing the risk of later faeces contamination. Then, repeating the movement similarly, this time with pressure on the region between anal fin and the anus, a faeces column of 1-2.5 cm (corresponding to the content of the intestine between anus and anal fin) was stripped into a slant basin. Water and urine stripped with faeces were thus separated immediately from faeces and could be decanted easily. 2.1.1.2. C o n t i n u o u s filtration m e t h o d . For faeces collection by con-

tinual filtration, experimental fish were transferred into a cylindrical tank with a funnel shaped b o t t o m . Two tanks with capacities of 600 litres and 100 litres were used. Water flow rate through the tanks was kept between 10-15 litres min -1. A drain pipe was attached to the b o t t o m . A b o u t half of the effluent water passed this tube. It was drained over a sieve or a cloth filter. The remaining effluent volume was drained through a flow over pipe (Fig. 1). In the initial trial faecal pellets carried by the effluent water were caught by a sieve and picked up with tweezers immediately. As this method of faeces collection was

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10

tO

Fig. 1. Faeces collection apparatus. 1, Lid; 2, cylindrical tank, 100 litres and 600 litres, respectively; 3, over-flow pipe; 4, rotating cloth filter (600/am mesh size); 5, driving shaft; 6, electric motor with gearing; 7, elastic scraper; 8, basin for collecting faeces; 9, stand; 10, afflux.

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efficient with respect to a rapid separation of water and faeces but labour intensive, faeces filtration was automated as shown in Fig. 1. The effluent water was passed over a cloth filter of 600/am mesh size. This filter was rotating over two rolls at a speed of 1 cm s-1. Faecal pellets caught by the moving cloth filter were separated quickly from the water and scraped into a store-basin after passing the top roll.
2.1.2. Rearing facilities

Three experiments were performed in order to optimize the methods of faeces collection by continuous filtration. 1. In the initial experiment 220 trout (mean wet weight 200 g) were kept in a 1.6 m 3 tank (flow rate about 20 litres min - 1 additionally aerated) and accustomed to the experimental diet HM for one week. The water temperature was 13C, and fish were fed twice a day at 9.00 and 15.00 hours to satiation (about 2% o f b o d y weight per day). After the adaption all fish were transferred to the 600 litre faeces collection tank for faeces collection. Faeces were collected in six intervals (lasting 3 0 - 4 5 rain; Table 2) within 24 h. Immediately before each sampling period a subsample of about 25 fish were caught and anaesthetized. Faeces were sampled from these fish by the stripping method. Stripped fish were discarded. The faeces of fish remaining in the tank were collected by continuous filtration. ~' In a second trial three circular tanks (400 litres; water flow rate 8 litres min -1) were stocked with 78 trout (average wet weight 193 g) each. Fish were fed the experimental diet HM once a day at 12.00 hours (1% body weight a day; temperature 15C). After an initial feeding period of one week fish were transferred from one circular tank to the lheces collection tank. Faeces were filtered on two days from 13.00 to 16.00 hours corresponding to 1-4 h after feeding, and from 07.00 to 10.00 hours corresponding to 19-22 h after feeding (Table 3). On the second day fish were fed in the faeces collection tank. Parallel to each faeces collection period the faeces of about 25% of the fish remaining in the circular tanks were sampled by the stripping method. Stripped fish were again discarded. 3. In the third trial five tanks (volume 180 litres; water flow rate 5 litres min -1) were stocked with 55 trout (average wet weight 91g: water temperature 15C). Fish were accustomed for one week to the commercial diet K40, which was fed once a day at 12.00 hours (2c:~ body weight). For faeces collection the total fish stock from one tank

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was transferred to the 100 litre faeces collection tank. Faeces were continuously collected in four intervals within 48 h as described in the previous experiment (Table 4). Parallel to each collection period faeces was stripped from the fish stock o f one remaining tank. Stripped fish were discarded. All faeces samples were dried in a vacuum drying oven at 75C and stored in an exsiccator for further analyses. 2.1.3. Experimental diets The test diet HM (composition is depicted in Table 1) was specifically developed to meet the following requirements: (a) faeces should remain cohesive in water, and (b) faeces should settle fast in order to facilitate its rapid separation from water. This was achieved by adding undigestible ballast materials (cellulose) and anorganic matter (bolus alba). A commercially available feed was used in addition to the test diet with a content o f 40% crude protein, 7.5% ether extract, 3% fibre and 15% ash. According to the information provided by the producer 1 kg feed contained 1 2 000 IU vitamin A, 400 IU vitamin D3 and 60 mg

TABLE 1

Digestion Trials with Trout. Composition of the Experimental Diet HM Specifically Developed to Produce Water-stable Faeces Ingredients Herring meal a Bolus alba Cellulose Water Lignin sulphonate Vitamin and mineral premix b Chromium(IIl)oxide % in diet 75.0 10-0 6-7 3.5 3.0 1.0 0.8

a 70% crude protein, 5-8% ether extract according to the producer. Deutsche Vilomix 12101, 1 kg premix contained 2 000 000 IU vitamin A, 200 000 IU vitamin D3, 2500 mg vitamin E, 6500 mg Mn, 4600 mg Fe, 3600 mg Zn, 500mgCu, 100mgJ.

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vitamin E. Both the experimental and the commercial feeds were supplemented with 0.8% c h r o m i u m ( I I I ) o x i d e (Cr203) for the determination of digestibility and were then pelleted to 3 mm pellets.

2.2. Analytical procedures

Protein content was calculated from Kjeldahl-nitrogen according to official methods (Naumann and Bassler, 1976). The energy content was determined with a micro-calorimeter (Iulius Peters, Berlin) according to Roth and Becker (1956). Amino acids in samples from feeds and faeces were determined after 24-h hydrolysis with 4N-methanesulphonic acid in an atmosphere of nitrogen at 105C using an amino acid analyser BiCal 200 (Horstmann, 1979). Chromium in feeds and faeces was determined photometrically after acid digestion (Petry and Rapp, 1970/71) using diphenylcarbacid reagent (Joshida, 1960). The apparent digestibility of protein, energy and amino acids was calculated using the formula (Castell and Tiews, 1980): % indicator in feed X % nutrient in faeces ) % indicator in faeces %. nutrient in feed

Da (%) = 100 X

2.3. Statistical analysis

Data were subjected to two-way variance analyses according to the following models:
x q = I.t + a~ + b~ + e~i

with xii = measured value, ja = mean of population, ai = effect of rows, b i = effect o f columns and e i / = residue error, and
xi/k = I.t + ai + b i + (ab)i i + ei/k

with xij k = measured value, /a = mean of population, a i = effect of rows, b / = effect o f columns, ( a b ) i / = interaction and eo.k = residue error. The effects are assumed fixed, and the errors are random (Sachs, 1974).

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3. RESULTS The experimental diet HM met all requirements with respect to which it had been composed. Faecal pellets obtained were soft and showed a smooth surface. They were cohesive in water and spherical in shape, and did not fall apart during filtration. A mean settling speed of 9.3 cm s-1 was determined. At a total height o f 1.2 m of the experimental tank, less than 15 s were required for the settling o f faecal pellets. The apparent protein digestion coefficients for the experimental diet HM are listed in Table 2, stratified according to methods and periods of faeces collection. With 89.5%, the mean digestion coefficient for protein derived from continuously filtered faeces was slightly but significantly (p < 0.05) higher when compared with the mean digestion coefficient obtained from stripped faeces (88.6%). No differences were found between digestion coefficients obtained from faeces samples in different sampling periods. The data derived from the second experimental series (Table 3) corresponded to those obtained during the first one. Therefore, automation o f faeces collection had no influence on the results. With filtered faeces a mean protein digestion coefficient of 89.6%, was determined as compared to 87.3% after application o f the stripping method. The difference between the two was significant (p < 0-05) and slightly greater than in the first experimental series. The mean digestion coefficients for energy were 86-4% and 84.8%,, respectively. The difference was not significant (p > 0.05), however. Sampling time had no influence on the digestion coefficients for protein and energy. The daily chromium intake rate was 64.5 mg Cr kg -1 fish. The intake rate is identical with the excretion rate, as chromium(III)oxide is not absorbed by the intestine. Assuming a continuous defaecation process, it can be estimated that 2.7 mg chromium were excreted by 1 kg fish h -1. The amount of faeces stripped from 1 kg fish per period averaged 175 mg ( - 1 9 mg standard deviation) dry matter, corresponding to 3.2 mg chromium. Under natural conditions 1 kg fish would have excreted this amount o f faeces and chromium, respectively, within 71 rain. The digestion coefficients for protein and energy of the commercial feed K40, which was fed in the third experimental series, are listed in Table 4. The mean protein digestion coefficients (84-6% after continuous filtration and 79-5% after stripping)differed significantly (p < 0.001)

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TABLE 2

Apparent Digestion Coefficients for Crude Protein (DaCP) of the Experimental Diet HM in Relation to Faeces Collection Method and Sampling Time
Period Time a Faeces collection method b CF, DaCP (%) ST, DaCP (%)

1 2 3 4 5 6 Mean Standard deviation

09.45-10.15 hours 13.30-14.00 hours 17.30-18.00 hours 21.30-22.00 hours 01.45-02.30 hours 07.00-07.30 hours

89.6 88.7 89.3 90.3 89"8 89.2 89.5 -+0-6

87.9 88.0 89.1 89.0 88.5 88-9 88.6 +-0.5

Two-way variance analysis, model without interaction: Cause o f variance DF MQ F

Periods Methods (CF or ST) Residue Total

5 1 5 11

0.39 2.52 0.185

2.1 13.64"

* Significant with p < 0.05. Stripping of faeces was started immediately before the beginning of the continuous filtration period. CF = continuous filtration of released faeces; ST = stripping faeces from lower intestine. and so did the digestion coefficients for energy (p < 0.01), which were 73.7% and 68.0%, respectively. In addition a poor but significant (p < 0.01) influence o f sampling time on protein digestion coefficients was detected. Digestion coefficients obtained with faeces sampled 19-22 h after feeding t e nde d to be 1-2% higher than those obtained with faeces sampled 1 - 4 h after feeding. The a m o u n t o f stripped faeces averaged 262 mg (-+ 29 mg standard

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TABLE 3
Apparent Digestion Coefficients for Crude Protein (DaCP) and Energy (DaE) of the Experimental Diet HM in Relation to Faeces Collection Method and Sampling Time

Period

Timea

Faeces collection method ~ CF DaCe (%) DaE (%)

86.2 86.7 86.4-+ 0.4

ST DaCe (%)
88.2 86.9 87.6 86.5 87-9 86.7 87.3 - 0.8

DaE (%)
84.3 85-4 84.8-+ 0.8

I 2 1 2

13.00-16.00 hours 07.00-10.00 hours 13.00-16.00 hours 07.00-10.00 hours

89.9 89.7 88.9 89.7 89.4 89-7 89.6-+ 0.4

Mean o f period 1 Mean of period 2 Total mean -+ standard deviation

Two-way variance analyses, model with and without interaction:

Digestion coefficients o f protein Digestion coefficients o f energy

Cause o f variance DF
Methods (CF or ST) Periods Interaction o f methods x periods Residue Total 1 1 1 4 7

MQ
10.125 0-405 1.125 0.19

F
53.3** 2.1 5.9

Cause of variance DF MQ
M e t h o d s ( C F or ST) Periods Residue Total 1 1 1 3

2.56 28.2 0-64 7.1 0.09

** Significant with p < 0-01. a Stripping of faeces was started immediately after the beginning o f the continuous filtration period. b CF = continuous filtration; ST = stripping.

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TABLE 4

Apparent Digestion Coefficients for Crude Protein (DaCP) and Energy (DaE) of the Commercial Feed K40 in Relation to Faeces Collection Method and Sampling Time

Period

Time a

Faeces collection method b CF DaCP ( %) OaE ( %)

74.4 75.3 70.8 74.2

ST DaCP ( %)
78.3 81.0 78.3 80.3

DaE ( %)
66.3 70.1 66.5 69.1

1 2 1 2

13.00-16.00 hours 07.00-10.00 hours 13.00-16.00 hours 07.00-10.00 hours

84-4 85.4 84.1 84.5

Mean of period 1 Mean of period 2 Total mean -+ standard deviation

84.2 72.6 85.0 74-8 84.6 _+0-6 73.7 -+2-0

78.3 66.4 80.6 69-6 79-5 -+ 1.4 68.0 -+ 1.9

Two-way variance analyses, models with interaction."

Digestion coefficients of protein Digestion coefficients of energy

Cause o f variance
Methods (CF or ST) Periods Interaction of methods x periods Residue Total

DF
1 1 1 4 7

MQ

Cause o f variance

MQ

52.53 302-3*** 4.65 26.8** 1.36 0.173 7.8*

Methods(CF or ST) 64.41 33.9** Periods 14.31 7.5 Interaction of methods periods 0.55 0.3 Residue 1.901

*** Significant with p < 0.001; ** significant with p < 0.01; * significant with p < 0.05. a Stripping of faeces was started immediately after the beginning of the continuous filtration period. b CF = continuous filtration; ST = stripping.

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deviation) kg-1 fish, corresponding to 3.4 mg Cr kg-1 fish. The daily chromium rate was 108-4 mg Cr kg-1 fish. Assuming a continuous defaecation process, it can be estimated that 1 kg fish excreted, on average, 4-5 mg Cr h -1. Thus, it can be calculated that the amount of chromium stripped (3.4 mg kg-1 fish) and the corresponding amount of faeces, respectively, would have been excreted within 45 rain under natural conditions. The faecal pellets of the commercial feed K40 disinegrated quickly in water. It was therefore not possible to measure the settling speed correctly. Within two days of continuous filtration only 40% of the chromium administered had been recovered. When feeding the experimental diet HM, this rate increased to 60%. It must be emphasized, however, that the objective of the faeces collection apparatus was not to filter faeces quantitatively but to separate them from water intact and as quickly as possible. Therefore, the given recovery rates represent only a relative measure of water-stability of faeces. The influence of the method of faeces collection on the digestion coefficients of amino acids is summarized in Table 5. The determination of digestibility of amino acids was performed with the sample taken between 07.00 and 10.00 hours on the second day (Table 4). The digestion coefficients obtained for the sum o f amino acids were 87-4% (faeces filtration) and 85.6% (faeces stripping). These values differed less than those for crude protein (84.5 and 80.3%, respectively). An exception was found for the apparent digestion coefficients of aspartic acid, which differed considerably between 86.7% (faeces filtration) and 78.6% (faeces stripping). The two-way variance analysis revealed significant differences (p ~ 0.01) between the mean digestion coefficients for individual amino acids. The same holds for the average digestion coefficients for amino acids obtained when using the two different faeces collection methods. 4. DISCUSSION Previous experiments performed by different authors (Possompes, 1973; Windell et al., 1978; Cho and Slinger, 1979)revealed, as well as the study presented here, that faeces collection from water resulted in higher digestion coefficients as compared to faeces collection directly from the intestine. Three hypotheses have to be taken into consideration in order to explain these results: 1. The faeces filtered from water is leached whereby an increased digestibility is simulated.

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TABLE 5 Influence of Faeces Collection Method a on the Apparent Digestion Coefficients for Amino Acids and Protein of the Commercial Feed K40
A m i n o acids Apparent digestion coefficients CF ST

Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Sum of amino acids Crude protein

86.7 86.0 87.0 90.0 86.4 82.6 86.4 90.3 87.6 85.6 87.7 87-7 84.2 86.8 88.8 86.1 90.4 87.4 84.5

78-6 83-9 83-6 88.2 84.6 82.7 86.5 86-6 86.4 85.0 86.4 86.6 83-4 87.2 87.0 83-7 90-2 85.6 80.3

Two-way variance analysis, model without interaction: Cause o f variance DF MQ F

Amino acids Methods (CF or ST) Residue Total

16 1 16 33

9.06 25.9 2.01

4.5** 12.9"*

** Significant with p < 0.01. a CF = continuous filtration; ST = stripping.

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2. The stripped faeces contains residues of not yet digested or absorbed nutrients resulting in an underestimation of digestibility. 3. The stripped faeces contains traces of urine, slime, serum, epithel of intestine and, eventually, sexual products, artefacts, which also simulate a decreased digestibility. The results presented in this paper indicate that the first hypothesis possesses the greatest power of explanation without disproving completely the two other hypotheses. When the experimental diet HM was fed, faeces leaching could evidently be minimized due to the waterstability of the faeces and their high settling speed. In consequence the digestion coefficients for protein and energy derived from faeces collected by continuous filtration and by stripping differed only by 1-2.5%. In contrast, the commercial feed K40 produced a faeces of poor coherence in water. Already a contact with water of 20-30 s (speed of sedimentation could not be determined exactly) resulted in a considerable increase in the measured protein and energy digestion coefficients of at least 5% due to leaching of faecal nitrogen and energy into water. It is evident that the diverging results obtained with the experimental and commercial diets cannot be interpreted adequately using hypotheses 2 and 3. Adulterations of stripped faeces by yet undigested materials and artefacts like urine, slime, etc., must have occurred with both the experimental and the commercial diets. Accordingly the decrease of the measured digestion coefficient due to such adulterations should be independent of the type of feed. As this is not true, it must be concluded that leaching of faeces collected from water resulted in an overestimation of digestibility rather than that adulterations of stripped faeces caused an underestimation of digestion coefficients. The reliability of faeces collection from water basically depends on the question: is there an important fraction of soluble materials in naturally defaecated faeces? If that is true, a contact of faeces with water of a few seconds would be sufficient to leach this soluble fraction. Possompes (1973), who studied the kinetics of leaching for faecal nitrogen, could demonstrate that the crude protein content of a stripped faeces sample decreased from 35 to 20% when it was exposed to water for 5 rain. The crude protein content of filtered faeces samples decreased from 22.8 to 16.3% when the time of water contact of the faeces was increased from 5 to 60 rain. Accordingly, Windell et al. (1978) demonstrated that most leaching of faecal materials occurred within the first

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hour, when faeces dissected from the lower intestine was exposed to water. Smith e t al. (1980), who constructed a metabolism chamber enabling the quantitative and separate collection of excretions from gills, bladder and intestine, found the portion of soluble nitrogen to come up to 50% of the total faecal nitrogen. However, the occurrence of leaching of faecal materials and the resulting objections against faeces collection from water do not invalidate the objections against the different methods of faeces collection from the intestine. It is difficult to determine to which extent artefacts and undigested nutrients in faeces samples collected from the intestine simulate a poor digestibility. There seems to be no direct experimental approach to solving the problem, as it is in principle impossible to study naturally defaecated faeces which has not been in contact with water. Even the method of total collection of metabolic products using a metabolism chamber (Smith e t a l . , 1980) does not give a definite answer, as fish are subjected to considerable stress, which might also provoke a depression of digestibility as compared with natural conditions. Results obtained with the metabolism chamber, however, seem to agree fairly well with those obtained with the stripping method, whereas digestion coefficients obtained with faeces collected from water tend to be higher (Cho and Slinger, 1979). The methodical objections against the stripping method hold true for other methods of faeces collection from the intestine (i.e. dissecting or suction). Windell e t al. (1978), however, found considerably lower digestion coefficients for protein and lipids when applying the stripping method as compared with the dissecting or suction method. These results, which are in contrast to those reported by Austreng (1978), seem to be due to contaminations of the stripped faeces with milt, as running ripe males of 636 g average weight were used in these experiments. Eliminating this source of error, the results obtained with the stripping and suction method should agree, as the differences between the two methods only consist of the use of external pressure for stripping and of internal vacuum for suction.

5. CONCLUSIONS Among the methods used for faeces collection from the intestine, the stripping method can be applied with the least experimental effort. The results obtained are reliable and can be reproduced. However, some precautions have to be considered: (a) fish should be anaesthetized

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prior to stripping; (b) the a m o u n t of faeces stripped should be controlled. According to the results of the study presented here, the quantity o f stripped faeces should be limited to the volume excreted under natural conditions within about 1 h. The continuous filtration m e t h o d seems to be the most reliable, amongst all methods tested, when collecting faeces from water. The technique minimizes the contact time with water down to some 10 s. The digestion coefficients obtained with the stripping m e t h o d as well as the continuous filtration m e t h o d differ by 1-2.5%, if faecal pellets are stable in the water after defaecation. Greater differences in digestion coefficients are due to disintegration o f faecal pellets filtered from water. In these cases values should be obtained by using the stripping method. ACKNOWLEDGEMENTS The author wishes to express his gratitude to: Professor H.-J. Horstmann, Institut ftir Physiologische Chemie der Universiffit Er!angen-Nfirnberg, for performing the amino acid analyses; Mr Mehler, Mela-Silver-Cup, for supplying the experimental diets; Dr Reichle, Fachberatung ffir Fischerei des Bezirks Oberpfalz, for material and financial support; and Professor Dr Rosenthal, Biologische Anstalt Helgoland, Hamburg, for reviewing this manuscript.

REFERENCES Austreng, E. (1978). Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture, 13,265-72. Bergot, F. (1979). Problemes particulieres pos6s par l'utilisation des glucides chez la truite arc-en-ciel. Annales de la nutrition et de l'alimentation, 33,247-57. Castell, J. D. & Tiews, K. (eds) (1980). Report of the EIFAC, IUNS and ICES
Working Group on Standardization of Methodology in Fish Nutrition Research,

Hamburg. (EIFAC Technical Paper No. 36.) Cho, C. Y. & Slinger, S. J. (1979). Apparent digestibility measurement in feedstuffs for rainbow trout. In: Finfish Nutrition and Fishfeed Technology. Proceedings of a World Symposium, Vol. 2, eds J. E. Halver and K. Tiews, Heenemann Verlagsgesellschaft, Berlin.

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