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Feed intake measurements in fish using radioactive isotopes
70 (1988) 277-288 Elsevier Science Publishers B.V.. Amsterdam -
Aquaculture,
277 Printed in The Netherlands
Feed Intake Measurements in Fish Using Radioactive Isotopes II. Experiments with Atlantic Salmon and Rainbow Trout in Sea-Pens TROND STOREBAKKEN and ERLAND AUSTRENG The Agricukural NLH (Norway)
Research Council of Norway, Institute of Aquaculture
Research, N-1432 As-
(Accepted 6 July 1987)
ABSTRACT Storebakken, T. and Austreng, E., 1988. Feed intake measurements in fish using radioactive isotopes. II. Experiments with Atlantic salmon and rainbow trout in sea-pens. Aquaculture, 70: 277-288.
Short-term feed intake was measured in 2419 Atlantic salmon (Salmo salar L. ) and 1232 rainbow trout (Salmo gairdneri Richardson), weighing 0.1-4.0 kg. The fish were kept in net-pens in the sea. They were hand-fed with a moist diet containing radioactive Ag13iIfor a 2-h period. The radioactive diet was either fed to fish which had been deprived of feed for 12-15 h to assess the “maximum meal size” or to fed fish to assess “the influence of previous feeding on subsequent feed intake”. The amount of radioactive feed ingested by the fish was measured with a portable gamma scintillation counter placed under their stomachs. The accuracy of this method when using large fish was checked, and the fish were monitored for possible radioactive contamination. The feed intake was further assessed by weighing the stomach contents of 16 salmon. The results showed that feed intake can be measured in large fish using radioactive isotopes. No radioactive contamination was found in the fish. When the water temperature was lo-13°C the intake of moist feed amounted to 6-12% of the fish weight during the first 2-h period after the deprivation period. When feed intake during this first period was high, it became low during the rest of the day. When the water temperature was 5-6” C, feed intake after the deprivation period was only 2-3% of the fish weight, and the fish ingested relatively more feed in later meals. There was no significant difference in feed intake for the two species of fish. A mean feed intake of 4.1% moist feed was recorded from the weight of stomach contents in the separate experiment which was carried out at a water temperature of 14-15°C.
INTRODUCTION
A method to assess feed intake in fish using radioactive isotopes has been published previously (Storebakken et al., 1981). This method permits measurement of feed intake without disturbing, hurting or killing the fish. This
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eliminates the stress caused by bringing the fish to the laboratory. The method was developed while working with rainbow trout (Salmo gairdneri Richardson) weighing less than 1 kg. One aim of the present experiments was to study whether the method could also be utilized on larger Atlantic salmon (Salmo salur L.) and rainbow trout. A second aim was to find out whether the fish absorbed any of the orally administered Ag1311.A previous study on rainbow trout in fresh water demonstrated considerable variation in connection with feed intake measurements (Storebakken and Austreng, 1988). The method, however, has been a valuable tool in experiments comparing different feeding treatments (Storebakken, 1985; Storebakken and Austreng, 1987). Feed intake is one of the important regulators of fish growth. Vahl (1979) described a model in which only two parameters are necessary to design a feeding regime which might result in maximum growth in an aquaculture system. These are the maximum voluntary feed intake in one meal and the evacuation rate of the stomach. Hence, a third aim was to measure one of these parameters, namely the maximum voluntary feed intake. According to Ishiwata (1968) both the acclimatization and the hunger of the fish should be emphasized in feeding experiments. To study variations in appetite, feed intake was measured in both feed-deprived and fed fish which were kept in different types of netpens at different water temperatures. MATERIALS
AND METHODS
The present study consists of four different experiments, all of which were carried out at the Institute of Aquaculture Research at Averpry. The four experiments were conducted 26-28 June 1979 (Expt. 1) ,23-25 September 1980 (Expt. 2), 2-4 December 1981 (Expt. 3), and 15-23 July 1985 (Expt. 4). Daylength was 18 h in Expts. 1 and 4 (long day), 12 h in Expt. 2 (decreasing daylength), and 6 h in Expt. 3 (short day). Atlantic salmon and rainbow trout weighing between 0.1 and 4.0 kg were used in the experiments. The fish were kept in net-pens which had a volume of either 27 m3 or 500 m3 in the sea (salinity: 30-32%0). The fish in each pen were sorted by weight. The mean coefficient of variation (C.V. = 100~ s/x, s= standard deviation, X=mean value) of the fish weight within pens was 35%. The fish were transferred from larger to smaller pens at least one month prior to the experiments to allow them to adapt to the more confined rearing conditions. The fish utilized in Expts. 1,2 and 3 were accustomed to hand-feeding more than 10 times a day with a wet feed consisting of 10% binder meal and 90% trash fish. During a preparation period of at least 2 weeks and throughout the experiments the fish were fed a moist diet which was commercially available (“Tess Salmomix loo”, T. Skretting A/S, Stavanger, Norway). The formulation and chemical composition of this diet are presented in Table 1. The diet
279
TABLE 1 Formulation and chemical composition silver iodide
Ingredients (g ) Capelin meal (Nor Sea Mink) Soybean meal (extracted) Wheat (cooked) Capelin oil (Nor Salm Oil)’ Vitamin and micromineral premix’ Alginate and guar gum mixture Water Chemical analyses (g) Dry matter (DM) Content in DM Crude protein Crude fat Ash Crude fibre Nitrogen-free extract Metabolizable energy (ME) ( MJ ) ’ ME from protein ( % )
of the diet. This diet was supplemented
with radioactive
247 137 99 122 17 28 350 562 459
240 84 37 176 18.0 42
“Supplemented with 200 mg ethoxyquin per kg oil. bMinimum content per kg diet according to the producer’s specifications: vitamin A, 2500 i.u.; vitamin Kt, 10 mg; vitamin E, 60 mg; thiamine, 10 mg; riboflavin, 25 mg; pyridoxin, 15 mg; pantothenate, 40 mg; niacin, 150 mg; folic acid, 5 mg; biotin, 0.2 mg; choline, 600 mg; inoeitol, 100 mg; ascorbic acid, 200 mg. The vitamins B12, D3 and p-aminobenzoic acid and microminerals are added in unspecified amounts. “ME from protein and fat calculated according to Phillips and Brockway (1959) (16.3 kJ ME/g protein; 33.5 kJ ME/g fat). ME from NFE calculated according to Singh and Nose (1967) (13.8 kJ ME/g cooked starch).
was pelleted using a lo-mm die for fish with a mean weight up to 1 kg. For fish larger than this a 15-mm die was used. The moist diet was analysed for dry matter ( 105 ’ C, overnight ) , ash (550 oC, overnight), crude fat (Folch et al., 1957 ), crude protein (Kjeldahl method) and crude fibre (AOAC, 1975) content. Nitrogen-free extract was calculated by difference. Radioactive feed and feed intake measurements Radioactive silver iodide (Ag1311)was added to the diets in amounts of 0.45 mCi/kg moist pellet per 26 June 1979 in Expt. 1,0.34 mCi/kgper ‘23September 1980 in Expt. 2, and 0.35 mCi/kg per 2 December 1981 in Expt. 3. In Expt. 1 the homogeneity of the moist diet was checked by adding 0.20% Cr,O, to the
280
diet by the same procedures as for the Ag13’I. The chrome content in two samples of feed was analysed by atomic absorption (Williams et al., 1962). The homogeneity of the blend in Expt. 2 was checked in four samples by measuring the radioactivity with a “1280 UltraGamma” well gamma scintillation counter ( LKB-Wallac, Finland). The reproducibility of the readings with the “NE 5013” gamma scintillation counter was checked with moist pellet standard containing Ag1311in Expt. 2. The radioactivity of the moist pellet standard was measured in a plastic bag placed on the top of the NaI crystal under field conditions. One week after Expt. 2 the procedure was repeated with the same standard and a “Mock iodine-131 gamma counter checking source” (Radiochemical Centre, Amersham, England) in the laboratory. The radioactivity contained in the checking source was equivalent to 0.1 mCi’ 1311.The instrument was checked with this checking source under field conditions in Expt. 3. The radioactive diet was fed every 10 min for 2 h. A feeding period of only 2 h was chosen to facilitate the measurement of a large number of fish. The measuring period was limited to 4 h after feeding in Expts. 1 and 2 to ensure that all radioactive material was present in the anterior part of the gastrointestinal tract (Storebakken et al., 1981). The measuring period was set 2 h longer in Expt. 3 due to a lower water temperature and, thus, a slower rate of gastric evacuation. Feed intake measurements were started immediately after completion of the feeding period with the radioactive diet. The fish were randomly sampled from the pens, anaesthetized with chlorobutane and weighed. Radioactivity measurements were conducted on all fish from the 27-m3 pens, while up to 420 fish were utilized from the 500-m3 pens. Radioactivity was measured under the stomach for two 10-s periods with a Scalar Spectrometer “NE 5013” gamma scintillation counter (Nuclear Enterprises, England) as described by Storebakken et al. (1981). The counts were corrected for background irradiation. The amount of radioactive material ingested was calculated as described by Storebakken et al. ( 1981) by means of the following equation:
F=-0.0494+ 0.00023M where F represents the amount of radioactivity ingested Q.&i) and M is the radioactivity measuredunder the fish stomach (counts/l0 s). Feed intake then was calculated based on the amount of radioactivity added to the feed. Radioactive feed was offered to different groups of fish according to three feeding procedures:
281 Procedure 1 Deprivation 12-15 h Radioactive diet 2h Counting
Procedure 2 Deprivation 12-15 h Radioactive-free diet 4h Radioactive diet 2h Counting
Procedure 3 Deprivation 12-15 h Radioactive-free diet 6h Radioactive diet 2h Counting
Procedure 1 was carried out to measure the “maximum meal size”, while procedures 2 and 3 were carried out to measure “the influence of previous feeding on subsequent feed intake”. The numbers of fish and pens used for the different feeding procedures are presented in Table 2. The stomach contents from 25 rainbow trout which weighed between 0.3 and 3.5 kg were sampled after counting the ingested amount of radioactive material in Expts. 1 and 2. This was done in order to ascertain whether the feed intake measurements necessitated any corrections for fish size. The radioactivity in the stomach contents was measured in the well counter as described by Storebakken et al. (1981). In addition, four salmon from Expt. 2, which weighed between 1.4 and 2.9 kg, were sampled and treated in the same manner as described for the rainbow trout to determine whether there was any need for a separate procedure for the salmon. Fifty fish from the groups which ingested most radioactive feed were randomly sampled and slaughtered 2 days after the feed intake measurements to determine whether they were contaminated by Ag1311. The fish were gutted and washed, and carcass radioactivity was measured with the “NE 5013” counter. Further, four rainbow trout were slaughtered 10 h after feeding the radioactive diets. Duplicate 2.5-g samples of flesh taken near the dorsal fin, intestine adipose tissue, and the liver were removed from each fish for measurement of radioactivity using a well counter. The counting was carried out after the samples had been stored frozen for 5 days. The fourth experiment was conducted to measure the feed intake of salmon which were accustomed to hourly feedings of moist pellets using an automatic feeder. Salmon in a 500-m3 sized pen were treated as described above in “procedure l”, but without radioactivity in the diet. The salmon were fed a moist pellet composed of 55% fish silage and 45% binder meal. When the feeding was finished, 16 salmon which varied in weight between 1.1 and 2.9 kg were sampled randomly. One week later, 12 salmon weighing between 0.7 and 1.7 kg were sampled randomly from the same pen after a food deprivation procedure identical to the one outlined above for ‘Lprocedure 1”. The stomach contents were weighed and expressed as a percentage of body weight.
282 RESULTS
Evaluation of the radioisotope method Tests run on the homogeneity of the blend of the radioactive diet revealed only minor variations in the samples. Both feed samples in Expt. 1 contained 0.21% Cr,O,. The radioactivity in the four samples from Expt. 2 averaged 219 000 counts mine1 g-l, with a C.V. of 2.6%. Results concerning the reproducibility of repeated countings with the “NE 5013” are presented in Fig. 1. They show that the instrument was very unstable during the first 24 h of Expt. 2. Results from this period are indicated with brackets in Table 2. After adjustments were made to stabilize the instrument, the readings from the moist pellet standard were slightly above 80 000 counts/l0 s. One week later the instrument in the laboratory read 81000 counts/l0 s (corrected for decay). The “gamma counter checking source” read 2600 counts/l0 sunder the same conditions. The instrument was stable during Expt. 3. The mean counting value obtained by using the “checking source” in Expt. 3 was 3100 counts/l0 s, which was 21% higher than the values obtained in Expt. 2. The counting efficiency, thus, varied from one experiment to another. The regression of the measurement of feed intake made with the “NE 5013” on the values obtained with the corresponding stomach samples was:
Experiment Moot
2
pe~fef
standard
Expert men t 3 Gnmma
0212
0312
checkrng source
0412
0512
Dafe
Fig. 1. Test results on the reproducibility of repeated countings with the “NE 5013” gamma scintillation counter.
283
FS= -3.04+1.36FC+O.40
W
(R2=0.96)
where FS is the amount of radioactive feed ingested (g) calculated from the radioactivity in the stomach samples, FC is the feed intake (g) calculated from the readings under the stomach, and W is the fish weight (kg). FC was the only parameter which contributed significantly (P IO.001 ) to the regression. The regression shows that the FS values were slightly lower than the calculated FC. The mean ratio between FS and FC for the four salmon from Expt. 2 was 0.78 (s=O.12), which was within the range of variation noted in the rainbow trout material. The feed intake was accordingly calculated by the same equation for both species. In Expts. 2 and 3, respectively, the mean FS/FC ratios were 0.96 (n=18 fish, s=O.17) and084 (rz=8, s=O.14). No radioactivity was detected in any of the 50 carcasses sampled in the study for radioactive contamination. In the samples taken from the four trout, mean amounts of 0.04 nCi/g flesh (s=O.Ol), 0.00 nCi/g adipose tissue, and 0.35 nCi/g liver (~~0.14) were found. Feed intake measurements During the first 15-30 min of feeding in the “procedure 1” treatment, the fish were very active when the feed was offered. Feeding activity then decreased. During “procedure 2” and “procedure 3” the fish seemed less interested in the feed. The counting results normally were within the range of 200 counts/l0 s to 100 000 counts/l0 s, and the background irradiation represented 200 to 600 counts/l0 s. A regression analysis of feed intake on weight of fish for each pen explained only a fraction of the total variation present in the data. In the regressions for rainbow trout, the R2 values were 0.0-0.1. For salmon, they were 0.0-0.4, with those in only three of the 16 pens exceeding 0.2, and that in one pen approaching 0.4. The different feeding procedures did not result in different R2 values. Therefore, within each pen, other factors appeared to be more important than fish size in controlling short-term feed intake. The results from the feed intake measurements are presented as mean values in relation to the various feeding procedures and net-pen sizes in Table 2. In Expt. 1, a high feed intake was obtained for both rainbow trout and salmon by “procedure 1”. The feed intakes of fish obtained by procedures 2 and 3 were much lower. Both rainbow trout and salmon in the 27-m3 pens exhibited more active feeding behaviour when they were fed in Expt. 1 than in Expt. 2 and this was reflected in the feed intake measurements. During Expt. 3, the feed intake was reduced, and the number of fish which did not eat during “procedure 1” also increased. In Expt. 3, the feed intake of fish during “procedure 2” relative to “procedure 1” also increased when compared with the two other experiments. Neither of the results revealed significant differences between the two
284 TABLE 2 Feed intake of the fish presented as % of fish weight ingested in a P-hour feeding period” Temp. (“C)
Fish number
Sea-pen Size (m3)
Number
Fish size (kg) X
Rainbow trout Expt. 1 11.6 Proc. 1 11.7 Proc. 2 11.8 Proc. 3 10.8 Proc. 1 Expt. 2 12.9 Proc. 1 12.9 Proc. 2 Expt. 3 5.1 Proc. 1 4.8 Proc. 2 Salmon Expt. 1 Proc. 1 Proc. 2 Proc. 3 Proc. 1 Expt. 2 Proc. 1 Proc. 2 (Proc. 1 Expt. 3 Proc. 1 Proc. 2
27 27 27 500
75 71 76 320
1.56 1.55 1.74 1.71
Feed intake f%) s
-
Percent of fish with no intake
X
s
8
s
12.5 0.6 0.5 10.3
-
2.7 49.3 67.1 8.8
-
27 27
2 2
241 98
1.93 1.84
0.05 0.04
6.3 0.9
1.3 0.1
9.5 44.9
4.9 0.0
27 27
2 3
206 145
1.85 2.24
0.02 0.03
1.9 0.9
0.0 0.1
19.3 31.0
5.8 4.5
74 74 75 337
1.98 1.98 2.10 1.50
11.1 0.3 0.5 9.6
-
2.7 75.7 67.1 0.9
-
-
11.6 11.7 11.8 11.6
27 27 27 500
12.9 12.9 12.9
27 27 500
4 4 1
195 197 422
0.97 1.00 1.24
0.05 0.05 -
7.4 4.5 17.9
1.4 0.7 -
19.8 48.2 3.3
4.4 7.2 -_)
5.6 5.5
500 500
1 2
355 690
1.44 1.47
0.04
2.8 1.4
0.3
31.0 33.0
1.1
“The results are presented as means (x) and standard deviations (s) between different pens.
species or between the two pen sizes as far as the feed intake was concerned. The number of fish which had not eaten was inversely related to the feed intake values for each pen. The mean C.V. values for the feed intake of fish obtained by “procedure 1” in Expts. 1, 2 and 3, respectively were: 70.1, 88.4 and 85.9% for the rainbow trout and 50.9, 79.9 and 102.6% for the salmon. No differences between procedures 2 and 3 were observed and the mean C.V. values, in the same order as listed above, were 188.7, 155.1 and 117.4% for the rainbow trout and 269.1, 108.8 and 118.2% for the salmon. These results show that both decreased water
temperature and a previous feeding resulted in increased variation in feed intake. Water temperature was 14-15’ C during Expt. 4. The feed intake of salmon varied from 1.7 to 6.6% of their body weight. There was no significant effect of fish size on feed intake, but the fish which had eaten the least were sexually maturing males. No feed was present in the stomachs of the group of fish which had been starved for 12-15 h. DISCUSSION
The comparison between the amount of radioactive material measured outside the fish (FC) to that in the stomach contents (J’S) indicates that the radioisotope method (Storebakken et al., 1981) can be applied for feed intake studies on large fish. A non-systematic bias, however, is indicated by the high standard deviation of the mean FS/FC ratios and the instability of the gamma scintillation counter during the first 24 h of Expt. 2. This bias may have been caused by several factors in addition to variation in the counting efficiency of the instrument. But efforts were made to eliminate those factors. The background irradiation was compensated for. The blending of the diets was homogeneous. The results in Fig. 1 indicate that the variation in the counting efficiency within experiments was small when the instrument was stabilized. Variation in the shape of the fish is another possible source of error since the stomach of a fish with a wide, U-shaped belly is relatively closer to the NaI crystal of the counter than the stomach of a fish with a straighter belly. Fish size is another possible source of error, but this was not found to be an important factor in this study. A systematic bias in the counting results is indicated by the higher counting results from the checking source in Expt. 3 relative to Expt. 2. Based upon the radioactivity measurements of the carcasses and tissue samples, it can be concluded that the fish did not accumulate radioactive silver iodide into body tissues. The observed levels of radioactivity of 0.0-0.4 nCi/g tissue were within the tolerance limit for “unrestricted waters” (IAEA, 1962). These amounts of radioactivity are so small that they may even represent a contamination from gutting the fish. The 1311isotope has a half-life of 8.3 days. Within a few days the measured amounts of radioactivity would have decayed beyond the limits of detection. The maximum feed intake obtained by “procedure 1” was high but consistent with the values obtained by Ishiwata (1968) and Grove et al. (1978) and with the critical stomach volumes found by Kariya et al. (1968)) all in rainbow trout. The fact that fish weight did not significantly influence the feed intake in the pens was surprising since fish size is an important factor in determining growth (Brett, 1979). A possible explanation is that the effect of fish size on
286
feed intake was masked by other factors than the ones that were monitored. This is suggested by the high non-systematic variation found for feed intake in this study. Austreng (1979) found that rainbow trout require about 15.5 MJ ME/kg growth. Based on this, the maximum feed intake found in Expts. 1 and 2 should have been sufficient to support a growth of 4-8%/day. Salmon weighing 150200 g can grow at a rate approaching 2%/day at a water temperature of 14”C, while the growth of rainbow trout and salmon maintained in sea-pens has been observed to be slower ( Austreng et al., 1987 ). The maximum ration consumed in one meal, thus, far exceeds the requirement for growth under a regime of constant feed intake. This high feed intake capacity, however, is consistent with results obtained with rainbow trout in freshwater tanks (Storebakken, 1985; Storebakken and Austreng, 1987). The feed intake obtained by “procedure 1” was higher in Expts. 1 and 2 than in Expt. 3. This concurs with the well-documented relationship between the water temperature and feed intake, reviewed by Brett and Groves (1979). Despite similar fish sizes and comparable water temperatures in Expts. 1 and 2, the results of the two experiments were considerably different. The water temperature in Expt. 2 was slightly higher than that in Expt. 1, but it remained below the optimum temperature for growth found for juvenile rainbow trout and salmon (Hokanson et al., 1977; Farmer et al., 1983). Accordingly, the increased water temperature should have resulted in an increase rather than a decrease in growth. The reduced feed intake of fish in Expt. 2 is likely to be attributed to factors other than water temperature. One of the most probable of these is photoperiod, since the general effect of decreasing daylength on freshwater fish is reduced growth (Brett, 1979). The reduced feed intake of fish noted in Expt. 4, in comparison with fish in Expts. 1 and 2, probably is an effect of acclimatization (Ishiwata, 1968). The salmon in Expt. 4 were adapted to hourly feeding, whereas the others were adapted to meal-feeding about ten times a day. The difference in available energy content and bulkiness between a wet and a moist diet in the previous feeding of the fish may also have contributed to a relatively lower feed intake capacity in the salmon of Expt. 4 (Grove et al., 1978). There are, however, factors other than diets and feeding strategy complicating the comparisons between Expts. l-3 and 4. The salmon of Expts. l-3 originated from the first generation of a family selection programme (Gjedrem, 1983). Those of Expt. 4 came from the second generation of this selection programme and the genetic growth potential was different. Moreover, the salmon utilized in Expt. 4 were the survivors of an outbreak of “Hitra disease” (Poppe et al., 1984). The others had no history of major health problems. The effects of the foregoing factors on the feed intake are difficult to assess. The feed intakes of the two species did not differ significantly during the study. This is consistent with the growth results obtained for the two species
of fish reared in the sea but in contrast to the results obtained in fresh water ( Austreng et al., 1987). The experiments show that the feed intakes of fish in this study were strongly influenced by previous feeding or feed deprivation time. This is consistent with what Adron et al. (1973) and Grove et al. (1978) found with rainbow trout. The differences found between Expts. 1 and 2 for the ratios between the feed intake obtained by “procedure 2” and “procedure l”, however, indicate that factors other than fish size, deprivation period and water temperature influence the appetite and the time required for the fish to reach satiation. Several factors may have caused these differences, such as the light intensity and photoperiod (see review by Brett, 1979) (Expt. 1, clouded, near maximum daylength; Expt. 2, sunny, decreasing daylength), and degree of adaptation to the pens, the time of the year and the stage of sexual maturation. ACKNOWLEDGEMENTS
We thank Mr. Torkjell Brers, Ms. Anne Dullum, Mr. Helge Lien and Mr. Georg Ostby for skillful technical assistance, and the Agricultural University of Norway, Department of Poultry and Fur Animal Science for helpful collaboration.
REFERENCES Adron, J.W., Grant, P.T. and Cowey, C.B., 1973. A system for the quantitative study of food preferences and behaviour. J. Fish Biol., 5: 625-636. AOAC, 1975. Official Methods of Analysis of the Association of Official Analytical Chemists, 12th Edn. Association of Official Analytical Chemists, Washington, DC, 1094 pp. Austreng, E., 1979. Fett og protein i f& til laksefisk. VI. Fordrayelighet og fi%utnyttelse hos regnbueaure (S&no gairdneri, Richardson) ved ulikt fettinnhold i fGret. Meld. Nor. Landbrukhegsk., 58(6), 12 pp. Austreng, E., Storebakken, T. and Asglrd, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: 157-160. Brett, J.R., 1979. Environmental factors and growth. In: W.S. Hoar, D.J. Randall and J.R. Brett (Editors), Bioenergetics and Growth. Fish Physiology, Vol. 8. Academic Press, New York, NY, pp. 599-675. Brett, J.R. and Groves, T.D.D., 1979. Physiological energetics. In: W.S. Hoar, D.J. Randall and J.R. Brett (Editors), Bioenergetics and Growth. Fish Physiology, Vol. 8. Academic Press, New York, NY, pp. 279-352. Farmer, G.J., Ashfield, D. and Goff, T.R., 1983. A feeding guide for juvenile Atlantic salmon. Can. Manuscr. Rep. Aquat. Sci., No. 1718,13 pp. Folch, J., Lees, M. and Sloane Stanley, S.J., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Gjedrem, T., 1983. Genetic variation in quantitative traits and selective breeding in fish and shellfish. Aquaculture, 33: 51-72. Grove, D.J., Loizides, L.G. and Nott, J., 1978. Satiation amount, frequency of feeding and gastric emptying rate in Salmo guirdneri. J. Fish Biol., 12: 507-516.
Hokanson, K.E.F., Kleiner, C.F. and Thorsland, T.W., 1977. Effect of constant temperature and die1 fluctuation on growth, mortality, and yield of juvenile rainbow trout, Salmo gairdneri (Richardson). J. Fish Res. Board Can., 34: 639-648. IAEA, 1962. Basic Safety Standards for Radiation Protection. Safety Series No. 9. International Atomic Energy Agency, Vienna, 59 pp. Ishiwata, N., 1968. Ecologicai studies on the feeding of fishes. IV. Satiation curve. Bull. Jpn. Sot. Sci. Fish., 34: 691-693. Kariya, T., Shirahata, S. and Nakumura, Y., 1968. An experiment to estimate the satiation rate of feeding in fish. Bull. Jpn. Sot. Sci. Fish., 34: 29-34. Phillips, A.M. and Brockway, D.R., 1959. Dietary calories and the production of trout in hatcheries. Prog. Fish. Cult., 21: 3-16. Poppe, T., H&stein, T. and Salte, R., 1984. Hitra disease (Haemorrhagic Syndrome ) in Norwegian salmon farming: present status. In: A.E. Ellis (Editor), Fish and Shellfish Pathology. Academic Press, London, pp. 223-229. Singh, R.P. and Nose, T., 1967. Digestibility of carbohydrates in young rainbow trout. Bull. Freshwater Fish Res. Lab., Tokyo, 17: 21-25. Storebakken, T., 1985. Binders in fish feeds. I. Effect of alginate and guar gum on growth, digestibility, feed intake and passage through the gastrointestinal tract of rainbow trout. Aquaculture, 47: 11-26. Storebakken, T. and Austreng, E., 1987. Ration level for salmonids. II. Growth, feed intake, protein digestibility, body composition, and feed conversion in rainbow trout weighing 0.5-1.0 kg. Aquaculture, 60: 207-221. Storebakken, T. and Austreng, E., 1988. Feed intake measurements in fish using radioactive isotopes. I. Experiments with rainbow trout in freshwater. Aquaculture, 70: 269-276. Storebakken, T., Austreng, E. and Steenberg, K., 1981. A method for determination of feed intake in salmonids using radioactive isotopes. Aquaculture, 24: 133-142. Vahl, O., 1979. An hypothesis on the control of food intake in fish. Aquaculture, 17: 221-229. Williams, C.H., David, D.J. and Iismaa, O., 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci., 59: 381-386.
Aquaculture,
277 Printed in The Netherlands
Feed Intake Measurements in Fish Using Radioactive Isotopes II. Experiments with Atlantic Salmon and Rainbow Trout in Sea-Pens TROND STOREBAKKEN and ERLAND AUSTRENG The Agricukural NLH (Norway)
Research Council of Norway, Institute of Aquaculture
Research, N-1432 As-
(Accepted 6 July 1987)
ABSTRACT Storebakken, T. and Austreng, E., 1988. Feed intake measurements in fish using radioactive isotopes. II. Experiments with Atlantic salmon and rainbow trout in sea-pens. Aquaculture, 70: 277-288.
Short-term feed intake was measured in 2419 Atlantic salmon (Salmo salar L. ) and 1232 rainbow trout (Salmo gairdneri Richardson), weighing 0.1-4.0 kg. The fish were kept in net-pens in the sea. They were hand-fed with a moist diet containing radioactive Ag13iIfor a 2-h period. The radioactive diet was either fed to fish which had been deprived of feed for 12-15 h to assess the “maximum meal size” or to fed fish to assess “the influence of previous feeding on subsequent feed intake”. The amount of radioactive feed ingested by the fish was measured with a portable gamma scintillation counter placed under their stomachs. The accuracy of this method when using large fish was checked, and the fish were monitored for possible radioactive contamination. The feed intake was further assessed by weighing the stomach contents of 16 salmon. The results showed that feed intake can be measured in large fish using radioactive isotopes. No radioactive contamination was found in the fish. When the water temperature was lo-13°C the intake of moist feed amounted to 6-12% of the fish weight during the first 2-h period after the deprivation period. When feed intake during this first period was high, it became low during the rest of the day. When the water temperature was 5-6” C, feed intake after the deprivation period was only 2-3% of the fish weight, and the fish ingested relatively more feed in later meals. There was no significant difference in feed intake for the two species of fish. A mean feed intake of 4.1% moist feed was recorded from the weight of stomach contents in the separate experiment which was carried out at a water temperature of 14-15°C.
INTRODUCTION
A method to assess feed intake in fish using radioactive isotopes has been published previously (Storebakken et al., 1981). This method permits measurement of feed intake without disturbing, hurting or killing the fish. This
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers B.V.
278
eliminates the stress caused by bringing the fish to the laboratory. The method was developed while working with rainbow trout (Salmo gairdneri Richardson) weighing less than 1 kg. One aim of the present experiments was to study whether the method could also be utilized on larger Atlantic salmon (Salmo salur L.) and rainbow trout. A second aim was to find out whether the fish absorbed any of the orally administered Ag1311.A previous study on rainbow trout in fresh water demonstrated considerable variation in connection with feed intake measurements (Storebakken and Austreng, 1988). The method, however, has been a valuable tool in experiments comparing different feeding treatments (Storebakken, 1985; Storebakken and Austreng, 1987). Feed intake is one of the important regulators of fish growth. Vahl (1979) described a model in which only two parameters are necessary to design a feeding regime which might result in maximum growth in an aquaculture system. These are the maximum voluntary feed intake in one meal and the evacuation rate of the stomach. Hence, a third aim was to measure one of these parameters, namely the maximum voluntary feed intake. According to Ishiwata (1968) both the acclimatization and the hunger of the fish should be emphasized in feeding experiments. To study variations in appetite, feed intake was measured in both feed-deprived and fed fish which were kept in different types of netpens at different water temperatures. MATERIALS
AND METHODS
The present study consists of four different experiments, all of which were carried out at the Institute of Aquaculture Research at Averpry. The four experiments were conducted 26-28 June 1979 (Expt. 1) ,23-25 September 1980 (Expt. 2), 2-4 December 1981 (Expt. 3), and 15-23 July 1985 (Expt. 4). Daylength was 18 h in Expts. 1 and 4 (long day), 12 h in Expt. 2 (decreasing daylength), and 6 h in Expt. 3 (short day). Atlantic salmon and rainbow trout weighing between 0.1 and 4.0 kg were used in the experiments. The fish were kept in net-pens which had a volume of either 27 m3 or 500 m3 in the sea (salinity: 30-32%0). The fish in each pen were sorted by weight. The mean coefficient of variation (C.V. = 100~ s/x, s= standard deviation, X=mean value) of the fish weight within pens was 35%. The fish were transferred from larger to smaller pens at least one month prior to the experiments to allow them to adapt to the more confined rearing conditions. The fish utilized in Expts. 1,2 and 3 were accustomed to hand-feeding more than 10 times a day with a wet feed consisting of 10% binder meal and 90% trash fish. During a preparation period of at least 2 weeks and throughout the experiments the fish were fed a moist diet which was commercially available (“Tess Salmomix loo”, T. Skretting A/S, Stavanger, Norway). The formulation and chemical composition of this diet are presented in Table 1. The diet
279
TABLE 1 Formulation and chemical composition silver iodide
Ingredients (g ) Capelin meal (Nor Sea Mink) Soybean meal (extracted) Wheat (cooked) Capelin oil (Nor Salm Oil)’ Vitamin and micromineral premix’ Alginate and guar gum mixture Water Chemical analyses (g) Dry matter (DM) Content in DM Crude protein Crude fat Ash Crude fibre Nitrogen-free extract Metabolizable energy (ME) ( MJ ) ’ ME from protein ( % )
of the diet. This diet was supplemented
with radioactive
247 137 99 122 17 28 350 562 459
240 84 37 176 18.0 42
“Supplemented with 200 mg ethoxyquin per kg oil. bMinimum content per kg diet according to the producer’s specifications: vitamin A, 2500 i.u.; vitamin Kt, 10 mg; vitamin E, 60 mg; thiamine, 10 mg; riboflavin, 25 mg; pyridoxin, 15 mg; pantothenate, 40 mg; niacin, 150 mg; folic acid, 5 mg; biotin, 0.2 mg; choline, 600 mg; inoeitol, 100 mg; ascorbic acid, 200 mg. The vitamins B12, D3 and p-aminobenzoic acid and microminerals are added in unspecified amounts. “ME from protein and fat calculated according to Phillips and Brockway (1959) (16.3 kJ ME/g protein; 33.5 kJ ME/g fat). ME from NFE calculated according to Singh and Nose (1967) (13.8 kJ ME/g cooked starch).
was pelleted using a lo-mm die for fish with a mean weight up to 1 kg. For fish larger than this a 15-mm die was used. The moist diet was analysed for dry matter ( 105 ’ C, overnight ) , ash (550 oC, overnight), crude fat (Folch et al., 1957 ), crude protein (Kjeldahl method) and crude fibre (AOAC, 1975) content. Nitrogen-free extract was calculated by difference. Radioactive feed and feed intake measurements Radioactive silver iodide (Ag1311)was added to the diets in amounts of 0.45 mCi/kg moist pellet per 26 June 1979 in Expt. 1,0.34 mCi/kgper ‘23September 1980 in Expt. 2, and 0.35 mCi/kg per 2 December 1981 in Expt. 3. In Expt. 1 the homogeneity of the moist diet was checked by adding 0.20% Cr,O, to the
280
diet by the same procedures as for the Ag13’I. The chrome content in two samples of feed was analysed by atomic absorption (Williams et al., 1962). The homogeneity of the blend in Expt. 2 was checked in four samples by measuring the radioactivity with a “1280 UltraGamma” well gamma scintillation counter ( LKB-Wallac, Finland). The reproducibility of the readings with the “NE 5013” gamma scintillation counter was checked with moist pellet standard containing Ag1311in Expt. 2. The radioactivity of the moist pellet standard was measured in a plastic bag placed on the top of the NaI crystal under field conditions. One week after Expt. 2 the procedure was repeated with the same standard and a “Mock iodine-131 gamma counter checking source” (Radiochemical Centre, Amersham, England) in the laboratory. The radioactivity contained in the checking source was equivalent to 0.1 mCi’ 1311.The instrument was checked with this checking source under field conditions in Expt. 3. The radioactive diet was fed every 10 min for 2 h. A feeding period of only 2 h was chosen to facilitate the measurement of a large number of fish. The measuring period was limited to 4 h after feeding in Expts. 1 and 2 to ensure that all radioactive material was present in the anterior part of the gastrointestinal tract (Storebakken et al., 1981). The measuring period was set 2 h longer in Expt. 3 due to a lower water temperature and, thus, a slower rate of gastric evacuation. Feed intake measurements were started immediately after completion of the feeding period with the radioactive diet. The fish were randomly sampled from the pens, anaesthetized with chlorobutane and weighed. Radioactivity measurements were conducted on all fish from the 27-m3 pens, while up to 420 fish were utilized from the 500-m3 pens. Radioactivity was measured under the stomach for two 10-s periods with a Scalar Spectrometer “NE 5013” gamma scintillation counter (Nuclear Enterprises, England) as described by Storebakken et al. (1981). The counts were corrected for background irradiation. The amount of radioactive material ingested was calculated as described by Storebakken et al. ( 1981) by means of the following equation:
F=-0.0494+ 0.00023M where F represents the amount of radioactivity ingested Q.&i) and M is the radioactivity measuredunder the fish stomach (counts/l0 s). Feed intake then was calculated based on the amount of radioactivity added to the feed. Radioactive feed was offered to different groups of fish according to three feeding procedures:
281 Procedure 1 Deprivation 12-15 h Radioactive diet 2h Counting
Procedure 2 Deprivation 12-15 h Radioactive-free diet 4h Radioactive diet 2h Counting
Procedure 3 Deprivation 12-15 h Radioactive-free diet 6h Radioactive diet 2h Counting
Procedure 1 was carried out to measure the “maximum meal size”, while procedures 2 and 3 were carried out to measure “the influence of previous feeding on subsequent feed intake”. The numbers of fish and pens used for the different feeding procedures are presented in Table 2. The stomach contents from 25 rainbow trout which weighed between 0.3 and 3.5 kg were sampled after counting the ingested amount of radioactive material in Expts. 1 and 2. This was done in order to ascertain whether the feed intake measurements necessitated any corrections for fish size. The radioactivity in the stomach contents was measured in the well counter as described by Storebakken et al. (1981). In addition, four salmon from Expt. 2, which weighed between 1.4 and 2.9 kg, were sampled and treated in the same manner as described for the rainbow trout to determine whether there was any need for a separate procedure for the salmon. Fifty fish from the groups which ingested most radioactive feed were randomly sampled and slaughtered 2 days after the feed intake measurements to determine whether they were contaminated by Ag1311. The fish were gutted and washed, and carcass radioactivity was measured with the “NE 5013” counter. Further, four rainbow trout were slaughtered 10 h after feeding the radioactive diets. Duplicate 2.5-g samples of flesh taken near the dorsal fin, intestine adipose tissue, and the liver were removed from each fish for measurement of radioactivity using a well counter. The counting was carried out after the samples had been stored frozen for 5 days. The fourth experiment was conducted to measure the feed intake of salmon which were accustomed to hourly feedings of moist pellets using an automatic feeder. Salmon in a 500-m3 sized pen were treated as described above in “procedure l”, but without radioactivity in the diet. The salmon were fed a moist pellet composed of 55% fish silage and 45% binder meal. When the feeding was finished, 16 salmon which varied in weight between 1.1 and 2.9 kg were sampled randomly. One week later, 12 salmon weighing between 0.7 and 1.7 kg were sampled randomly from the same pen after a food deprivation procedure identical to the one outlined above for ‘Lprocedure 1”. The stomach contents were weighed and expressed as a percentage of body weight.
282 RESULTS
Evaluation of the radioisotope method Tests run on the homogeneity of the blend of the radioactive diet revealed only minor variations in the samples. Both feed samples in Expt. 1 contained 0.21% Cr,O,. The radioactivity in the four samples from Expt. 2 averaged 219 000 counts mine1 g-l, with a C.V. of 2.6%. Results concerning the reproducibility of repeated countings with the “NE 5013” are presented in Fig. 1. They show that the instrument was very unstable during the first 24 h of Expt. 2. Results from this period are indicated with brackets in Table 2. After adjustments were made to stabilize the instrument, the readings from the moist pellet standard were slightly above 80 000 counts/l0 s. One week later the instrument in the laboratory read 81000 counts/l0 s (corrected for decay). The “gamma counter checking source” read 2600 counts/l0 sunder the same conditions. The instrument was stable during Expt. 3. The mean counting value obtained by using the “checking source” in Expt. 3 was 3100 counts/l0 s, which was 21% higher than the values obtained in Expt. 2. The counting efficiency, thus, varied from one experiment to another. The regression of the measurement of feed intake made with the “NE 5013” on the values obtained with the corresponding stomach samples was:
Experiment Moot
2
pe~fef
standard
Expert men t 3 Gnmma
0212
0312
checkrng source
0412
0512
Dafe
Fig. 1. Test results on the reproducibility of repeated countings with the “NE 5013” gamma scintillation counter.
283
FS= -3.04+1.36FC+O.40
W
(R2=0.96)
where FS is the amount of radioactive feed ingested (g) calculated from the radioactivity in the stomach samples, FC is the feed intake (g) calculated from the readings under the stomach, and W is the fish weight (kg). FC was the only parameter which contributed significantly (P IO.001 ) to the regression. The regression shows that the FS values were slightly lower than the calculated FC. The mean ratio between FS and FC for the four salmon from Expt. 2 was 0.78 (s=O.12), which was within the range of variation noted in the rainbow trout material. The feed intake was accordingly calculated by the same equation for both species. In Expts. 2 and 3, respectively, the mean FS/FC ratios were 0.96 (n=18 fish, s=O.17) and084 (rz=8, s=O.14). No radioactivity was detected in any of the 50 carcasses sampled in the study for radioactive contamination. In the samples taken from the four trout, mean amounts of 0.04 nCi/g flesh (s=O.Ol), 0.00 nCi/g adipose tissue, and 0.35 nCi/g liver (~~0.14) were found. Feed intake measurements During the first 15-30 min of feeding in the “procedure 1” treatment, the fish were very active when the feed was offered. Feeding activity then decreased. During “procedure 2” and “procedure 3” the fish seemed less interested in the feed. The counting results normally were within the range of 200 counts/l0 s to 100 000 counts/l0 s, and the background irradiation represented 200 to 600 counts/l0 s. A regression analysis of feed intake on weight of fish for each pen explained only a fraction of the total variation present in the data. In the regressions for rainbow trout, the R2 values were 0.0-0.1. For salmon, they were 0.0-0.4, with those in only three of the 16 pens exceeding 0.2, and that in one pen approaching 0.4. The different feeding procedures did not result in different R2 values. Therefore, within each pen, other factors appeared to be more important than fish size in controlling short-term feed intake. The results from the feed intake measurements are presented as mean values in relation to the various feeding procedures and net-pen sizes in Table 2. In Expt. 1, a high feed intake was obtained for both rainbow trout and salmon by “procedure 1”. The feed intakes of fish obtained by procedures 2 and 3 were much lower. Both rainbow trout and salmon in the 27-m3 pens exhibited more active feeding behaviour when they were fed in Expt. 1 than in Expt. 2 and this was reflected in the feed intake measurements. During Expt. 3, the feed intake was reduced, and the number of fish which did not eat during “procedure 1” also increased. In Expt. 3, the feed intake of fish during “procedure 2” relative to “procedure 1” also increased when compared with the two other experiments. Neither of the results revealed significant differences between the two
284 TABLE 2 Feed intake of the fish presented as % of fish weight ingested in a P-hour feeding period” Temp. (“C)
Fish number
Sea-pen Size (m3)
Number
Fish size (kg) X
Rainbow trout Expt. 1 11.6 Proc. 1 11.7 Proc. 2 11.8 Proc. 3 10.8 Proc. 1 Expt. 2 12.9 Proc. 1 12.9 Proc. 2 Expt. 3 5.1 Proc. 1 4.8 Proc. 2 Salmon Expt. 1 Proc. 1 Proc. 2 Proc. 3 Proc. 1 Expt. 2 Proc. 1 Proc. 2 (Proc. 1 Expt. 3 Proc. 1 Proc. 2
27 27 27 500
75 71 76 320
1.56 1.55 1.74 1.71
Feed intake f%) s
-
Percent of fish with no intake
X
s
8
s
12.5 0.6 0.5 10.3
-
2.7 49.3 67.1 8.8
-
27 27
2 2
241 98
1.93 1.84
0.05 0.04
6.3 0.9
1.3 0.1
9.5 44.9
4.9 0.0
27 27
2 3
206 145
1.85 2.24
0.02 0.03
1.9 0.9
0.0 0.1
19.3 31.0
5.8 4.5
74 74 75 337
1.98 1.98 2.10 1.50
11.1 0.3 0.5 9.6
-
2.7 75.7 67.1 0.9
-
-
11.6 11.7 11.8 11.6
27 27 27 500
12.9 12.9 12.9
27 27 500
4 4 1
195 197 422
0.97 1.00 1.24
0.05 0.05 -
7.4 4.5 17.9
1.4 0.7 -
19.8 48.2 3.3
4.4 7.2 -_)
5.6 5.5
500 500
1 2
355 690
1.44 1.47
0.04
2.8 1.4
0.3
31.0 33.0
1.1
“The results are presented as means (x) and standard deviations (s) between different pens.
species or between the two pen sizes as far as the feed intake was concerned. The number of fish which had not eaten was inversely related to the feed intake values for each pen. The mean C.V. values for the feed intake of fish obtained by “procedure 1” in Expts. 1, 2 and 3, respectively were: 70.1, 88.4 and 85.9% for the rainbow trout and 50.9, 79.9 and 102.6% for the salmon. No differences between procedures 2 and 3 were observed and the mean C.V. values, in the same order as listed above, were 188.7, 155.1 and 117.4% for the rainbow trout and 269.1, 108.8 and 118.2% for the salmon. These results show that both decreased water
temperature and a previous feeding resulted in increased variation in feed intake. Water temperature was 14-15’ C during Expt. 4. The feed intake of salmon varied from 1.7 to 6.6% of their body weight. There was no significant effect of fish size on feed intake, but the fish which had eaten the least were sexually maturing males. No feed was present in the stomachs of the group of fish which had been starved for 12-15 h. DISCUSSION
The comparison between the amount of radioactive material measured outside the fish (FC) to that in the stomach contents (J’S) indicates that the radioisotope method (Storebakken et al., 1981) can be applied for feed intake studies on large fish. A non-systematic bias, however, is indicated by the high standard deviation of the mean FS/FC ratios and the instability of the gamma scintillation counter during the first 24 h of Expt. 2. This bias may have been caused by several factors in addition to variation in the counting efficiency of the instrument. But efforts were made to eliminate those factors. The background irradiation was compensated for. The blending of the diets was homogeneous. The results in Fig. 1 indicate that the variation in the counting efficiency within experiments was small when the instrument was stabilized. Variation in the shape of the fish is another possible source of error since the stomach of a fish with a wide, U-shaped belly is relatively closer to the NaI crystal of the counter than the stomach of a fish with a straighter belly. Fish size is another possible source of error, but this was not found to be an important factor in this study. A systematic bias in the counting results is indicated by the higher counting results from the checking source in Expt. 3 relative to Expt. 2. Based upon the radioactivity measurements of the carcasses and tissue samples, it can be concluded that the fish did not accumulate radioactive silver iodide into body tissues. The observed levels of radioactivity of 0.0-0.4 nCi/g tissue were within the tolerance limit for “unrestricted waters” (IAEA, 1962). These amounts of radioactivity are so small that they may even represent a contamination from gutting the fish. The 1311isotope has a half-life of 8.3 days. Within a few days the measured amounts of radioactivity would have decayed beyond the limits of detection. The maximum feed intake obtained by “procedure 1” was high but consistent with the values obtained by Ishiwata (1968) and Grove et al. (1978) and with the critical stomach volumes found by Kariya et al. (1968)) all in rainbow trout. The fact that fish weight did not significantly influence the feed intake in the pens was surprising since fish size is an important factor in determining growth (Brett, 1979). A possible explanation is that the effect of fish size on
286
feed intake was masked by other factors than the ones that were monitored. This is suggested by the high non-systematic variation found for feed intake in this study. Austreng (1979) found that rainbow trout require about 15.5 MJ ME/kg growth. Based on this, the maximum feed intake found in Expts. 1 and 2 should have been sufficient to support a growth of 4-8%/day. Salmon weighing 150200 g can grow at a rate approaching 2%/day at a water temperature of 14”C, while the growth of rainbow trout and salmon maintained in sea-pens has been observed to be slower ( Austreng et al., 1987 ). The maximum ration consumed in one meal, thus, far exceeds the requirement for growth under a regime of constant feed intake. This high feed intake capacity, however, is consistent with results obtained with rainbow trout in freshwater tanks (Storebakken, 1985; Storebakken and Austreng, 1987). The feed intake obtained by “procedure 1” was higher in Expts. 1 and 2 than in Expt. 3. This concurs with the well-documented relationship between the water temperature and feed intake, reviewed by Brett and Groves (1979). Despite similar fish sizes and comparable water temperatures in Expts. 1 and 2, the results of the two experiments were considerably different. The water temperature in Expt. 2 was slightly higher than that in Expt. 1, but it remained below the optimum temperature for growth found for juvenile rainbow trout and salmon (Hokanson et al., 1977; Farmer et al., 1983). Accordingly, the increased water temperature should have resulted in an increase rather than a decrease in growth. The reduced feed intake of fish in Expt. 2 is likely to be attributed to factors other than water temperature. One of the most probable of these is photoperiod, since the general effect of decreasing daylength on freshwater fish is reduced growth (Brett, 1979). The reduced feed intake of fish noted in Expt. 4, in comparison with fish in Expts. 1 and 2, probably is an effect of acclimatization (Ishiwata, 1968). The salmon in Expt. 4 were adapted to hourly feeding, whereas the others were adapted to meal-feeding about ten times a day. The difference in available energy content and bulkiness between a wet and a moist diet in the previous feeding of the fish may also have contributed to a relatively lower feed intake capacity in the salmon of Expt. 4 (Grove et al., 1978). There are, however, factors other than diets and feeding strategy complicating the comparisons between Expts. l-3 and 4. The salmon of Expts. l-3 originated from the first generation of a family selection programme (Gjedrem, 1983). Those of Expt. 4 came from the second generation of this selection programme and the genetic growth potential was different. Moreover, the salmon utilized in Expt. 4 were the survivors of an outbreak of “Hitra disease” (Poppe et al., 1984). The others had no history of major health problems. The effects of the foregoing factors on the feed intake are difficult to assess. The feed intakes of the two species did not differ significantly during the study. This is consistent with the growth results obtained for the two species
of fish reared in the sea but in contrast to the results obtained in fresh water ( Austreng et al., 1987). The experiments show that the feed intakes of fish in this study were strongly influenced by previous feeding or feed deprivation time. This is consistent with what Adron et al. (1973) and Grove et al. (1978) found with rainbow trout. The differences found between Expts. 1 and 2 for the ratios between the feed intake obtained by “procedure 2” and “procedure l”, however, indicate that factors other than fish size, deprivation period and water temperature influence the appetite and the time required for the fish to reach satiation. Several factors may have caused these differences, such as the light intensity and photoperiod (see review by Brett, 1979) (Expt. 1, clouded, near maximum daylength; Expt. 2, sunny, decreasing daylength), and degree of adaptation to the pens, the time of the year and the stage of sexual maturation. ACKNOWLEDGEMENTS
We thank Mr. Torkjell Brers, Ms. Anne Dullum, Mr. Helge Lien and Mr. Georg Ostby for skillful technical assistance, and the Agricultural University of Norway, Department of Poultry and Fur Animal Science for helpful collaboration.
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Hokanson, K.E.F., Kleiner, C.F. and Thorsland, T.W., 1977. Effect of constant temperature and die1 fluctuation on growth, mortality, and yield of juvenile rainbow trout, Salmo gairdneri (Richardson). J. Fish Res. Board Can., 34: 639-648. IAEA, 1962. Basic Safety Standards for Radiation Protection. Safety Series No. 9. International Atomic Energy Agency, Vienna, 59 pp. Ishiwata, N., 1968. Ecologicai studies on the feeding of fishes. IV. Satiation curve. Bull. Jpn. Sot. Sci. Fish., 34: 691-693. Kariya, T., Shirahata, S. and Nakumura, Y., 1968. An experiment to estimate the satiation rate of feeding in fish. Bull. Jpn. Sot. Sci. Fish., 34: 29-34. Phillips, A.M. and Brockway, D.R., 1959. Dietary calories and the production of trout in hatcheries. Prog. Fish. Cult., 21: 3-16. Poppe, T., H&stein, T. and Salte, R., 1984. Hitra disease (Haemorrhagic Syndrome ) in Norwegian salmon farming: present status. In: A.E. Ellis (Editor), Fish and Shellfish Pathology. Academic Press, London, pp. 223-229. Singh, R.P. and Nose, T., 1967. Digestibility of carbohydrates in young rainbow trout. Bull. Freshwater Fish Res. Lab., Tokyo, 17: 21-25. Storebakken, T., 1985. Binders in fish feeds. I. Effect of alginate and guar gum on growth, digestibility, feed intake and passage through the gastrointestinal tract of rainbow trout. Aquaculture, 47: 11-26. Storebakken, T. and Austreng, E., 1987. Ration level for salmonids. II. Growth, feed intake, protein digestibility, body composition, and feed conversion in rainbow trout weighing 0.5-1.0 kg. Aquaculture, 60: 207-221. Storebakken, T. and Austreng, E., 1988. Feed intake measurements in fish using radioactive isotopes. I. Experiments with rainbow trout in freshwater. Aquaculture, 70: 269-276. Storebakken, T., Austreng, E. and Steenberg, K., 1981. A method for determination of feed intake in salmonids using radioactive isotopes. Aquaculture, 24: 133-142. Vahl, O., 1979. An hypothesis on the control of food intake in fish. Aquaculture, 17: 221-229. Williams, C.H., David, D.J. and Iismaa, O., 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci., 59: 381-386.