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Crude and pre-extruded products of wheat as nutrient sources in extruded diets for Atlantic salmon (Salmo salar, L) grown in sea water
Aquaculture, 118 (1993) 105-l 17 Elsevier Science Publishers B.V.. Amsterdam
105
AQUA 50109
Crude and pre-extruded products of wheat as nutrient sources in extruded diets for Atlantic sallmon (Salmo salar, L) grown in sea water Petter Arnesen and Ashild Krogdahl’ AKVAFORSK (Institute ofAquaculture Research), Aas. Norway (Accepted 8 June 1993)
ABSTRACT Atlantic salmon with initial mean weight 175 g, grown in sea water for 10 1 days, were fed extruded diets containing either 0, 15, 30 or 45% crude whole wheat (WC), pre-extruded whole wheat (WE) or pre-extruded after-meal (AE). After-meal is a protein-rich wheat by-product. In the WE diet series, we found growth (weight increase) to be significantly lower in the 45% diet than in the control diet, but although not statistically significant, there was a tendency towards reduced growth also in the other diet series, with increasing dietary inclusions of wheat. Condition factors were not affected by diet, and no significant differences were found in proximate carcass composition between fish fed the control diet or the diet contributing the highest amount of energy as carbohydrates. Plasma glucose, liver glycogen and hepatosomatic indices were within the normal ranges for all diets. No significant effects of wheat inclusion level were found on digestibilities of protein or lipid in any of the diet series. Starch digestibility was significantly higher in the diet containing 45% pre-extruded after-meal than in the diets containing 45% pre-extruded whole wheat or 45% crude whole wheat. The highest inclusions of wheat resulted in reduced dry matter digestibility in the preextruded whole wheat series and in the crude whole wheat series.
INTRODUCTION
Evaluation of feedstuffs of plant origin as feed ingredients for Atlantic salmon and rainbow trout has been the main objective of a recent research programme undertaken at our institute. Whole ground wheat has for many years been the main carbohydrate ingredient in fish feeds in Norway. It is used partly due to its binding properties and partly because the wheat carbohydrates are believed to spare protein by reducing the amount of protein used to supply metabolic energy (Bergot, 1979). The productive energy value of carbohydrate (i.e. how much of the carbohydrate that is actually utilized for energy) for Atlantic salmon is not known, and even though carbohydrate-rich Correspondence to; Petter Arnesen, BIOMAR A.S., P.O.Box 309, Sentrum, N-0103 Oslo, Norway. (Present address). ‘Present address: Norwegian College of Veterinary Medicine, Oslo, Norway.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved,
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P. ARNESEN AND A. KROGDAHL
feedstuffs have been used in fish feeds for many years, we have found only one scientific work dealing with the value of carbohydrate feedstuffs as nutrient sources for Atlantic salmon (Hughes, 1990). There are a number of studies dealing with feeding high-carbohydrate diets to rainbow trout, all carried out in fresh water. Atlantic salmon grown in sea water is the most important aquaculture species in Norway, and the present study was conducted in order to determine: ( 1) the effects of 3 different wheat products in practical diets for Atlantic salmon on health, growth and nutrient digestibilities; and (2) the maximum tolerable level of digestible carbohydrates from wheat in practical diets. MATERIALS
AND METHODS
Experimentaldiets Three wheat products, crude whole wheat (WC), pre-extruded whole wheat (WE) and pre-extruded after-meal (AE), were tested. After-meal is a protein-rich fraction which is removed after the bran in the milling process, i.e. layers of the kernel located immediately beneath the bran. Pre-extruded wheat products were subjected to extrusion before diet preparation. Thus, these products were extruded twice as all diets were extrusion-pelleted. All wheat products (produced from the same batch of wheat) were ground and screened ( < 1 mm) prior to diet preparation. The chemical composition of the wheat products is shown in Table 1. The control diet contained 25% wheat bran, The other three products were incorporated at levels of 15,30 and 45%, in exchange for bran, fish meal and capelin oil. Nutrient composition and proximate chemical analysis of the diets are shown in Table 2. When preparing the diets, adjustments were made for the content of fish meal and capelin oil in such a manner that estimated digestible energy from wheat carbohydrate mainly substituted energy from lipid. TABLE 1 Proximate composition
of wheat products (O/o)
Wheat product
Water
Crude protein
Lipid
Ash
Dietary fibre
Whole wheat Crude Extruded
15.7 11.2
13.5 14.0
2.8 2.8
1.6 1.6
12.0 11.4
8.0
18.3
4.5
2.7
6.9
10.8
15.7
4.3
6.4
40.0
After-meal Extruded Bran
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WHEAT CA.RBOHYDRATE DIETS FOR ATLANTIC SALMON TABLE
2
Composition of control diet, whole crude wheat diets (WC), whole extruded wheat diets (WE) and aftermeal diets (AE) Diet Control Ingredient (g/kg) Bran 250 Whole 0 Crude Extr. 0 A-meal 0 Extr. Fishm. 544 Fish oil 160 Binder 20 Finnst. 10 V.M. mix 10 1 Vit. C 1 Asta 4 Chr. ox DE (MJ)’ 15.5 Chemical analysis (%) 89 DM Protein 40 24 Lipid Starch 8 Ash 8.2 10.0 Fibre 0.34 Chr. ox 18.2 DE (MJ):’ % from: protein 45 lipid 49 starch 6 P/E ratio 22
WC15
WC30
WC45
170
80
0
150 0
300 0 0 434 140 20 10 10 1 1 4 14.0
0
486 148 20 10 10 1
1 4 14.7
WE15
WE30
WE45
AEl5
AE30
AE45
170
80
0
170
80
0
450 150
0 300
0 450
0
0 0
0 0
0
0 376 128 20 10 10 1 1 4 13.1
0 486 148 20 10 10 1 1 4 14.7
0 434 140 20 10 10 1 1 4 14.0
150 486 148 20 10 10 1 1 4 14.7
300 434 140 20 10 10 1 1 4 13.9
450 376 128 20 10 10 1 1 4 13.0
0
0
376 128 20 10 10 1 1 4 13.1
92 40 20 12 7.7 8.6 0.31 16.3
93 38 19 20 7.1 6.8 0.34 17.6
92 35 17 28 5.2 5.4 0.33 17.2
92 40 20 12 7.6 8.6 0.31 15.9
92 36 17 21 6.4 6.8 0.31 14.4
93 34 17 29 6.1 5.4 0.35 14.5
89 38 19 12 7.4 7.8 0.35 15.8
92 36 21 19 6.7 5.2 0.29 18.1
92 35 18 26 5.7 3.0 0.34 18.0
49 45 6 24
45 41 14 21
42 37 21 20
48 46 6 25
46 44 10 25
46 44 10 23
48 44 8 24
40 43 17 20
39 37 24 19
‘Theoretical value, ‘Real value. Extr.=extruded; A-meal = after meal; Fishm. = Norse-LT94 fish meal; Finnst. = Finnstim@; Asta=astaxanthin; Chr. ox=Cr203; DM=dry matter; DE=digestible energy; P/E ratio=protein/energy ratio.
Digestible energy (DE) values used were 39.5 kJ/g for lipid, 23.6 kJ/g for protein and 17.3 kJ/g for carbohydrate (Phillips and Brockway, 1959). When preparing the diet formulas, the digestibility coefficients used for lipid, protein and starch were 90, 85 and 40%, respectively (values based on empirical data from our research stations). Real estimations of DE in the diets were based on the apparent digestibility coefficients calculated in the study. Theoretical values for DE content, and real estimations, are shown in Table 2. The experimental diets were prepared by a commercial feed producer. All diets were extruded into 5-mm pellets. Daily feed rations supplied 120% of
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P. ARNESEN AND A. KROGDAHL
calculated energy requirement needed to support daily specific growth rate (Austreng et al., 1987). Automatic feeders, active during the day-light hours, were used to deliver the feed. Chromic oxide was included in the diets (0.4%) as an indicator in the digestibility calculations. In the following, each series of diets (0, 15, 30 and 45%) containing the same wheat product. is referred to as a series. Fish and experimental conditions Atlantic salmon (initial mean weight 175 g) were kept in 27 m3 net pens in sea water, 285 fish (50 kg) per pen. Water temperature was measured daily. It decreased from 13.5 to 7.O”C during the feeding trial. Four pens of fish were fed the control diet. The other diets were fed to two pens. Fish were weighed at the start, after 3 8 days and at the end of the experiment, after 10 1 days of feeding. All fish in each pen were weighed in groups (around 20 kg of fish in each group), thereby making it possible to derive the correct mean weight of the fish. Daily feed requirement was adjusted according to estimations of daily specific growth rate based on water temperature and body weight (Austreng et al., 1987). Two weeks before the termination of the experiment, blood samples were collected (heparin used as anticoagulant and fluoride as a glycolysis inhibitor) from 10 fish in each pen. The normal feeding routine was upheld during sampling, in order to keep the fish in the absorptive state. Fish without stomach contents were discarded and replaced. In addition to sampling blood, we also removed and weighed livers and viscera from 5 fish per group. After weighing, livers were immediately frozen at - 96’ C, using a freeze clamp, and kept at - 80°C until analysed for glycogen content. The 5 sampled fish from each of the four control diet pens and from the two 45% AE diet pens, together with the corresponding viscera, were kept for proximate analysis. After weighing and sampling, feeding continued for 2 weeks before feces were stripped as described by Austreng ( 1978). Feces from each pen were pooled. Chemical analyses Wheat products, diets and feces were analysed for dry matter content, nitrogen, total lipid, and ash. Diets and feces were also analysed for starch. Dry matter content was analysed by freeze-drying; crude protein according to the micro-Kjeldahl method (N x 6.25 ); total lipid by the micro-method according to Zollner and Kirsch ( 1962), modified by extracting the lipids with a mixture of chloroform and methanol (2 : 1); and ash by combustion for 15 h at 550°C. Chromic oxide in diets and feces was analysed using ICP spectroscopy, and dietary libre in wheat products using the AOAC method (Prosky et al., 1985). Analyses of chemical composition of fish and viscera were conducted after homogenizing samples in a food processor and freeze-drying. Crude protein
109
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
and ash were analysed as described above, and total lipid according to the Soxhlet-method after hydrolysis with 4 M hydrochloric acid for 30 min at 100°C. Dry matter content was determined by drying for at least 18 h at 20 mmHg and 72°C. Starch was gelatinized in a boiling water bath in the presence of a heatstable a.mylase (Termamyl 120 L, Novo A/S). This resulted in a partial degradation of starch to glucose. Complete hydrolysis of starch to glucose was accomplished by adding an amyloglucosidase (Boehringer Mannheim, kit no. 737 160). Amount of glucose was then assessed quantitatively using a Beckman glucose hexokinase assay (Dri-STAT@’ Reagent) and an Encore automatic analyser. Liver glycogen was converted to glucose according to the method of Dalrymple and Hamm ( 1973). Glucose analysis was then carried out as described above. Design and statisticalanalyses One-way ANOVA was used to test effects of wheat products. Least-square means (LSMEANS ) were calculated and used to test the least significant difference (LSD) between means. LSD tests were carried out on differences between means within the same series, using inclusion level as class variable, and between mean values within the same inclusion level, using wheat product as class variable. A two-way ANOVA with wheat product and inclusion level as class variables was employed to examine possible interaction effects between wheat product and inclusion level. Models and differences between means were considered statistically significant when P< 0.05. Calculations Nutrient digestibility:as described by Maynard et al. ( 1979) Dressing percentage: [Wet carcass weight (g)/whole wet body weight
(!3)1x100
Hepatosomatic index: [Wet liver weight (g)/whole wet body weight (g) 1x 100 Conditionfactor: [Whole wet body weight (g ) / (fork length (cm) ) 3] x 100 Absorbed starch: [Starch content (g/ 100 g diet) x starch digestibility (%) ] / 100
Growth:Final mean fish weight (g)-initial
mean fish weight (g )
RESULTS
Water temperatures were higher than normal for the season, and consequently the calculated growth (expressed as increase in total body mass) and feed requirement were higher than expected. In order to avoid an early termination of the experiment due to lack of feed, we restricted feeding by 20% during the last month. This might have influenced growth rate during the last
P. ARNESEN AND A. KROGDAHL
110
part of the experiment. We expect, however, the magnitude of such an influence to be equal for all diets, and not to have had any significant bearing on the conclusions of the investigation, The control fish had a weight increase of about 300 g, which was 80% of that calculated by using tables for daily specific growth rate ( Austreng et al., 1987). We had expected growth to be faster due to high water temperatures, and thus it was evident that there had been a considerable discrepancy between our estimations of daily weight increase (on which the daily feed ration sizes were based) and the actual increase. As a result, we had operated with a too large biomass, and had most probably overfed the fish during the first phase of the experiment. Although there seemed to be a tendency toward a reduction in growth performance as wheat inclusion increased (Table 3 ) , it was only in the WE series that comparison of means revealed a significant difference in growth between the control and the 45% wheat diet. There were no significant within-level differences in growth performance, and no interaction effects between wheat product and inclusion level. Feed efficiency ratios were not calculated in the present study since we did not have control of feed wastage. Whole body and viscera composition of fish fed the 45% AE diet was not different from that of fish fed the control diet (Table 4). Furthermore, neither diet nor inclusion level was found to influence condition factors, which TABLE 3 Growth (increase in body weight), plasma glucose, liver glycogen and hepatosomatic
index (HSI)
Diet/level
Growth (g)
Plasma glucose (mmol/l)
Liver glycogen (O/O)
HSI
Control WC 15 30 45
301 -t 305 + 285 k 264 +
6.7A 17.0Aa 7SAa 7.0Aa
6.0i0.4A 6.5k06A” 5.2? O.OAa 6.3f0.6Aa
1.9 2.0 2.6
1.8+0.2A l.5f0.0Aa 1.9fO.O*= l.7f0.1AB
Control WE 15 30 45
301 f 290 f 274+ 267 f
6.7* 4.0ABa l.SABa 3.0Ba
6.0 f 60.4A 5.4 k 0.4Aa 6.3 2 O.OAa 7.0+0.5*=
1.9 1.9 2.7 2.4
1.8 + 0.2* 1.9*0.4*= 1.7?0.2*’ 1.6 k O.O*”
Control AE 15 30 45
3Olk 6.7A 302+ 1.5~~~ 278 275 + O.OA”
6.0i0.4A 6.3+0.4*= 5.9 7.1 f0.2AB
1.9 2.0 2.3 1.9
1.8i0.2* 1.6 + O.O*= 1.5 1.7f0.1Aa
Mean valueskstandard error of mean (s.e.m.). s.e.m. co.05 are given as 0.0 in the table. Superscripts in capital letters refer to differences between mean values within the same dietary series. Superscripts in small letters refer to differences between mean values within the same inclusion level. -, missing value.
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
111
TABLE 4 Proximak
composition
(%) of wet fish and viscera
Diet/level
Fat
Crude protein
Ash
DM
Fish Control AE45
10.9kO.4 10.3kO.2
18.4kO.2 18.7kO.3
2.6fO.O 2.7fO.O
30.9f0.4 30.3f0.7
Viscera Control AE45
23.2f 1.9 25.0f2.8
10.6kO.l 1 l.OkO.4
1.3fO.l 1.3kO.l
36.6& 1.1 38.6k3.2
Mean values f standard error of mean (s.e.m). s.e.m. < 0.05 are given as 0.0 in the table. No superscripts have been assigned since no significant differences were found.
ranged from 1.08 to 1.16, or dressing percentages, which ranged from 87 to 89%. Plasma glucose levels, hepatosomatic indices and liver glycogen levels are shown in Table 3. Levels of all three variables were within the range considered to be normal, i.e. around 5-7 mmol/l, l-2 and 2-3%, respectively. ANOVA d:id not reveal significant effects of inclusion level on plasma glucose or hepatosomatic indices in any of the series, and there were no within-level differences. Because we had not analysed duplicate samples of liver for glycogen, we could not test differences between means for this variable. With. the exception of the AE series, where starch digestibility was found to be significantly lower in the 15% diet than in the other diets, there were no within-series differences in digestibility values for protein, lipid or starch (Table 5 ) . However, at the 45% inclusion level, mean starch digestibility was significantly higher in the AE series than in the WC and WE series. No significant interactions were found between wheat product and inclusion level for the above digestibilities. Comparisons of within-series means revealed a significantly higher dry matter digestibility in the 30% WC diet than in the 45% WC diet. In the WE series, (dry matter digestibility was significantly higher in the control diet than in any of the other diets, and it was higher in the 15O/bthan in the 45% diet. Dry matter digestibility in the AE series was significantly lower in the 15% diet than in the 45% diet. At the 45% wheat inclusion level, all series had significantly different dry matter digestibility, highest in the 45% AE diet and lowest in the 45% WE diet. There was a significant interaction effect between inclusion level and wheat product for dry matter digestibility (P= 0.000 1) . There was a significantly higher dry matter content (DM) in feces from the 45% WC fish (DM = 2 1%) than from the control group (DM = 17%). Furthermore, the dry matter content of feces from fish fed the AE 45% and WE
112
P. ARNESEN AND
A. KROGDAHL
TABLE 5 Digestibility of nutrients and dry matter (DM), and amount of absorbed starch Diet/level
Protein (%)
Lipid (%)
Starch (%)
DM (O/O)
Control WC 15 30 45
86f l.l* 84f 1.2Aa 89f3.1A” 88f0.6A”
94 k 0.6* 93 k 0.5*a 95 + 1.8*’ 95f0.2A=
86 *0.7* 5orf: 3.9Aa 7334+0.9*b
68 f l.OAB 63 f 2.5AB” 73 + 6.4A” 57 *0.3na
6.9 6.0 7.2 9.5
Control WE 15 30 45
86+ l.l* SO+ 1.8*” 78+4.8*’ 84+0.8*
94 + 0.6* 93 f l.OA’ 94 f 0.2*= 95 f 0.6Aa
86f0.7* 48 f 9.6*’ 41 f2.5*’ 28 ?I 5.6Ab
68k 1.0* 59 k 3.6na 55+0.3nca 49k 1.8cb
6.9 5.8 8.6 8.1
Control AE 15 30 45
8611.1* 84+ 1.9Aa 85 85+0.1A8
94&0.6* 95kO.lA”
86+_0.7* 6Ok 5.3B” 93 95 k 2.5*”
68i: l.OAB 61 f 3.5Ba 78 74 f 0.3AC
6.9 7.2 17.7 24.7
95 kO.gA”
Absorbed starch (g/100 g diet)
Mean valuesf standard error of mean (s.e.m.). s.e.m. ~0.05 are given as 0.0 in the table. Superscripts in capital letters refer to differences between mean values within the same dietary series. Superscripts in small letters refer to differences between mean values within the same inclusion level. -3 missing value.
45% diets (DM= 15% and 17%, respectively) that of fish fed the WC 45% diet (DM = 2 1%). matter content of all feces collected was within mal for salmonids ( 13-2 1o/o), based on results sen, 1992).
was significantly lower than However, the variation in dry a range we consider to be norfrom previous studies (Arne-
DISCUSSION
Mortality rates were negligible, and fish growth, which is a good indicator of the general condition of the fish, was satisfactory compared to the control, even at the highest incorporations of wheat. We have found only one study (Hughes, 1990) in the literature where Atlantic salmon have been fed diets high in wheat. In that study, small fish performed as well on diets containing either 18% wheat middlings or triticale, and neither of the two grains seemed to have any negative effect on fish health. There are a number of reports dealing with growth performance of rainbow trout fed diets high in carbohydrate. Hilton et al. ( 1982) found digestible carbohydrates in excess of 25% to cause growth reduction, a result which was confirmed by Hilton and Slinger ( 1983 ), who found significantly reduced growth when increasing the amount of digestible carbohydrates from approximately 25% to around 30%. Bergot ( 1979)) however, found that increasing
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
113
digestible carbohydrates in the diet from 15 to 30% had no adverse effects on growth, and Hilton et al. ( 1987 ) found no differences in growth performance between ad libitum feeding of diets containing 25% maize starch or a fishmeal-based control diet. In a recent study, Pfeffer et al. ( 199 1) found growth of rainbow trout fed diets containing around 29% maize starch per kg dry matter to be reduced when the maize was extruded prior to mixing. In the present study, growth performance of Atlantic salmon seemed to be affected by dietary wheat inclusion in a similar manner to what we have previously observed in rainbow trout (unpublished results by Krogdahl and Arnesen) in fresh water fed diets containing extruded wheat. A slight growth reduction compared to the control was observed in fish fed the highest wheat inclusion; however, this was significant only in the WE series. It is interesting to note that growth rate of fish fed the control diet was not significantly different from those fed the 45% AE diet, even though there was quite a large difference in energy contribution by protein in the two diets (45 and 39%, respectively). Total digestible energy in the control diet and the 45% AE diet were almost identical (18.2 MJ/kg and 18.0 MJ/kg, respectively ). Although our data analysis did not reveal major differences in growth between the different dietary treatment groups, inspection of the growth data suggest that the diets containing the highest inclusions of wheat did not support maximal growth. It is also possible that differences in growth between dietary treatments might have become more pronounced if the study had been longer than 101 days. The economic implications of including high levels of wheat in diets for salmon have not been considered in this study. To do so, we would have had to calculate feed costs and we would also need data on feed efticiency ratios. Ingestion of carbohydrate-rich diets did not cause a rise in plasma glucose levels in Atlantic salmon. In a similar study with rainbow trout in fresh water (unpulblished results by Krogdahl and Arnesen) we found that plasma glucose levels rose steeply when dietary inclusions of extruded wheat exceeded 30%. Elevated plasma glucose levels in rainbow trout ingesting large amounts of digestible carbohydrates has been observed by a number of workers (Bergot, 1979; Hilton and Slinger, 1983; Walton, 1986). Hilton and Atkinson ( 1982 ) and Hilton et al. ( 1982) did not, however, find differences in plasma glucose by feeding increasing levels of dietary glucose. It is possible, however, that the maximum glucose incorporation (2 1% and 25%) in these studies was too low to cause a rise in plasma glucose. According to the “glucostatic theory” of appetite control, a continuous elevation of blood sugar can lead to loss of appetite. Hilton and Slinger ( 1983 ) suggested this to be one explanation for the improved growth rate obtained in rainbow trout when dietary digestible carbohydrates were reduced. A high plasma glucose level in fish fed diets containing extruded starch might have contributed to a lower appetite and thereby the observed reduction in feed
114
P. ARNESEN AND A. KROGDAHL
consumption and weight gain observed in rainbow trout in the previously mentioned study by Pfeffer et al. ( 199 1). No mention of plasma glucose concentrations was made. No significant effect was observed in the present study of either wheat product or inclusion level on the hepatosomatic indices and liver glycogen levels, indicating that Atlantic salmon may differ from rainbow trout. The calculated glucose absorption was between 6 and 25 g per 100 g of diet (calculated values are shown in Table 5 ) . If we anticipate an ingestion of 1 g feed per g increase in growth, the total ingestion of feed during the experiment was around 300 g per fish. This brings the total absorption of glucose to between 18 and 75 g, which could not be traced either to the blood or to the liver. There was no excessive accumulation of glycogen in the liver. Increased deposition of glycogen in liver when feeding carbohydrate-rich diets has been shown in rainbow trout (Hilton and Atkinson, 1982; Hilton et al., 1982; Hilton and Slinger 1983; Walton, 1986; Pfeffer et al., 1991) and carp, red sea bream and yellowtail (Yone, 1979). Hemre et al. ( 1989), on the other hand, found that feeding carbohydrate-rich diets to cod resulted in an increase in hepatosomatic indices which was caused by fat deposition, not glycogen. Large depositions of glycogen in liver of rainbow trout have been shown to alter liver function and possibly upset normal excretion of toxic substances (Hilton and Dixon, 1982). Our results indicate that liver dysfunction, due to excessive glycogen deposition, did not occur in sea grown Atlantic salmon fed carbohydrate-rich diets. Neither protein nor lipid digestibility was affected by dietary carbohydrate level, a finding that is in agreement with studies on rainbow trout (Takeuchi et al., 1990; Kaushik et al., 1989; Pfeffer et al., 1991) and cod (Hemre et al., 1989). Starch digestibilities were high in the after-meal diets and increased with increased dietary level. Digestibility values of 95% are of a magnitude usually seen for glucose in rainbow trout (Kaushik et al., 1989) and most other monogastric animals (Maynard et al., 1979 ), suggesting that the aftermeal starch is very soluble in the intestine. We did not analyse the chemical composition of the available carbohydrate fraction of after-meal, but judging by its high digestibility, the ratio between amylose and amylopectin in the starch is probably high. The presence of sugars is also known to be high in the aleurone layer (Saunders, 1978) of which the after-meal largely consists. Starch and sugars might have been subjected to some degree of degradation by bacterial enzymes in the hind-gut, something that would have influenced their recovery in the feces. We are not aware of any study, however, that points to any major bacterial breakdown of carbohydrates in the hind-gut of salmonids. Microflora of coldwater fish living in sea water are dominated by IG’ibriobacteria (Cahill, 1990; Onarheim and Raa, 1990). The observation that there were no differences in starch digestibilities between whole crude wheat and whole extruded wheat
WHEAT CARBOHYDRATE
DIETS FOR ATLANTIC SALMON
115
diets suggests that pre-extrusion did not have any beneficial effect on starch digestibility in this study. Dry matter digestibility values varied considerably, and since there were no significant effects of wheat incorporation level on the digestibility of protein or lipid, most of the variation in dry matter was probably due to variations in starch digestibility and dietary Iibre content. The accuracy of the digestibility measurements might have been influenced by the rather low chromic oxide inclusion (0.4% included) in the diets. Lied and Julshamn ( 1989), in a study with cod, showed that 1% chromic oxide was necessary in order to minimize the non-systematic variation. However, this should not affect the relative effects of our study. Moreover, the variation between replicates in the recovery of chromic oxide in feces was small. The general effects of feeding Atlantic salmon carbohydrate-rich diets seemed to be somewhat different to those seen in rainbow trout fed similar diets. In many studies, rainbow trout have responded to carbohydrate-rich diets by exhibiting elevated plasma glucose levels and reduced growth rate. Results from the present study indicate that the handling of digestible carbohydrates is different in Atlantic salmon grown in salt water than in rainbow trout in fresh water. Whether this is a species difference or an environmental effect i(water salinity) is not known. Fish size must also be considered, since most studies with rainbow trout have been carried out with smaller fish than in the present study. Differences between studies may also be related to the type of dietary carbohydrate ingredient used. It is well known that highly digestible carbohydrate sources, such as glucose, are known to cause a rapid rise in blood sugar level, thereby possibly reducing the appetite and growth. In rainbow trout (initial mean weight 290 g, final mean weight 5 10 g), we (unpublished results by Krogdahl and Arnesen) found a two-fold increase in blood sugar and reduced growth when diets contained 45% white flour (60% extraction ofwheat), compared to a fishmeal-based-control diet. The present study indicates that, provided diets are nutritionally well balanced, Atlantic salmon accept diets where digestible wheat carbohydrate contributes as much as 24% of the digestible energy. A wheat incorporation of this magnitude in practical diets does not seem to inflict any negative effects on either health (judged by mortality rate) or nutrient digestibilities, and there is only a minor negative effect on growth. Pre-extrusion of wheat is not likely to improve starch digestibility in extruded diets. If energy in the form of carbohydrate is only accumulated as liver glycogen or abdominal fat, or lost through excretory organs, there is no direct nutritional reason for incorporating this nutrient in fish feeds. The suggestion that energy from carbohydrates is not utilized by salmonids is not supported by the present results. From general physiological understanding, it seems unlikely that glucose should escape through excretory organs to any great extent at normal plasma glucose levels, as found in this investigation. Moreover,
116
P. ARNESEN AND
A.KROGDAHL
glucose did not accumulate in liver as glycogen. The starch digested and absorbed therefore appears to have been utilized for metabolic energy. ACKNOWLEDGEMENTS
This work was funded by AKVAFORSK, A/S Denofa og Lilleborg Fabriker, The Norwegian Grain Corporation and The Royal Norwegian Council for Scientific and Industrial Research. The authors wish to thank Skretting A/S for preparing the diets, Mrs. Kjellrun H. Gannestad and Mrs. Sissel Nergaard for feeding and management of the fish and Mrs. Inger 0ien Kristiansen for doing most of the lab work. Petter Arnesen was supported by a grant from the Agricultural Research Council of Norway.
REFERENCES Amesen. P., 1992. Various carbohydrate feedstuffs in diets for Atlantic salmon (Salmo salar, L) and rainbow trout (Oncorhynchus mykiss, Walbaum). D. Sci, Thesis, 1992: 21, Agricultural University of Norway, 152 pp. Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture, 13: 265272. Austreng, E., Storebakken, T. and Asgird, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: 157-160. Bergot, F., 1979. Carbohydrate in rainbow trout diets. Effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture, 18: 157167. Cahill, M.M., 1990. Bacterial flora of fishes: A review. Microb. Ecol., 19: 2 l-4 1. Dalrymple, R.H. and Hamm, R., 1973. A method for the extraction of glycogen and metabolites from a single muscle sample. J. Food Technol., 8: 439-444. Hemre, G.I., Lie, O., Lied, E. and Lambertsen, G., 1989. Starch as an energy source in feed for cod (Gadus morhua): Digestibility and retention. Aquaculture, 80: 261-270. Hilton, J.W. and Atkinson, J.L., 1982. Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets. Br. J. Nutr., 47: 597-697. Hilton, J.W. and Dixon, D.G., 1982. Effect of increased liver glycogen and liver weight on liver function in rainbow trout (Salmo gairdneri, R): recovery from anaesthesia and plasma “Ssulphobromophthalein clearance. J. Fish Dis., 5: 185-195. Hilton, J.W. and Slinger, S.J., 1983. Effect of wheat bran replacement of wheat middlings in extrusion processed (floating) diets on the growth of juvenile rainbow trout (Salmo gairdneri) . Aquaculture, 3 5: 20 l-2 10. Hilton, J.W., Atkinson, J.L. and Slinger, S.J., 1982. Maximum tolerable level, digestion and metabolism, of D-glucose (cerelose) in rainbow trout (Salmo gairdneri) reared on a practical trout diet. Can. J. Fish. Aquat. Sci., 39: 1229-1234. Hilton, J.W., Atkinson, J.L. and Slinger, S.J., 1987. Evaluation ofthe net energy value ofglucose (cerelose) and maize starch in diets for rainbow trout (Salmo gairdneri). Br. J. Nutr., 58: 453-461. Hughes, S.G., 1990. Use of triticale as a replacement for wheat middlings in diets for Atlantic salmon. Aquaculture, 90: 173- 178.
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Kaushik, S.J., Medale, F., Fauconneau, B. and Blanc, D., 1989. Effect of digestive carbohydrates on protein/energy utilization and on glucose metabolism in rainbow trout (Safmo gairdneri, R). Aquaculture, 79: 63-74. Lied, E. and Julshamn, K., 1989. Chromic oxide in digestibility experiments. In: Abstracts, 3rd International Symposium on Feeding and Nutrition in Fish, Toba, Japan, 28 Aug.-l Sept., 1989. Maynard, L.A., Loosli, J.K., Hintz, H.F. and Warner, R.G. (Editors), 1979. Animal Nutrition. McGraw-Hill, Inc., New Delhi, 602 pp. Onarheim, A.M. and Raa, J., 1990. Characteristics and possible biological significance of an autoc hthonous flora in the intestinal mucosa of sea-water fish. In: R. LCsel (Editor), Microbiology in Poicilotherms. Elsevier Science Publishers, B.V., Amsterdam, pp. 197-20 1. Pfeffer, ES.,Beckmann-Toussaint, J., Heinrichfreise, B. and Jansen, H.D., 1991. Effect of extrusion on efficiency of utilization of maize starch by rainbow trout (Oncorhynchus mykiss). Aquaculture, 96: 293-303. Phillips, A.M. and Brockway, D.R., 1959. Dietary calories and the production of trout in hatcheries. Prog. Fish-Cult., 2 1: 3- 16. Prosky, I,., Asp, N.G., Furda, I., De Wries, J.W., Schweizer, T.F. and Harland, B., 1985. Determination of total dietary fiber in foods and food products: Collaborative study. J. Assoc. Off. Anal. Chem., 68: 677-679. Saunders, R.M., 1978. Wheat bran: composition and digestibility. In: G.A. Spiller (Editor), Topics in Dietary Fiber Research. Plenum Press, London, pp. 43-58. Takeuchi, T., Jeong, K.-S. and Watanabe, T., 1990. Availability of extruded carbohydrate ingredients to rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Nippon Suisan Gakkaishi, 56: 1839-1845. Walton, M.J., 1986. Metabolic effects of feeding a high protein/low carbohydrate diet as compared to a low protein/high carbohydrate diet to rainbow trout (Salmo gairdneri). Fish Physiol. Biochem., 1: 7- 15. Yone, Y ,, 1979. The utilization of carbohydrates by fishes. In: Proceedings, 7th Japan-Soviet Joint Symposium on Aquaculture, Sept. 1978, Tokyo. Zollner, N. and Kirsch, K., 1962. uber die quantitative Bestimmung von Lipoiden (mikromethode) mittels der vielen nattlrlichen Lipoiden (alles bekannten Plasmalipoiden) gemeinsamen Sulfophosphovanillin-Reaktion. Z. Ges. Med. (Res. Exp. Med.), 135: 545.
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AQUA 50109
Crude and pre-extruded products of wheat as nutrient sources in extruded diets for Atlantic sallmon (Salmo salar, L) grown in sea water Petter Arnesen and Ashild Krogdahl’ AKVAFORSK (Institute ofAquaculture Research), Aas. Norway (Accepted 8 June 1993)
ABSTRACT Atlantic salmon with initial mean weight 175 g, grown in sea water for 10 1 days, were fed extruded diets containing either 0, 15, 30 or 45% crude whole wheat (WC), pre-extruded whole wheat (WE) or pre-extruded after-meal (AE). After-meal is a protein-rich wheat by-product. In the WE diet series, we found growth (weight increase) to be significantly lower in the 45% diet than in the control diet, but although not statistically significant, there was a tendency towards reduced growth also in the other diet series, with increasing dietary inclusions of wheat. Condition factors were not affected by diet, and no significant differences were found in proximate carcass composition between fish fed the control diet or the diet contributing the highest amount of energy as carbohydrates. Plasma glucose, liver glycogen and hepatosomatic indices were within the normal ranges for all diets. No significant effects of wheat inclusion level were found on digestibilities of protein or lipid in any of the diet series. Starch digestibility was significantly higher in the diet containing 45% pre-extruded after-meal than in the diets containing 45% pre-extruded whole wheat or 45% crude whole wheat. The highest inclusions of wheat resulted in reduced dry matter digestibility in the preextruded whole wheat series and in the crude whole wheat series.
INTRODUCTION
Evaluation of feedstuffs of plant origin as feed ingredients for Atlantic salmon and rainbow trout has been the main objective of a recent research programme undertaken at our institute. Whole ground wheat has for many years been the main carbohydrate ingredient in fish feeds in Norway. It is used partly due to its binding properties and partly because the wheat carbohydrates are believed to spare protein by reducing the amount of protein used to supply metabolic energy (Bergot, 1979). The productive energy value of carbohydrate (i.e. how much of the carbohydrate that is actually utilized for energy) for Atlantic salmon is not known, and even though carbohydrate-rich Correspondence to; Petter Arnesen, BIOMAR A.S., P.O.Box 309, Sentrum, N-0103 Oslo, Norway. (Present address). ‘Present address: Norwegian College of Veterinary Medicine, Oslo, Norway.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved,
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P. ARNESEN AND A. KROGDAHL
feedstuffs have been used in fish feeds for many years, we have found only one scientific work dealing with the value of carbohydrate feedstuffs as nutrient sources for Atlantic salmon (Hughes, 1990). There are a number of studies dealing with feeding high-carbohydrate diets to rainbow trout, all carried out in fresh water. Atlantic salmon grown in sea water is the most important aquaculture species in Norway, and the present study was conducted in order to determine: ( 1) the effects of 3 different wheat products in practical diets for Atlantic salmon on health, growth and nutrient digestibilities; and (2) the maximum tolerable level of digestible carbohydrates from wheat in practical diets. MATERIALS
AND METHODS
Experimentaldiets Three wheat products, crude whole wheat (WC), pre-extruded whole wheat (WE) and pre-extruded after-meal (AE), were tested. After-meal is a protein-rich fraction which is removed after the bran in the milling process, i.e. layers of the kernel located immediately beneath the bran. Pre-extruded wheat products were subjected to extrusion before diet preparation. Thus, these products were extruded twice as all diets were extrusion-pelleted. All wheat products (produced from the same batch of wheat) were ground and screened ( < 1 mm) prior to diet preparation. The chemical composition of the wheat products is shown in Table 1. The control diet contained 25% wheat bran, The other three products were incorporated at levels of 15,30 and 45%, in exchange for bran, fish meal and capelin oil. Nutrient composition and proximate chemical analysis of the diets are shown in Table 2. When preparing the diets, adjustments were made for the content of fish meal and capelin oil in such a manner that estimated digestible energy from wheat carbohydrate mainly substituted energy from lipid. TABLE 1 Proximate composition
of wheat products (O/o)
Wheat product
Water
Crude protein
Lipid
Ash
Dietary fibre
Whole wheat Crude Extruded
15.7 11.2
13.5 14.0
2.8 2.8
1.6 1.6
12.0 11.4
8.0
18.3
4.5
2.7
6.9
10.8
15.7
4.3
6.4
40.0
After-meal Extruded Bran
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WHEAT CA.RBOHYDRATE DIETS FOR ATLANTIC SALMON TABLE
2
Composition of control diet, whole crude wheat diets (WC), whole extruded wheat diets (WE) and aftermeal diets (AE) Diet Control Ingredient (g/kg) Bran 250 Whole 0 Crude Extr. 0 A-meal 0 Extr. Fishm. 544 Fish oil 160 Binder 20 Finnst. 10 V.M. mix 10 1 Vit. C 1 Asta 4 Chr. ox DE (MJ)’ 15.5 Chemical analysis (%) 89 DM Protein 40 24 Lipid Starch 8 Ash 8.2 10.0 Fibre 0.34 Chr. ox 18.2 DE (MJ):’ % from: protein 45 lipid 49 starch 6 P/E ratio 22
WC15
WC30
WC45
170
80
0
150 0
300 0 0 434 140 20 10 10 1 1 4 14.0
0
486 148 20 10 10 1
1 4 14.7
WE15
WE30
WE45
AEl5
AE30
AE45
170
80
0
170
80
0
450 150
0 300
0 450
0
0 0
0 0
0
0 376 128 20 10 10 1 1 4 13.1
0 486 148 20 10 10 1 1 4 14.7
0 434 140 20 10 10 1 1 4 14.0
150 486 148 20 10 10 1 1 4 14.7
300 434 140 20 10 10 1 1 4 13.9
450 376 128 20 10 10 1 1 4 13.0
0
0
376 128 20 10 10 1 1 4 13.1
92 40 20 12 7.7 8.6 0.31 16.3
93 38 19 20 7.1 6.8 0.34 17.6
92 35 17 28 5.2 5.4 0.33 17.2
92 40 20 12 7.6 8.6 0.31 15.9
92 36 17 21 6.4 6.8 0.31 14.4
93 34 17 29 6.1 5.4 0.35 14.5
89 38 19 12 7.4 7.8 0.35 15.8
92 36 21 19 6.7 5.2 0.29 18.1
92 35 18 26 5.7 3.0 0.34 18.0
49 45 6 24
45 41 14 21
42 37 21 20
48 46 6 25
46 44 10 25
46 44 10 23
48 44 8 24
40 43 17 20
39 37 24 19
‘Theoretical value, ‘Real value. Extr.=extruded; A-meal = after meal; Fishm. = Norse-LT94 fish meal; Finnst. = Finnstim@; Asta=astaxanthin; Chr. ox=Cr203; DM=dry matter; DE=digestible energy; P/E ratio=protein/energy ratio.
Digestible energy (DE) values used were 39.5 kJ/g for lipid, 23.6 kJ/g for protein and 17.3 kJ/g for carbohydrate (Phillips and Brockway, 1959). When preparing the diet formulas, the digestibility coefficients used for lipid, protein and starch were 90, 85 and 40%, respectively (values based on empirical data from our research stations). Real estimations of DE in the diets were based on the apparent digestibility coefficients calculated in the study. Theoretical values for DE content, and real estimations, are shown in Table 2. The experimental diets were prepared by a commercial feed producer. All diets were extruded into 5-mm pellets. Daily feed rations supplied 120% of
108
P. ARNESEN AND A. KROGDAHL
calculated energy requirement needed to support daily specific growth rate (Austreng et al., 1987). Automatic feeders, active during the day-light hours, were used to deliver the feed. Chromic oxide was included in the diets (0.4%) as an indicator in the digestibility calculations. In the following, each series of diets (0, 15, 30 and 45%) containing the same wheat product. is referred to as a series. Fish and experimental conditions Atlantic salmon (initial mean weight 175 g) were kept in 27 m3 net pens in sea water, 285 fish (50 kg) per pen. Water temperature was measured daily. It decreased from 13.5 to 7.O”C during the feeding trial. Four pens of fish were fed the control diet. The other diets were fed to two pens. Fish were weighed at the start, after 3 8 days and at the end of the experiment, after 10 1 days of feeding. All fish in each pen were weighed in groups (around 20 kg of fish in each group), thereby making it possible to derive the correct mean weight of the fish. Daily feed requirement was adjusted according to estimations of daily specific growth rate based on water temperature and body weight (Austreng et al., 1987). Two weeks before the termination of the experiment, blood samples were collected (heparin used as anticoagulant and fluoride as a glycolysis inhibitor) from 10 fish in each pen. The normal feeding routine was upheld during sampling, in order to keep the fish in the absorptive state. Fish without stomach contents were discarded and replaced. In addition to sampling blood, we also removed and weighed livers and viscera from 5 fish per group. After weighing, livers were immediately frozen at - 96’ C, using a freeze clamp, and kept at - 80°C until analysed for glycogen content. The 5 sampled fish from each of the four control diet pens and from the two 45% AE diet pens, together with the corresponding viscera, were kept for proximate analysis. After weighing and sampling, feeding continued for 2 weeks before feces were stripped as described by Austreng ( 1978). Feces from each pen were pooled. Chemical analyses Wheat products, diets and feces were analysed for dry matter content, nitrogen, total lipid, and ash. Diets and feces were also analysed for starch. Dry matter content was analysed by freeze-drying; crude protein according to the micro-Kjeldahl method (N x 6.25 ); total lipid by the micro-method according to Zollner and Kirsch ( 1962), modified by extracting the lipids with a mixture of chloroform and methanol (2 : 1); and ash by combustion for 15 h at 550°C. Chromic oxide in diets and feces was analysed using ICP spectroscopy, and dietary libre in wheat products using the AOAC method (Prosky et al., 1985). Analyses of chemical composition of fish and viscera were conducted after homogenizing samples in a food processor and freeze-drying. Crude protein
109
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
and ash were analysed as described above, and total lipid according to the Soxhlet-method after hydrolysis with 4 M hydrochloric acid for 30 min at 100°C. Dry matter content was determined by drying for at least 18 h at 20 mmHg and 72°C. Starch was gelatinized in a boiling water bath in the presence of a heatstable a.mylase (Termamyl 120 L, Novo A/S). This resulted in a partial degradation of starch to glucose. Complete hydrolysis of starch to glucose was accomplished by adding an amyloglucosidase (Boehringer Mannheim, kit no. 737 160). Amount of glucose was then assessed quantitatively using a Beckman glucose hexokinase assay (Dri-STAT@’ Reagent) and an Encore automatic analyser. Liver glycogen was converted to glucose according to the method of Dalrymple and Hamm ( 1973). Glucose analysis was then carried out as described above. Design and statisticalanalyses One-way ANOVA was used to test effects of wheat products. Least-square means (LSMEANS ) were calculated and used to test the least significant difference (LSD) between means. LSD tests were carried out on differences between means within the same series, using inclusion level as class variable, and between mean values within the same inclusion level, using wheat product as class variable. A two-way ANOVA with wheat product and inclusion level as class variables was employed to examine possible interaction effects between wheat product and inclusion level. Models and differences between means were considered statistically significant when P< 0.05. Calculations Nutrient digestibility:as described by Maynard et al. ( 1979) Dressing percentage: [Wet carcass weight (g)/whole wet body weight
(!3)1x100
Hepatosomatic index: [Wet liver weight (g)/whole wet body weight (g) 1x 100 Conditionfactor: [Whole wet body weight (g ) / (fork length (cm) ) 3] x 100 Absorbed starch: [Starch content (g/ 100 g diet) x starch digestibility (%) ] / 100
Growth:Final mean fish weight (g)-initial
mean fish weight (g )
RESULTS
Water temperatures were higher than normal for the season, and consequently the calculated growth (expressed as increase in total body mass) and feed requirement were higher than expected. In order to avoid an early termination of the experiment due to lack of feed, we restricted feeding by 20% during the last month. This might have influenced growth rate during the last
P. ARNESEN AND A. KROGDAHL
110
part of the experiment. We expect, however, the magnitude of such an influence to be equal for all diets, and not to have had any significant bearing on the conclusions of the investigation, The control fish had a weight increase of about 300 g, which was 80% of that calculated by using tables for daily specific growth rate ( Austreng et al., 1987). We had expected growth to be faster due to high water temperatures, and thus it was evident that there had been a considerable discrepancy between our estimations of daily weight increase (on which the daily feed ration sizes were based) and the actual increase. As a result, we had operated with a too large biomass, and had most probably overfed the fish during the first phase of the experiment. Although there seemed to be a tendency toward a reduction in growth performance as wheat inclusion increased (Table 3 ) , it was only in the WE series that comparison of means revealed a significant difference in growth between the control and the 45% wheat diet. There were no significant within-level differences in growth performance, and no interaction effects between wheat product and inclusion level. Feed efficiency ratios were not calculated in the present study since we did not have control of feed wastage. Whole body and viscera composition of fish fed the 45% AE diet was not different from that of fish fed the control diet (Table 4). Furthermore, neither diet nor inclusion level was found to influence condition factors, which TABLE 3 Growth (increase in body weight), plasma glucose, liver glycogen and hepatosomatic
index (HSI)
Diet/level
Growth (g)
Plasma glucose (mmol/l)
Liver glycogen (O/O)
HSI
Control WC 15 30 45
301 -t 305 + 285 k 264 +
6.7A 17.0Aa 7SAa 7.0Aa
6.0i0.4A 6.5k06A” 5.2? O.OAa 6.3f0.6Aa
1.9 2.0 2.6
1.8+0.2A l.5f0.0Aa 1.9fO.O*= l.7f0.1AB
Control WE 15 30 45
301 f 290 f 274+ 267 f
6.7* 4.0ABa l.SABa 3.0Ba
6.0 f 60.4A 5.4 k 0.4Aa 6.3 2 O.OAa 7.0+0.5*=
1.9 1.9 2.7 2.4
1.8 + 0.2* 1.9*0.4*= 1.7?0.2*’ 1.6 k O.O*”
Control AE 15 30 45
3Olk 6.7A 302+ 1.5~~~ 278 275 + O.OA”
6.0i0.4A 6.3+0.4*= 5.9 7.1 f0.2AB
1.9 2.0 2.3 1.9
1.8i0.2* 1.6 + O.O*= 1.5 1.7f0.1Aa
Mean valueskstandard error of mean (s.e.m.). s.e.m. co.05 are given as 0.0 in the table. Superscripts in capital letters refer to differences between mean values within the same dietary series. Superscripts in small letters refer to differences between mean values within the same inclusion level. -, missing value.
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
111
TABLE 4 Proximak
composition
(%) of wet fish and viscera
Diet/level
Fat
Crude protein
Ash
DM
Fish Control AE45
10.9kO.4 10.3kO.2
18.4kO.2 18.7kO.3
2.6fO.O 2.7fO.O
30.9f0.4 30.3f0.7
Viscera Control AE45
23.2f 1.9 25.0f2.8
10.6kO.l 1 l.OkO.4
1.3fO.l 1.3kO.l
36.6& 1.1 38.6k3.2
Mean values f standard error of mean (s.e.m). s.e.m. < 0.05 are given as 0.0 in the table. No superscripts have been assigned since no significant differences were found.
ranged from 1.08 to 1.16, or dressing percentages, which ranged from 87 to 89%. Plasma glucose levels, hepatosomatic indices and liver glycogen levels are shown in Table 3. Levels of all three variables were within the range considered to be normal, i.e. around 5-7 mmol/l, l-2 and 2-3%, respectively. ANOVA d:id not reveal significant effects of inclusion level on plasma glucose or hepatosomatic indices in any of the series, and there were no within-level differences. Because we had not analysed duplicate samples of liver for glycogen, we could not test differences between means for this variable. With. the exception of the AE series, where starch digestibility was found to be significantly lower in the 15% diet than in the other diets, there were no within-series differences in digestibility values for protein, lipid or starch (Table 5 ) . However, at the 45% inclusion level, mean starch digestibility was significantly higher in the AE series than in the WC and WE series. No significant interactions were found between wheat product and inclusion level for the above digestibilities. Comparisons of within-series means revealed a significantly higher dry matter digestibility in the 30% WC diet than in the 45% WC diet. In the WE series, (dry matter digestibility was significantly higher in the control diet than in any of the other diets, and it was higher in the 15O/bthan in the 45% diet. Dry matter digestibility in the AE series was significantly lower in the 15% diet than in the 45% diet. At the 45% wheat inclusion level, all series had significantly different dry matter digestibility, highest in the 45% AE diet and lowest in the 45% WE diet. There was a significant interaction effect between inclusion level and wheat product for dry matter digestibility (P= 0.000 1) . There was a significantly higher dry matter content (DM) in feces from the 45% WC fish (DM = 2 1%) than from the control group (DM = 17%). Furthermore, the dry matter content of feces from fish fed the AE 45% and WE
112
P. ARNESEN AND
A. KROGDAHL
TABLE 5 Digestibility of nutrients and dry matter (DM), and amount of absorbed starch Diet/level
Protein (%)
Lipid (%)
Starch (%)
DM (O/O)
Control WC 15 30 45
86f l.l* 84f 1.2Aa 89f3.1A” 88f0.6A”
94 k 0.6* 93 k 0.5*a 95 + 1.8*’ 95f0.2A=
86 *0.7* 5orf: 3.9Aa 7334+0.9*b
68 f l.OAB 63 f 2.5AB” 73 + 6.4A” 57 *0.3na
6.9 6.0 7.2 9.5
Control WE 15 30 45
86+ l.l* SO+ 1.8*” 78+4.8*’ 84+0.8*
94 + 0.6* 93 f l.OA’ 94 f 0.2*= 95 f 0.6Aa
86f0.7* 48 f 9.6*’ 41 f2.5*’ 28 ?I 5.6Ab
68k 1.0* 59 k 3.6na 55+0.3nca 49k 1.8cb
6.9 5.8 8.6 8.1
Control AE 15 30 45
8611.1* 84+ 1.9Aa 85 85+0.1A8
94&0.6* 95kO.lA”
86+_0.7* 6Ok 5.3B” 93 95 k 2.5*”
68i: l.OAB 61 f 3.5Ba 78 74 f 0.3AC
6.9 7.2 17.7 24.7
95 kO.gA”
Absorbed starch (g/100 g diet)
Mean valuesf standard error of mean (s.e.m.). s.e.m. ~0.05 are given as 0.0 in the table. Superscripts in capital letters refer to differences between mean values within the same dietary series. Superscripts in small letters refer to differences between mean values within the same inclusion level. -3 missing value.
45% diets (DM= 15% and 17%, respectively) that of fish fed the WC 45% diet (DM = 2 1%). matter content of all feces collected was within mal for salmonids ( 13-2 1o/o), based on results sen, 1992).
was significantly lower than However, the variation in dry a range we consider to be norfrom previous studies (Arne-
DISCUSSION
Mortality rates were negligible, and fish growth, which is a good indicator of the general condition of the fish, was satisfactory compared to the control, even at the highest incorporations of wheat. We have found only one study (Hughes, 1990) in the literature where Atlantic salmon have been fed diets high in wheat. In that study, small fish performed as well on diets containing either 18% wheat middlings or triticale, and neither of the two grains seemed to have any negative effect on fish health. There are a number of reports dealing with growth performance of rainbow trout fed diets high in carbohydrate. Hilton et al. ( 1982) found digestible carbohydrates in excess of 25% to cause growth reduction, a result which was confirmed by Hilton and Slinger ( 1983 ), who found significantly reduced growth when increasing the amount of digestible carbohydrates from approximately 25% to around 30%. Bergot ( 1979)) however, found that increasing
WHEAT CARBOHYDRATE DIETS FOR ATLANTIC SALMON
113
digestible carbohydrates in the diet from 15 to 30% had no adverse effects on growth, and Hilton et al. ( 1987 ) found no differences in growth performance between ad libitum feeding of diets containing 25% maize starch or a fishmeal-based control diet. In a recent study, Pfeffer et al. ( 199 1) found growth of rainbow trout fed diets containing around 29% maize starch per kg dry matter to be reduced when the maize was extruded prior to mixing. In the present study, growth performance of Atlantic salmon seemed to be affected by dietary wheat inclusion in a similar manner to what we have previously observed in rainbow trout (unpublished results by Krogdahl and Arnesen) in fresh water fed diets containing extruded wheat. A slight growth reduction compared to the control was observed in fish fed the highest wheat inclusion; however, this was significant only in the WE series. It is interesting to note that growth rate of fish fed the control diet was not significantly different from those fed the 45% AE diet, even though there was quite a large difference in energy contribution by protein in the two diets (45 and 39%, respectively). Total digestible energy in the control diet and the 45% AE diet were almost identical (18.2 MJ/kg and 18.0 MJ/kg, respectively ). Although our data analysis did not reveal major differences in growth between the different dietary treatment groups, inspection of the growth data suggest that the diets containing the highest inclusions of wheat did not support maximal growth. It is also possible that differences in growth between dietary treatments might have become more pronounced if the study had been longer than 101 days. The economic implications of including high levels of wheat in diets for salmon have not been considered in this study. To do so, we would have had to calculate feed costs and we would also need data on feed efticiency ratios. Ingestion of carbohydrate-rich diets did not cause a rise in plasma glucose levels in Atlantic salmon. In a similar study with rainbow trout in fresh water (unpulblished results by Krogdahl and Arnesen) we found that plasma glucose levels rose steeply when dietary inclusions of extruded wheat exceeded 30%. Elevated plasma glucose levels in rainbow trout ingesting large amounts of digestible carbohydrates has been observed by a number of workers (Bergot, 1979; Hilton and Slinger, 1983; Walton, 1986). Hilton and Atkinson ( 1982 ) and Hilton et al. ( 1982) did not, however, find differences in plasma glucose by feeding increasing levels of dietary glucose. It is possible, however, that the maximum glucose incorporation (2 1% and 25%) in these studies was too low to cause a rise in plasma glucose. According to the “glucostatic theory” of appetite control, a continuous elevation of blood sugar can lead to loss of appetite. Hilton and Slinger ( 1983 ) suggested this to be one explanation for the improved growth rate obtained in rainbow trout when dietary digestible carbohydrates were reduced. A high plasma glucose level in fish fed diets containing extruded starch might have contributed to a lower appetite and thereby the observed reduction in feed
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consumption and weight gain observed in rainbow trout in the previously mentioned study by Pfeffer et al. ( 199 1). No mention of plasma glucose concentrations was made. No significant effect was observed in the present study of either wheat product or inclusion level on the hepatosomatic indices and liver glycogen levels, indicating that Atlantic salmon may differ from rainbow trout. The calculated glucose absorption was between 6 and 25 g per 100 g of diet (calculated values are shown in Table 5 ) . If we anticipate an ingestion of 1 g feed per g increase in growth, the total ingestion of feed during the experiment was around 300 g per fish. This brings the total absorption of glucose to between 18 and 75 g, which could not be traced either to the blood or to the liver. There was no excessive accumulation of glycogen in the liver. Increased deposition of glycogen in liver when feeding carbohydrate-rich diets has been shown in rainbow trout (Hilton and Atkinson, 1982; Hilton et al., 1982; Hilton and Slinger 1983; Walton, 1986; Pfeffer et al., 1991) and carp, red sea bream and yellowtail (Yone, 1979). Hemre et al. ( 1989), on the other hand, found that feeding carbohydrate-rich diets to cod resulted in an increase in hepatosomatic indices which was caused by fat deposition, not glycogen. Large depositions of glycogen in liver of rainbow trout have been shown to alter liver function and possibly upset normal excretion of toxic substances (Hilton and Dixon, 1982). Our results indicate that liver dysfunction, due to excessive glycogen deposition, did not occur in sea grown Atlantic salmon fed carbohydrate-rich diets. Neither protein nor lipid digestibility was affected by dietary carbohydrate level, a finding that is in agreement with studies on rainbow trout (Takeuchi et al., 1990; Kaushik et al., 1989; Pfeffer et al., 1991) and cod (Hemre et al., 1989). Starch digestibilities were high in the after-meal diets and increased with increased dietary level. Digestibility values of 95% are of a magnitude usually seen for glucose in rainbow trout (Kaushik et al., 1989) and most other monogastric animals (Maynard et al., 1979 ), suggesting that the aftermeal starch is very soluble in the intestine. We did not analyse the chemical composition of the available carbohydrate fraction of after-meal, but judging by its high digestibility, the ratio between amylose and amylopectin in the starch is probably high. The presence of sugars is also known to be high in the aleurone layer (Saunders, 1978) of which the after-meal largely consists. Starch and sugars might have been subjected to some degree of degradation by bacterial enzymes in the hind-gut, something that would have influenced their recovery in the feces. We are not aware of any study, however, that points to any major bacterial breakdown of carbohydrates in the hind-gut of salmonids. Microflora of coldwater fish living in sea water are dominated by IG’ibriobacteria (Cahill, 1990; Onarheim and Raa, 1990). The observation that there were no differences in starch digestibilities between whole crude wheat and whole extruded wheat
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diets suggests that pre-extrusion did not have any beneficial effect on starch digestibility in this study. Dry matter digestibility values varied considerably, and since there were no significant effects of wheat incorporation level on the digestibility of protein or lipid, most of the variation in dry matter was probably due to variations in starch digestibility and dietary Iibre content. The accuracy of the digestibility measurements might have been influenced by the rather low chromic oxide inclusion (0.4% included) in the diets. Lied and Julshamn ( 1989), in a study with cod, showed that 1% chromic oxide was necessary in order to minimize the non-systematic variation. However, this should not affect the relative effects of our study. Moreover, the variation between replicates in the recovery of chromic oxide in feces was small. The general effects of feeding Atlantic salmon carbohydrate-rich diets seemed to be somewhat different to those seen in rainbow trout fed similar diets. In many studies, rainbow trout have responded to carbohydrate-rich diets by exhibiting elevated plasma glucose levels and reduced growth rate. Results from the present study indicate that the handling of digestible carbohydrates is different in Atlantic salmon grown in salt water than in rainbow trout in fresh water. Whether this is a species difference or an environmental effect i(water salinity) is not known. Fish size must also be considered, since most studies with rainbow trout have been carried out with smaller fish than in the present study. Differences between studies may also be related to the type of dietary carbohydrate ingredient used. It is well known that highly digestible carbohydrate sources, such as glucose, are known to cause a rapid rise in blood sugar level, thereby possibly reducing the appetite and growth. In rainbow trout (initial mean weight 290 g, final mean weight 5 10 g), we (unpublished results by Krogdahl and Arnesen) found a two-fold increase in blood sugar and reduced growth when diets contained 45% white flour (60% extraction ofwheat), compared to a fishmeal-based-control diet. The present study indicates that, provided diets are nutritionally well balanced, Atlantic salmon accept diets where digestible wheat carbohydrate contributes as much as 24% of the digestible energy. A wheat incorporation of this magnitude in practical diets does not seem to inflict any negative effects on either health (judged by mortality rate) or nutrient digestibilities, and there is only a minor negative effect on growth. Pre-extrusion of wheat is not likely to improve starch digestibility in extruded diets. If energy in the form of carbohydrate is only accumulated as liver glycogen or abdominal fat, or lost through excretory organs, there is no direct nutritional reason for incorporating this nutrient in fish feeds. The suggestion that energy from carbohydrates is not utilized by salmonids is not supported by the present results. From general physiological understanding, it seems unlikely that glucose should escape through excretory organs to any great extent at normal plasma glucose levels, as found in this investigation. Moreover,
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glucose did not accumulate in liver as glycogen. The starch digested and absorbed therefore appears to have been utilized for metabolic energy. ACKNOWLEDGEMENTS
This work was funded by AKVAFORSK, A/S Denofa og Lilleborg Fabriker, The Norwegian Grain Corporation and The Royal Norwegian Council for Scientific and Industrial Research. The authors wish to thank Skretting A/S for preparing the diets, Mrs. Kjellrun H. Gannestad and Mrs. Sissel Nergaard for feeding and management of the fish and Mrs. Inger 0ien Kristiansen for doing most of the lab work. Petter Arnesen was supported by a grant from the Agricultural Research Council of Norway.
REFERENCES Amesen. P., 1992. Various carbohydrate feedstuffs in diets for Atlantic salmon (Salmo salar, L) and rainbow trout (Oncorhynchus mykiss, Walbaum). D. Sci, Thesis, 1992: 21, Agricultural University of Norway, 152 pp. Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture, 13: 265272. Austreng, E., Storebakken, T. and Asgird, T., 1987. Growth rate estimates for cultured Atlantic salmon and rainbow trout. Aquaculture, 60: 157-160. Bergot, F., 1979. Carbohydrate in rainbow trout diets. Effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture, 18: 157167. Cahill, M.M., 1990. Bacterial flora of fishes: A review. Microb. Ecol., 19: 2 l-4 1. Dalrymple, R.H. and Hamm, R., 1973. A method for the extraction of glycogen and metabolites from a single muscle sample. J. Food Technol., 8: 439-444. Hemre, G.I., Lie, O., Lied, E. and Lambertsen, G., 1989. Starch as an energy source in feed for cod (Gadus morhua): Digestibility and retention. Aquaculture, 80: 261-270. Hilton, J.W. and Atkinson, J.L., 1982. Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets. Br. J. Nutr., 47: 597-697. Hilton, J.W. and Dixon, D.G., 1982. Effect of increased liver glycogen and liver weight on liver function in rainbow trout (Salmo gairdneri, R): recovery from anaesthesia and plasma “Ssulphobromophthalein clearance. J. Fish Dis., 5: 185-195. Hilton, J.W. and Slinger, S.J., 1983. Effect of wheat bran replacement of wheat middlings in extrusion processed (floating) diets on the growth of juvenile rainbow trout (Salmo gairdneri) . Aquaculture, 3 5: 20 l-2 10. Hilton, J.W., Atkinson, J.L. and Slinger, S.J., 1982. Maximum tolerable level, digestion and metabolism, of D-glucose (cerelose) in rainbow trout (Salmo gairdneri) reared on a practical trout diet. Can. J. Fish. Aquat. Sci., 39: 1229-1234. Hilton, J.W., Atkinson, J.L. and Slinger, S.J., 1987. Evaluation ofthe net energy value ofglucose (cerelose) and maize starch in diets for rainbow trout (Salmo gairdneri). Br. J. Nutr., 58: 453-461. Hughes, S.G., 1990. Use of triticale as a replacement for wheat middlings in diets for Atlantic salmon. Aquaculture, 90: 173- 178.
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Kaushik, S.J., Medale, F., Fauconneau, B. and Blanc, D., 1989. Effect of digestive carbohydrates on protein/energy utilization and on glucose metabolism in rainbow trout (Safmo gairdneri, R). Aquaculture, 79: 63-74. Lied, E. and Julshamn, K., 1989. Chromic oxide in digestibility experiments. In: Abstracts, 3rd International Symposium on Feeding and Nutrition in Fish, Toba, Japan, 28 Aug.-l Sept., 1989. Maynard, L.A., Loosli, J.K., Hintz, H.F. and Warner, R.G. (Editors), 1979. Animal Nutrition. McGraw-Hill, Inc., New Delhi, 602 pp. Onarheim, A.M. and Raa, J., 1990. Characteristics and possible biological significance of an autoc hthonous flora in the intestinal mucosa of sea-water fish. In: R. LCsel (Editor), Microbiology in Poicilotherms. Elsevier Science Publishers, B.V., Amsterdam, pp. 197-20 1. Pfeffer, ES.,Beckmann-Toussaint, J., Heinrichfreise, B. and Jansen, H.D., 1991. Effect of extrusion on efficiency of utilization of maize starch by rainbow trout (Oncorhynchus mykiss). Aquaculture, 96: 293-303. Phillips, A.M. and Brockway, D.R., 1959. Dietary calories and the production of trout in hatcheries. Prog. Fish-Cult., 2 1: 3- 16. Prosky, I,., Asp, N.G., Furda, I., De Wries, J.W., Schweizer, T.F. and Harland, B., 1985. Determination of total dietary fiber in foods and food products: Collaborative study. J. Assoc. Off. Anal. Chem., 68: 677-679. Saunders, R.M., 1978. Wheat bran: composition and digestibility. In: G.A. Spiller (Editor), Topics in Dietary Fiber Research. Plenum Press, London, pp. 43-58. Takeuchi, T., Jeong, K.-S. and Watanabe, T., 1990. Availability of extruded carbohydrate ingredients to rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Nippon Suisan Gakkaishi, 56: 1839-1845. Walton, M.J., 1986. Metabolic effects of feeding a high protein/low carbohydrate diet as compared to a low protein/high carbohydrate diet to rainbow trout (Salmo gairdneri). Fish Physiol. Biochem., 1: 7- 15. Yone, Y ,, 1979. The utilization of carbohydrates by fishes. In: Proceedings, 7th Japan-Soviet Joint Symposium on Aquaculture, Sept. 1978, Tokyo. Zollner, N. and Kirsch, K., 1962. uber die quantitative Bestimmung von Lipoiden (mikromethode) mittels der vielen nattlrlichen Lipoiden (alles bekannten Plasmalipoiden) gemeinsamen Sulfophosphovanillin-Reaktion. Z. Ges. Med. (Res. Exp. Med.), 135: 545.