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Utilization of free glycerol as a source of dietary energy in rainbow trout (Salmo gairdneri)
Aquaculture, 56 (1986) 215-227 Elsevier Science Publishers B.V., Amsterdam
215 -
Printed
in The Netherlands
Utilization of Free Glycerol as a Source of Dietary Energy in Rainbow Trout (Sulmo guirdneri) D.J. MENTON’,
S.J. SLINGER
and J.W. HILTON’
Group for Advancement of Fish Studies, Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ont. NlG 2Wl (Canada) ‘This study represents part of the dissertation submitted by D.J. Menton of the requirements for the M.Sc. degree, University of Guelph. *To whom reprint requests should be sent. (Accepted
in partial fulfillment
9 June 1986)
ABSTRACT Menton, D.J., Slinger, S.J. and Hilton, J.W., 1986. Utilization of free glycerol as a source of dietary energy in rainbow trout (Salmo gairdneri) tdquaculture, 56: 215-227. Three feeding trials were conducted in which free glycerol was added at levels ranging from 1 to 12% to both high and low energy (lipid) diets to determine the utilization of free glycerol as an energy source in rainbow trout. The supplementation of free glycerol had no significant effect on the final body weight, fee&gain ratio, carcass dressing percent, liver: body weight ratio or liver glycogen content of the trout. The inclusion of free glycerol in high or low energy trout diets had no significant effect on the final carcass composition of the trout or on the efficiency of dietary protein conversion into tissue protein. Glycerol was not an effective precursor for lipogenesis nor was it stored as liver glycogen. Trout reared on 6 and 12% glycerol-supplemented diets had significantly higher blood glucose levels than control fish, indicating that the glycerol was being converted to glucose which is not an efficient energy source in rainbow trout. The feeding response in fish consuming the glycerol-supplemented diets was slow and/or sluggish in comparison to the control groups. This may be related to hyperglycaemia which was noted in these trout receiving the glycerol-supplemented diets. It was concluded that free glycerol was not an effective source of dietary energy in rainbow trout.
INTRODUCTION
The natural diet of rainbow trout is high in proteins and lipids while low in digestible carbohydrates (Walton and Cowey, 1982). Practical diets for rainbow trout require very high levels of protein per unit gain relative to other commercial meat-producing animals (Pieper and Pfeffer, 1979). Protein supplements are often the most expensive ingredients in commercial trout diets (Hilton and Slinger, 1981). Fish-feed formulators therefore have attempted to
0044-8486/86/$03.50
0 1986 Elsevier Science Publishers
B.V.
216
spare the catabolism of dietary proteins as energy sources by using more cost effective energy sources such as lipids (Reinitz et al., 1978; Takeuchi et al., 1978; Cho, 1982). In contrast to most domestic animal diets in which carbohydrates are the most economical energy source in sparing dietary proteins, carnivorous fish such as rainbow trout have difficulty either efficiently digesting or metabolizing dietary carbohydrates for energy purposes (Cowey et al., 1977; Hilton and Atkinson, 1982; Hilton et al., 1982). Considering that only the glycerol portion of a fat molecule can be used as a precursor for gluconeogenesis, in a manner similar to the carbon backbone of most amino acids, it remains to be determined just how much of the protein sparing effect seen with lipid supplementation to trout diets comes from glycerol. The addition of 1% free glycerol is comparable to approximately the amount of bound glycerol found in a 10% dietary supplement of lipid ( Eggstein and Kuhlmann, 1974). Some aspects of glycerol metabolism have been studied in rainbow trout (Newsholme and Taylor, 1969; Lech, 1970; Holub et al., 1975). However, there appear to have been no studies conducted on utilization of free glycerol as a dietary energy source in any species of fish. Renaud and Moon (1980) indicated that glycerol is metabolized into glucose in the hepatocytes of the eel (Anguillu rostruta) . Considering that glycerol is a component (via lipids) of natural trout diets, it may be possible that trout can effectively utilize glycerol in energy metabolism and thereby spare the use of dietary proteins (amino acids) for tissue synthesis. The purpose of this study was to determine the effect, on the growth and physiological response of rainbow trout, of including free glycerol, at both low and high levels of supplementation, into low and high energy (lipid) practicaltype diets. MATERIALS AND METHODS
General design Three feeding trials were conducted on juvenile trout reared at 15°C for either a 12- or 16-week growth period. Practical trout diets containing an optimum level of protein and all other essential nutrients, according to the National Research Council (1981) , were supplemented with different levels of free glycerol and/or fish oil. In all experiments, the calculated minimum total w3 fatty acid content of the test diets would be in excess of 1.5% of the diet. In experiment 1, the effects of including 1 and 2% free glycerol (approximately the amount of bound glycerol present in diets supplemented with 10 and 20% fish oil, respectively, w/w) to a low energy trout diet were compared with the effects of a high energy (lipid) diet on the growth and physiological response of the trout. In experiment 2, the effects of adding 1% free glycerol to either a low or high energy (low or high lipid level) diet on the growth and physiological
217 TABLE 1 Formulation and composition of the test diets Ingredient
Diet number Experiment 1 12
Capelin meal Soybean meal Wheat middlings Bentonite Vitamin premix’ Mineral premix’ Glycerol Capelin oil Analysis’ Protein Lipid Ash Glycerol
Experiment 2
Experiment 3
3
4
1
2
3
4
12
3
4
35 20
35 15
35 15
35 15
25
25
25
25
35
35
35
35
11
12
25
42 2
41 2
57 -
55 -
19 37.5
20 25
15 43
18 34
20 26
2
43 2
18 39.5 -
-
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1 1
1 2
1
1 1
1
1 1
1
-
-
1 6
1 12
2
2
2
4
4
14.5
14.5
15
2
2
2
39.9
41.5
41.7
41.5
37.4
37.2
37.5
37.9
40.5
41.7
41.4
42.0
22.4
9.6
9.2
9.2
10.2
9.7
20.0
20.3
21.9
9.7
9.1
9.4
10.9
10.4
0.1
0.2
10.1 1.4
9.7 2.0
6.2 ND3
6.1 ND
6.0 ND
6.1 ND
9.7 0.1
10.3 0.2
10.1 4.8
10.2 11.5
1 -
-
15
-
1 -
‘As described in Hilton and Slinger (1981) . ‘Results expressed on a dry matter basis. “Not determined.
response of trout were studied. In experiment 3, the effect was determined of including 6 and 12% free glycerol in a low energy diet on the growth and physiological response of rainbow trout. Diet formulation and processing Test diets for the different experiments were formulated as described in Table 1. After mixing, test diets were processed by steam pelleting on a laboratory pellet mill and stored in a cooler ( - 5°C) until required for feeding. Dried samples were collected from each test diet and analyzed for dietary protein (N x 6.25)) moisture and ash (Horwitz, 1980)) and lipid by the method of Bligh and Dyer (1959). Free glycerol was extracted from test diets in the well mixed water: methanol supernatant of the lipid extraction column and measured according to the method of Eggstein and Kuhlmann (1974). Supply and maintenance of fish Young rainbow trout were obtained from two local commercial trout farmers and conditioned to laboratory conditions for approximately 2-4 weeks prior to
utilization in the experiments. Between 1 to 2 weeks before commencing the experiments, trout were randomly distributed into one of three different 12tank aquatic systems that consisted of either baked enamelled metal (volume 40 or 70 1) or fiberglass tanks (volume 45 1). Aquatic systems were supplied by either a one-pass flow-through water supply or a recirculated system with 5-20% replacement water (biological filter). Water supplies consisted of a 50 : 50 blend of city of Guelph municipal and University of Guelph well water that was dechlorinated by passing through an activated charcoal system. Water flow rates into tanks varied from 1.5 to 2 l/min. Individual aquatic tanks were aerated and water temperatures of each aquatic system were thermostatically maintained at approximately 15 ‘C. Water temperature was measured daily while dissolved oxygen concentration (YSI calibrated probe), pH and total ammonia ( Nessler’s reagent, experiment 3 only) were measured weekly. These parameters ranged from 15.02 0.2”C, pH 7.4 2 0.2 for experiment 1; 14.8? 0.9”C, pH 7.2 kO.4, 8.8 kO.8 mg/dl oxygen for experiment 2; and 15.1 -t 0.5 oC, pH 7.3 2 0.4,8.4 2 0.4 mg/dl oxygen, 0.3 ? 0.0 mg/l total ammonia for experiment 3. The aquatic systems were housed in a windowless laboratory and maintained on a 12 h light: 12 h dark photoperiod. Fish were fed to satiety, initially four and later three times per day, as described by Hilton and Slinger (1981). Growth and biochemical analysis Test diets were fed to triplicate groups of 75 or 100 fish per tank for a period of 12 weeks (experiments 2 and 3) or 16 weeks (experiment 1). Initial weight of the fish was 4.8 + 0.1 g/fish in experiment 1,2.1? 0.1 g/fish in experiment 2 and 3.12 0.2 g/fish in experiment 3. Fish were monitored daily for mortalities, feed consumption was measured weekly, and the weight of fish per tank was determined at the end of each 28-day period. At the end of 16 weeks in experiment 1 and 12 weeks in experiment 2, six fish were sampled at random from each tank following an 18-h fast and anaesthetized with tricaine methanesulphonate (MS222,lOO mg/l) . The fish were individually weighed, and then sacrificed by severing the spinal cord behind the head. Thereafter, the livers were removed, individually weighed and quickly frozen with liquid nitrogen. The frozen livers were stored at - 20°C until required for glycogen analysis by the method of Murat and Serfaty (1974). Blood was collected from these same fish in heparinized centrifuge tubes by amputation of the caudal peduncle. Blood was then centrifuged at 4000 xg for 5 min and the plasma stored at - 20°C until required for analysis. Plasma protein was then determined by the method of Lowry et al. (1951) , plasma glucose by the glucose oxidase method (Trinder, 1969) and plasma glycerol by the method of Eggstein and Kuhlmann (1974). In experiment 3, liver and blood samples were collected by these same procedures from four fish per tank after
219
an 18-h fast and 3 h post-feeding. Liver glycogen, plasma glucose and plasma protein levels were then determined by the previously described methods. An additional six fish were sampled at random from each tank in all three experiments, sacrificed as previously described, ground in a meat grinder, frozen, freeze-dried and then stored at - 5 ‘C until required for analysis. In addition, at the beginning of each experiment, 12-24 fish were sampled, ground, frozen, freeze-dried and then also stored at - 5 “C until required for analysis. Carcass ash, moisture and protein contents were determined by the methods of Horwitz (1980) and lipid by the method of Bligh and Dyer (1959 ) . An additional four fish per tank were sampled by the same procedure in experiments 2 and 3 for determination of dressing percentage. Sampled fish were immediately sacrificed and individually weighed. The visceral contents plus kidney and gills were removed, the eviscerated carcass weight determined, and dressing percentage calculated as Dressing percentage
= eviscerated
carcass wt. x loo/total
fish wt.
The eviscerated carcass was ground, frozen, freeze-dried and analyzed for ash, moisture, protein and lipid content as previously described. The productive protein value ( PPV ) was calculated as described by Pfeffer (1982 ) where PPV (%)
= nitrogen
gained x loo/nitrogen
fed
Statistical analysis The results were subjected to analysis of variance and statistical differences were determined at the 5% probability level using the Tukey’s Honestly Significant Difference procedure as described by Steel and Torrie (1980). Factorial analyses with significance determined at the 5% probability level were conducted to determine dietary lipid and glycerol effects ( experiment 2 ) . RESULTS
Experiment 1 At the end of 16 weeks, trout reared on diet 1 (high energy - 22% lipid) had a significantly higher body weight and lower feed: gain ratio than trout reared on the low energy (9% lipid) diets 2-4, unsupplemented or with 1 and 2% free glycerol added (Table 2). Trout fed the higher energy diet 1 (22% lipid) showed a significantly higher productive protein value (PPV) than trout reared on the low energy diets (diets 2-4, Table 2). Final carcass composition data indicated that trout reared on the high energy diet 1 ( 22% ) had a significantly higher lipid content and a significantly lower protein and ash content than trout reared on the low energy diets (diets 2-4,
3 1
4 2
SEM*
78.1b
1.2b 0.0
32.6b
91.p
1.0” 1.0
39.9”
32.6b
1.2b 0.1
77.7b
32.5b
1.2b 0.4
74.3b
0.5
1.4
24.5” 1.2” 0.3 29.8”
25.3” 1.2” 0.2 31.2”
2 1
1 0
2 0
1 0
37.7b
l.lb 1.1
25.8”
3 0
37.0b
l.lb 1.3
25.0”
4 1
0.6
to.1 (0.1
0.7
SEM
‘Initial body weight: experiment 1 - 4.8 g/fish, experiment 2 - 2.1 g/fish, experiment 3 - 3.1 g/fish. 2Standarderror of the mean. 3BesuItain rows with the same letter superscript for each experiment, not significantly different (P10.05) 4Productive protein value = nitrogen gained x lOO/nitrogen fed.
Body weight (g/fish) Feed : gain Mortalities (%I Productive protein value’ ( % )
(%I Parameter
Free glycerol
Experiment 2
Experiment 1
Diet num~r
31.0b
31.9b
G
36.5”
1.1” 1.0
1.2” 2.7
0.9” 1.7
37.8b
3 6
41.4b
2 0
44.4”
1 0
Experiment 3
31.0b
1.1” 1.7
38.5b
4 12
0.4
0.3
SEM
Final body weight’, feed:gain ratios, mortalities, and productive protein values for the trout after 12 and 16 weeks on the different test diets at 15°C
TABLE 2
3 1
4 2 SEM
ND
ND3
ND ND ND
Eviscerated carcass analysis Protein Lipid Ash
ND ND ND
ND
57.1b 33.gb 8.0b
ND ND ND
ND
57.gb 32.8b 8.3b
0.5 0.4 0.2
63.7”b 26.8” 9.0”
81.7”
54.7” 34.3” 7.8”
59.6’ 32.4b 8.0b
78.2b
81.3*
64.4” 26.7” 8.7”
47.6b 44.1b 5.gb
54.4’ 34.5” 7.7”
3 0
60.3& 31.6b 8.0b
79.0b
46.8b 44.Sb 6.1b
4 1
0.7 0.6 0.1
0.4
0.5 0.3 0.1
SEM
‘F&.ulta in rows with the same letter superscript for each experiment, not significantly different (Pr0.05). 2Dressingpercentage = (eviscerated carcass weight/total fish weight) x 100. 3Not determined.
ND ND ND
57.1b 33.9b 7.8b
48.4” 44.3’ 6.3”
2 1
1 0
2 0
1 0
Carcassanalysis Protein Lipid Ash Dressing percentage’
Parameter
Freeglycerol
Experiment 2
Experiment 1
Diet number
58.7” 30.0” 8.8”
76.7”
48.3” 40.6” 7.3”
1 0
65.2b 22.0b 10.ob
80.3b
59.0b 29.2b 8.5b
2 0
Experiment 3
62.7b 24.5b 9.80b
79.2h
58.2b 29.0b 8.5b
3 6
62.8b 23.8b 9.3”b
78.5b
57.2b 28.5b 8.gb
4 12
1.0 1.0 0.3
0.7
1.6 0.7 0.2
SEM
Final carcass composition and dressing percentage and eviscerated carcass composition of the trout after completion of experiments 1,2 and 3
TABLE 3
222
Table 3). Data analyses also demonstrated no significant effect of dietary treatment on the liver: body weight ratios, liver glycogen content, plasma glucose levels or plasma protein levels of the trout after 16 weeks on the test diets (data not presented). Experiment 2 At the end of 12 weeks there was no significant difference in the final body weights of the trout reared on the different test diets (Table 2). Trout reared on the high energy diets (20% lipid, diets 3 and 4) had a significantly lower feed: gain ratio than trout reared on the low energy diets (10% lipid, diets 1 and 2 ) and the inclusion of 1% free glycerol to diets 2 and 4 had no significant effect on feed: gain ratio. Similarly, trout reared on the high energy diets had a significantly higher PPV compared to trout reared on the low energy diets (Table 2 ) . Addition of 1% free glycerol to test diets 2 and 4 exerted no significant effects. Both whole and eviscerated carcass composition of trout reared on the high energy diets 3 and 4 showed significantly higher lipid and lower protein and ash levels than trout reared on the low energy diets 1 and 2 (Table 3 ) . The addition of 1% free glycerol to either the low or high energy diets (diets 2 and 4, respectively) had no significant effect on either the whole or eviscerated final carcass composition of the trout. In addition there was a significantly higher dressing percentage in trout reared on the low energy diets in comparison to trout reared on the high energy diets. However, free glycerol inclusion resulted in no significant effect on the dressing percentage of trout reared on either the high or low energy diets. There was no significant effect of either dietary energy level or free glycerol supplementation level on the liver: body weight ratio, liver glycogen content, plasma glucose, plasma glycerol (0.54 5 0.05 mg/dl) or plasma protein content of the trout after 12 weeks on the test diets. The dietary lipid level was the only factor significantly affecting the carcass dressing percentage, feed: gain ratios, PPV and the proportions ofprotein, lipid and ash in the whole and eviscerated carcass. Experiment 3 At the end of 12 weeks, trout reared on the high energy diet (diet 1, 22% lipid) had a significantly higher final body weight and lower feed: gain ratio than trout reared on the low energy diets (Tables 1 and 2). Similarly, PPV of the trout was significantly higher in trout reared on the high energy as compared to the low energy diets. Mortalities were low and apparently not related to the different dietary treatments. Final carcass analysis indicated that trout reared on the high energy diet (diet 1, 22% lipid) had a significantly lower carcass protein and ash content
223 TABLE 4 The fed and fasted liver :body weight ratio@, liver glycogen content*, plasma protein’ and plasma glucose levels’ of the trout after 12 weeks on the test diets - experiment 3 Diet number
Free glycerol ( % ) Feeding condition Parameter Liver: body wt. ratio Liver glycogen (So) Plasma protein ( mg/ml) Plasma glucose (mg/dl)
1 0
2 0
Fed
Fasted
3 6
Fed
Fasted
4 12
Fed
Fasted
Fed
Fasted
SEM
1.0
1.2
1.0
1.2
1.3
1.2
1.4
1.4
0.1
8.2
9.3
6.9
7.5
7.7
9.6
8.3
9.2
0.9
43.2
36.2
42.6
40.3
44.0
41.5
45.6
35.2
2.9
91.5”
87.5”
92.8”
96.3
117.2’
87.4”
107.5b
107Jb
4.8
‘(Liver weight/body weight) x 100. Results based upon the mean of three replicates (four to six fish/replicate) per treatment. “Results in rows with the same letter superscript not significantly different (PI 0.05).
and a higher carcass lipid content than did trout reared on the low energy diets (Table 3). In contrast, there was a significantly higher dressing percentage and eviscerated carcass protein content in trout reared on the low energy diets (diets 2-4, 9% lipid) in comparison to trout reared on the high energy diet (diet 1,22% lipid; Table 3). In addition, eviscerated carcass lipid content was significantly lower in trout reared on the low energy diets than in trout reared on the high energy diet. There was no significant diet-related trend in the eviscerated carcass ash content. As in experiments 1 and 2, there was no significant difference in the liver: body weight ratio and liver glycogen content of the trout reared on the different test diets. Furthermore, there was no significant difference in these same parameters in either the fed (3 h post-feeding) or fasted state (18 h postfeeding, Table 4). As in experiments 1 and 2, there was no significant difference in the plasma protein levels of the trout reared on the various test diets (Table 4). However, plasma glucose levels were significantly different in the trout reared on the various test diets in the fed or fasted state. Trout reared on either the high energy diet (diet 1, 22% lipid) or the low energy diet (diet 2, 9% lipid) containing no free glycerol had similar plasma glucose levels that were not significantly affected by the fish being in the fed or fasted state (Table 4). In contrast, trout reared on the 6% free glycerol-supplemented diet (diet 3,9% lipid) showed a significantly higher plasma glucose level in the fed, but
224
not the fasted, state compared with trout reared on the previously described high (diet 1) and low (diet 2) energy diets which were not supplemented with free glycerol. Trout reared on diet 4 containing 12% free glycerol (9% lipid) had a significantly higher plasma glucose level than the other groups of trout with the exception of trout reared on diet 3 in the fed state. However, in contrast to the trout reared on the 6% free glycerol-supplemented diet (diet 3,9% lipid), there was no significant difference in plasma glucose levels of trout reared on the 12% free glycerol-supplemented diet in either the fed or fasted state. DISCUSSION
The results of these studies indicate that free glycerol is not an effective source of dietary energy for the rainbow trout. Supplementation with free glycerol in the range of l-12% of the diet exerted no significant effect on the growth, feed:gain ratio, liver weight, liver glycogen content or PPV of the trout. In contrast, trout reared on the high energy (lipid) diets of all three experiments consistently had significantly higher PPV than did trout reared on the low energy diets. This would indicate that glycerol did not spare the utilization and/or catabolism of dietary proteins as an energy source as did the lipid in the high energy diets. It was also noted that free glycerol addition to either low energy diets or high energy diets exerted no significant effect on the final carcass composition of the trout. The lack of any effect on the final carcass lipid levels would appear to indicate that dietary free glycerol had no significant effect on lipogenic activity in the trout. Considering that the addition of 1% free glycerol was comparable to approximately the amount of bound glycerol found in a 10% addition of lipid to these diets, suggests that the sparing effect of amino acids produced by the lipid trout diets comes from the fatty acid portion of the lipid molecule. Why glycerol was not an effective source of dietary energy cannot be determined with certainty from this study. Although a digestibility study on the absorption of free glycerol in rainbow trout was not conducted, subsequent studies involving the intraperitoneal injection of radiolabelled glycerol into rainbow trout indicated that a portion of the glycerol was being quickly absorbed into the blood and tissues of this fish (Menton, 1985). In mammals, glycerol is readily absorbed in intestinal sacs at rates of about one-fourth that of glucose (Lin, 1977). The elevated plasma glucose levels of trout reared on 6% free glycerol ( 3 h post-feeding) and 12% free glycerol ( 3 and 18 h post-feeding) in experiment 3, indicates that at least some of the absorbed free glycerol ingested by the trout is being converted into glucose, but with no apparent effect on liver glycogen levels. Renaud and Moon (1980) similarly found that glycerol was principally incorporated into glucose in the hepatocytes of the eel with lesser amounts metabolized into lipid, carbon dioxide and glycogen in that
225
order. The maintenance of high levels of plasma glucose in trout reared on the 12% glycerol diet, even with an 18-h fast, appears to indicate that trout have considerable difficulty in metabolizing any glucose produced by gluconeogenesis from high levels of dietary glycerol. Indeed, even with the highly efficient absorption of glucose by the trout (National Research Council, 1981; Hilton et al., 1982)) the utilization of dietary glucose for energy by these fish is very poor. In experiments 2 and 3, it was consistently observed that in consecutive daily feedings, trout reared on the l-12% glycerol-supplemented diets showed a definite reduction in their feeding response, but with no significant reduction in the final body weight after 12 weeks on experiment. The cause of the reduction in feeding response may be related to the conversion of glycerol to glucose and the hyperglycaemia in these fish. Previous research on the feeding of high carbohydrate diets to trout has indicated that these fish show a reduction in feeding response with consecutive daily feedings, due in part to hyperglycaemia (Hilton and Atkinson, 1982) which may produce some satiating effect in the trout. The higher productive protein values (PPV) of trout fed the high energy (lipid) diets indicated that significantly more dietary protein was being converted into tissue protein, but with a concurrent significant reduction in the carcass dressing percentage compared to fish fed the low energy diets. In addition, trout fed the high energy diets had higher lipid and lower protein levels in the tissues of both the whole and eviscerated carcasses when compared to trout fed the low energy diets. This would indicate that while excess dietary energy in the form of lipid may increase dietary protein retention (Cho et al., 1976; Reinitz et al., 1978; Watanabe et al., 1979)) it also increases the fat content of the muscle and mesenteric fat pad (Takeuchi et al., 1978) ; the latter tissue being the principle site of lipid deposition in trout (Henderson and Sargent, 1981). It remains to be determined whether the economic advantage of feeding high energy (lipid) diets to trout to increase protein retention in the muscle is offset by the wastage of dietary fat deposited in the mesenteric fat pad while processing the fish for market. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Debbie Conrad, Mr. Martin Hodgson, Mr. Richard Pope, Mr. Keith Were and Mr. Andrew Pharazyn for technical support; and the Ontario Ministry of Natural Resources, the Ontario Ministry of Agriculture and Food, and the Natural Sciences and Engineering Research Council of Canada for financial support.
226 REFERENCES Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 911-917. Cho, C.Y., 1982. Effects of dietary protein and lipid levels on energy metabolism of rainbow trout (Sahogairdneri). Proc. 9th Symp. on Energy Metabolism. Lillehammer, Norway, pp. 176-179. Cho, C.Y., Bayley, H.S. and Slinger, S.J., 1976. Energy metabolism in growing rainbow trout: partition of dietary energy in high protein and high fat diets. In: M. Vermorel (Editor), Energy Metabolism of Farm Animals. Clermont-Ferrand, France, pp. 299-302. Cowey, C.B., De la Higuera, M. and Adron, J.W., 1977. The effect of dietary composition and of insulin on gluconeogenesis in rainbow trout (Salmo gairdneri) . Br. J. Nutr., 38: 385-395. Eggstein, M. and Kuhlmann, E., 1974. Triglycerides and glycerol: determination after alkaline hydrolysis. In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 4. Academic Press, Inc., New York, NY, pp. 1825-1831. Henderson, R.J. and Sargent, J.R., 1981. Lipid biosynthesis in rainbow trout, Salmo gairdnerii, fed diets of differing lipid content. Comp. Biochem. Physiol. C, 69: 31-37. 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: 597-607. Hilton, J.W. and Slinger, S.J., 1981. Nutrition and feeding of rainbow trout. Canadian Special Publication of Fisheries and Aquatic Sciences 55,15 pp. Hilton, J.W., Atkinson, J.L. and Slinger, S.J. 1982. Maximum tolerable level, digestion andmetabolism of D-glucose (cerelose) in rainbow trout (Salmo gairdneri) reared on a practical trout diet. Can. J. Fish. Aquat. Sci., 39: 1229-1234. Holub, B.J., Connor, J.T.H. and Slinger, S.J., 1975. Incorporation of glycerol-3-phosphate into the hepatic lipids of rainbow trout, Salmo gairdneri. J. Fish. Res. Board Can., 32: 61-64. Horwitz, W., 1980. Official Methods of Analysis of the Association of Official Analytical Chemists, 13th edition. AOAC, Washington, DC., 1018 pp. Lech, J., 1970. Glycerol kinase and glycerol utilization in trout (Salmo gairdneri) liver. Comp. Biochem. Physiol., 34: 117-124. Lin, E.C.C., 1977. Glycerol utilization and its regulation in mammals. Annu. Rev. Biochem., 46: 765-795. Lowry, O.H., Rosebrough, N.J., Fare, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Menton, D.J., 1985. Glycerol as an energy substrate for the rainbow trout (Salmo gairdneri Richardson) . M.Sc. Thesis, University of Guelph, Guelph, Ont. NlG 2W1, 167 pp. Murat, J.C. and Serfaty, A., 1974. Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin. Chem., 20: 1576-1577. National Research Council, 1981. Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC, 63 pp. Newsholme, E.A. and Taylor, K., 1969. Glycerol kinase activities in muscles from vertebrates and invertebrates. Biochem. J., 112: 465-474. Pfeffer, E., 1982. Utilization of dietary protein by salmonid fish. Comp. Biochem. Physiol. B, 73: 51-57. Pieper, A. and Pfeffer, E., 1979. Carbohydrates as possible sources of dietary energy for rainbow trout (Salmo gairdneri Richardson). In: J.E. Halver and K. Tiews (Editors), Proc. World Symp. on Finfish Nutrition and Fishfeed Technology, Vol. 1, Heenemann, Berlin, pp. 209-219. Reinitz, G.L., Orme, L.E., Lemm, C.A. and Hitzel, F.N., 1978. Influence of varying lipid concentrations with two protein concentrations in diets for rainbow trout (Salmo gairdneri) . Trans. Am. Fish. Sot., 107: 751-754. Renaud, J.M. and Moon, T.W., 1980. Characterization of gluconeogenesis in hepatocytes isolated from the American eel, Anguilla rostrata LeSueur. J. Comp. Physiol., 135: 115-125.
227 Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics: A Biometrical Approach, 2nd edition. McGraw-Hill Book Company, New York, NY, 633 pp. Takeuchi, T., Watanabe, T. and Ogino, C., 1978. Supplementary effect of lipids in a high protein diet of rainbow trout. Bull. Jpn. Sot. Sci. Fish., 44: 677-681. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem., 6: 24-27. Walton, M.J. and Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. B, 73: 59-79. Watanabe, T., Takeuchi, T. and Ogino, Ch., 1979. Studies on the sparing effect of lipids on dietary protein in rainbow trout (Salmo gairdneri) . In: J.E. Halver and K. Tiews (Editors), Proc. World Symp. on Finfish Nutrition and Fishfeed Technology. Vol. 1, Heenemann, Berlin, pp. 113-125.
215 -
Printed
in The Netherlands
Utilization of Free Glycerol as a Source of Dietary Energy in Rainbow Trout (Sulmo guirdneri) D.J. MENTON’,
S.J. SLINGER
and J.W. HILTON’
Group for Advancement of Fish Studies, Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ont. NlG 2Wl (Canada) ‘This study represents part of the dissertation submitted by D.J. Menton of the requirements for the M.Sc. degree, University of Guelph. *To whom reprint requests should be sent. (Accepted
in partial fulfillment
9 June 1986)
ABSTRACT Menton, D.J., Slinger, S.J. and Hilton, J.W., 1986. Utilization of free glycerol as a source of dietary energy in rainbow trout (Salmo gairdneri) tdquaculture, 56: 215-227. Three feeding trials were conducted in which free glycerol was added at levels ranging from 1 to 12% to both high and low energy (lipid) diets to determine the utilization of free glycerol as an energy source in rainbow trout. The supplementation of free glycerol had no significant effect on the final body weight, fee&gain ratio, carcass dressing percent, liver: body weight ratio or liver glycogen content of the trout. The inclusion of free glycerol in high or low energy trout diets had no significant effect on the final carcass composition of the trout or on the efficiency of dietary protein conversion into tissue protein. Glycerol was not an effective precursor for lipogenesis nor was it stored as liver glycogen. Trout reared on 6 and 12% glycerol-supplemented diets had significantly higher blood glucose levels than control fish, indicating that the glycerol was being converted to glucose which is not an efficient energy source in rainbow trout. The feeding response in fish consuming the glycerol-supplemented diets was slow and/or sluggish in comparison to the control groups. This may be related to hyperglycaemia which was noted in these trout receiving the glycerol-supplemented diets. It was concluded that free glycerol was not an effective source of dietary energy in rainbow trout.
INTRODUCTION
The natural diet of rainbow trout is high in proteins and lipids while low in digestible carbohydrates (Walton and Cowey, 1982). Practical diets for rainbow trout require very high levels of protein per unit gain relative to other commercial meat-producing animals (Pieper and Pfeffer, 1979). Protein supplements are often the most expensive ingredients in commercial trout diets (Hilton and Slinger, 1981). Fish-feed formulators therefore have attempted to
0044-8486/86/$03.50
0 1986 Elsevier Science Publishers
B.V.
216
spare the catabolism of dietary proteins as energy sources by using more cost effective energy sources such as lipids (Reinitz et al., 1978; Takeuchi et al., 1978; Cho, 1982). In contrast to most domestic animal diets in which carbohydrates are the most economical energy source in sparing dietary proteins, carnivorous fish such as rainbow trout have difficulty either efficiently digesting or metabolizing dietary carbohydrates for energy purposes (Cowey et al., 1977; Hilton and Atkinson, 1982; Hilton et al., 1982). Considering that only the glycerol portion of a fat molecule can be used as a precursor for gluconeogenesis, in a manner similar to the carbon backbone of most amino acids, it remains to be determined just how much of the protein sparing effect seen with lipid supplementation to trout diets comes from glycerol. The addition of 1% free glycerol is comparable to approximately the amount of bound glycerol found in a 10% dietary supplement of lipid ( Eggstein and Kuhlmann, 1974). Some aspects of glycerol metabolism have been studied in rainbow trout (Newsholme and Taylor, 1969; Lech, 1970; Holub et al., 1975). However, there appear to have been no studies conducted on utilization of free glycerol as a dietary energy source in any species of fish. Renaud and Moon (1980) indicated that glycerol is metabolized into glucose in the hepatocytes of the eel (Anguillu rostruta) . Considering that glycerol is a component (via lipids) of natural trout diets, it may be possible that trout can effectively utilize glycerol in energy metabolism and thereby spare the use of dietary proteins (amino acids) for tissue synthesis. The purpose of this study was to determine the effect, on the growth and physiological response of rainbow trout, of including free glycerol, at both low and high levels of supplementation, into low and high energy (lipid) practicaltype diets. MATERIALS AND METHODS
General design Three feeding trials were conducted on juvenile trout reared at 15°C for either a 12- or 16-week growth period. Practical trout diets containing an optimum level of protein and all other essential nutrients, according to the National Research Council (1981) , were supplemented with different levels of free glycerol and/or fish oil. In all experiments, the calculated minimum total w3 fatty acid content of the test diets would be in excess of 1.5% of the diet. In experiment 1, the effects of including 1 and 2% free glycerol (approximately the amount of bound glycerol present in diets supplemented with 10 and 20% fish oil, respectively, w/w) to a low energy trout diet were compared with the effects of a high energy (lipid) diet on the growth and physiological response of the trout. In experiment 2, the effects of adding 1% free glycerol to either a low or high energy (low or high lipid level) diet on the growth and physiological
217 TABLE 1 Formulation and composition of the test diets Ingredient
Diet number Experiment 1 12
Capelin meal Soybean meal Wheat middlings Bentonite Vitamin premix’ Mineral premix’ Glycerol Capelin oil Analysis’ Protein Lipid Ash Glycerol
Experiment 2
Experiment 3
3
4
1
2
3
4
12
3
4
35 20
35 15
35 15
35 15
25
25
25
25
35
35
35
35
11
12
25
42 2
41 2
57 -
55 -
19 37.5
20 25
15 43
18 34
20 26
2
43 2
18 39.5 -
-
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1 1
1 2
1
1 1
1
1 1
1
-
-
1 6
1 12
2
2
2
4
4
14.5
14.5
15
2
2
2
39.9
41.5
41.7
41.5
37.4
37.2
37.5
37.9
40.5
41.7
41.4
42.0
22.4
9.6
9.2
9.2
10.2
9.7
20.0
20.3
21.9
9.7
9.1
9.4
10.9
10.4
0.1
0.2
10.1 1.4
9.7 2.0
6.2 ND3
6.1 ND
6.0 ND
6.1 ND
9.7 0.1
10.3 0.2
10.1 4.8
10.2 11.5
1 -
-
15
-
1 -
‘As described in Hilton and Slinger (1981) . ‘Results expressed on a dry matter basis. “Not determined.
response of trout were studied. In experiment 3, the effect was determined of including 6 and 12% free glycerol in a low energy diet on the growth and physiological response of rainbow trout. Diet formulation and processing Test diets for the different experiments were formulated as described in Table 1. After mixing, test diets were processed by steam pelleting on a laboratory pellet mill and stored in a cooler ( - 5°C) until required for feeding. Dried samples were collected from each test diet and analyzed for dietary protein (N x 6.25)) moisture and ash (Horwitz, 1980)) and lipid by the method of Bligh and Dyer (1959). Free glycerol was extracted from test diets in the well mixed water: methanol supernatant of the lipid extraction column and measured according to the method of Eggstein and Kuhlmann (1974). Supply and maintenance of fish Young rainbow trout were obtained from two local commercial trout farmers and conditioned to laboratory conditions for approximately 2-4 weeks prior to
utilization in the experiments. Between 1 to 2 weeks before commencing the experiments, trout were randomly distributed into one of three different 12tank aquatic systems that consisted of either baked enamelled metal (volume 40 or 70 1) or fiberglass tanks (volume 45 1). Aquatic systems were supplied by either a one-pass flow-through water supply or a recirculated system with 5-20% replacement water (biological filter). Water supplies consisted of a 50 : 50 blend of city of Guelph municipal and University of Guelph well water that was dechlorinated by passing through an activated charcoal system. Water flow rates into tanks varied from 1.5 to 2 l/min. Individual aquatic tanks were aerated and water temperatures of each aquatic system were thermostatically maintained at approximately 15 ‘C. Water temperature was measured daily while dissolved oxygen concentration (YSI calibrated probe), pH and total ammonia ( Nessler’s reagent, experiment 3 only) were measured weekly. These parameters ranged from 15.02 0.2”C, pH 7.4 2 0.2 for experiment 1; 14.8? 0.9”C, pH 7.2 kO.4, 8.8 kO.8 mg/dl oxygen for experiment 2; and 15.1 -t 0.5 oC, pH 7.3 2 0.4,8.4 2 0.4 mg/dl oxygen, 0.3 ? 0.0 mg/l total ammonia for experiment 3. The aquatic systems were housed in a windowless laboratory and maintained on a 12 h light: 12 h dark photoperiod. Fish were fed to satiety, initially four and later three times per day, as described by Hilton and Slinger (1981). Growth and biochemical analysis Test diets were fed to triplicate groups of 75 or 100 fish per tank for a period of 12 weeks (experiments 2 and 3) or 16 weeks (experiment 1). Initial weight of the fish was 4.8 + 0.1 g/fish in experiment 1,2.1? 0.1 g/fish in experiment 2 and 3.12 0.2 g/fish in experiment 3. Fish were monitored daily for mortalities, feed consumption was measured weekly, and the weight of fish per tank was determined at the end of each 28-day period. At the end of 16 weeks in experiment 1 and 12 weeks in experiment 2, six fish were sampled at random from each tank following an 18-h fast and anaesthetized with tricaine methanesulphonate (MS222,lOO mg/l) . The fish were individually weighed, and then sacrificed by severing the spinal cord behind the head. Thereafter, the livers were removed, individually weighed and quickly frozen with liquid nitrogen. The frozen livers were stored at - 20°C until required for glycogen analysis by the method of Murat and Serfaty (1974). Blood was collected from these same fish in heparinized centrifuge tubes by amputation of the caudal peduncle. Blood was then centrifuged at 4000 xg for 5 min and the plasma stored at - 20°C until required for analysis. Plasma protein was then determined by the method of Lowry et al. (1951) , plasma glucose by the glucose oxidase method (Trinder, 1969) and plasma glycerol by the method of Eggstein and Kuhlmann (1974). In experiment 3, liver and blood samples were collected by these same procedures from four fish per tank after
219
an 18-h fast and 3 h post-feeding. Liver glycogen, plasma glucose and plasma protein levels were then determined by the previously described methods. An additional six fish were sampled at random from each tank in all three experiments, sacrificed as previously described, ground in a meat grinder, frozen, freeze-dried and then stored at - 5 ‘C until required for analysis. In addition, at the beginning of each experiment, 12-24 fish were sampled, ground, frozen, freeze-dried and then also stored at - 5 “C until required for analysis. Carcass ash, moisture and protein contents were determined by the methods of Horwitz (1980) and lipid by the method of Bligh and Dyer (1959 ) . An additional four fish per tank were sampled by the same procedure in experiments 2 and 3 for determination of dressing percentage. Sampled fish were immediately sacrificed and individually weighed. The visceral contents plus kidney and gills were removed, the eviscerated carcass weight determined, and dressing percentage calculated as Dressing percentage
= eviscerated
carcass wt. x loo/total
fish wt.
The eviscerated carcass was ground, frozen, freeze-dried and analyzed for ash, moisture, protein and lipid content as previously described. The productive protein value ( PPV ) was calculated as described by Pfeffer (1982 ) where PPV (%)
= nitrogen
gained x loo/nitrogen
fed
Statistical analysis The results were subjected to analysis of variance and statistical differences were determined at the 5% probability level using the Tukey’s Honestly Significant Difference procedure as described by Steel and Torrie (1980). Factorial analyses with significance determined at the 5% probability level were conducted to determine dietary lipid and glycerol effects ( experiment 2 ) . RESULTS
Experiment 1 At the end of 16 weeks, trout reared on diet 1 (high energy - 22% lipid) had a significantly higher body weight and lower feed: gain ratio than trout reared on the low energy (9% lipid) diets 2-4, unsupplemented or with 1 and 2% free glycerol added (Table 2). Trout fed the higher energy diet 1 (22% lipid) showed a significantly higher productive protein value (PPV) than trout reared on the low energy diets (diets 2-4, Table 2). Final carcass composition data indicated that trout reared on the high energy diet 1 ( 22% ) had a significantly higher lipid content and a significantly lower protein and ash content than trout reared on the low energy diets (diets 2-4,
3 1
4 2
SEM*
78.1b
1.2b 0.0
32.6b
91.p
1.0” 1.0
39.9”
32.6b
1.2b 0.1
77.7b
32.5b
1.2b 0.4
74.3b
0.5
1.4
24.5” 1.2” 0.3 29.8”
25.3” 1.2” 0.2 31.2”
2 1
1 0
2 0
1 0
37.7b
l.lb 1.1
25.8”
3 0
37.0b
l.lb 1.3
25.0”
4 1
0.6
to.1 (0.1
0.7
SEM
‘Initial body weight: experiment 1 - 4.8 g/fish, experiment 2 - 2.1 g/fish, experiment 3 - 3.1 g/fish. 2Standarderror of the mean. 3BesuItain rows with the same letter superscript for each experiment, not significantly different (P10.05) 4Productive protein value = nitrogen gained x lOO/nitrogen fed.
Body weight (g/fish) Feed : gain Mortalities (%I Productive protein value’ ( % )
(%I Parameter
Free glycerol
Experiment 2
Experiment 1
Diet num~r
31.0b
31.9b
G
36.5”
1.1” 1.0
1.2” 2.7
0.9” 1.7
37.8b
3 6
41.4b
2 0
44.4”
1 0
Experiment 3
31.0b
1.1” 1.7
38.5b
4 12
0.4
0.3
SEM
Final body weight’, feed:gain ratios, mortalities, and productive protein values for the trout after 12 and 16 weeks on the different test diets at 15°C
TABLE 2
3 1
4 2 SEM
ND
ND3
ND ND ND
Eviscerated carcass analysis Protein Lipid Ash
ND ND ND
ND
57.1b 33.gb 8.0b
ND ND ND
ND
57.gb 32.8b 8.3b
0.5 0.4 0.2
63.7”b 26.8” 9.0”
81.7”
54.7” 34.3” 7.8”
59.6’ 32.4b 8.0b
78.2b
81.3*
64.4” 26.7” 8.7”
47.6b 44.1b 5.gb
54.4’ 34.5” 7.7”
3 0
60.3& 31.6b 8.0b
79.0b
46.8b 44.Sb 6.1b
4 1
0.7 0.6 0.1
0.4
0.5 0.3 0.1
SEM
‘F&.ulta in rows with the same letter superscript for each experiment, not significantly different (Pr0.05). 2Dressingpercentage = (eviscerated carcass weight/total fish weight) x 100. 3Not determined.
ND ND ND
57.1b 33.9b 7.8b
48.4” 44.3’ 6.3”
2 1
1 0
2 0
1 0
Carcassanalysis Protein Lipid Ash Dressing percentage’
Parameter
Freeglycerol
Experiment 2
Experiment 1
Diet number
58.7” 30.0” 8.8”
76.7”
48.3” 40.6” 7.3”
1 0
65.2b 22.0b 10.ob
80.3b
59.0b 29.2b 8.5b
2 0
Experiment 3
62.7b 24.5b 9.80b
79.2h
58.2b 29.0b 8.5b
3 6
62.8b 23.8b 9.3”b
78.5b
57.2b 28.5b 8.gb
4 12
1.0 1.0 0.3
0.7
1.6 0.7 0.2
SEM
Final carcass composition and dressing percentage and eviscerated carcass composition of the trout after completion of experiments 1,2 and 3
TABLE 3
222
Table 3). Data analyses also demonstrated no significant effect of dietary treatment on the liver: body weight ratios, liver glycogen content, plasma glucose levels or plasma protein levels of the trout after 16 weeks on the test diets (data not presented). Experiment 2 At the end of 12 weeks there was no significant difference in the final body weights of the trout reared on the different test diets (Table 2). Trout reared on the high energy diets (20% lipid, diets 3 and 4) had a significantly lower feed: gain ratio than trout reared on the low energy diets (10% lipid, diets 1 and 2 ) and the inclusion of 1% free glycerol to diets 2 and 4 had no significant effect on feed: gain ratio. Similarly, trout reared on the high energy diets had a significantly higher PPV compared to trout reared on the low energy diets (Table 2 ) . Addition of 1% free glycerol to test diets 2 and 4 exerted no significant effects. Both whole and eviscerated carcass composition of trout reared on the high energy diets 3 and 4 showed significantly higher lipid and lower protein and ash levels than trout reared on the low energy diets 1 and 2 (Table 3 ) . The addition of 1% free glycerol to either the low or high energy diets (diets 2 and 4, respectively) had no significant effect on either the whole or eviscerated final carcass composition of the trout. In addition there was a significantly higher dressing percentage in trout reared on the low energy diets in comparison to trout reared on the high energy diets. However, free glycerol inclusion resulted in no significant effect on the dressing percentage of trout reared on either the high or low energy diets. There was no significant effect of either dietary energy level or free glycerol supplementation level on the liver: body weight ratio, liver glycogen content, plasma glucose, plasma glycerol (0.54 5 0.05 mg/dl) or plasma protein content of the trout after 12 weeks on the test diets. The dietary lipid level was the only factor significantly affecting the carcass dressing percentage, feed: gain ratios, PPV and the proportions ofprotein, lipid and ash in the whole and eviscerated carcass. Experiment 3 At the end of 12 weeks, trout reared on the high energy diet (diet 1, 22% lipid) had a significantly higher final body weight and lower feed: gain ratio than trout reared on the low energy diets (Tables 1 and 2). Similarly, PPV of the trout was significantly higher in trout reared on the high energy as compared to the low energy diets. Mortalities were low and apparently not related to the different dietary treatments. Final carcass analysis indicated that trout reared on the high energy diet (diet 1, 22% lipid) had a significantly lower carcass protein and ash content
223 TABLE 4 The fed and fasted liver :body weight ratio@, liver glycogen content*, plasma protein’ and plasma glucose levels’ of the trout after 12 weeks on the test diets - experiment 3 Diet number
Free glycerol ( % ) Feeding condition Parameter Liver: body wt. ratio Liver glycogen (So) Plasma protein ( mg/ml) Plasma glucose (mg/dl)
1 0
2 0
Fed
Fasted
3 6
Fed
Fasted
4 12
Fed
Fasted
Fed
Fasted
SEM
1.0
1.2
1.0
1.2
1.3
1.2
1.4
1.4
0.1
8.2
9.3
6.9
7.5
7.7
9.6
8.3
9.2
0.9
43.2
36.2
42.6
40.3
44.0
41.5
45.6
35.2
2.9
91.5”
87.5”
92.8”
96.3
117.2’
87.4”
107.5b
107Jb
4.8
‘(Liver weight/body weight) x 100. Results based upon the mean of three replicates (four to six fish/replicate) per treatment. “Results in rows with the same letter superscript not significantly different (PI 0.05).
and a higher carcass lipid content than did trout reared on the low energy diets (Table 3). In contrast, there was a significantly higher dressing percentage and eviscerated carcass protein content in trout reared on the low energy diets (diets 2-4, 9% lipid) in comparison to trout reared on the high energy diet (diet 1,22% lipid; Table 3). In addition, eviscerated carcass lipid content was significantly lower in trout reared on the low energy diets than in trout reared on the high energy diet. There was no significant diet-related trend in the eviscerated carcass ash content. As in experiments 1 and 2, there was no significant difference in the liver: body weight ratio and liver glycogen content of the trout reared on the different test diets. Furthermore, there was no significant difference in these same parameters in either the fed (3 h post-feeding) or fasted state (18 h postfeeding, Table 4). As in experiments 1 and 2, there was no significant difference in the plasma protein levels of the trout reared on the various test diets (Table 4). However, plasma glucose levels were significantly different in the trout reared on the various test diets in the fed or fasted state. Trout reared on either the high energy diet (diet 1, 22% lipid) or the low energy diet (diet 2, 9% lipid) containing no free glycerol had similar plasma glucose levels that were not significantly affected by the fish being in the fed or fasted state (Table 4). In contrast, trout reared on the 6% free glycerol-supplemented diet (diet 3,9% lipid) showed a significantly higher plasma glucose level in the fed, but
224
not the fasted, state compared with trout reared on the previously described high (diet 1) and low (diet 2) energy diets which were not supplemented with free glycerol. Trout reared on diet 4 containing 12% free glycerol (9% lipid) had a significantly higher plasma glucose level than the other groups of trout with the exception of trout reared on diet 3 in the fed state. However, in contrast to the trout reared on the 6% free glycerol-supplemented diet (diet 3,9% lipid), there was no significant difference in plasma glucose levels of trout reared on the 12% free glycerol-supplemented diet in either the fed or fasted state. DISCUSSION
The results of these studies indicate that free glycerol is not an effective source of dietary energy for the rainbow trout. Supplementation with free glycerol in the range of l-12% of the diet exerted no significant effect on the growth, feed:gain ratio, liver weight, liver glycogen content or PPV of the trout. In contrast, trout reared on the high energy (lipid) diets of all three experiments consistently had significantly higher PPV than did trout reared on the low energy diets. This would indicate that glycerol did not spare the utilization and/or catabolism of dietary proteins as an energy source as did the lipid in the high energy diets. It was also noted that free glycerol addition to either low energy diets or high energy diets exerted no significant effect on the final carcass composition of the trout. The lack of any effect on the final carcass lipid levels would appear to indicate that dietary free glycerol had no significant effect on lipogenic activity in the trout. Considering that the addition of 1% free glycerol was comparable to approximately the amount of bound glycerol found in a 10% addition of lipid to these diets, suggests that the sparing effect of amino acids produced by the lipid trout diets comes from the fatty acid portion of the lipid molecule. Why glycerol was not an effective source of dietary energy cannot be determined with certainty from this study. Although a digestibility study on the absorption of free glycerol in rainbow trout was not conducted, subsequent studies involving the intraperitoneal injection of radiolabelled glycerol into rainbow trout indicated that a portion of the glycerol was being quickly absorbed into the blood and tissues of this fish (Menton, 1985). In mammals, glycerol is readily absorbed in intestinal sacs at rates of about one-fourth that of glucose (Lin, 1977). The elevated plasma glucose levels of trout reared on 6% free glycerol ( 3 h post-feeding) and 12% free glycerol ( 3 and 18 h post-feeding) in experiment 3, indicates that at least some of the absorbed free glycerol ingested by the trout is being converted into glucose, but with no apparent effect on liver glycogen levels. Renaud and Moon (1980) similarly found that glycerol was principally incorporated into glucose in the hepatocytes of the eel with lesser amounts metabolized into lipid, carbon dioxide and glycogen in that
225
order. The maintenance of high levels of plasma glucose in trout reared on the 12% glycerol diet, even with an 18-h fast, appears to indicate that trout have considerable difficulty in metabolizing any glucose produced by gluconeogenesis from high levels of dietary glycerol. Indeed, even with the highly efficient absorption of glucose by the trout (National Research Council, 1981; Hilton et al., 1982)) the utilization of dietary glucose for energy by these fish is very poor. In experiments 2 and 3, it was consistently observed that in consecutive daily feedings, trout reared on the l-12% glycerol-supplemented diets showed a definite reduction in their feeding response, but with no significant reduction in the final body weight after 12 weeks on experiment. The cause of the reduction in feeding response may be related to the conversion of glycerol to glucose and the hyperglycaemia in these fish. Previous research on the feeding of high carbohydrate diets to trout has indicated that these fish show a reduction in feeding response with consecutive daily feedings, due in part to hyperglycaemia (Hilton and Atkinson, 1982) which may produce some satiating effect in the trout. The higher productive protein values (PPV) of trout fed the high energy (lipid) diets indicated that significantly more dietary protein was being converted into tissue protein, but with a concurrent significant reduction in the carcass dressing percentage compared to fish fed the low energy diets. In addition, trout fed the high energy diets had higher lipid and lower protein levels in the tissues of both the whole and eviscerated carcasses when compared to trout fed the low energy diets. This would indicate that while excess dietary energy in the form of lipid may increase dietary protein retention (Cho et al., 1976; Reinitz et al., 1978; Watanabe et al., 1979)) it also increases the fat content of the muscle and mesenteric fat pad (Takeuchi et al., 1978) ; the latter tissue being the principle site of lipid deposition in trout (Henderson and Sargent, 1981). It remains to be determined whether the economic advantage of feeding high energy (lipid) diets to trout to increase protein retention in the muscle is offset by the wastage of dietary fat deposited in the mesenteric fat pad while processing the fish for market. ACKNOWLEDGEMENTS
The authors wish to thank Ms. Debbie Conrad, Mr. Martin Hodgson, Mr. Richard Pope, Mr. Keith Were and Mr. Andrew Pharazyn for technical support; and the Ontario Ministry of Natural Resources, the Ontario Ministry of Agriculture and Food, and the Natural Sciences and Engineering Research Council of Canada for financial support.
226 REFERENCES Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 911-917. Cho, C.Y., 1982. Effects of dietary protein and lipid levels on energy metabolism of rainbow trout (Sahogairdneri). Proc. 9th Symp. on Energy Metabolism. Lillehammer, Norway, pp. 176-179. Cho, C.Y., Bayley, H.S. and Slinger, S.J., 1976. Energy metabolism in growing rainbow trout: partition of dietary energy in high protein and high fat diets. In: M. Vermorel (Editor), Energy Metabolism of Farm Animals. Clermont-Ferrand, France, pp. 299-302. Cowey, C.B., De la Higuera, M. and Adron, J.W., 1977. The effect of dietary composition and of insulin on gluconeogenesis in rainbow trout (Salmo gairdneri) . Br. J. Nutr., 38: 385-395. Eggstein, M. and Kuhlmann, E., 1974. Triglycerides and glycerol: determination after alkaline hydrolysis. In: H.U. Bergmeyer (Editor), Methods of Enzymatic Analysis, Vol. 4. Academic Press, Inc., New York, NY, pp. 1825-1831. Henderson, R.J. and Sargent, J.R., 1981. Lipid biosynthesis in rainbow trout, Salmo gairdnerii, fed diets of differing lipid content. Comp. Biochem. Physiol. C, 69: 31-37. 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: 597-607. Hilton, J.W. and Slinger, S.J., 1981. Nutrition and feeding of rainbow trout. Canadian Special Publication of Fisheries and Aquatic Sciences 55,15 pp. Hilton, J.W., Atkinson, J.L. and Slinger, S.J. 1982. Maximum tolerable level, digestion andmetabolism of D-glucose (cerelose) in rainbow trout (Salmo gairdneri) reared on a practical trout diet. Can. J. Fish. Aquat. Sci., 39: 1229-1234. Holub, B.J., Connor, J.T.H. and Slinger, S.J., 1975. Incorporation of glycerol-3-phosphate into the hepatic lipids of rainbow trout, Salmo gairdneri. J. Fish. Res. Board Can., 32: 61-64. Horwitz, W., 1980. Official Methods of Analysis of the Association of Official Analytical Chemists, 13th edition. AOAC, Washington, DC., 1018 pp. Lech, J., 1970. Glycerol kinase and glycerol utilization in trout (Salmo gairdneri) liver. Comp. Biochem. Physiol., 34: 117-124. Lin, E.C.C., 1977. Glycerol utilization and its regulation in mammals. Annu. Rev. Biochem., 46: 765-795. Lowry, O.H., Rosebrough, N.J., Fare, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Menton, D.J., 1985. Glycerol as an energy substrate for the rainbow trout (Salmo gairdneri Richardson) . M.Sc. Thesis, University of Guelph, Guelph, Ont. NlG 2W1, 167 pp. Murat, J.C. and Serfaty, A., 1974. Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin. Chem., 20: 1576-1577. National Research Council, 1981. Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC, 63 pp. Newsholme, E.A. and Taylor, K., 1969. Glycerol kinase activities in muscles from vertebrates and invertebrates. Biochem. J., 112: 465-474. Pfeffer, E., 1982. Utilization of dietary protein by salmonid fish. Comp. Biochem. Physiol. B, 73: 51-57. Pieper, A. and Pfeffer, E., 1979. Carbohydrates as possible sources of dietary energy for rainbow trout (Salmo gairdneri Richardson). In: J.E. Halver and K. Tiews (Editors), Proc. World Symp. on Finfish Nutrition and Fishfeed Technology, Vol. 1, Heenemann, Berlin, pp. 209-219. Reinitz, G.L., Orme, L.E., Lemm, C.A. and Hitzel, F.N., 1978. Influence of varying lipid concentrations with two protein concentrations in diets for rainbow trout (Salmo gairdneri) . Trans. Am. Fish. Sot., 107: 751-754. Renaud, J.M. and Moon, T.W., 1980. Characterization of gluconeogenesis in hepatocytes isolated from the American eel, Anguilla rostrata LeSueur. J. Comp. Physiol., 135: 115-125.
227 Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics: A Biometrical Approach, 2nd edition. McGraw-Hill Book Company, New York, NY, 633 pp. Takeuchi, T., Watanabe, T. and Ogino, C., 1978. Supplementary effect of lipids in a high protein diet of rainbow trout. Bull. Jpn. Sot. Sci. Fish., 44: 677-681. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem., 6: 24-27. Walton, M.J. and Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. B, 73: 59-79. Watanabe, T., Takeuchi, T. and Ogino, Ch., 1979. Studies on the sparing effect of lipids on dietary protein in rainbow trout (Salmo gairdneri) . In: J.E. Halver and K. Tiews (Editors), Proc. World Symp. on Finfish Nutrition and Fishfeed Technology. Vol. 1, Heenemann, Berlin, pp. 113-125.