Starch as an energy source in feed for cod (Gadus morhua): Digestibility and retention

Aquaculture, 80 (1989) 261-270 Elsevier Science Publishers B.V., Amsterdam

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Starch as an Energy Source in Feed for Cod (Gadus morhua): Digestibility and Retention GRO-INGUNN

HEMRE’,

OYVIND LIE, EINAR LIED and GEORG LAMBERTSEN

Institute of Nutrition, Directorate of Fisheries, P.O. Box 1900, N-5024 Bergen (Norway) ‘To whom correspondence (Accepted

should be addressed.

13 October 1988)

ABSTRACT 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. An experiment was set up to determine whether cod, Godus morhua, could use pre-cooked potato starch as an energy source. Four energy levels (O%, lo%, 20% and 30%) of carbohydrate with a constant fat content were fed for 8 weeks. The digestion capacity for precooked potato starch decreased from 40% to 26% with increasing amount of carbohydrate, the average being 33%. The digestibilities of protein and fat were independent of the starch content in the feed. The protein intake was similar for all groups. Energy intake increased when the level of carbohydrate in the feed increased. Neither growth values nor retention values for protein and fat indicated that carbohydrate was utilized to any significant degree as a source of energy. Plasma glucose increased from 350 mg/l to 800 mg/l as the carbohydrate increased from 0 to 30%, and the average was 530 mg/l. The glycogen deposition in muscle tissue seemed to reach a level of ca. 0.4% of muscle wet weight in all groups. The glycogen deposition in the liver attained a maximum of 3.5% of liver wet weight.

INTRODUCTION

Strictly carnivorous fish species have a very low capacity to digest complex carbohydrates, even at a low dietary level, as shown by experiments with yellowtail (Setiofu quinqueradiata) (Shimeno et al., 1977, 1978). The digestion capacity of complex carbohydrates decreases with increasing amounts of carbohydrate in the feed (Pieper and Pfeffer, 1978; Rychly and Spannhof, 1979; Bergot and Breque, 1983 ). Easily digestible carbohydrates such as glucose, however, gave an absorption above 90% in experiments with rainbow trout (Salmo gairdrwri) (Bergot and Breque, 1983 ). A rapid increase in blood glucose was found whereas the return to initial levels took about 24 h (Bergot, 1979a, b). Cowey et al. ( 1977) suggested that the inability of rainbow trout to regulate glucose in the blood was partly due to a lack of glucose phosphorylating capacity. High blood glucose

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levels were also found in yellowtail given a high carbohydrate diet (Shimeno et al., 1977). Furuichi et al. (1987) found that the glycogen content in the liver and in the muscle of carp (Cyprinus carpio), and the liver glycogen content in red sea bream (Chrysophrys major) were reduced when these fish were given a poorly absorbable starch compared to a highly absorbable one. Cowey et al. (1975) found that the liver glycogen deposition in plaice (Pleuronectes plutessa) was elevated when diets containing carbohydrate were given. Several authors have claimed an increase in liver glycogen deposition above normal levels when the carbohydrate content in the diet was increased (Phillips et al., 1948; Buhler and Halver, 1961; Lee and Putnam, 1973; Pieper and Pfeffer, 1978; Shimeno et al., 1978). Cod is a carnivorous species and its natural prey provides mainly protein and fat (Jobling, 1982). As a consequence, protein is used not only for growth, but also serves to meet energy needs. Feeds used in fish farming usually contain some starch as a cheap component, and as an aid to stabilizing the feed pellet. The aim of the present investigation was to obtain digestibility values for dietary starch in cod, and further, by determining the values for protein retention, protein productive value (PPV) and protein efficiency ratio (PER), to determine whether dietary starch had a protein-sparing effect. Blood glucose as well as glycogen deposition in liver and muscle were determined to give an indication of the retention of carbohydrate in well-fed cod under tank conditions. MATERIALS AND METHODS

Fish and diets Cod hatched and reared at the Aquaculture Station Austevoll (Directorate of Fisheries, Bergen, Norway) were used in this study. Four groups with replicates of 50 cod (initial weight: 311+- 18 g) were kept in eight 240-l tanks and fed ad libitum three times a week. Water renewal was 4 l/min, salinity and temperature were 30% and 8 + 0.5’ C, respectively. Four experimental diets with graded amounts of pre-cooked potato starch were used. Frozen squid mantle was used as the protein source and capelin oil as the lipid source. Crz03 was added as an indicator for digestibility measurements. The composition and chemical analyses of the feeds are given in Table 1.

At the end of the experiment the cod were killed with a sharp blow on the head. Blood was collected from five fish in each tank, centrifuged and the serum subsequently stored at - 20 oC. Each fish was then weighed before muscle and liver samples were dissected and immediately frozen. The gastrointestinal tract was divided into four segments by ligations immediately behind the pyloric

263 TABLE 1 Feed composition

and analytical values (g/kg ) Group

Squid mantle Capelin oil (Norsalmoil ) Dextrinized potato starch Binder (guar-gum) Vitamin mixture” Mineral mixtureb Cr,O,, Analysis Dry weight Protein Fat Digestible carbohydrate Ash Energy (MJ/kg)

I

2

3

4

974 14 10 1 1 1

940 15 33 10 1 1 1

896 18 74 10 1 1 1

841 22 124 10 1 1 1

250 180 20

270 170 20 30 20 5.1

300 170 20 70 20 5.8

340 150 20 120 20 6.4

20 4.9

“Composition, see Lie et al. (1988). hCommercial mineral mixture for poultry and swine, Lied and Rosenlund

(1984).

ceca, at the first caudal and the second cranial bend of the intestine, at the ileorectal valve, and at the anus. The segments were frozen, and the gut content was removed for analyses. Pooled samples from five fish were used for these analyses. Analytical methods Samples of feed, muscle, liver and gut contents were analysed for dry matter, protein, fat and glycogen. Protein (N x 6.25) was analysed according to Crooke and Simpson (1971) and fat was measured gravimetrically using ethyl acetate extraction. The chromium contents in diet and faeces samples were determined by atomic absorption spectrophotometry as described by Lied and Njaa ( 1982). Digestible carbohydrate in the feed and faeces and glycogen in the liver and muscle were analysed using an enzymatic method modified from Murat and Serfaty (1974) and Holm et al. (1986). The enzymes used were a heatstable amylase (Termamyl-120L; Novo-Industries, Denmark) and amyloglucosidase (EC 3.2.1.3; Boehringer Mannheim). The samples of feed, gut contents and muscle were freeze-dried before analyses, whereas liver samples were analysed directly. Serum samples were deproteinized (using 0.33 M trichloroacetic acid) before glucose was measured. Glucose was measured spectrophotometrically as NADPH at 340 nm after a hexokinase reaction in an automated analyser.

264

Statistics One-way ANOVA analysis of variance weight gain and hepatosomatic index.

was used for statistical

evaluation

of

RESULTS AND DISCUSSION

Feeds Earlier experiments have shown that squid mantle is well accepted by the cod (Lie et al., 1986), as was also confirmed in the present experiment. Lie et al. (1988) found that an increase in the fat energy level above 20% in the feed resulted in an increase in the hepatosomatic index. To avoid accumulation of fat in the liver the lipid energy level in the feed was kept at 20% in the present study. Increasing amounts of carbohydrate also increased the dry weight and therefore the energy concentration of the wet feed. Weight gain and feed consumption Increasing the amount of carbohydrate energy in the diet from 0 to 30% did not influence the weight gain significantly, except for one of the replicates in group 4 (Table 2 ) . However, a marked increase in the amount of energy consumption was found with increasing carbohydrate content in the feeds. Furuichi et al. (1987) concluded from feeding experiments with carp that a low carbohydrate intake was preferred even in this herbivorous species. However, Degani and Viola (1987) found that growth was improved in the eel (Anguilla anguilla) given a high carbohydrate diet (38% wheat meal). Cowey et al. (1975) reported that weight gain was superior in plaice fed a diet including carbohydrates compared to a carbohydrate;free diet with a high fat content, but the energy consumption was higher in the carbohydrate group. Increasing demands of energy with increasing carbohydrate levels in the feed have been shown for mullet (Mugil capita) (Alexis and Papaparaskeva-Papoutsoglou, 1986) and for rainbow trout (Lee and Putnam, 1973). In the present experiment the total amounts of protein ingested per fish during the 8 weeks were: 52.8 g, 47.2 g, 49.6 g and 51.2 gin groups 1,2,3 and 4, respectively. These values suggest that carbohydrate incorporation had no protein-sparing effect, which is in accordance with the findings for mullet by Alexis and PapaparaskevaPapoutsoglou ( 1986). Shimeno et al. ( 1978)) however, noticed some proteinsparing effect of carbohydrate incorporation’in the feed to yellowtail. Protein productive value (PPV) was generally low, except for group 4a, showing a mean PPV value of 0.26. Protein efficiency ratio (PER) was 25% higher in group 4 than in the other groups, whereas no differences were found between groups 1, 2 and 3. Pfeffer (1982) suggested that feed waste was the reason for low PER values in his experiments with rainbow trout. Degani and Viola (1987) found decreasing PER values as the carbohydrate content in the diets to eel increased. Since the fish in the present experiment were fed by

TABLE 2 Growth, feed intake, hepatosomatic index, PER and PPV of cod fed diets with four different carbohydrate levels for 8 weeks (a and b are replicates) Group la

lb

2a

2b

3a

3b

4a

4b

Initial weight (g ) SD (n=25) Final weight (g) SD” Weight gain (g) Weight gain ( % )

293.3 76 383.4 116 90.1 31

339.2 73 422.6 105 83.2 25

323.6 55 414.8 79 91.2 28

292.4 69 367.4 100 75.0 26

302.4 58 393.6 78 91.2 30

316.4 73 408.5 105 92.1 29

325.7 55 450.2 97 124.5 38

292.6 69 389.4 97 96.8 33

Feed consumption (g/fish)

294.8

293.3

305.5

248.7

307.1

288.7

359.4

308.9

Feed conversion (g dry feed/g weight gain)

0.8

0.9

0.9

0.9

1.0

0.9

1.0

1.1

Hepatosomatic index ( % ) b SD PER’ PPVd

6.5 1.5 1.71 0.17

6.8 1.0 1.59 0.13

6.7 1.2 1.77 0.12

5.9 1.0 1.78 0.13

7.3 1.2 1.79 0.14

7.2 1.1 1.92 0.13

7.7 1.2 2.29 0.26

7.4 1.1 2.06 0.18

‘n was reduced to 24 in group 2a, 3b and 4b, and to 21 in group 2b. “Liver weight/final weight x 100, hepatosomatic index at start: 7.9 ? 2.0 (n = 10). ‘Protein efficiency ratio = g weight gain/g protein intake. dProtein productive value = g protein retention/g protein intake.

hand, feed waste does not account for the low values. Cowey et al. (1975) reported increasing PER values in plaice with increasing energy levels in the diet. The feed used in the present experiment had a low fat energy content and this probably explains the low PER values. However, Buhler and Halver (1961) found increasing PER values in chinook salmon (Oncorhynchus tshawytscha) fed increasing levels of carbohydrates and concluded that this species was able to use carbohydrate as an energy source. Digestibility There was a linear decrease in carbohydrate digestibility when the starch content of the diet increased: 40%, 33% and 26% in groups 2,3 and 4, respectively. This indicates that cod has a limited capacity to digest starch. According to Nagayama and Saito (1968) the amylase activity in the fish intestine is low. Hofer and Sturmbauer (1985) found that starch exerts a negative feed-back on amylase. Both Spannhof and Plantikow (1983) and Hofer and Sturmbauer (1985) found decreasing digestibilities in rainbow trout as the amount of carbohydrate in the diets increased. With a mean digestibility for starch of 33% in the present experiment, and total energy for starch of 17.2 kJ/g, the avail-

able energy was 5.7 kJ/g when starch was included in the feed to cod. In all groups there was a marked cumulative increase in the digestibility along the intestine. This indicates that glucose released from starch is absorbed over the entire length of the intestine. A direct study on the glucose absorption capacity in cod showed that glucose absorption took place along the whole length of the intestine (Buddington and Diamond, 1987); nevertheless, given an easily digestible carbohydrate, 70% of the released glucose was absorbed in the pylorus and the upper part of the intestine. Fat digestibility averaged 85% and did not vary between groups. The amount of protein digested was approximately 90% in all four groups. These results show that increasing amounts of dietary starch do not affect the digestibility of fat and protein in cod. De novo synthesis of fat from carbohydrate The hepatosomatic index was significantly (P < 0.05 ) higher in groups 3 and 4 than in groups 1 and 2 (Table 2 ) . The amount of fat intake varied from 5.9 g/fish in group 1 to 6.7 g/fish in group 4. The increase in the hepatosomatic index was not caused by accumulation of glycogen in the liver, but by an increased fat content (Table 3 ). Some of this fat may have come from a de novo synthesis of fat from carbohydrate, as reported by Shimeno and Hosokawa (1975) in yellowtail and Degani and Viola (1987) in eel. However, Nagai and Ikeda (1971,1972) could not detect any increase in the liver fat content in carp when glucose-U-i4C was added to the feed. TABLE 3 Dry matter, protein, fat and carbohydrate in the liver and muscle, and the concentration of glucose in the blood, of cod fed diets with four different levels of carbohydrate for 8 weeks (a and b are replicates) Group Initial

la

lb

2a

2b

3a

3b

4a

4b

69.7

Liver Dry matter (% ) Protein (% ) Fat (%) Glycogen (% )

4.6 60.9 3.6

72.1 5.3 62.5 1.4

72.4 5.9 64.3 1.3

71.0 5.2 61.9 2.3

71.0 5.6 62.1 1.4

70.7 4.8 60.8 3.5

71.3 4.8 62.4 1.56

71.2 5.0 61.6 1.0

71.3 4.9 61.7 1.1

Muscle Dry matter (% ) Protein (% ) Fat (%) Glycogen (% )

21.0 l&,3 0.2 0.1

20.9 17.8 0.3 0.4

20.9 18.2 0.3 0.3

21.0 18.2 0.3 0.4

20.7 17.7 0.3 0.3

20.3 17.4 0.3 0.7

20.8 18.1 0.3 0.4

20.9 18.1 0.3 0.3

21.1 18.1 0.7 0.7

350

360

480

500

520

540

700

800

Blood glucose (mg/l)

267

Glycogen deposition in liver The initial glycogen stores were about 3.5% (Table 3), which is about the maximum level of glycogen formed in cod liver (G.I. Hemre, unpublished results, 1987). After 8 weeks only one of the replicates of group 3a retained this level. There was no relationship between dietary carbohydrate and liver glycogen deposition in this experiment. Both plaice (Cowey et al., 1975 ) and rainbow trout (Hilton and Atkinson, 1982) increased their liver glycogen stores as the amount of carbohydrate in the feed increased. Shatunovskii and Denisove (1968) and Shatunovskii et al. (1975) showed that during maturation cod used deposits of fat and of glycogen for the build-up of gonads. An increased gonad index was noticed in our experiment, and this may be a possible explanation for the low liver glycogen levels observed. Glycogen deposition in muscle Glycogen deposited in the muscle tissues is used there as an energy reserve, while glycogen deposited in the liver is mainly a reserve of blood glucose. Initially the amount of muscle glycogen was 0.17 g/fish, or 0.1% of the total muscle weight (Table 3). The low content could be due to high muscle activity during fish transport and to the experimental set-up. Black’et al. (1961) measured a rapid conversion of muscle glycogen to lactic acid in stressed fish. In the present study there were no marked differences between the groups after 8 weeks but a marked increase from the initial glycogen values. Even in the fish of group 1 without any dietary carbohydrate, the average glycogen level was 0.4%. This indicates a gluconeogenetic activity using triglycerides and/or amino acids to build up glycogen reserves. Shimeno et al. (1977) noted that glucose from gluconeogenetic activity was used to build up glycogen stores in yellowtail fed no carbohydrate in the diet. Groups 3 and 4 showed high individual variations in the amount of glycogen deposited in the muscle. Both Edwards et al. (1977) and Refstie and Austreng (1981) showed differences between different families of rainbow trout in the ability to utilize carbohydrates. Dietary carbohydrate and blood glucose The mean level of blood glucose in the present study was 531 mg/l, but wide individual variation was found. In addition, there was a significant increase in the concentration of glucose in blood with the carbohydrate content in the diet. This effect was also reported in rainbow trout by Bergot (1979a), and in yellowtail, red sea bream and carp by Yone (1979 ). Larsson et al. (1976) observed that wild cod from the Skagerrak showed considerable differences in blood glucose levels with a mean level of 500 mg glucose/l blood. Distribution of absorbed carbohydrate In the present experiment only 1% of the absorbed carbohydrate was recovered as blood glucose, assuming that the intestine was the only source of

268

glucose after a meal. About 17% of the absorbed carbohydrate was deposited as liver glycogen, and 7% as muscle glycogen, provided the levels of glycogen in these organs were 3.5% and 0.4%, respectively. Thus only 25% of the absorbed carbohydrate could be accounted for as either glucose or glycogen. Neither growth values nor retention values for protein and fat gave significant indications of the use of carbohydrate as an energy source. Shimeno and Hosokawa (1975) postulated that some blood glucose was secreted in the urine and/or over the gills in yellowtail fed high carbohydrate diets. According to Cowey et al. (1975) fish are able to use some carbohydrate for energy. These suggestions are conflicting, and further work is needed to elucidate the fate and metabolism of absorbed carbohydrate in this species.

REFERENCES

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270 Shimeno, S. and Hosokawa, H., 1975. How much carbohydrate can fish utilize? Their metabolic characteristics and adaptability. Kagaku To Seibutsu (Chemistry and Living Things), 13: 365-367. Shimeno, S., Hosokawa, H., Hirata, H. and Takeda, M., 1977. Comparative studies on carbohydrate metabolism of yellowtail and carp. Bull. Jpn. Sot. Sci. Fish., 43: 213-217. Shimeno, S., Hosokawa, H. and Takeda, M., 1978. The importance of carbohydrate in the diet of a carnivorous fish. FAO JFN: EIFAC/78/Symp: Ef 5, pp. l-31. Spannhof, L. and Plantikow, H., 1983. Studies on carbohydrate digestion in rainbow trout. Aquaculture, 30: 95-108. Yone, Y., 1979. The utilization of carbohydrate by fishes. Proc. 7th Japan-Soviet Joint Symp. Aquaculture, Tokyo, Sept. 78, pp. 39-48.