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Stability in fish feed and bioavailability to rainbow trout of two ascorbic acid forms
Aquaculture,
84 (1990)
335-343
Elsevier Science Publishers
335
B.V., Amsterdam
-
Printed
in The Netherlands
Stability in Fish Feed and Bioavailability to Rainbow Trout of two Ascorbic Acid Forms T. SKELBAEK”,
N.G. ANDERSENb,
“Danochemo
A/S, Malmparken
bThe Danish
Institute
DK-9850
(Accepted
Hirtshals
M. WINNING”
5, DK-2750
for Fisheries
Ballerup
and Marine
and S. WESTERGAARD” (Denmark)
Research,
North Sea Centre,
(Denmark)
10 June 1989)
ABSTRACT Skelbaek, T., Andersen, N.G., Winning, M. and Westergaard, S., 1990. Stability in fish feed and bioavailability to rainbow trout of two ascorbic acid forms. Aquaculture, 84: 335-343. The stability in warm pelleted fish feed and the bioavailability to rainbow trout of crystalline ascorbic acid (AA) and a synthetic polymer-coated AA product (PCAA) were compared. The AA loss during pelleting was 29% for crystalline AA and 19% for PCAA. After 6 weeks at room temperature 73% of PCAA was still retained whereas less than 10% of crystalline AA was left. Rainbow trout were deprived of AA for 8 weeks, and then supplemented with AA from crystalline AA and PCAA, respectively. At the same time, two groups of trout were supplemented with a constant level of AA from either crystalline AA or PCAA. The concentration of free AA in the liver was used as an index of the AA status of the fish. The bioavailability of PCAA turned out to be the same as that of crystalline AA. After 8 weeks some of the fish given an AA-free diet showed dilated swim bladders, and/or inflammation, and haemorrhage in the gastrointestinal system. This was interpreted as vitamin C deficiency symptoms. We did not observe any difference in growth between fish given a diet containing AA and those given an AA-free diet.
INTRODUCTION
In recent years, the importance of supplementing ascorbic acid (AA) to fish and the problem of stability of AA in fish feed have been the objects of a great deal of attention (Murai et al., 1978; Sandnes and Utne, 1982; Soliman et al., 1987). In a pelleted catfish diet, Love11 and Lim (1978) found that the half times for uncoated AA and ethylcellulose-coated ascorbic acid (ECAA) were approx. 2.3 months and 2.6 months, respectively. However, Hilton et al. (1977a) showed that all ECAA in laboratory-pelleted fish feed was lost after 6 weeks of storage at room temperature whereas the loss was not quite as serious in commercially produced feed, in which 25% was left after 5 weeks at room temperature. Sandnes and Utne (1982) reported that commercial trout feed contained only 17% of ECAA after 4 weeks at 20’ C. In cold pelleted feed stored at
0044.8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
336
T. SKELBAEK ET AL.
room temperature for 50 days, Soliman et al. (1987) showed that less than 10% of uncoated AA and its sodium salt was retained whereas 35% of glyceridecoated ascorbic acid (GCAA) was left, and even 85% of ascorbic acid 2-sulphate ( AABS),a chemically modified form. However, great differences in biological activity of AA2S have been demonstrated. Halver et al. (1975) found equimolar activities of AABS and AA given to rainbow trout. In catfish, the activity of AABS was four times less than AA (Murai et al., 1978)) and similar results were reported by Sandnes et al. (1987) for Atlantic salmon. In the present study, the stability was determined in warm pelleted fish feed of crystalline AA, and three forms of coated AA: synthetic polymer-coated AA (PCAA), ECAA and GCAA. Furthermore, the bioavailability to rainbow trout of PCAA and crystalline AA were compared. MATERIALS
AND METHODS
Stability of crystalline AA and PCAA during pelleting and storage Production of feed. Pelleted fish feeds contained 500-600 ppm AA in the form of crystalline AA (BASF or Merck, Fed. Rep. Germany), synthetic polymercoated ascorbic acid, 80% (PCAA) (Davitin C80, Danochemo, Denmark), ethylcellulose-coated ascorbic acid, 97.5% (ECAA) (Hoffmann la Roche, Switzerland) and glyceride-coated ascorbic acid, 50% (GCAA) ( Ascorbidan 50, Grindsted Products, Denmark), respectively. They were produced at Bioteknisk Institut (The Biotechnical Institute), Denmark, in a pilot plant, processing 100 kg per batch (Moeller et al., 1984). The feed formula is shown in Table 1. The feed ingredients were mixed for 20 min, and 4-5% steam was added, causing heating to 80-84°C. The conditioning time was approx. 26 s. The steamed mixture was pressed through a matrix (diameter 6~ 50 mm), cooled, and dried at ambient temperature in 20 min to a water content of maximum 10%. Stability test. In three different runs, a total of 10 batches of PCAA were tested. Each run included one feed with crystalline AA. Additionally, other types of coated AA were included once. The samples were stored at 22°C in dark, closed containers (6 g in each). Triple determinations of AA were carried out at different intervals for 3 months and for a few batches extended to 6 ( n = 4 ) and 9 months (n = 3 ) according to the method below. The feed sample (6 g) and 10 ml of isopropanol were boiled on a magnetic stirrer. This was followed by addition of approx. 100 ml of nitrogen-saturated extraction solution (4% metaphosphoric acid solution with 0.1% ethylenediaminetetraacetic acid disodium (EDTA) ). Under a nitrogen atmosphere, the sample was heated on a magnetic stirrer until vigorously boiling, and cooled
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
337
TABLE 1 Composition
of fish feed Ingredient
% of diet
Raw material
Fish meal, type A Wheat, heat-treated Soy meal, toasted Dry yeast, MAK 35 Fish oil, tobis Vitamin/mineral mix”
55 10 18 10 5 2
Proximal analysi$
Raw protein Total fats Carbohydrates Ash Water
50 20 10 8 12
“Composition of vitamin/mineral mix (mg/kg): menadione sodium bisulphite, 114; retinyl acetate, 588; cholecalciferol, 0.725; a-tocopherylacetate, 429; thiamine hydrochloride, 114; vitamin B-12,0.57; D-pantothenic acid, 286; niacin, 571; Ca, 200 g/kg; P, 90 g/kg; Fe, 2286; Zn, 2860; Cu, 718; Mn, 1697; I, 29; Se, 8.57. bAccording to label claim.
immediately afterwards. The sample volume was adjusted to 200 ml with extraction solution. After vigorous shaking and sedimentation for a few minutes, an aliquot of the sample was filtered (0.45 pm) and injected on a reversedphase column (Lichrosorb C18,5 pm 250 x 4 mm). The mobile phase was 0.04 A4 acetate buffer with 2.2% tetrahexylammonia bromide as the ion-pairing agent. The ascorbic acid was quantitated at 243 nm by an external standard technique. Bioavailability
of crystalline AA and PCAA to rainbow trout
Experimental design. The bioavailability was investigated both to vitamin Cdeficient trout and to trout with a normal vitamin C level. During one part of the experiment, trout were given an AA-deficient diet for 8 weeks (tanks A and B ) . Then the fish were redistributed into the two holding tanks and were supplemented for 4 weeks with crystalline AA (tank A) or PCAA (tank B). During the other part of the experiment, trout were supplemented for 8 weeks with a constant level of AA in the form of crystalline AA (tank C) or PCAA (tank D). The water volume in each tank was 1.5 x 1.5x 0.3 m3, the water temperature was 11.5”C + 0.5”C, and there was continuous lighting in the room. Initially the average weight of the trout was 22.8 2 3.1 g (s.d. ), and the number of fish was 325 in each of tanks A and B, and 250 in each of tanks C and D. The fish
338
T.SKELBAEKETAL.
TABLE 2 Concentration of AA (ppm) in the feed used for the bioavailability study
n
Mean + s.e.m.
Tank A AA
Tank B PCAA
Tank C AA
Tank D PCAA
15 14829
18 128&B
21 1142 10
15 156t 12
were fed twice a day. In order to avoid overstocking, the numbers of fish in the tanks were gradually reduced (range of biomass: lo-33 kg/m3). The feeding level was 0.8-0.9 times the maximum ration (From and Rasmussen, 1984 ) and was adjusted daily according to the biomass and expected growth. Feed. Vitamin C-deficient feed and feed containing approx. 150 ppm AA of crystalline AA and PCAA, respectively, were manufactured similarly to those of the stability test. The pellet diameter was 3 mm, and the fat content was raised to a total of 20% by fish oil. In order to minimize AA degradation during the experimental period, feed was kept at - 18°C before the fish were fed. Analyses of feed AA concentrations during the experimental period showed no degradation (P > 0.05 ) (Table 2 ) . Sampling and analysis. The concentration of free AA in the liver was used as an index of the bioavailability of AA in the feed. Th liver was removed from the fish and kept at - 79 oC until analysis, approx. l-2 days later. Analyses were carried out by Levnedsmiddelstyrelsen, Denmark (The National Food Agency of Denmark) by a method in principle similar to that of the feed assay (method SL AB 1131 slightly modified). Initially 20 livers were analysed individually, and then the number of livers tested was 10 from each tank. Fish from tanks A and B were analysed after 0,8,9,10,11, and 12 weeks, and those from tanks C and D after 0,3,5, and 7 weeks. Fish weights were registered in connection with the liver analyses. Fifty fish were weighed initially, and every 4th week 30 fish from each tank were weighed. RESULTS
Stability of crystalline AA and PCAA during pelleting and storage The amount of AA retained after the pelleting process was 70.9% ? 9.4% (x+s.e.m., n=3) for crystalline AA and 80.5%?2.6% (n?s.e.m., n=lO) for PCAA. After 6 weeks there was less than 10% left in the feed produced with crystalline AA whereas 72.6% ? 2.6% (X? s.e.m., n= 10) was left in the feed produced with PCAA. After 9 months there was still about 15% AA left of the
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
AA in feed
1
:339
(% of added)
3
5
7
9
Months
Fig. 1. AA concentration (% of added) in pelleted fish feed with crystalline AA, PCAA and other coated products, versus time (a? s.e.m.).
PCAA (Fig. 1). The other coated AA products tested appear to have a stability in pelleted fish feed equal to that of crystalline AA. Bioavailability of crystalline AA and PCAA in trout Liver AA. The liver AA concentration in fish from tanks A and B decreased during a period of 8 weeks from 1585 3.1 to 37 t 2.4 ppm (%+s.e.m., n=20) when fish received an AA-free feed. After 4 weeks on an AA-sufficient diet, it increased again to 162-173 ppm. The liver AA concentration in fish from tanks C and D, receiving normal level AA feed, was 128-158 ppm during the whole test period (Fig. 2). The AA concentration was a little higher in fish from tank A and tank D compared with fish from tank B and tank C, respectively. The differences were analogous to those of the AA concentrations in the feed given to the groups. Over the whole period there was no significant difference in either liver or feed data between tanks A and B (t-test, P> 0.05) whereas both liver and feed data from tank C were significantly lower than those from tank D (t-test, P< 0.01). Clinical observations. After 7-8 weeks on an AA-deficient diet, 11 fish developed dilated swim bladders. At that time, inflammation and haemorrhage in the gastrointestinal system were noticed. Four days after starting the AA supplementation, some fish still showed the above-mentioned symptoms, but after
T. SKELBAEK ET Al
340 AA in liver
(PPM)
/ o lank
C
x Tank
D
I
\ \
IOO-
\ \ \ \
---
AA-free
-
Normal AA level diet
diet
\ \
60s
\
2
4
6
\
8
10
Fig. 2. Liver AA concentration Fish
weight
12
Weeks
(ppm) versus time (Xk s.e.m.)
(g)
1
‘40j 120.
100.
80
i
60-
40.
20
t
ABCD 0
-
-e Wee
4
Fig. 3. Fish weight in tanks A, B, C, and D (f+s.e.m.
).
s
STABILITY
AND
BIOAVAILABILITY
OF ASCORBIC
ACID
FORMS
TO RAINBOW
TROUT
341
that time none of the symptoms were observed. During the rest of the experiment, however, several fish developed fibrous tissue between the organs in the body cavity. The fish given the AA-containing diet (tanks C and D) showed none of the symptoms mentioned. They were observed for a total of 9 weeks. Growth. After 8 weeks there was no difference in the mean weight of the fish between the four groups (P> 0.05)) and after 13 weeks there was no difference between tanks A and B (P> 0.05) (Fig. 3). DISCUSSION
The present study showed that the synthetic polymer-coated product PCAA is more stable than crystalline AA and other coated forms tested, viz. ethylcellulose-coated AA and glyceride-coated AA. The processing loss was only 29% of uncoated AA and 19% of PCAA, whereas degradation during storage was rapid for crystalline AA, unlike PCAA. The pelleting loss compares well with results of Love11 and Lim (1978) who found, however, much lower degradation rates after pelleting. The difference may partly be caused by the higher processing temperature in the present experiment, 80-84’ C, compared to 6672 “C! used by Love11and Lim (1978). Hilton et al. (1977a) found that different pelleting procedures have great influence on the stability of AA. This may also be the reason why these experiments failed to find better stability of ECAA and GCAA compared to crystalline AA, as found by Hilton et al. (1977a) for ECAA and by Soliman et al. (1987) for GCAA in cold pelleted diet. The clinical symptoms such as dilated swim bladder, inflammation, and haemorrhage in the gastrointestinal system, observed in the group of fish deprived of AA, are presumed to be vitamin C deficiency symptoms. The symptoms appeared after only 7 or 8 weeks and at liver concentrations of 37 ppm AA (range = 25-68 ppm), and the fish seemed to show an earlier stage of deficiency than the more advanced symptoms observed by Hilton et al. (1977b) who found that trout given an AA-free diet suffered from overt deficiency symptoms such as anorexia, lethargy, and prostration at a liver concentration of < 20 ppm AA after 16-20 weeks. Lim and Love11 (1978) observed AA deficiency symptoms in channel catfish after 8-12 weeks at liver concentrations of < 30 ppm. A high growth rate, as occurred in the present experiment, is known to result in a higher depletion rate of the AA stored in the fish. Absence of AA for 8 weeks did not impair the growth rate of young trout. Hilton et al. (1978) claim that AA requirement varies with age and growth rate, being highest in young fish. The present observations agree well with those of Lim and Love11 (1978) who observed that the growth rate in channel catfish was not disturbed by an AA-free diet for 8 weeks. The concentration of free AA in the liver was used as an index of the AA status in the fish. The anterior kidney was preferred by Halver et al. (1975),
342
T. SKELBAEK ET AL.
though Lim and Love11 (1978) established that the anterior kidney of channel catfish did not reflect differences in the AA concentration in the diet. Furthermore, the anterior kidney is very small and difficult to isolate. Lim and Love11 (1978) and Murai et al. (1978) found good correlation between the diet and liver AA concentration, and Hilton et al. (1977b) found this correlation better than that between diet and anterior kidney. The results of the present experiment show that even a minor difference in the AA concentration of the diet is reflected in the liver AA. Comparison of the liver AA concentrations in the fish given crystalline AA (tanks A and C ) and in the fish given PCAA (tanks B and D ) shows that AA in the form of PCAA is available to the trout to the same extent as crystalline AA. The liver AA did not reach a steady level within 4 weeks of AA supplementation, in agreement with Sandnes (1984) who found the period to be 4-6 weeks for trout fry. In summary, the present results indicate that PCAA has superior stability, and at the same time full bioavailability. ACKNOWLEDGEMENT
This work was partly supported by Industri- og Handelsstyrelsen (The National Agency of Industry and Trade), Denmark.
REFERENCES From, J. and Rasmussen,
G., 1984. A growth model, gastric evacuation,
and body composition
rainbow trout, Salmo gairdneri Richardson, 1836. Dana, 3: 61-139. Halver, J.E., Smith, R.R., Tolbert, B.M. and Baker, E.M., 1975. Utilization
of ascorbic
in
acid in
fish. Ann. N.Y. Acad. Sci., 258: 81-102. Hilton, .J.W., Cho, C.Y. and Slinger, S.J., 1977a. Factors affecting the stability of supplemental ascorbic acid in practical trout diets. J. Fish. Res. Board Can., 34: 683-687. Hilton, d.W.. Cho, C.Y. and Slinger, S.J., 197713. Evaluation of ascorbic acid status of rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 34: 2207-2210. Hilton, J.W., Cho, C.Y. and Sl inger, S.J., 1978. Effect of graded levels of supplemental ascorbic acid in practical diets fed to rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 35: 431-436. Lim, C. and Lovell, R.T., 1978. Pathology of vitamin (Zctaluruspunctatus). J. Nutr., 108: 1137-1146.
C deficiency
syndrome
in channel catfish
Lovell. R.T. and Lim, C., 1978. Vitamin C in pond diets for channel catfish. Trans. Am. Fish. Sot., 107: 321-325. Moeller, J., Busk, J. and Bentsen, H., 1984. Pelletering af Fedt- og Proteinrige Foderblandinger (Pelleting of Fat and Protein-rich Feed Rations). Beretning no. 117, Biotekniskinstitut, Kolding, ISSN 0106-0082,60 pp. Murai, T., Andrews, J.W. and Bauernfeind,
J.C., 1978. Use of L-ascorbic
acid, ethocel-coated
ascorbic acid and ascorbate 2-sulfate in diets for channel catfish, Zctaluruspunctatus. 108: 1761-1766.
J. Nutr.,
Sandnes, K., 1984. Some aspects of ascorbic acid and reproduction in fish. In: I. Wegger, F.J. Tagwerker and J. Moustgaard (Editors), Ascorbic Acid in Domestic Animals, Proc. Workshop, 1st Meeting Date 1983. Royal Danish Agricultural Society, Copenhagen, p. 206-12.
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
343
Sandnes, K. and Utne, F., 1982. Processing loss and storage stability of ascorbic acid in dry fish feed. Fiskeridir. Skr., Ser. Ernaering, 2: 39-44. Sandnes, K., Hansen, T. and Waagboe, R., 1987. Ascorbate-2-sulfate as vitamin C source for Atlantic salmon (Salmo salar). Int. Symp. Feeding and Nutrition in Fish, Bergen, 23-27 August 1987 (abstract, unpublished). Solimann, A.K., Jauncey, K. and Roberts, R.J., 1987. Stability of L-ascorbic acid (vitamin C) and its forms in fish feeds during processing, storage and leaching. Aquaculture, 60: 73-83.
84 (1990)
335-343
Elsevier Science Publishers
335
B.V., Amsterdam
-
Printed
in The Netherlands
Stability in Fish Feed and Bioavailability to Rainbow Trout of two Ascorbic Acid Forms T. SKELBAEK”,
N.G. ANDERSENb,
“Danochemo
A/S, Malmparken
bThe Danish
Institute
DK-9850
(Accepted
Hirtshals
M. WINNING”
5, DK-2750
for Fisheries
Ballerup
and Marine
and S. WESTERGAARD” (Denmark)
Research,
North Sea Centre,
(Denmark)
10 June 1989)
ABSTRACT Skelbaek, T., Andersen, N.G., Winning, M. and Westergaard, S., 1990. Stability in fish feed and bioavailability to rainbow trout of two ascorbic acid forms. Aquaculture, 84: 335-343. The stability in warm pelleted fish feed and the bioavailability to rainbow trout of crystalline ascorbic acid (AA) and a synthetic polymer-coated AA product (PCAA) were compared. The AA loss during pelleting was 29% for crystalline AA and 19% for PCAA. After 6 weeks at room temperature 73% of PCAA was still retained whereas less than 10% of crystalline AA was left. Rainbow trout were deprived of AA for 8 weeks, and then supplemented with AA from crystalline AA and PCAA, respectively. At the same time, two groups of trout were supplemented with a constant level of AA from either crystalline AA or PCAA. The concentration of free AA in the liver was used as an index of the AA status of the fish. The bioavailability of PCAA turned out to be the same as that of crystalline AA. After 8 weeks some of the fish given an AA-free diet showed dilated swim bladders, and/or inflammation, and haemorrhage in the gastrointestinal system. This was interpreted as vitamin C deficiency symptoms. We did not observe any difference in growth between fish given a diet containing AA and those given an AA-free diet.
INTRODUCTION
In recent years, the importance of supplementing ascorbic acid (AA) to fish and the problem of stability of AA in fish feed have been the objects of a great deal of attention (Murai et al., 1978; Sandnes and Utne, 1982; Soliman et al., 1987). In a pelleted catfish diet, Love11 and Lim (1978) found that the half times for uncoated AA and ethylcellulose-coated ascorbic acid (ECAA) were approx. 2.3 months and 2.6 months, respectively. However, Hilton et al. (1977a) showed that all ECAA in laboratory-pelleted fish feed was lost after 6 weeks of storage at room temperature whereas the loss was not quite as serious in commercially produced feed, in which 25% was left after 5 weeks at room temperature. Sandnes and Utne (1982) reported that commercial trout feed contained only 17% of ECAA after 4 weeks at 20’ C. In cold pelleted feed stored at
0044.8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
336
T. SKELBAEK ET AL.
room temperature for 50 days, Soliman et al. (1987) showed that less than 10% of uncoated AA and its sodium salt was retained whereas 35% of glyceridecoated ascorbic acid (GCAA) was left, and even 85% of ascorbic acid 2-sulphate ( AABS),a chemically modified form. However, great differences in biological activity of AA2S have been demonstrated. Halver et al. (1975) found equimolar activities of AABS and AA given to rainbow trout. In catfish, the activity of AABS was four times less than AA (Murai et al., 1978)) and similar results were reported by Sandnes et al. (1987) for Atlantic salmon. In the present study, the stability was determined in warm pelleted fish feed of crystalline AA, and three forms of coated AA: synthetic polymer-coated AA (PCAA), ECAA and GCAA. Furthermore, the bioavailability to rainbow trout of PCAA and crystalline AA were compared. MATERIALS
AND METHODS
Stability of crystalline AA and PCAA during pelleting and storage Production of feed. Pelleted fish feeds contained 500-600 ppm AA in the form of crystalline AA (BASF or Merck, Fed. Rep. Germany), synthetic polymercoated ascorbic acid, 80% (PCAA) (Davitin C80, Danochemo, Denmark), ethylcellulose-coated ascorbic acid, 97.5% (ECAA) (Hoffmann la Roche, Switzerland) and glyceride-coated ascorbic acid, 50% (GCAA) ( Ascorbidan 50, Grindsted Products, Denmark), respectively. They were produced at Bioteknisk Institut (The Biotechnical Institute), Denmark, in a pilot plant, processing 100 kg per batch (Moeller et al., 1984). The feed formula is shown in Table 1. The feed ingredients were mixed for 20 min, and 4-5% steam was added, causing heating to 80-84°C. The conditioning time was approx. 26 s. The steamed mixture was pressed through a matrix (diameter 6~ 50 mm), cooled, and dried at ambient temperature in 20 min to a water content of maximum 10%. Stability test. In three different runs, a total of 10 batches of PCAA were tested. Each run included one feed with crystalline AA. Additionally, other types of coated AA were included once. The samples were stored at 22°C in dark, closed containers (6 g in each). Triple determinations of AA were carried out at different intervals for 3 months and for a few batches extended to 6 ( n = 4 ) and 9 months (n = 3 ) according to the method below. The feed sample (6 g) and 10 ml of isopropanol were boiled on a magnetic stirrer. This was followed by addition of approx. 100 ml of nitrogen-saturated extraction solution (4% metaphosphoric acid solution with 0.1% ethylenediaminetetraacetic acid disodium (EDTA) ). Under a nitrogen atmosphere, the sample was heated on a magnetic stirrer until vigorously boiling, and cooled
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
337
TABLE 1 Composition
of fish feed Ingredient
% of diet
Raw material
Fish meal, type A Wheat, heat-treated Soy meal, toasted Dry yeast, MAK 35 Fish oil, tobis Vitamin/mineral mix”
55 10 18 10 5 2
Proximal analysi$
Raw protein Total fats Carbohydrates Ash Water
50 20 10 8 12
“Composition of vitamin/mineral mix (mg/kg): menadione sodium bisulphite, 114; retinyl acetate, 588; cholecalciferol, 0.725; a-tocopherylacetate, 429; thiamine hydrochloride, 114; vitamin B-12,0.57; D-pantothenic acid, 286; niacin, 571; Ca, 200 g/kg; P, 90 g/kg; Fe, 2286; Zn, 2860; Cu, 718; Mn, 1697; I, 29; Se, 8.57. bAccording to label claim.
immediately afterwards. The sample volume was adjusted to 200 ml with extraction solution. After vigorous shaking and sedimentation for a few minutes, an aliquot of the sample was filtered (0.45 pm) and injected on a reversedphase column (Lichrosorb C18,5 pm 250 x 4 mm). The mobile phase was 0.04 A4 acetate buffer with 2.2% tetrahexylammonia bromide as the ion-pairing agent. The ascorbic acid was quantitated at 243 nm by an external standard technique. Bioavailability
of crystalline AA and PCAA to rainbow trout
Experimental design. The bioavailability was investigated both to vitamin Cdeficient trout and to trout with a normal vitamin C level. During one part of the experiment, trout were given an AA-deficient diet for 8 weeks (tanks A and B ) . Then the fish were redistributed into the two holding tanks and were supplemented for 4 weeks with crystalline AA (tank A) or PCAA (tank B). During the other part of the experiment, trout were supplemented for 8 weeks with a constant level of AA in the form of crystalline AA (tank C) or PCAA (tank D). The water volume in each tank was 1.5 x 1.5x 0.3 m3, the water temperature was 11.5”C + 0.5”C, and there was continuous lighting in the room. Initially the average weight of the trout was 22.8 2 3.1 g (s.d. ), and the number of fish was 325 in each of tanks A and B, and 250 in each of tanks C and D. The fish
338
T.SKELBAEKETAL.
TABLE 2 Concentration of AA (ppm) in the feed used for the bioavailability study
n
Mean + s.e.m.
Tank A AA
Tank B PCAA
Tank C AA
Tank D PCAA
15 14829
18 128&B
21 1142 10
15 156t 12
were fed twice a day. In order to avoid overstocking, the numbers of fish in the tanks were gradually reduced (range of biomass: lo-33 kg/m3). The feeding level was 0.8-0.9 times the maximum ration (From and Rasmussen, 1984 ) and was adjusted daily according to the biomass and expected growth. Feed. Vitamin C-deficient feed and feed containing approx. 150 ppm AA of crystalline AA and PCAA, respectively, were manufactured similarly to those of the stability test. The pellet diameter was 3 mm, and the fat content was raised to a total of 20% by fish oil. In order to minimize AA degradation during the experimental period, feed was kept at - 18°C before the fish were fed. Analyses of feed AA concentrations during the experimental period showed no degradation (P > 0.05 ) (Table 2 ) . Sampling and analysis. The concentration of free AA in the liver was used as an index of the bioavailability of AA in the feed. Th liver was removed from the fish and kept at - 79 oC until analysis, approx. l-2 days later. Analyses were carried out by Levnedsmiddelstyrelsen, Denmark (The National Food Agency of Denmark) by a method in principle similar to that of the feed assay (method SL AB 1131 slightly modified). Initially 20 livers were analysed individually, and then the number of livers tested was 10 from each tank. Fish from tanks A and B were analysed after 0,8,9,10,11, and 12 weeks, and those from tanks C and D after 0,3,5, and 7 weeks. Fish weights were registered in connection with the liver analyses. Fifty fish were weighed initially, and every 4th week 30 fish from each tank were weighed. RESULTS
Stability of crystalline AA and PCAA during pelleting and storage The amount of AA retained after the pelleting process was 70.9% ? 9.4% (x+s.e.m., n=3) for crystalline AA and 80.5%?2.6% (n?s.e.m., n=lO) for PCAA. After 6 weeks there was less than 10% left in the feed produced with crystalline AA whereas 72.6% ? 2.6% (X? s.e.m., n= 10) was left in the feed produced with PCAA. After 9 months there was still about 15% AA left of the
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
AA in feed
1
:339
(% of added)
3
5
7
9
Months
Fig. 1. AA concentration (% of added) in pelleted fish feed with crystalline AA, PCAA and other coated products, versus time (a? s.e.m.).
PCAA (Fig. 1). The other coated AA products tested appear to have a stability in pelleted fish feed equal to that of crystalline AA. Bioavailability of crystalline AA and PCAA in trout Liver AA. The liver AA concentration in fish from tanks A and B decreased during a period of 8 weeks from 1585 3.1 to 37 t 2.4 ppm (%+s.e.m., n=20) when fish received an AA-free feed. After 4 weeks on an AA-sufficient diet, it increased again to 162-173 ppm. The liver AA concentration in fish from tanks C and D, receiving normal level AA feed, was 128-158 ppm during the whole test period (Fig. 2). The AA concentration was a little higher in fish from tank A and tank D compared with fish from tank B and tank C, respectively. The differences were analogous to those of the AA concentrations in the feed given to the groups. Over the whole period there was no significant difference in either liver or feed data between tanks A and B (t-test, P> 0.05) whereas both liver and feed data from tank C were significantly lower than those from tank D (t-test, P< 0.01). Clinical observations. After 7-8 weeks on an AA-deficient diet, 11 fish developed dilated swim bladders. At that time, inflammation and haemorrhage in the gastrointestinal system were noticed. Four days after starting the AA supplementation, some fish still showed the above-mentioned symptoms, but after
T. SKELBAEK ET Al
340 AA in liver
(PPM)
/ o lank
C
x Tank
D
I
\ \
IOO-
\ \ \ \
---
AA-free
-
Normal AA level diet
diet
\ \
60s
\
2
4
6
\
8
10
Fig. 2. Liver AA concentration Fish
weight
12
Weeks
(ppm) versus time (Xk s.e.m.)
(g)
1
‘40j 120.
100.
80
i
60-
40.
20
t
ABCD 0
-
-e Wee
4
Fig. 3. Fish weight in tanks A, B, C, and D (f+s.e.m.
).
s
STABILITY
AND
BIOAVAILABILITY
OF ASCORBIC
ACID
FORMS
TO RAINBOW
TROUT
341
that time none of the symptoms were observed. During the rest of the experiment, however, several fish developed fibrous tissue between the organs in the body cavity. The fish given the AA-containing diet (tanks C and D) showed none of the symptoms mentioned. They were observed for a total of 9 weeks. Growth. After 8 weeks there was no difference in the mean weight of the fish between the four groups (P> 0.05)) and after 13 weeks there was no difference between tanks A and B (P> 0.05) (Fig. 3). DISCUSSION
The present study showed that the synthetic polymer-coated product PCAA is more stable than crystalline AA and other coated forms tested, viz. ethylcellulose-coated AA and glyceride-coated AA. The processing loss was only 29% of uncoated AA and 19% of PCAA, whereas degradation during storage was rapid for crystalline AA, unlike PCAA. The pelleting loss compares well with results of Love11 and Lim (1978) who found, however, much lower degradation rates after pelleting. The difference may partly be caused by the higher processing temperature in the present experiment, 80-84’ C, compared to 6672 “C! used by Love11and Lim (1978). Hilton et al. (1977a) found that different pelleting procedures have great influence on the stability of AA. This may also be the reason why these experiments failed to find better stability of ECAA and GCAA compared to crystalline AA, as found by Hilton et al. (1977a) for ECAA and by Soliman et al. (1987) for GCAA in cold pelleted diet. The clinical symptoms such as dilated swim bladder, inflammation, and haemorrhage in the gastrointestinal system, observed in the group of fish deprived of AA, are presumed to be vitamin C deficiency symptoms. The symptoms appeared after only 7 or 8 weeks and at liver concentrations of 37 ppm AA (range = 25-68 ppm), and the fish seemed to show an earlier stage of deficiency than the more advanced symptoms observed by Hilton et al. (1977b) who found that trout given an AA-free diet suffered from overt deficiency symptoms such as anorexia, lethargy, and prostration at a liver concentration of < 20 ppm AA after 16-20 weeks. Lim and Love11 (1978) observed AA deficiency symptoms in channel catfish after 8-12 weeks at liver concentrations of < 30 ppm. A high growth rate, as occurred in the present experiment, is known to result in a higher depletion rate of the AA stored in the fish. Absence of AA for 8 weeks did not impair the growth rate of young trout. Hilton et al. (1978) claim that AA requirement varies with age and growth rate, being highest in young fish. The present observations agree well with those of Lim and Love11 (1978) who observed that the growth rate in channel catfish was not disturbed by an AA-free diet for 8 weeks. The concentration of free AA in the liver was used as an index of the AA status in the fish. The anterior kidney was preferred by Halver et al. (1975),
342
T. SKELBAEK ET AL.
though Lim and Love11 (1978) established that the anterior kidney of channel catfish did not reflect differences in the AA concentration in the diet. Furthermore, the anterior kidney is very small and difficult to isolate. Lim and Love11 (1978) and Murai et al. (1978) found good correlation between the diet and liver AA concentration, and Hilton et al. (1977b) found this correlation better than that between diet and anterior kidney. The results of the present experiment show that even a minor difference in the AA concentration of the diet is reflected in the liver AA. Comparison of the liver AA concentrations in the fish given crystalline AA (tanks A and C ) and in the fish given PCAA (tanks B and D ) shows that AA in the form of PCAA is available to the trout to the same extent as crystalline AA. The liver AA did not reach a steady level within 4 weeks of AA supplementation, in agreement with Sandnes (1984) who found the period to be 4-6 weeks for trout fry. In summary, the present results indicate that PCAA has superior stability, and at the same time full bioavailability. ACKNOWLEDGEMENT
This work was partly supported by Industri- og Handelsstyrelsen (The National Agency of Industry and Trade), Denmark.
REFERENCES From, J. and Rasmussen,
G., 1984. A growth model, gastric evacuation,
and body composition
rainbow trout, Salmo gairdneri Richardson, 1836. Dana, 3: 61-139. Halver, J.E., Smith, R.R., Tolbert, B.M. and Baker, E.M., 1975. Utilization
of ascorbic
in
acid in
fish. Ann. N.Y. Acad. Sci., 258: 81-102. Hilton, .J.W., Cho, C.Y. and Slinger, S.J., 1977a. Factors affecting the stability of supplemental ascorbic acid in practical trout diets. J. Fish. Res. Board Can., 34: 683-687. Hilton, d.W.. Cho, C.Y. and Slinger, S.J., 197713. Evaluation of ascorbic acid status of rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 34: 2207-2210. Hilton, J.W., Cho, C.Y. and Sl inger, S.J., 1978. Effect of graded levels of supplemental ascorbic acid in practical diets fed to rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 35: 431-436. Lim, C. and Lovell, R.T., 1978. Pathology of vitamin (Zctaluruspunctatus). J. Nutr., 108: 1137-1146.
C deficiency
syndrome
in channel catfish
Lovell. R.T. and Lim, C., 1978. Vitamin C in pond diets for channel catfish. Trans. Am. Fish. Sot., 107: 321-325. Moeller, J., Busk, J. and Bentsen, H., 1984. Pelletering af Fedt- og Proteinrige Foderblandinger (Pelleting of Fat and Protein-rich Feed Rations). Beretning no. 117, Biotekniskinstitut, Kolding, ISSN 0106-0082,60 pp. Murai, T., Andrews, J.W. and Bauernfeind,
J.C., 1978. Use of L-ascorbic
acid, ethocel-coated
ascorbic acid and ascorbate 2-sulfate in diets for channel catfish, Zctaluruspunctatus. 108: 1761-1766.
J. Nutr.,
Sandnes, K., 1984. Some aspects of ascorbic acid and reproduction in fish. In: I. Wegger, F.J. Tagwerker and J. Moustgaard (Editors), Ascorbic Acid in Domestic Animals, Proc. Workshop, 1st Meeting Date 1983. Royal Danish Agricultural Society, Copenhagen, p. 206-12.
STABILITY AND BIOAVAILABILITY OF ASCORBIC ACID FORMS TO RAINBOW TROUT
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Sandnes, K. and Utne, F., 1982. Processing loss and storage stability of ascorbic acid in dry fish feed. Fiskeridir. Skr., Ser. Ernaering, 2: 39-44. Sandnes, K., Hansen, T. and Waagboe, R., 1987. Ascorbate-2-sulfate as vitamin C source for Atlantic salmon (Salmo salar). Int. Symp. Feeding and Nutrition in Fish, Bergen, 23-27 August 1987 (abstract, unpublished). Solimann, A.K., Jauncey, K. and Roberts, R.J., 1987. Stability of L-ascorbic acid (vitamin C) and its forms in fish feeds during processing, storage and leaching. Aquaculture, 60: 73-83.