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Digestive processes in silver carp (Hypophthalmichthys molitrix) studied in vitro
123
Aquaculture, 50 (1985) 123-131 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
DIGESTIVE PROCESSES IN SILVER CARP (HYPOPHTHALMICHTHYS STUDIED IN VITRO
MOLITRIX)
G. BITTERLICH Institut fiir Zoologie, Abteilung Zoophysiologie, la, A-6020 Innsbruck (Austria) (Accepted
Universitiit Innsbruck,
Peter-Mayr Str.
20 August 1985)
ABSTRACT Bitterlich, G., 1985. Digestive processes in silver carp (Hypophthalmichthys studied in vitro. Aquaculture, 50: 123-131.
molitrix)
In-vitro simulations of the digestive process in silver carp were used to study the digestibility of a filamentous blue-green alga (Anabaena flos-aquae) and of heterotrophic aerobic bacteria, and to explore their interrelations in the alimentary tract. After 9 h of incubation with the gut juice, only 9.5% of the algal protein was delivered as free amino acids. Bacteria were apparently not utilized. The loss of free amino acids and an increased ammonia production in samples containing algae or algae-bacteria mixtures indicated enhanced growth of the bacterial population during gut passage at the expense of the fish.
INTRODUCTION
The utilization of food by silver carp has been investigated by several methods (Bitterlich, 1985a, 198513; Bitterlich and Gnaiger, 1984). Results indicated that algae and bacteria can hardly be digested by silver carp. Indirect evidence suggested that zooplankton and amorphous organic detritus are of primary importance in meeting the energy requirements of stomachless filter-feeding fish. In view of the low protein content of detritus, the problem of nitrogen resources is especially critical. Therefore, an in-vitro method developed by Grabner (1985) was applied to study the availability of protein from Anabaena flos-aquae, which was presumed to be a digestible algal species (unpubl. observations on gut contents), and heterotrophic bacteria. MATERIAL
Principle
AND METHODS
of in-vitro simulated
process
(Grabner, 1985)
Food and gut fluid supernatant are mixed in equal proportions and concentrations to that of the species being investigated. The sample is
0044-8486/85/$03.30
o 1985 Elsevier Science Publishers B.V.
124
~ont~uously stirred at a constant temperature for a time equal to the duration of the gut passage. The hydrolysis products of protein may cause a pH shift. This change in pH can be monitored and compensated by automatic titration with specific buffers. In order to prevent the outflow of the titration system from blocking, COz-free air was continuously bubbled through the titration tube. At intervals, 100-~1 subsamples are taken out of the test tube and pipetted into an equal volume of the following “HPLCbuffer”: 0.2 N NazIIP04, 0.6 I? NaCl, 0.5% EDTA, 40% ethanol, pH 5.5. At this pH no significant tryptic activity can be detected. Subsequently the sample is stored in the deep freeze pending further investigations. Test set-up based on preliminu~
studies on silver carp
Silver carp (~~PophthaZmi~hth~s molitrix, age 2+) were obtained from an intensive fish culture in Szarvas (Hungary) and immediately dissected for the collection of gut contents. Previous investigations of the digestive enzymes of silver carp (Bitterlich, 1985b)revealed high tryptic activities (250-400 U/ml) in the fore-gut, which decreased quickly from mid-gut to hind-gut (measured at optimum pH of trypsin after Rick, 19’74). For the in-vitro incubation a mean tryptic activity of 160 U/ml was used. The anterior parts of the gut contents of several silver carp were harvested, pooled and stored in a deep freeze for the in-vitro experiment. Tryptic activity in the gut fluid supematant concentrate remained constant throughout the invitro series. Sub~quently gut contents were centrifuged at 12 000 g and the supernatant diluted with distilled water to reach a final concentration of 160 U/ml in the mixture of enzyme test material. Earlier studies of pH in thawed gut fluid supernatants showed that silver carp maintains a relatively low pH of 6.2 in the fore-gut, which increases continuously by one unit from the first to the eighth tenth of the gut, after which point, a slight decrease was observed (Bitterlich, 1985). According to this result the pH was simulated in the experiments, manually by addition of 1 N HCl or automatically by triethanolamine (50 or 250 mmol). Algae and gut fluid supernatants were mixed in a proportion of 2 : 3 which corresponds to the mean ratio of sediment and supernatant in the gut contents of silver carp after centr~ugation at 12 000 g. Bacteria could not be weighed and specified, either in the intestine of silver carp or for the experiment; therefore volumes of cultured microorganisms were pipetted into the sample vial. Approximate gut passage time of silver carp was 11 h at the ambient water temperature of 25°C (Bitterlich, unpubl.). Hence invitro samples were incubated for between 9 and 13 h at 25°C. The fresh weight of food in each subsample was corrected for dilutions during the titrations.
125
Algal culture Anabaena flos-aquae was cultured in Bold’s Basal Medium at 25°C and using cool white light. Algae were stirred and aerated continuously by filtered air. In order to keep the culture as sterile as possible, culture tubes and medium were sterilized. Prior to the experiment, algae were centrifuged at 2000 g for 10 min and the fresh weight determined on a Sartorius balance. Bacteria culture Heterotrophic aerobic bacteria, isolated from a dying Anabaena culture, were cultured in Columbia medium at room temperature. The culture was stirred continuously by aeration. For the experiment bacteria were washed in physiological sodium chloride solution and centrifuged at 2000 g. Aliquots of the culture were used for the in-vitro test. Amino acid analysis Samples were centrifuged
at 11300
g for 5 min.
(a) Free amino acids In the supernatant the release of free amino acids during the digestive process was evaluated. 60 ~1 of the supernatant were mixed with 40 ~1 of lithium citrate buffer (0.2 M). Norleucin was used as an internal standard. Samples were stored in the deep freeze. Prior to the analysis of amino acids, samples were centrifuged again at 11300 g for some minutes. Triethanolamine buffer and “HPLC-buffer” were analysed as a blank and contained neither amino acids nor ammonia. (b) Total amino acids Undissolved amino acids were analysed in the sediment fraction of the food. The test residues were washed twice in “HPLC-buffer” (half concentrated) and lyophylised. For the acid hydrolysis this sample was suspended in HCl (6 N), sealed under nitrogen gas and kept in an oven at 105°C for 24 h. The vial was opened, the HCl evaporated under heat and nitrogen gas, and resuspended in lithium citrate buffer. Prior to the injection of the samples into the amino acid analyser they were centrifuged at 11300 g for some minutes. (c) Amino acid analysis The amino acid analysis was carried out on a Labotron Liquimat 3 amino acid analyser (Geretsried, Germany) after a method of Moore and Stein (1951) and Spackman et al. (1958) by using a Durrum DC 6A cation-exchange resin and the Pica-Buffer-System IV Lithium (Pierce).
126 RESULTS
In-vitro tests with algae and bacteria yielded different results. In the course of the incubation of Anabaena flosuquae with the gut juice of silver carp, only a slight increase of the total quantity of detectable free amino acids was observed (Fig. 1). The calculation of b,,, (maximum slope) yielded a mean value of 0.1 /Imol*gfw-‘-he’. In contrast, a loss of free amino acids was recorded during the in-vitro experiments with bacteria. b max was -0.001. Results obtained from the incubation of gut juice with algae-bacteria mixtures were not significantly different from those acquired with algal incubations alone. However, variability between the individual algae and algae-bacteria samples was high, which may have been due to heterogenous stages of growth or different levels of impurity of the Anabaena cultures. At given time intervals the release of free amino acids was analysed. A loss of free amino acids such as threonine, serine, aspartic acid and glutamic acid during in-vitro digestion was observed. At the same time ammonia production was enhanced (Fig. 2). A correlation between the two latter results will be discussed below. The increasing ammonia concentration might account for the unexpected basifying trend observed in most of the samples after hydrolysis of digestible protein.
IN-VITRO INCUBATION TIME [h 1 Fig. 1. Release of free amino acids during in-vitro incubation. Substrates (pH range in brackets): A=algae (6.7-7.1), B=algae (6.3-7), C=algae (6.8-B), D=algae (B-8.1), E=algae (B), F = algae (6.3-7), G = algae + bacteria (6.4-7.7), H = algae + bacteria (6.3-B), I = bacteria (6.5-7.7), K= bacteria (B), L = bacteria (B-8.2), M = bacteria (6.2-7.3).
127
n ALGAE q ALG.+ BAC. I
AMI NO
Cl BACTERIA
ACIDS
Fig. 2. Amount of free amino acids released from food after 9 h of in-vitro incubation. For abbreviations of amino acids see legend to Fig. 3.
Light microscopic investigation of the condition of the algae at the beginning and at the end of the in-vitro test indicated that Anabaena flosaquae was digested (Figs. 4A and B). In contrast, a comparison of the pattern of amino acids in the food with the release of free amino acids after 9 h of incubation suggested low exploitation of Anubaena and bacteria (Figs. 2 and 3). Only 9.5% of the protein in algae and none of the bacterial protein was liberated as free amino acids. However, protein degradation to peptides was not considered in this assay. DISCUSSION
The results presented indicate that Anubaena flos-uquue was hardly digested by silver carp gut fluid during the in-vitro tests (Fig. 2, Table 1). However, the variability between different samples was high (Fig. 1). Several factors might have contributed to this effect. The low velocity during the centrifugation of food prior to the experiments rendered the separation of algae and culture medium difficult. Therefore, a considerable weighing error may have been involved. Moreover, the algal culture might have been in different growth phases.
128
800
t
W ALGAE •l ALG.+
BAC.
q BACTERIA
'3 G600 z E 1 Fi LOO i= 2 IZ
ki z 0 0
200
O--
!?
a
L
=
AMINO
ACIDS
Fig. 3. Amino acid pattern in the food. Detectible amino acids: Asp = aspartic acid, Thr = threonine, Ser serine, Glu = glutamic acid, Pro = proline, Gly = glycine, Ala = alanine, Val = valine, Met = methionine, Ile = isoleucine, Leu = leucine, Tyr = tyrosine, Phe = phenylalanine, Orn = ornithine, Lys = lysine, His = histidine, Arg = arginine. NH, = Ammonia. During acid hydrolysis asparagine and glutamine are transformed into aspartic and glutamic
Fig. test.
acids respectively.
4. The condition of algae and the beginning 200 x (slide 24 X 36 mm). Photos: P. Flory.
(A) and at the end (B)
of the in-vitro
129
Peptides, as well as the undigested proteins, were not investigated. Studies on the in-vitro digestib~ity of a bacterial protein (Pruteen) and of leguminose protein by Grabner (1985) showed that essential amino acids were predominantly liberated, whereas non-essential amino acids remained largely in peptide bonds. In the present in-vitro tests the pattern of hydrolysed free amino acids also shifted towards a predominance of the essential amino acids compared to the amino acid pattern in the food (compare Figs. 2 and 3). Grabner and Hofer (1985) suggested that the peptides degraded and absorbed at the wall of the intestine may constitute a considerable nutrient source. After the in-vitro digestion of soya bean protein by the gut fluid supernatants of common carp, 93.2% of the protein was solubilized as free amino acids (30.6%) and soluble peptides (62.6%) (Grabner and Hofer, 1985). In the present investigation, 9.5% of the protein in algae and none of the bacterial protein was hydrolysed to free amino acids. TABLE 1 Utilization of food Nutrient
Amount of amino acids in the nutrient (,umol/gfw)
Release of free amino acids after 9 h of in-vitro incubation (as % of amino acids in nutrient)
Algae Algae-Bacteria Bacteria
380.49 475.80 286.96
9.52 7.05 -0.45
Originally the amino acids in the food were determined in freeze-dried material (Fig. 1). To obtain the amounts of amino acids per g fresh weight (gfw), the results were divided by 10 (approximate dry-weight to fresh-weight ratio).
The energy demand as well as the specific amino acid requirements of silver carp have not been defined. Thus the question whether silver carp could survive on an Anabaena diet cannot yet be answered. Nevertheless, previous investigations into the utilization of algae by stomachless filterfeeding fish fully support the present results (Bitterlich, 1985a). In contrast, Herodek et al. (in prep.) found good growth of young silver carp on Anu~ae~u flos~q~ue. However, noticeable changes of feeding habits and digestion mechanisms take place throughout maturation of silver carp (Antalfi and T6lg, 1971). On the other hand, the feeding experiments by Pekar and Olah (pers. commun., 1984) corroborate the negative result obtained in the invitro tests. An increasing production of ammonia simultaneously with the loss of free amino acids was observed during the incubation of algae-bacteria mixtures and to a minor extent in alga incubations {Fig. 2). The disappearance of free amino acids from the solution is particularly obvious with threonine, serine, aspartic acid and glutamic acid (Fig. 2). Although they
130
constituted a relatively high proportion of the protein in the food (Fig. 3), only a few percents were detected in the enzyme hydrolysate, which were removed totally during gut passage (Fig. 2). Due to the higher concentration of essential ammo acids, their possible degradation by bacteria is difficult to establish. Bacteria were apparently not digested by silver carp gut juice. Studies should be extended to include bacteria naturally associated with the food. The increasing ammonia production and the loss of free amino acids during gut passage indicate enhanced growth of the bacterial population at the expense of the fish. It is uncertain whether this a~umption holds true for invivo digestion, where hydrolysed protein breakdown products are absorbed quickly. It can be assumed that the production of ammonia was the reason for the unexpected increase of pH in the sample. In this situation, stoichiometric evaluation of pH is complex and could not be accomplished in this study. Many microscopic investigations of the digestibility of algae (Bitterlich, 1985a) are based on a comparison of their appearance before and after gut passage (Fig. 4). However, quantitative conclusions have to be interpreted with care, as indicated by the contradictory biochemical results presented in this study. ACKNOWLEDGEMENTS
Supported by the Fonds zur Fiirderung der wissens~haftlichen Fo~chung in osterreich, project no. 4716, and by a &terreich-Ungarn Austauschstipendium in Szarvas, Hungary. I thank R. Hofer for initiating the project and M. Grabner for placing the technical equipment at my disposal and for valuable advice. I am grateful to J. Olah, A. Varadi, A. Peteri, Janci (HAKI; Hungary), E. Schaber and C. Heufler (Inst. fur Hygiene, Innsbruck) to J. Dalla Via and R. Lackner (Inst. fiir Zoophysiologie, Innsbruck) and Cyclobios for their help. I thank E. Gnaiger, D. Robins and W. Wieser for reading the manuscript.
REFERENCES Antalfi, A. and Tolg, I., 1971. Graskarpfen (translated into German from Hungarian by I. Bogsch). DonauVerlag, Gunsburg. Bitterlich, G., 1985a. The nutrition of stomachless phytoplanktivorous fish in comparison with Tilapia. Hydrobiologia, 121(Z): 173-179. Bitterlich, G., 198Sb. Digestive enzyme pattern of two stomachless filterfeeders, silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). J. Fish Biol., 26 (in press). Bitterlich, G. and Gnaiger, E., 1984. Phytoplanktivorous or omnivorous fish? Digestibility of zooplankton by silvercarp, Hypophtha~michthys molitrix (Val.). Aquaculture, 40: 26X-263.
131 Grabner, M., 1985. An in vitro method for measuring protein digestibility of fish feed components. Aquaculture, 48: 97-110. Grabner, M. and Hofer, R., 1985. The digestibility of the proteins of broad bean (Vicia tuba) and soya bean (Glycine mu) under in vitro conditions simulating the alimentary tracts of rainbow trout (Salmo gairdneri) and carp (Cyprinus carpio). Aquaculture, 48: 111-122. Herodek, S., Tbtrai, I., V6ros, L. and 01&h, J. Effect of silver carp (Hypophtholmichthys molitrix Val.) on the eutrophication and fish production (in prep.) Moore, S. and Stein, W.H., 1951. Chromatography of amino acids on sulfonated polystyrene resins. Analyt. Chem., 192: 663-681. Rick, W., 1974. Trypsin. In: H.U. Bergmeyer (Editor), Methoden der Enzymatischen Analyse. Verlag Chemie, Weinheim, pp. 1060-1063. Spackman, D., Stein, W.H. and Moore, S., 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem., 30: 1190-1206.
Aquaculture, 50 (1985) 123-131 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
DIGESTIVE PROCESSES IN SILVER CARP (HYPOPHTHALMICHTHYS STUDIED IN VITRO
MOLITRIX)
G. BITTERLICH Institut fiir Zoologie, Abteilung Zoophysiologie, la, A-6020 Innsbruck (Austria) (Accepted
Universitiit Innsbruck,
Peter-Mayr Str.
20 August 1985)
ABSTRACT Bitterlich, G., 1985. Digestive processes in silver carp (Hypophthalmichthys studied in vitro. Aquaculture, 50: 123-131.
molitrix)
In-vitro simulations of the digestive process in silver carp were used to study the digestibility of a filamentous blue-green alga (Anabaena flos-aquae) and of heterotrophic aerobic bacteria, and to explore their interrelations in the alimentary tract. After 9 h of incubation with the gut juice, only 9.5% of the algal protein was delivered as free amino acids. Bacteria were apparently not utilized. The loss of free amino acids and an increased ammonia production in samples containing algae or algae-bacteria mixtures indicated enhanced growth of the bacterial population during gut passage at the expense of the fish.
INTRODUCTION
The utilization of food by silver carp has been investigated by several methods (Bitterlich, 1985a, 198513; Bitterlich and Gnaiger, 1984). Results indicated that algae and bacteria can hardly be digested by silver carp. Indirect evidence suggested that zooplankton and amorphous organic detritus are of primary importance in meeting the energy requirements of stomachless filter-feeding fish. In view of the low protein content of detritus, the problem of nitrogen resources is especially critical. Therefore, an in-vitro method developed by Grabner (1985) was applied to study the availability of protein from Anabaena flos-aquae, which was presumed to be a digestible algal species (unpubl. observations on gut contents), and heterotrophic bacteria. MATERIAL
Principle
AND METHODS
of in-vitro simulated
process
(Grabner, 1985)
Food and gut fluid supernatant are mixed in equal proportions and concentrations to that of the species being investigated. The sample is
0044-8486/85/$03.30
o 1985 Elsevier Science Publishers B.V.
124
~ont~uously stirred at a constant temperature for a time equal to the duration of the gut passage. The hydrolysis products of protein may cause a pH shift. This change in pH can be monitored and compensated by automatic titration with specific buffers. In order to prevent the outflow of the titration system from blocking, COz-free air was continuously bubbled through the titration tube. At intervals, 100-~1 subsamples are taken out of the test tube and pipetted into an equal volume of the following “HPLCbuffer”: 0.2 N NazIIP04, 0.6 I? NaCl, 0.5% EDTA, 40% ethanol, pH 5.5. At this pH no significant tryptic activity can be detected. Subsequently the sample is stored in the deep freeze pending further investigations. Test set-up based on preliminu~
studies on silver carp
Silver carp (~~PophthaZmi~hth~s molitrix, age 2+) were obtained from an intensive fish culture in Szarvas (Hungary) and immediately dissected for the collection of gut contents. Previous investigations of the digestive enzymes of silver carp (Bitterlich, 1985b)revealed high tryptic activities (250-400 U/ml) in the fore-gut, which decreased quickly from mid-gut to hind-gut (measured at optimum pH of trypsin after Rick, 19’74). For the in-vitro incubation a mean tryptic activity of 160 U/ml was used. The anterior parts of the gut contents of several silver carp were harvested, pooled and stored in a deep freeze for the in-vitro experiment. Tryptic activity in the gut fluid supematant concentrate remained constant throughout the invitro series. Sub~quently gut contents were centrifuged at 12 000 g and the supernatant diluted with distilled water to reach a final concentration of 160 U/ml in the mixture of enzyme test material. Earlier studies of pH in thawed gut fluid supernatants showed that silver carp maintains a relatively low pH of 6.2 in the fore-gut, which increases continuously by one unit from the first to the eighth tenth of the gut, after which point, a slight decrease was observed (Bitterlich, 1985). According to this result the pH was simulated in the experiments, manually by addition of 1 N HCl or automatically by triethanolamine (50 or 250 mmol). Algae and gut fluid supernatants were mixed in a proportion of 2 : 3 which corresponds to the mean ratio of sediment and supernatant in the gut contents of silver carp after centr~ugation at 12 000 g. Bacteria could not be weighed and specified, either in the intestine of silver carp or for the experiment; therefore volumes of cultured microorganisms were pipetted into the sample vial. Approximate gut passage time of silver carp was 11 h at the ambient water temperature of 25°C (Bitterlich, unpubl.). Hence invitro samples were incubated for between 9 and 13 h at 25°C. The fresh weight of food in each subsample was corrected for dilutions during the titrations.
125
Algal culture Anabaena flos-aquae was cultured in Bold’s Basal Medium at 25°C and using cool white light. Algae were stirred and aerated continuously by filtered air. In order to keep the culture as sterile as possible, culture tubes and medium were sterilized. Prior to the experiment, algae were centrifuged at 2000 g for 10 min and the fresh weight determined on a Sartorius balance. Bacteria culture Heterotrophic aerobic bacteria, isolated from a dying Anabaena culture, were cultured in Columbia medium at room temperature. The culture was stirred continuously by aeration. For the experiment bacteria were washed in physiological sodium chloride solution and centrifuged at 2000 g. Aliquots of the culture were used for the in-vitro test. Amino acid analysis Samples were centrifuged
at 11300
g for 5 min.
(a) Free amino acids In the supernatant the release of free amino acids during the digestive process was evaluated. 60 ~1 of the supernatant were mixed with 40 ~1 of lithium citrate buffer (0.2 M). Norleucin was used as an internal standard. Samples were stored in the deep freeze. Prior to the analysis of amino acids, samples were centrifuged again at 11300 g for some minutes. Triethanolamine buffer and “HPLC-buffer” were analysed as a blank and contained neither amino acids nor ammonia. (b) Total amino acids Undissolved amino acids were analysed in the sediment fraction of the food. The test residues were washed twice in “HPLC-buffer” (half concentrated) and lyophylised. For the acid hydrolysis this sample was suspended in HCl (6 N), sealed under nitrogen gas and kept in an oven at 105°C for 24 h. The vial was opened, the HCl evaporated under heat and nitrogen gas, and resuspended in lithium citrate buffer. Prior to the injection of the samples into the amino acid analyser they were centrifuged at 11300 g for some minutes. (c) Amino acid analysis The amino acid analysis was carried out on a Labotron Liquimat 3 amino acid analyser (Geretsried, Germany) after a method of Moore and Stein (1951) and Spackman et al. (1958) by using a Durrum DC 6A cation-exchange resin and the Pica-Buffer-System IV Lithium (Pierce).
126 RESULTS
In-vitro tests with algae and bacteria yielded different results. In the course of the incubation of Anabaena flosuquae with the gut juice of silver carp, only a slight increase of the total quantity of detectable free amino acids was observed (Fig. 1). The calculation of b,,, (maximum slope) yielded a mean value of 0.1 /Imol*gfw-‘-he’. In contrast, a loss of free amino acids was recorded during the in-vitro experiments with bacteria. b max was -0.001. Results obtained from the incubation of gut juice with algae-bacteria mixtures were not significantly different from those acquired with algal incubations alone. However, variability between the individual algae and algae-bacteria samples was high, which may have been due to heterogenous stages of growth or different levels of impurity of the Anabaena cultures. At given time intervals the release of free amino acids was analysed. A loss of free amino acids such as threonine, serine, aspartic acid and glutamic acid during in-vitro digestion was observed. At the same time ammonia production was enhanced (Fig. 2). A correlation between the two latter results will be discussed below. The increasing ammonia concentration might account for the unexpected basifying trend observed in most of the samples after hydrolysis of digestible protein.
IN-VITRO INCUBATION TIME [h 1 Fig. 1. Release of free amino acids during in-vitro incubation. Substrates (pH range in brackets): A=algae (6.7-7.1), B=algae (6.3-7), C=algae (6.8-B), D=algae (B-8.1), E=algae (B), F = algae (6.3-7), G = algae + bacteria (6.4-7.7), H = algae + bacteria (6.3-B), I = bacteria (6.5-7.7), K= bacteria (B), L = bacteria (B-8.2), M = bacteria (6.2-7.3).
127
n ALGAE q ALG.+ BAC. I
AMI NO
Cl BACTERIA
ACIDS
Fig. 2. Amount of free amino acids released from food after 9 h of in-vitro incubation. For abbreviations of amino acids see legend to Fig. 3.
Light microscopic investigation of the condition of the algae at the beginning and at the end of the in-vitro test indicated that Anabaena flosaquae was digested (Figs. 4A and B). In contrast, a comparison of the pattern of amino acids in the food with the release of free amino acids after 9 h of incubation suggested low exploitation of Anubaena and bacteria (Figs. 2 and 3). Only 9.5% of the protein in algae and none of the bacterial protein was liberated as free amino acids. However, protein degradation to peptides was not considered in this assay. DISCUSSION
The results presented indicate that Anubaena flos-uquue was hardly digested by silver carp gut fluid during the in-vitro tests (Fig. 2, Table 1). However, the variability between different samples was high (Fig. 1). Several factors might have contributed to this effect. The low velocity during the centrifugation of food prior to the experiments rendered the separation of algae and culture medium difficult. Therefore, a considerable weighing error may have been involved. Moreover, the algal culture might have been in different growth phases.
128
800
t
W ALGAE •l ALG.+
BAC.
q BACTERIA
'3 G600 z E 1 Fi LOO i= 2 IZ
ki z 0 0
200
O--
!?
a
L
=
AMINO
ACIDS
Fig. 3. Amino acid pattern in the food. Detectible amino acids: Asp = aspartic acid, Thr = threonine, Ser serine, Glu = glutamic acid, Pro = proline, Gly = glycine, Ala = alanine, Val = valine, Met = methionine, Ile = isoleucine, Leu = leucine, Tyr = tyrosine, Phe = phenylalanine, Orn = ornithine, Lys = lysine, His = histidine, Arg = arginine. NH, = Ammonia. During acid hydrolysis asparagine and glutamine are transformed into aspartic and glutamic
Fig. test.
acids respectively.
4. The condition of algae and the beginning 200 x (slide 24 X 36 mm). Photos: P. Flory.
(A) and at the end (B)
of the in-vitro
129
Peptides, as well as the undigested proteins, were not investigated. Studies on the in-vitro digestib~ity of a bacterial protein (Pruteen) and of leguminose protein by Grabner (1985) showed that essential amino acids were predominantly liberated, whereas non-essential amino acids remained largely in peptide bonds. In the present in-vitro tests the pattern of hydrolysed free amino acids also shifted towards a predominance of the essential amino acids compared to the amino acid pattern in the food (compare Figs. 2 and 3). Grabner and Hofer (1985) suggested that the peptides degraded and absorbed at the wall of the intestine may constitute a considerable nutrient source. After the in-vitro digestion of soya bean protein by the gut fluid supernatants of common carp, 93.2% of the protein was solubilized as free amino acids (30.6%) and soluble peptides (62.6%) (Grabner and Hofer, 1985). In the present investigation, 9.5% of the protein in algae and none of the bacterial protein was hydrolysed to free amino acids. TABLE 1 Utilization of food Nutrient
Amount of amino acids in the nutrient (,umol/gfw)
Release of free amino acids after 9 h of in-vitro incubation (as % of amino acids in nutrient)
Algae Algae-Bacteria Bacteria
380.49 475.80 286.96
9.52 7.05 -0.45
Originally the amino acids in the food were determined in freeze-dried material (Fig. 1). To obtain the amounts of amino acids per g fresh weight (gfw), the results were divided by 10 (approximate dry-weight to fresh-weight ratio).
The energy demand as well as the specific amino acid requirements of silver carp have not been defined. Thus the question whether silver carp could survive on an Anabaena diet cannot yet be answered. Nevertheless, previous investigations into the utilization of algae by stomachless filterfeeding fish fully support the present results (Bitterlich, 1985a). In contrast, Herodek et al. (in prep.) found good growth of young silver carp on Anu~ae~u flos~q~ue. However, noticeable changes of feeding habits and digestion mechanisms take place throughout maturation of silver carp (Antalfi and T6lg, 1971). On the other hand, the feeding experiments by Pekar and Olah (pers. commun., 1984) corroborate the negative result obtained in the invitro tests. An increasing production of ammonia simultaneously with the loss of free amino acids was observed during the incubation of algae-bacteria mixtures and to a minor extent in alga incubations {Fig. 2). The disappearance of free amino acids from the solution is particularly obvious with threonine, serine, aspartic acid and glutamic acid (Fig. 2). Although they
130
constituted a relatively high proportion of the protein in the food (Fig. 3), only a few percents were detected in the enzyme hydrolysate, which were removed totally during gut passage (Fig. 2). Due to the higher concentration of essential ammo acids, their possible degradation by bacteria is difficult to establish. Bacteria were apparently not digested by silver carp gut juice. Studies should be extended to include bacteria naturally associated with the food. The increasing ammonia production and the loss of free amino acids during gut passage indicate enhanced growth of the bacterial population at the expense of the fish. It is uncertain whether this a~umption holds true for invivo digestion, where hydrolysed protein breakdown products are absorbed quickly. It can be assumed that the production of ammonia was the reason for the unexpected increase of pH in the sample. In this situation, stoichiometric evaluation of pH is complex and could not be accomplished in this study. Many microscopic investigations of the digestibility of algae (Bitterlich, 1985a) are based on a comparison of their appearance before and after gut passage (Fig. 4). However, quantitative conclusions have to be interpreted with care, as indicated by the contradictory biochemical results presented in this study. ACKNOWLEDGEMENTS
Supported by the Fonds zur Fiirderung der wissens~haftlichen Fo~chung in osterreich, project no. 4716, and by a &terreich-Ungarn Austauschstipendium in Szarvas, Hungary. I thank R. Hofer for initiating the project and M. Grabner for placing the technical equipment at my disposal and for valuable advice. I am grateful to J. Olah, A. Varadi, A. Peteri, Janci (HAKI; Hungary), E. Schaber and C. Heufler (Inst. fur Hygiene, Innsbruck) to J. Dalla Via and R. Lackner (Inst. fiir Zoophysiologie, Innsbruck) and Cyclobios for their help. I thank E. Gnaiger, D. Robins and W. Wieser for reading the manuscript.
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