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Nitrogen utilization by rainbow trout (Salmo gairdneri) fed on the yeast Hansenula anomala
NITROGEN UTILIZ,ITION BY RAINBOW TROUT (SALMO GAZRDNERZ) FED ON THE YEAST HANSENULA ANOMALA M. DE LA HIGUERA. F. J. SANCHEZ-MUNIZ. F. J. MATAIX and G. VARELA Departamento
de Flsiologia
Animal.
Facultad (Received
de Farmacia. 18 Nooember
Universidad
Complutense.
Madrid-3.
Spain
1980)
Abstract--l. Nutritive and metabolic consequences of feeding rainbow trout with a single cell protein diet were studied. Experimental diet was adjusted by using yeast Hansenula anomala as the only protein source, compared to white fish meal for the controls. 2. Food intake, weight increments and consequently protein efficiency ratio (PER) significantly decreased in trout fed on yeast diet. Furthermore, body nitrogen increase was lower and protein productive value (PPV) higher for the same diet, although in a nonsignificant way. 3. Plasma ammonia increased significantly in the experimental animals. Plasma urea levels, although increased in about 90%, were not significantly altered. Plasma uric acid levels, unaltered in plasma and liver, suffered a three-fold increase in kidney.
INTRODUCTION
In relation to new protein sources, yeast is of great interest because of its high vitamin B-complex content and also because yeast appears to be a promising future supply of protein. Furthermore, its high thermostability and economical aspects related to world protein supplies (Abbot, 1974), present yeast as a source with a great nutritional future (further details can be obtained from Davis 1974). On the other hand, the use of single cells as the only protein source produces some physiological alterations in the blood of rainbow trout as has been already published in a previous paper (SBnchezMuniz. et al., 1979). The use of single cell proteins in human and animal nutrition requires tests to evaluate its safety and nutritional qualities (Groot, 1974). This paper deals with nutritive utilization of yeast Hansenula anomala protein and also with the alterations on some nitrogen metabolic end products related to protein and nucleic acids metabolism. Furthermore, as yeast contains a great proportion of nucleic acids whose catabolism could lead to kidney damage (Groot, 1974), results are also discussed about the alterations found in the blood of rainbow trout fed with H. anomala (SBnchezMuniz rt al.. 1979). MATERIAL AND METHODS Animals
and maintenance
Rainbow trout obtained from a local fish farm, of an average weight of 100 g; were kept at IS + 1°C in tanks (80 x 35 x 40cm) divided into compartments in order to maintain uniform conditions. After 1 I days of acclimation to the new conditions and food, rainbow trout were individually weighed and placed into individual compartments where they were kept for three week experiments. One group was to evaluate nutritive utilization of protein and the other to measure nitrogen metabolism end products in plasma, liver and kidney. 583
Each fish was fed once daily, one pellet was offered at a time and when all the food offered had been consumed, more was added until satiation. Diets
The composition of the experimental diets to give a protein content of 50% (N x 6.25) is shown in Table 1. Yeast (Hansenula anomala strain 926 grown on synthetic ethanol) was prepared at the Instituto de Fermentaciones Industriales (C.S.I.C. Madrid). The dry components of each diet were blended with sodium alginate (5Og per kg) as a quelant and then thoroughly stirred with distilled water to get a homogeneous humidified mixture. Pellets were made by passing the diet mixture through an electric meat grinder equipped with dies of 2.5 mm hole size. After drying the diets were kept in a deep freeze at -20°C. Assays on protein
utilization
As concluded in a previous paper (de la Higuera et al., 1977), protein productive value (PPV) is a suitable way of determining protein utilization since it is more accurate and less time consuming than biological value (BV) determinations in fish, because of the problem of collecting and separating fish excreta from water. PPV was calculated as follows: animals were kept in individual compartments inside the same tank to maintain uniform conditions. Before the experiment began, two groups of I2 animals were adapted for I I days in the cages, as well as to each diet. They were then weighed and divided into two groups of 6 fish each. A blank group for each diet was killed to give initial body nitrogen and the other two groups were fed for 3 wk on each experimental diet. After the experimental period, the animals were weighed, killed and analyzed for final body nitrogen. Initial body nitrogen for the experimental lot was calculated from the blank. This method for PPV determination is an adaptation of Cremer‘s technique (1963) for rats, the mathematical expression which defines this index being: ppv
= Final body nitrogen Nitrogen
- Initial body nitrogen
x loo
ingested
In these three week assays on nutritive utilization. protein efficiency ratio (PER) was also calculated as the ratio of body weight increase (g): ingested protein (g).
584
M. DE LA HIGUERA rr al. Table
I. Composition
of the experimental
diets (g/kg dry diet)
Component
Fish meal diet
White fish meal
Yeast diet
704
__
Yeast meal
_-
812
Dextrine
38
__
130
__
Cod liver oil
20
20
Maize oil
__
60
28
28
premix*
30
30
Sodium alginate
SO
50
Crude protein
510
soo
Total lipid
100
Cellulose
Vitamin premix Mineral
*
95
* Cowey et trl. ( 1972). Blood
sanlphg
was extracted with an extractor using ethyl-ether as solvent. Moisture was measured in an oven at 105 k 1 C until constant weight. Nitrogen free extract (NFE) was calculated by difference. Plasma ammonia and urea nitrogen were determined calorimetrically at 630 nm by the phenolhypochlorite method of Chaney & Marbach (1962). Urea was previously treated with urease to give free ammonia and then determined calorimetrically as above. Plasma, liver and kidney uric acid was determined calorimetrically at 7OOnm according to the method of Henry (I 969).
After stunning the trout by a blow on the head, blood was collected from the caudal vessel with a heparinized syringe. Plasma was obtained by centrifugation for IO min at 3000 rev/min. Aliquots were used for ammonia. urea and uric acid determinations. Preparation
of tissue e.wacts
Liver and kidney extracts were obtained by homogenization (using an Ultraturrax homogenizer at 20,000 U per min for I5 set) in ice-cold 0.6 N HC104, in order to obtain total deproteinization and at a w/v ratio of l/4. Liver and kidney homogenates were centrifuged at 7OOOrev/min for 5 min. Aliquots were used for uric acid determinations. Amlyrical
RESULTS
methods
To determine animal body composition the following methods and techniques were used. Nitrogen determinations were done using the Kjeldahl’s technique. The conversion factor of nitrogen into protein was 6.25. Total lipids were measured gravimetrically. Lipid Table 2. Diet influence
The influence of the experimental diet (prepared with yeast as the only protein source) on nutritive utilization of protein is shown in Table 2. Table 3 shows diet effects on ammonia, urea and uric acid
on nutritive
utilization
Fish meal diet Food intake (g dry matter) Weight increment (g) Body N increase (g) PER PPV Values given are mean Table
3. Influence
46.3 36.5 1.06 1.52 27.7
f + + + k
Student’s r-test
Yeast diet
3.5 4.2 0.09 0.1 I 0.9
30.5 17.0 0.79 1.10 32.9
+ + f f +
2.3 3.0 0.12 0.14 3.7
P < 0.01 P < 0.05
NS P < 0.05
NS
f SE of 6 fish. NS = Not significant. of the diet on some nitrogen
metabolism
Fish meal diet Food intake (g dry matter) Weight increment (g) Plasma ammonia (mg/lOO ml) Plasma urea (mg/lOO ml) Plasma uric acid (mg/lOO I) Kidney uric acid (pmol/g wet wt) Liver uric acid (pmol/g wet wt) Hepato-somatic index ( x 1000) Values given are mean
of protein
40.8 27.5 3.4 41.4 1.5 0.8 1.2 13.4
+ + f + f i * +
5.8 6.0 0.3 3.6 0.6 0.1 0.1 1.3
+ SE of 6 fish. NS = Not significant
end products
Yeast diet 26.4 14.0 5.2 77.6 1.3 2.4 0.9 12.3
_t 2.6 + 1.3 + 0.4 + 16.0 * 0.1 + 0.5 +_ 0.1 + 1.5
Student’s f-test P < 0.05 P i 0.05 P < 0.05
NS NS P < 0.01 NS NS
Yeast utilization by trout levels in plasma as well as liver and kidney uric acid contents. Results for the experimental diet were tested against those for fish meal diet taken as control by Student’s t-test. Significant differences were established at the 0.05 level. Food intake, body weight changes and heparosomaricindex After 21 days under the experimental diet a significant decrease in food intake was observed in the two experimental lots fed on yeast diet (Tables 2 and 3) which represents more than 30% for both. The lesser intake for yeast diet was reflected in the weight increments that showed a proportional and significant decrease (P < 0.05) for both lots of about 50%. On the other hand hepatosomatic index did not suffer any change (Table 3). Protein utilizution indices Body nitrogen increment tended to decrease according to the forementioned weight changes, although it was not significant (Table 2). PER however, showed a significant decrease (P < 0.05) when trout were fed a yeast diet. When body nitrogen increase is related to nitrogen intake (PPV) the values are not significant although trout fed on yeast diet showed a tendency towards a better utilization of dietary protein. Ammonia, urea and uric acid levels Plasma ammonia values (Table 3) showed a significant increase of about 53% (P < 0.05) for the experimental diet. Plasma urea levels were also increased by 87% although not significantly. Table 3 shows there were no modifications of uric acid levels in plasma and in liver but kidney showed a significant increase (P < O.Ol), about 3-fold, when trout were fed on yeast protein diet. DISCUSSION
Food intake decrease (Table 2) can be attributed, among other possible factors, to a lower acceptability of trout of the yeast protein diet compared to white fish meal. These results due to the foodstuff, which is “new” for the trout, may also be a consequence of the alterations found in this and previous studies (Sinchez-Muniz, 1977, SLnchez-Muniz et al., 1979). In any case, yeast diet intake is acceptable as it is inside the intakes given for trout fed on normal diets. Assuming that according to Cowey et al. (1974), proteins differ in their ability to support growth even when given at high dietary concentrations, it remains possible that the differences found would depend on the biological availability of some essential amino acids in the protein. The relative body nitrogen increase, as well as the higher PER for the fish meal diet, points to the forementioned theory. Nevertheless, the lower body weight increment of trout fed on yeast diet is firstly a direct consequence of a lower food or total protein intake (Gbmez-Jarabo, 1976). D’Mello et al. (1976) showed that in growing pigs the intake is not a decisive factor in obtaining a better nitrogen utilization for single cell protein compared to that of white fish meal. In any case, and independently of the
585
nutritional value of both diets, a significant food intake decrease must be reflected in body growth. If the different PER results can be mostly explained in terms of food intake, the values for protein productive value (PPV) can also be a consequence of a better net utilization of yeast protein. In this sense and apart from extreme cases, protein utilization decreases with increasing dietary protein levels, mostly because more protein is being used as an energy source (Miller & Payne, 1961). Nevertheless, other possibilities should be taken into account when discussing these results. Amino acid availability is also an important factor and D’Mello et al. (1976)have reported a better digestibility of certain amino acids after feeding pigs with microbial cells compared to white fish meal protein. Nevertheless, when considering high protein diets it remains unresolved whether a better protein utilization could be explained on the basis of essential amino acid content or amino acid disposibilities (Cowey et (II.. 1974). Furthermore, the better protein retention found by d’Mello et al. (1976) is, according to these authors, presumably due to the higher intakes of lysine. Provisional results have shown a great lysine content of H. unomala protein (Varela et al., 1976), that could contribute to the results obtained here for trout fed on this yeast. In relation to diet effects on some nitrogen metabolism end products (Table 3), yeast diet entails an increase of catabolites mainly related to nucleic acids catabolism. These results are a consequence of dietary nucleic acids content (about 9%) of the yeast diet. The results found are logical. Nevertheless, so little information about dietary use of single cells by fish is available that the present work will be compared with information available obtained from mammals. In the growing pig ammonia production is increased together with allantoin, when pigs are fed on diets containing microbial cells grown on methanol. D’Mello et crl. (1976) think that it is a consequence of the degradation of pyrimidines and purines from dietary nucleic acids. Similar results were reported by Heaf & Davies (1976) for rats fed on RNA supplemented diet. Plasma urea levels (that increased in about 87% although not significantly) would not agree with the results of D’Mello et al. (1976) who found a decrease of urea excretion under a single cell diet compared to white fish meal, results that correlate with a higher biological value of the bacterial product. The increased urea levels under yeast diet were not due to protein intake that decreased significantly, but to a higher nucleotide catabolism. Goldstein & Foster (1965) showed the importance of purine catabolism as a source of urea. Enzyme assays show that uricase and probably allantoinase and allantoicase are present in sufficient quantities to account for the amount of urea produced by most fish, uric acid being the main source (Vellas & Serfaty, 1974). Uricase or its induction could be the reason for the nonsignificant changes found in plasma uric acid levels under a single cell protein diet. Yeast diet seems not to have influenced the trout liver uric acid content. This suggests that liver uricase must be present and active in quantities sufficient to metabolize “extra” uric acid. Furthermore, the hepa-
M. UE LA HIGUERAet al.
586
tosomatic ratio, taken as an index of liver da-nage, was not affected by the experimental conditions. Kidney uric acid showed a three-fold increase in those trout fed on yeast diet, although optical microscopy did not show any damage. These results could be attributed either to low kidsey uricase conccntrations, or to inhibition of this enzyme by increasing accumulation of substrate or reaction product. Norriind & Kihleberg (1973) found after uricase inhibition, an increase of plasma, urine and kidney uric acid. Kidney uric acid significantly increased from 0.4mg for a 0% RNA diet to 6.7mg for rats fed on a 3% RNA diet. A hypocromic and microcytic anaemia appeared in those trout fed on the same yeast diet (Sanchez-Muniz ef al., 1979), the negative correlation found between plasma uric acid, urea, ammonia and plasma fl-globulins fraction was partly responsible. Heaf & Davies (1976) showed that RNA supplemented diets increased plasma uracil levels about 20-fold and as uracil might be expected to cause changes in the internal environment of cells around which they circulate, the erythrocyte alterations could be a direct consequence. The mammalian erythrocytes have the capacity for interconverting purine bases, their nucleosides and their nucleotides (Bishop, 1964). but there is little evidence that uracil in blood is harmful. Although yeast protein utilization by rainbow trout is acceptable, caution should be taken when planning large-scale use of “single cell” proteins, at least as the only protein source. Acknowledgements-The authors wish to express their gratitude to the Departamento de Anatomia Patol6gica de la Facultad de Veterinaria de la Universidad de Madrid for performing the histological studies. REFERENCES
ABBOTJ. C. (1974) Economics of single cell protein in relation to world protein supplies. In Single Cell Protein (Edited by DAVIS P.), pp. 2145. Academic Press, London. BISHOPC. (1964) The Red Blood Cell (Edited by BISHOPC. & SURGENORSD. M.), p. 148. Academic Press. New York. CHANEYA. L. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. C/in. Chem. 8, 130-132. COWEY C. B., POPE J. A., ADRONJ. W. & BLAIRA. (1972) Studies on the nutrition of marine flatfish. The protein requirement of plaice (Pleuronecfes plutessu). Br. j. Nutr. 28, 447456.
COWEY C. B., ADRON J. & BLAIR A. (1974) Studies on the
nutrition of marine flatfish. Utilization of various dietary protein by plaice (Pleuronecres plaressa). Br. J. NW. 31, 297-306. CREMER H. D. (1963) Evaluation of‘ Protein Quality. National Academy of Sciences: National Research Council Washing&. 1100,32-38. DAVISP. (1974) Sinole Cell Protein (Edited bv DAVIS P.I. Academic Prkss, London. ’ D’MELLOJ. P. F., PEERSD. G. B WHITTEMORE C. T. (1976). Utilization of dried microbial cells grown on methanol in a semipurified diet for growing pigs. Br. J. Nutr. 36, 403-410. GOLDSTEINL. & FORSTERR. P. (1965) The role of uricolysis in the production of urea in fishes and other aquatic vertebrates. Comp. Biochem. Physiol. 14, 567-576. GOMEZ-JARABO G. A. (1976) Utilizucidn nutririuu de /u proreinu en lu rrucha (Sulmo yuirdneri). Injuenciu v de/ nitjel Droteic’ode la dietu. Ph.D. Thesis.
de lu edud
Facultad de
Geterinaria, University of Madrid. GROOTA. P. (1974) Minimal test necessary to evaluate the
nl;:ritional qualities and the safety by single cell proteins. In Single Cell Protein (Edited by DAVIS P.), pp. 75-92. Academic Press, London. HEAFD. J. & DAVIESJ. I. (1976) The effect of RNA supplementation of rat diets on the ccmposition of body fluids. Br. J. Nutr.
36, 381-402.
HENRY R. J. (1969) Determinacibn del Bcido ljrico mediante reacci6n con fosfotungstato alcalino. In Quimica Clinica. Principios y tknicus. Vol. I, (Edited by 5~s) pp. 337-343. Barcelona. HIGUERAM. DE LA. MURILLOA.. VARELAG. & ZAMORAS. (1977) The influence of high dietary fat levels on protein utilization by the trout (Salmo gairdnerii). Camp. Biothem. Physiol. ?%A, 3741. MILLERD. S. & PAYNEP. R. (1961) Problems in the prediction of protein values in diets. The influence of protein concentration. Br. J. Nutr. 15, 1I-19. NORRLINDB. & KIHLBERGR. (1973) A short assay for the estimation of dietary purine compounds, using a rat model system with inhibition of uricase. J. Nutr. 103, 1262- 1269. SANCHEZ-MUNIZF. J. (1977) Algunos uspectos sobre la urilizacidn nutritit;a de lu letladura (Hansenula unomalu) por la trucha (Salmo gairdneri). Escuela de Bromatologia. Uni-
versity of Madrid. SANCHEZ-MUNIZF. J.. HIGUERAM. DE LA, MATAIXF. J. & VARELAG. (1979) The yeast Hansen& unomulu as a protein source for rainbow trout (Salmo guirdneri). Haematological aspects. Comp. Biochem. Physiol. 63A. 153-157. VARELAG., MATAIXF. J. & NAVARROM. P. (1976) Estudio del valor nutritivo de la proteina de la levadura Hunsenulu anomala crecida sobre etanol de sintesis. ATA. 16, 265-272.
VELLASF. & SERFATYA. (1974) Metabolism of ammonia and urea in the carp. J. Physiol., Paris 68, 591-614.
de Flsiologia
Animal.
Facultad (Received
de Farmacia. 18 Nooember
Universidad
Complutense.
Madrid-3.
Spain
1980)
Abstract--l. Nutritive and metabolic consequences of feeding rainbow trout with a single cell protein diet were studied. Experimental diet was adjusted by using yeast Hansenula anomala as the only protein source, compared to white fish meal for the controls. 2. Food intake, weight increments and consequently protein efficiency ratio (PER) significantly decreased in trout fed on yeast diet. Furthermore, body nitrogen increase was lower and protein productive value (PPV) higher for the same diet, although in a nonsignificant way. 3. Plasma ammonia increased significantly in the experimental animals. Plasma urea levels, although increased in about 90%, were not significantly altered. Plasma uric acid levels, unaltered in plasma and liver, suffered a three-fold increase in kidney.
INTRODUCTION
In relation to new protein sources, yeast is of great interest because of its high vitamin B-complex content and also because yeast appears to be a promising future supply of protein. Furthermore, its high thermostability and economical aspects related to world protein supplies (Abbot, 1974), present yeast as a source with a great nutritional future (further details can be obtained from Davis 1974). On the other hand, the use of single cells as the only protein source produces some physiological alterations in the blood of rainbow trout as has been already published in a previous paper (SBnchezMuniz. et al., 1979). The use of single cell proteins in human and animal nutrition requires tests to evaluate its safety and nutritional qualities (Groot, 1974). This paper deals with nutritive utilization of yeast Hansenula anomala protein and also with the alterations on some nitrogen metabolic end products related to protein and nucleic acids metabolism. Furthermore, as yeast contains a great proportion of nucleic acids whose catabolism could lead to kidney damage (Groot, 1974), results are also discussed about the alterations found in the blood of rainbow trout fed with H. anomala (SBnchezMuniz rt al.. 1979). MATERIAL AND METHODS Animals
and maintenance
Rainbow trout obtained from a local fish farm, of an average weight of 100 g; were kept at IS + 1°C in tanks (80 x 35 x 40cm) divided into compartments in order to maintain uniform conditions. After 1 I days of acclimation to the new conditions and food, rainbow trout were individually weighed and placed into individual compartments where they were kept for three week experiments. One group was to evaluate nutritive utilization of protein and the other to measure nitrogen metabolism end products in plasma, liver and kidney. 583
Each fish was fed once daily, one pellet was offered at a time and when all the food offered had been consumed, more was added until satiation. Diets
The composition of the experimental diets to give a protein content of 50% (N x 6.25) is shown in Table 1. Yeast (Hansenula anomala strain 926 grown on synthetic ethanol) was prepared at the Instituto de Fermentaciones Industriales (C.S.I.C. Madrid). The dry components of each diet were blended with sodium alginate (5Og per kg) as a quelant and then thoroughly stirred with distilled water to get a homogeneous humidified mixture. Pellets were made by passing the diet mixture through an electric meat grinder equipped with dies of 2.5 mm hole size. After drying the diets were kept in a deep freeze at -20°C. Assays on protein
utilization
As concluded in a previous paper (de la Higuera et al., 1977), protein productive value (PPV) is a suitable way of determining protein utilization since it is more accurate and less time consuming than biological value (BV) determinations in fish, because of the problem of collecting and separating fish excreta from water. PPV was calculated as follows: animals were kept in individual compartments inside the same tank to maintain uniform conditions. Before the experiment began, two groups of I2 animals were adapted for I I days in the cages, as well as to each diet. They were then weighed and divided into two groups of 6 fish each. A blank group for each diet was killed to give initial body nitrogen and the other two groups were fed for 3 wk on each experimental diet. After the experimental period, the animals were weighed, killed and analyzed for final body nitrogen. Initial body nitrogen for the experimental lot was calculated from the blank. This method for PPV determination is an adaptation of Cremer‘s technique (1963) for rats, the mathematical expression which defines this index being: ppv
= Final body nitrogen Nitrogen
- Initial body nitrogen
x loo
ingested
In these three week assays on nutritive utilization. protein efficiency ratio (PER) was also calculated as the ratio of body weight increase (g): ingested protein (g).
584
M. DE LA HIGUERA rr al. Table
I. Composition
of the experimental
diets (g/kg dry diet)
Component
Fish meal diet
White fish meal
Yeast diet
704
__
Yeast meal
_-
812
Dextrine
38
__
130
__
Cod liver oil
20
20
Maize oil
__
60
28
28
premix*
30
30
Sodium alginate
SO
50
Crude protein
510
soo
Total lipid
100
Cellulose
Vitamin premix Mineral
*
95
* Cowey et trl. ( 1972). Blood
sanlphg
was extracted with an extractor using ethyl-ether as solvent. Moisture was measured in an oven at 105 k 1 C until constant weight. Nitrogen free extract (NFE) was calculated by difference. Plasma ammonia and urea nitrogen were determined calorimetrically at 630 nm by the phenolhypochlorite method of Chaney & Marbach (1962). Urea was previously treated with urease to give free ammonia and then determined calorimetrically as above. Plasma, liver and kidney uric acid was determined calorimetrically at 7OOnm according to the method of Henry (I 969).
After stunning the trout by a blow on the head, blood was collected from the caudal vessel with a heparinized syringe. Plasma was obtained by centrifugation for IO min at 3000 rev/min. Aliquots were used for ammonia. urea and uric acid determinations. Preparation
of tissue e.wacts
Liver and kidney extracts were obtained by homogenization (using an Ultraturrax homogenizer at 20,000 U per min for I5 set) in ice-cold 0.6 N HC104, in order to obtain total deproteinization and at a w/v ratio of l/4. Liver and kidney homogenates were centrifuged at 7OOOrev/min for 5 min. Aliquots were used for uric acid determinations. Amlyrical
RESULTS
methods
To determine animal body composition the following methods and techniques were used. Nitrogen determinations were done using the Kjeldahl’s technique. The conversion factor of nitrogen into protein was 6.25. Total lipids were measured gravimetrically. Lipid Table 2. Diet influence
The influence of the experimental diet (prepared with yeast as the only protein source) on nutritive utilization of protein is shown in Table 2. Table 3 shows diet effects on ammonia, urea and uric acid
on nutritive
utilization
Fish meal diet Food intake (g dry matter) Weight increment (g) Body N increase (g) PER PPV Values given are mean Table
3. Influence
46.3 36.5 1.06 1.52 27.7
f + + + k
Student’s r-test
Yeast diet
3.5 4.2 0.09 0.1 I 0.9
30.5 17.0 0.79 1.10 32.9
+ + f f +
2.3 3.0 0.12 0.14 3.7
P < 0.01 P < 0.05
NS P < 0.05
NS
f SE of 6 fish. NS = Not significant. of the diet on some nitrogen
metabolism
Fish meal diet Food intake (g dry matter) Weight increment (g) Plasma ammonia (mg/lOO ml) Plasma urea (mg/lOO ml) Plasma uric acid (mg/lOO I) Kidney uric acid (pmol/g wet wt) Liver uric acid (pmol/g wet wt) Hepato-somatic index ( x 1000) Values given are mean
of protein
40.8 27.5 3.4 41.4 1.5 0.8 1.2 13.4
+ + f + f i * +
5.8 6.0 0.3 3.6 0.6 0.1 0.1 1.3
+ SE of 6 fish. NS = Not significant
end products
Yeast diet 26.4 14.0 5.2 77.6 1.3 2.4 0.9 12.3
_t 2.6 + 1.3 + 0.4 + 16.0 * 0.1 + 0.5 +_ 0.1 + 1.5
Student’s f-test P < 0.05 P i 0.05 P < 0.05
NS NS P < 0.01 NS NS
Yeast utilization by trout levels in plasma as well as liver and kidney uric acid contents. Results for the experimental diet were tested against those for fish meal diet taken as control by Student’s t-test. Significant differences were established at the 0.05 level. Food intake, body weight changes and heparosomaricindex After 21 days under the experimental diet a significant decrease in food intake was observed in the two experimental lots fed on yeast diet (Tables 2 and 3) which represents more than 30% for both. The lesser intake for yeast diet was reflected in the weight increments that showed a proportional and significant decrease (P < 0.05) for both lots of about 50%. On the other hand hepatosomatic index did not suffer any change (Table 3). Protein utilizution indices Body nitrogen increment tended to decrease according to the forementioned weight changes, although it was not significant (Table 2). PER however, showed a significant decrease (P < 0.05) when trout were fed a yeast diet. When body nitrogen increase is related to nitrogen intake (PPV) the values are not significant although trout fed on yeast diet showed a tendency towards a better utilization of dietary protein. Ammonia, urea and uric acid levels Plasma ammonia values (Table 3) showed a significant increase of about 53% (P < 0.05) for the experimental diet. Plasma urea levels were also increased by 87% although not significantly. Table 3 shows there were no modifications of uric acid levels in plasma and in liver but kidney showed a significant increase (P < O.Ol), about 3-fold, when trout were fed on yeast protein diet. DISCUSSION
Food intake decrease (Table 2) can be attributed, among other possible factors, to a lower acceptability of trout of the yeast protein diet compared to white fish meal. These results due to the foodstuff, which is “new” for the trout, may also be a consequence of the alterations found in this and previous studies (Sinchez-Muniz, 1977, SLnchez-Muniz et al., 1979). In any case, yeast diet intake is acceptable as it is inside the intakes given for trout fed on normal diets. Assuming that according to Cowey et al. (1974), proteins differ in their ability to support growth even when given at high dietary concentrations, it remains possible that the differences found would depend on the biological availability of some essential amino acids in the protein. The relative body nitrogen increase, as well as the higher PER for the fish meal diet, points to the forementioned theory. Nevertheless, the lower body weight increment of trout fed on yeast diet is firstly a direct consequence of a lower food or total protein intake (Gbmez-Jarabo, 1976). D’Mello et al. (1976) showed that in growing pigs the intake is not a decisive factor in obtaining a better nitrogen utilization for single cell protein compared to that of white fish meal. In any case, and independently of the
585
nutritional value of both diets, a significant food intake decrease must be reflected in body growth. If the different PER results can be mostly explained in terms of food intake, the values for protein productive value (PPV) can also be a consequence of a better net utilization of yeast protein. In this sense and apart from extreme cases, protein utilization decreases with increasing dietary protein levels, mostly because more protein is being used as an energy source (Miller & Payne, 1961). Nevertheless, other possibilities should be taken into account when discussing these results. Amino acid availability is also an important factor and D’Mello et al. (1976)have reported a better digestibility of certain amino acids after feeding pigs with microbial cells compared to white fish meal protein. Nevertheless, when considering high protein diets it remains unresolved whether a better protein utilization could be explained on the basis of essential amino acid content or amino acid disposibilities (Cowey et (II.. 1974). Furthermore, the better protein retention found by d’Mello et al. (1976) is, according to these authors, presumably due to the higher intakes of lysine. Provisional results have shown a great lysine content of H. unomala protein (Varela et al., 1976), that could contribute to the results obtained here for trout fed on this yeast. In relation to diet effects on some nitrogen metabolism end products (Table 3), yeast diet entails an increase of catabolites mainly related to nucleic acids catabolism. These results are a consequence of dietary nucleic acids content (about 9%) of the yeast diet. The results found are logical. Nevertheless, so little information about dietary use of single cells by fish is available that the present work will be compared with information available obtained from mammals. In the growing pig ammonia production is increased together with allantoin, when pigs are fed on diets containing microbial cells grown on methanol. D’Mello et crl. (1976) think that it is a consequence of the degradation of pyrimidines and purines from dietary nucleic acids. Similar results were reported by Heaf & Davies (1976) for rats fed on RNA supplemented diet. Plasma urea levels (that increased in about 87% although not significantly) would not agree with the results of D’Mello et al. (1976) who found a decrease of urea excretion under a single cell diet compared to white fish meal, results that correlate with a higher biological value of the bacterial product. The increased urea levels under yeast diet were not due to protein intake that decreased significantly, but to a higher nucleotide catabolism. Goldstein & Foster (1965) showed the importance of purine catabolism as a source of urea. Enzyme assays show that uricase and probably allantoinase and allantoicase are present in sufficient quantities to account for the amount of urea produced by most fish, uric acid being the main source (Vellas & Serfaty, 1974). Uricase or its induction could be the reason for the nonsignificant changes found in plasma uric acid levels under a single cell protein diet. Yeast diet seems not to have influenced the trout liver uric acid content. This suggests that liver uricase must be present and active in quantities sufficient to metabolize “extra” uric acid. Furthermore, the hepa-
M. UE LA HIGUERAet al.
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tosomatic ratio, taken as an index of liver da-nage, was not affected by the experimental conditions. Kidney uric acid showed a three-fold increase in those trout fed on yeast diet, although optical microscopy did not show any damage. These results could be attributed either to low kidsey uricase conccntrations, or to inhibition of this enzyme by increasing accumulation of substrate or reaction product. Norriind & Kihleberg (1973) found after uricase inhibition, an increase of plasma, urine and kidney uric acid. Kidney uric acid significantly increased from 0.4mg for a 0% RNA diet to 6.7mg for rats fed on a 3% RNA diet. A hypocromic and microcytic anaemia appeared in those trout fed on the same yeast diet (Sanchez-Muniz ef al., 1979), the negative correlation found between plasma uric acid, urea, ammonia and plasma fl-globulins fraction was partly responsible. Heaf & Davies (1976) showed that RNA supplemented diets increased plasma uracil levels about 20-fold and as uracil might be expected to cause changes in the internal environment of cells around which they circulate, the erythrocyte alterations could be a direct consequence. The mammalian erythrocytes have the capacity for interconverting purine bases, their nucleosides and their nucleotides (Bishop, 1964). but there is little evidence that uracil in blood is harmful. Although yeast protein utilization by rainbow trout is acceptable, caution should be taken when planning large-scale use of “single cell” proteins, at least as the only protein source. Acknowledgements-The authors wish to express their gratitude to the Departamento de Anatomia Patol6gica de la Facultad de Veterinaria de la Universidad de Madrid for performing the histological studies. REFERENCES
ABBOTJ. C. (1974) Economics of single cell protein in relation to world protein supplies. In Single Cell Protein (Edited by DAVIS P.), pp. 2145. Academic Press, London. BISHOPC. (1964) The Red Blood Cell (Edited by BISHOPC. & SURGENORSD. M.), p. 148. Academic Press. New York. CHANEYA. L. & MARBACHE. P. (1962) Modified reagents for determination of urea and ammonia. C/in. Chem. 8, 130-132. COWEY C. B., POPE J. A., ADRONJ. W. & BLAIRA. (1972) Studies on the nutrition of marine flatfish. The protein requirement of plaice (Pleuronecfes plutessu). Br. j. Nutr. 28, 447456.
COWEY C. B., ADRON J. & BLAIR A. (1974) Studies on the
nutrition of marine flatfish. Utilization of various dietary protein by plaice (Pleuronecres plaressa). Br. J. NW. 31, 297-306. CREMER H. D. (1963) Evaluation of‘ Protein Quality. National Academy of Sciences: National Research Council Washing&. 1100,32-38. DAVISP. (1974) Sinole Cell Protein (Edited bv DAVIS P.I. Academic Prkss, London. ’ D’MELLOJ. P. F., PEERSD. G. B WHITTEMORE C. T. (1976). Utilization of dried microbial cells grown on methanol in a semipurified diet for growing pigs. Br. J. Nutr. 36, 403-410. GOLDSTEINL. & FORSTERR. P. (1965) The role of uricolysis in the production of urea in fishes and other aquatic vertebrates. Comp. Biochem. Physiol. 14, 567-576. GOMEZ-JARABO G. A. (1976) Utilizucidn nutririuu de /u proreinu en lu rrucha (Sulmo yuirdneri). Injuenciu v de/ nitjel Droteic’ode la dietu. Ph.D. Thesis.
de lu edud
Facultad de
Geterinaria, University of Madrid. GROOTA. P. (1974) Minimal test necessary to evaluate the
nl;:ritional qualities and the safety by single cell proteins. In Single Cell Protein (Edited by DAVIS P.), pp. 75-92. Academic Press, London. HEAFD. J. & DAVIESJ. I. (1976) The effect of RNA supplementation of rat diets on the ccmposition of body fluids. Br. J. Nutr.
36, 381-402.
HENRY R. J. (1969) Determinacibn del Bcido ljrico mediante reacci6n con fosfotungstato alcalino. In Quimica Clinica. Principios y tknicus. Vol. I, (Edited by 5~s) pp. 337-343. Barcelona. HIGUERAM. DE LA. MURILLOA.. VARELAG. & ZAMORAS. (1977) The influence of high dietary fat levels on protein utilization by the trout (Salmo gairdnerii). Camp. Biothem. Physiol. ?%A, 3741. MILLERD. S. & PAYNEP. R. (1961) Problems in the prediction of protein values in diets. The influence of protein concentration. Br. J. Nutr. 15, 1I-19. NORRLINDB. & KIHLBERGR. (1973) A short assay for the estimation of dietary purine compounds, using a rat model system with inhibition of uricase. J. Nutr. 103, 1262- 1269. SANCHEZ-MUNIZF. J. (1977) Algunos uspectos sobre la urilizacidn nutritit;a de lu letladura (Hansenula unomalu) por la trucha (Salmo gairdneri). Escuela de Bromatologia. Uni-
versity of Madrid. SANCHEZ-MUNIZF. J.. HIGUERAM. DE LA, MATAIXF. J. & VARELAG. (1979) The yeast Hansen& unomulu as a protein source for rainbow trout (Salmo guirdneri). Haematological aspects. Comp. Biochem. Physiol. 63A. 153-157. VARELAG., MATAIXF. J. & NAVARROM. P. (1976) Estudio del valor nutritivo de la proteina de la levadura Hunsenulu anomala crecida sobre etanol de sintesis. ATA. 16, 265-272.
VELLASF. & SERFATYA. (1974) Metabolism of ammonia and urea in the carp. J. Physiol., Paris 68, 591-614.