Pyridoxine requirements of the gilthead bream, Sparus aurata

Aquaculture, 23 (1981) 243-255 Elsevier Scientific Publishing Company,

PYRIDOXINE REQUIREMENTS A URA TA

G.Wm. KISSIL*,

C.B. COWEY**,

243 Amsterdam

- Printed

in The Netherlands

OF THE GILTHEAD BREAM, SPAR US

J.W. ADRON**

and R.H. RICHARDS***

*Mariculture Laboratory, Israel Oceanographic & Limnological Research, P.O. Box 1212, Elat (Israel) **N.E.R.C. Institute of Marine Biochemistry, St. Fittick’s Road, Aberdeen, ABl 3RA (U.K.) *** Department of Aquatic Pathobiology, University of Stirling, Stirling, FK9 4LA (U.K.) Contribution (Accepted

No. 231, Israel Oceanographic 19 August

& Limnological

Research

1980)

ABSTRACT Kissil, G.Wm., Cowey, C.B., Adron, J.W. and Richards, R.H., 1981. Pyridoxine ments of the gilthead bream, Sparus aurata. Aquaculture, 23: 243-255.

require-

Minimal dietary pyridoxine requirements of the gilthead bream (Sparus aurata Linnaeus) were determined. Partially defined test diets containing graded levels of pyridoxine were fed to groups of fish of average initial weight 2.7 g and 70 g. The criteria used to measure pyridoxine requirements were growth, mortality, food conversion, fish behavior, liver alanine aminotransferase (AAT; EC 2.6.1.2) activity and histopathology. At the lowest dietary pyridoxine level used with the small fish (1.15 mg/kg dry diet) and at the two lowest levels used with the bigger fish (1.15 and 1.46 mg/kg dry diet), vitamin deficiency symptoms were evident in all parameters examined. Signs of deficiency were manifested as growth retardation, high mortality, poor food conversion, hyperirritability coupled with erratic swimming behavior and degenerative changes in peripheral nerves. In both experiments, the dietary level at and above which no deficiency signs appeared was 1.97 mg/kg dry diet. Liver AAT activity was a more conservative parameter, indicating pyridoxine insufficiencies in both groups of fish also at the level of 1.97 mg/kg dry diet.

INTRODUCTION

A dietary requirement for vitamin B,, pyridoxine, has been demonstrated and in certain cases quantified in several species of freshwater and marine fish. In general, these experiments involved the use of diets completely or partially deficient in pyridoxine. The criteria used for measuring pyridoxine requirements included rate of growth, mortality, concentration ,of pyridoxine in liver, activity of various aminotransferases in body tissues, appearance of characteristic behavior patterns or development of pathological changes in body tissues or organs, and food conversion efficiency (Ogino, 1965; Dupree, 1966; Sakaguchi et al., 1969; Takeda and ,Yone, 1971; Arai et al., 1972; Halver, 1972; Smith et al., 1974; Adron et al., 1978). 0044~8486/81/0000-0000/$02.50

o 1981 Elsevier

Scientific

Publishing

Company

244

The gilthead bream (Sparus auruta) is presently being developed for commercial cultivation at the Mariculture Laboratory in Elat, Israel, and the experiments described here were carried out to define minimal requirements of pyridoxine for this species. This paper represents a complete analysis of all data obtained in these experiments including preliminary results previously published (Kissil, 1978). MATERIALS

AND METHODS

Diets The basal diet (Table I) was prepared at the Institute of Marine Biochemistry, Aberdeen, U.K. and air-freighted in dry form to Elat. Diets containing different concentrations of pyridoxine (Table II) were then made from the basal diet by adding to it appropriate quantities of an aqueous solution containing pyridoxine HCI. For experiment 1, diets containing five different levels of pyridoxine (1.15,1.97, 3.00,11.23 and 21.51 mg/kg dry diet) were used. For experiment 2, six dietary levels of pyridoxine (1.15, 1.46,1.97, 5.06, 8.68 and 26.65 mg/kg dry diet) were used. Diets were given to the fish by extrusion as a moist paste from veterinary syringes. The paste diets were prepared by adding 1200 ml water (including the appropriate quantity of pyridoxine HCl solution) to 800 g of the dry basal diet. These complete test diets were prepared in quantities of 2 kg at a time and stored at -15” C in the form of sausages (200-300 g each) until used. TABLE I Composition of basal diet given to gilthead bream Components

Quantity (g/kg dry diet)

Casein - vitamin freesqb Dextrin Glucose Cod liver oil’ Capelin oild Mineral premix e Vitamin premixf Cellofas-binder g a-Cellulose * Attractant mixturen

500 100 50 30 90 20 28 50 32 100

aNutritional biochemicals, Cleveland, Ohio. bBaaal level of 1.15 mg vitamin B,/ 500 g casein as determined by laboratory analysis. cSolvitax, British Cod Liver Oils Ltd., Hull. dskretting, P.O.B. 319,400l Stavanger, Norway. e*fAs in Kiss& (1978). sIC1 Ltd., Ardossan, Ayrshire. “Mixture composition based on squid muscle extract as reported by Mackie (1973) minus trimethylamine oxide and trimethylamine.

245

Fish Sparus aurutu obtained from the wild (Mediterranean coast of Israel) were acclimatized to the experimental conditions in stock tanks of 2-4 m3 for 3-8 months before being used in the two experiments that were carried out consecutively. The experimental system consisted of cylindrical fiberglass tanks (170 liter capacity) having conical bottoms; seawater flowed through the tanks at the rate of one exchange/hour and thence to waste; no recirculation of water occurred. For experiment 1, fish 2.69 f 0.07 g (2 ? SE.) were distributed between 15 tanks, 25 fish per tank. Thus the five dietary treatments were replicated three times over the 133 day experimental period. In this experiment, fish were fed twice per day to satiation. At each feeding a small quantity of food was extruded in short lengths into the tank; this was eaten before it reached the bottom of the tank. Then a further small quantity of food was added to the tank; feeding was continued in this manner until a first portion of uneaten food reached the bottom of the tank. Feeding was then stopped for that meal. The quantity of food eaten each day was recorded. Experiment 2 was similar to experiment 1 except that fish from the stock tank were now larger, 69.26 + 0.90 g and an additional dietary pyridoxine level was included (18 experimental tanks were used with 15 fish per tank); in this experiment, fish were given a daily ration equal to 16 g dry diet/kg biomass fish spread over two feedings each day for 136 days. In each experiment, fish were weaned onto the experimental diets for a lo-day period. Initial weight measurements were then made; thereafter fish were weighed approximately every 21 days. Daily observations were made during feeding to record any behavioral abnormalities among fish. Mortalities were recorded and any dead fish removed for pathological examination. Calculations Food conversion was calculated from food consumption and live weight gain of fish after both were adjusted for mortalities. Adjustment was made by subtracting the weight of a dead fish from the biomass of its tank at the previous weighing. The food it consumed between its last weighing and the day of death was subtracted from the total food consumption of the tank. Mortality included those deaths resulting from fish jumping out of tanks as the behavior causing this jumping was associated with pyridoxine deficiency. Food conversion and mortality for each tank was calculated for the whole experimental period. Mean values calculated for each treatment were then compared by the analysis of variance and Duncan’s new multiple range test where necessary (Steel and Torrie, 1960). An average relative growth rate

246

accumulated from day 1 was calculated ment. These values were also compared methods.

after each weighing for every treatfor equality by the above mentioned

His tology Fish were anesthetized in quinaldine (l:lO,OOO) and their spinal columns severed. Samples of brain, eye, heart, kidney, liver, muscle, skin, spinal cord and spleen were fixed in 10% buffered neutral formalin and then processed by routine histological methods, embedded in paraffin wax and sectioned (5 pm thickness). These sections were stained using haematoxylin and eosin, Gram-Humberstone (Humberstone, 1963) and chloranilic acid (Disbrey and Rack, 1970). In experiment 1,17 fish, 4.5% of the total, were sampled for histology on two occasions, namely at 5 weeks after onset of behavior typical of pyridoxine deficiency (days 53-59 of the experiment) and again3 weeks later (days 80-82). In experiment 2,16 fish, or 5.9% of the test animals, were sampled 1 week after the appearance of deficiency symptoms (day 44) and again at the termination of the experiment (days 137-138). Alanine amino transferase (EC 2.6.1.2)

measurement

Near the end of the experiments, day 113 in experiment 1 and days 98 and 136 in experiment 2, alanine aminotransferase (AAT) activity was measured in the livers of three fish from each treatment. Liver homogenates were prepared and AAT activity measured at 25°C by an interrupted assay procedure (Swick et al., 1965). One half of the homogenate was incubated with 0.3 mM pyridoxal-5-phosphate (final concentration) in 0.1 M phosphate buffer, pH 7.4, for 30 min at 5°C; the other half of the homogenate was incubated in 0.1 M phosphate buffer, pH 7.4, without added pyridoxal-5phosphate but under otherwise identical conditions. AAT activity was then measured in replicate 1 ml portions of these incubated homogenates. By means of this procedure, the percentage stimulation of AAT activity resulting from preincubation with pyridoxal-5-phosphate was obtained. RESULTS

Weight gains of the fish in experiments 1 and 2 are shown in Figs. 1 and 2. Comparisons of the relative growth rates (RGR) within each experiment indicated the following: In experiment 1, there was no significant difference in RGR among fish receiving diets 2 to 5 (1.97 to 21.51 mg pyridoxine/kg dry diet) while fish receiving diet 1 (1.15 mg pyridoxine/kg dry diet) showed

247 180

> 0

I

20

t

1

60

TIME-days

100

140

60 1

20

60

100

140

TIME-days

Fig.1. The growth of gilthead bream in experiment 1, fed on test diets containing five different levels of pyridoxine. Each point on the curves represents the average weight of all fish in that treatment at the time of weighing. Fig. 2. The growth of gilthead bream in experiment 2, fed on test diets containing six different levels of pyridoxine. Each point on the curves represents the average weight of all fish in that treatment at the time of weighing.

a significantly (P < 0.01) lower RGR by day 43. In experiment 2, fish given diets 1 and 2 (1.15 and 1.46 mg pyridoxine/kg dry diet) exhibited significantly lower (P < 0.01) RGR by days 62 and 97 respectively than the remaining treatments (1.97, 5.06, 8.68 and 26.65 mg pyridoxine/kg dry diet). Mortality calculated for the two experiments paralleled the pattern found in the growth data (Table II). Those treatments which displayed significantly poorer growth, lower RGR, suffered significantly (P < 0.05) higher mortalities. This same pattern was also evident in the food conversion data shown in Table II. Poor conversion of food occurred in those same treatments which had lower RGR and higher fish mortality, i.e. in the fish given diet 1 in experiment 1 and diets 1 and 2 in experiment 2. Behavior of a type that has been associated with a pyridoxine deficiency was observed in those fish which collectively displayed poor growth and high mortalities. After 28 days of feeding, (day 18 of the experiment) some of the fish in experiment 1, diet 1, showed signs of hyperirritability and erratic swimming which culminated in some fish jumping out of tanks. Those erratic swimmers which did not jump out died within a few days. This same behavior was also observed in experiment 2; it was first seen in fish given diet 1 after 45 days of feeding (day 35 of the experiment) and later in fish given diet 2 after 60 days of feeding (day 50 of experiment).

1.15 1.97 3.00 11.23 21.51 -

7.0 5.9 7.0 5.8 0.9

1.15 1.46 1.97 5.06 8.68 26.65

f f + + r

93.0 20.8 19.6 10.1 15.4 -

0.2 0.1 0.2 0.2 0.2

8.1 3.7 4.1 3.6 3.8 -

f f f + f

Pyridoxine in diet Lz;;“g dry

% MortalityC (mean f S.E.)

Pyridoxine in diet (dn$g dry Food conversionb (mean f S.E.)

Experiment 2

Experiment 1

5.3 3.2 2.1 2.0 2.2 2.0

f f f f * *

0.2 0.3 0.1 0.1 0.2 0.1

Food conversiond (mean + S.E.)

24.4 32.7 4.5 6.7 6.7 8.9

+ f f * f r

1.8 0 0.02 0.01 0.01 0

% Mortalitye (mean ?r S.E.)

‘Dry weight food consumed (g)/Live weight gain (g). b*c*d*e Underlined diet parameters are not significantly different from each other (P < 0.05). b13524. ‘12354; d125B; e21634A.

1 2 3 4 5 6

Diet no.

Food conversiona and mortality of gilthead bream fed diets containing different amounts of pyridoxine.

TABLE II

249

The percentage stimulation of liver AAT (i.e. the increase in activity of AAT following preincubation of the homogenate with pyridoxal phosphate compared with the activity of the homogenate preincubated without added pyridoxal phosphate) is shown in Fig. 3. In experiment 1 the first two treatments (1.15 and 1.97 mg pyridoxine/kg dry diet) indicate a deficiency of pyridoxine, as preincubation led to a significant increase in activity (pyruvate production) presumably because not all the apoenzyme present in the tissue was saturated with the co-enzyme pyridoxal phosphate. In experiment 2, the first three dietary treatments (1.15, 1.46 and 1.97 mg pyridoxine/kg dry diet) in both assay series (days 98 and 137), also showed large increases in liver AAT activity following preincubation of the homogenate with pyridoxal phosphate. Pathological changes were first evident in experiment 1 on day 53 in fish given diet 1; in experiment 2 pathology first appeared on day 44, again in fish given diet 1. The changes were much more pronounced by day 80 in experiment 1 and by day 137 in experiment 2 (fish given diets 1 and 2 with either 1.15 or 1.46 mg pyridoxine/kg). In all such samples, there was a degeneration of peripheral nerves. The earliest changes consisted of localised necrosis of nerve fibers in main nerve tracts with eventual associated inflammatory responses (Fig. 4A) which principally involved cells resembling the eosinophilic granule cells of salmonids (Roberts, 1979). In samples taken at the end of experiment 2 (day 137), this degeneration was very pronounced (Fig. .erp.l

2

5

PVRIDOXINE

8

doyll3

.erp.Z

day 137

~erp.2

day 98

11

a3

LEVEL-mgjkgdry

26

29 diet

Fig. 3. Percent stimulation of alanine aminotransferase activity in liver extracts of gilthead bream fed diets containing different pyridoxine levels, after preincubation of the extracts with pyridoxal phosphate (see text for details). Values for experiment 1 (0) represent single livers, while those for experiment 2 ( n and A) are means for three separate livers each.

250

251

4B) and occurred in association with focal necrosis in brain and spinal cord (Fig. 4C). Eosinophilic granule cells were also present in large numbers in the adipose tissue surrounding the pancreas in such fish (Fig. 4D) and were also noted in occasional fish on apparently adequate pyridoxine levels. Another change particularly noticeable after day 80 in experiment 1 was destruction of red blood cells in the haemopoietic areas of the kidney, with extensive melanin dispersal and lack of melanomacrophage cells in such areas (Fig. 5A). Congestion of the spleen (Fig. 5B) and meninges was evident in the majority of deficient fish, and in many fish a pronounced infiltration of the submucosa of the intestine by inflammatory cells occurred (Fig. 5C). DISCUSSION

The data obtained in experiment 1 relating the effects of dietary pyridoxine level to weight gain, mortality, food conversion, behavior and histology seemed to indicate that a minimum requirement for pyridoxine is not more than 1.97 mg/kg dry diet. In an attempt to define the pyridoxine requirement of Sparus auratu more closely, experiment 2 included an additional dietary pyridoxine level, 1.46 mg/kg, and fish were given a restricted ration as there was some evidence that satiation feeding with this species led to excessive food intake. At the feeding rate adopted (16 g dry diet/kg biomass/ day), this additional dietary pyridoxine level was not sufficient to meet the pyridoxine of the fish as judged by the criteria listed above. Thus the lowest dietary pyridoxine level that gave rise to healthy fish growing normally during the experimental periods was 1.97 mg/kg. This is higher than that previously reported for the same fish, 1.25 mg/kg (Kissil, 1978) due to background pyridoxine in the casein of the test diets which was unknown at the time of publication of Kissil(l978). The 1.97 mg/kg level is also higher than that required for turbot, Scophthalmk maximus, 0.5-1.0 mg/kg (Adron et al., 1978) but much lower than the range of values, 5-20 mg/kg given by Halver (1972) as required for a number of freshwater fish. Mortality caused by fish jumping out of tanks, previously discour ted (Kissil, 1978), was included in the re-evaluated mortality data of experiments 1 and 2 as such behavior was associated with pyridoxine deficiency. This coupled with additional data for experiment 2 resulted in differing values for mortality given in Table II and Kissil(l978). In both cases higher mortality paralleled the trend of poorer growth and food conversion at pyridoxine levels below 1.97 mg/kg. Fig. 4. Histological changes observed in gilthead bream fed test diets with insufficient pyridoxine levels. A - Spinal nerve with focal necrotic areas (n) and inflammatory infiltration (arrow). H & E, X 320. B - Pronounced degeneration of peripheral nerve (p) in pancreas. H & E, x 1250. C - Necrotic area in cortex of brain. H & E, x 320. D - Large numbers of eosinophilic cells (arrows) in pancreas. Note normal acinar cells (a). H & E, x 125.

252

Fig. 5. Histological changes observed in gilthead bream fed test diets with insufficient pyridoxine levels. A - Decreased haematopoietic tissue and melanin dispersal in kidney. H & E, x 320. B - Congested spleen with relatively few areas of white pulp. H & E, x 125. C - Intestine with marked cellular infiltration of submucosa. H & E, x 125.

If the data from the liver AAT activity are used to estimate the pyridoxine requirement, a higher value than 1.97 mg/kg is obtained. Assuming that an increase in alanine aminotransferase activity due to preincubation with pyridoxal phosphate is an indicator of an insufficiency of pyridoxine in the liver, then levels of 3.00 and 5.06 mg pyridoxine/kg dry diet for the fish in experiments 1 and 2 appear necessary. Similar trends of a greater need for dietary pyridoxine when enzyme activity rather than weight gain criteria are used have been demonstrated in red sea bream (Takeda and Yone, 1971) and turbot (Adron et al., 1978). This raises the question of which criteria should be used when providing diets for the culture of these fishes. As practical diets may be subject to harsh treatments (high temperature and high humidity) for short periods during pelleting, it is prudent to incorporate a safety factor in the levels of B vitamins incorporated in these diets. Thus there is a case for using the higher value in practical diets. The occurrence of abnormal behavior at an earlier stage in the fish of experiment 1 than in those of experiment 2 is probably a function of fish size. The larger fish of experiment 2 are growing at a slower rate than are those of experiment 1; therefore, vitamin deficiency symptoms would take longer to appear. Nervous disorders associated with pyridoxine deficiency in fish have been reported by Halver (1957), Ogino (1965), Sakaguchi et al. (1969) and Smith et al. (1974). A similar association was observed in pyridoxine deficient Sparus aurata in the present study. Previous work has not usually included histological descriptions of any effects on nervous tissue, but in the present study degenerative changes in the peripheral nerves as well as brain and spinal cord were prominent. In animals, pyridoxine deficiency has been associated with disturbances in metabolism of both a! amino butyric acid and phospholipids. The histological changes described in the present paper may represent the terminal effects of such changes. Anaemia has also been found in pyridoxine deficient fish by Halver (1957) and Smith (1967). Smith et al. (1974) described a normochromic, normocytic anaemia in rainbow trout (Sulmo guirdneri Richardson), and Adron et al. (1978) have noted a reduction in packed cell volume and plasma proteins when the pyridoxine antagonist 4-deoxypyridoxine hydrochloride was administered to turbot. In a variety of animal species, a microcytic hypochromic anaemia has been noted, as indicated by Shen et al. (1964). Although haematological indices were not measured in the present study, evidence of destruction of haematopoietic tissue was observed in certain of the pyridoxine deficient fish, and this may well have caused an anaemic condition. Kidney degeneration has been noted by a number of workers. Adron et al. (1978) described atrophy of tubule cells with sloughing and blocking of tubular lumina in turbot treated with a pyridoxine antagonist. Smith et al. (1974) noted ,hydropic degeneration and the deposition of calcium oxalate crystals in kidney tubules of rainbow trout. The oxalate production was

254

thought to have been associated with a high glycine intake resulting from the presence in the diet of gelatin. Other changes noted by Smith et al. (1974) have included hyperplasia of adrenal tissue, diffuse degenerative changes in the liver, and degeneration and necrosis of pancreatic tissues. No such changes were noted in our work on Spar-us auratu, but the sloughing of intestinal mucosa and peripancreatic fat necrosis noted by Smith and colleagues may be associated with the cellular infiltration into the submucosa and fat reported in the present work. Sakaguchi et al. (1969) noted scoliosis in pyridoxine deficient yellowtail (Seriola quinquerudiutu, Temminck & Schlegel), a pathology not seen in pyridoxine deficient Spurus uuru ta. ACKNGWLEDGEMENTS

The authors wish to thank Kelvin Posner and Philip Shilco for their technical assistance, Dr. I. Paperna for his help in sampling fish for histological preparation, and Ian MacRae for preparation of histological material. This work was partially supported by a travel grant provided by the Nuffield Foundation, London.

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255 Shen, S.C., Yong, P.I. and Oguro, O., 1964. Experimental production of pyridoxine deficiency anaemia in rats. Blood J. Hematol., 23: 679-687. Smith, C.E., 1967. Progress in sport fishery research. U.S. Fish. Wildl. Serv., Bur. Sport Fish. Wildl., FGL 54, 52 pp. Smith, C.E., Brin, M. and Halver, J.E., 1974. Biochemical, physiological and pathological changes in pyridoxine deficient rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can., 31: 1893-1898. Steel, R.G.D.,and Torrie, J.H., 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, NY, 481 pp. Swick, R.W., Barnstein, P.L. and Stange, J.L., 1965. The metabolism of mitochondrial protein. 1. Distribution and characterization of the isozymes of alanine aminotransferase in rat liver. J. Biol. Chem., 240: 3334-3340. Takeda, T. and Yone, Y., 1971. Studies of nutrition of red sea bream. 2. Comparison of vitamin B, requirement level between fish fed a synthetic diet and fish fed beef liver during prefeeding period. Rep. Fish. Res. Lab., Kyushu Univ., 1: 37-47.