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Fish silage as a dietary ingredient for salmon. II. Preliminary growth findings and nutritional pathology
Aquaculdm, 40 (lY84) 283-291 Elsevier Science Publishers B.V..
283 Amsterdam
-
Printed
in The Netherlands
FISH S’[LAGE AS A DIETARY INGREDIENT FOR SALMON. II. PRELIMINARY GROWTH FINDINGS AND NUTRITIONAL PATHC’LOGY
A.J.
JACKSON,
Dunstaffnage (Accepted
A.K. Marine
20 March
KERR
and A.M.
Research
BULLOCK
Laboratory,
P.O. Box
3, Oban,
Argyll
(Great
Britain)
1984)
ABSTRACT Jackson, A.J., Kerr, A.K. and for salmon. II. Preliminary 40: 2!S3-291.
Bullock, growth
A.M., 1984. Fish silage as a dietary ingredient findings and nutritional pathology. Aquaculture,
Sprat silage with and without the antioxidant ethoxyquin was stored at 10 and 20°C for 8 weeks to produce four silages of different quality (10, l.O+E, 20 and 20+E). The lipid in the silages without ethoxyquin contained high levels of hydroperoxides and secondary breakdown products. The silages were mixed 1 : 1 with a binder meal and fed to salmon (mean weight 425 g) held in seawater cages. A commercial dry pellet was used for comparison. The five diets were fed for 16 weeks during which time the water temperature increased from 5.4”C to 12.8”C. Growth, food consumption and food conversion ratios were calculated. There were no significant differences in the final weights of the fish fed any of the diets, although the commercial diet was consumed least and gave the slowest growth rate while the water temperature was below 10°C. The most rancid silage (20) proved to be very palatable and produced the most rapid growth during the early part of the trial although growth was reduced latterly. At the end of the trial histological examination of 21 different. tissues from four fish in each group showed few differences. However, the fish fed the silages without ethoxyquin showed consistent changes in the morphological appearance and distribution of the eosinophilic granule cells. The nature and possible reasons for these changes are discussed.
INTRODUCTION
The use of fish meal as a major ingredient in commercial fish diets is still a common practice despite extensive research into the possibility of replacing it with cheaper plant or single-cell proteins. Fish meal production is, however, both capital and energy intensive (60---70 kg fuel/tonne of raw fish, Windsor and Barlow, 1981), mainly due to the necessity of separating the raw material into the three fractions of oil, protein and water. In contrast the manufacture of commercial fish diets often requires the recombining of fish protein with fish oil. The use of whole fish is therefore
0044-8486/84/$03.00
0 1984
Elsevier
Science
Publishers
B.V.
284
still widely used on marine farms despite the problems of continuity in supply and the expense of frozen storage. Fish silage, whose production from whole-fish or fishery wastes is neither capital nor energy intensive, possesses good storage characteristics if correctly treated (Jackson et al., 1984). Moreover, it is widely used as a dietary ingredient for poultry and pigs (see review, Raa and Gildberg, 1982). While the potential for including it in fish diets has been recognised (Asgard and Austreng, 1981), few formal studies have been conducted to assess its suitability as a dietary ingredient. Growth trials were therefore conducted using the sprat silages already described in a previous paper (Jackson et al., 1984) and combining them into moist diets for salmon. In addition to growth performance particular attention was paid to the nutritional pathology of the fish following the report by Asgard (1981) that salmon fed silage diets showed small internal inflammations. MATERIALS
AND METHODS
Chemical analyses All analyses were conducted
according
to Jackson
et al. (1982).
Diets The four sprat silages used have already been described (Jackson et al., 1984) and were 8 weeks old at the start of the feeding trial. Two of the silages had been stored at lO”C, one without and one with the antioxidant ethoxyquin (10 and lO+E), and two had been stored at 20°C one without and one with ethoxyquin (20 and 20+E). During the trial all the silages were stored at ambient room temperature (8--14°C). The silages were mixed with an equal weight of binder meal (Table I) and as the alginate started to gel they were passed through a worm-driven TABLE
I
The composition
of the binder
meal
Ingredient
g/100
Herring meal Pre-pressed soya meal Blood meal Alginate based bindera Vitamin and mineral mixb
42 25 25 4 4
g
aProtan A/S (Norway) AG66, mixture of sodium alginate and sodium phosphate. bVitamin mix according to Tacon et al. (1983); mineral premix No. 5 according National Academy of Sciences (1973).
to
285
extrude]. fitted with a 7 mm die. The resulting pellets which were stored ready for feeding. 3 to 4 clays. A commercially available salmon used as a comparative control. The proximate are shown in Table II. TABLE
lengths broke to form discrete Fresh pellets were made every diet (BP Nutrition, U.K.) was compositions of the five diets
II:
The results of the proximate analyses of the five diets (g/100 g). Protein, lipid and ash are given on a dry weight basis. Gross energy values (kcaI/lOO g) were calculated from the dietary composition Diet
Moisture Nx 6.25 Lipid Ash Gross energy
10
lO+E
20
20+E
Comm.
36.3 60.7 15.2 8.6 549.4
36.0 60.6 15.1 8.4 549.5
32.3 59.7 16.6 9.3 552.5
31.9 59.6 16.4 9.2 551.7
9.8 51.4 17.6 9.1 545.6
Fish
In March 1982 100 Atlantic salmon Salmo salar (mean weight 424.8 g) which had been on-grown in the sea for 9 months were distributed between five nylon net cages (3.4 m3) with 20 fish in each. The cages were moored in Dunstaffnage Bay where the salinity ranged from 21-28 p.p.t. The tempera14
1
4
Fig. 1. Weekly
8 Time (weeks]
seawater
12
temperatures
16
(“C)
taken during the feeding
trial.
ture variation during the trial is shown in Fig. 1; note the increasing temperature during the trial. The fish were fed once a day to satiation as judged by the majority of the fish ceasing to rise to the slow-sinking pellets. The food containers were weighed after each feed so that daily consumptions could be calculated. Every 4 weeks the salmon were anaesthetised with benzocaine and individually weighed. Food conversion ratios (FCR) could then be calculated for each group of fish (Jackson et al., 1982). During the trial there were three deaths; two from the cage on the control diet and one from the cage on the 10°C silage diet. In each instance the cause of death was not clear but did not appear to be diet related. Nutritional
pathology
For histological studies four fish were sampled from each group within the trial. Because of variable growth rates within each group the fish were selected in order to represent a range of sizes. The following tissues were sampled: head, body and fin skin, gill, red and white muscle, heart, spleen, kidney (anterior and posterior), liver, thymus, gall bladder, pancreas, gonad, brain, spinal cord, naris, eye and the alimentary tract including stomach and pyloric caecae. All tissues were fixed for 48 h in 10% buffered formalin (Drury and Wallington, 1967) prior to histological processing. Sections were cut at 6 I.trn and the following staining techniques applied: Haematoxylin and Eosin, Mallory’s PTAH, and Masson’s trichrome stain. In order to avoid bias, histological examination of the tissues was conducted by one of us without prior knowledge of the composition of each diet being fed. Each of the five groups was identified by the prefix A-E and only after microscopical examination was complete were dietary data compared (A - commercial diet; B - 10; C -- lO+E; D - 20; E - 20+E). RESULTS
The four silages produced satisfactory pellets following mixing and mincing with the binder meal. The pellets were water stable for at least 5 min which was adequate when being fed to salmon in cages. If the diets were stored at room temperature (approximately 15°C) no fungal or bacterial spoilage was noted for at least 2 weeks. Fresh pellets were, however, made at least twice a week and stored at 4” C. All the diets proved palatable to the salmon and, despite the initial low water temperatures, reasonable weight gains were maintained (Fig. 2). There were no significant differences between the weight gains of the fish on the different treatments, although the commercial diet gave the slowest growth for the first 2 months. The rancid nature of the 20°C silage with no ethoxyquin did not appear to have any detrimental effect on growth. The dry weight intake of food is shown in Fig. 3. Apart from a general increase in the amount of food eaten as the water temperature increased and
287
as the f:.sh grew larger, no obvious differences between groups were observed. The commercial diet was initially consumed least, but the feeding gradually improved and when the water temperature had reached 10°C the fish were eating a comparable amount when compared to fish ingesting the other diets. The diet containing the 20°C silage proved very palatable at the start, but as the tria: continued the amount taken decreased in relation to the others and during the final period it was the least consumed. The food conversion ratios calculated on a dry weight basis (dry weight of food to wet weight of fish) showed no differences with time or between treatments and ranged from 1.55 to 1.74.
-
comm
-
10
- lO+E - 20 - i'O+E
T/me (weeks)
Fig. 2. Mean weight increase (total increase the five different diets for 16 weeks.
in weight
g/number
of fish) of the salmon
fed
After 16 weeks samples of the various tissues were taken for histological examination. Gross observations showed a lack of muscular pigmentation in the fish fed the diets containing ethoxyquin-free silages. By contrast, the fish fed the diets containing the ethoxyquin protected silage had strongly pigmen ted flesh. The most prominent and constant finding throughout the histological investigation was the variation in distribution amd morphological appearance of these cells is not of the eosinophilic granule cells. The presence uncommon within a variety of fish tissues; they are, however, most commonly located in the peripheral tissue of the digestive tract. In the control group (A) on the commercial diet their distribution and appearance were considered to be similar to those observed in any farmed
288
salmonid. Within the oesophagus of the control group considerable numbers were present in the loose connective tissue of the lamina propria and the submucosa. In the stomach, few were observed within the connective tissue surrounding the epithelium, but a greater concentration in the interstices of the collagenous network associated with both the internal and external surfaces of the stratum compactum was noted. Throughout the pyloric caeca, intestine and rectum the majority of eosinophilic granule cells were contained within the external surface of the stratum compactum (stratum granulosum). Within this zone they were often composed of a layer three to four cells in depth. The internal layer of the stratum compactum and the subepithelial layers of the propria contained significantly fewer cells. 120
1
20 L
4 Time
Fig. 3. Mean dietary
12
8 (weeks1
intake
(total
dry weight
16
intake
g/number
of fish),
Comparative histological analysis of groups B, C, D and E revealed differences in regional distribution and morphological appearance of the granule cells. This observation was most pronounced within group D (20” C) where a marked alteration in their morphology was evident throughout the relevant areas of the digestive tract; the cells often exhibiting swelling and vacuolation. Degranulation was not an uncommon feature. Due to the unevenness of their distribution in normal tissue a quantitative analysis of the numbers within each group in relation to normal tissue was not possible. However, by selective staining (Mallory’s trichrome) a subjective assessment indicated that within groups D, B, C and E (20,10, lO+E, 20+E) respectively, there was a decreasing tendency towards swelling and degranulation. In group D, and to a lesser extent in group B, granule cell swelling and degranulation was not limited to the digestive tract. In two fish within group
289
D and one fish within group B a proliferation and degranulation of the cells surrounding the optic nerve was noted in association with a localised inflammatory response. A similar cell response in the interstices of the atria1 muscle in the same fish was also evident. Cellular changes were not limited to the eosinophilic granule cells. A prominent feature also, principally within group D and to a lesser extent in group B both fed diets without ethoxyquin, was a progressive degeneration and vacuolation of the supportive cells of the gut epithelium. This finding was constant within group D and thus appeared to represent a comcomitant and progressive necrotic response within the digestive tract directly related to the severity of granule cell degranulation. Only minimal changes in the digestive tract histology were noted in groups C and E (both with ethoxyq.uin) when compared to control group A. Apart from occasional hepatocyte vacuolation within the livers of all groups, a common feature of farmed fish, no other histopathological changes were observed in the range of tissues examined. DISCUSSION
The silages proved to be acceptable ingredients in salmon diets despite their acidic nature. Indeed there was some evidence to suggest that at water temperatures below 10°C the moist silage pellets were more palatable than the dry commercial pellet. This finding is in agreement with the observations of Norwegian salmon farmers that moist food produces a better growth rate than dry food during the winter months (Edwards, 1978; Bull-Berg, 1981). The rancid silage (20) did not initially inhibit food intake, and may have proved to be an attractant for the first 6 weeks of the trial since it then produced the most vigorous feeding response and highest food intake. The growth of the salmon would indicate that none of the diets was nutritionally limiting which agrees with the biochemical analyses of the silages showing adequate levels of essential amino acids and fatty acids (Jackson et al., 1984). No growth inhibition was apparent from feeding diets high in hydroperoxides and secondary breakdown products. Fatty acid oxidatic’n products have been shown to be toxic when fed to carp (Wantan.abe and Hashimoto, 1968) and channel catfish (Murai and Andrews, 1974) elthough, the most acute effects were only observed when a-tocopherol was absent. In the trials reported here ol-tocopherol acetate was added to all the diets at 400 mg/kg. Rungruangsak and Utne (1981) reported decreasing growth rates of rainbow trcut when they progressively replaced minced-fish with silage. However, ncl mention was made of the pellet quality and the diets containing 97% silsge bound with 1% guar gum must have had a very poor consistency. At a 5Ci% inclusion level there was no significant difference in the growth rate when compared to the control diet which agrees with the results reported here. Crampton et al. (1982) found salmon in sea cages had signifi-
290
cantly faster growth rates when fed a 25% silage diet as compared to feeding them a commercial dry pellet. The major histopathological finding, namely the swelling, vacuolation and degranulation of the eosinophilic granule cells, appears to be related to the absence of the antioxidant in the original silage and the consequent formation of fatty acid oxidation products. The exact function or functions of this cell type are not as yet clear although Baldo and Fletcher (1975) have reported localised degranulation of this cell type in plaice skin undergoing a hypersensitivity response. Ellis (1982) found a similar degranulation of these cells in rainbow trout intestine, coincidental with a decrease in the histamine content of the tissue following intraperitoneal injection of furunculosis toxin. Degranulation of EGC’s is not a common phenomenon in cells under normal physiological conditions in healthy fish (Bergeron and Woodward, 1983) and the results obtained from the control group fed the commercial diet confirms this. The evidence would therefore indicate that some dietary factor present in the rancid silages was causing a cellular reaction which has been associated with the immune response of fish. From these experiments the exact nature of the toxic factor is not evident, although Wantanabe and Hashimoto (1968) reported that there were several toxic factors in oxidised saury oil which could cause muscular degeneration when fed to carp. The progressive degeneration of the columnar epithelial cells noted in the gut of the antioxidant deficient groups provides further evidence of a generalised initiation of cell necrosis within the gut. Ezeanor and Stokoe (1980) postulated that the gut wall of rainbow trout constitutes a composite defence mechanism, both humoral and mechanical, which develops in response to environmental demands, a hypothesis in agreement with the results of this study. Fish silage proved to be an acceptable dietary ingredient for salmon. Diets produced by mixing it in equal quantities with a binder meal proved palatable and promoted good growth when compared to a commercial dry diet. The presence of fatty acid oxidation products in the diets resulted in cellular changes which are often associated with the immune response in fish. Long-term studies need to be conducted to ascertain whether these oxidation products can cause serious clinical problems in fish. Although the silage based diets did not appear to have had any effect on the fish muscle, it is important in the future to check the muscle texture and flavour following long term feeding of such diets because of the importance of these factors on the eventual sale of the salmon. ACKNOWLEDGEMENTS
The authors wish to acknowledge the financial assistance provided by the Highlands and Islands Development Board and the Ministry of Agriculture and Fisheries. Much valuable technical assistance was provided by Mrs. Sheila Phillips.
291 REFERENCES Asgard, T., 1981. Acid preservation could make offals a source of income. Fish Farmer, 4 (2): !)-11. Asgard, T. and Austreng, E., 1981. Fish silage for salmonids: a cheap way of utilising waste as feed. Feedstuffs, 53 (27): 22--24. Baldo, B.A. and Fletcher, T.C., 1975. Phylogenetic aspects of hypersensitivity reactions in flatfish. In: W.H. Hildeman and A.A. Benedict (Editors), Immunologic Phylogeny. Plenum Press, London, pp. 365-372. Bergeron, T. and Woodward, B., 1983. Ultrastructure of the granule cells in the small intestine of the rainbow trout before and after stratum granulosum formation. Can. J. Zool., 61: 133-138. Bull-Berg, L., 1981. The development of fish farming in Norway. In: Proc. of the S.M.B.A.IH.1.D.B. Fish Farming Meeting, Oban, Feb. 1981. S.M.B.A., Oban, pp. l-17. Crampton, V., Bromage, N. and Watret, R., 1982. Moist feeds for salmon. Fish Farmer, 5 (8): :.l. Drury, R.A.B. and Wallington, E.A., 1967. Carleton’s Histological Technique. Oxford University Press, London, 432 pp. Edwards, D.J., 1978. Salmon and Trout Farming in Norway. Fishing News Books, Farnham, 195 pp. Ellis, A.E ., 1982. Differences between the immune mechanisms of fish and higher vertebrates. In: R.J. Roberts (Editor), Microbial Diseases of Fish. Academic Press, London, pp. l-29. Ezeanor, D.N. and Stokoe, W.M., 1980. A cytochemical light and electron microscopic study Iof the eosinophilic granule cells in the gut of the rainbow trout. J. Fish Biol., 17 : 61!3%634. Jackson, .4.J., Capper, B.S. and Matty A.J., 1982. Evaluation of some plant proteins in complete diets for the tilapia Sarotherodon mossambicus. Aquaculture, 27: 97-109. Jackson, .4.J., Kerr, A.K. and Cowey, C.B., 1984. Fish silage as a dietary ingredient for salmon. I. Nutritional and storage characteristics. Aquaculture, 38: 211~-220. Murai, T. and Andrews, J.W., 1974. Interactions of dietary a-tocopherol, oxidised menhaden oil and ethoxyquin on channel catfish. J. Nutr., 104: 1416-1431. National Academy of Sciences, 1973. Nutrient requirements of trout salmon and channel catfish. Nutrient Requirements of Domestic Animals No. 11. Natl. Acad. Sci., Washington, DC, 57 pp. Raa, J. and Gildberg, A., 1982. Fish silage: A review. CRC Critical Rev. Food Sci. Nutr., April: 383-419. Rungruansak, K. and Utne, F., 1981. Effect of different acidified wet feeds on protease activitilss in the digestive tract and on the growth rate of rainbow trout. Aquaculture, 22: 67.-79. Tacon, A.G.J., Stafford, E.A. and Edwards, C.A., 1983. A preliminary investigation of nutriti1.e value of three terrestrial lumbricid worms for rainbow trout. Aquaculture, 35: 18’7-199. Watanabe, T. and Hashimoto, Y., 1968. Toxic components of oxidised saury oil inducing muscular dystrophy in carp. Bull. Jpn. Sot. Sci. Fish., 34: 1131-1140. Windsor, M. and Barlow, S., 1981. Introduction to Fishery By-Products. Fishing News Books, Farnham, 187 pp.
283 Amsterdam
-
Printed
in The Netherlands
FISH S’[LAGE AS A DIETARY INGREDIENT FOR SALMON. II. PRELIMINARY GROWTH FINDINGS AND NUTRITIONAL PATHC’LOGY
A.J.
JACKSON,
Dunstaffnage (Accepted
A.K. Marine
20 March
KERR
and A.M.
Research
BULLOCK
Laboratory,
P.O. Box
3, Oban,
Argyll
(Great
Britain)
1984)
ABSTRACT Jackson, A.J., Kerr, A.K. and for salmon. II. Preliminary 40: 2!S3-291.
Bullock, growth
A.M., 1984. Fish silage as a dietary ingredient findings and nutritional pathology. Aquaculture,
Sprat silage with and without the antioxidant ethoxyquin was stored at 10 and 20°C for 8 weeks to produce four silages of different quality (10, l.O+E, 20 and 20+E). The lipid in the silages without ethoxyquin contained high levels of hydroperoxides and secondary breakdown products. The silages were mixed 1 : 1 with a binder meal and fed to salmon (mean weight 425 g) held in seawater cages. A commercial dry pellet was used for comparison. The five diets were fed for 16 weeks during which time the water temperature increased from 5.4”C to 12.8”C. Growth, food consumption and food conversion ratios were calculated. There were no significant differences in the final weights of the fish fed any of the diets, although the commercial diet was consumed least and gave the slowest growth rate while the water temperature was below 10°C. The most rancid silage (20) proved to be very palatable and produced the most rapid growth during the early part of the trial although growth was reduced latterly. At the end of the trial histological examination of 21 different. tissues from four fish in each group showed few differences. However, the fish fed the silages without ethoxyquin showed consistent changes in the morphological appearance and distribution of the eosinophilic granule cells. The nature and possible reasons for these changes are discussed.
INTRODUCTION
The use of fish meal as a major ingredient in commercial fish diets is still a common practice despite extensive research into the possibility of replacing it with cheaper plant or single-cell proteins. Fish meal production is, however, both capital and energy intensive (60---70 kg fuel/tonne of raw fish, Windsor and Barlow, 1981), mainly due to the necessity of separating the raw material into the three fractions of oil, protein and water. In contrast the manufacture of commercial fish diets often requires the recombining of fish protein with fish oil. The use of whole fish is therefore
0044-8486/84/$03.00
0 1984
Elsevier
Science
Publishers
B.V.
284
still widely used on marine farms despite the problems of continuity in supply and the expense of frozen storage. Fish silage, whose production from whole-fish or fishery wastes is neither capital nor energy intensive, possesses good storage characteristics if correctly treated (Jackson et al., 1984). Moreover, it is widely used as a dietary ingredient for poultry and pigs (see review, Raa and Gildberg, 1982). While the potential for including it in fish diets has been recognised (Asgard and Austreng, 1981), few formal studies have been conducted to assess its suitability as a dietary ingredient. Growth trials were therefore conducted using the sprat silages already described in a previous paper (Jackson et al., 1984) and combining them into moist diets for salmon. In addition to growth performance particular attention was paid to the nutritional pathology of the fish following the report by Asgard (1981) that salmon fed silage diets showed small internal inflammations. MATERIALS
AND METHODS
Chemical analyses All analyses were conducted
according
to Jackson
et al. (1982).
Diets The four sprat silages used have already been described (Jackson et al., 1984) and were 8 weeks old at the start of the feeding trial. Two of the silages had been stored at lO”C, one without and one with the antioxidant ethoxyquin (10 and lO+E), and two had been stored at 20°C one without and one with ethoxyquin (20 and 20+E). During the trial all the silages were stored at ambient room temperature (8--14°C). The silages were mixed with an equal weight of binder meal (Table I) and as the alginate started to gel they were passed through a worm-driven TABLE
I
The composition
of the binder
meal
Ingredient
g/100
Herring meal Pre-pressed soya meal Blood meal Alginate based bindera Vitamin and mineral mixb
42 25 25 4 4
g
aProtan A/S (Norway) AG66, mixture of sodium alginate and sodium phosphate. bVitamin mix according to Tacon et al. (1983); mineral premix No. 5 according National Academy of Sciences (1973).
to
285
extrude]. fitted with a 7 mm die. The resulting pellets which were stored ready for feeding. 3 to 4 clays. A commercially available salmon used as a comparative control. The proximate are shown in Table II. TABLE
lengths broke to form discrete Fresh pellets were made every diet (BP Nutrition, U.K.) was compositions of the five diets
II:
The results of the proximate analyses of the five diets (g/100 g). Protein, lipid and ash are given on a dry weight basis. Gross energy values (kcaI/lOO g) were calculated from the dietary composition Diet
Moisture Nx 6.25 Lipid Ash Gross energy
10
lO+E
20
20+E
Comm.
36.3 60.7 15.2 8.6 549.4
36.0 60.6 15.1 8.4 549.5
32.3 59.7 16.6 9.3 552.5
31.9 59.6 16.4 9.2 551.7
9.8 51.4 17.6 9.1 545.6
Fish
In March 1982 100 Atlantic salmon Salmo salar (mean weight 424.8 g) which had been on-grown in the sea for 9 months were distributed between five nylon net cages (3.4 m3) with 20 fish in each. The cages were moored in Dunstaffnage Bay where the salinity ranged from 21-28 p.p.t. The tempera14
1
4
Fig. 1. Weekly
8 Time (weeks]
seawater
12
temperatures
16
(“C)
taken during the feeding
trial.
ture variation during the trial is shown in Fig. 1; note the increasing temperature during the trial. The fish were fed once a day to satiation as judged by the majority of the fish ceasing to rise to the slow-sinking pellets. The food containers were weighed after each feed so that daily consumptions could be calculated. Every 4 weeks the salmon were anaesthetised with benzocaine and individually weighed. Food conversion ratios (FCR) could then be calculated for each group of fish (Jackson et al., 1982). During the trial there were three deaths; two from the cage on the control diet and one from the cage on the 10°C silage diet. In each instance the cause of death was not clear but did not appear to be diet related. Nutritional
pathology
For histological studies four fish were sampled from each group within the trial. Because of variable growth rates within each group the fish were selected in order to represent a range of sizes. The following tissues were sampled: head, body and fin skin, gill, red and white muscle, heart, spleen, kidney (anterior and posterior), liver, thymus, gall bladder, pancreas, gonad, brain, spinal cord, naris, eye and the alimentary tract including stomach and pyloric caecae. All tissues were fixed for 48 h in 10% buffered formalin (Drury and Wallington, 1967) prior to histological processing. Sections were cut at 6 I.trn and the following staining techniques applied: Haematoxylin and Eosin, Mallory’s PTAH, and Masson’s trichrome stain. In order to avoid bias, histological examination of the tissues was conducted by one of us without prior knowledge of the composition of each diet being fed. Each of the five groups was identified by the prefix A-E and only after microscopical examination was complete were dietary data compared (A - commercial diet; B - 10; C -- lO+E; D - 20; E - 20+E). RESULTS
The four silages produced satisfactory pellets following mixing and mincing with the binder meal. The pellets were water stable for at least 5 min which was adequate when being fed to salmon in cages. If the diets were stored at room temperature (approximately 15°C) no fungal or bacterial spoilage was noted for at least 2 weeks. Fresh pellets were, however, made at least twice a week and stored at 4” C. All the diets proved palatable to the salmon and, despite the initial low water temperatures, reasonable weight gains were maintained (Fig. 2). There were no significant differences between the weight gains of the fish on the different treatments, although the commercial diet gave the slowest growth for the first 2 months. The rancid nature of the 20°C silage with no ethoxyquin did not appear to have any detrimental effect on growth. The dry weight intake of food is shown in Fig. 3. Apart from a general increase in the amount of food eaten as the water temperature increased and
287
as the f:.sh grew larger, no obvious differences between groups were observed. The commercial diet was initially consumed least, but the feeding gradually improved and when the water temperature had reached 10°C the fish were eating a comparable amount when compared to fish ingesting the other diets. The diet containing the 20°C silage proved very palatable at the start, but as the tria: continued the amount taken decreased in relation to the others and during the final period it was the least consumed. The food conversion ratios calculated on a dry weight basis (dry weight of food to wet weight of fish) showed no differences with time or between treatments and ranged from 1.55 to 1.74.
-
comm
-
10
- lO+E - 20 - i'O+E
T/me (weeks)
Fig. 2. Mean weight increase (total increase the five different diets for 16 weeks.
in weight
g/number
of fish) of the salmon
fed
After 16 weeks samples of the various tissues were taken for histological examination. Gross observations showed a lack of muscular pigmentation in the fish fed the diets containing ethoxyquin-free silages. By contrast, the fish fed the diets containing the ethoxyquin protected silage had strongly pigmen ted flesh. The most prominent and constant finding throughout the histological investigation was the variation in distribution amd morphological appearance of these cells is not of the eosinophilic granule cells. The presence uncommon within a variety of fish tissues; they are, however, most commonly located in the peripheral tissue of the digestive tract. In the control group (A) on the commercial diet their distribution and appearance were considered to be similar to those observed in any farmed
288
salmonid. Within the oesophagus of the control group considerable numbers were present in the loose connective tissue of the lamina propria and the submucosa. In the stomach, few were observed within the connective tissue surrounding the epithelium, but a greater concentration in the interstices of the collagenous network associated with both the internal and external surfaces of the stratum compactum was noted. Throughout the pyloric caeca, intestine and rectum the majority of eosinophilic granule cells were contained within the external surface of the stratum compactum (stratum granulosum). Within this zone they were often composed of a layer three to four cells in depth. The internal layer of the stratum compactum and the subepithelial layers of the propria contained significantly fewer cells. 120
1
20 L
4 Time
Fig. 3. Mean dietary
12
8 (weeks1
intake
(total
dry weight
16
intake
g/number
of fish),
Comparative histological analysis of groups B, C, D and E revealed differences in regional distribution and morphological appearance of the granule cells. This observation was most pronounced within group D (20” C) where a marked alteration in their morphology was evident throughout the relevant areas of the digestive tract; the cells often exhibiting swelling and vacuolation. Degranulation was not an uncommon feature. Due to the unevenness of their distribution in normal tissue a quantitative analysis of the numbers within each group in relation to normal tissue was not possible. However, by selective staining (Mallory’s trichrome) a subjective assessment indicated that within groups D, B, C and E (20,10, lO+E, 20+E) respectively, there was a decreasing tendency towards swelling and degranulation. In group D, and to a lesser extent in group B, granule cell swelling and degranulation was not limited to the digestive tract. In two fish within group
289
D and one fish within group B a proliferation and degranulation of the cells surrounding the optic nerve was noted in association with a localised inflammatory response. A similar cell response in the interstices of the atria1 muscle in the same fish was also evident. Cellular changes were not limited to the eosinophilic granule cells. A prominent feature also, principally within group D and to a lesser extent in group B both fed diets without ethoxyquin, was a progressive degeneration and vacuolation of the supportive cells of the gut epithelium. This finding was constant within group D and thus appeared to represent a comcomitant and progressive necrotic response within the digestive tract directly related to the severity of granule cell degranulation. Only minimal changes in the digestive tract histology were noted in groups C and E (both with ethoxyq.uin) when compared to control group A. Apart from occasional hepatocyte vacuolation within the livers of all groups, a common feature of farmed fish, no other histopathological changes were observed in the range of tissues examined. DISCUSSION
The silages proved to be acceptable ingredients in salmon diets despite their acidic nature. Indeed there was some evidence to suggest that at water temperatures below 10°C the moist silage pellets were more palatable than the dry commercial pellet. This finding is in agreement with the observations of Norwegian salmon farmers that moist food produces a better growth rate than dry food during the winter months (Edwards, 1978; Bull-Berg, 1981). The rancid silage (20) did not initially inhibit food intake, and may have proved to be an attractant for the first 6 weeks of the trial since it then produced the most vigorous feeding response and highest food intake. The growth of the salmon would indicate that none of the diets was nutritionally limiting which agrees with the biochemical analyses of the silages showing adequate levels of essential amino acids and fatty acids (Jackson et al., 1984). No growth inhibition was apparent from feeding diets high in hydroperoxides and secondary breakdown products. Fatty acid oxidatic’n products have been shown to be toxic when fed to carp (Wantan.abe and Hashimoto, 1968) and channel catfish (Murai and Andrews, 1974) elthough, the most acute effects were only observed when a-tocopherol was absent. In the trials reported here ol-tocopherol acetate was added to all the diets at 400 mg/kg. Rungruangsak and Utne (1981) reported decreasing growth rates of rainbow trcut when they progressively replaced minced-fish with silage. However, ncl mention was made of the pellet quality and the diets containing 97% silsge bound with 1% guar gum must have had a very poor consistency. At a 5Ci% inclusion level there was no significant difference in the growth rate when compared to the control diet which agrees with the results reported here. Crampton et al. (1982) found salmon in sea cages had signifi-
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cantly faster growth rates when fed a 25% silage diet as compared to feeding them a commercial dry pellet. The major histopathological finding, namely the swelling, vacuolation and degranulation of the eosinophilic granule cells, appears to be related to the absence of the antioxidant in the original silage and the consequent formation of fatty acid oxidation products. The exact function or functions of this cell type are not as yet clear although Baldo and Fletcher (1975) have reported localised degranulation of this cell type in plaice skin undergoing a hypersensitivity response. Ellis (1982) found a similar degranulation of these cells in rainbow trout intestine, coincidental with a decrease in the histamine content of the tissue following intraperitoneal injection of furunculosis toxin. Degranulation of EGC’s is not a common phenomenon in cells under normal physiological conditions in healthy fish (Bergeron and Woodward, 1983) and the results obtained from the control group fed the commercial diet confirms this. The evidence would therefore indicate that some dietary factor present in the rancid silages was causing a cellular reaction which has been associated with the immune response of fish. From these experiments the exact nature of the toxic factor is not evident, although Wantanabe and Hashimoto (1968) reported that there were several toxic factors in oxidised saury oil which could cause muscular degeneration when fed to carp. The progressive degeneration of the columnar epithelial cells noted in the gut of the antioxidant deficient groups provides further evidence of a generalised initiation of cell necrosis within the gut. Ezeanor and Stokoe (1980) postulated that the gut wall of rainbow trout constitutes a composite defence mechanism, both humoral and mechanical, which develops in response to environmental demands, a hypothesis in agreement with the results of this study. Fish silage proved to be an acceptable dietary ingredient for salmon. Diets produced by mixing it in equal quantities with a binder meal proved palatable and promoted good growth when compared to a commercial dry diet. The presence of fatty acid oxidation products in the diets resulted in cellular changes which are often associated with the immune response in fish. Long-term studies need to be conducted to ascertain whether these oxidation products can cause serious clinical problems in fish. Although the silage based diets did not appear to have had any effect on the fish muscle, it is important in the future to check the muscle texture and flavour following long term feeding of such diets because of the importance of these factors on the eventual sale of the salmon. ACKNOWLEDGEMENTS
The authors wish to acknowledge the financial assistance provided by the Highlands and Islands Development Board and the Ministry of Agriculture and Fisheries. Much valuable technical assistance was provided by Mrs. Sheila Phillips.
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