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Performance of broiler chicks fed on low and high oil fish silages in relation to changes taking place in lipid and protein components
Animal Feed Srience and Technology, 28 (1990) 199-223
199
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
P e r f o r m a n c e of Broiler Chicks Fed on L o w and High Oil Fish Silages in Relation to Changes T a k i n g Place in Lipid and Protein Components D.H. MACHIN 1'*, S. PANIGRAHI 1, JULIET BAINTON 1and T.R. MORRIS 2
1Overseas Development and Natural Resource Institute, Central Avenue, Chatham ME4 4TB (Gt. Britain) 2Department o/Agriculture and Horticulture, University o/ Reading, Earley Gate, Reading RG6 2AT (Gt. Britain) (Received 3 August, 1988; accepted for publication 8 August 1989)
ABSTRACT Machin, D.H., Panigrahi, S., Bainton, J. and Morris, T.R., 1990. Performance of broiler chicks fed on low and high oil fish silages in relation to changes taking place in lipid and protein components. Anita. Feed Sci. Technol., 28: 199-223. Changes in the protein and lipid components of fish silages made from low oil content fish (LOF; whiting, Gadus merlinger) and high oil content fish (HOF; mackerel, Scromber scombus) were studied during ensiling, drying and subsequent storage. Chicks were fed on diets containing the silages dried on to cassava meal, or fish meal produced from the same batch offish. The HOF silages had > 50% of total N in the form of non-protein N, compared with 18% in the LOF silages; both levels were higher than in corresponding fish meals. Cystine levels in all silages were lower than in the original fish material, the greatest reduction occurring in HOF silage. Tryptophan levels were lower in all processed materials, but the most marked reductions were in LOF silages. Levels of lysine and methionine fell equally in all processed fish materials. The levels of histamine remained low in all fish products. Marked changes occurred in the fatty acid levels of the ensiled fish materials, especially in the HOF silage. The changes were mostly associated with losses of unsaturated fatty acids, in particular C18-3, C18-4, C20-3, C20-4, C20-5, C22-5 and C22-6. Malonaldehyde levels indicated that marked oxidation of the lipid occurred in the HOF silage, this occurred to a much lower extent in LOF silage and to an even lesser extent in fish meals. Addition of antioxidant did not appear to affect these changes. Weight gains of chicks fed on balanced diets containing 4.7 or 9.4% crude protein from HOF silage (9.8 and 19.6% of the dietary dry matter) and 5.2 or 10.4% crude protein from LOF silage (6.8 and 13.5% of the dietary dry matter) were 99, 85 and 98 and 91%, respectively, of the gains achieved with corresponding fish meals. Food conversion ratios for the above dietary treatments were respectively, 97, 103, 101 and 99% of those of chicks fed on fish meals. It was concluded that the differences in chick performance reflected the changes that occurred in the lipid and protein components and the interactions between these components. *Present address: Food and Agriculture Organization of the United Nations, Via delle Terme di Caracalla, 00100 Rome, Italy.
037%8401/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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D.H~ MACHIN ET AL.
INTRODUCTION
Ensiling has been used for many years to preserve waste fish. Material processed in this way has been shown to be a good feed for pigs (Green, 1984). However, feeding trials with chicks to evaluate fish silage dried on to starchy fillers have given variable results. Where fish silage formed up to 10% of the dietary dry matter, broiler performance was mostly similar to or better than controls (Rattagool et al., 1980a-c ). In contrast, though Poulter et al. (1980) included up to 46% fish silage in the diet without impairing broiler performance, the performance of broilers given more than 10% fish silage has generally been inferior to that of controls (Disney et al., 1978; Kompiang et al., 1979, 1980; Hall, 1983 ). In most broiler experiments, little attention has been given to balancing nutritional content, except for crude protein, or occasionally specific nutrients such as tryptophan (Hall, 1983 ), vitamin E (Disney et al., 1978), or thiamine (Kompiang et al., 1979 ). It is likely that the poorer performance of broilers fed on fish silages is partly because of inadequate understanding of changes that take place during the ensilage process and subsequent drying, and partly because of failure to formulate diets with adequate levels of known nutrients. During autolysis, about 80% of the protein in fish silages made with formic acid becomes soluble. Many of the amino acids are then exposed to damage by reaction with other components of the silage. Amino acids likely to be affected in this way include tryptophan, tyrosine, lysine, methionine and, if spoilage has taken place, histidine. Silage production and air drying on to dry feeds has been shown to be highly conducive to the breakdown and rapid oxidation of the highly unsaturated fatty acids of fish materials ( Hall, 1983). The nutritional significance of this will be a loss of essential fatty acids and the production of highly reactive breakdown products. Carpenter et al. (1963) suggested that these products could have an adverse effect on palatability, cause protein damage and induce deficiencies through the destruction of vitamins or increased vitamin requirements. This study was therefore carried out to assess: (1) the production and use for feeding of fish silages made from fish of low oil content (whiting, Gadus merlinger) and fish of high oil content (mackerel, Scromber scrombus); (2) high and low levels of silage inclusion in diets; (3 } the fate of the silage fats during silage production, drying and storage and the effect of an antioxidant on such changes; (4) the fate of amino acids during ensilage and the effects of this and subsequent amino-acid supplementation on performance of chicks; (5) the comparison of fish materials made into fish meal or into fish silage.
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
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MATERIALSAND METHODS
Production of dried meals containing fish silages Fresh whole whiting and mackerel with guts intact were obtained direct from the UK ports of Torbay and Hull, respectively. All fish were blast frozen in blocks of 20 kg on landing and consigned directly to the laboratory in transport maintained at - 2 0 ° C . The fish were maintained at this temperature for a further 4 days, then thawed overnight. At the time of processing, loose ice was still located between individual fish and fish were still part frozen. The fish were minced through a KS Mince-master (R) (13.5 kW) using a 6-mm plate and then through the same machine using a 4-mm plate. Formic acid (85%) was added to the fish as it entered the mincer on the second pass, to give a final concentration of 3% by weight of fish. Sixty kilograms of each fish type was then placed in polythene bins and remixed manually. The fish and acid mixtures were then maintained at a temperature of 23 ° C until dried; liquefaction took place after 3 days. Butylated hydroxy toluene (BHT) dissolved in a solution of methanol and water was added as an antioxidant to one aliquot of each fish silage type at 200 mg B H T kg-1 of the fat content. The antioxidant was added to the minced fish with the formic acid to prevent oxidation occurring from inception. The wet silages (60 kg) were mixed with dry ground cassava meal (30 kg) 7 days after liquefaction. These proportions had been shown by an initial study to be the minimum necessary for the production of meals that could be easily dried. The mixing was begun manually in polythene bins with a wooden paddle and completed with an electrically powered concrete mixer. The resultant mixtures were spread on drying trays to a depth of 2.5 cm in a forced-air oven at 50°C, turned daily, and dried for 3 days. All lumps of material were crumbled by hand during turning. After drying, the materials were ground through the 4-mm screen of a hammer mill and stored at - 20 ° C in air-tight plastic sacks until used.
Production offish meals Aliquots (40 kg) of each type of fish were processed as described for the fish silages except that no formic acid was added. The minced fish was then placed in steam-heated kettles and brought to 100°C over 75 min. The fish was maintained at this temperature for 15 min, then mixed with 20 kg of cassava meal and dried, ground and stored as described for fish silage meals.
Chemical analyses Samples of fresh minced fish of each type and all dried meals were analysed for the following: (1) crude protein, ether extract, crude fibre, ash, calcium,
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D.H. MACHINET AL.
phosphorus and chloride expressed as salt equivalent, using methods given in the UK fertilisers and feedingstuffs (amendment) regulations (Ministry of Agriculture, Fisheries and Food, 1976); (2) non-protein nitrogen ( N P N ) by the method described by Backoff (1976); (3) histamine by the method of Hardy and Smith (1976); (4) fatty acid composition by the method of Hofstetter et al. ( 1965 ); (5) formic acid by the procedure of Canale et al. ( 1984 ); (6) amino acids by using a Biotronik LC5000 analyser. Hydrolysis of the samples was carried out by open flask refiuxing to 6 M HC1 for 24 h. Tryptophan was determined by the method of Lucas and Sotelo (1980) and available lysine by the method of Roach et al. (1967).
Monitoring of lipid oxidation The state of oxidation of the fish lipid component was monitored from receipt of the fresh fish through to consumption of the processed meals. MoniTABLE 1
Experimental 'incomplete block design' Treatments
Period 1
Period 2
Large
Small
Large
X X X X X
X X X X X
X X X X
F2, O2, LbAI, S2
X
F2, 02, L:, A2, S 1 F2, 02, L : , A : , S 1 F1, O2, L2, A2, S2 F:, 02, L1, A2, S 2 F2, O1, L2, A2, S2 F2, O1, L2, A1, S2 F2, 01, L2, A2, S1 F2, Ol, L2, Al, Sl F2, 01, L1, A2, S2 F2, O:,L1, A1, S2 F2, O1, L1, A2, Sl F2, O:,L1, A1, S1 FI, OI, L2, A2,$2 FI, O:,L:,A2, S2
X X X X X X X X X X
F2, 02, L2, A2, S2 F2,02, L2, A:,S2 F2,02, L2, A~,S: F2, O2, L2, A1, S 1 F2, 02, LI, A2,$2
Period 3 Small
Large
Small
X
X
X X X X
X
X
X
X X X X -
X X X X X X X X X X X X X X
X X X X X X -
X X X X X X
X X X X X X X X X X X X
X X X X X X
X X
Abbreviations: 01 = low oil fish; 02= high oil fish; L~ = low level inclusion; L2=high level inclusion; F1 -- fish meal; F2 = fish silage; A: = without antioxidant; A2 = with antioxidant; $1 -- without amino acid supplementation; $2 = with amino acid supplementation. S m a l l = s m a l l cage size (4 chicks); Large=large cage size (8 chicks).
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
203
toring was based on the level of malonaldehyde, an oxidative breakdown product of lipid, in the fish lipid, determined by the technique described by Hall (1983) using 2°thiobarbituric acid (TBA). Malonaldehyde was determined in the fresh fish, wet ensiled and wet cooked fish, and the dried products throughout the storage and feeding period. Owing to difficulties in obtaining the whiting and mackerel at the same time the malonaldehyde determinations for the former were carried out over 203 days and the latter over 294 days, from receipt of fresh fish. It was also not possible to carry out determinations on exactly equivalent days though attempts were made to match these as far as possible. The times of malonaldehyde determinations are given in Table 10.
Experimental design To evaluate the effects of high and low oil fish silages at high and low levels of inclusion, treated with and without antioxidants, in diets supplemented or not supplemented with the limiting amino acids, tryptophan, lysine and methionine, the experimental incomplete block design shown in Table 1 was prepared. Complete blocking did not extend to fish meals, but involved giving the 20 experimental diets to groups of broiler chicks (four replicates per treatment) over three experimental periods. All combinations of fish type, antioxidant treatment, amino-acid supplementation and level of inclusion were produced in the design and the effects compared with fish meals which were given at both levels of inclusion and were treated with antioxidant.
Formulation and preparation of diets Details of diet formulation, and the calculated and determined composition are shown in Tables 2-5. The dried cassava acid fish silages or fish meal mixtures were prepared so that 22.5 and 45% of the mixtures would provide approximately 5 and 10% fish protein in the broiler diets, respectively. However, in practice, the dried mackerel products in the experimental diets provided 4.7% crude protein (9.8% fish dry matter) in the low inclusion diets and 9.4% crude protein (19.6% fish dry matter) in the high inclusion diets, whilst the dried whiting products provided 5.2% crude protein (6.8% fish dry matter) in the low inclusion diets and 10.4% crude protein (13.5% fish dry matter) in the high inclusion diets. Metabolizable energy values for the dried meals were calculated by using the equations of Lodhi et al. (1976) (see Table 7). The remainder of the diets were formulated by using a linear programme to commercial broiler starter specifications, from maize meal, soya bean meal, maize gluten meal, meat and bone meal, wheatfeed, sunflower meal, molasses, dicalcium phosphate, limestone, salt (NaC1), maize oil and a vitamin-trace mineral premix (Vitamealo, UK ) (Table 2 ). All feeds were in the form of meals.
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D.H. MACHINET AL.
TABLE 2 Formulation of experimental diets (based on fish meals) (%) Raw materials
Maize Soya bean meal Maize gluten meal Meat and bone meal Wheatfeed Sunflower meal Molasses Dicalcium phosphate Limestone Salt (NaCl) Maize oil L-Lysine DL-Methionine Premix 1 High oil fish material (dried on cassava ) Low oil fish material (dried on cassava)
High oil fish materials
Low oil fish materials
Low fish inclusion diet
High fish inclusion diet
Low fish inclusion diet
High fish inclusion diet
34.91 22.96 10.00 4.27
0.56 10.43 10.00
48.51 21.69 6.07 0.11 0.55 0.12 0.15 0.30 -
28.26 14.98 10.00 0.52 0.25 0.09 0.39 0.08 0.13 0.30 -
22.50
45.00
4.00 0.59 0.23 0.06 0.07 0.11 0.30 22.50
16.77 10.00 4.00 0,81 0.94 0.10 0.87 0.10 0.12 0.30 45.00
' P r e m i x (vitamin a n d trace mineral) supplied the following per kilogram of feed: vitamin A, 10 000 iu; vitamin D3, 2500 iu; vitamin E, 10.0 iu; v i t a m i n K, 2.0 mg; vitamin B1, 0.50 mg; vitamin B2, 6.0 mg; nicotinic acid, 20.0 mg; v i t a m i n B6, 1.0 mg; p a n t o t h e n i c acid, 10.0 mg; biotin, 0.08 mg; folic acid, 1.0 mg; vitamin B~2, 5.0 tLg; Mn, 80.0 mg; Zn, 60.0 mg; Cu, 5.0 mg; Se, 0.10 mg; I, 1.0 mg. TABLE 3 Calculated composition of experimental diets Nutrients (g kg -1 )
Crude protein Crude fibre Crude fat Lysine Methionine + cystine Calcium Phosphorus Salt (NaC1) Linoleic acid Metabolizable energy ( M J k g - ' ) Dry matter
High oil fish materials
Low oil fish materials
Low fish inclusion diet
High fish inclusion diet
Low fish inclusion diet
High fish inclusion diet
230 40 66 14 9 9 7 4 10
230 68 108 14 10 10 8 4 10
220 35 37 14 9 9 7 4 15
220 36 30 14 9 9 7 4 10
12.75 903
12.75 907
12.70 897
12.75 901
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
205
TABLE 4
Composition of diets (g kg -~ dry matter) Fish type
Process- Inclus- AntiAmino Moisture Crude Crude Crude Calcium Phosing ion oxidants acids content of protein fibre fat phorus fresh matter (gkg -1)
High Fish
oil
Low oil
High
+ +
+ +
82 83 82 74
234 248 243 247
72 79 81 75
111 143 125 138
12.0 11.7 12.0 12.7
8.3 8.4 8.5 8.3
Low
+ + -
+ + -
100 97 97 95
258 252 248 270
50 50 50 48
66 49 65 69
12.3 11.3 11.1 10.7
8.9 8.2 8.1 7.8
silage
Fish meal
High
+
-
Low
+
-
68 100
248 260
85 46
115 74
11.3 11.3
8.0 8.2
Fish silage
High
+
+ + -
85 86 84 86
243 242 249 239
44 43 38 39
39 39 39 37
10.5 10.1 9.7 9.8
7.4 7.4 7.6 7.7
--
+ + -
104 102 101 97
242 249 255 246
39 37 39 39
49 49 42 43
11.9 10.6 11.6 10.9
7.9 7.7 7.8 7.8
+ +
-
95 106
251 260
42 41
34 41
11.0 11.6
7.4 8.1
+ -
Low
+
+
Fish meal
High
Low
Amino acid supplementation Following amino acid analysis of the dried fish silage and fish meals, diets designated for amino acid supplementation received additions of synthetic lysine, methionine and tryptophan to raise their levels to those of the equivalent fish meals. Synthetic methionine was added to balance total sulphur amino acid (methionine + cystine ) levels. The amino acid supplementation levels are shown in Table 6.
Feeding trial The trial was carried out in a controlled environment experimental house using 32 wire-floored cages, each equipped with its own feeder and drinker.
206
D.H. MACHIN ET AL.
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TABLE 6 Amino-acid supplementation of fish silages 1 (To) High oil fish silage
Low oil fish silage
+Antioxidant -Antioxidant
÷Antioxidant -Antioxidant
1.70 1.70
1.79 1.79
1.75 1.75
1.58 0.11 1.58
Methionine ÷ lysine A Silage content 0.72 B Addition, DL. Methionine 0.27 C Final level2 0.99
0.81 0.18 0.99
0.85 0.02 0.80
0.71 0.16 0.87
Tryptophan A Silage content B Addition, Tryptophan C Final level2
0.23 0.05 0.28
0.17 0.05 0.22
0.14 0.08 0.22
Lysine A Silage content B Addition, L. Lysine C Final level2
0.26 0.02 0.28
1Amino acid addition to silage to equate with fish meal levels. 2Amino acid levels brought to levels in corresponding fish meals. H a l f t h e cages h o u s e d e i g h t chicks p e r cage a n d t h e o t h e r cages h o u s e d four chicks p e r cage. T h e e x p e r i m e n t w a s c a r r i e d o u t o v e r t h r e e successive experim e n t a l periods, so t h a t e a c h t r e a t m e n t w a s i m p o s e d as s h o w n in t h e experim e n t a l design o n t w o g r o u p s o f chicks h o u s e d in e a c h t y p e o f cage. E q u a l n u m b e r s of m a l e a n d f e m a l e d a y - o l d chicks were a l l o c a t e d a t r a n d o m to t h e e x p e r i m e n t a l cages a n d fed On e x p e r i m e n t a l diets till 21 d a y s o f age. D u r i n g t h i s p e r i o d food c o n s u m p t i o n w a s recorded, a n d e a c h c h i c k w a s w e i g h e d a t t h e end of the experiment. At the end of each experimental period a male and f e m a l e c h i c k w e r e selected f r o m e a c h pen, i n d i v i d u a l l y weighed, killed a n d t h e liver a n d p a n c r e a s r e m o v e d a n d weighed.
Statistical analysis T h e r e s u l t s were a n a l y s e d b y u s i n g a g e n e r a l l i n e a r i n t e r a c t o r m o d e l ( G l i m 3) ( B a k e r a n d Nelder, 1978). RESULTS All silages w e r e liquefied w i t h i n 3 d a y s a n d t h e s e a n d c o o k e d fish for fish m e a l were dried on to c a s s a v a m e a l w i t h no p r o b l e m .
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D.H. MACHIN ET AL.
Composition o[fish products Proximate composition Table 7 shows that all material produced from mackerel (high oil fish; HOF) had high protein and fat content whilst fish products from whiting (low oil fish; LOF) had high protein content, but low fat content, as expected. Silages and fish meals of each fish type dried on to cassava were similar in composition. Ash content and calcium and phosphorus contents were higher for LOF than for HOF, reflecting the bony skeletons of LOF and the cartilaginous nature of the skeletons of HOF,
Non-protein nitrogen Table 7 shows marked differences between the products obtained from the two types of fish, although the N P N contents of the raw fish were similar. The N P N content of the HOF, as a percentage of total N, increased from 5.3 in the raw fish to 30.6 in the dried fish meal and to over 50 in the dried silages, with little difference between silages treated or not treated with antioxidant. In the case of the LOF the N P N content of the raw fish and that of the dried fish TABLE 7
Composition of raw fish, silages and fish meals (g k g - 1 d r y matter) High oil fish material3 R a w minced
L o w oil fish m a t e r i a l a
Silages
Fish meal
d r i e d on to
d r i e d o n to
Raw minced Silages dried on to
Fish meal dried o n to
cassava
cassava
cassava
cassava
+AO +AO
Moisture Crude protein C r u d e fibre C r u d e fat
Ash Calcium Phosphorus Salt ( N a C l ) Non-protein nitrogen (%total N ) ME (MJ kg-1) -' F o r m i c acid ( % )
Histamine content ( m g per 100 g sample) 2
654 477
+AO
-AO
488 58 10.4 9.0 8.7 5.3
36 36 29 220 238 238 24 22 25 198 210 204 58 60 59 5.8 6.2 6,1 4.4 5.1 4.6 3.9 4.0 4.1 54,3 56.6 30.6
Not detected <5
17,1 1,9 <5
+AO 804 760 107 143 33.7 24.5 14.8 6.3
17.1 17.0 2.2 N o t d e t e c t e d N o t d e t e c t e d <5 <5 <5
1ME = Metabolizable energy calculated using the equation of L o d h i et al. (1976). 2Limit of sensitivity of method = 5 m g per 100 g sample. 3 + AO or - AO = w i t h or w i t h o u t a d d e d antioxidant,
-AO
67 57 70 247 229 246 28 29 29 28 30 27 80 80 88 12.0 10.8 13.9 8.1 7.2 8.8 4.5 4.3 4.8 17.1 19.1 4.9 14.8 3.8 <5
14.6 14.4 3.4 N o t detected <5 <5
CHICKSFEDONLOWANDHIGHOILFISHSILAGE
209
meal were similar, whilst the NPN content of the dried silages increased to only 17-19%.
Formic acid Levels in the silage meals were similar for materials treated or not treated with antioxidant (Table 7). The LOF and HOF silages should have contained 4.6% and 3.4% formic acid, respectively, in the final dried product, but the actual levels were much lower than those, and showed that formic acid was lost during the drying of the silage products. Histamine All the fish products (Table 7) contained < 5 mg per 100 g histamine, which is the limit of sensitivity of the method used. Amino acids As would be expected from material of fish origin the fish silages and meals have a good balance of amino acids and high levels of essential amino acids (Table 8). There were, however, several notable differences between the two types of fish material and the product type. The raw fish and the fish meals of both fish types were very similar in amino acid composition except that the fish meals were in general lower in lysine than the raw fish. However, the reactive lysine contents of both materials and fish types were similar. The cystine content of silages made from both types of fish material was markedly lower than that of raw fish. This was most pronounced with HOF in which the silage cystine levels were about half of the non-silage materials. Methionine contents of both fish silage and fish-meal products were slightly less than those of raw fish. There was a noticeable difference between the two fish types in histidine content, the HOF products having nearly twice as much as the LOF. There was, however, very little difference between the histidine levels of products derived from each type of fish. The reactive lysine contents of all materials indicate that the lysine of all the fish products was highly available and that in general the lysine of the processed fish products was slightly more available than lysine in the raw fish. The tryptophan contents of the antioxidant-treated HOF silage and fish meal were unchanged during processing, whilst the silage without antioxidant had slightly lower levels than that of the raw fish. In the case of LOF products the tryptophan contents of both fish silages were markedly reduced during processing, whilst that of the fish meal was only slightly less than that of raw fish.
210
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Fatty acids Table 9 shows that although the two fish materials differed markedly in overall fat content, they were similar in the composition of fatty acids. There were, however, considerable differences in fatty acid composition between raw and processed HOF though only small differences between similar LOF materials. The levels of many of the saturated fatty acids in the raw HOF were lower than similar fatty acids in processed HOF materials though the levels of polyunsaturated fatty acids in HOF processed materials were much lower in all processed materials than in the raw fish. The dried fish silages also had slightly lower levels of certain unsaturated fatty acids than dried fish meal; this loss TABLE 9
Fatty acid composition of raw fish, fish silage and fish meals (%) Fatty acids
High oil fish material Raw minced
C12:0 C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C18:4 C20:1 C20:2 C20:3, C20:4 C20:5 C20:0 C22:1 C23:1 C24:0 C24:1 C22:5 C22:6
<0.1 4.9 0.4 0.6 <0.1 18.2 6.0 1.5 0.7 3.6 20.5 1.5 1.5 2.9 4.7 0.3 1.3 6.8 <0.1 7.8 0.1 0.5 1.9 1.5 12.9
Silages dried on to cassava
Low oil fish material
+ AO
- AO
Fish meal dried on to cassava + AO
<0.1 6.4 0.5 0.9 0.1 26.6 7.8 1.8 0.9 5.5 28.5 1.1 0.3 0.6 5.8 <0.1 <0.1 0.4 <0.1 7.8 0.4 <0.1 1.7 0.3 2.0
<0.1 6.2 0.5 0.9 0.1 25.3 7.6 1.8 0.8 5.4 27.8 1.4 0.9 0.7 5.7 0.2 <0.1 1.3 0.5 7.7 0.5 <0.1 1.7 0.3 2.7
<0.1 6.0 0.5 0.9 0.1 23.7 7.4 1.9 1.0 5.4 27.0 1.7 0.9 0.9 5.7 0.2 <0.1 1.8 0.7 8.0 0.5 <0.1 1.8 0.4 3.5
Raw minced
<0.1 3.4 0.3 0.5 <0.1 14.1 5.2 1.4 0.7 3.6 18.3 2.3 1.0 2.0 8.5 0.7 0.9 6.1 0.8 11.7 0.8 0.5 1.7 1.5 14.8
Silages dried on to cassava + AO
- AO
<0.1 3.6 0.3 0.4 <0.1 14.8 6.2 1.3 0.7 3.3 19.9 3.4 1.3 1.9 9.4 0.4 0.8 6.6 <0.1 10.8 0.4 1.3 1.4 11.8
<0.1 4.4 <0.1 0.4 <0.1 14.9 5.8 1.1 0.6 3.1 19.9 3.2 1.3 1.8 9.0 0.3 0.8 7.1 <0.1 10.9 0.3 1.0 1.2 12.9
Fish meal dried on to cassava + AO <0.1 4.0 <0.1 0.4 <0.1 13.5 6.0 1.2 0.7 3.2 18.7 4.5 1.3 2.0 8.1 0.5 0.9 7.4 0.4 9.0 0.4 0.7 1.1 1.6 14.8
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D.H. MACHIN ET AL.
was greatest with the most unsaturated fatty acids and also appeared greater in the silage treated with antioxidant t h a n the untreated silage. These two effects were most marked with C18: 3, C18: 4, C20: 5 and C22: 6 fatty acids.
Changes in TBA values during processing and feeding offish products The results of monitoring TBA values as an indication of lipid oxidation are given in Table 10. W i t h both LOF and HOF there was a rapid increase in TBA values up to Day 3 followed by a fall by Day 9-10 after which the TBA values remained constant. The TBA values for HOF material were much higher t h a n those for a similar quantity of LOF material, and were also much greater per unit of lipid in HOF t h a n LOF materials. The TBA values were also greater in the fish silages t h a n the fish meals, though it was not possible to detect differences between silages treated with antioxidant and those which were untreated.
Composition of diets The composition of feeds shown in Tables 4 and 5 was within acceptable limits of the formulated specifications apart from the low oil, high level fish silage diet treated with antioxidant and supplemented with amino acids (F2, 01, L2, A2, $2) which had a slightly lower level of tryptophan t h a n formulated. This level was, however, considered to be within the normally acceptable range of experimental error for the technique used. TABLE 10 Changes in TBA valuesof fish materials over time (mg malonaldehydekg- 1dry matter) Storage (days)
0 3 9 10 14 17 88 179 203 294
High oil fish material
Low oil fish material
Silage + AO
Silage - AO
Fish meal + AO
Silage + AO
Silage - AO
Fish Meal + AO
77.3 271.3 153.1
77.3 271.2 196.1
77.3 83.9 138.8
3.3 22.5
3.3 25.0
3.3 7.0
10.5 3.9
13.4 3.1
1.4
3.3
2.7
3.0
7.8
6.3
4.0
181.5
186.1
128.4
125.2
83.0
164.7
181.0
139.0
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
213
Performance of broiler chicks The live weight gains, feed-conversion ratios and pancreas and liver weights of chicks fed on the experimental diets are shown in Table 11. Statistical analyses are given in Table 12. The chicks remained healthy throughout all three experiments and mortality was low ( 4.1% overall).
Weight gain Table 12 and Fig. 1 show that the oil content of the fish, level of inclusion and the processing of fish as fish meal or silage were significant sources of variation, but that treatment of materials with antioxidant or supplementation with amino acids did not have significant main effects. Chicks fed on LOF materials or fish materials at low levels of inclusion or fish meals had significantly greater weight gains than those fed on HOF material or fish materials at high levels of inclusion or fish silage. There were significant interactions between level of inclusion and processing as silage or meal, between level of inclusion and treatment with antioxidant and between level of inclusion and amino acid supplementation. Addition of TABLE
11
The performance of broiler chicks Fish type
ProInclusion AntiAmino Live weight gain Food conversion Pancreas Liver cessing oxidant Acids (0-3 week) (g) ratio (g) (g)
High oil
Fish silage
High
Low
Low
oil
Fish meal Fish silage
High Low High
Low
+
+
419
1.71
1.74
-
+
415
1.63
1.65
11.71
+
-
415
1.70
1.87
11.74
-
--
458
1.63
1.79
13.89
+
+
508
1.60
2.03
13.79
-
+
529
1.57
2.19
14.50
+
-
492
1.64
1.84
13.17
+
-
484 503
1.60 1.61
2.03 1.87
13.71 13.43
+ +
+
506 463
1.65 1.43
2.18 1.75
13.42 12.57
-
÷
509
1.42
1.75
13.54
+
-
480
1.44
1.77
13.42
+ +
+ + -
504 560 546 554
1.43 1.39 1.42 1.42
1.90 2.09 1.98 1.85
13.17 13.48 14.61 13.97
+ +
-
545 537 564
1.43 1.42 1.43
2.00 2.00 2.07
14.42 15.99 14.36
0.03
0.03
0.22
-
Fish meal S E o f means
High Low
10.8
12.99
D.H. MACHINET AL.
214 TABLE 12
Statistical analyses of factors assessed Source of variation
Variance ratios Weight gain
Cage factors Size Period Size × Period
Food conversion
Pancreas weight 1
1.0 46.1"** 30.1"**
19.2"** 61.1"** 13.0"**
2.7 1.2 25.5***
51.9"** 51.1"** 20.1"** 2.7 0.1
725.0*** 7.5 0.8 8.5** 2.0
5.9* 2.9 5.8* 0.1 <0.1
22.1"** 3.9 1.6 1.5 0.4 8.6** 0.1
2.4 1.1 4.8* 1.0 0.1 0.1 0.1
0.0012
0.028
Liver
weight I
0.1 15.5"** 8.3***
Nutritional factors 0 L F A S
3.3 9.2** 0.4 <0.1 1.3
Interaction 1 LxF LXA LXS O×L OXF O×A OXS Error mean square
7.5** 3.9* 4.1" <0.1 0.4 <0.1 0.2 886
<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 1.1 1.20
1Adjusted by covariance for differences in live weight: Abbreviations: 0 = oil content of fish; L = level of inclusion of fish material; F = processing of fish as silage or meal; A -- antioxidant treatment; S -- amino-acid supplementation. * = P < 0.05; ** = P < 0.01; * * * - P < 0.001.
antioxidant caused a reduction in weight gain at the high level of fish silage inclusion, but an increase at the low level, and supplementation with amino acids caused a reduction in weight gain at high levels of inclusion, but an increase in weight gain at low levels of inclusion (see Fig. 1). No significant interactions were noted between level of inclusion and oil content of fish, oil content of fish and processing of fish as silage or meal, antioxidant treatment and supplementation of diets with amino acids. Cage size had no significant effect on weight gain, but there was a highly significant effect of period and a size X period interaction.
CHICKS FED ON LOWAND HIGH OIL FISH SILAGE
215
(A) 700]
•
Silage
600/1
m Meal
|
500 -' Live weight gain (g)
400300200
1000¸
High Low inclusion inclusion High oil content
High Low inclusion inclusion Low oil content
fish
fish
(B) 7007
• +AO
• +AA
600 |1
E -AO
[ ] -AA
t
500" Live 400 weight gain (g) 300-
200100O"
High
Low
inclusion
inclusion
(DAntiox[dant addition
High
Low inclusion qi) Amino odd supplementation inclusion
Fig. 1. (A) Main effects on weight gain (also illustrates level of inclusion and method of processing interactions). ( B ) Interaction between level of inclusion and (i) antioxidant and (ii) amino acid supplementation ( + A O or - A O = w i t h or without added anti-oxidant, + A A or - A A = w i t h or without added amino acid).
Feed conversion ratios Table 12 and Fig. 2 show that the oil content of fish, level of inclusion and antioxidant treatment each had significant effects. Processing fish as fish meal or silage and supplementation of diets with amino acids had no significant effect on feed conversion ratios (FCR). Chicks fed on LOF materials at low levels of inclusion and material not treated with antioxidant had better FCR than those fed on HOF material at high levels of inclusion and fish treated with antioxidant. There were significant interactions between levels of inclusion and processing of fish material as silage or meal and between oil content
216
D.H. MACHINET AL.
(A) 1.8 1.6 1.4 1.2 Food conversion ratio
1.0 0.8 0.6 0.4 0.2 0 High inclusion
Low inclusion
High inclusion
High oil content fish (a)
(i) LEVEL OF INCLUSION AND METHOD OF PROCESSING
•
Silage
Low inclusion
Low oil content fish (ii) OIL CONTENT OF FISH AND ANTIOXIDANT ADDITION
•
+AO
1.8 1.6 1.4 1.2 Food conversion ratio
1.0 O. 8 0.6 0.4 0.2 0 High inclusion
Low inclusion
High oil content fish
Low content fish
Fig. 2. Main effects on food conversion ratio (also illustrates level of inclusion and antioxidant addition interactions). (A) Oil content of fish, antioxidant addition and inclusion effects. (B) Interaction between (i) level of inclusion and method of processing, (ii) oil content of fish and antioxidant addition.
of fish material and antioxidant addition. Other interactions were not significant. All cage factors assessed showed that the F C R of chicks were very markedly affected by cage size and period and that there was a highly significant size × period interaction.
Pancreas weights These were analysed with chick live weight as a covariate. Only the oil content of the fish and processing as fish meal or silage had significant effects on
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
217
pancreas weight. Pancreas weights for chicks fed on the HOF materials and those fed on fish meal were greater than those for chicks fed on LOF and fish silages, respectively. Of all interactions studied, only that between level of inclusion and amino-acid supplementation was significant. Cage factors, size and period, had no significant effects on pancreas weight though there was a significant size X period interaction.
Liver weights Statistical analyses similar to those for pancreas weight (Table 12) were carried out for chick liver weights. Of all nutritional factors examined only level of inclusion had a significant effect on liver weight. Diets with the high inclusion of fish material produced heavier livers, relative to live weight. None of the interactions studied was significant. Cage size had no significant effect, but there was a significant effect of period on liver weight and a size X period interaction. DISCUSSION
The results of this experiment show that fish silage made from both oily and non-oily fish can be made into dry meals with cassava and that both products can be fed to chicks with varying degrees of success. There were, however, considerable differences in performance between chicks given the various fish products, which relate significantly to the various treatments the products underwent.
Level of inclusion The results of this experiment are in line with those of previous workers (Disney et al., 1978; Kompiang et al., 1979, 1980; Rattagool et al., 1980a-c; Hall, 1983) and show that where fish silage products form more than 10% of the diet, performance is inferior. However, in this experiment the higher level of inclusion resulted in a much smaller reduction in performance than those shown in most previous studies. Weight gains of chicks fed on the HOF and LOF silages at high rates of inclusion were 85 and 91%, respectively, of those of chicks fed on the fish meals. Previous workers (Disney et al., 1978; Kompiang et al., 1980) have reported levels of performance of less than 66% of those of controls. Considerable care was taken to establish the chemical composition of the fish products, which had not been possible in other experiments, and hence it was possible to formulate diets to a greater degree of accuracy than in most earlier studies. This would account for a large proportion of the improved livestock performance in this study. However, there was a considerable difference in the performance of chicks given diets with low and high levels of inclusion, which cannot be explained by simple formulation differences.
218
D.H. MACHIN ET AL.
Changes in the protein component offish products The HOF material had much higher levels of N P N than the LOF material and in both cases N P N levels of fish silages were much higher than those of fish meals. The breakdown of the protein reflected in the N P N values could have considerable significance with regard to the lower performance of chicks receiving high levels of N P N in silages or fish products in general at high inclusion levels. Gilberg and Raa (1977) reported that only 8% of ammonia was produced in acid-treated cod viscera silage stored at 27°C for 220 days. It can therefore be assumed that since the material used in this experiment was stored at - 20 ° C for most of the time between drying and feeding, little ammonia would have been produced which could have had a detrimental effect on chick performance. The histidine contents of the HOF fish products were much greater than those of the LOF materials. During spoilage, decomposition of histidine occurs in fish products (Disney et al., 1978; Raa and Gilberg, 1982) to produce toxic products such as histamine. In this experiment considerable care was taken to ensure that all fish were fresh and to prevent decomposition. As a result, histidine levels of all fish products in this study showed little change from raw fish. Clearly if spoilage had occurred in the fish products the HOF materials with their high histidine content would be more likely to produce high histamine levels than would LOF materials. The histamine content of both raw fish and the processed silages and meals was very low and indicates that any lowered chick performance could not be directly associated with high levels of histamine or fish spoilage. Several changes in the amino acid composition of the fish products were identified in this study and in particular lowered levels of cystine in the dried HOF silages, lowered lysine and methionine in all processed fish products and lowered tryptophan levels in the dried LOF silages. Disney and Hoffman (1976) noted an increase in food intake when silages dried on to substrates were supplemented with lysine and methionine though no amino acid determinations were carried out. The reduction in these two amino acids in the processed fish products in this experiment provides an explanation for these findings. The loss of tryptophan in acid medium is well documented (Backoff, 1976; Jensen and Schmidtsdorff, 1977; Johnsen and Skrede, 1981 ). Kompiang et al. (1980) further report that this amino acid is stable at low pH when present in proteins but degrades when free. Clearly, in silages with high levels of NPN, tryptophan is likely to be seriously degraded. In this experiment tryptophan losses were apparent in the LOF silages, but surprisingly not in the HOF silages, which had the greatest N P N levels. In all previous cases of high tryptophan losses the fish materials were maintained at low pH for longer than in
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
219
this study. The low tryptophan losses in this study reflect the short time the fish was ensiled in acid medium. Several authors have indicated that small deficiencies in tryptophan have greater significance than would be expected (Gorbatov, 1976; Lougnon et al., 1985). However, the slightly low tryptophan levels in unsupplemented fish silage diets did not appear to affect livestock performance. To assess the significance of these amino acid losses in the fish products and also to offset any lowered availability of lysine and methionine, as suggested by Carpenter et al. (1963), diets in this experiment containing fish silages were supplemented with the amino acids, lysine, methionine and tryptophan to raise them to the levels of the fish-meal diets. The results of the subsequent feeding trial showed that amino acid supplementation produced only a small improvement in the performance of chicks given silage in their diets at low levels of inclusion and no improvement at high levels of inclusion. It is therefore concluded that the lowered performance must be associated with other factors.
Changes in the lipid component offish products The fatty acids of the lipid component of fish products were markedly changed during processing. These changes were mostly associated with the unsaturated fatty acids and, as would be expected, were most pronounced in the HOF products. To monitor oxidative changes taking place in the fish products, malonaldehyde was measured throughout processing, storage and feeding of the fish products. The production of malonaldehyde followed a similar path to that reported by Hall (1983) for peroxide levels in fish silages; in ensiled fish these rapidly increased during the first 3 days, after which they declined and stabilised by Day 7. He did not, however, note a similar effect with malonaldehyde. These results do, however, indicate marked oxidative changes taking place in the HOF silages. A similar effect, but to a much lower extent, took place in the LOF silages and to an even lesser extent in the fish meals. Additions of antioxidant to fish silages did not appear to affect levels of malonaldehyde. The fatty acid composition of raw fish, silages and fish meals dried onto cassava was measured before the feeding trial commenced. A considerable reduction in the unsaturated fatty acids of the HOF materials and especially of the polyunsaturated fatty acids, C18: 3, C18: 4, C20: 3, C20: 4, C20: 5, C22: 5 and C22: 6 had taken place, but only a slight reduction in the unsaturated fatty acid levels of LOF materials. Levels of these unsaturated fatty acids were also slightly lower in silages than fish meals and in the cases of HOF materials the levels of C18:3, C20:5 and C22:6 were slightly lower in silages treated with antioxidant than in those not treated. All these results are in agreement with those of Hall and Ledward (1986) who found similar effects with silages made from silver-belly fish (Leiognathus spp. ).
220
D.H. MACHIN ET AL.
A consequence of these losses is a possible deficiency in the essential fatty acids, linolenic acid (C18:3) and arachidonic acid (C20:4), in diets limiting in those nutrients. In this study, diets were formulated to provide at least 1% linoleic acid from non-fish sources and the fish products were only mixed into diets immediately prior to feeding. It is therefore very unlikely that deficiencies in these fatty acids occurred, though the possibility that the oxidized fatty acids of the fish materials could have caused the rapid oxidation of non-fish fatty acids might be considered. A further consequence of the considerable loss of unsaturated fatty acids and production of oxidation products is a reduction in palatability of the meals. Both Dugan (1975) and Barlow and Pike (1977) implicated oxidized fats in loss of appetite and reduction in weight gain though since they monitored only peroxide values it is not possible to compare their results directly with these. That oxidized fats could have adverse effects on palatability and nutritive value of foods through protein and vitamin damage or through increased vitamin requirements has also been proposed by Carpenter et al. ( 1963 ). Clearly these effects could be contributing to the reduction in performance of chicks fed on HOF materials. Chicks fed on fish meals had significantly greater weight gains than those fed on high levels of fish silage, but similar FCRs. The lipid in fish meal was also less oxidized than that in fish silages. From these results it would appear that the fish silages were less palatable than the fish meals without the nutritive value of materials being particularly damaged, and that this could relate to higher levels of lipid oxidation products in fish silages. The fish meal was produced by boiling fish and then drying it on to cassava. As a consequence of boiling, lipases would be inactivated and free fatty acid production reduced (Reece, 1980). Oxidation of free fatty acids could also be reduced by the production of antioxidant materials during boiling (Sen and Bhandary, 1980). It is possible that the lowered oxidation of the fish meals was a consequence of these two effects. Hall and Ledward (1986) reported that TBA values in antioxidant-treated silages were much lower than in untreated silages but that the levels of polyunsaturated fatty acids in treated silages decreased much more than in untreated silages. This effect is hard to explain. In this study the TBA values of both the treated and untreated silages in HOF and LOF materials showed little difference, but the levels of several of the unsaturated fatty acids in antioxidant-treated HOF silage (in particular C18:3, C20:5 and C20:6) were lower than those in the untreated silage. These results give part support to those of Hall and Ledward (1986), although not demonstrating the anomaly of low TBA values whilst at the same time showing greater unsaturated fatty acid loss.
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
221
Changes in pancreas and liver weights Pancreas weights were significantly affected by both oil content of the fish material and types of processing, whilst liver weight was affected only by level of inclusion. It is possible that the effects on the pancreas were mostly associated with the greater lipase and protease production required for the digestion of materials with high oil contents and of fish meals than for low oil materials and highly soluble silages. The heavier liver weights of chicks receiving the high inclusion-rate diets could be associated with possible subclinical detoxification of formic acid or greater protein storage associated with high intakes of fish material. CONCLUSION The reduced performance of chicks fed on fish silages dried on to substrates is largely due to the oxidation of lipids. This conclusion is in line with those of Kompiang et al. (1980) and Hall (1983). The fish silage products for this study were produced with care to minimise oxidation by the use of fresh fish materials, storage of materials at low temperature, minimum ensilage periods and use of antioxidants. Diets were also carefully balanced to meet the chicks' requirements. These precautions yielded a better performance of chicks fed on the silages at high levels of inclusion than that reported by other workers. However, it is difficult to consider how further improvements could be achieved through this approach, though there is clearly considerable potential for improvement. Possible alternative approaches to improving the performance of chicks given silage include the use of preheating treatments to deactivate lipases (and, as a consequence, other enzymes) followed by preservation of fish material using acids without autolysis. In this way, both free fatty acid production and fatty acid oxidation would be reduced and protein would remain intact so that amino acids would be less open to damage. ACKNOWLEDGEMENTS The authors thank Prof. R. Mead for his assistance with experimental design and analysis of data, Mr. B. Waite for assistance with care of experimental birds and Mrs. P. Carlaw for analysis of ingredients and diets. REFERENCES Backoff,H.P., 1976. Somechemicalchangesin fish silage.J. FoodTechnol.,11: 353-363. Baker, R.J. and Nelder,J.A., 1978.The GlimSystem (Release3). NumericalAlgorithmsGroup., Oxford, 150 pp. Barlow, S.M. and Pike, I.H., 1977. The role of fat in fish meal,in pig and poultrynutrition. Int. Assoc.Fish Meal Manufacturers,Tech. Bull., No. 4, 38 pp. Canale, A., Valente, M.E. and Ciotti, A., 1984. Determinationof volatile carboxylicacid (C1 to
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C5) and lactic acid in aqueous acid extracts of silage by high performance liquid chromatography. J. Sci. Food Agric., 35: 1178-1182. Carpenter, K.J., Lea, C.H. and Parr, L.J., 1963. Chemical and nutritional changes in stored herring meal. 4. Nutritional significance of oxidation of the oil. Br. J. Nutr., 17: 151-169. Disney, J.G. and Hoffman, A., 1976. A dried fish silage product. In: Proceedings of the Torry Research Station Symposium on Fish Silage. Aberdeen, Scotland, pp. 1-14. Disney, J.G., Hoffman, A., Olley, J., Clucas, I.J., Barranco, A. and Francis, B.J., 1978. Development of a fish silage/carbohydrate animal feed for use in the Tropics. Trop. Sci., 20: 129-144. Dugan, L., 1975. Lipids. VI. Chemical properties and reactions. In: O.R. Fennema (Editor), Principles of Food Science. Vol. 1. Marcel Dekker, New York, NY. Gilberg, A. and Raa, J., 1977. Properties of propionic acid/formic acid preserved silage of cod viscera. J. Sci. Food Agric., 28: 47-53. Green, S., 1984. The use of fish silage in pig nutrition. Ph.D. Thesis, University of Nottingham. Gorbatov, V.M., 1976. The utilisation of blood and other slaughter by-products. Proceedings of the European Meeting of Meat Research Workers, 1976. Vses N. auchno-Issled. Inst. Myasnoi Promyshlennosti Moscow, 22: 10.1. Hall, G.M., 1983. Silage from tropical fish. Ph. D. Thesis, University of Nottingham. Hall, G.M. and Ledward, D.A., 1986. Silage from tropical fish 3. Lipid behaviour. J. Food Technol., 21: 45-54. Hardy, R. and Smith, J.G.M., 1976. The storage of mackerel (Scromber scrombus ). Development of histamines and rancidity. J. Sci. Food Agric., 27: 595-599. Hofstetter, H.H., Sen, N. and Holman, R.T., 1965. Characterisation of unsaturated fatty acids by gas liquid chromatography. J. Am. {Oilseeds) Chem. Soc., 42: 537-540. Jensen, J. and Schmidtsdorff, W., 1977. Fish silage, low fat and soluble fish protein products. International Association of Fish Meal Manufacturers Symposium. Production and Use of Fish Meal. Szczecin, Poland, pp. 23-35. Johnsen, F. and Skrede, A., 1981. Evaluation of fish viscera silage as a feed resource - chemical characteristics. Acta Agric. Scand., 31: 21- 28. Kompiang, I.P., Arifudin, R. and Raa, J., 1979. Nutritional value of ensilaged by-catch fish from Indonesian shrimp trawlers. In: J.J. Connell (Editor), Advances in Fish Science and Technology. Farmhouse Fishing News Books, Farnham, 349 pp. Kompiang, I.P., Darwanto, A. and Arifudin, R., 1980. Nutritional value of fish silage. In: Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 44-47. Lodhi, G.N., Daulat, Singh and Ichhponani, J.S., 1976. Variation in nutrient content of feedingstufts rich in protein and reassessment of the chemical method for metabolizable energy estimation for poultry. J. Agric. Sci., 86: 293-303. Lougnon, J., Bertrand, S., Mathe, J. and Pieri, A., 1985. Nutritional value and originality of tryptophan. Communication presented at 16th Journ~es de la Rdcherche Porcine en France, Paris. Lucas, B. and Sotelo, A., 1980. Effect of different alkalies, temperature and hydrolysis times on tryptophan determination of pure protein and of foods. Anal. Biochem., 109: 192-197. Ministry of Agriculture, Fisheries and Food, 1976. The Fertilisers and Feeding Stuffs (Amendment ) Regulations. Statutory Instrument No. 840, Her Majesty's Stationery Office, London. Poulter, R.G., Jayawardena, K.M., Ganegoda, P. and Ranaweera, K.N.P., 1980. Studies on fish silage in Sri Lanka - a summary. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 64-68. Raa, J. and Gilberg, A., 1982. Fish silage: A review. CRC Crit. Rev. Food Sci. Nutr., 16: 383-419. Rattagool, P.P., Swachathamwongratana, S. and Wongchinda, N., 1980a. Fish silage in Thailand: further trials on broiler chicken. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 59-62. Rattagool, P.P., Wongchinda, N. and Swachathamwongratana, S., 1980b. Studies on the nutritive
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value of fish silage for broiler chickens. Proceedings Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 48-54. Rattagool, P.P., Wongchinda, N. and Swachathamwongratana, S., 1980c. Fish silage in Thailand. Trial feeding on broiler chicken. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 55-58. Reece, P., 1980. Control and reduction of free fatty acid concentration in oil recovered from fish silage prepared from sprat. J. Sci. Food Agric., 31: 147-155. Roach, A.G., Sanderson, P. and Williams, D.R., 1967. Comparison of methods for the determination of available lysine value in animal and vegetable protein sources. J. Sci. Food Agric., 18: 274-278. Sen, D.P. and Bhandary, C.S., 1980. Lipid oxidation in raw and cooked oil sardine, SardineUa longiceps. In: Fats and Oils in Relation to Food Products and their Preparations. Report of the Central Food Technology Research Institute, Mysore, India, pp. 132-133.
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
P e r f o r m a n c e of Broiler Chicks Fed on L o w and High Oil Fish Silages in Relation to Changes T a k i n g Place in Lipid and Protein Components D.H. MACHIN 1'*, S. PANIGRAHI 1, JULIET BAINTON 1and T.R. MORRIS 2
1Overseas Development and Natural Resource Institute, Central Avenue, Chatham ME4 4TB (Gt. Britain) 2Department o/Agriculture and Horticulture, University o/ Reading, Earley Gate, Reading RG6 2AT (Gt. Britain) (Received 3 August, 1988; accepted for publication 8 August 1989)
ABSTRACT Machin, D.H., Panigrahi, S., Bainton, J. and Morris, T.R., 1990. Performance of broiler chicks fed on low and high oil fish silages in relation to changes taking place in lipid and protein components. Anita. Feed Sci. Technol., 28: 199-223. Changes in the protein and lipid components of fish silages made from low oil content fish (LOF; whiting, Gadus merlinger) and high oil content fish (HOF; mackerel, Scromber scombus) were studied during ensiling, drying and subsequent storage. Chicks were fed on diets containing the silages dried on to cassava meal, or fish meal produced from the same batch offish. The HOF silages had > 50% of total N in the form of non-protein N, compared with 18% in the LOF silages; both levels were higher than in corresponding fish meals. Cystine levels in all silages were lower than in the original fish material, the greatest reduction occurring in HOF silage. Tryptophan levels were lower in all processed materials, but the most marked reductions were in LOF silages. Levels of lysine and methionine fell equally in all processed fish materials. The levels of histamine remained low in all fish products. Marked changes occurred in the fatty acid levels of the ensiled fish materials, especially in the HOF silage. The changes were mostly associated with losses of unsaturated fatty acids, in particular C18-3, C18-4, C20-3, C20-4, C20-5, C22-5 and C22-6. Malonaldehyde levels indicated that marked oxidation of the lipid occurred in the HOF silage, this occurred to a much lower extent in LOF silage and to an even lesser extent in fish meals. Addition of antioxidant did not appear to affect these changes. Weight gains of chicks fed on balanced diets containing 4.7 or 9.4% crude protein from HOF silage (9.8 and 19.6% of the dietary dry matter) and 5.2 or 10.4% crude protein from LOF silage (6.8 and 13.5% of the dietary dry matter) were 99, 85 and 98 and 91%, respectively, of the gains achieved with corresponding fish meals. Food conversion ratios for the above dietary treatments were respectively, 97, 103, 101 and 99% of those of chicks fed on fish meals. It was concluded that the differences in chick performance reflected the changes that occurred in the lipid and protein components and the interactions between these components. *Present address: Food and Agriculture Organization of the United Nations, Via delle Terme di Caracalla, 00100 Rome, Italy.
037%8401/90/$03.50
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D.H~ MACHIN ET AL.
INTRODUCTION
Ensiling has been used for many years to preserve waste fish. Material processed in this way has been shown to be a good feed for pigs (Green, 1984). However, feeding trials with chicks to evaluate fish silage dried on to starchy fillers have given variable results. Where fish silage formed up to 10% of the dietary dry matter, broiler performance was mostly similar to or better than controls (Rattagool et al., 1980a-c ). In contrast, though Poulter et al. (1980) included up to 46% fish silage in the diet without impairing broiler performance, the performance of broilers given more than 10% fish silage has generally been inferior to that of controls (Disney et al., 1978; Kompiang et al., 1979, 1980; Hall, 1983 ). In most broiler experiments, little attention has been given to balancing nutritional content, except for crude protein, or occasionally specific nutrients such as tryptophan (Hall, 1983 ), vitamin E (Disney et al., 1978), or thiamine (Kompiang et al., 1979 ). It is likely that the poorer performance of broilers fed on fish silages is partly because of inadequate understanding of changes that take place during the ensilage process and subsequent drying, and partly because of failure to formulate diets with adequate levels of known nutrients. During autolysis, about 80% of the protein in fish silages made with formic acid becomes soluble. Many of the amino acids are then exposed to damage by reaction with other components of the silage. Amino acids likely to be affected in this way include tryptophan, tyrosine, lysine, methionine and, if spoilage has taken place, histidine. Silage production and air drying on to dry feeds has been shown to be highly conducive to the breakdown and rapid oxidation of the highly unsaturated fatty acids of fish materials ( Hall, 1983). The nutritional significance of this will be a loss of essential fatty acids and the production of highly reactive breakdown products. Carpenter et al. (1963) suggested that these products could have an adverse effect on palatability, cause protein damage and induce deficiencies through the destruction of vitamins or increased vitamin requirements. This study was therefore carried out to assess: (1) the production and use for feeding of fish silages made from fish of low oil content (whiting, Gadus merlinger) and fish of high oil content (mackerel, Scromber scrombus); (2) high and low levels of silage inclusion in diets; (3 } the fate of the silage fats during silage production, drying and storage and the effect of an antioxidant on such changes; (4) the fate of amino acids during ensilage and the effects of this and subsequent amino-acid supplementation on performance of chicks; (5) the comparison of fish materials made into fish meal or into fish silage.
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
201
MATERIALSAND METHODS
Production of dried meals containing fish silages Fresh whole whiting and mackerel with guts intact were obtained direct from the UK ports of Torbay and Hull, respectively. All fish were blast frozen in blocks of 20 kg on landing and consigned directly to the laboratory in transport maintained at - 2 0 ° C . The fish were maintained at this temperature for a further 4 days, then thawed overnight. At the time of processing, loose ice was still located between individual fish and fish were still part frozen. The fish were minced through a KS Mince-master (R) (13.5 kW) using a 6-mm plate and then through the same machine using a 4-mm plate. Formic acid (85%) was added to the fish as it entered the mincer on the second pass, to give a final concentration of 3% by weight of fish. Sixty kilograms of each fish type was then placed in polythene bins and remixed manually. The fish and acid mixtures were then maintained at a temperature of 23 ° C until dried; liquefaction took place after 3 days. Butylated hydroxy toluene (BHT) dissolved in a solution of methanol and water was added as an antioxidant to one aliquot of each fish silage type at 200 mg B H T kg-1 of the fat content. The antioxidant was added to the minced fish with the formic acid to prevent oxidation occurring from inception. The wet silages (60 kg) were mixed with dry ground cassava meal (30 kg) 7 days after liquefaction. These proportions had been shown by an initial study to be the minimum necessary for the production of meals that could be easily dried. The mixing was begun manually in polythene bins with a wooden paddle and completed with an electrically powered concrete mixer. The resultant mixtures were spread on drying trays to a depth of 2.5 cm in a forced-air oven at 50°C, turned daily, and dried for 3 days. All lumps of material were crumbled by hand during turning. After drying, the materials were ground through the 4-mm screen of a hammer mill and stored at - 20 ° C in air-tight plastic sacks until used.
Production offish meals Aliquots (40 kg) of each type of fish were processed as described for the fish silages except that no formic acid was added. The minced fish was then placed in steam-heated kettles and brought to 100°C over 75 min. The fish was maintained at this temperature for 15 min, then mixed with 20 kg of cassava meal and dried, ground and stored as described for fish silage meals.
Chemical analyses Samples of fresh minced fish of each type and all dried meals were analysed for the following: (1) crude protein, ether extract, crude fibre, ash, calcium,
202
D.H. MACHINET AL.
phosphorus and chloride expressed as salt equivalent, using methods given in the UK fertilisers and feedingstuffs (amendment) regulations (Ministry of Agriculture, Fisheries and Food, 1976); (2) non-protein nitrogen ( N P N ) by the method described by Backoff (1976); (3) histamine by the method of Hardy and Smith (1976); (4) fatty acid composition by the method of Hofstetter et al. ( 1965 ); (5) formic acid by the procedure of Canale et al. ( 1984 ); (6) amino acids by using a Biotronik LC5000 analyser. Hydrolysis of the samples was carried out by open flask refiuxing to 6 M HC1 for 24 h. Tryptophan was determined by the method of Lucas and Sotelo (1980) and available lysine by the method of Roach et al. (1967).
Monitoring of lipid oxidation The state of oxidation of the fish lipid component was monitored from receipt of the fresh fish through to consumption of the processed meals. MoniTABLE 1
Experimental 'incomplete block design' Treatments
Period 1
Period 2
Large
Small
Large
X X X X X
X X X X X
X X X X
F2, O2, LbAI, S2
X
F2, 02, L:, A2, S 1 F2, 02, L : , A : , S 1 F1, O2, L2, A2, S2 F:, 02, L1, A2, S 2 F2, O1, L2, A2, S2 F2, O1, L2, A1, S2 F2, 01, L2, A2, S1 F2, Ol, L2, Al, Sl F2, 01, L1, A2, S2 F2, O:,L1, A1, S2 F2, O1, L1, A2, Sl F2, O:,L1, A1, S1 FI, OI, L2, A2,$2 FI, O:,L:,A2, S2
X X X X X X X X X X
F2, 02, L2, A2, S2 F2,02, L2, A:,S2 F2,02, L2, A~,S: F2, O2, L2, A1, S 1 F2, 02, LI, A2,$2
Period 3 Small
Large
Small
X
X
X X X X
X
X
X
X X X X -
X X X X X X X X X X X X X X
X X X X X X -
X X X X X X
X X X X X X X X X X X X
X X X X X X
X X
Abbreviations: 01 = low oil fish; 02= high oil fish; L~ = low level inclusion; L2=high level inclusion; F1 -- fish meal; F2 = fish silage; A: = without antioxidant; A2 = with antioxidant; $1 -- without amino acid supplementation; $2 = with amino acid supplementation. S m a l l = s m a l l cage size (4 chicks); Large=large cage size (8 chicks).
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
203
toring was based on the level of malonaldehyde, an oxidative breakdown product of lipid, in the fish lipid, determined by the technique described by Hall (1983) using 2°thiobarbituric acid (TBA). Malonaldehyde was determined in the fresh fish, wet ensiled and wet cooked fish, and the dried products throughout the storage and feeding period. Owing to difficulties in obtaining the whiting and mackerel at the same time the malonaldehyde determinations for the former were carried out over 203 days and the latter over 294 days, from receipt of fresh fish. It was also not possible to carry out determinations on exactly equivalent days though attempts were made to match these as far as possible. The times of malonaldehyde determinations are given in Table 10.
Experimental design To evaluate the effects of high and low oil fish silages at high and low levels of inclusion, treated with and without antioxidants, in diets supplemented or not supplemented with the limiting amino acids, tryptophan, lysine and methionine, the experimental incomplete block design shown in Table 1 was prepared. Complete blocking did not extend to fish meals, but involved giving the 20 experimental diets to groups of broiler chicks (four replicates per treatment) over three experimental periods. All combinations of fish type, antioxidant treatment, amino-acid supplementation and level of inclusion were produced in the design and the effects compared with fish meals which were given at both levels of inclusion and were treated with antioxidant.
Formulation and preparation of diets Details of diet formulation, and the calculated and determined composition are shown in Tables 2-5. The dried cassava acid fish silages or fish meal mixtures were prepared so that 22.5 and 45% of the mixtures would provide approximately 5 and 10% fish protein in the broiler diets, respectively. However, in practice, the dried mackerel products in the experimental diets provided 4.7% crude protein (9.8% fish dry matter) in the low inclusion diets and 9.4% crude protein (19.6% fish dry matter) in the high inclusion diets, whilst the dried whiting products provided 5.2% crude protein (6.8% fish dry matter) in the low inclusion diets and 10.4% crude protein (13.5% fish dry matter) in the high inclusion diets. Metabolizable energy values for the dried meals were calculated by using the equations of Lodhi et al. (1976) (see Table 7). The remainder of the diets were formulated by using a linear programme to commercial broiler starter specifications, from maize meal, soya bean meal, maize gluten meal, meat and bone meal, wheatfeed, sunflower meal, molasses, dicalcium phosphate, limestone, salt (NaC1), maize oil and a vitamin-trace mineral premix (Vitamealo, UK ) (Table 2 ). All feeds were in the form of meals.
204
D.H. MACHINET AL.
TABLE 2 Formulation of experimental diets (based on fish meals) (%) Raw materials
Maize Soya bean meal Maize gluten meal Meat and bone meal Wheatfeed Sunflower meal Molasses Dicalcium phosphate Limestone Salt (NaCl) Maize oil L-Lysine DL-Methionine Premix 1 High oil fish material (dried on cassava ) Low oil fish material (dried on cassava)
High oil fish materials
Low oil fish materials
Low fish inclusion diet
High fish inclusion diet
Low fish inclusion diet
High fish inclusion diet
34.91 22.96 10.00 4.27
0.56 10.43 10.00
48.51 21.69 6.07 0.11 0.55 0.12 0.15 0.30 -
28.26 14.98 10.00 0.52 0.25 0.09 0.39 0.08 0.13 0.30 -
22.50
45.00
4.00 0.59 0.23 0.06 0.07 0.11 0.30 22.50
16.77 10.00 4.00 0,81 0.94 0.10 0.87 0.10 0.12 0.30 45.00
' P r e m i x (vitamin a n d trace mineral) supplied the following per kilogram of feed: vitamin A, 10 000 iu; vitamin D3, 2500 iu; vitamin E, 10.0 iu; v i t a m i n K, 2.0 mg; vitamin B1, 0.50 mg; vitamin B2, 6.0 mg; nicotinic acid, 20.0 mg; v i t a m i n B6, 1.0 mg; p a n t o t h e n i c acid, 10.0 mg; biotin, 0.08 mg; folic acid, 1.0 mg; vitamin B~2, 5.0 tLg; Mn, 80.0 mg; Zn, 60.0 mg; Cu, 5.0 mg; Se, 0.10 mg; I, 1.0 mg. TABLE 3 Calculated composition of experimental diets Nutrients (g kg -1 )
Crude protein Crude fibre Crude fat Lysine Methionine + cystine Calcium Phosphorus Salt (NaC1) Linoleic acid Metabolizable energy ( M J k g - ' ) Dry matter
High oil fish materials
Low oil fish materials
Low fish inclusion diet
High fish inclusion diet
Low fish inclusion diet
High fish inclusion diet
230 40 66 14 9 9 7 4 10
230 68 108 14 10 10 8 4 10
220 35 37 14 9 9 7 4 15
220 36 30 14 9 9 7 4 10
12.75 903
12.75 907
12.70 897
12.75 901
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
205
TABLE 4
Composition of diets (g kg -~ dry matter) Fish type
Process- Inclus- AntiAmino Moisture Crude Crude Crude Calcium Phosing ion oxidants acids content of protein fibre fat phorus fresh matter (gkg -1)
High Fish
oil
Low oil
High
+ +
+ +
82 83 82 74
234 248 243 247
72 79 81 75
111 143 125 138
12.0 11.7 12.0 12.7
8.3 8.4 8.5 8.3
Low
+ + -
+ + -
100 97 97 95
258 252 248 270
50 50 50 48
66 49 65 69
12.3 11.3 11.1 10.7
8.9 8.2 8.1 7.8
silage
Fish meal
High
+
-
Low
+
-
68 100
248 260
85 46
115 74
11.3 11.3
8.0 8.2
Fish silage
High
+
+ + -
85 86 84 86
243 242 249 239
44 43 38 39
39 39 39 37
10.5 10.1 9.7 9.8
7.4 7.4 7.6 7.7
--
+ + -
104 102 101 97
242 249 255 246
39 37 39 39
49 49 42 43
11.9 10.6 11.6 10.9
7.9 7.7 7.8 7.8
+ +
-
95 106
251 260
42 41
34 41
11.0 11.6
7.4 8.1
+ -
Low
+
+
Fish meal
High
Low
Amino acid supplementation Following amino acid analysis of the dried fish silage and fish meals, diets designated for amino acid supplementation received additions of synthetic lysine, methionine and tryptophan to raise their levels to those of the equivalent fish meals. Synthetic methionine was added to balance total sulphur amino acid (methionine + cystine ) levels. The amino acid supplementation levels are shown in Table 6.
Feeding trial The trial was carried out in a controlled environment experimental house using 32 wire-floored cages, each equipped with its own feeder and drinker.
206
D.H. MACHIN ET AL.
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TABLE 6 Amino-acid supplementation of fish silages 1 (To) High oil fish silage
Low oil fish silage
+Antioxidant -Antioxidant
÷Antioxidant -Antioxidant
1.70 1.70
1.79 1.79
1.75 1.75
1.58 0.11 1.58
Methionine ÷ lysine A Silage content 0.72 B Addition, DL. Methionine 0.27 C Final level2 0.99
0.81 0.18 0.99
0.85 0.02 0.80
0.71 0.16 0.87
Tryptophan A Silage content B Addition, Tryptophan C Final level2
0.23 0.05 0.28
0.17 0.05 0.22
0.14 0.08 0.22
Lysine A Silage content B Addition, L. Lysine C Final level2
0.26 0.02 0.28
1Amino acid addition to silage to equate with fish meal levels. 2Amino acid levels brought to levels in corresponding fish meals. H a l f t h e cages h o u s e d e i g h t chicks p e r cage a n d t h e o t h e r cages h o u s e d four chicks p e r cage. T h e e x p e r i m e n t w a s c a r r i e d o u t o v e r t h r e e successive experim e n t a l periods, so t h a t e a c h t r e a t m e n t w a s i m p o s e d as s h o w n in t h e experim e n t a l design o n t w o g r o u p s o f chicks h o u s e d in e a c h t y p e o f cage. E q u a l n u m b e r s of m a l e a n d f e m a l e d a y - o l d chicks were a l l o c a t e d a t r a n d o m to t h e e x p e r i m e n t a l cages a n d fed On e x p e r i m e n t a l diets till 21 d a y s o f age. D u r i n g t h i s p e r i o d food c o n s u m p t i o n w a s recorded, a n d e a c h c h i c k w a s w e i g h e d a t t h e end of the experiment. At the end of each experimental period a male and f e m a l e c h i c k w e r e selected f r o m e a c h pen, i n d i v i d u a l l y weighed, killed a n d t h e liver a n d p a n c r e a s r e m o v e d a n d weighed.
Statistical analysis T h e r e s u l t s were a n a l y s e d b y u s i n g a g e n e r a l l i n e a r i n t e r a c t o r m o d e l ( G l i m 3) ( B a k e r a n d Nelder, 1978). RESULTS All silages w e r e liquefied w i t h i n 3 d a y s a n d t h e s e a n d c o o k e d fish for fish m e a l were dried on to c a s s a v a m e a l w i t h no p r o b l e m .
208
D.H. MACHIN ET AL.
Composition o[fish products Proximate composition Table 7 shows that all material produced from mackerel (high oil fish; HOF) had high protein and fat content whilst fish products from whiting (low oil fish; LOF) had high protein content, but low fat content, as expected. Silages and fish meals of each fish type dried on to cassava were similar in composition. Ash content and calcium and phosphorus contents were higher for LOF than for HOF, reflecting the bony skeletons of LOF and the cartilaginous nature of the skeletons of HOF,
Non-protein nitrogen Table 7 shows marked differences between the products obtained from the two types of fish, although the N P N contents of the raw fish were similar. The N P N content of the HOF, as a percentage of total N, increased from 5.3 in the raw fish to 30.6 in the dried fish meal and to over 50 in the dried silages, with little difference between silages treated or not treated with antioxidant. In the case of the LOF the N P N content of the raw fish and that of the dried fish TABLE 7
Composition of raw fish, silages and fish meals (g k g - 1 d r y matter) High oil fish material3 R a w minced
L o w oil fish m a t e r i a l a
Silages
Fish meal
d r i e d on to
d r i e d o n to
Raw minced Silages dried on to
Fish meal dried o n to
cassava
cassava
cassava
cassava
+AO +AO
Moisture Crude protein C r u d e fibre C r u d e fat
Ash Calcium Phosphorus Salt ( N a C l ) Non-protein nitrogen (%total N ) ME (MJ kg-1) -' F o r m i c acid ( % )
Histamine content ( m g per 100 g sample) 2
654 477
+AO
-AO
488 58 10.4 9.0 8.7 5.3
36 36 29 220 238 238 24 22 25 198 210 204 58 60 59 5.8 6.2 6,1 4.4 5.1 4.6 3.9 4.0 4.1 54,3 56.6 30.6
Not detected <5
17,1 1,9 <5
+AO 804 760 107 143 33.7 24.5 14.8 6.3
17.1 17.0 2.2 N o t d e t e c t e d N o t d e t e c t e d <5 <5 <5
1ME = Metabolizable energy calculated using the equation of L o d h i et al. (1976). 2Limit of sensitivity of method = 5 m g per 100 g sample. 3 + AO or - AO = w i t h or w i t h o u t a d d e d antioxidant,
-AO
67 57 70 247 229 246 28 29 29 28 30 27 80 80 88 12.0 10.8 13.9 8.1 7.2 8.8 4.5 4.3 4.8 17.1 19.1 4.9 14.8 3.8 <5
14.6 14.4 3.4 N o t detected <5 <5
CHICKSFEDONLOWANDHIGHOILFISHSILAGE
209
meal were similar, whilst the NPN content of the dried silages increased to only 17-19%.
Formic acid Levels in the silage meals were similar for materials treated or not treated with antioxidant (Table 7). The LOF and HOF silages should have contained 4.6% and 3.4% formic acid, respectively, in the final dried product, but the actual levels were much lower than those, and showed that formic acid was lost during the drying of the silage products. Histamine All the fish products (Table 7) contained < 5 mg per 100 g histamine, which is the limit of sensitivity of the method used. Amino acids As would be expected from material of fish origin the fish silages and meals have a good balance of amino acids and high levels of essential amino acids (Table 8). There were, however, several notable differences between the two types of fish material and the product type. The raw fish and the fish meals of both fish types were very similar in amino acid composition except that the fish meals were in general lower in lysine than the raw fish. However, the reactive lysine contents of both materials and fish types were similar. The cystine content of silages made from both types of fish material was markedly lower than that of raw fish. This was most pronounced with HOF in which the silage cystine levels were about half of the non-silage materials. Methionine contents of both fish silage and fish-meal products were slightly less than those of raw fish. There was a noticeable difference between the two fish types in histidine content, the HOF products having nearly twice as much as the LOF. There was, however, very little difference between the histidine levels of products derived from each type of fish. The reactive lysine contents of all materials indicate that the lysine of all the fish products was highly available and that in general the lysine of the processed fish products was slightly more available than lysine in the raw fish. The tryptophan contents of the antioxidant-treated HOF silage and fish meal were unchanged during processing, whilst the silage without antioxidant had slightly lower levels than that of the raw fish. In the case of LOF products the tryptophan contents of both fish silages were markedly reduced during processing, whilst that of the fish meal was only slightly less than that of raw fish.
210
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CHICKS FED ON LOWAND HIGH OIL FISH SILAGE
211
Fatty acids Table 9 shows that although the two fish materials differed markedly in overall fat content, they were similar in the composition of fatty acids. There were, however, considerable differences in fatty acid composition between raw and processed HOF though only small differences between similar LOF materials. The levels of many of the saturated fatty acids in the raw HOF were lower than similar fatty acids in processed HOF materials though the levels of polyunsaturated fatty acids in HOF processed materials were much lower in all processed materials than in the raw fish. The dried fish silages also had slightly lower levels of certain unsaturated fatty acids than dried fish meal; this loss TABLE 9
Fatty acid composition of raw fish, fish silage and fish meals (%) Fatty acids
High oil fish material Raw minced
C12:0 C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C18:4 C20:1 C20:2 C20:3, C20:4 C20:5 C20:0 C22:1 C23:1 C24:0 C24:1 C22:5 C22:6
<0.1 4.9 0.4 0.6 <0.1 18.2 6.0 1.5 0.7 3.6 20.5 1.5 1.5 2.9 4.7 0.3 1.3 6.8 <0.1 7.8 0.1 0.5 1.9 1.5 12.9
Silages dried on to cassava
Low oil fish material
+ AO
- AO
Fish meal dried on to cassava + AO
<0.1 6.4 0.5 0.9 0.1 26.6 7.8 1.8 0.9 5.5 28.5 1.1 0.3 0.6 5.8 <0.1 <0.1 0.4 <0.1 7.8 0.4 <0.1 1.7 0.3 2.0
<0.1 6.2 0.5 0.9 0.1 25.3 7.6 1.8 0.8 5.4 27.8 1.4 0.9 0.7 5.7 0.2 <0.1 1.3 0.5 7.7 0.5 <0.1 1.7 0.3 2.7
<0.1 6.0 0.5 0.9 0.1 23.7 7.4 1.9 1.0 5.4 27.0 1.7 0.9 0.9 5.7 0.2 <0.1 1.8 0.7 8.0 0.5 <0.1 1.8 0.4 3.5
Raw minced
<0.1 3.4 0.3 0.5 <0.1 14.1 5.2 1.4 0.7 3.6 18.3 2.3 1.0 2.0 8.5 0.7 0.9 6.1 0.8 11.7 0.8 0.5 1.7 1.5 14.8
Silages dried on to cassava + AO
- AO
<0.1 3.6 0.3 0.4 <0.1 14.8 6.2 1.3 0.7 3.3 19.9 3.4 1.3 1.9 9.4 0.4 0.8 6.6 <0.1 10.8 0.4 1.3 1.4 11.8
<0.1 4.4 <0.1 0.4 <0.1 14.9 5.8 1.1 0.6 3.1 19.9 3.2 1.3 1.8 9.0 0.3 0.8 7.1 <0.1 10.9 0.3 1.0 1.2 12.9
Fish meal dried on to cassava + AO <0.1 4.0 <0.1 0.4 <0.1 13.5 6.0 1.2 0.7 3.2 18.7 4.5 1.3 2.0 8.1 0.5 0.9 7.4 0.4 9.0 0.4 0.7 1.1 1.6 14.8
212
D.H. MACHIN ET AL.
was greatest with the most unsaturated fatty acids and also appeared greater in the silage treated with antioxidant t h a n the untreated silage. These two effects were most marked with C18: 3, C18: 4, C20: 5 and C22: 6 fatty acids.
Changes in TBA values during processing and feeding offish products The results of monitoring TBA values as an indication of lipid oxidation are given in Table 10. W i t h both LOF and HOF there was a rapid increase in TBA values up to Day 3 followed by a fall by Day 9-10 after which the TBA values remained constant. The TBA values for HOF material were much higher t h a n those for a similar quantity of LOF material, and were also much greater per unit of lipid in HOF t h a n LOF materials. The TBA values were also greater in the fish silages t h a n the fish meals, though it was not possible to detect differences between silages treated with antioxidant and those which were untreated.
Composition of diets The composition of feeds shown in Tables 4 and 5 was within acceptable limits of the formulated specifications apart from the low oil, high level fish silage diet treated with antioxidant and supplemented with amino acids (F2, 01, L2, A2, $2) which had a slightly lower level of tryptophan t h a n formulated. This level was, however, considered to be within the normally acceptable range of experimental error for the technique used. TABLE 10 Changes in TBA valuesof fish materials over time (mg malonaldehydekg- 1dry matter) Storage (days)
0 3 9 10 14 17 88 179 203 294
High oil fish material
Low oil fish material
Silage + AO
Silage - AO
Fish meal + AO
Silage + AO
Silage - AO
Fish Meal + AO
77.3 271.3 153.1
77.3 271.2 196.1
77.3 83.9 138.8
3.3 22.5
3.3 25.0
3.3 7.0
10.5 3.9
13.4 3.1
1.4
3.3
2.7
3.0
7.8
6.3
4.0
181.5
186.1
128.4
125.2
83.0
164.7
181.0
139.0
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
213
Performance of broiler chicks The live weight gains, feed-conversion ratios and pancreas and liver weights of chicks fed on the experimental diets are shown in Table 11. Statistical analyses are given in Table 12. The chicks remained healthy throughout all three experiments and mortality was low ( 4.1% overall).
Weight gain Table 12 and Fig. 1 show that the oil content of the fish, level of inclusion and the processing of fish as fish meal or silage were significant sources of variation, but that treatment of materials with antioxidant or supplementation with amino acids did not have significant main effects. Chicks fed on LOF materials or fish materials at low levels of inclusion or fish meals had significantly greater weight gains than those fed on HOF material or fish materials at high levels of inclusion or fish silage. There were significant interactions between level of inclusion and processing as silage or meal, between level of inclusion and treatment with antioxidant and between level of inclusion and amino acid supplementation. Addition of TABLE
11
The performance of broiler chicks Fish type
ProInclusion AntiAmino Live weight gain Food conversion Pancreas Liver cessing oxidant Acids (0-3 week) (g) ratio (g) (g)
High oil
Fish silage
High
Low
Low
oil
Fish meal Fish silage
High Low High
Low
+
+
419
1.71
1.74
-
+
415
1.63
1.65
11.71
+
-
415
1.70
1.87
11.74
-
--
458
1.63
1.79
13.89
+
+
508
1.60
2.03
13.79
-
+
529
1.57
2.19
14.50
+
-
492
1.64
1.84
13.17
+
-
484 503
1.60 1.61
2.03 1.87
13.71 13.43
+ +
+
506 463
1.65 1.43
2.18 1.75
13.42 12.57
-
÷
509
1.42
1.75
13.54
+
-
480
1.44
1.77
13.42
+ +
+ + -
504 560 546 554
1.43 1.39 1.42 1.42
1.90 2.09 1.98 1.85
13.17 13.48 14.61 13.97
+ +
-
545 537 564
1.43 1.42 1.43
2.00 2.00 2.07
14.42 15.99 14.36
0.03
0.03
0.22
-
Fish meal S E o f means
High Low
10.8
12.99
D.H. MACHINET AL.
214 TABLE 12
Statistical analyses of factors assessed Source of variation
Variance ratios Weight gain
Cage factors Size Period Size × Period
Food conversion
Pancreas weight 1
1.0 46.1"** 30.1"**
19.2"** 61.1"** 13.0"**
2.7 1.2 25.5***
51.9"** 51.1"** 20.1"** 2.7 0.1
725.0*** 7.5 0.8 8.5** 2.0
5.9* 2.9 5.8* 0.1 <0.1
22.1"** 3.9 1.6 1.5 0.4 8.6** 0.1
2.4 1.1 4.8* 1.0 0.1 0.1 0.1
0.0012
0.028
Liver
weight I
0.1 15.5"** 8.3***
Nutritional factors 0 L F A S
3.3 9.2** 0.4 <0.1 1.3
Interaction 1 LxF LXA LXS O×L OXF O×A OXS Error mean square
7.5** 3.9* 4.1" <0.1 0.4 <0.1 0.2 886
<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 1.1 1.20
1Adjusted by covariance for differences in live weight: Abbreviations: 0 = oil content of fish; L = level of inclusion of fish material; F = processing of fish as silage or meal; A -- antioxidant treatment; S -- amino-acid supplementation. * = P < 0.05; ** = P < 0.01; * * * - P < 0.001.
antioxidant caused a reduction in weight gain at the high level of fish silage inclusion, but an increase at the low level, and supplementation with amino acids caused a reduction in weight gain at high levels of inclusion, but an increase in weight gain at low levels of inclusion (see Fig. 1). No significant interactions were noted between level of inclusion and oil content of fish, oil content of fish and processing of fish as silage or meal, antioxidant treatment and supplementation of diets with amino acids. Cage size had no significant effect on weight gain, but there was a highly significant effect of period and a size X period interaction.
CHICKS FED ON LOWAND HIGH OIL FISH SILAGE
215
(A) 700]
•
Silage
600/1
m Meal
|
500 -' Live weight gain (g)
400300200
1000¸
High Low inclusion inclusion High oil content
High Low inclusion inclusion Low oil content
fish
fish
(B) 7007
• +AO
• +AA
600 |1
E -AO
[ ] -AA
t
500" Live 400 weight gain (g) 300-
200100O"
High
Low
inclusion
inclusion
(DAntiox[dant addition
High
Low inclusion qi) Amino odd supplementation inclusion
Fig. 1. (A) Main effects on weight gain (also illustrates level of inclusion and method of processing interactions). ( B ) Interaction between level of inclusion and (i) antioxidant and (ii) amino acid supplementation ( + A O or - A O = w i t h or without added anti-oxidant, + A A or - A A = w i t h or without added amino acid).
Feed conversion ratios Table 12 and Fig. 2 show that the oil content of fish, level of inclusion and antioxidant treatment each had significant effects. Processing fish as fish meal or silage and supplementation of diets with amino acids had no significant effect on feed conversion ratios (FCR). Chicks fed on LOF materials at low levels of inclusion and material not treated with antioxidant had better FCR than those fed on HOF material at high levels of inclusion and fish treated with antioxidant. There were significant interactions between levels of inclusion and processing of fish material as silage or meal and between oil content
216
D.H. MACHINET AL.
(A) 1.8 1.6 1.4 1.2 Food conversion ratio
1.0 0.8 0.6 0.4 0.2 0 High inclusion
Low inclusion
High inclusion
High oil content fish (a)
(i) LEVEL OF INCLUSION AND METHOD OF PROCESSING
•
Silage
Low inclusion
Low oil content fish (ii) OIL CONTENT OF FISH AND ANTIOXIDANT ADDITION
•
+AO
1.8 1.6 1.4 1.2 Food conversion ratio
1.0 O. 8 0.6 0.4 0.2 0 High inclusion
Low inclusion
High oil content fish
Low content fish
Fig. 2. Main effects on food conversion ratio (also illustrates level of inclusion and antioxidant addition interactions). (A) Oil content of fish, antioxidant addition and inclusion effects. (B) Interaction between (i) level of inclusion and method of processing, (ii) oil content of fish and antioxidant addition.
of fish material and antioxidant addition. Other interactions were not significant. All cage factors assessed showed that the F C R of chicks were very markedly affected by cage size and period and that there was a highly significant size × period interaction.
Pancreas weights These were analysed with chick live weight as a covariate. Only the oil content of the fish and processing as fish meal or silage had significant effects on
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
217
pancreas weight. Pancreas weights for chicks fed on the HOF materials and those fed on fish meal were greater than those for chicks fed on LOF and fish silages, respectively. Of all interactions studied, only that between level of inclusion and amino-acid supplementation was significant. Cage factors, size and period, had no significant effects on pancreas weight though there was a significant size X period interaction.
Liver weights Statistical analyses similar to those for pancreas weight (Table 12) were carried out for chick liver weights. Of all nutritional factors examined only level of inclusion had a significant effect on liver weight. Diets with the high inclusion of fish material produced heavier livers, relative to live weight. None of the interactions studied was significant. Cage size had no significant effect, but there was a significant effect of period on liver weight and a size X period interaction. DISCUSSION
The results of this experiment show that fish silage made from both oily and non-oily fish can be made into dry meals with cassava and that both products can be fed to chicks with varying degrees of success. There were, however, considerable differences in performance between chicks given the various fish products, which relate significantly to the various treatments the products underwent.
Level of inclusion The results of this experiment are in line with those of previous workers (Disney et al., 1978; Kompiang et al., 1979, 1980; Rattagool et al., 1980a-c; Hall, 1983) and show that where fish silage products form more than 10% of the diet, performance is inferior. However, in this experiment the higher level of inclusion resulted in a much smaller reduction in performance than those shown in most previous studies. Weight gains of chicks fed on the HOF and LOF silages at high rates of inclusion were 85 and 91%, respectively, of those of chicks fed on the fish meals. Previous workers (Disney et al., 1978; Kompiang et al., 1980) have reported levels of performance of less than 66% of those of controls. Considerable care was taken to establish the chemical composition of the fish products, which had not been possible in other experiments, and hence it was possible to formulate diets to a greater degree of accuracy than in most earlier studies. This would account for a large proportion of the improved livestock performance in this study. However, there was a considerable difference in the performance of chicks given diets with low and high levels of inclusion, which cannot be explained by simple formulation differences.
218
D.H. MACHIN ET AL.
Changes in the protein component offish products The HOF material had much higher levels of N P N than the LOF material and in both cases N P N levels of fish silages were much higher than those of fish meals. The breakdown of the protein reflected in the N P N values could have considerable significance with regard to the lower performance of chicks receiving high levels of N P N in silages or fish products in general at high inclusion levels. Gilberg and Raa (1977) reported that only 8% of ammonia was produced in acid-treated cod viscera silage stored at 27°C for 220 days. It can therefore be assumed that since the material used in this experiment was stored at - 20 ° C for most of the time between drying and feeding, little ammonia would have been produced which could have had a detrimental effect on chick performance. The histidine contents of the HOF fish products were much greater than those of the LOF materials. During spoilage, decomposition of histidine occurs in fish products (Disney et al., 1978; Raa and Gilberg, 1982) to produce toxic products such as histamine. In this experiment considerable care was taken to ensure that all fish were fresh and to prevent decomposition. As a result, histidine levels of all fish products in this study showed little change from raw fish. Clearly if spoilage had occurred in the fish products the HOF materials with their high histidine content would be more likely to produce high histamine levels than would LOF materials. The histamine content of both raw fish and the processed silages and meals was very low and indicates that any lowered chick performance could not be directly associated with high levels of histamine or fish spoilage. Several changes in the amino acid composition of the fish products were identified in this study and in particular lowered levels of cystine in the dried HOF silages, lowered lysine and methionine in all processed fish products and lowered tryptophan levels in the dried LOF silages. Disney and Hoffman (1976) noted an increase in food intake when silages dried on to substrates were supplemented with lysine and methionine though no amino acid determinations were carried out. The reduction in these two amino acids in the processed fish products in this experiment provides an explanation for these findings. The loss of tryptophan in acid medium is well documented (Backoff, 1976; Jensen and Schmidtsdorff, 1977; Johnsen and Skrede, 1981 ). Kompiang et al. (1980) further report that this amino acid is stable at low pH when present in proteins but degrades when free. Clearly, in silages with high levels of NPN, tryptophan is likely to be seriously degraded. In this experiment tryptophan losses were apparent in the LOF silages, but surprisingly not in the HOF silages, which had the greatest N P N levels. In all previous cases of high tryptophan losses the fish materials were maintained at low pH for longer than in
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
219
this study. The low tryptophan losses in this study reflect the short time the fish was ensiled in acid medium. Several authors have indicated that small deficiencies in tryptophan have greater significance than would be expected (Gorbatov, 1976; Lougnon et al., 1985). However, the slightly low tryptophan levels in unsupplemented fish silage diets did not appear to affect livestock performance. To assess the significance of these amino acid losses in the fish products and also to offset any lowered availability of lysine and methionine, as suggested by Carpenter et al. (1963), diets in this experiment containing fish silages were supplemented with the amino acids, lysine, methionine and tryptophan to raise them to the levels of the fish-meal diets. The results of the subsequent feeding trial showed that amino acid supplementation produced only a small improvement in the performance of chicks given silage in their diets at low levels of inclusion and no improvement at high levels of inclusion. It is therefore concluded that the lowered performance must be associated with other factors.
Changes in the lipid component offish products The fatty acids of the lipid component of fish products were markedly changed during processing. These changes were mostly associated with the unsaturated fatty acids and, as would be expected, were most pronounced in the HOF products. To monitor oxidative changes taking place in the fish products, malonaldehyde was measured throughout processing, storage and feeding of the fish products. The production of malonaldehyde followed a similar path to that reported by Hall (1983) for peroxide levels in fish silages; in ensiled fish these rapidly increased during the first 3 days, after which they declined and stabilised by Day 7. He did not, however, note a similar effect with malonaldehyde. These results do, however, indicate marked oxidative changes taking place in the HOF silages. A similar effect, but to a much lower extent, took place in the LOF silages and to an even lesser extent in the fish meals. Additions of antioxidant to fish silages did not appear to affect levels of malonaldehyde. The fatty acid composition of raw fish, silages and fish meals dried onto cassava was measured before the feeding trial commenced. A considerable reduction in the unsaturated fatty acids of the HOF materials and especially of the polyunsaturated fatty acids, C18: 3, C18: 4, C20: 3, C20: 4, C20: 5, C22: 5 and C22: 6 had taken place, but only a slight reduction in the unsaturated fatty acid levels of LOF materials. Levels of these unsaturated fatty acids were also slightly lower in silages than fish meals and in the cases of HOF materials the levels of C18:3, C20:5 and C22:6 were slightly lower in silages treated with antioxidant than in those not treated. All these results are in agreement with those of Hall and Ledward (1986) who found similar effects with silages made from silver-belly fish (Leiognathus spp. ).
220
D.H. MACHIN ET AL.
A consequence of these losses is a possible deficiency in the essential fatty acids, linolenic acid (C18:3) and arachidonic acid (C20:4), in diets limiting in those nutrients. In this study, diets were formulated to provide at least 1% linoleic acid from non-fish sources and the fish products were only mixed into diets immediately prior to feeding. It is therefore very unlikely that deficiencies in these fatty acids occurred, though the possibility that the oxidized fatty acids of the fish materials could have caused the rapid oxidation of non-fish fatty acids might be considered. A further consequence of the considerable loss of unsaturated fatty acids and production of oxidation products is a reduction in palatability of the meals. Both Dugan (1975) and Barlow and Pike (1977) implicated oxidized fats in loss of appetite and reduction in weight gain though since they monitored only peroxide values it is not possible to compare their results directly with these. That oxidized fats could have adverse effects on palatability and nutritive value of foods through protein and vitamin damage or through increased vitamin requirements has also been proposed by Carpenter et al. ( 1963 ). Clearly these effects could be contributing to the reduction in performance of chicks fed on HOF materials. Chicks fed on fish meals had significantly greater weight gains than those fed on high levels of fish silage, but similar FCRs. The lipid in fish meal was also less oxidized than that in fish silages. From these results it would appear that the fish silages were less palatable than the fish meals without the nutritive value of materials being particularly damaged, and that this could relate to higher levels of lipid oxidation products in fish silages. The fish meal was produced by boiling fish and then drying it on to cassava. As a consequence of boiling, lipases would be inactivated and free fatty acid production reduced (Reece, 1980). Oxidation of free fatty acids could also be reduced by the production of antioxidant materials during boiling (Sen and Bhandary, 1980). It is possible that the lowered oxidation of the fish meals was a consequence of these two effects. Hall and Ledward (1986) reported that TBA values in antioxidant-treated silages were much lower than in untreated silages but that the levels of polyunsaturated fatty acids in treated silages decreased much more than in untreated silages. This effect is hard to explain. In this study the TBA values of both the treated and untreated silages in HOF and LOF materials showed little difference, but the levels of several of the unsaturated fatty acids in antioxidant-treated HOF silage (in particular C18:3, C20:5 and C20:6) were lower than those in the untreated silage. These results give part support to those of Hall and Ledward (1986), although not demonstrating the anomaly of low TBA values whilst at the same time showing greater unsaturated fatty acid loss.
CHICKS FED ON LOW AND HIGH OIL FISH SILAGE
221
Changes in pancreas and liver weights Pancreas weights were significantly affected by both oil content of the fish material and types of processing, whilst liver weight was affected only by level of inclusion. It is possible that the effects on the pancreas were mostly associated with the greater lipase and protease production required for the digestion of materials with high oil contents and of fish meals than for low oil materials and highly soluble silages. The heavier liver weights of chicks receiving the high inclusion-rate diets could be associated with possible subclinical detoxification of formic acid or greater protein storage associated with high intakes of fish material. CONCLUSION The reduced performance of chicks fed on fish silages dried on to substrates is largely due to the oxidation of lipids. This conclusion is in line with those of Kompiang et al. (1980) and Hall (1983). The fish silage products for this study were produced with care to minimise oxidation by the use of fresh fish materials, storage of materials at low temperature, minimum ensilage periods and use of antioxidants. Diets were also carefully balanced to meet the chicks' requirements. These precautions yielded a better performance of chicks fed on the silages at high levels of inclusion than that reported by other workers. However, it is difficult to consider how further improvements could be achieved through this approach, though there is clearly considerable potential for improvement. Possible alternative approaches to improving the performance of chicks given silage include the use of preheating treatments to deactivate lipases (and, as a consequence, other enzymes) followed by preservation of fish material using acids without autolysis. In this way, both free fatty acid production and fatty acid oxidation would be reduced and protein would remain intact so that amino acids would be less open to damage. ACKNOWLEDGEMENTS The authors thank Prof. R. Mead for his assistance with experimental design and analysis of data, Mr. B. Waite for assistance with care of experimental birds and Mrs. P. Carlaw for analysis of ingredients and diets. REFERENCES Backoff,H.P., 1976. Somechemicalchangesin fish silage.J. FoodTechnol.,11: 353-363. Baker, R.J. and Nelder,J.A., 1978.The GlimSystem (Release3). NumericalAlgorithmsGroup., Oxford, 150 pp. Barlow, S.M. and Pike, I.H., 1977. The role of fat in fish meal,in pig and poultrynutrition. Int. Assoc.Fish Meal Manufacturers,Tech. Bull., No. 4, 38 pp. Canale, A., Valente, M.E. and Ciotti, A., 1984. Determinationof volatile carboxylicacid (C1 to
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C5) and lactic acid in aqueous acid extracts of silage by high performance liquid chromatography. J. Sci. Food Agric., 35: 1178-1182. Carpenter, K.J., Lea, C.H. and Parr, L.J., 1963. Chemical and nutritional changes in stored herring meal. 4. Nutritional significance of oxidation of the oil. Br. J. Nutr., 17: 151-169. Disney, J.G. and Hoffman, A., 1976. A dried fish silage product. In: Proceedings of the Torry Research Station Symposium on Fish Silage. Aberdeen, Scotland, pp. 1-14. Disney, J.G., Hoffman, A., Olley, J., Clucas, I.J., Barranco, A. and Francis, B.J., 1978. Development of a fish silage/carbohydrate animal feed for use in the Tropics. Trop. Sci., 20: 129-144. Dugan, L., 1975. Lipids. VI. Chemical properties and reactions. In: O.R. Fennema (Editor), Principles of Food Science. Vol. 1. Marcel Dekker, New York, NY. Gilberg, A. and Raa, J., 1977. Properties of propionic acid/formic acid preserved silage of cod viscera. J. Sci. Food Agric., 28: 47-53. Green, S., 1984. The use of fish silage in pig nutrition. Ph.D. Thesis, University of Nottingham. Gorbatov, V.M., 1976. The utilisation of blood and other slaughter by-products. Proceedings of the European Meeting of Meat Research Workers, 1976. Vses N. auchno-Issled. Inst. Myasnoi Promyshlennosti Moscow, 22: 10.1. Hall, G.M., 1983. Silage from tropical fish. Ph. D. Thesis, University of Nottingham. Hall, G.M. and Ledward, D.A., 1986. Silage from tropical fish 3. Lipid behaviour. J. Food Technol., 21: 45-54. Hardy, R. and Smith, J.G.M., 1976. The storage of mackerel (Scromber scrombus ). Development of histamines and rancidity. J. Sci. Food Agric., 27: 595-599. Hofstetter, H.H., Sen, N. and Holman, R.T., 1965. Characterisation of unsaturated fatty acids by gas liquid chromatography. J. Am. {Oilseeds) Chem. Soc., 42: 537-540. Jensen, J. and Schmidtsdorff, W., 1977. Fish silage, low fat and soluble fish protein products. International Association of Fish Meal Manufacturers Symposium. Production and Use of Fish Meal. Szczecin, Poland, pp. 23-35. Johnsen, F. and Skrede, A., 1981. Evaluation of fish viscera silage as a feed resource - chemical characteristics. Acta Agric. Scand., 31: 21- 28. Kompiang, I.P., Arifudin, R. and Raa, J., 1979. Nutritional value of ensilaged by-catch fish from Indonesian shrimp trawlers. In: J.J. Connell (Editor), Advances in Fish Science and Technology. Farmhouse Fishing News Books, Farnham, 349 pp. Kompiang, I.P., Darwanto, A. and Arifudin, R., 1980. Nutritional value of fish silage. In: Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 44-47. Lodhi, G.N., Daulat, Singh and Ichhponani, J.S., 1976. Variation in nutrient content of feedingstufts rich in protein and reassessment of the chemical method for metabolizable energy estimation for poultry. J. Agric. Sci., 86: 293-303. Lougnon, J., Bertrand, S., Mathe, J. and Pieri, A., 1985. Nutritional value and originality of tryptophan. Communication presented at 16th Journ~es de la Rdcherche Porcine en France, Paris. Lucas, B. and Sotelo, A., 1980. Effect of different alkalies, temperature and hydrolysis times on tryptophan determination of pure protein and of foods. Anal. Biochem., 109: 192-197. Ministry of Agriculture, Fisheries and Food, 1976. The Fertilisers and Feeding Stuffs (Amendment ) Regulations. Statutory Instrument No. 840, Her Majesty's Stationery Office, London. Poulter, R.G., Jayawardena, K.M., Ganegoda, P. and Ranaweera, K.N.P., 1980. Studies on fish silage in Sri Lanka - a summary. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 64-68. Raa, J. and Gilberg, A., 1982. Fish silage: A review. CRC Crit. Rev. Food Sci. Nutr., 16: 383-419. Rattagool, P.P., Swachathamwongratana, S. and Wongchinda, N., 1980a. Fish silage in Thailand: further trials on broiler chicken. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 59-62. Rattagool, P.P., Wongchinda, N. and Swachathamwongratana, S., 1980b. Studies on the nutritive
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value of fish silage for broiler chickens. Proceedings Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 48-54. Rattagool, P.P., Wongchinda, N. and Swachathamwongratana, S., 1980c. Fish silage in Thailand. Trial feeding on broiler chicken. Proceedings of Indo Pacific Fisheries Commission Workshop on Fish Silage. FAO Fish. Rep. No. 230, pp. 55-58. Reece, P., 1980. Control and reduction of free fatty acid concentration in oil recovered from fish silage prepared from sprat. J. Sci. Food Agric., 31: 147-155. Roach, A.G., Sanderson, P. and Williams, D.R., 1967. Comparison of methods for the determination of available lysine value in animal and vegetable protein sources. J. Sci. Food Agric., 18: 274-278. Sen, D.P. and Bhandary, C.S., 1980. Lipid oxidation in raw and cooked oil sardine, SardineUa longiceps. In: Fats and Oils in Relation to Food Products and their Preparations. Report of the Central Food Technology Research Institute, Mysore, India, pp. 132-133.