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Genotypic and phenotypic fatty acid composition in the tissues of salmon, Salmo salar
Comp. Biochem. Physiol. Vol. 96B,No. 4, pp. 721-727, 1990
0305-0491/90$3.00+ 0.00 Pergamon Press plc
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GENOTYPIC A N D PHENOTYPIC FATTY ACID COMPOSITION IN THE TISSUES OF SALMON, SALMO SALAR* ANITA VIGAt and OTTO GRAHL-NIELSEN~§ tZoological Laboratory and :~Department of Chemistry, University of Bergen, Allegt. 41, N-5007 Bergen, Norway (Received I I January 1990) Abstract--l. Two-year-old salmon were fed for 8 months with three diets with small, but significant, differences in lipid content and fatty acid composition. 2. The fatty acid composition in abdominal fat, red muscle, white muscle, liver and heart of the fish was determined at the end of the feeding period by a chemometric method. 3. The fatty acid composition of all tissues differed from that of the diets, least in the abdominal fat, most in the liver and heart tissue. 4. The fatty acid composition of the tissues, the heart tissue in particular, was independent of the fatty acid composition of the diets. 5. Large differences in the fatty acid compositions were observed between different fish. 6. The advantage of multivariate interpretation of fatty acid comfaositions is demonstrated.
INTRODUCTION It is commonly acknowledged that the composition of fatty acids of neutral lipids, and to a lesser degree, of phospholipids, in the tissues of fish is influenced by the composition of fatty acids in the dietary fat (Sargent et al., 1988). This knowledge has been mainly reached on the basis of investigations into the dietary requirements of species reared in captivity. In such investigations diets with extreme compositions are often used (Anderson and Arthington, 1989; Leger et al., 1981; Leray and Pelletier, 1985; Lie et aL, 1986). From the data presented in these, and several other publications, there is no doubt that a large change in the fatty acid composition of the diet results in changes in tissue fatty acid composition, although compositions identical to that in the diet is never experienced. Fish do have the ability to synthesize fatty acids de novo, and also to selectively metabolize, absorb or discharge fatty acids (Sargent et al., 1988). Apparently, fresh water species carry out these abilities more efficiently than marine species (Owen et al., 1975). They do this to obtain an optimal fatty acid composition (Ackman, 1980), which must be characteristic for each species, i.e. genotypic. The fatty acid composition might therefore be used to gain systematic information, i.e. distinguishing between different stocks of the same species (Grahl-Nielsen and U1vund, 1990). However, under natural conditions the fatty acid composition will be influenced by nongenetic phenomena, like changes in temperature, (Malak et al., 1989), salinity (Daikoku et al., 1982), growth (Muje et aL, 1989), the reproductive cycle (Henderson et al., 1984), pollution (Morris et al., 1982). In addition comes the influence of the diet *Submitted in partial fulfillment of the requirements of Anita Viga for the degree of Cand. Scient. §Author to whom correspondence should be addressed.
(Henderson and Tocher, 1987). The lipids of fish are composed of 20 or more fatty acids, and it is reasonable that different external influences affect different fatty acids. For example, temperature changes are mainly manifested in the polyunsaturated fatty acids. Use of the fatty acid composition to gain genotypic or phenotypic information therefore requires knowledge of the natural variation. The fatty acid composition varies markedly between different tissues, and also between different classes of lipids within the same tissue. Depending on the purpose, investigations may span from fatty acid composition in total body lipids, e.g. in fish oils (Lambertsen and Br~ekkan, 1965; Ackman and Eaton, 1966) to composition in different lipid classes, e.g. in various phospholipids in selected tissues (Castledine and Buckley, 1982). Selection of the target for the analysis depends on the purpose of the investigation, but the broader the target the more will superposition of fatty acids from different lipid classes and/or tissues complicate the evaluation of the results. Large differences in the fatty acid composition between individual specimens were demonstrated as far back as in 1967 by Jangaard et al. (1967). Still, almost all investigations are carried out on pooled samples from several fish. In most investigations evaluation of the analytical results have been done by simply listing tables of fatty acid compositions, followed by statements of similarity or dissimilarity, without any quantitation of these differences. Still, in a few investigations the Student's t-test has been used to test the significance of the difference between samples when one fatty acid is compared at a time. However, covariation of several fatty acids is most likely under these circumstances, and quantitative evaluation of the data should call for multivariate statistics.
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ANITA VIOA and OTTO GRAHL-NIELSEN
The purpose o f this investigation was to see if small, b u t significant, differences in the fatty acid c o m p o s i t i o n in the diet will influence the fatty acid c o m p o s i t i o n o f various tissues of salmon. It was considered necessary to use a relatively long time for equilibration to the diets, to avoid effects carried over from lipids stored in the fish. F u r t h e r more, differences between individual fish should be studied. It would have been desirable to study the fatty acid c o m p o s i t i o n o f all the different lipid classes in different tissues. However, since several fish are needed to statistically evaluate the differences between the individuals, the total n u m b e r of analyses a n d l a b o u r involved would have become prohibitive. Therefore, it was chosen to examine the fatty acid c o m p o s i t i o n of several, highly different tissues, ranging from intestinal fat, considered to be pure storage fat, or triglycerides, via red a n d white muscle a n d liver to h e a r t tissue, the lipids of the latter consisting mainly o f phospholipids (Grahl-Nielsen a n d U l v u n d , 1990). The objective was to use a statistical multivariate method, based o n Principal C o m p o n e n t Analysis, for evaluation of the differences/similarities in the results. Hereby the c o m b i n e d effect o f all the fatty acids in the diets a n d tissues could be determined.
MATERIALS AND METHODS Smolt of Atlantic Salmon, S a l m o salar, in May 1986 were distributed randomly in nine net pens, each of 100m 3, 2-300m from shore at Holmane, Sirev~.g, in southern Norway. They were fed a commercial dry diet, Tess Elite from Skretting, A/S, Norway, which is widely used in salmon-farming in Norway. In August 1987, the salmon had reached an average weitht of I kg. At this time the feeding experiment started. Salmon in three of the pens were given Edel, in three other pens Edel Lt. These are both commercial dry diets, based on herring flour ca 60%, capelin oil ca 15%, carbohydrates ca 20%, and additives like vitamins, minerals and binding substances ca 5%. The herring flour of the two diets had been produced in different ways, in the Edel by a normal process, and in the Edel Lt by a low temperature process. The two diets also differed in that the Edel Lt had ca 0.5% less capelin oil. These differences resulted in that Edel had a lipid content of 18.5% and Edel Lt 14°, The salmon in the last three pens were fed with an experimental diet based on the same ingredients, but in different proportions: ca 54% herring flour from the low temperature process, ca 23% capelin oil, ca 30% carbohydrates and ca 4% additives. The resulting lipid content of this diet was 21%. The lipid contents of the three diets, 14%, 18.5% and 21%, respectively, were determined as fatty acid content by using the fatty acid 17 : 0 as internal standard in the analyses described below. The accuracy of this determination was not very high, but this is not elaborated on, since it is the relative amounts of the various fatty acids which are of importance for this investigation. The three diets are referred to as low, medium and high fat diet. Fish were collected for determination of tissue fatty acids in May 1988, i.e. after they had been fed for ca 8 months on the experimental diets. The average weight of the fish were 3.4 + 0.9 kg. There was no significant difference between the weights of the fish fed the different diets, although those fed the high fat diet tended to weigh less than the others. A significant difference between the three groups was
the absence of intestinal fat in those fed the low fat diet. Those fed the high fat diet had more intestinal fat than those fed the medium fat diet. Samples were retrieved from the following tissues: white and red muscle were cut dorsally from a caudal section, the liver was cut in two halves and samples taken from the middle, the heart tissue was then taken from the tip of the ventricle, the intestinal fat, only present in fish fed medium and high fat diet, was collected from the area around the pylorus. Between 10 and 20 mg were used for each determination, and up to five parallel samples from each tissue in each fish were analyzed. The analysis of the fatty acids was based on direct methanolysis (Stoffel et al., 1959) of the tissue samples. The main difference from established methods for determination of fatty acids in tissues of marine animals are the small sample amounts needed, and the exclusion of extraction of lipids prior to methanolysis (Grahl-Nielsen and Barnung, 1985). The samples were transferred directly to thick-walled glass tubes, 0.8 ml anhydrous 2 N HCI in methanol was added and the tubes were securely tightened with Teflonlined screw caps. The reagents contained 0.05% of the antioxidant 4 - m e t h y l - 2 , 6 - d i t e r t b u t y l p h e n o l (BHT). Methanolysis was complete after 2 hr at ll0°C. The fatty acid composition of the diets was determined by the traditional method based on extraction with methanol/chloroform by the method of Folch et al. (1957). Aliquots of the washed chloroform extract were transferred to similar tubes as those used for the tissue analyses, the chloroform was thoroughly evaporated under a ~tream of nitrogen gas, and the methanolysis performed aff-'described above. The methanol/HC1 was evaporated by nitrogen gas down to 0.4 ml and an equivalent amount of water was added. The fatty acid methyl esters were then extracted twice with 2 ml hexane. After adjustments of concentrations in the extracts by partial evaporation of the hexane for the tissues with low lipid levels, or dilution with hexane for the adipose tissue and the red muscle tissue, 1 #1 of the final solution was gas chromatographed on a 30 m x 0.32 mm fused silica column, from J&W Scientific, with 50% cyanopropylmethyl-50% methyl-phenyl polysiloxane, of 0.25 micron thickness, as stationary phase, and helium as mobil phase. The components eluting from the column were detected by a flame ionization detector, and the detector output was coupled to a VG Multichrome lab-data system for storage and treatment of the chromatograms. The 16 fatty acids, from 14:0 to 22:6n3, as methyl esters, given in Table 1, were identified by comparison with standards. The amount of the various acids in the samples was found by determination of the areas of the peaks in the chromatograms, and the amounts were normalized by expressing each as the percentage of the sum of the 16 acids. For multivariate treatment, the resulting relative numbers, given in Table 1, were loaded into a Sperry PC. To level out the large differences in numerical values between the least and the most abundant fatty acids, the data were scaled by taking the logarithms. Various combinations of the data, as discussed later, were then subjected to principal component analysis by the program SIMCA (Albano et al., 1981), available in the software package SIRIUS (Kvalheim and Karstang, 1987). The program places the samples in a 16-dimensional space, i.e. with one co-ordinate for each of the 16 acids. The dimensionality is then reduced from 16 to 2, which represents the directions of the largest and second largest variation between the samples in the 16-dimensional space. The two new dimensions are represented by two principal components, and the samples are plotted in a co-ordinate system of these, giving the PC-plots shown in Figs 1-3. Separate PC-models of groups of samples belonging to the same diet were formed, as discussed below.
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The composition of the fatty acids in the three diets is given in Table 1. The largest difference is in 18:4 n3, which is 1.5 times higher in the medium fat diet than in the low fat diet. For the other fatty acids the differences between the highest and lowest value is in the order of 5-25%. When the SD of the determinations of 5% is taken into consideration, it is clear that the fatty acid composition of the three diets are quite similar. It is likely that salmon encounters differences in this order of magnitude when preying on different organisms under natural conditions. This is in contrast to the much larger differences between experimental diets used in other investigations, many times with some fatty acids completely absent from one of the diets (Leger etal., 1981; Leray and Pelletier, 1985; Lie etal., 1986). The common way of evaluating differences between diets, as carried out above, takes only one fatty acid into account at a time. To make a comparison between the three diets on the basis of all the fatty acids, principal component analysis was undertaken. This resulted in the PC-plot in Fig. 1. The plot represents 98% of the total variance between the 12 diet samples. It is demonstrated here that even if the differences between the three diets are small when one fatty acid is considered at a time, the difference based on all fatty acids is considerable. To enumerate the differences between the diets, statistical class models were formed. A minimum of five samples is required to form a model. Therefore, a model of the low fat diet, with only two replicates, could not be formed. The models are based on the relative position of the replicates in the space with one dimension for each of the fatty acids, i.e. 16 dimensions. The outer limit of the models are determined in terms of relative SD of its samples, within a chosen limit of significance, in this case 95%. This is the RSDm~ for the model. The distances of all the 12 samples from the two models in the 16-dimensional space are given in Table 2. The RSDm~ for the two models are also shown in the table. It is seen that the five replicates for the medium fat diet fall within the statistical
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AN1TA V1GA and OTTO GRAHL-NIELSEN
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% PC 1 Fig. 2. Principal component plot of the samples of the high fat diet and of the tissues from the fish fed this diet; H, high fat diet; F, intestinal fat; R, red muscle; W, white muscle; L, liver; A, heart. model for that diet. One of the replicates of the high fat diet falls slightly outside its own model. This is because the model has the form of a long, narrow cylinder, leaving the one sample just outside. It is, however, seen from the table that the samples from the other diets fall far outside the model. Furthermore, it is seen that the two low fat samples are less different from the high fat samples than from the medium fat samples. This is in consistence with the PC-plot of the diet samples in Fig. 1. It is thus demonstrated that multivariate data-analysis offers an alternative method for evaluation of the traditional tables of fatty acid compositions. Similarities and differences between samples and/or groups of samples are quantitatively determined by RSD~PC 2
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values, while PC-plots visualizes the relationship between the samples. The results of the determinations of the fatty acids in the tissues of the salmon fed the three diets are given in Table 1. Three features are obvious: (1) the spread of the data within each tissue is considerable, i.e. relative SD are usually in the order of 50% or more; (2) for each tissue, the differences between diets are small; (3) the differences between the various tissues are large. This feature will be discussed first. The values in the table indicate that the fatty acid composition of intestinal fat is least different from that in the diet, and that the composition in the liver and the heart tissue differs mostly. Similar to the evaluation of the diets above, the tissue data are more easily perceived by performing principal component analysis. In the case of the high fat diet this resulted in the PC-plot in Fig. 2. The plot shows that the fatty acid composition of the diet has been metabolically altered by the salmon upon deposition of lipids in the various tissues. The smallest alteration was for the intestinal fat and the red muscle. Nevertheless, the differences are significant. Larger differences between the diet and the Table 2. Relative distances, expressed as residual SD, of the diet samples to the statistical models of the medium and high fat diets Model for medium fat diet
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tissue, respectively. The symbols for the diet samples are the same as in Fig. 1. Numbers in italics represent samples from fish fed the low fat diet, plain numbers represent samples from fish fed the medium fat and bold numbers represent samples from fish fed the high fat diet. Within each group, similar numbers represent different parallel samples from the same fish.
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Salmo tissue fatty acids
725
Table 3. Relativedistances,expressedas residualSD, of the samplesfrom the high fat diet and from the tissuesof salmon fed this diet, to the statisticalmodel of the high fat diet High fat diet Intestinalfat Red muscle Whitemuscle Liver Heart 2 267 273 330 876 642 8 266 275 382 853 879 11 230 230 357 870 888 3 237 317 384 896 852 3 242 282 388 934 884 291 270 478 901 944 311 260 452 1057 717 275 240 289 975 776 235 342 712 886 210 362 761 882 371 773 755 white muscle are obvious, with the largest difference for the liver and the heart. Similar to the case of the diets above, the PC-plot in Fig. 2 is supported by quantitative measurements of the distances of the various samples to the model of the diet, Table 3. The numbers are in accordance with the plot: the samples of the intestinal fat and of the red muscle all have approximately the same distance from the diet. The white muscle samples have larger distances, and the liver and heart samples clearly the largest. There is a much larger spread between the liver samples than between the samples of the other tissues. The livers from the different fish did have different appearances, with respect to size and colour. In the case of the liver, compared to the other tissues, it was more difficult to dissect out a small piece of homogeneous tissue free of blood and other substances. Intestinal fat is considered to function as a store of reserve lipid (Henderson and Tother, 1987), mainly made up of triglycerides. The relative amounts of triglycerides in the tissue lipids are expected to be smaller in red muscle than in intestinal fat, and still smaller in white muscle and liver, i.e. Leger et al. (1981) found 70-90% neutral lipids in red muscle of trout, 65-80% in white muscle and 20-30% in the liver. The variations between species, and the dependance on condition, temperature and other variables are, however, large (Henderson and Tocher, 1987). While the muscle and liver tissue, and also the adipose tissue of fish have been subjected to numerous investigations of lipid and fatty acid composition (Henderson and Tocher, 1987), we are only aware of two investigations on the lipids of heart tissue of fish (AI-Tai et al., 1984; Henderson and Sargent, 1984). In these the amount of phospholipids was not determined, although it was stated in the former that the lipids of the heart contained free fatty acids and triglycerides in addition to phospholipids. This corresponds to findings in our laboratory which showed that lipids from the heart of herring contained 70% phospholipids, 19% free fatty acids and 11% neutral lipids (Grahl-Nielsen and Ulvund, 1990). Even if many investigations have been concerned with the fatty acid composition of phospholipids and triglycerides in different tissues of fish, no effort has been done to quantify observed differences. Still, by inspection of published tables, it is apparent that the composition of phospholipids varies more between the tissues than does the composition of
triglycerides (Cowey et aL, 1976, 1983; Sheridan et al., 1985). This is reasonable since phospholipids have more important physiological functions than triglycerides. It is also clear that the phospholipids and triglycerides of the same tissue have large differences in composition. Since the phospholipids and triglycerides were not separated in the present investigation, the observed, and quantified differences between the tissues are due both to differences in the ratio between phospholipids and triglycerides as well as to differences in the phospholipids. By comparing the compositions of the liver and white muscle tissues in Table l, one acid at a time, it was found that the differences between the two tissues had exactly the same pattern as observed by Lie et al. (1988) for the same tissues of salmon. Very similar patterns of differences have also been observed by Leger et al. (1981) and by Braekkan et al. (1971). It must again be emphasized that the multivariate treatment improves the interpretation of the analytical results over the classical monovarite evaluation of one fatty acid at a time, both by displaying the differences in a PC-plot, by ranking the importance of the fatty acids and by quantification of the differences in terms of class and sample distances. This holds true even in cases like heart tissue vs diet, where the differences can easily be seen in the table of fatty acid compositions. It is particularly valuable when the differences become smaller, as in the case of abdominal fat vs diet. Now, to the question if the differences in the fatty acid composition of the diets have induced differences in the fatty acid composition of the tissues after 8 months? The data in Table 1 do not indicate differences: within each tissue there are only small differences between the three average values for each fatty acid. In addition, the deviations from the averages are large. Principal component analysis of the data were undertaken to see if multivariate treatment would give a different conclusion. The resulting PC-plots for the liver and heart tissues are shown in Fig. 3. For the liver tissue there is a slight indication, shown by the dashed line in Fig. 3, that the fatty acid composition in the fish fed the low fat diet is somewhat different from the compositions in the other two groups of fish. In the figure the tissue samples from the fish fed the low fat diet lie to the left of the other samples, just as the two samples of the low fat diet lies to the left of the other diet samples. This shows that the same fatty acids are responsible for the differences
726
ANITAVIGAand OTTOGRAHL-NIELSEN
in both cases, implying that the fatty acid composition of the diet may have had an influence on the fatty acid composition of the liver tissue. However, since the differences were small, and since no differences were observed between the liver tissues of the fish fed the medium and high fat diets, further elaboration upon this will be left for another investigation. For the intestinal fat and the two muscle tissues the multivariate treatment gave no clearcut differences between samples from fish fed the three diets. In the case of the heart, samples from fish fed the three diets are apparently completely intermingled. Since the difference between the diets, on one hand, and the tissues, on the other, dominates the plot, it was checked whether the three tissues were separated along the third PC, perpendicular to the plane of the plot in Fig. 3. This was not the case, neither were they separated from each other when the calculations were done on the tissue samples alone. The control of the metabolic conversion of the lipids in the diet to those of the heart tissue, which are assumed to be dominated by phospholipids, see above, is strong enough to overrule differences in the diets. The large analytical variance demonstrated by the high relative SD of the results in Table 1, is displayed by the large distances between the sample points in Fig. 3. There are variations between the parallel samples from each fish, but an even higher variation between different fish. This large individual difference corresponds with the finding of Jangaard et aL (1967). Even if such large individual differences have been known for a long time, it is peculiar that only five out of 22 investigations referred to in the present paper have used individual specimens. (The other 16 references are either reviews or do not contain comparisons of fatty acid compositions.) Of the 17 investigations with determinations on pooled samples, 10 have only one determination, while the remaining seven have two or three replicate determinations. Statistical monovariate Student's t-test was used in five of the 22 investigations for comparison between different groups. Only one of these was based on analyses of individual specimens. The other four were based replicate analyses of pooled, homogenized samples. It ought to be clear that when testing the significance of the difference between the two groups, of e.g. fish that have been given different diets, it is the variance between fish within each group which should be considered, not the analytical variance of replicates from the same, pooled and homogenized sample, no matter how many fish have been pooled. A phrase often used in the literature in this field of research is that the fatty acid composition of tissue "reflects" the fatty acid composition in the diet (Owen et al., 1972; Henderson et al., 1984; Lie et al., 1988; Martin et al., 1984; Yu et al., 1977; Leray and Pelletier, 1985; Pagliarani et al., 1986; Cowey et al., 1983; Yingst and Stickney, 1979). This is an ambiguous term without statistical significance. It leaves the impression that the tissue composition is similar to that of the diet and will consequently follow changes in the diet. Unfortunately, this is a common view. It has emerged from investigations with large differences in experimental diets where some consequences for the fatty acid composition of tissues were observed,
although not quantified (Anderson and Arthington, 1989; Leger et al., 1981; Leray and Pelletier, 1985; Lie et al., 1986). In fact, the possibility of tailoring of the fatty acid composition of cultured fish before they go to the market has been suggested (Sargent et al., 1988). The present results suggest that, when encountered with less dramatic changes in the diet, the fatty acid composition of the tissues is not controlled by the diet. This is particularly the case of the heart tissue, with a possible exception for the liver tissue. In accordance with this, several investigations indicate that fish living in their normal habitat with an adequate supply of food have genotypic fatty acid profiles in their tissue. Henderson et al. (1982) suggest that the species related composition becomes less obvious as the lipid level in the diet increases, since levels in excess of 10% depress the fatty acid synthesis in fish, at least in Coho salmon. Ackman and Takeuchi (1986) have shown that wild Atlantic salmon parr maintains a typical fatty acid composition in the tissue by selective accumulation of certain fatty acids, as 20:4n6, from the diet. Rainbow trout possesses the ability to control to a large extent its fatty acid composition (Boggio et al., 1985). They observed a smaller overall net change in the fatty acid composition of the muscle of the trout than the differences in the diet. Muje et al. (1989) have found that vendace developed the ability to metabolically modify the fatty acid composition with age, while Sheridan et al. (1985) found that the fatty acid composition of the smolt of steeihead trout was independent of the diet. Linko et al. (1985) observed that the available plankton feed had no effect on the fatty acid composition of the phospholipids of Baltic herring. Acknowledgement--We thank T. Skretting A/S for making
salmon available for this investigation. REFERENCES
Ackman R. G. (1980) Fish lipids. In Advances in Fish Science and Technology (Edited by Connell J. J.), pp. 86-103. Fishing New Books, Farnham, Surrey, UK. Ackman R. G~ and Takeuchi T. (1986) Comparison of fatty acids and lipids of smolting hatchery-fed and wild Atlantic salmon Salmo salar. Lipids 21, 117-120. Albano C., Blomquist G., Coomans D., Dunn W. J., Edlund U., Eliasson B., Hellberg S., Johansson E., Nord6n B., Sj~str~m M., Soderman B., Wold H. and Wold S. (1981) Pattern recognition by means of disjoint principal component models (SIMCA). Philosophy and methods. In Proc. Syrup. Appl. Stat. (Edited by H~skuldson et al.), pp. 182-217. NEUCC, RECAU ans RECKU, Copenhagen, Denmark. A1-Tai W. F., Jones M. G. and Wilson M. T. (1984) The phospholipids associated with cytochrome C oxidase isolated from beef, dogfish and cod heart. Comp. Biochem. Physiol. 77B, 6094i16. Anderson A. J. and Arthington A. H. (1989) Effect of dietary lipid on the fatty acid composition of silver perch (Leiopotherapon bidyanus) lipids. Comp. Biochem. Physiol. 93B, 715-720. Bell M. V., Henderson R. J. and Sargent J. R. (1985) Changes in the fatty acid composition of phospholipids from turbot (Scophthalmus maximus) in relation to dietary polyunsaturated fatty acid deficiences. Comp. Biochem. Physiol. 81B, 193-198.
Salmo tissue fatty acids Boggio S. M., Hardy R. W., Babbitt J. K. and Brannon E. L. (1985) The influence of dietary lipid source and alpha-tocopheryl acetate level on product quality of rainbow trout (Salmo gairdneri). Aquaculture 51, 13-24. Br~ekkan O. R., Lambertsen G. and Andresen J. (1971) Influence of dietary fat on the fatty acid pattern of muscle and liver lipids in rainbow trout (Salmo gairdneri). Fisk. Dir. Skr., Ser. Tekn. Unders. 5, No. 8 (Dir. of Fish., Bergen, Norway), 12 pp. Casteldine A. J. and Buckley J. T. (1982) Incorporation and turnover of essential fatty acids in phospholipids and neutral lipids of rainbow trout. Comp. Biochem. Physiol. 71B, 119-126. Cowey C. B., Adron J. W., Owen J. M. and Roberts R. J. (1976) The effect of different dietary oils on tissue fatty acids and tissue pathology in turbot Scophthalmus maximus. Comp. Biochem. Physiol. 53B, 399-403. Cowey C. B., Wee K. L. and Tacon A. G. J. (1983) Effect of fatty acid intake on growth and fatty acid composition of liver and muscle of snakehead. Bull. Jap. Soc. scient. Fish. 49, 1573-1577. Daikoku T., Yano I. and Masui M. (1982) Lipid and fatty acid compositions and their changes in the different organs and tissues of guppy, Poecilia reticulata, on sea water adaptation. Comp. Biochem. Physiol. 73A, 167-174. Folch J., Lees M. and Stanley G. H. S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. 3". biol. Chem. 226, 497-509. Grahl-Nielsen. O. and Barnung T. B. (1985) Variations in the fatty acid profile of marine animals caused by environmental and developmental changes. Mar. Envir. Res. 17, 218-221. Grahl-Nielsen. O. and Ulvund K. A. (1990) Distinguishing between different populations of herring by chemometry of fatty acids. In Proc. of the Int. Symp. and Educ. Workshop on Fish-Marking Techniques, Seattle, WA. Am. Fish. Soc. Bethesda ML, (in press). Henderson R. J. and Sargent J. R. (1984) Lipid metabolism in rainbow trout (Salmo gairdnerff) fed diets containing partially hydrogenated fish oil. Comp. Biochem. Physiol. 78B, 557-564. Henderson R. J. and Tocher D. R. (1987) The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26, 281-347. Henderson R. J., Sargent J. R. and Hopkins C. C. E. (1984) Changes in the content and fatty acid composition of lipid in an isolated population of the capelin Mallotus villosus during sexual maturation and spawning. Mar. Biol. 78, 255-263. Henderson R. J., Sargent J. R. and Pirie B. J. S. (1982) Peroxisomal oxidation of fatty acids in livers of rainbow trout (Salmo gairdnerii) fed diets of marine zooplankton. Comp. Biochem. Physiol. 73B, 565-570. Jangaard P. M., Ackman R. G. and Sipos J. C. (1967) Seasonal changes in fatty composition of cod liver, flesh, roe and milt lipids. J. Fish. Res. Bd. Can. 24, 613-627. Kvalheim O. M. and Karstang T. (1987) A general-purpose program for multivariate data analysis. Chemom. Intel. Lab. Syst. 2, 235-237. Lambertsen G. and Br~ekkan O. R. (1965) The fatty acid composition of herring oils. Fisk. Dir. Skr., Ser.
727
Tekn. Unders. 4, No. 13 (Dir. of Fish., Bergen, Norway), 15pp. Leger C., Fremont L. and Boudon M. (1981) Fatty acid composition of lipids in the trout--I. Influence of dietary fatty acids on the trigiyceride fatty acid desaturation in serum, adipose tissue, liver, white and red muscle. Comp. Biochem. Physiol. 69B, 99-105. Leray C. and Pelletier X. (1985) Fatty acid composition of trout phospholipids: Effect of (n-3) essential fatty acid deficiency. Aquaculture 50, 51-59. Lie O, Lied E. and Lambertsen G. (1986) Liver retention of fatty acids in cod (Gadus morhua) fed different oils. Aquaculture 59, 187-196. Lie O, Waagb~ R. and Sandnes K. (1988) Growth and chemical composition of adult Atlantic salmon (Salmo salar) fed dry and silage-based diets. Aquaculture 69, 343-353. Linko R. R., Kaitaranta J. K. and Vuorela R. (1985) Comparison of the fatty acids in Baltic herring and available plankton feed. Comp. Biochem. Physiol. 82B, 699-705. Malak N. A., Brichnon G., Meister R and Zwingelstein G. (1989) Environmental temperature and metabolism of the molecular species of phosphatidylcholine in the tissues of the rainbow trout. Lipids 24, 318-324. Martin F. D., Wright D. A. and Means J. C, (1984) Fatty acids and starvation in larval striped bass (Morone saxatilis). Comp. Biochem. Physiol. 77B, 785-790. Morris R. J., Lockwood A. P. M. and Dawson M. E. (1982) Changes in the fatty acid composition of the gill phopholipids in Gammarus duebeni with degree of gill contamination. Mar. Pollut. Bull. 13, 345-348. Muje P., Agren J. J., Lindquist O. V. and H~nninen O. (1989) Fatty acid composition of vendaee (Coregonus albula L.) muscle and its plankton feed. Comp. Biochem. Physiol. 92B, 75-79. Owen J. M., Adron J. W., Sargent J. R. and Cowey C. B. (1972) Studies on the nutrition of marine flatfish. The effect of dietary fatty acids on the tissue fatty-acids of the plaice Pleuronectes platessa. Mar. Biol. 13, 160-166. Pagliarani A., Pirini M., Trigari G. and Ventrella V. (1986) Effect of diets containing different oils on brain fatty acid composition in sea bass (Dicentrarchus labrax L.) Comp. Biochem. Physiol. 83B, 277-282. Sargent J. R., Henderson R. J. and Tocher D. R. (1988) The lipids. In Fish Nutrition (Edited by Halver J. E.), 2nd edn, pp. 153-218. Academic Press, San Diego, CA. Sheridan M. A., Allen W. V. and Kerstetter T. H. (1985) Changes in the fatty acid composition of Steelhead trout, Salmo gairdnerii Richardson, associated with parr-smolt transformation. Comp. Biochem. Physiol. 80B, 671-675. Stoffel W., Chu F. and Ahrens E. H. (1959) Analysis of long-chain fatty acids by gas-liquid chromatography. Micromethod for preparation of methyl esters. Analyt. Chem. 31, 307-308. Yingst III W. I. and Stickney R. R. (1979) Effects on dietary lipids on fatty acid composition of Channel catfish fry. Proc. Am. Fish. Soc. 108, 620-625. Yu T. C., Sinnhuber R. O. and Putnam G. B. (1977) Effect of dietary lipids on fatty acid composition of body lipid in rainbow trout (Salmo gairdneri). Lipids 12, 495--499.
0305-0491/90$3.00+ 0.00 Pergamon Press plc
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GENOTYPIC A N D PHENOTYPIC FATTY ACID COMPOSITION IN THE TISSUES OF SALMON, SALMO SALAR* ANITA VIGAt and OTTO GRAHL-NIELSEN~§ tZoological Laboratory and :~Department of Chemistry, University of Bergen, Allegt. 41, N-5007 Bergen, Norway (Received I I January 1990) Abstract--l. Two-year-old salmon were fed for 8 months with three diets with small, but significant, differences in lipid content and fatty acid composition. 2. The fatty acid composition in abdominal fat, red muscle, white muscle, liver and heart of the fish was determined at the end of the feeding period by a chemometric method. 3. The fatty acid composition of all tissues differed from that of the diets, least in the abdominal fat, most in the liver and heart tissue. 4. The fatty acid composition of the tissues, the heart tissue in particular, was independent of the fatty acid composition of the diets. 5. Large differences in the fatty acid compositions were observed between different fish. 6. The advantage of multivariate interpretation of fatty acid comfaositions is demonstrated.
INTRODUCTION It is commonly acknowledged that the composition of fatty acids of neutral lipids, and to a lesser degree, of phospholipids, in the tissues of fish is influenced by the composition of fatty acids in the dietary fat (Sargent et al., 1988). This knowledge has been mainly reached on the basis of investigations into the dietary requirements of species reared in captivity. In such investigations diets with extreme compositions are often used (Anderson and Arthington, 1989; Leger et al., 1981; Leray and Pelletier, 1985; Lie et aL, 1986). From the data presented in these, and several other publications, there is no doubt that a large change in the fatty acid composition of the diet results in changes in tissue fatty acid composition, although compositions identical to that in the diet is never experienced. Fish do have the ability to synthesize fatty acids de novo, and also to selectively metabolize, absorb or discharge fatty acids (Sargent et al., 1988). Apparently, fresh water species carry out these abilities more efficiently than marine species (Owen et al., 1975). They do this to obtain an optimal fatty acid composition (Ackman, 1980), which must be characteristic for each species, i.e. genotypic. The fatty acid composition might therefore be used to gain systematic information, i.e. distinguishing between different stocks of the same species (Grahl-Nielsen and U1vund, 1990). However, under natural conditions the fatty acid composition will be influenced by nongenetic phenomena, like changes in temperature, (Malak et al., 1989), salinity (Daikoku et al., 1982), growth (Muje et aL, 1989), the reproductive cycle (Henderson et al., 1984), pollution (Morris et al., 1982). In addition comes the influence of the diet *Submitted in partial fulfillment of the requirements of Anita Viga for the degree of Cand. Scient. §Author to whom correspondence should be addressed.
(Henderson and Tocher, 1987). The lipids of fish are composed of 20 or more fatty acids, and it is reasonable that different external influences affect different fatty acids. For example, temperature changes are mainly manifested in the polyunsaturated fatty acids. Use of the fatty acid composition to gain genotypic or phenotypic information therefore requires knowledge of the natural variation. The fatty acid composition varies markedly between different tissues, and also between different classes of lipids within the same tissue. Depending on the purpose, investigations may span from fatty acid composition in total body lipids, e.g. in fish oils (Lambertsen and Br~ekkan, 1965; Ackman and Eaton, 1966) to composition in different lipid classes, e.g. in various phospholipids in selected tissues (Castledine and Buckley, 1982). Selection of the target for the analysis depends on the purpose of the investigation, but the broader the target the more will superposition of fatty acids from different lipid classes and/or tissues complicate the evaluation of the results. Large differences in the fatty acid composition between individual specimens were demonstrated as far back as in 1967 by Jangaard et al. (1967). Still, almost all investigations are carried out on pooled samples from several fish. In most investigations evaluation of the analytical results have been done by simply listing tables of fatty acid compositions, followed by statements of similarity or dissimilarity, without any quantitation of these differences. Still, in a few investigations the Student's t-test has been used to test the significance of the difference between samples when one fatty acid is compared at a time. However, covariation of several fatty acids is most likely under these circumstances, and quantitative evaluation of the data should call for multivariate statistics.
721
722
ANITA VIOA and OTTO GRAHL-NIELSEN
The purpose o f this investigation was to see if small, b u t significant, differences in the fatty acid c o m p o s i t i o n in the diet will influence the fatty acid c o m p o s i t i o n o f various tissues of salmon. It was considered necessary to use a relatively long time for equilibration to the diets, to avoid effects carried over from lipids stored in the fish. F u r t h e r more, differences between individual fish should be studied. It would have been desirable to study the fatty acid c o m p o s i t i o n o f all the different lipid classes in different tissues. However, since several fish are needed to statistically evaluate the differences between the individuals, the total n u m b e r of analyses a n d l a b o u r involved would have become prohibitive. Therefore, it was chosen to examine the fatty acid c o m p o s i t i o n of several, highly different tissues, ranging from intestinal fat, considered to be pure storage fat, or triglycerides, via red a n d white muscle a n d liver to h e a r t tissue, the lipids of the latter consisting mainly o f phospholipids (Grahl-Nielsen a n d U l v u n d , 1990). The objective was to use a statistical multivariate method, based o n Principal C o m p o n e n t Analysis, for evaluation of the differences/similarities in the results. Hereby the c o m b i n e d effect o f all the fatty acids in the diets a n d tissues could be determined.
MATERIALS AND METHODS Smolt of Atlantic Salmon, S a l m o salar, in May 1986 were distributed randomly in nine net pens, each of 100m 3, 2-300m from shore at Holmane, Sirev~.g, in southern Norway. They were fed a commercial dry diet, Tess Elite from Skretting, A/S, Norway, which is widely used in salmon-farming in Norway. In August 1987, the salmon had reached an average weitht of I kg. At this time the feeding experiment started. Salmon in three of the pens were given Edel, in three other pens Edel Lt. These are both commercial dry diets, based on herring flour ca 60%, capelin oil ca 15%, carbohydrates ca 20%, and additives like vitamins, minerals and binding substances ca 5%. The herring flour of the two diets had been produced in different ways, in the Edel by a normal process, and in the Edel Lt by a low temperature process. The two diets also differed in that the Edel Lt had ca 0.5% less capelin oil. These differences resulted in that Edel had a lipid content of 18.5% and Edel Lt 14°, The salmon in the last three pens were fed with an experimental diet based on the same ingredients, but in different proportions: ca 54% herring flour from the low temperature process, ca 23% capelin oil, ca 30% carbohydrates and ca 4% additives. The resulting lipid content of this diet was 21%. The lipid contents of the three diets, 14%, 18.5% and 21%, respectively, were determined as fatty acid content by using the fatty acid 17 : 0 as internal standard in the analyses described below. The accuracy of this determination was not very high, but this is not elaborated on, since it is the relative amounts of the various fatty acids which are of importance for this investigation. The three diets are referred to as low, medium and high fat diet. Fish were collected for determination of tissue fatty acids in May 1988, i.e. after they had been fed for ca 8 months on the experimental diets. The average weight of the fish were 3.4 + 0.9 kg. There was no significant difference between the weights of the fish fed the different diets, although those fed the high fat diet tended to weigh less than the others. A significant difference between the three groups was
the absence of intestinal fat in those fed the low fat diet. Those fed the high fat diet had more intestinal fat than those fed the medium fat diet. Samples were retrieved from the following tissues: white and red muscle were cut dorsally from a caudal section, the liver was cut in two halves and samples taken from the middle, the heart tissue was then taken from the tip of the ventricle, the intestinal fat, only present in fish fed medium and high fat diet, was collected from the area around the pylorus. Between 10 and 20 mg were used for each determination, and up to five parallel samples from each tissue in each fish were analyzed. The analysis of the fatty acids was based on direct methanolysis (Stoffel et al., 1959) of the tissue samples. The main difference from established methods for determination of fatty acids in tissues of marine animals are the small sample amounts needed, and the exclusion of extraction of lipids prior to methanolysis (Grahl-Nielsen and Barnung, 1985). The samples were transferred directly to thick-walled glass tubes, 0.8 ml anhydrous 2 N HCI in methanol was added and the tubes were securely tightened with Teflonlined screw caps. The reagents contained 0.05% of the antioxidant 4 - m e t h y l - 2 , 6 - d i t e r t b u t y l p h e n o l (BHT). Methanolysis was complete after 2 hr at ll0°C. The fatty acid composition of the diets was determined by the traditional method based on extraction with methanol/chloroform by the method of Folch et al. (1957). Aliquots of the washed chloroform extract were transferred to similar tubes as those used for the tissue analyses, the chloroform was thoroughly evaporated under a ~tream of nitrogen gas, and the methanolysis performed aff-'described above. The methanol/HC1 was evaporated by nitrogen gas down to 0.4 ml and an equivalent amount of water was added. The fatty acid methyl esters were then extracted twice with 2 ml hexane. After adjustments of concentrations in the extracts by partial evaporation of the hexane for the tissues with low lipid levels, or dilution with hexane for the adipose tissue and the red muscle tissue, 1 #1 of the final solution was gas chromatographed on a 30 m x 0.32 mm fused silica column, from J&W Scientific, with 50% cyanopropylmethyl-50% methyl-phenyl polysiloxane, of 0.25 micron thickness, as stationary phase, and helium as mobil phase. The components eluting from the column were detected by a flame ionization detector, and the detector output was coupled to a VG Multichrome lab-data system for storage and treatment of the chromatograms. The 16 fatty acids, from 14:0 to 22:6n3, as methyl esters, given in Table 1, were identified by comparison with standards. The amount of the various acids in the samples was found by determination of the areas of the peaks in the chromatograms, and the amounts were normalized by expressing each as the percentage of the sum of the 16 acids. For multivariate treatment, the resulting relative numbers, given in Table 1, were loaded into a Sperry PC. To level out the large differences in numerical values between the least and the most abundant fatty acids, the data were scaled by taking the logarithms. Various combinations of the data, as discussed later, were then subjected to principal component analysis by the program SIMCA (Albano et al., 1981), available in the software package SIRIUS (Kvalheim and Karstang, 1987). The program places the samples in a 16-dimensional space, i.e. with one co-ordinate for each of the 16 acids. The dimensionality is then reduced from 16 to 2, which represents the directions of the largest and second largest variation between the samples in the 16-dimensional space. The two new dimensions are represented by two principal components, and the samples are plotted in a co-ordinate system of these, giving the PC-plots shown in Figs 1-3. Separate PC-models of groups of samples belonging to the same diet were formed, as discussed below.
Salmo tissue fatty acids
723
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The composition of the fatty acids in the three diets is given in Table 1. The largest difference is in 18:4 n3, which is 1.5 times higher in the medium fat diet than in the low fat diet. For the other fatty acids the differences between the highest and lowest value is in the order of 5-25%. When the SD of the determinations of 5% is taken into consideration, it is clear that the fatty acid composition of the three diets are quite similar. It is likely that salmon encounters differences in this order of magnitude when preying on different organisms under natural conditions. This is in contrast to the much larger differences between experimental diets used in other investigations, many times with some fatty acids completely absent from one of the diets (Leger etal., 1981; Leray and Pelletier, 1985; Lie etal., 1986). The common way of evaluating differences between diets, as carried out above, takes only one fatty acid into account at a time. To make a comparison between the three diets on the basis of all the fatty acids, principal component analysis was undertaken. This resulted in the PC-plot in Fig. 1. The plot represents 98% of the total variance between the 12 diet samples. It is demonstrated here that even if the differences between the three diets are small when one fatty acid is considered at a time, the difference based on all fatty acids is considerable. To enumerate the differences between the diets, statistical class models were formed. A minimum of five samples is required to form a model. Therefore, a model of the low fat diet, with only two replicates, could not be formed. The models are based on the relative position of the replicates in the space with one dimension for each of the fatty acids, i.e. 16 dimensions. The outer limit of the models are determined in terms of relative SD of its samples, within a chosen limit of significance, in this case 95%. This is the RSDm~ for the model. The distances of all the 12 samples from the two models in the 16-dimensional space are given in Table 2. The RSDm~ for the two models are also shown in the table. It is seen that the five replicates for the medium fat diet fall within the statistical
724
AN1TA V1GA and OTTO GRAHL-NIELSEN
PC 2
% PC 1 Fig. 2. Principal component plot of the samples of the high fat diet and of the tissues from the fish fed this diet; H, high fat diet; F, intestinal fat; R, red muscle; W, white muscle; L, liver; A, heart. model for that diet. One of the replicates of the high fat diet falls slightly outside its own model. This is because the model has the form of a long, narrow cylinder, leaving the one sample just outside. It is, however, seen from the table that the samples from the other diets fall far outside the model. Furthermore, it is seen that the two low fat samples are less different from the high fat samples than from the medium fat samples. This is in consistence with the PC-plot of the diet samples in Fig. 1. It is thus demonstrated that multivariate data-analysis offers an alternative method for evaluation of the traditional tables of fatty acid compositions. Similarities and differences between samples and/or groups of samples are quantitatively determined by RSD~PC 2
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values, while PC-plots visualizes the relationship between the samples. The results of the determinations of the fatty acids in the tissues of the salmon fed the three diets are given in Table 1. Three features are obvious: (1) the spread of the data within each tissue is considerable, i.e. relative SD are usually in the order of 50% or more; (2) for each tissue, the differences between diets are small; (3) the differences between the various tissues are large. This feature will be discussed first. The values in the table indicate that the fatty acid composition of intestinal fat is least different from that in the diet, and that the composition in the liver and the heart tissue differs mostly. Similar to the evaluation of the diets above, the tissue data are more easily perceived by performing principal component analysis. In the case of the high fat diet this resulted in the PC-plot in Fig. 2. The plot shows that the fatty acid composition of the diet has been metabolically altered by the salmon upon deposition of lipids in the various tissues. The smallest alteration was for the intestinal fat and the red muscle. Nevertheless, the differences are significant. Larger differences between the diet and the Table 2. Relative distances, expressed as residual SD, of the diet samples to the statistical models of the medium and high fat diets Model for medium fat diet
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tissue, respectively. The symbols for the diet samples are the same as in Fig. 1. Numbers in italics represent samples from fish fed the low fat diet, plain numbers represent samples from fish fed the medium fat and bold numbers represent samples from fish fed the high fat diet. Within each group, similar numbers represent different parallel samples from the same fish.
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Salmo tissue fatty acids
725
Table 3. Relativedistances,expressedas residualSD, of the samplesfrom the high fat diet and from the tissuesof salmon fed this diet, to the statisticalmodel of the high fat diet High fat diet Intestinalfat Red muscle Whitemuscle Liver Heart 2 267 273 330 876 642 8 266 275 382 853 879 11 230 230 357 870 888 3 237 317 384 896 852 3 242 282 388 934 884 291 270 478 901 944 311 260 452 1057 717 275 240 289 975 776 235 342 712 886 210 362 761 882 371 773 755 white muscle are obvious, with the largest difference for the liver and the heart. Similar to the case of the diets above, the PC-plot in Fig. 2 is supported by quantitative measurements of the distances of the various samples to the model of the diet, Table 3. The numbers are in accordance with the plot: the samples of the intestinal fat and of the red muscle all have approximately the same distance from the diet. The white muscle samples have larger distances, and the liver and heart samples clearly the largest. There is a much larger spread between the liver samples than between the samples of the other tissues. The livers from the different fish did have different appearances, with respect to size and colour. In the case of the liver, compared to the other tissues, it was more difficult to dissect out a small piece of homogeneous tissue free of blood and other substances. Intestinal fat is considered to function as a store of reserve lipid (Henderson and Tother, 1987), mainly made up of triglycerides. The relative amounts of triglycerides in the tissue lipids are expected to be smaller in red muscle than in intestinal fat, and still smaller in white muscle and liver, i.e. Leger et al. (1981) found 70-90% neutral lipids in red muscle of trout, 65-80% in white muscle and 20-30% in the liver. The variations between species, and the dependance on condition, temperature and other variables are, however, large (Henderson and Tocher, 1987). While the muscle and liver tissue, and also the adipose tissue of fish have been subjected to numerous investigations of lipid and fatty acid composition (Henderson and Tocher, 1987), we are only aware of two investigations on the lipids of heart tissue of fish (AI-Tai et al., 1984; Henderson and Sargent, 1984). In these the amount of phospholipids was not determined, although it was stated in the former that the lipids of the heart contained free fatty acids and triglycerides in addition to phospholipids. This corresponds to findings in our laboratory which showed that lipids from the heart of herring contained 70% phospholipids, 19% free fatty acids and 11% neutral lipids (Grahl-Nielsen and Ulvund, 1990). Even if many investigations have been concerned with the fatty acid composition of phospholipids and triglycerides in different tissues of fish, no effort has been done to quantify observed differences. Still, by inspection of published tables, it is apparent that the composition of phospholipids varies more between the tissues than does the composition of
triglycerides (Cowey et aL, 1976, 1983; Sheridan et al., 1985). This is reasonable since phospholipids have more important physiological functions than triglycerides. It is also clear that the phospholipids and triglycerides of the same tissue have large differences in composition. Since the phospholipids and triglycerides were not separated in the present investigation, the observed, and quantified differences between the tissues are due both to differences in the ratio between phospholipids and triglycerides as well as to differences in the phospholipids. By comparing the compositions of the liver and white muscle tissues in Table l, one acid at a time, it was found that the differences between the two tissues had exactly the same pattern as observed by Lie et al. (1988) for the same tissues of salmon. Very similar patterns of differences have also been observed by Leger et al. (1981) and by Braekkan et al. (1971). It must again be emphasized that the multivariate treatment improves the interpretation of the analytical results over the classical monovarite evaluation of one fatty acid at a time, both by displaying the differences in a PC-plot, by ranking the importance of the fatty acids and by quantification of the differences in terms of class and sample distances. This holds true even in cases like heart tissue vs diet, where the differences can easily be seen in the table of fatty acid compositions. It is particularly valuable when the differences become smaller, as in the case of abdominal fat vs diet. Now, to the question if the differences in the fatty acid composition of the diets have induced differences in the fatty acid composition of the tissues after 8 months? The data in Table 1 do not indicate differences: within each tissue there are only small differences between the three average values for each fatty acid. In addition, the deviations from the averages are large. Principal component analysis of the data were undertaken to see if multivariate treatment would give a different conclusion. The resulting PC-plots for the liver and heart tissues are shown in Fig. 3. For the liver tissue there is a slight indication, shown by the dashed line in Fig. 3, that the fatty acid composition in the fish fed the low fat diet is somewhat different from the compositions in the other two groups of fish. In the figure the tissue samples from the fish fed the low fat diet lie to the left of the other samples, just as the two samples of the low fat diet lies to the left of the other diet samples. This shows that the same fatty acids are responsible for the differences
726
ANITAVIGAand OTTOGRAHL-NIELSEN
in both cases, implying that the fatty acid composition of the diet may have had an influence on the fatty acid composition of the liver tissue. However, since the differences were small, and since no differences were observed between the liver tissues of the fish fed the medium and high fat diets, further elaboration upon this will be left for another investigation. For the intestinal fat and the two muscle tissues the multivariate treatment gave no clearcut differences between samples from fish fed the three diets. In the case of the heart, samples from fish fed the three diets are apparently completely intermingled. Since the difference between the diets, on one hand, and the tissues, on the other, dominates the plot, it was checked whether the three tissues were separated along the third PC, perpendicular to the plane of the plot in Fig. 3. This was not the case, neither were they separated from each other when the calculations were done on the tissue samples alone. The control of the metabolic conversion of the lipids in the diet to those of the heart tissue, which are assumed to be dominated by phospholipids, see above, is strong enough to overrule differences in the diets. The large analytical variance demonstrated by the high relative SD of the results in Table 1, is displayed by the large distances between the sample points in Fig. 3. There are variations between the parallel samples from each fish, but an even higher variation between different fish. This large individual difference corresponds with the finding of Jangaard et aL (1967). Even if such large individual differences have been known for a long time, it is peculiar that only five out of 22 investigations referred to in the present paper have used individual specimens. (The other 16 references are either reviews or do not contain comparisons of fatty acid compositions.) Of the 17 investigations with determinations on pooled samples, 10 have only one determination, while the remaining seven have two or three replicate determinations. Statistical monovariate Student's t-test was used in five of the 22 investigations for comparison between different groups. Only one of these was based on analyses of individual specimens. The other four were based replicate analyses of pooled, homogenized samples. It ought to be clear that when testing the significance of the difference between the two groups, of e.g. fish that have been given different diets, it is the variance between fish within each group which should be considered, not the analytical variance of replicates from the same, pooled and homogenized sample, no matter how many fish have been pooled. A phrase often used in the literature in this field of research is that the fatty acid composition of tissue "reflects" the fatty acid composition in the diet (Owen et al., 1972; Henderson et al., 1984; Lie et al., 1988; Martin et al., 1984; Yu et al., 1977; Leray and Pelletier, 1985; Pagliarani et al., 1986; Cowey et al., 1983; Yingst and Stickney, 1979). This is an ambiguous term without statistical significance. It leaves the impression that the tissue composition is similar to that of the diet and will consequently follow changes in the diet. Unfortunately, this is a common view. It has emerged from investigations with large differences in experimental diets where some consequences for the fatty acid composition of tissues were observed,
although not quantified (Anderson and Arthington, 1989; Leger et al., 1981; Leray and Pelletier, 1985; Lie et al., 1986). In fact, the possibility of tailoring of the fatty acid composition of cultured fish before they go to the market has been suggested (Sargent et al., 1988). The present results suggest that, when encountered with less dramatic changes in the diet, the fatty acid composition of the tissues is not controlled by the diet. This is particularly the case of the heart tissue, with a possible exception for the liver tissue. In accordance with this, several investigations indicate that fish living in their normal habitat with an adequate supply of food have genotypic fatty acid profiles in their tissue. Henderson et al. (1982) suggest that the species related composition becomes less obvious as the lipid level in the diet increases, since levels in excess of 10% depress the fatty acid synthesis in fish, at least in Coho salmon. Ackman and Takeuchi (1986) have shown that wild Atlantic salmon parr maintains a typical fatty acid composition in the tissue by selective accumulation of certain fatty acids, as 20:4n6, from the diet. Rainbow trout possesses the ability to control to a large extent its fatty acid composition (Boggio et al., 1985). They observed a smaller overall net change in the fatty acid composition of the muscle of the trout than the differences in the diet. Muje et al. (1989) have found that vendace developed the ability to metabolically modify the fatty acid composition with age, while Sheridan et al. (1985) found that the fatty acid composition of the smolt of steeihead trout was independent of the diet. Linko et al. (1985) observed that the available plankton feed had no effect on the fatty acid composition of the phospholipids of Baltic herring. Acknowledgement--We thank T. Skretting A/S for making
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