Sampling. Methods. Holding and Diet. 454 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986

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2 454 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986 milder forms of lesions in nonmigratory, spawning rainbow trout (S. gairdnerii) compared with migratory, spawning steelhead trout. They speculated that the rise in 17 OHcorticosteroids, which occurs during upstream migration, might be implicated in lesion formation. However, in a subsequent study, intraperitoneal pellet implants of the glucocorticoid into juvenile, starving S. gairdnerii failed to produce coronary lesions. 12 Instead, the elevation in 17 OH-corticosteroids is probably associated with improving energy mobilization, and the glucocorticoids may even be antjatherogenic The basis for differences in lesion severity between spawning steelhead and rainbow trout remains an enigma. Because lesions can be found in immature fish, the roles of diet and blood cholesterol are of current concern in studies of salmonid arteriosclerosis. Moore et al. 7 suggest that early forms of coronary lesions in salmonids may be related to diet alone. The implication of diet may, in turn, be related to high blood cholesterol levels in fish, which are up to five times greater than those normally found in mammals. 14 ' 1S Cholesterol and, specifically, the low density IIpoprotein (LDL) fraction are central factors in some forms of coronary disease in mammals. 18 A preliminary study 10 showed that a dietary cholesterol supplement was associated with an increase in the number of lesions in brook trout (Salvelinus fontinalis). More recently, lesion proliferation was found to parallel the natural oscillations in plasma LDL levels of maturing Chinook salmon (Oncorhynchus tshawytscha) in the Great Lakes. 11 However, the role of cholesterol in lesion formation has not been examined experimentally in any detail in fishes. Our study examines the effect of a dietary cholesterol supplement and the resulting increase in blood cholesterol level on the development of coronary arteriosclerosis during sexual maturation of Atlantic salmon. The influence of ambient salinity was also investigated. Holding and Diet Methods The experiments were performed on post-smolt S. salar, which were grown in sea cages (off Deer Island, New Brunswick) to eliminate the confounding influence of seaward and river migrations. Replicate experiments were performed in 1982 and 1983 using different year classes from the same strain. In both years, the fish were brought from sea cages to the laboratory in June and placed in 3000-liter fiberglass tanks supplied with either filtered sea water or dechlorinated freshwater. The flow rate was 15 liters/min" 1, and the water was continuously aerated. The photoperiod simulated that for 45 N latitude, and the water temperature approximated the natural temperature profile through the aid of supplemental heating or cooling by a heat exchanger between the salt- and freshwater supplies. Some fish were maturing and became ripe during the experimental period; others were immature and at least 1 year from maturity. However, neither the state of maturity nor the sex was discernable when the experimental subgroups were established. Consequently, each subgroup contained variable sex and maturation ratios. The fish were subdivided into salt- and freshwater groups (two tanks per group) to examine whether ambient salinity was a factor in lesion development. The freshwater group was gradually acclimated by mixing freshwater and saltwater over a 3- to 4-week period, and during this period all fish were fed the control diet, a modified version of a moist commercial chow 17 (Table 1). Afterward, half of the fish (one tank of fish from each salinity group) were fed a cholesterol supplement in their diet (a 3% substitution of USP cholesterol for the herring oil in the control diet). It was anticipated that the dietary cholesterol supplement would elevate the blood cholesterol level. All animals were fed ad libitum three times a day except on weekends when they were fed once a day. The fish were maintained on these diets for about 5 months between June and December. Sampling A total of 133 and 160 fish were sampled in 1982 and 1983, respectively. In 1982, the majority of fish were sampled in early December. In 1983, all the fish were sampled in December. Smaller subsamples provided a preliminary index of lesion incidence and cholesterol levels and were taken at the outset of the study (June 1982; n = 8) and after 3 months on the dietary regime (September 1982; n = 12 from each of the four groups). The main results were derived from fish sampled in December, after a full 5 months on the dietary regime. The data for 1982 and 1983 were pooled to compensate for the low level of natural maturation among females in this stock. Each fish was killed by a sharp blow to the head, and was weighed and measured (fork length); a heparinized blood sample was taken by caudal puncture. Plasma was collected after centrifugation. The ventricle and bulbus arteriosus were removed in toto and placed in a buffered formalin solution. 18 Liver samples were also taken from some fish in 1982 and placed on dry ice for later enzymatic analysis. The fish were visually assessed for sexual maturity or the gonads were weighed to determine the gonadosomatic index. The fish were categorized as immature or mature. The significant differences between plasma sex hormone levels in mature and immature fish from both sexes were determined by using Student's f test. Whether the Table 1. Table of Constituents of Modified Commercial Grower Diet Constituent Whole herring (Clupea harengus) Herring meal Herring oil Cholesterol (USP) (Sigma Chemical Co.) Vitamin premix Ascorbic acid Control diet (%) Cholesterol supplemented (%) Values are expressed as percentage by weight. Carophyll red (a synthetic contaxanthin) was added to the food at a rate of 0.5 g/kg of food to obtain proper flesh coloration.

3 ARTERIOSCLEROSIS IN SALMON Farrell et al. 455 cholesterol diet affected plasma triglyceride and free fatty acid (FFA) levels or liver enzyme levels was also tested with Student's /test. For plasma total cholesterol and LDL levels, significant differences between control and cholesterol diets were determined for each subgroup according to sex, maturation, and salinity by using Student's r test. Muttifactorial analysis of variance (ANOVA) testing was used to identify significant F values resulting from the main effects of diet, salinity, sex, and maturation on lesion frequency and lesion severity index. The significant interaction effects were identified by using the Newman-Keuls test for post-hoc comparisons of subgroups. Arcsine transformation of the percent of lesion data was used for ANOVA testing. Inspection of data on lesion incidence revealed that specific subgroups might be more prominent in the differences produced by maturation and diet. These hypotheses were tested by using paired analysis with the Mann-Whitney U test to detect differences in location and by using the Kolmogorov-Smimov two-sample test to detect differences In the distribution for lesion incidence. Histology The hearts were routinely dehydrated, cleared, and embedded in paraffin wax (Tissueprep) for sectioning at 5 ^m thickness on a 820 AO rotary microtome. The coronary artery carries blood from the gills to the ventricle and lies on the ventral surface of the ventral aorta and bulbus arteriosus. Its first bifurcation, into the right and left coronary arteries, usually occurs close to the ventricle. Most of the arterial sections were taken from sites located on the bulbus arteriosus and occasionally on the ventricle. Most of the sedons were, therefore, from the main coronary artery and upstream of the major branch point. The sections were stained with Verhoeffs elastic stain and counter-stained with picro-ponceau. 18 Approximately 10 serial sections of the coronary arteries were taken from five or more sites, and a minimum of 50, but up to 150, arterial cross sections were graded for each fish. Most of the observations were made on surface distribution arteries because: 1) previous work has found lesions predominantly in larger arteries; 5 ' ) lesion formation in large distribution vessels may have a greater impact on coronary flow; and 3) these vessels were easier to align for cross sections. Cryostat sectioning and staining with Sudan Black B 18 were used to determine whether lesions in some mature fish contained lipid deposits. An artery without lesions was assigned a grade of 0, and lesion severity was ranked from 1 to 4, based on the system of Moore et al. 5 ' 6 With this system, grades 1 to 3 represent a progressive increase in lesion severity and grade 4 is a catch-all for severe lesions. Typical arterial sections for each grade are shown in Figure 1. Lesion incidence (%) was calculated for each fish from 100 (the number of cross sections with a grade greater than 0) ^ (the total number of cross sections graded). The lesion severity index (LSI) for each fish was determined by the formula: ((1 x n,) + (2 x n 2 ) + (3 x rtj (4 x n«))/(n, + riz + rig + n 4 ) (1) where n = the number of cross sections with a grade equivalent to the subscript. Plasma and Tissue Analyses In all fish, plasma samples were usually analyzed within 24 hours (stored on ice or occasionally frozen) for total cholesterol content and the high density lipoprotein (HDL) fraction by using an enzymatic test kit (Sigma No HDL). The LDL fraction was approximated by using the total cholesterol -HDL). The results are expressed as milligrams of cholesterol per deciliter of plasma. In 1982, the sex hormones testosterone and 11 -ketotestosterone were assayed in duplicate on frozen plasma samples by using radioimmunoassay. 19 This analysis served to confirm our visual assessment of sexual maturity. Frozen plasma samples were also used for the enzymatic analysis of triglycerides 20 and FFA 21 In triplicates on 100 /il or 10 fi\ of plasma, respectively. This analysis assessed whether other lipid components of the blood were affected by the diet or salinity regimes. The liver samples taken in 1982 were stored at - 80 C. The tissue was homogenized in 10 volumes of imidazole buffer (50 mm, ph 7.4), and was centrifuged for 5 minutes at 5000 g. The supernatant was used for the enzyme assays. All determinations were performed under saturating conditions in duplicates at 15 C in a final volume of 1 ml. Citrate synthase (E.C ), a key enzyme of the Krebs cycle 22 and therefore a good measure of overall oxidative capacity of a tissue, was measured with 5,5'dithiobis-2- nitrobenzoic acid (DTNB) as the optically active substance in an assay consisting of: 0.1 mm DTNB, 0.3 mm acetyl- CoA, 0.5 mm oxatoacetate (omitted for control) in 50 mm Tris-HCI buffer (ph 8.1 at 20 C). HydroxyacylcoenzymeA dehydrogenase (E.C ), a committing step in the oxidation of fatty acids, was determined by monitoring the decrease in NADH concentrations under the following conditions: 0.05 mm acetoacetyl-coa (omitted for control), 0.08 mm NADH in 50 mm imidazole buffer (ph 7.4 at 20 C). Activities are given in units (^mol/min~ 1 ) per gram of fresh weight of hepatic tissue. A change in the ratio of these two key liver enzymes would suggest that the capacity for fat metabolism had been modified. All procedures complied with the Animal Care Guidelines required by the Natural Sciences and Engineering Research Council of Canada. Results Effect of Cholesterol Supplement Fish fed the 3% cholesterol supplement in place of herring oil had significantly (p < 0.05; Student's /test) higher total plasma cholesterol levels than fish on the control diet. This was true for all experimental subgroups (Figure 2), even though individual fish showed large variations in total cholesterol. The greatest elevation of plasma cholesterol that resulted from the cholesterol-enriched diet was observed in immature fish. Most of the plasma cholesterol was in the HDL fraction; HDL levels were typically 80% or greater (Figure 2). The

4 45S ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986 ^ v Figure 1. Examples of coronary arteries from Atlantic salmon. A. Leslonless artery graded 0. B-O. Arteries with a progressively greater lesion involvement in the intima, which were graded 1 through 4, respectively. Bars = 50 /xm. All sections were stained with Verhoeff s elastic stain and picro-ponceau.

5 ARTERIOSCLEROSIS IN SALMON Farrell et al. 457 >ENRICHED DIET -NORMAL DIET MATURE FISH FRESHWATER IMMATURE FISH MATURE FISH SALTWATER IMMATURE FISH Figure 2. Plasma cholesterol levels in Atlantic salmon fed nonnai and cholesterol-enriched diets in freshwater and saltwater. Within each subgroup (sex and maturation status), the bar for the nonnai diet partially overlies the corresponding bar for the enriched diet. The high density Iipid (HDL) fraction of the total cholesterol is indicated by the stippled area within each bar, and the low density Iipid (LDL) fraction is the open area. Standard errors are shown, and the number of fish within each subgroup Is presented below the corresponding bar. cholesterol-enriched diet increased the HDL level and, In addition, produced important changes in the fraction of the total cholesterol as HDL. As a consequence, the LDL fraction was increased significantly (p < 0.05; Student's nest) in all subgroups fed the cholesterol-enriched diet with the exception of mature males in saltwater. The increase of LDL level was two- to fivefold. Levels up to 250 mg/dl~ 1 were observed for certain subgroups. Neither diet nor environment significantly affected plasma triglycerides or liver enzymes associated with fat metabolism (Table 2). Plasma FFAs were significantly (p < 0.05; Student's t test) reduced by the cholesterol supplement in saltwater fish, but were not affected in freshwater fish. The plasma sex hormones testosterone and 11 -ketotestosterone showed elevations typical of maturing fish (Table 3), and were not affected by the dietary supplement. Mature males were characterized by elevated levels of both hormones. Mature females were characterized by elevated levels of testosterone only. These data supported our visual assessment of maturation status. Lesions In the Coronary Artery The extent of lesion formation was high. In all, 94.5% of the fish had at least one identifiable arteriosclerotic lesion in the 50 or more arterial sections graded for each fish. The overall incidence of lesions was about 61%, which meant that a lesion was observed in three of every five arterial sections examined. Many fish (14%, 33 of 236) had a lesion in every arterial section graded, i.e., a lesion incidence of 100%. The LSI was usually between 2 and 3, and the lesions were morphologically similar to previous observations on salmonids. 6 Thus, the typical lesion in most fish involved single or multiple sites of smooth muscle proliferation in-

6 458 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986 Table 2. Trlglycerldes and Free Fatty Acids In Plasma and Liver Activities of 3-HydroxyacylcoenzymeA Dehydrogenase and Citrate Synthetase in Liver Plasma constituent Triglycerides (/imol/liter~ 1 ) Free fatty acids (^mol/liter" 1 ) 3-OH acylcoenzymea dehydrogenase 1 1 ( l / l ) Saltwater (n = 18) 4.03 (1.07) (0.048) 1.05 (0.13)t Control diet Freshwater (n = 20) 3.98 (1.81) (0.047) 1-27 (0.08) Cholesterol supplement Saltwater (n = 19) 4.30 (1.34) (0.038)* 1.26 (0.10)* Freshwater (n = 20) 2.92 (1.13) (0.050) 1.46 (0.12) Citrate synthetase (unlts/g" 1 ) (0.078)t (0.031) (0.050)* (0.024) Fish were sampled in 12/82 and 12/83. Values are presented as means (SEM) for n fish. *p < 0.05 by Student's t test, a significant difference from other values in the same row. tn = 15. *n = 18. volving six or more smooth muscle cells and disruption of the elastica (Figure 1). Numerous lesions that produced more than a 50% lumenal narrowing were also observed. We found no fatty deposits in the lesions, although this point was not rigorously tested. The lesion incidence and severity data are presented in Table 4 for the various experimental subgroups. An outstanding feature of this analysis was that all subgroups had an average incidence of lesions of at least 48%, an unexpected finding in immature fish that were at least 1 year from spawning. Therefore, lesions had developed to a significant degree in immature fish, and sexual maturation could not be the primary factor for the formation of coronary lesions in Atlantic salmon. The high lesion incidence in all subgroups also meant that the possible effects of diet, maturation, environment, and sex on lesion formation were superimposed on a high background frequency of lesions. Nevertheless, multjfactorial ANOVA revealed statistically significant main effects for diet and maturation. Thus, fish fed the cholesterol-enriched diet had a higher incidence of lesions than fish fed the control diet. Likewise, lesion incidence was higher in mature fish compared to immature fish. These observations clearly implicate both diet and maturation as secondary factors in the development of coronary lesions in Atlantic salmon. Salinity was not identified as a statistically significant main effect, but sex had a trend (p < 0.1) toward a main effect, with males tending to have more lesions than females. Table 3. Plasma Sex Hormone Levels In Mature and Immature Atlantic Salmon Atlantic salmon Mature males (n = 22) Immature males (n = 10) Mature females (n = 3) Immature females (n = 42) Testosterone (ng/ml) (3.16) a 6.10 (3.16) (19.05) a 1.66 (0.67) 11-Ketotestosterone (ng/ml) (5.16) (9.81) 3.41 (3.32) b 1.68 (0.95) b Values presented as means (SEM) for duplicate samples on n fish. Significant differences between the two dietary regimes were not evident, and so the data from the two diets were pooled. Values bearing the same letter were not significantly different (p > 0.05; Student's t test). Table 4. Effect of Diet, Environment, Maturity, and Sex on Lesion Incidence (%) and the Lesion Severity Index for 180 Atlantic Salmon Atlantic salmon Mature Male Female Lesion incidence % Lesions LSI (n) % Lesions LSI (n) Saltwater 71.2 (8.84) 2.38 (0.287) (11) 54.7 (10.74) 1.72 (0.330) (4) Control diet Freshwater 57.7 (7.25) 2.42 (0.245) (19) 60.8 (8.32) 2.34 (0.194) (9) Cholesterol supplement Saltwater 76.5 (5.68) 2.70 (0.198) (10) (13.29) 1.96 (0.339) (5) Freshwater 74.9 (5.02) 2.53 (0.144) (18) 91.8 (6.61) 2.85 (0.466) (4) Immature Male Female % Lesions LSI (n) % Lesions LSI (n) 47.8 (9.51) 1.98 (0.268) (12) 57.3 (7.99) 2.24 (0.238) (14) 52.5 (13.83) 2.05 (0.345) (6) 56.6 (7.27) 2.43 (0.244) (19) Fish were sampled in 12/82 and 12/83 after 5 months on each regime. Values are presented as means (SEM) (15.4) 2.34 (0.412) (5) 59.7 (5.64) 2.43 (.181) (22) 69.5 (9.55) 2.53 (0.391) (6) 50.6 (6.68) 2.62 (.201) (16)

7 ARTERIOSCLEROSIS IN SALMON Farrell et al. 459 Inspection of the lesion incidence data for the various subgroups (Table 4) revealed that differences for diet and maturation were more prominent in certain subgroups. The hypothesis that the increase in lesion incidence associated with the cholesterol diet was more prominent in certain subgroups was tested by using paired analysis for the control and cholesterol-enriched diets. Of the eight paired subgroups, significant increases were present in mature males and mature females held in freshwater (p < 0.05, Kolmogorov-Smirnov; p < 0.1, Mann-Whitney). Thus, feeding of a cholesterol supplement to mature Atlantic salmon in freshwater not only significantly elevated the plasma cholesterol and LDL levels, but was also associated with an increase in the incidence of coronary lesions. Immature males in saltwater showed a trend toward a significant difference (p < 0.1 Mann-Whitney; p > 0.1, Kolmogorov-Smimov). A similar hypothesis to examine for prominent subgroups in the maturation effect on lesion incidence was tested by using paired analysis for the mature and immature fish. Of the eight paired subgroups, there was a significant increase in females fed the cholesterol diet and held in freshwater (p < 0.05, Kolmogorov-Smimov; p < 0.05 Mann-Whitney). Also, males fed the control diet and held in saltwater had a trend toward a significant difference (p < 0.1, Kolmogorov-Smimov; p < 0.1, Mann-Whitney). Multifactorial ANOVA testing revealed no significant main effects on LSI. Some interactions were present. Lesions were significantly (p < 0.05) more severe in all mature males as a group compared with all immature males, and in all immature females as a group compared with all immature males. Lesions also tended (p < 0.1) to be more severe in freshwater fish compared with saltwater fish. Other interactions for LSI were not statistically significant with ANOVA testing. These observations implicate maturation and freshwater as factors that contribute toward more severe lesions in Atlantic salmon. Discussion Our understanding of coronary arteriosclerosis in fish Is in its developmental stage. This was illustrated by the unexpected finding that the sea-cage-reared immature fish had a high frequency of lesions. The incidence of lesions In some subgroups of immature fish was equivalent to that in certain subgroups of mature fish. The high incidence of lesions in immature fish is unlikely to be an artifact. Lesion data were collected on fish in 2 different years. Visually, the fish appeared to be in good condition. Also, the low sex hormone levels confirmed that the fish were indeed immature and at least 1 year from spawning. The stress of seacage rearing and/or commercial diets for 2 years may have promoted the high incidence of lesions in immature salmon. However, the fish had not faced the rigors of upstream migration and long-term residence in open sea, both of which have been previously regarded as important in promoting lesions. 1 Perhaps the strongest evidence we have that the high incidence of lesions in immature Atlantic salmon does, in fact, reflect a natural situation is a study in progress with S. sa/arcaught at sea off Greenland. Twenty-three such fish were at least 1 year from spawning; these had lesions with an average LSI of 2.1 (SEM 0.22) and a lesion incidence of 56.4% (SEM 6.7) (Saunders and Farrell, unpublished data). Comparable information on coronary artery lesions In immature fish is limited and sometimes contradictory. Van Citters and Watson 3 rarely found lesions in sea-caught, immature steelhead trout (26% of 30 fish). Robertson et al. 2 noted, without quantifying their observations, that lesion formation in Pacific salmon (Oncortiynchus species) began long before spawning and even occasionally at sea. In the only other study of arteriosclerosis in Atlantic salmon, 4 a control group of 2- to 3-year-old immature fish had a low incidence of lesions (8%). These fish had been reared in freshwater to the post-smolt stage, and were fed an artificial diet with relatively high protein and low lipid levels. In contrast, McKenzie et al. 8 found that freshwater, juvenile steelhead trout had lesions at ages as low as 11 months. Likewise, Moore et al. 6 stressed that immature Oncortiynchus have lesions, and that extensive lesion formation could be found in fish at sea as many as 5 months before returning to freshwater for spawning. While this is not an extensive data base, the observations clearly support the idea that lesions can devebp in immature salmonids. The present study provides definitive evidence that lesion formation can be extensive in immature S. salar. Implicit with this conclusion is that maturation is not the primary cause of arteriosclerosis in S. salar. The development of arteriosclerosis must clearly have its origins in immature fish. This is true whether the lesions are as extensive as in the present study or at a somewhat lower level as in previous studies. 2 ' 6-8 What factors initiate the disease are still unclear. The suggestion by Moore et al. 7 that diet is implicated has received experimental support from the present study. Lesion incidence was significantly higher in S. salar fed the cholesterol-enriched diet compared with the control diet. This observation clearly supports the view that diet can play at least a secondary role in enhancing the development of coronary lesions. The dietary influence can also be presumed to be rapid, since these experiments lasted only 5 months. Whether diet is the primary factor in lesion formation must await more definitive studies. Some of the dietary effect on lesion incidence may be related to high total plasma cholesterol and LDL levels. The total plasma cholesterol level in salmonids is certainly extremely high in comparison with mammals. 14 ' 15 Moreover, the LDL level exceeds that commonly recognized to predispose mammals to arterial damage, even though most of the plasma cholesterol in salmonids exists as HDL. Our cholesterol-enriched diet successfully increased total plasma cholesterol levels and the LDL fraction. This occurred without any major disturbance to plasma triglycerides, FFA, or the key enzyme involved in liver fat metabolism. Furthermore, the elevated plasma cholesterol level was associated with an increase in the incidence of lesions in two of the subgroups. The fact that these two subgroups were mature males and mature females in freshwater is extremely significant in view of comparable observations made with a natural population of Chinook salmon (O. tshawytscha) in Lake Michigan. 11 In mature Chinook salm-

8 460 ARTERIOSCLEROSIS VOL 6, No 4, JULY/AUGUST 1986 on in this normal freshwater environment, lesion proliferation paralleled natural fluctuations in plasma LDL levels (no sex distinction was attempted for the study with Chinook salmon). The characteristics of coronary artery disease in Atlantic salmon were split or fragmented elastica and multifocal myointimal proliferation. These characteristics resemble the initial stages of arteriosclerosis in mammals 23 as noted previously in numerous studies with salmonids. 3 " 8 ' 11 A tempting hypothesis is, therefore, that a corollary exists between cholesterol-induced lesion formation in mammals and those found in fish. However, only an association, and not a cause-effect relationship, was established between high plasma cholesterol levels and coronary lesions in salmon. The present study does provide a sound foundation for further study of a cause and effect relationship. We have demonstrated that plasma cholesterol levels can be manipulated through diet, so studies using low lipid diets from the early juvenile stage might yield valuable information. Another important consideration is the difference between lipid deposits in lesions in salmonids and mammals. In mammals, extensive lipid deposits lead to the characteristic fatty streaks found with atherosclerosis. Lipid is apparently absent in the coronary lesions of fishes, 3 ' 5 ' 8 ' 9 which may reflect a preferred use of lipid as a metabolic fuel, a high investment of lipid in gonad development in maturing fish, or the high HDL levels. Our cholesterol-enriched diet actually produced the greatest increase in plasma cholesterol in immature fish (see Figure 2), which were not diverting large amounts of energy to gonad development. Consequently, the coronary lesions observed in mature salmonids are unlikely ever to resemble the severe forms of atherosclerosis found in mammals. Maturation was a secondary factor in the etiology of the disease, since lesion incidence was greater in mature fish compared with immature fish at least 1 year from maturation. Therefore, it is possible that sex hormones are implicated in the disease, as initially suggested by House et al. 9 Males tended to have more lesions than females (an exception was the very high incidence in mature females in freshwater who were fed the cholesterol supplement). As well, the lesions in mature males were more severe than in their immature counterparts. In this regard, it would be worth examining the effects of 11-ketotestosterone implants rather than testosterone implants, as previously examined, 8 since female salmon have similar levels of testosterone as males (Table 3). It seems unlikely that the sex difference is due to a protective effect of estrogen, as has been postulated in humans, 24 because estrogen levels in male and female salmon are similar. 25 ' M The reason for a greater severity of lesions in immature females compared with immature males is unclear at this time. The role of environmental salinity in the etiology of arterial disease is apparently only minor. There was no main effect of salinity on the incidence of lesions. The LSI tended to be greater in fish in freshwater. It is also not clear whether a difference in total dietary intake between the freshwater and seawater groups or salinity per se was a factor. It is hoped that sampling S. salar at all stages of their life cycle will provide additional insights into this and other aspects of arteriosclerosis in this Atlantic salmon. The importance of hemodynamic factors in the initiation of lesions was not examined here and therefore cannot be ruled out. In mammals, the endothelium apparently acts as a protective barrier. Intimal thickening can be produced by experimental disruption of the endothelium with intravascular balloons, and there is increased endothelial permeability in association with altered hemodynamics at arterial branch points. 23 The present study focused on the main trunk of the coronary artery and avoided branch points. Therefore, the data do not to any degree reflect intimal thickening at branch points. Coronary artery blood pressure has not been measured in fish, but it is likely to be two to three times lower than in the mammalian coronary artery. 27 Other than this, too little is known about coronary physiology in fish to predict whether hemodynamic factors could promote lesion development in the main coronary artery. In summary, clear evidence is presented that coronary arteriosclerosis is substantially initiated in immature fish. The primary cause of the disease was not established. Diet, specifically a cholesterol enrichment that increased total plasma cholesterol levels, was a secondary factor in promoting arteriosclerotic changes in mature and immature fish. Maturation was also an important secondary factor in the disease and promoted a greater incidence and severity of lesions. Salinity had no effect on lesion incidence, but the severity of lesions tended to be greater in freshwater fish. Coronary arteriosclerosis is therefore a fact of life for mature S. salar. Acknowledgments The technical assistance of Wayne K. Anderson and Derek Knox was invaluable. Paul R. Harmon and Eugene B. Henderson assisted with the culture of the fish in the laboratory and helped with the collection of cage-reared fish. References 1. Robertson OH, Wextor BC, Miller BF. Degenerative changes in the cardiovascular system of spawning Pacific salmon (Oncorhynchus tshawytscha). Circ Res 1961 ;9: Robertson OH, Krupp MA, Thomas SF, Favour CB, Have S, Wexler BC. Hyperadrenocroticism in spawning migratory and non-migratory rainbow trout (Salmo gairdneri); Comparison with Pacific salmon (Genus Oncorhynchus). Gen Comp Endocrinol 1961; 1: Van Clttera RL, Watson NW. Coronary disease in spawning steethead trout, Salmo gairdnerii. Science, 1968;159: Manectie HC, Woodhouse SP, Elton PF, Klassen GA. Coronary artery lesions in Atlantic salmon (Salmo salar). Exp Mol Pathol 1973;17: Moore JF, Mayr W, Houghle C. Number, location and severity of coronary arterial changes in spawning Pacific salmon (Oncorhynchus). J Comp Pathol 1976:86: Moore JF, Mayr W, Houghle C. Number, location and severity of coronary arterial changes in steelhead trout (Salmo gairdneri). Atherosclerosis 1976:24: Moore JF, Mayr W, Houghle C. infrastructure of coronary arterial changes in spawning pacific salmon (Genus Oncorhynchus) and steelhead trout (Salmo gairdnerii). J Comp Pathol 1976:86: McKenzle JE, House EW, McWIIIIam JG, Johnston DW.

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