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Experimental dietary production of aortic atherosclerosis in Japanese quail
Atherosclerosis, 17 (1973) 63-70 0 Elsevier Scientific Publishing Company,
63 Amsterdam
- Printed in The Netherlands
EXPERIMENTAL DIETARY PRODUCTION SCLEROSIS IN JAPANESE QUAIL
OF AORTIC
ATHERO-
ROBERT L. SMITH AND DORIS M. HILKER Department of Food and Nutritional Sciences, University of Hawaii, Honolulu, Hawaii 96822 (U.S.A.) (Received March 31st, 1972)
SUMMARY
Japanese quail (Coturnix coturnix,japonicu) were evaluated as laboratory animals for use in atherosclerosis research by measuring plasma cholesterol along with macroscopic examination of the large arteries for atheromatous lesions. Eighteen-day old quail chicks were placed on a series of 11 varied diets to elicit response to sugar, fats and cholesterol. Birds were sacrificed at the end of 3, 6 and 9 months. Atheromas were found in birds given diets with high saturated fat and added cholesterol. Well defined indications of early stage arterial damage was observed in quail on several of the diets containing cholesterol, high sucrose and high saturated fatty acids (SAFA).
Key words: Animal model - Atheromas - Atherosclerosis - Cholesterol - Diet effects - Japanese quail
INTRODUCTION
Since the time that Anitschkowi produced vascular changes in rabbits, many animals have been used to demonstrate atherosclerosis?. Some are extremely resistant to the development of the disease such as the rat, hamster, ground squirrel and dog. Other animals including the rabbit, pig and chicken develop lipemia and atherosclerosis rather easily in response to simple cholesterol feeding. The quest has continued over the years to find an experimental animal that will best fulfill a long list of ideal qualities and all of the candidates to date have required considerable compromise from the ideal. Short life cycle, omnivorous eating habits, ease of housing and care, ability to produce a rapid and characteristic lesion are among the qualities desired. This investigation
was supported
in part by a grant from the Hawaii Heart Association.
64
ROBERT L. SMITH, DORIS M. HILKER
Starting in the early 1960’s, there arose considerable interest in the use of Japanese quail as a laboratory animal. They have been employed in embryologys-5, genetic&’ and nutrition*-lo. It has been notedll that Japanese quail thrive in small laboratory cages (200 cm2 per bird) and are simple, economical and easy to maintain. The cost per animal is small and the reproductivity is high with the female laying up to 300 eggs per year. Of special interest is the short life cycle: 16 to 17 days are required for hatching and sexual maturity is reached at approximately 42 days of age. They are omnivorous and consume about 1.5g of feed per day. The quail are hardy and have a high metabolic rate with a short life spanla. In short, they would appear to possess many of the attributes we would look for in a laboratory model for atherosclerosis research. The purposeaf the present study was to determine whether atherosclerotic lesions would develop in Japanese quail in response to diet. The effect of diet on plasma cholesterol was also determined.
MATERIALS AND METHODS
Eighteen-day old mixed male and female Japanese quail chicks were obtained from the University of Hawaii, Department of Animal Sciences. Both feed and water were presented ad libitum throughout the experiment. They were housed in modified standard rat cages with 2 or 3 birds to the cage. The room was lighted to a 14-hour day and the temperature was maintained at 75 “F. Eleven isocaloric and isoproteinic diets differing in levels and types of fat and carbohydrates were fed with and without added cholesterol as follows: C (control), PUFA (40% of calories from polyunsaturated oil), SAFA (40% of calories from saturated fat-beef tallow), HSLF (50% of calories from sucrose), LSHF (53 % of calories from beef tallow), ATH (53 % of calories from beef tallow plus 12 % of calories from sucrose and with 1% added cholesterol (Table 1). With the exception of the ATH diet, all of the diets with cholesterol (/c) at 0.5 % were the same as the corresponding diet without cholesterol. At the termination of 3 different feeding periods (I, II and III) corresponding to approximately 3, 6 and 9 months, some of the birds from each dietary group were sacrificed. Blood was drawn via heart puncture using a heparinized syringe. The blood sample was centrifuged and the plasma layer was used for cholesterol determinations using the method of Henlyr3 as modified by Henry14. The heart with the attached aortic tree (the brachiocephalic arteries to their bifurcations and the aorta to the iliac branching) was dissected from the body. The longitudinally opened arteries were examined under a 10 x dissecting binocular microscope for gross lesions. Student’s t testis316 was used to determine the significance of plasma cholesterol differences between the control groups and the various other dietary groups.
450 28.7 50 40 40 9 15 0 30 18 19.3 5
450 28.7 50 40 40 9 15 0 30 18 19.3 0
300 100 100 213.7 40 40 9 160 0 0 18 19.3 0
PUFA
300 95 100 213.7 40 40 9 160 0 0 18 19.3 5
PlJFA/c
300 100 100 213.7 40 40 9 0 160 0 18 19.3 0
SAFA
300 95 100 213.7 40 40 9 0 160 0 18 19.3 5
SAFAIc
300 0 478.7 50 40 40 9 1.5 0 30 18 19.3 0
HSLF 300 100 0 273.7 40 40 9 0 200 0 18 19.3 0
473.7 50 40 40 9 15 0 30 18 19.3 5
LSHF
300 0
HSLF/c
18 19.3 5
0
0 200
40 40 9
0 273.7
300 95
LSHF/c
10
18 19.3
0
0 200
40 40 9
90 273.7
300 0
ATH
Nutritional Biochemicals Corp. Mineral mix %: KC1 44.9; NaCl 28.1; MgS04 22.45; Al~(S0a)a 1.41; MnS04 1.12; Fez03 0.12; CuSO4 0.44; ZnS04 0.29; Co(CHsCOO)a 0.11; KI 0.06. Microingredient mix %: calcium gluconate 59.87; &methionine 26.94; choline chloride 11.97; niacin 0.48; d-a-tocopherol acetate (1360 IU/g) 0.20; riboflavin 0.24; thiamine-HCl 0.12; calcium pantothenate 0.12; pyridoxine 0.03; folic acid 0.006; biotin 0.0005; vitamin Brz (0.1 “/, in gelatin) 0.018; vitamin A 780 USPU/g mix; vitamin D 72 ICU/g mix.
300
c/c
300
C
(g/kg)
Soy protein Starch Sucrose AlphaceP Calcium gluconate Calcium phosphate Sodium phosphate Corn oil Beef tallow Glycerol Mineral mixb Micro nutrient mixC Cholesterol
DIET FORMULATION
TABLE 1
i:
s
:
ii! ci
2
5:
5 t;
B B mp
ti
66
ROBERT L. SMITH, DORIS M. HILKER
TABLE
2
THE RATING FROM
OF VARIOUS
OBSERVED
LESIONS
Dietary groups
Period
C
DIETARIES AND
ACCORDING
PRB-LESION
TO A RELATIVE
INDEX
OF
TRIN
0 0 0
19
1.06
0 0 1
0 0 0
25
1.39
Ratings (R) a 1s
2s
3s
4s
I II III
18
6 4 7
0
0 0 0
I II III
18
I II III
15
PUFA/c
I II III
SAFA
7 4 1
CALCULATED
TRS
No. of quail
c/c
ATHEROGENICITY
TISSUE
1 0 0 1
4
6 3 0
0 1 0
0 0 0
22
1.47
1 4
15
1 3 1
2 1 3
3 1 0
0 0 0
29
1.93
I II III
17
2 0 0
1 3 5
4 0 2
0 0 0
38
2.24
SAFA/c
I II III
16
2 0 0
3 0 0
2 3 2
0 1 3
47
2.94
HSLF
I II III
15
2 0 0
2 4 3
0 1 2
1 0 0
33
2.20
HSLF/c
I II III
16
0 0 0
4 2 2
2 1 5
0 0 0
40
2.50
LSHF
I II III
20
3 1 0
1 4 5
2 3 1
0 0 0
42
2.10
LSHF/c
I II III
16
4 0 0
2 3 1
0 2 1
0 0 3
37
2.31
ATH
III
7
0
0
0
7
28
4.00
PUFA
0
a 1s = no pre-lesion; 2s = slight pre-lesion, pink; 3s = considerable fatty streaks, red; 4s = atheromas observed. T = total each rating; R = rating each aorta.
RESULTS
Morphological observations
The gross examination of the dissected arterial tree revealed wide differences
AORTICATHEROSCLEROSIS IN JAPANESE QUAIL
Fig. 1. Typical lesions in quail aorta,
x
67
3.
among the various dietary groups as well as between individuals within a group. Table 2 shows a rating of the various diets based on a subjective evaluation of the excised aortas examined. The controls were free of lesions at all 3 sacrifice periods and the opened arterial tissue was firm and elastic with a smooth white intima. At the opposite end of the scale, all of the ATH group and most of the SAFA/c and LSHF/c groups showed lesions in Period III. Fig. 1 illustrates a typical lesion. Certain lesions had progressed almost to a point of totally occluding the arterial lumen. The heavily diseased arteries were pink to red in color, soft and non-elastic. They were often fragile and difficult to remove intact from the bird. Certain of these arteries were atheromatous as well, with the lesions being white, relatively soft, compact and raised without taking the form of bands or streaks. Others had fatty streaks alone. The lesions appeared to develop from scattered foci with the encephalobrachial arteries bearing the greatest number of lesions. No ulcerated lesion was observed but notice was taken on several occasions of a thrombus adhering to a lesion. The color of the intima varied from white in the controls, through shades of pink to a dark red in the case of the ATH, LSHF/c and SAFA/c dietary groups. Between the extremes of the various dietary groups, there was not only color and elasticity differences as already mentioned, but also observed was a longitudinally striated thickening of the tissue. When the artery was opened and flattened the interior appeared rough or wrinkled. Such longitudinal wrinkling was not seen in the controls or any of the first period quail. In the third period, the roughened striations varied from barely discernable to very apparent in the cholesterol fed birds. The quail on the HSLF, LSHF, SAFA and ATH diets showed this condition. The severity was increased by the inclusion of cholesterol in the diet.
ROBERT L. SMITH, DORIS M. HILKER
68 TABLE PLASMA
3 CHOLESTEROL
Dietary groups
Period
No. of quail
PIasma cholesterol (mg/IOO ml)
C
I II III
6 5 I
257 + 33 292 f 107 301 f 98
w
I II III
I 5 6
341 f 40 336 f 46 193 f 51
PUFA
I II III
6 5 4
269 f 257 f 290 f
PUFA/c
I II III
6 5 4
334 f 44 325 f 47 380 f 164
SAFA
I II III
I 3 7
359 f 222 f 383 f
SAFA/c
I II III
7 4 5
506 & 126 546 f 69s 1904 f 828&
HSLF
I II III
4 5 6
444 f 280 268 f 89 398 + 136
HSLF/c
I II III
6 3 7
669 f 302 f 556 f
218& 142 183
LSHF
1 II III
6 8 6
410 f 357 f 617 f
112b 105 343
LSHF/c
I II III
6 5 5
778 i 661 793 & 425” 1778 f 411a
ATH
III
7
1036 % 367a
25 54 82
165 14 134
* Probability when compared to control group < 0.01. b Probability when compared to control group < 0.05.
Plasma cholesterol
Plasma cholesterol levels are shown in Table 3. Plasma cholesterol levels in Japanese quail on SAFA, HSLF and LSHF diets showed a sharp increase when cholesterol was added to the diet. The mean plasma cholesterol levels were significantly higher than controls on the SAFA, ATH and LSHF diets; there were also wide
AORTIC
ATHEROSCLEROSIS
IN JAPANESE
QUAIL
69
deviations from the mean which were further exaggerated by dietary cholesterol. The inclusion of cholesterol in the control and PUFA diets had little effect on plasma cholesterol, whereas in the SAFA diet it stimulated the production of a very high plasma cholesterol level in Period III. The inclusion of cholesterol in any of the diets, other than the control, tended to raise the plasma cholesterol level. The length of time on the diet had a marked effect in some cases such as SAFA/c and the LSHF/c groups in the third period while the other diets showed no consistent pattern in this regard.
DISCUSSION
The usefulness of Japanese quail as an animal model for the study of atherosclerosis was demonstrated. Results indicate that this animal is capable of producing atheromatous lesions as well as the biochemical patterns expected to be consistent with this. Fatty streaks were produced in a relatively short time without the employment of drastic measures. Since the histochemical characteristics of the lesions were not studied, it would be presumptive to directly compare the lesions produced by quail to the human disease except to say that both suffer occluding arterial lesions. Parallelism was visually noted in some aspects of the lesion genesis observed such as the initial irritation or damage to the arterial wall, this being followed by a thickening of the tissue with the eventual appearance of lesions as described in Constantinides’ monographs outlining the disease progression as seen in man. Arterial lesions were apparent on several of the dietaries especially SAFA/c, LSHF/c and ATH compared to the controls and PUFA. On these diets the apparent damage became more intense with increased time, i.e., from Periods I to II to III. This tended to lead to a thickened and roughened arterial wall which in some groups was followed by the development of atheromatous lesions. Plasma cholesterol is a useful indicator widely used in atherosclerosis research. In Japanese quail it would appear that the normal plasma cholesterol range is between 200 to 300 mg %. This closely approximates the levels found in man for both North America and Western Europe. The observed plasma cholesterol responses to dietary influences are shared by manr’~r*. Dietary manipulations involved fats, sucrose and cholesterol as dietary stress factors. SAFA tended to raise plasma cholesterol while at the same time revealing considerable individual variation. Dietary cholesterol increased plasma cholesterol particularly in groups where the diet was already lipogenic. PUFA did not raise plasma cholesterol, and in the presence of dietary cholesterol it exhibited a protective or depressive effect against high plasma cholesterol levels as compared to diets with SAFA and sucrose. The several diets (SAFA, LSHF, ATH) that were high in saturated fats along with sucrose (HSLF), produced elevated plasma cholesterol levels, even when cholesterol was not included in the diet. This is in agreement with the observations of others17 that lipogenie dietary substances tend to raise plasma cholesterolrs~ra.
70
ROBERT L. SMITH, DORIS M. HILKER
ACKNOWLEDGMENTS
Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Journal Series No. 1362.
REFERENCES
1 ANITSCHKOW,N., Ueber die VerPnderung der Kaninchenaorta bei experimenteller Cholesterinsteatose, B&r. Puthol. Anat., 56 (1913) 379. 2 CONSTANTINIDES, P., Experimental Atherosclerosis, Elsevier, Amsterdam, 1965. 3 MCFARLAND,L. Z., AND V. 0. WILSON,Brain growth in Japanese quail, Anat. Rec., 151 (1965)465. 4 PAGGE~, C. S., AND W. D. IVEY, Coturnix quail as a laboratory research animal, Science, 129 (1959) 267. 5 RAUSCHER,F. J., AND J. A. REYNIERS,Japanese quail egg embryo as a host for viruses, J. Bacterial., 84 (1962) 1134. 6 POOLE, H. K., Egg shell pigmentation in Japanese quail. Genetic control of the egg trait, J. Hered., 55 (1964) 136. 7 SITTMANN,K. H., AND H. ABPLANALP,Inbreeding depression in Japanese quail, Genetics, 45 (1966) 371. 8 REYNIERS,J. A., AND M. R. SACHSTEDER, Nutrition of germ free and conventional Coturnix coturmx japonica, Abstr. 5th Znt. Congr. Nutr., Washington, D.C., 1960, p. 25. 9 HOWES, J. R., Energy, protein, methionine and lysine requirements for growing and laying Coturnix quail, Proc. Sou. Agr. Wkrs. Conv., Dallas, 1965, p. 258.
10 ALFORD, R. D., D. SCHULTZ AND T. THORNTON,The development Coturnix
of vitamin deficiencies in
quail, Poultry
Sci., 46 (1967) 1227. IVEY, W. D., AND J. R. HOWES,The Coturnix quail, U.F.A. W. Handbook, 3rd edition, 1967, p. 281. Coturnix, National Academy of Sciences Pub/. No. 1703, Washington, D.C., 1969. HENLY, A. A., Determination of cholesterol, Analyst, 82 (1957) 286. HENRY, R. J., AND Y. CHIAMORI,Cholesterol determination, Clin. Chim. Acta, 6 (1961) 1. KALTON, G., Introduction to Statistical Ideas, Chapman and Hall, London, 1966. ARKIN, H., AND R. COLTON,Statistical Methods, Barnes and Noble, New York, 1959.
11 12 13 14 15 16 17 FRIEDMAN,G. J., Nutrition in relation to atherosclerosis. In: M. G. WOHL AND R. S. G~~DHART (Eds.), Modern Nutrition in Health and Disease, 4th edition, Lea and Febiger, Philadelphia, Pa., 1969, pp. 877-931. between 18 ANTAR, M. A., J. A. LITTLE,C. LUCAS,G. C. BUCKLEYAND A. CSIMA,Interrelationship kinds of dietary carbohydrate and fat in hyperliproteinemic patients, Part 3 (Synergistic effect of sucrose and animal fat on serum lipid), J. Atheroscler. Res., 11 (1970) 191. 19 DAVIDSON,S., AND R. PASSMORE, Human Nutrition and Dietetics, 4th edition, Williams and Wilkins, Baltimore, Md., 1969, pp. 100-105.
63 Amsterdam
- Printed in The Netherlands
EXPERIMENTAL DIETARY PRODUCTION SCLEROSIS IN JAPANESE QUAIL
OF AORTIC
ATHERO-
ROBERT L. SMITH AND DORIS M. HILKER Department of Food and Nutritional Sciences, University of Hawaii, Honolulu, Hawaii 96822 (U.S.A.) (Received March 31st, 1972)
SUMMARY
Japanese quail (Coturnix coturnix,japonicu) were evaluated as laboratory animals for use in atherosclerosis research by measuring plasma cholesterol along with macroscopic examination of the large arteries for atheromatous lesions. Eighteen-day old quail chicks were placed on a series of 11 varied diets to elicit response to sugar, fats and cholesterol. Birds were sacrificed at the end of 3, 6 and 9 months. Atheromas were found in birds given diets with high saturated fat and added cholesterol. Well defined indications of early stage arterial damage was observed in quail on several of the diets containing cholesterol, high sucrose and high saturated fatty acids (SAFA).
Key words: Animal model - Atheromas - Atherosclerosis - Cholesterol - Diet effects - Japanese quail
INTRODUCTION
Since the time that Anitschkowi produced vascular changes in rabbits, many animals have been used to demonstrate atherosclerosis?. Some are extremely resistant to the development of the disease such as the rat, hamster, ground squirrel and dog. Other animals including the rabbit, pig and chicken develop lipemia and atherosclerosis rather easily in response to simple cholesterol feeding. The quest has continued over the years to find an experimental animal that will best fulfill a long list of ideal qualities and all of the candidates to date have required considerable compromise from the ideal. Short life cycle, omnivorous eating habits, ease of housing and care, ability to produce a rapid and characteristic lesion are among the qualities desired. This investigation
was supported
in part by a grant from the Hawaii Heart Association.
64
ROBERT L. SMITH, DORIS M. HILKER
Starting in the early 1960’s, there arose considerable interest in the use of Japanese quail as a laboratory animal. They have been employed in embryologys-5, genetic&’ and nutrition*-lo. It has been notedll that Japanese quail thrive in small laboratory cages (200 cm2 per bird) and are simple, economical and easy to maintain. The cost per animal is small and the reproductivity is high with the female laying up to 300 eggs per year. Of special interest is the short life cycle: 16 to 17 days are required for hatching and sexual maturity is reached at approximately 42 days of age. They are omnivorous and consume about 1.5g of feed per day. The quail are hardy and have a high metabolic rate with a short life spanla. In short, they would appear to possess many of the attributes we would look for in a laboratory model for atherosclerosis research. The purposeaf the present study was to determine whether atherosclerotic lesions would develop in Japanese quail in response to diet. The effect of diet on plasma cholesterol was also determined.
MATERIALS AND METHODS
Eighteen-day old mixed male and female Japanese quail chicks were obtained from the University of Hawaii, Department of Animal Sciences. Both feed and water were presented ad libitum throughout the experiment. They were housed in modified standard rat cages with 2 or 3 birds to the cage. The room was lighted to a 14-hour day and the temperature was maintained at 75 “F. Eleven isocaloric and isoproteinic diets differing in levels and types of fat and carbohydrates were fed with and without added cholesterol as follows: C (control), PUFA (40% of calories from polyunsaturated oil), SAFA (40% of calories from saturated fat-beef tallow), HSLF (50% of calories from sucrose), LSHF (53 % of calories from beef tallow), ATH (53 % of calories from beef tallow plus 12 % of calories from sucrose and with 1% added cholesterol (Table 1). With the exception of the ATH diet, all of the diets with cholesterol (/c) at 0.5 % were the same as the corresponding diet without cholesterol. At the termination of 3 different feeding periods (I, II and III) corresponding to approximately 3, 6 and 9 months, some of the birds from each dietary group were sacrificed. Blood was drawn via heart puncture using a heparinized syringe. The blood sample was centrifuged and the plasma layer was used for cholesterol determinations using the method of Henlyr3 as modified by Henry14. The heart with the attached aortic tree (the brachiocephalic arteries to their bifurcations and the aorta to the iliac branching) was dissected from the body. The longitudinally opened arteries were examined under a 10 x dissecting binocular microscope for gross lesions. Student’s t testis316 was used to determine the significance of plasma cholesterol differences between the control groups and the various other dietary groups.
450 28.7 50 40 40 9 15 0 30 18 19.3 5
450 28.7 50 40 40 9 15 0 30 18 19.3 0
300 100 100 213.7 40 40 9 160 0 0 18 19.3 0
PUFA
300 95 100 213.7 40 40 9 160 0 0 18 19.3 5
PlJFA/c
300 100 100 213.7 40 40 9 0 160 0 18 19.3 0
SAFA
300 95 100 213.7 40 40 9 0 160 0 18 19.3 5
SAFAIc
300 0 478.7 50 40 40 9 1.5 0 30 18 19.3 0
HSLF 300 100 0 273.7 40 40 9 0 200 0 18 19.3 0
473.7 50 40 40 9 15 0 30 18 19.3 5
LSHF
300 0
HSLF/c
18 19.3 5
0
0 200
40 40 9
0 273.7
300 95
LSHF/c
10
18 19.3
0
0 200
40 40 9
90 273.7
300 0
ATH
Nutritional Biochemicals Corp. Mineral mix %: KC1 44.9; NaCl 28.1; MgS04 22.45; Al~(S0a)a 1.41; MnS04 1.12; Fez03 0.12; CuSO4 0.44; ZnS04 0.29; Co(CHsCOO)a 0.11; KI 0.06. Microingredient mix %: calcium gluconate 59.87; &methionine 26.94; choline chloride 11.97; niacin 0.48; d-a-tocopherol acetate (1360 IU/g) 0.20; riboflavin 0.24; thiamine-HCl 0.12; calcium pantothenate 0.12; pyridoxine 0.03; folic acid 0.006; biotin 0.0005; vitamin Brz (0.1 “/, in gelatin) 0.018; vitamin A 780 USPU/g mix; vitamin D 72 ICU/g mix.
300
c/c
300
C
(g/kg)
Soy protein Starch Sucrose AlphaceP Calcium gluconate Calcium phosphate Sodium phosphate Corn oil Beef tallow Glycerol Mineral mixb Micro nutrient mixC Cholesterol
DIET FORMULATION
TABLE 1
i:
s
:
ii! ci
2
5:
5 t;
B B mp
ti
66
ROBERT L. SMITH, DORIS M. HILKER
TABLE
2
THE RATING FROM
OF VARIOUS
OBSERVED
LESIONS
Dietary groups
Period
C
DIETARIES AND
ACCORDING
PRB-LESION
TO A RELATIVE
INDEX
OF
TRIN
0 0 0
19
1.06
0 0 1
0 0 0
25
1.39
Ratings (R) a 1s
2s
3s
4s
I II III
18
6 4 7
0
0 0 0
I II III
18
I II III
15
PUFA/c
I II III
SAFA
7 4 1
CALCULATED
TRS
No. of quail
c/c
ATHEROGENICITY
TISSUE
1 0 0 1
4
6 3 0
0 1 0
0 0 0
22
1.47
1 4
15
1 3 1
2 1 3
3 1 0
0 0 0
29
1.93
I II III
17
2 0 0
1 3 5
4 0 2
0 0 0
38
2.24
SAFA/c
I II III
16
2 0 0
3 0 0
2 3 2
0 1 3
47
2.94
HSLF
I II III
15
2 0 0
2 4 3
0 1 2
1 0 0
33
2.20
HSLF/c
I II III
16
0 0 0
4 2 2
2 1 5
0 0 0
40
2.50
LSHF
I II III
20
3 1 0
1 4 5
2 3 1
0 0 0
42
2.10
LSHF/c
I II III
16
4 0 0
2 3 1
0 2 1
0 0 3
37
2.31
ATH
III
7
0
0
0
7
28
4.00
PUFA
0
a 1s = no pre-lesion; 2s = slight pre-lesion, pink; 3s = considerable fatty streaks, red; 4s = atheromas observed. T = total each rating; R = rating each aorta.
RESULTS
Morphological observations
The gross examination of the dissected arterial tree revealed wide differences
AORTICATHEROSCLEROSIS IN JAPANESE QUAIL
Fig. 1. Typical lesions in quail aorta,
x
67
3.
among the various dietary groups as well as between individuals within a group. Table 2 shows a rating of the various diets based on a subjective evaluation of the excised aortas examined. The controls were free of lesions at all 3 sacrifice periods and the opened arterial tissue was firm and elastic with a smooth white intima. At the opposite end of the scale, all of the ATH group and most of the SAFA/c and LSHF/c groups showed lesions in Period III. Fig. 1 illustrates a typical lesion. Certain lesions had progressed almost to a point of totally occluding the arterial lumen. The heavily diseased arteries were pink to red in color, soft and non-elastic. They were often fragile and difficult to remove intact from the bird. Certain of these arteries were atheromatous as well, with the lesions being white, relatively soft, compact and raised without taking the form of bands or streaks. Others had fatty streaks alone. The lesions appeared to develop from scattered foci with the encephalobrachial arteries bearing the greatest number of lesions. No ulcerated lesion was observed but notice was taken on several occasions of a thrombus adhering to a lesion. The color of the intima varied from white in the controls, through shades of pink to a dark red in the case of the ATH, LSHF/c and SAFA/c dietary groups. Between the extremes of the various dietary groups, there was not only color and elasticity differences as already mentioned, but also observed was a longitudinally striated thickening of the tissue. When the artery was opened and flattened the interior appeared rough or wrinkled. Such longitudinal wrinkling was not seen in the controls or any of the first period quail. In the third period, the roughened striations varied from barely discernable to very apparent in the cholesterol fed birds. The quail on the HSLF, LSHF, SAFA and ATH diets showed this condition. The severity was increased by the inclusion of cholesterol in the diet.
ROBERT L. SMITH, DORIS M. HILKER
68 TABLE PLASMA
3 CHOLESTEROL
Dietary groups
Period
No. of quail
PIasma cholesterol (mg/IOO ml)
C
I II III
6 5 I
257 + 33 292 f 107 301 f 98
w
I II III
I 5 6
341 f 40 336 f 46 193 f 51
PUFA
I II III
6 5 4
269 f 257 f 290 f
PUFA/c
I II III
6 5 4
334 f 44 325 f 47 380 f 164
SAFA
I II III
I 3 7
359 f 222 f 383 f
SAFA/c
I II III
7 4 5
506 & 126 546 f 69s 1904 f 828&
HSLF
I II III
4 5 6
444 f 280 268 f 89 398 + 136
HSLF/c
I II III
6 3 7
669 f 302 f 556 f
218& 142 183
LSHF
1 II III
6 8 6
410 f 357 f 617 f
112b 105 343
LSHF/c
I II III
6 5 5
778 i 661 793 & 425” 1778 f 411a
ATH
III
7
1036 % 367a
25 54 82
165 14 134
* Probability when compared to control group < 0.01. b Probability when compared to control group < 0.05.
Plasma cholesterol
Plasma cholesterol levels are shown in Table 3. Plasma cholesterol levels in Japanese quail on SAFA, HSLF and LSHF diets showed a sharp increase when cholesterol was added to the diet. The mean plasma cholesterol levels were significantly higher than controls on the SAFA, ATH and LSHF diets; there were also wide
AORTIC
ATHEROSCLEROSIS
IN JAPANESE
QUAIL
69
deviations from the mean which were further exaggerated by dietary cholesterol. The inclusion of cholesterol in the control and PUFA diets had little effect on plasma cholesterol, whereas in the SAFA diet it stimulated the production of a very high plasma cholesterol level in Period III. The inclusion of cholesterol in any of the diets, other than the control, tended to raise the plasma cholesterol level. The length of time on the diet had a marked effect in some cases such as SAFA/c and the LSHF/c groups in the third period while the other diets showed no consistent pattern in this regard.
DISCUSSION
The usefulness of Japanese quail as an animal model for the study of atherosclerosis was demonstrated. Results indicate that this animal is capable of producing atheromatous lesions as well as the biochemical patterns expected to be consistent with this. Fatty streaks were produced in a relatively short time without the employment of drastic measures. Since the histochemical characteristics of the lesions were not studied, it would be presumptive to directly compare the lesions produced by quail to the human disease except to say that both suffer occluding arterial lesions. Parallelism was visually noted in some aspects of the lesion genesis observed such as the initial irritation or damage to the arterial wall, this being followed by a thickening of the tissue with the eventual appearance of lesions as described in Constantinides’ monographs outlining the disease progression as seen in man. Arterial lesions were apparent on several of the dietaries especially SAFA/c, LSHF/c and ATH compared to the controls and PUFA. On these diets the apparent damage became more intense with increased time, i.e., from Periods I to II to III. This tended to lead to a thickened and roughened arterial wall which in some groups was followed by the development of atheromatous lesions. Plasma cholesterol is a useful indicator widely used in atherosclerosis research. In Japanese quail it would appear that the normal plasma cholesterol range is between 200 to 300 mg %. This closely approximates the levels found in man for both North America and Western Europe. The observed plasma cholesterol responses to dietary influences are shared by manr’~r*. Dietary manipulations involved fats, sucrose and cholesterol as dietary stress factors. SAFA tended to raise plasma cholesterol while at the same time revealing considerable individual variation. Dietary cholesterol increased plasma cholesterol particularly in groups where the diet was already lipogenic. PUFA did not raise plasma cholesterol, and in the presence of dietary cholesterol it exhibited a protective or depressive effect against high plasma cholesterol levels as compared to diets with SAFA and sucrose. The several diets (SAFA, LSHF, ATH) that were high in saturated fats along with sucrose (HSLF), produced elevated plasma cholesterol levels, even when cholesterol was not included in the diet. This is in agreement with the observations of others17 that lipogenie dietary substances tend to raise plasma cholesterolrs~ra.
70
ROBERT L. SMITH, DORIS M. HILKER
ACKNOWLEDGMENTS
Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Journal Series No. 1362.
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