Dietary Cholesterol and Fat Saturation Effects on Plasma Esterified ...

Dietary Cholesterol and Fat Saturation Effects on Plasma Esterified and Unesterified Cholesterol in Selected Lines of Japanese Quail Females H. S. SIEGEL, S. M. HAMMAD, R. M. LEACH, and G. F. BARBATO Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802 M. H. GREEN Nutrition Department, The Pennsylvania State University, University Park, Pennsylvania 16802 H. L. MARKS USDA, Agricultural Research Service, Southeast Poultry Research Laboratory, Athens, Georgia 30602 ABSTRACT Three lines of Japanese quail females, randombred controls (CL), high response (HL), and low response (LL) lines, selected for plasma total cholesterol for 18 generations, were fed all-plant-source, nonatherogenic diets to which 0 or .5% cholesterol were added from 6 to 18 wk of age. In all three lines, plasma cholesterol increased when cholesterol was fed; however, responses were greater in the HL than in the LL line, with CL intermediate. In a second experiment, females of the three lines were fed, from 6 to 14 wk of age, four isocaloric, isonitrogenous plant-source diets to which were added: 1) 10% glucose monohydrate (cerelose); 2) 10% cerelose + .1% cholesterol; 3) 4% corn oil; or 4) 4% coconut oil. Baseline data obtained before feeding experimental diets indicated that the HL had significantly higher plasma total, esterified (EC) and unesterified (UEC) cholesterol than LL and that nonovulating females had higher concentrations of esterified cholesterol than ovulating females. Diets used did not affect cholesterol fractions in the ovulating females, although there were significant differences among lines. Dietary cholesterol significantly increased the ratio of EC to UEC. Sclerotic lesion scores were higher in the HL than the LL birds and in birds fed the coconut oil diet. (Key words: Japanese quail, cholesterol, fat saturation, cholesteryl esters, atherosclerosis) 1995 Poultry Science 74:1370-1380

terol and the incidence of atherosclerotic lesions in birds, most of the cholesterol Although nutritional (Stamler et ah, accumulating in the arterial wall during 1958; Fisher and Kaunitz, 1964; Yokode et the natural history of atherosclerosis is ah, 1990), disease (Fabricant, 1985; Shih from endogenous hepatic synthesis (Bortz, and Pyrzak, 1987), and endocrine factors 1973). Inheritance appears to account for (Stamler et ah, 1958; Lupien, 1967) in- most of the individual differences in fluence concentrations of plasma choles- cholesterol metabolism in birds (Leveille and Sauberlich, 1964). Animal species differ in levels of plasma cholesterol and susceptibility to Received for publication December 15, 1994. Accepted for publication March 31, 1995. atherosclerosis (Carver et ah, 1958; Smith INTRODUCTION

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CHOLESTEROL FRACTIONS IN COTURNIX FEMALE PLASMA

and Hilker, 1973) and within species, serum or plasma cholesterol can be altered by genetic selection (Bruell, 1963). In avian species, significant differences in plasma total cholesterol have been produced by selection in Leghorn cocks and hens (Wilcox et ah, 1963; Estep et ah, 1969), pigeons (Clarkson and Lofland, 1961), and Japanese quail (Day et ah, 1977; Marks and Siegel, 1980; Shih et ah, 1983; Siegel et ah, 1984; Marks et ah, 1990). Divergent selection of Japanese quail for plasma cholesterol is accompanied by changes in body weight, plasma triglycerides and glucose, and yolk cholesterol (Siegel et ah, 1984; Marks and Washburn, 1989; Marks et ah, 1990; Marks and Washburn, 1991). Diets high in saturated fat or cholesterol may increase plasma cholesterol and, in turn, cause significant atherosclerotic changes in arteries of humans (Grundy, 1991). Birds also respond to dietary cholesterol by increased plasma cholesterol and concomitant atherosclerosis (Dauber and Katz, 1942; Finlayson and Hirchinson, 1961; Ojerio et ah, 1972; Smith and Hilker, 1973; Morrissey and Donaldson, 1977). The objectives of this research were to investigate some of the mechanisms that might account for the differences in plasma cholesterol previously determined in the females of the genetically selected lines (Marks and Siegel, 1980; Siegel et ah, 1984). Potential mechanisms were intestinal absorption of dietary cholesterol and transfer of cholesterol to the egg yolk. Not directly examined in the present report were possible differences in endogenous hepatic synthesis of cholesterol, and possible differences in the excretion or reabsorption of bile acids. We evaluated the absorption of cholesterol in the selected lines by comparing the effects of plant-source diets containing no cholesterol to those containing known amounts of cholesterol. In Experiment 2, the effects of dietary fat saturation were also considered. It has been reported that the selected lines differed in yolk cholesterol concentration (Marks and Washburn, 1989), which suggested that the lines differed in ability to transfer cholesterol into the egg. The structure of lipoprotein particles sug-

1371

gested that the relationship of cholesterol esters to unesterified cholesterol (CE:UEC ratio) may be important. This relationship may, in part, reflect the size of the very low density lipoprotein (VLDL) particles that transport cholesterol and other lipids to the ovary (Griffin, 1992). If heredity or diet influence this relationship, and thereby VLDL particle size, transfer of cholesterol to the developing ova may be altered. However, measurements of cholesterol fractions at fixed times are static, whereas measurement of atherosclerotic severity represents a cumulative effect. In Experiment 2, the effects of genetic and nutritional factors on atherosclerotic severity were evaluated. MATERIALS AND METHODS Two experiments were performed in which three lines of Japanese quail (Coturnix japonica) were hatched and reared at the Pennsylvania State University Poultry Research Farm from stocks developed at the Southern Regional Genetics Laboratory, Athens, GA 30602 (Marks and Siegel, 1980). The lines had originally been divergently selected from a randombred control line (CL) for 18 generations for high (HL) and low (LL) plasma total cholesterol response to injections of adrenocorticotropin (ACTH). In the 18th generation plasma total cholesterol levels before ACTH administration were 28% above and 30% below the CL for the HL and LL lines, respectively (Marks et ah, 1990). Selection ceased in the 19th generation, but the HL and LL lines were maintained as closed populations by random matings within lines to reproduce the 19th to the 25th generations. In both experiments chicks were grown from hatch to sexual maturity in galvanized metal brooder batteries under standard rearing conditions, with 18 h light:6 h dark. Sex was determined at 4 wk of age. An all-plantsource starter diet (Table 1) and water were provided for ad libitum consumption. Experiment 1 At 40 d of age, the females were randomly assigned by line, diet, and repli-

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SIEGEL ET Ah. TABLE 1. Composition of experimental diets Experiment 1

Ingredients and analysis

Starter diet

Experiment 2

Basal + cholesterol

Cerelose

52.8

52.8

43.8

43.8

46.4

46.2

5.0

5.0

5.0

5.0

5.3

5.3

30.0

30.0

31.0

31.0

32.9

32.7

.5 1.0 .4 .2 6.0 2.0 .1

.5 1.0 .4 .2 6.0 2.0 .1

.5 1.0 .4 .2 6.0 2.0 .1

.5 1.0 .4 .2 6.0 2.0 .1

.5 1.1 .4 .2 6.4 2.1 .1

.5 1.0 .4 .2 6.3 2.1 .1

10.0

10.0

2.0

.3 4.0

.9

2.0

Basal

Cerelose + cholesterol

Corn oil

Coconut oil

(°',\ Yellow corn 39.3 Corn gluten (60% CP) 5.0 Soybean meal (50% CP) 48.0 Trace mineral 1 mix .5 Vitamin mix 2 1.0 Sodium chloride .4 Choline chloride .2 Limestone 1.5 Dicalcium phosphate 2.0 DL-methionine .1 Glucose mono3 hydrate Corn oil 2.0 Coconut oil 4 Cholesterol Calculated analysis ME, kcal/kg 2,902.00 Protein 29.80 Lysine 1.67 Methionine .62 Cystine .46 Calcium 1.09

4.0 .5 2,902.00 22.30 1.13 .52 .35 2.85

2,902.00 22.30 1.13 .52 .35 2.85

.1 2,813.00 22.00 1.14 .51 .35 2.85

2,813.00 22.00 1.14 .51 .35 2.85

2,814.00 22.00 1.14 .51 .35 2.85

2,808.00 22.00 1.14 .51 .35 2.85

iProvided (in milligrams per kilogram of diet): FeS04-7H20, 330; MnS04-H20, 330; KI, 2.6; CuS04-5H20, 16.7; ZnC03, 115; Na2Mo04-2H20, 8.3; NaSe03-5H20, 3.3; Cr(S04)212H20, 20; sucrose diluent. 2 Provided (in milligrams per kilogram of diet): inositol, 250; niacin, 50; Ca pantothenate, 20; pyridoxine-HCl, 4.5; folic acid, 4; menadione, 1.52; biotin, .2; thiamine, 11; riboflavin, 11; vitamin B12, .02; vitamin A, 5,010 IU; cholecalciferol, 1,500 IU; vitamin E, 66 IU; sucrose diluent. 3 Cerelose, CPC International, Summit-Argo, IL 60501. 4 Sigma USP Grade, St. Louis, MO 63178-9916.

cate to 72 cages (20 cm wide x 17 cm deep x 20 cm high) of a quail battery. Diet treatments consisted of a basal all-plantsource breeder diet and the basal diet + .5% crystalline cholesterol without predissolving in fat, as in birds the efficiency of cholesterol absorption is unaffected by solvation (Berrio and Hebert, 1990). Feed and water continued to be consumed ad libitum and the 18 h light:6 h dark photoperiod schedule was maintained. At 28, 56, and 84 d after the start of cholesterol feeding (10, 14, and 18 wk of age), .75 mL of blood was drawn in heparinized syringes from each bird by cardiac puncture and the plasma was stored at -20 C for subsequent analysis. Cholesterol was determined colorimetri-

cally (Zak et ah, 1954) after saponification (Abell et ah, 1952). Experiment 2 At 40 d of age, females that were brooded under the same conditions as Experiment 1 were randomly assigned by line, diet, and replicate to 120 cages of the quail layer battery. Diet treatments began at 42 d of age (Table 1) and consisted of: 1) plant-source basal supplemented with 10% glucose monohydrate (cerelose), no added fat; 2) basal plus 10% cerelose with .1% crystalline cholesterol; 3) basal plus 4% corn oil; 4) basal plus 4% coconut oil. All diets were calculated to provide 2,800 kcal ME/kg and 22% CP.

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CHOLESTEROL FRACTIONS IN COTURNIX FEMALE PLASMA 1

TABLE 2. Effect of dietary cholesterol on plasma total cholesterol of females on lines of Japanese quail selected for high or low plasma cholesterol levels, Experiment 1 Weeks on diet 4

12

8 Dietary cholesterol

Lines

1

0%

.5%

0%

.5%

0%

.5%

(mi- M l ^

i||

358* 267b

SEM

190cd

261b 249bc 178c

26

357> 335* 267b

234* 227bc 208c

** 28

CO CO CS

HL CL LL

** 30

a d

- Means within weeks with no common superscript differ significantly (P < .05). *HL, CL, LL = High, control, and low plasma cholesterol lines, respectively. **Diet differences within weeks are significant (P < .01).

Immediately before beginning dietary treatments (42 d), .25 mL of blood was obtained from each bird from an ulnar vein puncture and drawn into heparin-coated capillary tubes. Plasma was recovered after 5 min centrifugation. Subsequent samples were taken at 4 (70 d of age) and 8 wk (98 d of age) after starting diets. Plasma was stored at -20 C. Measurements of EC and UEC were by HPLC (Duncan et al., 1979; Vercaemst et al, 1982) as modified by Hammad et al. (1992). The EC:UEC ratio was calculated. In this experiment, feed was removed from the troughs during the 8-h dark period immediately preceding the blood sampling. After the final blood sample, the birds were exsanguinated and the aortic arch at the junction of the brachiocephalic artery was removed and preserved in 10% formalin. Aortic atherosclerotic plaque formation scoring was performed on dissected aortae, split longitudinally and evaluated under a dissecting microscope on a 1 to 4 scale for deformation of the endothelium (Shih et al., 1983).

divided into two nominal sex categories: ovulating females and nonovulating females that laid no eggs for 7 d following the blood sample (Bacon et al, 1973). Diet was not considered in the first sample, as experimental dietary treatments had not yet started. For the 4 and 8 wk samples, plasma cholesterol and aorta data were analyzed as 2 (replicate) x 3 (line) x 4 (feed) factorials in completely randomized designs, with 5 birds per experimental unit (120 birds). Data were evaluated by ANOVA using General Linear Models (GLM) procedures (SAS Institute, 1990). The EC:UEC ratio data were converted to arc sine square-root percentages for analysis. Where appropriate, least squares means were computed (SAS Institute, 1990). RESULTS Experiment 1

Preliminary ANOVA indicated a nonsignificant replicate effect, therefore data were combined for presentation in Table 2. Feeding .5% cholesterol significantly inStatistical Analysis creased plasma total cholesterol in the HL In Experiment 1, plasma cholesterol data line at each of the measurement times, but were analyzed within sampling period as 3 did not increase cholesterol levels in the LL (replicate) x 3 (line) x 2 (diet) factorials in a line at the 4- or 12-wk measurements. In the completely randomized design, with 4 CL line, the difference was significant only birds per experimental group (72 birds). In at the 4-wk measurement. These results Experiment 2, for the first sample (i.e., 42 d were reflected in significant line by treatof age), because all birds had not yet begun ment interactions at 4 and 12 wk (P < .05). to ovulate, plasma cholesterol data were Although not significant at all sampling

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SIEGEL ET AL.

TABLE 3. Total esterified and unesterified (free) cholesterol in plasma of 42-d-old ovulating (O) and nonovulating (N) Japanese quail selected for plasma total cholesterol, prediet baseline

Variable Total cholesterol, mg/100 mL Esterified cholesterol, mg/100 mL Unesterified cholesterol, mg/100 mL

Sex classification1 (n)

HL*

(n)

CL

(n)

LL

Sex classification means

O N Line means

(6) (23)

345a 355= 353*

(19) (15)

200b 262" 228Y

(4) (24)

206" 222 b 219Y

231 281 SEM = 28

O N Line means

(6) (23)

110" 192= 175*

(19) (15)

88" 151= 116V

(4) (24)

91b 123=" 118V

93y 155" SEM = 12

O N Line means

(7) (26)

216= 158" 170*

(19) (15)

H2bc Ulbc

(4) (26)

114bc

136 123 SEM = 20

a_c

mY

96' 98V

Means within cholesterol category with no common superscript differ significantly (P < .05). -ySex classification or line means within cholesterol category with no common superscript differ significantly (P < .05). xY ' Sex classification or line means within cholesterol category with no common superscript differ significantly (P < .01). 1 0, N = Ovulating and nonovulating females, respectively. 2 HL, CL, LL = High, control, and low plasma cholesterol lines, respectively. x

times, plasma cholesterol levels were also consistently higher in HL than in LL quail in the absence of dietary cholesterol. Experiment 2 Plasma Cholesterol. As in Experiment 1, significant replicate effects were not present. Prefeed treatment plasma total, EC, and UEC concentrations were significantly higher in HL than in CL or LL quail (Table 3). Differences between ovulating and nonovulating females were not significant for total cholesterol or UEC; however, plasma of nonovulating females had significantly more EC than that of ovulating females. There was a significant line by sex classification interaction for the UEC fraction. Ovulating HL birds had significantly higher UEC cholesterol than nonovulating birds; the differences between nonovulating and ovulating females of the CL and LL were not significant. The effects of feeding experimental diets for 4 and 8 wk are shown in Tables 4 and 5, respectively. Throughout this 8-wk period almost all females were ovulating consistently. Total, EC, and UEC were significantly higher in the HL than in the CL or LL birds at both sampling times. Diet did not have an effect on the total or EC fractions at

4 wk; however, for the UEC fraction, plasma levels were higher in those receiving the basal or coconut oil diets than in those receiving . 1 % cholesterol. After 8 wk on diets, there were no differences among the cholesterol fractions of these females receiving the four diets and there were no interactions. The EC:UEC ratios are shown in Table 6. At both sampling periods, the ratio was highest in birds fed . 1 % cholesterol and at 98 d of age the ratio was lowest in birds fed the cerelose basal diet. There were no line effects and the interactions were not significant. Aorta Plaque Scores. Scores were significantly higher in the HL than in the LL quail, with the CL intermediate and not different from either selected line (Table 7). Birds fed 4% coconut oil had significantly more severe sclerotic lesions than those receiving the basal or corn oil diets, but were not different from those receiving . 1 % cholesterol. Interactions were not significant. DISCUSSION In contradistinction to lines susceptible to experimental atherosclerosis (SEA) [the susceptible-resistant (SUS-RES) lines,


1375

TABLE 4. Total, esterified and unesterified (free) cholesterol in plasma of 70-d-old female Japanese quail 1 selected for plasma total cholesterol and fed four different isocaloric, isonitrogenous diets for 4 wk Line 1 Variable

Diet 2

(n)

HL

(n)

Total cholesterol

1 2 3 4 Line means 1 2 3 4 Line means 1 2 3 4 Line means

(8) (9) (10) (10)

306" 271*1' 228 bc 268 ab 268 x 92* 127" 92* 93 a b 101 x 214" 144" 136 bc 175 ab 165 x

(10) (10) (11) (10)

Esterified cholesterol

Unesterified cholesterol

(8) (9) (10) (10) (8) (9) (10) (10)

(10) (10) (11) (10) (10) (10) (11) (10)

CL

(n)

LL

(mg/dL) 169«* (10) 189cd 1 183 cd (10) 148* bcd 174cd (9) 202 171 cd (10) 2Hbcd 174V 186* 57bc 67** (10) 68^ (10) 63^ 54= 64bc (9) 49c 72bc (10) 57V 66* H2bc (10) 122bc 115* 86' (10) 120bc (9) l 39 bc 122bc (10) 139bc 117* 121*

Diet means 216 198 200 217 SEM = 26 71 84 70 71 SEM = 12 145* 114y 131*y 145" SEM = 18

a_d

Means within cholesterol category with no common superscript differ significantly (P < .05). "-yDiet and line means with no common superscript differ significantly (P < .05). x -*Diet and line means with no common superscript differ significantly (P < .01). 1 HL / CL, LL = High, control, and low plasma cholesterol lines, respectively. 2 Diets were 2,800 kcal/kg, 22% CP corn-soybean, all-plant-source; 1) basal + 10% cerelose; 2) basal + 10% cerelose + . 1 % cholesterol; 3) basal + 4% corn oil; 4) basal + 4% coconut oil.

which were selected for atherosclerotic responses to atherogenic diets (Soret et al, 1976; Shih et al, 1983)], the lines in the present research were the result of 18 generations of divergent selection for plasma total cholesterol response to ACTH. Their selection resulted in high and low cholesterol lines in the absence of atherogenic diets, and eventually, in the absence of ACTH stimulation (Marks and Washburn, 1991). Thus in Experiment 1, HL females maintained higher plasma total cholesterol than LL quail when fed an all-plant-source, nonatherogenic diet. This result confirms the observation that inheritance influences endogenous cholesterol metabolism (Leveille and Sauberlich, 1964). Whether this effect is due to changes in hepatic cholesterol synthesis or to changes in rate of sterol excretion (Marks and Washburn, 1989) remains to be determined for these lines. It has been reported that a higher intake of soy protein increases excretion of neutral sterols, primarily cholesterol, in randombred quail (McClelland and Shih, 1988).

The increase in plasma total cholesterol following the addition of .5% cholesterol to the diet agrees with previous reports on cholesterol-sensitive avian species (Dauber and Katz, 1942; Clarkson and Lofland, 1961; Finlayson and Hirchinson, 1961; q e r i o et al, 1972; Soret et al, 1976; Shih et al, 1983; Radcliffe and Liebsch, 1985); however, it was clear that HL females were more responsive than LL females to dietary cholesterol. Similar results have been reported for Japanese quail in the degree of atherosclerotic responses to dietary cholesterol (Soret et al, 1976; McCormick et al, 1982; Shih et al, 1983), although most of these responses were measured in males. Baseline data of Experiment 2 from blood sampled before feeding experimental diets confirm results of Experiment 1 that HL females have higher total cholesterol than LL females; however, the distribution between EC and UEC (i.e., free) cholesterol fractions depends on the ovulating condition of the birds. Nonovulating females had significantly more EC than ovulating females, but the differ-

1376

SIEGEL ET AL.

TABLE 5. Total, esterified, and unesterified (free) cholesterol in plasma of 98-d-old female Japanese quail selected for plasma total cholesterol and fed four different isocaloric, isonitrogenous diets for 8 wk Line1 2

Diet

Variable Total cholesterol

Esterified cholesterol

Unesterified cholesterol

(n)

1 2 3 4 Line means 1 2 3 4 Line means 1 2 3 4 Line means

(10) (9) (10) (10) (10) (9) (10) (10) (10) (9) (10) (10)

(n)

HL 252* 204* 234* 213* 226 x 118" 105" 116* 94ab 110* 134" 99abc 118* H 9 ab 118*

(10) (8) (10) (10) (10) (8) (10) (10) (10) (8) (10) (10)

CL

(n)

LL

(mg/dL) 200* (10) 132bc 161** (10) 118c 138>* (10) 150bc 172b (10) 125c 168* 1322 77bc (10) 60= 90b 60< (10) 54' (10) 51 c (10) 59= 7 1 bc 70* 56 z 72bc 123* (10) 59"= 7Qbc (10) 99abc 84* c (10) 67bc lOlabc (10) 74* 95**

Diet means 195 161 174 170 S E M = 16

85 85 74 74 SEM = 9

109 76 99 96 S E M = 18

"-•Means within cholesterol category with no common superscript differ significantly (P < .05). x z - Diet and line means with no common superscript differ significantly (P < .01). 1 HL, CL, LL = High, control and low plasma cholesterol lines, respectively. 2 Diets were 2,800 kcal/kg, 22% CP corn-soybean, all-plant-source; 1) basal + 10% cerelose; 2) basal + 10% cerelose + .1% cholesterol; 3) basal + 4% corn oil; 4) basal + 4% coconut oil.

ence in the UEC fraction was not significant. The low number of ovulators in the selected lines (Table 3) reflect the later sexual maturity in these lines; however, despite these low numbers, differences

between the HL and LL lines were significant. We have already reported that males have higher plasma EC levels than females (Hammad et al, 1992). Lipoproteins in mammalian and avian plasma have a general common structure,

TABLE 6. Ratios of esterified to unesterified cholesterol in the plasma of 70- and 98-d-old female Japanese quail selected for plasma total cholesterol and fed four different isocaloric, isonitrogenous diets Line1 Age (d) 70

98

Diet 2 1 2 3 4 Line means 1 2 3 4 Line means

HL .43 c .88* .68* .53* .61 .88 bc 1.06" .98* .79c .93

CL .51" .59" .45" .40" .49 .62" 1.28" .64b .70 b .81

LL .55* c .73" .46c .52 bc .56 .83 b 1.01" .52' .88* .81

Diet means .49Y .73* .53y .49y SEM = .07 .58z 1.11" .7iy z .79X SEM = .05

"-•Means within age with no common superscript differ significantly (P < .05). x-z Diet means with no common superscript differ significantly (P < .05). J HL, CL, LL = High, control and low plasma cholesterol lines, respectively. 2 Diets were 2,800 kcal/kg, 22% CP corn-soybean, all-plant-source; 1) basal + 10% cerelose; 2) basal + 10% cerelose + .1% cholesterol; 3) basal + 4% corn oil; 4) basal + 4% coconut oil.


1377

1

TABLE 7. Atherosclerotic plaque formation in 98-d-old female Japanese quail selected for plasma total cholesterol and fed four different isocaloric, isonitrogenous diets for 8 wk Line 2 Diet 3

HL

CL

1 2 3 4 Diet means

2.1* 2.6» 1.81* 2.5" 2.3*

1.8bc 1.8bc 2.0* 2.3 ab 2.0xy

LL scores) 1.61* 1.3' 1.6^ 2.1* 1.6V

Line means 1.8/ i.9*y 1.8Y 2.3" SEM = .2

a_c

Means with no common superscript differ significantly (P < .05). MTJiet or line means with no common superscript differ significantly (P < .05). iPlaque formation in the aorta at the brachiocephalic junction was scored on a 1 to 4 scale for increasing severity on 10% formalin fixed tissue (Shih et al., 1983). 2 HL, CL, LL = High, control and low plasma cholesterol lines, respectively. 3 Diets were 2,800 kcal/kg, 22% CP corn-soybean, all-plant-source; 1) basal + 10% cerelose; 2) basal + 10% cerelose + .1% cholesterol; 3) basal + 4% corn oil; 4) basal + 4% coconut oil.

which is a particle consisting of a core of triglycerides and cholesteryl esters and a surface layer of phospholipids, apoproteins, and UEC (Brown and Goldstein, 1986; Grundy, 1991; Griffin, 1992). The VLDL particles of ovulating birds are unusually small and uniform in size (35 to 38 nm) compared to those of mammals, avian males, and nonovulating females (30 to 80 nm). This adaptation, which is due to the presence of the apoprotein VLDL-II found in the livers of ovulating birds and produced under estrogen control, ensures delivery to the oocyte membrane particles that are rich in triglyceride (Griffin and Perry, 1985; Nimpf and Schneider, 1991; Pinchasov et al, 1994) and apparently lower in EC (present research). The higher concentration of EC in the plasma of nonovulating females may reflect the greater average size of the VLDL particles compared to those of ovulating females because in principle, the core of the particle, which contains the cholesteryl esters, will be increased by the cube of the radius (r3) compared to the r2 for the shell (Griffin, 1992). The second part of Experiment 2 was conducted to determine whether a low level of cholesterol (.1%) or degree of saturation of fat source in the diet would have effects on plasma cholesterol fractions and atherogenesis in the selected lines. The levels of both the EC and UEC fractions were higher in the HL than in the

CL or LL quail females in the 4- and 8-wk samples; however there was relatively little effect of diet in these females. Only in the 4-wk sample was there a significant diet effect, in which the unesterified fraction of birds fed the coconut oil and basal diets were higher than those fed .1% cholesterol. Although diet did not affect plasma cholesterol fractions, both line and diet did significantly influence atherosclerotic plaque scores, suggesting that the genetic and nutritional effects were cumulative. Higher scores were found in HL quail fed .1% cholesterol or 4% coconut oil than in those fed the cerelose basal or 4% corn oil diets. By contrast, scores of LL birds fed .1% cholesterol were lowest. Several factors, alone or in combination, may affect results. 1) Cholesterol excretion — Cholesterol may be more actively excreted by the LL than the HL either via the egg or bile. In these lines, total cholesterol concentrations are higher in the yolk of LL than in the HL at 22 and 28 wk of age (Marks and Washburn, 1989). However, we have recently shown that by 14 wk of age, the egg yolks of the HL hens are significantly larger than those of the LL (Hammad et al, 1995), and thus, differences between lines in yolk cholesterol may be due to dilution. The plasma EC:UEC ratio was highest in both test periods in birds fed .1%

1378

SIEGEL ET AL.

cholesterol, and tended to be lowest in the birds fed the cerelose basal diet. The result indicates that dietary cholesterol has a greater effect on EC than on UEC. On the other hand, although numerically higher in the HL than in the CL or LL lines, differences between lines were not significant. Thus, if particle size is more affected by the core constituents than those of the surface layer (Griffin, 1992), then an increase in EC should increase particle size, and possibly exclude some VLDL particles from passing through the ovarian basal lamina. The results suggest that although dietary factors may increase VLDL particle size, genetic effects are minimal. 2) Cholesterol synthesis — Cholesterol in the diet may have a greater suppressive effect on hepatic cholesterol synthesis in the LL than in the HL by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase activity (Bortz, 1973). Inhibition of HMG-CoA reductase has been described in genetically restricted ovulator chickens (Mitchell et ah, 1979). In birds, low density lipoproteins (LDL), which contain high concentrations of EC are capable of down-regulating HMGCoA reductase activity through the LDLreceptor pathway (Brown and Goldstein, 1986). Low density lipoproteins are not secreted by the liver, but are produced in circulation by conversion from plasma VLDL. However, as indicated above, although in high concentration in ovulating avian female plasma, VLDL is protected by the apoprotein-VLDL II for delivery to the oocyte (Nimpf and Schneider, 1991) and there is little or no LDL. 3) Macrophages and Atherosclerosis — Early atherosclerotic lesions are characterized by the presence of macrophagederived foam cells that take u p lipoproteins by receptor-mediated endocytosis. In rabbits, macrophage colony-stimulating factor (M-CSF) plays an important role in moderating the atherogenic process by increasing the net hydrolytic activity of CE (Watanabe et al, 1995). If M-CSF activity is genetically influenced, LL quail, which already have lower plasma EC concentrations than HL quail, may also accumulate less EC in arterial walls.

In conclusion, the results show that sexually mature Japanese quail females divergently selected for plasma total cholesterol levels differ significantly in plasma cholesterol even in the absence of dietary cholesterol and that the effect is accentuated by adding even low levels of cholesterol or saturated fat to the diet. Atherosclerosis is more evident in the HL than in the LL quail and is increased by dietary cholesterol or saturated fat. Although plasma EC and UEC both increase in the HL, the effect of cholesterol in the diet appears greater in the EC. This effect may result in changes in lipoprotein particle size and be related to severity of atherosclerosis. The results do not preclude the possibility that excretion via the bile may differ among lines or that the other component of the lipoprotein particle core, triglycerides, may be influenced. ACKNOWLEDGMENTS The authors thank B. C. Ford for typing the manuscript. This research was conducted under Institutional Animal Care and Use Committee No. 1424 and No. 1690, The Pennsylvania State University, and was supported by Pennsylvania Department of Agriculture Grant ME 449024 to H.S.S.

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