550 Express,. Diagnostics Corp., Oberlin, OH 44074). Insulin, gluca- .... proportion of BW (data not shown) indicated allomet- ric growth of organs in all four ...
Response to dietary fat and cholesterol in young adult boars genetically selected for high or low plasma cholesterol C. D. Lu, W. G. Pond, H. J. Mersmann, D. R. Su, L. Krook, J. J. Harris and J. W. Savell J ANIM SCI 1995, 73:2043-2049.
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Response to Dietary Fat and Cholesterol in Young Adult Boars GeneticallySelectedfor High or Low Plasma Cholesterol1 C. D. Lu293, W. G. P ~ n d ~H. , ~ J. , Mersmann2, D.-R. Su2, L.Kr00k5, J. W. Save116
J. J. Harris6*',and
ABSTRACT: Twenty-eight young adult boars (age 7 to 8 mol genetically selected for four generations for high ( H G ) or low ( L G ) plasma cholesterol were studied t o assess dietary and genetic effects and their interactions on cholesterol metabolism. Boars within a genetic group were paired according t o their plasma cholesterol concentration at 8 wk of age and were fed for 15 wk (2.7 kg/d) one of two diets (16.5% fat and 1,156 mg of cholesterolkg diet, HD; or 3.1% fat and 0 cholesterol diet, LD) in a 2 x 2 factorial arrangement. Plasmatotal cholesterol ( P < .O 1) and HDLcholesterol ( P < .O 1) concentrations were higher in boars fed HS ( P < .01) and in HG boars ( P < .01). There was a trend toward a diet x genotype interaction for plasma HDL-cholesterol ( P < .06). Plasma Key Words:
insulin concentration tended to be lower in boars fed HD ( P < .09) or HG boars ( P < . l o ) . There was a diet x genotype interaction for plasma glucagon ( P < .04) concentration and a trend toward an interaction for insulin ( P < .07). After 15 wk, all boars were killed by electrocution and exsanguination and measurements were taken. Backfat thickness was greater ( P < .01) and carcass length ( P < . O l ) and weights of the four leancuts ( P < .02) were lower in LG than in HG were boars. No microscopic atheroscleroticplaques observed inaorta or coronary arteries. Cholesterol concentration in subcutaneous fat was lower ( P < .04) in LG boars,suggesting that cholesterol content of depot fat can be reduced by genetic selection in swine.
Atherosclerosis, Cholesterol, Fat, Diet,Genetics,Pigs
J. Anim. Sci. 1995. 73:2043-2049
Introduction Plasma cholesterol is a heritabletraitin swine. Genetic selection within a crossbred (Chester White x
'This work is a publication of theUSDNARS Children's NutritionResearchCenter, Dept. of Pediat., Baylor College of Medicine, Houston, T X , in collaboration with Sam Houston State Univ., Huntsville, TX. This project has been funded in part with federal funds from the USDA, ARS under Cooperative Agreement no. 58-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement from the U.S. Government. The authors thankWilliam Harrell for supervision of the management of the animals; Doug Blackard, John Nelson, and associates, Sam Houston State Univ. for animal care; Ray Riley and co-workers, Texas A&M Univ., College Station, for animal slaughter and carcass measurements; Julia Redmond for manuscriptpreparation;and Leslie Loddeke for editorial advice. 2USDA/ARS Children's Nutrition Research Center, Dept. of Pediatr., Baylor College of Medicine, Houston, TX 77030. 'Present address: College of Agric., Sultan Qaboos Univ., P.O. Box 34, Al-Khod Postal Code 123, Sultanate of Oman. 4T0 whom correspondenceshould be addressed. 5Dept. of Pathol., Cornel1 Univ., Ithaca, NY 14853. 'Dept. of Anim. Sci., Texas A&M University, College Station 77843. 'Beef Council, Austin, TX 78731. Received September 23, 1994. Accepted March 20, 1995.
Landrace x Large White x Yorkshire) population resulted in differences of more than 50 mg/dL in mean total plasma cholesterol concentration at 8 wkof age in the low ( LG) and high ( HG) cholesterol groups in the fourth generation of selection (Pond et al., 1993). Harris et al. ( 1993) reported that cholesterol concentration of liver, but not of cerebrum, heart, muscle, and subcutaneous fat was higher at 25 wk of age in HG thanin LG females from generation 3. The addition of 4% dried egg yolk and 11%edible tallow to an all-plant, corn-soybean meal-based diet of half the pigs of each genetic group also increased plasma and liver cholesterol concentration, but not that of other tissues. Atherosclerosis has been reported in some genetic lines of pigs fed atherogenic diets (Lee et al., 1986) and in mutant pigs with defective catabolism and abnormal composition of low-density lipoproteins (Rapacz et al., 1977, 1989; Checovich et al., 1988; Lee et al.,1990). The purpose of the work reported here was to test the hypothesis that ingestion of ahigh-fat ( 16%), high-cholesterol (.12%), high-energy (3,800 kcal ME/ kg) diet by young adult boars genetically selected for four generations for high ( H G ) or low ( L G ) plasma cholesterol produces differential metabolic responses. Measurements were madeof cholesterol concentration inplasma, adipose tissue, muscle, liver andbrain;
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LU ET AL.
Table 1. Composition of experimental diets, as-fed basis
degree of atherosclerosis of coronary arteriesand aorta; and plasma concentrations of total cholesterol, HDL-cholesterol, insulin, glucagon, and thyroidhormones. High
Materials and Methods Twenty-eight young adult boars from thefourth generation of selection for high ( H G ) ( n = 1 4 ) or low ( L G ) ( n = 14) plasma cholesterol at 8 wk of age were used. Boars ( 8 mo of age) within their respective HG and LG geneticgroups were paired according to plasma cholesterol concentration (mean of 148 and 62 mg/dL for HG and LG, respectively) measured at 8 wk of age and were assigned randomly withinpairs to two diets in a 2 x 2 factorial arrangement of treatments (two diets, two genotypes). Diets (Table 1) consisted of a corn-soybean meal-cornstarch formula supplementedwithvitamins and minerals (low-fat, nocholesterol diet, LD, 3,328 kcal ME/kg) and the same formula modified by thesubstitution of 15% cornstarch with 11%beef tallow and 4% dried egg yolk and a shift in the ratio of corn to soybean meal (high-fat, high-cholesterol diet, HD, 3,800 kcal ME/kg) to make the two diets isonitrogenous. Daily intakes of all nutrients werecalculated to be above recommended allowances for young adult boars (NCR, 1988). All boars had been kept together in a .4-ha dirt lot and given adlibitum access to a standard cornsoybean meal-baseddietcontaining no cholesterol from weaning (4 wk of age) to 7 mo of age, at which time they were moved in assigned pairs and kept two boars per pen (12 pens) or one boar per pen ( 4 pens) in 2-m x 8-m pens (half of surface concrete, half gravel) in an open pole barn with a corrugated metal rooffor shadeandshelter. All boars were fed a standard corn-soybean meal diet once daily on the concrete floor at 2.27 kg-boar-l-d-l (4.54 kgid for paired boars) for 4 wk; at 8 moof age, feeding of the experimental diets was started (2.2 7 kg.boar-l&l), initial BW of each boar was recorded, and beginning at 1 wk, blood was sampled following a 16-h fast from theanterior vena cava intofluoridatedtubes from which the plasma was separated by centrifugation at 3,000 x g for 10 min and frozen at -5°Cfor later analyses. Body weight was recorded and blood sampled at 5-wk intervals (wk 0 [wk 1 for blood sample], 5 , 10, and15). of the experiment, all boars were On 105 d transported to Texas A&M University and killed by electrical stunning and exsanguination. Live BW, hot and chilled carcass weight, carcass length (first rib to public bone),subcutaneousbackfatdepth (mean of first rib, 10th rib, last rib, and last lumbar vertebrae), weight of the four lean cuts (Boston butt,picnic, loin, and ham), and cross-sectional area of the longissimus muscle at the 10th rib were recorded for all 28 boars.
Item Ingredient, g/lOO g Corn, no. 2 dent yellow Soybeanmeal (44% CP) Beef tallowa Dried egg yolk Cornstarch Dicalcium phosphate Ground limestone .4 Iodized salt Vitamin premix' Choline chloride Trace mineral premix' Chemical composition CP, % Cholesterol, mg/kg Ether extract, % ME, kcal/kg
High fat Low fat cholesterol No cholesterol 64 16.8 11 4
0 2.4 .8 .4 .2 .2 .2
15 1,156 16.5 3,800
60.6 20.2 0 0 15
2.4 .8
.2 .2 .2
15 0 3.1 3,328
aPreserved with 100 ppm of ethoxyquin. 'Supplied the following units per kilogram of diet vitamin A, 5,280 IU; vitamin D3,704 IU; vitaminE, 70.4 IU; menadione sodium bisulfite, 3.2 mg; vitamin B12, 26.4 pg; riboflavin, 5.28 mg; niacin, 28.16 mg; d-pantothenic acid, 21.12 mg; d-biotin, 88 pg; thiamin, 2.2 mg. Obtained from International Nutrition, Omaha, NE. CSupplied the following in milligrams per kilogram of complete diet: Cu (as cupric oxide), 10; Fe (as ferrous sulfate heptahydrate), 160; Mn ( a s manganese oxide), 20; Zn ( a s zinc oxide), 100; CaCog used as carrier (.30%of diet). Obtained from International Nutrition, Omaha, NE.
Cerebrum, heart, liver, and kidneys were removed and weighed. Samples ( 5 to 10 g ) of cerebrum,liver, longissimus muscle, and semitendinosus muscle from four boars in eachgroup (total of 16boars) were frozen at -5°C and later analyzed for concentration of total cholesterol (Rhee et al. 1983). The left descending coronary artery, a section of the left ventricle, and the aorta from the arch to the ileac artery bifurcation from the same four boars in each group were fixed in 10% buffered formalin. The aorta was opened longitudinally, scored for visible raised atherosclerotic = one or more plaques (1 = no visible lesions,2 whitishspotsless than .5 cm diameterresembling plaques, 3 = one or more whitish spots .5 to 1.0cm diameterresembling plaques), pinned to a wooden board, and fixed in 10% buffered formalin.Tissues were later sectioned at 6 pm andstainedwith hematoxylin and eosin for light microscopy. Blood plasma was analyzedfor glucose (hexokinase reaction), total cholesterol (colorimetric assay following hydrolysis of cholesterol esters), HDL-cholesterol (supernatant following precipitation of LDL and VLDL from plasma is assayed for cholesterol concentration), triglycerides(colorimetricassay following 550 Express, enzymatichydrolysis)(Ciba-Corning Diagnostics Corp., Oberlin, OH 44074). Insulin,glucagon, triiodothyronine ( T3), and thyroxine ( T4) were determined by radioimmunoassaywith 1251-labeled
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DIET AND GENETIC EFFECTS ON CHOLESTEROL
antigens (ICN Biomedical, Diagnostics Division, Costa Mesa, CA 92626; Insulin IMMUCHEM double antibody 1251RIA Kit, Glucagen Kit, Triiodothyronine 1251IMMUCHEM Kit,and Thyroxine 1251RIA Kit, respectively). Analysis of variance of the carcass andtissue composition data was performed according to the GLM complete ranprocedures of SAS ( 1985) usinga x 2 arrangement of domized block design in a2 treatments. The experimental unit was an individual boar. Blood metabolites and live BW were treated as a nesteddesignwithdiet,geneticgroup, anddiet x geneticgroup inthemain plot andtimeandall interactionsinthe sub-plot. Orthogonal polynomials were employed to testlinear,quadratic,and cubic trends over time.
Results and Discussion Mean BW at wk (of experiment) 0, 5 , 10, and 15 were 137,153,170, and 188 kg, respectively; mean overall weights for HG-HD, HG-LD, LG-HD, and LGLD were 177, 154, 166, and 150 kg, respectively; overall mean BW across time and group was 162 k 7 kg and did not differ significantly among groups. Final live BW ( LBW) was not affected by diet or genotype (Table 2), but therewas a diet x genotype interaction ( P < . O l ) , reflecting a largerweight at all time periods in HD compared with LD boars in the HG group vs a larger weight at all time periods in LD than in HD boars in the LG group. This unexpected interaction is of boars probably related to randomassignment within each genetic groupto diet; the observation is of doubtful biological importance. The assignment to diet was made on the basis of 8-wk plasma cholesterol and BW was ignored. Although BW during the growing period up to 154 d of age is positively correlated with plasma cholesterol concentration at 8 wk of age (Pond et al., 19931, there is no evidence t o indicate such a relationship in young adult boars. There was a diet x
time interaction ( P < .01) resulting from a greater BW gain in boars fed HD than in those fed LD. This effect was expected because of thegreater energy density of HD (16% fat) than of LD (3% fat) and the consequent greater daily caloric intake of the former group of boars. All boars were fed 2.27 kg of their respective diets daily, no attempt havingbeen made to equalize energy intake between the two dietary groups. Plasma total cholesterol concentration (Table 3 1 was higher inHG than in LG boars (84.8 vs. 74.5 mg/ dL, respectively, P < . O l ) , inagreementwiththe of plasma cholesterol in reportedhighheritability swine (Pond, 1986; Reetz et al., 1975; Rothschild and Chapman, 19761, and washigher in boars fed HD than in those fed LD (92.5 vs. 66.8 mg/dL, respeccholesterol tively, ( P .01). Theincreaseinplasma associatedwith consumption of the high-fat,highcholesterol dietwassimilar in H and L genotypes. However, there was an effect of time on plasma cholesterol ( P < .O 1) and time x diet and time x genotype interactions ( P < .O 1): plasma cholesterol increased over time in response to HD in HG boars but not in LG boars;plasma cholesterol decreased over time in response t o LD in HG but not in LG boars. Plasma HDL-cholesterol (Table 3 ) tended to follow thesamepatternastotal cholesterol inthat HD increased HDL-cholesterol ( P < .O 1) and HG boars had higher HDL-cholesterol than LG boars ( P < .O 1). However, the increase in HDL-cholesterol in response to HD tended to be greater in HG than in LG boars, whereas HDL-cholesterol was similar in HG and LG boars fed LD, resulting in a diet x genotype interaction ( P < .06). Plasma triglycerides (Table 3 ) were generally low in all boars. A time effect was observed ( P < . O l ) that seemed t o be due to a decline in triglycerides in all groups from wk 0 to wk 5, particularly in boarsfed HD compared with those fed LD, resulting in a timeX diet interaction ( P < .03). These differences may be related to the change from ad libitum to limited feed
Table 2. Effects of diet (HD, high fat and cholesterol; LD, lowfat and cholesterol) and genotype (HG, high cholesterol; LG, low cholesterol) on body and organ weights, and dressing percentage HG Item
No. of animals LBW, kgb Cerebrum, g Heart, g Spleen,g Liver, g Kidney,g Dressing %
LG LD
7
7 211 107 586 258 308 3,210 629 72.7 74.8
7 171 105 508 253 2,573 49 1 74
HD 177 101 511
2,730 56 1
Probabilitya LD
SD
D
G
7 194 205 59 1 296 2,683 588 73.7
-
-
-
__
19 12 69 32 356 74 1.4
NSC NS NSC NS .05 .l NS
NS NS NS NS NS NS
.01 NS .01 .01 .04 .01
0
D x G
.l
aD, diet; G, genotype; D x G, diet and genotype interaction; TQ, quadratic effect of time; TC, cubic effect of time; NS, P > bLBW = live body weight. CNS = not statistically significant. Downloaded from jas.fass.org by guest on July 14, 2011
.lo.
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LU ET AL.
Table 3. Effects of diet (HD, highfat; LD, low fat) and genotype (HG, high cholesterol; LG, low cholesterol) on plasmatotalcholesterol,HDL-cholesterol, and triglycerides of young adult boars LG
HG HD
LD
HD
LD
SD
Probabilitva
91.1 96.4 98.3 109 98.8
65.6 71.1 76.6 69.9 70.8
130.4 71.3 76 67.3 86.3
62.9 63.9 58.9 60.2 62.8
-
G < .01, D < .01, T < .01, T x D < .01, T x D x G < .01.
HDL-Cholesterol, mg/dLC Time ( T I , wk 1 5 10 15 Overall
43.4 64.1 65.1 61.6 58.6
33 46.3 52.3 39.7 42.8
40.1 35.9 38.9 30.7 36.4
31.5 32.4 33.9 26.8 31.7
6.4
T < .01, TD < .01.
Triglycerides, mg/dLd Time (T),wk 1 5 10 15 Overall
29.4 19.6 26 20.8 24
30 22.4 31.4 27.8 27.9
25.4 23 23.3 26.8 24.6
26.1 21.9 22.5 29.8 25.9
-
T < .01, T x D .03.
Constituent Cholesterol, mg/dLb Time (T), wk 1 5 10
15 Overall
-
-
10.8
-
D < .01, G < .01,
6.6
aD, diet; G, genotype; D x G, diet x genotype interaction; TL, linear effect of time; TQ, quadratic effect of time; TC, cubic effect of time. ~ T L < .01, TQ < .ox TL .Ol; TQ c .03, TC < .01. ~ T L < .OI.
intake of all boars beginning at about 6 mo of age (6 t o 8 wk before the experiment started), although one would not have predicted such a long time delay in the adaptive response of plasma triglycerides to reduced feed intake. Plasma glucose was similar in HG and LG boars and was not responsive to dietary fat-cholesterol content. Overall means were 87, 88, 88, and 82 mg/dL (SEM = 10 mg/dL) for HG-HD, HG-LD, LG-HD, and LG-LD, respectively. There was an effect of time ( P < .01) on plasma glucose that seemed to be related to a higher mean concentration in all groups at wk 5 than at other times, and a trend toward a diet x genotype interaction ( P < .07). The physiological significance of thesetrendsis unclear. Plasmainsulin, glucagon, T3, and T 4 dataare summarized in Table 4. Plasma insulin tended t o be higher in LG than in HG boars ( P < . l o ) and in LD than in HD boars ( P < .09). The HG boars tended to respond to high dietaryfat and cholesterol ( H D ) with a reduction in plasma insulin, whereas the LG boars did not, resulting in a diet x genotype interaction ( P .07). Neither diet nor genotype affected plasma glucagon concentration. However, adiet x genotype interaction was apparent ( P < .04),indicating that HG boars were less responsive to dietaryfatand cholesterol than LG boars in altering plasma glucagon Concentration. The LG boars tended to have lower T 3 concentration ( P < .lo) than the HG boars; T3 tended
to increase form wk 0 t o wk 5 in boars fed HD but to decline inthose fedLD, andthen to decline inall groups between wk 5 and 15, resulting in a dietx time interaction ( P < .O 1) and a quadratic effect over time ( P < . O l ) . A similar overall pattern was observed for T4, although there was no significant effect of genotype on T 4 concentration, despite a 10% difference between the means of the two groups. There was a diet x time interaction ( P < .Ol), resulting from an increase in T4 concentration from wk 0 to wk 5 in HD boars but not in LD boars, and a genotype x time interaction ( P < .03), probably resulting from a greater increase in HG than in LG boars from wk 10 t o wk 15 (26 vs 13%, respectively). The data suggest the possibility of differentialmodulation of genetic expression of endocrine traits related to cholesterol metabolism in HG and LG boars. Cerebrum, heart, spleen, and kidney weights (Table 2 ) were not significantly different among groups, but there was a diet x genotype interaction ( P < .Ol) for allorgans ( P .04 for liver). Liver weight was increased by high dietary fat and cholesterol (LD < HD, P
