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Effects of Dietary Biotin and Linoleate on Polyunsaturated Fatty Acids in Tissue Phospholipids
Effects of Dietary Biotin and Linoleate on Polyunsaturated Fatty Acids in Tissue Phospholipids B. A. WATKINS1 and F. H. KRATZER Department of Avian Science, University of California, Davis, Davis, California 95616 (Received for publication March 23, 1987)
1987 Poultry Science 66:2024-2031 INTRODUCTION
The energy value of fats and oils for broiler chick growth has been evaluated by a number of investigators (Renner and Hill, 1960; Young, 1961; Sibbald and Kramer, 1978; Halloran and Sibbald, 1979). The utilization of fats and oils by chickens is dependent upon the composition of these energy sources (Curtin and Raper, 1956; Sunde, 1956; Donaldson et al., 1957; Sibbald and Kramer, 1978; Halloran and Sibbald, 1979). Linoleic acid is an important factor in determining the feeding value of oils for the chick (Young, 1961; Hill, 1966; Menge, 1970). Halloran and Sibbald (1979) presented a compositional fatty acid analysis of several poultry feedgrade fats and determined feeding values, which are dependent upon many factors in addition to the essential fatty acid content. The linoleate requirement for growing chicks was estimated by Hill (1966) to be 2% of total dietary metabolizable energy (ME) kilocalories, but Bieri and Prival (1966) suggested the requirement is 1 to 1.8% of total dietary kilocalories (linoleate at .4 to .8% of diet). Hopkins and Nesheim (1967) suggested that
linoleate be fed at 2.2 to 3.8% of total ME kilocalories for straight-run chicks. Menge (1970), using a purified diet, estimated the linoleate requirement of female and male chicks to be .6 and 1.2% of the diet, respectively. Hill et al. (1967) determined that the linoleate requirement is .8% of the diet from growth data and analyses of heart and liver lipids. Currently, the recommended level of dietary linoleate for growing chicks is set at 1.0% of the diet (National Research Council, 1984). Essential fatty acid (EFA) deficiency symptoms in chicks appear to be limited to depressed growth and elevated ratios of 20:3w9:20:4co6 in tissue lipids. Metabolism of fatty acids is greatly influenced by biotin status in the chick (Bannister, 1967; Pearson et al., 1976; Hood, 1984; Watkins and Kratzer 1987a,b). Biotin deficiency symptoms in chicks (dermatitis, depressed growth) resemble EFA deficiency symptoms in rats. The purpose of the present investigation was to determine the linoleate requirement of young chicks fed graded levels of d-biotin and associated changes in omega-6 fatty acids in tissue phospholipids. MATERIALS AND METHODS
'Present address: Department of Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Animals and Diets. Day-old male broiler chicks were weighed and wing banded. Diets and water were provided ad libitum. Each die-
2024
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ABSTRACT Two experiments with factorial designs were conducted to study the interaction between dietary biotin and linoleate in male broiler chicks. Chicks were fed a purified basal diet containing varying levels of d-biotin (0, 200, or 400 p-g/kg diet) and linoleate. In Experiment 1, chicks were fed the basal diet containing three levels of added linoleate (.5, 3.1, or 4.1% of diet) for each biotin level and in Experiment 2, four levels of linoleate were fed (.27, .98, 2.00, or 2.20% of diet). The average body weights of chicks fed biotin at 200 |xg/kg of diet and .27 or .5% added linoleate were not different (P>.05) from those of chicks fed higher levels of biotin or linoleate supplements to the basal diets. Linoleate deficiency resulted in elevated omega-9 fatty acids (18: la>9 and 20:3u>9) in liver and heart phospholipids. For liver phospholipids, linoleate deficiency led to reduced 18:2w6, 20:3u)6, and 20:4co6 but for heart phospholipids only 20:4
BIOTIN AND LINOLEATE ON FATTY ACIDS
Lipid Analyses. Lipids from all tissues analyzed for fatty acid composition were extracted by methods described earlier (Watkins and Kratzer, 1987a). Phospholipids (PL) from the liver and heart were isolated by thin-layer chromatography (TLC) on Silica Gel G plates (Sigma Chemical Company, St. Louis, MO). The TLC plates were developed in a solvent system containing petroleum ethendiethyl ethenglacial acetic acid (90:10:1, v/v/v). The TLC standards were obtained from Nu-ChekPrep (Elysian, MN). Areas containing PL were scraped off the TLC plate, the silica gel inactivated (.04 JV HC1), and PL extracted with chloroform:methanol (2:1, v/v). Fatty acid preparation and analysis were performed by methods described earlier (Watkins and Kratzer, 1987a).
TABLE 1. Basal diet used in chick Ingredients
experiments Amount (g/kg)
Isolated soy protein Corn starch Lipid source 1 Dried egg albumen 2 DL-Methionine CaC0 3 CaHP04-2H20 Mineral mix 3 Vitamin mix (biotin free)4 Cellulose
250.0 558.0 55.0 30.0 7.5
13.0 22.0 24.5 10.0 30.0
Total
1,000.0
Calculated composition Metabolizable energy, kcal/kg Crude protein, % Available biotin, Mg/kg
3,560 23 0
'Total lipid fed in Experiment 1 was 55 g/kg and 30 g/kg in Experiment 2. Corn starch was fed at 583 g/kg in Experiment 2. 2 Analyzed avidin binding of dried egg albumen was 7.25 units/g (1 unit of avidin binds 1 microgram of biotin). 3 Provided in milligrams per kilogram of diet: Co ( C 2 H 3 0 ) - 4 H 2 0 , 20;CuSO 4 -5H 2 O,97;FeSO 4 -7H 2 O, 640; K I 0 3 , 9; K 2 H P 0 4 , 4,950; KCl, 2,970; MgS0 4 • 7H a O, 5,500; MnSO„-H 2 0, 297; NaCl, 9,900; Na 2 MoO„-2H 2 0, 9: Na 2 Se0 3 -SHjO, .66; ZnO, 120.
"Provided in milligrams per kilogram of diet (except as noted): vitamin A, 4,500 IU; vitamin D 3 , 500 ICU; vitamin E, 50 IU; menadione, 1.5; thiamine, 15; riboflavin, 15; niacin, 50; calcium pantothenate, 20; folic acid, 6; pyridoxine, 6; vitamin B-12, 20 Mg; choline chloride (70%), 2,000, butylated hydroxytoluene, 200.
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tary treatment contained duplicate lots of eight chicks that were reared on raised wire floors in temperature-controlled battery brooders in both experiments. The basal diet fed in the experiments (Table 1) was formulated to be adequate for chick growth (National Research Council, 1984) but was deficient in biotin or linoleate (Table 2). Biotin (d-biotin, United States Biochemical Corporation, Cleveland, OH) was added to the basal diet at 0, 200, and 400 |xg/kg of diet. Dietary lipid sources were fed at 5.5 and 3.0% of the basal diets for Experiments 1 and 2, respectively (Table 1). Linoleate treatments for the basal diets were made by adding triolein or safflower oils (United States Biochemical Corporation, Cleveland, OH) to provide needed linoleate levels (Table 2). A gas chromatographic analysis of methylated fatty acids from the triolein and safflower oils is shown in Table 3. The triolein oil did contain some linoleate, so it was not possible to formulate a dietary treatment with the basal ingredients that would be completely devoid of linoleate. Chick body weights and feed consumptions were measured for all groups to calculate weight gains and feed conversions (total feed:total gain). At the termination of each experiment or during tissue collection, chicks were humanely killed by cervical dislocation or with a general anesthetic. Biotin Deficiency Symptoms and Scoring. Perosis and dermatitis symptoms were noted and scored according to severity at 21 d during both experiments. Deficiency symptoms were scored from 0 to 4 according to severity in all chicks, with 0 indicating no symptoms and 4 indicating the worst signs of dermatitis or perosis. Collection and Preparation ofSamples. Prostaglandin E2 levels were measured in four chick hearts per dietary treatment. Tissue collection was performed by methods described earlier (Watkins and Kratzer, 1987b). Liver and heart tissues used for fatty acid analysis were surgically removed from three chicks per dietary treatment at 21 d after killing by cervical dislocation. Tissues were immediately placed on ice, frozen, and stored at -20 C. Liver microsomes were obtained from fresh tissue of three chicks per dietary treatment in .25 M sucrose in a 10,000 x g supernatant and were isolated by a single centrifugation at 100,000 x g for 60 min (Hansen et al., 1981). Fatty acid compositions of liver microsomes were determined at 21 and 28 d.
2025
2026
WATKINS AND KRATZER TABLE 2. Amounts of triolein and safflower oils and linoleate levels in basal diets Linoleate Safflower
Actual %
55 15 0
0 40 55
3.15 4.12
30 19 4 0
0 11 26 30
Experiment
Triolein
1
-
2
1
% of total kcal ME/kg1
(g/Kg) 1.23 7.65 10.06
.50
.67
.27 .98
2.43 4.84 5.48
2.00 2.20
ME = Metabolizable energy.
biotin (0 (jug/kg of diet). Average body weights of chicks were significantly (P<.01) influenced by dietary biotin. Maximum growth was observed in chicks fed biotin at 200 |xg/kg of diet and above (Tables 4 and 5) in both experiments. Chick growth was not improved by the addition of linoleate at 3.1 or 4.1% of the basal diet (Table 4) in Experiment 1 or during Experiment 2 when linoleate was provided at .9, 2.0, and 2.2% of the diet, except when 0 |i.g of biotin and 2.2% linoleate were fed. Feed conversions were not significantly different in Experiment 1. Perosis scores were highest in the low-biotin groups and appeared unrelated to the levels of linoleate fed. Dermatitis was highest in the lowbiotin groups and linoleate levels appeared to have no affect on the symptom.
TABLE 3. Fatty acid composition (weight %) of dietary lipids fed to chicks1 Fatty acid
Triolein oil
• , „,* 16:0 16:1 17:0 18:0 18:lco9 tl8:2 18:2CJ6
Chick mortalities, average weights, feed conversions (total feed:total gain), and deficiency symptoms are shown in Tables 4 and 5 for Experiments 1 and 2, respectively. Mortality in Experiment 1 was high for chicks fed no added
1.29 74.60
1
0
2.60 15.28 0
.47
9.05
73.48
.20 .16
.34 .14 .29 .12
1.12 .38 .38
20:2OJ6
22:0
.11
.21
18:3CJ3
20:lu;9 21:0
6.86
5.37 6.52
20:0 RESULTS
Safflower oil
0
Values are means from four samples.
0 .26
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Prostaglandin Determination. Extraction of prostaglandins from freeze-clamped heart tissues of chicks was previously described (Watkins and Kratzer, 1987b). The recovery of PGE2 was measured from heart homogenate samples spiked with 3H-PGE2 (New England Nuclear, Boston, MA) after column liquid chromatography. Approximately 91% of H-PGE2 was re covered. Radioactivity was measured by liquid scintillation counting as described earlier. The PGE2 was quantified by radioimmunoassay from heart tissue extracts. Radioactivity in samples prepared from a PGE2 kit that used 125 I-PGE2 as the labeled antigen (NEK-020, New England Nuclear, Boston, MA) was counted with a Micromedic Systems gamma counter, model 4/200 (Micromedic Assay CompuCenter, Horsham, PA). Statistical Analyses. Data were subjected to an analysis of variance (Snedecor and Cochran, 1967) of a two-way design to detect treatment differences. All analyses were performed on a VAX-11/780 Alcor computer using SAS statistical programs (SAS Institute Inc., 1983). When significant F values were obtained, differences in treatment means were determined by Bonneferonni's or Tukey's methods. Due to a general lack of significant interactions, means of the main effects, linoleate and biotin, for fatty acid values are reported.
BIOTIN AND LINOLEATE ON FATTY ACIDS
2027
TABLE 4. Performance characteristics and incidence of deficiency symptoms of chicks fed the dietary treatments for 3 wk in Experiment I
Biotin
Linoleate
Chick mortality
Average b o d y weight'
Feed:gain
(Mg/kg)
(%)
(no.)
(g)
(g/g)
0 0 0
.5 3.1 4.1
5 2 1
202 ± 1 4 3 b 228 ± 9 5 b 264 ± 8 1 b
200 200 200
.5 3.1 4.1
1 0 0
400 400 400
.5 3.1 4.1
1 0 1
Perosis score 2
Dermatitis score2
1.63 1.67 1.65
3.54 3.42 3.00
2.36 2.71 2.57
364 ± 1 0 9 a 405 ± 6 8 a 405 ± 6 0 a
1.42 1.48 1.52
.13 .25 .37
.53 .06 .18
390 ± 1 1 8 a 381 ± 3 1 a 380± 123a
1.41 1.40 1.44
0 0 0
0 0 0
1
Values are means ± standard deviations; duplicate lots of eight chicks each per treatment.
2
Values are mean scores: 0 = normal; 4 = severe perosis or dermatitis.
The PL fatty acid values from livers and hearts of chicks in Experiment 1 are listed in Tables 6 and 7. Under biotin deficiency, 16:1 was higher, whereas 18:0 was lower in liver and heart (Tables 6 and 7, respectively). Also, liver PL from biotin-deficient chicks contained elevated 22:5w3 and decreased 20:3w6. In liver of chicks fed .5% linoleate, 16:1, 18:lw9, 20:lw9,
and 20:3w9 were higher and 18:2w6, 20:2co6, 20:3w6, 20:4w6, and 22:5w6 were lower. Heart PL from chicks fed .5% linoleate contained lower 18:0 and 20:4w6 and higher 20:3w9 than PL of chicks fed 3.15% linoleate (Table 7). The PL fatty acids values in chick liver and heart tissues from chicks in Experiment 2 are listed in Tables 8 and 9. Biotin deficiency re-
TABLE 5. Performance characteristics and incidence of deficiency symptoms of chicks fed the dietary treatments for 3 wk in Experiment 2
Biotin
Linoleate
Chick mortality
Average b o d y weight 1
Feed:gain
(Mg/kg)
(%)
(no.)
(g)
(g/g)
0 0 0 0
.27 .98 2.00 2.20
0 2 1 0
238 150 225 292
± 102b ± 138c ± 115b ± 42a
200 200 200 200
.27 .98 2.00 2.20
0 0 2 0
341 332 287 314
400 400 400 400
.27 .98 2.00 2.20
0 0 1 1
359 263 286 360
Perosis score 2
Dermatitis score 2
1.91bc 1.75 e 1.89bc 1.93b
2.14 .55 .88 1.18
3.28 3.44 3.23 3.28
± 68a ± 55a ± 187a ± 127a
1.73e 2.06a 1.76 e 1.84cd
.93 .31 .58 .14
.28 .03 .16 .10
± 69a ± 166ab ± 156a ± 112a
1.84cd 1.92bc 1.84cd 1.79de
.81 .37 .30 .36
Values in columns with no common superscripts are significantly different (P<.01). 1
Values are means ± standard deviations; duplicate lots of eight chicks each per treatment.
1
Values are mean scores: 0 = normal; 4 = severe perosis or dermatitis.
0 .04 0 0
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ab ' Values in columns with different superscripts are significantly different (P<.01).
2028
WATKINS AND KRATZER
TABLE 6. Average weight percent of liver phospholipid fatty acids from 21-day-old chicks in Experiment Linoleate (% diet) Fatty acid
.50
16:0 16:1 17:0 18:0 18:ltj9 18:2^6
24.25 2.04 a ND
3
23.36 20.49 a 9.38 b .39 .05
20:lo;9 20:2u)6
.63 a .32 b 1.48a 1.69 b 7.01 b
20:3CJ9
20:3w6 20:4co6 22:4co6 22:5w6 22:5o;3 22:6^3 20:5OJ3 22:1U>9
21:0
.81
.41b 1.81 2.49 .97 ND .08
Biotin (jug/kg diet)
3.15
0
200
400
Pooled SEM2
23.82 .87 b
25.31 3.00 a
22.48 .71b
24.32 .65b
1.63
ND
ND
ND
ND
25.97 12.06 b 14.67 a .41 .04
.26 b .71a .47 b 2.05 a 11.15 a 6.18 1.37 a 1.47 2.25 ND .15 .06
b
18.17 15.96 12.98 a .52 ND .57 .82
.32 b 1.40 b 8.02 b 2.42 .70
3.22 1.78 1.20 .19 .06
a
ab
25.82 16.28 11.05 b
a
30.00 16.59 12.05 a b
.30 .06 .33 .31
.37 .08 .43 .41
1.33 a 1.84 ab 11.08 a 7.72 1.33 1.67 ab 3.66
1.28 a 2.37 a 8.14 b
.10 ND .03
2.97 1.38 .61 .10 .06 .08 .16 .24 .17 .79
6.04
.34 .64
.03 1.69
.36
b
.15 .04 .12
.45 .70
1.85 .45 .15 .04
ab ' Values in rows within treatments with different superscripts are significantly different (P<.05). 1 Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick livers. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03).
suited in lower levels of 20:3w6, 20:3w9 and 20:4w6 and higher levels of 16:0 in both tissues. Heart PL also contained higher 16:1 and 18:0. Linoleate fed at .27% of the diet produced higher 18:lco9 and 20:3w9 and lower 18:0, 18:2oo6, 20:2w6, and 20:4w6 in PL of both tissues. The fatty acid compositions of liver microsomes from chicks were determined in Experiment 1 (Table 10). Twenty-one-day-old chicks fed biotin at 0 M-g/kg of diet had significantly higher 18:2w6 and 20:0 and lower 16:0, 18:0, and 20:3w6 than chicks fed biotin at 400 |Jig/kg of diet. At 28 days, significant differences were also observed in chicks fed biotin at 0 (xg/kg of diet. The 16:1 and 18:2a)6 were elevated and 18:0, 20:3w6, 20:4w6, 22:4w6, 22:5w6, and 22:6w3 were lower compared with levels in chicks fed biotin at 400 u,g/kg of diet. Prostaglandin E2 in freeze-clamped heart tissues from 14-day-old chicks was determined during Experiment 2 (Table 11). Chicks maintained on diets containing 0 fig/kg biotin and either .27 or 2.00% linoleate showed lower
levels of PGE2 than chicks fed adequate biotin. Chicks fed 200 ixg of biotin had PGE2 levels intermediate to those of chicks fed 0 or 400 (xg biotin. DISCUSSION
An interesting and consistent trend is observed during biotin deficiency in the chick; the present experiments show that 20:3w6 is decreased in liver PL. Similar changes were observed in liver lipids of biotin-deficient chicks in previous studies that included elevated 18:2co6 (Watkins and Kratzer, 1987a,b). Liver microsomes isolated from biotin-deficient chicks in Experiment 1 contained higher linoleate and lower 20:3w6 than their biotinadequate counterparts. Clearly, a biochemical block in EFA metabolism occurs in the chick during biotin deficiency (Watkins and Kratzer, 1987b). Elongation of EFA is probably depressed; however, desaturation may also be impaired. Occasionally, 20:4w6 is decreased in heart tissue of the biotin-deficient chick (Experiment
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18:3CJ6 18:3CJ3
l1
BIOTIN AND LINOLEATE ON FATTY ACIDS
2029
TABLE 7. Average weight percent of heart phospholipid fatty acids from 21-day-old chicks in Experiment Linoleate (% diet)
Biotin (Mg/kg diet)
Fatty acid
.50
3.15
16:0 16:1 17:0 18:0
20.88 1.67
19.29 1.51
18:1CJ9
18:2u;6 18:3w6 18:3to3 20:1CJ9
1.78 .47
20:3CJ6
20:4u;6 22:5w3 22:6co3 20:5OJ3
22:lto9 21:0
.11
1.63
2.38 2.49 7.02 b .58 .25 .47 .67 .25 .60 .15
.45 2.04 11.62 a .89 .65 ND3 .05 .79 .09
20.37 .94 b
18.93 .95b
1.15 .23
26.41 b 17.57 a 15.34
.06
28.99 a 17.48 a 13.93
.03 .69
1.17 1.04
.52 .09
.66 .03
1.70
1.55
1.85
.58
.40
.50
1.32 2.37 9.78 1.18
1.50 2.32 9.80
1.43 2.11 8.38
1.38
.55 .50 ND .89 .08 .60 .11
.48 .09 .71
.44 .65 .58
2.19
1.01
.31 .65 .25
.18 .41 .16
.75 ND .06 .07 .84 ND
1.41
Pooled SEM2
.47 .11
.52 b
400
.11
26.23 b 13.36 b 16.59
.57 .03
a
200
.12 .09 .36 .29 .22 .65
' Values in rows within treatments with different superscripts are significantly different (P<.01). 1
Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick hearts. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03).
TABLE 8. Average weight percent of liver phospholipid fatty acids from 21-day-old chicks in Experiment 21 Linoleate (% diet) Fatty acid
.27
2.00
16:0 16:1 17:0 18:0 18:lu>9 18:2a>6 18:3a>6 18:3ld3
26.22 1.37
26.69 1.89
20:1CJ9 20:2W6
20:3a>9 20:3CJ6
20:4o;6 22:4o>6 22:5u>6 22:5^3 22:6to3 20:5co3 22:1OJ9
21:0
ND3
Biotin (Mg/kg diet) 0 — (weight %) 28.32 a 2.99
23.52 23.25 a 9.24 b .30 b
a
26.91 15.95 b 13.48 a ,49 a .06
.06 a
b
.72 .12b 1.44a 1.75 b 4.14 b
.37 .46 a .52 b 2.24 a 7.05 a
.20
.67
.17b
.63 a
.80 .13 .50 .06 .16
.98 .30 .48 .19 .12
22.46 18.95 9.85
b
.35 .03 .66 .25
.49b 1.58 b 4.01b .21 .36 ND ND .63 ND .10
400
Pooled SEM2
26.91ab 1.23
24.12 b
1.17
ND
ND
ND b
200
27.98 20.52 10.66 .39 .06 .44 .25
a
.68 ND
25.21 19.33 13.58
.91 ab
.45 .10 .54 .38
.87b 1.99 ab 5.44 a b
1.59 a 2.42 a 7.33 a
.43 .37
.67 .47
1.20
1.47
.25 .17 .18 .21
.40 .67 .17 .10
1.30 1.09 1.48 .05 .03 .07 .04 .14 .20 .72 .33 .18 .32 .12 .52 .15 .09
' Values in rows within treatments with different superscripts are significantly different (P<.05). 'Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick livers. 2
SEM = Standard error or the mean.
3
ND = Not detected (levels below .03).
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22:4OJ6 22:5CJ6
(weight %) 20.96 2.89 a
28.40 a 13.52 b 15.78
.54 .12
20:2u>6 20:3u)9
0
.08
.10
26.01 b 18.75 a 14.79
l1
2030
WATKINS AND KRATZER
TABLE 9. Average weight percent of heart phospholipid fatty acids from 21-day-old chicks in Experiment 2 1 Linoleate (% diet)
Biotin (jug/kg diet)
Fatty acid
.27
2.00
16:0 16:1 17:0 18:0
24.86 1.27 a
24.80 .30 b
b
18:2^6
.40 ND3
20:1CJ9
1 5 9
20:4a>6
.61
.74
22:5u>6 22:5w3 =
.04 b
.26 a
22:6W3 20:5CJ3
22:lu>9 21:0
ND .03 .23 .52 .13
Pooled SEM2
25.56 a .06 b
20.42 b ,66 b
1.43
1.41
1.77
.47 1.32 b 4.71b .12 b ND .03 ND ND
b
28.93 18.31 16.24 b .62 .04
b
•04 a
.31 ND .25
.05 .03 ND .35 .11
400
•
2 9
23.54 16.03 21.46 a .35 ND
1.20 .39
ub
1.53 a 2.12 a 9.01 a 1.54a
.62 1.25 b 4.58 b ,36 b .15 ND .04 ND
.31 .04 .04
.12 b
.83 a
• 3 5 aub .34
.12
.11
.13
.30 .07 .98 .78 .91 .12 .02 .20 .10 .16 .10 .68 .13 .07 .04 .04 .12 .17 .09
' Values in rows within treatments with different superscripts are significantly different (P<.05). 1
Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick hearts. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03). TABLE 10. Average weight percent of fatty acid in microsomes from chick livers in Experiment
1'
21 days
28 days
Dietary biotin (/ug/kg diet)
Dietary biotin 0/g/kg diet)
Fatty acid
0
400
16:0 16:1 18:0 18:lu9 18:2o;6 20:0 20:1^9
22.44 b 3.20 16.76 b 16.40 27.81 a .56 a
25.83 a 3.18 20.32 a 16.89 19.89 b ,22 b
ND2 .34 ND
ND .23 ND
0
400
20.75 1.76a 19.00 b 10.95 27.83 a
20.48 .87 b 23.43 a 10.86 19.35 b
(weight %)
20:2CJ6 20:3CJ9 20:3CJ6 20:4CJ6 22:4W6
22:5w6 22:5o;3 22:6CJ3
1.54 b 8.55 1.15 * .70
ND .17
2.26 a 8.67 .94 .68 ND .67
.42 ND .60 ND
1.22 b 11.89 b 1.25 b .81b 0 9
K
.44 b
.19 .22 .66 .55
2.23 a 16.18 a 1.35 a 2.13 a .17
1.30a
' Values in rows with different superscripts (21 or 28 days) are significantly different (P<.05). 1
Mean values of three samples per dietary treatment. Each sample contained pooled microsomes from two chick livers. 2
ND = Not detected (levels below .03).
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1.33 .42 a .41b 1.41 b 7.25 a
K
22:4CJ6
20:3CJ6
29.32 17.32 13.98 b
.46 .04
.20 b 1.33 a 1.71 a 4.94 b
20:2w6 20:3w9
a
29.07 13.18 b 19.39 a
200
.07
.10 a
25.46 21.25 a 15.07 b
18:3u>3
18:3CJ6
(weight %) 28.52 a 1.64a
.12
.03
18-.1CJ9
0
BIOTIN AND LINOLEATE ON FATTY ACIDS TABLE 11. Prostaglandin (PGE2) levels in heart tissue of 14-day-old chicks in Experiment 2 1 Dietary biotin
Linoleate
PGE 2
(Mg/kg)
(% diet)
(pg/g wet tissue)
.27 2.00 .27 2.00 2.00
23.7 33.7 43.2 49.7 87.5
0 200 400
± ± ± ± ±
12.2 6.5 11.0 7.6 15.7
'Mean values ± standard deviations from four chick hearts per dietary treatment.
REFERENCES Bannister, D. W., 1976. The biochemistry of fatty liver and kidney syndrome. Biochem. J. 156:167-173. Bieri, J. G., and E. L. Prival, 1966. Linoleic acid requirement of the chick. J. Nutr. 90:428^132. Curtin, L. V., and J. T. Raper, 1956. Feeding value of hydrolyzed vegetable fats in broiler rations. Poultry Sci. 35:273-278. Donaldson, W. E., G. F. Combs, G. L. Romoser, and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chicks to dietary fat. Poultry Sci. 36:807-815. Halloran, H. R., and I. R. Sibbald, 1979. Metabolizable energy values of fats measured by several procedures. Poultry Sci. 58:1299-1307. Hansen, G., J. J. Strik, J. H. Koeman, and C. A. Kan, 1981. Biological activity of technical aroclor 1254 compared to aroclor 1254 residues: swine fat residues fed to broiler cockerels. Toxicology 21:203-212. Hill, E. G., 1966. Effects of dietary linoleate on chick liver fatty acids: dietary linoleate requirement. J. Nutr. 89:465-470. Hill, E. G., C. L. Silbemick, and E. McMeans, 1967. Dietary linoleate and methionine in chicks. Poultry Sci. 46:523-526. Hood, R. L., 1975. A radiochemical assay for biotin in biological materials. J. Sci. Food.Agric. 26:18471852. Hood, R. L., 1984. Cellular and biochemical aspects of fat deposition in the broiler chicken. World Poult. Sci. J. 40:160-169. Hopkins, D. T., and M. C. Nesheim, 1967. The linoleic acid requirement of chicks. Poultry Sci. 46:872-881. Kramer, T. R., M. Briske-Anderson, S. B. Johnson, and R. T. Holman, 1984. Effects of biotin deficiency on polyunsaturated fatty acid metabolism in rats. J. Nutr. 114:2047-2052. Menge, H., 1970. Comparative requirements of linoleic acid for male and female chicks. Poultry Sci. 49:178183. National Research Council, 1984. Nutrient requirements of poultry, No. 1. Nutrient Requirements of Domestic Animals, 8th rev. ed. Natl. Acad. Sci., Washington, DC. Pearson, J. A., A. R. Johnson, R. L. Hood, and A. C. Fogerty, 1976. Fatty liver and kidney syndrome in chicks. I. Effect of Biotin in diet. Aust. J. Biol. Sci. 29:419^128. Renner, R., and F. W. Hill, 1960. The utilization of corn oil, lard and tallow by chickens of various ages. Poultry Sci. 39:849-854. SAS Institute, Inc., 1983. SAS Introductory Guide. SAS Inst. Inc., Cary, NC. Sibbald, I. R., and J.K.G. Kramer, 1978. The effect of the basal diet on the true metabolizable energy of fat. Poultry Sci. 57:685-691. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th ed. Iowa State Univ. Press, Ames, IA. Sunde, M. L., 1956. The effect of fats and fatty acids in chick rations. Poultry Sci. 35:362-368. Watkins, B. A., and F. H. Kratzer, 1987a. Tissue lipid fatty acid compositions of biotin-adequate and biotindeficient chicks. Poultry Sci. 66:306-313. Watkins, B. A., and F. H. Kratzer, 1987b. Dietary biotin effects on polyunsaturated fatty acids in chick tissue lipids and prostaglandin E2 levels in freeze-clamped hearts. Poultry Sci. 66:1835-1845. Young, R. J., 1961. The energy value of fats and fatty acids for chicks. 1. Metabolizable energy. Poultry Sci. 40:1225-1233.
Downloaded from http://ps.oxfordjournals.org/ at NERL on May 24, 2015
2; Watkins and Kratzer, 1987a) but 20:3w6 is usually lowered. The decrease in 20:4w6 observed in Experiment 2 may be due to the lower levels of linoleate fed. The eicosanoic fatty acids, 20:4co6 and 20:3co6, are important precursors of prostaglandins (PG). Decreased levels of substrates in membrane PL may lower PG production. This was observed in heart tissue of the biotin-deficient chick in Experiment 2. Both 20:4w6 and PGE2 were lower in heart tissue of chicks fed biotin at 0 and 200 u.g/kg of diet than in tissue of chicks fed 400 (xg of biotin. In this case, biotin deficiency may indirectly affect PG biosynthesis in the chick by reducing PG precursors in PL. An absence of biotin (0 u-g/kg of diet) resulted in elevated 17:0 in total lipids of chick liver (data not shown); this observation was reported in rat liver fatty acids during a biotin deficiency (Kramer et al., 1984). Biotin deficiency also elevated 16:1 in chick heart PL. It is apparent that biotin deficiency impairs linoleate metabolism (omega-6 pathway) in the chick; possibly other polyenoic fatty acid pathways are also affected. Chicks fed .5 or .27% linoleate (1.23 and .67% of total ME/kcal per kg) and 200 jjLg of biotin in Experiments 1 and 2, respectively, maintained body weights that were not significantly different from those of chicks fed higher levels of linoleate. However, the low levels of dietary linoleate elevated 20:3w9 in PL. Feeding chicks diets that are low in linoleate for a longer period of time may be necessary to produce growth depression. The data in this study provide evidence that the biotin status of the chick will influence EFA metabolism and levels of eicosanoic fatty acids in PL of liver and heart.
2031
1987 Poultry Science 66:2024-2031 INTRODUCTION
The energy value of fats and oils for broiler chick growth has been evaluated by a number of investigators (Renner and Hill, 1960; Young, 1961; Sibbald and Kramer, 1978; Halloran and Sibbald, 1979). The utilization of fats and oils by chickens is dependent upon the composition of these energy sources (Curtin and Raper, 1956; Sunde, 1956; Donaldson et al., 1957; Sibbald and Kramer, 1978; Halloran and Sibbald, 1979). Linoleic acid is an important factor in determining the feeding value of oils for the chick (Young, 1961; Hill, 1966; Menge, 1970). Halloran and Sibbald (1979) presented a compositional fatty acid analysis of several poultry feedgrade fats and determined feeding values, which are dependent upon many factors in addition to the essential fatty acid content. The linoleate requirement for growing chicks was estimated by Hill (1966) to be 2% of total dietary metabolizable energy (ME) kilocalories, but Bieri and Prival (1966) suggested the requirement is 1 to 1.8% of total dietary kilocalories (linoleate at .4 to .8% of diet). Hopkins and Nesheim (1967) suggested that
linoleate be fed at 2.2 to 3.8% of total ME kilocalories for straight-run chicks. Menge (1970), using a purified diet, estimated the linoleate requirement of female and male chicks to be .6 and 1.2% of the diet, respectively. Hill et al. (1967) determined that the linoleate requirement is .8% of the diet from growth data and analyses of heart and liver lipids. Currently, the recommended level of dietary linoleate for growing chicks is set at 1.0% of the diet (National Research Council, 1984). Essential fatty acid (EFA) deficiency symptoms in chicks appear to be limited to depressed growth and elevated ratios of 20:3w9:20:4co6 in tissue lipids. Metabolism of fatty acids is greatly influenced by biotin status in the chick (Bannister, 1967; Pearson et al., 1976; Hood, 1984; Watkins and Kratzer 1987a,b). Biotin deficiency symptoms in chicks (dermatitis, depressed growth) resemble EFA deficiency symptoms in rats. The purpose of the present investigation was to determine the linoleate requirement of young chicks fed graded levels of d-biotin and associated changes in omega-6 fatty acids in tissue phospholipids. MATERIALS AND METHODS
'Present address: Department of Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Animals and Diets. Day-old male broiler chicks were weighed and wing banded. Diets and water were provided ad libitum. Each die-
2024
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ABSTRACT Two experiments with factorial designs were conducted to study the interaction between dietary biotin and linoleate in male broiler chicks. Chicks were fed a purified basal diet containing varying levels of d-biotin (0, 200, or 400 p-g/kg diet) and linoleate. In Experiment 1, chicks were fed the basal diet containing three levels of added linoleate (.5, 3.1, or 4.1% of diet) for each biotin level and in Experiment 2, four levels of linoleate were fed (.27, .98, 2.00, or 2.20% of diet). The average body weights of chicks fed biotin at 200 |xg/kg of diet and .27 or .5% added linoleate were not different (P>.05) from those of chicks fed higher levels of biotin or linoleate supplements to the basal diets. Linoleate deficiency resulted in elevated omega-9 fatty acids (18: la>9 and 20:3u>9) in liver and heart phospholipids. For liver phospholipids, linoleate deficiency led to reduced 18:2w6, 20:3u)6, and 20:4co6 but for heart phospholipids only 20:4
BIOTIN AND LINOLEATE ON FATTY ACIDS
Lipid Analyses. Lipids from all tissues analyzed for fatty acid composition were extracted by methods described earlier (Watkins and Kratzer, 1987a). Phospholipids (PL) from the liver and heart were isolated by thin-layer chromatography (TLC) on Silica Gel G plates (Sigma Chemical Company, St. Louis, MO). The TLC plates were developed in a solvent system containing petroleum ethendiethyl ethenglacial acetic acid (90:10:1, v/v/v). The TLC standards were obtained from Nu-ChekPrep (Elysian, MN). Areas containing PL were scraped off the TLC plate, the silica gel inactivated (.04 JV HC1), and PL extracted with chloroform:methanol (2:1, v/v). Fatty acid preparation and analysis were performed by methods described earlier (Watkins and Kratzer, 1987a).
TABLE 1. Basal diet used in chick Ingredients
experiments Amount (g/kg)
Isolated soy protein Corn starch Lipid source 1 Dried egg albumen 2 DL-Methionine CaC0 3 CaHP04-2H20 Mineral mix 3 Vitamin mix (biotin free)4 Cellulose
250.0 558.0 55.0 30.0 7.5
13.0 22.0 24.5 10.0 30.0
Total
1,000.0
Calculated composition Metabolizable energy, kcal/kg Crude protein, % Available biotin, Mg/kg
3,560 23 0
'Total lipid fed in Experiment 1 was 55 g/kg and 30 g/kg in Experiment 2. Corn starch was fed at 583 g/kg in Experiment 2. 2 Analyzed avidin binding of dried egg albumen was 7.25 units/g (1 unit of avidin binds 1 microgram of biotin). 3 Provided in milligrams per kilogram of diet: Co ( C 2 H 3 0 ) - 4 H 2 0 , 20;CuSO 4 -5H 2 O,97;FeSO 4 -7H 2 O, 640; K I 0 3 , 9; K 2 H P 0 4 , 4,950; KCl, 2,970; MgS0 4 • 7H a O, 5,500; MnSO„-H 2 0, 297; NaCl, 9,900; Na 2 MoO„-2H 2 0, 9: Na 2 Se0 3 -SHjO, .66; ZnO, 120.
"Provided in milligrams per kilogram of diet (except as noted): vitamin A, 4,500 IU; vitamin D 3 , 500 ICU; vitamin E, 50 IU; menadione, 1.5; thiamine, 15; riboflavin, 15; niacin, 50; calcium pantothenate, 20; folic acid, 6; pyridoxine, 6; vitamin B-12, 20 Mg; choline chloride (70%), 2,000, butylated hydroxytoluene, 200.
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tary treatment contained duplicate lots of eight chicks that were reared on raised wire floors in temperature-controlled battery brooders in both experiments. The basal diet fed in the experiments (Table 1) was formulated to be adequate for chick growth (National Research Council, 1984) but was deficient in biotin or linoleate (Table 2). Biotin (d-biotin, United States Biochemical Corporation, Cleveland, OH) was added to the basal diet at 0, 200, and 400 |xg/kg of diet. Dietary lipid sources were fed at 5.5 and 3.0% of the basal diets for Experiments 1 and 2, respectively (Table 1). Linoleate treatments for the basal diets were made by adding triolein or safflower oils (United States Biochemical Corporation, Cleveland, OH) to provide needed linoleate levels (Table 2). A gas chromatographic analysis of methylated fatty acids from the triolein and safflower oils is shown in Table 3. The triolein oil did contain some linoleate, so it was not possible to formulate a dietary treatment with the basal ingredients that would be completely devoid of linoleate. Chick body weights and feed consumptions were measured for all groups to calculate weight gains and feed conversions (total feed:total gain). At the termination of each experiment or during tissue collection, chicks were humanely killed by cervical dislocation or with a general anesthetic. Biotin Deficiency Symptoms and Scoring. Perosis and dermatitis symptoms were noted and scored according to severity at 21 d during both experiments. Deficiency symptoms were scored from 0 to 4 according to severity in all chicks, with 0 indicating no symptoms and 4 indicating the worst signs of dermatitis or perosis. Collection and Preparation ofSamples. Prostaglandin E2 levels were measured in four chick hearts per dietary treatment. Tissue collection was performed by methods described earlier (Watkins and Kratzer, 1987b). Liver and heart tissues used for fatty acid analysis were surgically removed from three chicks per dietary treatment at 21 d after killing by cervical dislocation. Tissues were immediately placed on ice, frozen, and stored at -20 C. Liver microsomes were obtained from fresh tissue of three chicks per dietary treatment in .25 M sucrose in a 10,000 x g supernatant and were isolated by a single centrifugation at 100,000 x g for 60 min (Hansen et al., 1981). Fatty acid compositions of liver microsomes were determined at 21 and 28 d.
2025
2026
WATKINS AND KRATZER TABLE 2. Amounts of triolein and safflower oils and linoleate levels in basal diets Linoleate Safflower
Actual %
55 15 0
0 40 55
3.15 4.12
30 19 4 0
0 11 26 30
Experiment
Triolein
1
-
2
1
% of total kcal ME/kg1
(g/Kg) 1.23 7.65 10.06
.50
.67
.27 .98
2.43 4.84 5.48
2.00 2.20
ME = Metabolizable energy.
biotin (0 (jug/kg of diet). Average body weights of chicks were significantly (P<.01) influenced by dietary biotin. Maximum growth was observed in chicks fed biotin at 200 |xg/kg of diet and above (Tables 4 and 5) in both experiments. Chick growth was not improved by the addition of linoleate at 3.1 or 4.1% of the basal diet (Table 4) in Experiment 1 or during Experiment 2 when linoleate was provided at .9, 2.0, and 2.2% of the diet, except when 0 |i.g of biotin and 2.2% linoleate were fed. Feed conversions were not significantly different in Experiment 1. Perosis scores were highest in the low-biotin groups and appeared unrelated to the levels of linoleate fed. Dermatitis was highest in the lowbiotin groups and linoleate levels appeared to have no affect on the symptom.
TABLE 3. Fatty acid composition (weight %) of dietary lipids fed to chicks1 Fatty acid
Triolein oil
• , „,* 16:0 16:1 17:0 18:0 18:lco9 tl8:2 18:2CJ6
Chick mortalities, average weights, feed conversions (total feed:total gain), and deficiency symptoms are shown in Tables 4 and 5 for Experiments 1 and 2, respectively. Mortality in Experiment 1 was high for chicks fed no added
1.29 74.60
1
0
2.60 15.28 0
.47
9.05
73.48
.20 .16
.34 .14 .29 .12
1.12 .38 .38
20:2OJ6
22:0
.11
.21
18:3CJ3
20:lu;9 21:0
6.86
5.37 6.52
20:0 RESULTS
Safflower oil
0
Values are means from four samples.
0 .26
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Prostaglandin Determination. Extraction of prostaglandins from freeze-clamped heart tissues of chicks was previously described (Watkins and Kratzer, 1987b). The recovery of PGE2 was measured from heart homogenate samples spiked with 3H-PGE2 (New England Nuclear, Boston, MA) after column liquid chromatography. Approximately 91% of H-PGE2 was re covered. Radioactivity was measured by liquid scintillation counting as described earlier. The PGE2 was quantified by radioimmunoassay from heart tissue extracts. Radioactivity in samples prepared from a PGE2 kit that used 125 I-PGE2 as the labeled antigen (NEK-020, New England Nuclear, Boston, MA) was counted with a Micromedic Systems gamma counter, model 4/200 (Micromedic Assay CompuCenter, Horsham, PA). Statistical Analyses. Data were subjected to an analysis of variance (Snedecor and Cochran, 1967) of a two-way design to detect treatment differences. All analyses were performed on a VAX-11/780 Alcor computer using SAS statistical programs (SAS Institute Inc., 1983). When significant F values were obtained, differences in treatment means were determined by Bonneferonni's or Tukey's methods. Due to a general lack of significant interactions, means of the main effects, linoleate and biotin, for fatty acid values are reported.
BIOTIN AND LINOLEATE ON FATTY ACIDS
2027
TABLE 4. Performance characteristics and incidence of deficiency symptoms of chicks fed the dietary treatments for 3 wk in Experiment I
Biotin
Linoleate
Chick mortality
Average b o d y weight'
Feed:gain
(Mg/kg)
(%)
(no.)
(g)
(g/g)
0 0 0
.5 3.1 4.1
5 2 1
202 ± 1 4 3 b 228 ± 9 5 b 264 ± 8 1 b
200 200 200
.5 3.1 4.1
1 0 0
400 400 400
.5 3.1 4.1
1 0 1
Perosis score 2
Dermatitis score2
1.63 1.67 1.65
3.54 3.42 3.00
2.36 2.71 2.57
364 ± 1 0 9 a 405 ± 6 8 a 405 ± 6 0 a
1.42 1.48 1.52
.13 .25 .37
.53 .06 .18
390 ± 1 1 8 a 381 ± 3 1 a 380± 123a
1.41 1.40 1.44
0 0 0
0 0 0
1
Values are means ± standard deviations; duplicate lots of eight chicks each per treatment.
2
Values are mean scores: 0 = normal; 4 = severe perosis or dermatitis.
The PL fatty acid values from livers and hearts of chicks in Experiment 1 are listed in Tables 6 and 7. Under biotin deficiency, 16:1 was higher, whereas 18:0 was lower in liver and heart (Tables 6 and 7, respectively). Also, liver PL from biotin-deficient chicks contained elevated 22:5w3 and decreased 20:3w6. In liver of chicks fed .5% linoleate, 16:1, 18:lw9, 20:lw9,
and 20:3w9 were higher and 18:2w6, 20:2co6, 20:3w6, 20:4w6, and 22:5w6 were lower. Heart PL from chicks fed .5% linoleate contained lower 18:0 and 20:4w6 and higher 20:3w9 than PL of chicks fed 3.15% linoleate (Table 7). The PL fatty acids values in chick liver and heart tissues from chicks in Experiment 2 are listed in Tables 8 and 9. Biotin deficiency re-
TABLE 5. Performance characteristics and incidence of deficiency symptoms of chicks fed the dietary treatments for 3 wk in Experiment 2
Biotin
Linoleate
Chick mortality
Average b o d y weight 1
Feed:gain
(Mg/kg)
(%)
(no.)
(g)
(g/g)
0 0 0 0
.27 .98 2.00 2.20
0 2 1 0
238 150 225 292
± 102b ± 138c ± 115b ± 42a
200 200 200 200
.27 .98 2.00 2.20
0 0 2 0
341 332 287 314
400 400 400 400
.27 .98 2.00 2.20
0 0 1 1
359 263 286 360
Perosis score 2
Dermatitis score 2
1.91bc 1.75 e 1.89bc 1.93b
2.14 .55 .88 1.18
3.28 3.44 3.23 3.28
± 68a ± 55a ± 187a ± 127a
1.73e 2.06a 1.76 e 1.84cd
.93 .31 .58 .14
.28 .03 .16 .10
± 69a ± 166ab ± 156a ± 112a
1.84cd 1.92bc 1.84cd 1.79de
.81 .37 .30 .36
Values in columns with no common superscripts are significantly different (P<.01). 1
Values are means ± standard deviations; duplicate lots of eight chicks each per treatment.
1
Values are mean scores: 0 = normal; 4 = severe perosis or dermatitis.
0 .04 0 0
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ab ' Values in columns with different superscripts are significantly different (P<.01).
2028
WATKINS AND KRATZER
TABLE 6. Average weight percent of liver phospholipid fatty acids from 21-day-old chicks in Experiment Linoleate (% diet) Fatty acid
.50
16:0 16:1 17:0 18:0 18:ltj9 18:2^6
24.25 2.04 a ND
3
23.36 20.49 a 9.38 b .39 .05
20:lo;9 20:2u)6
.63 a .32 b 1.48a 1.69 b 7.01 b
20:3CJ9
20:3w6 20:4co6 22:4co6 22:5w6 22:5o;3 22:6^3 20:5OJ3 22:1U>9
21:0
.81
.41b 1.81 2.49 .97 ND .08
Biotin (jug/kg diet)
3.15
0
200
400
Pooled SEM2
23.82 .87 b
25.31 3.00 a
22.48 .71b
24.32 .65b
1.63
ND
ND
ND
ND
25.97 12.06 b 14.67 a .41 .04
.26 b .71a .47 b 2.05 a 11.15 a 6.18 1.37 a 1.47 2.25 ND .15 .06
b
18.17 15.96 12.98 a .52 ND .57 .82
.32 b 1.40 b 8.02 b 2.42 .70
3.22 1.78 1.20 .19 .06
a
ab
25.82 16.28 11.05 b
a
30.00 16.59 12.05 a b
.30 .06 .33 .31
.37 .08 .43 .41
1.33 a 1.84 ab 11.08 a 7.72 1.33 1.67 ab 3.66
1.28 a 2.37 a 8.14 b
.10 ND .03
2.97 1.38 .61 .10 .06 .08 .16 .24 .17 .79
6.04
.34 .64
.03 1.69
.36
b
.15 .04 .12
.45 .70
1.85 .45 .15 .04
ab ' Values in rows within treatments with different superscripts are significantly different (P<.05). 1 Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick livers. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03).
suited in lower levels of 20:3w6, 20:3w9 and 20:4w6 and higher levels of 16:0 in both tissues. Heart PL also contained higher 16:1 and 18:0. Linoleate fed at .27% of the diet produced higher 18:lco9 and 20:3w9 and lower 18:0, 18:2oo6, 20:2w6, and 20:4w6 in PL of both tissues. The fatty acid compositions of liver microsomes from chicks were determined in Experiment 1 (Table 10). Twenty-one-day-old chicks fed biotin at 0 M-g/kg of diet had significantly higher 18:2w6 and 20:0 and lower 16:0, 18:0, and 20:3w6 than chicks fed biotin at 400 |Jig/kg of diet. At 28 days, significant differences were also observed in chicks fed biotin at 0 (xg/kg of diet. The 16:1 and 18:2a)6 were elevated and 18:0, 20:3w6, 20:4w6, 22:4w6, 22:5w6, and 22:6w3 were lower compared with levels in chicks fed biotin at 400 u,g/kg of diet. Prostaglandin E2 in freeze-clamped heart tissues from 14-day-old chicks was determined during Experiment 2 (Table 11). Chicks maintained on diets containing 0 fig/kg biotin and either .27 or 2.00% linoleate showed lower
levels of PGE2 than chicks fed adequate biotin. Chicks fed 200 ixg of biotin had PGE2 levels intermediate to those of chicks fed 0 or 400 (xg biotin. DISCUSSION
An interesting and consistent trend is observed during biotin deficiency in the chick; the present experiments show that 20:3w6 is decreased in liver PL. Similar changes were observed in liver lipids of biotin-deficient chicks in previous studies that included elevated 18:2co6 (Watkins and Kratzer, 1987a,b). Liver microsomes isolated from biotin-deficient chicks in Experiment 1 contained higher linoleate and lower 20:3w6 than their biotinadequate counterparts. Clearly, a biochemical block in EFA metabolism occurs in the chick during biotin deficiency (Watkins and Kratzer, 1987b). Elongation of EFA is probably depressed; however, desaturation may also be impaired. Occasionally, 20:4w6 is decreased in heart tissue of the biotin-deficient chick (Experiment
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18:3CJ6 18:3CJ3
l1
BIOTIN AND LINOLEATE ON FATTY ACIDS
2029
TABLE 7. Average weight percent of heart phospholipid fatty acids from 21-day-old chicks in Experiment Linoleate (% diet)
Biotin (Mg/kg diet)
Fatty acid
.50
3.15
16:0 16:1 17:0 18:0
20.88 1.67
19.29 1.51
18:1CJ9
18:2u;6 18:3w6 18:3to3 20:1CJ9
1.78 .47
20:3CJ6
20:4u;6 22:5w3 22:6co3 20:5OJ3
22:lto9 21:0
.11
1.63
2.38 2.49 7.02 b .58 .25 .47 .67 .25 .60 .15
.45 2.04 11.62 a .89 .65 ND3 .05 .79 .09
20.37 .94 b
18.93 .95b
1.15 .23
26.41 b 17.57 a 15.34
.06
28.99 a 17.48 a 13.93
.03 .69
1.17 1.04
.52 .09
.66 .03
1.70
1.55
1.85
.58
.40
.50
1.32 2.37 9.78 1.18
1.50 2.32 9.80
1.43 2.11 8.38
1.38
.55 .50 ND .89 .08 .60 .11
.48 .09 .71
.44 .65 .58
2.19
1.01
.31 .65 .25
.18 .41 .16
.75 ND .06 .07 .84 ND
1.41
Pooled SEM2
.47 .11
.52 b
400
.11
26.23 b 13.36 b 16.59
.57 .03
a
200
.12 .09 .36 .29 .22 .65
' Values in rows within treatments with different superscripts are significantly different (P<.01). 1
Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick hearts. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03).
TABLE 8. Average weight percent of liver phospholipid fatty acids from 21-day-old chicks in Experiment 21 Linoleate (% diet) Fatty acid
.27
2.00
16:0 16:1 17:0 18:0 18:lu>9 18:2a>6 18:3a>6 18:3ld3
26.22 1.37
26.69 1.89
20:1CJ9 20:2W6
20:3a>9 20:3CJ6
20:4o;6 22:4o>6 22:5u>6 22:5^3 22:6to3 20:5co3 22:1OJ9
21:0
ND3
Biotin (Mg/kg diet) 0 — (weight %) 28.32 a 2.99
23.52 23.25 a 9.24 b .30 b
a
26.91 15.95 b 13.48 a ,49 a .06
.06 a
b
.72 .12b 1.44a 1.75 b 4.14 b
.37 .46 a .52 b 2.24 a 7.05 a
.20
.67
.17b
.63 a
.80 .13 .50 .06 .16
.98 .30 .48 .19 .12
22.46 18.95 9.85
b
.35 .03 .66 .25
.49b 1.58 b 4.01b .21 .36 ND ND .63 ND .10
400
Pooled SEM2
26.91ab 1.23
24.12 b
1.17
ND
ND
ND b
200
27.98 20.52 10.66 .39 .06 .44 .25
a
.68 ND
25.21 19.33 13.58
.91 ab
.45 .10 .54 .38
.87b 1.99 ab 5.44 a b
1.59 a 2.42 a 7.33 a
.43 .37
.67 .47
1.20
1.47
.25 .17 .18 .21
.40 .67 .17 .10
1.30 1.09 1.48 .05 .03 .07 .04 .14 .20 .72 .33 .18 .32 .12 .52 .15 .09
' Values in rows within treatments with different superscripts are significantly different (P<.05). 'Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick livers. 2
SEM = Standard error or the mean.
3
ND = Not detected (levels below .03).
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22:4OJ6 22:5CJ6
(weight %) 20.96 2.89 a
28.40 a 13.52 b 15.78
.54 .12
20:2u>6 20:3u)9
0
.08
.10
26.01 b 18.75 a 14.79
l1
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WATKINS AND KRATZER
TABLE 9. Average weight percent of heart phospholipid fatty acids from 21-day-old chicks in Experiment 2 1 Linoleate (% diet)
Biotin (jug/kg diet)
Fatty acid
.27
2.00
16:0 16:1 17:0 18:0
24.86 1.27 a
24.80 .30 b
b
18:2^6
.40 ND3
20:1CJ9
1 5 9
20:4a>6
.61
.74
22:5u>6 22:5w3 =
.04 b
.26 a
22:6W3 20:5CJ3
22:lu>9 21:0
ND .03 .23 .52 .13
Pooled SEM2
25.56 a .06 b
20.42 b ,66 b
1.43
1.41
1.77
.47 1.32 b 4.71b .12 b ND .03 ND ND
b
28.93 18.31 16.24 b .62 .04
b
•04 a
.31 ND .25
.05 .03 ND .35 .11
400
•
2 9
23.54 16.03 21.46 a .35 ND
1.20 .39
ub
1.53 a 2.12 a 9.01 a 1.54a
.62 1.25 b 4.58 b ,36 b .15 ND .04 ND
.31 .04 .04
.12 b
.83 a
• 3 5 aub .34
.12
.11
.13
.30 .07 .98 .78 .91 .12 .02 .20 .10 .16 .10 .68 .13 .07 .04 .04 .12 .17 .09
' Values in rows within treatments with different superscripts are significantly different (P<.05). 1
Mean values of three samples per dietary treatment. Each sample contained pooled phospholipids from two chick hearts. 2
SEM = Standard error of the mean.
3
ND = Not detected (levels below .03). TABLE 10. Average weight percent of fatty acid in microsomes from chick livers in Experiment
1'
21 days
28 days
Dietary biotin (/ug/kg diet)
Dietary biotin 0/g/kg diet)
Fatty acid
0
400
16:0 16:1 18:0 18:lu9 18:2o;6 20:0 20:1^9
22.44 b 3.20 16.76 b 16.40 27.81 a .56 a
25.83 a 3.18 20.32 a 16.89 19.89 b ,22 b
ND2 .34 ND
ND .23 ND
0
400
20.75 1.76a 19.00 b 10.95 27.83 a
20.48 .87 b 23.43 a 10.86 19.35 b
(weight %)
20:2CJ6 20:3CJ9 20:3CJ6 20:4CJ6 22:4W6
22:5w6 22:5o;3 22:6CJ3
1.54 b 8.55 1.15 * .70
ND .17
2.26 a 8.67 .94 .68 ND .67
.42 ND .60 ND
1.22 b 11.89 b 1.25 b .81b 0 9
K
.44 b
.19 .22 .66 .55
2.23 a 16.18 a 1.35 a 2.13 a .17
1.30a
' Values in rows with different superscripts (21 or 28 days) are significantly different (P<.05). 1
Mean values of three samples per dietary treatment. Each sample contained pooled microsomes from two chick livers. 2
ND = Not detected (levels below .03).
Downloaded from http://ps.oxfordjournals.org/ at NERL on May 24, 2015
1.33 .42 a .41b 1.41 b 7.25 a
K
22:4CJ6
20:3CJ6
29.32 17.32 13.98 b
.46 .04
.20 b 1.33 a 1.71 a 4.94 b
20:2w6 20:3w9
a
29.07 13.18 b 19.39 a
200
.07
.10 a
25.46 21.25 a 15.07 b
18:3u>3
18:3CJ6
(weight %) 28.52 a 1.64a
.12
.03
18-.1CJ9
0
BIOTIN AND LINOLEATE ON FATTY ACIDS TABLE 11. Prostaglandin (PGE2) levels in heart tissue of 14-day-old chicks in Experiment 2 1 Dietary biotin
Linoleate
PGE 2
(Mg/kg)
(% diet)
(pg/g wet tissue)
.27 2.00 .27 2.00 2.00
23.7 33.7 43.2 49.7 87.5
0 200 400
± ± ± ± ±
12.2 6.5 11.0 7.6 15.7
'Mean values ± standard deviations from four chick hearts per dietary treatment.
REFERENCES Bannister, D. W., 1976. The biochemistry of fatty liver and kidney syndrome. Biochem. J. 156:167-173. Bieri, J. G., and E. L. Prival, 1966. Linoleic acid requirement of the chick. J. Nutr. 90:428^132. Curtin, L. V., and J. T. Raper, 1956. Feeding value of hydrolyzed vegetable fats in broiler rations. Poultry Sci. 35:273-278. Donaldson, W. E., G. F. Combs, G. L. Romoser, and W. C. Supplee, 1957. Studies on energy levels in poultry rations. 2. Tolerance of growing chicks to dietary fat. Poultry Sci. 36:807-815. Halloran, H. R., and I. R. Sibbald, 1979. Metabolizable energy values of fats measured by several procedures. Poultry Sci. 58:1299-1307. Hansen, G., J. J. Strik, J. H. Koeman, and C. A. Kan, 1981. Biological activity of technical aroclor 1254 compared to aroclor 1254 residues: swine fat residues fed to broiler cockerels. Toxicology 21:203-212. Hill, E. G., 1966. Effects of dietary linoleate on chick liver fatty acids: dietary linoleate requirement. J. Nutr. 89:465-470. Hill, E. G., C. L. Silbemick, and E. McMeans, 1967. Dietary linoleate and methionine in chicks. Poultry Sci. 46:523-526. Hood, R. L., 1975. A radiochemical assay for biotin in biological materials. J. Sci. Food.Agric. 26:18471852. Hood, R. L., 1984. Cellular and biochemical aspects of fat deposition in the broiler chicken. World Poult. Sci. J. 40:160-169. Hopkins, D. T., and M. C. Nesheim, 1967. The linoleic acid requirement of chicks. Poultry Sci. 46:872-881. Kramer, T. R., M. Briske-Anderson, S. B. Johnson, and R. T. Holman, 1984. Effects of biotin deficiency on polyunsaturated fatty acid metabolism in rats. J. Nutr. 114:2047-2052. Menge, H., 1970. Comparative requirements of linoleic acid for male and female chicks. Poultry Sci. 49:178183. National Research Council, 1984. Nutrient requirements of poultry, No. 1. Nutrient Requirements of Domestic Animals, 8th rev. ed. Natl. Acad. Sci., Washington, DC. Pearson, J. A., A. R. Johnson, R. L. Hood, and A. C. Fogerty, 1976. Fatty liver and kidney syndrome in chicks. I. Effect of Biotin in diet. Aust. J. Biol. Sci. 29:419^128. Renner, R., and F. W. Hill, 1960. The utilization of corn oil, lard and tallow by chickens of various ages. Poultry Sci. 39:849-854. SAS Institute, Inc., 1983. SAS Introductory Guide. SAS Inst. Inc., Cary, NC. Sibbald, I. R., and J.K.G. Kramer, 1978. The effect of the basal diet on the true metabolizable energy of fat. Poultry Sci. 57:685-691. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods, 6th ed. Iowa State Univ. Press, Ames, IA. Sunde, M. L., 1956. The effect of fats and fatty acids in chick rations. Poultry Sci. 35:362-368. Watkins, B. A., and F. H. Kratzer, 1987a. Tissue lipid fatty acid compositions of biotin-adequate and biotindeficient chicks. Poultry Sci. 66:306-313. Watkins, B. A., and F. H. Kratzer, 1987b. Dietary biotin effects on polyunsaturated fatty acids in chick tissue lipids and prostaglandin E2 levels in freeze-clamped hearts. Poultry Sci. 66:1835-1845. Young, R. J., 1961. The energy value of fats and fatty acids for chicks. 1. Metabolizable energy. Poultry Sci. 40:1225-1233.
Downloaded from http://ps.oxfordjournals.org/ at NERL on May 24, 2015
2; Watkins and Kratzer, 1987a) but 20:3w6 is usually lowered. The decrease in 20:4w6 observed in Experiment 2 may be due to the lower levels of linoleate fed. The eicosanoic fatty acids, 20:4co6 and 20:3co6, are important precursors of prostaglandins (PG). Decreased levels of substrates in membrane PL may lower PG production. This was observed in heart tissue of the biotin-deficient chick in Experiment 2. Both 20:4w6 and PGE2 were lower in heart tissue of chicks fed biotin at 0 and 200 u.g/kg of diet than in tissue of chicks fed 400 (xg of biotin. In this case, biotin deficiency may indirectly affect PG biosynthesis in the chick by reducing PG precursors in PL. An absence of biotin (0 u-g/kg of diet) resulted in elevated 17:0 in total lipids of chick liver (data not shown); this observation was reported in rat liver fatty acids during a biotin deficiency (Kramer et al., 1984). Biotin deficiency also elevated 16:1 in chick heart PL. It is apparent that biotin deficiency impairs linoleate metabolism (omega-6 pathway) in the chick; possibly other polyenoic fatty acid pathways are also affected. Chicks fed .5 or .27% linoleate (1.23 and .67% of total ME/kcal per kg) and 200 jjLg of biotin in Experiments 1 and 2, respectively, maintained body weights that were not significantly different from those of chicks fed higher levels of linoleate. However, the low levels of dietary linoleate elevated 20:3w9 in PL. Feeding chicks diets that are low in linoleate for a longer period of time may be necessary to produce growth depression. The data in this study provide evidence that the biotin status of the chick will influence EFA metabolism and levels of eicosanoic fatty acids in PL of liver and heart.
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