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Fat Absorption by Germ-Free Chicks1
Fat Absorption by Germ-Free Chicks1 FRANK M. BOYD2 AND HARDY M. EDWARDS, JR. Poultry Disease Research Center, and Poultry Science Department, University of Georgia, Athens, Georgia 30601 (Received for publication March 26, 1967) TABLE 1.—Percent of the diet of the foutmajor fatty acids
INTRODUCTION
L
EXPERIMENTAL
Four plastic germ-free isolators were employed with 2 cages, each containing 6 chicks, in each isolator. Operational procedures for the isolators have been described (Edwards and Boyd, 1963a). The fact that the isolator was sterile was established by 3 days after the chicks hatched and then, fresh chicken feces from battery-housed hens were placed in the drinking water of 1 Journal Series Publication No. 513 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations. Partly supported by U.S.P.H.S. Grants AM 07532, AM 6338 and research career programs Award 18, 411 from Nat. Inst, of Arthritis & Metab. Dis. 2 Present Address: Biology Dept., Northeast Louisiana State College, Monroe, Louisiana.
Diet Fatty acid 16:0* 18:0 18:1 18:2 Total Lipid
Tallow (%)
Corn oil (%)
2.14 1.19 2.01 1.65 8.57
0.85 0.18 2.12 4.15 7.41
* Number of carbons: Number of double bonds.
two isolators to produce the conventionalized treatment. The diets utilized were practical type corn-soybean meal rations with the addition of 0.5% chromic oxide and 6% beef tallow or 6% corn oil. Fecal samples for analyses were collected from individual cages from the 12 th through the 15 th day. Analyses were performed for Cr 2 0 3 by the method of Brisson (1956). Fats were extracted with chloroform and methanol (Folch et al., 1957); methyl esters were prepared, and relative concentrations of the fatty acids were determined by gas-liquid chromatography, with a disc integrator (Edwards, 1964). RESULTS AND DISCUSSION The levels of the four major fatty acids in each diet are given in Table 1. The retention values for these component fatty acids and for total lipid are given in Table 2 for tallow and Table 3 for the corn oil diets. A power failure caused the loss of the chicks in one isolator in the corn oil trial, so these data are omitted. It can be seen that retention of total fat and of palmitic (16:0) and stearic (18:0)
1481
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
ITTLE information is available on the J effect of intestinal flora on absorption of fat. Luckey (1963) reported that germfree rats retained 96.8% of the fat from a synthetic diet containing 6.8% corn oil, but no data were given for the retention of fat or specific fatfy acids from these diets by conventionally reared rats. Young et al. (1963) found that fatty acids were absorbed better by chicks when reared in a fumigated laboratory or when fed antibiotics than in a contaminated environment. This environmental effect on growth rate and requirement was also reported by Edwards and Boyd (1963a, b) for magnesium, phosphorus and calcium. The experiments described here were performed to compare the relative retentions of the major fatty acids of two diets by germ-free and conventionalized chicks.
1482
F. M. BOYD AND H. M. EDWARDS, JR.
TABLE 2.—Percent retention of fat and fatty acids by germ-free and conventionalized chicks fed the diet containing beef tallow Environment pv**
(mean) GF
16:0*
18:0
18:1
18:2
78.1 74.4 75.0 72.6
71.3 61.9 69.8 69.9
60.8 49.8 57.9 54.9
73.9 60.3 72.9 72.4
80.1 73.5 81.5 81.9
75.0
68.2
55.8
69.9
79.3
84.2 85.9 84.3 86.0
69.9 72.7 74.5 78.5
63.6 66.5 69.0 77.1
62.4 63.3 72.9 73.9
75.0 69.1 80.8 78.5
85.1
73.9
69.1
68.1
75.9
* Number of carbon atoms: Number of double bonds. ** CV = Conventionalized; GF = Germ-free.
acids was lower in conventional chicks than in germ-free chicks, while environment did not affect the retention of the unsaturated fatty acids, oleic (18:1) and linoleic (18:2). This was true both with the beef tallow diet, where these two saturated fatty acids constituted 38.9% of the total fatty acid content, and with the corn oil diet where they were only 13.9% of the total fatty acid content. The data obtained with chicks maintained in the conventional environment compare very closely with those obtained by Renner and Hill (1961), who reported absorption values from beef tallow of 57% for palmitic acid, 53% for stearic acid, and 8 1 % for the unsaturated fatty acids. The use of a purified low fat control diet in their study enabled them to correct for endogeneous fat, which makes their values slightly lower than those reported in the present studies. Purified diets were not used in these experiments because of the difficulties encountered in sterilizing these diets for germ-free studies. The improved absorption of stearic acid and palmitic acid from corn oil as compared to beef tallow is similar to the data of Young
TABLE 3.—Percent retention of fat and fatty acids by germ-free and conventionalized chicks fed the diet containing corn oil Fatty acid
Environment
Total fat
16:0*
18:0
18:1
18:2
py**
85.4 85.9
75.4 80.8
66.9 67.4
86.3 87.6
89.0 88.6
85.6
78.1
67.1
87.0
88.8
87.9 86.0 88.5 88.0
83.7 81.3 85.9 84.3
79.6 78.7 79.4 77.6
81.9 87.2 89.6 89.3
89.7 88.3 89.7 89.9
87.6
83.8
78.8
87.0
89.4
(mean) GF
(mean)
* Number of carbon atoms: Number of double bonds. ** CV = Conventionalized; GF = Germ-free.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
(mean)
Fatty acid
Total fat
and Garrett (1963) who demonstrated that a high ratio of palmitic acid to stearic acid as well as a high ratio of oleic acid or linoleic acid to the saturated acids would improve absorption of palmitic and stearic acids. The germ-free environment appears to increase the retention of palmitic and stearic acids from both corn oil and beef tallow. This might be explained by a change in chemical and physical properties of the lumen content caused by microflora. These changes may cause poor stability of micelles containing large amounts of palmitic acid and stearic acid, thus decreasing the absorption of these fatty acids. In view of the reports of Bloch et al. (1961), it is unlikely that the differences in absorption are due to hydrogenation of double bonds by intestinal microflora, since they were unable to produce saturated fatty acids with intestinal microflora. Another possibility is that the brush border of the intestinal mucosal cell of the germ-free animal has a higher content of enzymes that esterify fatty acids and thus more palmitic acid and stearic acid gain entrance to the cell through this pathway. The results obtained
FAT ABSORPTION
justify the use of the germ-free chicken for studies on the mechanism of fat absorption. SUMMARY The germ-free chick retained greater amounts of palmitic and stearic acid than chicks in conventional environments. This difference in absorption was apparent whether stearic acid was present at a relatively high level in the diet (1.19%) or low level (0.18%). The environment did not appear to influence the chickens' absorption of oleic acid or linoleic acid.
Bloch, K., P. Baronowsky, H. Goldfine, W. J. Lennarz, R. Light, A. T. Norris and G. Scheuerbrandt, 1961. Biosynthsis and metabolism of unsaturated fatty acids. Federation Proc. 20: 921-927. Brisson, G. J., 1956. On the routine determination of chromic oxide in feces. Canadian J. Agri. Sci. 36:210-211.
Edwards, H. M., Jr., 1964. The influence of breed and/or strain on the fatty acid composi.ion of egg lipids. Poultry Sci. 43: 751-754. Edwards, H. M., Jr., and F. M. Boyd, 1963a. The effect of microbial contamination on the requirement of chicks for certain nutrients. Poultry Sci. 42 : 235-240. Edwards, H. M., Jr., and F. M. Boyd, 1963b. Effect of germ-free environment on CA4' metabolism. Poultry Sci. 42: 1030-1031. Folch, J., M. Lees, and G. H. Sloan-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Luckey, T. D., 1963. Germfree Life and Gnotobiology. Academic Press, N.Y. Renner, R., and F. W. Hill, 1961. Factors affecting the absorbability of saturated fatty acids in the chick. J. Nutrition, 74: 254-258. Young, R. J., and R. L. Garrett, 1963. Effect of oleic and linoleic acids on the absorption of saturated fatty acids in the chick. J. Nutrition, 8 1 : 321-329. Young, R. J., R. L. Garrett and M. Griffith, 1963. Factors affecting the absorbability of fatty acid mixtures high in saturated fatty acids. Poultry Sci. 42: 1146-1154.
A Negative Genetic Correlation Between Bursa Weight at Hatching and Post-hatching Body Growth of Chickens F. V. MUIR AND R. G. JAAP Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (Received for publication March 27, 1967)
W
EIGHT of the bursa of Fabricius and body weight of the baby chick are positively correlated phenotypically, (Hammond and Bird, 1942). Jaap (1960) reported that this correlation was 0.47 and 0.79 at hatching and at eleven days of age, respectively. Early body growth is accompanied by rapid hypertrophy of the bursa (Glick, 1956). Glick (19S6) had reported that the phenotypic correlations between body weight and bursa weight reached a peak of 0.76 at 4& weeks in White Leghorns and 0.92 at 6 weeks in Rhode Is-
land Reds. Therefore, a negative genetic correlation between bursa weight at hatching and post-hatching body growth was unsuspected. The bursa is larger at hatching in the Leghorn baby chick than either the Rhode Island Red (Glick, 1956) or the broilertype chick (Jaap, 1958) which attained larger adult body size. Since the Leghorn appears to be particularly adapted to resist disease as well as stress conditions (Hutt, 1958), the suggestion was made that part of this advantage of the smaller-bodied
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
REFERENCES
1483
INTRODUCTION
L
EXPERIMENTAL
Four plastic germ-free isolators were employed with 2 cages, each containing 6 chicks, in each isolator. Operational procedures for the isolators have been described (Edwards and Boyd, 1963a). The fact that the isolator was sterile was established by 3 days after the chicks hatched and then, fresh chicken feces from battery-housed hens were placed in the drinking water of 1 Journal Series Publication No. 513 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations. Partly supported by U.S.P.H.S. Grants AM 07532, AM 6338 and research career programs Award 18, 411 from Nat. Inst, of Arthritis & Metab. Dis. 2 Present Address: Biology Dept., Northeast Louisiana State College, Monroe, Louisiana.
Diet Fatty acid 16:0* 18:0 18:1 18:2 Total Lipid
Tallow (%)
Corn oil (%)
2.14 1.19 2.01 1.65 8.57
0.85 0.18 2.12 4.15 7.41
* Number of carbons: Number of double bonds.
two isolators to produce the conventionalized treatment. The diets utilized were practical type corn-soybean meal rations with the addition of 0.5% chromic oxide and 6% beef tallow or 6% corn oil. Fecal samples for analyses were collected from individual cages from the 12 th through the 15 th day. Analyses were performed for Cr 2 0 3 by the method of Brisson (1956). Fats were extracted with chloroform and methanol (Folch et al., 1957); methyl esters were prepared, and relative concentrations of the fatty acids were determined by gas-liquid chromatography, with a disc integrator (Edwards, 1964). RESULTS AND DISCUSSION The levels of the four major fatty acids in each diet are given in Table 1. The retention values for these component fatty acids and for total lipid are given in Table 2 for tallow and Table 3 for the corn oil diets. A power failure caused the loss of the chicks in one isolator in the corn oil trial, so these data are omitted. It can be seen that retention of total fat and of palmitic (16:0) and stearic (18:0)
1481
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
ITTLE information is available on the J effect of intestinal flora on absorption of fat. Luckey (1963) reported that germfree rats retained 96.8% of the fat from a synthetic diet containing 6.8% corn oil, but no data were given for the retention of fat or specific fatfy acids from these diets by conventionally reared rats. Young et al. (1963) found that fatty acids were absorbed better by chicks when reared in a fumigated laboratory or when fed antibiotics than in a contaminated environment. This environmental effect on growth rate and requirement was also reported by Edwards and Boyd (1963a, b) for magnesium, phosphorus and calcium. The experiments described here were performed to compare the relative retentions of the major fatty acids of two diets by germ-free and conventionalized chicks.
1482
F. M. BOYD AND H. M. EDWARDS, JR.
TABLE 2.—Percent retention of fat and fatty acids by germ-free and conventionalized chicks fed the diet containing beef tallow Environment pv**
(mean) GF
16:0*
18:0
18:1
18:2
78.1 74.4 75.0 72.6
71.3 61.9 69.8 69.9
60.8 49.8 57.9 54.9
73.9 60.3 72.9 72.4
80.1 73.5 81.5 81.9
75.0
68.2
55.8
69.9
79.3
84.2 85.9 84.3 86.0
69.9 72.7 74.5 78.5
63.6 66.5 69.0 77.1
62.4 63.3 72.9 73.9
75.0 69.1 80.8 78.5
85.1
73.9
69.1
68.1
75.9
* Number of carbon atoms: Number of double bonds. ** CV = Conventionalized; GF = Germ-free.
acids was lower in conventional chicks than in germ-free chicks, while environment did not affect the retention of the unsaturated fatty acids, oleic (18:1) and linoleic (18:2). This was true both with the beef tallow diet, where these two saturated fatty acids constituted 38.9% of the total fatty acid content, and with the corn oil diet where they were only 13.9% of the total fatty acid content. The data obtained with chicks maintained in the conventional environment compare very closely with those obtained by Renner and Hill (1961), who reported absorption values from beef tallow of 57% for palmitic acid, 53% for stearic acid, and 8 1 % for the unsaturated fatty acids. The use of a purified low fat control diet in their study enabled them to correct for endogeneous fat, which makes their values slightly lower than those reported in the present studies. Purified diets were not used in these experiments because of the difficulties encountered in sterilizing these diets for germ-free studies. The improved absorption of stearic acid and palmitic acid from corn oil as compared to beef tallow is similar to the data of Young
TABLE 3.—Percent retention of fat and fatty acids by germ-free and conventionalized chicks fed the diet containing corn oil Fatty acid
Environment
Total fat
16:0*
18:0
18:1
18:2
py**
85.4 85.9
75.4 80.8
66.9 67.4
86.3 87.6
89.0 88.6
85.6
78.1
67.1
87.0
88.8
87.9 86.0 88.5 88.0
83.7 81.3 85.9 84.3
79.6 78.7 79.4 77.6
81.9 87.2 89.6 89.3
89.7 88.3 89.7 89.9
87.6
83.8
78.8
87.0
89.4
(mean) GF
(mean)
* Number of carbon atoms: Number of double bonds. ** CV = Conventionalized; GF = Germ-free.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
(mean)
Fatty acid
Total fat
and Garrett (1963) who demonstrated that a high ratio of palmitic acid to stearic acid as well as a high ratio of oleic acid or linoleic acid to the saturated acids would improve absorption of palmitic and stearic acids. The germ-free environment appears to increase the retention of palmitic and stearic acids from both corn oil and beef tallow. This might be explained by a change in chemical and physical properties of the lumen content caused by microflora. These changes may cause poor stability of micelles containing large amounts of palmitic acid and stearic acid, thus decreasing the absorption of these fatty acids. In view of the reports of Bloch et al. (1961), it is unlikely that the differences in absorption are due to hydrogenation of double bonds by intestinal microflora, since they were unable to produce saturated fatty acids with intestinal microflora. Another possibility is that the brush border of the intestinal mucosal cell of the germ-free animal has a higher content of enzymes that esterify fatty acids and thus more palmitic acid and stearic acid gain entrance to the cell through this pathway. The results obtained
FAT ABSORPTION
justify the use of the germ-free chicken for studies on the mechanism of fat absorption. SUMMARY The germ-free chick retained greater amounts of palmitic and stearic acid than chicks in conventional environments. This difference in absorption was apparent whether stearic acid was present at a relatively high level in the diet (1.19%) or low level (0.18%). The environment did not appear to influence the chickens' absorption of oleic acid or linoleic acid.
Bloch, K., P. Baronowsky, H. Goldfine, W. J. Lennarz, R. Light, A. T. Norris and G. Scheuerbrandt, 1961. Biosynthsis and metabolism of unsaturated fatty acids. Federation Proc. 20: 921-927. Brisson, G. J., 1956. On the routine determination of chromic oxide in feces. Canadian J. Agri. Sci. 36:210-211.
Edwards, H. M., Jr., 1964. The influence of breed and/or strain on the fatty acid composi.ion of egg lipids. Poultry Sci. 43: 751-754. Edwards, H. M., Jr., and F. M. Boyd, 1963a. The effect of microbial contamination on the requirement of chicks for certain nutrients. Poultry Sci. 42 : 235-240. Edwards, H. M., Jr., and F. M. Boyd, 1963b. Effect of germ-free environment on CA4' metabolism. Poultry Sci. 42: 1030-1031. Folch, J., M. Lees, and G. H. Sloan-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Luckey, T. D., 1963. Germfree Life and Gnotobiology. Academic Press, N.Y. Renner, R., and F. W. Hill, 1961. Factors affecting the absorbability of saturated fatty acids in the chick. J. Nutrition, 74: 254-258. Young, R. J., and R. L. Garrett, 1963. Effect of oleic and linoleic acids on the absorption of saturated fatty acids in the chick. J. Nutrition, 8 1 : 321-329. Young, R. J., R. L. Garrett and M. Griffith, 1963. Factors affecting the absorbability of fatty acid mixtures high in saturated fatty acids. Poultry Sci. 42: 1146-1154.
A Negative Genetic Correlation Between Bursa Weight at Hatching and Post-hatching Body Growth of Chickens F. V. MUIR AND R. G. JAAP Ohio Agricultural Research and Development Center, Columbus, Ohio 43210 (Received for publication March 27, 1967)
W
EIGHT of the bursa of Fabricius and body weight of the baby chick are positively correlated phenotypically, (Hammond and Bird, 1942). Jaap (1960) reported that this correlation was 0.47 and 0.79 at hatching and at eleven days of age, respectively. Early body growth is accompanied by rapid hypertrophy of the bursa (Glick, 1956). Glick (19S6) had reported that the phenotypic correlations between body weight and bursa weight reached a peak of 0.76 at 4& weeks in White Leghorns and 0.92 at 6 weeks in Rhode Is-
land Reds. Therefore, a negative genetic correlation between bursa weight at hatching and post-hatching body growth was unsuspected. The bursa is larger at hatching in the Leghorn baby chick than either the Rhode Island Red (Glick, 1956) or the broilertype chick (Jaap, 1958) which attained larger adult body size. Since the Leghorn appears to be particularly adapted to resist disease as well as stress conditions (Hutt, 1958), the suggestion was made that part of this advantage of the smaller-bodied
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 4, 2015
REFERENCES
1483