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Essential fatty acid status of colostrum-deprived gnotobiotic and conventional lambs: Organ fatty acid composition
NUTRITION RESEARCH, Vol. 4, pp. 503-509, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) ]984 Pergamon Press Ltd. A]] r i g h t s reserved.
ESSI!NT/AL FAqTY ACID ST2fIITS OF C O ~ - D E P R / V E D GNOTOBIOTIC AND CONVENTIONAL LAMBS: ORGAN FATTY ACID COMPOSITION G. Bruckner, Ph.D. 1, R. E. Tucker3, Ph.D., K. K.3Grun~ald, Ph.D. 2, and G. E. Mitchell, Jr. Ph.D. 1 .... . . . . . Department of2Cl~nlCal Nutrltlon, Uruiverslty of Kentucky, Lexzngton Kentucky 40536, Department of Food Science and Nu~ition, Justice Hall, Kansas State University, Manhattan, Kansas 66506, Department of Animal Science, University of Kentucky, Lexington, Kentucky 40536
ABSTRACP Gnotobiotic (GN) and conventional (CV) colostrum-deprived lambs were fed diets deficient (D) or supplemented with .32% of the total calories as linoleic acid (L). Plasma and organ fatty acid compositions were assessed after 2 too. of the dietary and environmental regimens. Linoleic acid deficiency in GN and CV lambs generally resulted in decreases of linoleic acid in plasma, liver and kidney samples and increases in oleic and eicosatrienoic acids. The triene/tetraene ratios (20:3n9/20:4n6) for plasma, kidney and liver increased in the D vs L groups. However, the elevated triene/tetraene ratios observed for the L groups (<. 4) indicated a slight overall essential fatty acid (EFA) deficiency condition. The presence of the microflora apparently increased the linoelic acid requir~nent of CV lambs. The data suggest that the requir~nents of the neonatal colostrumdeprived ruminant are in excess of .32% of the total calories and that changes in EFA status of the rtminant are reflected in tissue fatty acid composition. Key Words:
kk~ninant, Lamb, Essential Fatty Acids, Fatty Acid, Gnotobiotic INTg~DUC~ION
Essential fatty acids (EFA) were discovered by Burr and Burr (I, 2). These investigators showed that linoleic acid was one of the EFA. Man and most animals require a minimum supply of EFA (i to 2% of dietary calories) to prevent overt essential fatty acid deficiency (EFAD) symptc~s (3), however Cunninghan and Loosli (4) suggested that the ruminant animals requirement for EFA was .32 energy percent. While a great deal of research related to lipid metabolism in the rt~ninant has been reported (5), s c ~ controversy still exists as to the role of the intestinal microflora and the EFA status of the rtmlinant. Patton et al. (6) and others (7) have d~nonstrated the synthesis of linoleic acid by the rumen microflora, which in the neonatal ruminant may contribute to its EFA status. However, in an adult ruminant, predcndnantly biohydrogenation by the microflora has been shown to occur (8, 9, i0). To elucidate various interactions between the intestinal microflora and the rnmtinant animal, a limited number of gnotobiotic ruminant animals have been utilized (ii, 12, 13). While these studies were not related to EFA metaboli~n, increases in depot unsaturated fatty acids in gnotobiotic vs conventional lambs fed milk diets were noted (13). Therefore, the following experiment was conducted to study, in gnotobiotic and conventional colostrum deprived
503
504
G. BRUCKNs
eta].
lambs, the influence of the intestinal microflora on the lambs' EFA status, i.e., the resultant fatty acid cc~position of various organs after dietary intervention. MATERI_ALS AND METHODS The procurement, housing, maintenance, diet formulation and gro1~p regimens of the neonatal lambs have been previously described (14). The lambs, delivered by caesarean section or hysterectc~y, were reared either as gnotobiotes (~) or as conventional (CV) lambs. They were fed milk diets that were either deficient (D) or supplemented (L) isocalorically with .32% of calories as linoleic acid. To the skim milk diets (hot process extraction <.01% total fat) were added 6% by weight hydrogenated coconut oil (HCO) and vitamins E, A and D to meet requir~nent levels. The diets were then mixed, pasteurized, hcmogenized, bottled and sterilized. The colostrum-deprived GN and CV lambs were fed the sterile milk solution initially four times a day at approximately 6-h intervals (24 h/d) and later, three times daily. Jugular blood samples were taken frc~ the G~ and CV lambs 2 h after their afternoon feeding (.3 ml sodium heparin/5 ml blood, 1,000 USP/ml) 4 and, after centrifugation (1500 x g, 15 rain), the plasma was stored frozen at -20 degrees C under nitrogen. The lambs were sacrificed at 2 mo of age and the organs excised, weighed and frozen at -20 degrees C in saline (.9% NaCI) for subsequent fatty acid analysis (14). Fatty AcidAnalysis Plasma and tissue samples fr~n 2 mo old lambs (kidney, liver) were extracted for fatty acid analysis using established methods (15). Total lipid samples were saponified and methylated w~th boron trifluoride 14% in methanQl (16). The methyl esters were .qL~ntified by gas licglid chromatography (GLC)o using known reference standards~b. The methyl 5, 8, ii eicosatrienoic acid standard was kindly provided by Dr. Howard Specher, Columbus, Ohio. A 183-cm glass column packed with 10% EGSSX-Gas Chrcm QI00-120 mesh was used; the N flow rate was 41 ml/min and temperature p r o g r ~ at 2 degrees c/min, 165 degrees to 205 degrees C. Statistical Analysis C
FATTY ACIDS IN COLOSTRUM-DEPRIVATION
505
established itself in the CV animals was different from that observed for normal CV lambs that were mother suckled (14). In the liver samples (Table 2), decreases were noted for linoleic and arachidonic acids and increases for oleic and eicosatrienoic acids in the D vs L groups. However, unlike the decreases noted for linoleic acid in the plasma samples of the CVL vs GNL lambs, the liver 18:2n6 levels were elevated in CVL. The reason(s) for these differences is not apparent, but may be related to the different growth rates noted for GNL vs CVL (14). Medium chain fatty acids were not significantly affected. The inverse relationship between liver 18:1 and 18:2 fatty acids was again evident. The tri/tetra ratios in all liver samples were above .4. The microflora effects on fatty acid composition were more pronounced in the liver samples than in the plasma or kidney. Significant reductions in 14-carbon fatty acids were seen in the CV vs GN group. The liver, which is the first organ to receive the absorbed intestinal nutrients (short chain fatty acids) and microbial-metabolites, might exhibit more striking differences. In the kidney (Table 3) no microfloral effects were evident in the medium chain fatty acids. The relationships between the distribution percentage of the long chain fatty acids was similar to that noted for plasma. The kidney tri/ tetra values for the deficient groups [both ~ and CV) were not as elevated as in plasma and liver. I~ is apparent that a slight EFA deficiency was present in all of the lambs even in the presence of .32% of the total calories as linoleic acid. This suggests that this level of linoleic acid, cited by Cunningham and Loosli (4) to be adequate, is not sufficient for meeting the EFA requirements of colostrz~n-deprived neonatal lambs. Furthemmore, the slightly increased tri/ tetra ratios in plasma and kidney of CV vs ~ lambs, as well as the decreased growth rates for the CV vs GN lambs (14) indicates that the microflora might contribute a "nutritional" stress either by decreasing the availability of linoleic acid or by increasing its requirem~_nt through other stress mechanisms. These findings are in apparent conflict with those of Sklan et al (7), who imply that the neonatal ruminant's intestinal microflora may synthesize EFA. However, the CV lambs under the circumstances of our protocol, i.e., deprived of maternal physical microbial contact, probably did not develop a "normal" microflora {14) and, therefore, the "normal" microflora's contributions to the neonates EFA status has yet to be clarified. The use of the GN neonatal r~ninant se6~s to be an extremely useful model to elucidate microbe-host EFA interactions. The use of this model should shed light on the apparently complex influence of the gastro-intestinal microflora on EFA metabolism as well as on other nutrients required by the ruminant. ACKNOW7 m X ~ E N T S The investigation reported in this paper (No. 82-5-111) is in connection with a project of the Kentucky Agric. Expt. Sta. and is published with approval of the director. The help and advice of Dr. H. A. Gordon is gratefully acknowledged. A special thanks to Dr. Bobert Stuart for his surgical assistance and friendly advice.
506
G. BRUCKNERet al.
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FATTY ACIDS IN COLOSTRUM-DEPRIVATION
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G. BRUCKNER et al. REFERENCES
i.
BURR, G.O. and M.M. BURR. A new deficiency disease produced by the rigid exclusion of fat from the diet. J. Biol. Chem. 82:345-367, 1929.
2.
BURR, G.O. and M.M. BURR. On the nature and role of the fatty acids essential in nutrition. J. Biol. Chin. 86:587-621, 1930.
3.
HOIMAN, R.T. Essential fatty acid deficiency. In: R.T. Holman (ed.) Progress in ChemiStry of Fats and Other Lipids. IX(2) , pp. 279-348, Pergamon Press, New York, 1968.
4.
C~TNNINGHAM, H.M. and J.K. LOOSLI. The effect of fat free diets on young dairy calves with observations on metabolic fecal fat and digestion coefficients for lard and hydrogenated coconut oil. J. Dairy Sci. 37: 453-461, 1954.
5.
CHRISTIE, W.W. Lipid Metabolis~ in Ruminant Animals. New York, 1981.
6.
PATION, R.A., R.D. MCCARTHY and L.C. GRIEL. Lipid synthesis by rumen microorganisms. II. Further characterization of the effects of methionine. J. Dairy Sci. 53:460-465, 1970.
7.
SKI/IN, D., R. VOLCANI and P.U. BUDOWSKI. Formation of octadecadienoic acid by rumen liquor of calves, cows and sheep in vitro. Brit. J. h~Itr. 28:239-248, 1972.
8.
REISER, R. Hydrogenation of polyunsaturated fatty acids by the Inmlinant. Fed. Proc. 10:236, 1951.
9.
BICKERSTAFFE, R., D.E. NC[AKES and E.F. ANNISON. Quantitative aspects of fatty acid biohydrogenation, absorption and transfer into milk fat in the lactating goat, with special reference to the cis and trans iscrners of octadecenoate and linoleate. Bioch~n. J. 130:607-617, 1972.
Pergamon Press,
i0.
HARFOOT, C.G. Lipid metabolism in the rumen. In: W.W. Christie (ed.) Lipid Metabolism in the Ruminant Animal, pp. 21-55, Pergamon Press, New York, 1981.
ii.
ELI.TOT, L.M., T.J.L. ALEXANDER, P.D. W~LT.qTEAD and R.J. LYSONS. monitoring of gnotobiotic lambs. Lab. Anim. 8:51-59, 1974.
12.
PONTO, K.H. and W.G. BERGEN. Developmental aspects of glucose and VFA metabolism in the geunfree and conventional ruminant. J. Anita. Sci. 38: 893-899, 1974.
13.
LYSONS, R.J. and T.L.J. ~ E R . Depot fatty acids of gnotobiotic lambs. Proc. Nutr. Soc. 32:97A, 1973.
14.
BRUCKNER, G.G., R.E. ~CKER, K.K. G ~ , and G.E. MITCHETZ., JR. Essential fatty acid status and characteristics associated with a o l o s t ~ deprived gnotobiotic and conventional lambs: Growth, organ develolxnent, cell membrane integrity and parameters associated with lower bowel function. J. Anita. Sci. (In press).
15.
BLIGH, E.G. and W.J. DYER. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-920, 1959.
Microbial
FATTY ACIDS IN COLOSTRUM-DEPRIVATION
509
16.
MDRRISON, W.R. and L.M. SMITH. Preparation of fatty acidmethyl esters and dimethylacetals frc~ lipids with boron trifluoridemethanol. J. Lipid ires. 5:600-608, 1964.
17.
SNEDECDR, G.W. and W.G. COCHRAN. State Univ. Press, Ames, 1967.
Statistical Methods (6thEd.)
Accepted for publication March 4, 1984.
Iowa
ESSI!NT/AL FAqTY ACID ST2fIITS OF C O ~ - D E P R / V E D GNOTOBIOTIC AND CONVENTIONAL LAMBS: ORGAN FATTY ACID COMPOSITION G. Bruckner, Ph.D. 1, R. E. Tucker3, Ph.D., K. K.3Grun~ald, Ph.D. 2, and G. E. Mitchell, Jr. Ph.D. 1 .... . . . . . Department of2Cl~nlCal Nutrltlon, Uruiverslty of Kentucky, Lexzngton Kentucky 40536, Department of Food Science and Nu~ition, Justice Hall, Kansas State University, Manhattan, Kansas 66506, Department of Animal Science, University of Kentucky, Lexington, Kentucky 40536
ABSTRACP Gnotobiotic (GN) and conventional (CV) colostrum-deprived lambs were fed diets deficient (D) or supplemented with .32% of the total calories as linoleic acid (L). Plasma and organ fatty acid compositions were assessed after 2 too. of the dietary and environmental regimens. Linoleic acid deficiency in GN and CV lambs generally resulted in decreases of linoleic acid in plasma, liver and kidney samples and increases in oleic and eicosatrienoic acids. The triene/tetraene ratios (20:3n9/20:4n6) for plasma, kidney and liver increased in the D vs L groups. However, the elevated triene/tetraene ratios observed for the L groups (<. 4) indicated a slight overall essential fatty acid (EFA) deficiency condition. The presence of the microflora apparently increased the linoelic acid requir~nent of CV lambs. The data suggest that the requir~nents of the neonatal colostrumdeprived ruminant are in excess of .32% of the total calories and that changes in EFA status of the rtminant are reflected in tissue fatty acid composition. Key Words:
kk~ninant, Lamb, Essential Fatty Acids, Fatty Acid, Gnotobiotic INTg~DUC~ION
Essential fatty acids (EFA) were discovered by Burr and Burr (I, 2). These investigators showed that linoleic acid was one of the EFA. Man and most animals require a minimum supply of EFA (i to 2% of dietary calories) to prevent overt essential fatty acid deficiency (EFAD) symptc~s (3), however Cunninghan and Loosli (4) suggested that the ruminant animals requirement for EFA was .32 energy percent. While a great deal of research related to lipid metabolism in the rt~ninant has been reported (5), s c ~ controversy still exists as to the role of the intestinal microflora and the EFA status of the rtmlinant. Patton et al. (6) and others (7) have d~nonstrated the synthesis of linoleic acid by the rumen microflora, which in the neonatal ruminant may contribute to its EFA status. However, in an adult ruminant, predcndnantly biohydrogenation by the microflora has been shown to occur (8, 9, i0). To elucidate various interactions between the intestinal microflora and the rnmtinant animal, a limited number of gnotobiotic ruminant animals have been utilized (ii, 12, 13). While these studies were not related to EFA metaboli~n, increases in depot unsaturated fatty acids in gnotobiotic vs conventional lambs fed milk diets were noted (13). Therefore, the following experiment was conducted to study, in gnotobiotic and conventional colostrum deprived
503
504
G. BRUCKNs
eta].
lambs, the influence of the intestinal microflora on the lambs' EFA status, i.e., the resultant fatty acid cc~position of various organs after dietary intervention. MATERI_ALS AND METHODS The procurement, housing, maintenance, diet formulation and gro1~p regimens of the neonatal lambs have been previously described (14). The lambs, delivered by caesarean section or hysterectc~y, were reared either as gnotobiotes (~) or as conventional (CV) lambs. They were fed milk diets that were either deficient (D) or supplemented (L) isocalorically with .32% of calories as linoleic acid. To the skim milk diets (hot process extraction <.01% total fat) were added 6% by weight hydrogenated coconut oil (HCO) and vitamins E, A and D to meet requir~nent levels. The diets were then mixed, pasteurized, hcmogenized, bottled and sterilized. The colostrum-deprived GN and CV lambs were fed the sterile milk solution initially four times a day at approximately 6-h intervals (24 h/d) and later, three times daily. Jugular blood samples were taken frc~ the G~ and CV lambs 2 h after their afternoon feeding (.3 ml sodium heparin/5 ml blood, 1,000 USP/ml) 4 and, after centrifugation (1500 x g, 15 rain), the plasma was stored frozen at -20 degrees C under nitrogen. The lambs were sacrificed at 2 mo of age and the organs excised, weighed and frozen at -20 degrees C in saline (.9% NaCI) for subsequent fatty acid analysis (14). Fatty AcidAnalysis Plasma and tissue samples fr~n 2 mo old lambs (kidney, liver) were extracted for fatty acid analysis using established methods (15). Total lipid samples were saponified and methylated w~th boron trifluoride 14% in methanQl (16). The methyl esters were .qL~ntified by gas licglid chromatography (GLC)o using known reference standards~b. The methyl 5, 8, ii eicosatrienoic acid standard was kindly provided by Dr. Howard Specher, Columbus, Ohio. A 183-cm glass column packed with 10% EGSSX-Gas Chrcm QI00-120 mesh was used; the N flow rate was 41 ml/min and temperature p r o g r ~ at 2 degrees c/min, 165 degrees to 205 degrees C. Statistical Analysis C
FATTY ACIDS IN COLOSTRUM-DEPRIVATION
505
established itself in the CV animals was different from that observed for normal CV lambs that were mother suckled (14). In the liver samples (Table 2), decreases were noted for linoleic and arachidonic acids and increases for oleic and eicosatrienoic acids in the D vs L groups. However, unlike the decreases noted for linoleic acid in the plasma samples of the CVL vs GNL lambs, the liver 18:2n6 levels were elevated in CVL. The reason(s) for these differences is not apparent, but may be related to the different growth rates noted for GNL vs CVL (14). Medium chain fatty acids were not significantly affected. The inverse relationship between liver 18:1 and 18:2 fatty acids was again evident. The tri/tetra ratios in all liver samples were above .4. The microflora effects on fatty acid composition were more pronounced in the liver samples than in the plasma or kidney. Significant reductions in 14-carbon fatty acids were seen in the CV vs GN group. The liver, which is the first organ to receive the absorbed intestinal nutrients (short chain fatty acids) and microbial-metabolites, might exhibit more striking differences. In the kidney (Table 3) no microfloral effects were evident in the medium chain fatty acids. The relationships between the distribution percentage of the long chain fatty acids was similar to that noted for plasma. The kidney tri/ tetra values for the deficient groups [both ~ and CV) were not as elevated as in plasma and liver. I~ is apparent that a slight EFA deficiency was present in all of the lambs even in the presence of .32% of the total calories as linoleic acid. This suggests that this level of linoleic acid, cited by Cunningham and Loosli (4) to be adequate, is not sufficient for meeting the EFA requirements of colostrz~n-deprived neonatal lambs. Furthemmore, the slightly increased tri/ tetra ratios in plasma and kidney of CV vs ~ lambs, as well as the decreased growth rates for the CV vs GN lambs (14) indicates that the microflora might contribute a "nutritional" stress either by decreasing the availability of linoleic acid or by increasing its requirem~_nt through other stress mechanisms. These findings are in apparent conflict with those of Sklan et al (7), who imply that the neonatal ruminant's intestinal microflora may synthesize EFA. However, the CV lambs under the circumstances of our protocol, i.e., deprived of maternal physical microbial contact, probably did not develop a "normal" microflora {14) and, therefore, the "normal" microflora's contributions to the neonates EFA status has yet to be clarified. The use of the GN neonatal r~ninant se6~s to be an extremely useful model to elucidate microbe-host EFA interactions. The use of this model should shed light on the apparently complex influence of the gastro-intestinal microflora on EFA metabolism as well as on other nutrients required by the ruminant. ACKNOW7 m X ~ E N T S The investigation reported in this paper (No. 82-5-111) is in connection with a project of the Kentucky Agric. Expt. Sta. and is published with approval of the director. The help and advice of Dr. H. A. Gordon is gratefully acknowledged. A special thanks to Dr. Bobert Stuart for his surgical assistance and friendly advice.
506
G. BRUCKNERet al.
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G. BRUCKNER et al. REFERENCES
i.
BURR, G.O. and M.M. BURR. A new deficiency disease produced by the rigid exclusion of fat from the diet. J. Biol. Chem. 82:345-367, 1929.
2.
BURR, G.O. and M.M. BURR. On the nature and role of the fatty acids essential in nutrition. J. Biol. Chin. 86:587-621, 1930.
3.
HOIMAN, R.T. Essential fatty acid deficiency. In: R.T. Holman (ed.) Progress in ChemiStry of Fats and Other Lipids. IX(2) , pp. 279-348, Pergamon Press, New York, 1968.
4.
C~TNNINGHAM, H.M. and J.K. LOOSLI. The effect of fat free diets on young dairy calves with observations on metabolic fecal fat and digestion coefficients for lard and hydrogenated coconut oil. J. Dairy Sci. 37: 453-461, 1954.
5.
CHRISTIE, W.W. Lipid Metabolis~ in Ruminant Animals. New York, 1981.
6.
PATION, R.A., R.D. MCCARTHY and L.C. GRIEL. Lipid synthesis by rumen microorganisms. II. Further characterization of the effects of methionine. J. Dairy Sci. 53:460-465, 1970.
7.
SKI/IN, D., R. VOLCANI and P.U. BUDOWSKI. Formation of octadecadienoic acid by rumen liquor of calves, cows and sheep in vitro. Brit. J. h~Itr. 28:239-248, 1972.
8.
REISER, R. Hydrogenation of polyunsaturated fatty acids by the Inmlinant. Fed. Proc. 10:236, 1951.
9.
BICKERSTAFFE, R., D.E. NC[AKES and E.F. ANNISON. Quantitative aspects of fatty acid biohydrogenation, absorption and transfer into milk fat in the lactating goat, with special reference to the cis and trans iscrners of octadecenoate and linoleate. Bioch~n. J. 130:607-617, 1972.
Pergamon Press,
i0.
HARFOOT, C.G. Lipid metabolism in the rumen. In: W.W. Christie (ed.) Lipid Metabolism in the Ruminant Animal, pp. 21-55, Pergamon Press, New York, 1981.
ii.
ELI.TOT, L.M., T.J.L. ALEXANDER, P.D. W~LT.qTEAD and R.J. LYSONS. monitoring of gnotobiotic lambs. Lab. Anim. 8:51-59, 1974.
12.
PONTO, K.H. and W.G. BERGEN. Developmental aspects of glucose and VFA metabolism in the geunfree and conventional ruminant. J. Anita. Sci. 38: 893-899, 1974.
13.
LYSONS, R.J. and T.L.J. ~ E R . Depot fatty acids of gnotobiotic lambs. Proc. Nutr. Soc. 32:97A, 1973.
14.
BRUCKNER, G.G., R.E. ~CKER, K.K. G ~ , and G.E. MITCHETZ., JR. Essential fatty acid status and characteristics associated with a o l o s t ~ deprived gnotobiotic and conventional lambs: Growth, organ develolxnent, cell membrane integrity and parameters associated with lower bowel function. J. Anita. Sci. (In press).
15.
BLIGH, E.G. and W.J. DYER. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-920, 1959.
Microbial
FATTY ACIDS IN COLOSTRUM-DEPRIVATION
509
16.
MDRRISON, W.R. and L.M. SMITH. Preparation of fatty acidmethyl esters and dimethylacetals frc~ lipids with boron trifluoridemethanol. J. Lipid ires. 5:600-608, 1964.
17.
SNEDECDR, G.W. and W.G. COCHRAN. State Univ. Press, Ames, 1967.
Statistical Methods (6thEd.)
Accepted for publication March 4, 1984.
Iowa