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Changes in Plasma Triglyceride Fatty Acids During Lactation1
Changes in Plasma T r i g l y c e r i d e F a t t y A c i d s D u r i n g L a c t a t i o n ~ R. W. WALLENIUS and R. E. WHITCHURCH Department of Animal Sciences Washington State University Pullman 99163 ABSTRACT
differences (2, 5) as well as uptake and incorporation of labeled fatty acids into milk fat (2, 6, 9). Plasma from lactating cows probably contains about 8 to 12 mg/dl TG (5, 14) although Raptaael et al. (13) did not detect TG for all groups and Hartmann and Lascelles observed values of 15 to 17 mg/dl (10). Duncan and Garton (7) found that TG accounted for an average of 10% of total fatty acids in plasma in the dry period and about 2% twenty days into lactation. The fatty acid composition of the triglyceride fraction varied markedly between cows on the first and 22nd day of lactation. Studies of lipid and lipoprotein classes have compared (a) dry and lactating cows (10, 13, 16), (b) ketotic or sub-clinically ketotic with normal cows (11, 19), and (c) normal cows with those receiving restricted roughage-high grain (RRHG) rations to stimulate the milk fat depression syndrome (1, 3). There is evidence that methionine or methionine h y d r o x y analog (MHA) could influence lipid metabolism in lactating cows, either in the host tissue or by influencing rumen microbial lipid synthesis (12). As part of a study concerning the effect of MHA and sulfate on milk and milk fat production, we examined triglyceride fatty acids of milk and blood plasma at varying stages of lactation. Production and milk lipids have been reported (17). This paper reports changes in plasma T G F A during the first half of lactation in normal cows.
Total blood plasma lipid and plasma triglyceride fatty acids were analyzed at an average of 33, 89, and 139 days of lactation (ranges 20 to 57, 52 to 129, and 122 to 157 days for periods I, 11, and l i d for 35 Holstein cows in their second or later Lactation. Average milk production in the test periods was 35.7, 30.3, and 25.5 kg/day. Lipid analysis was part of a study comparing methionine hydroxy analog or sulfur for lactating cows with effects of treatment and time separated statistically. Cows fed methionine analog had a lower percent palmitate in the triglyceride fatty acid, but there was no other treatment effect. There were significant changes with time in percent total lipid and triglyceride fatty acid. Total lipid averaged 424, 526, and 529 mg/dl. Average percents of measured triglyceride fatty acid and differences with stage of lactation were: myristic, 2.96; palmitic, 27.52, I>1I or II1; palmitoleic, 2.83, 1>11 or 111; stearic, 38.58, 111 or III ; and linoleic, 4.59. INTRODUCTION
There are many reports concerning the controlling factors in milk fat synthesis, fat test depression, and ketosis, all of which potentially involve dietary, storage, and circulating lipids. Studies of lipids in plasma have demonstrated significant changes in total lipids as well as in specific lipid fractions during normal lactations (7, 10, 13, 16). Plasma triglycerides (TG) are the primary source of long-chain fatty acids from dietary or adipose sources for milk fat synthesis. Evidence for this is based on arterial-venous (A-V)
MATERIALS AND METHODS
Thirty-six Holstein cows were assigned to one of three rations: control, methionine hydroxy analog (MHA), or sulfate supplementation. Cows supplemented with MHA received 25 g/day in the concentrate part of their ration. Cows supplemented with sodium sulfate received 17.5 g/day. Experimental design, methods of management, and effects of treatment on production have been reported (17). Thirty milliliters of blood were collected via jugular vein into heparinized vacutainer tubes during
Received May 9, 1975. 1Scientific paper no. 4438. College of Agriculture Research Center, Washington State University, Pullman. Proj. 0126. 85
86
WALLENIUS AND WHITCHURCH
peak lactation (range 20 to 57 days), and again at an average of 89 (range 52 to 129) and 139 (range 122 to 157) days into lactation. These sampling times are referred to as 1, II, and 1II, respectively. Plasma was frozen at - 2 0 C for subsequent analysis of triglyceride fatty acid and total lipid. Concentrate averaged 48% of the total ration dry m a t t e r during the experim e n t and provided an estimated 60% of digestible energy (DE) in I, 53 to 60% of DE in II, and 53% of DE in III. Blood lipids were extracted by the m e t h o d of Folch et al. (8) as modified by Hartmann and Lascelles (10), except that 5 ml of plasma were extracted rather than 10 ml. All reagents in subsequent steps in the extraction procedure were reduced by half. Total lipids of plasma were determined gravimetrically and stored in 1 ml of c h l o r o f o r m under nitrogen at I C until further analysis. Triglycerides were separated from other plasma lipid fractions by thin layer chromatography. Further preparation and gas c h r o m a t o graphic analysis have been described (17). Effects of ration and stage of lactation on total lipids of plasma and percent of triglyceride fatty acids of plasma were separated by analysis of variance (15).
RESULTS A N D DISCUSSION
Rations had no effect on total lipids of plasma, and the pooled results (Table 1) show a trend similar to that r e p o r t e d by others. Total lipids of plasma were lower (P'(.O1) during 1 than in II and 111. Varman and Schultz (16)
showed plasma total lipids similar during the dry period and 30 days into lactation b u t increased by a b o u t 60%, 260 days into lactation. Most of this difference was associated with the phospholipid and cholesteryl ester fraction. Raphael et al. (13) f o u n d total lipid at 0 to 4 wk p o s t p a r t u m averaged 292 mg/dl and increased to 508 mg/dl in w k 5 to 10 with a slight decrease to 458 mg/dl during weeks 16 to 33 prepartum. Hartmann and Lascelles (10) did not observe as marked a change b e t w e e n dry and lactating cows and f o u n d a slight but n o t significant increase in total lipid in late as compared with early lactation. Duncan and Garton observed a m i n i m u m total lipid at parturition followed by an increase in early lactation (7). Percents of plasma T G F A are in Table 1. Averages are pooled for the three treatments (MHA, Sulfate, and Control). The percent palmitic acid in T G F A of plasma was lower (P<.01) for cows receiving MHA. Percents palmitate for the MHA, sulfate, and control treatments were 26.8, 27.3, and 28.5% of T G F A . The importance of this is n o t k n o w n immediately. Discussions by Dimick et al. (6), Askew et al. (3), and Benson and Emery (4) suggest that the T G F A pool available to the m a m m a r y gland may be significant in efficiency of milk fat synthesis and perhaps MHA plays a role in this control, possibly via microbial lipid synthesis in the rumen (12). Plasma T G F A did vary significantly in s o m e instances as lactation progressed. Effects of ration were removed statistically before comparisons of time. Myristic and linoleic acid did
TABLE 1. Average plasma total lipid and triglyceride long chain fatty acids at 33, 89, and 139 days of lactationa, b.
Lipid Total C 14 C 16 C16:1 C 18 C 18:1 C 18:2
Period 11
I 424 c 3.0 28.8 c 3.9 c 30.8 c 26.4 c 4.4
-+
13.1 .15 .45 .20 1.19 1.09 .27
526 d
-+
3.1 27.2 d 2.3 d 41.3 d 18.7 d 4.6
Ill 19.2 .13 .40 .13 .82 .63 .22
aTotal lipid as mg/dl plasma -+ SE, TGFA as weight % of those determined. bDifferent superscripts within rows are significantly different. Journal of Dairy Science VoI. 59, No. 1
539 d 2.8 26.5 d 2.2 d 43.9 e 16.9 d 4.9
+-
18.9 .13 .34 .15 .55 .39 .30
BLOOD PLASMA FATTY ACIDS n o t change. Palmitic, palmitoleic, and oleic acids decreased in II and III as c o m p a r e d with I ( P < . 0 1 ) . Stearic acid increased in II ( P < . 0 1 ) and again in III ( P < . 0 5 ) . Higher p e r c e n t a g e s of palmitic a n d oleic acids can be associated with an increased m o b i l i z a t i o n o f adipose tissue f a t t y acids (18), w h i c h is e x p e c t e d for high p r o d u c i n g cows in early lactation. The changes f r o m p e a k l a c t a t i o n (I) t o II and III also agree w i t h changes observed by Y a m d a g n i and Schdltz (19) and W a t e r m a n and Schultz (18) for normal, subclinically ketotic, and ketotic cows. This suggests t h a t t h e degree of mobilization o f adipose tissue fatty acids and lipid m e t a b o l i s m in general, may be similar for n o r m a l high p r o d u c i n g c o w s and c o w s t h a t are clinically or subclinically ketotic. REFERENCES
1 Annison, E. F., R. Bickerstaffe, and J. L. Linzell. 1974. Glucose and fatty acid metabolism in cows producing milk of low fat content. J. Agr. Sci. 82:87. 2 Annison, E. F., J. L. Linzell, S. Fazakerley, and B. W. Nichols. 1967. The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis. Biochem. J. 102:637. 3 Askew, E. W., J. D. Benson, J. W. Thomas, and R. S. Emery. 1971. Metabolism of fatty acids by mammary glands of cows fed normal, restricted roughage, or magnesium oxide supplemented rations. J. Dairy Sci. 54:854. 4 Benson, J. D., and R. S. Emery. 1971. Fatty acid esterification by homogenates of bovine liver and adipose tissue. J. Dairy Sci. 54:1034. 5 Bickerstaffe, R., E. F. Annison, and J. L. LinzeU. 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J. Agr. Sci. 82:71. 6 Dimick, P. S., R. D. McCarthy, and S. Patton. 1966. Paths of palmitic acid incorporation into milk fat trigtycerides. Biochim. Biophys. Acta
87
116:159. 7 Duncan, W. R. H., and G. A. Garton. 1963. Blood lipids. 3. Plasma lipids of the cow during pregnancy and lactation. Biochem. J. 89:414. 8 Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497. 9 Glascock, R. F., and V. A. Welch. 1974. Contribution of the fatty acids of three low density serum lipoproteins to bovine milk fat. J. Dairy Sci. 57:1364. 10 Hartmann, P. E., and A. L. Lascelles. 1965. Variation in the concentration of lipids and some other constituents in the blood plasma of cows at various stages of lactation. Aust. J. Biol. Sci. 18:114. 11 McCarthy, R. D., P. T. Chandler, L. C. Grid, Jr., and G. A. Porter. 1968. Fatty acid composition of blood serum lipoproteins from normal and ketotic cows. J. Dairy Sci. 51:392. 12 Patton, R. A., R. D. McCarthy, and L. C. Griel, Jr. 1970. Lipid synthesis by rumen microorganisms. II. Further characterization of the effects of methionine. J. Dairy Sci. 53:460. 13 Raphael, B. C., P. S. Dimick, and D. L. Puppione. 1973. Lipid characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56:1025. 14 Rindsig, R. B., and L. H. Schuhz. 1974. Effect of amount and frequency of feeding safflower oil on related milk, blood, and rumen components. J. Dairy Sci. 57:1037. 15 Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Co., Inc., NY. 16 Van-nan, P. N., and L. H. Schultz. 1968. Blood lipids of cows at different stages of lactation. J. Dairy Sci. 51:1971. 17 Wallenius, R. W., and R. E. Whitchurch. 1975. Methionine hydroxy analog or sulfate supplementation for high producing dairy cows. J. Dairy Sci. 58:1314. 18 Waterman, R., and L. H. Schuhz. 1972. Nicotinic acid treatment of bovine ketosis II. Effects on long-chain fatty acid compositions of plasma lipid fractions. J. Dairy. Sci. 55:1454. 19 Yamdagni, S., and L. H. Schuhz. 1970. Fatty acid composition of blood plasma lipids of normal and ketotic cows. J. Dairy Sci. 53:1046.
Journal of Dairy Science Vul. 59, No. 1
differences (2, 5) as well as uptake and incorporation of labeled fatty acids into milk fat (2, 6, 9). Plasma from lactating cows probably contains about 8 to 12 mg/dl TG (5, 14) although Raptaael et al. (13) did not detect TG for all groups and Hartmann and Lascelles observed values of 15 to 17 mg/dl (10). Duncan and Garton (7) found that TG accounted for an average of 10% of total fatty acids in plasma in the dry period and about 2% twenty days into lactation. The fatty acid composition of the triglyceride fraction varied markedly between cows on the first and 22nd day of lactation. Studies of lipid and lipoprotein classes have compared (a) dry and lactating cows (10, 13, 16), (b) ketotic or sub-clinically ketotic with normal cows (11, 19), and (c) normal cows with those receiving restricted roughage-high grain (RRHG) rations to stimulate the milk fat depression syndrome (1, 3). There is evidence that methionine or methionine h y d r o x y analog (MHA) could influence lipid metabolism in lactating cows, either in the host tissue or by influencing rumen microbial lipid synthesis (12). As part of a study concerning the effect of MHA and sulfate on milk and milk fat production, we examined triglyceride fatty acids of milk and blood plasma at varying stages of lactation. Production and milk lipids have been reported (17). This paper reports changes in plasma T G F A during the first half of lactation in normal cows.
Total blood plasma lipid and plasma triglyceride fatty acids were analyzed at an average of 33, 89, and 139 days of lactation (ranges 20 to 57, 52 to 129, and 122 to 157 days for periods I, 11, and l i d for 35 Holstein cows in their second or later Lactation. Average milk production in the test periods was 35.7, 30.3, and 25.5 kg/day. Lipid analysis was part of a study comparing methionine hydroxy analog or sulfur for lactating cows with effects of treatment and time separated statistically. Cows fed methionine analog had a lower percent palmitate in the triglyceride fatty acid, but there was no other treatment effect. There were significant changes with time in percent total lipid and triglyceride fatty acid. Total lipid averaged 424, 526, and 529 mg/dl. Average percents of measured triglyceride fatty acid and differences with stage of lactation were: myristic, 2.96; palmitic, 27.52, I>1I or II1; palmitoleic, 2.83, 1>11 or 111; stearic, 38.58, 1
There are many reports concerning the controlling factors in milk fat synthesis, fat test depression, and ketosis, all of which potentially involve dietary, storage, and circulating lipids. Studies of lipids in plasma have demonstrated significant changes in total lipids as well as in specific lipid fractions during normal lactations (7, 10, 13, 16). Plasma triglycerides (TG) are the primary source of long-chain fatty acids from dietary or adipose sources for milk fat synthesis. Evidence for this is based on arterial-venous (A-V)
MATERIALS AND METHODS
Thirty-six Holstein cows were assigned to one of three rations: control, methionine hydroxy analog (MHA), or sulfate supplementation. Cows supplemented with MHA received 25 g/day in the concentrate part of their ration. Cows supplemented with sodium sulfate received 17.5 g/day. Experimental design, methods of management, and effects of treatment on production have been reported (17). Thirty milliliters of blood were collected via jugular vein into heparinized vacutainer tubes during
Received May 9, 1975. 1Scientific paper no. 4438. College of Agriculture Research Center, Washington State University, Pullman. Proj. 0126. 85
86
WALLENIUS AND WHITCHURCH
peak lactation (range 20 to 57 days), and again at an average of 89 (range 52 to 129) and 139 (range 122 to 157) days into lactation. These sampling times are referred to as 1, II, and 1II, respectively. Plasma was frozen at - 2 0 C for subsequent analysis of triglyceride fatty acid and total lipid. Concentrate averaged 48% of the total ration dry m a t t e r during the experim e n t and provided an estimated 60% of digestible energy (DE) in I, 53 to 60% of DE in II, and 53% of DE in III. Blood lipids were extracted by the m e t h o d of Folch et al. (8) as modified by Hartmann and Lascelles (10), except that 5 ml of plasma were extracted rather than 10 ml. All reagents in subsequent steps in the extraction procedure were reduced by half. Total lipids of plasma were determined gravimetrically and stored in 1 ml of c h l o r o f o r m under nitrogen at I C until further analysis. Triglycerides were separated from other plasma lipid fractions by thin layer chromatography. Further preparation and gas c h r o m a t o graphic analysis have been described (17). Effects of ration and stage of lactation on total lipids of plasma and percent of triglyceride fatty acids of plasma were separated by analysis of variance (15).
RESULTS A N D DISCUSSION
Rations had no effect on total lipids of plasma, and the pooled results (Table 1) show a trend similar to that r e p o r t e d by others. Total lipids of plasma were lower (P'(.O1) during 1 than in II and 111. Varman and Schultz (16)
showed plasma total lipids similar during the dry period and 30 days into lactation b u t increased by a b o u t 60%, 260 days into lactation. Most of this difference was associated with the phospholipid and cholesteryl ester fraction. Raphael et al. (13) f o u n d total lipid at 0 to 4 wk p o s t p a r t u m averaged 292 mg/dl and increased to 508 mg/dl in w k 5 to 10 with a slight decrease to 458 mg/dl during weeks 16 to 33 prepartum. Hartmann and Lascelles (10) did not observe as marked a change b e t w e e n dry and lactating cows and f o u n d a slight but n o t significant increase in total lipid in late as compared with early lactation. Duncan and Garton observed a m i n i m u m total lipid at parturition followed by an increase in early lactation (7). Percents of plasma T G F A are in Table 1. Averages are pooled for the three treatments (MHA, Sulfate, and Control). The percent palmitic acid in T G F A of plasma was lower (P<.01) for cows receiving MHA. Percents palmitate for the MHA, sulfate, and control treatments were 26.8, 27.3, and 28.5% of T G F A . The importance of this is n o t k n o w n immediately. Discussions by Dimick et al. (6), Askew et al. (3), and Benson and Emery (4) suggest that the T G F A pool available to the m a m m a r y gland may be significant in efficiency of milk fat synthesis and perhaps MHA plays a role in this control, possibly via microbial lipid synthesis in the rumen (12). Plasma T G F A did vary significantly in s o m e instances as lactation progressed. Effects of ration were removed statistically before comparisons of time. Myristic and linoleic acid did
TABLE 1. Average plasma total lipid and triglyceride long chain fatty acids at 33, 89, and 139 days of lactationa, b.
Lipid Total C 14 C 16 C16:1 C 18 C 18:1 C 18:2
Period 11
I 424 c 3.0 28.8 c 3.9 c 30.8 c 26.4 c 4.4
-+
13.1 .15 .45 .20 1.19 1.09 .27
526 d
-+
3.1 27.2 d 2.3 d 41.3 d 18.7 d 4.6
Ill 19.2 .13 .40 .13 .82 .63 .22
aTotal lipid as mg/dl plasma -+ SE, TGFA as weight % of those determined. bDifferent superscripts within rows are significantly different. Journal of Dairy Science VoI. 59, No. 1
539 d 2.8 26.5 d 2.2 d 43.9 e 16.9 d 4.9
+-
18.9 .13 .34 .15 .55 .39 .30
BLOOD PLASMA FATTY ACIDS n o t change. Palmitic, palmitoleic, and oleic acids decreased in II and III as c o m p a r e d with I ( P < . 0 1 ) . Stearic acid increased in II ( P < . 0 1 ) and again in III ( P < . 0 5 ) . Higher p e r c e n t a g e s of palmitic a n d oleic acids can be associated with an increased m o b i l i z a t i o n o f adipose tissue f a t t y acids (18), w h i c h is e x p e c t e d for high p r o d u c i n g cows in early lactation. The changes f r o m p e a k l a c t a t i o n (I) t o II and III also agree w i t h changes observed by Y a m d a g n i and Schdltz (19) and W a t e r m a n and Schultz (18) for normal, subclinically ketotic, and ketotic cows. This suggests t h a t t h e degree of mobilization o f adipose tissue fatty acids and lipid m e t a b o l i s m in general, may be similar for n o r m a l high p r o d u c i n g c o w s and c o w s t h a t are clinically or subclinically ketotic. REFERENCES
1 Annison, E. F., R. Bickerstaffe, and J. L. Linzell. 1974. Glucose and fatty acid metabolism in cows producing milk of low fat content. J. Agr. Sci. 82:87. 2 Annison, E. F., J. L. Linzell, S. Fazakerley, and B. W. Nichols. 1967. The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis. Biochem. J. 102:637. 3 Askew, E. W., J. D. Benson, J. W. Thomas, and R. S. Emery. 1971. Metabolism of fatty acids by mammary glands of cows fed normal, restricted roughage, or magnesium oxide supplemented rations. J. Dairy Sci. 54:854. 4 Benson, J. D., and R. S. Emery. 1971. Fatty acid esterification by homogenates of bovine liver and adipose tissue. J. Dairy Sci. 54:1034. 5 Bickerstaffe, R., E. F. Annison, and J. L. LinzeU. 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J. Agr. Sci. 82:71. 6 Dimick, P. S., R. D. McCarthy, and S. Patton. 1966. Paths of palmitic acid incorporation into milk fat trigtycerides. Biochim. Biophys. Acta
87
116:159. 7 Duncan, W. R. H., and G. A. Garton. 1963. Blood lipids. 3. Plasma lipids of the cow during pregnancy and lactation. Biochem. J. 89:414. 8 Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497. 9 Glascock, R. F., and V. A. Welch. 1974. Contribution of the fatty acids of three low density serum lipoproteins to bovine milk fat. J. Dairy Sci. 57:1364. 10 Hartmann, P. E., and A. L. Lascelles. 1965. Variation in the concentration of lipids and some other constituents in the blood plasma of cows at various stages of lactation. Aust. J. Biol. Sci. 18:114. 11 McCarthy, R. D., P. T. Chandler, L. C. Grid, Jr., and G. A. Porter. 1968. Fatty acid composition of blood serum lipoproteins from normal and ketotic cows. J. Dairy Sci. 51:392. 12 Patton, R. A., R. D. McCarthy, and L. C. Griel, Jr. 1970. Lipid synthesis by rumen microorganisms. II. Further characterization of the effects of methionine. J. Dairy Sci. 53:460. 13 Raphael, B. C., P. S. Dimick, and D. L. Puppione. 1973. Lipid characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56:1025. 14 Rindsig, R. B., and L. H. Schuhz. 1974. Effect of amount and frequency of feeding safflower oil on related milk, blood, and rumen components. J. Dairy Sci. 57:1037. 15 Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Co., Inc., NY. 16 Van-nan, P. N., and L. H. Schultz. 1968. Blood lipids of cows at different stages of lactation. J. Dairy Sci. 51:1971. 17 Wallenius, R. W., and R. E. Whitchurch. 1975. Methionine hydroxy analog or sulfate supplementation for high producing dairy cows. J. Dairy Sci. 58:1314. 18 Waterman, R., and L. H. Schuhz. 1972. Nicotinic acid treatment of bovine ketosis II. Effects on long-chain fatty acid compositions of plasma lipid fractions. J. Dairy. Sci. 55:1454. 19 Yamdagni, S., and L. H. Schuhz. 1970. Fatty acid composition of blood plasma lipids of normal and ketotic cows. J. Dairy Sci. 53:1046.
Journal of Dairy Science Vul. 59, No. 1