Glucose Metabolism in Cows Fed L o w - and High-Roughage Diets ESSI EVANS, J. G. B U C H A N A N - S M I T H , G. K. MacLEOD, and J. B. STONE Department of Animal and Poultry Science University of Guelph Guelph, Ontario, Canada N1G 2W1
INTRODUCTION
ABSTRACT
Increasing the proportion of grain or decreasing the proportion of roughage in a lactating cow's diet is a primary cause of depressed milk fat secretion. Reasons are usually related to the fact that decreased roughage intake affects carbohydrate digestion which increases availability of glucogenic precursors and reduces concentrations of circulating precursors required for milk fat synthesis. Although low-roughage diets increase plasma glucose and insulin in lactating cows, until recently there has been no evidence to show that low-roughage diets also increase glucose utilization (or entry) rates in lactating cows or ruminants in general. Evans and BuchananSmith (9) have reported that low-roughage diets increased plasma concentration, pool size, and utilization rate of glucose, and depressed halftime in sheep. Bickerstaffe et al. (4) have reported that low-roughage diets increased glucose utilization rates in lactating cows. Decreasing the roughage content of a ruminant's diet is usually associated with increased digestible energy (DE) concentration, which is correlated positively with rates of glucose utilization in ruminants (16). Therefore, the effect of a low-roughage diet on glucose metabolism in ruminants may be attributed simply to i n - . creasing intake of digestible energy alone, rather than any effect on carbohydrate digestion. The purpose of our experiment was to compare the effects of isocaloric intakes (DE basis) of a low- and high-roughage diet on glucose metabolism in lactating cows. Glucose metabolism was evaluated using both [2-3H] glucose as a tracer and with a clearance test.
Six lactating first-calf Holstein cows were used to test the effect of dietary roughage on glucose metabolism. Cows were fed either a low-roughage or highroughage diet at isocaloric digestible energy intakes in a double changeover design experiment. Mean values (+ standard deviation) for milk yield (kg/day), fat (%), lactose (%), and protein (%) for cows fed low-roughage were 19.0 + 4.4, 3.11 + .78, 5.19 + .27, 3 . 4 4 -+ .48;values for cows fed high-roughage were 17.5 + 5.1, 3.99 + .58, 4.94 + .25, and 2.78 + .33. One hour post-feeding on the 20th day of each period 2 mCi of tritiated glucose were administered to each cow by single injection to measure glucose kinetics. Mean values (+ standard deviation) for plasma concentration (mg/lO0 ml) pool size (mg/kg), half-time (min), and utilization rate (mg/kg -75 per min) of glucose, and plasma insulin concentration (~tU/ml) for cows fed low-roughage were 63.1 + 3.9, 17.9 + 3.4, 30.4-+ 5.2, 8.55 -+ 2.44, and 22.0 + 3.9; for cows fed high-roughage values were 54.9 -+ 2.2, 114.5 + 17.2, 40.0 + 2.2, 4.06 + .38, and 16.2 -+ 2.4. A glucose load was administered intravenously to each cow on the last day of each period. Glucose halftimes and mean plasma insulin following the clearance test were not affected by diet. Compared to high-roughage, lowroughage diets greatly affect metabolism in lactating cows when isocaloric intakes of each are fed. Fat depression, however, may or may not occur simultaneously.
EXPERIMENTAL PROCEDURES Animals and Diets
Six lactating first-calf Holstein cows between 7 and 14 wk postpartum were used in an
Received October 8, 1974. 672
DIET AND GLUCOSE METABOLISM IN COWS experiment with changeover design in which two dietary treatments were imposed alternately on each cow for three test periods. Cows were paired by calving dates, and one cow of each pair was assigned randomly to one of the two dietary treatments for the first period. Treatments consisted of feeding a low-roughage (LR) or high-roughage (HR) diet for 18 days of each 21 day period (Table 1). An intermediate diet (Table 1) was fed for 1 wk before the experiment and for the first 3 days of each period to avoid metabolic disturbances from abrupt ration changes. Diets were fed at isocaloric digestible energy (DE) intakes estimated to meet the requirements for body weight maintenance, an additional 20% for growth, and for milk production (22). Diets containing 18.2% crude protein were formulated following nitrogen analysis of feedstuffs (1). Calcium phosphate was supplied to meet the requirements for calcium and phosphorus, and cobalt-iodized salt and water were available free choice. Cows were confined to stanchion stalls in a room where temperature was constant (21 to 23 C). Rations were equally divided into two meals and offered at milking times each day (0830 h and 1830 h). Method of Sampling
Glucose kinetics were studied with a single injection of [2 -3 H] glucose. On the 19th day of each period, cows were provided with 16 gauge 8 cm catheter (Angiocath, Deseret Pharmaceutical Co., Sandy, UT) inserted into a jugular vein. On the following morning 10 ml blood TABLE 1. Compositions of low-(LR), high-(HR), and intermediate-roughage (IR) diets. Diets were formulated to contain 18.2% crude protein.
Feedstuff
Estimated DE
LRa
Cracked corn Legume hay Soybean meal
(kcal/g) 3.85 2.51 3.21
(% of ration as fed) 60.0 20.0 40.0 20.0 67.0 43.5 20.0 13.0 16.5
HR a IRb
aTest rations. bFed between periods as a precaution to keep animals from going off-feed.
673
samples were withdrawn 30 min pre- and post-feeding. At 1 h post feeding, 1 c mCi [2 -3 HI glucose (New England Nuclear, Boston, MA) in 10 ml .9% saline solution were injected into the catheter and rinsed with additional saline. Subsequent blood samples were removed every 20 min until 180 rain post-injection of isotope, again at 220 min and then for three 60 min intervals post-injection of isotope. On the last day of each period glucose clearance was tested. One blood sample was withdrawn 30 rain after AM feeding. One to 1.5 h post-feeding, a saturated glucose solution equivalent to .5 g glucose/kg body weight was infused through the catheter. After infusion, blood samples were taken every 10 min for 120 min. All blood samples taken throughout the experiment were immediately placed in tubes containing heparin. Milk weights were recorded, and samples were retained from both milkings on the 18th and 19th days of each period. These were composited for analysis. Analytical Techniques
Milk samples were analyzed for fat, protein, and lactose content by the infrared milk analyzer method (5). Blood samples were centrifuged at 5000 × g for 20 min within 6 h after removal. The plasma portion was extracted and placed in plastic tubes. All plasma except that used to determine plasma glucose concentration and glucose specific radioactivity (SR) was immediately quick frozen in a methanol bath ( - 7 0 C) and stored at - 1 8 C. Concentration of plasma glucose was determined from all samples by the glucose oxidase method with commercially prepared kits (Sigma Chemical Corp., St. Louis, MO). Mean concentrations of plasma glucose were calculated from nine samples taken approximately 1 h apart beginning 30 min before feeding. Values from blood samples taken up to 180 min after isotope injection were used to measure glucose kinetics. Determination of the SR of plasma glucose was accomplished by synthesizing glucose pentaacetate (15) as discussed (9). Pool sizes and utilization rates of glucose were calculated from monoexponential regression analysis of the log SR glucose vs. time dilution curves (17, 27). Biological half times of glucose were calculated as described by Ginochio and Journal of Dairy Science Vol. 5B, No. 5
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EVANS ET AL.
TABLE 2. Milk yield and composition from cows fed either low-(LR) or high roughage (HR) diets (Means +- SD). Diet
obs
LR HR Significance (P < .05)*
9 9
Yield (kg/day) 19.0 _+4.4 17.5 + 5.1 NS
Fat
Protein
Lactose
3.11 -+ ,78 3 . 9 9 +- .58
(%) 3.44 -+ .48 2.78 + .33
5.19 -+ .27 4.94 -+ .25
NS
S
NS
*Significance ; NS, non-significant.
Evans (12). Half times of glucose for clearance tests were calculated f r o m m o n o e x p o n e n t i a l regression analysis of log plasma glucose vs. time after glucose administration. Plasma glucose values that exceeded the value determined prior to glucose infusion were used in the clearance test regression. Plasma insulin c o n c e n t r a t i o n s were d e t e r m i n e d by the single a n t i b o d y back titration m e t h o d of Wright et al. (28). Mean differences b e t w e e n dietary treatments were c o m p a r e d by Students t to establish significance (6).
RESULTS AND DISCUSSION Milk Yield and Composition
Results for milk yield and c o m p o s i t i o n are Table 2. Milk yield was u n a f f e c t e d by diet (P > .05). Milk yield may increase when the proportion of grain in the diet is increased (7). However, increases in milk yield may reflect increased DE intake f r o m feed with greater DE c o n c e n t r a t i o n (3). In the present e x p e r i m e n t
b o t h diets were fed at isocaloric intakes (DE basis) which might explain similar milk yields f r o m either diet. High-concentrate diets cause a reduction in the percentage of milk fat. The lack of a significant milk fat r e d u c t i o n w h e n the LR diet was fed w o u l d have resulted f r o m an insufficient period o f adaptation. This is improbable because concentrations of r u m e n and blood constituents that serve as milk precursors equilibrate within 10 days after a diet change (23, 25). In our experiment, cows were adapted gradually to changes in diet, and the test diets were fed for 15 days before milk sampling. Therefore, the failure to p r o d u c e a significant milk fat reduction could not be due to insufficient conditioning. Large variations in milk fat secretion b e t w e e n cows fed high grain rations have occurred (20). Data in the left c o l u m n of Table 3 express the effect of L R diet on the fat test in individual cows. One cow p r o d u c e d a consistently high milk fat test (4.5%) w i t h o u t any fat depression whereas fat was depressed m o r e t h a n 2.0% w h e n a n o t h e r cow (M) was fed the LR
TABLE 3. Individual cow data showing mean change in characteristics with low-roughage feeding.
Cow
Milk fat a
M D A P B X
-2.16 -1.26 -.43 -.50 -.48 +.11
(%)
Glucose utilization rate (mg/kg.75/min) +6.37 +4.94 +4.76 +3.01 +3.25 +4.64
Glucose pool size
Glucose half-time
(mg/kg)
(min)
+58.1 +98.5 +70.3 +52.6 +41.9 +41.3
-17.9 -4.3 -13.5 -7.9 -3.4 -14.0
a(+) symbolizes increase and (--) symbolizes decrease when compared to values from the high roughage diet. Journal of Dairy Science Vol. 58, No. 5
DIET AND GLUCOSE METABOLISM IN COWS TABLE 4. Correlation coefficients between time after a single injection of 2-3H glucose and specific radioactivity of plasma glucose in cows fed low-(LR) and high-roughage (HR) diets. Period Cow
1
2
3
M D A
LR .996 .986 .995
HR .998 .996 .998
LR .982 .994 .994
P B X
HR .995 .996 .985
LR .994 .999 .998
HR .976 .989 .998
diet. Milk fat test was depressed in each of the o t h e r four cows by the LR diet. High grain feeding raises the protein c o n t e n t of milk (13, 24). Milk protein c o n t e n t was greater (P < .05) for cows given the L R diet (Table 3).
Glucose Kinetics, Clearance, and Plasma Insulin
Tritium recycling is minimal with glucose labeled in the 2 position (17, 18), making it a suitable tracer for studying glucose m e t a b o l i s m in ruminants. Erroneous estimates o f glucose metabolism could be p r o d u c e d by fluctuations in the pool sizes or half-times of glucose during the sampling period. These fluctuations w o u l d
675
be evident as deviations in the linearity of log rate of loss of labeled glucose with time, producing multi- rather than m o n o e x p o n e n t i a l rate functions and high coefficients of variation a b o u t regression. Correlation coefficients of the m o n o e x p o n e n t i a l c o m p o n e n t s were above .97 for all m e a s u r e m e n t s (Table 4), and the m e a n coefficient of variation a b o u t all regressions was 1.16%. This indicates that glucose utilization rates were stable for the three hours in which they were measured. Table 5 shows kinetic data and mean plasma glucose and insulin for the two diets. All measures were different (P < .05) b e t w e e n the t w o diets. Elevations in plasma glucose and insulin have occurred with high grain feeding in mid- but not early lactation (14). The lack of effect of high-grain feeding on these measures in early stages of lactation was explained by the fact that the cows were f r e q u e n t l y in negative energy balance (14). Our results represent cases where sufficient intake was provided according to requirements. Because the cows were not e x c e p t i o n a l l y high producers, t h e y were m o s t likely never in negative energy balance. This might explain why plasma glucose and insulin when the L R diet was fed may explain the increased milk protein o u t p u t because insulin stimulates protein synthesis and inhibits gluconeogenesis f r o m an±too acids (9). Increased p o o l sizes (P < .05) in cows fed LR c o m p a r e d to the H R diet are attributed to elevations in c o n c e n t r a t i o n of plasma glucose. For the LR diet, associated increases in insulin c o n c e n t r a t i o n s are p r o b a b l y responsible for the
TABLE 5. Effects of feeding a low-(LR) or high-roughage (HR) diet on mean plasma glucose and insulin concentration and kinetic measures of glucose in lactating cows (Means ± SD).
Diet
obs
LR HR Significance (P < .05) c
9 9
Plasma glucose a
Plasma insulin a
Pool size
Half time
Utilization rate b
(mg/lO0 ml)
(uU/ml)
(mg/kg B.W.)
(min)
(mg/kg.75/min)
63.1 + 3.9 54.9 ± 2.1
22.0 ± 3.9 16.2 ± 2.4
179.1 ± 34.3 114.5 + 17.2
30.4 -+ 5.2 40.0 ± 2.2
8.55 -+ 2.44 4.06 ± 0.38
S
S
S
S
S
acalculated from nine samples taken at hourly intervals initiating 30 min before feeding for each individual observation. bCalculated over 3 h after injection of [2-3H] glucose. Cs, significant. Journal of Dairy Science Vol. 58, No. 5
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EVANS ET AL.
decreased half-times. Glucose utilization rates for cows fed the LR diet were increased, therefore, by both a larger pool size and a decreased half-time of glucose. The fact that glucose utilization rates were increased by alterations in both of its component parts indicates that metabolism of glucose is affected substantially by high-grain intake. (Table 5). Effects of the LR diet upon glucose metabolism in individual cows, which are in Table 3, show that increased plasma concentration, utilization rate, and pool size of glucose and depressed half-time occurred in every cow. Bickerstaffe et al. (4) have reported that glucose utilization rates in lactating cows were increased by feeding a low- compared to a high-roughage diet. In the experiment of Bickerstaffe et al. (4), the low- and high-roughage diet were both fed several weeks apart once to each cow. Thus, the milk production and intake of available energy from both diets differed within cows. In our experiment, milk production and intake of digestible energy from both diets were similar within cows. Therefore, the effect of the LR diet on glucose metabolism in our experiment may be attributed to altered digestion of carbohydrate rather than a greater intake of available energy. There was no effect of diet upon clearance half time (P > .05) or mean concentrations of plasma insulin (P > .05) in the cows after a glucose load (Table 6). Mean glucose half times were approximately the same (P > .05) with either labeled glucose or a clearance test when
cows were fed the LR diet (30.4 and 30.2 min respectively), but they were significantly (P < .05) different when the HR diet was fed (40.0 and 33.1 min). This strongly suggests that when the cows were given the LR diet, metabolic pathways for glucose removal from the peripheral circulation were operating at a maximum rate whereas with the HR diet the rate of glucose removal was less than maximal. It is possible that the greater glucose availability in cows fed the LR diet was sufficient conditioning along to elicit the changes in glucose metabolism which were observed, and sustained dietary conditioning was not required to induce a response. High-grain feeding of lactating cattle does affect their glucose metabolism substantially. Although milk fat production was depressed by the LR diet in some cows, it was not depressed in others. A similar conclusion can be made from data by Bickerstaffe et al. (4). Glucose metabolism may be an important focus for studies to explain milk fat depression in cows fed high-grain diets. However, there must be other considerations such as glucose availabilities to specific tissues or interactions with other areas of intermediary metabolism.
ACKNOWLEDGMENT The authors are indebted to the Kraftco Corp., Ontario Ministry of Agriculture and Food, and the National Research Council of Canada for funds for this project. Technical assistance was provided by Y. T. Yao.
TABLE 6. Comparisons of differences between half times and mean plasma insulin concentrations determined during glucose clearance test and isotope dilution studies in lactating cows given Iow-(LR) and high-roughage (HR) diets (± SD).
Diet
LR HR Significance (P < .05)d
Clearance half timea
Isotope dilution half timeb
Clearance insulina
Isotope dilution insulina
30.2 -+6.2 33.1-+ 3.3
(min) 30.4 ± 5.2 40.0± 2.2
49.6 +- 10.6 45.5-+ 7.6
~ (uU/ml) 22.0 -+ 3.9 16.2± 2.4
NS
S
NS
aComputed from samples taken after the infusion of .5g glucose/kg body weight. bcomputed after the injection of [2-3H] glucose. Ccomputed from daily average of 9 samples taken at hourly intervals, beginning 30 min before feeding. ds, significant; NS, non-significant. Journal of Dairy Science Vol. 58, No. 5
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DIET AND GLUCOSE METABOLISM IN COWS REFERENCES
1. A.O.A.C. 1970. Official m e t h o d of analysis (11th Ed.) Ass. of Offic. Agr. Chem. Washington, DC. 2. 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. 3. Baumgardt, B. R. 1970. Regulation of feed intake and energy balance. Page 235 in A. T. Phillipson, ed. Physiology of digestion and metabolism in the ruminant. Oriel Press, Newcastle u p o n Tyne, England. 4. Bickerstaffe, R., E. F. Annison, and J. L. Linzell. 1974. The metabolism of glucose, acetate, lipids and amino acids in the lactating dairy cow. J. Agr. Sci. 82:71. 5. Biggs, D. A. 1967. Milk analysis with the infrared milk analyzer. J. Dairy Sci. 50:799. 6. Brandt, A. E. 1938. The extension of S t u d e n t ' s T test to reversal or switchback trials involving 2 or more test periods. Iowa Agr. Exp. Sta. Res. Bull. 234:1938. 7. Brown, L. D., J. W. Thomas, R. S. Emery, L. D.. McGilliard, D. V. Armstrong, and C. A. Lassiter. 1964. Effects of high level grain feeding on milk production response of lactating dairy cows. J. Dairy Sci. 4:1184. 8. Church, D. C. 1970. Digestive physiology and nutrition of ruminants. Vol 1. Oregon State Press. 9. Evans, E, and J. G. Buchanan-Smith. 1975. Effects u p o n glucose metabolism of feeding a lowor high-roughage diet at two levels of intake to sheep. Brit. J. Nutr. 33 : 33. 10. Felig, P., and J. Wahren. 1974. Protein turnover and amino acid metabolism in the regulation of gluconeogenesis. Fed. Proc. 33 : 1092. 11. Fisher, L. J., and J. M. Elliot. 1966. Effect of intravenous infusion of propionate or glucose on bovine milk composition. J. Dairy Sci. 49:826. 12. Ginochio, R. J., and J. W. Evans. 1973. Glucose 2-T turnover in Shetland ponies. J. Anim. Sci. 37:484. 13. Haenlein, G. F. W., L. If. Schultz, and L. R. Hansen. 1968. Relation of milk fat-depressing rations and subclinical mastitis to milk proteins. J. Dairy Sci. 51:535. 14. Jenny, B. F., C. E. Polan, and F. W. Thye. 1974. Effect of high grain feeding and stage of lactation on serum insulin, glucose, and milk fat percentage in lactating cows. J. Nutr. 104:379.
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15. Jones, G. B., 1965. Determination of the specific activity of labelled glucose by liquid scintillation using glucose pentaacetate. Anal. Biochem. 12:249. 16. Judson, G. J., and R. A. Leng. 1968. Effect of diet on glucose synthesis in sheep. Proc. Aust. Soc. Anita. Prod. 7:354. 17. Judson, G. J., and R. A. Leng. 1972. Estimation of the total entry rate and resynthesis of glucose in sheep u n i f o r m l y labelled with 14 C and variously labelled with 3 H. Aust. J. Biol. Sci. 25:1313. 18. Katz, J., and A. Dunn. 1967. Glucose 2-T as a tracer for glucose metabolism. Biochemistry 6:1. 19. Kleiber, M. 1954. Precursor-product relationship for milk formation in the intact dairy cow. Rev. Can. Biol. 13:333. 20. Lathan, M. J., J. D. Sutton, and M. E. Sharpe. 1974. Fermentation and microorganisms in the r u m e n and the c o n t e n t of fat in the milk of cows given low roughage rations. J. Dairy Sci. 57:803. 21. McClymont, G. L., and S. Vallance. 1962. Depression of blood glycerides and milk fat synthesis by glucose infusion. Proc. Nutr. Soc. 21 :xii. 22. N. R. C. 1971. Nutrient requirements of domestic animals No. 3. Nutrient requirements of dairy cattle. National Research Council, Washington, DC. 23. Satter, L. D., and A. N. Bringe. 1969. Effect of abrupt ration changes on milk and blood components. J. Dairy Sci. 52:1776. 24. Schingoethe, D. J., P. E. Stake, and M. J. Owens. 1973. Whey c o m p o n e n t s in restricted-roughage rations, milk composition, and r u m e n volatile fatty acids. J. Dairy Sci. 56:909. 25. Storry, J. E., and J. D. Sutton. 1969. The effect of change from a low-roughage to high-roughage diet on r u m e n fermentation, blood composition and milk fat secretion in the cow. Brit. J. Nutr. 23:511. 26. Walker, C. K., and J. M. Elliot. 1973. Effect of roughage restriction on serum insulin in the dairy cow. J. Dairy Sci. 56:375. 27. White, R. G., J. w. Steel, R. A. Leng, and J. R. Luick. 1969. Evaluation of three isotope dilution techniques for studying the kinetics of glucose metabolism in sheep. Biochem J. 114:203. 28. Wright, P. H., D. R. Makulu, D. Vichick, and K. E. Sussman. 1971. Insulin i m m u n o a s s a y by back titration: Some characteristics of the technic and the insulin precipitant action of alcohol. Diabetes 20:33.
Journal of Dairy Science Vol. 58, No. 5
INTRODUCTION
ABSTRACT
Increasing the proportion of grain or decreasing the proportion of roughage in a lactating cow's diet is a primary cause of depressed milk fat secretion. Reasons are usually related to the fact that decreased roughage intake affects carbohydrate digestion which increases availability of glucogenic precursors and reduces concentrations of circulating precursors required for milk fat synthesis. Although low-roughage diets increase plasma glucose and insulin in lactating cows, until recently there has been no evidence to show that low-roughage diets also increase glucose utilization (or entry) rates in lactating cows or ruminants in general. Evans and BuchananSmith (9) have reported that low-roughage diets increased plasma concentration, pool size, and utilization rate of glucose, and depressed halftime in sheep. Bickerstaffe et al. (4) have reported that low-roughage diets increased glucose utilization rates in lactating cows. Decreasing the roughage content of a ruminant's diet is usually associated with increased digestible energy (DE) concentration, which is correlated positively with rates of glucose utilization in ruminants (16). Therefore, the effect of a low-roughage diet on glucose metabolism in ruminants may be attributed simply to i n - . creasing intake of digestible energy alone, rather than any effect on carbohydrate digestion. The purpose of our experiment was to compare the effects of isocaloric intakes (DE basis) of a low- and high-roughage diet on glucose metabolism in lactating cows. Glucose metabolism was evaluated using both [2-3H] glucose as a tracer and with a clearance test.
Six lactating first-calf Holstein cows were used to test the effect of dietary roughage on glucose metabolism. Cows were fed either a low-roughage or highroughage diet at isocaloric digestible energy intakes in a double changeover design experiment. Mean values (+ standard deviation) for milk yield (kg/day), fat (%), lactose (%), and protein (%) for cows fed low-roughage were 19.0 + 4.4, 3.11 + .78, 5.19 + .27, 3 . 4 4 -+ .48;values for cows fed high-roughage were 17.5 + 5.1, 3.99 + .58, 4.94 + .25, and 2.78 + .33. One hour post-feeding on the 20th day of each period 2 mCi of tritiated glucose were administered to each cow by single injection to measure glucose kinetics. Mean values (+ standard deviation) for plasma concentration (mg/lO0 ml) pool size (mg/kg), half-time (min), and utilization rate (mg/kg -75 per min) of glucose, and plasma insulin concentration (~tU/ml) for cows fed low-roughage were 63.1 + 3.9, 17.9 + 3.4, 30.4-+ 5.2, 8.55 -+ 2.44, and 22.0 + 3.9; for cows fed high-roughage values were 54.9 -+ 2.2, 114.5 + 17.2, 40.0 + 2.2, 4.06 + .38, and 16.2 -+ 2.4. A glucose load was administered intravenously to each cow on the last day of each period. Glucose halftimes and mean plasma insulin following the clearance test were not affected by diet. Compared to high-roughage, lowroughage diets greatly affect metabolism in lactating cows when isocaloric intakes of each are fed. Fat depression, however, may or may not occur simultaneously.
EXPERIMENTAL PROCEDURES Animals and Diets
Six lactating first-calf Holstein cows between 7 and 14 wk postpartum were used in an
Received October 8, 1974. 672
DIET AND GLUCOSE METABOLISM IN COWS experiment with changeover design in which two dietary treatments were imposed alternately on each cow for three test periods. Cows were paired by calving dates, and one cow of each pair was assigned randomly to one of the two dietary treatments for the first period. Treatments consisted of feeding a low-roughage (LR) or high-roughage (HR) diet for 18 days of each 21 day period (Table 1). An intermediate diet (Table 1) was fed for 1 wk before the experiment and for the first 3 days of each period to avoid metabolic disturbances from abrupt ration changes. Diets were fed at isocaloric digestible energy (DE) intakes estimated to meet the requirements for body weight maintenance, an additional 20% for growth, and for milk production (22). Diets containing 18.2% crude protein were formulated following nitrogen analysis of feedstuffs (1). Calcium phosphate was supplied to meet the requirements for calcium and phosphorus, and cobalt-iodized salt and water were available free choice. Cows were confined to stanchion stalls in a room where temperature was constant (21 to 23 C). Rations were equally divided into two meals and offered at milking times each day (0830 h and 1830 h). Method of Sampling
Glucose kinetics were studied with a single injection of [2 -3 H] glucose. On the 19th day of each period, cows were provided with 16 gauge 8 cm catheter (Angiocath, Deseret Pharmaceutical Co., Sandy, UT) inserted into a jugular vein. On the following morning 10 ml blood TABLE 1. Compositions of low-(LR), high-(HR), and intermediate-roughage (IR) diets. Diets were formulated to contain 18.2% crude protein.
Feedstuff
Estimated DE
LRa
Cracked corn Legume hay Soybean meal
(kcal/g) 3.85 2.51 3.21
(% of ration as fed) 60.0 20.0 40.0 20.0 67.0 43.5 20.0 13.0 16.5
HR a IRb
aTest rations. bFed between periods as a precaution to keep animals from going off-feed.
673
samples were withdrawn 30 min pre- and post-feeding. At 1 h post feeding, 1 c mCi [2 -3 HI glucose (New England Nuclear, Boston, MA) in 10 ml .9% saline solution were injected into the catheter and rinsed with additional saline. Subsequent blood samples were removed every 20 min until 180 rain post-injection of isotope, again at 220 min and then for three 60 min intervals post-injection of isotope. On the last day of each period glucose clearance was tested. One blood sample was withdrawn 30 rain after AM feeding. One to 1.5 h post-feeding, a saturated glucose solution equivalent to .5 g glucose/kg body weight was infused through the catheter. After infusion, blood samples were taken every 10 min for 120 min. All blood samples taken throughout the experiment were immediately placed in tubes containing heparin. Milk weights were recorded, and samples were retained from both milkings on the 18th and 19th days of each period. These were composited for analysis. Analytical Techniques
Milk samples were analyzed for fat, protein, and lactose content by the infrared milk analyzer method (5). Blood samples were centrifuged at 5000 × g for 20 min within 6 h after removal. The plasma portion was extracted and placed in plastic tubes. All plasma except that used to determine plasma glucose concentration and glucose specific radioactivity (SR) was immediately quick frozen in a methanol bath ( - 7 0 C) and stored at - 1 8 C. Concentration of plasma glucose was determined from all samples by the glucose oxidase method with commercially prepared kits (Sigma Chemical Corp., St. Louis, MO). Mean concentrations of plasma glucose were calculated from nine samples taken approximately 1 h apart beginning 30 min before feeding. Values from blood samples taken up to 180 min after isotope injection were used to measure glucose kinetics. Determination of the SR of plasma glucose was accomplished by synthesizing glucose pentaacetate (15) as discussed (9). Pool sizes and utilization rates of glucose were calculated from monoexponential regression analysis of the log SR glucose vs. time dilution curves (17, 27). Biological half times of glucose were calculated as described by Ginochio and Journal of Dairy Science Vol. 5B, No. 5
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EVANS ET AL.
TABLE 2. Milk yield and composition from cows fed either low-(LR) or high roughage (HR) diets (Means +- SD). Diet
obs
LR HR Significance (P < .05)*
9 9
Yield (kg/day) 19.0 _+4.4 17.5 + 5.1 NS
Fat
Protein
Lactose
3.11 -+ ,78 3 . 9 9 +- .58
(%) 3.44 -+ .48 2.78 + .33
5.19 -+ .27 4.94 -+ .25
NS
S
NS
*Significance ; NS, non-significant.
Evans (12). Half times of glucose for clearance tests were calculated f r o m m o n o e x p o n e n t i a l regression analysis of log plasma glucose vs. time after glucose administration. Plasma glucose values that exceeded the value determined prior to glucose infusion were used in the clearance test regression. Plasma insulin c o n c e n t r a t i o n s were d e t e r m i n e d by the single a n t i b o d y back titration m e t h o d of Wright et al. (28). Mean differences b e t w e e n dietary treatments were c o m p a r e d by Students t to establish significance (6).
RESULTS AND DISCUSSION Milk Yield and Composition
Results for milk yield and c o m p o s i t i o n are Table 2. Milk yield was u n a f f e c t e d by diet (P > .05). Milk yield may increase when the proportion of grain in the diet is increased (7). However, increases in milk yield may reflect increased DE intake f r o m feed with greater DE c o n c e n t r a t i o n (3). In the present e x p e r i m e n t
b o t h diets were fed at isocaloric intakes (DE basis) which might explain similar milk yields f r o m either diet. High-concentrate diets cause a reduction in the percentage of milk fat. The lack of a significant milk fat r e d u c t i o n w h e n the LR diet was fed w o u l d have resulted f r o m an insufficient period o f adaptation. This is improbable because concentrations of r u m e n and blood constituents that serve as milk precursors equilibrate within 10 days after a diet change (23, 25). In our experiment, cows were adapted gradually to changes in diet, and the test diets were fed for 15 days before milk sampling. Therefore, the failure to p r o d u c e a significant milk fat reduction could not be due to insufficient conditioning. Large variations in milk fat secretion b e t w e e n cows fed high grain rations have occurred (20). Data in the left c o l u m n of Table 3 express the effect of L R diet on the fat test in individual cows. One cow p r o d u c e d a consistently high milk fat test (4.5%) w i t h o u t any fat depression whereas fat was depressed m o r e t h a n 2.0% w h e n a n o t h e r cow (M) was fed the LR
TABLE 3. Individual cow data showing mean change in characteristics with low-roughage feeding.
Cow
Milk fat a
M D A P B X
-2.16 -1.26 -.43 -.50 -.48 +.11
(%)
Glucose utilization rate (mg/kg.75/min) +6.37 +4.94 +4.76 +3.01 +3.25 +4.64
Glucose pool size
Glucose half-time
(mg/kg)
(min)
+58.1 +98.5 +70.3 +52.6 +41.9 +41.3
-17.9 -4.3 -13.5 -7.9 -3.4 -14.0
a(+) symbolizes increase and (--) symbolizes decrease when compared to values from the high roughage diet. Journal of Dairy Science Vol. 58, No. 5
DIET AND GLUCOSE METABOLISM IN COWS TABLE 4. Correlation coefficients between time after a single injection of 2-3H glucose and specific radioactivity of plasma glucose in cows fed low-(LR) and high-roughage (HR) diets. Period Cow
1
2
3
M D A
LR .996 .986 .995
HR .998 .996 .998
LR .982 .994 .994
P B X
HR .995 .996 .985
LR .994 .999 .998
HR .976 .989 .998
diet. Milk fat test was depressed in each of the o t h e r four cows by the LR diet. High grain feeding raises the protein c o n t e n t of milk (13, 24). Milk protein c o n t e n t was greater (P < .05) for cows given the L R diet (Table 3).
Glucose Kinetics, Clearance, and Plasma Insulin
Tritium recycling is minimal with glucose labeled in the 2 position (17, 18), making it a suitable tracer for studying glucose m e t a b o l i s m in ruminants. Erroneous estimates o f glucose metabolism could be p r o d u c e d by fluctuations in the pool sizes or half-times of glucose during the sampling period. These fluctuations w o u l d
675
be evident as deviations in the linearity of log rate of loss of labeled glucose with time, producing multi- rather than m o n o e x p o n e n t i a l rate functions and high coefficients of variation a b o u t regression. Correlation coefficients of the m o n o e x p o n e n t i a l c o m p o n e n t s were above .97 for all m e a s u r e m e n t s (Table 4), and the m e a n coefficient of variation a b o u t all regressions was 1.16%. This indicates that glucose utilization rates were stable for the three hours in which they were measured. Table 5 shows kinetic data and mean plasma glucose and insulin for the two diets. All measures were different (P < .05) b e t w e e n the t w o diets. Elevations in plasma glucose and insulin have occurred with high grain feeding in mid- but not early lactation (14). The lack of effect of high-grain feeding on these measures in early stages of lactation was explained by the fact that the cows were f r e q u e n t l y in negative energy balance (14). Our results represent cases where sufficient intake was provided according to requirements. Because the cows were not e x c e p t i o n a l l y high producers, t h e y were m o s t likely never in negative energy balance. This might explain why plasma glucose and insulin when the L R diet was fed may explain the increased milk protein o u t p u t because insulin stimulates protein synthesis and inhibits gluconeogenesis f r o m an±too acids (9). Increased p o o l sizes (P < .05) in cows fed LR c o m p a r e d to the H R diet are attributed to elevations in c o n c e n t r a t i o n of plasma glucose. For the LR diet, associated increases in insulin c o n c e n t r a t i o n s are p r o b a b l y responsible for the
TABLE 5. Effects of feeding a low-(LR) or high-roughage (HR) diet on mean plasma glucose and insulin concentration and kinetic measures of glucose in lactating cows (Means ± SD).
Diet
obs
LR HR Significance (P < .05) c
9 9
Plasma glucose a
Plasma insulin a
Pool size
Half time
Utilization rate b
(mg/lO0 ml)
(uU/ml)
(mg/kg B.W.)
(min)
(mg/kg.75/min)
63.1 + 3.9 54.9 ± 2.1
22.0 ± 3.9 16.2 ± 2.4
179.1 ± 34.3 114.5 + 17.2
30.4 -+ 5.2 40.0 ± 2.2
8.55 -+ 2.44 4.06 ± 0.38
S
S
S
S
S
acalculated from nine samples taken at hourly intervals initiating 30 min before feeding for each individual observation. bCalculated over 3 h after injection of [2-3H] glucose. Cs, significant. Journal of Dairy Science Vol. 58, No. 5
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EVANS ET AL.
decreased half-times. Glucose utilization rates for cows fed the LR diet were increased, therefore, by both a larger pool size and a decreased half-time of glucose. The fact that glucose utilization rates were increased by alterations in both of its component parts indicates that metabolism of glucose is affected substantially by high-grain intake. (Table 5). Effects of the LR diet upon glucose metabolism in individual cows, which are in Table 3, show that increased plasma concentration, utilization rate, and pool size of glucose and depressed half-time occurred in every cow. Bickerstaffe et al. (4) have reported that glucose utilization rates in lactating cows were increased by feeding a low- compared to a high-roughage diet. In the experiment of Bickerstaffe et al. (4), the low- and high-roughage diet were both fed several weeks apart once to each cow. Thus, the milk production and intake of available energy from both diets differed within cows. In our experiment, milk production and intake of digestible energy from both diets were similar within cows. Therefore, the effect of the LR diet on glucose metabolism in our experiment may be attributed to altered digestion of carbohydrate rather than a greater intake of available energy. There was no effect of diet upon clearance half time (P > .05) or mean concentrations of plasma insulin (P > .05) in the cows after a glucose load (Table 6). Mean glucose half times were approximately the same (P > .05) with either labeled glucose or a clearance test when
cows were fed the LR diet (30.4 and 30.2 min respectively), but they were significantly (P < .05) different when the HR diet was fed (40.0 and 33.1 min). This strongly suggests that when the cows were given the LR diet, metabolic pathways for glucose removal from the peripheral circulation were operating at a maximum rate whereas with the HR diet the rate of glucose removal was less than maximal. It is possible that the greater glucose availability in cows fed the LR diet was sufficient conditioning along to elicit the changes in glucose metabolism which were observed, and sustained dietary conditioning was not required to induce a response. High-grain feeding of lactating cattle does affect their glucose metabolism substantially. Although milk fat production was depressed by the LR diet in some cows, it was not depressed in others. A similar conclusion can be made from data by Bickerstaffe et al. (4). Glucose metabolism may be an important focus for studies to explain milk fat depression in cows fed high-grain diets. However, there must be other considerations such as glucose availabilities to specific tissues or interactions with other areas of intermediary metabolism.
ACKNOWLEDGMENT The authors are indebted to the Kraftco Corp., Ontario Ministry of Agriculture and Food, and the National Research Council of Canada for funds for this project. Technical assistance was provided by Y. T. Yao.
TABLE 6. Comparisons of differences between half times and mean plasma insulin concentrations determined during glucose clearance test and isotope dilution studies in lactating cows given Iow-(LR) and high-roughage (HR) diets (± SD).
Diet
LR HR Significance (P < .05)d
Clearance half timea
Isotope dilution half timeb
Clearance insulina
Isotope dilution insulina
30.2 -+6.2 33.1-+ 3.3
(min) 30.4 ± 5.2 40.0± 2.2
49.6 +- 10.6 45.5-+ 7.6
~ (uU/ml) 22.0 -+ 3.9 16.2± 2.4
NS
S
NS
aComputed from samples taken after the infusion of .5g glucose/kg body weight. bcomputed after the injection of [2-3H] glucose. Ccomputed from daily average of 9 samples taken at hourly intervals, beginning 30 min before feeding. ds, significant; NS, non-significant. Journal of Dairy Science Vol. 58, No. 5
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