Glucagon, Insulin, Growth Hormone, and Glucose Concentrations in Blood Plasma of Lactating Dairy Cows 1 J. H. HERBEIN, R. J. AIELLO, 2 L. I. ECKLER, R. E. PEARSON, and R. M. AKERS Dairy Science Department Virginia Polytechnic Institute and State University Blacksburg 24061 ABSTRACT Concentrations of glucose, growth hormone, insulin, and glucagon in blood plasma relative to days in milk, milk production, type of housing, and season were measured. Blood samples were obtained from 133 to 150 lactating Holstein cows on 3 consecutive days in July, October, January, and April. Glucose, insulin, and ratio of insulin to glucagon increased with increasing days in milk. Growth hormone and ratio of growth hormone to insulin decreased with increasing days in milk. Glucagon concentrations were similar throughout lactation. Above average milk production throughout lactation was associated with above average glucagon, lower insulin, and lower glucose. The relationship was not significant between growth hormone concentration and milk production, how- ever. Cows on summer pasture with limited grain supplement had higher growth hormone and lower glucose, insulin, and glucagon than cows eating ad libitum in barns or feedlot. In general, however, all cows had higher glucose and lower growth hormone, insulin, and ratio of insulin to glucagon in July than in cooler months. INTRODUCTION Glucose availability has been implicated as a limiting factor for milk production (12). Received May 14, 1984. Supported in part by the John Lee Pratt Animal Nutrition Program at Virginia Polytechnic Institute and State University, Blacksburg 24061. :Department of Dairy Science, University of Wisconsin, Madison 53706. Growth hormone (GH), insulin (INS), and glucagon (GLN) concentrations in blood plasma appeared to be related to glucose availability in lactating dairy cows (4, 9, 14). It was hypo- thesized that ratio of GH to INS may regulate the supply of glucose and other nutrients for milk synthesis (4). Nonruminant studies have emphasized the importance of GLN in hepatic glucose production and homeorrhetic control of glucose metabolism (8). Ruminant de- pendence on hepatic gluconeogenesis suggests GLN may play an important role in glucose metabolism and possibly milk production (5). Changes of GLN concentration during lactation have not been determined for dairy cows. Plasma GLN concentrations in sheep and steers ranged from 100 pg/ml (3) to 410 pg/ml (2). Variation due to diet and physiological status was observed. The objective of this study was to measure changes of GLN, INS, GH, and glucose in blood plasma of lactating cows with respect to environmental and lactational characteristics. MATERIALS AND METHODS All lactating Holstein cows at the Virginia Tech Dairy Cattle Research Center were sampled by jugular venipuncture on 3 consecutive days in each of four seasons. Sampling began in summer and ended the following spring. Number of cows sampled and mean daily temperatures were: 133 and 20.5°C for summer (July), 139 and 5.0°C for fall (October), 150 and -2.8°C for winter (January), and 148 and 5.6°C for spring (April). Milk production, body weight, age, days in milk (DIM), and type of housing were recorded each season. Types of housing were: tie-stall barn, three-sided free-stall barn open to the south, and no housing (pasture or feed lot without shelter). All cows were sampled after milking (between 1200 and 1700 h). Blood (10 ml) was collected 1985 J Dairy Sci 68:320-325 320
Glucagon, Insulin, Growth Hormone, and Glucose Concentrations in Blood Plasma of Lactating Dairy Cows
Mar 10, 2023
Concentrations of glucose, growth
hormone, insulin, and glucagon in blood
plasma relative to days in milk, milk
production, type of housing, and season
were measured. Blood samples were
obtained from 133 to 150 lactating
Holstein cows on 3 consecutive days in
July, October, January, and April.
Glucose, insulin, and ratio of insulin to
glucagon increased with increasing days
in milk. Growth hormone and ratio of
growth hormone to insulin decreased
with increasing days in milk.
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Concentrations of glucose, growth hormone, insulin, and glucagon in blood plasma relative to days in milk, milk production, type of housing, and season were measured. Blood samples were obtained from 133 to 150 lactating Holstein cows on 3 consecutive days in July, October, January, and April. Glucose, insulin, and ratio of insulin to glucagon increased with increasing days in milk. Growth hormone and ratio of growth hormone to insulin decreased with increasing days in milk.
Transcript
Glucagon, Insulin, Growth Hormone, and Glucose Concentrations in Blood Plasma of Lactating Dairy Cows1Glucagon, Insulin, Growth Hormone, and Glucose Concentrations in Blood Plasma of Lactating Dairy Cows 1
J. H. HERBEIN, R. J. A IELLO, 2 L. I. ECKLER, R. E. PEARSON, and R. M. AKERS
Dairy Science Department Virginia Polytechnic Institute
and State University Blacksburg 24061
ABSTRACT
Concentrations of glucose, growth hormone, insulin, and glucagon in blood plasma relative to days in milk, milk production, type of housing, and season were measured. Blood samples were obtained from 133 to 150 lactating Holstein cows on 3 consecutive days in July, October, January, and April. Glucose, insulin, and ratio of insulin to glucagon increased with increasing days in milk. Growth hormone and ratio of growth hormone to insulin decreased with increasing days in milk. Glucagon concentrations were similar throughout lactation. Above average milk production throughout lactation was associated with above average glucagon, lower insulin, and lower glucose. The relationship was not significant between growth hormone concentration and milk production, how- ever. Cows on summer pasture with limited grain supplement had higher growth hormone and lower glucose, insulin, and glucagon than cows eating ad libitum in barns or feedlot. In general, however, all cows had higher glucose and lower growth hormone, insulin, and ratio of insulin to glucagon in July than in cooler months.
I N T R O D U C T I O N
Glucose availability has been implicated as a limiting factor for milk production (12).
Received May 14, 1984. Supported in part by the John Lee Pratt Animal
Nutrition Program at Virginia Polytechnic Institute and State University, Blacksburg 24061.
:Department of Dairy Science, University of Wisconsin, Madison 53706.
Growth hormone (GH), insulin (INS), and glucagon (GLN) concentrations in blood plasma appeared to be related to glucose availability in lactating dairy cows (4, 9, 14). It was hypo- thesized that ratio of GH to INS may regulate the supply of glucose and other nutrients for milk synthesis (4). Nonruminant studies have emphasized the importance of GLN in hepatic glucose production and homeorrhetic control of glucose metabolism (8). Ruminant de- pendence on hepatic gluconeogenesis suggests GLN may play an important role in glucose metabolism and possibly milk production (5). Changes of GLN concentration during lactation have not been determined for dairy cows. Plasma GLN concentrations in sheep and steers ranged from 100 pg/ml (3) to 410 pg/ml (2). Variation due to diet and physiological status was observed. The objective of this study was to measure changes of GLN, INS, GH, and glucose in blood plasma of lactating cows with respect to environmental and lactational characteristics.
M A T E R I A L S A N D METHODS
All lactating Holstein cows at the Virginia Tech Dairy Cattle Research Center were sampled by jugular venipuncture on 3 consecutive days in each of four seasons. Sampling began in summer and ended the following spring. Number of cows sampled and mean daily temperatures were: 133 and 20.5°C for summer (July), 139 and 5.0°C for fall (October), 150 and -2 .8°C for winter (January), and 148 and 5.6°C for spring (April). Milk production, body weight, age, days in milk (DIM), and type of housing were recorded each season. Types of housing were: tie-stall barn, three-sided free-stall barn open to the south, and no housing (pasture or feed lot without shelter).
All cows were sampled after milking (between 1200 and 1700 h). Blood (10 ml) was collected
1985 J Dairy Sci 68:320-325 320
GLUCOREGULATORY HORMONES DURING LACTATION 321
in syringes containing .2 ml K-EDTA (potas- sium-ethylenediaminetetraacetate) (4 m/l/I) and benzamidine hydrochloride (30 mM). Plasma was separated by centrifugation and stored at - 2 0 ° C until analyzed for GLN, INS, GH, and glucose. Plasma glucose was measured by the glucose oxidase method (Sigma Technical Bulletin No. 510, 1973). Plasma GLN, INS, and GH were quantified by standard homologous double ant ibody radioimmunoassay procedures (16). Glucagon antiserum (30K, Lot No. 615, R. H. Unger, Dallas, T X ) d i l u t e d 1:50,000 bound 30% of the radiolabeled glucagon. Specific INS antiserum (Research Products International Corp., Elk Grove, IL) diluted 1:1,000 bound 35% of radiolabeled insulin. Specific bovine GH antiserum (Rabbit 98J, R. M. Akers) diluted 1:1,000 bound 30% of the radiolabeled GH. There was no crossreactivity between INS and GLN (<2%) by standards prepared from purified bovine INS and GLN (Eli Lilly, Indianapolis, IN). The GH antisera did not crossreact (<2%) with other pi tui tary hormones with NIH GH-B18 as a reference standard. Standard curves were linear over a range of .015 to 2.0 ng GLN, .125 to 4.0 ng INS, and .5 to 9 ng GH. All samples were assayed in duplicate. Intraassay and interassay coefficients of variation were 6.1 and 13.2% for GLN, 4.7 and 7.4% for INS, and 4.5 and 5.2% for GH. Plasma samples taken during summer, fall, and winter were quantified for INS, GLN, and glucose after winter samples were taken. Spring samples were analyzed within 1 mo after sampling. Concentration of GH in all samples was measured after spring sampling.
Statistical Analysis
Repeatabil i ty of daily hormone and glucose concentrations for cow within season was determined from variance components of nested analysis of variance. Daily observations for blood variables were averaged for each cow within season and analysis begun with the following model:
Yijkl =/~ + Ai + Sj + L k + b l ( D - D ) + b2(D--D) 2 + b3(M-M) + b4(B-B)
+ bs (B-B) 2 + eijkl
where: Yijkl = observation for the I th cow with
the i th age in the j th season and the k th location,
/J = overall mean, A = effect of age in years at t ime of
sampling, S = effect of season of sampling, L = effect of location or type of housing, D = days in milk on 2 nd day of sampling, M = average daily milk production during
the 3 days of sampling, B = body weight the day after sampling
was completed, bx to bs = partial regression coefficients, and
eijkl = residual.
The complete model was used for GLN data. Nonsignificant terms were omit ted from models for other variables. The b s ( B - B ) 2 term was omit ted for INS and glucose, and both squared terms (D and B) were omitted for GH. The model, with both squared terms omitted, also was used for analysis of ratios of INS to GLN and GH to INS.
Primary predict ion equations to estimate changes of hormone or glucose concentration across the range of DIM (300 days) were developed from results of analyses. Because milk yield and body weight were held constant at their means (26.3 and 603 kg), the resulting plot of concentratiori ~/ersus DIM was not the best representation of changes as cows progress through lactation. A more realistic estimate of change of concentration was obtained by alternate prediction equations with average DIM, milk yield, and body weight for ten 30-day intervals (Table 1). Estimates of hormone or glucose concentration at each interval also were calculated with the alternate equations and plotted.
RESULTS AND DISCUSSION
Diurnal variation, stress, sampling time postfeeding, diet composition, and intake affected plasma substrate and hormone concen- trations (3, 7, 9, 14). Repeatabili t ies of daily hormone and glucose concentrations in our herd were low. Repeatabili t ies of glucose, INS, and GH were all .21. Repeatabi l i ty of GLN was .35. Cow groups from different locations entered the milk parlor at approxi- mately the same time each afternoon, but cows within group were not sampled in the same
Journal of Dairy Science Vol. 68, No. 2, 1985
322 HERBEIN ET AL.
TABLE 1. Means for days in milk (DIM), milk production (MILK), and body weight (BW) for all cows sampled in all seasons.
Postpartum Means interval DIM MILK BW
(days) (days) (kg) 0--30 16 26.5 571
31--60 44 29.7 561 61--90 73 29.2 563 91--120 107 27.2 569
121--150 136 25.3 580 151--180 165 23.9 592 1 8 1 - - 2 1 0 195 22.4 587 211--240 223 19.6 622 241--270 257 19.7 628 271--300 286 18.2 618
order each day. In addition, a variety of diets were fed as part of other research projects. Therefore, daily observations were averaged to estimate hormone or glucose concentration for each cow within season.
Changes of plasma glucose versus DIM (solid line; Figure 1) indicated a significant (P<.05) quadratic relationship when data for all seasons were combined. Glucose estimates (dashed line, Figure 1) for the ten 30-day intervals were lower in early lactation and higher in late lactation than predicted by the equation (solid line) for overall means for milk yield and body weight. Lactation and pregnancy increased glucose requirements in ruminants (1). Blood glucose concentration was related inversely to glucose requirement for lactation in goats (6). Low blood glucose during the first 20 days of lactation in dairy cattle followed by a significant increase the second 20 days was reported by Smith et al. (13). Our data indicated a continual increase of blood glucose during the first 7 mo of lactation. The tendency to stabilize or decrease during the last 2 mo possibly was related to increased glucose requirement for pregnancy.
Predicted GH concentrations from DIM are in Figure 2. The linear decrease during advancing lactation, similar to that reported by Koprowski and Tucker (10), was significant (P<.05). Decreasing GH during a short interval in early lactation has been reported (13, 14). Because daily injections of exogenous GH increased milk production (4, 11), a high correlation
between GH and milk production might be expected. Koprowski and Tucker (10), however, found no positive correlation in 26 cows sampled throughout lactation. Our data also indicated no significant relationship.
Concentrations of INS and GLN versus DIM (30-day intervals) are in Figure 3. Insulin and GLN are primary regulators of blood glucose
59 g
/ ~ " - - ~ I . _ 1
510 I I I I I 0 I00 150 200 250 300
DAYS IN MILK
Figure 1. Predicted glucose concentrations in blood plasma during lactation. Calculations included overall averages for milk production and body weight (solid line) or average milk production and body weight at 30-day intervals (I). Glucose = 57.5 -- .15805M + .00118B + .02989D + .0000729D2; for milk pro- duction (M), body weight (B), and days in milk (D).
Journal of Dairy Science Vol. 68, No. 2, 1985
GLUCOREGULATORY HORMONES DURING LACTATION 32 3
E
O I~ I ID
0 50 I O0 150 200 250 300
DAYS IN M I L K
Figure 2. Predicted growth hormone concentra- tions in blood plasma during lactation. Calculations included overall averages for milk production and body weight (solid line) or average milk production and body weight at 30-day intervals (u). Growth hormone = 16.7 + .04607M -- .00964B -- .00567D; for milk production (M), body weight (B), and days in milk (D).
c o n c e n t r a t i o n in n o n r u m i n a n t s (8) and ru- m i n a n t s (5). In sheep, hepa t i c g lucose o u t p u t was decreased b y exogenous INS (15) . G lucagon s t imula tes hepa t i c glucose o u t p u t , b u t rumi- nan t s secre te b o t h INS and G L N in response to a meal (2, 5). H igh -concen t r a t e diets also increased b o t h INS and GLN, as c o m p a r e d wi th feeding high roughage (3). Data in Figure 3 ind ica te t h a t hepa t i c syn thes i s of g lucose to m e e t l ac t a t ion r e q u i r e m e n t s was fac i l i t a ted b y depressed INS c o n c e n t r a t i o n r a the r t h a n e levated GLN. Similar to glucose versus DIM (Figure 1), DIM had a s igni f icant ( P < . 0 5 ) quadra t i c e f fec t on p lasma INS. Plasma GLN was n o t in f luenced by DIM. A role for b o t h INS and GLN, however , was ind ica ted b y the i r re la t ionsh ip wi th mi lk p r o d u c t i o n . With b o d y weight , DIM, and all o t h e r fac tors in the s ta t is t ical mode l held cons t an t , above average mi lk p r o d u c t i o n was associated wi th signifi- can t ly ( P < . 0 5 ) h igher GLN and lower INS.
Ra t ios o f GH to INS and INS to GLN were ca lcula ted for 30-day intervals (Figure 4). A di rec t e f fec t of GH on hepa t i c glucose synthes i s in r u m i n a n t s has n o t been d e m o n s t r a t e d , b u t increased GH c o n c e n t r a t i o n relat ive to INS in
80
7O
E
~ 60
50
m / A / / / /K/xl NSULIN
0 50 I00 150 200 250 300
DAYS IN M I L K
Figure 3. Predicted glucagon and insulin concen- trations in blood plasma during lactation. Calculations were based on average milk production and body weight at 30-day intervals. Glucagon = .910 + .00514M - - . 0 0 1 7 3 B + .O000017B 2 + .00066D --.0000012D 2. Insulin = .419 -- .01106M + .00067B + .00134D -- .OOO0030D2; for milk production (M), body weight (B), and days in milk (D).
o
z o Z ._~ INS:GLN 12::
Z _ i O I J j
0 I 0 0 150 200 250 300 DAYS IN MILK
Figure 4. Predicted ratios of growth hormone to insulin and insulin to glucagon during lactation. Calculations were based on average milk production and body weight at 30-day intervals. Ratios of growth hormone to insulin = 32.7 + .30461M - . 0 3 0 2 1 B - .01723D. Ratio of insulin to glucagon = 1.41 - .02301M + .00027B + .00014D; for milk production (M), body weight (B), and days in milk (D). GH = Growth hormone; INS = insulin.
Journal of Dairy Science Vol. 68, No. 2, 1985
324 HERBEIN ET AL.
TABLE 2. Seasonal least squares means for glucose and hormone concentrations in blood plasma.
Season Variable Summer Fall Winter Spring SE
Glucose, mg/dl 57.5 a 55.6 b 55.6 b 58.4 a Growth hormone, ng/ml 10.2 a 11.6 b 10.9 ab 11.6 b Insulin, ng/ml .581 a .652 ab .744 b .725 b Glucagon, ng/ml .764 a .560 b .724 a .646 c Insulin:glucagon .81a 1.20 b 1.07 b 1.15 b
.4
.3
.03
.02
.05
a'b'CMeans in rows without a common superscript differ (P<.05).
early lactation might increase glucogenic substrate (amino acids and glycerol) availability from peripheral tissues and transport of amino acids into liver cells (8). The lower ratio of INS to GLN in early lactation then would facilitate substrate utilization to meet lactational glucose requirements during negative energy balance (13). In contrast, the ratios of GH to INS and INS to GLN in later lactation gradually favor deposition of nutrients in peripheral tissues. Injections of GH increased the ratio of GH to INS by increasing plasma GH with no change of INS (11) or by increasing GH to a greater extent than INS (4). If INS is increased in response to GH injections, our data suggest that an increase of GLN also would be required to maintain the ratio of INS to GLN and assure adequate glucose availability for milk production.
Differences (P<.05) due to age and location were minimal. Age was not a significant factor in determining plasma hormone concentrations, but glucose concentration decreased with succeeding lactations. Differences due to location were associated with cows on pasture during summer. Pasture cows had higher GH and lower glucose, INS, and GLN than other groups. As a result, the ratio of GH to INS was higher. The differences may have been related to energy balance. Cows housed in barns or feedlot were fed silage-based diets for ad libitum intake. Cows on summer pasture received a limited amount of corn grain to supplement their grazing. Energy intake at or slightly below lactational requirements may have resulted because glucose and hormone concentrations were similar to those for cows in early lactation. The additional energy require- ment for grazing activity tended to lower body
weight but not milk production as compared with cows in other locations.
Mean daily temperature during summer sampling was 15 to 23°C higher than temper- atures in other seasons. A difference of ap- proximately 8°C separated temperatures in fall, winter, and spring. In general, plasma glucose was higher and GH and INS lower in summer than in other seasons (Table 2). The only exception was similar glucose in summer and spring. Differences between ratios of GH to INS (data not shown) were relatively small and not related to seasonal differences in glucose concentration. Glucagon was the only variable possibly related to seasonal temperature. During extremes of summer and winter, GLN was elevated. Despite significant differences in seasonal GLN, however, ratios of INS to GLN were. similar in fall, winter, and spring. The lower ratio in summer versus fall and winter may have been responsible for stimulating glucose output by liver and elevating plasma glucose. This relationship between plasma glucose and ratio of INS to GLN, however, was not substantiated by data for spring.
A C K N O W L E D G M E N T S
Appreciation is expressed to Lilly Research Laboratories, Eli Lilly and Company, for donating purified bovine insulin and glucagon for radioimmunoassays.
REFERENCES
1 Armstrong, D. G. 1965. Carbohydrate metabolism in ruminants and energy supply. Pages 272-288 in Physiology of digestion in the ruminant. R. W. Dougherty, R. S. Allen, W. Burroughs, N. L. Jacobson, and A. D. McGilliard, ed. Butterworths, Inc., Washington, DC.
Journal of Dairy Science Vol. 68, No. 2, 1985
G L U C O R E G U L A T O R Y HORMONES DURING LACTATION 325
2 Bassett, J. M. 1972. Plasma glucagon concentrat ions in sheep: Their regulation and relation to con- centrat ions o f insulin and growth hormone . Aust . J. Biol. Sci. 25:1277.
3 Berzins, R., and J. G. Manns. 1979. How con- centrate feeding affects glucoregulatory ho rmones in ruminants : Implications in bovine ketosis. J. Dairy Sci. 62:1739.
4 Bines, J. A., I. C. Hart, and S. V. Morant. 1980. Endocrine control o f energy metabol ism in the cow: the effect on milk yield and levels of some blood const i tuents of injecting growth hormone and growth hormone fragments. Br. J. Nutr. 43:179.
5 Brockman, R. P. 1978. Roles o f glucagon and insulin in the regulation of metabol i sm in ruminants : A…
J. H. HERBEIN, R. J. A IELLO, 2 L. I. ECKLER, R. E. PEARSON, and R. M. AKERS
Dairy Science Department Virginia Polytechnic Institute
and State University Blacksburg 24061
ABSTRACT
Concentrations of glucose, growth hormone, insulin, and glucagon in blood plasma relative to days in milk, milk production, type of housing, and season were measured. Blood samples were obtained from 133 to 150 lactating Holstein cows on 3 consecutive days in July, October, January, and April. Glucose, insulin, and ratio of insulin to glucagon increased with increasing days in milk. Growth hormone and ratio of growth hormone to insulin decreased with increasing days in milk. Glucagon concentrations were similar throughout lactation. Above average milk production throughout lactation was associated with above average glucagon, lower insulin, and lower glucose. The relationship was not significant between growth hormone concentration and milk production, how- ever. Cows on summer pasture with limited grain supplement had higher growth hormone and lower glucose, insulin, and glucagon than cows eating ad libitum in barns or feedlot. In general, however, all cows had higher glucose and lower growth hormone, insulin, and ratio of insulin to glucagon in July than in cooler months.
I N T R O D U C T I O N
Glucose availability has been implicated as a limiting factor for milk production (12).
Received May 14, 1984. Supported in part by the John Lee Pratt Animal
Nutrition Program at Virginia Polytechnic Institute and State University, Blacksburg 24061.
:Department of Dairy Science, University of Wisconsin, Madison 53706.
Growth hormone (GH), insulin (INS), and glucagon (GLN) concentrations in blood plasma appeared to be related to glucose availability in lactating dairy cows (4, 9, 14). It was hypo- thesized that ratio of GH to INS may regulate the supply of glucose and other nutrients for milk synthesis (4). Nonruminant studies have emphasized the importance of GLN in hepatic glucose production and homeorrhetic control of glucose metabolism (8). Ruminant de- pendence on hepatic gluconeogenesis suggests GLN may play an important role in glucose metabolism and possibly milk production (5). Changes of GLN concentration during lactation have not been determined for dairy cows. Plasma GLN concentrations in sheep and steers ranged from 100 pg/ml (3) to 410 pg/ml (2). Variation due to diet and physiological status was observed. The objective of this study was to measure changes of GLN, INS, GH, and glucose in blood plasma of lactating cows with respect to environmental and lactational characteristics.
M A T E R I A L S A N D METHODS
All lactating Holstein cows at the Virginia Tech Dairy Cattle Research Center were sampled by jugular venipuncture on 3 consecutive days in each of four seasons. Sampling began in summer and ended the following spring. Number of cows sampled and mean daily temperatures were: 133 and 20.5°C for summer (July), 139 and 5.0°C for fall (October), 150 and -2 .8°C for winter (January), and 148 and 5.6°C for spring (April). Milk production, body weight, age, days in milk (DIM), and type of housing were recorded each season. Types of housing were: tie-stall barn, three-sided free-stall barn open to the south, and no housing (pasture or feed lot without shelter).
All cows were sampled after milking (between 1200 and 1700 h). Blood (10 ml) was collected
1985 J Dairy Sci 68:320-325 320
GLUCOREGULATORY HORMONES DURING LACTATION 321
in syringes containing .2 ml K-EDTA (potas- sium-ethylenediaminetetraacetate) (4 m/l/I) and benzamidine hydrochloride (30 mM). Plasma was separated by centrifugation and stored at - 2 0 ° C until analyzed for GLN, INS, GH, and glucose. Plasma glucose was measured by the glucose oxidase method (Sigma Technical Bulletin No. 510, 1973). Plasma GLN, INS, and GH were quantified by standard homologous double ant ibody radioimmunoassay procedures (16). Glucagon antiserum (30K, Lot No. 615, R. H. Unger, Dallas, T X ) d i l u t e d 1:50,000 bound 30% of the radiolabeled glucagon. Specific INS antiserum (Research Products International Corp., Elk Grove, IL) diluted 1:1,000 bound 35% of radiolabeled insulin. Specific bovine GH antiserum (Rabbit 98J, R. M. Akers) diluted 1:1,000 bound 30% of the radiolabeled GH. There was no crossreactivity between INS and GLN (<2%) by standards prepared from purified bovine INS and GLN (Eli Lilly, Indianapolis, IN). The GH antisera did not crossreact (<2%) with other pi tui tary hormones with NIH GH-B18 as a reference standard. Standard curves were linear over a range of .015 to 2.0 ng GLN, .125 to 4.0 ng INS, and .5 to 9 ng GH. All samples were assayed in duplicate. Intraassay and interassay coefficients of variation were 6.1 and 13.2% for GLN, 4.7 and 7.4% for INS, and 4.5 and 5.2% for GH. Plasma samples taken during summer, fall, and winter were quantified for INS, GLN, and glucose after winter samples were taken. Spring samples were analyzed within 1 mo after sampling. Concentration of GH in all samples was measured after spring sampling.
Statistical Analysis
Repeatabil i ty of daily hormone and glucose concentrations for cow within season was determined from variance components of nested analysis of variance. Daily observations for blood variables were averaged for each cow within season and analysis begun with the following model:
Yijkl =/~ + Ai + Sj + L k + b l ( D - D ) + b2(D--D) 2 + b3(M-M) + b4(B-B)
+ bs (B-B) 2 + eijkl
where: Yijkl = observation for the I th cow with
the i th age in the j th season and the k th location,
/J = overall mean, A = effect of age in years at t ime of
sampling, S = effect of season of sampling, L = effect of location or type of housing, D = days in milk on 2 nd day of sampling, M = average daily milk production during
the 3 days of sampling, B = body weight the day after sampling
was completed, bx to bs = partial regression coefficients, and
eijkl = residual.
The complete model was used for GLN data. Nonsignificant terms were omit ted from models for other variables. The b s ( B - B ) 2 term was omit ted for INS and glucose, and both squared terms (D and B) were omitted for GH. The model, with both squared terms omitted, also was used for analysis of ratios of INS to GLN and GH to INS.
Primary predict ion equations to estimate changes of hormone or glucose concentration across the range of DIM (300 days) were developed from results of analyses. Because milk yield and body weight were held constant at their means (26.3 and 603 kg), the resulting plot of concentratiori ~/ersus DIM was not the best representation of changes as cows progress through lactation. A more realistic estimate of change of concentration was obtained by alternate prediction equations with average DIM, milk yield, and body weight for ten 30-day intervals (Table 1). Estimates of hormone or glucose concentration at each interval also were calculated with the alternate equations and plotted.
RESULTS AND DISCUSSION
Diurnal variation, stress, sampling time postfeeding, diet composition, and intake affected plasma substrate and hormone concen- trations (3, 7, 9, 14). Repeatabili t ies of daily hormone and glucose concentrations in our herd were low. Repeatabili t ies of glucose, INS, and GH were all .21. Repeatabi l i ty of GLN was .35. Cow groups from different locations entered the milk parlor at approxi- mately the same time each afternoon, but cows within group were not sampled in the same
Journal of Dairy Science Vol. 68, No. 2, 1985
322 HERBEIN ET AL.
TABLE 1. Means for days in milk (DIM), milk production (MILK), and body weight (BW) for all cows sampled in all seasons.
Postpartum Means interval DIM MILK BW
(days) (days) (kg) 0--30 16 26.5 571
31--60 44 29.7 561 61--90 73 29.2 563 91--120 107 27.2 569
121--150 136 25.3 580 151--180 165 23.9 592 1 8 1 - - 2 1 0 195 22.4 587 211--240 223 19.6 622 241--270 257 19.7 628 271--300 286 18.2 618
order each day. In addition, a variety of diets were fed as part of other research projects. Therefore, daily observations were averaged to estimate hormone or glucose concentration for each cow within season.
Changes of plasma glucose versus DIM (solid line; Figure 1) indicated a significant (P<.05) quadratic relationship when data for all seasons were combined. Glucose estimates (dashed line, Figure 1) for the ten 30-day intervals were lower in early lactation and higher in late lactation than predicted by the equation (solid line) for overall means for milk yield and body weight. Lactation and pregnancy increased glucose requirements in ruminants (1). Blood glucose concentration was related inversely to glucose requirement for lactation in goats (6). Low blood glucose during the first 20 days of lactation in dairy cattle followed by a significant increase the second 20 days was reported by Smith et al. (13). Our data indicated a continual increase of blood glucose during the first 7 mo of lactation. The tendency to stabilize or decrease during the last 2 mo possibly was related to increased glucose requirement for pregnancy.
Predicted GH concentrations from DIM are in Figure 2. The linear decrease during advancing lactation, similar to that reported by Koprowski and Tucker (10), was significant (P<.05). Decreasing GH during a short interval in early lactation has been reported (13, 14). Because daily injections of exogenous GH increased milk production (4, 11), a high correlation
between GH and milk production might be expected. Koprowski and Tucker (10), however, found no positive correlation in 26 cows sampled throughout lactation. Our data also indicated no significant relationship.
Concentrations of INS and GLN versus DIM (30-day intervals) are in Figure 3. Insulin and GLN are primary regulators of blood glucose
59 g
/ ~ " - - ~ I . _ 1
510 I I I I I 0 I00 150 200 250 300
DAYS IN MILK
Figure 1. Predicted glucose concentrations in blood plasma during lactation. Calculations included overall averages for milk production and body weight (solid line) or average milk production and body weight at 30-day intervals (I). Glucose = 57.5 -- .15805M + .00118B + .02989D + .0000729D2; for milk pro- duction (M), body weight (B), and days in milk (D).
Journal of Dairy Science Vol. 68, No. 2, 1985
GLUCOREGULATORY HORMONES DURING LACTATION 32 3
E
O I~ I ID
0 50 I O0 150 200 250 300
DAYS IN M I L K
Figure 2. Predicted growth hormone concentra- tions in blood plasma during lactation. Calculations included overall averages for milk production and body weight (solid line) or average milk production and body weight at 30-day intervals (u). Growth hormone = 16.7 + .04607M -- .00964B -- .00567D; for milk production (M), body weight (B), and days in milk (D).
c o n c e n t r a t i o n in n o n r u m i n a n t s (8) and ru- m i n a n t s (5). In sheep, hepa t i c g lucose o u t p u t was decreased b y exogenous INS (15) . G lucagon s t imula tes hepa t i c glucose o u t p u t , b u t rumi- nan t s secre te b o t h INS and G L N in response to a meal (2, 5). H igh -concen t r a t e diets also increased b o t h INS and GLN, as c o m p a r e d wi th feeding high roughage (3). Data in Figure 3 ind ica te t h a t hepa t i c syn thes i s of g lucose to m e e t l ac t a t ion r e q u i r e m e n t s was fac i l i t a ted b y depressed INS c o n c e n t r a t i o n r a the r t h a n e levated GLN. Similar to glucose versus DIM (Figure 1), DIM had a s igni f icant ( P < . 0 5 ) quadra t i c e f fec t on p lasma INS. Plasma GLN was n o t in f luenced by DIM. A role for b o t h INS and GLN, however , was ind ica ted b y the i r re la t ionsh ip wi th mi lk p r o d u c t i o n . With b o d y weight , DIM, and all o t h e r fac tors in the s ta t is t ical mode l held cons t an t , above average mi lk p r o d u c t i o n was associated wi th signifi- can t ly ( P < . 0 5 ) h igher GLN and lower INS.
Ra t ios o f GH to INS and INS to GLN were ca lcula ted for 30-day intervals (Figure 4). A di rec t e f fec t of GH on hepa t i c glucose synthes i s in r u m i n a n t s has n o t been d e m o n s t r a t e d , b u t increased GH c o n c e n t r a t i o n relat ive to INS in
80
7O
E
~ 60
50
m / A / / / /K/xl NSULIN
0 50 I00 150 200 250 300
DAYS IN M I L K
Figure 3. Predicted glucagon and insulin concen- trations in blood plasma during lactation. Calculations were based on average milk production and body weight at 30-day intervals. Glucagon = .910 + .00514M - - . 0 0 1 7 3 B + .O000017B 2 + .00066D --.0000012D 2. Insulin = .419 -- .01106M + .00067B + .00134D -- .OOO0030D2; for milk production (M), body weight (B), and days in milk (D).
o
z o Z ._~ INS:GLN 12::
Z _ i O I J j
0 I 0 0 150 200 250 300 DAYS IN MILK
Figure 4. Predicted ratios of growth hormone to insulin and insulin to glucagon during lactation. Calculations were based on average milk production and body weight at 30-day intervals. Ratios of growth hormone to insulin = 32.7 + .30461M - . 0 3 0 2 1 B - .01723D. Ratio of insulin to glucagon = 1.41 - .02301M + .00027B + .00014D; for milk production (M), body weight (B), and days in milk (D). GH = Growth hormone; INS = insulin.
Journal of Dairy Science Vol. 68, No. 2, 1985
324 HERBEIN ET AL.
TABLE 2. Seasonal least squares means for glucose and hormone concentrations in blood plasma.
Season Variable Summer Fall Winter Spring SE
Glucose, mg/dl 57.5 a 55.6 b 55.6 b 58.4 a Growth hormone, ng/ml 10.2 a 11.6 b 10.9 ab 11.6 b Insulin, ng/ml .581 a .652 ab .744 b .725 b Glucagon, ng/ml .764 a .560 b .724 a .646 c Insulin:glucagon .81a 1.20 b 1.07 b 1.15 b
.4
.3
.03
.02
.05
a'b'CMeans in rows without a common superscript differ (P<.05).
early lactation might increase glucogenic substrate (amino acids and glycerol) availability from peripheral tissues and transport of amino acids into liver cells (8). The lower ratio of INS to GLN in early lactation then would facilitate substrate utilization to meet lactational glucose requirements during negative energy balance (13). In contrast, the ratios of GH to INS and INS to GLN in later lactation gradually favor deposition of nutrients in peripheral tissues. Injections of GH increased the ratio of GH to INS by increasing plasma GH with no change of INS (11) or by increasing GH to a greater extent than INS (4). If INS is increased in response to GH injections, our data suggest that an increase of GLN also would be required to maintain the ratio of INS to GLN and assure adequate glucose availability for milk production.
Differences (P<.05) due to age and location were minimal. Age was not a significant factor in determining plasma hormone concentrations, but glucose concentration decreased with succeeding lactations. Differences due to location were associated with cows on pasture during summer. Pasture cows had higher GH and lower glucose, INS, and GLN than other groups. As a result, the ratio of GH to INS was higher. The differences may have been related to energy balance. Cows housed in barns or feedlot were fed silage-based diets for ad libitum intake. Cows on summer pasture received a limited amount of corn grain to supplement their grazing. Energy intake at or slightly below lactational requirements may have resulted because glucose and hormone concentrations were similar to those for cows in early lactation. The additional energy require- ment for grazing activity tended to lower body
weight but not milk production as compared with cows in other locations.
Mean daily temperature during summer sampling was 15 to 23°C higher than temper- atures in other seasons. A difference of ap- proximately 8°C separated temperatures in fall, winter, and spring. In general, plasma glucose was higher and GH and INS lower in summer than in other seasons (Table 2). The only exception was similar glucose in summer and spring. Differences between ratios of GH to INS (data not shown) were relatively small and not related to seasonal differences in glucose concentration. Glucagon was the only variable possibly related to seasonal temperature. During extremes of summer and winter, GLN was elevated. Despite significant differences in seasonal GLN, however, ratios of INS to GLN were. similar in fall, winter, and spring. The lower ratio in summer versus fall and winter may have been responsible for stimulating glucose output by liver and elevating plasma glucose. This relationship between plasma glucose and ratio of INS to GLN, however, was not substantiated by data for spring.
A C K N O W L E D G M E N T S
Appreciation is expressed to Lilly Research Laboratories, Eli Lilly and Company, for donating purified bovine insulin and glucagon for radioimmunoassays.
REFERENCES
1 Armstrong, D. G. 1965. Carbohydrate metabolism in ruminants and energy supply. Pages 272-288 in Physiology of digestion in the ruminant. R. W. Dougherty, R. S. Allen, W. Burroughs, N. L. Jacobson, and A. D. McGilliard, ed. Butterworths, Inc., Washington, DC.
Journal of Dairy Science Vol. 68, No. 2, 1985
G L U C O R E G U L A T O R Y HORMONES DURING LACTATION 325
2 Bassett, J. M. 1972. Plasma glucagon concentrat ions in sheep: Their regulation and relation to con- centrat ions o f insulin and growth hormone . Aust . J. Biol. Sci. 25:1277.
3 Berzins, R., and J. G. Manns. 1979. How con- centrate feeding affects glucoregulatory ho rmones in ruminants : Implications in bovine ketosis. J. Dairy Sci. 62:1739.
4 Bines, J. A., I. C. Hart, and S. V. Morant. 1980. Endocrine control o f energy metabol ism in the cow: the effect on milk yield and levels of some blood const i tuents of injecting growth hormone and growth hormone fragments. Br. J. Nutr. 43:179.
5 Brockman, R. P. 1978. Roles o f glucagon and insulin in the regulation of metabol i sm in ruminants : A…
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