Hormonal Responses to Bovine Somatotropin and Dietary Protein in Early Lactation Dairy Cows G. de BOER,1 P. H. ROBINSON? and J. J. KENNEllY Department of Animal Science University of Alberta Edmonton, AB, Canada T6G 2P5 ABSTRACT
tions and producing equal amounts of milk. as control cows did not show major physiological differences in hormones and metabolites with the exception of insulin-like growth factor-I. (Key words: somatotropin, protein. hormones, dairy cows)
The effects of bST injection and dietary protein level on blood hormone and metabolite concentrations were examined in four mature Holstein cows in a double crossover design. Cows were assigned at d 5 to 9 postpartum to receive daily injections of either a control (saline) solution or 20.6 mg of bST. Four 3-wk. periods were used during which one cow from each group was fed a medium protein diet (17.1% CP), and the other received a high protein diet (23.6% CP). Injections of bST or control solutions began on d 0 of the second period. intakes of OM were not influenced by dietary protein or bST injection. Milk. yield tended to increase with increased CP level but was not affected by bST injection. Based on the rate and extent of decline in milk. production after cessation of bST injection, the cows assigned to bST had lower milk. production potential than control cows. Thus, the effect of bST injection apparently was to enhance milk yield to levels similar to those of controls. There were no significant CP level or bST injection effects on glucose, FFA, somatostatin, or somatotropin concentrations. Glucagon concentrations were higher in bST-treated cows. Concentrations of insulin-like growth factor-I were increased with increased CP level and also with bST injection. Significant effects of days on bST were observed for insulin, insulin-like growth factor-I, glucose, and FFA. Cows given bST injec-
Abbreviation key: DIP = degraded intake protein, UP = high protein, IGF-I = insulinlike growth factor-I, MP = medium protein, VIP = undegraded intake protein. INTRODUCTION
Received August 16. 1990. Accepted March 4, 1991. lChampion Peed Services Ltd., 12805-97 Street, Grande Prairie, AB, Canada TSV 6K1. 2Agriculture Canada Research Station, Fredricton, NB. Canada E3B 4Z7. 1991 1 Dairy Sci 74:2623-2632
Daily injections of recombinantly derived bST improved milk. yield during short-term (9, 23) and long-term administration (3, 29). In addition. it has been suggested that cows have a greater ability to respond to bST in mid to late lactation than in early lactation (23). The physiological mechanisms underlying this phenomenon are unknown. Bovine somatotropin is thought to be a homeorhetic hormone responsible for the partition of nutrients within the dairy cow (2). Hormones such as insulin, glucagon. somatostatin, and insulin-like growth factor-! (lGF-I) are involved intimately in the regulation of metabolism as directed by somatotropin. insulin and glucagon are responsible for homeostasis of blood glucose (6). Glucose serves as an essential substrate for synthesis of milk. lactose, the osmotic regulator of milk. volume, and for synthesis of milk. fat and protein (4). Somatostatin may have a role in inhibition of somatotropin release from the anterior pituitary but probably has a greater role in regulation of insulin and glucagon secretion and of digestive functions within the intestinal tract (6, 7). insulin-like growth factors play a major role in mammalian development and growth and appear to mediate many of the effects of somatotropin on growth (13). Evidence for a direct stimulation of milk. secretion by IGF-I has
2623
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de BOER ET AL.
been demonstrated (13). Blood concentrations of IGF-l increase after bST injection (8, 14), and this has been associated with a concomitant increase in lGF-l concentrations in mammary tissue (14). Because attempts to identify somatotropin receptors in bovine mammary tissue preparations have not been successful, it generally has been accepted that bST exerts its effect indirectly through intermediaries such as lGF-I. However, recent work in our laboratory using molecular biology techniques has demonstrated the presence of somatotropin receptor mRNA in mammary cells (15). These data provide strong evidence of a direct action of bST on the mammary gland. Dairy cows show imprOVed performance with increasing content of protein in the diet (17, 19); however, there are limited data on interactive effects of bST and dietary protein. Cows show a greater response to bST when fed diets adequate in protein (16% CP) than when fed those diets deficient in protein (11 % CP) (9). Decreased lGF-l concentrations associated with diets deficient in nutrients (22) partly may explain the lower response of dairy cows on low protein diets. The objectives of this study were to examine the effects of bST injection and dietary protein level on blood hormone and metabolite concentrations and to relate any changes to responses in milk yield and milk composition of cows in early lactation. MATERIALS AND METHODS Cows and Experimental Design
Four lactating Holstein cows fitted with rumen cannulas (10 cm Ld, Bar Diamond Inc., Parma, ill) and duodenal cannulas (27) were tethered in stalls and had free access to water. Cows were allocated to one of two total mixed rations, which differed in CP content [medium protein (MP) = 17.1 % CP; high protein (UP) = 23.6% CP], 5 to 9 d postpartum. At this time, cows also were assigned to receive either recombinantly derived bST (20.6 mg/d, American Cyanamid Inc., Princeton, NJ) or excipient [2 ml of sterile saline (.9% NaCl)]; however, no cow received bST or excipient until d 1 of period 2. Excipient or bST was injected subcutaneously in the shoulder area at 1730 h. The design of the experiment was a double cross]owna) of Dairy Science Vol. 74, No.8, 1991
over, with diet protein level as the variable, within either bST or excipient (control) blocks. The experiment consisted of four periods of 21 d each, allowing evaluation of the influence of duration of bST treatment on response characteristics. The first 16 d of each period constituted the diet adjustment period, and d 17 to 21 were for measurement of response parameters. Thus, the cows were examined in wk 2 to 13 of lactation. The four cows averaged 644, 527, 591, and 655 kg BW during the study. Diets and Feeding
Cows were offered one of the two total mixed rations at 0800 (67% of total fed) and 1800 h (33%). Both diets were 25% second- .05). 2No significant diet, bST, or diet by bST interaction (P :> .05),
3Significant diet by bST interaction (P < .05).
*p < .05.
number of animals assigned to each treatment. Animal numbers were limited due to the requirement for duodenal cannulation and the intensive nature of the sampling schedule (24). Although the original objective of comparing average cows not treated with bST with average cows stimulated for superior performance with bST was not realized, this study does provide a unique opportunity to compare the responses in digestive physiology and intermediary metabolism of bST-treated and control cows at similar levels of milk production. Therefore, the question of whether bSTtreated cows can be considered to be of superior genetic merit or must be treated as metabolically different animals can be addressed. Milk yields (Table 4) tended (P = .061) to increase with increased CP content of the diet. The milk production response to the HP diet suggests that cows in early lactation will show positive benefits to CP levels in the diet greater than those recommended by NRC (20).
Although there were no significant diet by bST or days on bST by bST-effects for milk yield, the dietary CP-induced increases in milk yield of 4.3 kg in the bST-treated cows versus the 2.2-kg increase in the control cows (Table 4) IIIlI
- *.-
/
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·····0····'
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o Figure 1. Effect of duration of bST treatment on plasma somatotropin (ST ng/ml) and serum insulin-like growth factor-l (lGF ng/ml) concentrations in dairy cows. Journal of Dairy Science Vol. 74, No.8, 1991
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de BOER ET AL.
TABLE 5. Hormone and metabolite concentrations in control and bST-treated cows fed medium (MP) and high (lIP) protein diets.
Somatotropin (ST), og/ml Insulin (1), og/ml Glucagon (G), pg/ml Somatostatin, pg/ml Insulin-like growth factor-I (1GF), og/ml FFA, roM Glucose, mgldl Molar ratios I:G I:ST IGF:ST
Significance1
bST
Control MP
lIP
MP
lIP
SEM
Diet
bST
9.82 1.01 112.6 348.0
8.93 1.14 129.9 343.4
11.09 1.04 92.1 406.9
10.74 1.20 99.6 371.4
1.21 .11 6.2 27.9
NS NS NS NS
NS NS
114.6 .267 71.5
123.9 .259 72.5
301.7 .279 77.1
348.0 .333 79.5
5.41 .38 41.1
5.21 .51 49.8
6.85 .35 96.0
6.86 .42 112.6
9.7 .051 .9 .46 .08 13.7
*
NS
*
*
NS NS
NS NS
NS NS NS
NS NS
*
lNS (P > .05). *p < .05.
suggests the existence of an associative effect of dietary CP and bST. An associative effect of dietary protein and bST has been observed previously (9). Milk data coupled with a negative change in BW of the cows fed the lIP diet and injected with bST suggests that these cows were mobilizing more body reserves as energy for milk synthesis than control cows. Milk composition and yields of components (Table 4) were not influenced by diet or bST treatment with the exception of milk protein percentage, which was increased by higher protein content in the diet. Milk composition usually is unaffected by bST treatment (29). There were no significant days on bST treatment by bST interactions nor days on bST treatment effects for milk composition. Intake. Dry matter intake was not influenced by dietary protein level (Table 4), although it was slightly higher for cows fed the lIP diet within both the control and bST blocks. There were no days on bST or days on bST by bST treatment effects. However, cows tended to reach peak feed intake at 21 and 42 d on bST, followed by a slight decrease at d 63 of bST treatment. Feed intake patterns within each day were not affected by dietary protein level or bST treatment, although, in general, cows tended to consume feed less rapidly when fed the MP diet (data not shown). Body weights (Table 4) were not significantly affected by bST injection or by dietary CP; Journal of Dairy Science Vol. 74, No.8, 1991
however, there was a significant diet by bST interaction for BW change. Hormone and Metabolite Responses
Dietary Protein Effects. Only IGF-I concentrations (Table 5) were influenced by dietary CP levels. Concentrations of IGF-I increased in cows fed the lIP diet. Previous research suggests that animals fed diets deficient in protein and energy have lower IGF-I concentrations in blood (22). In addition, IGF-I concentrations have been shown to be positively
40 , . . - - - - - - - - - - - - - - - - - - - .
BST &_._ . . . ---8
---._~
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21
- , - - - , - - - - - r - - , - -.. .,-----1 ~
~
0
1
2
a
WEEKS AFTER LAST INJECTlON
Figure 2. Mean weekly milk yields of cows given control or bST injections. The arrow indicates termination of control and bST injections.
2629
HORMONE RESPONSES TO SOMATOTROPIN AND PR01EIN
TABLE 6. Hormone and metabolite concentrations in control and bST-treated cows fed medium (MP) and high (HP) protein diets averaged by days on bST. SigniIlClIIlce l
Days on bST treatment 0 Somatotropin, ngfml Control bST Average
42
21
63
SEM
bST
Days
bST x Days
10.84 9.37 10.11
10.32 13.65 11.98
8.80 10.25 9.52
7.54 10.39 8.97
1.71 1.71 1.21
NS
NS
NS
.73 .50 .61
.91 .91 .91
1.12 1.53 1.33
1.55 1.53 1.54
.15 .15 .11
NS
L
NS
Insulin, ng/ml
Control bST Average Glucagon. pg/ml Control bST Average
121.5 99.2 110.4
134.2 86.2 lJO.2
120.8 105.5 113.2
108.6 92.4 100.5
8.8 8.8 6.2
•
NS
NS
Somatostatin, pg/ml Control bST Average
321.5 414.8 368.2
362.2 405.7 384.0
327.8 380.9 354.3
371.3 355.2 363.3
39.4 39.4 27.9
NS
NS
NS
Insulin-like growth factor-I, ng/ml Control bST Average
106.5 134.2 120.3
120.9 362.3 241.6
124.3 412.0 268.2
125.3 391.0 258.2
13.7 13.7 9.7
•
L, Q
•
NS
L
NS
NS
L, Q, C ..
FFA, mM Control bST Average Glucose, mg/dl Control bST Average
.377 .465 .421
70.9 70.8 70.9
INS (P> .05), .p < .05, L, Q. or C treatment.
.206 .395 .300 73.5 83.5 78.5
.216 .209 .212 71.5 77.3 74.4
.254 .155 .205 72.0 81.5 76.8
JJ72 .072 .051 1.3 1.3 .9
= significant (P < .OS) linear, quadratic, or cubic contrasts for days on bST
correlated to N balance in dairy cows (5). The increase in IGF-I concentrations coupled with the trend for cows consuming the HP diet to produce more milk: suggests that the MP diet was marginal in protein and that there was an improvement in the nutritional status of the cows fed the HP diet. The lack of effect of dietary protein on plasma glucose, insulin, and glucagon is in agreement with previous results (10). Somatotropin Effects. Somatotropin concentrations (Table 5) were not influenced by bST treatment, suggesting that the injected bST was cleared from the circulatory system within 17 h after subcutaneous injection. Small but significant increases in plasma somatotropin were observed by de Boer and Kennelly (11) in
cows given 20.6 mg bST/d when sampled 23 h postinjection. Insulin, glucose, FFA, and somatostatin (Table 5) concentrations were not affected by bST treatment. Our data compare favorably with those of previous studies examining the effect of bST on insulin, glucose (lO, 21), and FFA (21). Glucagon concentrations (Table 5) were lower in the bST-treated cows of this study. This effect was not observed in previous work (10, 21). Examination of the data at d 0 of bST injection showed that glucagon concentrations already were different between the two groups. These differences remained throughout the experiment and may be related to BW. Concentrations of IGF-I were increased by bST treatment. The two- to threefold increase is consistent with responses 00Journal of Dairy Science Vol. 74, No.8, 1991
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de BOER ET AL.
TABLE 7. Hormone and metabolite concenttations in control and bST-treated cows fed medium (MP) and high (lIP) protein diets averaged by time after feeding. SignificanceI
Time after feeding, h 1.5
0 Somatotropin, nglml Control bST Average Insulin, nglml Control bST Average
3.5
6
SEM
9
10.20 10.42 10.31
9.14 9.94 9.54
7.74 9.22 8.48
8.68 9.88 9.28
11.21 7.83 9.52
.91 .91 .64
.74 .88 .81
.93 .94 .93
J.lO 1.23 J.l6
1.34 1.23 1.29
.90 1.03 .97
.09 .09 .07
Time
bST x Time
NS
NS
L, Q
NS
L
NS
Glucagon, pglml Control bST Average
107.9 89.4 98.7
111.2 90.0 100.6
120.7 9J.l 105.9
136.4 88.8 112.6
114.0 112.3 113.1
7.5 7.5 5.3
Somatostatin, pglml Control bST Average
381.7 410.7 396.2
361.9 346.8 354.4
388.6 376.7 382.7
371.5 408.6 390.0
351.0 388.8 369.9
28.1 28.1 19.8
NS
NS
Insulin-like growth factor-I, nglml Control bST MP2 ~ Average
132.5 295.1 188.7 238.9 213.8
122.7 298.3 192.3 228.8 210.5
120.9 278.6 184.5 214.9 199.7
119.9 263.4 182.1 201.1 191.6
122.2 270.3 177.2 215.5 196.3
11.6 11.6 11.6 11.6 8.2
NS
NS
L, Q
NS
NS
NS
FFA, roM Control bST ~
W
Average Glucose, mgldl Control bST Average
.346 .462
.271 .537 .404 71.7 77.7 74.7
.204 .309 .222 .291 .256 66.6 78.7 72.6
.147 .221 .188 .180 .184 67.9 77.8 72.8
.157 .200 .169 .188 .178 72.3 78.8 75.5
.187 .248 .195 .240 .218 70.9 79.5 75.2
.042 .042 .042 .042 .029 1.7 1.7 1.2
INS (P > .05), *p < .05, Lor Q = significant (P < .05) linear or quadratic contrasts for time relative to feeding. 2Significant diet and diet by time interaction effects (P < .05).
served previously (11. 14). The general lack of difference in blood metabolite and hormone concentrations. with the exception of IGF-I. between control and bST-treated cows may reflect the similarity of production between the groups. These data support the concept that bST-stimulated cows are similar metabolically to cows of equal actual production not stimulated with bST. Effects of Days on bSI'. Of the hormones and metabolites measured. only insulin. IGF-I. glucose. and FFA showed significant effects of days on bST treatment. The linear increase in Journal of Dairy Science Vol. 74, No.8, 1991
insulin and glucose concentrations and the linear decrease in FFA concentrations (Table 6) with increasing days on bST are consistent with a shift to a more positive energy balance with advancing lactation. Similar changes have been observed previously (11, 12. 29). Glucose and IGF-I also showed significant interactions for days on bST treatment by bST treatment. The quadratic response in IGF-I concentrations reflects the response of IGF to bST treatment after d 0 (Figure 1. Table 6). The minimal changes in insulin. glucagon. somatostatin. glucose. and FFA were expected because the
HORMONE RESPONSES TO SOMATOTROPIN AND PROTEIN
bST-treated cows were stimulated to productivity equal to that of cows not treated with bST. Feeding Effects. Postprandial effects were evident for insulin, glucagon, and FFA (Table 7). Insulin concentrations increased in a linear and quadratic fashion with time after feeding. The pattern essentially suggests that a peak occurred 3.5 to 6 h postfeeding. This effect has been observed previously (1, 12). The FFA concentrations were lowest at the same time as insulin concentrations peaked, as has been shown previously (12). Glucagon concentrations increased linearly with time after feeding, although the absolute changes were small. Changes in glucagon concentrations after feeding have been observed previously (12). The changes in insulin and glucagon reflect their responsiveness to feed intake and subsequent production of glucose (1). CONCLUSIONS
An associative effect of dietary CP and bST for milk yield was apparent but did not reach statistical significance. Cows assigned to bST treatment appeared to have a lower genetic potential for milk production than controls such that bST treatment increased milk production to a level similar to that of the control group. Data presented here suggest that the nutrient requirements of bST-treated cows are similar to controls at similar levels of milk production. With the exception of IGF-I, the data demonstrate that bST had a minimal (on the basis of plasma concentrations) impact on specific hormones and metabolites when bSTtreated cows are stimulated to productivity equal to that of untreated cows. Plasma concentrations of hormones and metabolites reflect a balance between secretion and clearance rates. Secretion rates are dependent upon the rates of synthesis and degradation of the hormone or metabolite within the primary tissue and the actions of regulatory factors on that primary tissue. Clearance rates are dependent upon the tissues responsible for metabolizing the hormone or metabolite. Because milk production and feed intakes were similar between the treatment groups, rates of secretion and clearance of insulin, glucagon, somatostatin, glucose, and FFA are not likely to have been
2631
different between those groups. The rate of somatotropin clearance probably was increased, and the rate of secretion probably was decreased in response to bST injection. Serum concentrations of IGF-I increased as a result of bST injection, demonstrating the direct effect of bST on serum IGF-I concentrations, likely through an enhancement of IGF-I secretion from various tissues. Evidence for existence of both bST and IGF-I receptors in the mammary gland (15) suggests that exogenous bST can have both direct and indirect effects on the mammary gland. Down regulation of receptors with increasing concentrations of hormones or releasing factors may be responsible for the lack of a direct correlation between concentration and effect. Further research is needed to understand better the interrelationship of bST, IGF-I, and other factors (tissues, receptors, hormones, metabolites, and undetermined compounds) in the regulation of milk synthesis and secretion by the dairy cow. ACKNOWLEDGMENTS
The authors thank G. van Doesburg, 1. Collier, H. Lehman, T. Huwiler, and 1. Gorde of the University of Alberta Dairy Research Unit for care of the cows; R. Khorasani and C. Streeter for sample collection; and 1. Moffat, J. Dietrich, and L. Wright for sample analysis. This study was funded partially by the Alberta Agricultural Research Institute and Cyanamid Canada Inc., Markham, ON, Canada. REFERENCES
1 Armentano, L. E., S. E. Mills, G. de Boer, and 1. W. Young. 1984. Effects of feeding frequency on glucose concentration, glucose twnover, and insulin concentration in steers. 1. Dairy Sci. 67: 1445. 2 Bauman, D. E., and W. B. Currie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. 1. Dairy Sci. 63: 1514. 3 Bauman, D. E., P. 1. Eppard, M. J. DeGeeter, and G. M Lanza. 1985. Responses of high-producing dairy cows to long-term treatment with pituitary somatotropin and recombinant somatotropin. 1. Dairy Sci. 68: 1352. 4 Bickerslllffe, R., E. F. ADDison, and 1. L. Linzen. 1974. The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. 1. Agric. Sci. (Camb.) 82:71. 5 Binnerts, W. J., P.W.M van Adrichem, C.P.1. Oudenaarden, 1. E. Vogt, and 1. E. Wassenaar. 1982. Plasma somatomedin in dairy cows: effect of manageIournal of Dairy Science Vol. 74, No.8, 1991
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ment and feeding level. Neth. Milk Dairy 1. 36: 149. 6 Brockman, R. P., and B. Laarveld. 1986. Hormonal regulation of metabolism in ruminants: a review. Livest. Prod. Sci. 14:313. 7 Christensen, R. A., R. J. Christopherson, and 1. 1. Kennelly. 1990. Effect of somatostatin and chronic cold exposure on hormonal and metabolite concentration, metabolic rate, thermoregulation, and gut motility in sheep. Can. 1. Anim. Sci. 70:1073. 8 Davis, S. R., P. D. Gluckman, I. C. Hart, and H. V. Henderson. 1987. Effects of injecting growth hormone or thyroxine on milk production and blood plasma concentrations of insulin-like growth factors I and II in dairy cows. 1. Endocrinol. 114:17. 9 de Boer, G., and 1. 1. Kennelly. 1989. Effect of somatotropin injection and dietary protein on milk yield, and kinetics of hormones in dairy cows. 1. Dairy Sci. 72:419. 10 de Boer, G., and 1. 1. Kennelly. 1989. Effect of somatotropin and dietary protein concentration on hormone and metabolite responses to single injections of hormones and glucose. 1. Dairy Sci. 72:429. 11 de Boer, G., and 1. 1. Kennelly. 1990. Insulin-lilre growth factor-I induction in response to exogenous somatotropin. 1. Dairy Sci. 73(Suppl. 1):136.(Abstr.) 12 de Boer, G., A. Trenkle, and 1. W. Young. 1985. Glucagon, insulin, growth hormone, and some blood metabolites during energy restriction ketonemia of lactating cows. 1. Dairy Sci. 68:326. 13 Gilmour, R. S., C. G. Prosser, I. R. Fleet, L. Cocco, 1. C. Saunders, K. D. Brown, and A. N. Corps. 1988. From animal to molecule: aspects of the biology of insulin-like growth factors. Br. 1. Cancer 58(Suppl. 1X):23. 14 Glimm, D. R., V. E. Baracos, and 1. 1. Kennelly. 1988. Effect of bovine somatotropin on the distribution of immunoreactive insulin-like growth factor-I in lactating bovine mammary tissue. 1. Dairy Sci. 71: 2923. 15 Glimm, D. R., V. E. Baracos, and 1. 1. Kennelly. 1990. Molecular evidence for the presence of growth hormone receptors in the bovine mammary gland. 1. Endocrinol. 126:R5. 16 Harris, V., G. R. Faloona, and R. H. Unger. 1979. Glucagon. Page 643 in Methods of hormone radioimmunoassay. 2nd ed. B. M. Iaffe and H. R. Behrman, ed. Academic Press, Inc., New York, NY. 17 Holter, 1. B., 1. A. Byrne, C. G. Schwab. 1982. Crude
Journal of Dairy Science Vol. 74, No.8, 1991
protein for high milk production. 1. Dairy Sci. 65: 1175. 18 Krishnamoorthy, U., C.I. Sniffen, M. D. Stern, and P. 1. Van Soest. 1983. Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen undegraded nitrogen content of feedstuffs. Br. 1. Nutr. 50:555. 19 MacLeod, G. K., D. G. Grieve, I. McMillan, and G. C. Smith. 1984. Effect of varying protein and energy densities in complete rations fed to cows in first lactation. 1. Dairy Sci. 69:713. 20 National Research Council. 1989. Nutrient requirements of dairy cattle. 6th rev. ed. Nat!. Acad. Sci., Washington, DC. 21 Peel, C. I., T. 1. Fronk, D. E. Bauman, and R. C. Gorewit. 1982. Lactational response to exogenous growth hormone and abomasal infusion of glucosesodium caseinate. 1. Nutr. 112:1770. 22 Phillips, S., and T. G. Unterman. 1984. Somatomedin activity in disorders of nutrition and metabolism. Clin. Endocrinol. Metab. 13:145. 23 Richard, A. L., S. N. McCutcheon, and D. E. Bauman. 1985. Responses of dairy cows to exogenous bovine growth hormone administered during early lactation. 1. Dairy Sci. 68:2385. 24 Robinson, P. H., G. de Boer, and 1. J. Kennelly. 1991. Influence of bovine somatotropin treatment on rumen fermentation, as well as forestomach and whole tract digestion, in dairy cows fed medium or high protein diets. 1. Dairy Sci. 74:(in press). 25 Robinson, P. H., and 1. J. Kennelly. 1988. Influence of ammoniation of high moisture barley on its in situ rumen degradation and influence on rumen fermentation in dairy cows. Can. J. Anim. Sci. 68:839. 26 Robinson, P. H., and J. J. Kennelly. 1989. Influence of ammoniation of high-moisture barley on digestibility, kinetics of rumen ingesta turnover, and milk production of dairy cows. Can. 1. Anim. Sci. 69:195. 27 Robinson, P. H., and 1. J. Kennelly. 1990. Evaluation of a duodenal cannula for dairy cattle. 1. Dairy Sci. 73:3146. 28 SASilll User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 29 Soderholm., C. G., D. E. Onerby, 1. G. Linn, F. R. Ehle, J. E. Wheaton, W. P. Hansen, and R. J. Annexstad. 1988. Effects of recombinant bovine somatotropin on milk production, body composition, and physiological parameters. 1. Dairy Sci. 71:355.
tions and producing equal amounts of milk. as control cows did not show major physiological differences in hormones and metabolites with the exception of insulin-like growth factor-I. (Key words: somatotropin, protein. hormones, dairy cows)
The effects of bST injection and dietary protein level on blood hormone and metabolite concentrations were examined in four mature Holstein cows in a double crossover design. Cows were assigned at d 5 to 9 postpartum to receive daily injections of either a control (saline) solution or 20.6 mg of bST. Four 3-wk. periods were used during which one cow from each group was fed a medium protein diet (17.1% CP), and the other received a high protein diet (23.6% CP). Injections of bST or control solutions began on d 0 of the second period. intakes of OM were not influenced by dietary protein or bST injection. Milk. yield tended to increase with increased CP level but was not affected by bST injection. Based on the rate and extent of decline in milk. production after cessation of bST injection, the cows assigned to bST had lower milk. production potential than control cows. Thus, the effect of bST injection apparently was to enhance milk yield to levels similar to those of controls. There were no significant CP level or bST injection effects on glucose, FFA, somatostatin, or somatotropin concentrations. Glucagon concentrations were higher in bST-treated cows. Concentrations of insulin-like growth factor-I were increased with increased CP level and also with bST injection. Significant effects of days on bST were observed for insulin, insulin-like growth factor-I, glucose, and FFA. Cows given bST injec-
Abbreviation key: DIP = degraded intake protein, UP = high protein, IGF-I = insulinlike growth factor-I, MP = medium protein, VIP = undegraded intake protein. INTRODUCTION
Received August 16. 1990. Accepted March 4, 1991. lChampion Peed Services Ltd., 12805-97 Street, Grande Prairie, AB, Canada TSV 6K1. 2Agriculture Canada Research Station, Fredricton, NB. Canada E3B 4Z7. 1991 1 Dairy Sci 74:2623-2632
Daily injections of recombinantly derived bST improved milk. yield during short-term (9, 23) and long-term administration (3, 29). In addition. it has been suggested that cows have a greater ability to respond to bST in mid to late lactation than in early lactation (23). The physiological mechanisms underlying this phenomenon are unknown. Bovine somatotropin is thought to be a homeorhetic hormone responsible for the partition of nutrients within the dairy cow (2). Hormones such as insulin, glucagon. somatostatin, and insulin-like growth factor-! (lGF-I) are involved intimately in the regulation of metabolism as directed by somatotropin. insulin and glucagon are responsible for homeostasis of blood glucose (6). Glucose serves as an essential substrate for synthesis of milk. lactose, the osmotic regulator of milk. volume, and for synthesis of milk. fat and protein (4). Somatostatin may have a role in inhibition of somatotropin release from the anterior pituitary but probably has a greater role in regulation of insulin and glucagon secretion and of digestive functions within the intestinal tract (6, 7). insulin-like growth factors play a major role in mammalian development and growth and appear to mediate many of the effects of somatotropin on growth (13). Evidence for a direct stimulation of milk. secretion by IGF-I has
2623
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de BOER ET AL.
been demonstrated (13). Blood concentrations of IGF-l increase after bST injection (8, 14), and this has been associated with a concomitant increase in lGF-l concentrations in mammary tissue (14). Because attempts to identify somatotropin receptors in bovine mammary tissue preparations have not been successful, it generally has been accepted that bST exerts its effect indirectly through intermediaries such as lGF-I. However, recent work in our laboratory using molecular biology techniques has demonstrated the presence of somatotropin receptor mRNA in mammary cells (15). These data provide strong evidence of a direct action of bST on the mammary gland. Dairy cows show imprOVed performance with increasing content of protein in the diet (17, 19); however, there are limited data on interactive effects of bST and dietary protein. Cows show a greater response to bST when fed diets adequate in protein (16% CP) than when fed those diets deficient in protein (11 % CP) (9). Decreased lGF-l concentrations associated with diets deficient in nutrients (22) partly may explain the lower response of dairy cows on low protein diets. The objectives of this study were to examine the effects of bST injection and dietary protein level on blood hormone and metabolite concentrations and to relate any changes to responses in milk yield and milk composition of cows in early lactation. MATERIALS AND METHODS Cows and Experimental Design
Four lactating Holstein cows fitted with rumen cannulas (10 cm Ld, Bar Diamond Inc., Parma, ill) and duodenal cannulas (27) were tethered in stalls and had free access to water. Cows were allocated to one of two total mixed rations, which differed in CP content [medium protein (MP) = 17.1 % CP; high protein (UP) = 23.6% CP], 5 to 9 d postpartum. At this time, cows also were assigned to receive either recombinantly derived bST (20.6 mg/d, American Cyanamid Inc., Princeton, NJ) or excipient [2 ml of sterile saline (.9% NaCl)]; however, no cow received bST or excipient until d 1 of period 2. Excipient or bST was injected subcutaneously in the shoulder area at 1730 h. The design of the experiment was a double cross]owna) of Dairy Science Vol. 74, No.8, 1991
over, with diet protein level as the variable, within either bST or excipient (control) blocks. The experiment consisted of four periods of 21 d each, allowing evaluation of the influence of duration of bST treatment on response characteristics. The first 16 d of each period constituted the diet adjustment period, and d 17 to 21 were for measurement of response parameters. Thus, the cows were examined in wk 2 to 13 of lactation. The four cows averaged 644, 527, 591, and 655 kg BW during the study. Diets and Feeding
Cows were offered one of the two total mixed rations at 0800 (67% of total fed) and 1800 h (33%). Both diets were 25% second- .05). 2No significant diet, bST, or diet by bST interaction (P :> .05),
3Significant diet by bST interaction (P < .05).
*p < .05.
number of animals assigned to each treatment. Animal numbers were limited due to the requirement for duodenal cannulation and the intensive nature of the sampling schedule (24). Although the original objective of comparing average cows not treated with bST with average cows stimulated for superior performance with bST was not realized, this study does provide a unique opportunity to compare the responses in digestive physiology and intermediary metabolism of bST-treated and control cows at similar levels of milk production. Therefore, the question of whether bSTtreated cows can be considered to be of superior genetic merit or must be treated as metabolically different animals can be addressed. Milk yields (Table 4) tended (P = .061) to increase with increased CP content of the diet. The milk production response to the HP diet suggests that cows in early lactation will show positive benefits to CP levels in the diet greater than those recommended by NRC (20).
Although there were no significant diet by bST or days on bST by bST-effects for milk yield, the dietary CP-induced increases in milk yield of 4.3 kg in the bST-treated cows versus the 2.2-kg increase in the control cows (Table 4) IIIlI
- *.-
/
/ /
... B- ..
1Ir.a:JHlML 1Ir.wr
·····0····'
IlII4lIIN11llll
- '" -
IlIFlIIIr
/ /
c···, ... ~
1Q.D ;:...
·0
. __ 0 ••..•..
.-
o Figure 1. Effect of duration of bST treatment on plasma somatotropin (ST ng/ml) and serum insulin-like growth factor-l (lGF ng/ml) concentrations in dairy cows. Journal of Dairy Science Vol. 74, No.8, 1991
2628
de BOER ET AL.
TABLE 5. Hormone and metabolite concentrations in control and bST-treated cows fed medium (MP) and high (lIP) protein diets.
Somatotropin (ST), og/ml Insulin (1), og/ml Glucagon (G), pg/ml Somatostatin, pg/ml Insulin-like growth factor-I (1GF), og/ml FFA, roM Glucose, mgldl Molar ratios I:G I:ST IGF:ST
Significance1
bST
Control MP
lIP
MP
lIP
SEM
Diet
bST
9.82 1.01 112.6 348.0
8.93 1.14 129.9 343.4
11.09 1.04 92.1 406.9
10.74 1.20 99.6 371.4
1.21 .11 6.2 27.9
NS NS NS NS
NS NS
114.6 .267 71.5
123.9 .259 72.5
301.7 .279 77.1
348.0 .333 79.5
5.41 .38 41.1
5.21 .51 49.8
6.85 .35 96.0
6.86 .42 112.6
9.7 .051 .9 .46 .08 13.7
*
NS
*
*
NS NS
NS NS
NS NS NS
NS NS
*
lNS (P > .05). *p < .05.
suggests the existence of an associative effect of dietary CP and bST. An associative effect of dietary protein and bST has been observed previously (9). Milk data coupled with a negative change in BW of the cows fed the lIP diet and injected with bST suggests that these cows were mobilizing more body reserves as energy for milk synthesis than control cows. Milk composition and yields of components (Table 4) were not influenced by diet or bST treatment with the exception of milk protein percentage, which was increased by higher protein content in the diet. Milk composition usually is unaffected by bST treatment (29). There were no significant days on bST treatment by bST interactions nor days on bST treatment effects for milk composition. Intake. Dry matter intake was not influenced by dietary protein level (Table 4), although it was slightly higher for cows fed the lIP diet within both the control and bST blocks. There were no days on bST or days on bST by bST treatment effects. However, cows tended to reach peak feed intake at 21 and 42 d on bST, followed by a slight decrease at d 63 of bST treatment. Feed intake patterns within each day were not affected by dietary protein level or bST treatment, although, in general, cows tended to consume feed less rapidly when fed the MP diet (data not shown). Body weights (Table 4) were not significantly affected by bST injection or by dietary CP; Journal of Dairy Science Vol. 74, No.8, 1991
however, there was a significant diet by bST interaction for BW change. Hormone and Metabolite Responses
Dietary Protein Effects. Only IGF-I concentrations (Table 5) were influenced by dietary CP levels. Concentrations of IGF-I increased in cows fed the lIP diet. Previous research suggests that animals fed diets deficient in protein and energy have lower IGF-I concentrations in blood (22). In addition, IGF-I concentrations have been shown to be positively
40 , . . - - - - - - - - - - - - - - - - - - - .
BST &_._ . . . ---8
---._~
'&'" ·,',······,ti-----.- . ----i::.
21
- , - - - , - - - - - r - - , - -.. .,-----1 ~
~
0
1
2
a
WEEKS AFTER LAST INJECTlON
Figure 2. Mean weekly milk yields of cows given control or bST injections. The arrow indicates termination of control and bST injections.
2629
HORMONE RESPONSES TO SOMATOTROPIN AND PR01EIN
TABLE 6. Hormone and metabolite concentrations in control and bST-treated cows fed medium (MP) and high (HP) protein diets averaged by days on bST. SigniIlClIIlce l
Days on bST treatment 0 Somatotropin, ngfml Control bST Average
42
21
63
SEM
bST
Days
bST x Days
10.84 9.37 10.11
10.32 13.65 11.98
8.80 10.25 9.52
7.54 10.39 8.97
1.71 1.71 1.21
NS
NS
NS
.73 .50 .61
.91 .91 .91
1.12 1.53 1.33
1.55 1.53 1.54
.15 .15 .11
NS
L
NS
Insulin, ng/ml
Control bST Average Glucagon. pg/ml Control bST Average
121.5 99.2 110.4
134.2 86.2 lJO.2
120.8 105.5 113.2
108.6 92.4 100.5
8.8 8.8 6.2
•
NS
NS
Somatostatin, pg/ml Control bST Average
321.5 414.8 368.2
362.2 405.7 384.0
327.8 380.9 354.3
371.3 355.2 363.3
39.4 39.4 27.9
NS
NS
NS
Insulin-like growth factor-I, ng/ml Control bST Average
106.5 134.2 120.3
120.9 362.3 241.6
124.3 412.0 268.2
125.3 391.0 258.2
13.7 13.7 9.7
•
L, Q
•
NS
L
NS
NS
L, Q, C ..
FFA, mM Control bST Average Glucose, mg/dl Control bST Average
.377 .465 .421
70.9 70.8 70.9
INS (P> .05), .p < .05, L, Q. or C treatment.
.206 .395 .300 73.5 83.5 78.5
.216 .209 .212 71.5 77.3 74.4
.254 .155 .205 72.0 81.5 76.8
JJ72 .072 .051 1.3 1.3 .9
= significant (P < .OS) linear, quadratic, or cubic contrasts for days on bST
correlated to N balance in dairy cows (5). The increase in IGF-I concentrations coupled with the trend for cows consuming the HP diet to produce more milk: suggests that the MP diet was marginal in protein and that there was an improvement in the nutritional status of the cows fed the HP diet. The lack of effect of dietary protein on plasma glucose, insulin, and glucagon is in agreement with previous results (10). Somatotropin Effects. Somatotropin concentrations (Table 5) were not influenced by bST treatment, suggesting that the injected bST was cleared from the circulatory system within 17 h after subcutaneous injection. Small but significant increases in plasma somatotropin were observed by de Boer and Kennelly (11) in
cows given 20.6 mg bST/d when sampled 23 h postinjection. Insulin, glucose, FFA, and somatostatin (Table 5) concentrations were not affected by bST treatment. Our data compare favorably with those of previous studies examining the effect of bST on insulin, glucose (lO, 21), and FFA (21). Glucagon concentrations (Table 5) were lower in the bST-treated cows of this study. This effect was not observed in previous work (10, 21). Examination of the data at d 0 of bST injection showed that glucagon concentrations already were different between the two groups. These differences remained throughout the experiment and may be related to BW. Concentrations of IGF-I were increased by bST treatment. The two- to threefold increase is consistent with responses 00Journal of Dairy Science Vol. 74, No.8, 1991
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de BOER ET AL.
TABLE 7. Hormone and metabolite concenttations in control and bST-treated cows fed medium (MP) and high (lIP) protein diets averaged by time after feeding. SignificanceI
Time after feeding, h 1.5
0 Somatotropin, nglml Control bST Average Insulin, nglml Control bST Average
3.5
6
SEM
9
10.20 10.42 10.31
9.14 9.94 9.54
7.74 9.22 8.48
8.68 9.88 9.28
11.21 7.83 9.52
.91 .91 .64
.74 .88 .81
.93 .94 .93
J.lO 1.23 J.l6
1.34 1.23 1.29
.90 1.03 .97
.09 .09 .07
Time
bST x Time
NS
NS
L, Q
NS
L
NS
Glucagon, pglml Control bST Average
107.9 89.4 98.7
111.2 90.0 100.6
120.7 9J.l 105.9
136.4 88.8 112.6
114.0 112.3 113.1
7.5 7.5 5.3
Somatostatin, pglml Control bST Average
381.7 410.7 396.2
361.9 346.8 354.4
388.6 376.7 382.7
371.5 408.6 390.0
351.0 388.8 369.9
28.1 28.1 19.8
NS
NS
Insulin-like growth factor-I, nglml Control bST MP2 ~ Average
132.5 295.1 188.7 238.9 213.8
122.7 298.3 192.3 228.8 210.5
120.9 278.6 184.5 214.9 199.7
119.9 263.4 182.1 201.1 191.6
122.2 270.3 177.2 215.5 196.3
11.6 11.6 11.6 11.6 8.2
NS
NS
L, Q
NS
NS
NS
FFA, roM Control bST ~
W
Average Glucose, mgldl Control bST Average
.346 .462
.271 .537 .404 71.7 77.7 74.7
.204 .309 .222 .291 .256 66.6 78.7 72.6
.147 .221 .188 .180 .184 67.9 77.8 72.8
.157 .200 .169 .188 .178 72.3 78.8 75.5
.187 .248 .195 .240 .218 70.9 79.5 75.2
.042 .042 .042 .042 .029 1.7 1.7 1.2
INS (P > .05), *p < .05, Lor Q = significant (P < .05) linear or quadratic contrasts for time relative to feeding. 2Significant diet and diet by time interaction effects (P < .05).
served previously (11. 14). The general lack of difference in blood metabolite and hormone concentrations. with the exception of IGF-I. between control and bST-treated cows may reflect the similarity of production between the groups. These data support the concept that bST-stimulated cows are similar metabolically to cows of equal actual production not stimulated with bST. Effects of Days on bSI'. Of the hormones and metabolites measured. only insulin. IGF-I. glucose. and FFA showed significant effects of days on bST treatment. The linear increase in Journal of Dairy Science Vol. 74, No.8, 1991
insulin and glucose concentrations and the linear decrease in FFA concentrations (Table 6) with increasing days on bST are consistent with a shift to a more positive energy balance with advancing lactation. Similar changes have been observed previously (11, 12. 29). Glucose and IGF-I also showed significant interactions for days on bST treatment by bST treatment. The quadratic response in IGF-I concentrations reflects the response of IGF to bST treatment after d 0 (Figure 1. Table 6). The minimal changes in insulin. glucagon. somatostatin. glucose. and FFA were expected because the
HORMONE RESPONSES TO SOMATOTROPIN AND PROTEIN
bST-treated cows were stimulated to productivity equal to that of cows not treated with bST. Feeding Effects. Postprandial effects were evident for insulin, glucagon, and FFA (Table 7). Insulin concentrations increased in a linear and quadratic fashion with time after feeding. The pattern essentially suggests that a peak occurred 3.5 to 6 h postfeeding. This effect has been observed previously (1, 12). The FFA concentrations were lowest at the same time as insulin concentrations peaked, as has been shown previously (12). Glucagon concentrations increased linearly with time after feeding, although the absolute changes were small. Changes in glucagon concentrations after feeding have been observed previously (12). The changes in insulin and glucagon reflect their responsiveness to feed intake and subsequent production of glucose (1). CONCLUSIONS
An associative effect of dietary CP and bST for milk yield was apparent but did not reach statistical significance. Cows assigned to bST treatment appeared to have a lower genetic potential for milk production than controls such that bST treatment increased milk production to a level similar to that of the control group. Data presented here suggest that the nutrient requirements of bST-treated cows are similar to controls at similar levels of milk production. With the exception of IGF-I, the data demonstrate that bST had a minimal (on the basis of plasma concentrations) impact on specific hormones and metabolites when bSTtreated cows are stimulated to productivity equal to that of untreated cows. Plasma concentrations of hormones and metabolites reflect a balance between secretion and clearance rates. Secretion rates are dependent upon the rates of synthesis and degradation of the hormone or metabolite within the primary tissue and the actions of regulatory factors on that primary tissue. Clearance rates are dependent upon the tissues responsible for metabolizing the hormone or metabolite. Because milk production and feed intakes were similar between the treatment groups, rates of secretion and clearance of insulin, glucagon, somatostatin, glucose, and FFA are not likely to have been
2631
different between those groups. The rate of somatotropin clearance probably was increased, and the rate of secretion probably was decreased in response to bST injection. Serum concentrations of IGF-I increased as a result of bST injection, demonstrating the direct effect of bST on serum IGF-I concentrations, likely through an enhancement of IGF-I secretion from various tissues. Evidence for existence of both bST and IGF-I receptors in the mammary gland (15) suggests that exogenous bST can have both direct and indirect effects on the mammary gland. Down regulation of receptors with increasing concentrations of hormones or releasing factors may be responsible for the lack of a direct correlation between concentration and effect. Further research is needed to understand better the interrelationship of bST, IGF-I, and other factors (tissues, receptors, hormones, metabolites, and undetermined compounds) in the regulation of milk synthesis and secretion by the dairy cow. ACKNOWLEDGMENTS
The authors thank G. van Doesburg, 1. Collier, H. Lehman, T. Huwiler, and 1. Gorde of the University of Alberta Dairy Research Unit for care of the cows; R. Khorasani and C. Streeter for sample collection; and 1. Moffat, J. Dietrich, and L. Wright for sample analysis. This study was funded partially by the Alberta Agricultural Research Institute and Cyanamid Canada Inc., Markham, ON, Canada. REFERENCES
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Journal of Dairy Science Vol. 74, No.8, 1991
protein for high milk production. 1. Dairy Sci. 65: 1175. 18 Krishnamoorthy, U., C.I. Sniffen, M. D. Stern, and P. 1. Van Soest. 1983. Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen undegraded nitrogen content of feedstuffs. Br. 1. Nutr. 50:555. 19 MacLeod, G. K., D. G. Grieve, I. McMillan, and G. C. Smith. 1984. Effect of varying protein and energy densities in complete rations fed to cows in first lactation. 1. Dairy Sci. 69:713. 20 National Research Council. 1989. Nutrient requirements of dairy cattle. 6th rev. ed. Nat!. Acad. Sci., Washington, DC. 21 Peel, C. I., T. 1. Fronk, D. E. Bauman, and R. C. Gorewit. 1982. Lactational response to exogenous growth hormone and abomasal infusion of glucosesodium caseinate. 1. Nutr. 112:1770. 22 Phillips, S., and T. G. Unterman. 1984. Somatomedin activity in disorders of nutrition and metabolism. Clin. Endocrinol. Metab. 13:145. 23 Richard, A. L., S. N. McCutcheon, and D. E. Bauman. 1985. Responses of dairy cows to exogenous bovine growth hormone administered during early lactation. 1. Dairy Sci. 68:2385. 24 Robinson, P. H., G. de Boer, and 1. J. Kennelly. 1991. Influence of bovine somatotropin treatment on rumen fermentation, as well as forestomach and whole tract digestion, in dairy cows fed medium or high protein diets. 1. Dairy Sci. 74:(in press). 25 Robinson, P. H., and 1. J. Kennelly. 1988. Influence of ammoniation of high moisture barley on its in situ rumen degradation and influence on rumen fermentation in dairy cows. Can. J. Anim. Sci. 68:839. 26 Robinson, P. H., and J. J. Kennelly. 1989. Influence of ammoniation of high-moisture barley on digestibility, kinetics of rumen ingesta turnover, and milk production of dairy cows. Can. 1. Anim. Sci. 69:195. 27 Robinson, P. H., and 1. J. Kennelly. 1990. Evaluation of a duodenal cannula for dairy cattle. 1. Dairy Sci. 73:3146. 28 SASilll User's Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 29 Soderholm., C. G., D. E. Onerby, 1. G. Linn, F. R. Ehle, J. E. Wheaton, W. P. Hansen, and R. J. Annexstad. 1988. Effects of recombinant bovine somatotropin on milk production, body composition, and physiological parameters. 1. Dairy Sci. 71:355.
