Temporal pattern of insulin-like growth factor-I response to exogenous bovine somatotropin in lactating cows

Temporal pattern of insulin-like growth factor-I response to exogenous bovine somatotropin in lactating cows

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 6(3):263-274, 1989 TEMPORAL PATTERN OF INSULIN-LIKE GROWTH FACTOR-I RESPONSE TO EXOGENOUS BOVINE SOMATOTROPIN IN ...

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DOMESTIC ANIMAL ENDOCRINOLOGY

Vol. 6(3):263-274, 1989

TEMPORAL PATTERN OF INSULIN-LIKE GROWTH FACTOR-I RESPONSE TO EXOGENOUS BOVINE SOMATOTROPIN IN LACTATING COWS 1 W.S. Cohick, K. Plaut, S.J. Sechen2, and D.E. Bauman3 Department of Animal Science Cornell University Ithaca, NY 14853-4801 Received January 3, 1989

ABSTRACT The effect of exogenous bovine somatotropin (bST) treatment on the temporal pattern of insulin-like growth factor-I (IGF-I) in serum of four multiparous Holstein cows was examined. Cows (190 -+ 24 days postpartum) were treated with daily subcutaneous injections of recombinant bST (40 mg) or excipient for 12-day periods in a crossover experimental design. During excipient treatment, concentrations of IGF-I in serum were relatively constant throughout the day and averaged 70 ng/ml. Following the first bST injection, serum IGF-I began increasing after a lag of 5 to 7 hr and progressively increased over the first 2 days of treatment. Serum IGF-I levels were approximately 2fold greater than control values at the end of day 1 of bST treatment, with a 3-fold elevation observed at the end of day 2. Concentrations of IGF-I in serum plateaued by day 3 of bST treatment. Serum concentrations of IGF-I did not follow the oscillating pattern of bST in serum resulting from daily bST injections. Milk yield (3.5% fatcorrected) plateaued after 6 days of bST treatment and was increased 61% ( + 15.3 kg). Both IGF-I and milk yield remained essentially constant across days for the remainder of treatment. Following cessation of treatment, serum IGF-I and milk yield gradually declined, returning to control values after approximately 4 days. The temporal pattern of circulating concentrations of IGF-I is consistent with a role for IGF-I in mediating a portion of the effects of exogenous bST in lactating cows. INTRODUCTION Administration of e x o g e n o u s bovine s o m a t o t r o p i n (bST) to lactating cows results in a substantial increase in milk p r o d u c t i o n (1). While specific mechanisms are not well-defined, it is clear that treatment with e x o g e n o u s ST results in a series of orchestrated changes involving m a n y tissues and physiological processes (1,2,3). A p o r t i o n of the effects involve alterations in metabolism of a n u m b e r o f tissues (e.g., liver and adipose) so that a greater p o r t i o n of nutrients are partitioned for milk synthesis (lactation) or lean tissue accretion (growth). In the g r o w i n g animal, m a n y of the mitogenic, g r o w t h - p r o m o t i n g activities o f ST (e.g., effects on b o n e and muscle) are t h o u g h t to be mediated by insulinlike g r o w t h factor-I (IGF-I) (2,3,4). Circulating concentrations of IGF-I are d e p e n d e n t on ST, as s h o w n by their r e d u c e d levels during ST deficiency and elevated c o n c e n t r a t i o n s d u r i n g ST excess (5). IGF-I may also affect m a m m a r y tissue of lactating cows, because lactation increased IGF-I r e c e p t o r n u m b e r s in m a m m a r y tissue from lactating as c o m p a r e d to non-lactating cows (6). However, information on c o n c e n t r a t i o n s o f IGF-I in serum and their regulation by ST are limited in lactating dairy cows. The objective o f the present study was to d e t e r m i n e the t e m p o r a l pattern of IGF-I response to e x o g e n o u s som a t o t r o p i n in serum o f lactating cows. Copyright © 1989 by DOMENDO, INC.

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MATERIALS AND METHODS Four multiparous Holstein cows, averaging 25 + 5 (mean _+ SD) kg milk per day and 190 _+ 24 days postpartum, were used in a crossover experimental design. Cows (650 kg body weight) were housed in a tie-stall barn beginning two weeks prior to the start of the experiment. A total mixed diet was offered to provide 120% NRC (7) recommendations for net energy and crude protein intake. Cows were fed hourly to minimize fluctuations in hormones and metabolites associated with meal feeding. Long-stem grass hay (approximately 2 kg/day) was offered daily to maintain normal rumen function. Orts were recorded at 0600 and 1800 hr. Feed was sampled daily and composited at 2week intervals for nutrient analysis (Forage Testing Laboratory, NYDHIC, Ithaca, NY). Results are presented in Table 1. Cows were milked at 0600 and 1800 hr. Samples were taken at each milking for determination of fat and protein content, and somatic cell count (8). Milk energy was estimated according to Tyrrell and Reid (9). Treatment periods were 12 days in length with an intervening 13-day period. Cows received daily subcutaneous injections (4 ml) of 40 mg methionyl bST (sometribove, Lot #M-91-07226; Monsanto Company, St. Louis, MO) or excipient (75 mM sodium bicarbonate) in the shoulder region at 1900 hr. Jugular catheters were inserted at least 24 hr prior to the first day of each treatment period and maintained throughout the study. Blood samples were taken at 60, 30, and 0 min prior to the first injection of each period, then every other hr for 48 hr beginning 1 hr after the first injection. On days 3,4,5 and 12 of treatment, 3 blood samples were taken approximately 20 min apart beginning at 1400 hr (i.e., 19 hr post-injection) to monitor daily changes in serum concentrations of IGF-I. Following the last injection of the second period, samples were taken every 6 hr for 9 days, then every 12 hr for 2.5 days. Blood was held at room temperature for 2 to 4 hr, then refrigerated for approximately 20 hr. Serum was harvested following centrifugation and stored at --20 C until analysis. Milk yield and composition data for days 6 to 12 of each treatment period were averaged and compared between control and bST treatment by use of analysis of variance for crossover design (10). Somatotropin analysis: Concentrations of ST in serum were determined by double antibody radioimmunoassay (RIA) using antibody supplied by NIADDK (anti-oGH-2 [AFP-CO123080]). Bovine ST from Miles Laboratory (Lot 12; 1.3 IU/mg protein) was used for iodination and standards. For iodination, 5 I~g bST was solubilized in Tris buffer (25 mM Tris-HC1, .15 M NaCI, .02% Na azide; pH 7.2) and reacted with Na~25I (Amersham, Arlington Heights, IL) in the presence of iodogen (1,3,4,6-tetrachloro-3ct, 6¢z diphenylglycoluril, Pierce Chemical Co., St. Louis, MO) for 2 min. Radiolabeled protein was separated from free Na~25I by gel filtration on a Sephadex G-75 column (1)< 20 cm) equilibrated with Tris buffer containing .5% bovine serum albumin (BSA; RIA grade, Sigma Chemical Co., St. Louis, MO). Fractions were collected into polypropylene tubes containing 1 ml cold Tris buffer with .5% BSA. Specific activity of iodinated hormone was 120 to 140 ~tCi/~tg. Precipitation of iodinated trace with trichloroacetic acid (TCA) was > 98%. Recovery of an added mass was 95.8%, with parallelism exhibited from 50 to 300 ~tl of serum. Sensitivity of the assay was .2 to 8.0 ng/tube. Intra- and inter-assay CV (n----4) were 5.4 and 6.5%.

IGF-I RESPONSE TO bST IN COWS

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IGF-I a n a l y s i s : IGF-I binding proteins were dissociated and inactivated by incubation of serum with acid, as described by Underwood et al. (11). Serum (100 ~tl) and .1 M glycyl-glycine HCI, pH 2.1 (120 ~tl) were incubated at pH 3.6 to 3.8 in polystyrene tubes for 48 hr at 37 C. Incubated samples were neutralized with l O~tl 1M NaOH prior to RIA. Concentrations of IGF-I in incubated samples were determined by RIA under equilibrium conditions. Recombinant n-methionyl human IGF°I (SmC, Lot 74244; provided by Dr. B.D. Burleigh, Pitman-Moore, Northbrook, IL) was used for iodination and standards. The sequences of human and bovine IGF-I have been shown to be identical (12). IGF-I (5 ttg) was iodinated using iodogen as described for ST. Reaction time was 20 to 30 sec, and a Sephadex G-50 column (1 >( 20 cm) was used to separate iodinated hormone from free Na~25I. Specific activity was 100 to 130 ~tCi/~tg and TCA precipitation was > 98%. Assay buffer was 150 mM sodium phosphate, .02% protamine sulfate, .25% BSA, and .02% sodium azide, pH 7.5. Monoclonal antibody to human IGF-I (3D1/2/1) was provided by Dr. R.C. Baxter (Royal Prince Alfred Hospital, Camperdown, N.S.W., Australia). Specificity of this antibody has been reported by Baxter et al. (13). First antibody was diluted 1:15,000 in assay buffer containing 1:13.5 normal mouse serum (Cambridge Medical Diagnostics, Billerica, MA). Final dilution of the first antibody was 1:75,000. Incubated sample (20 or 40 ~tl) or IGF-I standard (100 ~tl), assay buffer, 100 ttl first antibody, and 100 ~tl 125I-IGF-I (14 to 18,000 cpm/tube) were added to polystyrene tubes to provide a final assay volume of .5 ml. After incubation for 16 hr at 4 C, 10 !~1 goat anti-mouse IgG (Pel-Freez Biologicals, Rogers, AK) were added to each tube to separate bound from free hormone. Following a 30-min incubation at 4 C, 1 ml cold 6% (w/v) polyethylene glycol 8000 (PEG) in .15 M NaCI was added to each tube. Tubes were centrifuged at 2050 X g at 4 C for 20 min, decanted, and blotted. One ml cold 4% PEG (w/v) in . 15 M NaCI was then added to each tube. Following a second 20-min centrifugation at 2050 X g at 4 C, tubes were decanted, blotted, and counted in a gamma counter. Recovery of unlabeled IGF-I (.6 or 1.0 ng/assay tube) added to serum after glycyl-glycine treatment averaged 107.5 -+ 14.7% (mean _+ SD; n----5). Recovery of unlabeled IGF.I (1.3 or 2.2 ng/assay tube) added to serum and incubated with glycyl-glycine prior to measurement by RIA averaged 87.3 -+ 7.1% (mean _+ SD; n----5). In the later case, serum was pre-incubated with unlabeled IGFI for 18 hr at 4 C prior to glycyl-glycine treatment to allow for equilibration between the added mass of hormone and IGF-I binding proteins. Parallelism was exhibited from 10 to 50 ~tl of glycyl-glycine treated serum. Sensitivity of the assay was .2 to 8 ng/tube. Intra- and inter-assay CV (n----5) were 5.2 and 10.6%. Six pools of serum (3 from control cows and 3 from cows chronically treated with bST) were used to determine optimum time, temperature, and pH of the glycyl-glycine incubation. An extensive validation was done in our laboratory to ensure that concentrations of IGF-I in glycyl-glycine-treated sera were not affected by binding proteins in the RIA (14). Briefly, serum samples were incubated with glycyl-glycine HCI as previously described (37 C; 48 hr). One aliquot of each incubated sample was directly assayed by RIA. A second aliquot was eluted with 1 M acetic acid by gel filtration on a Sephadex G-50 column (.7 X 30 cm) to remove the binding proteins, and the fraction corresponding

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to free h o r m o n e was assayed b y RIA. The r e l a t i o n s h i p b e t w e e n the values o b t a i n e d b y the t w o m e t h o d s was linear (r 2 = .96). The e q u a t i o n of the line was y = .98x -- 7.23, w h e r e y ---- c o n c e n t r a t i o n of IGF-I in glycyl-glycine treated serum, and x = c o n c e n t r a t i o n of IGF-I in glycyl-glycine treated acid c h r o m a t o g r a p h e d serum. Thus, the slope was essentially 1, and the y - i n t e r c e p t was not different f r o m zero. The 25 s a m p l e s used for this c o m p a r i s o n w e r e o b t a i n e d f r o m c o w s u n d e r various conditions w h i c h altered circulating concentrations of IGF-I (i.e., u n d e r n u t r i t i o n , e x o g e n o u s bST t r e a t m e n t ) so that a w i d e range (15-fold) of IGF-I c o n c e n t r a t i o n s was r e p r e s e n t e d . RESULTS Yields of m i l k and m i l k c o m p o n e n t s during control and bST t r e a t m e n t are p r e s e n t e d in Table 2. Daily administration of bST increased fat-corrected m i l k yield 61% a b o v e c o n t r o l values, b u t had no effect on v o l u n t a r y intake. Therefore, cows treated w i t h bST w e r e in negative e n e r g y and p r o t e i n balance (Table 2). Milk fat p e r c e n t a g e increased during bST t r e a t m e n t (P<.O05), so that fat yield increased (77%) to a greater e x t e n t than m i l k yield (41%). Milk p r o t e i n p e r c e n t was slightly d e p r e s s e d (not significantly; P > . I ) ; thus, the increase in m i l k p r o t e i n yield (30%) was less than the increase in m i l k yield. Profiles of s e r u m c o n c e n t r a t i o n s of ST and IGF-I during the first 2 days of t r e a t m e n t are s h o w n in Figures 1 and 2. During e x c i p i e n t treatment, concentrations of ST in s e r u m averaged 2.2 _+ .3 n g / m l ( m e a n +_ SD), and w e r e relatively constant o v e r the s a m p l i n g p e r i o d (SD for individual c o w s averaged -+ .5 n g / m l ) . The average c o n c e n t r a t i o n of IGF-I during e x c i p i e n t t r e a t m e n t was 70 n g / m l , w i t h 3 c o w s averaging b e t w e e n 76 and 80 n g / m l , w h i l e the fourth a v e r a g e d 44 n g / m l . C o n c e n t r a t i o n s of IGF-I in s e r u m w e r e quite constant o v e r the day (SD for individual c o w s averaged + 5.5 n g / m l ) . Following the first s u b c u t a n e o u s injection of bST, c o n c e n t r a t i o n s of ST in s e r u m p e a k e d a p p r o x i m a t e l y 2 to 4 hr post-injection, d e c l i n i n g o v e r the r e m a i n d e r of the day t o w a r d baseline values. The s e r u m profile of ST was similar on day 2 of t r e a t m e n t e x c e p t that p e a k values w e r e slightly higher and persisted o v e r a longer p e r i o d of t i m e ( a p p r o x i m a t e l y 10 hr) (Figure 1). C o n c e n t r a t i o n s of IGF-I in s e r u m b e g a n increasing a p p r o x i m a t e l y 5 to 7 hr after the first bST injection and c o n t i n u e d to increase o v e r the r e m a i n d e r of the day. By the e n d of day 1, c o n c e n t r a t i o n s of IGF-I in s e r u m of bST-treated cows w e r e e l e v a t e d a p p r o x i m a t e l y 2-fold ( + 96 n g / m l ) a b o v e control values. Following the s e c o n d TABLE 1. COMPOSITION AND NUTRIENT CONTENT OF DIET. t

Variable Ingredient Cracked corn Soybean meal Chopped hay Mineral mix Nutrient content Crude protein Acid detergent fiber Calcium Phosphorus

Complete mixed diet Hay ...................... (% of dry matter) ...................... 56.2 17.2 23.8 2,8 12.4 10.7 23.5 49.7 .36 .63 .34 .22 .................... (Mcal/kg dry matter) ....................

Energy content 1.54 .84 Net energy (lactation) tCows were offered the complete mixed diet at a level to provide 120% of net energy and crude protein requirements plus 2 kg/day long hay.

IGF-I R E S P O N S E TABLE

2.

TO bST

PERFORMANCE

AND

IN COWS

NUTmENT

267

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OF

SOMATOTROPIN

DAIRY OR

COWS

TREATED

WITH

EXOGENOUS

BOVINE

EXCIPIENT.

Treatmentt Pooled Significance2 Variable Control bST SE Level Milk yield, kg/d 3 24.9 40.2 1.1 .005 fat yield, kg/d .88 1.56 .04 .005 protein yield, kg/d .82 1.07 .08 .025 Intake dry matter, kg/d 22.1 21.1 1.1 NS net energy, Mcal/d 33.4 32.1 1.8 NS protein, kg/d 2.78 2.68 .15 NS Net balance energy, Mcal/d4 5.5 - 5.4 1.2 .005 protein, kg/d s .24 - 1.01 .09 .005 tCows were treated with excipient (control) or bovine somatotropin (bST; 40 rag/day) for 12 days. Values are averages of days 6 through 12 of treatment. 2NS = nonsignificant at P<. 1. aMilk yield expressed as 3.5% fat-corrected. (Net energy balance = net energy intake - [milk energy + maintenance requirement]. 5Net protein balance = protein intake -- protein requirement. bST i n j e c t i o n , c o n c e n t r a t i o n s o f IGF-I c o n t i n u e d to i n c r e a s e , r e s u l t i n g i n a 3f o l d e l e v a t i o n ( + 134 n g / m l ) a b o v e c o n t r o l v a l u e s at t h e e n d o f t h e s e c o n d d a y o f t r e a t m e n t ( F i g u r e 2). T h e effects o f bST t r e a t m e n t o n t h e t e m p o r a l p a t t e r n o f IGF-I i n s e r u m a n d m i l k p r o d u c t i o n o v e r t h e e n t i r e 1 2 - d a y t r e a t m e n t i n t e r v a l are s h o w n i n F i g u r e 3. M a x i m a l c o n c e n t r a t i o n s o f IGF°I w e r e o b s e r v e d b y d a y 3 o f bST t r e a t m e n t ; t h e m i l k p r o d u c t i o n r e s p o n s e w a s m o r e g r a d u a l , t a k i n g a p p r o x i m a t e l y 6 day to r e a c h a p l a t e a u . T h e r e a f t e r , b o t h m i l k y i e l d a n d c o n c e n t r a t i o n s o f IGF-I i n s e r u m r e m a i n e d r e l a t i v e l y c o n s t a n t across days for t h e r e m a i n d e r o f bST treatment. Following cessation ofbST injections, milk production and concentrations o f IGF-I i n s e r u m d e c l i n e d g r a d u a l l y to b a s e l i n e v a l u e s o v e r t h e n e x t 4 days ( F i g u r e 4 ) . C o n c e n t r a t i o n s o f bST i n s e r u m h a d r e t u r n e d to b a s e l i n e v a l u e s w i t h i n 4 8 h r after t h e last bST i n j e c t i o n ( d a t a n o t p r e s e n t e d ) .

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Fig. 1. Profile of concentrations of somatotropin in serum of cows during initial two days of treatment period. Cows ( n = 4 ) received daily subcutaneous injections of bovine somatotropin (open circles) or excipient (closed circles) as indicated by arrows.

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DISCUSSION Administration of e x o g e n o u s bST to lactating dairy c o w s increased m i l k p r o d u c t i o n in the p r e s e n t study. The 15 kg increase in fat-corrected m i l k yield during bST t r e a t m e n t w i t h o u t a c o n c o m i t a n t increase in feed intake caused c o w s to b e in negative e n e r g y and p r o t e i n b a l a n c e (Table 2). Milk fat c o n t e n t increased w h i l e p e r c e n t a g e of m i l k p r o t e i n t e n d e d to decline, resulting in a greater p r o p o r t i o n a l increase in fat yield and smaller increase in p r o t e i n yield relative to the increase in m i l k yield. Similar changes in m i l k c o m p o s i t i o n have b e e n o b s e r v e d in p r e v i o u s studies in w h i c h cows treated w i t h bST w e r e in negative e n e r g y and p r o t e i n b a l a n c e (1). C o n c e n t r a t i o n s of IGF-I in b l o o d have b e e n s h o w n to be d e p e n d e n t on ST in m a n y species. S o m a t o t r o p i n deficiency d u e to h y p o p i t u i t a r i s m in h u m a n s ( 1 5 , 1 6 , 1 7 ) or h y p o p h y s e c t o m y in rats ( 1 8 , 1 9 ) resulted in d e c r e a s e d c o n c e n trations of IGF-I w h i c h w e r e restored b y t r e a t m e n t w i t h e x o g e n o u s ST. Hyp o p h y s e c t o m i z e d pigs also e x h i b i t e d d e c r e a s e d s e r u m IGF-I ( 2 0 ) . Under c o n d i t i o n s of ST excess, s u c h as a c r o m e g a l y , c o n c e n t r a t i o n s of IGF-I w e r e e l e v a t e d in h u m a n s ( 5 , 2 1 ) and dogs ( 2 2 ) . Administration of e x o g e n o u s ST has also b e e n s h o w n to increase c i r c u l a t i n g c o n c e n t r a t i o n s of IGFoI u n d e r n o r m a l c o n d i t i o n s in rats ( 2 3 ) , pigs ( 2 0 , 2 4 , 2 5 ) , and g r o w i n g steers ( 2 6 ) . H o w e v e r , i n f o r m a t i o n on the r e l a t i o n s h i p b e t w e e n e x o g e n o u s ST and c i r c u l a t i n g concentrations o f IGF-I is l i m i t e d for lactating cows. In the p r e s e n t investigation, c o n c e n t r a t i o n s o f IGF-I in s e r u m w e r e increased a p p r o x i m a t e l y 3-fold b y e x o g e n o u s a d m i n i s t r a t i o n of ST in lactating dairy c o w s (Figure 2). Davis et al. ( 2 7 ) r e p o r t e d e l e v a t e d c o n c e n t r a t i o n s o f IGF-I in p l a s m a of dairy cows treated w i t h bST for 4 days. Peel et al. ( 2 8 ) treated pairs of identical t w i n dairy c o w s w i t h bST or e x c i p i e n t and r e p o r t e d that c o n c e n t r a t i o n s of s o m a t o m e d i n s in s e r u m of bST-treated animals w e r e not different f r o m controls after 8 w e e k s

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Day 2

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Fig. 2. Profile of concentrations of IGF-I in serum of cows during initial two days of treatment period. Cows (n=4) received daily subcutaneous injections of bovine somatotropin (open circles) or excipient (closed circles) as indicated by arrows.

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of treatment, but were elevated by week 20 to 22 of treatment. However, Peel et al. (28) utilized a bioassay for determination of somatomedin (radioactive glucose incorporation into lipid by isolated rat adipocytes) which would be less sensitive and not specific for IGF-I. Concentrations of IGF-I in serum began increasing approximately 5 to 7 hr after the first subcutaneous injection of bST (Figure 2). A similar time lag of 4 to 8 hr has been observed following a single intramuscular injection of ST in hypopituitary children (16,29) and normal pigs (25), a single intraperitoneal injection in hypophysectomized rats (18,30), or a single intravenous injection in growing steers (26). Tl'ds lag phase suggests that IGF-I is not released from a storage pool, but is synthesized d e n o v o . The lag phase also suggests that it takes some time for the IGF-I gene to be turned on, and for the requisite splicing events that occur during processing of the message to take place. Concentrations of IGF-I in serum continued to increase over the 2-day sampling interval, resulting in a 3-fold elevation above control values by the end of day 2 (Figure 2). This finding contrasts with the pattern observed for concentrations of bST in serum which oscillated each day following the exogenous injection (Figure 1). Clearly, circulating concentrations of IGF-I did not follow the pattern observed for bST. Maximal concentrations of IGF-I were achieved after 2 to 3 days of bST treatment (Figure 3), a finding which agrees with reports in hypopituitary children (17,31) and in normal pigs treated with multiple injections of ST (32). The decline in concentrations of IGF-I following cessation of bST treatment closely paralleled the decline in milk production (Figure 4). The similarity

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Fig. 3. Daily 3.5% fat-corrected milk yield and concentrations of IGF-I in serum. Cows received daily injections of bovine snmatotropin (open circles) or excipient (closed circles) for 12 days. For each cow ( n = 4 ) , daily concentrations of IGF-I represent averages of 3 samples taken prior to injection (day 0), 12 samples taken every 2 hr (days 1 and 2), or 3 samples taken 19 to 21 hr post-injection (days 3,4,5, and 12).

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50 0 -1 Day Post Treatment

Fig. 4. Decline in 3.5% fat-corrected milk yield and concentrations of IGF-I in serum of cows ( n = 2 ) following cessation of somatotropin treatment.

in the shape o f the curves likely reflects the time it takes for the m e t a b o l i c m a c h i n e r y of the m a m m a r y gland, as well as of o t h e r tissues, to return to pretreatment conditions, the time for synthesis of IGF-I in response to bST to cease, and its slow clearance from blood. Overall, the similarity in the temporal pattern b e t w e e n milk yield response and serum concentrations of IGF-I is consistent With a role for IGF-I in mediating a p o r t i o n of the effects of e x o g e n o u s bST in lactating cows. However, the role of IGF-I during lactation has not b e e n clearly established. Although r e c e p t o r s for IGF-I have b e e n r e p o r t e d in bovine m a m m a r y tissue ( 6 , 3 3 ) , results of in v i t r o studies on the effect o f IGF-I on galactopoiesis have conflicted ( 3 4 , 3 5 ) . Conflicting results have also b e e n obtained for lactational responses observed during in v i v o administration of IGF-I in goats ( 3 6 , 3 7 ) . Nutritional status has b e e n shown to influence IGF-I. During fasting, circulating concentrations of IGF-I decreased in humans ( 3 8 , 3 9 , 4 0 ) , rats ( 4 1 , 4 2 , 4 3 ) , dogs ( 4 4 ) , and sheep (45). Chronic u n d e r f e e d i n g has also b e e n s h o w n to decrease concentrations o f IGF-I in growing rats ( 4 6 , 4 7 ) and cattle ( 2 6 , 4 8 , 4 9 ) . The IGF-I response to e x o g e n o u s ST was depressed in rats fed low p r o t e i n diets ( 2 3 , 5 0 ) , in h y p o p i t u i t a r y humans subjected to a 5-day fast (39), and in growing steers w h i c h w e r e chronically u n d e r f e d (26). In the present investigation, treatment w i t h e x o g e n o u s bST increased circulating concentrations of IGF-I even t h o u g h cows w e r e in substantial negative energy and p r o t e i n balance. This e n e r g y deficit was equal to about 20% of the milk e n e r g y output, but the increase in IGF-I c o n c e n t r a t i o n was o f the same magnitude that we had previously observed for bST-treated cows in positive e n e r g y balance ( 5 1 ) . Thus, the ability of nutritional status to alter concentrations of IGF-I, or the

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IGF-I response to ST, may d e p e n d on the severity of the nutritional deprivation. In addition, cows in the present study w e r e in positive e n e r g y and p r o t e i n balance at the initiation of ST treatment. The IGF-I response c o u l d differ w h e n cows are in negative e n e r g y and p r o t e i n balance prior to the initial treatment w i t h ST. Studies are n e e d e d in lactating ruminants to d e t e r m i n e the impact of nutritional status on the IGF-I/ST axis. ACKNOWLEDGEMENTS/FOOTNOTES 1Supported in part by Cornel! University Agricultural Experiment Station, USDABiotechnology Program (GAM-8502654) and Monsanto Agricultural Company (St. Louis, MO). 2present address: HFV-126 Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 20857. 3Reprint requests. REFERENCES 1. Peel CJ, Bauman DE. Somatotropin and lactation. J Dairy Sci 70:474-486, 1987. 2. Boyd RD, Bauman DE. Mechanisms of action for somatotropin in growth. In: Animal Growth Regulation, Campion DR, Hausman GJ, and Martin RJ (eds). Plenum Press, New York, p. 257-293, 1989. 3. Etherton TD. The mechanisms by which porcine growth hormone improves pig growth performance. In: Biotechnology in Growth Regulation, Heap RB, Prosser CG, and Lamming GE (eds). Butterworth, London, p. 97-105, 1989. 4. Clemmons DR, Dehoff M, McCusker R, Elgin R, Busby W. The role of insulin-like growth factor I in the regulation of growth. J Anim Sci 65 (Suppl 2):168-179, 1987. 5. Zapf J, Walter H, Froesch ER. Radioimmunological determination of insulinlike growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia. J Clin Invest 68:1321-1330, 1981. 6. Dehoff MH, Elgin RG, Collier RJ, Clemmons DR. Both type I and II insulin-like growth factor receptor binding increase during lactogenesis in bovine mammary tissue. Endocrinology 122:2412-2417, 1988. 7. National Research Council. Nutrient requirements of domestic animals. No 3. Nutrient requirements of dairy cattle. 5th rev ed, Natl Acad Sci, Washington DC, 1978. 8. Eppard PJ, Bauman DE, McCutcheon SN. Effect of close of bovine growth hormone on lactation of dairy cows. J Dairy Sci 68:1109-1115, 1985. 9. Tyrrell HF, Reid JT. Prediction of the energy value of cow's milk. J Dairy Sci 48:1215-1223, 1965. 10. Cochran WG, Cox GM. Completely randomized block, and latin square designs. In: Experimental Designs, J Wiley & Sons, New York, p. 95-147, 1957. 11. Underwood LE, D'Ercole AJ, Copeland KC, Van Wyk JJ, Hurley T, Handwerger S. Development of a heterologous radioimmunoassay for somatomedin C in sheep blood. J Endocrinol 93:31-39, 1982. 12. Honegger A~ Humbel RE. Insulin-like growth factors I and II in fetal and adult bovine serum: purification, primary structures, and immunological cross-reactivities. J Biol Chem 261:569-575, 1986. 13. Baxter RC, Axiak S, Raison RL. Monoclonal antibody against human somatomedinC/insulin-like growth factor-I. J Clin Endocrinol Metab 54:474-476, 1982. 14. Plaut K. Endocrine regulation of lactation: prolactin, somatotropin, and insulinlike growth factor-I. Ph.D. Dissertation, Cornell University, Ithaca, NY, 1989. 15. Furlanetto RW, Underwood LE, Van WykJJ, D'Ercole AJ. Estimation of somatomedinC levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest 60:648-657, 1977. 16. Copeland KC, Underwood LE, Van Wyk JJ. Induction of immunoreactive somatomedin C in human serum by growth hormone: dose-response relationships and effect on chromatographic profiles. J Clin Endocrinol Metab 50:690-697, 1980. 17. Blethen SL, Daughaday WH, and Weldon VV. Kinetics of the somatomedin C/insulinlike growth factor I: response to exogenous growth hormone (GH) in GH-deficient children. J Clin Endocrinol Metab 54:986-990, 1982.

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