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Effects of winter nutrition and summer pasture or a feedlot diet on plasma Insulin-like Growth Factor I (IGF-I) and the relationship between circulating concentrations of IGF-I and thyroid hormones in steers
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 7(4):465-476, 1990
EFFECTS OF WINTER NUTRITION AND SUMMER PASTURE OR A FEEDLOT DIET ON PLASMA INSULIN-LIKE GROWTH FACTOR I (IGF-I) AND THE RELATIONSHIP BETWEEN CIRCULATING CONCENTRATIONS OF IGF-I AND THYROID HORMONES IN STEERS A.C. Hammond.1, T.H. Elsasser**, W.E. Kunkle***, T.S. Rumsey**, M.J. Williams* and W.T. Butts* U.S. Department of Agriculture, Agricultural Research Service *Subtropical Agricultural Research Station, Brooksville, FL 34605 and **Ruminant Nutrition Laboratory, Beltsville, MD 20705 ***Animal Science Department, Institute of Food and Agricultural Sciences University of Florida, Gainesville, FL 32611 Received June 9, 1989
ABSTRACT Effects of two winter nutritional levels (LOW, MOD) and two summer pastures (bahiagrass, BG; perennial peanut, PP) on plasma IGF-I, and the relationship between IGF-I and average daily gain (ADG), thyroid hormones, plasma urea, packed cell volume (PCV) and steer type were determined in 101 steers (217 kg) varying in breed composition, frame size and initial condition. Relationships between body composition or composition of gain and IGF-I were determined in 11 contemporary steers assigned directly to the feedlot. Initial IGF-I (57.9 -+ 3.5 ng/ml) was positively correlated (P<.05) to initial condition, estimated percentage of Brahman and plasma T3, but not related to subsequent ADG. During the 126-day wintering period, ADG was .21 kg for the LOW winter treatment and .47 kg for the MOD winter treatment. Concentration of IGF-I in the wintering period was affected (P<.O1) by nutritional level (LOW--71.8 ng/ml, MOD = 150.6 ng/ml) and was positively related to winter ADG in MOD steers (P<.O1) but not in LOW steers. Concentration of IGF-I in winter was also positively related to condition at the end oftbe winter period (P<.O1), T3 (P<.05) and T~ (P<.05). There were no effects of winter treatment on IGF-I during the subsequent summer pasture period. During the 145-d summer period, ADG was .53 kg for BG and .68 kg for PP. Concentration of IGF-I during the summer period was affected (P<.05) by pasture treatment (BG----138.6 ng/ml, PP= 181.9 ng/ml), was positively related (P<.O 1) to PCV and percentage of Brahman, and was negatively related (P<.05) to estimated percentage of English breeding. In steers assigned directly to the feedlot, IGF-I was correlated with empty body (EB) weight (r=-.59, P<.IO), EB water (r=-.59, P<.lO) and EB protein (r-----.60, P<.lO) at slaughter, and with days on feed (r=-.65, P<.05), but was not correlated with ADG or rate of component gain. These data indicate that IGF-I is related to nutritional status in steers as in other species, that there may be significant breed or cattle type differences in circulating concentrations of IGF-I, and that circulating concentration of IGF-I may be functionally related to plasma concentration of thyroid hormones. INTRODUCTION Insulin-like g r o w t h factor I (IGF-I) is a p o t e n t g r o w t h h o r m o n e - d e p e n d e n t m i t o g e n that has b e e n s h o w n to mediate m a n y o f the s o m a t o g e n i c effects o f g r o w t h h o r m o n e . These actions and the role o f IGF-I in the regulation o f g r o w t h have b e e n r e v i e w e d (1). Although some o f the actions o f IGF-I may be paracrine or a u t o c r i n e in nature, circulating c o n c e n t r a t i o n s o f IGF-I are Copyright © 1990 by DOMENDO, INC.
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related to growth rate, nitrogen balance and body size in adult animals and humans. Circulating concentrations of IGF-I are dependent on growth hormone and, in at least rats (2-6), dogs (7), pigs (8), cattle (9-15) and humans (1620), are dependent on nutritional status. In addition, although there is some evidence with dairy cows that circulating IGF-I may display a diurnal rhythm (9), other work with cattle has shown no periodicity in plasma IGF-I concentrations (10). Generally, circulating IGF-I is not considered to be episodic. These characteristics of IGF-I along with recent advances in the ability to quantitatively analyze IGF-I in plasma of major domestic livestock species make it of particular interest to the study of growth in food producing animals. A steer backgrounding study was initiated in 1985 at the Subtropical Agricultural Research Station, Brooksville, Florida to investigate a number of nutritional, genetic, agronomic and economic factors involved in growing-finishing systems (21-23). Steers were selected with variation in type, primarily frame size, condition score and consequently estimated breed composition. The steers were subjected to five systems of backgrounding and finishing involving two winter feeding regimens followed by two summer grazing systems prior to feedlot finishing or direct to feedlot. This report presents data obtained on the effects of the two winter nutrition levels and two summer pastures on plasma IGF-I concentration, and the relationship between IGF-I and growth rate, steer type, plasma thyroxine (T4) concentration, plasma triiodothyronine (T3) concentration, plasma urea nitrogen (PUN) concentration and blood packed cell volume (PCV). In a small group of feedlot steers, relationships between IGFI and body composition or composition of gain were also investigated. MATERIALS AND METHODS Steers (217 kg) were purchased by order buyers in November of 1985 and ranged from small to large in frame size and thin to fleshy in condition. Initial handling of steers included vaccination (IBR, PI3, 7-Way Clostridium), vitamin A injection (1 million IU/head), anthelmintic treatment (Tramisol Injectable, Cyanamid, Wayne, NJ) and implanting (Compudose, Elanco Products Company, Indianapolis, IN). Steers were revaccinated for IBR and PI3 12 days after the first vaccination, and were treated a second time for parasites (IVOMEC, MSD AGVET, Rahway, NJ) 27 days after initial handling. For a preliminary period of 4 weeks, all steers were given access to bahiagrass pasture and for the first 3 weeks were fed 2.3 kg/head/day of a medicated receiving diet (Customized Pre-Conditioning Pellets Medicated, Lakeland Cash Feed Company, Inc., Lakeland, FL). Guaranteed crude protein content of this supplemental receiving diet was 11.5% and contained 193 mg chlortetracycline/kg. Prior to starting the trial, steers were visually evaluated by two of the authors (Kunkle, Butts) for frame size and condition. Each steer then was assigned to one of five systems to achieve a wide range in frame and condition within each system. Several other characteristics of these cattle also were visually evaluated and scored including breed composition (21). The systems were two levels of winter nutrition, low or moderate, and two summer pastures prior to finishing in the feedlot, or direct to feedlot. Steers on the low wintering plane of nutrition (LOW) were grazed on residual bahiagrass ( P a s p a l u m n o t a t u m Flugge.) pasture, offered ad libitum amounts of bahiagrass hay and fed 0.45 kg/head/day of soybean meal (44% crude protein). Steers on the moderate wintering plane of nutrition (MOD) were grazed on similar residual bahiagrass pasture, offered ad libitum amounts of bahiagrass hay and fed 3.2 kg/head/day of an 80%
EFFECTS OF NUTRITION QN IGF-I IN STEERS
467
blackstrap molasses - 20% soybean meal slurry. The winter period lasted for 126 days from December 1985 to April 1986. For 145 days from April to September 1986, half of the steers on each wintering level were grazed on bahiagrass pasture (BP) and the other half were grazed on a mixed sward of grass (predominantly Cynodon and Paspalum spp.) and rhizoma perennial peanut (PP, Arachis glabrata Benth.). Summer pastures were divided into two replicates per treatment and stocking rate was 1.4 head/hectare. Prior to being turned out on summer pasture, steers were reimplanted (Compudose, Elanco Products Company, Indianapolis, IN). Rates of live weight gain in the backgrounded steers were determined by obtaining shrunk weights (live weight following an overnight fast) prior to start of the trial, between the winter and summer phases and at the end of the summer phase. In addition to the initial condition scores obtained prior to the trial, condition scores were obtained between the winter and summer phases and at the end of the summer phase. Jugular blood samples were obtained in the backgrounded steers the day prior to the initial shrunk weight at the start of the winter phase, and at 56-day intervals throughout the winter and summer phases for analysis of packed cell volume (PCV), plasma urea nitrogen (PUN), plasma IGF-I, plasma thyroxine (T4) and plasma triiodothyronine (T3). This sampling schedule provided at least two blood samples for each backgrounding period, winter and summer. The anticoagulant used was EDTA and plasma was stored at -- 20 C in polypropylene vials prior to analysis. Urea nitrogen was determined by a modification (Industrial Method 339-01, Technicon Industrial Systems, Tarrytown, NY) of the automated diacetyl-monoxime method of Marsh et al. (24). Plasma concentrations of T 3 and T4 were measured by solid phase radioimmunoassay using commercial kit assays (Immunochem. Inc.) as previously described (25) and validated for use with bovine plasma. Quantitation of plasma concentrations of each hormone were made using standards included in the kits which consisted of calibrated amounts of T3 and T4 in human serum. Similar bovine serum standards were prepared by treating a pool of bovine serum with 8-anilinonapthanesulphonic acid (8.ANS) to dissociate thyroid hormones from serum carrier proteins and then subjecting the pooled serum to extraction with activated charcoal to remove all thyroid hormones. Amounts of T3 and T4 were added to the extracted serum to obtain serum concentrations of hormones equal to those of the human serum standards in each type kit. Displacement of tracer counts by each standard curve for T3 or T4 were identical as were the recoveries of cold hormone (>95% for each assay). Increasing volumes of plasma from a standard bovine plasma pool displaced tracer counts in a dose-related fashion yielding a slope parallel to that obtained with the standard curve for either T3 or T4. Intraassay and interassay coefficients of variation were less than 7%. Plasma IGF-I was measured by a double antibody radioimmunoassay based on the glycyl-glycine acidification procedure described initially by Underwood, et al. (26) and modified and validated for bovine plasma by Elsasser, et al. (27). Recombinant human IGF-I with the threonine substitution in the 59 position was iodinated by the iodogen method for tracer and similarly used as standard for the assay. Because the primary and tertiary structure of native human and bovine IGF-I are identical (28) and because the recombinant, natural and synthetic forms of the peptide behave identically in the assay (29), this assay was essentially a homologous assay. Plasma was acidified with an
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HAMMOND ET AL.
equal volume of .2 M glycyl-glycine HCI (pH 2.1) for 36 hr. At this time each sample was individually and rapidly diluted with assay buffer (50 mM TRIS, 10 mM EDTA, .25% BSA, pH 7.6); 50 ~tl added immediately to duplicate assay tubes followed by an additional 150 Ixl of assay buffer. To complete the assay volume, both tracer and primary antibody were added in 100 ul volumes. Assays were run as nonequilibrium assays (24 hr with primary antibody alone then an additional 24 hr in the presence of tracer). At the completion of the incubation (at 4 C), bound counts were precipitated with goat antirabbit gamma globulin in polyethylene glycol (PEG) followed by centrifugation and aspiration. To accomplish the secondary precipitation in the separation of bound and free tracer, goat antirabbit gamma globulin was diluted 1:60 in BSA-free assay buffer. This was mixed into a solution of 6% (w/v) PEG (8000 mw) in the proportion of 2 parts antibody solution plus 5 parts PEG solution. The resulting PEG-antibody solution was mixed at 4C for 18 hr prior to addition to the assay. To the 400 1~1 assay tube volume, 450 ~tl of the PEG-antibody solution was added. This was incubated at 4C for 1 hr at which time 100 ~tl of normal rabbit serum (1:100) was added to each tube. This was incubated an additional 30 min prior to centrifugation at 1850 X g. Pellets were counted in an LKB automatic gamma count er for 1 min. Dilutions of acidified plasma displaced counts parallel to the regression line obtained by displacement of counts by the standard curve. Recovery of cold IGF-I added to plasma prior to extraction averaged 92% across a range of hormone mass (5 through 100 ng/ml added). All samples were analyzed in 4 assays with all reagents prepared in the same lot. Intraassay and interassay coefficients of variation were 8 and 10 %, respectively. Minimum detectable quantity at this plasma dilution was 4 ng/ml. Statistical analysis of the relationship between initial IGF-I and other initial variables or subsequent rates of gain was by simple linear regression using the GLM Procedure of the Statistical Analysis System (30). In subsequent analyses involving blood data, the mean of the last two samples for each of the two phases, winter and summer, were analyzed and taken to be indicative of the respective treatments during these times. Statistical analysis of the relationship b etween plasma IGF-I in winter and each independent variable also used a regression approach, the GLM Procedure (30), with the main effect of wintering level included as a class variable along with the wintering level X independent variable interaction. There were no carryover effects of winter plane of nutrition on plasma IGF-I in summer so a similar regression approach was utilized to analyze relationships bet w e e n IGF-I concentrations in the summer period and each independent summer variable with summer pasture and pasture replicate within summer pasture included as class variables. The error term for the summer pasture effect was replicate within summer pasture. Independent variable and independent variable X summer pasture interaction were tested using independent variable X replicate within summer pasture as the error term. Steers assigned directly to the feedlot were fed a high energy diet of 72.5% whole shelled corn, 15% cottonseed hulls and 12.5% of a commercial protein, vitamin and mineral premix (Custom Beef Concentrate 52 Pellets Medicated, Lakeland Cash Feed Company, Inc., Lakeland, FL) which contained not less than 52.0% crude protein, 74,800 USP Units vitamin A/kg, 3.0% calcium, 1.5% phosphorous, not more than 7.5% salt, and 294 mg monensin/kg. Over 3 weeks this diet was changed to 82.5% whole shelled corn, 10% cottonseed hulls and
EFFECTS OF NUTRITION ON IGF-I IN STEERS
469
7.5% premix. Steers were group-fed in 4 pens, 6 steers/pen, to a visually estimated thickness of 11 to 12 mm of fat over the ribeye. Rates of live weight gain in the direct.to-feedlot steers were determined by the difference between shrunk weights taken just prior to the start of feeding and just prior to slaughter. Blood samples and estimates of body composition using urea dilution (31-32) were obtained from a subset of 12 of these steers every 42 days beginning with day 14 in the feedlot. One of these steers was subsequently dr oppe d from the experiment for reasons unrelated to treatment. Blood was analyzed for PCV and blood plasma for PUN, IGF-I, T4 and T 3 as described for backgrounding steers. Rates of empty body c o m p o n e n t (water, protein and fat) gain were determined by regression over time. There was no significant effect of time in the feedlot on IGF-I so further statistical analysis utilized means for each steer. The relationship between IGF-I and all other variables analyzed with these steers was determined by simple linear regression as described for initial IGF-I analysis in the backgrounding steers. RESULTS Mean and SD for independent variables tested during backgrounding of steers and for the direct to feedlot steers are given in Tables 1 and 2. For the directto-feedlot steers, time on feed (203.9 _+ 43.5 days) and empty body weight at slaughter (436.9 + 57.7 kg) also were analyzed as independent variables. Mean IGF-I by system, and effects of winter plane of nutrition or summer pasture on IGF-I are given in Table 3. Plasma IGF-I was higher (P<.01) for the MOD plane of nutrition compared to LOW and plasma IGF-I was higher (P<.05) for the higher quality summer grass-legume pasture, PP, compared to BG. Although not tested statistically, mean plasma IGF-I ranked highest in the direct to feedlot steers on a high energy concentrate diet compared to any of the backgrounding treatments. Correlations between initial plasma IGF-I concentrations (57.9 -+ 3.5 ng/ ml) and initial independent variables or subsequent rate of live weight gain during winter or summer are given in Table 4. Correlations between initial TABLE 1. MEAN + SD OF INDEPENDENTVARIABLESSTUDIED WITH RESPECT TO EFFECTS ON IGF-I IN BACKGROUNDED AND DIRECT-TO-FEEDLOTSTEERS Independent variable Rate of gain (kg/day) Condition score = Frame score b Muscle score ~ Estimated b r e e d c o m p o s i t i o n Brahman (%) English (%) Continental (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%) T3 ( n g / m l ) T4 ( n g / m l )
Backgrounded Steers ('n= 101) Initial Winter Summer 8.0 _+ 1.2 8.8 + 2.1 1.84 _+ .33 39.9 55.3 4.8 8.6 36.8 1.64 68.1
_+ -+ + _+ + _+ +
.35 -+ 7.4 +
22.2 20.9 14.0 2.30 11.8 3.9" 39.0 .48 d 1.61 18.00 66.0
.20 1.0
+ 3.9 -+ 3.1 -+ .4 + 16.7
Direct to Feedlot Steers ( n = 11) 1.19 8.0 9.1 1.71
+ + -+ +
26.1 56.1 17.7 11.4 6.1 8.4 36.6 -+ 3.5 38.4 2.07 +.32 3.23 76.2 + 19.4 116.6
_+ + -+ -+ + + +
.60 + 9.0 +
.15 1.1
.14 1.4 = 2.7 r .38 f 19.2 f 20.0 f 25.5 f 1.1 2.8 .41 10.5
•Body condition scoring system: 4 , 5 , 6 = r e l a t i v e degrees of emaciation; 7 = 1 m m fat over the ribeye; 8 = 2 m m fat over the ribeye; 9 = 3 m m fat; etc. bFrame score defined as slaughter w e i g h t r e q u i r e d to finish w i t h .5" fat over the ribeye; 2 = 8 0 0 lb.; 5 = 9 0 0 lb.; 8 = 1 , 0 0 0 lb.; 1 1 = 1 , 1 0 0 lb., 1 4 = 1 , 2 0 0 lb. "Muscle s c o r e : 1 . 2 = h e a v y + ; 1 . 5 = h e a v y ; 1 . 8 = h e a v y - - ; 2 . 2 = m e d i u m + ; 2.5=medium; 2 . 8 = m e d i u m - - ; 3.2 = l i g h t + ; 3.5 = l i g h t ; 3 . 8 = l i g h t - . dn=93. ~n = 70. qnitial.
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TABLE 2. COMPOSITION OF GAIN AND BODY COMPOSITION AT SLAUGHTEROF DIRECT-TO-FEEDLOT STEERS Water
Component" Protein
Fat
.41 + .08 226.3 _+ 25.2 52.0 + 2.7
.14 +_ .02 70.4 + 8.2 16.2 _+ .7
.57 + .11 119.4 _+ 26.0 27.2 -+ 3.9
Item C o m p o s i t i o n of gain (kg/day) C o m p o s i t i o n at slaughter (kg) C o m p o s i t i o n at slaughter (%) •Mean _+ SD
IGF-I and initial condition score, initial plasma T 3, and estimated percentage of Brahman breeding were each positive and significant, but correlation coefficients were low. Initial IGF-I and initial plasma T4 only tended to be correlated, and correlations between initial plasma IGF-I and all other initial independent variables were not significant. In addition, initial plasma IGF-I was not correlated with subsequent rates of gain in either the winter or summer backgrounding phases. TABLE 3. MEAN IGF-I BY MAIN EFFECTWITHIN SYSTEMIN BACKGROUNDEDSTEERSAND IN DIRECT-TO-FEEDLOT STEERS Treatment Winter plane of nutrition LOW ( n = 4 9 ) MOD ( n = 5 2 ) S u m m e r pasture BG ( n = 5 1 ) PP ( n = 5 0 ) Direct to feedlot (n = 11)
IGF-I ( n g / m l )
SEM
71.8" 150.6
4.4 5.6
138.6 b 181.9 203.4
5.8 4.7 7.0
"Winter plane of nutrition effect ( P < . 0 1 ) . bSummer pasture effect ( P < . 0 5 ) .
During the wintering phase, there was a significant interaction (P<.01) between plane of nutrition and rate of live weight gain on plasma IGF-I (Table 5). At the LOW plane of nutrition, with rates of gain averaging .21 kg/day (23), there was no significant relationship between plasma IGF-I and rate of gain. With the MOD plane of nutrition, rate of gain averaged .47 kg/day (23) and was positively related to plasma IGF-I (P<.O1). There were no other significant interactions between winter treatment and other independent variables studied. With the significant effects of winter plane of nutrition removed, plasma IGF-I was positively related to condition score at the end of the winter period (P<.O1), positively related to plasma T 3 (P<.05) and positively related TABLE 4. CORRELATIONBETWEEN INITIALPLASMAIGF-I AND EACH INITIALINDEPENDENTVARIABLEOR SUBSEQUENT RATE OF GMN IN BACKGROUNDEDSTEERS Independent variable Condition score Frame score Muscle score Estimated b r e e d c o m p o s i t i o n Brahman (%) English (%) Continental (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%)
T3 (ng/ml) T4 (ng/ml) Winter gain (kg/day) S u m m e r gain (kg/day) •Not significant, P > . I O.
r
Prob.
.21 .10 .03
<.05 NS" NS
.22 - . 14 - . 12 .08 .19
<.05
.30 .19
<.01 <.10
- . 13 - . 11
NS NS NS NS NS NS
EFFECTS OF NUTRITION ON IGF-I IN STEERS TABLE5.
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RELATIONSHIP BETWEEN PLASMA IGF-I AND AVERAGE DAILY GAIN IN BACKGROUNDED STEERS ON TWO WINTERING PLANES OF NUTRITION Sy-x
Treatment X LOW ADG• (kg) MOD ADG (kg) •Average daily gain.
Slope 28.0 148.7
Intercept 65.9 80.3
(ng/ml) 31.1 33.9
rz .02 .32
Prob. NS <.01
tO plasma T4 ( P < . 0 5 ) , but was not related to plasma urea N or p a c k e d cell v o l u m e (Table 6). There was no significant replicate w i t h i n s u m m e r pasture effect on plasma IGF-I ( P > . I O ) . Plasma IGF-I in s u m m e r was significantly higher ( P < . 0 5 ) on PP pasture than on BG pasture (Table 3) and associated rates of live w e i g h t gain also w e r e significantly higher on PP pastures than on BG pastures, .60 v s . . 5 3 kg/day, respectively (23). However, with significant effects o f s u m m e r pasture removed, there was no significant additional variation in plasma IGFI due to rate o f live w e i g h t gain (P>. 10). O f all i n d e p e n d e n t variables tested, plasma IGF-I in s u m m e r was significantly related only to estimated p e r c e n t a g e o f Brahman and English breeding, and to p a c k e d cell volume. Because there were no significant interactions associated with these effects, a simple regression m o d e l was used to d e t e r m i n e the relationships b e t w e e n each o f these i n d e p e n d e n t variables using s u m m e r pasture as a class variable. C o m m o n regressions from p o o l e d data derived from these analyses are given in Table 7. Correlation b e t w e e n mean circulating IGF-I in the 1 1 direct to feedlot steers and each i n d e p e n d e n t variable studied is given in Table 8. In these steers, IGF-I was negatively correlated w i t h EB weight, EB water and EB p r o t e i n at slaughter, and negatively correlated w i t h days on feed r e q u i r e d to reach an estimated e n d p o i n t of 1 1 to 12 m m backfat. Circulating IGF-I was not correlated with live w e i g h t gain or individual c o m p o n e n t gain in these steers. DISCUSSION In the present study with steers, circulating IGF-I c o n c e n t r a t i o n s w e r e positively affected by plane o f nutrition in the w i n t e r and quality of pasture in the summer. In addition, IGF-I c o n c e n t r a t i o n s ranked highest in plasma from steers o n a c o n c e n t r a t e based (relatively high energy) feedlot diet. Furthermore, the relationship b e t w e e n IGF-I and average daily gain o f steers interacted w i t h TABLE 6. RELATIONSHIPSt BETWEEN PLASMAIGF-I AND EACH INDEPENDENT VARIABLE IN BACKGROUNDED STEERS DURING WINTERING PHASE
Sy'x
X Slope Intercept (ng/ml) rz Prob. Condition score 16.3 -8.9 30.4 .60 <.01 Initial frame score 3.6 79.3 35.7 .57 NSb Initial muscle score -22.2 152.0 35.7 .56 NS Estimated breed composition Brahman (%) -1.2 116.1 36.4 .55 NS English (%) 1.O 105.7 36.4 .55 NS Continental (%) .83 110.8 36.5 .55 NS Plasma urea N (mg/dl) .23 108.5 36.5 .55 NS Packed cell volume (%) 1.6 49.0 36.2 .55 NS Plasma T3 (ng/ml) 27.0 67.9 35.5 .55 <.05 Plasma T4 (ng/ml) .72 63.9 35.4 .58 <.05 •Effects due to winter plane of nutrition (LOW vs. MED) on plasma IGF-I were determined by using winter plane or nutrition as a class variable (for results see Table 2). Common regressions from pooled data were derived from these analyses for each independent variable and are presented here. bNot significant, P>. 10.
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TABLE 7. SIGNIFICANT RELATIONSHIPS BETWEEN PLASMAIGF-I AND INDIVIDUALINDEPENDENT VARIABLESIN BACKGROUNDEDSTEERS DURING SUMMER PASTURE PHASE Independent variable
Slope
Intercept
Sy.x (ng/ml)
r2
Prob
Estimated b r e e d composition B r a h m a n (%) English (%) Packed cell v o l u m e (%)
.42 -.49 2.4
143.5 165.0 72.5
36.3 36.0 36.6
.30 .31 .29
<.05 <.01 <.05
level of winter nutrition. Plasma IGF-I was lower on the low plane of winter nutrition (adequate protein but low energy) and was not related to average daily gain. With supplemental energy (molasses) in addition to protein supplementation in the winter, plasma IGF-I concentrations were higher and were positively related to average daily gain of steers. As mentioned in the materials and methods, there was no carryover effect of winter treatment on IGF-I in summer. In contrast, Granger e t al. (14) found in heifers that plasma IGF-I concentrations that were depressed due to a lower quality of wintering diet, continued to be lower during a subsequent summer pasture phase when heifers were on a com m on diet. Early work with growing cattle using bioassay techniques for serum somatomedin activity demonstrated significantly higher mean serum IGF-I in high rate of gain groups compared to low rate of gain groups of bulls (33-34). In more recent studies with cattle, circulating concentrations of IGF-I also were shown to be affected by plane of nutrition in heifers (14-15), cows (9) and steers (1 O- 13). Both energy (9-13) and protein (13) intake influenced plasma IGF-I concentrations. As with other species, there have been reported positive relationships between circulating IGF-I and nitrogen balance (1 O, 13) or growth rate (11). One recent report of work with sheep demonstrated higher IGF-I TABLE 8. CORRELATIONBETWEEN PLASMAIGF-P AND EACH INDEPENDENT VARIABLESTUDIED IN DIRECT TO FEEDLOT STEERS Independent variable Rate of gain (kg/day) Condition score Frame s c o r e Muscle score Estimated b r e e d c o m p o s i t i o n B r a h m a n (%) English (%) C o n t i n e n t a l (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%) T, ( n g / m l ) T4 ( n g / m l ) T i m e on feed (days) C o m p o s i t i o n of gain Water ( k g / d a y ) Protein (kg/day) Fat ( k g / d a y ) Body c o m p o s i t i o n at s l a u g h t e r E m p t y b o d y w e i g h t (kg) Water (kg) Protein (kg) Fat (kg) Water (%) Protein (%) Fat (%)
r
Prob.
.33 .48 -.51 -.02
NSb NS NS NS
.34 .33 -.51 .75 .18 .15 .03 -.65
NS NS NS <.01 NS NS NS <.05
.05 .08 .13
NS NS MS
-.55 -.59 -.60 -.43 .09 .02 -.13
<. 10 <. 10 <.10 NS NS NS NS
•Mean o f 5 to 7 b l o o d s a m p l e s o b t a i n e d every 42 days b e g i n n i n g day 14 in t h e feedlot. "Not significant, P>. 10.
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concentrations in ewes given abomasal infusions of glucose and/or casein compared to controls; and a positive relationship between circulating IGF-I concentrations in ewes bearing triplets and birth weight of their lambs (35). A relatively small number of steers assigned directly to the feedlot were investigated in our study, so data from these steers should be considered preliminary and subject to confirmation. Nevertheless, the results indicated that IGF-I was negatively correlated to total empty body and lean body mass at slaughter at a constant fat endpoint. Days on feed in the feedlot which were required to reach the constant fat endpoint also showed a negative correlation with circulating IGF-I. These data suggest that IGF-I may be higher in earlier maturing but not necessarily larger cattle types. It may be important to note that steers in this study varied with respect to size, type, condition, breed, etc., and may have expressed variability in circulating IGF-I that may not be seen in studies with more homogeneous groups of experimental animals. One such study was that of Anderson et al. (36) in which significant correlations were found between IGF-I concentrations and percentage of carcass fat (r=--.60), carcass fat accretion rate (r = - . 5 7 ) , total carcass fat (r = - . 5 2 ) , fat thickness (r = --.73) and percentage of carcass protein (r = .60) in Simmental crossbred bulls. From among the other independent variables measured in the present study, the relationship between circulating concentrations of IGF-I and thyroid hormones may be the most interesting. There were significant positive relationships between IGF-I and T 3 initially, and between IGF-I and both T 3 and T4 at the end of the winter feeding phase. Serum T 3 and IGF-I were both reduced in calorie-restricted rats and were associated with reduced growth rate, i.e., lower body weight in calorie-restricted rats compared to controls of the same age (6). In one trial with steers, plasma IGF-I and plasma T4 both increased with increasing dietary protein level (13). In another trial with steers, IGF-I and T4 were reduced in response to restricted feeding but T 3 was unchanged (12). An effect of thyroid hormones on the regulation of growth hormone receptors was alluded to by Maes e t al. (3-4) which suggests that the effects of nutrition on circulating IGF-I may be mediated through mechanisms that modulate the effect of growth hormone on IGF-I production at the growth hormone receptor level, but investigation of these possibilities was beyond the scope of the present trial. Throughout both the winter and summer periods, plasma IGF-I was positively correlated with estimated percentage of Brahman breeding. There also was a positive correlation between plasma IGF-I and packed cell volume during the summer grazing period. The relationships between IGF-I and packed cell volume, and between IGF-I and breed type may be an autocorrelation effect. In one study, Brahman cattle were shown to have higher packed cell volume compared to an English breed type (37) and this has been a consistent observation of ours at Brooksville, as well (unpublished). We have previously pointed out that it is difficult to describe cause and effect relationships between various aspects of growth, the apparent plane of nutrition and plasma concentrations of IGF-I (13). Whether plasma concentrations of IGF-I are involved directly in the regulation of growth and metabolism or simply reflect changes in metabolism that coincide with other nutritional and hormonal events associated with growth cannot be determined from the present data. We conclude from these data, however, that circulating concentrations of IGF-I are related to nutritional status in steers as with other species,
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t h a t t h e r e m a y b e s i g n i f i c a n t b r e e d or c a t t l e t y p e d i f f e r e n c e s i n 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 o f IGF-I, a n d t h a t 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 o f IGF-I m a y b e f u n c t i o n a l l y r e l a t e d to 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 o f t h y r o i d h o r m o n e s . ACKNOWLEDGEMENTS/FOOTNOTES The authors thank M.J. Schoppman, E.L. Adams and M.T. Pello for technical assistance and V.E. Rooks and the STARSfarm staff for animal handling and care. Mention of companies or products is for the benefit of readers and does not constitute an endorsement or preferential treatment by the authors or the USDA. Published as Florida Agricultural Experiment Station Journal Series No. R-00579. ~Address correspondence and requests for reprints to: A. C. Hammond, Research Leader, USDAARS-STARS, P.O. Box 46, Brooksville, FL 34605-0046.
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REFERENCES 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. Prewitt TE, D'Ercole AJ, Switzer BR, Van Wyk JJ. Relationship of serum immunoreactive somatomedin-C to dietary protein and energy in growing rats. J Nutr 112:144-150, 1982. Maes M, Underwood LE, Ketelslegers J-M. Plasma somatomedin-C in fasted and refed rats: close relationship with changes in liver somatogenic but not lactogenic b i n d i n g sites. J Endocrinol 97:243-252, 1983. Maes M, Underwood LE, Gerard G, Ketelslegers J-M. Relationship between plasma somatomedin-C and liver somatogenic b i n d i n g sites in neonatal rats during malnutrition and after short and long term refeeding. Endocrinology 115:786-792, 1984. Bolze MS, Reeves RD, Lindbeck FE, Elders MJ. Influence of selected amino acid deficiencies on somatomedin, growth and glycosaminoglycan metabolism in weanling rats. J Nutr 115:782-787, 1985. Schalch DS, Cree TC. Protein utilization in growth: effect of calorie deficiency on serum growth hormone, somatomedins, total thyroxine (T4) and triiodothyronine, free T4 index, and total corticosterone. Endocrinology 117:2307-2312, 1985. Eigenmann JE, de Bruijne JJ, Froesch ER. Insulin-like growth factor I and growth hormone in canine starvation. Acta Endocrinol 108:161-166, 1985. White ME, Allen CE, Dayton WR. Effect of sera from fed and fasted pigs on proliferation and protein turnover in cultured myogenic cells. J Anim Sci 66:3440, 1988. Binnerts WT, van Adrichem PWM, Ondenaavden CPJ, Vogt JE, Wassenaar JE. Plasma somatomedin in dairy cows: effect of management and feeding level. Neth Milk DairyJ 36:149-152, 1982. Breier BH, Bass JJ, Butler JH, Gluckman PD. The somatotrophic axis in young steers: influence of nutritional status on pulsatile release of growth hormone and circulating concentrations of insulin-like growth factor 1. J Endocrinol 111:209-215, 1986. Breier BH, Gluckman PD, Bass JJ. Influence of nutritional status and oestradiol-179 on plasma growth hormone, insulin-like growth factors-I and --II and the response to exogenous growth hormone in young steers. J Endocrinol 118:243-250, 1988. Ellenburger MA, Johnson DE, Carstens GE, Hossner KL, Holland MD, Nett TM, Nockels, CF. Endocrine and metabolic changes during altered growth rates in beef cattle. J Anim Sci 67:1446-1454, 1989. Elsasser TH, Rumsey TS, Hammond AC. Influence of diet on basal and growth hormone-stimulated plasma concentrations of IGF-I in beef cattle. J Anim Sci 67:128141, 1989. Granger AL, Wyatt WE, Craig WM, Thompson DL Jr., Hembry FG. Effects of breed and wintering diet on growth, puberty and plasma concentrations of growth hormone and insulin-like growth factor 1 in heifers. Domest Anim Endocrinol 6:253-262, 1989. Houseknecht KL, Boggs DL, Campion DR, Sartin JL, Kiser TE, Rampacek GB, Amos HE. Effect of dietary energy source and level on serum growth hormone, insulinlike growth factor 1, growth and body composition in beef heifers. J Anim Sci 66:2916-2923, 1988.
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16. Clemmons DR, Klinbansld A, Underwood LE, Me_ArthurJW, Ridgway EL, Beitins IZ, Van Wyk JJ. Reduction of plasma immunoreactive somatomedin C during fasting in humans. J Clin Endocrinol Metab 53:1247-1250, 1981. 17. Clemmons DR, Seek MM, Underwood LE. Supplemental essential amino acids augment the somatomedin-C/insulin-like growth factor I response to refeeding after fasting. Metabolism 34:391-395, 1985. 18, Merimee TJ, Zapf J, Froesch ER. Insulin-like growth factors in the fed and fasted states. J Clin Endocrinol Metab 55:999-1002, 1982. 19. Isley WL, Underwood LE, Clemmons DR. Dietary components that regulate serum somatomedin-C concentrations in humans. J Clin Invest 71 : 175-182, 1983. 20. Isley WL, Underwood LE, Clemmons DR. Changes in plasma somatomedin-C in response to ingestion of diets with variable protein and energy content. J Parenteral Enteral Nutr 8:407-411, 1984. 21. Kunkle WE, Hammond AC, Butts WT, Williams MJ, Baker FS Jr, Palmer AZ, Spreen TH. Evaluation of the STARS stockering and feeding study. In: Proceedings of the Thirty-Seventh Annual Beef Cattle Short Course. University of Florida, Gainesville, p. 67-74, 1988. 22. Williams MJ, Hammond AC, Butts WT, Kunkle WE, Palmer AZ, Baker FS, Spreen TH. Backgrounding systems for Florida: Pasture dynamics and steer performance. In: International Conference on Livestock and Poultry in the Tropics. University of Florida, Gainesville, p. 15-26, 1988. 23. Kunkle WE, Hammond AC, Butts WT, Williams MJ, Baker FS Jr, Palmer AZ, Spreen TH. Effects of backgrounding and finishing system on performance and carcass characteristics of steers. J Anim Sci 66 (Suppl 1): 297-298, 1988. 24. Marsh WH, Fingerhut B, Miller H. Automated and manual direct methods for the determination of blood urea. J Clin Chem 11:624-627, 1965. 25. Kahl S, Bitman J, Rumsey TS. Effect of Synovex-S on growth rate and plasma thyroid hormone concentrations in cattle. J Anim Sci 46:232-239, 1978. 26. 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. 27. Elsasser TH, Rumsey TS, Hammond AC, Fayer R. Influence of parasitism on plasma concentrations of growth hormone, somatomedin-C and somatomedin binding proteins in calves. J Endocrinol 116:191-200, 1988. 28. Honneger A, Humbel RE. Insulin-like growth factors I and II in fetal and bovine serum. Purification, primary structures, and immunological cross-reactivities. J Biol Chem 261:569-575, 1986. 29. Schalch D, Reismann D, Emler C, Humbel R, Li CH, Peters M, Lau E. Insulin-like growth factor-I/somatomedin C (IGF-I/Sm C): Comparison of natural, solid phase synthetic and recombinant DNA analog peptides in two radioligand assays. Endocrinology 115:2490-2492, 1984. 30. SAS Institute Inc. SAS/STATGuide for Personal Computers , Version 6 Edition, SAS Institute Inc., Cary, NC, 1985. 31. Hammond AC, Rumsey TS, Haaland GL. Estimation of empty body water in steers by urea dilution. Growth 48:29-34, 1984. 32. Hammond AC, Rumsey TS, Haaland GL. Prediction of empty body components in steers by urea dilution. J Anita Sci 66:354-360, 1988. 33. Lund-Larsen TR, Sundby A, Kruse V, Velle W. Relation between growth rate, serum somatomedin and plasma testosterone in young bulls. J. Anim Sci 44:189-194, 1977. 34. Ringberg T. Serum somatomedinDa measure of prospective growth capacity in bull calves. Acta Agric Scand 29:216-218, 1979. 35. Gluckman OD, Barry TN. Relationships between plasma concentrations of placental lactogen, insulin-like growth factors, metabolites and lamb size in late gestation ewes subject to nutritional supplementation and in their lambs at birth. Domest Anim Endocrinol 5:209-217, 1988. 36. Anderson PT, Bergen WG, Merkel RA, Enright WJ, Zinn SA, Refsal KR, Hawkins DR. The relationship between composition of gain and circulating hormones in growing beef bulls fed three dietary crude protein levels. J Anita Sci 66:3059-3067, 1988. 37. Gutierrez-De La R JH, Warnick AC, Cowley JJ, Hentges Jr. JF. Environmental physiology in the sub-tropics. I. Effect of continuous environmental stress on some hematological values of beef cattle. J Anim Sci 32:968-973, 1971.
Vol. 7(4):465-476, 1990
EFFECTS OF WINTER NUTRITION AND SUMMER PASTURE OR A FEEDLOT DIET ON PLASMA INSULIN-LIKE GROWTH FACTOR I (IGF-I) AND THE RELATIONSHIP BETWEEN CIRCULATING CONCENTRATIONS OF IGF-I AND THYROID HORMONES IN STEERS A.C. Hammond.1, T.H. Elsasser**, W.E. Kunkle***, T.S. Rumsey**, M.J. Williams* and W.T. Butts* U.S. Department of Agriculture, Agricultural Research Service *Subtropical Agricultural Research Station, Brooksville, FL 34605 and **Ruminant Nutrition Laboratory, Beltsville, MD 20705 ***Animal Science Department, Institute of Food and Agricultural Sciences University of Florida, Gainesville, FL 32611 Received June 9, 1989
ABSTRACT Effects of two winter nutritional levels (LOW, MOD) and two summer pastures (bahiagrass, BG; perennial peanut, PP) on plasma IGF-I, and the relationship between IGF-I and average daily gain (ADG), thyroid hormones, plasma urea, packed cell volume (PCV) and steer type were determined in 101 steers (217 kg) varying in breed composition, frame size and initial condition. Relationships between body composition or composition of gain and IGF-I were determined in 11 contemporary steers assigned directly to the feedlot. Initial IGF-I (57.9 -+ 3.5 ng/ml) was positively correlated (P<.05) to initial condition, estimated percentage of Brahman and plasma T3, but not related to subsequent ADG. During the 126-day wintering period, ADG was .21 kg for the LOW winter treatment and .47 kg for the MOD winter treatment. Concentration of IGF-I in the wintering period was affected (P<.O1) by nutritional level (LOW--71.8 ng/ml, MOD = 150.6 ng/ml) and was positively related to winter ADG in MOD steers (P<.O1) but not in LOW steers. Concentration of IGF-I in winter was also positively related to condition at the end oftbe winter period (P<.O1), T3 (P<.05) and T~ (P<.05). There were no effects of winter treatment on IGF-I during the subsequent summer pasture period. During the 145-d summer period, ADG was .53 kg for BG and .68 kg for PP. Concentration of IGF-I during the summer period was affected (P<.05) by pasture treatment (BG----138.6 ng/ml, PP= 181.9 ng/ml), was positively related (P<.O 1) to PCV and percentage of Brahman, and was negatively related (P<.05) to estimated percentage of English breeding. In steers assigned directly to the feedlot, IGF-I was correlated with empty body (EB) weight (r=-.59, P<.IO), EB water (r=-.59, P<.lO) and EB protein (r-----.60, P<.lO) at slaughter, and with days on feed (r=-.65, P<.05), but was not correlated with ADG or rate of component gain. These data indicate that IGF-I is related to nutritional status in steers as in other species, that there may be significant breed or cattle type differences in circulating concentrations of IGF-I, and that circulating concentration of IGF-I may be functionally related to plasma concentration of thyroid hormones. INTRODUCTION Insulin-like g r o w t h factor I (IGF-I) is a p o t e n t g r o w t h h o r m o n e - d e p e n d e n t m i t o g e n that has b e e n s h o w n to mediate m a n y o f the s o m a t o g e n i c effects o f g r o w t h h o r m o n e . These actions and the role o f IGF-I in the regulation o f g r o w t h have b e e n r e v i e w e d (1). Although some o f the actions o f IGF-I may be paracrine or a u t o c r i n e in nature, circulating c o n c e n t r a t i o n s o f IGF-I are Copyright © 1990 by DOMENDO, INC.
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related to growth rate, nitrogen balance and body size in adult animals and humans. Circulating concentrations of IGF-I are dependent on growth hormone and, in at least rats (2-6), dogs (7), pigs (8), cattle (9-15) and humans (1620), are dependent on nutritional status. In addition, although there is some evidence with dairy cows that circulating IGF-I may display a diurnal rhythm (9), other work with cattle has shown no periodicity in plasma IGF-I concentrations (10). Generally, circulating IGF-I is not considered to be episodic. These characteristics of IGF-I along with recent advances in the ability to quantitatively analyze IGF-I in plasma of major domestic livestock species make it of particular interest to the study of growth in food producing animals. A steer backgrounding study was initiated in 1985 at the Subtropical Agricultural Research Station, Brooksville, Florida to investigate a number of nutritional, genetic, agronomic and economic factors involved in growing-finishing systems (21-23). Steers were selected with variation in type, primarily frame size, condition score and consequently estimated breed composition. The steers were subjected to five systems of backgrounding and finishing involving two winter feeding regimens followed by two summer grazing systems prior to feedlot finishing or direct to feedlot. This report presents data obtained on the effects of the two winter nutrition levels and two summer pastures on plasma IGF-I concentration, and the relationship between IGF-I and growth rate, steer type, plasma thyroxine (T4) concentration, plasma triiodothyronine (T3) concentration, plasma urea nitrogen (PUN) concentration and blood packed cell volume (PCV). In a small group of feedlot steers, relationships between IGFI and body composition or composition of gain were also investigated. MATERIALS AND METHODS Steers (217 kg) were purchased by order buyers in November of 1985 and ranged from small to large in frame size and thin to fleshy in condition. Initial handling of steers included vaccination (IBR, PI3, 7-Way Clostridium), vitamin A injection (1 million IU/head), anthelmintic treatment (Tramisol Injectable, Cyanamid, Wayne, NJ) and implanting (Compudose, Elanco Products Company, Indianapolis, IN). Steers were revaccinated for IBR and PI3 12 days after the first vaccination, and were treated a second time for parasites (IVOMEC, MSD AGVET, Rahway, NJ) 27 days after initial handling. For a preliminary period of 4 weeks, all steers were given access to bahiagrass pasture and for the first 3 weeks were fed 2.3 kg/head/day of a medicated receiving diet (Customized Pre-Conditioning Pellets Medicated, Lakeland Cash Feed Company, Inc., Lakeland, FL). Guaranteed crude protein content of this supplemental receiving diet was 11.5% and contained 193 mg chlortetracycline/kg. Prior to starting the trial, steers were visually evaluated by two of the authors (Kunkle, Butts) for frame size and condition. Each steer then was assigned to one of five systems to achieve a wide range in frame and condition within each system. Several other characteristics of these cattle also were visually evaluated and scored including breed composition (21). The systems were two levels of winter nutrition, low or moderate, and two summer pastures prior to finishing in the feedlot, or direct to feedlot. Steers on the low wintering plane of nutrition (LOW) were grazed on residual bahiagrass ( P a s p a l u m n o t a t u m Flugge.) pasture, offered ad libitum amounts of bahiagrass hay and fed 0.45 kg/head/day of soybean meal (44% crude protein). Steers on the moderate wintering plane of nutrition (MOD) were grazed on similar residual bahiagrass pasture, offered ad libitum amounts of bahiagrass hay and fed 3.2 kg/head/day of an 80%
EFFECTS OF NUTRITION QN IGF-I IN STEERS
467
blackstrap molasses - 20% soybean meal slurry. The winter period lasted for 126 days from December 1985 to April 1986. For 145 days from April to September 1986, half of the steers on each wintering level were grazed on bahiagrass pasture (BP) and the other half were grazed on a mixed sward of grass (predominantly Cynodon and Paspalum spp.) and rhizoma perennial peanut (PP, Arachis glabrata Benth.). Summer pastures were divided into two replicates per treatment and stocking rate was 1.4 head/hectare. Prior to being turned out on summer pasture, steers were reimplanted (Compudose, Elanco Products Company, Indianapolis, IN). Rates of live weight gain in the backgrounded steers were determined by obtaining shrunk weights (live weight following an overnight fast) prior to start of the trial, between the winter and summer phases and at the end of the summer phase. In addition to the initial condition scores obtained prior to the trial, condition scores were obtained between the winter and summer phases and at the end of the summer phase. Jugular blood samples were obtained in the backgrounded steers the day prior to the initial shrunk weight at the start of the winter phase, and at 56-day intervals throughout the winter and summer phases for analysis of packed cell volume (PCV), plasma urea nitrogen (PUN), plasma IGF-I, plasma thyroxine (T4) and plasma triiodothyronine (T3). This sampling schedule provided at least two blood samples for each backgrounding period, winter and summer. The anticoagulant used was EDTA and plasma was stored at -- 20 C in polypropylene vials prior to analysis. Urea nitrogen was determined by a modification (Industrial Method 339-01, Technicon Industrial Systems, Tarrytown, NY) of the automated diacetyl-monoxime method of Marsh et al. (24). Plasma concentrations of T 3 and T4 were measured by solid phase radioimmunoassay using commercial kit assays (Immunochem. Inc.) as previously described (25) and validated for use with bovine plasma. Quantitation of plasma concentrations of each hormone were made using standards included in the kits which consisted of calibrated amounts of T3 and T4 in human serum. Similar bovine serum standards were prepared by treating a pool of bovine serum with 8-anilinonapthanesulphonic acid (8.ANS) to dissociate thyroid hormones from serum carrier proteins and then subjecting the pooled serum to extraction with activated charcoal to remove all thyroid hormones. Amounts of T3 and T4 were added to the extracted serum to obtain serum concentrations of hormones equal to those of the human serum standards in each type kit. Displacement of tracer counts by each standard curve for T3 or T4 were identical as were the recoveries of cold hormone (>95% for each assay). Increasing volumes of plasma from a standard bovine plasma pool displaced tracer counts in a dose-related fashion yielding a slope parallel to that obtained with the standard curve for either T3 or T4. Intraassay and interassay coefficients of variation were less than 7%. Plasma IGF-I was measured by a double antibody radioimmunoassay based on the glycyl-glycine acidification procedure described initially by Underwood, et al. (26) and modified and validated for bovine plasma by Elsasser, et al. (27). Recombinant human IGF-I with the threonine substitution in the 59 position was iodinated by the iodogen method for tracer and similarly used as standard for the assay. Because the primary and tertiary structure of native human and bovine IGF-I are identical (28) and because the recombinant, natural and synthetic forms of the peptide behave identically in the assay (29), this assay was essentially a homologous assay. Plasma was acidified with an
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equal volume of .2 M glycyl-glycine HCI (pH 2.1) for 36 hr. At this time each sample was individually and rapidly diluted with assay buffer (50 mM TRIS, 10 mM EDTA, .25% BSA, pH 7.6); 50 ~tl added immediately to duplicate assay tubes followed by an additional 150 Ixl of assay buffer. To complete the assay volume, both tracer and primary antibody were added in 100 ul volumes. Assays were run as nonequilibrium assays (24 hr with primary antibody alone then an additional 24 hr in the presence of tracer). At the completion of the incubation (at 4 C), bound counts were precipitated with goat antirabbit gamma globulin in polyethylene glycol (PEG) followed by centrifugation and aspiration. To accomplish the secondary precipitation in the separation of bound and free tracer, goat antirabbit gamma globulin was diluted 1:60 in BSA-free assay buffer. This was mixed into a solution of 6% (w/v) PEG (8000 mw) in the proportion of 2 parts antibody solution plus 5 parts PEG solution. The resulting PEG-antibody solution was mixed at 4C for 18 hr prior to addition to the assay. To the 400 1~1 assay tube volume, 450 ~tl of the PEG-antibody solution was added. This was incubated at 4C for 1 hr at which time 100 ~tl of normal rabbit serum (1:100) was added to each tube. This was incubated an additional 30 min prior to centrifugation at 1850 X g. Pellets were counted in an LKB automatic gamma count er for 1 min. Dilutions of acidified plasma displaced counts parallel to the regression line obtained by displacement of counts by the standard curve. Recovery of cold IGF-I added to plasma prior to extraction averaged 92% across a range of hormone mass (5 through 100 ng/ml added). All samples were analyzed in 4 assays with all reagents prepared in the same lot. Intraassay and interassay coefficients of variation were 8 and 10 %, respectively. Minimum detectable quantity at this plasma dilution was 4 ng/ml. Statistical analysis of the relationship between initial IGF-I and other initial variables or subsequent rates of gain was by simple linear regression using the GLM Procedure of the Statistical Analysis System (30). In subsequent analyses involving blood data, the mean of the last two samples for each of the two phases, winter and summer, were analyzed and taken to be indicative of the respective treatments during these times. Statistical analysis of the relationship b etween plasma IGF-I in winter and each independent variable also used a regression approach, the GLM Procedure (30), with the main effect of wintering level included as a class variable along with the wintering level X independent variable interaction. There were no carryover effects of winter plane of nutrition on plasma IGF-I in summer so a similar regression approach was utilized to analyze relationships bet w e e n IGF-I concentrations in the summer period and each independent summer variable with summer pasture and pasture replicate within summer pasture included as class variables. The error term for the summer pasture effect was replicate within summer pasture. Independent variable and independent variable X summer pasture interaction were tested using independent variable X replicate within summer pasture as the error term. Steers assigned directly to the feedlot were fed a high energy diet of 72.5% whole shelled corn, 15% cottonseed hulls and 12.5% of a commercial protein, vitamin and mineral premix (Custom Beef Concentrate 52 Pellets Medicated, Lakeland Cash Feed Company, Inc., Lakeland, FL) which contained not less than 52.0% crude protein, 74,800 USP Units vitamin A/kg, 3.0% calcium, 1.5% phosphorous, not more than 7.5% salt, and 294 mg monensin/kg. Over 3 weeks this diet was changed to 82.5% whole shelled corn, 10% cottonseed hulls and
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7.5% premix. Steers were group-fed in 4 pens, 6 steers/pen, to a visually estimated thickness of 11 to 12 mm of fat over the ribeye. Rates of live weight gain in the direct.to-feedlot steers were determined by the difference between shrunk weights taken just prior to the start of feeding and just prior to slaughter. Blood samples and estimates of body composition using urea dilution (31-32) were obtained from a subset of 12 of these steers every 42 days beginning with day 14 in the feedlot. One of these steers was subsequently dr oppe d from the experiment for reasons unrelated to treatment. Blood was analyzed for PCV and blood plasma for PUN, IGF-I, T4 and T 3 as described for backgrounding steers. Rates of empty body c o m p o n e n t (water, protein and fat) gain were determined by regression over time. There was no significant effect of time in the feedlot on IGF-I so further statistical analysis utilized means for each steer. The relationship between IGF-I and all other variables analyzed with these steers was determined by simple linear regression as described for initial IGF-I analysis in the backgrounding steers. RESULTS Mean and SD for independent variables tested during backgrounding of steers and for the direct to feedlot steers are given in Tables 1 and 2. For the directto-feedlot steers, time on feed (203.9 _+ 43.5 days) and empty body weight at slaughter (436.9 + 57.7 kg) also were analyzed as independent variables. Mean IGF-I by system, and effects of winter plane of nutrition or summer pasture on IGF-I are given in Table 3. Plasma IGF-I was higher (P<.01) for the MOD plane of nutrition compared to LOW and plasma IGF-I was higher (P<.05) for the higher quality summer grass-legume pasture, PP, compared to BG. Although not tested statistically, mean plasma IGF-I ranked highest in the direct to feedlot steers on a high energy concentrate diet compared to any of the backgrounding treatments. Correlations between initial plasma IGF-I concentrations (57.9 -+ 3.5 ng/ ml) and initial independent variables or subsequent rate of live weight gain during winter or summer are given in Table 4. Correlations between initial TABLE 1. MEAN + SD OF INDEPENDENTVARIABLESSTUDIED WITH RESPECT TO EFFECTS ON IGF-I IN BACKGROUNDED AND DIRECT-TO-FEEDLOTSTEERS Independent variable Rate of gain (kg/day) Condition score = Frame score b Muscle score ~ Estimated b r e e d c o m p o s i t i o n Brahman (%) English (%) Continental (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%) T3 ( n g / m l ) T4 ( n g / m l )
Backgrounded Steers ('n= 101) Initial Winter Summer 8.0 _+ 1.2 8.8 + 2.1 1.84 _+ .33 39.9 55.3 4.8 8.6 36.8 1.64 68.1
_+ -+ + _+ + _+ +
.35 -+ 7.4 +
22.2 20.9 14.0 2.30 11.8 3.9" 39.0 .48 d 1.61 18.00 66.0
.20 1.0
+ 3.9 -+ 3.1 -+ .4 + 16.7
Direct to Feedlot Steers ( n = 11) 1.19 8.0 9.1 1.71
+ + -+ +
26.1 56.1 17.7 11.4 6.1 8.4 36.6 -+ 3.5 38.4 2.07 +.32 3.23 76.2 + 19.4 116.6
_+ + -+ -+ + + +
.60 + 9.0 +
.15 1.1
.14 1.4 = 2.7 r .38 f 19.2 f 20.0 f 25.5 f 1.1 2.8 .41 10.5
•Body condition scoring system: 4 , 5 , 6 = r e l a t i v e degrees of emaciation; 7 = 1 m m fat over the ribeye; 8 = 2 m m fat over the ribeye; 9 = 3 m m fat; etc. bFrame score defined as slaughter w e i g h t r e q u i r e d to finish w i t h .5" fat over the ribeye; 2 = 8 0 0 lb.; 5 = 9 0 0 lb.; 8 = 1 , 0 0 0 lb.; 1 1 = 1 , 1 0 0 lb., 1 4 = 1 , 2 0 0 lb. "Muscle s c o r e : 1 . 2 = h e a v y + ; 1 . 5 = h e a v y ; 1 . 8 = h e a v y - - ; 2 . 2 = m e d i u m + ; 2.5=medium; 2 . 8 = m e d i u m - - ; 3.2 = l i g h t + ; 3.5 = l i g h t ; 3 . 8 = l i g h t - . dn=93. ~n = 70. qnitial.
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TABLE 2. COMPOSITION OF GAIN AND BODY COMPOSITION AT SLAUGHTEROF DIRECT-TO-FEEDLOT STEERS Water
Component" Protein
Fat
.41 + .08 226.3 _+ 25.2 52.0 + 2.7
.14 +_ .02 70.4 + 8.2 16.2 _+ .7
.57 + .11 119.4 _+ 26.0 27.2 -+ 3.9
Item C o m p o s i t i o n of gain (kg/day) C o m p o s i t i o n at slaughter (kg) C o m p o s i t i o n at slaughter (%) •Mean _+ SD
IGF-I and initial condition score, initial plasma T 3, and estimated percentage of Brahman breeding were each positive and significant, but correlation coefficients were low. Initial IGF-I and initial plasma T4 only tended to be correlated, and correlations between initial plasma IGF-I and all other initial independent variables were not significant. In addition, initial plasma IGF-I was not correlated with subsequent rates of gain in either the winter or summer backgrounding phases. TABLE 3. MEAN IGF-I BY MAIN EFFECTWITHIN SYSTEMIN BACKGROUNDEDSTEERSAND IN DIRECT-TO-FEEDLOT STEERS Treatment Winter plane of nutrition LOW ( n = 4 9 ) MOD ( n = 5 2 ) S u m m e r pasture BG ( n = 5 1 ) PP ( n = 5 0 ) Direct to feedlot (n = 11)
IGF-I ( n g / m l )
SEM
71.8" 150.6
4.4 5.6
138.6 b 181.9 203.4
5.8 4.7 7.0
"Winter plane of nutrition effect ( P < . 0 1 ) . bSummer pasture effect ( P < . 0 5 ) .
During the wintering phase, there was a significant interaction (P<.01) between plane of nutrition and rate of live weight gain on plasma IGF-I (Table 5). At the LOW plane of nutrition, with rates of gain averaging .21 kg/day (23), there was no significant relationship between plasma IGF-I and rate of gain. With the MOD plane of nutrition, rate of gain averaged .47 kg/day (23) and was positively related to plasma IGF-I (P<.O1). There were no other significant interactions between winter treatment and other independent variables studied. With the significant effects of winter plane of nutrition removed, plasma IGF-I was positively related to condition score at the end of the winter period (P<.O1), positively related to plasma T 3 (P<.05) and positively related TABLE 4. CORRELATIONBETWEEN INITIALPLASMAIGF-I AND EACH INITIALINDEPENDENTVARIABLEOR SUBSEQUENT RATE OF GMN IN BACKGROUNDEDSTEERS Independent variable Condition score Frame score Muscle score Estimated b r e e d c o m p o s i t i o n Brahman (%) English (%) Continental (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%)
T3 (ng/ml) T4 (ng/ml) Winter gain (kg/day) S u m m e r gain (kg/day) •Not significant, P > . I O.
r
Prob.
.21 .10 .03
<.05 NS" NS
.22 - . 14 - . 12 .08 .19
<.05
.30 .19
<.01 <.10
- . 13 - . 11
NS NS NS NS NS NS
EFFECTS OF NUTRITION ON IGF-I IN STEERS TABLE5.
471
RELATIONSHIP BETWEEN PLASMA IGF-I AND AVERAGE DAILY GAIN IN BACKGROUNDED STEERS ON TWO WINTERING PLANES OF NUTRITION Sy-x
Treatment X LOW ADG• (kg) MOD ADG (kg) •Average daily gain.
Slope 28.0 148.7
Intercept 65.9 80.3
(ng/ml) 31.1 33.9
rz .02 .32
Prob. NS <.01
tO plasma T4 ( P < . 0 5 ) , but was not related to plasma urea N or p a c k e d cell v o l u m e (Table 6). There was no significant replicate w i t h i n s u m m e r pasture effect on plasma IGF-I ( P > . I O ) . Plasma IGF-I in s u m m e r was significantly higher ( P < . 0 5 ) on PP pasture than on BG pasture (Table 3) and associated rates of live w e i g h t gain also w e r e significantly higher on PP pastures than on BG pastures, .60 v s . . 5 3 kg/day, respectively (23). However, with significant effects o f s u m m e r pasture removed, there was no significant additional variation in plasma IGFI due to rate o f live w e i g h t gain (P>. 10). O f all i n d e p e n d e n t variables tested, plasma IGF-I in s u m m e r was significantly related only to estimated p e r c e n t a g e o f Brahman and English breeding, and to p a c k e d cell volume. Because there were no significant interactions associated with these effects, a simple regression m o d e l was used to d e t e r m i n e the relationships b e t w e e n each o f these i n d e p e n d e n t variables using s u m m e r pasture as a class variable. C o m m o n regressions from p o o l e d data derived from these analyses are given in Table 7. Correlation b e t w e e n mean circulating IGF-I in the 1 1 direct to feedlot steers and each i n d e p e n d e n t variable studied is given in Table 8. In these steers, IGF-I was negatively correlated w i t h EB weight, EB water and EB p r o t e i n at slaughter, and negatively correlated w i t h days on feed r e q u i r e d to reach an estimated e n d p o i n t of 1 1 to 12 m m backfat. Circulating IGF-I was not correlated with live w e i g h t gain or individual c o m p o n e n t gain in these steers. DISCUSSION In the present study with steers, circulating IGF-I c o n c e n t r a t i o n s w e r e positively affected by plane o f nutrition in the w i n t e r and quality of pasture in the summer. In addition, IGF-I c o n c e n t r a t i o n s ranked highest in plasma from steers o n a c o n c e n t r a t e based (relatively high energy) feedlot diet. Furthermore, the relationship b e t w e e n IGF-I and average daily gain o f steers interacted w i t h TABLE 6. RELATIONSHIPSt BETWEEN PLASMAIGF-I AND EACH INDEPENDENT VARIABLE IN BACKGROUNDED STEERS DURING WINTERING PHASE
Sy'x
X Slope Intercept (ng/ml) rz Prob. Condition score 16.3 -8.9 30.4 .60 <.01 Initial frame score 3.6 79.3 35.7 .57 NSb Initial muscle score -22.2 152.0 35.7 .56 NS Estimated breed composition Brahman (%) -1.2 116.1 36.4 .55 NS English (%) 1.O 105.7 36.4 .55 NS Continental (%) .83 110.8 36.5 .55 NS Plasma urea N (mg/dl) .23 108.5 36.5 .55 NS Packed cell volume (%) 1.6 49.0 36.2 .55 NS Plasma T3 (ng/ml) 27.0 67.9 35.5 .55 <.05 Plasma T4 (ng/ml) .72 63.9 35.4 .58 <.05 •Effects due to winter plane of nutrition (LOW vs. MED) on plasma IGF-I were determined by using winter plane or nutrition as a class variable (for results see Table 2). Common regressions from pooled data were derived from these analyses for each independent variable and are presented here. bNot significant, P>. 10.
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HAMMOND ET AL.
TABLE 7. SIGNIFICANT RELATIONSHIPS BETWEEN PLASMAIGF-I AND INDIVIDUALINDEPENDENT VARIABLESIN BACKGROUNDEDSTEERS DURING SUMMER PASTURE PHASE Independent variable
Slope
Intercept
Sy.x (ng/ml)
r2
Prob
Estimated b r e e d composition B r a h m a n (%) English (%) Packed cell v o l u m e (%)
.42 -.49 2.4
143.5 165.0 72.5
36.3 36.0 36.6
.30 .31 .29
<.05 <.01 <.05
level of winter nutrition. Plasma IGF-I was lower on the low plane of winter nutrition (adequate protein but low energy) and was not related to average daily gain. With supplemental energy (molasses) in addition to protein supplementation in the winter, plasma IGF-I concentrations were higher and were positively related to average daily gain of steers. As mentioned in the materials and methods, there was no carryover effect of winter treatment on IGF-I in summer. In contrast, Granger e t al. (14) found in heifers that plasma IGF-I concentrations that were depressed due to a lower quality of wintering diet, continued to be lower during a subsequent summer pasture phase when heifers were on a com m on diet. Early work with growing cattle using bioassay techniques for serum somatomedin activity demonstrated significantly higher mean serum IGF-I in high rate of gain groups compared to low rate of gain groups of bulls (33-34). In more recent studies with cattle, circulating concentrations of IGF-I also were shown to be affected by plane of nutrition in heifers (14-15), cows (9) and steers (1 O- 13). Both energy (9-13) and protein (13) intake influenced plasma IGF-I concentrations. As with other species, there have been reported positive relationships between circulating IGF-I and nitrogen balance (1 O, 13) or growth rate (11). One recent report of work with sheep demonstrated higher IGF-I TABLE 8. CORRELATIONBETWEEN PLASMAIGF-P AND EACH INDEPENDENT VARIABLESTUDIED IN DIRECT TO FEEDLOT STEERS Independent variable Rate of gain (kg/day) Condition score Frame s c o r e Muscle score Estimated b r e e d c o m p o s i t i o n B r a h m a n (%) English (%) C o n t i n e n t a l (%) Plasma urea N ( m g / d l ) Packed cell v o l u m e (%) T, ( n g / m l ) T4 ( n g / m l ) T i m e on feed (days) C o m p o s i t i o n of gain Water ( k g / d a y ) Protein (kg/day) Fat ( k g / d a y ) Body c o m p o s i t i o n at s l a u g h t e r E m p t y b o d y w e i g h t (kg) Water (kg) Protein (kg) Fat (kg) Water (%) Protein (%) Fat (%)
r
Prob.
.33 .48 -.51 -.02
NSb NS NS NS
.34 .33 -.51 .75 .18 .15 .03 -.65
NS NS NS <.01 NS NS NS <.05
.05 .08 .13
NS NS MS
-.55 -.59 -.60 -.43 .09 .02 -.13
<. 10 <. 10 <.10 NS NS NS NS
•Mean o f 5 to 7 b l o o d s a m p l e s o b t a i n e d every 42 days b e g i n n i n g day 14 in t h e feedlot. "Not significant, P>. 10.
EFFECTS OF NUTRITION ON IGF-I IN STEERS
473
concentrations in ewes given abomasal infusions of glucose and/or casein compared to controls; and a positive relationship between circulating IGF-I concentrations in ewes bearing triplets and birth weight of their lambs (35). A relatively small number of steers assigned directly to the feedlot were investigated in our study, so data from these steers should be considered preliminary and subject to confirmation. Nevertheless, the results indicated that IGF-I was negatively correlated to total empty body and lean body mass at slaughter at a constant fat endpoint. Days on feed in the feedlot which were required to reach the constant fat endpoint also showed a negative correlation with circulating IGF-I. These data suggest that IGF-I may be higher in earlier maturing but not necessarily larger cattle types. It may be important to note that steers in this study varied with respect to size, type, condition, breed, etc., and may have expressed variability in circulating IGF-I that may not be seen in studies with more homogeneous groups of experimental animals. One such study was that of Anderson et al. (36) in which significant correlations were found between IGF-I concentrations and percentage of carcass fat (r=--.60), carcass fat accretion rate (r = - . 5 7 ) , total carcass fat (r = - . 5 2 ) , fat thickness (r = --.73) and percentage of carcass protein (r = .60) in Simmental crossbred bulls. From among the other independent variables measured in the present study, the relationship between circulating concentrations of IGF-I and thyroid hormones may be the most interesting. There were significant positive relationships between IGF-I and T 3 initially, and between IGF-I and both T 3 and T4 at the end of the winter feeding phase. Serum T 3 and IGF-I were both reduced in calorie-restricted rats and were associated with reduced growth rate, i.e., lower body weight in calorie-restricted rats compared to controls of the same age (6). In one trial with steers, plasma IGF-I and plasma T4 both increased with increasing dietary protein level (13). In another trial with steers, IGF-I and T4 were reduced in response to restricted feeding but T 3 was unchanged (12). An effect of thyroid hormones on the regulation of growth hormone receptors was alluded to by Maes e t al. (3-4) which suggests that the effects of nutrition on circulating IGF-I may be mediated through mechanisms that modulate the effect of growth hormone on IGF-I production at the growth hormone receptor level, but investigation of these possibilities was beyond the scope of the present trial. Throughout both the winter and summer periods, plasma IGF-I was positively correlated with estimated percentage of Brahman breeding. There also was a positive correlation between plasma IGF-I and packed cell volume during the summer grazing period. The relationships between IGF-I and packed cell volume, and between IGF-I and breed type may be an autocorrelation effect. In one study, Brahman cattle were shown to have higher packed cell volume compared to an English breed type (37) and this has been a consistent observation of ours at Brooksville, as well (unpublished). We have previously pointed out that it is difficult to describe cause and effect relationships between various aspects of growth, the apparent plane of nutrition and plasma concentrations of IGF-I (13). Whether plasma concentrations of IGF-I are involved directly in the regulation of growth and metabolism or simply reflect changes in metabolism that coincide with other nutritional and hormonal events associated with growth cannot be determined from the present data. We conclude from these data, however, that circulating concentrations of IGF-I are related to nutritional status in steers as with other species,
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t h a t t h e r e m a y b e s i g n i f i c a n t b r e e d or c a t t l e t y p e d i f f e r e n c e s i n 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 o f IGF-I, a n d t h a t 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 o f IGF-I m a y b e f u n c t i o n a l l y r e l a t e d to 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 o f t h y r o i d h o r m o n e s . ACKNOWLEDGEMENTS/FOOTNOTES The authors thank M.J. Schoppman, E.L. Adams and M.T. Pello for technical assistance and V.E. Rooks and the STARSfarm staff for animal handling and care. Mention of companies or products is for the benefit of readers and does not constitute an endorsement or preferential treatment by the authors or the USDA. Published as Florida Agricultural Experiment Station Journal Series No. R-00579. ~Address correspondence and requests for reprints to: A. C. Hammond, Research Leader, USDAARS-STARS, P.O. Box 46, Brooksville, FL 34605-0046.
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