Effect of exogenous estradiol on plasma concentrations of somatotropin, insulin-like growth factor-I, insulin-like growth factor binding protein activity, and metabolites in ovariectomized angus and braham cows

Effect of exogenous estradiol on plasma concentrations of somatotropin, insulin-like growth factor-I, insulin-like growth factor binding protein activity, and metabolites in ovariectomized angus and braham cows

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 14(6):367-380, 1997 ELSEVIER EFFECT OF EXOGENOUS ESTRADIOL ON PLASMA CONCENTRATIONS OF SOMATOTROPIN, INSULIN-LIKE...

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DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 14(6):367-380, 1997

ELSEVIER

EFFECT OF EXOGENOUS ESTRADIOL ON PLASMA CONCENTRATIONS OF SOMATOTROPIN, INSULIN-LIKE GROWTH FACTOR-I, INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN ACTIVITY, AND METABOLITES IN OVARIECTOMIZED ANGUS AND BRAHMAN COWS R.B. Simpson,*,t C.C. Chase, Jr.,*, 1 L.J. Spicer,** J.A. Carroll,***,$ A.C. Hammond,* and T.H. Welsh, Jr.*** *Agricultural Research Service, USDA, Brooksville, FL 34601-4672, **Department of Animal Science, Oklahoma State University, Stillwater, OK 74078-0425, and ***Departments of Animal Science and Veterinary Anatomy, Texas A & M University System, College Station, TX 77843 Received December13, 1997 Accepted June29, 1997 To determine the effect of breed and estradiol-17f3 on selected hormones and metabolites, ovariectomized (>13 mo) Angus (n = 14) and Brahman (n = 12) cows were paired by age and body weight and randomly assigned as either nonimplanted controls (CON) or implanted with estradiol (E2) for 45 d. After Day 7 and through Day 42, plasma concentration of somatotropin was greater for E2 than CON cows (treatment X day, P < 0.05). During an intensive blood sampling on Day 36, E2 cows tended (P < 0.10) to have greater somatotropin pulse amplitudes than CON cows, but other parameters of somatotropin release were not affected (P > 0.10) by E2 treatment. The effect of breed was apparent on Day 36 as Brahman cows had greater (P < 0.05) somatotropin pulse amplitude, basal secretion, and mean concentration than Angus cows. Overall, plasma concentration of IGF-I was greater (P < 0.01) for E2 than CON cows (158.3 vs. 104.2 ng/ml) and was greater for Brahman than Angus cows (164.1 vs. 98.4 ng/ml). However, there was a trend (P < 0.10) for a treatment × breed × day interaction for IGF-I (i.e., the magnitude of increase in IGF-I concentration was greater in E2-Angus than E2-Brahman cows). After Day 7 and through Day 42, total plasma IGF binding protein (IGFBP) activity was greater (P < 0.01) for E2 than CON cows. Ligand blotting revealed at least five forms of IGFBP activity, and E2 cows had greater (P < 0.05) binding activity of IGFBP-3 and the 30- and 32-kDa IGFBP than CON cows. Brahman cows had greater (P < 0.05) IGFBP-3 and the 32-kDa IGFBP than Angus cows. After Day 14 and through Day 42, concentration of urea nitrogen (PUN) was greater (P < 0.001) for CON than E2 cows (treatment × day, P < 0.001). Brahman had greater (P < 0.01) PUN than Angus cows (16.6 vs. 14.2 mg/dl). Plasma concentration of glucose was greater (P < 0.01) for E2 than CON cows (78.9 vs. 76.4 mg/dl) but was not affected (P > 0.10) by breed. In summary, these data suggest that some, but not all, of the positive effects of estradiol on peripheral concentration of IGF-I and IGFBP activity can be attributed to increased somatotropin. Moreover, breed influenced basal and E2-induced secretion of somatotropin and IGF-I such that differences between Brahman and Angus cows in plasma IGF-I concentrations were abated within 3 wk of estradiol implantation. Thus, breed influences the metabolite and hormonal response of cattle to estrogenic implants. © Elsevier Science Inc. 1997 INTRODUCTION P r e v i o u s studies suggest that estradiol f r o m ovaries o f cyclic c o w s (1,2) and e w e s (3) m a y e n h a n c e systemic secretion o f insulin-like g r o w t h factor-I (IGF-I), and that p o o r nutrition m a y inhibit this positive effect o f estradiol on I G F - I secretion (1).

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368

SIMPSON ET AL.

Unequivocally, long-term treatment with exogenous estradiol increases IGF-I secretion in steers (4-6). However, the effect of exogenous estradiol on IGF-I secretion and IGF-I binding proteins (IGFBP) in ovariectomized cows is not well characterized. Because IGFBPs may alter the responsiveness of target tissue to IGF-I (7), estradiol regulation of IGFBPs may play an important role in an animal's response to exogenous estradiol. Additional studies have shown that estrogens potentiate somatotropin-stimulated hepatic IGF-I secretion via increasing the number of hepatic somatotropin receptors (5,8,9). On the other hand, estrogens stimulate somatotropin secretion in castrated sheep (10,11) and cattle ( 4 - 6 ) as well as stimulate somatotropin release from cultured anterior pituitary cells of sheep (12) but not of cattle (13). Thus, the stimulatory effect of estrogens on IGF-I secretion may or may not be direct. We have reported previously that intact Brahman (Bos indicus) cows have greater systemic concentrations of IGF-I and IGFBP activity than Angus (Bos taurus) cows (14). Whether these differences in IGF-I concentrations and IGFBP activity between Brahman and Angus cows are attributable to differences in somatotropin secretion or to differences in responsiveness to endogenous estradiol is unknown. Therefore, the specific objectives of the present study were to 1) determine the effect of exogenous estradiol on plasma concentrations of somatotropin, IGF-I, IGF-I binding protein activity and metabolites in ovariectomized cows and 2) compare selected hormonal and metabolic responses to exogenous estradiol between ovariectomized Angus and Brahman cows.

MATERIALS AND METHODS Animals and Treatments. At the Subtropical Agricultural Research Station near Brooksville, Florida, mature Angus (n = 14; 448 + 16.4 kg) and Brahman (n = 12; 467 _+ 13.0 kg) cows were used to study the effects of exogenous estradiol on plasma concentrations of somatotropin, IGF-I, IGFBP activity, glucose, and urea nitrogen (PUN). At least 3 mos before the start of the study, cows were ovariectomized as described previously (14). Cows were placed in a drylot 18 d before the initiation of the study and were gradually adapted to receive 6.8 kg/d (as-fed) of a total mixed ration (53.9% corn, 30.3% cottonseed hulls, 15.2% soybean meal, and 0.6% dicalcium phosphate; 14% CP and 70% TDN estimated from NRC; 15). Cows were paired by age and body weight into seven pens (six pens housed two Angus and two Brahman cows/pen and one pen housed two Angus cows). Within each pen, treatments were randomly assigned within each breed. Treatments were non-implanted control (CON) or estradiol ear-implant (E2; 24 mg estradiol1713, Compudose ® 200, Elanco Products Co., Indianapolis, IN). Data and Sample Collection. At 7-day intervals, cows were weighed and fat thickness over the ribeye, between the 12th and 13th ribs, was measured by ultrasonography (Aloka 210 equipped with a 3.5 MHz probe, Corometrics Medical Systems, Wallingford, CT). Blood samples collected via coccygeal vessel puncture were obtained daily from Days - 2 to 7, and once weekly from Days 14 to 42 (Day 0 = day of implant administration). Blood samples were processed to yield plasma, and plasma was stored ( - 2 0 ° C) for somatotropin, IGF-I, IGFBP activity, glucose, and PUN analyses. On Day 35, cows were fitted with jugular cannulae and on Day 36, blood samples were collected at 20-rain intervals for 6 hr, processed to yield plasma and plasma was stored ( - 2 0 ° C) for somatotropin analysis. On Day 45, cows were slaughtered (Central Packing Co., Center Hill, FL), and pituitary glands were collected and dissected carefully to obtain weights of the anterior and posterior pituitary glands. Assays. Plasma concentration of somatotropin was determined by a double-antibody radioimmunoassay as previously described (16,17). Intra- and interassay coefficients of

BREED AND ESTRADIOL AFFECTS SOMATROPIC AXIS

369

variation for the somatotropin assays were 5.9 and 8.1%, respectively. For data from samples collected every 20 min for 6 hr on Day 36, basal somatotropin and frequency and amplitude of somatotropin pulses were determined for each animal as defined previously (18). Plasma concentration of IGF-I was determined by radioimmunoassay after acidethanol extraction as described previously (19). Intra- and interassay coefficients of variation for the IGF-I assays were 10.6 and 20.2%, respectively. Sensitivity of the assay defined as 90% of total binding was 11.5 pg/tube. Total IGFBP activity in plasma was determined after incubation with [~25I]IGF-I by the method of Moses et al. (20). Briefly, 10-1xl aliquots of plasma were incubated overnight at 4 ° C with 100 txl of [125I]IGF-I (15,000 cpm; counter efficiency was 75%) and 150 Ixl of assay buffer (phosphate buffered saline containing 2.5 mg of bovine serum albumin/ml, pH 7.5). Activated charcoal (500 Ixl; 5% w/v in assay buffer) was added to each tube to separate bound from free [125I]IGF-I, the tubes were incubated for 30 min at 4 ° C, and then centrifuged at 1,200 × g for 20 min at 4 ° C. Intra- and interassay coefficients of variation were 2.8 and 7.7%, respectively. Plasma IGFBPs were analyzed by one-dimensional SDS-PAGE for samples collected on Day 14 and 42 of treatment, as described previously (21). Briefly, 4 txl samples in 21 Ixl of denaturing buffer were heat denatured and then separated on a 12% polyacrylamide gel via electrophoresis. Samples were stratified by treatment and breed across four gels. After separation, proteins in gels were electrophoretically transferred to nitrocellulose, and ligand-blotted overnight with [t25I]IGF-I. After washing the nitrocellulose and then exposure to X-ray film at - 7 0 ° C for 24 hr, band intensity on autoradiographs was determined using a PDI Model DNA 35 scanner and Quality One (version 2.4) software for quantification by scanning densitometry (PDI, Inc., Huntington Station, NY). Plasma concentration of glucose was determined by an automated colorimetric method (Technicon AutoAnalyzer II Industrial Method 339-19, Technicon Industrial Systems, Tarrytown, NY) based on the glucose oxidase procedure described by Gochman and Schmitz (22). Plasma concentration of urea nitrogen was determined by an automated colorimetric procedure (Technicon AutoAnalyzer II Industrial Method 339-01, Technicon Industrial Systems, Tarrytown, NY) based on the diacetyl monoxime method of Marsh et al. (23). Statistical Analyses. Data were analyzed by least squares analysis of variance using the GLM Procedure of SAS (24). The model used to analyze average daily body weight gain and pituitary gland weight included treatment, breed and treatment × breed as sources of variation. For data collected over time (i.e., fat thickness over the ribeye and plasma concentrations of hormones and metabolites) the model was expanded to also include pen within treatment × breed, day or time, treatment × day or time, breed X day or time, and treatment X breed X day or time. Effects of treatment, breed and treatment × breed were tested using the pen within treatment × breed mean square as the error term. For analysis of IGFBP data from ligand blotting, the effect of gel number was included in the model. Data are presented as least-squares means _+ standard error. RESULTS

Average Daily Body Weight Gain, Fat Thickness Over the Ribeye, and Pituitary Gland Weights. At the start of the study, body weight averaged 448 +_ 16.4 kg and 467 - 13.0 kg for mature, ovariectomized Angus and Brahman cows, respectively. Average daily body weight gain over the 42 d of treatment, was small and not affected (P > 0.10) by treatment or treatment × breed (Table 1). However, Angus cows had greater (P < 0.01) average daily body weight gain than Brahman cows (403 vs. 21 g). Neither treatment, breed, day, nor any interactions affected (all P > 0. I0) fat thickness

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SIMPSON ET AL.

TABLE 1. EFFECTSOFTREATMENT(T), CONTROLANDESTRADIOL(E2) IMPLANT,ANDBREED(B), ANGUSAND BRAHMAN,ONAVERAGEDAILYBODYWEIGHTGAIN(ADG, DAY 0-42) ANDPITUITARYGLANDWEIGHTS (DAY45 POSTIMPLANT).a Control

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No. of cows ADG, g Pituitary gland weight Anterior, g mg/kg body weight Posterior, g mg/kg body weight Total, g mg/kg body weight

7 270 -+ 137.1

6 38 + 148.1

7 535 -+ 137.1

1.62 + 0.t62 3.76 -+ 0.373 0.29 -+ 0.029 0.68 +- 0.068 1.90 -+ (/.165 4.42 ± 0.386

1.34± 0.175 2.80 + 0.403 0.16 ± 0.031 0.35 + 0.074 1.50-+ 0.178 3.14 ± 0.417

2.32 _+0.162 4.77 -+ 0.373 0.29 ± 0.029 0.60 -+ 0.060 2.66 + 0.165 5.46 -+ 0.386

Probability Brahman

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a Data are least-squares means -+ SE.

over the ribeye, and the main effects of treatment and breed averaged 6.7 _+ 0.67 m m for both CON and E2 cows, and 7.2 _+ 0.64 mm and 6.2 + 0.69 m m for Angus and Brahman cows, respectively. After 45 d of treatment, anterior pituitary gland weight from E2 cows was greater (P < 0.01) than that of CON cows (1.97 vs. 1.48 g), even when adjusted for cow body weight (BW; 4.16 vs. 3.28 mg/kg BW; P < 0.05; Table 1). Treatment did not affect (P > 0.10) actual or adjusted weight of the posterior pituitary gland. Therefore, the larger actual (P < 0.01) and adjusted (P < 0.05) total pituitary gland weights from E2 cows compared with CON cows (2.22 vs. 1.71 g and 4.69 vs. 3.79 mg/kg BW) was attributable to larger weight of the anterior pituitary gland. Angus cows had greater (P < 0.01) actual and adjusted weights of anterior pituitary gland (1.97 vs. 1.48 g and 4.27 vs. 3.17 mg/kg BW), posterior pituitary gland (0.29 vs. 0.18 g and 0.64 vs. 0.48 mg/kg BW) and total pituitary gland (2.26 vs. 1.66 g and 4.91 vs. 3.57 mg/kg BW) than Brahman cows. Plasma Concentration of Hormones and Metabolites. During the 47-day study, treatment and day interacted (P < 0.05) to affect plasma concentration of somatotropin. After Day 7 and through Day 42, plasma concentration of somatotropin was greater (Days 14 and 35, P < 0.10; Days 21, 28 and 42, P < 0.05) for E2 than CON cows (treatment × day, P < 0.05; Figure 1). Breed and breed x day did not affect (P > 0.10) somatotropin concentrations which averaged 3.1 ± 0.36 ng/ml and 3.5 ± 0.39 ng/ml for Angus and Brahman cows, respectively. On Day 36, during the intensive blood sampling, E2 treatment did not affect (P > 0.10) basal or mean concentration of somatotropin or the number of pulses of somatotropin determined in samples collected every 20 min for 6 hr (Table 2). However, E2 cows tended (P < 0.10) to have greater somatotropin pulse amplitude than CON cows (5.08 vs. 2.96 ng/ml). Also on Day 36, Brahman cows had greater basal (P < 0.05; 4.22 vs. 2.31 ng/ml) and mean (P < 0.01; 5.26 vs. 2.72 ng/ml) concentration of somatotropin than Angus cows. Breed did not affect (P > 0.10) the number of somatotropin pulses detected during the 6-hr sampling, but the amplitude of the somatotropin pulses was greater (P < 0.05) for Brahman than Angus cows (5.30 vs. 2.74 ng/ml). During the 47-day study, plasma concentration of IGF-I was greater (P < 0.01) for E2 (158.3 _+ 12.79 ng/ml) than CON cows (104.2 _+ 12.79 ng/ml), and was greater (P < 0.01) for Brahman (164.1 ± 13.27 ng/ml) than Angus cows (98.4 ± 12.29 ng/ml). However, for plasma concentration of IGF-I, there was a trend for a treatment x breed X day interaction (P < 0.10) and a treatment × breed interaction (P < 0.101 and there was (P < 0.001) both a treatment X day and breed x day interaction (Figure 2). Throughout the study, Brahman

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Figure 1. Effect of treatment, control (CON) versus estradiol implant (E2) administeredon Day 0 of the study, and day of treatmenton plasma concentrationsof somatotropin(ng/ml) in ovariectomizedAngus and Brahman cows. Data are least-squares means ± SE. cows had greater plasma concentration of IGF-I than Angus cows (all days, P < 0.05, except Day 35, P < 0.12). During the first week after treatment, plasma concentration of IGF-I increased in E2 but not CON cows (from Day 4 through Day 42, all P < 0.01). The magnitude of the increase in IGF-I concentration in E2-Angus cows was great enough that the concentration of IGF-I was similar to that in E2-Brahman cattle after 14 days of treatment. The E2-Brahman cows tended to have greater plasma concentration of IGF-I than CON-Brahman cows on each of the sampling days, but these differences were not always significant. Total plasma IGFBP activity was greater (P < 0.05) for E2 (19.65 _ .536 %/10 I~1) than CON cows (17.76 _ .524 %/10 Ixl) during the 47-day study. Day of the study affected (P < 0.001) total IGFBP activity and treatment X day tended (P < 0.10) to affect total IGFBP activity (Figure 3). On Day 0, total IGFBP activity did not differ (P > 0.10) between treatments; however, E2 cows had greater total IGFBP activity on Day 7 (P < 0.08) and from Day 14 to 42 (all days P < 0.01) than CON cows. Neither breed, treatment X breed nor breed x day affected (P > 0.10) total IGFBP activity that averaged 18.88 _ .552 %/10 txl for Brahman and 18.54 - .507 %/10 Ixl for Angus cows. Ligand blotting revealed at least five forms of IGFBP activity in plasma of Angus and TABLE 2. EFFECTS OF TREATMENT (T), CONTROL AND ESTRADIOL(E2) IMPLANT, AND BREED (B), ANGUS AND BRAHMAN, ON SECRETION OF SOMATOTROPINFROM PLASMA COLLECTEDEVERY 20 MIN FOR 6 HR ON DAY

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Angus

Somatotropin Basal, ng/ml Mean, ng/ml No. of pulses/6 hr Amplitude, ng/ml

2.22 - 0.727 2.53 ± 0.840 2.43 +- 0.443 2.12 ± 1.069

a Data are least-squares means.

E2

Brahman 3.23 ± 4.08 ± 3.17 ± 3.80±

0.882 0.907 0.479 1.155

Angus 2.39 ± 2.91 ± 2.14± 3.36±

0.771 0.840 0.443 1.069

Probability Brahman 5.176.44± 2.83 ± 6.79 ±

0.884 0.907 0.479 1.155

T 0.22 0.14 0.51 0.10

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0.29 0.27 0.96 0.44

372

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Brahman cows (Figure 4). These included a 95-kDa singlet, a 40- to 44-kDa doublet, 34-kDa and 22-kDa singlets, and a triplet between 28- and 32-kDa (one at 28-, 30- and 32-kDa). The 40- to 44-kDa doublet and the 34-kDa singlet were identified as IGFBP-3 and IGFBP-2, respectively. The identity of the 95-kDa, 28- to 32-kDa and 22-kDa IGFBPs were not identified in this study. Estradiol treatment increased (P < 0.05) total IGFBP activity (i.e., sum of all IGFBPs), the binding activity of IGFBP-3, the 30- and 32-kDa IGFBP, whereas day had no effect (P > 0.10). Breed affected (P < 0.05) IGFBP-3 and

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the 32-kDa IGFBP, such that Brahman cows had greater activity of these two IGFBP than Angus cows. Treatment and breed had no effect (P > 0.10) on the binding activity of the 95-kDa IGFBP, IGFBP-2, the 28-kDa IGFBP or the 22-kDa IGFBP (Table 3). Treatment X day affected (P < 0.05) all IGFBP except for IGFBP-2 and the 32- and 22-kDa IGFBP. The breed x day interaction was significant for only the 28-kDa IGFBP. The treatment × breed × day interaction was significant for the 95-kDa, 32-kDa, and 28-kDa IGFBP. During the 47-day study, treatment and day interacted (P < 0.001) to affect plasma concentration of PUN (Figure 5A). Before treatment and on many days up to Day 14, PUN concentrations did not differ between CON and E2 cows. However, after Day 14 and through Day 42 concentration of PUN was greater in CON than E2 cows (all days, P < 0.001). Average concentration of PUN was greater (P < 0.01) for Brahman (16.6 ± 0.59 mg/dl) than Angus cows (14.2 ± 0.55 mg/dl), and there was a trend (P < 0.10) for a breed X day interaction. Concentrations of PUN were greater (all days, P < 0.05) for Brahman than Angus cows on all days of the study except Days 1 and 2 (Figure 5B). Plasma concentration of glucose was greater (P < 0.01) for E2 than CON cows and during the 47-day study averaged 78.9 and 76.4 --- 0.80 mg/dl, respectively. There was a trend (P < 0.10) for a treatment × day interaction for plasma concentration of glucose. Treatment did not affect plasma concentration of glucose until after Day 5 of the study (Figure 6). Plasma concentration of glucose tended to be greater for E2 than CON cows from Days 2 to 42 of the study, with differences greatest from Days 7 to 28 (all days, P < 0.01 ). Neither breed nor breed x day affected (P > 0.10) plasma concentration of glucose which averaged 77.5 ± 0.77 mg/dl and 78.8 ± 0.84 mg/dl for Angus and Brahman cows, respectively. DISCUSSION These results revealed that 1) Brahman cows had a greater plasma concentration of IGF-I than Angus cows and estradiol implants had a greater impact on plasma IGF-I in Angus than in Brahman cows; 2) estradiol implants increased plasma concentrations of somatotropin, glucose and IGFBP activity in both breeds; 3) estradiol implants decreased concentrations of PUN in both breeds of cattle but Brahman cows had higher levels of PUN than Angus cows.

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Figure 6. Effect of treatment, control (CON) versus estradiol implant (E2) administered on Day 0, and day of treatment on plasma concentrations of glucose (mg/dl) in ovariectomizedAngus and Brahman cows. Data are least-squares means -+ SE. somatotropin (by 54%) and pulse amplitude of somatotropin without affecting pulse frequency of somatotropin (5). Reasons for discrepancies between those studies are unclear but may be attributable, in part, to the different methods used to identify secretory pulses of somatotropin and may be influenced by duration of exposure to the implant. In the present and previous studies ( 4 - 6 ) , baseline concentrations of somatotropin were not significantly influenced by estradiol treatment. Collectively, estradiol treatment increases overall mean concentration of somatotropin in gonadectomized cattle by 40 to 70%; this increase in mean concentration of somatotropin appears to be attributable to an increase in pulse amplitude of somatotropin and not to a change in pulse frequency or baseline concentration of somatotropin. Concentrations of somatotropin also seemed to be influenced by breed such that Brahman cows had greater mean concentration of somatotropin and pulse amplitude of somatotropin than Angus cows, an observation not previously reported. However, breed differences in concentration of somatotropin are not without precedence. For example, Simmental steers had greater concentration of somatotropin than Angus steers (4). Additionally, Brangus steers had greater somatotropin pulse amplitudes than did Angus steers (25). Why secretion of somatotropin is less in Angus than Brahman cows is unclear. In the present study, Brahman cows had smaller weights of anterior and posterior pituitary glands than Angus cows. Whether differences in weights of pituitary glands directly reflect changes in somatotropin secretion/metabolic disposition will require further study. Based on immunocytochemical analysis, 3/4 Brahman X 1/4 Angus steers had greater numbers of somatotrophs than 3/4 Angus × 1/4 Brahman steers, but size of somatotrophs did not differ between genotypes (26). Thus, in spite of a smaller anterior pituitary gland, Brahman cows may have greater somatotropin concentration because a greater number of somatotrophs exist within the pituitary gland. Estradiol treatment also increased mean plasma concentration of IGF-I in both Angus and Brahman cows, but this effect was much greater in Angus (i.e., 257% increase) than Brahman (i.e., 14% increase) cows. The reason for this breed difference in IGF-I response to estradiol is unclear but likely is attributable in part to the lower initial concentration of

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IGF-I in Angus versus Brahman cows. Previously, estradiol treatment in steers has been shown to increase plasma/serum concentration of IGF-I by 18 to 50% (5,27,28). Also, estradiol injection increased serum concentration of IGF-I in maintenance-fed but not restrict-fed ovariectomized Hereford cows (1). Whether the greater levels of IGF-I in E2 versus CON cows in the present study is attributable to increased somatotropin concentration or to factors in addition to if not independent of increased somatotropin remains to be determined. Others have observed that estradiol treatment in steers increases the number of hepatic somatotropin receptors in addition to increased concentration of somatotropin (29,30). Moreover, both plasma IGF-I concentrations and growth rate stimulated by a combined treatment of estradiol and exogenous somatotropin in steers were additive to that observed with either treatment alone (5), supporting the idea that estradiol enhances the stimulatory effects of somatotropin on IGF-I concentration and subsequent growth. Similar to a previous report using intact Brahman and Angus cows (14), in the present study concentration of IGF-I was greater in ovariectomized Brahman than Angus cows. Other studies have documented that breed/genotype differences in concentration of IGF-I exist among cattle (31-34). Interestingly, estradiol treatment increased concentration of IGF-I in Angus cows such that on several sampling days, estradiol-treated Angus and Brahman cows had similar concentrations of IGF-I, a new finding. If estradiol treatment increases levels of IGF-I via an increase in hepatic and (or) other tissue somatotropin receptors, then it is possible that Brahman cattle have a constituitively higher level of expression of somatotropin receptors than Angus cattle. Further studies will be required to verify this suggestion. Alternatively, greater concentrations of somatotropin in control Brahman than Angus cows may have resulted in the greater concentration of IGF-I in Brahman than Angus cows in the present study. In support of this latter suggestion, plasma somatotropin and IGF-I concentrations in weekly samples were positively correlated across breeds in control cows (r = 0.37, P < 0.001). Similar to that observed for somatotropin and IGF-I, plasma IGFBP activity and concentration of glucose were increased by estradiol treatment. Although previous studies have not evaluated the effects of estradiol on IGFBP activity, estradiol treatment has been reported to increase plasma glucose in steers (5). Perhaps, the higher glucose concentration in E2-treated cattle are the result of greater hepatic gluconeogenesis. In contrast to two previous studies where Brangus steers had lower plasma levels of glucose than Angus steers (25,35), we found no significant difference in plasma glucose concentration between ovariectomized Brahman and Angus cows. Although intact Brahman cows had 11% greater levels of IGFBP activity than Angus cows during superovulation (14), we found no difference in total IGFBP activity between ovariectomized Brahman and Angus cows in the present study using a charcoal-exchange assay or Western ligand blotting. However, using Western ligand blotting procedures, we found that Brahman cows had greater IGFBP-3 binding activity than Angus cows. Also, estradiol treatment increased IGFBP-3 binding activity in plasma that agrees with results for feedlot steers that indicated that a combined estradiol-trenbolone acetate implant increased IGFBP-3 levels above nonimplanted controls (36). This previous report (36) did not quantify treatment effects on other IGFBPs. We observed that binding activities of the 30- and 32-kDa IGFBP were also increased with estradiol treatment, a new finding. The other IGFBPs isolated in this study, IGFBP-2, a 95-kDa, 28-kDa and 22-kDa IGFBP, were not significantly affected by estradiol treatment, although there was a significant treatment x day interaction for the 28-kDa IGFBP. Based on other studies, the binding of the 30- and 32-kDa region corresponds to the immunoreactivity for IGFBP-5 in cattle (37,38), the 28-kDa region corresponds to immunoreactivity for IGFBP-4 in sheep (39) and cattle (38) while the binding activity in the 22-kDa region may represent a deglycosylated form of IGFBP-4

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because the molecular weight is similar to proteins identified as IGFBP-4 in sheep (39) and cattle (38). The relative binding activities of IGFBP-3, IGFBP-2, and the other IGFBP in the present study are similar to the binding activities of those reported by Stewart et al. (40), except that the 95-kDa IGFBP was not identified previously in cattle. However, a 96-kDa IGFBP has been found in horse serum (41). This 95-kDa IGFBP likely represents a proteolytic fragment of the soluble form (i.e., extracellular domain) of the IGF-II receptor (42). Similar to previous studies in steers (5,28), in the present study with ovariectomized cows, estradiol treatment decreased levels of PUN by 10 to 30%. A decrease in levels of PUN is thought to be related directly to an increase in nitrogen utilization/retention and protein deposition. Whether this effect of estradiol treatment on nitrogen retention was translated into increases in body protein in the present study is unclear because average daily body weight gain and fat thickness over the ribeye were not affected by treatment. However in yearling steers, estradiol treatment increases muscle protein synthesis and accretion (6). Brahman cows also had greater concentrations of PUN and lower average daily body weight gain than Angus cows in the present study. Whether the greater nitrogen retention in E2 versus CON cows is attributable to increased IGF-I is uncertain, but concentrations of PUN and IGF-I were negatively correlated in the present study (r = - 0 . 1 8 ; P < 0.05) and a previous study in steers (43), Furthermore, exogenous IGF-I treatment increases protein accretion in fasted lambs (44). Why nitrogen retention and average daily body weight gain is lower in Brahman versus Angus cows in spite of the greater plasma concentrations of 1GF-I in Brahman cows will require further study, but perhaps the greater IGFBP-3 and 32-kDa IGFBP activity in Brahman versus Angus cows negated the potential positive effect of the greater IGF-I concentrations. In summary, these data suggest that some but not all of the positive effects of estradiol on peripheral concentration of IGF-I and IGFBP activity can be attributed to increased somatotropin. Moreover, breed influenced basal and E2-induced secretion of somatotropin and IGF-I such that the differences between Brahman and Angus cows in plasma IGF-I concentration were abated within 3 wk of estradiol implantation. Thus, breed influences the metabolite and hormonal response of cattle to estrogenic implants. ACKNOWLEDGMENTS/FOOTNOTES Research conducted at USDA, ARS, Subtropical AgriculturalResearch Station is in cooperation with the University of Florida, Institute of Food and AgriculturalSciences. This manuscriptwas published as Florida Agricultural Experiment Station Journal Series No. R-05523, and approved for publication by the Director, Oklahoma AgriculturalExperiment Station; this research was supported in part under project H-2088 (to L.J. Spicer). Appreciationis expressed to: E.L. Adams, E.J. Bowers, S.C. Howard, B.E. Keefer, E.V. Rooks, and M.L. Rooks for technicalassistance;and the National Hormoneand PituitaryProgram (Baltimore,MD) for the IGF-I antiserum. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product to the exclusion of others that may also be suitable. 1-Present Address: Universityof Tennessee, KnoxvilleExperiment Station, Knoxville,TN 37996. :1:Present Address: USDA, ARS, Universityof Missouri, Columbia,MO 65221. ~Correspondenceshould be addressed to C.C. Chase, Jr., USDA, ARS, STARS, 22271 ChinsegutHill Road, Brooksville, FL 34601-4672.

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