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Am J Physiol Endocrinol Metab 285: E252–E261, 2003.
First published April 1, 2003; 10.1152/ajpendo.00557.2002.
Effects of dexamethasone and colostrum intakeon the somatotropic axis in neonatal calves Stephanie N. Sauter,1 Edgar Ontsouka,1 Bettina Roffler,1
Yolande Zbinden,1 Chantal Philipona,1 Michael Pfaffl,2
Bernhard H. Breier,3 Ju
¨ rg W. Blum,1 and Harald M. Hammon1
1Division of Animal Nutrition and Physiology, Faculty of Veterinary Medicine, Institute of AnimalGenetics, Nutrition, and Housing, University of Berne, CH-3012 Berne, Switzerland; 2Centre of Lifeand Food Sciences, Institute of Physiology, Weihenstephan, Technical University of Munich,D-85354 Freising-Weihenstephan, Germany; and 3Department of Pediatrics, Research Centrefor Developmental Medicine and Biology, University of Auckland, NZ-92019 Auckland, New Zealand Submitted 19 December 2002; accepted in final form 28 March 2003 Sauter, Stephanie N., Edgar Ontsouka, Bettina Rof-
fetal to the postnatal status and function (7, 11, 19).
fler, Yolande Zbinden, Chantal Philipona, Michael
Cortisol stimulates hepatic GH receptor (GHR) and Pfaffl, Bernhard H. Breier, Ju
¨ rg W. Blum, and Harald
insulin-like growth factor I (IGF-I) mRNA levels in the M. Hammon. Effects of dexamethasone and colostrum in-
sheep fetus (30), and dexamethasone (Dexa) increases take on the somatotropic axis in neonatal calves. Am J hepatic GHR mRNA levels in rabbits (24). Studies with Physiol Endocrinol Metab 285: E252–E261, 2003. First pub- hepatocytes indicate an increase of GHR and IGF-I lished April 1, 2003; 10.1152/ajpendo.00557.2002.—Gluco-corticoids and colostrum feeding influence postnatal matura- expression and enhanced IGF-I response to GH after tion of the somatotropic axis. We have tested the hypothesis Dexa administration (5). In neonatal pigs, Dexa treat- that dexamethasone (Dexa) affects the somatotropic axis in ment increases postnatal growth and stimulates func- neonatal calves dependent on colostrum intake. Calves were tioning of the somatotropic axis immediately after fed either with colostrum or with a milk-based formula (n ⫽ birth (10). However, findings concerning effects of glu- 14/group), and, in each feeding group, one-half of the calves cocorticoids on the somatotropic axis are inconsistent, were treated with Dexa (30 g 䡠 kg body wt⫺1 䡠 day⫺1). Pre- perhaps because of different species or age (9, 38).
and postprandial blood samples were taken on days 1, 2, 4, Cortisol reduces the prenatal growth rate in sheep and 5, and liver samples were taken on day 5 of life. Dexa fetus during late gestation (17), and glucocorticoids increased insulin-like growth factor (IGF)-I, but decreased inhibit postnatal growth, in part, by direct interaction growth hormone (GH) and IGF-binding protein (IGFBP)-1 with the somatotropic axis (9, 48, 49). Obviously, the and -2 plasma concentrations and increased GH receptor(GHR) mRNA levels in liver. Dexa increased IGF-I mRNA gestational stage and the time around birth modify levels only in formula-fed calves and increased hepatic GHR glucocorticoid effects on the somatotropic axis. There- binding capacity, but only in colostrum-fed calves. Colostrum fore, one could speculate that glucocorticoids stimulate feeding decreased IGFBP-1 and -2 plasma concentrations the functioning of the somatotropic axis in the neonatal and hepatic IGFBP-2 and -3 mRNA levels. In conclusion, calf at birth.
Dexa and colostrum feeding promoted maturation of the Several studies have recently shown that the soma- somatotropic axis. Dexa effects partly depended on whether totropic axis in neonatal calves works in principle and colostrum was fed or not.
is influenced by colostrum feeding, nutrient density, glucocorticoids; growth hormone; growth hormone receptor; and age (13, 20, 47). Although colostral IGF-I did not insulin-like growth factor I; insulin-like growth factor-bind- affect systemic IGF-I levels in a significant manner, colostrum intake increased IGF-I plasma concentra-tions and hepatic IGF-I expression in neonatal calves,whereas GH plasma concentrations behaved inconsis- THE SOMATOTROPIC AXIS is the main endocrine system tently with respect to colostrum feeding (13, 20, 23, 50).
regulating postnatal growth in mammals, but prenatal Besides the large nutrient supply by colostrum feeding, growth is rather independent of growth hormone (GH; nonnutritive components of colostrum seem to affect see Refs. 7 and 16). Glucocorticoids are supposed to the IGF-I status in neonatal calves (13).
enhance the maturation of the somatotropic axis There is minor knowledge in precocious species like around birth in ruminants and pigs. The prepartum cattle about effects of glucocorticoids on the somato- cortisol surge may play an important role in initiating tropic axis at birth and about interactions of glucocor- the perinatal switch of the somatotropic axis from the ticoid effects with colostrum feeding. We have found Address for reprint requests and other correspondence: H. M.
The costs of publication of this article were defrayed in part by the Hammon, Div. of Animal Nutrition and Physiology, Univ. of Berne, payment of page charges. The article must therefore be hereby Route de la Tioleyre 4, CH-1725 Posieux, Switzerland (E-mail: marked ‘‘advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0193-1849/03 $5.00 Copyright 2003 the American Physiological Society DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES high endogenous cortisol levels around birth in calves bottle two times daily. To ensure that all calves received the (21, 42). Furthermore, glucocorticoids influenced the same amounts of formula or colostrum, calves with reduced somatotropic axis in newborn sheep and pigs (10, 30).
appetite were tube fed their planned amounts of formula or On the basis of these premises, we administered Dexa colostrum. Calves received their first meal at 3.1 ⫾ 0.04 h to colostrum- and formula-fed neonatal calves, starting (GrFD⫺), 2.2 ⫾ 0.02 h (GrFD⫹), 2.4 ⫾ 0.03 h (GrCD⫺), and3.4 ⫾ 0.04 h (GrCD⫹) after birth. The following feedings were the treatment immediately after birth to simulate a at 8, 24, and 32 h after the first feeding. From day 3 on, calves high glucocorticoid status. The objective of this study were fed daily at 0800 and at 1600.
was to clarify the effects of glucocorticoids on the so- Colostrum was from cows of the Federal Research Station matotropic axis and whether these effects depend on for Animal Production. Cows were milked two times daily, feeding colostrum or milk-based formulas that con- and the colostrums of milkings 1 (day 1), 3 (day 2), and 5 (day tained nutrients in similar amounts to colostrum, but 3) after parturition were stored separately in plastic bottles much fewer growth-promoting substances like hor- at ⫺20°C. Individual pools of milkings 1, 3, and 5 were mones and growth factors (3, 42). We have tested the prepared at the beginning of the study and then stored in hypothesis that Dexa influences the maturation of the plastic bottles at ⫺20°C until used. Before feeding, colostrum somatotropic axis in neonatal calves and that Dexa was warmed to 40°C and then fed immediately.
effects are modified by colostrum feeding. Therefore, Three formulas for days 1, 2, and 3 were created that contained nutrients (protein, fat, lactose) in comparable we have measured various components of the somato- amounts as colostrum milkings 1, 3, and 5 after parturition tropic axis in blood and mRNA concentrations in liver and were fed on days 1, 2, and 3 after birth. Formulas were as well as GHR binding sites in liver.
produced by UFA (Sursee, Switzerland) and consisted ofcalcium-caseinate (Emmi Milch, Lucerne, Switzerland), lact- MATERIALS AND METHODS
albumin (Emmi Milch), milk fat (double cream; Institut Ag- Animals, husbandry, feeding, and experimental proce- ricole de l'Etat de Fribourg, Grangeneuve, Switzerland), and dures. The experimental procedures were approved by the a vitamin and mineral premix (Provimi, Cossonay-Gare, Cantonal Committee for the Permission of Animal Experi- Switzerland). The three formulas were dissolved by adding mentation (Granges-Paccot, Canton of Fribourg, Switzer- water and were stored in plastic bottles at ⫺20°C until used.
land) and followed the actual law of animal protection. They Before feeding, formulas were warmed to 40°C and then fed were supervised by the Federal Veterinary Administration.
immediately. The milk replacer (UFA 200 natura, without Twenty-eight male calves (11 Holstein-Friesian, 12 Simmen- antibiotics) was prepared as a 100 g/l solution. Contents of tal ⫻ Red Holstein, 4 Brown Swiss, and 1 Red Holstein ⫻ different colostrum milkings, formulas, and milk replacer are Limousin) were studied. They were born at the Federal shown in Table 2.
Research Station for Animal Production (Posieux, Switzer- To protect against infections, all calves were subcutane- land) or at neighboring farms. Calves were born spontane- ously injected with 2 g of a bovine colostral immunoglobulin ously and were separated immediately after birth from their preparation (Gammaserin; Gra¨ub, Berne, Switzerland) be- dams and held on straw in boxes for 5 days.
fore the first feed intake. Additionally, all calves were fed Four treatment groups were created randomly (GrFD⫺, chicken egg-derived immunoglobulins containing high-anti- GrFD⫹, GrCD⫺, GrCD⫹), each consisting of seven calves body titers against rotavirus and pathogenic Escherichia coli (Table 1). Calves of GrCD⫺ and GrCD⫹ received colostrum of type K 99 (Globigen 88; kindly donated by Lohmann Animal milkings 1, 3, and 5 (days 1, 2, and 3 after parturition, Health). Fed amounts were 10 g (day 1), 8 g (day 2), 6 g (day respectively) on the first 3 days. Calves of GrFD⫺ and GrFD⫹ 3), and 4 g (day 4)/meal. On days 1, 2, and 3, calves were were fed three different milk-based formulas for days 1, 2, subcutaneously injected antibiotics (25 mg Enrofloxacin/10 and 3 that represented colostral milkings 1, 3, and 5, respec- kg body wt; 5% Baytril; Bayer, Leverkusen, Germany). To tively. Calves of GrCD⫹ and GrFD⫹ were treated with 30 avoid different treatment protocols, drugs were given to both g䡠kg body wt⫺1䡠day⫺1 Dexa (Dexa TAD; Lohmann Animal formula- and colostrum-fed calves.
Health status. The health status was evaluated daily based grouped with the goal to obtain similar body weight and on the following clinical traits: rectal temperature, heart breed distributions in all four groups. Calves were fed by rate, respiratory rate, behavior, nasal discharge, respiratory Table 1. Experimental groups g䡠kg body wt⫺1䡠day⫺1 Days 1–4 Twenty-eight calves were randomly grouped and were fed either colostrum (GrCD⫺ and GrCD⫹) or formula (GrFD⫺ and GrFD⫹). In each feeding group, one-half of the calves were treated with dexamethasone (Dexa) (GrCD⫹ and GrFD⫹). With Dexa treatment, calves weretreated two times daily at feeding times; dose of DEXA was chosen according to previous studies in neonatal calves (14). Day 1, colostrumand formula were fed in amounts of 6% of body wt; Day 2, colostrum and formula were fed in amounts of 8% of body wt; Days 3 and 4,colostrum and formula were fed in amounts of 10% of body wt.
AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Table 2. Composition of colostrum milkings, formula, and milk replacer fed to neonatal calves Gross energy, MJ/kg (MJ/kg dry matter) Crude protein, g/kg (g/kg dry matter) Crude lipids, g/kg (g/kg dry matter) Nitrogen free extracts, g/kg (g/kg dry matter) Crude ashes, g/kg (g/kg dry matter) Formula (per kg) was composed of calcium-caseinate (86, 208, and 282 g on days 1, 2, and 3, respectively), lactalbumin (576, 245, and 80 g on days 1, 2, and 3, respectively), milk fat (267, 262, and 353 g double cream on days 1, 2, and 3, respectively), lactose (57, 271, and 271g on days 1, 2, and 3, respectively), and a mineral premix (14 g on days 1–3). The mineral premix (per kg) contained calcium (186 g),magnesium (224 g), sodium (31 g), phosphorus (92 g), chlorine (48 g), iron (12 g), manganese (8.1 mg), copper (1.6 mg), zinc (7.8 mg), iodine(0.03 mg), cobalt (0.02 mg), and selenium (0.02 mg). Milk replacer was fed as 10% solution (wt/vol) and was composed (per kg) of skim milkpowder (550 g), whey (40 g), corn-derived products (dextrose, glucose, oat cream, starch; 172 g), tallow (145 g), lard (44 g/kg), lecithin (asemulgator; 19 g), calcium (12 g), phosphorus (7.5 g), magnesium (1.6 g), sodium (4.9 g), zinc (80 mg), manganese (60 mg), iron (20 mg), copper(8 mg), iodine (3 mg), selenium (0.5 mg), cobalt (0.5 mg), vitamin A (26.25 mol retinol equivalent/kg), cholecalciferol (195 nmol/kg), vitaminE (360 mol ␣-tocopherol/kg;), thiamine (57 mol/kg), riboflavin (21 mol/kg), vitamin B6 (59 mol/kg), and vitamin B12 (37 nmol/kg). Milkreplacer included 30 g lactose/kg milk (320 g/kg dry matter). NM, not measurable (below detection limit). ND, not determined.
sounds, appetite, fecal consistency, and navel adspection.
and GHR mRNA, were recently described in detail (18, 40).
Body weight was determined on days 1 and 5 of life.
RT-PCR quantification was performed with the LightCycler Blood samples. Blood samples were taken from the jugular system (Roche Molecular Biochemicals, Rotkreuz, Switzer- vein with evacuated tubes on days 1, 2, and 5 and with a land), using the software package 3.3 (Roche Molecular Bio- catheter on day 4. Tubes containing dipotassium-EDTA (1.8 chemicals). Absolute quantification was based on external g/l blood) were used for the determination of pre- and post- recombinant DNA standards (40) and was related to 18S prandial values of IGF-I and IGF-binding protein (IGFBP)-1 RNA to compensate for RNA loading differences. Values were at 0, 1, 2, 4, and 8 h after the 1st, 3rd, and 7th feeding and on expressed on a molar basis.
day 5 and for the determination of preprandial values of For 125I-GH-binding studies, liver samples were thawed IGFBP-2 and IGFBP-3 on days 1 and 4, respectively. The and transferred to receptor buffer (in mM: 50 Tris 䡠 HCl, 6 same tubes were used to measure plasma concentrations of MgCl2, and 1 EGTA; pH 7.4; 4°C). The tissue was homoge- GH in pre- and postprandial samples on days 1 and 2, in 25 nized four times for 20 s at low speed (8,000 U/min) with an samples on day 4 taken before (0 h) and every 20 min after Ultra-Turrax homogenizer (T25; Janke & Kunkel, Staufen, the morning feeding for 8 h for the evaluation of secretory Germany). The homogenate was centrifuged at 800 g for 10 patterns, and on day 5. Tubes were put on crushed ice and min, and the supernatant was centrifuged at 10,000 g for 10 centrifuged at 1,000 g for 20 min. Supernatants were sepa- min; thereafter, the resulting supernatant was centrifuged rated into aliquots and stored at ⫺20°C.
again at 100,000 g for 1 h (22). The remaining pellet was Blood analyses. Plasma GH, IGF-I, and IGFBP-1 concen- resuspended in ice-cold buffer (as above) by a motor-driven trations were measured by RIA, as described previously (20, glass-Teflon homogenizer and stored at ⫺80°C until assayed.
26). Plasma concentrations of IGFBP-2 and IGFBP-3 were The protein concentration was determined using a kit (BCA measured by ligand blots using 125I-labeled IGF-I. The pro- Protein Assay Reagent; Pierce, Rockford, IL), and then the cedure was described recently (20, 23).
membrane suspensions were adjusted to a final protein con- Analyses in liver. Calves were slaughtered on day 5 of life, centration of 4 mg/ml. For receptor measurements, mem- and liver samples were put either in liquid nitrogen (GHR branes were suspended in incubation buffer (as above). Lin- binding studies) or in TRIzol (GIBCO-BRL, Basel, Switzer- earity of binding dependent on protein concentration was land) and then frozen in liquid nitrogen. Liver samples were tested, and the final protein concentration in assays was stored at ⫺80°C until analyzed for mRNA of IGF-I, IGF-II, within the linear range. Binding of 125I-recombinant bovine IGFBP-2, IGFBP-3, and GHR and for GHR binding sites.
GH (rbGH) was measured as previously described (1, 22).
For mRNA measurements, total RNA was extracted using The rbGH was from Monsanto (St. Louis, MO), and prolactin TRIzol reagent (GIBCO-BRL) and resuspended in RNase- was from the United States Department of Agriculture Ani- free water that was treated with diethyl pyrocarbonate mal Hormone Program (Dr. D. Bolt, Beltsville, MD). Compet- (Sigma-Aldrich, Deisendorf, Germany). RNA integrity and itive binding studies for the characterization of 125I-rbGH purity were tested by measurement of optical density and by binding sites were performed in pooled membrane suspen- electrophoresis using ethidium bromide staining. Total RNA sions with 0.35 ng of the radiolabeled ligand and increasing was reverse transcribed into cDNA with hexamer primers concentrations of unlabeled rbGH and prolactin. For the (Pharmacia Biotech, Buckinghamshire, UK), as described determination of the number of 125I-rbGH binding sites, the previously (40). Materials and procedures for RT-PCR, in- radiolabeled rbGH (0.35 ng) was incubated with increasing cluding primers for bovine IGF-I, IGF-II, IGFBP-2, IGFBP-3, concentrations of the unlabeled rbGH (1, 22).
AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org
DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Analyses in formula, colostrum, and milk replacer. Sam- the general linear model (GLM) and differences were local- ples of formulas 1, 2, and 3 and samples of the individual ized by Bonferroni t-test (P ⬍ 0.05; see Ref. 45).
pools of colostrum of milkings 1, 3, and 5 were lyophilized to Concentrations of mRNA as well as number (Bmax) and determine dry matter, crude protein (by the Kjeldahl 50% inhibition (IC50) of GH binding sites were evaluated method), crude fat (by the Berntrop method), and ashes (after using the GLM (45) with Dexa treatment and feeding as combustion at 550°C) using standard procedures at the Swiss main effects. The Dexa ⫻ feeding interaction was included in Federal Research Station for Animal Production. Contents of the model. Dexa treatment and feeding effects were localized nitrogen-free extracts and gross energy (based on energy by the Bonferroni t-test (P ⬍ 0.05).
equivalents of 36.6, 17.0, and 24.2 MJ/kg fat, nitrogen-freeextracts, and crude protein) were calculated. Information on contents of the milk replacer was given by the producer.
Feed intake, body weight, and health status. Feed Concentrations of insulin and IGF-I in formula and co- intake was the same in all four groups. The mean body lostrum were analyzed as described by Hammon and Blum weights of all four groups before the first meal (46.7 ⫾ Statistical procedures. Values of body weight, blood traits, 3.8 kg) were the same and did not change until day 5 mRNA concentrations, and GH binding sites were expressed (46.5 ⫾ 3.8 kg). There were no differences in health as means ⫾ SE or pooled SE. For plasma concentrations of traits among the different groups (data not shown).
GH, IGF-I, and IGFBP-1, areas under the concentration However, two calves of GrFD⫺ and GrFD⫹ had loose curves were computed for each day as measures of mean feces for 2 days.
concentrations between 0 and 8 h. The ratios of GH to IGF-I Blood hormone concentrations. Preprandial plasma plasma concentrations were calculated as means for describ- GH concentrations (Fig. 1) did not change from day 1 to ing the maturation of the GH-IGF-I system, as defined by day 5 but were higher (P ⬍ 0.05) before the first feed Gluckman et al. (19). Data were evaluated using the RAN- intake in Dexa-treated than in nontreated calves. Con- DOM and REPEATED methods of the MIXED procedure centrations of GH on day 1 changed with time in an (45). Separate models were applied for the calculation of bodyweight as well as preprandial and mean concentrations of inconsistent manner but on day 2 remained low in GH, IGF-I, and IGFBPs during the whole experimental pe- Dexa-treated groups and transiently increased (P ⬍ riod and for the calculation of postprandial effects on days 1, 0.1) after feed intake in nontreated groups. On day 4, 2, and 4, respectively. Dexa treatment, feeding, and time concentrations postprandially increased (P ⬍ 0.01) in were used as fixed effects, and the individual calves were nontreated groups, whereas GH concentrations re- used as random effects. For the evaluation of differences in mained low in Dexa-treated groups. Mean and basal Dexa responses with regard to different feeding and for the concentrations as well as amplitudes of secretory peaks evaluation of differences in the time pattern within Dexa or were lower (P ⬍ 0.05) in Dexa-treated than in non- feeding groups, interactions (Dexa ⫻ feeding; Dexa ⫻ time; treated groups.
feeding ⫻ time) were included in the model. Treatment, Preprandial plasma concentrations of IGF-I (Fig. 2) feeding, and time differences were localized by the Bonfer-roni t-test (P ⬍ 0.05). Episodic secretion of GH on day 4 decreased (P ⬍ 0.01) in nontreated calves from day 1 to (mean concentrations, basal concentrations, peak ampli- day 5. Mean concentrations remained unchanged in tudes, and peak frequencies) was analyzed according to Mer- Dexa-treated calves from day 1 to day 2 but decreased riam and Wachter (36). Group differences were analyzed by (P ⬍ 0.01 in GrFD⫹; P ⬍ 0.1 in GrCD⫹) from day 2 to Fig. 1. Plasma pre- and postprandialgrowth hormone (GH) concentrationson days 1 and 2, secretory patternsmeasured during 8 h every 20 min af-ter feed intake on day 4, and prepran-dial concentrations on day 5 in neona-tal calves fed with formula or co-lostrum GrCD⫺ (‚)] and with Dexa injections[GrFD⫹ (F) and GrCD⫹ (Œ)]. On day 4,all calves were fed with a milk re-placer. Values are presented as means,and pooled SE is shown at bottom (n ⫽7). ␦, Dexa effect (P ⬍ 0.05); , feedingeffect (P ⬍ 0.05); , time effect (P ⬍0.05); ␦ ⫻ , Dexa ⫻ feeding interac-tion (P ⬍ 0.05); ␦ ⫻ , Dexa ⫻ timeinteraction (P ⬍ 0.05); ⫻ , feeding ⫻time interaction (P ⬍ 0.05). Arrowsmark times of feed intake.
AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org
DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Fig. 2. Plasma pre- and postprandial IGF-I concen-trations on days 1, 2, and 4 and preprandial concen-trations on day 5 in neonatal calves fed with formulaor colostrum without Dexa injections [GrFD⫺ (E)and GrCD⫺ (‚)] and with Dexa injections [GrFD⫹ (F)and GrCD⫹ (Œ)]. On day 4, all calves were fed with amilk replacer. For further details see legend toFig. 1.
day 4. Postprandial concentrations changed (P ⬍ 0.05) after feed intake in colostrum-fed calves and responded with time after feed intake on days 1, 2, and 4, de- (P ⬍ 0.05) to feed intake on days 2 and 4 in nontreated creased in GrFD⫺ (P ⬍ 0.01) on day 2, and decreased calves. Preprandial IGFBP-1 concentrations on day 1 (P ⬍ 0.05) in colostrum-fed groups on day 4. Prepran- were higher (P ⬍ 0.05) in colostrum-fed than in for- dial IGF-I concentrations on day 1 were lower (P ⬍ mula-fed calves. IGFBP-1 concentrations from day 2 to 0.05) in Dexa-treated than in nontreated calves. Dexa day 5 were lower (P ⬍ 0.05) in Dexa-treated than in treatment increased (P ⬍ 0.05) IGF-I concentrations nontreated calves. On days 2 and 5, IGFBP-1 concen- compared with nontreated calves on days 4 and 5. The trations tended to be higher in GrFD⫺ than in GrCD⫺.
ratios of mean GH to mean IGF-I plasma concentra- Plasma concentrations of IGFBP-2 increased (P ⬍ tions on day 4 were lower (P ⬍ 0.001) in Dexa-treated 0.05) in GrFD⫺ and decreased (P ⬍ 0.05) in GrCD⫹ than in nontreated calves and tended to be lower (P ⬍ from day 1 to day 4 (Table 3). Plasma concentrations of 0.1) in colostrum-fed than in formula-fed calves (ratios IGFBP-2 on day 4 were lower (P ⬍ 0.05) in Dexa- were 0.68 ⫾ 0.1, 0.3 ⫾ 0.03, 0.53 ⫾ 0.1, and 0.14 ⫾ treated than in nontreated calves and were lower in 0.004 for GrFD⫺, GrFD⫹, GrCD⫺, and GrCD⫹, respec- colostrum-fed than in formula-fed calves, and the Dexa-induced reduction was greater (P ⬍ 0.01) in for- Preprandial plasma concentrations of IGFBP-1 (Fig.
mula-fed than in colostrum-fed calves. Plasma concen- 3) decreased in GrFD⫹, GrCD⫺, and GrCD⫹ (P ⬍ 0.05) trations of IGFBP-3 decreased (P ⬍ 0.05) in GrFD⫺ from day 1 to day 2 and remained unchanged until day from day 1 to day 4 (Table 3). There were no differences 5. Plasma concentrations decreased on day 1 (P ⬍ 0.05) concerning Dexa treatment or feeding.
Fig. 3. Plasma pre- and postprandialIGF-binding protein (IGFBP)-1 con-centrations on days 1, 2, and 4 andpreprandial concentrations on day 5 inneonatal calves fed with formula orcolostrum [GrFD⫺ (E) and GrCD⫺ (‚)] and withDexa injections [GrFD⫹ (F) and GrCD⫹(Œ)]. On day 4, all calves were fed witha milk replacer. For further details seelegend to Fig. 1.
AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Table 3. Plasma concentrations of IGFBP-2 and IGFBP-3 in newborn calves fed with formula or colostrumwith (GrFD⫹, GrCD⫹) or without Dexa injections (GrFD⫺, GrCD⫺) ANOVA (P values) Values are means; n ⫽ 7 animals/group. Units are optical density ⫻ 103. IGFBP, IGF-binding protein; SE, pooled standard error; ␦, Dexa effect; , feeding effect; ␦⫻, Dexa ⫻ feeding interaction. NS, statistically not significant. * P ⬍ 0.05 and † P ⬍ 0.01.
Hepatic mRNA concentrations on day 5 of life. Ex- However, concentrations of biologically active factors pression of IGF-I tended to be higher (P ⬍ 0.1) in like IGF-I or insulin were much lower in the three Dexa-treated than in nontreated calves and was higher formulas than in colostrum and possibly affected neo- (P ⬍ 0.05) in GrFD⫹ than in GrFD⫺ and in GrCD⫹ natal development because receptors for IGF-I and (Table 4). Expression of IGF-II tended to be higher (P ⬍ insulin are present in the gut mucosa of neonatal 0.1) in formula-fed than in colostrum-fed calves. Dexa calves (18, 22). Body weight remained stable during treatment resulted (P ⬍ 0.1) in different responses to the experimental period, and Dexa treatment or differ- feed intake in formula-fed and colostrum-fed calves.
ent feeding did not affect growth performance, yet the Expression of IGFBP-2 and IGFBP-3 was higher (P ⬍ experimental period of 5 days was too short to get 0.001 and P ⬍ 0.01) in formula-fed than in colostrum- reliable data on effects of Dexa on growth performance, fed calves. Calves treated with Dexa showed higher as was the case in 18-day-old pigs (10).
expression (P ⬍ 0.01) of GHR than nontreated calves.
Feeding and treatment effects on GH and GHR. Hepatic GH binding sites on day 5 of life. In 125I-GH Plasma GH concentrations in our study were not influ- binding studies (Fig. 4A), IC50 of unlabeled rbGH was enced by different feeding but were reduced by Dexa 4.1 ⫻ 10⫺10 M, and IC50 of prolactin was 5.4 ⫻ 10⫺6 M.
treatment. Plasma GH concentrations were barely reg- Dexa treatment resulted in different effects on Bmax in ulated by feeding in neonatal calves (20, 23, 42), formula-fed and colostrum-fed calves (P ⬍ 0.1 for whereas they were influenced by feeding intensity in Dexa ⫻ feeding interaction), and Bmax tended to be older cattle (7, 43). Because nutrient intake was the higher (P ⬍ 0.1) in GrCD⫹ than in GrCD⫺ (Fig. 4B).
same in the present study, nonnutritive factors such as IC50 of GH binding sites were similar in different IGFs, IGFBPs, and lactoferrin, which are present in high amounts in colostrum (3) but not in the formulas,obviously did not influence GH secretion. In cattle, plasma GH concentrations decrease during ontoge- Feed intake, body weight, and health status. De- netic development (8, 14). Because glucocorticoids are creases in dry matter, gross energy, crude protein, important for neonatal maturation (7), reduced GH crude fat, crude ashes, insulin, and IGF-I concentra- concentrations after Dexa treatment in our study tions but an increase in nitrogen-free extracts (mainly might indicate enhanced maturation of the somato- of lactose) in colostrum of milkings 1, 3, and 5 fed to tropic axis, as shown in neonatal pigs (10). On the neonatal calves have also been observed in earlier other hand, cortisol treatment in 1-mo-old calves did studies (42). Because formulas of days 1, 2, and 3 not affect plasma GH concentrations (15). Because contained similar amounts of nutrients as the co- glucocorticoids have stimulatory and inhibitory effects lostrum of milkings 1, 3, and 5 and because on day 4 all on pituitary GH production and release (38), effects of calves received the same milk replacer, nutrient in- glucocorticoids on GH secretion may depend on the takes were comparable in different feeding groups.
developmental status. In addition, reduced GH concen- Table 4. Expression of IGF-I, IGF-II, IGFBP-2, IGFBP-3, and GHR on day 5 of life in liver of newborn calvesfed with formula or colostrum with (GrFD⫹, GrCD⫹) or without Dexa injections (GrFD⫺, GrCD⫺) ANOVA (P Values) IGF-I (⫻10⫺6) IGF-II (⫻10⫺3) IGFBP-2 (⫻10⫺6) IGFBP-3 (⫻10⫺6) Values are means; n ⫽ 7 animals/group. Units are fmol/g total RNA. GHR, growth hormone receptor NS, statistically not significant. * P ⬍ 0.1, † P ⬍ 0.01, and ‡ P ⬍ 0.001. Means with different capital letter superscripts (A, B) are significantly different (P ⬍ 0.05) within Dexatreatment; means with different small letter superscripts (a, b) are significantly different (P ⬍ 0.05) within feeding.
AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES nied by colostrum feeding, supports maturation of thesomatotropic axis by stimulating hepatic GHR synthe-sis. Possibly, glucose might have caused the increase ofGHR expression because glucose stimulated GHRmRNA in porcine hepatocytes (4) and because we foundthe highest glucose plasma concentrations in GrCD⫹(Sauter SN, Blum JW, and Hammon HM, unpublishedobservation). Higher hepatic GH binding sites are animportant basis for enhanced IGF-I responses to GH,and the response usually increases with age (8). Theeffects of Dexa treatment on GHR correspond to theview that parturition-associated mechanisms (such ashigh cortisol levels) are necessary for the postnatalincrease of hepatic GH binding sites (6). Obviously,Dexa administration mimics the cortisol surge at birthand causes this postnatal hepatic GHR stimulation.
Because the stimulation of GHR expression by Dexawas observed in both feeding groups but stimulation ofGH binding by Dexa was only noticed in colostrum-fedcalves, feeding of colostrum may regulate posttran-scriptional GHR synthesis in Dexa-treated calves.
Feeding and Dexa effects on IGF-I expression and plasma concentrations. Although IGF-I plasma concen-trations were numerically higher in colostrum-fed thanin formula-fed calves and concentrations of hepaticIGF-I mRNA tended to increase in GrCD⫺ comparedwith GrFD⫺, colostrum effects on neonatal IGF-I levelswere not significant in the present study. These find-ings are contrary to our previous results (13, 20), butcalves of those studies differed in age of the calvesand/or feeding protocol.
Treatment with Dexa increased IGF-I plasma con- centrations in this study, although glucocorticoids areknown to inhibit postnatal growth in association with Fig. 4. 125I-labeled GH binding to 4 mg of pooled membrane suspen-sions dependent on increasing concentrations of unlabeled GH (F) reduced IGF-I plasma concentrations (9, 48, 49). How- and prolactin (PRL; ‚; A) and the number of 125I-GH binding sites in ever, our data correspond to findings in neonatal pigs the liver of neonatal calves fed formula or colostrum without Dexa (10) and growing bulls (43) treated with Dexa. How- injections (GrFD⫺ and GrCD⫺) and with Dexa injections (GrFD⫹ and ever, Coxam et al. (15) did not find an effect of cortisol GrCD⫹) on day 5 of life (B).
on plasma IGF-I concentrations in calves, and Dexadecreased plasma IGF-I concentrations in cows and trations in Dexa-treated calves possibly resulted from finishing calves (2, 33). Therefore, effects of glucocorti- elevated IGF-I concentrations because of negative coids on IGF-I plasma concentrations are variable and feedback mechanisms of IGF-I on pituitary GH secre- may depend on treatment-dose (34) and age (9). In any tion (38). We have demonstrated a negative feedback case, plasma IGF-I concentrations increase during mechanism in neonatal calves resulting from Long-R3- postnatal development in cattle (7, 28, 44). Therefore, elevated IGF-I concentrations in Dexa-treated calves, Hepatic GHR mRNA concentrations (especially the lower GH-to-IGF-I ratios in Dexa-treated calves, and GHR 1A variant) and GHR binding in cattle are low at the trend of lower GH-to-IGF-I ratios in colostrum-fed birth and increase with age, indicating ontogenetic than in formula-fed calves indicate enhanced matura- maturation of the somatotropic axis (1, 7, 31, 43). GHR- tion of the somatotropic axis, as defined by Gluckman binding studies revealed an almost fivefold increase of et al. (19).
Bmax in bulls (450 kg body wt) compared with neonatal Increased IGF-I plasma concentrations in Dexa- calves (Zbinden Y, Blum JW, and Hammon HM, un- treated calves might result from different clearance published data). In our study, colostrum intake did not rates of IGF-I. Our study has demonstrated reduced influence GHR expression and GH binding in liver, but plasma concentrations of IGFBP-1 and -2 in Dexa- Dexa treatment increased hepatic GHR expression.
treated calves. These binding proteins are able to leave The greatest GH binding was seen in calves fed co- the blood circulation together with IGF-I. Because we lostrum and treated by Dexa. Glucocorticoids stimu- found no differences in plasma concentrations of lated hepatic GHR mRNA in fetal sheep (30), neonatal IGFBP-3, which (together with its acid-labile subunit) pig (10), rabbit (24), and porcine (5) hepatocytes. Our retains most IGF-I in circulation (25), and because the findings indicate that Dexa, especially when accompa- ratios of small IGFBPs to IGFBP-3 were lower in AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Dexa-treated calves, clearance of IGF-I from the circu- plasma concentrations in Dexa-treated calves. We did lation might have been reduced in Dexa-treated calves.
not measure hepatic IGFBP-1 gene expression in this Furthermore, insulin, which was elevated in Dexa- study, but plasma concentrations of IGFBP-1 were treated calves (Sauter SN, Blum JW, and Hammon regulated directly at the transcriptional level (12), and HM, unpublished observation) and enhances plasma insulin overcame stimulatory effects of glucocorticoids IGF-I concentrations in cows (35), might have caused on hepatic IGFBP-1 mRNA levels (37, 39). Further- increased plasma IGF-I concentrations.
more, insulin suppressed IGFBP-2 plasma concentra- Hepatic production of IGF-I mainly determines cir- tions in cows (35), which is in accord with the present culating IGF-I concentrations (9, 13, 25). Hepatic IGF-I study. However, Dexa treatment did not influence he- expression increased after Dexa treatment in GrFD⫹ patic expression of IGFBP-2, indicating that it did not but decreased in GrCD⫹. Thus, in formula-fed calves, regulate IGFBP-2 at the transcriptional level.
there was a positive association of IGF-I mRNA con- In addition, plasma IGFBP-2 concentrations in neo- natal calves are regulated by the amount and time whereas there was a negative association in colostrum- point of colostrum intake (20, 23). In our study, de- fed calves. An inverse behavior of IGF-I plasma con- creased IGFBP-2 plasma concentrations in colostrum- centrations and hepatic expression was also seen in fed calves were associated with reduced hepatic neonatal Dexa-treated and colostrum-fed pigs (10). Dif- IGFBP-2 mRNA levels. Because nutrient intake was ferent Dexa effects on IGF-I expression in formula- and the same in formula- and colostrum-fed calves, other colostrum-fed calves were surprising, because we have factors than nutrient density were responsible for re- previously shown a significant relationship between duced IGFBP-2 levels. Overall, decreasing levels of hepatic IGF-I mRNA and plasma concentrations in IGFBP-1 and IGFBP-2 induced by Dexa treatment 8-day-old calves (13). One could speculate that colos- and/or colostrum feeding in the present study support tral IGF-I was intestinally absorbed and that Dexa the hypothesis of an accelerated maturation of the soma- treatment might have stimulated the absorption of totropic axis, as seen in postnatal development (7).
colostral IGF-I, thus resulting in reduced hepatic IGF-I Furthermore, we found reduced hepatic IGFBP-3 expression in colostrum-fed calves because of feedback mRNA levels after colostrum feeding, but this was not inhibition (20, 38). However, there was no evidence for associated with reduced plasma concentrations. Con- increased absorption of macromolecules like IgG in centrations of IGFBP-3 mRNA did not go along with Dexa-treated calves (Sauter SN, Blum JW, and Ham- hepatic IGF-I mRNA. Because effects of GH on the mon HM, unpublished observation), and we could not hepatic IGF system were weak, expression of IGFBP-3 demonstrate significant absorption of IGF-I in neona- was likely not regulated by GH and IGF-I as it is in the tal calves (20, 50), although receptors for IGF-I are case of growing and adult animals (7, 12, 41). Previous present in intestinal mucosa (18, 22). Different feeding findings support lacking colostral effects on IGFBP-3 may influence hepatic IGF-I mRNA stability after plasma concentrations (20). Surprisingly, hepatic ex- Dexa treatment and may regulate IGF-I production atthe posttranscriptional level. Dexa depressed hepatic pression and plasma concentrations of IGFBP-3 were IGF-I mRNA levels in rats but increased IGF-I plasma not affected by Dexa treatment. We expected increased concentrations in a dose-dependent manner (32). Inter- IGFBP-3 concentrations in Dexa-treated calves, be- estingly, the hepatic IGF-II mRNA responses to feed- cause IGF-I and insulin were elevated and are known ing and Dexa treatment were comparable to IGF-I to stimulate IGFBP-3 production (12, 27). However, mRNA responses, pointing at similar regulatory mech- glucocorticoids reduced IGFBP-3 plasma concentra- anisms for hepatic IGF-I and IGF-II mRNA.
tions in finishing calves (2), and Dexa did not influence Feeding and Dexa effects on IGFBPs. Biological ef- hepatic IGFBP-3 mRNA in neonatal pigs (10). There- fects of IGF-I and IGF-II greatly depend on the pres- fore, IGFBP-3 levels in neonatal calves do not support ence of IGFBPs. Plasma concentrations of IGFBP-1 the view of an accelerated maturation of the somato- and -2 are high during fetal development and decrease tropic axis by colostrum feeding or Dexa treatment.
after birth, whereas IGFBP-3, which (together with an In conclusion, the ratios of GH to IGF-I plasma acid-labile subunit) binds most of the IGF-I in blood, concentrations in the present study were reduced by increases in blood after birth (7, 12, 27, 41). These Dexa treatment and by colostrum feeding. Because a patterns were also shown in calves (46). Plasma con- low GH-to-IGF-I ratio mirrors maturation of the soma- centrations of IGFBP-1 and -2 were reduced by Dexa totropic axis in neonates (19), both colostrum intake treatment in the present study. Effects of glucocorti- and Dexa treatment promoted maturation of the GH- coids on IGFBP-1 plasma concentrations are variable; IGF-I axis, particularly if they were combined. Vari- inhibitory and stimulatory effects are reported (12, 41).
able hepatic IGF-I mRNA responses after Dexa treat- On the other hand, glucocorticoids consistently depress ment depended on different feeding, indicating differ- IGFBP-2 plasma concentrations (12, 27, 41). Because ences in IGF-I regulation. The higher number of GHR insulin (Sauter SN, Blum JW, and Hammon HM, un- binding in colostrum-fed and Dexa-treated calves published observation) and IGF-I plasma concentra- might have induced greater hepatic GH sensitivity and tions were elevated in Dexa-treated calves and both confirms the view of an accelerated maturation of the factors depress IGFBP-1 levels (12, 29), insulin and/or somatotropic axis. Furthermore, because nutrient in- IGF-I may have caused the reduction of IGFBP-1 take was the same for all calves in the present study, AJP-Endocrinol Metab • VOL 285 • AUGUST 2003 • www.ajpendo.org DEXAMETHASONE AND SOMATOTROPIC AXIS IN NEONATAL CALVES Dexa effects on the GH-IGF-I system were not influ- 15. Coxam V, Davicco MJ, Durand D, Bauchart D, Opmeer F,
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Mobile audio assistance in bus transportation for the blind J H Sánchez and C A Oyarzún Department of Computer Science, University of Chile, Blanco Encalada 2120, Santiago, CHILE ABSTRACT People with visual disabilities have serious difficulties when mobilizing through the city on the public transportation system. We introduce AudioTransantiago, a handheld application that allows users to plan trips and provide contextual information during the journey through the use of synthesized voices. The usability and cognitive evaluation of AudioTransantiago was performed using a prototype evaluation in order to identify and solve usability issues, ending up with an intuitive and simple interface. Finally, a cognitive impact evaluation administered during bus trips taken with the assistance of AudioTransantiago demonstrated that the software provides more autonomy and effectiveness for users' trips, improving their orientation and mobility.