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Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves

Open ArchivePublished:December 13, 2017DOI:https://doi.org/10.3168/jds.2017-13603

      ABSTRACT

      Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.

      Key words

      INTRODUCTION

      Intensive milk or milk replacer (MR) feeding programs could improve energy and nutrient intake, body growth (
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      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
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      ). A greater growth and development early in life could potentially affect the lifetime performance of dairy cows (
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      ). Intensive or ad libitum milk feeding, starting in the colostral period, may enhance the synthesis and release of IGF-I; IGF-I belongs to the somatotropic axis and is an important factor stimulating postnatal growth (
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      ;
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      • Weary D.M.
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      ;
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      • Blum J.W.
      Energy metabolism in the newborn farm animal with emphasis on the calf: endocrine changes and responses to milk-born and systemic hormones.
      ). The postnatal interaction of growth hormone (GH) and IGF-I, together with its binding proteins (IGFBP), affect body growth and organ development in mammals (
      • Etherton T.D.
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      Biology of somatotropin in growth and lactation of domestic animals.
      ;
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      • Oliver M.H.
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      ), including the development of the mammary gland (
      • Akers R.M.
      Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows.
      ;
      • Weaver S.R.
      • Hernandez L.L.
      Autocrine-paracrine regulation of the mammary gland.
      ) and immune function (
      • Clark R.
      The somatogenic hormones and insulin-like growth factor-1: Stimulators of lymphopoiesis and immune function.
      ). Because the plasma concentrations of IGF-I and IGFBP depend on nutrient intake (
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ), the increased protein and energy intake from elevated milk or MR feeding might affect the blood and tissue level of IGF-I and its binding proteins (
      • Bartlett K.S.
      • McKeith F.K.
      • VandeHaar M.J.
      • Dahl G.E.
      • Drackley J.K.
      Growth and body composition of dairy calves fed milk replacers containing different amounts of protein at two feeding rates.
      ;
      • Maccari P.
      • Wiedemann S.
      • Kunz H.-J.
      • Piechotta M.
      • Sanftleben P.
      • Kaske M.
      Effects of two different rearing protocols for Holstein bull calves in the first 3weeks of life on health status, metabolism and subsequent performance.
      ;
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      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). Therefore, changes of the components of the somatotropic axis due to an intensive MR feeding program may at least partly explain the accelerated mammary gland development in preweaning calves (
      • Geiger A.J.
      • Parsons C.L.M.
      • James R.E.
      • Akers R.M.
      Growth, intake, and health of Holstein heifer calves fed an enhanced preweaning diet with or without postweaning exogenous estrogen.
      ;
      • Soberon F.
      • Van Amburgh M.E.
      Effects of preweaning nutrient intake in the developing mammary parenchymal tissue.
      ).
      Butyrate is known as a natural growth-stimulating substrate that enhances growth performance in young mammals and has the potential to interfere with parameters of the somatotropic axis (e.g., by stimulation of GH and IGFBP release;
      • Tsubaki J.
      • Choi W.K.
      • Ingermann A.R.
      • Twigg S.M.
      • Kim H.S.
      • Rosenfeld R.G.
      • Oh Y.
      Effects of sodium butyrate on expression of members of the IGF-binding protein superfamily in human mammary epithelial cells.
      ;
      • Guilloteau P.
      • Martin L.
      • Eeckhaut V.
      • Ducatelle R.
      • Zabielski R.
      • van Immerseel F.
      From the gut to the peripheral tissues: The multiple effects of butyrate.
      ;
      • Miletta M.C.
      • Petkovic V.
      • Eblé A.
      • Ammann R.A.
      • Flück C.E.
      • Mullis P.-E.
      Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.
      ). In preweaning calves, butyrate supplementation in MR increases BW, structural growth, and health and affects insulin-dependent glucose metabolism (
      • Guilloteau P.
      • Martin L.
      • Eeckhaut V.
      • Ducatelle R.
      • Zabielski R.
      • van Immerseel F.
      From the gut to the peripheral tissues: The multiple effects of butyrate.
      ;
      • Górka P.
      • Kowalski Z.M.
      • Pietrzak P.
      • Kotunia A.
      • Jagusiak W.
      • Holst J.J.
      • Guilloteau P.
      • Zabielski R.
      Effect of method of delivery of sodium butyrate on rumen development in newborn calves.
      ;
      • Kato S.
      • Sato K.
      • Chida H.
      • Roh S.G.
      • Ohwada S.
      • Sato S.
      • Guilloteau P.
      • Katoh K.
      Effects of Na-butyrate supplementation in milk formula on plasma concentrations of GH and insulin, and on rumen papilla development in calves.
      ). Recent findings in preweaning calves indicates that ad libitum MR feeding stimulates body growth and anabolic metabolism, but butyrate supplementation did not further improve postnatal growth either in ad libitum or restrictive MR-fed calves (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ). The objective of the present study was to verify the previously published effects on body growth due to ad libitum MR feeding and butyrate supplementation by investigating the systemic and hepatic IGF-I system including IGFBP as main regulators of postnatal body growth in calves.

      MATERIALS AND METHODS

      Animals, Feeding, and Diets

      The animal experiment at the Educational and Research Centre for Animal Husbandry, Hofgut Neumuehle, Germany, was recently described in a companion paper (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ). The experimental procedures were performed in accordance with the German Animal Welfare Act () and were approved by the relevant Department for Animal Welfare Affairs [Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany (23 177–07/G 13–20–069)]. Briefly, 64 Holstein calves (n = 32 each for male and female) were used from birth until 80 ± 2 (mean ± SD) days of life. After birth, the calves were brought to individual straw-bedded calf hutches. This husbandry lasted for 10 ± 3 d (mean ± SD) before the calves were finally housed in an open, straw-bedded stable. In each pen, young calves were separated from the older group for 2 to 3 wk, with a self-feeding station and separate access to concentrate, hay, and water. The health of the calves was evaluated by daily rectal temperature measurement and navel and feces examination until 3 wk of age. Health maladies, such as diarrhea, pneumonia, and navel ill, were documented and treated by a veterinarian. Clinical data from this study will be presented in a companion paper.
      During the first 2 h after birth, all calves received 2.5 ± 0.09 kg (mean ± SD) of colostrum via bottle. Subsequently, the calves were allocated to 1 of the 4 feeding groups regarding their sex, birth weight, and cow parity to create equal groups. For the following 5 meals (until d 3 of life), calves were fed acidified transition milk (2 mL of acidifier/L of milk, H. W. Schaumann GmbH, Pinneberg, Germany) with teat buckets either in amounts of 3 L per meal (Res; n = 32) or ad libitum (Adl; n = 32). From d 4 on, all calves were fed MR (12.5% solids, 21.9% CP, 18.6% crude fat; Trouw Nutrition Deutschland GmbH, Burgheim, Germany) either without (B-) or with (B+) supplementation of calcium-sodium butyrate (0.24% as fixed to the MR powder; Benelux GmbH, Amel, Belgium) in amounts of either 6 L/d (ResB- and ResB+; n = 16, respectively) or ad libitum (maximum 25 L/d; AdlB- and AdlB+; n = 16, respectively). The ingredients and the chemical composition of the MR were presented in the companion paper (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ) and are given in the Supplemental Table S1 (https://doi.org/10.3168/jds.2017-13603). The dose for butyrate supplementation in MR was adapted from the work of
      • Górka P.
      • Kowalski Z.M.
      • Pietrzak P.
      • Kotunia A.
      • Jagusiak W.
      • Holst J.J.
      • Guilloteau P.
      • Zabielski R.
      Effect of method of delivery of sodium butyrate on rumen development in newborn calves.
      and the butyrate intake in ResB- and ResB+ is presented in the Results section. The MR allowance to all calves was reduced linearly between d 57 and 70, and thereafter calves continue to receive 2 L of MR/d until the end of the trial. In the calf hutches, feeding took place twice a day at 0700 and 1700 h. To guarantee ad libitum feed intake, buckets for Adl calves were maintained and calves had the chance to drink MR all the time. Buckets were refilled at noon, if necessary. Teat buckets were covered with a lid to ensure that no rain or dirt contaminated the milk. In the stable, feeding was conducted using automatic feeding systems for MR and concentrate (Förster-Technik GmbH, Engen, Germany). Milk replacer was fed in small portions, with a maximum of 2 L per meal for Res calves and 5 L per meal for Adl calves, followed by an off time of 2 h for Res calves and 30 min for Adl calves after the end of the meal. Water and hay were freely available, and concentrate as pelleted starter (Raiffeisen Waren-Zentrale Rhein-Main eG, Köln, Germany; Supplemental Table S1) was offered ad libitum in the stable.

      Measurement of Performance Data

      During the period in the calf hutches, the daily milk and MR intake was documented by weighting residues with an electronic scale (Sartorius AG, Göttingen, Germany). In the stable, data of MR and concentrate intake were sent automatically from the automatic feeding system to the connected computer program (Förster-Technik GmbH). The nutrient compositions of MR and concentrate were analyzed by an accredited external laboratory (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) according to the Weender standard procedure (
      • Naumann C.
      • Bassler R.
      ).
      The birth weight was measured on a platform scale after the first colostrum intake. The exact amount of ingested colostrum was subtracted from the initial weight. The BW in wk 7 and 11 was recorded using a mobile scale (Tru-Test Ltd., Auckland, New Zealand). The ADG was calculated from BW with the precise number of days between weekly weight measurements.

      Blood and Liver Sampling and Analyses

      Blood samples were taken from the jugular vein before first colostrum intake (d 1), 24 h after colostrum intake (d 2), before first MR intake (d 4), and then weekly from d 7 to 77, or once every other week from d 21 for IGFBP. All calves were fasted for at least 1 h before blood sampling. The blood was collected in evacuated tubes (Greiner Bio-One GmbH, Frickenhausen, Germany) containing potassium-EDTA (1.8 mg/mL) to determine plasma concentrations of insulin (on d 49 and 77), IGF-I, and IGFBP-2, -3, and -4; and tubes containing sodium fluoride (2–4 mg/mL) and potassium oxalate (1–3 mg/mL) to determine plasma glucose concentration on d 49 and 77. Blood samples were cooled in ice water until centrifugation at 3,500 × g for 10 min at room temperature. The supernatants from plasma were pipetted into aliquots and stored at −20°C until analyzed.
      The glucose concentration in blood plasma was analyzed spectrophotometrically (HORIBA ABX SAS, Montpellier, France) using the kit #A11A01667 (Axon Lab AG, Baden, Switzerland). Plasma concentration of insulin (#RIA-1257) was determined by RIA using kits from DRG Instruments GmbH (Marburg, Germany), which were adapted to bovine (
      • Hammon H.M.
      • Stürmer G.
      • Schneider F.
      • Tuchscherer A.
      • Blum H.
      • Engelhard T.
      • Genzel A.
      • Staufenbiel R.
      • Kanitz W.
      Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding dairy cows with high and low fat content in liver after calving.
      ). Intra- and interassay coefficients of variation for insulin were 3.7 and 5.5%, respectively. Plasma concentration of IGF-I was measured by ELISA adapted from
      • Daxenberger A.
      • Breier B.H.
      • Sauerwein H.
      Increased milk levels of insulin-like growth factor 1 (IGF-1) for the identification of bovine somatotropin (bST) treated cows.
      using an anti-human IGF-I polyclonal antiserum (GroPep, Adelaide, Australia) that showed 100% cross-reactivity to bovine IGF-I. For standard preparation, recombinant human IGF-I (receptor grade, GroPep) was used and IGFBP were blocked with excess of human IGF-II (GroPep). Biotinyl human IGF-I was obtained from Ibt GmbH (Binzwangen, Germany). Intra- and interassay coefficients of variation were 6.4 and 9.1%, respectively. Recoveries of 3 IGF-I concentrations added to different samples were 93.4%± 1.7, 90.8 ± 1.8, and 90.8 ± 2.7%. The IGFBP-2, -3, and -4 were analyzed in plasma using quantitative Western ligand blot analysis, as previously described (
      • Laeger T.
      • Wirthgen E.
      • Piechotta M.
      • Metzger F.
      • Metges C.C.
      • Kuhla B.
      • Hoeflich A.
      Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ).
      On d 50 ± 2 and 80 ± 2 (mean ± SD; d 80 females only), about 80 to 100 mg of liver tissue was collected from each calf by biopsy (modified from
      • Swanson K.S.
      • Merchen N.R.
      • Erdman Jr., J.W.
      • Drackley J.K.
      • Orias F.
      • Douglas G.N.
      • Huhn J.C.
      Technical note: a technique for multiple liver biopsies in neonatal calves.
      ) with a Bard Magnum biopsy instrument and Bard Magnum core tissue biopsy needle (C.R. Bard Inc., Covington, GA). The sample notch of a 12-gauge biopsy needle contained about 20 to 30 mg of liver tissue. To collect several biopsies while avoiding additional stress and abdominal pain for the calves, we used a Bard TruGuide coaxial biopsy needle that remained in the abdomen for the duration of biopsy. Liver samples of male calves on d 80 ± 2 (mean ± SD) were collected after calves were harvested at the end of the study. Liver samples were flushed in ice-cold 0.9% NaCl and frozen in liquid nitrogen. For further analysis, the liver tissue was pulverized in liquid nitrogen. The relative mRNA abundance of genes related to the IGF system was quantified as previously described (
      • Saremi B.
      • Sauerwein H.
      • Dänicke S.
      • Mielenz M.
      Technical note: Identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). Primer sequences and PCR conditions for reference genes [hippocalcin-like 1 (HPCAL1), low-density lipoprotein 10 (LRP10), and RNA polymerase II (POLR2A)] and target genes [growth hormone receptor 1A (GHR1A), IGF-I (IGF1), IGFBP-1 (IGFBP1), -2 (IGFBP2), -3 (IGFBP3), and -4 (IGFBP4), IGF Type 1 receptor (IGF1R), and insulin receptor (INSR)] were recently published (
      • Kendall P.E.
      • Auchtung T.L.
      • Swanson K.S.
      • Radcliff R.P.
      • Lucy M.C.
      • Drackley J.K.
      • Dahl G.E.
      Effect of photoperiod on hepatic growth hormone receptor 1A expression in steer calves.
      ;
      • Saremi B.
      • Sauerwein H.
      • Dänicke S.
      • Mielenz M.
      Technical note: Identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). The primer products were verified by sequencing applied with the BigDye Terminator v1.1 Cycle Sequencing kit and an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific Inc., Waltham, MA). Real-time PCR was performed with the use of a LightCycler (F. Hoffman-La Roche AG, Basel, Switzerland); SYBR Green I (F. Hoffman-La Roche AG) was used as the fluorescent dye. Melting curve analysis and agarose gel electrophoresis were used to confirm the specificity of the PCR products. Quantification cycle values and amplification efficiencies obtained with the use of LinRegPCR version 2013.0 (
      • Ruijter J.M.
      • Pfaffl M.W.
      • Zhao S.
      • Spiess A.N.
      • Boggy G.
      • Blom J.
      • Rutledge R.G.
      • Sisti D.
      • Lievens A.
      • De Preter K.
      • Derveaux S.
      • Hellemans J.
      • Vandesompele J.
      Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications.
      ) were imported into qBASE+ version 2.6.1 (Biogazelle, Zwijnaarde, Belgium) for all subsequent calculations and quality controls (
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). The geometric mean of the reference gene abundances was applied for normalization. The data are presented as the ratio of the copy number of the respective gene of interest to the geometric mean of the reference gene abundance.

      Statistical Analyses

      Data were evaluated by repeated-measures ANOVA using PROC MIXED in SAS for Windows (release 9.4; SAS Institute Inc., Cary, NC). The ANOVA model contained the fixed effects of feeding regimen (milk; Res vs. Adl), butyrate supplementation, time (wk 1–7 and 8–11 for performance data; d 49 and 77 for BW and for plasma glucose and insulin; d 50 and 80 for tissue data; time of frequent blood samples for plasma IGF-I and IGFBP), sex, and respective interactions. Repeated measures on each calf were considered using the repeated statement of the MIXED procedure with an unstructured type of the block diagonal residual covariance matrix structure (plasma IGF-I and IGFBP) or with an autoregressive residual covariance structure (performance data, plasma glucose and insulin, gene expression data; SAS Institute Inc.). Least squares means and their standard errors were computed for each fixed effect, and all pair-wise differences of least squares means were tested with the Tukey-Kramer procedure. The SLICE statement of the MIXED procedure was used to conduct partitioned analyses of the least squares means for interactions. Differences in data with P-values <0.05 were defined as significant, and P-values <0.1 were considered as trends. Values are presented as least squares means ± standard error if not declared otherwise in the text.

      RESULTS

      Feed Intake and Growth Performance

      Data for feed intake and growth performance are presented in Table 1 and focused on the time periods of wk 1 to 7 (before MR reduction) and 8 to 11 (MR reduction), respectively. In both time periods, milk intake was greater in Adl than in Res calves (P < 0.001). On the contrary, concentrate intake in wk 8 to 11 was greater in Res than in Adl calves (P < 0.001). Total DMI in wk 1 to 7 was greater (P < 0.001) in Adl than in Res calves. The DMI was not affected by butyrate supplementation and by sex. In B-supplemented groups, B intake was greater (P < 0.001) in AdlB+ than in ResB+. Butyrate intake was 3.3 ± 0.1 g/d for wk 1 to 7 and 2.6 ± 0.09 g/d for wk 8 to 11 in AdlB+, and was 1.7 ± 0.1 g/d for wk 1 to 7 and 1.1 ± 0.09 g/d for wk 8 to 11 in ResB+. The BW was greater (P < 0.001) at the end of wk 7 (d 49) and at the end of wk 11 (d 77) in Adl than in Res (Table 1). The ADG was higher from wk 1 to 7 (P < 0.001) but lower from wk 8 to 11 in Adl than Res groups (P < 0.001). Body weight (P < 0.06) and ADG (P = 0.1) at wk 11 tended to be higher in B- than B+ calves (Table 1).
      Table 1Dry matter intake of liquid and concentrate feed, BW, and ADG of calves fed milk and milk replacer (MR) either ad libitum or restrictively and supplemented MR without (ResB-; AdlB-) or with 0.24% butyrate (ResB+; AdlB+)
      Modified from Frieten et al., 2017.
      Item
      Values are presented as LSM.
      Dietary treatmentSEMFixed effect, P-value
      Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.
      ResB-AdlB-ResB+AdlB+MilkButyrateTimeSex
      Milk × timeMilk × butyrateButyrate × time
      Liquid intake, kg
       wk 1–734.5
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      68.8
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      34.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      65.8
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1.36<0.0010.7<0.0010.8
       wk 8–1112.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      29.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      13.1
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      30.2
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      <0.0010.50.2
      Concentrate intake, kg
       wk 1–76.851.898.933.112.07<0.0010.6<0.0010.4
       wk 8–1139.7
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      18.6
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      38.1
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      20.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      <0.0010.70.6
      Total DMI, kg
       wk 1–741.4
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      70.6
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      43.8
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      68.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      2.44<0.0010.8<0.0010.4
       wk 8–1152.648.551.251.0<0.0011.00.9
      BW, kg
       d 4972.7
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      92.5
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      70.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      88.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      2.30<0.0010.11<0.0010.12
       d 77106.2
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      120.6
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      102.0
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      115.7
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      <0.0010.80.12
      ADG, g
       wk 1–7572.6
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1,015.1
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      591.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      941.8
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      37.0<0.010.2<0.0010.2
       wk 8–111,212.1
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      997.6
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1,121.0
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      966.4
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      <0.0010.80.4
      a,b Different letters within the same row indicate significant differences (P ≤ 0.05).
      1 Modified from
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      .
      2 Values are presented as LSM.
      3 Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.

      Plasma Concentrations of Glucose, Insulin, IGF-I, and IGF Binding Proteins

      The results of glucose and insulin concentrations in blood plasma are presented in detail in the companion paper (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ). Plasma glucose and insulin concentrations decreased in Adl calves but increased in Res calves from d 49 to 77 (P < 0.001; Table 2). Plasma glucose concentration was higher on d 49 (P < 0.001) in Adl than in Res calves, but was lower on d 77 (P < 0.05) in Adl calves than in ResB-. Plasma insulin concentration was much higher in Adl than in Res calves at d 49 (P < 0.001), but did not differ between Adl and Res calves on d 77 of age.
      Table 2Blood plasma concentrations of glucose and insulin in calves fed milk and milk replacer (MR) either ad libitum or restrictively and supplemented MR without (ResB-; AdlB-) or with 0.24% butyrate (ResB+; AdlB+)
      Modified from Frieten et al., 2017.
      Item
      Values are presented as LSM.
      Dietary treatmentSEMFixed effect, P-value
      Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.
      ResB-AdlB-ResB+AdlB+MilkButyrateTimeSex
      Milk × timeMilk × butyrateButyrate × time
      Glucose, mmol/L
       d 495.07
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      5.79
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      4.99
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      6.13
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      0.180.30.80.010.7
       d 775.66
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      4.84
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      5.39
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      4.97
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      0.18<0.0010.140.3
      Insulin, μg/L
       d 490.34
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1.24
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      0.28
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1.19
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      0.17<0.010.80.030.3
       d 770.580.390.540.410.17<0.0010.90.8
      a,b Different letters within the same row indicate significant differences (P ≤ 0.05).
      1 Modified from
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      .
      2 Values are presented as LSM.
      3 Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.
      The plasma concentration of IGF-I slightly increased (P < 0.05) from d 1 to 2 and decreased (P < 0.001) from d 4 to 14 of age in all groups. Plasma IGF-I increased (P < 0.001) in Adl groups from wk 3 on, but in Res calves increased from wk 7 on (P < 0.001; Figure 1A). We noted a distinct decrease of plasma IGF-I in Adl calves from wk 9 to 10 (P < 0.001). Plasma IGF-I was higher from wk 1 to 9 (P < 0.01), but was lower (P = 0.02) at wk 11 in Adl than in Res calves. Butyrate feeding depressed plasma IGF-I from wk 1 to 4 and at wk 9 (P < 0.05). Plasma IGFBP-2 concentration decreased (P < 0.001) during first week of age and then increased (P < 0.01) until wk 2 in all groups (Figure 1B). Thereafter, plasma IGFBP-2 remained high in Res calves, but decreased (P < 0.1) and remained low until wk 9 in Adl calves. Plasma IGFBP-2 concentration significantly differed from wk 3 until the end of the trial, with higher concentrations in Res calves up to wk 9. At wk 11, plasma IGFBP-2 decreased (P < 0.1) in Res calves and increased (P < 0.001) in Adl calves, indicating higher concentrations in Adl than Res calves at wk 11 (P < 0.01). Plasma IGFBP-3 concentration decreased (P < 0.001) during first week of age in all groups, and was greater (P < 0.01) in Adl than in Res calves from wk 3 to 9 (Figure 1C). We observed a trend for a greater IGFBP-3 plasma concentration at wk 11 in Adl than Res calves (P = 0.07). Calves fed butyrate had a lower IGFBP-3 plasma concentration on d 1 and 7 of age (P ≤ 0.05). Plasma IGFBP-4 concentration increased (P < 0.001) after birth, but decreased (P < 0.001) from d 4 to 21 in all groups (Figure 1D) and was greater (P < 0.05) in Res than in Adl calves on d 1, 63, and 77 of age.
      Figure thumbnail gr1
      Figure 1Blood plasma concentrations of IGF-I (A), IGF binding protein (IGFBP)-2 (B), IGFBP-3 (C), and IGFBP-4 (D) in calves fed milk and milk replacer (MR) either ad libitum or restrictively and supplemented MR without (△, AdlB-; ○, ResB-) or with 0.24% butyrate (▴, AdlB+; •, ResB+). Arrows mark the start of weaning. Data are presented as LSM ± SEM; an asterisk (*) indicates effect of feeding regimen (P < 0.05); a section mark (§) indicates effect of butyrate supplementation (P < 0.05).

      Hepatic Gene Expression Related to the IGF System

      The hepatic gene expression of GHR1A, IGF1, IGFBP1, -2, -3, and -4, IGF1R, and INSR are presented in Table 3. The mRNA abundance of GHR1A and IGF1 on d 50 was greater (P < 0.02) in Adl than in Res calves, with highest abundance for IGF1 in AdlB-, but did not differ on d 80. The abundance of IGF1 mRNA was greater (P = 0.03) in male than in female calves. Male calves had a greater (P < 0.001) IGFBP1 mRNA abundance and tended to have a greater (P = 0.07) IGFBP3 mRNA abundance than female calves. The mRNA encoding for IGFBP2 changed with time (P = 0.02), and was greater (P < 0.001) on d 50 but was lower (P = 0.01) on d 80 in Res than in Adl calves. On d 50, IGFBP2 mRNA was greatest (P < 0.05) in ResB+ and on d 80 butyrate supplementation affected IGFBP2 mRNA, with greatest expression in AdlB+. The abundance of IGFBP4 mRNA increased with time (P < 0.01) in all feeding groups. The mRNA abundance of IGF1R tended (P = 0.07) to be greater in Res than in Adl calves on d 50, and the mRNA abundance of INSR showed a tendency (P = 0.06) for the butyrate × time interaction with an increasing abundance from d 50 to 80 in butyrate-fed calves.
      Table 3Relative mRNA expression (log2) of liver samples in calves fed milk and milk replacer (MR) either ad libitum or restrictively and supplemented MR without (ResB-; AdlB-) or with 0.24% butyrate (ResB+; AdlB+)
      Relative mRNA expression related to reference genes
      Values are presented as LSM.
      GHR1A = growth hormone receptor 1A; IGFBP1–4 = IGF binding protein-1 to −4; IGFR1 = IGF-I receptor; INSR = insulin receptor.
      Dietary treatmentSEMFixed effect, P-value
      Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.
      ResB-AdlB-ResB+AdlB+MilkButyrateTimeSex
      Milk × timeMilk × butyrateButyrate × time
      GHR1A
       d 500.550.690.570.730.060.030.40.90.3
       d 800.590.630.630.680.060.20.90.8
      IGF1
       d 5015.3
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      24.9
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      16.3
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      21.0
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      2.220.150.60.20.03
       d 8024.618.320.521.62.20<0.010.70.7
      IGFBP1
       d 502282393423721570.30.20.2<0.001
       d 803613893316551250.50.41.0
      IGFBP2
       d 507.07
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      4.25
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      7.86
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      4.21
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      0.900.70.090.020.7
       d 804.98
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      7.02
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      6.78
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      9.92
      Different letters within the same row indicate significant differences (P ≤ 0.05).
      1.00<0.0010.90.07
      IGFBP3
       d 503.653.924.224.060.460.50.40.20.07
       d 803.193.913.733.950.340.30.50.9
      IGFBP4
       d 502.712.812.692.710.220.50.9<0.010.6
       d 802.903.153.033.200.260.60.90.6
      IGF1R
       d 502.742.694.092.540.430.20.20.50.6
       d 803.082.983.433.170.420.20.20.5
      INSR
       d 506.436.535.565.500.610.70.50.30.9
       d 806.505.916.626.410.580.50.90.06
      a,b Different letters within the same row indicate significant differences (P ≤ 0.05).
      1 Values are presented as LSM.
      2 GHR1A = growth hormone receptor 1A; IGFBP1–4 = IGF binding protein-1 to −4; IGFR1 = IGF-I receptor; INSR = insulin receptor.
      3 Main fixed effects are presented in 2 rows: first row indicates P-value for milk (ad libitum versus restrictive), butyrate supplementation, time, and sex; second row indicates P-values for interaction milk × time, milk × butyrate, and butyrate × time.

      DISCUSSION

      Calves with unlimited milk and MR intake for 8 wk postnatal more or less doubled their MR intake compared with Res calves fed 6 L of MR/d. Such a great liquid feed intake was previously reported in other studies with dairy calves that were allowed to drink milk or MR ad libitum for a distinct time period after calving (
      • Hepola H.P.
      • Hänninen L.
      • Raussi S.
      • Pursiainen P.
      • Aarnikoivu A.-M.
      • Saloniemi H.
      Effects of providing water from a bucket or a nipple on the performance and behavior of calves fed ad libitum volumes of acidified milk replacer.
      ;
      • Maccari P.
      • Wiedemann S.
      • Kunz H.-J.
      • Piechotta M.
      • Sanftleben P.
      • Kaske M.
      Effects of two different rearing protocols for Holstein bull calves in the first 3weeks of life on health status, metabolism and subsequent performance.
      ;
      • Korst M.
      • Koch C.
      • Kesser J.
      • Müller U.
      • Romberg F.-J.
      • Rehage J.
      • Eder K.
      • Sauerwein H.
      Different milk feeding intensities during the first 4 weeks of rearing in dairy calves: Part 1: Effects on performance and production from birth over the first lactation.
      ). On the other hand, concentrate intake was lower in Adl than in Res calves and the increase in concentrate intake was delayed in Adl calves (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ). An impaired concentrate intake in calves due to unrestricted milk feeding (
      • Jasper J.
      • Weary D.M.
      Effects of ad libitum milk intake on dairy calves.
      ) or enhanced MR feeding (
      • Kristensen N.B.
      • Sehested J.
      • Jensen S.K.
      • Vestergaard M.
      Effect of milk allowance on concentrate intake, ruminal environment, and ruminal development in milk-fed Holstein calves.
      ;
      • Davis Rincker L.E.
      • VandeHaar M.J.
      • Wolf C.A.
      • Liesman J.S.
      • Chapin L.T.
      • Weber Nielsen M.S.
      Effect of intensified feeding of heifer calves on growth, pubertal age, calving age, milk yield, and economics.
      ) was previously observed in some, but not in all studies (
      • Maccari P.
      • Wiedemann S.
      • Kunz H.-J.
      • Piechotta M.
      • Sanftleben P.
      • Kaske M.
      Effects of two different rearing protocols for Holstein bull calves in the first 3weeks of life on health status, metabolism and subsequent performance.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ;
      • Korst M.
      • Koch C.
      • Kesser J.
      • Müller U.
      • Romberg F.-J.
      • Rehage J.
      • Eder K.
      • Sauerwein H.
      Different milk feeding intensities during the first 4 weeks of rearing in dairy calves: Part 1: Effects on performance and production from birth over the first lactation.
      ). In addition, calves receiving elevated amounts of milk, but no ad libitum milk feeding, during the first weeks of life had a greater concentrate intake after the intensive milk feeding period and after weaning (
      • Khan M.A.
      • Lee H.J.
      • Lee W.S.
      • Kim H.S.
      • Kim S.B.
      • Ki K.S.
      • Ha J.K.
      • Lee H.G.
      • Choi Y.J.
      Pre- and postweaning performance of Holstein female calves fed milk through step-down and conventional methods.
      ). Therefore, the management of the intensive milk feeding period might have an important effect on the concentrate intake in preruminant calves. In any case, ad libitum milk feeding (
      • Maccari P.
      • Wiedemann S.
      • Kunz H.-J.
      • Piechotta M.
      • Sanftleben P.
      • Kaske M.
      Effects of two different rearing protocols for Holstein bull calves in the first 3weeks of life on health status, metabolism and subsequent performance.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ;
      • Korst M.
      • Koch C.
      • Kesser J.
      • Müller U.
      • Romberg F.-J.
      • Rehage J.
      • Eder K.
      • Sauerwein H.
      Different milk feeding intensities during the first 4 weeks of rearing in dairy calves: Part 1: Effects on performance and production from birth over the first lactation.
      ) or enhanced milk feeding programs using MR with a greater CP content (
      • Diaz M.C.
      • Van Amburgh M.E.
      • Smith J.M.
      • Kelsey J.M.
      • Hutten E.L.
      Composition of growth of Holstein calves fed milk replacer from birth to 105-kilogram body weight.
      ;
      • Bartlett K.S.
      • McKeith F.K.
      • VandeHaar M.J.
      • Dahl G.E.
      • Drackley J.K.
      Growth and body composition of dairy calves fed milk replacers containing different amounts of protein at two feeding rates.
      ;
      • Khan M.A.
      • Weary D.M.
      • von Keyserlingk M.A.G.
      Invited review: Effects of milk ration on solid feed intake, weaning, and performance in dairy heifers.
      ) resulted in an elevated body growth during the preweaning period in calves. On the other hand, the supplementation of butyrate with the MR did not affect MR or concentrate intake in a consistent manner (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ).
      The enhanced growth in Adl calves during the ad libitum milk feeding period coincided with elevated plasma concentrations of insulin, IGF-I, and IGFBP-3 and reduced plasma concentrations of IGFBP-2 during this time period. The stimulation of the postnatal somatotropic axis depends on the nutrient supply, starting in the neonatal period (
      • Breier B.H.
      • Oliver M.H.
      • Gallaher B.W.
      ;
      • Hammon H.M.
      • Steinhoff-Wagner J.
      • Schonhusen U.
      • Metges C.C.
      • Blum J.W.
      Energy metabolism in the newborn farm animal with emphasis on the calf: endocrine changes and responses to milk-born and systemic hormones.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ), and reflects the glucose and insulin status of the animal (
      • Breier B.H.
      • Gluckman P.D.
      • Bass J.J.
      Plasma concentrations of insulin-like growth factor-I and insulin in the infant calf: ontogeny and influence of altered nutrition.
      ;
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Brameld J.M.
      • Gilmour R.S.
      • Buttery P.J.
      Glucose and amino acids interact with hormones to control expression of insulin-like growth factor-I and growth hormone receptor mRNA in cultured pig hepatocytes.
      ). The nutrient intake was much greater in Adl than in Res calves during the ad libitum milk feeding period. Elevated nutrient intake affects the plasma IGF-I concentration in mammalian (
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ), including the preweaning calf (
      • Smith J.M.
      • Van Amburgh M.E.
      • Díaz M.C.
      • Lucy M.C.
      • Bauman D.E.
      Effect of nutrient intake on the development of the somatotropic axis and its responsiveness to GH in Holstein bull calves.
      ;
      • Daniels K.M.
      • Hill S.R.
      • Knowlton K.F.
      • James R.E.
      • McGilliard M.L.
      • Akers R.M.
      Effects of milk replacer composition on selected blood metabolites and hormones in preweaned Holstein heifers.
      ) and ad libitum milk feeding (
      • Maccari P.
      • Wiedemann S.
      • Kunz H.-J.
      • Piechotta M.
      • Sanftleben P.
      • Kaske M.
      Effects of two different rearing protocols for Holstein bull calves in the first 3weeks of life on health status, metabolism and subsequent performance.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). The stimulating effect of elevated nutrient supply by milk or MR intake on the IGF-I plasma concentration starts postpartum (
      • Hammon H.
      • Blum J.W.
      The somatotropic axis in neonatal calves can be modulated by nutrition, growth hormone, and Long-R3-IGF-I.
      ;
      • Hammon H.M.
      • Zanker I.A.
      • Blum J.W.
      Delayed colostrum feeding affects IGF-I and insulin plasma concentrations in neonatal calves.
      ;
      • Sauter S.N.
      • Ontsouka E.
      • Roffler B.
      • Zbinden Y.
      • Philipona C.
      • Pfaffl M.
      • Breier B.H.
      • Blum J.W.
      • Hammon H.M.
      Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves.
      ) and is related to the greater nutrient availability due to milk feeding (
      • Breier B.H.
      • Gluckman P.D.
      • Bass J.J.
      Plasma concentrations of insulin-like growth factor-I and insulin in the infant calf: ontogeny and influence of altered nutrition.
      ).
      The stimulation of the somatotropic axis due to ad libitum milk feeding in the present study was supported by the elevated hepatic gene expression of the liver-specific GHR1A and IGF1. Both were higher expressed in liver of Adl than Res calves at d 50 of age. The IGF-I originating from the liver contributes in a significant manner to the IGF-I in the blood plasma (
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ), hepatic IGF1 gene expression correlated with IGF-I in blood plasma of calves (
      • Cordano P.
      • Hammon H.M.
      • Morel C.
      • Zurbriggen A.
      • Blum J.W.
      mRNA of insulin-like growth factor (IGF) quantification and presence of IGF binding proteins, and receptors for growth hormone, IGF-I and insulin, determined by reverse transcribed polymerase chain reaction, in the liver of growing and mature male cattle.
      ), and its gene expression depends on the GH action in the liver (
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ;
      • Renaville R.
      • Hammadi M.
      • Portetelle D.
      Role of the somatotropic axis in the mammalian metabolism.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ). The elevated glucose and insulin status at the time of ad libitum milk feeding may have stimulated GHR1A gene expression in the liver of Adl calves, as insulin treatment promotes hepatic GHR1A mRNA abundance in dairy cows (
      • Butler S.T.
      • Marr A.L.
      • Pelton S.H.
      • Radcliff R.P.
      • Lucy M.C.
      • Butler W.R.
      Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A.
      ;
      • Weber C.
      • Schäff C.T.
      • Kautzsch U.
      • Börner S.
      • Erdmann S.
      • Bruckmaier R.M.
      • Röntgen M.
      • Kuhla B.
      • Hammon H.M.
      Variable liver fat concentration as a proxy for body fat mobilization postpartum has minor effects on insulin-induced changes in hepatic gene expression related to energy metabolism in dairy cows.
      ).
      The reduced insulin and IGF-I concentrations in blood plasma after the MR reduction period reflected the lower concentrate intake in Adl calves (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ). The gene expression of IGF1 as well as GHR1A in liver was not affected by the nutrient intake on d 80 of age in a significant manner. The increase in hepatic IGF1 mRNA from d 50 to 80 in Res, but not in Adl calves, indicated that Res calves gained on nutrient intake with time due to elevated concentrate intake; Adl calves reached an elevated IGF1 gene expression level on d 50 of age and did not further increase IGF1 mRNA abundance in liver on d 80 of age. The differences in concentrate intake at the end of the study between groups (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ) were, however, not large enough to cause changes in hepatic GHR1A and IGF1 mRNA abundances between groups, but the greater concentrate intake in Res calves at the end of the study resulted in a greater plasma IGF-I concentration. Therefore, tissues other than liver that synthesize IGF-I (e.g., muscle tissue) may contribute to the elevated plasma IGF-I concentrations in Res calves on d 80 of age (
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ).
      The IGFBP-3, which is part of the 150-kDa complex and binds most of the IGF-I in blood plasma (
      • Jones J.I.
      • Clemmons D.R.
      Insulin-like growth factors and their binding proteins: biological actions.
      ;
      • Murphy L.J.
      Insulin-like growth factor-binding proteins: functional diversity or redundancy?.
      ), did not show an association between hepatic gene expression and plasma concentration at d 50 and 80 of age. In general, IGFBP are important regulators of the IGF-I action, and inhibitory and stimulating effects of IGFBP have been reported (
      • Jones J.I.
      • Clemmons D.R.
      Insulin-like growth factors and their binding proteins: biological actions.
      ;
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ). After a decrease during the first week of postnatal life, the plasma IGFBP-3 concentration increased with age during the MR feeding period (
      • Skaar T.C.
      • Baumrucker C.R.
      • Deaver D.R.
      • Blum J.W.
      Diet effects and ontogeny of alterations of circulating insulin-like growth factor binding proteins in newborn dairy calves.
      ;
      • Hammon H.M.
      • Zbinden Y.
      • Sauerwein H.
      • Breier B.H.
      • Blum J.W.
      • Donkin S.S.
      The response of the hepatic insulin-like growth factor system to growth hormone and dexamethasone in calves.
      ;
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ). The increase of plasma IGFBP-3 was more dominant in Adl than in Res calves, and the dynamics of plasma IGFBP-3 concentration were similar to those of plasma IGF-I during the ad libitum milk feeding period. Although plasma GH was not measured in our study, it is well known that the IGFBP-3 status depends on GH action (
      • Jones J.I.
      • Clemmons D.R.
      Insulin-like growth factors and their binding proteins: biological actions.
      ;
      • Murphy L.J.
      Insulin-like growth factor-binding proteins: functional diversity or redundancy?.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ). Obviously, GH does not directly stimulate IGFBP3 gene expression, because IGFBP3 gene expression is located in nonparenchymal hepatic cells, but not in hepatocytes, and these cells do not express GH receptors (
      • Murphy L.J.
      Insulin-like growth factor-binding proteins: functional diversity or redundancy?.
      ;
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ). However, both GH and IGFBP-3 are stimulated by enhanced nutrient intake in calves and growing ruminants (
      • Renaville R.
      • Hammadi M.
      • Portetelle D.
      Role of the somatotropic axis in the mammalian metabolism.
      ;
      • Daniels K.M.
      • Hill S.R.
      • Knowlton K.F.
      • James R.E.
      • McGilliard M.L.
      • Akers R.M.
      Effects of milk replacer composition on selected blood metabolites and hormones in preweaned Holstein heifers.
      ). The fact that IGFBP3 gene expression in liver did not correspond to IGFBP-3 plasma concentration is probably due to IGFBP-3 synthesis in other tissues than the liver (e.g., fibroblast, endothelial cells, bone;
      • Jones J.I.
      • Clemmons D.R.
      Insulin-like growth factors and their binding proteins: biological actions.
      ), and possibly due to the low number of cells in the liver (nonparenchymal cells) with IGFBP3 gene expression (
      • Murphy L.J.
      Insulin-like growth factor-binding proteins: functional diversity or redundancy?.
      ;
      • Le Roith D.
      • Bondy C.
      • Yakar S.
      • Liu J.-L.
      • Butler A.
      The somatomedin hypothesis: 2001.
      ). In addition, the IGFBP-3 plasma concentration depends on the proteolytic cleavage activity in the blood plasma and is probably less regulated at the transcriptional level (
      • Jones J.I.
      • Clemmons D.R.
      Insulin-like growth factors and their binding proteins: biological actions.
      ). Overall, the greater plasma IGFBP-3 concentration in Adl calves corresponded to the elevated IGF-I concentration in these calves and mirrored their greater nutrient intake, but was not related to the hepatic IGFBP3 gene expression.
      The IGFBP-2 status in the calves indicated corresponding changes of plasma concentration and hepatic gene expression with respect to nutrient intake. The concentration of IGFBP-2 in blood plasma and liver was lower in the Adl than in the Res calves during the ad libitum milk feeding period, but increased to a higher level in Adl calves at the end of the study when concentrate intake was greater in Res than Adl calves. The plasma IGFBP-2 concentration indicated an inverse relationship with respect to the insulin and IGF-I status in calves (
      • Hammon H.
      • Blum J.W.
      The somatotropic axis in neonatal calves can be modulated by nutrition, growth hormone, and Long-R3-IGF-I.
      ;
      • Hammon H.M.
      • Zanker I.A.
      • Blum J.W.
      Delayed colostrum feeding affects IGF-I and insulin plasma concentrations in neonatal calves.
      ;
      • Daniels K.M.
      • Hill S.R.
      • Knowlton K.F.
      • James R.E.
      • McGilliard M.L.
      • Akers R.M.
      Effects of milk replacer composition on selected blood metabolites and hormones in preweaned Holstein heifers.
      ) and dairy cows (
      • McGuire M.A.
      • Dwyer D.A.
      • Harrell R.J.
      • Bauman D.E.
      Insulin regulates circulating insulin-like growth factors and some of their binding proteins in lactating cows.
      ), and IGFBP-2 is elevated during the catabolic state (
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Breier B.H.
      • Oliver M.H.
      • Gallaher B.W.
      ;
      • Renaville R.
      • Hammadi M.
      • Portetelle D.
      Role of the somatotropic axis in the mammalian metabolism.
      ). Therefore, elevated plasma IGFBP-2 concentration may reflect the inadequate nutrient supply in Res calves during the ad libitum MR feeding period and the impaired nutrient status in Adl calves after the MR step-down period, which corresponds to findings in humans (
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ). Surprisingly, hepatic IGFBP1 gene expression did not behave in the same way as hepatic IGFBP2 gene expression, but elevated IGFBP-1 status during impaired nutrient supply was shown in human patients (
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Savage M.O.
      Insulin-like growth factors, nutrition and growth.
      ) and in neonatal calves (
      • Sauter S.N.
      • Ontsouka E.
      • Roffler B.
      • Zbinden Y.
      • Philipona C.
      • Pfaffl M.
      • Breier B.H.
      • Blum J.W.
      • Hammon H.M.
      Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves.
      ).
      Plasma concentration of IGFBP-4 increased from d 1 to 2 of life, as recently reported in calves (
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ), but decreased thereafter and remained more or less constant in all calves until d 63 of age, indicating no effect of ad libitum MR feeding on plasma IGFBP-4; this was in contrast to previous findings (
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ), but in agreement with findings of
      • Daniels K.M.
      • Hill S.R.
      • Knowlton K.F.
      • James R.E.
      • McGilliard M.L.
      • Akers R.M.
      Effects of milk replacer composition on selected blood metabolites and hormones in preweaned Holstein heifers.
      in calves. In addition, ad libitum MR feeding did not affect hepatic IGFBP4 gene expression, but IGFBP4 gene expression increased from d 50 to 80 of age in all calves. A corresponding increase of plasma IGFBP-4 during the last weeks of the study was only seen in Res calves. The IGFBP-4 is one of the smaller IGFBP, with a significant plasma concentration that is decreased by IGF-I and inhibits IGF-I action (
      • Clemmons D.R.
      Insulin-like growth factor binding proteins and their role in controlling IGF actions.
      ;
      • Duan C.
      Specifying the cellular responses to IGF signals: roles of IGF-binding proteins.
      ;
      • Firth S.M.
      • Baxter R.C.
      Cellular actions of the insulin-like growth factor binding proteins.
      ). Therefore, the simultaneous increase of IGFBP-4 and IGF-I in blood plasma at the end of the study in Res calves may indicate a modulation of the IGF-I activity by IGFBP-4. In addition, the MR feeding levels did not affect hepatic gene expression of IGF1R and INSR in the current trial.
      Ad libitum MR-fed calves supplemented with butyrate partly indicated a lower plasma IGF-I concentration. The body growth was not stimulated by butyrate, in contrast to the finding of
      • Górka P.
      • Kowalski Z.M.
      • Pietrzak P.
      • Kotunia A.
      • Jagusiak W.
      • Holst J.J.
      • Guilloteau P.
      • Zabielski R.
      Effect of method of delivery of sodium butyrate on rumen development in newborn calves.
      , who reported greater BW in calves fed MR supplemented with butyrate. A recent review of studies with butyrate treatment in preweaning calves indicated inconsistent effects on body growth when butyrate was supplied by MR (
      • Niwińska B.
      • Hanczakowska E.
      • Arciszewski M.B.
      • Klebaniuk R.
      Review: Exogenous butyrate: implications for the functional development of ruminal epithelium and calf performance.
      ). Reasons for these different findings are not obvious, but the age of the calf when starting the butyrate supplementation could be of importance (
      • Niwińska B.
      • Hanczakowska E.
      • Arciszewski M.B.
      • Klebaniuk R.
      Review: Exogenous butyrate: implications for the functional development of ruminal epithelium and calf performance.
      ). Hepatic gene expression of GHR1A and IGF1 did not respond to butyrate supplementation in a consistent manner, but IGFBP2 gene expression was elevated and INSR gene expression increased considerably in butyrate-treated groups at the end of the study. These changes on the parameters of the somatotropic axis due to butyrate supplementation may suggest that nutrient supply in butyrate-treated calves was impaired (
      • Thissen J.P.
      • Ketelslegers J.M.
      • Underwood L.E.
      Nutritional regulation of the insulin-like growth factors.
      ;
      • Breier B.H.
      • Oliver M.H.
      • Gallaher B.W.
      ;
      • Renaville R.
      • Hammadi M.
      • Portetelle D.
      Role of the somatotropic axis in the mammalian metabolism.
      ). Reasons for these findings are rather speculative; because butyrate treatment did not result in elevated plasma butyrate concentration in these calves (
      • Frieten D.
      • Gerbert C.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Kanitz E.
      • Weitzel J.M.
      • Hammon H.M.
      Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.
      ), a direct effect of butyrate on the GH-IGF-axis is not likely, as described in previous studies (
      • Tsubaki J.
      • Choi W.K.
      • Ingermann A.R.
      • Twigg S.M.
      • Kim H.S.
      • Rosenfeld R.G.
      • Oh Y.
      Effects of sodium butyrate on expression of members of the IGF-binding protein superfamily in human mammary epithelial cells.
      ;
      • Miletta M.C.
      • Petkovic V.
      • Eblé A.
      • Ammann R.A.
      • Flück C.E.
      • Mullis P.-E.
      Butyrate increases intracellular calcium levels and enhances growth hormone release from rat anterior pituitary cells via the G-protein-coupled receptors GPR41 and 43.
      ). Studies in calves and heifers could not find a stimulating effect of butyrate on plasma GH and IGF-I concentrations (
      • Nosbush B.B.
      • Linn J.G.
      • Eisenbeisz W.A.
      • Wheaton J.E.
      • White M.E.
      Effect of concentrate source and amount in diets on plasma hormone concentrations of prepubertal heifers.
      ;
      • Kato S.
      • Sato K.
      • Chida H.
      • Roh S.G.
      • Ohwada S.
      • Sato S.
      • Guilloteau P.
      • Katoh K.
      Effects of Na-butyrate supplementation in milk formula on plasma concentrations of GH and insulin, and on rumen papilla development in calves.
      ). In agreement with the findings on the somatotropic axis, the results on growth performance in the current study do not support data from literature that propose an improved growth rate in calves after butyrate feeding (
      • Guilloteau P.
      • Martin L.
      • Eeckhaut V.
      • Ducatelle R.
      • Zabielski R.
      • van Immerseel F.
      From the gut to the peripheral tissues: The multiple effects of butyrate.
      ), but do support previous findings on the lack of a growth promoting effect due to butyrate feeding in calves (
      • Kato S.
      • Sato K.
      • Chida H.
      • Roh S.G.
      • Ohwada S.
      • Sato S.
      • Guilloteau P.
      • Katoh K.
      Effects of Na-butyrate supplementation in milk formula on plasma concentrations of GH and insulin, and on rumen papilla development in calves.
      ). However, intestinal mucosa growth was stimulated by butyrate supplementation in the male calves of the current study (
      • Gerbert C.
      • Frieten D.
      • Koch C.
      • Dusel G.
      • Eder K.
      • Zitnan R.
      • Hammon H.M.
      Impact of ad libitum milk feeding and butyrate supplementation on organ and epithelial growth in the gastrointestinal tract of dairy calves.
      ). Interestingly, this local growth-promoting effect in the small intestine did not cause an enhanced body growth in the calves of the present study.
      We found no sex effects on DMI in these calves, and sex effects on growth performance were of minor importance. The lack of sex effects on plasma IGF-I concentration fits into this context. The lack of sex effects on growth performance and plasma IGF-I was recently described in another study of our laboratory (
      • Schäff C.T.
      • Gruse J.
      • Maciej J.
      • Mielenz M.
      • Wirthgen E.
      • Hoeflich A.
      • Schmicke M.
      • Pfuhl R.
      • Jawor P.
      • Stefaniak T.
      • Hammon H.M.
      Effects of feeding milk replacer ad libitum or in restricted amounts for the first five weeks of life on the growth, metabolic adaptation, and immune status of newborn calves.
      ), but disagrees with earlier findings of sex effects on growth and plasma IGF-I in calves (
      • Kerr D.E.
      • Laarveld B.
      • Fehr M.I.
      • Manns J.G.
      Profiles of serum IGF-I concentrations in calves from birth to eighteen months of age and in cows throughout the lactation cycle.
      ;
      • Kertz A.F.
      • Reutzel L.F.
      • Barton B.A.
      • Ely R.L.
      Body weight, body condition score, and wither height of prepartum Holstein cows and birth weight and sex of calves by parity: a database and summary.
      ;
      • Egli C.P.
      • Blum J.W.
      Clinical, haematological, metabolic and endocrine traits during the first three months of life of suckling simmentaler calves held in a cow-calf operation.
      ). However, main sex effects on growth and plasma IGF-I as well as plasma IGFBP-3 may occur when calves are older than 12 wk (
      • Govoni K.E.
      • Hoagland T.A.
      • Zinn S.A.
      The ontogeny of the somatotropic axis in male and female Hereford calves from birth to one year of age.
      ). In this context, it was surprising to find a greater hepatic gene expression of IGF1, IGFBP1, and IGFBP3 in male than female calves, which corresponds to the overall picture of a greater somatotropic activity in males than in females (
      • Gatford K.L.
      • Egan A.R.
      • Clarke I.J.
      • Owens P.C.
      Sexual dimorphism of the somatotrophic axis.
      ). Female gonadal steroids, such as estrogen, inhibit gene expression of IGF1 and IGFBP in liver (
      • Leung K.-C.
      • Johannsson G.
      • Leong G.M.
      • Ho K.K.
      Estrogen regulation of growth hormone action.
      ) and may explain the lower hepatic gene expression of IGF1 and IGFBP1 and IGFBP3 in female calves; however, the interaction of gonadal sex steroids and the somatotropic axis might become more clear in older calves.

      CONCLUSIONS

      Feeding milk and MR ad libitum for the first 8 wk of life affected IGF-I and IGFBP plasma concentration and hepatic gene expression. These changes mirrored the greater growth rate in Adl calves during MR feeding. At the end of the study, the greater IGF-I and lower IGFBP-2 in blood plasma in Res calves corresponded with the greater ADG in these calves. Butyrate supplementation did not stimulate growth performance but partly depressed the IGF-I status in the calves. As a consequence, the combination of an ad libitum milk-feeding program and allocation of butyrate with the MR did not result in an additional stimulation of IGF-I and IGFBP in calves.

      ACKNOWLEDGMENTS

      We gratefully thank Claudia Reiko [Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany] and Christine and Patrick Höflich (Ligandis Gbr, Gülzow, Germany). For practical help throughout the study, we also thank the staff of the Educational and Research Centre for Animal Husbandry, Hofgut Neumuehle, and the team of Animal Nutrition and students at the University of Applied Sciences Bingen.

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