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Pre- and post-weaning injections of bovine somatotropin to optimize puberty achievement of Bos indicus beef heifers

Pre- and post-weaning injections of bovine somatotropin to optimize puberty achievement of Bos... Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Pre- and post-weaning injections of bovine somatotropin to optimize puberty achievement of Bos indicus beef heifers *2 # # P. Moriel , B. I. Cappellozza§, M. B. Piccolo*, R. F. Cooke‡, M. F. Miranda , L. F. D. Batista , R. S. # # # # Carvalho , E. A. Colombo , F. V. Santili , R. V. O. Filho , V. S. M. Ferreira†, and J. L. M. Vasconcelo s† *University of Florida, IFAS – Range Cattle Research & Education Center, Ona, FL 33865 §Elanco Animal Health, São Paulo 04703-002, Brazil ‡Department of Animal Science, Texas A&M University, College Station, TX 77843 † e a en o n al od c on, o a lo a e n e ity s , Botucatu 18168-000, Brazil #Department of Animal Production, Sao Paulo State University, Botucatu 18168-000, Brazil The study was funded by Elanco Animal Health (Brooklin, São Paulo, Brazil). Corresponding author: P. Moriel. Phone: (863) 735-1314, pmoriel@ufl.edu © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Abstract. The present study evaluated the growth and puberty attainment of Bos indicus heifers administered recombinant bovine ST (bST) or saline injections during pre- and/or post-weaning. On d 0, 177 suckling Nellore heifers were stratified by initial age and BW (80 ± 10 d; 97 ± 16 kg), and randomly assigned, in a 2 × 2 factorial design (n = 44 to 45 heifers/treatment), to receive s.c. injections of saline (5 0.75 mL 0.9% NaCl) or sometribove zinc ( os lac™; Elanco, G een eld, IN 6.1 ; 4 mg/kg of BW ) on d 0 and 10 (PRE) and/or d 167 and 177 (POS). All heifers were managed as a single group in Brachiaria decumbens pastures from d 0 until 24 d post-weaning (d 191), and then provided a corn silage-based TMR from d 191 to 380 to achieve 65 to 70% of mature BW at the end of the study (d 380). Heifer full BW was collected on d 0, 10, 167, 177, and monthly from d 191 to 380. Transrectal ultrasonography of ovaries was performed on d 1 and 10 of each month from d 229 to 380 to assess the percentage of pubertal heifers. Liver biopsies and blood samples from jugular vein were collected on d 0, 10, 167, 177, and 380. Additional blood samples were collected monthly from d 259 to 380 (n = 10 to 15 heifers/treatment). No interactions among day of the study, PRE, and POS injections of saline or bST were detected (P ≥ 0.11 ). Pre-weaning bST injections increased heifer ADG from d 0 to 10 and plasma IGF-1 on d 10 (P ≤ 0.03), did not impact ADG from d 0 to 177, plasma IGF-1 from d 259 to 380, and any liver gene mRNA expression (P ≥ 0.19), b tended to decrease ADG from d 191 to 380 (P = 0.07) and percentage of pubertal heifers on d 349 (P = 0.07), 359 (P = 0.002), and 380 (P = 0.0001) compared to saline injections. Post-weaning bST injections increased plasma IGF-1 on d 177 and overall liver mRNA expression of GHR-1A (P ≤ 0.05) , decreased plasma IGF-1 from d 259 to 380 (P = 0.03), tended to decrease liver mRNA expression of GHR- 1B on d 177 (P = 0.08), but did not impact ADG from d 167 to 177 and 191 to 380, and puberty attainment from d 229 to 380 (P ≥ 0.12) compared to saline injections. Thus, pre- and post-weaning injections of bST successfully increased heifer plasma IGF-1 concentrations 10 d after first injection. Post-weaning injections of bST had no impact on puberty attainment, whereas pre-weaning bST injections of bST impaired puberty attainment of Nellore beef heifers. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Key Words: beef heifers, Bos indicus, insulin-like growth factor 1, metabolic imprinting, puberty, somatotropin. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 INTRODUCTION Although post-weaning injections of bovine somatotropin (bST) hastened puberty attainment of Bos taurus heifers (Cooke et al., 2013), less emphasis has been placed on pre-weaning management strategies despite their greater impact on heifer puberty attainment compared to post-weaning management practices (Roberts et al., 2007). This greater impact of pre-weaning strategies may be attributed to metabolic imprinting, which is the concept that body physiological outcomes to early-life nutritional challenges can persist for long periods, even after the removal of such challenges (Lucas, 1991; Du et al., 2010). Beef heifers early-weaned at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar BW, but hastened puberty attainment than heifers weaned at 270 d of age (Moriel et al., 2014). In that study, heifer ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability on age at puberty (Moriel et al., 2014). Likewise, Piccolo et al. (2018) reported that 3 pre-weaning injections of bST (250 mg of sometribove zinc/heifer), administered every 14 d and starting at 132 d of age, hastened puberty attainment in Angus × Brahman beef heifers. Bos indicus beef heifers are known for achieving puberty at older ages than B. taurus-influenced heifers (Short et al., 1994). However, the impact of pre- and post-weaning injections of bST on attainment of puberty of B. indicus beef heifers is unknown. It was hypothesized that bST injections would enhance heifer growth and puberty attainment compared to saline injections, as observed by Piccolo et al. (2018). Moreover, heifer puberty achievement would be enhanced at greater magnitude by pre- vs. post-weaning injections of bST due to metabolic imprinting effects. Thus, this study evaluated the growth and puberty attainment of Nellore heifers administered injections of bST or saline solution during pre-weaning only, post-weaning only, or both phases. MATERIALS AND METHODS Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 The experiment described herein utilized heifers that remained at a commercial cow-calf operation (Sítio Boa Vista do Rio Claro, São Manuel, São Paulo Brazil) from February 2016 to March 2017. All animals were cared for by acceptable practices as outlined in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010). Animals and treatments Pre-weaning phase (d 0 to 167). On d 0, cow-calf pairs (n = 177; cow BW = 470 ± 57 kg; cow BCS = 2.9 ± 0.2, scale 1 to 9) of Nellore dams and their suckling Nellore heifers were stratified by heifer age and initial heifer BW (age = 80 ± 10 d; heifer BW = 97 ± 16 kg), and randomly assigned to 1 of 4 treatments, in a 2 × 2 factorial design (n = 44 to 45 heifers/treatment). Treatments were administered to heifers only and consisted of two injections of saline or bovine somatotropin (bST; Posilac™, Elanco, Greenfield, IN) administered 10 d apart during pre-weaning phase only (d 0 and 10; PRE), post-weaning phase only (d 167 and 177; POS), or during both phases (d 0, 10, 167, and 177). Hence, all treatment combinations included: pre- and post-weaning injections of saline (SAL-SAL); pre- and post-weaning injections of bST (BST-BST); pre-weaning injections of bST and post-weaning injections of saline (BST- SAL); and pre-weaning injections of saline and post-weaning injections of bST (SAL-BST). All injections were administered subcutaneously on the right side of the neck. Saline injections consisted of 5 mL of a 0.75 0.9% NaCl solution, whereas bST injections were administered at 6.14 mg/kg of BW . The bST dosage was selected according to Piccolo et al. (2018) who successfully reported an increase in plasma IGF-1 concentrations after similar dosage of bST without any detrimental effects to growth and physiological parameters of Brangus crossbred heifers. The number of injections was selected based on 2 criteria: (1) to increase the plasma IGF-1 concentrations for 20 to 28 d after the first bST injection during (PRE heifers) or not (POS heifers) the age window that heifers were susceptible to nutrition-induced impacts on puberty (70 to 180 d of age; Moriel et al., 2014); and (2) to minimize the number of animal handlings Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 and reduce labor. During the pre-weaning phase, all cow-calf pairs were managed as a single group, rotated monthly among 6 pastures of Brachiaria decumbens (54 ha/pasture), and provided free choice access to commercial trace mineral salt (Fosbovi Reprodução, DSM Produtos Nutricionais, São Paulo, Brazil; DM basis: 11.1% Ca, 9.0% P, 1.8% S, 14.1% Na, 60 mg/kg Co, 1,500 mg/kg Cu, 1,800 mg/kg Fe, 75 mg/kg I, 1,800 mg/kg Mn, 17 mg/kg Se, and 4,500 mg/kg Zn) from d 0 to 167. Post-weaning phase (d 167 to 380). Heifers were transferred into a single 40-ha Brachiaria decumbens pasture (50.0% TDN, 7.5% CP; DM basis) immediately after weaning (d 167). From d 167 to 191, heifers were provided free choice access to water, ground mombaça (Panicum maximum) grass silage (Table 1), and 1 g/kg of BW (as-fed) of a commercial mixture of trace minerals and vitamins (Fosbovinho Proteico ADE, DSM Produtos Nutricionais; 26.6% CP, 60.2% TDN, 4.9% Ca, 3.4% P, 1.7% S, 1.3% Na, 2.6 mg/kg Co, 250 mg/kg Cu, 20 mg/kg I, 500 mg/kg Mn, 2.5 mg/kg Se, 750 mg/kg Zn, 31,000, IU/kg vitamin A, 4,000 IU/kg vitamin D , and 525 IU/kg vitamin E). On d 191, heifers were transferred to a 6-ha Brachiaria decumbens pasture with free choice access to water. A TMR was formulated using NRC (2016) and provided in amounts to ensure that all heifers would achieve between 65 to 70% of mature BW on d 380 (assuming a mature BW of 450 kg). Hence, heifers were limit-fed a corn silage-based TMR at 2.39% of BW (DM basis) from d 191 to 380. The TMR consisted of 68.7% corn-silage and 31.3% soybean meal (DM basis). Heifers were provided free choice access to a commercial trace mineral and vitamin mixture (Bovipac Plus, MCassab, São Paulo, Brazil; DM basis: 19.1% Ca, 8.9% P, 2.5% S, 10.5% Na, 1,094 mg/kg Cu, 5,855 mg/kg Fe, 39 mg/kg I, 1,003 mg/kg Mn, 19 mg/kg Se, 2,625 mg/kg Zn, 206 IU/kg vitamin A, 58,800 IU/kg vitamin D , and 784 mg/kg vitamin E) from d 191 to 380. Sample and data collection Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Individual full BW was collected from all heifers at 0800 h on d 0, 10, 167, and 177, whereas individual BW and BCS of all cows were collected on d 0 and 167. Full post-weaning BW and BCS of heifers were obtained on d 229, 259, 289, 320, 349, and 380. From d 229 to 380, transrectal ultrasonography of ovaries (Probe UST 5561-7.5 MHz linear array transducer, Aloka Prosound 2, Corimetrics Medical Systems, Inc., Wallingford, CT) was performed in all heifers twice monthly (d 1 and 10 of each month), by the same trained veterinarian, to determine the percentage of pubertal heifers. Age at puberty was set as the day when corpus luteum (CL) was detected. Body weight at puberty attainment was determined using the monthly ADG (calculated using BW obtained on d 1 of each month), and initial and final 30-d BW measurements of the respective month when CL was detected [BW at puberty = initial BW of the respective month + (ADG of the respective month  number of days between the day at CL detection and initial BW collection)]. A sub-group of heifers were randomly selected on d 0, and used for collections of blood (n = 15 heifers/treatment) and liver samples (n = 10 heifers/treatment) on d 0, 10, 167, 177, and 380. Additional blood samples were collected from the same heifers on d 259, 289, 320, and 350. Blood samples were collected (10 mL) via jugular venipuncture in sodium-heparin (158 USP) containing tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), placed on ice immediately after collection, and then centrifuged at 1,200  g for 25 min at 4°C. Plasma was stored frozen at -20°C until later analysis of plasma concentrations of IGF-1. All liver samples were collected via needle biopsy, following the procedure described by Arthington and Corah (1995). Immediately following collection, 100 mg of wet liver tissue/heifer was stored into 1.5 mL of RNA stabilization solution (RNAlater, Ambion Inc., Austin, TX), kept on ice for 8 h, and stored at -20°C until later analyses of mRNA expression of cyclophilin, GH receptor 1A and 1B (GHR- 1A and GHR-1B), IGF-1, IGF binding protein 3 (IGFBP-3), and 40S ribosomal protein S9 (RSP9). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Laboratory analyses Hand-plucked samples of pastures, grass silage, trace mineral and vitamin mixtures, and TMR were collected monthly, and sent in duplicate to a commercial laboratory (3R LAB, Lavras, Minas Gerais, Brazil) for concentrations of DM (method 930.15; AOAC, 2006), CP (method 984.13; AOAC, 2006), TDN (Weiss et al., 1992), NEm, and NEg (NRC, 2000). Concentrations of NDF and ADF were determined using the method of Van Soest et al. (1991) adapted for an ANKOM 200 Fiber Analyzer (ANKOM Technology, Macedon, NY). Nutritional composition of pasture, grass silage, and TMR samples is shown in Table 1. Plasma concentrations of IGF-I were determined using a human-specific commercial ELISA kit (SG100; R&D Systems, Inc., Minneapolis, MN) with 100% cross-reactivity with bovine IGF-I and previously validated for bovine samples (Moriel et al., 2012). Inter- and intra-assay CV for IGF-1 assay were 1.31 and 2.65%, respectively. A detailed description of procedures for mRNA isolation and tissue gene expression was described by Cappellozza et al. (2014). Briefly, total RNA was extracted from liver tissue samples using the TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA). Extracted RNA was quantified via UV absorbance (UV Mini 1240; Shimadzu Scientific Instruments, Inc., Columbia, MD) at 260 nm, incubated (2.5 μg) a 37°C o 30 in n the presence of RNase-free (DNase; New England Biolabs Inc., Ipswich, MA), and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA). Real-time PCR was completed using the SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers (20 pM each) with the StepOne Real-time PCR system (Applied Biosystems). At the end of each real-time PCR, amplified products were subjected to a dissociation gradient (95°C for 15 s, 60°C for 30 s, and 95°C for 15 s) to verify the amplification of a single product by denaturation at the anticipated temperature. The amplified products were purified with the QIAquick PCR purification kit (Qiagen Inc., Valencia, CA) and sequenced at the Department of Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Animal Science from University of Florida to verify the specificity of amplification. All amplified products represented only the genes of interest. Primer sequence of target genes are shown in Table 2 and were validated by previous studies, except for GHR-1B, which was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer Express v.3.0.1 software (Applied Biosystems, Foster City, CA). Responses were quantified based on the threshold cycle (CT) and were normalized to the geometrical mean CT values of cyclophilin and RSP9 (ΔCT) exa ned n he sa e sa le and assessed at the same time as the targets. Within each target –ΔΔCT gene, results are expressed as relative fold change (2 ) using the average ΔCT of all samples (Ocón- Grove et al., 2008). Inter- and intra-assay CV for mRNA expression of IGF-1, IGFBP-3, GHR-1A, and GHR- 1B were 2.77 and 3.77%, 2.94 and 3.11%, 2.33 and 4.27%, and 2.59 and 3.89%, respectively (Bustin et al., 2009). Statistical analyses All data were analyzed as a 2 × 2 factorial design using SAS (SAS Institute Inc., Cary, NC, USA, version 9.4) with Satterthwaite approximation to determine the denominator degrees of freedom for the test of fixed effects. Heifer was the experimental unit, whereas heifer(PRE × POS) was included as random effect in all analyses. Growth performance data were analyzed using the MIXED procedure, whereas puberty attainment was analyzed using the GLIMMIX procedure. Heifer ADG, BW and age at puberty, and mature BW on d 380 were tested for fixed effects of pre-weaning injections (PRE), post- weaning injections (POS), and all resulting interactions. Heifer BW, BCS, plasma IGF-1, liver mRNA expression, and puberty attainment were analyzed as repeated measures and tested for fixed effects of PRE, POS, day of the study, and all resulting interactions using heifer(PRE × POS) as the subject. Heifer BW and age on d 0 were included as covariates in all analyses, but removed from the model if P > 0.10. Proper covariance structure for each statistical analysis was selected based on the lowest Akaike Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 information criterion. Compound symmetry covariance structure was used for statistical analyses of pre- and post-weaning heifer BW, cow BW and BCS, post-weaning heifer BCS, and liver mRNA expression of IGF-1 and IGFBP-3. Autoregressive 1 covariance structure was used for the statistical analyses of liver mRNA expression of GHR-1A and -1B, and puberty attainment. All results are reported as least-squares means. Data were separated using PDIFF if a significant F-test was detected. Significance was set at P ≤ 0.05, and tendencies at P > 0.05 and ≤ 0.10 . RESULTS Effects of PRE × POS × day and PRE × POS were not detected for any variable in the study (P ≥ 0.11), and hence, effects of PRE and POS treatments were reported separately. Heifer BW and age on d 0 did not differ among treatments (P ≥ 0.75). However, heifer BW and age on d 0 were included as covariates in the analyses of heifer BW and ADG from d 0 to 177 (P ≤ 0.001), except for ADG from d 0 and 177 that was covariate-adjusted to age on d 0 only (P = 0.04). Effects of PRE × day of the study were (P = 0.02) detected for heifer BW from d 0 to 177. However, BW on d 10, 167 and 177 did not differ between heifers administered pre-weaning injections of bST or saline (P ≥ 0.12 ; Table 3). Effects of POS × day of the study tended (P = 0.07) to be detected for heifer BW from d 0 to 177, which did not differ between saline and bST heifers on d 10 and 167 (P ≥ 0.20), but tended to be greater on d 177 following post-weaning injections of bST vs. saline (P = 0.10; Table 3). Pre-weaning injections of saline or bST did not affect heifer ADG from d 0 to 177 and 167 to 177 (P ≥ 0.22). However, heifer ADG from d 0 to 10 was greater for those administered pre-weaning injections of bST vs. saline (P = 0.03), whereas ADG from d 10 to 167 was greater for heifers administered pre-weaning injections of saline vs. bST (P = 0.05; Table 3). Heifer pre- or post-weaning injections of saline and bST did not affect cow BW, BCS, and BW and BCS change from d 0 to 167 (P ≥ 0.17; Table 3). Post-weaning injections of saline and bST did not affect heifer ADG from d 167 to 177, Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 and overall ADG from d 0 to 177 (P ≥ 0.12; Table 3). Effects of PRE and POS were not detected (P ≥ 0.31) for heifer ADG from d 177 to 191 (0.27, 0.26, 0.23, and 0.29 ± 0.057 kg/d for PRE-bST, PRE-saline, POS- bST, and POS-saline heifers, respectively). Heifer BW from d 191 to 380 was covariate-adjusted to heifer BW and age on d 0 (P ≤ 0.002), whereas overall ADG from d 191 to 380 was covariate-adjusted to heifer BW on d 0 only (P = 0.02). Body weight and age on d 0 were included as covariates in the analyses of BW and age at puberty (P ≤ 0.06). Effects of PRE × day of the study and POS × day of the study were detected (P ≤ 0.0 3) for heifer post- weaning BW. Heifers administered pre-weaning injections of bST were lighter on d 349 and 380 (P ≤ 0.04), and tended to be lighter on d 259 (P = 0.06) compared to heifers administered pre-weaning injections of saline (Table 4). Heifer BCS did not differ following pre- or post-weaning injections of saline and bST (P ≥ 0.11). Overall heifer ADG from d 191 to 380 tended to be greater (P = 0.07) for heifers administered pre-weaning injections of saline vs. bST, and did not differ (P = 0.12) between heifers administered post-weaning injections of saline vs. bST (Table 4). Heifers administered pre-weaning injections of saline had greater (P = 0.03) mature BW on d 380, but similar BW and age at puberty (P ≥ 0.11) compared to heifers administered pre-weaning injections of bST (Table 4). Mature BW on d 380, and BW and age at puberty did not differ between heifers given post-weaning injections of bST or saline (P ≥ 0.39; Table 4 ). Effects of PRE × day of the study and POS × day of the study were detected (P ≤ 0.01) for plasma concentrations of IGF-1 collected from d 0 to 177. Heifers administered pre-weaning injections of bST had greater plasma IGF-1 concentrations on d 10 compared to heifers administered pre-weaning injections of saline (P = 0.0001; Table 5). Heifers given post-weaning injections of bST had greater plasma IGF-1 concentrations on d 177 compared to saline (P = 0.005; Table 5). Interactions among day of the study, PRE, and POS injections were not detected (P ≥ 0.19) for plasma concentrations of IGF-1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 collected from d 259 to 380, except for main effects of POS and day of the study (P ≤ 0.03). Pre-weaning injections of bST or saline did not impact overall plasma IGF-1 concentrations (P = 0.19), whereas heifers administered post-weaning injections of bST had less overall plasma IGF-1 concentrations from d 259 to 380 compared to heifers administered post-weaning injections of saline (P = 0.03; Table 5). Interactions among day of the study, PRE, and POS, as well as, main effects of pre-weaning injections were not detected (P ≥ 0.14) for liver mRNA expression of GHR-1A. However, main effects of day of the study and post-weaning injections were detected (P ≤ 0.03). Liver mRNA expression of GHR- 1A did not differ between d 0 and 10 (1.39 and 1.28 ± 0.210-fold increase, respectively; P = 0.64), decreased on d 167 (0.86 ± 0.210-fold increase; P ≤ 0.01), achieved greatest values on d 177 (1.82 ± 0.210-fold increase, respectively; P ≤ 0.07), and returned to baseline levels on d 380 (1.37 ± 0.210-fold increase, respectively; P = 0.92). Overall liver mRNA expression of GHR-1A was greater for heifers administered post-weaning injections of bST vs. saline (P = 0.03; Table 6). Liver mRNA expression of GHR-1B and IGF-1 on d 0 was included as covariate (P ≤ 0.01) in the analyses of liver mRNA expression of GHR-1B and IGF-1, respectively. Effects of POS × day of the study (P = 0.02), but not PRE, POS, and PRE × day of the study (P ≥ 0.16), were detected for liver mRNA expression of GHR-1B. Heifers administered post-weaning injections of bST had greater (P = 0.02) mRNA expression of GHR-1B on d 167, but tended to have less (P = 0.08) liver GHR-1B mRNA expression on d 177 compared to heifers administered post-weaning injections of saline (Table 6). Interactions among day of the study, PRE, and POS, as well as, main effects of PRE and POS were not detected (P ≥ 0.12) for liver mRNA expression of IGF-1 (Table 6). However, effect of day of the study was detected (P < 0.0001) for liver mRNA expression of IGF-1, which was greatest on d 10 (2.55 ± 0.170-fold increase; P < 0.0001), decreased on d 167 and 177 (0.84 and 1.03 ± 0.170-fold increase; P ≤ 0.01), andwe re intermediate on d 380 (1.27 ± 0.170-fold increase, respectively). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Effects of PRE × day of the study, POS × day of the study, PRE, and POS were not detected (P ≥ 0.20) for liver mRNA expression of IGFBP-3 (Table 6). However, effect of day of the study was detected (P < 0.0001) for liver mRNA expression of IGFBP-3, which was greatest on d 10 (2.22 ± 0.209-fold increase; P ≤ 0.06), decreased on d 0, 167, and 380 (0.95, 1.12, and 1.10 ± 0.209-fold increase, respectively), and were intermediate on d 177 (1.27 ± 0.170-fold increase, respectively; P ≤ 0.08). A tendency for effects of PRE × day of the study (P = 0.09), but not POS × day of the study (P = 0.18), was detected for heifer puberty attainment. A greater percentage of heifers administered pre- weaning injections of saline attained puberty on d 349 (P = 0.07), 359 (P = 0.002), and 380 (P = 0.0001) compared to heifers administered pre-weaning injections of bST (Figure 1a). DISCUSSION Day and Anderson (1998) proposed that the period from birth to puberty in beef heifers could be divided into infantile, developmental, static, and peripubertal periods. During the developmental phase (2 to 6 mo of age), GnRH secretion and follicular growth increases, with LH secretion and number of ovarian follicles peaking at 3 to 4 mo of age. Enhancing heifer ADG and nutrient intake during this developmental phase led to hastened follicle growth (Gasser et al., 2006a,b) and puberty attainment of beef heifers (Moriel et al., 2014). The exact nutrition-mediated mechanisms involved in this early activation of the reproductive axis in beef heifers are unknown. However, circulating IGF-I can impact gonadotropin secretion and activity required for the first ovulation and subsequent puberty achievement in beef heifers by influencing hypothalamic-pituitary secretory activity (Butler and Smith, 1989; Schillo et al., 1992) and augmenting the effects of gonadotropins in ovarian follicular cells (Spicer and Echternkamp, 1995). In agreement, heifer ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability on age at puberty (Moriel et al., 2014). Thus, Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 metabolic imprinting may be explored by identifying strategies to increase heifer ADG and plasma IGF-1 during the developmental phase leading to optimized future reproductive performance. Systemic IGF-1 has been positively correlated with muscle skeletal growth (Jiang and Ge, 2014). Piccolo et al. (2018) reported that Brangus crossbred heifers administered pre-weaning bST injections (250 mg every 14 d from 132 to 174 d of age) had an 8.6 ng/mL increase in plasma IGF-1 concentrations and 7.2% increase on ADG from d 0 to 42, relative to first injection, but no differences on ADG from first injection to weaning (167-d period) compared to saline heifers. Likewise, pre-weaning bST injections in the present study increased plasma IGF-1 concentrations by 51.7 ng/mL and ADG from d 0 to 10 by 35%, but did not impact ADG from d 0 to 177. The increase in BW gain and circulating IGF-1 concentrations following bST injections varied from 0 to 45% compared to control vehicles (Dalke et al., 1992; Houseknecht et al., 1992), and several factors, such as plane of nutrition, age, and size of treated animals may explain this large variation (Rausch et al., 2002). Also, multiple mechanisms may be involved in post-bST BW gain including repartitioning of nutrients toward muscle rather than adipose tissue deposition (Breier, 1999), and enhanced long-bone growth (Buskirk et al., 1996), nitrogen retention (Eisemann et al., 1986), and circulating IGF-1-induced protein synthesis of muscle (Jiang and Gee, 2014) and non-carcass tissues (Early et al., 1990). Multiple 14-d apart administrations of bST, during the post-weaning phase, reduced subcutaneous fat thickness by 9.2% without impacting LM depth, marbling scores, and BW gain (Cooke et al., 2013). Although body composition was not evaluated in the present study, it is unlikely that only two 10-d apart injections of bST substantially affected body composition and nutrient requirements of heifers. Thus, our results perhaps indicate that the increment on bST-induced ADG from d 0 to 10 may be the result of increased feed intake and gut fill, as reported by Enright et al. (1990), or that muscle protein deposition from d 0 to 10 was not sufficient to impact heifer BW at weaning. Nevertheless, pre-weaning bST injections in the present study successfully Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 increased plasma IGF-1 concentrations and ADG of heifers during the developmental phase of the reproductive axis in beef heifers (Day and Anderson, 1998). In contrast, post-weaning bST injections increased plasma IGF-1 concentrations by 37.2 ng/mL, but did not impact ADG during the injection period (d 167 to 177). Other studies also demonstrated that post-weaning bST injections increased plasma IGF-1 concentrations (Cooke et al., 2013), but did not increase post-weaning ADG of Angus × Holstein administered 500 mg of bST every 14 d from 6 to 10 mo of age (Carstens et al., 1997) and Angus × Hereford heifers injected with 250 mg of bST every 14 d from 6 to 13 mo of age (Cooke et al., 2013). Body weight gain and circulating IGF-1 responses to bST are influenced by cattle age and nutritional status (Rausch et al., 2002; Radcliff et al., 2004). Cattle somatotropic axis is functional at birth (Granz et al., 1997), and the response to bST begins as early as 1 d of age (Govoni et al., 2004), gradually increasing as age increases (Velayudhan et al., 2007). Likewise, plasma IGF-1 concentrations following bST injection were greater for Holstein heifers gaining 1.2 vs. 0.8 kg/d (Radcliff et al., 2004). In the current study, heifers were weaned on d 167 and provided free choice access to grass silage and pastures of relatively poor nutritional composition, which resulted in BW loss from d 167 to 177 and reduced ADG from d 177 to 191 compared to the pre- weaning period. Hence, despite the more advanced age at the time of bST injections, the lack of positive impacts of post-weaning injections of bST on BW gain from d 167 to 177 and 177 to 191 may be related to the reduced magnitude of increase in plasma IGF-1 concentrations, which was likely suppressed by the weaning-induced physiological stress (Arthington et al., 2008) and the poorer nutritional status of heifers during post- vs. pre-weaning periods (Radcliff et al., 2004). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 The binding of GH to GHR-1A stimulates hepatic synthesis of IGF-1 (Smith et al., 2002), and is highly correlated with the hepatic mRNA expression of GHR-1A and IGF-1 (Lucy et al., 2001). Transcription of the growth hormone receptor (GHR) gene is initiated from multiple transcription start sites, generating GHR-1A, -1B, and -1C RN ha d e n h e un5 tran ′ slated region but still encode the same amino acid sequence (Jiang and Lucy, 2001). The GHR-1A mRNA is only expressed in the liver (Lucy et al., 1998), whereas GHR-1B and -1C mRNA are expressed in a wide array of tissues, including liver, skeletal muscle, adipose tissue, and mammary gland (Jiang et al., 1999; Jiang and Lucy, 2001). Hepatic synthesis of IGF-1 is regulated primarily at the transcriptional level (Thissen et al., 1994), and is the major source of circulating IGF-1 (Yakar et al., 1999), which is also responsible for stimulating the hepatic expression of IGFBP-3 mRNA (Thissen et al., 1994). Thus, an increased hepatic expression of GHR-1A mRNA enhances the capacity for GH binding (Lapierre et al., 1982) and the hepatic synthesis of IGF-1 (Radcliff et al., 2004). Nutrient intake and BW gain positively affects the hepatic abundance of GHR-1A, IGF-1, and IGFBP-3 (Thissen et al., 1994; Smith et al., 2002; Radcliff et al., 2004). Holstein heifers administered daily injections of bST (25 µg/kg of BW from 120 to 247 d of age, in average) had greater mRNA expression of IGF-1, but similar mRNA expression of GHR-1A and IGFBP-3 (Radcliff et al., 2004). Piccolo et al. (2018) demonstrated that 3 pre-weaning injections of bST did not impact liver mRNA expression of GHR-1A and IGFBP-3 throughout the study, but increased liver mRNA expression of GHR-1B and IGF-1 at approximately 220 d after the last pre-weaning injection of bST, suggestive of metabolic imprinting effects causing long-term changes to gene expression despite the similar nutritional status of those heifers. In contrast, pre-weaning injections of bST did not impact the mRNA expression of any gene measured in the present study, whereas post-weaning bST injections increased overall liver mRNA expression of GHR-1A and decreased liver GHR-1B mRNA on d 177. Several reasons may be responsible for this lack of effects of pre-weaning bST injections. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Sartori et al. (2010; 2016) reported that Bos indicus cattle naturally have greater circulating IGF-I concentrations compared to Bos taurus cohorts. Moreover, Mendonça et al. (2013) demonstrated that even under the same environment and diet, Bos taurus-influenced dairy cows have less circulating concentrations of IGF-I compared to Bos indicus cows, which might be related to the different organ sensitivity to IGF-1. Despite the lack of published evidence, it is possible that a breed × bST effect on liver gene mRNA expression exists, but further studies comparing effects of breed on physiological and growth responses to bST are warranted to confirm this rationale. Also, due to differences on nutritional status, liver mRNA expression of IGF-1 and GHR-1A of all heifers were greater on d 0 vs. 167 (data not shown). Therefore, liver mRNA expression of GHR-1A and IGF-1 were perhaps at maximum during pre- weaning phase, which may have prevented further increments on mRNA expression of these genes. Plasma IGF-1 concentrations increase after 3 d, peak at approximately 7 to 8 d, and gradually return to baseline levels starting 12 d post-injection of bST (Bilby et al., 1999, 2004). Hence, it is possible that the timing of liver sample collection was not optimal to detect the peak expression of liver mRNA of IGFBP- 3, IGF-1, GHR-1B and GHR-1A. The detection of greater plasma IGF-1 concentrations on d 10 and 177, but similar liver mRNA expression of IGF-1 on those days between bST vs. saline heifers supports this rationale. Nevertheless, the greater overall mRNA expression of GHR-1A following post-weaning bST injections provide evidence that even post-weaning injections of bST can cause metabolic imprinting effects and alter long-term gene expression despite the similar post-weaning nutritional status. The impact of bST injections on post-treatment circulating concentrations of IGF-1 and puberty attainment of beef heifers has been variable. Injections of bST (250 mg every 14 d from 120 to 232 d of age) did not impact post-treatment serum concentrations of IGF-1 and puberty attainment of Angus × Simmental crossbred heifers (Buskirk et al., 1996). Pre-weaning injections of bST (250 mg every 14 d from 132 to 174 d of age) hastened puberty attainment of Brangus heifers at the start of the breeding season compared to saline injections, despite their similar nutritional management, ADG, and BW during Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 breeding season (Piccolo et al., 2018). Post-weaning bST injections (250 mg of bST every 14 d from 6 to 13 mo of age) increased puberty attainment of Angus × Hereford heifers at the start of breeding season (Cooke et al., 2013), but had no impact on puberty achievement of Angus heifers administered bST (350 mg every 14 d from 7 to 14.5 mo of age) compared to vehicle-treated heifers (Hall et al., 1994). In the present study, pre-weaning injections of bST decreased post-weaning growth performance compared to saline injections, whereas post-weaning injections of bST did not impact post- weaning growth performance of heifers. Age at puberty in cattle typically decreases as BW gain increases (Schillo et al., 1992). Hence, the similar post-weaning ADG likely explains the similar puberty achievement of heifers given post-weaning injections of bST and saline, whereas the decreased puberty attainment of heifers administered pre-weaning injections of bST vs. saline may be at least partially explained by the observed differences in post-weaning growth rate and BW. The reason for the decreased post-weaning growth performance of heifers following pre-weaning bST injections is unknown. Moriel et al. (2014) observed that during similar nutritional management, liver mRNA expression of IGF-1 and puberty attainment at start of breeding season were greater for heifers early- weaned at 60 d of age and placed on high-concentrate diets until 150 d of age compared to heifers normally weaned at 270 d of age, suggestive of metabolic imprinting effects. Hence, it was expected that the long-term post-weaning plasma concentrations of IGF-1 would be positively affected by pre- rather than post-weaning injections of bST due to the greater susceptibility of younger animals to metabolic imprinting effects. However, overall plasma IGF-1 concentrations collected from d 259 to 380 was decreased by post-weaning injections of bST, and not impacted by pre-weaning injections of bST. Regardless of the reasons for such contradiction, these results reinforce our rationale that the timing of bST injecting can have profound and diverse long-term impacts on growth, reproductive, and physiological parameters of beef heifers. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Additional factors beyond BW gain may have contributed to the variable responses on heifer growth performance and puberty achievement following bST treatment, including body composition, timing of injections, duration of injections, dosage, breed, and potentially the interactions among all of these factors. Although bST injections may stimulate lipolytic activity in adipose tissue (Lanna et al., 1995) and reduce backfat thickness in cattle (Vesteergard et al., 1993; Cooke et al., 2013), neither pre- weaning nor post-weaning injections of bST altered heifer BCS throughout the post-weaning phase, indicating that the pre-weaning bST-induced reduction in post-weaning ADG was likely not be attributed to altered body composition and nutrient requirements. One could argue that the period duration of bST injections (2 injections 10-d apart) may have been insufficient to impact puberty attainment of heifers. This rationale supports the observed results of post-weaning, but not the pre-weaning bST- induced effects on puberty attainment. Despite the lack of evidence to support this rationale, potential candidates are the effects of breed or the interaction between breed and bST dosage. The bST dosage 0.75 used in the present study (6.14 mg/kg ) was obtained from previous work that successfully hastened percentage of pubertal heifers at start of breeding season following pre-weaning bST administration (Piccolo et al., 2018). However, in that study, pre-weaning plasma IGF-1 concentrations increased by 8.6 ng/mL after bST injections (94.8 and 103.4 ng/mL for saline and bST heifers, respectively). In the present study, baseline plasma concentrations of IGF-1 were 186 and 88.2 ng/mL on d 0 and 167, respectively, and increased by 25.4 and 9.9 ng/mL on d 10 and 177, respectively, following first bST injection. Brahman (Simpson et al., 1997) and Nellore cows (Roberts et al., 2005) have greater circulating concentrations of IGF-1 than Angus cattle. Furthermore, circulating concentrations of IGF-1 in straight- bred Brahman cows were greater than in the crossbred cows (Roberts et al., 2005), which likely explains the greater baseline IGF-1 levels observed in the present study compared to Piccolo et al. (2018). However, it is also possible that the greater increment on plasma IGF-1 concentrations following bST injection, in combination with interval between bST injection, were detrimental to the development of Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 the reproductive axis. Further studies investigating the effects of breed on ovarian activity and gene expression in reproductive tissue organs and brain, following bST injections, are warranted to confirm this rationale. In conclusion, pre-weaning injections of bST, administered 10-d apart starting at approximately 2 to 3 mo of age, successfully increased pre-weaning plasma concentrations of IGF-1 and growth performance of Nellore heifers during the period of bST treatment. 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Roberts, A. J., S. I. Paisley, T. W. Geary, E. E. Grings, R. C. Waterman, and M. D. MacNeil. 2007. Effects of restricted feeding of beef heifers during the postweaning period on growth, ef ciency, and ultrasound carcass characteristics. J. Anim. Sci. 85:2740-2745. Sartori, R., M. R. Bastos, P. S. Baruselli, L. U. Gimenes, R. L. Ereno, and C. M. Barros. 2010. Physiological differences and implications to reproductive management of Bos taurus and Bos indicus cattle in a tropical environment. In: Reproduction in Domestic Ruminants VII. Ed. Nottingham University Press, UK. Pp. 357-375. Sartori, R., L. U, Gimenes, P. L. J. Monteiro Jr., L. F. Melo, P. S. Baruselli, and M. R. Bastos. 2016. Metabolic and endocrine differences between Bos taurus and Bos indicus females that impact the interaction of nutrition and reproduction. Theriogenology. 86:32-40. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Schillo, K. K., J. B. Hall, and S. M. Hileman. 1992. Effects of nutrition and season on the onset of puberty in the beef heifer. J. Anim. Sci. 70:3994–4005. Short, R. E., R. B. Staigmiller, R. A. Bellows, and R. C. Greer. 1994. Breeding heifers at one year of age: Biological and economical considerations. In: M. J. Fields and R. S. Sand(ed.) Factors Affecting Calf Crop. p. 55-68. CRC Press, Boca Raton, FL. Simpson, R. B., C. C. Chase, Jr., L. J. Spicer, J. A. Carroll, A. C. Hammond, and T. H. Welsh, Jr. 1997. Effect of exogenous estradiol on plasma concentrations of somatotropin, insulin-like growth factor-I, insulin-like growth factor binding protein activity, and metabolites in ovariectomized Angus and Brahman cows. Domest. Anim. Endocrinol. 14:367–380. Smith, J. M., M. E. Van Amburgh, M. C. Diaz, M. C. Lucy, and D. E. Bauman. 2002. Effect of nutrient intake on the development of the somatotropic axis and its responsiveness to GH in Holstein bull calves. J. Anim. Sci. 80:1528–1537. Spicer, L. J., and S. E. Echternkamp. 1995. The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals. Domest. Anim. Endocrinol. 12:223–245. Thissen, J. P., J. M. Ketelslegers, and L. E. Underwood. 1994. Nutritional regulation of the insulin-like growth factors. Endocr. Rev. 15:80–101. Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597. Velayudhan, B. T., K. E. Govoni, T. A. Hoagland, and S. A. Zinn. 2007. Growth rate and changes of the somatotropic axis in beef cattle administered exogenous bovine somatotropin beginning at two hundred, two hundred fifty, and three hundred days of age. J. Anim. Sci. 85:2866–2872. Vestergaard, M., K. Sejrsen, J. Foldager, S. Klastrup, and D. E. Bauman. 1993. The effect of bovine growth hormone on growth, carcass composition and meat quality of dairy heifers. Acta Agric. Scand., Sect. A 43:165–172. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Wagner, J. J., K. S. Lusby, J. W. Oltjen, J. Rakestraw, R. P. Wettemann, and L. E. Walters. 1988. Carcass composition in mature Hereford cows: Estimation and effect on daily metabolizable energy requirement during winter. J. Anim. Sci. 66:603–612. Weiss, W. P., H. R. Conrad, and N. R. St. Pierre. 1992. A theoretically-based model for predicting total digestible nutrient values of forages and concentrates. Anim. Feed Sci. Technol. 39:95-110. Wiltbank J. N., K. E. Gregory, L. A. Swiger, J. E. Ingalls, J. A. Rothlisberger, and R. M. Koch. 1966. Effects of heterosis on age and weight at puberty in beef heifers. J. Anim. Sci. 25:744-751. Yakar, S., J. Liu, B. Stannard, A. Butler, D. Accili, B. Sauer, and D. LeRoith. 1999. Normal growth and development in the absence of hepatic insulin-like growth factor. Proc. Natl. Acad. Sci. USA 96:7324–7329. Ye, J., G. Coulouris, I. Zaretskaya, I. Cutcutache, S. Rozen, and T. L. Madden. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinf. 13:134. doi:10.1186/1471-2105-13-134. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 1. Average nutritional composition (DM basis) of pastures, grass silage, and corn silage-based TMR provided to heifers throughout the study. Pre-weaning Post-weaning Grass Item TMR 1 1 2 pasture pasture silage DM, % 39.0 22.7 31.0 50.8 CP, % 9.8 15.8 5.9 20.9 NDF, % 68.3 56.6 78.0 37.1 ADF, % 40.8 29.3 48.6 23.3 TDN , % 54.6 60.6 45.9 70.0 NEm , Mcal/kg 1.13 1.38 0.76 1.69 NEg , Mcal/kg 0.57 0.80 0.22 1.08 Ca, % 0.32 0.62 0.49 0.29 K, % 1.65 2.76 1.05 1.25 Mg, % 0.25 0.37 0.27 0.24 P, % 0.25 0.35 0.15 0.36 S, % 0.17 0.24 0.19 0.40 Samples of brachiaria pastures collected monthly during pre- (d 0 to 167) and post-weaning phases (d 192 to 380). Samples of mombaça grass silage collected from d 167 to 191. Samples of TMR collected monthly from d 191 to 380. Total mixed ration consisted of 68.7% corn- silage and 31.3% soybean meal (DM basis). Calculated as described by Weiss et al. (1992). Calculated using the equations proposed by the NRC (2000). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 2. Primer sequences and accession number for all gene transcripts analyzed by quantitative real- time PCR . Target gene Primer sequence Accession no. Cyclophilin Forward 5'-GGTACTGGTGGCAA GTCCAT-3' NM_178320.2 Reverse 5'-GCCATCCAACCACTCAGTCT-3' IGF-1 Forward 5'-CTCCTCGCATCT CTTCTATCT-3' NM_001077828 Reverse 5'-ACTCATCCACGATTCCTGTCT-3' IGFBP-3 Forward 5'-AATGGCAGTGA GTCGGAAGA-3' NM_174556.1 Reverse 5'-AAGTTCTGGGTGTCTGTGCT-3' GHR-1A Forward 5'-CCAGCCTCTGTT TCAGGAGTGT-3' AY74 8827 Reverse 5'-TGCCACTGCCAAGGTCAAC-3' GHR-1B Forward 5'-AGCCTGGAGGA ACCATACGA-3' - Reverse 5'-TAGCCCCATCTGTCCAGTGA-3' RSP9 Forward 5'-CCTCGACCAA GAGCTGAAG-3' DT86 0044 Reverse 5'-CCTCCAGACCTCACGTTTGTTC-3' Primer sequence for IGF-1, IGFBP-3, and GHR-1A were obtained from Coyne et al. (2011 ), whereas those for cyclophilin and RSP9 were obtained from Cooke et al. (2008) and Janovick-Guretzky et al. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 (2007), respectively. Primer sequence for GHR-1B was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer Express v. 3.0.1 software (Applied Biosystems, Foster City, CA). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 3. Growth performance (d 0 to 177) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Post-weaning Pre-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Heifer BW , kg d 10 109 11 1 1.35 0.21 0.02 11 0 10 9 1.36 0.48 0.07 d 167 187 185 1.35 0.24 185 187 1.36 0.20 d 177 187 184 1.35 0.12 184 187 1.36 0.10 Heifer ADG , kg/d d 0 to 10 0.48 0.65 0.06 1 0.03 0.62 0.51 0.061 0.14 d 10 to 167 0.47 0.45 0.008 0.05 0.45 0.47 0.009 0.12 d 167 to 177 -0.01 -0.07 0.068 0.54 -0.06 -0.01 0.069 0.59 d 0 to 177 0.47 0.45 0.009 0.24 0.45 0.46 0.009 0.28 Cow BW, kg d 0 487 48 2 5.68 0.61 0.22 48 3 48 6 5.7 0.63 0.41 d 167 465 456 5.68 0.25 460 461 5.7 0.95 Cow BCS Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 0 2.89 2.87 0.020 0.47 0.80 2.88 2.88 0.020 0.89 0.95 d 167 2.83 2.82 0.020 0.63 2.83 2.82 0.020 0.93 BW change, kg -21.5 -27.1 2.84 0.17 -22.9 -25.6 2.84 0.50 BCS change -0.06 -0.05 0.019 0.87 -0.06 -0.05 0.019 0.84 1 0.75 The study began on d 0 and heifer calves were weaned on d 167. Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck and 10 d apart during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). P-value for comparison of treatment within day. Covariate-adjusted to BW and age on d 0 (P ≤ 0.001), except for ADG from d 0 and 177 that was covariate-adjusted to age on d 0 only (P = 0.04). Table 4. Post-weaning growth performance (d 191 to 380) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Heifer BW , kg d 191 190 18 8 3.06 0.56 0.02 18 8 19 0 3.11 0.91 0.03 d 229 197 192 3.06 0.25 194 195 3.11 0.99 d 259 220 212 3.06 0.06 217 215 3.11 0.46 d 289 240 235 3.06 0.15 240 235 3.11 0.20 d 320 266 260 3.06 0.13 265 262 3.11 0.33 d 349 291 283 3.06 0.03 289 286 3.11 0.45 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 380 317 309 3.06 0.04 316 310 3.11 0.24 3,4 Heifer BCS d 229 5.12 5.08 0.046 0.51 0.19 5.12 5.10 0.046 0.88 0.27 d 259 5.10 5.02 0.046 0.20 5.08 5.02 0.046 0.48 d 289 5.46 5.50 0.046 0.45 5.54 5.40 0.046 0.23 d 320 5.86 5.90 0.046 0.66 5.92 5.84 0.046 0.15 d 349 5.96 5.86 0.046 0.16 5.92 5.90 0.046 0.62 d 380 6.32 6.26 0.046 0.42 6.36 6.24 0.046 0.08 ADG d 191 to 380 , kg/d 0.83 0.80 0.014 0.07 - 0.83 0.80 0.014 0.12 - Mature BW on d 380 , % 70.7 68.8 0.63 0.03 - 70.1 69.4 0.65 0.48 - BW at puberty , kg 311 306 3.4 0.30 - 311 307 3.4 0.39 - Age at puberty , d 453 458 3.2 0.11 - 456 455 3.2 0.89 - 1 0.75 Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value for comparison of treatment within day. Covariate-adjusted to BW and age on d 0 (P ≤ 0.002), except for overall ADG from d 191 to 380 that was covariate-adjusted to BW on d 0 only (P = 0.02). Using a 1 to 9 scale according to Wagner et al. (1988). Assuming a mature cow BW of 450 kg. Body weight at puberty attainment was determined using the ADG, and initial and final BW measurements of the respective month when CL was detected [BW at puberty = initial BW of the respective month + (ADG of the respective month  number of days between the day at CL detection and initial BW collection)]. Age at puberty was set as the day when corpus luteum (CL) was detected (d 1 or 10 of each month from d 192 to 380). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 5. Pre- and post-weaning plasma IGF-1 concentrations of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Plasma IGF-1, ng/mL d 0 193.0 186.0 9.3 0.59 0.00 1 198.9 179.9 9.3 0.15 0.01 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 10 159.7 211.4 9.3 0.0001 186.8 184.4 9.3 0.85 d 167 82.4 82.2 9.3 0.99 76.4 88.2 9.3 0.37 d 177 83.0 76.1 9.3 0.60 60.9 98.1 9.3 0.005 Overall (d 259 to 380) 197.7 210.8 7.1 0.19 - 216.0 192.5 7.1 0.03 - 1 0.75 Injections of saline (5 mL 0.9 % NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value for comparison of treatment within day. Average plasma IGF-1 concentrations of blood samples collected on d 259, 289, 319, 349, and 380. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 6. Pre- and post-weaning liver mRNA expression (fold increase ) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 3 3 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day -- Fold increase -- -- Fold increase -- GHR-1A 1.37 1.32 0.17 4 0.78 0.98 1.17 1.52 0.17 1 0.03 0.87 IGF-1 1.37 1.48 0.129 0.53 0.86 1.37 1.48 0.131 0.52 0.12 IGFBP-3 1.50 1.31 0.122 0.29 0.64 1.46 1.36 0.122 0.54 0.69 GHR-1B d 10 1.75 1.64 0.17 9 0.71 0.46 1.56 1.84 0.22 2 0.34 0.02 d 167 1.14 1.12 0.179 0.93 0.77 1.49 0.222 0.02 d 177 1.36 0.79 0.179 0.07 1.35 0.81 0.222 0.08 d 380 1.31 1.31 0.179 0.99 1.14 1.47 0.222 0.28 Responses were quantified based on the threshold cycle (CT) and were normalized to average CT of cyclophilin and RSP9 (ΔCT) examined in –ΔΔCT the same sample and assessed at the same time as the targets. Within each target gene, results are expressed as relative fold change (2 ) using the average ΔCT of all samples as reference, as described by Ocón-Grove et al. (2008). 2 0.75 Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value represents the main effects of treatment for overall mRNA expression of GHR-1A, IGF-1, and IGFBP-3 obtained on d 0, 10, 167, 177 and 380. For mRNA expression of GHR-1B, the P-value represents the comparison of treatment within day for. Liver mRNA expression of IGF-1 and GHR-1B were covariate-adjusted to respective mRNA expression on d 0 (P ≤ 0.02), whereas liver mRNA expression of GHR-1A was covariate-adjusted to BW on d 0 only (P = 0.05). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1. Puberty attainment of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10; Figure 1a) or post-weaning phases (d 167 and 177; Figure 1b). Effects of PRE × day of the study, but not POS × day of the study (P = 0.22), tended to be detected (P = 0.09) for puberty attainment during the study. *P ≤ 0.05, †0.05 < P ≤ 0.10. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1a PRE-Saline PRE-bST 40 * 229 239 259 269 289 299 320 349 359 380 Day of the study Accepted Manuscript Pubertal heifers, % of total heifers Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1b POS-Saline POS-bST 229 239 259 269 289 299 320 349 359 380 Day of the study Accepted Manuscript Pubertal heifers, % of total heifers http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Pre- and post-weaning injections of bovine somatotropin to optimize puberty achievement of Bos indicus beef heifers

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Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Pre- and post-weaning injections of bovine somatotropin to optimize puberty achievement of Bos indicus beef heifers *2 # # P. Moriel , B. I. Cappellozza§, M. B. Piccolo*, R. F. Cooke‡, M. F. Miranda , L. F. D. Batista , R. S. # # # # Carvalho , E. A. Colombo , F. V. Santili , R. V. O. Filho , V. S. M. Ferreira†, and J. L. M. Vasconcelo s† *University of Florida, IFAS – Range Cattle Research & Education Center, Ona, FL 33865 §Elanco Animal Health, São Paulo 04703-002, Brazil ‡Department of Animal Science, Texas A&M University, College Station, TX 77843 † e a en o n al od c on, o a lo a e n e ity s , Botucatu 18168-000, Brazil #Department of Animal Production, Sao Paulo State University, Botucatu 18168-000, Brazil The study was funded by Elanco Animal Health (Brooklin, São Paulo, Brazil). Corresponding author: P. Moriel. Phone: (863) 735-1314, pmoriel@ufl.edu © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Abstract. The present study evaluated the growth and puberty attainment of Bos indicus heifers administered recombinant bovine ST (bST) or saline injections during pre- and/or post-weaning. On d 0, 177 suckling Nellore heifers were stratified by initial age and BW (80 ± 10 d; 97 ± 16 kg), and randomly assigned, in a 2 × 2 factorial design (n = 44 to 45 heifers/treatment), to receive s.c. injections of saline (5 0.75 mL 0.9% NaCl) or sometribove zinc ( os lac™; Elanco, G een eld, IN 6.1 ; 4 mg/kg of BW ) on d 0 and 10 (PRE) and/or d 167 and 177 (POS). All heifers were managed as a single group in Brachiaria decumbens pastures from d 0 until 24 d post-weaning (d 191), and then provided a corn silage-based TMR from d 191 to 380 to achieve 65 to 70% of mature BW at the end of the study (d 380). Heifer full BW was collected on d 0, 10, 167, 177, and monthly from d 191 to 380. Transrectal ultrasonography of ovaries was performed on d 1 and 10 of each month from d 229 to 380 to assess the percentage of pubertal heifers. Liver biopsies and blood samples from jugular vein were collected on d 0, 10, 167, 177, and 380. Additional blood samples were collected monthly from d 259 to 380 (n = 10 to 15 heifers/treatment). No interactions among day of the study, PRE, and POS injections of saline or bST were detected (P ≥ 0.11 ). Pre-weaning bST injections increased heifer ADG from d 0 to 10 and plasma IGF-1 on d 10 (P ≤ 0.03), did not impact ADG from d 0 to 177, plasma IGF-1 from d 259 to 380, and any liver gene mRNA expression (P ≥ 0.19), b tended to decrease ADG from d 191 to 380 (P = 0.07) and percentage of pubertal heifers on d 349 (P = 0.07), 359 (P = 0.002), and 380 (P = 0.0001) compared to saline injections. Post-weaning bST injections increased plasma IGF-1 on d 177 and overall liver mRNA expression of GHR-1A (P ≤ 0.05) , decreased plasma IGF-1 from d 259 to 380 (P = 0.03), tended to decrease liver mRNA expression of GHR- 1B on d 177 (P = 0.08), but did not impact ADG from d 167 to 177 and 191 to 380, and puberty attainment from d 229 to 380 (P ≥ 0.12) compared to saline injections. Thus, pre- and post-weaning injections of bST successfully increased heifer plasma IGF-1 concentrations 10 d after first injection. Post-weaning injections of bST had no impact on puberty attainment, whereas pre-weaning bST injections of bST impaired puberty attainment of Nellore beef heifers. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Key Words: beef heifers, Bos indicus, insulin-like growth factor 1, metabolic imprinting, puberty, somatotropin. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 INTRODUCTION Although post-weaning injections of bovine somatotropin (bST) hastened puberty attainment of Bos taurus heifers (Cooke et al., 2013), less emphasis has been placed on pre-weaning management strategies despite their greater impact on heifer puberty attainment compared to post-weaning management practices (Roberts et al., 2007). This greater impact of pre-weaning strategies may be attributed to metabolic imprinting, which is the concept that body physiological outcomes to early-life nutritional challenges can persist for long periods, even after the removal of such challenges (Lucas, 1991; Du et al., 2010). Beef heifers early-weaned at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar BW, but hastened puberty attainment than heifers weaned at 270 d of age (Moriel et al., 2014). In that study, heifer ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability on age at puberty (Moriel et al., 2014). Likewise, Piccolo et al. (2018) reported that 3 pre-weaning injections of bST (250 mg of sometribove zinc/heifer), administered every 14 d and starting at 132 d of age, hastened puberty attainment in Angus × Brahman beef heifers. Bos indicus beef heifers are known for achieving puberty at older ages than B. taurus-influenced heifers (Short et al., 1994). However, the impact of pre- and post-weaning injections of bST on attainment of puberty of B. indicus beef heifers is unknown. It was hypothesized that bST injections would enhance heifer growth and puberty attainment compared to saline injections, as observed by Piccolo et al. (2018). Moreover, heifer puberty achievement would be enhanced at greater magnitude by pre- vs. post-weaning injections of bST due to metabolic imprinting effects. Thus, this study evaluated the growth and puberty attainment of Nellore heifers administered injections of bST or saline solution during pre-weaning only, post-weaning only, or both phases. MATERIALS AND METHODS Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 The experiment described herein utilized heifers that remained at a commercial cow-calf operation (Sítio Boa Vista do Rio Claro, São Manuel, São Paulo Brazil) from February 2016 to March 2017. All animals were cared for by acceptable practices as outlined in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010). Animals and treatments Pre-weaning phase (d 0 to 167). On d 0, cow-calf pairs (n = 177; cow BW = 470 ± 57 kg; cow BCS = 2.9 ± 0.2, scale 1 to 9) of Nellore dams and their suckling Nellore heifers were stratified by heifer age and initial heifer BW (age = 80 ± 10 d; heifer BW = 97 ± 16 kg), and randomly assigned to 1 of 4 treatments, in a 2 × 2 factorial design (n = 44 to 45 heifers/treatment). Treatments were administered to heifers only and consisted of two injections of saline or bovine somatotropin (bST; Posilac™, Elanco, Greenfield, IN) administered 10 d apart during pre-weaning phase only (d 0 and 10; PRE), post-weaning phase only (d 167 and 177; POS), or during both phases (d 0, 10, 167, and 177). Hence, all treatment combinations included: pre- and post-weaning injections of saline (SAL-SAL); pre- and post-weaning injections of bST (BST-BST); pre-weaning injections of bST and post-weaning injections of saline (BST- SAL); and pre-weaning injections of saline and post-weaning injections of bST (SAL-BST). All injections were administered subcutaneously on the right side of the neck. Saline injections consisted of 5 mL of a 0.75 0.9% NaCl solution, whereas bST injections were administered at 6.14 mg/kg of BW . The bST dosage was selected according to Piccolo et al. (2018) who successfully reported an increase in plasma IGF-1 concentrations after similar dosage of bST without any detrimental effects to growth and physiological parameters of Brangus crossbred heifers. The number of injections was selected based on 2 criteria: (1) to increase the plasma IGF-1 concentrations for 20 to 28 d after the first bST injection during (PRE heifers) or not (POS heifers) the age window that heifers were susceptible to nutrition-induced impacts on puberty (70 to 180 d of age; Moriel et al., 2014); and (2) to minimize the number of animal handlings Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 and reduce labor. During the pre-weaning phase, all cow-calf pairs were managed as a single group, rotated monthly among 6 pastures of Brachiaria decumbens (54 ha/pasture), and provided free choice access to commercial trace mineral salt (Fosbovi Reprodução, DSM Produtos Nutricionais, São Paulo, Brazil; DM basis: 11.1% Ca, 9.0% P, 1.8% S, 14.1% Na, 60 mg/kg Co, 1,500 mg/kg Cu, 1,800 mg/kg Fe, 75 mg/kg I, 1,800 mg/kg Mn, 17 mg/kg Se, and 4,500 mg/kg Zn) from d 0 to 167. Post-weaning phase (d 167 to 380). Heifers were transferred into a single 40-ha Brachiaria decumbens pasture (50.0% TDN, 7.5% CP; DM basis) immediately after weaning (d 167). From d 167 to 191, heifers were provided free choice access to water, ground mombaça (Panicum maximum) grass silage (Table 1), and 1 g/kg of BW (as-fed) of a commercial mixture of trace minerals and vitamins (Fosbovinho Proteico ADE, DSM Produtos Nutricionais; 26.6% CP, 60.2% TDN, 4.9% Ca, 3.4% P, 1.7% S, 1.3% Na, 2.6 mg/kg Co, 250 mg/kg Cu, 20 mg/kg I, 500 mg/kg Mn, 2.5 mg/kg Se, 750 mg/kg Zn, 31,000, IU/kg vitamin A, 4,000 IU/kg vitamin D , and 525 IU/kg vitamin E). On d 191, heifers were transferred to a 6-ha Brachiaria decumbens pasture with free choice access to water. A TMR was formulated using NRC (2016) and provided in amounts to ensure that all heifers would achieve between 65 to 70% of mature BW on d 380 (assuming a mature BW of 450 kg). Hence, heifers were limit-fed a corn silage-based TMR at 2.39% of BW (DM basis) from d 191 to 380. The TMR consisted of 68.7% corn-silage and 31.3% soybean meal (DM basis). Heifers were provided free choice access to a commercial trace mineral and vitamin mixture (Bovipac Plus, MCassab, São Paulo, Brazil; DM basis: 19.1% Ca, 8.9% P, 2.5% S, 10.5% Na, 1,094 mg/kg Cu, 5,855 mg/kg Fe, 39 mg/kg I, 1,003 mg/kg Mn, 19 mg/kg Se, 2,625 mg/kg Zn, 206 IU/kg vitamin A, 58,800 IU/kg vitamin D , and 784 mg/kg vitamin E) from d 191 to 380. Sample and data collection Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Individual full BW was collected from all heifers at 0800 h on d 0, 10, 167, and 177, whereas individual BW and BCS of all cows were collected on d 0 and 167. Full post-weaning BW and BCS of heifers were obtained on d 229, 259, 289, 320, 349, and 380. From d 229 to 380, transrectal ultrasonography of ovaries (Probe UST 5561-7.5 MHz linear array transducer, Aloka Prosound 2, Corimetrics Medical Systems, Inc., Wallingford, CT) was performed in all heifers twice monthly (d 1 and 10 of each month), by the same trained veterinarian, to determine the percentage of pubertal heifers. Age at puberty was set as the day when corpus luteum (CL) was detected. Body weight at puberty attainment was determined using the monthly ADG (calculated using BW obtained on d 1 of each month), and initial and final 30-d BW measurements of the respective month when CL was detected [BW at puberty = initial BW of the respective month + (ADG of the respective month  number of days between the day at CL detection and initial BW collection)]. A sub-group of heifers were randomly selected on d 0, and used for collections of blood (n = 15 heifers/treatment) and liver samples (n = 10 heifers/treatment) on d 0, 10, 167, 177, and 380. Additional blood samples were collected from the same heifers on d 259, 289, 320, and 350. Blood samples were collected (10 mL) via jugular venipuncture in sodium-heparin (158 USP) containing tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), placed on ice immediately after collection, and then centrifuged at 1,200  g for 25 min at 4°C. Plasma was stored frozen at -20°C until later analysis of plasma concentrations of IGF-1. All liver samples were collected via needle biopsy, following the procedure described by Arthington and Corah (1995). Immediately following collection, 100 mg of wet liver tissue/heifer was stored into 1.5 mL of RNA stabilization solution (RNAlater, Ambion Inc., Austin, TX), kept on ice for 8 h, and stored at -20°C until later analyses of mRNA expression of cyclophilin, GH receptor 1A and 1B (GHR- 1A and GHR-1B), IGF-1, IGF binding protein 3 (IGFBP-3), and 40S ribosomal protein S9 (RSP9). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Laboratory analyses Hand-plucked samples of pastures, grass silage, trace mineral and vitamin mixtures, and TMR were collected monthly, and sent in duplicate to a commercial laboratory (3R LAB, Lavras, Minas Gerais, Brazil) for concentrations of DM (method 930.15; AOAC, 2006), CP (method 984.13; AOAC, 2006), TDN (Weiss et al., 1992), NEm, and NEg (NRC, 2000). Concentrations of NDF and ADF were determined using the method of Van Soest et al. (1991) adapted for an ANKOM 200 Fiber Analyzer (ANKOM Technology, Macedon, NY). Nutritional composition of pasture, grass silage, and TMR samples is shown in Table 1. Plasma concentrations of IGF-I were determined using a human-specific commercial ELISA kit (SG100; R&D Systems, Inc., Minneapolis, MN) with 100% cross-reactivity with bovine IGF-I and previously validated for bovine samples (Moriel et al., 2012). Inter- and intra-assay CV for IGF-1 assay were 1.31 and 2.65%, respectively. A detailed description of procedures for mRNA isolation and tissue gene expression was described by Cappellozza et al. (2014). Briefly, total RNA was extracted from liver tissue samples using the TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA). Extracted RNA was quantified via UV absorbance (UV Mini 1240; Shimadzu Scientific Instruments, Inc., Columbia, MD) at 260 nm, incubated (2.5 μg) a 37°C o 30 in n the presence of RNase-free (DNase; New England Biolabs Inc., Ipswich, MA), and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA). Real-time PCR was completed using the SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers (20 pM each) with the StepOne Real-time PCR system (Applied Biosystems). At the end of each real-time PCR, amplified products were subjected to a dissociation gradient (95°C for 15 s, 60°C for 30 s, and 95°C for 15 s) to verify the amplification of a single product by denaturation at the anticipated temperature. The amplified products were purified with the QIAquick PCR purification kit (Qiagen Inc., Valencia, CA) and sequenced at the Department of Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Animal Science from University of Florida to verify the specificity of amplification. All amplified products represented only the genes of interest. Primer sequence of target genes are shown in Table 2 and were validated by previous studies, except for GHR-1B, which was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer Express v.3.0.1 software (Applied Biosystems, Foster City, CA). Responses were quantified based on the threshold cycle (CT) and were normalized to the geometrical mean CT values of cyclophilin and RSP9 (ΔCT) exa ned n he sa e sa le and assessed at the same time as the targets. Within each target –ΔΔCT gene, results are expressed as relative fold change (2 ) using the average ΔCT of all samples (Ocón- Grove et al., 2008). Inter- and intra-assay CV for mRNA expression of IGF-1, IGFBP-3, GHR-1A, and GHR- 1B were 2.77 and 3.77%, 2.94 and 3.11%, 2.33 and 4.27%, and 2.59 and 3.89%, respectively (Bustin et al., 2009). Statistical analyses All data were analyzed as a 2 × 2 factorial design using SAS (SAS Institute Inc., Cary, NC, USA, version 9.4) with Satterthwaite approximation to determine the denominator degrees of freedom for the test of fixed effects. Heifer was the experimental unit, whereas heifer(PRE × POS) was included as random effect in all analyses. Growth performance data were analyzed using the MIXED procedure, whereas puberty attainment was analyzed using the GLIMMIX procedure. Heifer ADG, BW and age at puberty, and mature BW on d 380 were tested for fixed effects of pre-weaning injections (PRE), post- weaning injections (POS), and all resulting interactions. Heifer BW, BCS, plasma IGF-1, liver mRNA expression, and puberty attainment were analyzed as repeated measures and tested for fixed effects of PRE, POS, day of the study, and all resulting interactions using heifer(PRE × POS) as the subject. Heifer BW and age on d 0 were included as covariates in all analyses, but removed from the model if P > 0.10. Proper covariance structure for each statistical analysis was selected based on the lowest Akaike Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 information criterion. Compound symmetry covariance structure was used for statistical analyses of pre- and post-weaning heifer BW, cow BW and BCS, post-weaning heifer BCS, and liver mRNA expression of IGF-1 and IGFBP-3. Autoregressive 1 covariance structure was used for the statistical analyses of liver mRNA expression of GHR-1A and -1B, and puberty attainment. All results are reported as least-squares means. Data were separated using PDIFF if a significant F-test was detected. Significance was set at P ≤ 0.05, and tendencies at P > 0.05 and ≤ 0.10 . RESULTS Effects of PRE × POS × day and PRE × POS were not detected for any variable in the study (P ≥ 0.11), and hence, effects of PRE and POS treatments were reported separately. Heifer BW and age on d 0 did not differ among treatments (P ≥ 0.75). However, heifer BW and age on d 0 were included as covariates in the analyses of heifer BW and ADG from d 0 to 177 (P ≤ 0.001), except for ADG from d 0 and 177 that was covariate-adjusted to age on d 0 only (P = 0.04). Effects of PRE × day of the study were (P = 0.02) detected for heifer BW from d 0 to 177. However, BW on d 10, 167 and 177 did not differ between heifers administered pre-weaning injections of bST or saline (P ≥ 0.12 ; Table 3). Effects of POS × day of the study tended (P = 0.07) to be detected for heifer BW from d 0 to 177, which did not differ between saline and bST heifers on d 10 and 167 (P ≥ 0.20), but tended to be greater on d 177 following post-weaning injections of bST vs. saline (P = 0.10; Table 3). Pre-weaning injections of saline or bST did not affect heifer ADG from d 0 to 177 and 167 to 177 (P ≥ 0.22). However, heifer ADG from d 0 to 10 was greater for those administered pre-weaning injections of bST vs. saline (P = 0.03), whereas ADG from d 10 to 167 was greater for heifers administered pre-weaning injections of saline vs. bST (P = 0.05; Table 3). Heifer pre- or post-weaning injections of saline and bST did not affect cow BW, BCS, and BW and BCS change from d 0 to 167 (P ≥ 0.17; Table 3). Post-weaning injections of saline and bST did not affect heifer ADG from d 167 to 177, Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 and overall ADG from d 0 to 177 (P ≥ 0.12; Table 3). Effects of PRE and POS were not detected (P ≥ 0.31) for heifer ADG from d 177 to 191 (0.27, 0.26, 0.23, and 0.29 ± 0.057 kg/d for PRE-bST, PRE-saline, POS- bST, and POS-saline heifers, respectively). Heifer BW from d 191 to 380 was covariate-adjusted to heifer BW and age on d 0 (P ≤ 0.002), whereas overall ADG from d 191 to 380 was covariate-adjusted to heifer BW on d 0 only (P = 0.02). Body weight and age on d 0 were included as covariates in the analyses of BW and age at puberty (P ≤ 0.06). Effects of PRE × day of the study and POS × day of the study were detected (P ≤ 0.0 3) for heifer post- weaning BW. Heifers administered pre-weaning injections of bST were lighter on d 349 and 380 (P ≤ 0.04), and tended to be lighter on d 259 (P = 0.06) compared to heifers administered pre-weaning injections of saline (Table 4). Heifer BCS did not differ following pre- or post-weaning injections of saline and bST (P ≥ 0.11). Overall heifer ADG from d 191 to 380 tended to be greater (P = 0.07) for heifers administered pre-weaning injections of saline vs. bST, and did not differ (P = 0.12) between heifers administered post-weaning injections of saline vs. bST (Table 4). Heifers administered pre-weaning injections of saline had greater (P = 0.03) mature BW on d 380, but similar BW and age at puberty (P ≥ 0.11) compared to heifers administered pre-weaning injections of bST (Table 4). Mature BW on d 380, and BW and age at puberty did not differ between heifers given post-weaning injections of bST or saline (P ≥ 0.39; Table 4 ). Effects of PRE × day of the study and POS × day of the study were detected (P ≤ 0.01) for plasma concentrations of IGF-1 collected from d 0 to 177. Heifers administered pre-weaning injections of bST had greater plasma IGF-1 concentrations on d 10 compared to heifers administered pre-weaning injections of saline (P = 0.0001; Table 5). Heifers given post-weaning injections of bST had greater plasma IGF-1 concentrations on d 177 compared to saline (P = 0.005; Table 5). Interactions among day of the study, PRE, and POS injections were not detected (P ≥ 0.19) for plasma concentrations of IGF-1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 collected from d 259 to 380, except for main effects of POS and day of the study (P ≤ 0.03). Pre-weaning injections of bST or saline did not impact overall plasma IGF-1 concentrations (P = 0.19), whereas heifers administered post-weaning injections of bST had less overall plasma IGF-1 concentrations from d 259 to 380 compared to heifers administered post-weaning injections of saline (P = 0.03; Table 5). Interactions among day of the study, PRE, and POS, as well as, main effects of pre-weaning injections were not detected (P ≥ 0.14) for liver mRNA expression of GHR-1A. However, main effects of day of the study and post-weaning injections were detected (P ≤ 0.03). Liver mRNA expression of GHR- 1A did not differ between d 0 and 10 (1.39 and 1.28 ± 0.210-fold increase, respectively; P = 0.64), decreased on d 167 (0.86 ± 0.210-fold increase; P ≤ 0.01), achieved greatest values on d 177 (1.82 ± 0.210-fold increase, respectively; P ≤ 0.07), and returned to baseline levels on d 380 (1.37 ± 0.210-fold increase, respectively; P = 0.92). Overall liver mRNA expression of GHR-1A was greater for heifers administered post-weaning injections of bST vs. saline (P = 0.03; Table 6). Liver mRNA expression of GHR-1B and IGF-1 on d 0 was included as covariate (P ≤ 0.01) in the analyses of liver mRNA expression of GHR-1B and IGF-1, respectively. Effects of POS × day of the study (P = 0.02), but not PRE, POS, and PRE × day of the study (P ≥ 0.16), were detected for liver mRNA expression of GHR-1B. Heifers administered post-weaning injections of bST had greater (P = 0.02) mRNA expression of GHR-1B on d 167, but tended to have less (P = 0.08) liver GHR-1B mRNA expression on d 177 compared to heifers administered post-weaning injections of saline (Table 6). Interactions among day of the study, PRE, and POS, as well as, main effects of PRE and POS were not detected (P ≥ 0.12) for liver mRNA expression of IGF-1 (Table 6). However, effect of day of the study was detected (P < 0.0001) for liver mRNA expression of IGF-1, which was greatest on d 10 (2.55 ± 0.170-fold increase; P < 0.0001), decreased on d 167 and 177 (0.84 and 1.03 ± 0.170-fold increase; P ≤ 0.01), andwe re intermediate on d 380 (1.27 ± 0.170-fold increase, respectively). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Effects of PRE × day of the study, POS × day of the study, PRE, and POS were not detected (P ≥ 0.20) for liver mRNA expression of IGFBP-3 (Table 6). However, effect of day of the study was detected (P < 0.0001) for liver mRNA expression of IGFBP-3, which was greatest on d 10 (2.22 ± 0.209-fold increase; P ≤ 0.06), decreased on d 0, 167, and 380 (0.95, 1.12, and 1.10 ± 0.209-fold increase, respectively), and were intermediate on d 177 (1.27 ± 0.170-fold increase, respectively; P ≤ 0.08). A tendency for effects of PRE × day of the study (P = 0.09), but not POS × day of the study (P = 0.18), was detected for heifer puberty attainment. A greater percentage of heifers administered pre- weaning injections of saline attained puberty on d 349 (P = 0.07), 359 (P = 0.002), and 380 (P = 0.0001) compared to heifers administered pre-weaning injections of bST (Figure 1a). DISCUSSION Day and Anderson (1998) proposed that the period from birth to puberty in beef heifers could be divided into infantile, developmental, static, and peripubertal periods. During the developmental phase (2 to 6 mo of age), GnRH secretion and follicular growth increases, with LH secretion and number of ovarian follicles peaking at 3 to 4 mo of age. Enhancing heifer ADG and nutrient intake during this developmental phase led to hastened follicle growth (Gasser et al., 2006a,b) and puberty attainment of beef heifers (Moriel et al., 2014). The exact nutrition-mediated mechanisms involved in this early activation of the reproductive axis in beef heifers are unknown. However, circulating IGF-I can impact gonadotropin secretion and activity required for the first ovulation and subsequent puberty achievement in beef heifers by influencing hypothalamic-pituitary secretory activity (Butler and Smith, 1989; Schillo et al., 1992) and augmenting the effects of gonadotropins in ovarian follicular cells (Spicer and Echternkamp, 1995). In agreement, heifer ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability on age at puberty (Moriel et al., 2014). Thus, Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 metabolic imprinting may be explored by identifying strategies to increase heifer ADG and plasma IGF-1 during the developmental phase leading to optimized future reproductive performance. Systemic IGF-1 has been positively correlated with muscle skeletal growth (Jiang and Ge, 2014). Piccolo et al. (2018) reported that Brangus crossbred heifers administered pre-weaning bST injections (250 mg every 14 d from 132 to 174 d of age) had an 8.6 ng/mL increase in plasma IGF-1 concentrations and 7.2% increase on ADG from d 0 to 42, relative to first injection, but no differences on ADG from first injection to weaning (167-d period) compared to saline heifers. Likewise, pre-weaning bST injections in the present study increased plasma IGF-1 concentrations by 51.7 ng/mL and ADG from d 0 to 10 by 35%, but did not impact ADG from d 0 to 177. The increase in BW gain and circulating IGF-1 concentrations following bST injections varied from 0 to 45% compared to control vehicles (Dalke et al., 1992; Houseknecht et al., 1992), and several factors, such as plane of nutrition, age, and size of treated animals may explain this large variation (Rausch et al., 2002). Also, multiple mechanisms may be involved in post-bST BW gain including repartitioning of nutrients toward muscle rather than adipose tissue deposition (Breier, 1999), and enhanced long-bone growth (Buskirk et al., 1996), nitrogen retention (Eisemann et al., 1986), and circulating IGF-1-induced protein synthesis of muscle (Jiang and Gee, 2014) and non-carcass tissues (Early et al., 1990). Multiple 14-d apart administrations of bST, during the post-weaning phase, reduced subcutaneous fat thickness by 9.2% without impacting LM depth, marbling scores, and BW gain (Cooke et al., 2013). Although body composition was not evaluated in the present study, it is unlikely that only two 10-d apart injections of bST substantially affected body composition and nutrient requirements of heifers. Thus, our results perhaps indicate that the increment on bST-induced ADG from d 0 to 10 may be the result of increased feed intake and gut fill, as reported by Enright et al. (1990), or that muscle protein deposition from d 0 to 10 was not sufficient to impact heifer BW at weaning. Nevertheless, pre-weaning bST injections in the present study successfully Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 increased plasma IGF-1 concentrations and ADG of heifers during the developmental phase of the reproductive axis in beef heifers (Day and Anderson, 1998). In contrast, post-weaning bST injections increased plasma IGF-1 concentrations by 37.2 ng/mL, but did not impact ADG during the injection period (d 167 to 177). Other studies also demonstrated that post-weaning bST injections increased plasma IGF-1 concentrations (Cooke et al., 2013), but did not increase post-weaning ADG of Angus × Holstein administered 500 mg of bST every 14 d from 6 to 10 mo of age (Carstens et al., 1997) and Angus × Hereford heifers injected with 250 mg of bST every 14 d from 6 to 13 mo of age (Cooke et al., 2013). Body weight gain and circulating IGF-1 responses to bST are influenced by cattle age and nutritional status (Rausch et al., 2002; Radcliff et al., 2004). Cattle somatotropic axis is functional at birth (Granz et al., 1997), and the response to bST begins as early as 1 d of age (Govoni et al., 2004), gradually increasing as age increases (Velayudhan et al., 2007). Likewise, plasma IGF-1 concentrations following bST injection were greater for Holstein heifers gaining 1.2 vs. 0.8 kg/d (Radcliff et al., 2004). In the current study, heifers were weaned on d 167 and provided free choice access to grass silage and pastures of relatively poor nutritional composition, which resulted in BW loss from d 167 to 177 and reduced ADG from d 177 to 191 compared to the pre- weaning period. Hence, despite the more advanced age at the time of bST injections, the lack of positive impacts of post-weaning injections of bST on BW gain from d 167 to 177 and 177 to 191 may be related to the reduced magnitude of increase in plasma IGF-1 concentrations, which was likely suppressed by the weaning-induced physiological stress (Arthington et al., 2008) and the poorer nutritional status of heifers during post- vs. pre-weaning periods (Radcliff et al., 2004). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 The binding of GH to GHR-1A stimulates hepatic synthesis of IGF-1 (Smith et al., 2002), and is highly correlated with the hepatic mRNA expression of GHR-1A and IGF-1 (Lucy et al., 2001). Transcription of the growth hormone receptor (GHR) gene is initiated from multiple transcription start sites, generating GHR-1A, -1B, and -1C RN ha d e n h e un5 tran ′ slated region but still encode the same amino acid sequence (Jiang and Lucy, 2001). The GHR-1A mRNA is only expressed in the liver (Lucy et al., 1998), whereas GHR-1B and -1C mRNA are expressed in a wide array of tissues, including liver, skeletal muscle, adipose tissue, and mammary gland (Jiang et al., 1999; Jiang and Lucy, 2001). Hepatic synthesis of IGF-1 is regulated primarily at the transcriptional level (Thissen et al., 1994), and is the major source of circulating IGF-1 (Yakar et al., 1999), which is also responsible for stimulating the hepatic expression of IGFBP-3 mRNA (Thissen et al., 1994). Thus, an increased hepatic expression of GHR-1A mRNA enhances the capacity for GH binding (Lapierre et al., 1982) and the hepatic synthesis of IGF-1 (Radcliff et al., 2004). Nutrient intake and BW gain positively affects the hepatic abundance of GHR-1A, IGF-1, and IGFBP-3 (Thissen et al., 1994; Smith et al., 2002; Radcliff et al., 2004). Holstein heifers administered daily injections of bST (25 µg/kg of BW from 120 to 247 d of age, in average) had greater mRNA expression of IGF-1, but similar mRNA expression of GHR-1A and IGFBP-3 (Radcliff et al., 2004). Piccolo et al. (2018) demonstrated that 3 pre-weaning injections of bST did not impact liver mRNA expression of GHR-1A and IGFBP-3 throughout the study, but increased liver mRNA expression of GHR-1B and IGF-1 at approximately 220 d after the last pre-weaning injection of bST, suggestive of metabolic imprinting effects causing long-term changes to gene expression despite the similar nutritional status of those heifers. In contrast, pre-weaning injections of bST did not impact the mRNA expression of any gene measured in the present study, whereas post-weaning bST injections increased overall liver mRNA expression of GHR-1A and decreased liver GHR-1B mRNA on d 177. Several reasons may be responsible for this lack of effects of pre-weaning bST injections. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Sartori et al. (2010; 2016) reported that Bos indicus cattle naturally have greater circulating IGF-I concentrations compared to Bos taurus cohorts. Moreover, Mendonça et al. (2013) demonstrated that even under the same environment and diet, Bos taurus-influenced dairy cows have less circulating concentrations of IGF-I compared to Bos indicus cows, which might be related to the different organ sensitivity to IGF-1. Despite the lack of published evidence, it is possible that a breed × bST effect on liver gene mRNA expression exists, but further studies comparing effects of breed on physiological and growth responses to bST are warranted to confirm this rationale. Also, due to differences on nutritional status, liver mRNA expression of IGF-1 and GHR-1A of all heifers were greater on d 0 vs. 167 (data not shown). Therefore, liver mRNA expression of GHR-1A and IGF-1 were perhaps at maximum during pre- weaning phase, which may have prevented further increments on mRNA expression of these genes. Plasma IGF-1 concentrations increase after 3 d, peak at approximately 7 to 8 d, and gradually return to baseline levels starting 12 d post-injection of bST (Bilby et al., 1999, 2004). Hence, it is possible that the timing of liver sample collection was not optimal to detect the peak expression of liver mRNA of IGFBP- 3, IGF-1, GHR-1B and GHR-1A. The detection of greater plasma IGF-1 concentrations on d 10 and 177, but similar liver mRNA expression of IGF-1 on those days between bST vs. saline heifers supports this rationale. Nevertheless, the greater overall mRNA expression of GHR-1A following post-weaning bST injections provide evidence that even post-weaning injections of bST can cause metabolic imprinting effects and alter long-term gene expression despite the similar post-weaning nutritional status. The impact of bST injections on post-treatment circulating concentrations of IGF-1 and puberty attainment of beef heifers has been variable. Injections of bST (250 mg every 14 d from 120 to 232 d of age) did not impact post-treatment serum concentrations of IGF-1 and puberty attainment of Angus × Simmental crossbred heifers (Buskirk et al., 1996). Pre-weaning injections of bST (250 mg every 14 d from 132 to 174 d of age) hastened puberty attainment of Brangus heifers at the start of the breeding season compared to saline injections, despite their similar nutritional management, ADG, and BW during Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 breeding season (Piccolo et al., 2018). Post-weaning bST injections (250 mg of bST every 14 d from 6 to 13 mo of age) increased puberty attainment of Angus × Hereford heifers at the start of breeding season (Cooke et al., 2013), but had no impact on puberty achievement of Angus heifers administered bST (350 mg every 14 d from 7 to 14.5 mo of age) compared to vehicle-treated heifers (Hall et al., 1994). In the present study, pre-weaning injections of bST decreased post-weaning growth performance compared to saline injections, whereas post-weaning injections of bST did not impact post- weaning growth performance of heifers. Age at puberty in cattle typically decreases as BW gain increases (Schillo et al., 1992). Hence, the similar post-weaning ADG likely explains the similar puberty achievement of heifers given post-weaning injections of bST and saline, whereas the decreased puberty attainment of heifers administered pre-weaning injections of bST vs. saline may be at least partially explained by the observed differences in post-weaning growth rate and BW. The reason for the decreased post-weaning growth performance of heifers following pre-weaning bST injections is unknown. Moriel et al. (2014) observed that during similar nutritional management, liver mRNA expression of IGF-1 and puberty attainment at start of breeding season were greater for heifers early- weaned at 60 d of age and placed on high-concentrate diets until 150 d of age compared to heifers normally weaned at 270 d of age, suggestive of metabolic imprinting effects. Hence, it was expected that the long-term post-weaning plasma concentrations of IGF-1 would be positively affected by pre- rather than post-weaning injections of bST due to the greater susceptibility of younger animals to metabolic imprinting effects. However, overall plasma IGF-1 concentrations collected from d 259 to 380 was decreased by post-weaning injections of bST, and not impacted by pre-weaning injections of bST. Regardless of the reasons for such contradiction, these results reinforce our rationale that the timing of bST injecting can have profound and diverse long-term impacts on growth, reproductive, and physiological parameters of beef heifers. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Additional factors beyond BW gain may have contributed to the variable responses on heifer growth performance and puberty achievement following bST treatment, including body composition, timing of injections, duration of injections, dosage, breed, and potentially the interactions among all of these factors. Although bST injections may stimulate lipolytic activity in adipose tissue (Lanna et al., 1995) and reduce backfat thickness in cattle (Vesteergard et al., 1993; Cooke et al., 2013), neither pre- weaning nor post-weaning injections of bST altered heifer BCS throughout the post-weaning phase, indicating that the pre-weaning bST-induced reduction in post-weaning ADG was likely not be attributed to altered body composition and nutrient requirements. One could argue that the period duration of bST injections (2 injections 10-d apart) may have been insufficient to impact puberty attainment of heifers. This rationale supports the observed results of post-weaning, but not the pre-weaning bST- induced effects on puberty attainment. Despite the lack of evidence to support this rationale, potential candidates are the effects of breed or the interaction between breed and bST dosage. The bST dosage 0.75 used in the present study (6.14 mg/kg ) was obtained from previous work that successfully hastened percentage of pubertal heifers at start of breeding season following pre-weaning bST administration (Piccolo et al., 2018). However, in that study, pre-weaning plasma IGF-1 concentrations increased by 8.6 ng/mL after bST injections (94.8 and 103.4 ng/mL for saline and bST heifers, respectively). In the present study, baseline plasma concentrations of IGF-1 were 186 and 88.2 ng/mL on d 0 and 167, respectively, and increased by 25.4 and 9.9 ng/mL on d 10 and 177, respectively, following first bST injection. Brahman (Simpson et al., 1997) and Nellore cows (Roberts et al., 2005) have greater circulating concentrations of IGF-1 than Angus cattle. Furthermore, circulating concentrations of IGF-1 in straight- bred Brahman cows were greater than in the crossbred cows (Roberts et al., 2005), which likely explains the greater baseline IGF-1 levels observed in the present study compared to Piccolo et al. (2018). However, it is also possible that the greater increment on plasma IGF-1 concentrations following bST injection, in combination with interval between bST injection, were detrimental to the development of Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 the reproductive axis. Further studies investigating the effects of breed on ovarian activity and gene expression in reproductive tissue organs and brain, following bST injections, are warranted to confirm this rationale. In conclusion, pre-weaning injections of bST, administered 10-d apart starting at approximately 2 to 3 mo of age, successfully increased pre-weaning plasma concentrations of IGF-1 and growth performance of Nellore heifers during the period of bST treatment. 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Effects of heterosis on age and weight at puberty in beef heifers. J. Anim. Sci. 25:744-751. Yakar, S., J. Liu, B. Stannard, A. Butler, D. Accili, B. Sauer, and D. LeRoith. 1999. Normal growth and development in the absence of hepatic insulin-like growth factor. Proc. Natl. Acad. Sci. USA 96:7324–7329. Ye, J., G. Coulouris, I. Zaretskaya, I. Cutcutache, S. Rozen, and T. L. Madden. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinf. 13:134. doi:10.1186/1471-2105-13-134. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 1. Average nutritional composition (DM basis) of pastures, grass silage, and corn silage-based TMR provided to heifers throughout the study. Pre-weaning Post-weaning Grass Item TMR 1 1 2 pasture pasture silage DM, % 39.0 22.7 31.0 50.8 CP, % 9.8 15.8 5.9 20.9 NDF, % 68.3 56.6 78.0 37.1 ADF, % 40.8 29.3 48.6 23.3 TDN , % 54.6 60.6 45.9 70.0 NEm , Mcal/kg 1.13 1.38 0.76 1.69 NEg , Mcal/kg 0.57 0.80 0.22 1.08 Ca, % 0.32 0.62 0.49 0.29 K, % 1.65 2.76 1.05 1.25 Mg, % 0.25 0.37 0.27 0.24 P, % 0.25 0.35 0.15 0.36 S, % 0.17 0.24 0.19 0.40 Samples of brachiaria pastures collected monthly during pre- (d 0 to 167) and post-weaning phases (d 192 to 380). Samples of mombaça grass silage collected from d 167 to 191. Samples of TMR collected monthly from d 191 to 380. Total mixed ration consisted of 68.7% corn- silage and 31.3% soybean meal (DM basis). Calculated as described by Weiss et al. (1992). Calculated using the equations proposed by the NRC (2000). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 2. Primer sequences and accession number for all gene transcripts analyzed by quantitative real- time PCR . Target gene Primer sequence Accession no. Cyclophilin Forward 5'-GGTACTGGTGGCAA GTCCAT-3' NM_178320.2 Reverse 5'-GCCATCCAACCACTCAGTCT-3' IGF-1 Forward 5'-CTCCTCGCATCT CTTCTATCT-3' NM_001077828 Reverse 5'-ACTCATCCACGATTCCTGTCT-3' IGFBP-3 Forward 5'-AATGGCAGTGA GTCGGAAGA-3' NM_174556.1 Reverse 5'-AAGTTCTGGGTGTCTGTGCT-3' GHR-1A Forward 5'-CCAGCCTCTGTT TCAGGAGTGT-3' AY74 8827 Reverse 5'-TGCCACTGCCAAGGTCAAC-3' GHR-1B Forward 5'-AGCCTGGAGGA ACCATACGA-3' - Reverse 5'-TAGCCCCATCTGTCCAGTGA-3' RSP9 Forward 5'-CCTCGACCAA GAGCTGAAG-3' DT86 0044 Reverse 5'-CCTCCAGACCTCACGTTTGTTC-3' Primer sequence for IGF-1, IGFBP-3, and GHR-1A were obtained from Coyne et al. (2011 ), whereas those for cyclophilin and RSP9 were obtained from Cooke et al. (2008) and Janovick-Guretzky et al. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 (2007), respectively. Primer sequence for GHR-1B was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer Express v. 3.0.1 software (Applied Biosystems, Foster City, CA). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 3. Growth performance (d 0 to 177) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Post-weaning Pre-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Heifer BW , kg d 10 109 11 1 1.35 0.21 0.02 11 0 10 9 1.36 0.48 0.07 d 167 187 185 1.35 0.24 185 187 1.36 0.20 d 177 187 184 1.35 0.12 184 187 1.36 0.10 Heifer ADG , kg/d d 0 to 10 0.48 0.65 0.06 1 0.03 0.62 0.51 0.061 0.14 d 10 to 167 0.47 0.45 0.008 0.05 0.45 0.47 0.009 0.12 d 167 to 177 -0.01 -0.07 0.068 0.54 -0.06 -0.01 0.069 0.59 d 0 to 177 0.47 0.45 0.009 0.24 0.45 0.46 0.009 0.28 Cow BW, kg d 0 487 48 2 5.68 0.61 0.22 48 3 48 6 5.7 0.63 0.41 d 167 465 456 5.68 0.25 460 461 5.7 0.95 Cow BCS Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 0 2.89 2.87 0.020 0.47 0.80 2.88 2.88 0.020 0.89 0.95 d 167 2.83 2.82 0.020 0.63 2.83 2.82 0.020 0.93 BW change, kg -21.5 -27.1 2.84 0.17 -22.9 -25.6 2.84 0.50 BCS change -0.06 -0.05 0.019 0.87 -0.06 -0.05 0.019 0.84 1 0.75 The study began on d 0 and heifer calves were weaned on d 167. Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck and 10 d apart during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). P-value for comparison of treatment within day. Covariate-adjusted to BW and age on d 0 (P ≤ 0.001), except for ADG from d 0 and 177 that was covariate-adjusted to age on d 0 only (P = 0.04). Table 4. Post-weaning growth performance (d 191 to 380) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Heifer BW , kg d 191 190 18 8 3.06 0.56 0.02 18 8 19 0 3.11 0.91 0.03 d 229 197 192 3.06 0.25 194 195 3.11 0.99 d 259 220 212 3.06 0.06 217 215 3.11 0.46 d 289 240 235 3.06 0.15 240 235 3.11 0.20 d 320 266 260 3.06 0.13 265 262 3.11 0.33 d 349 291 283 3.06 0.03 289 286 3.11 0.45 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 380 317 309 3.06 0.04 316 310 3.11 0.24 3,4 Heifer BCS d 229 5.12 5.08 0.046 0.51 0.19 5.12 5.10 0.046 0.88 0.27 d 259 5.10 5.02 0.046 0.20 5.08 5.02 0.046 0.48 d 289 5.46 5.50 0.046 0.45 5.54 5.40 0.046 0.23 d 320 5.86 5.90 0.046 0.66 5.92 5.84 0.046 0.15 d 349 5.96 5.86 0.046 0.16 5.92 5.90 0.046 0.62 d 380 6.32 6.26 0.046 0.42 6.36 6.24 0.046 0.08 ADG d 191 to 380 , kg/d 0.83 0.80 0.014 0.07 - 0.83 0.80 0.014 0.12 - Mature BW on d 380 , % 70.7 68.8 0.63 0.03 - 70.1 69.4 0.65 0.48 - BW at puberty , kg 311 306 3.4 0.30 - 311 307 3.4 0.39 - Age at puberty , d 453 458 3.2 0.11 - 456 455 3.2 0.89 - 1 0.75 Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value for comparison of treatment within day. Covariate-adjusted to BW and age on d 0 (P ≤ 0.002), except for overall ADG from d 191 to 380 that was covariate-adjusted to BW on d 0 only (P = 0.02). Using a 1 to 9 scale according to Wagner et al. (1988). Assuming a mature cow BW of 450 kg. Body weight at puberty attainment was determined using the ADG, and initial and final BW measurements of the respective month when CL was detected [BW at puberty = initial BW of the respective month + (ADG of the respective month  number of days between the day at CL detection and initial BW collection)]. Age at puberty was set as the day when corpus luteum (CL) was detected (d 1 or 10 of each month from d 192 to 380). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 5. Pre- and post-weaning plasma IGF-1 concentrations of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 2 2 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day Plasma IGF-1, ng/mL d 0 193.0 186.0 9.3 0.59 0.00 1 198.9 179.9 9.3 0.15 0.01 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 d 10 159.7 211.4 9.3 0.0001 186.8 184.4 9.3 0.85 d 167 82.4 82.2 9.3 0.99 76.4 88.2 9.3 0.37 d 177 83.0 76.1 9.3 0.60 60.9 98.1 9.3 0.005 Overall (d 259 to 380) 197.7 210.8 7.1 0.19 - 216.0 192.5 7.1 0.03 - 1 0.75 Injections of saline (5 mL 0.9 % NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value for comparison of treatment within day. Average plasma IGF-1 concentrations of blood samples collected on d 259, 289, 319, 349, and 380. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 6. Pre- and post-weaning liver mRNA expression (fold increase ) of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10) or post-weaning phases (d 167 and 177) . Pre-weaning Post-weaning injections P-value injections P-value 3 3 Item Saline BST SEM P-value PRE × day Saline BST SEM P-value POS × day -- Fold increase -- -- Fold increase -- GHR-1A 1.37 1.32 0.17 4 0.78 0.98 1.17 1.52 0.17 1 0.03 0.87 IGF-1 1.37 1.48 0.129 0.53 0.86 1.37 1.48 0.131 0.52 0.12 IGFBP-3 1.50 1.31 0.122 0.29 0.64 1.46 1.36 0.122 0.54 0.69 GHR-1B d 10 1.75 1.64 0.17 9 0.71 0.46 1.56 1.84 0.22 2 0.34 0.02 d 167 1.14 1.12 0.179 0.93 0.77 1.49 0.222 0.02 d 177 1.36 0.79 0.179 0.07 1.35 0.81 0.222 0.08 d 380 1.31 1.31 0.179 0.99 1.14 1.47 0.222 0.28 Responses were quantified based on the threshold cycle (CT) and were normalized to average CT of cyclophilin and RSP9 (ΔCT) examined in –ΔΔCT the same sample and assessed at the same time as the targets. Within each target gene, results are expressed as relative fold change (2 ) using the average ΔCT of all samples as reference, as described by Ocón-Grove et al. (2008). 2 0.75 Injections of saline (5 mL 0.9% NaCl) or bST (6.14 mg/kg of BW ) were administered subcutaneously in the neck during pre- (d 0 and 10) and/or post-weaning (d 167 and 177). Heifers were weaned on d 167 and limit-fed corn-silage based diets from d 191 to 380. P-value represents the main effects of treatment for overall mRNA expression of GHR-1A, IGF-1, and IGFBP-3 obtained on d 0, 10, 167, 177 and 380. For mRNA expression of GHR-1B, the P-value represents the comparison of treatment within day for. Liver mRNA expression of IGF-1 and GHR-1B were covariate-adjusted to respective mRNA expression on d 0 (P ≤ 0.02), whereas liver mRNA expression of GHR-1A was covariate-adjusted to BW on d 0 only (P = 0.05). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1. Puberty attainment of Nellore heifers administered, in a 2 × 2 factorial design, injections of saline solution or bovine somatotropin (BST) during pre- (d 0 and 10; Figure 1a) or post-weaning phases (d 167 and 177; Figure 1b). Effects of PRE × day of the study, but not POS × day of the study (P = 0.22), tended to be detected (P = 0.09) for puberty attainment during the study. *P ≤ 0.05, †0.05 < P ≤ 0.10. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1a PRE-Saline PRE-bST 40 * 229 239 259 269 289 299 320 349 359 380 Day of the study Accepted Manuscript Pubertal heifers, % of total heifers Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy125/5193117 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1b POS-Saline POS-bST 229 239 259 269 289 299 320 349 359 380 Day of the study Accepted Manuscript Pubertal heifers, % of total heifers

Journal

Translational Animal ScienceOxford University Press

Published: Nov 19, 2018

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