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Effects of low-moisture, sugarcane molasses-based block supplementation on growth, physiological parameters, and liver trace mineral status of growing beef heifers fed low-quality, warm-season forage

Effects of low-moisture, sugarcane molasses-based block supplementation on growth, physiological... Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Effects of low-moisture, sugarcane molasses-based block supplementation on growth, physiological parameters, and liver trace mineral status of growing beef heifers fed low- quality, warm-season forage P. Moriel* , L. F. A. Artioli, ‡M. B. Piccolo*, M. Miranda*, J. Ranches*, V. S. M. Ferreira§, L. Q. Antunes§, A. M. Bega§, V. F. B. Miranda§, J. F. R. L. Vieira§, and J. L. M. Vasconcelos§ *University of Florida – Range Cattle Research & Education Center, Ona, 33865-9706, USA ‡De Heus MBU Brazil Animal Nutrition Industry, Guararapes, São Paulo 16700-000, Brazil D rt t o A i l ro tio o lo t t i r it ot t -00 0 , Braz il Correponding author: 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/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 ABSTRACT The objectives of the study were to evaluate the growth, physiological parameters, and liver trace mineral status of beef heifers provided low-quality warm-season forage and different forms (meal vs. block) of trace mineral-fortified supplementation. One hundred yearling Nellore heifers were blocked by initial BW (184 ± 2.5 kg) and randomly assigned into 1 of 20 drylot pens (5 heifers/pen). Treatments were randomly assigned to pens (5 pens/treatment) and consisted of heifers receiving: (1) a loose meal trace mineral supplement (TM; De Heus Animal Nutrition Industry); (2) free choice access to a low- moisture, cooked sugarcane molasses-based protein block (LMB); (3) isocaloric and isonitrogenous, loose meal protein supplement pair-fed to LMB supplement DM intake (PSPF); and (4) loose meal protein supplement offered at 0.2% of BW (PS). Supplements were formulated to achieve same daily intake of supplemental trace mineral among treatments. Hence, TM supplement was offered at 66.6% of the supplement DMI of LMB heifers. Heifers were offered free choice access to water and ground brachiaria (Brachiaria brizantha) hay from d 0 to 45. Overall ADG from d 0 to 45 was the least for TM heifers (P  0.05) and did not differ among LMB, PSPF, and PS heifers (P ≥ 0. 0 ). Daily hay DMI did not differ among treatments (P ≥ 0. 3) . Total intake of DM and TDN were least for TM heifers (P  0.03) and did not differ (P ≥ 0. ) o g LM, PSPF, and PS heifers. Total supplemental intake of CP and RDP and total intake of CP and RDP (supplement + hay) were least for TM and greatest for PS heifers (P  0.05), and intermediate for LMB and PSPF heifers (P ≥ 0.70). Effects of treatment × day and treatment were not detected (P ≥ 0. ) o prlasma concentrations of IGF-1, insulin, and NEFA. Effects of treatment were detected for plasma concentrations of PUN (P = 0.005) and tended to be detected for plasma concentrations of glucose (P = 0.08), which were least for TM heifers (P  0.03) and did not differ (P ≥ 0.17) among LMB, PSPF, and PS heifers. Trace mineral intake and liver concentrations of all trace minerals did not differ (P ≥ 0. 3 ) among treatments. Hence, the use LMB supplementation resulted in positive effects on growth without impacting trace mineral status compared to a loose meal trace Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 mineral salt, and similar growth performance and trace mineral status compared to a conventional protein supplementation offered at 0.2% of body weight. Key words: block, heifers, molasses, Nellore, protein supplement, trace mineral Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 INTRODUCTION Low-moisture, cooked molasses blocks (LMB) for forage-fed cattle is a popular supplementation strategy due to its convenience, decreased production costs (labor and fuel), and potential for improving forage intake and digestion (Löest et al., 2001) and grazing of underutilized pastures (Bailey and Welling, 1999). The improved forage digestion can be attributed to the supply of RDP, which is often the most limiting nutrient under grazing of low-quality grasses (Köster et al., 1996; Titgemeyer et al., 2004). It is also possible to use mineral-fortified LMB as an efficient strategy to improve the trace mineral status of beef calves (Ranches et al., 2018). For instance, beef calves grazing bahiagrass pastures and fed trace mineral-fortified LMB had greater liver concentrations of Co, Cu, Mn, Se, and Zn compared to a non- fortified LMB, despite the less supplement DMI of fortified vs. non-fortified LMB calves (Ranches et al., 2018). The manufacturing process of LMB, particularly extreme heat or pH, may alter the bioavailability of nutrients, such as ammonia release in the rumen (Trater et al., 2003; Katulski et al., 2017), whereas the delivery form of supplements (loose meal vs. block form) may alter supplement consumption patterns and nutrient utilization (Katulski et al., 2017). Nevertheless, LMB supplementation for growing beef heifers fed low-quality forage may lead to improved nutrient consumption and trace mineral status, and consequently growth, compared to trace mineral salt offered in loose meal form. In addition, due to improved nutrient utilization, LMB supplementation may lead to similar performance compared to conventional supplementation strategies offering greater amounts of protein supplements. The objectives of the study were to evaluate growth, physiological parameters, and liver trace mineral status of beef heifers fed low-quality forage and offered different forms (meal vs. block) of trace mineral- fortified supplementation. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 MATERIALS AND METHODS Animals and experiment design The study described herein was conducted at São Paulo State University (São Manuel, São Paulo, Brazil) from November to December 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) and approved by the São Paulo State University Animal Care and Use Committee. One hundred Nellore heifers were stratified and blocked by initial BW (184 ± 2.5 kg; 12-13 mo of age), and then randomly assigned into 1 of 20 drylot pens (5 blocks; 4 pens/block; 200 m and 5 heifers/pen). Treatments were randomly assigned to pens within each block (1 pen/treatment/block; 5 pens/treatment), and consisted of heifers receiving: (1) a complete trace mineral mix supplement offered in a loose meal form (TM; De Heus Animal Nutrition Industry, Rio Claro, São Paulo); (2) free choice access to a low-moisture, cooked sugarcane molasses-based protein block (LMB; MUB , De Heus MBU Brazil Animal Nutrition Industry, Guararapes, São Paulo); (3) protein supplement offered in a loose meal form and pair-fed to achieve isocaloric and isonitrogenous supplement intake compared to LMB heifers (PSPF); and (4) a commercial protein supplement offered in a loose meal form and at levels recommended by manufacturer (PS; DM basis; De Heus Animal Nutrition Industry, Rio Claro, Sao Paulo). Treatments were offered from d 0 to 45 and all supplements included the same vitamin/trace mineral premix (DM basis: 3% Ca, 10% Mg, 23.5% S, 600 mg/kg Co, 20,000 mg/kg Cu, 264 mg/kg Se, 1,200 mg/kg I, 80,000 mg/kg Zn, 53,200 mg/kg Mn, 4,000,000 mg/kg vitamin A, 400,000 mg/kg vitamin D , 20,000 mg/kg vitamin E, and 40,000 mg/kg monensin, Poulcox 40, Peshteria, Bulgaria). All supplements were formulated to achieve same daily supplemental trace mineral intake among treatments. Hence, within each block of pens: (1) TM supplement was offered daily at 66.6% (DM basis) of the supplement DMI of LMB heifers obtained in the previous day; (2) PSPF supplement DM offered was adjusted daily to Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 achieve similar daily intake of supplemental TDN and CP compared to LMB heifers; and (3) PS supplement was offered at 0.2% of heifer initial BW (DM basis), which is the manufacturer recommended level offered in commercial beef cattle operations. Supplement DM offered to PS heifers was adjusted accordingly to the average BW obtained on d 24 and 25. Each pen assigned to LMB treatment received a single 50-kg supplement block from d 0 to 45, but each LMB block was weighed daily at 0730 h to calculate supplement DMI from previous day. Rainfall precipitation was observed on 2 d, and on these days, supplement DMI was not calculated and removed from statistical analyses. All remaining supplements were hand-fed daily at 0800 h from d 0 to 45. Heifers were offered free choice access to water and ground brachiaria (Brachiaria brizantha) hay from d 0 to 45. Hay was chopped to achieve between 2 to 5 cm of length. Hay and supplements were offered in separated feed bunks. Chemical composition of hay and supplements are shown in Table 1. Data collection Except for LMB, supplements were consumed entirely within 1 h after feeding. Hay DM offered and refused were obtained daily for each pen by drying samples of hay offered and refused in a forced- air oven at 56°C for 48 h. Daily DMI of LMB supplement was estimated by multiplying the daily DM concentration of the supplement by the weight disappearance of each supplement block obtained between consecutive days. Daily total DMI was determined by subtracting the daily hay DM refused from the total daily hay and supplement DM offered. Samples of supplement offered were collected daily and pooled within each week, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients (Table 1). Samples of hay offered were collected daily and pooled within every 15 d, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients (Table 2). Samples were Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 analyzed for concentrations of CP (method 984.13; AOAC, 2006), and ADF (method 973.18 modified for use in an Ankom 200 fiber analyzer; Ankom Technology Corp., Fairport, NY; AOAC, 2006). Concentrations of TDN were calculated as proposed by Weiss et al. (1992). Individual full BW of heifers were assessed at 0730 h on 2 consecutive days (d 0 and 1, 24 and 25, and 45 and 46), immediately before morning feeding. Shrunk BW were not obtained during the study to avoid shrink-induced stress effects on forage and supplement DMI and blood physiological parameters that could interfere with data interpretation. Blood samples (10 mL) were collected from jugular vein on d 0, 24, and 45, immediately before feeding into 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 laboratory analyses. On d 0, three heifers from each pen were randomly selected and assigned to liver tissue biopsies on d 0 and 45. Liver samples (100 mg of tissue wet weight) were collected via needle biopsy following the procedure described by Arthington and Corah (1995) th tor t −2 0 Sa °C. mples were then assessed for trace mineral concentrations at Michigan State University Diagnostic Center for Population & Animal Health (Lansing, MI). Liver trace mineral concentrations on d 0 were initially included as covariate to adjust liver trace mineral concentrations on d 45, but later removed from statistical model (P  0.22). Liver samples were collected only on d 0 and 45: (1) because our goal was to evaluate the final liver trace mineral concentrations of heifers after receiving their respective supplement for 45 d, and (2) to avoid a surgery-induced inflammatory response in the middle term of the study that could interfere with growth performance and physiological parameters. Average total trace mineral consumption from d 0 to 45 was calculated by multiplying the total DMI of hay and supplement by the average weekly mean concentration of each trace mineral present in hay and supplement. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Laboratory analyses Plasma concentrations of insulin were determined using a single chemiluminescent enzyme immunoassay (Immulite 1000; Siemens Medical Solutions Diagnostics, Los Angeles, CA). Intra- and inter- assay CV for insulin were 1.9 and 2.8%, respectively. Commercial quantitative colorimetric kits were used to determine the plasma concentrations of glucose (G7521; Pointe Scientific, Inc., Canton, MI), PUN (B7551; Pointe Scientific Inc., Canton, MI), and NEFA (HR Series NEA-2; Wako Pure Chemical Industries Ltd. USA, Richmond, VA). Inter- and intra-assay CV for assays of glucose, PUN, and NEFA were 2.7% and 3.4%, 3.2% and 5.8%, and 3.9 and 4.2%, respectively. Plasma IGF-1 concentrations were analyzed in duplicate samples using commercial enzyme-linked immunosorbent assay kits (SG100; R&D Systems Inc., Minneapolis, MN) previously validated for bovine samples (Moriel et al., 2012). Inter- and intra-assay CV for IGF-1 assay were 1.81% and 2.35%, respectively.
 Statistical analyses All data were analyzed as randomized complete block design using the MIXED procedure of 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. Pen was the experimental unit, whereas pen(treatment) and heifer(pen) were included as random effects in all analyses. Heifer BW, blood parameters, liver concentrations of trace minerals, and daily DMI of hay and supplement were analyzed as repeated measures, and tested for fixed effects of treatment, time, and resulting interaction, using pen(treatment) as the subject. The covariance structure was chosen using the lowest Akaike information criterion. Compound symmetry was used as the covariance structure in all statistical analyses, except for Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 statistical analyses of plasma concentrations of glucose, PUN, and NEFA that used the autoregressive 1 covariance structure. Heifer BW on d 0 and plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin on d 0 did not differ between treatments (P ≥ 0.40) but were included as covariates (P  0.02) in the analyses of heifer BW and plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin, respectively. Liver concentrations of all trace minerals were not included as covariates (P  0.22). Heifer overall ADG and total DM intake of hay and supplement (d 0 to 45) were tested for fixed effects of treatment using pen(treatment) as random effect. Effects of block were included in all statistical analyses but removed from model if P > 0.10. 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 tendencies were noted if P > 0.05 ≤ 0. 0 . RESULTS Effects of day (P < 0.0001), but not treatment and treatment × day (P ≥ 028 . ), were detected for heifer BW. Effects of treatment tended to be detected (P = 0.10) for overall ADG from d 0 to 45, which was the least for TM heifers (P  0.05) and did not differ among LMB, PSPF, and PS heifers (P ≥ 0. 0; Table 3). Effects of treatment × wk and treatment were detected for average daily supplement DMI (P < 0.0001), but not daily hay DMI (P ≥ 0. 3 ). From wk 1 to 7, daily supplement DMI was always least for TM and greatest for PS heifers (P  0.05) and did not differ (P ≥ 0.5 ) b tw L MB and PSPF heifers (Figure 1). However, supplement DMI of PS heifers did not differ from wk 1 to 7 (P ≥ 0. 2) wh r l t DMI of TM, PSPF, and LMB tended to increase on wk 7 vs. all previous wk (P  0.10; Figure 1). Mean daily Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 hay DMI across treatments from d 0 to 45 was 4.04  0.117 kg, whereas overall daily supplement DMI was least for TM and greatest for PS heifers (P  0.001) and did not differ (P = 0.84) between LMB and PSPF heifers (60, 92, 91, and 386  4.9 g/d for TM, LMB, PSPF, and PS heifers, respectively). Effects of treatment were detected for total intake of supplement CP and TDN, and total intake of DM, CP, and TDN (supplement + hay) from d 0 to 45 (P  0.01), but not for total intake of hay DM, CP, TDN, RDP, and RUP, and G:F from d 0 to 45 (P ≥ 0.52 ; Table 4). Total intake of supplemental DM and TDN were least for TM and greatest for PS heifers (P  0.0005) and did not differ (P ≥ 0.91) between LMB and PSPF heifers (Table 4). Total intake of DM and TDN were least for TM heifers (P  0.03) and did not differ (P ≥ 0. 6) among LMB, PSPF, and PS heifers (Table 4). Total supplemental intake of CP and RDP and total intake of CP and RDP (supplement + hay) were least for TM and greatest for PS heifers (P  0.05) and did not differ (P ≥ 0.70) between LMB and PSPF heifers (Table 4). Total intake of RUP was greatest for PS heifers (P < 0.0001) and did not differ (P ≥ 01. 6) among LMB, PSPF, and TM heifers (Table 4). Plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin did not differ (P ≥ 0.57) o g treatments on d 0 but were included as covariates (P  0.02) to adjust the plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin, respectively, obtained on d 24 and 45. Effects of treatment × day and treatment were not detected (P ≥ 0. ) o p r lasma concentrations of IGF-1, insulin, and NEFA. Effects of treatment, but not treatment × day (P ≥ 0. 2) w r t t or l con centrations of PUN (P = 0.005) and tended to be detected for plasma concentrations of glucose (P = 0.08), which were both least for TM heifers (P  0.03) and did not differ (P ≥ 017 . ) among LMB, PSPF, and PS heifers (Table 5). Effects of treatment were not detected (P ≥ 0.45) or l t tot l i t k trace o minerals from d 0 to 45 (Table 6). Liver concentrations of trace minerals were not included as covariates (P ≥ Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 0.22) to adjust the liver concentrations of trace minerals on d 45. Effects of treatment and treatment × day were not detected (P ≥ 0. 3) or li r o tr tions of trace minerals (Table 6). DISCUSSION Aubel et al. (2011) observed that LMB consumption of mature beef cows declined over time (0.30 to 0.12 kg/d) as the forage transitioned from winter dormancy to active spring growth. Similarly, Bailey et al. (2008) reported that LMB consumption of mature beef cows increased from 0.14 to 0.36 kg/d as forage chemical composition decreased. Heifers assigned to LMB supplementation did not receive concentrate supplementation before the start of the study, and hence, the greater consumption of LMB supplement on wk 1 likely reflects the adaptation period to LMB supplementation. After adaptation, LMB consumption remained constant and at the manufacturer recommendations until wk 6. In contrast to studies described above, LMB consumption increased on wk 7, despite the greater forage quality during the last 15 d of the study. The exact reasons for this response is unknown but it demonstrates that other factors beyond forage quality may impact LMB consumption, potentially rainfall, season, animal category, BW, and supplement composition. For instance, beef calves grazing bahiagrass pastures and fed trace mineral-fortified LMB had greater supplement DMI compared to non- fortified LMB calves (272 vs. 395 g/d, respectively; Ranches et al., 2018), whereas LMB supplement in Aubel et al. (2011) and Bailey et al. (2008) contained significantly less CP compared to the present study (4 and 30 vs. 46% CP, respectively). Positive effects of LMB supplementation on intake of low-quality forages have been previously reported (Badurdeen et al., 1994; Greenwood et al., 1998; Greenwood et al., 2000). Badurdeen et al. (1994) observed a 10% increase in forage intake when bull calves were offered a 56% CP molasses-based LMB, and Greenwood et al. (1998) reported a 13% increase in forage intake when a 30% CP molasses- based LMB was provided. However, forage DMI did not differ among treatments in the present study. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Moore et al. (1999) reported that supplements decreased voluntary forage intake when supplemental TDN intake was > 0.7% BW, when forage TDN:CP ratio was < 7 (adequate CP), or when voluntary forage intake was > 1.75% BW. Supplemental TDN intake ranged from 0.02 to 0.11% of BW and forage TDN:CP ratio was between 7.3 and 9.2, whereas TM heifers had a voluntary forage consumption of 4.04 kg/d, which represents 2.13% of the average BW from d 0 to 45. Depressions in NDF digestion have been reported when sugarcane molasses was supplemented at levels of at least 15% of the dietary DM to cattle fed low-quality forage (Brown, 1993; Kalmbacher et al., 1995). In the present study, the contribution of sugarcane molasses was 1.1% of total diet DMI of LMB heifers. Consequently, the lack of treatment effects on forage DMI may not be attributed to sugar-induced depression in NDF digestion or to the supplementation levels utilized in the present study. An increase on forage intake was expected as RDP supplementation generally improves utilization of low-quality warm-season forages (Köster et al. 1996). Daily RDP requirements for cattle fed low-quality forage is approximately 11% of TDN intake (Köster et al. 1996). Hence, TM heifers consumed slight less RDP than the daily requirement (197 vs. 206 g/d of RDP, respectively), which suggests that responses to supplementation may not have been maximized, and LMB, PSPF, and PS supplementation did not cause dramatic changes to RDP consumption and potentially forage digestion. According to NRC (2016) and using the observed hay and supplement DMI of each treatment, heifers required 188 g/d of MP and 7.85 Mcal/d of ME for an ADG of 0.1 kg/d. However, estimated MP consumption were 217, 262, 262, and 292 g/d, whereas ME consumption were 6.29, 7.83, 7.82, and 8.81 Mcal/d for TM, LMB, PSPF, and PS heifers, respectively. Therefore, protein consumption was not the limiting factor for any treatment group to experience the observed ADG in the present study. In addition, TM heifers were energy-deficient and consumed 1.56 Mcal/d less than their daily ME requirements, which explains the less ADG compared to all remaining treatments. Heifers assigned to PS treatment consumed an additional 1 Mcal/d of ME compared to LMB and PSPF, which perhaps was not sufficient to induce Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 significant greater ADG in a 45-d feeding period. It is possible that differences in growth performance between LMB and PSPF vs. PS heifers would be observed with greater supplementation periods (perhaps >60 d). Only plasma concentrations of glucose and PUN differed among treatments and were greater for LMB, PSPF, and PS vs. TM heifers, which reflects the differences on ADG and can be associated with differences in energy and protein intake among treatments. Insulin and IGF-1 synthesis is directly influenced by energy intake and circulating glucose concentrations (Vizcarra et al., 1998), whereas plasma concentrations of PUN are positively associated with intake of CP, RDP, and concentrations of ruminal ammonia (Hammond, 1997). Optimal PUN concentration in beef heifers ranges from 11 to 15 mg/dL (Byers and Moxon, 1980), indicating that all heifers in the present study consumed adequate amounts of CP and RDP, except for TM heifers which were slightly below the optimum PUN levels. Despite the greatest total intake of TDN and CP, plasma concentrations of glucose and IGF-1 of PS heifers were only numerically greater, whereas plasma concentrations of insulin and PUN did not differ compared with LMB heifers. Although plasma concentrations of NEFA did not differ among treatments, NEFA may increase expression of gluconeogenic enzymes and decrease the uptake of glucose by body tissues (White et al., 2011), which may explain the numerical increase of plasma NEFA concentrations of LMB, PSPF, and PS vs. TM heifers. Blood samples were collected immediately before morning feeding at the time of BW collection in order to minimize gut fill effects on BW results and avoid disruption of diurnal feed intake. Thus, it is possible that the peak of release of all physiological parameters were missed. For instance, plasma concentrations of insulin generally peak between 1 to 2 h after feeding (Moriel et al., 2008). Pre-weaning supplementation of mineral-fortified LMB is an efficient strategy to improve the trace mineral status of calves (Ranches et al., 2018). Beef calves grazing bahiagrass pastures and Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 supplemented with trace mineral-fortified LMB had greater liver concentrations of Co, Cu, Mn, Se, and Zn compared to control non-fortified LMB calves, despite the less supplement DMI of fortified vs. non- fortified LMB calves (272 vs. 395 g/d, respectively; Ranches et al., 2018). Except for LMB, supplements offered in loose meal form were consumed entirely within 1 h after feeding. Although number of visits to LMB blocks were not measured in the present study, others have reported that cows spent more than 1 h per day visiting the sites where LMB was placed (Bailey and Welling, 2007; Bailey et al., 2008). Hence, it was expected that nutrient utilization and trace mineral status would be enhanced in LMB heifers due to slower supplement consumption pattern compared to those offered supplements in a loose meal form. In the current study, TM and PSPF heifers were limit-fed their respective supplements to achieve similar trace mineral premix compared to LMB heifers and avoid confounding effects on trace mineral intake, which would allow the proper comparison of the impact of supplement delivery form on trace mineral status of heifers. As designed, total intake (hay + supplement) of trace mineral premix and each trace mineral element did not differ among treatments, but contrary to our hypothesis, liver concentrations of all trace minerals also did not differ among heifers. These results indicate that (1) heifers consumed adequate amounts of trace minerals and were not deficient in any trace mineral element, according to NRC (2005); and (2) bioavailability and/or absorption of trace minerals was likely not impacted by supplement delivery form. Similarly, Katulski et al. (2017) observed that tissue mineral content was proportionate to mineral intake of forage-fed heifers offered LMB or free choice mineral supplement, and that differences in mineral availability between loose mineral and LMB supplements were not evident. Another potential partial explanation for the lack of treatment effects on liver trace mineral status is the impact of trace mineral antagonists (Arthington, 2017). Dietary S concentrations above 0.30% of DM may reduce Cu and Se bioavailability by associating with Mo in the rumen (Suttle, 1974; Mason, 1990; NRC, 2005). Estimated dietary S concentrations (hay + supplements) for all treatments Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 were between 0.25 to 0.28%, which is slightly below the levels reported to induce Cu and Se deficiency. However, estimated dietary concentrations of Fe ranged from 285 to 302 mg/kg, which is within the dietary concentrations linked to Cu deficiency (250 to 500 mg of Fe/kg of diet DM; NRC, 2005). Iron is found in nearly all sources of cattle feed, water, and soil (Arthington, 2017), and hence, the relatively high levels of dietary Fe can be likely attributed to soil contamination of harvested forage offered to all heifers. In conclusion, trace mineral-fortified LMB supplementation enhanced growth performance and had minor impact on physiological parameters of beef heifers fed low-quality warm-season forage compared to a non-protein, trace mineral supplement offered in loose meal form. However, supplement delivery form (block vs. loose meal protein supplement) did not impact growth performance, physiological parameters, and liver trace mineral status when heifers experience similar but adequate intake of trace minerals. Hence, the use LMB supplements led to positive effects on growth without impacting trace mineral status compared to a loose meal trace mineral salt and led to similar growth performance and trace mineral status compared to a conventional protein supplementation offered at 0.2% of body weight. References AOAC. 2006. Official methods of analysis. 18th ed. AOAC Int., Arlington, VA. Arthington, J. D. 2017. Trace mineral supplementation of grazing beef cattle. Proc. Appl. Repro. Strat. Beef Cattle. 2017:136-148. Arthington, J. D., and L. R. Corah. 1995. Liver biopsy procedures for determining the trace mineral status in beef cows. Part II. Video, AI 9134. Extension TV. Dep. Commun. Coop. Ext. Serv., Kansas State Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Univ., Manhattan, KS. Aubel, N. A., J. R. Jaeger, J. S. Drouillard, M. D. Schlegel, L. A. Pacheco, D. R. Linden, J. W. Bolte, J. J. Higgins, and K. C. Olson. 2011. Effects of mineral-supplement delivery system on frequency, duration, and timing of supplement use by beef cows grazing topographically rugged, native rangeland in the Kansas Flint Hills. J. Anim. Sci. 89:3699–3706. doi:10.2527/jas.2010-3808 Badurdeen, A. L., M.N.M. Ibrahim, and S.S.E. Ranawana. 1994. Methods to improve utilization of rice straw. III. 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T. J. Ellerman, H. C. Muller, and J. S. Drouillard. 2017. Effects of low-moisture molasses block supplements on tissue concentrations of trace elements and growth performance of forage-fed beef cattle. J. Anim. Sci. 95(Suppl. 1):310-310. doi:10.2527/asasann.2017.633 Köster, H. H., R. C. Cochran, E. C. Titgemeyer, E. S. Vanzant, I. Abdelgadir, and G. St-Jean. 1996. Effect of increasing degradable intake protein on intake and digestion of low-quality, tall-grass-prairie forage by beef cows. J. Anim. Sci. 74:2473–2481. Leupp, J. L., J. S. Caton, S. A. Soto-Navarro, and G. P. Lardy. 2005. Effects of cooked molasses blocks and fermentation extract or brown seaweed meal inclusion on intake, digestion, and microbial efficiency in steers fed low-quality hay. J. Anim. Sci. 2005. 83:2938–2945. doi.org/10.2527/2005.83122938x Löest, C. A., E. C. Titgemeyer, J. S. Drouillard, B. D. Lambert, and A. M. Trater. 2001. Urea and biuret as nonprotein nitrogen sources in cooked molasses blocks for steers fed prairie hay. Anim. Feed Sci. Technol. 94:115–126. doi.org/10.1016/S0377-8401(01)00312-1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Mason, J. 1990. The biochemical pathogenesis of molybdenum-induced copper deficiency syndromes in ruminants: Towards the final chapter. Ir. Vet. J. 43:18–22. Moore, J. E., M. H. Brant, W. E. Kunkle, and D. I. Hopkins. 1999. Effects of supplementation on voluntary forage intake, diet digestibility, and animal performance. J. Anim. Sci. 77:122– 135. doi.org/10.2527/1999.77suppl_2122x Mori l . . . t . . Filho . F. Cook J. L. M s. . 2 o 008. C o n lo centrations of progesterone and insulin in serum of nonlactating dairy cows in response to carbohydrate source and processing. J. Dairy Sci. 91:4616–4621 doi:10.3168/jds.2008-1286 Moriel, P., R. F. Cooke, D. W. Bohnert, J. M. B. Vendramini, and J. D. Arthington. 2012. Effects of energy supplementation frequency and forage quality on performance, reproductive, and physiological responses of replacement beef heifers. J. Anim. Sci. 90:2371–2380. doi:10.2527/jas.2011-4958 NRC. 2005. Mineral tolerance of animals. 2nd rev. ed. Natl. Acad. Press, Washington, DC. NRC. 2016. Nutrient requirements of beef cattle. Rev. 8th ed. Natl. Acad. Press, Washington, DC. Ranches, J., J. S. Drouillard, L. D. Silva, G. Zylberlicht, A. D. Moreira, J. S. Heldt, and J. D. Arthington. 2018. Low moisture, cooked molasses blocks for limit-creep: a method for supplementing trace minerals to pre-weaned beef calves. J. Anim. Sci. 96(Suppl. 1):51-52. doi:10.1093/jas/sky027.097 Suttle, N. F. 1974. Effects of organic and inorganic sulfur on the availability of dietary copper to sheep. Br. J. Nutr. 32:559–568. doi:10.1079/BJN19740109 Titgemeyer, E. C., J. S. Drouillard, R. H. Greenwood, J. W. Ringler, D. J. Bindel, R. D. Hunter, and T. Nutsch. 2004. Effect of forage quality on digestion and performance responses of cattle to Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 supplementation with cooked molasses blocks. J. Anim. Sci. 82:487–494. doi.org/10.2527/2004.822487x Trater, A. M., E. C. Titgemeyer, J. S. Drouillard, and J. N. Pike. 2003. Effects of processing factors on in vitro ammonia release from cooked molasses blocks containing urea. Anim. Feed Sci. Technol. 107:173–190. doi.org/10.1016/S0377-8401(03)00070-1 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 nutrition animal. J. Dairy Sci. 74:3583–3597. doi:10.3168/jds.S0022-0302(91)78551-2 Vizcarra, J. A., R. P. Wettemann, J. C. Spitzer, and D. G. Morrison. 1998. Body condition at parturition and postpartum weight gain influence luteal activity and concentrations of glucose, insulin, and nonesterified fatty acids in plasma of primiparous beef cows. J. Anim Sci. 76:927–936. doi.org/10.2527/1998.764927x 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. doi:10.1016/0377-8401(92)90034-4 White, H. M., S. L. Koser, and S. S. Donkin. 2011. Characterization of bovine pyruvate carboxylase promoter 1 responsiveness to serum from control and feed-restricted cows. J. Anim. Sci. 89:1763- 1768. doi.org/10.2527/jas.2010-3407 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 1. Average chemical composition of supplements provided from d 0 to 45. Supplement Item TM LMB PSPF PS Ingredients, % (as-fed basis) Dried sugarcane molasses - 53 .0 - - Ground corn - - 54.2 45.0 Cottonseed meal - 14.3 10.3 - Soybean meal - - - 16.0 Urea - 13.0 13.0 5.0 Kaolin - - 4.6 3.5 Limestone 40.5 - 0.80 10.8 Ca phosphate 15.0 8.7 8.8 4.0 NaCl 40.0 5.0 5.0 15.0 Trace mineral/vitamin premix 3.8 2.5 2.5 0.63 Mg oxide 0.67 0.79 0.80 0.25 Soybean oil - 2.7 - - Palatability enhancer 0.05 - - - ---------------------- DM basis ---------------------- TDN , % - 50.7 50.7 53.3 CP, % - 46.0 46.0 25.0 RDP, % of CP - 93.2 95.5 83.6 ADF, % - 7.5 9.1 9.9 Ca, % 18.6 2.3 2.3 5.0 P, % 3.0 2.0 2.0 1.0 Mg, % 1.3 0.93 0.93 1.8 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 K, % 0.03 2.41 0.30 0.32 Na, % 21.0 2.0 1.9 4.7 S, % 1.7 0.86 0.74 0.95 Cu, mg/kg 712 606 612 121 Fe, mg/kg 3214 2179 2088 435 Mn, mg/kg 3738 2467 2299 459 Se, mg/kg 119 79 81 15 Zn, mg/kg 2864 1907 1982 389 TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Samples of supplements were collected daily and pooled within each week, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients. Rumen-inert indigestible substance included. DM basis: 3% Ca, 10% Mg, 23.5% S, 600 mg/kg Co, 20,000 mg/kg Cu, 264 mg/kg Se, 1,200 mg/kg I, 80,000 mg/kg Zn, 53,200 mg/kg Mn, 4,000,000 mg/kg vitamin A, 400,000 mg/kg vitamin D , 20,000 mg/kg vitamin E, and 40,000 mg/kg monensin, Poulcox 40, Peshteria, Bulgaria). Tecnaroma zta sweet note fruit red 4W/10638 powder (New Products Comercial Agricola e Veterinária, Campinas, São Paulo, Brazil). Calculated as described by Weiss et al. (1992). Table 2. Average chemical composition of bahiagrass hay offered from d 0 to 15, 16 to 30, and 31 to 45. Hay Item d 0 to 15 d 16 to 30 d 31 to 45 ----------------- DM basis ----------------- TDN , % 42.5 45.6 60.2 CP, % 5.8 4.9 6.6 RDP, % of CP 83.7 84.5 86.0 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 ADF, % 47.9 49.4 41.6 Ca, % 0.21 0.22 0.33 P, % 0.11 0.10 0.08 Mg, % 0.25 0.21 0.22 K, % 0.33 0.21 0.85 Na, % 0.04 0.06 0.12 S, % 0.28 0.24 0.15 Cu, mg/kg 5.5 5.0 6.4 Fe, mg/kg 216 391 179 Mn, mg/kg 93 88 119 Se, mg/kg 0.10 0.08 0.10 Zn, mg/kg 30 32 41 Samples of hay were collected daily and pooled within each 15-d period, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 3. Growth performance of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Treatment Item TM LMB PSPF PS SEM × day 2,3 BW , kg d 24 186 188 188 188 0.95 0.60 0.28 d 45 194 196 195 196 0.95 ADG, kg/d a b b b d 0 to 45 0.18 0.26 0.25 0.27 0.029 - 0.10 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Average of full BW collected on d -1 e 0 was included as covariate (P < 0.0001). Full BW on d 24 and 45 represent the average of full BW collected on d 24 and 25, and 45 and 46, respectively. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 4. Total nutrient intake and overall feed efficiency of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Item TM LMB PSPF PS SEM Treatment Total intake d 0 to 45 , kg DM Hay 182 18 0 18 4 19 1 5.7 0.61 a b b c Supplement 2.56 3.94 3.87 17.4 0.208 <0.0001 a a a b Total 185 184 188 208 5.8 0.04 TDN Hay 84.3 83.5 85.1 88.3 2.63 0.60 a b b c Supplement 0 1.99 1.97 9.27 0.156 <0.0001 a a a b Total 84.3 85.4 87.0 97.6 2.48 0.009 CP Hay 10.5 10.4 10.6 11.0 0.33 0.62 a b b c Supplement 0 1.81 1.79 4.35 0.145 <0.0001 a b b c Total 10.5 12.2 12.4 14.4 0.37 <0.0001 RDP Hay 8.9 8.9 9.0 9.4 0.28 0.59 a b b c Supplement 0 1.69 1.71 3.64 0.138 <0.0001 a b b c Total 8.9 10.6 10.7 12.0 0.33 <0.0001 RUP Hay 1.58 1.56 1.59 1.65 0.049 0.62 a c b d Supplement 0 0.12 0.08 0.71 0.009 <0.0001 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 a a a b Total 1.58 1.69 1.67 2.37 0.052 <0.0001 Overall G:F d 0 to 45 0.049 0.063 0.058 0.055 0.007 0.52 a-c Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Calculated using sum of the average intake of each respective nutrient from hay and supplement at each 15-d interval. Calculated by dividing total BW gain by total DMI from d 0 to 45. Table 5. Plasma concentrations of glucose, IGF-1, insulin, urea N (PUN), and non-esterified fatty acids (NEFA) of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Plasma TM LMB PSPF PS SEM Treatment × day a b b b Glucose, mEq/L 83.6 90.9 96.1 95.5 3.54 0.88 0.08 IGF-1, ng/mL 33.1 35.1 34.5 37.8 2.6 0.95 0.61 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Insulin, IU/mL 7.11 6.69 7.02 6.97 0.474 0.78 0.93 a b b b PUN, mg/dL 9.6 11.1 11.9 11.1 0.40 0.12 0.005 NEFA, mEq/L 0.148 0.153 0.173 0.154 0.023 0.87 0.78 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Plasma concentrations of glucose, IGF-1, insulin, PUN, and NEFA were included as covariates (P  0.02). Means shown above represent the average plasma concentration of each respective parameter obtained on d 24 and 45. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 6. Total intake of premix and trace minerals from d 0 to 45 and liver concentrations of trace minerals of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Item TM LMB PSPF PS SEM Treatment × day Total intake , d 0 to 45 ------- g (DM basis) ------- Trace mineral premix 95 98 96 106 13 - 0.92 Cu 2.58 2.60 2.61 2.83 0.122 - 0.45 Fe 55.9 55.6 56.4 59.3 1.75 - 0.47 Mn 3.12 3.13 3.15 3.35 1.08 - 0.42 Se 0.04 0.04 0.04 0.05 0.001 - 0.56 Zn 13.8 14.0 13.9 15.0 0.62 - 0.55 Liver concentration ------ mg/kg (DM basis) ------ Cu 535 502 562 529 41 0.40 0.78 Fe 714 621 657 656 42 0.96 0.47 Mn 8.9 9.3 8.8 9.2 0.29 0.23 0.59 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Mo 2.8 3.1 3.1 2.9 0.10 0.96 0.13 Se 0.59 0.64 0.61 0.65 0.036 0.13 0.61 Zn 166 171 170 188 11.4 0.55 0.49 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Calculated as the total supplement and hay DMI from d 0 to 45 multiplied by the respective average concentration of each trace mineral from supplement and hay obtained every 15-d period. Liver concentrations of all trace minerals were not included as covariates (P  0.22). Means shown above represent the average liver concentration of the respective trace mineral obtained on d 0 and 45. Figure 1. Daily supplement DMI from wk 1 to 7 (d 0 to 45) of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment). Effects treatment × wk were detected for average Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 daily supplement DMI (P < 0.0001). Supplement DMI of PS heifers did not differ from wk 1 to 7 (P ≥ 0.62), whereas supplement DMI of TM, PSPF, and LMB tended to increase on wk 7 vs. all previous wk (P a-c  0.10) Within a week, means without a common superscript differed (P  0.05). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1 TM LMB PSPF PS c c c c c c c 1 2 3 4 5 6 7 Week of the study b b b b b b b b a a a Accepted Manuscript Supplement DMI, g/d http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Effects of low-moisture, sugarcane molasses-based block supplementation on growth, physiological parameters, and liver trace mineral status of growing beef heifers fed low-quality, warm-season forage

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Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Effects of low-moisture, sugarcane molasses-based block supplementation on growth, physiological parameters, and liver trace mineral status of growing beef heifers fed low- quality, warm-season forage P. Moriel* , L. F. A. Artioli, ‡M. B. Piccolo*, M. Miranda*, J. Ranches*, V. S. M. Ferreira§, L. Q. Antunes§, A. M. Bega§, V. F. B. Miranda§, J. F. R. L. Vieira§, and J. L. M. Vasconcelos§ *University of Florida – Range Cattle Research & Education Center, Ona, 33865-9706, USA ‡De Heus MBU Brazil Animal Nutrition Industry, Guararapes, São Paulo 16700-000, Brazil D rt t o A i l ro tio o lo t t i r it ot t -00 0 , Braz il Correponding author: 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/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 ABSTRACT The objectives of the study were to evaluate the growth, physiological parameters, and liver trace mineral status of beef heifers provided low-quality warm-season forage and different forms (meal vs. block) of trace mineral-fortified supplementation. One hundred yearling Nellore heifers were blocked by initial BW (184 ± 2.5 kg) and randomly assigned into 1 of 20 drylot pens (5 heifers/pen). Treatments were randomly assigned to pens (5 pens/treatment) and consisted of heifers receiving: (1) a loose meal trace mineral supplement (TM; De Heus Animal Nutrition Industry); (2) free choice access to a low- moisture, cooked sugarcane molasses-based protein block (LMB); (3) isocaloric and isonitrogenous, loose meal protein supplement pair-fed to LMB supplement DM intake (PSPF); and (4) loose meal protein supplement offered at 0.2% of BW (PS). Supplements were formulated to achieve same daily intake of supplemental trace mineral among treatments. Hence, TM supplement was offered at 66.6% of the supplement DMI of LMB heifers. Heifers were offered free choice access to water and ground brachiaria (Brachiaria brizantha) hay from d 0 to 45. Overall ADG from d 0 to 45 was the least for TM heifers (P  0.05) and did not differ among LMB, PSPF, and PS heifers (P ≥ 0. 0 ). Daily hay DMI did not differ among treatments (P ≥ 0. 3) . Total intake of DM and TDN were least for TM heifers (P  0.03) and did not differ (P ≥ 0. ) o g LM, PSPF, and PS heifers. Total supplemental intake of CP and RDP and total intake of CP and RDP (supplement + hay) were least for TM and greatest for PS heifers (P  0.05), and intermediate for LMB and PSPF heifers (P ≥ 0.70). Effects of treatment × day and treatment were not detected (P ≥ 0. ) o prlasma concentrations of IGF-1, insulin, and NEFA. Effects of treatment were detected for plasma concentrations of PUN (P = 0.005) and tended to be detected for plasma concentrations of glucose (P = 0.08), which were least for TM heifers (P  0.03) and did not differ (P ≥ 0.17) among LMB, PSPF, and PS heifers. Trace mineral intake and liver concentrations of all trace minerals did not differ (P ≥ 0. 3 ) among treatments. Hence, the use LMB supplementation resulted in positive effects on growth without impacting trace mineral status compared to a loose meal trace Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 mineral salt, and similar growth performance and trace mineral status compared to a conventional protein supplementation offered at 0.2% of body weight. Key words: block, heifers, molasses, Nellore, protein supplement, trace mineral Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 INTRODUCTION Low-moisture, cooked molasses blocks (LMB) for forage-fed cattle is a popular supplementation strategy due to its convenience, decreased production costs (labor and fuel), and potential for improving forage intake and digestion (Löest et al., 2001) and grazing of underutilized pastures (Bailey and Welling, 1999). The improved forage digestion can be attributed to the supply of RDP, which is often the most limiting nutrient under grazing of low-quality grasses (Köster et al., 1996; Titgemeyer et al., 2004). It is also possible to use mineral-fortified LMB as an efficient strategy to improve the trace mineral status of beef calves (Ranches et al., 2018). For instance, beef calves grazing bahiagrass pastures and fed trace mineral-fortified LMB had greater liver concentrations of Co, Cu, Mn, Se, and Zn compared to a non- fortified LMB, despite the less supplement DMI of fortified vs. non-fortified LMB calves (Ranches et al., 2018). The manufacturing process of LMB, particularly extreme heat or pH, may alter the bioavailability of nutrients, such as ammonia release in the rumen (Trater et al., 2003; Katulski et al., 2017), whereas the delivery form of supplements (loose meal vs. block form) may alter supplement consumption patterns and nutrient utilization (Katulski et al., 2017). Nevertheless, LMB supplementation for growing beef heifers fed low-quality forage may lead to improved nutrient consumption and trace mineral status, and consequently growth, compared to trace mineral salt offered in loose meal form. In addition, due to improved nutrient utilization, LMB supplementation may lead to similar performance compared to conventional supplementation strategies offering greater amounts of protein supplements. The objectives of the study were to evaluate growth, physiological parameters, and liver trace mineral status of beef heifers fed low-quality forage and offered different forms (meal vs. block) of trace mineral- fortified supplementation. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 MATERIALS AND METHODS Animals and experiment design The study described herein was conducted at São Paulo State University (São Manuel, São Paulo, Brazil) from November to December 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) and approved by the São Paulo State University Animal Care and Use Committee. One hundred Nellore heifers were stratified and blocked by initial BW (184 ± 2.5 kg; 12-13 mo of age), and then randomly assigned into 1 of 20 drylot pens (5 blocks; 4 pens/block; 200 m and 5 heifers/pen). Treatments were randomly assigned to pens within each block (1 pen/treatment/block; 5 pens/treatment), and consisted of heifers receiving: (1) a complete trace mineral mix supplement offered in a loose meal form (TM; De Heus Animal Nutrition Industry, Rio Claro, São Paulo); (2) free choice access to a low-moisture, cooked sugarcane molasses-based protein block (LMB; MUB , De Heus MBU Brazil Animal Nutrition Industry, Guararapes, São Paulo); (3) protein supplement offered in a loose meal form and pair-fed to achieve isocaloric and isonitrogenous supplement intake compared to LMB heifers (PSPF); and (4) a commercial protein supplement offered in a loose meal form and at levels recommended by manufacturer (PS; DM basis; De Heus Animal Nutrition Industry, Rio Claro, Sao Paulo). Treatments were offered from d 0 to 45 and all supplements included the same vitamin/trace mineral premix (DM basis: 3% Ca, 10% Mg, 23.5% S, 600 mg/kg Co, 20,000 mg/kg Cu, 264 mg/kg Se, 1,200 mg/kg I, 80,000 mg/kg Zn, 53,200 mg/kg Mn, 4,000,000 mg/kg vitamin A, 400,000 mg/kg vitamin D , 20,000 mg/kg vitamin E, and 40,000 mg/kg monensin, Poulcox 40, Peshteria, Bulgaria). All supplements were formulated to achieve same daily supplemental trace mineral intake among treatments. Hence, within each block of pens: (1) TM supplement was offered daily at 66.6% (DM basis) of the supplement DMI of LMB heifers obtained in the previous day; (2) PSPF supplement DM offered was adjusted daily to Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 achieve similar daily intake of supplemental TDN and CP compared to LMB heifers; and (3) PS supplement was offered at 0.2% of heifer initial BW (DM basis), which is the manufacturer recommended level offered in commercial beef cattle operations. Supplement DM offered to PS heifers was adjusted accordingly to the average BW obtained on d 24 and 25. Each pen assigned to LMB treatment received a single 50-kg supplement block from d 0 to 45, but each LMB block was weighed daily at 0730 h to calculate supplement DMI from previous day. Rainfall precipitation was observed on 2 d, and on these days, supplement DMI was not calculated and removed from statistical analyses. All remaining supplements were hand-fed daily at 0800 h from d 0 to 45. Heifers were offered free choice access to water and ground brachiaria (Brachiaria brizantha) hay from d 0 to 45. Hay was chopped to achieve between 2 to 5 cm of length. Hay and supplements were offered in separated feed bunks. Chemical composition of hay and supplements are shown in Table 1. Data collection Except for LMB, supplements were consumed entirely within 1 h after feeding. Hay DM offered and refused were obtained daily for each pen by drying samples of hay offered and refused in a forced- air oven at 56°C for 48 h. Daily DMI of LMB supplement was estimated by multiplying the daily DM concentration of the supplement by the weight disappearance of each supplement block obtained between consecutive days. Daily total DMI was determined by subtracting the daily hay DM refused from the total daily hay and supplement DM offered. Samples of supplement offered were collected daily and pooled within each week, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients (Table 1). Samples of hay offered were collected daily and pooled within every 15 d, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients (Table 2). Samples were Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 analyzed for concentrations of CP (method 984.13; AOAC, 2006), and ADF (method 973.18 modified for use in an Ankom 200 fiber analyzer; Ankom Technology Corp., Fairport, NY; AOAC, 2006). Concentrations of TDN were calculated as proposed by Weiss et al. (1992). Individual full BW of heifers were assessed at 0730 h on 2 consecutive days (d 0 and 1, 24 and 25, and 45 and 46), immediately before morning feeding. Shrunk BW were not obtained during the study to avoid shrink-induced stress effects on forage and supplement DMI and blood physiological parameters that could interfere with data interpretation. Blood samples (10 mL) were collected from jugular vein on d 0, 24, and 45, immediately before feeding into 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 laboratory analyses. On d 0, three heifers from each pen were randomly selected and assigned to liver tissue biopsies on d 0 and 45. Liver samples (100 mg of tissue wet weight) were collected via needle biopsy following the procedure described by Arthington and Corah (1995) th tor t −2 0 Sa °C. mples were then assessed for trace mineral concentrations at Michigan State University Diagnostic Center for Population & Animal Health (Lansing, MI). Liver trace mineral concentrations on d 0 were initially included as covariate to adjust liver trace mineral concentrations on d 45, but later removed from statistical model (P  0.22). Liver samples were collected only on d 0 and 45: (1) because our goal was to evaluate the final liver trace mineral concentrations of heifers after receiving their respective supplement for 45 d, and (2) to avoid a surgery-induced inflammatory response in the middle term of the study that could interfere with growth performance and physiological parameters. Average total trace mineral consumption from d 0 to 45 was calculated by multiplying the total DMI of hay and supplement by the average weekly mean concentration of each trace mineral present in hay and supplement. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Laboratory analyses Plasma concentrations of insulin were determined using a single chemiluminescent enzyme immunoassay (Immulite 1000; Siemens Medical Solutions Diagnostics, Los Angeles, CA). Intra- and inter- assay CV for insulin were 1.9 and 2.8%, respectively. Commercial quantitative colorimetric kits were used to determine the plasma concentrations of glucose (G7521; Pointe Scientific, Inc., Canton, MI), PUN (B7551; Pointe Scientific Inc., Canton, MI), and NEFA (HR Series NEA-2; Wako Pure Chemical Industries Ltd. USA, Richmond, VA). Inter- and intra-assay CV for assays of glucose, PUN, and NEFA were 2.7% and 3.4%, 3.2% and 5.8%, and 3.9 and 4.2%, respectively. Plasma IGF-1 concentrations were analyzed in duplicate samples using commercial enzyme-linked immunosorbent assay kits (SG100; R&D Systems Inc., Minneapolis, MN) previously validated for bovine samples (Moriel et al., 2012). Inter- and intra-assay CV for IGF-1 assay were 1.81% and 2.35%, respectively.
 Statistical analyses All data were analyzed as randomized complete block design using the MIXED procedure of 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. Pen was the experimental unit, whereas pen(treatment) and heifer(pen) were included as random effects in all analyses. Heifer BW, blood parameters, liver concentrations of trace minerals, and daily DMI of hay and supplement were analyzed as repeated measures, and tested for fixed effects of treatment, time, and resulting interaction, using pen(treatment) as the subject. The covariance structure was chosen using the lowest Akaike information criterion. Compound symmetry was used as the covariance structure in all statistical analyses, except for Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 statistical analyses of plasma concentrations of glucose, PUN, and NEFA that used the autoregressive 1 covariance structure. Heifer BW on d 0 and plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin on d 0 did not differ between treatments (P ≥ 0.40) but were included as covariates (P  0.02) in the analyses of heifer BW and plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin, respectively. Liver concentrations of all trace minerals were not included as covariates (P  0.22). Heifer overall ADG and total DM intake of hay and supplement (d 0 to 45) were tested for fixed effects of treatment using pen(treatment) as random effect. Effects of block were included in all statistical analyses but removed from model if P > 0.10. 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 tendencies were noted if P > 0.05 ≤ 0. 0 . RESULTS Effects of day (P < 0.0001), but not treatment and treatment × day (P ≥ 028 . ), were detected for heifer BW. Effects of treatment tended to be detected (P = 0.10) for overall ADG from d 0 to 45, which was the least for TM heifers (P  0.05) and did not differ among LMB, PSPF, and PS heifers (P ≥ 0. 0; Table 3). Effects of treatment × wk and treatment were detected for average daily supplement DMI (P < 0.0001), but not daily hay DMI (P ≥ 0. 3 ). From wk 1 to 7, daily supplement DMI was always least for TM and greatest for PS heifers (P  0.05) and did not differ (P ≥ 0.5 ) b tw L MB and PSPF heifers (Figure 1). However, supplement DMI of PS heifers did not differ from wk 1 to 7 (P ≥ 0. 2) wh r l t DMI of TM, PSPF, and LMB tended to increase on wk 7 vs. all previous wk (P  0.10; Figure 1). Mean daily Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 hay DMI across treatments from d 0 to 45 was 4.04  0.117 kg, whereas overall daily supplement DMI was least for TM and greatest for PS heifers (P  0.001) and did not differ (P = 0.84) between LMB and PSPF heifers (60, 92, 91, and 386  4.9 g/d for TM, LMB, PSPF, and PS heifers, respectively). Effects of treatment were detected for total intake of supplement CP and TDN, and total intake of DM, CP, and TDN (supplement + hay) from d 0 to 45 (P  0.01), but not for total intake of hay DM, CP, TDN, RDP, and RUP, and G:F from d 0 to 45 (P ≥ 0.52 ; Table 4). Total intake of supplemental DM and TDN were least for TM and greatest for PS heifers (P  0.0005) and did not differ (P ≥ 0.91) between LMB and PSPF heifers (Table 4). Total intake of DM and TDN were least for TM heifers (P  0.03) and did not differ (P ≥ 0. 6) among LMB, PSPF, and PS heifers (Table 4). Total supplemental intake of CP and RDP and total intake of CP and RDP (supplement + hay) were least for TM and greatest for PS heifers (P  0.05) and did not differ (P ≥ 0.70) between LMB and PSPF heifers (Table 4). Total intake of RUP was greatest for PS heifers (P < 0.0001) and did not differ (P ≥ 01. 6) among LMB, PSPF, and TM heifers (Table 4). Plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin did not differ (P ≥ 0.57) o g treatments on d 0 but were included as covariates (P  0.02) to adjust the plasma concentrations of glucose, PUN, IGF-1, NEFA, and insulin, respectively, obtained on d 24 and 45. Effects of treatment × day and treatment were not detected (P ≥ 0. ) o p r lasma concentrations of IGF-1, insulin, and NEFA. Effects of treatment, but not treatment × day (P ≥ 0. 2) w r t t or l con centrations of PUN (P = 0.005) and tended to be detected for plasma concentrations of glucose (P = 0.08), which were both least for TM heifers (P  0.03) and did not differ (P ≥ 017 . ) among LMB, PSPF, and PS heifers (Table 5). Effects of treatment were not detected (P ≥ 0.45) or l t tot l i t k trace o minerals from d 0 to 45 (Table 6). Liver concentrations of trace minerals were not included as covariates (P ≥ Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 0.22) to adjust the liver concentrations of trace minerals on d 45. Effects of treatment and treatment × day were not detected (P ≥ 0. 3) or li r o tr tions of trace minerals (Table 6). DISCUSSION Aubel et al. (2011) observed that LMB consumption of mature beef cows declined over time (0.30 to 0.12 kg/d) as the forage transitioned from winter dormancy to active spring growth. Similarly, Bailey et al. (2008) reported that LMB consumption of mature beef cows increased from 0.14 to 0.36 kg/d as forage chemical composition decreased. Heifers assigned to LMB supplementation did not receive concentrate supplementation before the start of the study, and hence, the greater consumption of LMB supplement on wk 1 likely reflects the adaptation period to LMB supplementation. After adaptation, LMB consumption remained constant and at the manufacturer recommendations until wk 6. In contrast to studies described above, LMB consumption increased on wk 7, despite the greater forage quality during the last 15 d of the study. The exact reasons for this response is unknown but it demonstrates that other factors beyond forage quality may impact LMB consumption, potentially rainfall, season, animal category, BW, and supplement composition. For instance, beef calves grazing bahiagrass pastures and fed trace mineral-fortified LMB had greater supplement DMI compared to non- fortified LMB calves (272 vs. 395 g/d, respectively; Ranches et al., 2018), whereas LMB supplement in Aubel et al. (2011) and Bailey et al. (2008) contained significantly less CP compared to the present study (4 and 30 vs. 46% CP, respectively). Positive effects of LMB supplementation on intake of low-quality forages have been previously reported (Badurdeen et al., 1994; Greenwood et al., 1998; Greenwood et al., 2000). Badurdeen et al. (1994) observed a 10% increase in forage intake when bull calves were offered a 56% CP molasses-based LMB, and Greenwood et al. (1998) reported a 13% increase in forage intake when a 30% CP molasses- based LMB was provided. However, forage DMI did not differ among treatments in the present study. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Moore et al. (1999) reported that supplements decreased voluntary forage intake when supplemental TDN intake was > 0.7% BW, when forage TDN:CP ratio was < 7 (adequate CP), or when voluntary forage intake was > 1.75% BW. Supplemental TDN intake ranged from 0.02 to 0.11% of BW and forage TDN:CP ratio was between 7.3 and 9.2, whereas TM heifers had a voluntary forage consumption of 4.04 kg/d, which represents 2.13% of the average BW from d 0 to 45. Depressions in NDF digestion have been reported when sugarcane molasses was supplemented at levels of at least 15% of the dietary DM to cattle fed low-quality forage (Brown, 1993; Kalmbacher et al., 1995). In the present study, the contribution of sugarcane molasses was 1.1% of total diet DMI of LMB heifers. Consequently, the lack of treatment effects on forage DMI may not be attributed to sugar-induced depression in NDF digestion or to the supplementation levels utilized in the present study. An increase on forage intake was expected as RDP supplementation generally improves utilization of low-quality warm-season forages (Köster et al. 1996). Daily RDP requirements for cattle fed low-quality forage is approximately 11% of TDN intake (Köster et al. 1996). Hence, TM heifers consumed slight less RDP than the daily requirement (197 vs. 206 g/d of RDP, respectively), which suggests that responses to supplementation may not have been maximized, and LMB, PSPF, and PS supplementation did not cause dramatic changes to RDP consumption and potentially forage digestion. According to NRC (2016) and using the observed hay and supplement DMI of each treatment, heifers required 188 g/d of MP and 7.85 Mcal/d of ME for an ADG of 0.1 kg/d. However, estimated MP consumption were 217, 262, 262, and 292 g/d, whereas ME consumption were 6.29, 7.83, 7.82, and 8.81 Mcal/d for TM, LMB, PSPF, and PS heifers, respectively. Therefore, protein consumption was not the limiting factor for any treatment group to experience the observed ADG in the present study. In addition, TM heifers were energy-deficient and consumed 1.56 Mcal/d less than their daily ME requirements, which explains the less ADG compared to all remaining treatments. Heifers assigned to PS treatment consumed an additional 1 Mcal/d of ME compared to LMB and PSPF, which perhaps was not sufficient to induce Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 significant greater ADG in a 45-d feeding period. It is possible that differences in growth performance between LMB and PSPF vs. PS heifers would be observed with greater supplementation periods (perhaps >60 d). Only plasma concentrations of glucose and PUN differed among treatments and were greater for LMB, PSPF, and PS vs. TM heifers, which reflects the differences on ADG and can be associated with differences in energy and protein intake among treatments. Insulin and IGF-1 synthesis is directly influenced by energy intake and circulating glucose concentrations (Vizcarra et al., 1998), whereas plasma concentrations of PUN are positively associated with intake of CP, RDP, and concentrations of ruminal ammonia (Hammond, 1997). Optimal PUN concentration in beef heifers ranges from 11 to 15 mg/dL (Byers and Moxon, 1980), indicating that all heifers in the present study consumed adequate amounts of CP and RDP, except for TM heifers which were slightly below the optimum PUN levels. Despite the greatest total intake of TDN and CP, plasma concentrations of glucose and IGF-1 of PS heifers were only numerically greater, whereas plasma concentrations of insulin and PUN did not differ compared with LMB heifers. Although plasma concentrations of NEFA did not differ among treatments, NEFA may increase expression of gluconeogenic enzymes and decrease the uptake of glucose by body tissues (White et al., 2011), which may explain the numerical increase of plasma NEFA concentrations of LMB, PSPF, and PS vs. TM heifers. Blood samples were collected immediately before morning feeding at the time of BW collection in order to minimize gut fill effects on BW results and avoid disruption of diurnal feed intake. Thus, it is possible that the peak of release of all physiological parameters were missed. For instance, plasma concentrations of insulin generally peak between 1 to 2 h after feeding (Moriel et al., 2008). Pre-weaning supplementation of mineral-fortified LMB is an efficient strategy to improve the trace mineral status of calves (Ranches et al., 2018). Beef calves grazing bahiagrass pastures and Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 supplemented with trace mineral-fortified LMB had greater liver concentrations of Co, Cu, Mn, Se, and Zn compared to control non-fortified LMB calves, despite the less supplement DMI of fortified vs. non- fortified LMB calves (272 vs. 395 g/d, respectively; Ranches et al., 2018). Except for LMB, supplements offered in loose meal form were consumed entirely within 1 h after feeding. Although number of visits to LMB blocks were not measured in the present study, others have reported that cows spent more than 1 h per day visiting the sites where LMB was placed (Bailey and Welling, 2007; Bailey et al., 2008). Hence, it was expected that nutrient utilization and trace mineral status would be enhanced in LMB heifers due to slower supplement consumption pattern compared to those offered supplements in a loose meal form. In the current study, TM and PSPF heifers were limit-fed their respective supplements to achieve similar trace mineral premix compared to LMB heifers and avoid confounding effects on trace mineral intake, which would allow the proper comparison of the impact of supplement delivery form on trace mineral status of heifers. As designed, total intake (hay + supplement) of trace mineral premix and each trace mineral element did not differ among treatments, but contrary to our hypothesis, liver concentrations of all trace minerals also did not differ among heifers. These results indicate that (1) heifers consumed adequate amounts of trace minerals and were not deficient in any trace mineral element, according to NRC (2005); and (2) bioavailability and/or absorption of trace minerals was likely not impacted by supplement delivery form. Similarly, Katulski et al. (2017) observed that tissue mineral content was proportionate to mineral intake of forage-fed heifers offered LMB or free choice mineral supplement, and that differences in mineral availability between loose mineral and LMB supplements were not evident. Another potential partial explanation for the lack of treatment effects on liver trace mineral status is the impact of trace mineral antagonists (Arthington, 2017). Dietary S concentrations above 0.30% of DM may reduce Cu and Se bioavailability by associating with Mo in the rumen (Suttle, 1974; Mason, 1990; NRC, 2005). Estimated dietary S concentrations (hay + supplements) for all treatments Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 were between 0.25 to 0.28%, which is slightly below the levels reported to induce Cu and Se deficiency. However, estimated dietary concentrations of Fe ranged from 285 to 302 mg/kg, which is within the dietary concentrations linked to Cu deficiency (250 to 500 mg of Fe/kg of diet DM; NRC, 2005). Iron is found in nearly all sources of cattle feed, water, and soil (Arthington, 2017), and hence, the relatively high levels of dietary Fe can be likely attributed to soil contamination of harvested forage offered to all heifers. In conclusion, trace mineral-fortified LMB supplementation enhanced growth performance and had minor impact on physiological parameters of beef heifers fed low-quality warm-season forage compared to a non-protein, trace mineral supplement offered in loose meal form. However, supplement delivery form (block vs. loose meal protein supplement) did not impact growth performance, physiological parameters, and liver trace mineral status when heifers experience similar but adequate intake of trace minerals. Hence, the use LMB supplements led to positive effects on growth without impacting trace mineral status compared to a loose meal trace mineral salt and led to similar growth performance and trace mineral status compared to a conventional protein supplementation offered at 0.2% of body weight. References AOAC. 2006. Official methods of analysis. 18th ed. AOAC Int., Arlington, VA. Arthington, J. D. 2017. Trace mineral supplementation of grazing beef cattle. Proc. Appl. Repro. Strat. 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Titgemeyer, C. A. Lo t J. . Dro ill r . 99 . t o l t strategy on intake and digestion of prairie hay by beef steers and plasma amino acid o tr tio . ro . A i . i. 4:5 − d o. i.org/10.15232/S1080-7446(15)31791-5 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Greenwood, R. H., E. C. Titgemeyer, and J. S. Drouillard. 2000. Effects of base ingredient in cooked molasses blocks on intake and digestion of prairie hay by beef steers. J. Anim. Sci. 78:167–172. doi.org/10.2527/2000.781167x Hammond, A. C. 1997. Update on BUN and MUN as a guide for protein supplementation in cattle. Pages 43–52 in Proc. Florida Ruminant Nutr. Symp., Univ. Florida, Gainesville. Kalmbacher, R. S., W. F. Brown, and F. M. Pate. 1995. Effect of molasses-based liquid supplements on digestibility of creeping bluestem and performance of mature cows on winter range. J. Anim. 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T. J. Ellerman, H. C. Muller, and J. S. Drouillard. 2017. Effects of low-moisture molasses block supplements on tissue concentrations of trace elements and growth performance of forage-fed beef cattle. J. Anim. Sci. 95(Suppl. 1):310-310. doi:10.2527/asasann.2017.633 Köster, H. H., R. C. Cochran, E. C. Titgemeyer, E. S. Vanzant, I. Abdelgadir, and G. St-Jean. 1996. Effect of increasing degradable intake protein on intake and digestion of low-quality, tall-grass-prairie forage by beef cows. J. Anim. Sci. 74:2473–2481. Leupp, J. L., J. S. Caton, S. A. Soto-Navarro, and G. P. Lardy. 2005. Effects of cooked molasses blocks and fermentation extract or brown seaweed meal inclusion on intake, digestion, and microbial efficiency in steers fed low-quality hay. J. Anim. Sci. 2005. 83:2938–2945. doi.org/10.2527/2005.83122938x Löest, C. A., E. C. Titgemeyer, J. S. Drouillard, B. D. Lambert, and A. M. Trater. 2001. Urea and biuret as nonprotein nitrogen sources in cooked molasses blocks for steers fed prairie hay. Anim. Feed Sci. Technol. 94:115–126. doi.org/10.1016/S0377-8401(01)00312-1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Mason, J. 1990. The biochemical pathogenesis of molybdenum-induced copper deficiency syndromes in ruminants: Towards the final chapter. Ir. Vet. J. 43:18–22. Moore, J. E., M. H. Brant, W. E. Kunkle, and D. I. Hopkins. 1999. Effects of supplementation on voluntary forage intake, diet digestibility, and animal performance. J. Anim. Sci. 77:122– 135. doi.org/10.2527/1999.77suppl_2122x Mori l . . . t . . Filho . F. Cook J. L. M s. . 2 o 008. C o n lo centrations of progesterone and insulin in serum of nonlactating dairy cows in response to carbohydrate source and processing. J. Dairy Sci. 91:4616–4621 doi:10.3168/jds.2008-1286 Moriel, P., R. F. Cooke, D. W. Bohnert, J. M. B. Vendramini, and J. D. Arthington. 2012. Effects of energy supplementation frequency and forage quality on performance, reproductive, and physiological responses of replacement beef heifers. J. Anim. Sci. 90:2371–2380. doi:10.2527/jas.2011-4958 NRC. 2005. Mineral tolerance of animals. 2nd rev. ed. Natl. Acad. Press, Washington, DC. NRC. 2016. Nutrient requirements of beef cattle. Rev. 8th ed. Natl. Acad. Press, Washington, DC. Ranches, J., J. S. Drouillard, L. D. Silva, G. Zylberlicht, A. D. Moreira, J. S. Heldt, and J. D. Arthington. 2018. Low moisture, cooked molasses blocks for limit-creep: a method for supplementing trace minerals to pre-weaned beef calves. J. Anim. Sci. 96(Suppl. 1):51-52. doi:10.1093/jas/sky027.097 Suttle, N. F. 1974. Effects of organic and inorganic sulfur on the availability of dietary copper to sheep. Br. J. Nutr. 32:559–568. doi:10.1079/BJN19740109 Titgemeyer, E. C., J. S. Drouillard, R. H. Greenwood, J. W. Ringler, D. J. Bindel, R. D. Hunter, and T. Nutsch. 2004. Effect of forage quality on digestion and performance responses of cattle to Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 supplementation with cooked molasses blocks. J. Anim. Sci. 82:487–494. doi.org/10.2527/2004.822487x Trater, A. M., E. C. Titgemeyer, J. S. Drouillard, and J. N. Pike. 2003. Effects of processing factors on in vitro ammonia release from cooked molasses blocks containing urea. Anim. Feed Sci. Technol. 107:173–190. doi.org/10.1016/S0377-8401(03)00070-1 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 nutrition animal. J. Dairy Sci. 74:3583–3597. doi:10.3168/jds.S0022-0302(91)78551-2 Vizcarra, J. A., R. P. Wettemann, J. C. Spitzer, and D. G. Morrison. 1998. Body condition at parturition and postpartum weight gain influence luteal activity and concentrations of glucose, insulin, and nonesterified fatty acids in plasma of primiparous beef cows. J. Anim Sci. 76:927–936. doi.org/10.2527/1998.764927x 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. doi:10.1016/0377-8401(92)90034-4 White, H. M., S. L. Koser, and S. S. Donkin. 2011. Characterization of bovine pyruvate carboxylase promoter 1 responsiveness to serum from control and feed-restricted cows. J. Anim. Sci. 89:1763- 1768. doi.org/10.2527/jas.2010-3407 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 1. Average chemical composition of supplements provided from d 0 to 45. Supplement Item TM LMB PSPF PS Ingredients, % (as-fed basis) Dried sugarcane molasses - 53 .0 - - Ground corn - - 54.2 45.0 Cottonseed meal - 14.3 10.3 - Soybean meal - - - 16.0 Urea - 13.0 13.0 5.0 Kaolin - - 4.6 3.5 Limestone 40.5 - 0.80 10.8 Ca phosphate 15.0 8.7 8.8 4.0 NaCl 40.0 5.0 5.0 15.0 Trace mineral/vitamin premix 3.8 2.5 2.5 0.63 Mg oxide 0.67 0.79 0.80 0.25 Soybean oil - 2.7 - - Palatability enhancer 0.05 - - - ---------------------- DM basis ---------------------- TDN , % - 50.7 50.7 53.3 CP, % - 46.0 46.0 25.0 RDP, % of CP - 93.2 95.5 83.6 ADF, % - 7.5 9.1 9.9 Ca, % 18.6 2.3 2.3 5.0 P, % 3.0 2.0 2.0 1.0 Mg, % 1.3 0.93 0.93 1.8 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 K, % 0.03 2.41 0.30 0.32 Na, % 21.0 2.0 1.9 4.7 S, % 1.7 0.86 0.74 0.95 Cu, mg/kg 712 606 612 121 Fe, mg/kg 3214 2179 2088 435 Mn, mg/kg 3738 2467 2299 459 Se, mg/kg 119 79 81 15 Zn, mg/kg 2864 1907 1982 389 TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Samples of supplements were collected daily and pooled within each week, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients. Rumen-inert indigestible substance included. DM basis: 3% Ca, 10% Mg, 23.5% S, 600 mg/kg Co, 20,000 mg/kg Cu, 264 mg/kg Se, 1,200 mg/kg I, 80,000 mg/kg Zn, 53,200 mg/kg Mn, 4,000,000 mg/kg vitamin A, 400,000 mg/kg vitamin D , 20,000 mg/kg vitamin E, and 40,000 mg/kg monensin, Poulcox 40, Peshteria, Bulgaria). Tecnaroma zta sweet note fruit red 4W/10638 powder (New Products Comercial Agricola e Veterinária, Campinas, São Paulo, Brazil). Calculated as described by Weiss et al. (1992). Table 2. Average chemical composition of bahiagrass hay offered from d 0 to 15, 16 to 30, and 31 to 45. Hay Item d 0 to 15 d 16 to 30 d 31 to 45 ----------------- DM basis ----------------- TDN , % 42.5 45.6 60.2 CP, % 5.8 4.9 6.6 RDP, % of CP 83.7 84.5 86.0 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 ADF, % 47.9 49.4 41.6 Ca, % 0.21 0.22 0.33 P, % 0.11 0.10 0.08 Mg, % 0.25 0.21 0.22 K, % 0.33 0.21 0.85 Na, % 0.04 0.06 0.12 S, % 0.28 0.24 0.15 Cu, mg/kg 5.5 5.0 6.4 Fe, mg/kg 216 391 179 Mn, mg/kg 93 88 119 Se, mg/kg 0.10 0.08 0.10 Zn, mg/kg 30 32 41 Samples of hay were collected daily and pooled within each 15-d period, and then sent in duplicate to a commercial laboratory (3rlab, Lavras, Minas Gerais, Brazil) for wet chemistry analysis of all nutrients. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 3. Growth performance of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Treatment Item TM LMB PSPF PS SEM × day 2,3 BW , kg d 24 186 188 188 188 0.95 0.60 0.28 d 45 194 196 195 196 0.95 ADG, kg/d a b b b d 0 to 45 0.18 0.26 0.25 0.27 0.029 - 0.10 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Average of full BW collected on d -1 e 0 was included as covariate (P < 0.0001). Full BW on d 24 and 45 represent the average of full BW collected on d 24 and 25, and 45 and 46, respectively. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 4. Total nutrient intake and overall feed efficiency of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Item TM LMB PSPF PS SEM Treatment Total intake d 0 to 45 , kg DM Hay 182 18 0 18 4 19 1 5.7 0.61 a b b c Supplement 2.56 3.94 3.87 17.4 0.208 <0.0001 a a a b Total 185 184 188 208 5.8 0.04 TDN Hay 84.3 83.5 85.1 88.3 2.63 0.60 a b b c Supplement 0 1.99 1.97 9.27 0.156 <0.0001 a a a b Total 84.3 85.4 87.0 97.6 2.48 0.009 CP Hay 10.5 10.4 10.6 11.0 0.33 0.62 a b b c Supplement 0 1.81 1.79 4.35 0.145 <0.0001 a b b c Total 10.5 12.2 12.4 14.4 0.37 <0.0001 RDP Hay 8.9 8.9 9.0 9.4 0.28 0.59 a b b c Supplement 0 1.69 1.71 3.64 0.138 <0.0001 a b b c Total 8.9 10.6 10.7 12.0 0.33 <0.0001 RUP Hay 1.58 1.56 1.59 1.65 0.049 0.62 a c b d Supplement 0 0.12 0.08 0.71 0.009 <0.0001 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 a a a b Total 1.58 1.69 1.67 2.37 0.052 <0.0001 Overall G:F d 0 to 45 0.049 0.063 0.058 0.055 0.007 0.52 a-c Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Calculated using sum of the average intake of each respective nutrient from hay and supplement at each 15-d interval. Calculated by dividing total BW gain by total DMI from d 0 to 45. Table 5. Plasma concentrations of glucose, IGF-1, insulin, urea N (PUN), and non-esterified fatty acids (NEFA) of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Plasma TM LMB PSPF PS SEM Treatment × day a b b b Glucose, mEq/L 83.6 90.9 96.1 95.5 3.54 0.88 0.08 IGF-1, ng/mL 33.1 35.1 34.5 37.8 2.6 0.95 0.61 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Insulin, IU/mL 7.11 6.69 7.02 6.97 0.474 0.78 0.93 a b b b PUN, mg/dL 9.6 11.1 11.9 11.1 0.40 0.12 0.005 NEFA, mEq/L 0.148 0.153 0.173 0.154 0.023 0.87 0.78 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Plasma concentrations of glucose, IGF-1, insulin, PUN, and NEFA were included as covariates (P  0.02). Means shown above represent the average plasma concentration of each respective parameter obtained on d 24 and 45. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Table 6. Total intake of premix and trace minerals from d 0 to 45 and liver concentrations of trace minerals of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment) . Treatment P-value Treatment Item TM LMB PSPF PS SEM Treatment × day Total intake , d 0 to 45 ------- g (DM basis) ------- Trace mineral premix 95 98 96 106 13 - 0.92 Cu 2.58 2.60 2.61 2.83 0.122 - 0.45 Fe 55.9 55.6 56.4 59.3 1.75 - 0.47 Mn 3.12 3.13 3.15 3.35 1.08 - 0.42 Se 0.04 0.04 0.04 0.05 0.001 - 0.56 Zn 13.8 14.0 13.9 15.0 0.62 - 0.55 Liver concentration ------ mg/kg (DM basis) ------ Cu 535 502 562 529 41 0.40 0.78 Fe 714 621 657 656 42 0.96 0.47 Mn 8.9 9.3 8.8 9.2 0.29 0.23 0.59 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Mo 2.8 3.1 3.1 2.9 0.10 0.96 0.13 Se 0.59 0.64 0.61 0.65 0.036 0.13 0.61 Zn 166 171 170 188 11.4 0.55 0.49 a-b Within a row, means without a common superscript differed (P  0.05). TM = a complete vitamin/trace mineral mix supplement offered in a loose meal form (66.6% of LMB supplement DMI); LMB = free choice access to a low-moisture, cooked sugarcane molasses-based block; PSPF = protein supplement offered in a loose meal form and pair-fed to achieve similar supplement intake of DM, TDN and CP of LMB heifers; PS = a commercial protein supplement offered in a loose meal form at 0.2% of BW (DM basis). Calculated as the total supplement and hay DMI from d 0 to 45 multiplied by the respective average concentration of each trace mineral from supplement and hay obtained every 15-d period. Liver concentrations of all trace minerals were not included as covariates (P  0.22). Means shown above represent the average liver concentration of the respective trace mineral obtained on d 0 and 45. Figure 1. Daily supplement DMI from wk 1 to 7 (d 0 to 45) of Nellore heifers offered free choice access to warm-season grass hay and receiving trace mineral supplement (TM), sugarcane molasses cooked block (LMB), pair-fed protein supplement (PSPF), and a commercial protein supplement (PS) from d 0 to 45 (n = 100 heifers; 5 heifers/pen; 5 pens/treatment). Effects treatment × wk were detected for average Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 daily supplement DMI (P < 0.0001). Supplement DMI of PS heifers did not differ from wk 1 to 7 (P ≥ 0.62), whereas supplement DMI of TM, PSPF, and LMB tended to increase on wk 7 vs. all previous wk (P a-c  0.10) Within a week, means without a common superscript differed (P  0.05). Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txy123/5183292 by Ed 'DeepDyve' Gillespie user on 20 November 2018 Figure 1 TM LMB PSPF PS c c c c c c c 1 2 3 4 5 6 7 Week of the study b b b b b b b b a a a Accepted Manuscript Supplement DMI, g/d

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Translational Animal ScienceOxford University Press

Published: Nov 14, 2018

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