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Effects of cyclic monensin feeding on ruminal function in cannulated beef steers consuming low-quality forage

      ABSTRACT

      Objective

      The aim of this research was to evaluate the effects of cyclically feeding monensin (Rumensin 90, Elanco Animal Health) to ruminally cannulated steers (12 Bos taurus; 260 kg of BW) consuming low-quality forage (4.9% CP) on measures of intake, digestion, and ruminal function.

      Materials and Methods

      Twelve steers were randomly assigned to 1 of 3 treatments in a completely randomized design: (1) no monensin (CON); (2) 200 mg·head−1·d−1 monensin (MON); or (3) 200 mg·head−1·d−1 monensin for 14 d and 0 mg·head−1·d−1 monensin for the subsequent 14 d (CYC; recurrent cycle). Hay and ort samples were collected d 10 through 13 for intake determination during each of the four 28-d replicated cycles. Rumen fluid was collected with a suction strainer 0, 2, 4, 8, and 12 h after feeding on d 14 for pH and VFA analysis. Data were analyzed using the MIXED procedure of SAS 9.4 (SAS Institute Inc.).

      Results and Discussion

      Treatment × cycle interactions or treatment effects were not observed for any measure of intake (OM intake, NDF intake; P ≥ 0.17) or pH (P = 0.13). A tendency for a treatment × cycle interaction was observed (P = 0.08) for OM digestion but not for NDF digestion (P = 0.37). Treatment × cycle interactions were observed (P < 0.01) for molar proportions of acetate, propionate, and acetate:propionate ratio. Because these treatment × cycle interactions occurred, treatment × hour interactions, treatment effects, and hour effects were evaluated within cycles for these parameters. Treatment × hour interactions (P ≤ 0.02) occurred for molar proportion of propionate in cycle 1 and propionate and acetate:propionate ratio in cycle 3, and there was a tendency (P = 0.09) for a treatment × hour interaction for acetate:propionate in cycle 4. Although treatment effects were observed for molar proportions of acetate, propionate, and acetate:propionate ratio in cycle 1 (P ≤ 0.01), these effects had diminished by cycle 4 (P ≥ 0.39). An effect of hour after feeding was observed (P ≤ 0.02) in all 4 cycles for acetate, propionate, acetate:propionate, and ruminal pH.

      Implications and Applications

      Further evaluation of monensin feeding methods that vary duration of feeding and withdrawal periods may be warranted. Additionally, further study concerning differences in the effect of these methods on a diet bases on concentrate versus low-quality forage warrant investigation.

      Key words

      INTRODUCTION

      Agents that alter ruminal fermentation can improve diet utilization and increase animal productivity in terms of meat and milk yield or efficiency of yield per unit of resources consumed. Monensin, a carboxylic polyether ionophore antibiotic, is extensively used in the feedlot sector to achieve this goal (
      • Goodrich R.D.
      • Garrett J.E.
      • Gast D.R.
      • Kirick M.A.
      • La4son D.A.
      • Meiske J.C.
      Influence of monensin on the performance of cattle..
      ;
      • Beauchemin K.A.
      • McGinn S.M.
      Methane emissions from feedlot cattle fed barley or corn diets..
      ;
      • Guan H.
      • Wittenberg K.M.
      • Ominski K.H.
      • Krause D.O.
      Efficacy of ionophores in cattle diets for mitigation of enteric methane..
      ). Conversely, its effectiveness has been documented to a lesser extent in grazing systems (
      • Lemenager R.P.
      • Owens F.N.
      • Lusby K.S.
      • Totusek R.
      Monensin, forage intake and lactation of range beef cows..
      ;

      Crosthwait, G. L., S. E. Coleman, and R. D. Wyatt. 1979. Effect of monensin on weight gain and forage intake by replacement heifers on native range. Anim. Sci. Res. Report, pages 87–90. Oklahoma Agric. Exp. Stn.

      ;

      Ellis, W. C., G. W. Horn, D. Delaney, and K. R. Pond. 1983. Effects of ionophores on grazed forage utilization and their economic value for cattle on wheat pasture. Pages 343–355 in Proc. Nat. Wheat Past. Symp. MP-115. Oklahoma Agric. Exp. Stn.

      ) because widespread use of monensin in grazing systems has been constrained by the difficulty associated with providing monensin to these animals on a daily basis.
      Loss of monensin efficacy when fed continuously has been previously observed by
      • Rumpler W.V.
      • Johnson D.E.
      • Bates D.B.
      The effect of high dietary cation concentration of methanogensis by steers fed diets with and without ionophores..
      and
      • Guan H.
      • Wittenberg K.M.
      • Ominski K.H.
      • Krause D.O.
      Efficacy of ionophores in cattle diets for mitigation of enteric methane..
      , and the likelihood of microbial populations becoming refractory to monensin has been considered by several other researchers (
      • Dawson K.A.
      • Boling J.A.
      Monensin-resistant bacteria in the rumen of calves on monensin-containing and unmedicated diets..
      ;
      • Russell J.B.
      • Strobel H.J.
      Effect of ionophore on ruminal fermentation..
      ;
      • Callaway T.R.
      • Edrington T.S.
      • Rychilk J.L.
      • Genovese K.J.
      • Poole T.L.
      • Jung Y.S.
      • Bischoff K.M.
      • Anderson R.C.
      • Nisbet D.J.
      Ionophores: Their use as ruminant growth promotants and impact on food safety..
      ;
      • Simjee S.
      • Heffron A.L.
      • Pridmore A.
      • Shryock T.R.
      Reversible monensin adaptation in Enterococcus faeciumEnterococcus faecalis and Clostridium perfringens of cattle origin: Potential impact on human food safety..
      ). The concept of ionophore rotation (alternating the specific molecule supplied) has been suggested to alleviate loss of effectiveness from continuous use of a single ionophore (

      Galyean, M. L., and M. E. Hubbert. 1989. Rationale for use and selection of ionophores in ruminant production. Pages 64–81 in Proc. Southwest Nutr. Manage. Conf. Univ. Arizona, Tucson.

      ;
      • Morris F.E.
      • Branine M.E.
      • Galyean M.L.
      • Hubbert M.E.
      • Freeman A.S.
      • Lofgreen L.P.
      Effect of rotating monensin plus tylosin and lasalocid on performance, ruminal fermentation, and site and extent of digestion in feedlot cattle..
      ;
      • Guan H.
      • Wittenberg K.M.
      • Ominski K.H.
      • Krause D.O.
      Efficacy of ionophores in cattle diets for mitigation of enteric methane..
      ;

      Crossland, W., L. Tedeschi, T. R. Callaway, M. Miller, B. W. Smith, and M. Cravey. 2015. Effects of rotating antibiotic and ionophore feed additives on enteric methane and volatile fatty acid production of steers consuming a high forage diet. Accessed Dec. 14, 2020. http://m.jtmtg.org/abs/t/63995.

      ); however, each of these authors determined that ionophore rotation was unsuccessful in prolonging the effects of ionophores.
      According to
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      , the benefits of monensin are most pronounced during the second week (d 7–14) of monensin inclusion and remain evident for at least 7 d after withdrawal (
      • Bell N.L.
      • Callaway T.R.
      • Anderson R.C.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin withdrawal on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      ); yet, by d 14 after withdrawal, all measured parameters (intake, digestion, VFA, pH, ruminal ammonia, and methane) returned to their control values. Thus, it was hypothesized that if provided cyclically (14 d of monensin followed by 14 d without) rather than continually (providing monensin daily), monensin would continue to improve nutrient utilization each time it was added to the diet, therefore prolonging its beneficial effects. The specific objectives of this study were to (1) evaluate the effects of cyclic monensin feeding on intake, digestion, and ruminal fermentation parameters when beef cattle are consuming a low-quality forage basal diet and (2) quantify the persistence of monensin effects on intake, digestion, and ruminal fermentation parameters when fed cyclically to beef cattle consuming a low-quality-forage diet to determine whether the benefits would be observed to the same extent with each subsequent addition or whether they would diminish over time as with continual feeding.

      MATERIALS AND METHODS

      Animals and Facilities

      The experimental protocol was approved by the Institutional Animal Care and Use Committee at Texas A&M University–Kingsville and included the use of anesthesia when surgical procedures were performed (IACUC Approval No. 2017-03-28).
      Twelve steers (Bos taurus; 260 ± 18 kg of BW) were fitted with ruminal cannulas and randomly assigned to a pen in a completely randomized design. Steers were individually housed in covered soil-surfaced pens (3.05 m × 3.05 m) for the duration of the study.

      Diet and Treatment

      Steers had continuous access to fresh water and trace-mineral-salt blocks (≥96% NaCl, 1.00% S, 0.15% Fe, 0.25% Zn, 0.30% Mn, 0.009% I, 0.015% Cu, 0.0025% Co, and 0.001% Se; United Salt Corporation). Bluestem grass hay (Andropogon gerardii; Table 1) was offered at 0600 h daily at 130% of the previous 3-d average consumption.
      Table 1Chemical composition of low-quality forage (bluestem grass hay) provided ad libitum and dried distillers grains with solubles (DDGS) provided as monensin carrier (1 kg·head−1·d−1) fed to ruminally cannulated beef steers
      Item (% CM)Bluestem hayDDGS
      OM93.1294.28
      CP4.8629.56
      NDF83.8151.86
      ADF42.0720.72
      The experimental treatments were as follows:
      • 1
        CON: 0 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health) for the entirety of the study.
      • 2
        MON: 200 mg·head−1·d−1 monensin for the entirety of the study.
      • 3
        CYC: 200 mg·head−1·d−1 monensin for 14 d and 0 mg·head−1·d−1 of monensin for the subsequent 14 d. This cycle was repeated 4 times, resulting in four 28-d cycles.
      Treatments were provided to steers individually at 0600 h daily within a carrier of 1 kg·head−1·d−1 dried distillers grains with solubles (DDGS). Steers received their assigned treatment for the entirety of the 112-d study.

      Experimental Protocol and Sampling

      The study consisted of four 28-d cycles. Days 1 through 9 of each cycle allowed for treatment adaptation and d 10 through 14 for sampling. Treatment protocols continued on d 15 to 28 of each cycle without sampling. This 14-d period allowed time for withdrawal of monensin in the CYC treatment group.
      Measures of intake and digestion occurred between d 10 and 14 of each cycle. Hay and DDGS samples were collected on d 10 through 13 and composited within each cycle to correspond with ort and fecal samples collected on d 11 through 14. Orts were collected immediately before daily feeding and composited within animal for each cycle.
      Beginning at 0600 h on d 11, fecal grab samples were obtained 3 times daily. Fecal samples were collected every 8 h for 4 d with initial sample delayed by 2 h daily. Fecal samples were composited within animal and cycles, and then frozen at −20°C until further analysis.
      Rumen fluid (20 mL) was collected via suction strainer from 3 different locations within the ventral and dorsal sacs of the rumen. Rumen fluid was collected for VFA and pH analysis at 0, 2, 4, 8, and 12 h after feeding on d 14. A portable pH meter (Symphony, VWR) was used to measure pH at the time of sampling. Subsequently, an 8-mL ruminal fluid subsample was combined with 2 mL of freshly prepared 25% (wt/vol) metaphosphoric acid in a scintillation vial. This mixture was frozen at −20°C for subsequent VFA analysis.

      Laboratory Analyses

      Intake and Digestion.

      Hay, DDGS, ort, and fecal samples were dried in a forced-air oven for 96 h at 55°C. Samples were allowed to air equilibrate for partial DM determination. Samples were ground (No. 4 Wiley Mill, Thomas Scientific) to allow passage through 1- and 2-mm sieves. Chemical analyses were performed using samples ground to 1 mm. Hay, DDGS, ort, and fecal samples were dried at 105°C for DM determination and then combusted for 8 h at 450°C for OM determination. Crude protein was determined by an independent laboratory (Cumberland Valley Analytical Services Inc.). Indigestible NDF (iNDF) was used as an internal marker and determined on hay, DDGS, ort, and fecal samples. Previously ground samples (2 mm) were weighed into ANKOM 57 filter bags, heat sealed, and incubated for 264 h (
      • Casali A.O.
      • Detmann E.
      • De Campos Valadares Filho S.
      • Pereira J.C.
      • Henriques L.T.
      • de Freitas S.G.
      • Paulino M.F.
      Influence of incubation time and particle size on indigestible compound in cattle feeds and feces obtained by in situ procedures..
      ) in a ruminally cannulated Bos taurus steer fed an ad libitum diet of bermudagrass (Cynodon dactylon) hay. Samples were then removed from the rumen, submerged in ice water to stop microbial activity, and rinsed with tap water until the water ran clear. After washing, samples were dried at 60°C for 96 h. An ANKOM fiber analyzer (ANKOM Technology) was used to accomplish NDF analysis according to

      Goering, H. K., and P. J. Van Soest. 1975. Forage Fiber Analysis. (Apparatus, Reagents, Procedures and Some Applications). Agriculture Handbook, 379. USDA ARS.

      , with sodium sulfite and α-amylase omitted, and final weights were used to calculate iNDF according to
      • Valente T.N.P.
      • Detmann E.
      • Filho S.C.V.
      • Cunha M.D.
      • de Queiroz A.C.
      • Sampaio C.B.
      In situ estimation of indigestible compounds contents in cattle feed and feces using bags made from different textiles..
      . Total iNDF intake was calculated based on iNDF values for hay, DDGS, and ort samples. These values were multiplied by the iNDF value of feces to calculate total fecal production and, ultimately, apparent digestibility.

      VFA.

      Previously prepared ruminal fluid samples were thawed and centrifuged at 15,000 × g for 10 min at 22°C. Supernatant fluid was collected and stored at −20°C for VFA analysis. After thawing, VFA concentrations were determined using gas chromatography [Varian Model 3800; Varian Inc.; equipped with a glass column (180 cm × 4 mm i.d.)] packed with GP 10% SP-1200/1% H3PO4 on 80/100 Chromosorb WAW (Supelco), with N2 used as a carrier gas at a flow rate of 85 mL∙min−1.

      Statistical Analysis

      Intake and digestion were analyzed as CRD with repeated measures using the MIXED procedure of SAS 9.4 (SAS Institute Inc.). Fixed effects in the model included treatment, cycle, and treatment × cycle. The repeated measures factor was cycle with animal nested within treatment as the subject. Ruminal fermentation parameters (VFA and pH) were analyzed as CRD with repeated measures using the MIXED procedure. Fixed effects included treatment, cycle, hour, and their interactions; random effects included animal nested within treatment, and the interaction between cycle and animal nested within treatment. Hour was analyzed as a repeated measures factor with the interaction between cycle and animal nested within treatment as a subject. We modeled nonindependence for the repeated measured factor in these analyses with the following variance-covariance structures: first-order autoregressive, compound symmetry, and Toeplitz and their heteroscedastic forms; variance components; first-order autoregressive moving average, and unstructured. We used corrected Akaike information criteria to select the most appropriate variance-covariance structure. Normality of residuals was assessed with the Shapiro-Wilk test (
      • Shapiro S.S.
      • Wilk M.B.
      An analysis of variance test for normality (Complete Samples)..
      ). When residuals were skewed, we applied a version of the Box-Cox transformation (
      • Box G.E.P.
      • Cox D.R.
      An analysis of transformations..
      ), with exponents for a power transformation ranging from −2 to 2 (by increments of 0.5) and including a log-transformation; the objective was to maximize the Shapiro-Wilk test statistic. All inferential statistics are based on transformed data (when needed); we present back-transformed means ± SE (

      Sokal, R. R., and J. F. Rohlf. 2012. Biometry. 4th ed. W.H. Freeman and Co.

      ). Because treatment × cycle interactions were observed for several VFA, we tested hypotheses about treatment, hour, and their interaction within each cycle with contrast statements. When significant effects of treatment were observed, pairwise comparisons were made using t-tests. When analyzing data, P ≤ 0.05 was considered significant, and P > 0.05 and ≤0.10 was considered a tendency.

      RESULTS AND DISCUSSION

      To meet nutrient demands, forages are an essential component of cattle production operations. The nutritional quality of these forages, however, can vary significantly throughout the year and, at times, may require supplementation to improve utilization. In an effort to study the effectiveness of monensin on improving productivity of grazing animals during a time of low-quality-forage consumption, this study observed its effects over a 112-d period (a common supplementation period for grazing cattle). Evidence (
      • Rumpler W.V.
      • Johnson D.E.
      • Bates D.B.
      The effect of high dietary cation concentration of methanogensis by steers fed diets with and without ionophores..
      ;
      • Guan H.
      • Wittenberg K.M.
      • Ominski K.H.
      • Krause D.O.
      Efficacy of ionophores in cattle diets for mitigation of enteric methane..
      ) suggests that the effects of monensin do not provide a long-term means of improved nutrient utilization. For example, methane production in a study by
      • Guan H.
      • Wittenberg K.M.
      • Ominski K.H.
      • Krause D.O.
      Efficacy of ionophores in cattle diets for mitigation of enteric methane..
      returned to baseline levels by the third week of monensin inclusion when cattle were fed a high-forage diet (13.1% CP). In the present study, the effect of monensin fed cyclically was evaluated to determine whether improved nutrient utilization could be prolonged with cyclic feeding.

      Intake

      No treatment × cycle (P ≥ 0.43) or treatment (P ≥ 0.17) effects were observed for any intake parameter measured (Table 2), including forage OM intake, total OM intake, total digestible OM intake, forage NDF intake, total NDF intake, and total digestible NDF intake. Cycle effects (P ≤ 0.01) were observed for all intake parameters measured. Intake increased from cycle 1 to 2, remained stable between cycle 2 and 3, and increased from cycle 3 to 4. Monensin supplementation to cattle consuming forage-based diets has produced various responses. The majority of studies report no effect of monensin on forage intake (

      Crosthwait, G. L., S. E. Coleman, and R. D. Wyatt. 1979. Effect of monensin on weight gain and forage intake by replacement heifers on native range. Anim. Sci. Res. Report, pages 87–90. Oklahoma Agric. Exp. Stn.

      ;
      • Ward M.G.
      • Adams D.C.
      • Wallace J.D.
      • Galyean M.I.
      • Knapp B.W.
      Supplementation and monensin effects on digesta kinetics. I. Cattle grazing summer range..
      ,
      • Ward M.G.
      • Adams D.C.
      • Wallace J.D.
      • Galyean M.I.
      • Volesky J.D.
      Supplementation and monensin effects on digesta kinetics. II. Cattle grazing winter range..
      ;
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      ). However, few reported a reduction in forage intake when monensin was applied (
      • Turner H.A.
      • Raleigh R.J.
      • Young D.C.
      Effect of monensin on feed efficiency for maintaining gestating mature cows wintered on meadow hay..
      ;
      • Lemenager R.P.
      • Owens F.N.
      • Lusby K.S.
      • Totusek R.
      Monensin, forage intake and lactation of range beef cows..
      ,
      • Lemenager R.P.
      • Owens F.N.
      • Shockey B.J.
      • Lusby K.S.
      • Totusek R.
      Monensin effects on rumen turnover rate, twenty-four-hour VFA pattern, nitrogen components and cellulose disappearance..
      ) and even fewer reported an increase (

      DeLaney, D. S. 1980. Effects of monensin on intake, digestibility, and turnover of organic matter and bacterial protein in grazing cattle. MS Thesis. Texas A&M Univ., College Station.

      ).

      Ellis, W. C., G. W. Horn, D. Delaney, and K. R. Pond. 1983. Effects of ionophores on grazed forage utilization and their economic value for cattle on wheat pasture. Pages 343–355 in Proc. Nat. Wheat Past. Symp. MP-115. Oklahoma Agric. Exp. Stn.

      suggests that the direction and magnitude of intake response to monensin is related to forage digestibility and the animal’s energy requirements. Feeding monensin continuously or cyclically was not different from the control. Although inconsistent with the hypothesis that cyclic monensin feeding would continue to improve overall nutrient utilization each time it is added to the diet, the lack of intake response was anticipated as it is in agreement with the majority of previous work in cattle consuming forage-based diets. Improvements resulting from monensin inclusion are generally most notable in ruminal fermentation parameters, such as VFA concentrations, rather than intake or digestion.
      Table 2Effects of exclusion, continual, and cyclic monensin feeding on nutrient intake by ruminally cannulated beef steers consuming low-quality forage (bluestem grass hay) supplemented with dried distillers grains with solubles (DDGS) as monensin carrier
      ItemTreatment
      CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      SEMP-value
      T × C = treatment × cycle interaction.
      CONMONCYCTreatmentCycleT × C
      OM intake (g/kg of BW)
       Forage16.0418.1514.071.410.17<0.010.83
       DDGS3.433.433.43
       Total19.4821.5817.501.410.17<0.010.82
       Digestible12.4513.0011.590.650.34<0.010.35
      NDF intake (g/kg of BW)
       Forage14.2916.1612.511.260.17<0.010.86
       DDGS1.871.871.87
       Total16.1918.0314.381.260.17<0.010.85
       Digestible11.2611.8210.130.710.27<0.010.67
      1 CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      2 T × C = treatment × cycle interaction.

      Digestion

      Similar to intake, others (
      • Cochran R.C.
      • Vanzant E.S.
      • Riley J.G.
      • Owensby C.E.
      Influence of intraruminal monensin administration on performance and forage use in beef cattle grazing early-summer bluestem range..
      ;
      • Fredrickson E.L.
      • Galyean M.L.
      • Branine M.E.
      • Sowell B.
      • Wallace J.D.
      Influence of ruminally dispensed monensin and forage maturity on intake and digestion..
      ;
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      ) have reported minimal effects of monensin on diet digestibility when cattle are consuming a forage-based diet. In cattle grazing early-summer bluestem range (12% CP),
      • Cochran R.C.
      • Vanzant E.S.
      • Riley J.G.
      • Owensby C.E.
      Influence of intraruminal monensin administration on performance and forage use in beef cattle grazing early-summer bluestem range..
      observed no difference in OM digestibility (OMD) between cattle provided monensin (100 mg·steer−1·d−1 via ruminal delivery device) and control (71.4 vs. 68.7% OMD, respectively).
      • Fredrickson E.L.
      • Galyean M.L.
      • Branine M.E.
      • Sowell B.
      • Wallace J.D.
      Influence of ruminally dispensed monensin and forage maturity on intake and digestion..
      reported no effect of monensin on OMD (56.5 vs. 57.7% OMD, respectively) in cattle grazing native blue grama rangeland (7.1% CP, 85.4% NDF, and 53.0% ADF), with monensin (68 mg·steer−1·d−1 via ruminal delivery device) or without. Furthermore,
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      reported forage OMD to be similar for control and monensin (200 mg·steer−1·d−1) steers (60.5 vs. 58.9%, respectively) consuming bermudagrass hay (13.7% CP).
      In contrast to these reports, a tendency (P < 0.08) toward an interaction between treatment and cycle for OMD was observed (Figure 1) in the present study, where OMD was lower (P < 0.03) in the MON treatment than in the CYC treatment during cycle 2; however, similar to previous reports, during cycles 1, 3, and 4, no treatment effects (P ≥ 0.39) were observed. No treatment × cycle (P = 0.37; Table 3) or treatment (P = 0.32) effects were observed for NDF digestibility; however, a cycle effect (P < 0.01) was observed. Neutral detergent fiber digestion was not different among cycles 1, 2, and 4 but was lower in cycle 3.
      Figure 1
      Figure 1Effects of continual and cyclic monensin inclusion on mean OM digestion (OMD; ±1 SEM) in steers consuming bluestem grass hay. CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d. The OMD means within a cycle followed by the same lowercase letter (a, b) are not significantly different (P > 0.05, protected LSD).
      Table 3Effects of exclusion, continual, and cyclic monensin feeding on nutrient apparent digestibility by ruminally cannulated beef steers consuming low-quality forage (bluestem grass hay) supplemented with dried distillers grains with solubles as monensin carrier
      Total-tract digestion (%)Treatment
      CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      P-value
      T × C = treatment × cycle interaction.
      CONMONCYCTreatmentCycleT × C
      OM65.3860.8866.810.41<0.010.08
       (Mean ± SEM)
      Back-transformed means have asymmetric SE (Sokal and Rohlf, 2012).
      (62.26, 68.66)(57.98, 63.93)(63.83, 70.16)
      NDF71.0066.1371.060.32<0.010.37
       (Mean ± SEM)(68.49, 73.61)(63.79, 68.56)(68.54, 76.67)
      1 CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      2 T × C = treatment × cycle interaction.
      3 Back-transformed means have asymmetric SE (

      Sokal, R. R., and J. F. Rohlf. 2012. Biometry. 4th ed. W.H. Freeman and Co.

      ).

      Rumen pH

      There were no 3- or 2-way interactions (P ≥ 0.16) or treatment (P = 0.13) effects observed for rumen pH in the present study. These results were as expected and are in agreement with previous work with monensin in cattle consuming forage-based diets (
      • Ward M.G.
      • Adams D.C.
      • Wallace J.D.
      • Galyean M.I.
      • Volesky J.D.
      Supplementation and monensin effects on digesta kinetics. II. Cattle grazing winter range..
      ;
      • Fredrickson E.L.
      • Galyean M.L.
      • Branine M.E.
      • Sowell B.
      • Wallace J.D.
      Influence of ruminally dispensed monensin and forage maturity on intake and digestion..
      ; and
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      ). Ward observed no difference in ruminal pH between steers provided monensin (101 mg·steer−1·d−1 via ruminal delivery device) and control (6.49 vs. 6.45, respectively) steers grazing native range (8.3% CP in trial 1 and 4.9% CP in trial 2). In a similar trial by
      • Fredrickson E.L.
      • Galyean M.L.
      • Branine M.E.
      • Sowell B.
      • Wallace J.D.
      Influence of ruminally dispensed monensin and forage maturity on intake and digestion..
      , monensin (68 mg·steer−1·d−1 via ruminal delivery device) had no effect on ruminal pH in steers grazing native range (7.1% CP; 6.4 vs. 6.5, for monensin vs. control, respectively). Similarly,
      • Bell N.L.
      • Anderson R.C.
      • Callaway T.R.
      • Franco M.O.
      • Sawyer J.E.
      • Wickersham T.A.
      Effect of monensin inclusion on intake, digestion, and ruminal fermentation parameters by Bos taurus indicus and Bos taurus taurus steers consuming bermudagrass hay..
      observed no effect of monensin (200 mg·head−1·d−1) on rumen pH (6.68 vs. 6.62 for monensin vs. control, respectively) of steers consuming bermudagrass hay (13.7% CP). An effect of hour after feeding on ruminal pH was observed (P ≤ 0.01), with an increase in pH observed until 8 h after feeding. A reduction in pH occurred 12 h after feeding and was followed by an increase 16 h after feeding. This change in pH was simply a result of feeding as it was consistent across treatments and cycles.

      VFA

      The primary effects of feeding monensin are typically observed on ruminal fermentation. A lack of response on intake, digestion, and ruminal pH were not unexpected due to observations in previous work with cattle consuming forage-based diets; however, it was anticipated that effects would be observed in measures of VFA. Ruminal VFA production is of particular importance because of its role in metabolic pathways. Acetate and butyrate are precursors for long-chain fatty acid synthesis and result in the production of H2, which can subsequently be used to produce CH4 by methanogenic archaea (
      • Moss A.R.
      • Jouany J.P.
      • Newbold J.
      Methane production by ruminants: Its contribution to global warming..
      ). Propionate serves as a substrate for gluconeogenesis and is therefore a primary source of glucose for ruminant animals. As an end product of fermentation, propionate is a principle hydrogen sink after CH4. Therefore, the acetate:propionate ratio is of importance because of its association with energy balance (

      Thomas, P. C., and P. A. Martin. 1988. The influence of nutrient balance on milk yield and composition. Pages 97–118 in Nutrition and Lactation in the Dairy Cow. P. C. Garnsworthy, ed. Butterworths.

      ) and CH4 formation by methanogenic archaea.
      As anticipated, a treatment × cycle interaction (P ≤ 0.01) was observed for acetate:propionate ratio and molar proportions of acetate and propionate (Table 4). Because of these interactions, treatment effects were evaluated within cycles for these parameters, and cycle means are displayed in Table 5.
      Table 4Effects of exclusion, continual, and cyclic monensin feeding on VFA and pH in ruminal fluid of cannulated beef steers consuming low-quality forage (bluestem grass hay) supplemented with dried distillers grains with solubles as monensin carrier
      ItemTreatment
      CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      SEMP-value
      T × C = treatment × cycle interaction; T × H = treatment × hour interaction; T × H × C = treatment × hour × cycle interaction.
      CONMONCYCTreatmentCycleHourT × CT × HT × H × C
      Total VFA (mM)63.1661.4061.372.800.88<0.01<0.010.700.630.12
      Acetate:propionate3.883.233.100.08<0.01<0.01<0.01<0.010.020.16
      Molar percentage (%)
       Acetate70.9367.6366.95<0.01<0.01<0.01<0.010.670.56
        (Mean ± SEM)
      Back-transformed means have asymmetric SE (Sokal and Rohlf, 2012).
      (70.36, 71.49)(67.03, 68.22)(66.34, 67.55)
       Propionate18.2720.9521.66<0.01<0.01<0.01<0.01<0.010.12
        (Mean ± SEM)(18.01, 18.54)(20.58, 21.34)(21.26, 22.07)
       Butyrate8.708.458.260.550.01<0.010.480.220.65
        (Mean ± SEM)(8.41, 9.01)(8.18, 8.74)(8.00, 8.54)
       Isobutyrate0.600.740.750.02<0.01<0.010.830.400.76
        (Mean ± SEM)(0.57, 0.63)(0.70, 0.77)(0.71, 0.79)
       Valerate0.510.560.630.05<0.01<0.010.870.060.63
        (Mean ± SEM)(0.49, 0.54)(0.54, 0.59)(0.60, 0.66)
       Isovalerate0.610.870.870.01<0.01<0.010.480.030.11
        (Mean ± SEM)(0.57, 0.66)(0.80, 0.93)(0.81, 0.94)
      pH5.595.835.700.080.13<0.01<0.010.160.670.74
      1 CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      2 T × C = treatment × cycle interaction; T × H = treatment × hour interaction; T × H × C = treatment × hour × cycle interaction.
      3 Back-transformed means have asymmetric SE (

      Sokal, R. R., and J. F. Rohlf. 2012. Biometry. 4th ed. W.H. Freeman and Co.

      ).
      Table 5Effects by cycle of exclusion, continual, and cyclic monensin feeding on VFA in ruminal fluid of cannulated beef steers consuming low-quality forage (bluestem grass hay) supplemented with dried distillers grains with solubles as monensin carrier
      ItemTreatment
      CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      SEMP-value
      T × H = treatment × hour interaction.
      CONMONCYCTreatmentHourT × H
      Cycle 1
       Acetate:propionate3.922.452.170.12<0.01<0.010.11
       Molar percentage (%)
        Acetate71.0662.0060.17<0.01<0.010.80
         (Mean ± SEM)
      Back-transformed means have asymmetric SE (Sokal and Rohlf, 2012).
      (70.27, 71.84)(61.11, 62.89)(59.24, 61.09)
        Propionate18.1225.2527.75<0.010.020.01
         (Mean ± SEM)(17.73, 18.54)(24.36, 26.22)(26.63, 28.99)
      Cycle 2
       Acetate:propionate3.803.123.060.12<0.01<0.010.81
       Molar percentage (%)
        Acetate69.3766.7966.640.04<0.010.72
         (Mean ± SEM)(68.56, 70.17)(65.95, 67.61)(65.79, 67.47)
        Propionate18.2821.3721.78<0.01<0.010.94
         (Mean ± SEM)(17.88, 18.71)(20.78, 22.01)(21.16, 22.45)
      Cycle 3
       Acetate:propionate4.033.713.590.120.04<0.010.02
       Molar percentage (%)
        Acetate72.1570.7870.080.18<0.010.44
         (Mean ± SEM)(71.37, 72.91)(69.99, 71.57)(69.28, 70.87)
        Propionate17.9219.1219.540.04<0.01<0.01
         (Mean ± SEM)(17.54, 18.33)(18.67, 19.60)(19.06, 20.04)
      Cycle 4
       Acetate:propionate3.793.653.600.120.51<0.010.09
       Molar percentage (%)
        Acetate71.1170.5670.400.80<0.010.28
         (Mean ± SEM)(70.32, 71.89)(69.76, 71.34)(69.61, 71.19)
        Propionate18.7919.3519.700.39<0.010.17
         (Mean ± SEM)(18.36, 19.25)(18.89, 19.85)(19.22, 20.22)
      1 CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d.
      2 T × H = treatment × hour interaction.
      3 Back-transformed means have asymmetric SE (

      Sokal, R. R., and J. F. Rohlf. 2012. Biometry. 4th ed. W.H. Freeman and Co.

      ).

      Acetate and Propionate.

      During cycle 1, a treatment × hour interaction was observed for molar proportion of propionate (P = 0.01) but was not observed (P ≥ 0.11) for molar proportion of acetate or the acetate:propionate ratio. In cycle 1, monensin fed by either method increased (P < 0.01) molar proportion of propionate by ≥ 39.3% (Figure 2). Molar proportion of propionate for steers consuming monensin cyclically was not different (P ≥ 0.15) than that of steers continuously fed, at 0, 2, or 4 h after feeding, but tended to be greater (≥12.0%; P ≤ 0.10) for steers consuming monensin cyclically 8 and 12 h after feeding. Feeding monensin by either method reduced (P < 0.01) molar proportion of acetate by ≥12.7% (Figure 3). Effects on acetate and propionate caused a reduction (P < 0.01) in the acetate:propionate ratio by ≥37.5% when monensin was fed in any manner (Figure 4).
      Figure 2
      Figure 2Effects of continual and cyclic monensin inclusion on mean propionate (±1 SEM) in ruminal fluid of steers consuming bluestem grass hay. CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d. Effect of hour after feeding—cycle 1: P < 0.02, cycle 2: P ≤ 0.01, cycle 3: P ≤ 0.01, cycle 4: P < 0.01.
      Figure 3
      Figure 3Effects of continual and cyclic monensin inclusion on mean acetate (±1 SEM) in ruminal fluid of steers consuming bluestem grass hay. CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d. There was an effect of hour after feeding for each cycle (P < 0.01).
      Figure 4
      Figure 4Effects of continual and cyclic monensin inclusion on mean acetate:propionate (±1 SEM) in ruminal fluid of steers consuming bluestem grass hay. CON = 0 mg·head−1·d−1 monensin; MON = 200 mg·head−1·d−1 monensin (Rumensin 90; Elanco Animal Health); CYC = 200 mg·head−1·d−1 monensin for 14 d followed by 0 mg·head−1·d−1 monensin for the subsequent 14 d. There was an effect of hour after feeding for each cycle (P < 0.01).
      An effect of hour after feeding (P ≤ 0.01) was observed in cycle 1 for molar proportions of acetate and propionate. Molar proportion of acetate declined until 2 h after feeding and then steadily increased until 12 h after feeding. Inversely, molar proportions of propionate increased until 2 h after feeding and then declined until 4 h after feeding and remained relatively stable through 12 h after feeding.
      During cycle 2, no treatment × hour interactions (P ≥ 0.72) were observed for molar proportions of acetate, propionate, or the acetate:propionate ratio. In cycle 2, treatment effects were observed for molar proportions of acetate (P = 0.04) and propionate (P < 0.01) and the acetate:propionate ratio (P < 0.01). Feeding monensin by either method reduced (P ≤ 0.03) the molar proportion of acetate by ≥3.8%, increased (P < 0.01) the molar proportion of propionate by ≥16.9%, and reduced (P < 0.01) the acetate:propionate ratio by ≥17.9%, but there was no difference (P ≥ 0.24; data not shown) between continuous and cyclic feeding of monensin for any of these measures of fermentation.
      An effect of hour after feeding (P < 0.01) was observed for molar proportions of acetate and propionate and the acetate:propionate ratio during cycle 2. Molar proportion of acetate declined after feeding and then increased from 2 through 12 h after feeding. Inversely, molar proportion of propionate increased until 2 h after feeding and then declined through 12 h after feeding. Similar to molar proportion of acetate, the acetate:propionate ratio declined until 2 h after feeding and then increased through 12 h after feeding.
      During cycle 3, a treatment × hour interaction was observed for molar proportion of propionate (P < 0.01) and the acetate:propionate ratio (P = 0.02). Feeding monensin by either method increased (P ≤ 0.05) molar proportion of propionate at 0, 4, 8, and 12 h after feeding by ≥6.01%. This increase in propionate reduced (P ≤ 0.05) the acetate:propionate ratio by ≥7.42% during these specific time measurements. However, 2 h after feeding, no effect (P ≥ 0.74) of treatment was observed for propionate; therefore, the acetate:propionate ratio was also unaffected (P ≥ 0.81). Although a treatment × hour interaction was not observed (P = 0.44) for molar proportion of acetate during cycle 3, an effect of hour after feeding (P < 0.01) was observed. Molar proportion of acetate declined following feeding before increasing from h 2 to 12 after feeding. An effect of treatment was not observed (P = 0.18) for molar proportion of acetate during cycle 3.
      During cycle 4, a tendency for treatment × hour interaction (P = 0.09) was observed for the acetate:propionate ratio. At h 12, feeding monensin cyclically reduced (P < 0.01) the acetate:propionate ratio by 11.9% versus control, whereas there were no differences (P ≥ 0.27) between treatments from h 0 through 8. No treatment effects (P ≥ 0.39) were observed for molar proportions of acetate or propionate during cycle 4, but an effect of hour after feeding (P < 0.01) was observed for molar proportions of both acetate and propionate. Acetate and the acetate:propionate ratio declined until 2 h after feeding and then increased thereafter. Conversely, propionate increased until 2 h following feeding and then declined thereafter.

      Total VFA, Butyrate, and the Minor VFA.

      Because no treatment × cycle interactions (P ≥ 0.48) were observed for total VFA concentration or molar proportions of butyrate, isobutyrate, valerate, or isovalerate, treatment effects are discussed across, rather than within, cycles for these parameters.
      Concentration of total VFA was not affected by treatment (P = 0.88) nor were any interactions involving treatment significant (treatment × cycle: P = 0.70; treatment × hour: P = 0.63; treatment × cycle × hour: P = 0.12). Treatment means ranged from 61.4 (MON) to 63.2 mM (CON). An effect of cycle (P < 0.01) was observed for total VFA concentration. A 36.26% reduction in total VFA concentration was observed from cycle 1 to 2, followed by a 20.90% increase from cycle 2 to 3 and a 10.84% decrease from cycle 3 to 4. An effect of hour after feeding (P < 0.01) was observed for total VFA concentration, with an increase from h 0 through 4 followed by a decrease from h 4 through 12.
      Similar to total VFA, molar proportion of butyrate was unaffected by treatment (P = 0.55) or any interactions involving treatment (treatment × cycle: P = 0.48; treatment × hour: P = 0.22; treatment × cycle × hour: P = 0.65). An effect of cycle (P < 0.01) was observed for molar proportion of butyrate. Molar proportion of butyrate decreased from cycle 1 through cycle 4 for a total 15.57% reduction. An effect of hour after feeding (P < 0.01) was observed, with molar proportion of butyrate increasing following feeding and then decreasing from h 2 through 12 after feeding.
      No interactions (treatment × cycle: P = 0.83; treatment × hour: P = 0.40; treatment × cycle × hour: P = 0.76) were observed for isobutyrate. A treatment effect (P = 0.02) was observed for molar proportion of isobutyrate. Feeding monensin by either method increased the proportion of isobutyrate (P ≤ 0.02) by ≥23.22%; however, there was no difference between CYC and MON feeding (P = 0.80). An effect of cycle (P < 0.01) was observed for molar proportion of isobutyrate. Molar proportion of isobutyrate increased from cycle 1 to 2 and then decreased from cycle 2 through 4. An effect of hour after feeding (P < 0.01) was observed for molar proportion of isobutyrate, with an increase observed after feeding and then a decrease from h 2 through 12 after feeding.
      A treatment × cycle × hour interaction was not observed (P = 0.63) for valerate. However, a tendency (P = 0.06) for a treatment × hour interaction was observed. At 0 h after feeding, molar proportion of valerate tended to be 16.74% greater (P = 0.07) for MON than CON steers. In h 2, cyclic monensin feeding tended to (P = 0.10) increase molar proportion of valerate compared with control but was not different (P = 0.32) than continuous feeding. During h 4 and 8, cyclic monensin feeding resulted in a greater (P ≤ 0.05) molar proportion of valerate than in both control and continuous feeding. By 12 h after feeding, cyclic feeding of monensin caused a greater (P = 0.01) molar proportion of valerate than control but was not different (P = 0.14) from continuous feeding.
      A treatment × cycle × hour interaction was not observed (P = 0.11) for isovalerate. However, a treatment × hour interaction was observed (P = 0.03). Molar proportion of isovalerate at feeding was ≥27.96% greater (P ≤ 0.05) in steers fed monensin by either method. At 2 h after feeding, cyclic feeding of monensin resulted in a greater (P = 0.05) molar proportion and continuous feeding tended to (P = 0.07) result in greater molar proportion of isovalerate than control. From h 4 through 12 after feeding, feeding monensin by either method increased (P ≤ 0.01) the molar proportion of isovalerate by ≥43.58%. An effect of cycle (P < 0.01) was observed for isovalerate, with a 27.11% increase in molar proportion between cycle 1 and 2, followed by a 36.39% decrease from cycle 2 to 3 and a 9.50% increase from cycle 3 to 4.
      Monensin inhibits ruminal CH4 production by inhibiting hydrogen-producing bacteria. Hydrogen-producing bacteria generally produce acetate and butyrate more than propionate (
      • Russell J.B.
      • Houlihan A.J.
      Ionophore resistance of ruminal bacteria and its potential impact on human health..
      ); therefore, monensin-induced inhibition of hydrogen-producing bacteria alters the ruminal VFA profile. Upon monensin consumption, reducing equivalents shift to increased propionate and reduced CH4 production, leading to increased energy retention by the animal.
      Ultimately, when analyzing VFA concentrations across cycles, the magnitude of effect of monensin feeding diminished over time until effects were no longer detectable. In cycle 1, monensin fed by either method was different than CON. In cycle 2, CYC and MON remained similar and were generally different than CON, but the magnitude of the effect had noticeably declined. By cycle 3, the magnitude of the effect had significantly declined, but MON and CYC were still generally the same and different from CON. In cycle 4, the trend was still numerically visible, but the size of the effect was so small that it was no longer significant. This confirms the hypothesis that monensin effects diminish over time; however, feeding monensin cyclically did not prolong the effects on fermentation as previously expected. Ultimately, providing monensin cyclically produced similar results to providing monensin continuously. Thus, providing monensin after a cycle of withdrawal did not amplify its utility compared with continuous feeding. This suggests that length of withdrawal time may affect response, and further investigation may be warranted.

      APPLICATIONS

      Effects of monensin (fed continuously or cyclically) were not sustained throughout the course of the trial. These data suggest that monensin is effective at favorably altering fermentation profiles in steers fed diets based on low- to moderate-quality forages, at least over the first 84 d of application. However, by the end of the 112-d trial, no monensin feeding strategy resulted in fermentation profiles substantially different than controls. Feeding method may have some effect, and further evaluations that vary duration of feeding and withdrawal periods may be warranted. In conclusion, although the cyclic monensin feeding strategy may not prolong the benefits of monensin, it may reduce overall cost of feeding this feed additive by reducing product and labor cost.

      ACKNOWLEDGMENTS

      The authors are grateful for the funding support provided by the USDA National Institute of Food and Agriculture (NIFA). Financial support for research supplies and graduate student funding were provided by USDA NIFA Foundational Program Animal, Nutrition, Growth and Lactation Grant project 2017-67016-26585 and USDA NIFA Hispanic Serving Institutions Grant Project 2017-38422-27298. Financial support for undergraduate student support was provided by USDA NIFA Research and Extension Experiences for Undergraduates Grant Project 2018-67032-27813. The help of undergraduate students Ariel Perez, Valerie Ruiz, and Lorena Villanueva was greatly appreciated.

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