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Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on risk of biohydrogenation-induced milk fat depression

  • Author Footnotes
    1 Current address: Provimi North America, 10 Nutrition Way, Brookville, OH 45309.
    M. Baldin
    Footnotes
    1 Current address: Provimi North America, 10 Nutrition Way, Brookville, OH 45309.
    Affiliations
    Department of Animal Science, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    2 Current address: USDA-Agricultural Research Service, U. S. Dairy Forage Research Center, Madison, WI 53706.
    G.I. Zanton
    Footnotes
    2 Current address: USDA-Agricultural Research Service, U. S. Dairy Forage Research Center, Madison, WI 53706.
    Affiliations
    Novus International Inc., St. Charles, MO 63304
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  • K.J. Harvatine
    Correspondence
    Corresponding author
    Affiliations
    Department of Animal Science, The Pennsylvania State University, University Park 16802
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  • Author Footnotes
    1 Current address: Provimi North America, 10 Nutrition Way, Brookville, OH 45309.
    2 Current address: USDA-Agricultural Research Service, U. S. Dairy Forage Research Center, Madison, WI 53706.
Open ArchivePublished:November 08, 2017DOI:https://doi.org/10.3168/jds.2017-13446

      ABSTRACT

      Diet-induced milk fat depression (MFD) is a multifactorial condition resulting from the interaction of numerous risk factors, including diet fermentability and unsaturated fatty acids concentration, feed additives, and individual cow effects. 2-Hydroxy-4-(methylthio)butanoate (HMTBa) is a methionine analog that has been observed to increase milk fat in some cases, and interactions with MFD risk factors may exist. The objective was to evaluate the effect of HMTBa supplementation on milk fat synthesis in cows with different levels of milk production and fed diets with increasing risk of biohydrogenation-induced MFD. Sixteen high-producing cows (44.1 ± 4.5 kg of milk/d; mean ± SD) and 14 low-producing (31.4 ± 4.3 kg of milk/d) were used in a randomized block design. Treatments were unsupplemented control and HMTBa fed at 0.1% of diet dry matter (25 g/d at 25 kg of dry matter intake). The experiment was 70 d and included a 14-d covariate period followed by 3 phases whereby diets were fed with increasing risk of MFD to determine the interaction of treatment and diet-induced MFD. During the low-risk phase, the base diet was balanced to 33.5% neutral detergent fiber (NDF) and had no exogenous oil (28 d); during the moderate-risk phase, the diet was balanced to 31% NDF and contained 0.75% soybean oil (14 d); and, during the high-risk phase, the diet was balanced to 28.5% NDF and contained 1.5% soybean oil (14 d). An interaction of treatment, production-level, and dietary phase was observed. Low producing cows neither experienced substantial biohydrogenation-induced MFD nor a response in milk fat to HMTBa supplementation. In high-producing cows, HMTBa maintained higher milk fat concentration during the moderate- (2.94 vs. 3.49%) and high-risk (2.38 vs. 3.11%) phases. High-producing cows receiving HMTBa also had greater milk fat yield (0.94 vs. 1.16 kg/d) and lower trans-10 C18:1 (6.11 vs. 1.50) during the high-risk phase. In conclusion, HMTBa increased milk fat in situations with a high risk of biohydrogenation-induced MFD by decreasing absorption of alternate biohydrogenation intermediates.

      Key words

      INTRODUCTION

      Fat is one of the most important components of milk, as it significantly affects the value of milk, the yield and quality of dairy products, and because of the recent associations between consumption of milk bioactive fatty acids (FA) and human health (
      • Bainbridge M.L.
      • Cersosimo L.M.
      • Wright A.-D.G.
      • Kraft J.
      Content and composition of branched-chain fatty acids in bovine milk are affected by lactation stage and breed of dairy cow.
      ). Consequently, the mechanisms regulating milk fat synthesis in the mammary gland, as well as nutritional and management strategies that improve milk fat synthesis, have been studied extensively. Low-milk fat syndrome, contemporarily called diet-induced milk fat depression (MFD), is a condition in which milk fat yield decreases up to 50% with generally no change in yields of milk and other milk components (
      • Harvatine K.J.
      • Perfield J.W.
      • Bauman D.E.
      Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during cla-induced milk fat depression in dairy cows.
      ). Milk fat synthesis is affected by several factors (e.g., genetics, physiological state, and environment), but is especially responsive to nutrition. The biohydrogenation (BH) theory (
      • Bauman D.E.
      • Griinari J.M.
      Nutritional regulation of milk fat synthesis.
      ) of MFD is a unifying concept that mechanistically explains the inhibition of milk fat synthesis when feeding highly fermentable and high-UFA diets. This model attributes the causal mechanism to changes in ruminal BH pathways, leading to increased formation and rumen outflow of specific bioactive FA that inhibit mammary lipid synthesis.
      The understanding of dietary regulation of milk fat synthesis has improved, yet varying levels of MFD still commonly occur on dairy farms. This is primarily because MFD is a multifactorial condition resulting from the interaction of several factors, such as dietary FA level, FA profile and availability, diet fermentability, feeding strategies, rumen modifiers, and individual cow effects (
      • Harvatine K.J.
      Lipid and fat nutrition.
      ). Diet UFA level and carbohydrate fermentability have been characterized as central risk factors for BH-induced MFD (
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      ;
      • Rico D.E.
      • Holloway A.
      • Harvatine K.
      Effect of monensin on recovery from diet-induced milk fat depression.
      ). Additionally, an association between level of milk production and risk of BH-induced MFD has been proposed. For instance, supplementation with calcium salts of UFA decreased milk fat in high-producing cows, whereas no MFD was observed in the low-producing cows (
      • Harvatine K.J.
      • Allen M.
      Effects of fatty acid supplements on milk yield and energy balance of lactating dairy cows.
      ;
      • Rico D.E.
      • Ying Y.
      • Harvatine K.
      Effect of a high-palmitic acid fat supplement on milk production and apparent total-tract digestibility in high-and low-milk yield dairy cows.
      ).
      Varying levels of evidence support the effectiveness of some rumen modifiers in attenuating BH-induced MFD. Early work observed that the methionine analog, 2-hydroxy-4-(methylthio)butanoate (HMTBa), increased milk fat when feeding highly fermentable diets (
      • Rosser R.
      • Polan C.
      • Chandler P.
      • Bibb T.
      Effects of whey components and methionine analog on bovine milk fat production.
      ;
      • Huber T.J.
      • Emery R.
      • Bergen W.
      • Liesman J.
      • Kung L.
      • King K.
      • Gardner R.
      • Checketts M.
      Influences of methionine hydroxy analog on milk and milk fat production, blood serum lipids, and plasma amino acids.
      ). A recent meta-analysis observed increased milk fat yield with HMTBa supplementation (
      • Zanton G.I.
      • Bowman G.
      • Vázquez-Añón M.
      • Rode L.
      Meta-analysis of lactation performance in dairy cows receiving supplemental dietary methionine sources or postruminal infusion of methionine.
      ), but it was inconclusive on the role of HMTBa in circumstances of MFD. As BH-induced MFD is the result of the interaction of numerous factors, studying one single factor at a time will be less informative and more inconsistent than studying multiple factors simultaneously. The current study was conducted to evaluate the interaction between dietary risk factors (UFA and carbohydrate fermentability), cow milk production level, and supplementation with a rumen modifier (HMTBa). The objective was to evaluate the effect of HMTBa supplementation on milk fat synthesis in 2 groups of cows with different levels of milk production and fed diets with increasing risk of BH-induced MFD. Our hypothesis was that high-producing cows are at higher risk of BH-induced MFD and that HMTBa would reduce the extent of BH-induced MFD.

      MATERIALS AND METHODS

      Experimental Design and Treatments

      All experimental procedures were approved by the Pennsylvania State University Institutional Animal Care and Use Committee. The experiment was conducted from October to December 2013 at the Pennsylvania State University Dairy Production Research and Teaching Center (University Park). Thirty multiparous Holstein cows were used in a randomized block design that tested the effect of treatment during 3 dietary phases that progressively increased risk for diet-induced MFD. Animals were housed individually in tiestalls with rubber mattresses and sawdust bedding and had continuous access to water. Cows were blocked by milk production (high or low) at the end of a 14-d pretrial period (Table 1). The high-producing group (n = 16; 166 ± 69 d postpartum; mean ± SD) averaged 44.1 ± 4.5 kg/d of milk and the low-producing group (n = 14; 267 ± 82 d postpartum) averaged 31.4 ± 4.3 kg/d of milk. Cows were paired within block (high and low) and randomly assigned to 1 of the 2 treatments: control (CON) or HMTBa (0.1% of diet DM, targeting 25 g of HMTBa per cow/d at 25 kg of DMI; Table 2). The HMTBa (Alimet, Novus International Inc., St. Charles, MO) was provided in a corn carrier and mixed in the TMR. An equivalent amount of the same ground corn carrier was added to the control treatment. The experiment was split into 3 phases that fed diets formulated to have low, moderate, and high risk of BH-induced MFD, respectively (Table 3). Risk for altered BH was increased by reducing diet NDF and increasing UFA and starch content (Table 3). Diet UFA were increased using a combination of rapidly available FA from soybean oil and more slowly available FA from roasted soybeans. The low-risk phase was 28 d to allow complete adaptation of the rumen to HMTBa, and the moderate- and high-risk phases were 14 d, as previous time course work has demonstrated that changes in BH occur within 10 to 14 d (
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      ). Importantly, phase is the repeated variable and the effect of treatment is only compared within phase, as phase and treatment are confounded. Diets were fed as a TMR once daily at 0700 h at 110% of expected daily intake. Cows were treated with rbST (Posilac, Elanco Animal Health, Greenfield, IN) every 14 d.
      Table 1Characteristics of cows in the low- and high-production block at the end of the pretrial period
      ItemBlock
      Milk production level at the end of the pretrial period (low = 14 cows, high = 16 cows).
      SEMP-value
      LowHigh
      DIM26716619.4<0.01
      DMI (kg/d)24.726.80.620.02
      Milk yield (kg/d)31.444.11.30<0.001
      Milk fat (%)3.943.200.16<0.01
      Milk fat (kg/d)1.121.370.060.01
      Milk protein (%)3.403.260.080.22
      Milk protein (kg/d)0.971.400.04<0.001
      Milk FA, g/100 g of total FA
      Fatty acids (FA) <16 C originate from de novo synthesis in the mammary gland; FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      trans-10 C18:10.480.770.120.11
       ∑ <16 C25.425.80.620.57
       ∑ 16 C25.926.90.520.20
       ∑ >16 C41.139.20.830.13
      1 Milk production level at the end of the pretrial period (low = 14 cows, high = 16 cows).
      2 Fatty acids (FA) <16 C originate from de novo synthesis in the mammary gland; FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      Table 2Overview of experimental design
      ItemBH-induced MFD risk
      Risk of biohydrogenation (BH)-induced milk fat depression (MFD).
      Low, d 0 to 28Moderate, d 29 to 42High, d 43 to 56
      Block
      Milk production level: low-producing cows (low) averaged 31.4 ± 4.3 kg/d and high-producing cows (high) averaged 44.1 ± 4.5 kg/d at the end of the pretrial period.
      LowControlControlControl
      HMTBa
      HMTBa = 2-hydroxy-4-(methylthio)butanoate.
      HMTBaHMTBa
      HighControlControlControl
      HMTBaHMTBaHMTBa
      1 Risk of biohydrogenation (BH)-induced milk fat depression (MFD).
      2 Milk production level: low-producing cows (low) averaged 31.4 ± 4.3 kg/d and high-producing cows (high) averaged 44.1 ± 4.5 kg/d at the end of the pretrial period.
      3 HMTBa = 2-hydroxy-4-(methylthio)butanoate.
      Table 3Ingredient and chemical composition of basal diets with increasing risk of biohydrogenation (BH)-induced milk fat depression (MFD)
      ItemRisk of BH-induced MFD
      LowModerateHigh
      Ingredient (% of DM)
       Corn silage40.538.135.0
       Alfalfa haylage14.713.612.5
       Processed grass hay2.92.72.5
       Ground corn7.712.317.2
       Canola meal11.611.110.9
       Cottonseed hulls4.13.52.8
       Roasted soybeans
      Whole soybeans were roasted in a mobile roaster and were approximately 158°C when removed from the roaster. Beans were rolled into approximately quarters before feeding.
      3.55.66.6
       Minerals and vitamins mix
      Composition (DM basis): 11% CP; 18% NDF; 5.2% fat; 14.9% Ca; 0.35% P; 4.58% Mg; 0.41% K; 0.31% S; 357 mg/kg of Cu; 1,085 mg/kg of Zn; 181 mg/kg of Fe; 6.67 mg/kg of Se; 262,105 IU/kg of vitamin A; 65,421 IU/kg of vitamin D; and 1,970 IU/kg of vitamin E (Cargill Animal Nutrition, Roaring Spring, PA).
      3.33.33.3
       Soybean oil0.00.71.5
       Corn gluten meal4.34.24.0
       Soybean hulls5.02.51.4
       Molasses2.52.42.3
      Composition (% of DM, unless noted)
       CP17.117.317.3
       RDP (% of CP)62.061.661.1
       ADF23.521.219.3
       NDF33.930.928.5
       Forage NDF20.218.317.5
       Starch24.426.829.2
       Fatty acids4.14.95.6
       PUFA2.22.73.1
       MUFA0.91.11.2
       peNDF
      peNDF = physically effective NDF. Analyzed using the Penn State Particle Separator (Nasco, Fort Atkinson, WI). Particles remaining (% of total) in the upper sieve, middle sieve, lower sieve, and bottom pan were 3, 37, 16, and 44, respectively, for the low-risk diet; 3, 35, 18, and 44, respectively, for the moderate-risk diet; 3, 35, 15, and 47, respectively, for the high-risk diet.
      19.017.315.1
      1 Whole soybeans were roasted in a mobile roaster and were approximately 158°C when removed from the roaster. Beans were rolled into approximately quarters before feeding.
      2 Composition (DM basis): 11% CP; 18% NDF; 5.2% fat; 14.9% Ca; 0.35% P; 4.58% Mg; 0.41% K; 0.31% S; 357 mg/kg of Cu; 1,085 mg/kg of Zn; 181 mg/kg of Fe; 6.67 mg/kg of Se; 262,105 IU/kg of vitamin A; 65,421 IU/kg of vitamin D; and 1,970 IU/kg of vitamin E (Cargill Animal Nutrition, Roaring Spring, PA).
      3 peNDF = physically effective NDF. Analyzed using the Penn State Particle Separator (Nasco, Fort Atkinson, WI). Particles remaining (% of total) in the upper sieve, middle sieve, lower sieve, and bottom pan were 3, 37, 16, and 44, respectively, for the low-risk diet; 3, 35, 18, and 44, respectively, for the moderate-risk diet; 3, 35, 15, and 47, respectively, for the high-risk diet.

      Sampling and Measurements

      Feed intake was measured daily. Cows were milked twice daily at 0500 and 1700 h and milk yield determined by an integrated milk meter (AfiMilk, SAE Afikim, Afikim, Israel). The parlor was calibrated using a stall deviation calculated using data from the entire herd (>200 cows) over 7 d. Stall deviations were determined by modeling the effect of day, milking (a.m./p.m.), cow, and stall, excluding observations of experimental cows during treatment periods. Milk was sampled at both milkings once per week and was composited based on yield at each milking. Body weight was recorded as cows exited the parlor throughout the trial (AfiFarm 3.04E scale system, SAE Afikim). Feed ingredients were sampled once per week and stored at −20°C, thawed at room temperature, dried at 55°C in a forced-air oven for 72 h, and ground in a Wiley mill through a 1-mm screen (A. H. Thomas, Philadelphia, PA). Feed samples were composited within dietary phase (equal dry weight basis). Blood samples were collected from the tail vein using potassium EDTA vacuum tubes (Greiner Bio-One North America Inc., Monroe, NC) at 0600, 1400, and 2000 h. Blood was immediately placed on ice, centrifuged within 30 min at 1,300 × g for 15 min at 4°C, and plasma was harvested and stored at −20°C until laboratory analysis. Prior to analysis, plasma samples from 1400 and 2000 h were composited by cow to represent a postabsorptive state.

      Sample Analysis

      Feed samples were analyzed for DM, CP, and starch by wet chemistry procedures according to
      • AOAC International
      Official Methods of Analysis.
      and for NDF and ADF according to
      • Van Soest P.J.
      • Robertson J.
      • Lewis B.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      . Total FA concentration and FA profile of feed samples was determined by GC after direct methylation (
      • Sukhija P.S.
      • Palmquist D.
      Rapid method for determination of total fatty acid content and composition of feedstuffs and feces.
      ), as described by
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      . Plasma samples were analyzed for insulin (Coat-a-count insulin kit, Siemens Healthcare Diagnostics, Los Angeles, CA), glucose (PGO Enzyme procedure no. P 7119, Sigma-Aldrich, St. Louis, MO), BUN [Modified Enzymatic Urea Nitrogen (Procedure No. 2050); Stanbio Laboratory, Boerne, TX], and FA (Wako HR Series NEFA-HR kit, Wako Chemicals USA Inc., Richmond, VA), as modified by
      • Ballou M.A.
      • Gomes R.
      • Juchem S.
      • DePeters E.
      Effects of dietary supplemental fish oil during the peripartum period on blood metabolites and hepatic fatty acid compositions and total triacylglycerol concentrations of multiparous Holstein cows.
      .
      One milk sample was stored at 4°C with preservative (Bronolab-WII, Advanced Instruments Inc., Norwood, MA) until analyzed for fat and protein by Fourier transform infrared spectroscopy (Fossomatic 4000 Milko-Scan and 400 Fossomatic, Foss Electric, Hillerød, Denmark; at Dairy One Laboratory, Ithaca, NY). A second milk sample was immediately centrifuged at 3,000 × g for 15 min at 4°C and fat cake was stored at −20°C before analysis of FA composition, as described by
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      . In short, lipid extraction was performed according to
      • Hara A.
      • Radin N.S.
      Lipid extraction of tissues with a low-toxicity solvent.
      using hexane:isopropanol. Fatty acid methyl esters were prepared by base-catalyzed transmethylation according to
      • Chouinard P.Y.
      • Corneau L.
      • Barbano D.M.
      • Metzger L.E.
      • Bauman D.E.
      Conjugated linoleic acids alter milk fatty acid composition and inhibit milk fat secretion in dairy cows.
      . Fatty acid methyl esters were quantified by GC using an Agilent 6890A gas chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a fused-silica capillary column (SP-2560, 100 m × 0.25 mm i.d. with 0.2-μm film thickness; Supelco Inc., Bellefonte, PA) and a flame ionization detector with hydrogen as the carrier gas. Initial oven temperature was 80°C, which was increased by 2°C/min to 190°C and held for 15 min. Inlet and detector temperatures were 250°C with a 100:1 split ratio. Constant gas flows were 1 mL/min for hydrogen carrier, 25 mL/min for detector hydrogen, 400 mL/min for detector airflow, and 40 mL/min for detector nitrogen plus carrier. Fatty acid peaks were identified using FAME standards (GLC 461, GLC 780, and pure trans-10,cis-12 CLA and cis-9,trans-11 CLA, NuChek Prep Inc., Elysian, MN; Bacterial Acid Methyl Ester Mix, 47080-U, Sigma-Aldrich; and GLC 110 mixture, Matreya LLC., State College, PA). Recovery of individual FA were determined using an equal weight reference standard (GLC 461; NuChek Prep Inc.). Correction factors for individual FA and calculation of milk FA yield were carried out as described by
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      .

      Statistical Analysis

      Data were analyzed as a randomized block design using PROC MIXED of SAS with repeated measures (version 9.3; SAS Institute Inc., Cary, NC). An interaction of treatment, production level (block), and experimental day (dietary phase) was observed; thus, data were analyzed within block (high or low) with the following reduced model
      Yijkl=μ+Ti+Cj(Ti)+Dk+TiDk+βl+ɛijkl,


      where Yijkl is the variable of interest, μ is the overall mean, Ti is the fixed effect of treatment (control or HMTBa), Cj(Ti) is the random effect of cow nested within treatment i, Dk is the fixed effect of experimental day (k = 1 to 8), TiDk is the interaction of treatment and experimental day, βl is the fixed effects of pretrial measurements (covariate), and εijkl is the residual error. The ARH(1) or AR(1) covariance structures were used depending on model fit and the Kenward-Roger denominator degrees of freedom adjustment was employed. The preplanned contrast tested the effect of HMTBa at each day. In the time-course data, the treatment effect was similar within dietary phase (low, moderate, and high risk of MFD); therefore, for simplicity, effect of treatment was tested by 2-wk intervals that matched dietary phases. Data points with Studentized residuals outside of ±3.0 were considered outliers and excluded from analysis, which typically included less than 4 data points per variable. Significant differences were declared at P < 0.05 for main effects and P < 0.10 for interactions and tendencies were declared at P < 0.10 for main effects and P < 0.15 for interactions.

      RESULTS

      Effect of Production Level

      Milk yield and composition differed between the low- and high-producing blocks at the end of the pretrial period (Table 1). Low-producing cows had 95 d greater DIM, 2.1 kg/d lower DMI, and 12.7 kg/d lower milk yield compared with high-producing cows (Table 1). Additionally, low-producing cows had 0.74 percentage points higher milk fat concentration, but had lower yield of milk fat (248 g/d; P = 0.01) and milk protein (427 g/d; P < 0.001). No difference between the 2 blocks was observed for FA by source (de novo, mixed source, and preformed), but trans-10 C18:1 was numerically higher in high-producing cows (0.77 vs. 0.48; P = 0.11; Table 1).

      Performance Responses

      Body weight was not affected by treatment and averaged 740 ± 30 kg in the high-producing group and 703 ± 19 kg in the low-producing group (P = 0.90 and P = 0.70, respectively). Dry matter intake, milk yield, and milk protein concentration and yield were not affected by treatment in either block (Table 4). Moreover, no treatment by day interaction was observed for any of these variables (Table 4). A tendency for higher milk protein concentration (3.63 vs. 3.52%, P = 0.05) in low-producing cows in the control treatment was observed.
      Table 4Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on performance and milk composition in high- and low-producing cows
      Item (kg/d, unless noted)Block
      Milk production level: high-producing cows (H) averaged 44.1 ± 4.5 kg/d and low-producing cows (L) averaged 31.4 ± 4.3 kg/d at the end of the pretrial period.
      Treatment (Trt)
      Treatment was control (ground corn) or HMTBa (0.1% of diet DM provided in a ground corn carrier).
      SEMP-value
      Effect of treatment (Trt), experimental day, and their interaction.
      ControlHMTBaTrtDayTrt × day
      DMIH27.325.60.740.10<0.010.53
      L24.524.10.860.70<0.010.60
      Milk yieldH40.839.53.850.740.010.47
      L21.523.81.340.24<0.010.81
      Milk fat (%)H2.983.420.10<0.01<0.01<0.05
      L3.943.960.090.920.020.46
      Milk fatH1.261.270.050.87<0.010.02
      L0.800.980.070.10<0.010.96
      Milk protein (%)H3.333.360.080.46<0.010.64
      L3.633.520.070.05<0.010.58
      Milk proteinH1.401.280.060.170.540.38
      L0.730.850.040.06<0.010.16
      1 Milk production level: high-producing cows (H) averaged 44.1 ± 4.5 kg/d and low-producing cows (L) averaged 31.4 ± 4.3 kg/d at the end of the pretrial period.
      2 Treatment was control (ground corn) or HMTBa (0.1% of diet DM provided in a ground corn carrier).
      3 Effect of treatment (Trt), experimental day, and their interaction.
      We found no effect of treatment or treatment by day interaction for milk fat concentration and yield in the low-producing group (Table 4). Milk fat concentration remained relatively constant and milk fat yield decreased slightly over the 3 dietary phases in low-producing cows on both treatments (Figure 1A and B). On the other hand, a treatment by day interaction was observed for milk fat concentration and yield in the high-producing group (Table 4). In the high-producing cows, milk fat concentration did not differ between treatments during the low-risk phase; but, during the moderate- and high-risk phases, control cows progressively decreased milk fat content, whereas HMTBa cows maintained higher milk fat concentration (0.55 and 0.73 percentage points, respectively; Figure 1A and Supplemental Table S1, https://doi.org/10.3168/jds.2017-13446). Milk fat yield did not differ between treatments during low and moderate risk phases, but was higher by 220 g/d in HMTBa during the high-risk phase (Supplemental Table S1 and Figure 1B).
      Figure thumbnail gr1
      Figure 1Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) supplementation (0.1% of DM) on milk fat concentration (A) and yield (B) in high- (Con-H and HMTBa-H) and low-producing (Con-L and HMTBa-L) cows receiving diets with low, moderate, and high risk for biohydrogenation-induced milk fat depression. High-producing cows averaged 44.1 ± 4.5 kg/d and low-producing cows averaged 31.4 ± 4.3 kg/d. Effect of treatment (control vs. HMTBa) within each dietary phase in high-producing cows is shown (*P < 0.05 and **P < 0.01). Treatments (Trt) did not differ at any time point in low-producing cows (P > 0.05). Pooled SEM for milk fat concentration was 0.22 and 0.20 in low- and high-producing cows, respectively. Pooled SEM for fat yield was 0.12 and 0.09 in low-and high-producing cows, respectively.
      No overall effect of treatment was observed on the concentration of BUN, nonesterified FA, glucose, and insulin in plasma of high- or low-producing cows in either fasting or postabsorptive state (P > 0.12 for all; Supplemental Table S2, https://doi.org/10.3168/jds.2017-13446). The treatment by time by production level interaction was not significant for the plasma variables (P > 0.50 for all).

      Milk FA

      We observed no overall effect of treatment or treatment by day interactions for trans-11 C18:1, trans-10 C18:1, FA by source (de novo, mixed, and preformed), and total odd- and branched-chain FA (OBCFA) in the low-producing block (Table 5). In the high-producing cows we found a treatment by day interactions for trans-11 C18:1, trans-10 C18:1, and sum of de novo synthesized FA (<16 C; Table 5). During the high-risk phase, high-producing cows supplemented with HMTBa had 35.3% higher trans-11 C18:1, 2.3-fold lower trans-10 C18:1, and higher de novo FA (<16 C) concentration in milk fat (Figure 2, Figure 3). Individual FA <16 carbons generally followed a similar pattern (Supplemental Tables S3, S4, and S5; https://doi.org/10.3168/jds.2017-13446). Although a treatment by day interaction was not observed for OBCFA, in high-producing cows during the high-risk phase HMTBa increased C15:0 and C17:0 (Supplemental Figure S1, https://doi.org/10.3168/jds.2017-13446), and resulted in greater total OBCFA concentration (3.04 vs. 2.79 g/100 g of total FA, P = 0.04; Supplemental Table S2).
      Table 5Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on selected milk fatty acids (FA) in high- and low-producing cows
      FA (g/100 g of total FA)Block
      Milk production level: high-producing cows (H) averaged 44.1 ± 4.5 kg/d and low-producing cows (L) averaged 31.4 ± 4.3 kg/d at the end of the pretrial period.
      Treatment
      Treatment was control (ground corn) or HMTBa (0.1% of diet DM provided in a corn carrier).
      SEMP-values
      Effect of treatment (Trt), experimental day, and their interaction.
      ControlHMTBaTrtDayTrt × day
      trans-11 C18:1H1.341.560.100.16<0.001<0.001
      L1.601.900.160.23<0.0010.15
      trans-10 C18:1H2.461.000.340.01<0.001<0.001
      L0.660.660.030.99<0.0010.50
      ∑ <16 C
      FA <16 C originate from de novo synthesis in the mammary gland, FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      H21.922.31.110.80<0.0010.01
      L23.423.40.300.96<0.0010.88
      ∑ 16 C
      FA <16 C originate from de novo synthesis in the mammary gland, FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      H25.624.31.650.590.050.39
      L24.523.90.380.30<0.0010.17
      ∑ >16 C
      FA <16 C originate from de novo synthesis in the mammary gland, FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      H41.840.31.540.53<0.0010.58
      L44.645.20.570.49<0.0010.48
      ∑ OBCFA
      Sum of all odd- and branched-chain fatty acids in milk.
      H3.343.440.040.17<0.00010.11
      L3.423.430.030.74<0.00010.97
      1 Milk production level: high-producing cows (H) averaged 44.1 ± 4.5 kg/d and low-producing cows (L) averaged 31.4 ± 4.3 kg/d at the end of the pretrial period.
      2 Treatment was control (ground corn) or HMTBa (0.1% of diet DM provided in a corn carrier).
      3 Effect of treatment (Trt), experimental day, and their interaction.
      4 FA <16 C originate from de novo synthesis in the mammary gland, FA >16 C originate from extraction from plasma, and 16 C FA originate from both sources.
      5 Sum of all odd- and branched-chain fatty acids in milk.
      Figure thumbnail gr2
      Figure 2Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) supplementation (0.1% DM) on the milk fat concentration of the predominant isomers of the normal (trans-11 C18:1; A) and the alternate (trans-10 C18:1; B) biohydrogenation pathways in high- (Con-H and HMTBa-H) and low-producing (Con-L and HMTBa-L) cows receiving diets with low, moderate, and high risk for biohydrogenation-induced milk fat depression. High-producing cows averaged 44.1 ± 4.5 kg/d and low-producing cows averaged 31.4 ± 4.3 kg/d. The treatment (Trt) by day interaction for high- (H) and low-producing (L) cows is shown. Significant differences between control and HMTBa in high-producing cows within each dietary phase are shown (***P < 0.01). Treatments did not differ (P > 0.05) at any time point in low-producing cows. Pooled SEM for trans-11 C18:1 was 0.40 and 0.20 in low- and high-producing cows, respectively. Pooled SEM for trans-10 C18:1 was 0.06 and 0.73 in low- and high-producing cows, respectively. FA = fatty acid.
      Figure thumbnail gr3
      Figure 3Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) supplementation (0.1% diet DM) on milk fatty acid (FA) concentration by source in high-producing cows (44.1 ± 4.5 kg/d) receiving diets with low, moderate, and high risk for biohydrogenation-induced milk fat depression. Fatty acids <16 C originate from de novo synthesis in the mammary gland (A), FA >16 C are extracted from plasma (C), and 16-C FA from both sources (B). Significance (*P < 0.05) is presented for the control versus HMTBa within each dietary phase.

      DISCUSSION

      The sequentially increasing risk factor design allowed the efficient investigation of the interaction between HMTBa, susceptibility to MFD due to production level, and dietary challenge (low, moderate, and high risk for BH-induced MFD). Decreasing diet NDF and increasing starch and UFA is a well-characterized model to alter ruminal BH pathways and induce the formation of bioactive FA that inhibit milk fat synthesis (
      • Bauman D.E.
      • Griinari J.M.
      Nutritional regulation of milk fat synthesis.
      ;
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      ;
      • Rico D.E.
      • Holloway A.
      • Harvatine K.
      Effect of monensin on recovery from diet-induced milk fat depression.
      ). Increasing starch concentration from 24.4 to 26.8 to 29.2% of the diet is expected to have decreased rumen pH and altered microbial populations to those adaptable to starch digestion and lower pH, although neither were measured in the current experiment. The physically effective NDF during the moderate- and high-risk diets (17.3 and 15.1%DM, respectively) was below the suggested threshold of 21% required to maintain rumen pH below 6.0 (
      • Zebeli Q.
      • Tafaj M.
      • Weber I.
      • Dijkstra J.
      • Steingass H.
      • Drochner W.
      Effects of varying dietary forage particle size in two concentrate levels on chewing activity, ruminal mat characteristics and passage in dairy cows.
      ). Of note, responses to HMTBa have been demonstrated to be independent of rumen pH (
      • Vázquez-Añón M.
      • Cassidy T.
      • McCullough P.
      • Varga G.
      Effects of alimet on nutrient digestibility, bacterial protein synthesis, and ruminal disappearance during continuous culture.
      ;
      • Lee C.
      • Oh J.
      • Hristov A.
      • Harvatine K.
      • Vazquez-Anon M.
      • Zanton G.
      Effect of 2-hydroxy-4-methylthio-butanoic acid on ruminal fermentation, bacterial distribution, digestibility, and performance of lactating dairy cows.
      ). Increasing diet PUFA from 2.2 to 2.7 and 3.1% of the diet increased the amount of rumen-available UFA requiring BH and is also expected to have modified the microbial population by selecting against populations sensitive to UFA.
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      characterized the time-course of BH-induced MFD and demonstrated that milk fat content and yield were decreased by d 3 and 5, respectively, and the maximal response occurred in approximately 7 to 10 d. Additionally, concentration of trans-10 C18:1 and trans-10,cis-12 CLA in milk fat increased progressively and were higher than control on d 1 and 3, respectively. Changes in key microbial populations have also been demonstrated to occur over a similar rapid time-course during BH-induced MFD (
      • Rico D.E.
      • Preston S.
      • Risser J.
      • Harvatine K.
      Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.
      ). In summary, we highlighted the fast adaptation of rumen microbes to changes in diet fermentability and UFA and supported the use of 10- to 14-d experimental periods to investigate BH-induced MFD.
      Cows were grouped based on level of milk production, but DIM, DMI, and milk fat and protein concentrations and yields also differed between the blocks, as expected. A 3-way interaction of treatment, dietary risk, and production level for milk fat concentration required the analysis within production blocks. Briefly, BH-induced MFD did not occur in low-producing cows, and the effects of HMTBa supplementation would have to occur through a different mechanism to affect milk fat production in this block. The low-producing group was composed primarily by late-lactation cows. Energy status of the cow may be a more important determinant of hormonal profile than stage of lactation (
      • Peel C.J.
      • Fronk T.J.
      • Bauman D.E.
      • Gorewit R.C.
      Effect of exogenous growth hormone in early and late lactation on lactational performance of dairy cows.
      ), and cows in the high-producing group were mid-lactation and also expected to be in a positive energy balance. We did not observe any treatment by production level interaction for plasma variables. Therefore, the differential response of low- and high-producing cows to dietary BH-induced MFD risk factors and HMTBa supplementation in the current study was not expected to have been mediated by hormonal status. Furthermore, as reviewed by
      • Harvatine K.J.
      • Boisclair Y.
      • Bauman D.
      Recent advances in the regulation of milk fat synthesis.
      , trans-10,cis-12 CLA (a potent bioactive trans FA formed during altered ruminal BH) reduces milk fat yield during all phases of the lactation cycle. This indicates that, in the current study, the absence of MFD in low-producing cows was not related to a lower responsiveness of the mammary gland to bioactive trans FA that inhibit milk fat synthesis and is likely due to a reduced flow of bioactive FA from the rumen.
      The high-producing cows in the current experiment had higher DMI.
      • Jenkins T.C.
      • Wallace R.
      • Moate P.
      • Mosley E.
      Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem.
      proposed that the kinetics of ruminal passage of feed particles directly affects the rates of lipolysis and BH. Rumen passage rate is expected to increase as DMI increases, which may modify the microbial population and increase ruminal outflow of trans intermediate FA before complete BH has occurred. Additionally, high-producing cows may differ in feeding and ruminating behavior (e.g., increased meal size, slug feeding), which may result in subclinical rumen acidosis and further alteration of normal microbial populations and BH pathways (
      • Harvatine K.J.
      Lipid and fat nutrition.
      ). Milk trans-10 C18:1, a proxy of altered ruminal BH, was 38% higher in high-producing cows at the end of the pretrial period, indicating presence of the alternate ruminal BH pathway before initiation of the trial. Furthermore, it suggests that high-producing cows may be at higher risk of BH-induced MFD even under a low-risk diet, which has been reported in 2 other studies with similar high- and low-producing blocks (
      • Harvatine K.J.
      • Allen M.
      Effects of fatty acid supplements on milk yield and energy balance of lactating dairy cows.
      ;
      • Rico D.E.
      • Ying Y.
      • Harvatine K.
      Effect of a high-palmitic acid fat supplement on milk production and apparent total-tract digestibility in high-and low-milk yield dairy cows.
      ).
      Increasing dietary risk factors resulted in pronounced BH-induced MFD in high-producing cows in the control treatment, as evidenced by a 14 and 30% decrease in milk fat concentration during the moderate- and high-risk phases, respectively. The extent of MFD in the high-risk phase is within the range of 25 to 38% observed using similar changes in diet fermentability and UFA content (
      • Peterson D.G.
      • Matitashvili E.A.
      • Bauman D.E.
      Diet-induced milk fat depression in dairy cows results in increased trans-10,cis-12 cla in milk fat and coordinate suppression of mrna abundance for mammary enzymes involved in milk fat synthesis.
      ;
      • Harvatine K.J.
      • Bauman D.E.
      Srebp1 and thyroid hormone responsive spot 14 (s14) are involved in the regulation of bovine mammary lipid synthesis during diet-induced milk fat depression and treatment with cla.
      ;
      • Rico D.E.
      • Harvatine K.J.
      Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration.
      ). The phenotype of the response observed in the current experiment, including decreased milk fat with no changes in milk yield or yield of other components, is characteristic of classical BH-induced MFD (
      • Harvatine K.J.
      • Perfield J.W.
      • Bauman D.E.
      Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during cla-induced milk fat depression in dairy cows.
      ). This is further supported by the substantial 7.9-fold increase in trans-10 C18:1 in milk fat of high-producing cows in the control treatment. Trans-10 C18:1 is a major intermediate formed during altered BH (
      • Jenkins T.C.
      • Wallace R.
      • Moate P.
      • Mosley E.
      Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem.
      ), and the concentration of trans-10 C18:1 in milk can serve as a proxy to characterize the condition. This is mostly because absorbed FA are rapidly incorporated into milk fat (
      • Harvatine K.J.
      • Bauman D.
      Characterization of the acute lactational response to trans-10, cis-12 conjugated linoleic acid.
      ) and because the majority (>85%) of preformed milk FA come from absorption in cows in a positive energy balance (
      • Palmquist D.
      • Mattos W.
      Turnover of lipoproteins and transfer to milk fat of dietary (1-carbon-14) linoleic acid in lactating cows.
      ).
      We observed a 3-way interaction of treatment, dietary phase, and production level, where HMTBa supplementation resulted in greater milk fat concentration during the moderate- and high-risk phases and greater milk fat yield in the high-risk phase in high-producing cows. Greater milk fat synthesis in HMTBa-supplemented high-producing cows resulted primarily from maintenance of the de novo FA synthesis pathway in the mammary gland. Supplementation with HMTBa, which is a hydroxyl analog of methionine, was initially intended to provide additional metabolizable methionine to the cow. However, with a reported ruminal degradability of 50% or more (
      • Koenig K.M.
      • Rode L.
      • Knight C.
      • McCullough P.
      Ruminal escape, gastrointestinal absorption, and response of serum methionine to supplementation of liquid methionine hydroxy analog in dairy cows.
      ;
      • Noftsger S.
      • St-Pierre N.
      • Sylvester J.
      Determination of rumen degradability and ruminal effects of three sources of methionine in lactating cows.
      ), HMTBa may also affect rumen microbial composition or growth by providing methionine or through a rumen modifier mechanism. In fact, HMTBa has been suggested to increase bacterial N synthesis in vitro and in vivo, which could be associated with increased microbial protein yield (
      • Vázquez-Añón M.
      • Cassidy T.
      • McCullough P.
      • Varga G.
      Effects of alimet on nutrient digestibility, bacterial protein synthesis, and ruminal disappearance during continuous culture.
      ;
      • Lee C.
      • Oh J.
      • Hristov A.
      • Harvatine K.
      • Vazquez-Anon M.
      • Zanton G.
      Effect of 2-hydroxy-4-methylthio-butanoic acid on ruminal fermentation, bacterial distribution, digestibility, and performance of lactating dairy cows.
      ). Early work proposed that the positive effect of HMTBa on milk fat was due to a direct effect of HMTBa on rumen lipid metabolism (
      • Patton R.A.
      • McCarthy R.
      • Griel L.
      Observations on rumen fluid, blood serum, and milk lipids of cows fed methionine hydroxy analog 1, 2.
      ). However, the effect of HMTBa on milk fat is variable, but more apparent when feeding diets with increased risk of MFD. For example,
      • Rosser R.
      • Polan C.
      • Chandler P.
      • Bibb T.
      Effects of whey components and methionine analog on bovine milk fat production.
      reported that supplementation with a methionine analog maintained milk fat synthesis when cows were fed a highly fermentable diet (85% pelleted concentrate). On the contrary, no increase in milk fat was observed when low-risk diets (>33% NDF and <3% ether extract) were supplemented with HMTBa (
      • Johnson-VanWieringen L.M.
      • Harrison J.
      • Davidson D.
      • Swift M.
      • Von Keyserlingk M.
      • Vazquez-Anon M.
      • Wright D.
      • Chalupa W.
      Effects of rumen-undegradable protein sources and supplemental 2-hydroxy-4-(methylthio)-butanoic acid and lysine· hcl on lactation performance in dairy cows.
      ;
      • Lee C.
      • Oh J.
      • Hristov A.
      • Harvatine K.
      • Vazquez-Anon M.
      • Zanton G.
      Effect of 2-hydroxy-4-methylthio-butanoic acid on ruminal fermentation, bacterial distribution, digestibility, and performance of lactating dairy cows.
      ).
      In high-producing cows, HMTBa supplementation during the high-risk phase decreased the concentration of alternate BH isomers (trans-10 C18:1) and increased isomers of the normal pathway (trans-11 C18:1). This indicates a role for HMTBa in stabilizing rumen BH and reducing the shift to the altered trans-10 pathways that occurs during BH-induced MFD. This positive effect of HMTBa on ruminal BH could be mediated through 2 mechanisms. First, if an increase in microbial mass occurs with HMTBa supplementation (see discussion above), an increase in BH capacity would be expected. Second, HMTBa may modify the microbial population and maintain key ruminal microbial species important to BH.
      • Rico D.E.
      • Preston S.
      • Risser J.
      • Harvatine K.
      Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.
      characterized the shift in common culturable microbial populations during BH-induced MFD and reported an increase in the abundance of Streptococcus bovis (amylolytic) and Megasphaera elsdenii and Selenomonas ruminantium (lactate-utilizing bacteria) and a decrease in Fibrobacter succinogenes (fibrolytic) and Butyrivibrio fibrisolvens/Pseudobutyrivibrio group (BH-involved). Several studies have indicated that bacteria other than Butyrivibrio spp. might be involved in BH (
      • Shingfield K.
      • Wallace R.
      Synthesis of conjugated linoleic acid in ruminants and humans.
      ), but the key populations in vivo are not clear. Additionally,
      • Martin C.
      • Mirande C.
      • Morgavi D.
      • Forano E.
      • Devillard E.
      • Mosoni P.
      Methionine analogues hmb and hmbi increase the abundance of cellulolytic bacterial representatives in the rumen of cattle with no direct effects on fiber degradation.
      observed an increase in the abundance of Fibrobacter succinogenes in rumen contents of cows supplemented with a methionine analog. Although F. succinogenes has been described primarily as fibrolytic, a role for this species in the BH of linoleic acid (C18:2) has also been proposed (
      • Shingfield K.
      • Wallace R.
      Synthesis of conjugated linoleic acid in ruminants and humans.
      ).
      • Baldin M.
      • Ying Y.
      • Zanton G.
      • Tucker H.
      • Vasquez-Anon M.
      • Harvatine K.
      2-hydroxy-4-(methylthio) butanoate (hmtba) supplementation increases milk fat and decreases synthesis of alternative biohydrogenation intermediates in diets with risk for milk fat depression.
      evaluated the effect of HMTBa on microbial populations when feeding diets with increased risk of MFD. Even though there was an increase in the abundance of rumen protozoa, those authors reported no changes in 9 selected microbial taxa with well-characterized functions, including 2 species (Butyrivibrio/Pseudobutyrivibrio, Butyrivibrio hungatei) involved in trans-11 production. Of note, the bacterial species measured in the study of
      • Baldin M.
      • Ying Y.
      • Zanton G.
      • Tucker H.
      • Vasquez-Anon M.
      • Harvatine K.
      2-hydroxy-4-(methylthio) butanoate (hmtba) supplementation increases milk fat and decreases synthesis of alternative biohydrogenation intermediates in diets with risk for milk fat depression.
      represented only a small fraction of the total rumen microbiome that can be potentially important to ruminal BH. Characterization of microbial population was beyond the scope of the current experiment.
      A postrumen effect of methionine and analog compounds on milk fat synthesis has also been proposed (
      • Patton R.A.
      • McCarthy R.
      • Griel L.
      Observations on rumen fluid, blood serum, and milk lipids of cows fed methionine hydroxy analog 1, 2.
      ;
      • Huber T.J.
      • Emery R.
      • Bergen W.
      • Liesman J.
      • Kung L.
      • King K.
      • Gardner R.
      • Checketts M.
      Influences of methionine hydroxy analog on milk and milk fat production, blood serum lipids, and plasma amino acids.
      ;
      • Zanton G.I.
      • Bowman G.
      • Vázquez-Añón M.
      • Rode L.
      Meta-analysis of lactation performance in dairy cows receiving supplemental dietary methionine sources or postruminal infusion of methionine.
      ). Methionine plays an important role in choline synthesis, and methionine and choline both act as methyl donors, thereby potentially influencing milk volume and milk fat (
      • Zanton G.I.
      • Bowman G.
      • Vázquez-Añón M.
      • Rode L.
      Meta-analysis of lactation performance in dairy cows receiving supplemental dietary methionine sources or postruminal infusion of methionine.
      ). Additionally, methionine is involved in the transport of lipids (
      • Patton R.A.
      • McCarthy R.
      • Griel L.
      Observations on rumen fluid, blood serum, and milk lipids of cows fed methionine hydroxy analog 1, 2.
      ) and its supplementation has been shown to increase blood triglycerides (
      • Huber T.J.
      • Emery R.
      • Bergen W.
      • Liesman J.
      • Kung L.
      • King K.
      • Gardner R.
      • Checketts M.
      Influences of methionine hydroxy analog on milk and milk fat production, blood serum lipids, and plasma amino acids.
      ), which are a key substrate for milk fat synthesis. Contributions of postrumen effects to the milk fat response observed in the current study cannot be ruled out, but we expect that the rumen mechanism played the predominant role.
      The experimental design used in our study verified the existence of interactions between dietary risk factors for MFD, animal characteristics, and a feed additive. Our approach provided an efficient way to investigate these interactions and demonstrated the potential of HMTBa to reduce BH-induced MFD. This important interaction could not have not been identified if the study had looked at a single factor at a time and should also represent on farm scenarios where gradual modifications in the diet increase the risk of BH-induced MFD.

      CONCLUSIONS

      High-producing cows had lower milk fat concentration than low-producing cows. During the treatment period, increasing diet UFA and carbohydrate fermentability caused MFD in high-producing cows receiving the control treatment, but HMTBa supplementation maintained higher milk fat concentration and yield, especially during the high-risk phase. Additionally, HMTBa maintained lower concentration of trans-10 C18:1 in milk; which suggested a role for HMTBa in stabilizing rumen BH and preventing the shift to the altered BH pathway. Low-producing cows neither experienced substantial BH-induced MFD nor a response in milk fat to HMTBa supplementation. Characterization of the interaction of dietary risk factors, milk production level, and HMTBa supplementation provides a mechanism to prevent or attenuate BH-induced MFD on farms and reduces its occurrence in high-producing cows within herds with normal milk fat. The experimental model offers a framework to further study other rumen modifiers that can combat BH-induced MFD.

      ACKNOWLEDGMENTS

      Gratitude is expressed to Y. Ying and N. Urrutia (Penn State University) for technical assistance and to the Pennsylvania State University Dairy Cattle Research and Education Center for continuous care of animals. We also thank Novus International and Penn State for support of the project.

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