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Comparison of the effects of short-term feeding of sodium acetate and sodium bicarbonate on milk fat production

  • C. Matamoros
    Affiliations
    Department of Animal Science, Pennsylvania State University, University Park 16802
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  • Author Footnotes
    * Current address: Drug Metabolism and Pharmacokinetics, Genentech, 1 DNA Way, South San Francisco, CA 94080.
    J. Cai
    Footnotes
    * Current address: Drug Metabolism and Pharmacokinetics, Genentech, 1 DNA Way, South San Francisco, CA 94080.
    Affiliations
    Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park 16802
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  • A.D. Patterson
    Affiliations
    Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park 16802
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  • K.J. Harvatine
    Correspondence
    Corresponding author
    Affiliations
    Department of Animal Science, Pennsylvania State University, University Park 16802
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  • Author Footnotes
    * Current address: Drug Metabolism and Pharmacokinetics, Genentech, 1 DNA Way, South San Francisco, CA 94080.
Open ArchivePublished:April 01, 2021DOI:https://doi.org/10.3168/jds.2020-19526

      ABSTRACT

      Supplementation with sodium acetate (NaAcet) increases milk fat production through an apparent stimulation of de novo lipogenesis in the mammary gland. Sodium acetate increases acetate supply to the mammary gland, but it also increases dietary cation-anion difference, which can also increase milk fat yield. The objective of this study was to determine if the effect of NaAcet on milk fat production was due to an increase in acetate supply or an increase in dietary cation-anion difference. The study included 12 multiparous cows in a replicated 3 × 3 Latin square design balanced for carryover effects, with 14-d experimental periods. Treatments were a basal total mixed ration (31.8% neutral detergent fiber, 14.8% crude protein, 25.5% starch, and 4.4% fatty acids on a dry matter basis) as a no-supplement control, acetate supplemented at 3.25% of dry matter as NaAcet, and sodium bicarbonate (NaHCO3) providing an equal amount of sodium to the NaAcet treatment. The NaAcet and NaHCO3 were mixed into the basal diet before feeding. Milk samples were taken at each milking during the last 3 d of each period. Plasma samples were taken every 9 h during the last 3 d (a total of 8 times) to determine concentrations of plasma metabolites and hormones. Eating behavior was monitored during the last week of each period using an automated system. The NaAcet and NaHCO3 treatments increased milk fat concentration and yield compared to the no-supplement control. The NaAcet treatment increased milk fat production predominantly by increasing the yield of de novo and mixed-source fatty acids. The NaHCO3 treatment increased the yield of preformed and de novo fatty acids, suggesting different mechanisms for the 2 treatments. The NaAcet treatment increased plasma acetate concentration in a period of the day concurrent with the highest dry matter intake. The NaAcet treatment increased milk fat production by stimulating the production of de novo fatty acids, a mechanism consistent with previous reports, possibly by increasing acetate supply to the mammary gland. The NaHCO3 treatment increased milk fat production by increasing the production of all biological categories of fatty acids, except for odd and branched-chain fatty acids, possibly by increasing overall diet digestibility.

      Key words

      INTRODUCTION

      Milk fat is a major contributor to the economic value of milk, and it provides an opportunity to increase farm income, because it is the most variable milk component and is highly responsive to nutrition and management. Many efforts have focused on biohydrogenation-induced milk fat depression and minimizing the reductions in milk fat yield caused by bioactive products of ruminal biohydrogenation (
      • Bauman D.E.
      • Griinari J.M.
      Nutritional regulation of milk fat synthesis.
      ). However, strategies that increase milk fat outside of biohydrogenation-induced milk fat depression provide opportunities to increase production on a large number of farms. Acetate deficiency has been disproven as the cause of classic diet-induced milk fat depression (summarized by
      • Harvatine K.J.
      • Boisclair Y.R.
      • Bauman D.E.
      Recent advances in the regulation of milk fat synthesis.
      ), but recent work has demonstrated that increasing acetate supply through the ruminal infusion of neutralized sodium acetate (NaAcet) increases milk fat production in the absence of biohydrogenation-induced milk fat depression (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ). Most recently,
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      reported that feeding NaAcet in a TMR increased milk fat concentration and yield, suggesting the possibility of NaAcet as a feed additive.
      Acetate is the main substrate for de novo lipogenesis in the mammary gland of dairy cows; it provides carbon units for the synthesis of malonyl-CoA and is a substrate for the production of approximately half of the reducing equivalents needed for de novo lipogenesis (
      • Palmquist D.L.
      • Davis C.L.
      • Brown R.E.
      • Sachan D.S.
      Availability and metabolism of various substrates in ruminants. V. Entry rate into the body and incorporation into milk fat of d(−)β-hydroxybutyrate.
      ;
      • Bauman D.E.
      • Brown R.E.
      • Davis C.L.
      Pathways of fatty acid synthesis and reducing equivalent generation in mammary gland of rat, sow, and cow.
      ).
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      reported that increasing ruminal infusion of neutralized acetate linearly increased milk fat yield. This rise was driven predominantly by an increase in milk fatty acids (FA) of less than 16 carbons that originate from de novo synthesis in the mammary gland, and 16-carbon FA that can originate from both de novo synthesis and preformed FA uptake from the plasma.
      Investigation of the effect of acetate supply is difficult experimentally, because non-neutralized acetate drastically decreases feed intake (reviewed by
      • Forbes J.M.
      • Barrio J.P.
      Abdominal chemo- and mechanosensitivity in ruminants and its role in the control of food intake.
      , and most recently reported by
      • Gualdrón-Duarte L.B.
      • Allen M.S.
      Effects of acetic acid or sodium acetate infused into the rumen or abomasum on feeding behavior and metabolic response of cows in the postpartum period.
      ), so supplementing acetate neutralized by sodium or potassium hydroxide is preferred. The 2 common sodium controls used are sodium chloride or sodium bicarbonate (NaHCO3). No perfect control is available to account for the sodium, because sodium chloride does not control for the increase in DCAD, and NaHCO3 controls for DCAD but provides additional buffering capacity to the rumen. It is important to consider the DCAD effect:
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      observed a positive linear relationship between DCAD and milk fat concentration and yield, and
      • Hu W.
      • Murphy M.R.
      Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis.
      observed a quadratic relationship between DCAD and milk fat yield, with peak milk fat yield at approximately 550 mEq/kg of DM in meta-analyses. Although the mechanism is not known,
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      attributed the effects to improved NDF digestibility and acid–base homeostasis, possibly resulting in a lower presence of bioactive biohydrogenation intermediates.
      Ruminal infusion of neutralized NaAcet increased milk fat compared with a sodium chloride control (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ), and feeding NaAcet increased milk fat yield compared with a NaHCO3 control, but the magnitude of the response was smaller (
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ). The objective of the current study was to determine the effect on milk fat production of NaAcet supplementation (as part of a TMR) compared with a NaHCO3 DCAD control and a no-supplement control. Our hypothesis was that both NaAcet and NaHCO3 would increase milk fat production compared with the no-supplement control, but via different mechanisms. Specifically, NaAcet would increase milk fat production by increasing de novo FA production through increased supply of acetate, and NaHCO3 would increase milk fat production by decreasing alternate biohydrogenation intermediates.

      MATERIALS AND METHODS

      Experimental Design and Animals

      All experimental procedures were approved by The Pennsylvania State University Institutional Animal Care and Use Committee (#46398). Twelve lactating multiparous Holstein cows (115 ± 16 DIM and 47.2 ± 7.7 kg/d of milk production, both immediately before the start of the experiment; mean ± SD) were randomly assigned to 1 of 6 treatment sequences in a 3 × 3 Latin square design with 14-d periods balanced for carryover effects. Cows were housed in a tie-stall barn at The Pennsylvania State University Dairy Production Research and Teaching Center. Ten stalls were equipped with a feed intake monitoring system that consisted of hanging feed tubes as described by
      • Rottman L.W.
      • Ying Y.
      • Zhou K.
      • Bartell P.A.
      • Harvatine K.J.
      The effects of feeding rations that differ in neutral detergent fiber and starch concentration within a day on production, feeding behavior, total-tract digestibility, and plasma metabolites and hormones in dairy cows.
      . The number and characteristics of meals and feed intake across the day were analyzed as described by
      • Niu M.
      • Ying Y.
      • Bartell P.A.
      • Harvatine K.J.
      The effects of feeding time on milk production, total-tract digestibility, and daily rhythms of feeding behavior and plasma metabolites and hormones in dairy cows.
      .

      Diets and Treatments

      Treatments were a no-supplement control (CON); a TMR containing 3.25% NaAcet (DM basis) targeting an intake of 10 mol/d of acetate; and a TMR containing 3.36% NaHCO3 (DM basis) as a DCAD control (sodium concentration equal to the NaAcet treatment). A combination of NaAcet trihydrate and anhydrous was used (65:35 wt/wt) due to availability. The NaAcet and NaHCO3 treatments were added to the CON TMR and mixed with an I. H. Rissler 1050 Mobile TMR Mixer (Zartman Farms) for a minimum of 5 min before feeding. The basal TMR was formulated to have 17% CP, 32% NDF, and 25% starch on a DM basis. Diets were not balanced to be isocaloric, because this would have required modification of the fermentation characteristics of the diet and caused possible confounding factors. The addition of NaAcet and NaHCO3 slightly diluted all nutrients in the treatment diets (Table 1) but did not change diet fermentability or the ratio of fermentable carbohydrate to protein.
      Table 1Ingredient and nutrient composition (% of DM unless otherwise noted) of experimental diets
      The sodium acetate (NaAcet) treatment delivered at 8.4 mol/d based on actual average DMI. The sodium bicarbonate (NaHCO3) treatment was a DCAD control to balance for the sodium in the NaAcet.
      ItemControlNaAcetNaHCO3
      Ingredient
       Corn silage
      Corn silage contained on average 7.3% CP, 2.8% fatty acids, 35.4% NDF, and 12.5% ADF on a DM basis.
      39.538.238.2
       Alfalfa haylage
      Alfalfa haylage contained on average 16.3% CP, 1.7% fatty acids, 44.7% NDF, and 23.2% ADF on a DM basis.
      16.916.416.4
       Ground corn14.113.713.6
       Canola meal11.310.910.9
       Roasted soybeans5.65.55.5
       Molasses3.83.63.6
       Whole cottonseed3.83.63.6
       Grass hay
      Grass hay contained on average 80.4% NDF and 27.2% ADF on a DM basis.
      2.82.72.7
       Mineral-vitamin mix
      Contained (%, as fed basis): 45.8 dried corn distillers grains with solubles; 35.8 limestone (38% Ca); 8.3 magnesium oxide (54% Mg); 6.4 salt; 1.73 vitamin ADE premix; 1.09 selenium premix (0.06% selenium); and 0.88 trace mineral mix. Composition (DM basis): 11% CP; 40% NDF; 6.9% ADF; 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; 125,875 IU/kg of vitamin A (retinyl acetate); 31,418 IU/kg of vitamin D (activated 7-dehydrocholesterol); and 946 IU/kg of vitamin E (dl-α tocopheryl acetate).
      1.91.91.9
       NPN
      Fed as a slow-release urea (Optigen, Alltech Inc.; 259% CP on a DM basis).
      0.240.240.24
       NaAcet
      Blend of sodium acetate trihydrate and anhydrous (35:65 wt/wt).
      3.3
       NaHCO33.4
      Nutrient composition
       NDF31.830.830.8
       ADF13.012.612.6
       CP14.814.414.3
       Fatty acids4.44.24.2
       Starch25.524.724.6
       Ash6.28.18.1
       NEL,
      Estimated with NDS Professional 3.9.7.07 (RUM&N Sas; https://www.rumen.it/en) based on CNCPS 6.55 (Cornell University; https://blogs.cornell.edu/cncps/). NEL was estimated with actual intake of each treatment, and the energy provided by NaAcet was considered to be as described by Urrutia and Harvatine (2017b).
      Mcal/kg
      1.701.711.64
       DCAD,
      Estimated with NDS Professional 3.9.7.07 (RUM&N Sas; https://www.rumen.it/en) based on CNCPS 6.55 (Cornell University; https://blogs.cornell.edu/cncps/). NEL was estimated with actual intake of each treatment, and the energy provided by NaAcet was considered to be as described by Urrutia and Harvatine (2017b).
      mEq/kg
      176572575
      1 The sodium acetate (NaAcet) treatment delivered at 8.4 mol/d based on actual average DMI. The sodium bicarbonate (NaHCO3) treatment was a DCAD control to balance for the sodium in the NaAcet.
      2 Corn silage contained on average 7.3% CP, 2.8% fatty acids, 35.4% NDF, and 12.5% ADF on a DM basis.
      3 Alfalfa haylage contained on average 16.3% CP, 1.7% fatty acids, 44.7% NDF, and 23.2% ADF on a DM basis.
      4 Grass hay contained on average 80.4% NDF and 27.2% ADF on a DM basis.
      5 Contained (%, as fed basis): 45.8 dried corn distillers grains with solubles; 35.8 limestone (38% Ca); 8.3 magnesium oxide (54% Mg); 6.4 salt; 1.73 vitamin ADE premix; 1.09 selenium premix (0.06% selenium); and 0.88 trace mineral mix. Composition (DM basis): 11% CP; 40% NDF; 6.9% ADF; 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; 125,875 IU/kg of vitamin A (retinyl acetate); 31,418 IU/kg of vitamin D (activated 7-dehydrocholesterol); and 946 IU/kg of vitamin E (dl-α tocopheryl acetate).
      6 Fed as a slow-release urea (Optigen, Alltech Inc.; 259% CP on a DM basis).
      7 Blend of sodium acetate trihydrate and anhydrous (35:65 wt/wt).
      8 Estimated with NDS Professional 3.9.7.07 (RUM&N Sas; https://www.rumen.it/en) based on CNCPS 6.55 (Cornell University; https://blogs.cornell.edu/cncps/). NEL was estimated with actual intake of each treatment, and the energy provided by NaAcet was considered to be as described by
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      .

      Feed Sampling and Analysis

      Cows were fed individually once a day (0600 h) at 110% of expected intake. Orts were measured daily to determine DMI. Feedstuff DM was determined weekly for diet adjustment (48 h in a forced-air oven at 55°C). All individual ingredients were sampled daily during the last 3 d of each period, composited by period, and dried (48 h in a forced-air oven at 55°C). Samples were ground using a Wiley mill with a 1-mm screen (A. H. Thomas). Absolute DM was determined by drying for 48 h at 105°C; CP was determined with an elemental analyzer (Costech ECS 4010; Costech Analytical Technologies Inc.); and starch was determined using an enzymatic method as described in
      • Karkalas J.
      An improved enzymic method for the determination of native and modified starch.
      after sample gelatinization for 15 min with sodium hydroxide. Both NDF and ADF were determined as described by
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      using sodium sulfite and a heat-stable amylase (Ankom 200 Fiber Analyzer; Ankom Technology). The total FA concentration of each experimental diet was determined by GC after direct acid methylation (
      • Sukhija P.S.
      • Palmquist D.L.
      Rapid method for determination of total fatty acid content and composition of feedstuffs and feces.
      ) as described by
      • Rico D.E.
      • Ying Y.
      • Harvatine K.J.
      Effect of a high-palmitic acid fat supplement on milk production and apparent total-tract digestibility in high- and low-milk yield dairy cows.
      . Ash content was determined by complete combustion of OM of the sample with a furnace at 600°C for 6 h. All feed analyses were conducted on individual feed components, and dietary nutrient composition was estimated according to formulation.

      Milk Sample and Analysis

      Cows were milked twice daily at 0600 and 1800 h, and milk yield was determined by an integrated milk meter (AfiMilk; SAE Afikim). Milk yield was corrected using a stall deviation, calculated using data from the rest of the milking herd (>200 cows) as described by
      • Andreen D.M.
      • Salfer I.J.
      • Ying Y.
      • Reinemann D.J.
      • Harvatine K.J.
      Technical note: Method for improving precision of in-parlor milk meters and adjusting milk weights for stall effects.
      . Briefly, individual stall deviations were calculated by modeling the effect of day, milking, cow, and stall, excluding observations from cows in the current experiment. Milk was sampled at both milkings during the last 3 d of the experimental period. One sample was stored at 4°C with preservative (Bronolab-WII; Advanced Instruments Inc.) until it was analyzed for fat, protein, and MUN by Fourier-transform infrared spectroscopy (Fossomatic Milko-Scan FT+ and FC; Foss Electric; Dairy One). Another milk sample was composited by cow within period according to milk yield, and a fat cake was extracted by centrifugation at 1,300 × g for 15 min at 4°C. The milk FA profile was determined by GC as described by
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      . Briefly, milk lipids were extracted with hexane:isopropanol (
      • Hara A.
      • Radin N.S.
      Lipid extraction of tissues with a low-toxicity solvent.
      ) and transmethylated with sodium methoxide (
      • 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.
      ). Then, FA methyl esters were quantified with a GC equipped with a flame ionization detector.

      Plasma Sample and Analysis

      Blood samples were collected from the tail vessel using vacuum tubes containing sodium heparin (Greiner Bio-One North America Inc.) 8 times over the last 3 d of each experimental period. Blood was immediately placed on ice and plasma was collected after centrifugation for 1,300 × g for 15 min at 4°C. Plasma samples were stored in a freezer at −20°C until further analysis. Plasma glucose was determined using the glucose oxidase-peroxidase method (PGO enzyme P 7119; Sigma-Aldrich;
      • Raabo E.
      • Terkildsen T.C.
      On the enzymatic determination of blood glucose.
      ), nonesterified fatty acids (NEFA) were measured as described by
      • Ballou M.A.
      • Gomes R.C.
      • Juchem S.O.
      • DePeters E.J.
      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.
      ; Wako HR Series NEFA-HR kit; Wako Chemicals USA), and BHB was measured using a BHB dehydrogenase colorimetric method (β-Hydroxybutyrate LiquiColor; Stanbio Chemistry). Plasma insulin was determined using a commercially available radioimmunoassay that is 90% cross-reactive with bovine insulin (PI-12K Radioimmunoassay; Millipore Corp.).
      Plasma acetate concentration was determined based on the method of
      • Cai J.
      • Zhang J.
      • Tian Y.
      • Zhang L.
      • Hatzakis E.
      • Krausz K.W.
      • Smith P.B.
      • Gonzalez F.J.
      • Patterson A.D.
      Orthogonal comparison of GC–MS and 1 H NMR spectroscopy for short chain fatty acid quantitation.
      after optimization for bovine plasma. Briefly, 300 µL of plasma were mixed with 500 µL of 0.005 M aqueous sodium hydroxide containing 10 µg/mL of hexanoic-6,6,6-d3 acid (C/D/N Isotopes Inc.). Samples were extracted and derivatized into propyl esters in a 1-step procedure using 500 µL of propanol:pyridine (vol/vol = 3:2) mixture and propyl chloroformate as the derivatizing agent, and incubated at 60°C for 1 h. Derivatized samples were extracted twice with hexane to a total volume of 500 µL (300 µL + 200 µL), placed in a glass autosampler vial, and stored at −20°C until GC-MS quantitation. Experimental conditions for GC-MS were as described in
      • Cai J.
      • Zhang J.
      • Tian Y.
      • Zhang L.
      • Hatzakis E.
      • Krausz K.W.
      • Smith P.B.
      • Gonzalez F.J.
      • Patterson A.D.
      Orthogonal comparison of GC–MS and 1 H NMR spectroscopy for short chain fatty acid quantitation.
      . All MS data were processed using MS-Dial (ver. 2.9; http://prime.psc.riken.jp/Metabolomics_Software/MS-DIAL/) for identification and normalized to the internal standard (hexanoic-6,6,6-d3 acid) for quantitation.

      Statistical Analysis

      Milk production variables were analyzed with JMP Pro 13.2.1 (SAS Institute Inc.) in the Fit Model module, with a standard least square personality and a model that included the fixed effect of treatment and the random effects of cow and period. Treatment means were separated using a protected least significant difference test. The effect of milking time (a.m. vs. p.m.) on milk production variables was assessed using repeated measures in PROC MIXED of SAS 9.4 (SAS Institute Inc.). The model included the fixed effects of milking time, treatment, and their interaction, and the random effects of cow and period. Subject was cow by period, denominator degrees of freedom were adjusted using the Kenwood Rogers method, and the heterogeneous autoregressive covariance structure was used. Plasma metabolite data were analyzed similarly using repeated measures and the heterogeneous autoregressive structure. Preplanned contrasts within time points for repeated data were control versus NaAcet and NaAcet versus NaHCO3. High-resolution figures were created using Daniel's XL Toolbox (
      • Kraus D.
      Consolidated data analysis and presentation using an open-source add-in for the Microsoft Excel ® spreadsheet software.
      ).
      Differences for main effects were declared at P ≤ 0.05, and tendencies at 0.05 < P ≤ 0.10; differences for interactions were declared at P ≤ 0.10, and tendencies at 0.10 < P ≤ 0.15. One cow had diet-induced milk fat depression during 2 of the 3 experimental periods, as defined by a high milk fat C18:1 trans-10 concentration (1.02 and 11.1% of FA on NaAcet and CON, respectively; individual data not shown). The data points for this cow were included in the analysis, because they were not outliers (within ±3 Studentized residuals). However, conclusions did not change whether those data points were included or not.

      RESULTS AND DISCUSSION

      Intake and Feeding Behavior

      The NaAcet treatment was designed to provide approximately 10 mol/d of acetate as NaAcet, based on a previous dose titration study (
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ), but it provided 8.4 mol/d at the actual average DMI. We found an effect of treatment (P = 0.02; Table 2) on DMI, whereby NaAcet was not different from CON but NaHCO3 decreased DMI by 1.4 kg/d compared with CON. Recent work suggested that ruminally infused NaAcet at a dose similar to that fed in the current experiment did not decrease DMI (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ). However,
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      reported that DMI was 2.5 kg/d higher when feeding NaAcet at a similar dose compared with a NaHCO3 sodium control. It is apparent from our results that NaHCO3 at the high rates fed in our experiment decreased DMI; it might not be the optimal control when assessing the effect of NaAcet on DMI. Based on the quadratic relationship between DMI and DCAD reported in a meta-analysis by
      • Hu W.
      • Murphy M.R.
      Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis.
      , it is reasonable to expect a decrease in DMI at the NaHCO3 level fed in the current experiment. However, in a meta-analysis that consisted mostly of NaHCO3 supplementation studies,
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      reported a curvilinear relationship between DCAD and DMI, in contrast to our results. It is important to note that the estimated DCAD levels in our experimental diets were on the high end of the reported literature: very few treatment means were greater than 500 mEq/kg of DM. Our inclusion rate for NaHCO3 was also considerably higher than that reported in the literature; for example, the highest NaHCO3 inclusion in the meta-analysis of
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      was 2.41% of DM (
      • Wildman C.D.
      • West J.W.
      • Bernard J.K.
      Effects of dietary cation-anion difference and potassium to sodium ratio on lactating dairy cows in hot weather.
      ), 28% lower than the feeding rate in the current experiment. Importantly, the NaHCO3 treatment in the current experiment was designed as a control for the NaAcet treatment and is not a recommended feeding rate.
      Table 2Effect of feeding sodium acetate (NaAcet) and sodium bicarbonate (NaHCO3) on DMI and feeding behavior
      VariableTreatment
      Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI.
      SEMP-value: Treatment
      P-value for the fixed effect of treatment in a mixed model that included the random effect of period and cow; n = 12 cows per treatment.
      ControlNaAcetNaHCO3
      DMI, kg/d23.4
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      23.5
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      22.0
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      1.410.02
      Meals, bouts/d10.010.59.80.400.06
      Meal length, min/meal35.234.437.31.160.06
      Meal weight, kg/meal2.292.472.320.100.28
      a,b Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      1 Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI.
      2 P-value for the fixed effect of treatment in a mixed model that included the random effect of period and cow; n = 12 cows per treatment.
      The number of meal bouts per day tended to decrease by 0.7 meals/d in NaHCO3 compared with NaAcet (P = 0.06; Table 2). Meal length tended to increase 2.9 min per meal in NaHCO3 compared with NaAcet (P = 0.06; Table 2), but meal size did not differ between treatments. We found no effect of treatment, but we did find an effect of time (P < 0.001) on eating rate when calculated as kilograms per hour or percent per hour (Figure 1A and Figure 1B, respectively). As expected, intake was increased after feed delivery at 0700 h and during the afternoon, and it was lower overnight.
      Figure thumbnail gr1
      Figure 1Effect of feeding sodium acetate (NaAcet) and sodium bicarbonate (NaHCO3) on rate of feed intake across the day, as observed by an automated system over 2-h intervals. Data shown as eating rate in (A) kilograms per hour and (B) percent of daily intake per hour. Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI. The effects of treatment (Trt), time of day (time), and their interaction are shown in each panel. Data are least squares means with standard error of the mean.
      Technical issues precluded the estimation of total-tract digestibility in this experiment (data not shown). It is important to consider the effect of the increase in sodium intake as a positive linear relationship that has been reported between apparent DM digestibility and DCAD (
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      ). Although the mechanism has not been demonstrated, it may be due to increases in ruminal pH, changes in ruminal dilution rate, or associative effects on the rumen microbiome, warranting further research on the effect of diets with a high DCAD on feeding behavior, ruminal microbial function, and apparent total-tract digestibility (
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      ;
      • Mao S.
      • Huo W.
      • Liu J.
      • Zhang R.
      • Zhu W.
      In vitro effects of sodium bicarbonate buffer on rumen fermentation, levels of lipopolysaccharide and biogenic amine, and composition of rumen microbiota.
      ). Fiber digestibility might have been limited by RDP supply, because the dietary CP content was lower than expected in the experimental diets (
      • Soliva C.R.
      • Amelchanka S.L.
      • Kreuzer M.
      The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources.
      ).

      Milk Fat Production and Milk FA Profile

      We found effects of treatment on milk fat concentration and yield as hypothesized (both P = 0.001; Table 3). Both NaAcet and NaHCO3 treatment increased milk fat yield by 0.20 and 0.16 kg/d and milk fat concentration by 0.43 or 0.29 percentage points, respectively, compared with CON. Milk fat concentration and yield did not differ between NaAcet and NaHCO3. Feeding an average dose of 500 g/d (8.4 mol/d) of acetate in the NaAcet treatment resulted in an increase of 200 g/d of milk fat compared with CON, so the apparent transfer rate of carbon from acetate to milk FA was approximately 40%. This finding was in accordance with recent NaAcet ruminal infusions:
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      reported a 42% apparent transfer rate with a dose of 7 mol/d of acetate, and
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      reported a 36% apparent transfer rate with a dose of 10 mol/d of acetate. Synthesis of milk fat and lactose are partially under shared regulation. Recent publications have not reported an effect of NaAcet supplementation on milk yield similar to the current study, suggesting that increased NaAcet supply likely stimulates the lipogenic capacity of the mammary gland specifically, rather than generally stimulating all milk components (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ;
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ).
      Table 3Effect of feeding sodium acetate (NaAcet) and sodium bicarbonate (NaHCO3) on milk, milk components, and fatty acid (FA) composition
      VariableTreatment
      Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI.
      SEMP-value: Treatment
      P-value for the fixed effect of treatment in a mixed model that included the random effect of period and cow; n = 12 cows per treatment.
      ControlNaAcetNaHCO3
      Milk yield, kg/d
       Milk44.445.645.73.340.41
       Fat1.46
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      1.66
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      1.62
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.090.001
       Protein1.291.331.330.090.48
      Milk composition, %
       Fat3.31
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      3.74
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      3.59
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.150.001
       Protein2.942.952.940.070.89
       MUN, mg/dL9.72
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      8.17
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      9.15
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.690.002
      FA yield by source,
      Calculated with milk fat yield and milk fatty acid composition and corrected for glycerol content in milk fat. Straight-chain even-carbon FA <16C originate from de novo synthesis in the mammary gland; straight-chain 16C FA originated from plasma and de novo synthesis; and FA >16C originate from plasma; OBCFA is the summation of odd and branched-chain FA.
      g/d
       Σ <16C373
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      431
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      416
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      25.100.01
       Σ 16C362
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      458
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      411
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      29.06<0.001
       Σ >16C55859461129.040.07
       Σ OBCFA43.243.245.62.80.24
      FA composition, % of FA
       Σ <16C27.327.427.30.550.99
       Σ 16C26.4
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      29.5
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      27.2
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.54<0.001
       Σ >16C41.3
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      38.3
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      40.4
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.60<0.001
       Σ OBCFA3.2
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      2.8
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      3.0
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.07<0.001
      Specific FA of interest, % of FA
       C16:025.5
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      28.5
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      26.3
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.52<0.001
       C16:1 cis-90.94
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.99
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.92
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.060.01
       C18:1 trans-100.64
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.58
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.57
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.050.02
       C18:1 cis-917.9
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      16.9
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      17.6
      Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      0.430.04
      a–c Least squares means within a row with different superscript letters differ (P ≤ 0.05).
      1 Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI.
      2 P-value for the fixed effect of treatment in a mixed model that included the random effect of period and cow; n = 12 cows per treatment.
      3 Calculated with milk fat yield and milk fatty acid composition and corrected for glycerol content in milk fat. Straight-chain even-carbon FA <16C originate from de novo synthesis in the mammary gland; straight-chain 16C FA originated from plasma and de novo synthesis; and FA >16C originate from plasma; OBCFA is the summation of odd and branched-chain FA.
      We found an effect of treatment on mixed and preformed FA concentration in milk fat (P < 0.001 for both; Table 3). Sodium acetate increased the concentration of mixed-source FA (16-carbon FA) by 3.1 and 2.3 percentage points compared with CON and NaHCO3, respectively, but decreased preformed (>16 carbons) FA by 3.0 and 2.1 percentage points. We found no effect of treatment on concentrations of de novo FA in milk FA (P = 0.99; Table 3). Treatment did affect de novo FA yield: NaAcet increased de novo yield by 58 g/d compared with CON but was not different from NaHCO3. We found an effect of treatment on mixed-source FA yield: NaAcet increased yield by 96 and 47 g/d compared with CON and NaHCO3, respectively (P ≤ 0.01 for both; Table 3). We found a tendency for an effect of treatment on preformed FA yield (P = 0.07): NaHCO3 numerically increased preformed FA yield by 53 and 17 g/d compared with CON and NaAcet treatment, respectively (Table 3).
      The increase in milk fat production by NaAcet treatment appears to have been driven mainly by an increase in production of de novo and mixed-source FA compared with CON, consistent with previous experiments (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ;
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ). Interestingly, within individual FA, NaAcet increased the concentration of C16:0 by 3.0 and 2.2 percentage points compared with CON and NaHCO3 (P < 0.001; Table 3), suggesting that increasing the acetate supply to the mammary gland drives de novo lipogenesis and de novo FA elongation toward completion. The regulation of chain termination reactions and acyltransferases in mammary de novo lipogenesis in ruminants is not well understood (reviewed by
      • Palmquist D.L.
      Milk Fat: Origin of Fatty Acids and Influence of Nutritional Factors Thereon.
      ); the mechanism by which an increase in acetate supply appears to change the catalytic activity of these reactions is unknown and requires further research.
      Both NaAcet and NaHCO3 decreased the concentration of C18:1 trans-10 in milk FA by 0.06 and 0.07 percentage points, respectively, compared with CON (P = 0.02; Table 3). This small change was likely not biologically relevant and does not explain the magnitude of the increase in milk fat we observed. The experimental diet was formulated to minimize the risk of diet-induced milk fat depression, and all treatments were well below the reported mean and median of milk C18:1 trans-10 from a recent metanalysis of more than 500 treatment means of milk C18:1 trans-10 in the literature (mean = 1.39 and median = 0.7 C18:1 trans-10 g/100 g of FA;
      • Matamoros C.
      • Klopp R.N.
      • Moraes L.E.
      • Harvatine K.J.
      Meta-analysis of the relationship between milk trans-10 C18:1, milk fatty acids <16 C, and milk fat production.
      ). Furthermore, using the equation reported by
      • Matamoros C.
      • Klopp R.N.
      • Moraes L.E.
      • Harvatine K.J.
      Meta-analysis of the relationship between milk trans-10 C18:1, milk fatty acids <16 C, and milk fat production.
      ; milk fat percentage = 2.51 + 1.55e(−0.503 × C18:1 trans-10 g/100 g of FA)], the changes in milk C18:1 trans-10 g/100 g of FA would represent a change of approximately 1% in milk fat percentage for NaAcet and NaHCO3 compared with CON. Future work is needed to understand the effect of NaAcet supplementation on milk fat production that includes diets with varying risks of diet-induced milk fat depression.
      We found an effect of treatment on odd and branched-chain FA (OBCFA) concentrations in milk fat, but not on OBCFA yield (P < 0.001 and P = 0.24, respectively; Table 3). The NaAcet treatment decreased OBCFA concentration in milk by 0.4 and 0.2 percentage points compared with CON and NaHCO3, respectively. Milk OBCFA and C18:1 trans content and ruminal microbial function have a clear relationship (
      • Griinari J.M.
      • Bauman D.E.
      Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants.
      ;
      • Vlaeminck B.
      • Fievez V.
      • Cabrita A.R.J.
      • Fonseca A.J.M.
      • Dewhurst R.J.
      Factors affecting odd- and branched-chain fatty acids in milk: A review.
      ). It is important to note that the current experiment had low levels of alternate biohydrogenation intermediates, and future work should test the ability of both NaAcet and NaHCO3 to decrease alternate biohydrogenation during challenging situations.
      A positive linear relationship between DCAD and milk fat production was reported in a meta-analysis by
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      . The relationship between milk fat yield or fat-corrected milk yield has also been characterized as a quadratic relationship, with maximal milk fat production occurring at approximately 550 mEq/kg of DM, in agreement with our results at our estimated DCAD (
      • Hu W.
      • Murphy M.R.
      Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis.
      ;
      • Iwaniuk M.E.
      • Weidman A.E.
      • Erdman R.A.
      The effect of dietary cation-anion difference concentration and cation source on milk production and feed efficiency in lactating dairy cows.
      ). The specific mechanism for how increasing dietary DCAD by increasing sodium leads to an increase milk fat is unknown, but
      • Iwaniuk M.E.
      • Weidman A.E.
      • Erdman R.A.
      The effect of dietary cation-anion difference concentration and cation source on milk production and feed efficiency in lactating dairy cows.
      proposed that it might be due to a decrease in the risk of diet-induced milk fat depression through improved ruminal fermentation. In vitro work has shown lower daily production of C18:1 trans-10 in continuous culture fermenters by increasing DCAD with the addition of potassium or sodium as K2CO3 or NaOH, respectively (
      • Jenkins T.C.
      • Bridges Jr., W.C.
      • Harrison J.H.
      • Young K.M.
      Addition of potassium carbonate to continuous cultures of mixed ruminal bacteria shifts volatile fatty acids and daily production of biohydrogenation intermediates.
      ). In the current experiment, the mechanism by which NaHCO3 increased milk fat production was not obvious, given that both treatments had low milk concentrations of C18:1 trans-10 (indicating an absence of biohydrogenation-induced milk fat depression) and that no ruminal parameters were measured. Considering that NaHCO3 increased milk preformed FA, it is possible that NaHCO3 stabilizes ruminal function resulting in increased FA digestibility, but we did not measure this; it is also possible that increased overall nutrient digestibility had a sparing effect of circulating preformed FA by peripheral tissues other than the mammary gland (
      • Iwaniuk M.E.
      • Erdman R.A.
      Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
      ). It is important to note that the NaHCO3 treatment was designed to provide an amount of sodium equal to that of the NaAcet treatment, so the NaHCO3 feeding rate was considerably higher than that of a commercial diet.

      Milk Yield, Milk Protein, and MUN

      We found no effect of treatment on milk yield or on milk protein concentration and yield (P = 0.41, 0.53, and 0.48, respectively; Table 3). This was consistent with other reports, in which dosages ranging from 5 to 15 mol/d of acetate supplemented as NaAcet did not affect milk yield or milk protein production (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ;
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ). We also found an effect of treatment on MUN (P < 0.05; Table 3): NaAcet decreased MUN by 1.55 and 0.98 mg/dL compared with CON and NaHCO3, respectively. The increase in sodium intake in the NaAcet treatment may have led to an increase in water intake and urine production, thus decreasing BUN and consequently MUN (
      • Spek J.W.
      • Dijkstra J.
      • Van Duinkerken G.
      • Bannink A.
      A review of factors influencing milk urea concentration and its relationship with urinary urea excretion in lactating dairy cattle.
      ).

      Plasma Metabolites

      Feeding NaAcet in a TMR is expected to result in more dynamic absorption of acetate across the day compared with continuous infusions, because acetate delivery is affected by the daily pattern of feed intake (i.e., feed intake was more than 3-fold higher during the day from approximately 0800 to 1700 h than overnight from approximately 2000 to 0500 h;
      • Niu M.
      • Ying Y.
      • Bartell P.A.
      • Harvatine K.J.
      The effects of feeding time on milk production, total-tract digestibility, and daily rhythms of feeding behavior and plasma metabolites and hormones in dairy cows.
      ). Supplementation with NaAcet increased plasma acetate concentrations (treatment P < 0.001; Figure 2A). Plasma acetate concentrations peaked in the latter portion of the day (~2000 h), although we found no treatment × time interaction. This peak was concomitant with the period of highest intake during the day, and the highest rate of acetate absorption across the rumen wall. The peak of plasma acetate concentrations was also in accordance with the diurnal variations in plasma acetate concentrations of cows fed ad libitum reported by
      • Allen M.S.
      • Bradford B.J.
      • Harvatine K.J.
      The cow as a model to study food intake regulation.
      . The diurnal variations of plasma acetate did not change treatment responses between milkings: we found no interaction between treatment and milking time in the current experiment (Supplemental Figure S1; https://scholarsphere.psu.edu/resources/13f2add6-d337-43e3-820d-b5954902ac38). However, cows were only milked twice per day; a higher milking frequency might have allowed observation of a treatment × time interaction, because higher plasma acetate is expected to change intracellular acetate across the day.
      Figure thumbnail gr2
      Figure 2Effect of feeding sodium acetate (NaAcet) and sodium bicarbonate (NaHCO3) on select plasma metabolites over the day: (A) acetate, (B) BHB, (C) glucose, (D) insulin, and (E) nonesterified fatty acids (NEFA). Treatments were a no-treatment control, 3.25% NaAcet, and 3.36% NaHCO3 (sodium equivalent to NaAcet treatment) on a DM basis. The NaAcet treatment was delivered at 8.4 mol/d based on actual average DMI. The effects of treatment (Trt), time of day (time), and their interaction are shown in each panel. Data are least squares means with standard error of the mean. Symbols denote a significant difference in treatments within a time point (P ≤ 0.05; *NaAcet vs. control and ×NaAcet vs. NaHCO3) or a tendency (0.05 < P ≤ 0.10; †NaAcet vs. control and ‡NaAcet vs. NaHCO3).
      Acetate can be converted to butyrate in the rumen, which is converted to BHB in the rumen wall (
      • Sutton J.D.
      • Dhanoa M.S.
      • Morant S.V.
      • France J.
      • Napper D.J.
      • Schuller E.
      Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets.
      ). We found an interaction of treatment × time for plasma BHB (P < 0.05; Figure 2B). Feeding NaAcet increased plasma BHB from 1700 to 2300 h compared with NaHCO3 and CON, and tended to increase plasma BHB from 1400 to 0200 h compared with CON. Plasma BHB concentration was increased during the same period that we observed the largest increases in plasma acetate. This increase in plasma BHB was in agreement with other recent NaAcet infusion (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ) and feeding (
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ) experiments in dairy cows. The effect of increased plasma BHB on milk fat is of interest, because BHB provides half of the first 4 carbons for de novo lipogenesis in the bovine mammary gland (
      • Palmquist D.L.
      Milk Fat: Origin of Fatty Acids and Influence of Nutritional Factors Thereon.
      ). Milk fat production was not changed with approximately 330 g/d of butyrate supplemented as calcium butyrate; NaAcet increased milk fat, when supplemented at an equal carbon mass (
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ). However,
      • Izumi K.
      • Fukumori R.
      • Oikawa S.
      • Oba M.
      Short communication: Effects of butyrate supplementation on the productivity of lactating dairy cows fed diets differing in starch content.
      recently reported that sodium butyrate fed at a lower rate and 2 levels of dietary starch (approximately 250 g/d of butyrate supplemented as sodium butyrate) increased milk fat yield by approximately 95 g/d.
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      reported that calcium butyrate decreased milk yield and DMI, possibly overriding the potential effects of butyrate supplementation on milk fat production. Additional work on the effect of butyrate dose and form are required to understand these responses.
      We found an interaction of treatment and time on plasma insulin (P = 0.04; Figure 2D) and plasma glucose (P = 0.09; Figure 2B) concentrations. Plasma insulin concentrations peaked in CON around 1700 h, when they were more than 40% higher than NaAcet and NaHCO3, but concentrations for NaAcet and NaHCO3 did not differ. Feeding NaAcet increased plasma insulin concentrations by 18% at 0500 h compared with CON, but concentrations were not different from NaHCO3. Plasma glucose concentrations were decreased by NaAcet during the latter portion of the day. Plasma glucose concentrations for NaAcet were different from CON at 1400, 2000, and 2300 h, different from NaHCO3 at 2300 h, and tended to be lower than NaHCO3 at 1700 and 2000 h. The greatest difference between treatments was at 2300 h, when plasma glucose concentrations for NaAcet were 5.1 and 4.9 mg/dL lower than for NaHCO3 and CON, respectively.
      Plasma NEFA tended to differ between treatments (P = 0.06). Overall plasma NEFA was numerically 16.3 and 12.4 µEq/L higher in NaAcet and NaHCO3 than in CON, respectively. Plasma NEFA peaked in all treatments just before feeding, in agreement with the diurnal variations in feeding of lactating cows fed ad libitum once a day (
      • Allen M.S.
      • Bradford B.J.
      • Harvatine K.J.
      The cow as a model to study food intake regulation.
      ). Findings were also in agreement with previous reports of infusions of NaAcet, which showed no effect on plasma NEFA concentrations compared to the control treatment. (
      • Urrutia N.
      • Harvatine K.J.
      Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
      ,
      • Urrutia N.L.
      • Harvatine K.J.
      Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
      ;
      • Urrutia N.
      • Bomberger R.
      • Matamoros C.
      • Harvatine K.J.
      Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
      ).
      Interestingly, peak plasma acetate concentrations occurred at the same time as the nadir of plasma glucose in the NaAcet treatment. Acetate has been reported to increase insulin secretion by directly stimulating pancreatic β cells (
      • Priyadarshini M.
      • Villa S.R.
      • Fuller M.
      • Wicksteed B.
      • Mackay C.R.
      • Alquier T.
      • Poitout V.
      • Mancebo H.
      • Mirmira R.G.
      • Gilchrist A.
      • Layden B.T.
      An acetate-specific GPCR, FFAR2, regulates insulin secretion.
      ) or indirectly by increasing the production of incretins such as glucagon-like peptide 1 (
      • Tolhurst G.
      • Heffron H.
      • Lam Y.S.
      • Parker H.E.
      • Habib A.M.
      • Diakogiannaki E.
      • Cameron J.
      • Grosse J.
      • Reimann F.
      • Gribble F.M.
      Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.
      ) in murine models. It is unlikely that these mechanisms played a role in the decrease in plasma glucose, because we found no increase in plasma insulin concentrations. Considering that glucose is a major source of reducing equivalents for de novo lipogenesis (
      • Ingle D.L.
      • Bauman D.E.
      • Garrigus U.S.
      Lipogenesis in the ruminant: In vitro study of tissue sites, carbon source and reducing equivalent generation for fatty acid synthesis.
      ), it is possible that the increase in de novo lipogenesis in the mammary gland with NaAcet supplementation increased the overall rate of peripheral glucose utilization, most likely from the mammary gland. This would agree with recent observations by
      • Danes M.A.C.
      • Hanigan M.D.
      • Arriola Apelo S.I.
      • Dias J.D.L.
      • Wattiaux M.A.
      • Broderick G.A.
      Post-ruminal supplies of glucose and casein, but not acetate, stimulate milk protein synthesis in dairy cows through differential effects on mammary metabolism.
      , in which an abomasal infusion of an average 925 g/d of acetate increased milk fat production and glucose uptake by the mammary gland. It would also agree with the observations of
      • Purdie N.G.
      • Trout D.R.
      • Poppi D.P.
      • Cant J.P.
      Milk synthetic response of the bovine mammary gland to an increase in the local concentration of amino acids and acetate.
      , in which infusions of acetate to the external iliac artery increased glucose uptake by 18%. This mechanism would be insulin-independent, because glucose uptake by the mammary gland is not regulated by insulin-dependent glucose transporters (
      • Bell A.W.
      • Bauman D.E.
      Adaptations of glucose metabolism during pregnancy and lactation.
      ), in accordance with our observations for milk fat production and plasma insulin and glucose concentrations.

      CONCLUSIONS

      Both NaAcet and NaHCO3 increased milk fat production in lactating dairy cows that were not experiencing biohydrogenation-induced milk fat depression. Supplementation with NaAcet increased milk fat production mainly by increasing the production of FA that are synthesized de novo in the mammary gland, including C16:0. This mechanism is consistent with recent publications that observed a similar apparent transfer of acetate carbon to milk FA of approximately 40%. Sodium acetate supplementation in a TMR was effective in increasing plasma acetate, but peaked during the latter portion of the day, likely driven by the pattern of feed intake across the day. Interestingly, increasing plasma acetate appears to increase peripheral glucose utilization, lowering plasma glucose when plasma acetate concentration is high, independent of plasma insulin concentrations. We hypothesized that this was due to an increase glucose utilization by the mammary gland to support the energy requirements of de novo lipogenesis. Supplementation with NaHCO3 also increased milk fat production, but through a different mechanism than NaAcet: it increased the production of all biological categories of FA, except for OBCFA. Because the diets were designed to have a low risk of diet-induced milk fat depression, we suspect that an increase in digestibility supported increased milk fat synthesis. Supplementation with NaAcet increased acetate supply and supported de novo lipogenesis in the lactating mammary gland, and NaHCO3 increased milk fat concentration in the absence of diet-induced milk fat depression, possibly by increasing overall diet digestibility.

      ACKNOWLEDGMENTS

      Funding was provided in part by Agriculture and Food Research Initiative Competitive Grant No. 2019-67015-29577 from the USDA National Institute of Food and Agriculture (Washington, DC) and Penn State University, including the USDA National Institute of Food and Agriculture Federal Appropriations under project number PEN04664, accession number 1017181, and NIH Grant T32GM108563 (CIM). The authors thank Isaac Salfer, Rebecca Bomberger, Elaine Barnoff, Richard Shepardson II, Danielle Andreen, and Penn State Dairy Barn staff—all of The Pennsylvania State University—for their assistance with data collection. The authors also thank Anitha Vijay, Yuan Tian, Imhoi Koo, Wei Gui, and Philip B. Smith from the Patterson Laboratory and Metabolomics Core Facility at The Pennsylvania State University for their assistance with plasma acetate analysis. The authors have not stated any conflicts of interest.

      REFERENCES

        • Allen M.S.
        • Bradford B.J.
        • Harvatine K.J.
        The cow as a model to study food intake regulation.
        Annu. Rev. Nutr. 2005; 25 (16011477): 523-547
        • Andreen D.M.
        • Salfer I.J.
        • Ying Y.
        • Reinemann D.J.
        • Harvatine K.J.
        Technical note: Method for improving precision of in-parlor milk meters and adjusting milk weights for stall effects.
        J. Dairy Sci. 2020; 103 (32307171): 5162-5169
        • Ballou M.A.
        • Gomes R.C.
        • Juchem S.O.
        • DePeters E.J.
        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.
        J. Dairy Sci. 2009; 92 (19164678): 657-669
        • Bauman D.E.
        • Brown R.E.
        • Davis C.L.
        Pathways of fatty acid synthesis and reducing equivalent generation in mammary gland of rat, sow, and cow.
        Arch. Biochem. Biophys. 1970; 140 (4394114): 237-244
        • Bauman D.E.
        • Griinari J.M.
        Nutritional regulation of milk fat synthesis.
        Annu. Rev. Nutr. 2003; 23 (12626693): 203-227
        • Bell A.W.
        • Bauman D.E.
        Adaptations of glucose metabolism during pregnancy and lactation.
        J. Mammary Gland Biol. Neoplasia. 1997; 2 (10882310): 265-278
        • Cai J.
        • Zhang J.
        • Tian Y.
        • Zhang L.
        • Hatzakis E.
        • Krausz K.W.
        • Smith P.B.
        • Gonzalez F.J.
        • Patterson A.D.
        Orthogonal comparison of GC–MS and 1 H NMR spectroscopy for short chain fatty acid quantitation.
        Anal. Chem. 2017; 89 (28650151): 7900-7906
        • 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.
        J. Nutr. 1999; 129 (10419994): 1579-1584
        • Danes M.A.C.
        • Hanigan M.D.
        • Arriola Apelo S.I.
        • Dias J.D.L.
        • Wattiaux M.A.
        • Broderick G.A.
        Post-ruminal supplies of glucose and casein, but not acetate, stimulate milk protein synthesis in dairy cows through differential effects on mammary metabolism.
        J. Dairy Sci. 2020; 103 (32418692): 6218-6232
        • Forbes J.M.
        • Barrio J.P.
        Abdominal chemo- and mechanosensitivity in ruminants and its role in the control of food intake.
        Exp. Physiol. 1992; 77 (1543591): 27-50
        • Griinari J.M.
        • Bauman D.E.
        Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants.
        in: Yurawecz M.P. Mossoba M.M. Kramer J.K.G. Pariza M.W. Nelson G.J. Advance in Conjugated Linoleic Acid Research. AOCS Press, 1999: 180-200
        • Gualdrón-Duarte L.B.
        • Allen M.S.
        Effects of acetic acid or sodium acetate infused into the rumen or abomasum on feeding behavior and metabolic response of cows in the postpartum period.
        J. Dairy Sci. 2018; 101 (29398027): 2016-2026
        • Hara A.
        • Radin N.S.
        Lipid extraction of tissues with a low-toxicity solvent.
        Anal. Biochem. 1978; 90 (727482): 420-426
        • Harvatine K.J.
        • Boisclair Y.R.
        • Bauman D.E.
        Recent advances in the regulation of milk fat synthesis.
        Animal. 2009; 3 (22444171): 40-54
        • Hu W.
        • Murphy M.R.
        Dietary cation-anion difference effects on performance and acid-base status of lactating dairy cows: A meta-analysis.
        J. Dairy Sci. 2004; 87 (15328236): 2222-2229
        • Ingle D.L.
        • Bauman D.E.
        • Garrigus U.S.
        Lipogenesis in the ruminant: In vitro study of tissue sites, carbon source and reducing equivalent generation for fatty acid synthesis.
        J. Nutr. 1972; 102 (5063218): 609-616
        • Iwaniuk M.E.
        • Erdman R.A.
        Intake, milk production, ruminal, and feed efficiency responses to dietary cation-anion difference by lactating dairy cows.
        J. Dairy Sci. 2015; 98 (26409960): 8973-8985
        • Iwaniuk M.E.
        • Weidman A.E.
        • Erdman R.A.
        The effect of dietary cation-anion difference concentration and cation source on milk production and feed efficiency in lactating dairy cows.
        J. Dairy Sci. 2015; 98 (25557895): 1950-1960
        • Izumi K.
        • Fukumori R.
        • Oikawa S.
        • Oba M.
        Short communication: Effects of butyrate supplementation on the productivity of lactating dairy cows fed diets differing in starch content.
        J. Dairy Sci. 2019; 102 (31629511): 11051-11056
        • Jenkins T.C.
        • Bridges Jr., W.C.
        • Harrison J.H.
        • Young K.M.
        Addition of potassium carbonate to continuous cultures of mixed ruminal bacteria shifts volatile fatty acids and daily production of biohydrogenation intermediates.
        J. Dairy Sci. 2014; 97 (24359822): 975-984
        • Karkalas J.
        An improved enzymic method for the determination of native and modified starch.
        J. Sci. Food Agric. 1985; 36: 1019-1027
        • Kraus D.
        Consolidated data analysis and presentation using an open-source add-in for the Microsoft Excel ® spreadsheet software.
        Medical Writing. 2014; 23: 25-28
        • Mao S.
        • Huo W.
        • Liu J.
        • Zhang R.
        • Zhu W.
        In vitro effects of sodium bicarbonate buffer on rumen fermentation, levels of lipopolysaccharide and biogenic amine, and composition of rumen microbiota.
        J. Sci. Food Agric. 2017; 97 (27339112): 1276-1285
        • Matamoros C.
        • Klopp R.N.
        • Moraes L.E.
        • Harvatine K.J.
        Meta-analysis of the relationship between milk trans-10 C18:1, milk fatty acids <16 C, and milk fat production.
        J. Dairy Sci. 2020; 103 (32921467): 10195-10206
        • Niu M.
        • Ying Y.
        • Bartell P.A.
        • Harvatine K.J.
        The effects of feeding time on milk production, total-tract digestibility, and daily rhythms of feeding behavior and plasma metabolites and hormones in dairy cows.
        J. Dairy Sci. 2014; 97 (25306274): 7764-7776
        • Palmquist D.L.
        Milk Fat: Origin of Fatty Acids and Influence of Nutritional Factors Thereon.
        Springer, US2009
        • Palmquist D.L.
        • Davis C.L.
        • Brown R.E.
        • Sachan D.S.
        Availability and metabolism of various substrates in ruminants. V. Entry rate into the body and incorporation into milk fat of d(−)β-hydroxybutyrate.
        J. Dairy Sci. 1969; 52: 633-638
        • Priyadarshini M.
        • Villa S.R.
        • Fuller M.
        • Wicksteed B.
        • Mackay C.R.
        • Alquier T.
        • Poitout V.
        • Mancebo H.
        • Mirmira R.G.
        • Gilchrist A.
        • Layden B.T.
        An acetate-specific GPCR, FFAR2, regulates insulin secretion.
        Mol. Endocrinol. 2015; 29 (26075576): 1055-1066
        • Purdie N.G.
        • Trout D.R.
        • Poppi D.P.
        • Cant J.P.
        Milk synthetic response of the bovine mammary gland to an increase in the local concentration of amino acids and acetate.
        J. Dairy Sci. 2008; 91 (18096943): 218-228
        • Raabo E.
        • Terkildsen T.C.
        On the enzymatic determination of blood glucose.
        Scand. J. Clin. Lab. Invest. 1960; 12 (13738785): 402-407
        • Rico D.E.
        • Ying Y.
        • Harvatine K.J.
        Effect of a high-palmitic acid fat supplement on milk production and apparent total-tract digestibility in high- and low-milk yield dairy cows.
        J. Dairy Sci. 2014; 97 (24731645): 3739-3751
        • Rottman L.W.
        • Ying Y.
        • Zhou K.
        • Bartell P.A.
        • Harvatine K.J.
        The effects of feeding rations that differ in neutral detergent fiber and starch concentration within a day on production, feeding behavior, total-tract digestibility, and plasma metabolites and hormones in dairy cows.
        J. Dairy Sci. 2015; 98 (25935247): 4673-4684
        • Soliva C.R.
        • Amelchanka S.L.
        • Kreuzer M.
        The requirements for rumen-degradable protein per unit of fermentable organic matter differ between fibrous feed sources.
        Front. Microbiol. 2015; 6 (26236297): 715
        • Spek J.W.
        • Dijkstra J.
        • Van Duinkerken G.
        • Bannink A.
        A review of factors influencing milk urea concentration and its relationship with urinary urea excretion in lactating dairy cattle.
        J. Agric. Sci. 2013; 151: 407-423
        • Sukhija P.S.
        • Palmquist D.L.
        Rapid method for determination of total fatty acid content and composition of feedstuffs and feces.
        J. Agric. Food Chem. 1988; 36: 1202-1206
        • Sutton J.D.
        • Dhanoa M.S.
        • Morant S.V.
        • France J.
        • Napper D.J.
        • Schuller E.
        Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets.
        J. Dairy Sci. 2003; 86 (14672193): 3620-3633
        • Tolhurst G.
        • Heffron H.
        • Lam Y.S.
        • Parker H.E.
        • Habib A.M.
        • Diakogiannaki E.
        • Cameron J.
        • Grosse J.
        • Reimann F.
        • Gribble F.M.
        Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.
        Diabetes. 2012; 61 (22190648): 364-371
        • Urrutia N.
        • Bomberger R.
        • Matamoros C.
        • Harvatine K.J.
        Effect of dietary supplementation of sodium acetate and calcium butyrate on milk fat synthesis in lactating dairy cows.
        J. Dairy Sci. 2019; 102 (30981489): 5172-5181
        • Urrutia N.
        • Harvatine K.J.
        Effect of conjugated linoleic acid and acetate on milk fat synthesis and adipose lipogenesis in lactating dairy cows.
        J. Dairy Sci. 2017; 100 (28501397): 5792-5804
        • Urrutia N.L.
        • Harvatine K.J.
        Acetate dose-dependently stimulates milk fat synthesis in lactating dairy cows.
        J. Nutr. 2017; 147 (28331053): 763-769
        • Van Soest P.J.
        • Robertson J.B.
        • Lewis B.A.
        Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
        J. Dairy Sci. 1991; 74 (1660498): 3583-3597
        • Vlaeminck B.
        • Fievez V.
        • Cabrita A.R.J.
        • Fonseca A.J.M.
        • Dewhurst R.J.
        Factors affecting odd- and branched-chain fatty acids in milk: A review.
        Anim. Feed Sci. Technol. 2006; 131: 389-417
        • Wildman C.D.
        • West J.W.
        • Bernard J.K.
        Effects of dietary cation-anion difference and potassium to sodium ratio on lactating dairy cows in hot weather.
        J. Dairy Sci. 2007; 90 (17235174): 970-977

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