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Potassium carbonate as a cation source for early-lactation dairy cows fed high-concentrate diets

Open ArchivePublished:December 30, 2016DOI:https://doi.org/10.3168/jds.2016-11776

      ABSTRACT

      Previous studies reported that addition of K2CO3 to high-concentrate diets improved milk fat synthesis, although the mechanism is yet to be established. The objective of the current experiment was to investigate the effects of dietary cation-anion difference (DCAD), cation source, and buffering ability of the mineral supplement on rumen biohydrogenation of fatty acids and production performance in dairy cows fed a high-concentrate diet. Thirty-five early-lactation Holstein cows (25 multiparous ruminally fistulated and 10 primiparous nonfistulated) were used in a randomized complete block design (7 blocks) with 33-d periods, including a 5-d pre-treatment collection period used as a covariate. Diets were (1) control, a basal diet [47% nonfibrous carbohydrates, DCAD (Na + K – Cl – S) = 65 mEq/kg of dry matter (DM)] containing 40% forage (including 60% corn silage) and 60% concentrate, (2) K2CO3 (control + K2CO3, 1.8% of DM, DCAD = 326 mEq/kg of DM), (3) KHCO3 (control + KHCO3, 2.6% of DM, DCAD = 324 mEq/kg of DM), (4) KCl (control + KCl, 2.0% of DM, DCAD = 64 mEq/kg of DM), and (5) Na2CO3 (control + Na2CO3, 1.4% of DM, DCAD = 322 mEq/kg of DM). Pre-planned orthogonal contrasts were used to assess the effects of K2CO3 (control vs. K2CO3), buffering ability (K2CO3 vs. KHCO3), DCAD (K2CO3 vs. KCl), and cation type (K2CO3 vs. Na2CO3). Supplementing K2CO3 in a high-concentrate diet did not improve milk fat yield or 4% fat-corrected milk yield. Milk fat concentration was greater in cows fed K2CO3 compared with control (4.03 vs. 3.26%). Milk yield tended to decrease (34.5 vs. 38.8 kg/d) and lactose yield decreased in cows fed K2CO3 as compared with KCl (1.64 vs. 1.87 kg/d). Milk fat concentration of trans-10 18:1 was increased when cows were fed Na2CO3 as compared with K2CO3. A positive relationship was observed between concentrations of anteiso 15:0 and trans-10,cis-12 18:2 in milk fat from cows receiving K2CO3. Milk Na concentration was increased, whereas milk Cl was decreased with K2CO3 as compared with KHCO3 or KCl. A positive relationship was established between milk Cl concentration and milk yield (R2 = 0.34) across all dietary treatments. Cation-anion difference (Na + K – Cl – S) in ruminal fluid was increased with K2CO3 as compared with control or KCl. Blood pH tended to decrease in cows fed KCl compared with K2CO3. Our results suggest that mineral supplementation tends to affect milk and milk fat synthesis and that factors other than DCAD, potassium ion, or buffer ability may be implicated. The variations observed in mineral composition of milk suggest an allostatic process to maintain an ionic equilibrium in mammary epithelial cells in response to mineral composition of the diet.

      Key words

      INTRODUCTION

      Negative energy balance typically appears in early-lactation dairy cows as a consequence of a reduction of DMI, as well as an increase in energy demand for milk production (
      • Grummer R.R.
      Etiology of lipid-related metabolic disorders in periparturient dairy cows.
      ). To compensate for this energy deficit, cows are fed high-concentrate diets. However, highly fermentable carbohydrates introduced in diets can result in a decreased rumen pH, and consequently lead to subacute ruminal acidosis (
      • Nocek J.E.
      Bovine acidosis: Implications on laminitis.
      ;
      • Krause K.M.
      • Oetzel G.R.
      Understanding and preventing subacute ruminal acidosis in dairy herds: A review.
      ). Under these conditions, the pattern of fermentation is altered, increasing rumen appearance of biohydrogenation intermediates derived from dietary PUFA, such as trans-10,cis-12 CLA (
      • Baumgard L.H.
      • Corl B.A.
      • Dwyer D.A.
      • Saebø A.
      • Bauman D.E.
      Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis.
      ) and trans-9,cis-11 CLA (
      • Perfield II, J.W.
      • Lock A.L.
      • Griinari J.M.
      • Sæbø A.
      • Delmonte P.
      • Dwyer D.A.
      • Bauman D.E.
      Trans-9, cis-11 conjugated linoleic acid reduces milk fat synthesis in lactating dairy cows.
      ), which are recognized for their inhibitory effects on milk fat synthesis.
      • Davis C.L.
      • Brown R.E.
      Low-fat milk syndrome.
      characterized “low-fat milk syndrome” as a consequence of diets with a high ratio of readily digestible carbohydrates to fibrous constituents that can create unfavorable conditions within the rumen. Based on these observations, different types of minerals were proposed to help stabilize the rumen pH and thus reduce the incidence of milk fat depression (
      • Emery R.S.
      • Brown L.D.
      Effect of feeding sodium and potassium bicarbonate on milk fat, rumen pH, and volatile fatty acid production.
      ). More recently, it has been demonstrated that increasing the DCAD through mineral supplementation of diets containing 20 or 40% (DM basis) of concentrates increased the synthesis of milk and milk fat (
      • Apper-Bossard E.
      • Faverdin P.
      • Meschy F.
      • Peyraud J.L.
      Effects of dietary cation-anion difference on ruminal metabolism and blood acid-base regulation in dairy cows receiving 2 contrasting levels of concentrate in diets.
      ). The authors suggested that responses were related to the capacity to maintain blood pH via an increase in blood HCO3, as well as a localized rumen buffering effect. In vitro studies have shown that the addition of K2CO3 promotes the predominant pathway of fatty acid (FA) biohydrogenation, which is characterized by the formation of trans-11 18:1 and cis-9,trans-11 CLA as intermediates (
      • 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.
      ). Moreover, K2CO3 supplementation reduced outflows of isomers used as markers for altered rumen biohydrogenation pathways (trans-10 18:1), or linked directly with milk fat depression (e.g., trans-10,cis-12 CLA;
      • 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.
      ). Likewise, it has been reported that K2CO3 may counteract the negative effects of high-concentrate diets on milk fat synthesis (
      • West J.W.
      • Coppock C.E.
      • Nave D.H.
      • Schelling G.T.
      Effects of potassium buffers on feed intake in lactating dairy cows and on rumen fermentation in vivo and in vitro.
      ;
      • Harrison J.
      • White R.
      • Kincaid R.
      • Block E.
      • Jenkins T.
      • St-Pierre N.
      Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.
      ). Our hypothesis is that the effects observed on milk fat synthesis when K2CO3 is fed to dairy cows could originate from changes in the rumen environment and the acid-base status of the animal, given that supplementation of dairy cow diets with K2CO3 increases K ion concentration, DCAD, and buffer ability. However, the mechanism by which K2CO3 supplementation might decrease the effect of high-concentrate diets on milk fat depression is yet to be established.
      The objective of the current study was to investigate the effects of K2CO3, buffering ability of the mineral supplement, DCAD, and cation source on milk production and composition in early-lactation dairy cows fed a high-concentrate diet.

      MATERIALS AND METHODS

      Cows, Experimental Design, and Treatments

      The experiment was carried out at the Centre de Recherche en Sciences Animales de Deschambault (Deschambault, Quebec, Canada) and all the procedures with cows were approved by the animal care committee of Université Laval in accordance with the guidelines of the
      Canadian Council on Animal Care
      .
      Thirty-five lactating Holstein cows (25 multiparous ruminally fistulated and 10 primiparous nonfistulated) averaging 37 ± 13 DIM (mean ± SD), 618 ± 59 kg of BW, and producing 39.6 ± 8.0 kg/d of milk were used in a randomized complete block design. Cows were blocked by expecting calving date, parity (primiparous and multiparous), and cannulation. Blocks were completed successively from January to July 2013 and consisted of groups of 5 cows calving over an interval of 25 ± 18 d. Within each block, cows were randomly assigned to the experimental diets. The experiment started with a 5-d pretreatment collection period, used as a covariate. During this time, cows were fed a diet with a forage-to-concentrate ratio of 53:47 (Table 1). The treatment period lasted 28 d, of which 23 d were for adaptation and the last 5 d were used for data and sample collections. All cows were housed in individual tie stalls and had free access to water at all time.
      Table 1Ingredients and chemical composition of the experimental TMR
      Item, % of DM (unless otherwise stated)PretreatmentDietary treatment
      ControlK2CO3KHCO3KClNa2CO3
      Ingredient
       Grass silage32.514.814.514.414.414.5
       Corn silage20.324.524.324.124.224.2
       Ground corn30.424.923.823.623.723.9
       Ground barley24.824.624.424.524.7
       Soybean meal8.6
       Corn gluten meal5.19.49.39.29.39.3
       Mineral-vitamin mix
      Optima 20–4, La Coop, St-Romuald, QC, Canada.
      3.11.11.21.11.11.2
       NaCl0.20.30.30.30.3
       Limestone0.40.40.40.40.4
       K2CO31.8
       KHCO32.6
       KCl2.0
       Na2CO31.4
      Chemical composition
       DM, % as fed40.9541.0740.3541.4741.5639.89
       OM95.3795.5495.8395.8195.6595.84
       CP16.3015.1514.8414.5514.6914.79
       Amylase-treated NDF27.0824.0024.4123.8423.3325.32
       ADF18.9314.5114.8314.0413.9814.81
       Starch27.2334.6732.2732.8730.9429.96
      NEL,
      Calculated according to NRC (2001).
      Mcal/kg of DM
      1.651.641.631.631.631.63
      Minerals
       Na0.230.280.230.240.240.79
       K1.370.971.701.831.800.91
       Cl0.330.440.420.431.110.41
       S0.240.220.210.210.220.21
       Ca0.740.610.580.610.660.57
       Mg0.330.210.170.180.170.17
       P0.400.390.380.390.390.38
      DCAD,
      DCAD = [Na + K] − [Cl + S].
      mEq/kg of DM
       Formulated2406532632464322
       Actual209107285320120326
      Fatty acids, mg/g of DM
       12:00.050.030.030.030.030.04
       14:00.080.080.090.080.080.08
       15:00.020.020.020.020.020.02
       16:04.174.444.324.234.344.30
       16:10.060.070.070.070.070.08
       17:00.040.040.040.040.040.04
       18:00.801.301.281.121.251.20
      cis-9 18:16.607.006.836.506.976.67
      cis-11 18:10.250.340.330.290.350.31
       18:215.5614.4913.5013.4414.4813.44
       18:33.611.871.751.661.941.73
       Total31.2429.7128.2527.4929.5827.93
      1 Optima 20–4, La Coop, St-Romuald, QC, Canada.
      2 Calculated according to
      NRC
      .
      3 DCAD = [Na + K] − [Cl + S].
      Treatment diets consisted of (1) a control basal diet formulated to contain, on a DM basis, 40% forage (including 60% corn silage) and 60% concentrate (total diet containing 47% NFC and 24% amylase-treated NDF, with a DCAD of 65 mEq/kg of DM; control); (2) the control diet + 1.8% K2CO3 with a DCAD of 326 mEq/kg of DM (K2CO3); (3) control diet + 2.6% KHCO3 with a DCAD of 324 mEq/kg of DM (KHCO3); (4) the control diet + 2.0% KCl with a DCAD of 64 mEq/kg of DM (KCl); and (5) the control diet + 1.4% Na2CO3 with a DCAD of 322 mEq/kg of DM (Na2CO3). Based on initial feed ingredient composition, diets were formulated to meet or exceed the
      NRC
      requirements (Table 1). Diets were fed as TMR at 1000 h daily and the amounts of feed offered were adjusted at 110% of expected intake according to the previous day consumption. The forages were sampled every week and dried for 3 d in a forced-air oven at 55°C to determine DM concentration and adjust the as-fed forage proportions in the diets.

      Experimental Measurements and Sampling

      In each collection period, BW was registered at 0930 h for the last 3 d. Samples of TMR and orts were collected for the last 5 consecutive days during the pre-treatment and the experimental periods and stored at −20°C. Prior to analysis, samples of TMR and orts were thawed at room temperature and dried in a forced–air oven for 72 h at 55°C to determine DM concentration. Dried samples were ground to 2 mm using a Wiley mill (model 4, Arthur M. Thomas Co., Philadelphia, PA), pooled by cow and period, ground again to 1 mm using a Cyclotec Sample Mill (model 1093, Tecator Inc., Höganäs, Sweden), and kept frozen at −20°C until further analyses.
      All TMR and orts were analyzed as described by
      • Fauteux M.-C.
      • Gervais R.
      • Rico D.E.
      • Lebeuf Y.
      • Chouinard P.Y.
      Production, composition, and oxidative stability of milk highly enriched in polyunsaturated fatty acids from dairy cows fed alfalfa protein concentrate or supplemental vitamin E.
      , including ash (method 942.05;
      AOAC International
      ) and starch (
      • Hall M.B.
      Determination of starch, including maltooligosaccharides, in animal feeds: Comparison of methods and a method recommended for AOAC collaborative study.
      ).
      Additionally, subsamples of TMR and orts were subjected to a digestion process in HNO3 (70%) + H2O2 (30%), according to a procedure adapted from
      • Mills H.A.
      • Jones Jr, J.B.
      using a digestion block (DigiPREP MS, SCP Science, Baie d'Urfé, QC, Canada) for CP and mineral (except Cl) determinations. Subsequently, CP (N × 6.25) was assessed using an autoanalyzer (QuikChem 8000 Lachat Zellweger Analytics Inc., Lachat Instruments, Milwaukee, WI; method 13–107–06–2–D;
      Lachat Instruments
      Methods list for automated ion analyzers (flow injection analyses - ion chromatography).
      ). Concentrations of P, K, Ca, Mg, Na, and S were determined by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 4300DV, Perkin Elmer, Shelton, CT). Finally, Cl in TMR and orts was extracted using a method adapted from
      • Liu L.
      Determination of chloride in plant tissue.
      . Briefly, 0.25 g of dried and ground samples were mixed with 20 mL of H2SO4 (0.007 M) for 60 min and then centrifuged at 32,570 × g for 30 min at room temperature. The concentration of Cl in the last supernatant was determined using an ion chromatograph (model ICS-2100, Dionex Corp., Sunnyvale, CA) equipped with an AS11 HC capillary column (Dionex Corp.). Determination of dietary FA composition was carried out by gas chromatography according to the method described by
      • Fauteux M.-C.
      • Gervais R.
      • Rico D.E.
      • Lebeuf Y.
      • Chouinard P.Y.
      Production, composition, and oxidative stability of milk highly enriched in polyunsaturated fatty acids from dairy cows fed alfalfa protein concentrate or supplemental vitamin E.
      .
      Cows were milked twice daily at 0700 and 1700 h, and milk yield was determined using calibrated milk meters (Flowmaster Pro, DeLaval, Tumba, Sweden) for the last 3 d in each collection period. Milk samples were composited daily from evening and morning milking proportionately to respective milk yields. A first composited subsample was stored at 4°C with bronopol (2-bromo-2-nitropropane-1,3-diol) until further determination of milk fat, protein, lactose, and MUN by the method 972.160 (
      AOAC International
      ) at Valacta (Ste-Anne-de-Bellevue, QC, Canada), using a Foss MilkoScan FT 6000 (Foss, Hillerød, Denmark). Somatic cell count was also determined at Valacta using a Fossomatic FC (Foss). A second composite milk subsample was pooled by cow and period without preservative for subsequent determination of mineral and FA composition.
      Lipid extraction of milk samples and methylation of FA were performed according to procedures described by
      • Chouinard P.Y.
      • Lévesque J.
      • Girard V.
      • Brisson G.J.
      Dietary soybeans extruded at different temperatures: Milk composition and in situ fatty acid reactions.
      . Milk FA profile was determined according to
      • Boivin M.
      • Gervais R.
      • Chouinard P.Y.
      Effect of grain and forage fractions of corn silage on milk production and composition in dairy cows.
      , using a gas chromatograph (Agilent 7890A, Agilent Technologies, Santa Clara, CA) equipped with a 100-m CP-Sil-88 capillary column (0.25 mm i.d., 0.20 mm film thickness; Agilent Technologies Canada Inc., Mississauga, ON, Canada) and a flame ionization detector.
      Milk concentrations of P, K, Ca, Mg, Na, and S were determined according to methods described above for feed and ort samples. For Cl concentration, a 1-mL sample was ashed in a muffle furnace at 550°C for 5 h and then solubilized in 1 mL of 0.5 N HNO3 and diluted with distilled water to a final volume of 50 mL. Milk concentration of Cl was determined as described by
      • Gaucheron F.
      • Le Graët Y.
      • Piot M.
      • Boyaval E.
      Determination of anions of milk by ion chromatography.
      , using an ion chromatograph (model ICS-2100, Dionex Corp., Sunnyvale, CA) equipped with an AS11 HC capillary column (Dionex Corp.).
      Rumen fluid was collected from the ventral sac of rumen fistulated cows on 2 consecutive days of each collection period at 0, 1, 2, 4, 6, and 8 h relative to feeding time. Rumen fluid was collected and filtered using a sampler tube (Bar-Diamond Inc., Parma, ID). Following an immediate pH measurement (pHTestr 30, Oakton Instruments, Vernon Hills, IL), 10 mL of rumen fluid was stabilized with 0.2 mL of H2SO4 (50%, vol/vol) and stored at −20°C until determination of VFA and NH3-N concentrations. A second sample (15 mL) of rumen fluid was collected and stored at −20°C before analyses of mineral composition. Ruminal cation-anion difference (RCAD) was calculated as follows:
      RCAD(mEq/L)=[Na+K][Cl+S].


      At the time of analysis, acidified samples of rumen fluid were thawed and centrifuged at 16,060 × g for 15 min at 4°C. The supernatant was analyzed for NH3-N with the indophenol-blue method (
      • Novozamsky I.
      • van Eck R.
      • van Schouwenburg J.C.
      • Walinga I.
      Total nitrogen determination in plant material by means of the indophenol-blue method.
      ) using a spectrophotometer (Spectronic 1201, Milton Roy Company, Miami, FL) at 630 nm. The profile of ruminal VFA was determined with a gas chromatograph (Clarus 680, Perkin Elmer, Waltham, MA) equipped with a polar capillary column (HP-Innowax 30-m length, 0.320 mm i.d., 0.25 μm film thickness; Agilent Technologies Canada Inc.) and a flame ionization detector. The split ratio was 25:1. At the time of the sample injection, the column temperature was 80°C, maintained for 0.5 min followed by a first increase to 180°C at 10°C/min, and a second increase to 220°C at 30°C/min. The temperature was then maintained at 220°C for 2 min. The second sample of rumen fluid was thawed and centrifuged at 13,000 × g for 30 min at 4°C, the supernatants were then composited by day and cow, and concentrations of P, K, Ca, Mg, Na, and S were determined according to the same procedure used for milk and dietary samples. Prior to the determination of Cl concentration in rumen fluid, samples were centrifuged a second time at 32,570 × g for 30 min at 4°C and diluted in water with a 1:3 rumen fluid-to-water ratio (
      • Adriano D.C.
      • Doner H.E.
      Bromine, chlorine, and fluorine.
      ;
      • Johnson G.V.
      • Fixen P.E.
      Testing soils for sulfur, boron, molybdenum, and chlorine.
      ). Determination of Cl was performed using ion chromatography, as described above.
      On the last 2 d of each collection period, blood samples were taken at 0930 h (preprandial) and 1400 h (postprandial) from a jugular vein into evacuated tubes without preservative (Vacutainer 366430, Becton Dickinson, Franklin Lakes, NJ) and immediately transferred to 1.7-mL syringes (Radiometer, Copenhagen, Denmark) containing 80 IU of electrolyte-balanced heparin. Blood hematocrits, concentrations of electrolytes (Na+, K+, Ca2+, and Cl) and HCO3, and partial pressure of CO2 and O2 were immediately determined by potentiometry using a blood gas and mineral analyzer (ABL 77, Radiometer).

      Statistical Analysis

      Data for milk production and composition, DMI, BW, and minerals in milk and rumen fluid were analyzed as a randomized block design using the GLIMMIX procedure of SAS (version 9.3; SAS Institute Inc., Cary, NC). The model used was
      Yij=μ+Ti+Bj+C+ɛij


      where Yij is the variable observed, μ is the overall mean, Ti denotes the fixed effect of treatment (i = 1 to 5), Bj is the random effect of block (j = 1 to 7), C is the covariable adjustment for each cow (C = 1 to 35), and εij denotes the residual error. Blood and rumen fermentation parameters were analyzed using the REPEATED statement in the MIXED procedure of SAS, using the following model:
      Yijkl=μ+Ti+Hj+(T×H)ij+A(k)l+Bl+C+εijkl


      where Yijkl is the variable observed, μ is the overall mean, Ti denotes the fixed effect of treatment (i = 1 to 5), Hj refers to fixed effect of sampling time (j = 0, and 4 h postprandial for blood parameters and j = 0, 1, 2, 4, 6, and 8 h postprandial for rumen parameters), (T × H)ij is the fixed effect of the interaction, A(k)l is the random effect of cow k (k = 1 to 5) within block (l = 1 to 7), Bl is the random effect of block, C is the covariate adjustment for blood and rumen parameters of each cow (C = 1 to 35), and εijkl denotes the residual error. Cow within block was included in the model as the subject of the repeated statement. For blood parameters, covariance structures were selected between compound symmetry and first-order autoregressive based on the Akaike's information criterion. For rumen parameters, the spatial covariance structure was used to estimate covariances. Because no treatment × sampling time interaction was observed for blood parameters, as well as for rumen pH and VFA, values were combined by day and analyzed according to the model previously mentioned. Pre-planned orthogonal contrasts were used to assess the effects of K2CO3 (control vs. K2CO3), buffer ability (K2CO3 vs. KHCO3), DCAD (K2CO3 vs. KCl), and cation source (K2CO3 vs. Na2CO3). Simple linear regressions were conducted using the REG procedure of SAS to determine relationships between milk yield and milk minerals, and the association between milk fat concentration of trans-10, cis-12 18:2, and anteiso 15:0. Differences were declared significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10.

      RESULTS AND DISCUSSION

      Diets, DMI, and BW

      The actual DCAD were 107, 285, 320, 120, and 326 mEq/kg of DM for control, K2CO3, KHCO3, KCl, and NaCO3 experimental TMR, respectively (Table 1). Therefore, mineral supplementation allowed a modulation of DCAD, as formulated among treatments. Also, isokalemic conditions were maintained between K2CO3, KHCO3, and KCl treatments, as shown by the minimal variations observed in dietary K concentrations of these 3 treatments.
      Supplementing cows with K2CO3 tended to decrease BW compared with the control diet (Table 2). However, no significant effect of mineral supplementation was observed on DMI (Table 2). Conversely, using a mineral mixture to increase DCAD from 0 to 300 mEq/kg of DM of TMR,
      • Apper-Bossard E.
      • Faverdin P.
      • Meschy F.
      • Peyraud J.L.
      Effects of dietary cation-anion difference on ruminal metabolism and blood acid-base regulation in dairy cows receiving 2 contrasting levels of concentrate in diets.
      observed a linear increase in DMI up to 1.9 and 4.0 kg/d when cows received 20 and 40% of concentrate, respectively. Similarly, increasing DCAD (196 to 544 mEq/kg of DM) by increasing levels of K2CO3 in diets of dairy cows in established lactation (95 DIM) led to a concomitant increase in DMI up to 1.3 kg/d (
      • 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.
      ). A systematic review conducted by
      • Hu W.
      • Kung L.
      • Murphy M.R.
      Relationships between dry matter intake and acid–base status of lactating dairy cows as manipulated by dietary cation–anion difference.
      also reported a positive relationship between DMI and the acid-base status of lactating dairy cows. On the other hand, the same group of researchers reported no effect of 2 DCAD levels (220 vs. 470 mEq/kg of DM) on DMI of cows in early lactation (1 to 47 DIM;
      • Hu W.
      • Murphy M.R.
      • Constable P.D.
      • Block E.
      Dietary cation-anion difference effects on performance and acid-base status of dairy cows postpartum.
      ). These authors attributed the discrepancies between studies due to tremendous physiological changes occurring in early lactation, which attenuated the effect of dietary treatments. Another possible implication of DCAD on DMI could be related to ion availability, because their manipulation always affects the regulation of cell volume, and consequently the transport of substrates into and out of the cell as well as regulation of osmotic pressure (
      • McDowell L.R.
      Potassium.
      ;
      • Hoffmann E.K.
      • Lambert I.H.
      • Pedersen S.F.
      Physiology of cell volume regulation in vertebrates.
      ). Potassium contributes to 50% of the osmolality of intracellular fluid, whereas Na and Cl contribute to 80% of extracellular osmolality (
      • McDowell L.R.
      Potassium.
      ). Although the cell has the capacity to adjust its requirements through ionic homeostasis, long-term exposure to anisosmotic conditions exerts changes that affect signaling events which control cell growth, proliferation, and death (
      • Hoffmann E.K.
      • Lambert I.H.
      • Pedersen S.F.
      Physiology of cell volume regulation in vertebrates.
      ). In this regard, it is important to notice that transport of nutrients across the rumen epithelium may be impaired. Studies support the existence of a short-chain FA transport mechanism in the epithelial cells that involves ions (
      • Gäbel G.
      • Bestmann M.
      • Martens H.
      Influences of diet, short-chain fatty acids, lactate and chloride on bicarbonate movement across the reticulo-rumen wall of sheep.
      ;
      • Sehested J.
      • Andersen J.B.
      • Aaes O.
      • Kristensen N.B.
      • Diernæs L.
      • Møller P.D.
      • Skadhauge E.
      Feed-induced changes in the transport of butyrate, sodium and chloride ions across the isolated bovine rumen epithelium.
      ), and this transport mechanism can be the cause of a feed-induced regulation (
      • Sehested J.
      • Andersen J.B.
      • Aaes O.
      • Kristensen N.B.
      • Diernæs L.
      • Møller P.D.
      • Skadhauge E.
      Feed-induced changes in the transport of butyrate, sodium and chloride ions across the isolated bovine rumen epithelium.
      ). In the current experiment, despite a variation in systemic acid-base status reflected by a tendency for lower blood pH (Table 3), cows fed KCl had a numerically higher DMI (1.9 kg/d) compared with cows fed K2CO3 diet. Given that increasing DCAD do not necessary implicate keeping the ideal cell ionic balance of K, Na, and Cl, we speculate that KCl as compared with K2CO3 may provide possibly a better balance of ions in the rumen, allowing fewer feed-induced regulation effects.
      Table 2Dry matter intake, BW, and milk yield and composition of early-lactation cows receiving high-concentrate diets supplemented with different minerals
      ItemDietary treatmentSEMP-value, K2CO3 vs.
      ControlK2CO3KHCO3KClNa2CO3ControlKHCO3KClNa2CO3
      DMI, kg/d24.823.423.625.323.61.20.260.960.140.88
      BW, kg63561662062563110.00.070.710.380.14
      Milk yield, kg/d
       Actual37.734.534.738.838.11.80.300.840.060.11
       4% FCM
      4% FCM = [0.4 × milk yield (kg/d)] + [15 × fat yield (kg/d)].
      33.334.733.636.936.02.00.610.680.400.60
       ECM
      ECM = 0.327 × milk yield (kg/d) + 12.95 × fat yield (kg/d) + 7.2 × protein yield (kg/d).
      33.333.832.736.135.11.90.810.640.340.60
      Milk fat
       Concentration, %3.264.033.883.723.660.220.020.620.310.22
       Yield, kg/d1.221.391.311.431.380.10.210.550.800.95
      Milk protein
       Concentration, %3.053.122.993.032.930.090.470.190.320.04
       Yield, kg/d1.151.071.021.171.110.060.250.490.170.56
      Lactose
       Concentration, %4.844.754.844.804.830.040.090.110.310.12
       Yield, kg/d1.821.641.681.871.840.090.130.710.040.08
      MUN, mg/dL8.59.09.58.310.10.50.420.510.280.12
      SCC, 1,000/mL646835255025.00.900.310.180.56
      Milk minerals, mmol/L
       Na15.1016.3015.4214.9115.460.480.020.09<0.010.10
       K43.4245.0545.1144.8543.261.280.370.970.910.34
       Cl17.9218.8317.2018.4217.261.250.590.340.810.36
       S9.9410.4310.2910.209.610.320.190.700.530.03
       Ca32.8034.3233.4831.8330.471.130.270.530.07<0.01
       Mg4.955.255.215.165.010.180.170.850.670.27
       P36.2937.4038.5137.5535.041.190.410.420.920.09
      1 4% FCM = [0.4 × milk yield (kg/d)] + [15 × fat yield (kg/d)].
      2 ECM = 0.327 × milk yield (kg/d) + 12.95 × fat yield (kg/d) + 7.2 × protein yield (kg/d).
      Table 3Blood parameters of early-lactation cows receiving high-concentrate diets supplemented with different minerals
      Parameter
      Anion gap = [K+ + Na+] – [Cl− + HCO3−]; HCO3−= bicarbonate; pCO2= partial pressure of CO2; pO2 = partial pressure of O2.
      Dietary treatmentSEMP-value, K2CO3 vs.
      ControlK2CO3KHCO3KClNa2CO3ControlKHCO3KClNa2CO3
      pH7.437.427.427.407.430.010.740.920.100.84
      Electrolytes, mmol/L
       Na+139.7140.1140.0140.4139.60.40.460.850.580.36
       K+3.694.013.793.923.680.07<0.010.010.27<0.01
       Cl105.9106.1106.5108.0105.30.80.840.610.040.41
       Ca2+1.221.241.201.231.200.020.410.030.810.19
      Anion gap, mmol/L10.510.510.510.29.90.60.990.980.690.45
      HCO3, mmol/L27.127.626.826.128.00.80.670.480.220.73
      Hematocrit, %27.527.427.626.627.30.70.920.840.390.90
      Blood gas, mm Hg
       pCO241.943.141.542.543.40.90.380.240.660.82
       pO236.937.938.936.938.61.80.600.530.560.66
      1 Anion gap = [K+ + Na+] – [Cl + HCO3]; HCO3= bicarbonate; pCO2= partial pressure of CO2; pO2 = partial pressure of O2.
      • 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.
      evaluated the effect of cation sources [Na3H(CO3)2 vs. K2CO3] on performance of dairy cows fed TMR with similar DCAD (approximately 380 mEq/kg of DM) and observed no difference on DMI. These results are in agreement with our findings where cation sources modulated through the supply of Na2CO3 or K2CO3 did not affect DMI.
      Investigating dietary buffers effects on high-producing dairy cows,
      • Erdman R.A.
      Dietary buffering requirements of the lactating dairy cow: A review.
      compiled literature data and showed that, as opposed to KHCO3, which did not affect DMI, adding K2CO3 did increase DMI compared with diets without K supplementation. The author suggested that the observed effects of mineral supplementation on DMI could be partly explained by a higher rumen pH, associated with a more favorable rumen environment, allowing greater DM digestibility.
      • West J.W.
      • Coppock C.E.
      • Nave D.H.
      • Schelling G.T.
      Effects of potassium buffers on feed intake in lactating dairy cows and on rumen fermentation in vivo and in vitro.
      also observed a greater DMI for cows supplemented with K2CO3 compared with KHCO3 or NaHCO3. In contrast, in the current trial, DMI was similar for cows fed K2CO3 and KHCO3 diets (23.5 ± 1.1 kg/d). This level of intake allowed a daily consumption of 3.05 mol of CO32− ions and 6.11 mol of HCO3 ions for K2CO3 and KHCO3 treatments, respectively.
      • West J.W.
      • Coppock C.E.
      • Nave D.H.
      • Schelling G.T.
      Effects of potassium buffers on feed intake in lactating dairy cows and on rumen fermentation in vivo and in vitro.
      reported that, in the rumen, CO32− ions contained in K2CO3 has twice the capacity to absorb hydrogen ions compared with HCO3 ions found in KHCO3. Therefore, the isokalemic conditions maintained between K2CO3 and KHCO3 treatments led to an equivalent buffer ability of these 2 diets and could explain the lack of difference in DMI between these 2 treatments.
      A trend to decreased BW was observed in cows fed K2CO3 as compared with control. This tendency could possibly be explained in part by a numerically lower DMI (−1.4 kg/d) for K2CO3 as compared with the control treatment.

      Rumen Fermentation Attributes and Minerals

      As expected, concentration of K in the rumen was similar for cows fed K2CO3 and KHCO3 or KCl (Table 4). However, despite that K2CO3, KHCO3, and KCl treatments were isonatremic (Na = 0.23%, DM basis), concentration of Na in the rumen increased with K2CO3 as compared with KHCO3 (+13%), and KCl (+17%). As expected, Cl concentration in the rumen was decreased when cows were fed K2CO3 compared with KCl (−41%). But surprisingly, despite similar dietary Cl concentrations, KHCO3 increased Cl concentration by 19% as compared with K2CO3. These results provide further evidence that mineral interactions exist in the rumen, probably as a consequence of transport mechanisms of cations and anions across forestomach epithelia (
      • Leonhard-Marek S.
      • Stumpff F.
      • Martens H.
      Transport of cations and anions across forestomach epithelia: Conclusions from in vitro studies.
      ). Two active pathways contribute to Na absorption: an electroneutral Na+/H+ exchange and an electrogenic Na+ conductance (
      • Martens H.
      • Gabel G.
      Transport of Na and Cl across the epithelium of ruminant forestomachs: Rumen and omasum. A review.
      ). By incubating ruminal epithelia of sheep,
      • Leonhard-Marek S.
      • Becker G.
      • Breves G.
      • Schröder B.
      Chloride, gluconate, sulfate, and short-chain fatty acids affect calcium flux rates across the sheep forestomach epithelium.
      showed that the presence of Cl on the luminal side, which corresponds to the rumen cavity, increased electrogenic Na absorption. This increase was mediated via a greater Cl/HCO3 or Cl/OH luminal exchange, a higher pH in the microclimate of the epithelia surface, and a pH effect on the nonselective cation conductance responsible for Na absorption (
      • Leonhard-Marek S.
      • Becker G.
      • Breves G.
      • Schröder B.
      Chloride, gluconate, sulfate, and short-chain fatty acids affect calcium flux rates across the sheep forestomach epithelium.
      ). According to this information, we speculated that lower Cl concentration observed in the rumen of cows fed K2CO3 might have decreased Na transport efficiency, which would explain the greater rumen Na concentrations observed with K2CO3 compared with KCl and KHCO3 treatments.
      Table 4Rumen pH, volatile fatty acid (VFA), and mineral concentrations of rumen fluid from early-lactation cows receiving high-concentrate diets supplemented with different minerals
      ItemDietary treatmentSEMP-value, K2CO3 vs.
      ControlK2CO3KHCO3KClNa2CO3ControlKHCO3KClNa2CO3
      pH6.256.396.366.426.390.080.170.730.820.98
      VFA, mol/100 mol
       Acetate56.5758.0555.3655.4358.281.650.490.210.220.91
       Propionate24.6823.2523.2825.8522.361.950.610.990.350.75
       Butyrate12.4912.6015.2112.0012.401.150.940.080.700.89
       Isobutyrate1.061.051.061.100.970.140.950.910.700.60
       Valerate1.891.721.761.901.880.340.660.920.650.67
       Isovalerate2.732.222.542.462.780.470.370.570.680.31
       Caproate0.910.891.040.861.330.380.950.740.950.32
       Acetate:propionate2.402.572.462.252.740.240.630.740.350.62
      Mineral, mM
       Na180.76191.20169.49162.87197.267.960.340.050.010.58
       K37.9963.0161.8162.9034.444.88<0.010.840.98<0.01
       Cl11.8811.6014.3219.8011.561.050.800.02<0.010.97
       S1.541.601.491.371.290.150.730.510.170.08
       Ca1.921.651.661.821.310.170.270.970.490.15
       Mg4.864.153.923.812.870.510.250.710.590.04
       P23.0723.9821.9422.6221.201.950.700.380.550.26
      RCAD,
      RCAD (ruminal cation-anion difference) = ([Na + K] − [Cl + S]).
      mEq/L
      20824321720421711.640.030.090.020.09
      1 RCAD (ruminal cation-anion difference) = ([Na + K] − [Cl + S]).
      Magnesium concentration in the rumen was significantly greater when cows received K2CO3 compared with Na2CO3 diet (Table 4). Absorption of Mg in the rumen epithelium and subsequent transport to blood (basolateral extrusion) occurs against an electrochemical gradient (
      • Schweigel M.
      • Vormann J.
      • Martens H.
      Mechanisms of Mg2+ transport in cultured ruminal epithelial cells.
      ). An elevated K concentration in the rumen is associated with a decrease in Mg absorption, whereas higher absorption and transport efficiency for Mg is reported when Na concentration in the rumen is increased (
      • Schweigel M.
      • Kolisek M.
      • Nikolic Z.
      • Kuzinski J.
      Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.
      ). No difference was observed between Mg concentration in the rumen of cows fed control and K2CO3 treatments. Therefore, the difference in ruminal Mg concentration observed between cows fed K2CO3 and Na2CO3 diets could possibly be a consequence of treatment differences in dietary Na rather than K concentrations.
      No effect of treatment or treatment × time interaction, under any of the studied contrasts, was observed on rumen VFA concentrations and pH (Table 4). Similarly,
      • West J.W.
      • Coppock C.E.
      • Nave D.H.
      • Schelling G.T.
      Effects of potassium buffers on feed intake in lactating dairy cows and on rumen fermentation in vivo and in vitro.
      found no difference in rumen pH when cows were fed diets containing no buffer, 1.8% KHCO3, 1.2% K2CO3, or 1.5% NaHCO3 (DM basis). Conversely,
      • Fraley S.E.
      • Hall M.B.
      • Nennich T.D.
      Effect of variable water intake as mediated by dietary potassium carbonate supplementation on rumen dynamics in lactating dairy cows.
      demonstrated that feeding dairy cows with diets containing up to 3.2% of K2CO3 increased rumen pH linearly. In the current experiment, rumen pH was depressed for all treatments from 0 until 8 h (6.59 to 6.17) postfeeding (Figure 1).
      Figure thumbnail gr1
      Figure 1Postfeeding temporal pattern of rumen pH (a), and NH3-N concentration (b) of early-lactation cows fed high-concentrate diets unsupplemented (□, control) or supplemented with different minerals (○, K2CO3; Δ, KHCO3; ◊, KCl; x, Na2CO3).

      Systemic Acid-Base Status

      Compared with K2CO3, blood K concentration was lower for cows receiving control and Na2CO3 diets and similar for cows fed KCl treatment (Table 3). However, despite the isokalemic nature of the diets, cows fed KHCO3 had lower blood K concentration compared with cows fed K2CO3 diet. However, to our knowledge, no study has specifically assessed the K bioavailability from these 2 mineral supplements in lactating dairy cows.
      Relative to KCl, adding K2CO3 in the diet tended to increase blood pH, but decreased blood Cl concentration (Table 3). It is well known that DCAD, has a direct effect on the blood acid-base status, and the metabolic condition of the cows (
      • Block E.
      Manipulation of dietary cation-anion difference on nutritionally related production diseases, productivity, and metabolic responses of dairy cows.
      ). Indeed, blood HCO3 concentration and pH have been shown to increase with increasing DCAD (
      • Roche J.R.
      • Petch S.
      • Kay J.K.
      Manipulating the dietary cation-anion difference via drenching to early-lactation dairy cows grazing pasture.
      ;
      • Apper-Bossard E.
      • Peyraud J.L.
      • Faverdin P.
      • Meschy F.
      Changing dietary cation-anion difference for dairy cows fed with two contrasting levels of concentrate in diets.
      ), whereas decreased DCAD though anionic diets seems to reduce blood pH (
      • Charbonneau E.
      • Chouinard P.Y.
      • Tremblay G.F.
      • Allard G.
      • Pellerin D.
      Timothy silage with low dietary cation-anion difference fed to nonlactating cows.
      ). Therefore, in agreement with
      • Charbonneau E.
      • Chouinard P.Y.
      • Tremblay G.F.
      • Allard G.
      • Pellerin D.
      Hay to reduce dietary cation-anion difference for dry dairy cows.
      , increased dietary concentration of Cl may explain the tendency for a lower blood pH found in cows fed KCl, compared with K2CO3 diet. In contrast, blood anion gap (K+ + Na+ – Cl – HCO3), partial pressure of CO2 and O2, concentrations of HCO3, and hematocrits were similar among treatments.

      Milk Production and Composition

      Milk yield tended to decrease and lactose yield decreased when cows were fed K2CO3 compared with the KCl diet (Table 2). Because of a high metabolic rate, the cellular environment of cows in lactation tends to be acidotic (
      • Block E.
      Manipulation of dietary cation-anion difference on nutritionally related production diseases, productivity, and metabolic responses of dairy cows.
      ).
      • Shire J.
      • Beede D.
      Influence of DCAD on lactational performance: A review of some practical considerations.
      suggested that supplementing cationic salts might improve lactation performance by affecting several biological mechanisms such as ruminal buffer ability, blood pH, rumen microbial synthesis, bioactive intermediates of ruminal FA biohydrogenation, and reactions to environmental stressors. Also, it was reported that, when compared with cows in any other stage of lactation and because they typically receive high-concentrate diets with rapidly degradable starch, early-lactation dairy cows may be more prone to metabolic acidosis (
      • Chan P.S.
      • West J.W.
      • Bernard J.K.
      • Fernandez J.M.
      Effects of dietary cation-anion difference on intake, milk yield, and blood components of the early lactation cow.
      ). However, the tendency for a lower milk yield observed in the current study, when early-lactation dairy cows were fed K2CO3 compared with the KCl diet, suggests that the effects of mineral supplementation on milk synthesis may involve other factors than DCAD, K ion, and buffer ability. Although numerically cows fed KCl as compared with K2CO3 ate more (1.9 kg/d), this could be partially explain the trend to increase milk yield using KCl instead of K2CO3.
      The cellular mechanisms involved in the transport of milk constituents out of and across the mammary secretory cell is composed of 4 transcellular and one paracellular known routes (
      • Shennan D.B.
      • Peaker M.
      Transport of milk constituents by the mammary gland.
      ). These mechanisms require that a constant concentration of gradients in body fluids be maintained to support transport of substrates or metabolites in and out the cells, as well as regulation of osmotic pressure (
      • McDowell L.R.
      Potassium.
      ). Mineral concentrations in milk are recognized to reflect their cellular levels (
      • Holt C.
      The milk salts: their secretion, concentrations and physical chemistry.
      ). Approximately 95% of milk K and Na, and 100% of milk Cl are found in the aqueous phase, and contribute substantially to milk osmolality (
      • Holt C.
      The milk salts: their secretion, concentrations and physical chemistry.
      ). However, the mammary gland is able to generate and maintain large K, Na, and Cl gradients between milk and plasma (
      • Shennan D.B.
      • Peaker M.
      Transport of milk constituents by the mammary gland.
      ), suggesting that active ion movements are involved in the secretory mechanisms of milk constituents (
      • Linzell J.L.
      • Peaker M.
      Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism.
      ).
      In the current study, Cl concentration was similar in milk from cows fed K2CO3 and KCl diets, despite a decreased blood Cl concentration in cows fed K2CO3 diet. This result suggests that Cl concentration in the milieu intérieur (
      • Holmes F.L.
      Claude Bernard, the “milieu intérieur”, and regulatory physiology.
      ) of mammary epithelial cells was maintained constant. The lack of difference in milk Cl concentration between K2CO3 and KCl treatments (Table 2) is in agreement with Bernard's constancy (
      • Holmes F.L.
      Claude Bernard, the “milieu intérieur”, and regulatory physiology.
      ), a mechanism that allows to protect cells from external (blood) conditions to maintain homeostasis of, in this case, the mammary gland.
      • Shennan D.B.
      • Peaker M.
      Transport of milk constituents by the mammary gland.
      suggested that, similar to many secretory epithelia, intracellular accumulation of Cl, via Na+-K+-Cl cotransport across the basolateral membrane, is the driving force for the secretion of ions and water across the apical membrane of the mammary epithelial cell. More specifically, in mice, it has been previously observed that Cl transport in HC11 mammary epithelial cells was achieved by the coordinated action of symporters such as Na+-K+-2Cl cotransporter isoform 1, cystic fibrosis transmembrane conductance regulator, or Cl channels 1 and 2 (
      • Selvaraj N.G.
      • Omi E.
      • Gibori G.
      • Rao M.C.
      Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl cotransporter.
      ;
      • Anantamongkol U.
      • Ao M.
      • Sarathy (nee Venkatasubramanian) J.
      • Devi Y.S.
      • Krishnamra N.
      • Rao M.C.
      Prolactin and dexamethasone regulate second messenger-stimulated Cl secretion in mammary epithelia.
      ). Accordingly, using a simple linear regression to assess the association between milk yield and milk concentrations of minerals and lactose, we observed a positive relationship between Cl concentration and milk yield (Figure 2). In contrast, milk yield was not significantly associated with milk concentrations of Na or K. Given that lactose is exclusively synthesized in mammary epithelial cells (
      • Kuhn N.J.
      • Linzell J.L.
      Measurement of the quantity of lactose passing into mammary venous plasma and lymph in goats and in a cow.
      ), lactose synthesis could then be considered to depend mostly on the availability of precursors, the latter being in turn dependent on their transport into the cell and their metabolic fate. A positive relation was also observed between lactose yield and milk Cl concentration (R2 = 0.29; Figure 2). Previous studies suggested that ions are involved indirectly (
      • Shennan D.B.
      • Peaker M.
      Transport of milk constituents by the mammary gland.
      ) or directly (
      • McManaman J.L.
      • Neville M.C.
      Mammary physiology and milk secretion.
      ) in transport of nutrients across cell membranes, factors that may influence the synthesis of milk. Consequently, we hypothesized that the tendency for increased milk yield and for increased lactose yield when cows were supplemented with KCl as compared with K2CO3 could partly be explained by a potential role of Cl in transport of nutrients and metabolites into and out of mammary epithelial cells.
      Figure thumbnail gr2
      Figure 2Association between milk yield and milk concentration of (a) sodium, (b) potassium, (c) chlorine, (d) calcium, and (e) sulfur, and between lactose yield and milk concentration of chlorine (f), in early-lactation cows fed high-concentrate diets unsupplemented (□, control) or supplemented with different minerals (○, K2CO3; Δ, KHCO3; ◊, KCl; x, Na2CO3).
      Despite similar dietary concentrations (Table 1), Ca and S increased in milk when cows received K2CO3 as compared with Na2CO3 diet (Table 2). Both minerals could play an important role in the allostatic process required to maintain ionic equilibrium of the mammary epithelial cell; however, more research is needed to establish the exact mechanism.
      Milk fat concentration was increased by 24% in cows fed K2CO3 compared with the control diet (Table 2). Consistently,
      • Harrison J.
      • White R.
      • Kincaid R.
      • Block E.
      • Jenkins T.
      • St-Pierre N.
      Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.
      reported a 9% increase in milk fat concentration when cows were fed a diet supplemented with 3.2 vs. 0% K2CO3 (DM basis). Likewise, augmenting dietary K2CO3 from 0 up to 2.46% (DM basis) increased milk fat concentration by 10% (
      • 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.
      ). However, in contrast with these 2 previous studies, in the current experiment, the 4% FCM was similar in cows fed K2CO3 and control diets, possibly due to a numerical decrease in milk yield (−3.2 kg/d) when cows were supplemented with K2CO3.
      When compared with the K2CO3, the control, KHCO3, and KCl treatments did not affect milk protein concentration or yield. However, cows receiving the K2CO3 produced milk with a greater protein concentration as compared with cows receiving Na2CO3 diet. When studying mineral supplements with differing buffering capacities,
      • Mooney C.S.
      • Allen M.S.
      Effect of dietary strong ions on chewing activity and milk production in lactating dairy cows.
      did not observe any variation in milk protein concentration among cows fed NaCl, KCl, NaHCO3, or KHCO3 supplemented diets. However, in a more recent study by
      • Martins C.M.M.R.
      • Arcari M.A.
      • Welter K.C.
      • Netto A.S.
      • Oliveira C.A.F.
      • Santos M.V.
      Effect of dietary cation-anion difference on performance of lactating dairy cows and stability of milk proteins.
      , increasing DCAD from −71 to +290 mEq/kg of DM decreased milk protein concentration by 5%. These changes were related to a linear reduction of casein concentration in milk, which was also reported by
      • Harrison J.
      • White R.
      • Kincaid R.
      • Block E.
      • Jenkins T.
      • St-Pierre N.
      Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.
      . In the current study, DCAD did not affect milk protein synthesis. Moreover, the decrease in milk protein concentration observed with Na2CO3 compared with K2CO3 diet did not lead to a decrease in milk protein yield, suggesting a dilution effect due to a numerical increase in milk yield (+3.6 kg/d).
      Also, one should not ignore that, in the current study, a small number of animals was assigned to each treatment (n = 7) and a substantial variation existed among cows in their response to high-concentrate diets. These conditions could have affected the statistical power of the current experiment, and consequently masked potential treatment effects on milk performance.

      Milk Fatty Acid Composition

      Dietary mineral supplementation had some effects on milk FA profile (Table 5). When Na2CO3 was added to the diet as compared with K2CO3, milk fat concentrations of trans-6–8 18:1, trans-10 18:1, and cis-6–8 18:1, as well as the trans-10/trans-11 18:1 ratio were increased by 18%, 140%, 55%, and 106%, respectively. A tendency for similar increases was observed for milk fat concentrations of trans-9 18:1, cis-13 18:1, and cis-14 18:1, whereas a tendency for a decrease in trans-13–14 18:1 was observed when Na2CO3 was compared with the K2CO3 diet. Ruminal bacteria are largely responsible for rumen biohydrogenation of PUFA (
      • Harfoot C.G.
      Lipid metabolism in the rumen.
      ). It may be possible that differences in cation source (K vs. Na) affected a niche of bacteria involved in this process, leading to differences in milk fat concentrations of various biohydrogenation intermediates. When comparing cows supplemented with KCl and K2CO3, along with a decrease in DCAD, milk fat concentrations of trans-15 18:1 (+13%), cis-6–8 18:1 (+44%), cis-13 18:1 (+60%), and cis-15 18:1 (+25%; tendency), as well as the trans-10/trans-11 18:1 ratio (+85%), were increased with KCl.
      Table 5Fatty acid composition of milk fat from early-lactation cows receiving high-concentrate diets supplemented with different minerals
      Item, %Dietary treatmentSEMP-value, K2CO3 vs.
      ControlK2CO3KHCO3KClNa2CO3ControlKHCO3KClNa2CO3
      4:02.6432.642.6482.6822.8530.1260.990.970.820.24
      6:01.7261.7961.7511.7411.8310.0830.560.710.640.76
      8:01.1031.1851.1081.0951.1700.0640.390.410.340.88
      10:02.8823.0832.7852.7002.9640.2060.500.320.200.69
      10:10.2250.2580.2520.2290.2250.0180.170.810.200.17
      11:00.0990.1090.0930.1090.0940.0230.740.571.000.60
      12:03.6103.7623.5343.2183.6310.2610.680.530.140.72
      iso 13:00.0190.0190.0210.0190.0220.0020.930.720.870.29
      anteiso 13:00.0130.0140.0130.0110.0160.0010.690.780.340.28
      12:10.0920.1040.1020.0860.0880.0090.290.890.130.19
      13:00.1590.1500.1360.1580.1410.0280.780.720.820.80
      iso 14:00.0530.0620.0500.0570.0750.0120.500.380.740.30
      14:011.50610.76510.77510.19311.3560.3760.170.980.280.27
      cis-9 14:10.8750.8710.8600.9450.8270.0670.960.410.900.64
      cis-11 14:10.0230.0260.0210.0240.0220.0020.360.580.170.24
      iso 15:00.5850.5010.5470.5210.5530.0590.080.330.640.22
      anteiso 15:00.3570.3070.3750.3330.3830.0170.030.010.26<0.01
      15:01.3801.1641.2101.3751.1650.2020.430.870.441.00
      iso 16:00.1560.1640.1640.1930.1730.0330.821.000.420.79
      16:029.42829.35130.93228.75728.480.8940.940.150.580.43
      trans-9 16:10.0310.0330.0390.0310.0630.0140.930.760.950.13
      cis-9 16:11.1351.2421.3681.2971.2050.0870.390.310.650.77
      cis-11 16:10.0380.0390.0310.0430.0350.0040.810.190.530.42
      cis-13 16:10.1850.2060.2030.1790.1690.0190.340.890.230.11
      iso 17:0
      Coelution with minor concentration of trans-10 16:1.
      0.2570.2360.2640.2660.2600.0170.310.160.180.26
      anteiso 17:0
      Coelution with minor concentration of cis-10 16:1.
      0.3620.3450.3930.3920.3960.0210.590.140.120.11
      17:00.2540.2380.2550.2740.2430.0210.600.560.230.86
      cis-7 17:00.0130.0140.0140.0140.0130.0010.780.680.590.44
      cis-8 17:10.0090.0100.0090.0100.0100.0010.390.430.790.88
      cis-9 17:10.0970.0990.1020.1150.1060.0110.860.870.320.66
      iso 18:00.0170.0150.0170.0190.0210.0020.750.590.410.20
      18:06.4636.6386.0156.7756.5410.5230.780.320.820.87
      trans-4 18:10.0160.0180.0160.0160.0200.0020.570.610.660.44
      trans-5 18:10.0130.0140.0140.0140.0160.0010.710.820.890.38
      trans-6–8 18:10.1780.1650.1720.2060.2000.0150.430.690.020.04
      trans-9 18:10.1560.1550.1610.1780.1790.0110.910.630.080.06
      trans-10 18:10.2540.2220.2990.3950.5270.0980.790.540.160.02
      trans-11 18:10.5280.5020.5480.5620.5520.0850.680.470.350.43
      trans-12 18:10.1890.1850.1770.1960.1680.0150.820.660.550.34
      trans-13–14 18:10.3180.3060.2920.2820.2200.0340.790.580.750.06
      trans-15 18:10.2440.2280.2270.2570.2390.0120.320.930.050.49
      trans-16 18:10.1890.1720.1960.1660.1780.0090.210.720.100.64
      cis-6–8 18:10.1180.0850.1160.1250.1390.0120.050.060.03<0.01
      cis-9–10 18:114.99315.2514.64616.40115.3630.9610.850.640.370.93
      cis-11 18:10.7110.6490.5690.7820.6230.0640.480.370.130.77
      cis-12 18:10.2060.2190.2120.2370.2290.0150.490.700.390.61
      cis-13 18:10.0490.0490.0450.0810.0750.0110.980.780.030.07
      cis-14 18:10.0420.0360.0380.0400.0470.0040.360.670.480.09
      cis-15 18:10.0380.0360.0440.0480.0470.0050.700.230.080.11
      cis-9,trans-12 18:20.0450.0420.0390.0460.0440.0020.420.400.330.66
      trans-9,trans-12 18:20.0110.0120.0100.0100.0120.0010.640.530.380.99
      cis-9,trans-13 18:20.2770.2580.2560.2590.2640.0230.550.930.990.84
      trans-8,cis-13 18:20.0820.0740.0820.0820.0800.0040.260.250.230.36
      trans-9,cis-12 18:20.0220.0190.0230.0250.0210.0010.190.130.010.47
      trans-11,cis-15 18:20.0350.0300.0370.0380.0470.0060.350.160.09<0.01
      cis-9,12 18:22.0462.1522.0282.2782.1530.1850.340.270.270.99
      cis-9,trans-11 18:20.2630.2490.2950.2820.2790.0340.640.120.270.31
      trans-10,cis-12 18:20.0120.0150.0130.0140.0120.0010.220.300.730.14
      cis-9,12,15 18:30.2780.2680.2750.2740.2740.0300.800.870.890.88
      cis-6,9,12 18:30.0320.0360.0310.0310.0330.0030.170.120.120.33
      cis-9,trans-11,cis-15 18:30.0200.0170.0220.0240.0190.0020.210.050.010.59
      cis-6,9,12,15 18:40.0220.0180.0190.0210.0240.0030.370.940.530.17
      19:00.0650.0680.0610.0610.0660.0050.700.370.380.76
      20:00.0950.0980.0870.0970.0940.0060.700.170.870.65
      cis-9 20:10.0330.0310.0310.0340.0320.0010.530.830.270.72
      cis-11 20:10.0360.0370.0310.0400.0400.0030.860.260.680.70
      cis-11,14 20:20.0250.0270.0230.0290.0260.0020.560.280.620.67
      cis-11,14,17 20:30.0080.0060.0080.0080.0080.0010.240.230.140.18
      cis-8,11,14 20:30.0900.0850.0750.0850.0890.0100.660.330.990.75
      cis-8,11,14,17 20:40.0080.0080.0080.0100.0110.0010.920.880.150.06
      cis-5,8,11,14 20:40.1350.1390.1330.1520.1260.0120.690.600.330.28
      cis-5,8,11,14,17 20:50.0370.0270.0290.0340.0300.0020.020.580.080.42
      22:00.0170.0170.0140.0160.0120.0010.900.160.830.02
      cis-13 22:10.0070.0080.0070.0090.0080.0010.250.380.640.95
      cis-13,16 22:20.0080.0080.0070.0080.0090.0010.810.810.850.52
      cis-13,16,19 22:30.0040.0040.0050.0060.0040.0010.780.320.600.97
      cis-7,10,13,16 22:40.0150.0170.0160.0140.0120.0020.440.180.650.04
      cis-7,10,13,16,19 22:50.0350.0330.0340.0300.0280.0070.750.590.910.32
      cis-4,7,10,13,16,19 22:60.0100.0090.0090.0100.0090.0010.840.440.590.50
      24:00.0090.0080.0090.0080.0080.0010.390.870.600.79
      24:10.0060.0070.0070.0060.0070.0010.840.900.700.59
      Glycerol11.59811.61811.54611.60711.6580.0650.830.910.440.65
      Others
      Represent unidentified chromatogram peaks.
      0.6800.6750.7240.5460.6130.0610.950.110.560.47
      Sum
       De novo fatty acids
      Sum of straight even-chain fatty acids from C6 to C14.
      22.02221.84021.29020.19822.1140.9570.890.680.220.84
       C1630.81830.86532.55430.31429.9690.9000.970.140.630.43
       Preformed fatty acids
      Sum of branched-chain fatty acids (iso 13:0, anteiso 13:0, iso 14:0, iso 15:0, anteiso 15:0, iso 16:0, iso 17:0, and anteiso 17:0), odd-chain fatty acids (13:0 and 15:0), and all fatty acids with a chain length of 17C or more.
      32.36032.10331.01034.46832.8701.4030.890.540.190.67
      Ratio
      trans-10/trans-11 18:10.5200.4730.6180.8670.9720.1880.840.550.100.04
      1 Coelution with minor concentration of trans-10 16:1.
      2 Coelution with minor concentration of cis-10 16:1.
      3 Represent unidentified chromatogram peaks.
      4 Sum of straight even-chain fatty acids from C6 to C14.
      5 Sum of branched-chain fatty acids (iso 13:0, anteiso 13:0, iso 14:0, iso 15:0, anteiso 15:0, iso 16:0, iso 17:0, and anteiso 17:0), odd-chain fatty acids (13:0 and 15:0), and all fatty acids with a chain length of 17C or more.
      Moreover, a numerical (−44%) yet not significant (P = 0.16) decrease in milk fat concentration of trans-10 18:1 was observed when K2CO3 was compared with the KCl diet. When DCAD was increased (from 320 to 530 mEq/kg of DM) through K2CO3 supplementation,
      • Harrison J.
      • White R.
      • Kincaid R.
      • Block E.
      • Jenkins T.
      • St-Pierre N.
      Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.
      observed a 40% decrease in concentration of trans-10 18:1 in milk fat of dairy cows. In contrast, other studies showed no effect on milk trans-10 18:1 when mineral supplements were combined to achieve different DCAD (
      • Roche J.R.
      • Petch S.
      • Kay J.K.
      Manipulating the dietary cation-anion difference via drenching to early-lactation dairy cows grazing pasture.
      ;
      • Apper-Bossard E.
      • Peyraud J.L.
      • Faverdin P.
      • Meschy F.
      Changing dietary cation-anion difference for dairy cows fed with two contrasting levels of concentrate in diets.
      ). Discrepancies between studies could be related to different factors such as levels of mineral supplementation, basal diet composition, and individual variation in animal response to high-concentrate diets.
      Additionally, a decreased concentration of anteiso 15:0 in milk fat was observed when K2CO3 was added to the diet, compared with control, KHCO3, or Na2CO3. Lipid membrane composition of ruminal bacteria is characterized by a large proportion of branched-chain FA (iso 15:0, iso 17:0, anteiso 15:0, anteiso 17:0;
      • Vlaeminck B.
      • Dufour C.
      • van Vuuren A.M.
      • Cabrita A.R.J.
      • Dewhurst R.J.
      • Demeyer D.
      • Fievez V.
      Use of odd and branched-chain fatty acids in rumen contents and milk as a potential microbial marker.
      ). In particular, amylolytic bacteria are reported to have greater proportions of anteiso FA in their lipid membranes (
      • Vlaeminck B.
      • Fievez V.
      • Cabrita A.
      • Fonseca A.
      • Dewhurst R.
      Factors affecting odd-and branched-chain fatty acids in milk: A review.
      ). Diets with high inclusion of concentrate, which increase the relative abundance of amylolytic bacteria in the rumen, can be expected to lead to greater proportions of rumen anteiso FA and, consequently, to greater concentrations of these FA in milk fat (
      • Vlaeminck B.
      • Fievez V.
      • Cabrita A.
      • Fonseca A.
      • Dewhurst R.
      Factors affecting odd-and branched-chain fatty acids in milk: A review.
      ).
      A decrease in melting point of bacterial membrane lipid composition has been previously reported as a mechanism to modulate and maintain membrane fluidity and transport functions (homeoviscous adaptation) in response to pH (
      • Giotis E.S.
      • McDowell D.A.
      • Blair I.S.
      • Wilkinson B.J.
      Role of branched-chain fatty acids in pH stress tolerance in Listeria monocytogenes..
      ) and osmotic stress (
      • Chihib N.-E.
      • Ribeiro da Silva M.
      • Delattre G.
      • Laroche M.
      • Federighi M.
      Different cellular fatty acid pattern behaviours of two strains of Listeria monocytogenes Scott A and CNL 895807 under different temperature and salinity conditions.
      ). Because anteiso FA have low melting points compared with iso FA, synthesis of anteiso FA is stimulated under stress conditions (
      • Annous B.A.
      • Becker L.A.
      • Bayles D.O.
      • Labeda D.P.
      • Wilkinson B.J.
      Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures.
      ). Consequently, it could be suggested that adding K2CO3 to the diet, compared with other source of minerals, induced modifications in the rumen environment, which prevented bacteria to resort to modulation of their membrane FA profile to resist stressing conditions normally related to high-concentrate diets.
      Of note, when cows were fed the K2CO3 diet, milk fat concentrations of anteiso 15:0 and trans-10,cis-12 18:2 were positively correlated (Figure 3). Because concentrations of milk branched-chain FA were not determined when mineral supplementation was evaluated in earlier studies, the link between anteiso 15:0 and trans-10,cis-12 18:2 has not been previously reported. Further research is then needed to elucidate the potential link between these 2 FA involved in rumen lipid metabolism and the effect of mineral supplementation on this relationship.
      Figure thumbnail gr3
      Figure 3Association between milk fat concentration of trans-10, cis-12 18:2, and anteiso 15:0 in early-lactation cows fed a high-concentrate diet containing 1.8% of K2CO3 (DM basis).

      CONCLUSIONS

      In the current experiment, and as opposed to previous studies, supplementing high-concentrate diets with K2CO3 did not increase milk or milk fat yield in early-lactation cows. Adding K2CO3 even led to a tendency for a decreased milk yield when compared with KCl. Overall, using K2CO3 as a mineral supplement to modulate DCAD, K ion, or buffer ability of diets, affected the rumen environment, but did not stimulate synthesis of milk components. Our results suggest that increasing dietary K through the addition of K2CO3 could lead to a disequilibrium in cellular ion composition that can impair nutrient transport into and out of the mammary epithelial cells, and consequently affect milk synthesis. Further research is needed to establish under which conditions dietary K2CO3 supplementation can contribute to rumen stability of early-lactation cows, and to determine how dietary mineral supplementation affects the metabolism of mammary epithelial cells.

      ACKNOWLEDGMENTS

      This experiment was funded through the Programme de recherche en partenariat pour l'innovation en production et en transformation laitières supported by Agriculture and Agri-Food Canada, the Fonds de Recherche du Québec–Nature et Technologies (FRQNT; Québec, QC, Canada), the Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec, and Novalait Inc. The authors thank Gilles Bélanger, Danielle Mongrain, and Mario Laterrière from Agriculture and Agri-Food Canada, (Québec, QC, Canada), as well as Micheline Gingras and Yolaine Lebeuf from Université Laval (Québec, QC, Canada) for professional and technical assistance, Yan Martel-Kennes and Annie Dumas for their help in organizing the experiment at the farm, and André Perreault, Philippe Cantin, Denis Lefebvre, and Sébastien Coursol for all their work in the barn at the Centre de Recherche en Sciences Animales de Deschambault (Deschambault, QC, Canada).

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