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The objective of this study was to evaluate the effects of amino resin-treated soybean meal (SBM) on ruminal fermentation, nutrient digestion, and N partitioning. The treatments were (1) untreated solvent-extracted SBM, (2) amino resin-treated SBM (AR-SBM), and (3) heat-treated SBM (HT-SBM). The experimental design was arranged as a replicated 3 × 3 Latin square with 6 fermentors in a dual-flow continuous culture system. Treatments were randomly assigned to fermentors within a Latin square for each period. Each fermentor was fed 106 g/d of diet DM equally distributed in 2 feeding times daily at 0800 and 1800 h. Diets were formulated to contain 16% CP, 30% NDF, and 30% starch across treatments. The experiment consisted of 3 experimental periods, each lasting for 10 d. The first 7 d of each period were considered adaptation, and the last 3 d were used for sampling and data collection. On d 8 and 9, samples were collected for analysis of diurnal variation in concentrations of NH 3 -N, pH, and VFA during the first 8 h after feeding. On d 8, 9, and 10, samples were collected from the liquid and solid effluents accumulated over 24 h for analysis of daily averages of NH 3 -N and VFA pools, and true ruminal digestibility estimates. Data were analyzed using the MIXED procedure of SAS, and significance was declared when P ≤ 0.05. The model included the fixed effect of treatment and random effects of square, period, and fermentor within square, whereas time and interaction treatment × time were included for analyses of diurnal variation, with time as repeated measures. Compared with SBM, the cultured ruminal contents of AR-SBM and HT-SBM had lower NH 3 -N concentrations, indicating lower microbial fermentation of protein. Molar proportions of isovalerate and isobutyrate were greater in SBM than AR-SBM and HT-SBM, with greater molar proportion of isobutyrate for SBM, particularly during the first 2 h after feeding. The flow of NH 3 -N was greater for SBM compared with AR-SBM and HT-SBM, whereas NAN flow, bacterial N flow, and N efficiency were greater for AR-SBM and HT-SBM compared with SBM. Our results indicate that both the amino resin and heat treatments of SBM allow for similar decreases in microbial degradation of CP without limiting microbial protein synthesis in diets with 16% CP. Amino resin treatment may be effective in reducing microbial fermentation of protein in the rumen without adverse effects on digestibility or fermentation parameters as compared with SBM., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to compare cashew nutshell extract (CNSE) to monensin and evaluate changes in in vitro mixed ruminal microorganism fermentation, nutrient digestibility, and microbial nitrogen outflow. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square design with 4 experimental periods of 10 d (7 d for diet adaptation and 3 d for sample collection). Basal diets contained 43.5:56.5 forage: concentrate ratio and each fermenter was fed 106 g of DM/d divided equally between 2 feeding times. Treatments were control (CON, basal diet without additives), 2.5 μM monensin (MON), 0.1 mg CNSE granule/g DM (CNSE100), and 0.2 mg CNSE granule/g DM (CNSE200). On d 8 to10, samples were collected for pH, lactate, NH 3 -N, volatile fatty acids (VFA), mixed protozoa counts, organic matter (OM), and neutral detergent fiber (NDF) digestibility. Data were analyzed with the GLIMMIX procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). We observed that butyrate concentration in all treatments was lower compared with CON and the concentration for MON was lower compared with CNSE treatments. Protozoal population in all treatments was lower compared with CON. No effects were observed for pH, lactate, NH 3 -N, total VFA, OM, or N utilization. Within the 24-h pool, protozoal generation time, tended to be lower, while NDF digestibility tended to be greater in response to all additives. Furthermore, the microbial N flow, and the efficiency of N use tended to be lower for the monensin treatment compared with CNSE treatments. Overall, our results showed that both monensin and CNSE decreased butyrate synthesis and protozoal populations, while not affecting OM digestibility and tended to increase NDF digestibility; however, such effects are greater with monensin than CNSE nutshell., (© 2024, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to evaluate the effects of dietary replacement of magnesium oxide (MgO) with calcium-magnesium hydroxide [CaMg(OH) 2 ] and its interaction with ruminal buffer (sodium sesquicarbonate) supplementation on production, Ca and Mg balance, and overall physiological response of mid-lactation Holstein dairy cows. Sixty cows averaging 40.5 ± 7.0 kg of milk/d were used. Treatments were assigned following a 2 × 2 factorial arrangement: (1) MgO, (2) MgO + buffer, (3) CaMg(OH) 2 , or (4) CaMg(OH) 2 + buffer. Diets were formulated to have 16.5% of crude protein, 1.82 Mcal/kg of net energy for lactation, 0.67% Ca, 0.39% P, and 0.25% Mg, all on a dry matter (DM) basis. Treatments were individually top dressed. Milk production, composition, and DM intake were evaluated. A subsample of 20 cows were randomly selected for the evaluation of Ca and Mg balance, blood gases, and electrolytes. Ruminal fluid was also collected for evaluation of pH and Ca and Mg solubility. Effects of Mg source, buffer, and the interaction Mg source × buffer were analyzed through orthogonal contrasts. An interaction of Mg source × buffer was found for DM intake and feed efficiency, in which cows fed CaMg(OH) 2 had a similar feed efficiency regardless of ruminal buffer inclusion; however, when cows were fed MgO, the inclusion of buffer reduced feed efficiency. No effects on body weight and milk yield were observed. Buffer addition tended to increase the concentrations of fat, protein, and solids-not-fat, without affecting the yields of these milk components. Magnesium source and buffer did not affect ruminal fluid, blood, urine, or fecal pH; however, buffer supplementation increased urinary pH. Treatment with CaMg(OH) 2 increased blood concentration of HCO 3 - , total CO 2 , and base excess compared with cows fed MgO. No differences were observed in the ruminal solubility of Ca and Mg or on milk or urinary Ca and Mg excretion. Greater plasma Mg concentration was observed for animals fed MgO compared with cows fed CaMg(OH) 2 ; however, both sources were above the threshold recommended in the literature for dairy cows. Also, a reduction in fecal Mg excretion was observed in animals fed CaMg(OH) 2 . In summary, we provide evidence that CaMg(OH) 2 could replace MgO without affecting performance, overall physiological response, or Ca and Mg balance of mid-lactating dairy Holstein cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH 3 -N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH 3 -N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH 3 -N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH 3 -N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to evaluate the effects of replacing magnesium oxide (MgO) with calcium-magnesium carbonate [CaMg(CO 3 ) 2 ] on ruminal fermentation with or without the addition of sodium bicarbonate (NaHCO 3 ). Eight fermentors of a dual-flow continuous-culture system were distributed in a replicated (2) 4 × 4 Latin square design in a 2 × 2 factorial arrangement of treatments (magnesium sources × NaHCO 3 ). The treatments tested were 0.21% MgO [MgO; dry matter (DM) basis; 144.8 mEq of dietary cation-anion difference (DCAD)]; 0.21% MgO + 0.50% NaHCO 3 (MgO+NaHCO 3 ; DM basis; 205.6 mEq of DCAD); 1.00% CaMg(CO 3 ) 2 [CaMg(CO 3 ) 2 ; DM basis; 144.8 mEq of DCAD]; and 1.00% CaMg(CO 3 ) 2 + 0.50% NaHCO 3 [CaMg(CO 3 ) 2 +NaHCO 3 ; DM basis; 205.6 mEq of DCAD]. Diets were formulated to have a total of 0.28% of Mg (DM basis). The experiment consisted of 40 d, which was divided into 4 periods of 10 d each, where 7 d were used for adaptation and 3 d for sampling to determine pH, volatile fatty acids (VFA), ammonia (NH 3 -N), lactate, mineral solubility, N metabolism, and nutrient digestibility. The effects of Mg source [MgO vs. CaMg(CO 3 ) 2 ], NaHCO 3 (with vs. without), and the interaction were tested with the MIXED procedure of SAS version 9.4 (SAS Institute). There was no Mg source × NaHCO 3 interaction in the pH variables and mineral solubility, and Mg sources evaluated did not affect the variables related to ruminal pH and solubility of Mg. On the other hand, the inclusion of NaHCO 3 increased the pH daily average, independent of Mg source, which led to a reduced time that pH was below 5.8 and decreased area under the curve. Total VFA and lactate concentration were similar among treatments regardless of NaHCO 3 and Mg source; however, the molar proportion of isobutyrate and NH 3 -N concentration were lower in diets with CaMg(CO 3 ) 2 compared with MgO. Moreover, NaHCO 3 inclusion increased NH 3 -N, total daily NH 3 -N flow, isobutyrate concentration, and acid detergent fiber digestibility. Our results showed that CaMg(CO 3 ) 2 leads to a lower NH 3 -N concentration and isobutyrate proportion. Therefore, because most of the tested variables were not significantly different between MgO and CaMg(CO 3 ) 2 when combined or not with NaHCO 3 , CaMg(CO 3 ) 2 can be a viable alternative source to replace MgO in dairy cow diets without affecting mineral solubility, ruminal pH, nutrient digestibility, total VFA, and the main ruminal VFA. Although Mg sources are known to have an alkalizing effect, NaHCO 3 inclusion in diets with Mg supplementation allowed an increase in ruminal pH, as well as an increase in isobutyrate and NH 3 -N flow., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to evaluate ruminal microbiome changes associated with feeding Lactobacillus plantarum GB-LP1 as direct-fed microbials (DFM) in high-producing dairy cow diets. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin square design. A basal diet was formulated to meet the requirements of a cow producing 45 kg of milk per day (16% crude protein and 28% starch). There were 4 experimental treatments: the basal diet without any DFM (CTRL); a mixture of Lactobacillus acidophilus, 1 × 10 9 cfu/g, and Propionibacterium freudenreichii, 2 × 10 9 cfu/g [MLP = 0.01% of diet dry matter (DM)]; and 2 different levels of L. plantarum, 1.35 × 10 9 cfu/g (L1 = 0.05% and L2 = 0.10% of diet DM). Bacterial samples were collected from the fluid and particulate effluents before feeding and at 2, 4, 6, and 8 h after feeding; a composite of all time points was made for each fermentor within their respective fractionations. Bacterial community composition was analyzed through sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequenced data were analyzed on DADA2, and statistical analyses were performed in R (RStudio 3.0.1, https://www.r-project.org/) and SAS 9.4 (SAS Institute Inc.); orthogonal contrasts were used to compare treatments. Different than in other fermentation scenarios (e.g., silage or beef cattle high-grain diets), treatments did not affect pH or lactic acid concentration. Effects were mainly from overall DFM inclusion, and they were mostly observed in the fluid phase. The relative abundance of the phylum Firmicutes, family Lachnospiraceae, and 6 genera decreased with DFM inclusion, with emphasis on Butyrivibrio_2, Saccharofermentans, and Ruminococcus_1 that are fibrolytic and may display peptidase activity during fermentation. Lachnospiraceae_AC2044_group and Lachnospiraceae_XPB1014_group also decreased in the fluid phase, and their relative abundances were positively correlated with NH 3 -N daily outflow from the fermentors. Specific effects of MLP and L. plantarum were mostly in specific bacteria associated with proteolytic and fibrolytic functions in the rumen. These findings help to explain why, in the previous results from this study, DFM inclusion decreased NH 3 -N concentration without altering pH and lactic acid concentration., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Magnesium oxide (MgO) is the most common supplemental source of Mg for dairy cows and a proven ruminal alkalizer when supplemented above NRC (2001) recommendations. However, overfeeding MgO may increase feeding costs, whereas the effects of alternative sources of Mg on ruminal fermentation are not well known. Moreover, it is still unclear if Mg supplementation influences the effects of bicarbonate-based buffers on ruminal fermentation. We aimed to evaluate the effect of Mg source on ruminal fermentation with diets formulated to a final concentration of 0.25% Mg, and to determine if the effect of sodium sesquicarbonate as a buffer varies with the source of Mg. We used 8 fermentors in a duplicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments, by combining 2 factors: (1) Mg source: using either MgO or an alternative source consisting of a blend of CaMg(OH) 4 and CaMg(CO 3 ) 2 (BLN) and (2) sodium sesquicarbonate buffer inclusion, at 0 or 0.6% of dry matter intake. Based on preliminary tests of reactivity, we hypothesized that BLN plus buffer would allow for greater ruminal pH, acetate molar proportion, and NDF digestibility than diets with MgO or without buffer. Four 10-d periods were completed, where the last 3 d were used for pH measurements and collection of samples for volatile fatty acids (VFA), ammonia (NH 3 -N), Mg solubility, N metabolism, and nutrient digestibility. Effects of Mg source (source), sodium sesquicarbonate inclusion (buffer), and their interaction (source × buffer) were tested with the MIXED procedure of SAS (SAS Institute Inc.). We did not find an effect of Mg source on ruminal fermentation variables; however, concentration of soluble Mg in ruminal fluid was greater for MgO compared with BLN. On the other hand, buffer supplementation increased average ruminal pH, acetate molar proportion, and branched-chain VFA molar proportion; tended to increase NDF digestibility; and decreased both area under the curve and time below pH 6.0. An interaction of source × buffer was found for propionate, butyrate, and NH 3 -N, the first one decreasing and the 2 others increasing only when buffer was supplemented to the BLN diet. Our results indicate that supplementing Mg with either MgO or BLN promotes similar ruminal fermentation in diets with total concentration of 0.25% Mg. Further evaluations are needed to assess Mg availability and animal performance in dairy cows fed BLN., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Choline is usually supplemented as ruminally protected choline chloride to prevent its degradation in the rumen, but the effects of unprotected choline on ruminal fermentation are unclear. Some research indicates a possible role of dietary fiber on microbial degradation of choline; therefore we aimed to evaluate the effects of unprotected choline chloride on ruminal fermentation and to investigate whether those effects depend on dietary neutral detergent fiber (NDF) concentration. Our hypothesis was that dietary NDF concentration would influence choline chloride effects on microbial ruminal fermentation. We used 8 fermentors in a duplicated 4 × 4 Latin square with a 2 × 2 factorial arrangement, combining 2 factors: (1) dietary NDF concentration and (2) unprotected choline chloride supplementation. Resulting treatments are (1) 30%NDF/Ctrl [30% NDF control diet without supplemental choline (Cho)]; (2) 30%NDF/Cho [30% NDF diet plus 1.9 g of choline ion per kg of dry matter (DM)]; (3) 40%NDF/Ctrl (40% NDF control diet without supplemental choline); and (4) 40%NDF/Cho (40% NDF diet plus 1.9 g of choline ion per kg of DM). Four 10-d periods were completed, each consisting of 7 d for adaptation and 3 d for collection of samples for estimation of nutrient disappearance and daily average concentrations of volatile fatty acids and NH 3 -N. In addition, kinetics of pH, acetate, and propionate were evaluated at 0, 1, 2, 4, 6, and 8 h after morning feeding. On the last day of each period, bacteria pellets were harvested for 15 N analysis and N metabolism. Fixed effects of dietary NDF concentration, unprotected choline chloride supplementation, and their interaction (NDF × Cho) were tested using the MIXED procedure of SAS version 9.4 (SAS Institute Inc., Cary, NC). Choline tended to increase total volatile fatty acid concentrations and decreased acetate molar proportion regardless of dietary NDF concentration, but it increased propionate molar proportion and decreased acetate to propionate ratio only with the 30% NDF diet. Supplementing choline decreased NDF disappearance regardless of dietary NDF; however, organic matter disappearance tended to be reduced only when choline was added to 40% NDF. Our data indicate that unprotected choline chloride effects on ruminal fermentation depend on dietary NDF concentration, allowing for a greater propionate synthesis without decreasing organic matter disappearance when fed with a 30% NDF diet., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Undesirable interactions between trace mineral elements and ruminal contents may occur during digestion when mineral salts are supplemented. Antimicrobial effects of copper sulfate (CuSO 4 ) may affect ruminal digestibility of nutrients when fed as a source of copper (Cu), while sodium selenite (Na 2 SeO 3 ) may be reduced in the rumen to less available forms of selenium (Se). Our objective was to evaluate if protection of CuSO 4 and Na 2 SeO 3 by lipid-microencapsulation would induce changes on ruminal microbial fermentation. We used 8 fermentors in a dual-flow continuous-culture system in a 4 × 4 duplicated Latin square with a 2 × 2 factorial arrangement of treatments. Factors were CuSO 4 protection (unprotected and protected by lipid-microencapsulation) and Na 2 SeO 3 protection (unprotected and protected by lipid-microencapsulation). Treatments consisted of supplementation with 15 mg/kg of Cu and 0.3 mg/kg of Se from either unprotected or protected (lipid-microencapsulated) sources, as follows: (1) Control (unprotected CuSO 4 + unprotected Na 2 SeO 3 ); (2) Cu-P (protected CuSO 4 + unprotected Na 2 SeO 3 ); (3) Se-P (unprotected CuSO 4 + protected Na 2 SeO 3 ); (4) (Cu+Se)-P (protected CuSO 4 + protected Na 2 SeO 3 ). All diets had the same nutrient composition and fermentors were fed 106 g of dry matter/d. Each experimental period was 10 d (7 d of adaptation and 3 d for sample collections). Daily pooled samples of effluents were analyzed for pH, NH 3 -N, nutrient digestibility, and flows (g/d) of total N, NH 3 -N, nonammonia N (NAN), bacterial N, dietary N, and bacterial efficiency. Kinetics of volatile fatty acids was analyzed in samples collected daily at 0, 1, 2, 4, 6, and 8 h after feeding. Main effects of Cu protection, Se protection, and their interaction were tested for all response variables. Kinetics data were analyzed as repeated measures. Protection of Cu decreased acetate molar proportion, increased butyrate proportion, and tended to decrease acetate:propionate ratio in samples of kinetics, but did not modify nutrient digestibility. Protection of Se tended to decrease NH 3 -N concentration, NH 3 -N flow, and CP digestibility; and to increase flows of nonammonia N and dietary N. Our results indicate that protection of CuSO 4 may increase butyrate concentration at expenses of acetate, while protection of Na 2 SeO 3 tended to reduce ruminal degradation of N. Further research is needed to determine the effects of lipid-microencapsulation on intestinal absorption, tissue distribution of Cu and Se, and animal performance., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)