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Branched-chain amino acids are deaminated by amylolytic bacteria to branched-chain volatile fatty acids (BCVFA), which are growth factors for cellulolytic bacteria. Our objective was to determine the dietary conditions that would increase the uptake of BCVFA by rumen bacteria. We hypothesized that increased forage would increase cellulolytic bacterial abundance and incorporation of BCVFA into their structure. Supplemental polyunsaturated fatty acids, supplied via corn oil (CO), should inhibit cellulolytic bacteria growth, but we hypothesized that additional BCVFA would alleviate that inhibition. Further, supplemental BCVFA should increase neutral detergent fiber degradation and efficiency of bacterial protein synthesis more with the high forage and low polyunsaturated fatty acid dietary combination. The study was an incomplete block design with 8 dual-flow continuous cultures used in 4 periods with 8 treatments (n = 4 per treatment) arranged as a 2 × 2 × 2 factorial. The factors were: high forage (HF) or low forage (LF; 67 or 33%), without or with supplemental CO (3% dry matter), and without or with 2.15 mmol/d (which included 5 mg/d of 13 C each of BCVFA isovalerate, isobutyrate, and 2-methylbutyrate). The isonitrogenous diets consisted of 33:67 alfalfa:orchardgrass pellet, and was replaced with a concentrate pellet that mainly consisted of ground corn, soybean meal, and soybean hulls for the LF diet. The main effect of supplementing BCVFA increased neutral detergent fiber (NDF) degradability by 7.6%, and CO increased NDF degradability only in LF diets. Supplemental BCVFA increased bacterial N by 1.5 g/kg organic matter truly degraded (6.6%) and 0.05 g/g truly degraded N (6.5%). The relative sequence abundance decreased with LF for Fibrobacter succinogenes, Ruminococcus flavefaciens, and genus Butyrivibrio compared with HF. Recovery of the total 13 C dose in bacterial pellets decreased from 144 µg/ mg with HF to 98.9 µg/ mg with LF. Although isotope recovery in bacteria was greater with HF, BCVFA supplementation increased NDF degradability and efficiency of microbial protein synthesis under all dietary conditions. Therefore, supplemental BCVFA has potential to improve feed efficiency in dairy cows even with dietary conditions that might otherwise inhibit cellulolytic bacteria., (© 2023, 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 leverage a frequentist (ELN) and Bayesian learning (BLN) network analyses to summarize quantitative associations among variables measured in 4 previously published dual-flow continuous culture fermentation experiments. Experiments were originally designed to evaluate effects of nitrate, defaunation, yeast, and/or physiological shifts associated with pH or solids passage rates on rumen conditions. Measurements from these experiments that were used as nodes within the networks included concentrations of individual volatile fatty acids, mM and nitrate, NO3-,%; outflows of non-ammonia nitrogen (NAN, g/d), bacterial N (BN, g/d), residual N (RN, g/d), and ammonia N (NH3-N, mg/dL); degradability of neutral detergent fiber (NDFd, %) and degradability of organic matter (OMd, %); dry matter intake (DMI, kg/d); urea in buffer (%); fluid passage rate (FF, L/d); total protozoa count (PZ, cells/mL); and methane production (CH4, mmol/d). A frequentist network (ELN) derived using a graphical LASSO (least absolute shrinkage and selection operator) technique with tuning parameters selected by Extended Bayesian Information Criteria (EBIC) and a BLN were constructed from these data. The illustrated associations in the ELN were unidirectional yet assisted in identifying prominent relationships within the rumen that were largely consistent with current understanding of fermentation mechanisms. Another advantage of the ELN approach was that it focused on understanding the role of individual nodes within the network. Such understanding may be critical in exploring candidates for biomarkers, indicator variables, model targets, or other measurement-focused explorations. As an example, acetate was highly central in the network suggesting it may be a strong candidate as a rumen biomarker. Alternatively, the major advantage of the BLN was its unique ability to imply causal directionality in relationships. Because the BLN identified directional, cascading relationships, this analytics approach was uniquely suited to exploring the edges within the network as a strategy to direct future work researching mechanisms of fermentation. For example, in the BLN acetate responded to treatment conditions such as the source of N used and the quantity of substrate provided, while acetate drove changes in the protozoal populations, non-NH3-N and residual N flows. In conclusion, the analyses exhibit complementary strengths in supporting inference on the connectedness and directionality of quantitative associations among fermentation variables that may be useful in driving future studies., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To investigate the effects of acetate, propionate, and pH on thermodynamics of volatile fatty acids (VFA) in the rumen, a dual-flow continuous culture study was conducted to quantify production of major VFA, interconversions among the VFA, and H 2 and CH 4 emissions in a 4 × 4 Latin square design. The 4 treatments were (1) control: pH buffered to an average of 6.75; (2) control plus 20 mmol/d of infused acetate (InfAc); (3) control plus 7 mmol/d of infused propionate (InfPr); and (4) a 0.5-unit decline in pH elicited by adjustment of the buffer (LowpH). All fermentors were fed 40 g of a pelleted diet containing whole alfalfa pellets and concentrate mix pellets (50:50) once daily. After 7 d of treatment, sequential, continuous infusions of [2- 13 C] sodium acetate (3.5 mmol/d), [U- 13 C] sodium propionate (2.9 mmol/d), and [1- 13 C] sodium butyrate (0.22 mmol/d) were carried out from 12 h before feeding for 36 h. Filtered liquid effluent (4 mL) was sampled at 0, 2, 4, 6, 8, 12, 16, and 22 h after feeding, and assessed for VFA concentrations, with another filtered sample (20 mL) used to quantify aqueous concentrations of CH 4 and H 2 . Headspace CH 4 and H 2 gases were monitored continuously. Ruminal microbes were isolated from the mixed effluent samples, and the microbial community structure was analyzed using the 16S rRNA amplicon sequencing technique. The digestibility of neutral detergent fiber, acid detergent fiber, and starch and microbial C sequestrated from VFA were not affected by treatments. The LowpH treatment increased net propionate production and decreased H 2 and CH 4 headspace emissions, primarily due to shifts in metabolic pathways of VFA formation, likely due to the observed changes in bacterial community structure. Significant interconversions occurred between acetate and butyrate, whereas interconversions of other VFA with propionate were relatively small. The InfAc and InfPr treatments increased net acetate and propionate production, respectively; however, interconversions among VFA were not affected by pH, acetate, or propionate treatments, suggesting that thermodynamics might not be a primary influencer of metabolic pathways used for VFA formation., (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/).)

Previous research revealed a potential effect of dietary trace mineral source on both ruminal and fecal microbiota. However, the effect of Zn source, specifically, has not previously been considered. Based on reported postruminal solubility, we hypothesized that Zn hydroxychloride would decrease Treponema spp. fecal excretion relative to cows fed Zn sulfate. To test this hypothesis, lactating Holstein cows (n = 24; 685 ± 9 kg of body weight; 159 ± 8 d in milk; parity 3 ± 0.2) were randomly assigned to 1 of 2 dietary treatments: control (75 mg/kg Zn from ZnSO 4 ) or Zn hydroxychloride (HYD; 75 mg/kg IntelliBond Z; Micronutrients USA LLC). Single fecal grab samples were collected on d 1 before dietary treatments and on d 27 after dietary treatments were applied. Fecal microbial DNA was extracted and sequenced to establish taxonomy using a universal primer for the 16S rRNA gene. Supplementation of HYD decreased the relative abundance of Treponema 2 by 3-fold (14.7% vs. 4.9%). Poor sequencing resolution at the species level limited inference of Treponema spp. toward management or gut health implications of HYD supplementation. However, the inclusion of pathogenic species among Treponema spp. indicates a potential implication of HYD feeding to reduce environmental exposure of the dairy cow to Treponema spp., (© 2022.)

To support improving genetic potential for increased milk production, intake of digestible carbohydrate must also increase to provide digestible energy and microbial protein synthesis. We hypothesized that the provision of exogenous branched-chain volatile fatty acids (BCVFA) would improve both neutral detergent fiber (NDF) degradability and efficiency of microbial protein synthesis. However, BCVFA should be more beneficial with increasing efficiency of bacterial protein synthesis associated with increasing passage rate (k p ). We also hypothesized that decreasing pH would increase the need for isobutyrate over 2-methylbutyrate. To study these effects independent from other sources of variation in vivo, we evaluated continuous cultures without (control) versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate), low versus high k p of the particulate phase (2.5 vs. 5.0%/h), and high versus low pH (ranging from 6.3 to 6.8 diurnally vs. 5.7 to 6.2) in a 2 × 2 × 2 factorial arrangement of treatments. Diets were 50% forage pellets and 50% grain pellets administered twice daily. Without an interaction, NDF degradability tended to increase from 29.7 to 35.0% for main effects of control compared with BCVFA treatments. Provision of BCVFA increased methanogenesis, presumably resulting from improved NDF degradability. Decreasing pH decreased methane production. Total volatile fatty acid (VFA) and acetate production were decreased with increasing k p , even though true organic matter degradability and bacterial nitrogen flow were not affected by treatments. Decreasing pH decreased acetate but increased propionate and valerate production, probably resulting from a shift in bacterial taxa and associated VFA stoichiometry. Decreasing pH decreased isobutyrate and isovalerate production while increasing 2-methylbutyrate production on a net basis (subtracting doses). Supplementing BCVFA improved NDF degradability in continuous cultures administered moderate (15.4%) crude protein diets (excluding urea in buffer) without major interactions with culture pH and k p ., (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-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Our objectives were to evaluate potential interactions in culture conditions that influence how exogenously dosed branched-chain VFA (BCVFA) would be recovered as elongated fatty acids (FA) or would affect bacterial populations. A 2 × 2 × 2 factorial arrangement of treatments evaluated 3 factors: (1) without versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate; each dose was partially substituted with 13 C-enriched tracers before and during the collection period); (2) high versus low pH (ranging diurnally from 6.3 to 6.8 vs. 5.7 to 6.2); and (3) low versus high particulate-phase passage rate (k p ; 2.5 vs. 5.0%/h) in continuous cultures administered a 50:50 forage:concentrate diet twice daily. Samples of effluent were collected and composited before harvesting bacteria from which FA and DNA were extracted. Profiles and enrichments of FA in bacteria were evaluated by gas chromatography and isotope-ratio mass spectrometry. The 13 C enrichment in bacterial FA was calculated as percentage recovery of dosed 13 C-labeled BCVFA. Dosing BCVFA increased the even-chain iso-FA, preventing the reduced concentration at higher k p and potentially as a physiological response to decreased pH. However, decreasing pH decreased recovery of 13 C in these even-chain FA, suggesting greater reliance on isobutyrate produced from degradation of dietary valine. The iso-FA were decreased, whereas anteiso-FA and 16:0 increased with decreasing pH. Thus, 2-methylbutyrate still appeared to be important as a precursor for anteiso-FA to counter the increased rigidity of bacterial membranes that had more saturated straight-chain FA when pH decreased. Provision of BCVFA stimulated the relative sequence abundance of Fibrobacter and Treponema, both of which require isobutyrate and 2-methylbutyrate. Numerous bacterial community members were shifted by low pH, including increased Prevotella and genera within the phylum Proteobacteria, at the expense of members within phylum Firmicutes. Because of relatively few interactions with pH and k p , supplementation of BCVFA can stimulate neutral detergent fiber degradability via key fibrolytic bacteria across a range of conditions. Decreasing pH shifted bacterial populations and their FA composition, suggesting that further research is needed to distinguish pH from dietary changes., (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-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Within the rumen, nitrate can serve as an alternative sink for aqueous hydrogen [H 2 (aq)] accumulating during fermentation, producing nitrite, which ideally is further reduced to ammonium but can accumulate under conditions not yet explained. Defaunation has also been associated with decreased methanogenesis in meta-analyses because protozoa contribute significantly to H 2 production. In the present study, we applied a 2 × 2 factorial treatment arrangement in a 4 × 4 Latin square design to dual-flow continuous culture fermentors (n = 4). Treatments were control without nitrate (-NO 3 - ) versus with nitrate (+NO 3 - ; 1.5% of diet dry matter), factorialized with normal protozoa (faunated, FAUN) versus defaunation (DEF) by decreasing the temperature moderately and changing filters over the first 4 d of incubation. We detected no main effects of DEF or interaction of faunation status with +NO 3 - . The main effect of +NO 3 - increased H 2 (aq) by 11.0 µM (+117%) compared with -NO 3 - . The main effect of +NO 3 - also decreased daily CH 4 production by 8.17 mmol CH 4 /d (31%) compared with -NO 3 - . Because there were no treatment effects on neutral detergent fiber digestibility, the main effect of +NO 3 - also decreased CH 4 production by 1.43 mmol of CH 4 /g of neutral detergent fiber degraded compared with -NO 3 - . There were no effects of treatment on other nutrient digestibilities, N flow, or microbial N flow per gram of nutrient digested. The spike in H 2 (aq) after feeding NO 3 - provides evidence that methanogenesis is inhibited by substrate access rather than concentration, regardless of defaunation, or by direct inhibition of NO 2 - . Methanogens were not decreased by defaunation, suggesting a compensatory increase in non-protozoa-associated methanogens or an insignificant contribution of protozoa-associated methanogens. Despite adaptive reduction of NO 3 - to NH 4 + and methane inhibition in continuous culture, practical considerations such as potential to depress dry matter intake and on-farm ration variability should be addressed before considering NO 3 - as an avenue for greater sustainability of greenhouse gas emissions in US dairy production., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Concern over the carbon footprint of the dairy industry has led to various dietary approaches to mitigate enteric CH 4 production. One approach is feeding the electron acceptor NO 3 - , thus outcompeting methanogens for aqueous H 2 . We hypothesized that a live yeast culture (LYC; Saccharomyces cerevisiae from Yea-Sacc 1026, Alltech Inc., Nicholasville, KY) would stimulate the complete reduction of NO 3 - to NH 3 by selenomonads, thus decreasing the quantity of CH 4 emissions per unit of energy-corrected milk production while decreasing blood methemoglobin concentration resulting from the absorbed intermediate, NO 2 - . Twelve lactating Jersey cows (8 multiparous and noncannulated; 4 primiparous and ruminally cannulated) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Cattle were fed diets containing 1.5% NO 3 - (from calcium ammonium nitrate) or an isonitrogenous control diet (containing additional urea) and given a top-dress of ground corn without or with LYC, with the fourth week used for data collection. Noncannulated cows were spot measured for CH 4 emission by mouth using GreenFeed (C-Lock Inc., Rapid City, SD). The main effect of NO 3 - decreased CH 4 by 17% but decreased dry matter intake by 10% (from 19.8 to 17.8 kg/d) such that CH 4 :dry matter intake numerically decreased by 8% and CH 4 :milk net energy for lactation production was unaffected by treatment. Milk and milk fat production were not affected, but NO 3 - decreased milk protein from 758 to 689 g/d. Ruminal pH decreased more sharply after feeding for cows fed diets without NO 3 - . Acetate:propionate was greater for cows fed NO 3 - , particularly when combined with LYC (interaction effect). Blood methemoglobin was higher for cattle fed NO 3 - than for those fed the control diet but was low for both treatments (1.5 vs. 0.5%, respectively; only one measurement exceeded 5%), indicating minimal risk for NO 2 - accumulation at our feeding level of NO 3 - . Although neither apparent organic matter nor neutral detergent fiber digestibilities were affected, apparent N digestibility had an interaction for NO 3 - × LYC such that apparent N digestibility was numerically lowest for diets containing both NO 3 - and LYC compared with the other 3 diets. Under the conditions of this study, NO 3 - mitigated ruminal methanogenesis but also depressed dry matter intake and milk protein yield. Based on the fact that few interactions were detected, LYC had a minimal role in attenuating negative cow responses to NO 3 - supplementation., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Diet composed of smaller particles can improve feed intake, digestibility, and animal growth or health, but in ruminant species can reduce rumination and buffering-the loss of which may inhibit fermentation and digestibility. However, the explicit effect of particle size on the rumen microbiota remains untested, despite their crucial role in digestion. We evaluated the effects of reduced particle size on rumen microbiota by feeding long-stem (loose) alfalfa hay compared to a ground and pelleted version of the same alfalfa in yearling sheep wethers during a two-week experimental period. In situ digestibility of the pelleted diet was greater at 48 h compared with loose hay; however, distribution of residual fecal particle sizes in sheep did not differ between the dietary treatments at any time point (day 7 or 14). Both average daily gain and feed efficiency were greater for the wethers consuming the pelleted diet. Observed bacterial richness was very low at the end of the adaptation period and increased over the course of the study, suggesting the rumen bacterial community was still in flux after two weeks of adaptation. The pelleted-hay diet group had a greater increase in bacterial richness, including common fibrolytic rumen inhabitants. The pelleted diet was positively associated with several Succiniclasticum, a Prevotella, and uncultured taxa in the Ruminococcaceae and Rickenellaceae families and Bacteroidales order. Pelleting an alfalfa hay diet for sheep does shift the rumen microbiome, though the interplay of diet particle size, retention and gastrointestinal transit time, microbial fermentative and hydrolytic activity, and host growth or health is still largely unexplored., Competing Interests: The authors have declared that no competing interests exist.

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH 4 emissions. In the NO 3 - reduction process, NO 2 - can accumulate, which could directly inhibit methanogens and possibly other microbes in the rumen. Saccharomyces cerevisiae yeast was hypothesized to decrease NO 2 - through direct reduction or indirectly by stimulating the bacterium Selenomonas ruminantium, which is among the ruminal bacteria most well characterized to reduce both NO 3 - and NO 2 - . Ruminal fluid was incubated in continuous cultures fed diets without or with NaNO 3 (1.5% of diet dry matter; i.e., 1.09% NO 3 - ) and without or with live yeast culture (LYC) fed at a recommended 0.010 g/d (scaled from cattle to fermentor intakes) in a 2 × 2 factorial arrangement of treatments. Treatments with LYC had increased NDF digestibility and acetate:propionate by increasing acetate molar proportion but tended to decrease total VFA production. The main effect of NO 3 - increased acetate:propionate by increasing acetate molar proportion; NO 3 - also decreased molar proportions of isobutyrate and butyrate. Both NO 3 - and LYC shifted bacterial community composition (based on relative sequence abundance of 16S rRNA genes). An interaction occurred such that NO 3 - decreased valerate molar proportion only when no LYC was added. Nitrate decreased daily CH 4 emissions by 29%. However, treatment × time interactions were present for both CH 4 and H 2 emission from the headspace; CH 4 was decreased by the main effect of NO 3 - until 6 h postfeeding, but NO 3 - and LYC decreased H 2 emission up to 4 h postfeeding. As expected, NO 3 - decreased methane emissions in continuous cultures; however, contrary to expectations, LYC did not attenuate NO 2 - accumulation., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH 4 emissions. In the NO 3 - reduction process, NO 2 - can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO 2 - accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO 3 - , NO 2 - , or NH 4 + replacing peptone in the medium. Gas head phase, NO 3 - , or NH 4 + did not influence culture growth, but increasing NO 2 - concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO 3 - , NO 2 - , or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO 2 - concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO 2 - and does not seem to be a viable mechanism to prevent NO 2 - accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO 2 - influencing signal transduction requires further research., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Supplements investigated throughout the present study are produced by fermenting lactose that is present in whey to lactate, yielding products differing in ammonium relative to lactate concentrations and in physical form (liquid or dry). Trials 1 and 2 investigated Lacto-Whey (LW; Fermented Nutrition Corp., Luxemburg, WI) and GlucoBoost (GB; Fermented Nutrition Corp.), respectively, using dual-flow continuous culture systems (n = 4), each with a 4 × 4 Latin square design. A greater proportion of nonprotein nitrogen was present in GB than in LW. In trial 1, the treatment with LW was isonitrogenously dosed against soybean meal (SBM) as a control (no LW) and factorialized with either a wheat- or corn-based concentrate (55% inclusion rate, dry matter basis). We hypothesized that LW would increase propionate production and that the combination of +LW with wheat would increase bacterial assimilation of NH 3 -N into cellular N. No differences were observed for total volatile fatty acid (VFA) production per day. However, treatment × time interactions revealed that +LW increased lactate concentration at 0, 0.5, and 1 h and tended to increase molar percentage of propionate at 1 and 1.5 h postfeeding, documenting the immediate availability of lactate converted to propionate in the +LW treatments. The main effect of corn increased the proportion of bacterial N derived from NH 3 -N. Trial 2 was designed to investigate GB; isonitrogenous treatments included an SBM control, crystal GB, liquid GB (LGB), and LGB with yeast culture, which were dosed twice daily. We hypothesized that GB would increase propionate production and bacterial assimilation of NH 3 -N; the combination of LGB and yeast culture was expected to have a positive additive effect, yielding the greatest VFA production and bacterial NH 3 -N assimilation. No differences were observed for total VFA production; however, LGB decreased molar percentage of acetate and increased propionate and butyrate molar percentages. There were no differences in non-NH 3 -N flow or microbial N flow. Under the conditions of our studies, lactate in LW and GB was fermented extensively to propionate, and microbial protein synthesis in these treatments was comparable with that in SBM controls., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to apply digital imaging to improve quantification of rumen protozoal biomass and distinguish treatment differences in cell motility and volume among ruminal protozoa. Observations of protozoa in rumen fluid treated with essential oils (CinnaGar, CIN; Provimi North America, Brookville, OH) or an ionophore (monensin, MON) indicated possible cell shrinkage. We hypothesized that MON would decrease protozoal motility and interact with CIN on cell volume. In addition, we hypothesized that analysis of still frames from video of swimming protozoa would improve volume prediction accuracy. Flocculated rumen fluid was incubated in batch culture dosed with N-free feed only (control), MON, CIN, or a combination of MON+CIN. Samples were taken at 0, 3, or 6 h post-treatment and wet-mounted on a microscope fitted with a high-definition camera. At 3 h post-inoculation, there was a treatment interaction for average speed such that CIN attenuated the effect of MON, with treatment means of 243, 138, 211, and 183 µm/s for control, MON, CIN, and MON+CIN, respectively. At 6 h post-inoculation, MON decreased average speed by 79.2 µm/s compared with the main effect mean without MON. We measured both minimum and maximum diameters (depth and width, respectively) perpendicular to the longitudinal axis of swimming protozoa, yielding a 3-dimensional estimate of protozoal volume. The ellipsoid formula (4/3)πabc, where a = 1/2 length, b = 1/2 width, and c = 1/2 depth, was compared with previously published volume estimations using genera-specific coefficients (genera-specific coefficient × length × width 2 ). Residuals (genera-specific coefficients - ellipsoid) were plotted against predicted (ellipsoid) and centered to the mean (X i -x¯) to evaluate both mean and slope biases. For Entodinium spp., Y = 0.248 (±0.037) (X i - 7.98 × 10 4 ) + 1.97 × 10 4 (±1.48 × 10 3 ); n = 100; r 2 [coefficient of determination (squared correlation coefficient)] = 0.31, with significant slope and mean biases. For family Isotrichidae, Y = -0.124 (±0.068) (X i - 2.54 × 10 6 ) - 1.21 × 10 4 (±4.86 × 10 4 ); n = 32; r 2 = 0.10, where slope tended to be different from zero but with no mean bias. For Epidinium spp., Y = 0.375 (±0.056) (X i - 2.45 × 10 5 ) + 6.65 × 10 4 (±0.28 × 10 4 ); n = 64; r 2 = 0.43, with both mean and slope biases. The present regression analyses demonstrate that the genera-specific coefficient-based method more likely overestimates volume for Entodinium and Epidinium than for the teardrop-shaped Isotrichidae. Based on simulations derived from previous literature reporting treatments that depress protozoal populations or among-animal changes in protozoal population structures, our proposed ellipsoid method offers potential to advance the prediction of treatment effects on protozoal volume and to shift focus from the number of cells present to the diversity, function, and biomass of protozoa under various treatment conditions., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effects of altering pH and solids passage rate (k p ) on concentration of aqueous H 2 [H 2 (aq)], CH 4 production, volatile fatty acids (VFA) production, and fiber digestibility in a continuous culture fermentation system. The present study was conducted as a 2 × 2 factorial treatment arrangement in a Latin square design using continuous culture fermentors (n = 4). Our continuous culture system was converted to a closed system to measure CH 4 and H 2 emission while measuring H 2 (aq) concentration and VFA production for complete stoichiometric assessment of fermentation pattern. Treatments were control pH (CpH; ranging from 6.3 to 6.9) or low pH (LpH; 5.8 to 6.4) factorialized with solids k p that was adjusted to be either low (Lk p ; 2.5%/h) or high (Hk p ; 5.0%/h); liquid dilution was maintained at 7.0%/h. Fermentors were fed once daily (40 g of dry matter; 50:50 concentrate:forage diet). Four periods lasted 10 d each, with 3 d of sample collection. The main effect of LpH increased nonammonia nitrogen flow, and both LpH and Hk p increased nonammonia nonbacterial N flow. We observed a tendency for Hk p to increase bacterial N flow per unit of nonstructural carbohydrates and neutral detergent fiber degraded. The main effect of LpH decreased H 2 (aq) by 4.33 µM compared with CpH. The main effect of LpH decreased CH 4 production rate from 5 to 9 h postfeeding, and Hk p decreased CH 4 production rate from 3 to 9 h postfeeding. We found no effect of LpH on daily CH 4 production or CH 4 produced per gram of neutral detergent fiber degraded, but Hk p decreased daily CH 4 production by 33.2%. Both the main effects of LpH and Hk p decreased acetate molar percentage compared with CpH and Lk p , respectively. The main effect of both LpH and Hk p increased propionate molar percentage, decreasing acetate-to-propionate ratio from 2.62 to 2.34. We noted no treatment effects on butyrate molar percentage or total VFA production. The results indicate increasing k p and decreasing pH decreased acetate-to-propionate ratio, but only increasing k p decreased CH 4 production; lack of differences for LpH might be a result of compensatory methanogenesis during the second half of the day postfeeding., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Appropriate trace mineral supplementation can improve immune response and hoof health in cattle and at much higher rates of supplementation to swine and poultry can alter microbial colonization of the gut, resulting in improved gut health. Diet can influence fecal microbial excretion in cattle, and the fecal microbiome may serve as a means for assessing gastrointestinal microbial changes. We hypothesized that feeding diets that differed in source of supplemental Cu, Zn, or Mn would alter the relative abundance of fecal microbes in lactating dairy cattle and that organic Zn would have the greatest effect. Twenty-four cows were fed diets devoid of supplemental Cu, Zn, and Mn for a 16-d preliminary phase (basal diet provided 9, 29, and 32 mg/kg of Cu, Zn, and Mn, respectively), and then were randomly assigned to 1 of 3 treatment diets (n = 8 cows/treatment): one group of cows was fed supplemental Cu, Zn, and Mn from sulfate minerals; the second group was fed glycinate minerals; and the third group was fed Cu and Mn sulfate with glycinate Zn. Assayed total dietary concentrations were approximately 21, 73, and 72 mg/kg for Cu, Zn, and Mn, respectively. Milk production (averaged 38.8 kg/d), DMI (averaged 25.8 kg/d), and milk components were not affected by treatment. Fecal DNA was extracted, amplified using a universal primer targeting the V4-V5 hypervariable region of the 16S rRNA gene, and sequenced to compare microbial community composition between treatments. Relative abundances of Treponema species-level operational taxonomic units (OTU) were less for animals fed Cu and Mn sulfate with glycinate Zn compared with sulfates alone, but were similar to animals fed glycinate mineral sources. Relative abundances for exclusive glycinate mineral and sulfate mineral treatments were similar. Treponema OTU and cultured representatives are often associated with bovine digital dermatitis. These data may provide an additional link between organic Zn supplementation and improved hoof health. To our knowledge this is the first report of a dietary treatment decreasing the relative abundance of Treponema OTU in cattle feces; however, the potential benefits of this response on overall animal health and the mechanism for the observed responses are unknown and warrant further investigation., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The present study was conducted as a 3 × 2 factorial experiment in 7 pen replications conducted using progeny (n = 360) of successive farrowings of 1 Landrace breeding female population. Grower-finisher dietary treatments included the addition of Bio-Mos (BM; at 0.2, 0.1 and 0.05% inclusion rates for phase 1 (30.2 to 63.5 kg), 2 (63.5 to 90.5 kg), and 3 (90.5 to 113.6 kg), respectively), the inclusion of a subtherapeutic antibiotic (AB; tetracycline; at 0.0055% inclusion rate in all dietary phases) and a no additive control (CON) diet. Housing systems were a conventional, indoor (IN) facility providing 1.0 m(2)/pig solid concrete and 0.3 m(2)/pig slatted floor with 12 pigs per pen and an outdoor access (OUT) system providing 1.1 m(2)/pig indoor, bedded concrete, and 1.9 m(2)/pig outdoor solid concrete with 6 pigs per pen. Housing systems analyses acknowledge confounding of space with number of pigs per pen. Daily growth rate (ADG), feed intake (ADFI), feed conversion (G:F), ultrasonic carcass composition, blood hematocrit (1 group), and observed illnesses were measured. Dietary × housing treatment interactions were not observed. Pigs reared in OUT had greater ADFI (0.1 kg/d; P = 0.01) resulting in greater ADG (0.04 kg/d; P < 0.0005) and required fewer days to reach a standard 113.6 kg endpoint (4.0 d; P < 0.0005) but had reduced (poorer) G:F (0.01 kg gain/kg feed; P = 0.05) when compared to IN. Pigs fed BM and CON diets had greater ADG (0.02 kg/d; P < 0.05) and required 3 fewer days to 113.6 kg (P < 0.05) when compared with pigs fed AB. Carcass composition measures were not different across dietary or housing treatments. Hematocrit was 2 units greater (P < 0.05) at the end of the trial (d 84) for OUT housed pigs but not different at the start, d 28, or d 56 of the trial. In the present study, the addition of a subtherapeutic antibiotic in swine finisher diets did not improve pig growth, efficiency, or health whereas the addition of BM did not increase growth performance compared to the CON diet in both housing systems, a finding suggesting the potential for improved gut health and or improved appetite in pigs not fed an antibiotic. Whereas pigs reared OUT had greater growth rate and a more desirable hematocrit level, the observed differences may be attributed to stocking density, number of pigs per pen, or outdoor access, effects that are not able to be fully described under the experimental design.