The purpose of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), a potent methane inhibitor, on total and metabolically active methanogens in the rumen of dairy cows over the course of the day and over a 12-wk period. Rumen contents of 8 ruminally cannulated early-lactation dairy cows were sampled at 2, 6, and 10 h after feeding during wk 4, 8, and 12 of a randomized complete block design experiment in which 3-NOP was fed at 60 mg/kg of feed dry matter. Cows (4 fed the control and 4 fed the 3-NOP diet) were blocked based on their previous lactation milk yield or predicted milk yield. Rumen samples were extracted for microbial DNA (total) and microbial RNA (metabolically active), PCR amplified for the 16S rRNA gene of archaea, sequenced on an Illumina platform, and analyzed for archaea diversity. In addition, the 16S copy number and 3 ruminal methanogenic species were quantified using the real-time quantitative PCR assay. We detected a difference between DNA and RNA (cDNA)-based archaea communities, revealing that ruminal methanogens differ in their metabolic activities. Within DNA and cDNA components, methanogenic communities differed by sampling hour, week, and treatment. Overall, Methanobrevibacter was the dominant genus (94.3%) followed by Methanosphaera, with the latter genus having greater abundance in the cDNA component (14.5%) compared with total populations (5.5%). Methanosphaera was higher at 2 h after feeding, whereas Methanobrevibacter increased at 6 and 10 h in both groups, showing diurnal patterns among individual methanogenic lineages. Methanobrevibacter was reduced at wk 4, whereas Methanosphaera was reduced at wk 8 and 12 in cows supplemented with 3-NOP compared with control cows, suggesting differential responses among methanogens to 3-NOP. A reduction in Methanobrevibacter ruminantium in all 3-NOP samples from wk 8 was confirmed using real-time quantitative PCR. The relative abundance of individual methanogens was driven by a combination of dietary composition, dry matter intake, and hydrogen concentrations in the rumen. This study provides novel information on the effects of 3-NOP on individual methanogenic lineages, but further studies are needed to understand temporal dynamics and to validate the effects of 3-NOP on individual lineages of ruminal methanogens.

Asparagopsis taxiformis (AT) is a source of multiple halogenated compounds and, in a limited number of studies, has been shown to decrease enteric CH4 emission in vitro and in vivo. Similarly, oregano has been suggested as a potential CH4 mitigating agent. This study consisted of 2 in vitro and 2 in vivo experiments. Experiment (Exp.) 1 was aimed at establishing the effect of AT on CH4 emission in vitro. Two experiments (Exp. 2 and 3) with lactating dairy cows were conducted to determine the antimethanogenic effect of AT and oregano (Exp. 3) in vivo. Another experiment (Exp. 4) was designed to investigate stability of bromoform (CHBr3) in AT over time. In Exp. 3, 20 Holstein cows were used in a replicated 4 × 4 Latin square design with four 28-d periods. Treatments were basal diet (control) or basal diet supplemented with (dry matter basis) 0.25% AT (LowAT), 0.50% AT (HighAT), or 1.77% oregano (Origanum vulgare L.) leaves. Enteric gas emissions were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD), and rumen samples were collected for fermentation analysis using the ororuminal technique. In Exp.1 (in vitro), relative to the control, AT (at 1% dry matter basis, inclusion rate) decreased CH4 yield by 98%. In Exp. 3, HighAT decreased average daily CH4 emission and CH4 yield by 65% and 55%, respectively, in experimental periods 1 and 2, but had no effect in periods 3 and 4. The differential response to AT among experimental periods was likely a result of a decrease in CHBr3 concentration in AT over time, as observed in Exp. 4 (up to 84% decrease in 4 mo of storage). In Exp. 3, H2 emission was increased by AT and, as expected, the proportion of acetate in the total volatile fatty acids in the rumen was decreased and those of propionate and butyrate were increased by HighAT compared with the control. Compared with the control, HighAT decreased dry matter intake, milk yield, and energy-corrected milk yield in Exp. 3. Milk composition was not affected by treatment, except lactose percentage and yield were decreased by HighAT. Concentrations of iodine and bromide in milk were increased by HighAT compared with the control. Milk CHBr3 concentration and its organoleptic characteristics were not different between control and HighAT. Oregano had no effect on CH4 emission or lactational performance of the cows in Exp. 3. Overall, AT included at 0.50% in the ration of dairy cows can have a large mitigation effect on enteric CH4 emission, but dry matter intake and milk production may also decrease. There was a marked decrease in the CH4 mitigation potential of AT in the second half of Exp. 3, likely resulting from CHBr3 decay over time.

T137 Effects of Ascophyllum nodosum meal and monensin on ruminal fermentation and microbiota. L. H. P. Silva*1, A. T. O. Melo1, S. F. Reis2, B. P. Jackson3, F. Evans4, and A. F. Brito1, 1University of New Hampshire, Durham, NH, 2Faculdade de Ciências Biomédicas de Cacoal, Cacoal, RO, Brazil, 3Dartmouth College, Hanover, NH, 4Acadian Seaplants Ltd, Dartmouth, NS, Canada. Ascophyllum nodosum meal (ASCO) is known to contain high concentrations of phlorotannins and iodine, which can impact the ruminal microbiota. However, there are few studies that have evaluated how ASCO affect ruminal microbiota. Five ruminally-cannulated Jersey cows were used in a 5 × 5 Latin square design to evaluate the effects of incremental levels of ASCO (0, 57, 113, and 170 g/d) or monensin (MON; 300 mg/d) on ruminal fermentation and microbiota. Each experimental period lasted 28 d with 21 d for diet adaptation and 7 d for sample collection. Cows were housed in a freestall equipped with Calan doors. A basal diet with a 65:35 forage:concentrate ratio (DM basis) was offered twice daily. Ruminal fluid was collected 3 h after the morning feeding for 3 consecutive days (d 23–25) of each sampling period and pooled for protozoa count, total bacteria, and microbiota analysis. Ruminal fluid was also collected at 0, 1, 2, 4, 6, and 8 h after feeding on the d 27 for ruminal pH, NH3-N, and VFA analysis. Total bacteria were quantified by RT-qPCR. Microbiota analysis was performed on a MiSeq platform and processed using QIIME2. All data were checked for normality by Shapiro-Wilk test, and when necessary log-transformed. Data were analyzed in SAS following a 5 × 5 Latin square design with repeated measurements for ruminal pH, NH3-N, and VFA. Contrasts were used to 1) compare linear and 2) quadratic effects of ASCO levels, 3) ASCO vs. MON, and 4) control vs. MON. Ruminal pH and NH3-N were not affected by treatments. Total VFA concentration in the rumen was not affected by MON but decreased linearly with ASCO supplementation (P = 0.02). Ruminal butyrate and the acetate:propionate ratio showed quadratic responses to ASCO (P < 0.05). Protozoa count and total bacteria were not changed by treatments, as well as bacterial and archaeal diversity. The protozoal Simpson diversity index and the protozoal genera Entodinium and Isotricha reduced linearly with incremental levels of ASCO (P < 0.05). Overall, ASCO did not affected protozoa count and total bacteria but reduced protozoal diversity with minimal effect on ruminal fermentation parameters.

Rumen microbes play an important role in the conversion of indigestible plant material to energy and protein in dairy cows. Sampling for ruminal contents via cannula is considered the gold standard technique for microbial analysis, but the technique requires ruminally cannulated animals and specialized animal facilities. The purpose of this study was to determine whether other sampling methods and locations along the digestive tract may serve as noninvasive proxies to the cannula method for microbial analysis. Six ruminally cannulated lactating Holstein dairy cows were adapted to a standard total mixed ration for 2 wk and sampled during the third week. Sampling locations and methods included salivary content, rumination bolus (regurgitated digesta collected from the cow's mouth), feces, and rumen contents via stomach tube and cannula. Stomach tube and cannula samples differ in proportions of solid and liquid material and were therefore separated into whole (as collected), liquid, and solid fractions. Samples were collected at 0 (before feeding), 2, 4, 6, 8, and 12 h after feeding over 2 d. All samples were extracted for total genomic DNA and selected samples for metabolically active DNA (RNA), PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, and analyzed for bacterial diversity using the QIIME2 pipeline followed by statistical analysis in R (https://www.R-project.org/). In DNA-based analysis, at the community level, saliva, rumination bolus, and fecal samples clustered in separate groups, whereas all fractions of stomach tube and cannula samples clustered together, indicating that microbial communities of stomach tube and cannula samples were homogeneous. Rumination bolus samples at 6, 8, and 12 h after feeding clustered with stomach tube and cannula samples, indicating that rumination bolus samples may be an alternative for cannula samples; however, time of sampling is critical for sampling of bolus digesta. Results of the RNA-based analysis of rumination bolus samples and solid samples from cannula and stomach tube at 0 and 6 h after feeding were similar. We concluded that the solid fraction of samples obtained via the stomach tube method may serve as a proxy for the solid fraction of whole ruminal contents obtained via cannula for DNA-based microbial investigations. Both rumination bolus and stomach tube solid samples may serve as proxies for cannula solid samples for RNA-based microbial analysis.

In response to the need for updated and affordable dual-flow continuous culture fermenter systems within the scientific community, modifications were made to the classic design for new construction. These modifications are summarized in 4 categories. First, simplification and performance optimization of the base unit with updated electrical components, modernized gearing, and self-calibrating thermostats. Second, changes to the shape of the fermenter jar to decrease construction cost and improve anaerobic stability. Third, extended reach and angle to the stirring mechanism for improved mixing and solids turnover. And lastly, simplification of a 2-stage filter system to reduce failure rate. Our hypothesis was that fermenter experiments with the updated fermenter system would decrease variance estimates for common dependent variables: NDF digestibility, VFA production, ammonia N flow, and bacterial N flow. To test this hypothesis, data were collected from 3 published fermenter experiments (n = 48) utilizing the old system and 2 fermenter experiments with the updated units (n = 48). Experiments utilized common diets, dilution rates, feeding rates, and sampling times. Variances were calculated within treatment, then pooled to provide a ratio of new versus old system variances for the 4 dependent variables. An F-test was conducted on the new:old variance ratio and a significant decrease in experimental variance was declared at P < 0.05. Updated fermenter systems decreased (P < 0.01) variance of NDFd estimates by 55% and decreased (P < 0.01) variance of bacterial N flow estimates by 55% compared with the original fermenter system. However, variance for VFA production was increased (P < 0.01) by 279% and variance for ammonia N flow tended to be increased (P = 0.09) by 46% in new versus old fermenter units. These data indicate the potential for new, more affordable dual-flow continuous culture systems to improve reliability of fiber digestibility and microbial growth research but more work is needed to reduce variance of concentration-based fermentation parameters.

BackgroundThe faecal microbiota is emerging as potentially important in intestinal disease. More research is needed to characterise the faecal microbiota from horses with colic. ObjectivesTo compare the relative abundance of bacterial populations comprising the faecal microbiota in horses presenting for colic compared with an elective surgical procedure. Study designProspective observational clinical study. MethodsAdmission faecal samples were collected from horses presenting for colic and elective surgical procedures. Faecal samples were extracted for genomic DNA, PCR- amplified, sequenced and analysed using QIIME. Species richness and Shannon diversity were estimated for each faecal sample. The extent of the relationship between bacterial communities (beta diversity) was quantified using pairwise UniFrac distances, visualised using principal coordinate analysis (PCoA) and statistically analysed using PERMANOVA. The relative abundance of bacterial populations between the two treatment groups were compared using ANCOM. ResultsFaecal bacterial communities in horses presenting for colic had fewer species (P<0.001) and lower diversity (P<0.001) compared with horses presenting for elective surgical procedures. Based on the PERMANOVA analysis, there was a significant difference in the bacterial community composition between horses admitted for colic vs. elective procedures (P = 0.001). Based on ANCOM test, at the genus level, 14 bacterial lineages differed between the two groups. The relative abundance of known commensal bacteria including Prevotella, Clostridia, Lachnospiraceae were reduced whereas Christenellaceae, Streptococcus and Sphaerochaeta were increased in horses with colic when compared with elective cases. Main limitationsRelative low numbers and a diverse population of horses. ConclusionsChanges in bacterial populations in the faecal microbiota of horses presenting for colic observed in this study concurs with previous studies in veterinary and human patients with gastrointestinal disease. Future studies focusing on different causes of colic, chronic or recurrent disease, and the association with histological changes within the intestine are needed. The Summary is available in Portuguese - see Supporting Information

T193 Partial replacement of maize meal and molasses for cracked maize in supplements of grazing dual-purpose cows during the dry season. I. G. Salas-Reyes1,2, C. M. Arriaga-Jordán1,3, A. García-Martínez1,2, J. G. Estrada-Flores1,3, B. Albarrán-Portillo*1,2, and R. Rojo-Rubio1,2, 1Universidad Autónoma del Estado de México, Toluca, México, 2Centro Universitario UAEM Temascaltepec, Temascaltepec, México, 3Instituto de Investigación en Ciencias Agropecuarias y Rurales, Toluca, México.

Ten ruminally cannulated Holstein cows were used in a crossover design that investigated changes in ruminal bacterial populations in response to induction and recovery from diet-induced milk fat depression (MFD). Further, the effect on the ruminal microbiota of the cows with diet-induced milk fat depression inoculated with rumen contents from non-milk fat-depressed donor cows was evaluated. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber, high-starch, and high-polyunsaturated fatty acid diet (26.1% neutral detergent fiber, 28.1% starch, 5.8% total fatty acids, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. Induction was followed by a recovery phase, where all cows were switched to a high-fiber, low-starch, and low-polyunsaturated fatty acid diet (31.8% neutral detergent fiber, 23% starch, 4.2% total fatty acids, and 1.2% C18:2) and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Ruminal samples were collected at the end of induction (d 10) and during recovery (d 13, 16, and 28), separated to solid and liquid fractions, extracted for DNA, PCR- amplified for the V1-V2 region of the 16S rRNA gene, and analyzed for bacterial diversity. Results indicated that bacterial communities were different between fractions. In each fraction, differences were significant between the induction (d 10) and recovery (d 13, 16, and 28) periods; however, differences were less apparent with time during the recovery period. The MFD (d 10) was typified by a reduction in the relative sequence abundance of Bacteroidetes and an increase in the relative sequence abundance of Firmicutes and Actinobacteria across both fractions. At the genus level, relative sequence abundance of unclassified Lachnospiraceae, Butyrivibrio, Bulleidia, and Coriobacteriaceae were higher on d 10 and were positively correlated with trans-10,cis-12 CLA and the trans-10 isomer, suggesting their potential role in altered biohydrogenation reactions. A switch to the recovery diet resulted in a sharp increase in the Bacteroidetes lineages and a decrease in Firmicutes members on d 13; however, this shift appears to stabilize by d 28, indicating the restoration process for ruminal bacteria from an altered state is gradual and complex. Inoculation of 10% of rumen contents from non-MFD donor cows to MFD cows revealed this procedure had transient effects on only a few bacterial populations, and such effects disappeared after d 16 following cessation of inoculation. It can be concluded that alterations in milk FA profiles at induction are preceded by microbial alterations in the rumen driven by dietary changes.

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

Frothy bloat is a serious metabolic disorder that affects stocker cattle grazing hard red winter wheat forage in the Southern Great Plains causing reduced performance, morbidity, and mortality. We hypothesize that a microbial dysbiosis develops in the rumen microbiome of stocker cattle when grazing on high quality winter wheat pasture that predisposes them to frothy bloat risk. In this study, rumen contents were harvested from six cannulated steers grazing hard red winter wheat (three with bloat score "2" and three with bloat score "0"), extracted for genomic DNA and subjected to 16S rDNA and shotgun sequencing on 454/Roche platform. Approximately 1.5 million reads were sequenced, assembled and assigned for phylogenetic and functional annotations. Bacteria predominated up to 84% of the sequences while archaea contributed to nearly 5% of the sequences. The abundance of archaea was higher in bloated animals (P < 0.05) and dominated by Methanobrevibacter. Predominant bacterial phyla were Firmicutes (65%), Actinobacteria (13%), Bacteroidetes (10%), and Proteobacteria (6%) across all samples. Genera from Firmicutes such as Clostridium, Eubacterium, and Butyrivibrio increased (P < 0.05) while Prevotella from Bacteroidetes decreased in bloated samples. Co-occurrence analysis revealed syntrophic associations between bacteria and archaea in non-bloated samples, however; such interactions faded in bloated samples. Functional annotations of assembled reads to Subsystems database revealed the abundance of several metabolic pathways, with carbohydrate and protein metabolism well represented. Assignment of contigs to CaZy database revealed a greater diversity of Glycosyl Hydrolases dominated by oligosaccharide breaking enzymes (>70%) in non-bloated samples. However, the abundance and diversity of CaZymes were greatly reduced in bloated samples indicating the disruption of carbohydrate metabolism. We conclude that mild to moderate frothy bloat results from tradeoffs both within and between microbial domains due to greater competition for substrates that are of limited availability as a result of biofilm formation.