Influence of empirically derived filtering parameters, ASV, and OTU pipelines on assessing rumen microbial diversity.

Microbes play an important role in human and animal health as well as animal productivity. The host microbial interactions within ruminants play a critical role in animal health and productivity and provide up to 70% of the animal's energy need in the form of fermentation products. As such, many studies have investigated microbial community composition to understand microbial community changes and factors that affect microbial colonization and persistence. The advances in next generation sequencing (NGS) technologies and low cost of sequencing have gravitated many studies to utilize 16S rDNA-based analysis tools for interrogation of microbiomes at a much finer scale than traditional culturing. However, such methods that rely on single base pair differences for bacterial taxa clustering may inflate or underestimate diversity leading to inaccurate identification of bacterial diversity. Therefore, in this study, we sequenced mock communities of known membership and abundance to establish filtration parameters to reduce inflation of microbial diversity due to PCR and sequencing errors. Additionally, we evaluated the effect of the resulting filtering parameters proposed using established bioinformatic pipelines on a study consisting of Holstein and Jersey cattle to identify bread and treatment effects on the bacterial community composition and the impact of the filtering on global microbial community structure analysis and results. Filtration resulted in a sharp reduction in bacterial taxa identified, yet retain most sequencing data (retaining > 79% of sequencing reads) when analyzed using 3 different microbial analysis pipelines (DADA2, Mothur, USEARCH). After filtration, conclusions from α and ß-diversity tests show very similar results across all analysis methods. The mock community-based filtering parameters proposed in this study help provide a more realistic estimation of bacterial diversity. Additionally, the filtration reduced the variation between microbiome analysis methods and help identify microbial community differences that could have been missed due to large animal to animal variation observed in the unfiltered data. As such, we believe, the new filtering parameters described in this study will help obtain diversity estimates closer to realistic values and will improve the ability of detecting microbial community differences and help better understand microbial community changes in 16S rDNA-based studies.

The effects of 2-hydroxy-4-methylthio-butanoic acid supplementation on the rumen microbial population and duodenal flow of microbial nitrogen.

Four multiparous, lactating Holstein cows (average DIM 169.5 ± 20.5 d), fitted with ruminal and duodenal cannulas, were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments to investigate the effects of 2-hydroxy-4-methylthio-butanoic acid (HMTBA) when fed with diets differing in metabolizable protein (MP) supply and equal levels of crude protein on milk production and composition, rumen microbial activity, duodenal protein flow, and rumen bacterial community composition in vivo and in vitro. Experimental periods were 28 d in length. Cows were housed in individual tie stalls and were randomly assigned to 4 dietary treatments: low MP or high MP, supplemented with or without 25 g of HMTBA, which was top-dressed once daily at 0930 h. No interactions were observed between HMTBA and level of dietary MP, with the exception of ruminal acetate-to-propionate ratio. Milk yield was not affected by treatment and averaged 23.8 ± 2.06 kg/d. There was a tendency for increased milk protein percent in cows receiving low MP diets, averaging 3.30 ± 0.09% and 3.21 ± 0.09% for low MP and high MP, respectively. The total-tract apparent digestibility of organic matter, neutral detergent fiber, and nitrogen were greater in cows consuming the low MP diet. Rumen pH was lower in cows consuming high MP diets as well as in those consuming HMTBA. Rumen ammonia concentrations tended to be greater in cows consuming HMTBA, and volatile fatty acid concentrations were greater in cows consuming HMTBA. Duodenal dry matter flow, nitrogen flow, and microbial nitrogen flow did not differ between treatments. The bacterial community structure of cows receiving HMTBA was not affected at the phylum level. The relative abundance of bacterial phyla in vivo differed when compared with in vitro conditions for Firmicutes, Bacteroidetes, Proteobacteria, TM7, Tenericutes, Spirochaetes, SR1, and Verrucomicrobia.

Increasing the concentration of linolenic acid in diets fed to Jersey cows in late lactation does not affect methane production.

Although the inclusion of fat has reduced methane production in ruminants, relatively little research has been conducted comparing the effects of source and profile of fatty acids on methane production in lactating dairy cows. A study using 8 multiparous (325 ± 17 DIM; mean ± SD) lactating Jersey cows was conducted to determine effects of feeding canola meal and lard versus extruded byproduct containing flaxseed as a high-C18:3 fat source on methane production and diet digestibility in late-lactation dairy cows. A crossover design with 32-d periods (28-d adaptation and 4-d collections) was used to compare 2 different fat sources. Diets contained approximately 50% forage mixture of corn silage, alfalfa hay, and brome hay; the concentrate mixture changed between diets to include either (1) a conventional diet of corn, soybean meal, and canola meal with lard (control) or (2) a conventional diet of corn and soybean meal with an extruded byproduct containing flaxseed (EXF) as the fat source. Diets were balanced to decrease corn, lard, and canola meal and replace them with soybean mean and EXF to increase the concentration of C18:3 (0.14 vs. 1.20% of DM). Methane production was measured using headbox-style indirect calorimeters. Cattle were restricted to 95% ad libitum feed intake during collections. Milk production (17.4 ± 1.04 kg/d) and dry matter intake (15.4 ± 0.71 kg/d) were similar among treatments. Milk fat (5.88 ± 0.25%) and protein (4.08 ± 0.14%) were not affected by treatment. For methane production, no difference was observed for total production (352.0 vs. 349.8 ± 16.43 L/d for control vs. EXF, respectively). Methane production per unit of dry matter intake was not affected and averaged 23.1 ± 0.57 L/kg. Similarly, methane production per unit of energy-corrected milk was not affected by fat source and averaged 15.5 ± 0.68 L/kg. Heat production was similar, averaging 21.1 ± 1.02 Mcal/d. Digestibility of organic matter, neutral detergent fiber, and crude protein was not affected by diet and averaged 69.9, 53.6, and 73.3%, respectively. Results indicated that increasing C18:3 may not affect methane production or digestibility of the diet in lactating dairy cows.

Reducing methane production with corn oil and calcium sulfate: Responses on whole-animal energy and nitrogen balance in dairy cattle.

The addition of fat and calcium sulfate to diets fed to ruminants has resulted in a reduction in methane production, but the effects on energy balance have not been studied. A study using indirect calorimetry and 16 multiparous (8 Holstein and 8 Jersey; 78 ± 15 d in milk; mean ± standard deviation) lactating dairy cows was conducted to determine how mitigating methane production by adding corn oil or calcium sulfate to diets containing reduced-fat distillers grains affects energy and nitrogen balance. A replicated 4 × 4 Latin square design with 35-d periods (28 d of adaption and 4 d of collections) was used to compare 4 different dietary treatments. Treatments were composed of a control (CON) diet, which did not contain reduced-fat distillers grain and solubles (DDGS), and treatment diets containing 20% (dry matter basis) DDGS (DG), 20% DDGS with 1.38% (dry matter basis) added corn oil (CO), and 20% DDGS with 0.93% (dry matter basis) added calcium sulfate (CaS). Compared with CON, dry matter intake was not affected by treatment, averaging 29.6 ± 0.67 kg/d. Milk production was increased for diets containing DDGS compared with CON (26.3 vs. 27.8 ± 0.47 kg/d for CON vs. DDGS, respectively), likely supported by increased energy intake. Compared with CON, energy-corrected milk was greater in DG and CO (30.1 vs. 31.4, 31.7, and 31.0 ± 0.67 kg/d for CON, DG, CO, and CaS, respectively). Compared with CON, the addition of calcium sulfate and corn oil to diets containing DDGS reduced methane production per kg of dry matter intake (22.3, 19.9, and 19.6 ± 0.75 L/kg per d for CON, CO, and CaS, respectively). Similarly, methane production per kilogram of energy-corrected milk was reduced with the addition of calcium sulfate and corn oil to diets containing DDGS (14.2, 12.5, and 12.4 ± 0.50 L/kg per d for CON, CO, and CaS, respectively). Compared with CON and CaS, the intake of digestible energy was greater for DG and CO treatments (57.7, 62.1, 62.0, and 59.0 ± 1.38 Mcal/d for CON, DG, CO, and CaS, respectively). Intake of metabolizable energy was greater in all treatments containing DDGS compared with CON (50.5 vs. 54.0 ± 1.08 Mcal/d for CON vs. DDGS, respectively). Net balance (milk plus tissue energy) per unit of dry matter was greater in CO (containing DDGS and oil) than CON (1.55 vs. 1.35 ± 0.06 Mcal/kg for CO vs. CON, respectively). Tissue energy was greater in DG and CO compared with CON (6.08, 7.04, and 3.16 ± 0.99 Mcal/d for DG, CO, and CON, respectively. Results of this study suggest that the addition of oil and calcium sulfate to diets containing DDGS may be a viable option to reduce methane production and in the case of oil also improve net energy balance in lactating dairy cows.

Energy balance and diurnal variation in methane production as affected by feeding frequency in Jersey cows in late lactation.

Methane (CH4) production of ruminants typically increases with increased dry matter intake (DMI). However, few studies have observed the effects of feeding multiple times a day and its effects on diurnal variation in CH4 production and energy balance in late-lactation dairy cattle. A study using headbox-style indirect calorimetry and 12 multiparous (225 ± 16.2 d in milk; mean ± SD) lactating Jersey cows was conducted to determine the effects of feeding twice daily on diurnal variation in CH4 production and total energy balance. A crossover design with 14-d periods (10 d of adaption and 4 d of collection) was used to compare 2 treatments. Treatments consisted of either once a day feeding (1×; 100% of feed given at 1000 h) or twice a day feeding (2×; 50% of feed given at 1000 h and the final 50% at 2000 h) with a common diet fed in both treatments. Dry matter intake was not different between treatments, with a mean of 16.9 ± 0.88 kg/d. Once a day feeding tended to have greater milk yield compared with twice a day feeding (21.2 vs. 20.4 ± 1.59 kg/d, respectively). Milk fat and milk protein percentage were not different, with means of 6.18 ± 0.20% and 3.98 ± 0.08%, respectively. Total CH4 production did not differ between treatments, with a mean of 402.1 ± 20.8 L/d. Similarly, CH4 per unit of milk yield and DMI was not different between treatments, with means of 20.5 ± 1.81 and 23.8 ± 1.21 L/kg, respectively. Feeding frequency did not affect diurnal variation of hourly CH4 production, with a mean of 17.1 ± 0.74 L/h. A trend was observed for a treatment × hour interaction. Methane production per hour increased after the second feeding for cattle fed twice versus once daily. Gross energy, digestible energy, metabolizable energy, and balance (milk plus tissue) per kilogram of DMI did not differ by feeding frequency, with means of 4.41 ± 0.01, 3.05 ± 0.03, 2.63 ± 0.03, and 1.32 ± 0.08 Mcal/kg of DM, respectively. Metabolizable energy for maintenance was 146 kcal/kg of metabolic body weight, with an efficiency of converting metabolizable energy to net energy balance (milk plus tissue) of 76%. Nitrogen balance did not differ among treatments, with a mean balance of 17.3 ± 13.0 g/d. Therefore, total CH4 production and energy maintenance were not affected by feeding frequency. However, CH4 was variable throughout the day, and caution should be exercised when collecting CH4 samples at a limited number of time points because this may under- or overestimate total production.

The influence of fat and hemicellulose on methane production and energy utilization in lactating Jersey cattle.

Feeding fat to lactating dairy cows may reduce methane production. Relative to cellulose, fermentation of hemicellulose is believed to result in less methane; however, these factors have not been studied simultaneously. Eight multiparous, lactating Jersey cows averaging (±SD) 98 ± 30.8 d in milk and body weight of 439.3 ± 56.7 kg were used in a twice-replicated 4 × 4 Latin square to determine the effects of fat and hemicellulose on energy utilization and methane production using a headbox-type indirect calorimetry method. To manipulate the concentration of fat, porcine tallow was included at either 0 or 2% of the diet dry matter. The concentration of hemicellulose was adjusted by manipulating the inclusion rate of corn silage, alfalfa hay, and soybean hulls resulting in either 11.3 or 12.7% hemicellulose (dry matter basis). The resulting factorial arrangement of treatments were low fat low hemicellulose (LFLH), low fat high hemicellulose (LFHH), high fat low hemicellulose (HFLH), and high fat high hemicellulose (HFHH). Neither fat nor hemicellulose affected dry matter intake, averaging 16.2 ± 1.18 kg/d across treatments. Likewise, treatments did not affect milk production, averaging 23.0 ± 1.72 kg/d, or energy-corrected milk, averaging 30.1 ± 2.41 kg/d. The inclusion of fat tended to reduce methane produced per kilogram of dry matter intake from 24.9 to 23.1 ± 1.59 L/kg, whereas hemicellulose had no effect. Increasing hemicellulose increased neutral detergent fiber (NDF) digestibility from 43.0 to 51.1 ± 2.35%. Similarly, increasing hemicellulose concentration increased total intake of digestible NDF from 6.62 to 8.42 ± 0.89 kg/d, whereas fat had no effect. Methane per unit of digested NDF tended to decrease from 64.8 to 49.2 ± 9.60 L/kg with increasing hemicellulose, whereas fat had no effect. An interaction between hemicellulose and fat content on net energy balance (milk plus tissue energy) was observed. Specifically, increasing hemicellulose in low-fat diets tended to increase net energy balance, but this was not observed in high-fat diets. These results confirm that methane production may be reduced with the inclusion of fat, whereas energy utilization of lactating dairy cows is improved by increasing hemicellulose in low-fat diets.

Effects of dietary supplementation of formaldehyde and crystalline amino acids on gut microbial composition of nursery pigs.

Formaldehyde-based feed additives are approved in the US for Salmonella control and reducing bacterial contamination in animal feed. However, we hypothesize formaldehyde inclusion in swine diets may influence gut microbial composition due to its antimicrobial properties which might negatively influence microbial populations and pig growth performance. Also, formaldehyde inclusion in diets is known to reduce the dietary availability of amino acids. Therefore, our study was conducted to characterize if the effects of feed formaldehyde-treatment are due to influences on microbial population or diet amino acid (AA) sources. Dietary treatments were arranged in a (2 × 2) + 1 factorial with formaldehyde treatment (none vs. 1000 ppm formaldehyde) and crystalline AA inclusion (low vs. high) with deficient AA content plus a positive control diet to contain adequate AA content without dietary formaldehyde. Treating diets with formaldehyde reduced growth rate (P = 0.001) while the AA inclusion had no evidence of impact. Formaldehyde reduced feed bacterial content and altered fecal microbial communities (P < 0.05). Therefore, we conclude that the negative influence on growth was due to the impact on the fecal microbial community. Implications are that strategies for feed pathogen control need to take into account potential negative impacts on the gut microbial community.

Effect of partially replacing a barley-based concentrate with flaxseed-based products on the rumen bacterial population of lactating Holstein dairy cows.

AIMS: The effects of partial replacement of a barley-based concentrate with flaxseed-based products on the rumen bacterial population of lactating Holstein dairy cows were evaluated. METHODS AND RESULTS: Treatments fed were CONT, a normal diet that included barley silage, alfalfa hay and a barley-based concentrate that contained no flaxseed or faba beans; FLAX, inclusion of a nonextruded flaxseed-based product containing 55·0% flaxseed, 37·8% field peas and 6·9% alfalfa; EXT, similar to FLAX, but the product was extruded and EXTT, similar to FLAX, but product was extruded and field peas were replaced by high-tannin faba beans. The rumen bacterial population was evaluated by utilizing 16S rRNA gene sequencing. Most abundant phyla, families and genera were unaffected. However, some taxa were affected; for example, unsaturated fatty acid content was negatively correlated with Clostridiaceae, and tannin content was negatively correlated with BS11 and Paraprevotellaceae. CONCLUSIONS: Predominant rumen bacterial taxa were not affected, but the abundance of some taxa found in lower proportions shifted, possibly due to sensitivity to unsaturated fatty acids or tannins. SIGNIFICANCE AND IMPACT OF THE STUDY: Flaxseed-based products were effective for partially replacing barley-based concentrate in rations of lactating dairy cows. No negative effects of these products were observed on the abundance of predominant rumen bacterial taxa, with only minor shifts in less abundant bacteria.

The effect of regular or reduced-fat distillers grains with solubles on rumen methanogenesis and the rumen bacterial community.

AIMS: The effect of feeding dried distillers grains with solubles (DDGS) or reduced-fat DDGS (RFDG) on ruminal methanogenesis and the rumen bacterial community of dairy cattle was evaluated. METHODS AND RESULTS: Treatments were CONT, a diet with no distillers grains; DG, inclusion of 20% DDGS; rfDG, inclusion of 20% RFDG; and MIX, inclusion of 10% DDGS and 10% RFDG. Methane emission was measured; rumen bacterial community was evaluated by sequencing the V4 region of the 16S rRNA gene. Total methane production remained unaffected. However, feeding distillers grains tended to reduce methanogenesis per unit of feed intake, decreased the abundance of the phylum Bacteroidetes and tended to increase Firmicutes. The abundance of Prevotellaceae positively correlated with feed intake; methane emission was positively correlated with the abundance of Prevotellaceae and was negatively correlated with the abundance of Succinivibrionaceae. CONCLUSIONS: DDGS or RFDG may reduce methanogenesis per unit of feed intake; shifts in the abundance of predominant ruminal bacterial families may influence methane formation, likely because of their role on hydrogen liberation and utilization pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: Replacing corn and soybean meal with DDGS or RFDG in dairy rations may reduce the proportion of dietary energy wasted as methane, without detrimental effects on the overall bacterial population.

Rumen bacterial communities can be acclimated faster to high concentrate diets than currently implemented feedlot programs.

AIMS: Recent studies have demonstrated RAMP, a complete starter feed, to have beneficial effects for animal performance. However, how RAMP may elicit such responses is unknown. To understand if RAMP adaptation results in changes in the rumen bacterial community that can potentially affect animal performance, we investigated the dynamics of rumen bacterial community composition in corn-adapted and RAMP-adapted cattle. METHODS AND RESULTS: During gradual acclimation of the rumen bacterial communities, we compared the bacterial community dynamics in corn and RAMP-adapted using 16S rRNA gene amplicon sequencing. Significant shifts in bacterial populations across diets were identified. The shift in corn-adapted animals occurred between adaptation step3 and step4, whereas in RAMP-adapted cattle, the shift occurred between step2 and step3. As the adaptation program progressed, the abundance of OTUs associated with family Prevotellaceae and S24-7 changed in corn-adapted animals. In RAMP-adapted animals, OTUs belonging to family Ruminococcaceae and Lachnospiraceae changed in abundance. CONCLUSIONS: Rumen bacteria can be acclimated faster to high concentrate diets, such as RAMP, than traditional adaptation programs and the speed of bacterial community acclimation depends on substrate composition. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may have implications for beef producers to reduce feedlot costs, as less time adapting animals would result in lower feed costs. However, animal feeding behavior patterns and other factors must be considered.

Reduced-fat dried distillers grains with solubles reduces the risk for milk fat depression and supports milk production and ruminal fermentation in dairy cows.

Twenty Holstein cows, 12 primiparous and 8 multiparous, with (mean ± SD) 91 ± 19 d in milk and 595 ± 81 kg were used in replicated 4 × 4 Latin squares to compare the effects of feeding conventional dried distillers grains with solubles (DDGS) and reduced-fat DDGS (RFDDGS) in combination with rumen-inert fat (RIF) on milk production and rumen fermentation; one square contained rumen cannulated animals for rumen measurements. In each 21-d period, cows were randomly assigned to 1 of 4 dietary treatments (values on a dry matter basis): (1) control (CON) that contained 0% DDGS; (2) DG contained 30% DDGS; (3) RFDG contained 30% RFDDGS in substitution of DDGS; and (4) RFDG+RIF was similar to RFDG with the addition of 1.9% RIF. Unlike most practical diets in the dairy field, our diets had <22% forage neutral detergent fiber and >18.0% crude protein. Dry matter intake was similar across treatments with any form of DDGS averaging 26.0 ± 0.6 kg/d, whereas the CON diet resulted in less dry matter intake, 21.6 ± 0.6 kg/d. Milk yield tended to be 1.7 kg/d greater for diets with either type of DDGS. Concentration of milk protein was greatest for the DG and RFDG diets, intermediate for the RFDG+RIF diet, and least for the CON diet, namely 3.22, 3.21, 3.12, and 3.07 ± 0.05%. Reduced milk fat percentage and yield were observed when cows consumed the DG diet, 3.27 ± 0.10% and 1.11 ± 0.04 kg/d, respectively, whereas these responses were similar among CON, RFDG, and RFDG+RIF, which averaged 3.68 ± 0.10% and 1.22 ± 0.04 kg/d. The presence of trans-10,cis-12 conjugated linoleic acid was only detected in milk from cows consuming the DG diet; similarly, concentration and yield of trans-10 18:1 were greater for cows consuming this diet. Rumen ammonia was similar across treatments averaging 27.0 ± 2.1mg/dL. The CON and RFDG+RIF diets had similar mean pH, 6.1 ± 0.11, whereas DG and RFDG resulted in lower pH averaging 5.79 ± 0.11. No effect on total concentration of volatile fatty acids was observed; the overall mean was 121 ± 4.11 mM; molar proportion of acetate was affected by treatment resulting in 67.3, 63.2, 61.4, and 60.9 ± 0.93 mol/100 mol for CON, RFDG+RIF, RFDG, and DG, respectively. Results from DNA sequencing showed that rumen bacterial community structure was relatively stable with minor changes at the family and genus levels; these changes may be associated with low starch diets, and hence reduced amylolytic bacteria populations. Feeding high proportions of RFDDGS resulted in greater dry matter intake with low risk for milk fat depression while supporting ruminal fermentation.

Ruminal degradation and intestinal digestibility of protein and amino acids in high-protein feedstuffs commonly used in dairy diets.

A study was conducted to determine the rumen degradation and intestinal digestibility of crude protein (CP) and AA, and AA composition of the rumen-undegradable protein (RUP) from 3 sources of blood meal (BM1, BM2, and BM3), canola meal (CM), low-fat distillers dried grains with solubles (LFDG), soybean meal (SBM), and expeller soybean meal (ESBM). Two Holstein cows fitted with ruminal and proximal duodenal cannulas were used for in situ incubation of 16h and for the mobile bag technique. To correct for bacterial contamination of the RUP, 2 methods were used: purines and DNA as bacterial markers. Ruminal degradations of CP were 85.3, 29.8, 40.7, 75.7, 76.9, 68.8, and 37.0 ± 3.93% for BM1, BM2, BM3, CM, LFDG, SBM, and ESBM, respectively. Ruminal degradation of both total essential AA and nonessential AA followed a similar pattern to that of CP across feedstuffs. Based on the ratio of AA concentration in the RUP to AA concentration in the original feedstuff, ruminal incubation decreased (ratio <1) the concentrations of His, Lys, and Trp, and increased (ratio >1) the concentrations of Ile and Met across feedstuffs. Compared with purines, the use of DNA as bacterial marker resulted in a higher estimate of bacterial CP contamination for CM and lower estimates for LFDG and ESBM. Intestinal digestibility of RUP could not be estimated for BM1, BM3, and SBM due to insufficient recovery of residue. For the remaining feedstuffs, intestinal digestibility of RUP was highest for ESBM, followed by BM2 and LFDG, and lowest for CM: 98.8, 87.9, 89.7, and 72.4 ± 1.40%, respectively. Intestinal absorbable dietary protein was higher for BM2 compared with CM and LFDG, at 61.7, 17.9, and 20.7 ± 2.73% CP, respectively. As prices fluctuate, intestinal absorbable protein or AA may be used as a tool to aid in the selection among feedstuffs with different protein quality.

Effect of feeding dried distillers grains with solubles on ruminal biohydrogenation, intestinal fatty acid profile, and gut microbial diversity evaluated through DNA pyro-sequencing.

The objectives of this study were to evaluate the effect of dried distillers grains with solubles (DDGS) on ruminal biohydrogenation and duodenal flow of fatty acids, and to evaluate effects on the ruminal and duodenal microbial community using Roche 454 pyro-sequencing. Three crossbred steers (average BW 780 ± 137 kg) fitted with ruminal and duodenal cannulae were used in a 3-diet, 6-period crossover design. Animals were housed in individual free stalls and fed twice daily at 0700 and 1900 h. Diets (DM basis) were 1) CONTROL, 19.5% corn bran, 20% sorghum silage, 60% brome hay, 0.5% trace minerals, and 0.25% urea, but no DDGS; 2) LOW DDGS, inclusion of 9.75% DDGS replacing equal percentage of corn bran; 3) HIGH DDGS, inclusion of 19.5% DDGS completely replacing corn bran. Feed ingredients and duodenal digesta samples were analyzed for fatty acid composition. The DNA was extracted from isolated mixed ruminal bacterial samples and from intestinal digesta samples. The V1-V3 region of the 16S rRNA gene was sequenced, and bacterial phylogenetic analysis was conducted. Data were analyzed using the MIXED procedure of SAS. Biohydrogenation of C18:1 increased (P < 0.01) with DDGS inclusion; means were 68.3, 75.6, and 79.3 ± 4.3% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of biohydrogenation of C18:2 (P < 0.05) were 84.1, 91.5, and 93.3 ± 3.4%. Duodenal flow of total fatty acids increased (P < 0.01) with DDGS inclusion; means were 134, 168, and 223 ± 33 g/d for CONTROL, LOW DDGS, and HIGH DDGS, respectively. In the same order, means of C18:0 flow (P < 0.01) were 51, 86, and 121 ± 18 g/d. DDGS did not affect the predominant bacterial phyla in the gut, which were Bacteroidetes (P = 0.62) and Firmicutes (P = 0.71). However, the phylum Fibrobacteres decreased (P < 0.01) when DDGS was fed with means of 5.5, 6.0 and 3.7 ± 0.6% for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Fibrobacteres were lower (P < 0.01) in isolated ruminal bacterial samples compared to duodenal digesta samples with means of 0.1 and 10.1 ± 0.6%, respectively. Overall, the inclusion of DDGS in diets increased ruminal biohydrogenation of C18:1 and C18:2, which increased duodenal flow of C18:0. In addition, the bacterial community of the rumen clustered separately from that of the duodenum suggesting different bacterial diversity between isolated ruminal bacteria and duodenal digesta.

On the notion of causality in medicine: addressing Austin Bradford Hill and John L. Mackie / Sobre a noção de causalidade na medicina: aproximando Austin Bradford Hill e John L. Mackie

Almost 50 years ago appeared the seminal article by Austin Bradford Hill where he presented parameters for inferring causes from statistical associations, which became known as Hill’s causal criteria. This was a milestone for the renewal of the idea of cause in medicine. Our article revisits his contribution in light of the ideas from the Australian philosopher John L. Mackie, whose important works on causality reached an audience distinct from Hill’s. We suggest that both the British epidemiologist and the Australian philosopher share the purpose of articulating probabilistic determinism and multi-causality, the first with a predominantly probabilistic model and the second with an analytical approach. This article explores the possible consequences of addressing these authors jointly in regard to causal inferences in medicine, especially in respect to mental disorders...

Há quase 50 anos era publicado o artigo seminal de Austin Bradford Hill, no qual ele apresenta parâmetros para inferência de causas com base em associações estatísticas, que ficaram conhecidas como critérios causais de Hill. Aquele foi um marco para a renovação da ideia de causa na medicina. Nosso trabalho revisita suas ideias, articulando-as com o trabalho do filósofo australiano John L. Mackie, também autor de importante obra sobre causalidade, mas que teve uma audiência distinta da que Hill alcançou. Sugerimos que tanto o epidemiologista britânico quanto o filósofo australiano têm em comum o fato de que ambos procuram articular determinismo probabilístico e multicausalidade, o primeiro com um modelo predominantemente probabilista e o segundo com uma abordagem analítica. Este artigo explora as possíveis consequências da aproximação desses dois autores no que diz respeito a inferências causais na medicina, com foco particular nos transtornos mentais...

Causas "fracas" e redes causais complexas em psiquiatria / Weak' causes and complex causal nets in psychiatry / Causes "faibles" et réseaux causaux complexes / Causas "sencillas" y redes causales complejas en psiquiatría

OBJETIVOS: Realizamos uma análise do conceito de causalidade aplicado em psiquiatria e propomos sua abordagem baseada nas ideias de influências causais fracas e de redes causais complexas. MÉTODO: Análise conceitual de artigos e livros selecionados. RESULTADOS: Identificamos o modelo de causalidade INUS, proposto pelo filósofo J. L. Mackie que contorna algumas das dificuldades relacionadas à presença de múltiplas causas interdependentes, como parece ocorrer no caso dos transtornos mentais e mostramos que tal modelo se aplica bem ao TEPT. CONCLUSÃO: Sugerimos que o fracasso na busca de causas "fortes" para os transtornos mentais talvez indique a necessidade de revisão de nossas expectativas, com a adoção de modelos alternativos de causalidade.(AU)

OBJECTIVES: We briefly analyze the concept of causality as applied to psychiatry and put forward an approach based on the notions of weak causal influences and complex causal networks. METHOD: a conceptual analysis of selected articles and books. RESULTS: We present the INUS causation model proposed by the philosopher J.L.Mackie that gets around some of the difficulties related to the presence of multiple interdependent causes, as appears to be the case in mental disorders. This model seemingly works well in PTSD, as shown. CONCLUSION: We suggest that the failure to find "strong" causes for mental disorders may indicate the need to revise our expectations by adopting alternative models of causality.(AU)

OBJECTIFS: Nous avons effectué une analyse de la notion de causalité appliquée en psychiatrie et proposons une approche basée sur les notions des influences causales faibles et des réseaux causaux complexes. MÉTHODE: Analyse conceptuelle d'articles et de livres sélectionnés. RÉSULTATS: Nous avons identifié le modèle de causalité INUS, proposé par le philosophe J. L. Mackie, qui contourne certaines des difficultés liées à la présence de multiples causes interdépendantes, comme cela semble se produire dans le cas des troubles mentaux, et nous montrons que ce modèle s'applique bien au TSPT. CONCLUSION: Nous suggérons que l'impossibilité de trouver des causes "fortes" de troubles mentaux peut indiquer le besoin d'une révision de nos attentes et l'adoption de modèles alternatifs de causalité.(AU)

OBJETIVOS: se realizó un análisis del concepto de causalidad aplicado en psiquiatría y propusimos su enfoque basado en las ideas de las influencias causales sencillas y redes sociales complejas. MÉTODO: Análisis conceptuales de artículos y libros seleccionados. RESULTADOS: Se identificó el modelo de causalidad INUS propuesto por el filósofo JL Mackie que evita algunos de los problemas relacionados con la presencia de múltiples causas interdependientes, como parece ocurrir en el caso de los trastornos mentales y mostramos que este modelo se aplica también al TEPT. CONCLUSIÓN: Se sugiere que el fracaso en encontrar las causas "complejas" para los trastornos mentales puede indicar la necesidad de revisar nuestras expectativas y la adopción de modelos alternativos de causalidad.(AU)

Causas "fracas" e redes causais complexas em psiquiatria / Weak' causes and complex causal nets in psychiatry / Causes "faibles" et réseaux causaux complexes / Causas "sencillas" y redes causales complejas en psiquiatría

OBJETIVOS: Realizamos uma análise do conceito de causalidade aplicado em psiquiatria e propomos sua abordagem baseada nas ideias de influências causais fracas e de redes causais complexas. MÉTODO: Análise conceitual de artigos e livros selecionados. RESULTADOS: Identificamos o modelo de causalidade INUS, proposto pelo filósofo J. L. Mackie que contorna algumas das dificuldades relacionadas à presença de múltiplas causas interdependentes, como parece ocorrer no caso dos transtornos mentais e mostramos que tal modelo se aplica bem ao TEPT. CONCLUSÃO: Sugerimos que o fracasso na busca de causas "fortes" para os transtornos mentais talvez indique a necessidade de revisão de nossas expectativas, com a adoção de modelos alternativos de causalidade.

OBJECTIVES: We briefly analyze the concept of causality as applied to psychiatry and put forward an approach based on the notions of weak causal influences and complex causal networks. METHOD: a conceptual analysis of selected articles and books. RESULTS: We present the INUS causation model proposed by the philosopher J.L.Mackie that gets around some of the difficulties related to the presence of multiple interdependent causes, as appears to be the case in mental disorders. This model seemingly works well in PTSD, as shown. CONCLUSION: We suggest that the failure to find "strong" causes for mental disorders may indicate the need to revise our expectations by adopting alternative models of causality.

OBJECTIFS: Nous avons effectué une analyse de la notion de causalité appliquée en psychiatrie et proposons une approche basée sur les notions des influences causales faibles et des réseaux causaux complexes. MÉTHODE: Analyse conceptuelle d'articles et de livres sélectionnés. RÉSULTATS: Nous avons identifié le modèle de causalité INUS, proposé par le philosophe J. L. Mackie, qui contourne certaines des difficultés liées à la présence de multiples causes interdépendantes, comme cela semble se produire dans le cas des troubles mentaux, et nous montrons que ce modèle s'applique bien au TSPT. CONCLUSION: Nous suggérons que l'impossibilité de trouver des causes "fortes" de troubles mentaux peut indiquer le besoin d'une révision de nos attentes et l'adoption de modèles alternatifs de causalité.

OBJETIVOS: se realizó un análisis del concepto de causalidad aplicado en psiquiatría y propusimos su enfoque basado en las ideas de las influencias causales sencillas y redes sociales complejas. MÉTODO: Análisis conceptuales de artículos y libros seleccionados. RESULTADOS: Se identificó el modelo de causalidad INUS propuesto por el filósofo JL Mackie que evita algunos de los problemas relacionados con la presencia de múltiples causas interdependientes, como parece ocurrir en el caso de los trastornos mentales y mostramos que este modelo se aplica también al TEPT. CONCLUSIÓN: Se sugiere que el fracaso en encontrar las causas "complejas" para los trastornos mentales puede indicar la necesidad de revisar nuestras expectativas y la adopción de modelos alternativos de causalidad.

Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.

Sixteen multiparous lactating Holstein cows were used in 2 experiments to evaluate the effects of reduced-fat dried distillers grains with solubles (RFDG) on milk production, rumen fermentation, intestinal microbial N flow, and total-tract nutrient digestibility. In experiment 1, RFDG was fed at 0, 10, 20, or 30% of diet dry matter (DM) to 12 noncannulated Holstein cows (mean ± standard deviation: 89 ± 11 d in milk and 674 ± 68.2 kg of body weight) to determine effects on milk production. In experiment 2, the same diets were fed to 4 ruminally and duodenally cannulated Holstein cows (mean ± standard deviation: 112 ± 41 d in milk; 590 ± 61.14 kg of body weight) to evaluate the effects on rumen fermentation, intestinal flow of microbial N, and total-tract nutrient digestibility. In both experiments, cows were randomly assigned to 4 × 4 Latin squares over 21-d periods. Treatments (DM basis) were (1) control (0% RFDG), (2) 10% RFDG, (3) 20% RFDG, and (4) 30% RFDG. Feed intake and milk yield were recorded daily. In both experiments, milk samples were collected on d 19 to 21 of each period for analysis of milk components. In experiment 2, ruminal pH was measured; samples of rumen fluid, duodenal digesta, and feces were collected on d 18 to 21. Microbial N was estimated by using purines and DNA as microbial markers. Milk yield was not affected by treatment and averaged 34.0 ± 1.29 kg/d and 31.4 ± 2.81 kg/d in experiments 1 and 2, respectively. Percentage of milk protein tended to increase in experiment 1; estimates were 3.08, 3.18, 3.15, and 3.19 ± 0.06% when RFDG increased from 0 to 30% in the diets. However, milk protein concentration was not affected in experiment 2 and averaged 3.02 ± 0.07%. Percentage of milk fat was not affected and averaged 3.66 ± 0.05% and 3.25 ± 0.14% in experiments 1 and 2, respectively. Total ruminal volatile fatty acids and ammonia concentrations were not affected by treatment and averaged 135.18 ± 6.45 mM and 18.66 ± 2.32 mg/dL, respectively. Intestinal microbial N flow was not affected by treatment; however, purines yielded higher estimates of flow compared with DNA markers. When averaged across treatments, intestinal flow of microbial N was 303 and 218 ± 18 g of N/d, using purines and DNA as the markers. Dry matter, organic matter, neutral detergent fiber, and nonfiber carbohydrate digestibility tended to increase with increasing inclusion of RFDG. Results from these experiments indicate that dairy rations can be formulated to include up to 30% RFDG while maintaining lactation performance, volatile fatty acids concentration, and intestinal supply of microbial N.

In vivo determination of rumen undegradable protein of dried distillers grains with solubles and evaluation of duodenal microbial crude protein flow.

The objectives of this trial were to determine the rumen undegradable protein (RUP) of dried distillers grains with solubles (DDGS), to compare the estimates of duodenal bacterial CP (BCP) flow using diaminopumelic acid (DAPA) or DNA as bacterial markers, and to estimate duodenal protozoal CP (PCP) and yeast CP (YCP) flow when DDGS are fed. Three crossbred steers fitted with ruminal and double L-shaped duodenal cannulae (average BW 780 ± 137 kg) were used in a 3 treatment, 6 period crossover design. Animals were housed in individual free stalls and fed twice daily at 0700 and 1900 h. Diets (DM basis) were 1) CONTROL, which is 0% DDGS but with 19.5% corn bran, 20% sorghum silage, 60% brome hay, 0.5% trace minerals, and 0.25% urea, 2) LOW DDGS, which is inclusion of 9.75% DDGS replacing equal percentage of corn bran, and 3) HIGH DDGS, which is inclusion of 19.5% DDGS completely replacing corn bran. Duodenal BCP flow was estimated using DAPA and DNA as bacterial markers. In addition, duodenal PCP and YCP flow were estimated using DNA markers. The value of DDGS RUP as a percent of CP was determined to be 63.0 ± 0.64%. Estimates of duodenal BCP flow using DAPA were 473, 393, and 357 ± 78 g/d (P = 0.09) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Estimates of duodenal BCP flow using DNA were 479, 397, and 368 ± 74 g/d (P = 0.14), respectively. Average BCP flow across treatments was unaffected (P = 0.71) by marker type and were 404 and 417 ± 83 g/d for DAPA and DNA markers, respectively. Estimates of duodenal PCP flow were 82, 80, and 78 ± 12 g/d (P = 0.64) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Estimates of duodenal YCP flow were 0.15, 1.94, and 4.80 ± 0.66 g/d (P < 0.01) for CONTROL, LOW DDGS, and HIGH DDGS, respectively. Duodenal BCP flow tended to decrease with DDGS inclusion, but estimates were not affected by marker type. In addition, DDGS did not affect duodenal PCP supply and provided small amounts of duodenal YCP. Overall, the value of DDGS RUP determined in this study will contribute to a better understanding of the effect of this coproduct in ruminant nutrition.

Rumen microbial population dynamics during adaptation to a high-grain diet.

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.