Effects of Bacillus subtilis on in vitro ruminal fermentation and methane production.

The objective of this study was to evaluate the effect of a proprietary strain of a Bacillus subtilis on in vitro ruminal fermentation and methane production in batch culture serum bottles. One hundred forty-nine batch culture bottles were used in a complete randomized block design. The arrangement of treatments was a 3 × 3 × 4 factorial to evaluate the effects of inoculum, time, diet, and their respective interactions. There were three experimental runs total, where the run was used as block. Inoculum treatments were 1.85 mg/mL of microcrystalline cellulose (CON); 10 billion B. subtilis plus microcrystalline cellulose (A1); and 60 billion B. subtilis plus microcrystalline cellulose (A2). Diet treatments were 0.50 g of early lactation diet (E, 30% starch), mid-lactation diet (M, 25% starch), or dry cow diet (D, 18% starch). The combination resulted in total of nine treatments. Each treatment had five replicates, two of which were used to determine nutrient degradability at 24 and 48 h after inoculation, and three were used to determine pH, ammonia nitrogen (NH3-N), volatile fatty acids, lactate, total gas, and methane production at 3, 6, 24, and 48 h after inoculation. Fixed effects of inoculum, diet, and their interaction were tested using the GLIMMIX procedure of SAS. Significance was declared at P ≤ 0.05. We observed that, compared to control, the supplementation of B. subtilis, decreased the production of acetate and propionate, while increasing the production of butyrate, iso-butyrate, valerate, iso-valerate, and caproate within each respective diet. Additionally, the total methane production exhibited mixed responses depending on the diet type. Overall, the inclusion of B. subtilis under in vitro conditions shows the potential to reduce ruminal methane production when supplemented with a mid-lactation diet, constituting a possible methane mitigation additive for dairy cattle diets.

Effects of monensin and cashew nut-shell extract on bacterial community composition in a dual-flow continuous culture system.

The objective of this study was to evaluate the effects of including monensin and two doses of CNSE in a high producing dairy cow diet on ruminal bacterial communities. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin Square design. A basal diet was formulated to meet the requirements of a cow producing 45 kg of milk per d (17% crude protein and 27% starch). There were four experimental treatments: the basal diet without any feed additive (CON), 2.5 µM monensin (MON), 100 ppm CNSE granule (CNSE100), and 200 ppm CNSE granule (CNSE200). Samples were collected from the fluid and solid effluents at 3, 6, and 9 h after feeding; a composite of all time points was made for each fermenter within their respective fractions. Bacterial community composition was analyzed by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Treatment responses for bacterial community structure were analyzed with the PERMANOVA test run with the R Vegan package. Treatment responses for correlations were analyzed with the CORR procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). Significance was declared at P ≤ 0.05. We observed that the relative abundance of Sharpea (P < 0.01), Mailhella (P = 0.05), Ruminococcus (P = 0.03), Eubacterium (P = 0.01), and Coprococcus (P < 0.01) from the liquid fraction and the relative abundance of Ruminococcus (P = 0.03) and Catonella (P = 0.02) from the solid fraction decreased, while the relative abundance of Syntrophococcus (P = 0.02) increased in response to MON when compared to CNSE treatments. Our results demonstrate that CNSE and monensin have similar effects on the major ruminal bacterial genera, while some differences were observed in some minor genera. Overall, the tested additives would affect the ruminal fermentation in a similar pattern.

Megasphaera elsdenii and Saccharomyces cerevisiae as direct fed microbials and their impact on ruminal microbiome during an acute acidosis challenge in continuous culture.

Our objective was to evaluate the effects of combinations of Saccharomyces cerevisiae and Megasphaera elsdenii as direct-fed microbials (DFM) on ruminal microbiome during an acute acidosis challenge in a continuous culture system. Treatments provided a DFM dose of 1 × 108 colony-forming unit (CFU)/mL, as follows: control (no DFM), YM1 (S. cerevisiae and M. elsdenii strain 1), YM2 (S. cerevisiae and M. elsdenii strain 2), and YMM (S. cerevisiae and half of the doses of M. elsdenii strains 1 and 2). We conducted four experimental periods of 11 d, which consisted of non-acidotic days (1 to 8) and acidotic challenge days (9 to 11) to establish acute ruminal acidosis conditions with a common basal diet containing 12% neutral detergent fiber and 58% starch. Treatments were applied from days 8 to 11, and samples of liquid and solid-associated bacteria were collected on days 9 to 11. Overall, 128 samples were analyzed by amplification of the V4 region of bacterial 16S rRNA, and data were analyzed with R and SAS for alpha and beta diversity, taxa relative abundance, and correlation of taxa abundance with propionate molar proportion. We observed a lower bacterial diversity (Shannon index, P = 0.02) when YM1 was added to the diet in comparison to the three other treatments. Moreover, compared to control, addition of YM1 to the diet increased relative abundance of phylum Proteobacteria (P = 0.05) and family Succinivibrioceae (P = 0.05) in the solid fraction and tended to increase abundance of family Succinivibrioceae (P = 0.10) and genus Succinivibrio (P = 0.09) in the liquid fraction. Correlation analysis indicated a positive association between propionate molar proportion and relative abundance of Proteobacteria (r = 0.36, P = 0.04) and Succinivibrioceae (r = 0.36, P = 0.05) in the solid fraction. The inclusion of YM1 in high-grain diets with a high starch content resulted in greater abundance of bacteria involved in succinate synthesis which may have provided the substrate for the greater propionate synthesis observed.

In vitro evaluation of microencapsulated organic acids and pure botanicals as a supplement in lactating dairy cows diet on in vitro ruminal fermentation.

The utilization of microencapsulated organic acids and pure botanicals (mOAPB) is widely used in the monogastric livestock industry as an alternative to antibiotics; in addition, it can have gut immunomodulatory functions. More recently, an interest in applying those compounds in the ruminant industry has increased; thus, we evaluated the effects of mOAPB on ruminal fermentation kinetics and metabolite production in an in vitro dual-flow continuous-culture system. For this study, two ruminal cannulated lactating dairy Holstein cows were used as ruminal content donors, and the inoculum was incubated in eight fermenters arranged in a 4 × 4 Latin square design. The basal diet was formulated to meet the nutritional requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk and supplemented with increasing levels of mOAPB (0; 0.12; 0.24; or 0.36% of dry matter [DM]), which contained 55.6% hydrogenated and refined palm oil, 25% citric acid, 16.7% sorbic acid, 1.7% thymol, and 1% vanillin. Diet had 16.1 CP, 30.9 neutral detergent fiber (NDF), and 32.0 starch, % of DM basis, and fermenters were fed 106 g/d split into two feedings. After a 7 d adaptation, samples were collected for 3 d in each period. Samples of the ruminal content from the fermenters were collected at 0, 1, 2, 4, 6, and 8 h postmorning feeding for evaluation of the ruminal fermentation kinetics. For the evaluation of the daily production of total metabolites and for the evaluation of nutrient degradability, samples from the effluent containers were collected daily at days 8 to 10. The statistical analysis was conducted using MIXED procedure of SAS and treatment, time, and its interactions were considered as fixed effects and day, Latin square, and fermenter as random effects. To depict the treatment effects, orthogonal contrasts were used (linear and quadratic). The supplementation of mOAPB had no major effects on the ruminal fermentation, metabolite production, and degradability of nutrients. The lack of statistical differences between control and supplemented fermenters indicates effective ruminal protection and minor ruminal effects of the active compounds. This could be attributed to the range of daily variation of pH, which ranged from 5.98 to 6.45. The pH can play a major role in the solubilization of lipid coat. It can be concluded that mOAPB did not affect the ruminal fermentation, metabolite production, and degradability of dietary nutrients using an in vitro rumen simulator.

Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium-magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3-N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3-N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.

Magnesium oxide (MgO) is extensively used as a dietary magnesium (Mg) source in dairy cow diets. However, dairy operations can benefit from other Mg sources. Thus, we evaluated the replacement of dietary MgO with calcium­magnesium hydroxide (CaMg(OH)2) in diets with and without ruminal buffer and their effects on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. The study used 20 lactating Holstein cows that were blocked in groups of four and randomly assigned to one of the four treatments. The ruminal content, feed, feces, and urine were collected for analysis of the microbiota composition, ruminal fermentation, nitrogen metabolism, and apparent nutrient digestibility. There was no interaction effect of dietary buffer use and Mg source, while buffer improved total tract apparent digestibility of the dry matter and fiber components; CaMg(OH)2 increased the ruminal concentration of butyrate and the abundance of butyrate-producing bacteria. In summary, we conclude that using CaMg(OH)2 can improve ruminal fermentation regardless of buffer use, which indicates that we can take advantage of the mineral formulation in the diet to modulate the ruminal microbiota composition.

Effects of starch sources varying in particle sizes on ruminal fermentation, nutrient flow, starch digestibility, and lactation performance of dairy cows.

Cereal grains are the predominant starch source (SS) for dairy cows; however, starch digestibility varies greatly depending on source, grain processing, and potentially interactions between these factors. The objective was to study the effects of the interactions between SS, and particle sizes (PS) on ruminal fermentation, nutrient flow, starch digestibility, and lactation performance of dairy cows. Four ruminally cannulated multiparous Holstein cows were used in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Two SS (corn or sorghum) used in this study were either finely or coarsely ground (using a 1- or 4-mm screen sieve). Digesta flow was quantified using the reticular sampling technique, applying the triple-marker method. Data were analyzed using the GLIMMIX procedure of SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). For ruminal pH, data were analysed with time as repeated measure. There were no interactions between SS and PS on production or intake, flow, and digestibility of nutrients. Dry matter intake was greater for the corn diet compared to the sorghum diet (25.15 vs. 21.98 kg/d), which consequently affected nutrient intake, however, PS did not affect intake. Milk yield was not affected by SS; however, it was greater for cows fed fine grains than cows fed coarser grains (25.32 vs. 23.16 kg/d). Milk fat and milk protein were not affected by SS or PS. Interactions (SS × PS) were observed for ruminal pH, reticular pH, and volatile fatty acids (VFA) concentrations but not for ruminal NH3-N concentration. Ruminal and reticular pH were greater for sorghum when coarsely ground and the total VFA concentration was decreased, compared to coarse corn and fine sorghum; however, coarsely grinding corn did not affect ruminal or reticular pH nor VFA concentration. Acetate concentration was lower for corn when finely ground; however, finely grinding sorghum did not affect acetate. Decreasing PS increased ruminal digestibility of starch (87.18% vs. 83.43%), reduced the flow of starch to the reticulum (0.79 vs. 0.96 kg/d) but decreased neutral detergent fiber digestibility in the rumen (30.23% vs. 34.88%). Although SS were differently affected by processing, the effects of PS on production, intake, flow, and digestibility of nutrients were observed regardless of the SS. Furthermore, the effects of decreasing PS on pH and VFA concentrations were more pronounced in sorghum compared to corn.

Starch digestibility varies greatly depending on starch source (SS), grain processing, and potentially interactions between these factors. Four ruminally cannulated lactating Holstein cows were fed a total mixed ration that varied in SS and particle sizes (PS) to evaluate the interactions between SS and PS on ruminal fermentation, nutrient flow, starch digestibility, and lactation performance of dairy cows. There were no interactions between SS, and PS on production, intake, flow, and digestibility of nutrients; however, interactions were observed for ruminal pH, reticular pH, volatile fatty acids (VFA) concentrations, and in some VFA molar proportions.

The Effects of Incremental Doses of Aflatoxin B1 on In Vitro Ruminal Nutrient Digestibility and Fermentation Profile of a Lactating Dairy Cow Diet in a Dual-Flow Continuous Culture System.

Aflatoxin B1 (AFB1) is a mycotoxin known to impair human and animal health. It is also believed to have a deleterious effect on ruminal nutrient digestibility under in vitro batch culture systems. The objective of this study was to evaluate the effects of increasing the dose of AFB1 on ruminal dry matter and nutrient digestibility, fermentation profile, and N flows using a dual-flow continuous culture system fed a diet formulated for lactating dairy cows. Eight fermenter vessels were used in a replicated 4 × 4 Latin square design with 10 d periods (7 d adaptation and 3 d sample collection). Treatments were randomly applied to fermenters on diet DM basis: (1) 0 µg of AFB1/kg of DM (Control); (2) 50 µg of AFB1/kg of DM (AF50); (3) 100 µg of AFB1/kg of DM (AF100); and (4) 150 µg of AFB1/kg of DM (AF150). Treatments did not affect nutrient digestibility, fermentation, and N flows. Aflatoxin B1 concentration in ruminal fluid increased with dose but decreased to undetectable levels after 4 h post-dosing. In conclusion, adding incremental doses of AFB1 did not affect ruminal fermentation, digestibility of nutrients, and N flows in a dual-flow continuous culture system fed diets formulated for lactating dairy cows.

Unraveling the pros and cons of various in vitro methodologies for ruminant nutrition: a review.

To decrease the time and cost of experiments as well as the use of animals in nutrition research, in vitro methodologies have become more commonplace in the field of ruminant nutrition. Therefore, the objectives of this review are 1) to describe the development of different in vitro methodologies, 2) to discuss the application, utilization, and advantages of in vitro methodologies, 3) to discuss shortcomings of in vitro methodologies, and 4) to describe the potential developments that may be able to improve in vitro methods. Having been used for decades, some in vitro methodologies such as pure, batch, and continuous cultures have been very well documented and utilized to investigate a wide array of different aspects of nutrition, including the effects of different dietary compositions, individual fermentation end products, and impacts on the microbiome of the rumen. However, both batch and pure cultures can result in a build-up of end products that may inhibit fermentation, as they culture ruminal contents or defined strains of bacteria, respectfully. Continuous culture; however, allows for the removal of end products but, similar to pure and batch cultures, is applicable only to ruminal fermentation and cannot provide information regarding intestinal digestion and bioavailability. This information for in vitro can only be provided using an assay designed for total tract digestibility, which is the three-step procedure (TSP). The TSP may be improved by coupling it with cell culture to investigate the absorption of nutrients in both the ruminal and intestinal phases of the methodology; however, the TSP needs further development to investigate all nutrients and the methodologies available for cell culture are still relatively new to ruminant nutrition. Therefore, while in vitro methodologies provide useful data in the field of ruminant nutrition without the continuous use of animals, there is still much work to be done to improve the methodologies to further apply them.

Effects of ruminal lipopolysaccharides on growth and fermentation end products of pure cultured bacteria.

Elevated levels of ruminal lipopolysaccharides (LPS) have been linked to ruminal acidosis; however, they result in reduced endotoxicity compared to LPS derived from species like Escherichia coli. Additionally, there is a knowledge gap on the potential effect of LPS derived from ruminal microbiome on ruminal bacteria species whose abundance is associated with ruminal acidosis. The objective of this study was to evaluate the effects of LPS-free anaerobic water (CTRL), E. coli-LPS (E. COLI), ruminal-LPS (RUM), and a 1:1 mixture of E. coli and ruminal-LPS (MIX) on the growth characteristics and fermentation end products of lactate-producing bacteria (Streptococcus bovis JB1, Selenomonas ruminantium HD4) and lactate-utilizing bacterium (Megasphaera elsdenii T81). The growth characteristics were predicted based on the logistic growth model, the ammonia concentration was determined by the phenolic acid/hypochlorite method and organic acids were analyzed with high performance liquid chromatography. Results indicate that, compared to the CTRL, the maximum specific growth rate of S. bovis JB1 decreased by approximately 19% and 23% when RUM and MIX were dosed, respectively. In addition, acetate and lactate concentrations in Se. ruminantium HD4 were reduced by approximately 30% and 18%; respectively, in response to MIX dosing. Compared to CTRL, lactate concentration from S. bovis JB1 was reduced approximately by 31% and 22% in response to RUM and MIX dosing; respectively. In summary, RUM decreased the growth and lactate production of some lactate-producing bacteria, potentially mitigating the development of subacute ruminal acidosis by restricting lactate availability to some lactate-utilizing bacteria that metabolize lactate into VFAs thus further contributing to the development of acidosis. Also, RUM did not affect Megasphaera elsdenii T81 growth.

Effects of calcium-magnesium carbonate and calcium-magnesium hydroxide as supplemental sources of magnesium on ruminal microbiome.

Our objective was to evaluate the inclusion of calcium-magnesium carbonate [CaMg(CO3)2] and calcium-magnesium hydroxide [CaMg(OH)4] in corn silage-based diets and their impact on ruminal microbiome. Our previous work showed a lower pH and molar proportion of butyrate from diets supplemented with [CaMg(CO3)2] compared to [CaMg(OH)4]; therefore, we hypothesized that ruminal microbiome would be affected by Mg source. Four continuous culture fermenters were arranged in a 4 × 4 Latin square with the following treatments defined by the supplemental source of Mg: 1) Control (100% MgO, plus sodium sesquicarbonate as a buffer); 2) CO 3 [100% CaMg(CO3)2]; 3) OH [100% CaMg(OH)4]; and 4) CO 3 /OH [50% Mg from CaMg(CO3)2, 50% Mg from CaMg(OH)4]. Diet nutrient concentration was held constant across treatments (16% CP, 30% NDF, 1.66 MCal NEl/kg, 0.67% Ca, and 0.25% Mg). We conducted four fermentation periods of 10 d, with the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 16 solid and 16 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R and SAS to determine treatment effects on taxa relative abundance of liquid and solid fractions. Correlation of butyrate molar proportion with taxa relative abundance was also analyzed. Treatments did not affect alpha and beta diversities or relative abundance of phylum, class and order in either liquid or solid fractions. At the family level, relative abundance of Lachnospiraceae in solid fraction was lower for CO3 and CO3/OH compared to OH and Control (P < 0.01). For genera, abundance of Butyrivibrio (P = 0.01) and Lachnospiraceae ND3007 (P < 0.01) (both from Lachnospiraceae family) was lower and unclassified Ruminococcaceae (P = 0.03) was greater in CO3 than Control and OH in solid fraction; while abundance of Pseudobutyrivibrio (P = 0.10) and Lachnospiraceae FD2005 (P = 0.09) (both from Lachnospiraceae family) and Ruminobacter (P = 0.09) tended to decrease in CO3 compared to Control in liquid fraction. Butyrate molar proportion was negatively correlated to Ruminococcaceae (r = -0.55) in solid fraction and positively correlated to Pseudobutyrivibrio (r = 0.61) and Lachnospiraceae FD2005 (r = 0.61) in liquid. Our results indicate that source of Mg has an impact on bacterial taxa associated with ruminal butyrate synthesis, which is important for epithelial health and fatty acid synthesis.

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge.

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1-8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9-11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, D-, L-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3-N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3-N concentration was reduced to approximately 2 mg/dL as the challenge progressed.

The effects of course format, sex, semester, and institution on student performance in an undergraduate animal science course.

The transition of courses from in-person to an online format due to the COVID-19 pandemic could have potentially affected overall student performance in lecture-based courses. The objective of this case study was to determine the impact of course format, as well as the effects of student sex, time of year at which the course was taken, and the institution it was taken at on student performance in an undergraduate animal science course. The course used for this study was taught at two institutions (University of Florida; UF and University of Nevada, Reno; UNR) over 7 yr (2014-2017 at UNR and 2018-2021 at UF). Student's performance (n = 911) was evaluated using both quizzes and exams from 2014 through the spring semester 2020 and only exams were used for summer and fall semesters of 2020 and the spring and summer semesters of 2021. The final score (out of 100%) for each student was used to evaluate student's performance. In addition, students were classified as high-performing students, if they scored ≥95% and low-performing students, if they scored ≤70%. The variables evaluated were the effects of semester (spring, summer, or fall), institution (UF or UNR), sex (male or female), number of teaching assistants (TAs; 0-13), and course format (online or in-person). The course was taught in-person at UNR and in-person and online at UF. The spring semester of 2020 was taught in-person until March but was switched to online approximately 9 wk after the semester started and was considered an online semester for this analysis. As the course was only taught online at UF, the variable course format was assessed using UF records only. Data were analyzed using both linear models and logistic regressions. The probability that students were high performing was not affected by sex or institution. Interestingly, both fall semester and the online format had a positive, desirable effect on the probability that students were high performing. The probability that students were low performing was not affected by sex. However, if a student performed poorly in the class, they were more likely to have taken the course at UNR, or at UF with many TAs. Thus, student's performance was impacted by changing the course format, as well as institution, the number of TAs, and the semester in which the course was taken.

Ruminal Phages - A Review.

The rumen ecosystem is a complex and dynamic environment, which hosts microorganisms including archaea, bacteria, protozoa, fungi, and viruses. These microorganisms interact with each other, altering the ruminal environment and substrates that will be available for the host digestion and metabolism. Viruses can infect the host and other microorganisms, which can drive changes in microorganisms' lysis rate, substrate availability, nutrient recycling, and population structure. The lysis of ruminal microorganisms' cells by viruses can release enzymes that enhance feedstuff fermentation, which may increase dietary nutrient utilization and feed efficiency. However, negative effects associated to viruses in the gastrointestinal tract have also been reported, in some cases, disrupting the dynamic stability of the ruminal microbiome, which can result in gastrointestinal dysfunctions. Therefore, the objective of this review is to summarize the current knowledge on ruminal virome, their interaction with other components of the microbiome and the effects on animal nutrition.

Effects of neutral detergent fiber digestibility estimation method on calculated energy concentration of canola meals from 12 Canadian processing plants.

Our aim was to determine whether the method used to estimate truly digestible neutral detergent fiber (tdNDF) affects calculated concentrations of total digestible nutrients (TDN1x) and net energy of lactation (NEL3x) of canola meal (CM). Samples were collected from 12 CM processing plants in Canada over 4 yr (2011 to 2014, n = 47) and analyzed for dry matter (DM), crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (ADL), and neutral detergent insoluble CP (NDICP). Ruminal in situ incubation of CM samples was performed at 0, 24, 48, 96, and 288 h to determine NDF fractions (A, B, and C), effective ruminal NDF digestibility (ERNDFD), and indigestible NDF (iNDF) of CM. Three tdNDF-estimation methods were evaluated: 1) National Research Council (NRC) = 0.75 × (NDF - NDICP - ADL) × {1- [ADL/ (NDF - NDICP)]0.667}; 2) iNDF = 0.75 × (NDF - NDICP - NDF remaining after 288 h in situ); and 3) ERNDFD estimated from in situ NDF digestion kinetics. Resulting tdNDF values were used for calculation of TDN1x and NEL3x according to NRC (2001) equations. Data were analyzed with MIXED procedure of SAS 9.4 to determine the effect of processing plant on chemical composition, NDF degradation kinetics and NEL3x of CM. Effect of tdNDF estimation method on calculated TDN1x and NEL3x of CM was also evaluated. Model for analysis of processing plant included the fixed effect of plant and the random effect of year (plant) as replication, while analysis of tdNDF methods included the fixed effect of tdNDF estimation method and the random effects of processing plant and of year(plant) as replication. There was an effect of processing plant on DM (P = 0.03), CP (P < 0.01), EE (P < 0.01), and NDF (P < 0.01) of CM. Processing plant also had an effect on NDF fractions A (P < 0.01) and B (P = 0.02) but did not affect fraction C and ERNDFD. The tdNDF estimation method had an effect on tdNDF (P < 0.01), TDN1x (P < 0.01), and NEL3x (P < 0.01) of CM, yielding average NEL3x values of 1.72, 1.87, and 2.07 Mcal/kg for NRC, iNDF, and ERNDFD, respectively. Our results indicate that calculated energy concentration of CM according to NRC (2001) equations varies depending on the method used for estimation of tdNDF. Further research will be needed to determine the most accurate estimation method.

Effects of lignocellulolytic enzymes on the fermentation profile, chemical composition, and in situ ruminal disappearance of whole-plant corn silage.

The objective of this study was to examine the enzyme activities of an enzymatic complex produced by Pleurotus ostreatus in different pH and the effects of adding increased application rates of this enzymatic complex on the fermentation profile, chemical composition, and in situ ruminal disappearance of whole-plant corn silage (WPCS) at the onset of fermentation and 30 d after ensiling. The lignocellulolytic enzymatic complex was obtained through in vitro cultivation of P. ostreatus. In the first experiment, the activities of laccase, lignin peroxidase (LiP), manganese peroxidase, endo- and exo-glucanase, xylanase, and mannanase were determined at pH 3, 4, 5, and 6. In the second experiment, five application rates of enzymatic complex were tested in a randomized complete block design (0, 9, 18, 27, and 36 mg of lignocellulosic enzymes/kg of fresh whole-plant corn [WPC], corresponding to 0, 0.587, 1.156, 1.734, and 2.312 g of enzymatic complex/kg of fresh WPC, respectively). There were four replicates per treatment (vacuum-sealed bags) per opening time. Bags were opened 1, 2, 3, and 7 d after ensiling (onset of fermentation period) and 30 d after ensiling to evaluate the fermentation profile, chemical composition, and in situ dry matter and neutral fiber detergent disappearance of WPCS. Laccase had the greatest activity at pH 5 (P < 0.01), whereas manganese peroxidase and LiP had the greatest activity at pH 4 (P < 0.01; P < 0.01). There was no effect of the rate of application of enzymatic complex, at the onset of fermentation, on the fermentation profile (P > 0.21), and chemical composition (P > 0.36). The concentration of water-soluble carbohydrate quadratically decreased (P < 0.01) over the ensiling time at the onset of fermentation, leading to a quadratic increase of lactic acid (P = 0.02) and a linear increase of acetic acid (P = 0.02) throughout fermentation. Consequently, pH quadratically decreased (P < 0.01). Lignin concentration linearly decreased (P = 0.04) with the enzymatic complex application rates at 30 d of storage; however, other nutrients and fermentation profiles did not change (P > 0.11) with the enzymatic complex application rates. Addition of lignocellulolytic enzymatic complex from P. ostreatus cultivation to WPC at ensiling decreased WPCS lignin concentration 30 d after ensiling; however, it was not sufficient to improve in situ disappearance of fiber and dry matter.

Adaptation of in vitro methodologies to estimate the intestinal digestion of lipids in ruminants.

The objective of this study was to adapt existing in vitro methodologies to determine the extent of intestinal digestion of corn oil (CO), canola oil (CA), and beef tallow (BT) via manipulation of incubation length and concentrations of lipase, bile, and calcium within a buffer solution. Unless otherwise stated, 0.5 g of each lipid source were incubated separately and in triplicate, with triplicate batch culture runs for each treatment in 40 mL of 0.5 M KH2PO4 (pH = 7.6) for 24 h with pancreatin (8 g/L), bovine bile (2.5 g/L), and CaCl2 (10 mM). Individually, concentrations of pancreatin, bile, and CaCl2, as well as incubation length were tested. To examine the use of this assay to estimate in vitro total tract digestion, a KH2PO4 solution with concentrated amounts to reach the same final concentrations of pancreatin, bile, and Ca were used as the third step in a three-step total tract digestibility procedure. Free glycerol and free fatty acid (FFA) concentrations were measured using colorimetric assays as indicators of digestion. Data wereanalyzed as a completely randomized block design (block = run), using the Glimmix procedure of SAS. For each lipid source, free glycerol increased with increasing pancreatin; however, FFA was lowest at 0 g/L pancreatin but was similar at 6, 8, and 10 g/L. Both glycerol and FFA were greater for 2.5 and 5 g/L of bile than for 0 g/L for each lipid source. Calcium concentration did not affect glycerol or FFA for either CO or CA; however, glycerol and FFA for BT were greater when calcium was included at 5 and 10 mM than at 0 mM. For all fat sources, free glycerol and FFA increased after 1 h until 12 h, but did not increase from 12 to 24 h. When a concentrated mixture was used following fermentation and acidification steps, digestibility using FFA concentration increased as compared to just adding buffer; however, free glycerol concentration was indeterminable. Thus, free glycerol and FFA can be used as indicators of lipid digestion when a lipid source is incubated for at least 12 h in a buffer solution containing 8 g/L pancreatin, 2.5 g/L bile, and 5 mM Ca when only estimating in vitro intestinal digestion; however, when utilizing this assay in a three-step in vitro total tract digestibility procedure, only FFA can be used.

Effects of dietary inclusion of yerba mate (Ilex paraguariensis) extract on lamb muscle metabolomics and physicochemical properties in meat.

This study aimed to evaluate the effect of dietary yerba mate (Ilex paraguariensis) extract (YME) on muscle metabolomics and physicochemical properties of lamb meat. Thirty-six uncastrated male lambs (90 d old) were fed experimental diets, which treatments consisted of 0%, 1%, 2%, and 4% inclusion of YME. Animals were fed for 50 d before slaughter. Muscle and meat samples were collected for metabolomics and meat quality analysis, respectively. The experiment was carried out in a randomized block design and analyzed using orthogonal contrasts. There was a quadratic effect of YME inclusion in tenderness (P < 0.05) and a positive linear effect on meat lightness (P < 0.05). No qualitative changes (P > 0.05) on individual metabolites were observed; however, changes in the quantitative metabolic profile were observed, showing that animals fed 1% and 2% of YME have a greater concentration of desirable endogenous muscle antioxidants, with direct impact on metabolic pathways related to beta-alanine metabolism and glutathione metabolism. Therefore, YME dietary supplementation up to 2% of the diet to lambs had little to no effects on the majority of meat quality traits evaluated; moreover, 4% of YME inclusion negatively affected feed intake and meat quality traits.

Ruminal Lipopolysaccharides Analysis: Uncharted Waters with Promising Signs.

The objective of this review is to present the need for the development of a comprehensive ruminal lipopolysaccharide (LPS) extraction, purification and analysis protocol and state hypotheses that could contribute to planning novel strategies against ruminal acidosis. Lipopolysaccharide is an immunostimulatory molecule of Gram-negative bacterial outer membranes and has been reported to contribute to ruminal acidosis in cattle. Bacterial death and lysis are normal processes, and thus LPS is normally present in ruminal fluid. However, ruminal LPS concentration is much greater during subacute ruminal acidosis (SARA). Contrary to the widely known LPSs, ruminal LPS seems to be composed of a variety of LPS chemotypes that may interact with each other resulting in an LPS "mixture". Hypotheses regarding the influence of each specific ruminal bacterial specie to innate immunity during SARA, and the representativeness of the exclusive use of the Escherichia coli LPS to rumen epithelial tissue challenges, could expand our knowledge regarding SARA. In addition, possible correlation between the monomeric Toll-like Receptor 4 (TRL4) and the antagonistic penta-acylated lipid A of LPS could contribute to novel strategies to tackle this nutrition disorder.

Ruminal acidosis, bacterial changes, and lipopolysaccharides.

Acute and subacute ruminal acidosis (SARA) are common nutritional problems in both beef and dairy cattle. Therefore, the objective of this review is to describe how ruminal Gram-negative bacteria could contribute to the pathogenesis of ruminal acidoses, by releasing lipopolysaccharides (LPS; a component of their cell wall) in the ruminal fluid. When cattle consume excessive amounts of highly fermentable carbohydrates without prior adaptation, normal fermentation become disrupted. The fermentation of these carbohydrates quickly decreases ruminal pH due to the accumulation of short-chain fatty acids and lactate in the rumen. As a consequence, ruminal epithelium may be damaged and tissue function could be impaired, leading to a possible translocation of pathogenic substances from the rumen into the bloodstream. Such changes in fermentation are followed by an increase in Gram-positive bacteria while Gram-negative bacteria decrease. The lyses of Gram-negative bacteria during ruminal acidosis increase LPS concentration in the ruminal fluid. Because LPS is a highly proinflammatory endotoxin in the circulatory system, past studies have raised concerns regarding ruminal LPS contribution to the pathogenesis of ruminal acidosis. Although animals that undergo these disorders do not always have an immune response, recent studies showed that different Gram-negative bacteria have different LPS composition and toxicity, which may explain the differences in immune response. Given the diversity of Gram-negative bacteria in the rumen, evaluating the changes in the bacterial community during ruminal acidosis could be used as a way to identify which Gram-negative bacteria are associated with LPS release in the rumen. By identifying and targeting ruminal bacteria with possible pathogenic LPS, nutritional strategies could be created to overcome, or at least minimize, ruminal acidosis.

Effects of Flaxseed Oil and Vitamin E Supplementation on Digestibility and Milk Fatty Composition and Antioxidant Capacity in Water Buffaloes.

This study aimed to evaluate the effects of the supplementation of flaxseed oil and/or vitamin E on dry matter (DM) and nutrient digestibility, milk composition, fatty acid composition, and antioxidant capacity in buffalo milk. Four crossbred female dairy water buffaloes (97 ± 22 days in milk; 6.57 ± 2.2 kg of milk/day, mean ± SD) were distributed in a 4 × 4 Latin square design, with a 2 × 2 factorial arrangement (with or without flaxseed oil at 25 g/kg dry matter; with or without vitamin E at 375 IU/kg dry matter). The experimental period was divided into four periods of 21 days each (16 days for adaptation; five days for data collection). There were four treatments: control diet (no flaxseed oil and no added vitamin E); flaxseed oil diet (flaxseed oil at 25 g/kg DM); vitamin E diet (vitamin E at 375 IU/kg DM), and a combination of both flaxseed oil and vitamin E. The animals were fed total mixed ratios. For all response variables, there was no interaction between flaxseed oil and vitamin E. Flaxseed oil supplementation reduced neutral detergent fiber (NDF) and acid detergent fiber (ADF) apparent total tract digestibility, increased the n-3 fatty acid concentration in milk approximately three-fold while reducing the n-6/n-3 ratio from 9.3:1 to 2.4:1. Vitamin E supplementation increased NDF apparent total tract digestibility and milk total antioxidant capacity. Although there was no interaction between the treatments; flaxseed oil supplementation in lactating buffaloes increased polyunsaturated fatty acid, while vitamin E supplementation increased antioxidant capacity and decreased oxidation products.