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. Methane emissions from ruminants are an important environmental pollutant in the dairy industry. Direct-fed microbials have been reported to reduce methane emissions, improve ruminal fermentation, and benefit from improving energy efficiency. We evaluated the effect of a Bacillus subtilis on in vitro ruminal fermentation and methane production. Overall, our results indicate that the inclusion 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. Moreover, decreased methane production in ruminal fermentation, when supplemented with a mid-lactation diet but not when supplemented with an early or dry cow diet.

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.

Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli.

Summary: Short-stature corn hybrids that carry the brachytic2 (br2) mutation have increased plant standability compared with conventional tall and brown midrib (BMR) hybrids but similar dry matter yields to tall hybrids. Diets containing silage from 3 br2 hybrids were compared with diets containing silage from tall and BMR hybrids for their effects on cows in mid lactation when nutrient composition was similar. Cows fed diets with silage from the short hybrids produced as much or more energy-corrected milk (ECM) as cows fed diets with silage from tall or BMR hybrids. Intake was greater and feed efficiency was lower in cows fed short hybrids than in those fed BMR. We conclude that short-stature corn hybrids could be valuable sources of silage to improve production and profits on dairy farms. Created in BioRender.

The objective of this study was to determine the impacts of feeding a rumen-protected matrix of fat and protein from flaxseed and peas (LinPRO-R, LIN; approximately 21% fat, 24% CP, and 10.07% omega-3 FA; % of DM) on ruminal fermentation, nutrient degradability, and methane emissions, in vitro. Treatments were: 0 (0P), 2.5 (2.5P), 5 (5P), and 7.5% (7.5P) of DM inclusion of LIN, with LIN replacing portions of dried distiller's grains and calcium salts of long-chain fatty acids in the diet to maintain similar levels of CP and EE. In Experiment 1, 3 independent runs with 4 replicates per treatment were used, and 0.5 g of each TMR (16% CP, 33% NDF, 28% starch, 4.2% EE; as % of DM) was incubated in buffered ruminal content for 24 and 48 h, for a total of 6 runs, in glass serum bottles. Gas pressure was measured at 0, 3, 6, 9, 12, 24, and 48 h to estimate total gas and methane productions in the headspace and VFA and lactate concentrations in the fermentation liquid phase. Furthermore, OM degradation was measured at 24 and 48 h after bottles were opened. For Experiment 2, twice daily, 53 g of DM of the same treatments was provided to each fermenter. The treatments were arranged in a replicated 4 × 4 Latin square using 8 fermenters. Four 10-d experimental periods (7 d adaptation and 3 d sample collection) were done. Samples were collected from fermenter effluents at 3, 6, 9, and 24 h after morning feeding and composited to determine lactate and VFA concentrations and nutrient degradation. Experiment 1 data was analyzed using PROC GLIMMIX of SAS where treatment was a fixed effect and run was a random effect. Experiment 2 data were analyzed similarly, with square, fermenter within square, period, and the fermenter by treatment interaction being random effects. For Experiment 1 with increasing LIN, total gas volume and methane produced per g of OM degraded decreased linearly, whereas lactate concentration and OM degradability linearly increased. There was no effect of LIN inclusion level on any VFA proportion. For Experiment 2 Butyrate and isobutyrate proportions linearly decreased, and NDF degradation quadratically increased, with increasing inclusion of LIN. There were no differences for any other VFA proportions, the total concentration of all VFA, lactate concentration, or the degradation of DM, OM, or CP. Thus, inclusion of LIN, with its higher concentration of omega-3, can occur without impairing ruminal fermentation and nutrient degradation, and alters the fermentation profile to reduce total gas volume and methane produced per g of OM degraded as well as favor other VFA rather than butyrate or isobutyrate and increasing fiber degradation.

The objective of this study was to evaluate the effects of different slow-release urea (SRU) compounds on ruminal fermentation, nutrient degradation, and nitrogen (N) utilization in a dual-flow continuous-culture system (experiment 1), as well as ammonia-N (NH₃-N) release rate in a batch culture system (experiment 2). In experiment 1, 8 fermenters were used in a replicated 4 × 4 Latin square design with 4 treatments and 4 experimental periods. The treatments were formulated to contain the same amount of N, differing in the source of nonprotein N, such as control (CON), with noncoated urea at 0.62% DM; partial inclusion of SRU compound 1 (SRU1) at 0.51% DM; partial inclusion of SRU compound 2 (SRU2) at 0.51% DM; and partial inclusion of SRU compound 3 (SRU3) at 0.51% DM. Each period lasted 10 d. The last 3 d of each period were used for sample collection. Samples were collected for pH, lactate, VFA, NH₃-N kinetics, nutrient degradability, and N metabolism. In experiment 2, a batch culture incubation was conducted as a complete randomized block design, using 3 Erlenmeyer flasks per treatment in 3 runs. Each treatment contained 1 of the 4 NPN sources used in experiment 1, (CL, S1, S2, S3) or without any NPN (blank, BK), and samples were collected at different time points for NH₃ analysis. All flasks, except for BK, contained equal amounts of 21.56 mg N, and all flasks were inoculated with 260 mL of a 1:2 mixture of ruminal fluid and nutritive solution. Data of both experiments 1 and 2 were analyzed using the MIXED procedure of SAS. In experiment 1, there were no effects of treatment on pH or NH₃-N kinetics. There were no effects of treatments on lactate kinetics; however, there was an interaction between treatment and time. For 24-h VFA pool, there were treatment effects on acetate, propionate, acetate:propionate ratio (A:P), and branched-chain (BC)VFA proportion. Compared with CON, SRU1 had lower A:P ratio and acetate proportion, with greater proportion of propionate, which could represent a favorable fermentation partner compared with CON. Treatment SRU1 had lower BCVFA proportion than the other treatments, which indicates less protein degradation. There were no treatment effects for nutrient degradability and N flow. Based on observations in experiment 1, SRU1 could have the potential of improving ruminal fermentation and N utilization. Moreover, the different SRU compounds had different fermentation patterns according to their VFA profiles. In experiment 2, there were significant effects for treatment and time, and a tendency for treatment by time interaction effect. The N release rate of S1 was similar to CL and faster than S2 and 3, and the differences in N release rates could be detected as early as at 0.75 h of incubation. Thus, S1 may not be as slow degrading when compared with S2 and S3. In conclusion, no effects were found on nutrient degradability and N utilization. However, the different SRU compounds had different N release rates, which could affect ruminal fermentation pattern in different diets.