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.

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.

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.

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.

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.

Optimum grape pomace proportion in feedlot cattle diets: ruminal fermentation, total tract nutrient digestibility, nitrogen utilization, and blood metabolites.

Because of its high content of polyphenolic compounds, the dietary inclusion of grape pomace (GP) in ruminant diets can reduce reactive nitrogen (N) and methane emissions and enhance the shelf life and beneficial fatty acids (FAs) content of meat. However, the dietary inclusion of GP beyond a threshold that is still to be determined for feedlot cattle can also compromise nutrient supply and, thus, growth performance. This study investigated the optimum proportion of GP in finishing cattle diets. Nutrient intake and apparent total tract digestion, ruminal pH and fermentation, estimated microbial protein synthesis, route of N excretion, and blood metabolites were measured. Six ruminally fistulated crossbred beef heifers (mean initial body weight ± SD: 714 ± 50.7 kg) were used in a replicated 3 × 3 Latin square with 21-d periods. Dietary treatments were 0%, 15%, and 30% of dietary dry matter (DM) as GP, with diets containing 84%, 69%, and 54% dry-rolled barley grain, respectively. There was a linear increase (P = 0.07) in DM intake and quadratic change (P ≤ 0.01) in neutral detergent fiber (NDF) intake. There was a quadratic change (P ≤ 0.04) in apparent total tract DM, NDF, and crude protein digestibility as dietary GP content increased. However, there were no treatment effects (P ≥ 0.18) on total ruminal short-chain FA concentration and duration and area pH < 6.2, 5.8, and 5.5. Although N intake did not differ (269, 262, 253 g/d; P = 0.33) across dietary treatments, feeding GP led to a tendency for a quadratic change (P ≤ 0.07) in ruminal ammonia-N and plasma urea-N concentrations. Total N excretion also changed (quadratic, P = 0.03) because of changes (quadratic, P = 0.02) in fecal N excretion as urinary excretion of N and urea-N did not differ (P ≥ 0.15) across treatments. Feeding GP led to quadratic changes (P ≤ 0.01) in fecal excretion of fiber-bound N. Microbial N flow and apparent N retention also changed (quadratic, P ≤ 0.04) as dietary GP proportion increased. In conclusion, responses to dietary GP proportion were mostly quadratic with indications that nutrient supply as reflected by changes in apparent total tract nutrient digestibility, microbial N supply, and apparent N retention could be compromised beyond a 15% dietary inclusion level.

Effects of maturity at harvest on the nutritive value and ruminal digestion of Eragrostis tef (cv. Moxie) when fed to beef cattle.

Teff (Eragrostis tef cv. Moxie), a warm-season annual grass, could be an excellent forage for beef cattle. However, there is limited information on its nutritive value to cattle when harvested at different stages of maturity. Thus, the objective of this research was to determine the effect of feeding teff hay harvested at the boot (BT), early-heading (EH), or late-heading (LH) stages of maturity on nutrient intake, ruminal fermentation characteristics, omasal nutrient flow, and N utilization in beef cattle. Six ruminally cannulated beef heifers (mean initial BW ± SD, 476 ± 32.6) were used in a replicated 3 × 3 Latin square design with 28-d periods (18 d for adaptation and 10 d for measurements). Dry matter intake was measured daily. Indwelling pH loggers were used to measure ruminal pH from days 21 to 28. Ruminal fluid and omasal digesta were collected from days 26 to 28 to determine fermentation characteristics and omasal nutrient flow. Fecal and urine samples to quantify N excretion were also collected (days 26 to 28). Blood samples for plasma urea-N (PUN) determination were collected 3 h post-feeding on day 28. There were no changes (P > 0.28) in the ADF or NDF content of teff with advancing maturity, but indigestible NDF increased (P < 0.01) with increasing maturity. Maturity had no effect (P ≥ 0.14) on DMI, and ruminal total short-chain fatty acid (SCFA) concentration, pH, digestibility, and outflow of DM, OM, NDF, ADF, and CP. However, the CP content of BT hay was greater (P < 0.01) than for EH and LH hay (18.1, 14.1, and 11.5%, respectively, DM basis), and this resulted in the higher CP intake (P < 0.01) for heifers fed the BT than the EH and LH hay. Consequently, ruminal ammonia-N (NH3-N) concentration was greater (P < 0.01) for heifers fed BT than EH and LH hay, thereby possibly explaining the tendency for a decrease (P = 0.08) in PUN concentration, and a decrease (P < 0.01) in the excretion of total N, urine N, and urea-N (UUN) with advancing maturity. However, fecal N excretion (g/d) did not differ (P = 0.76). In conclusion, despite a decrease in CP intake and ruminal NH3-N concentration, feeding beef heifers EH and LH compared to BT teff hay did not compromise ruminal digestion and outflow of DM, OM, NDF, ADF, and CP, and microbial protein synthesis. Advancing maturity in teff hay also resulted in a decrease in the excretion of total N and urine N and UUN when fed to cattle.