Rumen Inoculum Research Articles

A systematic study of sheep faeces to be used as an alternative inoculum source in the Hohenheim gas test

ABSTRACT In vitro studies of gas production (GP) using rumen fluid obtained from rumen-cannulated animals are common in feed evaluation for ruminants. The objective was to compare sheep faeces with rumen fluid from dairy cows as inocula in the Hohenheim gas test (HGT) using a large number of different-quality feeds. It then was evaluated whether GP obtained by using rumen inoculum (RI-GP) can be estimated from GP using faecal inoculum (FI-GP). Ninety feeds for ruminants including roughages (n = 27), total mixed rations (n = 6), commercial compound feeds for dairy cows (n = 18), cereal grains (n = 10), energy-rich by-products (n = 5), legume grains (n = 6), oilseed meals (n = 6), and other protein-rich feeds (n = 12) were incubated with either rumen inoculum (RI) or faecal inoculum (FI), and GP was recorded at 2, 4, 8, 12, 24, 48, and 72 h. Nonlinear equations were fitted to the GP data to determine GP kinetic parameters. Correlations between RI-GP and FI-GP were calculated for different time points. Linear regressions were calculated to estimate RI-GP from FI-GP using the entire data set or subsets of roughages and concentrates. GP kinetic parameters were lower for all feed categories when incubated with FI instead of RI. On average of all feed samples, the potential GP was 9 mL/200 mg dry matter (DM) lower and the rate of GP was 3.1%/h lower with FI than RI. The estimation of kinetic data for FI included a lag phase of 1.51 h on average, whereas no lag phase was estimated for RI. Estimated parameters indicated an overall lower fermentation activity of FI than RI. However, there was a very similar progression of GP curves of RI-GP and FI-GP. RI-GP after 24 h was significantly correlated with FI-GP in the time period of 24 to 60 h (r = 0.973 – 0.982, including all feed samples). Linear regression analysis showed that RI-GP after 24 h can be estimated from FI-GP after 24 h (Slope = 1.02, R2 = 0.97) and 48 h (Slope = 1.1, R2 = 0.97) including the complete data set. Splitting the data set into roughages and concentrate feeds did not lead to distinctly higher estimation accuracy. In conclusion, sheep faeces can be used as an alternative inoculum for studying a wide range of different-quality feedstuffs. The standard gas test may be modified without the need for rumen-cannulated animals.

Impacts of Protein and Energy Levels on Rumen Fermentation and Microbial Activity Under Different Incubation Temperatures

This study aimed to explore the effects of different incubation temperatures on ruminal fermentation and rumen microorganisms and determine the appropriate protein and energy levels to enhance microbial protein synthesis using an in vitro system. Rumen inoculum was collected from two fistulated Holstein heifers (trial 1: BW: 652.3 kg ± 25.2; trial 2: BW: 683.3 kg ± 30.2) and assessed using a closed-batch culture system. The experimental model employed a 2 × 5 factorial arrangement using incubation temperatures set to 39 and 41 °C, with protein levels set to 12.0, 13.5, 15.0, 16.5, and 18.0% of DM in trial 1 or with energy levels set to 2.4, 2.5, 2.6, 2.7, and 2.8 Mcal/kg of DM in trial 2. The data were analyzed using the MIXED procedure. The results showed increased (p < 0.05) NH3-N concentrations and total volatile fatty acids (TVFAs) with higher incubation temperatures, while the liquid-associated bacterial (LAB) amounts decreased (p < 0.05) in trials 1 and 2. The interaction between the energy level and incubation temperature affected (p < 0.05) the LAB protein levels in trial 2. Higher protein levels led to increased (p < 0.05) NH3-N and acetate concentrations, but it decreased (p < 0.05) the propionate percentage. Conversely, higher energy levels decreased (p < 0.05) the amount of acetate and increased the propionate concentration, altering the acetate-to-propionate ratio. However, no interaction involving TVFA and LAB was observed between the incubation temperature and the protein or energy levels. Changes in the NH3-N, TVFAs, and LAB protein amounts were observed under different incubation temperatures and energy levels. In conclusion, these findings provide insight into the metabolic adaptation under different ruminal temperatures and the impacts of dietary adjustments on rumen fermentation and microbial activity. However, there are limitations to replicating the complex physiological responses that occur within the whole body solely through in vitro experiments.

The Anti-Methanogenic Activity of Lovastatin in Batch Cultures Using Rumen Inoculum from Sheep, Goats, and Cows

Enteric methanogenesis in ruminants is identified as one of the primary anthropogenic sources of total atmospheric methane. Recent evidence suggests that rumen methanogenesis is significantly suppressed by lovastatin. Nevertheless, it has not been reported whether the methane reduction by lovastatin depends on ruminant livestock type, nor has fiber degradability been examined. The current research aimed to analyze the in vitro effect of lovastatin on the major fermentation end-products, gas production (GP) kinetics, and fiber degradation of a forage-based diet using rumen inoculum from sheep, goats, and cows. The experiment was conducted as a 3 × 3 factorial arrangement of treatments (dose of lovastatin: 0, 80, and 160 mg/L and three inoculum sources) in a completely randomized design. The results suggested that lovastatin did not affect the GP kinetics parameters. The anti-methanogenic properties of lovastatin were variable depending on dose and inoculum source. Lovastatin demonstrated a superior methane-lowering effect in sheep rumen inoculum compared with goat and cow inocula. The total volatile fatty acid (VFA) production was unaffected by lovastatin, but changes in acetate and valerate proportions were registered. Remarkably, lovastatin decreased the NH3-N concentration with goat and sheep inocula and the in vitro neutral fiber detergent (NDF) degradation for all inoculum sources.

In vitro evaluation of ruminal digestibility and fermentation characteristics of local feedstuff-based beef cattle ration

Background: Optimum productivity of beef cattle is achieved with adequate feed supply, both in quality and quantity. Consumption of local feedstuff Neptunia plena L. Benth and Leersia hexandra Swartz as a ration by the animal subject is expected to promote cost efficiency and production, as well as provide essential nutrition needs. Therefore, this research aimed to evaluate dry matter digestibility (DMD), organic matter digestibility (OMD), N-NH3 production, and volatile fatty acid (VFA) in beef cattle. Methods: Feed and rumen inoculum samples were prepared and analyzed for their proximate contents. There were five treatment groups based on the diet received by beef cattle. In vitro approaches were used to determine the DMD, OMD, N-NH3 production, and VFA in beef cattle. The research was conducted in the Laboratory of Feed Nutrient Science, Faculty of Animal Husbandry and Agriculture, Diponegoro University, Semarang. The data were analyzed using ANOVA at a significance level of 95%, and a Duncan Multiple Range Test. Results: The results showed that the highest DMD (P<0.05) was derived from T5 (56.47%), followed by T4 (56.45%) and T3 (55.90%). T5=62.40% significantly (P<0.05) generated the highest OMD followed by T4=61.95% and T3=60.82%. This treatment had the highest N-NH3 value, namely 5.02 mM, compared with T3=4.55 mM, T4=4.50 mM, T2=4.22 mM, and T1=3.99 mM. Furthermore, T5 had the highest VFA (P<0.05) compared with T4, T3, T2, and T1 with the value of 150.5, 133.0, 130.5, 130.0, and 123.5 mM, respectively. Conclusions: The local feedstuff-based ration contributed to beef cattle production.

Relationship between predicted in vivo and observed in vivo methane production from dairy cows fed a grass-silage based diet with barley, oats, or dehulled oats as a concentrate supplement

The objectives of this study were to compare predicted in vivo methane (CH4) values based on data from an in vitro gas production experiment with observed in vivo values measured by the GreenFeed system, and to investigate the effect of the diet fed to donor animals of rumen inoculum (RI) on predicted in vivo CH4 production. For this purpose, we conducted an in vivo experiment simultaneously with an in vitro gas production experiment. The in vivo experiment (previously published) was a 4 × 4 Latin square design including 16 Nordic Red dairy cows. Cows were fed 60% grass silage and 40% concentrate which consisted of either barley, oats with hulls (hulled oats), dehulled oats, or a 50:50 mixture of hulled and dehulled oats on dry matter basis. The in vitro experiment was a 2 × 4 factorial design replicated in 4 runs. The in vitro diets were incubated for 48 h in two types of RI and formulated according to the diets fed in vivo. The RI was obtained from cows fed either barley (two cows) or hulled oats (two cows) as concentrate in the in vivo experiment. A set of models were applied to the gas and CH4 data obtained from the in vitro system to predict in vivo total gas and CH4 production. For the comparison between predicted in vivo and observed in vivo CH4, two different mean retention times (MRT of 35 and 50 h) in the rumen were used for the predictions. In the in vitro experiment, incubation residues were determined for organic matter digestibility and volatile fatty acids at 48 h of incubation. Assuming a MRT of 35 h in the rumen resulted in a significant relationship (P = 0.04) between predicted in vivo and observed in vivo CH4 yield (g/kg dry matter) with an R-square of 0.91 and a root mean square error of 0.20. Ranking of the diets in terms of their CH4 production was consistent between the in vitro and in vivo experiment. There were no significant interactions between diet and RI for any of the investigated parameters (P = 0.40). Rumen inoculum did not affect organic matter digestibility or total volatile fatty acid production. In conclusion, there was a good agreement between predicted in vivo and observed in vivo CH4 values. In addition, the diet of RI donor animal did not influence the comparison of diets in terms of CH4 production.

Flavonoids from citrus peel display potential synergistic effects on inhibiting rumen methanogenesis and ammoniagenesis: a microbiome perspective.

Flavonoids have been recognized as potential phytochemicals to reduce enteric methane (CH4) production and improve rumen nitrogen efficiency in ruminants. We evaluated whether naringin, hesperidin, their combination, or a mixed citrus flavonoid extract (CFE) as additives can inhibit methanogenesis and ammoniagenesis in dairy cows using an in vitro rumen batch refermentation system. The rumen inocula from dairy cows were incubated in batch cultures with five groups: no addition (CON), hesperidin (20 g/kg DM), naringin (20 g/kg DM), hesperidin + naringin (10 g/kg DM of hesperidin + 10 g/kg DM of naringin), and CFE (20 g/kg DM). The combination of naringin plus hesperidin and CFE achieved greater reductions in CH4 and ammonia production compared to either naringin or hesperidin alone. Microbiome analysis revealed that the decrease in CH4 emissions may have been caused by both the direct inhibitory impact of citrus flavonoids on Methanobrevibacter and a simultaneous decrease in protozoa Isotricha abundance. The relatively lower proportion of Entodinium in naringin plus hesperidin or CFE was responsible for the lower ammonia concentration. These results suggest that citrus flavonoids possess potential synergistic effects on mitigating ruminal CH4 emissions by cows and improving nitrogen utilization.

The influence of virginiamycin on digestion and ruminal parameters under feedlot conditions.

This experiment aimed to assess the impact of virginiamycin on in vitro gas production dynamics, rumen kinetics, and nutrient digestibility in beef steers fed a grain-based diet. Nine ruminally cannulated British-crossbred steers (596 ± 49kg) were assigned to this experiment. Animals were housed in three pens (n = 3/pen) equipped with a Calan gate feed system and water troughs. Pens were enrolled in a 3 × 3 Latin square design containing three periods of 16 d, and a 5-d washout interval between periods. Dietary treatments consisted of virginiamycin (VM) administration at 0 (VM0), 180 (VM180), or 240mg/d (VM240). During days 15 and 16 of each period, about 600mL of rumen fluid and urine samples were collected before (0h), and at 4, 8, 12, and 16h after the morning feed (0730 hours), rumen inoculum was used to take pH and redox potential measurements immediately after collection using a portable pH and redox meter, and subsamples were taken for volatile fatty acids (VFA) and NH3-N analyses, and urine samples were composited daily and analyzed for creatinine and purine derivatives (PD) content to estimate microbial crude protein flow. During the 4-h post-morning feed rumen collection, rumen inoculum was utilized to perform in vitro gas production measurements. Fecal samples were collected on day 16 of each period to estimate nutrient digestibility using acid detergent insoluble ash as an internal marker. Animals were considered the experimental unit for the statistical analyses, and periods and squares were included as random variables. The total and rate of gas production were similar among treatments (P ≥ 0.17). The second-pool (i.e., fiber) gas production increased linearly as VM inclusion increased (P = 0.01), with VM240 being greater compared to VM180 and VM0 (7.84, 6.94, and 6.89mL, respectively). Ruminal pH linearly increased as VM increased, with VM240 being greater than VM0 and VM180 intermediate (5.90, 5.82, and 5.86, respectively; P = 0.03). The VFA concentrations did not differ (P ≥ 0.13), but the acetate-to-propionate ratio was the highest in VM240 (P = 0.005). Branched-chain VFA increased (P ≤ 0.03) while lactate concentrations decreased (P = 0.005) linearly with VM. The ruminal NH3-N concentration was the lowest in the VM0 (P = 0.006). The estimated absorbed PD, purine derivative to creatinine index, and microbial N flow increased linearly with VM (P ≤ 0.07). The provision of VM influenced rumen dynamics in a dose-dependent manner.

Effects of ratios of yak to cattle inocula on methane production and fiber digestion in rumen in vitro cultures1

Yaks are well-adapted to the harsh environment of the Tibetan plateau, and they emit less enteric methane (CH4) and digest poor-quality forage better than cattle. To examine the potential of yak rumen inoculum to mitigate CH4 production and improve digestibility in cattle, we incubated substrate with rumen inoculum from yak (YRI) and cattle (CRI) in vitro in five ratios (YRI: CRI): 0:100 (control), (2) 25:75, (3) 50:50, (4) 75:25 and (5) 100:0 for 72 h. The YRI: CRI ratios of 50:50, 75:25 and 100:0 produced less total gas and CH4 and accumulated less hydrogen (H2) than 0: 100 (control) at most time points. From 12 h onwards, there was a linear decrease (P < 0.05) in carbon dioxide (CO2) production with increasing YRI: CRI ratio. At 72 h, the ratios of 50:50 and 75:25 had higher dry matter (+7.71% and +4.11%, respectively), as well as higher acid detergent fiber digestibility (+15.5% and +7.61%, respectively), when compared to the 0:100 ratio (P < 0.05). Increasing the proportion of YRI generally increased total VFA concentrations, and, concomitantly, decreased the proportion of metabolic hydrogen ([2H]) incorporated into CH4, and decreased the recovery of [2H]. The lower [2H] recovery indicates unknown [2H] sinks in the culture. Estimated Gibbs free energy changes (∆G) for reductive acetogenesis were negative, indicating the thermodynamic feasibility of this process. It would be beneficial to identify: 1) the alternative [2H] sinks, which could help mitigate CH4 emission, and 2) core microbes involved in fiber digestion. This experiment supported lower CH4 emission and greater nutrient digestibility of yaks compared to cattle. Multi-omics combined with microbial culture technologies developed in recent years could help to better understand fermentation differences among species.

Effect of sodium sulfate concentration in drinking water for beef heifers, and the in vitro effect of bismuth subsalicylate on H2S production and fiber disappearance

This study evaluated the effects of ( i) increasing the concentration of sulfate in drinking water on dry matter intake (DMI), water intake, ruminal fermentation, and apparent total-tract digestibility, and ( ii) water sulfate concentration and bismuth subsalicylate (BSS) dose on in vitro ruminal fermentation. Eight ruminally cannulated beef heifers (382 ± 45 kg) were used in a replicated incomplete 3 × 3 Latin square design. Water treatments contained low (342 ± 29 mg/L; LS), medium (2785 ± 72 mg/L; MS), or high (4948 ± 163 mg/L; HS) sulfate. Ruminal digesta was collected to evaluate water sulfate and BSS dose (0.0%, 0.2%, 0.4%, and 0.6% dry matter) on ruminal H2S production in vitro. Water intake increased linearly as water sulfate concentration increased ( p = 0.002), but DMI was not affected. Heifers drinking MS and HS had greater ruminal H2S at 10.5 h after water provision than LS (sulfate × time, p &lt; 0.001). In vitro H2S production (µg and µg/mL of rumen inoculum) increased and plateaued with increasing sulfate ( p &lt; 0.001) and was linearly reduced ( p &lt; 0.001) by increasing BSS dose. Increasing water sulfate concentrations did not negatively affect water or feed intake but increased ruminal H2S concentrations. Bismuth subsalicylate may reduce H2S production.

PSVI-27 Evaluation of Coated and Non-Coated Seed Species Pertinent to the Great Basin in Response to in Vitro Ruminal Incubation

Abstract Target grazing has exhibited itself as an effective method of invasive weed mitigation across degraded rangelands in the Great Basin. Additionally, if the seeds of target species are supplemented into the diets of ruminants, they can serve as vectors for the dispersal of native seed species through fecal pats, which may prove to be a useful tactic for rangeland rehabilitation. However, a challenge associated with seed supplementation, is that the seeds of many target species are sufficiently degraded in the rumen. Therefore, the aim of this study was to evaluate the effects of seed coating technologies on germination rates post ruminal in vitro incubation. In vitro incubations were performed on seven species of seeds pertinent to the Great Basin: Achnatherum hymenoides (ACHY), Achillea millefolium (ACMI), Agropyron cristatum (AGCR), Atriplex canascens (ATCA), Elymus elymoides (ELEL), Pseudoregneria spicata (PSSP), and Sphaeralcea grossulariifolia (SPRT) uncoated (control) or coated with either Polyvinylpyrrolidone (PVP) or PVP+ ethocel, as well as uncoated Bromus tectorum (BRTE) seeds. A Daisy II incubator (Ankom Technology, Macedon, NY) was used for the in vitro analysis with rumen inoculum from two rumen-cannulated crossbred Angus steers (body weight = 910.5 ± 34.5 kg) and a buffer ratio of 1:4. Two F57 filter bags containing 1 g of seeds per treatment (control, PVP or PVP+ethocel) were then placed inside of the Daisy II incubator and kept at 39°C for 12 and 24 h for the control treatments, and for 12, 24, and 48 h for the PVP and PVP+ethocel coating treatments. For uncoated seeds, ACHY germination by 24 h was greater when compared with 0 h (P &amp;lt; 0.05), whereas ATCA germination rate decreased linearly by 12 h (P &amp;lt; 0.05). No differences were observed over time on germination rates of BRTE, as well as all of the other uncoated seeds (P &amp;gt; 0.05). When seeds were coated with PVP, ATCA germination rates started to linearly decrease by 12 h (P &amp;lt; 0.05), whereas PVP coated ACMI germination rates only started to decrease by 24 h (P &amp;lt; 0.05). The germination rates of PVP coated ELEL and PSSP seeds only started to significantly decrease by 48 h (P &amp;lt; 0.05). When coated with PVP+ethocel, the observed germination rates were similar to those observed for PVP coated seeds. However, when compared with uncoated seeds, coating with PVP+ethocel seem to decrease germination rates of ACMI and ATCA by 0 h (P &amp;lt; 0.05). No changes on germination rates were observed over time for coated and uncoated AGCR and SPRT (P &amp;gt; 0.05). In conclusion, seed germination rates after in vitro incubation were not increased by coating the seeds with PVP or PVP+ethocel.

342 Effectiveness of Liver Abscess-Controlling Antibiotic on Rumen Kinetics of Beef Steers Consuming a High-Grain Diet

Abstract Virginiamycin (VM) possesses antimicrobial properties due to its blocking of protein synthase in Gram-positive bacteria, allowing it to reduce lactic acidosis and the incidence of liver abscesses in ruminants. Ruminal acidosis is a common metabolic disorder that affects feedlot and dairy cattle and occurs when the supply of organic acids from fermentation exceeds its absorption and degradation, accumulating acid content in the rumen. The objective of this study was to evaluate the effects of three different doses of VM administration on in vivo and in vitro ruminal digestion kinetics of beef steers consuming a high-grain diet [metabolizable energy (ME): 2.99 Mcal/kg; Crude Protein (CP): 15.2 % dry matter basis (DM)]. Nine ruminally cannulated British-crossbred steers (596 ± 49 kg) were assigned to this experiment. Animals were housed in three pens (n = 3/pen) equipped with a Calan gate feed system and water trough. Pens were enrolled in a 3×3 Latin square design containing three periods of 16 d, and a 5-d washout interval between periods. Dietary treatments consisted of VM administration at 0 mg/d (VM0), 180 mg/d (VM180), and 240 mg/d (VM240). During d 15 and 16 of each period, about 600 mL of rumen fluid was collected before (0 h) and at 4, 8, 12, and 16 h relative to the morning feed (0730 h) pH and redox potential (Eh) measurements were taken immediately after collection using a portable pH and redox meter, and subsamples were taken for volatile fatty acids (VFA), and NH3-N analyses. During the 4-h post-morning feed rumen collection, rumen inoculum was utilized to perform in vitro gas production (IVGP) measurements. All statistical procedures were performed using SAS software where steer was considered the experimental unit, and period and square were included as random. Acetate, propionate, and total VFA did not differ among treatments (P ≥ 0.50), whereas butyrate increased linearly (P = 0.033) as the VM dose increased. Acetate:propionate ratio did not differ among treatments (P = 0.273). Lactate concentration decreased linearly (P = 0.027) as the VM dose increased; likewise, pH increased linearly (P = 0.019) as the VM dose increased. Branched-chain VFA and NH3-N concentrations increased linearly (P ≤ 0.056) as the VM dose increased. The total and rate of gas production were similar among treatments (P ≥ 0.161). However, second-pool gas production increased linearly as VM inclusion increased (P = 0.023). The in vitro neutral detergent fiber digestibility did not differ among treatments (P = 0.984). The provision of VM altered the rumen dynamics in a dose-dependent manner. Animals consuming high-grain diets will likely promote rumen health through a more stable pH and fermentation profile.

Rumen Content from Slaughterhouse as an Alternative Inoculum Source for In Vitro Analysis of Feeds: A Multivariate Approach

The objective of the current study was to compare the rumen inoculum of slaughtered cattle with the ruminal inoculum of cannulated cattle; additionally, determine its reliability in the in vitro analysis of ruminant feeds throughout a multivariate approach. Five male bovines (weight 320 ± 9.4 kg; mean ± standard deviation) provided with ruminal cannula and between five and seven bovines slaughtered in slaughterhouse were used. The evaluations were carried out following a completely randomized design. The data obtained were subjected to different multivariate analyzes to determine the reliability of the ruminal inoculum of animals slaughtered in commercial slaughterhouses compared to that obtained from cannulated animals. The relative contribution indicated that the in vitro dry matter digestibility (IVDMD, 50.75%) and in vitro neutral detergent fiber digestibility (IVNDFD, 29.83%) analysis influence 80.13% of the results to determine the reliability of the ruminal inoculum from slaughtered cattle. Furthermore, it was determined that the first two principal components (IVNDFD and acetic acid production) are the ones that influence the results by 89.87%. The grouping of diets using the Tocher optimization method and the dendrogram shows the formation of six groups and two groups, respectively. The grouping shows that the ruminal inoculum source was not the limiting parameter in the evaluation. Rumen inoculum from cattle slaughtered in a commercial slaughterhouse (with unknown diet) has potential as an alternative for the in vitro analysis of cattle feed, provided that the lignin concentration in the diet is less than 35.5 g/kg DM.

PSIV-25 Investigating the Use of Cover Crop Inclusion and Preservation in Silages with Addition of Molasses, Homofermentative and Heterofermentative Bacterial Mixes

Abstract The use of cover crops in agriculture has had positive effects on soil health and sustainability. In the western US, utilizing cover crops for silage production provides many of the same soil health benefits while providing forage for ruminants, serving multiple sectors with the same water costs. Therefore, the aim of this study was to determine the effects of mixed grass and legume silage combined with different inoculants on silage quality and in vitro rumen fermentation. Four silage mixes (100% sorghum [0SB], 67% sorghum + 33% soybean [33SB], 33% sorghum + 67% soybean [67SB], and 100% soybean [100SB]) were prepared and 6 treatments were applied to each mix 1) homofermentative lactic acid producing bacteria (LB), 2) heterofermentative lactic and acetic acid producing bacteria (LB+), 3) molasses, 4) molasses and LB (MLB), 5) molasses and LB+ (MLB+), and 6) no inoculant (control). Silage mixes plus inoculants were made in triplicate, vacuum sealed in plastic bags, and ensiled for 60 days at 25°C. Once bags were opened, 200 mg air-dried of each silage treatment were incubated in 14 mL of buffering media and 4 mL of rumen inoculum. Each incubation spanned for 48 h with gas production measured at 0, 2, 4, 8, 12, 18, 24, 36, and 48-h post-incubation. Total gas production (TGP), pH, ammonia and total volatile fatty acids (VFA) were determined. Ammonia concentrations were greater when samples were inoculated with LB, and the least for MLB+ (P &amp;lt; 0.05); however, no differences were observed between silage mixes (P &amp;gt; 0.05). Total VFA concentrations were similar across treatments (P &amp;gt; 0.05). However, butyrate was greater for 33SB silages mixes inoculated with MLB (P &amp;gt; 0.05). Valerate concentration was greater for all treatments inoculated with molasses (P &amp;lt; 0.001). Isobutyrate and Isovalerate concentrations were only different when comparing between silage concentrations, where 0SB silages had the greatest concentration (P &amp;lt; 0.05). Although no differences were observed on TGP (P &amp;gt; 0.05), pH of samples incubated with no inoculant, LB, and LB+ were higher than all samples inoculated with molasses (P &amp;lt; 0.001). In conclusion, the use of mixed grass and legume silages with up to 33% soybean inclusion appears to be beneficial to ruminal fermentation. However, variable results might be produced depending on the inoculant used. The incorporation of these cover crop varietals and their subsequent inclusion in silos for livestock feed represents a viable and effective application for producers currently utilizing cover crops in their operations.

PSXIII-20 The Effect of Hemp Varieties and Cannabidiol Concentrations on the in Vitro Ruminal Microbiome

Abstract This study aimed to evaluate the effect of hemp varieties and cannabidiol (CBD) concentrations on in vitro ruminal microbiota diversity and abundance relative do alfalfa (Medicago sativa). Two ruminally cannulated Angus steers were used as inoculum donors for in vitro incubation. Two 160-mL serum bottles containing 200 mg air-dried of either alfalfa, fedora 17 (F17) and felina 32 after late harvest (F32S) or without seeds (F32O) were incubated in 14 mL of buffering media and 4 mL of rumen inoculum. A stock solution of 1mg/mL of CBD was prepared and added in increasing concentrations into each bottle to reach a final concentration of 0.001%, 0.002%, 0.003%, and 0.004% of CBD. After a period of 48 hours, the whole content inside of the serum bottles was used immediately for DNA extraction followed by 16S rRNA sequencing with an Oxford Nanopore technologies MiniON. Amplicon sequence data were bioinformatically processed in QIIME2 version 2022.2. Alpha diversity was measured by the Shannon diversity index. Bray-Curtis’s dissimilarity matrix was constructed to estimate the differences among the samples and CBD levels, and differential abundance was then evaluated using analysis of compositions of microbiomes with bias correction (ANCOM) testing. Statistical significance was established at P &amp;lt; 0.05. All data were visualized in R version 4.1.2 (R Core Team, 2022) using the qiime2R package. Shannon diversity index was the highest for alfalfa (P &amp;lt; 0.05), but no difference was observed among the different CBD inclusion levels. When looking at beta diversity through Bray-Curtis, differences were only observed when comparing the different alfalfa and hemp varieties (P &amp;lt; 0.05), where alfalfa grouped separately from all the hemp samples. The most abundant phyla included Firmicute, Bacteroidetes, Proteobacteria, and Fusobacteria (P &amp;lt; 0.05). At family level, alfalfa samples had greater Aerococcaceae and Enterobacteriaceae than all the hemp treatments, but less Lactobacillales (unclassified), Streptococcaceae, Succinivibrionaceae, and Bacilli (unclassified) than F32S and F17 (P &amp;lt; 0.05). In conclusion, these results suggest that sample type might play a larger role on microbiome than CBD lconcentrations. Further studies are required to determine if greater concentrations of CBD than those examined herein could also lead to a shift on rumen microbes.

The Dose-Dependent Role of Sage, Clove, and Pine Essential Oils in Modulating Ruminal Fermentation and Biohydrogenation of Polyunsaturated Fatty Acids: A Promising Strategy to Reduce Methane Emissions and Enhance the Nutritional Profile of Ruminant Products

The livestock industry significantly contributes to greenhouse gas emissions, with ruminant animals, including cows, sheep, and goats, being responsible for a substantial share of these emissions due to methane production. Reducing methane emissions from ruminants is crucial for mitigating the environmental impact of livestock production. Additionally, there has been a growing interest in improving the nutritional quality of ruminant products through modifying their profile of fatty acids. The current study aimed to investigate the potential of sage (SAG), pine (PIN), and clove (CLO) essential oils as natural additives for modulating in vitro ruminal fermentation characteristics and biohydrogenation of polyunsaturated fatty acids (PUFA). Within the current experiment, three dose levels (300, 600, and 900 mg/L) of essential oils were evaluated using rumen inoculum from three mature Dalagh ewes (58 ± 2.84 kg body weight). The results revealed that the essential oils had a significant impact on gas production, methane and carbon dioxide production, ruminal fermentation parameters, and ruminal biohydrogenation of dietary PUFAs. The essential oil treatments resulted in reduced gas production compared with the control group. Methane production was significantly reduced by all doses of the essential oils, with the highest dose of CLO resulting in the lowest methane production. In addition, the essential oils affected ruminal fermentation parameters, including pH, ammonia concentration, and production of total volatile fatty acids. Promising modifications in ruminal biohydrogenation of PUFAs and the profile of fatty acids were also observed in the current study. These findings suggest that SAG, Pin, and CLO hold promise in mitigating methane emissions and improve the nutritional value of ruminant products. Further investigation is required to evaluate their effectiveness in practical feeding strategies for livestock.

Technical note: Evaluation of an alternative automatic heating-stirring system in the determination of in vitro ruminal dry matter digestibility of forages using the Tilley and Terry method

This study evaluated an alternative heating-stirring system in the determination of in vitro ruminal digestibility of dry matter (IVRDMD) of forages using the Tilley and Terry (TT) method. For this purpose, the IVRDMD of three forage species (Marandu, Tifton 85, and Mombasa) was determined by incubating 500 mg of each dried and ground (1 mm) forage in 50-mL rumen inoculum during 48h, followed by quantification of the incubation residue. Two heating-stirring systems were used: i) heating in a water bath at 39°C with manual stirring every two hours (i.e., traditional system); and ii) heating in an oven with controlled temperature at 39°C and automatic agitation (44 rpm; alternative system); there was no effect of the interaction between the heating-stirring system and the type of forage (p = 0.829) on the IVRDMD of forages. The type of heating-stirring system (p = 0.422) did not affect the IVRDMD of forages. Nevertheless, the IVRDMD values of Marandu grass (system i = 598.7 g kg-1 vs system ii = 599.4 g kg-1) were greater (p &lt; 0.001) than Tifton 85 (system i = 392.1 vs g kg-1 vs system ii = 370.7 g kg-1) and Mombasa (system i = 397.4 g kg-1; system ii = 369.7 g kg-1) grasses. In conclusion, the obtained data indicate that the alternative heating-stirring system produces similar results to those obtained using the traditionally heating-stirring system during the determination of the IVRDMD of forages.

In vitro evaluation of ruminal digestibility and fermentation characteristics of local feedstuff-based beef cattle ration

Background: Consumption of local feedstuff Neptunia plena L. Benth and Leersia hexandra Swartz as a ration by the animal subject is expected to promote cost efficiency and production, as well as provide essential nutrition needs. Therefore, this study aimed to evaluate ruminal dry matter digestibility (DMD), organic matter digestibility (OMD), ammonia (NH 3) production, and volatile fatty acid (VFA) in beef cattle. Methods: Feed and rumen inoculum samples were prepared and analyzed for their proximate contents. There were five treatment groups based on the diet received by beef cattle, namely: T 1 ( Leersia hexandra Swartz 100 %); T 2 ( Neptunia plena L. Benth 100%); T 3 ( Leersia hexandra Swartz 15% + ( Neptunia plena L. Benth 15% + 70 % Other Feedstuffs); T 4 ( Leersia hexandra Swartz 20% + ( Neptunia plena L. Benth 20% + 60% Other Feedstuffs); T 5 ( Leersia hexandra Swartz 25% + ( Neptunia plena L. Benth 25% + 50% Other Feedstuffs). In vitro approaches were used to determine the DMD, OMD, NH 3 production, and VFA in beef cattle. Results: The results showed that the highest DMD (P&lt;0.05) was derived from T 5 (56.47%), followed by T 4 (56.45%) and T 3 (55.90%). T 5=62.40% significantly (P&lt;0.05) generated the highest OMD followed by T 4=61.95% and T 3=60.82%. This treatment had the highest NH 3 value, namely 5.02 mM, compared with T 3=4.55 mM, T 4=4.50 mM, T 2=4.22 mM, and T 1=3.99 mM. Furthermore, T 5 had the highest VFA (P&lt;0.05) compared with T 4, T 3, T 2, and T 1 with the value of 150.5, 133.0, 130.5, 130.0, and 123.5 mM, respectively. Conclusions: The local feedstuff-based ration can be used to ensure the sustainable production of beef cattle

Lactiplantibacillus plantarum BX62 reduces methane production, and improves antioxidant capacity and rumen fermentation in vitro

This study was conducted to evaluate the effects of Lactiplantibacillus plantarum BX62 with high-antioxidant activity on the dynamics of antioxidant capacity, rumen fermentation, methane (CH4) emission, and ruminal bacterial diversity during incubation of alfalfa substrate by in vitro fermentation. The rumen inocula from sheep were incubated in batch cultures in three groups: (1) control group without inoculation, (2) L. plantarum MTD/1 (MTD) inoculated-group, and (3) L. plantarum BX62 (BX) inoculated-group. The incubations were maintained for 3, 6, 12, 24 and 36 h, with 3 replicates per group and incubation time point. Results showed that the groups × incubation time interactions (P < 0.05) were significant for gas production (GP), CH4 production, and dry matter digestibility (DMD). The BX-inoculated group increased GP (P < 0.05) and DMD (P = 0.001) at 3 h and 6 h of fermentation versus control, whereas decreased the CH4 production (P < 0.05) at 24 h and 36 h of fermentation. In addition, compared to control, two inoculation groups increased total volatile fatty acid (P = 0.028) and acetate (P = 0.034) concentrations at 3 h of fermentation. Total antioxidant capacity during fermentation, except after 6 h, was higher (P < 0.001) for both inoculated groups than control. Higher (P < 0.05) superoxide dismutase activity during fermentation was also found with both inoculated groups than control. BX- and MTD-inoculated groups notably decreased NAD+ content (P < 0.05) and NAD+ to NADH ratio (P < 0.05) versus control at 3 h and 6 h of fermentation. Bacteroidetes abundance increased (P < 0.05) while Firmicutes decreased (P < 0.05) for both inoculated groups compared to control. The relative abundances of fibrolytic bacteria such as Prevotella_1 and Lachnospiraceae, and of H2-incorporating bacteria, such as Succinivibrio, were higher (P < 0.05) for BX- and MTD-inoculated groups than control. In conclusion, the BX strain with high-antioxidant activity could be used as a candidate strain to improve the in vitro antioxidant status of ruminants and might have the potential to mitigate CH4 emissions by competing for hydrogen utilization with methanogens, but it had little difference with the MTD strain.

Bioprospecting essential oils of exotic species as potential mitigations of ruminant enteric methanogenesis

The inclusion of essential oils (EOs) in the diet of ruminants is one of the strategies used to alter ruminal microbial fermentation, improving feed efficiency, while simultaneously reducing enteric methane (CH4) production. This study aimed to evaluate the effects of three new EOs from plants exotic to the Azores, Pittosporum undulatum (PU), Hedychium gardnerianum (HG), and Cryptomeria japonica (CJ), on biogas production kinetics and in vitro CH4 production.Three levels of EOs (40, 80, and 120 μL/g dry matter (DM) were added to the basal diet to evaluate ruminal fermentation using the in vitro gas production technique. Added 800 mL of rumen inoculum with 5 g DM of the basal diet (BD) to all experimental units for 96 h, except for the blanks, to which only the inoculum was added. The total gas and CH4 produced by treatment incubation were recorded every hour after incubation until 96 h. The results showed that the gas production decreased significantly (P < 0.001) at 24 and 96 h after incubation, in the medium and high levels, with the essential oils PU, CJ, and HG treatment, when compared to the control treatment. The same significant differences (P < 0.001) were observed in in vitro CH4 production. The greatest reduction was noted with the addition of PUEO at the highest concentration (120 μL), which allowed a reduction in CH4 production at 24 h of 47% (P < 0.01). There was an interaction effect between EOs and concentration levels for all variables (P < 0.001). A decrease in total volatile fat acid (VFA) concentration (P < 0.05) was recorded compared to control, as well as the insoluble fraction and the potential degradation of the BD when EOs were included in the diet. In conclusion, the addition of EOs to the BD effectively reduced total enteric gas emissions and mitigated CH4 production. The most significant reduction of CH4 (47% in 24 h of incubation) occurs when 120 μL PUEO is added to each gram DM. The inclusion of OEs in the BD also affected the gas production kinetics and fermentation parameters.

In Vitro Rumen Fermentation and Post-Ruminal Digestibility of Sorghum-Soybean Forage as Affected by Ensiling Length, Storage Temperature, and Its Interactions with Crude Protein Levels.

The study aimed to evaluate the effects of ensiling length, storage temperature, and its interaction with crude protein (CP) levels in sorghum−soybean forage mixtures on in vitro rumen fermentation and post-ruminal digestibility of nutrients. The dietary treatments consisted of fresh forages (d 0) and silages of sorghum and soybean stored indoors or outdoors for 75 and 180 d with additional ingredients to make two dietary CP levels, 90 and 130 g/kg dry matter (DM) and a forage-to-concentrate ratio of 80 to 20. An in vitro procedure was conducted using the ANKOM RF technique to study rumen fermentation. The dietary treatments were incubated in duplicate for 8 and 24 h in three runs. After each incubation time, in vitro rumen fermentation parameters were measured, and the protozoa population was counted using a microscope. Post-ruminal digestibility was determined using the pepsin and pancreatic solubility procedure. Cumulative gas production (GP) increased quadratically with ensiling length (8 h, p < 0.01; 24 h, p = 0.02), and the GP differed between CP levels at both incubation times (p < 0.01). However, total short-chain fatty acid (SCFA) concentrations in rumen inoculum increased quadratically with ensiling length (p < 0.01; for both incubation times), and interaction between ensiling length and CP levels was observed in proportions of acetate and propionate after 24 h of incubation (p < 0.01; for both incubation times). Similarly, an interaction between ensiling length and CP levels was found in the proportion of valerate after 24 h of incubation (p < 0.01). There was a quadratic response to ensiling length in the NH4−N concentration after 8 h (p < 0.01) and 24 h (p < 0.05), and the CP level also differed (p < 0.01) at both incubation times. The ciliate protozoa count after 24 h was higher in low CP diets than in high CP diets (p = 0.04). The amount of CP in the undegraded substrate at both incubation times differed between CP levels (p < 0.01; for both incubation times). An interaction effect between ensiling length and storage temperature after 8 h (p = 0.02) and 24 h (p < 0.01) was observed for intestinal CP digestibility. The effect of CP levels on intestinal CP digestibility differed after 8 h (p < 0.01) and 24 h (p < 0.01). In conclusion, increasing ensiling length beyond 75 d reduced CP digestibility, and additional CP inclusion did not ameliorate this.