Total Volatile Fatty Acid Production Research Articles

Application of propionate-producing bacterial consortium in ruminal methanogenesis inhibited environment with bromoethanesulfonate as a methanogen direct inhibitor.

Methane production in ruminants is primarily due to the conversion of metabolic hydrogen (H2), produced during anaerobic microbial fermentation, into methane by ruminal methanogens. While this process plays a crucial role in efficiently disposes of H2, it also contributes to environmental pollution and eliminating methane production in the rumen has proven to be challenging. This study investigates the use of probiotics, specifically propionate-producing bacteria, to redirect accumulated H2 in a methane-mitigated environment. For this objective, we supplemented experimental groups with Lactiplantibacillus plantarum and Megasphaera elsdenii for the reinforced acrylate pathway (RA) and Selenomonas ruminantium and Acidipropionibacterium thoenii for the reinforced succinate pathway (RS), as well as a consortium of all four strains (CB), with the total microbial concentration at 1.0 × 1010 cells/mL. To create a methane-mitigated environment, 2-bromoethanesulfonate (BES) was added to all experimental groups at a dose of 15 mg/0.5 g of feed. BES reduced methane production by 85% in vitro, and the addition of propionate-producing bacteria with BES further decreased methane emission by up to 94% compared with the control (CON) group. Although BES did not affect the alpha diversity of the ruminal bacteriome, it reduced total volatile fatty acid production and altered beta diversity of ruminal bacteriota, indicating microbial metabolic adaptations to H2 accumulation. Despite using different bacterial strains targeting divergent metabolic pathways (RA and RS), a decrease in the dominance of the [Eubacterium] ruminantium group suggesting that both approaches may have a similar modulatory effect. An increase in the relative abundance of Succiniclasticum in the CB group suggests that propionate metabolism is enhanced by the addition of a propionate-producing bacterial consortium. These findings recommend using a consortium of propionate-producing bacteria to manage H2 accumulation by altering the rumen bacteriome, thus mitigating the negative effects of methane reduction strategies.

Rapid Screening of Methane-Reducing Compounds for Deployment in Livestock Drinking Water Using In Vitro and FTIR-ATR Analyses

Several additives have been shown to reduce enteric methane emissions from ruminants when supplied in feed. However, utilising this method to deliver such methane-reducing compounds (MRCs) in extensive grazing systems is challenging. Use of livestock drinking water presents a novel method to deliver MRCs to animals in those systems. This work evaluated 13 MRCs for suitability to be deployed in this manner. Compounds were analysed for solubility and stability in aqueous solution using Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy. Furthermore, aqueous solutions of MRCs were subjected to variations in temperature and starting pH of water used to assess solubility and stability of the MRCs in simulated water trough conditions, also using FTIR-ATR spectroscopy. In vitro batch culture fermentations were carried out using a medium-quality tropical grass feed substrate, to simulate pastures consumed by cattle in extensive grazing systems. Measurements were made of total gas and methane production, in vitro dry matter digestibility (IVDMD), and volatile fatty acid (VFA) concentration. Of the MRCs tested, 12 were found to be soluble and stable in water using the FTIR method employed, whilst the other could not be measured. Of the 12 soluble and stable MRCs, one containing synthetic tribromomethane (Rumin8 Investigational Veterinary Product) reduced methane production by 99% (p = 0.001) when delivered aqueously in vitro, without a reduction in IVDMD (p = 0.751), with a shift towards decreased acetate and increased propionate production and decreased total VFA production (p < 0.001). Other compounds investigated also appeared suitable, and the methods developed in this study could be used to guide future research in the area.

94 Associative effects of two potent anti-methanogenic Moringa oleifera accessions mixed at various proportions on in vitro rumen fermentation and methane inhibition

Abstract Moringa is an important anti-methanogenic plant with a potential to develop as multipurpose feed additives. Previous studies identified two potent accessions (accession 07633 designated as A3 and accession collected from Pretoria designated as A11) with high methane (CH4) inhibition characteristics. Two metabolite ion features (MIFs) 4.44_609.1462 and MIF 4.53_433.1112 were identified as bioactive MIFs associated with greater methane inhibition in Moringa accession A3 and A11, respectively. Two separate studies were conducted with an aim to name the MIF associated with methane inhibition and evaluate the benefit of mixing plant extract from A3 and A11 in different proportions to exploit their additive or synergistic effect in inhibiting CH4 emission. In study 1, software and online tools such as MS-DIAl, MS-FINDER, and the mass bank database were used to identify the MIF 4.44_609.1462 and MIF 4.53_433.1112, and the two MIFs were tentatively named as Hesperidin and Isovitexin, respectively (Figure 1). In study 2, the Moringa plant extracts from A3 and A11 were mixed in the following proportion A3100:A110, A380:A1120, A360:A1140, A350:A1150, A340:A1160, A320:A1180, and A30:A11100 proportions (A3:A11), and applied at a rate 50 mg/kg DM of Eragrostis curvula hay incubated in an Incoshake incubator to determine their effect on in vitro gas production and CH4, and in vitro organic matter digestibility (IVOMD) of the hay. In each incubation run, the sole or a mixture of the Moringa plant extract treatments were added to the test feed and incubated in 125 mL serum bottles with four replications per treatment, and the incubation was repeated for three runs. A Chi–square (☐2) test were used to determine the presence of an associative effect for mixtures combined at different proportion. Generally, the mixture resulted in less (P < 0.05) CH4 volume, slightly improved or no effect on IVOMD, propionate concentration decreased acetate while increasing propionate concentration, with higher CH4 inhibition, and % CH4 inhibition was significantly greater when the two accessions were mixed in 50% proportion (Figure 2). Specifically, associative effects were recorded when the two accessions are mixed in 50% proportion in terms of greater CH4 inhibition, propionic acid concentration and total volatile fatty acid production (TVFA), as well as reduction in CH4 per unit TVFA and C2:C3 ratio (Table 1). Therefore a 50% binary cocktail of M. oleifera accession A3 and A11 (A350:A1150) plant extracts can be used as feed additive for CH4 inhibition without affecting the feed fermentation adversely when compared with the sole accession leaf extracts or other proportions of binary cocktails. Detailed mechanisms of action, however, need to be confirmed.

Volatile Fatty Acids from Household Food Waste: Production and Kinetics

Household food waste (HFW), which is rich in organic matter, is a good candidate for producing added-value bio-based chemicals, such as volatile fatty acids (VFAs), by acidogenic fermentation processes. However, the lack of design tools, such as appropriate kinetic models, hinders the implementation of this technology because the results of these processes are affected by operational factors. In this work, VFA production by the acidogenic fermentation of HFW under uncontrolled pH levels (4–5) was studied at thermophilic (55 °C) and mesophilic (35 °C) temperature conditions. Batch reactors were used to digest HFW, and VFA production and the individual acid distributions were measured at different fermentation times from 0 to 624 h. The results showed higher individual and total VFA production at 35 °C and 120 h of fermentation time as a consequence of the competition between the VFA production and decomposition reactions. Acetic and valeric acids were VFAs mainly produced as a result of a high content of proteins in the initial substrate, and a small amount of propionic and butyric acids were present. A simplified kinetic model was successfully developed to represent the complex process of VFA formation from the acidogenic fermentation of HFW. A simple mechanism for the production–decomposition of VFAs, corresponding to a zero-order reaction for the first 48 h and a single consecutive reaction from that time on, was proposed. For both mesophilic and thermophilic conditions, the suggested kinetic model was able to predict the individual and total concentrations of VFAs along the fermentation time.

Long-chain fatty acids facilitate acidogenic fermentation of food waste: Attention to the microbial response and the change of core metabolic pathway under saturated and unsaturated fatty acids loading

Long-chain fatty acids (LCFAs) are recognized as a significant inhibitory factor in anaerobic digestion of food waste (FW), yet they are inevitably present in FW due to lipid hydrolysis. Given their distinct synthesis mechanism from traditional anaerobic digestion, little is known about the effect of LCFAs on FW acidogenic fermentation. This study reveals that total volatile fatty acids (VFAs) production increased by 9.98 % and 4.03 % under stearic acid and oleic acid loading, respectively. Acetic acid production increased by 20.66 % under stearic acid loading compared to the control group (CK). However, the LCFA stress restricted the degradation of solid organic matter, particularly under oleic acid stress. Analysis of microbial community structure and quorum sensing (QS) indicates that LCFA stress enhanced the relative abundance of Lactobacillus and Klebsiella. In QS system, the relative abundance of luxS declined from 0.157 % to 0.116 % and 0.125 % under oleic acid and stearic acid stress, respectively. LCFA stress limited the Autoinducer-2 (AI-2) biosynthesis, suggesting that microorganisms cannot use QS to resist the LCFA stress. Metagenomic sequencing showed that LCFA stress promoted acetic acid production via the conversion of pyruvate and acetyl-CoA to acetate. Direct conversion of pyruvate to acetic acid increased by 47.23 % compared to the CK group, accounting for the enhanced acetic acid production under stearic acid loading. The abundance of β-oxidation pathway under stearic acid loading was lower than under oleic acid loading. Overall, the stimulating direct conversion of pyruvate plays a pivotal role in enhancing acetic acid biosynthesis under stearic acid loading, providing insights into the effect of LCFA on mechanism of FW acidogenic fermentation.

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.

The Impact of Three White-Rot Fungi on Nutrient Availability, Greenhouse Gas Emissions, and Volatile Fatty Acid Production in Myceliated Sorghum.

Our study employed Pleurotus ostreatus, P. djamor, and Trametes versicolor (white rot fungi = WRF) in the process of solid-state fermentation (SSF) to convert sorghum grains into myceliated sorghum (MS). The MS was then used for in vitro studies to assess changes in nutrient content compared to untreated sorghum (control). The results demonstrated a significant (p < 0.001) increase in dry matter (DM), crude protein (CP), ash, neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents of MS. Specifically, CP and ash values saw a remarkable increase from 68 to 330% and 40 to 190% in MS, respectively. Additionally, NDF and ADF degradability values increased significantly (p < 0.001) by 81.5% and 56.2% in P. djamor-treated MS at 24 h post-incubation. The treatment × time interaction was also significant (p < 0.001) for greenhouse gas (GHG) emissions. T. versicolor MS exhibited the highest total volatile fatty acid (TVFA) and propionate production. The use of WRF in the SSF process led to a significant improvement in the nutritional value of sorghum. Despite the varying effects of different WRF on the nutritional parameters in MS, they show potential for enhancing the feed value of sorghum in animal feed.

Effects of Centella asiatica Extracts on Rumen In Vitro Fermentation Characteristics and Digestibility.

Two in vitro experiments were conducted to evaluate the effects of Centella asiatica extract (CAE) supplementation on the rumen's in vitro fermentation characteristics. In the first experiment, CAE with five concentrations (C: 0%; T1: 3.05%; T2: 6.1%; T3: 12.2%; and T4: 24.4% CAE in diet) was supplemented in the rumen fluid and incubated for 6, 24, and 48 h to determine the optimal dosage. The total gas and methane production increased in all incubation times, and the total volatile fatty acids increased at 6 and 48 h. Ammonia nitrogen, branched chain volatile fatty acids, acetate, and butyrate were increased by CAE supplementation. T1 was chosen as the optimal dosage based on the total volatile fatty acids, branched chain volatile fatty acids, and ammonia nitrogen production. The CAE with the identified optimal dosage (T1) was incubated to identify its effect on the rumen's in vitro degradability in the second experiment. The CAE supplementation did not influence the in vitro dry matter, crude protein, or neutral detergent fiber degradability. In conclusion, CAE has no CH4 abatement or digestion promotion effects. However, CAE could be utilized as a feed additive to increase the rumen's total volatile fatty acid production without an adverse effect on the in vitro dry matter, crude protein, or neutral detergent fiber degradability.

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.

Ruminal inocula with distinct fermentation profiles differentially affect the in vitro fermentation pattern of a commercial algal blend.

The in vitro rumen batch technique is widely used for screening novel feed sources; however, it remains unclear to what extent the in vitro fermentability of non-conventional feed sources is affected by non-adapted ruminal inocula. Thus, in this study, we evaluated the effects of distinct ruminal inocula on the in vitro fermentation parameters of a sustainable non-conventional feed, a commercially available algal blend composed of microalgae (Chlorella vulgaris and Nannochloropsis oceanica) and seaweeds (Ulva sp. and Gracilaria gracilis). First, four late-lactation Holstein cows were fed four forage-based diets varying only in the proportions of basal forage (100% corn silage, 70% corn silage and 30% haylage, 30% corn silage and 70% haylage, and 100% haylage) in a 4 × 4 Latin square design with the last square omitted. After 3 weeks of adaptation, haylage-based diets resulted in ruminal fermentation parameters distinct from those promoted by corn silage-based diets, as reflected in increased pH, ammonia-N contents, and acetate proportions. Individual ruminal fluids derived from each of the four diets were further used as inocula in in vitro incubations. Here, a 1:1 mixture of corn silage and haylage was supplemented with 0, 5, 10, or 15% algal blend and incubated with each inoculum for 24 h in a 4 × 4 factorial design. Total gas and methane production decreased with inocula from cows fed haylage-based diets and with increasing algal blend supplementation levels. The fermentation pH increased and the ammonia-N contents decreased with inocula from cows fed haylage-based diets; however, these parameters were not affected by algal blend inclusion levels. The interaction between the ruminal inoculum source and the algal blend supplementation level affected the total volatile fatty acids (VFA) and the proportions of most individual VFA. Total VFA production decreased with increasing algal supplementation levels, particularly with inocula from cows fed 30% corn silage and 70% haylage; the acetate, propionate, and valerate proportions were only affected by algal blend levels under incubation with 100% corn silage inocula. Overall, our findings highlight the importance of the ruminal inoculum source when assessing the fermentability of non-conventional feed as well as the potential of the algal blend as a natural modulator of ruminal fermentation.

The Effect of Saponite Clay on Ruminal Fermentation Parameters during In Vitro Studies.

Reducing the emission of global warming gases currently remains one of the strategic tasks. Therefore, the objective of our work was to determine the effect of saponite clay on fermentation in the rumen of cows. The pH, total gas production, CH4, and volatile fatty acid (VFA) production in ruminal fluid was determined in vitro. Saponite clay from the Tashkiv deposit (Ukraine) has a high content of silicon, iron, aluminum, and magnesium. The addition of 0.15 and 0.25 g of saponite clay to the incubated mixture did not change the pH but reduced the total production (19% and 31%, respectively) and CH4 (24% and 46%, respectively) in the ruminal fluid compared to the control group and had no significant effect on the total VFA levels, but propionic acid increased by 15% and 21% and butyric acid decreased by 39% and 32%, respectively. We observed a decrease in the fermentation rates, with a simultaneous increase in the P:B ratio and an increase in the fermentation efficiency (FE) in the groups fermented with saponite clay, probably a consequence of the high efficiency in the breakdown of starch in the rumen. Therefore, further in vivo studies to determine the effective dose and effect of saponite clay on cow productivity and the reduction of gas emissions are promising and important.

The addition of anaerobic fungi isolates from buffalo rumen to increase fiber digestibility, fermentation, and microbial population in ruminants

Abstract. Agustina S, Wiryawan KG, Suharti S, Meryandini A. 2024. The addition of anaerobic fungi isolates from buffalo rumen to increase fiber digestibility, fermentation, and microbial population in ruminants. Biodiversitas 25: 107-115. Rumen microbes have an important role in the rumen. Anaerobic fungi are microbes needed in the forage digestion process in the rumen. The addition of microbes, particularly anaerobic fungi is essential to increase the digestibility of forage within rumen. Therefore, this study aimed to evaluate the addition of anaerobic fungi isolates from buffalo rumen to increase fiber digestibility, fermentation, and microbial population in sheep rumen. The in vitro tests were carried out using the Tilley and Terry method, using elephant grass and rice straw as tested forage. Piromyces sp. (F1, and F3), Caecomyces sp. (F2, and F5), and Neocallimastix frontalis (F4) isolates from buffalo rumen were used as tested anaerobic fungi. The result showed that the addition of anaerobic fungi isolates from buffalo rumen significantly affected fiber digestibility (Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), cellulose and hemicellulose) except lignin digestibility. The addition of N. frontalis had higher fiber digestibility which is 49.02% NDF digestibility, 42.11% ADF digestibility, 44.28% hemicellulose digestibility, and 38.60% cellulose digestibility. Furthermore, N. frontalis also significantly increased In vitro Dry Matter Digestibility (IVDMD), In vitro Organic Matter Digestibility (IVOMD), ammonium (NH3) production, total Volatile Fatty Acid (VFA) production, and microbial population compared to Piromyces sp., and Caecomyces sp. In conclusion, anaerobic fungus type N. frontalis showed promising potential to be used as a ruminant probiotic due to its superior effect on fiber digestibility, fermentation, and microbial population compared to Caecomyces sp. and Piromyces sp.

Effects of yeast culture and oxalic acid supplementation on in vitro nutrient disappearance, rumen fermentation, and bacterial community composition.

Hemicellulose is an important polysaccharide in ruminant nutrition, but it has not been studied as thoroughly as cellulose. Further research is needed to explore supplements that can improve its digestibility and ruminal buffering effects. Our previous research demonstrated the efficacy of oxalic acid (OA) as an essential nutrient in yeast culture (YC) for improving rumen fermentation performance. Consequently, we conducted in vitro rumen digestion experiments to examine the effects of YC and OA on rumen fermentation and bacterial composition. Two diets containing different levels of hemicellulose were formulated: diet 1 with 10.3% and diet 2 with 17% hemicellulose. Three levels of YC (0.00, 0.625, and 1.25 g/kg) and three doses of OA (0.0, 0.4, and 0.8 g/kg, DM) were added into each diet with a 3 × 3 factorial design. A comprehensive assessment was conducted on a total of 18 experimental treatments at fermentation periods of 0, 6, 12, 24, and 48 h. In the first experiment (diet 1), the supplementation of YC, OA, and their interaction significantly increased in vitro DM disappearance (IVDMD) and NDF disappearance (IVNDFD; p < 0.001). In the second experiment (diet 2), the supplementation of OA and the interaction between YC and OA (p < 0.001) increased IVDMD and IVCPD, but had no significant effects on IVNDFD. The interactions of YC and OA significantly increased ammonia nitrogen (p < 0.001). The production of acetic acid, propionic acid, and total volatile fatty acids (TVFA), and pH levels were significantly higher in treatments supplemented with YC and OA (p < 0.001). YC and OA in both diets significantly altered the rumen bacterial community leading to increased Shannon and Simpson diversity indices (p < 0.001). In both diets, OA supplementation significantly increased the relative abundance of the phylum Bacteroidetes and Prevotella genus. The result also showed a positive correlation between the Prevotella and Selenomonas genera with IVDMD, IVNDFD, propionic acid, and TVFA production, suggesting that these dominant bacteria enhanced nutrient disappearance in the rumen. In conclusion, adding YC and OA resulted in modifications to the bacterial community's composition and diversity, and improved nutrient disappearance. These changes indicate improved rumen fermentation efficiency, which is promising for future in vivo studies.

Supplementation of complex natural feed additive containing (C. militaris, probiotics and red ginseng by-product) on rumen-fermentation, growth performance and carcass characteristics in Korean native steers.

This study evaluated the effects of a complex natural feed additive on rumen fermentation, carcass characteristics and growth performance in Korean-native steers. In this study, in vitro and in vivo experiment were conducted. Seven different levels of complex natural feed additive (CA) were added to the buffered rumen fluid using AnkomRF gas production system for 12, 24 and 48 h. All experimental data were analyzed by mixed procedure of SAS. Total gas production increased in the CA groups, with the highest response observed in the 0.06% group at 48 h of incubation (linear, p = 0.02; quadratic, p < 0.01). Regarding rumen fermentation parameters, the total volatile fatty acid (TVFA) tended to increase in all the CA groups (p = 0.07). The concentrations of butyrate, iso-butyrate, and iso-valerate significantly increased in all treatment groups (p < 0.05). In the in vivo experiment, 23 Korean-native steers were allocated to two groups: (1) Control and (2) Treatment; control +0.07% CA (DM basis), in a randomized complete-block design and blocked by body weight (ave. body weight = 641.96 kg ± 62.51 kg, p = 0.80) and feed intake (ave. feed intake = 13.96 kg ± 0.74 kg, p = 0.08) lasted for 252 days. Average daily gain decreased in the treatment group (p < 0.01). Backfat thickness significantly decreased in the CA group (p = 0.03), whereas meat color tended to increase (p = 0.07). In conclusion, in the in vitro experiment, the inclusion of complex natural feed additive decreased methane proportion and tended to increase TVFA production, but supplementation to Korean native steers decreased average daily gain and backfat thickness.

Enteric methane emission reduction potential of natural feed supplements in ewe diets.

Research into the potential use of various dietary feed supplements to reduce methane (CH4) production from ruminants has proliferated in recent years. In this study, two 8-wk long experiments were conducted with mature ewes and incorporated the use of a variety of natural dietary feed supplements offered either independently or in combination. Both experiments followed a randomized complete block design. Ewes were offered a basal diet in the form of ad libitum access to grass silage supplemented with 0.5kg concentrates/ewe/d. The entire daily dietary concentrate allocation, incorporating the respective feed supplement, was offered each morning, and this was followed by the daily silage allocation. In experiment 1, the experimental diets contained 1) no supplementation (CON), 2) Ascophyllum nodosum (SW), 3) A. nodosum extract (EX1), 4) a blend of garlic and citrus extracts (GAR), and 5) a blend of essential oils (EO). In experiment 2, the experimental diets contained 1) no supplementation (CON), 2) A. nodosum extract (EX2), 3) soya oil (SO), and 4) a combination of EX2 and SO (EXSO). Twenty ewes per treatment were individually housed during both experiments. Methane was measured using portable accumulation chambers. Rumen fluid was collected at the end of both experiments for subsequent volatile fatty acid (VFA) and ammonia analyses. Data were analyzed using mixed models ANOVA (PROC MIXED, SAS v9.4). Statistically significant differences between treatment means were considered when P < 0.05. Dry matter intake was not affected by diet in either experiment (P > 0.05). Ewes offered EO tended to have an increased feed:gain ratio relative to CON (P < 0.10) and SO tended to increase the average daily gain (P < 0.10) which resulted in animals having a higher final body weight (P < 0.05) than CON. Ewes offered EX1 and SO emitted 9% less CH4 g/d than CON. The only dietary treatment to have an effect on rumen fermentation variables relative to CON was SW, which enhanced total VFA production (P < 0.05). In conclusion, the A. nodosum extract had inconsistent results on CH4 emissions whereby EX1 reduced CH4 g/d while EX2 had no mitigating effect on CH4 production, likely due to the differences in PT content reported for EX1 and EX2. SO was the only dietary feed supplement assessed in the current study that enhanced animal performance whilst mitigating daily CH4 production.

EFFECT OF NITRO COMPOUNDS ON in vitro RUMINAL METHANE, CARBON DIOXIDE, HYDROGEN AND DRY MATTER DEGRADABILITY

This study determined the in vitro effects of ethyl nitroacetate (ENA), ethyl 2-nitropropionate (E-2-NPP) and 3-nitropropionic acid (NPA) on ruminal production of CH4, H2, volatile fatty acids (VFAs) and in vitro dry matter disappearance of ground alfalfa. In vitro incubations were carried out over 24-h succeeding days (3 batches). Total gas and methane production decreased with all nitro compounds (P 0.05) in all the batches. Total production of VFAs was reduced by ENA and E-2-NPP (P 0.05), but not by NPA. DM disappearance was similar between treatments but decreased across batches. Further studies should be aimed at understanding the impact of NPA on ruminal metabolism, as well as its effect on in vivo models.

Enhancing Anaerobic Digestion with an UASB Reactor of the Winery Wastewater for Producing Volatile Fatty Acid Effluent Enriched in Caproic Acid

The production of Volatile Fatty Acids (VFAs) from wastewater holds significant importance in the context of biorefinery concepts due to their potential as valuable precursors for various bio-based processes. Therefore, the primary objective of this research is to investigate the fermentation of Winery Wastewater (WW) in an Upflow Anaerobic Sludge Blanket (UASB) reactor to generate VFAs, with particular emphasis on Caproic Acid (HCa) production and the dynamics of the microbiota, under varying Hydraulic Retention Time (HRT) periods (8, 5, and 2.5 h). The change from an 8 h to a 5 h HRT period resulted in an approximately 20% increase in total VFA production. However, when the HRT was further reduced to 2.5 h, total VFA production decreased by approximately 50%. Concerning the specific production of HCa, expressed in grams of Chemical Oxygen Demand (gCOD), the maximum yield was observed at around 0.9 gCOD/L for a 5-h HRT. Microbial population analysis revealed that Eubacteria outnumbered Archaea across all HRTs. Population dynamics analysis indicated that the Firmicutes Phylum was predominant in all cases. Within this phylum, bacteria such as Clostridium kluyveri and Clostridium sp., known for their ability to produce HCa, were identified. Based on the results obtained, the application of the UASB reactor for WW treatment, within the biorefinery framework, has the potential to provide a practical alternative for HCa production when operated with a 5 h HRT.

Essential Oil Blends with or without Fumaric Acid Influenced In Vitro Rumen Fermentation, Greenhouse Gas Emission, and Volatile Fatty Acids Production of a Total Mixed Ration

The growing interest in improving rumen fermentation and mitigating methane emissions necessitates the use of essential oil blends (EOB) and fumaric acid (FA). This study evaluated the synergistic effect of four EOB with or without FA supplementation on in vitro dry matter digestibility, greenhouse gas emission, and total volatile fatty acid production using inoculum from three rumen-cannulated Black Angus beef cows. The study was arranged in a 4 × 2 + 1 factorial design to evaluate the effects of the four EOB and two FA levels on a total mixed ration (TMR). The EOB dosage was 100 µL while FA was added at 3% of total mixed ration. The EOB × FA interaction (p &lt; 0.05) influenced the dry matter, neutral detergent fiber, and hemicellulose degradabilities. All the EOB and FA (EFA) treatments decreased (p &lt; 0.001) the dry matter degradability compared to the control (TMR substrate only). The EFA4 treatment reduced the neutral detergent fiber and hemicellulose degradabilities compared to the control. The ruminal pH was influenced (p &lt; 0.001) by both the EOB and FA inclusion, and the EOB × FA interaction was significant. The microbial mass was higher (p &lt; 0.001) in the EFA1, EFA4, and EOB4 compared to the control and the EOB3 treatments. The EFA1 and EOB1 produced less (p &lt; 0.001) gas than the control by 29.1 and 32.1%, respectively. Compared with the control, the EFA1 and EOB1 treatments decreased (p &lt; 0.001) methane gas by 90.8% and 86.4%, respectively, while the carbon dioxide was reduced (p = 0.004) by 65.7 and 57.9%, respectively. The EOB × FA interaction was significant (p &lt; 0.001) for the total and individual volatile fatty acid concentrations. The inclusion of FA increased the propionate concentration by 9.5% and decreased (p = 0.02) the acetate concentration by 4%. In summary, the synergistic effect of the EOB and FA offers an effective way to reduce greenhouse gas emission and enhance total volatile fatty acids.

Microencapsulation of Mitragyna leaf extracts to be used as a bioactive compound source to enhance in vitro fermentation characteristics and microbial dynamics.

Mitragyna speciosa Korth is traditionally used in Thailand. They have a high level of antioxidant capacities and bioactive compounds, the potential to modulate rumen fermentation and decrease methane production. The aim of the study was to investigate the different levels of microencapsulated-Mitragyna leaves extracts (MMLE) supplementation on nutrient degradability, rumen ecology, microbial dynamics, and methane production in an in vitro study. A completely randomized design was used to assign the experimental treatments, MMLE was supplemented at 0%, 4%, 6%, and 8% of the total dry matter (DM) substrate. The addition of MMLE significantly increased in vitro dry matter degradability both at 12, 24, and 48 h, while ammonia-nitrogen (NH3-N) concentration was improved with MMLE supplementation. The MMLE had the greatest propionate and total volatile fatty acid production when added with 6% of total DM substrate, while decreased the methane production (12, 24, and 48 h). Furthermore, the microbial population of cellulolytic bacteria and Butyrivibrio fibrisolvens were increased, whilst Methanobacteriales was decreased with MMLE feeding. The results indicated that MMLE could be a potential alternative plant-based bioactive compound supplement to be used as ruminant feed additives.

PSI-19 A Saccharomyces Cerevisiae-Derived Postbiotic Stimulates in Vitro Volatile Fatty Acid Production and Influences Hindgut Microbial Population Characteristics

Abstract Horses rely on volatile fatty acids (VFA) to provide a source of energy and fermentative end-products for their health and performance. Improvement in VFA production, has been associated in various species with improved feed conversion and availability of nutrients necessary to maintain epithelial cell growth, blood flow, and the normal secretory and absorptive functions of the intestine and positive changes in the microbial ecology of the gut. In this study, we characterized the effect of a dietary Saccharomyces cerevisiae fermentation postbiotic (SCFP; Diamond V, Cedar Rapids, IA) using an in vitro equine intestinal model to estimate the effects on VFA production and microbiome changes in the horse hindgut. Treatments contained inoculated medium containing a predigested blend of alfalfa and timothy hay substrate and a dilution of fresh equine excreta. The test article (SCFP) was evaluated at 150 mg (TEC) and 225 mg (TECP), in addition to a negative control containing inoculated medium alone, and the positive control containing inulin. The full array of treatments and replicates were incubated anaerobically at 37.2 C for 24 h. VFA concentrations were analyzed by gas chromatography and the microbiome was characterized through shotgun sequencing. Both levels of SCFP supported greater (P &amp;lt; 0.05) acetate, propionate, butyrate, and total VFA production versus the negative control. Greater production of acetate, propionate, butyrate and total VFA was observed for TECP compared with the positive control (P &amp;lt; 0.05). Between the two SCFP treatments, TECP promoted more acetate, propionate and total VFA (P &amp;lt; 0.01), and 18.3% more butyrate than TEC. Significant microbiome changes were also observed, with TEC and TECP promoting greater microbiome diversity (P &amp;lt; 0.05) compared with both negative and positive controls. Compared with the negative control, both levels of SCFP influenced an increased abundance (P &amp;lt; 0.05) of lactic acid producers (Veillonella), butyrate producers (Butyricicoccus, Anaerobutyricum), and an increased abundance of multiple Bacteroides species, all known for their enrichment of carbohydrate-degrading enzymes. Interestingly, our data show a distinct effect of inulin in microbial communities by promoting an increased abundance of Streptococcus species. This study provides evidence that a SCFP can stimulate VFA production, and this effect is incremental with the level of administration which could potentially provide gut health benefits in horses.