Search Results (374)

This study aimed to evaluate the effect of bromoform (CHBr₃) dose on in vitro rumen fermentation and on CHBr₃ and dibromomethane (CH₂Br₂) concentrations in solution and the gas cap. In vitro treatments consisted of CHBr₃ (DOSE: 0, 1, 10, 100, 1000, 10,000 µg of CHBr₃), with five replicates per DOSE at each time-point (TIME: 0, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, and 72 h). The 10,000 µg CHBr₃ DOSE inhibited fermentation completely and was removed from the dataset. The acetate:propionate ratio, nitrogen, and methane (CH₄) produced per gram of DMD decreased as DOSE increased (p = 0.01). As the DOSE increased, CH₄ decreased, and H₂ increased in a dose-dependent manner (p < 0.01). The CHBr₃ concentration dropped below the detection limit within 3 h of incubation. Dibromomethane concentrations for DOSE 1000 and 100 µg of CHBr₃ increased in solution and gas cap beginning at 0.25 h and 1 h post incubation and plateaued by hour 3 and 5, respectively (p < 0.01). The addition of CHBr₃ alters the molar proportion of volatile fatty acids, decreases CH₄, and increases H₂ production, and it is dehalogenated to CH₂Br₂ within 3 h of incubation in an in vitro system. Full article

Salmonella in gut habitats have traditionally been thought to conserve energy for growth via fermentation. However, recent reports indicate that ingested Salmonella can stimulate host-derived nitrate accumulation in the mucosal microenvironment, thereby enabling growth through nitrate respiration. Sodium tungstate is an effective treatment that inhibits the growth of certain nitrate-respiring bacteria, including Escherichia coli, Paracoccus and Proteus, when cultured under gut simulating conditions or within the gut of experimentally treated mice. This inhibitory effect is hypothesized to occur by inactivation of molybdenum-containing enzymes required for nitrate metabolism. Information is lacking on whether tungstate can inhibit the growth of Salmonella, particularly in the presence of culturable gut microbiota. Therefore, the objectives of this study were to evaluate the effects of sodium tungstate on Salmonella during pure culture or when cultured with freshly collected bovine rumen microbiota and to assess its impact on fermentation as well as nitrate and nitrite metabolism within the rumen microbial cultures. Our results indicate that 50 mM sodium tungstate treatment, whether alone or in combination with 5 mM nitrate, markedly increased the growth of Salmonella serovars Newport, Dublin and Typhimurium during pure culture. Moreover, during in vitro incubation, increased growth of experimentally inoculated S. Newport as well as wildtype E. coli and lactic acid bacteria was observed with ruminal microbiota treated with 100 mM tungstate when compared to non-tungstate-treated controls. Effects of tungstate on nitrate and nitrite metabolism were as expected during pure and mixed culture. When cultured with reduced tungsten rather than tungstate, the latter being bound to four oxygen atoms, an inhibitory effect on the growth of S. Newport was observed and effects on nitrate and nitrite metabolism were consistent with those observed with tungstate. These results suggest that, under conditions used in the present experiments, tungstate may have served as a source of oxygen for respiration above that achieved with nitrate alone. While this hypothesis has yet to be proven, it is supported by an adverse effect of tungstate, whether alone or in combination with 5 mM nitrate, on methane and volatile fatty acid production by the ruminal microbiota when compared to untreated or nitrate-only-treated microbiota. Full article

Essential oil-based feed additives have been proposed as a practical strategy to mitigate enteric methane emissions in ruminants, but their effects are not always consistent. The objective of this study was to evaluate the effects of dietary supplementation with an essential oil blend on in vitro methane production, rumen fermentation, nutrient digestibility, growth performance, carcass traits, and meat quality in Lithuanian Blackface lambs. We hypothesized that supplementation would induce measurable changes in in vitro methane production and selected rumen fermentation variables, while growth performance and technological meat quality would remain comparable between treatments. Sixty Lithuanian Blackface lambs were allocated to control (C) and treatment (T) groups (30 lambs per group). The C group received a basal diet, and the T group received the same diet supplemented with an essential oil blend, Agolin Ruminant, at a dose rate of 0.1 g/animal/day, consisting of linalool, eugenol, geranyl acetate, and geraniol. An in vitro rumen fermentation assay was performed using rumen fluid pooled within both dietary groups from multiple lambs and incubated as a single batch with four replicate fermentation flasks per treatment (n = 4 fermenters per group) to quantify methane production and in vitro nutrient digestibility. In vivo apparent nutrient digestibility was evaluated in a dedicated sub-trial (n = 6 animals per group). Growth performance in the main trial was analyzed using the pen as the experimental unit (n = 3 pens per group), and slaughter-based measurements—including slaughter and carcass traits, rumen volatile fatty acids and protozoal counts, and Longissimus dorsi meat quality and intramuscular fatty acids—were determined in 10 lambs per treatment (n = 10 animals per group). In vitro methane production did not differ between groups (p = 0.366); in vitro crude fiber digestibility showed a tendency to increase with supplementation (p = 0.066). Fermentation end-products were largely unchanged, although propionate tended to be higher (p = 0.063), and the acetate:propionate ratio was lower (p = 0.043) in the supplemented group; protozoal counts were not different between groups. In vivo apparent nutrient digestibility was comparable between treatments. Growth performance was lower in the supplemented group, resulting in an overall mean ADG 19.0% lower. Slaughter and carcass traits were comparable between treatments. Meat proximate composition, cholesterol concentration, pH, color, water losses, and instrumental texture/shear parameters were not affected by supplementation. Intramuscular lauric (C12:0), myristic (C14:0), and pentadecanoic (C15:0) fatty acids were lower (p < 0.05), while C14:1 n-7 tended to decrease (p = 0.050); however, total saturated, monounsaturated, and polyunsaturated fatty acids and nutritional ratios were unchanged. Overall, under the study conditions and dose used, the essential oil blend did not significantly reduce in vitro methane production and elicited only limited rumen fermentation responses; ADG was 19.0% lower in the supplemented group, whereas carcass traits and technological meat quality were unaffected, and only specific intramuscular fatty acids were altered. Full article

Pasture plants can contribute to ruminant nutrition and may, depending on composition, influence rumen fermentation and methane production. This study evaluated the nutritional composition, bioactive compounds, and methane production potential of 32 terrestrial plant species commonly foraged by goats in Malta. Dried plant samples were analysed for proximate composition using near-infrared spectroscopy, total polyphenols using the Folin–Ciocalteu assay, antioxidant activity using the DPPH assay, and methane production using an in vitro rumen fermentation system incubated for 48 h, with rumen fluid pooled from three goats (analyses performed in triplicate). Crude protein ranged from 1.16 to 31.97% DM, neutral detergent fibre from 12.29 to 48.89%, and ash from 9.69 to 17.20% across species. Total polyphenolic content varied from 0.07 to 1.30% (w/w), while antioxidant activity (IC₅₀) ranged from 0.37 to 55.9 mg/mL. Methane production after 48 h ranged from 30.39 to 198.26 L CH₄ kg⁻¹, indicating variation in fermentation characteristics among species. These results indicate that Rumex bucephalophorus and Urtica pilulifera demonstrated relatively high protein or bioactive values and comparatively lower in vitro methane-related parameters under the conditions tested. Full article

This study evaluated the effects of Nypa fruticans fruit pellet supplementation combined with different CP levels on rumen fermentation characteristics and CH₄ production using an in vitro gas production technique. A 3 × 4 factorial arrangement was used, consisting of three CP levels (12, 14, and 16%) and four levels of Nypa fruticans fruit pellet supplementation (0, 0.5, 1.0, and 1.5% of substrate dry matter), with incubation run included as a random effect in the statistical model. Rumen fluid from Thai native beef cattle was incubated under anaerobic conditions. Gas production kinetics, ruminal pH, ammonia–nitrogen (NH₃–N), protozoal populations, digestibility, volatile fatty acids (VFA), and CH₄ production were determined. Significant interactions between CP level and Nypa fruticans fruit pellet supplementation were observed for gas production kinetics. Ruminal pH was influenced by CP level at 24 h, while NH₃–N increased with higher CP levels but decreased with increasing supplementation. Protozoal populations were reduced by Nypa fruticans fruit pellets. Methane production was affected by CP level, Nypa fruticans fruit pellet supplementation, and their interaction. A clearer reduction was observed at 24 h, particularly at higher supplementation levels. At 24 h of incubation, total VFA, propionate, and butyrate concentrations increased with supplementation, whereas no clear effects were observed at 12 h. In vitro dry matter digestibility was affected at 24 h (p < 0.05), but no effect was observed at 48 h, while organic matter digestibility remained unchanged. In conclusion, Nypa fruticans fruit pellets, in combination with CP level, modified rumen fermentation patterns and were associated with lower CH₄ production under in vitro conditions, without negatively affecting digestibility. These findings suggest potential for further in vivo evaluation. Full article

This study aims to evaluate the blanching process of wasted Undaria pinnatifida as a ruminant feed source by assessing chemical compositions, in vitro nutrient digestibility, rumen fermentation characteristics, greenhouse gas emissions, and rumen microbes. The blanching process was conducted at different temperatures (15 vs. 80 vs. 90 °C) and times (2 vs. 4 min) to assess the chemical and mineral contents. Supplementation levels of U. pinnatifida (0 vs. 0.5 vs. 1 vs. 2%) were observed with the blanching process (non-blanching (NBL) vs. blanching (LOS)). Increasing blanching temperature and time decreased (p < 0.05) dry matter, crude ash, and the mineral contents, including sodium, phosphorus, and arsenic. Moreover, LOS treatment increased (p < 0.01) in vitro dry matter and neutral detergent fiber digestibility, ruminal pH, and the acetate-to-propionate ratio, but reduced (p < 0.01) CH₄ (mL/g NDFD). Additionally, 2% of LOS treatment reduced the abundance of protozoa, fungi, fibrolytic microbes, methanogenic archaea, Methanobrevibacter ruminantium, Methanosarcina barkeri, and Methanosphaera stadtmanae (p < 0.01). Therefore, blanching at 80 °C for 2 min improved the nutritional profile by reducing antinutritional minerals. Subsequent in vitro fermentation suggested that supplementing the diet with 0.5–1% of LOS improved digestibility and altered fermentation, potentially reducing methane yield (per NDFD). Full article

This study investigated the interactive effects of corn silage and ramie silage on in vitro rumen fermentation characteristics, aiming to provide a scientific basis and empirical evidence for the rational incorporation of ramie into ruminant diets. Four binary substrate mixtures were formulated based on dry matter (DM) mass ratios of corn silage to ramie silage: 100:0 (CON), 60:40 (R40), 20:80 (R80), and 0:100 (R100). Rumen fluid was collected from three adult Liuyang black goats surgically fitted with permanent rumen cannulas, and a standardized 48 h in vitro batch culture assay was conducted. Results demonstrated that increasing the proportion of ramie silage significantly decreased (p < 0.05) the DM degradation rate, neutral detergent fiber (NDF) degradation rate, acid detergent fiber (ADF) degradation rate, and total gas production per gram of substrate DM. Specifically, CON and R40 exhibited significantly higher values for all four parameters than R80 and R100 (p < 0.05). Methane production was significantly reduced in all ramie-containing treatments relative to CON (p < 0.05), whereas hydrogen production increased progressively with ramie inclusion level, with CON yielding significantly less H₂ than both R80 and R100 (p < 0.05). Regarding fermentation parameters, increasing ramie proportion elevated (p < 0.05) both fermentation fluid pH and the acetate-to-propionate ratio, while total volatile fatty acid (TVFA) concentration declined linearly (p < 0.05). TVFA concentrations did not differ significantly between CON and R40, yet both were significantly greater than those in R80 and R100 (p < 0.05). Collectively, these findings indicate that ramie silage is a nutritionally valuable forage with potential as a high-quality partial replacement for conventional silages in ruminant feeding systems; however, its inclusion in corn–ramie mixed silages should not exceed 40% (on a DM basis) to maintain optimal fermentative efficiency and nutrient degradability. Full article

To explore the appropriate supplementation rate of yeast culture (YC) in Kazakh sheep during fattening, the effects of different YC supplementation rates on rumen fermentation parameters and microbial community were studied through in vitro rumen fluid fermentation experiments. A 0.40 g high-concentrate diet was used as the fermentation substrate, and five groups were added with YC at 0% (CK), 1.25% (YC1), 2.5% (YC2), 3.75% (YC3) and 5% (YC4) of dietary dry matter, respectively. Anaerobic fermentation was carried out for 48 h in 60 mL fermentation broth. The results showed that the 48 h GP and microbial crude protein (MCP) concentration in all YC supplementation groups were significantly higher than those in the CK group (p < 0.05). The concentrations of total volatile fatty acids (TVFA) and propionate in the YC1 and YC2 groups were significantly increased and the A/P ratio in the two groups was significantly decreased (p < 0.05). The Multi-factor Comprehensive Evaluation Index (MFAEI) calculation indicated that 1.25% was appropriate. The YC1 and YC2 groups significantly increased the richness and diversity of rumen bacterial communities (Chao1 and Shannon indices, p < 0.05), and significantly increased the relative abundance of Bacteroidota and NK4A214_group (p < 0.05), while significantly decreasing the relative abundance of the potential pathogenic bacterium Campylobacter (p < 0.05). Ustilago abundance was significantly suppressed in all the YC-supplemented groups (p < 0.05). The most effective YC supplementation rate among the tested doses was 1.25% according to the MFAEI and key microbial indicators. The results suggest that dietary supplementation of 1.25% YC (dry matter basis) may beneficially modulate rumen fermentation parameters under in vitro conditions, providing a reference for further in vivo studies on its application in fattening Kazakh sheep. Full article

Milk fat synthesis in dairy cows is a complex process affected by ruminal fermentation, systemic metabolism, and mammary gland activity. To explore the metabolic interplay across these systems, a multi-tissue metabolomics approach (rumen fluid, plasma, and milk) using ultra-high-performance liquid chromatography–mass spectrometry was used to identify metabolic differences between Chinese Holstein cows with high (H-MF, 5.82 ± 0.41%) and low (L-MF, 3.60 ± 0.12%) milk fat content under the same diet. The bovine mammary epithelial cells (BMECs) were also cultured to evaluate the impact of a key metabolite, malic acid (MA), on lipid metabolism. Our findings reveal distinct metabolic profiles across rumen fluid, plasma, and milk, with 96, 109, and 79 differential metabolites, respectively, between the L-MF and H-MF groups. In rumen fluid, H-MF cows showed higher levels of lauric acid and succinic acid, linked to fatty acid biosynthesis, while the L-MF cows had elevated citraconic and orotic acids, associated with amino acid metabolism and liver stress. Plasma from the H-MF cows contained higher β-hydroxybutyric acid, methionine sulfoxide, and phosphatidylcholine, supporting lipogenesis, whereas L-MF plasma showed increased 3-hydroxy-L-proline, indicating tissue catabolism. In milk, the L-MF cows had higher MA, while the H-MF cows exhibited elevated L-carnitine, linked to fatty acid β-oxidation. Metabolite trend analysis during rumen fluid–plasma–milk showed that 211 metabolites were classified into 8 profiles. Profile 1 had the largest number of metabolites whose levels were down-regulated from rumen to plasma and enriched in lipid metabolism. Profile 3 (mainly related to amino acid metabolism) and profile 4 (mainly related to energy metabolism) exhibited opposite trends from plasma to milk. In vitro, 200 μM of MA reduced the triglyceride content in BMECs and down-regulated lipogenic genes and their protein expression levels (fatty acid synthase, stearoyl-CoA desaturase and sterol regulatory element binding protein 1). These results highlight how rumen fluid, plasma, and milk metabolites collectively influence milk fat synthesis, with MA acting as a key regulator of lipid metabolism in mammary epithelial cells. Full article

Livestock represent a key asset in the livelihood of smallholder farmers and play a critical role in the social dynamics and nutritional security of resource-poor communities. However, within these resource-poor communities, livestock productivity remains low. This is often due to seasonal changes in the quantity and quality of available feed from the natural veld, which in turn also contributes to methane production. This study aimed to evaluate the effects of supplementing Eragrostis curvula hay with Searsia lancea leaf or silage meal on in vitro fermentation efficiency and gas and methane production. Therefore, an in vitro study using a semi-automated pressure transducer technique was conducted on grass hay alone (control) and grass hay supplemented with 15% or 30% of either S. lancea leaf or silage meal. The dietary treatments were arranged in a complete randomized design, with each treatment replicated four times. Total gas and methane production was recorded at 3, 6, 12, 24 and 48 h using a pressure transducer attached to a data logger. After incubation, samples were collected to determine volatile fatty acids. Supplementing grass hay with 15% S. lancea leaf meal increased gas production by 76%, 52%, 32% and 12% in the first 24 h of incubation. Similarly, increasing the supplementation level to 30% increased gas production by 75%, 63%, 45% and 14%. However, supplementing grass hay with silage meal at 15% significantly reduced gas production by 37% during the first 3 h of incubation, whereas supplementation at 30% had no effect. Supplementing grass hay with S. lancea meals effectively reduced methane production at 24 and 48 h. Grass hay supplemented with 15% or 30% silage meal reduced methane by 46% and 39% at 24 h, while at 48 h, methane was reduced by 39% and 49%, respectively. Supplementing grass hay with S. lancea meals, however, did not affect volatile fatty acids. In conclusion, S. lancea can be strategically used as a supplementary feed source to modulate the rumen ecosystem by attenuating enteric methane production. Further studies are required to determine the effect of S. lancea on rumen microbial composition and its metabolic function. Full article

Nigella sativa meal (NSM) is a by-product of the oil extraction process with potential use as a functional feed ingredient in ruminant nutrition due to its rich bioactive compounds and nutrient content. Therefore, this replicated (n = 6) completely randomized design (CRD) study aimed to firstly characterize NSM for its the bioactive compounds by chromatography–mass spectrometry (GC–MS) and liquid chromatography–MS (LC–MS). The effects of its dietary inclusion at 0%, 5%, 7.5%, and 10% NSM on in vitro rumen fermentation, gas production, CH₄ production, and rumen degradability were analyzed. Rumen fluid was collected orally from five sheep (body weight 20 ± 2 kg/head) and incubated with basal diets supplemented with pre-determined levels of NSM. The identified bioactive compounds identified included palmitic acid, oleic acid, linoleic acid, phenolic, flavonoid, thymoquinone, and saponin. The results showed that the dietary NSM supplementation in the diet significantly increased (p < 0.05) gas production at 18 and 48 h, NH₃–N concentration, IVDMD (In vitro dry matter degradability), and IVOMD (In vitro organic matter degradability). However, there was no significant effect (p > 0.05) on gas production at 12 and 24 h, and CH₄ production, pH, acetate, propionate, iso-butyrate, butyrate, iso-valerate, valerate, the acetate-to-propionate ratio (A:P), or total VFA concentration. Rumen fermentation was optimally modulated up to 10% without adverse effects on digestibility or CH₄ production. In this context, NSM acted as a functional feed ingredient in vitro. Therefore, in vivo analyses are required to confirm the efficacy under practical feeding conditions. Full article

The rumen simulation technique (RUSITEC^®) is a known model for research in rumen microbiology and fermentation. However, our research group observed inconsistencies in gas production across trials. This study investigated the effects of different gas outlet locations on digestibility, ruminal fermentation, gas production, and microbial protein synthesis. Fifteen fermenters tested three different gas outlet locations within the RUSITEC^® equipment: (1) gas outlet directly on the effluent vessel for output liquid (EV); (2) gas outlet directly on fermenter cap (F); and (3) gas outlet on both effluent vessel and fermenter cap (EVF). Data were analyzed using a completely randomized design in SAS (v. 9.4) with the MIXED procedure, and significance was set at p < 0.10. Results showed that altering the gas outlet location did not affect nutrient digestibility (p > 0.10), microbial protein synthesis (p > 0.10), and volatile fatty acid (VFA) production when expressed on a molar basis (p > 0.10). However, total gas production (p = 0.108) was higher in the EVF group and ammonia nitrogen produced in the fermenter was higher in group F (p = 0.081). Furthermore, methane (CH₄) production was underestimated when the gas outlet location was in just one of the locations when compared to the EVF group (p = 0.006). VFA proportion was also affected, with lower acetate (p = 0.005) and higher butyrate (p = 0.014) for group EV. These results indicate that the location of the gas outlet is an important methodological factor affecting fermentation measurements in the RUSITEC system, with outlets positioned in both the effluent and fermenter vessels enhancing gas recovery. Full article

In ruminant nutrition, the lignocellulosic complex is a primary constraint limiting the utilization of dietary fiber. The objective of this study was to evaluate the effects of inoculating lignin-degrading bacteria (LDB) isolated from the ruminant rectum on in vitro rumen fermentation characteristics. Rectal fecal samples were collected from healthy beef cattle, dairy cattle, buffaloes, and goats (n = 4 per species) using the grab sampling technique. Twenty-eight bacterial colonies were isolated through enrichment and screening on media containing sodium lignosulfonate. Lignin degradation efficiency was assessed spectrophotometrically, while laccase activity was determined using a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation assay. Seven isolates exhibiting ligninolytic activity (1.4–5.6% degradation efficiency) were selected to evaluate their effects on in vitro rumen fermentation using a completely randomized design with four replicates. LDB treatments were standardized to a concentration of 2.4 × 10⁵ colony-forming units/mL of rumen fluid medium, while the control received an equal volume of a 0.85% sterile NaCl solution. A rice straw-based total mixed ration served as the substrate, with rumen fluid collected from beef cattle. All treatments were incubated for 48 h. Notably, isolate BC3 consistently enhanced in vitro dry matter digestibility (23.1%), total gas production (18.6%), and total volatile fatty acid concentrations (13.2%) relative to the control and other LDB isolates (p < 0.01). All seven LDB isolates were identified as Gram-negative, rod-shaped, facultative anaerobic bacteria that exhibit catalase activity and tolerate moderately acidic conditions. Phylogenetic tree analysis based on 16S rRNA gene sequencing identified isolate BC3 as being closely related to Escherichia coli strains. These findings demonstrate that the ruminant hindgut is a promising source of LDB with the functional potential to enhance feed digestibility and fermentation end-products in the rumen. Future research should prioritize in vivo trials to evaluate the safety and efficacy of LDB as a direct-fed microbial, specifically focusing on its impact on animal performance and health. Full article

The present study was conducted to demonstrate the effects of Codium fragile extract on methane production using in vitro and in vivo experiments. An in vitro batch experiment was conducted to evaluate different inclusion levels of Codium fragile extract (0, 0.25, and 0.5% of substrate dry matter). Methane production significantly decreased in the 0.5% treatment (p < 0.05), whereas dry matter digestibility and total volatile fatty acid concentration were not significantly affected (p > 0.05). Based on the in vitro results, an in vivo feeding experiment was conducted using a 0.5% inclusion level of Codium fragile extract on Hanwoo steers. Methane emissions were significantly decreased by approximately 10% in steers fed Codium fragile extract (p < 0.05). In contrast, rumen fermentation characteristics, feed intake, average daily gain, and blood parameters were not significantly different between the treatments (p > 0.05). These results demonstrate that a dietary additive with 0.5% Codium fragile extract effectively reduced methane emissions without negatively affecting rumen fermentation and growth performance in Hanwoo steers. Full article

Few studies have investigated the effects of L-valine (L-Val) supplementation on in vitro rumen fermentation parameters and methane (CH₄) production in low-nitrogen diets for ruminants. Therefore, we examined the impact of L-Val supplementation in low-protein diets on in vitro rumen fermentation parameters, CH₄ production, and microbial community structure. Two crude protein (CP) levels and 4 L-Val levels were tested as follows: CON group (Control group with 14.05% CP), LD group (low-nitrogen diets with 11.26% CP), LVA group (LD group + 0.25% L-Val), LVB group (LD group + 0.5% L-Val), LVC group (LD group + 0.75% L-Val), and LVD group (LD group + 1% L-Val). The experiment was conducted at five time points (2 h, 4 h, 8 h, 12 h, 24 h), with three replicates per treatment at each time point. Results indicated the following: (1) Fermentation pH decreased overall with time; at each time point, the LVB group exhibited the highest pH, significantly higher than the LD, LVC, and LVD groups (p < 0.05). (2) Ammonia nitrogen (NH₃-N) concentration increased over time, with LVA~LVD groups showing higher levels than the LD group at 24 h, while showing no difference compared to the CON group (p > 0.05). (3) Microbial protein (MCP) trends aligned with NH₃-N, with the LVB group exhibiting higher MCP than the LD group, while showing no difference compared to the CON group (p > 0.05). (4) Compared to the LD group, adding 0.5~1% L-Val increased acetic acid, total VFA (TVFA), and isobutyric acid concentrations at 4 h, 8 h, and 24 h fermentation (p < 0.05). (5) The LVB group exhibited higher proportions of protozoa and Fibrobacter succinogenes (F. succinogenes) compared to the LD group (p < 0.05). The proportion of F. succinogenes showed no significant difference from the CON group (p > 0.05), while the proportion of Butyrivibrio fibrisolvens (B. fibrisolvens) decreased when L-Val addition exceeded 0.5%. (6) Correlation analysis revealed positive correlation between protozoa and TVFA (R = 0.512, p = 0.030). Isobutyric acid showed positive correlations with protozoa, B. fibrisolvens, and F. succinogenes (p < 0.05). In summary, under the present experimental conditions, the addition of 0.5% L-Val to a low-nitrogen diet did not affect predicted CH₄ production, but improved other in vitro rumen fermentation parameters, including acetate, isobutyrate and MCP. Meanwhile, it favored the growth and proliferation of the fibrolytic bacteria (B. fibrisolvens and F. succinogenes). This provides a theoretical basis for the rational formulation of low-nitrogen diets for sheep. Full article