Branched-chain Volatile Fatty Acids Research Articles

Impact of oil type and savory plant on nutrient digestibility and rumen fermentation, milk yield, and milk fatty acid profile in dairy cows

Fat supplements are well known for their multiple beneficial effects on ruminant health, reproduction and productivity, and as a source for certain bioactive compounds in ruminant products. On the other hand, numerous phytochemicals have demonstrated the potential to improve rumen fermentation through modifying the volatile fatty acid (VFA) pattern to favour those with greater energy efficiency, boosting microbial protein synthesis, and decreasing methane emission and ruminal ammonia concentration. Savory is an aromatic plant rich in various phytochemicals (mainly carvacrol and flavonoids) that can alter ruminal metabolism of dietary fatty acids, potentially increasing the production of some bioactive compounds such as conjugated linoleic acids (CLAs). This study aimed to investigate combined effects of oil type (fish oil (FO) versus soybean oil (SO)) and the inclusion of savory (Satureja khuzistanica) plant (SP) in the diet on total tract digestibility of nutrients, rumen fermentation, milk yield and milk fatty acid profile in dairy cattle. Eight multiparous lactating Holstein cows were used in a replicated 4 × 4 Latin square design experiment with four diets and four 21-d periods. During each experimental period consisted of 14 days of adaptation and a 7-day sampling period, cows were randomly assigned to one of the four dietary treatments: the diet supplemented with 2% (DM basis) fish oil (FOD) or soybean oil (SOD), the FOD or SOD plus 370 g DM/d/head SP (FODs, SODs, respectively). The experimental diets were arranged in a 2 × 2 factorial design, with the fat sources as the first and SP as the second factor. The FO-supplemented diets had lower dry matter intake (DMI) and higher total tract digestibility than SO-supplemented diets (P < 0.05), and including SP in the diet improved total tract digestibility of dry matter (DM), organic matter (OM), ether extract (EE), and non-fibrous carbohydrates (NFC) (P < 0.05) without negatively affecting DMI. Rumen pH was lower with SO than with FO diets (P < 0.01) and increased with SP inclusion in the diet (P < 0.05). Total protozoa count and ruminal ammonia concentration decreased, and the branched-chain VFA (BCVFA) proportion increased with SP inclusion in the diet (P < 0.05). Milk production, as well as the concentration and yield of milk components (except lactose concentration) were higher with SO than with FO diets (P < 0.05), but these variables remained unaffected by SP. The milk concentrations of both non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) were lower with SO compared to FO diets, and these variables were reduced by SP (P < 0.01). The proportions of both mono- and polyunsaturated FA (MUFA and PUFA, respectively) in milk were higher with FO than with SO diets (P < 0.01), and their proportions increased by SP at the expense of saturated FA (SFA) (P < 0.01). Including SP in the diet increased the proportions of all the milk n-3 FA (C18:3c, C20:5, and C22:6) by 21%, 40%, and 97%, respectively, and those of conjugated linoleic acids (C18:2 (c9,t11-CLA) and C18:2 (t10,c12-CLA)) by 23% and 62%, respectively. There was no interaction between oil type and SP for the assessed variables. Fish oil, despite reducing milk production and milk components, was more effective than soybean oil in enriching milk with healthy FA. These findings also show promise for SP as a feed additive with the potential to improve total tract digestibility, rumen fermentation and milk FA composition.

Effects of Storage Time and Temperature on the Fermentation Characteristics of Rumen Fluid from a High-Forage Diet

The objective of this experiment was to investigate the effects of storage temperature and preservation time on the fermentation characteristics of rumen fluid collected from six Hu sheep fed a high-forage diet. The storage temperatures were set at −80 °C and −20 °C, and the preservation times were labelled as follows: 0 d (fresh rumen fluid, D0), 7 d (D7), 14 d (D14), 30 d (D30), 60 d (D60), 120 d (D120), and 240 d (D240). A repeated-measures design was applied to analyze the fermentation characteristics of rumen fluid across each preservation time point and storage temperature. The results showed that storage temperature had no significant effects on pH value, ammonia nitrogen (NH3-N), microbial protein (MCP), and volatile fatty acid (VFA) concentration and proportion (p &gt; 0.05). Specifically, the pH value increased on D7, D14, and D60 (p &lt; 0.05), while the MCP concentration decreased on D7, D14, D30, D120, and D240 but increased on D60 (p &lt; 0.05), and the concentration of NH3-N decreased on D14 (p &lt; 0.05). The acetate concentration increased on D30, while the concentrations of propionate, butyrate, valerate, and total VFA increased on D60 (p &lt; 0.05). The concentration of isovalerate decreased on D60, and isobutyrate and branched-chain VFA concentrations decreased on D120 (p &lt; 0.05). The proportion of acetate increased on D30, while the butyrate and valerate proportions increased on D60, and the proportions of isovalerate, isobutyrate, and branched-chain VFA decreased on D60 (p &lt; 0.05). For rumen fluid from a high-forage diet, the storage temperature (−80 °C and −20 °C) did not affect its fermentation characteristics, suggesting that rumen fluid could be preserved at −80 °C or −20 °C without altering its fermentation characteristics. The fermentation parameters of rumen fluid changed significantly after 7 days of preservation; hence, it is recommended to finish determining the fermentation parameters of rumen fluid within 7 days of collection. The concentrations and proportions of most VFA changed after 30 days of preservation; therefore, it is recommended that VFA determination should be completed within 30 days of rumen fluid collection.

Improving dairy manure hydrolysis and acidification through microbial community restructuring by adaptation to hyperthermophilic conditions

Dairy manure (DM) contributes significantly to greenhouse gas emissions and ecosystem degradation, yet its resistance to biodegradation hinders widespread bioprocessing applications. Lignocellulosic materials in DM pose a particular challenge because of their recalcitrance. Bioprocessing under hyperthermophilic (≥70 °C) conditions potentially offers an advantage over traditional fermentation temperatures due to enhanced activity of enzymes and the kinetics of enzymatic reactions. This can lead to a higher conversion rate and a greater extent of biomass hydrolysis and acidification. To test the validity of this hypothesis, the current study evaluated the efficacy of anaerobic hydrolysis and acidogenic fermentation of DM under mesophilic, thermophilic, and hyperthermophilic conditions. All inocula were adapted to corresponding temperatures but were derived from the same mesophilic source. Hyperthermophilic conditions resulted in superior DM hydrolysis efficiency (53%) compared to mesophilic (34%) and thermophilic (42%) conditions. The hyperthermophilic environment was particularly favorable to the decomposition of crude proteins and hemicellulose, which were reduced by 64% and 54%, respectively. Furthermore, hyperthermophilic fermentation also yielded the highest volatile fatty acid (VFA) production rate of 460 mg/L/day during the first four days, representing improvements of 50% and 90% over mesophilic and thermophilic conditions. In part, this was attributed to the enhanced production of branched-chain VFAs, including an increase of 6–10% in isobutyric acid and 12–13% in isovaleric acid. At hyperthermophilic conditions, however, there was no accumulation of VFAs during the days 5–8 of fermentation, which could be due to acetate conversion by the syntrophic acetate-oxidizing bacteria. A considerable gain in hydrolysis efficiency and VFA production rate were accompanied by a reduction in microbial diversity, which suggests that hyperthermophilic temperature is a favorable environment for the selection of organisms with enhanced DM hydrolysis and fermentation capabilities. A significantly increased relative abundance of xylanolytic Caldicoprobacter (23% of population) and proteolytic Thermovirga (9% of population) could be the major contributors to improved decomposition of hemicellulose and protein. As revealed by the techno-economic analysis, acidogenic fermentation of DM at 70 °C and a retention period of 4 days provides the greatest positive net present value, highest internal rate of return of 9.2%, and shortest investment payback period of 9 years. This study demonstrates that hyperthermophilic conditions enable superior deconstruction and bioconversion of lignocellulose-containing biomass into VFAs under reduced retention times, offering a promising approach for improving DM management and generating bioproducts.

Assessing the effects of high-carvacrol oregano oil on rumen microbial fermentation, gas production, and methane production in vitro

The objective of this in vitro study was to assess the effects of high-carvacrol oregano oil (ORE; 50 g/kg oregano oil, 800-802 g/kg carvacrol), on microbial fermentation and CH4 production. In the experiment (a complete randomized block design), treatments included a negative control (CTL, no additive), a positive control (monensin, MON, 10 mg/L), and ORE (20, 40, 80, 120, 200, 160, and 1000 mg/L). Compared with CTL, MON shifted (p &lt; 0.05) volatile fatty acid (VFA) profile from acetate and butyrate to propionate production, thereby reducing (p &lt; 0.05) CH4 production (-26%). Monensin decreased (p &lt; 0.05) NH3 and branched-chain VFA concentrations. Of the doses evaluated, only the highest dose (1000 mg/L) affected ruminal microbial fermentation and CH4 production. At this dose, ORE reduced (p &lt; 0.05) gas production, total VFA, acetate, propionate and NH3 concentrations, and CH4 production (-22%). The reduced gas production and total VFA is an indication of feed digestion inhibition. These results suggest that ORE may decrease CH4 production and improve ruminal N utilization. However, these findings need to be validated in vivo to determine the optimal dose to benefit from the positive effects while avoiding the negative impact of ORE on feed digestion.

Organic acid preservation of cereal grains improves grain quality, growth performance, and intestinal health of post-weaned pigs

This study investigated the effect of preserving wheat and barley grains with an organic acid liquid surfactant mould inhibitor compared to conventional artificial drying and assessed its effects on the health and performance of pigs post-weaning (PW). A 2 × 2 factorial arrangement was conducted to investigate the interaction between grain preservation method (dried vs. preserved) and zinc oxide (ZnO) inclusion (yes vs. no) on growth performance, diarrhoea scores and incidence, and total tract nutrient digestibility. One hundred and ninety-two pigs (3 pigs/pen; 16 replicates/treatment) were assigned to one of four experimental diets for 35 days: (1) dried grain diet, (2) preserved grain diet, (3) dried grain diet with ZnO and (4) preserved grain diet with ZnO. Diets were formulated to contain similar levels of net energy and standardised ileal digestible lysine. On day 35 PW, 24 pigs (12 replicates/treatment) from the non-ZnO supplemented groups were euthanised and digesta was collected for coefficient of apparent ileal digestibility (CAID), gut microbial population and volatile fatty acid analysis. Stomach tissue was also collected for gene expression analysis, as well as small intestine samples for gut morphology. Notable improvements in grain quality were observed in the preserved grain, including a reduction in grain pH, mould presence, and contamination by mycotoxins, specifically deoxynivalenol in wheat and barley, and ochratoxin A and HT-2 toxin in barley. The preserved grain diet improved average daily gain (ADG; P < 0.01) and body weight (BW; P < 0.01) compared to the dried grain diet. The incorporation of ZnO increased average daily feed intake (ADFI; P < 0.01) and BW (P < 0.05) in the dried grain diet, however, ZnO did not affect ADFI (P > 0.05), and reduced ADG and BW in the preserved grain diet (P < 0.05). Pigs offered the preserved grain diet had reduced faecal scores and diarrhoea incidence compared to those offered the dried grain diet (P < 0.05). Supplementation of ZnO reduced faecal scores and diarrhoea incidence compared to non-supplemented pigs (P < 0.001). On day 35 PW, the preserved grain diet had improved CAID of nitrogen and gross energy (P < 0.05), lower levels of colonic branched-chain volatile fatty acids (P < 0.05), and beneficial shifts in gut microbial populations. Specifically, there was a reduction in ileal Streptococcus (P < 0.001) and an increased abundance of colonic Faecalibacterium (P < 0.05). In conclusion, organic acid preservation improved grain quality, benefiting post-weaned pigs through improved nutrient digestibility and gut health, thus enhancing overall growth performance PW.

Production Responses of Multiparous Dairy Cattle with Differing Prepartum Muscle Reserves and Supplementation of Branched-Chain Volatile Fatty Acids

Periparturient dairy cattle undergo physiological adaptations to support fetal growth and colostrum synthesis in late gestation and milk production in early lactation. To support energy and protein demands dairy cattle mobilize body tissue reserves. The objective of this study was to determine the effects of prepartum skeletal muscle reserves and supplementation of branched-chain volatile fatty acids (BCVFA) on body composition measurements, metabolic markers related to health, protein, and energy status, and subsequent milk yield in multiparous dairy cows. Skeletal muscle reserves were assessed by 3 ultrasounds of the longissimus dorsi muscle depth (LDD) measured 42 d before expected calving (BEC), and cows (n = 48) were assigned to either high muscle (HM; > 4.6 cm) or low muscle (LM; ≤ 4.6 cm) groups. Cows were then randomly assigned to either control (CON) of soyhull pellets (80 g/d) or BCVFA treatment which contained isobutyrate (40 g/d), isovalerate (20 g/d), and 2-methylbutyrate (20 g/d) calcium salt products. Treatments were top dressed from 42 BEC until parturition, resulting in 4 combinations of muscle groups and treatments: HM-CON (n = 13), HM-BCVFA (n = 13), LM-CON (n = 11), and LM-BCVFA (n = 11). Measurements of the LDD, BW, and BCS were taken on the following days relative to calving -42, -35, -21, -7, 0, 7, 14, 21, 28. Weekly blood samples were taken to measure glucose, BHB, and insulin concentrations, and 5 of the blood sample time points were utilized to determine 3-methylhistidine and creatinine blood concentrations. Milk yield was recorded daily for the first 28 d of lactation, and samples were taken from both milkings once a wk for the first 4 wk to determine components. The statistical model included the fixed effects of treatment, group, time, their interactions, and the random effect of cow nested within group and treatment. Prepartum muscle mobilization varied between muscle groups, as LM cows accreted muscle prepartum, and HM cows mobilized muscle. The HM cows had higher milk fat, protein, lactose, and energy corrected milk yields. The BCVFA supplementation tended to increase blood glucose concentrations both prepartum and postpartum and decreased milk urea nitrogen concentrations. Higher prepartum skeletal muscle reserves improve productivity of early lactation cows likely due to differences in muscle mobilization, and BCVFA supplementation improves glucose dynamics during the transition period, which may improve the metabolic health of the periparturient dairy cow.

Rumen microbiota helps Tibetan sheep obtain energy more efficiently to survive in the extreme environment of the Qinghai-Tibet Plateau.

T-sheep and H-sheep exhibit different environmental adaptability and production performance. The rumen microbiome has co-evolved with hosts and plays a vital role in nutrient digestion and energy metabolism. In our previous study, we found that T-sheep have a higher efficiency in energy metabolism than H-sheep, but the rumen microbial community remains unclear. In this study, we determined the rumen bacterial profile and rumen fermentation parameters to reveal the bacterial profiles and predictive functions among breeds and diets with four different energy levels, as well as the correlation between bacterial profiles and rumen fermentation characteristics. The results showed that the rumen total volatile fatty acids (VFAs), acetate, butyrate, total branched-chain VFAs, iso-butyrate, and iso-valerate were higher in T-sheep than H-sheep. The alpha diversity of ruminal bacteria is not affected by dietary energy, but it shows a distinction between the sheep breeds. Specifically, T-sheep rumen bacteria exhibit higher alpha diversity than H-sheep. The beta diversity of ruminal bacteria is not influenced by dietary energy or sheep breeds, indicating similar communities of ruminal bacteria between different diets and sheep breeds. The phyla of Bacteroidetes and Firmicutes predominate in the rumen, with a higher relative abundance of Firmicutes observed in T-sheep than H-sheep. The two most abundant genera in the rumen were Prevotella 1 and Rikenellaceae RC9 gut group. Prevotella 1 is the predominant bacterial genus in the rumen of H-sheep, while the Rikenellaceae RC9 gut group dominates in the rumen of T-sheep. Microbial co-occurrence network analysis reveals that variations in rumen fermentation characteristics result from differences in module abundance, with a higher abundance of VFA-producing modules observed in the rumen of T-sheep. Microbial function prediction analysis showed that dietary energy rarely alters the functional composition of rumen bacteria. However, there were differences in the functions of rumen bacteria between sheep breeds, with T-sheep showing a greater emphasis on energy metabolism-related functions, while H-sheep showed a greater emphasis on protein metabolism-related functions. These findings provide evidence of the special rumen microbial community that helps T-sheep efficiently obtain energy from low-protein and low-energy diets, enabling them to survive in the extreme environment of the Qinghai-Tibet Plateau.

Molecular signatures of longissimus dorsi differ between dairy cattle based on prepartum muscle reserves and branched-chain volatile fatty acid supplementation.

Dairy cattle with high (HM) versus low muscle (LM) reserves exhibit distinct temporal changes in longissimus dorsi muscle depth (LDD) in late gestation. Branched-chain volatile fatty acids (BCVFA) supplementation increased blood glucose levels. We hypothesized that differences in HM and LM reflect distinct muscle metabolism and BCVFA supplementation altered metabolic pathways. At 42 d before expected calving (BEC) Holstein dairy cows were enrolled in a 2 x 2 factorial study of diet and muscle reserves, by assigning to control (CON) or BCVFA supplemented diets and LDD of HM (>4.6 cm) or LM (≤4.6 cm) groups: HM-CON (n=13), HM-BCVFA (n=10), LM-CON (n=9), and LM-BCVFA (n=9). Longisumus dorsi was biopsied at 21 d BEC, total RNA isolated and protein coding gene expression measured with RNA-seq. Between HM and LM 713 genes were differentially expressed and 481 between BCVFA and CON (P<0.05). Transcriptional signatures indicated differential distribution of Type II fibers between groups, with MYH1 greater in LM and MYH2 greater in HM cattle (P<0.05). Signatures of LM cattle relative to HM indicated greater activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle displayed greater expression of genes that encode extracellular matrix proteins and factors that regulate their proteolysis and turnover. BCVFA modified transcriptomes by increasing expression of genes that regulate fatty acid degradation and flux of carbons into the TCA cycle as acetyl CoA. Molecular signatures support distinct metabolic strategies between LM and HM cattle, and that BCVFA supplementation increased substrates for energy generation.

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.

PSI-20 Isobutyrate supplementation improved energy and nitrogen digestibility in prepubertal gilts fed a high insoluble, but not soluble, fiber diet

Abstract Isoacids are short, branched-chain volatile fatty acids produced by microbial degradation of branched-chain amino acids. While there is a lack of information on isoacids supplementation in monogastric animals, in ruminants, isoacids are growth factors for cellulolytic bacteria which leads to improved digestibility of fibrous ingredients. The objective was to determine if feeding isoacids to prepubertal gilts could improve digestibility of high fiber diets. Two groups of 45 prepubertal gilts (body weight = 139.10 ± 0.68 kg) were individually housed and allotted to 1 of 9 dietary treatments for a 28-d trial (n = 10 per treatment). Dietary treatments consisted of a 3 × 3 factorial arrangement with factors of diet type [corn-soybean meal (CSBM), CSBM with 40% inclusion of distiller’s dried grains with solubles (DDGS), or CSBM with 40% inclusion of sugar beet pulp (SBP)] and isoacids inclusion [no isoacid (NO), 0.50% inclusion of isobutyrate (IB), or 0.88% inclusion of an isoacid mixture (isobutyrate, 2-methylbutyrate, isovalerate; 1:1:1 ratio; MX). Isobutyrate inclusion was the same between IB and MX diets. Gilts were limit fed approximately 2.2 kg per day. Overall growth performance, apparent total tract digestibility (ATTD) of energy and nitrogen (N), and fecal volatile fatty acids (VFA) profile (d 26) were determined. Average daily feed intake (ADFI) was reduced and gain:feed ratio (GF) was increased in gilts fed DDGS (2.14 ± 0.07 kg/d and 0.20 ± 0.02 kg/kg, respectively) or SBP (2.07 ± 0.07 kg/d and 0.19 ± 0.02 kg/kg, respectively) compared with gilts fed CSBM (2.30 ± 0.07 kg/d and 0.15 ± 0.02 kg/kg, respectively), regardless of isoacids inclusion (P &amp;lt; 0.05). Gilts fed DDGS-IB had increased energy (86.5 vs. 83.5 ± 0.42%) and N (84.7 vs. 81.3 ± 0.85%) ATTD compared with gilts fed DDGS-NO (P &amp;lt; 0.05). Gilts fed CSBM-MX had reduced energy (86.9 vs. 88.3 ± 0.50%) and N (75.4 vs. 78.7 ± 0.39%) ATTD compared with gilts fed CSBM-NO (P &amp;lt; 0.05). Moreover, gilts fed SBP-NO had increased energy (85.9 vs. 83.6 ± 0.44%) ATTD compared with the other groups and increased N (71.8 vs. 67.2 ± 0.51%) ATTD compared with SBP-IB (P &amp;lt; 0.05). Fecal content of isobutyrate was decreased in NO compared with the other groups (2.10 vs. 3.00 ± 0.31 mM/L), while fecal content of heptanoate was increased in MX compared with NO (0.06 vs. 0.03 ± 0.01 mM/L), regardless of diet type (P &amp;lt; 0.05). Gilts fed SBP-MX had increased fecal content of total VFA compared with gilts fed SBP-NO (108.90 vs. 67.60 ± 11.50 mM/L; P &amp;lt; 0.05). These results suggest that IB may positively impact the digestibility of diets with a high content of insoluble fiber.

Optimizing dietary rumen-degradable starch to rumen-degradable protein ratio improves lactation performance and nitrogen utilization efficiency in mid-lactating Holstein dairy cows.

The dietary rumen-degradable starch (RDS) to rumen-degradable protein (RDP) ratio, denoted as the RDS-to-RDP ratio (SPR), has been proven to enhance in vitro rumen fermentation. However, the effects of dietary SPR in vivo remain largely unexplored. This study was conducted to investigate the effect of dietary SPR on lactation performance, nutrient digestibility, rumen fermentation patterns, blood indicators, and nitrogen (N) partitioning in mid-lactating Holstein cows. Seventy-two Holstein dairy cows were randomly assigned to three groups (24 head/group), balanced for (mean ± standard deviation) days in milk (116 ± 21.5), parity (2.1 ± 0.8), milk production (42 ± 2.1 kg/d), and body weight (705 ± 52.5 kg). The cows were fed diets with low (2.1, control), medium (2.3), or high (2.5) SPR, formulated to be isoenergetic, isonitrogenous, and iso-starch. The study consisted of a one-week adaptation phase followed by an eight-week experimental period. The results indicated that the high SPR group had a lower dry matter intake compared to the other groups (p < 0.05). A quadratic increase in milk yield and feed efficiency was observed with increasing dietary SPR (p < 0.05), peaking in the medium SPR group. The medium SPR group exhibited a lower milk somatic cell count and a higher blood total antioxidant capacity compared to other groups (p < 0.05). With increasing dietary SPR, there was a quadratic improvement (p < 0.05) in the total tract apparent digestibility of crude protein, ether extract, starch, neutral detergent fiber, and acid detergent fiber. Although no treatment effect was observed in rumen pH, the rumen total volatile fatty acids concentration and microbial crude protein synthesis increased quadratically (p < 0.05) as dietary SPR increased. The molar proportion of propionate linearly increased (p = 0.01), while branched-chain volatile fatty acids linearly decreased (p = 0.01) with increasing dietary SPR. The low SPR group (control) exhibited higher concentration of milk urea N, rumen ammonia N, and blood urea N than other groups (p < 0.05). Despite a linear decrease (p < 0.05) in the proportion of urinary N to N intake, increasing dietary SPR led to a quadratic increase (p = 0.01) in N utilization efficiency and a quadratic decrease (p < 0.05) in the proportion of fecal N to N intake. In conclusion, optimizing dietary SPR has the potential to enhance lactation performance and N utilization efficiency. Based on our findings, a medium dietary SPR (with SPR = 2.3) is recommended for mid-lactating Holstein dairy cows. Nevertheless, further research on rumen microbial composition and metabolites is warranted to elucidate the underlying mechanisms of the observed effects.

Evaluation of direct-fed microbials on in vitro ruminal fermentation, gas production kinetic, and greenhouse gas emissions in different ruminants’ diet

IntroductionThree in vitro experiments were conducted to evaluate the effects of increasing levels of Enterococcus faecium and Saccharomyces cerevisiae (DFM1) and increasing levels of Bacillus licheniformis and Bacillus subtilis (DFM2) on in vitro ruminal fermentation parameters in three different dietary scenarios.MethodsFor Exp. 1, the basal diet consisted of 25:75 roughage:concentrate ratio (R:C) and was composed by 5 treatments: control (no additive), 2 levels of DFM1 (1X = 1.9 mg and 5X = 9.0 mg), and 2 levels of DFM2 (1X = 3.8 mg and 5X = 19 mg). The Exp. 2 consisted of a 41:59 R:C diet and was composed by 5 treatments: control (no additive) and 2 levels of DFM1 (1X = 3.8 mg and 5 X = 19 mg) and 2 levels of DFM2 (1X = 5.6 mg and 5X = 28 mg). The Exp. 3 consisted of a 100:0 R:C diet [Brachiaria (syn. Urochloa brizantha)] and was composed by the same treatments described in Exp. 1. The DFM1 contained 3.5 × 109 CFU per g of Enterococccus faecium and Saccharomyces cerevisiae, whereas the DFM2 contained Bacillus licheniformis and Bacillus subtilis at 3.2 × 109 CFU per g. In each Exp., an in vitro gas production (GP) system with 43-bottles (AnkomRF) was used in four consecutive 48 or 72-h fermentation batches to evaluate total GP (TGP), kinetics and fermentation profiles, methane (CH4), and carbon dioxide (CO2).ResultsFor Exp 1, DFM1 increased quadratically TGP at 24 and 48-h, which reflected in a greater in vitro organic matter digestibility (IVOMD). The concentrations of ammonia-N, CH4, and CO2 (mmol/g of IVOMD) reduced quadratically as DFM1 increased. For Exp. 2, DFM1 inclusion reduced butyrate concentration and acetate to propionate ratio. Regarding GHG emissions, DFM1 and DFM2 quadratically reduced CH4 and CO2 emission per IVOMD (mmol/g of IVOMD). For Exp. 3, DFM1 increased quadratically TGP at 48h with no impact on IVOMD. Otherwise, DFM2 increased linearly TGP at 24 and 48h which reflected in a greater IVOMD. The inclusion of DFM1 increased linearly iso-valerate and branched-chain volatile fatty acids (BCVFA) concentration and DFM2 addition increased BCVFA quadratically.DiscussionOverall, addition of DFM1 [Enterococccus faecium (5 × 109 CFU per g) + Saccharomyces cerevisiae (5 × 109 CFU per g)] or DFM2 [Bacillus licheniformis + Bacillus subtilis (3.2 × 109 CFU per g)] might enhance the fermentation process in the rumen and decrease greenhouse gas emissions in a dose-dependent manner, though the results are contingent on the specific type of diet.

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.

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.

Evaluation of yeast-based additives, as an alternative to ionophores, on rumen fermentation of ruminant diets using an in vitro gas production system

IntroductionThe study aimed to assess yeast-based additives' effects, as monensin alternatives, on rumen fermentation parameters, greenhouse gas emissions, and ruminal kinetics of ruminant diets using an in vitro system. Three experiments were conducted, each individually evaluating escalating levels of three yeast-based additives.MethodsThree experiments were designed: Experiment 1 evaluated prebiotic blend 1—yeast culture [Saccharomyces cerevisiae (Scer)], beta-glucans, fructooligosaccharides, galactooligosaccharides, and mannanoligosaccharides; Experiment 2 investigated prebiotic blend 2—beta-glucan fractions and mannanoligosaccharides from Scer; Experiment 3 examined yeast cells—hydrolyzed, inactivated, and spray-dried yeast (Scer) cells. Uniform experimental design and procedures were employed across the three experiments. Each experiment had six treatments: monensin (Rumensin®, 25 mg/kg DM) as positive control, and yeast additive levels (0, 533, 1,067, 1,600, and 2,133 mg/kg on DM basis) added to ruminant diets (60% corn silage and 40% concentrate). An in vitro gas production (GP) system with 50 AnkomRF bottles assessed total GP (at 24 and 48 hours), kinetics, fermentation profiles, methane (CH4), and carbon dioxide (CO2) concentrations. Comparison with monensin utilized Dunnett’s test (5%). Yeast additive levels were analyzed for linear and quadratic responses.ResultsIn Experiment 1, the 1,600 mg/kg yeast additive had lower concentrations of propionate, isobutyrate, valerate, and branched-chain volatile fatty acids (BCVFAs), and a higher acetate concentration and acetate-to-propionate ratio than monensin. In Experiment 2, the 1,600 mg/kg yeast additive led to lower total VFA and isovalerate concentrations than monensin. Additionally, compared to the 1,067 mg/kg yeast additive, monensin showed lower isovalerate concentration and higher NH3-N concentration. In Experiment 3, the 533 mg/kg yeast additive resulted in lower valerate and BCVFA concentrations, and higher CH4 and CO2 concentrations than monensin. Monensin had lower total VFA, butyrate, and acetate-to-propionate ratio, and higher propionate concentration compared to the 2,133 mg/kg yeast additive.DiscussionCollectively, these findings suggest yeast-based additives could be monensin alternatives, enhancing animal nutrient utilization efficiency and contributing to improved livestock sustainability.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. III: Protein metabolism and incorporation into bacterial protein

Some cellulolytic bacteria cannot transport branched-chain AA (BCAA) and do not express complete synthesis pathways, thus depending on cross-feeding for branched-chain volatile fatty acid (BCVFA) precursors for membrane lipids or for reductive carboxylation to BCAA. Our objective was to assess BCVFA uptake for BCAA synthesis in continuous cultures administered high forage (HF) and low forage (LF) diets without or with corn oil (CO). We hypothesized that BCVFA would be used for BCAA synthesis more in the HF than in LF diets. To help overcome bacterial inhibition by polyunsaturated fatty acids in CO, BCVFA usage for bacterial BCAA synthesis was hypothesized to decrease when CO was added to HF diets. The study was an incomplete block design with 8 dual-flow fermenters used in 4 periods with 8 treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were: HF or LF (67 or 33% forage, 33:67 alfalfa:orchardgrass pellets), without or with supplemental CO (3% of dry matter), and without or with 2.15 mmol/d (5 mg/d 13C) each of isovalerate, isobutyrate, and 2-methylbutyrate for one combined BCVFA treatment. The flow of bacterial BCAA increased by 10.7% by supplementing BCVFA and 9.14% with LF versus HF; similarly, dosing BCVFA versus without BCVFA increased BCAA by 1.98% in total bacterial AA, whereas LF increased BCAA by 1.92% versus HF. Additionally, BCVFA supplementation increased bacterial AA flow by 16.6% when supplemented in HF - CO and 12.4% in LF + CO diets, but not in the HF + CO (-1.5%) or LF - CO (+6.7%) diets (Diet × CO × BCVFA interaction). The recovery of 13C in bacterial AA flow was 31% lower with LF than with HF. Of the total 13C recovered in bacteria, 13.8, 17.3, and 30.2% were recovered in Val, Ile, and Leu, respectively; negligible 13C was recovered in other AA. When fermenters were dosed with BCVFA, nonbacterial and total effluent flows of AA, particularly of alanine and proline, suggest decreased peptidolysis. Increased ruminal outflow of bacterial AA, especially BCAA, but also nonbacterial AA could potentially support postabsorptive responses from BCVFA supplementation to dairy cattle.

Extrusion of lupines with or without addition of reducing sugars: Effects on the formation of Maillard reaction compounds, partition of nitrogen and Nε-carboxymethyl-lysine, and performance of dairy cows

The extrusion of leguminous seeds induces the formation of Maillard reaction compounds (MRC) as a product of protein advanced glycation and oxidation, which lowers protein degradability in the rumen. However, the quantitative relationship between the parameters of pretreatment (i.e., addition of reducing sugars) and extrusion, and the formation of MRC has not been established yet. Moreover, the fate of the main stable MRC, Nε-carboxymethyl-lysine (CML), in the excretory routes has never been investigated in ruminants. We aimed to test the effects of the temperature of extrusion of white lupines with or without addition of reducing sugars on the formation of MRC, crude protein (CP) degradability in the rumen, N use efficiency for milk production (milk N/N intake), and performance of dairy cows. Two experiments with a replicated 4 × 4 Latin square design were conducted simultaneously with 16 (3 rumen-cannulated) multiparous Holstein cows to measure indicators of ruminal CP degradability (ruminal NH3 concentration, branched-chain volatile fatty acids), metabolizable protein supply (plasma essential AA concentration), N use efficiency (N isotopic discrimination), and dairy performance. In parallel, apparent total-tract digestibility of dry matter, organic matter, neutral detergent fibers, N, total Lys and CML, and partition of N and CML were measured with 4 cows in both experiments. The diets consisted on a DM basis of 20% raw or extruded lupines and 80% basal mixed ration of corn silage, silage and hay from permanent grasslands, pelleted concentrate, and a vitaminized mineral mix. Expected output temperatures of lupine extrusion were 115°C, 135°C, and 150°C, without and with the addition of reducing sugars before extrusion. The extrusion numerically reduced the in vitro ruminal CP degradability of the lupines, and consequently increased the predicted supply of CP to the small intestine. Nitrogen balance and urinary N excretion did not differ among dietary treatments in either experiment. Milk yield and N use efficiency for milk production increased with extrusion of lupines at 150°C without addition of reducing sugars compared with raw lupines. Nitrogen isotopic discrimination between dietary and animal proteins (the difference between δ15N in plasma and δ15N in the diet) were lower with lupines extruded at 150°C without and with addition of reducing sugars. Regardless of sugar addition, milk true protein yield was not affected, but milk urea concentration and fat:protein ratio were lower with lupines extruded at 150°C than with raw lupines. In the CML partition study, we observed that on average 26% of the apparently digested CML was excreted in urine, and a much lower proportion (0.63% on average) of the apparently digested CML was secreted in milk, with no differences among dietary treatments. In conclusion, we showed that the extrusion of white lupines without or with addition of reducing sugars numerically reduced enzymatic CP degradability, with limited effects on N partition, but increased milk yield and N use efficiency at the highest temperature of extrusion without addition of reducing sugars.

Effect of replacing corn straw by sweet sorghum silage or whole plant corn silage in sheep diets on rumen fermentation and bacterial flora.

Sweet sorghum silage (SS; Sorghum dochna 'Dochna') has been extensively studied in recent years as a supplementary forage-to-corn silage (CS; Zea mays L.), but there are still relatively few studies on its effects on the rumen environment of sheep. Determining the short-term impact of converting roughage from corn straws to SS compared to CS on rumen fermentation and bacterial population dynamics was the main goal of the current study. Twelve female thin-tailed Han sheep (29.8±1.34kg) were randomly divided into one of two treatments: concentrate supplemented with SS or CS, respectively. During the 15-day pretest period, concentrate was fed in two separate feedings at 0800h and 1800h, and ensure that the animals were all consumed within an hour of being fed. Thereafter, the animals had free access to corn straw. The feeding procedures during the pretest period were the same as during the measurement period. Rumen fluid was collected via sheep esophageal tube on the last day of adaptation phase (1-7 days) and stabilisation phase (8-30 days), respectively. The results showed that there was a similarity in the total concentration of VFA (volatile fatty acid) and the proportions of acetate, propionate, butyrate, and branched-chain VFA (P>0.05) and microbial diversity indices (P>0.05) between the two silage groups throughout the experimental period. The concentration of Ammonia nitrogen (P=0.001) and proportion of valerate (P=0.028) decreased in the CS and SS groups, respectively. The abundance and predicted function of rumen bacteria in the SS group did not differ significantly (P>0.05) between the two measurement phases. However, the abundance of Prevotella_1 (P=0.038) was higher in the CS group than in the SS group at 7d. The abundances of Firmicutes (P=0.005) and Ruminococcaceae_NK4A214_group (P=0.002) increased, while the abundances of Bacteroidetes (P=0.044), Proteobacteria (P=0.046), and Prevotella_1 (P=0.009) decreased in the CS group at 30d. Genes related to pyruvate metabolism (P=0.020) were significantly higher at 30d than at 7d, whereas purine metabolism (P=0.007), pyrimidine metabolism (P=0.007), and metabolic pathways (P=0.010) were lower at 30d in the CS group. In conclusion, this study indicated that SS maintained a steady rumen environment, while CS caused high fluctuations in bacterial abundance and predicted function for sheep.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. I: Digestibility, microbial protein, and prokaryotic community structure

Branched-chain amino acids are deaminated by amylolytic bacteria to branched-chain volatile fatty acids (BCVFA), which are growth factors for cellulolytic bacteria. Our objective was to determine the dietary conditions that would increase the uptake of BCVFA by rumen bacteria. We hypothesized that increased forage would increase cellulolytic bacterial abundance and incorporation of BCVFA into their structure. Supplemental polyunsaturated fatty acids, supplied via corn oil (CO), should inhibit cellulolytic bacteria growth, but we hypothesized that additional BCVFA would alleviate that inhibition. Further, supplemental BCVFA should increase neutral detergent fiber degradation and efficiency of bacterial protein synthesis more with the high forage and low polyunsaturated fatty acid dietary combination. The study was an incomplete block design with 8 dual-flow continuous cultures used in 4 periods with 8 treatments (n = 4 per treatment) arranged as a 2 × 2 × 2 factorial. The factors were: high forage (HF) or low forage (LF; 67 or 33%), without or with supplemental CO (3% dry matter), and without or with 2.15 mmol/d (which included 5 mg/d of 13C each of BCVFA isovalerate, isobutyrate, and 2-methylbutyrate). The isonitrogenous diets consisted of 33:67 alfalfa:orchardgrass pellet, and was replaced with a concentrate pellet that mainly consisted of ground corn, soybean meal, and soybean hulls for the LF diet. The main effect of supplementing BCVFA increased neutral detergent fiber (NDF) degradability by 7.6%, and CO increased NDF degradability only in LF diets. Supplemental BCVFA increased bacterial N by 1.5 g/kg organic matter truly degraded (6.6%) and 0.05 g/g truly degraded N (6.5%). The relative sequence abundance decreased with LF for Fibrobacter succinogenes, Ruminococcus flavefaciens, and genus Butyrivibrio compared with HF. Recovery of the total 13C dose in bacterial pellets decreased from 144 µg/ mg with HF to 98.9 µg/ mg with LF. Although isotope recovery in bacteria was greater with HF, BCVFA supplementation increased NDF degradability and efficiency of microbial protein synthesis under all dietary conditions. Therefore, supplemental BCVFA has potential to improve feed efficiency in dairy cows even with dietary conditions that might otherwise inhibit cellulolytic bacteria.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. II: Biohydrogenation and incorporation into bacterial lipids

To maintain membrane homeostasis, ruminal bacteria synthesize branched-chain fatty acids (BCFA) or their derivatives (vinyl ethers) that are recovered during methylation procedures as branched-chain aldehydes (BCALD). Many strains of cellulolytic bacteria require 1 or more branched-chain volatile fatty acid (BCVFA). Therefore, the objective of this study was to investigate BCVFA incorporation into bacterial lipids under different dietary conditions. The study was an incomplete block design with 8 continuous culture fermenters used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were high (HF) or low forage (LF, 67 or 33% forage, 33:67 alfalfa:orchardgrass), without or with supplemental corn oil (CO; 3% dry matter, 1.5% linoleic fatty acid), and without or with 2.15 mmol/d (5 mg/d 13C each of isovalerate, isobutyrate, and 2-methylbutyrate). After methylation of bacterial pellets collected from each fermenter's effluent, fatty acids and fatty aldehydes were separated before analysis by gas chromatography and isotope ratio mass spectrometry. Supplementation of BCVFA did not influence biohydrogenation extent. Label was only recovered in branched-chain lipids. Lower forage inclusion decreased BCFA in bacterial fatty acid profile from 9.45% with HF to 7.06% with LF and decreased BCALD in bacterial aldehyde profile from 55.4% with HF to 51.4% with LF. Supplemental CO tended to decrease iso even-chain BCFA and decreased iso even-chain BCALD in their bacterial lipid profiles. The main 18:1 isomer was cis-9 18:1, which increased (P < 0.01) by 25% from CO (data not shown). Dose recovery in bacterial lipids was 43.3% lower with LF than HF. Supplemental CO decreased recovery in the HF diet but increased recovery with LF (diet × CO interaction). Recovery from anteiso odd-chain BCFA and BCALD was the greatest; therefore, 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Recovery in iso odd-chain fatty acids (isovalerate as primer) was greater than label recovery in iso even-chain fatty acids (isobutyrate as primer). Fatty aldehydes were less than 6% of total bacterial lipids, but 26.0% of 13C recovered in lipids were recovered in BCALD because greater than 50% of aldehydes were branched-chain. Because BCFA and BCALD are important in the function and growth of bacteria, especially cellulolytics, BCVFA supplementation can support the rumen microbial consortium, increasing fiber degradation and efficiency of microbial protein synthesis.