Branched-chain Volatile Fatty Acids Research Articles

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

IntroductionT-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.MethodsIn 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.ResultsThe 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.DiscussionThese 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.

The effect of sainfoin (Onobrychis viciifolia Scop.) drying temperature and pelleting on in vitro fermentation characteristics using equine faecal inocula

This study was conducted to determine the effects of drying temperature and pelleting of the tanniniferous forage legume sainfoin (Onobrychis viciifolia Scop.) on the in vitro fermentation characteristics by equine faecal inocula. The sainfoin used for the experiment was air dried on the land and oven dried at 30, 70 and 130 °C for 2.5 h. Part of the dried sainfoin samples was left as dried hay, another part was pelleted, resulting in 6 different samples. The hay and pellets were tested for particle size distribution and pellets were tested for their pellet quality in terms of hardness, durability and bulk density. The gas production technique was used to simulate fermentation and faecal samples of eight randomly selected horses were used to create four independent inocula mixtures that were examined in two subsequent gas production runs. Per inocula mixture, the hay and pellet samples were incubated in triplicate as is, or in combination with polyethylene glycol to inactivate the tannins. Cumulative gas production, a proxy for the organic matter (OM) fermentation, was measured for 72 h, whereafter fermentation fluids were sampled and analysed for volatile fatty acids (VFA) and pH. The results of the pellet quality measurements showed that the particle sizes of the sainfoin particles in the pellets were decreased by a higher drying temperature or by the pelleting process, indicating a loss of fibre physical effectiveness, a factor that may be indicative for chewing activity and hence saliva production. The gas production results showed a significant (P < 0.05) effect of drying temperature and the addition of polyethylene glycol on the cumulative gas production, the VFA production and the percentage of branched chain VFA produced. Hay dried at 130 °C had a lower (P < 0.05) gas production compared to hay dried at 30 and 70 °C, which indicates a lower OM fermentability. The addition of polyethylene glycol increased the gas and total VFA production, which indicates that tannins in sainfoin decrease the production of gas and VFA in hay pellets compared to the hay product. The pelleting process had no effect on the fermentation characteristics, but pelleting conditions strongly decreased the level of the amino acid lysine and its reactivity. The results indicate the increase in drying temperature to cause a decrease in the OM hindgut fermentation and the total and reactive lysine contents of sainfoin. The positive effects of the tannins (a higher proportion of propionic acid relative to acetic acid and a lower protein fermentation) remain, despite of technological treatments.

Assessing milk response to different combinations of branched-chain volatile fatty acids and valerate in Jersey cows

Some cellulolytic bacteria require 1 or more branched-chain volatile fatty acids (BCVFA) for the synthesis of branched-chain AA and branched-chain long-chain fatty acids because they are not able to uptake branched-chain AA or lack 1 or more enzymes to synthesize branched-chain AA de novo. Supplemental BCVFA and valerate were included previously as a feed additive that was later removed from the market; these older studies and more current studies have noted improvements in neutral detergent fiber digestibility and milk efficiency. However, most studies provided a single BCVFA or else isobutyrate (IB), 2-methylbutyrate (MB), isovalerate, and valerate altogether without exploring optimal combinations. Our objective was to determine a combination of isoacids that is optimal for milk production. Sixty (28 primiparous and 32 multiparous) lactating Jersey cows (106 ± 54 days in milk) were blocked and assigned randomly to either a control (CON) treatment without any isoacids, MB [12.3 mmol/kg dry matter (DM)], MB + IB (7.7 and 12.6 mmol/kg DM of MB and IB, respectively), or all 4 isoacids (6.2, 7.3, 4.2, and 5.1 mmol/kg DM of MB, IB, isovalerate, and valerate, respectively). Cattle were fed the CON treatment for a 2-wk period, then were assigned randomly within a block to treatments for 8 wk (n = 15). There was a trend for an interaction of supplement and parity for milk components. There were no differences in components for primiparous cows, whereas MB + IB tended to increase protein concentration by 0.04 and 0.08 percentage units in multiparous cows compared with the CON and MB treatments, respectively. Feeding MB + IB increased fat concentration by 0.23 to 0.31 percentage units compared with all other treatments in multiparous cows. Milk yield and dry matter intake (DMI) did not change with treatment. Treatment interacted with week for milk net energy for lactation/DMI; MB + IB tended to increase milk net energy of lactation/DMI by 0.10 Mcal/kg compared with MB and approached a trend for CON, mainly during the early weeks of the treatment period, whereas differences decreased during the last 2 wk of the treatment period. Cows fed MB had the highest 15:0 anteiso fatty acids in the total milk fatty acid profile, which was greater than that for CON or MB + IB cows, but not cows supplemented with isoacids. Cows fed MB alone had the numerically lowest milk net energy for lactation/DMI. The combination of MB + IB appeared optimal for increasing feed efficiency in our study and was not at the expense of average daily gain. Further research is needed for evaluating how potential changes in supplemental isoacid dosage should vary under differing dietary conditions.

Evaluation of a Natural Phytogenic Formulation as an Alternative to Pharmaceutical Zinc Oxide in the Diet of Weaned Piglets.

A natural phytogenic formulation (NPF) was tested as an alternative to pharmaceutical zinc oxide (ZnO) in weaned piglets with respect to growth performance, apparent total tract digestibility and faecal microbiota composition and metabolic activity. Two dietary NPF levels (NPF: 1000 and 2000 mg/kg diet) were compared to a positive control (ZnO: 3000 mg ZnO/kg diet) and a negative control (CON: no added ZnO or NPF) using 84 weaned piglets from 29 d to 78 d (days of age). Feed conversion ratio was improved (p < 0.05) in ZnO and NPF piglets were compared to CON at 50 d. Dry matter, organic matter and crude protein (p < 0.05) digestibility was improved in NPF piglets compared to CON at 57 d. Compared to CON, NPF inclusion reduced E. coli (p < 0.05) and increased C. leptum subgroup (p < 0.01) at 57 d and 78 d, and reduced C. perfringens subgroup (p < 0.05; at 78 d). The ZnO reduced (p < 0.001) E. coli and C. perfringens subgroup (p < 0.01) compared to CON at 78 d. Moreover, ZnO and NPF reduced molar ratios of branched chain volatile fatty acids (p < 0.05) compared to CON, while NPF also increased butyric acid (p < 0.05) at 78 d. In conclusion, the NPF appeared to be a promising alternative to pharmaceutical doses of ZnO.

Biotin and Leucine Alone or in Combination Promoted the Synthesis of Odd- and Branched-Chain Fatty Acids in the Rumen In Vitro

The odd- and branched-chain fatty acids (OBCFA) accumulated in ruminant products are a class of beneficial fatty acids for human health. Since biotin and leucine are involved in OBCFA synthesis, this study aimed to evaluate their effect on OBCFA synthesis in vitro. There were four treatments: the control group that only provided the basal diet, or the basal diet supplemented with biotin (4 mg/kg dry matter, DM), leucine (4 g/kg DM), or a combination of biotin (4 mg/kg DM) and leucine (4 g/kg DM). The results showed that biotin promoted the degradation of DM (p &lt; 0.10), while leucine significantly increased the concentration of branched-chain volatile fatty acids and valerate (p &lt; 0.05). The concentrations of total odd-chain fatty acids, total iso, total anteiso, total branched-chain fatty acids, total OBCFA, and total fatty acids were significantly increased by the supplementation of biotin or leucine (p &lt; 0.05). Biotin and leucine significantly stimulated the activities of acetyl-CoA carboxylase, fatty acid synthase, and malonyl-CoA, with a significant interaction effect (p &lt; 0.05). In conclusion, the results of this study suggested that biotin and leucine can be used as effective nutrition strategies to promote OBCFA synthesis.

Effects of exogenous amylolytic or fibrolytic enzymes inclusion on in vitro fermentation of lactating dairy cow diets in a dual-flow continuous-culture system

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH3-N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH3-N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH3-N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH3-N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows.

Dynamic Variations in Rumen Fermentation Characteristics and Bacterial Community Composition during In Vitro Fermentation

This study aimed to explore the dynamic variations of rumen fermentation characteristics and bacterial community composition during a 24 h in vitro fermentation. A total of twenty-three samples were collected from original rumen fluid (ORF, n = 3), fermentation at 12 h (R12, n = 10), and fermentation at 24 h (R24, n = 10). Results showed that gas production, concentrations of microbial crude protein, ammonia nitrogen, and individual volatile fatty acids (VFA), as well as total VFA and branched-chain VFA concentrations, were higher in R24 when compared with R12 (p &lt; 0.05). However, no significant differences were observed in acetate to propionate ratio and fermentation efficiency between R12 and R24 (p &gt; 0.05). Bacterial diversity analysis found that Shannon index and Simpson index were higher in R24 (p &lt; 0.05), and obvious clusters were observed in rumen bacterial community between R12 and R24. Taxonomic analysis at the phylum level showed that the abundances of Proteobacteria and Fibrobacteres were higher in R12 than that in R24, and inverse results were observed in Bacteroidetes, Firmicutes, Cyanobacteria, Verrucomicrobia, Lentisphaerae, and Synergistetes abundances. Taxonomic analysis at the genus level revealed that the abundances of Rikenellaceae RC9 gut group, Succiniclasticum, Prevotellaceae UCG-003, Christensenellaceae R-7 group, Ruminococcaceae UCG-002, Veillonellaceae UCG-001, and Ruminococcaceae NK4A214 group were higher in R24, whereas higher abundances of Succinivibrionaceae UCG-002, Ruminobacter, and Fibrobacter, were found in R12. Correlation analysis revealed the negative associations between gas production and abundances of Proteobacteria, Succinivibrionaceae UCG-002, and Ruminobacter. Moreover, the abundances of Firmicutes, Rikenellaceae RC9 gut group, Christensenellaceae R-7 group, and Ruminococcaceae UCG-002 positively correlated with VFA production. These results indicate that both rumen fermentation characteristics and bacterial community composition were dynamic during in vitro fermentation, whereas the fermentation pattern, efficiency, and bacterial richness remained similar. This study provide insight into the dynamics of rumen fermentation characteristics and bacterial composition during in vitro fermentation. This study may also provide a reference for decision-making for the sampling time point when conducting an in vitro fermentation for bacterial community investigation.