Dual-flow Continuous Culture System Research Articles

Effects of monensin and cashew nut-shell extract on bacterial community composition in a dual-flow continuous culture system.

The objective of this study was to evaluate the effects of including monensin and two doses of CNSE in a high producing dairy cow diet on ruminal bacterial communities. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin Square design. A basal diet was formulated to meet the requirements of a cow producing 45kg of milk per d (17% crude protein and 27% starch). There were four experimental treatments: the basal diet without any feed additive (CON), 2.5 μM monensin (MON), 100ppm CNSE granule (CNSE100), and 200ppm CNSE granule (CNSE200). Samples were collected from the fluid and solid effluents at 3, 6, and 9h after feeding; a composite of all time points was made for each fermenter within their respective fractions. Bacterial community composition was analyzed by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Treatment responses for bacterial community structure were analyzed with the PERMANOVA test run with the R Vegan package. Treatment responses for correlations were analyzed with the CORR procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). Significance was declared at P ≤ 0.05. We observed that the relative abundance of Sharpea (P < 0.01), Mailhella (P = 0.05), Ruminococcus (P = 0.03), Eubacterium (P = 0.01), and Coprococcus (P < 0.01) from the liquid fraction and the relative abundance of Ruminococcus (P = 0.03) and Catonella (P = 0.02) from the solid fraction decreased, while the relative abundance of Syntrophococcus (P = 0.02) increased in response to MON when compared to CNSE treatments. Our results demonstrate that CNSE and monensin have similar effects on the major ruminal bacterial genera, while some differences were observed in some minor genera. Overall, the tested additives would affect the ruminal fermentation in a similar pattern.

PSVI-30 Effects of Different Cobalt Sources on Ruminal Fermentation of Lactating Dairy Cow Diets in a Dual-Flow Continuous Culture System

Abstract The objective of this study was to evaluate the effects of different sources of cobalt in a lactating dairy cow diet on in vitro ruminal fermentation. All diets provided the same amount of supplemental Co (1 mg per kg of diet DM) included in a premix with a targeted inclusion level of 2.5% of diet DM. Treatments were cobalt carbonate (CON), and three alternative Co sources; alternative source 1 (AS1), alternative source 2 (AS2), and alternative source 3 (AS3). Eight dual-flow continuous culture fermenters were used in a replicated 4 × 4 Latin square. Experimental periods were 10 d (7 d adaptation and 3 d sample collection). Composite samples of daily effluents were collected and analyzed for NH3-N, lactate, and VFA concentration as well as for nutrient digestibility. Samples of ruminal content were collected from each fermenter at 0, 1, 2, 4, 6, and 8 h after feeding to determine lactate, NH3-N, and pH kinetics. All data were analyzed using PROC MIXED of SAS and the repeated variable of time was included for pH, lactate, and NH3-N. There were neither treatment effects nor interactions between treatment and time for pH, NH3-N, and lactate concentrations, indicating a similar Co kinetics across treatments. With regards to lactate pool, we did not observe treatment effects for L-lactate; however, we observed treatment effects for D-lactate and total-lactate as seen in Table 1. Analyses of total and individual VFA, N utilization, and nutrient degradation are still undergoing, hence, it is possible that Co sources have effects on ruminal fermentation.

Effects of Microencapsulated Blend of Organic Acids and Pure Botanicals on the Ruminal Microbiota in an In Vitro Dual-Flow Continuous Culture System

The objective of the study was to evaluate the effects of dietary supplementation of a microencapsulated blend of organic acids and pure botanicals (mOAPBs) on the solid- and liquid-associated microenvironment (SAM and LAM, respectively) of the ruminal microbiome using an in vitro dual-flow continuous culture system. Ruminal content was incubated in eight fermenters and the basal diet was supplemented with increasing levels of mOAPBs (0; 0.12; 0.24; or 0.36% DM) which contained 55.6% hydrogenated and refined palm oil, 25% citric acid, 16.7% sorbic acid, 1.7% thymol, and 1% vanillin. All diets had a similar nutritional composition (16.1 CP, 30.9 NDF, and 32.0 starch, % DM basis). After 7 days of adaptation, a pooled sample across the days was collected in each period for identification of the microbiome of SAM and LAM. There was no effect of mOAPB on alpha-, beta-diversity, and microbial abundance. The SAM had a greater bacterial diversity and the principal component analysis demonstrated that it had a divergent bacterial profile from LAM. Additionally, SAM had an increased abundance of carbohydrate-degrading microorganisms. In summary, mOAPBs did not modulate the ruminal microbiome. The microenvironment microbiome of solid- and liquid-associated microenvironments were different, with SAM having a greater carbohydrate-degrading microorganism population.

The Effects of Incremental Doses of Aflatoxin B1 on In Vitro Ruminal Nutrient Digestibility and Fermentation Profile of a Lactating Dairy Cow Diet in a Dual-Flow Continuous Culture System.

Aflatoxin B1 (AFB1) is a mycotoxin known to impair human and animal health. It is also believed to have a deleterious effect on ruminal nutrient digestibility under in vitro batch culture systems. The objective of this study was to evaluate the effects of increasing the dose of AFB1 on ruminal dry matter and nutrient digestibility, fermentation profile, and N flows using a dual-flow continuous culture system fed a diet formulated for lactating dairy cows. Eight fermenter vessels were used in a replicated 4 × 4 Latin square design with 10 d periods (7 d adaptation and 3 d sample collection). Treatments were randomly applied to fermenters on diet DM basis: (1) 0 μg of AFB1/kg of DM (Control); (2) 50 μg of AFB1/kg of DM (AF50); (3) 100 μg of AFB1/kg of DM (AF100); and (4) 150 μg of AFB1/kg of DM (AF150). Treatments did not affect nutrient digestibility, fermentation, and N flows. Aflatoxin B1 concentration in ruminal fluid increased with dose but decreased to undetectable levels after 4 h post-dosing. In conclusion, adding incremental doses of AFB1 did not affect ruminal fermentation, digestibility of nutrients, and N flows in a dual-flow continuous culture system fed diets formulated for lactating dairy cows.

Effects of partially replacing dietary corn with sugars in a dual-flow continuous culture system on the ruminal microbiome.

The objective of this study was to evaluate the effects of feeding sugars as a replacement for starch on the ruminal microbiome using a dual-flow continuous culture system. Four periods of 10 days each were conducted with 8 fermenters in a 4 × 4 replicated Latin square design. Treatments included: 1) control with corn-CON, 2) molasses-MOL, 3) untreated condensed whey permeate-CWP, and 4) CWP treated with a caustic agent-TCWP as a partial substitute for corn. Sugars were defined as the water-soluble carbohydrates (WSC) concentration. Diets were formulated by replacing 4% of the diet DM in the form of starch from corn with the sugars in byproducts. Microbial samples for DNA analysis were collected from the solid and liquid effluent containers at 3, 6, and 9 h after feeding. Bacterial community composition was analyzed with sequencing the V4 region of the 16S rRNA gene using Illumina MiSeq platform. Data were analyzed with R 4.1.3 packages vegan, lmer, and ggplot to determine the effects of treatment on the relative abundance of taxa in the solid and liquid fractions, as well as the correlation of Acetate: Propionate ratio and pH to taxa relative abundance. Treatments did not affect alpha or beta diversity. At the phylum level the relative abundance of Proteobacteria was increased in CON compared to sugars in the solid fraction. In the liquid fraction, Firmicutes had greater relative abundance in sugar treatments while Bacteroidota and Spirochaetota were present in lower relative abundance in CWP. For solid and liquid samples, the family Lachnospiraceae had greater relative abundance in sugar treatments compared to CON. The decreased relative abundance of Christensenellaceae and Rikenellaceae paired with the greater relative abundance of Selenomonadaceae in CWP could help explain greater propionate molar proportion and decreased ruminal pH previously observed for this treatment. The genera Olsenella a lactic acid-producing bacterium, had the greatest relative abundance in MOL. Incorporating TCWP or MOL as a partial replacement for starch was more conservative of fibrolytic bacterial taxa compared to CWP. Additionally, TCWP did not increase bacterial taxa associated with synthesis of lactate as compared to MOL. Overall, replacing starch with sugars is mostly conservative of the ruminal microbiome; however, changes observed coincide with differences observed in acetate and propionate proportions and ruminal pH.

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.

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1–8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9–11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, d-, l-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3–N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3–N concentration was reduced to approximately 2 mg/dL as the challenge progressed.

Effects of different protein sources on nutrient disappearance, rumen fermentation parameters and microbiota in dual-flow continuous culture system

Scarce high-quality protein feed resources has limited the development of animal husbandry. In this study, we used a dual-flow continuous culture system to evaluate effects of difference dietary protein sources including soybean meal (SBM), cottonseed meal (CSM), and rapeseed meal (RSM), on nutrient disappearance, rumen fermentation, and microbiota of XiongDong black goats. Dietary proteins of either CSM, RSM or SBM had no effect on nutrient disappearance (P > 0.05). CSM or RSM significantly reduced (P < 0.01) the pH and enhanced (P < 0.01) the ammonia nitrogen (NH3-N) concentration in fermentation liquid compared to SBM. The short-chain fatty acids (SCFAs) contents were greater (P = 0.05) and acetate was lower (P < 0.01) in SBM than those in RSM and CSM, whereas propionate was greater (P < 0.01) in RSM than that in SBM, consequently reducing the acetate to propionate ratio (A/P) in RSM. Bacteroidetes, Firmicutes, and Proteobacteria were detected as the dominant phyla, and the relative abundances of Spirochaetae (P < 0.01) and Chlorobi (P < 0.05) declined in the CSM and RSM groups as compared to those in the SBM group. At the genus level, Prevotella_1 was the dominant genus; as compared to SBM, its relative abundance was greater (P < 0.01) in CSM and RSM. The abundances of Prevotellaceae_Ga6A1 and Christensenellaceae_R7 were lower (P < 0.05) in CSM, whereas Eubacterium_oxidoreducens_group, and Treponema_2 were lower (P < 0.01) in both CSM and RSM, and other genera were not different (P > 0.10). Although the bacterial community changed with different dietary protein sources, the disappearances of nutrients were not affected, suggesting that CSM and RSM could be used by rumen bacteria, as in case with SBM, and are suitable protein sources for ruminant diets.

Effects of lactic acid-producing bacteria as direct-fed microbials on the ruminal microbiome

The objective of this study was to evaluate ruminal microbiome changes associated with feeding Lactobacillus plantarum GB-LP1 as direct-fed microbials (DFM) in high-producing dairy cow diets. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin square design. A basal diet was formulated to meet the requirements of a cow producing 45 kg of milk per day (16% crude protein and 28% starch). There were 4 experimental treatments: the basal diet without any DFM (CTRL); a mixture of Lactobacillus acidophilus, 1 × 109 cfu/g, and Propionibacterium freudenreichii, 2 × 109 cfu/g [MLP = 0.01% of diet dry matter (DM)]; and 2 different levels of L. plantarum, 1.35 × 109 cfu/g (L1 = 0.05% and L2 = 0.10% of diet DM). Bacterial samples were collected from the fluid and particulate effluents before feeding and at 2, 4, 6, and 8 h after feeding; a composite of all time points was made for each fermentor within their respective fractionations. Bacterial community composition was analyzed through sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequenced data were analyzed on DADA2, and statistical analyses were performed in R (RStudio 3.0.1, https://www.r-project.org/) and SAS 9.4 (SAS Institute Inc.); orthogonal contrasts were used to compare treatments. Different than in other fermentation scenarios (e.g., silage or beef cattle high-grain diets), treatments did not affect pH or lactic acid concentration. Effects were mainly from overall DFM inclusion, and they were mostly observed in the fluid phase. The relative abundance of the phylum Firmicutes, family Lachnospiraceae, and 6 genera decreased with DFM inclusion, with emphasis on Butyrivibrio_2, Saccharofermentans, and Ruminococcus_1 that are fibrolytic and may display peptidase activity during fermentation. Lachnospiraceae_AC2044_group and Lachnospiraceae_XPB1014_group also decreased in the fluid phase, and their relative abundances were positively correlated with NH3-N daily outflow from the fermentors. Specific effects of MLP and L. plantarum were mostly in specific bacteria associated with proteolytic and fibrolytic functions in the rumen. These findings help to explain why, in the previous results from this study, DFM inclusion decreased NH3-N concentration without altering pH and lactic acid concentration.

Effects of partially replacing dietary corn with molasses, condensed whey permeate, or treated condensed whey permeate on ruminal microbial fermentation

Corn is a feedstuff commonly fed to dairy cows as a source of energy. The objective of this study was to evaluate whether partially replacing dietary corn with molasses or condensed whey permeate, in lactating dairy cow diets in a dual-flow continuous culture system, can maintain nutrient digestibility by ruminal microorganisms. Furthermore, this study evaluated whether treating condensed whey permeate before feeding could aid the fermentation of the condensed whey permeate in the rumen. Eight fermentors were used in a 4 × 4 replicated Latin square with 4 periods of 10 d each. The control diet (CON) was formulated with corn grain, and the other diets were formulated by replacing corn grain with either sugarcane molasses (MOL), condensed whey permeate (CWP), or treated condensed whey permeate (TCWP). Diets were formulated by replacing 4% of the diet dry matter (DM) in the form of starch from corn with sugars from the byproducts. Sugars were defined as water-soluble carbohydrates (WSC) in the rations. The fermentors were fed 52 g of DM twice daily of diets containing 17% crude protein, 28% neutral detergent fiber, and 45% nonfiber carbohydrates. Liquid treatments were pipetted into each fermentor. After 7 d of adaptation, samples were collected for analyses of volatile fatty acids (VFA), lactate, and ammonia, and fermentors' pH were measured at time points after the morning feeding for 3 d. Pooled samples from effluent containers were collected for similar analyses, nutrient flow, and N metabolism. Data were statistically analyzed using Proc MIXED of SAS version 9.4 (SAS Institute Inc.); fixed effects included treatment and time, and random effects included fermentor, period, and square. The interaction of treatment and time was included for the kinetics samples. The TCWP and MOL treatments maintained greater fermentor pH compared with CWP. Total VFA concentration was increased in CWP compared with MOL. The acetate:propionate ratio was increased in TCWP compared with CON, due to tendencies of increased acetate molar proportion and decreased propionate molar proportion in TCWP. Lactate concentration was increased in MOL. Digestibility of WSC was increased in the diets that replaced corn with byproducts. The partial replacement of 4% of DM from corn starch with the sugars in byproducts had minimal effects on ruminal microbial fermentation and increased pH. Treated CWP had similar effects to molasses.

Effects of bacterial cultures, enzymes, and yeast-based feed additive combinations on ruminal fermentation in a dual-flow continuous culture system.

Bacterial cultures, enzymes, and yeast-derived feed additives are often included in commercial dairy rations due to their effects on ruminal fermentation. However, the effects of these additives when fed together are not well understood. The objective of this study was to evaluate the changes in ruminal fermentation when a dairy ration is supplemented with combinations of bacterial probiotics, enzymes and yeast. Our hypotheses were that ruminal fermentation would be altered, indicated through changes in volatile fatty acid profile and nutrient digestibility, with the inclusion of (1) an additive, (2) yeast, and (3) increasing additive doses. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square with four 10 d experimental periods, consisting of 7 d for diet adaptation and 3 d for sample collection. Basal diets contained 52:48 forage:concentrate and fermenters were fed 106 g of dry matter per day divided equally between two feeding times. Treatments were: control (CTRL, without additives); bacterial culture/enzyme blend (EB, 1.7 mg/d); bacterial culture/enzyme blend with a blend of live yeast and yeast culture (EBY, 49.76 mg/d); and a double dose of the EBY treatment (2×, 99.53 mg/d). The bacterial culture/enzyme blend contained five strains of probiotics (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtilis, and Enterococcus faecium) and three enzymes (amylase, hemicellulase, and xylanase). On d 8–10, samples were collected for pH, redox, volatile fatty acids, lactate, ammonia N, and digestibility measurements. Statistical analysis was performed using the GLIMMIX procedure of SAS. Repeated measures were used for pH, redox, VFA, NH3-N, and lactate kinetics data. Orthogonal contrasts were used to test the effect of (1) additives, ADD (CTRL vs. EB, EBY, and 2X); (2) yeast, YEAST (EB vs. EBY, and 2X); and (3) dose, DOSE (EBY vs. 2X). No effects (P > 0.05) were observed for pH, redox, NH3-N, acetate, isobutyrate, valerate, total VFA, acetate:propionate, nutrient digestibility or N utilization. Within the 24 h pool, the molar proportion of butyrate increased (P = 0.03) with the inclusion of additives when compared to the control while the molar proportion of propionate tended to decrease (P = 0.07). In conclusion, the inclusion of bacterial cultures, enzymes and yeast in the diet increased butyrate concentration; but did not result in major changes in ruminal fermentation.

Antimicrobial supplementation alters digestibility and ruminal fermentation in a continuous culture model

ABSTRACT A dual-flow continuous culture system was used to evaluate the effects of laidlomycin propionate and bacitracin zinc on fermentation, nutrient digestibility, and microbial efficiency. Factors were laidlomycin propionate (LP; 2 mg/L of culture volume) and bacitracin zinc (BAC; 1.4 mg/L), and treatments were as follows: (1) no BAC or LP (CON), (2) LP without BAC (LP), (3) BAC without LP (BAC), and (4) LP and BAC (LP/BAC). A fifth treatment was supplemented with monensin sodium (MON; 6 mg/L) to act as a positive control. Both LP and LP/BAC had significantly greater pH than MON (P < 0.05). Antibiotic treatment did not affect NH 3-N concentration (P = 0.62), but did influence total VFA production (P = 0.02). Monensin fermenters had a greater proportion of total VFA than did LP fermenters (P < 0.05), and improved VFA production by 7.2% compared to CON. Laidlomycin suppressed the production of both acetate (P < 0.01) and butyrate (P = 0.05), and acetate was further reduced when LP was fed in combination with BAC (P = 0.01). Laidlomycin numerically reduced the acetate:propionate ratio (P = 0.12). These results suggest that bacitracin may produce ionophore-like effects on pH and VFA production in vitro.

183 A blend of plant extracts (Actifor Pro) potentially reduced methane production in a dual-flow continuous culture system fed bermudagrass hay with or without corn gluten feed

Abstract Twelve dual-flow continuous culture fermenters (1.95 L) were used to evaluate the effect of a phytogenic feed additive (Actifor Pro, Delacon, Engerwitzdorf, Austria; ACT) on ruminal fermentation and methane production when fed high fiber diets, comprised of bermudagrass hay (BGH) with or without corn gluten feed (CGF). Fermenters were utilized in a generalized randomized block design with a 2 × 2 factorial arrangement of treatments (n = 6): 1) diet (with or without CGF at 22% of diet DM) and 2) additive (with or without ACT at 1.0 g/L ACT). Two 10-d periods were conducted. Overall, comparing to no CGF, supplementation with CGF resulted in lower dry matter, organic matter, crude protein digestibility, microbial efficiency of nitrogen utilization, and methane production (mL of CH4/mol of total VFA), but increased neutral detergent fiber digestibility (all P ≤ 0.05). Diet × additive interactions were observed for molar proportion of acetate and propionate, and acetate-to-propionate ratio (A:P, all interactions P &amp;lt; 0.05), where inclusion of ACT increased acetate molar proportion and A:P (both P &amp;lt; 0.05), and decreased propionate molar proportion in diets with CGF (P = 0.05). A diet × additive interaction was also observed for methane production (ml of CH4/mol of total VFA; P = 0.08), where ACT decreased CH4 production per mol of VFA by 42% (P = 0.04), when only BGH was fed to the fermenters. In conclusion, CGF supplementation in BGH diets reduced methane production and improved fiber digestibility. The decrease in methane production per mol of VFA observed with ACT in the BGH without CGF diet warrants further investigation.

Copper sulfate and sodium selenite lipid-microencapsulation modifies ruminal microbial fermentation in a dual-flow continuous-culture system

Undesirable interactions between trace mineral elements and ruminal contents may occur during digestion when mineral salts are supplemented. Antimicrobial effects of copper sulfate (CuSO4) may affect ruminal digestibility of nutrients when fed as a source of copper (Cu), while sodium selenite (Na2SeO3) may be reduced in the rumen to less available forms of selenium (Se). Our objective was to evaluate if protection of CuSO4 and Na2SeO3 by lipid-microencapsulation would induce changes on ruminal microbial fermentation. We used 8 fermentors in a dual-flow continuous-culture system in a 4 × 4 duplicated Latin square with a 2 × 2 factorial arrangement of treatments. Factors were CuSO4 protection (unprotected and protected by lipid-microencapsulation) and Na2SeO3 protection (unprotected and protected by lipid-microencapsulation). Treatments consisted of supplementation with 15 mg/kg of Cu and 0.3 mg/kg of Se from either unprotected or protected (lipid-microencapsulated) sources, as follows: (1) Control (unprotected CuSO4 + unprotected Na2SeO3); (2) Cu-P (protected CuSO4 + unprotected Na2SeO3); (3) Se-P (unprotected CuSO4 + protected Na2SeO3); (4) (Cu+Se)-P (protected CuSO4 + protected Na2SeO3). All diets had the same nutrient composition and fermentors were fed 106 g of dry matter/d. Each experimental period was 10 d (7 d of adaptation and 3 d for sample collections). Daily pooled samples of effluents were analyzed for pH, NH3-N, nutrient digestibility, and flows (g/d) of total N, NH3-N, nonammonia N (NAN), bacterial N, dietary N, and bacterial efficiency. Kinetics of volatile fatty acids was analyzed in samples collected daily at 0, 1, 2, 4, 6, and 8 h after feeding. Main effects of Cu protection, Se protection, and their interaction were tested for all response variables. Kinetics data were analyzed as repeated measures. Protection of Cu decreased acetate molar proportion, increased butyrate proportion, and tended to decrease acetate:propionate ratio in samples of kinetics, but did not modify nutrient digestibility. Protection of Se tended to decrease NH3-N concentration, NH3-N flow, and CP digestibility; and to increase flows of nonammonia N and dietary N. Our results indicate that protection of CuSO4 may increase butyrate concentration at expenses of acetate, while protection of Na2SeO3 tended to reduce ruminal degradation of N. Further research is needed to determine the effects of lipid-microencapsulation on intestinal absorption, tissue distribution of Cu and Se, and animal performance.

Estimating degradation of individual essential amino acids in fish meal and blood meal by rumen microbes in a dual-flow continuous-culture system

Current feeding systems are based on the assumption that the AA profile of rumen undegraded protein is similar to that of the original feed. The objective of this experiment was to determine rumen bacterial degradation of individual essential AA in fish meal (FM) and blood meal (BM). Eight dual-flow continuous-culture fermentors were used in a completely randomized block design with a factorial arrangement of treatments and 3 replicated periods. Fermentors were supplied with 95 g of dry matter/d of isonitrogenous diets. Treatments contained a nonprotein N source (urea and tryptone) that was substituted with increasing proportions of FM or BM (0, 33, 67, or 100%). Diets consisted of 22.0% crude protein, 35.2% neutral detergent fiber, 34.6% nonfiber carbohydrates, 2.0% ether extract, and 9.2% ash. We hypothesized that the increase in the flow of individual AA would be attributed to the increase in the supply of the AA from each protein supplement. True organic matter degradation was decreased by increasing levels of FM or BM, but did not affect degradation of neutral detergent fiber and acid detergent fiber, total volatile fatty acids (VFA) concentration, or the molar proportion of propionate. There was a substrate by level of inclusion interaction in acetate molar proportion and branched-chain VFA. Butyrate concentration decreased linearly with increasing levels of FM and BM in treatment. Changes in branched-chain VFA reflected differences in content of branched-chain AA between supplements and the level of inclusion, although the quadratic effect suggests that other factors were involved. Ammonia-N concentration showed a substrate by level of inclusion interaction. Total dietary N and AA flows increased with increasing levels of FM or BM in treatment. The efficiency of bacterial crude protein synthesis was not affected by treatment, but the flow of bacterial N decreased in FM diets as the level of FM increased. Flows of AA increased linearly with increasing levels of the respective AA from supplements. Arginine, Ile, Met and Phe were more degradable, while His was more resistant to bacterial degradation. Results suggest that the resistance to rumen bacterial degradation of individual AA varies within FM and BM protein and may affect the estimates of dietary supply of individual AA to the small intestine.

Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

Comparison of microbial fermentation data from dual-flow continuous culture system and omasal sampling technique: A meta-analytical approach

Although the omasal sampling technique (OST) has been successfully used to estimate ruminal fermentation and nutrient flow, alternatives to invasive animal trials should be pursued and evaluated. The objective of this study was to evaluate carbohydrate and N metabolisms using a meta-analytical approach to compare 2 methods: dual-flow continuous culture system (DFCCS) and OST. To be included, studies needed to report diet chemical composition and report at least 1 of the dependent variables of interest. A total of 155 articles were included, in which 97 used the DFCCS and 58 used the OST. The independent variables used were dietary nonfiber carbohydrate concentration, neutral detergent fiber (NDF) degradability, true crude protein (CP) degradability, and efficiency of microbial protein synthesis (EMPS). In addition, 12 dependent variables were used. Statistical analyses were performed using the Mixed procedure of SAS (SAS Institute Inc., Cary, NC). A random coefficients model was used considering study as a random effect and including the possibility of covariance between the slope and the intercept. The effect of method (DFCCS or OST) was included and tested in the estimates of the intercept, linear, and quadratic effects of the independent variable. There was no method effect when NDF degradability was regressed with total volatile fatty acids concentration, true CP degradability, and EMPS. Molar proportions of acetate and propionate were quadratically associated with NDF degradability. When NDF degradability was regressed with acetate and propionate there was a method effect, differing only in the intercept (β0) estimate. True organic matter digestibility, bacterial N/total N, efficiency of N utilization, total volatile fatty acid concentration, and molar proportion of butyrate linearly increased as dietary nonfiber carbohydrate concentration increased, and none of these variables were affected by method. Concentration of ammonia N had a linear and positive association with true CP degradability. This was the only variable that had a method effect when regressed with true CP degradability, differing only in the estimate of the intercept (β0). As EMPS increased, efficiency of N utilization also increased, and it was affected by method. Overall, the majority of DFCCS responses were similar to OST. When a method effect was observed, it was mainly on the estimate of the intercept, demonstrating that the magnitude of these responses was different. However, the relationships between independent and dependent variables were similar across methods.

In vitro evaluation of Lactobacillus plantarum as direct-fed microbials in high-producing dairy cows diets.

The objectives of this study were: 1) to compare the effects of live yeast (LY), yeast fermentation product (YFP), a mix of Lactobacillus acidophilus and Propionibacterium freudenreichii (MLP), and Lactobacillus plantarum included as additives in dairy cows’ diets on in vitro ruminal fermentation and gas production (GP); and 2) to evaluate the effects of L. plantarum as direct-fed microbials (DFM) in dairy cows’ diets on in vitro ruminal fermentation, GP, nutrient digestibility, and N metabolism. Three experiments were carried out: Exp. 1 had the objective to compare all additives regarding ruminal fermentation parameters: an Ankom GP system was used in a completely randomized design, consisting of four 48 h incubations, and eight replications per treatment. There were eight treatments: a basal diet without additive (CTRL) or with one of the following additives: LY, YFP, MLP, or L. plantarum at four levels (% of diet Dry Matter (DM)): 0.05% (L1), 0.10% (L2), 0.15% (L3), and 0.20% (L4). In Exp. 2, a batch culture was used to evaluate ruminal fermentation, and CO2 and CH4 production using the same treatments and a similar experimental design, except for having 16 replications per treatment. Based on Exp. 1 and 2 results, Exp. 3 aimed at evaluating the effects of the L. plantarum on ruminal true nutrient digestibility and N utilization in order to evaluate the use of L. plantarum as DFM. The treatments CTRL, MLP, L1, and L2 were used in a replicated 4 × 4 Latin square design using a dual-flow continuous culture system. Data were analyzed using linear and nonlinear regression; treatment means were compared through contrasts, and L treatments in Exp. 1 and 2 were tested for linear, quadratic, and cubic effects. In Exp. 1, all treatments containing additives tended to reduce OM digestibility as well as reduced total volatile fatty acids (VFA) concentration and total GP. The YFP had greater OM digestibility than LY, and MLP treatment had greater total VFA concentration compared to L. plantarum treatments. In Exp. 2, additives reduced CO2 production, and there were no major differences in CH4. In Exp. 3, all additives reduced NH3-N concentration. In conclusion, pH and lactate concentration were not affected in all three experiments regardless of additive tested, suggesting that these additives may not improve ruminal fermentation by pH modulation; and L. plantarum may improve ruminal N metabolism when used as DFM in high-producing dairy cows’ diets, mainly by reducing NH3-N concentration.

In vitro Digestibility, In situ Degradability, Rumen Fermentation and N Metabolism of Camelina Co-Products for Beef Cattle Studied with A Dual Flow Continuous Culture System of Camelina Co-Products for Beef Cattle.

Simple SummaryCurrently, vegetable protein sources such as soybean meal and rapeseed meal are expensive and with volatile prices. These economic circumstances are driving the research of potential new protein resources for beef cattle diets that can reduce the ration cost without compromising animal productive yields. As possible candidates, camelina meal and camelina expeller have been studied; they are co-products with a high protein percentage, obtained after oil extraction from the oil seeds of Camelina sativa. The objectives of this study were to characterize these camelina co-products and ascertain if they could be useful ingredients for beef cattle diets. The results indicate that the diets formulated with camelina meal and camelina expeller do not show differences in the efficiency of microbial protein synthesis compared to the current reference proteins, camelina meal diet being the most similar to soybean meal and rapeseed meal diets, and camelina expeller the diet with the highest fermentation potential. The results of soybean meal as an individual ingredient reveal more differences with camelina co-products. In vivo studies are necessary to draw conclusions, but in vitro results obtained suggest that camelina meal and camelina expeller are potential substitutes for rapeseed meal in beef cattle diets.Camelina meal (CM) and camelina expeller (CE) were compared with soybean meal (SM) and rapeseed meal (RM). Trial 1 consisted of a modified Tilley and Terry in vitro technique. Trial 2 was an in situ technique performed by incubating nylon bags within cannulated cows. Trial 3 consisted in dual-flow continuous culture fermenters. In Trial 1, CM, CE and RM showed similar DM digestibility and OM digestibility, and SM was the most digestible ingredient (p < 0.05). Trial 2 showed that CE had the numerically highest DM degradability, but CP degradability was similar to RM. Camelina meal had a DM degradability similar to SM and RM and had an intermediate coefficient of CP degradability. In Trial 3, CE diet tended to present a higher true OM digestibility than SM diet (p = 0.06). Total volatile fatty acids (VFA) was higher in CE and CM diets than in SM diet (p = 0.009). Crude protein degradation tended to be higher (p = 0.07), and dietary nitrogen flow tended to be lower (p = 0.06) in CE diet than in CM diet. The efficiency of microbial protein synthesis was not affected by treatment (p > 0.05). In conclusion, CE and CM as protein sources differ in CP coefficient of degradability but their results were similar to RM. More differences were detected with regard to SM.

Nutrients digestibility, metabolic parameters and milk production in postpartum Holstein cows fed pomegranate (Punica granatum L.) by-products silage under heat stress condition

Two experiments were conducted to determine the best level of pomegranate by-products silage (PBS) in dairy cows ration and then its effect on transition dairy cows under heat stress condition was studied. In the first experiment, corn silage (CS) was replaced with the incremental levels of PBS [0, 60, 120 and 180 g PBS/kg dry matter (DM)] in total mixed ration (TMR) and then the nutrient digestibility and rumen fermentation parameters were studied using a dual flow continuous culture system. In the second experiment, effects of feeding PBS (120 g PBS addition per kg DM of TMR) on in vivo nutrient digestibility, ruminal fermentation, blood metabolites and milk yield were investigated in 20 postpartum transition dairy cows under heat stress condition [average of temperature-humidity index (THI); 74.3 ± 2.58]. Fresh pomegranate by-products (PB) including peel and seed were ensiled in a bunker silo with addition 20 g urea/kg DM. Results of the first experiment showed that PBS had about 49 g phenolic compounds/kg DM. Replacing of CS in TMR with 60 g and 120 g PBS improved neutral detergent fiber (aNDF) and acid detergent fiber (ADF) digestibility. TMR supplemented with 120 g PBS produced greatest total volatile fatty acids (VFA) and molar proportion of propionate after fermentation, but molar proportion of acetate and acetate:propionate ratio were lowest. Results of second experiment showed that replacing of CS with 120 g PBS increased total milk production and energy corrected milk (ECM), but it decreased milk protein and lactose content. Feeding fresh cows with 120 g PBS increased their lying behavior compared to the control group (712.1 vs. 572.6 min. d). Cows fed 120 g PBS had higher ADF digestibility and lower crude protein (CP) digestibility than that the control group. Cows fed 120 g PBS numerically had greater serum glucose. It was concluded that replacing of CS with 120 g PBS had potential to increase milk yield in Holstein dairy cows under heat stress condition without any adverse effect on their blood metabolites or health.