Dual-flow Continuous Culture Fermenters Research Articles

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.

Evaluation of a new commercial source of monensin in lactating dairy cows and continuous culture fermenters

The objective of this experiment was to evaluate a new commercial source of monensin (MON) on performance of mid-lactation dairy cows and fermentation parameters of dual-flow continuous culture fermenters. In Experiment 1, 43 Holstein cows (25 multiparous and 18 primiparous; 175 ± 97 DIM; 45.3 ± 10 kg/d milk yield; 692 ± 68 kg BW; mean ± SD) were used in a randomized block design experiment with a 15-d covariate and a 9-wk treatment period. The first 3 wk of the treatment period were considered adaptation and the last 6 wk were used for data collection and analysis. Treatments were: Control (CTR; no MON added), Rumensin® 90 (RUM; 350 mg/d MON from Elanco Animal Health Inc.), and Monovet® 90 (MVet; 350 mg/d MON from Huvepharma® Inc. US). All cows were fed the same base diet throughout the experiment and treatments were top-dressed during the treatment period. Orthogonal contrasts were used to evaluate CTR vs. MON (RUM + MVet) and RUM vs. MVet. Compared with CTR, MON tended to increase milk yield (43.8 vs. 42.3 kg/d) but did not affect DMI or feed efficiency. The MVet treatment improved feed efficiency compared with RUM (1.70 vs. 1.58). Treatments did not affect milk fat content and yield, milk protein yield, MUN concentration, or energy-corrected milk yield. In Experiment 2, nine continuous culture fermenters were used in a randomized block design experiment with two 10-d periods. Fermenters were fed once a day approximately 100 g DM of the same diet fed in Experiment 1. Treatments were: CTR, RUM (2 mg/L MON from Rumensin® 90), and MVet (2 mg/L MON from Monovet® 90). Monensin did not affect DM, OM, NDF, ADF, and CP degradability or total VFA production (mmol/d), but decreased acetate, butyrate, and isobutyrate molar proportions and increased propionate molar proportion compared with CTR. Both sources of MON decreased acetate to propionate ratio compared with CTR (1.61 vs. 2.40). Adding MON increased daily production of propionate (74.1 vs. 108 mmol/d) and decreased that of butyrate and isobutyrate compared with CTR. Monensin supplementation decreased ammonia-N production (433 vs. 545 mg/d) compared with CTR. Overall, results indicate MON tended to increase milk yield and altered VFA profile in the rumen and in vitro, and that MVet is a suitable source of MON for dairy cows.

PSVII-8 Effects of Processed Saccharina Latissima and Ascophyllum Nodosum on Ruminal Fermentation and Methane Production in Continuous Culture

Abstract The aim of this study was to evaluate the effects of processed dietary supplements derived from two seaweeds, Saccharina latissima (SL) and Ascophyllum nodosum (AN), on microbial methane production and fermentation profile using in vitro methods. Four dual-flow continuous culture fermenters were utilized in a 4x4 Latin Square design to assess these 4 supplements: unwashed, coarsely milled SL (UNW), 3-min rinsed SL (3MR), 20-sec blanched SL (20SB), and a crude phlorotannin extract from AN (PHLT). Treatments were randomly assigned to fermenters and were top dressed on the total mixed ration (TMR) at 5% of total dry matter (DM). Four, 10-d experimental periods were conducted, consisting of a 7-d equilibration period followed by a 3-d sampling period. Vessel pH was continually measured, and effluent samples were collected for volatile fatty acid (VFA) analysis and calculation of DM, organic matter (OM), neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibilities. Methane was measured prior to the first AM and PM feedings during the 3-d sampling period. Data were analyzed using the PROC MIXED procedure of SAS (9.4) with fermenter as the random effect and period and treatment as fixed effects. Processed treatments (3MR= 7.86, 20SB= 5.75, PHLT= 10.46 mg/dL) yielded less methane (P < 0.02) than unprocessed (UNW= 24.06 mg/dL). Apparent digestibilities of DM, OM, NDF and ADF were not affected by treatment. Total VFA (mM) was greater (P = 0.02) in 3MR than PHLT. The acetate to propionate ratio (A:P) was greater (P < 0.01) in PHLT compared with 20SB, 3MR and UNW (5.62 vs 1.47, 1.27 and 1.54, respectively). Time spent below pH 5.8 during fermentation was not affected by treatment. These results indicate that seaweed processing method can affect methane production in vitro without negatively impacting rumen fermentation and should be further investigated using in vivo models.

Ruminal Microbial Degradation of Individual Amino Acids from Heat-Treated Soyabean Meal and Corn Gluten Meal in Continuous Culture.

Simple SummaryDietary rumen undegradable protein provides almost half of the amino acids available for absorption. Current feeding systems assume that the amino acid profile of a protein reaching the small intestine is similar to that of the original protein. However, the results from the present experiment confirm that the degradability of individual amino acids from heat-processed soybean meal and corn gluten meal was different between protein sources and among amino acids within each protein source. The degradability of essential amino acids in general, and that of lysine in particular, was higher in both protein supplements, and the degradability of methionine was also higher in corn gluten meal compared with the average degradability of amino acids. Therefore, the flow of essential amino acids, lysin and methionine may be overestimated. Therefore, the results suggest the need to include the degradability of individual amino acids in precision feeding strategies.Eight dual-flow continuous culture fermenters were used in three periods to study the effects of diets containing heat-treated soyabean meal (HSBM) or corn gluten meal (CGM) on ruminal microbial fermentation and the degradation of individual amino acids (AA). Treatments were a mix of non-protein nitrogen (N; urea and tryptone) that were progressively substituted (0, 33, 67 and 100%) for HSBM or CGM. Ruminal escape of AA was calculated with the slope ratio technique. Total volatile fatty acids (95.0 mM) and molar proportions (mol/100 mol) of acetate (59.3), propionate (21.8) and butyrate (10.5) were not affected by the treatments. As the level of HSBM or CGM increased, the concentration of ammonia-N and the degradation of protein decreased (p < 0.01), and the flows of nonammonia and dietary N increased (p < 0.01) quadratically. Compared with HSBM, CGM provided the highest flow (g/d) of total (20.6 vs. 18.3, p < 0.01), essential (9.04 vs. 8.25, p < 0.04) and nonessential (11.5 vs. 10.0, p < 0.01) AA, and increased linearly (p < 0.01) as the level of supplemental protein increased. Ruminal degradation of essential AA was higher (p < 0.04) than nonessential AA in CGM, but not in HSBM. Degradation of lysine was higher (p < 0.01) in both proteins, and degradation of methionine was higher in CGM. Ruminal degradation of individual AAs differ within and between protein sources and needs to be considered in precision feeding models.

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.

Evaluation of iso-α-acid and β-acid extracts from hops (Humulus lupulus L.) on fermentation by rumen microbes in dual-flow continuous culture fermenters

Hops (Humulus lupulus L.), primarily used in the brewing industry, have been shown to have bacteriostatic properties against Gram-positive bacteria. Active antimicrobial compounds contained within hop flowers include iso-α-acids (isohumulones) and β-acids (lupulones). Previous experiments have demonstrated effects of hop extracts on rumen fermentation in batch culture. The objective of this study was to determine the direct effects of iso-α-acids and β-acids on rumen fermentation in continuous culture fermenters. Two experiments were conducted using dual-flow continuous culture fermenters. Each experiment utilized eight fermenters in two consecutive 10 d periods. Within both experiments, the same basal diet consisting of 44 % corn silage, 14 % alfalfa hay, 13 % ground corn, 11 % protein mix, 10 % corn gluten feed, 5 % cottonseed and 3 % liquid vitamin and mineral supplements on a DM basis was provided to the fermenters at a rate of 75 g of DM/L of fermenter volume/day. In experiment 1, hop beta-extract was added daily to the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED), or 1800 (HIGH) mg of β-acids/kg of diet DM/day. In experiment 2, hop iso-α extract was provided to fermenters via the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED), or 1800 (HIGH) mg of iso-α-acids/kg of diet DM/day. Data in both experiments were analyzed as a randomized complete block design with experimental period serving as a block and all treatments equally represented within each block. Data were statistically analyzed using GLM procedure of SAS with a model including the fixed effects of experimental period (block), treatment, and the interaction of treatment and period. In experiment 1, mean and maximum fermentation pH increased (P = 0.005) linearly with increasing levels of beta extract inclusion. Additionally, time spent below pH 5.8 increased and time between pH 5.8 and 6.2 decreased (P = 0.007) linearly with greater beta extract inclusion (P = 0.08). However, beta extract did not affect DM, OM, NDF or ADF digestion. Furthermore, beta extract did not modify VFA production or N metabolism. In experiment 2, iso-alpha extract also increased average pH, and tended to increase time above pH 6.2. Iso-α extract did not affect DM, OM, NDF and ADF digestion, VFA production, or N metabolism. Results indicate that increasing the concentration of beta extract or iso-α extract increase rumen pH within continuous culture, but neither extract affects rumen nutrient digestion.

390 Effects of dietary-induced pH changes on feed digestion, nitrogen metabolism, fermentation, and biohydrogenation of polyunsaturated fatty acids in dual-flow continuous culture fermenters

Abstract Ruminal pH decreases as greater concentrations of grain are consumed, and as a result, reduces biohydrogenation of polyunsaturated fatty-acids. Our objective was to investigate effects of diet on pH and biohydrogenation activity in vitro. Eight dual-flow, continuous culture fermenters were blocked by side of the room and randomly assigned, within block, to 1 of 4 treatments. Treatments consisted of 100:0 roughage-to-concentrate (Grass), 50:50 roughage-to-concentrate plus flax oil supplement (Mix+F), 10:90 roughage-to-concentrate plus corn oil supplement (Con+C), or 10:90 roughage-to-concentrate plus flax oil supplement (Con+F). Fermenter pH, area under the curve, and maximum pH were lowest for Con+C and Con+F (P &amp;lt; 0.05). Apparent and true DM, OM, and NDF digestibility were greater with grain inclusion (P &amp;lt; 0.05) while ADF digestibility was not affected by treatment (P &amp;gt; 0.10). Protein degradation was lowered as grain inclusion increased (P &amp;lt; 0.05). Total VFA concentrations were lowest for Grass, highest for Con+C and Con+F, and intermediate for Mix+F (P &amp;lt; 0.05). Acetate-to-propionate was lowest for Con treatments (P &amp;lt; 0.05). Biohydrogenation of 18:2n-6 and 18:3n-3 fatty-acids were reduced (P &amp;lt; 0.01) with inclusion of concentrate regardless of roughage-to-concentrate ratio. Concentrations of stearic acid recovered from effluent were greatest for Grass, intermediate for Mix+F, and lowest for both Con+C and Con+F (P &amp;lt; 0.05). Linoleic acid proportions in effluent were greatest for Con+C and Con+F and intermediate for Mix+F (P &amp;lt; 0.05), and inclusion of concentrate, regardless of amount, increased the amount linoleic acid recovered from fermenter overflow (P &amp;lt; 0.01). Between Con+C and Con+F concentrations of alpha-linolenic acid (ALA) was greater when flax oil was supplemented (P &amp;lt; 0.01). Proportions of ALA in effluent was similar (P &amp;gt; 0.10) between Grass and Con+C; however, between Mix+F and Con+F ALA concentrations tended (P = 0.06) to be greater for Con+F. Omega6-to-omega3 ratios were similar across Grass, Mix+F, and Con+F (P &amp;gt; 0.10) and greatest for Con+C (P &amp;lt; 0.05).

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.

Effects of live yeast (Saccharomyces cerevisiae) and type of cereal on rumen microbial fermentation in a dual flow continuous culture fermentation system.

Live yeast additives may help optimize ruminal fermentation of high-grain diets, especially when the starch of the ration is highly fermentable. Eight dual flow continuous culture fermenters were used in a 2×2 factorial design in two replicated periods of 9days (six for adaptation and three for sampling) to determine the effect of live yeast and type of cereal on rumen microbial fermentation and nutrient digestibility. Main factors were the addition of live yeast: no yeast (NY) vs. 2×107 CFU of yeast/g of diet (LY); and type of cereal in the diet: corn (CO) vs. barley (BA). All fermenters were fed 80g dry matter/day of a 10-90 forage to concentrate diet and pH was allowed to fluctuate with an upper (6.6) and lower (5.5) limit. Treatment BA increased OM digestion, valerate proportion, peptides and ammonia N fractions, ammonia N flow, crude protein degradation and target copies of Megasphaera elsdenii; and decreased NDF digestion, propionate proportion, branched-chain VFA (BCVFA) concentration, AA-N fraction and nonammonia N flow. Treatment LY increased BCVFA and decreased ammonia N fraction and flow, and the target copies of Streptococcus bovis. Treatment LY decreased the slope of pH drop, the area under pH 6.0 and the gas production. These results suggest potential benefits of LY in stabilizing the fermentation of BA-based diets.

Comparison of warm season and cool season forages for dairy grazing systems in continuous culture.

The objective of this study was to compare warm-season annual grasses to cool-season perennial (CSP) grasses for ruminal nutrient digestibility and N metabolism in a dual-flow continuous culture fermentation system. Dietary treatments were 1) fresh alfalfa, 2) CSP grasses and legumes, 3) brown-midrib sorghum-sudangrass (BMRSS), and 4) teff grass from an organic dairy production system. Eight dual-flow continuous culture fermenters were used during two consecutive 10-d periods consisting of 7 d for stabilization followed by 3 d of sampling. Fermenter samples were collected on days 8, 9, and 10 for analysis of pH, NH3-N, and VFA. Apparent DM, OM, NDF, and ADF digestibility were on average lesser (P < 0.05) in CSP grasses and legumes and warm-season annual grasses compared with alfalfa. True DM and OM digestibility were lesser (P < 0.05) for CSP grasses and legumes and warm-season annual grasses compared with fresh alfalfa. Total VFA were not affected (P > 0.05) by forage. The NH3-N concentrations were highest (P < 0.05) with alfalfa compared with the other CSP grasses and legumes and warm-season annual grasses. CP digestibility was not affected (P > 0.05) by forage treatment. Flow of NH3-N was greatest (P < 0.05) for alfalfa, reflecting the greatest NH3-N concentration. Flow of total N was greatest (P < 0.05) for alfalfa, intermediate for teff, and lowest for CSP grasses and legumes and BMRSS. Flows of bacterial N, efficiency of bacterial N, non-NH3-N, and dietary N were not affected (P > 0.05) by forage source. Overall, fermentation of warm-season grasses was similar to the cool-season grasses and legumes which indicate dairy producers may use warm-season grasses without concerns about negative impact on rumen health.

Comparisons of bacterial and archaeal communities in the rumen and a dual-flow continuous culture fermentation system using amplicon sequencing.

Dual-flow continuous culture (CC) fermenters are commonly used to study rumen fermentation in vitro. Research using culture-based and oligonucleotide techniques has shown that certain microbial populations within fermenters may be maintained at abundances similar to those observed in vivo. In this study, bacterial and archaeal communities in the rumen of dairy cattle and in a dual-flow CC fermentation system were compared using high-throughput amplicon sequencing targeting the V4 hypervariable region of 16S rRNA. We hypothesized that the in vitro system harbored a comparable bacterial and archaeal community to that observed in the rumen. Members of the Bacteroidetes and Firmicutes made up the 2 most abundant phyla in the rumen, inoculum, and fermenters and did not differ among sample types (P > 0.10). Similarly, Prevotellaceae, the most abundant family in all 3 sample types, did not differ based on source (P = 0.80). However, beta diversity analyses revealed that bacterial and archaeal communities differed between fermenters and rumen samples (P ≤ 0.001), but fermenter bacterial and archaeal communities stabilized by day 4 of each period. While the overall bacterial and archaeal community differs between natural rumens and those detected in in vitro fermenter systems, several prominent taxa were maintained at similar relative abundances suggesting that fermenters may provide a suitable environment in which to study shifts among the predominant members of the microbial community.

Effects of bismuth subsalicylate and dietary sulfur level on fermentation by ruminal microbes in continuous culture.

In ruminants, excess dietary sulfur can be associated with a reduction in DM intake, poor feedlot performance and sulfur-associated polioencephalomalacia. Bismuth subsalicylate (BSS) has been shown to decrease hydrogen sulfide in vitro. The objective of this experiment was to evaluate effects of BSS inclusion (0 or 0.5% of diet DM) and dietary sulfur (0.21 or 0.42% of diet DM) on microbial fermentation in continuous culture. Treatments were arranged in a 2 × 2 factorial design. Eight dual-flow continuous culture fermenters were used during 2 consecutive 10-d periods consisting of 7 d for stabilization followed by 3 d of sampling. A pelleted feedlot diet containing 39% dry rolled corn, 32% earlage, 21% wet distillers grains, 3.2% corn silage, 1.5% soybean meal, 0.6% urea and 2.7% mineral premix (DM basis) was provided as substrate for microbes at a rate of 75 g of DM × fermenter–1 × d–1. Effluents from sampling days were composited by fermenter within period, resulting in 4 replicates/treatment. Bismuth subsalicylate inclusion decreased (P < 0.01) true OM digestion, while no effects were observed for NDF and ADF digestion. Total VFA concentrations, molar proportions of acetic, propionic, and branched-chained VFA decreased (P < 0.01) with BSS addition. The ratio of acetic to propionic acid and the molar proportion of butyric acid increased (P < 0.01) with BSS addition. In regard to nitrogen metabolism, BSS increased NH3–N concentration, NH3–N and dietary-N flows (P < 0.01), and decreased non-NH3–N flow, microbial-N flow, CP degradation, and efficiency of microbial protein synthesis (P < 0.01). Inclusion of BSS increased mean, minimum, and maximum fermentation pH (P < 0.01). Amount of dietary sulfur and BSS inclusion influenced flows of amino acids and fatty acids from fermenters. Influences on fatty acid biohydrogenation and amino acid flows demonstrated an overall suppression of microbial fermentation. Results from this experiment indicate that BSS inclusion at 0.5% of diet DM has detrimental effects on in vitro rumen fermentation in continuous culture.

Effect of dried fermentation biomass on microbial fermentation in continuous culture and in vitro intestinal digestibility

The objective of the current experiment was to determine if fermentation biomass (FB), a dried bacterial by-product derived from lysine production (Ajinomoto Heartland, Inc.) can be used as a protein source in ruminant diets. Eight dual-flow continuous culture fermenters were inoculated with rumen fluid and used during one experimental period consisting of a 7-d adaptation period followed by 3 sampling days. Microbial substrate was provided by one of two isonitrogenous diets, CON or DFB. In CON, soybean meal (SBM) provided 57% of total CP, and in DFB, SBM and FB provided 12 and 45% of total CP, respectively. CON contained 3% molasses, 16% ground corn, 13% grass hay, 48% corn silage, and 20% SBM on a DM basis; DFB contained 3% molasses, 18.4% ground corn, 13% grass hay, 50% corn silage, 8.5% SBM, and 6.7% FB. On sampling days, liquid and solid effluent were collected, combined, and homogenized to be used for chemical analysis and in vitro estimation of intestinal digestibility (ID). Treatment did not affect average, maximum, or minimum fermenter pH. There was no effect on apparent or true OM, NDF, or ADF digestibility (%). Total and branched-chain VFA as well as acetate (mM) were higher in CON, and isobutyrate concentration (mol/100mol) tended to increase with CON treatment. Source of N had no effect on total, dietary, or bacterial-N flows. Addition of FB decreased NH3-N flow from 0.4 to 0.2±0.05g/d and tended to decrease effluent NH3-N concentration from 17.1 to 9.7±2.21mg/100mL. His and Met flows increased from 0.48 to 0.53±0.012 and 0.18–0.20±0.005g/d, respectively, when FB partially replaced SBM in the diet, but there were no effects on other AA or total AA flows. There was a trend in percent non-essential AA input (CON=73.6% vs. DFB=82.2%; SE=2.83) in effluent; however, there was no effect on percent of essential or total AA input in effluent. Effluent from the DFB treatment was higher in ID than CON (CON=70.4% vs. DFB=79.6%; SE=1.64), although there was no difference in estimated amount of protein available for intestinal absorption (g). These results indicate that FB elicited a similar response in N metabolism and AA flows to SBM but had a greater estimated ID and depressed VFA production, and has potential use as a protein source in ruminant diets.

Effects of dietary roughage and sulfur in diets containing corn dried distillers grains with solubles on hydrogen sulfide production and fermentation by rumen microbes in vitro.

Dried distillers grains with solubles (DDGS) have been used in production animal diets; however, overuse of DDGS can cause toxic concentrations of ruminal hydrogen sulfide gas (HS), resulting in polioencephalomalacia, a deleterious brain disease. Because HS gas requires an acidic rumen environment and diet can influence ruminal pH, it has been postulated that dietary manipulation could help mitigate HS production. The objective of this study was to assess the effect of dietary roughage and sulfur concentrations on HS production and rumen fermentation. In Exp. 1, 7 dual-flow continuous culture fermenters were used in 4 consecutive 9-d periods consisting of 6 d of adaptation followed by 3 d of sampling. At the conclusion of each 9-d continuous culture period, adapted rumen fluid was used for inoculation of 24-h batch culture incubations for Exp. 2. For both experiments, 6 dietary treatments were formulated to consist of 0.3%, 0.4%, or 0.5% dietary sulfur (LS, MS, and HS, respectively) and 3% or 9% dietary roughage (LR and MR, respectively), using grass hay as the roughage source. A corn-based diet without DDGS was used as a control diet. Headspace gas was sampled to determine HS production and concentration. In Exp. 1, greater dietary roughage had no effect ( = 0.14) on HS production but did create a less acidic environment because of an increase ( < 0.01) in the in vitro pH. In Exp. 2, an increase in dietary sulfur caused an increase ( = 0.04) in ruminal HS production, but there was no direct effect ( = 0.25) of dietary roughage on HS production. Greater dietary roughage resulted in a less ( = 0.01) acidic final batch culture pH but a lower ( < 0.01) total VFA concentration. Further investigation is needed to determine a more effective way to mitigate ruminal HS production using dietary manipulation, which could include greater inclusion of dietary roughage or the use of different roughage sources.

Effects of flaxseed and chia seed on ruminal fermentation, nutrient digestibility, and long-chain fatty acid flow in a dual-flow continuous culture system.

Flaxseed (FS) and chia seed (CS) are oilseeds rich in omega-3 fatty acids, which may change meat and milk composition when added to ruminants' diets and may have health benefits for humans. Literature on the effects of CS supplementation on ruminal metabolism is nonexistent. A dual-flow continuous culture fermenter system consisting of 6 fermenters was used to assess the effect of FS and CS supplementation in an alfalfa hay-based diet on ruminal fermentation, nutrient digestibility, microbial protein synthesis, and long-chain fatty acid flow. Diets were randomly assigned to fermenters in a replicated 3 × 3 Latin square design, with 3 consecutive periods of 10 d each, consisting of 7 d for diet adaptation and 3 d for sample collection. Each fermenter was fed a total of 72 g of DM/d divided in 6 equal portions. Treatments were 1) alfalfa hay + calcium soaps of palm oil fatty acid (MEG; 69.3 g DM/d of alfalfa hay plus 2.7 g DM/d of calcium soaps of palm oil fatty acid), 2) alfalfa hay + FS (FLAX; 68.4 g DM/d of alfalfa hay plus 3.6 g DM/d of ground FS), and 3) alfalfa hay + CS (CHIA; 68.04 g DM/d of alfalfa hay plus 3.96 g DM/d of ground CS). Dietary treatments had similar amounts of total fat, and fat supplements were ground to 2-mm diameter. Effluents from the last 3 d of incubation were composited for analyses. Data were analyzed using the MIXED procedure of SAS. Ruminal apparent and true nutrient digestibility of all nutrients did not differ ( > 0.05) among treatments. Compared with MEG, FLAX and CHIA increased the flows of C18:3 -3, C20:4 -6, and total PUFA ( < 0.01). Both CHIA and FLAX treatments had greater ruminal concentrations of C18:0, indicating that both CS and FS fatty acids were extensively biohydrogenated in the rumen. The NH-N concentration, microbial N flow, and efficiency of microbial protein synthesis were not affected ( > 0.05) by treatments. Lastly, there were no differences ( > 0.05) among diets for total VFA concentration and molar proportions of individual VFA. Results from this study indicate that FS and CS supplementation did not impair ruminal fermentation, digestibility, microbial efficiency, and ruminal N metabolism. Overall, CS appears to be as effective as FS as a fat source when added to ruminants' diets using a dual-flow continuous culture system.

The effect of diet supplemented with vegetable oils and/or monensin on the vaccenic acid production in continuous culture fermenters.

Studies have shown that supplementing ruminant diets with vegetable oils modulated the rumen biohydrogenation and increased polyunsaturated fatty acid in their products. These positive values are often accompanied by a marginal loss of supplemented unsaturated fatty acids and rise in the concentrations of saturated fatty acids. This study were carried out mainly to investigate the effect of supplementing diets with sunflower oil, olive oil with or without monensin on the production and accumulation of vaccenic acid (VA) in continuous culture fermenters as a long term in vitro rumen simulation technique. Eight dual-flow continuous culture fermenters were used in an 8 replication experiment lasted 10 days each (first 7 days for adaptation and last 3 days for samples collection). Supplementing diets with plant oils and monensin in the present experiment increased VA and conjugated linoleic acids (P > 0.05) in ruminal cultures. The results suggest that supplementing diets with both olive oil and sunflower oil and monensin increased VA accumulation compared to plant oils supplemented alone without affecting the rumen dry matter and organic matter digestibility.

Mitigation of in vitro hydrogen sulfide production using bismuth subsalicylate with and without monensin in beef feedlot diets.

The objective of this study was to determine if a sulfur binder, bismuth subsalicylate (BSS), alone or combined with monensin (MON) could decrease the production of HS by rumen microbes. In Exp. 1, two 24-h batch culture incubations were conducted using a substrate consisting of 50% corn, 40% distillers grains, 9.75% hay, and 0.25% mineral premix, on a DM basis. Five treatments including BSS concentrations of 0% (control), 0.5%, 1%, 2%, and 4% of DM were assigned in 5 replicates to 120-mL serum bottles containing rumen fluid, buffer, and 0.5 g of dietary substrate. Addition of 2% and 4% BSS decreased ( < 0.05) gas production, whereas all concentrations of BSS reduced ( < 0.05) HS production by 18%, 24%, 82%, and 99% for 0.5%, 1%, 2%, and 4% BSS, respectively. Final pH increased ( < 0.05) with 2% and 4% BSS treatments. At 4% of DM, BSS decreased ( < 0.05) total VFA concentration (m) and propionate (mol/100 mol) but increased acetate (mol/100 mol) and acetate to propionate ratio. Concentration of branched-chain VFA increased ( < 0.05) with the addition of 0.5% BSS, compared with the control. On the basis of these results, addition of BSS (1% of DM) and MON (5 mg/kg) were used to assess their effects on metabolism and HS release by rumen microbes in 8 dual flow continuous culture fermenters during two 10-d periods (Exp. 2). Treatments were arranged in a 2 × 2 factorial design. Substrate similar to that used in Exp. 1 was provided at 75 g DM/fermenter daily. Headspace HS concentration was reduced ( < 0.05) by 99% with BSS treatment but was not affected ( = 0.21) by MON. An overall increase ( < 0.05) in fermentation pH was found following addition of BSS. Addition of BSS increased ( < 0.05) digestion of NDF and ADF but decreased ( < 0.05) nonfiber carbohydrate digestion and total VFA concentration. Acetate and propionate (mol/100 mol) increased ( < 0.05) with BSS, whereas butyrate (mol/100 mol) and branched-chain VFA (m) decreased ( < 0.05). Addition of BSS increased ( < 0.05) NH-N concentration and NH-N outflow but decreased ( < 0.05) microbial N outflow. Results from this study showed no response to monensin addition, but BSS markedly reduced HS production and altered microbial fermentation during in vitro rumen fluid incubations.

The effects of a garlic oil chemical compound, propyl-propane thiosulfonate, on ruminal fermentation and fatty acid outflow in a dual-flow continuous culture system

The ban on the use of antibiotics as growth promoters in animal feeds in the European Union has stimulated research on potential alternatives. Recently, propyl-propane thiosulfonate (PTSO), a stable organosulfurate compound of garlic, was purified. The objectives of the current study were to investigate the potential effects of PTSO on rumen microbial fermentation and to define effective doses. Two experiments were conducted using dual-flow continuous culture fermenters in 2 replicated periods. Each experimental period consisted of 5 d for adaptation of the ruminal fluid and 3 d for sampling. Temperature (39°C), pH (6.4), and liquid (0.10 h−1) and solid (0.05 h−1) dilution rates were maintained constant. Samples were taken 2 h after feeding and from the 24-h effluent. Samples were analyzed for volatile fatty acids (VFA) and nitrogen fractions, and degradation of nutrients was calculated. In addition, 24-h effluents from experiment 2 were analyzed for their fatty acid (FA) profile. Treatments in experiment 1 included a negative control without additive, a positive control with monensin (12mg/L), and PTSO at 30 and 300mg/L. The addition of 30mg/L did not affect any of the measurements tested. The addition of 300mg/L reduced microbial fermentation, as suggested by the decreased total VFA concentration, true degradation of organic matter and acid detergent fiber, and a tendency to decrease neutral detergent fiber degradation. Experiment 2 was conducted to test increasing doses of PTSO (0, 50, 100, and 150mg/L) on rumen microbial fermentation. At 2 h postfeeding, total VFA and molar proportion of propionate responded quadratically, with higher values in the intermediate doses. Molar proportions of butyrate increased and branched-chain VFA decreased linearly as the dose of PTSO increased. In the 24-h effluents, total VFA, acetate, and branched-chain VFA concentrations decreased linearly and those of propionate responded cubically with the highest value at 100mg/L. Saturated FA decreased and unsaturated FA increased linearly with increasing dose of PTSO. The concentration of trans-10,cis-12 conjugated linoleic acid decreased by 78.5% with addition of PTSO at the highest dose (150mg/L). Results suggest the potential of PTSO to modify ruminal fermentation in a direction consistent with higher propionate molar proportion, higher outflow of unsaturated FA, and low trans-10,cis-12 conjugated linoleic acid in an effective dose between 50 and 100mg/L.

Effect of pH on in vitro microbial fermentation and nutrient flow in diets containing barley straw or non-forage fiber sources

Roughage in beef diets could be reduced or removed if replaced with less costly non-forage fiber sources, but there is little information about their effects on microbial fermentation and nutrient flow in different ruminal conditions. In the present experiment, eight dual-flow continuous culture fermenters (1320ml) were used in three periods (5 days adaptation, 3 days sampling) to study the effect of pH and different sources of non-forage fiber in beef diets on rumen microbial fermentation. In each period, fermenters were randomly assigned to treatments. Temperature (39°C), solid (0.05h−1) and liquid (0.10h−1) dilution rates were maintained constant. Fermenters were fed 97g of DM/day in three equal portions. A factorial arrangement of treatments was used, the factors being the culture pH (5.5 and 6.2) and the main fiber source in the diet: barley straw (BS; 100g/kg DM), beet pulp (BP; 170g/kg DM), soybean hulls (SH; 170g/kg DM) and whole cottonseed (WCS; 160g/kg DM). Diets were formulated to have similar crude protein (150g/kg DM), metabolizable energy (2.95Mcal/kg of DM) and NFC level (550g/kg DM). Low pH (5.5) reduced apparent DM and OM digestibility, total and branched chained volatile fatty acid concentrations, and acetate proportion, and increased propionate proportions, as expected. Replacement of BS with BP and SH increased OM, aNDF and ADF digestibility. An interaction diet×pH was detected for aNDF and ADF digestibility because the level of reduction in fiber digestibility at low pH was different among diets. Total volatile fatty acid concentration was lower in BS than in BP, SH and WCS and acetate proportion was higher in BP compared with WCS (50.3 vs 44.4mol/100mol). Low pH decreased NH3-N concentration (2.24 vs 4.10mg/100ml) and increased non-ammonia N flow (2.77 vs 2.70g/day) compared with high pH. There were no differences among treatments in the flow of bacterial N and the efficiency of microbial protein synthesis. These results suggest that the effect of low pH on fiber and true OM digestibility was different among fiber sources. Replacing straw with non-forage fiber had no detrimental effects on in vitro fermentation, suggesting that these sources could be potentially useful in beef diets. However, many important differences exist between in vitro and in vivo experiments, which can influence the digestion and fermentation processes, so data need to be interpreted with the understanding of the nature and limitations of the technique used.

Effect of time of cutting and maceration on nutrient flow, microbial protein synthesis, and digestibility in dual-flow continuous culture1

Maceration and evening-cutting are 2 forage management techniques that have independently improved forage quality and nutrient utilization in ruminants, but have not been evaluated in combination. Using a dual-flow continuous culture fermenter system, this preliminary study was designed to evaluate the individual and combined effects of time of cutting and maceration on in vitro ruminal digestion, nutrient flows, and microbial protein synthesis. Forages were harvested as hay from a timothy (Phleum pratense L.)-birdsfoot trefoil (Lotus corniculatus L.) stand in the morning (AM) or evening (PM). Half of each morning- and evening-cut treatment was macerated (AM-M, PM-M). The chemical composition (DM, OM, CP, NDF, ADF), including nonstructural carbohydrates (NSC) and water-soluble carbohydrates (WSC), was determined for each of the 4 treatments (AM, AM-M, PM, PM-M). Forages were ground to 2 mm and allocated to separate fermenters at 60 g of DM/d in a 4 × 4 Latin square design. Fermenters were operated over four 10-d periods with the first 7 d for adaptation followed by 3 d of sampling. Evening-cutting enhanced the apparent digestibility of NDF (P = 0.02) and ADF (P = 0.006), with a tendency (P < 0.10) for improved true DM digestibility and microbial protein synthesis. Molar proportions of individual VFA were not affected (P > 0.10) by time of cutting, though evening-cutting increased (P = 0.02) total concentration of VFA. Maceration had no effect (P > 0.10) on true nutrient digestibility or microbial protein synthesis. An interaction of time of cutting and maceration (P < 0.05) was observed whereby maceration decreased true DM and OM digestibilities in evening-cut treatments, but had no effect in morning-cut treatments. Similarly, maceration reduced total N supply (P < 0.001) and molar proportions of acetate (P = 0.04) and increased molar proportions of propionate (P = 0.01) in evening-cut treatments with no effect on morning-cut treatments. These results indicate that independent use of evening cutting increased fiber digestibility and total VFA concentration, and independent use of maceration shifted molar proportions of VFA toward glucogenic fermentation. The combined use of these management techniques afforded no improvement for in vitro digestibility or metabolism when applied to morning-cut hay, and decreased nutrient digestibility when applied to evening-cut hay. Due to inherent limitations of in vitro systems, the results of this study should be interpreted with caution. Further in vivo studies are needed to support our conclusions.