M.D. Stern

Evaluation of a dual flow continuous culture system for estimating bacterial fermentation in vivo of mixed diets containing various soya bean products

Abstract A dual flow continuous culture system was evaluated for its ability to simulate rumen fermentation in vivo. Soya bean meal, whole soya beans and whole soya beans extruded at 132 or 149° C, provided 50% of total crude protein in diets comprised of 52% grain mix, 36% maize silage and 12% alfalfa hay (dry matter basis). These four diets had previously been studied with four ruminal and intestinal cannulated lactating Holstein cows in a trial of 4 × 4 Latin square design. To conform with the results of this trial, fermenters were maintained at a liquid dilution rate of 0.096 h−1, solids dilution rate of 0.055 h−1 and pH of 6.25. There were three experimental periods, each lasting 8 days with effluent collected on the last 3 days. A Bonferroni t-test was used to compare results obtained in vitro with those obtained in vivo. Results from the two studies were similar in true organic matter digestibility and crude protein and amino acid degradability. Individual amino acid flows were higher with the diets containing heat treated (extruded) soya beans in both studies. Results indicate that the dual flow continuous culture system can provide a reasonable estimate of rumen fermentation.

Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture

Four ruminal and duodenal cannulated lactating dairy cows and four dual flow continuous culture fermenters were used in a replicated 4 × 4 Latin square to compare fermentation and microbial ecology of in vivo and in vitro systems. Diets contained 32% maize silage, 19.8% alfalfa-grass hay, and 48.2% concentrate, and were arranged in a 2 × 2 factorial with two levels of non-fibrous carbohydrate (40%, 25%) and two levels of ruminally degradable intake protein (12%, 9%). Concentrations of viable bacteria were greater (P = 0.01) in vitro compared with in vivo, averaging 5.04 × 109 and 2.75 × 109 cells per ml, respectively. Cellulolytic bacterial concentrations (cells per ml) were lower (P = 0.04) in vitro (4.09 × 107) compared with in vivo (6.15 × 107), but concentrations of proteolytic and amylolytic bacteria were similar (P > 0.1). Protozoal concentrations (cells per ml) were greater (P = 0.0001) in vivo (3.72 × 105) compared with in vitro (2.8). Digestion of neutral detergent fiber (NDF) and total non-structural carbohydrate (TNC) exhibited square × NFC interactions (P < 0.002), and comparison of these interaction means revealed that the difference in NDF and TNC digestion between ruminal and in vitro fermentations occurred only for the 40% NFC diets. Mean TNC digestion was 87.6, 61.7, 60.9, and 60.4% whereas NDF digestion was 33.8, 55.9, 57.6, and 58.4% for the in vitro-40% NFC, in vitro-25% NFC, in vivo-25% NFC, and in vivo-40% NFC treatments, respectively. These data suggest that the continuous culture system had difficulty simulating digestion of high TNC diets. Other differences between fermentation in continuous culture and in vivo can be attributed mainly to lack of absorption from the fermenters, defaunation of the in vitro system, and endogenous protein contamination in vivo.

Evaluation of various methods for protecting soya-bean protein from degradation by rumen bacteria

A dual-flow continuous-culture system was used to study the effects of protection method on protein degradation of soya-bean meal (SBM) by rumen bacteria. Treatments included solvent extraction (control), sodium hydroxide, ethanol, formaldehyde, expeller processing, propionic acid, extrusion and lignosulfonate. Diets contained approximately 17.0% crude protein, with 50% of the crude protein coming from the respective treated SBM and were fed to rumen bacteria in fermenters at a rate of 75 g dry matter day−1. Crude protein degradation of formaldehyde-treated, expeller processed, propionic acid-treated, extruded and lignosulfonate-treated SBM diets were lower (P < 0.05) than the control diet. Sodium hydroxide and ethanol treatments did not affect (P < 0.05) crude protein degradation. Total bacterial N output was lowest (P < 0.05) for SBM protected by formaldehyde, expeller processing and lignosulfonate treatments. Undegraded dietary N in the effluent was highest (P < 0.05) for SBM protected by formaldehyde, expeller processing, propionic acid and lignosulfonate treatments. Protection by formaldehyde, expeller processing, propionic acid and lignosulfonate treatments increased (P < 0.05) total amino-acid flow compared with the control. Formaldehyde and expeller processing treatments also increased (P < 0.05) essential amino-acid flow compared with the control. An in sacco study was also conducted which showed that treatment of SBM with formaldehyde, lignosulfonate, or expeller processing resulted in the greatest (P < 0.05) reduction in protein degradation.

Variation in ruminal degradation and intestinal digestion of animal byproduct proteins

Abstract In situ and in vitro procedures were used to determine ruminal degradation and intestinal digestion of crude protein (CP) from animal byproducts. Seven samples of meat and bone meal (MBM), hydrolyzed feather meal (HFM), ring dried blood meal (RBM), and batch dried blood meal (BBM) were obtained from various rendering and meat packing plants. Ruminal undegradable CP determined using in situ procedures ranged from 51.3 to 60.8% ( x = 55.4 ± 1.3% ), 53.6–87.9% ( x = 73.6 ± 3.8% ), 76.4–86.4% ( x = 81.0 ± 1.2% ), and 77.6–94.4% ( x = 84.3 ± 2.1% ), for MBM, HFM, RBM and BBM, respectively. Estimates of intestinal digestion of ruminal undegradable CP, determined using a three-step in vitro procedure, ranged from 40.9 to 70.1% ( x = 56.0 ± 4.0% ), 59.2–75.2% ( x = 65.3 ± 2.1% ), 72.0–90.3% ( x = 79.6 ± 2.5% ) and 28.8–79.2% ( x = 61.4 ± 6.8% ), for MBM, HFM, RBM, and BBM, respectively. Total intestinally absorbable dietary protein (IADP) was calculated as ruminal undegradable protein multiplied by intestinal digestion of the ruminal undegradable protein fraction. Intestinally absorbable dietary protein ranged from 21.6 to 39.3% ( x = 31.2 ± 2.7% ), 35.8–59.4% ( x = 47.6 ± 3.1% ), 57.5–75.2% ( x = 64.6 ± 2.6% ), and 24.8–62.7% ( x = 51.3 ± 5.3% ), for MBM, HFM, RBM, and BBM, respectively. Results demonstrate that large variations in ruminal degradation, intestinal digestion and IADP can occur among and within different rendering byproducts. These variations should be considered when determining the quality of animal byproducts as protein supplements for ruminants. Measurements of ruminal degradation and intestinal digestion of CP should be used by manufacturers to guarantee the quality of various batches of byproduct that are produced.

Intestinal digestibility of amino acids in rumen-undegraded protein estimated using a precision-fed cecectomized rooster bioassay: II. Distillers dried grains with solubles and fish meal.

The objectives of this experiment were to measure intestinal digestibility of AA in the rumen-undegraded protein fraction (RUP-AA) of distillers dried grains with solubles (DDGS) and fish meal (FM) samples and to determine whether these feeds contain a constant protein fraction that is undegradable in the rumen and indigestible in the small intestine, as assumed in the French Institut National de la Recherche Agronomique (Paris, France) and Scandinavian AAT-PBV (AAT = AA absorbed from small intestine; PBV = protein balance in the rumen) models. Five sources of DDGS and 5 sources of FM were obtained from Feed Analysis Consortium, Inc. (Champaign, IL). To obtain the rumen-undegradable protein fraction, samples were ruminally incubated in situ for 16 h in 4 lactating cows, and the collected rumen-undegraded residues (RUR) were pooled by sample. Subsamples of the intact feeds and RUR were crop-intubated to 4 cecectomized roosters, and total excreta were collected for 48 h. Intact feeds, RUR, and excreta were analyzed for AA. Basal endogenous AA loss estimates were obtained from fasted birds and were used to calculate standardized digestibility of RUP-AA and AA in the intact feeds. Indigestibility coefficients of the intact feeds were calculated as (100 - % standardized AA digestibility), and indigestibility of the RUR was calculated as [(100 - % ruminal degradation of AA) x (100 - % standardized RUP-AA digestibility)/100]. Results indicate that standardized digestibility of feed-AA differs from RUP-AA for DDGS samples but not for FM samples, and that standardized digestibility of individual AA differs within samples. For the DDGS samples, standardized feed-AA and RUP-AA digestibility values were most often lowest for His and Lys and highest for Met and Trp. For FM samples, standardized feed-AA and RUP-AA digestibility values were most often lowest for His and highest for Trp. Results also indicate that DDGS and most FM samples do not contain a constant protein fraction that is both undegradable in the rumen and indigestible in the small intestine. Indigestibility values of RUR were lower than in intact feeds, suggesting that the feed ingredients used in this experiment contain a protein fraction that is indigestible in the intestine but partly degradable in the rumen or digestible in the intestine after rumen incubation, or both.

Evaluation of the fermentation dynamics of soluble crude protein from three protein sources in continuous culture fermenters

Eight dual-flow continuous culture fermenters (1.03 +/- 0.05 L) were used to assess differences in microbial degradation of the soluble CP fraction of canola meal (CMSCP), soybean meal (SBMSCP), and fish meal (FMSCP) using a completely randomized design with two 9-d experimental periods and a solution of tryptone as a control treatment (control). All fermenters received the same basal diet (58% ground corn, 40% canary grass hay, 0.4% vitamin-mineral premix, 1% CaCO(3), 0.6% salt on a DM basis) in 8 equal portions daily. During sampling on the last 3 d of each period, 90-mL doses containing soluble CP were infused into the fermenters 30 min after the beginning of the first and last feedings of the day. The total amount of soluble CP supplied by the infusions of FMSCP, CMSCP, and SBMSCP was 3.2 g/d, representing 24% of the daily dietary CP intake. Infusion of FMSCP resulted in the greatest (P < 0.05) NH(3)-N concentration (4.6 +/- 0.40 mg/dL) compared with the other treatments (0.5 +/- 0.40 mg/dL). Microbial N flow (g/d) from the fermenters was also greatest (P < 0.05) with FMSCP (1.42 +/- 0.062) compared with the other soluble CP fractions (1.08 +/- 0.062). The efficiency of microbial protein synthesis tended to be lowest with the control diet, and the efficiency of N utilization was lowest with FMSCP treatment. These results indicate that N was limiting microbial growth in the control diet, and there was more rumen-available N with the FMSCP diet compared with the other dietary treatments. The extent of degradation of the soluble CP fraction from fish meal, soybean meal, and canola meal was determined to be 99, 30, and 37% of soluble CP, respectively. These results indicate that the soluble CP fraction is not 100% degraded in all feeds and that assuming a high degradation extent of the soluble CP fraction from soybean meal and canola meal may result in an underestimation of the supply of undegradable protein from these protein sources.

In vitro digestibility of individual amino acids in rumen-undegraded protein: The modified three-step procedure and the immobilized digestive enzyme assay

Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA value of the RUR. In conclusion, the modified TSP and IDEA kits are good approaches for estimating RUP-AA digestibility in soybean meal products, distillers dried grains with solubles, and fish meal samples.

In vitro and in vivo comparisons of diaminopimelic acid and purines for estimating protein synthesis in the rumen

Abstract Means analyses were used to compare estimates of crude protein (CP) degradation (%), true organic matter (OM) digestion (%), efficiency of microbial synthesis (g N kg −1 OM truly digested) and microbial nitrogen (N) flow (g d −1 ) using diaminopimelic acid (DAPA) and purines as microbial markers for 12 in vitro and four in vivo experiments. In vitro experiments included 268 determinations and in vivo experiments included 80 determinations of microbial activity by each marker. In vitro estimates of ruminal CP degradation, true OM digestion, efficiency of microbial synthesis and microbial N flow by purines (60.5, 52.4, 33.1 and 1.3) were lower ( P P Using purines as the microbial marker resulted in lower microbial activity and similar coefficients of variation compared with DAPA. Unrealistic values of CP degradation were observed more frequently using DAPA as the microbial marker.

Intestinal digestibility of amino acids in rumen undegradable protein estimated using a precision-fed cecectomized rooster bioassay: I. Soybean meal and SoyPlus

The objectives of this experiment were to measure intestinal digestibility of AA in rumen undegradable protein (RUP-AA) in soybean meal (SBM) and expeller SBM (SoyPlus, West Central, Ralston, IA; SP) and to determine if these feeds contain a constant protein fraction that is undegradable in the rumen and indigestible in the small intestine, as assumed in the French Institut National de la Recherche Agronomique (Paris, France) and Scandinavian AAT-PBV (AAT = AA absorbed from small intestine; PBV = protein balance in the rumen) models. Three samples of SBM and 3 samples of SP were obtained from the Feed Analysis Consortium Inc. (Savoy, IL). To obtain the RUP fraction, samples were ruminally incubated in situ for 16 h in 4 lactating cows, and the collected rumen undegraded residues (RUR) were pooled by sample. Subsamples of the intact feeds and RUR were crop intubated to 4 cecectomized roosters, and total excreta were collected for 48 h. Intact feeds, RUR, and excreta were analyzed for AA. Basal endogenous AA loss estimates were obtained from fasted birds and were used to calculate standardized digestibility of AA in the intact feeds and RUP-AA. Indigestibility coefficients of the intact feeds were calculated as (100 - % standardized AA digestibility), and indigestibility of the RUR was calculated as [(100 - % ruminal degradation of AA) x [(100 - % standardized RUP-AA digestibility)]/100]. Results indicated that standardized digestibility of feed-AA was similar to standardized digestibility of RUP-AA for SBM and SP samples and that standardized digestibility of individual AA differed within samples. Standardized feed-AA and RUP-AA digestibility values were lowest for Lys and Cys and highest for Trp and Met. Results also indicated that SBM and SP did not contain a constant protein fraction that was both undegradable in the rumen and indigestible in the small intestine. Indigestibility values of RUR were lower than in intact feeds, suggesting that SBM and SP contain a protein fraction that is indigestible in the intestine but partly degradable in the rumen, digestible in the intestine after ruminal incubation, or both.

Measuring resistance to ruminal degradation and bioavailability of ruminally protected methionine

The objectives of this study were to evaluate ruminal degradation and intestinal digestion of two ruminally protected methionine (RPM) products and to assess the potential use of changes in plasma methionine concentrations as an indication of methionine availability to the animal. Ruminal degradation of the protected methionine was assessed using the in situ technique. The intestinal availability of methionine after ruminal incubation was determined in vitro using an enzymatic procedure. Four Holstein cows receiving a typical mid-lactation ration (16.5% CP, 1.6 Mcal NEL/kg) were supplemented with 0, 30, and 60 g per day of a slowly degraded ruminally protected methionine (SDM), or 60 g per day of a moderately slowly degradable ruminally protected methionine (MSDM) in a 44 Latin square design. Blood samples were collected from the jugular vein at 0, 6, 12, 18, 24, and 36 h after feeding the RPM sources. Ruminal degradation rates of SDM and MSDM were 0.03 h ˇ1 and 0.07 h ˇ1 , respectively. The calculated amount of methionine available for absorption, based on the in situ and in vitro results, was 17.9, 11.9 and 23.8 g per day when dosing 60 g of MSDM, 30 and 60 g of SDM, respectively. The highest (p<0.05) methionine plasma concentration (133.9 mM) was measured with 60 g of SDM, followed by 30 g of SDM, and 60 g of MSDM. Plasma methionine concentrations were affected by an interaction (p<0.05) between time after dosing methionine and rate of ruminal degradation of the methionine dosed. Methionine plasma concentration peaked 12 h after dosing SDM, whereas methionine plasma concentration appeared to peak between 6 and 12 h after feeding MSDM. There was a good relationship (r 2 a0.86) between the grams of methionine escaping from the RPM products and the greatest area under the curve describing plasma methionine concentration. Data from this study show that the lower the ruminal degradation rate, the later the maximum plasma concentration of methionine will occur, and that the plasma methionine concentrations or their area