Short communication: a novel system to estimate protein degradability in legume and grass hays

Previous research from our laboratory has demonstrated that near-infrared reflectance spectroscopy has utility in predicting rumen-undegradable protein (RUP) contents of legume and grass silages. This study was conducted to evaluate whether application of previous research techniques could yield a useful near-infrared reflectance spectroscopy RUP prediction system for legume and grass hays. Legume and grass hays (n = 106) were evaluated for RUP content by in situ techniques in four ruminally cannulated cows. In situ RUP for legume and grass hays averaged 25.9% CP and ranged from 14.6 to 45.5% CP, respectively. We developed a near-infrared reflectance spectroscopy RUP calibration equation for the legume and grass hay data set using in situ RUP contents as base data. This procedure resulted in an acceptable (R2 = 0.87, SE = 2.46% CP) near-infrared reflectance spectroscopy equation to predict RUP content of legume and grass hays. Data suggest that near-infrared spectroscopy predicts RUP contents of legume and grass hays with accuracies similar to legume and grass silages.     

Degradation of Crude Protein in Forages Determined by In Vitro and In Situ Procedures

Objectives were to determine in selected forages fiber and protein fractions, in vitro and in situ CP degradability, and to compare in vitro methods of estimating rumen CP degradability with the in situ bag technique. Forages analyzed (five samples per treatment except alfalfa hays, which had four) included alfalfa as baled hay, alfalfa ensiled in conventional upright silos, alfalfa ensiled in oxygen-limiting silos, ammonia-treated corn silage, untreated corn silage, and orchardgrass hay. Untreated corn silages had the greatest protease insoluble CP at 48 h, indicating protein in corn silage is not degraded well by protease enzyme. In situ CP degradability was greater than 80% for all ensiled forages. Ensiled forages had the greatest estimated A fraction (rapidly degraded in the rumen), alfalfa hays had the greatest B fraction (intermediate rate of degradation), and orchardgrass hays had the greatest C fraction (not degraded in rumen).High correlations between in situ degradability and some in vitro measurements suggest laboratory techniques of estimating CP degradability of forages are possible. For silages, buffer-soluble CP had the greatest correlation (.58) with in situ degradability; for hays, NDIN had the greatest correlation (−.83).     

Technical note: Effects of filter bags on neutral detergent fiber recovery and fiber digestion in vitro

Filter bags facilitate the measurement of amylase-treated neutral detergent fiber (aNDF) and in vitro (IV) undigested aNDF (uNDF) by eliminating the transfer of residues from beakers into filtration crucibles. The objectives of this study were to (1) determine effects of filter bags on recovery of aNDF and (2) evaluate effects of filter bags on IV uNDF. For study 1, 6 samples each of grass hay (GR), alfalfa (AL), and corn silage (CS) were selected. Large standard deviations (SD) of ash-free aNDF (aNDFom) for samples in each forage type indicated compositional diversity (15.1, 7.45, and 12.9% of DM for GR, AL, and CS, respectively), and starch SD for CS was 16.4% of DM. Samples were weighed into Berzelius beakers or filter bags [25-µm pores (F57) or 6-µm pores (F58); Ankom Technology, Macedon, NY] for measurement of aNDF and aNDFom. All samples were extracted with neutral detergent, thermostable α-amylase, and sodium sulfite, and then soaked in boiling water and then acetone. Residues from beakers were filtered through a sea sand–covered GF/D filter (Whatman, Marlborough, MA) in Gooch crucibles (CR). Filter bags were extracted in a pressurized chamber at 100°C. The aNDF values did not differ between F57 and CR, but F58 was greater than CR for CS and AL. For GR, F58 was greater than CR for aNDFom. For study 2, diverse samples with large SD of aNDFom (20.7, 7.45, and 12.9% of DM for GR, AL, and CS, respectively) were weighed as loose powder into medium bottles (LS) or F57 bags, which were weighted to prevent floating. Blended ruminal fluid from 3 steers fed a 30% aNDFom diet was used as inoculum. Three samples of 1 forage type were randomly assigned to 1 of 6 IV runs using both treatments (LS and F57), and 3 bottles of each sample–treatment combination were removed after 12 h and 2 were removed after 120 h to measure uNDF. For LS, residues were extracted as in study 1 for CR. For F57, bags were rinsed in cold water and extracted as described in study 1. After 12 h, uNDF of F57 was greater than LS in CS, AL, and overall types. Ash-free uNDF (uNDFom) after 12 h of F57 was greater than LS in CS and overall types. After 120 h, F57 was greater than LS for uNDF of CS, but no differences were detected for uNDFom. The SD of uNDFom, but not uNDF, was higher after 12 and 120 h for F57 compared with LS. From 6 to 96 h, overall gas production of F57 was less than LS, and F57 was less than LS for CS from 3 to 96 h. Overall, LS gave greater maximum and faster rates of gas production than F57, as did AL and CS, but lag did not vary. Results indicate that filter bags affected aNDF and aNDFom measurement and inhibited fermentation for some materials.     

Effects of sample size on neutral detergent fiber digestibility of triticale forages using the Ankom DaisyII Incubator system

Accurate and precise determinations of fiber digestibility are essential for proper diet formulation for dairy cows. Our objectives were 3-fold: (1) regress in vitro neutral detergent fiber digestibility (NDFD) values from 48 triticale forages determined at multiple endpoints ranging from 12 to 240 h with Ankom Daisy^II Incubator system (Ankom Technology Corp., Macedon, NY) methods using 0.25- or 0.50-g sample sizes on concentrations of fiber-related analytes or growth stage; (2) directly compare NDFD values determined with 0.25- or 0.50-g sample sizes by Ankom methods after 12-, 24-, 30-, 48-, 144-, or 240-h incubations; and (3) compare NDFD values determined by Ankom methods after 30 and 48 h of incubation with those determined by traditional sealed-tube procedures obtained from a commercial laboratory. Generally, plant growth stage, which was quantified with a linear model suitable for serving as an independent regression variable, proved to be a better predictor variable for NDFD than neutral detergent fiber or acid detergent lignin. For direct comparisons of 0.25- and 0.50-g sample sizes using Ankom methods, the regression relationship for a 30-h incubation was explained by a linear model [Y = 1.206x – 1.1; coefficient of determination (R²) = 0.933], in which the slope differed from unity, but the intercept did not differ from 0. After a 48-h incubation, a linear model (Y = 1.014x + 7.1; R² = 0.964) indicated that the slope did not differ from unity, but the intercept was >0. A linear regression (Y = 1.040x – 1.8; R² = 0.861) of the 30-h incubation results obtained by Ankom methods using the 0.25-g sample size on those from the commercial laboratory indicated the slope and intercept did not differ from unity or 0, respectively. A similar relationship was obtained from the 48-h incubation (Y = 1.021x – 3.4; R² = 0.866). Relationships were poorer when the 0.50-g sample size was used by Ankom methods, particularly for the 30-h incubation, where the slope (0.824) was less than unity. Generally, NDFD values were greater for the 0.25-g sample size by Ankom methods, especially with 24-, 30-, and 48-h incubation times, and agreement with traditional sealed-tube methods was improved with the smaller sample size. Synchronization of results between Ankom and traditional methods needs to be further verified across a wider range of forages and harvest/preservation methods before definitive recommendations can be made.     

Fermentation and aerobic stability of rehydrated corn grain silage treated with different doses of Lactobacillus buchneri or a combination of Lactobacillus plantarum and Pediococcus acidilactici

We investigated the effects of different types and doses of inoculants for ensiling rehydrated corn grain. Shelled corn was finely ground and rehydrated to 35% moisture. Treatments were as follows: (1) control (no additives); (2) Lactobacillus plantarum and Pediococcus acidilactici (LPPA) at a theoretical application rate of 1 × 10⁵ cfu/g; (3) LPPA at 5 × 10⁵ cfu/g; (4) LPPA at 1 × 10⁶ cfu/g; (5) Lactobacillus buchneri (LB) at 1 × 10⁵ cfu/g; (6) LB at 5 × 10⁵ cfu/g; and (7) LB at 1 × 10⁶ cfu/g. We detected no effect of inoculant dose. Gas losses were greater in silages treated with LB compared with control and LPPA silages. Treating silages with LB reduced the concentrations of lactic acid and ethanol and increased silage pH and concentrations of acetic acid, propionic acid, and 1,2-propanediol. At silo opening, silages treated with LB had higher counts of lactic acid bacteria but lower yeast counts than the control silage. Aerobic stability was greater for silages treated with LB and lower for silages treated with LPPA compared with the control. The LB reduced dry matter (DM) losses during aerobic exposure, whereas LPPA increased them. Prolamin content was lower in silages treated with LB compared with the control, resulting in greater ruminal in situ DM degradability. Inoculating LB to a dose of 1 × 10⁵ cfu/g increased aerobic stability and ruminal in situ DM degradability of rehydrated corn grain silage. The addition of LPPA did not alter the fermentation process and worsened the aerobic stability of rehydrated corn grain silage. Further studies are warranted to confirm these conclusions in other corn hybrids, inoculants, and their combinations.     

Development of a novel system to estimate protein degradability in legume and grass silages

Five trials were conducted to develop a system to estimate rumen-undegradable protein (RUP) of legume and grass silages using near infrared reflectance spectroscopy. In situ procedures were the reference method used to determine silage RUP content. Trials 1, 2, and 3 were devoted to improving in situ procedures. In trial 1, alfalfa silage with and without heat treatment was incubated ruminally in 30 cows. The standard deviation of in situ RUP attributable to cow and diet was 0.82 and 3.80 g/10(-1) kg of crude protein (CP) for the unheated and heated alfalfa, respectively. Based on trial 1, it was determined that 8 cows would be required to establish RUP standards. In trial 2, low (13.3 g/10(-1) kg of CP) and high (44.5 g/10(-1) kg of CP) RUP standards were developed using eight ruminally cannulated cows. In trial 3, 11 new RUP standards were developed by mixing trial 2 RUP standards together. The RUP standards were used to employ a calibration curve technique in ruminally cannulated cows. The technique was employed in four ruminally cannulated cows to estimate RUP contents of 121 silages, and RUP values were used for near-infrared reflectance spectroscopic analysis in trial 4. Trial 4 procedures yielded a calibration for RUP content of silages with an R2 of 0.84 and a standard error of calibration of 1.55 g/10(-1) kg of CP. In trial 5, the equation was tested on 300 silage samples. The mean predicted RUP content was 21.8 g/10(-1) kg of CP. Data suggest near-infrared reflectance spectroscopy can predict RUP content of silages.     

Effects of spontaneous heating on forage protein fractions and in situ disappearance kinetics of crude protein for alfalfa-orchardgrass hays packaged in large round bales

During 2006 and 2007, forages from 3 individual hay harvests were used to assess the effects of spontaneous heating on concentrations of crude protein (CP), neutral detergent insoluble CP (NDICP), acid detergent insoluble CP (ADICP), and in situ disappearance kinetics of CP and NDICP for large round bales of mixed alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.). Over the 3 harvests, 96 large round bales were made at preset bale diameters of 0.9, 1.2, or 1.5 m and at moisture concentrations ranging from 9.3 to 46.6%. Internal bale temperatures were monitored daily during an outdoor storage period. The change in concentrations of NDICP (poststorage − prestorage) increased with heating degree days (HDD) >30°C in a relationship best explained with a nonlinear model {Y = 24.9 - [22.7 × (e^{−0.000010 × x × x})]; R² = 0.892} that became asymptotic at +24.9 percentage units of CP, thereby indicating that NDICP increases rapidly within bales that heat spontaneously. When maximum internal bale temperature (MAX) was used as the independent variable, the best regression model was quadratic and the coefficient of determination was still relatively high (R² = 0.716). The change in concentrations of ADICP (poststorage − prestorage; ΔADICP) also increased with HDD and was best fitted to a nonlinear model {Y = 14.9 - [15.7 × (e^{−0.0000019 × x × x})]} with a very high coefficient of determination (R² = 0.934). A similar quartic response was observed for the regression of ΔADICP on MAX (R² = 0.975). Increases in ΔADICP as a result of heating (HDD or MAX) were paralleled by concurrent increases in hemicellulose at relatively low increments of heating, but the inverse relationship was observed as hemicelluloses likely became reactive and concentrations decreased in more severely heated hays. Changes in ruminal disappearance rate of CP were best fitted to cubic models for regressions on both HDD (R² = 0.939) and MAX (R² = 0.876); these changes represented an approximate 50% rate reduction in severely heated hays relative to prestorage controls. Within ranges of heating most commonly encountered under field conditions, changes in rumen-degradable protein decreased in a primarily linear relationship with HDD or MAX. However, the mean change in rumen-degradable protein for the 4 most severely heated hays was only −2.6 percentage units of CP, which represents a minimal reduction from prestorage controls and is far less than the maximum of −7.9 percentage units of CP observed with less-severe heating. Interpretation of these results was complicated by poor recovery of NDICP from our most severely heated hays following machine rinsing of 0-h Dacron bags; theoretically, and by definition, this unrecovered pool of NDICP is assumed to be entirely degradable in the rumen. It remains unclear whether these responses could be corroborated in vivo or by other analytical techniques, or whether the magnitude of HDD or MAX for our most severely heated hays exceeds the reliable limits for estimating RDP via in situ methodology.     

Development of an in vitro method to determine rumen undigested aNDFom for use in feed evaluation

A portion of the forage cell wall, defined as neutral detergent fiber (NDF), is indigestible to anaerobic microbial digestion in ruminants. This fraction has been characterized by surface area relationships between acid detergent lignin, but recently, data have been published describing the dynamic nature of this relationship. In situ approaches have been described to estimate indigestible NDF, recovering the undigested NDF after long-term fermentations (uNDF). To be applicable to nutritionists and diet formulation, determining uNDF needs to be conducted in a commercial laboratory similar to other routine analyses of forage chemistry. A series of studies were conducted to evaluate an in vitro approach, to describe uNDF, which is repeatable and adaptable for routine feed evaluation. One hundred and two forages of several species were analyzed for NDF, acid detergent lignin, and uNDF. The uNDF was estimated by several approaches involving long-term fermentations and filtration steps to evaluate the length of time necessary to exhaust the digestible NDF and a filtration method necessary to maintain sample integrity by ensuring low sample loss and uniform recovery with residues from long-term in vitro fermentation. To determine uNDF, in vitro fermentations were conducted on 0.50 or 0.75 g of dry matter samples, in triplicate, at multiple time points up to 504 h and initially used Gooch crucibles with Celite (Thermo Fisher Scientific, Waltham, MA) as a filtering aid. The final method utilized a 1.5-µm pore size glass microfiber filter, which allowed for increased repeatability and improved sample recovery (lowest standard deviation). In this study, in vitro fermentations of 240 h were adequate to characterize and identify uNDF, which was repeatable among conventional forages provided the samples, after NDF analyses, were filtered through the same glass fiber filter. This approach could be adapted by commercial laboratories and would provide opportunities to develop near-infrared reflectance spectroscopy equations and calibrations.     

Effect of applying inoculants with heterolactic or homolactic and heterolactic bacteria on the fermentation and quality of corn silage

This study examined the effect of applying different bacterial inoculants on the fermentation and quality of corn silage. Corn plants were harvested at 35% DM, chopped, and ensiled in 20-L mini silos after application of (1) deionized water (CON) or inoculants containing (2) 1 × 10⁵ cfu/g of Pediococcus pentosaceus 12455 and Propionibacteria freudenreichii (B2); (3) 4 × 10⁵ cfu/g of Lactobacillus buchneri 40788 (BUC); or (4) 1 × 10⁵ cfu/g of Pediococcus pentosaceus 12455 and 4 × 10⁵ cfu/g of L. buchneri 40788 (B500). Four replicates of each treatment were weighed into polyethylene bags within 20-L mini silos. Silos were stored for 575 d at ambient temperature (25°C) in a covered barn. After silos were opened, aerobic stability, chemical composition, and yeast and mold counts were determined. The DNA in treated and untreated silages was extracted using lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform and used as a template for a conventional PCR with primers designed on the 16S rRNA gene to detect the presence of L. buchneri in all silage samples. Acetic acid concentration was greater in B2 silages versus others (6.46 vs. 4.23% DM). Silages treated with BUC and B500 had lower pH and propionic acid concentration and greater lactic acid concentration than others. The B500 silage had the greatest lactic:acetic acid ratio (1.54 vs. 0.41), and only treatment with BUC reduced DM losses (5.0 vs. 14.3%). Yeast and mold counts were less than the threshold (10⁵) typically associated with silage spoilage and did not differ among treatments. Consequently, all silages were very stable (>250 h). Aerobic stability was not improved by any inoculant but was lower in B500 silages versus others (276 vs. 386 h). The conventional PCR confirmed the presence of similar populations of L. buchneri in all silages. This may have contributed to the prolonged aerobic stability of all silages.     

Models to predict dry feed intake in Holstein calves to 4 months of age

Voluntary daily dry feed intake (DFI) in Holstein calves was predicted using 60,761 individual daily observations collected from 1,235 Holstein calves in 30 experiments from 4 research stations in the United States and Europe. Consumption of dry feed (calf starter and hay, kg/d or percent of body weight) was measured from 3 to 114 d of age. Linear models and 2- and 3-parameter nonlinear models were evaluated to predict DFI using age of calf, intake of milk replacer, ambient temperature, percent forage, and neutral detergent fiber concentration in ration dry matter (DM) as independent variables. The initial data set was randomly divided within study location into development (80% of all observations) and validation data sets, and initial screening was conducted using the development data set. Five nonlinear models and 3 linear models (candidate models) were identified and used in further model evaluation. Cross-validation studies (n = 20) with the validation data set were conducted by linear regression of DFI with predicted DFI as independent variable. Candidate models were subsequently evaluated with data from 12 published studies in 2 analyses. The exponential model that best predicted daily DFI in Holstein calves in original and external data sets was DFI (kg/d) = 1.3207 × e^{[(?5.3892 + 0.6376 × MEgap) × EXP(?0.0392 × Age)]} – 0.0013 × Temp + 0.0032 × NDFDM + 0.0026 × Age × MEgap – 0.3646 × PctForage [coefficient of determination (R²) = 0.92, concordance correlation coefficient (CCC) = 0.96, and mean square error of prediction (MSEP) = 0.10 kg]; where MEgap (Mcal/d) = difference of daily metabolizable energy (ME) requirement and ME intake from milk replacer; Age = age of calf (d) from 3 to 114, Temp = mean daily ambient temperature (°C), NDFDM = ration neutral detergent fiber (% DM); PctForage = percent forage in ration DM. The linear model that best predicted DFI was DFI (kg/d = ?0.1349 + 0.0106 × Age + 0.1808 × MEgap + 0.0013 × Age × MEgap + 0.0001 × Temp + 0.00002 × Age × Temp (R² = 0.93, CCC = 0.96, and MSEP = 0.10 kg). When Temp and ration characteristics were not included, optimal models were 1.4362 × e^{[(?4.6646 + 0.5234 × MEgap) × EXP(?0.0361 × Age)]} + 0.0025 × Age × MEgap (R² = 0.92, CCC = 0.96, and MSEP = 0.11 kg) and ?0.1344 + 0.0102 × Age + 0.1810 × MEgap + 0.0013 × Age × MEgap [R² = 0.93, CCC = 0.96, and MSEP = 0.10 kg]. Models of daily DFI may improve prediction of nutrient supply to young Holstein calves to approximately 4 mo of age, thereby increasing prediction of growth performance.     

Effects of spontaneous heating on estimates of total digestible nutrients for alfalfa-orchardgrass hays packaged in large round bales

Large round or large square hay packages are more likely to heat spontaneously during storage than hay packaged in conventional (45 kg) bales, and the effects of this phenomenon on the associated energy estimates for these hays can be severe. Our objectives for this project were to assess the relationship between estimates of total digestible nutrients (TDN) and spontaneous heating and to describe any important differences in energy estimates that may result specifically from 2 methods of estimating truly digestible fiber (TD-Fiber). Using the summative approach to estimate TDN, TD-Fiber can be estimated from inputs of protein-corrected neutral detergent fiber (NDFn) and acid detergent lignin (TD-FiberLIG) or from NDFn and 48-h neutral detergent fiber digestibility (TD-FiberNDFD). Throughout 2006 and 2007, mixed alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.) hays from 3 individual harvests were obtained from the same 8.2-ha research site near Stratford, Wisconsin. Both options for estimating TD-Fiber (TD-FiberLIG or TD-FiberNDFD) were then used independently via the summative approach to estimate the total TDN concentrations (TDN-LIG or TDN-NDFD, respectively) within these hays. Estimates of both TDN-LIG and TDN-NDFD then were related to heating degree days >30°C accumulated during storage by various regression techniques. Changes (poststorage - prestorage) in TDN-LIG that occurred during storage (ΔTDN-LIG) were best fitted with a nonlinear decay model in which the independent variable was squared [Y = (11.7 × e^{−0.0000033×x×x}) − 11.6; R² = 0.928]. For changes in TDN-NDFD (ΔTDN-NDFD), a quadratic regression model provided the best fit (Y = 0.0000027x² − 0.010x + 0.4; R² = 0.861). Generally, ΔTDN-LIG estimates were 2.0 to 4.0 percentage units lower than ΔTDN-NDFD estimates when heating exceeded 500 HDD. For regressions on maximum internal bale temperature, both ΔTDN-LIG (Y = −0.38x + 16.3; R² = 0.954) and ΔTDN-NDFD (Y = −0.25x + 10.2; R² = 0.848) were best fitted by linear models with heterogeneous (P < 0.001) slopes and intercepts. In both cases, coefficients of determination were high, suggesting that simple measures of spontaneous heating are excellent predictors of energy losses in heated forages. Regardless of method, reductions in TDN were associated primarily with losses of nonfiber carbohydrate, which is known to occur via oxidation of sugars during spontaneous heating. For heated forages, some discrepancy between TDN-LIG and TDN-NDFD existed because the relationship between NDFD and spontaneous heating was shown previously to be very poor, resulting in minimal changes for estimates of TD-FiberNDFD as a consequence of heating. In contrast, TD-FiberLIG declined in close association with heating, largely because TD-FiberLIG was sensitive to changes in concentrations of both NDFn and acid detergent lignin. Discrepancies between TDN-LIG and TDN-NDFD were exacerbated further when neutral detergent fiber rather than NDFn was used to estimate TD-FiberNDFD. Estimates of TDN declined by as much as 13.0 percentage units within severely heated hays, and this is a serious consequence of spontaneous heating.     

Predicting the effect of proteolysis on ruminal crude protein degradation of legume and grass silages using near-infrared reflectance spectroscopy

Two studies were conducted to assess whether routine applications of near infrared reflectance spectroscopy could predict the effects of silage proteolysis on ruminal crude protein (CP) degradation of legume and grass silages. A preliminary study was conducted to assess the effect of laboratory drying method on ruminal CP degradation of silages. Thirty legume and grass silages were freeze-, oven-, or microwave-dried and incubated in situ in the ventral rumen of three ruminally cannulated cows for 24 h. Freeze-drying was considered least likely to alter ruminal CP degradation of the silages; therefore, oven- and microwave-drying were compared using first-order regression with freeze-drying. Oven-drying for 48 h at 55 degrees C compared favorably (R2 = 0.84) with freeze-drying. Microwave-drying resulted in a large bias (2.84 g/10(-1) kg of CP) and was poorly related (R2 = 0.48) to freeze-drying. In a second study, alfalfa and timothy were cut at three maturities and allowed to wilt for 0, 10, 24, 32, 48, and 54 h. Forages were ensiled in triplicate cylindrical mini silos and allowed to ferment for 120 d. After fermentation, silages were oven-dried, ground, and scanned on a near-infrared reflectance spectrophotometer. Duplicate, dried, 2-mm ground silage samples were incubated in the ventral rumen of three ruminally cannulated cows for 24 h. Forage species, maturity, and wilting time significantly affected 24-h ruminal CP degradation of the silages. Near-infrared reflectance spectroscopy accurately predicted (R2 = 0.91) 24-h ruminal CP degradation of silages. Data suggest near-infrared reflectance spectroscopy can accurately assess the effects of forage species, maturity, and wilting time (proteolysis) on 24-h ruminal CP degradation of legume and grass silages.     

Technical note: Near infrared reflectance spectroscopy to predict fecal indigestible neutral detergent fiber for dairy cows

In vitro and in situ procedures performed to estimate indigestible neutral detergent fiber (iNDF) in forage or fecal samples are time consuming, costly, and limited by intrinsic factors. In contrast, near infrared reflectance spectroscopy (NIRS) has become widely recognized as a valuable tool for accurately determining chemical composition and digestibility parameters of forages. The aim of this study was to build NIRS calibrations and equations for fecal iNDF. In total, 1,281 fecal samples were collected to build a calibration data set, but only 301 were used to develop equations. Once dried, samples were ground and chemically analyzed for crude protein, ash, amylase and sodium sulfite–treated NDF corrected for ash residue (aNDFom), acid detergent fiber, acid detergent lignin, and in vitro digestion at 240 h to estimate iNDF (uNDF240). Each fecal sample was scanned using a NIRSystem 6500 instrument (Perstorp Analytical Inc., Silver Spring, MD). Spectra selection was performed, resulting in 301 sample spectra used to develop regression equations with good accuracy and low standard error of prediction. The standard error of calibration (SEC), cross validation (SECV), and coefficients of determination for calibration (R²) and for cross validation (1 ? VR, where VR = variance ratio) were used to evaluate calibration and validation results. Moreover, the ratio performance deviation (RPD) and ratio of the range of the original data to SECV (range/SECV; range error ratio, RER) were also used to evaluate calibration and equation performance. Calibration data obtained on fiber fractions aNDFom (R² = 0.92, 1 ? VR = 0.87, SEC = 1.48, SECV = 1.89, RPD = 2.80, and RER = 20.19), uNDF240 (R² = 0.92, 1 ? VR = 0.86, SEC = 1.65, SECV = 2.24, RPD = 2.57, and RER = 14.30), and in vitro rumen aNDFom digestibility at 240 h (R² = 0.90, 1 ? VR = 0.85, SEC = 2.68, SECV = 3.43, RPD = 2.53, and RER = 14.0) indicated the predictive equations had good predictive value.     

Incidence of Penicillium roqueforti and roquefortine C in silages

Wilted grass and whole-crop maize silages taken from farm silos in northern Germany were analysed for fermentation pattern, mould counts and composition of mycoflora as well as for roquefortine C. In general, increasing DM contents of visibly unmoulded silages resulted in decreasing amounts of volatile fatty acids and a greater portion of samples with a high number of mould propagules. The average mould count of these silages was found to be 1·4×10⁴ cfu g⁻¹, whereas visibly moulded samples contained about 1×10⁸ cfu g⁻¹. Penicillium roqueforti was the predominating fungal species in silages occasionally accompanied by species of the genera Aspergillus, Mucor, Monascus and/or Geotrichum. Penicillium roqueforti was detected in 89% of the visibly moulded and in 85% of the visibly unmoulded samples. Of 24 visibly moulded silages tested, 21 samples contained roquefortine C, a mycotoxin known to be produced by P roqueforti. The highest level of roquefortine C found was 36 mg kg⁻¹ DM. Even 6 of 24 visibly unmoulded samples analysed for this mycotoxin were contaminated with roquefortine C but only in trace amounts. Roquefortine C is considered as a model compound for the biosynthesis of toxic fungal metabolites produced by P roqueforti in silages. The P roqueforti-count can be employed as a criterion to predict the contamination of silages with mycotoxins produced by this fungal species. © 1998 SCI.     

Activity of trisodium phosphate compared with sodium hydroxide wash solutions against Listeria monocytogenes attached to chicken skin during refrigerated storage

The potential of using trisodium phosphate (TSP) and sodium hydroxide (NaOH) to reduce Listeria monocytogenes populations in chicken skin was studied. Raw chicken legs inoculated with L. monocytogenes were dipped in water (control), in 8% (pH=12·59), 10% (pH=12·68) and 12% (pH=12·75) (w/v) TSP solutions, or in NaOH solutions of equal pH values to those of TSP: 0·175%, 0·200% and 0·220% (w/v). Surface pH values and L. monocytogenes counts of chicken skin were determined immediately after treatment (day 0) and after 1, 3 and 5 days of storage at 2°C. Compared with water dipping, TSP and NaOH treatments significantly (P<0·05) reduce Listeria populations at days 0, 1, 3 and 5 of refrigerated storage. Bacterial reductions varied between 1·12 and 3·34 log₁₀ cycles for TSP-treated samples and between 1·80 and 3·28 log₁₀ cycles for NaOH treated samples. The observed reductions in all treated samples were significantly (P<0·05) greater following storage. The concentration of the TSP solution was a significant factor in reducing the populations of L. monocytogenes. However, bacterial reductions were similar in samples treated with different concentrations of NaOH. The TSP or NaOH treatments resulted in relatively high residual surface pH values (8–9) initially and throughout storage. The pH values were significantly higher in the samples treated with TSP than in those treated with NaOH.     

Technical note: In vitro digestibility of amylase-treated,ash-corrected neutral detergent fiber,with addition of sodium sulfite,at 240 hours with or without rumen fluid reinoculation

Long-term in vitro fermentation (240 h) evaluating amylase-treated, ash-corrected neutral detergent fiber, with addition of sodium sulfite (aNDFom) digestibility is required to quantify the indigestible fiber fraction. It is commonly accepted to inoculate rumen fluid more than one time during such fermentations, every 96 h or at 120 h. However, no studies have been conducted to verify if the reinoculation is actually required to properly carry out the fermentation process. The current study aims to evaluate the effects of these procedures on aNDFom digestibility at 240 h. The study was conducted on a total of 24 forage samples (8 alfalfa hays, 8 grass hays, and 8 corn silages). Samples were digested in triplicate at 240 h in vitro. Rumen fluid was added twice (at 96 and 192 h) in treatment 1, after 120 h in treatment 2, whereas no addition was made in treatment 3. At the end of the fermentations, residual aNDFom was quantified to calculate digestibility. Among treatments, no difference was found in digestibility of aNDFom. Moreover, treatment 1 resulted in higher variability compared with other treatments. Results obtained in the current study show that subsequent addition of rumen fluid is not necessary for a proper estimation of aNDFom digestibility, and can be avoided.     

Inoculation with Lactobacillus plantarum of Alfalfa,Corn, Sorghum,and Wheat Silages

Alfalfa, corn, sorghum, and wheat forages were harvested for ensiling. Effects of inoculation of the forages with Lactobacillus plantarum (10⁷/g silage) were studied in small experimental 55-kg drum silos. Control and treated silages were prepared, and drums from each treatment were opened on days 1, 2, 4, 8, 16, and 33. Proximate analysis, pH, neutral detergent fiber, acid detergent fiber, soluble carbohydrate, lactic acid, and volatile fatty acid contents were measured. Total faculative anaerobic microflora were increased in all silages by addition of L. plantarum, and numbers of lactobacilli, were increased in all but corn silage. Yeasts and molds were lower only in inoculated alfalfa and wheat silages. The effect of the inoculum was observed on chemical composition only for alfalfa and wheat silages. These treated silages had a lower pH, higher lactic acid content, and great recovery of dry matter, crude protein, and detergent fiber (alfalfa) and nitrogen-free extract (wheat). Inoculation has an effect on microflora in all silages and a positive beneficial effect on nutrient recovery in alfalfa and wheat silages.     

Dry matter and nutritional losses during aerobic deterioration of corn and sorghum silages as influenced by different lactic acid bacteria inocula

The economic damage that results from aerobic deterioration of silage is a significant problem for farm profitability and feed quality. This paper quantifies the dry matter (DM) and nutritional losses that occur during the exposure of corn and sorghum silages to air over 14 d and assesses the possibility of enhancing the aerobic stability of silages through inoculation with lactic acid bacteria (LAB). The trial was carried out in Northern Italy on corn (50% milk line) and grain sorghum (early dough stage) silages. The crops were ensiled in 30-L jars, without a LAB inoculant (C), with a Lactobacillus plantarum inoculum (LP), and with a Lactobacillus buchneri inoculum (LB; theoretical rate of 1 × 10⁶ cfu/g of fresh forage). The pre-ensiled material, the silage at silo opening, and the aerobically exposed silage were analyzed for DM content, fermentative profiles, yeast and mold count, starch, crude protein, ash, fiber components, 24-h and 48-h DM digestibility and neutral detergent fiber (NDF) degradability. The yield and nutrient analysis data of the corn and sorghum silages were used as input for Milk2006 to estimate the total digestible nutrients, net energy of lactation, and milk production per Mg of DM. The DM fermentation and respiration losses were also calculated. The inocula influenced the in vitro NDF digestibility at 24 h, the net energy for lactation (NE_L), and the predicted milk yield per megagram of DM, whereas the length of time of air exposure influenced DM digestibility at 24 and 48 h, the NE_L, and the predicted milk yield per megagram of DM in the corn silages. The inocula only influenced the milk yield per megagram of DM and the air exposure affected the DM digestibility at 24 h, the NE_L, and the milk yield per megagram of DM in the sorghum silages. The milk yield, after 14 d of air exposure, decreased to 1,442, 1,418, and 1,277 kg/Mg of DM for C, LB, and LP corn silages, respectively, compared with an average value of 1,568 kg of silage at opening. In the sorghum silages, the milk yield, after 14 d of air exposure, decreased to 1,226, 1,278, and 1,250 kg/Mg of DM for C, LB, and LP, respectively. When the estimated milk yield per megagram of harvested DM of corn and sorghum silage were related to mold count, it was shown that the loss of potential milk production occurred when the mold count exceeded 4 log cfu/g of silage, and it was almost halved when the mold count reached values greater than 8 log cfu/g of silage. Inoculation with L. buchneri, at a rate of 1 × 10⁶ cfu/g of fresh forage, enhanced the stability of the silage after exposure to air, and, consequently, contributed to maintaining the nutritional value of the harvested forage over time, for air exposure up to 7 d.     

Elimination of aggregates of (1→3) (1→4)-β-D-glucan in dilute solutions for light scattering and size exclusion chromatography study

Methods for eliminating aggregates of cereal (1→3) (1→4)-β-D-glucan in dilute solutions were investigated using dynamic light scattering and size exclusion chromatography. Wheat β-D-glucan samples were selected and dissolved in various solvents under different preparation conditions. The molecular size distribution was monitored by dynamic light scattering measurement. In most of the solutions, there were two well separated species of different average sizes. It appeared that the specie with smaller average size represented the un-aggregated molecules (unimers) and the specie with larger particle size corresponded to the aggregates. The results showed that heat treatment, filtration, ultrasonication, and the use of urea solution (up to 6 M) could not eliminate aggregates completely. However, the percentage of aggregates in aqueous NaOH solution decreased significantly with the increase of NaOH concentration. In 0.5 M NaOH solution, no aggregation was detectable by dynamic light scattering measurement. Both dynamic light scattering and HPSEC data showed that wheat β-D-glucan was stable in 0.5 M NaOH solution without any noticeable degradation when stored at 25 °C for 12 h. The results of present study suggested that 0.5 M NaOH solution is a suitable solvent for cereal β-D-glucans. Using this solvent, the molecular characteristics of wheat β-D-glucan was studied by both dynamic and static light scattering. The weight average molecular weight (M_w), radius of gyration (R_g), hydrodynamic radius (R_h), and the second virial coefficient (A₂) were obtained with the values of 3.29×10⁵ g/mol, 45.6 nm, 26.2 nm, and 1.04×10⁻³ cm³ mol/g² respectively. This study also confirmed that wheat β-glucan in solution exhibited a random coil conformation.