Comparison of two in situ reference methods to estimate indigestible NDF by near infrared reflectance spectroscopy in alfalfa.

Undigested forage neutral detergent fiber (uNDF) from long-term ruminal in situ incubations are used to estimate indigestible neutral detergent fiber (iNDF). Measurement of iNDF is important in forage evaluation because it defines the potentially digestible pool of neutral detergent fiber (NDF). Near-infrared reflectance spectroscopy (NIRS) can be calibrated to in situ reference sets to rapidly predict uNDF. Our objective was to compare uNDF estimates after 240 h of incubation when two types of bags were used in the in situ reference method. The bags compared were 4 cm × 5 cm Ankom F57 bags (25 micron pore size), and 5 cm × 10 cm Ankom in situ bags (50 micron pore size). Alfalfa samples from Pennsylvania and Wisconsin (n = 144) of different varieties and harvest intervals were used. One-half or two gram samples, respectively, were weighed into the small and large bags in triplicate. Mass to surface area was 0.05 and 0.02 g/cm2 for the small and large bags, respectively. The iNDF content after 240 h incubation was evaluated by two types bags in three rumen-cannulated Holstein cows. Each dried and ground forage was also scanned to determine the visible-near-infrared-reflectance spectra with a FOSS 6500 spectrophotometer. Prediction equations were developed for each bag type using modified partial least square regressions. The estimated iNDF fraction from small and large bags were 13.75% and 9.97%, respectively (SED = 0.39, P < 0.001). The coefficient of determination for calibration (R2), cross-validation (1 - VR), calibration standard deviation (SEC), and interactive authentication standard deviation (SECV) was 0.94, 0.92, 0.85 and 0.98 for values determined with the small bag and 0.88, 0.85, 1.12 and 1.27 for iNDF for values determined with the large bag, respectively. Results indicate that iNDF varies among alfalfa cultivars and NIRS can be used to quickly and quantitatively estimate iNDF content in alfalfa. Bag type influences 240h NDF residues. NIRS predictions of iNDF from the small bag calibration set had higher R2 and lower SEC and SECV than the large bag calibrations.

Effect of Lactobacillus inoculants and forage dry matter on the fermentation and aerobic stability of ensiled mixed-crop tall fescue and meadow fescue.

This study evaluated the effects of Lactobacillus plantarum with or without Lactobacillus buchneri on the fermentation and aerobic stability of mixed tall fescue (Festuca arundinacea Schreb) and meadow fescue (Festuca pratensis Huds.) silage ensiled at different dry matter (DM) contents. The first cut was harvested at boot stage and second-cut grasses were harvested when 30- to 35-cm tall. Four DM content treatments of the first cut were 17.9, 24.9, 34.6, and 48.7%; and of the second cut were 29.1, 36.3, 44.1, and 49.2%. Chopped grasses at each DM content were treated with (1) deionized water (control), (2) Lb. plantarum MTD-1 (LP), or (3) a combination of Lb. plantarum MTD-1 and Lb. buchneri 40788 (LP+LB). The application amount of each inoculant to the fresh forage was 1 × 10(6) cfu/g. Grasses were ensiled in vacuum-sealed polyethylene bags containing 150 g of DM for 60 d, with 4 replicates for each treatment. Silages inoculated with LP+LB had greater pH compared with untreated or LP-treated silages. Lactate was greater in LP silage than control or LP+LB silages. As silage DM increased, lactate in untreated and LP-treated silages decreased, but increased in LP+LB-treated silage. Acetate concentration decreased with increased DM in all silages. The LP+LB-treated silage had the longest and control silage the shortest aerobic stability for both harvests. The greatest values in aerobic stability were observed in silages with highest DM content. In this study, aerobic stability of grass mixes ensiled between 18 and 44% DM content increased as the percentage of DM increased. The LP and LP+LB inoculants improved aerobic stability of silages harvested between 18 and 44% DM content.

Short communication: Characteristics of proteolytic activities of endo- and exopeptidases in alfalfa herbage and their implications for proteolysis in silage.

The pH optimum and thermostability of both exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.

Reducing phosphorus inputs for grazing Holstein steers.

A 2-yr study was conducted to confirm that managed pastures can provide Holstein steers adequate P to meet their daily requirement. Treatments offered were trace mineralized salt with or without additional P. In the first year, 80 Holstein steers (248 kg of BW) were assigned to 4 grazing groups. Treatments were trace mineralized salt only or a 67:33 mixture of trace mineralized salt and dicalcium phosphate. Steers rotationally grazed a cool-season grass/legume mixture for 137 d. Fecal bags were placed on 3 steers from each grazing group (n = 12) over a 4-d period for estimation of forage DMI and forage contribution to daily P intake twice during the grazing season. Analyzed pasture samples contained 3.28 mg of P/g of DM. During the second year, 72 Holstein steers (297 kg of BW) were blocked into 2 BW groups and subsequently assigned to 1 of 4 pasture groups. Steers rotationally grazed the same forage base as the first year for 126 d. Pasture samples contained 3.27 mg of P/g of DM. No significant differences (P > 0.10) were detected for BW, ADG, or free-choice supplemental mineral intake. Forage provided 126% of the recommended NRC P requirement. Thus, supplemental phosphorous was not required for Holstein steers grazing mixed, cool-season, grass/legume pastures.

Virulence of Aphanomyces euteiches Isolates from Iowa and Wisconsin and Benefits of Resistance to A. euteiches in Alfalfa Cultivars.

Aphanomyces euteiches has become recognized as an important root rot pathogen of alfalfa in the north-central United States, and resistant cultivars are now commonly planted. Recent evidence indicates the existence of A. euteiches strains, designated as race 2, that are virulent on resistant cultivars, but there is little information on the prevalence of such strains or their impact on the performance of A. euteiches-resistant cultivars. The purpose of this study was to assess the virulence of A. euteiches isolates obtained from Iowa and Wisconsin soils and to determine the frequency of isolates virulent on race 1-resistant alfalfa populations. In addition, the yield performance of susceptible and resistant alfalfa populations was compared in four Iowa locations and one Wisconsin location. Fourteen isolates of A. euteiches from different Iowa locations were used to challenge two race 1-resistant cultivars (Paramount and Quantum), a susceptible cultivar (Agate or Vernal), and two resistant breeding populations (WAPH-1 and WAPH-2). Fifty-nine isolates of A. euteiches from one location in Wisconsin were used to challenge one susceptible cultivar (Saranac) and WAPH-1 and WAPH-2. Every isolate was virulent to one or more alfalfa cultivars or populations. Emergence of seedlings in growth chamber experiments did not differ significantly among isolates or alfalfa populations. Alfalfa population and A. euteiches isolate had significant effects on disease severity index (DSI, 1-5 scale), but there were significant interactions (P < 0.05) between these two effects. All 14 Iowa isolates of A. euteiches were virulent (DSI ≥ 3.0) on Agate (mean DSI = 4.4, range 3.8 to 4.9), WAPH-1 (mean DSI = 3.9, range 3.0 to 4.4), and the two commercial resistant cultivars (mean DSI = 3.9 and 4.1, range 3.2 to 4.4). On WAPH-2, only three isolates were virulent (mean DSI = 2.5, range 1.8 to 3.2). Of 59 Wisconsin isolates, all were virulent on Saranac (mean DSI = 4.6, range 3.9 to 5.0), 21 were virulent on WAPH-1 (mean DSI = 2.9, range 1.8 to 4.8), and only four were virulent on WAPH-2 (mean DSI = 2.3, range 1.8 to 3.4). In field studies, we compared yield performance of alfalfa cultivars that were resistant or susceptible to A. euteiches or Phytophthora medicaginis at four Iowa locations for one to three harvest years, and one Wisconsin location for two harvest years. Mean yields of cultivars with resistance to one or both pathogens were significantly higher than those of susceptible cultivars in only one of the four Iowa locations. In Wisconsin, WAPH-4, a Race 2-resistant alfalfa population, expressed a significant yield advantage when compared with both WAPH-1, a Race 1-resistant alfalfa population, and Columbia 2000, a cultivar susceptible to both race 1 and 2 of A. euteiches. These results indicate that race 2 of A. euteiches is prevalent in Iowa and Wisconsin soils and may be limiting the yield benefits of currently available race 1-resistant alfalfa cultivars. Incorporation of race 2 resistance is likely to improve the performance of alfalfa cultivars in A. euteiches-infested soils.

Regression Analyses for Evaluating the Influence of Bacillus cereus on Alfalfa Yield Under Variable Disease Intensity.

ABSTRACT We developed and tested regression methods to exploit the variability in disease inherent in field experiments, and applied the methods to evaluate strains of Bacillus cereus for biocontrol efficacy. Four B. cereus strains were tested for their effect on alfalfa (Medicago sativa) performance in 16 field trials planted during 1993 to 1996 at multiple sites in Wisconsin. To evaluate performance of the strains, we used the ratio of (metalaxyl response)/(untreated control response) as a measure of disease intensity within the experiments. The ratio of (Bacillus response)/(untreated control response) was then regressed as a function of disease intensity. The slope of the resulting line provides a statistical test to compare performance of the Bacillus strain with that of the untreated seed (H(o): slope = 0) and metalaxyl controls (H(o): slope = 1). Under conditions in which disease occurred, forage yield of plots planted with seed treated with B. cereus strain AS4-12 exceeded yield from the untreated control plots (P = 0.002) and was similar to yield of plots planted with metalaxyl-treated seed (P = 0.14). Yield gain associated with AS4-12 and metalaxyl seed treatment averaged 6.1 +/- 2.8% (+/-standard error) and 3.0 +/- 2.8%, respectively. In contrast to the regression approach, means analysis by analysis of variance did not detect differences among treatments. Three other B. cereus strains either did not increase alfalfa yield or increased yield less than did AS4-12. Metalaxyl and three of the Bacillus strains increased seedling emergence, but the improved stands were not predictive of increased forage yield. In six additional studies conducted for one season in 1997, AS4-12 enhanced yield of two cultivars at diverse locations in Wisconsin, but there was an apparent cultivar-location interaction. A strong correlation between response to AS4-12 and metalaxyl treatment suggests that these treatments controlled similar pathogens, most likely the oomycete pathogens Phytophthora medicaginis and Pythium spp.

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.

Prediction of laboratory and in situ protein fractions in legume and grass silages using near-infrared reflectance spectroscopy.

Legume and grass silage samples (n = 121) were collected from commercial forage testing laboratories (trial 1). Samples were dried at 55 degrees C for 48 h, ground, scanned on a near-infrared reflectance spectrophotometer, and analyzed for crude protein (CP), soluble CP, acid detergent fiber (ADF) CP, and neutral detergent fiber (NDF) CP by wet chemistry methods. Sixty samples were selected for calibration development, and the remaining samples were used for equation validation. Near-infrared reflectance spectroscopy accurately predicted the CP content of the silages (R2 = 0.96), but prediction of soluble CP, ADF CP, and NDF CP was markedly less accurate. The coefficients of determination and standard errors of calibration for CP, ADF CP, NDF CP (percentage of DM), and soluble CP (percentage of CP) were as follows (0.96 and 0.80, 0.77 and 0.24, 0.72 and 0.71, and 0.82 and 4.40). In a second study, legume and grass silage samples (n = 32) were dried at 55 degrees C and ground (2 mm). Duplicate dacron bags containing 5 g of silage were incubated in the ventral rumen of three ruminally cannulated cows for 0, 3, 6, 12, 24, 48, and 72 h. In situ protein fractions, including rapidly degraded protein, slowly degraded protein, undegradable protein, degradation rate, and rumen-undegradable protein, were determined. Original samples were reground (1 mm) and scanned. Previously defined near-infrared spectroscopy calibration procedures were conducted. Coefficients of determination for in situ CP fractions were R2 > 0.92 with the exception of degradation rate (R2 = 0.87). Data suggest that in situ protein fractions are better predicted by near-infrared reflectance spectroscopy than by laboratory protein fractions.

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.

Digestibility by lambs of water-stressed alfalfa as determined by total collection or internal markers.

A lamb digestion trial was conducted to compare the ability of internal markers to predict digestibility of alfalfa. Lambs were fed alfalfa hay grown with varying amounts of water stress where water per harvest ranged from 10 to 20 cm/ha and yield ranged 1400 to 4200 kg/ha. In vivo dry matter digestibility was most highly correlated with digestibility determined using acid detergent fiber insoluble ash (r = .80) followed by acid insoluble ash (r = .69). In vivo digestibility of the fibrous components was most highly correlated with digestibility determined by acid detergent insoluble ash and indigestible neutral detergent fiber followed by acid lignin and acid insoluble ash.

Digestibility and rate of passage by lambs of water-stressed alfalfa.

Two lamb digestion experiments were conducted to evaluate the effect of alfalfa [Medicago sativa (L.)] grown under varying levels of water deficiency (stress) on the rate of passage and digestibility of various fibrous components. Experiment 1 consisted of a randomized complete block design in which 12 Suffolk X Hampshire crossbred wethers averaging 40 kg were fed alfalfa hay grown at three (10, 15 or 20 cm water/ha) levels of water per harvest. Experiment 2 consisted of a switchback design in which four Hampshire wethers averaging 45 kg were fed alfalfa hay grown at two (5 or 20 cm/ha) levels of water per harvest. Forage yields ranged from 1,420 (10 cm/ha in Exp. 1) to 4,200 (20 cm/ha in Exp. 2) kg/ha. In both experiments, water stress reduced cell wall constituents (neutral detergent fiber), acid detergent fiber, lignin and cellulose content of the alfalfa hay. Organic matter digestibility was decreased when the percentage of leaves fell below 60% at the highest yield. Digestibility of N and the rate of NDF digestibility were not affected by water stress. The second experiment additionally included nutrient balance and rate of passage measurements. Greater (P less than .10) amounts of N and P were absorbed from water-stressed than nonstressed hay. Ruminal retention time of particulate markers tended (P less than .10) to increase with greater water stress. The results of this study are interpreted to indicate that while moderate water stress may have little effect on in vivo digestibility of alfalfa, severe stress may reduce digestibility of fibrous fractions and total organic matter.