Evaluating the effects of Lactobacillus animalis and Propionibacterium freudenreichii on performance and rumen and fecal measures in lactating dairy cows.

Two experiments evaluated the effect of supplementation with a bacterial direct-fed microbial on performance and apparent total-tract nutrient digestion of dairy cows. In experiment 1, 30 multiparous cows (75 ± 32 d in milk) were randomly assigned to 1 of 2 treatments fed for 10 wk. All cows were fed a diet containing 23.8% starch. Treatments were top dressed to rations twice daily and consisted of a combination of Lactobacillus animalis (1 × 109 cfu/d) and Propionibacterium freudenreichii (2 × 109 cfu/d; LAPF) or carrier alone (CON). In experiment 2, 6 ruminally cannulated cows (123 ± 129 d in milk) were randomly assigned to a crossover design with two 6-wk periods. Cows received the same CON or LAPF treatment as in experiment 1. Cows were fed the same 23.8% starch diet as experiment 1 during wk 1 through 5 of each period, and then cows were abruptly switched to a 31.1% starch diet for wk 6. For both experiments, intake and milk yield were measured daily, and milk samples were collected weekly. In experiment 1, fecal grab samples were collected every 6 h on d 7 of experimental wk 1, 2, 4, 6, 8, and 10. Fecal consistency was scored, and fecal starch was measured in daily composite samples. Fecal composites from a subset of 7 cows per treatment were used to measure apparent total-tract nutrient digestion. In experiment 2, rumen pH was continuously recorded during wk 5 and 6. On d 7 of wk 5 (the final day of feeding the 23.8% starch ration), d 1 of wk 6 (the day of diet transition), and d 7 of wk 6 (the final day of feeding the 31.1% starch ration), rumen in situ digestion was determined. Samples of rumen fluid and feces were collected every 6 h on those days for measurement of fecal starch (composited by cow within day), rumen volatile fatty acids, and fecal pH. Rumen and fecal samples were collected at one time point on those days for microbiota assessment. In experiment 1, treatment did not affect intake, milk yield, milk composition, or fecal score. The LAPF treatment decreased fecal starch percentage and tended to increase starch digestion compared with CON, but the differences were very small (0.59 vs. 0.78% and 98.74 vs. 98.46%, respectively). Digestion of other nutrients was unaffected. In experiment 2, LAPF increased rumen pH following the abrupt switch to the high-starch diet, but milk yield was lower for LAPF compared with CON (35.7 vs. 33.2 kg/d). Contrary to the decrease in fecal starch with LAPF observed in experiment 1, fecal starch tended to be increased by LAPF following the abrupt ration change in experiment 2 (2.97 vs. 2.15%). Few effects of treatment on rumen and fecal microbial populations were detectable. Under the conditions used in our experiments, addition of the bacterial direct-fed microbials did not have a marked effect on animal performance, ruminal measures, or total-tract nutrient digestion.

The effects of air stress during storage and low packing density on the fermentation and aerobic stability of corn silage inoculated with Lactobacillus buchneri 40788.

We determined if a microbial inoculant could improve the fermentation and aerobic stability of corn silage subjected to various challenges during storage that included an air stress challenge and low packing density. In Experiment 1, whole-plant corn was untreated (CTR) or treated (INO, Lactobacillus buchneri 40788 and Pediococcus pentosaceus 12455. Five individually replicated 7.5-L silos, at a density of 240 kg of dry matter (DM)/m3, for each treatment were kept sealed (NAS) for 19 wk, air stressed early (ES, 3 h/wk for wk 1-9), or air stressed late during storage (LS, 3 h/wk for wk 10-19). Inoculation increased the number of agar-culturable lactic acid bacteria regardless of air stress status, but it did not affect the relative abundance of Lactobacillus. Early, but not late air stress, resulted in silages with a higher relative abundance of Acetobacter when compared with NAS. Silages treated with INO had greater concentrations of acetic acid than CTR. Numbers of yeasts were lowest for INO regardless of air stress and CTR-LS had the most yeasts among all treatments. Silages that were not air stressed had a higher relative abundance of Candida tropicalis than air stressed silages. Monascus purpureus was detected in ES and LS but not in NAS, and its relative abundance was numerically higher in CTR-ES than in INO-ES and statistically higher in CTR-LS compared with INO-LS. Early air stress numerically reduced aerobic stability compared with NAS, and there was a statistical tendency for lower stability in LS compared with NAS. Inoculation improved aerobic stability regardless of when the air stress occurred. In Experiment 2, corn silage was prepared with the same primary treatments of CTR and INO but was packed at a low (LD; 180 kg of DM/m3) or a normal (ND; 240 kg of DM/m3) density and sealed (NAS) or air stressed (AS; 24 h on d 28, 42, and 89) for 92 d of storage. The concentration of acetic acid was greater in INO compared with CTR and in AS compared with NAS. Numbers of yeasts were lower in NAS compared with AS regardless of inoculation and they were lower in INO-AS compared with CTR-AS. Treatment with INO improved aerobic stability but the improvement was better in NAS versus AS and better in ND versus LD. Overall, our experiments corroborate past findings showing that INO markedly improves the aerobic stability of corn silage but they are the first to show that improvement can be sustained even when the silage was exposed to regular air stresses and when packed at a low density.

Effects of Lactobacillus hilgardii 4785 and Lactobacillus buchneri 40788 on the bacterial community, fermentation and aerobic stability of high-moisture corn silage.

AIMS: To evaluate the capacity of Lactobacillus hilgardii and Lactobacillus buchneri on modifying the bacterial community and improving fermentation and aerobic stability of high-moisture corn (HMC). METHODS AND RESULTS: High-moisture corn was untreated (CTR), treated with L. hilgardii (LH) or L. buchneri (LB) at 600 000 CFU per gram fresh weight, or with L. hilgardii and L. buchneri at 300 000 CFU per gram fresh weight each (LHLB), and stored for 10, 30 or 92 days. Compared to CTR, inoculated silages had higher Lactobacillaceae relative abundance, lower yeasts numbers and higher aerobic stability. Treatment with LHLB resulted in a higher acetic acid concentration than LH and higher 1,2 propanediol concentration than LB, such differences were numerically greater at 10 and 30 days but statistically greater at 92 days. At 10 days, all inoculated silages were more stable than CTR, but LHLB was even more stable than LB or LH. CONCLUSIONS: The combination of L. hilgardii and L. buchneri had a synergistic effect on yeast inhibition, leading to greater improvements in aerobic stability as early as 10 days after ensiling. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus hilgardii, especially in combination with L. buchneri, can improve the aerobic stability of HMC after a very short period of ensiling.

The Risk Factors of mTORi-Associated Posttransplant Proteinuria.

Treatment with mammalian target of rapamycin inhibitors (mTORi) has been associated with an increased incidence of proteinuria after kidney transplantation as compared to other immunosuppressive agents. Proteinuria after mTORi use may occur in different clinical conditions and the precise mechanism remains unclear. The objective of this study was to investigate the related risk factors for proteinuria after mTORi treatment in kidney transplant recipients. This retrospective observational study population consisted of kidney transplant recipients followed up in a medical center in Southern Taiwan from January 1999 to April 2016. The baseline characteristics and transplantation-related profiles were collected at the time of enrollment. We examined risk factors for mTORi-associated proteinuria using a multivariate logistic regression analysis. P < .05 was considered as statistically significant. Hyperlipidemia and obesity at the initiation of mTORi treatment were strong predictors for proteinuria. Earlier identification of these risk factors may assist physicians in deciding the best candidate for mTORi conversion in order to optimize transplantation outcomes.

Silage review: Recent advances and future uses of silage additives.

Additives have been available for enhancing silage preservation for decades. This review covers research studies published since 2000 that have investigated the efficacy of silage additives. The review has been divided into 6 categories of additives: homofermentative lactic acid bacteria (LAB), obligate heterofermentative LAB, combination inoculants containing obligate heterofermentative LAB plus homofermentative LAB, other inoculants, chemicals, and enzymes. The homofermentative LAB rapidly decrease pH and increase lactic acid relative to other fermentation products, although a meta-analysis indicated no reduction in pH in corn, sorghum, and sugarcane silages relative to untreated silages. These additives resulted in higher milk production according to the meta-analysis by mechanisms that are still unclear. Lactobacillus buchneri is the dominant species used in obligate heterofermentative LAB silage additives. It slowly converts lactic acid to acetic acid and 1,2-propanediol during silo storage, improving aerobic stability while having no effect on animal productivity. Current research is focused on finding other species in the Lb. buchneri group capable of producing more rapid improvements in aerobic stability. Combination inoculants aim to provide the aerobic stability benefits of Lb. buchneri with the silage fermentation efficiency and animal productivity benefits of homofermentative LAB. Research indicates that these products are improving aerobic stability, but feeding studies are not yet sufficient to make conclusions about effects on animal performance. Novel non-LAB species have been studied as potential silage inoculants. Streptococcus bovis is a potential starter species within a homofermentative LAB inoculant. Propionibacterium and Bacillus species offer improved aerobic stability in some cases. Some yeast research has focused on inhibiting molds and other detrimental silage microorganisms, whereas other yeast research suggests that it may be possible to apply a direct-fed microbial strain at ensiling, have it survive ensiling, and multiply during feed out. Chemical additives traditionally have fallen in 2 groups. Formic acid causes direct acidification, suppressing clostridia and other undesired bacteria and improving protein preservation during ensiling. On the other hand, sorbic, benzoic, propionic, and acetic acids improve silage aerobic stability at feed out through direct inhibition of yeasts and molds. Current research has focused on various combinations of these chemicals to improve both aerobic stability and animal productivity. Enzyme additives have been added to forage primarily to breakdown plant cell walls at ensiling to improve silage fermentation by providing sugars for the LAB and to enhance the nutritive value of silage by increasing the digestibility of cell walls. Cellulase or hemicellulase mixtures have been more successful at the former than the latter. A new approach focused on Lb. buchneri producing ferulic acid esterase has also had mixed success in improving the efficiency of silage digestion. Another new enzyme approach is the application of proteases to corn silage to improve starch digestibility, but more research is needed to determine the feasibility. Future silage additives are expected to directly inhibit clostridia and other detrimental microorganisms, mitigate high mycotoxin levels on harvested forages during ensiling, enhance aerobic stability, improve cell wall digestibility, increase the efficiency of utilization of silage nitrogen by cattle, and increase the availability of starch to cattle.

Utility of circulating serum miRNAs as biomarkers of early cartilage degeneration in animal models of post-traumatic osteoarthritis and inflammatory arthritis.

OBJECTIVE: The purpose of this study was to determine if serum microRNA (miRNA) signatures were biomarkers of early cartilage degeneration in preclinical mouse models of post-traumatic osteoarthritis (OA) and inflammatory arthritis. METHODS: Cartilage degeneration was induced in 10-12 week old male C57BL6 mice by destabilization of the medial meniscus (DMM) or intra-articular injection of methylated-bovine-serum-albumin (AIA), with sham-operated or saline-injected control animals (n = 6/treatment/time). Total serum RNA and knee joints were isolated at 1, 4 and 16 weeks post-induction. Cartilage degeneration was scored histologically. Serum miRNA expression profiling was performed using Agilent microarrays and validated by qPCR. RESULTS: DMM-operated and AIA mice had characteristic cartilage degeneration (proteoglycan loss, chondrocyte hypertrophy, structural damage), that increased significantly with time compared with controls, and with distinct temporal differences between arthritis models. However, expression profiling revealed no statistically significant dysregulation of serum miRNAs between AIA vs saline-injected or DMM vs sham-operated control mice at the critical early disease stages. The inability to detect DMM or AIA serum miRNA signatures compared with controls was not due to the insensitivity of the expression profiling approach since significant changes were observed in miRNA expression between the arthritis models and between time points. CONCLUSION: While distinct patterns of progressive cartilage degradation were induced in the arthritis models, we were unable to identify any serum miRNAs that were significantly dysregulated in early stages of disease compared with controls. This suggests circulating serum miRNAs may not be useful as cartilage biomarkers in distinguishing the early or progressive stages of arthritis cartilage degeneration.

Identification of the major yeasts isolated from high moisture corn and corn silages in the United States using genetic and biochemical methods.

The objective of this study was to identify species of yeasts in samples of high moisture corn (HMC) and corn silage (CS) collected from farms throughout the United States. Samples were plated and colonies were isolated for identification using DNA analysis. Randomly selected colonies were also identified by fatty acid methyl esters (FAME) and by physiological substrate profiling (ID 32C). For CS, Candida ethanolica, Saccharomyces bulderi, Pichia anomala, Kazachstania unispora, and Saccharomyces cerevisiae were the predominant yeasts. Pichia anomala, Issatchenkia orientalis, S. cerevisiae, and Pichia fermentans were the prevalent species in HMC. The 3 identification methods were in agreement at the species level for 16.6% of the isolates and showed no agreement for 25.7%. Agreement in species identification between ID 32C and DNA analysis, FAME and ID 32C, and FAME and DNA analysis was 41.1, 14.4, and 2.2%, respectively. Pichia anomala and I. orientalis were able to grow on lactic acid, whereas S. cerevisiae metabolized sugars (galactose, sucrose, and glucose) but failed to use lactic acid. The yeast diversity in CS and HMC varied due to type of feed and location. Differences in species assignments were seen among methods, but identification using substrate profiling generally corresponded with that based on DNA analysis. These findings provide information about the species that may be expected in silages, and this knowledge may lead to interventions that control unwanted yeasts.

Factors affecting the numbers of expected viable lactic acid bacteria in inoculant applicator tanks.

The application of correct numbers of viable microorganisms to forages at the time of ensiling is one of the most important factors affecting the probability of a beneficial effect from an inoculant. The objective of this study was to determine relationships between numbers of expected lactic acid bacteria (LAB) from silage inoculants in application tanks and various factors that might affect their viability. The pH and temperature of inoculant-water mixes were measured in applicator tanks (n=53) on farms in Wisconsin, Minnesota, South Dakota, and California during the corn harvest season of 2012. Samples were collected on-farm and plated on de Man, Rogosa, and Sharpe agar to enumerate LAB and establish the number of viable LAB (cfu/mL). Expected numbers of LAB were calculated from the minimum label guarantees for viable bacteria and mixing rates with water. In addition, the pH of the inoculant-water mixes at sampling, the ambient temperature at sampling, and the length of time that the samples had been in the tank were measured and obtained. The log difference between the measured and expected numbers of LAB was calculated and expressed as ΔM - E in log scale. Ambient temperature at sampling had no relationship with time in the tank or ΔM - E. Most (83%) of the inoculants had been mixed with water in the applicator tanks for <10h. For these samples, a negative linear correlation (R2=0.36) existed between time that the inoculant-water mixes were in the applicators tanks and ΔM - E. The pH of the inoculant-water mixes was also negatively correlated (R2=0.28) with time in the applicator tank, but pH was not related to ΔM - E. The temperatures of the inoculant-water mixtures were negatively correlated with ΔM - E (R2=0.39). Seven of 8 samples whose ΔM - E were at least -0.95 or more lower than expected (equivalent of about 1 or more log concentration less than expected) had water temperatures above 35°C. These data support our previous laboratory findings and suggest that high temperatures of inoculant-water mixes have the potential to negatively affect the final application rate of some inoculants, which may affect their overall effectiveness to improve silage fermentation.

Short communication: The effects of dry matter and length of storage on the composition and nutritive value of alfalfa silage.

During the ensiling of feeds, various processes result in chemical changes that can affect their ultimate nutritive value at feed out. The primary objective of this study was to evaluate the effect of prolonged ensiling times on potential changes in in vitro digestibility of neutral detergent fiber (NDF-D) of alfalfa ensiled at about 33% [low dry matter (DM), LDM] or 45% (high DM, HDM) whole-plant DM. Alfalfa from the same field (direct chopped or wilted) was chopped with a conventional forage harvester set for a theoretical length of cut of 0.95 cm and ensiled in mini silos for 45, 180, 270, and 360 d. Fresh forages and silages were analyzed for nutrient content, fermentation end-products, and 30-h NDF-D. The pH of the fresh forages ranged from 6.1 to 6.2 and decreased to approximately 4.7 and 4.3 in HDM and LDM silages, respectively. Production of acids and alcohols were less in HDM compared with LDM as expected. Concentrations of soluble protein and NH3-N also increased with time of storage as expected but soluble protein was greater, whereas NH3-N was lower in HDM compared with LDM silage. The effect of length of storage and DM on hemicellulose and NDF concentrations were very small, whereas DM content at harvest tended to slightly increase the concentration of acid detergent fiber in HDM compared with LDM up to 270 d of storage. The NDF-D was greater in fresh forage compared with corresponding silages. However, time of storage between 45 and 360 d had no effect on the NDF-D of alfalfa silage, regardless of DM concentration at ensiling.

Risk Factors Associated With Mammalian Target of Rapamycin Inhibitor Withdrawal in Kidney Transplant Recipients.

BACKGROUND: Mammalian target of rapamycin inhibitors (mTORi) play an essential role as novel immunosuppressive agents in kidney transplantation (KT). Treatment cessation usually occurs after adverse effects occur. We investigated the risk factors associated with withdrawal of mTORi in KT recipients and evaluated the outcomes related to the withdrawal. METHODS: The study enrolled KT recipients being followed up in a medical center in southern Taiwan from January 1999 through December 2014. RESULTS: Risk factors associated with mTORi withdrawal were initial proteinuria level, higher initial serum creatinine level posttransplantation, and history of glomerulonephritis as the primary etiology of renal failure. mTORi withdrawal was associated with increased risk of graft failure (hazard ratio [HR], 9.97 [95% confidence interval (CI), 1.03-96.8]; P = .047). Higher body mass index (HR, 11.2 [95% CI, 1.63-76.6]; P = .01) and tacrolimus usage (HR, 8.30 [95% CI, 1.14-60.7]; P = .037) were associated with increased risk of new-onset diabetes after transplantation in mTORi withdrawal groups. CONCLUSIONS: Proteinuria, poor graft function, and primary glomerulonephritis were associated with cessation of mTORi treatment. Earlier identification of these risk factors may prevent further adverse events and optimize transplantation outcomes after mTORi conversion.

The effect of a chemical additive on the fermentation and aerobic stability of high-moisture corn.

The objective of this experiment was to evaluate the effect of a chemical additive on the fermentation and aerobic stability of high-moisture corn (HMC). Ground HMC (~63% dry matter) was untreated, or treated with an additive containing sodium benzoate, potassium sorbate, and sodium nitrite as active ingredients, at 0, 2, 3, or 4 L/t of fresh matter. Laboratory silos (7.5 L) were prepared and ensiled for 21 and 90d (4 silos/treatment per d of ensiling). Small bag silos were prepared for untreated HMC and HMC treated with 4 L/t of the additive and analyzed for nitrate-N and nitrite-N after 0, 3, and 7d of ensiling. The concentration of nitrate-N was similar between these 2 treatments and was below levels considered problematic for ruminants. Nitrite-N was greater in HMC treated with the high level of additive but was also very low for both treatments. Numbers of yeasts were similar among treatments in fresh HMC and decreased substantially after ensiling. Numbers of yeasts were similar among treatments after 21d of ensiling but after 90d they were lower in treated versus untreated HMC. Concentrations of organic acids (lactic, acetic, and propionic) and pH were not different among treatments at any time of ensiling. In contrast, treatment with the additive markedly decreased the concentration of ethanol in HMC after 21 and 90d when compared with untreated HMC. Treatment with all levels of the additive markedly improved the aerobic stability and improved the recovery of dry matter compared with untreated HMC. Overall, our findings suggest that the chemical additive used in this study has the potential to improve the fermentation and aerobic stability of HMC after a relatively short period (21d) and after a moderate length (90d) of ensiling.

Effects of a spoilage yeast from silage on in vitro ruminal fermentation.

Feeding silages with high concentrations of yeasts from aerobic spoilage is often implicated as a cause of poor animal performance on dairies. Our objective was to determine if a commonly found spoilage yeast, isolated from silage, had the potential to alter in vitro ruminal fermentations. A single colony of Issatchenkia orientalis, isolated from high-moisture corn, was grown in selective medium. The yeast culture was purified and added to in vitro culture tubes containing a total mixed ration (43% concentrate, 43% corn silage, 11% alfalfa haylage, and 3% alfalfa hay on a dry matter basis), buffer, and ruminal fluid to achieve added theoretical final concentrations of 0 (CTR), 4.40 (low yeast; LY), 6.40 (medium yeast; MY), and 8.40 (high yeast; HY) log10 cfu of yeast/mL of in vitro fluid. Seven separate tubes were prepared for each treatment and each time point and incubated for 12 and 24h at 39 °C. At the end of the incubation period, samples were analyzed for pH, yeast number, neutral detergent fiber (NDF) digestibility, volatile fatty acids (VFA), and fatty acids (FA). We found that total viable yeast counts decreased for all treatments in in vitro incubations but were still relatively high (5.3 log10 cfu of yeasts/mL) for HY after 24h of incubation. Addition of HY resulted in a lower pH and higher concentration of total VFA in culture fluid compared with other treatments. Moreover, additions of MY and HY decreased in vitro NDF digestibility compared with CTR, and the effect was greatest for HY. Overall, the biohydrogenation of dietary unsaturated FA was not altered by addition of I. orientalis and decreased over time with an increase in the accumulation of saturated FA, especially palmitic and stearic acids. We conclude that addition of I. orientalis, especially at high levels, has the potential to reduce in vitro NDF digestion and alter other aspects of ruminal fermentations.

Chemical composition and nutritive value of corn silage harvested in the northeastern United States after Tropical Storm Irene.

In the fall of 2011, Hurricane (Tropical Storm) Irene caused significant damage to the forage corn crop in the northeastern United States. Compromised crops were subjected to various degrees of flooding, lodging, and contamination with sediment. The objective of this study was to determine if compromised plants harvested for silage fermented normally and if the nutritive value of these silages was adversely affected. The chemical and nutrient composition of compromised silages was compared with that from silages made from unaffected plants from the same region. The concentration of NEL and in vitro digestibility of NDF were lower in plants compromised by the hurricane. In addition, the ash content of compromised silages was higher than that of unaffected silages. Specifically, concentrations of Al, Co, Fe, and Mn were higher in compromised silages. Overall, silage fermentation appeared to be normal; the final silage pH, and concentrations of fermentation acids, alcohols, and esters were similar between compromised and unaffected silages. Numbers of yeasts (but not molds) tended to be higher in compromised silage than in unaffected silage. Pathogenic microorganisms were not detected in any silage. The incidences and concentrations of mycotoxins were similar between compromised and normal silage. Several farms that fed compromised silage reported subsequent health issues with their animals.

The effect of hybrid type and dietary proportions of corn silage on the lactation performance of high-producing dairy cows.

We evaluated the effects of corn silage hybrids [control vs. brown midrib (BMR)] and the proportion of corn silage in rations on the performance of high-producing dairy cows. The chemical composition of the corn silages was similar except for lignin, which was higher in the control hybrid [3.09%, dry matter (DM) basis] compared with the BMR hybrid (2.19%). The 30-h in vitro neutral detergent fiber (NDF) digestibility was also higher (62.8% of NDF) in the BMR hybrid than in the control hybrid (52.2%). Twenty-seven Holstein cows were fed 1 of 3 diets comprising 62% forage and 38% concentrate (DM basis) containing 35% (DM basis) corn silage from the control hybrid (NLO), 35% of the BMR hybrid (BLO), or 50% of the BMR (BHI). Cows were fed the diets in a replicated 3×3 Latin square design with 28-d periods. Intake of DM was similar among treatments but milk production was greater for cows fed BLO (50.1kg/d) and BHI (51.1kg/d) than for NLO (47.9kg/d). Milk fat percentage was lower for cows fed BHI (3.37%) than for those fed BLO (3.55%) and NLO (3.56%) but yield of milk fat was similar among treatments. Yield and percentage of milk protein was higher for cows in BHI compared with NLO. The concentration of milk urea N was lower in cows fed BHI (14.0mg/dL) than in those fed NLO (14.7mg/dL) and intermediate for BLO (14.5mg/dL). The yield of 3.5% fat-corrected milk was higher in cows fed BLO (50.2kg/d) than in NLO (48.2kg/d) and was intermediate for BHI (49.8kg/d). The total-tract digestibility of dietary DM, organic matter, starch, and crude protein was lower for cows in NLO compared with the other treatments. The total-tract digestibility of NDF was highest for BHI (54.4%), intermediate for BLO (50.9%), and lowest for NLO (43.2%). We conclude that BMR corn silage can be included in rations at moderate and high proportions of a total ration, resulting in high levels of milk production.

Effects of an exogenous protease on the fermentation and nutritive value of corn silage harvested at different dry matter contents and ensiled for various lengths of time.

The objective of this experiment was to evaluate the effects of adding an experimental protease to corn plants harvested at different maturities on silage fermentation and in vitro ruminal starch digestibility (IVSD). Corn plants were harvested at maturities resulting in plants with 31 or 40% dry matter (DM). Plants were chopped, kernel processed, and treated with (1) only a 0.1 M phosphate buffer (pH 5.5, 5% vol/wt of fresh forage), (2) buffer with protease to obtain a final concentration of 20mg of protease/kg of wet forage, and (3) buffer with protease to obtain a final concentration of 2,000 mg of protease/kg of wet forage. Treated forages (about 500 g) were ensiled in nylon-polyethylene pouches and stored between 21 and 23°C for 0, 45, 90, and 150 d. Data were analyzed as a 2 × 3 × 4 factorial arrangement of treatments, with the main effects of harvest DM, dose of protease, days of ensiling, and their interactions. The treatment with the highest dose of protease resulted in more robust fermentations across harvest DM with higher concentrations of lactic and acetic acids compared with untreated silage. Concentrations of soluble protein (% of crude protein) increased with time of ensiling, regardless of DM content at harvest. However, averaged over both harvest DM contents, it increased by 37% for silages treated with the high dose of protease compared with an average 11% increase for untreated silages and silage treated with the low dose of protease, between d 0 and 45. Averaged over both harvest DM contents, the concentration of soluble protein peaked in silages treated with the high dose of protease after 45 d of ensiling, whereas it peaked at d 90 in untreated silages and silage treated with the low dose of protease. Similar changes occurred in the concentration of NH3-N due to length of ensiling and treatment with protease. In fresh forages, the concentration of starch for early- and late-harvested forages was similar, but IVSD was lower in the latter. After 45 d of ensiling, IVSD was highest in both early- and late-harvested silages that were treated with the high level of protease. After 150 d of ensiling, IVSD was similar among silages treated with protease, regardless of DM at harvest. Treating corn plants with a high dose of an experimental protease at harvest accelerated proteolysis during ensiling, resulting in corn silages with levels of IVSD after 45 d of ensiling that were only obtained in untreated corn silages after 150 d of ensiling.

The effect of an exogenous protease on the fermentation and nutritive value of high-moisture corn.

The objective of this study was to determine if treating high-moisture corn at harvest with an exogenous protease could accelerate the increase in in vitro ruminal starch degradation that is normally found with advancing times of ensiling. Ground high-moisture corn (HMC; 73% dry matter) was untreated or treated with an exogenous protease to achieve a final concentration of 2,000 mg of protease/kg of fresh corn. Corn was ensiled in laboratory-scale bags (approximately 500 g) that were evacuated of air, heat-sealed, and stored at 22 to 23°C for 70 and 140 d. Samples of freshly treated corn samples were collected to represent d 0 samples. Treatment of HMC with protease did not affect the gross populations of lactic acid bacteria or yeasts throughout the ensiling period. Treatment of HMC with protease resulted in higher concentrations of lactic acid and ethanol after 70 but not 140 d of ensiling. Concentrations of crude protein, water-soluble carbohydrates, and starch were unaffected by treatment with protease within each sampling day. After 70 or 140 d of ensiling, HMC that was treated with protease had higher concentrations of soluble protein (as a % of crude protein) and NH3-N, and had lower concentrations of prolamin protein, compared with untreated corn. In vitro rumen degradability (7-h incubation) of starch was greater in protease-treated versus untreated corn at all sampling days but the difference was more pronounced after 70 and 140 d compared with d 0. Concentrations of soluble protein and NH3-N were positively correlated with in vitro starch degradation. Conversely, the concentrations of prolamin protein in HMC were negatively correlated with in vitro starch degradation. Treating HMC with an exogenous protease could be a method to obtain greater potential for ruminal starch fermentation after a relatively short period of ensiling.

Effect of exogenous protease enzymes on the fermentation and nutritive value of corn silage.

The objective of this study was to evaluate the effects of adding experimental formulations of exogenous protease enzymes on the fermentation and nutritive value of whole-plant corn ensiled in laboratory silos. Chopped and processed whole-plant corn (36.8% DM) was ensiled without enzymes or treated with 1 of 2 experimental proteases (E85 or E86; AB Vista, Wiltshire, UK) at 20 or 2,000 mg/kg (wet-weight basis). Forages were packed in vacuumed and heat-sealed bags and ensiled for 45 and 150 d at 23±1°C. When compared with untreated silage, addition of proteases and length of ensiling time had no effect on silage pH or concentration of crude protein. The results were similar for the concentrations of acid detergent fiber, neutral detergent fiber, and starch, although protease × time interactions were observed for these components, which were biologically minor. When compared with untreated silages, only treatment with the 2,000-mg/kg application amount of E 0425 resulted in lower neutral detergent fiber after 45 d of ensiling. Proteases did not affect NDF digestibility after 150 d of ensiling when compared with untreated silage. Similarly, treatment with enzymes did not affect the concentrations of lactic and acetic acids or ethanol when compared with untreated silage. Concentrations of NH(3)-N and soluble protein (% of crude protein) increased with storage time compared with concentrations at harvest and were greater for the 2,000-mg/kg doses of proteases when compared with untreated silage at both 45 and 150 d. In vitro ruminal digestibility of starch after 7 h of incubation was 66.3% for freshly chopped corn plants. After 45 d of ensiling, starch digestion was greater for E 0430 applied at the 2,000-mg/kg dose (80.6%) than in all other treatments, with the exception that it was similar to the 2,000-mg/kg dose of E85. After 150 d of ensiling, the 20-mg/kg dose of E 0425 (81.9%), the 2,000-mg/kg dose of E 0425 (82.9%), and the 2,000-mg/kg dose of E 0430 (88.6%) had greater starch digestibility than untreated silage (74.0%). To the best of our knowledge, this is the first study to show that addition of exogenous proteases added to corn forage at the time of harvest can increase in vitro ruminal starch digestibility during silage fermentation. Data suggests that adding exogenous sources of protease enzymes at ensiling may be a method to obtain a high degree of ruminal starch digestibility in corn silage that would normally require longer periods of time to obtain from prolonged storage.

The effects of hybrid, maturity, and length of storage on the composition and nutritive value of corn silage.

The objective of this study was to evaluate the effect of hybrid, maturity at harvest [dry matter (DM) content], and length of storage on the composition and nutritive value of corn silage. The plants used in this study included a normal (NORM) and a brown midrib (BMR) hybrid, harvested at 32 or 41% DM and ensiled for various lengths of time (0 to 360 d) without inoculation. Measurements included nutrient analysis, fermentation end products, in vitro digestion of NDF (NDF-D, 30 h), and in vitro digestion of starch (7h). The concentration of acetic acid increased with length of storage for all treatments, specifically increasing as much as 140% between d 45 to 360 for 32% DM BMR silage. Small changes in lactic acid and ethanol were noted but varied by DM and hybrid. When averaged across maturities and length of storage, compared with NORM, BMR silage was lower in concentrations of lignin, crude protein, neutral detergent fiber, and acid detergent fiber, but higher in starch. On average, NDF-D of both hybrids was not affected by length of storage between 45 and 270 d. The NDF-D was markedly greater for BMR than NORM after all times of storage. Increasing maturity at harvest generally did not affect the NDF-D of NORM, with the exception that it was slightly lower for the more mature plants at 270 and 360 d. In contrast, the NDF-D of BMR was lower in more mature silage by approximately 5 percentage units from 45 to 360 d. The concentration of starch for 32% DM NORM was lower (21%) than other treatments (31±3%; mean±SD) at harvest. This finding was probably the cause for starch digestibility to be highest in 32% DM NORM samples atd 0 (about 80%) and lower (65 to 68%) for other treatments. Concentrations of soluble N and ammonia-N increased with length of storage, indicating that proteolytic mechanisms were active beyond 2 to 3 mo of storage. The in vitro digestion of starch generally increased with length of storage, probably as a result of proteolysis. Although active fermentation occurs for only a relatively short time in the silo, many metabolic processes remain active during long-term storage. Changes in the nutritive value of corn silage during storage should be accounted for during ration formulations.

Effect of physical damage to ears of corn before harvest and treatment with various additives on the concentration of mycotoxins, silage fermentation, and aerobic stability of corn silage.

We studied the effects of damaging ears of corn in the field prior to harvest and the use of various additives on the production of selected mycotoxins, silage fermentation, and aerobic stability of whole plant corn. In experiment 1, ears of corn were undamaged or were slashed with a knife 7 d before harvesting, exposing damaged kernels to the environment. Corn plants were harvested (about 35% DM) and treated in a 2 × 2 factorial arrangement of treatments. Treatments were undamaged or damaged plants, untreated or treated with Lactobacillus buchneri 40788 (400,000 cfu/g of fresh forage) and Pediococcus pentosaceus (100,000 cfu/g). Damaging ears prior to harvest increased the amount of fumonisin but decreased the amount of starch in harvested corn plants. After ensiling, corn silage made from plants damaged before harvest had lower starch but greater concentrations of deoxynivalenol and fumonisin than silage made from plants that were undamaged. Microbial inoculation resulted in fewer yeasts and lower concentrations of zearalenone in silage when compared to uninoculated silage. Inoculated silage also had more acetic acid and 1,2-propanediol than did uninoculated silage. In experiment 2, ears of corn were undamaged or were slashed with a knife 27 d or 9 d before harvesting for corn silage. Whole plants were harvested at about 36% DM in a 2 × 3 factorial arrangement of treatments. Factors were time of damaging the ears (27 d, 9 d, or no damage) relative to harvest and no additive or 0.1% (fresh weight) potassium sorbate. Damaging plants 9 d prior to harvest did not affect the concentrations of deoxynivalenol, fumonisin, and zearalenone in plants at harvest. However, concentrations of deoxynivalenol and fumonisin were increased in fresh forage that had ears damaged at 27 d when compared to corn plants that were undamaged. Corn plants damaged for 27 d prior to harvest also had a lower concentration of starch than corn damaged for 9 d but was higher in acid detergent fiber than other treatments. The addition of potassium sorbate at harvest had no effect on the concentrations of mycotoxins in the resulting silage, but concentrations of mycotoxins were still greatest in silage made from plants that were damaged the longest prior to harvest (27 d). Silages treated with potassium sorbate had fewer yeasts and molds than silages without the additive. Damaging ears of corn before harvest had no effects on the aerobic stability of silages in both experiments. In contrast, the addition of the inoculant and potassium sorbate improved aerobic stabilities of silages when compared to untreated silages. These studies showed that physical damage to ears of corn prior to harvest can result in the production of mycotoxins in the field. This finding suggests that producers should test corn silage for mycotoxins prior to feeding especially if the forage has been subjected to physical damage prior to ensiling.