Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

Effects of direct-fed microbial supplementation on performance and immune parameters of lactating dairy cows.

We evaluated the effects of supplementing bacterial direct-fed microbial (DFM) on performance, apparent total-tract digestibility, rumen fermentation, and immune parameters of lactating dairy cows. One hundred fourteen multiparous Holstein cows (41 ± 7 DIM) were used in a randomized complete block design with an experiment comprising 14 d of a covariate (pre-experimental sample and data collection) and 91 d of an experimental period. Cows were blocked based on energy-corrected milk (ECM) yield during the covariate period and the following treatments were randomly assigned within each block: (1) control (CON), corn silage-based total mixed ration without DFM; (2) PRO-A, basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d; and 3) PRO-B, basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d. Milk yield, dry matter intake (DMI), and body weight were measured daily, while milk samples for component analysis were taken on 2 consecutive days of each week of data collection. Feces, urine, rumen, and blood samples were taken during the covariate period, wk 4, 7, 10, and 13 for estimation of digestibility, N-partitioning, rumen fermentation, plasma nutrient status and immune parameters. Treatments had no effect on DMI and milk yield. Fat-corrected milk (3.5% FCM) and milk fat yield were improved with PRO-B, while milk fat percent and feed efficiency (ECM/DMI) tended to increase with PRO-B compared with PRO-A and CON. Crude fat digestibility was greater with PRO-B compared with CON. Feeding CON and PRO-A resulted in higher total volatile fatty acid concentration relative to PRO-B. Percentage of neutrophils tended to be reduced with PRO-A compared with CON and PRO-B. The mean fluorescence intensity (MFI) of anti-CD44 antibody on granulocytes tended to be higher in PRO-B compared with CON. The MFI of anti-CD62L antibody on CD8+ T cells was lower in PRO-A than PRO-B, with PRO-A also showing a tendency to be lower than CON. This study indicates the potential of DFM to improve fat digestibility with consequential improvement in fat corrected milk yield, feed efficiency and milk fat yield by lactating dairy cows. The study findings also indicate that dietary supplementation with DFM may augment immune parameters or activation of immune cells, including granulocytes and T cells; however, the overall effects on immune parameters are inconclusive.

Silage review: Mycotoxins in silage: Occurrence, effects, prevention, and mitigation.

Ensiled forage, particularly corn silage, is an important component of dairy cow diets worldwide. Forages can be contaminated with several mycotoxins in the field pre-harvest, during storage, or after ensiling during feed-out. Exposure to dietary mycotoxins adversely affects the performance and health of livestock and can compromise human health. Several studies and surveys indicate that ruminants are often exposed to mycotoxins such as aflatoxins, trichothecenes, ochratoxin A, fumonisins, zearalenone, and many other fungal secondary metabolites, via the silage they ingest. Problems associated with mycotoxins in silage can be minimized by preventing fungal growth before and after ensiling. Proper silage management is essential to reduce mycotoxin contamination of dairy cow feeds, and certain mold-inhibiting chemical additives or microbial inoculants can also reduce the contamination levels. Several sequestering agents also can be added to diets to reduce mycotoxin levels, but their efficacy varies with the type and level of mycotoxin contamination. This article gives an overview of the types, prevalence, and levels of mycotoxin contamination in ensiled forages in different countries, and describes their adverse effects on health of ruminants, and effective prevention and mitigation strategies for dairy cow diets. Future research priorities discussed include research efforts to develop silage additives or rumen microbial innocula that degrade mycotoxins.

Silage review: Foodborne pathogens in silage and their mitigation by silage additives.

Silage is one of the main ingredients in dairy cattle diets and it is an important source of nutrients, particularly energy and digestible fiber. Unlike properly made and managed silage, poorly made or contaminated silage can also be a source of pathogenic bacteria that may decrease dairy cow performance, reduce the safety and quality dairy products, and compromise animal and human health. Some of the pathogenic bacteria that are frequently or occasionally associated with silage are enterobacteria, Listeria, Bacillus spp., Clostridium spp., and Salmonella. The symptoms caused by these bacteria in dairy cows vary from mild diarrhea and reduced feed intake by Clostridium spp. to death and abortion by Listeria. Contamination of food products with pathogenic bacteria can cause losses of millions of dollars due to recalls of unsafe foods and decreases in the shelf life of dairy products. The presence of pathogenic bacteria in silage is usually due to contamination or poor management during the fermentation, aerobic exposure, or feed-out stages. Silage additives and inoculants can improve the safety of silage as well as the fermentation, nutrient recovery, quality, and shelf life. This review summarizes the literature on the main foodborne pathogens that occasionally infest silage and how additives can improve silage safety.

Effects of homolactic bacterial inoculant on the performance of lactating dairy cows.

The objective of this experiment was to determine the effect of applying a homofermentative bacterial inoculant to corn silage on the performance of dairy cows. After harvesting, corn forage was treated with nothing (CON) or with an inoculant containing a mixture of Lactococcus lactis, Lactobacillus plantarum, and Enterococcus faecium at 1.5 × 105 cfu/g of fresh forage (MC; SiloSolve MC, Chr. Hansen A/S, Hørsholm, Denmark). After 186 d of storage in Ag-Bags (A Miller-St. Nazianz Inc., St. Nazianz, WI), silages were fed as part of a total mixed ration containing 55% concentrates, 10% alfalfa hay, and 35% CON or MC corn silage. Sixty early-lactation Holstein dairy cows (30 multiparous and 30 primiparous) housed in a freestall barn with Calan gates (American Calan Inc., Northwood, NH) were assigned to the dietary treatments from 20 to 100 d in milk. Silage inoculated with MC had a more homofermentative pattern evidenced by greater lactic acid concentration (3.83 vs. 4.48% of DM) and lower concentrations of acetic (2.34 vs. 1.68% of DM) and propionic (0.37 vs. 0.10% of DM) acids and ammonia (9.11 vs. 7.82% of N) for CON and MC, respectively. Dry matter intake (23.1 vs. 23.2 kg/d) did not differ among treatments, but the MC silage had greater apparent digestibility of DM (68.8 vs. 70.8%), which led to greater yields of milk (37.7 vs. 38.5 kg/d), fat-corrected milk (37.6 vs. 38.4 kg/d), milk fat (1.30 vs. 1.33 kg/d), and lactose (1.83 vs. 1.92 kg/d) for CON and MC cows, respectively. Milk from cows fed MC silage had higher lactose (4.86 vs. 4.93%), lower protein (2.93 vs. 2.83%), and similar contents of fat (3.47 vs. 3.44%) compared with CON cows. Feed efficiency (fat-corrected milk/dry matter intake) was not affected by treatment (1.69 vs. 1.72 for CON and MC, respectively). Inoculation of corn silage with the homofermentative inoculant increased digestibility of the total mixed ration and increased milk yield by lactating dairy cows.

Maternal nutritional restriction during late gestation impairs development of the reproductive organs in both male and female lambs.

Maternal nutritional restrictions during late gestation could lead to fetal hypoglycemia. Glucose levels in the fetal sheep regulate circulating insulin-like growth factor 1 (IGF1) levels, which stimulate cell proliferation and differentiation of reproductive organs after binding to its own receptor or estrogen receptors. The objective of this study was to determine the effects of subnutrition of ewes during the last trimester of gestation on the serum glucose/IGF1 levels and development of reproductive organs in their lambs. Pregnant ewes carrying singletons were randomly assigned to restricted (R ewes, n = 8) or control (C ewes, n = 8) groups (4 lambs of each gender/group) and fed with 50% or 100% of metabolic energy requirements from ∼100 days of gestation to term (∼147 days), respectively. Blood samples from lambs were taken on the first day after born and once at week for serum glucose and IGF1 determination. Lambs were euthanatized at 2 months of age, reproductive organs were weighted and tissue samples were collected from them for histology and to measure mRNA expression of IGF1 and its receptor (IGF1R) by qRT-PCR. Pre-partum glucose levels in R ewes were significantly lower compared to C ewes (p < .05). Compared to lambs born from C ewes, lambs born from R ewes showed lower serum levels of glucose and IGF1 during the first week of age (p < .05). At 2 month of age, these lambs had significant lower uterine and testicular weight and lower ovarian, uterine and testicular mRNA expressions of IGF1 and its receptor (p < .05). Histological findings showed that diameter of secondary and tertiary follicles in ovaries and number of endometrial glands in uterus of females, or number of Sertoli cells and seminiferous tubules and diameter, perimeter and tubular area in testicles of males were significantly lower in lambs born from R ewes compared to the respective organs of lambs born from the C ewes (p < .05). In conclusion, these results demonstrate that maternal subnutrition during late gestation affects IGF1 levels during fetal life and impairs reproductive development in the neonatal lamb, which could have permanent negative consequences in the future reproductive performance of the offspring.

Effects of maturity at ensiling of bermudagrass and fibrolytic enzyme application on the performance of early-lactation dairy cows.

The objective of this study was to examine effects of adding fibrolytic enzymes to diets containing bermudagrass ensiled after 4 or 7wk of regrowth on the diet digestibility, ruminal fermentation and performance of lactating cows, and the interaction of the treatments. In experiment 1, 64 Holstein cows (22±4d in milk) were assigned to an experiment with a 2×2 factorial treatment arrangement and a 56-d duration. Treatments were diets containing 4 or 7wk regrowth bermudagrass silage without or with an exogenous fibrolytic enzyme cocktail. The cellulase-xylanase enzyme was applied at 2.33g/kg of total mixed ration dry matter (DM) during mixing immediately before feeding. Experiment 2 was aimed at examining treatment effects on the ruminal fermentation profile. Four ruminally cannulated cows were assigned to the 4 treatments using a 4×4 Latin square design with 14-d periods. No enzyme by maturity interaction was detected for any measurement. Regardless of forage maturity, applying the fibrolytic enzyme did not affect DM intake, milk yield, apparent digestibility, feed efficiency, energy balance, and ruminal fermentation though it tended to increase milk lactose concentration (4.88 vs. 4.81%). Feeding the 4-wk diet instead of the 7-wk diet increased DM intake (22.4 vs. 21.3kg/d), digestibility of DM, neutral detergent fiber, and acid detergent fiber, and tended to increase 3.5%-fat corrected milk yield (47.2 vs. 44.3kg/d) and milk fat yield (1.88 vs. 1.73kg/d). Therefore, daily intake of net energy and secretion of milk energy were greater for the 4-wk diet. In addition, the 4-wk diet increased the ruminal concentrations of acetate, propionate, valerate, lactate, and total volatile fatty acids, and decreased ruminal pH, without affecting the acetate:propionate ratio. Feeding fibrolytic enzymes did not improve the performance of early-lactation dairy cows, but harvesting the forage earlier tended to improve milk production.

Effect of microbial inoculants on the quality and aerobic stability of bermudagrass round-bale haylage.

The objective of this study was to compare the efficacy of using 4 commercially available microbial inoculants to improve the fermentation and aerobic stability of bermudagrass haylage. We hypothesized that the microbial inoculants would increase the fermentation and aerobic stability of the haylages. Bermudagrass (4-wk regrowth) was harvested and treated with (1) deionized water (control); (2) Buchneri 500 (B500; Lallemand Animal Nutrition, Milwaukee, WI) containing 1×10(5) of Pediococcus pentosaceus and 4×10(5) of Lactobacillus buchneri 40788; (3) Biotal Plus II (BPII; Lallemand Animal Nutrition) containing 1.2×10(5) of P. pentosaceus and Propionibacteria freudenreichii; (4) Silage Inoculant II (SI; AgriKing Inc., Fulton, IL) containing 1×10(5) of Lactobacillus plantarum and P. pentosaceus; and (5) Silo King (SK; AgriKing Inc.), containing 1×10(5) of L. plantarum, Enterococcus faecium, and P. pentosaceus, respectively. Forty round bales (8 per treatment; 441±26kg; 1.2×1.2 m diameter) were made and each was wrapped with 7 layers of plastic. Twenty bales were stored for 112 d and the remaining 20 were stored for 30 d and sampled by coring after intermediary storage periods of 0, 3, 7, and 30 d. The pH of control and inoculated haylages sampled on d 3 did not differ. However, B500 and BPII had lower pH (5.77±0.04 vs. 6.16±0.04; 5.06±0.13 vs. 5.52±0.13) than other treatments by d 7 and 30, respectively. At final bale opening on d 112, all treatments had lower pH than the control haylage (4.77±0.07 vs. 5.37±0.07). The B500, BPII, and SI haylages had greater lactic acid and lactic-to-acetic acid ratios than SK and control haylages. No differences were detected in neutral detergent fiber digestibility, dry matter losses, dry matter, lactic and acetic acid concentrations, and yeast and coliform counts. The SK haylage had lower clostridia counts compared with the control (1.19±0.23 vs. 1.99±0.23 cfu/g). Treatments B500, BPII, SI, and SK tended to reduce mold counts and they improved aerobic stability by 236, 197, 188, and 95%, respectively, compared with the control (276±22 vs. 99±22h).

Effects of 8 chemical and bacterial additives on the quality of corn silage.

This project aimed to evaluate the effects 8 additives on the fermentation, dry matter (DM) losses, nutritive value, and aerobic stability of corn silage. Corn forage harvested at 31% DM was chopped (10mm) and treated with (1) deionized water (control); (2) Buchneri 500 (BUC; 1×10(5) cfu/g of Pediococcus pentosaceus 12455 and 4×10(5) cfu/g of Lactobacillus buchneri 40788; Lallemand Animal Nutrition, Milwaukee, WI); (3) sodium benzoate (BEN; 0.1% of fresh forage); (4) Silage Savor acid mixture (SAV: 0.1% of fresh forage; Kemin Industries Inc., Des Moines, IA); (5) 1×10(6) cfu/g of Acetobacter pasteurianus-ATCC 9323; (6) 1×10(6) cfu/g of Gluconobacter oxydans-ATCC 621; (7) Ecosyl 200T (1×10(5) cfu/g of Lactobacillus plantarum MTD/1; Ecosyl Products Inc., Byron, IL); (8) Silo-King WS (1.5×10(5) cfu/g of L. plantarum, P. pentosaceus and Enterococcus faecium; Agri-King, Fulton, IL); and (9) Biomax 5 (BIO; 1×10(5) cfu/g of L. plantarum PA-28 and K-270; Chr. Hansen Animal Health and Nutrition, Milwaukee, WI). Treated forage was ensiled in quadruplicate in mini silos at a density of 172 kg of DM/m(3) for 3 and 120 d. After 3 d of ensiling, the pH of all silages was below 4 but ethanol concentrations were least in BEN silage (2.03 vs. 3.24% DM) and lactic acid was greatest in SAV silage (2.97 vs. 2.51% DM). Among 120-d silages, additives did not affect DM recovery (mean=89.8% ± 2.27) or in vitro DM digestibility (mean=71.5% ± 0.63). The SAV silage had greater ammonia-N (0.85 g/kg of DM) and butyric acid (0.22 vs. 0.0% DM) than other treatments. In contrast, BEN and Silo-King silages had the least ammonia-N concentration and had no butyric acid. The BEN and A. pasteurianus silages had the lowest pH (3.69) and BEN silage had the least ethanol (1.04% DM) and ammonia nitrogen (0.64 g/kg DM) concentrations, suggesting that fermentation was more extensive and protein degradation was less in BEN silages. The BUC and BIO silages had greater acetic acid concentrations than control silages (3.19 and 3.19 vs. 2.78% DM), but yeast counts did not differ. Aerobic stability was increased by 64% by BUC (44.30 h) and by 35% by BEN (36.49 h), but other silages had similar values (27.0±1.13 h).

Fibrolytic enzyme and ammonia application effects on the nutritive value, intake, and digestion kinetics of bermudagrass hay in beef cattle.

The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass (Cynodon dactylon cultivar Coastal) hay harvested after 2 maturities (5- and 13-wk regrowths). Six individually housed, ruminally cannulated Brangus steers (BW 325 ± 10 kg) were used in an experiment with a 6 × 6 Latin square design with a 3 (additives) × 2 (maturities) factorial arrangement of treatments. Each period consisted of 14 d of adaptation and 7, 4, 1, 1, and 4 d for measuring in vivo digestibility, in situ degradability, no measurements, rumen liquid fermentation and passage indices, and rate of solid passage, respectively. Steers were fed hay for ad libitum intake and supplemented with sugarcane molasses and distillers grain (supplement total of 2.88 kg DM/d). Enzyme did not affect the nutritional composition of hay but ammonia treatment decreased hay NDF, hemicellulose, and ADL concentrations and increased the CP concentration particularly for the mature lignified 13-wk hay. The enzyme increased NDF and hemicellulose digestibility of the 5-wk hay but decreased those of the 13-wk hay. Ammoniation decreased intake of hay but increased digestibility of DM, OM, NDF, hemicellulose, ADF, and cellulose and increased the ruminal in situ soluble and potentially digestible fractions and the rate of DM degradation of the 13-wk hay. Also, ammoniation increased the concentrations of ruminal NH3, total VFA, acetate, and butyrate but enzyme treatment did not. Neither enzyme addition nor ammoniation affected rate of liquid and solid passage. In conclusion, ammoniation decreased the concentration of most fiber fractions, decreased the intake of hays, and increased their CP concentration, in vivo digestibility, and in situ degradability at both maturities whereas enzyme application increased fiber digestibility of the 5-wk hay but decreased it in the case of the 13-wk hay.

Effect of treatment with a mixture of bacteria and fibrolytic enzymes on the quality and safety of corn silage infested with different levels of rust.

This project aimed to determine if a dual-purpose bacterial inoculant could mitigate potential adverse effects of increasing levels of rust infestation on the quality, aerobic stability, and safety of corn silage. Corn plants with no rust infestation (NR), or medium (all leaves on the lower half of the plant affected, MR), or high (all leaves affected, HR) levels of southern rust infestation were harvested at random locations on a field, chopped, and ensiled without (control, CON) or with a dual-purpose inoculant applied at a rate that supplied 1×10(5) cfu/g of Pediococcus pentosaceus 12455 and 4×10(5) cfu/g of Lactobacillus buchneri 40788. Each treatment was prepared in quadruplicate in 20-L mini silos and ensiled for 97 d. As the level of rust infestation increased, the concentrations of dry matter (DM) and neutral detergent fiber increased, whereas DM digestibility decreased by up to 16%. Control HR silages also had lower 24-h neutral detergent fiber digestibility (NDFD; 36.2% of DM) than CON MR (39.8%) or NR silages (38.1%). Inoculation increased the NDFD of NR (43.4%) and MR silages (45.7%) but not HR silages (33.0%). Concentrations of lactate and volatile fatty acids decreased with increasing rust infestation in CON silages, but this trend was absent in inoculated silages. In HR silages, inoculation increased aerobic stability by 75% (77.3 vs. 44 h), and prevented production of aflatoxin (5.2 vs. 0 mg/kg). The concentration of aflatoxin in uninoculated HR silages exceeded action levels stipulated by the US Food and Drug Administration. In conclusion, increasing rust infestation was associated with reductions in the nutritive value and fermentation of corn silage. Inoculation reduced adverse effects of rust infestation on the fermentation, increased 24-h NDFD of NR and MR silages, and decreased aerobic spoilage and aflatoxin production in HR silages.

Effect of adding a mycotoxin-sequestering agent on milk aflatoxin M1 concentration and the performance and immune response of dairy cattle fed an aflatoxin B1-contaminated diet.

This project aimed to examine the effects of adding 2 doses of a montmorillonite-based mycotoxin adsorbent on milk aflatoxin M(1) (AFM(1)) concentrations and the performance and innate immune response of dairy cows fed an aflatoxin B(1) (AFB(1))-contaminated diet. Eight lactating cows were used in a duplicated 4×4 Latin square design with 12-d periods. Treatments included the following: (1) control diet (C), (2) aflatoxin diet (T) containing C and 75 µg of AFB(1)/kg, 3) low-clay (LC) diet containing T and Calibrin A (Amlan International, Chicago, IL) added at 0.2% of the diet dry matter (DM), and 4) high-clay diet (HC) containing T and Calibrin A added at 1% of the diet DM. Milk production and DM intake were recorded daily and milk was sampled twice daily on d 5, 9, 10, 11, and 12 in each period. Blood samples were collected on d 5 and 9 of each period. Dietary treatments did not affect DM intake, milk yield, or feed efficiency. Even though cows were limit fed, feeding T instead of C reduced milk fat yield (0.67 vs. 0.74 kg/d) and milk protein concentration (3.28 vs. 3.36%). Concentrations of AFM(1) in milk of cows fed the T and LC diets were similar (0.57 and 0.64 µg/kg) and greater than those of cows fed the HC diet (0.46 µg/kg). Haptoglobin concentration was greater (22.0 vs. 14.4) and ß(2)-integrin expression (220 vs. 131) tended to be greater in cows fed diet T instead of C, but values for cows fed LC, HC, and C did not differ. In comparison to C, feeding T increased the innate immune response and decreased milk fat yield and milk protein concentration, but feeding LC and HC did not affect these measures. Only the HC diet reduced milk AFM(1) concentration.

Effect of a dual-purpose inoculant on the quality and nutrient losses from corn silage produced in farm-scale silos.

This project aimed to determine effects of applying an inoculant containing homofermentative and heterofermentative bacteria on the fermentation, nutritive value, aerobic stability, and nutrient losses from corn silage produced in farm-scale silos. Corn forage was harvested at 34% dry matter (DM) and treated without (control) or with 5 × 105 cfu/g of Lactobacillus buchneri and Pediococcus pentosaceus. The inoculant was sprayed on alternate 8-row-wide swaths of forage, and the untreated and inoculated forages were alternately packed into 3.6-m-wide bag silos. Forty-five tonnes of corn forage were packed into each of 4 replicate bags per treatment and ensiled for 166 d. Silage removed from the bags (500 kg/d) was separated into good and spoiled (visibly moldy or darker) silage portions, and weighed for 35 d. Weekly composites were analyzed for chemical composition, aerobic stability, and fungal counts. Aerobic stability was measured using data loggers that recorded sample and ambient temperature every 30 min for 7 d. Inoculation did not affect the chemical composition of the spoiled or good silage but decreased the quantity (5.7 vs. 12.9 kg/d) and percentage (3.4 vs. 7.8) of spoiled silage in the bags by over 50%. Losses of crude protein (0.28 vs. 0.92 kg/d), gross energy (6.0 × 104 vs. 1.8 × 105 kJ/d), and neutral detergent fiber (1.34 vs. 4.12 kg/d) in spoiled silage were less in inoculated versus control silages. Inoculated silages had lower pH (3.91 vs. 3.99), lactate concentration (7.63 vs. 7.86%), lactate:acetate ratio (1.58 vs. 2.53%), and a greater acetate (5.11 vs. 3.56%) concentration than the control silage. Inoculated silages tended to have fewer yeasts (2.59 vs. 4.62 log cfu/g) than control silages, but aerobic stability was not different across treatments (14.7 vs. 9.5 h). Applying the inoculant made the fermentation more heterolactic, inhibited the growth of yeasts, and substantially reduced the amount of spoilage and the associated energy and nutrient losses.

Control of Escherichia coli O157:H7 in corn silage with or without various inoculants: efficacy and mode of action.

This study aimed to evaluate the effectiveness of 3 commercial bacterial inoculants at controlling Escherichia coli O157:H7 in corn silages during ensiling and feedout phases of silage production. A second objective was to determine whether the inoculants exhibited and transferred antibacterial activity against E. coli O157:H7 to the silages. Chopped corn forage was ensiled after treatment with the following: distilled water (control); 5 x 10(5) cfu/g of E. coli O157:H7 (EC); EC and 1 x 10(6) cfu/g of Pediococcus pentosaceus and Propionibacterium freudenreichii (EC+BII); EC and 1 x 10(6) cfu/g of Lactobacillus buchneri (EC+LB); and EC and 1 x 10(6) cfu/g of L. buchneri and P. pentosaceus (EC+B500). Each treatment was ensiled in triplicate in mini silos for 3, 7, 31, and 82 d and analyzed for pH and E. coli O157:H7 counts. Samples from d 82 were also analyzed for volatile fatty acids, lactate, and aerobic stability. Antibacterial activity of inoculants and silages was determined by the Kirby-Bauer disc diffusion test. The pH of silages from all treatments decreased below 4 within 3 d of ensiling and remained low until d 82. Therefore, E. coli O157:H7 was not detected in silages after any of the ensiling durations. Applying inoculants containing L. buchneri resulted in less lactate, more acetate, and greater aerobic stability compared with the control. Applying EC+BII containing P. freudenreichii did not increase propionate or aerobic stability. Subsamples of d 82 silages were reinoculated with 1 x 10(5) cfu/g of E. coli O157:H7 either immediately after silo opening on d 82 or after 144h of aerobic exposure (d 88), and E. coli were enumerated 24h later. All silages reinoculated with the pathogen on d 82 had similar, low pH values (<4) and no E. coli were detected 24h later. Control, EC, and EC+BII silages reinoculated with the pathogen after 144h of aerobic exposure had relatively greater pH values (4.71, 5.67, and 6.03, respectively) and E. coli counts (2.87, 6.73, and 6.87 log cfu/g, respectively) 24h later, whereas those treated with L. buchneri had low pH values (<4) and undetectable (EC+B500) or 10-fold lower (1.97, cfu/g; EC+LB) E. coli counts. All pure cultures of commercial bacterial inoculants exhibited antibacterial activity independent of pH against E. coli O157:H7, but the pH-independent activity did not persist in the treated silages, suggesting that E. coli elimination from silages was mediated by pH reduction.

Multiple levels in the control of rhythms in enzyme synthesis and activity by circadian clocks: recent trends.

It is now well established that circadian clocks can control rhythmicity at different stages of the sequence of events leading from gene activity to a functional enzyme molecule. Conceptual and experimental distinction of the effective control targets is emphasized, with particular attention to recent results obtained on circadian clock control of transcription and to data indicating that proteins can behave as conformational oscillators. Thus circadian rhythmicity both in gene expression and in the dynamics of the enzyme molecule would contribute to the temporal compartmentation of processes such as metabolic co-ordination and channeling necessary for the adaptive efficiency of physiological programmes.

Phytochrome-mediated responses of cells and protoplasts of green calli obtained from the leaves of a CAM plant.

Green callus obtained from leaves of the CAM-inducible plant Kalanchoe blossfeldiana cv. Montezuma has previously been shown to perform C3-type photosynthesis under 16-h days and to shift to crassulacean acid metabolism (CAM) under 9-h days. The utilization of photoperiodic regimes (i.e. night interruptions by 30 min red light) established that CAM induction in the callus was under the control of phytochrome, as shown by measurements of CAM criteria: phosphoenolpyruvate carboxylase activity and malic acid pools. Short-term responsiveness of the callus cells to phytochrome modulations by monochromatic radiations was also established by the rapid changes observed in the diameter of the callus-derived protoplasts. These results provide further evidence that whole plant correlations are not necessary for phytochrome operativity.

Are spontaneous conformational interconversions a molecular basis for long-period oscillations in enzyme activity?

An unconventional hypothesis to the molecular basis of enzyme rhythms is that the intrinsic physical instability of the protein molecules which, in an aqueous medium, tend to move continuously from one conformational state to another could lead, in the population of enzyme molecules, to sizeable long-period oscillations in affinity for substrate and sensitivity to ligands and regulatory effects. To investigate this hypothesis, malate dehydrogenase was extracted and purified from leaves of the plant Kalanchoe blossfeldiana. The enzyme solutions were maintained under constant conditions and sampled at regular intervals for up to 40 or 70 h for measurements of activity as a function of substrate concentration, Km for oxaloacetic acid and sensitivity to the action of 2,3-butanedione, a modifier of active site arginyl residues. The results show that continuous slow oscillations in the catalytic capacity of the enzyme occur in all the extracts checked, together with fluctuations in Km. Apparent circadian periodicities were observed in accordance with previous data established during long run (100 h) experiments. The saturation curves for substrate showed multiple kinetic functions, with various pronounced intermediary plateaus and "bumps" depending on the time of sampling. Variation in the response to the effect of butanedione indicated fluctuation in the accessibility to the active site. Taken together, the results suggest that, under constant conditions, the enzyme in solution shifts continuously and reversibly between different configurations. This was confirmed by parallel studies on the proton-NMR spectrum of water aggregates in the enzyme solution and proton exchange rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism.

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using (14)C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by (14)CO2 fumigations and NaH(14)CO3 injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO2 in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.

Phosphorylation-dephosphorylation process as a probable mechanism for the diurnal regulatory changes of phosphoenolpyruvate carboxylase in CAM plants.

Day and night forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) were extracted from leaves of the CAM plants Kalanchoe daigremontiana, K. tubiflora and K. blossfeldiana previously fed with [32P] labelled phosphate solution. A one-step immunochemical purification followed by SDS polyacrylamide gel electrophoresis and autoradiography showed that, in all species, the night form of the enzyme was phosphorylated and not the day form. Limited acid hydrolysis of the night form and two-dimensional separation identified predominantly labelled phosphoserine and phosphothreonine. In vitro addition of exogenous acid phosphatase (EC 3.1.3.2) to desalted night form-containing extracts resulted within 30 min in a shift in PEPC enzymic properties similar to the in vivo changes from night to day form. It is suggested that phosphorylation-dephosphorylation of the enzyme could be the primary in vivo process which might explain the observed rhythmicity of enzymic properties.