Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed.

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Review: Feed residues of glyphosate - potential consequences for livestock health and productivity.

Glyphosate is the active ingredient in a wide range of herbicides used for weed control, including weed control in genetically modified, glyphosate-insensitive crops. In addition, glyphosate herbicides are used for pre-harvest desiccation of glyphosate-sensitive crops. Together, the use of glyphosate leads to residues in livestock feed. In addition to its herbicidal property, glyphosate has documented antimicrobial and mineral-chelating properties. The aim of the present paper is to address, based on the published literature and own observations, whether dietary glyphosate residues may affect livestock gut microbiota and/or mineral status potentially with derived unfavourable effects on animal health and productivity. However, and as reported, literature on the potential effects of glyphosate on livestock is very scarce and mainly reporting in vitro studies; hence, a solid basis of in vivo studies with livestock in physiological and productive phases, particularly sensitive to disorders in mineral status and in the gut microbiota, is needed for drawing final conclusions.

Effect of dried oregano (Origanum vulgare L.) plant material in feed on methane production, rumen fermentation, nutrient digestibility, and milk fatty acid composition in dairy cows.

Essential oils (EO) from oregano may have antimicrobial properties, potentially representing a methane mitigation strategy suitable for organic production. This study aimed to (1) examine the potential of oregano in lowering enteric methane production of dairy cows fed differing levels of dried oregano (Origanum vulgare ssp. hirtum) plant material containing high levels of EO; (2) determine whether differing levels of dried oregano plant material of another subspecies (Origanum vulgare ssp. vulgare) with naturally low levels of EO in feed affected enteric methane production; and (3) evaluate the effect of various levels of the 2 oregano subspecies (containing high or low levels of EO) in feed on rumen fermentation, nutrient digestibility, and milk fatty acids. Each experiment had a 4 × 4 Latin square design using 4 lactating Danish Holstein dairy cows that had rumen, duodenal, and ileal cannulas and were fed 4 different levels of oregano. Experiment 1 used low EO oregano [0.12% EO of oregano dry matter (DM)] and evaluated a control (C) diet with no oregano and 3 oregano diets with 18 (low; L), 36 (medium; M), and 53 g of oregano DM/kg of dietary DM (high; H). Experiment 2 used high EO oregano (4.21% EO of oregano DM) with 0, 7, 14, and 21 g of oregano DM/kg of dietary DM for C, L, M, and H, respectively. Oregano was added to the diets by substituting grass/clover silage on a DM basis. Low or high EO oregano in feed did not affect dry matter intake (DMI) or methane production (grams per day, grams per kilogram of DMI, grams per kilogram of energy-corrected milk, and percentage of gross energy intake). Rumen fermentation was slightly affected by diet in experiment 1, but was not affected by diet in experiment 2. In both experiments, the apparent total-tract digestibility of DM, organic matter, and neutral detergent fiber decreased linearly and cubically (a cubic response was not observed for neutral detergent fiber) with increasing dietary oregano content, while milk fatty acids were slightly affected. In conclusion, dried oregano plant material with either high or low levels of EO did not lower the methane production of dairy cows over 4 consecutive days, and no substantial effects were observed on rumen fermentation or nutrient digestibility. This conclusion regarding methane production is in contrast with literature and requires further study.

Methane production, rumen fermentation, and diet digestibility of Holstein and Jersey dairy cows being divergent in residual feed intake and fed at 2 forage-to-concentrate ratios.

Improving feed efficiency of dairy cows through breeding is expected to reduce enteric methane production per unit of milk produced. This study examined the effect of 2 forage-to-concentrate ratios on methane production, rumen fermentation, and nutrient digestibility in Holstein and Jersey dairy cows divergent in residual feed intake (RFI). Before experimental onset, RFI was estimated using a random regression model on phenotypic herd data. Ten lactating Holstein and 10 lactating Jersey cows were extracted from the herd and allocated to a high or low pre-experimental RFI group of 5 animals each within breed. Cows were fed ad libitum with total mixed rations either low (LC) or high (HC) in concentrates during 3 periods in a crossover design with a back-cross and staggered approach. Forage-to-concentrate ratio was 68:32 for LC and 39:61 for HC. Cows adapted to the diets in 12 to 24 d and feces were subsequently collected on 2 d. Afterward, gas exchange was measured in respiration chambers and rumen liquid was collected once after cows exited the chambers. Pre-experimental RFI was included in the statistical analysis as a class (low and high RFI) or continuous variable. Methane per kilogram of dry matter intake (DMI) was lower for Holsteins than Jerseys and the response to increased concentrate level was more pronounced for Holsteins than Jerseys (27.2 vs.13.8%); a similar pattern was found for the acetate:propionate ratio. However, methane production per kilogram of energy-corrected milk (ECM) was unaffected by breed. Further, total-tract digestibility of neutral detergent fiber was higher for Jerseys than Holsteins. For RFI as a class variable, DMI, methane production regardless of the expression, and digestibility were unaffected by RFI. For RFI as a continuous variable, DMI was lower and methane per kilogram of DMI was higher for cows with negative (efficient) than positive (inefficient) RFI values, and neutral detergent fiber digestibility was higher for Holsteins with negative than positive RFI values, but not for Jerseys. Daily methane production and methane per kilogram of ECM were unaffected by RFI. In conclusion, methane per kilogram of DMI of Jerseys was lowered to a smaller extent in response to the HC diet than of Holsteins. When pre-experimental RFI was used as a continuous variable, higher methane per kilogram of DMI was found for cows with negative RFI than positive RFI values, but not for methane per kilogram of ECM. These findings call for validation in larger studies.

Effect of dietary nitrate level on enteric methane production, hydrogen emission, rumen fermentation, and nutrient digestibility in dairy cows.

Nitrate may lower methane production in ruminants by competing with methanogenesis for available hydrogen in the rumen. This study evaluated the effect of 4 levels of dietary nitrate addition on enteric methane production, hydrogen emission, feed intake, rumen fermentation, nutrient digestibility, microbial protein synthesis, and blood methemoglobin. In a 4×4 Latin square design 4 lactating Danish Holstein dairy cows fitted with rumen, duodenal, and ileal cannulas were assigned to 4 calcium ammonium nitrate addition levels: control, low, medium, and high [0, 5.3, 13.6, and 21.1g of nitrate/kg of dry matter (DM), respectively]. Diets were made isonitrogenous by replacing urea. Cows were fed ad libitum and, after a 6-d period of gradual introduction of nitrate, adapted to the corn-silage-based total mixed ration (forage:concentrate ratio 50:50 on DM basis) for 16d before sampling. Digesta content from duodenum, ileum, and feces, and rumen liquid were collected, after which methane production and hydrogen emissions were measured in respiration chambers. Methane production [L/kg of dry matter intake (DMI)] linearly decreased with increasing nitrate concentrations compared with the control, corresponding to a reduction of 6, 13, and 23% for the low, medium, and high diets, respectively. Methane production was lowered with apparent efficiencies (measured methane reduction relative to potential methane reduction) of 82.3, 71.9, and 79.4% for the low, medium, and high diets, respectively. Addition of nitrate increased hydrogen emissions (L/kg of DMI) quadratically by a factor of 2.5, 3.4, and 3.0 (as L/kg of DMI) for the low, medium, and high diets, respectively, compared with the control. Blood methemoglobin levels and nitrate concentrations in milk and urine increased with increasing nitrate intake, but did not constitute a threat for animal health and human food safety. Microbial crude protein synthesis and efficiency were unaffected. Total volatile fatty acid concentration and molar proportions of acetate, butyrate, and propionate were unaffected, whereas molar proportions of formate increased. Milk yield, milk composition, DMI and digestibility of DM, organic matter, crude protein, and neutral detergent fiber in rumen, small intestine, hindgut, and total tract were unaffected by addition of nitrate. In conclusion, nitrate lowered methane production linearly with minor effects on rumen fermentation and no effects on nutrient digestibility.

Dietary Nitrate for Methane Mitigation Leads to Nitrous Oxide Emissions from Dairy Cows.

Nitrate supplements to cattle diets can reduce enteric CH emissions. However, if NO metabolism stimulates NO emissions, the effectiveness of dietary NO for CH mitigation will be reduced. We quantified NO emissions as part of a dairy cow feeding experiment in which urea was substituted in nearly iso-N diets with 0, 5, 14 or 21 g NO kg dry matter (DM). The feeding experiment was a Latin square with repetition of Period 1. Each period lasted 4 wk, with CH emission measurements in Week 4 using respiration chambers. During Period 3, NO concentrations in chamber outlet air were monitored semicontinuously during 48 h. High, but fluctuating, NO concentrations were seen at the two highest NO levels (up to between 2 and 5 µL L), and dynamics were linked with recent feed intake. In Periods 4 and 5, NO concentrations and feed intake were determined from all four respiration chambers during two 7-h periods. Emissions of NO coincided with feed intake, again with NO concentrations in the microliter per liter range at the two highest NO intake levels. Neither feed nor excretion of NO via urine were significant sources of NO, indicating that emissions came from the animals. Leakages due to rumen fistulation could also not account for NO emissions. The possibility that NO is produced in the oral cavity is discussed. Nitrous oxide emission factors ranged between 0.7 and 1.0% except in one case at 21 g NO kg DM, where it was 3.4%. When accounting for NO emissions at the highest NO intake level, the overall GHG mitigation effect in two different animal-diet combinations changed from -47 to -40%, and from -19 to -17%, respectively, due to NO emissions.

Methanogenic community changes, and emissions of methane and other gases, during storage of acidified and untreated pig slurry.

AIMS: Acidification with concentrated H(2)SO(4) is a novel strategy to reduce NH(3) emissions from livestock slurry. It was recently found that also CH(4) emissions from acidified slurry are reduced. This study investigated the microbiological basis and temporal stability of these effects. METHODS AND RESULTS: Pig slurry from two farms, acidified by different techniques or untreated, was stored for 83 days in a pilot-scale facility. Methanogens were characterized before and after storage by T-RFLP and qPCR targeting mcrA. Emissions of NH(3) and CH(4) during storage were quantified. Acidified slurry pH was nearly constant at values of 5·5 and 6·5. Ammonia losses were reduced by 84 and 49%, respectively, while CH(4) emission with both acidification techniques was reduced by >90%. T-RFLP fingerprints showed little effect of acidification or storage time. A major T-RF of 105 bp could represent methanogens related to Thermoplasmata (Tp). No treatment effects on gene copy numbers were seen with universal methanogen primers, whereas effects were found with Tp-specific primers. CONCLUSION: Methane emissions were reduced >90% during storage. Thermoplasmata-related methanogens could be involved in CH(4) emissions from pig slurry. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of acidification on CH(4) emissions during storage of pig slurry was quantified for the first time. Acidification with sulphuric acid holds promise as a novel greenhouse gas mitigation strategy for confined livestock production.

Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract.

The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract.

Lyophilized Carnobacterium divergens AS7 bacteriocin preparation improves performance of broiler chickens challenged with Clostridium perfringens.

The present study aimed to investigate the effects of Carnobacterium divergens AS7 bacteriocin (divercin AS7) on growth performance, digestibility, fermentation processes, selected microbial populations, and histomorphology in broiler chickens challenged with a mixture of 3 Clostridium perfringens isolates. In total, 480 one-day-old male Ross 308 chicks were randomly assigned to 4 experimental groups (12 replicate pens of 10 birds per treatment). The diets were either nonsupplemented or supplemented with a lyophilized preparation of divercin AS7. On d 18, 19, and 20, half of the birds were challenged twice a day with the C. perfringens mixture. The C. perfringens challenge did not influence broiler BW gain but impaired feed conversion ratio from d 29 to 42 (P=0.023) and throughout the experimental period (P=0.038). Moreover, the C. perfringens challenge resulted in decreased pH levels of crop, gizzard, and ileum contents (P<0.05) and reduced the numbers of lactic acid bacteria in the ceca (P=0.01). Divercin supplementation decreased broiler feed intake from d 14 to 28 (P=0.001) but increased BW gain from d 29 to 42 (P=0.048). The divercin supplementation increased the AMEn level (P=0.015) and reduced digesta pH in crop and ileum (P=0.004 and P=0.042, respectively), but of nonchallenged birds only. Divercin supplementation, moreover, increased gizzard lactate concentrations (P=0.003). The crop concentrations of lactate and succinate and the ileum concentration of lactate were increased by divercin supplementation (P=0.005, P=0.027, and P=0.002, respectively) and C. perfringens challenge (P=0.034, P=0.053, and P=0.0002, respectively). Divercin supplementation decreased villus heights (P=0.0006) and crypt depths (P=0.044) in noninfected birds, whereas in challenged birds, villus heights (P<0.0001) were increased. In conclusion, the present study demonstrated a very complex response pattern of broilers exposed to C. perfringens challenge and dietary divercin AS7 supplementation, but it indicated that divercin AS7 may partly counterbalance the negative effects associated with C. perfringens.

Dietary divercin modifies gastrointestinal microbiota and improves growth performance in broiler chickens.

1. The aim of the present study was to investigate the effects of dietary administration of a divercin AS7 liquid preparation on broiler chicken performance, nutrient digestibility, counts of lactic acid bacteria (LAB) and coliform bacteria, as well as on the microbial activity in the gastrointestinal tract (GIT) as expressed by digesta pH and concentrations of short-chain fatty acids and lactic acid. 2. A total of 450 1-d-old male Ross 308 chickens were randomly distributed to three dietary treatments, with 15 pens per treatment and 10 birds per pen. The dietary treatments consisted of a positive control (PC) supplemented with 60 mg/kg salinomycin, a negative control (NC) without any additives, and the divercin (DIV) supplemented diet containing 0 x 2 mL/kg of the liquid divercin AS7 preparation. 3. The dietary divercin AS7 supplementation significantly increased body weight gain at 35 d compared to the NC group. Moreover, the pH of crop contents was higher and that of caecal contents lower in birds fed on the divercin supplemented diets. 4. Significantly lower counts of LAB were observed in the crops and caeca of the birds treated with divercin. Further, the divercin supplementation decreased lactic and succinic acid concentrations in the crop and ileum. 5. The present study demonstrates that the use of divercin supplemented diets can influence composition and activity of the microbiota in the broiler chicken GIT even in the lower parts that should otherwise not be targeted due to the peptide structure of the bacteriocin.

Effect of ß -glucanase and xylanase supplementation of barley- and rye-based diets on caecal microbiota of broiler chickens.

1. The aim was to investigate the effect of grain type (barley or rye) and exogenous enzymes (ß-glucanase or xylanase) on the composition of chicken caecal microbiota as examined by classical culturing and molecular techniques (fluorescent in-situ hybridisation (FISH) and terminal-restriction fragment-length polymorphism (T-RFLP)). 2. Plate counting revealed higher total numbers of anaerobic bacteria, lactic acid bacteria and yeasts in caecal contents of birds fed with rye-based diets than in birds fed with barley-based diets. 3. As assessed by FISH analysis, the most abundant bacterial groups in the broiler caeca were Clostridium coccoides-Eubacterium rectale followed by Bacteroides sp., Lactobacillus sp./Enterococcus sp., Bifidobacterium sp. and Enterobacteriaceae. For both cereal types, the enzyme supplementation significantly decreased the relative amount of Enterobacteriaceae. 4. The T-RFLP profiles indicated that the caecal microbiota of birds receiving rye-based diets was more diverse than that of birds fed on barley-based diets. 5. Irrespective of the method applied, the results indicate that the cereal type as well as the exogenous enzyme supplementation influence the microbiota in broiler chicken caeca, and may have the effect of reducing potentially pathogenic Enterobacteriaceae populations.

Effects of microbial inoculants on corn silage fermentation, microbial contents, aerobic stability, and milk production under field conditions.

The present study aimed to investigate the effects of 2 corn silage inoculation strategies (homofermentative vs. heterofermentative inoculation) under field conditions and to monitor responses in silage variables over the feeding season from January to August. Thirty-nine commercial dairy farms participated in the study. Farms were randomly assigned to 1 of 3 treatments: control (nonactive carrier; Chr. Hansen A/S, Hørsholm, Denmark), Lactisil (inoculation with 1 x 10(5)Lactobacillus pentosus and 2.5 x 10(4)Pediococcus pentosaceus per gram of fresh matter; Chr. Hansen A/S), and Lalsil Fresh (inoculation with 3 x 10(5)Lactobacillus buchneri NCIMB 40788 per gram of fresh matter; Lallemand Animal Nutrition, Blagnac, France). Inoculation with Lactisil had no effects on fermentation variables and aerobic stability. On the contrary, inoculation with Lalsil Fresh doubled the aerobic stability: 37, 38, and 80+/-8h for control, Lactisil, and Lalsil Fresh, respectively. The effect of Lalsil Fresh on aerobic stability tended to differ between sampling times, indicating a reduced difference between treatments in samples collected in April. Lalsil Fresh inoculation increased silage pH and contents of acetic acid, propionic acid, propanol, propyl acetate, 2-butanol, propylene glycol, ammonia, and free AA. The contents and ratios of DL-lactic acid, L-lactic acid relative to DL-lactic acid, free glucose, and DL-lactic acid relative to acetic acid decreased with Lalsil Fresh inoculation. Lalsil Fresh inoculation increased the silage counts of total lactic acid bacteria and reduced yeast counts. The Fusarium toxins deoxynivalenol, nivalenol, and zearalenone were detected in all silages at all collections, but the contents were not affected by ensiling time or by inoculation treatment. The effect of inoculation treatments on milk production was assessed by collecting test-day results from the involved farms and comparing the actual milk production with predicted milk production within farm based on test-day results from 2007 and 2008. The average milk production of lactating cows at test days during the study (January to September 2009) was 30.7+/-0.5 kg of energy-corrected milk/d. Milk production was 104.6+/-0.7% of the predicted yield and did not differ among treatments. In conclusion, the present study showed that homofermentative inoculants might not compete efficiently or might not deviate sufficiently from the epiphytic flora on whole-crop corn to affect fermentation in standard qualities of corn silage. Heterofermentative inoculation increased aerobic stability and numerous fermentation variables. None of the treatments affected milk production, and more-stable corn silage seemed to have a similar production value as compared with less-stable homofermented silage. Heterofermented silage can be evaluated for its properties to limit aerobic silage deterioration in the feed chain.

Influence of dietary peas and organic acids and probiotic supplementation on performance and caecal microbial ecology of broiler chickens.

1. The effect of dietary pea and addition of organic acid blend (OA) or probiotic (Pro) on performance and caecal microbial ecology of broiler chickens was studied. 2. A growth trial was conducted with 160 Ross 308 female broilers from d 1 to 35 of age. There were 8 treatment groups based on either control (S) or white pea (P). Both S and P were supplemented with OA (Galliacid - fumaric acid, calcium formate, calcium propionate and potassium sorbate coated with plant triglycerides, Vetagro) and or with Pro (LABYuc-Probio - lactic acid bacteria, Saccharomyces cerevisiae and Yucca schidigeri extract, Mifarmex GmbH). 3. Inclusion of peas in the diet increased feed intake and decreased gain:feed ratio in comparison to the control diet. Neither probiotic nor OA supplementations affected broiler performance. 4. The caecal microbiota was characterised in 37-d-old birds by fluorescent in situ hybridisation (FISH) and terminal-restriction fragment length polymorphism (T-RFLP). Total bacterial counts in caecal contents were slightly higher for birds fed the pea diets, but were not affected by OA or Pro supplements. 5. Neither pea nor Pro affected the Lactobacillus/Enterococcus and Streptococcus/Lactococcus counts in caecal contents, whereas OA supplementation slightly increased the Lactobacillus/Enterococcus counts. The composition of the Lactobacillus/Enterococcus population was altered by inclusion of peas as revealed by the T-RFLP patterns. 6. The DNA fingerprint further suggested that the caecal microbiota was dominated by the lactic acid bacterium Streptococcus alactolyticus. 7. In ileal contents, the concentration of short-chain fatty acids (SCFA) was decreased only by Pro supplementation. In caecal contents, the SCFA concentration was higher for birds fed on the pea diets, and increased significantly with Pro supplementation 8. In conclusion, the results indicate that the use of pea and probiotics in broiler feed may stimulate the caecal commensal microbiota (growth and/or activity) to some extent and hence prevent establishment of pathogenic and zoonotic enterobacteria in these segments of the gut.

Growth inhibition of bacterial isolates recovered from two types of Portuguese dry smoked sausages (chouriço).

Potassium sorbate (PS), sodium benzoate (SB) and methyl p-hydroxybenzoate (MHB) were investigated as surface treatments for their ability to inhibit the growth of 18 isolates of spoilage and pathogenic bacteria from two types of Portuguese dry smoked sausages (Chouriço). MHB significantly inhibited the growth rate of 12 of the isolates (p<0.05) whereas no effect was observed for four isolates of lactic acid bacteria, identified as Enterococcusfaecium, Pediococcusacidilactici and Lactobacilluscurvatus, and two isolates identified as Clostridiumaminovalericum and Staphylococcusepidermidis. PS and SB had less influence on the bacterial growth rates. It was concluded that MHB can be applied as surface treatment to improve the stability and safety of the product along shelf life period in modified atmosphere package.

Mycoflora of two types of Portuguese dry-smoked sausages and inhibitory effect of sodium benzoate, potassium sorbate, and methyl p-hydroxybenzoate on mold growth rate.

The mycoflora of chouriqo types Alentejano and Ribatejano, two varieties of Portuguese dry-smoked sausages, have been investigated after a producer-defined shelf life period (120 days at 20 +/- 5 degrees C) in modified atmosphere packaging (55% N2 and 45% CO2). On the basis of morphological and physiological characteristics, the isolates were identified as Penicillium, Aspergillus, Fusarium, Rhizopus, Monilia, Absidia, and Cephalosporium. The species identified were as follows: Penicillium terrestres (43.4%), Penicillium sp. (13.3%), Fusarium sp. (10%), Aspergillus glaucus (10%), Aspergillus versicolor (6.8%), Monilia fruticola (3.3%), Absidia sp. (3.3%), Cephalosporium sp. (3.3%), Rhizopus stolonifer (3.3%), and Fusarium tricinctum (3.3%). Additionally, the effects of three preservatives (potassium sorbate [PS], sodium benzoate [SB], and methyl p-hydroxybenzoate [MHB]) were studied on the growth rate of mold representative isolates. MHB showed a greater inhibitory effect than SB and PS in all fungi isolates, except in A. glaucus [Tm30(A)], in which the inhibitory effect of MHB was similar to PS. At 0.05% (wt/vol), all fungi were inhibited with MHB, except for R. stolonifer [Tm25(A)], which started to decrease the growth rate only at a concentration higher than 0.1%. PS was more effective at inhibiting mold growth than SB, except in Absidia sp. [Tm16(R)], in which both showed a similar inhibitory effect. MHB showed great promise as an application to the surface at 0.1% (wt/vol) to improve the stability and safety of the product through the inhibition of potential spoilage and toxigenic molds.

Effect of feeding fermented liquid feed and fermented grain on gastrointestinal ecology and growth performance in piglets.

To investigate the microbial and nutritional characteristics of dry feed, liquid feed containing fermented liquid cereal grains, and fermented liquid feed, and their effect on gastrointestinal ecology and growth performance, 120 piglets from 40 litters were used and housed in pens with 5 animals in each. The 3 dietary treatments (all nonheated and nonpelleted diets) were: a dry meal diet (DRY); a fermented, liquid cereal grain feed (FLG); and a fermented liquid feed (FLF). The FLG diet was prepared by storing the dietary cereals (barley and wheat) and water (1:2.5, wt/wt) in a closed tank at 20 degrees C and adding the remaining dietary ingredients immediately before feeding. The FLF diet was prepared by storing compound feed and water (1:2.5, wt/wt) in a closed tank at 20 degrees C. Three times daily, 50% of the fermented cereals or compound feed and water stored in the tanks was removed and replaced with an equal amount of fresh cereals or feed and water. On d 14, 1 piglet from each pen was killed and samples from the gastrointestinal tract were obtained. The pH of the fermented cereals was 3.85 (SD = 0.10), that of the FLG diet was 5.00 (SD = 0.18), and the pH of the FLF diet was 4.45 (SD = 0.11). The dietary concentration of lysine (g/16 g of N) pointed to a decreased concentration in the FLF (5.46, SD = 0.08) compared with the DRY (6.01) and FLG (6.21, SD = 0.27) diets, and the concentration of cadaverine was greater in the FLF diet (890 mg/kg, SD = 151.3) than in the DRY (32 mg/kg) or FLG (153 mg/kg, SD = 18.7) diets. Fermenting only the cereal component of the diet (FLG) promoted the growth of yeasts to a greater extent than fermenting the whole diet (FLF). Terminal RFLP profiles of diets and digesta from the stomach and midcolon showed differences among dietary groups. The number of yeasts able to grow at 37 degrees C in the stomach and caudal small intestine was greatest in the FLG group compared with the other 2 dietary groups (P < 0.01). In the cecum and colon, the differences were only significant between piglets fed the FLG and the FLF diets (P < 0.05). The greatest number of yeasts able to grow at 20 degrees C was detected in the animals fed the FLG diet. However, the values were different from the FLF-fed piglets only in the stomach (P < 0.05) and midcolon (P < 0.05). There was a tendency (P < 0.10) for greater ADG of the piglets fed the FLG compared with the FLF diet. Feeding liquid feed containing fermented, liquid cereal grains as a means of avoiding microbial decarboxylation of free amino acids in the feed and increasing feed intake by improving palatability seems promising but requires further investigation.

Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs.

The effect of feeding a coarsely ground meal (COARSE) and a finely ground pelleted diet with 1.8% (as-fed basis) added formic acid (ACID) was compared with feeding a standard finely ground pelleted diet (STD) on the gastrointestinal ecology of growing pigs at different intervals after feeding. One hundred five castrated male growing-finishing pigs (initial BW 27 kg) were used. At a BW of 63 kg, 60 pigs were killed 0.5, 2.5, 4.5, 6.5, and 8.5 h after feeding, and samples from the gastrointestinal tract (GIT) were obtained. The remaining 45 pigs were kept on the experimental diets to a BW of 99 kg. Feeding the three diets resulted in a similar pattern of gastric pH with time, (i.e., highest pH values 0.5 h after feeding and decreasing values at the following sampling times, to reach a value of 2.12 at 8.5 h after feeding). The pH of the gastric digesta of pigs fed the ACID diet was below 4 at all sampling times, whereas the digesta from the other two dietary groups had values above pH 4 at the first sampling times. Feeding the ACID diet decreased the counts of total anaerobes in the proximal GIT (P < or = 0.007), and of lactic acid bacteria (P < or = 0.001), enterobacteria (P < or = 0.02), and yeasts (P < or = 0.01) along the GIT compared with feeding the other two diets. Feeding the COARSE diet stimulated the growth of total anaerobes and lactic acid bacteria in the stomach and distal small intestine increased the microbial diversity mainly in the stomach (P = 0.001), compared with feeding the other two diets (P < or = 0.09), and decreased the number of enterobacteria in the cecum compared with the STD diet (P = 0.03), with the same tendency in the mid-colon (P = 0.07). The concentration of lactic acid in the stomach was highest in the pigs fed the COARSE diet compared with the other two groups (P < 0.05). The concentration of formic acid was highest in the stomach and all segments of the small intestine of the pigs fed the ACID diet compared with those fed the STD and COARSE diets (P < 0.05). The results from this study suggest that feeding a coarsely ground diet and a finely ground diet with added formic acid affect the gastrointestinal ecology of pigs mainly by changing the environment in the proximal GIT. The presence of organic acids in the proximal GIT is a crucial factor contributing to the decrease in the number of enterobacteria along the GIT. The time after feeding at which samples are taken to measure characteristics describing the gastrointestinal ecology affects the results from the stomach and small intestine.

Carbon limitation induces sigma(S)-dependent gene expression in Pseudomonas fluorescens in soil.

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the sigma(S)-dependent Escherichia coli promoter P(fic) and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57(pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of beta-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing beta-galactosidase from P(fic). Changes in cell size and expression of beta-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of beta-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In straw-amended soil beta-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.

Characterisation of the gastrointestinal bacterial community in pigs fed fermented liquid feed and dry feed: composition and fermentation capacity (phenotypic fingerprint).

Feeding pigs with fermented liquid feed (FLF) has been shown to reduce the number of enteropathogens such as Salmonella and Brachyospira hyodysenteriae as well as coliform bacteria in general in the gastrointestinal tract (GIT). Also the commensal bacterial populations have been shown to respond to the use of FLF, e.g. the total counts of anaerobes, including lactic acid bacteria are reduced. In the present work we demonstrate that the capacity to ferment a series of substrates (mainly low-molecular weight carbohydrates) is reduced in caecum, colon and faeces of pigs fed FLF compared to pigs fed dry feed. This reduction could be due to the fact that these substrates are partially depleted by fermentation in the liquid feed prior to entering the animal. Therefore nutrient availability may be limited in the large intestine of pigs fed FLF, which may again affect bacterial enzyme synthesis and growth and thus the possibility for pathogenic and zoonotic bacteria to establish.

Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil.

The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.