Concentration profiles of putrescine and cadaverine in inoculated vacuum packed beef

The potential of some Lactobacillus, Staphylococcus and Streptococcus strains for producing putrescine and cadavering during growth in vacuum packed beef was studied. Throughout the six weeks storage at 4 degrees C the samples were sensorily assessed at regular intervals, and their pH values and diamine contents were determined. The amines were quantified by means of capillary gas chromatography. The putrescine and cadaverine contents remained within the range of the initial values until overt spoilage at the end of the sixth week (at about 0.08 mg amine/100 g).     

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.     

Formation of cadaverine, histamine, putrescine and tyramine by bacteria isolated from meat, fermented sausages and cheeses

Enterobacteriaceae (EB, n = 149), Lactobacillus (LB, n = 162) and Leuconostoc sp. (LC, n = 89) and enterococci (EC, n = 137), isolated from raw meat (n = 65), fermented sausages (n = 50) and cheese (n = 55) samples, were cultivated in a broth containing precursor amino acids (each 3 g/l). After incubation, the liquid culture was chemically analysed for cadaverine (CAD), putrescine (PUT), histamine (HIS) and tyramine (TYR) formation at pH 5.2 and at pH 6.7. The majority of EB isolates (147 of 149) was capable of forming >100 mg/l of either CAD or PUT. Among the most frequently isolated species Hafnia alvei and Serratia liquefaciens, formation of >100 mg/l HIS occurred, but with low prevalence (1.6 and 6.5%, respectively). Twelve of 149 isolates (8%) were able to produce more than 10 mg/l HIS. One hundred forty-two isolates (95.3%) produced less than 10 mg/l TYR, and 7 isolates (4.7%) 10 mg/l to a maximum of 35.3 mg/l TYR. For LB + LC, one isolate (Leuconostoc mesenteroides ssp. mesenteroides) formed >100 mg/l PUT and one >100 mg/l CAD (of all 251 LB + LC isolates 0.4% each). Formation of >100 mg/l HIS and TYR was detected in 3.6 and 19% of the LB + LC isolates, respectively. For the EC isolates, maximum levels for PUT, CAD and HIS were 25.4 mg/l, 6.0 mg/l and 15.7 mg/l, respectively. TYR was formed in quantities of 100–1000 mg/l by 47.9% of EC faecalis (n = 75), and 59.7% of EC faecium (n = 62) isolates. More than 1000 mg/l TYR were formed by 50.7 and 35.5% of the isolates, respectively. A low initial pH of 5.2 compared to the initial pH of 6.7 favoured tyramine production by lactic acid bacteria, but was associated with lower CAD yield by EB. A considerable intra-species variability in amine formation was observed.     

The effect of ripening and storage conditions on the distribution of tyramine,putrescine and cadaverine in Edam-cheese

The aim of the work was to describe the development of selected biogenic amines (histamine, tyramine, putrescine and cadaverine) in 4 layers of Dutch-type cheese (Edam-cheese) depending on 3 ripening/storage regimes during a 98-day period. Biogenic amines were analysed by means of ion-exchange chromatography. A further goal was to identify microbial sources of biogenic amines in the material analysed. Phenotype characterization and repetitive sequence-based PCR fingerprinting were used to identify the isolated bacteria. The highest content of tyramine, putrescine and cadaverine was determined in cheeses stored in a ripening cellar at a temperature of 10 °C during the whole observation period. Lower biogenic amines content was determined in samples which were moved into a cold storage device (5 °C) after 38 days of storage in a ripening cellar (10 °C). The lowest concentrations of biogenic amines were detected in cheeses which were moved into a cold storage device (5 °C) after 23 days of storage in a ripening cellar (10 °C). During the 98-day period, histamine was not detected in any of the regimes. Within the cheeses analysed, non-starter lactic acid bacteria Lactobacillus curvatus, Lactobacillus casei/paracasei and Lactobacillus plantarum were detected as the main producers of the biogenic amines tested. In starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris the decarboxylase activity tested was not detected.     

Dietary exposure assessment of putrescine and cadaverine and derivation of tolerable levels in selected foods consumed in Austria

Biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermidine and spermine) are nitrogenous compounds. They occur naturally in living organisms and are involved in many biological processes. Nonetheless, high amounts in food may be hazardous to human health. The diamines putrescine and cadaverine in food can potentiate the effects of simultaneously ingested histamine. In protein-rich foods, high concentrations of these diamines are indicative for hygienic deficiencies in the food chain. Even though being formed endogenously and being essential for some physiological metabolic pathways, both diamines are known as precursors for carcinogenic nitrosamines. Putrescine also plays a certain role in tumour growth. Nevertheless, no tolerable levels in foods have been established so far. The present study suggests tolerable levels in cheese, fermented sausages, fish, sauerkraut and seasonings that are based on toxicological threshold levels, occurrence of diamines in foods and food consumption in Austria. Average daily intake of putrescine via fermented food was calculated to be 6.8 (female adults) and 8.8 (male adults) mg per person. Respective numbers for cadaverine were 9.8 and 11.6 mg per person and day. For putrescine, proposed maximum tolerable levels for sauerkraut, fish, cheese, fermented sausages and seasonings are 140, 170, 180, 360 and 510 mg/kg, respectively. Likewise, for cadaverine, in sauerkraut, fish, cheese, fermented sausages and seasonings, maximum tolerable levels are 430, 510, 540, 1,080 and 1,540 mg/kg, respectively. These limits can be met by current manufacturing practices, as ascertained from the results of our own studies and from literature. Admittedly, only few data are published on toxicological threshold levels of these diamines, which mean that these tolerable levels are associated with some uncertainty.     

Influence of putrescine,cadaverine, spermidine or spermine on the formation of N-nitrosamine in heated cured pork meat

The influence of biogenic amines (i.e. putrescine, cadaverine, spermidine and spermine) on the N-nitrosamine formation in heated cured lean meat was studied in the presence or absence of sodium nitrite and at different meat processing temperatures. Experimental evidence was produced using gas chromatography with thermal energy analysis detection (GC–TEA). Concentration of N-nitrosamines was modelled as a function of the temperature and the nitrite concentration for two situations, i.e. presence or absence of added biogenic amines to the meat. The significance of the influence of the changing parameters was evaluated by ANOVA (Analysis of Variance).     

A screening method for the simultaneous determination of putrescine, cadaverine, histamine, spermidine and spermine in fish by means of high pressure liquid chromatography of their 5-dimethylaminonaphthalene-l-sulphonyl derivatives

Summary A simple and rapid method is described for the extraction, derivatization and subsequent determination by means of high pressure liquid chromatography of putrescine, cadaverine, histamine, spermidine and spermine as their 5-dimethylaminonaphthalene-1-sulphonyl derivatives.The amines are extracted from the fish material by an aqueous trichloroacetic acid solution and derivatized by means of 5-dimethylaminonaphthalene-1-sulphonylchloride (dansyl chloride). The reaction mixture is then injected on a RP-8 column using gradient elution to separate the amine derivatives. Use of the method results in a considerable saving with respect to both time and costs.

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Eine Screening-Methode für die gleichzeitige Bestimmung von Putrescin, Cadaverin, Histamin, Spermidin und Spermin im Fisch mittels Hochdruck-Flüssigchromatographie ihrer 5-Dimethylaminonaphthalin-Sulfonyl-Derivate

Zusammenfassung Eine einfache und schnelle Methode für die Extraktion, Derivatisierung und Bestimmung von Putrescin, Cadaverin, Histamin, Spermidin und Spermin als 5-Dimethylaminonaphthalin-1-sulfonyl-Derivate mit Hilfe der Hochdruck-Flüssigchromatographie (HPLC).Die Amine werden mit einer wäßrigen Trichloressigsäure-Lösung aus dem Fisch extrahiert und mit Hilfe von 5-Dimethylaminonaphthalin-1-sulfonyl (Dansylchloride) derivatisiert. Die Reaktionsmischung wird auf eine RP-8-Säule injiziert und durch Gradientelution getrennt. Die beschriebene Methode bring einen beträchtlichen Zeitgewinn und eine Verringerung der Analysenkosten.

     

Aerobic deterioration in maize silage stored in a bunker silo under farm conditions in a subtropical climate

Whole maize plants at the milk and dough maturation stages were ensiled in August and October 1984, for periods of 45 and 40 days, respectively. Thirteen net bags containing herbage samples were buried at different locations within the silo to determine changes and losses during the conservation period. Thermocouple wires and small-diameter plastic pipes were connected to every bag for temperature and gas measurements. Air penetration in the upper layer was much faster than in the inner parts. Losses in dry matter of 3.9-7.4% were found in well sealed sites in the bunkers, and of up to 36% at locations where air penetrated. The rate of air penetration into the silage and temperature at various sites in the bunker were recorded. Correlation between CO₂, and N₂, contents in the silage was very high (r² = 0.995) whereas oxygen levels were close to zero.     

Low permeability to oxygen of a new barrier film prevents butyric acid bacteria spore formation in farm corn silage

The outgrowth of Clostridium spore-forming bacteria causes late blowing in cheeses. Recently, the role of air diffusion during storage and feed-out and the role of aerobic deterioration has been shown to indirectly favor butyric acid bacteria (BAB) growth and to determine the presence of high concentrations of BAB spores in farm tank milk. A new oxygen barrier (OB) film was tested and compared with conventional polyethylene (ST). The objective was to verify whether the OB film could prevent BAB spore formation in whole-crop corn silage during storage on 2 commercial farms with different potential silage spoilage risks. Two bunkers (farms 1 and 2) were divided into 2 parts along the length so that half the feed-out face would be covered with ST film and the other half with OB film. Plastic net bags with freshly chopped corn were buried in the upper layer and in the central part (CORE) of the bunkers. The silos were opened in summer and fed out at different removal rates (19 vs. 33 cm/d). Herbage at ensiling, silage at unloading, and silage after air exposure (6 and 15 d) were analyzed for pH, nitrate, BAB spores, yeasts, and molds. The BAB spores in herbages at ensiling were 2.84 log₁₀ most probable number (MPN)/g, with no differences between treatments or farms. Nitrate was below the detection limit on farm 1 and exceeded 2,300 mg/kg of fresh matter on farm 2. At unloading, the BAB spores in the ST silage on farm 1 were greater than 5 log₁₀ MPN/g, whereas in the CORE and the OB silages, they were approximately 2 log₁₀ MPN/g. The ST silage had the greatest pH (5.89), the greatest mold count (5.07 log₁₀ cfu/g), and the greatest difference between silage temperature and ambient temperature (dT_{section-ambient}). On farm 2, the ST silage had the greatest concentration of BAB spores (2.19 log₁₀ MPN/g), the greatest pH (4.05), and the least nitrate concentration compared with the CORE and the OB silages. Pooled data on BAB spores collected from aerobically deteriorated samples showed a positive relationship with pH, mold count, and dT_{section-ambient} and a negative relationship with nitrate concentration. A high concentration of BAB spores (>5 log MPN/g) was associated with visible spoilage, high pH values (>5.00), high mold counts (>5 log cfu/g), high dT_{section-ambient}, and nitrate below 1,000 mg/kg of fresh matter. We concluded that the use of a film with reduced oxygen permeability prevented the outgrowth of BAB spores during conservation and feed-out, and it could improve the microbiological quality of corn silage by eliminating the fractions of silage with high BAB spore concentrations.     

Effect of Lactobacillus buchneri LN4637 and Lactobacillus buchneri LN40177 on the aerobic stability, fermentation products, and microbial populations of corn silage under farm conditions

This study determined the efficacy of the use of 2 commercial inoculants containing Lactobacillus buchneri alone or in combination with homofermentative lactic acid bacteria in improving aerobic stability of corn silage stored in commercial farm silos in northern Italy. In the first survey, samples were collected from 10 farms that did not inoculate their silages and from 10 farms that applied a Pioneer 11A44 inoculant (L. buchneri strain LN4637; Pioneer Hi-Bred International, Des Moines, IA). In the second survey, corn silage samples were collected from 11 farms that did not inoculate their silages and from 11 farms that applied a Pioneer 11CFT inoculant (L. buchneri strain LN40177; Pioneer Hi-Bred International). Inoculants were applied directly through self-propelled forage harvesters, at the recommended rate of 1 g/t of fresh forage, to achieve a final application rate of 1.0 × 10⁵ cfu/g of L. buchneri. One corn bunker silo, which had been open for at least 10 d, was examined in detail on each farm. The silages inoculated with L. buchneri had lower concentrations of lactic acid, a lower lactic-to-acetic acid ratio, a lower yeast count, and higher aerobic stability compared with the untreated silages. Unexpectedly, concentrations of acetic acid and 1,2-propanediol, 2 hallmarks of L. buchneri activity, did not differ between treatments and were only numerically higher in the inoculated silages compared with untreated ones, in both surveys. Aerobic stability, on average, was 107 and 121 h in the inoculated silages and 64 and 74 h in the untreated silages, for surveys 1 and 2, respectively, and decreased exponentially as the yeast count in the silage at the time of sampling increased, regardless of treatment. Inoculation with L. buchneri proved to be effective in reducing the yeast count to <2 log cfu/g of silage in 16 of 21 of the studied farm silages, confirming the ability of this inoculum to enhance the aerobic stability of corn silages in farm bunker silos.     

Dairy farm management practices and the risk of contamination of tank milk from Clostridium spp. and Paenibacillus spp. spores in silage,total mixed ration,dairy cow feces,and raw milk

The occurrence of Paenibacillus and Clostridium spores in silage is of great concern for dairy producers because their spores can contaminate milk and damage processed milk and semi-hard cheeses. Spoiled silage is considered to be the main contamination source of the total mixed ration (TMR), feces of dairy cows, and consequently bulk tank milk via the contamination of cow teats by dirt during milking. The presence of an anaerobic and facultative anaerobic sporeformer population in different matrices (soil, corn silage, other feeds, TMR, feces, and milk) and its transmission pathway has been studied on 49 dairy farms by coupling plate count data with 16S-DNA identification. The different matrices have shown a high variability in the anaerobic and facultative anaerobic spore count, with the highest values being found in the aerobically deteriorated areas of corn silages. Clostridium tyrobutyricum, Paenibacillus macerans, and Paenibacillus thermophilus were detected in all the matrices. The TMR spore count was influenced by the amount of spoiled corn silage in the TMR and by the care taken when cleaning the spoiled silage before feed-out. Most of the farms that prevent the presence of visible moldy silage in the silo and carefully clean to remove molded spots were able to maintain their TMR spore counts below 4.0 log spores/g. When a level of 4.5 log spores/g of TMR was exceeded, the feces presented a greater contamination than 3.0 log spores/g. Moreover, the higher the number of spores in the feces was, the higher the number of spores in the milk. Most of the farms that presented a feces contamination greater than 5.0 log spores/g had a higher milk spore contamination than 1,000 spores/L. Careful animal cleaning and good milking practices have been found to be essential to maintain low levels of contamination in bulk tank milk, but it has emerged that only by coupling these practices with a correct silage management and cleaning during TMR preparation can the contamination of milk by spores be kept at a low level. It has been found that aerobically deteriorated silage has a great capacity to contaminate TMR and consequently to increase the risk of milk spore contamination, even when routine milking practices are adopted correctly.     

Chromatographic analysis of cadaverine to detect incipient spoilage in mutton

Suitability of measuring cadaverine for rapid assessment of bacterial quality of fresh and stale mutton (showing signs of incipient spoilage) was examined. HPTLC for rapid screening and detection and HPLC for accurate quantification was employed. Analysis of over 150 samples of varying degrees of freshness and postmortem age showed that in more than 90% of the samples, no cadaverine was detectable when meat was deemed acceptable with <10⁶ orgs/g. However, when meat showed incipient spoilage, as judged by off odour and dull colour, cadaverine was detected in the range of 1–5 ppm. Such meat samples had a bacterial load of 10⁷ orgs / g or more. HPTLC analysis allowed the simultaneous analysis of 5–6 samples and the total time including extraction was 1.5–2 h. As only easily available chemicals and equipment were required, it was a simple, rapid and inexpensive technique. HPTLC detection of cadaverine can therefore be recommended for the rapid assessment of bacterial quality of mutton and HPLC can be used for accurate quantification.     

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.     

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.     

Performance of lactating dairy cows fed ryegrass silage and corn silage with ground corn, steam-flaked corn, or hominy feed

Forty-eight mid-lactation Holstein cows were used in a 6-wk completely randomized block design trial with a 4 × 3 factorial arrangement of treatments to determine the effects of feeding different proportions of corn silage and ryegrass silage with supplemental ground corn (GC), steam-flaked corn (SFC), and hominy feed (HF) on the performance of lactating dairy cows. Forage provided 49% of the dietary dry matter in the experimental diets, which were formulated to meet National Research Council requirements. Ryegrass silage provided 100, 75, 50, or 25% of the total forage dry matter, with corn silage supplying the remainder. There were no interactions between the proportion of forage provided by ryegrass silage and energy supplement. Dry matter intake and milk protein percentage decreased linearly with increasing proportions of ryegrass silage, but milk protein yield was similar among forage treatments. There were no differences among forage treatments in milk yield, milk fat percentage and yield, and energy-corrected milk yield. Dry matter intake was higher and there was a tendency for increased milk fat percentage for GC compared with SFC or HF. No other differences were observed in milk yield or composition among energy supplements. Plasma urea nitrogen and glucose concentrations were similar among treatments. Under the conditions of this trial, our results indicate that feeding a combination of corn silage and ryegrass silage is more desirable than feeding ryegrass silage alone, whereas supplementation with GC, SFC, or HF supports similar levels of milk production.     

Effect of Lactobacillus hilgardii,Lactobacillus buchneri,or their combination on the fermentation and nutritive value of sorghum silage and corn silage

The objective of this study was to determine the effect of inoculation with Lactobacillus hilgardii with or without Lactobacillus buchneri on the fermentation, chemical composition, and aerobic stability of sorghum and corn silage after 2 ensiling durations. Sorghum forage was harvested at 27% dry matter (DM; experiment 1), and different corn hybrids were harvested at late (43.8% DM; experiment 2) or normal maturity (34% DM; experiment 3). All harvested forages were chopped and ensiled in quadruplicate in vacuum-sealed nylon-polyethylene bags (40 × 61 cm) for 30 and 90 d after treatment with (1) deionized water (uninoculated) or (2) L. buchneri (1.5 × 10⁵ cfu/g of fresh weight; LB); (3) L. hilgardii (1.5 × 10⁵ cfu/g of fresh weight; LH); or (4) L. buchneri and L. hilgardii (1.5 × 10⁵ cfu/g of fresh weight of each inoculant). Data for each experiment were analyzed separately accounting for the 2 × 2 × 2 factorial treatment arrangement. Inoculating sorghum forage with LB or LH separately increased acetate and 1,2 propanediol concentration, tended to increase DM loss, reduced lactate concentration and the lactate-to-acetate ratio, and increased aerobic stability after 90 but not after 30 d of ensiling. Inoculating late-harvested corn silage with LB or LH separately increased and decreased DM loss, respectively, increased 1,2 propanediol concentration, reduced lactate-to-acetate ratio and yeast counts but did not affect aerobic stability. Inoculating normal-harvested corn silage with LH reduced DM loss and increased 1,2 propanediol concentration and yeast counts; LB reduced lactate concentration, lactate-to-acetate ratio, and total acids. Either inoculant alone increased aerobic stability after 30 or 90 d. The main benefit of combining LB with LH was prevention of increases in DM losses by LH or LB separately. No improvement in aerobic stability resulted from applying LH instead of LB separately or from combining them. Application of LB or LH separately improved aerobic stability of sorghum silage after 90 d and normal-harvested corn silage after 30 or 90 d but did not affect that of late-harvested corn silage.     

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.     

Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition in dairy cows fed alfalfa silage or corn silage

Four Holstein cows fitted with ruminal cannulas were used in a 4 × 4 Latin square design (28-d periods) with a 2 × 2 factorial arrangement of treatments to investigate the effects of addition of a specific mixture of essential oil compounds (MEO; 0 vs. 750 mg/d) and silage source [alfalfa silage (AS) vs. corn silage (CS)] on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition. Total mixed rations containing either AS or CS as the sole forage source were balanced to be isocaloric and isonitrogenous. In general, no interactions between MEO addition and silage source were observed. Except for ruminal pH and milk lactose content, which were increased by MEO supplementation, no changes attributable to the administration of MEO were observed for feed intake, nutrient digestibility, end-products of ruminal fermentation, microbial counts, and milk performance. Dry matter intake and milk production were not affected by replacing AS with CS in the diet. However, cows fed CS-based diets produced milk with lower fat and higher protein and urea N concentrations than cows fed AS-based diets. Replacing AS with CS increased the concentration of NH₃-N and reduced the acetate-to-propionate ratio in ruminal fluid. Total viable bacteria, cellulolytic bacteria, and protozoa were not influenced by MEO supplementation, but the total viable bacteria count was higher with CS- than with AS-based diets. The apparent digestibility of crude protein did not differ between the AS and CS treatments, but digestibilities of neutral detergent fiber and acid detergent fiber were lower when cows were fed CS-based diets than when they were fed AS-based diets. Duodenal bacterial N flow, estimated using urinary purine derivatives and the amount of N retained, increased in cows fed CS-based diets compared with those fed AS-based diets. Feeding cows AS increased the milk fat contents of cis-9, trans-11 18:2 (conjugated linoleic acid) and 18:3 (n-3 fatty acid) compared with feeding cows CS. Results from this study showed limited effects of MEO supplementation on nutrient utilization, ruminal fermentation, and milk performance when cows were fed diets containing either AS or CS as the sole forage source.