Effects of the methane-inhibitors nitrate, nitroethane, lauric acid, Lauricidin and the Hawaiian marine algae Chaetoceros on ruminal fermentation in vitro.

The effects of several methane-inhibitors on rumen fermentation were compared during three 24h consecutive batch cultures of ruminal microbes in the presence of nonlimiting amounts of hydrogen. After the initial incubation series, methane production was reduced greater than 92% from that of non-treated controls (25.8+/-8.1 micromol ml(-1) incubation fluid) in cultures treated with nitroethane, sodium laurate, Lauricidin or a finely-ground product of the marine algae, Chaetoceros (added at 1, 5, 5 and 10 mg ml(-1), respectively) but not in cultures treated with sodium nitrate (1 mg m1(-1)). Methane production during two successive incubations was reduced greater than 98% from controls (22.5+/-3.2 and 23.5+/-7.9 micromol ml(-1), respectively) by all treatments. Reductions in amounts of volatile fatty acids and ammonia produced and amounts of hexose fermented, when observed, were most severe in sodium laurate-treated cultures. These results demonstrate that all tested compounds inhibited ruminal methane production in our in vitro system but their effects on fermentation differed.

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.

PCR detection and 16S rRNA sequence-based phylogeny of a novel Propionibacterium acidipropionici applicable for enhanced fermentation of high moisture corn.

AIMS: The aims of this study were to develop a sensitive and more rapid detection of Propionibacterium acidipropionici DH42 in silage and rumen fluid samples, and to explore its 16S rRNA sequence-based phylogeny. METHODS AND RESULTS: Nested polymerase chain reaction (PCR) was used with DH42-specific primers dhb1 and dhb2 for the secondary amplification of a 1267-bp fragment of 16S rRNA encoding gene. Using the established protocols for PCR amplification, as low as 10(2) and 10(3) CFU ml(-1) of strain DH42 in silage extracts and rumen fluid, respectively, were detected. To determine phylogenetic relationships between DH42 and other representatives of Propionibacterineae, a 1529-bp fragment of its 16S rRNA was amplified by PCR and sequenced. The propionibacterium DH42 formed a cluster with Eubacterium combesii, P. acidipropionici and P. microaerophilus. CONCLUSIONS: 16S rRNA-based PCR detection technique was developed for DH42 in silage and rumen fluid samples. The 16S rRNA sequence confirmed the earlier identification of strain DH42 as P. acidipropionici. However, variable nucleotide positions were revealed. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability of 16S rRNA sequence within the species P. acidipropionici, determined in this study, poses the need of re-sequencing for some species of the suborder Propionibacterineae for a more reliable classification.

Toxicity of ozonated animal manure to the house fly, Musca domestica.

Swine manure slurries were ozonated at a dosage of 1 g/L and tested for their toxicity to the house fly (Musca domestica). The observed toxicity of ozonated swine manure was consistent and independent of origin of the swine manure. A dose (dilution) response curve was performed. A 50% dilution in the ozonated swine manure slurry resulted in 90% reduction in toxicity. Neither the synthetic nor ozonated synthetic swine manure, both of which contained higher concentrations of formaldehyde and three other unidentified carbonyl compounds than the ozonated swine manure, were toxic to the flies. Ozonated swine manure slurry was centrifuged and passed through a 0.45-microm filter. The liquid phase was as toxic as the unfiltered slurry; as such, the toxicant appears to be present in liquid phase. Neither ammonia, hydrogen sulfide, formaldehyde, nor other simple aldehydes appeared to be the toxic agent. The toxic agent appears to be a polar chemical compound and is concentrated in the urine. Several possible compounds have been identified. The toxicity of untreated and ozonated manure slurries from different livestock was compared. Six animal manure slurries (beef and dairy cattle, horse, poultry, sheep, and swine) were ozonated (dosage of 1 g/L) and tested for toxicity to the house fly. Ozonated dairy cattle manure slurry showed 78% mortality after 72 h, whereas ozonated swine manure slurry achieved a 100% mortality rate in 48 h. Neither the unozonated dairy nor swine manure slurries, nor any of the other raw or ozonated manure slurries, were toxic to the flies.

Calcium level affects the efficacy of supplemental microbial phytase in corn-soybean meal diets of weanling pigs.

A 2 x 2 x 2 factorial experiment was conducted with 64 pigs (4 wk old, 8.04 +/- .50 kg BW) to determine the effect of various dietary concentrations of Ca, vitamin D, and microbial phytase (Aspergillus niger) on phytate-P utilization. A low-P, corn-soybean meal diet was supplemented with two levels of phytase (unit/gram), 750 (suboptimal) and 1,200 (optimal); of vitamin D (international unit/kilogram), 660 (normal) and 6,660 (high); and of Ca (percentage), .4 (low) and .8 (normal). Pen feed consumption and individual pig weights, plasma inorganic P and Ca concentrations, and plasma alkaline phosphatase (AP) activity were measured at d 10, 20, and 30. The normal dietary Ca concentration had an adverse effect (P < .05) on all the response measures. The depressive effect of the normal dietary Ca on performance was greater (P < .05) at the normal vitamin D level or at the optimal phytase level than at the other levels of these two factors. The elevation in plasma AP activity in pigs fed the normal dietary Ca was greater (P < .05) at the suboptimal than at the optimal phytase level. The decreases in plasma inorganic P concentration and increases in plasma Ca concentration associated with the normal dietary Ca were substantial. In conclusion, the normal level of Ca in the diet greatly reduced the efficacy of supplemental phytase. Raising vitamin D in the diet partially offset this adverse effect but did not produce further improvement when the Ca level was low.

Supplementing corn-soybean meal diets with microbial phytase linearly improves phytate phosphorus utilization by weanling pigs.

Two experiments were conducted with weanling pigs to determine the effectiveness of a dietary supplement of Aspergillus niger phytase in improving the availability of phytate-P in corn-soybean meal diets without supplemental inorganic P. Experiment 1 consisted of two P and Ca balance trials and two feeding trials. Twelve pigs (8.18 +/- .44 kg BW) were housed individually in stainless steel metabolism cages. Six pigs received 750 phytase units (PU)/g of basal diet and the other six pigs received the basal diet without supplemental phytase as control. In Exp. 2, 96 pigs (8.81 +/- .75 kg BW) were allotted to 16 partially slotted floor pens and their basal diets were supplemented with either 0, 250, 500, or 750 PU/g for 4 wk. Individual pig weights and pen feed consumption were measured weekly. Blood samples were taken from all pigs at the end of each trial in Exp. 1 and from three pigs per pen weekly in Exp. 2 to measure serum (plasma) inorganic P (P) and Ca concentrations and alkaline phosphatase (AP) activities. The results of Exp. 1 indicated that dietary phytase increased P retention by 50% (P < .0001) and decreased fecal P excretion by 42% (P < .0001). Pigs that received dietary phytase had serum P and Ca concentrations and serum AP activities that were nearly normal, whereas control pigs had values indicative of a moderate P deficiency. Favorable effects of phytase disappeared when the phytase was removed from the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Supplementing corn-soybean meal diets with microbial phytase maximizes phytate phosphorus utilization by weanling pigs.

Two experiments were conducted with crossbred weanling pigs to determine the optimal dietary supplement of Aspergillus niger phytase activity to a low-P, corn-soybean meal basal diet (BD). In Exp. 1, 50 pigs (7.61 +/- .56 kg BW) received the BD supplemented with 750, 1,050, 1,250, or 1,350 phytase units (PU)/g, or .21% P as mono-dibasic calcium phosphate (MDCaP) for 4 wk. In Exp. 2, 12 pigs (6.39 +/- .74 kg BW) were individually housed in metabolism cages and received BD, BD plus the optimal phytase activity (1,200 PU/g), or BD plus .21% P as MDCaP for 2 wk. In Exp. 1, additions of phytase > 1,050 PU/g of BD did not improve ADG, ADFI, gain/feed, or plasma AP activity. Quadratic relationships between dietary phytase activity and these measures were found and their stationary points were at approximately 1,200 PU/g of BD. Estimated maximum responses of these measures in pigs fed phytase were > or = 90% compared with MDCaP. Pigs fed 1,250 PU/g of BD maintained normal plasma P and Ca concentrations. In Exp. 2, pigs that received 1,200 PU/g of BD utilized dietary P more effectively (P < .05) than pigs fed the BD or the BD plus MDCaP. Although they consumed 44% less P per day, these pigs retained only 7% less P than pigs that received MDCaP. One thousand units of phytase activity supported retention of 1.1 mg of P from the BD, and this level of phytase supplementation was equivalent in effect to .91 mg of P from MDCaP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of microbial inoculation of high-moisture corn on fermentation characteristics, aerobic stability, and cattle performance.

Two trials were conducted to evaluate the effects of microbial inoculation for preservation of high-moisture corn (HMC). In Trial 1, three concrete, upright silos were filled with 83 t of HMC that was treated with either a dry microbial inoculant (DI), reconstituted liquid microbial inoculant (RLI), or an untreated control (CON). The CON or RLI-treated HMC were placed in two silos in Trial 2. Both inoculants were applied to supply 2 x 10(6) colony-forming units (cfu)/g of wet corn. Samples were collected from the bottom two doors of each silo for 21 d (Trial 1) or 40 d (Trial 2) postensiling and during feedout to evaluate fermentation changes over time. Dacron bags buried at four elevations within each silo were recovered during feedout to determine DM losses and chemical composition changes during the fermentation period. In both trials, concentrations of soluble N (SN) increased over time. Acetate content was higher in DI than in CON in Trial 1 and ethanol was greater (P < .05) for RLI than for CON in Trial 2. Acidity declined faster (P < .05) and soluble carbohydrate was reduced for RLI during the feedout phase in Trial 2. Dry matter recovery tended to be greater with CON (P < .10) than with RLI-treated HMC in Trial 2. Aerobic stability was evaluated on corn from silos in Trial 2 by placement in styrofoam containers. Temperature and microbial numbers increased (P < .05) during the 5-d aerobic incubation period for both HMC treatments. The population of microorganisms was greater for RLI on d 3 than for CON.(ABSTRACT TRUNCATED AT 250 WORDS)

Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs.

Two experiments were conducted to determine the effects of supplemental microbial phytase on utilization of dietary zinc by weanling pigs. Experiment 1 was a 2 x 3 factorial arrangement of treatments with 24 pigs for 4 wk. Two levels of phytase activity (0 and 1350 units/g) and three levels of zinc (0, 30 and 60 mg/kg as ZnSO4.7H2O) were added to a corn-soybean meal basal diet. Weekly measures included growth performance, plasma alkaline phosphatase activity and plasma mineral concentrations. In Experiment 2, mineral balances were determined in 12 pigs fed the basal diet or the diet with added zinc (30 mg/kg) or phytase (1350 units/g). The results indicated that either supplemental phytase or supplemental zinc increased plasma alkaline phosphatase activity and plasma zinc concentrations, but these increases were not additive. Supplemental phytase decreased plasma alkaline phosphatase activity in pigs supplemented with zinc. Supplemental phytase also significantly enhanced weight gain, feed intake, gain:feed ratio, plasma concentrations of inorganic phosphorus, and retention of phosphorus and calcium. Neither supplemental phytase nor zinc affected zinc retention. Supplementing corn-soybean meal diets with microbial phytase at 1350 units/g feed improves bioavailability of zinc as well as of phytate phosphorus to weanling pigs.

Effect of dietary copper on intestinal mucosa enzyme activity, morphology, and turnover rates in weanling pigs.

Twenty-four pigs from four litters weaned at 21 d of age (6.6 kg of BW) were used to evaluate the influence of 250 ppm of dietary Cu on intestinal mucosa glucose-6-phosphatase (GP), alkaline phosphatase (AP), and adenosine triphosphatase (ATPase) activity; mucosal morphology; and the turnover rate of the intestinal mucosa throughout the gastrointestinal tract. Pigs were allotted into four pens of six pigs each based on sex, litter, and weight. Pens were then assigned to one of two treatments: 1) corn-soybean meal-whey diet with no antimicrobials (CO), or 2) CO + 250 ppm of Cu. Pigs were fed twice daily an amount approximately equal to ad libitum intake for 14 d. On d 14, pigs were injected i.p. with [3H]thymidine (50 microCi/kg of BW) 10 h after the morning meal. One pig from each pen was euthanatized at 1, 6, 12, 20, 32, and 44 h postinjection, and intestinal tissue was collected from the duodenum, two jejunum sites (upper and lower), ileum, cecum, and colon. The activity of GP and AP in the lower jejunum tended to decrease in pigs fed Cu (P less than .11, P less than .08, respectively). The ATPase activity was not affected by treatment (P greater than .10). Crypt death, villus height, or epithelial cell size (P greater than .10) were not affected by feeding Cu. Migration rate of epithelial cells up the villus was also not affected by treatment (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological relationships between microbiological status and dietary copper levels in the pig.

Ten germ-free pigs and 10 conventionally reared pigs were fed one of two nutritionally balanced diets containing either 16 ppm Cu (basal) or 283 ppm Cu (high-Cu) to evaluate the physiological relationships between Cu and microbiological environment. Germ-free pigs tended to have higher ADG and average daily feed intake (ADFI) than conventionally reared pigs. Feeding the high-Cu diet tended to reduce ADG and ADFI in germ-free pigs but it increased ADG and ADFI in conventionally reared pigs. Hemoglobin and hematocrit were higher in germ-free pigs than in conventionally reared pigs (P less than .001), and hematocrit (P less than .01) and erythrocyte count (P less than .06) were reduced by feeding the high-Cu diet. Germ-free pigs had lower total leukocyte count (P less than .01) and the relative percentages of differentiated leukocytes were altered compared with conventionally reared pigs. Feeding the high-Cu diet increased the percentage of band neutrophils and monocytes in germ-free pigs but reduced the percentage of these cells in conventionally reared pigs (P less than .04). Germ-free pigs had higher concentrations of Cu and Zn in liver and plasma (P less than .001) and greater plasma ceruloplasmin oxidase activity (P less than .001) than conventionally reared pigs did. The high-Cu diet increased liver Cu and Zn (P less than .001) and plasma Cu (P less than .001) and reduced liver and plasma Fe (P less than .05). Organ weights (g/kg BW) differed between germ-free and conventionally reared pigs, and feeding the high-Cu diet reduced thymus weights (P less than .002). Intestinal weight and thickness were reduced in germ-free pigs, and feeding the high-Cu diet generally reduced villus height and width and crypt depth in germ-free pigs, whereas it increased these measurements in conventionally reared pigs.

Sensitivity of ruminal microorganisms to pentachlorophenol.

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.

Characteristics of live and killed brewer's yeast slurries and intoxication by intraruminal administration to cattle.

The physical and nutritional characteristics of live and killed brewer's yeast slurries and the possible toxicity of intraruminal administration of loading doses of these by-products were evaluated. Dry-matter (DM) percentages of live brewer's yeast slurry ranged from 10.5 to 29.0, and DM percentages of killed brewer's yeast slurry ranged from 14.6 to 18.5. Total crude protein (N X 6.25) percentages of live and killed yeast slurries (DM basis) were 44.1 and 43.1; ethanol percentages (wet basis) were 6.96 and 1.84, respectively. Phase contrast photomicrographs showed distinct differences in morphology between live and killed yeast cells. Intraruminal administration of loading doses (0, 2.3, 4.5 and 6.8 kg) of live and killed brewer's yeast slurries to bull calves induced clinical intoxication at the 4.5-kg and 6.9-kg dosages of live brewer's yeast slurry. No toxicity was induced either with the killed brewer's yeast or with the 0-kg or 2.3-kg dose levels of live brewer's yeast slurry. The clinical signs and plasma ethanol concentrations suggested ethanol intoxication. Ruminal NH3 concentrations increased to over 70 mg/dl with the 4.5-kg and 6.9-kg dosages of live brewer's yeast slurry, but they did not exceed 35 mg/dl with an equivalent dosage of killed brewer's yeast slurry.

Isolation from swine feces of a bacterium which decarboxylates p-hydroxyphenylacetic acid to 4-methylphenol (p-cresol).

An obligate anaerobe has been isolated from swine feces which decarboxylates p-hydroxyphenylacetic acid to 4-methylphenol (p-cresol). The bacterium was an ovoid rod, gram positive, nonsporeforming, and nonmotile. Lactate and acetate were major end products of glucose fermentation. Based on its characteristics, the bacterium is tentatively assigned to the genus Lactobacillus.

Effects of zeolite a or clinoptilolite in diets of growing swine.

Growth, nutrient balance, plasma ammonia levels and urinary p-cresol excretion were evaluated in growing pigs fed diets containing various levels of zeolite A or clinoptilolite. In one growth trial, crossbred pigs averaging 25 kg initial body weight were assigned to diets containing no zeolite, .3% zeolite A or .5% clinoptilolite for a 6-wk growing phase trial. Average daily gain (ADG), average daily feed intake (ADF) and feed/gain (F/G) were unaffected by supplementation of either zeolite in the diet, but metabolizable energy (ME) utilization was improved by feeding diets containing either zeolite. A second growth trial utilized the same crossbred pigs, which averaged 65 kg initial body weight, and were assigned to diets containing no zeolite, 1% zeolite A or 5% clinoptilolite for an 8-wk finishing phase trial. Average daily gain, ADF, and ME utilization were unaffected by feeding either zeolite diet, while F/G was increased in pigs fed the diet containing 5% clinoptilolite. In two nutrient balance trials, 16 crossbred pigs averaging 7.5 kg in initial body weight were fed diets containing 0, 1, 2 or 3% zeolite A in one trial and 16 crossbred pigs averaging 7.0 kg initial body weight were fed diets containing 0, 2.5, 5.0 or 7.5% clinoptilolite in a second trial. In both trials, digestible energy, ME, N-corrected ME and ME corrected for N balance and zeolite levels were linearly reduced as increasing amounts of either zeolite were fed. Daily fecal N increased and apparent digestibility of N was linearly reduced by feeding increasing amounts of zeolite A or clinoptilolite. Biological value of protein was improved linearly as higher levels of zeolite A were fed, indicating that there may be some ammonia binding to zeolite A in the gastrointestinal tract. Net protein utilization was reduced by feeding increasing levels of clinoptilolite in the diet. Calcium, P, Mg, Na, K and Fe retentions were linearly reduced by feeding increasing amounts of zeolite A in the diet, while increasing levels of clinoptilolite caused only P retention to be linearly reduced. Both free and conjugated forms of urinary p-cresol were linearly reduced by feeding increasing levels of clinoptilolite. Plasma ammonia levels were reduced at subsequent bleedings after a meal and by increasing levels of clinoptilolite.

The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites.

The purpose of this study was to investigate the effects of dietary antibiotic supplementation on the fecal urinary excretion of volatile phenolic and aromatic bacterial metabolites by the weanling pig, and to determine if a relationship exists between an exposure to these metabolites and growth performance. Wealing pigs were fed a basal diet, supplemented with either 110 ppm chlortetracycline, 110 ppm sulfamethazine and 55 ppm penicillin, 40 ppm lincomycin sulfate, or no antibiotics, for 30 days. Pigs on the chlortetracycline-sulfamethazine-penicillin diet on the average tended to grow at a faster rate, attained a higher percentage weight gain, and weighed slightly more than pigs on either the lincomycin sulfate or no antibiotic diets. Under all treatments, p-cresol was the predominant metabolite of the volatile phenolic and aromatic metabolites detected in feces and urine, with the urine accounting for 88% of its total daily excretion. Pigs on the chlortetracycline-sulfamethazine-penicillin diet excreted less urinary p-cresol than pigs on either the lincomycin sulfate or no antibiotic diets. Total p-cresol excretion expressed on the metabolic body size, resulted in significant treatment differences. Regression analysis of percentage body weight gain on urinary p-cresol excretion gave a negative correlation coefficient (r = -0.73). The results suggest that intestinal p-cresol production may be responsible for depressing the growth of the weanling pig.

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.

Gas-liquid chromatography for evaluating polysaccharide degradation by Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85.

Two predominant rumen cellulolytic bacteria, Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85, were incubated with ground filter paper (Whatman no. 1), cattle manure fiber, wheat straw, Kentucky bluegrass, alfalfa, and corn silage as substrates. Analyses of the initial substrate and the recovered residue after 48 h of static incubation showed that R. flavefaciens C94 was quantitatively more effective than B. succinogenes S85 in degrading total dry matter (32.3% versus 16.1%). However, B. succinogenes S85 demonstrated a qualitative advantage in degrading the hemicellulose and hemicellulosic sugars of particular substrates. R. flavefaciens degraded a mean 29.7% of the cellulose and 35.6% of the hemicellulose in the various substrates, whereas B. succinogenes degraded a mean 17.9 and 31.6% of these fractions, respectively. Gas-liquid chromatography was an important aid in characterizing the polysaccharide-degrading capabilities of these rumen species.