Impact of high-concentrate feeding and low ruminal pH on methanogens and protozoa in the rumen of dairy cows.
ABSTRACT Non-lactating dairy cattle were transitioned to a high-concentrate diet to investigate the effect of ruminal pH suppression, commonly found in dairy cattle, on the density, diversity, and community structure of rumen methanogens, as well as the density of rumen protozoa. Four ruminally cannulated cows were fed a hay diet and transitioned to a 65% grain and 35% hay diet. The cattle were maintained on an high-concentrate diet for 3 weeks before the transition back to an hay diet, which was fed for an additional 3 weeks. Rumen fluid and solids and fecal samples were obtained prior to feeding during weeks 0 (hay), 1, and 3 (high-concentrate), and 4 and 6 (hay). Subacute ruminal acidosis was induced during week 1. During week 3 of the experiment, there was a significant increase in the number of protozoa present in the rumen fluid (P=0.049) and rumen solids (P=0.004), and a significant reduction in protozoa in the rumen fluid in week 6 (P=0.003). No significant effect of diet on density of rumen methanogens was found in any samples, as determined by real-time PCR. Clone libraries were constructed for weeks 0, 3, and 6, and the methanogen diversity of week 3 was found to differ from week 6. Week 3 was also found to have a significantly altered methanogen community structure, compared to the other weeks. Twenty-two unique 16S rRNA phylotypes were identified, three of which were found only during high-concentrate feeding, three were found during both phases of hay feeding, and seven were found in all three clone libraries. The genus Methanobrevibacter comprised 99% of the clones present. The rumen fluid at weeks 0, 3, and 6 of all the animals was found to contain a type A protozoal population. Ultimately, high-concentrate feeding did not significantly affect the density of rumen methanogens, but did alter methanogen diversity and community structure, as well as protozoal density within the rumen of nonlactating dairy cattle. Therefore, it may be necessary to monitor the rumen methanogen and protozoal communities of dairy cattle susceptible to depressed pH when methane abatement strategies are being investigated.
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ABSTRACT: We used six ruminally cannulated steers in a two-period crossover design to study ruminal fermentative and microbial changes associated with induced subacute acidosis. Steers were adapted to either an 80% alfalfa hay (hay-adapted)- or corn grain (grain-adapted)-based concentrate diet. After feed was withheld for 24 h, steers were overfed with an all-grain diet at 3.5 x NEm daily for 3 d. Ruminal contents and jugular blood samples were collected before withholding feed and at 0 and 12 h daily for 3 d during the overfeeding period. Ruminal samples were analyzed for pH, lactate, VFA concentrations, and counts of total anaerobic, amylolytic, lactic acid-producing and -fermenting bacteria, and ciliated protozoa. Blood samples were analyzed to assess acid-base status. Ruminal pH declined to a range of 5.5 to 5.0 with increased VFA concentrations, but normal lactate concentrations (<5 mM) were indicative of subacute acidosis. Total viable and amylolytic bacterial counts were higher (P < .05) in grain-adapted than hay-adapted steers. Anaerobic lactobacilli counts increased over time (P < .01) in both groups and were generally higher in grain-adapted than hay-adapted steers. Lactate-utilizing bacteria were initially greater in grain-adapted than hay-adapted steers and increased over time in both groups following grain challenge. Total ciliates were initially higher (P < .05) in grain-adapted than hay-adapted steers and decreased after 48 h in both groups. Blood acid-base changes were minimal. Bacterial changes associated with subacute acidosis resemble those reported during adaptation to grain feeding, and the decline in ciliated protozoa may be the only microbial indicator of a potentially acidotic condition in the rumen.Journal of Animal Science 01/1998; 76(1):234-41. · 2.09 Impact Factor
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ABSTRACT: PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10(0) and 10(6) entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.Applied and Environmental Microbiology 02/2006; 72(1):200-6. · 3.68 Impact Factor
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ABSTRACT: The predicted 16S riboprint patterns of 10 restriction endonucleases for 26 diverse methanogens were compared to actual patterns produced on agarose gels. The observed patterns corroborated the expected riboprints. Our analyses confirmed that the endonuclease HaeIII gave the best results generating 15 different riboprint sets. Six of these 15 riboprints represented more than one strain. Of these, three riboprint sets were further differentiated: Methanomicrobium mobile, Methanolacinia paynteri, and Methanoplanus petrolearius were differentiated from each other by the endonuclease AluI; Methanofollis liminatans, Methanospirillum hungatei, and Methanoculleus bourgensis were differentiated from each other by HpaII; and the combination of FokI and MluNI was used to differentiate Methanobrevibacter sp. ZA-10, and Methanobrevibacter arboriphilicus strains DH-1, AZ, and DC from each other. We could not differentiate the following pairs of strains from each other: Methanosarcina mazeii S-6 and C16, Methanobacterium bryantii MoH and MoH-G, Methanobacterium thermoautotrophicum GC-1 and DeltaH, and Methanobrevibacter arborophillicus DC and A2. This riboprint strategy provided a simple and rapid method to presumptively identify 22 of the 26 diverse strains of methanogens belonging to 13 genera from a range of environments.Journal of Microbiological Methods 12/2003; 55(2):337-49. · 2.16 Impact Factor