Hisao Itabashi

The Effect of Protozoa on the Composition of Rumen Bacteria in Cattle Using 16S rRNA Gene Clone Libraries

The effect of the presence of protozoa on the composition of rumen bacteria was investigated in cattle. Seven castrated Holstein cattle were divided into two groups: four faunated and three unfaunated, and 16S ribosomal RNA gene (rDNA) clonal libraries were constructed. A total of 312 clones were sequenced across 1,500 bp. The 151 sequences of the faunated cattle were classified into 98 operational taxonomic units (OTUs) having at least 97% similarity. The sequences derived from the faunated cattle were classified into Firmicutes (59.7%), Bacteroidetes (34.4%), Spirochaetes (2.6%), Actinobacteria (2.0%), and Proteobacteria (1.3%). Bacteroides and Prevotella (34.4%) were the major groups in the faunated cattle. The 161 sequences in the unfaunated cattle were classified into 72 OTUs. The sequences derived from the unfaunated libraries were classified into Firmicutes (65.7%), Bacteroidetes (31.1%), Proteobacteria (1.9%), and Spirochaetes (1.2%). The Clostridium botulinum group and its relatives (36.0%) were the major groups in the unfaunated cattle. An analysis by the computer program LIBSHUFF clarified that the presence of ruminal protozoa markedly affected the composition of rumen bacteria.

Culture-independent analysis of fecal microbiota in cattle.

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.

Phylogenetic analysis of methyl coenzyme-M reductase detected from the bovine rumen.

Aims:  The object of the present study is isolation of methyl coenzyme‐M reductase (MCR) genes (mcrA) from the bovine rumen fluid and determination of phylogenetical placements of the genes to investigate mechanisms of methanogenesis in the rumen from a point of view of mcrA genes.

Real-Time PCR Detection of the Effects of Protozoa on Rumen Bacteria in Cattle

A real-time PCR approach was used in this study to clarify the populations of major bacterial species in the rumens of faunated and unfaunated cattle. The sensitivity of this novel real-time PCR assay was evaluated by using 101 to 108 plasmid copies of target bacteria. The numbers of plasmid copies of Ruminococcus albus, Ruminococcus flavefaciens, Prevotella ruminicola, and the CUR-E cluster were higher in the unfaunated than in the faunated rumens. The CUR-E cluster belongs to the Clostridium group. In contrast, Fibrobacter succinogenes was higher in the faunated than in the unfaunated rumens. Although it is well known that an absence of protozoa brings about an increase in the bacterial population, it was clarified here that an absence of protozoa exerted differential effects on the populations of cellulolytic bacteria in cattle rumens (i.e., F. succinogenes, R. albus, and R. flavefaciens). In addition, real-time PCR analysis suggested that the CUR-E cluster was more prevalent in the unfaunated rumens.

Supplementation of the diet of dairy cows with trehalose results in milk with low lipid peroxide and high antioxidant content

The objective of this study was to investigate the effect of dietary supplementation with the disaccharides trehalose and cellobiose on antioxidant activity in rumen fluid, blood, and milk of dairy cows. Nine Holstein dairy cows housed in a free-stall barn were divided into 3 groups, with each group receiving a different dietary treatment (a control diet, a 1% trehalose-supplemented diet, or a 1% cellobiose-supplemented diet) following a 3x3 Latin square design. Feed intake and milk production increased in cows receiving the trehalose-supplemented diet compared with those receiving the control and cellobiose-supplemented diets. The total protozoa numbers in the rumen fluid of cows fed trehalose- or cellobiose-supplemented diets were greater than those of the control group. The C18:0 and C18:1 fatty acid content was increased in the milk of cows fed the trehalose-supplemented diet compared with that of the control group, and the C18:3n-3 fatty acid content in the milk of cows fed the cellobiose-supplemented diet was less than that of the control group. Plasma biochemical parameters were unchanged among the different treatments. In rumen fluid, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and superoxide dismutase activity were increased 2h after feeding in cows receiving the cellobiose-supplemented diet compared with the control group, and the concentration of thiobarbituric acid reactive substances in the rumen fluid of cows fed the cellobiose-supplemented diet was decreased. In contrast, the values of these parameters measured in the milk of cows fed the cellobiose-supplemented diet were no different from those of control cows. Dietary supplementation with trehalose did, however, bring about an improvement of the oxidative status of milk and blood in these animals compared with controls. These results provide the first evidence supporting the use of dietary disaccharides to decrease lipid peroxide levels and increase the antioxidant content of dairy cow milk. The findings suggest that disaccharides, particularly trehalose, might be useful as supplements for reducing oxidative stress and improving the quality of milk for human consumption, as well as possibly impairing the processes that give rise to lipid oxidation odor in dairy cow milk.

The mcrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme-M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.

Influence of Rumen Ciliate Protozoa on the Concentration of Free Amino Acids in the Rumen Fluids

無繊毛虫ヤギでは,繊毛虫が存在する通常のヤギに比べ反芻胃内容液中のアンモニア濃度が低下するので,その要因をアンモニア生成の面から明らかにする目的で両区のヤギに乾草と濃厚飼料とを給与し,反芻胃内容液遊離アミノ酸の濃度と組成とにつき比較検討した.1) 反芻胃内容液中のα-アミノ態窒素濃度は,両区とも採食1時間後に増加しその後は低下する傾向を示したが,通常区の方が無繊毛虫区よりも明らかに高かった.2) 採食1時間後における総遊離アミノ酸中の個々のアミノ酸の割合は,通常区では,グルタミン酸:20.1%,アラニン:16.1%,プロリン:7.2%,δ-アミノバレリアン酸:11.1%であるのに対し,無繊毛虫区では,それぞれ13.9%, 7.9%, 2.7%, 36.1%であり,アミノ酸組成に両区の間で差異が認められた.3) 給与飼料中からは多種類の遊離アミノ酸が検出されたが,特にプロリン,アスパラギン酸,チロシンおよびグルタミン酸が多く,反芻胃内容液の遊離アミノ酸組成とは著しく相違し,さらにその含量は採食後の反芻胃内容液中の含量と比べると極めて少なかった.これらの結果から,無繊毛虫区でアンモニア濃度が低下する主な要因は,飼料タンパク質からのアミノ酸の生成が通常区に比べ少ないためであると考察された.

Fatty acid composition of ruminal bacteria and protozoa, and effect of defaunation on fatty acid profile in the rumen with special reference to conjugated linoleic acid in cattle

Objectives of this study were to compare fatty acid (FA) composition of ruminal bacterial (B) and protozoal (P) cells, and to investigate effect of protozoa on FA profile in the rumen of cattle. Three cows were used to prepare ruminal B and P cells. Four faunated and three defaunated cattle (half-siblings) were used to study effect of protozoa on ruminal FA profile. Proportions of C16:0 and C18:0 in total fatty acids in B cells were 20.7% and 37.4%, whereas those in P cells were 33.4% and 11.6%, respectively. Proportions of trans-vaccenic acid (VA) and cis-9, trans-11 conjugated linoleic acid (CLA) in B cells were 3.9% and 1.0%, and those in P cells were 5.5% and 1.6%, respectively, being higher in P cells. Proportions of C18:1, C18:2 and C18:3 in P cells were two to three times higher than in B cells. Proportions of unsaturated fatty acids, VA and CLA in B cells of faunated cattle were higher than those of defaunated. VA and CLA in the ruminal fluid of faunated were also 1.6 to 2.5 times higher than those of defaunated. This tendency was similar for cell-free fraction of ruminal fluid. These results indicate that protozoa contribute greatly in VA and CLA production in the rumen.

Rumen fermentation, milk production and conjugated linoleic acid in the milk of cows fed high fiber diets added with dried distillers grains with solubles

The effects of corn dried distillers grains with solubles (DDGS) feeding on rumen fermentation and milk production in cows were evaluated using diets high in neutral detergent fiber (NDF, 45.9-46.6%). The control diet (Control) consisted mainly of hay, corn silage and concentrates. In the experimental diets, the concentrates were replaced with DDGS as 10% dry matter (DM) (10%DDGS) and 20% DM (20%DDGS). Eight cows were used for each 14-day treatment period. Effect of DDGS feeding on DM intake was not significant. Ruminal volatile fatty acids and ammonia-N at 5 h after feeding of 20%DDGS were decreased compared to Control, whereas protozoal count at 2 h after feeding of 20%DDGS was higher than that of 10%DDGS. Milk yield of cows fed DDGS diets was greater than that of Control, although percentages of milk protein and solids-not-fat were decreased by DDGS diets. The proportions of C10:0, C12:0, C14:0 and C16:0 in the milk fat decreased, and those of C18:0, C18:1, C18:2 and cis-9, trans-11 conjugated linoleic acid (CLA) increased markedly with elevated DDGS. Increase in trans-11 C18:1 was observed in the rumen fluid at 5 h after feeding. These findings suggest that DDGS feeding enhanced milk yield, as well as CLA synthesis under a high dietary NDF condition.

Investigations of the protection of soybean meal and amino acids from rumen degradation with whole blood treatment

Abstract In order to clarify the mechanism by which whole blood protects feed protein from rumen degradation, time courses of the amino acid pattern similarities between soybean meal (SBM), whole blood treated SBM (WBTSBM), whole blood treated lysine (WBTLYS) and whole blood and their residual proteins during ruminal exposure were calculated. 1 kg of goat blood was mixed with 1 kg SBM, dried at 60 °C for 24 h and incubated in nylon bags that were suspended in the rumen of Holstein steers. After 24 h of incubation, about 100% and 65% of the nitrogen (N) had disappeared from the SBM and WBTSBM, respectively. Changes in the amino acid pattern similarities showed that the degradation of SBM protein in the WBTSBM is more uniform compared with untreated SBM protein. This means that the decrease in the rumen degradability of WBTSBM protein is caused by the decrease in the degradability of SBM protein per se. These results suggest that a mechanism other than physical encapsulation may play an important role in the prevention of ruminal degradation. Almost all the lysine was diffused from WBTLYS, being prepared using a 1:2 or 1:1 combination (weight basis) of whole blood:lysine, by washing with water. The amounts of N and the amino acid composition of the WBTLYS residues did not change during the 24 h incubation period. The treatment of lysine with whole blood could not protect the lysine from ruminal degradation.