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To construct a genomic library of Veillonella parvula H2, total DNA was extracted, sheared by a Hydroshear machine, and the DNA fragments ligated a into Smal-digested vector pUC18 before being transformed into E. coli DH5a. Colonies were selected on Luria-Bertani (LB) plates containing ampicillin, 5-bromo-4-chloro-3-indoyl-3-galactose (X-gal), and isopropy-β-D-thiogalactoside (IPTG) and proliferated. Recombinant plasmids were analysed for the presence of inserted DNA fragments of 3-4 kb by restriction mapping. The titre of the library was determined to be 10⁵ pfu/mL according to the formula N=ln(l-p)/(l-f). The genomic library consisted of 99% of the genome of Veillonella parvula, demonstrating a successful library construction.
Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85-89% similar to 16S rDNA database sequences. For the remaining 3.14%, the similarity was lower than 85%. Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomie units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of the Cytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actmobacteria (2.38%). These include 10 single-clone OTUs, so Good's coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.
The study was conducted on four beef cannulae put into forestomach of 500 kg mass in the Latin square system. The control group, apart from corn silage, was fed with hay, while experimental groups were fed trefoil silage, darnel or meadow trefoil-darnel mixture. The parameters characterizing changes taking place in the rumen as well as the number of bacteria and protozoa were determined in the rumen fluid collected before feeding and 1.5 h and 3 hours after feeding. The substitution of hay with silage caused the increase of N-NH3 concentration in the rumen and changed the fermentation profiles expressed by the increase of the amounts of the following acids: propanoic, pentanoic and iso-pentanoic, and the decrease of the following acids: acetic and butyric. The silage of meadow trefoil caused the increase of bacteria and protozoa count, while the darnel silage and meadow trefoil-darnel mixture only caused the increase of bacteria count, and reduced the protozoa count.
The aim of this work was to determine the influence of feeding sheep with frozen fodder on the profile of rumen fermentation and its reflection in blood acid-base equilibrium.
This study examined the potential of liquid wild dog rose (Rosa canina) seeds oil and solid seeds residue obtained after CO2 extraction in supercritical conditions to mitigate rumen methane production in vitro. Two experiments were carried out. The substrate comprised of a mixture of meadow hay and barley meal (60:40) for the control diets (CON1 in experiment with oil and CON2 in experiment with residue). The control diets were supplemented up to 5% in dry matter of rose seeds oil (RO) and 5% of rose seeds residue (RR). The following parameters were measured: pH, ammonia, volatile fatty acids, ciliate protozoa and bacteria count, methane concentration and methanogens population. In the RO treatment a decrease in methane production and an increase in the Archea population were observed. In the RR treatment no change in methane production was reported, whereas some variations in protozoal populations were detected in relation to CON2. The potential to mitigate methane production was reported only in wild dog rose seeds oil treatment. Besides, no negative effect of wild dog rose seeds residue on rumen processes was stated, what may predispose this protein and fibre containing by-product to be utilized as ruminants dietary ingredient.
Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.
The aim of the study was to investigate the effect of different levels of protein in a diet on the incorporation of endogenous urea nitrogen (EUN) into individual amino acids (AA) of the ruminal bacteria of goats fed a low- (LP), medium- (MP), or high-protein diet (HP) in a 3 × 3 Latin square design. Three Alpine goats of about 35 kg body weight fitted with cannula into the rumen and catheter into the jugular vein were fed three isoenergetic diets containing 11% (LP), 13% (MP), or 16% (HP) crude protein in dry matter. The goats were infused for 6 days continuously with an 15N urea solution into the jugular vein. Ruminal bacteria were hydrolysed with 6M HCl. Next, butyl derivatives of free bacterial AA were obtained using HCl in butanol, then N-acylated using trifluoroacetic acid anhydride and analysed by gas chromatography using a mass-selective detector. The concentration of urea in plasma was 178, 356 and 667 mg · l–1 in goats from groups LP, MP and HP, respectively. 15N-excess during the infusion of labelled urea was significantly higher (P < 0.05) in the vast majority of AA of ruminal bacteria from goats fed the LP diet in comparison with goats fed the HP diet. Therefore, the level of protein in the diets affects the incorporation of EUN into bacterial AA. With the LP diet, EUN was incorporated mostly into glutamic acid, isoleucine and arginine, while in the case of the HP diet, into glutamic acid and arginine, as well as methionine. Regardless of the level of nitrogen in the diets, the incorporation of 15N into proline was very low. Irrespective of the dietary nitrogen level, EUN appears to be predominantly used for synthesis of glutamic acid in ruminal bacteria.
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