Cloning and characterization of cobA, one of vitamin B12 biosynthesis pathway genes from Selenomonas ruminantium

Bacterial biosynthesis of vitamin B12 can occur via either aerobic or anaerobic route. While the aerobic pathway has been fully elucidated and understood, less is known about the anaerobic pathway. Selenomonas ruminantium is thought to be the main producer of this vitamin in rumen environment and must use the anaerobic pathway. In our work we found one of the genes of vitamin B12 biosynthetic pathway of S. ruminantium, encoding for the cobalamin adenosyltransferase, enzyme taking part at the last steps of the synthesis process. Deduced amino acid sequence showed the highest similarity to cobalamin adenosyltransferases of other ruminal anaerobic bacteria and that of species Selenomonas. Phylogenetic comparisons of CobA protein sequences of several anaerobic bacteria of Clostridiale order indicate possible horizontal transfer of this gene.

PSXIV-15 Metagenomic sequencing of rumen microorganisms of cattle fed a corn stover-based diet

Worldwide, there is a need to discover new microorganisms that efficiently degrade lignocellulosic complexes that would help to improve the digestibility of low-quality agricultural byproducts. The aim of the study was to evaluate the effects of a corn stover-based diet (CSD) on rumen bacteria. Ruminal fluid of 6 Holstein cows (595 ± 96 kg) was collected during two periods. During first period, animals were consuming a diet based on corn silage and oat hay (DB), mineral premix and water ad libitum (50:50, DM). In second period, animals were provided a CSD (100% DM), mineral premix and water ad libitum for 45 days. Ruminal fluid was collected through esophageal tube, filtered and stored at -80°C until DNA extraction. Rumen microorganisms were identified by sequencing the 16SrRNA gene using the Illumina Miseq platform and primers for V3 and V4 regions. Data were analyzed by QIIME 1.9. Analysis of variance was performed for a completely randomized design using the MIXED procedure of SAS 9.1. The taxonomic affiliation showed that both populations were mainly composed of Firmicutes, Bacteroidetes and Proteobacteria. The most abundant bacteria species in both diets were Ruminococcus flavefaciens, Prevotella copri, Prevotella ruminicola, Fibrobacter succinogenes, Bacillus coagulans, Bacteroides uniformis and Selenomonas ruminantium. Feeding a CSD, increased the relative abundance of Prevotella ruminicola (from 6.1 to 20.9%, P < 0.01), Streptococcus luteciae (from 0.05 to 0.78%, P < 0.01), Clostridium aminophilum (0.45 to 3.1%, P < 0.01), Selenomonas ruminantium (5.2 to 21.8%, P < 0.02) and Pantoea agglomerans (0.7 to 3.9%, P < 0.01) and decreased Propionibacterium acnes (0.7 to 0.1%, P < 0.02) and Bacteroides ovatus (0.9 to 0.1%, P < 0.01). Feeding cattle with a diet with a more lignified forage like CSD led to the proliferation of bacteria such as Prevotella ruminicula, Streptococcus luteciae, Clostridium aminophilum, Selenomonas ruminantium and Pantoea agglomerans.

Effect of Feeding Glycerin on Ruminal Environment and In Situ Degradability of Feedstuffs in Young Bulls

This work studied the effect of feeding glycerin in bulls that were fed high levels of concentrate on the ruminal environment and in situ degradability of feedstuffs. Four ruminally cannulated young bulls were assigned to a 4 × 4 Latin square arrangement of treatments. The diets consisted of 15% barley straw and 85% concentrate in dry matter (DM). There were four different concentrates: without glycerin, and with 20, 40 or 80 g of glycerin per kg of DM. Each diet was offered for 24 days, the ruminal fluid was sampled to evaluate the ruminal metabolism and to determine the ruminal bacterial population by quantitative real-time polymerase chain reaction analysis, and the in situ degradability of six different feedstuffs was measured. The treatment with the highest level of glycerin provided the lower pH (p < 0.001), and the acetic/propionic molar ratio decreased (p < 0.001) as glycerin increased. The incorporation of glycerin in the diet did not affect the DNA copies/µL of the total bacteria, Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus (p > 0.05) in the DNA extract of rumen fluid, but at high levels increased Selenomonas ruminantium (p < 0.01). Very few effects of glycerin incorporation were found for the in situ degradability. In young bulls that were fed high levels of concentrate, glycerin at 20 or 40 g/kg of feed could be included without affecting the ruminal pH and raising the propionic acid, but at 80 g/kg the ruminal pH dropped, despite the increase of Selenomonas ruminantium.

Impact of strain and dose of live yeast and yeast derivatives on in vitro ruminal fermentation of a high-grain diet at two pH levels

The objective of this study was to determine the effects of live yeast (LY) or yeast derivatives (YD) on gas production (GP), dry matter (DM) disappearance (DMD), fermentation characteristics, and microbial profiles in batch culture. The study was a completely randomized design with a factorial arrangement: 2 media pH × 5 yeasts × 4 dosages. An additional treatment of monensin (Mon) was added as a positive control for each pH level. Media pH was low (5.8) and high (6.5); the yeasts were three LY (LY1-3) and two YD (YD4-5); and doses were 0, 4 × 106, 8 × 106, and 1.6 × 107 cfu mL−1 for LY and 0, 15, 30, and 60 mg bottle−1 for YD. Substrate consisted of 10% silage and 90% concentrate (DM basis) and samples were incubated for 24 h. Media pH of 6.5 vs. 5.8, increased (P < 0.01) GP, DMD, and volatile fatty acid (VFA) concentrations but decreased (P < 0.01) NH3-N concentration and copy numbers of Ruminococcus albus, Ruminococcus flavefaciens, and Selenomonas ruminantium. Increasing dose of LY2 linearly (P < 0.05) increased DMD. Total VFA concentration was greater with LY2 (P < 0.01) than LY3 and YD5 at pH 6.5. Overall, adding yeast products improved (P < 0.05) DMD at pH 5.8, and increased VFA concentration compared with Mon. These results indicate that in vitro GP and DMD of a high-grain diet varied with source and dose of yeast supplementation. Some yeast products have the potential to improve fermentation of feedlot diets when supplemented at appropriate doses.

The effect of Saccharomyces cerevisiae on in vitro growth and fermentation of Selenomonas ruminantium and Megasphaera elsdenii

Stimulation of lactate utilization by Selenomonas ruminantium and Megasphaera elsdenii may help in reducing problems associated with rumen acidosis. The objective of this study was to determine the effect of a Saccharomyces cerevisiae live culture and Saccharomyces cerevisiae fermentation products on in vitro growth and fermentation of lactate-utilizing ruminal bacteria, S. ruminantium (ATCC 19205) and M. elsdenii (ATCC 25940). The cultures were run for 0, 6, 12, 24 and 48 h under anaerobic conditions on a growth medium supplemented with a yeast live culture (SC) or with yeast fermentation products (SCFP) and, as reference, on the same medium without supplementation (CON). Neither SC nor SCFP had a significant effect on the growth of S. ruminantium after 6, 12 and 24 h of incubation, but the live yeast culture significantly (P≤0.05) improved the growth of these bacteria after 48 h of incubation. The yeast fermentation products significantly (P≤0.05) decreased pH and increased lactate synthesis by S. ruminantium. The Saccharomyces cerevisiae live culture significantly improved the growth of M. elsdenii after 12 and 24 h of incubation, and the S. cerevisiae fermentation products increased its growth after 48 h. The After 24 and 48 h of incubation the Saccharomyces cerevisiae live culture reduced the concentration of total volatile fatty acids (VFA), while caproate was the main product of in vitro fermentation of M. elsdenii (P≤0.05). Saccharomyces cerevisiae live cultures may improve microbial fibre fermentation in the rumen by maintaining optimal pH conditions.