Dynamics of the ruminal microbial ecosystem, and inhibition of methanogenesis and propiogenesis in response to nitrate feeding to Holstein calves.

It is known that nitrate inhibits ruminal methanogenesis, mainly through competition with hydrogenotrophic methanogens for available hydrogen (H2) and also through toxic effects on the methanogens. However, there is limited knowledge about its effects on the others members of ruminal microbiota and their metabolites. In this study, we investigated the effects of dietary nitrate inclusion on enteric methane (CH4) emission, temporal changes in ruminal microbiota, and fermentation in Holstein calves. Eighteen animals were maintained in individual pens for 45 d. Animals were randomly allocated to either a control (CTR) or nitrate (NIT, containing 15 g of calcium nitrate/kg dry matter) diets. Methane emissions were estimated using the sulfur hexafluoride (SF6) tracer method. Ruminal microbiota changes and ruminal fermentation were evaluated at 0, 4, and 8 h post-feeding. In this study, feed dry matter intake (DMI) did not differ between dietary treatments (P > 0.05). Diets containing NIT reduced CH4 emissions by 27% (g/d) and yield by 21% (g/kg DMI) compared to the CTR (P < 0.05). The pH values and total volatile fatty acids (VFA) concentration did not differ between dietary treatments (P > 0.05) but differed with time, and post-feeding (P < 0.05). Increases in the concentrations of ruminal ammonia nitrogen (NH3-N) and acetate were observed, whereas propionate decreased at 4 h post-feeding with the NIT diet (P < 0.05). Feeding the NIT diet reduced the populations of total bacteria, total methanogens, Ruminococcus albus and Ruminococcus flavefaciens, and the abundance of Succiniclasticum, Coprococcus, Treponema, Shuttlewortia, Succinivibrio, Sharpea, Pseudobutyrivibrio, and Selenomona (P < 0.05); whereas, the population of total fungi, protozoa, Fibrobacter succinogenes, Atopobium and Erysipelotrichaceae L7A_E11 increased (P < 0.05). In conclusion, feeding nitrate reduces enteric CH4 emissions and the methanogens population, whereas it decreases the propionate concentration and the abundance of bacteria involved in the succinate and acrylate pathways. Despite the altered fermentation profile and ruminal microbiota, DMI was not influenced by dietary nitrate. These findings suggest that nitrate has a predominantly direct effect on the reduction of methanogenesis and propionate synthesis.

Changes in hematological, biochemical, and blood gases parameters in response to progressive inclusion of nitrate in the diet of Holstein calves.

BACKGROUND AND AIM: Nitrate (NO3 -) reduces enteric methane emissions and could be a source of non-protein nitrogen in ruminant feeds. Nonetheless, it has a potential toxic effect that could compromise animal health and production. The purpose of this study was to determine the effects of progressive inclusion of NO3 - in the diet on the hematological, biochemical, and blood gases parameters, in turn, the effects on feed intake and live weight gain (LWG) in Holstein calves. MATERIALS AND METHODS: Eighteen Holstein heifers and steers (nine animals/treatment) were maintained in individual pens for 45 days. Animals were randomly allocated to either a control or nitrate diet (ND) (containing 15 g of NO3 -/kg of dry matter [DM]). The biochemical parameters and blood gases were analyzed only in the NO3 - group on days: -1, 1, 7, 13, 19, and 25 corresponding to 0, 20, 40, 60, 80, and 100% of the total inclusion of NO3 - in the diet, respectively. In addition, DM intake (DMI) and LWG were evaluated among dietary treatments. RESULTS: Feeding the ND did not influence DMI or LWG (p>0.05). Methemoglobin (MetHb) and deoxyhemoglobin increased according to the NO3 - concentrations in the diet (p<0.05), while an opposite effect was observed for oxyhemoglobin and carboxyhemoglobin (p<0.05). Hematocrit levels decreased (p<0.05), while albumin, alanine aminotransferase, and gamma-glutamyl transpeptidase concentrations were not modified (p>0.05). However, glucose, urea, aspartate aminotransferase (AST), and retinol concentrations increased (p<0.05) according to the NO3 - concentrations in the diet. CONCLUSION: This study confirmed that the progressive inclusion of 123 g of NO3 -/animal/day in the diet could be safe without affecting DMI and LWG of Holstein calves. In turn, a dose-response effect of the MetHb, glucose, urea, AST, and retinol was observed, but these values did not exceed reference values. These results highlighted the importance of using a scheme of progressive inclusion of NO3 - in the diet of calves to reduce the risks of NO3 - toxicity.

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.(AU)

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR / Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR. / Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.