The effect of incremental levels of dietary nitrate on methane emissions in Holstein steers and performance in Nelore bulls.

Two experiments were conducted to study effects of dietary nitrate on enteric methane production, blood methemoglobin concentration, and growth rate in cattle. In Exp. 1, 36 Holstein steers (288 ± 25 kg BW) were fed increasing levels of dietary nitrate (6 levels; 0 to 3.0% of feed DM) in corn silage-based total mixed rations. Nitrate was introduced gradually in a 25-d adaptation period before methane production was determined in environmentally controlled rooms. In the rooms, feed intake was restricted and similar among all treatments. Methane production (g/d) decreased linearly as dietary nitrate concentration increased (P < 0.01). The apparent efficiency (measured methane reduction divided by potential methane reduction) with which enteric methane was mitigated was 49%. Blood methemoglobin levels increased with increasing nitrate dose. In Exp. 2, 300 Nelore bulls (392 ± 28 kg) were fed increasing levels of nitrate (6 levels; 0 to 2.4% of feed DM) in high-concentrate total mixed rations offered ad libitum. Feed intake decreased linearly with increasing level of dietary nitrate (P < 0.01). However, ADG was not affected by nitrate dose (P = 0.54), resulting in a linear improvement in G:F (P = 0.03) as dietary nitrate level increased. Carcass dressing percentage showed a quadratic response to incremental dietary nitrate, reaching the highest value at 0.96% of NO3/kg DM (P = 0.04).

Dietary nitrate supplementation reduces methane emission in beef cattle fed sugarcane-based diets.

The objective of this study was to determine the effect of dietary nitrate on methane emission and rumen fermentation parameters in Nellore × Guzera (Bos indicus) beef cattle fed a sugarcane based diet. The experiment was conducted with 16 steers weighing 283 ± 49 kg (mean ± SD), 6 rumen cannulated and 10 intact steers, in a cross-over design. The animals were blocked according to BW and presence or absence of rumen cannula and randomly allocated to either the nitrate diet (22 g nitrate/kg DM) or the control diet made isonitrogenous by the addition of urea. The diets consisted of freshly chopped sugarcane and concentrate (60:40 on DM basis), fed as a mixed ration. A 16-d adaptation period was used to allow the rumen microbes to adapt to dietary nitrate. Methane emission was measured using the sulfur hexafluoride tracer technique. Dry matter intake (P = 0.09) tended to be less when nitrate was present in the diet compared with the control, 6.60 and 7.05 kg/d DMI, respectively. The daily methane production was reduced (P < 0.01) by 32% when steers were fed the nitrate diet (85 g/d) compared with the urea diet (125 g/d). Methane emission per kilogram DMI was 27% less (P < 0.01) on the nitrate diet (13.3 g methane/kg DMI) than on the control diet (18.2 g methane/kg DMI). Methane losses as a fraction of gross energy intake (GEI) were less (P < 0.01) on the nitrate diet (4.2% of GEI) than on the control diet (5.9% of GEI). Nitrate mitigated enteric methane production by 87% of the theoretical potential. The rumen fluid ammonia-nitrogen (NH(3)-N()) concentration was significantly greater (P < 0.05) for the nitrate diet. The total concentration of VFA was not affected (P = 0.61) by nitrate in the diet, while the proportion of acetic acid tended to be greater (P = 0.09), propionic acid less (P = 0.06) and acetate/propionate ratio tended to be greater (P = 0.06) for the nitrate diet. Dietary nitrate reduced enteric methane emission in beef cattle fed sugarcane based diet.

Persistency of methane mitigation by dietary nitrate supplementation in dairy cows.

Feeding nitrate to dairy cows may lower ruminal methane production by competing for reducing equivalents with methanogenesis. Twenty lactating Holstein-Friesian dairy cows (33.2±6.0 kg of milk/d; 104±58 d in milk at the start of the experiment) were fed a total mixed ration (corn silage-based; forage to concentrate ratio 66:34), containing either a dietary urea or a dietary nitrate source [21 g of nitrate/kg of dry matter (DM)] during 4 successive 24-d periods, to assess the methane-mitigating potential of dietary nitrate and its persistency. The study was conducted as paired comparisons in a randomized design with repeated measurements. Cows were blocked by parity, lactation stage, and milk production at the start of the experiment. A 4-wk adaptation period allowed the rumen microbes to adapt to dietary urea and nitrate. Diets were isoenergetic and isonitrogenous. Methane production, energy balance, and diet digestibility were measured in open-circuit indirect calorimetry chambers. Cows were limit-fed during measurements. Nitrate persistently decreased methane production by 16%, whether expressed in grams per day, grams per kilogram of dry matter intake (DMI), or as percentage of gross energy intake, which was sustained for the full experimental period (mean 368 vs. 310±12.5 g/d; 19.4 vs. 16.2±0.47 g/kg of DMI; 5.9 vs.4.9±0.15% of gross energy intake for urea vs. nitrate, respectively). This decrease was smaller than the stoichiometrical methane mitigation potential of nitrate (full potential=28% methane reduction). The decreased energy loss from methane resulted in an improved conversion of dietary energy intake into metabolizable energy (57.3 vs. 58.6±0.70%, urea vs. nitrate, respectively). Despite this, milk energy output or energy retention was not affected by dietary nitrate. Nitrate did not affect milk yield or apparent digestibility of crude fat, neutral detergent fiber, and starch. Milk protein content (3.21 vs. 3.05±0.058%, urea vs. nitrate respectively) but not protein yield was lower for dietary nitrate. Hydrogen production between morning and afternoon milking was measured during the last experimental period. Cows fed nitrate emitted more hydrogen. Cows fed nitrate displayed higher blood methemoglobin levels (0.5 vs. 4.0±1.07% of hemoglobin, urea vs. nitrate respectively) and lower hemoglobin levels (7.1 vs. 6.3±0.11 mmol/L, urea vs. nitrate respectively). Dietary nitrate persistently decreased methane production from lactating dairy cows fed restricted amounts of feed, but the reduction in energy losses did not improve milk production or energy balance.

Tissue transglutaminase mRNA expression in apoptotic cell death.

INTRODUCTION: Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis. METHODS: Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro. RESULTS: The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8-2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo. CONCLUSION: t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.