Efficiency of Amino Acid Utilization in Nellore Cattle Grazing Low-Quality Forage Supplemented with Different Sources of Nitrogen.

This study aimed to evaluate the effects of supplementation with non-protein nitrogen (NPN) or ruminal undegradable protein (RUP) on intake, digestibility, and amino acid (AA) use efficiency of Nellore cattle grazing during the dry season. Eight Nellore steers (12 ± 2 months old) were used in quadruplicate Latin squares (2 × 2). The animals were placed on Urochloa brizantha cv. Xaraés under continuous grazing. The treatments included the following: (1) urea supplementation (NPN) and (2) supplementation of corn gluten meal 60 (CGM, RUP). Animals supplemented with CGM showed higher intakes of dry matter (DM) supplement, total AA, essential AA, and individual AA. The supplementation did not affect the total AA digestibility, total AA flux, and the AA fluxes of microbial origin and RUP from the diet (p > 0.05). The ruminal microorganism origin flux of total AA to the duodenum was 44.5% and 52.7% for animals supplemented with NPN and CGM, respectively. Animals supplemented with CGM showed an increase in blood concentrations of isoleucine (+19.09 µmol/L), cystine (+27.29 µmol/L), and albumin (+0.11 g/dL) (p < 0.05), but this increase was not accompanied by an improvement in N use efficiency of steers (p > 0.05). RUP supplementation via CGM can be an efficient nutritional strategy to enhance the intake and absorption of AA by Nellore cattle grazing low-quality forage during the dry season.

The Impact of Mineral and Energy Supplementation and Phytogenic Compounds on Rumen Microbial Diversity and Nitrogen Utilization in Grazing Beef Cattle.

The objective of this study was to evaluate the effect of the addition of a phytogenic compound blend (PHA) containing hydrolyzable tannins, carvacrol, and cinnamaldehyde oil to mineral salt or energy supplementation on the rumen microbiota and nitrogen metabolism of grazing Nellore cattle. Eight castrated Nellore steers were distributed in a double-Latin-square 4 × 4 design, with a 2 × 2 factorial arrangement (two types of supplements with or without the addition of the PHA), as follows: energy supplement without the PHA addition (EW); energy supplement with the PHA addition (EPHA); mineral supplement without the addition of the PHA (MW); mineral supplement with the PHA addition (MPHA). Steers that received supplements with the PHA have a lower ruminal proportion of valerate (with the PHA, 1.06%; without the PHA, 1.15%), a lower ruminal abundance of Verrucomicrobia, and a tendency for lower DM digestibility (with the PHA, 62.8%; without the PHA, 64.8%). Energy supplements allowed for higher ammonia concentrations (+2.28 mg of NH3-N/dL), increased the propionate proportion (+0.29% of total VFA), and had a higher ruminal abundance of Proteobacteria and Spirochaetae phyla in the rumen. The PHA addition in the supplement did not improve nitrogen retention, reduced the ruminal proportion of valerate, and had a negative impact on both the total dry-matter digestibility and the abundance of several ruminal bacterial groups belonging to the Firmicutes and Verrucomicrobia phyla.

Effect of Post-Ruminal Urea Supply on Growth Performance of Grazing Nellore Young Bulls at Dry Season.

The objective of this study was to evaluate the effect of the use of post-ruminal urea on performance, nitrogen metabolism and the ruminal environment of Nellore cattle reared on pasture during the dry season. In experiment 1 (Exp. 1), nine ruminal-cannulated Nellore steers, 30 ± 2 months old (651 ± 45 kg body weight (BW)), were allotted to a 3 × 3 Latin triple square. In experiment 2 (Exp. 2), 84 Nellore bulls, 18 ± 3 months old (315 ± 84 kg BW), were distributed in complete randomized blocks, by initial BW. Protein supplements were supplied daily, in the amount of 2 g/kg BW, and consisted of either CONT: protein + conventional urea (50% CP), PRU: protein + post ruminal urea (50% CP) and U + PRU: protein + urea conventional + post-ruminal urea (70% CP). The paddocks were composed of Urochloa brizantha cv. Marandu grass. In Exp. 1, there was no treatment effect for DM, OM, NDF, forage intake, and CP, but there was a higher intake for PRU (p < 0.005) and a higher digestibility for U+ PRU (p = 0.001). There was no effect on ruminal pH or NH3-N concentration (p ≥ 0.232), but there was an interaction between treatment and time for them (p < 0.039). Furthermore, there was a treatment effect on the total SCFA concentration, with CONT being higher than the others. A difference in the acetate:propionate ratio was found (p < 0.027), with a greater relationship for PRU and U + PRU. A treatment effect (p = 0.049) was found for the propionate proportion, with a higher proportion in the CONT. Nitrogen intake was consequently lower for the CONT and higher urinary excretion for the U + PRU (p = 0.002). Animals supplemented with CONT showed a tendency to have more Bacteria and fewer Archaea (p = 0.086). In Exp. 2, there was a treatment effect for the disappearance rate of the supplement (p < 0.001). Intake was faster for PRU and CONT, but performance was not affected by PRU (p = 0.311). The use of post-ruminal urea alters the microbial population, but does not affect performance. Therefore, supplementation with post-ruminal urea presented similar results compared to conventional urea. Ruminal and blood parameters and animal performance were not influenced by treatments.

Effects of Post-Ruminal Urea Supplementation during the Seasonal Period on Performance and Rumen Microbiome of Rearing Grazing Nellore Cattle.

The objective was to evaluate the effects of urea with post-ruminal absorption in the supplementation of growing Nellore cattle reared on pasture during a seasonal period. For the study, two experiments were conducted. In experiment 1, rumen and blood parameters were evaluated using eight rumen-cannulated Nellore bulls with initial body weight (BW) of 763 ± 44 kg, distributed in a double Latin square 4 × 4. In experiment 2, 120 Nellore steers with initial BW of 380 ± 35 kg were used for performance evaluation, distributed in a randomized block design (blocking factor or initial BW). The evaluated treatments were 1: (TP-U) (control) = supplement with 24% crude protein (CP) containing urea as a source of non-protein nitrogen (NPN; 3%) and soybean meal, 2: (TP-PRU) = 24% CP supplement containing post-ruminal urea (PRU; 3.6%) and soybean meal; 3: (NPN-U-PRU) = 24% CP supplement containing urea + post-ruminal urea (U = 3% and PRU = 3.9%), without soybean meal; 4: (NPN-PRU) = supplement with 24% CP containing post-ruminal urea (7.5%), without soybean meal. The supplement was offered at 3 g/kg BW per animal, daily, once a day. All animals were kept on Urochloa brizantha cv. Marandu pasture. Statistical analyses were performed using the SAS PROC MIXED, and the data were evaluated by the following contrasts: C1 = TP-U/TP-PRU vs. NPN-U-PRU/NPN-PRU (Soybean meal replacement by NPN); C2 = TP-U vs. TP-PRU (conventional urea vs. post-immune urea); C3 = NPN-U-PRU vs. NPN-PRU (low and high post-ruminal urea-PRU level). The digestibility of dry matter, organic matter, and NDF was lower when soybean meal was replaced by non-protein nitrogen, also being different between the levels of post-ruminal urea used in the supplement. Ruminal pH was different when soybean meal was replaced by NPN (p = 0.003). Total concentration of short-chain fatty acids, concentrations of isobutyrate (p = 0.003), valerate (p = 0.001), and isovalerate (p = 0.001) were different, and blood urea was different when soybean meal was replaced by NPN (p = 0.006). Simpson's diversity index was higher in the rumen of animals supplemented with TP-U than in those supplemented with TP-PRU (p = 0.05). A total of 27 phyla, 234 families, and 488 genera were identified. Nitrospirota and Gemmatimonadota phyla were detected just in the rumen of steers supplemented with TP-PRU. The performance (final BW, weight gain and gain per area) of the animals was different, being higher (p = 0.04) in animals supplemented with soybean meal, compared to NPN. The removal of soybean meal from the supplement and its replacement with either conventional urea plus post-ruminal urea or only post-ruminal urea compromises the performance of the animals. The lower the post-ruminal urea inclusion level, the lower the apparent digestibility of dry matter, organic matter, and NDF, when compared to animals supplemented with higher levels.

Effect of marker dosage frequency and spot fecal sampling frequency in the prediction accuracy of fecal output using chromic oxide and titanium dioxide in grazing BON steers.

This study aimed to evaluate the effect of two marker dosage frequencies and two spot fecal sampling frequencies on the variability of fecal output estimation and fecal recovery rate in grazing Blanco Orejinegro (BON) steers with chromium (Cr) or titanium (Ti) as external markers. Four steers (230.5 ± 14.4 kg BW) were used in a 4 × 4 Latin square design in a split-plot arrangement with two markers: Cr or Ti, two marker dosage frequencies: 10.0 g Cr2O3 or TiO2/steer once daily (DF1) or 5.0 g Cr2O3 or TiO2/steer twice daily (DF2), and two fecal sampling frequencies: spot sampling once daily (SF1) or spot sampling twice daily (SF2). Steers were equipped with fecal collection bags to evaluate the actual fecal output (FOR). Fecal marker concentration (FMC) was affected by marker dosage frequency × fecal sampling frequency interaction (P = 0.032): SF1DF1 allowed higher FMC (2.26 g/kg DM) than SF2DF1 (1.95 g/kg DM). Chromium marker allowed higher estimated fecal output (FOe) values when compared to FOR (P < 0.001). Both Ti mean and SD bias (+ 0.074 ± 0.160 kg) were lower than Cr mean and SD bias (+ 0.312 ± 0.272 kg). The Ti fecal output prediction showed lower both RSR (0.586 vs 1.401) and MSPE (0.029 vs 0.167) than Cr prediction. The titanium external marker allowed fecal output estimation with more accuracy in grazing beef cattle trials. A protocol including a once-daily marker dosage associated with a once-daily fecal sampling is plausible, allowing good fecal recovery rates and accurate fecal output estimation.

Rumen bacterial diversity in relation to nitrogen retention in beef cattle.

This study aimed to characterize the rumen bacterial diversity of beef steers differing in the efficiency of nitrogen retention (ENR). Eight castrated steers and fitted with ruminal silicone - and duodenal T-type cannulas were used in a cross-over design with three consecutive periods and three diets. During each experimental period, nitrogen balance was measured, and based on the efficiency of N utilization data, steers were split into three ENR groups: high (HNR, 56.6% ± 3.3%, n = 10), medium (MNR, 45.8% ± 2.2%, n = 6), and low (LNR, 37.7% ± 1.9%, n = 8) using the NbClust package version 2.0.4 in R. Prevotellaceae, Lactobacillaceae, Leuconostocaceae, Clostridiales_Incertae_Sedis_XIII, Lachnospiraceae, and Peptostreptococcaceae were more abundant in LNR (P < 0.05) compared to HNR or MNR. Negative correlations were found between N retention and Mogibacterium, Anaerofustis, Butyrivibrio, Coprococcus, Hespellia, Lactonifactor and Lachnospiraceae (r ≤ -0.61; P ≤ 0.05). Prevotella, Hespellia, Lactonifactor, Lachnospiraceae_other, and Anaerobiospirillum were positively correlated between urinary N excretion (r > 0.55; P < 0.01), and negative correlations were found with Elusimicrobia, Victivallis and Treponema (r < -0.41; P < 0.05). The adjustment of the rumen bacterial community differed significantly between the N use retention groups. The high N retention in beef cattle was associated with less abundant bacteria in the rumen; however, N fixation capacity and uncharacterized rumen microorganisms need to be elucidated in future studies. In contrast, lower N utilization was associated with high abundance of bacteria that promote greater urinary N excretion through ruminal protein degradation.

Corrigendum: Long-Term Encapsulated Nitrate Supplementation Modulates Rumen Microbial Diversity and Rumen Fermentation to Reduce Methane Emission in Grazing Steers.

[This corrects the article DOI: 10.3389/fmicb.2019.00614.].

Long-Term Encapsulated Nitrate Supplementation Modulates Rumen Microbial Diversity and Rumen Fermentation to Reduce Methane Emission in Grazing Steers.

This study investigated the long-term effects (13 months) of encapsulated nitrate supplementation (ENS) on enteric methane emissions, rumen fermentation parameters, ruminal bacteria, and diversity of archaea in grazing beef cattle. We used a total of thirty-two Nellore steers (initial BW of 197 ± 15.3 kg), 12 of which were fitted with rumen cannulas. For 13 months, the animals were maintained in 12 paddocks and fed a concentrate of ground corn, soybean meals, mineral supplements, and urea (URS) or encapsulated nitrate (EN) containing 70 g of EN/100 kg of BW (corresponding to 47 g NO3 -/100 kg BW). Encapsulated nitrate supplementation resulted in similar forage, supplement and total DMI values as URS (P > 0.05), but ENS tended to increase (+48 g/d; P = 0.055) average daily weight gain. Daily reductions in methane emissions (-9.54 g or 18.5%) were observed with ENS when expressed as g of CH4/kg of forage dry matter intake (fDMI) (P = 0.037). Lower concentrations of NH3-N and a higher ruminal pH were observed in ENS groups 6 h after supplementation (P < 0.05). Total VFA rumen concentration 6 h (P = 0.009) and 12 h after supplementation with EN resulted in lower acetate concentrations in the rumen (P = 0.041). Steers supplemented with EN had a greater ruminal abundance of Bacteroides, Barnesiella, Lactobacillus, Selenomonas, Veillonella, Succinimonas, Succinivibrio, and Duganella sp. (P < 0.05), but a lower abundance of Methanobrevibacter sp. (P = 0.007). Strong negative correlations were found between daily methane emissions and Proteobacteria, Erysipelotrichaceae, Prevotellaceae, and Roseburia, Kandleria, Selenomonas, Veillonella, and Succinivibrio sp. (P < 0.05) in the rumen of ENS steers. Encapsulated nitrate is a feed additive that persistently affects enteric methane emission in grazing steers, thereby decreasing Methanobrevibacter abundance in the rumen. In addition, ENS can promote fumarate-reducer and lactate-producer bacteria, thereby reducing acetate production during rumen fermentation.

Effect of soybean oil availabilities on rumen biohydrogenation and duodenal flow of fatty acids in beef cattle fed a diet with crude glycerine.

Soybean oil with different ruminal availability (whole soybeans (WS), soybean oil (SO) and calcium salts (CS)) was used to evaluate the fatty acid (FA) intake, rumen biohydrogenation (BH) and duodenal flow of FA in Nellore steers fed diets with crude glycerine (CG). Eight castrated Nellore steers were fitted with a ruminal and duodenal silicone cannula, and distributed in a double, simultaneous, Latin square 4 × 4 design with four diets and four experimental periods. Concentrates contained ground maize, urea, mineral salts, CG (100 g/kg DM) and soybean products with different availability of soybean oil: (1) no additional fat (CO), (2) WS, (3) SO or (4) CS. Fat supplementation was fixed to obtain 50 g ether extract/kg DM. Experimental treatments had no effect on DM intake, DM duodenal flow or ruminal turnover rate of C:16 FA. However, fat addition increased C:18 and turnover rates of total FA rumen (p < 0.05). CS resulted in lower C:18 turnover rates and lower ruminal BH of monounsaturated and unsaturated FA (UFA) than WS (p < 0.05). SO resulted in a greater duodenal flow of C18:0 (stearic acid), C18:1t-11 (vaccenic acid) and saturated FA than the WS and CS diets (p < 0.05). CS resulted in a higher duodenal flow of C18:3n-3 (linolenic acid) than WS (p < 0.05). The association of CG and calcium salts in Nellore steers was the best nutritional strategy to increase duodenal flow of healthier UFA, which may increase the deposition of these FA in meat. However, SO associated with CG association increased the duodenal flow of vaccenic acid, which is main precursor of endogenous synthesis of conjugated linoleic acids in tissues.

Effects of partial replacement of maize in the diet with crude glycerin and/or soyabean oil on ruminal fermentation and microbial population in Nellore steers.

The objective of this study was to determine whether a combination of crude glycerin (CG) and soyabean oil (SO) could be used to partially replace maize in the diet of Nellore steers while maintaining optimum feed utilisation. Eight castrated Nellore steers fitted with ruminal and duodenal cannulas were used in a double 4×4 Latin square design balanced for residual effects, in a factorial arrangement (A×B), when factor A corresponded to the provision of SO, and factor B to the provision of CG. Steers feed SO and CG showed similar DM intake, DM, organic matter and neutral-detergent fibre digestibility to that of steers fed diets without oil and without glycerine (P>0·05). Both diets with CG additions reduced the acetate:propionate ratio and increased the proportion of iso-butyrate, butyrate, iso-valerate and valerate (P<0·05). Steers fed diets containing SO had less total N excretion (P<0·001) and showed greater retained N expressed as % N intake (P=0·022). SO and CG diet generated a greater ruminal abundance of Prevotella, Succinivibrio, Ruminococcus, Syntrophococcus and Succiniclasticum. Archaea abundance (P=0·002) and total ciliate protozoa were less in steers fed diets containing SO (P=0·011). CG associated with lipids could be an energy source, which is a useful strategy for the partial replacement of maize in cattle diets, could result in reduced total N excretion and ruminal methanogens without affecting intake and digestibility.

Studies on bacterial community composition are affected by the time and storage method of the rumen content.

The objective of this study was to investigate three storage methods and four storage times for rumen sampling in terms of quality and yield of extracted metagenomic DNA as well as the composition of the rumen bacterial community. One Nellore steer fitted with a ruminal silicone-type cannula was used as a donor of ruminal contents. The experiment comprised 11 experimental groups: pellet control (PC), lyophilized control (LC), P-20: pellet stored frozen at -20°C for a period of 3, 6, and 12 months, P-80: pellet stored frozen at -80°C for a period of 3, 6, and 12 months, and L-20: lyophilized sample stored frozen at -20°C for a period of 3, 6, and 12 months. Metagenomic DNA concentrations were measured spectrophotometrically and fluorometrically and ion torrent sequencing was used to assess the bacterial community composition. The L-20 method could not maintain the yield of DNA during storage. In addition, the P-80 group showed a greater yield of metagenomic DNA than the other groups after 6 months of storage. Rumen samples stored as pellets (P-20 and P-80) resulted in lower richness Chao 1, ACE, and Shannon Wiener indices when compared to PC, while LC and PC were only different in richness ACE. The storage method and storage time influenced the proportions of 14 of 17 phyla identified by sequencing. In the P-20 group, the proportion of Cyanobacteria, Elusimicrobia, Fibrobacteres, Lentisphaerae, Proteobacteria, and Spirochaetes phyla identified was lower than 1%. In the P-80 group, there was an increase in the proportion of the Bacteroidetes phylum (p = 0.010); however, the proportion of Actinobacteria, Chloroflexi, SR1, Synergistetes, TM7, and WPS.2 phyla were unchanged compared to the PC group (p > 0.05). The class Clostridium was the most abundant in all stored groups and increased in its proportion, especially in the L-20 group. The rumen sample storage time significantly reduced the yield of metagenomic DNA extracted. Therefore, the storage method can influence the abundance of phyla, classes, and bacterial families studied in rumen samples and affect the richness and diversity index.