Liquid-associated Bacteria Research Articles

Changes in the Fermentation and Bacterial Community by Artificial Saliva pH in RUSITEC System.

The purpose of the study was to assess the artificial saliva (AS) pH on ruminal fermentation and rumen bacteria community in the rumen simulation technique (RUSITEC) system. The experiment was performed in two treatments (low AS pH vs. high AS pH) with four replicates. The low AS pH was sustained by altering the composition of the AS (NaHCO3 from 9.8 to 1.96 g/L, Na2HPO4 from 9.3 to 1.86 g/L) according to McDougall's method. The diets were supplemented with 16 g basic diets with forage to the concentrate ratio of 50:50. The experiments were conducted over 13-day incubation periods, with 9 days adaption and 4 days sample collection. The results showed low AS pH decreased dry matter (DM) degradability (64.37 vs. 58.67%), organic matter (OM) degradability (64.38 vs. 59.32%), neutral detergent fiber (NDF) degradability (46.87 vs. 39.94%), acid detergent fiber (ADF) degradability (38.16 vs. 31.13%), and crude protein (CP) degradability (70.33 vs. 62.99%), respectively. Compared with the high AS pH, the low AS pH increased the proportion of butyrate (P = 0.008) and decreased the proportion of propionate (P < 0.001). At the bacteria community, the low AS pH increased the abundances of Spirochaetes (P = 0.001) and Synergistetes (P = 0.004) and decreased the Verrucomicrobia abundance (P = 0.004) in solid-associated bacteria. At the genus level, the low AS pH increased the abundance of Lactobacillus (P = 0.050) and decreased the abundance of Schwartzia (P = 0.002) in solid-associated bacteria. The abundances of Prevotellaceae_YAB2003_group (P = 0.040), Schwartzia (P = 0.002), and Ruminobacter (P = 0.043) were lower in the low AS pH group compared with the high AS pH group in liquid-associated bacteria. Low AS pH decreased the number of Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes (P < 0.001) both in the solid- and liquid-associated bacteria, respectively. The results of the present study included three groups of bacteria communities according to the different sensitives to rumen pH: the abundances of Lactobacillus, Succinivibrio, and Prevotella_7 are increased with decreasing AS pH; the amounts of R. albus, R. flavefaciens, F. succinogenes as well as the abundances of Schwartzia and Ruminobacter decreased with the reducing AS pH; the abundances of Selenomonas_1, Rikenellaceae_RC9_gut_group, and Succiniclasticum were not affected by the AS pH in RUSTITEC.

The origin of N isotopic discrimination and its relationship with feed efficiency in fattening yearling bulls is diet-dependent.

Nitrogen (N) isotopic discrimination (i.e. the difference in natural 15N abundance between the animal proteins and the diet; Δ15N) is known to correlate with N use efficiency (NUE) and feed conversion efficiency (FCE) in ruminants. However, results from the literature are not always consistent across studies, likely due to isotopic discrimination pathways that may differ with the nature of diets. The objective of the present study was to assess at which level, from rumen to tissues, Δ15N originates and becomes related to NUE and FCE in fattening yearling bulls when they are fed two contrasted diets. Twenty-four Charolais yearling bulls were randomly divided into two groups and fed during 8 months, from weaning to slaughter, either 1) a high starch diet based on corn silage supplying a balanced N to energy ratio at the rumen level (starch) or 2) a high fiber diet based on grass silage supplying an excess of rumen degradable N (fiber). All animals were slaughtered and samples of different digestive pools (ruminal, duodenal, ileal and fecal contents), animal tissues (duodenum, liver and muscle), blood and urine were collected for each animal. Ruminal content was further used to isolate liquid-associated bacteria (LAB), protozoa and free ammonia, while plasma proteins were obtained from blood. All samples along with feed were analyzed for their N isotopic composition. For both diets, the digestive contribution (i.e. the N isotopic discrimination occurring before absorption) to the Δ15N observed in animal tissues accounted for 65 ± 11%, leaving only one third to the contribution of post-absorptive metabolism. Concerning the Δ15N in digestive pools, the majority of these changes occurred in the rumen (av. Δ15N = 2.12 ± 0.66‰), with only minor 15N enrichments thereafter (av. Δ15N = 2.24 ± 0.41‰), highlighting the key role of the rumen on N isotopic discrimination. A strong, significant overall relationship (n = 24) between Δ15N and FCE or NUE was found when using any post-absorptive metabolic pool (duodenum, liver, or muscle tissues, or plasma proteins; 0.52 < r < 0.73; P ≤ 0.01), probably as these pools reflect both digestive and post-absorptive metabolic phenomena. Fiber diet compared to starch diet had a lower feed efficiency and promoted higher (P ≤ 0.05) Δ15N values across all post-absorptive metabolic pools and some digestive pools (ruminal, duodenal, and ileal contents). The within-diet relationship (n = 12) between Δ15N and feed efficiency was not as strong and consistent as the overall relationship, with contrasted responses between the two diets for specific pools (diet x pool interaction; P ≤ 0.01). Our results highlight the contrasted use of N at the rumen level between the two experimental diets and suggests the need for different equations to predict FCE or NUE from Δ15N according to the type of diet. In conclusion, rumen digestion and associated microbial activity can play an important role on N isotopic discrimination so rumen effect related to diet may interfere with the relationship between Δ15N and feed efficiency in fattening yearling bulls.

Ruminal use of undegraded-feed soluble protein and accuracy of the estimations of the nutrient content in ruminal bacteria.

The possible escape from the rumen of undegraded-feed soluble proteins (SP) was investigated using nitrogen-15 (15 N) infusions (25 mg) in three rumen and duodenum cannulated wethers. Animals were fed three isoproteic diets differing in SP content and including protein concentrates either untreated (control) or treated with solutions of either malic acid (MHT) or orthophosphoric acid (OHT) and heat. Compared with control, MHT and OHT diets reduced ruminal concentrations of ammonia-nitrogen (NH3 -N) by 35.9% (P = 0.007), non-ammonia nitrogen (NAN) by 36.8% (P = 0.007), and SP-nitrogen (SP-N) by 45.2% (P = 0.072) over the post-feeding period. Both NAN and SP-N were lower (P ≤ 0.040) for OHT than for MHT diet. The 15 N enrichment of NAN and SP-N did not vary either among diets or with time, and both values were closely related (R2 = 0.965; P < 0.001). Estimations, either using solid-associated bacteria (SAB) or liquid-associated bacteria (LAB) as a reference, indicated that 0.983 and 0.894 of SP-N (values averaged across diets) was of microbial origin, respectively, which would indicate a practically negligible ruminal escape of feed SP-N. Values of 15 N-enrichment in SAB and LAB fitted well to previously published SAB-LAB relationships, indicating a 22% underevaluation of the N supply from SAB when only LAB is used as a reference. Both the negligible ruminal escape of feed SP and the underevaluation of the bacterial nutrient supply as a consequence of the use of LAB as the only bacterial reference should be considered to improve ruminant protein feeding systems. © 2019 Society of Chemical Industry.

Evaluation of 15N and purine bases as microbial markers to estimate ruminal bacterial nitrogen outflow in dairy cows

The present study aimed to evaluate 15N and purine bases (PB) as markers for ruminal bacterial nitrogen (N) outflow in dairy cows. Additionally, the effects of calcium salts of fatty acids (CSFA) in diets with different levels of rumen degradable protein (RDP) on PB omasal flow, urinary recovery of purine derivatives (PD), and endogenous excretion of PD were evaluated. Eight ruminal cannulated Holstein cows, with 207 ± 23.6 (mean ± SD) days in milk, 618 ± 39.0 kg of body weight, and 33.4 ± 2.45 kg/d of milk yield were used in a replicated 4 × 4 Latin square design. Four treatments were arranged in a 2 × 2 factorial design to evaluate the following factors: CSFA addition [-CS: basal diet without CSFA; and + CS: with 33.2 g/kg dry matter (DM) of CSFA]; and the level of RDP (LRDP: low RDP, with 98 g/kg DM of RDP; and HRDP: high RDP, with 110 g/kg DM of RDP). Particle-associated bacteria (PAB) showed lower (P ≤ 0.008) 15N enrichment and purine N to total bacterial N ratio than liquid associated bacteria (LAB). Increasing RDP concentration reduced (P = 0.007) 15N enrichment and tended (P ≤ 0.095) to reduce PB concentration in bacterial pellets. Purine bases overestimated (P ≤ 0.002) LAB-NAN and total microbial flow but, regarding PAB-NAN flow, there was a trend of interaction (P = 0.067) between marker and fat supplementation effects. In the absence of fat supplementation, PB underestimated (P ≤ 0.05) the flow of PAB-NAN compared to 15N and failed to detect the negative effect of fat supplementation evidenced by 15N labeling. Omasal PB flow tended to increase (P = 0.061) in low protein degradability diets, but PD excretion was constant (322 ± 5,9 mmol/d, mean ± SE) regardless of diet and, as a result, urinary recovery of PB decreased (P = 0.007). Daily excretion of creatinine and endogenous PD was also unaffected by the diet and averaged 0.262 mmol/kg BW and 0.640 mmol/kg BW0.75, respectively. In conclusion, the response of PB and its urinary derivatives to variation in diet composition compared to 15N reveals their weaknesses in the estimation of microbial synthesis.

Dietary protein reduction on microbial protein, amino acids digestibility, and body retention in beef cattle. I. Digestibility sites and ruminal synthesis estimated by purine bases and 15N as markers.

The objectives of this study were to evaluate the effect of reducing dietary CP contents on 1) total and partial nutrient digestion and nitrogen balance and 2) on microbial crude protein (MCP) synthesis and true MCP digestibility in the small intestine obtained with 15N and purine bases (PB) in beef cattle. Eight bulls (4 Nellore and 4 Crossbred Angus × Nellore) cannulated in the rumen and ileum were distributed in duplicated 4 × 4 Latin squares. The diets consisted of increasing CP contents: 100, 120, or 140 g CP/kg DM offered ad libitum, and restricted intake (RI) diet with 120 g CP/kg DM. The experiment lasted four 17-d periods, with 10 d for adaptation to diets and another 7 for data collection. Omasal digesta flow was obtained using Co-EDTA and indigestible NDF (iNDF) as markers, and to estimate ileal digesta flow only iNDF was used. From days 11 to 17 of each experimental period, ruminal infusions of Co-EDTA (5.0 g/d) and 15N (7.03 g of ammonium sulfate enriched with 10% of 15N atoms) were performed. There was no effect of CP contents (linear effect, P = 0.55 and quadratic effect, P = 0.11) on ruminal OM digestibility. Intake of CP linearly increased (P < 0.01) with greater dietary CP. The NH3-N (P < 0.01) and urinary N excretion (P < 0.01) increased in response to dietary CP, whereas retained N increased linearly (P = 0.03). Liquid-associated bacteria (LAB) in the omasum had greater N content (P < 0.05) in relation to the particle-associated bacteria (PAB). There was no difference between LAB and PAB (P = 0.12) for 15N:14N ratio. The 15N:14N ratio was greater (P < 0.01) in RI animals in relation to those fed at voluntary intake. Microbial CP had a quadratic tendency (P = 0.09) in response to CP increase. Microbial efficiency (expressed in relation to apparent ruminally degradable OM and true ruminally degradable OM) had a quadratic tendency (P = 0.07 and P = 0.08, respectively) to CP increasing and was numerically greatest at 120 g CP/kg DM. The adjusted equations for estimating true intestinal digestibility of MCP (Y1) and total CP (Y2) were, respectively, as follows: Y1 =--16.724(SEM = 40.06) + 0.86X(SEM = 0.05) and Y2 = -43.81(SEM = 49.19) + 0.75X(SEM = 0.05). It was concluded that diets with 120 g/kg of CP optimize the microbial synthesis and efficiency and ruminal ash and protein NDF digestibility, resulting in a better use of N compounds in the rumen. The PB technique can be used as an alternative to the 15N to estimate microbial synthesis.

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity

ABSTRACTThe objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Investigating the effects of sex of growing Nellore cattle and crude protein intake on the utilization of recycled N for microbial protein synthesis in the rumen by using intravenous 15N15N-urea infusion

The aim of this study was to investigate the effect of sex and levels of dietary crude protein (CP) on the recovery of 15N in microbial protein as well as to evaluate nitrogen balance compounds and variables related to urea renal handling in Nellore cattle. Four Nellore bulls and four Nellore heifers, fitted with rumen cannulas, were distributed in a two 4×4 Latin square design. The four experimental diets differed in levels of CP: 90.0, 110.0, 130.0, and 150.0g/kg on a dry matter basis. Continuous intravenous infusion of 15N15N-urea solution was provided at a rate of 0.30mmol urea/h followed by determination of 15N in ruminal bacteria, ammonia (NH3), and feces to determine the incorporation of recycled N in rumen bacteria. The increased dietary CP revealed a linear increase (P<0.05) in total digestible nutrients (TDN), total digestibility of CP and neutral detergent fiber (NDF), retained nitrogen, urinary excretion of urea-N, amount of filtered and absorbed urea (g/day), and plasmatic concentrations of urea-N. Fecal excretion of N presented a linear increase (P<0.05) in relation to dietary CP. Rumen NH3 concentration increased in a linear fashion (P<0.05), and rumen 15N-NH3 contents and fecal 15N contents decreased in a linear fashion (P<0.05) with increasing dietary CP. The regressions NH3=23.6183×e(0.0104×NI) and 15N-NH3=−0.00062×NI+0.1219 were obtained in order to calculate rumen NH3 concentrations (mg/l) and 15N-NH3 (atom percent excess), respectively, as a function of N intake (NI, g/day). The contents of total-N and 15N did not differ between liquid-associated bacteria (LAB) and particle-associated bacteria (PAB). There was no effect of sex (P>0.05) on the percentage of infused 15N that was recovered from feces and bacteria. There was no effect of dietary CP level on the percentage of 15N infused in plasma that was recovered from feces. However, there was a decreasing linear effect of the level of dietary CP (P<0.05) on the percentage of 15N infused in the jugular vein recovered from bacteria. We concluded that microbial protein synthesis was not affected by sex. The LAB and PAB had the same total-N content and recovery of infused 15N-urea. The greater recovery of infused intravenous 15N-urea on microbial protein synthesis in a low CP diet suggests greater urea recycling in association with this diet.

Quantification of ruminal microbiota and production of methane and carbonic dioxide from diets with inclusion of glycerin

The aim of this study was to quantify the liquid-associated bacteria and protozoa of the rumen and to evaluate the potential of in vitroproduction of gases CH4 and CO2 of diets with inclusion of glycerin for sheep. Two diets were formulated with a roughage:concentrate ratio of 20:80, according to the presence or absence of glycerin: G0 - control, without glycerin; and G10 - 10% glycerin as part of the concentrate. To quantify the liquid-associated bacteria and protozoa from the rumen and production of CH4 and CO2 gases, a completely randomized design was used. The diets had no effect (P&gt;0.05) on production and composition of liquid-associated protozoa and bacteria from the rumen of lambs. Likewise, a (P&gt;0.05) diet effect was not observed on production of CH4 and CO2in vitro, which presented average concentrations of 24.55mL/g MS and 51.52mL/g MS, respectively. The use of 10% glycerin did not alter production or composition of ruminal microflora, and moreover, did not negatively impact the production of CH4 and CO2 gases in vitro.

Comparison of three 15N methods to correct for microbial contamination when assessing in situ protein degradability of fresh forages.

The use of stable (15)N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using (15)N were compared: (15)N-labeled forage (dilution method, LF), (15)N enrichment of rumen solids-associated bacteria (SAB), and (15)N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The (15)N-labeled forages were obtained by fertilizing the plots where herbage was grown with (15)NH4 (15)NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that ((15)NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P < 0.001) between uncorrected N degradability values and those corrected for microbial contamination with all of the methods. Apparent N degradability of the low-N, high-fiber forage (late-cut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ disappearance curves is improved when values are corrected for microbial contamination of the bag residue.

A utilização de glicerina em dietas para bovinos altera a microbiota ruminal

Objetivou-se com este estudo avaliar a produção e composição das bactérias e protozoários ruminais de bovinos alimentados com glicerina. Cinco bovinos machos, providos de cânulas ruminais, foram distribuídos em delineamento experimental quadrado latino (5 x 5). As dietas foram formuladas com a inclusão de 0, 5, 10, 15 e 20% de glicerina na matéria seca (MS). Foram colhidas amostras de conteúdo ruminal nos tempos 0, 2, 5 e 8 horas após a alimentação e, em seguida, separaram-se as frações sólida e líquida para determinação das bactérias líquido-associadas (BLA), protozoários líquido-associados (PLA) e bactérias sólido-aderidas (BSA). Com o acréscimo da concentração de glicerina na dieta, houve redução linear na produção de MS das BLA e PLA (P&lt;0,05). No tratamento sem inclusão de glicerina, as quantidades de MS foram de 1.048,5 e 2.199,5 mg/L para BLA e PLA, respectivamente, enquanto no tratamento com 20% de glicerina foram, respectivamente, 756,9 e 1.619,9 mg/L para BLA e PLA. Além disso, houve redução nos teores de matéria orgânica (MO) e aumento linear do teor de nitrogênio (N) das BLA com o aumento da concentração de glicerina na dieta. A composição dos protozoários não foi alterada em função dos tratamentos (em média 47,3% MO e 7,2% N). A produção das BSA não foi alterada (P&gt;0,05) com o incremento da glicerina na dieta, e os valores médios para as quantidades de MS, MO e N foram, respectivamente, 3.131,0; 2.463,1 e 294,2mg/kg. Houve aumento linear no teor de N das BSA de 10,8 para 11,3% nos tratamentos com 0 e 20% de glicerina, respectivamente. Não foi verificado efeito do tempo de colheita para as BSA, ao passo que, para as BLA, ocorreu redução na produção de MO e nos teores de MO e N. A utilização de altas concentrações de glicerina na dieta de bovinos necessita de maiores estudos, pois há alteração da produção e composição dos microrganismos ruminais.

Microbial and chemical composition of liquid-associated bacteria in goats' rumen and fermenters

This study was undertaken to investigate the relationship between chemical composition and microbial profile of rumen liquid-associated bacteria (LAB) in vivo (Murciano-Granadina goats) and in a rumen simulation system (single-flow continuous-culture fermenters). To achieve this aim, analyses of purine bases along with some molecular techniques (quantitative PCR to assess abundance and DGGE to identify biodiversity and bacterial profile) were carried out. A control diet (AHC) based on alfalfa hay (AH) and concentrate (C) in a 1:1 ratio and two experimental diets (AHCBI and AHCBII), in which concentrate was partially replaced with multinutrient blocks, were used. Diets AHCBI and AHCBII included multinutrient blocks differing in the relative amount of two-stage olive cake and the source of protein (sunflower meal vs. fava beans). We aimed to investigate the effect of these blocks on rumen microbiota to evaluate their potential as safe substitutes of cereal-based concentrates. Similar patterns of response to diet were found for chemical composition, microbial abundances and diversity in LAB isolated from goat's rumen and fermenters. Whereas bacterial density (log10 gene copies/g FM: 11.6 and 9.4 for bacteria and methanogens, respectively, in rumen) and diversity indexes (Shannon index: 3.6) were not affected by diet, DGGE analyses showed that bacterial community profile was affected. The cluster analysis suggested differences in bacterial profile between LAB pellets isolated from the rumen of goat and fermenters. A relationship between chemical composition and bacterial community composition in LAB pellets seems to exist. Changes in the former were reflected in the bacterial community profile. Further research is needed to clarify the relationship between chemical and microbial composition of ruminal bacterial pellets with diets of different quality.

Effects of nitrogen underfeeding and energy source on nitrogen ruminal metabolism, digestion, and nitrogen partitioning in dairy cows1

This work aimed to investigate the effects of 2 levels of N (low or high) and 2 energy sources (starch or fiber) on N partitioning, N ruminal metabolism, and digestion in dairy cows. Four Holstein cows were used in a 4 × 4 Latin square design. The 4 cows (on average, 662 ± 62 kg and at 71 ± 10 d in milk at the beginning of the experiment) were fitted with rumen, proximal duodenum, and terminal ileum cannula. The cows received 4 diets having the same forage proportion on a DM basis. The high level of N supply met 110% of the protein requirements of cows with an adequate supply in rumen-degradable N. The low level covered 80% of these requirements with a shortage in rumen-degradable N. Energy sources differed by their nature (i.e., starch from barley, corn, and wheat or fiber from soybean hulls and dehydrated beet pulp). Duodenal digesta flow was determined using YbCl3 as a marker. Microbial duodenal N flow was determined using purine and pyrimidine bases as markers from liquid-associated bacteria and mixed bacteria samples. Microbial N flow and efficiency of microbial protein synthesis, calculated using mixed bacteria as a reference microbial sample, were not significantly modified by the N level (P = 0.19 and 0.29, respectively) and the energy source of the diet (P = 0.11 and 0.08, respectively). Total tract apparent digestibility of OM and total tact digestibility of NDF were lower at the low N level (P = 0.006 and 0.007, respectively). Total tract apparent digestibility of OM tended to be greater (P = 0.08) with high-starch diets than with high-fiber diets. Total tact digestibility of NDF was greater (P < 0.001) with high-fiber diets than with high-starch diets. Duodenal N flow was less (P = 0.001) at the low N level than high N level and tended to be greater (P = 0.06) with high-starch diets than with high-fiber diets. Daily output of N in urine was less (P < 0.001) at the low N level than at the high N level. Daily output of N in feces did not differ between low and high N levels (P = 0.24) and between high-starch and high-fiber diets (P = 0.17). Milk yield and protein yield were less (P = 0.002 and P = 0.013, respectively) at the low N level than at the high N level. Milk fat yield tended to be less (P = 0.09) at the low N level than at the high N level and with high-starch than with high-fiber diets (P = 0.06). In conclusion, a large reduction in dietary N led to reduced N excretion in urine and decreased milk production but did not affect N excretion in feces or microbial protein synthesis.

Use of a colon simulation technique to assess the effect of live yeast on fermentation parameters and microbiota of the colon of pig

The impact of 2 doses of a Saccharomyces cerevisiae were evaluated, 5 × 10(10) cfu/kg of feed (L1) and 5 × 10(11) cfu/kg of feed (L2) against a control (CON) with no added yeast, using an in vitro model [colon simulation technique (Cositec)] to mimic digestion in the pig colon. The L2 (but not L1) dose significantly improved DM digestibility compared to CON (61 v 58%) and increased NH(3) concentrations (+15%). Volatile fatty acid concentrations increased with L2 compared to CON--isobutyrate (+13.5%), propionate (+8.5%), isovalerate (+17.8%), and valerate (+25%)--but only valerate was increased with L1 (+14.2%). The analysis of microbiota from the liquid associated bacteria (LAB) and solid associated bacteria (SAB) revealed an interaction between the fraction and treatment (P < 0.05). Indeed, L2 had a significant impact on SAB and LAB (P < 0.01) whereas L1 only tended to change the structure of the population in the SAB (P < 0.1). Overall, this study showed that a live yeast probiotic could improve digestion in a colonic simulation model but only at the higher dose used and this effect was associated with a shift in the bacterial population therein.

Urinary excretion of purine derivatives, microbial protein synthesis, nitrogen use, and ruminal fermentation in sheep and goats fed diets of different quality1

The objective of this study was to compare N balance, microbial N flow (MNF) estimated from purine derivatives (PD) urinary excretion, and its variation when estimated using purine bases:N ratios in liquid associated bacteria (LAB) from models reported in the literature (MNF - response models) or measured ratios in liquid and solid-associated bacterial (SAB) pellets (MNF-LAB+SAB), diet digestibility, and rumen fermentation variables in sheep and goats fed 3 different practical, quality diets to study interspecies differences concerning N use as accurately as possible. Four mature female Merino sheep and 4 mature female Granadina goats, each fitted with a ruminal cannula, were used in 3 × 3 Latin square design with an extra animal. Two experimental diets had a forage-to-concentrate ratio of 70:30 (DM basis) with alfalfa hay (ALC) or grass hay (GRC) as forage, and the third diet contained 70% concentrate and 30% alfalfa hay (CAL). All animals were fed the diets at a daily rate of 56 g/kg BW(0.75) to minimize feed selection. Digestibility of nutrients was similar (P = 0.16 to 0.88) in the 2 species, but some animal species × diet interactions (P = 0.01 to 0.04) were detected. There were small differences between the fermentation patterns of both animal species. Goats showed decreased VFA concentrations (P = 0.005) and butyrate proportions (P = 0.04), and greater acetate proportions (P = 0.02) compared with sheep, whereas N intake and percentage of N intake excreted in feces were similar in both species (P = 0.58 and 0.15, respectively), the percentage excreted via the urine was greater in goats compared with sheep (P < 0.001). As a consequence, sheep had greater (P < 0.001) N retention than goats (averaged across diets, 32.6% and 16.1% of N intake, respectively). There were no differences (P = 0.95) between animal species in total PD excretion, but goats showed a greater excretion of allantoin (P = 0.01) and decreased excretion of xanthine (P = 0.008) and hypoxanthine (P = 0.007) compared with sheep. In general, differences between sheep and goats were more pronounced for the medium-quality diet (GRC) compared with those of high-quality diet (ALC and CAL). The greater urinary losses in goats would indicate a greater contribution of goats to N environmental contamination compared with sheep.

Effects of dietary non-ionic surfactant and forage to concentrate ratio on bacterial population and fatty acid composition of rumen bacteria and plasma of goats

This study was conducted to investigate effects of alkyl polyglycoside (APG) in diets that varied in forage to concentrate ratio (F:C) on ruminal microbial and plasma fatty acid (FA) composition and bacterial population in goats. A 2×2 factorial experiment was arranged within a 4×4 Latin square design using four wether goats fitted with permanent ruminal fistulae. Treatments included two levels of dietary F:C (40:60 vs. 60:40, on an air dry basis) and APG (no APG vs. 6.5g APG per day per animal). Dietary APG inclusion increased the proportion of iso-14:0 (P=0.03), and decreased the proportions of anteiso-17:0 (P=0.04) and monounsaturated fatty acids (MUFA) (P=0.04) in solid associated bacteria (SAB), but had no effect on FA in liquid associated bacteria (LAB) or in plasma. In addition, APG decreased (P<0.05) the Ruminococcus albus population. The major effects of APG were independent of F:C ratio. High dietary F:C decreased the proportions of 17:0 (P=0.01) and 9c12c18:2 (P=0.05) in LAB, decreased iso-13:0 (P=0.04) in SAB and 9c12c18:2 (P=0.05) in plasma, increased the proportions of anteiso-13:0 (P=0.04) in LAB, and 17:0 (P=0.0003) and 9c18:1 (P=0.03) in plasma, but had no effect on the ruminal bacterial population. These results indicate that, in both low and high F:C diets, dietary APG alter the FA composition of ruminal bacteria, which further imply a change in function and survival of rumen bacteria.

Composition of free and adherent ruminal bacteria: inaccuracy of the microbial nutrient supply estimates obtained using free bacteria as reference samples and 15N as the marker

Previous studies have indicated that 15N enrichment of solid-associated bacteria (SAB) may be predicted from the same value in liquid-associated bacteria (LAB). The aims of this study were to confirm this and to measure the error in the nutrient supply from SAB, when LAB are used as the reference sample. For this purpose, the chemical and amino acid (AA) compositions of both the bacterial populations were studied in four experiments carried out on different groups of three rumen cannulated wethers. Diets (one in Experiments 1 and 4 and three in Experiments 2 and 3) had forage-to-concentrate ratios (dry matter (DM) basis) between 2 : 1 and 40 : 60, and were consumed at intake levels between 40 and 75 g DM/kg (BW)0.75. The bacteria samples were isolated after continuous infusion of (15NH4)2SO4 (40, 18, 30 and 25 mg 15N/day, in Experiments 1 to 4, respectively) for at least 14 days. In all experiments, SAB had consistently higher concentrations of organic matter (826 v. 716 g/kg DM, as average) and total lipids (192 v. 95 g/kg DM, as average) than LAB. Similar CP concentrations of both populations were observed, except a higher concentration in SAB than in LAB in Experiment 3. A consistent (in Experiment 4 only as tendency) higher AA-N/total N ratio (on average 17.5%) was observed in SAB than in LAB. The 15N enrichment in SAB was systematically lower than in LAB. On the basis of the results of all studies a close relationship was found between the 15N enrichment in SAB and LAB, which was shown irrespective of experiments. This relationship was established from Experiments 1 and 2 and the above cited previous results (n = 20; P < 0.001; R2 = 0.996), and then confirmed from the results of Experiments 3 and 4. These relationships between SAB and LAB demonstrate that CP supply from SAB is underevaluated by, on average, 21.2% when LAB are used as the reference. This underevaluation was higher for true protein and even higher for the lipid supply (32.5% and 59.6%, respectively, as an average of the four experiments). Large differences in AA profile were observed between SAB and LAB. The prediction equation obtained using 15N as the marker may be used to correct the errors associated with the traditional use of LAB as the reference sample, and therefore to obtain more accurate estimates of the microbial nutrient supply to the ruminants.

Effects of alkyl polyglycoside, a nonionic surfactant, and forage-to-concentrate ratio on rumen fermentation, amino acid composition of rumen content, bacteria and plasma in goats

In the present study, the effects of different forage-to-concentrate ratios (F:C) and an alkyl polyglycoside (APG) supplementation on parameters of rumen and blood metabolism were investigated in goats. A 2 × 2 factorial experiment was arranged within a 4 × 4 Latin square design (four 22-day periods), using four wether goats equipped with permanent ruminal cannulas. The experimental diets included two F:C levels (40:60 vs. 60:40), and two APG supplementation levels (None or 13 ml APG daily per animal). Rumen contents and blood samples were collected at the end of each period. Dietary F:C alteration affected plasma urea and influenced the proportions of leucine, histidine, arginine, glycine, proline, alanine, valine, phenylalanine, cysteine and tyrosine in rumen content, and the proportions of methionine, threonine and proline in solid-associated bacteria (SAB) significantly. Dietary APG decreased the proportions of valine and phenylalanine in rumen content, and the histidine content of liquid-associated bacteria. The interaction between dietary F:C and APG was significant for the proportions of glycine and alanine in rumen content, and the proportions of lysine and threonine in SAB. The proportion of lysine was greater, but the proportion of threonine was less in SAB for goats fed high F:C diet without APG supplementation. The proportions of plasma free amino acids and glucose concentration were not affected by experimental treatments. These results indicated that dietary APG addition affected the amino acid composition of the rumen content and ruminal bacteria, but this depended on the dietary F:C ratio. It is necessary to validate the effectiveness of dietary APG supplementation in further studies with more animals.

Effect of chestnut and quebracho tannins on fatty acid profile in rumen liquid- and solid-associated bacteria: an in vitro study

Tannins are phenolic compounds that interfere with biohydrogenation (BH) of polyunsaturated fatty acids (FAs). The aim of the present in vitro study was to investigate the effects of two different sources of tannins on FA profiles of rumen bacteria, with particular reference to rumenic and vaccenic acid. A control diet (C; composed of 300 g/kg of wheat straw, 132 g/kg of soyabean meal, 96 g/kg of barley meal, 152 g/kg of maize meal, 300 g/kg of maize gluten and 20 g/kg of mineral vitamin premix, all expressed on dry matter (DM)) and four diets, obtained by adding to C two different types of tannins from chestnut (TC) and from quebracho (TQ) at two concentration levels (49 and 82 g/kg DM), were compared. The content of the main unsaturated FAs (C18:1 cis9, C18:1 trans11, C18:2 cis9, cis12 and C18:3 cis9, cis12, cis15) from solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was affected by the presence of tannins in the diets. In particular, C18:1 trans11 content was significantly increased, especially with TC1, whereas the decreasing of C18:1 cis9 was unaffected, regardless of the presence or the kind of tannins added to feeds. SAB contained higher amounts of intermediates of polyunsaturated FA BH (as C18:1 trans11 and C18:2 cis9, trans11) than LAB that were characterized by a higher amount of C18:0. In the concentration range adopted in this study, the effect of TC and TQ on changes of bacterial FA profile was comparable. Tannins seem to be a good means to modulate the FA profile of rumen bacteria, favouring the accumulation of C18:1 trans11 during in vitro rumen fermentation.

Effects of partial replacement of concentrate with feed blocks on nutrient utilization, microbial N flow, and milk yield and composition in goats

Two experiments were carried out to evaluate the effect of partial replacement of concentrate with 2 types of feed blocks (FB) on rumen protozoa numbers, nutrient utilization, microbial N flow to the duodenum, and milk yield and composition in goats. The concentrate included oat, corn grain, barley, soybean meal, salt, and vitamin-mineral mixture. The FB (types I and II) were composed of crude 2-stage olive cake (120 or 100g/kg), fava beans (0 or 400g/kg), barley (320 or 200g/kg), beet molasses (220 or 100g/kg), sunflower meal (180 or 0g/kg), quicklime (70 or 90g/kg), salt (60g/kg), urea (0 or 20g/kg), and vitamin-mineral mixture (30g/kg). In experiment 1, 6 adult, dry, nonpregnant, rumen-fistulated Granadina goats (46.9±2.15kg of BW) were used and 3 trials were carried out. In each trial, 2 goats were randomly assigned to receive 600g of alfalfa hay and 400g of concentrate (diet AC), 600g of alfalfa hay, 200g of concentrate, and FB I (diet ACBI), or 600g of alfalfa hay, 200g of concentrate, and FB II (diet ACBII) with 6 replications per diet. The FB were supplied ad libitum. The ratio of purine bases to N was higher in solid- and liquid-associated bacteria for FB goats than for AC goats. In experiment 2, 18 Granadina goats (39.6±1.89kg of BW) in the middle of the third lactation were used, and 3 trials were carried out by following a 3 × 3 Latin square experimental design. In every trial, 6 animals randomly received 1.0kg of alfalfa hay supplemented with 1.0kg of concentrate (diet AC) or 0.5kg of concentrate and FB I and II (diets ACBI and ACBII) with 18 replications per diet. The FB were supplied ad libitum. The intakes of organic matter and fat were higher with the AC diet than with the FB diets. The intake of acid detergent fiber was higher for FB-containing diets than for the AC diet. The neutral detergent fiber digestibility of FB diets was higher than that of the AC diet. Energy intake was higher for diets AC and ACBII than for ACBI. Microbial N flow was affected by diet. Milk yield was higher in goats fed the AC diet than in those receiving the FB diets. Conjugated linoleic acid content was higher in milk from FB than in milk from AC goats. Our study suggests that FB type II based on local ingredients could be used advantageously to reduce half of the amount of concentrate without detrimental effects on nutrient utilization, N value of the diet, and milk composition. The decrease of milk yield with ACBII compared with that obtained with the AC diet could be compensated by better quality of milk, decreased cost of feeding, and environmental advantage derived of including by-products in FB.

Effect of live yeast culture supplementation on hindgut microbial communities and their polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber or high-starch diet

Four cecum and right ventral colon-fistulated horses were assigned in a 4 x 4 Latin square design and fed a high-fiber (HF) or a high-starch (HS) diet with or without 10 g of Saccharomyces cerevisiae (SC; CBS 493.94) containing 4.5 x 10(9) cfu/g. The HF and HS diets consisted of pelleted feeds and long wheat straw (18.0 and 3.5 g of DM.kg(-1) of BW.d(-1), respectively) given in 2 equal meals to provide an NDF:starch ratio of 3.5 and 1.0, respectively. After a 21-d adaptation period intestinal contents were collected 4 h after the morning meal on d 23 and 25 to determine bacterial and SC concentrations. Polysaccharidase activities (CMCase, xylanase, amylase) and activities of glycoside hydrolases (alpha-l-arabinosidase, beta-d-cellobiosidase, beta-d-glucosidase, beta-d-xylosidase) were determined in liquid-associated bacteria (LAB) and solid-adherent bacteria (SAB) isolated from both compartments. Lactobacilli were increased in the cecum (P = 0.012) and colon (P = 0.086) when starch intake increased, whereas total anaerobes, cellulolytics, and streptococci did not change in either compartment. In yeast-supplemented horses, SC concentrations were greater in cecum (4.4 x 10(6) cfu/mL) than in right-ventral colon (5.6 x 10(4) cfu/mL) and did no change with diet. Concentrations of lactobacilli and lactic-acid utilizers were greater (P = 0.099 and 0.067, respectively) in the cecum but remained similar in the colon of SC-supplemented horses. The CMCase activities of SAB were not affected by diet. Colonic xylanase activities of SAB were reduced (P = 0.046) by starch addition, but no change was seen in the cecum. All SAB glucoside hydrolase activities in the cecum and colon, except beta-d-xylosidase in the cecum, were decreased when starch intake was increased. The LAB CMCase (P = 0.049 in the colon) and xylanase (P = 0.021 in the cecum; P < 0.001 in the colon) activities decreased with starch intake. No effect of starch on LAB or SAB amylase activity was observed. Addition of SC improved SAB CMCase in the cecum (P = 0.019) and colon (P = 0.037) as well as beta-d-cellobiosidase (P = 0.002) and beta-d-glucosidase (P = 0.041) in the cecum. Only xylanase in the cecum (P = 0.015) and beta-d-xylosidase in the cecum (P = 0.028) were improved with SC, whereas colonic LAB alpha-amylase activity was significantly decreased (P = 0.046). Most enzymes involved in plant cell wall digestion were increased after SC addition. This fact may contribute to explain a better digestion of fiber that has been previously reported in SC-supplemented horses.