Estimation of microbial nitrogen in nylon-bag residues by feed15N dilution

1985 ◽  
Vol 54 (2) ◽  
pp. 473-481 ◽  
Author(s):  
T. Varvikko ◽  
J. E. Lindberg

1. Rapeseed (Brussicu nupus), barley grain, ryegrass (Loliurnperenne) and barley straw were labelled with15N as an internal marker. The dilution of15N was used to estimate microbial N (RMN15N) in the feed residues in nylon bags incubated in the rumen for 5, 12 and 24 h. For comparative purposes, diaminopimelic acid (DAP) content of the residues was also determined and rumen microbial N (RMNDAP-N) in the feed residues calculated using DAP as a bacterial marker. The influence of two bag pore sizes (20 μm and 40 μm) with different sample sizes (1 g and 5 g respectively) was also studied.2. For all feeds, the average disappearance of15N was faster than that of total N, the difference between N and15N disappearance being marked with barley, ryegrass and barley straw. The disappearance of microbially corrected dry matter (DM; correction calculated from the15N values) was, accordingly, always faster than the uncorrected DM disappearance. Except for the bag pore/sample size effect for N disappearance, significant (P < 0.01–0.001) feed, pore/sample size and incubation-time effects were always found for the disappearance values.3. Errors (%) resulting from the microbial contamination (calculated from the15N values) in N-loss measurement with rapeseed, barley, ryegrass and barley straw, at 5, 12 and 24 h in 20 μm bags were respectively: –1.8, –3.9, –0.9; –3.8, –22.4, –3.8; –7.2, –4.1, –2.9; –164.5, –146.3, –204.6. In 40 μm bags the corresponding errors were respectively: –4.4, –1.2, –0.7; –26.1, –10.5, –3.9; –13.2, –6.4, –5.5; –221.2, –310.1, –1284.6.4. The largest residual proportions of RMN15N, RMNDAP-Nand DAP-N (% of total N) were found in barley straw, followed by barley, ryegrass and rapeseed, in that order. RMN15N(g/kg residual DM) followed the descending order: barley, ryegrass, straw, rapeseed. RMNDAP-N, (g/kg residual DM) and DAP (mg/kg residual DM) followed the descending order: barley, ryegrass, rapeseed and barley straw. Feed, pore/sample size and incubation-time effects were always significant (P < 0.001).5. With 40 μm bags RMN15Nvalues of barley, ryegrass and barley straw (expressed as % of total N or g/kg residual DM) were substantially higher than those of RMNDAP-NWith 20 μm bags the RMN15Nand RMNDAP-Nvalues were generally quite close for these feeds. With rapeseed residues, RMN15Nwas clearly lower than RMNDAP-Nwith 20 μm bags, but only small differences were found with 40 μm bags.6. A more diverse microbial colonization of feed samples was indicated in the 40 μm bags compared with the 20 μm bags. It was also noticed, consequently, that in most cases larger error resulted from the microbial contamination in 40 μm bags.

1978 ◽  
Vol 39 (1) ◽  
pp. 165-179 ◽  
Author(s):  
J. R. Ling ◽  
P. J. Buttery

1. Three sheep, each fitted with a ruminal cannula and duodenal re-entrant cannulas were given three isonitrogenous, isoenergetic diets in a Latin-Square design. Each diet contained (/kg) approximately 400 g N as white fish meal, soya-bean meal or urea and approximately 600 g dry matter (dm) was barley grain. The diets were fed continuously and supplied about 28 g N/d.2. Total duodenal digesta was collected manually for 72 h and the proportions of microbial N in that digesta were simultaneously estimated for all sheep using RNA, radioactive sulphur (35S), diaminopimelic acid (DAPA) and aminoethylphosphonic acid (AEPA) as markers.3. Three of the estimation methods showed that the variable source of dietary N had the greatest (RNA P < 0.05, 35S P < 0.005, DAPA P < 0.1) effect on the proportions of microbial N in duodenal digesta, though differences between sheep accounted for some variation.4. These methods also ranked the diets in the order: urea > soya-bean meal > fish meal with respect to the proportions of digesta N that were microbial in origin; the respective mean values for these diets with the different markers were: RNA 0.98, 0.70, 0.56; 35S 0.92, 0.64, 0.54; DAPA 0.80, 0.47, 0.42.5. AEPA was found to be present in substantial quantities not only in isolated rumen protozoa, but also in dietary and bacterial material; an observation that invalidated its further use as a protozoal marker.6. Calculations using values obtained from the 35S procedure showed that the proportions of dietary N degraded within the rumen were 0.38, 0.43 and 0.89 for the white fish meal, soya-bean meal and barley respectively.7. The marker methods are compared and their advantages and disadvantages (real and apparent) are discussed. It is concluded that where microbial N estimates of a more general and comparative nature are required, the use of RNA as a marker is probably adequate. Where information for more exacting purposes is required, the use of 35S appears to be more appropriate.


1984 ◽  
Vol 51 (1) ◽  
pp. 77-83 ◽  
Author(s):  
A. B. McAllan ◽  
R. H. Smith

1. Protozoa-free steers with simple rumen and abomasal cannulas were given basal diets consisting of a concentrate mixture of flaked maize and tapioca with barley straw (BS) or alkali-treated barley straw (BSA). Other diets were supplemented with urea (BSU and BSAU respectively) or with fish meal replacing the tapioca (BSF and BSAF respectively). The diets were isoenergetic and calculated to provide sufficient metabolizable energy (ME) to support a growth rate of 0.5 kg/d. Rumen-degradable nitrogen (RDN): ME values (g/MJ) were estimated to be 0.50, 1.20 and 0.80 for the basal diet, urea- and fish-meal-supplemented diets respectively. RNA and α, ε-diaminopimelic acid (DAP) were used as microbial markers. 103Ruthenium and polyethylene glycol (PEG) were given as flow markers and flows (g/24 h) at the abomasum of organic matter (OM) and nitrogenous constituents were calculated.2. Samples of mixed bacteria separated from rumen digesta from animals receiving N-supplemented diets contained significantly more N than those from animals receiving basal diets (approximately 74 and 62 mg/g dry matter (DM) respectively) but there were no other significant differences in total-N contents between treatments. RNA-N: total-N values were similar for all diets (approximately 0.13). DAP-N: total-N values were significantly lower in bacteria from animals receiving alkali-treated (AT) rather than untreated (UT) straw (approximately 0.008 and 0.011 respectively).3. The proportion of OM intake digested in the rumen (ADOM) was significantly higher for animals receiving AT straw rather than UT straw (approximately 0.54 and 0.43 respectively). N supplementation had no effect on OM digestibility.4. When basal rather than N-supplemented diets or AT-straw- rather than UT-straw-containing diets were given, there were significantly lower flows of ammonia-N, non-ammonia-N (NAN) and microbial-N (based on RNA flow, MN(RNA)) at the abomasum. Mean daily MN(RNA) flows (g) were 21, 30, 31, 16, 27 and 28 for diets BS, BSU, BSF, BSA, BSAU and BSAF respectively. These correspond to estimated efficiencies of microbial protein synthesis, expressed as g MN(RNA) /kg truly-digested OM, at 14, 22, 22, 12, 18 and 19 respectively. Values were significantly lower for basal as compared with corresponding N-supplemented diets and for AT-straw diets as compared with corresponding UT-straw diets.5. Estimated mean proportions of total feed-N intake degraded in the rumen, based on MN(RNA) as microbial marker, of diets BS, BSU and BSF were 0.60, 0.74 and 0.47 respectively; corresponding values for diets BSA, BSAU and BSAF were 0.72, 0.73 and 0.36 respectively. Making certain assumptions, the mean proportions of fish-meal-N digested in the rumen were calculated to be 0.23 and 0.14 respectively for UT- and AT-straw diets. The values were not significantly different. Values for microbial flows based on DAP as marker were significantly lower, by about 25%, than those based on RNA.


1986 ◽  
Vol 56 (1) ◽  
pp. 131-140 ◽  
Author(s):  
T. Varvikko

1. In the previous work (Varvikko & Lindberg, 1985), 15N-labelled rapeseed (Brassica napus), barley, ryegrass (Lolium perenne) and barley straw were incubated in the rumen in nylon bags for 5, 12 and 24 h and microbial nitrogen in the residues was quantified using the feed 15N-dilution method. In the present study, residual amino acids (AA) of these feeds were analysed, and microbially corrected AA of feed origin (feed AA) were estimated as the difference between total residual AA and respective microbial AA, assuming a constant AA composition for the microbial protein.2. In barley and barley-straw residues, and also in ryegrass incubated in the rumen for 24 h, very large enrich- ment by microbial N and AA-N was found. The microbial enrichment was rather small in rapeseed residues and ryegrass incubated for 5 or 12 h. During the rumen incubation, feed N and AA-N (g/kg feed dry matter (DM)) decreased very clearly in all the feeds, and feed and incubation time effects were always statistically significant (P < 0.001).3. The slow degradation of essential (E) feed AA compared with the respective non-essential (NE) AA degradation increased the proportion of feed EAA (g/kg determined feed AA) in barley and barley-straw residues. In rapeseed and ryegrass, residual feed EAA: NEAA remained very similar to the original. Branched-chain (Br) AA tended to increase proportionally in all the feed residues, suggesting these AA to be, on average, more resistant against microbial degradation in the rumen than other AA. Similarly, lysine was clearly increased in barley residues. A rumen degradation faster than the average rate caused decreased residual feed glutamic acid in rapeseed; methionine, alanine and glycine in barley; arginine and alanine in ryegrass; and methionine, asparagine and tyrosine in barley straw. Feed and incubation time effects were significant (P < 0.054–001) for feed AA (g/kg determined feed AA) grouped as EAA, BrAA or NEAA, and for most individual AA, as well as for feed AA disappearance (%) and relative amounts (%) of feed AA in the respective residual AA.4. According to present findings, AA composition of the rumen-undegraded vegetable feed residues may markedly differ, either quantitatively or qualitatively (or both), from their original AA composition. When determining the feed AA composition of nylon-bag residues, the microbial error may be very large with starchy or fibrous feeds of low protein content. The microbial AA do not, however, considerably confuse the AA determination of protein-rich feeds.


1997 ◽  
Vol 65 (2) ◽  
pp. 237-245 ◽  
Author(s):  
J. F. Pérez ◽  
J. Balcells ◽  
J. A. Guada ◽  
C. Castrillo

AbstractFour ewes fitted with ruminal and duodenalT-piece cannulas were given fourdietsin a 4 × 4 factorial design. Diets consisted of 700 (HF) or 400 (LF) g/day of ammonia-treated barley straw supplemented respectively with 150 or 600 g/day of concentrate made up with barley plus either soya-bean meal (SBM) or fishmeal (FM) as the protein source, offered at 2-h intervals. Duodenal flowsof digestawere estimated by the dual-phasetechniqueusing CoEDTAand Yb-acetate as markers and (15NH4)2SO4 was infusedinto the rumento label microbial N. Bacteria were isolated from the liquid (LAB) or solid (SAB) rumendigesta. Purinebases (PB) were isolated by precipitationin an acid solution of AgN03, and microbial contribution either to the duodenalnitrogen(N) or PB were determinedby 15N measurements induodenaldigesta and bacteria.Simultaneously, therumen degradation of Nand PB contained in SBM and FM was studiedby incubating supplements in polyesterbags in the rumen.PBcontent (mmol/g dry matter)and guanine: adenine(G/A) ratio of barley strawwas 2·89 and 5·23; barley grain,7·91 and 111;SBM, 18·8 and 1·26; and FM, 58·9 and 6·96, respectively. Duodenal flow ofPB(mmol/day)was significantly higher than PB intake on all diets and G/A ratio showed a meanvalue of 0·97, similarto the ratios determined in SAB(0·80) and LAB (1·04) and muchlower than diets(1·31 to 4·32). Microbial contribution to duodenal Nflow ranged from43·3% to 61·0%, beinghigherin SBM(59·0%)thanin FM(46·7%)diets. However, microbial contribution to duodenal PB was not affected by the experimentaltreatment, accounting for proportionately 0·77 of total PB at the duodenum. Rumen degradability of PB was much higher than that of total N and in both cases degradability was higher in SBM than FM. Direct measurements of non-microbialN were significantly higher than values determined by the polyester-bagmeasurements. However, once corrected forthe endogenousN (52 mgN per kg live weight)contribution, results show edan acceptable agreement. Duodenal flow of PB non-attributable to microbes (unlabelled PB) showed a mean value of 3·25 mmol/daywithouta significanteffect of dietary treatment. However, undegradablePBsupply determinedfor0·02, 0·05 and 0·08 per h fractional out flow rates were proportionately lower than 0·025 with SBM and 0·100 with FM diets of the estimated duodenalPB flow. Despite the magnitudeof the unlabelledduodenalPB, the close agreement between G/A ratios in duodenaldigesta and bacteria suggests thatthe contribution of dietary PB to the duodenalflow was low and seemsto confirm the reliability of values obtained from polyester-bag measurements.


1988 ◽  
Vol 60 (2) ◽  
pp. 339-353 ◽  
Author(s):  
J. M. Dawson ◽  
C. I. Bruce ◽  
P. J. Buttery ◽  
M. Gill ◽  
D. E. Beever

1. Ryegrass (Lolium perenne cv. Cropper) silage was given to four Friesian steers (initial live weight (LW) 172 kg) alone or with a fishmeal supplement (150 g fresh weight/kg silage dry matter (DM)) in a balanced two- period change-over design. The dietary components were the same as those used in a recent experiment by Gill et al. (1987). All diets were offered hourly at 24 g DM/kg LW.2. Fishmeal supplementation increased dietary nitrogen intake (P < 0.01) and significantly increased the flow of total N (P < 0.01), non-ammonia N (NAN) (P < 0.01) and amino acids (P < 0.05) at the duodenum. The increased supply of NAN to the duodenum was due largely (67%) to increased flow of undegraded dietary protein.3. Microbial protein production was estimated simultaneously with 15N, diaminopimelic acid (DAPA) and a novel technique using L-[4,5-3H]leucine. Estimates varied with the marker and source of microbial isolate but mean values indicated that microbial N flow was significantly increased by fishmeal supplementation (P < 0.05). The use of L-[4,5-3H]leucine as a microbial marker is justified and its possible advantages over other markers are discussed.4. The efficiency of microbial protein synthesis was significantly increased from 30.8 g N/kg organic matter apparently digested in the rumen (OMADR) to 54.3 gN/kg OMADR by fishmeal supplementation (P < 0.01). However, this indicates that relatively high efficiencies can be achieved with unsupplemented high quality silage supplied continuously. Rumen degradable N (RDN) supply was significantly increased by fishmeal supplementation (P < 0.05) but apparent efficiency of capture of RDN by rumen microbes was not significantly increased.5. Attempts were made to investigate the source of N utilized by the microbes on the two diets by intrarumen infusions of (15NH4)2SO4 and L-[4,5-3H]leucine but these were confounded by rumen-mixing problems. Findings obtained suggest that a lower proportion of microbial N may have been derived from rumen ammonia when the silage was supplemented with fishmeal but no differences in the extent of direct incorporation of leucine into microbial protein were observed. This could indicate an increase in microbial peptide uptake on the fishmeal-supplemented diet. However, evidence was also obtained suggesting that the improvement in microbial protein synthetic efficiency with supplementary fishmeal was also due to the provision of a more continuous supply of nitrogenous substrates for microbial growth, as a result of hourly feeding.6. The results are related to the increased growth responses attained by fishmeal supplementation of this silage in the experiment of Gill et al. (1987).


1983 ◽  
Vol 49 (3) ◽  
pp. 419-432 ◽  
Author(s):  
J. D. Sutton ◽  
R. Knight ◽  
A. B. McAllan ◽  
R. H. Smith

1. Six wether sheep were each provided with a permanent cannula in the rumen and re-entrant cannulas in the proximal duodenum.2. In a preliminary study, the sheep consumed 200 g hay and 400 g concentrates supplemented with up to 40 g linseed oil, coconut oil or cod-liver oil daily. Feed was refused at higher levels of supplementation.3. Five of the sheep were used in a 5 × 5 Latin-square experiment. They were given 200 g hay and 400 g concentrates alone (B) or supplemented with 40 g linseed oil (L), coconut oil (C), protected linseed oil or protected coconut oil daily. The protected oils were prepared by emulsifying the free oils with formaldehyde-treated sodium caseinate. Formaldehyde-treated sodium caseinate was also included in the other three diets.4. Digestion in the stomach was measured by spot sampling duodenal digesta, using chromic oxide-impregnated paper as the marker. Microbial flow at the duodenum was measured by use of both diaminopimelic acid (DAPA) and RNA as microbial markers.5. Both the free oils had broadly similar effects despite their very different fatty acid compositions. Digestion in the stomach of organic matter (OM) was reduced from 0·48 (diet B) to 0·29 (diets L and C) and that of neutral-detergent fibre from 0·50 (diet B) to 0·19 (diet L) and 0·12 (diet C). The molar proportions of acetic acid and n-butyric acid were decreased and that of propionic acid was increased. Protozoal numbers were reduced by 78% (diet L) and 90% (diet C). The flow of total nitrogen and microbial N was increased by both oils and the efficiency of microbial protein synthesis (g N/kg OM apparently digested in the rumen) was increased from 30 (diet B) to 85 (diet L) and 74 (diet C) when based on DAPA and from 41 (diet B) to 94 (diet L) and 81 (diet C) when based on RNA. The efficiency when based on true digestion of OM (g N/kg OM truly digested in the rumen) was increased from 23 (diet B) to 46 (diet L) and 44 (diet C) when based on DAPA and from 29 (diet B) to 49 (diet L) and 46 (diet C) when based on RNA. The amounts of microbial OM (g/d) at the duodenum were increased from 68 (diet B) to 124 (diet L) and 106 (diet C) when based on DAPA and from 92 (diet B) to 136 (diet L) and 115 (diet C, non-significant) when based on RNA.6. When the oils were given in the protected form, the effects on digestion in the stomach were reduced but not eliminated. No significant increases in the amount of total N or microbial N at the duodenum were established, though there was a tendency for an increase in the efficiency of microbial protein synthesis with protected linseed oil. The results suggested that the method of protection used reduced the effects of the oils on rumen digestion and synthesis but was only partially successful in preventing hydrogenation of the fatty acids.7. It is concluded that free oils can markedly increase the efficiency of microbial protein synthesis, possibly by their defaunating effect, and that this may enhance the potential for using non-protein-N on oil-supplemented diets.


2001 ◽  
Vol 86 (6) ◽  
pp. 717-724 ◽  
Author(s):  
M. J. Ranilla ◽  
M. D. Carro ◽  
S. López ◽  
C. J. Newbold ◽  
R. J. Wallace

Incubations were carried out with a batch culture system to study the effects of different N sources on the fermentation by ruminal micro-organisms from Merino sheep of two fibre substrates derived from feedstuffs that differed in their fermentation rate. The substrates were neutral-detergent fibre (NDF) from barley straw and sugarbeet pulp. N sources were ammonia (NH4Cl) and peptides (Trypticase). Three treatments were made by replacing ammonia-N with peptide-N at levels of 0 (AMMO), 33 (PEPLOW) and 66 % (PEPHIGH) of total N. There were no differences (P>0·05) between treatments in NDF degradation for both the barley straw and the sugarbeet pulp. Peptides increased (P<0·05) total volatile fatty acids daily production for both substrates, with greater values (P<0·001) for PEPHIGH than for PEPLOW for the sugarbeet pulp. The presence of peptides also increased (P<0·05) microbial N synthesis compared with AMMO, with PEPHIGH supporting more growth (P<0·001) than PEPLOW when the sugarbeet pulp NDF was fermented. The presence of peptides increased (P<0·01) the amount of solids-associated micro-organisms (SAM)-N for both the barley straw and the sugarbeet pulp fibres, values in the PEPHIGH treatment being higher (P<0·001) than those in PEPLOW. The proportion of SAM-N in the total microbial N was not affected (P>0·05) by the presence of peptides compared with the AMMO treatment, but values were greater for the PEPHIGH compared with the PEPLOW N source, reaching statistical significance (P<0·05) only for the sugarbeet pulp. For liquid-associated micro-organisms, the AMMO treatment resulted in the greatest (P<0·05) proportion of N derived from ammonia for both substrates, with a further decrease (P<0·01) for the PEPHIGH treatment compared with the PEPLOW for the sugarbeet pulp, indicating preferential uptake of peptides when they were available. Microbial growth efficiency (g microbial N/kg NDF degraded) was not affected (P>0·05) by N source. These results indicate that N forms other than ammonia are needed for maximal growth of fibre-digesting ruminal micro-organisms.


1971 ◽  
Vol 38 (1) ◽  
pp. 33-42 ◽  
Author(s):  
P. Jackson ◽  
J. A. F. Rook ◽  
K. G. Towers

Summary(1) Two wether sheep fitted with ruminal and duodenal re-entrant cannulas were used to study the influence of the physical form of a barley grain and barley straw diet and intraruminal addition of ammonium salts (mainly acetate) on digestibility of dietary constituents, the flow of digesta to the duodenum and the composition of digesta from the rumen and duodenum.(2). Grinding and pelleting of the diet depressed the digestibility of crude fibre and increased that of the nitrogen-free extract and addition of ammonium salts increased the digestibility of crude fibre. The effects of the physical form of the diet on the composition of the short-chain fatty acids of rumen liquor were not consistent and the addition of ammonium salts produced changes over and above those attributable to the small amounts of acids in the mixture. The extreme values observed for the molar proportion of propionic acid were 12·3 and 38·1% and the corresponding values for η-butyric acid were 28·1 and 9·0%.(3). Variations in the flow of nitrogenous materials to the duodenum were related more to the pattern of fermentation established in the rumen than to the experimental treatments. There was a highly significant relationship between the molar proportion of propionic acid and the abomasal output of nitrogen and also the abomasal output of α-ε-diaminopimelic acid and α-linked glucose polymers. The amino acid composition of duodenal digesta differed from that of the diet—in particular the proportion of glutamic acid was decreased and the proportions of aspartic acid, alanine, lysine and histidine were increased—but differences in composition between treatments and between animals were small.(4). The faecal output of nitrogen differed little between animals and between treatments, but nitrogen retention was significantly increased during the intraruminal infusion of ammonium salts.


2005 ◽  
Vol 81 (1) ◽  
pp. 57-65 ◽  
Author(s):  
A. R. Askar ◽  
J. A. Guada ◽  
J. Balcells ◽  
A. de Vega ◽  
C. Castrillo

AbstractThe origin of post-ruminal purine bases (PB) was studied in 24 growing lambs that were given a pelleted concentrate plus barley straw (C) or whole barley grain plus protein supplement (WB). Six lambs from each treatment were slaughtered at 10 and 30 days post weaning after15N labelling of microbial nitrogen (N) and PB. Microbial contribution to digesta non-ammonia N (NAN) and PB was lower (P< 0·01) when estimated from duodenal rather than abomasal samples (0·36 v. 0·52 (s.e.d. 0·021) for NAN and 0·47 v. 0·77 (s.e.d. 0·029) for PB) as a result of endogenous contamination. In comparison with15N, total PB/N led to higher estimates (P< 0·01) of microbial contribution to abomasal NAN in WB treatment (0·62 v. 0·46 s.e.d. 0·049). The difference was removed after correcting for microbial PB, while this effect was not observed with < the C diet, resulting in a marker by diet interaction (P< 0·05). Abomasal PB flow increased (P< 0·1) from 10 to 30 days after weaning mainly due to the higher proportion of microbial PB (0·70 v. 0·81 (s.e.d. 0·047)). Rumen apparent PB degradation did not differ between diets in older lambs, but it was proportionally 0·39 lower for WB treatment (P< 0·05) in younger lambs. When the microbial PB flow was estimated indirectly from labelled microbial N and the PB/N ratio of bacterial extracts the estimates were in agreement with those derived from PB-15N in the WB treatment but resulted in unrealistic values in lambs on diet C. Results suggest that significant proportions of dietary PB can escape rumen degradation which may lead to overestimation of microbial contribution to abomasal NAN when the PB/N ratio is used as marker. The extent of the overestimation is affected by the lamb age and grain processing.


1987 ◽  
Vol 67 (4) ◽  
pp. 1143-1148 ◽  
Author(s):  
B. A. CROOKER ◽  
J. H. CLARK ◽  
R. D. SHANKS ◽  
G. C. FAHEY Jr.

Seven feeds were individually incubated (12 h) in ruminally suspended polyester bags. Several alterations (P < 0.05) between individual amino acid compositions of feeds and their respective residues were detected after adjusting for microbial contamination of residues. Detection of diaminopimelic acid in feeds suggests that not all diaminopimelic acid in digesta originates from bacteria. Key words: Amino acid, ruminal degradation, microbial attachment, diaminopimelic acid


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