We review key findings of one the most cited papers in the 75-year history of BJN. We then identify important consequent developments, as well as opportunities to use analytical and molecular biology advances to maximise conversion of non-protein nitrogen into microbial protein.

The current study was carried out to examine the effect of cottonseed processing and chitosan supplementation on lamb performance, digestibility and nitrogen digestion. Eighty uncastrated Santa Inês lambs (23 ± 2.2 kg average weight, 4 months old) were distributed in a completely randomized design in a 2 × 2 factorial arrangement that consisted of two cottonseed processing forms (whole or ground) and two chitosan levels (0 or 136 mg/kg live weight). Higher dry matter and organic matter apparent digestibility coefficient (ADC) was achieved with the diets containing the whole cottonseed. Ether extract ADC was higher in the animals fed the chitosan-containing diet. There was an interaction effect on the ADC of neutral detergent fibre corrected for ash and protein, which increased with chitosan inclusion associated with the whole cottonseed. The lambs that received the treatment containing the whole cottonseed showed higher microbial protein synthesis. Chitosan addition increased nitrogen retention. The animals fed chitosan-containing diets showed higher microbial protein synthesis. There was an interaction effect on microbial protein synthesis. Whole cottonseed associated with chitosan in lamb diets increases ether extract ADC and microbial protein synthesis.

Non-decorticated sunflower meal (SFM) is a potential protein source for dairy cows with high-fibre content but high ruminal degradability. The effect of replacement of soybean meal (SBM) and wheat middlings (WM) with SFM on the intake, digestibility, microbial protein synthesis, nitrogen utilization and milk production of dairy cows was evaluated. Twelve Holstein cows were blocked by days in milk and distributed in three 4 × 4 Latin squares. Diets were formulated to be isonitrogenous and contained 550 g maize silage/kg dry matter (DM). Treatment diets were no SFM (CON) or 70, 140 and 210 g/kg DM of SFM replacing fixed mixture of SBM and WM (536 and 464 g/kg of the mixture, respectively). The inclusion of SFM in diet did not affect DM intake, but intake of rumen degradable protein increased linearly. Inclusion of SFM reduced or tended to reduce total-tract digestibility of non-fibre carbohydrate, total digestible nutrients and excretion of purine derivatives. Milk production, milk protein content and efficiency of nitrogen use for lactation were reduced with increasing levels of SFM in the diet. The use of non-decorticated SFM as a replacement for SBM–WM mixture in diet reduces performance and efficiency of nutrient use in lactating dairy cows. The outcome of the current study is attributed to reduced fibre digestibility in SFM hulls. Therefore, future studies should evaluate the use of decorticated SFM.

Diets combining herbage and total mixed rations (TMR) are increasingly used in temperate regions for feeding ruminants, but little information is available regarding the effects on nutrient intake and digestion of this feeding management in beef cattle. The aim of this study was to determine the effects of combining TMR (10% CP and 13% ADF), and legume-based herbage (14% CP and 27% ADF) on intake, nutrient digestion, ruminal fermentation, microbial N flow and glucose and nitrogen metabolism in heifers. The experiment was a 3×3 Latin square design replicated three times; each period lasted 18 days (10 adaptation days and 8 measurement days). Nine cross-bred (Aberdeen Angus×Hereford) heifers (214±18 kg) fitted with permanent rumen catheters and housed in individual metabolic cages were assigned to one of three treatments: 24 h access to TMR (T), 24 h access to herbage (H) or combined diets with 18 h access to TMR and 6 h access to herbage (T+H). Data were evaluated using a mixed model. Animals fed T+H (TMR 71% and herbage 29%) diets tended to have a higher dry matter intake as a proportion of their BW than animals fed T. The T+H diet did not change ruminal fermentation (pH, N–NH3 and volatile fatty acids) or the N metabolism relative to the T diet, but increased the glucagon concentration and altered glucose metabolism. Conversely, animals fed T+H had increased purine derivatives excretion, increased N use efficiency for microbial protein synthesis and decreased plasma urea and urinary N excretion relative to animals fed H diet. The use of combined diets led to consumption of nutrients similar to a TMR diet, without reducing nutrient use and could improve N utilization compared with the herbage-only diet.

The long-term effects of adding chestnut (CHE; Castanea sativa) and valonea (VAL; Quercus valonea) tannin-rich extracts to sheep feed were investigated. In Experiment 1, sheep (65 kg BW) were fed 842 g/day of a ryegrass-based hay. The control-treated animals (CON) received 464 g/day of concentrate, and tannin-treated animals received the same amount of concentrate additionally containing 20 g of the respective tannin-rich extract. Hay and concentrates were offered together in one meal. After the onset of treatment, methane release was measured in respiration chambers for 23.5-h intervals (nine times) in a 190-days period. Faeces and urine were collected three times (including once before the onset of the tannin treatment) to assess digestibility and urinary excretion of purine derivatives. Based on the results obtained from Experiment 1, a second experiment (Experiment 2) was initiated, in which the daily tannin dosage was almost doubled (from 0.9 (Experiment 1) to 1.7 g/kg BW0.75). With the exception of the dosage and duration of the treatment (85 days), Experiment 2 followed the same design as Experiment 1, with the same measurements. In an attempt to compare in vitro and in vivo effects of tannin supplementation, the same substrates and tannin treatments were examined in the Hohenheim gas test. In vitro methane production was not significantly different between treatments. None of the tannin-rich extract doses induced a reduction in methane in the sheep experiments. On the 1st day of tannin feeding in both experiments, tannin inclusion tended to decrease methane release, but this trend disappeared by day 14 in both experiments. In balance period 3 of Experiment 1, lower dry matter and organic matter digestibility was noted for tannin treatments. The digestibility of CP, but not NDF or ADF, was reduced in both experiments. A significant shift in N excretion from urine to faeces was observed for both tannin-rich extracts in both experiments, particularly in Experiment 2. In balance period 2 of Experiment 2, an increased intake of metabolisable energy for VAL was observed. The urinary excretion of purine derivatives was not significantly different between treatments, indicating that microbial protein synthesis was equal for all treatments. Thus, we concluded that both tannin-rich extracts temporary affect processes in the rumen but did not alter methane release over a longer period.

Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia-N accumulation in the Rusitec were reduced by tannin treatment. The amount and efficiency of microbial protein synthesis were not significantly affected by tannin supplementation. The results of this study indicated that some tannin-rich extracts are able to reduce methane production without altering microbial protein synthesis. We hypothesized that chestnut and valonea extract have the greatest potential to reduce methane production without negative side effects.

To determine whether non-fibrous carbohydrate (NFC) supplementation improves fiber digestibility and microbial protein synthesis, 18 Corriedale ewes with a fixed intake level (40 g dry matter (DM)/kg BW0.75) were assigned to three (n = 6) diets: F = 100% fresh temperate forage, FG = 70% forage + 30% barley grain and FGM = 70% forage + 15% barley grain + 15% molasses-based product (MBP, Kalori 3000). Two experimental periods were carried out, with late (P1) and early (P2) vegetative stage forage. For P2, ewes were fitted with ruminal catheters. Forage was distributed at 0900 h, 1300 h, 1800 h and 2300 h, and supplement added at 0900 h and 1800 h meals. Digestibility of the different components of the diets, retained N and rumen microbial protein synthesis were determined. At the end of P2, ruminal pH and N-NH3 concentration were determined hourly for 24 h. Supplementation increased digestibility of DM (P < 0.001) and organic matter (OM; P < 0.001) and reduced NDF digestibility (P = 0.043) in both periods, with greater values in P2 (P = 0.008) for the three diets. Daily mean ruminal pH differed (P < 0.05) among treatments: 6.33 (F), 6.15 (FG) and 6.51 (FGM). The high pH in FGM was attributed to Ca(OH)2 in MBP. Therefore, the decreased fiber digestibility in supplemented diets could not be attributed to pH changes. The mean ruminal concentration of N-NH3 was 18.0 mg/dl, without differences among treatments or sampling hours. Microbial protein synthesis was greater in P2 (8.0 g/day) than in P1 (6.1 g/day; P = 0.006), but treatments did not enhance this parameter. The efficiency of protein synthesis tended to be lower in supplemented groups (16.4, 13.9 and 13.4 in P1, and 20.8, 16.7 and 16.2 g N/kg digestible OM ingested in P2, for F, FG and FGM, respectively; P = 0.07) without differences between supplements. The same tendency was observed for retained N: 2.55, 1.38 and 1.98 in P1, and 2.28, 1.23 and 1.10 g/day in P2, for F, FG and FGM, respectively; P = 0.05). The efficiency of microbial protein synthesis was greater in P2 (P = 0.007). In conclusion, addition of feeds containing NFCs to fresh temperate forage reduced the digestibility of cell walls and did not improve microbial protein synthesis or its efficiency. An increase in these parameters was associated to the early phenological stage of the forage.

The objective of the present study was to delineate how interactions between feeding sunflower oil (SFO) to partially defaunate the rumen and altering dietary ruminally fermentable carbohydrate may alter urea-N kinetics and N metabolism in lambs. In a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments, four Suffolk ram lambs (61·5 (se 4·0) kg) were used. Treatments were 0 ( − SFO) v. 6 % (+SFO) SFO and dry-rolled barley (DRB) v. pelleted barley (PB). N balance was measured over 4 d, with concurrent measurement of urea-N kinetics using continuous intra-jugular infusions of [15N15N]urea. Feeding SFO decreased (P = 0·001) ruminal protozoa and NH3-N concentrations. Urinary N excretion was lower (P = 0·003), and retained N was higher (P = 0·002) in +SFO lambs compared with − SFO lambs. Endogenous production of urea-N (urea-N entry rate; UER) was similar across treatments. Urea-N transfer to the gastrointestinal tract (GIT) (GIT entry rate; GER), expressed as absolute amounts (16·4 v. 13·1 g/d) or as a proportion of the UER (0·693 v. 0·570), its anabolic use (9·0 v. 6·0 g/d) and microbial N supply (14·6 v. 10·9 g/d) were higher (P ≤ 0·001) in +SFO lambs compared with –SFO lambs. As a proportion of the UER, GER was higher, whereas urinary urea-N loss was lower, in lambs fed PB compared with those fed DRB (P = 0·01). In summary, feeding SFO increased urea-N recycling to the GIT and microbial non-NH3-N supply, thus providing new evidence that the improved efficiency of N utilization in partially defaunated ruminants could be partly mediated by an increase in urea-N recycling.

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products – meat, milk and dairy – contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.

Two incubation trials were carried out with the rumen-simulation technique (RUSITEC). In each trial, four vessels received a diet of grass hay and concentrate (600 and 400 g/kg DM, respectively; diet F), and the other four were fed a diet composed of concentrate and barley straw (900 and 100 g/kg DM, respectively; Diet C). Vessels were given 20 g of the corresponding diet daily, and half of them were supplemented with disodium malate to achieve a final concentration of 6.55 mM. There were no effects (P>0·05) of malate either on pH or on the daily production of NH3-N, but malate treatment increased (P<0·05) DM, neutral detergent and acid detergent fibre disappearance after 48 h incubation. The daily production of propionate and butyrate increased (P<0·001), and the ratio CH4:volatile fatty acids decreased (P<0·001) by supplementing both diets with malate. Whereas adding malate to the F diet produced an increase in acetate production (P=0·011) and the growth of solid-associated micro-organisms (P=0·037), no effects (P>0·05) were observed for diet C. For both diets, there were no differences (P>0·05) between treatments in the daily flow of liquid-associated micro-organisms measured using 15N as a microbial marker. These results indicate that malate stimulated the in vitro fermentation of both diets by increasing the apparent disappearance of the diet and decreasing the ratio of CH4:volatile fatty acids, but a greater response was observed with diet F. If these results are confirmed in vivo, malate could be used as a feed additive for ruminants fed diets containing medium proportions of forage (i.e. dairy animals) and not only in animals fed high-concentrate diets, as has so far been proposed.

The present study investigated the susceptibility of dietary n-3 PUFA to ruminal biohydrogenation, the stability of ingested vitamin E in the rumen and the subsequent uptake of PUFA and vitamin E into plasma. Six cannulated sheep were assigned to six diets over five 33d periods, in an incomplete 6×5 Latin square. The diets, based on dried grass, were formulated to supply 50g fatty acids/kg DM using three lipid sources: Megalac® (calcium soap of palm fatty acid distillate; Volac Ltd, Royston, Herts., UK), linseed (formaldehyde-treated; Trouw Nutrition, Northwich, Ches., UK) and linseed–fish oil (formaldehyde-treated linseed+fish oil). The diets were supplemented with 100 or 500mg α-tocopheryl acetate/kg DM. Fat source or level of vitamin E in the diet did not alter microbial activity in the rumen. Biohydrogenation of linoleic acid (18:3n-6; 85–90%), linolenic acid (18:3n-3; 88–93%), docosahexaenoic acid (22:6n-3; 91%) and EPA (20:5n-3; 92%) was extensive. Feeding formaldehyde-treated linseed elevated concentrations of 18:3n-3 in plasma, whilst 22:6n-3 and 20:5n-3 were only increased by feeding the linseed–fish oil blend. Duodenal recovery of ingested vitamin E was high (range 0·79–0·92mg/mg fed). High dietary vitamin E was associated with increased plasma α-tocopherol (2·57 v. 1·46μg/ml for 500 and 100mg α-tocopheryl acetate/kg DM respectively), although all concentrations were low. Plasma vitamin E levels, however, tended to decrease as the type and quantity of PUFA in the diet increased. The present study illustrates that nutritionally beneficial PUFA in both fish and linseed oils are highly susceptible to biohydrogenation in the rumen. Although α-tocopheryl acetate resisted degradation in the rumen, plasma vitamin E status remained deficient to borderline, suggesting either that uptake may have been impaired or metabolism post-absorption increased.

The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation. The energy source was fibre prepared from sugar-beet pulp. N was added as NH3 continuously infused (AC) or peptides (Bacto® Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS). Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment. Treatments had no effect on DM degradation. Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment. Microbial N flow tended to be higher with the peptide additions (P<0·061), with no significant difference between the two peptides treatments. The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0·05) and the proportion of LAM derived from NH3 lower (P<0·05). However, LAM only accounted for 20–30 % total microbial population. Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply. This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

Determination of microbial degradability of N is important in formulating a sound supplementation strategy for efficient utilisation of basal as well as supplementary diet components. In vitro degradability of N (IVDN) from tannin-containing browses (Acacia cyanophylla, Acacia albida, Acioa barteri and Quercus ilex) and two herbaceous legumes (Desmodium intortum andDesmodium uncinatum) was determined using the in vitro gas-production method coupled with NH3-N measurement in the presence and absence of a tannin-binding agent (polyethylene glycol (PEG), molecular mass 6000). Addition of PEG to tannin-containing feeds significantly (P < 0·05) increased in vitro gas and short-chain fatty acid (SCFA) production, and IVDN. The use of PEG as a tannin-binding agent increased IVDN from 28 to 59, 32 to 72, 19 to 40, 32 to 73, 40 to 80, and 26 to 77 % in A. cyanophylla, A. albida, A. barteri, D. intortum,D. uncinatum and Q. ilexrespectively. There was significant correlation between total phenolic compounds (total phenol, TP; total tannin, TT) in leguminous forages and percentage increase in IVDN on addition of PEG (P < 0·05; R2 0·70 and 0·82 for TP and TT respectively). The difference in IVDN observed in the absence and presence of PEG indicates the amount of protein protected from degradation in the rumen by tannins. When measured after 24 h incubation, tannin-containing feeds incubated in absence of PEG resulted in higher microbial protein synthesis than in the presence of PEG. Addition of PEG significantly (P < 0·05) reduced the efficiency of microbial protein synthesis expressed as μmol purine/mmol SCFA.

Incubation trials were carried out with the rumen simulation technique (RUSITEC) to study the effects of four forms of N on the growth of ruminal micro-organisms and the fermentation variables when an all-fibre basal diet was incubated. The basal diet consisted of 10 g neutral-detergent fibre (NDF) from grass hay plus 2 g NDF from sugarbeet pulp. N forms were isolated soyabean protein, soyabean peptides, amino acids blended to profile soyabean protein and NH3 as NH4Cl. Half of the daily N supply was infused as NH4Cl and the other half was infused as each of the four treatments described. Non-NH3 N (NAN) forms increased NDF (P = 0·006), acid-detergent fibre (P = 0·003) and cellulose (P = 0·015) disappearance after 48 h incubation, CO2 (P < 0·001), CH4 (P = 0·002) and total volatile fatty acids production (P < 0·001), as well as the molar percentages of isobutyrate, isovalerate and valerate, which reflected the fermentation of amino acid C skeletons. NAN treatments also increased microbial N flow (P < 0·001) compared with NH3, with peptides and protein supporting more (P = 0·036) than amino acids. The proportion of microbial N derived from NH3 decreased successively (P < 0·05) with NH3 > amino acids > peptides > protein treatments, indicating preferential uptake of peptides without passage through the NH3 pool. Microbial efficiency (g microbial N/kg organic matter apparent disappearance) was greater (P = 0·002) for the NAN forms than for the NH3 treatment, with peptides and protein treatments supporting higher (P = 0·009) values than amino acid treatment. These results indicate that N forms other than NH3 are required for optimal fibre digestion and microbial growth.

The effects of ammoniation of wheat straw with or without supplementation of protein sources of either high (casein) or relatively low (potato protein) rumen degradability on intake and digestion were studied with four sheep in a 4 × 4 Latin square design. Rations offered were: (1) untreated wheat straw (UWS), (2) ammoniated wheat straw (AWS), (3) AWS supplemented with 3·2 g casein/kg live weight (W)0·75 per d (AWSC) and (4) AWS supplemented with 3·9 g potato protein/kg W0·75per d (AWSP). Straw was offered ad lib. and all rations were supplemented with sugarbeet pulp and a mineral mixture. NH3 treatment increased intake and digestion. Supplementation of AWS with potato protein increased total digestible organic matter intake (DOMI) compared with AWS whereas supplementation with casein did not affect total DOMI. Protein supplementation of AWS significantly reduced rumen digestion of cellulose, and when the supplementation was with casein it reduced rumen digestion of neutral-detergent fibre and hemicellulose also. This lower rumen digestion was compensated by a higher proportion of digestion occurring in the hindgut for hemicellulose (P<0·05 for AWSC, P>0·05 for AWSP), but not for cellulose. Across all rations, rumen fluid volume increased with increasing cell-wall intake. The efficiencies of microbial protein synthesis were (average of three different methods of estimation) 23·3, 26·2, 34·8 and 31·7gN/kg apparently-rumen-degraded organic matter for UWS, AWS, AWSC and AWSP respectively. The difference between UWS and AWS was not significant, but values for AWSC and AWSP were significantly higher than that for AWS. The rumen digestion of feed aimno acid-N (AA-N) was significantly higher for AWSC than for the other rations. The apparent small-intestinal digestion of AA-N and N was significantly higher for AWSP than for the other rations. The true small-intestinal digestion values were 0·86, 0·84 and 0·68 for AA-N, N and non-protein-N respectively. Heal endogenous losses of AA-N were approximately 6 mg/g duodenal non-protein dry-matter flow. Linear relationships were observed between DOMI and N balance and truly absorbed AA-N, indicating that DOMI could have been limited by small-intestinal amino acid availability. Regression of N balance v. truly absorbed AA-N resulted in an estimate of net efficiency of utilization of truly absorbed AA-N of 0·54.