María José Ranilla

Effect of the addition of malate on in vitro rumen fermentation of cereal grains.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato(R); Norel & Nature S.A., Barcelona, Spain; composed of disodium malate-calcium malate (0.16:0.84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato(R) was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mm-malate. Gas production was measured at regular intervals up to 120 h. Malate increased (P<0.01) the average fermentation rate of all substrates, and the lag time decreased (P<0.05) linearly with increasing concentrations of malate for all substrates, with the exception of sorghum. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as malate concentration increased from 0 to 10 mm. Propionate and butyrate production increased (P<0.05), while the value of the acetate : propionate ratio and l-lactate concentrations decreased (P<0.05) linearly with increasing doses of malate. Malate treatment increased (P<0.05) the CO2 production and decreased the production of CH4, although this effect was not significant (P>0.05) for maize. Malate at 4 and 7 mm increased (P<0.05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH4 production and l-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mm-malate were observed.

Influence of different concentrations of disodium fumarate on methane production and fermentation of concentrate feeds by rumen micro-organisms in vitro

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of disodium fumarate on the fermentation of five concentrate feeds (maize, barley, wheat, sorghum and cassava meal). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-fumarate. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as fumarate concentration increased from 0 to 10 mm. Propionate and acetate production increased (P<0.05), while the value of the acetate:propionate ratio decreased (P<0.05) linearly with increasing doses of fumarate. In contrast, l-lactate and NH3-N concentrations in the cultures were not affected (P>0.05) by the addition of fumarate. For all substrates, fumarate treatment decreased (P<0.05) CH4 production, the mean values of the decrease being 2.3, 3.8 and 4.8 % for concentrations of 4, 7 and 10 mM-fumarate respectively. Addition of fumarate did not affect (P>0.05) the total gas production. If the results of the present experiment are confirmed in vivo, fumarate could be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH, acetate and propionate production and it decreased CH4 production.

Effects of disodium fumarate on in vitro rumen microbial growth, methane production and fermentation of diets differing in their forage:concentrate ratio

The effects of disodium fumarate on microbial growth, CH4 production and fermentation of three diets differing in their forage content (800, 500 and 200 g/kg DM) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from four Merino sheep. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mm-fumarate, and (15)N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation. Fumarate did not affect (P>0.05) any of the measured gas production parameters. In 17 h incubations, the final pH and the production of acetate and propionate were increased linearly (P<0.001) by the addition of fumarate. Fumarate tended to increase (P=0.076) the organic matter disappearance of the diets and to decrease (P=0.079) the amount of NH3-N in the cultures. Adding fumarate to batch cultures tended (P=0.099) to decrease CH4 production, the mean values of the decrease being 5.4 %, 2.9 % and 3.8 % for the high-, medium- and low-forage diet, respectively. Fumarate tended to increase (P=0.082) rumen microbial growth for the high-forage diet, but no differences (P>0.05) were observed for the other two diets. These results indicate that the effects of fumarate on rumen fermentation depend on the nature of the incubated substrate, the high-forage diet showing the greatest response.

Effect of forage to concentrate ratio in the diet on ruminal fermentation and digesta flow kinetics in sheep offered food at a fixed and restricted level of intake.

The effects of four diets differing in their for age: concentrate ratio (80:20, 60:40, 40: 60 and 20:80; g/100 g fresh matter) on rumen characteristics, digestibility and digesta flow kinetics were investigated. Alfalfa hay was used as forage and concentrate was composed of barley, soya-bean meal and maize. Diets were prepared by mixing all ingredients and offered to the animals as complete diets. Eight mature Merino sheep, each fitted with a rumen cannula, were offered 1·055 kg dry matter per day of the corresponding diet over two experimental periods. The daily evolution of ruminai pH, volatile fatty acids (VFA) and ammonia nitrogen (N) concentrations were measured. Digestibility was determined by total faecal collection and Cr and Co were used as markers to estimate digesta passage rates. Microbial nitrogen flow at the duodenum (MNDF) was estimated from the urinary excretion of purine derivatives (PD). The apparent digestibility of organic matter increased ( P P P P > 0·05). Both liquid and solid digesta outflow rates from the rumen decreased quadratically ( P P > 0·05) by changes in the diet. In contrast, the daily urinary excretion of both allantoin and total PD increased quadratically ( P P

Comparative digestibility and digesta flow kinetics in two breeds of sheep

A study was conducted to compare apparent digestibility and digesta flow kinetics in the whole digestive tract in two breeds of sheep (Churra and Merino) offered alfalfa hay at about maintenance. Ten mature sheep (five Churra and five Merino) each fitted with a rumen cannula were used in the study. Apparent digestibility was determined by total faecal collection. Liquid (Co-EDTA) and solid (Cr-mordanted fibre) markers were used to estimate rumen volumes and digesta flows. There were no significant differences between Churra and Merino sheep either in dry-matter or fibre apparent digestibility (P > 0·05). Liquid and solid passage rates did not differ between breeds and the estimated total mean retention times were similar for both genotypes. The volume of liquid in the rumen was proportionately 0·14 greater in Churra than in Merino sheep, although the difference was not significant. Estimated saliva secretion was greater in Churra (11·6 (s.e. 0·77) I/day) than in Merino sheep (10·5 (s.e. 0·72) I/day) but the difference did not reach statistical significance. Solid contents of the rumen did not differ between breeds. Rumen particle density was lowest at 2 h after feeding and remained stable at other sampling times, with no significant differences between breeds. There were no significant differences between breeds in the urinary excretion of total purine derivatives but urinary excretion of allantoin and microbial nitrogen supply tended to be higher in Merino than in Churra sheep ( P

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

Aims:  To assess the effect of protozoal species on rumen fermentation characteristics in vitro.

In vitro microbial growth and rumen fermentation of different substrates as affected by the addition of disodium malate

The effects of two concentrations of disodium malate on the in vitro fermentation of three substrates differing in their forage: concentrate ratio (0·8: 0·2, 0·5: 0·5 and 0·2: 0·8; g/g dry matter; low-, medium- and high-concentrate substrates, respectively) by rumen micro-organisms were studied using batch cultures. Rumen contents were collected from four Merino sheep offered lucerne hay ad libitum and supplemented daily with 400 g concentrate. Disodium malate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mmol/l malate and 15 N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation to study fermentation kinetics. When gas production values were corrected for gas released from added malate, no effects ( P > 0·05) of malate were detected for any of the estimated gas production parameters. In 17-h incubations, the final pH and total volatile fatty acid (VFA) production were increased ( P P > 0·05) were detected in the final amounts of ammonia-N and lactate. When net VFA productions were corrected for the amount of VFA produced from malate fermentation itself, adding malate did not affect ( P > 0·05) the production of acetate, propionate and total VFA. Malate reduced methane (CH 4 ) production by proportionately 0·058, 0·013 and 0·054 for the low-, medium- and high-concentrate substrates, respectively. Adding malate to batch cultures increased ( P P > 0·05) its efficiency of growth (55·5, 56·7 and 54·3 mg of microbial N per g of organic matter apparently fermented for malate at 0, 4 and 8 mmol/l, respectively). There were no interactions ( P > 0·05) malate × substrate for any of the measured variables, and no differences ( P > 0·05) in pH, CH 4 production and microbial growth were found between malate at 4 and 8 mmol/l. The results indicate that malate had a beneficial effect on in vitro rumen fermentation of substrates by increasing VFA production and microbial growth, and that only subtle differences in the effects of malate were observed between substrates. Most of the observed effects, however, seem to be due to fermentation of malate itself.

Influence of nitrogen source on the fermentation of fibre from barley straw and sugarbeet pulp by ruminal micro-organisms in vitro

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.

Effect of forage to concentrate ratio in complete diets offered to sheep on voluntary food intake and some digestive parameters.

Twelve mature ewes were used to study the effect of forage: concentrate ratio in complete diets on voluntary intake and some digestive characteristics. Diets consisted of four combinations of chopped lucerne hay and a concentrate (390 g cracked barley grains, 440 g cracked maize grains and 170 g soya-bean meal per kg of concentrate) in the following proportions (fresh matter basis): 0·8:0·2 (C20), 0·6:0·4 (C40), 0·4:0·6 (C60) and 0·2:0·8 (C80). Diets were offered over two 42-day periods and, in each of them, three sheep received one of the four diets, with the restriction that no animal received the same diet in both periods. Chromium Ill-mordanted fibre was used as a marker to estimate passage rate of digest a and microbial nitrogen supply (MNS) was estimated from the urinary excretion of purine derivatives. The increase in the proportion of concentrate affected linearly ( P P P P > 0·05) by the proportion of concentrate in the diet. Both particulate passage rate from the rumen and through the caecum and proximal colon decreased linearly ( P P > 0·05) by the diet, whereas its efficiency (g/kg digestible OM intake) tended ( P

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and in batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.