M. D. Carro

Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet

Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW(0.75). Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH(3)-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.

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.

Effect of DL-malate on mixed ruminal microorganism fermentation using the rumen simulation technique (RUSITEC)

The objective of this study was to investigate the effects of DL-malate on the in vitro fermentation of a 50 : 50 forage : concentrate diet using the rumen simulation technique (RUSITEC) and to compare these effects with those induced by the addition of propionate. The RUSITEC system consisted of eight vessels: three of them received daily 5.62 mmol of DL-malate, three vessels received daily 5.62 mmol of propionate and two vessels received no additives (control). After an adaptation period of 11 days the main fermentation parameters were determined for five consecutive days. There were no significant differences between treatments either in pH or in the daily production of NH3‐N. Compared to control diet, the addition of DL-malate resulted in an increase (p < 0.05) of hemicellulose disappearance and a trend (p < 0.10) towards a greater disappearance of dry matter, organic matter and neutral detergent fibre. Differences between DLmalate and propionate in diet degradation were not significant. DL-malate treatment resulted in an increase (p < 0.01) of daily propionate production, and a decrease (p < 0.05) in the amount of both methane (mmol/g DM fermented) and L-lactate (mg/day). Compared to propionate, DL-malate produced an increase (p < 0.05) of acetate production and tended to produce a lower amount of propionate (p < 0.10). In conclusion, these results seem to indicate that DL-malate enhanced the in vitro fermentation by increasing production of propionate and digestibility of hemicellulose. # 1999 Published by Elsevier Science B.V. All rights reserved.

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

Influence of exogenous fibrolytic enzymes and fumarate on methane production, microbial growth and fermentation in Rusitec fermenters.

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34.3 endoglucanase, 0.57 exoglucanase, 24.7 xylanase and 5.51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P 0.05). Supplementing with FUM increased (P 0.05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage.

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square to evaluate the effects of 4 diets on microbial synthesis, microbial populations, and ruminal digestion. The experimental diets had forage to concentrate ratios (F:C; DM basis) of 70:30 (HF) or 30:70 (HC) with alfalfa hay (A) or grass hay (G) as forage and were designated as HFA, HCA, HFG, and HCG. The concentrate was based on barley, gluten feed, wheat middlings, soybean meal, palmkern meal, wheat, corn, and mineral-vitamin premix in the proportions of 22, 20, 20, 13, 12, 5, 5, and 3%, respectively (as-is basis). Sheep were fed the diets at a daily rate of 56 g/kg of BW(0.75) to minimize feed selection. High-concentrate diets resulted in greater (P < 0.001) total tract apparent OM digestibility compared with HF diets, but no differences were detected in NDF digestibility. Ruminal digestibility of OM, NDF, and ADF was decreased by increasing the proportion of concentrate, but no differences between forages were detected. Compared with sheep fed HF diets, sheep receiving HC diets had less ruminal pH values and acetate proportions, but greater butyrate proportions. No differences among diets were detected in numbers of cellulolytic bacteria, but protozoa numbers were less (P = 0.004) and total bacteria numbers tended (P = 0.08) to be less for HC diets. Carboxymethylcellulase, xylanase, and amylase activities were greater for HC compared with HF diets, with A diets showing greater (P = 0.008) carboxymethylcellulase activities than G diets. Retained N ranged from 28.7 to 37.9% of N intake and was not affected by F:C (P = 0.62) or the type of forage (P = 0.31). Microbial N synthesis and its efficiency was greater (P < 0.001) for HC diets compared with HF diets. The results indicate that concentrates with low cereal content can be included in the diet of sheep up to 70% of the diet without detrimental effects on ruminal activity, microbial synthesis efficiency, and N losses.

Plasma pregnancy-associated glycoprotein and progesterone concentrations in pregnant Assaf ewes carrying single and twin lambs

Ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) concentrations were monitored weekly during the last three months of gestation and the first month postpartum in plasma of twelve Assaf ewes. The oPAG levels were determined with a heterologous RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG. The P4 levels were measured with a radioimmunological procedure with a sensitivity to less than 0.1 ng ml−1. The oPAG profiles in ewes carrying a single lamb and in those carrying twins were very similar from week 9 of gestation to lambing. Ewes which had twins presented numerically higher oPAG concentrations from week 12 to lambing, but that difference was only significant (P 0.05) was found between oPAG concentrations and lamb birth-weight at any week of gestation considered in this study. However, the lamb birth-weight was positively correlated (P < 0.10) with the P4 concentrations from weeks 9 to 19 of gestation. Results indicate that number of fetuses could influence the oPAG production in ewes during the last three months of gestation.

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