Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of three fibrolytic enzymes (xylanase from Trichoderma viride (XYL) and fibrolytic enzymes from Aspergillus niger (ASP) and Trichoderma longibrachiatum (TR)) on the fermentation of three substrates composed of grass hay:concentrate in the proportions (dry matter (DM) basis) of 0.7:0.3 (HF), 0.5:0.5 (MF) and 0.3:0.7 (LF). Enzymes were characterized for xylanase, endoglucanase, exoglucanase and amylase activities, and were supplied at rates of 40 and 80 enzymatic units/g substrate DM. In 8 h incubations, all enzymes increased (P=0.048 to P<0.001) the true degradability of substrate DM and the production of acetate, propionate, total volatile fatty acids (VFA) and gas. After 24 h incubation, some of the observed effects disappeared, but all enzymes still increased (P=0.028 to P<0.001) the degradability of substrate acid detergent fibre and the production of acetate, propionate and total VFA. For all enzymes, the effects on ruminal variables were less marked at 24 than at 8 h of incubation. Only few significant (P=0.044 to P=0.001) enzyme × substrate interactions were detected, although the magnitude of the response for each substrate varied with the enzyme. When considering the amount of organic matter apparently fermented (OMAF) and the methane:OMAF ratio as main variables, TR80 produced the greatest increase in OMAF (17.0%) for HF substrate, with ASP80 and TR40 having similar values (11.1 and 12.6%), and XYL and ASP40 showing no effects (P>0.05). A decrease (P<0.05) of methane:OMAF ratio was only found for TR80 at 8 h (17.4%). All enzymes, with the exception of ASP40, increased (P<0.05) OMAF at 8 h for MF substrate (11.3–25.4%), TR80 showing the greatest response. After 24 h of incubation, both doses of XYL and TR increased (P<0.05) OMAF (mean value 8.2%) and decreased methane:OMAF ratio (mean value 9.5%). All enzymes increased significantly OMAF with LF substrate at 8 h (7.5–19.9%), but after 24 h no effect (P>0.05) was detected on OMAF and methane:OMAF ratio. In general, few differences were detected between both doses of enzymes, which indicate than the used enzymes would be effective in enhancing ruminal degradation of substrates at a dose lower than 80 enzymatic units/g substrate DM.     

In vitro gas production and ruminal fermentation of glycerol, propylene glycol and molasses

Ruminal fermentation pattern and in vitro gas production was determined for three energy sources for ruminants, glycerol, propylene glycol and molasses with ruminal fluid from sheep. Substrates incubated were alfalfa, corn silage, glycerol (320 and 640 μl), propylene glycol (320 and 640 μl) and molasses (320 μl). The greater volume of gas produced was observed at the highest dose of glycerol which also showed the slowest rate of gas production and the longest lag time (P<0.05). Propylene glycol presented the minor volume of gas and was rapidly metabolized with short lag time. Molasses presented typical characteristics of a rapidly available substrate, with the fastest rate of gas production (P<0.05). Glycerol fermentation resulted in a reduction of acetate, a slight increase in propionate and an increment in percentage of butyrate. Incubations with propylene glycol also reduced acetate and butyrate, but increased propionate (P<0.05). Molasses fermentation reduced acetate and increased propionate and butyrate. Increasing dose of energy sources resulting in a greater volume of gas produced. In conclusion, glycerol fermentation reduced acetate and increased the molar proportion of butyrate and propionate was the main product of fermentation of propylene glycol.     

Effects of essential oils on in vitro ruminal fermentation and ammonia release

Ruminal inoculum enriched with particle-associated microorganisms was collected from two lactating dairy cows fed an alfalfa hay/cereal silage/concentrate diet 1 h before feeding and used to evaluate effects of essential oils (EO) on ruminal fermentation in short-term in vitro incubations. Ruminal ammonia N was labeled with ¹⁵N and native and hydrolyzed casein were provided as sources of amino acids. Forty EO were tested at 10 and 100 mg/l final medium concentration. Monensin-Na, and sodium laurate were also incubated at 5 and 2000 mg/l, respectively. Compared with blanks (i.e., no addition of EO), sodium laurate increased medium pH and a number of EO reduced medium pH. Both sodium laurate and monensin reduced ammonia concentrations compared to the blank. Only one of the tested EO (i.e., Caraway) slightly reduced ammonia concentration, by 8%, compared with the blank. Monensin and sodium laurate resulted in higher (i.e., 9–34%, monensin, and 29–47%, sodium laurate) ¹⁵N enrichment of ammonia N, an indication of reduced deamination of amino acids in these treatments versus the blank. Several EO (i.e., FrankMyrrh, Gardenia, Hibiscus, Eucaliptus, and Peppermint) had similar effects, but of a smaller magnitude (i.e., 5–12%). Some EO increased medium total VFA concentration, primarily through an increase in acetate concentration. Overall, effects of EO on fermentation were subtle, and it is unlikely that these moderate in vitro effects would correspond to any substantive impact on ruminal fermentation in vivo.     

Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P. thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P. thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics.     

Chemical composition and in vitro ruminal fermentation of common tree forages in the semi-arid rangelands of Swaziland

Browse plants play an important role in providing feed for livestock in semi-arid rangelands of Africa. Chemical composition and in vitro ruminal fermentation of leaves collected from Acacia burkei, Acacia tortilis, Acacia nilotica, Dichrostachys cinerea and Ehretia obtusifolia in communal grazing lands in the lowveld of Swaziland is presented. Leaves were collected from trees located on two soil types (i.e., lithosol and vertisol) in the communal land but it had no effect on the chemical composition of tree leaves. The NDFom and ADFom content were highest in D. cinerea and A. burkei and lowest in E. obtusifolia and A. nilotica. Crude protein (CP) contents ranged between 108 g/kg and 122 g/kg DM. D. cinerea had the highest Ca and Mg content, while A. tortilis had the lowest. There were marked variations in K level amongst browse species, with A. tortilis (9.1 g/kg DM) having the highest value. The P, Zn and Fe did not differ between browse species. Soil type and tree species interaction impacted in vitro fermentation parameters. Extent of fermentation, as measured by 48 h cumulative gas production, and organic matter degradability was highest in E. obtusifolia leaves and lowest in D. cinerea leaves within soil type. Fermentation efficiency, as measured by partitioning factors, was highest in A. nilotica leaves. Leaves of E. obtusifolia could be a valuable supplementary feedstuff for ruminant livestock due to its in vitro fermentation characteristics as well as low fibre and moderate CP levels.     

Effect of tea saponin on rumen fermentation in vitro

The present study was conducted to investigate the effect of tea saponins (TS) on ruminal fermentation in vitro using gas syringes as incubators. The TS were added at levels of 0, 2, 4, 6 and 8 mg against 200 mg mixture of corn meal and grass meal (1/1, w/w) in rumen fluid. In vitro gas production (GP) was recorded and methane concentration was determined at 3, 6, 9, 12 and 24 h incubation. After 24 h, the incubation was stopped and the inoculants were determined for pH, ammonia-N, volatile fatty acids (VFAs), protozoa counts and microbial protein yield. The GP was increased with the increasing level of TS except 8 mg at 24 h, which kept little change from that of the control. Methane concentration was decreased at all levels of TS at each incubation time. At 24 h incubation, inclusion with 2, 4, 6 and 8 mg of TS decreased methane concentration by 13, 22, 25 and 26%, respectively. The pH of ruminal fluid was slightly lower at 4 and 6 mg TS, but all values were in the normal range. Ammonia-N concentrations decreased significantly (P < 0.01) when the TS were included. Concentrations of individual and total VFAs were not significantly effected by TS addition. The TS significantly inhibited the protozoa growth in ruminal fluid (P < 0.01). At 24 h incubation, protozoa counts were reduced by 19, 25, 45 and 79%, respectively at levels of 2, 4, 6 and 8 mg of TS compared to that in control. The microbial protein was enhanced with the TS addition except 2 mg level, and reached 1.92, 2.36 and 2.61 mg/mL with addition of 4, 6 and 8 mg TS, compared to 1.50 mg/mL in control. It is suggested that TS could modify the rumen fermentation and inhibit the release of methane and ammonia, which may be beneficial for improving nutrient utilization and animal growth.     

Effect of monensin, fish oil or their combination on in vitro fermentation and conjugated linoleic acid (CLA) production by ruminal bacteria

An in vitro study was conducted to examine the effect of adding monensin, fish oil, or their combination on rumen fermentation and conjugated linoleic acid (CLA) production by mixed ruminal bacteria when incubated with safflower oil. Concentrate (1 g/100 ml) with safflower oil (0.2 g/100 ml) was added to a mixed solution (600 ml) of strained rumen fluid and buffer (control). Monensin (10 ppm), fish oil (0.02 g/100 ml), or monensin plus fish oil was also added into control mixture. All the culture solutions prepared were incubated anaerobically at 39 °C for 12 h. A higher pH and ammonia concentration were observed from the culture solution containing monensin at 12 h of incubation than those from the control or the culture containing fish oil. Monensin increased (P < 0.007) the C₃ content over all the collection times of culture solution while reducing the C₄ content at 6 h (P < 0.018) and 12 h (P < 0.001) of incubations. Supplementation of monensin, fish oil or their combination changed the content of C₁₈-fatty acids of ruminal culture. Monensin alone reduced (P < 0.021) the content of cis-9, trans-11 CLA compared to fish oil at all sampling times, but increased (P < 0.041) the trans-10, cis-12 CLA production compared to fish oil addition and the control which were similar at incubation for 12 h. The combination of monensin and fish oil increased the content of cis-9, trans-11 CLA (P < 0.023) and transvaccenic acid (TVA, P < 0.018) significantly compared to the control or monensin alone at incubation for 12 h.     

Seasonal chemical composition, in vitro fermentation and in sacco dry matter degradation of four indigenous multipurpose tree species in Nigeria

A study was carried out to estimate the nutritive value of four indigenous multi-purpose tree (MPT) species (Enterolobium cyclocarpum, Treculia africana, Gliricidia sepium and Millettia griffoniana) by the evaluation of their seasonal chemical composition, in vitro fermentation and in sacco dry matter (DM) degradation. The main objective of the study was to assess the potential of these indigenous MPT in supplementing the feed of ruminant animals during the dry season when grasses are scarce and their quality generally fall short of animal requirements. Leaf samples were randomly collected from the trees for estimation of DM, crude protein (CP), ether extract (EE), ash, neutral detergent fibre (NDFom), acid detergent fibre (ADFom), lignin (sa), in vitro fermentation and in sacco DM degradation. Samples were collected three times to represent seasonal variations as follows: November: early dry; February: mid-dry and April: late dry seasons. All samples had high CP (160–199 g/kg DM) and moderate fibre concentrations (NDFom, 380–580 g/kg DM; ADFom, 290–400 g/kg DM and lignin (sa), 75–107 g/kg DM). T. africana recorded the highest (180–199 g/kg DM) (P<0.001) CP content throughout the seasons. The values obtained for the in vitro fermentation characteristics and in sacco DM degradation of these indigenous MPT indicated the presence of potentially degradable nutrients in the MPT. Data from this study showed that E. cyclocarpum, T. africana, G. sepium and M. griffoniana have potentials that could be harnessed as feed supplements for ruminant animal production in Nigeria during the dry season.     

Influence of storage time and temperature on in vitro digestion of neutral detergent fibre at 48 h, and comparison to 48 h in sacco neutral detergent fibre digestion

The effect of within-day delays of 0.5, 2.5, 4.5 and 6.5 h between collection of rumen fluid from a cow and initiation of in vitro fermentation, as well as storage of rumen fluid for 48 h at either −24°C or 6, 22 and 39°C, on in vitro digestion of neutral detergent fibre (NDF) at 48 h was determined. In addition, the 48 h in vitro digestion of NDF, determined with a minimum time delay (i.e., 0.5 h) between collection from the cow and initiation of incubation, was compared to NDF digestion determined in sacco at 48 h. Rumen inoculum from a single cow was utilized in a thrice replicated incubation with whole crop alfalfa, corn, cereal and sudangrass forages of a lower and higher quality. The same cow was used as the host for the in sacco bags. The in vitro procedure used a bulk procedure with 5.0 cm × 5.5 cm multi-weave polyethylene polyester polymer bags that retained particles of 25 mm and larger. The in sacco procedure used the same bags retained in a large mesh bag. A within-day time delay of up to 6.5 h between collection of rumen fluid from the cow and initiation of in vitro fermentation had no impact on measured 48 h in vitro digestion of NDF. In contrast, no temperature dependent storage procedure maintained 48 h in vitro digestion of NDF at levels determined with no 48 h storage, although high quality alfalfa was least affected by any storage procedure. The 48 h in vitro digestion of NDF, determined using the minimum time delay between collection from the cow and initiation of the incubation, was higher than values obtained in sacco. Results show that this bulk in vitro procedure resulted in higher 48 h digestion of NDF than those determined with a similar in sacco procedure, thereby suggesting that laboratories located some distance from the donor animal can utilize in vitro procedures to accurately estimate 48 h digestion of NDF. However, storage of rumen fluid for 48 h, by any temperature dependent procedure examined, in order to facilitate fewer trips to the donor animal, or trips of substantially longer duration, will underestimate 48 h digestion of NDF to an extent that depends upon the forage incubated.     

Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardii

After transition to anaerobiosis an oscillatory fermentation of plastidic starch is found in the green alga Chlamydomonas reinhardii. Oscillations are investigated by analysis of fluctuations in starch, fermentation products and metabolites. Period length, amplitudes and phase relationships are determined by a biometrical processing of the biochemical signals, which allows statistical treatment even under limitation of data points. The mean period length is 59 min and oscillations are detectable within 3 h after the onset of anaerobiosis. Metabolized starch carbon is found by 90% in the accumulating fermentation products as formate, acetate and ethanol. From the intermediates only pyruvate increases during fermentative oscillations. A phase shift of 180° between ATP and both, AMP and ADP, indicates activity of adenylate kinase and values for energy charge fluctuates between 0.70 and 0.89. Phase relations of glucose-6-P to both AMP and pyruvate are 190° and 56°, respectively, whereas that of pyruvate to ATP is 134°. Glucose-6-P concentration does not follow the rate of starch degradation. Phase relations of fermentation products to pyruvate indicates enzyme activities of formate pathway. High period length as observed during algal fermentation is thought to be generated by a complex rate control in a different compartmentalized glycolytic pathway.     

Effect of tamarind (Tamarindus indica) seed husk tannins on in vitro rumen fermentation

This study was conducted to determine the effect of tamarind seed husk (TSH) as a source of tannin on various parameters of rumen fermentation in vitro. The TSH contained 14% tannin (DM basis). The biological interference of TSH tannin on rumen fermentation was assessed using polyethylene glycol (PEG) 6000 as an indicator. Three compound feed mixtures (CFM) were prepared either without TSH (CFM-I), with 2.5% TSH (CFM-II) and with 7.5% TSH (CFM-III). Parameters studied were in vitro gas production with PEG, rate of substrate degradation, and microbial protein synthesis. Addition of PEG to TSH resulted in an increase in gas production from 5.5 to 16.5 ml per 200 mg DM. Presence of TSH tannin depressed cumulative gas production by 16.8% in CFM-II, and by 29.2% in CFM-III during initial stages of fermentation (i.e. at 8 h). Rate of substrate disappearance (T^1/2) was 14.4, 17.6 and 20.5 h in CFM-I, CFM-II and CFM-III, respectively. Irrespective of the carbohydrate source, presence of TSH tannin improved the efficiency of microbial protein synthesis in vitro. Thus, TSH is a natural source of tannin that can be used to beneficially manipulate rumen fermentation.     

Fermentation of plant cell walls by ruminal bacteria,protozoa and fungi and their interaction with fibre particle size

Abstract

The objective of the experiment was to evaluate the contribution of various ruminal microbial groups to the fermentation of cell walls of corn stover with different particle sizes based on ruminal gas production in vitro. Physical, chemical, and antibiotical methods were used to differentiate groups of bacteria, protozoa and fungi in rumen fluid, offering following rumen microbial groups: whole rumen fluid (WRF), bacterial (B), protozoal (P), fungal (F), bacterial plus protozoal (B + P), bacterial plus fungal (B + F), protozoal plus fungal (P + F), and negative control (CON). Cell walls from corn stover were ground and ball milled to produce two different particle sizes. The results showed that digestion of the cell walls was undertaken by the interaction among ruminal bacteria, protozoa and fungi, and such co-actions seemed to fail alternation by one of three microbial groups or any combinations. However, B + P group showed a significant contribution to the degradation of milled cell walls, and B + F group revealed a great synergy effect on the ground cell walls degradation. Particle size of cell walls also had a considerable influence on their fermentation extent instead of the fermentative patterns by various rumen microbial groups.     

The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis

Tannins may reduce rumen degradability of protein, increase the proportion of feed protein reaching the lower digestive tract for enzymatic digestion and thereby increase the efficiency of protein utilization. The objective was to assess the effects of different types and levels of tannins on rumen in vitro gas production and its kinetics, in vitro true degradability (IVTD) and rumen degradability of protein (IVRDP), and microbial protein synthesis by incubating alfalfa (Medicago sativa L.) hay in buffered rumen fluid. Alfalfa was incubated in buffered rumen fluid with and without the addition of different levels of gallic acid (GA), quebracho tannin (QT), or tannic acid (TA). Tannins at the lower inclusion levels had minimal effects on fermentation products compared to the higher levels. Addition of QT and TA reduced ammonium-N (NH₄⁺-N) concentration. Addition of QT at 20, 40, and 60 g/kg DM decreased NH₄⁺-N by 2, 7, and 12% compared with control whereas addition of TA reduced NH₄⁺-N by 5, 6, and 12% when added at 20, 40 and 60 g/kg DM, respectively. In experiment 2, addition of QT at 50, 100, and 150 g/kg DM, resulted in reduction of NH₄⁺-N by 12, 30, and 51%, respectively, compared with the control. Addition of TA at 50, 100, and 150 g/kd DM reduced NH₄⁺-N by 14, 26, and 47% compared with control. Inclusion of QT at 50, 100, 150 DM reduced IVRDP by 13, 30, and 36% compared with control whereas at these levels of inclusion, TA resulted in reduction of IVRDP by 14, 25, and 48%. Rate of gas production decreased (P<0.001), while asymptotic gas production increased (P<0.0001) with increasing level of GA and TA. Quebracho tannin decreased (P<0.0001) both the rate and asymptotic of gas production. Gallic acid had a positive effect on fermentation as indicated by increased gas production and total short chain fatty acids (SCFA) production. Quebracho tannin decreased 24 h gas production, IVTD, and total SCFA production. Acetate to propionate ratio increased with the addition of GA and but decreased when QT was added. Addition of tannins did not markedly increase total purines but numerical values tended to be higher in the presence of tannins compared with the control. Efficiency of microbial growth was lower in the presence of GA and unaltered by TA, but higher in the presence of QT compared with the control. The effect of tannins on rumen fermentation and protein degradation varied with type and level of tannins. In vivo studies will be conducted to validate the in vitro results.     

Effects of inclusion of neutral detergent soluble fibre sources in diets varying in forage particle size on feed intake, digestive processes, and performance of mid-lactation Holstein cows

The aim of this study was to evaluate effects of partial replacement of neutral detergent soluble fibre (NDSF) for starch in diets varying in particle size (PS) of alfalfa hay on chewing activities, ruminal fermentation, nutrient digestibility and performance in mid-lactation dairy cows. Eight multiparous Holstein cows (146 ± 6.0 d in milk; 36.7 ± 2.57 kg milk/d) were used in a replicated 4 × 4 Latin square design with four 21 d periods with the last 7 d for data collection. The experiment was a 2 × 2 factorial arrangement with 2 levels of NDSF (low = 85 g/kg or high = 130 g/kg diet dry matter) each combined with 2 PS (short = 20 mm or long = 40 mm) of alfalfa hay. Results show that forage PS alone, or in combination with NDSF inclusion, had no effect on dry matter (DM) intake. Although total chewing, eating and ruminating times were not affected by treatments, eating time per kg of neutral detergent fibre (NDF) ingested was higher in long versus short alfalfa hay-based diets (P<0.05). Feeding long forage PS increased sorting of the diet against particles >19 mm, and in favor of those <8 mm (P<0.05). Feeding diets high in NDSF lowered DM intake (P<0.05), but increased apparent digestibility of all nutrients including NDF (P<0.05) independent of forage PS. Ruminal pH and concentrations of total volatile fatty acids were unaffected by dietary treatments, however the proportion of butyrate was higher in ruminal fluid of cows fed high NDSF diets (P<0.05). Changes in milk composition included lower milk crude protein content in high NDSF diets and higher lactose content for short hay-based diets (P<0.05). That milk yield and milk energy output were similar in low versus high NDSF diets suggests that high NDSF-fed cows had higher energy efficiency due to lower DM intake. Results suggest that, independent of forage PS, NDSF sources can be successfully included to partly replace starchy grains in diets exceeding minimum fibre recommendations.     

A new in vitro system to study the effect of liquid phase turnover and pH on microbial fermentation of concentrate diets for ruminants

A simple and inexpensive in vitro feed evaluation system which estimates microbial fermentation by measuring gas production and allows for adjustment of liquid dilution rate has been design. Working conditions, in terms of substrate and inoculum proportions (experiment 1, EXP 1) and the liquid replacement schedule (experiment 2, EXP 2) were initially set. In EXP 1, three substrate concentrations (10, 20 and 30 mg/ml: S1, S2 and S3) and three inoculum proportions (0.10, 0.20 and 0.30; I10, I20 and I30) were assayed in a 3 × 3 factorial design, in three incubations of 24 h. Gas production (ml/g organic matter) with S1 was the highest, avoiding to be in excess for microbial colonisation and activity and inoculum I20 limited inoculum contribution by self-fermentation; they were hence considered for further trials. In EXP 2, two liquid replacement schedules (every hour from 0 to 12 h and every 3 h from 12 to 24 h, HF, and every 2 h from 0 to 12 h and every 4 h from 12 to 24 h, LF) were compared with a non-replacement (NR) treatment, using three bottles per treatment. No differences among treatments were detected, and thus the LF schedule was chosen because it was easier to carry out. Once these working conditions were adjusted in EXP 1 and 2, the effect of fluid turnover and incubation pH on the extent of microbial fermentation in a mixed diet (experiment 3, EXP 3) was studied in two incubations. Two liquid turnover rates (0.06 and 0.10 h⁻¹) were compared with a non-replacement treatment at two different pH (6.8 and 6.1) in a 3 × 2 factorial design. Liquid outflow increased gas production, dry matter disappearance (DMd) and total volatile fatty acid (VFA) concentration at both pH (P>0.01), but protozoal numbers were unaffected. Liquid replacement at pH 6.8 did not affect the VFA profile, but the acetate proportion diminished (P=0.04) and the propionate increased (P<0.001), with replacement at pH 6.1. Increasing the fluid turnover rate from 0.06 to 0.10 h⁻¹ reduced gas production from 8 to 20 h of incubation (P<0.05) but it did not alter the VFA profile. The volume of gas produced decreased (P<0.05) with incubation pH, as did DMd (P=0.04) and protozoal (P=0.03) and total VFA (P=0.002) concentration. The acetate proportion was lower (P<0.001) and that of propionate and butyrate was higher (P<0.001) at pH 6.1 than at pH 6.8. Both liquid turnover and pH must be taken into consideration for in vitro evaluation of mixed and concentrate diets for ruminants and this system provides a simple and inexpensive method for doing this.     

Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Net photosynthetic rates (P _n) of easy (EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes were examined under the following culture conditions: (1) photoautotrophic [sugar-free medium, high photosynthetic photon flux (PPF), high vessel ventilation rates and CO₂ enrichment, (PA)]; (2) modified photomixotrophic [sugar-containing medium diluted with sugar-free medium over time, high PPF, and high vessel ventilation rates (PM)]; (3) modified photomixotrophic enriched [same as PM with CO₂ enrichment, (PME)]; or (4) conventional photomixotrophic [sugar-containing medium, low PPF, and low vessel ventilation rates (control)]. Regardless of genotype, plantlets cultured under PA conditions died within 2 wk, whereas under PM and PME conditions, plantlets increased their P _n. After 6 wk, P _n per gram dry weight was 1.7 times greater in EK 16-3 than EK 11-1 plantlets cultured under PME conditions. In vitro-produced leaves of EK 16-3 plantlets were elongated with expanded blades, whereas EK 11-1 produced short leaves without expanded blades, especially under control conditions. After in vitro culture, EK 16-3 PME plantlets exhibited the highest dry weights among treatments. EK 16-3 PME and EK 16-3 PM had similarly high survivability, shoot and root dry weights and leaf lengths ex vitro compared to EK 16-3 control and EK 11-1 PM and PME plantlets. Ex vitro growth, survivability and P _n per leaf area of either genotype were not affected by CO₂ enrichment under modified photomixotrophic conditions. These results suggest that growth and survivability of sea oats genotypes with different acclimatization capacities can be enhanced by optimizing culture conditions.     

Effects of condensed tannins in white clover flowers on their digestion in vitro

Protein in white clover (Trifolium repens L.) is poorly utilised by ruminants because of its extensive degradation to ammonia in the rumen. However, white clover produces condensed tannins (CT) in its flowers, which can reduce rumen proteolysis. Effects of increasing proportions of clover dry matter (DM) as flowers (and therefore floral CT) on soluble protein, ammonia and volatile fatty acid (VFA) concentrations were determined with in vitro incubations. Minced mixtures of 0, 250, 500, 750 and 1000 g/kg of DM as white clover flower (F) with the remainder as white clover leaf, were incubated in vitro and sampled after 0, 2, 4, 8, 12 and 24 h. Treatments contained 0, 13, 26, 39 and 52 g CT/kg DM, respectively. A further treatment with 500 g/kg DM as flower and 500 g/kg DM as leaf had polyethylene glycol added to remove effects of CT. Increasing the proportion of white clover as flowers from 0 to 1000 g/kg DM reduced net conversion of plant N to ammonia N from 290 to 120 mM/M at least partly due to reduced solubility of the protein. Treatments with 750 g/kg DM or more as clover flowers reduced ammonia concentrations to levels likely to limit microbial growth. Total VFA production was not affected by flower content, although the proportion of acetate to propionate increased. The contribution of CT to treatment effects was small compared to effects attributed to difference in chemical composition between flowers and leaves.     

Different fibrous ingredients and coarsely ground maize affect hindgut fermentation in the pig in vitro but not Salmonella Typhimurium survival

The fermentation kinetics of both fibre choice and maize particle size were studied in vitro from the ileal contents of cannulated pigs given five different experimental diets. Additionally, in vitro batch systems were used to study the quantitative effect of fibre choice and maize particle size on Salmonella Typhimurium growth. Freeze-dried ileal effluents obtained from five cannulated pigs given the five experimental diets in a latin square were used as incubation substrates. The experimental diets consisted of: (1) a standard maize-based diet (ST) where all the ingredients were milled to pass through a 2.5-ml screen, (2) the same diet with the maize ground using a 4.0-ml screen (CG), (3 and 4) two diets in which the maize was partially replaced by (3) sugar beet pulp (80 g/kg; BP) or (4) wheat bran (100 g/kg; WB), or (5) a combination of 80 g sugar beet pulp and 100 g wheat bran/kg (diet HF). Results showed that substrate from BP and HF generated more gas than substrate from ST (P<0.001). In addition, short chain fatty acids production during the entire incubation period was higher (P=0.001) for all the substrates compared to ST substrate (P<0.001) where lower acetate and higher propionate, valerate and branched-chain fatty acids molar ratios were measured. The molar ratio of acetate was highest and the branched-chain fatty acids was lowest for substrates from diets that included sugar beet pulp. The butyrate molar ratio was lower for all substrates compared to the ST diet except for the WB diet. After 12 h of incubation, HF substrate presented the lowest ammonia (91.7 mg/l vs. 125.3 mg/l, P=0.014) and the highest purine bases concentration (0.28 μmol/ml vs. 0.22 μmol/ml, P=0.009). None of the substrates showed any inhibitory effect on Salmonella growth when the batch systems were inoculated with S. Typhimurium. In summary, under the in vitro conditions used, the changes in hindgut fermentation promoted by the inclusion of different fibrous ingredients or by different maize particle size did not produce an inhibitory effect on the growth of S. Typhimurium.