Effect of chitosans on in vitro rumen digestion and fermentation of maize silage

The gas in vitro technique was used to study the effects of six types of chitosans, each having different molecular weights and acetylation degrees, on rumen microbial fermentation. In a first trial, a separate concentration of 750 mg/l of culture fluid for each of the six chitosans (CHI1, CHI2, CHI3, CHI4, CHI5, and CHI6) was incubated for 24 h in diluted ruminal fluid with maize silage as the substrate. The ionophore antibiotic monensin (MON) was used as a positive control, and a negative control with no chitosan (CTR) was also included. Each treatment was tested in triplicate for three different periods. At the end of the trial, samples were collected to determine volatile fatty acid (VFA) and ammonia N concentrations, and pH and gas production values were recorded. Methane concentration was estimated stoichiometrically. In vitro true organic matter digestibility (IVOMD) and partitioning factor (PF, mg OM truly degraded/ml gas produced) were also calculated. In a second trial, a separate concentration of 750 mg/l of each of the six chitosans was incubated for 144 h in diluted ruminal fluid with maize silage as the substrate, to study the effects of these compounds on fermentation kinetics.All six chitosans decreased the IVOMD and PF values. Chitosan inclusion did not affect the fermentation of the substrate's soluble fraction, but did reduce the fermentation kinetics of the insoluble but fermentable fraction. However, only CHI5 and CHI6 decreased total VFA concentration. CHI3 and CHI6 decreased the molar proportion of acetate and increased the molar proportion of propionate, thus increasing the propionate-to-acetate ratio. Chitosan inclusion did not affect molar proportions of butyrate. With the exception of CHI2, the molar proportion of branch-chained VFA was lowered by all of the chitosan treatments. Most of the treatments also decreased methane production, also with the exception of CHI2.In conclusion, chitosan extracts may enable the manipulation of rumen microbial fermentation, but further research is required to elucidate the effect of chitosans on ruminal fermentation parameters in commercial diets as well as the adaptability of rumen microflora to these additives.     

Effect of elemental nano-selenium on feed digestibility,rumen fermentation,and purine derivatives in sheep

The objective of this experiment was to study the effect of elemental nano-selenium (NS) on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Eight male ruminally cannulated sheep (42.5 ± 3.2 kg of body weight, BW) were used in a replicated 4×4 Latin square experiment in four 20 day periods. Depending on treatment designation, sheep were fed the basal diet supplemented with 0 (control), 0.3, 3 and 6 g of nano-Se/kg dry matter (DM). Ruminal pH (range of 6.68–6.80) and ammonia N concentration (range of 9.95–12.49 mg/100 mL) was decreased (P<0.01), and total VFA concentration (range of 73.63–77.72 mM) was increased linearly (P<0.01) and quadratically (P<0.01) with increasing nano-Se supplementation. The ratio of acetate to propionate was linearly (P<0.01) and quadratically (P<0.01) decreased due to the increasing of propionate concentration. In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis and crude protein (CP) of soybean meal were linearly (P<0.01) and quadratically (P<0.01) improved by feeding nano-Se. Similarly, nutrients digestibility in the total tract and urinary excretion of purine derivatives were also quadratically (P<0.01) changed by increasing nano-Se supplementation. The present results indicated that nano-Se supplementation in basal diet improved rumen fermentation and feed utilization. Nano-Se could also stimulate rumen microbial activity, digestive microorganisms or enzyme activity. The optimum dose of nano-Se was about 3.0 g/kg dietary DM in sheep.     

Effects of dietary whole cottonseed and crude protein level on rumen protozoal population and fermentation parameters

In this investigation in vitro and in vivo trials were performed to determine the efficacy of a cottonseed to limit protozoal population and fermentation parameters. The composition of diets given to the different treatments were as follow: (1) control (without whole cottonseed), 16% crude protein (CP), 3.2% ether extract (E.E.); (2) 20% whole cottonseed, 16% CP, 6.5% E.E.; (3) 20% whole cottonseed, 13% CP, 6.4% E.E. and (4) 20% crushed whole cottonseed, 13% CP, 6.4% E.E. DM disappearance (DMD) and fermentation characteristics of the treatments were determined by in vitro incubation studies. In the in vivo trial, ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1, 3, 5, 7, 9, 11, 14, 21 and 28 from four sheep fed about treatment diets. The pH and protozoal counts were determined in each sample, while ammonia nitrogen and volatile fatty acid (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD after 24 h incubation decreased (p < 0.01) with the addition of cottonseed in diets 3 and 4 and DMD after 72 h incubation was highest (p < 0.01) for the control diet. The fractional rate of gas production (c) for the control and diet 2 was higher (p < 0.05) than for the diets 3 and 4. Feeding crushed whole cottonseed decreased molar proportion of propionate (p < 0.05) and increased molar proportion of butyrate (p < 0.01). Low crude protein level increased the molar proportion of propionate (p < 0.05) and decreased molar proportion of butyrate (p < 0.05) and cellolytic protozoa population (p < 0.05). Feeding cottonseed decreased (p < 0.05) the total protozoa population from approximately 500,000 to 250,000 ml⁻¹ and Holotrich and cellulolytic protozoa disappeared from the rumen of sheep and only Entodinium sp., remained. This was associated with lower concentration of ammonia nitrogen in rumen fluid of sheep fed diets 4 (p < 0.05) and 2 (p < 0.01). It was concluded that cottonseed reduced rumen fauna and ammonia nitrogen, but had no effect on ruminal VFA while the crushed whole cottonseed decreased molar proportion of isovalerate only. In vivo molar proportion of propionate and butyrate and valerate were increase and decrease, respectively, by decreasing CP percentage in treatment diets.     

Influences of flavomycin,ropadiar, and saponin on nutrient digestibility,rumen fermentation,and methane emission from sheep

This study focused on the effects of three additives given together with a hay/concentrate-based diet on nutrient digestibility, rumen fermentation, and methane emission from sheep. The basal diet consisted of 1.29 kg mixed hay and 0.43 kg concentrate mixture based on dry matter (DM). Treatments consisted of control (no additive), flavomycin⁴⁰ (250 mg/d), ropadiar from an oregano extract (250 mg/d), and saponin in the form of a yucca schidigera extract (170 mg/d). Results indicated that intake and digestibility were unaffected by treatments (P>0.05). The NH₃-N concentration of rumen liquor was lower (P<0.05) for additive treatments versus the control treatment. Higher concentrations of volatile fatty acid (VFA) were observed in the saponin (75.8 mmol/L) and ropadiar (73.1 mmol/L) treatments. The proportion of individual fatty acid of rumen liquor was unchanged, whereas lower ratio of acetate to propionate in the saponin treatment was observed (P<0.05). The average methane production expressed on digested organic matter (OM) and neutral detergent fiber (aNDFom) basis were decreased by approximately 3.3 and 12.0 g/kg, respectively in saponin, and 4.2 and 11.9 g/kg in ropadiar treatment compared to the control. Methane production was positively correlated with the concentrations of NH₃-N, and negatively correlated with total VFA and the proportion of propionate of rumen liquor (P<0.05). The study found that saponin and ropadiar could have the potential to reduce rumen methanogenesis in sheep.     

Batch culture evaluation of carbohydrase inhibitors to moderate rumen fermentation

While carbohydrase inhibitors have been widely investigated for regulating human carbohydrate assimilation, their potential application to animal nutrition has been largely ignored. Batch culture fermentations were conducted to determine how commercially available α-amylase and α-glucosidase inhibitors affect rumen fermentation. Fermentations were with 0.5 g of ground corn grain, and 40 mL of buffered rumen fluid inoculum. Rumen fluid donors were fed a 0.5 concentrate 0.5 forage diet. Incubations were conducted in duplicate and replicated on consecutive days with pH and volatile fatty acid (VFA) concentrations measured. The microbial-derived inhibitors, acarbose (ACB) and trestatin (TRE), prevented the decreases in pH and VFA production observed in the control tubes at doses of 1.2–9.5 and 0.1–1.1 mg, respectively. Miglitol, glipizide, and the plant-derived amylase inhibitors failed to affect pH or reduce VFA concentrations with the same apparent potency as did ACB and TRE, which both show the potential to reduce the amount of starch fermented in the rumen. These compounds may be potentially useful for reducing ruminal acid production and allowing more starch to pass to the small intestine from the forestomachs.     

Effect of rare earth elements on in vitro rumen microbial fermentation and feed digestion

The objectives of this study were to investigate the effects of rare earth elements (REEs) on in vitro rumen fermentation, gas production, microbial protein synthesis and nutrient digestion using in vitro batch culture and continuous culture technique. A mixture of REE containing (g/kg) 380 g of LaCI₃·6H₂O, 521 g of CeCI₃·6H₂O, 30 g of PrCI₃·6H₂O and 69 g chlorides of other light REEs. The experimental diet consisted of 885 g/kg barley grain, 84 g/kg barley silage and 31 g/kg supplement (dry matter (DM) basis). Diet supplemented with different dosages of REE (control, no additional REE; low, 400 mg/kg REE; and high, 800 mg/kg REE, DM basis) were incubated for 4, 8, 14 and 24 h in diluted rumen fluid. At the end of 24 h of incubation, gas production and concentration of volatile fatty acid (VFA) linearly increased with increasing REE supplementation; whereas, influence of REE supplementation on VFA profile was marginal. Dry matter disappearance was not affected (P>0.10). Six dual-flow continuous culture fermenters were used in a replicated 3 × 3 Latin square with same treatments and same diet used in the batch culture. Mean ruminal pH (5.71) and total VFA (93.6 mM) concentration were not affected by supplementation of REE. The molar proportion (mol/100 mol) of acetate (39.1) and propionate (50.5) was similar among the treatments. However, the proportion (mol/100 mol) of butyrate was higher with the high REE (6.6) than with low REE (5.3) or the control (5.8). Ruminal true digestibilities of organic matter (OM) (0.785, 0.811 and 0.828), acid detergent fibre (0.360, 0.431 and 0.432) and crude protein (0.496, 0.590 and 0.589) for control, low and high REE, respectively, linearly increased with increasing REE supplementation, whereas, the increase in ruminal digestibility from low to high dosage of REE was minimal. Microbial nitrogen (N) production (g/day) and microbial efficiency (g N/kg of truly fermented OM) were not affected by treatments. Improvement of ruminal digestibility of OM due to REE supplementation was attributed to the increase in digestibility of fibre and degradability of protein. The results suggest that REE supplementation improved ruminal fibrolytic and proteolytic activities.     

Effect of peppermint (Mentha piperita) oil on in vitro methanogenesis and fermentation of feed with buffalo rumen liquor

The effect of inclusion of peppermint (Mentha piperita) oil (at 0, 0.33, 1.0 and 2.0 μl/ml of incubation medium) on gas and methane production, fermentation of feed and microbial profile was studied in in vitro gas production test, using 200 mg of wheat straw and concentrate mixture in equal proportion as substrate in a 100 ml graduated syringe. The buffalo rumen liquor was used as inoculum and the observations were recorded at 24 h of incubation. Methane emission was reduced (P<0.001) by 19.9%, 46.0% and 75.6% at 0.33, 1.0 and 2.0 μl levels, respectively. The concentration (mM/100 ml) of total volatile fatty acids was reduced (P<0.01) by inclusion of peppermint oil at higher levels (1.0 and 2.0 μl) whereas at 0.33 μl level there was no effect. The proportion of acetate increased (P<0.05) and that of propionate decreased (P<0.001) at 1.0 and 2.0 μl levels of peppermint oil. There was a fall (P<0.001) in carboxymethylcellulase and xylanase activities and the inhibition increased with the increasing level of peppermint oil which resulted in a dose dependent decrease (P<0.05) in in vitro true digestibility of feed. At 0.33 μl level of peppermint oil, the population density of total bacteria was similar to that of control but fungi, Ruminococcus flavefaciens and methanogens increased by 4-, 6- and 2-folds, respectively, as determined with real-time PCR. At 1.0 and 2.0 μl levels the population density of total bacteria, fungi, Fibrobacter succinogens and methanogens decreased drastically and fell below the control values. The numbers of holotrichs and spirotrichs were reduced (P<0.001) by increasing dose of peppermint oil. The higher doses of peppermint oil were toxic for the rumen microbes but the lower levels could be further explored in in vivo experiments as rumen modifier to reduce methanogenesis.     

Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages

Published analyses of enteric methane (CH₄) emissions from sheep and cattle show an inverse relationship between feed intake and CH₄ yield (g CH₄/kg dry matter (DM) intake), which suggests opportunities for reducing CH₄ emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH₄ yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH₄ emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6 × metabolizable energy requirements for maintenance; ME_m), 28 sheep in Experiment 2 (at 0.8 and 2.0 × ME_m), eight sheep and two measurement periods in Experiment 3 (at 1.6 × ME_m), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5 × ME_m). Prior to each experiment, sheep had a 10 d acclimatization period to diets. Apparent digestibility was measured over 7 d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH₄ yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60 kg/d, the predicted responses were an increase in CH₄ production (g/d) of 187% (12.4–35.6 g/d; P<0.001), and a decline in CH₄ yield of 21% (25.6–20.2 g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH₄ production, and molar proportion of propionate predicting 0.60 of the variation in CH₄ yield. Increasing feed intakes by 1 kg/d of DM reduced CH₄ yield by 4.5 g/kg DM intake. Plant chemical composition was weakly related to CH₄ yield. High intakes of fresh forages will lower CH₄ yield from fermentation, but effects of feed composition on CH₄ emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH₄ emissions.     

Effect of combined ensiling of sorghum and soybean with or without molasses and lactobacilli on silage quality and in vitro rumen fermentation

Two sorghum (Sorghum bicolor (L.) Moench) varieties (SG₁ & SG₂), with the former showing higher grain and total DM yield, but also increased tannin contents compared to the latter, and one soybean (Glycine max (L.) Merr.) variety (INCASOY-35) were sown, harvested (at pasty grain state), chopped and ensiled (CIAP, Cuba). Silages were made in following combinations: either SG₁ or SG₂ combined with soybean in two proportions (0.4 and 0.6). All silages were prepared with or without molasses (3.5% of fresh material) and Lactobacillus sp. as inoculant (3 × 10⁵ colony forming units/g). Silage quality parameters included pH, ammonia, lactate, acetate, butyrate and water soluble carbohydrates content. Further, both fresh and ensiled materials were incubated in vitro with buffered rumen fluid to study the fermentation characteristics. Silage of a good quality could be produced with both sorghum varieties alone, but combined silages showed improved quality compared to soybean silage (p<0.05 for all quality characteristics). Addition of molasses and bacterial inoculant further improved silage quality (p<0.05 for all quality characteristics). In vitro incubation (24 h) of ensiled material resulted in lower acetate and higher propionate proportion compared to fresh forages. However, ensiling without molasses and inoculant reduced in vitro short chain fatty acid production and hence the apparent rumen degradability of organic mater as well as the fermentation rate. As expected, a higher proportion of sorghum increased the molar propionate proportion and the fractional fermentation rate, whereas ammonia (mmol/L) concentrations were reduced. SG₁ silages produced higher molar propionate proportions, lower acetate proportions and ammonia concentrations.     

Influence of natural magnesium sources on the in vitro fermentation and protozoan population in the rumen fluid collected from sheep

The objective of this experiment was to determine the effect of two types of caustic calcinated magnesite (caustic magnesite (CM) and Agromag (AG)) upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, and VFA) and protozoan population in the rumen fluid collected from sheep. Both magnesium additives (CM and AG) as natural products in the dose of 0.01 g were added to the fermentation bottles containing rumen inoculum from sheep and different substrates. Meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG) were used as substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique during 72 h of incubation. The rumen protozoa, Entodinium spp., Trichostomatids and large Entodiniomorphids and the total protozoan concentration were counted after 24 h of incubation. The methane production was significantly decreased with CM or AG, respectively, by 58 or 62% (MH), by 65% (WS), by 52% (AC) and by 58% (BG). The total VFA concentration was significantly lower compared to control for CM plus MH, WS, AC, BG and AG plus WS. The total VFA concentration was significantly higher compared to control for AG plus AC. The effect of the both additives on ciliate population was not uniform and depended on the substrates used and protozoan type. Ciliate population was significantly increased in Entodinium spp. (AG plus BG) and Diploplastron affinae (CM or AG plus BG) compared to control. Tested additives significantly decreased population of Entodinium spp. (AG plus MH or AC), Dasytricha ruminantium (AG plus AC), Ophryoscolex c. tricoronatus, Eremoplastron dilobum and Polyplastron multivesiculatum (CM or AG plus BG). It can be concluded that both natural magnesium sources influenced rumen fermentation patterns and protozoan population in vitro depending on the type of the substrate used; therefore, the relative efficacy of individual tested additive cannot be determined from these experiments. In vivo experiments are required in future.     

Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation

Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermentation pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at ?20 °C (FRZ), frozen in liquid N (FLN) or added with 0.04 glycerol and frozen in liquid N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 ml per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilisation of added glycerol after thawing in FGL increased total gas production (P<0.05) and 24 h volatile fatty acid (VFA) production (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas production (as ml/g dry matter per hour), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermentation at 8 and 12 h, respectively. Differences in gas production did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690–0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas production studies does not affect microbial fermentation pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to volume ratio. Freezing in liquid N is more appropriate than at ?20 °C.     

The relationship between the volatile fatty acids supply and the nitrogen retention in growing sheep nourished by total intragastric infusions

Twelve four-month old Suffolk × Small-tail-Han male sheep (live weight 21–26 kg), fitted with rumen and abomasum fistulas and nourished by total intragastric infusions, were used to study the relationship between the volatile fatty acids (VFA) supply and the nitrogen (N) retention in sheep. The animals were randomly divided into four groups and four levels of mixed VFA energy (the molar proportion of acetic acid, propionic acid and butyric acid was 65:25:10), i.e. 333, 378, 423 and 468 kJ kg^?1 W^0.75 d^?1, were infused into the rumen, as treatments I, II, III and IV, respectively. The results showed that the N retention was significantly increased (P < 0.05) with the VFA infusion level. Significant regression relationship was found between the VFA supply (x, g d^?1) and the N retention (y, mg d^?1): y = 2.75x ? 403, r² = 0.86, n = 12, P < 0.01. It was concluded that to get efficient utilization of dietary N and high N retention in sheep, it is necessary to supply enough dietary energy.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

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 dl-malate 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.     

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G⁺, glucose in the Hungate's medium as a glucose control; G^?, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract + tryptone + (NH₄)₂SO₄ in Hungate's medium (control); N2: yeast extract + (NH₄)₂SO₄; N3: tryptone + (NH₄)₂SO₄; and N4: tryptone + yeast extract). In the G^? media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G⁺ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G^? medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r > 0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.     

Effects of type of ionophore and carrier on in vitro ruminal dry matter disappearance,gas production,and fermentation end products of a concentrate substrate

Effects of ionophore type and carrier on in vitro ruminal digestion and fermentation patterns of a concentrate substrate were evaluated at various incubation times. Treatments were: control (no ionophore); lasalocid sodium commercial premix (Bov); lasalocid sodium mycelium cake (LasBio); laidlomycin sodium salt (LaidNa); laidlomycin propionate commercial premix (LaidPro); monensin sodium salt (Mon); and monensin sodium commercial premix (Rum). The Bov, LasBio, Mon, and Rum treatments supplied 4 μg of ionophore/mL of culture volume, whereas the LaidNa and LaidPro treatments supplied 1.33 μg of ionophore/mL. Total gas and methane production did not differ among treatments at any of the incubation times (P>0.09). Similarly, in vitro dry matter disappearance (IVDMD) was not affected by treatment (P>0.28) at 6, 18, and 24 h of incubation; however, IVDMD (P=0.03) was greater for ionophores than for the control at 12 h of incubation. Molar proportions of acetate (P<0.01), acetate:propionate (P<0.01), and total volatile fatty acid (VFA) concentrations (P<0.01) were decreased and propionate was increased (P<0.001) for the average of all ionophore-containing substrates compared with the control. Total VFA were decreased by Bov, LaidNa, and Rum, contrasted with their specific counterparts (LasBio, LaidPro, and Mon, respectively; P<0.05). Differences were detected among ionophore types for acetate (lasalocid vs. laidlomycin; P<0.05), propionate (lasalocid vs. monensin; P<0.05), and butyrate (monensin vs. lasalocid or laidlomycin; P<0.05). Capture of metabolic hydrogen in end products of fermentation was greater for ionophore-containing treatments (P<0.01) than for the control. These data suggest limited unique effects of ionophore type or carrier on IVDMD, total gas production, and methane; however, VFA proportions varied among ionophore types and carriers, which deserves further study.     

Ruminal ochratoxin A degradation—Contribution of the different microbial populations and influence of diet

The mycotoxin ochratoxin A (OTA) is degraded extensively in the rumen. In this study, the relative contribution of different rumen microbial populations (MP) and the effect of diet on degradation of OTA were evaluated in a factorial design experiment. Degradation of OTA was quantified by using the Hohenheim gas test (HGT) in vitro fermentation system. Five different HGT diets were used (concentrate:forage proportions (C:F) – 10:90, 30:70, 50:50, 70:30, 90:10), and donor animals were fed diets with the respective ratio. Diets with the highest concentrate content were supplied with and without 10 g/kg sodium bicarbonate (70:30 BC and 90:10 BC). The MP included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi. Protozoa numbers were counted after 24 h and OTA and ochratoxin alpha (OTα) analysed at 0, 4, 8, 12, 24 h. Area under the curve (AUC) and half-life were calculated for the latter two. The short average OTA half-life for whole rumen fluid of 2.6 h (1.3–4.5 h) demonstrates the high OTA degradation capacity of the rumen MP (i.e., standard HGT inoculum) and corresponds well with published in vivo results. Both MP and diet affected OTA degradation. Interactions among factors occurred (P<0.001), which made it necessary to do further comparisons within factor levels. Among MP, those with bacteria (bacteria + fungi and bacteria + protozoa) had lower AUC values (P<0.001) for OTA (196–673 ng/ml h, meaning higher degradation capacity, than those without bacteria (fungi + protozoa and protozoa; 701–1206 ng/ml h). Whole rumen fluid had the lowest AUC values (146–249 ng/ml h; P<0.05). Diet had a quadratic effect (P=0.001) on protozoal numbers with minimum values for the lowest and highest C:F ratios, for bacteria + protozoa, fungi + protozoa and protozoa, but no corresponding effect was found for OTA degradation parameters. While the generally high capacity to degrade OTA was confirmed, results for the contribution of different microbial groups shed new light on ruminal OTA degradation.     

Effects of yeast culture (Saccharomyces cerevisiae) on ruminal digestion in non-lactating dairy cows

Six non-lactating dairy cows fitted with ruminal cannulas were used in a cross-over design, to investigate the effects of supplemental yeast culture (Saccharomyces cerevisiae) (YC) and interaction of YC by sampling time on ruminal fermentation and in situ fibre degradation. Cows were fed twice daily with a diet composed of 67% corn silage, 32% concentrate and 1% vitamin and mineral mixture, on a dry matter (DM) basis. Concentrates were not mixed with silage. YC (0.5% DM) significantly decreased rumen ammonia from 148.5 mg l⁻¹ to 103.1 mg l⁻¹ 3 h post-feeding, and significantly increased by about 20% the concentration of total volatile fatty acids before and 1 h after feeding. YC significantly increased molar percentage of propionate and decreased the acetate : propionate ratio before feeding. No significant effect was observed on ruminal pH and molar percentages of acetate or butyrate. Pattern of degradation of DM, neutral and acid detergent fibre from hay was affected, with a cubic effect of interaction of YC by incubation time. However, magnitude of degradation was not significantly different at any time. These results show that modifications of ruminal fermentation due to YC addition are time dependent when the diet is fed twice daily.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

Effect of natural dolomites on the in vitro fermentation and rumen protozoan population using rumen fluid and fresh faeces inoculum from sheep

The objective of the study was to determine the effect of dolomites from five different sources upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, hydrogen recovery) and protozoan population. Dolomites as natural products in the dose of 0.1 g were added to the fermentation bottles containing inoculum from sheep and substrates. Both rumen fluid (RF) and fresh faeces (FF) from sheep as the sources of inocula for in vitro fermentation were used. Meadow hay (MH) and barley grain (BG) were used as fermentation substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique in separate incubation during 72 h. Both inocula (RF and FF) and dolomites impact in vitro fermentation characteristics. The gas volume was significantly increased with dolomites with RF or FF, respectively, by 20% or 20–40% (MH) and by 10% or 10–30% (BG). The methane production was significantly decreased with dolomite additives with RF inocula by 15–32% (MH) and by 50–70% (BG). A significant effect of the dolomite additives on the rumen protozoan population was observed during fermentation of MH; the total protozoan concentration and the number of Entodinium spp. was decreased (P < 0.05). Populations of Isotrichids and large Entodiniomorphids were not influenced by experimental incubations. More studies are needed to optimize the combination of different diets with dolomite additives for practical feeding conditions.