Single-flow Continuous Culture Fermenters Research Articles

Supply of palmitic, stearic, and oleic acid changes rumen fiber digestibility and microbial composition

The concept that fat supplementation impairs total-tract fiber digestibility in ruminants has been widely accepted over the past decades. Nevertheless, the recent interest in the dietary fatty acid profile to dairy cows enlightened the possible beneficial effect of specific fatty acids (e.g., palmitic, stearic, and oleic acids) on total-tract fiber digestibility. Because palmitic, stearic, and oleic acids are the main fatty acids present in ruminal bacterial cells, we hypothesize that the dietary supply of these fatty acids will favor their incorporation into the bacterial cell membranes, which will support the growth and enrichment of fiber-digesting bacteria in the rumen. Our objective in this experiment was to investigate how dietary supply of palmitic, stearic, and oleic acid affect fiber digestion, bacterial membrane fatty acid profile, microbial growth, and composition of the rumen bacterial community. Diets were randomly assigned to 8 single-flow continuous culture fermenters arranged in a replicated 4 × 4 Latin square with four 11-d experimental periods. Treatments were (1) a control basal diet without supplemental fatty acids (CON); (2) the control diet plus palmitic acid (PA); (3) the control diet plus stearic acid (SA); and (4) the control diet plus oleic acid (OA). All fatty acid treatments were included in the diet at 1.5% of the diet (dry matter [DM] basis). The basal diet contained 50% orchardgrass hay and 50% concentrate (DM basis) and was supplied at a rate of 60 g of DM/d in 2 equal daily offers (0800 and 1600 h). Data were analyzed using a mixed model considering treatments as fixed effect and period and fermenter as random effects. Our results indicate that PA increased in vitro fiber digestibility by 6 percentage units compared with the CON, while SA had no effect and OA decreased fiber digestibility by 8 percentage units. Oleic acid decreased protein expression of the enzymes acetyl-CoA carboxylase compared with CON and PA, while fatty acid synthase was reduced by PA, SA, and OA. We observed that PA, but not SA or OA, altered the bacterial community composition by enhancing bacterial groups responsible for fiber digestion. Although the dietary fatty acids did not affect the total lipid content and the phospholipid fraction in the bacterial cell, PA increased the flow of anteiso C13:0 and anteiso C15:0 in the phospholipidic membrane compared to the other treatments. In addition, OA increased the flow of C18:1 cis-9 and decreased C18:2 cis-9,cis-12 in the bacterial phospholipidic membranes compared to the other treatments. Palmitic acid tended to increase bacterial growth compared to other treatments, whereas SA and OA did not affect bacterial growth compared with CON. To our knowledge, this is the first research providing evidence that palmitic acid supports ruminal fiber digestion through shifts in bacterial fatty acid metabolism that result in changes in growth and abundance of fiber-degrading bacteria in the microbial community.

395 Effects of supplemental protein and yeast on fermentation of low quality forage in single-flow continuous culture fermenters

Abstract Various anecdotes suggest the combination of supplemental protein and yeast improve utilization of low-quality forage. Two studies were conducted to evaluate effects of a commercially available supplement containing protein and yeast on fermentation in single-flow continuous culture fermenters. In Exp. 1, Bermuda grass with no supplement (NCON1), dried distillers grains (PCON1), and two commercially available supplements Diamond V XP (DV1) or SweetPro 16 (SP1) were compared. In Exp. 2, K31 tall fescue with no supplement (NCON2), supplemental dried distillers grains (PCON2), and two commercially available supplements Diamond V XP (DV2) or SweetPro 16 (SP2) were compared. Twenty-four single flow fermenters (1470mL) were randomly assigned to treatments and fed twice daily for 8 d, 5 d of adaptation and 3 d of collection. In both experiments, digestibility of DM, OM, NDF, and ADF were not influenced by supplementation regardless of source (P > 0.10). In Exp. 1, NH3-N concentration increased (P < 0.01) for DV1 over both NCON1 and PCON1 and was greatest for SP1 (P < 0.01). Greater amounts of NH3-N and microbial N were recovered from fermenter overflow when SP1 was supplemented (P < 0.01). Crude protein (CP) degradation was also greatest for SP1; however, when supplement was included, regardless of source, CP degradation was increased over NCON1. Acetate, propionate, butyrate, and total VFA levels were similar (P > 0.10) amongst NCON1, PCON1, and DV1 and greatest when SP1 was supplemented. In Exp. 2, fermenter NH3-N concentration was greatest for SP2 (P < 0.01) and similar amongst NCON2, PCON2, and DV2 (P > 0.01). No differences were detected (P > 0.10) between treatments with respects to CP degradation. Total VFA concentrations increased over time regardless of treatment during the second study.

Use of tomato and cucumber waste fruits in goat diets: effects on rumen fermentation and microbial communities in batch and continuous cultures

SUMMARYTwoin vitroexperiments were conducted to analyse the effects of replacing dietary barley grain with wastes of tomato and cucumber fruits and a 1 : 1 tomato : cucumber mixture on rumen fermentation characteristics and microbial abundance. The control (CON) substrate contained 250 g/kg of barley grain on a dry matter (DM) basis, and another 15 substrates were formulated by replacing 50, 100, 150, 200 or 250 g of barley grain/kg with the same amount (DM basis) of tomato or cucumber fruits or 1 : 1 tomato : cucumber mixture. In Expt 1, all substrates were incubated in batch cultures with rumen micro-organisms from goats for 24 h. Increasing amounts of tomato, cucumber and the mixture of both fruits in the substrate increased final pH and gas production, without changes in final ammonia-nitrogen (NH3-N) concentrations, substrate degradability and total volatile fatty acid (VFA) production, indicating that there were no detrimental effects of any waste fruits on rumen fermentation. Therefore, in Expt 2 the substrates including 250 g of waste fruits (T250, C250 and M250 for tomato, cucumber and the mixture of both fruits, respectively) and the CON substrate were incubated in single-flow continuous-culture fermenters for 8 days. Total VFA production did not differ among substrates, but there were differences in VFA profile. Molar proportions of propionate, isobutyrate and isovalerate were lower and acetate : propionate ratio was greater for T250 compared with CON substrate. Fermentation of substrates containing cucumber (C250 and M250) resulted in lower proportions of acetate, isobutyrate and isovalerate and acetate : propionate ratio, but greater butyrate proportions than the CON substrate. Carbohydrate degradability and microbial N synthesis tended to be lower for substrates containing cucumber than for the CON substrate, but there were no differences between CON and T250 substrates. Abundance of total bacteria,Fibrobacter succinogenesandRuminococcus flavefaciens, fungi, methanogenic archaea and protozoa were similar in fermenters fed T250 and CON substrates, but fermenters fed C250 and M250 substrates had lower abundances ofR. flavefaciens, fungi and protozoa than those fed the CON substrate. Results indicated that tomato fruits could replace dietary barley grain up to 250 g/kg of substrate DM without noticeable effects on rumen fermentation and microbial populations, but the inclusion of cucumber fruits at 250 g/kg of substrate DM negatively affected some microbial populations as it tended to reduce microbial N synthesis and changed the VFA profile. More studies are needed to identify the dietary inclusion level of cucumber which produces no detrimental effects on rumen fermentation and microbial growth.

Microbial and chemical composition of liquid-associated bacteria in goats' rumen and fermenters

This study was undertaken to investigate the relationship between chemical composition and microbial profile of rumen liquid-associated bacteria (LAB) in vivo (Murciano-Granadina goats) and in a rumen simulation system (single-flow continuous-culture fermenters). To achieve this aim, analyses of purine bases along with some molecular techniques (quantitative PCR to assess abundance and DGGE to identify biodiversity and bacterial profile) were carried out. A control diet (AHC) based on alfalfa hay (AH) and concentrate (C) in a 1:1 ratio and two experimental diets (AHCBI and AHCBII), in which concentrate was partially replaced with multinutrient blocks, were used. Diets AHCBI and AHCBII included multinutrient blocks differing in the relative amount of two-stage olive cake and the source of protein (sunflower meal vs. fava beans). We aimed to investigate the effect of these blocks on rumen microbiota to evaluate their potential as safe substitutes of cereal-based concentrates. Similar patterns of response to diet were found for chemical composition, microbial abundances and diversity in LAB isolated from goat's rumen and fermenters. Whereas bacterial density (log10 gene copies/g FM: 11.6 and 9.4 for bacteria and methanogens, respectively, in rumen) and diversity indexes (Shannon index: 3.6) were not affected by diet, DGGE analyses showed that bacterial community profile was affected. The cluster analysis suggested differences in bacterial profile between LAB pellets isolated from the rumen of goat and fermenters. A relationship between chemical composition and bacterial community composition in LAB pellets seems to exist. Changes in the former were reflected in the bacterial community profile. Further research is needed to clarify the relationship between chemical and microbial composition of ruminal bacterial pellets with diets of different quality.

Ruminal microbiota developing in different in vitro simulation systems inoculated with goats’ rumen liquor

The aim of this study was to evaluate the capability of different in vitro incubation systems to maintain a rumen microbiota similar to that inoculated to the system from the rumen. Batch cultures (DaisyII ANKOM incubator, DAI and Wheaton flasks, WB) and single-flow continuous-culture fermenters (CC) were tested with pooled rumen liquor from goats and using the same substrate across systems. Samples of filtered rumen content (0h) were kept and used to compare the microbial ecosystem with that obtained after incubation in the different systems for 48h in DAI; 24 and 72h in WB and 4 days in CC. In CC, samples from the effluents obtained on the 5th day of incubation were also taken to compare the microbiota with that developed in the fermenter contents the previous day. Microbial abundances and bacterial diversity were evaluated by real time-PCR (qPCR) and terminal-restriction fragment length polymorphism (T-RFLP), respectively. Total bacteria population declined (P≤0.015) in both batch cultures after 48h (DAI system) or 72h (WB) of incubation, but did not change (P≤0.134) after 24h of incubation in WB or 4 days of incubation in CC. Fibrobacter succinogenes, Ruminococcus flavefaciens and fungi decreased (P<0.001) in DAI and WB after 48 and 72h, respectively; however, there was an increase (P<0.001) of these microorganisms after 24h of incubation in WB and 4 days in CC. Protozoa density declined (P≤0.028) after 48h of incubation in DAI, after 72h of incubation in WB and after 4 days in CC. Methanogens increased (P=0.001) after 48h of incubation in DAI and WB and declined (P≤0.002) in numbers after 72h in WB or 4 days of incubation in CC. When the microbial abundances in fermenter effluents were compared to those in the contents, only declines (P≤0.05) in fibrolytic microorganisms and protozoa were observed. With regard to the overall bacterial diversity, the Shannon index and Pielou evenness were lower (P≤0.049) in the DAI and in WB, after 48 and 24h of incubation, respectively, compared to those in the original rumen liquor. On the other hand, no changes (P=0.469) in the Shannon index were observed among inoculum, fermenter contents and effluents in the CC system. The structure of the bacterial community that developed in the DAI and WB systems after 48 and 24h of incubation, respectively, was rather different compared to the population originally present in the inoculum. On the contrary, the bacterial community profile that developed in the CC was very similar to the original inoculum after 4 days of incubation. Effluents and fermenter contents had very similar profiles.The results of this study show that batch cultures displayed important changes in microbial abundances and bacterial species profile while continuous-culture fermenters maintain a more stable microbiota and bacterial community structure, throughout the incubation of the same type of rumen inoculum.

Changes in ruminal microbiota due to rumen content processing and incubation in single-flow continuous-culture fermenters

The aim of this study was to investigate the impact of rumen content manipulation and its incubation in an in vitro system on the abundance of some microbial groups and the bacterial diversity of goat rumens. Animals and single-flow continuous-culture fermenters were fed diets differing in forage to concentrate ratio (70 : 30; LC and 30 : 70; HC). Rumen contents were sampled after animals’ adaptation to the experimental diets, processed for inoculum preparation and inoculated into fermenters. Fermenter contents were sampled 1 and 7 days after inoculation. Total bacteria, Fibrobacter succinogenes, fungi and methanogen abundances were lower in the fermenter than in goat rumens, but no differences were found for Ruminococcus flavefaciens. The abundances of all these microorganisms were similar at 1 and 7 days of rumen content incubation in fermenters. Bacterial species richness did not change due to rumen content processing or the in vitro incubation. Shannon–Wiener index and Pielou evenness were lower in the fermenter than in rumen only when the enzyme HaeIII was used in terminal-restriction fragment length polymorphism analysis. Non-metric multidimensional scaling analysis, both in denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, showed a segregation of in vivo and in vitro samples, but no trends of grouping for fermenter samples was observed. The HC diet promoted higher abundance of total bacteria than LC in rumen but not in fermenters. Diet only had an effect on bacterial diversity when the enzyme HaeIII was considered. Rumen content processing and incubation in fermenters caused an important decline of the studied ruminal microbial groups although bacterial community structure and diversity did not significantly change.

The effect of the feed-to-buffer ratio on bacterial diversity and ruminal fermentation in single-flow continuous-culture fermenters

Eight single-flow continuous-culture fermenters were used in a completely randomized block design with a 2 × 4 factorial arrangement of treatments to investigate the effects of the feed-to-buffer ratio (F/B) on ruminal fermentation, the diversity and community structure of bacteria, nutrient digestibility, and N metabolism. Four diets with forage-to-concentrate ratios of 70:30 or 30:70 with alfalfa or grass hay as forage were supplied to fermenters twice per day at 2 different F/B (23.5 and 35 g of DM/L). The dilution rate was kept constant (5.3%) among all fermenters by infusing the same volume of buffer. An increase in the total volatile fatty acid (VFA) concentration and a decrease in the average pH were observed with an increased F/B. In addition, the molar proportions of all individual VFA found in fermenters differed, depending on the F/B. A terminal restriction fragment length polymorphism analysis showed that the community structure and diversity of bacteria were highly influenced by the F/B. Both diversity and the number of peaks in the electropherograms were lower in most fermenters receiving diets at a high F/B, whereas the similarity percentage of the bacterial communities across diets was higher as the F/B increased. Moreover, the high reduction of neutral detergent fiber digestibility (15.3% ± 3.65) in fermenters with high F/B suggested a pH-related decrease in the cellulolytic bacterial community as the F/B increased. The crude protein degradation found in fermenters receiving diets with a high F/B was lower compared with that from fermenters with a low F/B. The VFA concentration and purine bases flow response patterns to diets were similar to in vivo conditions only in the case of fermenters with a low F/B. The results suggested that the community structure and diversity of bacteria, as well as the in vitro fermentation parameters, may be affected by the F/B that is used, most likely through a pH effect. In addition, several fermentation parameters showed different response patterns to diets according to the F/B used. Therefore, the amount of feed supplied to single-flow continuous-culture fermenters in which pH is not under control should be carefully chosen according to the volume of buffer infused for the purpose of simulating ruminal fermentation.

Comparison of bacterial pellets and microbial markers for the estimation of the microbial nitrogen and amino acids flows from single flow continuous culture fermenters fed diets containing two‐stage olive cake

The effects of using effluent bacteria (EB) and solid- (SAB) and liquid- (LAB) associated bacteria and diaminopimelic acid (DAPA) or purine bases (PB) and partially substituting alfalfa hay (AH) by a concentrate including olive cake on the microbial N flow (MNF) and amino acids (AA) flow were investigated with continuous culture fermenters fed AH and a mixture of AH and a concentrate containing barley grains and two-stage olive cake (2:1 ratio) without (AHCO) or with polyethylene glycol (PEG) (AHCOP). The MNF was not different among diets with SAB or LAB (p = 0.302 and 0.203, respectively) and DAPA, but differed with PB (p = 0.021 and 0.014, respectively). With EB both markers detected similar differences, AHCOP showing a higher value (p < 0.05) than AH and AHCO. The MNF was higher (p < 0.001) with PB than DAPA. Daily flow of non-essential AA was not different (p = 0.356) among diets but essential AA flow was higher (p < 0.05) for AH and AHCOP than for AHCO. The SAB presented lower (p < 0.05) total AA than LAB and higher total AA (p < 0.05) for diet AH than AHCO. The AA profile of EB was similar to that of LAB, but alanine and leucine were higher (p < 0.05) in EB than in LAB. Microbial contribution to AA flow was 45.4%, 55.6% and 58.1% for diets AH, AHCO and AHCOP respectively. With both markers, microbial AA flow was higher (p < 0.05) for diet AHCOP compared with AH (451 and 355 mg/day, respectively), but not different (p > 0.05) for AHCOP and AHCO (389 mg/day). The results would indicate that olive cake could be used in the practical feeding of small ruminants without negatively affecting microbial AA N supply.

A comparison of different legume seeds as protein supplement to optimise the use of low quality forages by ruminants

The potential of different legume seeds species, including recently new developed varieties (Vicia faba: a commercial variety and varieties Alameda, Palacio and Baraka; Lupinus angustifolius; Pisum sativum and Cicer arietinum: varieties Fardon and Zegrí) as protein supplements to low quality forages was evaluated. First, the chemical composition, in vitro digestibility, in situ degradability and in vitro/in situ intestinal digestibility of legume seeds were determined. The chemical composition was rather similar within genus. Vicia faba beans contained more condensed tannins (35.8–56.4 g/kg DM) and less ether extract (12.8–9.5 g/kg DM) than the other legumes. The rumen degradability and intestinal digestibility of the undegraded protein in the rumen was very similar among the seeds, with exception of lupins, having a much higher degradation rate than the rest. Second, the fermentation characteristics of diets, which were based on two low quality forages (olive leaves and barley straw) and feed blocks without or with supplementation of legume seeds or soybean meal, were investigated by using single-flow continuous culture fermenters. In this trial, the fermentation parameters (ammonia, pH and volatile fatty acids), the microbial protein synthesis and the degradation of olive leaves and barley straw promoted by the different diets were studied. Compared to soybean meal, beans and peas showed similar suitability as protein supplements for sustaining in vitro fermentation of low-quality forages. However, our results suggest a significant interaction between the type of legume used as supplement and the type of forage used, which need to be further studied in vivo.

Ruminal fermentation, microbial growth and amino acid flow in single-flow continuous culture fermenters fed a diet containing olive leaves

Six single-flow continuous culture fermenters were used to determine fermentation profile, microbial growth and amino acid (AA) flow promoted by olive leaves supplemented with barley grains and faba beans (OLSUP), and alfalfa hay (AH). Two incubation runs were carried out with three fermenters inoculated with ruminal fluid from wethers and three from goats. The inoculum source did not affect (p = 0.059 to 0.980) any of the parameters. Daily volatile fatty acid (VFA) production and carbohydrate digestibility were greater (p = 0.009 and 0.024, respectively) for AH, therefore the pH values were lower (p = 0.015) than for OLSUP. Acetate was greater (p < 0.001) and isobutyrate, isovalerate and caproate lower (p < 0.001 to 0.006) for AH with greater acetate/propionate (p = 0.014) and 'VFA/digested carbohydrate' (p = 0.026) ratios. Daily microbial N flow and efficiency were greater (p = 0.016 and p = 0.041) for diet AH. Individual AA flows were greater (p < 0.001 to 0.016) for AH, but microbial essential AA proportion was greater for OLSUP (p = 0.015). The results indicate that OLSUP promoted lower bacterial growth and AA flow than AH, which could have been partially due to a limitation of N availability to ruminal microbes.

Comparison of microbial fermentation of high- and low-forage diets in Rusitec, single-flow continuous-culture fermenters and sheep rumen

Eight Rusitec and eight single-flow continuous-culture fermenters (SFCCF) were used to compare the ruminal fermentation of two diets composed of alfalfa hay and concentrate in proportions of 80 : 20 (F80) and 20 : 80 (F20). Results were validated with those obtained previously in sheep fed the same diets. Rusitec fermenters were fed once daily and SFCCF twice, but liquid dilution rates were similar in both types of fermenters. Mean values of pH over the 12 h postfeeding were higher (P < 0.001) in Rusitec than in SFCCF, with diet F80 showing higher values (P < 0.001) in both types of fermenters. Concentrations of total volatile fatty acids (VFA) were higher (P < 0.001) in SFCCF than in Rusitec, and in both systems were higher (P = 0.002) for diet F20 than for diet F80. There were significant differences between systems in the proportions of the main VFA, and a fermentation system × diet interaction (P < 0.001) was detected for all VFA with the exception of valerate. No differences (P = 0.145) between the two types of fermenters were detected in dry matter (DM) digestibility, but NDF, microbial N flow and its efficiency were higher (P = 0.001) in SFCCF compared to Rusitec. Whereas pH values and VFA concentrations remained fairly stable through the day in both in vitro systems, pH dropped and VFA increased shortly after feeding in sheep rumen reaching the minimum and maximal values, respectively, about 4 h after feeding. Both in vitro systems detected differences between diets similar to those found in sheep for liquid dilution rate, pH values, DM digestibility, microbial N flow and growth efficiency. In contrast, acetate/propionate ratios were lower for diet F20 than for F80 in sheep rumen (2.73 and 3.97) and SFCCF (3.07 and 4.80), but were higher for diet F20 compared to F80 (4.29 and 3.40) in Rusitec, with values considered to be unphysiological for high-concentrate diets. In vivo NDF digestibility was affected (P = 0.017) by diet, but no differences between diets (P > 0.05) were found in any in vitro system. A more precise control of pH in both types of fermenters and a reduction of concentrate retention time in Rusitec could probably improve the simulation of in vivo fermentation.

Effects of concentrate replacement by feed blocks on ruminal fermentation and microbial growth in goats and single-flow continuous-culture fermenters1

The effect of replacing concentrate with 2 different feed blocks (FB) on ruminal fermentation and microbial growth was evaluated in goats and in single-flow continuous-culture fermenters. Diets consisted of alfalfa hay plus concentrate and alfalfa hay plus concentrate with 1 of the 2 studied FB. Three trials were carried out with 6 rumen-fistulated Granadina goats and 3 incubation runs in 6 single-flow continuous-culture fermenters. Experimental treatments were assigned randomly within each run, with 2 repetitions for each diet. At the end of each in vivo trial, the rumen contents were obtained for inoculating the fermenters. For each incubation run, the fermenters were inoculated with ruminal fluid from goats fed the same diet supplied to the corresponding fermenter flask. The average pH values, total and individual VFA, and NH(3)-N concentrations, and acetate:propionate ratios in the rumen of goats were not affected (P >or= 0.10) by diet, whereas the microbial N flow (MNF) and efficiency were affected (P <or= 0.001), with the greatest values observed for the diet without FB. In fermenters, the diet affected pH (P<0.001), propionate concentrations (P=0.01), acetate:propionate ratio (P=0.03), carbohydrate digestibility (P >or= 0.05), and total (P=0.02), NH(3) (P=0.005), and non-NH(3) (P=0.02) N flows, whereas the efficiency of VFA production was not affected (P=0.75). The effect of diet on MNF and efficiency depended on the bacterial pellet used as a reference. An effect (P<0.05) of diet on the composition of solid- and liquid-associated bacteria was observed. The compositions of liquid-associated bacteria in the fermenter contents and effluent were similar (P=0.05). Differences (P<0.001) between in vivo and in vitro values for most fermentation variables and bacterial pellet compositions were found. Partial replacement of the concentrate with FB did not greatly compromise carbohydrate fermentation in unproductive goats. However, this was not the case for MNF and efficiency. Differences between the results obtained in vivo and in vitro indicate a need to identify conditions in fermenters that allow better simulation of fermentation, microbial growth, and bacterial pellet composition in vivo. Reduced feeding cost could be achieved with the inclusion of FB in the diets of unproductive goats without altering rumen fermentation.

Ruminal fermentation of spiny ( Opuntia amyclae) and spineless ( Opuntia ficus indica f. inermis) cactus cladodes and diets including cactus

Ruminal fermentation of spineless Opuntia ficus indica f. inermis, OFI) and spiny ( Opuntia amyclae, OA) cactus cladodes and of diets including them was determined in glass syringes and single-flow continuous culture fermenters, respectively. The OFI and OA chemical composition and gas production were similar. Two iso-nitrogenous and iso-energetic diets composed of barley straw, OFI (OFI-diet) or OA (OA-diet), Atriplex nummularia L., a mineral–vitamin mixture, and urea were supplied to four single-flow continuous culture fermenters. Digested carbohydrates (DCHO), volatile fatty acids (VFA) output and efficiency, non-ammonia (NAN) and bacterial N output and efficiency were similar ( P>0.05) for OFI- and OA-diets. We conclude that OFI and OA had similar nutritive value. In vivo studies are needed to confirm current results.

Patulin-producing molds in corn silage and high moisture corn and effects of patulin on fermentation by ruminal microbes in continuous culture

The objectives were to investigate the presence of patulin-producing Penicillium sp. in corn silage and high moisture corn as well as adverse effects of patulin on microbial fermentation in continuous culture fermenters. Eighty-three samples of corn silage or high moisture corn were cultured to determine the presence of molds. Penicillium sp. were isolated from 0.82 of samples. Of these Penicillium sp. isolates, 0.03 produced patulin on yeast extract sucrose and potato dextrose agar. The patulin-producing isolates belonged to the P. viridicatum group. The other molds identified were: Mucor sp. (0.45), Aspergillus sp. (0.41), and Fusarium sp. (0.25). Eight single-flow continuous culture fermenters were used to study effects of patulin on fermentation by ruminal microbes. Two 1-l fermenters were supplemented with 0, 10, 20 or 40 mg of patulin every 12 h for three consecutive days. Increasing patulin reduced neutral detergent and acid detergent fiber digestibility at a decreasing rate (linear, P < 0.01; quadratic, P < 0.05). True digestion of organic matter and total non-structural carbohydrates decreased linearly ( P < 0.05) as patulin concentration increased. Crude protein digestion and bacterial N flows decreased linearly ( P < 0.05). Conversely, there was a linear increase ( P < 0.05) in ammonia nitrogen with increased patulin. Total, ammonia and non-ammonia N flows were not affected by patulin. Efficiency of microbial protein synthesis was not affected by patulin but there was a linear decreased ( P < 0.05) in the efficiency of N utilization. Increasing patulin levels caused a linear decrease ( P < 0.001) of total volatile fatty acid concentration and a quadratic decrease of acetate and propionate molar proportions ( P < 0.05). Ten and 20 mg/l of patulin produced a decrease in acetate proportion and an increase in propionate proportion. Lactate concentration (mmol/l) increase from 0.0 to 216.5 mmol/l (linear, P < 0.05) with increasing patulin concentration. Penicillium sp. molds are common contaminants of corn silage and high moisture corn and they produce patulin that can adversely affect fermentation by ruminal microbes. Alterations in microbial digestion of dry matter, and production of microbial end products, impact the production and/or health of ruminants.

Effects of patulin on rumen microbial fermentation in continuous culture fermenters

Eight single-flow continuous culture fermenters were used to study the effects of different concentrations of patulin on rumen microbial fermentation. Two 1 l fermenters were spiked with 0, 30, 60 or 90 mg of patulin every 12 h for 3 consecutive days. True digestion of organic matter (TOM), acid detergent fiber (ADF) and crude protein (CP) decreased ( P<0.05, P<0.01 and P<0.01, respectively) with patulin addition ranging from 30 to 90 mg. Bacterial nitrogen flow was lower ( P<0.01) with patulin addition (30–90 mg) compared with the control treatment, whereas, the efficiency of bacterial growth (g of N/kg OMTD) was lowest ( P<0.05) when 90 mg of patulin were added to fermenter flasks. Total volatile fatty acid (VFA) concentration (mM) in fermenter effluents decreased ( P<0.05) from 180.1 to 119.5 with the addition of 90 mg of patulin but did not differ ( P>0.05) between the control treatment and 30–60 mg of patulin. Acetate (mol/100 mol) was depressed ( P<0.01) with patulin addition. Conversely, there was an increase ( P<0.05) in the molar proportion of butyrate and valerate in the fermenters treated with the toxin. At the highest level of patulin addition (60 and 90 mg), branched-chain VFA were lower ( P<0.01) probably due to a reduction in protein (branched-chain amino acid) degradation. It is concluded that patulin can alter metabolism of nutrients by ruminal microbes. These changes could potentially have a negative impact on animal health and performance.

Effects of type of carbohydrate supplementation to lush pasture on microbial fermentation in continuous culture.

Eight single-flow continuous culture fermenters were used to study the effects of the type of energy source on ruminal N utilization from high quality pasture. The four dietary treatments included high quality grass and legume pasture alone (50:50; wt/wt), pasture plus soybean hulls, pasture plus beet pulp, and pasture plus corn. Diets supplemented with additional sources of energy (soybean hulls, beet pulp, and corn) were isocaloric but differed in the type and rate of carbohydrate fermentation. Energy supplements constituted 45% of the total dietary dry matter and were fed twice daily at 12-h intervals in place of pasture, which is characteristic of grain feeding at milking when animals are in a grazing situation. Energy supplementation reduced pH, NH3 N flow, and NH3 N concentration and increased bacterial N flow (as a percentage of N intake). The supplementation of corn and soybean hulls resulted in the highest microbial N flow (as a percentage of N intake). Corn had a tendency to reduce fiber digestion because of excessively low NH3 N concentrations. Beet pulp was similar to corn in that it decreased NH3 N concentrations. Supplementation of soybean hulls resulted in a more synchronized fermentation, greater volatile fatty acid production, and greater fiber digestion. Nitrogen utilization by microbes was maximized by supplementation with soybean hulls or corn twice a day. With diets based on pasture, it may be more important to improve bacterial N flow and bacterial utilization of N than to maximize the efficiency of bacterial protein synthesis because better utilization of N by ruminal microorganisms results in higher bacterial N flow and higher fiber digestion.