Total Protozoa Numbers Research Articles

Weight and protozoa number but not bacteria diversity are associated with successful pair formation of dealates in the Formosan subterranean termite, Coptotermes formosanus.

New colonies of Formosan subterranean termites are founded by monogamous pairs. During swarming season, alates (winged reproductives) leave their parental colony. After swarming, they drop to the ground, shed their wings, and male and female dealates find suitable nesting sites where they mate and become kings and queens of new colonies. The first generation of offspring is entirely dependent on the nutritional resources of the founder pair consisting of the fat and protein reserves of the dealates and their microbiota, which include the cellulose-digesting protozoa and diverse bacteria. Since termite kings and queens can live for decades, mate for life and colony success is linked to those initial resources, we hypothesized that gut microbiota of founders affect pair formation. To test this hypothesis, we collected pairs found in nest chambers and single male and female dealates from four swarm populations. The association of three factors (pairing status, sex of the dealates and population) with dealate weights, total protozoa, and protozoa Pseudotrichonympha grassii numbers in dealate hindguts was determined. In addition, Illumina 16S rRNA gene sequencing and the QIIME2 pipeline were used to determine the impact of those three factors on gut bacteria diversity of dealates. Here we report that pairing status was significantly affected by weight and total protozoa numbers, but not by P. grassii numbers and bacteria diversity. Weight and total protozoa numbers were higher in paired compared to single dealates. Males contained significantly higher P. grassii numbers and bacteria richness and marginally higher phylogenetic diversity despite having lower weights than females. In conclusion, this study showed that dealates with high body weight and protozoa numbers are more likely to pair and become colony founders, probably because of competitive advantage. The combined nutritional resources provided by body weight and protozoa symbionts of the parents are important for successful colony foundation and development.

The effect of vegetable and animal oils added to different forages and concentrates on the in vitro fermentation parameters in ruminants

ABSTRACT This study aimed to determine the effects of fermentation parameters of sunflower oil, vegetable oil blend, palm oil and chicken oil addition to sunflower meal, wheat grain, alfalfa herbage and corn silage at different rates (4% and 6%, dry matter basis). The oil supplementation up to 6% rate to the alfalfa herbage and corn silage did not adversely affect the level of in vitro gas production, metabolic energy (ME), net energy lactation (NEL), organic matter digestion (OMD) and molarities of acetic, propionic, butyric, iso-valeric, iso-butyric and valeric acids and number of total protozoa (P > 0.05). The oil supplementation to alfalfa herbage increased molarities of butyric, iso-valeric, iso-butyric and valeric acids and number of total protozoa (except sunflower oil) in rumen fluid (P < 0.05). Palm oil decreased the in vitro gas production, ME, NEL and OMD values of sunflower meal (P < 0.05). Supplementation of vegetable oil blend to wheat grain increased ME, NEL and OMD values and butyric acid molarity in rumen fluid (P < 0.05). The in vitro ruminal ammonia–nitrogen concentrations of alfalfa herbage and corn silage increased by vegetable oil blend, palm oil and chicken oil up to 6% (P < 0.05). As a result, the nutrient content of the feedstuffs used changed the in vitro ruminal fermentation values of the oil additive to be added. The addition of sunflower oil, vegetable oil blend, palm oil and chicken oil at 4% and 6% rates to corn silage, alfalfa herbage, sunflower meal and wheat grain feed found to differ on in vitro ruminal fermentation parameters.

Influence of partial replacement of alfalfa with two Salicornia forages on digestion, rumen variables, blood biochemistry metabolites and antioxidant capacity in sheep

In a completely randomized design, 24 Shall rams (average initial body weight [BW]: 43 ± 1.4 kg, aged 1.5 years) were used to evaluate the effects of partial replacement of alfalfa (Medicago sativa) hay with 2 species of Salicornia forage (i.e. bigelovii and persica) on in vivo total tract apparent digestibility, estimated microbial N synthesis (EMNS), rumen fluid parameters, blood metabolites, total antioxidant capacity (TAC) and production of methane in vitro. The three diets offered to sheep (8 per diet) prepared on a dry matter basis were 1- alfalfa hay (990 g/kg DM) + 10 g/kg DM vitamin-mineral supplement (control; AD), 2- alfalfa hay + Salicornia bigelovii (SBF) + vitamin-mineral supplement at a ratio of 690:300:10 (SBD), and 3- alfalfa hay + Salicornia persica (SPF) + vitamin-mineral supplement at a ratio of 690:300:10 (SPD). Salicornia diets did not affect DM, organic matter, crude protein or neutral detergent fiber digestibility. There was no dietary effect on in vivo ruminal pH, volatile fatty acids, or in vitro methane production. Salicornia diets increased rumen concentration of ammonia-N, total numbers of protozoa, and the population of Entodiniinae, whereas the cellulolytic bacteria population and EMNS decreased. Feeding SBD and SPD had no effect on the concentration of plasma albumin, cholesterol, glucose, total protein, triglyceride, and the albumin-globulin ratio but urea-N and TAC increased. Overall, replacing part of the alfalfa hay ration with SBF or SPF improved TAC in sheep with no negative effects on nutrient digestibility or in vitro methane production. This trial showed that Salicornia could be used as a partial substitute for conventional forage in the diet of sheep.

The Eff ect of Total Mix Ration with Xylitol Supplementation on In Vitro Ruminal Total Gas and Methane Production, Digestion Values, Organic Acids, Ammonia-Nitrogen, and the Number of Total Protozoa in Dairy Cattle

The Eff ect of Total Mix Ration with Xylitol Supplementation on In Vitro Ruminal Total Gas and Methane Production, Digestion Values, Organic Acids, Ammonia-Nitrogen, and the Number of Total Protozoa in Dairy Cattle

Free and Microencapsulated Essential Oils Incubated In Vitro: Ruminal Stability and Fermentation Parameters.

Simple SummaryEssential oils are an alternative for replacing antibiotics in animal feeds, but their volatile nature demands a high degree of stability. The aim of this study was to test the in vitro ruminal degradation of two different forms (free and microencapsulated) of three commercial products (mixtures of essential oils based on cinnamaldehyde, named Olistat-Cyn, Olistat-G, and Olistat-P) using an in vitro technique. The products were incubated in filter bags using an inoculum (buffer plus rumen fluid) for 48 h at 39 °C. It was found that the microencapsulation (matrix based on vegetable hydrogenated fatty acids) was efficient to protect essential oils from ruminal degradation because of the low disappearance of the microencapsulated essential oils in comparison to the free ones that were almost completely degraded. Olistat-G caused not only a significant decrease in the pH and the total protozoa number but also a significant increase in the total volatile fatty acids. As a conclusion, microencapsulation was found to be effective to ensure rumen by-pass and to be used as an additive in ruminant feeding. Among the essential oils tested, Olistat-G (mixture of cinnamaldehyde and vitamins) was capable of changing rumen fermentation, potentially reducing methane emissions.Essential oils (EOs) are generally considered as an alternative to antibiotics because of their antimicrobial properties. Despite their vast variety, their volatile nature poses hindrance on their use in animal feeds, which demands a high degree of stability. This study aimed at testing the susceptibility of three EOs (mixtures of EOs based on cinnamaldehyde, named Olistat-Cyn, Olistat-G, and Olistat-P) in two forms (free: fEOs; and microencapsulated: mEOs) to in vitro ruminal degradation using the Ankom DaisyII technique. The microencapsulation was made using a matrix based on vegetable hydrogenated fatty acids. Compared to the fEOs, which were completely degraded within 48 h of in vitro incubation, the mEOs showed a low ruminal disappearance. In comparison to the fermentation profile at 0 h, Olistat-G significantly decreased the pH and the total protozoa number after 48 h, while the total VFAs increased. However, the other EOs (Olistat-Cyn and Olistat-P) had no effect on the rumen fermentation parameters. In conclusion, the protection of EOs from ruminal degradation by microencapsulation was found to be very effective to ensure rumen by-pass. Among the EOs, Olistat-G was capable of changing rumen fermentation, potentially reducing methane emissions.

Moderate Coconut Oil Supplement Ameliorates Growth Performance and Ruminal Fermentation in Hainan Black Goat Kids.

The study investigated amelioration effects of coconut oil (CO) on growth performance, nutrient digestibility, ruminal fermentation, and blood metabolites in Hainan Black goat kids. Twenty-four Hainan Black goat kids (10 days of age) were assigned randomly to four treatments for 90 days, including pre-weaning (10–70 d of age) and post-weaning (70-100 d of age) days. The treatment regimens were control (CON), low CO (LCO), medium CO (MCO), and high CO (HCO) with 0, 4, 6, 8 g CO per goat per day, respectively. During the pre-weaning period, the average daily gain (ADG) linearly and quadratically increased (P < 0.05), whereas the average daily feed intake (ADFI) linearly decreased, and the feed conversion ratio (FCR) also decreased linearly and quadratically by increasing CO supplementation (P < 0.05). During the post-weaning period, increasing CO supplementation linearly and quadratically increased the BW at 100 days and ADG (P < 0.05), but quadratically decreased the ADFI and FCR (P < 0.05). The digestibility of ether extract (EE) linearly and quadratically increased with increasing CO supplementation (P < 0.05). Supplementation of CO linearly increased ruminal pH (P < 0.05), but linearly decreased (P < 0.05) ammonia-N, total VFAs, molar proportions of acetate, ruminal microbial enzyme activity of carboxymethyl-cellulase, cellobiase, xylanase, pectinase and α-amylase, and number of total protozoa, the abundance of Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Prevotella ruminicola, and Ruminobacter amylophilus. The estimated methane emission decreased linearly and quadratically with increasing CO addition (P < 0.05). The serum concentration of triglycerides (TG), non-esterified fatty acids (NEFA) and growth hormone (GH) linearly (P < 0.05) increased by raising the CO supplementation. The present results indicate that CO supplementation at 6 g/day per goats is optimum due to improved growth performance and decreased estimated methane emission. Supplementation CO up to 8 g/day depressed growth and feed conversion due to its suppression of growth performance, rumen protozoa, cellulolytic bacteria and microbial enzyme activity, and reduced ADF and ADF digestibility.

Effects of pomegranate peel extract on ruminal and post-ruminal in vitro degradation of rumen inoculum of the dairy cow

This experiment was carried out to study the effect of water extracted pomegranate peel extract (PE) on ruminal protein degradation and post-ruminal digestion in the dairy cow. PE was added at six levels of total phenolics (g/kg of the basal diet); 3.75 (PE1); 4.4 (PE2); 5.05 (PE3); 5.70 (PE4); and 6.35 (PE5). Rumen degradable crude protein (rdCP) decreased with PE addition (L < 0.0001), but total CP degradability (tdCP) was not affected. Compared to PE0, PE2, and PE3 diets showed higher (L = 0.054, Q = 0.029) digestibility of bypass CP (dBCP). Increasing levels of PE resulted in a decrease in proteolytic bacteria numbers (p < 0.0001). At PE4 and PE5 levels, total VFA and acetate concentrations linearly decreased compared to PE0. PE inclusion lowered the acetate:propionate ratio (L = 0.0001) and Ammonia-N production after 24 h (L = 0.0008) of incubation. The total number of protozoa, genera Dasytricha and Isotricha, and subfamilies Entodiniinae, Diplodiniinae, and Ophrioscolecinae decreased with increasing dietary PE concentration (p < 0.0001). The results suggest that all levels of PE addition reduce the protozoal population and Ammonia-N concentration. All PE levels slowed down protein degradation in the rumen but PE2 and PE3 showed the greatest effect.

Effects of commercial aldehydes from green leaf volatiles (green odour) on rumen microbial population and fermentation profile in an artificial rumen (Rusitec)

The effects of plant metabolites on rumen metabolism vary greatly depending on their antimicrobial spectrum and applied doses. In this study, the minimum inhibitory concentrations (MICs) of commercial aldehydes, trans-2-hexenal (T2H), cis-3-hexenal (C3H), trans-2-nonenal (T2N), and trans-2-decenal (T2D) from green leaf volatiles, were tested on rumen bacteria. These compounds were found more effective on Gram-positive rumen bacteria than the Gram-negatives, and C3H was the most effective compound. Then, for 14 days, the in vitro effects of C3H compared with monensin (5 mg/day) on the rumen microbial population and ruminal fermentation at 187.5, 375 and 750 mg/day doses were tested based on the MIC value (500 μg/mL) by using the rumen simulation technique (Rusitec). Supplementation with C3H at 375 mg/day increased the cell numbers of Butyrivibrio fibrisolvens significantly. The addition of C3H at 375 and 750 mg/day doses also increased Streptecoccus bovis cell counts. The use of monensin did not affect the cell numbers of these bacteria. On the other hand, C3H did not change the counts of total bacteria, methanogens, or hyper-ammonia-producing (HAP) bacteria like monensin. The numbers of Ruminococcus albus and Ruminococcus flavefaciens were also stable in the presence of C3H but decreased significantly with the addition of monensin (P < 0.05). Fibrobacter succinogenes, Megasphaera elsdenii, and Selenomonas ruminantium cell counts were not affected by either application. In addition, C3H increased the acetate and methane production along with the acetate-to-propionate ratio at all tested concentrations, unlike monensin. Supplementation with C3H decreased propionate production significantly, except at the 187.5 mg/day dose. Butyrate production increased (P < 0.05) only in the presence of 187.5 and 375 mg/day doses of C3H. Production of total volatile fatty acids (VFA) and dry matter digestibility (DMD) did not change in treatment groups. Also, the total protozoa numbers and ammonia-N concentrations significantly decreased (P < 0.05) in C3H-treated samples, similar to monensin. Although C3H did not have favorable effects on energy efficiency, it suppressed rumen protozoa and mitigated rumen ammonia without adversely effecting ruminal fermentation in all applied doses. Based on the result, C3H has the potential to improve protein utilization in the rumen.

Effect of phenological stage on nutrient composition, in vitro fermentation and gas production kinetics of Plantago lanceolata herbage

This study was aimed at determining the nutrient composition and in vitro ruminal digestion values of Plantago lanceolata herbage in different phenological stages. The plant samples were gathered in the vegetative, flowering and early seed stages of the plant. The crude protein, diethyl ether extract, ash, non-fibre carbohydrates, and proanthocyanidins levels of the vegetative and flowering stages were higher than those of the early seed stage (P &amp;lt; 0.001). Structural carbohydrate levels (P &amp;lt; 0.05) were determined to have a higher value in the early seed stage. Glucose, fructose, Ca, K, Mg, P, Fe and Cu concentrations decreased as the plant matured (P &amp;lt; 0.001), but Na, Zn, and Mn concentrations increased (P &amp;lt; 0.05). The asymptote gas production, gas production rate (P &amp;lt; 0.001), total gas production at 24 h (P = 0.002), metabolic energy, net energy lactation and organic matter digestibility values and the number of Entodinium (P &amp;lt; 0.001) and total bacteria count (P = 0.026) of the flowering and vegetative stages were higher than those of the early seed stage. Methane produced by 0.2 g dry matter was similar in the three phenological stages (P = 0.078). The bound condensed tannins and saponin contents of plants and ammoniacal-N, number of total protozoa and pH value of rumen fluid were similar in the three different phenological stages (P &amp;gt; 0.05). The present study indicates that P. lanceolata in the vegetative and flowering stages has, owing to its chemical composition, energy content and digestibility, the potential to be used as a forage source for ruminants in areas affected by drought.

Synergism of Cattle and Bison Inoculum on Ruminal Fermentation and Select Bacterial Communities in an Artificial Rumen (Rusitec) Fed a Barley Straw Based Diet.

This study evaluated the effect of increasing the proportion of bison relative to cattle inoculum on fermentation and microbial populations within an artificial rumen (Rusitec). The experiment was a completely randomized design with a factorial treatment structure (proportion cattle:bison inoculum; 0:100, 33:67, 67:33, and 100:0) replicated in two Rusitec apparatuses (n = 8 fermenters). The experiment was 15 d with 8 d of adaptation and 7 d of sampling. Fermenters were fed a diet of 70:30 barley straw:concentrate (DM basis). True digestibility of DM was determined after 48 h of incubation from d 13 to 15, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured on d 9–12. Protozoa counts were determined at d 9, 11, 13, and 15 and particle-associated bacteria (PAB) from d 13 to 15. Select bacterial populations in the PAB were measured using RT-qPCR. Fermenter was considered the experimental unit and day of sampling as a repeated measure. Increasing the proportion of bison inoculum resulted in a quadratic effect (P < 0.05) on straw, concentrate and total true DM disappearance and on straw and total neutral detergent fiber (aNDF) disappearance, with greater disappearances observed with mixed inoculum. There were no effect of source or proportion of inoculum on ADF disappearance (P > 0.05). Increasing bison inoculum linearly increased (P < 0.05) concentrate aNDF disappearance, total and concentrate N disappearance as well as total daily VFA and acetate production. A positive quadratic response (P < 0.05) was observed for daily NH3-N, propionate, butyrate, valerate, isovalerate and isobutyrate production, as well as the acetate:propionate ratio. Increasing the proportion of bison inoculum linearly increased (P < 0.05) total protozoa numbers. No effects were observed on pH, total gas and methane production, microbial N synthesis, or copies of 16S rRNA associated with total bacteria, Selenomonas ruminantium or Prevotella bryantii. Increasing bison inoculum had a quadratic effect (P < 0.05) on Fibrobacter succinogenes, and tended to linearly (P < 0.10) increase Ruminococcus flavefaciens and decrease (P < 0.05) Ruminococcus albus copy numbers. In conclusion, bison inoculum increased the degradation of feed protein and fiber. A mixture of cattle and bison rumen inoculum acted synergistically, increasing the DM and aNDF disappearance of barley straw.

Positive consequences of maternal diet and post-natal rumen inoculation on rumen function and animal performance of Merino lambs

Neonatal modification of the microbial inoculum entering the gastrointestinal tract may alter the rumen microbiome and consequently alter pre- and post-weaning rumen fermentation and growth of lambs. This study aimed to determine (1) if modifying the rumen by providing ewes with lipids differing in rumen-availability in late gestation and lactation would affect performance of the lamb offspring and (2) whether cross-inoculation of neonatal lambs with digesta from sheep on an alternate diet would modify the fermentation, size, and characteristics of the developing rumen and performance of lambs. Two diets were offered ad libitum to 36 pregnant ewes (and to their lambs after lambing) from 1 month pre-lambing until 2 weeks after weaning, after which lambs were grazed in treatment groups in paddocks. Diets consisted of 92% of a blend of oaten and lucerne chopped hay, 4% molasses and 4% fat (coconut oil – CO or protected fat – PF). Newborn lambs were inoculated weekly while suckling (weeks one to eight postnatal) with fresh rumen fluid from donor ewes eating CO or PF diets, or were inoculated with water. Lamb body weight, condition score, wool growth, rumen fermentation, and rumen development were studied during the first 5 months of life of the lambs. Diet and inoculation affected dry matter intake after weaning (P<0.05), being lower in lambs eating CO or inoculated with water. Feeding of CO instead of PF reduced the protozoa population and daily methane production of lambs (P<0.05). Type of gut inoculum had an effect on the concentrations of acetate, propionate, butyrate, total volatile fatty acids, and total protozoa numbers in the rumen of lambs during lactation. By weaning, effects of post-natal inoculation were only apparent for the concentration of butyrate and the protozoal population. It is concluded that dietary fat can affect rumen development and fermentation of the lambs. In addition inoculation with exogenous rumen fluid can modulate some aspects of rumen fermentation. However, lamb performance evaluated at weaning and at 5 months after birth was not altered by either diet or early-life inoculum, suggesting that while the rumen microbiome may have plasticity in its composition, microbial changes do not necessarily result in improved animal performance.

Feeding increasing concentrate to Tifton 85 hay ratios modulated rumen fermentation and microbiota in Nellore feedlot steers

SUMMARYThe use of diets with increasing proportions of concentrate to fibre can ensure appropriate energy levels and result in greater efficiency in Nellore feedlot steers. It was hypothesized that higher proportions of concentrate in the diet of these Nellore steers may affect ruminal fermentation and microbiota as a consequence of ruminal pH reduction. The present study was conducted to evaluate the effect of diets with four different roughage (hay Tifton 85) : concentrate ratios on intake, digestibility, rumen fermentation and rumen microbiota of Nellore feedlot steers. Higher proportions of concentrate in the diet did not affect intake and digestibility of dry and organic matter. The concentration of N-NH3, total rumen volatile fatty acid, acetic (C2), butyric (C4), isobutyric, valeric and isovaleric acids, and microbial nitrogen did not differ among diets. However, increasing proportions of concentrate in the diet resulted in a linear reduction in average rumen pH and increased propionic acid (C3) concentration, resulting in lower relative C2 : C3. Bacterial population ofFibrobacter succinogenes, Ruminococus flavefaciensandRuminococcus albusdecreased in the rumen. However, bacteria that are consumers of lactic acid (Selenomonas ruminantiumandMegasphaera elsdenii) and producers of lactic acid (Lactobacillussp. andStreptococcus bovis) increased when animals were fed with high-concentrate diets. The total number of protozoa was similar for the different roughage : concentrate ratios. Protozoan counts were only influenced by diet for the genusDasytricha. The findings point to diets with increasing concentrate to Tifton 85 hay ratios as inhibiting the growth of some cellulolytic bacteria and reducing fibre digestibility, and indicate Tifton 85 hay as a possible modulated rumen fermentation in the Nellore steer feedlot.

Effect of different levels of raisin waste on performance, nutrients digestibility and protozoal population of Mehraban growing lambs

The aim of this study was to assess the effects of different inclusion levels of raisin waste (RW) in the diet on the animal performance and ruminal fermentation parameters of growing lambs. Four levels of RW inclusion (i.e., R0, R1, R2 and R3 for 0, 100, 200 and 300 g RW kg-1 dry matter of diet, respectively) were tested. The experimental diets were fed to 24 male lambs (six months old) and six animals were allocated to each treatment. In the first experiment, effects of different levels of RW on the animals’ performance, some rumen parameters and protozoa populations were studied. In the second experiment, the apparent total tract digestibility of diets and nitrogen balance were measured. The highest final body weights were observed for the R2 and R3 diets. The R3 diet had the lowest dry matter intake (1156 vs. 1303 g day-1 for R3 and R0, respectively) and feed conversion rate (6.4 vs. 8.7 for R3 and R0, respectively). Total number of protozoa increased with the addition of RW, but Epidinium spp. completely disappeared with the R3diet. Inclusion of RW at levels higher than 200 g RW kg-1 DM of diet significantly reduced crude protein (p=0.042) and neutral detergent fiber digestibility (p=0.049). Our findings showed that RW could be included in the diets of growing lambs up to 200 g kg-1 DM without compromising their production performance.

Altered Protozoan and Bacterial Communities and Survival of Escherichia coli O157:H7 in Monensin-Treated Wastewater from a Dairy Lagoon

Surviving predation is a fitness trait of Escherichia coli O157:H7 (EcO157) that provides ample time for the pathogen to be transported from reservoirs (e.g. dairies and feedlots) to farm produce grown in proximity. Ionophore dietary supplements that inhibit rumen protozoa may provide such a selective advantage for EcO157 to proliferate in lagoons as the pathogen is released along with the undigested supplement as manure washings. This study evaluated the fate of an outbreak strain of EcO157, protozoan and bacterial communities in wastewater treated with monensin. Although total protozoa and native bacteria were unaffected by monensin, the time for 90% decrease in EcO157 increased from 0.8 to 5.1 days. 18S and 16S rRNA gene sequencing of wastewater samples revealed that monensin eliminated almost all colpodean and oligohymenophorean ciliates, probably facilitating the extended survival of EcO157. Total protozoan numbers remained high in treated wastewater as monensin enriched 94% of protozoan sequences undetected with untreated wastewater. Monensin stimulated 30-fold increases in Cyrtohymena citrina, a spirotrichean ciliate, and also biflagellate bicosoecids and cercozoans. Sequences of gram-negative Proteobacteria increased from 1% to 46% with monensin, but gram-positive Firmicutes decreased from 93% to 46%. It is noteworthy that EcO157 numbers increased significantly (P<0.01) in Sonneborn medium containing monensin, probably due to monensin-inhibited growth of Vorticella microstoma (P<0.05), a ciliate isolated from wastewater. We conclude that dietary monensin inhibits ciliate protozoa that feed on EcO157. Feed supplements or other methods that enrich these protozoa in cattle manure could be a novel strategy to control the environmental dissemination of EcO157 from dairies to produce production environments.

Aerobic treatment of dairy wastewater in an industrial three-reactor plant: Effect of aeration regime on performances and on protozoan and bacterial communities

An industrial three-reactor plant treating 45 m3 d−1 of dairy wastewater was monitored to investigate the effect of different aeration regimes on performance efficiency and to find relationships with bacterial and protozoan communities in the activated sludge. During the study, the plant was maintained at six different “on/off” cycles of the blower (45/15, 15/15, 15/45, 30/30, 30/45 and 30/60 min), providing between 30.2 and 90.6 kg O2 d−1, and the main chemical/biochemical parameters (COD, BOD, NH4+, NO2−, NO3−, PO43−, etc.) were determined. When at least 45.4 kg O2 d−1 (30/45) were provided, COD removal efficiencies were always in the range 88–94% but decreased to about 70% under aeration regimes 15/45 and 30/60. Ammonium ion degradation performance was compromised only in the lowest aeration regime (15/45). Total number of protozoa and their species richness, and bacterial viable counts and denaturing gradient gel electrophoresis (DGGE) profiles were used to characterize the microbiota of the activated sludge. Cell abundances and community structures of protozoa and bacteria were very similar in the three aerated reactors but changed with the aeration regimes. In particular, the 15/45 and 30/60 regimes led to low protozoan diversity with prevalence of flagellates of the genus Trepomonas at the expense of the mobile and sessile forms and, thus, to a less efficient activated sludge as indicated by Sludge Biotic Index values (3 and 4.5 for the two regimes, respectively). The structure of the bacterial community strongly changed when the aeration regimes varied, as indicated by the low similarity values between the DGGE profiles. On the contrary, number of viable bacteria and values of the biodiversity index remained stable throughout the whole experimentation. Taken together, the results of the present study clearly indicate that aeration regime variations strongly influence the structure of both protozoan and bacterial communities and, above all, that a high biodiversity among protozoan populations in the activated sludge is prerequisite for high performances in dairy wastewater treatment.

Supplementation of the diet of dairy cows with trehalose results in milk with low lipid peroxide and high antioxidant content

The objective of this study was to investigate the effect of dietary supplementation with the disaccharides trehalose and cellobiose on antioxidant activity in rumen fluid, blood, and milk of dairy cows. Nine Holstein dairy cows housed in a free-stall barn were divided into 3 groups, with each group receiving a different dietary treatment (a control diet, a 1% trehalose-supplemented diet, or a 1% cellobiose-supplemented diet) following a 3 × 3 Latin square design. Feed intake and milk production increased in cows receiving the trehalose-supplemented diet compared with those receiving the control and cellobiose-supplemented diets. The total protozoa numbers in the rumen fluid of cows fed trehalose- or cellobiose-supplemented diets were greater than those of the control group. The C18:0 and C18:1 fatty acid content was increased in the milk of cows fed the trehalose-supplemented diet compared with that of the control group, and the C18:3n-3 fatty acid content in the milk of cows fed the cellobiose-supplemented diet was less than that of the control group. Plasma biochemical parameters were unchanged among the different treatments. In rumen fluid, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and superoxide dismutase activity were increased 2h after feeding in cows receiving the cellobiose-supplemented diet compared with the control group, and the concentration of thiobarbituric acid reactive substances in the rumen fluid of cows fed the cellobiose-supplemented diet was decreased. In contrast, the values of these parameters measured in the milk of cows fed the cellobiose-supplemented diet were no different from those of control cows. Dietary supplementation with trehalose did, however, bring about an improvement of the oxidative status of milk and blood in these animals compared with controls. These results provide the first evidence supporting the use of dietary disaccharides to decrease lipid peroxide levels and increase the antioxidant content of dairy cow milk. The findings suggest that disaccharides, particularly trehalose, might be useful as supplements for reducing oxidative stress and improving the quality of milk for human consumption, as well as possibly impairing the processes that give rise to lipid oxidation odor in dairy cow milk.

Earthworms change the abundance and community structure of nematodes and protozoa in a maize residue amended rice–wheat rotation agro-ecosystem

The influence of earthworms on nematodes and protozoan communities was determined during the wheat phase of a six year rice–wheat rotation agro-ecosystem. Experimental plots in the rotation had five treatments, i.e. incorporation or mulching of maize residues with or without added earthworms and a control. The addition of maize residues to soil strongly affected the abundance and community structure of nematodes and protozoa in the absence of earthworms. The presence of earthworms gave significantly lower total nematode numbers at all soil depths following maize residue incorporation than the same treatment without earthworms, and also gave lower (although not significantly) total nematode numbers in the upper soil layer following maize residue mulching than the same treatment without earthworms. This was mainly due to a significant decrease in bacterial-feeding nematode numbers. Earthworms also strongly affected the distribution of the number of total nematodes and two trophic groups (bacterial and plant feeders) with soil depth. In the presence of earthworms, total protozoan and flagellate numbers significantly increased at all soil depths following both incorporation and mulching of maize residues, while numbers of amoebae increased only when maize residues were mulched. Additionally, in earthworm casts total nematode numbers (mainly bacterial and fungal feeders) were significantly higher, whereas total protozoa numbers (mainly flagellates and amoebae) were significantly lower than that in soil from 0 to 5 cm layer. These results indicated that earthworm activity could affect the abundance and community structure of microfauna, and change their distribution between soil layers and cast material, depending on the mode of application of organic residues.

Effects of forage:concentrate ratio and forage type on apparent digestibility, ruminal fermentation, and microbial growth in goats1

The effects of forage type and forage:concentrate ratio (F:C) on apparent nutrient digestibility, ruminal fermentation, and microbial growth were investigated in goats. A comparison between liquid (LAB) and solid (SAB)-associated bacteria to estimate microbial N flow (MNF) from urinary purine derivative excretion was also examined. Treatments were a 2 x 2 factorial arrangement of forage type (grass hay vs. alfalfa hay) and high vs. low F:C (70:30 and 30:70, respectively). Four ruminally cannulated goats were fed, at maintenance intake, 4 experimental diets according to a 4 x 4 Latin square design. High-concentrate diets resulted in greater (P < 0.001) nutrient digestibility except for ADF. However, CP digestibility increased (P < 0.001) only for the high-concentrate diets including grass hay. Likewise, N retention, ruminal NH(3)-N concentration, and urinary excretion of purine derivatives increased (P < 0.05) with increasing concentrate in animals fed diets based on grass hay (0.23 vs. 0.13 g of retained N/g of digested N, 30.1 vs. 12.9 mg of NH(3)-N/100 mL, and 11.5 vs. 8.40 mmol/d, respectively), but not (P > 0.05) when diets included alfalfa hay. Total protozoa numbers and holotricha proportion were greater and less (P < 0.001), respectively, in high- than in low-concentrate diets. The F:C affected (P < 0.001) ruminal pH but not total VFA concentration (P = 0.12). Ammonia-N concentration was similar (P = 0.13) over time, whereas pH, VFA concentration, and protozoa numbers differed (P < 0.001) among diets. Estimated MNF was strongly influenced by using either the purine bases:N ratio obtained in our experimental conditions or values reported in the literature for small ruminants. There was a F:C effect (P = 0.006) on MNF estimated from LAB but not from SAB. The effect of F:C shifting from 70:30 to 30:70 in goat diets depends on the type of forage used. The MNF measured in goats fed different diets was influenced by the bacterial pellet (LAB or SAB). In addition, the purine bases:N ratio values found were different from those reported in the literature, which underlines the need for these variables to be analyzed directly in pellets isolated from specific animals and experimental conditions.

Efeito da monensina e do óleo de soja sobre os protozoários ciliados do rúmen e correlação dos protozoários com parâmetros da fermentação ruminal e digestivos

Quatro vacas lactantes canuladas no rúmen, com 155 dias em lactação, foram dispostas em um quadrado latino 4 × 4 e alimentadas, duas vezes ao dia, com silagem de milho e concentrado (relação 55:45, base matéria seca), para se avaliarem os efeitos da monensina e do óleo de soja na dieta de vacas lactantes sobre a contagem de protozoários ciliados, além de estabelecer correlações entre os protozoários com alguns parâmetros da digestão e da fermentação ruminal. Os tratamentos consistiram da inclusão de 33 ppm de monensina sódica ou de 4% de óleo de soja na dieta, assim representados: dieta controle, sem óleo ou monensina - CT; dieta com monensina - MN; dieta com óleo - OL; combinação de 33 ppm de monensina e 4% de óleo de soja - OM. Verificou-se a ocorrência de 11 gêneros de ciliados, sendo Entodinium o predominante em todos os tratamentos. Não foi observada interação significativa óleo de soja × monensina sobre a contagem de protozoários e gêneros. Os gêneros Entodinium, Dasytricha, Eremoplastron e Isotricha foram reduzidos nas dietas com óleo, enquanto a monensina diminuiu a contagem de Dasytricha, Eremoplastron e Epidinium. O número total de protozoários e de ciliados celulolíticos foi reduzido pelos efeitos de óleo de soja e monensina, indicando efeito aditivo defaunatório quando combinados o óleo e a monensina. Os ciliados celulolíticos foram reduzidos pelo consumo de ácido linoléico e positivamente relacionados à digestibilidade ruminal da FDN e amônia ruminal. O número total de protozoários foi correlacionado à proporção de propionato no líquido ruminal, indicando que monensina e óleo de soja, pelo seu efeito defaunatório, podem reduzir a perda de metano no rúmen.

The role of laboratory, glasshouse and field scale experiments in understanding the interactions between genetically modified crops and soil ecosystems: A review of the ECOGEN project

The interactions of genetically modified (GM) crops with soil species and ecosystems is complex, requiring both specific and broad spectrum assessments. In the ECOGEN project we undertook experiments at three scales of increasing complexity, using Bt maize expressing the Cry1Ab protein from Bacillus thuringiensis as an example. Test species were selected for laboratory-scale experiments to represent taxonomic groups that we could also monitor at glasshouse and field scales (e.g., nematodes, protozoa, micro-arthropods, earthworms, and snails). In the laboratory, single species were exposed to purified Cry1Ab protein or to Bt maize leaf powder incorporated into simplified diets under controlled conditions. In the glasshouse, multiple test species and soil microbial communities taken from ECOGEN's field sites were exposed to Bt maize plants growing under glasshouse or mesocosm conditions. In the field, evaluations were conducted on our selected indicator groups over multiple sites and growing seasons. Field evaluation included assessment of effects due to the local environment, crop type, seasonal variation and conventional crop management practice (tillage and pesticide use), which cannot be assessed in the glasshouse. No direct effects of Cry1Ab protein or Bt leaf residues were detected on our laboratory test organisms, but some significant effects were detected in the glasshouse. Total nematode and protozoan numbers increased in field soil under Bt maize relative to conventional maize, whilst microbial community structure and activity were unaffected. Field results for the abundance of nematodes and protozoa showed some negative effects of Bt maize, thus contradicting the glasshouse results. However, these negative results were specific to particular field sites and sampling times and therefore were transient. Taking the overall variation found in maize ecosystems at different sites into account, any negative effects of Bt maize at field scale were judged to be indirect and no greater than the impacts of crop type, tillage and pesticide use. Although the ECOGEN results were not predictive between the three experimental scales, we propose that they have value when used with feedback loops between the scales. This holistic approach can used to address questions raised by results from any level of experimentation and also for putting GM crop risk:benefit into context with current agricultural practices in regionally differing agro-ecosystems.