Ruminal Protozoa Research Articles

Effect of manila palm (Veitchia merrillii Becc.) on rumen fermentation and microbial population in beef heifers

The aim of the study was to evaluate the influence of manila palm (MP) on rumen fermentation and rumen microbial ecology in beef cattle. Four Brahman crossbred heifers with an initial body weight (BW) of 250 ± 40 kg (1–1.5 years old) were randomly assigned according to a 4 × 4 Latin square design to the following treatments: (1) Control (0% concentrate powder MP), (2) 1% concentrate powder MP, (3) 3% concentrate powder MP, and (4) 5% concentrate powder MP. The manila palm contained 12.54% of condense tannin. Cow were fed with a rice straw ad libitum and concentrate at 1%BW. There were no treatments effect on ruminal pH, NH3-N, microbial protein synthesis, nor ruminal temperature. Inclusion of MP did not affect (P > 0.12) blood urea N and hematocrit. However, ruminal concentration of acetic acid was linearly decreased (P < 0.01), while propionic acid was linearly increased (P < 0.01) as MP was increased in diet. In a such a manner that predicted ruminal CH4 production was decreased (P < 0.01) as MP was increased in diet. Inclusion of MP in diet decreased linearly (P = 0.02) protozoal population as MP level increased. The present study suggests that manila powder has modulatory effects on ruminal fermentation, decreasing acetate to propionate ratio, reducing estimated CH4 production without effects on ruminal pH, microbial synthesis nor blood parameters. Changes on ruminal fermentation parameters could be partially explained by decreases in the ruminal protozoa. Supplementation beyond 3% of concentrate portion (approximately 1.2% of total ration DM intake) did not increase significantly this positive effects, thus is recommended 1.2% of total ration as optimal use as an animal feed supplement.

Interactions between Entodinium caudatum and an amino acid-fermenting bacterial consortium: fermentation characteristics and protozoal population in vitro.

Ruminal protozoa, especially entodiniomorphs, engulf other members of the rumen microbiome in large numbers; and they release oligopeptides and amino acids, which can be fermented to ammonia and volatile fatty acids (VFAs) by amino acid-fermenting bacteria (AAFB). Studies using defaunated (protozoa-free) sheep have demonstrated that ruminal protozoa considerably increase intraruminal nitrogen recycling but decrease nitrogen utilization efficiency in ruminants. However, direct interactions between ruminal protozoa and AAFB have not been demonstrated because of their inability to establish axenic cultures of any ruminal protozoan. Thus, this study was performed to evaluate the interaction between Entodinium caudatum, which is the most predominant rumen ciliate species, and an AAFB consortium in terms of feed degradation and ammonia production along with the microbial population shift of select bacterial species (Prevotella ruminicola, Clostridium aminophilum, and Peptostreptococcus anaerobius). From an Ent. caudatum culture that had been maintained by daily feeding and transfers every 3 or 4 days, the bacteria and methanogens loosely associated with Ent. caudatum cells were removed by filtration and washing. An AAFB consortium was established by repeated transfers and enrichment with casamino acids as the sole substrate. The cultures of Ent. caudatum alone (Ec) and AAFB alone (AAFB) and the co-culture of Ent. caudatum and AAFB (Ec + AAFB) were set up in three replicates and incubated at 39°C for 72 h. The digestibility of dry matter (DM) and fiber (NDF), VFA profiles, ammonia concentrations, pH, and microscopic counts of Ent. caudatum were compared among the three cultures. The co-culture of AAFB and Ent. caudatum enhanced DM degradation, VFA production, and Ent. caudatum cell counts; conversely, it decreased acetate: propionate ratio although the total bacterial abundance was similar between Ec and the Ec + AAFB co-culture after 24 h incubation. The ammonia production and relative abundance of C. aminophilum and P. anaerobius did not differ between AAFB alone and the Ec + AAFB co-culture. Our results indicate that Ent. caudatum and AAFB could have a mutualistic interaction that benefited each other, but their interactions were complex and might not increase ammoniagenesis. Further research should examine how such interactions affect the population dynamics of AAFB.

Effects of ruminal protozoa on methane emissions in ruminants—A meta-analysis

The effects of different ruminal protozoa (RP) on CH4 emissions from ruminants were evaluated in a meta-analysis, using 64 publications reporting data from 79 in vivo experiments. Experiments included in the database reported CH4 emissions (g/d) and total RP (TRP, log10 cells/mL) from the same group of animals. The relationship between CH4 emissions and RP (TRP, entodiniomorphids, and isotrichids), and TRP-, entodiniomorphid-, and isotrichid-based CH4 emission prediction models, were evaluated as mixed models with experiment as a random effect and weighted by the reciprocal of the standard error of the mean and centered around one. Positive associations existed between TRP and isotrichids with CH4 emissions but not between entodiniomorphids and CH4 emissions. A reduction in CH4 emissions was observed, averaging 7.96 and 4.25 g/d, per log unit reduction in TRP and isotrichid concentrations, respectively. Total RP and isotrichids were important variables in predicting CH4 emissions from ruminants. Isotrichid CH4 prediction model was more robust than the TRP, evidenciated by lower predicted sigma hat study (%), and error (%), and with higher concordance correlation coefficient. Both TRP and isotrichid models can accurately predict CH4 emissions across different ruminant types, as shown by the low square root of the mean square prediction error, with 6.59 and 4.08% of the mean of root of the mean square prediction error in the TRP and isotrichid models, respectively. Our results confirm that isotrichids are more important than entodiniomorphids in methanogenesis. Distinguishing these 2 populations yielded a more robust CH4 prediction model than combining them as total protozoa.

Effect of 2-hydroxy-4-(methylthio)butanoate (HMTBa) on milk fat, rumen environment and biohydrogenation, and rumen protozoa in lactating cows fed diets with increased risk for milk fat depression.

Biohydrogenation-induced milk fat depression (MFD) is a reduction in milk fat synthesis caused by bioactive fatty acids (FA) produced during altered ruminal microbial metabolism of unsaturated FA. The methionine analog 2-hydroxy-4-(methylthio)butanoate (HMTBa) has been shown to reduce the shift to the alternate biohydrogenation pathway and maintain higher milk fat yield in high-producing cows fed diets lower in fiber and higher in unsaturated FA. The objective of this experiment was to verify the effect of HMTBa on biohydrogenation-induced MFD and investigate associated changes in rumen environment and fermentation. Twenty-two rumen cannulated high-producing Holstein cows [168 ± 66 d in milk; 42 ± 7 kg of milk/d (mean ± standard deviation)] were used in a randomized design performed in 2 blocks (1 = 14 cows, 2 = 8 cows). Treatments were control (corn carrier) and HMTBa (0.1% of diet dry matter). The experiment included a 7-d covariate period followed by 3 phases that fed diets with increasing risk of MFD. The diet during the covariate and low-risk phase (7 d) was 32% neutral detergent fiber with no additional oil. The diet during the moderate-risk phase (17 d) was 29% neutral detergent fiber with 0.75% soybean oil. Soybean oil was increased to 1.5% for the last 4 d. The statistical model included the random effect of block and time course data were analyzed with repeated measures including the random effect of cow and tested the interaction of treatment and time. There was no effect of block or interaction of block and treatment or time. There was no overall effect of treatment or treatment by time interaction for dry matter intake, milk yield, and milk protein concentration and yield. Overall, HMTBa increased milk fat percent (3.2 vs. 3.6%) and yield (1,342 vs. 1,543 g/d) and there was no interaction of treatment and dietary phase. Additionally, HMTBa decreased the concentration of trans-10 18:1 in milk fat and rumen digesta. Average total ruminal concentration of volatile FA across the day and total-tract dry matter and fiber digestibility were not affected by HMTBa, but HMTBa increased average rumen butyrate and decreased propionate concentration and increased total protozoa abundance. Additionally, HMTBa increased the fractional rate of α-linoleic acid clearance from the rumen following a bolus predominantly driven by a difference in the first 30 min. Plasma insulin was decreased by HMTBa. In conclusion, HMTBa prevented the increase in trans FA in milk fat associated with MFD through a mechanism that is independent of total volatile FA concentration, but involves modification of rumen biohydrogenation. Decreased propionate and increased butyrate and ruminal protozoa may also have functional roles in the mechanism.

Associative effects between Chlorella vulgaris microalgae and Moringa oleifera leaf silage used at different levels decreased in vitro ruminal greenhouse gas production and altered ruminal fermentation

Moringa oleifera leaf silage and Chlorella vulgaris microalgae mixture used at different levels replacing concentrate feed mixture in the diets of ruminant were evaluated using an in vitro gas production technique. C. vulgaris was included in rations at 1, 2, and 3% concentrations. The concentrate feed mixture was replaced by M. oleifera silage up to 100%. Productions of total gas, methane (CH4), and carbon dioxide (CO2) and ruminal fermentation were measured. Interactions between M. oleifera and C. vulgaris levels were observed for the rate of total gas production, lag time of CH4 production, pH, and concentrations of ammonia-N (NH3-N), total volatile fatty acid (VFA), and propionate. The lower level of C. vulgaris increased total gas production and decreased CH4 and CO2 production as well as improved nutrient degradability compared to the other levels of C. vulgaris which showed less improvement in these parameters. The replacement levels of concentrate at 10 to 40% with M. oleifera linearly increased the asymptotic total gas production and degradabilities of dry matter and acid detergent fiber (P<0.05), while the replacement levels of 80 to 100% lowered the asymptotic (P<0.01) for the ration containing 1% C. vulgaris. Rations containing M. oleifera linearly increased the lag time of total gas production (P<0.05), neutral detergent fiber degradability, and ruminal bacteria count and decreased the asymptotic CH4 and CO2 production and ruminal protozoal count (P<0.05). For the rations containing 2 and 3% C. vulgaris, M. oleifera linearly (P<0.01) decreased the asymptotic total gas, CH4 and CO2 production, and ruminal protozoal count. The lag time of CH4 production was not affected at 1% C. vulgaris, but reduced linearly at 2% and 3% C. vulgaris. Ruminal pH was not affected by M. oleifera, but was increased by C. vulgaris at 3% level. Overall, M. oleifera in the ration containing C. vulgaris at all levels increased ruminal NH3-N concentration; however, C. vulgaris at 2% level and M. oleifera at levels up to 40% lowered NH3-N concentration. M. oleifera rations with 1% and 2% C. vulgaris increased the concentrations of total VFA and propionate, whereas these variables were not affected at 3% C. vulgaris level. In conclusion, replacement of concentrate mixture with M. oleifera at 30% level and C. vulgaris at 1% in the diet due to associative effects may improve ruminal fermentation and feed degradability while decreasing CH4 production.

In vitro fermentation andproduction of methane and carbon dioxide from rations containing Moringa oleifera leave silage as a replacement of soybean meal: in vitro assessment.

Plant leaf meal of some forage trees such as Moringa oleifera has attracted an increasing interest as a good and cheap source of protein. The present in vitro experiment employed the in vitro wireless gas production (GP) technique to evaluate the inclusion of M. oleifera leaves ensiled for 45 days as a replacement for soybean meal in rations. A control basal ration was formulated to contain 17.5% soybean meal as a source of protein. Soybean meal in the control ration was replaced with silage (MOS) at increasing levels of 0 to 100%. Replacing soybean meal with MOS gradually increased (P < 0.001) GP kinetics (asymptotic GP, rate of GP, and lag time of GP). However, soybean meal replacement decreased (P < 0.001) asymptotic methane (CH4) and carbon dioxide (CO2) productions, and rate of CH4 production and increased the lag time of CH4 and CO2 production. Gradual increases (P < 0.001) in the digestibility of dry matter, neutral detergent fiber and acid detergent fiber, ruminal bacteria count, fermentation pH, and the concentrations of ruminal total volatile fatty acids, acetate, and propionate were observed with rations containing MOS. Decreases in the digestibility of crude protein, ruminal protozoal count, and the concentrations of ruminal ammonia-N were observed with MOS rations. It is concluded soybean meal can be completely replaced by MOS with desirable effects on ruminal fermentation.

Effect of Long-Term Supplementation With Silkworm Pupae Oil on the Methane Yield, Ruminal Protozoa, and Archaea Community in Sheep.

Supplementation with lipids and oils is one of the most efficient strategies for reducing enteric methane emission. However, high costs and adverse impacts on fiber degradation restrict the use of conventional oils. Silkworm pupae, a non-conventional oil source rarely used for human consumption in India, could be one of the cheaper alternatives for methane mitigation. The objective of this study was to investigate the effect on sheep of long-term supplementation (180 days) of silkworm pupae oil (SWPO) with two distinct supplementation regimes (daily and biweekly) on daily enteric methane emission, methane yield, nutrient digestibility, rumen fermentation, ruminal archaea community composition, and protozoal population. The effect of the discontinuation of oil supplementation on enteric methane emission was also investigated. Eighteen adult male sheep, randomly divided into three groups (n = 6), were provisioned with a mixed diet consisting of 10.1% crude protein (CP) and 11.7 MJ/kg metabolizable energy formulated using finger millet straw and concentrate in a 55:45 ratio. SWPO was supplemented at 2% of dry matter intake (DMI) in test groups either daily (CON) or biweekly (INT), while no oil was supplemented in the control group (CTR). DMI (p = 0.15) and CP (p = 0.16) in the CON and INT groups were similar to that of the CTR group; however, the energy intake (MJ/kg) in the supplemented groups (CON and INT) was higher (p < 0.001) than in CTR. In the CON group, body weight gain (kg, p = 0.02) and average daily gain (g, p = 0.02) were both higher than in the CTR. The daily methane emission in the CON (17.5 g/day) and INT (18.0 g/day) groups was lower (p = 0.01) than the CTR group (23.6 g/day), indicating a reduction of 23–25% due to SWPO supplementation. Similarly, compared with the CTR group, methane yields (g/kg DMI) in test groups were also significantly lower (p < 0.01). The transient nature of the anti-methanogenic effect of SWPO was demonstrated in the oil discontinuation study, where daily methane emission reverted to pre-supplementation levels after a short period. The recorded methanogens were affiliated to the families Methanobacteriaceae, Methanomassilliicoccaceae, and Methanosarcinaceae. The long-term supplementation of oil did not induce any significant change in the rumen archaeal community, whereas minor species such as Group3b exhibited differing abundance among the groups. Methanobrevibacter, irrespective of treatment, was the largest genus, while Methanobrevibacter gottschalkii was the dominant species. Oil supplementation in CON and INT compared with CTR decreased (p < 0.01) the numbers of total protozoa (× 107 cells/ml), Entodiniomorphs (× 107 cells/ml), and Holotrichs (× 106 cells/ml). SWPO continuous supplementation (CON group) resulted in the largest reduction in enteric methane emission and relatively higher body weight gain (p = 0.02) in sheep.

Can a blend of integrated feed additives modulate ruminal fermentation patterns and performance of growing lambs? In vitro and in vivo assessments

Two in vitro and in vivo experiments were accomplished to investigate the impacts of integrated feed additives (IFA, combination of protected fat, lysine, and methionine plus yucca extract, eucalyptus essential oil, and direct-fed microbial) on gas production (GP), ruminal fermentation and degradability parameters and lamb performance, digestibility, and nitrogen balance. In the in vitro experiment, responses of graded doses (0, 25, 50, 75, 100, and 125 g IFA/kg DM) were evaluated. In the in vivo experiment, 15 Barki male lambs (30.43 ± 0.74 kg BW ± SE) were individually allotted in complete randomized design into three treatments (five animal/treatment) as control (basal diet without additive), low dose (50 g/kg DM) and high dose (100 g/kg DM) for 120 days. In vitro results showed that both doses (100 and 125 g/kg DM) quadratically decreased (p < 0.001) GP and ammonia nitrogen (NH-3N) concentration. In vivo experiment revealed that dry matter intake was increased (p = 0.016) with low dose compared to high dose. Inclusion of high dose decreased blood serum glucose concentrations (p > 0.05) and ruminal protozoal populations (p = 0.094) compared with low dose and control diet. Both doses of IFA increased (p < 0.05) serum urea, creatinine, and triglyceride concentrations, while decreased (p < 0.001) ruminal NH3-N concentrations. These results suggested that, although IFA was effective to modify in vitro ruminal fermentation process and feed degradability, such aspects did not improve in vivo performance of growing lambs.

In Vitro Screening of Plant Materials to Reduce Ruminal Protozoal Population and Mitigate Ammonia and Methane Emissions

Alternative feed sources can be utilized to reduce enteric methane (CH4) emissions, a major greenhouse gas that contributes to global warming. This study aimed to evaluate the potential use of tropical plants to improve digestibility, reduce protozoal populations, improve rumen fermentation, and minimize methane emissions from ruminants. The plants considered herein grow in tropical climates, are easily accessible in large quantities, and are directly related to human food production. Nine plants that grow naturally in tropical climates were assessed. Plant supplementation substantially enhanced accumulative gas production at 24 h (p &lt; 0.05). The apparent organic matter digestibility (AOMDvt) of the diet was not affected by five of the nine plants. With the addition of the plant material, ammonia nitrogen concentrations were reduced by up to 47% and methane concentrations were reduced by 54%. Five of the nine plant materials reduced methane production in terms of CH4/dry matter and CH4/digestibility of the organic matter by 15–35% and 8–24%, respectively. In conclusion, supplementation with plants with high tannin contents was shown to be a viable strategy for improving rumen fermentation, reducing protozoal populations, and limiting methane emissions. In this regard, the leaves of Piper sarmentosum, Acmella oleracea, Careya arborea, and Anacardium occidentale were especially promising.

Ruminal Protozoal Populations of Angus Steers Differing in Feed Efficiency.

Simple SummaryThe rumen protozoa have been demonstrated to enhance methanogenesis and impact intraruminal recycling of microbial protein. However, they are also known to contribute to fiber degradation and the stabilization of ruminal pH changes. The apparent metabolic impact of ciliated protozoa in the rumen may contribute to the variation in feed efficiency. This study examined the relationship between the rumen protozoa and feed efficiency in beef steers. We monitored feed intake and body weight for a 70-day feed efficiency trial. Following the trial, rumen content was collected, protozoal DNA was extracted from the content, and the relationship between protozoa community diversity and species abundance with feed efficiency was examined. The ciliated protozoal community diversity differed between low- and high-feed efficient steers. Greater abundances of unidentified protozoa genera were detected in the low-feed efficient steers. These data suggest that unidentified protozoa and ciliated protozoal community diversity influence feed efficiency in beef steers. Feed accounts for as much as 70% of beef production costs, and improvement of the efficiency with which animals convert feed to product has the potential to have substantial financial impact on the beef industry. The rumen microbiome plays a key role in determining feed efficiency; however, previous studies of rumen microbiota have not focused on protozoal communities despite the estimation that these organisms represent approximately 50% of rumen content biomass. Protozoal communities participate in the regulation of bacterial populations and nitrogen cycling—key aspects of microbiome dynamics. The present study focused on identifying potential associations of protozoal community profiles with feed efficiency. Weaned steers (n = 50) 7 months of age weighing approximately 260 kg were adapted to a growing ration and GrowSafe for 2 weeks prior to a 70-day feed efficiency trial. The GrowSafe system is a feeding system that monitors feed intake in real time. Body weights were collected on the first day and then every 7 days of the feed efficiency trial, and on the final day, approximately 50 mL of rumen content were collected via orogastric tubing and frozen at −80 °C. Body weight and feed intake were used to calculate residual feed intake (RFI) as a measure of feed efficiency, and steers were categorized as high (n = 14) or low (n = 10) RFI based on ±0.5 standard deviations about the mean RFI. Microbial DNA was extracted, and the eukaryotic component profiled by amplification and sequencing of 18S genes using degenerate primers that can amplify this locus across a range of protists. The taxonomy of protozoal sequences was assigned using QIIME 1.9 and analyzed using QIIME and SAS 9.4 with significance determined at α ≤ 0.05. Greater abundances of unassigned taxa were associated with high-RFI steers (p = 0.03), indicating a need for further study to identify component protozoal species. Differences were observed between low- and high-RFI steers in protozoal community phylogenetic diversity, including weighted beta-diversity (p = 0.04), Faith’s phylogenetic diversity (p = 0.03), and observed Operational taxonomic unit (OTU) (p = 0.03). The unassigned taxa and differences in phylogenetic diversity of protozoal communities may contribute to divergences observed in feed efficiency phenotypes in beef steers.

PSII-17 Program Chair Poster Pick: Effect of ruminal protozoa on methane emissions in ruminants – a meta-analysis

Abstract The effects of ruminal protozoa (RP) concentration on methane emissions from ruminants were evaluated in a meta-analysis using 67 publications reporting data from 85 in vivo experiments. Experiments included in the database reported methane emissions (g/kg DMI) and RP (log10 cells/mL) from the same group of animals. Quantitative data including diet chemical composition, ruminal fermentation, total tract digestibility, and milk production; and qualitative information including methane mitigation strategies, animal type, and methane measurement methods were also collected. The studies were conducted in dairy cows (51%), beef steers (32%) and small ruminants (32%). 70% of the studies reported a reduction in methane emissions. Supplemental lipids reduced methane emissions 95% of the time. The relationship between methane emissions and RP concentration was evaluated as a random coefficient model with the experiment as a random effect and weighted by the inverse pooled SEM squared, including the possibility of covariance between the slope and the intercept. A quadratic effect of RP concentration on methane emissions was detected: CH4= -28.8 + 12.2 × RP-0.64 × RP2. To detect potential interfering factors in the relationship, the influence of several qualitative and quantitative factors were separately tested. Acetate, butyrate, and isobutyrate molar proportions had positive relationships with methane emissions and influenced the relationship between RP concentration and methane emissions, where the presence of ruminal fermentation variables reduced the effects of RP concentration in methane emissions. Total tract digestibility of DM, OM, and CP had negative relationships while NDF digestibility had a positive relationship with methane emissions; however, they only changed the magnitude of intercept and slope of RP and RP2 for the relationship. For dairy cows, milk fat and protein concentrations had positive relationships and milk yield had a negative relationship with methane emissions and changed the magnitude of intercept and slope of RP and RP2 for the relationship.

Methane emission, ruminal fermentation parameters and fatty acid profile of meat in Santa Inês lambs fed the legume macrotiloma

Context Using legumes in ruminant production system may provide benefits such as improvement in animal performance and decrease in methane (CH4) emissions, mainly based on nutritional characteristics of these plants and the presence of tannins. Macrotiloma (Macrotyloma axillare) is a tropical legume that still lacks studies regarding its effects on animal performance and CH4 emissions. Aims The objective of this experiment was to evaluate productive performance, CH4 emission, ruminal fermentation parameters, carcass characteristics and fatty acid profile of the meat from lambs fed tropical grass hay supplemented with the legume forage macrotiloma. Methods For a 90-day experimental period, 14 Santa Inês male lambs (aging 100 days; 18.06 ± 3.26 kg initial bodyweight) were allocated in individual pens and divided into two treatments: chopped aruana grass (Panicum maximum cv. Aruana) hay (CON); aruana hay supplemented with chopped macrotiloma hay at a ratio of 75:25 grass to legume (MAC). Lambs were weighed fortnightly for calculation of average daily bodyweight gain (ADG) and CH4 emission was measured on three occasions (days 28 to 32; 57 to 61; 85 to 89) using the sulfur hexafluoride (SF6) tracer technique. At the last day of CH4 collection, rumen fluid samples were collected for: determination of ammoniacal nitrogen (NH3-N) and short chain fatty acids (SCFA); ruminal protozoa count; and relative abundance determination of general bacteria (BACT); Ruminococcus flavefaciens (RUMI); Fibrobacter succinogenes (FIBRO); methanogenic archaea (METH) by real-time quantitative PCR (real-time-qPCR) analysis. Subsequently, after 16 h fasting, the animals were slaughtered; carcass characteristics were then evaluated and longissimus thoracis samples were collected for fatty acid profile analysis. Key results No significant difference (P &amp;gt; 0.05) was observed between MAC and CON for productive performance, CH4 emission, SCFA and carcass characteristics. Reduced protozoa count and relative abundance of METH were observed for MAC (P &amp;lt; 0.05). Conclusions Despite the lack of effects on productive performance and CH4 emission, the decreased relative abundance of METH and protozoa count indicated that macrotiloma may possibly present anti-methanogenic activity. Implications The inclusion of this legume in the diet of ruminants may affect rumen microbes and potentially decrease environmental impacts of the production system.

Evaluating Strategies to Reduce Ruminal Protozoa and Their Impacts on Nutrient Utilization and Animal Performance in Ruminants - A Meta-Analysis.

Several studies have evaluated the effects of complete or partial ruminal protozoa (RP) inhibition; however, to this date, no practical suppressant has been identified and used in large scale. This meta-analysis quantitatively evaluates the effectiveness of multiple strategies on inhibiting RP numbers and their influence on ruminal fermentation and animal performance. This study compared 66 peer-reviewed articles (16 manuscripts for complete and 50 manuscripts for partial RP inhibition that used supplemental phytochemicals and lipids, published from 2000 to 2018, to inhibit RP in vivo. Data were structured to allow a meta-analytical evaluation of differences in response to different treatments (complete RP inhibition, phytochemicals, and lipids). Data were analyzed using mixed models with the random effect of experiment and weighted by the inverse of pooled standard error of the mean (SEM) squared. Supplemental phytochemicals and LCFA had no effects on inhibiting RP numbers; however, supplemental MCFA had a potent antiprotozoal effect. Both complete and partial RP (supplemental phytochemicals and lipids) inhibition decreased methane production, total tract digestibility of OM and NDF, and ruminal NH3-N concentration and increased propionate molar proportion. Methane production, molar proportions of acetate and propionate, total tract NDF digestibility were affected by the interaction of treatment (supplemental phytochemicals and lipids) and RP numbers. Supplemental phytochemicals and lipids can be effective in reducing methane production when RP numbers is below 7 Log10 cells/mL, especially by supplemental saponins, tannins, and MCFA. In terms of animal performance, supplemental tannins could be recommended to control methane emissions without affecting animal performance. However, their negative effects on total tract digestibility could be a drawback when feeding tannins to ruminants. The negative effects of supplemental lipids on milk fat composition should be considered when feeding lipids to ruminants. In conclusion, ruminal protozoa play important roles on methanogenesis, fiber digestion, and ruminal NH3-N concentration, regardless of experimental diets and conditions; supplemental phytochemicals and lipids can be effective on reducing methane production when RP numbers is below 7 Log10 cells/mL. Among these partial RP inhibition strategies, supplemental tannins could be recommended to control methane production.

Diet and breed alter community structures of rumen protozoa in cattle subjected to different feeding systems

The objective were to identify and quantify the rumen protozoa from two genetic groups of beef cattle (Nellore and crossbred European beef cattle) subjected to three feeding systems (pasture, pasture supplement, and confined). Samples of rumen contents were obtained from the center of the ruminal mass after slaughter. Quantification and identification of rumen protozoa were performed with a Sedgewick-Rafter counting chamber by optic microscopy. Data were analyzed using general linear analysis methodology with a Poisson distribution (1 %). In addition, the data underwent of Analisys Principal Component. A effect on ruminal protozoa was observed for the diets and breeds analyzed. Was identified 14 genera, among which Entodinium was predominant in all animals analyzed. Ciliates belonging to Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, and Ostracodinium showed highest density in Nellore animals when compared with the crossbreed, Nellore × European. Larger numbers of protozoa from the family Isotrichidae (Dasytricha and Isotricha) were observed in animals maintained by pasture or pasture supplement feeding. According to the kind of feeding, those fed exclusively with pasture had a greater diversity of ciliates in comparison to those restricted to, or supplemented with pasture. In one animal, the occurrence of ciliates belonging to the genus Buetschlia, was observed (prevalence of 1.66 %), which is one of only a few records of this genus in ruminants.

Variables de la digesta en rumen de novillos y toros de lidia con diferencias en la procedencia y estación del año en las corridas taurinas del estado de Tlaxcala, México

El objetivo del estudio fue evaluar el peso corporal (PC), la edad y las variables digestivas en el rumen de los animales lidiados en eventos taurinos del estado de Tlaxcala durante un año. En veinte eventos taurinos se registró la procedencia (ganadería de Tlaxcala [GL]; ganadería fuera del estado de Tlaxcala [GF]) y la época de estudio (sequía y lluvia), registrándose las variables: PC, edad (dentición), peso de la digesta del rumen, contenido de la fibra detergente neutro (FDN), pH, ácidos grasos volátiles (AGV), conteo de protozoarios en rumen y la presencia de abscesos hepáticos. Los toros tuvieron mayor PC que los novillos (456 vs 361, p ≤ 0.05). En la estación de sequía se registró mayor PC (432 vs 405, p ≤ 0.05) que durante la estación de lluvia; presentando interacción estadística entre la clasificación (novillo o toro) y la estación (p ≤ 0.05). Los animales del GF tuvieron mayor contenido de la digesta que el GL (8.1 vs 7.25%, p ≤ 0.05). El pH ruminal fue menor en los novillos que en los toros (5.86 vs 6.54, p ≤ 0.05), al igual que el contenido de FDN (61.2 vs 64.7, p &lt; 0.05). En conclusión, los novillos presentaron mayores problemas de acidosis ruminal que los toros. El reglamento taurino, con base al peso y edad de los animales, no es aplicado correctamente por las autoridades.

Dietary supplementation with tannin and soybean oil on intake, digestibility, feeding behavior, ruminal protozoa and methane emission in sheep

Tannins and soybean oil are supplements used in diets that depending on concentration may promote beneficial or negative effects on animal productivity. This study aimed to evaluate the effect of supplementation with tannins extract or soybean oil, as well as their combination, on intake, digestibility, methane production, feeding behavior and rumen parameters in Santa Inês crossbred uncastrated male sheep. Eight sheep were assigned to a double 4 × 4 Latin square (4 treatments and 4 periods), and 4 sheep to a simple 4 × 4 Latin square (for ruminal fluid collection) and fed a basal diet of 60% elephant grass and 40% concentrate (dry matter (DM) basis). The treatments were: control (no tannins or soybean oil); tannins (30 g/kg DM); soybean oil (50 g/kg DM); and tannins plus soybean oil (30 g/kg DM of tannin and 50 g/kg DM of soybean oil). Intake did not differ between treatments. Tannins supplementation increased eating time (ET) (P < 0.01) and decreased DM intake rate (DMIR) (P = 0.02) and rumen fluid pH (P = 0.04), but did not affect digestibility. Supplementation with soybean oil decreased acid detergent fibre (P = 0.04) and neutral detergent fibre (NDF) (P = 0.02) digestibility, and increased number (P < 0.05) and time (P = 0.04) of chewing per ruminal bolus. The combination of tannins and soybean oil decreased digestibility of organic matter (DOM) (P = 0.04) and NDF (P = 0.01), increased ET (P < 0.01) and decreased DMIR (P < 0.01) and NDF intake rate (P = 0.02). The inclusion of tannins did not decrease methane production. However, the inclusion of soybean oil or soybean oil plus tannins reduced methane production (absolute value and per unit of ingested DM and digestible organic matter) (P < 0.01). The number of rumen protozoa decreased in the treatments with tannins, soybean oil and soybean oil plus tannins compared to control treatment (P < 0.01), and a greater proportion of reduction was obtained using only soybean oil. The inclusion of soybean oil alone is sufficient to reduce methane production, without affecting DM intake and DOM, although reducing fibre digestion.

Effect of feeding Acacia nilotica pods to sheep on nutrient digestibility, nitrogen balance, ruminal protozoa and rumen enzymes activity.

This study was conducted to evaluate the effect of supplying two levels of Acacia nilotica (A.nilotica) pods to rations of sheep on nutrient digestibility, nitrogen balance and rumen liquor parameters (pH, total protozoa count, protein concentration and enzymes activity). Twelve mature rams (50±1.25kg B.W.) were distributed into three groups, each with four rams. Animals in group one were considered as a control which fed a basal diet, consisting of concentrate mixture and Egyptian clover. The second group and the third one received the same basal diet with supplying the concentrate mixture by 1.5% and 3.0% of A.nilotica pods meal respectively. The experiment lasted for 3weeks. It was found that supplementation of A.nilotica pods to the concentrate mixture at a rate of 1.5% and 3.0% significantly improved the total feed intake compared to the control. The digestibility of dry matter and crude fibre was significantly reduced with A.nilotica supplements, whereas the digestibility of crude protein was significantly improved. All of nitrogen intake and N-retained were significantly increased in rams fed on concentrates with 1.5% and 3.0% A. nilotica pods when compared to the control. The pH of ruminal fluid was not affected by the dietary treatments. Nevertheless, the total rumen protozoa count was significantly decreased in A.nilotica pods supplemented groups. Also, the rumen protein concentration and the ruminal enzymes activity, especially α-amylase, cellulase and protease, were lower in A.nilotica pods supplemented treatments. In conclusions, inclusion of low levels of A.nilotica pods (1.5% and 3.0%) in the concentrates can be used as a natural protein protectant in ruminants by forming tannin protein complexes in the rumen to maximize the amino acids available in the lower digestive tract. Also, these levels can increase the protein digestibility as well as the N-retained in the body .

Using video microscopy to improve quantitative estimates of protozoal motility and cell volume

The objective of this study was to apply digital imaging to improve quantification of rumen protozoal biomass and distinguish treatment differences in cell motility and volume among ruminal protozoa. Observations of protozoa in rumen fluid treated with essential oils (CinnaGar, CIN; Provimi North America, Brookville, OH) or an ionophore (monensin, MON) indicated possible cell shrinkage. We hypothesized that MON would decrease protozoal motility and interact with CIN on cell volume. In addition, we hypothesized that analysis of still frames from video of swimming protozoa would improve volume prediction accuracy. Flocculated rumen fluid was incubated in batch culture dosed with N-free feed only (control), MON, CIN, or a combination of MON+CIN. Samples were taken at 0, 3, or 6 h post-treatment and wet-mounted on a microscope fitted with a high-definition camera. At 3 h post-inoculation, there was a treatment interaction for average speed such that CIN attenuated the effect of MON, with treatment means of 243, 138, 211, and 183 µm/s for control, MON, CIN, and MON+CIN, respectively. At 6 h post-inoculation, MON decreased average speed by 79.2 µm/s compared with the main effect mean without MON. We measured both minimum and maximum diameters (depth and width, respectively) perpendicular to the longitudinal axis of swimming protozoa, yielding a 3-dimensional estimate of protozoal volume. The ellipsoid formula (4/3)πabc, where a = 1/2 length, b = 1/2 width, and c = 1/2 depth, was compared with previously published volume estimations using genera-specific coefficients (genera-specific coefficient × length × width2). Residuals (genera-specific coefficients - ellipsoid) were plotted against predicted (ellipsoid) and centered to the mean (Xi-x¯) to evaluate both mean and slope biases. For Entodinium spp., Y = 0.248 (±0.037) (Xi - 7.98 × 104) + 1.97 × 104 (±1.48 × 103); n = 100; r2 [coefficient of determination (squared correlation coefficient)] = 0.31, with significant slope and mean biases. For family Isotrichidae, Y = -0.124 (±0.068) (Xi - 2.54 × 106) - 1.21 × 104 (±4.86 × 104); n = 32; r2 = 0.10, where slope tended to be different from zero but with no mean bias. For Epidinium spp., Y = 0.375 (±0.056) (Xi - 2.45 × 105) + 6.65 × 104 (±0.28 × 104); n = 64; r2 = 0.43, with both mean and slope biases. The present regression analyses demonstrate that the genera-specific coefficient-based method more likely overestimates volume for Entodinium and Epidinium than for the teardrop-shaped Isotrichidae. Based on simulations derived from previous literature reporting treatments that depress protozoal populations or among-animal changes in protozoal population structures, our proposed ellipsoid method offers potential to advance the prediction of treatment effects on protozoal volume and to shift focus from the number of cells present to the diversity, function, and biomass of protozoa under various treatment conditions.

An Investigation into Rumen Fungal and Protozoal Diversity in Three Rumen Fractions, during High-Fiber or Grain-Induced Sub-Acute Ruminal Acidosis Conditions, with or without Active Dry Yeast Supplementation.

Sub-acute ruminal acidosis (SARA) is a gastrointestinal functional disorder in livestock characterized by low rumen pH, which reduces rumen function, microbial diversity, host performance, and host immune function. Dietary management is used to prevent SARA, often with yeast supplementation as a pH buffer. Almost nothing is known about the effect of SARA or yeast supplementation on ruminal protozoal and fungal diversity, despite their roles in fiber degradation. Dairy cows were switched from a high-fiber to high-grain diet abruptly to induce SARA, with and without active dry yeast (ADY, Saccharomyces cerevisiae) supplementation, and sampled from the rumen fluid, solids, and epimural fractions to determine microbial diversity using the protozoal 18S rRNA and the fungal ITS1 genes via Illumina MiSeq sequencing. Diet-induced SARA dramatically increased the number and abundance of rare fungal taxa, even in fluid fractions where total reads were very low, and reduced protozoal diversity. SARA selected for more lactic-acid utilizing taxa, and fewer fiber-degrading taxa. ADY treatment increased fungal richness (OTUs) but not diversity (Inverse Simpson, Shannon), but increased protozoal richness and diversity in some fractions. ADY treatment itself significantly (P < 0.05) affected the abundance of numerous fungal genera as seen in the high-fiber diet: Lewia, Neocallimastix, and Phoma were increased, while Alternaria, Candida Orpinomyces, and Piromyces spp. were decreased. Likewise, for protozoa, ADY itself increased Isotricha intestinalis but decreased Entodinium furca spp. Multivariate analyses showed diet type was most significant in driving diversity, followed by yeast treatment, for AMOVA, ANOSIM, and weighted UniFrac. Diet, ADY, and location were all significant factors for fungi (PERMANOVA, P = 0.0001, P = 0.0452, P = 0.0068, Monte Carlo correction, respectively, and location was a significant factor (P = 0.001, Monte Carlo correction) for protozoa. Diet-induced SARA shifts diversity of rumen fungi and protozoa and selects against fiber-degrading species. Supplementation with ADY mitigated this reduction in protozoa, presumptively by triggering microbial diversity shifts (as seen even in the high-fiber diet) that resulted in pH stabilization. ADY did not recover the initial community structure that was seen in pre-SARA conditions.

Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified.