Rumen Protozoa Research Articles

Rumen ciliate biota of water buffalo (Bubalus bubalis Linnaeus, 1758) in Kastamonu, Turkey

Species composition and distribution of rumen ciliates were investigated in the rumen contents of six Anatolian water buffaloes living in Kastamonu, Turkey. Thirty-seven species and 19 morphotypes belonging to 11 genera were identified, all being first reports of rumen ciliates from Anatolian water buffaloes. The mean number of ciliates was 70.5 ± 62.3 × 104 cells in 1 mL of rumen contents, while the average number of ciliate species per host was 17.5 ± 3.6. Dasytricha ruminantium, Entodinium simulans, and En. longinucleatum were found to be the dominant species occurring in all animals. Entodinium ellipsoideum, Eudiplodinium dilobum, and Ostracodinium munham were isolated from buffaloes for the first time. No previously unknown species were observed.

Effect of Field and Experimentally-induced Cases of Frothy Bloat on Physical Examination, Rumen Fermentation Pattern and Blood Constituents in Baladi Egyptian Sheep

Rumen fermentation pattern of sheep is greatly affected by several factors. This research studied the effect of field and experimentally-induced cases of frothy bloat on physical examination, blood constituents and rumen fermentation efficiency in Baladi Egyptian sheep. Sixty-six native breed Baladi Egyptian sheep were classified according to clinical presentation into fifty-eight apparently healthy sheep and eight clinically diseased cases of frothy bloat divided into three field cases due to grazing on lush pasture of barseem and five experimental cases induced by ingestion of cabbage leaves. All animals were subjected to detailed case history, comprehensive clinical examination and sampling of blood and rumen liquor for analysis. Results revealed that field and experimentally-induced cases of rumen frothy bloat caused highly significant increase in pulse and respiratory rates with highly significant decrease in rumen motility. Field cases of frothy bloat caused highly significant increase in Holotricha % and monocytes % with highly significant decrease in TPC, Entodinium %, TVFAs, lactic acid, propionic acid, butyric acid, acetic acid, CH4, lymphocytes %, albumin and A/G ratio. Experimentally-induced cases of frothy bloat caused highly significant increase in Hb and MCHC with highly significant decrease in TVFAs, lactic, butyric and acetic acids, A/P ratio, CH4 and CH4/TVFAs %. It could be concluded that barseem lush-pasture bloat has more dramatic effect on rumen fermentation efficiency than bloat due to ingestion of cabbage leaves. Frothy bloat is still field problem of concern despite awareness of sheep farmers. Further investigations are highly recommended from aspects of rumen methane, rumen enzymes and rumen ciliates for therapeutic and productive purposes

Effect of Supplementing Streptococcus gallolyticus Strain TDGB 406 on Rumen Fermentation and Microbial Profile of Goats Fed on Oak Leaves

An experiment was conducted to study the effect of supplementation of tannin degrading bacteria (Streptococcus gallolyticus strain TDGB 406) on rumen fermentation and microbial profile of goats fed on oak (Quercus semicarpifolia) leaves. Three adult male goats (average body weight 27.5±1.0 kg) fitted with permanent fistula were distributed into three groups (switch over design). The animals of group 1 served as control (C) while animals of group T1 and T2 were given autoclaved and live culture of isolate TDGB 406 (106 cells/ml), respectively @ 5 ml/kg live weight. The animals were fed measured quantity of dry oak leaves as the main roughage source and ad libitum maize hay along with fixed quantity of concentrate mixture. The feeding of live culture of isolate TDGB 406 as probiotic did not show any effect on the rumen fermentation pattern viz. pH, total nitrogen, TCA ppt-N, NPN, lactic acid, total volatile fatty acids production and the activities of carboxymethylcellulase, xylanase, protease and tannase. However, the production of NH3-N was higher (P<0.05) in live culture fed animals as compared to control group. The population of rumen protozoa, total bacteria and fungi were similar in all the three groups, whereas, tannin degrading bacteria population was higher in T2 where the live culture of TDGB 406 was fed. The results of the study indicated that TDGB 406 can modify rumen microbial eco-system when used as probiotic in goats without disturbing the rumen environment.

Effects of Rumen Protozoa of Brahman Heifers and Nitrate on Fermentation and In vitro Methane Production.

Two experiments were conducted assessing the effects of presence or absence of rumen protozoa and dietary nitrate addition on rumen fermentation characteristics and in vitro methane production in Brahman heifers. The first experiment assessed changes in rumen fermentation pattern and in vitro methane production post-refaunation and the second experiment investigated whether addition of nitrate to the incubation would give rise to methane mitigation additional to that contributed by defaunation. Ten Brahman heifers were progressively adapted to a diet containing 4.5% coconut oil distillate for 18 d and then all heifers were defaunated using sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol). After 15 d, the heifers were given a second dose of Empicol. Fifteen days after the second dosing, all heifers were allocated to defaunated or refaunated groups by stratified randomisation, and the experiment commenced (d 0). On d 0, an oral dose of rumen fluid collected from unrelated faunated cattle was used to inoculate 5 heifers and form a refaunated group so that the effects of re-establishment of protozoa on fermentation characteristics could be investigated. Samples of rumen fluid collected from each animal using oesophageal intubation before feeding on d 0, 7, 14, and 21 were incubated for in vitro methane production. On d 35, 2% nitrate (as NaNO3) was included in in vitro incubations to test for additivity of nitrate and absence of protozoa effects on fermentation and methane production. It was concluded that increasing protozoal numbers were associated with increased methane production in refaunated heifers 7, 14, and 21 d after refaunation. Methane production rate was significantly higher from refaunated heifers than from defaunated heifers 35 d after refaunation. Concentration and proportions of major volatile fatty acids, however, were not affected by protozoal treatments. There is scope for further reducing methane output through combining defaunation and dietary nitrate as the addition of nitrate in the defaunated heifers resulted in 86% reduction in methane production in vitro.

The Role of Ciliate Protozoa in the Rumen.

First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear. Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult to obtain absolute quantification. Despite repeated attempts it has proven impossible to maintain rumen protozoa in axenic culture. Thus it has been difficult to establish conclusively a role of ciliate protozoa in rumen fiber degradation. The development of techniques to clone and express ciliate genes in λ phage, together with bioinformatic indices to confirm the ciliate origin of the genes has allowed the isolation and characterization of fibrolytic genes from rumen protozoa. Elimination of the ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11%. Our recent findings suggest that holotrich protozoa play a disproportionate role in supporting methanogenesis whilst the small Entodinium are responsible for much of the bacterial protein turnover. As yet no method to control protozoa in the rumen that is safe and practically applicable has been developed, however a range of plant extract capable of controlling if not completely eliminating rumen protozoa have been described.

Evaluation and validation of a paralumbar fossa trans-abdominal rumen fluid sampling technique

Evaluation of rumen fluid for pH and rumen microbes (in particular, rumen protozoa) is helpful for both diagnostic and treatment purposes. Evaluating the pH of the rumen fluid is used to confirm acute ruminal acidosis as well as subacute ruminal acidosis. Evaluation of the ruminal protozoa helps to confirm the necessity of ruminal transfaunation. Protozoa will be dead or dying if the ruminant has been ill and off-feed for some time (usually&gt;4 days). Passage of an oro-ruminal tube is 1 method for obtaining a rumen fluid sample. However, this method may yield rumen fluid that is mixed with saliva and may falsely elevate rumen pH. The tube method also requires more time and effort. A simple means of obtaining rumen fluid is trans-abdominal ruminal aspiration. This method has been used for over 20 years by the primary investigator (Roberson), but has never been evaluated in regards to safety and efficiency. The purpose of this study was to document the methodology, determine the actual time of obtaining the sample, and evaluate any negative consequences of the procedure. Secondary aims were to determine pH and number of protozoa/40x field.

Chitin as a source of energy for rumen ciliates

Chitin as a source of energy for rumen ciliates

New Primers Targeting Full-Length Ciliate 18S rRNA Genes and Evaluation of Dietary Effect on Rumen Ciliate Diversity in Dairy Cows.

Analysis of the full-length 18S rRNA gene sequences of rumen ciliates is more reliable for taxonomical classification and diversity assessment than the analysis of partial hypervariable regions only. The objective of this study was to develop new oligonucleotide primers targeting the full-length 18S rRNA genes of rumen ciliates, and to evaluate the effect of different sources of dietary fiber (corn stover or a mixture of alfalfa hay and corn silage) and protein (mixed rapeseed, cottonseed, and/or soybean meals) on rumen ciliate diversity in dairy cows. Primers were designed based on a total of 137 previously reported ciliate 18S rRNA gene sequences. The 3'-terminal sequences of the newly designed primers, P.1747r_2, P.324f, and P.1651r, demonstrated >99% base coverage. Primer pair D (P.324f and P.1747r_2) was selected for the cloning and sequencing of ciliate 18S rRNA genes because it produced a 1423-bp amplicon, and did not amply the sequences of other eukaryotic species, such as yeast. The optimal species-level cutoff value for distinguishing between the operational taxonomic units of different ciliate species was 0.015. The phylogenetic analysis of full-length ciliate 18S rRNA gene sequences showed that distinct ciliate profiles were induced by the different sources of dietary fiber and protein. Dasytricha and Entodinium were the predominant genera in the ruminal fluid of dairy cattle, and Dasytricha was significantly more abundant in cows fed with corn stover than in cows fed with alfalfa hay and corn silage.

Effect of forage level and replacing canola meal with dry distillers grains with solubles in precision-fed heifer diets: Digestibility and rumen fermentation

Objectives of this study were to determine the effects of feeding differing forage-to-concentrate ratios (F:C) and inclusion rates of corn dry distillers grain with solubles (DDGS) on digestion and rumen fermentation in precision-fed dairy heifer rations. A split-plot design with F:C as whole plot and DDGS inclusion level as sub-plot was administered in a 4-period (19 d) 4×4 Latin square. Eight rumen-cannulated Holstein heifers (12.5±0.5mo of age and 344±15kg of body weight) housed in individual stalls were allocated to 2 F:C [50:50, low forage, or 75:25 high forage; dry matter (DM) basis] and to a sequence of DDGS inclusion (0, 7, 14, and 21%; DM basis). Forage was a mix of 50% corn silage and 50% grass hay (DM basis). Diets were fed to allow for 800g/d of body weight gain and fed 1×/d. Rumen contents were sampled at –2, 0, 2, 4, 6, 8, 10, 12, and 20h after feeding for rumen fermentation measures. Low-forage rations had greater DM and organic matter apparent digestibility. We detected a quadratic effect for DM, organic matter, acid detergent fiber, and neutral detergent fiber apparent digestibility, with the 14% DDGS inclusion level having the highest values. Nitrogen retention decreased with increasing levels of DDGS. Molar proportions of acetate tended to be greater for HF and decreased as DDGS increased; propionate increased as DDGS increased, resulting in the opposite effect on acetate to propionate ratio. Rumen protozoa count decreased as DDGS increased. Moderate levels (14% of DM) of DDGS appear to enhance nutrient utilization and fermentation in precision-fed dairy heifers fed different F:C diets.

Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows.

The effect of long-term dietary supplementation with rutin on the lactation performance, ruminal fermentation and metabolism of dairy cows were investigated in this study. Twenty multiparous Chinese Holstein cows were randomly divided into four groups, and each was offered a basal diet supplemented with 0, 1.5, 3.0 or 4.5 mg rutin/kg of diet. The milk yield of the cows receiving 3.0 and 4.5 mg rutin/kg was higher than that of the control group, and the milk yield was increased by 10.06% and 3.37% (p < 0.05). On the basis of that finding, the cows supplemented with 0 or 3.0 mg rutin/kg of diet were used to investigate the effect of rutin supplementation on blood metabolites and hormone levels. Compared with the control group, the serum blood urea nitrogen (BUN) concentration of the 3.0 mg rutin/kg group is significantly decreased (p < 0.05). In another trial, four adult cows with permanent rumen fistula and duodenal cannulae were attributed in a self-control design to investigate the peak occurrence of rutin and quercetin in different parts of the gastrointestinal tract, ruminal fermentation and microbial population in dairy cows. The cows supplemented with 3.0 mg rutin/kg in the diet differed from the control period. Samples of rumen fluid, duodenal fluid and blood were collected at 1, 2, 3, 4, 5, 6, 7 and 8 h after morning feeding. Compared to the control group, the pH, ammonia nitrogen concentration, number and protein content of rumen protozoa and blood urea nitrogen were lower, but the concentration of total volatile fatty acid (TVFA), microbial crude protein (MCP) and serum lysozyme content were higher for the cows fed the rutin diets. The addition of 3.0 mg rutin/kg to diets for a long term tended to increase the milk yield and improve the metabolism and digestibility of the dairy cows.

Morphological Particularities of Population of Rumen Protozoa in Domestic Ruminants

Introduction: Rumen is a complex ecosystem in which food consumed by ruminants is digested by symbiotic microorganisms. Fermentations result at this level is volatile fatty acids and microbial biomass, which are used by the host as a nutrient substrate. Between ruminal microorganisms (bacteria, protozoa and fungi) and host are diverse interactions (adaptability and symbiosis) which provides advantages in the ability of ruminant digestion. Aims: The main objectives of this study were: development of new methods of fixing, staining and identification of rumen protozoa; morpho-physiological characterization of rumen protozoa subfamilies and genera; investigations of the structure of rumen protozoa populations in domestic ruminants. Materials and methods: Sampling of investigated animals (5 cattle, 5 sheep, and 5 goats) was collected by of ruminal survey method. Rumen fluid was preserved in formalin (18.5%) and MFS (Methyl green-formalin-sodium chlorate). Examination of samples was achieved by performing the native method and stained preparations and then exposed under a microscope (optical and confocal). Criteria for identification of protozoa were: number and location of ciliary areas; cell shape and size; location, number and size of skeletal plates; presence of caudal spines. Results: Rumen protozoa counting results revealed different mean values ​​for the three ruminant species, the highest levels were recorded in goats (7.39 x 106/ml rumen fluid ), followed by cattle (4,958 x 106/ml rumen fluid) and sheep, with much lower averages (1,814 x 106/ml rumen fluid). Conclusion: The analysis of rumen protozoa population identified in cattle, sheep and goats revealed predominance following genres: Isotricha , Dasytricha , Entodinium , Epidinium , Ophryoscolex and some types of subfamily Diplodiniinae ( Diplodinium , Eudiplodinium , Poliplastron ).

Response of the Rumen Microbiota of Sika Deer (Cervus nippon) Fed Different Concentrations of Tannin Rich Plants.

High throughput sequencing was used to examine the rumen microbiota of sika deer fed high (OLH) and low concentration (OLL) of tannin rich oak leaves. The results showed that Prevotella spp. were the most dominant bacteria. The most predominant methanogens were the members of the order Methanoplasmatales. The dominant rumen protozoa were Entodinium longinucleatum, Eudiplodinium maggii, and Epidinium caudatum, and the fungal communities were mostly represented by Piromyces spp. Moreover, the relative abundance of Pseudobutyrivibrio spp. (P=0.026), unidentified bacteria (P=0.028), and Prevotella spp. (P=0.022) was lower in the OLH group than in the OLL group. The concentration of propionate in the OLH group was greater than in the OLL group (P=0.006). Patterns of relationships showed that methanogens belonging to the order Methanoplasmatales were negatively correlated with Treponema spp., Ent. Longinucleatum, and acetate. Methanosphaera stadtmanae was positively correlated to propionate, while Methanobrevibacter ruminantium was negatively associated with Methanobrevibacter thaueri and Methanobrevibacter millerae. Tannins altered the rumen microbes and fermentation patterns. However, the response of the entire rumen microbiota and the relationship between rumen microorganisms and the fermentation parameters were not fully understood.

Determination of the Effects of Some Plant Extracts on Rumen Fermentation and Protozoal Counts by Hohenheim &lt;em&gt;In Vitro&lt;/em&gt; Gas Production Technique

The aim of the present study was to determine the effects of some plant extracts on rumen fermentation and protozoal counts by using Hohenheim in vitro gas production technique in cattle. In this study in vitro gas productions at varying doses of thymol, oregano, zingiber and syzygium essence oils were determined at 2, 4, 8, 12, 24, 36 and 48 (h), respectively. For all feed types, high doses (50 ppm) of thymol and oregano supplementations significantly decreased gas production at later hours of incubation (p<0.05). On the other hand, for all feed types, all doses of zingiber and syzygium supplementations significantly increased gas production at later hours of incubation (p<0.05). High total gas production quantity indicates that most of the substrates are converted to gas which results in decreased concentrations of volatile fatty acids and other beneficial end products. Varying doses of all essence oils were assessed within the same incubation periods and it was found that high doses of thymol and oregano supplementations resulted significant decrease in gas production (p<0.05). For all feed types, the highest protozoal counts were identified in Z. officinale 200 pmm group compared to positive and negative control groups, while the lowest protozoal count for TMR was recorded in T. vulgaris, O. vulgare and S. aromaticium groups. These essence oils can be utilized as rumen regulators. Similar effects are anticipated with the supplementation of these oils to ruminant rations (in vivo), which, therefore, will lead to improved ruminant performance.

RUMINANT NUTRITION SYMPOSIUM: Use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis.

Globally, methane (CH4) emissions account for 40% to 45% of greenhouse gas emissions from ruminant livestock, with over 90% of these emissions arising from enteric fermentation. Reduction of carbon dioxide to CH4 is critical for efficient ruminal fermentation because it prevents the accumulation of reducing equivalents in the rumen. Methanogens exist in a symbiotic relationship with rumen protozoa and fungi and within biofilms associated with feed and the rumen wall. Genomics and transcriptomics are playing an increasingly important role in defining the ecology of ruminal methanogenesis and identifying avenues for its mitigation. Metagenomic approaches have provided information on changes in abundances as well as the species composition of the methanogen community among ruminants that vary naturally in their CH4 emissions, their feed efficiency, and their response to CH4 mitigators. Sequencing the genomes of rumen methanogens has provided insight into surface proteins that may prove useful in the development of vaccines and has allowed assembly of biochemical pathways for use in chemogenomic approaches to lowering ruminal CH4 emissions. Metagenomics and metatranscriptomic analysis of entire rumen microbial communities are providing new perspectives on how methanogens interact with other members of this ecosystem and how these relationships may be altered to reduce methanogenesis. Identification of community members that produce antimethanogen agents that either inhibit or kill methanogens could lead to the identification of new mitigation approaches. Discovery of a lytic archaeophage that specifically lyses methanogens is 1 such example. Efforts in using genomic data to alter methanogenesis have been hampered by a lack of sequence information that is specific to the microbial community of the rumen. Programs such as Hungate1000 and the Global Rumen Census are increasing the breadth and depth of our understanding of global ruminal microbial communities, steps that are key to using these tools to further define the science of ruminal methanogenesis.

Methods for taxonomic studies of rumen ciliates (alveolata: ciliophora): a brief review.

This review presents the principal methods used in taxonomic studies of rumen ciliates: live observation, Lugol staining, fixation and staining with methyl-green formalin saline (MFS) solution, protargol staining, silver carbonate impregnation, scanning electron microscopy and molecular techniques. Mastering these techniques is essential for successful research on the taxonomy of rumen ciliates. No single technique reveals all of the characteristics required for a complete description of a rumen ciliate; therefore, it is necessary to combine the use of these techniques as appropriate to the rumen ciliate group under study. Tables are provided to summarize: 1) morphological methods more appropriate for revealing morphological structures of interest, 2) morphological methods indicated for each group of rumen ciliates, and 3) main primers used for PCR amplification of the 18S rDNA of rumen ciliates.

Milk yield and composition, body condition, rumen characteristics, and blood metabolites of dairy cows fed diet supplemented with palm oil

An addition of rumen-protected fat to the diet of cows may limit negative energy balance and/or shorten its duration, leading to increased milk production with reduced risk of metabolic disorders in dairy cows. The aim of the study was to test the effect of rumen-inert fat supplement of palm oil on milk production, milk composition, rumen characteristics, and metabolic variables of early lactating dairy cows. For this purpose, 24 Holstein-Friesian cows were divided into two equal groups and fed a corn silage-based diet, without palm oil supplementation (control) or with 300 g palm oil (Palm Fat 99, Noack & Co. GmbH, Vienna, Austria) per cow for 8 weeks starting from day 30 after parturition. Milk, rumen, and blood samples were taken three times during experiment at days 30, 58, and 86 of lactation. Body condition scores of cows were determined in the same time periods. Milk yields were measured at the morning and evening milking (600 and 1800). Milk samples were analyzed for milk fat and milk protein content. Rumen content was tested for electrochemical reaction. A native slide was prepared for microscopical examination of the rumen protozoa motility that was numerically estimated. Protozoa were counted in whole rumen contents by light microscopy. Blood samples were tested for total protein, albumin, urea, tryglicerides, cholesterol, total bilirubin, beta-hydroxybutyrate, Ca, and P. Compared with the control, palm oil supplementation resulted in an increase of the average milk yield and milk fat content. The loss in body condition was significantly lower in the group fed palm oil than in the control group. Rumen pH, total number, and motility of protozoa in the group fed palm oil were significantly higher than those in the control group. Palm oil supplementation did not influence blood metabolite concentrations except for urea and glucose which were significantly lower and Ca and cholesterol which were significantly higher in the palm oil-supplemented group. Our results indicate that supplementation with palm oil in weeks 4 to 12 postpartum spared postpartum body weight loss, increased milk yield and milk fat content, and had positive effects on rumen characteristics.

Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.

Study of methanogen communities associated with different rumen protozoal populations.

Protozoa-associated methanogens (PAM) are considered one of the most active communities in the rumen methanogenesis. This experiment investigated whether methanogens are sequestrated within rumen protozoa, and structural differences between rumen free-living methanogens and PAM. Rumen protozoa were harvested from totally faunated sheep, and six protozoal fractions (plus free-living microorganisms) were generated by sequential filtration. Holotrich-monofaunated sheep were also used to investigate the holotrich-associated methanogens. Protozoal size determined the number of PAM as big protozoa had 1.7–3.3 times more methanogen DNA than smaller protozoa, but also more endosymbiotic bacteria (2.2- to 3.5-fold times). Thus, similar abundance of methanogens with respect to total bacteria were observed across all protozoal fractions and free-living microorganisms, suggesting that methanogens are not accumulated within rumen protozoa in a greater proportion to that observed in the rumen as a whole. All rumen methanogen communities had similar diversity (22.2 ± 3.4 TRFs). Free-living methanogens composed a conserved community (67% similarity within treatment) in the rumen with similar diversity but different structures than PAM (P < 0.05). On the contrary, PAM constituted a more variable community (48% similarity), which differed between holotrich and total protozoa (P < 0.001). Thus, PAM constitutes a community, which requires further investigation as part of methane mitigation strategies.

EFFECT OF ROUGHAGE TO CONCENTRATE RATIO ON DIGESTIBILITY IN SMALL RUMINANTS

Three experimental rations differed in concentrate: roughage ratios {90:10 (R1), 80:20 (R2) or 70:30% (R3)} were designed to study chemical compositions and cell wall constituents analysis by in vitro at different incubation times, in vitro nutrient disappearance, in vitro fermentations, microbial protein, count and classification of ciliated protozoa, total and cellulolytic bacteria number. Also, utilization of energy and rumen protozoa in male sheep fed the experimental rations. Samples of rations were in vitro incubated with ruminal fluid from sheep fed concentrate feed mixture and good quality berseem hay for 2, 4, 6, 8, 24 and 48 h. the results indicated that R1 had the highest (P 0.01) CF content and cell wall constituents, whereas, R1 showed the lowest (P>0.01) CF content and it’s components. All contents and cell wall constituents showed gradual decrease (P 0.05) nutrient disappearance, followed by R2 then R1. The best cell wall content disappearance also was for R3. All values of nutrients disappearance showed gradual increase (P<0.05) by progressed incubation time. In vitro fermentations, microbial protein and ruminal protozoa showed the highest values for R3. While, R2 had the best utilization of energy. It can be concluded that rations containing high roughage ratio enhanced nutrient degradability, In vitro fermentations, microbial protein and increased ruminal protozoa.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.