Mitogenomic architecture and evolution of the soil ciliates Colpoda.

Colpoda are cosmopolitan unicellular eukaryotes primarily inhabiting soil and benefiting plant growth, but they remain one of the least understood taxa in genetics and genomics within the realm of ciliated protozoa. Here, we investigate the architecture of de novo assembled mitogenomes of six Colpoda species, using long-read sequencing and involving 36 newly isolated natural strains in total. The mitogenome sizes span from 43 to 63 kbp and typically contain 28-33 protein-coding genes. They possess a linear structure with variable telomeres and central repeats, with one Colpoda elliotti strain isolated from Tibet harboring the longest telomeres among all studied ciliates. Phylogenomic analyses reveal that Colpoda species started to diverge more than 326 million years ago, eventually evolving into two distinct groups. Collinearity analyses also reveal significant genomic divergences and a lack of long collinear blocks. One of the most notable features is the exceptionally high level of gene rearrangements between mitochondrial genomes of different Colpoda species, dominated by gene loss events. Population-level mitogenomic analysis on natural strains also demonstrates high sequence divergence, regardless of geographic distance, but the gene order remains highly conserved within species, offering a new species identification criterion for Colpoda species. Furthermore, we identified underlying heteroplasmic sites in the majority of strains of three Colpoda species, albeit without a discernible recombination signal to account for this heteroplasmy. This comprehensive study systematically unveils the mitogenomic structure and evolution of these ancient and ecologically significant Colpoda ciliates, thus laying the groundwork for a deeper understanding of the evolution of unicellular eukaryotes.IMPORTANCEColpoda, one of the most widespread ciliated protozoa in soil, are poorly understood in regard to their genetics and evolution. Our research revealed extreme mitochondrial gene rearrangements dominated by gene loss events, potentially leading to the streamlining of Colpoda mitogenomes. Surprisingly, while interspecific rearrangements abound, our population-level mitogenomic study revealed a conserved gene order within species, offering a potential new identification criterion. Phylogenomic analysis traced their lineage over 326 million years, revealing two distinct groups. Substantial genomic divergence might be associated with the lack of extended collinear blocks and relaxed purifying selection. This study systematically reveals Colpoda ciliate mitogenome structures and evolution, providing insights into the survival and evolution of these vital soil microorganisms.

Programmed chromosome fragmentation in ciliated protozoa: multiple means to chromosome ends.

SUMMARYCiliated protozoa undergo large-scale developmental rearrangement of their somatic genomes when forming a new transcriptionally active macronucleus during conjugation. This process includes the fragmentation of chromosomes derived from the germline, coupled with the efficient healing of the broken ends by de novo telomere addition. Here, we review what is known of developmental chromosome fragmentation in ciliates that have been well-studied at the molecular level (Tetrahymena, Paramecium, Euplotes, Stylonychia, and Oxytricha). These organisms differ substantially in the fidelity and precision of their fragmentation systems, as well as in the presence or absence of well-defined sequence elements that direct excision, suggesting that chromosome fragmentation systems have evolved multiple times and/or have been significantly altered during ciliate evolution. We propose a two-stage model for the evolution of the current ciliate systems, with both stages involving repetitive or transposable elements in the genome. The ancestral form of chromosome fragmentation is proposed to have been derived from the ciliate small RNA/chromatin modification process that removes transposons and other repetitive elements from the macronuclear genome during development. The evolution of this ancestral system is suggested to have potentiated its replacement in some ciliate lineages by subsequent fragmentation systems derived from mobile genetic elements.

Community assembly and co-occurrence network complexity of interstitial microbial communities in the Arctic (investigation of ciliates in the White Sea intertidal zone).

Arctic coastal ecosystems play a major role in global environmental system and have been altered significantly by climate changes. To better understanding the response of marine coastal ecosystems towards rapid Arctic climate changes, we examined the variation in diversity and community structure and provided insights into the co-occurrence network and community assembly of interstitial ciliates in the Kandalaksha Gulf of the White Sea from 2009 to 2019. Co-occurrence networks analysis indicated considerably high ration of positive correlations within a community that indicated low competition between interstitial ciliate species. Furthermore, we found that contribution of stochastic processes to the ciliate community assembly was insignificant. Compare with earlier data from the same ecosystem obtained in 1980s-1990s, the role of competitive factors is decreasing, and communities are becoming more spatially and temporally homogeneous. This community simplification is likely due to the response of the entire intertidal ecosystem to global climate change in Arctic.

Ciliate Morpho-Taxonomy and Practical Considerations before Deploying Metabarcoding to Ciliate Community Diversity Surveys in Urban Receiving Waters.

Disentangling biodiversity and community assembly effects on ecosystem function has always been an important topic in ecological research. The development and application of a DNA metabarcoding method has fundamentally changed the way we describe prokaryotic communities and estimate biodiversity. Compared to prokaryotes (bacteria and archaea), the eukaryotic microbes (unicellular eukaryotes) also fulfill extremely important ecological functions in different ecosystems regarding their intermediate trophic positions. For instance, ciliated microbes (accounting for a substantial portion of the diversity of unicellular eukaryotes) perform pivotal roles in microbial loops and are essential components in different ecosystems, especially in water purification processes. Therefore, the community composition of ciliated species has been widely utilized as a proxy for water quality and biological assessment in urban river ecosystems and WWTPs (wastewater treatment plants). Unfortunately, investigating the dynamic changes and compositions in ciliate communities relies heavily on existing morpho-taxonomical descriptions, which is limited by traditional microscopic approaches. To deal with this dilemma, we discuss the DNA-based taxonomy of ciliates, the relative merits and challenges of deploying its application using DNA metabarcoding for surveys of ciliate community diversity in urban waterbodies, and provide suggestions for minimizing relevant sources of biases in its implementation. We expect that DNA metabarcoding could untangle relationships between community assembly and environmental changes affecting ciliate communities. These analyses and discussions could offer a replicable method in support of the application of evaluating communities of ciliated protozoa as indicators of urban freshwater ecosystems.

The combined effects of microplastics and the heavy metal cadmium on the marine periphytic ciliate Euplotes vannus.

Microplastics could be grazed by marine organisms and possibly transferred to higher trophic levels along the microbial loop. Due to their size and capacity to concentrate heavy metals that trigger joint toxic effects, microplastics (MPs) have already become a severe threat to marine organisms. The detrimental effects of MPs on large marine organisms have been studied, but the combined toxicity of MPs and cadmium (Cd) on protozoan ciliates remains unclear. In the present study, we selected different diameters and concentrations of polystyrene microspheres (PS-MPs) and Cd2+ as model MPs and heavy metals to evaluate their single and combined effects on the periphytic marine ciliate Euplotes vannus in relation to carbon biomass and oxidative stress. The MPs were indeed ingested by Euplotes vannus and significantly reduced the abundance and carbon biomass of ciliate populations. Combined exposure to MPs and Cd2+ not only increased the bioaccumulation of Cd2+ in ciliates but also exacerbated the decrease in ciliate biomass by increasing oxidative stress and membrane damage. In comparison, the effects of nano-sized plastics (0.22 µm) were more harmful than those of micro-sized plastics (1.07 µm, 2.14 µm and 5.00 µm). A smaller size represents a higher potential for penetrating biological members and a stronger adsorption capacity for cadmium. These results provide new insight into the combined toxicity of microplastics and heavy metals on ciliated protozoa and lay a foundation for higher trophic levels and ecosystems.

Community assembly and co-occurrence network complexity of pelagic ciliates in response to environmental heterogeneity affected by sea ice melting in the Ross Sea, Antarctica.

In the Southern Ocean, the living environment of organisms has changed due to the dramatic increase in melting sea ice and the loss of glaciers, which have consequently caused substantial changes in biodiversity. Samples of pelagic ciliates from 13 sites were collected as bioindicators to demonstrate the relationship between spatial distribution patterns and environmental heterogeneity affected by sea ice melting and to reveal the community assembly mechanisms in the Ross Sea. Univariate analyses and multivariate analyses were effective tools demonstrating clear spatial patterns and providing a sufficient explanation to interpret strong correlations between pelagic ciliate communities and environmental variations, especially the distribution pattern of nutrients and Chl a. Moreover, environmental heterogeneity might affect the co-occurrence network complexity of ciliate communities. Furthermore, our results also indicated that stochastic processes play a significant role in the community assembly of pelagic ciliates. This study examined the controlling mechanisms of environmental heterogeneity affected by sea ice melting on pelagic ciliate communities and provided explanations for the community assembly of pelagic ciliates in polar marine ecosystems.

Absolute quantification of chromosome copy numbers in the polyploid macronucleus of Tetrahymena thermophila at the single-cell level.

Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by amitosis, making ciliates a potentially valuable model system to study this process. However, a method to accurately quantify the copy number of MAC chromosomes has not yet been established. Here, we used droplet digital PCR (ddPCR) to quantify the absolute copy number of the MAC chromosomes in Tetrahymena thermophila. We first confirmed that ddPCR is a sensitive and reproducible method to determine accurate chromosome copy numbers at the single-cell level. We then used ddPCR to determine the copy number of different MAC chromosomes by analyzing individual T. thermophila cells in the G1 and the amitotic (AM) phases. The average copy number of MAC chromosomes was 90.9 at G1 phase, approximately half the number at AM phase (189.8). The copy number of each MAC chromosome varied among individual cells in G1 phase and correlated with cell size, suggesting that amitosis accompanied by unequal cytokinesis causes copy number variability. Furthermore, the fact that MAC chromosome copy number is less variable among AM-phase cells suggests that the copy number is standardized by regulating DNA replication. We also demonstrated that copy numbers differ among different MAC chromosomes and that interchromosomal variations in copy number are consistent across individual cells. Our findings demonstrate that ddPCR can be used to model amitosis in T. thermophila and possibly in other ciliates.

Notes on Homalogastra similis nom. nov. (Ciliophora, Scuticociliatia), a replacement name for the junior homonym Homalogastra binucleata Liu et al., 2020.

We recently established a new species, Homalogastra binucleata, with a morphological description and phylogenetic analyses based on SSU rRNA gene sequence data (Liu et al., 2020). Helmut Berger (Salzburg, Austria) commented that Homalogastra binucleata Liu et al., 2020 is a junior primary homonym of H. binucleata Song, 1993, the latter having been transferred to the genus Uronemita by Liu et al. (2016). According to article 57.2 of the ICZN (1999), the junior primary homonym, Homalogastra binucleata Liu et al., 2020, is permanently invalid. It is replaced by Homalogastra similis nom. nov.

Disentangling the relative roles of natural and anthropogenic-induced stressors in shaping benthic ciliate diversity in a heavily disturbed bay.

Coastal areas are facing biodiversity loss and degradation of habitats due to intensified human activities. However, our understanding of the relative contribution of natural gradients and human induced disturbance to biodiversity is limited. Here, we investigated the response of three facets of alpha and beta diversity of benthic ciliates to environmental gradients in a highly disturbed estuarine bay in China. We used linear regression and distance-based redundancy analysis to determine the key driving factors for biodiversity. Variation partitioning was further used to examine the relative influence of natural gradients and anthropogenic disturbances on ciliate communities. Our results revealed that ciliate alpha diversity and functional composition remained similar despite notable variation in species composition along salinity gradient. Sediment grain size, together with heavy metals were the strongest determinants in shaping both alpha and beta diversity. After controlling for the effect of natural factors, heavy metals still had significant impacts on beta diversity. Human induced nitrogen enrichment was positively correlated with algivorous functional group with possible impacts on benthic food webs. These results suggest that beta diversity is overall more sensitive to anthropogenic stressors than alpha diversity and give important insights into the role of anthropogenic disturbance on coastal diversity, being also useful for developing ecosystem protection and conservation strategies.

Taxonomy and Molecular Phylogeny of Two New Species of Prostomatean Ciliates With Establishment of Foissnerophrys gen. n. (Alveolata, Ciliophora).

Prostomatean ciliates play important roles in the flow of material and energy in aquatic microbial food webs, and thus have attracted wide attention for over a century. Their taxonomy and systematics are, however, still poorly understood because of their relatively few taxonomically informative morphological characters. In this study, two new prostomateans, Lagynus binucleatus sp. n. and Foissnerophrys alveolata gen. n., sp. n., collected from a freshwater pool and the intertidal zone of a sandy beach, respectively, in Qingdao, China, are investigated using living observation, protargol staining, and SSU rRNA gene sequencing methods. The genus Lagynus is redefined, and the new species L. binucleatus sp. n. is established based on significant morphological differences with similar forms. Furthermore, a new genus, Foissnerophrys gen. n., is established based on a combination of morphological and molecular data with F. alveaolata sp. n. the type species by monotypy. The identities of intracellular prokaryotes of these two new species are discussed based on fluorescence in situ hybridization (FISH) data and newly obtained 16S rRNA gene sequences.

Balantidium coli apendicular como hallazgo incidental. Reporte de un caso / Apendicular balantidium coli as incidental finding during surgery. Report of a case

Resumen Balantidium coli es el único miembro de la familia Balantidiidae capaz de producir infección en seres humanos. Presentamos un caso en un hombre de 43 años que ingresa para corrección quirúrgica de hernia ventral durante la cual se realizó apendicetomía profiláctica. En el estudio histopatológico se observó apéndice cecal con arquitectura conservada, sin la presencia de apendicitis ni periapendicitis. En la luz se reconocieron estructuras grandes (aproximado de 50 μm) redondas con citoplasma amplio con vacuolas grandes, cilias periféricas y núcleos densos, los cuales correspondieron a trofozoitos de Balantidium coli.

Abstract Balantidium coli is the only member of the Balantidiiae family capable of infecting human beings. We present one in a 43 years-old male admitted for a surgical co rrection of an incisional hernia with prophylactic appendicectomy. Histopathological findings reported the cecal appendix within normal architecture, appendicitis and peri-appendicitis free. At the lumen big, rounded shape structures (aprox. 50 mm) were visible with broad cytoplasm, big vacuoles, peripheral cilia and dense nucleus, corresponding to Balantidium coli trophozoites.

Pathological findings and husbandry management in captive Chrysaora spp. medusae affected by umbrellar ulcerative lesions.

During a 12 month period, a group of 14 medusa-stage jellies of the genus Chrysaora, including Pacific sea nettle (Chrysaora fuscescens, n = 11) and Japanese sea nettle (Chrysaora pacifica, n = 3), that were maintained in a public aquarium developed progressive ulcerative umbrellar lesions. In 6 cases (42.9%), ulceration was deep, transmural, and perforated through the mesoglea and subumbrella. In 6 cases (42.9%), ciliated protozoa histomorphologically consistent with scuticociliates were observed in the mesoglea and gastrovascular cavity. In 2 cases (14.3%), commensal dinoflagellates (zooxanthellae) were in the mesoglea and in the cytoplasm of the scuticociliates. During this period, water quality parameters including temperature [°C], pH, oxidation-reduction potential (ORP) [mV], salinity [psu], dissolved oxygen [%], ammonia (NH3), and nitrite(NO2) levels were monitored daily or weekly. The main water quality abnormalities were increased NO2 and pH levels above recommended reference ranges for C. fuscescens and elevated temperature above recommended reference ranges for C. pacifica tank. After correction of water quality parameters, apparent improvement of jellies was observed. In this case, environmental factors were considered the most likely predisposing factors for the development of ulcerative lesions, and ciliated protozoa were considered secondary rather than primary pathogens.

A strategy for complete telomere-to-telomere assembly of ciliate macronuclear genome using ultra-high coverage Nanopore data.

Ciliates contain two kinds of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC) in a single cell. The MAC usually have fragmented chromosomes. These fragmented chromosomes, capped with telomeres at both ends, could be gene size to several megabases in length among different ciliate species. So far, no telomere-to-telomere assembly of entire MAC genome in ciliate species has been finished. Development of the third generation sequencing technologies allows to generate sequencing reads up to megabases in length that could possibly span an entire MAC chromosome. Taking advantage of the ultra-long Nanopore reads, we established a simple strategy for the complete assembly of ciliate MAC genomes. Using this strategy, we assembled the complete MAC genomes of two ciliate species Tetrahymena thermophila and Tetrahymena shanghaiensis, composed of 181 and 214 chromosomes telomere-to-telomere respectively. The established strategy as well as the high-quality genome data will provide a useful approach for ciliate genome assembly, and a valuable community resource for further biological, evolutionary and population genomic studies.

Corrigendum: Impact of Zinc and/or Herbal Mixture on Ruminal Fermentation, Microbiota, and Histopathology in Lambs.

[This corrects the article DOI: 10.3389/fvets.2021.630971.].

Impact of Zinc and/or Herbal Mixture on Ruminal Fermentation, Microbiota, and Histopathology in Lambs.

We investigated the effect of diets containing organic zinc and a mixture of medicinal herbs on ruminal microbial fermentation and histopathology in lambs. Twenty-eight lambs were divided into four groups: unsupplemented animals (Control), animals supplemented with organic zinc (Zn, 70 mg Zn/kg diet), animals supplemented with a mixture of dry medicinal herbs (Herbs, 100 g dry matter (DM)/d) and animals supplemented with both zinc and herbs (Zn+Herbs). Each lamb was fed a basal diet composed of meadow hay (700 g DM/d) and barley (300 g DM/d). The herbs Fumaria officinalis L. (FO), Malva sylvestris L. (MS), Artemisia absinthium L. (AA) and Matricaria chamomilla L. (MC) were mixed in equal proportions. The lambs were slaughtered after 70 d. The ruminal contents were used to determine the parameters of fermentation in vitro and in vivo and to quantify the microbes by molecular and microscopic methods. Samples of fresh ruminal tissue were used for histopathological evaluation. Quantitative analyses of the bioactive compounds in FO, MS, AA, and MC identified 3.961, 0.654, 6.482, and 12.084 g/kg DM phenolic acids and 12.211, 6.479, 0.349, and 2.442 g/kg DM flavonoids, respectively. The alkaloid content in FO was 6.015 g/kg DM. The diets affected the levels of total gas, methane and n-butyrate in vitro (P < 0.046, < 0.001, and < 0.001, respectively). Relative quantification by real-time PCR indicated a lower total ruminal bacterial population in the lambs in the Zn and Zn+Herbs groups than the Control group (P < 0.05). The relative abundances of Ruminococcus albus, R. flavefaciens, Streptococcus bovis, and Butyrivibrio proteoclasticus shifted in the Zn group. Morphological observation found a focally mixed infiltration of inflammatory cells in the lamina propria of the rumen in the Zn+Herbs group. The effect of the organic zinc and the herbal mixture on the parameters of ruminal fermentation in vitro was not confirmed in vivo, perhaps because the ruminal microbiota of the lambs adapted to the zinc-supplemented diets. Long-term supplementation of a diet combining zinc and medicinal herbs, however, may negatively affect the health of the ruminal epithelium of lambs.

Does microplastic ingestion dramatically decrease the biomass of protozoa grazers? A case study on the marine ciliate Uronema marinum.

Microplastic debris has become a significant global environmental issue. Yet, the effects on ingestion of microplastics by protozoan grazers-an important link in the microbial loop-are scant. Feeding experiments were conducted with the free-living marine ciliate Uronema marinum grazing on cultured bacteria Pseudoaltermonas sp., exposing them to different concentrations or sizes of polystyrene beads for 96 h. The number of beads decreased during exposure experiments. Under the microplastic influence, the ciliate cells were observed to decrease in abundance, body size, and biomass. It was noted that the ciliate biomass in the highest microplastic density treatment was significantly lower than that in the control (98.1% lower) and that microplastics can be ingested by ciliate protozoa which performed an important role in the transportation of energy across the microbial loop. Moreover, carbon biomass of ciliates exposed to microplastics of different particle diameters decreased significantly compared to the control. However, this effect does not seem to vary depending on microplastic sizes. This study is a first step in providing experimental insight into the feeding relationship between microplastics and marine protozoan grazers. Further research based on components of the microbial loop is needed to explore the impacts of microplastics in marine food webs.

Identification and Characterization of Base-Substitution Mutations in the Macronuclear Genome of the Ciliate Tetrahymena thermophila.

Polyploidy can provide adaptive advantages and drive evolution. Amitotic division of the polyploid macronucleus (MAC) in ciliates acts as a nonsexual genetic mechanism to enhance adaptation to stress conditions and thus provides a unique model to investigate the evolutionary role of polyploidy. Mutation is the primary source of the variation responsible for evolution and adaptation; however, to date, de novo mutations that occur in ciliate MAC genomes during these processes have not been characterized and their biological impacts are undefined. Here, we carried out long-term evolution experiments to directly explore de novo MAC mutations and their molecular features in the model ciliate, Tetrahymena thermophila. A simple but effective method was established to detect base-substitution mutations in evolving populations whereas filtering out most of the false positive base-substitutions caused by repetitive sequences and the programmed genome rearrangements. The detected mutations were rigorously validated using the MassARRAY system. Validated mutations showed a strong G/C→A/T bias, consistent with observations in other species. Moreover, a progressive increase in growth rate of the evolving populations suggested that some of these mutations might be responsible for cell fitness. The established mutation identification and validation methods will be an invaluable resource to make ciliates an important model system to study the role of polyploidy in evolution.

The Pathology of Cetacean Morbillivirus Infection and Comorbidities in Guiana Dolphins During an Unusual Mortality Event (Brazil, 2017-2018).

Cetacean morbillivirus (CeMV; Paramyxoviridae) is the most significant pathogen of cetaceans worldwide. The novel "multi-host" Guiana dolphin (Sotalia guianensis; GD)-CeMV strain is reported in South American waters and infects Guiana dolphins and southern right whales (Eubalaena australis). This study aimed to describe the pathologic findings, GD-CeMV viral antigen distribution and detection by RT-PCR (reverse transcriptase polymerase chain reaction), and infectious comorbidities in 29 Guiana dolphins that succumbed during an unusual mass-mortality event in Rio de Janeiro state, Brazil, between November 2017 and March 2018. The main gross findings were lack of ingesta, pulmonary edema, ascites, icterus, hepatic lipidosis, multicentric lymphadenomegaly, as well as pneumonia, polyserositis, and multiorgan vasculitis caused by Halocercus brasiliensis. Microscopically, the primary lesions were bronchointerstitial pneumonia and multicentric lymphoid depletion. The severity and extent of the lesions paralleled the distribution and intensity of morbilliviral antigen. For the first time in cetaceans, morbilliviral antigen was detected in salivary gland, optic nerve, heart, diaphragm, parietal and visceral epithelium of glomeruli, vulva, and thyroid gland. Viral antigen within circulating leukocytes suggested this as a mechanism of dissemination within the host. Comorbidities included disseminated toxoplasmosis, mycosis, ciliated protozoosis, and bacterial disease including brucellosis. These results provide strong evidence for GD-CeMV as the main cause of this unusual mass-mortality event.

The Encystment-Related MicroRNAs and Its Regulation Molecular Mechanism in Pseudourostyla cristata Revealed by High Throughput Small RNA Sequencing.

MicroRNAs (miRNAs) regulate the expression of target genes in diverse cellular processes and play important roles in different physiological processes. However, little is known about the microRNAome (miRNAome) during encystment of ciliated protozoa. In the current study, we first investigated the differentially expressed miRNAs and relative signaling pathways participating in the transformation of vegetative cells into dormant cysts of Pseudourostyla cristata (P. cristata). A total of 1608 known miRNAs were found in the two libraries. There were 165 miRNAs with 1217 target miRNAs. The total number of differential miRNAs screened between vegetative cells and dormant cysts databases were 449 with p < 0.05 and |log2 fold changes| > 1. Among them, the upregulated and downregulated miRNAs were 243 and 206, respectively. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that some of the differentially expressed miRNAs were mainly associated with oxidative phosphorylation, two-component system, and biosynthesis of amino acids. Combining with our bioinformatics analyzes, some differentially expressed miRNAs including miR-143, miR-23b-3p, miR-28, and miR-744-5p participates in the encystment of P. cristata. Based on these findings, we propose a hypothetical signaling network of miRNAs regulating or promoting P. cristata encystment. This study shed new lights on the regulatory mechanisms of miRNAs in encystment of ciliated protozoa.

Effect of dry medicinal plants (wormwood, chamomile, fumitory and mallow) on in vitro ruminal antioxidant capacity and fermentation patterns of sheep.

The objective of this study was to determine the effect of dry medicinal plants (wormwood, chamomile, fumitory and mallow) and dietary substrates containing a mix of the plants on the end products of in vitro ruminal and intestinal fermentation, rumen protozoan population and ruminal antioxidant capacity of sheep. The experiment consisted of fermentations with the four plants used individually as the sole substrate and fermentation of a mix of medicinal plants (Plants): meadow hay:barley grain (MH:B), 700/300 w/w and Plants:MH:B, 100/600/300 w/w/w. The experiment was conducted using the in vitro gas production technique (IVGPT) with 35 ml of buffered inocula and approximately 250 mg (DM basis) of substrate incubated for 24 hr at 39°C in anaerobic conditions. Quantitative analyses of the bioactive compounds by ultra-high-resolution mass spectrometry in Plants identified three main groups: flavonoids (22 mg/g DM), phenolic acids (15 mg/g DM) and alkaloids (3 mg/g DM). The total antioxidant capacity (TAC) of the plant extracts and rumen fluid was analysed using a ferric reducing antioxidant power assay. The values of total and individual short-chain fatty acids, acetate:propionate ratio, pH and total gas production were significantly affected by the single plant substrates and inocula (p < .001). Apart from these parameters, the values of ammonia N, methane production and total gas produced were decreased in Plants:MH:B in comparison with MH:B (p < .001). A positive correlation was recorded between total polyphenols content and TAC of plant extracts (R2  = .778, p < .001). The counts of the total ciliate protozoan population in rumen fluid after 24 hr of fermentation were not significantly different (p > .05). Results suggest that the dietary substrate containing the medicinal plant mix possessed strong ruminal antioxidant capacity, had the potential to reduce methane emission and ammonia concentration and caused desirable changes in the gastrointestinal ecosystem.