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Although the existence of rumen ciliates is not vital for the host animals, their contribution to the digestion of organic matter, including fiber, and to the physiological stability of the rumen environment was thought to be important for the host animals. Among the monophyletic ophryoscolecids, Entodinium was considered the ancestral genus within this group. This genus has been well-known for its omnipresence and predominance in the rumen of various host ruminants. Entodinium species are highly bacterivorous, and its growth dependence on the associated prokaryotes has been well documented. Unlike free-living ciliates such as Tetrahymena thermophila or Paramecium tetraurelia, however, repeated efforts have not succeeded in establishing an axenic culture of any rumen ciliates for any extended period. Some of the normal biological processes are probably compromised in the temporary axenic cultures of ruminal protozoa. To better understand this important group of ruminal microbes and manage their population to improve their contributions to rumen functions, multipronged technical approaches are needed.Many researchers attempted to establish axenic cultures of ruminal protozoa, especially Entodinium caudatum, but no maintainable axenic culture was ever developed. No study has been reported that investigated the reasons why axenic cultures of rumen ciliates are hard to establish or maintain. Thus, the first study was designed to investigate the toxicity of eight different antibiotics, which have been used to remove prokaryotes in attempts to establish an axenic culture of the ruminal protozoan, Ent. caudatum, which is the most predominant protozoal species in the rumen (chapter 3). As demonstrated by other researchers, only temporary axenic cultures of Ent. caudatum was possible even in the presence of multiple growth factors that have been proven useful in growing other ciliates in axenic cultures. We also showed that mixed rumen bacteria enabled the temporary axenic culture of Ent. caudatum resume normal growth. All the tested antibiotics inhibited the growth of Ent. caudatum and damaged the morphological structure, both internal and surficial, including accumulation of glycogen granules and damages to the macronuclei and other cellular structures. It was concluded that antibiotics are directly toxic to Ent. caudatum and also indirectly inhibit it by killing the prokaryotes that are essential for the survival of Ent. caudatum. This species may have to be cultured as monoxenic or multixenic cultures. The rumen microbiome consists of various guilds (or functional groups) of microbes, both prokaryotes and eukaryotes. Different interactions exit, including mutualistic, commensal, antagonistic, parasitic (between phages and cellular microbes), and prey-predator. The interaction between ruminal protozoa and amino acid-fermenting bacteria (AAFB) has important implication to nitrogen utilization inefficiency in ruminant, and thus chapter 4 was devoted to studies on the interaction between Ent. caudatum as a model of ruminal protozoa and an AAFB consortium that was previously enriched on casamino acids as the only substrates. Washed cells of Ent. caudatum and AAFB served as the inocula and cultures as single cultures or co-cultures of both. The interactions between Ent. caudatum and AAFB were examined with respect to Ent. caudatum growth, dry matter digestion, fermentation characteristics, and populations of select bacteria cable of proteolysis and amino acid fermentation. Even though the total bacterial abundance was comparable, the co-culture had improved dry matter digestibility, VFA production, Ent. caudatum growth compared to those of single culture of Ent. caudatum. However, ammonia-N and relative abundance of Clostridium aminophilum and Peptostreptococcus anaerobius, two hyper ammonia-producing bacteria, were not different between the co-culture and the Ent. caudatum single culture. These results indicate a mutualistic interaction between Ent. caudatum and AAFB, but their interaction probably may not enhance ammoniagenesis under the experimental conditions. However, the inability to remove all the prokaryotes associated with Ent. caudatum made it impossible to infer the interactions between Ent. caudatum and AAFB or other prokaryotes. Physical association between rumen ciliates and prokaryotes including methanogens has been previously demonstrated by molecular-based detection or by visual examination. However, it remains poorly defined which microbes are actually associated with ruminal protozoa. In the study reported in chapter 5, the prokaryotes associated with ruminal protozoa were analyzed by identifying and comparing the free-living prokaryotes and those recovered together with washed single ruminal protozoal cells. Amplicon libraries were prepared from metagenomic DNA extracted from single protozoal cells after washing and from free-living prokaryotes (FLP). After Illumina MiSeq amplicon sequencing, protozoa-associated prokaryotes (PAP) and FLP were identified and compared between the two fractions. Single protozoal cells of both monocultures of Ent. caudatum and Epi. caudatum and of fresh ruminal fluid (the above two groups and another six genera) were analyzed to help confirm the microbes associated with ruminal protozoa. Irrespective of the source of singles, the microbial community of PAP was significantly different from that of FLP. In addition, some bacterial taxa were PAP specific and were almost exclusively found in PAP across most of the protozoal genera. The result of the Illumina sequencing was validated and confirmed using quantitative qPCR for three groups of archaea including Methanobrevibacter, Thermoplasmata, and Methanosphaera stadtmanae. Selective predation can be difficult to distinguish from endosymbiosis, but the detection of PAP-specific bacteria across different ruminal protozoa and irrespective of sources will be possible candidates of true symbionts, and some of them may be essential for the survival of ruminal protozoa.Rumen ciliates are considered as strict anaerobes, and thus they are cultured under anaerobic conditions, which are typically created using gas manifolds or anaerobic chambers. The requirement for anaerobic media and for the equipment facilities to create anaerobic conditions makes cultivation of ruminal protozoa time consuming and difficult. Strong antioxidants have been shown to allow strict anaerobes, including methanogens, to grow in aerobic media. We hypothesized that rumen ciliates could also be cultured aerobically with antioxidants. This hypothesis was tested using Ent. caudatum and Epidinium caudatum as ruminal protozoa and ascorbic acid, glutathione and ¿-ketoglutarate as antioxidant added to media (chapter 6). In this study, monocultures of Ent. caudatum and Epi. caudatum were grown in respective media containing the above antioxidants. Both species were able to grow in aerobic media, and the antioxidants also improve revival of these two species from frozen stock cultures. The fermentation characteristics were altered to some extent, so were the prokaryotes present in the monocultures. Because the prokaryotic community and fermentation of monocultures of ruminal protozoa inevitably differ from those of the rumen, the observed alteration of fermentation and prokaryotic communities may not necessarily interfere in research using ruminal protozoa cultures. Nevertheless, these antioxidants can be used to facilitate research in which ruminal protozoa need to be cultured in vitro. As we demonstrated in the first study, it is extremely difficult to achieve an axenic culture of rumen ciliates (chapter 3). The lack of axenic cultures of ruminal protozoa has hampered research efforts to obtain direct evidence or definitive information that help elucidate the fundamental biology of ruminal protozoa, such as metabolism. The last study of this dissertation (chapter 7) aimed to sequence the metabolically active macronuclear genome and help understand the metabolism, physiology, and ecology of Ent. caudatum as a model species of ruminal protozoa. The cells of an actively growing Ent. caudatum culture were washed and lysed. The macronuclei were isolated and purified. The macronuclear genomic DNA was sequenced using the MiSeq Illumina MiSeq platform and the MinION system. From over 80 megabases sequence reads that provided about 79X coverage, 37,547 contigs were assembled. Telomeric repeats (CCCCAAT)n were predicted as which was different with those of free-living ciliates. More than 3,000 scaffolds were capped at both ends with these repeats. Based on the assembly statistics, the macronuclear genome of Ent. caudatum may have a large number of nanochromosomes. From the draft assembly, 13,491 protein-coding genes were predicted and annotated. The draft genome provided genomic evidence on the substrates, metabolic pathways, and some physiological features. This is the first genomic study on ruminal protozoa. The macronuclear genome of Ent. caudatum can be useful in comparative genomic studies of this ruminal protozoan and other ciliates. The methodological framework used in this study can also be useful to genome studies of the other rumen ciliates. Collectively, using multipronged approaches, this dissertation addressed several aspects of ruminal protozoa, and the findings shine new lights on the metabolism, physiology, and ecology of ruminal protozoa, particularly Ent. caudatum (chapter 8). The approaches used in the studies can help future studies on ruminal protozoa in general and their roles in ruminal functions.