Background: The horizontal transfer of expressed genes from Bacteria into Ciliates which live in close contact with each other in the rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). More than 4000 ESTs were sequenced from representatives of the two major groups of rumen Cilates: the order Entodiniomorphida (Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Metadinium medium, Diploplastron affine, Polyplastron multivesiculatum and Epidinium ecaudatum) and the order Vestibuliferida, previously called Holotricha (Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium).

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of ؊30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.Rumen microbial populations are characterized by a broad diversity of microorganisms, mainly bacteria, ciliate protozoa, and fungi. Protozoa represent up to 50% of the total rumen microbial biomass. They are involved particularly in the rumen digestion of cellulose (6,7,17,50), starch (7,16,45), and proteins (40), and they contribute actively to the control of the bacterial population (19) and to the formation of the end products of ruminal fermentation (21,44,45). They play an important role in the biodegradation of plant toxins and mycotoxins (22,49) and in the regulation of ruminal conditions such as pH and redox potential (1,27,39,44). They have been shown to eliminate certain pathogens from the digestive tract of ruminants, protecting them from disease and so improving the food safety of edible animal products (33).Although some rumen ciliates can usually be cultivated in vitro, it is difficult to maintain them for a long time, and most species die within a few months to a year (3,8,12,13,14,15,34,35,36). Another way to maintain rumen ciliate protozoa for long periods involves with the rumen of previously defaunated animals (18) into which a few isolated cells of single species are inoculated to obtain monofaunated animals (16,19,21). However, preparing and maintaining a large number of animals in a monofaunated state for a wide range of ...

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