The Cultivation of Sheep Rumen Oligotrich Protozoa in vitro

Abstract

SUMMARY : Oligotrich protozoa from the sheep rumen, principally Entodinium caudatum, were maintained in culture in vitro and dividing every 2 days at a density of 15,000-30,000 protozoa/ml. for 18 months on a medium of rice starch, dried grass, 10 % (v/v) rumen fluid (fresh or autoclaved) and 50 pg. chloramphenicollml. The effect on these organisms of varying each of these factors in turn and the use of different growth conditions is described. The protozoa have also been grown from an inoculum of 100-900/ml. to a density of over 50,00O/ml. in 10 days.Many unsuccessful attempts to cultivate rumen protozoa in vitro have been made in the last hundred years; the earlier attempts were summarized by Becker, Schultz & Emmerson (1929). Margolin (1930) and Westphal (1934) achieved some success in the cultivation of oligotrich protozoa, but the first real advance was made by Hungate (1942,1943) who maintained some species of cattle oligotrich ciliates alive for over a year. Sugden (1953) was unable to maintain oligotrich protozoa from the sheep alive for more than 15 days, whereas Kandatsu & Takahashi (1955a, b, 1956) kept Entodinia spp. from goats alive for over 30 days with some multiplication. The purpose of the present paper is to describe experiments in which Entodinia spp., principally Entodiniurn caudatum from sheep rumen, have been maintained with frequent division for over 18 months; a preliminary communication has already appeared (Coleman, 1958). METNODSMedia. The standard medium consisted of 30 ml. quantities of autoclaved mineral salts solution (Coleman, 1958) to which was added: Na,S . 9H20 to a final concentration of 0.01 yo (w/v); c. 15 mg. dried grass (55" for 3 days) finely ground in a Lee Attrition Mill (Lee Engineering Co., Milwaukee, Wis., U.S.A.); 15 mg. rice starch, added as a suspension of known concentration in water; 3.0 ml. prepared fresh rumen fluid; chlorarnphenicol to a final concentration of 50 ,ug./ml. The final volume of complete medium was c. 35 ml. Tubes of complete medium were gassed vigorously for 3 min. with a mixture of 95% (v/v) N, + 5 yo (v/v) CO, (which had been freed from oxygen by the method of Stone & Beeson, 1936) and stoppered immediately. , 411 media were warmed to 38" before use. Fresh rumen fluid was usually prepared and used the same day (see Coleman, 1958), except when it was inconvenient to obtain fresh rumen contents. A4t these times the prepared material was kept a t -15" under 95 yo (v/v) N, + 5 y% (v/v) CO, for not longer than 4 clays and warmed to 38" 35-2

The Cultivation of the Rumen Ciliates Epidinium ecaudatum caudatum and Polyplastron multivesiculatum in vitro

“…2 ) ; the presence of autoclaved rumen fluid had no effect on this rate. This mean generation time compares favourably with times of 1.8 and 1.6 days for Entodinium caudatum and E. simplex respectively (Coleman, 1960(Coleman, , 1969.…”

“…As a result of these observations stock cultures were maintained thereafter without rumen fluid. Epidinium ecaudatum caudatum therefore differs from both the entodinia studied previously (Coleman, 1960(Coleman, , 1969 where the presence of autoclaved rumen fluid increased the population density almost ten times. The effect of saIt concentration on the growth of Epidinium ecaudaturn caudatuni from a small inoculum in the presence of 10 :( prepared, fresh, rumen fluid.…”

“…Wholemeal flour was not replaceable by an equal quantity of a purified starch, such as rice starch, for the long-term maintenance of the protozoa, although some epidinia remainedalive for up to 20 days in the presence of rice starch as sole source of carbohydrate. Epidinium ecaudatum caudaturn therefore resembles Entodinium simplex (Coleman, I 969) rather than Entodinium caudatum (Coleman, 1960) which grew well on rice starch.…”

“…The first isolate, which was grown from a single organism on the first strain of Epidinium ecaudatum caudaturn, would not grow on Entodinium caudatum or E. simplex grown in vitro (Coleman, 1960(Coleman, ,1969, on mixed entodinia from the rumen or on the second strain of epidinia, and therefore died when the first strain of epidinia died. Experiments carried out during the first few months of the life of this isolate of Polyplastron showed that epidinia broken by sonication supported 25 % as many polyplastrons as intact epidinia.…”

Molecular cloning, expression, and characterization of an endo-.BETA.-1,4-glucanase cDNA from Epidinium caudatum.

“…Many papers in the literature describe be cultivation of rumen ciliate protozoa (Coleman, 1960(Coleman, , 1987Hino and Kametaka, 1977;Hino et al, 1973;Hungate, 1942Hungate, , 1943Michalowski et al, 1991;Onodera and Henderson, 1980); however, no media have yet been devised that will keep axenic cultures of rumen protozoa alive for a long time.…”