The rumens of sheep and cattle commonly contain ciliate Protozoa belonging to numerous genera. The biochemical study of these organisms has been hampered in the past by the difficulty of separating the different types from bacteria, and from one another, in sufficient quantity. The conventional bacterial pure-culture techniques have not so far proved applicable to these organisms, and investigators have therefore had to rely mainly upon alterations to the ruminant's diet, and various manipulations of the rumen liquor in vitro, in attempts to obtain suspensions of individual types of Protozoa. A technique of considerable value in this field was originated by Eadie & Oxford (1957), who found that many species of Protozoa could be eliminated from the sheep rumen by emptying and thorough washing of that organ. In a number of sheep treated in this way, only the smaller species of the oligotrich ciliate Entodinium survived the emptying procedure. It was further found (J. M. Eadie, personal communication, 1958) that when the diet of a treated sheep was altered by including a moderate amount of starchy foods, the numbers of entodinia present in the rumen increased to such an extent that suspensions of mixed Entodinium species, free from other Protozoa, and almost free from bacteria, could readily be prepared from the rumen liquor. Because Entodinium can, under many feeding regimes, be the predominant protozoal genus in the rumen, and because the biochemistry of these organisms has not been studied previously, the present investigation was undertaken. Some of our observations have already been reported briefly (Abou Akkada, Hobson & Howard, 1959). MATERIALS AND METHODSSheep as source of entodinia. The single sheep (no. 105) which served throughout these experiments had previously had its rumen fauna partially removed by the method of Eadie & Oxford (1957), and thereafter was kept isolated from other ruminants. Before the present work was commenced it had been inoculated with Dasytricha ruminantium, but during our experiments only very small numbers * Part 2: Howard (1959b). of this organism were present. In addition to a feed of hay (450 g.) morning and afternoon, the sheep was offered daily 450 g. of a concentrates ration, approximately threequarters of which consisted of ground maize and crushed oats.Preparation of protozoal suspensions. Samples of rumen liquor were withdrawn 3 hr. after the concentrates meal, strained through gauze and mixed in a separating funnel with an equal volume of the bicarbonate buffer solution described below (cf. Oxford, 1958). All manipulations and incubation of protozoa were carried out at 380. During incubation for 1 hr. the entodinia settled to the bottom of the funnel as a grey-white layer, which was run off into boiling tubes containing the buffer solution. From this stage onwards the buffer solutions used always contained chloramphenicol (50,ug./ml.) to suppress bacteria. After threefold washing by decantation, the protozoa formed a clean white layer in the bottom of the tubes. Micr...

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Egypt), AND K. EL-SHAZLY. Effect of absence of ciliate protozoa from the rumen on microbial activity and growth of lambs. Appl. Microbiol. 12:384-390. 1964.-A survey of the components of the rumen ciliate population in a series of adult sheep, raised in the Faculty of Agriculture, University of Alexandria, has shown that a mixture of Entodinium, Isotricha, Ophryoscolex, Diplodinium, and Polyplastron species was found in the rumen contents of Egyptian sheep; no Epidinium and a negligible number of Dasytricha ruminantium were also observed. The microbial population, reducing sugars, ammonia, volatile fatty acids (VFA) production, and growth rate of 14 lambs inoculated with whole rumen contents from a mature sheep were compared over a 6-month period with those of 13 lambs maintained under the same conditions, except that they were strictly isolated from other ruminants. Certain large oval organisms and large numbers of flagellates and Oscillospira were frequently observed in the rumen contents of the isolated lambs. The reducing sugars, ammonia, and VFA levels, measured before and at intervals after feeding, in the inoculated lambs showed a pronounced rise above the values found in the ciliate-free animals. The propionic acid-acetic acid ratio in the rumen contents of the faunated lambs was considerably higher than in the nonfaunated controls. The inoculated lambs grew faster than the isolated lambs.

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Washed suspensions of mixed rumen protozoa readily hydrogenated fatty acids in cottonseed, soya bean and corn oils in addition to free oleic and linoleic acids. Protozoa belonging to the family Ophryoscolecidae appeared to account for almost all the activity of mixed protozoa, protozoa of the family Isotrichidae possessing little or no ability to hydrogenate the added substrates. The hydrogenation of oleic acid was markedly increased by Fe and Mn. The addition of starch cellulose, urea and sodium formate greatly stimulated hydrogenation of oleic acid by rumen protozoa. Glucose, casein, ammonium sulphate, i-cysteine and n-valeric acid had no or little effect on extent of hydrogenation.When suspensions of mixed protozoa were incubated in a buffer solution, an increase in iodine value was observed. The desaturation in the protozoal cells was appreciably increased by DL-methionine and was not influenced by L-cysteine. No desaturation activity by the pure suspensions of Ophryoscolecidae or Isotrichidae was observed.The presence of protozoa in the rumen of sheep greatly increased the levels of saturated fatty acids over those in the ciliate-free animals. The concentrations of saturated fatty acids in the plasma of faunated sheep were significantly higher than those in the same animals after the removal of rumen ciliate protozoa.TNTRODTirTTOV present investigation was therefore, undertaken in order to study the lipid metabolism of rumen proThe ciliates of the rumen are of two families: the tozoa in fairly pure preparation and almost free of highly complex family Ophryoscolecidae (Oligo-bacteria, trichs) of which there are many species and the family Isotrichidae (holotrichs), consisting of the MATERIALS AND METHODS Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium (Abou Akkada, Bartley & In vitro studies Fina, 1969) . Considerable interest has been shownPreparation of protozoal suspension. Two fistuduring the last few years in the carbohydrate and lated sheep which had been fed a diet composed of nitrogen metabolism of these organisms (Abou 50% Berseem (Trifolium Alexandrinum); 24% Akkada, 1965). Furthermore, the contribution of undecorticated cottonseed cake; 24% rice bran; the rumen protozoa to the metabolism and growth 1-5 % calcium carbonate; and 0-5 % common salt, of the host animal has been recognized by a num-served as the source of protozoa.

1. Fourin vitrofermentation experiments, in duplicate, were carried out over 24 hr. periods, to estimate the production of v.f.a. and o.a from forages.2. A new technique for determining the total organic acids produced in the rumen is described.3. The o.a. produced in the earlier part of the fermentation are considerably higher than the v.f.a. but the difference then diminishes. There is practically no difference in the amounts produced after 24 hr.4. Highly significant correlations were found between thein vitroproduction of v.f.a. or o.a. and d.e./kg. d.m., for all the roughages tested. Nonlegumes gave a more highly significant correlation than legumes.5. Regression equations show that o.a. or v.f.a. were more accurate thanin vitrocellulose digestion for predicting the d.e./kg. d.m. of non-legume forages. It is concluded that V.F.A. productionin vitrois the best criterion in this respect.

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