The samples of rumen content were taken just before the morning feeding as well as at 4 and 8 h thereafter. The protozoa were purified as described above and lyophilized and stored at −80 °C (see above). The collection of material was repeated three times on three different days. The same animal was, however subjected to the sampling no more than 2 times a week.
The chitin content was assayed by quantification of N-acetylglucosoamine released after hydrolysis of lyophilized samples protozoa according to Chen and Johnson [5].
For this purpose the samples lyophilized of protozoa (200 mg) were hydrolyzed with 6 M hydrochloric acid at 105 °C for 12 h. After cooling to room temperature 150 mg of activated charcoal was added and all suspension was filtrated using the Filter Grade:390. The filter was washed with 20% ethanol solution. The filtrate was collected and evaporated to dryness at 45 °C and allowed to quantify N-acetylglucosoamine by the method Chen and Johnson [5]. The true content of this carbohydrate in the cells of protozoa was obtained by the comparison with standard curve of N-acetylglucosoamine. The content of chitin in the cell protozoa was calculated according to Volenc [31].
The chitin present in the zoospores was quantified by the same method.