The influence of microbial activity on the pattern of gas production and the possibility of developing a protocol for standardising the microbial activity of ruminal fluid were investigated (chapter 2). Firstly, ruminal fluid was diluted 1:2, 1:5, 1:9 and 1:20 (v/v) with buffer solution and the in vitro gas production from cellulose, glucose, hay and ryegrass studied. Gas production at 4, 24 and 120h were significantly decreased as the ratio of buffer solution to ruminal fluid increased. For each substrate, incubation with ruminal fluid diluted 1:2 (v/v) gave a higher (P<0.5) asymptotic value 'B', rate 'C' of gas production and lower lag time than when incubated in the 1:5, 1:9 and 1:20 dilutions. Secondly, the effect of frequency of ovine ruminal sampling on microbial activity and substrate fermentation was investigated. The bacterial DM, bacterial absorbance and blank's gas volume were significantly affected by daily sampling of ruminal fluid. Daily sampling of rumen fluid did not significantly affect the gas production degradability parameters (B,C and Lag) until the bacterial DM fell below 0.09 g DM/10 cm³ strained rumen fluid. Bacterial DM was strongly related to the absorbance of ruminal fluid (R² = 0.99, p<0.001), suggesting that standardisation of the level of microbial activity between days and laboratory can be achieved by estimating the bacterial DM from the absorbance of the strained ruminal fluid. The influence of donor diet on initial microbial concentration and gas production degradability was studied (chapter 3). Bacterial DM, bacterial absorbance and the blanks' gas volume increased significantly as the ratio of sheep pellet to hay increased. The gas production degradability parameters (B, C and Lag) were also affected by changing the donor diet ratio of sheep pellets and hay. NDF digestibility of cellulose and hay was not significantly affected by donor diet. Bacterial DM was strongly related to the absorbance of ruminal fluid and the blanks' gas volume (R² = 0.99, p<0.001). These results suggest that changing the ratio of concentrate to hay reduced the initial bacterial concentration and affected the gas production degradability parameters.