The study included two experiments with fresh pig manure as a main substrate (15.0%DM basis) in the completely randomized designs. In the Exp 1 the 50ml-syringes were used. The treatments were inoculum resources extracted from fresh pig manure (FPM), sludge of reactor of bio-digester (SR), sludge of removal tank (SRT) and rumen fluid (RF) of cattle. In Exp 2, two small experiments were done simultaneously on 50ml-syringes (Exp 2a) and 5 liter-flasks with six treatments and three replications. The treatments were inoculum resources including no inoculum [NC], fresh pig manure [FPM], sludge of reactor of biodigester [SR], sludge of 5liter-flasks incubated for 60 days with the ratio (g:v) of sludge and medium (1:5) [SF1:5], [SF2:5] and [SF3:5], respectively. The incubation period was 80 days with incubated temperature of 39^oC. In the Exp 2b corresponding to each treatment of the experiment 2a, one 5 liter-flask was used. It was concluded that better inoculum sources used for studying in vitro biogas production should be from the sludge of biogas reactors. Inoculum extracted from the sludge with the ratio of 1:5 was optimum for biogas production and quality. The syringes could be promisingly used for the in vitro biogas production research.