Abstract
In vitro gas production techniques (IVGPTs) are crucial in animal nutrition research for evaluating the fermentative characteristics and nutritional value of feed ingredients and diets. Gas production (GP) is a key parameter in fermentation. Rumen fluid (RF) and faeces (FA) are the primary sources of microbial inoculum for fermenting microorganisms. The storage methods used for these inocula present both advantages and disadvantages. Traditionally, rumen cannulation was used to collect RF samples. However, researchers are exploring better alternative methods, such as stomach tube (ST) collection, which offers comparable statistical power and feasibility to cannulation. However, this approach is also challenging due to animal stress and saliva contamination, which emphasize the need for more representative sampling methods. Using rumen fluid from slaughtered animals offers an ethical approach, with the advantages of cost and availability. FA provides a viable alternative, especially for hindgut fermentation studies; however, in ruminants, differences in the microbial compositions of FA and RF need to be considered. These differences may in turn affect the GP rates and fermentation kinetics. Storage of microbial inocula can standardise in vitro studies, ensuring repeatability and reliability. The use of cryoprotectants such as glycerol and dimethyl sulfoxide (DMSO) may help to preserve microbial activity during the freezing-thawing process, as they help promote bacterial recovery. This review provides an overview of the two main inocula used in IVGPTs and their preservation methods, highlighting both their advantages and limitations.