90 Awardee Talk: Shedding light into the black box: The dual-flow continuous culture system and ruminal fermentation
Authors: Faciola Antonio P 
Published: September 2024

Abstract
Nutrients derived from ruminal fermentation supply a substantial amount of metabolizable nutrients for ruminants. These include amino acids, carbohydrates, fatty acids, vitamins, and fermentation end products, such as ammonia and volatile fatty acids. Furthermore, ruminal fermentation modifies dietary nutrients in such a way that nutrients reaching the small intestine for absorption may substantially differ from those being consumed in the diet. Therefore, understanding ruminal fermentation kinetics and quantification of nutrients flowing out of the rumen is an important aspect of ruminant nutrition. Accurate quantification of these nutrients, using in vivo experiments, is laborious, costly, and invasive, requiring surgical procedures such as ruminal, abomasal, and intestinal cannulas. To decrease the time and cost of experiments as well as the use of animals, in vitro methodologies are important tools in ruminant nutrition. Therefore, the objectives of this presentation are i) describe different in vitro methodologies, ii) discuss the advantages of in vitro methodologies, iii) discuss shortcomings of in vitro methodologies, and iv) describe potential developments that may improve in vitro methodologies. Having been used for decades, in vitro methodologies such as pure, batch, and continuous cultures have been well documented to investigate a wide array of aspects of nutrition, including the effects of different dietary compositions, individual fermentation end products, and impacts on the microbiome. However, both batch and pure cultures can result in a build-up of end products that may inhibit fermentation. Continuous culture, however, allows for the removal of end products but, similar to pure and batch cultures, is applicable only to ruminal fermentation and cannot provide information regarding intestinal digestion. The dual-flow continuous culture system (DFCC), developed in the 1970s and used by various laboratories in the USA and Europe consists of fermentation vessels of approximately 1.5 L that are capable of simulating ruminal fermentation for periods longer than 10 d. It has been extensively used and evaluated by meta-analyses. When compared with other in vitro methodologies, the DFCC has longer fermentation time, larger volume, and the possibility of sampling from the liquid and the solid flows. When compared with in vivo experiments the DFCC is faster, less costly, less invasive, allows for greater range of treatments, allows for more controlled condition, and isolates ruminal function. Limitations of the DFCC include it does not predict in vivo response, it assumes equal saliva flow and concentration, no effects of intake, no ruminal absorption, less retention of protozoa, possible differences in microbiome, and limited gas emission data. Therefore, while in vitro methodologies provide useful data into the field of ruminant nutrition, some in vitro methodologies are more robust than others. Furthermore, in vivo experiments are still the ideal approach for assessing nutrient utilization and necessary to evaluate animal responses.