Unraveling the pros and cons of various in vitro methodologies for ruminant nutrition: a review

To decrease the time and cost of experiments as well as the use of animals in nutrition research, in vitro methodologies have become more commonplace in the field of ruminant nutrition. Therefore, the objectives of this review are 1) to describe the development of different in vitro methodologies, 2) to discuss the application, utilization, and advantages of in vitro methodologies, 3) to discuss shortcomings of in vitro methodologies, and 4) to describe the potential developments that may be able to improve in vitro methods. Having been used for decades, some in vitro methodologies such as pure, batch, and continuous cultures have been very well documented and utilized to investigate a wide array of different aspects of nutrition, including the effects of different dietary compositions, individual fermentation end products, and impacts on the microbiome of the rumen. However, both batch and pure cultures can result in a build-up of end products that may inhibit fermentation, as they culture ruminal contents or defined strains of bacteria, respectfully. 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 and bioavailability. This information for in vitro can only be provided using an assay designed for total tract digestibility, which is the three-step procedure (TSP). The TSP may be improved by coupling it with cell culture to investigate the absorption of nutrients in both the ruminal and intestinal phases of the methodology; however, the TSP needs further development to investigate all nutrients and the methodologies available for cell culture are still relatively new to ruminant nutrition. Therefore, while in vitro methodologies provide useful data in the field of ruminant nutrition without the continuous use of animals, there is still much work to be done to improve the methodologies to further apply them.