Interpretive Summary: Unraveling the pros and cons of various in vitro methodologies for ruminant nutrition: a review
By: James R Vinyard, Antonio P Faciola
In vitro methodologies have been used in animal nutrition research for decades to decrease the use of animals and experimental time and cost. Due to its lower cost, in vitro methodologies are commonly used for preliminary testing, prior to large in vivo trials. The use of in vitro also helps researchers to focus on the three R’s of animal research: reduce, refine, and replace (Curzer et al., 2016). When in vitro methodologies are used, they allow researchers to reduce both the number of animals used and their total use, as they only require the collection of inoculum rather than using them to complete lengthy digestibility or production trials. They refine their use, as tissues are either collected from animals that are being harvested for meat otherwise or ruminal cannulae are placed in a small number of animals rather than using a large group for in vivo study. In some instances, such as pure and cell culture, the use of animals is eventually even replaced, as the unit of study becomes refined strains of bacteria or cells. However, there are many different needs that can be met by in vitro methods, whether that be ruminal degradation, impacts on microbial communities and ruminal fermentation like batch or continuous cultures (Arce-Cordero et al., 2022), estimating total tract digestibility using three-step procedures (Calsamiglia and Stern, 1995), or determining specific microbial affects using pure bacterial cultures or epithelial cell cultures (Zhan et al., 2020).
Due to the wide variety of the investigatory capabilities of in vitro methodologies, there are various experimental designs that can achieve adequate statistical power, including completely randomized, completely randomized block, and Latin square; along with the factorial or split-plot arrangement of treatments. These different designs were reviewed in depth by Hristov et al. (2019) and thus will not be discussed in detail here. As a whole, in vitro is used in instances where the use of in vivo is inappropriate, due to risk to animals, costly, or as a preliminary experiment. For instance, in vitro can be used to test levels of toxin exposure (Dai et al., 2019; Kent-Dennis et al., 2020) to animals that may be of a level that would cause illness or death in vivo, without risking an animal’s wellbeing. In vitro is also a very useful tool when investigating the specific physiological mechanisms of a microbial species or for a pilot study to determine the efficacy of a new rumen-protected product.
While they are well documented and some have been evaluated meta-analytically, to our knowledge, there is no in-depth review of in vitro methodology that discusses the techniques and applications for pure, batch, continuous, and cell cultures and the three-step methods for determination of digestibility. Thus, to fulfill the gap in the literature, the objectives of this review are to 1) describe the development of different in vitro methodologies, 2) discuss their application, utilization, and advantages, 3) discuss their shortcomings, and 4) describe potential developments that may be able to improve these methodologies.
Read the full article in Translational Animal Science.