Alternative methods to animal experimentation in rabbit nutrition trials integrating the 3Rs principles

Animal studies are essential to nutrition research, particularly in investigating the effects of dietary changes on animal growth, reproduction, health and metabolism. These studies provide quantitative data on feedstuffs’ nutritive value and animal response to diets, indispensable for building accurate nutrient databases and defining animal nutrient requirements, respectively. However, advancements in (bio)technologies have encouraged the development of non-animal alternatives in rabbit nutrition research. Moreover, Europe’s commitment to replacing animals for scientific purposes emphasises the need to regulate and harmonise animal experimentation according to the principles of the 3Rs (Replacement, Reduction and Refinement). While animal methods remain necessary in some cases, attention must be paid to their reliability and validity, alongside the adoption of alternative methods. Alternative approaches in rabbit nutrition include prediction equations to estimate the nutritional value of feedstuffs based on their chemical composition, in vitro models to simulate the digestibility and fermentability of diets and feedstuffs, and the use of near-infrared spectroscopy (NIRS) to calculate feed composition and nutritive value. Other non-animal-based approaches using mathematical modelling and cell/tissue/organ culture models are also rapidly evolving to test animal responses to dietary changes. Reduction can be achieved through extensive literature searches, careful experimental design, statistical methods and data sharing to avoid unnecessary duplication of experiments. Refinement includes appropriate housing, care and enrichment to minimise the suffering of animals used in research. Additionally, integrating precision livestock farming technology into research practices and omics tools through non-invasive procedures can also contribute to refining rabbit trials. The aim of this work was to critically review these approaches following Replacement, Reduction and Refinement principles for rabbit nutrition. We first examine already existing possibilities and practical methods and later discuss their adequacy. Recommendations for designing rabbit nutrition trials and further research needs, opportunities and challenges for the use of alternative methods that pursue any of the 3Rs will also be reviewed in the light of rabbit nutrition trials.