A review on applying immunological methods in animal research

Research on animal nutrition, including the impact of diets on immune system development and function, continues to expand our understanding of nutritional immunology to improve animal health and performance. From the perspective of research conducted in mammalian and avian immunology, this manuscript highlights species-appropriate approaches for performing immune assessments in experimental animals. Emphasis is placed on the importance of basic immunology knowledge, attention to detail to avoid flawed experiments, and precision in reporting and interpreting results to avoid incorrect conclusions and health recommendations. Animal nutritionists who incorporate immunological approaches into their research must use standardized procedures, validated tools, appropriate controls, and collaborate closely with immunologists to gather valid, reliable immunological insights from their studies. Topics addressed include the uses of peripheral blood analysis as a window into systemic immune system activities, along with methodological approaches ranging from establishing profiles of circulating leukocyte populations to leukocyte isolation methods and commonly used procedures to gain insight into their functional capabilities. By highlighting the differences in mammalian and avian procedures, researchers will also be made aware of potential pitfalls when working with other animal species. For example, white blood cells in birds and fish also include thrombocytes that may make up the majority of white blood cells but are not considered part of the immune cell populations (lymphocytes, neutrophils [heterophils in birds], monocytes, eosinophils, and basophils) in the blood. The methodologies described for blood are also applicable to immune system-related studies in tissues (e.g., lymphoid organs and sites of inflammation, etc.), including establishment of leukocyte profiles and leukocyte isolation for in vitro assessment. In addition to in vitro assays, methodologies to assess cell-mediated immunity in vivo are addressed. These include using the skin as a window into responses taking place in a complex tissue. Among characteristic skin swelling responses following intradermal injection of antigens or immunostimulants, the unique in vivo growing feather pulp bioassay in poultry is highlighted, as it is a minimally invasive procedure that enables assessment of local tissue/cellular immune responses across time in an individual without invasive surgical procedure or euthanization of the birds. Overall, the objective is to assist nutritionists and animal health researchers in implementing and integrating scientifically sound immunological approaches and considerations in research studies to acquire important insights that will further expand knowledge in nutritional immunology.