Laboratory Animal Sciences 2021

The concept of the 3Rs remains more prevalent than ever in global discussions related to humane animal use in aspects of research, teaching and testing. The 3Rs serve as a touchstone, in terms of refinement, reduction, and replacement, for veterinary staff, ethical review committees and scientists. Russell and Burch established these words as part of a new applied science in 1959, aiming to improve animal welfare with the 3Rs as a launchpad for future development of scientific methods and medical discovery. In all aspects of animal use, the anticipated benefits of scientific inquiry are to be weighed against the potential detriments borne by the animals themselves; this ethical balance is referred to as the ‘harm-benefit’ analysis. Over time, aspects of the use of the 3Rs in study design (refinement), statistical analysis (reduction), and selection of alternatives to live animals (replacement), have strayed into the potential for abuse by groups that seek to constrain the spirit of their interpretation. For example, with the generation and availability of novel techniques (e.g., CRISPR) and development of human disease (e.g., COVID-19) in animal models, any rigid 3Rs interpretation of ‘reduction’ to minimal numbers until we achieve ‘replacement’ of all animals is incompatible with the necessity to support science and production of reliable and reproducible results. The 3Rs have remained ubiquitous in large part because of their benefit to animals, although importantly they are also of benefit to those stakeholders invested in the animal research enterprise.  The exquisiteness of the 3Rs is that, much like a classical piece of art, they are inherently subject to broad interpretation across the contemporary biomedical community. Reconsidering the foundational 3Rs with progressive interpretations, in combination with other ethical tools of analysis and validity assessments, will be essential in the ongoing intricate considerations of appropriate and future animal use.

Learning Objectives:

1. Review the foundational 3Rs for animal use and welfare and discuss limitations of rigid interpretation of the terminology

2. Provide expanded definitions of the 3Rs that better address the complexity of appropriate animal use and welfare in contemporary society

Primates have been essential in unlocking novel immune mechanisms that have driven development of groundbreaking new drugs in infectious disease and transplantation, amongst others related to the complexity of the primate immune system that is most similar to humans. As a result, primates have served as critical ‘gatekeepers’ in safety and efficacy evaluations of vaccines and immunotherapies.  Behind this success is a path with considerable examples of where primates have overestimated the effect, with an exaggerated immune response that is not present in clinical trials. The explanation for translational failures may partially relate to model-imposed stressors that affect the immune system. Common sampling practice in nonhuman primate (NHP) safety and toxicology studies involves serial sampling from a chemically or capture (mechanically) restrained animal. While sedation events may be required to protect researcher safety during studies of infectious disease and promote ease of sample collection, this comes at a cost to animal welfare and introduces confounding that reduces our ability to interpret data accurately. Study specific endpoints related to stress, immune response, and safety that are critical for vaccine development and toxicity studies are confounded by the effects of restraint stress and sedation on the research subjects. For example we have observed significant differences in cytokine levels by sex and cytokine levels were significantly blunted in sedated NHPs when compared to awake cooperating NHPs, highlighting the importance of model design on translational relevance for clinical settings. Acute and chronic stress alters immune responses by shifting immune cell populations or altering signaling, such that the model design must be carefully considered to be representative of the typical Phase I/II clinical studies in healthy immunocompetent adults to avoid misinterpretation of immune-based study endpoints. At the conclusion of this session, participants will develop an improved understanding of how model-related parameters affect study validity by influencing background immunologic and metabolic processes, impacting accurate disease characterization, and defining our ability to recapitulate the target disease state in ways that may confound outcome measures and impact translatability.

Learning Objectives:

1. An overview of the role of experimental design factors from animal selection to model handling in influencing animal welfare and scientific outcomes

2. Example strategies using behavioral management as a strategy to intentionally introduce variation for generalizability or reduce confounding to make an experiment more robust.

Primates have been essential in unlocking novel immune mechanisms that have driven development of groundbreaking new drugs in infectious disease and transplantation, amongst others related to the complexity of the primate immune system that is most similar to humans. As a result, primates have served as critical ‘gatekeepers’ in safety and efficacy evaluations of vaccines and immunotherapies.  Behind this success is a path with considerable examples of where primates have overestimated the effect, with an exaggerated immune response that is not present in clinical trials. The explanation for translational failures may partially relate to model-imposed stressors that affect the immune system. Common sampling practice in nonhuman primate (NHP) safety and toxicology studies involves serial sampling from a chemically or capture (mechanically) restrained animal. While sedation events may be required to protect researcher safety during studies of infectious disease and promote ease of sample collection, this comes at a cost to animal welfare and introduces confounding that reduces our ability to interpret data accurately. Study specific endpoints related to stress, immune response, and safety that are critical for vaccine development and toxicity studies are confounded by the effects of restraint stress and sedation on the research subjects. For example we have observed significant differences in cytokine levels by sex and cytokine levels were significantly blunted in sedated NHPs when compared to awake cooperating NHPs, highlighting the importance of model design on translational relevance for clinical settings. Acute and chronic stress alters immune responses by shifting immune cell populations or altering signaling, such that the model design must be carefully considered to be representative of the typical Phase I/II clinical studies in healthy immunocompetent adults to avoid misinterpretation of immune-based study endpoints. At the conclusion of this session, participants will develop an improved understanding of how model-related parameters affect study validity by influencing background immunologic and metabolic processes, impacting accurate disease characterization, and defining our ability to recapitulate the target disease state in ways that may confound outcome measures and impact translatability.

Learning Objectives:

1. An overview of the role of experimental design factors from animal selection to model handling in influencing animal welfare and scientific outcomes

2. Example strategies using behavioral management as a strategy to intentionally introduce variation for generalizability or reduce confounding to make an experiment more robust.

Aseptic technique, including the use of sterile drapes, is essential to reduce microbial contamination to the lowest practical level and improve surgical outcomes. In biomedical research, surgeons are often responsible for simultaneously conducting rodent surgical procedures, monitoring anesthesia, and adjusting nonsterile equipment. This presentation will cover agile and innovative techniques on how to improve rodent aseptic surgery using common household products: cling film and foil. Recently, some institutions have approved the use of Press’n Seal® cling film and Reynolds Wrap® aluminum foil as practical, cost-effective alternatives to sterile drapes and barriers during rodent surgeries. This presentation will also cover recent studies and publications that evaluated sterility properties for both Press’n Seal® cling film® and Reynolds Wrap® aluminum foil and other potential benefits for each product in rodent surgery.

Learning Objectives:

1. Methods on how to aseptically use foil (Reynolds Wrap) in rodent surgery

2. Methods on how aseptically use cling film (Press N' Seal) in rodent surgery

3. Long-term and short-term benefits to using both cling film and foil in aseptic rodent surgery

Handling laboratory animals can stimulate stress and anxiety that negatively impacts animal welfare and introduces a substantial confounding factor in many areas of scientific research. However, the method used to pick up laboratory mice is critical in determining whether handling induces stress and anxiety. While the traditional method of picking up mice by the tail induces strong aversion and high anxiety, picking up mice in a handling tunnel or scooping them on the open hand (known as cupping) is non-aversive and substantially reduces anxiety and handling stress. Differences between tunnel and tail handled mice are particularly dramatic. Importantly, mice familiarised with non-aversive handling methods continue to interact readily with handlers and show reduced anxiety even after routine procedures that may induce some pain or discomfort, such as scruff restraint, subcutaneous or intraperitoneal injection, oral gavage, anaesthesia, tattooing or ear-tagging. This underlines the very strong impact that non-aversive handling methods have in taming animals to accept human contact and restraint, providing a major refinement over picking up mice by the tail. Concern about practicability can be a major barrier to implementing non-aversive handling methods by those already trained to pick up mice by the tail. However, mice can be picked up just as quickly with non-aversive methods once the handler has developed good skills and I will share some tips for gaining good technique. New handlers find tunnel handling the easiest method to learn - mice are easier to catch, less likely to bite and easier to restrain. Replacing tail handling wherever possible with non-aversive methods avoids unnecessary handling stress and anxiety in animals, makes handling easier, reportedly improves job satisfaction for the handler, and removes an important confound in many areas of animal research.

Learning Objectives:

1. Scientific evidence that picking up mice by the tail induces strong handling aversion, stress and anxiety that negatively impacts animal welfare, makes animals harder to handle and can be a major confounding factor in a wide range of research that is sensitive to handling stress

2. Scientific evidence showing the benefits of using non-aversive methods to pick up mice, such as use of a handling tunnel or scooping on the open hand (cupping)

3. How to pick up mice efficiently using non-aversive methods and tips for implementation in animal facilities

Pain management includes the choice of analgesia agents, their dose, administration method, duration and frequency of treatment, and a pain-monitoring scheme for each individual animal. Insufficiently treated pain or inadequately chosen or insufficient reported anesthesia or analgesia protocols may carry the potential to hamper animal welfare and the reproducibility of animal experiments substantially. Respective protocols are therefore an important part of experimental design, and determining an optimal protocol is mandatory when planning animal experiments. Unfortunately, there is evidence that especially the most widely used laboratory species, the mouse, is often treated insufficiently or that protocols are applied that may not reflect the recent state of the art of veterinary analgesia/anesthesia. In this talk requirements for a good analgesia protocol in general, but also the challenges that we face when applying analgesia to small patients like laboratory mice are discussed. Important concepts of (mouse) analgesia including preventive and multimodal approaches and strategies to overcome specific challenges will be discussed. The focus of this talk will lie on stress-free or stress reducing, sustained release or voluntary oral administration protocols for analgesic substances that have a great potential to improve animal welfare in animal research.

Learning Objectives:

1. Define the requirements of a good analgesia protocol

2. Identify various challenges in designing an appropriate analgesia protocol

3. Identify and choose potential refinement opportunities for analgesia protocols

There is growing concern about the reliability of biomedical research results. Poor experimental design, inappropriate analysis methods and incomplete reporting have all contributed to inconsistent results. The NC3Rs has developed resources to assist researchers in designing rigorous experiments, selecting the appropriate analysis methods and reporting experiments thoroughly. One such resource is the Experimental Design Assistant (EDA). The EDA is free online software with a supporting website to help researchers design more rigorous in vivo experiments. Experiments designed in the EDA are represented as machine readable diagrams, which the system analyses using computer-based logical reasoning to provide feedback. This feedback highlights the implication of particular design features and enables users to make informed choices, tailored to the objective of their own experiments. The system also suggests statistical analysis methods compatible with the design of the experiment. It includes inbuilt power calculators to determine the appropriate sample size, and features to facilitate randomisation and blinding. The EDA is also a very useful tool to communicate experimental plans as the diagram contains detailed information for each step of the experiment and provides a visual overview, which is helpful to support discussions. The EDA is endorsed by major biomedical research funders worldwide, to help researchers design experiments that are more likely to yield robust and reproducible data while using the minimum number of animals consistent with the scientific objectives.

Learning Objectives:

1. Understand the impact of subjective bias on the reliability of animal research results

2. Identify the experimental design features that can be used to reduce effects of bias and make results more reliable

3. Understand how the EDA can be used to design rigorous in vivo experiments that generate reliable results