Laboratory Animal Sciences 2020

Concerns about the quality of published animal research include inefficient or flawed study design, inappropriate statistical analysis and incomplete reporting. The ARRIVE guidelines were developed in 2010 to promote transparent reporting. Ten years on, despite widespread endorsement of the guidelines by the scientific community, including over a thousand journals, funders and universities, there is limited evidence that the quality of reporting in publications describing in vivo experiments has improved. We have revised the guidelines to facilitate their uptake and their use in practice. The recommendations in ARRIVE 2.0 are prioritised to highlight the most important information: the ARRIVE Essential 10 constitute the minimum requirement to enable reviewers and readers to assess the reliability of the findings presented in a publication. It includes details on the study design, the sample size, measures to reduce subjective bias, outcome measures, statistical methods, the animals, experimental procedures and results. This presentation will introduce the ARRIVE guidelines 2.0 and explain the importance of reporting the critical information covered in the Essential 10.

Learning Objectives:

1. Identify the minimum information to include in manuscripts describing animal research

2. Understand how this information contributes to the reliability and reproducibility of research findings

Inherent in research, is the simplification of a complex world into a testing space to explore cause and effect. Across preclinical research, questions are being raised on the testing space and whether it is too narrow.  Within genomic studies, such as GWAS, significant issues have been raised that the underlying datasets are not ethically diverse. Whilst within in vivo, in vitro and clinical research, a sex bias has been highlighted as culturally embedded in our working practices. In this workshop, we will focus on the sex bias embedded in our research pipelines. There have been multiple calls for action, and the funders of biomedical research are actively pushing the inclusion of sex as a biological variable, yet little has changed in practice.  We will first explore the current standard practice, why there is a movement to include females and why the imbalance exists. Then in small groups we will explore the blockers that hinder the use of both sexes within our research.

Learning Objectives:

1. To understand the endemic sex bias our research pipelines and realise the implications of this to the generalisability of our research.

2. To understand that a number of misconceptions exist in the community that hinder the inclusion of both sexes in preclinical research.  For example, studying both sexes does not require you to double your sample size as you will use a factorial design which estimates the effect from both sexes simultaneously.   Another example: for the majority of variables, female mice are no more variable than male mice.  Inclusion of both sexes should be the norm and justification for not including both sexes required.

In the course of the so-called reproducibility crisis, various sources of poor reproducibility of animal research have been identified, including a lack of scientific rigor, low statistical power, analytical flexibility, and publication bias. However, the reproducibility of a finding depends also on its external validity. Unless a finding is robust against common sources of variation between independent replicate studies, it will not be reproducible. In animal research, effects of experimental treatments can vary depending on the animals’ phenotype, which can vary depending on the animals’ environment. Environmental differences between replicate studies can thus produce conflicting findings. As environmental differences between replicate studies are unavoidable, systematic variation (heterogenization) of the animals’ phenotype rather than rigorous standardization is needed to improve reproducibility. Heterogenization of study populations can be achieved in many ways, including variation in genotype (sex, strain), age, housing condition, or test procedure, by splitting experiments into several replicate batches or by multi-laboratory studies. Using a randomized complete (or incomplete) block design, we can analyze not only whether a treatment has an effect but also whether that effect is robust against environmental variation and thus more likely to be reproducible – without the need for a larger sample size. Incorporating biological variation into study design should thus be the default option for almost all study types of experimental animal research—including exploratory studies. Rigorous standardization to a single genotype, a single sex, and a specific environmental context can only be justified if either the outcome of interest is known to be robust against variation in these factors, or the research question is truly limited to that specific context. In all other cases, systematic heterogenization will be scientifically more valid, and when considering individual studies as parts of research programmes, will also minimize animal use und thus be ethically and economically preferable.

Learning Objectives:

1. Understand the relationship between external validity and reproducibility of results

2. Understand why standardization fails to guarantee reproducibility

3. Ability to explain how heterogenization can be achieved without a need for larger sample sizes

Tuberculosis (TB) is the No.1 cause of death due to a single infectious agent in the world today. According to the global report from World Health Organization, one third of world population is currently infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Approximately 90% of those infected have latent TB and therefore are not infectious and have no TB symptoms; however, the remaining ~10% will develop active tuberculosis at some time during their lifetime, resulting in an estimated 10 million new TB cases and ~2 million deaths worldwide annually. While the successful implementation of DOTS (Directly Observed Treatment, Short-course) strategy has significantly decreased the global TB spread in the past two decades, the development of multidrug-resistant (MDR) and extremely drug-resistant (XDR) TB is now becoming a serious concern in high burden populations. To accelerate the global control of TB and limit the spread of drug-resistant TB, more effective anti-TB vaccines and drug regimens are needed. Additionally, it’s clear that we need a better understanding of the interaction between the M. tuberculosis and its host at the cellular and organism level. To address these questions, suitable experimental animal models of TB are required that mimic the course of human TB as well as reproduce the human body responses to TB vaccines and drug regimens. In this Webinare, we will highlight the different animal models in TB medical research, and discuss the issues around the appropriate choice of animal model in understanding host-pathogen interaction mechanisms during an M. tuberculsis infection, and evaluation of anti-TB vaccines and drugs. 

Animal welfare is the most important issue in any in vivo laboratory. The ability to detect and intervene in cases where the conditions of animals may be deteriorating as well as the ability ensure that undue stress and anxiety are avoided is of paramount importance. Remembering that rodents, despite the fact that they are in the laboratory setting, are social creatures and respond adversely to disruption in their routines through undue handling and or disruption of circadian rhythm is important to ensuring the quality of subsequent in vivo studies in cognition, function and behaviour. It is for this reason that longitudinal monitoring of group housed animals is required. We have shown here salient examples of what occurs when animals are not being directly observed and how animal behaviour can be observed and monitored at in their home cage and at the times when they are most likely to manifest behaviours that are more than likely true to their behavioural phenotype. In addition, we show how grouped housing and minimal handling can be achieved in order to contribute to the 3R ethos as well as impact directly on the quality of the data that is generated from in vivo experiments.

Learning Objectives: 

1. How remote monitoring in the home-cage of grouped housed animal is achieved

2. How this technology can contribute to both animal welfare and the quality of in vivo studies through rapid intervention and earlier recognition of humane end-points

Nonterminal blood collection from mice is frequently performed in biomedical research. Multiple phlebotomy sites and restraint techniques are available to investigators. Methods that minimize pain and distress in mice can improve the accuracy and reproducibility of research data as well as improve animal welfare. Collection site can influence physiologic parameters, and phlebotomy techniques vary in feasibility and the amount of training required. The methods used to compare the impact of various phlebotomy techniques on animal welfare and data quality vary, making it difficult to compare between studies. Additionally, recommendations proposed by individual studies, institutions, and working groups vary. At Cornell, we recently compared phlebotomy via tail tip amputation to the more commonly used techniques of facial vein puncture and tail vein incision to guide our institutional policies (Moore, ES et al., JAALAS. 2017;56(3)). In this presentation, I will provide an overview of recent publications, including a review of our study, evaluating the impact of rodent phlebotomy technique on parameters of pain and distress.

Compassion Fatigue can affect all those that care for and work with laboratory animals, including husbandry staff, veterinary staff, researchers, and other support staff. It is known as the “cost of caring,” and is caused by the connection that we experience with those we are taking care of, and the absorption their pain and suffering. It depletes our emotional resources to constantly care for and about animals, and the effect is heightened with those that form strong bonds with the research animals. The effects of this condition manifest as mental, behavioral, and physical changes, such as apathy, irritability, heightened emotions, isolation, addictive behaviors, and worsened chronic health issues. This is a relatively new topic in the field of laboratory animal science, and information regarding it is not often taught in a basic training course. At the University of Michigan, we have recently started to increase awareness and provide resources and support for our staff to combat compassion fatigue. This includes building a relationship between our Employee Assistance Program counseling group and the Unit for Laboratory Animal Medicine (ULAM), adding language to our required animal user training, creating a Peer Support Group, providing animal memorial boards at our now annual Science of Enrichment Symposium, and creating a committee to plan an animal memorial event. We have received positive feedback regarding these additions, and continue to work to make changes to our program to foster a culture of openness regarding compassion fatigue, and to improve morale by improving work satisfaction. It is our belief that caring for the mental well-being of those that work with research animals helps maintain employees in our field that deeply care about animals, increases job satisfaction, and encourages new ideas to promote increased animal welfare.

Biomedical research suffers from a translation gap, where most treatments that seem effective when tested on animals turn out not to work in human patients. This is likely due in large part to differences in how animal and human experiments are performed, and a key step in overcoming this problem is to treat animals like we treat human patients. This means making their well-being a top priority and giving them control over stressors in their housing and testing environments.

Cognitive tests of learning and memory are typically performed in an operant chamber outside of the home cage. Many of the aversive features of these hand-run tests – handling by experimenters, temporary separation from social companions, and being placed in unfamiliar environments – can be eliminated by testing animals on a voluntary basis, inside their home cages. Automated home cage operant tests further help ensure that animals volunteer to be tested only when appropriately motivated. Properly designed tests that tap into animals’ intrinsic motivations and match the level of cognitive challenge to their level of skill can even be a valuable form of environmental enrichment.

Aversive features and low or fluctuating motivation associated with conventional hand-run operant tests can negatively impact cognitive performance. Automated home cage operant tests may then more accurately assess learning and memory. Large-scale replication studies further suggest that results of these tests are more consistent across different laboratories. By yielding control to experimental animals and allowing them to be tested voluntarily rather than through coercion, automated home cage operant tests may additionally pave the way towards improved translation from animal to human patients. If so, this would serve as a prime example of how good animal welfare can lead to good science, with improved quality of life for experimental animals and accelerated development of treatments for human patients.

The ability to rapidly and accurately identify pain in mice is critical for providing them optimal care and welfare. To meet this need, we developed and validated the Grooming Transfer Test (GTT) and Nest Consolidation Test (NCT). We assessed these novel tests along with electronic von Frey and ambulatory parameters at baseline, after isoflurane anesthesia +/- analgesia, and after laparotomy in adult CD1 and C57BL/6 mice of both sexes, housed singly with or without an existing nest or in pairs. While ambulatory parameters had no and von Frey responses minimal significant changes after surgery, GTT and NCT were significantly altered for 48 hours after surgery in both sexes, strains, and across the various housing conditions. Buprenorphine and carprofen each reduced post-operative pain, however only the combination of the two completely prevented delays in nesting behavior. Therefore, these two novel cage-side methods can be used to quickly and objectively identify mice from a variety of signalments and housing conditions with alleviated and unalleviated postoperative pain. 

Digital technology is improving the speed and accuracy of all the world’s work processes and activities, the including the research vivarium.   This presentation will address some of ways that modernization of animal studies has been occurring, from room monitoring and control systems, to racks and individual cages and, to the use of holding cages as part of the actual study segment.  Using advantages of these  of these technologies in this manner enables researchers to automatically gather and interpret data while allowing facility managers to improve operating efficiency and utilization of resources.   The use of this type of data can be very helpful in resolving concerns about study reproducibility with the goal of improved research outcomes.   We will discuss the value of the technology-enabled animal facilities to the administration, management and investigators.  

Experimental models that promise to replace animal tests that are currently required for drugs and chemicals are rapidly proliferating. This includes a number of novel solutions offered by biomedical engineers that are collectively known as microphysiological systems or tissue chips. These elegant models aim to recreate a tissue or a physiological function using human cells that are usually cultured in 3D structures and under media flow. However, while the advances in biomedical engineering and material science are truly encouraging, there has been a challenge of transferring these technologies from the developer labs to the end-users. One of the barriers to the technology transfer of tissue chips has been a concern that these devices are too difficult to operate as they require specialized equipment, knowledge and experience; hence, the confidence in how well tissue chips perform is yet to be established. To bridge the gap developments in tissue chips and their use for decision making in drug development and chemical safety evaluation, the National Center for Advancing Translational Sciences has funded several Centers to test tissue chips in an independent third-party environment. This presentation will detail the experiences of one such center at Texas A&M University. 

Learning Objectives: 

1. To become familiar with the diversity of current options for the Microphysiological Systems (also known as Tissue Chips)
2. To understand how these microphysiological systems may be tested with respect to reproducibility and technology transfer.