Inspiring Hope: How The 3Rs Framework and the CAM Assay Model Set New Standards in Animal Testing

The growing public concern about the use of animals in research causes scholars to reevaluate and optimize existing experimental models. This has led to the rise of the 3Rs concept in animal testing. Thus, alternative in vivo and in vitro models fully replacing research animals and enhancing research quality and ethics are gaining popularity.

We take a closer look at the use of the 3Rs framework in the context of the chick chorioallantoic membrane assay (CAM). The CAM assay has been widely recognized by the scientific community as a cost-effective and humane alternative to preclinical animal models, used to study tumor growth and metastasis, angiogenesis, toxicity, pharmacokinetics, and mechanism of action of new drug substances.

Find out how advanced AI image analysis software will help you run your CAM lab following the 3Rs principles. 

Understanding the three principles of the 3Rs framework

The Principles of Humane Experimental Technique were introduced by Russell and Burch in 1959. The 3Rs framework consists of 3 main principles for improving the treatment of laboratory animals: Replacement, Reduction, and Refinement. These animal testing alternatives are intended to protect the welfare of animals used in lab experiments while reducing the pain and distress caused to them.

The 3Rs principles deal with ethical constructs like “humanity” and “inhumanity” related to the use of sentient animals for research purposes (Tannenbaum & Bennett, 2015). Moral assessment typically involves a harm-benefit evaluation whereby the harm caused to test animals is measured up against the benefit of a given experiment provided to human society. By doing this evaluation researchers should be able to judge whether alternatives to the animal experiment are available and how opting for one of those will affect the validity of the results (Eggel & Würbel, 2021).

Putting international animal welfare standards into research practice

Research guidelines known as the 3Rs are subject to international legislation on the use of animals in research and are regulated by different legislative bodies.

The protection of animals used for scientific purposes in the EU operates under Directive 2010/63/EU of the European Parliament. Directive 2010/63/EU protects non-human vertebrates including larval and fetal forms,  as well as live cephalopods. It enforces careful benefit-harm assessments before experiments and minimal housing- and care standards for the test animals. The European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes covers aspects like the conduct of experiments and authorization processes. 

In the USA guidelines on the treatment of test animals are regulated under The Animal Welfare Act (AWA). This law sets standards for the handling of animals used in research, teaching, testing, exhibition, transport, and by dealers. Yet, AWA protects only certain mammal species like dogs, cats, primates, hamsters, and guinea pigs, while a significant number of species is not included in the list. Thus, farm animals used for food or fiber (fur, hide, etc.), coldblooded species (amphibians and reptiles), horses not used for research purposes, fish, invertebrates (crustaceans, insects, etc.), rats of the genus Rattus and mice of the genus Mus are not on the list of protected species. Bird species are covered by the AWA, but the regulatory standards regarding their handling are still under development.

Several publications provide extensive guidelines on the use of laboratory animals and humane research practices. These include:

• The Guide for the Care and Use of Laboratory Animals
• The International Guiding Principles for Biomedical Research Involving Animals by CIOMS 
• OECD Principles on Good Laboratory Practice (GLP)
• The NC3Rs resources for researchers on how to use alternative methods 
• The UFAW Handbook on Animal Welfare in Science
• The guidelines of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International)
• The ARRIVE guidelines (Animals in Research: Reporting In Vivo Experiments) 

Special databases and reports dedicated to the utilization of the 3R principles provide helpful information on the use of animals in research, existing 3R alternatives, and guidelines on conducting ethical and humane research. You can consult the following sources to improve the planning and implementation of your experiments:  

• The UFAW Animals in Research Information System (ARIS)
• The EC Summary Report on the statistics on the use of animals for scientific purposes in the Member States of the European Union and Norway
• The ALURES- Animal Use Reporting EU System
• The Office of Laboratory Animal Welfare (OLAW) Guidelines
• The Canadian Council on Animal Care (CCAC) Animal Use Protocol 
• The International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) 
• ALTBIB – Alternatives to Animal Testing 
• Animal Welfare Institute Refinement Database 
• EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM)

After you’ve consulted this documentation, you should be well-equipped to proceed with the planning of your study. 

Embracing 3R techniques for more humane animal research

The 3Rs principles of animal testing provide a sustainable framework for selecting alternative models, minimizing the number of animals used, and refining experimental procedures. Let’s have a look at how each of these principles works.

Key replacement methods transforming animal testing

Replacement refers to avoiding the use of animals in experimentation. There are two alternatives when it comes to animal replacement: absolute and relative replacement.

Some sources even propose using alternative organisms like prokaryotes, different types of bacteria, and fungi for molecular and genetic studies, cellular differentiation, and metabolic regulation research. For example, extracting insulin from pig or cow pancreas has been replaced by bacteria synthesizing this hormone, which offers a sustainable treatment for diabetic patients. More commonly lower vertebrate species e.g. frogs or fish are being used instead of mammals. (Doke & Dhawale, 2015).

Other works suggest the use of some invertebrate species, like Drosophila and nematode worms, assuming they have higher pain thresholds. Yet, how pain perception in invertebrates differs from that in vertebrate organisms is still a matter of debate (Graham & Prescott, 2015; Burell, 2017). 

Here are some examples of replacement with a lower vertebrate, invertebrate species, or fetal form with considering:

• Zebrafish model
• Drosophila model 
• Caenorhabditis elegans assays   
• Artemia salina assays
• Sea urchins assays 
• Hydra attenuata assays   
• Lymnaea stagnalis assay  
• Chick embryo model (CAM assay)  

In vitro and ex-vivo models provide a controlled environment to study cellular response. Such models offer insights into possible outcomes before conducting experiments in vivo (Doke & Dhawale, 2015, Graham & Prescott, 2015). Yet, one significant limitation of these methods is that they cannot fully replicate the complex tissue microenvironment and the workings of the immune system in living organisms In vitro techniques span a variety of approaches (Jean-Quartier et al., 2018; Lee et al., 2017) including:

• Organ-on-chip-models
• Spheroid systems
• Microfluidic systems
• Stem-cell models 
• Transwell models
• Tissue-engineered microvessel models
• Organotypic models
• In chemico assays 
• Cell-based assays 

In silico approaches, also referred to as non-testing methods, are often overlooked, yet they provide a great potential to refine experimental designs and reduce the number of animals used in testing. These models are advanced computational methods based on the analysis of information from past in vitro and in vivo studies (Jean-Quartier et al., 2018).

In silico models can be used to perform various analysis techniques like validation, classification, inference, prediction, and mathematical modeling. Using these methods you can for example predict the biochemical and physiological characteristics of potential therapeutic substances before testing them on living organisms. In silico models include a broad array of computation methods, ranging from simple structure-activity relationships to more complex tissue simulations (Jean-Quartier et al., 2018; Madden et al., 2020):  

• Sequence analysis
• Pathways analysis and network inference
• Pan-cancer analysis
• Chemical perturbation mapping 
• Pharmacogenomic mapping
• Genome-phenotype mapping
• Agent-based models (ABMs)
• Quantitative structure-activity relationship (QSAR) models  
• Machine learning models

Effective reduction strategies in animal testing

Reduction refers to minimizing the number of animals used in a given experiment while obtaining similar quality of information (Graham & Prescott, 2015). This can be achieved by carefully estimating the number of animals needed with statistical methods and thoroughly planning the study design (Doke & Dhawale, 2015).  

Advanced quantitative techniques like power analysis and Bayesian models can help you calculate the needed sample size by determining the minimum number of animals needed for a given experiment. Using non-invasive methods like magnetic resonance imaging (MRI), positron emission tomography (PET), and computer tomography (CT) to gather data allows to ensure the viability of the test animals for longer (Graham & Prescott, 2015). The focus should be on collecting more information with the help of modern technology. Yet, the amount of harm caused to individual animals is an aspect that should not be neglected.   

Minimizing test animal distress with refinement techniques

Refinement guidelines address the handling of animals during the research procedures as well as animal care and husbandry (Hubrecht & Carter, 2019). One central aspect of refinement focuses on minimizing animal pain and suffering during experimentation using adequate anesthesia and analgesia, reducing the duration of the procedures as well as establishing humane endpoints (Graham & Prescott, 2015).

Apart from pain management during and after the procedure, less invasive methods for data collection can be used to reduce animal discomfort: blood sampling, imaging, and behavioral observation. Modern technologies like CT scans, MRIs, ultrasound, and telemetric monitoring play a pivotal role in this regard (Graham & Prescott, 2015; Wachsmuth et al., 2021).  

Other factors in mitigating animal distress are human handling, providing suitable housing conditions, and enabling the animals to express species-specific behaviors (Graham & Prescott, 2015).

How to successfully run a CAM assay laboratory according to the 3Rs principles? 

The CAM assay is a well-established alternative animal model for drug discovery, toxicology testing, safety testing, and efficacy testing as well as for studying tumor formation, metastasis, angiogenesis, cell death, and tissue engineering. It makes use of the extraembryonic membrane (chorioallantoic membrane) of a developing chick embryo to test substance or engraft tissue culture.

The CAM model can be applied both ex-ovo and in-ovo and has been described in existing studies as a cost-effective, efficient in terms of results, and highly reproducible method (Naik et al., 2018). We delve deep into the different possibilities of using the CAM model in alignment with the 3Rs principles to help you responsibly plan animal testing studies.

CAM Assay Animal Replacement Techniques in Focus

In the context of relative replacement strategies, the CAM model turns out to be astonishingly effective. Applying the CAM methodology can successfully replace popular rodent models. At the same time, the use of chicken embryos is less restricted by animal welfare legislation as they are not considered fully developed animals yet (Chen et al., 2021). According to the current EU-legislation experiments on chick embryos do not require authorization provided testing ends before hatching (Ribatti, 2017; Fischer et al., 2022).

On the other hand, the CAM assay proves to be remarkably successful in replacing fully-developed animals in testing as the chick embryo is a living creature with developing organs, an isolated environment, and a circulatory system needed for administering drug substances. Avian embryos don’t have a fully functioning immune system yet, which ensures good biocompatibility. This makes it much easier to transplant tissues from different organisms (Chen et al., 2021; Fischer et al., 2022).

Thus, although it is advantageous during engraftment, the lack of a fully developed immune system in the avian embryos means that follow-up studies involving live animals are still necessary to validate the results (Chen et al., 2021; Fischer et al., 2022). 

The CAM assay is used for the replacement of several mammalian xenograft models for tumor research and drug testing. Thus, it is a viable alternative to experiments using, for example, mice or rabbits. Among the mammal models that can be replaced using the CAM assay (Pratheeshkumar et al., 2012; Lee et al., 2017; Ribatti, 2023) the following ones need to be mentioned:   

• the Draize Rabbit Eye Irritation Test   
• patient-derived xenograft (PDX) models 
• the rat aortic ring assay
• the matrigel plug assay

CAM Assay Animal Reduction Methods Unveiled

Animal reduction in the context of the CAM assay means optimizing the model through careful planning of the experimental design to reduce the number of chick embryos needed. How this will work in your case depends primarily on priorly assessing the trade-offs between embryo viability, ease of access to the CAM, and experiment efficiency (Naik et al., 2018).

Reducing the count of chick embryos typically works by carefully estimating the number needed for your CAM experiment. This depends on the research question you want to explore. Some studies suggest that to ensure the sufficient statistical power of your sample, you should have at least N=10 number of chick embryos in each experimental group (Naik et al., 2018). 

Conventional methods such as observation through a microscope, stereological techniques, and morphometric analysis require the removal of the extraembryonic membrane. Scanning electron microscopy (SEM) is also suitable for ex-vivo methods. Under these circumstances, the embryo is no longer vital (Chen et al., 2021). 

Using non-destructive techniques to capture images of the CAM vasculature such as X-rays, magnetic resonance, acoustic and optical imaging, light and fluorescent microscopy extended with the help of AI microscopy image analysis software secures the viability of the embryos for longer. Non-invasive imaging methods such as intravital microscopy and in-ovo visualization for example allow you to inspect real-time changes in the vasculature of the CAM and produce a large number of images in stacks which are perfectly suited for automated image analysis (Chen et al., 2021; Fischer et al., 2022).

Maintaining sterility during the procedures is vital to the survival of the embryos. Careful cleansing of the instruments must be performed before the intervention. However, using alcohol to clean the surface of the eggshells has been reported to increase embryo mortality. That is why the use of distilled water for this purpose is recommended (Naik et al., 2018).

AI-driven analysis methods provide great assistance in the animal reduction process, as they can help you produce in-depth and reproducible data, improve image quality, and perform complex bioimage analysis tasks like image segmentation and classification (Chen et al., 2021). If you are curious to learn how the automated analysis of CAM images works with the help of AI, have a look at our special case study on quantifying angiogenesis markers.

In the case of in-ovo methods, it is sometimes difficult to access the chorioallantoic membrane or perform some imaging procedures. To ensure better accessibility to the chorioallantoic membrane shell-less culture is often used. This means the incubation of the chicken embryos together with the yolk occurs outside of the eggshell (Dunn, 2023). Yet, the low viability of the chick embryos in this case reduces the reliability of the experiments (Naik et al., 2018).

To solve this issue the CAM-cup method has been proposed as an alternative that ensures a better survival rate of the embryos until later developmental stages. The CAM-cup method involves carefully transferring the chick embryos into plastic cups so that they and the vessels around them don’t get damaged. When it comes to ex-ovo methods (e.g. performed outside of the eggshell) the survivability of the embryos has been reported to be 50%-70%, in the case of the CAM-cup there is a chance of up to 90% survival rate (Naik et al., 2018; Merlos Rodrigo et al., 2021).

In a nutshell, you can reduce the number of embryos required for CAM assay studies through the following strategies:

• careful planning of the experimental design 
• using non-destructive imaging methods  
• ensuring consistent sterility during the procedures
• AI-powered image analysis software 
• applying the CAM-cup method

AI can be especially useful in minimizing the number of test animals as automated image analysis software can help you arrive at results faster and improve the statistical power of your data. We explored together with a Postdoctoral Researcher at the University of Liverpool Sarah Barnett how AI image analysis can enhance the capabilities of the CAM model.

Refining the CAM Assay: Approaches for improving animal welfare

In light of Russel and Burch’s (1959) 3Rs models, the minimizing of animal suffering, pain, and distress needs to be prioritized over human effort during animal testing. Certain steps need to be considered in CAM assay refinement to improve embryo survival rates to later stages of incubation while reducing suffering and distress. Let’s take a closer look at the possibilities of the CAM assay as a refinement model. 

Precise assessment of the incubation time can be used to control the growth of tumors and the degree of embryonic development needed for the experiment (Naik et al., 2018).  During the incubation providing constant temperature and humidity levels as well as agitation are necessary. The eggs need to be turned regularly in the incubator and provided with stimuli such as light and sound. The time point of transplantation also needs to be carefully considered. Some studies suggest that xenografting should be performed on day 9 or 10 of the incubation (Kunz et al., 2019; Merlos Rodrigo et al., 2021). 

One limitation of the CAM model when it comes to refinement strategies is that the point of onset of pain perception in the chick embryo is unclear. As regards the CAM itself, it is an extraembryonic organ and is not innervated. However, there is evidence that prerequisites for pain perception are present in avian embryos already at day 7 of their development (Fischer et al., 2022).

That is why proper pain relief has to be provided during the procedures. Anesthesia and analgesia need to be delivered to the chick embryos during and after experimentation. The use of adequate anesthetic protocols and transparent reporting ensures that the unjustifiable suffering of the test embryos is avoided. Through the close monitoring of the heart rate and movements of the chicken embryos, the onset of sedation can be determined. Another method to assess the effectiveness of sedation is introducing stressors like heat (Horr et al., 2023).

Only very few studies have dealt with anesthesia in the context of the CAM assay. The choice of the appropriate substance plays a central role in this regard. While some anesthetics may perform well on mammals, they are still not well-tested on birds. Medetomidine, Thiopental, Morphine, Xylazine, and Ketamine/Midazolam have been successfully applied to sedate chicken embryos to perform in-situ MRI scans (Waschkies et al., 2015; Horr et al., 2023).

Some protocols suggest the injection of anesthetics directly into the umbilical veins of the chicken embryos, although this procedure has been reported to be quite difficult to perform. It has been suggested that inoculation through the chorioallantoic membrane of the eggs is a much more reliable administration method (Horr et al., 2023).   

A different way to refine the CAM protocol is to make use of minimally invasive methods of tissue engraftment. The ex-ovo CAM assay for example allows for better accessibility as compared to the traditional in-ovo method which causes less distress to the embryo during the procedures. Further, non-invasive imaging methods to assess tumor growth and the perfusion of biomaterials on the CAM like MRI eliminate the need for unnecessary surgical procedures performed on the test embryos (Waschkies et al., 2015).

Another important aspect to consider is making humane decisions about endpoints when the chick embryos need to be sacrificed. Euthanasia of chicken embryos used in the context of the CAM assay has been performed through the injection of pentobarbital Narcoren into the CAM vasculature (Kunz et al., 2019). It is essential to have an exact idea about how long the experimentation should last. The incubation should last long enough to allow for sufficient growth of the embryos, but not for so long that it causes unnecessary suffering.  

Summing up, you can refine CAM assay protocol using these methods:

• planning the necessary incubation time  
• proper care and handling 
• using minimally invasive methods   
• adequate pain management 
• humane endpoints

Streamline CAM image analysis with AI-powered solutions for more responsible research practice

By embracing the CAM assay model you can transform animal testing practices for the better. Cutting-edge AI image analysis technology is your faithful ally in studying images generated in CAM experiments.

With the help of specialized AI bioimage analysis software like the IKOSA CAM Assay Application, you can study vast numbers of CAM images and various aspects of angiogenic growth in just a few clicks. On top of that, AI image analysis technology is highly sensitive to even the tiniest details on the CAM, which will require a smaller number of test embryos to arrive at statistically significant results.

Remember, by reducing the time needed for experimentation and improving the quality of your results, you are making a valuable contribution towards more humane and ethical animal research. Here’s how specialized AI software for analyzing CAM image data will help you:

• Arrive at more precise and replicable results with assistance from AI 
• Identify intricate vascular patterns on the CAM to improve the significance of your findings  
• Speed up analysis and reduce the overall time needed for experimentation by leveraging high-throughput analysis
• Automate estimations about blood vessel count, vessel branching points, vessel area, vessel length, and vessel thickness     
• Track changes to the CAM membrane over time after grafting
• Easily share your analysis results with research fellows  

Our practical use case teaches you how to utilize the IKOSA CAM Assay Application to access the hallmarks of angiogenesis in CAM experimentation.

Our authors:

Benjamin Obexer

Lead content writer, life science professional, and simply a passionate person about technology in healthcare

Elisa Opriessnig

Content writer focused on the technological advancements in healthcare such as digital health literacy and telemedicine.

Fanny Dobrenova

Health communications and marketing expert dedicated to delivering the latest topics in life science technology to healthcare professionals.