Animal Research

Animal research case studies


UCL is a leading centre for biomedical research in the UK. Scientific research is conducted not by shadowy figures in ivory towers, but by human beings working earnestly to address major issues facing society today.

UCL research mouse

Dr Clare Stanford: using mice to find treatments for ADHD

Dr Clare Stanford is a Reader in Experimental Psychopharmacology at UCL. Despite the intimidating title, Clare is a down-to-earth, compassionate researcher with a real commitment to animal welfare. She is chair of the Bloomsbury AWERB and does not hold back from questioning the ethics of research objectives , as well as the way it is carried out.

Clare is currently working on a mouse model for Attention Deficit Hyperactivity Disorder (ADHD). This is a strongly inherited psychiatric disorder, which causes problems for patients by making them hyperactive, excessively impulsive and inattentive. ADHD is often regarded as a childhood issue, but about 65% of people carry it through to adulthood where the associated problems are far worse. It has been associated with alcohol and drug misuse in later life, and an estimated 25% of the prison population have ADHD . There is also an increased risk of other health complications, including asthma and epilepsy.

Picture of 10-day old mice. The glowing mice had firefly genes injected into their brains at birth, designed to respond to different molecular processes important for cell development. The glow is not visible to the naked eye, so the image was taken…

Dr Simon Waddington and Rajvinder Karda: reducing mouse use with glowing firefly genes

Although animal research remains a necessary part of modern research, current methods are far from perfect. By injecting the genes that fireflies use to emit light into newborn mice, UCL scientists have developed a way to drastically reduce the numbers of mice needed for research into disease and development.

At the moment, researchers often need to cull and perform autopsies on animals to see how diseases develop on a molecular level. This means that an animal needs to be killed for every data point recorded, so some studies might use dozens of mice to get reliable data on disease progression.

The new technique could allow researchers to get molecular-level data by simply taking a picture with specialist equipment rather than killing an animal, allowing them to get data more regularly and ethically. An experiment that previously need 60 mice can be done with around 15, and the results are more reliable.


Dr Karin Tuschl: Using zebrafish to treat a rare form of childhood Parkinsonism

Using genetically modified zebrafish, UCL scientists have identified a novel gene affected in a devastating disorder with childhood-onset Parkinsonism. Indeed, when a drug that worked in the fish was given to one of the children, she regained the ability to walk.

The research studied a group of nine children who suffered from severely disabling neurological symptoms including difficulties in walking and talking. Dr Karin Tuschl and her team at the UCL Great Ormond Street Institute of Child Health and UCL Department of Cell and Developmental Biology used state of the art genome editing in zebrafish to validate the identity of the gene affected in these children.

The scientists disrupted a gene known as slc39a14 in the fish, which is important for transporting metals in the body. Disrupting the transporter in fish led to a build up of manganese in the brain and impaired motor behaviour. As similar symptoms were seen in the patients, this confirmed that slc39A14 is required to clear manganese from the body and protect it from manganese toxicity. It also confirmed that the scientists had found the gene causing the disease in the patients.

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Examples of Animal Behavior Research

Examples of Animal Behavior Research

Animal behavior research can involve a wide range of animal species and research subjects. In ethology, animal behavior is studied under natural conditions. Ethology has its roots in the scientific work of Charles Darwin (1809-1882), as well as Dutch biologist Nikolaas Tinbergen (1907-1988).

Ethology combines laboratory and field science, and also has strong relations to other disciplines, such as neuroanatomy, ecology, and evolutionary biology. Researchers in this field of study are interested in understanding the functions, causes, development, and evolution of animal behavior.

In this blog post we describe several examples of animal behavior research. Want to read even more? Please visit our Behavioral Research Blog !

Table of contents

Interactive enrichment for great apes

Behavioral observation of new zoo habitat for elephants, neuroscience research with guinea pigs, domesticated vs wild animals, chicken welfare, spatial behaviors in sheep, a rat model for parkinson's disease, how to monitor rat social behavior.

Zebrafish studies

Studying shrimp feeding behavior.

Observing monkey behavior and their use of tools

We already know some monkeys display above average intelligence. Behavioral studies have shown that capuchin monkeys use tools such as boulders and logs as anvils upon which they can crush nuts. Fragaszy et al. studied a group of wild capuchin monkeys in Brazil, specifically the placement of nuts prior to striking them. An interesting detail: they studied this same behavior in humans as well.

Keep reading:  Observing monkey behavior – cracking the nut

There is growing empirical support demonstrating improved welfare in captive animals when they can exert control over their environment. Research shows that great apes can successfully interact with digital media devices and can demonstrate behavioral changes when presented with digital enrichments.

Nicky Kim-McCormack and her colleagues from Australian National University studied the effects of digital enrichment on animal welfare. They included Seoul Zoo’s six orangutans and four chimpanzees in their study.

Continue reading:  Measuring changes in captive great ape welfare

Orang utang monkey ape food

The Oregon Zoo (Portland, OR, US) houses a herd of 5 Asian elephants. The zoo wanted to improve their welfare with evidence-based approaches. This meant creating a complex new habitat and monitoring the elephants closely by measuring hormone levels, activity, and behavior before, during, and after construction. 

They used The Observer XT behavioral analysis software to monitor the transition of the elephant herd to their new zoo habitat at the Oregon Zoo. This study, recently described in Animals, is a great example illustrating why behavioral observation matters. 

Continue reading: Behavioral research shows how elephants like their new habitat at the Oregon Zoo

oregon zoo elephants 2

Photo: Oregon Zoo

According to Kiera-Nicole Lee and her colleagues, guinea pigs differ from mice and rats, and that just might make them more suitable for some neuroscientific studies. This is due to the fact that results from studies with guinea pigs are more easily translated to humans.

Find out more :  Why guinea pigs are just like us

Guinea pigs on grass

Not many studies have compared the behaviors of wild and domesticated animals. When they do, such as with guinea pigs, they mostly compare adults.

Zipser et al. were curious to find out whether the differences in behavior between domesticated guinea pigs and the wild cavies were also found earlier on, and so they compared these species during the early and late phase of adolescence.

Read more:  How wild cavies and domesticated guinea pigs differ

There are almost 100 million chickens in the Netherlands — that's about 17 times as many chickens as people. Their welfare has improved enormously in the past few years, but there is still plenty of room for improvement. 

In the ChickenStress project, we work with different partners to improve chicken welfare and reduce problems like feather pecking.

Read more:  Research aims of the ChickenStress project

Chicken poultry looking into camera

The study of movement, activity, and behavior is valuable in research on animal health and welfare, specifically in livestock research . Understanding animal behavior in different environments provides insights that can help improve their living conditions.

There is still a lot to learn about the behavioral and social patterns in sheep. At the Aberystwyth University in Wales, UK, researchers used TrackLab for tracking and detailed analysis of spatial behaviors in sheep.

Tracking sheep to learn their behavior .

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Request a demo to obtain real-time insights into the activity, welfare, and health of your livestock.

Parkinson’s disease is a neurodegenerative disorder that affects mobility in a life-changing way. Slow movement, shuffling of the feet, and difficulties initiating movement are all impairments we recognize as typical for this disease.

Researchers Jordi Boix and his colleagues, from the University of Auckland in New Zealand, used two rat models to investigate a number of gait parameters. Their aim was to find resemblance to human symptoms, and to specifically find them early on.

Read more:  Using gait analysis in research on Parkinson's disease

How to measure spatial learning in rodents: 5 proven ways

Spatial learning basically refers to the association or representation of an organism in a three-dimensional environment. If we translate this to basic animal research terms: An animal learning its position in a given space. This task highly relies on visual cues and/or landmarks, whether in humans or animals.

Why is it important and how do we measure it? Here we dive into 5 different behavioral tests that specifically measure spatial learning and memory in rodents.

Monitoring and analyzing the social behavior of group housed rodents is something that many researchers find extremely challenging. It can also be very time consuming. However, including social behavior as part of a phenotypic screen has important benefits, and eventually leads to better translational value of rodent models . During her PhD research, Suzanne Peters developed an automated analysis that allows for the monitoring of socially interacting rats.

Read more:  Into the lab: how to monitor rat social behavior

Aither recent study by Bartal et al drove our curiousity to write this blog about altruistic behavior in rats . This study shows how this type of behavior is neurally linkend to the social functioning of humans. 

two rats white grey close together social

Request a free trial and find out what EthoVision XT can do for your research!

How fruit flies find your food (and mates!)

Those tiny flies that take over your garbage cans in the summer? They are called fruit flies for a reason! They have a fantastic sense of smell. Drosophila melanogaster (or fruit flies) are a popular animal model for researchers , because they have a fairly similar genetic makeup to our human genome, and they are easily manipulated to create genetically different strains. For neuroscientific studies, this makes them a really good model to learn about the effects of genes on behavior.

Find out what researchers discovered about  fruit flies and their smell .

fruit fly on a leaf

The zebrafish has become an important vertebrate model to study (developmental) neurobiology and behavior, not in the least because of recent improvements in transgenic, optogenetic and imaging techniques, and behavioral assays.

More and more studies with zebrafish are conducted. Some examples:

animal research studies examples

Request a free demo for your zebrafish research.

Shrimp is a popular dish, and these crustaceans are a very large part of commercial aquaculture. Especially Pacific white-leg shrimp, as they grow fast and are able to adapt to a wide range of environments. Finding the optimal way to feed shrimp can make or break profitable farming. 

This is why investigating the best feeding protocols has gained more interest lately, and a big part of that is looking at feeding behavior. In fact, understanding the basics of shrimp behavior is a crucial fundament to the refinement of feeding practices. 

Learn more about studying shrimp feeding behavior and why it’s important for aquaculture .

studying shrimp feeding behavior

Using the Observer XT to measure aggressive behavior in Dolphins

Atlantic spotted dolphins (Stenella frontalis) and bottlenose dolphins (Tursiops truncates) are a sympatric species, meaning they are very closely related while also inhabiting the same geographic region, in this case, the northern Bahamas. These species are known to travel and forage together but they also engage in aggressive interactions. 

Volker and Herzing, from the Florida Atlantic University, recently published an article in ‘Animal behavior and cognition’ on aggressive interactions between dolphins . 

Spotted dolphins

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Making the impossible possible – Tracking under water in the dark


Mixing sows: aggression and stress of group housing on first-time sow mother


Bed bug behavior - What smell can tell

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animal research studies examples

5 Horrific Experiments on Animals Occurring Right Now—Help End Them!

Knowing what we know about human biology today, you’d think tormenting another animal for “research” would be inconceivable in laboratories. Ninety percent of basic research, most of which involves animals, fails to lead to treatments for humans. At least 95% of all drugs that are shown to be safe and effective in experiments on animals fail in human trials. The evidence is clear: Other animals are poor models for researching diseases that affect humans.

Yet other primates, along with millions of cats, dogs, mice, rats, rabbits, and other animals, are still trapped in laboratories across the country right now . Experimenters subject them to painful, invasive procedures, confine them to lonely and barren cages, and deny them important social relationships—all for unreliable and useless “research.” Then, after experimenters are done exploiting them, they often kill them as if these sentient beings were just disposable lab equipment.

animal research studies examples

Elisabeth Murray’s ‘Monkey Fright’ Experiments at NIH

National Institutes of Health (NIH) experimenter Elisabeth Murray burns through taxpayers’ dollars to traumatize intelligent, social monkeys . In her twisted “emotional responsiveness” tests , she saws open and injects toxins into monkeys’ skulls to burn brain cells or suctions out parts of their brain. Then, after placing the animals inside a cage with no room to hide or escape, she torments them with their worst fears—a fake but realistic-looking rubber snake or spider— just to see how they’ll react . When she’s through with them, she kills them.

Records obtained by PETA document that in 2019 alone, Murray used 132 monkeys in these experiments. While her cruel tests have caused extreme harm and trauma to  all  the animals she has used, the records specify that 72 of these monkeys were used in experiments that are known to cause pain .

Sepsis Experiments on Mice

Laboratories across the U.S. subject mice to barbaric sepsis tests reminiscent of the horror film The Human Centipede . To induce sepsis in mice, experimenters may stitch mice together along the length of their bodies and inject toxins into them, puncture the animals’ intestines so that fecal matter and accompanying bacteria leak into their stomachs, insert a stent into the animals’ colons so that fecal matter leaks out continuously into their bodies, or use other egregiously cruel methods. This is despite the fact that a landmark 2013 study revealed that the results of sepsis experiments done on mice don’t apply to humans, because sepsis is a different disease in mice.

Urge NIH to Shut Down Murray’s Monkey Torment Lab

Menopause Tests on Marmosets at UMass

A University of Massachusetts (UMass) laboratory performs invasive surgeries on female marmosets in a ridiculous attempt to study menopause —which marmosets don’t even experience —in humans. Experimenters drill holes into the skulls of marmosets, thread electrode leads through their abdomens, and zip-tie them into restraining devices. To simulate menopause in these sensitive monkeys, experimenters surgically remove their ovaries and then use hand warmers on them to mimic hot flashes. UMass abruptly shut down good-faith negotiations with PETA that were aimed at reducing the use of animals in experiments and modernizing its research program.

Urge UMass to Stop Mutilating Marmosets

Brain-Mangling Tests on Owls at Johns Hopkins University

Experimenters at Johns Hopkins University cut into the skulls of barn owls, insert electrodes into their brains, force them to look at screens for hours a day, and bombard them with noises and lights . The laboratory pretends that doing this will tell us something about attention-deficit/hyperactivity disorder in humans.

Urge Johns Hopkins to End Cruel Tests on Owls

Sex Experiments on Sparrows at LSU

At Louisiana State University, experimenter Christine Lattin removes sparrows from their natural homes, pumps them full of sex hormones, releases them, and then torments them with terrifying calls from predators. These sounds can leave the highly social birds—who just want to raise and care for their young—in serious distress. At the end of the breeding season, Lattin recaptures and kills all the birds and their babies and then removes their brains for analysis.

Urge LSU to End Lattin’s Experiments on Sparrows

Help All Animals Suffering in Laboratories

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6 Ways to Help Animals Suffering in Experiments

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Research using animals: an overview

Around half the diseases in the world have no treatment. Understanding how the body works and how diseases progress, and finding cures, vaccines or treatments, can take many years of painstaking work using a wide range of research techniques. There is overwhelming scientific consensus worldwide that some research using animals is still essential for medical progress.

Animal research in the UK is strictly regulated. For more details on the regulations governing research using animals, go to the UK regulations page .

mouse being handled

Why is animal research necessary?

There is overwhelming scientific consensus worldwide that some animals are still needed in order to make medical progress.

Where animals are used in research projects, they are used as part of a range of scientific techniques. These might include human trials, computer modelling, cell culture, statistical techniques, and others. Animals are only used for parts of research where no other techniques can deliver the answer.

A living body is an extraordinarily complex system. You cannot reproduce a beating heart in a test tube or a stroke on a computer. While we know a lot about how a living body works, there is an enormous amount we simply don’t know: the interaction between all the different parts of a living system, from molecules to cells to systems like respiration and circulation, is incredibly complex. Even if we knew how every element worked and interacted with every other element, which we are a long way from understanding, a computer hasn’t been invented that has the power to reproduce all of those complex interactions - while clearly you cannot reproduce them all in a test tube.

While humans are used extensively in Oxford research, there are some things which it is ethically unacceptable to use humans for. There are also variables which you can control in a mouse (like diet, housing, clean air, humidity, temperature, and genetic makeup) that you could not control in human subjects.

Is it morally right to use animals for research?

Most people believe that in order to achieve medical progress that will save and improve lives, perhaps millions of lives, limited and very strictly regulated animal use is justified. That belief is reflected in the law, which allows for animal research only under specific circumstances, and which sets out strict regulations on the use and care of animals. It is right that this continues to be something society discusses and debates, but there has to be an understanding that without animals we can only make very limited progress against diseases like cancer, heart attack, stroke, diabetes, and HIV.

It’s worth noting that animal research benefits animals too: more than half the drugs used by vets were developed originally for human medicine. 

Aren’t animals too different from humans to tell us anything useful?

No. Just by being very complex living, moving organisms they share a huge amount of similarities with humans. Humans and other animals have much more in common than they have differences. Mice share over 90% of their genes with humans. A mouse has the same organs as a human, in the same places, doing the same things. Most of their basic chemistry, cell structure and bodily organisation are the same as ours. Fish and tadpoles share enough characteristics with humans to make them very useful in research. Even flies and worms are used in research extensively and have led to research breakthroughs (though these species are not regulated by the Home Office and are not in the Biomedical Sciences Building).

What does research using animals actually involve?

The sorts of procedures research animals undergo vary, depending on the research. Breeding a genetically modified mouse counts as a procedure and this represents a large proportion of all procedures carried out. So does having an MRI (magnetic resonance imaging) scan, something which is painless and which humans undergo for health checks. In some circumstances, being trained to go through a maze or being trained at a computer game also counts as a procedure. Taking blood or receiving medication are minor procedures that many species of animal can be trained to do voluntarily for a food reward. Surgery accounts for only a small minority of procedures. All of these are examples of procedures that go on in Oxford's Biomedical Sciences Building. 

Mouse pups

How many animals are used?

Figures for 2022 show numbers of animals that completed procedures, as declared to the Home Office using their five categories for the severity of the procedure.

# NHPs - Non Human Primates * Badgers are caught, tagged and released for monitoring in the wild as part the  work of the Wildlife Conservation Research Unit (WildCRU) .

Oxford also maintains breeding colonies to provide animals for use in experiments, reducing the need for unnecessary transportation of animals.

Figures for 2017 show numbers of animals bred for procedures that were killed or died without being used in procedures:

Why must primates be used?

Primates account for under half of one per cent (0.5%) of all animals housed in the Biomedical Sciences Building. They are only used where no other species can deliver the research answer, and we continually seek ways to replace primates with lower orders of animal, to reduce numbers used, and to refine their housing conditions and research procedures to maximise welfare.

However, there are elements of research that can only be carried out using primates because their brains are closer to human brains than mice or rats. They are used at Oxford in vital research into brain diseases like Alzheimer’s and Parkinson’s. Some are used in studies to develop vaccines for HIV and other major infections.

Primate in lab

What is done to primates?

The primates at Oxford spend most of their time in their housing. They are housed in groups with access to play areas where they can groom, forage for food, climb and swing.

Primates at Oxford involved in neuroscience studies would typically spend a couple of hours a day doing behavioural work. This is sitting in front of a computer screen doing learning and memory games for food rewards. No suffering is involved and indeed many of the primates appear to find the games stimulating. They come into the transport cage that takes them to the computer room entirely voluntarily.

After some time (a period of months) demonstrating normal learning and memory through the games, a primate would have surgery to remove a very small amount of brain tissue under anaesthetic. A full course of painkillers is given under veterinary guidance in the same way as any human surgical procedure, and the animals are up and about again within hours, and back with their group within a day. The brain damage is minor and unnoticeable in normal behaviour: the animal interacts normally with its group and exhibits the usual natural behaviours. In order to find out about how a disease affects the brain it is not necessary to induce the equivalent of full-blown disease. Indeed, the more specific and minor the brain area affected, the more focussed and valuable the research findings are.

The primate goes back to behavioural testing with the computers and differences in performance, which become apparent through these carefully designed games, are monitored.

At the end of its life the animal is humanely killed and its brain is studied and compared directly with the brains of deceased human patients. 

Primates at Oxford involved in vaccine studies would simply have a vaccination and then have monthly blood samples taken.

Housing for primates

How many primates does Oxford hold?

* From 2014 the Home Office changed the way in which animals/ procedures were counted. Figures up to and including 2013 were recorded when procedures began. Figures from 2014 are recorded when procedures end.

What’s the difference between ‘total held’ and ‘on procedure’?

Primates (macaques) at Oxford would typically spend a couple of hours a day doing behavioural work, sitting in front of a computer screen doing learning and memory games for food rewards. This is non-invasive and done voluntarily for food rewards and does not count as a procedure. After some time (a period of months) demonstrating normal learning and memory through the games, a primate would have surgery under anaesthetic to remove a very small amount of brain tissue. The primate quickly returns to behavioural testing with the computers, and differences in performance, which become apparent through these carefully designed puzzles, are monitored. A primate which has had this surgery is counted as ‘on procedure’. Both stages are essential for research into understanding brain function which is necessary to develop treatments for conditions including Alzheimer’s, Parkinson’s and schizophrenia.

Why has the overall number held gone down?

Numbers vary year on year depending on the research that is currently undertaken. In general, the University is committed to reducing, replacing and refining animal research.

You say primates account for under 0.5% of animals, so that means you have at least 16,000 animals in the Biomedical Sciences Building in total - is that right?

Numbers change daily so we cannot give a fixed figure, but it is in that order.

Aren’t there alternative research methods?

There are very many non-animal research methods, all of which are used at the University of Oxford and many of which were pioneered here. These include research using humans; computer models and simulations; cell cultures and other in vitro work; statistical modelling; and large-scale epidemiology. Every research project which uses animals will also use other research methods in addition. Wherever possible non-animal research methods are used. For many projects, of course, this will mean no animals are needed at all. For others, there will be an element of the research which is essential for medical progress and for which there is no alternative means of getting the relevant information.

How have humans benefited from research using animals?

As the Department of Health states, research on animals has contributed to almost every medical advance of the last century.

Without animal research, medicine as we know it today wouldn't exist. It has enabled us to find treatments for cancer, antibiotics for infections (which were developed in Oxford laboratories), vaccines to prevent some of the most deadly and debilitating viruses, and surgery for injuries, illnesses and deformities.

Life expectancy in this country has increased, on average, by almost three months for every year of the past century. Within the living memory of many people diseases such as polio, tuberculosis, leukaemia and diphtheria killed or crippled thousands every year. But now, doctors are able to prevent or treat many more diseases or carry out life-saving operations - all thanks to research which at some stage involved animals.

Each year, millions of people in the UK benefit from treatments that have been developed and tested on animals. Animals have been used for the development of blood transfusions, insulin for diabetes, anaesthetics, anticoagulants, antibiotics, heart and lung machines for open heart surgery, hip replacement surgery, transplantation, high blood pressure medication, replacement heart valves, chemotherapy for leukaemia and life support systems for premature babies. More than 50 million prescriptions are written annually for antibiotics. 

We may have used animals in the past to develop medical treatments, but are they really needed in the 21st century?

Yes. While we are committed to reducing, replacing and refining animal research as new techniques make it possible to reduce the number of animals needed, there is overwhelming scientific consensus worldwide that some research using animals is still essential for medical progress. It only forms one element of a whole research programme which will use a range of other techniques to find out whatever possible without animals. Animals would be used for a specific element of the research that cannot be conducted in any alternative way.

How will humans benefit in future?

The development of drugs and medical technologies that help to reduce suffering among humans and animals depends on the carefully regulated use of animals for research. In the 21st century scientists are continuing to work on treatments for cancer, stroke, heart disease, HIV, malaria, tuberculosis, diabetes, neurodegenerative diseases like Alzheimer's and Parkinson’s, and very many more diseases that cause suffering and death. Genetically modified mice play a crucial role in future medical progress as understanding of how genes are involved in illness is constantly increasing. 

Ethical care for research animals


The use of animals in some forms of biomedical research remains essential to the discovery of the causes, diagnoses, and treatment of disease and suffering in humans and in animals., stanford shares the public's concern for laboratory research animals..

Many people have questions about animal testing ethics and the animal testing debate. We take our responsibility for the ethical treatment of animals in medical research very seriously. At Stanford, we emphasize that the humane care of laboratory animals is essential, both ethically and scientifically.  Poor animal care is not good science. If animals are not well-treated, the science and knowledge they produce is not trustworthy and cannot be replicated, an important hallmark of the scientific method .

There are several reasons why the use of animals is critical for biomedical research: 

••  Animals are biologically very similar to humans. In fact, mice share more than 98% DNA with us!

••  Animals are susceptible to many of the same health problems as humans – cancer, diabetes, heart disease, etc.

••  With a shorter life cycle than humans, animal models can be studied throughout their whole life span and across several generations, a critical element in understanding how a disease processes and how it interacts with a whole, living biological system.

The ethics of animal experimentation

Nothing so far has been discovered that can be a substitute for the complex functions of a living, breathing, whole-organ system with pulmonary and circulatory structures like those in humans. Until such a discovery, animals must continue to play a critical role in helping researchers test potential new drugs and medical treatments for effectiveness and safety, and in identifying any undesired or dangerous side effects, such as infertility, birth defects, liver damage, toxicity, or cancer-causing potential.

U.S. federal laws require that non-human animal research occur to show the safety and efficacy of new treatments before any human research will be allowed to be conducted.  Not only do we humans benefit from this research and testing, but hundreds of drugs and treatments developed for human use are now routinely used in veterinary clinics as well, helping animals live longer, healthier lives.

It is important to stress that 95% of all animals necessary for biomedical research in the United States are rodents – rats and mice especially bred for laboratory use – and that animals are only one part of the larger process of biomedical research.

Our researchers are strong supporters of animal welfare and view their work with animals in biomedical research as a privilege.

Stanford researchers are obligated to ensure the well-being of all animals in their care..

Stanford researchers are obligated to ensure the well-being of animals in their care, in strict adherence to the highest standards, and in accordance with federal and state laws, regulatory guidelines, and humane principles. They are also obligated to continuously update their animal-care practices based on the newest information and findings in the fields of laboratory animal care and husbandry.  

Researchers requesting use of animal models at Stanford must have their research proposals reviewed by a federally mandated committee that includes two independent community members.  It is only with this committee’s approval that research can begin. We at Stanford are dedicated to refining, reducing, and replacing animals in research whenever possible, and to using alternative methods (cell and tissue cultures, computer simulations, etc.) instead of or before animal studies are ever conducted.

brown mouse on blue gloved hand

Organizations and Resources

There are many outreach and advocacy organizations in the field of biomedical research.

Two researchers in lab looking through microscopes

Stanford Discoveries

What are the benefits of using animals in research? Stanford researchers have made many important human and animal life-saving discoveries through their work. 

Small brown mouse - Stanford research animal

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Animal Study Proposal


The use of this sample animal study proposal is not required and is provided for the convenience of Institutional Animal Care and Use Committees (IACUC) at Assured institutions. Sections may be added, deleted, or modified to meet the needs of individual programs.

The sample animal study proposal is provided in response to requests from many institutions that wish to develop or revise an animal care and use protocol form intended for internal institutional use. It is based on a form used by intramural NIH investigators, and was modified as the result of review of many different extramural institutional forms in order to anticipate a variety of research scenarios. Institutions may download the form and modify it to suit their own institutional program and needs.

Most institutions have instituted an animal care and use protocol form that investigators are required to complete and submit to the IACUC. There is great variation in the length, format, content, and use of these forms, and a form that serves one institution well may not necessarily prove successful at another institution. However, in general, many IACUCs have found that use of a protocol form helps research investigators to delineate the information that the IACUC requires in order to review a proposal, and also helps the IACUC to achieve greater consistency in its review. Many of these forms are available from institutional websites.

We are interested in your comments on the content of this sample animal study proposal and in your suggestions for additions, deletions, or revisions. We anticipate changes to this document as institutional comments are received and as animal research and the policies that govern it evolve. Comments should be sent to: [email protected] .

Individuals not familiar with the PHS Policy are encouraged to visit the PHS Policy Tutorial .

View the animal study proposal

Download the sample animal study proposal

If you wish to use the sample animal study proposal as a template, click one of the formats below to download.

PDF (89 KB) MS Word (123 KB)

Contact the Division of Policy and Education by phone at 301-496-7163 or e-mail to [email protected] .

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Every year, hundreds of millions of animals, including dogs, cats, horses, monkeys, rabbits, mice, and rats, live in factory farm conditions and endure pain and suffering in laboratory experiments.

But as well as being cruel, animal testing is fundamentally flawed, due to species differences – the way that animal species are physiologically different and respond in different ways substances. Substances that are safe in monkeys or mice can be lethal to humans.

Here are seven times when animal testing has led to confusion and failure:

An anti-inflammatory drug, used to treat pain from arthritis, had been tested safe at least eight different animal studies , with species including African green monkeys.  But the effects in humans were catastrophic, causing a huge number of cardiovascular problems. It has been reported that 88-140,000 extra heart attacks may have been caused by Vioxx in the five years following its introduction.   None of these effects were seen in animals. Vioxx is estimated to have killed 60,000 people.

2. Tamoxifen

Tamoxifen is one of the oldest and most prescribed breast cancer drugs.  But it was originally patented as an oral contraceptive – which it is in rats. Unfortunately, in women it has completely the opposite effect , actually making them more fertile!  Today it is used as hormone therapy in breast cancer patients, acting as an anti-oestrogen, intercepting the hormone before it reaches its active site to promote cancer growth. In mice – the most commonly used animal in cancer research – Tamoxifen has the opposite effect behaving as an oestrogen. In experiments on rats, it has caused liver cancer ! Despite all of this confusion, human clinical studies ensured its successful use in humans.

3. Blood transfusions delayed over 200 years

Animal experiments took place as long ago as the 1600s , but led to blood transfusions from animals to people and deaths of recipients. Test tube methods, however, discovered that blood took up oxygen and identified certain salts that could prevent clotting. The 19 th century saw animal experiments spreading more confusion, including the that the addition of sodium citrate to prevent clotting was unsafe. Years later, in 1914, clinical researchers studying human blood in the test tube revealed this to be false, laying the foundations of the modern blood bank. While animal experiments had made blood transfusions appear lethal, it was the clinical discovery of the blood groups that made it safe.  All of the key discoveries were made either in clinical studies or using test tube methods and blood transfusions have gone on to save millions of lives.

4. Fialuridine

A clinical trial for a new Hepatitis B vaccine ended in tragedy when patients developed severe toxicity, with five people out of the 15 in the trial dying . Two others were likely saved only by having liver transplants. The vaccine was tested on dogs, rats, and monkeys , who did not suffer any similar effects.  

The heart drug Eraldin was thoroughly studied in animals and satisfied the regulatory authorities’ requirements. However, when humans began taking the drug, thousands became seriously ill, experiencing severe effects such as growths, stomach troubles, joint pain, and even blindness. None of these effects were detected in the animal trials. The drug ended up being withdrawn from the market.

Avandia, a diabetes drug, was suspended across Europe after data showed that its benefits no longer outweighed the risks . About 90,000 patients in Britain alone had been taking the drug, but there has been growing evidence of an increased risk of heart attack or stroke. These effects were not shown when Avandia was tested on rats – in fact, it had the opposite effect , protecting their hearts from damage.

TGN1412 was an experimental drug that caused human volunteers to suffer serious, permanent, and life-threatening damage within two hours of taking it. Prior to the human trials, the drug was tested in monkeys at 500 times the human dose; the monkeys did not experience the side effects suffered by the humans. Several studies regarding TGN1412 have highlighted the crucial differences between the human and monkey immune systems.

Ethical Alternatives Are the Way To Go

Animal experimenters try to lay claim to almost every medical advance, yet animal research represents on a fraction of medical research, and there is a long history of clinical medical discoveries without animal experiments, including life-saving drugs, surgical techniques, and causes of disease – studying people, their environment and lifestyle, showed the breakthrough links between smoking and cancer, and causes of heart disease.

Animal testing is an unreliable and misleading way to predict outcomes in human beings.  There are numerous alternatives available , that provide data of direct relevance to our species , including human tissue models, bioprinting 3D organ models, using computer modeling, and, of course, using modern imaging and other techniques to study human patients.

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244 Awesome Animal Topics for Research Papers

animal topics for research papers

So, did your professor just asked you to write an exceptional animals research paper? You may think that it is an easy assignment, but it may not be. Don’t wait until the last possible moment to write this essay because you may not be able to do a good job on it. Even though you know how to write the paper, there is another problem you need to take into consideration: finding a great topic.

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The 10 Most Inhumane and Bizarre Animal Experiments in History

“It’s alive! It’s alive!” screamed Dr. Frankenstein, as his bold experiment to bring the dead back to life succeeded, much to his ultimate dismay. As we know, tragedy ensued, but it couldn’t be helped, right? Human beings are a curious lot, always attempting to unlock the door to the unknown, searching for the answers we believe are lurking behind it. This innate desire to discover has often led us to the brink of both wonder and despair. It has also led us, much like the good Dr. F, to do some bizarre things, many of them quite brutal and inhumane. Many of our experiments are conducted not on dead corpses, but on living, feeling animals. Here are 10 of the strangest animal experiments human beings have ever conducted:

1. The Great American Monkey Head Transplant

Perhaps trying to pave the way towards a future when human heads and bodies are interchangeable, in 1970, neuroscientist Robert White took the decapitated head of a monkey and attached it to the decapitated body of another monkey. You might say that the experiment was successful, in a way. The monkey head did actually wake up, and, not in a good mood, due perhaps to its unfortunate predicament, tried to bite one of the members of White’s team. The poor creature managed to stay alive for a day and a half, although it was unable to move its body (which would have involved attaching the brain to the spinal cord, a procedure still out of our skill set). White, who died in 2010, never tried his methods on a human subject, although some speculated he had hoped to transplant the head of Stephen Hawking or Christopher Reeve onto a healthy body. Some scientists today believe the head transplant scenario is inevitable before the end of the century.

2. Two-Headed Dog

Dog head transplants predated monkey head transplants by several decades; American Charles Guthrie accomplished the feat in the early 1900s (score another one for good old American know-how!). It was during the Cold War, however, when dog head transplantation reached its zenith, and it was the Russians who took home the gold. Vladimir Demikhov, a Soviet scientist, performed twenty such operations on canine subjects and perfected the art of dog head grafting. Demikhov would graft the head and front paws of a puppy on to the neck of an adult dog. There are fascinating/horrid/heartbreaking videos of the experiments that show the two-headed pets drinking milk, the adult looking fairly miserable, the puppy seemingly more adjusted to its lot in life. One of the experimental duos actually lived for a month afterwards.

Here's the disturbing video:

3. Frankencat         

Nineteeth-century German scientist Karl August Weinhold was of the belief that the brain was more or less a battery, and the spinal cord akin to wires connecting the battery to the rest of the body. To prove his point, Weinhold conducted an experiment on an unsuspecting kitten. Weinhold basically scooped out the feline’s brain and spinal cord, waited for it to die, and then replaced the brain and spinal cord with an amalgam of zinc and silver. Then, Dr. Frankenstein-like, he sent a charge of electricity into the amalgam, and lo and behold, the cat, according to Weinhold, “got into such a life-tension that it raised its head, opened its eyes . . . finally got up with obvious effort, hopped around, and sank down exhausted.” A year after this cruel experiment, in 1818, Mary Shelley’s “Frankenstein” was published.

4. Charlotte’s Urine Web

Bugs don’t get a free pass when it comes to animal experiments. In 1948, a pharmaceutical researcher Peter Witt discovered that spiders build crazily-shaped webs when under the influence of certain drugs.

Some Swiss scientists took that information and ran with it. These scientists were aware that when healthy people were under the influence of psychedelic drugs like LSD and mescaline, the subjects exhibited symptoms similar to schizophrenic patients. Extrapolating from that, they theorized that schizophrenics were basically on a permanent high, their bodies producing LSD-like chemicals that kept their brains off-kilter. If that were true, then the scientists wanted to find out what that chemical was. They chose the urine of schizophrenics as the medium to study, “so that we ‘d never be stuck for large quantities to work on.” Was there some substance in the urine that carried schizophrenia? To find out, they concentrated the urine down and fed it to spiders. Their reasoning was that if the spiders produced strange, hallucinogenic webs, then there was indeed something to their theory. As a control group, they fed healthy urine concentrate to a second set of spiders. To their dismay, the scientists found no discernible difference between the webs of the two spider groups. What they did discover was that urine concentrate, “must taste extremely unpleasant, despite all the sugar that was added... After taking just a sip, the spiders exhibited a marked abhorrence for any further contact with this solution; they left the web, rubbed any residual drops off on the wooden frame, only returned to the web after having given their pedipalps and mouthparts a thorough cleaning, and could scarcely be persuaded to take another drop of the stuff.”

And this is why science is endlessly amazing.

5. Elephants Never Forget 

Spiders weren’t the only recipients of psychedelic drugs. In 1962, Oklahoma scientists Louis Jolyon West and Chester M. Pierce were curious what would happen if an elephant were tripping. They convinced a local zoo to volunteer an elephant by the name of Tusko to participate in the study. Preparing a syringe containing 297 milligrams of LSD (3000 times a normal human dose), the zoo director shot it into the unsuspecting Tusko. What would happen, they wondered? The scientists got their answer quickly. "Five minutes after the injection he trumpeted, collapsed, fell heavily onto his right side, defecated, and went into status epilepticus . The limbs on the left side were hyperextended and held stiffly out from the body; the limbs on the right side were drawn up in partial flexion; there were tremors throughout. The eyes were closed and showed a spasm of the orbicularis occuli; the eyeballs were turned sharply to the left, with markedly dilated pupils. The mouth was open, but breathing was extremely labored and stertorous, giving the impression of high respiratory obstruction due to laryngeal spasm. The tongue, which had been bitten, was cyanotic. The picture was that of a tonic left-sided seizure in which, mild clonic movements were present." Tusko died an hour and forty minutes later, despite efforts to revive him. The results published in a journal four months later informed readers that elephants are highly sensitive to LSD… Who would have thought?

6. Dogs Have No Souls

In the early 20th century, Duncan MacDougall decided to mix a little religion into his science experiments. Specifically, he decided to demonstrate that the soul had weight. MacDougall headed to an old age home where he procured the “services” of six patients dying of tuberculosis. Being able to easily identify the last hours of a tubercular victim, MacDougall placed the patient on a table scale as they were about to expire and weighed them before death and then at the moment of death. The first patient who died lost a total of 21 grams. The remaining five patients lost varied amounts of mass, but the 21 grams number stuck. MacDougall proceeded to then “volunteer” fifteen dogs to repeat the experiment. Since it was more difficult to predict the moment of a dog’s death, MacDougall basically weighed them, killed them, and then weighed them. The poor pooches apparently displayed no loss of weight before and after. MacDougall’s conclusion? People have souls that weigh 21 grams, dogs have no souls. The New York Times even ran a story about MacDougall’s experiments ("Soul has Weight, Physician Thinks"). Critics quickly pointed out that only one patient lost the 21 grams, that the others were all over the map, and that the weight loss could be accounted for by the patient perspiring at the moment of death since the lungs were no longer cooling the blood. Dogs, having no sweat glands, do not perspire.

7. The Walking Dead Dogs

Tearing another page out of the Mary Shelley playbook, Robert Cornish, a University of California researcher in the 1930s, decided to try his luck at the “reviving-the-dead” game. Cornish was convinced that, as long as major organ damage was limited, he could bring the dead back to life. His unwitting volunteers were four fox terriers. Perhaps displaying a streak of the messianic, he named all four terriers Lazurus, after the biblical character who rose from the dead after Jesus’ touch. Of course you have to die in order to be brought back to life, so the four Lazuri were all asphyxiated. Their blood was manually pumped to continue blood circulation, and Cornish then injected adrenaline and anticoagulants into their systems. Two of the Lazurus pups failed to live up to their name. Two, however, did indeed revive, with a couple of side effects. They were blind and severely brain-damaged. They did live for a few months after, though reportedly their presence terrified other dogs around them. Cornish continued his experiments, although he was kicked off the campus when word got out of his macabre doings. In 1947, he tried to get a human subject to revive, and actually got a death row inmate, scheduled for execution, to volunteer. Prison officials, however, fearful that if he was revived after execution, they would have to release him, promptly un-volunteered him. Cornish gave up his death-defying activities and turned to a life as a toothpaste salesman. 

8. Where’s the Rest of Me?

In 1928, a Soviet scientist, Sergei Brukhonenko, was the developer of the autoinjector, a primitive heart-lung machine. Seeking to prove it was possible to keep a disembodied head alive by circulating blood through it, he proceeded to disembody a head. Hooking the unfortunate canine paricipant up to his machine, he then filmed the head. He shined a light in its eyes and it blinked. He pounded the table and it flinched. He fed it cheese and it came out the other end of its esophagus. The veracity of Brukhonenko’s experiment has been questioned (some claiming it was staged), and the results have never independently been confirmed. Still, his work, while distasteful, contributed greatly to subsequent work on organ transplant and open heart surgery, and he was awarded the Lenin Prize by the Soviet Union after his death in 1960.

9. Rats and Coke and Ludwig and Miles

Three years ago, some researchers at the Albany Medical College decided to find out about the musical taste of rats. They were studying the link between pharmacology and neurology. They subjected some rats to a recording of Miles Davis’ “Four”, playing it on a loop for ninety minutes straight. After an hour and a half of Miles, they switched the recording to Beethoven’s “Fur Elise”, again for ninety minutes. When they switched off the music, the rats were happier with silence, but when forced to a choose, they chose the Beethoven over Miles. Then the researchers added a wild card. They fed the rats some cocaine, got them feeling good, and gave them the choice again. Lo and behold, Miles Davis became the rats’ choice of music when under the influence. Smooth...

10. Planet of the Ape Men

In 1927, Soviet scientist Il’ya Ivanov set out for Africa with an interesting objective: to capture a bunch of female chimpanzees, inseminate them with the sperm of (presumably) Soviet men, and create a man-ape hybrid. It didn’t work. Undaunted, Ivanov decided to switch it up. He returned to the motherland with a plan to inseminate female humans with ape sperm. He brought an orangutan (he named Tarzan) back with him as his donor. Ivanov actually got a few female volunteers to carry the proposed ape-child, but, sadly for science, before the experiment could be carried out, Tarzan died. Ivanov himself fell under suspicion by the Stalin-esque state, and was imprisoned in 1930. He died in a labor camp in 1932. His work was not totally without merit, however. Horse breeders used many of the insemination techniques Ivanov developed.

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Animal Research Sample Size Calculation (and Consequences)

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1. nature collection: statistics for biologists ., 2. krzywinski m, altman n. points of significance: power and sample size. nat methods. 2013; 10:1139-1140 ., 3. button ks, ioannidis jp, mokrysz c, nosek ba, flint j, robinson es, munafò mr. power failure: why small sample size undermines the reliability of neuroscience. nat rev neurosci. 2013 may;14(5):365-76 ., 4. dumas-mallet e, button ks, boraud t, gonon f, munafò mr. low statistical power in biomedical science: a review of three human research domains. r soc open sci. 2017 feb 1;4(2):160254 ..

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Animal studies in psychology

Undergraduates sometimes ask what the value of animal research is in psychology. The study of nonhuman animals has actually played a huge role in psychology, and it continues to do so today. If you’ve taken an introductory psychology class, then you have probably read about seminal psychological research that was done with animals: Skinner’s rats, Pavlov’s dogs, Harlow’s monkeys. Unfortunately, many introductory textbooks don’t give the full picture of animal research. Studies are often described without specifying that they were animal studies. When human studies are presented, there is rarely discussion of the basic animal research that enabled those studies to be done. Finally, information regarding the ethical and regulatory environments in which animal research is conducted is covered in a superficial manner or omitted altogether. These are important issues that deserve better understanding and broader discussion.

Why Nonhuman Animals are Studied in Psychology

Part of the justification for why nonhuman animals are studied in psychology has to do with the fact of evolution. Humans share common ancestry with the species most commonly studied in psychology: mice, rats, monkeys. To be sure, each species has its own specializations that enable it to fit into its unique ecological niche; but common ancestry results in structural (e.g., brain) and functional (e.g., memory) processes that are remarkably similar between humans and nonhumans. In addition, we can better understand fundamental processes because of the precise control enabled by animal research (e.g., living environments, experimental conditions, etc.). We can also ask and answer certain questions that would be difficult or impossible to do with humans. For example, we know what the connections are between the amygdala and other brain regions, but how does activity in the amygdala affect brain functioning? Using a new technique, it is now possible to temporarily inactivate the amygdala in a monkey and see how other brain areas (including those that are not directly connected to the amygdala) change their activity (Grayson et al., 2016). A study such as this not only helps us better understand how the brain works, but it also has enormous potential for developing treatments for people who have abnormal patterns of brain activity, such as those with epilepsy or Parkinson’s disease. Ten years from now, students may very well read in their textbooks about a “new treatment” to help people with Parkinson’s disease. Will this monkey study, which enabled such a discovery to be made, be described? Probably not, in much the same way that nonhuman research that permitted a significant human study to be conducted is rarely described in today’s textbooks.

Weighing Harm and Benefit

Researchers who study nonhumans recognize that their studies may involve certain harms that can range from the relatively minor (e.g., drawing a blood sample) to the more serious (e.g., neurosurgery). The research community tries to mitigate some of the harms by insuring, for example, that the animals’ psychological well-being is optimized; in fact, there is a large body of psychological research that focuses on animal welfare and identifying best practices to house and care for animals in captivity. Still, some harms will remain, and ethically, one must weigh those harms against the potential benefits (for humans and for the animals themselves) to be obtained from the research. Equally important is the consideration of the potential harms to humans of not doing the research. For example, without any animal research, effective treatments for human conditions like Alzheimer’s disease may very well be found, but it would certainly take decades longer to find them, and in the meantime, millions and millions of additional people would suffer.

Regulations for Animal Research

Finally, it’s important to note that animal research in the United States is very tightly regulated by a series of federal and state laws, policies and regulations, dating back to the landmark Animal Welfare Act from 1966. Oversight and inspection of facilities is provided by the U.S. Dept. of Agriculture, and, at the local level by Institutional Animal Care and Use Committees (IACUCs). Even procedures as simple as drawing a blood sample or testing an animal on a cognitive task must be approved by the local IACUC before the work can begin. Part of that approval process requires the scientist to identify whether there might be less invasive ways to do the same thing. In addition, the scientist must justify the numbers of animals that they use, insuring they are using the smallest number possible.

Animal research continues to play a vital role in psychology, enabling discoveries of basic psychological and physiological processes that are important for living healthy lives. You can learn more about some of this research, as well as the ethical and regulatory issues that are involved, by consulting online resources such as Speaking of Research . 

Grayson D.S., Bliss-Moreau E., Machado C.J., Bennett J., Shen K., Grant K.A., Fair D.A., Amaral, D.G. The rhesus monkey connectome predicts disrupted functional networks resulting from pharmacogenetic inactivation of the amygdala. Neuron . 2016 Jul 20;91(2):453-66. 

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animal research studies examples

Experimental design for animal research: proposal examples

An outline of examples to show the level of detail and type of information that the Medical Research Council (MRC) is looking for in grant proposals.

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Examples of justifications for experimental design and animal number in grant applications (PDF)

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animal research studies examples

Current ethical issues in animal research

The use of animals in research is a matter of substantial public interest and can generate impassioned debate which includes the ethics of using animals for experimentation. dominic wells reviews specific ethical issues in the scientific use of animals and puts the debate into context..

Dominic Wells Royal Veterinary College, UK


Ethics can be defined as a framework in which moral decisions (what is right or wrong) can be made. There are two main schools of thought: Consequential (utilitarian) or Deontological (intrinsic).

Within the animal rights movement two of the best-known philosophers are examples of these different schools of thought. Peter Singer is a utilitarian ethicist who argues that there is no valid reason for separating man from all the other animals, which he calls a speciesist view with close similarities to racism and sexism. Consequently animals have rights in a similar way to man. His seminal book, Animal Liberation , was published in 1975 (1) and he is regarded by many as the founding father of the animal rights movement. However, while animals have similar rights to man, the rights of the individual can in some cases be subsumed for the greater good, although this requires a very clear cost–benefit analysis. In contrast, Tom Reagan is a deontological ethicist who argues animals have intrinsic worth and rejects the concept that the ends can justify the means. Consequently animals have intrinsic value as do humans: for example, this argument is presented in (2). Thus, in this school of thought, the use of animals in research can never be justified.

Interestingly, the earliest clear statement on the ethics of animal experimentation occurred at the time of the debate about the rights of man. In his 1789 Introduction to the Principles of Morals and Legislation (3), the utilitarian philosopher Jeremy Bentham queried the use and abuse of animals. He wrote: “The question is not, Can they reason? nor, Can they talk? but, Can they suffer?”. It should be noted that Bentham had no fundamental objection to animal experiments provided that the goal was of benefit to humanity and that there was a reasonable prospect of achieving that goal.

In Animal Liberation (1), Singer codified the concept of animal rights in the context of human rights as: “Animal rights means that animals deserve certain kinds of consideration – consideration of what is in their own best interests regardless of whether they are cute, useful to humans, or an endangered species and regardless of whether any human cares about them at all (just as a mentally-challenged human has rights even if he or she is not cute or useful or even if everyone dislikes him or her). It means recognizing that animals are not ours to use – for food, clothing, entertainment, or experimentation”.

How do we relate these ethical views to the use of animals in research? Our attitude to ethical questions in animal research stems from the relationship of human society with all animals. Animals are used for food, transport and entertainment as well as research. In many societies ill-treatment of animals is not accepted, although this is by no means universal. Thus, in general we take a modified utilitarian attitude – ‘the end can justify the means’ or ‘the greatest good of the greatest number’, but crucially with humans given a greater worth than any other species – the speciesist view disparaged by Singer.

We seek to minimise the cost of the means to justify the end by minimising pain, suffering, distress and lasting harm in experimental animals. Thus, we aim to reduce the number of animals used in experiments to a minimum. We strive to refine the way experiments are carried out, to make sure animals suffer as little as possible. And we replace animal experiments with non-animal techniques wherever possible. These key tenets of humane experimental use of animals, often referred to as the 3Rs, were developed by Russell and Burch in their highly influential 1959 publication The Principles of Humane Experimental Technique .

animal research studies examples

The current Animals (Scientific Procedures) Act 1986 (4) relies on this modified utilitarian ethical judgement. The revised version that will come into force in January 2013, which incorporates changes associated with Directive 2010/63/EU, will continue the same approach. Each project must be assessed on a cost–benefit basis, by asking the question of whether the ends justify the means. Experimental design should aim to reduce the costs (by application of the 3Rs) and critically evaluate the likely benefits. A strong case needs to be made that the studies are necessary and that the experimental aims are well defined and are likely to yield clear answers. The benefits may be for humans and/or other animals but there is a clear hierarchy, with no protection for invertebrate animals other than octopus and with cats, dogs, horses and primates being given special status of greater protection compared with other non-human mammals.

animal research studies examples

Genetically modified (GM) mice raise additional ethical questions. GM animals are the most rapidly growing element of animal use with more than 1.6 million GM animals and harmful mutants bred in the UK without other manipulations in 2011 (5) and this trend appears likely to continue to increase. It has been argued that GM violates the integrity of the organism’s genome. This is of course unacceptable in the deontological and questionable from the strict utilitarian view. However, the modified utilitarian view would argue that, in the absence of a harmful phenotype, there is no difference from wild-type in terms of the welfare of the animals, i.e. the animal is unaware that its genome has been modified.

Other human uses of animal

It is reasonable to ask why there is so much focus on animal experiments. Much of this may be due to the lack of public understanding of other uses of animals. The use of shock tactics of antivivisectionists and the ‘Yuk factor’ of some of the images used are partly responsible for the exaggerated emphasis on animal experimentation. There are many non-experimental uses of animals, for example, as food, clothing, transport, pets, sport and exhibition. The numbers used in non-experimental activities are huge. The UK uses 3.6 million animals in research annually (78% rodents, 15% fish) but UK meat and fish eaters consume 2.5 billion animals every year (6). This is nearly 700 times the numbers used in research yet it could be argued that consumption of fish and meat is not essential for human wellbeing, whereas at least some of the animal research is essential. Both utilitarian and intrinsic ethical arguments would suggest this use of animals for meat is the more important problem that should be tackled ahead of the use of animals in research. This disparity between animals used for food and research is even greater when considered on a world-wide basis. It has been estimated that 140 billion animals are killed for food every year (3000 times the number estimated for use in research worldwide). While the slaughter of domestic mammals and birds may in many cases be reasonably humane, that cannot be said of most of the 90 billion fish killed worldwide each year, where suffocation is the most common cause of death.

Recreational uses of animals should also be considered in comparison with the use of animals in research. Fishing for game or coarse fish is a very popular pastime in the UK but, although it gives pleasure to many, it does not have major consequences in terms of human health. There is little doubt that fish feel pain and respond to it and so recreational fishing is less ethically justified than the use of fish in research. Sport involving animals often has a high attrition rate. As mentioned previously, horses receive special protection under ASPA legislation yet almost 50% of thoroughbred foals do not reach flat race training in the UK (7), as many suffer tendon injuries and fractures that impair their ability to perform. Again, the utilitarian argument would suggest that horse racing was ethically less acceptable than the use of horses in experimental research. Very large numbers of animals are kept as pets and this is not without ethical consequences. For example, based on a survey of over 600 cat owners (7) it can be estimated that cats kill over 220 million vertebrate wild animals per year in the UK, the majority of them being small mammals. This is 60 times the number used in research. So decreasing the cat population, or keeping them indoors on a permanent basis, would have a greater impact on the loss of life than reducing the numbers of animals used in research, but is keeping a cat indoors for life infringing its rights?

What is the ethical way forward? Both Singer and Regan argue that we should not eat meat or fish or use animals in any way that cause them harm. So we should all be vegetarian and limit our harmful interactions with animals. That is philosophically an entirely reasonable approach. However, given our current modified utilitarian (speciesist) use of animals in non-research areas, much of the ethical debate about the use of animals in research is redundant.

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The ethics of animal experimentation.

Many medical research institutions make use of non-human animals as test subjects. Animals may be subject to experimentation or modified into conditions useful for gaining knowledge about human disease or for testing potential human treatments. Because animals as distant from humans as mice and rats share many physiological and genetic similarities with humans, animal experimentation can be tremendously helpful for furthering medical science.

However, there is an ongoing debate about the ethics of animal experimentation. Some people argue that all animal experimentation should end because it is wrong to treat animals merely as tools for furthering knowledge. According to this point of view, an animal should have as much right as a human being to live out a full life, free of pain and suffering. Others argue that while it is wrong to unnecessarily abuse animals, animal experimentation must continue because of the enormous scientific resource that animal models provide. Proponents of continued animal experimentation often also point out that progress can still be made to improve the conditions of laboratory animals and they fully support efforts to improve living conditions in laboratories, to use anesthesia appropriately, and to require trained personnel to handle animals.

On closer scrutiny, there exists a wide range of positions on the debate over the ethics of animal testing. The two views mentioned above represent two common positions at the opposing ends of the spectrum. Others endorse a view closer to the middle of the spectrum. Usually, this middle view accepts experimentation on some, but not all, animals and aims to avoid unnecessary use of animals in scientific research by pursuing alternatives to animal testing.

The following sections briefly outline a few of the arguments for and against animal experimentation. They do not represent every possible argument, or even necessarily the best arguments. They also do not necessarily reflect the views of the HOPES team. They are simply our effort to review and raise awareness of the underlying issues.

The Case Against Animal Experimentation ^

An important part of the debate over animal rights centers on the question of the moral status of an animal. Most people agree that animals have at least some moral status – that is why it is wrong to abuse pets or needlessly hurt other animals. This alone represents a shift from a past view where animals had no moral status and treating an animal well was more about maintaining human standards of dignity than respecting any innate rights of the animal. In modern times, the question has shifted from whether animals have moral status to how much moral status they have and what rights come with that status.

The strongest pro animal rights answer to this question would be that non-human animals have exactly the same moral status as humans and are entitled to equal treatment. The ethicists who endorse this position do not mean that animals are entitled to the very same treatment as humans; arguing that animals should have the right to vote or hold office is clearly absurd. The claim is that animals should be afforded the same level of respectful treatment as humans; in short, we should not have the right to kill animals, force them into our service, or otherwise treat them merely as means to further our own goals.

One common form of this argument claims that moral status comes from the capacity to suffer or to enjoy life. In respect to his capacity, many animals are no different than humans. They can feel pain and experience pleasure. Therefore, they should have the same moral status and deserve equal treatment.

Supporters of this type of argument frequently claim that granting animals less moral status than humans is just a form of prejudice called “speciesism.” We have an innate tendency, they say, to consider the human species more morally relevant merely because it is the group to which we belong. However, we look upon past examples of this behavior as morally condemnable. Being of a particular race or gender does not give one any grounds for declaring outsiders to be of a lower moral status. Many animal rights advocates argue similarly—that just because we are human is not sufficient grounds to declare animals less morally significant.

The Case For Animal Experimentation ^

Defenders of animal experimentation usually argue that animals cannot be considered morally equal to humans. They generally use this claim as the cornerstone of an argument that the benefits to humans from animal experimentation outweigh or “make up for” the harm done to animals. The first step in making that argument is to show that humans are more important than animals. Below, I will outline one of the more common arguments used to reach this conclusion.

Some philosophers advocate the idea of a moral community. Roughly speaking, this is a group of individuals who all share certain traits in common. By sharing these traits, they belong to a particular moral community and thus take on certain responsibilities toward each other and assume specific rights. For example, in most human moral communities all individuals have the right to make independent decisions and live autonomous lives – and with that right comes the responsibility to respect others’ independence.

Although a moral community could theoretically include animals, it frequently does not. The human moral community, for instance, is often characterized by a capacity to manipulate abstract concepts and by personal autonomy. Since most animals do not have the cognitive capabilities of humans and also do not seem to possess full autonomy (animals do not rationally choose to pursue specific life goals), they are not included in the moral community. Once animals have been excluded from the moral community, humans have only a limited obligation towards them; on this argument, we certainly would not need to grant animals all normal human rights.

If animals do not have the same rights as humans, it becomes permissible to use them for research purposes. Under this view, the ways in which experimentation might harm the animal are less morally significant than the potential human benefits from the research.

One problem with this type of argument is that many humans themselves do not actually fulfill the criteria for belonging to the human moral community. Both infants and the mentally handicapped frequently lack complex cognitive capacities, full autonomy, or even both of these traits. Are those individuals outside the human moral community? Do they lack fundamental human rights and should we use them for experimentation? One philosophical position actually accepts those consequences and argues that those humans have the exact same rights (or lack of rights) as non-human animals. However, most people are uncomfortable with that scenario and some philosophers have put forth a variety of reasons to include all humans in the human moral community. A common way to “return” excluded individuals to the human moral community is to note how close these individuals come to meeting the criteria. In fact, some of them (the infants) will surely meet all of the criteria in the future. With that in mind, the argument runs, it is best practice to act charitably and treat all humans as part of the moral community.

In summary, defenders of animal experimentation argue that humans have higher moral status than animals and fundamental rights that animals lack. Accordingly, potential animal rights violations are outweighed by the greater human benefits of animal research.

A Middle Ground ^

There is a middle ground for those who feel uncomfortable with animal experimentation, but believe that in some circumstances the good arising out of experimentation does outweigh harm to the animal. Proponents of the middle ground position usually advocate a few basic principals that they believe should always be followed in animal research.

One principle calls for the preferential research use of less complex organisms whenever possible. For example bacteria , fruit flies, and plants would be preferred over mammals. This reflects a belief in a hierarchy of moral standing with more complex animals at the top and microorganisms and plants at the bottom. A philosophical grounding for this sort of hierarchy is the “moral worth as richness of life” model. This point of view suggests that more complicated organisms have richer, more fulfilling lives and that it is the richness of the life that actually correlates with moral worth.

Another principle is to reduce animal use as far as possible in any given study. Extensive literature searches, for instance, can ensure that experiments are not unnecessarily replicated and can ensure that animal models are only used to obtain information not already available in the scientific community. Another way to reduce animal use is to ensure that studies are conducted according to the highest standards and that all information collected will be useable. Providing high quality, disease-free environments for the animals will help ensure that every animal counts. Additionally, well designed studies and appropriate statistical analysis of data can minimize the number of animals required for statistically significant results.

A third principle is to ensure the best possible treatment of the animals used in a study. This means reducing pain and suffering as much as possible. When appropriate, anesthesia should be used; additionally, studies should have the earliest possible endpoints after which animals who will subsequently experience disease or suffering can be euthanized. Also, anyone who handles the animals should be properly trained.

The “bottom line” for the middle ground position is that animal experimentation should be avoided whenever possible in favor of alternative research strategies.

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– Adam Hepworth, 11-26-08

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50+ Inspiring Animal Research Topics

Why choose animal research topics for writing purposes .

Contrary to popular belief, animal research topics are not only used by veterinarians. They are also pursued by students majoring in Healthcare, Sound Engineering, and even subjects like Fashion Studies and Chemistry. Of course, it may require writing an excellent custom research paper because the trick here is to tailor things to what you need. The most challenging, however, is to choose your topic correctly and avoid being vague about what you must explore. Even if you would like to explore environmental issues, using animal research topics will be essential. You need to provide an explanation of your reasoning and the negative effects of human interaction with flora and fauna. 

Animal Research Topics

How To Choose Animals Research Topics? 

While there may be no universal topic that will reflect all sides of animal-related research, consider those subjects that you know well. It must inspire you and be an area where you feel comfortable. If you love marine life and can provide personal research examples, it would be good to choose something that will suit a reflection journal. Alternatively, consider animal topics for research papers that can be supported by reliable sources and statistical information. 

Start with an outline or a list of arguments that you would like to explore. Once done, continue with the wording for your topic that introduces the problem and offers a solution. You may also pose a research question about a problem or make a claim that will be supported by what you include in your paper. Always refer to your grading rubric and choose your research paper type accordingly. For example, your nursing research paper may talk about the use of animals for rehabilitation purposes, while a legal student may talk about animal rights in various countries. It all should be approached through the lens of what you learn as a primary subject! 

50+ Most Interesting & Easy Animal Research Topics 

- animal physiology research topics .

As you might already know, animal physiology studies anything related to the physical processes, changes in behaviors, breeding patterns, and more. As you think about choosing the animal physiology branch, always narrow things down if possible. 

- Controversial Animal Topics 

This aspect of animal research essay writing may not be everyone’s cup of tea, which is why it is necessary to explore the facts and provide information that represents both sides of the debate. Stay sensitive and avoid being too graphic unless it is necessary. Below are some ideas to consider: 

- Animal Rights Topics For Research Paper

The subject of animal rights is popular among students coming from all academic disciplines. Since you can approach it via the philosophical, legal, or medical lens, think about how to reflect your primary skills. It will make your research of animal right topics sound more confident. 

- Interesting Animal Research Topics 

- Veterinary Topics For Research Paper

In the majority of cases, you may refer to your veterinary branch first and proceed from there or take a look at the variety of veterinary research topics that we have presented below. Remember to quote every citation and idea that has been taken from other sources to avoid plagiarism.

- Animal Testing Research Topics

Even though this subject seems to be discussed everywhere these days, finding good animals topics to write about that deal with animal testing is not easy. Think about what are the underlying reasons for testing and what forces scientists to use it as a method. It will help you come up with ideas and better exploration strategies. 

- Animal Cruelty Topics

Warning: writing about animal cruelty subject is not for everyone, which is why you must be aware that the facts and statistics you may find will be shocking. It should be explored only if you are ready to embrace this disturbing subject. At the same time, you can explore milder animal cruelty cases like using pets as influencers on social media or the use of donkeys at the beaches to entertain tourists. There is always something to think about! 

- Research Questions About Animals

When you would like to take a general approach to animals research, it is good to come up with a research question as a part of your thesis statement or main argumentation. See these animals research paper examples: 

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Humanity often pays a high price for progress and understanding — at least, that seems to be the case in many famous psychological experiments. Human experimentation is a very interesting topic in the world of human psychology. While some famous experiments in psychology have left test subjects temporarily distressed, others have left their participants with life-long psychological issues . In either case, it’s easy to ask the question: “What’s ethical when it comes to science?” Then there are the experiments that involve children, animals, and test subjects who are unaware they’re being experimented on. How far is too far, if the result means a better understanding of the human mind and behavior ? We think we’ve found 20 answers to that question with our list of the most unethical experiments in psychology .

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Office of the Vice President for Research

Institutional animal care and use committee.

Ethics of Animal Use in Research

Laws & regulations, reporting concerns, other guidelines, pain & distress.

The use of animals in research, teaching and testing is a controversial ethical and political issue. Much of the discussion about this issue revolves around the relative value, often referred to as 'moral value', of humans and animals. When the needs of animals and humans come into conflict, which takes precedence? Today there exists a wide spectrum of views on this subject, ranging from those concerned with animal 'rights' to those who view animals only as a resource to be exploited. All of these viewpoints have contributed to the development of ethical principles of animal use. These in turn have shaped animal use regulations promulgated by the USDA and the Public Health Service, and reinforced by organizations such as AAALAC , AALAS and the AVMA .

Current legislation also recognizes that there are diverse viewpoints about the moral value of animals. Thus, all live animal use in research, teaching or testing must be reviewed by a committee (the IACUC) with diverse membership. This evaluation includes an emphasis on minimizing the overall use of animals.

Proposals for animal use are reviewed based on the potential for learning new information, or for teaching skills or concepts that cannot be obtained using an alternative. There are provisions for ensuring that animal use is performed in as humane a manner as possible, minimizing pain, distress or discomfort. These provisions include a requirement for a veterinarian to be employed at each institution, so that the needs of the animals are looked after by someone trained in, and sympathetic toward animals' needs. It is also required that all personnel with animal contact be trained in appropriate handling techniques and that they be skilled in any experimental procedures that will be performed. Finally, basic husbandry requirements are specified, ensuring that an animal's food, water and shelter will be provided for in an optimal manner. Deviations from the numerous requirements are granted by the IACUC only if adequate scientific justification is given that the proposed experiment is scientifically and socially important, and that any methods to alleviate pain or distress would frustrate the experimental objectives.

Animals have been used throughout history for anatomical and physiological research as well as for testing new medications and toxic substances. Many medical advances, including vaccines for polio and rabies, the development of certain antibiotics, cancer treating agents and transplant medicines, have been developed thanks to the use of animals in research.

The use of animals in research is a privilege and not a right. A research institution that receives money and support from the public is responsible for conducting research humanely and responsibly according to the limits set by society and regulatory bodies.  

Animal Welfare Act

The Animal Welfare Act (AWA) was passed in 1966. This act licenses dealers, exhibitors and breeders of animals, regulates research facilities that use animals, sets standards for the humane care and treatment of animals, and regulates the transportation of animals. The Act has been amended multiple times adding further protections for animals covered by the Act. The AWA specifically exempts birds, mice, rats, amphibians and reptiles used in research as well as agricultural animals that are used for agricultural production.

The United States Department of Agriculture is the government agency that is responsible for the enforcement of this act. Facilities must submit an annual report to the USDA. The USDA conducts unannounced inspections of research facilities at least once a year. If violations of the Act are found, fines can be imposed or research activities can be stopped.

Public Health Service Policy

The Public Health Service (PHS) Policy on the Humane Care and Use of Laboratory Animals is based on the United States Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. This policy covers all research that is funded by the National Institutes of Health (NIH) using vertebrate species of animals including birds, mice, and rats.

Institutions covered by this policy must follow the Guide for the Care and Use of Laboratory Animals (see below) and annually submit a written document called an Animal Welfare Assurance to NIH, which documents how the institution is complying with all the regulations covering animals used in research. The Office of Laboratory Animal Welfare (OLAW) at NIH is the agency that is responsible for enforcement of the PHS policy.

Guide for the Care and Use of Laboratory Animals

The Guide for the Care and Use of Laboratory Animals ("The Guide"), first developed in 1963, is a manual for research facilities receiving public funding for research using animals. The latest (2011) version of the Guide , sets specific standards for the care and use of laboratory animals. It addresses institutional responsibilities, husbandry and housing standards, veterinary care and physical plant specifications. It is written by experts in laboratory animal care and is published by the National Research Council.

AAALAC stands for the Association for the Assessment and Accreditation of Laboratory Animal Care. This is an independent (non-government) and voluntary accreditation organization. AAALAC accredits laboratory animal facilities through a process of intensive inspections (every 3 years) and reports (yearly). AAALAC follows the high standards put forth in the Guide. Accreditation, while voluntary, represents commitment to excellence in animal care and is an important factor to many funding agencies.

University of Minnesota Policy

The Regents Policy on Animal Care and Use addresses the use of all animals in research, teaching or display at the University of Minnesota. This policy follows from the federal and other laws and regulations. It addresses the roles and responsibilities of the Institutional Official, the Institutional Animal Care and Use Committee (IACUC), Research Animal Resources, and the University Community.

The Institutional Official (IO) is appointed by the University President and reports directly to him/her as well as to the federal authorities regarding compliance with all laws and regulations governing the use of laboratory animals in research and teaching. The President has formally delegated responsibility to appoint IACUC members to the Institutional Official.

The IACUC, which is a committee mandated by the AWA and the PHS policy, reviews and approves all activities involving animals at the University of Minnesota. The AWA and PHS policy have specific membership requirements for the committee. There must be at least:

University policy states that the committee should have at least 5 members, but the committee has many members, including several student members and ex-officio representatives from Occupational Health & Safety and the Department of Environmental Health and Safety.

The committee reviews all animal care and use protocols to ensure:

The committee also ensures the humane care of animals through the inspection of animal housing and use facilities twice a year and by investigating any complaints made regarding animal use. The committee is also responsible for reporting any instances of non-compliance and recommending corrective action.

Research Animal Resources (RAR) is designated by University policy as the program that provides the housing and husbandry as well as the veterinary care for the laboratory animals on the Twin Cities campus. They are also designated to serve as a consultation resource for the care and use of animals in research and teaching.

University policy also lists the roles and responsibilities of the University community. The University researchers and staff are to be appropriately trained and qualified to conduct activities with animals and are to abide by the decisions of the University and the IACUC.  

For serious questions or concerns about animal welfare, the process of review, or about committee decisions, contact:

Shashank Priya Institutional Official (612) 624-5054 [email protected]

Frances Lawrenz Deputy Institutional Official (612) 625-2046 [email protected]

You may also report animal welfare concerns or policy violations via the University of Minnesota's reporting system.  The UReport provides a way for University community members to report violations of rules, regulations and policies. The report can be made anonymously.

Note that, by federal law, no facility employee, Committee member, or laboratory personnel shall be discriminated against or be subject to any reprisal for reporting violations of any regulation or standards.  


The Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching is a text published by the Federation of Animal Sciences Societies. This Guide addresses standards for agricultural animal husbandry, housing and veterinary care. It does not apply to agricultural animals used for biomedical type research or teaching.

The standards are slightly different than those listed in the Guide for the Care and Use of Laboratory Animals. For example, cage space requirements may differ slightly between the two texts. Although this text is not regulatory, the University uses its provisions and principles as the basis for its care and use programs involving animals used for production or agricultural research.

There are several references available for the use of fish , amphibians and mammals in wildlife research . Again, these documents are not regulatory documents but are excellent references for the care and handling of these animals.  

The AWA defines a painful procedure in an animal as: "any procedure that would reasonably be expected to cause more than slight or momentary pain or distress in a human being to which that procedure was applied, that is, pain in excess of that caused by injections or other minor procedures." Pain can be acute, short lived - or chronic, lasting a long time. The signs manifesting acute or chronic pain may differ and may be different in different species. Prey species of animals can be adept at hiding signs of pain or illness and may be more difficult to assess discomfort.

Signs of Acute Pain in Animals

Signs of Chronic Pain

Distress is currently defined as "a state in which an animal cannot escape from or adapt to the external or internal stressors or conditions it experiences resulting in negative effects upon its well being…" Distress differs from stress, which is a physiological reaction that can lead to an adaptive response.

Principle IV of the US Government Principles states that unless the contrary is established, the assumption must be made that a procedure that causes pain or distress in a human being will cause pain and distress in an animal.


Current regulations stress the need to search for and utilize alternatives to procedures on animals that cause more than momentary pain or distress. The concept of the three "R"s has been used when thinking about alternatives to animal use. This concept was developed in 1959 by Russell and Burch in their book: The Principles of Humane Animal Experimental Techniques.

The three "R"s are Replacement, Reduction, and Refinement. Investigators at the University of Minnesota, who use animals that may undergo more than momentary pain or distress, should consider the three “R”s when conducting procedures which may be painful or distressful.

Replacement of animals with other systems may be an option. Computer modeling or in vitro testing may be a substitute for animal models. "Lower" or non-vertebrate animals, such as the fruit fly may be used in some situations rather than a higher order animal.

Reduction of the number of animals used for research is also an important concept. This is done mostly through experimental design and the use of statistics. The use of too few animals could result in statistically invalid results, which could necessitate the use of even more animals in subsequent experiments. Pilot studies to help determine statistical parameters can sometimes assist in determination of group sizes. Reduction of pain and distress may also actually require the use of more animals so that repeated procedures are not conducted on the same animal.

Refinement refers to methods that decrease the amount of pain and distress experienced by the animals that are actually needed to perform an experiment. This is not only done through the use of pain relieving measures such as anesthetics and analgesics whenever possible, but also through environmental enrichment.

The use of early endpoints can also be a form of refinement. For instance if an animal were to suffer from an early indicator of disease or a tumor reaches a certain measurable size, this could be used as the endpoint. The animals should be humanely euthanized at this point rather than waiting until the death of the animal.

For more examples of replacement/reduction/refinement and searches for alternatives, see IACUC's web page, “Finding Models and Alternatives”.

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Impact of ethical ideologies on students’ attitude toward animals—a pakistani perspective.

animal research studies examples

Simple Summary

1. introduction, 1.1. objectives.

1.2. Hypotheses

2. Methodology

2.1. research design, 2.2. instruments, 2.2.1. demographic sheet, 2.2.2. ethics position questionnaire (epq) [ 6 ], 2.2.3. animal attitude scale—10-item version (aas-10) [ 15 ], 2.2.4. animal issue scale (ais) [ 24 ], 2.3. sample and demographic characteristics, 2.4. procedure, 3.1. correlation among ethical ideologies (idealism and relativism), attitude toward animals, and concern for animal welfare, 3.2. difference among ethical ideologies, attitude toward animals, and concern for animal welfare for frequency of meat consumption in university students, 3.3. difference along semester/stage of program for idealism in university students, 3.4. predictive role of ethical position on attitude toward animals, 3.5. predictive role of ethical position on concern for animal welfare, 3.6. predictive role of idealism and relativism on attitude toward animals, 3.7. predictive role of idealism and relativism on concern for animal welfare, 4. discussion, 4.1. limitation and suggestion, 4.2. implications, 5. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

Share and Cite

Khalid, A.; Martens, P.; Khalid, A. Impact of Ethical Ideologies on Students’ Attitude toward Animals—A Pakistani Perspective. Animals 2023 , 13 , 927. https://doi.org/10.3390/ani13050927

Khalid A, Martens P, Khalid A. Impact of Ethical Ideologies on Students’ Attitude toward Animals—A Pakistani Perspective. Animals . 2023; 13(5):927. https://doi.org/10.3390/ani13050927

Khalid, Asiya, Pim Martens, and Aliya Khalid. 2023. "Impact of Ethical Ideologies on Students’ Attitude toward Animals—A Pakistani Perspective" Animals 13, no. 5: 927. https://doi.org/10.3390/ani13050927

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Ethical: relating to a person's moral principles.

United States Department of Agriculture: United States agency that is responsible for federal laws related to farming, agriculture, forestry, and food.

Animals Used in Research

Any animal can be used in research. But which animal will be chosen will depend on the research question. If a biologist is interested in the mating behavior of toads or zebras, then it will only make sense for her or him to study that specific animal.

Lab tech with rat

Scientists select the animal they use for research carefully. But in general mice and rats are the most commonly used. Image by metalgearsolid5.

This approach may have some limitations, for example when the species of interest is endangered. But, in general, scientists can study any animal of interest to their work. This is  basic research —studying an animal to learn more about it.

Choosing the Right Animal

Sometimes a scientist won’t pick an animal they are interested in studying, but one that helps them to answer a specific question. For example, if scientists are interested in studying a new medication or a pathway in the brain, they will use whatever animal will best allow them to do that.

For such research questions, scientists can use a wide range of animals. The United States Department of Agriculture  (USDA) is one agency in the U.S. that keeps track of how many animals are used each year for research. The animals that this agency reported being used in research in 2014 include a total of 834,453 animals. The table below shows the animals included in their data:

Other Animals Used in Research

Other animals not included in this list include birds, fish, mice and rats. It turns out that mice and rats are the most commonly used animals in research. Because of this, some argue that the numbers mentioned above don't properly report the total number of animals used in research. In fact, this number is likely to be much higher than it's currently reported by the USDA. 

Approving the Use of an Animal for Research

As mentioned above, there are some limitations for scientists when choosing the animal to best help them to answer a research question. And in addition to endangered animals, others such as chimpanzees and cephalopods, have special rules for use.  

If a scientist would like to use one of these animals for their work, she or he must clearly explain why this choice is important for the proposed work. This is in addition to the other documentation that must be filled out for using an animal in a research setting.

Human Participation in Research

Blood pressure

Humans will be used in place of animals for research purposes, at least at first, when there is no expected harm to the human. Image by Kris D.

While oftentimes an animal is used for a research study, there are also cases when humans are used. One example of this is when there is no harm to using a human for a study, such as taking blood pressure, heart rate, or other similar data. When there is little to no risk related to a study it is common for only humans to be used.

When the work may cause harm to humans, this testing  will often only occur after the work has been done using an animal model. Here scientists will study a particular topic in animals, and once they have successful results they will then apply their work to humans. There are many advantages to first using animals for a research study. Despite this, the decision to use animals first (before humans) is one that many scientists and non-scientists debate about. This is an ethical debate  related to animal research that is still ongoing. 

The requirement of both human  and  other animal studies relates to a  disadvantage of animal research . The physiology of humans and non-human animals can be very different, so the results of animal studies cannot always be directly compared to humans. One example of this is in drug research. A drug may have different effects on the body when given to a non-human animal versus when it is given to a human.

When human research occurs, scientists will ask people to volunteer to be a part of the study. The people who volunteer to particpate will be informed of any risks involved before the experiment begins. And similar to animal research, there are many guidelines in place to protect the safety of any person participating in a given research project. The guidelines for using humans in research are unique to those for animal use.

Additional images via Wikimedia Commons. Frog image via Fredlyfish4.

Read more about: Using Animals in Research

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Patrick McGurrin and Christian Ross. (2016, December 04). What Animals Are Used in Research?. ASU - Ask A Biologist. Retrieved February 28, 2023 from https://askabiologist.asu.edu/research-animals

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Patrick McGurrin and Christian Ross. "What Animals Are Used in Research?". ASU - Ask A Biologist. 04 December, 2016. https://askabiologist.asu.edu/research-animals

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Patrick McGurrin and Christian Ross. "What Animals Are Used in Research?". ASU - Ask A Biologist. 04 Dec 2016. ASU - Ask A Biologist, Web. 28 Feb 2023. https://askabiologist.asu.edu/research-animals

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Scientists use a variety of animals for research purposes. These include different types of reptiles, insects, mammals, and amphibians, among others.

Using Animals in Research

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Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0

Roles Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation NC3Rs, London, United Kingdom

ORCID logo

Roles Investigation, Visualization, Writing – original draft, Writing – review & editing

Affiliations The William Harvey Research Institute, London, United Kingdom, Barts Cardiovascular CTU, Queen Mary University of London, London, United Kingdom

Affiliation Taylor & Francis Group, London, United Kingdom

Affiliation Health Science Practice, ICF, Durham, North Carolina, United States of America

Affiliation Nature, San Francisco, California, United States of America

Affiliation School of Education, University of Bristol, Bristol, United Kingdom

Affiliation PLOS ONE, Cambridge, United Kingdom

Affiliation School of Biological Sciences, University of Bristol, Bristol, United Kingdom

Affiliation QUEST Center for Transforming Biomedical Research, Berlin Institute of Health & Department of Experimental Neurology, Charite Universitätsmedizin Berlin, Berlin, Germany

Affiliation National Heart and Lung Institute, Imperial College London, London, United Kingdom

Affiliation Centre for Evidence Synthesis in Global Health, Clinical Sciences Department, Liverpool School of Tropical Medicine, Liverpool, United Kingdom

Affiliation Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom

Affiliation Tasmanian School of Medicine, University of Tasmania, Hobart, Australia

Roles Investigation, Project administration, Visualization, Writing – original draft

Affiliation Data Sciences & Quantitative Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, United Kingdom

Roles Writing – review & editing

Affiliation Prioris.ai Inc, Ottawa, Canada

Roles Investigation, Project administration, Writing – original draft, Writing – review & editing

Affiliation Hindawi Ltd, London, United Kingdom

Affiliation Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

Roles Investigation, Visualization, Writing – review & editing

Affiliation Academia Europaea Knowledge Hub, Cardiff University, Cardiff, United Kingdom

Affiliation Medical Research Council, London, United Kingdom

Affiliation Statistics in Anesthesiology Research (STAR) Core, Department of Anesthesiology, College of Medicine, University of Florida, Gainesville, Florida, United States of America

Affiliation Discipline of Exercise and Sport Science, Faculty of Medicine and Health, University of Sydney, Sydney, Australia

Affiliation National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, United States of America

Affiliation Janssen Pharmaceutica NV, Beerse, Belgium

Affiliation Veterinary Public Health Institute, Vetsuisse Faculty, University of Bern, Bern, Switzerland


Published: July 14, 2020

Fig 1

Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication.

Citation: Percie du Sert N, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, et al. (2020) Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol 18(7): e3000411. https://doi.org/10.1371/journal.pbio.3000411

Academic Editor: Isabelle Boutron, University Paris Descartes, FRANCE

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: This work was supported by the National Centre of the Replacement, Refinement & Reduction on Animals in Research (NC3Rs, https://www.nc3rs.org.uk/ ). NPdS, KL, VH, and EJP are employees of the NC3Rs.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AA is the editor in chief of the British Journal of Pharmacology. WJB, ICC, and ME are authors of the original ARRIVE guidelines. WJB serves on the Independent Statistical Standing Committee of the funder CHDI foundation. AC is a Senior Editor for PLOS ONE. AC, CJM, MM, and ESS were involved in the IICARus trial. ME, MM, and ESS have received funding from NC3Rs. ME sits on the MRC ERPIC panel. STH is chair of the NC3Rs board; trusteeship of the BLF, Kennedy Trust, DSRU, and CRUK; member of Governing Board, Nuffield Council of Bioethics, member Science Panel for Health (EU H2020); founder and NEB Director Synairgen; consultant Novartis, Teva, and AZ; and chair MRC/GSK EMINENT Collaboration. VH, KL, EJP, and NPdS are NC3Rs staff; role includes promoting the ARRIVE guidelines. SEL and UD are on the advisory board of the UK Reproducibility Network. CJM has shareholdings in Hindawi, is on the publishing board of the Royal Society, and on the EU Open Science policy platform. UD, MM, NPdS, CJM, ESS, TS, and HW are members of EQIPD. MM is a member of the Animals in Science Committee and on the steering group of the UK Reproducibility Network. NPdS and TS are associate editors of BMJ Open Science. OHP is vice president of Academia Europaea, editor in chief of Function, senior executive editor of the Journal of Physiology, and member of the Board of the European Commission’s SAPEA (Science Advice for Policy by European Academies). FR is an NC3Rs board member and has shareholdings in GSK. FR and NAK have shareholdings in AstraZeneca. PR is a member of the University of Florida Institutional Animal Care and Use Committee and editorial board member of Shock. ESS is editor in chief of BMJ Open Science. SDS’s role is to provide expertise and does not represent the opinion of the NIH. TS has shareholdings in Johnson & Johnson. SA, MTA, MB, PG, DWH, and KR declared no conflict of interest.

Abbreviations: AAALAC, American Association for Accreditation of Laboratory Animal Care; ARRIVE, Animal Research: Reporting of In Vivo Experiments; AVMA, American Veterinary Medical Association; AWERB, Animal Welfare and Ethical Review Body; DOI, digital object identifier; EBI, European Bioinformatics Institute; EDA, Experimental Design Assistant; GLP, Good Laboratory Practice; IACUC, Institutional Animal Care and Use Committee; NC3Rs, National Centre for the 3Rs; NCBI, National Center for Biotechnology Information; PHISPS, Population; Hypothesis; Intervention; Statistical Analysis Plan; Primary; Outcome Measure; Sample Size Calculation; RRID, Research Resource Identifier; SAMPL, Statistical Analyses and Methods in the Published Literature; SPF, Specific Pathogen Free

See S1 Annotated byline for individual authors’ positions at the time this article was submitted . See S1 Annotated References for further context on the works cited in this article .


Transparent and accurate reporting is essential to improve the reproducibility of scientific research; it enables others to scrutinise the methodological rigour of the studies, assess how reliable the findings are, and repeat or build upon the work.

However, evidence shows that the majority of publications fail to include key information and there is significant scope to improve the reporting of studies involving animal research [ 1 – 4 ]. To that end, the UK National Centre for the 3Rs (NC3Rs) published the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines in 2010. The guidelines are a checklist of information to include in a manuscript to ensure that publications contain enough information to add to the knowledge base [ 5 ]. The guidelines have received widespread endorsement from the scientific community and are currently recommended by more than a thousand journals, with further endorsement from research funders, universities, and learned societies worldwide.

Studies measuring the impact of ARRIVE on the quality of reporting have produced mixed results [ 6 – 11 ], and there is evidence that in vivo scientists are not sufficiently aware of the importance of reporting the information covered in the guidelines and fail to appreciate the relevance to their work or their research field [ 12 ].

As a new international working group—the authors of this publication—we have revised the guidelines to update them and facilitate their uptake; the ARRIVE guidelines 2.0 are published alongside this paper [ 13 ]. We have updated the recommendations in line with current best practice, reorganised the information, and classified the items into two sets. The ARRIVE Essential 10 constitute the minimum reporting requirement, and the Recommended Set provides further context to the study described. Although reporting both sets is best practice, an initial focus on the most critical issues helps authors, journal staff, editors, and reviewers use the guidelines in practice and allows a pragmatic implementation. Once the Essential 10 are consistently reported in manuscripts, items from the Recommended Set can be added to journal requirements over time until all 21 items are routinely reported in all manuscripts. Full methodology for the revision and the allocation of items into sets is described in the accompanying publication [ 13 ].

A key aspect of the revision was to develop this explanation and elaboration document to provide background and rationale for each of the 21 items of ARRIVE 2.0. Here, we present additional guidance for each item and subitem, explain the importance of reporting this information in manuscripts that describe animal research, elaborate on what to report, and provide supporting evidence. The guidelines apply to all areas of bioscience research involving living animals. That includes mammalian species as well as model organisms such as Drosophila or Caenorhabditis elegans . Each item is equally relevant to manuscripts centred around a single animal study and broader-scope manuscripts describing in vivo observations along with other types of experiments. The exact type of detail to report, however, might vary between species and experimental setup; this is acknowledged in the guidance provided for each item.

We recognise that the purpose of the research influences the design of the study. Hypothesis-testing research evaluates specific hypotheses, using rigorous methods to reduce the risk of bias and a statistical analysis plan that has been defined before the study starts. In contrast, exploratory research often investigates many questions simultaneously without adhering to strict standards of rigour; this flexibility is used to develop or test novel methods and generate theories and hypotheses that can be formally tested later. Both study types make valuable contributions to scientific progress. Transparently reporting the purpose of the research and the level of rigour used in the design, execution, and analysis of the study enables readers to decide how to use the research, whether the findings are groundbreaking and need to be confirmed before building on them, or whether they are robust enough to be applied to other research settings.

To contextualise the importance of reporting information described in the Essential 10, this document also covers experimental design concepts and best practices. This has two main purposes: First, it helps authors understand the relevance of this information for readers to assess the reliability of the reported results, thus encouraging thorough reporting. Second, it supports the implementation of best practices in the design and conduct of animal research. Consulting this document at the start of the process when planning an in vivo experiment will enable researchers to make the best use of it, implement the advice on study design, and prepare for the information that will need to be collected during the experiment to report the study in adherence with the guidelines.

To ensure that the recommendations are as clear and useful as possible to the target audience, this document was road tested alongside the revised guidelines with researchers preparing manuscripts describing in vivo research [ 13 ]. Each item is written as a self-contained section, enabling authors to refer to particular items independently, and a glossary ( Box 1 ) explains common statistical terms. Each subitem is also illustrated with examples of good reporting from the published literature. Explanations and examples are also available from the ARRIVE guidelines website: https://www.arriveguidelines.org .

Box 1. Glossary

Bias: The over- or underestimation of the true effect of an intervention. Bias is caused by inadequacies in the design, conduct, or analysis of an experiment, resulting in the introduction of error.

Descriptive and inferential statistics: Descriptive statistics are used to summarise the data. They generally include a measure of central tendency (e.g., mean or median) and a measure of spread (e.g., standard deviation or range). Inferential statistics are used to make generalisations about the population from which the samples are drawn. Hypothesis tests such as ANOVA, Mann-Whitney, or t tests are examples of inferential statistics.

Effect size: Quantitative measure of differences between groups, or strength of relationships between variables.

Experimental unit: Biological entity subjected to an intervention independently of all other units, such that it is possible to assign any two experimental units to different treatment groups. Sometimes known as unit of randomisation.

External validity: Extent to which the results of a given study enable application or generalisation to other studies, study conditions, animal strains/species, or humans.

False negative: Statistically nonsignificant result obtained when the alternative hypothesis (H 1 ) is true. In statistics, it is known as the type II error.

False positive: Statistically significant result obtained when the null hypothesis (H 0 ) is true. In statistics, it is known as the type I error.

Independent variable: Variable that either the researcher manipulates (treatment, condition, time) or is a property of the sample (sex) or a technical feature (batch, cage, sample collection) that can potentially affect the outcome measure. Independent variables can be scientifically interesting, or nuisance variables. Also known as predictor variable.

Internal validity: Extent to which the results of a given study can be attributed to the effects of the experimental intervention, rather than some other, unknown factor(s) (e.g., inadequacies in the design, conduct, or analysis of the study introducing bias).

Nuisance variable: Variables that are not of primary interest but should be considered in the experimental design or the analysis because they may affect the outcome measure and add variability. They become confounders if, in addition, they are correlated with an independent variable of interest, as this introduces bias. Nuisance variables should be considered in the design of the experiment (to prevent them from becoming confounders) and in the analysis (to account for the variability and sometimes to reduce bias). For example, nuisance variables can be used as blocking factors or covariates.

Null and alternative hypotheses: The null hypothesis (H 0 ) is that there is no effect, such as a difference between groups or an association between variables. The alternative hypothesis (H 1 ) postulates that an effect exists.

Outcome measure: Any variable recorded during a study to assess the effects of a treatment or experimental intervention. Also known as dependent variable, response variable.

Power: For a predefined, biologically meaningful effect size, the probability that the statistical test will detect the effect if it exists (i.e., the null hypothesis is rejected correctly).

Sample size: Number of experimental units per group, also referred to as n .

Definitions are adapted from [ 14 , 15 ] and placed in the context of animal research.

ARRIVE Essential 10

The ARRIVE Essential 10 ( Box 2 ) constitute the minimum reporting requirement to ensure that reviewers and readers can assess the reliability of the findings presented. There is no ranking within the set; items are presented in a logical order.

Box 2. ARRIVE Essential 10

Item 1. Study design

For each experiment, provide brief details of study design including :

1a . The groups being compared, including control groups . If no control group has been used, the rationale should be stated .

Explanation. The choice of control or comparator group is dependent on the experimental objective. Negative controls are used to determine whether a difference between groups is caused by the intervention (e.g., wild-type animals versus genetically modified animals, placebo versus active treatment, sham surgery versus surgical intervention). Positive controls can be used to support the interpretation of negative results or determine if an expected effect is detectable.

It may not be necessary to include a separate control with no active treatment if, for example, the experiment aims to compare a treatment administered by different methods (e.g., intraperitoneal administration versus oral gavage) or animals that are used as their own control in a longitudinal study. A pilot study, such as one designed to test the feasibility of a procedure, might also not require a control group.

For complex study designs, a visual representation is more easily interpreted than a text description, so a timeline diagram or flowchart is recommended. Diagrams facilitate the identification of which treatments and procedures were applied to specific animals or groups of animals and at what point in the study these were performed. They also help to communicate complex design features such as whether factors are crossed or nested (hierarchical/multilevel designs), blocking (to reduce unwanted variation, see Item 4. Randomisation), or repeated measurements over time on the same experimental unit (repeated measures designs); see [ 16 – 18 ] for more information on different design types. The Experimental Design Assistant (EDA) is a platform to support researchers in the design of in vivo experiments; it can be used to generate diagrams to represent any type of experimental design [ 19 ].

For each experiment performed, clearly report all groups used. Selectively excluding some experimental groups (for example, because the data are inconsistent or conflict with the narrative of the paper) is misleading and should be avoided [ 20 ]. Ensure that test groups, comparators, and controls (negative or positive) can be identified easily. State clearly if the same control group was used for multiple experiments or if no control group was used.

Subitem 1a—Example 1

‘The DAV1 study is a one-way, two-period crossover trial with 16 piglets receiving amoxicillin and placebo at period 1 and only amoxicillin at period 2. Amoxicillin was administered orally with a single dose of 30 mg.kg -1 . Plasma amoxicillin concentrations were collected at same sampling times at each period: 0.5, 1, 1.5, 2, 4, 6, 8, 10 and 12 h’ [ 21 ].

Subitem 1a—Example 2



1b . The experimental unit (e.g., a single animal, litter, or cage of animals) .

Explanation. Within a design, biological and technical factors will often be organised hierarchically, such as cells within animals and mitochondria within cells, or cages within rooms and animals within cages. Such hierarchies can make determining the sample size difficult (is it the number of animals, cells, or mitochondria?). The sample size is the number of experimental units per group. The experimental unit is defined as the biological entity subjected to an intervention independently of all other units, such that it is possible to assign any two experimental units to different treatment groups. It is also sometimes called the unit of randomisation. In addition, the experimental units should not influence each other on the outcomes that are measured.

Commonly, the experimental unit is the individual animal, each independently allocated to a treatment group (e.g., a drug administered by injection). However, the experimental unit may be the cage or the litter (e.g., a diet administered to a whole cage, or a treatment administered to a dam and investigated in her pups), or it could be part of the animal (e.g., different drug treatments applied topically to distinct body regions of the same animal). Animals may also serve as their own controls, receiving different treatments separated by washout periods; here, the experimental unit is an animal for a period of time. There may also be multiple experimental units in a single experiment, such as when a treatment is given to a pregnant dam and then the weaned pups are allocated to different diets [ 23 ]. See [ 17 , 24 , 25 ] for further guidance on identifying experimental units.

Conflating experimental units with subsamples or repeated measurements can lead to artificial inflation of the sample size. For example, measurements from 50 individual cells from a single mouse represent n = 1 when the experimental unit is the mouse. The 50 measurements are subsamples and provide an estimate of measurement error and so should be averaged or used in a nested analysis. Reporting n = 50 in this case is an example of pseudoreplication [ 26 ]. It underestimates the true variability in a study, which can lead to false positives and invalidate the analysis and resulting conclusions [ 26 , 27 ]. If, however, each cell taken from the mouse is then randomly allocated to different treatments and assessed individually, the cell might be regarded as the experimental unit.

Clearly indicate the experimental unit for each experiment so that the sample sizes and statistical analyses can be properly evaluated.

Subitem 1b—Example 1

‘The present study used the tissues collected at E15.5 from dams fed the 1X choline and 4X choline diets ( n = 3 dams per group, per fetal sex; total n = 12 dams). To ensure statistical independence, only one placenta (either male or female) from each dam was used for each experiment. Each placenta, therefore, was considered to be an experimental unit’ [ 28 ].

Subitem 1b—Example 2

‘We have used data collected from high-throughput phenotyping, which is based on a pipeline concept where a mouse is characterized by a series of standardized and validated tests underpinned by standard operating procedures (SOPs)…. The individual mouse was considered the experimental unit within the studies’ [ 29 ].

Subitem 1b—Example 3

‘Fish were divided in two groups according to weight (0.7–1.2 g and 1.3–1.7 g) and randomly stocked (at a density of 15 fish per experimental unit) in 24 plastic tanks holding 60 L of water’ [ 30 ].

Subitem 1b—Example 4

‘In the study, n refers to number of animals, with five acquisitions from each [corticostriatal] slice, with a maximum of three slices obtained from each experimental animal used for each protocol (six animals each group)’ [ 31 ].

Item 2. Sample size

2a . Specify the exact number of experimental units allocated to each group, and the total number in each experiment . Also indicate the total number of animals used .

Explanation. The sample size relates to the number of experimental units in each group at the start of the study and is usually represented by n (see Item 1. Study design for further guidance on identifying and reporting experimental units). This information is crucial to assess the validity of the statistical model and the robustness of the experimental results.

The sample size in each group at the start of the study may be different from the n numbers in the analysis (see Item 3. Inclusion and exclusion criteria); this information helps readers identify attrition or if there have been exclusions and in which group they occurred. Reporting the total number of animals used in the study is also useful to identify whether any were reused between experiments.

Report the exact value of n per group and the total number in each experiment (including any independent replications). If the experimental unit is not the animal, also report the total number of animals to help readers understand the study design. For example, in a study investigating diet using cages of animals housed in pairs, the number of animals is double the number of experimental units.

Subitem 2a –example 1



2b . Explain how the sample size was decided . Provide details of any a priori sample size calculation, if done .

Explanation. For any type of experiment, it is crucial to explain how the sample size was determined. For hypothesis-testing experiments, in which inferential statistics are used to estimate the size of the effect and to determine the weight of evidence against the null hypothesis, the sample size needs to be justified to ensure experiments are of an optimal size to test the research question [ 33 , 34 ] (see Item 13. Objectives). Sample sizes that are too small (i.e., underpowered studies) produce inconclusive results, whereas sample sizes that are too large (i.e., overpowered studies) raise ethical issues over unnecessary use of animals and may produce trivial findings that are statistically significant but not biologically relevant [ 35 ]. Low power has three effects: first, within the experiment, real effects are more likely to be missed; second, when an effect is detected, this will often be an overestimation of the true effect size [ 24 ]; and finally, when low power is combined with publication bias, there is an increase in the false positive rate in the published literature [ 36 ]. Consequently, low-powered studies contribute to the poor internal validity of research and risk wasting animals used in inconclusive research [ 37 ].

Study design can influence the statistical power of an experiment, and the power calculation used needs to be appropriate for the design implemented. Statistical programmes to help perform a priori sample size calculations exist for a variety of experimental designs and statistical analyses, both freeware (web-based applets and functions in R) and commercial software [ 38 – 40 ]. Choosing the appropriate calculator or algorithm to use depends on the type of outcome measures and independent variables, and the number of groups. Consultation with a statistician is recommended, especially when the experimental design is complex or unusual.

When the experiment tests the effect of an intervention on the mean of a continuous outcome measure, the sample size can be calculated a priori, based on a mathematical relationship between the predefined, biologically relevant effect size, variability estimated from prior data, chosen significance level, power, and sample size (see Box 3 and [ 17 , 41 ] for practical advice). If you have used an a priori sample size calculation, report

Box 3. Information used in a power calculation

Sample size calculation is based on a mathematical relationship between the following parameters: effect size, variability, significance level, power, and sample size. Questions to consider are the following:

The primary objective of the experiment—What is the main outcome measure?

The primary outcome measure should be identified in the planning stage of the experiment; it is the outcome of greatest importance, which will answer the main experimental question.

The predefined effect size—What is a biologically relevant effect size?

The effect size is estimated as a biologically relevant change in the primary outcome measure between the groups under study. This can be informed by similar studies and involves scientists exploring what magnitude of effect would generate interest and would be worth taking forward into further work. In preclinical studies, the clinical relevance of the effect should also be taken into consideration.

What is the estimate of variability?

Estimates of variability can be obtained

Significance threshold—What risk of a false positive is acceptable?

The significance level or threshold (α) is the probability of obtaining a false positive. If it is set at 0.05, then the risk of obtaining a false positive is 1 in 20 for a single statistical test. However, the threshold or the p -values will need to be adjusted in scenarios of multiple testing (e.g., by using a Bonferroni correction).

Power—What risk of a false negative is acceptable?

For a predefined, biologically meaningful effect size, the power (1 − β) is the probability that the statistical test will detect the effect if it genuinely exists (i.e., true positive result). A target power between 80% and 95% is normally deemed acceptable, which entails a risk of false negative between 5% and 20%.

Directionality—Will you use a one- or two-sided test?

The directionality of a test depends on the distribution of the test statistics for a given analysis. For tests based on t or z distributions (such as t tests), whether the data will be analysed using a one- or two-sided test relates to whether the alternative hypothesis is directional or not. An experiment with a directional (one-sided) alternative hypothesis can be powered and analysed with a one-sided test with the goal of maximising the sensitivity to detect this directional effect. Controversy exists within the statistics community on when it is appropriate to use a one-sided test [ 42 ]. The use of a one-sided test requires justification of why a treatment effect is only of interest when it is in a defined direction and why they would treat a large effect in the unexpected direction no differently from a nonsignificant difference [ 43 ]. Following the use of a one-sided test, the investigator cannot then test for the possibility of missing an effect in the untested direction. Choosing a one-tailed test for the sole purpose of attaining statistical significance is not appropriate.

Two-sided tests with a nondirectional alternative hypothesis are much more common and allow researchers to detect the effect of a treatment regardless of its direction.

Note that analyses such as ANOVA and chi-squared are based on asymmetrical distributions (F-distribution and chi-squared distribution) with only one tail. Therefore, these tests do not have a directionality option.

There are several types of studies in which a priori sample size calculations are not appropriate. For example, the number of animals needed for antibody or tissue production is determined by the amount required and the production ability of an individual animal. For studies in which the outcome is the successful generation of a sample or a condition (e.g., the production of transgenic animals), the number of animals is determined by the probability of success of the experimental procedure.

In early feasibility or pilot studies, the number of animals required depends on the purpose of the study. When the objective of the preliminary study is primarily logistic or operational (e.g., to improve procedures and equipment), the number of animals needed is generally small. In such cases, power calculations are not appropriate and sample sizes can be estimated based on operational capacity and constraints [ 44 ]. Pilot studies alone are unlikely to provide adequate data on variability for a power calculation for future experiments. Systematic reviews and previous studies are more appropriate sources of information on variability [ 45 ].

If no power calculation was used to determine the sample size, state this explicitly and provide the reasoning that was used to decide on the sample size per group. Regardless of whether a power calculation was used or not, when explaining how the sample size was determined take into consideration any anticipated loss of animals or data, for example, due to exclusion criteria established upfront or expected attrition (see Item 3. Inclusion and exclusion criteria).

Subitem 2b—Example 1

‘The sample size calculation was based on postoperative pain numerical rating scale (NRS) scores after administration of buprenorphine (NRS AUC mean = 2.70; noninferiority limit = 0.54; standard deviation = 0.66) as the reference treatment… and also Glasgow Composite Pain Scale (GCPS) scores… using online software (Experimental design assistant; https://eda.nc3rs.org.uk/eda/login/auth ). The power of the experiment was set to 80%. A total of 20 dogs per group were considered necessary’ [ 46 ].

Subitem 2b—Example 2

‘We selected a small sample size because the bioglass prototype was evaluated in vivo for the first time in the present study, and therefore, the initial intention was to gather basic evidence regarding the use of this biomaterial in more complex experimental designs’ [ 47 ].

Item 3. Inclusion and exclusion criteria

3a . Describe any criteria used for including or excluding animals (or experimental units) during the experiment, and data points during the analysis . Specify if these criteria were established a priori . If no criteria were set, state this explicitly .

Explanation. Inclusion and exclusion criteria define the eligibility or disqualification of animals and data once the study has commenced. To ensure scientific rigour, the criteria should be defined before the experiment starts and data are collected [ 8 , 33 , 48 , 49 ]. Inclusion criteria should not be confused with animal characteristics (see Item 8. Experimental animals) but can be related to these (e.g., body weights must be within a certain range for a particular procedure) or related to other study parameters (e.g., task performance has to exceed a given threshold). In studies in which selected data are reanalysed for a different purpose, inclusion and exclusion criteria should describe how data were selected.

Exclusion criteria may result from technical or welfare issues such as complications anticipated during surgery or circumstances in which test procedures might be compromised (e.g., development of motor impairments that could affect behavioural measurements). Criteria for excluding samples or data include failure to meet quality control standards, such as insufficient sample volumes, unacceptable levels of contaminants, poor histological quality, etc. Similarly, how the researcher will define and handle data outliers during the analysis should also be decided before the experiment starts (see subitem 3b for guidance on responsible data cleaning).

Exclusion criteria may also reflect the ethical principles of a study in line with its humane endpoints (see Item 16. Animal care and monitoring). For example, in cancer studies, an animal might be dropped from the study and euthanised before the predetermined time point if the size of a subcutaneous tumour exceeds a specific volume [ 50 ]. If losses are anticipated, these should be considered when determining the number of animals to include in the study (see Item 2. Sample size). Whereas exclusion criteria and humane endpoints are typically included in the ethical review application, reporting the criteria used to exclude animals or data points in the manuscript helps readers with the interpretation of the data and provides crucial information to other researchers wanting to adopt the model.

Best practice is to include all a priori inclusion and exclusion/outlier criteria in a preregistered protocol (see Item 19. Protocol registration). At the very least, these criteria should be documented in a laboratory notebook and reported in manuscripts, explicitly stating that the criteria were defined before any data was collected.

Subitem 3a—Example 1

‘The animals were included in the study if they underwent successful MCA occlusion (MCAo), defined by a 60% or greater drop in cerebral blood flow seen with laser Doppler flowmetry. The animals were excluded if insertion of the thread resulted in perforation of the vessel wall (determined by the presence of sub-arachnoid blood at the time of sacrifice), if the silicon tip of the thread became dislodged during withdrawal, or if the animal died prematurely, preventing the collection of behavioral and histological data’ [ 51 ].

3b . For each experimental group, report any animals, experimental units, or data points not included in the analysis and explain why . If there were no exclusions, state so .

Explanation. Animals, experimental units, or data points that are unaccounted for can lead to instances in which conclusions cannot be supported by the raw data [ 52 ]. Reporting exclusions and attritions provides valuable information to other investigators evaluating the results or who intend to repeat the experiment or test the intervention in other species. It may also provide important safety information for human trials (e.g., exclusions related to adverse effects).

There are many legitimate reasons for experimental attrition, some of which are anticipated and controlled for in advance (see subitem 3a on defining exclusion and inclusion criteria), but some data loss might not be anticipated. For example, data points may be excluded from analyses because of an animal receiving the wrong treatment, unexpected drug toxicity, infections or diseases unrelated to the experiment, sampling errors (e.g., a malfunctioning assay that produced a spurious result, inadequate calibration of equipment), or other human error (e.g., forgetting to switch on equipment for a recording).

Most statistical analysis methods are extremely sensitive to outliers and missing data. In some instances, it may be scientifically justifiable to remove outlying data points from an analysis, such as obvious errors in data entry or measurement with readings that are outside a plausible range. Inappropriate data cleaning has the potential to bias study outcomes [ 53 ]; providing the reasoning for removing data points enables the distinction to be made between responsible data cleaning and data manipulation. Missing data, common in all areas of research, can impact the sensitivity of the study and also lead to biased estimates, distorted power, and loss of information if the missing values are not random [ 54 ]. Analysis plans should include methods to explore why data are missing. It is also important to consider and justify analysis methods that account for missing data [ 55 , 56 ].

There is a movement toward greater data sharing (see Item 20. Data access), along with an increase in strategies such as code sharing to enable analysis replication. These practices, however transparent, still need to be accompanied by a disclosure on the reasoning for data cleaning and whether methods were defined before any data were collected.

Report all animal exclusions and loss of data points, along with the rationale for their exclusion. For example, this information can be summarised as a table or a flowchart describing attrition in each treatment group. Accompanying this information should be an explicit description of whether researchers were blinded to the group allocations when data or animals were excluded (see Item 5. Blinding and [ 57 ]). Explicitly state when built-in models in statistics packages have been used to remove outliers (e.g., GraphPad Prism’s outlier test).

Subitem 3b—Example 1

‘Pen was the experimental unit for all data. One entire pen (ZnAA90) was removed as an outlier from both Pre-RAC and RAC periods for poor performance caused by illness unrelated to treatment…. Outliers were determined using Cook’s D statistic and removed if Cook’s D > 0.5. One steer was determined to be an outlier for day 48 liver biopsy TM and data were removed’ [ 58 ].

Subitem 3b—Example 2

‘Seventy-two SHRs were randomized into the study, of which 13 did not meet our inclusion and exclusion criteria because the drop in cerebral blood flow at occlusion did not reach 60% (seven animals), postoperative death (one animal: autopsy unable to identify the cause of death), haemorrhage during thread insertion (one animal), and disconnection of the silicon tip of the thread during withdrawal, making the permanence of reperfusion uncertain (four animals). A total of 59 animals were therefore included in the analysis of infarct volume in this study. In error, three animals were sacrificed before their final assessment of neurobehavioral score: one from the normothermia/water group and two from the hypothermia/pethidine group. These errors occurred blinded to treatment group allocation. A total of 56 animals were therefore included in the analysis of neurobehavioral score’ [ 51 ].

Subitem 3b—Example 3



3c . For each analysis, report the exact value of n in each experimental group .

Explanation. The exact number of experimental units analysed in each group (i.e., the n number) is essential information for the reader to interpret the analysis; it should be reported unambiguously. All animals and data used in the experiment should be accounted for in the data presented. Sometimes, for good reasons, animals may need to be excluded from a study (e.g., illness or mortality), or data points excluded from analyses (e.g., biologically implausible values). Reporting losses will help the reader to understand the experimental design process, replicate methods, and provide adequate tracking of animal numbers in a study, especially when sample size numbers in the analyses do not match the original group numbers.

For each outcome measure, indicate numbers clearly within the text or on figures and provide absolute numbers (e.g., 10/20, not 50%). For studies in which animals are measured at different time points, explicitly report the full description of which animals undergo measurement and when [ 33 ].

Subitem 3c—Example 1

‘Group F contained 29 adult males and 58 adult females in 2010 ( n = 87), and 32 adult males and 66 adult females in 2011 ( n = 98). The increase in female numbers was due to maturation of juveniles to adults. Females belonged to three matrilines, and there were no major shifts in rank in the male hierarchy. Six mid to low ranking individuals died and were excluded from analyses, as were five mid-ranking males who emigrated from the group at the beginning of 2011’ [ 60 ].

Subitem 3c—Example 2

‘The proportion of test time that animals spent interacting with the handler (sniffed the gloved hand or tunnel, made paw contact, climbed on, or entered the handling tunnel) was measured from DVD recordings. This was then averaged across the two mice in each cage as they were tested together and their behaviour was not independent…. Mice handled with the home cage tunnel spent a much greater proportion of the test interacting with the handler (mean ± s.e.m., 39.8 ± 5.2 percent time of 60 s test, n = 8 cages) than those handled by tail (6.4 ± 2.0 percent time, n = 8 cages), while those handled by cupping showed intermediate levels of voluntary interaction (27.6 ± 7.1 percent time, n = 8 cages)’ [ 61 ].

Item 4. Randomisation

4a . State whether randomisation was used to allocate experimental units to control and treatment groups . If done, provide the method used to generate the randomisation sequence .

Explanation. Using appropriate randomisation methods during the allocation to groups ensures that each experimental unit has an equal probability of receiving a particular treatment and provides balanced numbers in each treatment group. Selecting an animal ‘at random’ (i.e., haphazardly or arbitrarily) from a cage is not statistically random, as the process involves human judgement. It can introduce bias that influences the results, as a researcher may (consciously or subconsciously) make judgements in allocating an animal to a particular group, or because of unknown and uncontrolled differences in the experimental conditions or animals in different groups. Using a validated method of randomisation helps minimise selection bias and reduce systematic differences in the characteristics of animals allocated to different groups [ 62 – 64 ]. Inferential statistics based on nonrandomised group allocation are not valid [ 65 , 66 ]. Thus, the use of randomisation is a prerequisite for any experiment designed to test a hypothesis. Examples of appropriate randomisation methods include online random number generators (e.g., https://www.graphpad.com/quickcalcs/randomize1/ ) or a function like Rand() in spreadsheet software such as Excel, Google Sheets, or LibreOffice. The EDA has a dedicated feature for randomisation and allocation concealment [ 19 ].

Systematic reviews have shown that animal experiments that do not report randomisation or other bias-reducing measures such as blinding are more likely to report exaggerated effects that meet conventional measures of statistical significance [ 67 – 69 ]. It is especially important to use randomisation in situations in which it is not possible to blind all or parts of the experiment, but even with randomisation, researcher bias can pervert the allocation. This can be avoided by using allocation concealment (see Item 5. Blinding). In studies in which sample sizes are small, simple randomisation may result in unbalanced groups; here, randomisation strategies to balance groups such as randomising in matched pairs [ 70 – 72 ] and blocking are encouraged [ 17 ]. Reporting the precise method used to allocate animals or experimental units to groups enables readers to assess the reliability of the results and identify potential limitations.

Report the type of randomisation used (simple, stratified, randomised complete blocks, etc.; see Box 4 ), the method used to generate the randomisation sequence (e.g., computer-generated randomisation sequence, with details of the algorithm or programme used), and what was randomised (e.g., treatment to experimental unit, order of treatment for each animal). If this varies between experiments, report this information specifically for each experiment. If randomisation was not the method used to allocate experimental units to groups, state this explicitly and explain how the groups being compared were formed.

Box 4. Considerations for the randomisation strategy

Simple randomisation

All animals/samples are simultaneously randomised to the treatment groups without considering any other variable. This strategy is rarely appropriate, as it cannot ensure that comparison groups are balanced for other variables that might influence the result of an experiment.

Randomisation within blocks

Blocking is a method of controlling natural variation among experimental units. This splits up the experiment into smaller subexperiments (blocks), and treatments are randomised to experimental units within each block [ 17 , 66 , 73 ]. This takes into account nuisance variables that could potentially bias the results (e.g., cage location, day or week of procedure).

Stratified randomisation uses the same principle as randomisation within blocks, only the strata tend to be traits of the animal that are likely to be associated with the response (e.g., weight class or tumour size class). This can lead to differences in the practical implementation of stratified randomisation as compared with block randomisation (e.g., there may not be equal numbers of experimental units in each weight class).

Other randomisation strategies

Minimisation is an alternative strategy to allocate animals/samples to treatment group to balance variables that might influence the result of an experiment. With minimisation, the treatment allocated to the next animal/sample depends on the characteristics of those animals/samples already assigned. The aim is that each allocation should minimise the imbalance across multiple factors [ 74 ]. This approach works well for a continuous nuisance variable such as body weight or starting tumour volume.

Examples of nuisance variables that can be accounted for in the randomisation strategy

Implication for the analysis

If blocking factors are used in the randomisation, they should also be included in the analysis. Nuisance variables increase variability in the sample, which reduces statistical power. Including a nuisance variable as a blocking factor in the analysis accounts for that variability and can increase the power, thus increasing the ability to detect a real effect with fewer experimental units. However, blocking uses up degrees of freedom and thus reduces the power if the nuisance variable does not have a substantial impact on variability.

Subitem 4a—Example 1

‘Fifty 12-week-old male Sprague-Dawley rats, weighing 320–360g, were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and randomly divided into two groups (25 rats/group): the intact group and the castration group. Random numbers were generated using the standard = RAND() function in Microsoft Excel’ [ 75 ].

Subitem 4a—Example 2

‘Animals were randomized after surviving the initial I/R, using a computer based random order generator’ [ 76 ].

Subitem 4a—Example 3

‘At each institute, phenotyping data from both sexes is collected at regular intervals on age-matched wildtype mice of equivalent genetic backgrounds. Cohorts of at least seven homozygote mice of each sex per pipeline were generated…. The random allocation of mice to experimental group (wildtype versus knockout) was driven by Mendelian Inheritance’ [ 29 ].

4b . Describe the strategy used to minimise potential confounders such as the order of treatments and measurements, or animal/cage location . If confounders were not controlled, state this explicitly .

Explanation. Ensuring there is no systematic difference between animals in different groups apart from the experimental exposure is an important principle throughout the conduct of the experiment. Identifying nuisance variables (sources of variability or conditions that could potentially bias results) and managing them in the design and analysis increases the sensitivity of the experiment. For example, rodents in cages at the top of the rack may be exposed to higher light levels, which can affect stress [ 77 ].

Reporting the strategies implemented to minimise potential differences that arise between treatment groups during the course of the experiment enables others to assess the internal validity. Strategies to report include standardising (keeping conditions the same, e.g., all surgeries done by the same surgeon), randomising (e.g., the sampling or measurement order), and blocking or counterbalancing (e.g., position of animal cages or tanks on the rack), to ensure groups are similarly affected by a source of variability. In some cases, practical constraints prevent some nuisance variables from being randomised, but they can still be accounted for in the analysis (see Item 7. Statistical methods).

Report the methods used to minimise confounding factors alongside the methods used to allocate animals to groups. If no measures were used to minimise confounders (e.g., treatment order, measurement order, cage or tank position on a rack), explicitly state this and explain why.

Subitem 4b—Example 1

‘Randomisation was carried out as follows. On arrival from El-Nile Company, animals were assigned a group designation and weighed. A total number of 32 animals were divided into four different weight groups (eight animals per group). Each animal was assigned a temporary random number within the weight range group. On the basis of their position on the rack, cages were given a numerical designation. For each group, a cage was selected randomly from the pool of all cages. Two animals were removed from each weight range group and given their permanent numerical designation in the cages. Then, the cages were randomized within the exposure group’ [ 78 ].

Subitem 4b—Example 2

‘… test time was between 08.30am to 12.30pm and testing order was randomized daily, with each animal tested at a different time each test day’ [ 79 ].

Subitem 4b—Example 3

‘Bulls were blocked by BW into four blocks of 905 animals with similar BW and then within each block, bulls were randomly assigned to one of four experimental treatments in a completely randomized block design resulting in 905 animals per treatment. Animals were allocated to 20 pens (181 animals per pen and five pens per treatment)’ [ 80 ].

Item 5. Blinding

Describe who was aware of the group allocation at the different stages of the experiment (during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis) .

Explanation. Researchers often expect a particular outcome and can unintentionally influence the experiment or interpret the data in such a way as to support their preferred hypothesis [ 81 ]. Blinding is a strategy used to minimise these subjective biases.

Although there is primary evidence of the impact of blinding in the clinical literature that directly compares blinded versus unblinded assessment of outcomes [ 82 ], there is limited empirical evidence in animal research [ 83 , 84 ]. There are, however, compelling data from systematic reviews showing that nonblinded outcome assessment leads to the treatment effects being overestimated, and the lack of bias-reducing measures such as randomisation and blinding can contribute to as much as 30%–45% inflation of effect sizes [ 67 , 68 , 85 ].

Ideally, investigators should be unaware of the treatment(s) animals have received or will be receiving, from the start of the experiment until the data have been analysed. If this is not possible for every stage of an experiment (see Box 5 ), it should always be possible to conduct at least some of the stages blind. This has implications for the organisation of the experiment and may require help from additional personnel—for example, a surgeon to perform interventions, a technician to code the treatment syringes for each animal, or a colleague to code the treatment groups for the analysis. Online resources are available to facilitate allocation concealment and blinding [ 19 ].

Box 5. Blinding during different stages of an experiment

During allocation

Allocation concealment refers to concealing the treatment to be allocated to each individual animal from those assigning the animals to groups, until the time of assignment. Together with randomisation, allocation concealment helps minimise selection bias, which can introduce systematic differences between treatment groups.

During the conduct of the experiment

When possible, animal care staff and those who administer treatments should be unaware of allocation groups to ensure that all animals in the experiment are handled, monitored, and treated in the same way. Treating different groups differently based on the treatment they have received could alter animal behaviour and physiology and produce confounds.

Welfare or safety reasons may prevent blinding of animal care staff, but in most cases, blinding is possible. For example, if hazardous microorganisms are used, control animals can be considered as dangerous as infected animals. If a welfare issue would only be tolerated for a short time in treated but not control animals, a harm-benefit analysis is needed to decide whether blinding should be used.

During the outcome assessment

The person collecting experimental measurements or conducting assessments should not know which treatment each sample/animal received and which samples/animals are grouped together. Blinding is especially important during outcome assessment, particularly if there is a subjective element (e.g., when assessing behavioural changes or reading histological slides) [ 83 ]. Randomising the order of examination can also reduce bias.

If the person assessing the outcome cannot be blinded to the group allocation (e.g., obvious phenotypic or behavioural differences between groups), some, but not all, of the sources of bias could be mitigated by sending data for analysis to a third party who has no vested interest in the experiment and does not know whether a treatment is expected to improve or worsen the outcome.

During the data analysis

The person analysing the data should know which data are grouped together to enable group comparisons but should not be aware of which specific treatment each group received. This type of blinding is often neglected but is important, as the analyst makes many semisubjective decisions such as applying data transformation to outcome measures, choosing methods for handling missing data, and handling outliers. How these decisions will be made should also be decided a priori.

Data can be coded prior to analysis so that the treatment group cannot be identified before analysis is completed.

Specify whether blinding was used or not for each step of the experimental process (see Box 5 ) and indicate what particular treatment or condition the investigators were blinded to, or aware of.

If blinding was not used at any of the steps outlined in Box 5 , explicitly state this and provide the reason why blinding was not possible or not considered.

Item 5—Example 1

‘For each animal, four different investigators were involved as follows: a first investigator (RB) administered the treatment based on the randomization table. This investigator was the only person aware of the treatment group allocation. A second investigator (SC) was responsible for the anaesthetic procedure, whereas a third investigator (MS, PG, IT) performed the surgical procedure. Finally, a fourth investigator (MAD) (also unaware of treatment) assessed GCPS and NRS, mechanical nociceptive threshold (MNT), and sedation NRS scores’ [ 46 ].

Item 5—Example 2

‘… due to overt behavioral seizure activity the experimenter could not be blinded to whether the animal was injected with pilocarpine or with saline’ [ 86 ].

Item 5—Example 3

‘Investigators could not be blinded to the mouse strain due to the difference in coat colors, but the three-chamber sociability test was performed with ANY-maze video tracking software (Stoelting, Wood Dale, IL, USA) using an overhead video camera system to automate behavioral testing and provide unbiased data analyses. The one-chamber social interaction test requires manual scoring and was analyzed by an individual with no knowledge of the questions’ [ 87 ].

Item 6. Outcome measures

6a . Clearly define all outcome measures assessed (e.g., cell death, molecular markers, or behavioural changes) .

Explanation. An outcome measure (also known as a dependent variable or a response variable) is any variable recorded during a study (e.g., volume of damaged tissue, number of dead cells, specific molecular marker) to assess the effects of a treatment or experimental intervention. Outcome measures may be important for characterising a sample (e.g., baseline data) or for describing complex responses (e.g., ‘haemodynamic’ outcome measures including heart rate, blood pressure, central venous pressure, and cardiac output). Failure to disclose all the outcomes that were measured introduces bias in the literature, as positive outcomes (e.g., those statistically significant) are reported more often [ 88 – 91 ].

Explicitly describe what was measured, especially when measures can be operationalised in different ways. For example, activity could be recorded as time spent moving or distance travelled. When possible, the recording of outcome measures should be made in an unbiased manner (e.g., blinded to the treatment allocation of each experimental group; see Item 5. Blinding). Specify how the outcome measure(s) assessed are relevant to the objectives of the study.

Subitem 6a—Example 1

‘The following parameters were assessed: threshold pressure (TP; intravesical pressure immediately before micturition); post-void pressure (PVP; intravesical pressure immediately after micturition); peak pressure (PP; highest intravesical pressure during micturition); capacity (CP; volume of saline needed to induce the first micturition); compliance (CO; CP to TP ratio); frequency of voiding contractions (VC) and frequency of non-voiding contractions (NVCs)’ [ 92 ].

6b . For hypothesis-testing studies, specify the primary outcome measure, i.e., the outcome measure that was used to determine the sample size .

Explanation. In a hypothesis-testing experiment, the primary outcome measure answers the main biological question. It is the outcome of greatest importance, identified in the planning stages of the experiment and used as the basis for the sample size calculation (see Box 3 ). For exploratory studies, it is not necessary to identify a single primary outcome, and often multiple outcomes are assessed (see Item 13. Objectives).

In a hypothesis-testing study powered to detect an effect on the primary outcome measure, data on secondary outcomes are used to evaluate additional effects of the intervention, but subsequent statistical analysis of secondary outcome measures may be underpowered, making results and interpretation less reliable [ 88 , 93 ]. Studies that claim to test a hypothesis but do not specify a predefined primary outcome measure or those that change the primary outcome measure after data were collected (also known as primary outcome switching) are liable to selectively report only statistically significant results, favouring more positive findings [ 94 ].

Registering a protocol in advance protects the researcher against concerns about selective outcome reporting (also known as data dredging or p-hacking) and provides evidence that the primary outcome reported in the manuscript accurately reflects what was planned [ 95 ] (see Item 19. Protocol registration).

In studies using inferential statistics to test a hypothesis (e.g., t test, ANOVA), if more than one outcome was assessed, explicitly identify the primary outcome measure, state whether it was defined as such prior to data collection and whether it was used in the sample size calculation. If there was no primary outcome measure, explicitly state so.

Subitem 6b—Example 1

‘The primary outcome of this study will be forelimb function assessed with the staircase test. Secondary outcomes constitute Rotarod performance, stroke volume (quantified on MR imaging or brain sections, respectively), diffusion tensor imaging (DTI) connectome mapping, and histological analyses to measure neuronal and microglial densities, and phagocytic activity…. The study is designed with 80% power to detect a relative 25% difference in pellet-reaching performance in the Staircase test’ [ 96 ].

Subitem 6b—Example 2

‘The primary endpoint of this study was defined as left ventricular ejection fraction (EF) at the end of follow-up, measured by magnetic resonance imaging (MRI). Secondary endpoints were left ventricular end diastolic volume and left ventricular end systolic volume (EDV and ESV) measured by MRI, infarct size measured by ex vivo gross macroscopy after incubation with triphenyltetrazolium chloride (TTC) and late gadolinium enhancement (LGE) MRI, functional parameters serially measured by pressure volume (PV-)loop and echocardiography, coronary microvascular function by intracoronary pressure- and flow measurements and vascular density and fibrosis on histology’ [ 76 ].

Item 7. Statistical methods

7a . Provide details of the statistical methods used for each analysis, including software used .

Explanation. The statistical analysis methods implemented will reflect the goals and the design of the experiment; they should be decided in advance before data are collected (see Item 19. Protocol registration). Both exploratory and hypothesis-testing studies might use descriptive statistics to summarise the data (e.g., mean and SD, or median and range). In exploratory studies in which no specific hypothesis was tested, reporting descriptive statistics is important for generating new hypotheses that may be tested in subsequent experiments, but it does not allow conclusions beyond the data. In addition to descriptive statistics, hypothesis-testing studies might use inferential statistics to test a specific hypothesis.

Reporting the analysis methods in detail is essential to ensure readers and peer reviewers can assess the appropriateness of the methods selected and judge the validity of the output. The description of the statistical analysis should provide enough detail so that another researcher could reanalyse the raw data using the same method and obtain the same results. Make it clear which method was used for which analysis.

Analysing the data using different methods and selectively reporting those with statistically significant results constitutes p-hacking and introduces bias in the literature [ 90 , 94 ]. Report all analyses performed in full. Relevant information to describe the statistical methods include

The outcome measure is potentially affected by the treatments or interventions being tested but also by other factors, such as the properties of the biological samples (sex, litter, age, weight, etc.) and technical considerations (cage, time of day, batch, experimenter, etc.). To reduce the risk of bias, some of these factors can be taken into account in the design of the experiment, for example, by using blocking factors in the randomisation (see Item 4. Randomisation). Factors deemed to affect the variability of the outcome measure should also be handled in the analysis, for example, as a blocking factor (e.g., batch of reagent or experimenter) or as a covariate (e.g., starting tumour size at point of randomisation).

Furthermore, to conduct the analysis appropriately, it is important to recognise the hierarchy that can exist in an experiment. The hierarchy can induce a clustering effect; for example, cage, litter, or animal effects can occur when the outcomes measured for animals from the same cage/litter, or for cells from the same animal, are more similar to each other. This relationship has to be managed in the statistical analysis by including cage/litter/animal effects in the model or by aggregating the outcome measure to the cage/litter/animal level. Thus, describing the reality of the experiment and the hierarchy of the data, along with the measures taken in the design and the analysis to account for this hierarchy, is crucial to assessing whether the statistical methods used are appropriate.

For bespoke analysis—for example, regression analysis with many terms—it is essential to describe the analysis pipeline in detail. This could include detailing the starting model and any model simplification steps.

When reporting descriptive statistics, explicitly state which measure of central tendency is reported (e.g., mean or median) and which measure of variability is reported (e.g., standard deviation, range, quartiles, or interquartile range). Also describe any modification made to the raw data before analysis (e.g., relative quantification of gene expression against a housekeeping gene). For further guidance on statistical reporting, refer to the Statistical Analyses and Methods in the Published Literature (SAMPL) guidelines [ 98 ].

Subitem 7a—Example 1

‘Analysis of variance was performed using the GLM procedure of SAS (SAS Inst., Cary, NC). Average pen values were used as the experimental unit for the performance parameters. The model considered the effects of block and dietary treatment (5 diets). Data were adjusted by the covariant of initial body weight. Orthogonal contrasts were used to test the effects of SDPP processing (UV vs no UV) and dietary SDPP level (3% vs 6%). Results are presented as least squares means. The level of significance was set at P < 0.05’ [ 99 ].

Subitem 7a—Example 2

‘All risk factors of interest were investigated in a single model. Logistic regression allows blocking factors and explicitly investigates the effect of each independent variable controlling for the effects of all others…. As we were interested in husbandry and environmental effects, we blocked the analysis by important biological variables (age; backstrain; inbreeding; sex; breeding status) to control for their effect. (The role of these biological variables in barbering behavior, particularly with reference to barbering as a model for the human disorder trichotillomania, is described elsewhere …). We also blocked by room to control for the effect of unknown environmental variables associated with this design variable. We tested for the effect of the following husbandry and environmental risk factors: cage mate relationships (i.e. siblings, non-siblings, or mixed); cage type (i.e. plastic or steel); cage height from floor; cage horizontal position (whether the cage was on the side or the middle of a rack); stocking density; and the number of adults in the cage. Cage material by cage height from floor; and cage material by cage horizontal position interactions were examined, and then removed from the model as they were nonsignificant. N = 1959 mice were included in this analysis’ [ 100 ].

7b . Describe any methods used to assess whether the data met the assumptions of the statistical approach, and what was done if the assumptions were not met .

Explanation. Hypothesis tests are based on assumptions about the underlying data. Describing how assumptions were assessed and whether these assumptions are met by the data enables readers to assess the suitability of the statistical approach used. If the assumptions are incorrect, the conclusions may not be valid. For example, the assumptions for data used in parametric tests (such as a t test, z test, ANOVA, etc.) are that the data are continuous, the residuals from the analysis are normally distributed, the responses are independent, and different groups have similar variances.

There are various tests for normality, for example, the Shapiro-Wilk and Kolmogorov-Smirnov tests. However, these tests have to be used cautiously. If the sample size is small, they will struggle to detect non-normality; if the sample size is large, the tests will detect unimportant deviations. An alternative approach is to evaluate data with visual plots, e.g., normal probability plots, box plots, scatterplots. If the residuals of the analysis are not normally distributed, the assumption may be satisfied using a data transformation in which the same mathematical function is applied to all data points to produce normally distributed data (e.g., log e , log 10 , square root).

Other types of outcome measures (binary, categorical, or ordinal) will require different methods of analysis, and each will have different sets of assumptions. For example, categorical data are summarised by counts and percentages or proportions and are analysed by tests of proportions; these analysis methods assume that data are binary, ordinal or nominal, and independent [ 18 ].

For each statistical test used (parametric or nonparametric), report the type of outcome measure and the methods used to test the assumptions of the statistical approach. If data were transformed, identify precisely the transformation used and which outcome measures it was applied to. Report any changes to the analysis if the assumptions were not met and an alternative approach was used (e.g., a nonparametric test was used, which does not require the assumption of normality). If the relevant assumptions about the data were not tested, state this explicitly.

Subitem 7b—Example 1

‘Model assumptions were checked using the Shapiro-Wilk normality test and Levene’s Test for homogeneity of variance and by visual inspection of residual and fitted value plots. Some of the response variables had to be transformed by applying the natural logarithm or the second or third root, but were back-transformed for visualization of significant effects’ [ 101 ].

Subitem 7b—Example 2

‘The effects of housing (treatment) and day of euthanasia on cortisol levels were assessed by using fixed-effects 2-way ANOVA. An initial exploratory analysis indicated that groups with higher average cortisol levels also had greater variation in this response variable. To make the variation more uniform, we used a logarithmic transform of each fish’s cortisol per unit weight as the dependent variable in our analyses. This action made the assumptions of normality and homoscedasticity (standard deviations were equal) of our analyses reasonable’ [ 102 ].

Item 8. Experimental animals

8a . Provide species-appropriate details of the animals used, including species, strain and substrain, sex, age or developmental stage, and, if relevant, weight .

Explanation. The species, strain, substrain, sex, weight, and age of animals are critical factors that can influence most experimental results [ 103 – 107 ]. Reporting the characteristics of all animals used is equivalent to standardised human patient demographic data; these data support both the internal and external validity of the study results. It enables other researchers to repeat the experiment and generalise the findings. It also enables readers to assess whether the animal characteristics chosen for the experiment are relevant to the research objectives.

When reporting age and weight, include summary statistics for each experimental group (e.g., mean and standard deviation) and, if possible, baseline values for individual animals (e.g., as supplementary information or a link to a publicly accessible data repository). As body weight might vary during the course of the study, indicate when the measurements were taken. For most species, precise reporting of age is more informative than a description of the developmental status (e.g., a mouse referred to as an adult can vary in age from 6 to 20 weeks [ 108 ]). In some cases, however, reporting the developmental stage is more informative than chronological age—for example, in juvenile Xenopus , in which rate of development can be manipulated by incubation temperature [ 109 ].

Reporting the weight or the sex of the animals used may not feasible for all studies. For example, sex may be unknown for embryos or juveniles, or weight measurement may be particularly stressful for some aquatic species. If reporting these characteristics can be reasonably expected for the species used and the experimental setting but are not reported, provide a justification.

Subitem 8a—Example 1

‘One hundred and nineteen male mice were used: C57BL/6OlaHsd mice ( n = 59), and BALB/c OlaHsd mice ( n = 60) (both from Harlan, Horst, The Netherlands). At the time of the EPM test the mice were 13 weeks old and had body weights of 27.4 ± 0.4 g and 27.8 ± 0.3 g, respectively (mean ± SEM)’ [ 110 ].

Subitem 8a—Example 2

‘Histone Methylation Profiles and the Transcriptome of X . tropicalis Gastrula Embryos. To generate epigenetic profiles, ChIP was performed using specific antibodies against trimethylated H3K4 and H3K27 in Xenopus gastrula-stage embryos (Nieuwkoop-Faber stage 11–12), followed by deep sequencing (ChIP-seq). In addition, polyA-selected RNA (stages 10–13) was reverse transcribed and sequenced (RNA-seq)’ [ 111 ].

8b . Provide further relevant information on the provenance of animals, health/immune status, genetic modification status, genotype, and any previous procedures .

Explanation. The animals’ provenance, their health or immune status, and their history of previous testing or procedures can influence their physiology and behaviour, as well as their response to treatments, and thus impact on study outcomes. For example, animals of the same strain but from different sources, or animals obtained from the same source but at different times, may be genetically different [ 16 ]. The immune or microbiological status of the animals can also influence welfare, experimental variability, and scientific outcomes [ 112 – 114 ].

Report the health status of all animals used in the study and any previous procedures the animals have undergone. For example, if animals are specific pathogen free (SPF), list the pathogens that they were declared free of. If health status is unknown or was not tested, explicitly state this.

For genetically modified animals, describe the genetic modification status (e.g., knockout, overexpression), genotype (e.g., homozygous, heterozygous), manipulated gene(s), genetic methods and technologies used to generate the animals, how the genetic modification was confirmed, and details of animals used as controls (e.g., littermate controls [ 115 ]).

Reporting the correct nomenclature is crucial to understanding the data and ensuring that the research is discoverable and replicable [ 116 – 118 ]. Useful resources for reporting nomenclature for different species include

Subitem 8b—Example 1

‘A construct was engineered for knockin of the mi R-128 ( mi R-128-3p) gene into the Rosa26 locus. Rosa26 genomic DNA fragments (~1.1 kb and ~4.3 kb 5′ and 3′ homology arms, respectively) were amplified from C57BL/6 BAC DNA, cloned into the pBasicLNeoL vector sequentially by in-fusion cloning, and confirmed by sequencing. The mi R-128 gene, under the control of tetO-minimum promoter, was also cloned into the vector between the two homology arms. In addition, the targeting construct also contained a loxP sites flanking the neomycin resistance gene cassette for positive selection and a diphtheria toxin A (DTA) cassette for negative selection. The construct was linearized with ClaI and electroporated into C57BL/6N ES cells. After G418 selection, seven-positive clones were identified from 121 G418-resistant clones by PCR screening. Six-positive clones were expanded and further analyzed by Southern blot analysis, among which four clones were confirmed with correct targeting with single-copy integration. Correctly targeted ES cell clones were injected into blastocysts, and the blastocysts were implanted into pseudo-pregnant mice to generate chimeras by Cyagen Biosciences Inc. Chimeric males were bred with Cre deleted mice from Jackson Laboratories to generate neomycin-free knockin mice. The correct insertion of the mi R-128 cassette and successful removal of the neomycin cassette were confirmed by PCR analysis with the primers listed in Supplementary Table… ’ [ 119 ].

Subitem 8b—Example 2

‘The C57BL/6J (Jackson) mice were supplied by Charles River Laboratories. The C57BL/6JOlaHsd (Harlan) mice were supplied by Harlan. The α-synuclein knockout mice were kindly supplied by Prof…. (Cardiff University, Cardiff, United Kingdom.) and were congenic C57BL/6JCrl (backcrossed for 12 generations). TNFα−/− mice were kindly supplied by Dr…. (Queens University, Belfast, Northern Ireland) and were inbred on a homozygous C57BL/6J strain originally sourced from Bantin & Kingman and generated by targeting C57BL/6 ES cells. T286A mice were obtained from Prof…. (University of California, Los Angeles, CA). These mice were originally congenic C57BL/6J (backcrossed for five generations) and were then inbred (cousin matings) over 14 y, during which time they were outbred with C57BL/6JOlaHsd mice on three separate occasions’ [ 120 ].

Item 9. Experimental procedures

For each experimental group, including controls, describe the procedures in enough detail to allow others to replicate them, including :

9a . What was done, how it was done, and what was used .

Explanation. Essential information to describe in the manuscript includes the procedures used to develop the model (e.g., induction of the pathology), the procedures used to measure the outcomes, and pre- and postexperimental procedures, including animal handling, welfare monitoring, and euthanasia. Animal handling can be a source of stress, and the specific method used (e.g., mice picked up by tail or in cupped hands) can affect research outcomes [ 61 , 121 , 122 ]. Details about animal care and monitoring intrinsic to the procedure are discussed in further detail in Item 16. Animal care and monitoring. Provide enough detail to enable others to replicate the methods and highlight any quality assurance and quality control used [ 123 , 124 ]. A schematic of the experimental procedures with a timeline can give a clear overview of how the study was conducted. Information relevant to distinct types of interventions and resources are described in Table 1 .



When available, cite the Research Resource Identifier (RRID) for reagents and tools used [ 126 , 127 ]. RRIDs are unique and stable, allowing unambiguous identification of reagents or tools used in a study, aiding other researchers to replicate the methods.

Detailed step-by-step procedures can also be saved and shared online, for example, using Protocols.io [ 128 ], which assigns a digital object identifier (DOI) to the protocol and allows cross-referencing between protocols and publications.

Subitem 9a—Example 1



Subitem 9a—Example 2

‘For the diet-induced obesity (DIO) model, eight-week-old male mice had ad libitum access to drinking water and were kept on standard chow (SFD, 10.9 kJ/g) or on western high-fat diet (HFD; 22 kJ/g; kcal from 42% fat, 43% from carbohydrates and 15% from protein; E15721-34, Ssniff, Soest, Germany) for 15 weeks ( https://dx.doi.org/10.17504/protocols.io.kbacsie )’ [ 130 ].

Subitem 9a—Example 3

‘The frozen kidney tissues were lysed. The protein concentration was determined with the Pierce BCA assay kit (catalogue number 23225; Thermo Fisher Scientific, Rockford, IL, USA). A total of 100–150 μg total proteins were resolved on a 6–12% SDS-PAGE gel. The proteins were then transferred to a nitrocellulose membrane, blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against the following proteins: proliferating cell nuclear antigen (PCNA; Cat# 2586, RRID: AB_2160343), phospho-AMPK (Cat# 2531, RRID: AB_330330), phospho-mTOR (Cat# 2971, RRID: AB_330970)…. The β-actin (Cat# A5441, RRID: AB_476744) antibody was obtained from Sigma. The blots were subsequently probed with HRP-conjugated anti-mouse (Cat# A0216) or anti-rabbit IgG (Cat# A0208; Beyotime Biotechnology, Beijing, China) at 1:1000. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using ImageJ software (RRID: SCR_003070, Bio-Rad Laboratories)’ [ 131 ].

9b . When and how often .

Explanation. Clearly report the frequency and timing of experimental procedures and measurements, including the light and dark cycle (e.g., 12L:12D), circadian time cues (e.g., lights on at 8:00 AM), and experimental time sequence (e.g., interval between baseline and comparator measurements or interval between procedures and measurements). Along with innate circadian rhythms, these can affect research outcomes such as behavioural, physiological, and immunological parameters [ 132 , 133 ]. Also report the timing and frequency of welfare assessments, taking into consideration the normal activity patterns (see Item 16. Animal care and monitoring). For example, nocturnal animals may not show behavioural signs of discomfort during the day [ 134 ].

If the timing of procedures or measurements varies between animals, this information can be provided as a supplementary table listing each animal.

Subitem 9b—Example 1

‘Blood pressure, heart rate, oxygen saturation and amount of blood extracted were recorded every 5 minutes. Blood samples were drawn at baseline (pre injury), 0 minutes (immediately after injury), and after 30 and 60 minutes’ [ 135 ].

Subitem 9b—Example 2

‘After a 5-h fast (7:30–12:30am), awake and freely moving mice were randomized and subjected to three consecutive clamps performed in the same mice as described above, with a 2 days recovery after each hyperinsulinemic/hypoglycemic (mHypo, n = 6) or hyperinsulinemic/euglycemic (mEugly, n = 4) clamps’ [ 136 ].

9c . Where (including detail of any acclimatisation periods) .

Explanation. Physiological acclimatisation after a stressful event, such as transport (e.g., between supplier, animal facility, operating theatre, and laboratory), but before the experiment begins allows stabilisation of physiological responses of the animal [ 137 , 138 ]. Protocols vary depending on species, strain, and outcome; for example, physiological acclimatisation following transportation of different animals can take anywhere from 24 hours to more than 1 week [ 139 ]. Procedural acclimatisation immediately before a procedure allows stabilisation of the animals’ responses after unaccustomed handling, novel environments, and previous procedures, which otherwise can induce behavioural and physiological changes [ 140 , 141 ]. Standard acclimatisation periods may vary between research laboratories, and this information cannot be inferred by readers.

Indicate where studies were performed (e.g., dedicated laboratory space or animal facility, home cage, open field arena, water maze) and whether periods of physiological or procedural acclimatisation were included in the study protocol, including type and duration. If the study involved multiple sites, explicitly state where each experiment and sample analysis was performed. Include any accreditation of laboratories if appropriate (e.g., if samples were sent to a commercial laboratory for analysis).

Subitem 9c –example 1

‘Fish were singly housed for 1 week before being habituated to the conditioning tank over 2 consecutive days. The conditioning tank consisted of an opaque tank measuring 20 cm (w) 15 cm (h) 30 cm (l) containing 2.5L of aquarium water with distinct visual cues (spots or stripes) on walls at each end of the tank…. During habituation, each individual fish was placed in the conditioning apparatus for 20 minutes with free access to both compartments and then returned to its home tank’ [ 142 ].

9d . Why (provide rationale for procedures) .

Explanation. There may be numerous approaches to investigate any given research problem; therefore, it is important to explain why a particular procedure or technique was chosen. This is especially relevant when procedures are novel or specific to a research laboratory or constrained by the animal model or experimental equipment (e.g., route of administration determined by animal size [ 143 ]).

Subitem 9d—Example 1

‘Because of the very small caliber of the murine tail veins, partial paravenous injection is common if 18 F-FDG is administered by tail vein injection (intravenous). This could have significantly biased our comparison of the biodistribution of 18 F-FDG under various conditions. Therefore, we used intraperitoneal injection of 18 F-FDG for our experiments evaluating the influence of animal handling on 18 F-FDG biodistribution’ [ 144 ].

Subitem 9d—Example 2

‘Since Xenopus oocytes have a higher potential for homologous recombination than fertilized embryos… we next tested whether the host transfer method could be used for efficient HDR-mediated knock-in. We targeted the C-terminus of X . laevis Ctnnb1 (β-catenin), a key cytoskeletal protein and effector of the canonical Wnt pathway, because previous studies have shown that addition of epitope tags to the C-terminus do not affect the function of the resulting fusion protein (Fig …). CRISPR components were injected into X . laevis oocytes followed by host transfer or into embryos’ [ 145 ].

Item 10. Results

For each experiment conducted, including independent replications, report :

10a . Summary/descriptive statistics for each experimental group, with a measure of variability where applicable (e.g., mean and SD, or median and range) .

Explanation. Summary/descriptive statistics provide a quick and simple description of the data; they communicate quantitative results easily and facilitate visual presentation. For continuous data, these descriptors include a measure of central tendency (e.g., mean, median) and a measure of variability (e.g., quartiles, range, standard deviation) to help readers assess the precision of the data collected. Categorical data can be expressed as counts, frequencies, or proportions.

Report data for all experiments conducted. If a complete experiment is repeated on a different day or under different conditions, report the results of all repeats rather than selecting data from representative experiments. Report the exact number of experimental units per group so readers can gauge the reliability of the results (see Item 2. Sample size and Item 3. Inclusion and exclusion criteria). Present data clearly as text, in tables, or in graphs, to enable information to be evaluated or extracted for future meta-analyses [ 146 ]. Report descriptive statistics with a clearly identified measure of variability for each group. Fig 5 shows data summarised as means and standard deviations and, in brackets, ranges. Box plots are a convenient way to summarise continuous data, plotted as median and interquartile range, as shown in Fig 6 .

Subitem 10a—Example 1



Subitem 10a—Example 2



10b . If applicable, the effect size with a confidence interval .

Explanation. In hypothesis-testing studies using inferential statistics, investigators frequently confuse statistical significance and small p -values with biological or clinical importance [ 149 ]. Statistical significance is usually quantified and evaluated against a preassigned threshold, with p < 0.05 often used as a convention. However, statistical significance is heavily influenced by sample size and variation in the data (see Item 2. Sample size). Investigators must consider the size of the effect that was observed and whether this is a biologically relevant change.

Effect sizes are often not reported in animal research, but they are relevant to both exploratory and hypothesis-testing studies. An effect size is a quantitative measure that estimates the magnitude of differences between groups or strength of relationships between variables. It can be used to assess the patterns in the data collected and make inferences about the wider population from which the sample came. The confidence interval for the effect indicates how precisely the effect has been estimated and tells the reader about the strength of the effect [ 150 ]. In studies in which statistical power is low and/or hypothesis-testing is inappropriate, providing the effect size and confidence interval indicates how small or large an effect might really be, so a reader can judge the biological significance of the data [ 151 , 152 ]. Reporting effect sizes with confidence intervals also facilitates extraction of useful data for systematic review and meta-analysis. When multiple independent studies included in a meta-analysis show quantitatively similar effects, even if each is statistically nonsignificant, this provides powerful evidence that a relationship is ‘real’, although small.

Report all analyses performed, even those providing non-statistically significant results. Report the effect size to indicate the size of the difference between groups in the study, with a confidence interval to indicate the precision of the effect size estimate.

Subitem 10b—Example 1



Recommended Set

The Recommended Set ( Box 6 ) adds context to the study described, including further detail about the methodology and advice on what to include in the more narrative parts of a manuscript. Items are presented in a logical order; there is no ranking within the set.

Box 6. ARRIVE Recommended Set

Item 11. Abstract

Provide an accurate summary of the research objectives, animal species, strain and sex, key methods, principal findings, and study conclusions .

Explanation. A transparent and accurate abstract increases the utility and impact of the manuscript and allows readers to assess the reliability of the study [ 153 ]. The abstract is often used as a screening tool by readers to decide whether to read the full article or whether to select an article for inclusion in a systematic review. However, abstracts often either do not contain enough information for this purpose [ 11 ] or contain information that is inconsistent with the results in the rest of the manuscript [ 154 , 155 ]. In systematic reviews, initial screens to identify papers are based on titles, abstracts, and keywords [ 156 ]. Leaving out of the abstract information such as the species of animal used or the drugs being tested limits the value of preclinical systematic reviews as relevant studies cannot be identified and included. For example, in a systematic review of the effect of the MVA85A vaccine on tuberculosis challenge in animals, the largest preclinical trial did not include the vaccine name in the abstract or keywords of the publication; the paper was only included in the systematic review following discussions with experts in the field [ 157 ].

To maximise utility, include details of the species, sex, and strain of animals used and accurately report the methods, results, and conclusions of the study. Also describe the objectives of the study, including whether it was designed either to test a specific hypothesis or to generate a new hypothesis (see Item 13. Objectives). Incorporating this information will enable readers to interpret the strength of evidence and judge how the study fits within the wider knowledge base.

Item 11—Example 1

‘Background and Purpose

‘Asthma is an inflammatory disease that involves airway hyperresponsiveness and remodelling. Flavonoids have been associated to anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment of asthma. Our aim was to evaluate the effects of the sakuranetin treatment in several aspects of experimental asthma model in mice.

‘Experimental Approach

‘Male BALB/c mice received ovalbumin (i.p.) on days 0 and 14, and were challenged with aerolized ovalbumin 1% on days 24, 26 and 28. Ovalbumin-sensitized animals received vehicle (saline and dimethyl sulfoxide, DMSO), sakuranetin (20 mg kg –1 per mice) or dexamethasone (5 mg kg –1 per mice) daily beginning from 24th to 29th day. Control group received saline inhalation and nasal drop vehicle. On day 29, we determined the airway hyperresponsiveness, inflammation and remodelling as well as specific IgE antibody. RANTES, IL- 5, IL -4, Eotaxin, IL -10, TNF -α, IFN -γ and GMC-SF content in lung homogenate was performed by Bioplex assay, and 8-isoprostane and NF -kB activations were visualized in inflammatory cells by immunohistochemistry.

‘Key Results

‘We have demonstrated that sakuranetin treatment attenuated airway hyperresponsiveness, inflammation and remodelling; and these effects could be attributed to Th2 pro-inflammatory cytokines and oxidative stress reduction as well as control of NF -kB activation.

‘Conclusions and Implications

‘These results highlighted the importance of counteracting oxidative stress by flavonoids in this asthma model and suggest sakuranetin as a potential candidate for studies of treatment of asthma’ [ 158 ].

Item 11—Example 2

‘In some parts of the world, the laboratory pig (Sus scrofa) is often housed in individual, sterile housing which may impose stress. Our objectives were to determine the effects of isolation and enrichment on pigs housed within the PigTurn ® —a novel penning system with automated blood sampling—and to investigate tear staining as a novel welfare indicator. Twenty Yorkshire × Landrace weaner pigs were randomly assigned to one of four treatments in a 2 × 2 factorial combination of enrichment (non-enriched [NE] or enriched [E]) and isolation (visually isolated [I] or able to see another pig [NI]). Pigs were catheterised and placed into the PigTurns ® 48 h post recovery. Blood was collected automatically twice daily to determine white blood cell (WBC) differential counts and assayed for cortisol. Photographs of the eyes were taken daily and tear staining was quantified using a 0–5 scoring scale and Image-J software to measure stain area and perimeter. Behaviour was video recorded and scan sampled to determine time budgets. Data were analysed as an REML using the MIXED procedure of SAS. Enrichment tended to increase proportion of time standing and lying laterally and decrease plasma cortisol, tear-stain area and perimeter. There was a significant isolation by enrichment interaction. Enrichment given to pigs housed in isolation had no effect on plasma cortisol, but greatly reduced it in non-isolated pigs. Tear-staining area and perimeter were highest in the NE-I treatment compared to the other three treatments. Eosinophil count was highest in the E-NI treatment and lowest in the NE-I treatment. The results suggest that in the absence of enrichment, being able to see another animal but not interact may be frustrating. The combination of no enrichment and isolation maximally impacted tear staining and eosinophil numbers. However, appropriate enrichment coupled with proximity of another pig would appear to improve welfare’ [ 159 ].

Item 12. Background

12a . Include sufficient scientific background to understand the rationale and context for the study, and explain the experimental approach .

Explanation. Scientific background information for an animal study should demonstrate a clear evidence gap and explain why an in vivo approach was warranted. Systematic reviews of the animal literature provide the most convincing evidence that a research question has not been conclusively addressed, by showing the extent of current evidence within a field of research. They can also inform the choice of animal model by providing a comprehensive overview of the models used along with their benefits and limitations [ 160 – 162 ].

Describe the rationale and context of the study and how it relates to other research, including relevant references to previous work. Outline evidence underpinning the hypothesis or objectives and explain why the experimental approach is best suited to answer the research question.

Subitem 12a –example 1

‘For decades, cardiovascular disease has remained the leading cause of mortality worldwide… [and] cardiovascular research has been performed using healthy and young, non-diseased animal models. Recent failures of cardioprotective therapies in obese insulin-resistant …, diabetic …, metabolic syndrome-affected… and aged… animals that were otherwise successful in healthy animal models has highlighted the need for the development of animal models of disease that are representative of human clinical conditions…. The majority of laboratory-based studies investigating cardiovascular disease and myocardial tolerance to ischemia-reperfusion (I-R) are currently conducted using normogonadic models with either genetically-induced… or diet-induced… obesity and metabolic syndrome (MetS). In the clinical setting, elderly male patients often present with both testosterone deficiency (TD) and MetS…. A strong and compounding association exists between metabolic syndrome and testosterone deficiency which may have significant impact on cardiovascular disease and its outcomes which is not addressed by current models…. Although laboratory investigations generally rely on animal models of isolated metabolic syndrome or hypogonadism, their mutual presentation in the clinical setting warrants the development of appropriate animal models of the MetS with hypogonadism, especially in the context of cardiovascular disease research’ [ 163 ].

12b . Explain how the animal species and model used address the scientific objectives and, where appropriate, the relevance to human biology .

Explanation. Provide enough detail for the reader to assess the suitability of the animal model used to address the research question. Include information on the rationale for choosing a particular species and explain how the outcome measures assessed are relevant to the condition under study and how the model was validated. Stating that an animal model is commonly used in the field is not appropriate, and a well-considered, detailed rationale should be provided.

When the study models an aspect of a human disease, indicate how the model is appropriate for addressing the specific objectives of the study [ 164 ]. This can include a description of how the induction of the disease, disorder, or injury is sufficiently analogous to the human condition; how the model responds to known clinically effective treatments; how similar symptoms are to the clinical disease; and how animal characteristics were selected to represent the age, sex, and health status of the clinical population [ 14 ].

Subitem 12b—Example 1

‘For this purpose, we selected a pilocarpine model of epilepsy that is characterized by robust, frequent spontaneous seizures acquired after a brain insult …, well-described behavioral abnormalities …, and poor responses to antiepileptic drugs…. These animals recapitulate several key features of human temporal lobe epilepsy, the most common type of epilepsy in adults’ [ 165 ].

Subitem 12b—Example 2

‘Transplantation of healthy haematopoietic stem cells (HSCs) is a critical therapy for a wide range of malignant haematological and non-malignant disorders and immune dysfunction…. Zebrafish are already established as a successful model to study the haematopoietic system, with significant homology with mammals…. Imaging of zebrafish transparent embryos remains a powerful tool and has been critical to confirm that the zebrafish Caudal Haematopoietic Tissue (CHT) is comparable to the mammalian foetal haematopoietic niche…. Xenotransplantation in zebrafish embryos has revealed highly conserved mechanisms between zebrafish and mammals. Recently, murine bone marrow cells were successfully transplanted into zebrafish embryos, revealing highly conserved mechanism of haematopoiesis between zebrafish and mammals…. Additionally, CD34 enriched human cells transplanted into zebrafish were shown to home to the CHT and respond to zebrafish stromal-cell derived factors’ [ 166 ].

Item 13. Objectives

Clearly describe the research question, research objectives and, where appropriate, specific hypotheses being tested .

Explanation. Explaining the purpose of the study by describing the question(s) that the research addresses allows readers to determine whether the study is relevant to them. Readers can also assess the relevance of the model organism, procedures, outcomes measured, and analysis used.

Knowing whether a study is exploratory or hypothesis-testing is critical to its interpretation. A typical exploratory study may measure multiple outcomes and look for patterns in the data or relationships that can be used to generate hypotheses. It may also be a pilot study, which aims to inform the design or feasibility of larger subsequent experiments. Exploratory research helps researchers to design hypothesis-testing experiments by choosing what variables or outcome measures to focus on in subsequent studies.

Testing a specific hypothesis has implications for both the study design and the data analysis [ 16 , 167 ]. For example, an experiment designed to detect a hypothesised effect will likely need to be analysed with inferential statistics, and a statistical estimation of the sample size will need to be performed a priori (see Item 2. Sample size). Hypothesis-testing studies also have a predefined primary outcome measure, which is used to assess the evidence in support of the specific research question (see Item 6. Outcome measures).

In contrast, exploratory research investigates many possible effects and is likely to yield more false positive results because some will be positive by chance. Thus, results from well-designed hypothesis-testing studies provide stronger evidence than those from exploratory or descriptive studies. Independent replication and meta-analysis can further increase the confidence in conclusions.

Clearly outline the objective(s) of the study, including whether it is hypothesis-testing or exploratory, or if it includes research of both types. Hypothesis-testing studies may collect additional information for exploratory purposes; it is important to distinguish which hypotheses were prespecified and which originated after data inspection, especially when reporting unanticipated effects or outcomes that were not part of the original study design.

Item 13—Example 1

‘The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinson’s disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals’ [ 168 ].

Item 13—Example 2

‘In this exploratory study, we aimed to investigate whether calcium electroporation could initiate an anticancer immune response similar to electrochemotherapy. To this end, we treated immunocompetent balb/c mice with CT26 colon tumors with calcium electroporation, electrochemotherapy, or ultrasound-based delivery of calcium or bleomycin’ [ 169 ].

Item 13—Example 3

‘While characterizing a rab-6 . 2 -null C . elegans strain for another study, we observed that rab-6 . 2(ok2254) animals were fragile. We set out to analyze the fragile-skin phenotype in rab-6 . 2(ok2254) animals genetically…. We observed several ruptured animals on our rab-6 . 2(ok2254) culture plates during normal maintenance, a phenotype very rarely observed in wild-type cultures…. We hypothesized that RAB-6.2 is required for skin integrity’ [ 170 ].

Item 14. Ethical statement

Provide the name of the ethical review committee or equivalent that has approved the use of animals in this study and any relevant licence or protocol numbers (if applicable) . If ethical approval was not sought or granted, provide a justification .

Explanation. Authors are responsible for complying with regulations and guidelines relating to the use of animals for scientific purposes. This includes ensuring that they have the relevant approval for their study from an appropriate ethics committee and/or regulatory body before the work starts. The ethical statement provides editors, reviewers, and readers with assurance that studies have received this ethical oversight [ 12 ]. This also promotes transparency and understanding about the use of animals in research and fosters public trust.

Provide a clear statement explaining how the study conforms to appropriate regulations and guidelines. Include the name of the institution where the research was approved and the ethics committee who reviewed it (e.g., Institutional Animal Care and Use Committee [IACUC] in the United States or Animal Welfare and Ethical Review Body [AWERB] in the United Kingdom) and indicate protocol or project licence numbers so that the study can be identified. Also add any relevant accreditation, e.g., American Association for Accreditation of Laboratory Animal Care (AAALAC) [ 171 ] or Good Laboratory Practice (GLP).

If the research is not covered by any regulation and formal ethical approval is not required (e.g., a study using animal species not protected by regulations or law), demonstrate that international standards were complied with and cite the appropriate reference. In such cases, provide a clear statement explaining why the research is exempt from regulatory approval.

Item 14—Example 1

‘All procedures were conducted in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986, approved by institutional ethical review committees (Alderley Park Animal Welfare and Ethical Review Board and Babraham Institute Animal Welfare and Ethical Review Board) and conducted under the authority of the Project Licence (40/3729 and 70/8307, respectively)’ [ 172 ].

Item 14—Example 2

‘All protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Fuwai Hospital, Peking Union Medical College and the Beijing Council on Animal Care, Beijing, China (IACUC permit number: FW2010-101523), in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85-23, revised 1996)’ [ 173 ].

Item 14—Example 3

‘Samples and data were collected according to Institut de Sélection d’Animale (ISA) protocols, under the supervision of ISA employees. Samples and data were collected as part of routine animal data collection in a commercial breeding program for layer chickens in The Netherlands. Samples and data were collected on a breeding nucleus of ISA for breeding purposes only, and is a non-experimental, agricultural practice, regulated by the Act Animals, and the Royal Decree on Procedures. The Dutch Experiments on Animals Act does not apply to non-experimental, agricultural practices. An ethical review by the Statement Animal Experiment Committee was therefore not required. No extra animal discomfort was caused for sample collection for the purpose of this study’ [ 174 ].

Item 15. Housing and husbandry

Provide details of housing and husbandry conditions, including any environmental enrichment .

Explanation. The environment determines the health and wellbeing of the animals, and every aspect of it can potentially affect their behavioural and physiological responses, thereby affecting research outcomes [ 175 ]. Different studies may be sensitive to different environmental factors, and particular aspects of the environment necessary to report may depend on the type of study [ 176 ]. Examples of housing and husbandry conditions known to affect animal welfare and research outcomes are listed in Table 2 ; consider reporting these elements and any other housing and husbandry conditions likely to influence the study outcomes.



Environment, either deprived or enriched, can affect a wide range of physiological and behavioural responses [ 206 ]. Specific details to report include, but are not limited to, structural enrichment (e.g., elevated surfaces, dividers); resources for species-typical activities (e.g., nesting material, shelters, or gnawing sticks for rodents; plants or gravel for aquatic species); and toys or other tools used to stimulate exploration, exercise (e.g., running wheel), and novelty. If no environmental enrichment was provided, this should be clearly stated with justification. Similarly, scientific justification needs to be reported for withholding food and water [ 207 ] and for singly housing animals [ 208 , 209 ].

If space is an issue, relevant housing and husbandry details can be provided in the form of a link to the information in a public repository or as supplementary information.

Item 15—Example 1

‘Breeding colonies were kept in individually ventilated cages (IVCs; Tecniplast, Italy) at a temperature of 20°C to 24°C, humidity of 50% to 60%, 60 air exchanges per hour in the cages, and a 12/12-hour light/dark cycle with the lights on at 5:30 AM. The maximum caging density was five mice from the same litter and sex starting from weaning. As bedding, spruce wood shavings (Lignocel FS-14; J. Rettenmaier und Soehne GmbH, Rosenberg, Germany) were provided. Mice were fed a standardized mouse diet (1314, Altromin, Germany) and provided drinking water ad libitum . All materials, including IVCs, lids, feeders, bottles, bedding, and water were autoclaved before use. Sentinel mice were negative for at least all Federation of laboratory animal science associations (FELASA)-relevant murine infectious agents… as diagnosed by our health monitoring laboratory, mfd Diagnostics GmbH, Wendelsheim, Germany’ [ 210 ].

Item 15—Example 2

‘Same sex litter mates were housed together in individually ventilated cages with two or four mice per cage. All mice were maintained on a regular diurnal lighting cycle (12:12 light:-dark) with ad libitum access to food (7012 Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet) and water. Chopped corn cob was used as bedding. Environmental enrichment included nesting material (Nestlets, Ancare, Bellmore, NY, USA), PVC pipe, and shelter (Refuge XKA-2450-087, Ketchum Manufacturing Inc., Brockville, Ontario, Canada). Mice were housed under broken barrier-specific pathogen-free conditions in the Transgenic Mouse Core Facility of Cornell University, accredited by AAALAC (The Association for Assessment and Accreditation of Laboratory Animal Care International)’ [ 211 ].

Item 16. Animal care and monitoring

16a . Describe any interventions or steps taken in the experimental protocols to reduce pain, suffering, and distress .

Explanation. A safe and effective analgesic plan is critical to relieve pain, suffering, and distress. Untreated pain can affect the animals’ biology and add variability to the experiment; however, specific pain management procedures can also introduce variability, affecting experimental data [ 212 , 213 ]. Underreporting of welfare management procedures contributes to the perpetuation of noncompliant methodologies and insufficient or inappropriate use of analgesia [ 213 ] or other welfare measures. A thorough description of the procedures used to alleviate pain, suffering, and distress provides practical information for researchers to replicate the method.

Clearly describe pain management strategies, including

If analgesics or other welfare measures, reasonably expected for the procedure performed, are not performed for experimental reasons, report the scientific justification [ 214 ].

Subitem 16a—Example 1

‘If piglets developed diarrhea, they were placed on an electrolyte solution and provided supplemental water, and if the diarrhea did not resolve within 48 h, piglets received a single dose of ceftiofur (5.0 mg ceftiofur equivalent/kg of body weight i.m [Excede, Zoetis, Florham Park, NJ]). If fluid loss continued after treatment, piglets then received a single dose of sulfamethoxazole and trimethoprim oral suspension (50 mg/8 mg per mL, Hi-Tech Pharmacal, Amityville, NY) for 3 consecutive days’ [ 215 ].

Subitem 16a—Example 2

‘One hour before surgery, we administered analgesia to the mice by offering them nut paste (Nutella; Ferrero, Pino Torinese, Italy) containing 1 mg per kg body weight buprenorphine (Temgesic; Schering-Plough Europe, Brussels, Belgium) for voluntary ingestion, as described previously…. The mice had been habituated to pure nut paste for 2 d prior to surgery’ [ 216 ].

Subitem 16a—Example 3

‘If a GCPS score equal or greater than 6 (out of 24) was assigned postoperatively, additional analgesia was provided with methadone 0.1 mg kg −1 IM (or IV if required) … and pain reassessed 30 minutes later. The number of methadone doses was recorded’ [ 46 ].

16b . Report any expected or unexpected adverse events .

Explanation. Reporting adverse events allows other researchers to plan appropriate welfare assessments and minimise the risk of these events occurring in their own studies. If the experiment is testing the efficacy of a treatment, the occurrence of adverse events may alter the balance between treatment benefit and risk [ 34 ].

Report any adverse events that had a negative impact on the welfare of the animals in the study (e.g., cardiovascular and respiratory depression, central nervous system disturbance, hypothermia, reduction of food intake). Indicate whether they were expected or unexpected. If adverse events were not observed, or not recorded during the study, explicitly state this.

Subitem 16b—Example 1

‘Murine lymph node tumors arose in 11 of 12 mice that received N2-transduced human cells. The neo gene could be detected in murine cells as well as in human cells. Significant lymphoproliferation could be seen only in the murine pre-T cells. It took 5 months for murine leukemia to arise; the affected mice displayed symptoms of extreme sickness rapidly, with 5 of the 12 mice becoming moribund on exactly the same day (Figure …), and 6 others becoming moribund within a 1-month period…. Of the 12 mice that had received N2-transduced human cells, 11 had to be killed because they developed visibly enlarged lymph nodes and spleen, hunching, and decrease in body weight, as shown in Figure…. The 12th mouse was observed carefully for 14 months; it did not show any signs of leukemia or other adverse events, and had no abnormal tissues when it was autopsied…. The mice were observed at least once daily for signs of illness, which were defined as any one or more of the following: weight loss, hunching, lethargy, rapid breathing, skin discoloration or irregularities, bloating, hemi-paresis, visibly enlarged lymph nodes, and visible solid tumors under the skin. Any signs of illness were logged as “adverse events” in the experiment, the mouse was immediately killed, and an autopsy was performed to establish the cause of illness’ [ 217 ].

Subitem 16b—Example 2

‘Although procedures were based on those reported in the literature, dogs under Protocol 1 displayed high levels of stress and many experienced vomiting. This led us to significantly alter procedures in order to optimize the protocol for the purposes of our own fasting and postprandial metabolic studies’ [ 218 ].

16c . Describe the humane endpoints established for the study, the signs that were monitored, and the frequency of monitoring . If the study did not set humane endpoints, state this .

Explanation. Humane endpoints are predetermined morphological, physiological, and/or behavioural signs that define the circumstances under which an animal will be removed from an experimental study. The use of humane endpoints can help minimise harm while allowing the scientific objectives to be achieved [ 219 ]. Report the humane endpoints that were established for the specific study, species, and strain. Include clear criteria of the clinical signs monitored [ 134 ] and clinical signs that led to euthanasia or other defined actions. Include details such as general welfare indicators (e.g., weight loss, reduced food intake, abnormal posture) and procedure-specific welfare indicators (e.g., tumour size in cancer studies [ 50 ], sensory-motor deficits in stroke studies [ 220 ]).

Report the timing and frequency of monitoring, taking into consideration the normal circadian rhythm of the animal and timing of scientific procedures, as well as any increase in the frequency of monitoring (e.g., postsurgery recovery, critical times during disease studies, or following the observation of an adverse event). Publishing score sheets of the clinical signs that were monitored [ 221 ] can help guide other researchers to develop clinically relevant welfare assessments, particularly for studies reporting novel procedures.

This information should be reported even if no animal reached any of the humane endpoints. If no humane endpoints were established for the study, explicitly state this.

Subitem 16c—Example 1

‘Both the research team and the veterinary staff monitored animals twice daily. Health was monitored by weight (twice weekly), food and water intake, and general assessment of animal activity, panting, and fur condition…. The maximum size the tumors allowed to grow in the mice before euthanasia was 2000 mm 3 ’ [ 222 ].

Item 17. Interpretation/scientific implications

17a . Interpret the results, taking into account the study objectives and hypotheses, current theory, and other relevant studies in the literature .

Explanation. It is important to interpret the results of the study in the context of the study objectives (see Item 13. Objectives). For hypothesis-testing studies, interpretations should be restricted to the primary outcome (see Item 6. Outcome measures). Exploratory results derived from additional outcomes should not be described as conclusive, as they may be underpowered and less reliable.

Discuss the findings in the context of current theory, ideally with reference to a relevant systematic review, as individual studies do not provide a complete picture. If a systematic review is not available, take care to avoid selectively citing studies that corroborate the results or only those that report statistically significant findings [ 223 ].

When appropriate, describe any implications of the experimental methods or research findings for improving welfare standards or reducing the number of animals used in future studies (e.g., the use of a novel approach reduced the results’ variability, thus enabling the use of smaller group sizes without losing statistical power). This may not be the primary focus of the research, but reporting this information enables wider dissemination and uptake of refined techniques within the scientific community.

Subitem 17a—Example 1

‘This is in contrast to data provided by an ‘intra-renal IL-18 overexpression’ model …, and may reflect an IL-18 concentration exceeding the physiologic range in the latter study’ [ 224 ].

Subitem 17a—Example 2

‘The new apparatus shows potential for considerably reducing the number of animals used in memory tasks designed to detect potential amnesic properties of new drugs… approximately 43,000 animals have been used in these tasks in the past 5 years but with the application of the continual trials apparatus we estimate that this could have been reduced to 26,000 … with the new paradigm the number of animals needed to obtain reliable results and maintain the statistical power of the tasks is greatly reduced’ [ 225 ].

Subitem 17a—Example 3

‘In summary, our results show that IL-1Ra protects against brain injury and reduces neuroinflammation when administered peripherally to aged and comorbid animals at reperfusion or 3 hours later. These findings address concerns raised in a recent systematic review on IL-1Ra in stroke… and provide further supporting evidence for IL-1Ra as a lead candidate for the treatment of ischemic stroke’ [ 226 ].

17b . Comment on the study limitations, including potential sources of bias, limitations of the animal model, and imprecision associated with the results .

Explanation. Discussing the limitations of the work is important to place the findings in context, interpret the validity of the results, and ascribe a credibility level to its conclusions [ 227 ]. Limitations are unavoidable in scientific research, and describing them is essential to share experience, guide best practice, and aid the design of future experiments [ 228 ].

Discuss the quality of evidence presented in the study and consider how appropriate the animal model is to the specific research question. A discussion on the rigour of the study design to isolate cause and effect (also known as internal validity [ 229 ]) should include whether potential risks of bias have been addressed [ 9 ] (see Item 2. Sample size, Item 3. Inclusion and exclusion criteria, Item 4. Randomisation, and Item 5. Blinding).

Subitem 17b—Example 1

‘Although in this study we did not sample the source herds, the likelihood of these herds to be IAV positive is high given the commonality of IAV infections in the Midwest…. However, we cannot fully rule out the possibility that new gilts became infected with resident viruses after arrival to the herd. Although new gilts were placed into isolated designated areas and procedures were in place to minimize disease transmission (eg. isolation, vaccination), these areas or procedures might not have been able to fully contain infections within the designated areas’ [ 230 ].

Subitem 17b—Example 2

‘Even though our data demonstrates that sustained systemic TLR9 stimulation aggravates diastolic HF in our model of gene-targeted diastolic HF, there are several limitations as to mechanistic explanations of causality, as well as extrapolations to clinical inflammatory disease states and other HF conditions. First, our pharmacological inflammatory model does not allow discrimination between effects caused by direct cardiac TLR9 stimulation to that of indirect effects mediated by systemic inflammation. Second, although several systemic inflammatory conditions have disturbances in the innate immune system as important features, and some of these again specifically encompassing distorted TLR9 signalling… sustained TLR9 stimulation does not necessarily represent a clinically relevant inflammatory condition. Finally, the cardiac myocyte SERCA2a KO model does not adequately represent the molecular basis for, or the clinical features of, diastolic HF’ [ 231 ].

Item 18. Generalisability/translation

Comment on whether, and how, the findings of this study are likely to generalise to other species or experimental conditions, including any relevance to human biology (where appropriate) .

Explanation. An important purpose of publishing research findings is to inform future research. In the context of animal studies, this might take the form of further in vivo research or another research domain (e.g., human clinical trial). Thoughtful consideration is warranted, as additional unnecessary animal studies are wasteful and unethical. Similarly, human clinical trials initiated based on insufficient or misleading animal research evidence increase research waste and negatively influence the risk-benefit balance for research participants [ 229 , 232 ].

Consider the type of study conducted to assess the implications of the findings. Well-designed hypothesis-testing studies provide more robust evidence than exploratory studies (see Item 13. Objectives). Findings from a novel, exploratory study may be used to inform future research in a broadly similar context. Alternatively, enough evidence may have accumulated in the literature to justify further research in another species or in humans. Discuss what (if any) further research may be required to allow generalisation or translation. Discuss and interpret the results in relation to current evidence and, in particular, whether similar [ 233 ] or otherwise supportive [ 234 ] findings have been reported by other groups. Discuss the range of circumstances in which the effect is observed and factors that may moderate that effect. Such factors could include, for example, the population (e.g., age, sex, strain, species), the intervention (e.g., different drugs of the same class), and the outcome measured (e.g., different approaches to assessing memory).

Item 18—Example 1

‘Our results demonstrate that hDBS robustly modulates the mesolimbic network. This finding may hold clinical relevance for hippocampal DBS therapy in epilepsy cases, as connectivity in this network has previously been shown to be suppressed in mTLE. Further research is necessary to investigate potential DBS-induced restoration of MTLE-induced loss of functional connectivity in mesolimbic brain structures’ [ 235 ].

Item 18—Example 2

‘The tumor suppressor effects of GAS1 had been previously reported in cell cultures or in xenograft models, this is the first work in which the suppressor activity of murine Gas1 is reported for primary tumors in vivo . Recent advances in the design of safe vectors for transgene delivery… may result in extrapolating our results to humans and so a promising field of research emerges in the area of hepatic, neoplastic diseases’ [ 236 ].

Item 19. Protocol registration

Provide a statement indicating whether a protocol (including the research question, key design features, and analysis plan) was prepared before the study, and if and where this protocol was registered .

Explanation. Akin to the approach taken for clinical trials, protocol registration has emerged as a mechanism that is likely to improve the transparency of animal research [ 232 , 237 , 238 ]. Registering a protocol before the start of the experiment enables researchers to demonstrate that the hypothesis, approach, and analysis were planned in advance and not shaped by data as they emerged; it enhances scientific rigour and protects the researcher against concerns about selective reporting of results [ 239 , 240 ]. A protocol should consist of (1) the question being addressed and the key features of the research that is proposed, such as the hypothesis being tested, the primary outcome measure (if applicable), and the statistical analysis plan; and (2) the laboratory procedures to be used to perform the planned experiment.

Protocols may be registered with different levels of completeness. For example, in the Registered Report format offered by an increasing number of journals, protocols undergo peer review, and if accepted, the journal commits to publishing the completed research regardless of the results obtained [ 237 ].

Other online resources include the Open Science Framework [ 241 ], which is suitable to deposit PHISPS (Population; Hypothesis; Intervention; Statistical Analysis Plan; Primary; Outcome Measure; Sample Size Calculation) protocols [ 242 ] and provide researchers with the flexibility to embargo the preregistration, keep it from public view until the research is published, and selectively share it with reviewers and editors. The EDA can also be used to generate a time-stamped PDF, which sets out key elements of the experimental design [ 19 ]. This can be used to demonstrate that the study conduct, analysis, and reporting were not unduly driven by emerging data. As a minimum, we recommend registering protocols containing all PHISPS components as outlined above.

Provide a statement indicating whether or not any protocol was prepared before the study, and if applicable, provide the time-stamped protocol or the location of its registration. When there have been deviations from the protocol, describe the rationale for these changes in the publication so that readers can take this into account when assessing the findings.

Item 19—Example 1

‘A detailed description of all protocols can be found in the Registered Report (Kandela et al., 2015). Additional detailed experimental notes, data, and analysis are available on the Open Science Framework (OSF) (RRID: SCR_003238) ( https://osf.io/xu1g2/ )’ [ 243 ].

Item 19—Example 2

‘To maximize the objectivity of the presented research, we preregistered this study with its 2 hypotheses, its planned methods, and its complete plan of data analysis before the start of data collection ( https://osf.io/eb8ua/register/565fb3678c5e4a66b5582f67 , accessed 29 December 2017). We closely adhered to our plan…. All statistical analyses closely followed our preregistered analysis plan ( https://osf.io/eb8ua/ )’ [ 244 ].

Item 19—Example 3

‘We preregistered our analyses with the Open Science Framework which facilitates reproducibility and open collaboration in science research…. Our preregistration: Sheldon and Griffith (2017), was carried out to limit the number of analyses conducted and to validate our commitment to testing a limited number of a priori hypotheses. Our methods are consistent with this preregistration …’ [ 245 ].

Item 20. Data access

Provide a statement describing if and where study data are available .

Explanation. A data-sharing statement describes how others can access the data on which the paper is based. Sharing adequately annotated data allows others to replicate data analyses so that results can be independently tested and verified. Data sharing allows the data to be repurposed and new datasets to be created by combining data from multiple studies (e.g., to be used in secondary analyses). This allows others to explore new topics and increases the impact of the study, potentially preventing unnecessary use of animals and providing more value for money. Access to raw data also facilitates text and automated data mining [ 246 ].

An increasing number of publishers and funding bodies require authors or grant holders to make their data publicly available [ 247 ]. Journal articles with accompanying data may be cited more frequently [ 248 , 249 ]. Datasets can also be independently cited in their own right, which provides additional credit for authors. This practice is gaining increasing recognition and acceptance [ 250 ].

When possible, make available all data that contribute to summary estimates or claims presented in the paper. Data should follow the FAIR guiding principles [ 251 ]; that is, data are findable, accessible (i.e., do not use outdated file types), interoperable (can be used on multiple platforms and with multiple software packages), and reusable (i.e., have adequate data descriptors).

Data can be made publicly available via a structured, specialised (domain-specific), open-access repository such as those maintained by the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ ) or European Bioinformatics Institute (EBI, https://www.ebi.ac.uk/ ). If such a repository is not available, data can be deposited in unstructured but publicly available repositories (e.g., Figshare [ https://figshare.com/ ], Dryad [ https://datadryad.org/ ], Zenodo [ https://zenodo.org/ ], or Open Science Framework [ https://osf.io/ ]). There are also search platforms to identify relevant repositories with rigorous standards, e.g., FairSharing ( https://fairsharing.org/ ) and re3data ( https://www.re3data.org/ ).

Item 20—Example 1

‘Data Availability: All data are available from Figshare at http://dx.doi.org/10.6084/m9.figshare.1288935 ’ [ 252 ].

Item 20—Example 2

‘A fundamental goal in generating this dataset is to facilitate access to spiny mouse transcript sequence information for external collaborators and researchers. The sequence reads and metadata are available from the NCBI (PRJNA342864) and assembled transcriptomes (Trinity_v2.3.2 and tr2aacds_v2) are available from the Zenodo repository ( https://doi.org/10.5281/zenodo.808870 ), however accessing and utilizing this data can be challenging for researchers lacking bioinformatics expertise. To address this problem we are hosting a SequenceServer… BLAST-search website ( http://spinymouse.erc.monash.edu/sequenceserver/ ). This resource provides a user-friendly interface to access sequence information from the tr2aacds_v2 assembly (to explore annotated protein-coding transcripts) and/or the Trinity_v2.3.2 assembly (to explore non-coding transcripts)’ [ 253 ].

Item 21. Declaration of interests

21a . Declare any potential conflicts of interest, including financial and nonfinancial . If none exist, this should be stated .

Explanation. A competing or conflict of interest is anything that interferes with (or could be perceived as interfering with) the full and objective presentation, analysis, and interpretation of the research. Competing or conflicts of interest can be financial or nonfinancial, professional or personal. They can exist in institutions, in teams, or with individuals. Potential competing interests are considered in peer review, editorial, and publication decisions; the aim is to ensure transparency, and in most cases, a declaration of a conflict of interest does not obstruct the publication or review process.

Examples are provided in Box 7 . If unsure, declare all potential conflicts, including both perceived and real conflicts of interest [ 254 ].

Box 7. Examples of competing or conflicts of interest

Funding and other payments received or expected by the authors directly arising from the publication of the study, or funding or other payments from an organisation with an interest in the outcome of the work.


Research that may benefit the individual or institution in terms of goods in kind. This includes unpaid advisory position in a government, nongovernment organisation, or commercial organisations.


Employed by, on the advisory board, or a member of an organisation with an interest in the outcome of the work.

Intellectual property

Patents or trademarks owned by someone or their organisation. This also includes the potential exploitation of the scientific advance being reported for the institution, the authors, or the research funders.

Friends, family, relationships, and other close personal connections to people who may potentially benefit financially or in other ways from the research.

Beliefs or activism (e.g., political or religious) relevant to the work. Membership of a relevant advocacy or lobbying organisation.

Subitem 21a—Example 1

‘The study was funded by Gubra ApS. LSD, PJP, GH, KF and HBH are employed by Gubra ApS. JJ and NV are the owners of Gubra ApS. Gubra ApS provided support in the form of materials and salaries for authors LSD, PJP, GH, KF, HBH, JJ and NV’ [ 255 ].

Subitem 21a—Example 2

‘The authors have declared that no competing interests exist’ [ 256 ].

21b . List all funding sources (including grant identifier) and the role of the funder(s) in the design, analysis, and reporting of the study .

Explanation. The identification of funding sources allows the reader to assess any competing interests and any potential sources of bias. For example, bias, as indicated by a prevalence of more favourable outcomes, has been demonstrated for clinical research funded by industry compared with studies funded by other sources [ 257 – 259 ]. Evidence for preclinical research also indicates that funding sources may influence the interpretation of study outcomes [ 254 , 260 ].

Report the funding information including the financial supporting body(s) and any grant identifier(s). If the study was supported by several sources of funding, list them all, including internal grants. Specify the role of the funder in the design, analysis, reporting, and/or decision to publish. If the research did not receive specific funding but was performed as part of the employment of the authors, name the employer.

Subitem 21b—Example 1

‘Support was provided by the Italian Ministry of Health: Current research funds PRC 2010/001 [ http://www.salute.gov.it/ ] to MG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript’ [ 261 ].

Subitem 21b—Example 2

‘This study was financially supported by the Tuberculosis and Lung Research Center of Tabriz University of Medical Sciences and the Research Council of University of Tabriz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript’ [ 262 ].

Subitem 21b—Example 3

‘This work was supported by the salary paid to AEW. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript’ [ 263 ].

Supporting information

S1 annotated byline. individual authors’ positions at the time this article was submitted..


S1 Annotated References. Further context on the works cited in this article.



We would like to acknowledge the late Doug Altman’s contribution to this project. Doug was a dedicated member of the working group, and his input into the guidelines’ revision has been invaluable.

New Small Animal Teaching and Research Hospital Receives Major Gift Pledge

Linda and Dennis Clark

Linda and Dennis Clark ’68, ’71 have pledged a $20 million lead gift through the Texas A&M Foundation to support construction of a new Next-Generation Small Animal Teaching and Research Hospital at Texas A&M University. The cutting-edge facility will replace the current Small Animal Teaching Hospital and enable students, faculty and staff in the university’s School of Veterinary Medicine and Biomedical Sciences to further elevate its already world-renowned veterinary medicine program.

The couple hopes their gift will challenge and inspire others to support the construction project, which still requires a significant investment from private donors to reach fruition.

“The Clarks’ generosity is inspiring, and their lead gift will be truly transformational,” said Dr. M. Katherine Banks, Texas A&M president. “This new hospital will provide hands-on educational workspaces for veterinary students and state-of-the-art laboratories for animal health and translational research, enabling our researchers, faculty and outstanding students to continue their work and provide the best animal care in the world.”

While it was considered advanced and spacious when it opened in 1981, the current Small Animal Teaching Hospital has struggled to accommodate its ever-growing occupancy spurred by booming demand for veterinary medicine practitioners and a rise in caseloads. Increasingly complex procedures requiring more sophisticated equipment, training and staff have also pushed the hospital to its limits, making for crowded workspaces. Still, thanks to its talented students, faculty and staff, the school has established itself among the best in the country, ranking fourth in the nation according to U.S. News and World Report.

Dr. John August, the Carl B. King Dean of Veterinary Medicine, looks forward to the new facility’s ability to better facilitate outstanding educational experiences, exceptional patient care that supports the human-animal bond, and clinical trials that bring scientists together from across Texas A&M and around the world to solve medical mysteries that benefit both animals and human beings.

“Our primary goal is to provide exemplary companion animal primary care education for our veterinary students,” he said. “At the same time, we aspire to become a research-intensive tertiary-care center that is recognized as the best in the world, a place people come to because it is cutting-edge and because of the high level of compassionate care. The Clarks understand that’s the role Texas A&M should have in the care of companion animals, and we are so grateful for their generosity.”

The new, next-generation teaching hospital will match the abilities of the passionate faculty and staff within its walls by radically expanding in size, updating technological features, devoting space to house future advanced research equipment and offering welcoming spaces for clientele. In addition to private dollars, the project has received funding from the Texas Legislature and Texas’ Permanent University Fund.

“Updating this facility has been a university goal for some time,” said Tyson Voelkel ’98, president and CEO of the Texas A&M Foundation. “But it needed investment from outstanding former students and philanthropic partners like the Clarks, who were willing to make this monumental gift and build a brighter future for the university. They have seen what this school and its people are capable of, and they know that Aggies will fully utilize this new teaching hospital to push their field forward.”

Dennis graduated from Texas A&M in 1968 with a bachelor’s degree in political science and earned a master’s in management from the university in 1971. He was a member of the Corps of Cadets and was commissioned into the U.S. Army. After his active duty, he began a career in the restaurant industry, during which he met Linda. In 1986, the couple founded Encore Restaurants, eventually becoming franchise owners of 39 Sonic Drive-In locations throughout the Dallas-Fort Worth area. During this same period, they also developed a successful commercial real estate development business focusing on restaurant, retail and office projects.

“Coming to Texas A&M was a watershed event in my life,” Dennis said. “It taught me about personal discipline, leadership and taking pride in what I did. My experience in the Corps of Cadets was life-changing, and many of the relationships I made during that time continue today. Linda and I are deeply involved in this university’s academic and athletic programs because Texas A&M is part of who we are.”

Before their gift to support the new teaching hospital, the Clarks generously supported the 2015 Kyle Field Redevelopment campaign, named the Football Performance Nutrition addition to the Davis Player Development Center and created two endowed faculty chairs in the veterinary school.

The couple has also been longtime clients of the university’s veterinary school, experiencing Texas A&M veterinarians’ outstanding quality of care firsthand. Two of their dogs, Labrador retrievers Molly and Cadbury, underwent tibial plateau leveling osteotomy surgery — canine knee replacements — at the current Small Animal Teaching Hospital.

“Animals have always been an integral part of our lives,” Linda said. “This university has an extraordinary veterinary school with talented people doing exciting research that will not only improve animal care but may also impact humans down the road. This gift was a big decision for us, and it ultimately came from us asking ourselves, ‘How can we facilitate what’s going on and help make it be the best it can be?’”

Texas A&M Foundation The Texas A&M Foundation is a nonprofit organization that aspires to be among the most trusted philanthropies in higher education. It builds a brighter future for Texas A&M University, one relationship at a time. To learn more, visit txamfoundation.com .

Media contact : Dunae Reader, 979-845-8161, [email protected]

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Goats and Soda

Goats and Soda

What does the science say about the origin of the SARS-CoV-2 pandemic?

Michaeleen Doucleff 2016 square

Michaeleen Doucleff

animal research studies examples

Security guards stand in front of the Huanan Seafood Wholesale Market in Wuhan, China, on Jan. 11, 2020, after the market had been closed following an outbreak of COVID-19 there. Two studies document samples of SARS-CoV-2 from stalls where live animals were sold. Noel Celis/AFP via Getty Images hide caption

Security guards stand in front of the Huanan Seafood Wholesale Market in Wuhan, China, on Jan. 11, 2020, after the market had been closed following an outbreak of COVID-19 there. Two studies document samples of SARS-CoV-2 from stalls where live animals were sold.

Since the SARS-CoV-2 pandemic began three years ago, its origin has been a topic of much scientific — and political — debate. Two main theories exist: The virus spilled over from an animal into people, most likely in a market in Wuhan, China, or the virus came from the Wuhan Institute of Virology and spread due to some type of laboratory accident.

The Wall Street Journal added to that debate this week when they reported that the U.S. Department of Energy has shifted its stance on the origin of COVID. It now concludes, with "low confidence," that the pandemic most likely arose from a laboratory leak in Wuhan, China.

The agency based its conclusion on classified evidence that isn't available to the public. According to the federal government, "low confidence" means "the information used in the analysis is scant, questionable, fragmented, or that solid analytical conclusions cannot be inferred from the information."

And at this point, the U.S. intelligence community still has no consensus about the origin of SARS-CoV-2. Four of the eight intelligence agencies lean toward a natural origin for the virus, with "low confidence," while two of them — the DOE and the Federal Bureau of Investigation — support a lab origin, with the latter having "moderate confidence" about its conclusion.

But it's a theory that the agencies are definitely bringing into the public eye. On Tuesday, FBI director Christopher A. Wray reiterated his belief that COVID-19 "most likely" sprang from "a potential lab incident" in Wuhan, Chin

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Staff members of the Wuhan Hygiene Emergency Response Team investigate the shuttered Huanan Seafood Wholesale Market on Jan. 11, 2020, after it was linked to cases of COVID-19. Noel Celis/AFP via Getty Images hide caption

Staff members of the Wuhan Hygiene Emergency Response Team investigate the shuttered Huanan Seafood Wholesale Market on Jan. 11, 2020, after it was linked to cases of COVID-19.

But at the end of the day, the origin of the pandemic is also a scientific question. Virologists who study pandemic origins are much less divided than the U.S. intelligence community. They say there is " very convincing " data and " overwhelming evidence " pointing to an animal origin.

In particular, scientists published two extensive, peer-reviewed papers in Science in July 2022, offering the strongest evidence to date that the COVID-19 pandemic originated in animals at a market in Wuhan, China. Specifically, they conclude that the coronavirus most likely jumped from a caged wild animal into people at the Huanan Seafood Wholesale Market, where a huge COVID-19 outbreak began in December 2019.

Virologist Angela Rasmussen , who contributed to one of the Science papers, says the DOE's "low confident" conclusion doesn't "negate the affirmative evidence for zoonotic [or animal] origin nor do they add any new information in support of lab origin."

"Many other [news] outlets are presenting this as new conclusive proof that the lab origin hypothesis is equally as plausible as the zoonotic origin hypothesis," Rasmussen wrote in an email to NPR, "and that is a misrepresentation of the evidence for either."

So just what is the scientific evidence that the pandemic began at the seafood market?

Neither of the Science papers provide the smoking gun — that is, an animal infected with the SARS-CoV-2 coronavirus at a market.

But they come close. They provide photographic evidence of wild animals such as raccoon dogs and a red fox, which can be infected with and shed SARS-CoV-2, sitting in cages in the market in late 2019. What's more, the caged animals are shown in or near a stall where scientists found SARS-CoV-2 virus on a number of surfaces, including on cages, carts and machines that process animals after they are slaughtered at the market.

The data in the 2022 studies paints an incredibly detailed picture of the early days of the pandemic. Photographic and genetic data pinpoint a specific stall at the market where the coronavirus likely was transmitted from an animal into people. And a genetic analysis estimates the time, within weeks, when not just one but two spillovers occurred. It calculates that the coronavirus jumped into people once in late November or early December and then again few weeks later.

At this exact same time, a huge COVID outbreak occurred at the market. Hundreds of people, working and shopping at the market, were likely infected. That outbreak is the first documented one of the pandemic, and it then spilled over into the community, as one of the Science papers shows.

At the same time, the Chinese Center for Disease Control and Prevention found two variants of the coronavirus inside the market. And an independent study, led by virologists at the University of California, San Diego, suggests these two variants didn't evolve in people, because throughout the entire pandemic, scientists have never detected a variant linking the two together. Altogether, the new studies suggest that, most likely, the two variants evolved inside animals.

animal research studies examples

Michael Worobey is a top virus sleuth. He has tracked the origins of the 1918 flu, HIV and now SARS-CoV-2. Worobey is a research professor in the Department of Ecology and Evolutionary Biology at the University of Arizona. University of Arizona hide caption

Evolutionary biologist Michael Worobey helped lead two of the studies and has been at the forefront of the search for the origins of the pandemic. He has spent his career tracking down the origins of pandemics, including the origin of HIV and the 1918 flu.

Back in May 2021, Worobey signed a letter calling for an investigation into the lab-leak theory. But then, through his own investigation, he quickly found data supporting an animal origin.

When the studies were first published online, NPR spoke to Worobey, who's at the University of Arizona, to understand what the data tells us about the origin of SARS-CoV-2; how, he believes, the data may shift the debate about the lab-leak theory; and the significance of photos taken five years before the pandemic. Here are key points from the conversation, which has been edited for clarity and length.

Live animals that are susceptible to COVID-19 were in the market in December 2019

It's clear-cut these wild, live animals, including raccoon dogs and red foxes, were in the market. We have photographic evidence from December 2019. A concerned customer evidently took these photos and videos of the market on Dec. 3 and posted them on Weibo [because it was illegal to sell certain live animals]. The photos were promptly scrubbed. But a CNN reporter had communicated directly with the person who took the photos. I was able to get in touch with this reporter, and they passed on those photos from the source. So we don't completely verify the photos.

animal research studies examples

An anonymous user on the Chinese social media platform Weibo posted pictures of live animals for sale in the southwest corner of the Huanan Seafood Market in Wuhan, China, in 2019. Researchers investigating the origins of the SAR-CoV-2 virus are including these images in a forthcoming academic paper that pinpoints the southwest corner as the most probable origin point of the pandemic. Worobey and Holmes et al. hide caption

An anonymous user on the Chinese social media platform Weibo posted pictures of live animals for sale in the southwest corner of the Huanan Seafood Market in Wuhan, China, in 2019. Researchers investigating the origins of the SAR-CoV-2 virus are including these images in a forthcoming academic paper that pinpoints the southwest corner as the most probable origin point of the pandemic.

Live susceptible animals were held in a stall where SARS-CoV-2 was later detected on a machine that processed animals in the market

We analyzed a leaked report from the Chinese CDC detailing the results of this environmental sampling. Virtually all of the findings in the report matched what was in the World Health Organization's report. But there was some extra information in the leaked report. For example, there was information not just on which stalls had virus in them — or had samples positive for SARS-CoV-2 — but also how many samples in a given stall yielded positive results.

We found out that one stall actually had five positive samples — five surfaces in that stall had virus on them. And even better, in that particular stall, the samples were very animal-y. For example, scientists found virus on a feather/hair remover, a cart of the sort that we see in photographs that are used for transporting cages and, best of all, a metal cage in a back room.

So now we know that when the national public health authorities shut down the market and then sampled the surfaces there, one of the surfaces positive for SARS-CoV-2 was a metal cage in a back room.

What's even weirder — it turns out that one of the co-authors of the study, Eddie Holmes , had been taken to the Huanan market several years before the pandemic and shown raccoon dogs in one of the stalls. He was told, "This is the kind of place that has the ingredients for cross-species transmission of dangerous pathogens."

So he clicks photos of the raccoon dogs. In one photo, the raccoon dogs are in a cage stacked on top of a cage with some birds in it.

And at the end of our sleuth work, we checked the GPS coordinates on his camera, and we find that he took the photo at the same stall, where five samples tested positive for SARS-CoV-2.

So we connected all sorts of bizarre kinds of data. Together the data are telling a strong story.

animal research studies examples

These two photos, taken in 2014 by scientist Edward Holmes, show raccoon dogs and unknown birds caged in the southwest corner of the Huanan Seafood Market in Wuhan, China. GPS coordinates of these images confirm that the animals were housed in the southwest corner of the market where researchers found evidence of the virus in January 2020. Edward Holmes hide caption

These two photos, taken in 2014 by scientist Edward Holmes, show raccoon dogs and unknown birds caged in the southwest corner of the Huanan Seafood Market in Wuhan, China. GPS coordinates of these images confirm that the animals were housed in the southwest corner of the market where researchers found evidence of the virus in January 2020.

Earliest known cases of COVID-19, even those not directly related to individuals who had been in the market, radiate out from the market

With a virus, such as SARS-CoV-2, that causes no symptoms or mild symptoms in most people, you don't have any chance of linking all the early cases to the site where the outbreak started. Because the virus is going to quickly spread to people outside of wherever it started.

And yet, from the clinical observations in Wuhan, around half of the earliest known COVID cases were people directly linked to the seafood market. And the other cases, which aren't linked through epidemiological data, have an even closer geographical association to the market. That's what we show in our paper.

It's absurd how strong the geographical association is [to the market].

NPR: Absurd? How? In the sense that the seafood market is so clearly bull's-eye center of this outbreak?

Yes. And I don't understand how anyone could not be moved, at least somewhat, by that data and then take this idea [of an animal origin] seriously, especially given the other things we've found in these studies.

animal research studies examples

The Huanan Seafood Wholesale Market on July 16, 2021. Getty Images/ Stringer hide caption

The Huanan Seafood Wholesale Market on July 16, 2021.

The virus jumped into people right before the outbreak in the market

For example, our new genetic analysis tells us that this virus was not around for very long when the cases occurred at the market. For example, the earliest known patient at the market had an onset of symptoms on Dec. 10, 2019. And we can estimate that at that point in time, there were only about 10 people infected with the virus in the world and probably fewer than 70.

So if the pandemic didn't start at the market, one of the first five or 10 people infected in the world was at the market. And how do you explain that?

You have to remember: Wuhan is a city of 11 million people. And the Huanan market is only 1 of 4 places in Wuhan that sold live animals susceptible to SARS-CoV-2, such as raccoon dogs.

It's highly unlikely that the first COVID-19 outbreak would occur at the market if there weren't a source of the virus there

Step back and think, "Where is the first cluster of a new respiratory infection going to appear in this city?" It could appear at a market. But it could also appear at a school, a university or a meatpacking plant.

NPR: Or a biotech conference?

Yes. In Washington state, SARS-CoV-2 first appeared in a man who had traveled back from China. In Germany, it was at an auto-parts supplier.

There are thousands, perhaps 10,000, other places at least as likely, or even more likely, to be the place where a new pathogen shows up. And yet, in Wuhan, the first cluster of cases happens to be one of the four places that sells live animals, out of 10,000 other places. If you're not surprised by that, then I don't think you're understanding the unlikelihood that that presents.

NPR: So what is the likelihood of that coincidence happening — that the first cluster of cases occurs at a market that sells animals known to be susceptible to SARS-CoV-2, but the virus didn't actually come from the market?

I would put the odds at 1 in 10,000. But it's interesting. We do have one analysis where we show essentially that the chance of having this pattern of cases [clustered around the market] is 1 in 10 million [if the market isn't a source of the virus]. We consider that strong evidence in science.

The analyses that we've done are telling a very strong story.

The evidence is amongst the best we have for any emerging virus.

NPR: Really?

It's important to note we haven't found a related virus from the intermediate host. But we have a bunch of other evidence.

And the data zeroing in on the Huanan market, to me, is as compelling as the data that indicated to John Snow that the water pump was poisoning people who used it. [ John Snow was a doctor in London who helped launch the field of outbreak investigations by figuring out the source of a cholera outbreak in the city in the mid-19th century.]

Making these findings brought tears

Sometimes you have these rare moments where you're maybe the only person on Earth who has access to this kind of crucial information. As I just started to figure out that there were more cases around the market than you can expect randomly — I felt that way. And no exaggeration, that moment — those kinds of moments — bring a tear to your eye.

Correction Feb. 28, 2023

An earlier version of this story misstated the Federal Bureau of Investigation as the Federal Bureau of Information.


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