experiment on animals is not ethical

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• Future Perfect

Animal testing, explained

Is anything really “cruelty-free”?

by Celia Ford

It’s nearly impossible to go a day without benefitting from the suffering of animals. The ingredients in your toiletries and makeup; your medicine, vaccines, and implants; your cleaning supplies; the chemicals that helped grow your food — most of it was, at some point, tested on animals.

For centuries, the biological sciences have relied on animal testing. To figure out how a machine works, you need to disassemble it and check out its component parts. Understanding the living body, one of nature’s most complex and beautiful machines, is no different. Taking apart and fiddling with a toaster doesn’t hurt anyone, but dismantling a biological system certainly does.

Many scientists believe that experimenting on living animals is a necessary means of solving problems that affect both humans and animals. But these experiments often involve animals experiencing distress, whether from the side effects of an experimental drug, an intentionally inflicted illness, or simply their confined living situation. Some lucky lab animals get to spend their retirement in sanctuaries once they’re no longer needed. Most of the time, the animal dies, either as a direct consequence of the experiment or from euthanasia.

More often than not, animal research happens behind closed, locked, unmarked doors. That lack of transparency makes it difficult to know what to think about animal testing, and public opinion is tellingly divided. A 2018 Pew Research Center survey found that 47 percent of people in the US support the use of animals in scientific research, and 52 percent oppose it. Unlike climate change or reproductive health , where the parties are highly polarized, animal testing is one of few science-related policy issues where the attitudes of Republicans and Democrats are pretty similar: Both parties are split roughly 50-50.

Experimenting on animals places two seemingly good things — medical innovation and animal welfare — at odds. Even those who support animal research generally hold nuanced, conflicted beliefs about it, and questions about the nature and extent of animal testing are still hotly debated.

Inside this story

• What animal testing actually does
• Who is looking out for the welfare of animals
• The truth behind labels like “cruelty-free”
• The future of animal-free testing

Brands frequently mislead consumers about animal testing involving their products with vague labeling, and alternative research methods aren’t as broadly applicable as some activist organizations imply . Meanwhile, research facilities often ban employees from sharing photos of lab animals without institutional approval and rarely let the media observe experiments for themselves.

After spending six years as a neuroscience PhD student working in a lab with monkeys, I left academia with the impression that animal testing is neither as well-managed or justified as regulators claim, nor as malicious as others fear. Government agencies are starting to direct funding toward finding alternatives to animal testing, but the use of animals is deeply embedded in biological sciences.

A world without lab animals may be possible, but we don’t live in it yet. Here’s what’s actually going on.

What is animal testing?

Before humans invented microscopes, universities, or even paper, we were using animals for medical research. Over two millennia ago , ancient Greek philosopher Aristotle dissected dozens of animal species to better understand their anatomy and argued that studying their bodies could teach us a lot about our own biology. Over four centuries later, Galen of Pergamon , one of the most pivotal characters in Western medical history, performed public surgeries on animals ( especially monkeys ) for science, providing a spectacle that attracted curious audiences.

Today, animal experimentation is widespread and conducted far from the public eye. It falls under two broad, semi-overlapping umbrellas: biomedical research (which aims to understand, prevent, and treat diseases, as well as uncover fundamental information about how bodies work) and toxicology , or testing the effects of chemicals (including everything from toothpaste and makeup to pesticides) on living things.

Humans generally don’t want to be proverbial guinea pigs for new medicines or consumer products. We’d rather know that things are safe before we put them anywhere near our bodies. Companies, whether they deal in cosmetics or pharmaceuticals, also don’t want to be liable for poisoning their customers.

People can participate in experiments that might harm them, but historically, at best, such projects have been difficult to administer . At worst, they have involved illegal human experimentation that cast a long, dark shadow over the field of medical research.

The Tuskegee syphilis study , for example, put hundreds of poor Black men with untreated syphilis through decades of invasive tests in exchange for hot meals and basic medical treatment, just to see how the disease would progress if left untreated. Effective treatments became available during the study, but researchers withheld them. Once the experiment’s scandalous history was publicly disclosed in 1972 , the US government formalized basic ethical guidelines for human research and required Institutional Review Boards (IRBs) to approve studies on humans.

Today, many questions — like What is the lethal dose of this new drug? and Does this new surgical technique actually work? — can’t ethically be asked regarding humans without first being tested on a nonhuman subject.

For a long time, animals were the only alternative to humans available. To figure out the lethal dose of a new drug, scientists can give increasingly large amounts of it to mice and see what it takes to kill them. To test whether a brain implant actually relieves Parkinson’s symptoms, scientists do brain surgery on monkeys . Without computational models or cell cultures sophisticated enough to mimic the complicated interactions between organs, the options have historically been to use animals as a proxy or to drop or scale back your planned research.

We can only guess how many animals are being used in scientific experiments worldwide. The United States Department of Agriculture (USDA) publishes official reports on animal research every year, but they only include animals protected by the Animal Welfare Act (AWA), the federal law setting basic standards for the treatment and housing of certain farm animals and lab animals. The law covers dogs, cats, monkeys, guinea pigs, hamsters, pigs, rabbits, and sheep. In 2019, about 800,000 animals protected by AWA were used in research — 930,000, if you add those that lived in labs but were never included in a study.

Notably, the AWA doesn’t apply to mice and rats, which several studies estimate account for somewhere between 93 and 99 percent of all lab animals in the US. The AWA also excludes invertebrates like flies, worms, fish, and cephalopods like octopuses, whose intelligence makes them intriguing neuroscience subjects. The EU, which counts all vertebrates used in experiments, tallied about 10.6 million animals used in 2017. It’s harder to pin down a number in the US. Depending on who you ask , there might be 10 million rodents subjected to scientific experiments annually, or there might be 111 million. (Either way, it’s more than three times the number of rats in New York City.)

Rodents make appealing animal models for many scientists because they’re smart enough to learn simple tasks but are still socially regarded as pests; those who kill rats for a living don’t face the same kind of backlash as someone who, say, boasts about shooting a puppy . Nearly all mouse genes share functions with human genes, so at a basic level, their biology resembles ours. Mice only live for a year or two, enabling scientists to study things like chronic disease progression without waiting an entire human lifespan. And scientists can genetically alter mice in countless ways, knocking out or adding DNA to express diseases or make certain cell types glow under a microscope.

In some cases, a research question requires invasively studying a full, living biological system, but the gap between mice and humans is too wide. The USDA reported that 68,257 monkeys were used in 2019 to study subjects like SARS-CoV-2 , Parkinson’s disease , and HIV , where physiological and cognitive similarity to humans was a priority. Those primates were mostly macaques and marmosets; the use of chimpanzees (our closest ape relative) is now banned in many countries , including the US .

But monkey research may not be viable much longer. While hundreds of monkey experiments are being funded by the NIH , there aren’t enough long-tailed macaques to go around. In a desperate attempt to keep up with skyrocketing demand, thousands of wild-caught monkeys are illegally imported to US research institutions from countries like Cambodia. Two years ago, the long-tailed macaque was listed as endangered for the first time. PETA petitioned the US government to protect the species under the Endangered Species Act , which could end their use in research altogether, but the request has yet to be approved. Most people are uncomfortable with the idea of experimenting on an animal so similar to us, including some of the scientists who do it. However, many scientists and policymakers agree that we still don’t have non-animal alternatives that can answer tough research questions involving interactions between organs. Researchers worry that the looming primate shortage in the US — engendered by transportation restrictions and therapeutic testing requirements and exacerbated by pandemic-era demands — will limit our ability to respond to public health emergencies.

Monkeys are traditionally recognized as the only nonhuman animals that react to drugs with human-specific targets, meaning that in some cases, their body’s reactions could uniquely predict whether a drug will be safe and effective for humans. During the first years of the Covid-19 pandemic, monkeys were considered so crucial to SARS-CoV-2 research that when the rhesus macaque supply dried up, scientists didn’t turn to cell cultures or computer models — they just looked for different monkeys .

You might not agree that this research justifies the nonconsensual use of highly intelligent animals; many don’t, for both ethical and scientific reasons. But it’s happening, and if you’ve been vaccinated or take medications, you’ve likely benefited from it.

Who’s looking out for the welfare of lab animals?

The regulatory framework surrounding animal research is a tangled web of acronyms, committees, and working groups. Since the Animal Welfare Act was passed in 1966, the USDA has been in charge of enforcing it through inspections and annual reports.

In theory, researchers have to justify the use of animals in their work. To conduct an animal experiment, scientists in the US go through a review process with their Institutional Animal Care and Use Committee (IACUC), which decides whether animals are “necessary” and whether steps are being taken to minimize their pain.

IACUCs are mostly comprised of researchers who experiment on animals and the veterinarians who help them, strongly biasing committees toward approving animal experiments. In the US and elsewhere, scientists are subtly incentivized to use animals, even when they aren’t actually necessary. Academic journals tend to preferentially publish work with animal methods , and academic careers hinge on accumulating publications . These norms seep into the labs where animal experiments are performed. New animal researchers often receive explicit instructions on how to steer clear of animal rights activists, according to several researchers I spoke with while working as a neuroscientist (as well as my own experience).

This can make holding institutions accountable for animal welfare violations challenging. While researchers are required to report information about animals in their facilities, like what medical procedures they’ve received and when they’ve been fed, they are told to keep these reports “ minimal, but complete .” In other words: Avoid including photos, videos, or graphic descriptions that could enrage activists or entice the media.

There also isn’t a clear legal definition for “animal cruelty” in research settings beyond violations of the basic standards outlined by the Animal Welfare Act. This leaves some room for interpretation about what is acceptable and what would constitute illegal treatment. The EU’s Directive 2010/63/EU , its equivalent of the Animal Welfare Act, emphasizes that animals should only be used if there are no other options and if the potential benefits of the research outweigh the animals’ suffering.

This cost-benefit analysis is subjective. For example, a team of immunologists studying cancer in mice would probably say that the potential public health benefits of their work justify harming mice. A team of science policy experts at PETA would say that mice aren’t ours to use and that these experiments often don’t translate to human trials, anyway .

To bridge this ethical divide, research universities and private companies in the UK have signed a Concordat on Openness on Animal Research , pledging to proactively and transparently inform the public about their treatment of lab animals. In the decade since its launch, nine other countries have followed suit. It’s likely not a coincidence that these countries generally have the tightest restrictions on animal use. However, an independent review found that Concordat signatories in the UK are still struggling to be transparent about their animal research practices in the face of potential disapproval.

On top of the slow pace for necessary regulation, stigma obscures the true nature of what happens in these labs. In the late 2000s, the most extreme opponents of animal testing used violence to try to end the practice, sending poisoned razors and death threats to lab heads and, in at least one case, firebombing a neurobiologist’s car . But rather than encourage scientists to reconsider their methods, attacks like these cemented a culture of silence. While physical violence is not representative of activism against animal testing today — which usually centers around investigations , government advocacy , and direct care for animals and has shifted to become more inclusive — the threat of retaliation still haunts animal researchers , some of whom are encouraged by their institutions to hide their connections to animal testing from the public.

Scientists “don’t want to feel like they’re bad people,” said neuroscientist and author Garet Lahvis, who has written about primate research for Vox.

What if I want to avoid animal testing altogether? What does “cruelty-free” mean?

After learning about what lab animals go through, some people will want to find ways to avoid the products of animal testing. This is much easier said than done, however.

Animal testing is pervasive in health care. Many treatments we take for granted today, like anesthesia , flu shots , and allergy medications , went through preclinical trials in animals before reaching us. They are also valuable to your health, so please keep taking your medicine if you need it. We have more power to avoid animal testing elsewhere. Animal testing requirements are generally looser to nonexistent for cosmetics, cleaning supplies, and other household chemicals, so it’s possible to buy “cruelty-free” makeup or laundry detergent.

The legal distinction between “cosmetics” and “drugs” is blurry, though. Essentially, drugs claim to affect the body’s structure or function in some way, while cosmetics are things you apply to your body to change your appearance (like lipstick) or clean yourself (like deodorant — but not soap, which is neither a cosmetic nor a drug, but its own special category ). Many products we might think of as cosmetics are, in fact, also drugs, like anti-dandruff shampoo, tinted moisturizer with sunscreen, and other cosmetics that claim to treat some ailment. In the US, all of these items had to be tested on animals until the FDA Modernization Act 2.0 took effect in 2023 .

Cruelty-free claims used on product labels are often misleading, and differences in regulation across countries add to the confusion.

For years, the EU , Canada, Mexico, and 16 other countries (including South Korea, for the skincare girlies ) have had legislation in place banning animal testing for cosmetics or their ingredients (although last year, the UK changed their policy to allow testing for makeup ingredients again). But testing on final products or their ingredients has never been banned in the US. Even if a company doesn’t test its final product on animals, it may still run animal tests on raw ingredients. And even if those raw ingredients aren’t currently being tested on animals, they probably were when they were first introduced.

The US government doesn’t have a legal definition for the terms “cruelty-free” or “not tested on animals.” A product labeled “cruelty-free” likely earned voluntary certification from a private organization like Leaping Bunny or PETA’s Beauty Without Bunnies program by pledging to end animal testing at all stages of product development. The definition of “cruelty-free” isn’t standardized across animal protection groups, but earning a “bunny label” generally means that a brand attested to never conducting tests on animals during a product’s development.

Despite pressure from advocates and consumers, many US companies don’t bother with these pledges on animal testing. As of this year, approximately 310 brands globally still test their beauty and household cleaning products on animals. And some actively say they don’t test on animals at all but still sell their products to countries like China, which, until recently, required that all cosmetics (even imported ones) be tested on animals . Most certification programs exclude brands and products sold in China for this very reason.

To make it easier for US companies to sell truly cruelty-free products in China, US regulators and animal welfare advocates have been lobbying their Chinese counterparts for years to change their approach to animal testing for consumer products. Twenty years ago, Thomas Hartung, a toxicologist at the Johns Hopkins Center for Alternatives to Animal Testing, spoke with the National Medical Products Administration (China’s FDA) about regulating animal testing of chemicals and told me “it was like we were coming from Mars.”

In response to yearslong campaigns by organizations like PETA and the Institute for In Vitro Sciences, China recently lifted this requirement . It is now possible to buy Chinese cosmetics that weren’t tested on animals — kind of.

As of January 2021, China no longer requires pre-market or post-market animal testing for cosmetics, meaning that companies from the US and elsewhere can sell things like eyeliner or nail polish in China while still maintaining “cruelty-free” status. But certain “special cosmetics,” like sunscreen, teeth whiteners, and hair dye, or products made for children, are all still required to undergo animal testing. And if a product uses a raw ingredient that isn’t already approved in China, foreign companies have to either reformulate or get that ingredient approved, which requires more animal testing. So, it’s possible to sell US-made “cruelty-free” products in China, but it requires sifting through a confusing and ever-evolving swamp of documentation requirements.

We have made imperfect progress toward a world of cruelty-free cosmetics. While the number of animals used for cosmetic testing in the US has dropped by 90 percent since the 1980s, 44 of the largest 50 cosmetic brands in the world still are not cruelty-free . And without a consensus agreement on what “cruelty-free” actually means, consumers are left to guess which bunny labels are genuine and which are false advertising.

Since many brands can just slap on cruelty-free claims while still sending products abroad to animal testing labs, for now, if you want to avoid animal testing, Leaping Bunny and Beauty Without Bunnies are your best bets. These certifications consider post-market animal testing in other countries as part of their standards.

Alternative methods are (slowly) coming

In some places, like the UK, strict restrictions on animal research and a commitment to transparency have considerably improved lab conditions in recent decades. Companies like Neuralink , however, continue to perform high-risk, ethically dubious experiments hidden from the public eye.

While new alternative methods are under development, animal testing remains necessary in at least some circumstances. Tight regulation — and buy-in from scientists — will be key to minimizing harm in the meantime.

Nicole Kleinstreuer, acting director of the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) , told me that improving the current state of animal testing hinges on researchers gathering “the courage to admit that we can substantially improve upon how we’ve been doing things historically.”

Until relatively recently, alternatives to animal testing in many areas of science were very limited. But in the past decade, bioengineering and computer science have advanced rapidly. New tools like AI, organoids (balls of stem cells that grow into organ tissue), and CRISPR have made replacing animals, at least in certain experiments, more attainable.

For chemical testing, good animal-free research methods have been around for decades — long before most scientists considered using them. Even when well-validated animal alternatives exist, researchers can be slow to adopt them . Hartung, a toxicologist, said, “I turned 60 last year. The methods they’re using were introduced when I was in kindergarten.”

In 2007, the National Academies of Sciences, Engineering, and Medicine , a nonprofit that produces independent policy guidance for the US, laid out a strategy for researchers to move away from using animals in toxicity testing and to develop faster, more human-relevant models to take their place. Today, a number of working groups, both within the US and collaborating internationally, are still trying to put this principle into practice.

As the largest single public funder of biomedical research in the world, the National Institutes of Health (NIH) is uniquely positioned to influence animal testing. In 2023, the NIH spent an estimated $19 billion on US-based projects involving animals, according to Citizens for Alternatives to Animal Research and Experimentation. Between 2011 and 2021, they spent $2.2 billion on projects based in other countries — where oversight boils down to trusting self-generated, non-validated reports from foreign institutions.

Kleinstreuer said that changing the current state of animal research “really necessitates a sea change, and a dramatic investment on the part of funders, particularly the NIH.”

The people in charge of the money have the power to redistribute it and could choose to spend more of it on projects that don’t use animals and less on those that do. That’s the easy part. “It’s kind of the lowest-hanging fruit, and the easiest ask,” said Emily Trunnell , director of Science Advancement and Outreach at PETA. “Even people who are in support of animal testing are on board with the funding of different methods as well.”

NICEATM, led by Kleinstreuer, is doing the in-the-weeds work of figuring out how we’d know whether a replacement method is good enough to substitute for animal experiments. Earlier this year, the NIH also approved the Complement Animal Research in Experimentation (Complement-ARIE) Program , which will set up technology development centers for researchers to make better human-based models.

Non-animal methods can already outperform certain animal tests. Back in 2018, Hartung’s research group created algorithms mapping the relationships between 10,000 known chemical compounds. With this model and lots of data, they predicted the toxicity of 89 percent of the 48,000 toxic chemicals more accurately than animal tests could and for much less money — without endangering any living creatures. Since then, Hartung said things have only become better. But AI-driven research methods are still limited by what real-world data has already been collected. “When you have no data,” he said, “nothing is possible.”

In some cases, using animals is simply bad science. There are some questions “that absolutely necessitate a human cell-based approach,” Kleinstreuer said. “You can’t look at the efficacy of a drug whose target is not expressed in animals by using animal models,” she added. Certain cancer drugs target protein receptors that only exist in humans, and gene therapies often aim to rewrite human-specific DNA sequences. One emerging option: take a sample of human cells, reprogram them to behave like whatever cells you want them to be, and test your drug on the resulting tissue sample.

These tools offer exciting opportunities to personalize medicine to individual patients, but it’s still tough to extrapolate results from a small mass of lab-grown cells in a tightly controlled environment to a human body and the complex interactions of its organ systems. Cancer and embryonic development are incredibly complex biological processes, involving lots of different interconnected body parts that evolve over time. Without that capability, Kleinstreuer said it’s harder to argue that a substance is actually safe and ready to clear for human use.

Change happens one retirement at a time

As it stands, alternatives to animal tests are not being used as widely as they should be, especially in cosmetics. But if we want to study things like deep brain stimulation or run safety tests on new cancer drugs , animal tests are all we have.

While we are stuck with animal experiments, we can try to limit them and make them more humane. Lahvis believes that we should have extremely strict criteria for what animal experiments are funded. Strategically allocating grant funding could not only save millions of lives, but also inspire better science.

Convincing animal researchers to replace animals with other methods is still a huge challenge. Hartung joked that in academia, change happens “one retirement at a time.” Unfortunately, “it’s often been one graveyard at a time,” as retired scientists continue to serve as reviewers who help choose what new projects get funded and published.

The further along a scientist is in their career, the more challenging it becomes to pivot. Because scientists are pushed to maintain a constant level of productivity, Trunnell said, someone who builds their whole lab around their current use of animal models has no incentive to change, unless they have a strong desire to do so. Changing tactics could mean putting their job on the line.

“We’re highly leveraged by the system to keep doing what we’ve always done,” Lahvis agreed. And, Hartung said, turning against a tried-and-true method would require a scientist invalidating their existing body of work or at least acknowledging that it was either unethical, ineffective, or inefficient. Using past observations to inform future experiments is at the core of the scientific method, but, Hartung said, “We’re not trained to be very self-critical.”

That said, a growing number of scientists support the development of non-animal methods, even as they continue to work with animals themselves. People want new tools, whether for the sake of animal welfare or simply because it would make for better science. We might just have to wait another generation.

• Animal Welfare

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13.7 Cosmos & Culture

The 'necessity' of animal research does not mean it's ethical.

Samual Garner

Diane, a 4-year-old chimpanzee, relaxes in the trees at the Chimp Haven sanctuary in Keithville, La., on Aug. 25, 2014. She is one of many chimps who have been moved here from the New Iberia Research Center in Lafayette, La. Brandon Wade/AP hide caption

Diane, a 4-year-old chimpanzee, relaxes in the trees at the Chimp Haven sanctuary in Keithville, La., on Aug. 25, 2014. She is one of many chimps who have been moved here from the New Iberia Research Center in Lafayette, La.

A few weeks ago, two prominent scientists, Hollis Cline and Mar Sanchez, wrote a brief piece in The Hill newspaper arguing that animal research is "necessary." They were prompted by the recent National Institutes of Health (NIH) decision to phase out the use of primates in controversial maternal deprivation studies.

Scientists have long been fond of claims of necessity — in fact, justifications for animal research have remained largely the same since the writings of 19th century French physiologist Claude Bernard. However, this claim is problematic for a number of reasons.

If animal research is necessary, then it is not necessary in the sense that we have to do it. Rather, it is a choice that we make, a choice that its proponents believe is a necessary means to the end of further medical advances. Such advances are undoubtedly of significant moral importance, but even if we grant the assumption that animals are necessary for medical progress, this does not equate to a moral justification.

Research with humans is necessary to medical progress, but we have set strict limits on the extent to which humans can be exposed to risk and harm in research, even though doing so has undoubtedly slowed the rate of medical progress that might otherwise be achievable. Cline and Sanchez claim that animals in research are treated "humanely and with dignity," but the reality is that the level of protection afforded to research animals is far, far less than that afforded to human participants in research. Most animals involved in research are killed at the termination of the experiment, are kept in conditions not conducive to their welfare, and are otherwise harmed in myriad and significant ways, for example through the infliction of physical injuries, infectious diseases, cancers, or psychological distress.

While nonhuman animals cannot provide consent to research participation, we have reasoned in the case of humans that an inability to consent entitles an individual to greater protection and not lesser protection. What justifies our differential treatment of humans and nonhuman animals in research? For present purposes, it isn't necessary to rehearse every possible argument for and against animal research. It is sufficient to note that very few contemporary ethicists defend the status quo of animal research and, furthermore, that the burden of proof has now shifted to those who would defend invasive animal research.

Given the state of philosophical scholarship, meeting this burden of proof will not be easy or straightforward. Perhaps the most remarkable aspect of the scientific community's frequent claims of the necessity of animal research is how thoroughly they miss the moral point. For the most part, ethical criticisms of animal research aren't even addressed — as they aren't in Cline and Sanchez's piece — and when they are, they're usually dismissed with bad arguments, like this one , that have been refuted for decades.

Further, the claim that "animal research is necessary to medical progress" assumes a strong causal connection between the two, but what data we have available cast doubt upon the robustness of this connection. Despite strong claims about the historical benefits of animal research from the scientific community, the accuracy of animal models in predicting human responses has not been evaluated sufficiently, and the lack of certain kinds of data make this evaluation especially challenging . Based on existing data, however, numerous reviews have suggested that the accuracy of animal research in predicting human health outcomes appears to be far less than what we once assumed.

Animal studies also frequently appear to be poorly designed . The predictive value of animal research might increase if study design improved, but this isn't certain. Even NIH Director Francis Collins recognized these concerns in a forward-thinking 2011 commentary , stating that, "The use of animal models for therapeutic development and target validation...may not accurately predict efficacy in humans." Given these issues, systematic reviews should become routine and strong statements about the utility of animal models should be tempered. This does not mean that animal research has never produced any or even many important medical benefits, but these claims require empirical validation, not simply repeated assertion.

It also means that scientists and science agencies should be much more aggressive about seeking and funding alternatives to animals in research. Support has certainly grown, but investment of money and human labor into non-animal alternatives has been paltry. Even with this limited investment, some impressive advances are being made — witness the ongoing development of " organs on a chip " — but much more needs to be done, with more money behind it, and with more of a sense of haste.

Beyond funding, the scientific community simply needs to adopt a better attitude toward innovation in alternatives, or else their limitations will continue to be a self-fulfilling prophecy. This is science — a discipline with a remarkable history of achievement and innovation despite significant technical challenges. Where are the editorials galvanizing the scientific community to continue to innovate without animals? Where is the Human Genome Project-type investment in alternatives? To say that animal models are "necessary" when alternatives are not aggressively pursued seems a bit dishonest. And given the amount of harm caused to animals in research—whether you think it's justified or not—we should all want the alternatives field to grow.

Literally thousands of books and peer-reviewed papers have been written on the extent of our moral obligations to animals. As a field that is dedicated to rigorous inquiry and rational thought, the scientific community should take seriously the vast philosophy literature on these topics — the same field that gave rise to the conceptual foundations of science — rather than assertions and rhetoric. When it comes to animals and ethics, there have been very few serious attempts to engage the intellectual issues. Scientists can and should do better.

Samual Garner is a bioethicist living in Washington, DC. He is an associate fellow at the Oxford Centre for Animal Ethics and writes on human research ethics and animal ethics.

• medical research
• animal research
• animal ethics

Is animal research ethical?

Posted: by John Meredith on 16/02/22

More on these Topics:

How can it be right to use an animal for research where we could consider it unethical to use a human being? This is a fundamental question that confronts anybody who benefits from research using animals. If we claim that causing harm to animals is sometimes justifiable where it would be unacceptable to inflict a similar harm or risk on a person, then it seems we are assuming that animals must, in some sense, have less moral value. But is that a justifiable assumption, or is it just a self-serving prejudice? Are there solid rational arguments for treating humans differently from other animals, or are we simply falling back on outmoded habits of thought, a smokescreen that helps us avoid looking the ugly truth of our actions in the eye?

Moral status of animals

In the past, the moral status of animals did not merit a great deal of consideration; raising questions about whether humans were entitled to exploit animals would have struck most people as quaint or absurd. The great moral philosopher Rene Descartes, for example, the man famous for the phrase  cogito ergo sum  - ‘I think therefore I am’ - believed that animals had no inner life at all, that they were essentially as lifeless as clockwork dolls, incapable of emotion, self-awareness, or even feeling pain.  

Such ideas seem laughable to us now. We take it for granted that most animals experience pain and many have complex emotional lives that can depend on relationships with other animals and which can deliver feelings of pleasure and satisfaction. Since Descartes’ day, the growing study of animal behaviour makes this seem obvious, and cleverly designed experiments have confirmed what has been learned from observation, forcing us to acknowledge that sentience – inner life – exists in a great number of other species and sometimes at a very high level. 

But what implications does all this have for the moral consideration of animals? How should it affect the way we treat them? Philosopher Peter Singer, whose book  Animal Liberation  transformed the public debate on animal welfare, believes it should have deep and wide-reaching consequences. Singer argues that it is wrong to inflict harm on a person not because of any cosmic or biblical law about harm but because it is against that person’s interests as they themselves understand them. Considering moral questions in that light, he argues, explodes any idea that we can justify distinctions between individuals based on their sex or race, distinctions that have been passionately defended over many centuries. There are many differences between people of all kinds including, of course, both sexes, but they all have interests that are alike: an interest in avoiding pain or hunger for example. There is no rational basis for preferring the interests of any particular individual, or people of one race or sex class over those of another, that is simply racism and sexism. This is an idea has become widely accepted, if only recently, and it doesn’t seem particularly radical to us today, but Singer takes the idea a step further. 

If there is no non-arbitrary reason to prefer the interests of one human animal over another, how can there be any good reason to prefer the interests of a human animal over a non-human animal? Claims that humans are of special moral interest because of their intelligence or capacity for language or any of the many other things that have been suggested cut no ice.  A less intelligent human has as much interest in avoiding pain as a mathematical genius does, and the same goes for a dog, or a mouse, or a fish. To deny this, says Singer is to make a moral mistake akin to sexism or racism and he calls this way of thinking  speciesism .

One objection to the argument from speciesism is that it implies that there can never be a reason to prefer the welfare of a human being over any other animal where considerations of interest are the same. This strikes most people as counter-intuitive to say the least. Jean Kazez, philosopher and animal rights activist, suggests a thought experiment. Imagine a dedicated vegan responsible for the care of ten young children. It so happens that famine strikes and the children are all in danger of starvation except that our vegan carer owns a cow. Would it be morally acceptable for the vegan to stick by her principles and refuse to slaughter the cow to save the children? If the answer is no, then there seems to be some problem with the speciesist position. It would probably not be considered acceptable to slaughter one of the children to feed the others, after all. So, our intuition is that there must be some foundation for our moral preference for a human over an animal, at least in some extreme conditions. Perhaps the intuition is that there is moral value in feelings of kinship because this is a necessary feeling in order to be a fully healthy human, to flourish as a human being. If that is the case, then, kinship, for humans, is a kind of interest in the Singer sense and one that overrides other interests. That may be why we don’t find it reprehensible when a mother prefers the welfare of her child over that of another.

The moral value of ‘kinship’ overrides speciesism

If kinship carries moral weight, then the speciesist argument loses ground and a possible justification for preferring animals over human beings in research emerges.  Medical research is an attempt to save human lives and reduce human suffering (it has similar benefits for animal as well, of course, but we can set that aside for now, for the sake of simplicity). If, as scientists argue, this can only be achieved with the use of an animal model, then we are morally entitled to prefer the use of a non-human animal, so long as kinship has the moral value we are claiming for it and the suffering and distress of the animals is minimised as much as possible.

But what if this is all just a complicated exercise in justifying what we want to do anyway, what if our moral intuitions are just wrong? It is easy to imagine a Singerian arguing, in the case of our starving children and vegan nanny, that the cow has as much moral standing as any of the others: it has the same interest in living and not suffering the pain of hunger as the others and, what’s more, it may be better able to survive the famine given its ability to eat vegetation that cannot sustain humans. In that case, it seems the advocate of speciesism must argue that they all should starve together in the interests of admirable intellectual rigour, even if it feels a little hard on the children.

Using utility to resolve moral conflicts

As usual, though, the situation is more complicated. Peter Singer and his followers recognise that there is often a conflict of moral interests and so we need a framework for finding a resolution. This framework should not be  ad hoc or arbitrary or based on scripture or any other culturally specific text or tradition but should be rational. Within Singer’s argument the rational moral grounding is provided by utilitarianism the ethical doctrine first proposed by Jeremy Bentham in the 19th century. Utilitarianism argues that when two actions are in conflict, the morally correct one is the one that delivers the most happiness for the largest number (Bentham called this ‘utility’ for obscure reasons). In other words, the morality of an action is decided by its consequences, not by the intentions of the actor or anything else. Applied to the problem of our starving infants and their increasingly paranoid cow, a utilitarian might argue that killing the cow is justified despite it having a similar interest in living to the children because the slaughter would maximise future happiness (utility). If they all die, happiness would be at zero, and if a child was sacrificed to save the others, that would reduce overall happiness because of the distress of the survivors at their loss, the suffering endured by the child selected to die, and the indifference of the cow. 

How do you measure happiness?

Problems with utilitarian ways of thinking immediately suggest themselves: how can happiness be measured? How can the ‘happiness’ of a mouse, for example, be weighed against a person, or any other animal? Must we consider a well-intentioned action that has bad outcomes immoral instead of just unfortunate? The literature goes into all these problems and more at great depth, but for our purposes, it is at least clear that a utilitarian moral framework allows for the use of research animals in some circumstances. The human happiness delivered by a successful medical treatment can be great and long lasting while any pain or distress caused to the experimental animals is kept to a minimum and is of very limited duration. In the utilitarian scales, this tips firmly towards an ethical justification of animal research. It is a surprise to many people that Peter Singer, the father of the modern animal rights movement, comes to the same conclusion, although he argues for stricter controls and more work to reduce and mitigate the use of animals. Even without appealing to concepts such as kinship, in other words, the concept of speciesism, perhaps the most formidable intellectual weapon aimed against animal research by protest groups, does not carry the day. It is perfectly possible to allow the moral value of an animal’s interests and still justify its use in research – even if that research causes the animal harm or distress – so long as the future outcomes maximise happiness. 

Animal rights arguments

The only significant ethical argument against animal research that remains is based on the idea of rights. Just as humans have inalienable rights, the argument goes, so do animals. According to this view, the use of animals for research can never be justified for exactly the same reasons that we cannot justify using humans. But argument from rights has many more problems than argument from interests: from where are rights derived? What specific rights do animals have? Should rights be protected even when this is damaging to the welfare of the animal? This last point is perhaps the most salient. If we allow an animal has a right to its freedom, say, not to be kept in captivity (one of the key rights usually claimed by activists), then we are not only committed to ending all ownership of animals, but to the immediate release of all domestic animals into the wild even if that were to the detriment of the animals’ welfare as it surely would be. The problems mount at every step. How can it be possible to reconcile a vole’s right to life with a falcon’s right to eat? What possible mechanism could be constructed to resolve such conflicts and how much irreparable harm to natural ecosystems would follow if we built one? Without answers to questions like this it is hard to see animal rights arguments as much more than rhetoric.

Maximising future happiness and minimising present suffering is enough for an ethical justification of animal research

The case for ethical animal research, then, does not need as much building as it might at first appear. None of the major philosophical arguments for animal welfare exclude the possibility of ethical animal research. The harm that is done to animals in well-regulated research environments serves a higher moral purpose: the reduction of death and suffering by disease and other disorders. Of course, this is only true if pain, suffering and distress, are minimised – as they are through animal welfare regulations in the UK and EU for example. These regulations also require the application of the principles of the 3Rs – but it is quite obvious, all other things being equal, that the use of a mouse in an investigation into cancer development, for example, will create less suffering than using a person for the same purposes. 

So, a utilitarian calculation of maximising future happiness and minimising present suffering is enough for an ethical justification of animal research even for tough minded opponents of animal exploitation such as Professor Singer. But maybe justification is the wrong word. 

Are we not morally obliged to use animals in research?

If, as the biological sciences are almost unanimous in claiming, we cannot have new medicines without some animal research, and if there are hundreds of devastating human illnesses that will continue to cause misery, pain, and heartache without those new treatments, should we not think of animal research as a moral obligation instead? It is difficult science to do, both technically and emotionally, but if we choose not to carry it out, we are effectively choosing to allow human suffering to continue in the future that our efforts today have the potential to reduce or eliminate. We don’t know which suffering we will be successful in mitigating when, but we can be certain that progress is being made. Remove animal research and we don’t not remove suffering, we simply transfer it from the animals now (where it is carefully controlled and minimised, very often to nothing) to future humans. That is the heart of the ethical case for animal research and one that needs to be better addressed by those who oppose it.

Last edited: 7 April 2022 12:16

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Peter Singer: Are experiments on animals ethically justifiable?

Petra Stock

Professor Peter Singer does not take an absolutist position on the ethics of using animals in scientific research. 

The world-renowned ethicist and philosopher, based at Princeton University in the US, has been revisiting the issue of experimentation on animals, in updating and republishing his most famous book –  Animal Liberation Now – after more than 40 years.

In the book, Singer writes: “it will not do to say ‘Never!’” when it comes to scientific and medical research. 

In conversation with  Cosmos ,   Singer clarifies. He says there may be examples of lifesaving research, where even after giving full weight to the interests of animals, the research might still be justified by the very large number of people who will benefit, if it comes off – if there are genuinely no alternatives. 

“But I think it’s quite rare, and I don’t think the system that we have of assessing experiments is really rigorous enough to allow only those sorts of experiments to pass.”

Much of the research on animals today is not about developing life-saving drugs, he says. A lot of the substances being tested are not essential. They might be a new sunscreen or cleaning agent, or a rival pharmaceutical company working on an alternative to a tried-and-tested medicine in order to gain a slice of that lucrative market.

“A very substantial proportion of the research that is done on animals, is not for urgent, lifesaving conditions, and would not be justified if we were to consider the interests of the animals in a serious and significant way, as I think we should,” he says.

It continues to take place, he says, “because the animals become tools for research. The experimenter has no problem ordering another batch of a couple of 100 mice to do research on. I think that’s the problem.”

Significant numbers of animals are used in scientific and medical research in Australia and other countries. Based on available data, Singer’s book estimates as many as 15.6 million animals are experimented on in the US, and more than 52 million in China. 

“A very substantial proportion of the research that is done on animals, is not for urgent, lifesaving conditions, and would not be justified if we were to consider the interests of the animals in a serious and significant way, as I think we should.” Peter Singer

As  Cosmos  has previously reported , some 700,000 mice and 30,000 rats are used in research in Australia based on statistics from 3 states. Advocates say the national figure is likely in excess of 1 million rodents, in addition to other laboratory animals. 

In Australia, all research involving animals is required to seek approval through Animal Ethics Committees and must consider the “3Rs” of replacement, reduction and refinement.

Singer says he has previously served on an animal ethics committee at Monash University. He believes that while there is some value in the process, it doesn’t go far enough.

Lab rats and science mice: Why are we using animals in research?

“Too often the majority of the committee are scientists already trained and set in that way of doing research,” he says.

That makes it hard for researchers to see the alternatives, even though that could potentially lead to better scientific outcomes.

In his book, Singer highlights problems relating to the transferability of animal research. He quotes Richard Klausner, a former director of the US National Cancer Institute as saying: “We have cured mice of cancer for decades and it simply didn’t work in humans”.

Speaking with  Cosmos , Singer mentions a presentation from the 12 th  World Congress on Alternatives and Animal Use in the Life Sciences, an event at which he delivered the closing address.

Researchers in Canada tested substances on laboratory mice housed in two different set ups. One group of mice were kept in the standard way, in small containers the size of a shoebox, with a grid on top and bright lights, which he describes as “quite stressful conditions for mice”.

Meanwhile another group of mice were housed in quarters more suitable to their nature, with places they could hide, and tunnels to run through.

“The basic question is, are these animals who can suffer?” Peter Singer

Singer says, the researchers found when they tested substances on two groups, they got quite different reactions depending on the conditions the mice were kept in. 

This raises questions about the veracity of experimental results involving animals like mice, already stressed by the laboratory environment and practices.

Do mice and rats deserve greater ethical consideration in science? 

“The basic question is, are these animals who can suffer?,” Singer replies.

He says there’s no doubt that mice and rats – which represent the majority of laboratory animals used in science and research – can suffer. They’re mammals, vertebrates, with the same basic nervous system and brains like humans, albeit significantly smaller.

“In some respects, because they don’t understand this situation, things may be more terrifying to them than they would be to us,” he says.

“I certainly think that they count.”

Originally published by Cosmos as Peter Singer: Are experiments on animals ethically justifiable?

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Should Animals Be Used for Scientific or Commercial Testing?

• History of Animal Testing

Animals are used to develop medical treatments, determine the toxicity of medications, check the safety of products destined for human use, and other  biomedical , commercial, and health care uses. Research on living animals has been practiced since at least 500 BC.

Descriptions of the dissection of live animals have been found in ancient Greek writings from as early as circa 500 BC. Physician-scientists such as  Aristotle ,  Herophilus , and  Erasistratus  performed the experiments to discover the functions of living organisms.  Vivisection  (dissection of a living organism) was practiced on human criminals in ancient Rome and Alexandria, but prohibitions against mutilation of the human body in ancient Greece led to a reliance on animal subjects. Aristotle believed that animals lacked intelligence, and so the notions of justice and injustice did not apply to them.  Theophrastus , a successor to Aristotle, disagreed, objecting to the vivisection of animals on the grounds that, like humans, they can feel pain, and causing pain to animals was an affront to the gods. Read more background…

Pro & Con Arguments

Pro 1 Animal testing contributes to life-saving cures and treatments for humans and animals alike. Nearly every medical breakthrough in the last 100 years has resulted directly from research using animals, according to the California Biomedical Research Association. To name just a few examples, animal research has contributed to major advances in treating conditions including breast cancer, brain injury, childhood leukemia, cystic fibrosis, multiple sclerosis, and tuberculosis. Testing on animals was also instrumental in the development of pacemakers, cardiac valve substitutes, and anesthetics. [ 9 ] [ 10 ] [ 11 ] [ 12 ] [ 13 ] Scientists racing to develop a vaccine for coronavirus during the 2020 global pandemic needed to test on genetically modified mice to ensure that the vaccine did not make the virus worse. Nikolai Petrovsky, professor in the College of Medicine and Public Health at Flinders University in Australia, said testing a coronavirus vaccine on animals is “absolutely essential” and skipping that step would be “fraught with difficulty and danger.” [ 119 ] [ 133 ] Researchers have to test extensively to prevent “vaccine enhancement,” a situation in which a vaccine actually makes the disease worse in some people. “The way you reduce that risk is first you show it does not occur in laboratory animals,” explains Peter Hotez, Dean for the National School of Tropical Medicine at Baylor College. [ 119 ] [ 141 ] Further, animals themselves benefit from the results of animal testing. Vaccines tested on animals have saved millions of animals that would otherwise have died from rabies, distemper, feline leukemia, infectious hepatitis virus, tetanus, anthrax, and canine parvo virus. Treatments for animals developed using animal testing also include pacemakers for heart disease and remedies for glaucoma and hip dysplasia. [ 9 ] [ 21 ] Animal testing has also been instrumental in saving endangered species from extinction, including the black-footed ferret, the California condor and the tamarins of Brazil. The American Veterinary Medical Association (AVMA) endorses animal testing to develop safe drugs, vaccines, and medical devices. [ 9 ] [ 13 ] [ 23 ] Read More

Pro 2 Animals are appropriate research subjects because they are similar to human beings in many ways. Chimpanzees share 99% of their DNA with humans, and mice are 98% genetically similar to humans. All mammals, including humans, are descended from common ancestors, and all have the same set of organs (heart, kidneys, lungs, etc.) that function in essentially the same way with the help of a bloodstream and central nervous system. Because animals and humans are so biologically similar, they are susceptible to many of the same conditions and illnesses, including heart disease, cancer, and diabetes. [ 9 ] [ 17 ] [ 18 ] Animals often make better research subjects than humans because of their shorter life cycles. Laboratory mice, for example, live for only two to three years, so researchers can study the effects of treatments or genetic manipulation over a whole lifespan, or across several generations, which would be infeasible using human subjects. Mice and rats are particularly well-suited to long-term cancer research, partly because of their short lifespans. [ 9 ] [ 29 ] [ 30 ] Further, animals must be used in cases when ethical considerations prevent the use of human subjects. When testing medicines for potential toxicity, the lives of human volunteers should not be put in danger unnecessarily. It would be unethical to perform invasive experimental procedures on human beings before the methods have been tested on animals, and some experiments involve genetic manipulation that would be unacceptable to impose on human subjects before animal testing. The World Medical Association Declaration of Helsinki states that human trials should be preceded by tests on animals. [ 19 ] [ 20 ] A poll of 3,748 scientists by the Pew Research Center found that 89% favored the use of animals in scientific research. The American Cancer Society, American Physiological Society, National Association for Biomedical Research, American Heart Association, and the Society of Toxicology all advocate the use of animals in scientific research. [ 36 ] [ 37 ] [ 38 ] [ 39 ] [ 40 ] [ 120 ] Read More

Pro 3 Animal research is highly regulated, with laws in place to protect animals from mistreatment. In addition to local and state laws and guidelines, animal research has been regulated by the federal Animal Welfare Act (AWA) since 1966. As well as stipulating minimum housing standards for research animals (enclosure size, temperature, access to clean food and water, and others), the AWA also requires regular inspections by veterinarians. [ 3 ] All proposals to use animals for research must be approved by an Institutional Animal Care and Use Committee (IACUC) set up by each research facility. Most major research institutions’ programs are voluntarily reviewed for humane practices by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). [ 24 ] [ 25 ] Animal researchers treat animals humanely, both for the animals’ sake and to ensure reliable test results. Research animals are cared for by veterinarians, husbandry specialists, and animal health technicians to ensure their well-being and more accurate findings. Rachel Rubino, attending veterinarian and director of the animal facility at Cold Springs Harbor Laboratory, says, “Most people who work with research animals love those animals…. We want to give them the best lives possible, treat them humanely.” At Cedars-Sinai Medical Center’s animal research facility, dogs are given exercise breaks twice daily to socialize with their caretakers and other dogs, and a “toy rotation program” provides opportunities for play. [ 28 ] [ 32 ] Read More

Con 1 Animal testing is cruel and inhumane. Animals used in experiments are commonly subjected to force feeding, food and water deprivation, the infliction of burns and other wounds to study the healing process, the infliction of pain to study its effects and remedies, and “killing by carbon dioxide asphyxiation, neck-breaking, decapitation, or other means,” according to Humane Society International. The US Department of Agriculture reported in Jan. 2020 that research facilities used over 300,000 animals in activities involving pain in just one year. [ 47 ] [ 102 ] Plus, most experiments involving animals are flawed, wasting the lives of the animal subjects. A peer-reviewed study found serious flaws in the majority of publicly funded US and UK animal studies using rodents and primates: “only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used.” A 2017 study found further flaws in animal studies, including “incorrect data interpretation, unforeseen technical issues, incorrectly constituted (or absent) control groups, selective data reporting, inadequate or varying software systems, and blatant fraud.” [ 64 ] [ 128 ] Only 5% of animals used in experiments are protected by US law. The Animal Welfare Act (AWA) does not apply to rats, mice, fish, and birds, which account for 95% of the animals used in research. The types of animals covered by the AWA account for fewer than one million animals used in research facilities each year, which leaves around 25 million other animals without protection from mistreatment. The US Department of Agriculture, which inspects facilities for AWA compliance, compiles annual statistics on animal testing but they only include data on the small percentage of animals subject to the Act. [ 1 ] [ 2 ] [ 26 ] [ 28 ] [ 135 ] Even the animals protected by the AWA are mistreated. Violations of the Animal Welfare Act at the federally funded New Iberia Research Center (NIRC) in Louisiana included maltreatment of primates who were suffering such severe psychological stress that they engaged in self-mutilation, infant primates awake and alert during painful experiments, and chimpanzees being intimidated and shot with a dart gun. [ 68 ] Read More

Con 2 Animal tests do not reliably predict results in human beings. 94% of drugs that pass animal tests fail in human clinical trials. Over 100 stroke drugs and over 85 HIV vaccines failed in humans after succeeding in animal trials. Nearly 150 clinical trials (human tests) of treatments to reduce inflammation in critically ill patients have been undertaken, and all of them failed, despite being successful in animal tests. [ 57 ] [ 58 ] [ 59 ] Drugs that pass animal tests are not necessarily safe. The 1950s sleeping pill thalidomide, which caused 10,000 babies to be born with severe deformities, was tested on animals prior to its commercial release. Later tests on pregnant mice, rats, guinea pigs, cats, and hamsters did not result in birth defects unless the drug was administered at extremely high doses. Animal tests on the arthritis drug Vioxx showed that it had a protective effect on the hearts of mice, yet the drug went on to cause more than 27,000 heart attacks and sudden cardiac deaths before being pulled from the market. [ 5 ] [ 55 ] [ 56 ] [ 109 ] [ 110 ] Plus, animal tests may mislead researchers into ignoring potential cures and treatments. Some chemicals that are ineffective on (or harmful to) animals prove valuable when used by humans. Aspirin, for example, is dangerous for some animal species. Intravenous vitamin C has shown to be effective in treating sepsis in humans, but makes no difference to mice. Fk-506 (tacrolimus), used to lower the risk of organ transplant rejection, was “almost shelved” because of animal test results, according to neurologist Aysha Akhtar. A report on Slate.com stated that a “source of human suffering may be the dozens of promising drugs that get shelved when they cause problems in animals that may not be relevant for humans.” [ 105 ] [ 106 ] [ 127 ] Read More

Con 3 Alternative testing methods now exist that can replace the need for animals. Other research methods such as in vitro testing (tests done on human cells or tissue in a petri dish) offer opportunities to reduce or replace animal testing. Technological advancements in 3D printing allow the possibility for tissue bioprinting: a French company is working to bioprint a liver that can test the toxicity of a drug. Artificial human skin, such as the commercially available products EpiDerm and ThinCert, can be made from sheets of human skin cells grown in test tubes or plastic wells and may produce more useful results than testing chemicals on animal skin. [ 15 ] [ 16 ] [ 50 ] [ 51 ] Michael Bachelor, Senior Scientist and Product Manager at biotech company MatTek, stated, “We can now create a model from human skin cells — keratinocytes — and produce normal skin or even a model that mimics a skin disease like psoriasis. Or we can use human pigment-producing cells — melanocytes — to create a pigmented skin model that is similar to human skin from different ethnicities. You can’t do that on a mouse or a rabbit.” The Environmental Protection Agency is so confident in alternatives that the agency intends to reduce chemical testing on mammals 30% by 2025 and end it altogether by 2035. [ 61 ] [ 134 ] [ 140 ] Scientists are also able to test vaccines on humans volunteers. Unlike animals used for research, humans are able to give consent to be used in testing and are a viable option when the need arises. The COVID-19 (coronavirus) global pandemic demonstrated that researchers can skip animal testing and go straight to observing how vaccines work in humans. One company working on a COVID-19 vaccine, Moderna Therapeutics, worked on developing a vaccine using new technology: instead of being based on a weakened form of the virus, it was developed using a synthetic copy of the COVID-19 genetic code. [ 142 ] [ 143 ] Read More

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Ethical care for research animals

WHY ANIMAL RESEARCH?

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.

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

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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. 

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Animal Testing

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About Animal Testing

Humane Society International / Global

What is animal testing?

The term “animal testing” refers to procedures performed on living animals for purposes of research into basic biology and diseases, assessing the effectiveness of new medicinal products, and testing the human health and/or environmental safety of consumer and industry products such as cosmetics, household cleaners, food additives, pharmaceuticals and industrial/agro-chemicals. All procedures, even those classified as “mild,” have the potential to cause the animals physical as well as psychological distress and suffering. Often the procedures can cause a great deal of suffering. Most animals are killed at the end of an experiment, but some may be re-used in subsequent experiments. Here is a selection of common animal procedures:

• Forced chemical exposure in toxicity testing, which can include oral force-feeding, forced inhalation, skin or injection into the abdomen, muscle, etc.
• Exposure to drugs, chemicals or infectious disease at levels that cause illness, pain and distress, or death
• Genetic manipulation, e.g., addition or “knocking out” of one or more genes
• Ear-notching and tail-clipping for identification
• Short periods of physical restraint for observation or examination
• Prolonged periods of physical restraint
• Food and water deprivation
• Surgical procedures followed by recovery
• Infliction of wounds, burns and other injuries to study healing
• Infliction of pain to study its physiology and treatment
• Behavioural experiments designed to cause distress, e.g., electric shock or forced swimming
• Other manipulations to create “animal models” of human diseases ranging from cancer to stroke to depression
• Killing by carbon dioxide asphyxiation, neck-breaking, decapitation, or other means

What types of animals are used?

Many different species are used around the world, but the most common include mice, fish, rats, rabbits, guinea pigs, hamsters, farm animals, birds, cats, dogs, mini-pigs, and non-human primates (monkeys, and in some countries, chimpanzees). Video: Watch what scientists have to say about alternatives to animal testing .

It is estimated that more than 115 million animals worldwide are used in laboratory experiments every year. But because only a small proportion of countries collect and publish data concerning animal use for testing and research, the precise number is unknown. For example, in the United States, up to 90 percent of the animals used in laboratories (purpose-bred rats, mice and birds, fish, amphibians, reptiles and invertebrates) are excluded from the official statistics, meaning that figures published by the U.S. Department of Agriculture are no doubt a substantial underestimate.

Within the European Union, more than 12 million animals are used each year, with France, Germany and the United Kingdom being the top three animal using countries. British statistics reflect the use of more than 3 million animals each year, but this number does not include animals bred for research but killed as “surplus” without being used for specific experimental procedures. Although these animals still endure the stresses and deprivation of life in the sterile laboratory environment, their lives are not recorded in official statistics. HSI believes that complete transparency about animal use is vital and that all animals bred, used or killed for the research industry should be included in official figures. See some animal use statistics .

What’s wrong with animal testing?

For nearly a century, drug and chemical safety assessments have been based on laboratory testing involving rodents, rabbits, dogs, and other animals. Aside from the ethical issues they pose—inflicting both physical pain as well as psychological distress and suffering on large numbers of sentient creatures—animal tests are time- and resource-intensive, restrictive in the number of substances that can be tested, provide little understanding of how chemicals behave in the body, and in many cases do not correctly predict real-world human reactions. Similarly, health scientists are increasingly questioning the relevance of research aimed at “modelling” human diseases in the laboratory by artificially creating symptoms in other animal species.

Trying to mirror human diseases or toxicity by artificially creating symptoms in mice, dogs or monkeys has major scientific limitations that cannot be overcome. Very often the symptoms and responses to potential treatments seen in other species are dissimilar to those of human patients. As a consequence, nine out of every 10 candidate medicines that appear safe and effective in animal studies fail when given to humans. Drug failures and research that never delivers because of irrelevant animal models not only delay medical progress, but also waste resources and risk the health and safety of volunteers in clinical trials.

What’s the alternative?

If lack of human relevance is the fatal flaw of “animal models,” then a switch to human-relevant research tools is the logical solution. The National Research Council in the United States has expressed its vision of “a not-so-distant future in which virtually all routine toxicity testing would be conducted in human cells or cell lines”, and science leaders around the world have echoed this view.

The sequencing of the human genome and birth of functional genomics, the explosive growth of computer power and computational biology, and high-speed robot automation of cell-based (in vitro) screening systems, to name a few, has sparked a quiet revolution in biology. Together, these innovations have produced new tools and ways of thinking that can help uncover exactly how chemicals and drugs disrupt normal processes in the human body at the level of cells and molecules. From there, scientists can use computers to interpret and integrate this information with data from human and population-level studies. The resulting predictions regarding human safety and risk are potentially more relevant to people in the real world than animal tests.

But that’s just the beginning. The wider field of human health research could benefit from a similar shift in paradigm. Many disease areas have seen little or no progress despite decades of animal research. Some 300 million people currently suffer from asthma, yet only two types of treatment have become available in the last 50 years. More than a thousand potential drugs for stroke have been tested in animals, but only one of these has proved effective in patients. And it’s the same story with many other major human illnesses. A large-scale re-investment in human-based (not mouse or dog or monkey) research aimed at understanding how disruptions of normal human biological functions at the levels of genes, proteins and cell and tissue interactions lead to illness in our species could advance the effective treatment or prevention of many key health-related societal challenges of our time.

Modern non-animal techniques are already reducing and superseding experiments on animals, and in European Union, the “3Rs” principle of replacement, reduction and refinement of animal experiments is a legal requirement. In most other parts of the world there is currently no such legal imperative, leaving scientists free to use animals even where non-animal approaches are available.

If animal testing is so unreliable, why does it continue?

Despite this growing evidence that it is time for a change, effecting that change within a scientific community that has relied for decades on animal models as the “default method” for testing and research takes time and perseverance. Old habits die hard, and globally there is still a lack of knowledge of and expertise in cutting-edge non-animal techniques.

But with HSI’s help, change is happening. We are leading efforts globally to encourage scientists, companies and policy-makers to transition away from animal use in favour of 21st century methods. Our work brings together experts from around the globe to share knowledge and best practice, improving the quality of research by replacing animals in the laboratory.

Are animal experiments needed for medical progress?

It is often argued that because animal experiments have been used for centuries, and medical progress has been made in that time, animal experiments must be necessary. But this is missing the point. History is full of examples of flawed or basic practices and ideas that were once considered state-of-the-art, only to be superseded years later by something far more sophisticated and successful. In the early 1900’s, the Wright brothers’ invention of the airplane was truly innovative for its time, but more than a century later, technology has advanced so much that when compared to the modern jumbo jet those early flying machines seem quaint and even absurd. Those early ideas are part of aviation history, but no-one would seriously argue that they represent the cutting-edge of design or human achievement. So it is with laboratory research. Animal experiments are part of medical history, but history is where they belong. Compared to today’s potential to understand the basis of human disease at cellular and molecular levels, experimenting on live animals seems positively primitive. So if we want better quality medical research, safer more effective pharmaceuticals and cures to human diseases, we need to turn the page in the history books and embrace the new chapter—21st century science.

Independent scientific reviews demonstrate that research using animals correlates very poorly to real human patients. In fact, the data show that animal studies fail to predict real human outcomes in 50 to 99.7 percent of cases. This is mainly because other species seldom naturally suffer from the same diseases as found in humans. Animal experiments rely on often uniquely human conditions being artificially induced in non-human species. While on a superficial level they may share similar symptoms, fundamental differences in genetics, physiology and biochemistry can result in wildly different reactions to both the illness and potential treatments. For some areas of disease research, overreliance on animal models may well have delayed medical progress rather than advanced it. By contrast, many non-animal replacement methods such as cell-based studies, silicon chip biosensors, and computational systems biology models, can provide faster and more human-relevant answers to medical and chemical safety questions that animal experiments cannot match.

“The claim that animal experimentation is essential to medical development is not supported by proper, scientific evidence but by opinion and anecdote. Systematic reviews of its effectiveness don’t support the claims made on its behalf” (Pandora Pound et al. British Medical Journal 328, 514-7, 2004).

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Imagine a syringe being forced down your throat to inject a chemical into your stomach, or being restrained and forced to breathe sickening vapours for hours. That’s the cruel reality of animal testing for millions of mice, rabbits, dogs and other animals worldwide.

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What is ethical animal research? A scientist and veterinarian explain

Postdoctoral Research Fellow in Neuroscience, National Institutes of Health

Clinical Veterinarian, Emory National Primate Research Center, Emory University

Disclosure statement

Lana Ruvolo Grasser, Ph.D. is the 2022-2023 American College of Neuropsychopharmacology, Americans for Medical Progress Biomedical Research Awareness Day Fellow. She has previously received funding from the National Institute of Mental Health, Blue Cross Blue Shield Foundation of Michigan, and Wayne State University; none of which has supported the work described herein. She is a member of the Anxiety and Depression Association of America, International Society for Traumatic Stress Studies, International Society for Developmental Psychobiology, and Michigan Society for Neuroscience. Dr. Grasser contributed to this article in her personal capacity. The views expressed are her own and do not necessarily represent the views of the National Institutes of Health or the United States Government.

Rachelle Stammen works as a Clinical Veterinarian at the Emory National Primate Research Center. She is a member of the American Veterinary Medical Association, American Association of Laboratory Animal Science, Association of Primate Veterinarians, and a Diplomate of the American College of Laboratory Animal Medicine. This work is not affiliated with or reflect the opinions of Emory University or Emory National Primate Research Center.

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A proposed measure in Switzerland would have made that country the first to ban medical and scientific experimentation on animals. It failed to pass in February 2022, with only 21% of voters in favor. Yet globally, including in the United States , there is concern about whether animal research is ethical.

We are scientists who support ethical animal research that reduces suffering of humans and animals alike by helping researchers discover the causes of disease and how to treat it . One of us is a neuroscientist who studies behavioral treatments and medications for people with post-traumatic stress disorder – treatments made possible by research with dogs and rodents . The other is a veterinarian who cares for laboratory animals in research studies and trains researchers on how to interact with their subjects.

We both place high importance on ensuring that animal research is conducted ethically and humanely. But what counts as “ethical” animal research in the first place?

The 4 R’s of animal research

There is no single standard definition of ethical animal research. However, it broadly means the humane care of research animals – from their acquisition and housing to the study experience itself.

Federal research agencies follow guiding principles in evaluating the use and care of animals in research. One is that the research must increase knowledge and, either directly or indirectly, have the potential to benefit the health and welfare of humans and other animals. Another is that only the minimum number of animals required to obtain valid results should be included. Researchers must use procedures that minimize pain and distress and maximize the animals’ welfare. They are also asked to consider whether they could use nonanimal alternatives instead, such as mathematical models or computer simulations.

These principles are summarized by the “ 3 R’s” of animal research : reduction, refinement and replacement. The 3 R’s encourage scientists to develop new techniques that allow them to replace animals with appropriate alternatives.

Since these guidelines were first disseminated in the early 1960s , new tools have helped to significantly decrease animal research. In fact, since 1985, the number of animals in research has been reduced by half .

A fourth “R” was formalized in the late 1990s: rehabilitation , referring to care for animals after their role in research is complete.

These guidelines are designed to ensure that researchers and regulators consider the costs and benefits of using animals in research, focused on the good it could provide for many more animals and humans. These guidelines also ensure protection of a group – animals – that cannot consent to its own participation in research. There are a number of human groups that cannot consent to research, either, such as infants and young children, but for whom regulated research is still permitted, so that they can gain the potential benefits from discoveries .

Enforcing ethics

Specific guidelines for ethical animal research are typically established by national governments . Independent organizations also provide research standards.

In the U.S., the Animal Welfare Act protects all warmblooded animals except rats, mice and birds bred for research. Rats, mice and birds are protected – along with fish, reptiles and all other vertebrates – by the Public Health Service Policy .

Each institution that conducts animal research has an entity called the Institutional Animal Care and Use Committee , or IACUC. The IACUC is composed of veterinarians, scientists, nonscientists and members of the public. Before researchers are allowed to start their studies, the IACUC reviews their research protocols to ensure they follow national standards. The IACUC also oversees studies after approval to continually enforce ethical research practices and animal care. It, along with the U.S. Department of Agriculture , accreditation agencies and funding entities, may conduct unannounced inspections.

Laboratories that violate standards may be fined, forced to stop their studies, excluded from research funding, ordered to cease and desist, and have their licenses suspended or revoked. Allegations of misconduct are also investigated by the National Institutes of Health’s Office of Laboratory Animal Welfare .

Above and beyond the basic national standards for humane treatment, research institutions across 47 countries, including the U.S., may seek voluntary accreditation by a nonprofit called the Association for Assessment and Accreditation of Laboratory Animal Care , or AAALAC International. AAALAC accreditation recognizes the maintenance of high standards of animal care and use. It can also help recruit scientists to accredited institutes, promote scientific validity and demonstrate accountability.

Principles in practice

So what impact do these guidelines actually have on research and animals?

First, they have made sure that scientists create protocols that describe the purpose of their research and why animals are necessary to answer a meaningful question that could benefit health or medical care. While computer models and cell cultures can play an important role in some research, others studies, like those on Alzheimer’s disease , need animal models to better capture the complexities of living organisms. The protocol must outline how animals will be housed and cared for, and who will care for and work with the animals, to ensure that they are trained to treat animals humanely.

During continual study oversight, inspectors look for whether animals are provided with housing specifically designed for their species’ behavioral and social needs. For example, mice are given nesting materials to create a comfortable environment for living and raising pups . When animals don’t have environmental stimulation, it can alter their brain function – harming not only the animal, but also the science.

Monitoring agencies also consider animals’ distress. If something is known to be painful in humans, it is assumed to be painful in animals as well. Sedation, painkillers or anesthesia must be provided when animals experience more than momentary or slight pain.

For some research that requires assessing organs and tissues, such as the study of heart disease, animals must be euthanized. Veterinary professionals perform or oversee the euthanasia process. Methods must be in compliance with guidelines from the American Veterinary Medical Association , which requires rapid and painless techniques in distress-free conditions.

Fortunately, following their time in research, some animals can be adopted into loving homes , and others may be retired to havens and sanctuaries equipped with veterinary care, nutrition and enrichment.

Continuing the conversation

Animal research benefits both humans and animals. Numerous medical advances exist because they were initially studied in animals – from treatments for cancer and neurodegenerative disease to new techniques for surgery, organ transplants and noninvasive imaging and diagnostics .

These advances also benefit zoo animals, wildlife and endangered species. Animal research has allowed for the eradication of certain diseases in cattle , for example, leading not only to reduced farm cattle deaths and human famine, but also to improved health for wild cattle. Health care advances for pets – including cancer treatments , effective vaccines, nutritional prescription diets and flea and tick treatments – are also available thanks to animal research.

People who work with animals in research have attempted to increase public awareness of research standards and the positive effects animal research has had on daily life. However, some have faced harassment and violence from anti-animal research activists . Some of our own colleagues have received death threats.

Those who work in animal research share a deep appreciation for the creatures who make this work possible. For future strides in biomedical care to be possible, we believe that research using animals must be protected, and that animal health and safety must always remain the top priority.

Editor’s note: One photo depicting a species that is highly restricted for use in biomedical research has been removed from the article.

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Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research

Download the guidelines (PDF, 86KB)

February 2022

A foundational aspect of the discipline of psychology is teaching about and research on the behavior of nonhuman animals. Studying other animals is critical to understanding basic principles underlying behavior and to advancing the welfare of both human and nonhuman animals. While psychologists must conduct their teaching and research in a manner consonant with relevant laws and regulations, ethical concerns further mandate that psychologists consider the costs and benefits of procedures involving nonhuman animals before proceeding with these activities.

The following guidelines were developed by the American Psychological Association (APA) for use by psychologists working with nonhuman animals. The guidelines are informed by relevant sections of the Ethical Principles of Psychologists and Code of Conduct (APA, 2017).The acquisition, care, housing, use, and disposition of nonhuman animals in research must comply with applicable federal, state, and local laws and regulations, institutional policies, and with international conventions to which the United States is a party. APA members working outside the United States must also follow all applicable laws and regulations of the country in which they conduct research.

It is important to recognize that this document constitutes “guidelines,” which serve a different purpose than “standards.” Standards, unlike guidelines, require mandatory compliance, and may be accompanied by an enforcement mechanism. This document is meant to be aspirational and thereby provides recommendations for the professional conduct of specified activities. These guidelines are not intended to be mandatory, exhaustive, or definitive and should not take precedence over the professional judgment of individuals who have competence in the subject addressed.

Questions about these guidelines should be referred to the APA Committee on Animal Research and Ethics (CARE) via email at [email protected] , by phone at 202-336-6000, or in writing to the American Psychological Association, Science Directorate, Office of Research Ethics, 750 First St., NE, Washington, DC 20002-4242

These guidelines are scheduled to expire 10 years from (the date of adoption by the APA Council of Representatives). After this date users are encouraged to contact the APA Science Directorate to determine whether this document remains in effect.

• Research should be undertaken with a clear scientific purpose. There should be a reasonable expectation that the research will a) increase knowledge of the process underlying the evolution, development, maintenance, alteration, control, or biological significance of behavior; b) determine the replicability and generality of prior research; c) increase understanding of the species under study; or d) provide results that benefit the health or welfare of humans or other animals.
• The scientific purpose of the research should be of sufficient potential significance to justify the use of nonhuman animals. In general, psychologists should act on the assumption that procedures that are likely to produce pain in humans may also do so in other animals, unless there is species-specific evidence of pain or stress to the contrary.
• In proposing a research project, the psychologist should be familiar with the appropriate literature, consider the possibility of nonanimal alternatives, and use procedures that minimize the number of nonhuman animals in research. If nonhuman animals are to be used, the species chosen for the study should be the best suited to answer the question(s) posed.
• Research on nonhuman animals may not be conducted until the protocol has been reviewed and approved by an appropriate animal care committee; typically, an Institutional Animal Care and Use Committee (IACUC), to ensure that the procedures are appropriate and abide by the principles for humane experimental techniques embodied by the 3Rs – Replacement, Reduction, and Refinement (Russell & Burch, 1959).
• The researcher(s) should monitor the research and the subjects’ welfare throughout the course of an investigation to ensure continued justification for the research.
• Psychologists should ensure that personnel involved in their research with nonhuman animals be familiar with these guidelines.
• Investigators and personnel should complete all required institutional research trainings for the ethical conduct of such research.
• Research procedures with nonhuman animals should conform to the Animal Welfare Act (7 U.S.C. §2131 et. seq.) and when applicable, the Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS, 2015) and the Guide for the Care and Use of Laboratory Animals (National Resource Council, 2011), as well as other applicable federal regulations, policies, and guidelines, regarding personnel, supervision, record keeping, and veterinary care.
• As behavior is not only the focus of study of many experiments but also a primary source of information about an animal’s health and well-being, investigators should watch for and recognize deviations from normal, species-typical behaviors as indicators of potential health problems.
• Psychologists should assume it is their responsibility that all individuals who work with nonhuman animals under their supervision receive explicit instruction in experimental methods and in the care, maintenance, and handling of the species being studied. The activities that any individuals may engage in must not exceed their respective competencies, training, and experience in either the laboratory or the field setting

As a scientific and professional organization, APA recognizes the complexities of defining psychological well-being for both human and nonhuman animals. APA does not provide specific guidelines for the maintenance of psychological well-being of research animals, as procedures that are appropriate for a particular species may not be for others. Psychologists who are familiar with the species, relevant literature, federal guidelines, and their institution’s research facility should consider the appropriateness of measures such as social housing and enrichment to maintain or improve psychological well-being of those species.

• The facilities housing laboratory animals should meet or exceed current regulations and guidelines (USDA, 1990, 1991; NIH, 2015) and are required to be inspected twice a year (USDA, 1989; NIH, 2015).
• All procedures carried out on nonhuman animals are to be reviewed by an IACUC to ensure that the procedures are appropriate and humane. The committee must have representation from within the institution and from the local community. In the event that it is not possible to constitute an appropriate IACUC in the psychologist’s own institution, psychologists should seek advice and obtain review from a corresponding committee of a cooperative institution.
• Laboratory animals are to be provided with humane care and healthful conditions during their stay in any facilities of the institution. Responsibilities for the conditions under which animals are kept, both within and outside of the context of active experimentation or teaching, rests with the psychologist under the supervision of the IACUC (where required by federal regulations) and with individuals appointed by the institution to oversee laboratory animal care.
• Laboratory animals not bred in the psychologist’s facility are to be acquired lawfully. The USDA and local ordinances should be determined and followed prior to IACUC protocol submission.
• Psychologists should make every effort to ensure that those responsible for transporting the nonhuman animals to the facility provide adequate food, water, ventilation, and space, and impose no unnecessary stress on the animals (NRC, 2006).
• Nonhuman animals taken from the wild should be trapped in a humane manner and in accordance with applicable federal, state, and local regulations.
• Use of endangered, threatened, or imported nonhuman animals must only be conducted with full attention to required permits and ethical concerns. Information and permit applications may be obtained from the Fish and Wildlife Service website at www.fws.gov .

Consideration for the humane treatment and well-being of the laboratory animal should be incorporated into the design and conduct of all procedures involving such animals, while keeping in mind the primary goal of undertaking the specific procedures of the research project—the acquisition of sound, replicable data. The conduct of all procedures is governed by Guideline I (Justification of Research) above.

• Observational and other noninvasive forms of behavioral studies that involve no aversive stimulation to, or elicit no sign of distress from, the nonhuman animal are acceptable.
• Whenever possible behavioral procedures should be used that minimize discomfort to the nonhuman animal. Psychologists should adjust the parameters of aversive stimulation to the minimal levels compatible with the aims of the research. Consideration should be given to providing the research animals control over the potential aversive stimulation whenever it is consistent with the goals of the research. Whenever reasonable, psychologists are encouraged to first test on themselves the painful stimuli to be used on nonhuman animal subjects.
• Procedures in which the research animal is anesthetized and insensitive to pain throughout the procedure, and is euthanized (AVMA, 2020) before regaining consciousness are generally acceptable.
• Procedures involving more than momentary or slight aversive stimulation, which is not relieved by medication or other acceptable methods, should be undertaken only when the objectives of the research cannot be achieved by other methods.
• Experimental procedures that require prolonged aversive conditions or produce tissue damage or metabolic disturbances require greater justification and surveillance by the psychologist and IACUC. A research animal observed to be in a state of severe distress or chronic pain that cannot be alleviated and is not essential to the purposes of the research should be euthanized immediately (AVMA, 2020).
• Procedures that employ restraint must conform to federal regulations and guidelines.
• Procedures involving the use of paralytic agents without reduction in pain sensation require prudence and humane concern. Use of muscle relaxants or paralytics alone during surgery, without anesthesia, is unacceptable.
• All surgical procedures and anesthetization should be conducted under the direct supervision of a person who is trained and competent in the use of the procedures.
• Unless there is specific justification for acting otherwise, research animals should remain under anesthesia until all surgical procedures are ended.
• Postoperative monitoring and care, which may include the use of analgesics and antibiotics, should be provided to minimize discomfort, prevent infection, and promote recovery from the procedure.
• In general, laboratory animals should not be subjected to successive survival surgical procedures, except as required by the nature of the research, the nature of the specific surgery, or for the well-being of the animal. Multiple surgeries on the same animal must be justified and receive approval from the IACUC.
• To minimize the number of nonhuman animals used, investigators should maximize the amount of data collected from each subject in a manner that is compatible with the goals of the research, sound scientific practice, and the welfare of the animal.
• To ensure their humane treatment and well-being, nonhuman animals reared in the laboratory must not be released into the wild because, in most cases, they cannot survive, or they may survive by disrupting the natural ecology.
• Euthanasia must be accomplished in a humane manner, appropriate for the species and age, and in such a way as to ensure immediate death, and in accordance with procedures outlined in the latest version of the AVMA (American Veterinary Medical Association) Guidelines on Euthanasia of Animals (2020).
• Disposal of euthanized laboratory animals must be conducted in accordance with all relevant laws, consistent with health, environmental, and aesthetic concerns, and as approved by the IACUC. No animal shall be discarded until its death is verified.

Field research that carries a risk of materially altering the behavior of nonhuman animals and/or producing damage to sensitive ecosystems is subject to IACUC approval. Field research, if strictly observational, may not require animal care committee approval (USDA, 2000).

• Psychologists conducting field research should disturb their populations as little as possible, while acting consistent with the goals of the research. Every effort should be made to minimize potential harmful effects of the study on the population and on other plant and animal species in the area.
• Research conducted in populated areas must be done with respect for the property and privacy of the area’s inhabitants.
• Such research on endangered species should not be conducted unless IACUC approval has been obtained and all requisite permits are obtained (see section IV.D of this document). Included in this review should be a risk assessment and guidelines for prevention of zoonotic disease transmission (i.e., disease transmission between species, including human to nonhuman and vice versa).

Research on captive wildlife or domesticated animals outside the laboratory setting that materially alters the environment or behavior of the nonhuman animals should be subject to IACUC approval (Ng et al., 2019). This includes settings where the principal subjects of the research are humans, but nonhuman animals are used as part of the study, such as research on the efficacy of animal-assisted interventions (AAI) and research conducted in zoos, animal shelters, and so on. If it is not possible to establish an IACUC at the psychologists’ own institution, investigators should seek advice and obtain review from an IACUC of a cooperative institution.

• Researchers should minimize and mitigate any distress on the nonhuman animal subject caused by its involvement in the study. Qualifications for appropriate handling of animal subjects in AAI settings have been well described by the AVMA (2008). Psychologists studying the use of AAIs should have the expertise to recognize behavioral and/or physiological signs of stress and distress in the species involved in the study. However, when psychologists lack such expertise, they should ensure that the research team includes individuals with the necessary expertise to recognize and intervene to reduce the nonhuman animal subject’s distress. Any study that carries risk of experiencing, or being exposed to the experience of, another organism’s pain, fear, or distress requires greater justification and should be addressed in the IACUC protocol.
• When research is conducted in applied settings, such as hospitals, health clinics, and offices of doctors and mental health professionals, the investigator should understand the risk of, and declare mitigating strategies for, disease transmission between human and nonhuman participants. For example, studies of AAIs in health-care facilities offering mental health services may introduce risks for bi-directional zoonotic transmission of infectious diseases such as Methicillin-resistant Staphylococcus aureus (MRSA) (Lefebvre, et al., 2008). Investigators studying AAIs in health-care settings should therefore adhere to the guidelines for AAI management offered by the AVMA (2008).
• In all experimental circumstances, investigators should structure into the schedule the basic needs of the nonhuman animals such as food, water, and rest breaks.

Laboratory exercises as well as classroom demonstrations involving live animals are of great value as instructional aids. Psychologists are encouraged to include instruction and discussion of the ethics and values of nonhuman animal research in relevant courses.

• Nonhuman animals may be used for educational purposes only after review by an IACUC or other appropriate institutional committee.
• Consideration should be given to the possibility of using nonanimal alternatives. Procedures that may be justified for research purposes may not be so for educational purposes (e.g., animal models of pain that are used to develop safer analgesics would be in excess of what is needed to merely demonstrate the use of animal models in the study of behavior and cognition).
• All handlers of nonhuman animals in educational settings should adhere to the recommendations outlined above for personnel, housing, and acquisition of subjects. APA has adopted separate guidelines for the use of nonhuman animals in research and teaching at the pre-college level. A copy of the APA Guidelines for the Use of Nonhuman Animals in Behavioral Projects in Schools (K-12) can be obtained via email at [email protected] , by phone at 202-336-6000, or in writing to the American Psychological Association, Science Directorate, Office of Research Ethics, 750 First St., NE, Washington, DC 20002-4242 or downloaded at apa.org/science/leadership/care/animal-guide.pdf .

American Psychological Association. (2017). Ethical principles of psychologists and code of conduct (2002, amended effective June 1, 2010, and January 1, 2017). http://www.apa.org/ethics/code/

American Veterinary Medical Association. (2008). Guidelines for animal-assisted interventions in healthcare facilities. American Journal of Infection Control, 36(2), 78-85. https://doi.org/10.1016/j.ajic.2007.09.005

American Veterinary Medical Association. (2020). AVMA guidelines for the euthanasia of animals. https://www.avma.org/sites/default/files/2020-01/2020-Euthanasia-Final-1-17-20.pdf

Animal Welfare Act 7 U.S.C. § 2131 et seq. http://awic.nal.usda.gov/nal_display/index.php?info_center=3&tax_level=3&tax_subject=182&topic_id=1118&level3_id=6735

Institute for Laboratory Animal Research. (2011). Guide for the care and use of laboratory animals (8th ed.). Washington, DC: The National Academies Press

Lefebvre, S. L., Peregrine, A. S., Golab, G. C., Gumley, N. R., WaltnerToews, D., & Weese, J. S. (2008). A veterinary perspective on the recently published guidelines for animal-assisted interventions in health-care facilities. Journal of the American Veterinary Medical Association , 233(3), 394-402. https://doi.org/10.2460/javma.233.3.394

National Institutes of Health Office of Laboratory Animal Welfare. (2015). Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Bethesda, MD: NIH. https://olaw.nih.gov/policies-laws/phs-policy.htm

National Research Council. (2006). Guidelines for the humane transportation of research animals . Washington, DC: The National Academies Press.

Ng, Z., Morse, L., Albright, J., Viera, A., & Souza, M. (2019). Describing the use of animals in animal-assisted intervention research. Journal of Applied Animal Welfare Science , 22(4), 364-376.

Russell W.M.S., & Burch, R. L. (1959). The principles of humane experimental technique. Wheathampstead (UK): Universities Federation for Animal Welfare.

U.S. Department of Agriculture. (1989). Animal welfare; Final Rules. Federal Register , 54(168), (Aug 31, 1989), 36112-36163.

U.S. Department of Agriculture. (1990). Guinea pigs, hamsters, and rabbits; Final Rules. Federal Register , 55(136), (July 16, 1990), 28879- 28884.

U.S. Department of Agriculture. (1991). Animal welfare; Standards; Part 3, Final Rules. Federal Register , 55(32), (Feb 15, 1991), 6426-6505.

U.S. Department of Agriculture. (2000). Field study; Definition; Final Rules. Federal Register , 65(27), (Feb 9, 2000), 6312-6314.

U.S. Public Health Service. (2015). Public Health Service Policy on Humane Care and Use of Laboratory Animals. https://olaw.nih.gov/sites/default/ files/PHSPolicyLabAnimals.pdf

Additional Resources

Dess, N. K., & Foltin, R. W. (2004). The ethics cascade. In C. K. Akins, S. Panicker, & C. L. Cunningham (Eds.). Laboratory animals in research and teaching: Ethics, care, and methods (pp. 31-39). APA.

National Institutes of Mental Health. (2002). Methods and welfare considerations in behavioral research with animals: Report of a National Institutes of Health Workshop. Morrison, A. R., Evans, H. L., Ator, N. A., & Nakamura, R. K. (Eds.). NIH Publications No. 02-5083. Washington, DC: US Government Printing Office.

National Research Council. (2011). Guide for the care and use of laboratory animals. (8th ed.). Washington, DC: The National Academies Press.

National Research Council. (2003). Guidelines for the care and use of mammals in neuroscience and behavioral research. Washington, DC: The National Academies Press.

National Research Council. (2008). Recognition and alleviation of distress in laboratory animals. Washington, DC: The National Academies Press.

National Research Council. (2009). Recognition and alleviation of pain in laboratory animals. Washington, DC: The National Academies Press.

Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research was developed by the American Psychological Association Committee on Animal Research and Ethics in 2020 and 2021. Members on the committee were Rita Colwill, PhD, Juan Dominguez, PhD, Kevin Freeman, PhD, Pamela Hunt, PhD, Agnès Lacreuse, PhD, Peter Pierre, PhD, Tania Roth, PhD, Malini Suchak, PhD, and Sangeeta Panicker, PhD (Staff Liaison). Inquiries about these guidelines should be made to the American Psychological Association, Science Directorate, Office of Research Ethics, 750 First Street, NE, Washington, DC 20002, or via e-mail at [email protected].

Copyright © 2022 by the American Psychological Association. Approved by the APA Council of Representatives, February 2022.

Related Resources

• Some Friendly Advice for Responding to Requests for Information About Animal Research
• Resolution on the Use of Animals in Research, Testing and Education (PDF, 95KB)

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Harvard study on monkeys reignites ethical debate over animal testing

Updated on: November 21, 2022 / 12:54 PM EST / CBS/AFP

Mother monkeys permanently separated from their newborns sometimes find comfort in plush toys; this recent finding from Harvard experiments has set off intense controversy among scientists and reignited the ethical debate over animal testing.

The paper, "Triggers for mother love," was authored by neuroscientist Margaret Livingstone and appeared in the Proceedings of the National Academy of Sciences (PNAS) in September to little fanfare or media coverage.

But once news of the study began spreading on social media, it provoked a firestorm of criticism and eventually a letter to PNAS signed by over 250 scientists calling for a retraction.

Animal rights groups meanwhile recalled Livingstone's past work, which included temporarily suturing shut the eyelids of infant monkeys in order to study the impact on their cognition.

"We cannot ask monkeys for consent, but we can stop using, publishing, and in this case actively promoting cruel methods that knowingly cause extreme distress," wrote Catherine Hobaiter, a primatologist at the University of St. Andrews, who co-authored the retraction letter.

Hobaiter told AFP she was awaiting a response from the journal before further comment, but expected news soon.

Harvard and Livingstone, for their part, have strongly defended the research.

Livingstone's observations "can help scientists understand maternal bonding in humans and can inform comforting interventions to help women cope with loss in the immediate aftermath of suffering a miscarriage or experiencing a still birth," said Harvard Medical School in a statement .

The school added it was "deeply concerned about the personal attacks directed at scientists who conduct critically important research for the benefit of humanity."

Livingstone, in a separate statement , said: "I have joined the ranks of scientists targeted and demonized by opponents of animal research, who seek to abolish lifesaving research in all animals."

Such work routinely attracts the ire of groups such as People for the Ethical Treatment of Animals (PETA), which opposes all forms of animal testing.

In its statement, Harvard Medical School said PETA had published content regarding the study on its website that was "misleading and contains factual inaccuracies."

This controversy has notably provoked strong responses in the scientific community, particularly from animal behavior researchers and primatologists, said Alan McElligot of the City University of Hong Kong's Centre for Animal Health and a co-signer of the PNAS letter.

He told AFP that Livingstone appears to have replicated research performed by Harry Harlow, a notorious American psychologist, from the mid-20th century.

Harlow's experiments on maternal deprivation in rhesus macaques were considered groundbreaking, but may have also helped catalyze the early animal liberation movement.

"It just ignored all of the literature that we already have on attachment theory," added Holly Root-Gutteridge, an animal behavior scientist at the University of Lincoln in Britain.

McElligot and Root-Gutteridge argue the case was emblematic of a wider problem in animal research, in which questionable studies and papers continue to pass institutional reviews and are published in high impact journals.

McElligot pointed to a much-critiqued 2020 paper extolling the efficiency of foot snares to capture jaguars and cougars for scientific study in Brazil.

More recently, experiments on marmosets that included invasive surgeries have attracted controversy.

The University of Massachusetts Amherst team behind the work says studying the tiny monkeys, which have 10-year lifespans and experience cognitive decline in their old age, are essential to better understand Alzheimer's in people.

Opponents argue results rarely translate across species.

When it comes to testing drugs, there is evidence the tide is turning against animal trials.

In September, the Senate passed the bipartisan FDA Modernization Act, which would end a requirement that experimental medicines first be tested on animals before any human trials.

The vast majority of drugs that pass animal tests fail in human trials, while new technologies such as tissue cultures, mini organs and AI models are also reducing the need for live animals.

Opponents also say the vast sums of money that flow from government grants to universities and other institutes — $15 billion annually, according to watchdog group White Coat Waste — perpetuate a system in which animals are viewed as lab resources.

"The animal experimenters are the rainmaker within the institutions, because they're bringing in more money," said primatologist Lisa Engel-Jones, who worked as a lab researcher for three decades but now opposes the practice and is a science adviser for PETA.

"There's financial incentive to keep doing what you've been doing and just look for any way you can to get more papers published, because that means more funding and more job security," added Emily Trunnel, a neuroscientist who experimented on rodents and also now works for PETA.

Most scientists do not share PETA's absolutist stance, but instead say they adhere to the "three Rs" framework — refine, replace and reduce animal use.

On Livingstone's experiment, Root-Gutteridge said the underlying questions might have been studied on wild macaques who naturally lost their young, and urged neuroscientists to team up with animal behaviorists to find ways to minimize harm.

"Do I wish we lived in a world where generating this important knowledge were possible without the use of lab animals? Of course!" Livingstone said in her statement . "Alas, we are not there yet."

• Harvard Medical School

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Why Do Scientists Experiment on Animals?

Animal studies in science are experiments that control an animal's behaviour or physiology for study, often to serve as a model for human biology where testing on humans is impractical or unethical.

The species or classification of animals used in testing largely depends on the goal of the experiment.

For example, zebrafish are quick to breed, easy to house, and transparent as embryos - but they also carry 70 percent of the genes found in humans. All this makes them suitable for studies on human disease and embryological development.

Rodents have a long history of being used for science experiments, and today make up around three quarters of all animal subjects in testing. Easy to raise and breed, their mammalian physiology and genomes overlap even more considerably with those of humans, making them suitable models for studying behaviours, toxicology, and the effects of medical treatments.

Non-human primates , especially chimpanzees and rhesus monkeys, have also been used extensively in scientific testing. While harder to reproduce in large numbers and challenging to house comfortably, experiments on our closest evolutionary relatives can yield valuable information on a wide range of issues, from drug toxicity to neurology.

However, the close likeness of non-human primates to ourselves also means their use in experiments is the most controversial of all types of animal testing . Generally, data across different countries, including the European Union , show that non-human primate research constitutes less than 1 percent of all animal studies.

However, studies on monkeys aren't yet phased out: In 2017, the US had a record-high number of studies involving monkeys.

How useful are animal models in experiments?

If conducted under strict methods with appropriate protocols, animal experimentation can provide reliable evidence on how that animal's physiology or behaviour responds under the experiment's conditions; genetic studies are particularly effective, while behavioural studies can yield less firm conclusions.

Unfortunately, the nature of experiments that make use of animal models can often lend themselves to being poorly designed, conducted, or analysed. There can also be a sex imbalance, with much of rodent research done only on male mice , for example.

Experiments that apply the findings to human biology require significant assumptions on whether any differences between them are significant. Even where animals are genetically altered to better reflect human biochemistry, there is always the risk that an unidentified behaviour or function might mean the experimental results can't be applied to humans.

This doesn't make animal models useless. As with all experiments, the weight of replicated experiments performed critically under peer review determines how confident we should be in a set of results.

It does mean we ought to be cautious about how results from an experiment based on an animal model might apply to our own bodies.

What are the ethics of testing on animals?

Concerns surrounding experiments using animal models are often based on the morality of depriving animals of their liberty or subjecting them to pain or discomfort, to meet a human need or value.

At an extreme end of the ethics spectrum is the claim that all animals have rights equal to humans, and therefore any experiment that wouldn't ethically be conducted on humans shouldn't be conducted on any animal.

Ethics boards today tend to weigh up the potential benefits of an experiment with the risks of harm and suffering to the animal. However, what constitutes a benefit , as well as objective ways to define acceptable limits of harm, pain, and discomfort in different animals can make this more challenging than first appears.

What is the future of animal testing?

More than half a century ago, zoologists William Russell and Rex Burch suggested experimentation should become more humane by following the three Rs; restrict when to use animals; refine the kinds of experiments conducted on them; and replace as the technology becomes available.

Advances in computer modelling and in-vitro tissue culture design are continuing to provide alternatives to animal models that don't suffer from the same ethical and practical limitations.

Human tissue models, such as those making up 3D tissue conglomerates called organoids , are increasingly serving as appropriate models for studying growth and development.

These solutions might not make the way we conduct the experiments themselves more trustworthy. But with robust debate and reliable review procedures, they will steadily make animal testing - and the ethical and practical problems they bring - a thing of the past.

All Explainers are determined by fact checkers to be correct and relevant at the time of publishing. Text and images may be altered, removed, or added to as an editorial decision to keep information current.

Animal experimentation

Nonhuman animals are used in laboratories for a number of purposes. Examples of animal experimentation include product testing, use of animals as research models and as educational tools. Within each of these categories, there are also many different purposes for which they are used. For instance, some are used as tools for military or biomedical research; some to test cosmetics and household cleaning products, and some are used in class dissection to teach teenagers the anatomy of frogs or to have a subject for a Ph.D. dissertation.

The number of animals used in animal experimentation is certainly smaller than that of those used in others such as animal farming or the fishing industry. 1  Yet it has been estimated to be well above 100 million animals who are used every year. 2

The ways in which these animals can be harmed in experimental procedures, also known as vivisection, 3 vary. In almost all cases they are very significant and the majority of them end with the death of the animals.

There’s an important difference today between the consideration that is afforded to the potential and actual subjects used in experiments, depending on whether they are human or nonhuman animals. Few people today would condone experimenting on human beings in harmful ways, and in fact, indicative of this, such research is strongly restricted by law, when it isn’t just prohibited outright. When experimentation on humans is permitted it is always in a context of the individuals involved consenting to it, for whatever personal benefit that serves as an incentive for them. For nonhuman animals, this is not the case.

This is not because of any belief that experimentation on humans could not bring about important knowledge (in fact, it seems obvious that this practice would uncover far more useful and relevant knowledge than any experimentation on nonhuman animals ever can). Rather, the reason for this double standard is that nonhuman animals are not morally taken into account because the strong arguments against speciesism are not considered.

In the following sections the most important areas in which nonhuman animals are used in laboratories or classrooms, as well as the research methods that don’t use them, are addressed.

Animals used for experimentation

Environmental research.

Animals are made to suffer and are killed to test the impact that chemicals can have in the environment. Some of the most important environmentalist organizations have been lobbying for this practice and have often been successful despite the opposition of animal defenders.

Cosmetic and household products testing

While animal testing of new cosmetics and household products is now illegal in places such as the European Union and India, it’s still being carried out in the U.S. and other places, where many animals are blinded, caused extreme pain and killed.

Military experimentation

The use of animals to test new weaponry, bullets and warfare chemicals, as well as the effects of burns and poison for military purposes, remains mainly hidden today, but many animals die in terrible ways because of it.

Biomedical experimentation

Animals of a variety of species are harmed for numerous purposes in biomedical research because the non-animal methodologies aren’t implemented. Those animals are harmed in many ways that most people ignore.

Experimentation with new materials

When new materials are developed, they are often tested by using methods such as cell or tissue cultures, or computational models. However, materials are also commonly tested on animals who are killed afterwards.

Animals used in education

Animals used in primary and secondary education.

Dissecting animals and using them in other ways has been common practice in the U.S. and some other countries in primary and especially secondary education for many years. This means killing a huge number of animals and educating new generations in the idea that it’s acceptable to harm animals for our benefit.

Animals used in higher education

In the science departments of many different universities, research, teaching and training are successfully carried out without using animals as laboratory tools. However, animals are still subjected to all kind of procedures in many other places.

Towards a future without animals harmed in laboratories

Research methods that do not involve the use of nonhuman animals.

Defenders of animal experimentation often claim that there is no choice but to harm animals lest scientific progress be stopped, but this is not so. There are many non-harmful methods available today.

Companies that test on animals

Despite the fact that many other companies do not experiment on sentient animals, there are still companies that choose to continue carrying out animal tests out of a lack of will to implement new methods.

Companies that do not test on animals

Fortunately, although many companies today choose not to harm animals in product development, quality and safety isn’t affected in the least.

1 Every year tens of billions are killed in slaughterhouses and trillions are fished and killed in fish factories. For estimations regarding this see: Food and Agriculture Organization of the United Nations (2021) “ Livestock primary ”, FAO STAT , February 19 [accessed on 24 March 2013]. See also Mood, A. &  Brooke, P. (2010) “ Estimating the number of fish caught in global fishing each year ”, Fishcount.org.uk , July [accessed on 18 October 2020]; (2012) “ Estimating the number of farmed fish killed in global aquaculture each year ”, Fishcount.org.uk , July [accessed on 18 January 2021].

2  See Taylor, K.; Gordon, N.; Langley, G. & Higgins, W. (2008) “Estimates for worldwide laboratory animal use in 2005”,  Alternatives to Laboratory Animals , 36, pp. 327-342.

3 Although the term “vivisection” literally means “cutting a living animal,” this word has broadened its meaning in common language to denote any kind of laboratory invasive use of an animal. Defenders of animal experimentation prefer not to use it due to its negative connotations. Opponents of it claim that there shouldn’t be a problem with using this term given the meaning it already has in common language. They argue that its rejection is due to an intention to use language that is not explicit about how animals are used in this field.

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Right now, millions of mice, rats, rabbits, primates, cats, dogs, and other animals are locked inside cages in laboratories across the country. They languish in pain, ache with loneliness, and are deprived of everything that’s natural and important to them. All they can do is sit and wait in fear of the next terrifying and painful procedure that will be performed on them. A lack of environmental enrichment and the stress of their living situations cause some animals to develop neurotic behaviors, such as incessantly spinning in circles, rocking back and forth, pulling out their own fur, and even biting themselves. After enduring lives of pain, loneliness, and terror, almost all of them will be killed.

How PETA Helps Animals in Laboratories

Since PETA’s inception and the landmark  Silver Spring monkeys  case, we’ve been at the forefront of exposing and ending experiments on animals. Our scientists, campaigners, researchers, and other dedicated staff work hard to persuade universities, hospitals,  contract laboratories ,  other companies , and government agencies to abandon animal tests and embrace modern, non-animal methods.

Two teams lead PETA’s efforts to end tests on animals. Our Laboratory Investigations Department focuses on ending the use of animals in experiments not required by law, and our Regulatory Toxicology Department focuses on replacing the use of animals in tests required by law with human-relevant, animal-free toxicity testing approaches. With help from supporters like you, these teams and other hardworking staff at PETA win numerous  victories  for animals imprisoned in laboratories every year. Here’s how they do it:

• Promoting PETA’s Research Modernization Deal , the first comprehensive, science-backed plan to phase out tests on animals
• Conducting groundbreaking  eyewitness investigations and colorful advocacy campaigns to shut down laboratories and areas of animal experimentation
• Filing groundbreaking lawsuits to challenge public funding of wasteful, cruel animal experiments
• Working with members of Congress to enact laws to replace animals in laboratories
• Persuading government agencies to stop conducting and  requiring experiments on animals
• Encouraging  pharmaceutical, chemical , and  consumer product companies to replace tests on animals with more effective, non-animal methods
• Ending the use of animals in experiments at colleges and universities
• Helping  students and  teachers  end animal dissection in the classroom
• Developing and funding humane non-animal research methods
• Publishing scientific papers on reliable non-animal test methods and presenting them at scientific conferences
• Hosting free workshops and online seminars to share information about animal-free toxicity testing methods
• Urging  health charities not to invest in dead-end tests on animals

How Animals Are Exploited in Laboratories

More than 110 million animals suffer and die in the U.S. every year in cruel chemical, drug, food, and cosmetics tests. They also experience this fate in  medical training exercises , curiosity-driven  experiments at universities ,  classroom biology experiments , and  dissection even though modern, non-animal methods have repeatedly been shown to have more educational value, save teachers time, and save schools money. Exact numbers aren’t available, because mice, rats, birds, and cold-blooded animals—who make up more than 99% of animals used in experiments—aren’t covered by even the minimal protections of the federal Animal Welfare Act and therefore go uncounted.

Examples of chemical and toxicity tests on animals include forcing mice and rats to inhale toxic fumes, force-feeding dogs chemicals, and applying corrosive chemicals into rabbits’ sensitive eyes. Even if a product harms animals, it can still be marketed to consumers. Conversely, just because a product was shown to be safe in animals doesn’t guarantee that it will be safe to use in humans.

Much product testing conducted on animals today isn’t required by law. In fact, a number of countries have implemented bans on the testing of certain types of consumer goods on animals, such as the cosmetics testing bans in India, Israel, New Zealand, Norway, and elsewhere.

Meanwhile, at universities and other institutions, experimenters inflict suffering on and kill animals for little more than curiosity’s sake—even though the vast majority of their findings fail to advance human health . They tear baby monkeys away from their mothers , sew kittens’ eyes shut , mutilate owls’ brains , puncture the intestines of mice so that feces leak into their stomachs , and terrorize songbirds with the sounds of predators . At the end of experiments like these—which consume billions in taxpayer funds and charitable donations each year—almost all the animals are killed.

Animal Experiments Throughout History: A Century of Suffering

PETA created an interactive timeline, “ Without Consent ,” featuring almost 200 stories of animal experiments from the past century to open people’s eyes to the long history of suffering inflicted on nonconsenting animals in laboratories and to challenge them to rethink this exploitation. Visit “ Without Consent ” to learn more about harrowing animal experiments throughout history and how you can help create a better future for living, feeling beings.

Advancing Science Without Suffering: Animal-Free Testing

Testing on animals has been a spectacular failure that has resulted in the loss of trillions of dollars and has cost the lives of innumerable humans and other animals. Experiments on one species frequently fail to predict results in another. Even the National Institutes of Health, the world’s largest funder of biomedical research, acknowledges that 95% of all drugs that are shown to be safe and effective in animal tests fail in human trials.

Technologically advanced  non-animal research methods —such as those using human cells, computational models, or clinical studies—can be used in place of animal testing. These methods are more humane, have the potential to be faster, and are more relevant to humans.

Scientists in PETA’s Science Advancement & Outreach division , a part of the Laboratory Investigations Department, have developed a roadmap to phase out failing tests on animals with sophisticated, animal-free methods. Their Research Modernization Deal has gained the support of scientists, medical doctors, members of Congress, and thousands of others who care about ethical and effective science.

How You Can Help Animals Used in Experiments

Each of us can help prevent the suffering and deaths of animals in laboratories. Here are a few easy ways to get started:

• Sign up for PETA’s Action Team to be alerted when protests are taking place in your area.
• Urge your members of Congress to support PETA’s Research Modernization Deal .
• Search PETA’s Beauty Without Bunnies database to ensure that you’re buying only cruelty-free products.
• Donate only to charities that don’t experiment on animals .
• Request alternatives to animal dissection at your school.
• Call on your alma mater to stop experimenting on animals.
• Share information about animal experimentation issues with your friends and family—and invite them to join you in speaking up for animals.

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• Avicenna J Med Biotechnol
• v.4(3); Jul-Sep 2012

Regulations and Ethical Considerations in Animal Experiments: International Laws and Islamic Perspectives

Mohammad mehdi naderi.

1 Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

† These authors equally contribute to this work

Ali Sarvari

Alireza milanifar.

2 Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

Sara Borjian Boroujeni

Mohammad mehdi akhondi.

3 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

Growing usage of animals in the research projects has drawn more attention to their welfare and ethics surrounding this practice. Dissemination of information about the existing ethical consideration and alternatives in animal experiments has two important functions; first, it increases the researcher's awareness of the possible methods of using animals in the experiment, and second, to ensure that potential users are aware of the established alternatives. For example, legislations enacted in many countries during the 1980s state that laboratory animal applications should be reduced, refined and replaced wherever possible according to principles of the 3Rs. Thus, scientists around the world tried to apply the 3Rs in their biomedical researches regarding welfare of the laboratory animals. However, the Qur'an, the holy book of Muslims, and also Hadiths contain the obligatory ways to keep and treat animals since their revelations. According to Islamic viewpoint, animals represent Allah's ability and wisdom, and humans must pay attention to their health and living conditions. Several Islamic manuscripts state that animals have their own position in the creation hierarchy and humans are responsible for supplying minimal facilities and their welfare. This paper has tried to review ethical consideration in animal experiments and regarding Islamic resources in this case to encourage providing comprehensive ethical regulations in animal experiments which its establishment could be beneficial for animal ethics committees or research institutes.

Introduction

Increasingly use of animals in the scientific procedures has drawn more attention to the primary ethics of these valuable creatures. There are international guidelines for use and care of animals in scientific procedures, which references have been made to some of them in this paper. One of these guidelines is represented by the National Advisory Committee for Laboratory Animal Research (NAC LAR, Singapore), which seems to launch concise yet comprehensive considerations about the use and care of animals for scientific and research purposes. The NACLAR guidelines set out the responsibilities of all the sections involved in using and care of animals for research goals, according to accepted scientific, ethical and legal guidelines. It has been agreed that proposal to use animals for research goals must be assessed by an Institutional Animal Care and Use Committee (IAC UC) in approval of the Guidelines ( 1 ).

These international guidelines are classified into three parts that should be considered together as a comprehensive document:

The first part, “Guiding principles for the care and use of animals for scientific purposes”, explains all principles which promote the humane and responsible care and use of animals for research and scientific goals. The concept of the principles describes the 3Rs-Replacement, Reduction and Refinement. The limitation of the principles consist all aspects of the care and use of animals for research and scientific goals including their use in teaching, field trials, environmental studies, research, diagnosis, product testing, and the production of biological products ( 2 , 3 ). This part describes the responsibilities of institutions, scientists and persons who are involved in the care and use of animals for research and scientific goals. All scientific facilities which house and use animals for research goals will have to utilize according to the Guidelines to qualify for licensing from the Agri-Food and Veterinary Authority (AVA).

The second part, “Guidelines for institutional animal care and use committee”, includes the guiding principles for using and care of animals for research goals and explains in detail the operational aspects pertaining to the Institutional Animal Care and Use Committee (IACUC). The IACUC is responsible for the assessment and evaluation of animal care and use programs of an institution, and is responsible for confirming that the care and use of animals for research goals and all animal experimental methods are in compliance with the guidelines. Under the guidelines, all institutions with scientific facilities have to establish their own IACUC to assume this function.

The third part, “Training guidelines”, describes the training activities and requirements for users of animals and animal facilities personnel. This includes the scope of the core curriculum and the relevant core competencies, such as specific workshops for animal procedures. The Guidelines consider all animal users have to undergo appropriate training before initiation of any procedure using animals ( 2 , 4 ).

However, Islam is predominant culture (more than 95%) and religion in our country and this holy religion is not silent in any case of ethical and educational concepts and it has also profound teachings on how to deal with animals. The main animal welfare regulations in Islam include considering to their natural needs, such as water, food and a suitable place to live, their living and mental condition, good health and avoidance of causing them pain, distress, or harm and unnecessary termination of their lives. These should be considered carefully by the people who work with these creatures ( 5 ).

For this study, some international and Islamic resources which were relavant to dealing with animals were collected via searching online papers and eBooks, primarily or as borrowing the books from the library, secondarily (are mentioned in reference section). Thence, the resources based on subjects were classified and summarized, and finally, all the extracted points were reviewed by experts familiar with Islamic and ethical issues. Some international guidelines and Islamic considerations regarding welfare of the animals used in research or teaching, have been collected that the most important of them are outlined below.

International Guidelines of using Animals in Scientific Procedures

Animal experiments should be designed only after due consideration of animal health and the advancement of knowledge on humans or animals weighed against the potential impacts on the welfare of the animals.

Researchers should treat animals as sentient and must consider their proper care and use and the avoidance or minimization of discomfort, distress, or pain as imperatives. In this field, the 3 ‘R’ principles must be considered at all animal experiments:

• Replacement of animal experimentation with alternative methods such as mathematical models, computer simulation and in vitro biological systems, which replace or complement the use of animals must be considered before embarking on any procedure involving use of animals.
• Reduction in the number of animals used which means minimum number of animals required to obtain scientifically valid results. Furthermore, scientific projects involving the use of animals must not be repeated or duplicated unnecessarily.
• Refinement of projects and techniques used to minimize impact on animals which means: (a) Animals chosen must be of an appropriate species and quality for the scientific projects concerned taking into account their specific biological properties, including genetic constitution, behavior, and microbiological, nutritional and general health status ( 2 , 6 , 7 ).

Animal housing and management

Maintenance facilities of animals must be accurately designed, constructed, equipped and maintained to access a well standard of animal care and should follow acceptable standards of animal welfare for the particular species concerned and should fulfill scientific requirements.

In identifying the standards of animal care, the criterion should be animal well-being rather than mere ability to survive under the adverse conditions such as environmental extremes or high population densities ( 8 ).

Veterinary care

Institutions should establish and operate adequate veterinary care, prepared by the attending veterinarian which includes:

The presence of appropriate facilities, equipment, personnel, and services to execute the guiding principles; using appropriate procedures to control diseases ( e.g . vaccination and other prophylaxis, isolation and quarantine), diagnose, and treat diseases and injuries; daily observation of all animals to evaluate their health and well-being and finally certain manipulations or other tasks related to the care and use of animals must be performed only by the attending veterinarian ( 7 , 9 ).

Responsibilities of the Institutional Animal Care and Use Committees (IACUC)

The IACUC in each institute must assess the research procedures before granting approval for proposed projects or significant changes to on-going projects which the most important of its duties include:

The project proposals must describe a procedure designed to assure that discomfort, distress or pain to animals will be minimized or avoided. The researchers must provide written assurance that the activities do not unnecessarily duplicate previous experiments ( 10 ).

Projects which may cause more than momentary distress or pain to the animals will: (i) be done with appropriate analgesics, sedatives, or anesthetics; (ii) involve in their procedure designing, consultation with the attending veterinarian; (iii) not include the use of paralytics without anesthesia.

Personnel conducting procedures on the species being maintained or studied will be appropriately qualified and trained in those procedures. Procedures that involve surgery include appropriate provision for pre operative and post-operative care of the animals according to established veterinary medical practices. No animal will be used in more than one experiment unless justified for scientific reasons by the researcher ( 11 , 12 ).

Responsibilities of researchers

Researchers who use animals for scientific goals have a moral obligation to deal with the animals humanely and consider their welfare when designing the projects. Before any animal experiment begins, investigators should submit a proposal to the IACUC to demonstrate that the procedure will comply with the guiding principles. Moreover, the researchers must satisfy the IACUC of their competence to execute the techniques described in the experiment. The most important responsibilities of researchers in an animal experiment include:

• Minimize pain and distress: pain and distress cannot always be properly assessed in animals and researchers must assume that animals experience pain in a manner similar to humans. Investigators must be familiar with the normal behavior patterns of the animal species chosen.
• Prevent unacceptable study end-points: death as an end-point is often ethically unacceptable and should be fully justified. When death cannot be avoided, the procedures must be designed to result in the deaths of as few animals as possible ( 13 ).
• Avoid repeated use of animals in experiments: any animals should not be used in more than one experiment, either in the same or different projects, without the express approval of the IACUC.
• Minimize duration of experiments: experimental duration should be limited to that just sufficient to achieve the purpose of the experiment.
• Using appropriate euthanasia method: when it is necessary to kill an animal, human procedure must be used. These procedures must avoid distress, be reliable and produce rapid loss of consciousness without pain until death occurs ( 14 – 16 ).
• Pre-operative planning: pre-operative physical examination can often identify potential problems, such as increased anesthetic risk, which may compromise the surgical procedure. Sick animals should be rejected.
• Choosing surgical procedure: surgical procedures must be carried out under specific local or general anesthesia. There should be adequate monitoring of the depth of anesthesia and effects such as cardiovascular and respiratory depression and hypothermia.
• Post-operative care: attention to pain relief is the fundamental goal of post-operative care. Animal models of disease; animals must be used only if the disease in the animal can serve as a reliable model for research on the human disease.
• Experimental manipulation of animals’ genetic material: all proposals to manipulate the genetic material of animals, their germ cells or embryos must be submitted to the IACUC for approval.
• Experimental induction of tumors: the site for induction of neoplasia should be chosen accurately. Subcutaneous, intradermal and flank sites must be chosen wherever possible. Prior to the use of brain, footpad, and eye sites, specific justification as to the lack of any other alternative must be made to the IACUC ( 11 , 14 , 15 ).

Responsibilities of teachers

When animals are being used to obtain educational purpose, the person in charge of the class should: (i) accept responsibility for ensuring that the care and use of the animals is in compliance with all relevant legislation and NACLAR guidelines; (ii) allow students to anaesthetize animals or do surgery only if it is essential for their training; and (iii) be responsible for the humane killing of the animals, if required, bearing in mind that it is good practice to segregate manipulated animals from animals held under normal living conditions.

Persons supervising students who are training in research should ensure that the students are completely instructed prior to using animals and should be responsible for the ethics and the welfare of animals used by students ( 15 , 17 ).

Islamic Considerations for using Animals in Scientific Procedures

There are three sources of Islamic law which the first one is Qur'an Majeed; Hadiths or Traditions, is the second source; and Ijtihad, the endeavor of a Muslim scholar to derive a rule of divine law from Qur'an and Hadiths without relying on the views of other scholars, is the third one. Together, these three sources make up Islamic case law or “Jurisprudence” that is the guideline to be followed for any legal question. Many issues relating to animals, such as cruelty to animals, experimentation on animals and human/ animal relationship did not exist 14 centuries ago, and therefore, no specific laws were passed about them. To decide on issues developed in recent times, Islamic Jurisprudence (fiqh) has left it to Muslim Jurists (fuqaha'a) to use their judgment by inference and analogy, based on the three above mentioned sources.

Dominion over animals

The Holy Qur'an describes that man has dominion over animals: “He (God) it is Who made you vicegerents on earth” (Holy Qur'an, 35:39), but makes clear that this responsibility is not unconditional and states what happens to those who misuse their freedom of choice and fail to conform to the conditions that limit this responsibility: “Then We reduce him (to the status of) the lowest of the low” (Holy Qur'an, 95:4, 5). “They are those whom Allah has rejected and whom He has condemned... because they served evil” (Holy Qur'an, 5:63). “They have hearts wherewith they fail to comprehend, and eyes wherewith they fail to see, and ears wherewith they fail to hear. Such (humans) are far astray from the right path” (Holy Qur'an, 7:179). There are people who take the concept of man's dominion over animals as a licentious freedom to break all the established moral rules designed to protect animal rights. Again, the Holy Qur'an urges in remonstrance: “And be not like those who say, ‘We have heard,‘ while they do not hearken. Verily, the vilest of all creatures, in the sight of Allah, are those deaf and dumb ones who do not use their rationality” (Holy Qur’-an, 8:21, 22).

Experimentation on animals

Scientific and pharmaceutical experiments on animals are being done to find cures for diseases, most of which are self-induced by our own disorderly lifestyle. Many human problems physical, mental, or spiritual are of our own creation and our wounds self-inflicted. By no stretch of imagination can we blame animals for many of our troubles and make them suffer for it. All this (experiments), and much more, is being done to satisfy human needs, most of which are non-essential, fanciful, and wasteful and for which alternative, humane products are easily available. To kill animals to satisfy the human thirst for inessentials is a contradiction in terms within the Islamic tradition. Let us hope a day will dawn when the great religious teachings may at last begin to bear fruit; when we shall see the start of a new era, when man accords to animals the respect and status they have long deserved and for so long have been denied.

God in the Holy Qur'an, proposes all people to cerebrate regarding animals and their creation: do they not regard the camels, how do they created? (Holy Qur'an, 88:17). Have they not looked at the sky above them, how we have made and dressed it up? (Holy Qur'an, 50:6). Do they not look in the dominion of the skies and the earth and all things that God has created, (animals and plants) and may be the end of their lives to be close (Holy Qur'an, 7:185).

According to these verses, it is clear that Islam has a special point of view on all the creation aspects and none of the creatures is useless ( 18 ). Human beings are advised to deal with animals compassionately and moderately in Islamic teachings. As the prophet Mohammad said: “Only the compassionate will enter the heaven. Brothers! animals have subtle senses, so do not torture or over load them as they will be hurt” ( 19 ).

It is morally important to determine to what extent we are allowed to use animals, especially in researches and whether we can consider ourselves as their real author. “Have you thought that we have created you for fun, and you would not return back to us?” (Holy Qur'an 23:115). The Holy Qur'an's verses and narrations of the prophet Mohammad and the immaculate Imams demonstrate that we are not allowed to terminate animals’ lives; for i.e . there is a narration from the prophet Mohammad that He have said: If anybody kill a creature even a small bird without any reason, God will impeach him (Tarkol Atnaab 290, Nahjol Fasahe 250) because we are not their real owners ( 18 ). In other words, animals and other creatures are not as commodities and tools and God is all creatures’ real owner. Prophet Mohammad: “Do not hurt animals’ face because they praise God” ( 19 ). Based on Islamic teachings animal welfare must be provided and the most important cases in this regard include providing their natural needs, such as water, food and a suitable place to live, appropriate mental condition, good health and to prevent them from pain, distress, or harm and unnecessary termination of their lives ( 18 ).

Previous studies conducted in our country have demonstrated that since Islam is predominant religion in Iran and according to Islamic consideration, all creatures are in the divine presence, then nobody have authority to interfere animal's life without God's consent. Animals are part of our real life, so we can use them only with respect to the position for which are created. It must be in an ethical manner to benefit from the animals because they feel pain and distress certainly ( 18 ). Another study in our country has tried to introduce some codes about animals in four sections; maintenance of animals and transporting them, husbandry and the personnel and researcher's knowledge ( 20 ). Finally according to the Islamic believes and regarding to animal's positions in the nature we have to protect animal well being in the research and educational procedures.

Increasingly, humans transgress their ecological responsibilities. Instead of living within a circle of ecological interest, humans act in self-interest and at the expense of a relationship within nature that is caring and responsible. Millions of animals are used every year in many extremely painful and distressing scientific procedures. Legislation of animal experimentation in modern societies is based on the supposition that this is ethically acceptable when certain more-or less defined formal ( e.g . logistical, technical) demands and ethical principles are met ( 21 ).

In comparison with the other Abrahamic Judaic and Christian traditions, there are more teachings about the treatment, care and respecting animal's ethics in Islam. Islamic teachings exhibit an ingrained environmental ethic of stewardship and a way of life for Muslims that are rooted firmly in seeking harmony with the environment. Islamic considerations have recommended humans to provide water and food for animals and respect to their welfare and safety. The development of biomedical sciences further obligates researchers to respect principles of caring and using animals because of expanded animals’ utilization. It is hoped that utilization of ethical considerations in animal experiments improves the scientific design of the researches and related hygienic standards ( 14 , 15 ).

This paper has tried to review ethical consideration in animal experiments and regarding Islamic resources in this case to establish comprehensive ethical regulations in animal experiments, which its establishment could be beneficial and useful for animal ethics committees or research institutes.

• Open access
• Published: 04 September 2024

Early-life milk replacer feeding mediates lipid metabolism disorders induced by colonic microbiota and bile acid profiles to reduce body weight in goat model

• Ke Zhang 1   na1 ,
• Ting Zhang 1   na1 ,
• Mengmeng Guo 2   na1 ,
• Awang Cuoji 1 , 3   na1 ,
• Yangbin Xu 1   na1 ,
• Yitong Zhao 1   na1 ,
• Yuxin Yang 1   na1 ,
• Daniel Brugger 4   na1 ,
• Xiaolong Wang 1   na1 ,
• Langda Suo 3 , 5   na1 ,
• Yujiang Wu 3 , 5   na1 &
• Yulin Chen 1   na1  

Journal of Animal Science and Biotechnology volume  15 , Article number:  118 ( 2024 ) Cite this article

Metrics details

Dysregulation of lipid metabolism and its consequences on growth performance in young ruminants have attracted attention, especially in the context of alternative feeding strategies. This study aims to elucidate the effects of milk replacer (MR) feeding on growth, lipid metabolism, colonic epithelial gene expression, colonic microbiota composition and systemic metabolism in goat kids compared to breast milk (BM) feeding, addressing a critical knowledge gap in early life nutrition.

Ten female goat kids were divided into 2 groups: those fed breast milk (BM group) and those fed a milk replacer (MR group). Over a period of 28 d, body weight was monitored and blood and tissue samples were collected for biochemical, transcriptomic and metabolomic analyses. Profiling of the colonial microbiota was performed using 16S rRNA gene sequencing. Intestinal microbiota transplantation (IMT) experiments in gnotobiotic mice were performed to validate causality.

MR-fed pups exhibited reduced daily body-weight gain due to impaired lipid metabolism as evidenced by lower serum and liver total cholesterol (TC) and non-esterified fatty acid (NEFA) concentrations. Transcriptomic analysis of the colonic epithelium revealed upregulated genes involved in negative regulation of lipid metabolism, concomitant with microbiota shifts characterized by a decrease in Firmicutes and an increase in Actinobacteria. Specifically, genera such as Bifidobacterium and Prevotella were enriched in the MR group, while Clostridium and Faecalibacterium were depleted. Metabolomics analyses confirmed alterations in bile acid and fatty acid metabolic pathways. IMT experiments in mice recapitulated the metabolic phenotype observed in MR-fed goats, confirming the role of the microbiota in modulating host lipid metabolism.

Conclusions

Milk replacer feeding in goat kids disrupts lipid metabolism and gut microbiota dynamics, resulting in reduced growth rates and metabolic alterations. These findings highlight the importance of early nutritional intervention on metabolic programming and suggest that modulation of the gut microbiota may be a target for improving growth and metabolic health in ruminants. This study contributes to the understanding of nutritional management strategies in livestock and their impact on animal health and productivity.

In mammals, breastfeeding is universally recognized as the normative and superior nutritional foundation for newborns, primarily due to its synergistic combination of nutrients and bioactive compounds that confer significant biological effects [ 1 ]. The positive influence of breastfeeding on the health, growth and development of the offspring from a nutritional, physiological and developmental point of view is well established [ 2 ]. Breast milk supports the development of the immune system by providing age-appropriate nutrients, growth factors, antimicrobial peptides and proteins that meet the needs of the offspring [ 3 ]. However, in the context of intensive livestock production, particularly in goat production systems, the use of milk replacers (MR) is widespread due to limitations such as inadequate lactation, mastitis and postpartum paresis in ewes [ 4 ]. MR is used to compensate for the lack of maternal milk, improve litter survival and shorten the reproductive cycle of ewes [ 5 ]. Although current milk replacers are formulated to mimic the nutritional profile of breast milk using non-milk protein substitutes, they are notably lacking in many of the biologically active compounds found in natural milk. This disparity highlights the urgent need for systematic research aimed at identifying and selecting functional microorganisms and metabolites that promote the growth and maturation of offspring. These targeted microbial strains and metabolites could potentially be incorporated into novel milk replacers enriched with bioactive compounds.

The complex interplay between early life nutrition, gut microbiota composition, bile acid metabolism and lipid homeostasis have profound implications for the lifelong health and weight management of ruminants such as goats and other animals [ 6 ]. During the neonatal period, ruminants undergo a number of critical physiological adaptations that are essential for survival and future productivity. The process of ingestion of maternal milk involves the establishment and development of a diverse gut microbiota that plays an essential role in digestion, absorption [ 7 ] and metabolism of nutrients, particularly lipids [ 8 , 9 , 10 ]. When the natural supply of goat milk is limited or unavailable, feeding milk replacers is a standard practice to ensure adequate nutrition for rapidly growing young ruminants [ 11 ]. However, the extent to which this artificial feeding strategy affects the establishment and functionality of the gastrointestinal microbiota, particularly in the colon where bacterial fermentation is pronounced, remains an under-explored area of research. Bile acids, synthesized in the liver and secreted into the small intestine, play a central role in lipid digestion and absorption, while also serving as key regulatory signals involved in various aspects of host metabolism, including glucose-lipid balance and inflammatory pathways [ 12 , 13 ]. Perturbations in the gut microbiota can induce alterations in bile acid metabolism, often leading to perturbations in bile acid pools that are closely associated with lipid metabolism disorders in different species [ 14 , 15 ]. In this study, we aimed to unravel the complex interrelationships by investigating how early life milk replacer feeding affects the colonic microbiota and bile acid profiles, thereby affecting lipid metabolism and body weight in goat kids. We hypothesize that currently available commercial goat milk replacer formulae may exacerbate lipid metabolic disorders in young goats, thereby affecting their growth performance.

To address these knowledge gaps, we used milk replacer-fed goat models to systematically evaluate the effects of formula feeding on growth, microbiota composition and metabolic performance. In addition, we validated the causality of microbial dysbiosis-mediated disturbances in lipid metabolism using a pseudo germ-free mouse model. The overall aim of this investigation is to provide a comprehensive understanding of the proposed mechanisms by which milk replacers affect gut metabolism and physiology. Our research aims to contribute to the development of innovative milk replacer products and to improve the scientific understanding of milk replacer feeding practices.

Materials and methods

The experiment was approved by the Institutional Animal Care and Use Committee of the Northwest A&F University under permit number 2020-03-015.

Animals, diets, and experimental design

Prior to the start of this study, 50 female Tibetan cashmere goats were tested for estrus by allowing visual and olfactory contact with an intact male goat, and a total of 36 female goats were selected on the basis of number of litters (2 times). These 36 goats (aged 24 months; mean live weight 25.58 kg) were reared and maintained under the same conditions at the Lhasa Tibet Cashmere Goat Breeding Farm (Lhasa, China). The selected goats had no history of diarrhea or other digestive disorders before and during the study. In addition, they were not given any medications, including antibiotics and probiotics, during the study. The diets and nutrient composition fed to the goats during the study are shown in Additional file 1 : Table S1. The goats had free access to water and feed and the diet was not changed throughout the study. Following the pre-evaluation, 30 female goats were selected for treatment to induce concurrent estrus and subsequent mating, based on our standard pre-production procedure [ 16 ], due to the irregular estrus cycles observed in the other 20 female goats. Ultimately, 18 goats were successfully conceived and delivered, and a total of 10 singleton female goat kids of similar weight (mean weight 2.23 kg) were selected for subsequent experimental treatment. Ten female kids were fed breast milk ad libitum for 3 d after parturition, and on the 4 th d after birth, the kids were randomly divided into 2 groups ( n  = 5), with no difference in initial mean body weight between the two groups. To ensure consistency in the test environment, bulk milk samples from goats were used to make up the milk fed to the goat kids. The milk for the animals in the BM group was collected daily from the corresponding mothers of the kids. These mothers were milked daily to obtain the required bulk milk samples. Kids in the BM group were fed 4 times a day (at 8:00, 12:00, 18:00, and 22:00) with 250 mL of milk each time. Goat kids in the MR group were fed milk replacer 4 times a day (at 8:00, 12:00, 18:00 and 22:00) by mixing 40 g of commercial milk replacer (Beijing Precision Animal Nutrition Research Center, Beijing, China [ 8 ]) with 250 mL of warm water and bottle feeding each kid. This formulation meets or exceeds recommended levels for crude protein, crude fat, essential amino acids such as lysine, methionine, and threonine, as well as providing necessary vitamins and minerals including calcium, phosphorus, sodium chloride, and vitamin E. Each group of kids was housed in separate pens (6 m × 5 m; n  = 5) in the same environment. All goat kids had free access to clean water and were offered fresh dried alfalfa ad libitum every day. The kids were already accustomed to consuming forage from 14 d. Observations of their ingestive behavior indicated that they consumed the alfalfa regularly, which was a standard part of their diet. This inclusion of dry alfalfa did not have any noticeable adverse effects on the results, as it ensured a consistent and natural feeding behavior among the kids. Goat’s milk at 7 d contained 9.52% protein, 7.50% fat, 14.95% solids-non-fat, 3.99% lactose, 2.58% low lactose, 1.29% galactose and 6.36% casein. Goat’s milk at 14 d contained 5.91% protein, 6.70% fat, 12.14% solids-non-fat, 4.16% lactose, 2.46% low lactose, 1.01% galactose and 4.31% casein. The formula of the milk replacer is shown in Additional file 1 : Table S2. All kids were weighed at 0, 14, and 28 d after birth. At 29 days of age, after 12 h of fasting, 10 female goat kids from the experiment were sacrificed. Jugular vein blood was collected from the goats 1 h before slaughter, preserved in a vacuum tube containing coagulant, kept at 4 °C for 3 h, and then centrifuged at 2,000 ×  g at 4 °C for 10 min. They were euthanized by injection with thiopental (0.125 mg/kg body weight) and potassium chloride (5 to 10 mL) (Fig.  1 A). The homogenized digesta from the colon was then snap frozen in liquid nitrogen and stored at −80 °C for subsequent DNA analysis. Colonic epithelial tissue was excised from the mucosal layers using a glass slide, immediately washed in ice-cold phosphate-buffered saline (PBS) until the PBS was clear, and then transferred directly to liquid nitrogen until tissue RNA extraction.

Effects of formula feeding on body weight and serum parameters in goats. A Schematic representation of the experimental design. B Graphical representation of changes in body weight and average daily gain of goats subjected to formula feeding. C Serum glucose concentration in goats under different feeding conditions. D Comparison of triglyceride (TG) concentrations in both serum and liver tissues of goats in different feeding groups. E Comparison of total cholesterol (TC) concentrations in both serum and liver tissues of goats in different feeding groups. F Non-esterified fatty acid (NEFA) concentrations in the liver of goats under different feeding conditions. Data differences among goat subjects were statistically assessed using one-way analysis of variance (ANOVA) with Tukey’s test. Statistical significance levels are denoted as follows: ns (not significant) for P  > 0.05, * for P  < 0.05, and ** for P  < 0.01

Five goat kids (mix pool of colon content of each group) from BM and MR groups were used for colonic digesta transplantation. The colonic content (200 mg) from the donors was suspended in sterile 0.9% saline (2 mL) and centrifuged at 1,000 ×  g at 4 °C for 5 min to obtain the bacterial suspension. Thirty-two female C57/6 J mice (8 weeks) were divided into 4 groups: Con, a control group (without any treatment); Ab, an antibiotic group (which received sterile saline via gavage after antibiotic treatment stopped); BM_IMT, a group that was transplanted with group BM colonic microbiota after antibiotic treatment stopped; and MR_IMT, a group that was transplanted with MR colonic microbiota after antibiotic treatment stopped. Ab, MR_IMT, and BM_IMT groups were first treated with fresh composite antibiotics (containing 1 g/L ampicillin, 1 g/L streptomycin, 1 g/L gentamicin, and 0.5 g/L vancomycin; MACKLIN, Shanghai China) using water for 14 d [ 17 ]. Each recipient mouse received 200 μL of the supernatant by oral gavage once a day continuously for 21 d. To improve the colonic microbial survival rates during the cryopreservation, colonic content samples were diluted with sterile saline, homogenized and filtered. The formula of the mice diet was shown in Additional file 1 : Table S3. Subsequently, the resulting suspension was added to glycerol to get a final concentration of 10%. Finally, the colonic suspensions are labeled accurately, and stored in liquid nitrogen as soon as possible to ensure colonic microbial survival. When there is the need for IMT, the frozen colon suspension was thawed at 37 °C (water bath). Upon frozen colon suspension thawing, sterile saline solution can be added to obtain a required concentration (CFU = 1 × 10 9 ) and the infusion of colon suspension should be implemented as soon as possible at 37 °C. All colonic material preparation processes were carried out at an anaerobic incubator (LAI-3 T, Longyue, China). After transplantation, they were housed individually at room temperature (23 ± 1 °C) and light control (16L:8D) with free access to feed and drinking water. Body mass was measured every day. Mice were sacrificed after 7 d of IMT (at 10:00), and the serum, colon contents, and colon tissue were collected after the mice were euthanized via CO 2 inhalation then killed through cervical dislocation. All samples were stored at −80 °C for subsequent DNA analysis. Colonic tissue was stored in 4% paraformaldehyde in a refrigerator at 4 °C.

Serum and hepatic parameters

The levels of serum total cholesterol (TC), triglycerides (TG), glucose (Glu), cortisol (Cor), corticosterone (Cort), diamine oxidase (DAO) and hypoxia-inducible factor (HIF-α) were measured using the HY-50061, HY-50062, HY-50063, HY-10062, HY-10063, HY-M0046 and HY-NE021 kits (Huaying, Beijing, China) on an automatic biochemical analyzer (Hitachi7160, Tokyo, Japan). Serum immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were measured by the sandwich ELISA method using the IgG/A/M (HY-50094) kit (Huaying, Beijing, China). Serum levels of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured using ELISA kit in accordance with the manufacturer’s instructions (Huaying, Beijing, China). Around 0.10 g of semi-thawed liver were homogenized an equivalent amount (1/9) of homogenizing buffer (pH 7.4; 0.01 mol/L). Screw tubes filled with 0.1 mmol/L EDTA-Na 2 , 0.8% NaCl, and Tris–HCl. The tubes were then put in frozen water for 5 min, shaken for 5 min at 25 Hz/s with a TissueLyser, and the procedure was performed once more. Then, using assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the supernatant was collected after centrifuging it for 10 min at 2,500 ×  g at 4 °C to test the levels of hepatic TC, TG, and non-esterified fatty acid (NEFA) (A111-101, A110-1-1, A042-1-1). The findings from the hepatic lipid test were adjusted using the total protein concentration in accordance with the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

16S rRNA gene profiling

A total of 32 colonic content samples were collected from a mouse model after previous colonic digesta transplantation. Total DNA was then extracted from the cecum lumen content using the E.Z.N.A. stool DNA kit (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer’s instructions. The Nanodrop 2000 UV-VI spectrophotometer (Thermo Scientific, Wilmington, USA) was used to determine the DNA concentration and purity. 1% agarose gel electrophoresis was used to assess the quality of the extracted DNA. The primers 338F (5′-ACTCCTACGGGAGGCAG CAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) on a thermocycler PCR system (Gene Amp 9700, ABI, USA) were used to amplify the V3–V4 region of the DNA [ 18 ]. The paired-end sequencing (2 × 300 bp) on an Illumina MiSeq platform (Illumina, San Diego, USA) was conducted according to the standard Major Biobio-Pharm Technology Co., Ltd. (Shanghai, China) protocols on the pooled equimolar ratios of the purified amplicons.

Metagenomic sequencing, assembly and construction of the gene catalog

The DNA was extracted from colon content samples of goat using the E.Z.N.A Stool DNA kit (Omega Bio-tek, USA) according to the manufacturer’s instructions. The 1% agarose gel electrophoresis was used to evaluate the DNA quality and integrity, and DNA concentration was measured using the NanoDrop 2000 UV-VI spectrophotometers (Thermo Scientific, Wilmington, USA).

The DNA was fragmented by Covaris M220 (Genetics, China) to screen fragments of about 300 bp for subsequent construction of PE libraries. Metagenomic sequencing was conducted according to the standard protocols of Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Sequence reads were treated to remove low-quality reads, trim the read sequences, and remove the host genome sequences. Specifically, reads with sequence lengths less than 50 bp and low-quality bases (quality value ≤ 20). Host genomic sequences were removed by Bowtie2 (v 2.2.9) software [ 19 ]. MEGAHIT (v 1.1.2) [ 20 ] were used to align the clean data of each sample to contigs (≥ 300 bp). MetaGene [ 21 ] ( http://metagene.cb.k.u-tokyo.ac.jp/ ) was performed for predicting the open reading frames according to contigs (≥ 300 bp), and genes with nucleic acid lengths greater than 100 bp were selected and translated into amino acid sequences. Afterwards, CD-HIT (v 4.7) [ 22 ] was used to clustered (95% identity, 90% coverage), and the longest gene in each class was as the representative sequence to construct the non-redundant gene set. SOAPaligner (v 2.21) [ 23 ] was used to compare the high-quality reads obtained from the sequencing of single samples with the non-redundant gene set (95% identity), and to calculate the abundance data of each target gene in the corresponding samples.

After that, the constructed non-redundant (NR) gene was subjected to species and functional annotation, and the annotation process was conducted according to the standard protocols of Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) [ 24 ]. BLASTP (v 2.3.0) was used to blast the non-redundant gene set to the sequences of the NR database (e-value set to 1e-5) to obtain abundance data corresponding to each species. The non-redundant gene set sequences were aligned with KEGG database using BLASTP (v 2.3.0; e-value set to 1e-5). The annotation information of KEGG Ortholog (KO) from the KEGG database was acquired based on the relative abundance profile. HMMSCAN (v 3.1b2) [ 25 ] software was used to compare the amino acid sequence to the CAZyme (v 6.0) database (e-value set to 1e-5) and to obtain an annotation of the carbohydrate-active enzyme.

RNA isolation and RNA-seq analyses

Total RNA was extracted from colon epithelial tissues of goat kids using TRIzol reagent (Invitrogen) according the manufacturer’s instruction. The RNA quality was evaluated using the Nanodrop 2000 (NanoDrop Technologies) and Bioanalyzer 2100 (Agilent). The Agilent 2100 was used to determine the RNA integrity number (RIN) value of the sample RNA. All RNA sample had the RIN value greater than 8.0 (RIN number: 8.20, 9.30, 8.40, 9.60, 9.40 in BM group sample, respectively, 9.40, 9.50, 9.40, 9.30, 8.60 in MR group sample, respectively). The RNA-seq transcriptome library was prepared using 5 µg of total RNA, following the TruSeq stranded RNA sample preparation kit for Illumina (San Diego, CA, USA). RNA-Seq library was sequenced with the Illumina NovaSeq 6000 platform (2 × 150 bp read length). The raw reads were trimmed and quality controlled by the SEQPREP and Sickle software. The TopHat (v 2.1.1) [ 26 ] software was used to align the clean data with the Capra hircus reference genome (GCA001704415.1) to obtain mapped data.

Colonic content and serum metabolite measurements using LC–MS/MS

The colon content was homogenized by add ice-cold methanol/water (70%, v/v) in a ratio of 500 μL per 50 mg and then vortex for 3 min. The sample vortexed liquid was left at −20 °C for 30 min and centrifuge for 5 min at 4 °C and 12,000 ×  g . The collected supernatant was subjected to LC–MS/MS analysis (UPLC, Shim-pack UFLC SHIMADZU CBM A system; MS, QTRAP System). Serum samples were prepared by add 300 μL of methanol/water (70%, v/v) to 50 μL of serum, and vortexed for 3 min. The liquid was centrifuge for 10 min at 12,000 ×  g at 4 °C. The filtered samples were transferred to LC–MS/MS analysis.

Quantitative PCR (qPCR) analysis

Total RNA was extracted using Trizol reagent (CWBIO, China) from colon tissues, and qPCR was performed using LightCycler 96 (Roche, NC, USA). qPCR primers are shown in Additional file 1 : Table S4. All primers were obtained from Zhongke Biotechnology (Xi’an, China). The amplification efficiencies were 90%–110%. The reaction conditions were, 95 °C for 30 s, 95 °C for 10 s and 60 °C for 30 s for 40 cycles. Five biological replicates were set for each group. To minimize operational and technical errors, three technical replicates were performed for each sample. The expression of the target gene relative to the internal reference gene β-actin was calculated using the ΔΔCt method [ 27 ].

Statistical analyses

GraphPad Prism (v9.0.0, GraphPad, USA) was used to generate histograms. The two-group data were subjected to independent sample t -test via SPSS (v 21.0, IBM, USA) to evaluate significance. The data are expressed as mean with standard error. Beta diversity analysis used the Bray-Curtis distance algorithm, and analysis of Similarities (ANOSIM) test was used to performed significant differences between groups with 999 permutations. The Kruskal-Wallis H test was used to identify significant differences of the relative abundance at different taxonomic levels between groups. Spearman correlation network analysis used python (v.2.7) stats, and the absolute value of the correlation coefficient was set to 0.8 and P ≤ 0.05. Linear discriminant analysis effect size (LEfSe) was performed to identified the microbiome with higher relative abundance in the two groups. The non-parametric factorial Kruskal-Wallis rank sum test was used, followed by linear discriminant analysis (LDA) to evaluate the impact of each taxon abundance on the differential effect. A significant increase in microbiota abundance was defined as an LDA score (log10) greater than 3.0. Metabolites with VIP ≥ 1 and P value < 0.05 were generally considered to be significantly different. R package (heatmaply; Complex Heatmap) was used to draw heatmaps of significant metabolites, and R (igraph) was used for correlation analysis in metabolite correlation network diagrams. DESeq2 software [ 28 ] based on negative binomial distribution was used to analyze raw counts, and genes with comparative expression differences between groups were obtained based on the default parameters P -adjust < 0.05 and |log 2 FC| ≥ 1 filtering conditions. Goatools was used to perform GO enrichment analysis on the genes in the gene set [ 29 ]. The method used was Fisher’s exact test, when the corrected P -adjust < 0.05.

Dysregulation of lipid metabolism appears to be associated with the reduced daily weight gain observed in goat kids fed with milk replacer

Evaluating the effect of milk replacer on the growth performance of goat kids, we found that there was no significant difference in body weight between BM and MR groups on 0, 14 and 28 d after delivery, however, goats in the BM group gained weight significantly faster than those in the MR group, where kids in the BM group gained 104.64 g/d compared to 69.00 g/d in the MR group ( P  > 0.05; Fig.  1 B). Compared with BM kids, stress-related indicators showed a highly significant increase in serum concentrations of Cor, Cort, HIF-α and DAO in the MR group ( P  < 0.001; Additional file 1 : Fig. S1A), indicators related to pro-inflammation showed a highly significant increase in serum concentrations of IL-1β, IL-6 and TNF-α in the MR group, and a significant decrease in IL-10, an indicator related to anti-inflammation ( P  < 0.01; Additional file 1 : Fig. S1B), consistent with immune-related indicators including IgA, IgG and IgM showed a similar pattern of decrease ( P  < 0.01; Additional file 1 : Fig. S1B). The serum Glu index was also significantly decreased in the MR group ( P  < 0.05; Fig.  1 C). To clearly clarify the effect of milk replacer feeding on lipid metabolism in kids, TC, TG and NEFA indictors were measured in serum and liver samples and revealed that milk replacer feeding treatment decreased TC and NEFA concentrations ( P  < 0.05; Fig.  1 E and F), and no significant effect on TG concentrations in serum and liver ( P  > 0.05; Fig.  1 D). Taken together, these results revealed that milk replacer feeding attenuates weight gain of kids and the lipid metabolism associated with it.

Colonic epithelial lipid transport-related genes profile is influenced by milk replacer feeding

To further investigate the impact of milk replacer feeding on the expression profile of colonic epithelial lipid transport-related genes. We performed transcriptome sequencing using goat colonic epithelium samples. A total of 97.42 Gb clean data was obtained using RNA-seq of 10 colonic epithelium samples, with an average of 668,563,298 high-quality paired reads produced per sample. The alignment rate to the Capra hircus reference genome exceeded 94%. In total, 672 differentially expressed genes (DEGs) were screened, of which 403 were upregulated, and 269 were downregulated (Fig.  2 A; Additional file 2 : Tables S5). Among them, genes such as ABCG8 , ABCG5 , SCTR , SCT , CCL25 , PRAP1 , FABP2 , RBP2 , APOC3 , SLC5A12 , CLDN19 , SLC2A2 , SLC13A4 , LOC102172669, LOC102181858, LOC102186942 and LOC102186759 were significantly upregulated in the MR group. In contrast, the genes AQP5 , OSR2 , HOXD10 , MRAP2 , KRT4 and KRT6A were significantly downregulated in the MR group (|log 2 FC| ≥ 1 and P -adjust < 0.05; Fig.  2 B). Among them, this DEGs mainly enriched in cholesterol metabolism pathway (involved in APOC3 , ABCG8 , SOAT2 and ABCG5 ), steroid hormone biosynthesis pathway, bile secretion pathway (involved in SCT , ABCG8 , LOC102181069, ABCG5 and SCTR ), fat digestion and absorption pathway (involved in FABP2 , ABCG8 and ABCG5 ), insulin secretion pathway (involved in CREB3L3 , SLC2A2 and PDX1 ) and PPAR signaling pathway (involved in FABP2 and APOC3 ) (Fig.  2 C). They were also enriched in 336 Gene Ontology (GO) categories, mainly including negative regulation of cholesterol transport, negative regulation of sterol transport, negative regulation of intestinal lipid absorption, negative regulation of intestinal cholesterol absorption, and negative regulation of intestinal phytosterol absorption (Additional file 1 : Fig. S2A). Based on the functional enrichment analysis network, the significantly enriched pathways mainly involve downstream pathways related to negative regulation of cholesterol transport (Additional file 1 : Fig. S2B). Taken together, milk replacer feeding significantly upregulates the expression of genes associated with negative regulation of lipid metabolism in the colon, leading to lipid dysfunction in goat kids.

Transcriptional profiling of colonic epithelium in response to formula feeding. A Volcano plots illustrating the results of RNA-seq analyses comparing the colonic epithelium of goats in the BM (Breast Milk) and MR (Formula Feeding) groups. The red diamond indicates upregulated genes in the MR group, while the blue diamond represents downregulated genes in the MR group. B Heatmap displaying the differentially expressed genes (DEGs) in the MR and BM groups. C Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs in the BM vs. MR group. The genes associated with each pathway are listed in the respective bar

Milk replacer feeding altered the colonic microbiota and potential function in goat kids

To further investigate the effects of milk replacer feeding treatment on gut microbial composition and function in goat kids and the correlation between the colon microbes with lipid metabolism. The variety of the bacteria in the colon was examined in the two groups. According to the Shannon and Chao index, the MR group generally had less alpha diversity at the species level than the BM group ( P  = 0.02, Additional file 1 : Fig. S3A). Principal coordinate analysis (PCoA) at species level showed a clear separation of microbial community structure between the two groups (ANOSIM, r  = 0.996, P  = 0.001, Fig.  3 A). We also analyzed variations in microbial communities between the two groups at the phylum level, and we found that the abundance of Firmicutes was significantly lower in the MR groups than in the BM group. In contrast, the abundance of Actinobacteria was significantly higher in the MR groups than in the BM group (Fig.  3 B). At the genus level, the abundance of Megamonas , Bifidobacterium , Prevotella , and Parabacteroides were significantly increased in the MR group ( P  < 0.05, Additional file 1 : Fig. S2B), however, the abundance of Clostridium , Subdoligranulum , Faecalibacterium , Eubacterium and Lachnoclostridium were significantly decreased in the MR group ( P  < 0.05, Additional file 1 : Fig. S2B). Using LEfSe analysis to identify additional key signature species, it was discovered that MR goats had significantly higher levels of Bacteroides plebeius CAG 211, Lactobacillus mucosae , Bacteroides coprocola CAG 162, Bifidobacterium longum , Prevotella sp. 109, Ruminococcus gnavus CAG 126, and Megamonas funiformis CAG 377 (LDA > 3.5, Fig.  3 C; Additional file 3 : Table S6), and BM goats had significantly higher levels of Bacteroides vulgatus , Subdoligranulum variabile , Faecalibacterium prausnitzii , Ruminococcaceae bacterium AM2, Flavonifractor plautii , Clostridia bacterium UC5.1-1D1, Eubacterium desmolans , Staphylococcus sp. CAG 324, Lactobacillus reuteri , Pseudoflavonifractor capillosus , and Butyricimonas virosa (LDA > 3.5, Fig.  3 C). Further analysis of the differences in colonic microbial interaction networks between the two groups revealed that the MR group formed an interaction network with core species including Bacteroides plebeius , Escherichia coli , Bacteroides finegoldii , Bacteroides coprophilus , Parabacteroides distasonis , Olsenella sp. DNF00959, Ruminococcaceae bacterium GD1, Sutterella wadsworthensis , and Blautia sp. KLE 1732 (Degree Centrality > 0.45; Additional file 1 : Fig. S4), while the BM group formed an interaction network with core species including Clostridia bacterium UC5.1-1D1, Eubacterium desmolans , [Ruminococcus] gnavus , Lactobacillus amylovorus , Alistipes sp. HGB5, Alistipes finegoldii , Firmicutes bacterium CAG:424 , Clostridium sp. ATCC BAA-442, and Blautia sp. KLE 1732 (Degree Centrality > 0.45; Additional file 1 : Fig. S4). This further confirms the influence of milk replacer feeding on the core structure of colonic microbiota.

Effects of formula feeding on colonic microbiota and potential functional changes in goat kids. A Principal coordinate analysis (PCoA) plot based on relative species abundances, with box plots illustrating Bray–Curtis distances associated with groupings, assessed using the Wilcoxon rank-sum test. Analysis of similarity (ANOSIM) was employed to evaluate the dissimilarity of Bray–Curtis distances. B Relative abundance of bacterial phyla in the BM (Breast Milk) and MR (Formula Feeding) groups. Red asterisks indicate significant differences in bacterial abundance between the two groups. C Identification of bacterial species that exhibit differential abundance between groups, as determined by linear discriminant analysis effect size (LEfSe) analysis. The analysis employed a one-against-all multi-group comparison strategy, with a linear discriminant analysis (LDA) threshold set at > 3.0. D  and  E Variations in the abundance of KEGG (Kyoto Encyclopedia of Genes and Genomes) orthologs associated with the butyrate synthesis pathway and upstream pathways, including Gluconeogenesis, TCA cycle, and Fatty acid synthesis. Red color indicates significantly up-regulated enzyme commission (EC) numbers in the MR group, while green color represents significantly up-regulated EC numbers in the BM group. F Enrichment disparities in third-level metabolic pathways based on KEGG analysis in the goat colon. The red circle signifies metabolic pathways significantly enriched in the MR group, while the blue circle represents pathways significantly enriched in the BM group. Statistical significance was determined using the Dunn Test, with a significance level set at P  < 0.001. G Correlations between the composition of gut microbiota at the species level and the concentrations of serum and liver parameters. Positive correlations are denoted in red, while negative correlations are represented in green

At the functional level, the microbiota in the MR group was mainly enriched in pathways related to ‘starch and sucrose metabolism’, ‘fructose and mannose metabolism’, ‘biosynthesis of amino acids’, ‘phenylpropanoid biosynthesis’, ‘oxidative phosphorylation’, and ‘beta-Lactam resistance’, while the microbiota in the BM group was mainly enriched in ‘ABC transporters’, ‘pyruvate metabolism’, ‘aminoacyl-tRNA biosynthesis’, and ‘amino sugar and nucleotide sugar metabolism’ (Fig.  3 F). Further analysis of the differences in the abundance of CAZyme genes encoding carbohydrate enzymes in the colonic microbiota revealed that, compared to the BM group, the MR group showed significantly upregulated enzyme gene expression related to the synthesis of glucose from butyrate (Fig.  3 D), as well as enzymes involved in the degradation of L-leucine, L-valine, and L-isoleucine, which are precursors for gluconeogenesis, TCA cycle, and gatty acid synthesis (Fig.  3 E). This further confirms that disturbances in the core structure of colonic microbiota caused by milk replacer feeding may potentially affect the host’s absorption and utilization of energy and lipid nutrients.

The correlations between the level of serum triglyceride (TG) and cholesterol (TC) and liver triglyceride, cholesterol and free fatty acid (NEFA) and changes in microbial abundance were evaluated by Spearman’s correlation analysis. We found that liver cholesterol and liver free fatty acid concentrations positive correlated with Clostridia bacterium  UC5.1-1D1, Eubacterium desmolans , Flavonifractor plautii , Ruminococcaceae bacterium  AM2, Faecalibacterium prausnitzii , Lactobacillus reuteri and Subdoligranulum variabile , negatively correlated with Bacteroides plebeius  CAG:211, Bacteroides coprocola , Bacteroides plebeius . Besides, Bacteroides fragilis abundance positively correlated with liver NEFA, [Ruminococcus] gnavus negatively correlated with liver cholesterol concentrations, and Butyricimonas virosa positively correlated with liver cholesterol concentrations (Fig.  3 G). In conclusion, these results indicated that the changes in colonic microbial composition and function during milk replacer feeding may potentially impact the host’s lipid metabolism capabilities.

Milk replacer feeding affect the colonic content lipid metabolism pathways in goat kids

We analysed the effect of milk replacer feeding on differential metabolites in the colonic contents of goat kids using an LC–ESI–MS/MS system. A total of 272 metabolites were significantly altered in the MR group compared to the BM group, of which 56 metabolites were significantly downregulated and 216 metabolites were significantly upregulated (Additional file 4 : Table S7). Interesting, 44 metabolites were undetectable in the BM group, while they exhibited a high abundance in the MR group, primarily including carnitine and fatty acid-like compounds (Rs-mevalonic acid, salicylaldehyde, 3-hydroxypicolinic acid, nicotinuric acid, and 3-hydroxyhippuric acid) (Fig.  4 A; Additional file 4 : Table S7). Furthermore, 15 metabolites were exclusively abundant in the BM group and were not detectable in the MR group, mainly belonging to the bile acid class (lithocholic acid, hododeoxycholic acid, glycine deoxycholic acid, and deoxycholic acid) (Fig.  4 B). We further calculated Spearman’s rho coefficients between the DEG and colonic luminal metabolites, and coefficients greater than 0.8 with a P -value < 0.05 were considered significant. We found that the lipid metabolism genes ABCG5 , ABCG8 ,  SCTR  and SCT were positively correlated with the metabolites choline, spermidine, acetaminophen, glycocholic acid, glutathione, DL-carnitine and cyclic amp involved in the bile acid secretion pathway and negatively correlated with deoxycholic acid and lithocholic acid. LOC102172669, LOC102186942, LOC102181858 and LOC102186759 genes were positively correlated with the metabolites LTE4, 19(S)-HETE, 5,6-DiHETrE and 20-HETE involved in the arachidonic acid metabolism pathway and negatively correlated with 15-deoxy-δ-12,14-PGJ2 (Fig.  4 C).

Impact of formula feeding on colonic bile acid profile in goat kids. A  and  B Relative abundance of significantly different metabolites in the colon of the BM (Breast Milk) and MR (Formula Feeding) groups. C Correlations between representative metabolites and genes were analyzed, and the outcomes of differential gene and differential metabolite correlations with Pearson’s correlation coefficients exceeding 0.80 and P -values below 0.05. D Correlations between the composition of gut microbiota at the species level and representative metabolites. Positive correlations are depicted in red, while negative correlations are illustrated in green

To further analyze the correlation between differential metabolites and distinct colonic microbiota, Spearman’s coefficients between differential microbial and metabolites were then calculated, and coefficients greater than 0.8 with a P -value < 0.05 were considered significant. Interestingly, we found Bacteroides coprocola abundance positively correlated with carnitine C13:0 Isomer1, glycine linoleate, 2-methylbutyroylcarnitine, DL-carnitine, c is -9,10-epoxystearic acid, punicic acid, linoleic acid C18:2N6C, carnitine C18:2-OH and O-phosphorylethanolamine involved in the FA. Ruminococcus gnavus  CAG:126 abundance positively correlated with carnitine. Among the metabolites of oxidized lipid, Bacteroides plebeius  CAG:211 positively correlated with 9,10-EpOME, 12,13-EpOME, 9,10-DiHOME, 12,13-DiHOME and 6-keto-PGF1α. Lactobacillus reuteri , Megamonas funiformis and Bacteroides dorei negatively correlated with 9-HOTrE, 13(R)-HODE, 20-HETE and 9-HpODE. Lactobacillus reuter positively correlated with glycine deoxycholic acid. Faecalibacterium prausnitzii , Pseudoflavonifractor capillosus , Ruminococcaceae bacterium  D16, Eubacterium desmolans and Subdoligranulum variabile positively correlated with β-murine, taurodeoxycholic acid, deoxycholic acid and glycine deoxycholic acid involved in the bile acids. Ruminococcus gnavus  CAG:126 negatively correlated with hododeoxycholic acid and lithocholic acid (Fig.  4 D). These findings indicated that colon microbiota reshaping induced by milk replacer feeding leads to a deficiency in colonic secretion of certain secondary bile acids.

Milk replacer feeding affect the serum metabolism pathways in goat kids

Untargeted metabolome profiles were generated on goat serum samples using an LC–ESI–MS/MS system to assess the effect of milk replacer on differential metabolites in serum. We found 39 metabolites were significantly downregulated, and 20 metabolites were significantly upregulated in MR group (Fig.  5 A; Additional file 5 : Table S8). Among them, 1,7-dimethylxanthine, 9-HpODE, Lysope 14:0, 13-oxoODE, octapentaenoic acid, 9(S)-HpOTrE, 5,6-EET and 5,6-DiHETrE were the top 10 upregulated metabolites in the MR group. Lithocholic acid, carnitine C17:0, β-murine, urocanic acid, glycyl-L-proline, 1,2-dioctanoyl PC, 23-deoxydeoxycholic acid, 5-HETrE, capric acid (C10:0) and linoleylethanolamide were the top 10 downregulated metabolites in the MR group. Interestingly, we found that some metabolites were in high relative abundance in the BM group such as urocanic acid, 1,2-dioctanoyl PC, 5-HETrE, capric acid C10:0, linoleylethanolamide and 6-hydroxynicotinic acid, while phenylpyruvic acid, 1,7-Dimethylxanthine, 5,6-EET, methanesulfonic acid, tetracosaenoic acid and hydrocinnamic acid were in high abundance in the MR group (Fig.  5 B and C; Additional file 5 : Table S8).

Influence of formula feeding on serum metabolites in goat kids. A Volcano plots displaying the results of metabolite analyses comparing the serum of the BM vs. MR group. Red indicates upregulated metabolites in the MR group, while green represents downregulated metabolites. B Relative abundance of significantly different metabolites in the colon of the BM and MR groups. C The top 10 differentially expressed serum metabolites in the BM vs. MR comparison. D Significant alterations in metabolic pathways in the MR group. E Analysis of Differential Abundance (DA) scores for KEGG pathways in the BM vs. MR comparison

The KEGG compound database generated the KEGG enrichment to describe the effect of milk replacer on these responsive metabolites and revealed that the ‘arachidonic acid metabolism’, ‘biosynthesis of unsaturated fatty acids’, ‘cholesterol metabolism’, ‘fatty acid biosynthesis’, ‘fatty acid degradation’, ‘fatty acid elongation’, ‘fatty acid metabolism’, ‘linoleic acid metabolism’, and ‘alpha-linolenic acid metabolism’ were the most significantly affected pathways (Fig.  5 D). The metabolic pathways significantly downregulated were ‘fatty acid elongation’, ‘fatty acid metabolism’ and ‘epithelial cell signaling in helicobacter pylori infection’ in the MR group (Fig.  5 E). This further corroborates that milk replacer feeding significantly disrupts the concentrations of metabolites involved in lipid metabolism in the serum, thereby impacting the host’s lipid metabolism capacity.

Formula feeding affect the lipid metabolism profile is transferable by IMT

To validate the causal relationship between milk replacer feeding-induced colonic microbiota disruption and host lipid metabolism, we transferred the colon microbiota from BM and MR group to bacterial-restricted SPF C57/6 J mice (Fig.  6 A). Compared with BM_IMT mice, the weight of MR_IMT mice decreased from day 12 of transplantation, and the weight difference between the two groups was significant at d 20 of transplantation (Fig.  6 B). The organ indexes of liver and spleen of BM_IMT and MR_IMT mice showed no significant difference (Fig.  6 C). To clarify the causal relationship between hindgut microbes and lipid metabolism in goat, we evaluated the effects of microbiota on lipid metabolism ability of mice. The levels of TC, TG and NEFA indictor were measured in serum and liver samples and revealed that MR_IMT group significantly decreased TC concentrations in liver and NEFA concentrations in serum and liver compared with the BM_IMT group but no significant effect on TG concentration in serum and liver (Fig.  6 D–G). The mRNA expression level of genes associated with lipid metabolism in the NC, Ab, BM_IMT and MR_IMT treatment groups was further investigated. There was a significant increase in the mRNA expression of ABCG5 , ABCG8 and FABP2 in the MR_IMT group, while the mRNA expression of RBP2 and APOC3 showed no significant difference (Fig.  6 H).

Transference of altered lipid metabolism profile by formula feeding through intestinal microbiota transplantation. A Schematic representation of the experimental design. B Body weight of SPF C57/6 J mice. Data differences were assessed using one-way analysis of variance (ANOVA) with Tukey’s test. C Organ indexes of SPF C57/6 J mice. D Concentration of total cholesterol (TC) in the serum and liver of SPF C57/6 J mice. E Concentration of triglycerides (TG) in the serum and liver of SPF C57/6 J mice. F  and  G Concentration of non-esterified fatty acids (NEFA) in the serum and liver of SPF C57/6 J mice. H Expression of mRNA related to lipid metabolism in the colon of the mouse model. Data differences were analyzed using one-way ANOVA with Tukey’s test. Significance levels are indicated as follows: n.s. (not significant) for P  > 0.05, * for P  < 0.05, ** for P  < 0.01, and *** for P  < 0.001

We conducted 16S rRNA gene sequencing to examine the colon bacteria composition to confirm that fecal transplantation modulates the gut microbiota. Our results indicated that α-diversity (via Chao and Pd Index) and β-diversity were not significantly different in the BM_IMT and MR_IMT group (Additional file 1 : Fig. S5A and B). At the genus level, the abundance of Bacteroides , Colidextribacter , Parabacteroides and Escherichia-Shigella were enriched in the MR_IMT group, while u_f_Eggerthellaceae and Holdemania were enriched in the BM_IMT group (Additional file 1 : Fig. S5C). The ASV-level analysis showed that the relative abundance of ASV131 ( Bacteroidaceae ), ASV266 ( Lachnospiraceae ) were detected in the BM_IMT group. Moreover, the relative abundance of ASV398 ( Muribaculaceae ), ASV94 ( Muribaculaceae ), ASV6 ( Muribaculaceae ), ASV233 ( Clostridia_UCG-014 ), ASV472 ( Clostridia_vadinBB60 ), ASV993 ( Sutterellaceae ), ASV383 ( Sutterellaceae ) were not identified in the BM_IMT group (Additional file 1 : Fig. S5D). These results causally confirm that milk replacer feeding-induced colonic microbiota disruption is associated with a phenomenon that can affect the host’s lipid metabolism capacity, with potential cross-species implications.

Discussions

The purpose of this study was to understand how milk replacer feeding alters the colon microbiota in goat kids and affects their lipid metabolism, with a focus on the potential modulation of the host’s lipid metabolism pathways. Our results demonstrated that milk replacer feeding significantly reduced daily weight gain and decreased the concentration of cholesterol and free fatty acids in the serum and liver of goat kids. This was evident from multiple omics analyses, including metagenomic sequencing, metabolic profiling, RNA-seq analysis, and colonic digesta transplantation. These findings suggest that milk replacer feeding affects the microbial composition and metabolite characteristics, directly influencing the differential expression of colon lipid metabolism genes, thereby affecting the lipid metabolism pathway (Fig.  7 ).

Process of formula feeding-mediated disruption of goat kid lipid metabolism through modulation of gut microbiota and bile acid secretion. Figdraw software was used to create this illustration

A key observation was that milk replacer feeding led to significant changes in the gut microbiota composition. Specifically, we noted a disruption in the abundance of Bacteroides spp., which are known to play a crucial role in cholesterol metabolism [ 30 , 31 , 32 ]. This disruption likely contributed to the substantial changes in bile acid levels observed in our study. Correlation analyses between microbiota and bile acid metabolites supported this viewpoint, indicating that the presence of Bacteroides spp. could be beneficial in mitigating the negative effects on lipid metabolism seen with milk replacer feeding. Furthermore, our study found that the milk replacer feeding regimen did not alter the concentrations of serum and hepatic triglycerides but resulted in a reduction in cholesterol levels. This phenomenon may be attributed to the lower cholesterol content in milk replacer compared to breast milk, which may downregulate the generation of hepatic hydroxymethylglutaryl coenzyme A reductase, thereby reducing endogenous cholesterol synthesis [ 33 , 34 , 35 ].

The analysis of bile acids revealed the absence of certain secondary bile acids in the colon and serum of goats fed with milk replacer. This absence inhibited fat emulsification, reduced pancreatic lipolysis, and hindered the absorption of lipid substances in the intestine, ultimately impacting growth and development [ 36 ]. The lack of these bile acids likely led to an impaired lipid metabolic pathway, as indicated by the downregulation of genes associated with lipid metabolism in the colonic epithelium. Additionally, the study confirmed that milk replacer feeding induced an upregulation of ABCG5 and ABCG8 expression in the colonic epithelium. These genes are involved in cholesterol reverse transport, small intestine absorption, and bile acid secretion, interacting with dietary components to regulate blood cholesterol levels and maintain cholesterol metabolic homeostasis [ 37 , 38 , 39 ].

Overall, our findings highlight the impact of milk replacer feeding on the core composition of colonic microbiota and its subsequent effect on the expression profile of colonic lipid metabolism genes. These alterations influence the host’s lipid metabolic program and affect normal goat growth and development. Further investigation is needed to elucidate the mechanisms involving specific functional microbiota and the regulation of colonic epithelial lipid metabolism gene expression by key secondary bile acids.

Our study demonstrates that milk replacer feeding in goats profoundly influences colonic lipid metabolism and microbial ecology, which are crucial for overall metabolic health and development. We observed that milk replacer feeding reduced serum and liver concentrations of cholesterol and free fatty acids, thereby downregulating colonic lipid absorption and cholesterol transport pathways. These alterations are particularly impactful during the critical growth phase of kids, affecting their normal development. Furthermore, milk replacer feeding induced significant changes in the core microbial species composition in the colon, notably leading to the absence of secondary bile acids. Utilizing a germ-free mouse model validated a causal relationship between disrupted colonic microbiota induced by milk replacer feeding and key metabolic parameters such as daily weight gain, circulating cholesterol, free fatty acid levels, and the expression of colonic lipid metabolism genes. In summary, our findings underscore the detrimental effects of milk replacer feeding on colonic lipid metabolism through microbiota disruption in goats. This study provides novel insights into the mechanisms through which milk replacer feeding impacts colon metabolism and physiology in ruminants. These insights are pivotal for developing new milk replacer formulations enriched with active probiotics and prebiotics. Such formulations could potentially mitigate the adverse effects observed here by targeting specific bioactive substances and fostering beneficial bacterial strains.

Availability of data and materials

The metagenomic sequencing and RNA-seq data are available from the national center for biotechnology information (NCBI) under accessions PRJNA1029896 ( https://dataview.ncbi.nlm.nih.gov/object/PRJNA1029896?reviewer=f8oisljdbf1sdgvmu69er8df15 ) and PRJNA1030534 ( https://dataview.ncbi.nlm.nih.gov/object/PRJNA1030534?reviewer=l6t3793h9q753mangoer3cl3rm ), respectively.

Abbreviations

ATP-binding cassette

Breast milk

Corticosterone

Diamine oxidase

Hypoxia-inducible factor

Immunoglobulin A

Immunoglobulin G

Immunoglobulin M

Interleukin-6

Interleukin-10

Interleukin-1β

Intestinal microbiota transplantation

Linear discriminant analysis

Linear discriminant analysis effect size

Milk replacers

Non-esterified fatty acid

Principal coordinate analysis

Phosphate-buffered saline

RNA integrity numbers

Total cholesterol

Triglycerides

Tumor necrosis factor-α

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Acknowledgements

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This work was financially supported by National Natural Science Foundation of China (32160801), China Agriculture Research System (CARS-39-12), and Young Talent Fund of Association for Science and Technology in Shaanxi, China (2023-6-2-1) and “Double-chain” project on livestock breeding (2022GD-TSLD-46). None of the funders had any role in the design and conduct of the study, collection, management, analysis, and interpretation of the data, as well as preparation, revision, or approval of the manuscript.

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Ke Zhang and Ting Zhang contributed equally to this work.

Authors and Affiliations

Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China

Ke Zhang, Ting Zhang, Awang Cuoji, Yangbin Xu, Yitong Zhao, Yuxin Yang, Xiaolong Wang & Yulin Chen

College of Animal Engineering, Yangling Vocational and Technical College, Yangling , Shaanxi, 712100, China

Mengmeng Guo

Institute of Animal Sciences, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa, 850009, China

Awang Cuoji, Langda Suo & Yujiang Wu

Institute of Animal Nutrition and Dietetics, Vetsuisse-Faculty, University of Zurich, Zurich, 8057, Switzerland

Daniel Brugger

Key Laboratory of Animal Genetics and Breeding On Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lhasa, 850009, China

Langda Suo & Yujiang Wu

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Contributions

CYL, WXL, ZK, WYJ and YYX conceived, designed, and supervised the project; ZK, ZT, SLD, AC, GMM, and XYB collected samples and performed experiments; ZK, ZYT, ZT, and GMM carried out bioinformatic analyses; ZK and ZT drafted the paper. CYL, DB, WXL, WYJ and YYX revised the paper. All authors read, edited, and approved the final manuscript.

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Correspondence to Yujiang Wu or Yulin Chen .

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All subjects provided informed consent to participate in this study and agreed for publication of the research results.

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Supplementary Information

Additional file 1:.

Table S1 . Ingredients of the experimental diets of mother goats. Table S2 . Ingredients of the experimental diets of goat kids (DM basis). Table S3 . Ingredients of the experimental diets of mice (DM basis). Table S4 . qPCR primers used for gene expression analysis. Fig. S1 . Serum Stress markers, inflammatory cytokines and immune cytokines concentration in goats under different feeding conditions. Fig. S2 . Transcriptional profiling of colonic epithelium in response to formula feeding. A Gene Ontology (GO) enrichment analysis of genes in the differentially expressed gene set. B Network representation of GO enrichment analysis. Fig. S3 . Microbial diversity analysis and relative abundance of bacterial genera in Breast Milk (BM) and Formula Feeding (MR) Groups. A Alpha diversity analysis based on the species level. B Relative abundance of bacterial genera in the BM (Breast Milk) and MR (Formula Feeding) groups. Red asterisks indicate significant differences in bacterial abundance between the two groups. Fig. S4 . Microbial interaction network in the goat colon under different feeding additives. Fig. S5 . Impact of gut microbiota transplantation on mouse gut microbiome composition. A  Alpha diversity analysis at the ASV level. B  Principal Coordinate Analysis (PCoA) plot based on ASV level, with box plots illustrating Bray–Curtis distances associated with groupings, assessed using the Wilcoxon rank-sum test. Analysis of similarity (ANOSIM) was employed to evaluate the dissimilarity of Bray–Curtis distances. C  Relative abundance of bacterial genera in the four groups. Red asterisks indicate significant differences in bacterial abundance between the MR_IMT and MR_IMT groups. D  Selection of microbiota categories with significantly different abundance in the MR_IMT and MR_IMT groups.

Additional file 2:

Tables S5 . The information of DEGs between BM and MR group in goats.

Additional file 3:

Table S6 . Differential microbiota between BM and MR goat kids in species level.

Additional file 4:

Table S7 . Unique list of 272 colon metabolites altered comparing BM and MR goat kids.

Additional file 5: Table S8 . Unique list of 138 serum metabolites altered comparing BM and MR goat kids.

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Zhang, K., Zhang, T., Guo, M. et al. Early-life milk replacer feeding mediates lipid metabolism disorders induced by colonic microbiota and bile acid profiles to reduce body weight in goat model. J Animal Sci Biotechnol 15 , 118 (2024). https://doi.org/10.1186/s40104-024-01072-x

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• Published: 06 September 2024

Associating sensory cues with incoming seizures: developing an animal model of auras

• Ritwik Das 1 ,
• Carlos Howey 1 ,
• Autumn McFetridge 1 ,
• Valérie Lapointe 1 &
• Artur Luczak 1  

Scientific Reports volume  14 , Article number:  20881 ( 2024 ) Cite this article

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• Classical conditioning

For patients with epilepsy, one of the biggest problems is the unpredictability of the time when the next seizure will occur. Interestingly, some epileptic patients experience a sensory sensation preceding seizures, called aura, which helps them move to safety before a seizure. Here, we describe the development of the first animal model of auras, which could allow for a more detailed study of this phenomenon. Specifically, in mice, we presented sensory stimuli (sound and light cues) a few seconds before kindling an animal to induce seizures. Animals were kindled by electrical stimulation in the basolateral amygdalar nucleus. Over the course of stimulation sessions, animals started showing progressively stronger freezing behavior to sensory cues preceding kindling. Interestingly, seizures are known to cause retrograde amnesia, thus it was surprising that the association between seizures and preceding sensory cues developed in all experimental animals. In summary, our experiments show that similarly to auras, a sensory sensation can be associated with incoming generalized seizures and is not erased by retrograde amnesia.

Introduction

Epilepsy is a serious brain condition, affecting over 70 million people worldwide with the highest prevalence in infants and older age groups 1 . Epilepsy is a brain disorder, resulting in excessive and hypersynchronous neuronal activity leading to ‘seizures’ 2 . Seizures are often spontaneous and usually sudden events involving rapid onset of symptoms that can range from dramatic convulsive activity to subjective experiences or sensations that individuals perceive or encounter. During the initial part of certain seizures, a phenomenon known as "auras" can take place, wherein individuals retain consciousness and subsequently recall this as sensory sensations preceding behavioral seizures 3 . Auras are typically focal seizures manifested as ‘subjective’, internal events often described by patients as viscerosensory, somatic sensations, visions or related to autonomic functions such as flushing, sweating, piloerection 4 .

Mesial temporal lobe epilepsy (mTLE), the most common example of drug-resistant adult epilepsy, is clinically identified by prominent auras followed by episodes of staring, behavioral arrest, and frequent oro-facial automatisms 5 , 6 , 7 . Of the diverse manifestations of auras, those classified as viscerosensorial and affective are frequently linked to temporal lobe seizures 8 . Visceral sensations encompass bodily perceptions from the thorax, stomach, or bladder, including heaviness, constriction, ascending sensations, pain, flushing, nausea, tachycardia, and dyspnea 9 . Within the realm of affective auras, the sensation of fear stands out as the most prevalent symptom associated with epileptic discharges originating from the mesial temporal lobe 8 , 10 , 11 .

Fear auras, unlike the sensation of fear, are not associated with any perceived threats, including the imminent threat of a seizure. They are sudden in onset, occurring for a short duration with the individual being conscious during this ictal period. During fear sensation, an immediate threat activates the lateral nucleus of the amygdala leading to the central nucleus initiating defensive physiological reactions such as freezing 12 , 13 . Fear conditioning experiments suggest that the ventral hippocampus is involved in the manifestation of fear and anxiety, whereas the dorsal hippocampus is responsible for processing the temporal aspects and contextual cues associated with the fear response 14 , 15 , 16 . The hippocampus integrates contextual cues conveyed by the anterior cingulate cortex retro-splenial cortex (RSC), and post-rhinal cortex, subsequently influencing the anterior hypothalamus to generate an appropriate response, as extensively documented in the fear conditioning literature 17 . It has been postulated that in mTLE patients with amygdalar and hippocampal sclerosis, the occurrence of seizures with fear auras may stem from epileptic activity occurring in the brain circuits involved in defensive behavior 8 , 18 . In a stimulation study assessing visceral and emotional responses in drug-refractory epilepsy patients during pre-surgical evaluation, the amygdala and hippocampus emerged as the structures most frequently associated with these responses 9 . Despite the etiological distinctions between fear sensations and fear auras, there exists a similarity in the brain networks responsible for these disparate behaviors.

In our experimental study, we hypothesize that pairing auditory and visual cues with seizures originating from the amygdala in a rodent model of mTLE may allow for the identification of the associated aural phenomenon. Our study shows mice conditioned to auditory and light cues preceding seizures exhibiting a freezing response upon cue exposure. Notably, these associations develop despite seizure-induced retrograde amnesia 19 , 20 , resembling patient experiences of auras preceding generalized seizures.

Materials and methods

All experiments were carried out in accordance with protocols approved by the Animal Welfare Committees of the University of Lethbridge and followed the guidelines established by the Canadian Council on Animal Care. This study adheres to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for comprehensive reporting of animal research. The study cohort comprised of twelve male C57BL/6 J mice procured from Jackson Laboratories (Bar Harbor, USA). The mice were aged 6 months at the time the experiments started. The animals weighed between 25 and 35 g at the beginning of the experiment. They were housed in transparent plastic cages and kept in a room with a controlled environment, specific-pathogen free, and maintained on a 12-h light/dark cycle with lights on at 07:30 h. The animal cohort was categorized into two groups: the Light and Auditory Tone Conditioned (LAC) group (N = 6), and the Unconditioned (UC) control group (N = 6) (see Experimental Design “ Study design ”.

The mice were anaesthetized with 3% isoflurane and maintained between 1 and 2% for the duration of surgery. Lidocaine (2%) was administered subcutaneously at the incision site. The electrodes were chronically implanted under stereotaxic control. Bipolar electrodes were implanted bilaterally in the basolateral amygdalar nucleus, and a unipolar electrode was inserted into the dorsal hippocampus. In addition, a ground electrode was placed on the cerebellar surface which also served as reference. The inferior tips of the amygdalar electrodes were located at coordinates 1.3 mm posterior to bregma, 3.0 mm lateral to the midline, and 4.6 mm ventral to the skull. The hippocampal electrode was placed at coordinates 2.3 mm posterior to bregma, 1.5 mm lateral to the midline, and 1.3 mm ventral to the skull. In addition, in three out of six LAC mice (experimental results shown in supplementary “Introduction” and figure S1  in Supplementary Information ) bipolar electrodes were also implanted in the retrosplenial cortex, 3 mm posterior to the bregma, 0.3 mm lateral to the midline and 0.5 mm ventral to the cortical surface. The electrodes were constructed from PFA (perfluoroalkoxy) coated, stainless steel wire, 0.114 mm (0.0045 inches) in diameter (A-M Systems, Carlsborg, WA). In our experiments, we used unipolar and bipolar electrodes with a tip separation of 500 microns. The unipolar served only as a recording electrode while the bipolar was used for both stimulation and recording by using a switch. To ensure reliable connections, the wire ends were carefully stripped of Teflon insulation and securely attached to gold-plated stainless-steel female contact pins (Plastics1) to form the recording and stimulating electrodes. These contact pins were affixed to a 6-channel electrode pedestal implant which served as a stable base. During recording and stimulation, the pedestal implant was linked to a mating plug, which was connected to the pre-amplifier and than to the amplifier.

For electrophysiological recordings of local field potential (LFP) we used the hippocampal electrode, contralateral amygdalar electrode, and the stimulating electrode in the ipsilateral amygdala (which also served to record brain activity by a switch). All the electrodes were referenced to the cerebellar ground electrode. By referencing the hippocampal electrode to the cerebellum, we were able to reduce external electrical noise, which affects both locations equally. The rationale for using two electrodes at different depths in the amygdala was to achieve a more local signal and use these electrodes for stimulation. One of the tips of the bipolar amygdalar electrode was also referenced to the cerebellar electrode while recording to reduce the noise mentioned above from the recorded signal. The electrographic data was fed into a unitary gain preamplifier (Grass instruments 7P122 adapter), which then passed the signal into an analog amplifier (Grass Technologies 7P122G Low Level D.C. Amplifier). The signal was then digitized at a sampling rate of 2 kHz through Axon Digidata 1550 A Low-Noise Digitizer and saved on the recording computer using Axoscope software.

We also video recorded the mice (Fig.  1 ) to measure freezing behavior and assess the severity of the behavioral seizures using the Racine scale 21 . The video data was acquired at 30 frames/second through a Raspberry Pi 5-MP (mega-pixel) Camera connected to Raspberry Pi 3 Model B (Pi). The Pi also generated a one-second auditory tone at 12 kHz and activated light-emitting diodes (LEDs) for one second. Additionally, the Pi triggered the Master 8 stimulator to generate the kindling stimulus. To ensure time synchronization between the video data and electrophysiological signals, a custom Python script was used to send signals through the general-purpose input/output (GPIO) pins of the Pi to the electrophysiology recording system for each frame captured by the Raspberry Pi camera. Time stamps were also sent upon generation of the auditory tone, LED flash, and electrical kindling stimulus through the GPIOs.

Experimental apparatus. Raspberry Pi camera was positioned on the roof of the cage to capture the mouse's behavior during the session. The cage was equipped with a pair of LED bulbs placed on opposite walls, while speakers producing auditory tones were positioned outside the other two walls. A small hole in the roof of the cage allowed for the transmission of wired electrophysiology signals from the mouse's brain to an amplifier. The same wire was also used to deliver the electrical kindling stimulus to the animal’s brain.

Experimental design

Kindling protocol.

After 10 days of habituating mice to the recording apparatus, all the animals were kindled once daily to evoke seizures (Fig.  2 ). One of the amygdalar electrodes was electrically ‘kindled’ with 1 s train of 1 ms biphasic pulses at 60 Hz using a Master 8 Pulse Stimulator. The Master 8 produced a constant voltage stimulus, which was then converted to a constant current via a Stimulus isolator unit, A-385 (from World Precision Instruments). The current amplitude of stimulation was started at 50 microamperes and then increased by the same amount till seizures were evoked (electrographic seizures or afterdischarges of 5 s duration) 22 . Upon reaching this current amplitude, all animals received this stimulation for all subsequent training sessions.

Experimental design and timeline. ( a ) Timeline of the LAC group. In the light and auditory tone conditioned (LAC) group, the training sessions consisted of tone and LED flash presentations before a kindling stimulation. ( b ) Timeline of the UC group. In the unconditioned (UC) control group, the training days consisted of kindling stimulation alone. All other experimental procedures: surgery, recovery, habituation, and testing were similar for the two groups.

Sensory cue pairing—Auditory and visual stimulation

For sensory cue pairing with the seizures in the LAC animals, the kindling stimulus was preceded by the auditory tone (presented 4 s before kindling) and LED flash (3 s before kindling onset) (Fig.  2 a and 3 a). The auditory tone was 12 kHz in frequency for one second duration generated by a custom Python code output by 5 V twin speakers (1.2 watts per speaker) with a frequency range of 80 Hz to 20 kHz. The LED flash, lasting for one second, was produced by two LED bulbs triggered by the GPIO pins of a Raspberry Pi through the same code which produced the audio output. The UC animals only received the electrical kindling stimulus (without preceding sensory cues) (Fig.  2 b).

Analysis of electrophysiology signal. ( a ) Representative LFP signal from a LAC animal. The vertical bars represent the time stamps for the auditory stimulus and LED flash respectively. The lightning bolt indicates kindling stimulation following the sound and light presentation. ( b ) Measuring the duration of evoked seizures by thresholding the LFP amplitude. The last ictal spike with amplitude exceeding 10 standard deviations above the baseline activity (before kindling onset) was used to mark the end of seizures (green line). ( c ) Time frequency analysis. Continuous wavelet transform (CWT) of the LFP signal was used to identify the evoked seizures characterized by the increase in power of higher frequencies relative to the baseline activity.

Study design

The experimental sessions were divided into training (kindling) and testing sessions for the LAC and UC animal cohorts. To assess the progression of freezing behavior in the LAC animals across training sessions, where sensory cues (auditory cue followed by visual cue) were paired with amygdalar kindling-induced seizures, movement was quantified following the presentation of the auditory cue. Further details on the video analysis procedures used to quantify movement and movement changes across sessions can be found in “ Video analysis ”. We quantified this change across the training sessions (n = 18) for each LAC animal. The training sessions (n = 18) of the UC animals comprised only the kindling stimulus without any pairing with cues. Assessing freezing behavior across the training sessions enables the measurement of changes over time and allows for the consideration of any potential impact stemming from surgical procedures. Starting from no electrographic or behavioral seizures and progressing to advanced seizures with serial kindling, this evaluation measures the freezing effect attributable to cue pairing during evolving seizure activity in the LAC animals.

Furthermore, to assess the impact of the sensory cue association, we compared the freezing response of LAC animals with that of the UC group across two test sessions, which were interspersed between the training sessions. During these test sessions, animals were subjected solely to sensory cues. The test sessions aimed to determine whether the observed freezing behavior resulted from the pairing of cues with seizures (LAC) or if the repeated seizure exposure (LAC and UC) made the animals more fearful, causing them to freeze in response to sensory cues.

Analysis methods

Local field potential—quantifying the duration of evoked seizures.

The electrophysiology data was first normalized by z-scoring the data and then filtered using a Butterworth filter to remove low-frequency movement artifacts and highlight the slow-frequency band of evoked seizures (2–12 Hz) 23 , 24 . To identify periods of ictal activity, first we selected 60 s of baseline activity before sensory stimulation was applied (Fig.  3 b). As seizures are characterized by a heightened amplitude of local field potentials 25 , we use a threshold of 10 standard deviations calculated from the baseline period to detect ictal spikes during seizure. Once the kindling stimulus is presented, any electrical activity exceeding that threshold was identified as an electrographic seizure. The duration of the seizure was determined as time elapsed from the delivery of the electrical stimulus to the last ictal spike which exceeds the threshold level (Fig.  3 b).

To confirm that the duration of seizures was correctly marked, we also use spectrograms as illustrated in Fig.  3 c. For spectral analysis of the LFP signal, we applied a Butterworth filter with a high-pass frequency of 2 Hz to the normalized the signal, and used continuous wavelet transform (CWT) using complex valued Morse wavelets. As we were interested only in the power, we took the absolute value of the complex signal to measure the magnitude of a specific frequency at a particular time. We used CWT instead of the standard Fourier spectrogram because CWT is more suited for shorter time signals as in our case.

Video analysis

For the animal movement analysis, video frames were extracted 60 s before and after the auditory stimulus. Each frame was converted from red, green and blue (RGB) to grayscale and a thresholding was applied to detect the mouse in each frame, as illustrated in Fig.  4 a. For detecting movement, video was down sampled to 10 frames per second and then consecutive frames were subtracted from each other (Fig.  4 a, right panel). The difference in the number of pixels between consecutive frames was used as a measure of the mouse’s movement. To normalize this movement data, we z-scored each individual session. Following this, we convolved the z-scored data with a 500-ms duration Gaussian window and rescaled the resulting values to a range between zero and ten. Note that when mouse does not move then the number of pixels different between frames is close to zero. The freezing duration was determined by setting a threshold near zero (specifically, 1 on the rescaled movement values). A sample of movement measure during two session is presented in Figs.  4 b. We quantified changes in movement during the session by calculating the mean movement and freezing duration within a three-second window before and after the auditory stimulus presentation. The differences between post-cue and pre-cue mean movements, as well as post-cue and pre-cue freezing durations, were then used for statistical analysis. Due to technical problems, we missed data from few sessions for two animals, which resulted in a smaller number of points for those sessions in our plots.

Video analysis to quantify freezing behavior. ( a ) Video data pre-processing. The RGB frames were converted to grayscale and a thresholding was applied to detect the mice. Next, we did frame-differencing to quantify movement of mice between frames ( b ). Movement analysis of a LAC mouse during a training (kindling) session. This displays the mouse movement during a kindling evoked seizure paired with cues (auditory tone and LED flash). Post-cue freezing, represented by a red line, occurs in the three-second window after presentation of the auditory tone and before the kindling stimulus. After the kindling stimulus, the animal’s behavior progresses to a Racine Stage 5 seizure (see Video 1 in the supplementary materials showing animal during this session).

Kindling led to advanced seizures in all animals

Representative electrographic seizures recorded from the dorsal hippocampus (from the hemisphere ipsilateral to the stimulating amygdalar electrodes) in the LAC and UC animals are shown in Fig.  5 a,b respectively. In both groups of animals, the kindling procedure evoked seizures, with the duration of seizures increasing over stimulation sessions (Fig.  6 a). The duration plotted in Fig.  6 represents electrographic data recorded from the vicinity of the stimulating electrodes implanted in the amygdala. A Pearson correlation coefficient between the sessions and seizure duration was 0.446 with p-value = 3.207e-10. The rate of change in seizure duration was quantified by a linear regression analysis giving a positive slope of 0.978 between the sessions and the duration of the evoked seizures (Fig.  6 a). Across days, seizures developed similarly in both LAC and UC groups (see Video 1 in the supplementary material showing a representative LAC animal during the kindling sessions with behavioral seizures with Racine stage 5 (rearing and falling) 21 ). The Anova analysis did not reveal any difference in the seizure duration between LAC and UC groups as evidenced by a p-value of 0.181 (Fig.  6 b), and neither a difference between groups across days (p-value = 0.159). These results show that in both LAC and UC groups there was a similar progressive increase in the duration of seizures over days, thus providing validation that UC animals can be used as a proper control group for LAC animals.

Examples of recorded seizures. ( a ). Time frequency analysis of a training session for a LAC (auditory tone – LED – Kindling stimulus) animal. ( b ). Time frequency analysis of a training session for a UC (kindling stimulus alone) animal. Figure 5a and 5b show local field potentials (LFPs) and continuous wavelet transform (CWT) analyses for representative LAC ( a ) and UC animals ( b ) respectively. In the top panels of both ( a ) and ( b ), LFP recordings were thresholded at 10 standard deviations above baseline (see also Fig.  3 ) to measure the duration of evoked seizures following kindling. The wavelet analysis in the bottom panels shows seizure activity, as indicated by an increase in high-frequency power relative to baseline activity in both animal groups.

Progression of seizure duration across kindling sessions by group. ( a ). Each data point represents the seizure duration in a single animal during a single session. Linear regression highlights the positive correlation between session progression and seizure duration. ( b ) Boxplot comparing seizure durations between the LAC and UC groups. Statistical analysis using Anova shows no significant difference in seizure duration between the two groups.

Behavioral changes in the LAC group after sensory cue presentation

Using video analyses as described in the Methods section, we quantified the amount of change in animal movement following the presentation of sensory cues as a function of the kindling training sessions. The animals in the LAC group exhibited a reduction in post-cue movement as the kindling stimulation continued over sessions (Fig.  7 a). This change was quantified by measuring mean movement within a three-second window following the auditory cue presentation and subtracting the mean movement within the same period preceding the cue presentation (see Fig.  4 a). To examine the relationship between this difference in mean movement and the number of sessions, we calculated a squared correlation coefficient (R-squared = 0.165, Pearson’s correlation coefficient = -0.406). This R-squared value means that approximately 16.5% of the variance in the movement can be attributed to the number of training sessions (with cue exposure paired with kindling). Furthermore, we also calculated the F-statistic (18.8; p-value = 0.000036***) confirming that this effect was statistically significant (Fig.  7 a). The slope of the linear regression model that fit the data had a negative slope (regression coefficient = -0.1594) which provides information on how quickly the animal’s movement decreased after cue presentation across sessions.

Behavioral change in animals due to cue association with seizures. ( a ) Regression showing an association between sessions and post-cue movement in LAC animals. The progressive change in the difference in mean movement following sensory cue presentation across kindling sessions is plotted. The regression analysis illustrates a negative relation between the amount of movement and the session number. ( b ) Individual LAC Movement. Data for individual animals is shown here, where the progression of the change in movement following cue presentation from the initial stages of kindling (first three training sessions) to the last three training sessions are compared. The negative slope for all animals shows that movement reduction was consistent across all LAC animals. ( c ) Regression showing an association between sessions and post-cue freezing in LAC animals. Plot illustrating the increase in freezing behavior across sessions. Freezing duration was defined as time without movement within 3 s before or after cue presentation (Methods Sect. 2.5.2 and Fig.  4 ). ( d ) Group comparison of movement change due to cue association. Comparison of the difference in mean movement following cue presentation between LAC and UC groups during the test sessions (only cues). Animals in LAC group showed significantly fewer movements as compared to the control UC group in which kindling was not associated with sensory cues.

The decrease in movement after cue presentation was observed in all LAC animals. This is illustrated in Fig.  7 b, showing average post-cue movement difference during the first three training sessions and during the last three training sessions for each animal. We tested for statistical significance in post-cue movement difference across sessions by comparing these first three and last three training sessions, using a one-way Anova. The Anova analysis showed a significant F-statistic of 16.55 with a p-value of 0.000266(***), indicating a significant difference in movement patterns. Additionally, the Paired t-test was used, which also showed a significant t-statistic of 3.531 with p-value of 0.000257(***), further supporting the presence of an association between cues and seizures as evidenced by a behavioral alteration. In addition, a non-parametric test, namely Kolmogorov–Smirnov (KS) test was conducted. The D-statistic, which is the maximum diagonal distance between the first and last three training sessions, was 0.556 and the p-value was 0.0067 demonstrating significant statistical difference. All these analyses of the training sessions support the hypothesis that LAC animals learned to associate sensory cues with incoming seizure.

To ensure that our analyses are not sensitive to a specific definition of movement, we repeated analyses using animal freezing duration. The freezing duration was defined as the amount of time within 3 s after cue presentation, when movement was smaller than 1 unit (see Methods). We observed a positive relation between sessions and the difference in freezing duration after cue presentation (Fig.  7 c) which was consistent with our prior finding (Fig.  7 a). The squared correlation coefficient between freezing duration and session number: R-squared was 0.108 (Pearson’s correlation coefficient = 0.3279), showing that approximately 10.8% of the variation in freezing duration can be explained by the number of cue exposure sessions. Moreover, the linear regression model was found to be statistically significant, as evidenced by the F-statistic of 11.4 and a p-value of 0.00104. Thus, all the above analyses showed that pairing sensory cues with incoming seizures resulted in a significant change in behaviour in LAC animals.

We further verified this effect by comparing movement after cue presentation between LAC and UC groups during the test sessions. Note that in UC animals’ sensory cues were not presented during kindling sessions, however, after animals developed seizures, we had test sessions, where animals were only presented with sensory cues (not followed by electrical stimulation; Fig.  2 ). Similarly, in LAC animals, after seizure developed, we had test sessions where we presented only sensory cues. Out of the four sessions two were selected where the animal had previously experienced a generalized seizure in the training sessions. This choice was made so that test sessions with a similar seizure stage in all animals were used for making the comparison. Representative videos from such test sessions are included in Supplementary Materials: Video 2 for the LAC group and Video 3 for the UC group. For this comparison between LAC and UC group, we calculated the mean movement before and after the cue presentation over a longer time window: 20 s time window. We then performed a one-way Anova test on the difference between post-cue mean movement and pre-cue mean movement. The test yielded a F-value of 28.95 and a p-value of 0.000025 (Fig.  7 d). To ensure the robustness of our statistical findings, we employed the non-parametric Kolmogorov–Smirnov test. The obtained D-statistic of 0.75, accompanied by a p-value of 0.00078, supports the statistical significance of our results. Those results are consistent with the results presented in Fig.  7 a,b, confirming the behavioral changes in LAC animals due to association of sensory cues with seizures. Note that using UC animals as a control group, it allowed us to test that kindled animals are not becoming just more ‘fearful’ to sensory stimuli due to experiencing seizures, but probably because of the sensory cues being conditioned to seizure phenomenology.

In three out of these six LAC animals following the sensory cue pairing with evoked seizures, retrosplenial cortex (RSC) stimulation was paired with the evoked seizures (Supplementary Sect. 1 and Figure S1  in Supplementary Information ). All of these animals exhibited a progressive increase in freezing behavior following RSC stimulation across sessions where RSC stimulation was paired with the evoked seizures (RSC training sessions). For statistical analysis, refer to Supplementary Sect. 1.

We repeated our LAC experiments in a set of nine animals, where we paired the auditory and visual cues in a similar fashion (Supplementary Sect. 2 and figure S2  in Supplementary Information ). Here we observed a similar trend in the animals’ behavior, with the animals progressively freezing due to the pairing of sensory cues with evoked seizures across sessions displaying reproducibility of our experimental results. For further details refer to supplementary Sect. 2 (Supplementary Information).

In our experiments, continual temporal pairing of sensory cues with evoked seizures over several sessions in an animal model of temporal lobe epilepsy resulted in a progressive change in the animals' behavior. This change was quantified as an increase in freezing behavior across the sessions. These findings are interesting because amnesia is associated with seizures, specifically amnesia for events just preceding the seizure onset 20 , 26 , 27 , 28 . The ability of LAC animals to recall sensory cues presented prior to seizures raises an important question: how do they remember these cues?

A cardinal feature of generalized seizures, distinguished by abnormal synchronous activity in both cerebral hemispheres, is loss of consciousness followed by recovery with the individual having no memory of the episode 29 . This amnestic event is particularly prevalent in cases of secondarily generalized tonic–clonic seizures originating from the temporal lobe 30 . In a clinical study of mTLE patients, high seizure frequency was shown to be associated with greater deficits in autobiographical memory, relating to one’s own experiences, as reported in anterograde memory tests 31 . Clinical studies evaluating memory deficits in mTLE patients revealed amnesia for events preceding up to two years 32 , 33 , 34 . In many of these instances, patients were conscious during the seizure's onset and even made efforts to seek assistance from a nurse. However, they subsequently fail to recall the events associated with the ictal episode and even their attempt to seek help 30 .

Electroconvulsive therapy (ECT), a treatment inducing generalized tonic–clonic convulsions recommended for medically intractable affective disorders, usually causes retrograde amnesia affecting both episodic and semantic memory 19 , 35 , 36 , 37 , 38 , 39 . In an experimental study, mice subjected to pentylenetetrazole-induced seizure immediately after being trained on a spatial memory task demonstrated a notable decline in their subsequent performance on the following day, indicating that seizures induce retrograde amnesia 20 . This is further supported by a study in which rats were unable to create memories for a spatial task after being subjected to electroconvulsive seizures suggesting that seizures cause a deficit similar to bilateral hippocampal lesions 40 . Both the above-mentioned studies also found a correlation between impaired task performance and the effects of seizures on long-term potentiation (LTP) 20 , 40 .

However, there are also studies providing evidence of the epileptic subjects having some degree of recall regarding events associated with the epileptic attack. A study testing the association of environmental cues with amygdalar kindling displayed rats developing a preference for regions in the experimental apparatus where seizures were not evoked 41 . Building upon the previous study, further investigations were conducted by the same group to explore the conditioning effects to other cues associated with kindling, specifically the consumption of flavored solutions 42 . Their study revealed that the association of kindling with flavored solutions resulted in an aversive response in the rats. It is relevant to mention that in these studies, kindling brain structures in the limbic cortex, namely the basolateral amygdala and dorsal hippocampus were more strongly associated with the behavioral changes when compared to the effects observed due to kindling non-limbic brain areas.

A similar change in behavior is seen in our current experiments, where mice exposed to an auditory tone and a LED flash seconds before the electrical kindling (to evoke seizures), were able to remember this association as demonstrated by freezing behavior. This freezing behavior increased in intensity across the training sessions. We propose that as in classical conditioning, the sensory cues (conditioned stimulus) associate with the aural stage of seizures (unconditioned response) during which the mice retain consciousness and have memory preservation 43 , 44 , 45 . Consequently, the LAC animals demonstrated an ability to retain these cues in memory, leading to the development of an associated conditioned response that could potentially activate the brain networks associated with the epileptic circuit 46 . Even if sensory cues do not cause the entire seizure epoch as during kindling, our experiments suggest conditioning to the aural event.

The experiments described in Supplementary Sect. 1 ( Supplementary Information ), where RSC stimulation paired with evoked seizures led to progressive freezing behavior, have interesting implications. Initially, these animals had already experienced sensory cue pairing with seizures, which also resulted in progressive freezing. The RSC encodes contextual information and integrates various sensory inputs, including visual, auditory, and somatosensory stimuli 47 , 48 , 49 . Because RSC stimulation occurred in the same environment where cue-seizure pairing previously took place, it suggests that this stimulation might activate associated sensory networks, potentially eliciting sensations without distinct sensory percepts. Additionally, these interoceptive sensations, if present, paired with evoked seizures, precede the behavioral responses, resembling an aura-like phenomenon. However, since this phenomenon was observed in a limited sample set, it is important to replicate the experiment with a larger cohort of animals.

In summary, our experiments provide an important advancement toward developing an animal model of auras. Firstly, like auras, sensory sensation take place before the start of behavioral seizures. Secondly, analogous to clinical auras, our experimental animals remembered sensory sensations as demonstrated by freezing response. Thirdly, supplementing our experiments by additional electrical stimulation to sensory association cortex (RSC) led to a similar freezing response, which could be due to a phenomenon resembling clinical auras.

Establishing an animal model of auras can lead to multiple benefits. Firstly, seizures are unpredictable events, thus being able to study auras in more details could help to more reliably identify incoming seizure onset 50 . Secondly, model of auras could allow us to test new treatments to interrupt epileptic networks exactly before seizure onset. Thirdly, auras help to localize the epileptogenic zone 51 and we could use single neuron recordings and 2-photon imaging in rodents to better understand this concept. The identification of the epileptogenic zone is extremely crucial in drug resistant seizures where surgical resection is the gold standard. Finally, the presence of specific auras serves as prognostic markers for the surgical outcome in epileptic patients with drug resistant epilepsy 52 . Combining electrophysiology with imaging in animal models to identify brain activity associated with auras could help detect epileptogenic regions often overlooked during resection surgeries, thereby significantly improving patient prognosis.

Data availability

All data supporting described findings can be obtained from the corresponding authors (R.D. and A.L.) upon reasonable request, and sample videos used for our analysis are provided in Supplementary Materials.

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Acknowledgements

The authors thank I Esteves, H Chang, R Pais, D Pahwa, A Neumann, Y Kaushik, M Ello, R Singh, J Higham, M Pearson, B Ponech, T Miller and K Iyer for help on this project. We also thank Ian Q. Whishaw, Bryan Kolb, Bruce L. McNaughton, and G. Campbell (Cam) Teskey for inspiring discussion on the relation between seizures and physiological brain functions.

This work was supported by a CIHR Project grant and NSERC DG to AL and Alberta Innovates –Data Enabled Innovations Graduate Student Scholarship awarded to RD.

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R.D. and A.L. designed the experiment. R.D., A.M. and C.H. conducted the experiments and collected the data. R.D. and C.H. performed the surgeries. V.L., A.M., and C.H. performed the histology for the brain sections. R.D. and A.L. analyzed the data and wrote the manuscript, which all authors helped to revised.

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Das, R., Howey, C., McFetridge, A. et al. Associating sensory cues with incoming seizures: developing an animal model of auras. Sci Rep 14 , 20881 (2024). https://doi.org/10.1038/s41598-024-71885-3

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DOI : https://doi.org/10.1038/s41598-024-71885-3

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